Development of VHH antibodies against dengue virus type 2 NS1 and comparison with monoclonal antibodies for use in immunological diagnosis.

PubMed

Fatima, Aneela; Wang, Haiying; Kang, Keren; Xia, Liliang; Wang, Ying; Ye, Wei; Wang, Jufang; Wang, Xiaoning

2014-01-01

The possibility of using variable domain heavy-chain antibodies (VHH antibodies) as diagnostic tools for dengue virus (DENV) type 2 NS1 protein was investigated and compared with the use of conventional monoclonal antibodies. After successful expression of DENV type 2 NS1 protein, the genes of VHH antibodies against NS1 protein were biopanned from a non-immune llama library by phage display. VHH antibodies were then expressed and purified from Escherichia coli. Simultaneously, monoclonal antibodies were obtained by the conventional route. Sequence analysis of the VHH antibodies revealed novel and long complementarity determining regions 3 (CDR3). Epitope mapping was performed via a phage display peptide library using purified VHH and monoclonal antibodies as targets. Interestingly, the same region of NS1, which comprises amino acids 224HWPKPHTLW232, was conserved for both kinds of antibodies displaying the consensus motif histidine-tryptophan-tryptophan or tryptophan-proline-tryptophan. The two types of antibodies were used to prepare rapid diagnostic kits based on immunochromatographic assay. The VHH antibody immobilized rapid diagnostic kit showed better sensitivity and specificity than the monoclonal antibody immobilized rapid diagnostic kit, which might be due to the long CDR3 regions of the VHH antibodies and their ability to bind to the pocket and cleft of the targeted antigen. This demonstrates that VHH antibodies are likely to be an option for developing point-of-care tests against DENV infection.

Development of VHH Antibodies against Dengue Virus Type 2 NS1 and Comparison with Monoclonal Antibodies for Use in Immunological Diagnosis

PubMed Central

Fatima, Aneela; Wang, Haiying; Kang, Keren; Xia, Liliang; Wang, Ying; Ye, Wei; Wang, Jufang; Wang, Xiaoning

2014-01-01

The possibility of using variable domain heavy-chain antibodies (VHH antibodies) as diagnostic tools for dengue virus (DENV) type 2 NS1 protein was investigated and compared with the use of conventional monoclonal antibodies. After successful expression of DENV type 2 NS1 protein, the genes of VHH antibodies against NS1 protein were biopanned from a non-immune llama library by phage display. VHH antibodies were then expressed and purified from Escherichia coli. Simultaneously, monoclonal antibodies were obtained by the conventional route. Sequence analysis of the VHH antibodies revealed novel and long complementarity determining regions 3 (CDR3). Epitope mapping was performed via a phage display peptide library using purified VHH and monoclonal antibodies as targets. Interestingly, the same region of NS1, which comprises amino acids 224HWPKPHTLW232, was conserved for both kinds of antibodies displaying the consensus motif histidine-tryptophan-tryptophan or tryptophan-proline-tryptophan. The two types of antibodies were used to prepare rapid diagnostic kits based on immunochromatographic assay. The VHH antibody immobilized rapid diagnostic kit showed better sensitivity and specificity than the monoclonal antibody immobilized rapid diagnostic kit, which might be due to the long CDR3 regions of the VHH antibodies and their ability to bind to the pocket and cleft of the targeted antigen. This demonstrates that VHH antibodies are likely to be an option for developing point-of-care tests against DENV infection. PMID:24751715

Structural Correlates of Antibodies Associated with Acute Reversal of Amyloid β-related Behavioral Deficits in a Mouse Model of Alzheimer Disease

PubMed Central

Basi, Guriqbal S.; Feinberg, Hadar; Oshidari, Farshid; Anderson, John; Barbour, Robin; Baker, Jeanne; Comery, Thomas A.; Diep, Linnea; Gill, Davinder; Johnson-Wood, Kelly; Goel, Amita; Grantcharova, Katerina; Lee, Mike; Li, Jingzhi; Partridge, Anthony; Griswold-Prenner, Irene; Piot, Nicolas; Walker, Don; Widom, Angela; Pangalos, Menelas N.; Seubert, Peter; Jacobsen, J. Steven; Schenk, Dale; Weis, William I.

2010-01-01

Immunotherapy targeting of amyloid β (Aβ) peptide in transgenic mouse models of Alzheimer disease (AD) has been widely demonstrated to resolve amyloid deposition as well as associated neuronal, glial, and inflammatory pathologies. These successes have provided the basis for ongoing clinical trials of immunotherapy for treatment of AD in humans. Acute as well as chronic Aβ-targeted immunotherapy has also been demonstrated to reverse Aβ-related behavioral deficits assessing memory in AD transgenic mouse models. We observe that three antibodies targeting the same linear epitope of Aβ, Aβ3–7, differ in their ability to reverse contextual fear deficits in Tg2576 mice in an acute testing paradigm. Reversal of contextual fear deficit by the antibodies does not correlate with in vitro recognition of Aβ in a consistent or correlative manner. To better define differences in antigen recognition at the atomic level, we determined crystal structures of Fab fragments in complex with Aβ. The conformation of the Aβ peptide recognized by all three antibodies was highly related and is also remarkably similar to that observed in independently reported Aβ:antibody crystal structures. Sequence and structural differences between the antibodies, particularly in CDR3 of the heavy chain variable region, are proposed to account for differing in vivo properties of the antibodies under study. These findings provide a structural basis for immunotherapeutic strategies targeting Aβ species postulated to underlie cognitive deficits in AD. PMID:19923222

Single-dose Universal Hepatitis A Immunization in One-year-old Children in Argentina: High Prevalence of Protective Antibodies up to 9 Years After Vaccination.

PubMed

Urueña, Analía; González, Jorge E; Rearte, Analía; Pérez Carrega, María E; Calli, Rogelio; Pagani, María F; Uboldi, Andrea; Vicentín, Rosalía; Caglio, Patricia; Cañero-Velasco, María C; Gentile, Angela; Ramonet, Margarita; Vizzotti, Carla

2016-12-01

Single-dose hepatitis A virus (HAV) vaccination was implemented in all Argentinean children 12 months of age in 2005. Previous studies demonstrated high prevalence of protective antibody response 4 years after single-dose vaccination. This study assessed long-term seroprotection against HAV after vaccination. Children who received 1 dose of HAV vaccine at 1 year of age at least 6 years before enrollment were included at 5 centers in Argentina between 2013 and 2014. Demographic and socioeconomic characteristics were collected through a questionnaire. Blood samples were tested for anti-HAV antibodies. Antibody values ≥10 mIU/mL were considered seroprotective. Logistic regression analysis was performed to evaluate the association between demographic and socioeconomic variables and seroprotection. A total of 1088 children were included, with a median postvaccination interval of 7.7 years (range 6.3-9.2 years). Of these children, 97.4% (95% confidence interval: 96.3%-98.3%) had protective antibodies against HAV. No association between demographic or socioeconomic variables and seroprotection was found. Geometric mean concentration of antibody levels against HAV was 170.5 mUI/mL (95% confidence interval: 163.2-178.2 mUI/mL). Single-dose universal hepatitis A immunization in 1-year-old children resulted in sustained immunologic protection for up to 9 years in Argentina. These findings, along with the low current disease burden, confirm the success of the intervention.

Structure-Guided Combinatorial Engineering Facilitates Affinity and Specificity Optimization of Anti-CD81 Antibodies.

PubMed

Nelson, Bryce; Adams, Jarrett; Kuglstatter, Andreas; Li, Zhijian; Harris, Seth F; Liu, Yang; Bohini, Sandya; Ma, Han; Klumpp, Klaus; Gao, Junjun; Sidhu, Sachdev S

2018-07-06

Hepatitis C viral infection is the major cause of chronic hepatitis that affects as many as 71 million people worldwide. Rather than target the rapidly shifting viruses and their numerous serotypes, four independent antibodies were made to target the host antigen CD81 and were shown to block hepatitis C viral entry. The single-chain variable fragment of each antibody was crystallized in complex with the CD81 large extracellular loop in order to guide affinity maturation of two distinct antibodies by phage display. Affinity maturation of antibodies using phage display has proven to be critical to therapeutic antibody development and typically involves modification of the paratope for increased affinity, improved specificity, enhanced stability or a combination of these traits. One antibody was engineered for increased affinity for human CD81 large extracellular loop that equated to increased efficacy, while the second antibody was engineered for cross-reactivity with cynomolgus CD81 to facilitate animal model testing. The use of structures to guide affinity maturation library design demonstrates the utility of combining structural analysis with phage display technologies. Copyright © 2018 Elsevier Ltd. All rights reserved.

Comparison of humanized IgG and FvFc anti-CD3 monoclonal antibodies expressed in CHO cells.

PubMed

Serpieri, Flavia; Inocencio, Andre; de Oliveira, Jose Marcelino; Pimenta, Alécio A; Garbuio, Angélica; Kalil, Jorge; Brigido, Marcelo M; Moro, Ana Maria

2010-07-01

Two humanized monoclonal antibody constructs bearing the same variable regions of an anti-CD3 monoclonal antibody, whole IgG and FvFc, were expressed in CHO cells. Random and site-specific integration were used resulting in similar expression levels. The transfectants were selected with appropriate selection agent, and the surviving cells were plated in semi-solid medium for capture with FITC-conjugated anti-human IG antibody and picked with the robotic ClonePix FL. Conditioned media from selected clones were purified by affinity chromatography and characterized by SDS-PAGE, Western-blot, SEC-HPLC, and isoelectric focusing. Binding to the target present in healthy human mononuclear cells was assessed by flow cytometry, as well as by competition between the two constructs and the original murine monoclonal antibody. The humanized constructs were not able to dislodge the murine antibody while the murine anti-CD3 antibody could dislodge around 20% of the FvFc or IgG humanized versions. Further in vitro and in vivo pre-clinical analyses will be carried out to verify the ability of the humanized versions to demonstrate the immunoregulatory profile required for a humanized anti-CD3 monoclonal antibody.

Potent Inhibition of Human Immunodeficiency Virus Type 1 Replication by an Intracellular Anti-Rev Single-Chain Antibody

NASA Astrophysics Data System (ADS)

Duan, Lingxun; Bagasra, Omar; Laughlin, Mark A.; Oakes, Joseph W.; Pomerantz, Roger J.

1994-05-01

Human immunodeficiency virus type 1 (HIV-1) has a complex life cycle, which has made it a difficult target for conventional therapeutic modalities. A single-chain antibody moiety, directed against the HIV-1 regulatory protein Rev, which rescues unspliced viral RNA from the nucleus of infected cells, has now been developed. This anti-Rev single-chain construct (SFv) consists of both light and heavy chain variable regions of an anti-Rev monoclonal antibody, which, when expressed intracellularly within human cells, potently inhibits HIV-1 replication. This intracellular SFv molecule is demonstrated to specifically antagonize Rev function. Thus, intracellular SFv expression, against a retroviral regulatory protein, may be useful as a gene therapeutic approach to combat HIV-1 infections.

Site-directed antibody immobilization using a protein A-gold binding domain fusion protein for enhanced SPR immunosensing.

PubMed

de Juan-Franco, Elena; Caruz, Antonio; Pedrajas, J R; Lechuga, Laura M

2013-04-07

We have implemented a novel strategy for the oriented immobilization of antibodies onto a gold surface based on the use of a fusion protein, the protein A-gold binding domain (PAG). PAG consists of a gold binding peptide (GBP) coupled to the immunoglobulin-binding domains of staphylococcal protein A. This fusion protein provides an easy and fast oriented immobilization of antibodies preserving its native structure, while leaving the antigen binding sites (Fab) freely exposed. Using this immobilization strategy, we have demonstrated the performance of the immunosensing of the human Growth Hormone by SPR. A limit of detection of 90 ng mL(-1) was obtained with an inter-chip variability lower than 7%. The comparison of this method with other strategies for the direct immobilization of antibodies over gold surfaces has showed the enhanced sensitivity provided by the PAG approach.

Measles virus–specific plasma cells are prominent in subacute sclerosing panencephalitis CSF

PubMed Central

Owens, G.P.; Ritchie, A.M.; Gilden, D.H.; Burgoon, M.P.; Becker, D.; Bennett, J.L.

2012-01-01

Objective To demonstrate the specificity of expanded CD138+ plasma cell clones recovered from the CSF of a patient with subacute sclerosing panencephalitis (SSPE) for measles virus (MV). Methods IgG variable region sequences of single-antibody-secreting CD138+ cells sorted from SSPE CSF were amplified by single-cell PCR and analyzed. Human IgG1 recombinant antibodies (rAbs) were produced from four expanded CD138+ clones and assayed for immunoreactivity against MV proteins. Results Clonal expansion was a prominent feature of the SSPE plasma cell repertoire, and each of the four rAbs assayed was specific for either the MV fusion or the MV nucleocapsid protein. Conclusions Expanded plasma cell clones in the CSF of patients with subacute sclerosing panencephalitis produce disease-relevant antibodies. Recombinant antibodies derived from CSF B cells could provide a tool to identify target antigens in idiopathic inflammatory disorders. PMID:17515543

Modular Construction of Large Non-Immune Human Antibody Phage-Display Libraries from Variable Heavy and Light Chain Gene Cassettes.

PubMed

Lee, Nam-Kyung; Bidlingmaier, Scott; Su, Yang; Liu, Bin

2018-01-01

Monoclonal antibodies and antibody-derived therapeutics have emerged as a rapidly growing class of biological drugs for the treatment of cancer, autoimmunity, infection, and neurological diseases. To support the development of human antibodies, various display techniques based on antibody gene repertoires have been constructed over the last two decades. In particular, scFv-antibody phage display has been extensively utilized to select lead antibodies against a variety of target antigens. To construct a scFv phage display that enables efficient antibody discovery, and optimization, it is desirable to develop a system that allows modular assembly of highly diverse variable heavy chain and light chain (Vκ and Vλ) repertoires. Here, we describe modular construction of large non-immune human antibody phage-display libraries built on variable gene cassettes from heavy chain and light chain repertoires (Vκ- and Vλ-light can be made into independent cassettes). We describe utility of such libraries in antibody discovery and optimization through chain shuffling.

Obligate multivalent recognition of cell surface tomoregulin following selection from a multivalent phage antibody library.

PubMed

Heitner, Tara; Satozawa, Noboru; McLean, Kirk; Vogel, David; Cobb, Ronald R; Liu, Bing; Mahmoudi, Mithra; Finster, Silke; Larsen, Brent; Zhu, Ying; Zhou, Hongxing; Müller-Tiemann, Beate; Monteclaro, Felipe; Zhao, Xiao-Yan; Light, David R

2006-12-01

A therapeutic antibody candidate (AT-19) isolated using multivalent phage display binds native tomoregulin (TR) as a mul-timer not as a monomer. This report raises the importance of screening and selecting phage antibodies on native antigen and reemphasizes the possibility that potentially valuable antibodies are discarded when a monomeric phage display system is used for screening. A detailed live cell panning selection and screening method to isolate multivalently active antibodies is described. AT-19 is a fully human antibody recognizing the cell surface protein TR, a proposed prostate cancer target for therapeutic antibody internalization. AT-19 was isolated from a multivalent single-chain variable fragment (scFv) antibody library rescued with hyperphage. The required multivalency for isolation of AT-19 is supported by fluorescence activated cell sorting data demonstrating binding of the multivalent AT-19 phage particles at high phage concentrations and failure of monovalent particles to bind. Pure monomeric scFv AT-19 does not bind native receptor on cells, whereas dimeric scFv or immunoglobulin G binds with nanomolar affinity. The isolation of AT-19 antibody with obligate bivalent binding activity to native TR is attributed to the use of a multivalent display of scFv on phage and the method for selecting and screening by alternate use of 2 recombinant cell lines.

Enhancement and Analysis of Human Antiaflatoxin B1 (AFB1) scFv Antibody-Ligand Interaction Using Chain Shuffling.

PubMed

Rangnoi, Kuntalee; Choowongkomon, Kiattawee; O'Kennedy, Richard; Rüker, Florian; Yamabhai, Montarop

2018-06-06

A human antiaflatoxin B1 (AFB1) scFv antibody (yAFB1-c3), selected from a naı̈ve human phage-displayed scFv library, was used as a template for improving and analysis of antibody-ligand interactions using the chain-shuffling technique. The variable-heavy and variable-light (VH/VL)-shuffled library was constructed from the VH of 25 preselected clones recombined with the VL of yAFB1-c3 and vice versa. Affinity selection from these libraries demonstrated that the VH domain played an important role in the binding of scFv to free AFB1. Therefore, in the next step, VH-shuffled scFv library was constructed from variable-heavy (VH) chain repertoires, amplified from the naı̈ve library, recombined with the variable-light (VL) chain of the clone yAFB1-c3. This library was then used to select a specific scFv antibody against soluble AFB1 by a standard biopanning method. Three clones that showed improved binding properties were isolated. Amino acid sequence analysis indicated that the improved clones have amino acid mutations in framework 1 (FR1) and the complementarity determining region (CDR1) of the VH chain. One clone, designated sAFH-3e3, showed 7.5-fold improvement in sensitivity over the original scFv clone and was selected for molecular binding studies with AFB1. Homology modeling and molecular docking were used to compare the binding of this and the original clones. The results confirmed that VH is more important than VL for AFB1 binding.

A mutagenesis study of a catalytic antibody

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jackson, D.Y.; Prudent, J.R.; Baldwin, E.P.

1991-01-01

The authors have generated seven site-specific mutations in the genes encoding the variable region of the heavy chain domain (V{sub H}) of the phosphocholine-binding antibody S107.S107 is a member of a family of well-characterized highly homologous antibodies that bind phosphorylcholine mono- and diesters. Two of these antibodies, MOPC-167 and T15, have previously been shown to catalyze the hydrolysis of 4-nitrophenyl N-trimethylammonioethyl carbonate. Two conserved heavy-chain residues, Tyr-33 and Arg-52, were postulated to be involved in binding and hydrolysis of 4-nitrophenylcholine carbonate esters. To more precisely define the catalytic roles of these residues, three Arg-52 mutants (R52K, R52Q, R52C) and fourmore » Tyr-33 mutants (Y33H, Y33F, Y33E, Y33D) of antibody S107 were generated. The genes encoding the V{sub H} binding domain of S107 were inserted into plasmid pUC-fl, and in vitro mutagenesis was performed. These results not only demonstrate the importance of electrostatic interactions in catalysis by antibody S107 but also show that catalytic side chains can be introduced into antibodies to enhance their catalytic efficiency.« less

Principles for computational design of binding antibodies

PubMed Central

Pszolla, M. Gabriele; Lapidoth, Gideon D.; Norn, Christoffer; Dym, Orly; Unger, Tamar; Albeck, Shira; Tyka, Michael D.; Fleishman, Sarel J.

2017-01-01

Natural proteins must both fold into a stable conformation and exert their molecular function. To date, computational design has successfully produced stable and atomically accurate proteins by using so-called “ideal” folds rich in regular secondary structures and almost devoid of loops and destabilizing elements, such as cavities. Molecular function, such as binding and catalysis, however, often demands nonideal features, including large and irregular loops and buried polar interaction networks, which have remained challenging for fold design. Through five design/experiment cycles, we learned principles for designing stable and functional antibody variable fragments (Fvs). Specifically, we (i) used sequence-design constraints derived from antibody multiple-sequence alignments, and (ii) during backbone design, maintained stabilizing interactions observed in natural antibodies between the framework and loops of complementarity-determining regions (CDRs) 1 and 2. Designed Fvs bound their ligands with midnanomolar affinities and were as stable as natural antibodies, despite having >30 mutations from mammalian antibody germlines. Furthermore, crystallographic analysis demonstrated atomic accuracy throughout the framework and in four of six CDRs in one design and atomic accuracy in the entire Fv in another. The principles we learned are general, and can be implemented to design other nonideal folds, generating stable, specific, and precise antibodies and enzymes. PMID:28973872

Ultrasensitive direct impedimetric immunosensor for detection of serum HER2.

PubMed

Sharma, Shikha; Zapatero-Rodríguez, Julia; Saxena, Rahul; O'Kennedy, Richard; Srivastava, Sudha

2018-05-30

Assesment of human epidermal growth factor receptor 2 status is a key factor prompting definitive treatment decisions that help in reducing mortality rates associated with breast cancer. In this article, highly sensitive and low-cost impedimetric immunosensor using single-chain fragment variable antibody fragments was developed for quantitative detection of human epidermal growth factor receptor 2 from serum employing gold nanoparticle-modified disposable screen-printed carbon electrodes. The gold nanoparticles facilitate fast electron transfer and offer a biocompatible surface for immobilization of small antibody fragments in an oriented manner, resulting in improved antigen binding efficiency. The single-chain fragment variable antibody fragment-modified screen printed immunosensor exhibits wide dynamic range of 0.01-100 ng mL -1 and detection limit of 0.01 ng mL -1 . The advantages offered by this platform in terms of high sensitivity, broad dynamic range and low-cost demonstrates great potential for improved monitoring of human epidermal growth factor receptor 2 levels for the management of breast and other cancers. Copyright © 2018 Elsevier B.V. All rights reserved.

The constant region affects antigen binding of antibodies to DNA by altering secondary structure.

PubMed

Xia, Yumin; Janda, Alena; Eryilmaz, Ertan; Casadevall, Arturo; Putterman, Chaim

2013-11-01

We previously demonstrated an important role of the constant region in the pathogenicity of anti-DNA antibodies. To determine the mechanisms by which the constant region affects autoantibody binding, a panel of isotype-switch variants (IgG1, IgG2a, IgG2b) was generated from the murine PL9-11 IgG3 autoantibody. The affinity of the PL9-11 antibody panel for histone was measured by surface plasmon resonance (SPR). Tryptophan fluorescence was used to determine wavelength shifts of the antibody panel upon binding to DNA and histone. Finally, circular dichroism spectroscopy was used to measure changes in secondary structure. SPR analysis revealed significant differences in histone binding affinity between members of the PL9-11 panel. The wavelength shifts of tryptophan fluorescence emission were found to be dependent on the antibody isotype, while circular dichroism analysis determined that changes in antibody secondary structure content differed between isotypes upon antigen binding. Thus, the antigen binding affinity is dependent on the particular constant region expressed. Moreover, the effects of antibody binding to antigen were also constant region dependent. Alteration of secondary structures influenced by constant regions may explain differences in fine specificity of anti-DNA antibodies between antibodies with similar variable regions, as well as cross-reactivity of anti-DNA antibodies with non-DNA antigens. Copyright © 2013 Elsevier Ltd. All rights reserved.

Enhanced ADCC Activity of Affinity Maturated and Fc-Engineered Mini-Antibodies Directed against the AML Stem Cell Antigen CD96

PubMed Central

Kellner, Christian; Bräutigam, Joachim; Staudinger, Matthias; Schub, Natalie; Peipp, Matthias; Gramatzki, Martin; Humpe, Andreas

2012-01-01

CD96, a cell surface antigen recently described to be preferentially expressed on acute myeloid leukemia (AML) leukemic stem cells (LSC) may represent an interesting target structure for the development of antibody-based therapeutic approaches. The v-regions from the CD96-specific hybridoma TH-111 were isolated and used to generate a CD96-specific single chain fragment of the variable regions (scFv). An affinity maturated variant resulting in 4-fold enhanced CD96-binding was generated by random mutagenesis and stringent selection using phage display. The affinity maturated scFv CD96-S32F was used to generate bivalent mini-antibodies by genetically fusing an IgG1 wild type Fc region or a variant with enhanced CD16a binding. Antibody dependent cell-mediated cytotoxicity (ADCC) experiments revealed that Fc engineering was essential to trigger significant effector cell-mediated lysis when the wild type scFv was used. The mini-antibody variant generated by fusing the affinity-maturated scFv with the optimized Fc variant demonstrated the highest ADCC activity (2.3-fold enhancement in efficacy). In conclusion, our data provide proof of concept that CD96 could serve as a target structure for effector cell-mediated lysis and demonstrate that both enhancing affinity for CD96 and for CD16a resulted in mini-antibodies with the highest cytolytic potential. PMID:22879978

Detection of specific antibody responses to vaccination in variable flying foxes (Pteropus hypomelanus).

PubMed

Wellehan, James F X; Green, Linda G; Duke, Diane G; Bootorabi, Shadi; Heard, Darryl J; Klein, Paul A; Jacobson, Elliott R

2009-09-01

Megachiropteran bats are biologically important both as endangered species and reservoirs for emerging human pathogens. Reliable detection of antibodies to specific pathogens in bats is thus epidemiologically critical. Eight variable flying foxes (Pteropus hypomelanus) were immunized with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA). Each bat received monthly inoculations for 2 months. Affinity-purified IgG was used for production of polyclonal and monoclonal anti-variable flying fox IgG antibodies. ELISA and western blot analysis were used to monitor immune responses and for assessment of polyclonal and monoclonal antibody species cross-reactivity. Protein G, polyclonal antibodies, and monoclonal antibodies detected specific anti-DNP antibody responses in immunized variable flying foxes, with protein G being the most sensitive, followed by monoclonal antibodies and then polyclonal antibodies. While the polyclonal antibody was found to cross-react well against IgG of all bat species tested, some non-specific background was observed. The monoclonal antibody was found to cross-react well against IgG of six other species in the genus Pteropus and to cross-react less strongly against IgG from Eidolon helvum or Phyllostomus hastatus. Protein G distinguished best between vaccinated and unvaccinated bats, and these results validate the use of protein G for detection of bat IgG. Monoclonal antibodies developed in this study recognized immunoglobulins from other members of the genus Pteropus well, and may be useful in applications where specific detection of Pteropus IgG is needed.

Monovalent IgG4 molecules

PubMed Central

Wilkinson, Ian C.; Fowler, Susan B.; Machiesky, LeeAnn; Miller, Kenneth; Hayes, David B.; Adib, Morshed; Her, Cheng; Borrok, M. Jack; Tsui, Ping; Burrell, Matthew; Corkill, Dominic J.; Witt, Susanne; Lowe, David C.; Webster, Carl I.

2013-01-01

Antibodies have become the fastest growing class of biological therapeutics, in part due to their exquisite specificity and ability to modulate protein-protein interactions with a high biological potency. The relatively large size and bivalency of antibodies, however, limits their use as therapeutics in certain circumstances. Antibody fragments, such as single-chain variable fragments and antigen binding-fragments, have emerged as viable alternatives, but without further modifications these monovalent formats have reduced terminal serum half-lives because of their small size and lack of an Fc domain, which is required for FcRn-mediated recycling. Using rational engineering of the IgG4 Fc domain to disrupt key interactions at the CH3-CH3 interface, we identified a number of point mutations that abolish Fc dimerization and created half-antibodies, a novel monovalent antibody format that retains a monomeric Fc domain. Introduction of these mutations into an IgG1 framework also led to the creation of half-antibodies. These half-antibodies were shown to be soluble, thermodynamically stable and monomeric, characteristics that are favorable for use as therapeutic proteins. Despite significantly reduced FcRn binding in vitro, which suggests that avidity gains in a dimeric Fc are critical to optimal FcRn binding, this format demonstrated an increased terminal serum half-life compared with that expected for most alternative antibody fragments. PMID:23567207

Generation and characterization of protective antibodies to Marburg virus.

PubMed

Froude, Jeffrey W; Pelat, Thibaut; Miethe, Sebastian; Zak, Samantha E; Wec, Anna Z; Chandran, Kartik; Brannan, Jennifer Mary; Bakken, Russell R; Hust, Michael; Thullier, Philippe; Dye, John M

Marburg virus (MARV) and Ebola virus (EBOV) have been a source of epidemics and outbreaks for several decades. We present here the generation and characterization of the first protective antibodies specific for wild-type MARV. Non-human primates (NHP), cynomolgus macaques, were immunized with viral-replicon particles expressing the glycoproteins (GP) of MARV (Ci67 isolate). An antibody fragment (single-chain variable fragment, scFv) phage display library was built after four immunogen injections, and screened against the GP 1-649 of MARV. Sequencing of 192 selected clones identified 18 clones with distinct V H and V L sequences. Four of these recombinant antibodies (R4A1, R4B11, R4G2, and R3F6) were produced in the scFv-Fc format for in vivo studies. Mice that were challenged with wild-type Marburg virus (Ci67 isolate) receiving 100 µg of scFv-Fc on days -1, 1 and 3 demonstrated protective efficacies ranging from 75-100%. The amino-acid sequences of the scFv-Fcs are similar to those of their human germline counterparts, sharing an identity ranging between 68 and 100% to human germline immunoglobulin. These results demonstrate for the first time that recombinant antibodies offer protection against wild-type MARV, and suggest they may be promising candidates for further therapeutic development especially due to their human homology.

Relationship of health-related variables to levels of anti-polyribosylribitol phosphate antibodies in adults.

PubMed Central

McDonnell, W M; Gilsdorf, J R; Kent, J B; Sheagren, J N

1988-01-01

To identify adults who may be at risk for Haemophilus influenzae type b disease by virtue of low levels of antibody against the H. influenzae b capsule and who may thus benefit from receiving H. influenzae b vaccine, we correlated serum anticapsular antibody levels of 388 adult patients with 26 health-related variables, including 3 personal characteristics, 10 laboratory values, 4 drug use categories, and 8 disease categories. Steroid use was consistently associated with low levels of anticapsular antibody; associations were also found between low antibody levels and both non-Caucasian race and increasing age. However, less than 4% of the antibody variability could be attributed to these factors, and they were not predictive of low antibody levels. Thus, although H. influenzae b infections are seen in adults with predisposing medical conditions, on the basis of the present findings, use of the H. influenzae b vaccine in adults cannot be recommended. PMID:3260243

Fermentanomics: Relating quality attributes of a monoclonal antibody to cell culture process variables and raw materials using multivariate data analysis.

PubMed

Rathore, Anurag S; Kumar Singh, Sumit; Pathak, Mili; Read, Erik K; Brorson, Kurt A; Agarabi, Cyrus D; Khan, Mansoor

2015-01-01

Fermentanomics is an emerging field of research and involves understanding the underlying controlled process variables and their effect on process yield and product quality. Although major advancements have occurred in process analytics over the past two decades, accurate real-time measurement of significant quality attributes for a biotech product during production culture is still not feasible. Researchers have used an amalgam of process models and analytical measurements for monitoring and process control during production. This article focuses on using multivariate data analysis as a tool for monitoring the internal bioreactor dynamics, the metabolic state of the cell, and interactions among them during culture. Quality attributes of the monoclonal antibody product that were monitored include glycosylation profile of the final product along with process attributes, such as viable cell density and level of antibody expression. These were related to process variables, raw materials components of the chemically defined hybridoma media, concentration of metabolites formed during the course of the culture, aeration-related parameters, and supplemented raw materials such as glucose, methionine, threonine, tryptophan, and tyrosine. This article demonstrates the utility of multivariate data analysis for correlating the product quality attributes (especially glycosylation) to process variables and raw materials (especially amino acid supplements in cell culture media). The proposed approach can be applied for process optimization to increase product expression, improve consistency of product quality, and target the desired quality attribute profile. © 2015 American Institute of Chemical Engineers.

Molecular characterization of the monoclonal antibodies composing ZMAb: a protective cocktail against Ebola virus.

PubMed

Audet, Jonathan; Wong, Gary; Wang, Han; Lu, Guangwen; Gao, George F; Kobinger, Gary; Qiu, Xiangguo

2014-11-06

Ebola virus (EBOV) causes severe viral hemorrhagic fever in humans and non-human primates, with a case fatality rate of up to 88% in human outbreaks. Over the past 3 years, monoclonal antibody (mAb) cocktails have demonstrated high efficacy as treatments against EBOV infection. One such cocktail is ZMAb, which consists of three mouse antibodies, 1H3, 2G4, and 4G7. Here, we present the epitope binding properties of mAbs 1H3, 2G4, and 4G7. We showed that these antibodies have different variable region sequences, suggesting that the individual mAbs are not clonally related. All three antibodies were found to neutralize EBOV variant Mayinga. Additionally, 2G4 and 4G7 were shown to cross-inhibit each other in vitro and select for an escape mutation at the same position on the EBOV glycoprotein (GP), at amino acid 508. 1H3 selects an escape mutant at amino acid 273 on EBOV GP. Surface plasmon resonance studies showed that all three antibodies have dissociation constants on the order of 10(-7). In combination with previous studies evaluating the binding sites of other protective antibodies, our results suggest that antibodies targeting the GP1-GP2 interface and the glycan cap are often selected as efficacious antibodies for post-exposure interventions against EBOV.

Variable epitope libraries: new vaccine immunogens capable of inducing broad human immunodeficiency virus type 1-neutralizing antibody response.

PubMed

Charles-Niño, Claudia; Pedroza-Roldan, Cesar; Viveros, Monica; Gevorkian, Goar; Manoutcharian, Karen

2011-07-18

The extreme antigenic variability of human immunodeficiency virus (HIV) leads to immune escape of the virus, representing a major challenge in the design of effective vaccine. We have developed a novel concept for immunogen construction based on introduction of massive mutations within the epitopes targeting antigenically variable pathogens and diseases. Previously, we showed that these immunogens carrying large combinatorial libraries of mutated epitope variants, termed as variable epitope libraries (VELs), induce potent, broad and long lasting CD8+IFN-γ+ T-cell response. Moreover, we demonstrated that these T cells recognize more than 50% of heavily mutated variants (5 out of 10 amino acid positions were mutated in each epitope variant) of HIV-1 gp120 V3 loop-derived cytotoxic T lymphocyte epitope (RGPGRAFVTI) in mice. The constructed VELs had complexities of 10000 and 12500 individual members, generated as plasmid DNA or as M13 phage display combinatorial libraries, respectively, and with structural composition RGPGXAXXXX or XGXGXAXVXI, where X is any of 20 natural amino acids. Here, we demonstrated that sera from mice immunized with these VELs are capable of neutralizing 5 out of 10 viral isolates from Tier 2 reference panel of subtype B envelope clones, including HIV-1 isolates which are known to be resistant to neutralization by several potent monoclonal antibodies, described previously. These data indicate the feasibility of the application of immunogens based on VEL concept as an alternative approach for the development of molecular vaccines against antigenically variable pathogens. Copyright © 2011 Elsevier Ltd. All rights reserved.

Selection of specific interactors from phage display library based on sea lamprey variable lymphocyte receptor sequences.

PubMed

Wezner-Ptasinska, Magdalena; Otlewski, Jacek

2015-12-01

Variable lymphocyte receptors (VLRs) are non-immunoglobulin components of adaptive immunity in jawless vertebrates. These proteins composed of leucine-rich repeat modules offer some advantages over antibodies in target binding and therefore are attractive candidates for biotechnological applications. In this paper we report the design and characterization of a phage display library based on a previously proposed dVLR scaffold containing six LRR modules [Wezner-Ptasinska et al., 2011]. Our library was designed based on a consensus approach in which the randomization scheme reflects the frequencies of amino acids naturally occurring in respective positions responsible for antigen recognition. We demonstrate general applicability of the scaffold by selecting dVLRs specific for lysozyme and S100A7 protein with KD values in the micromolar range. The dVLR library could be used as a convenient alternative to antibodies for effective isolation of high affinity binders.

A large observational study to concurrently assess persistence of measles specific B-cell and T-cell immunity in individuals following two doses of MMR vaccine

PubMed Central

Haralambieva, Iana H.; Ovsyannikova, Inna G.; O’Byrne, Megan; Pankratz, V. Shane; Jacobson, Robert M.; Poland, Gregory A.

2011-01-01

The measurement of measles-specific neutralizing antibodies, directed against the surface measles virus hemagglutinin and fusion proteins, is considered the gold standard in measles serology. We assessed functional measles-specific neutralizing antibody levels in a racially diverse cohort of 763 young healthy adolescents after receipt of two doses of measles-mumps-rubella vaccine, by the use of an automated plaque reduction microneutralization (PRMN) assay, and evaluated their relevance to protective antibody levels, as well as their associations with demographic and clinical variables. We also concurrently assessed measles-specific IFNγ Elispot responses and their relation to the observed antibody concentrations. The geometric mean titer for our cohort was 832 mIU/mL (95% CIs: 776; 891). Sixty-eight subjects (8.9%) had antibody concentrations of less than the protective threshold of 210 mIU/mL (corresponding to PRMN titer of 120; suggesting protection against symptomatic disease), and 177 subjects (23.2%) demonstrated persisting antibody concentrations above 1,841 mIU/mL (corresponding to PRMN titer of 1,052; suggesting total protection against viral infection), 7.4 years after vaccination, in the absence of wild-type virus boosting. The mean measles-specific IFNγ Elispot response for our cohort was 46 (95% CIs: 43; 49) IFNγ-positive spots per 200,000 cells with no relation of cellular immunity measures to the observed antibody concentrations. No significant associations between antibody titers and demographic and clinical variables, including gender and race, were observed in our study. In conclusion, in a large observational study of measles immunity, we used an automated high-throughput measles virus-specific neutralization assay to measure humoral immunity, and concurrently determined measles-specific cellular immunity to aid the assessment of potential susceptibility to measles in vaccinated populations. PMID:21539880

Strain-Specific V3 and CD4 Binding Site Autologous HIV-1 Neutralizing Antibodies Select Neutralization-Resistant Viruses

DOE PAGES

Moody, M.  Anthony; Gao, Feng; Gurley, Thaddeus  C.; ...

2015-09-09

The third variable (V3) loop and the CD4 binding site (CD4bs) of the viral envelope are frequently targeted by neutralizing antibodies (nAbs) in HIV-1-infected individuals. In chronic infection, virus escape mutants repopulate the plasma and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize, but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tiermore » 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses.« less

Strain-Specific V3 and CD4 Binding Site Autologous HIV-1 Neutralizing Antibodies Select Neutralization-Resistant Viruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moody, M.  Anthony; Gao, Feng; Gurley, Thaddeus  C.

The third variable (V3) loop and the CD4 binding site (CD4bs) of the viral envelope are frequently targeted by neutralizing antibodies (nAbs) in HIV-1-infected individuals. In chronic infection, virus escape mutants repopulate the plasma and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize, but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tiermore » 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses.« less

Monoclonal antibody disulfide reduction during manufacturing

PubMed Central

Hutterer, Katariina M.; Hong, Robert W.; Lull, Jonathon; Zhao, Xiaoyang; Wang, Tian; Pei, Rex; Le, M. Eleanor; Borisov, Oleg; Piper, Rob; Liu, Yaoqing Diana; Petty, Krista; Apostol, Izydor; Flynn, Gregory C.

2013-01-01

Manufacturing-induced disulfide reduction has recently been reported for monoclonal human immunoglobulin gamma (IgG) antibodies, a widely used modality in the biopharmaceutical industry. This effect has been tied to components of the intracellular thioredoxin reduction system that are released upon cell breakage. Here, we describe the effect of process parameters and intrinsic molecule properties on the extent of reduction. Material taken from cell cultures at the end of production displayed large variations in the extent of antibody reduction between different products, including no reduction, when subjected to the same reduction-promoting harvest conditions. Additionally, in a reconstituted model in which process variables could be isolated from product properties, we found that antibody reduction was dependent on the cell line (clone) and cell culture process. A bench-scale model using a thioredoxin/thioredoxin reductase regeneration system revealed that reduction susceptibility depended on not only antibody class but also light chain type; the model further demonstrates that the trend in reducibility was identical to DTT reduction sensitivity following the order IgG1λ > IgG1κ > IgG2λ > IgG2κ. Thus, both product attributes and process parameters contribute to the extent of antibody reduction during production. PMID:23751615

Evaluation of Disulfide Bond Position to Enhance the Thermal Stability of a Highly Stable Single Domain Antibody

PubMed Central

Zabetakis, Dan; Olson, Mark A.; Anderson, George P.; Legler, Patricia M.; Goldman, Ellen R.

2014-01-01

Single domain antibodies are the small recombinant variable domains derived from camelid heavy-chain-only antibodies. They are renowned for their stability, in large part due to their ability to refold following thermal or chemical denaturation. In addition to refolding after heat denaturation, A3, a high affinity anti-Staphylococcal Enterotoxin B single domain antibody, possesses a melting temperature of ∼84°C, among the highest reported for a single domain antibody. In this work we utilized the recently described crystal structure of A3 to select locations for the insertion of a second disulfide bond and evaluated the impact that the addition of this second bond had on the melting temperature. Four double-disulfide versions of A3 were constructed and each was found to improve the melting temperature relative to the native structure without reducing affinity. Placement of the disulfide bond at a previously published position between framework regions 2 and 3 yielded the largest improvement (>6°C), suggesting this location is optimal, and seemingly provides a universal route to raise the melting temperature of single domain antibodies. This study further demonstrates that even single domain antibodies with extremely high melting points can be further stabilized by addition of disulfide bonds. PMID:25526640

Immunoglobulin Heavy Chain Variable Region and Major Histocompatibility Region Genes Are Linked to Induced Graves' Disease in Females From Two Very Large Families of Recombinant Inbred Mice

PubMed Central

Aliesky, Holly; Banuelos, Bianca; Magana, Jessica; Williams, Robert W.; Rapoport, Basil

2014-01-01

Graves' hyperthyroidism is caused by antibodies to the TSH receptor (TSHR) that mimic thyroid stimulation by TSH. Stimulating TSHR antibodies and hyperthyroidism can be induced by immunizing mice with adenovirus expressing the human TSHR A-subunit. Prior analysis of induced Graves' disease in small families of recombinant inbred (RI) female mice demonstrated strong genetic control but did not resolve trait loci for TSHR antibodies or elevated serum T4. We investigated the genetic basis for induced Graves' disease in female mice of two large RI families and combined data with earlier findings to provide phenotypes for 178 genotypes. TSHR antibodies measured by inhibition of TSH binding to its receptor were highly significantly linked in the BXD set to the major histocompatibility region (chromosome 17), consistent with observations in 3 other RI families. In the LXS family, we detected linkage between T4 levels after TSHR-adenovirus immunization and the Ig heavy chain variable region (Igvh, chromosome 12). This observation is a key finding because components of the antigen binding region of Igs determine antibody specificity and have been previously linked to induced thyroid-stimulating antibodies. Data from the LXS family provide the first evidence in mice of a direct link between induced hyperthyroidism and Igvh genes. A role for major histocompatibility genes has now been established for genetic susceptibility to Graves' disease in both humans and mice. Future studies using arrays incorporating variation in the complex human Ig gene locus will be necessary to determine whether Igvh genes are also linked to Graves' disease in humans. PMID:25051451

Fab is the most efficient format to express functional antibodies by yeast surface display.

PubMed

Sivelle, Coline; Sierocki, Raphaël; Ferreira-Pinto, Kelly; Simon, Stéphanie; Maillere, Bernard; Nozach, Hervé

2018-04-30

Multiple formats are available for engineering of monoclonal antibodies (mAbs) by yeast surface display, but they do not all lead to efficient expression of functional molecules. We therefore expressed four anti-tumor necrosis factor and two anti-IpaD mAbs as single-chain variable fragment (scFv), antigen-binding fragment (Fab) or single-chain Fabs and compared their expression levels and antigen-binding efficiency. Although the scFv and scFab formats are widely used in the literature, 2 of 6 antibodies were either not or weakly expressed. In contrast, all 6 antibodies expressed as Fab revealed strong binding and high affinity, comparable to that of the soluble form. We also demonstrated that the variations in expression did not affect Fab functionality and were due to variations in light chain display and not to misfolded dimers. Our results suggest that Fab is the most versatile format for the engineering of mAbs.

Application of histone modification-specific interaction domains as an alternative to antibodies.

PubMed

Kungulovski, Goran; Kycia, Ina; Tamas, Raluca; Jurkowska, Renata Z; Kudithipudi, Srikanth; Henry, Chisato; Reinhardt, Richard; Labhart, Paul; Jeltsch, Albert

2014-11-01

Post-translational modifications (PTMs) of histones constitute a major chromatin indexing mechanism, and their proper characterization is of highest biological importance. So far, PTM-specific antibodies have been the standard reagent for studying histone PTMs despite caveats such as lot-to-lot variability of specificity and binding affinity. Herein, we successfully employed naturally occurring and engineered histone modification interacting domains for detection and identification of histone PTMs and ChIP-like enrichment of different types of chromatin. Our results demonstrate that histone interacting domains are robust and highly specific reagents that can replace or complement histone modification antibodies. These domains can be produced recombinantly in Escherichia coli at low cost and constant quality. Protein design of reading domains allows for generation of novel specificities, addition of affinity tags, and preparation of PTM binding pocket variants as matching negative controls, which is not possible with antibodies. © 2014 Kungulovski et al.; Published by Cold Spring Harbor Laboratory Press.

Generation of human anti-MUC3 IgG antibodies after in vitro immunization of naive peripheral blood B-lymphocytes.

PubMed

Baritaki, S; Zafiropoulos, A; Georgopoulos, E; Souris, S; Krambovitis, E

2001-04-01

It has been demonstrated that IgG antibodies can be generated to self-antigen peptides as well as against viral antigens by an antigen-specific in vitro immunization system of resting human peripheral B-lymphocytes. Using a synthetic peptide from the consensus variable tandem-repeat region of the MUC3 mucin (TSSITTTGTTSHSTPSP) as the B cell epitope, we immunized blood donor B-lymphocytes in vitro and tested for MUC3-specific antibodies by ELISA. After the primary activation step all antibodies were IgM. At the end of the secondary immunization step we obtained 1.8% (21/1138) of the cultures with IgG-switched antibodies. In a competitive inhibition ELISA using the MUC1, MUC2, MUC3, MUC4 and PIP2 peptides, only one culture (F8.1) gave satisfactory specific inhibition. Using this antibody in fluorometric studies, it stained cells from two colon carcinoma cell lines predominantly in the cytoplasm, whereas those from a breast cancer cell line stained predominantly the cell surface. In a preliminary immunohistological evaluation with formalin-fixed sections, the antibody appeared to moderately stain colon sections, but not breast sections or lymph node. This method of in vitro immunization may be a useful tool in generating IgG antibodies specific to self-antigens and could find applications in tumour targeting and immunotherapy.

Identification and verification of hybridoma-derived monoclonal antibody variable region sequences using recombinant DNA technology and mass spectrometry

USDA-ARS?s Scientific Manuscript database

Antibody engineering requires the identification of antigen binding domains or variable regions (VR) unique to each antibody. It is the VR that define the unique antigen binding properties and proper sequence identification is essential for functional evaluation and performance of recombinant antibo...

Functional anergy in a subpopulation of naive B cells from healthy humans that express autoreactive immunoglobulin receptors.

PubMed

Duty, J Andrew; Szodoray, Peter; Zheng, Nai-Ying; Koelsch, Kristi A; Zhang, Qingzhao; Swiatkowski, Mike; Mathias, Melissa; Garman, Lori; Helms, Christina; Nakken, Britt; Smith, Kenneth; Farris, A Darise; Wilson, Patrick C

2009-01-16

Self-reactive B cells not controlled by receptor editing or clonal deletion may become anergic. We report that fully mature human B cells negative for surface IgM and retaining only IgD are autoreactive and functionally attenuated (referred to as naive IgD(+)IgM(-) B cells [B(ND)]). These B(ND) cells typically make up 2.5% of B cells in the peripheral blood, have antibody variable region genes in germline (unmutated) configuration, and, by all current measures, are fully mature. Analysis of 95 recombinant antibodies expressed from the variable genes of single B(ND) cells demonstrated that they are predominantly autoreactive, binding to HEp-2 cell antigens and DNA. Upon B cell receptor cross-linkage, B(ND) cells have a reduced capacity to mobilize intracellular calcium or phosphorylate tyrosines, demonstrating that they are anergic. However, intense stimulation causes B(ND) cells to fully respond, suggesting that these cells could be the precursors of autoantibody secreting plasma cells in autoimmune diseases such as systemic lupus erythematosus or rheumatoid arthritis. This is the first identification of a distinct mature human B cell subset that is naturally autoreactive and controlled by the tolerizing mechanism of functional anergy.

Human IgG repertoire of malaria antigen-immunized human immune system (HIS) mice.

PubMed

Nogueira, Raquel Tayar; Sahi, Vincent; Huang, Jing; Tsuji, Moriya

2017-08-01

Humanized mouse models present an important tool for preclinical evaluation of new vaccines and therapeutics. Here we show the human variable repertoire of antibody sequences cloned from a previously described human immune system (HIS) mouse model that possesses functional human CD4+ T cells and B cells, namely HIS-CD4/B mice. We sequenced variable IgG genes from single memory B-cell and plasma-cell sorted from splenocytes or whole blood lymphocytes of HIS-CD4/B mice that were vaccinated with a human plasmodial antigen, a recombinant Plasmodium falciparum circumsporozoite protein (rPfCSP). We demonstrate that rPfCSP immunization triggers a diverse B-cell IgG repertoire composed of various human VH family genes and distinct V(D)J recombinations that constitute diverse CDR3 sequences similar to humans, although low hypermutated sequences were generated. These results demonstrate the substantial genetic diversity of responding human B cells of HIS-CD4/B mice and their capacity to mount human IgG class-switched antibody response upon vaccination. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Development and Evaluation of Single Domain Antibodies for Vaccinia and the L1 Antigen

PubMed Central

Walper, Scott A.; Liu, Jinny L.; Zabetakis, Daniel; Anderson, George P.; Goldman, Ellen R.

2014-01-01

There is ongoing interest to develop high affinity, thermal stable recognition elements to replace conventional antibodies in biothreat detection assays. As part of this effort, single domain antibodies that target vaccinia virus were developed. Two llamas were immunized with killed viral particles followed by boosts with the recombinant membrane protein, L1, to stimulate the immune response for envelope and membrane proteins of the virus. The variable domains of the induced heavy chain antibodies were selected from M13 phage display libraries developed from isolated RNA. Selection via biopanning on the L1 antigen produced single domain antibodies that were specific and had affinities ranging from 4×10−9 M to 7.0×10−10 M, as determined by surface plasmon resonance. Several showed good ability to refold after heat denaturation. These L1-binding single domain antibodies, however, failed to recognize the killed vaccinia antigen. Useful vaccinia binding single domain antibodies were isolated by a second selection using the killed virus as the target. The virus binding single domain antibodies were incorporated in sandwich assays as both capture and tracer using the MAGPIX system yielding limits of detection down to 4×105 pfu/ml, a four-fold improvement over the limit obtained using conventional antibodies. This work demonstrates the development of anti-vaccinia single domain antibodies and their incorporation into sandwich assays for viral detection. It also highlights the properties of high affinity and thermal stability that are hallmarks of single domain antibodies. PMID:25211488

Peptide-based protein capture agents with high affinity, selectivity, and stability as antibody replacements in biodetection assays

NASA Astrophysics Data System (ADS)

Coppock, Matthew B.; Farrow, Blake; Warner, Candice; Finch, Amethist S.; Lai, Bert; Sarkes, Deborah A.; Heath, James R.; Stratis-Cullum, Dimitra

2014-05-01

Current biodetection assays that employ monoclonal antibodies as primary capture agents exhibit limited fieldability, shelf life, and performance due to batch-to-batch production variability and restricted thermal stability. In order to improve upon the detection of biological threats in fieldable assays and systems for the Army, we are investigating protein catalyzed capture (PCC) agents as drop-in replacements for the existing antibody technology through iterative in situ click chemistry. The PCC agent oligopeptides are developed against known protein epitopes and can be mass produced using robotic methods. In this work, a PCC agent under development will be discussed. The performance, including affinity, selectivity, and stability of the capture agent technology, is analyzed by immunoprecipitation, western blotting, and ELISA experiments. The oligopeptide demonstrates superb selectivity coupled with high affinity through multi-ligand design, and improved thermal, chemical, and biochemical stability due to non-natural amino acid PCC agent design.

A highly functional synthetic phage display library containing over 40 billion human antibody clones.

PubMed

Weber, Marcel; Bujak, Emil; Putelli, Alessia; Villa, Alessandra; Matasci, Mattia; Gualandi, Laura; Hemmerle, Teresa; Wulhfard, Sarah; Neri, Dario

2014-01-01

Several synthetic antibody phage display libraries have been created and used for the isolation of human monoclonal antibodies. The performance of antibody libraries, which is usually measured in terms of their ability to yield high-affinity binding specificities against target proteins of interest, depends both on technical aspects (such as library size and quality of cloning) and on design features (which influence the percentage of functional clones in the library and their ability to be used for practical applications). Here, we describe the design, construction and characterization of a combinatorial phage display library, comprising over 40 billion human antibody clones in single-chain fragment variable (scFv) format. The library was designed with the aim to obtain highly stable antibody clones, which can be affinity-purified on protein A supports, even when used in scFv format. The library was found to be highly functional, as >90% of randomly selected clones expressed the corresponding antibody. When selected against more than 15 antigens from various sources, the library always yielded specific and potent binders, at a higher frequency compared to previous antibody libraries. To demonstrate library performance in practical biomedical research projects, we isolated the human antibody G5, which reacts both against human and murine forms of the alternatively spliced BCD segment of tenascin-C, an extracellular matrix component frequently over-expressed in cancer and in chronic inflammation. The new library represents a useful source of binding specificities, both for academic research and for the development of antibody-based therapeutics.

A Highly Functional Synthetic Phage Display Library Containing over 40 Billion Human Antibody Clones

PubMed Central

Weber, Marcel; Bujak, Emil; Putelli, Alessia; Villa, Alessandra; Matasci, Mattia; Gualandi, Laura; Hemmerle, Teresa; Wulhfard, Sarah; Neri, Dario

2014-01-01

Several synthetic antibody phage display libraries have been created and used for the isolation of human monoclonal antibodies. The performance of antibody libraries, which is usually measured in terms of their ability to yield high-affinity binding specificities against target proteins of interest, depends both on technical aspects (such as library size and quality of cloning) and on design features (which influence the percentage of functional clones in the library and their ability to be used for practical applications). Here, we describe the design, construction and characterization of a combinatorial phage display library, comprising over 40 billion human antibody clones in single-chain fragment variable (scFv) format. The library was designed with the aim to obtain highly stable antibody clones, which can be affinity-purified on protein A supports, even when used in scFv format. The library was found to be highly functional, as >90% of randomly selected clones expressed the corresponding antibody. When selected against more than 15 antigens from various sources, the library always yielded specific and potent binders, at a higher frequency compared to previous antibody libraries. To demonstrate library performance in practical biomedical research projects, we isolated the human antibody G5, which reacts both against human and murine forms of the alternatively spliced BCD segment of tenascin-C, an extracellular matrix component frequently over-expressed in cancer and in chronic inflammation. The new library represents a useful source of binding specificities, both for academic research and for the development of antibody-based therapeutics. PMID:24950200

Camel heavy chain antibodies against prostate-specific membrane antigen.

PubMed

Evazalipour, Mehdi; Tehrani, Bahram Soltani; Abolhassani, Mohsen; Morovvati, Hamid; Omidfar, Kobra

2012-12-01

Prostate-specific membrane antigen (PSMA), a type II integral membrane glycoprotein, is highly overexpressed in all forms of prostate cancer tissues. It has also been demonstrated in a wide range of neovasculature of non-prostatic solid tumors, including bladder, pancreas, lung, kidney, colorectal, and gastric cancers. Given the unique expression of PSMA, it is considered an alluring target for antibody-based imaging and therapy of cancer. In the present study, the production and characterization of camel heavy chain antibodies (HCAbs) specific for the external domain of the PSMA are reported. Due to the absence of the CH1 domain, HCAbs are smaller than their counterparts in conventional antibodies. In this study, camel antibodies were generated through immunization of Camelus dromedarius with a synthetic 28 amino acid peptide corresponding to the external surface domain of antigen and PSMA-expressing cell lines. Different binding properties to protein A and protein G affinity columns were deployed to separate three subclasses of camel IgG. The affinity purified HCAbs bound selectively to the synthetic peptide in enzyme linked immunosorbent assay (ELISA) and reacted specifically with PSMA-expressing cell line through immunocytochemistry study. Currently, we are attempting to develop recombinant variable domain of these heavy chain antibodies (VHH or nanobody) for tumor imaging and cancer therapy.

Enhanced Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Virus-Like Particles of Human Papillomavirus

PubMed Central

Studentsov, Yevgeniy Y.; Schiffman, Mark; Strickler, Howard D.; Ho, Gloria Y. F.; Pang, Yuk-Ying Susana; Schiller, John; Herrero, Rolando; Burk, Robert D.

2002-01-01

Measurement of antibodies to human papillomavirus (HPV) is complicated by many factors. Although enzyme-linked immunosorbent assays (ELISAs) that use virus-like particles (VLPs) have proved useful, the assays have, in general, had moderate sensitivities and low signal-to-noise ratios. To enhance the performance of the assay, a systematic investigation was undertaken to examine key variables used in ELISAs for the detection of antibodies to VLPs of HPV. Incorporation of two vinyl polymers, polyvinyl alcohol (molecular weight, 50,000) (PVA-50) and polyvinylpyrrolidone (molecular weight, 360,000) (PVP-360), was found to increase the sensitivity as well as the specificity of the assay for the detection of antibodies to VLPs of HPV. In particular, the addition of PVA-50 to the blocking solution reduced the amount of nonspecific binding of antibodies to VLPs and the microplate surface, whereas the addition of PVP-360 increased the sensitivity of antibody detection. The new ELISA demonstrated increased sensitivity and specificity for the detection of cervical HPV type 16 infection compared to those of a prototype assay with coded clinical serum samples from women with known cervicovaginal HPV infection status. It is anticipated that the enhanced ELISA conditions will have wide application to a large number of clinical diagnostic assays. PMID:11980956

Strain-Specific V3 and CD4 Binding Site Autologous HIV-1 Neutralizing Antibodies Select Neutralization-Resistant Viruses.

PubMed

Moody, M Anthony; Gao, Feng; Gurley, Thaddeus C; Amos, Joshua D; Kumar, Amit; Hora, Bhavna; Marshall, Dawn J; Whitesides, John F; Xia, Shi-Mao; Parks, Robert; Lloyd, Krissey E; Hwang, Kwan-Ki; Lu, Xiaozhi; Bonsignori, Mattia; Finzi, Andrés; Vandergrift, Nathan A; Alam, S Munir; Ferrari, Guido; Shen, Xiaoying; Tomaras, Georgia D; Kamanga, Gift; Cohen, Myron S; Sam, Noel E; Kapiga, Saidi; Gray, Elin S; Tumba, Nancy L; Morris, Lynn; Zolla-Pazner, Susan; Gorny, Miroslaw K; Mascola, John R; Hahn, Beatrice H; Shaw, George M; Sodroski, Joseph G; Liao, Hua-Xin; Montefiori, David C; Hraber, Peter T; Korber, Bette T; Haynes, Barton F

2015-09-09

The third variable (V3) loop and the CD4 binding site (CD4bs) of the HIV-1 envelope are frequently targeted by neutralizing antibodies (nAbs) in infected individuals. In chronic infection, HIV-1 escape mutants repopulate the plasma, and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3 and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tier 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses. Copyright © 2015 Elsevier Inc. All rights reserved.

Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library.

PubMed

Liu, Jinny L; Anderson, George P; Goldman, Ellen R

2007-11-19

Shark heavy chain antibody, also called new antigen receptor (NAR), consists of one single Variable domain (VH), containing only two complementarity-determining regions (CDRs). The antigen binding affinity and specificity are mainly determined by these two CDRs. The good solubility, excellent thermal stability and complex sequence variation of small single domain antibodies (sdAbs) make them attractive alternatives to conventional antibodies. In this report, we construct and characterize a diversity enhanced semi-synthetic NAR V display library based on naturally occurring NAR V sequences. A semi-synthetic shark sdAb display library with a complexity close to 1e9 was constructed. This was achieved by introducing size and sequence variations in CDR3 using randomized CDR3 primers of three different lengths. Binders against three toxins, staphylococcal enterotoxin B (SEB), ricin, and botulinum toxin A (BoNT/A) complex toxoid, were isolated from panning the display library. Soluble sdAbs from selected binders were purified and evaluated using direct binding and thermal stability assays on the Luminex 100. In addition, sandwich assays using sdAb as the reporter element were developed to demonstrate their utility for future sensor applications. We demonstrated the utility of a newly created hyper diversified shark NAR displayed library to serve as a source of thermal stable sdAbs against a variety of toxins.

Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library

PubMed Central

Liu, Jinny L; Anderson, George P; Goldman, Ellen R

2007-01-01

Background Shark heavy chain antibody, also called new antigen receptor (NAR), consists of one single Variable domain (VH), containing only two complementarity-determining regions (CDRs). The antigen binding affinity and specificity are mainly determined by these two CDRs. The good solubility, excellent thermal stability and complex sequence variation of small single domain antibodies (sdAbs) make them attractive alternatives to conventional antibodies. In this report, we construct and characterize a diversity enhanced semi-synthetic NAR V display library based on naturally occurring NAR V sequences. Results A semi-synthetic shark sdAb display library with a complexity close to 1e9 was constructed. This was achieved by introducing size and sequence variations in CDR3 using randomized CDR3 primers of three different lengths. Binders against three toxins, staphylococcal enterotoxin B (SEB), ricin, and botulinum toxin A (BoNT/A) complex toxoid, were isolated from panning the display library. Soluble sdAbs from selected binders were purified and evaluated using direct binding and thermal stability assays on the Luminex 100. In addition, sandwich assays using sdAb as the reporter element were developed to demonstrate their utility for future sensor applications. Conclusion We demonstrated the utility of a newly created hyper diversified shark NAR displayed library to serve as a source of thermal stable sdAbs against a variety of toxins. PMID:18021450

Structure-Based Design of Hepatitis C Virus Vaccines That Elicit Neutralizing Antibody Responses to a Conserved Epitope

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pierce, Brian G.; Boucher, Elisabeth N.; Piepenbrink, Kurt H.

Despite recent advances in therapeutic options, hepatitis C virus (HCV) remains a severe global disease burden, and a vaccine can substantially reduce its incidence. Due to its extremely high sequence variability, HCV can readily escape the immune response; thus, an effective vaccine must target conserved, functionally important epitopes. Using the structure of a broadly neutralizing antibody in complex with a conserved linear epitope from the HCV E2 envelope glycoprotein (residues 412 to 423; epitope I), we performed structure-based design of immunogens to induce antibody responses to this epitope. This resulted in epitope-based immunogens based on a cyclic defensin protein, asmore » well as a bivalent immunogen with two copies of the epitope on the E2 surface. We solved the X-ray structure of a cyclic immunogen in complex with the HCV1 antibody and confirmed preservation of the epitope conformation and the HCV1 interface. Mice vaccinated with our designed immunogens produced robust antibody responses to epitope I, and their serum could neutralize HCV. Notably, the cyclic designs induced greater epitope-specific responses and neutralization than the native peptide epitope. Beyond successfully designing several novel HCV immunogens, this study demonstrates the principle that neutralizing anti-HCV antibodies can be induced by epitope-based, engineered vaccines and provides the basis for further efforts in structure-based design of HCV vaccines. IMPORTANCEHepatitis C virus is a leading cause of liver disease and liver cancer, with approximately 3% of the world's population infected. To combat this virus, an effective vaccine would have distinct advantages over current therapeutic options, yet experimental vaccines have not been successful to date, due in part to the virus's high sequence variability leading to immune escape. In this study, we rationally designed several vaccine immunogens based on the structure of a conserved epitope that is the target of broadly neutralizing antibodies.In vivoresults in mice indicated that these antigens elicited epitope-specific neutralizing antibodies, with various degrees of potency and breadth. These promising results suggest that a rational design approach can be used to generate an effective vaccine for this virus.« less

Cocaine-specific Antibodies Blunt the Subjective Effects of Smoked Cocaine in Humans

PubMed Central

Haney, Margaret; Gunderson, Erik W.; Jiang, Huiping; Collins, Eric D.; Foltin, Richard W.

2012-01-01

Background Rates of relapse among cocaine-dependent patients are high, and new treatment approaches are needed. Clinical data demonstrate that a cocaine vaccine (TA-CD: Celtic Pharmaceutical) produces selective anti-cocaine antibodies, yet the impact of these antibodies on cocaine’s direct effects is unknown. The objective of this human laboratory study was to measure the relationship between antibody titers and the effects of smoked cocaine on ratings of intoxication, craving and cardiovascular effects. Methods Ten cocaine-dependent men not seeking drug treatment spent 2 nights per week for 13 weeks inpatient where the effects of cocaine (0, 25, 50 mg) were determined prior to vaccination and at weekly intervals thereafter. Two doses of TA-CD (82 µg, n=4; 360 µg, n=6) were administered at weeks 1, 3, 5 and 9. Results Peak plasma antibody levels, which were highly variable, significantly predicted cocaine’s effects. Those individuals in the upper half of antibody production had an immediate (within 4 minutes of cocaine smoking) and robust (55–81%) reduction in ratings of Good Drug Effect and Cocaine Quality, while those in the lower half showed only a nonsignificant attenuation (6–26%). Self-reported cocaine use while participants were outpatient tended to decrease as a function of antibody titer (p < 0.12). By contrast, higher antibody levels predicted significantly greater cocaine-induced tachycardia. Conclusions The TA-CD vaccine substantially decreased smoked cocaine’s intoxicating effects in those generating sufficient antibody. These data support further testing of cocaine immunotherapy as a treatment for cocaine dependence. PMID:19846066

An affinity improved single-chain antibody from phage display of a library derived from monoclonal antibodies detects fumonisins by immunoassay.

PubMed

Hu, Zu-Quan; Li, He-Ping; Wu, Ping; Li, Ya-Bo; Zhou, Zhu-Qing; Zhang, Jing-Bo; Liu, Jin-Long; Liao, Yu-Cai

2015-03-31

Fumonisin B analogs, particularly FB1, FB2, and FB3, are major mycotoxins found in cereals. Single-chain fragment variable (scFv) antibodies represent a promising alternative immunoassay system. A phage-displayed antibody library derived from four monoclonal antibodies (mAbs) generated against FB1 was used to screen high binding affinity scFv antibodies; the best candidate was designated H2. Surface plasmon resonance measurements confirmed that the H2 scFv displayed a 82-fold higher binding affinity than its parent mAb. Direct competitive enzyme-linked immunosorbent assay demonstrated that the H2 antibody could competitively bind to free FB1, FB2, and FB3, with an IC50 of 0.11, 0.04, and 0.10 μM, respectively; it had no cross-reactivity to deoxynivalenol, nivalenol and aflatoxin. Validation assays with naturally contaminated samples revealed a linear relationship between the H2 antibody-based assay results and chemical analysis results, that could be expressed as y=1.7072x+5.5606 (R(2)=0.8883). Homology modeling of H2 revealed a favorable binding structure highly complementary to the three fumonisins. Molecular docking analyses suggested that the preferential binding of the H2 scFv to FB2 was due to the presence of a hydrogen radical in its R1 position, leading to a proper electrostatic matching and hydrophobic interaction. The H2 scFv antibody can be used for the rapid, accurate, and specific detection of fumonisin contamination in agricultural samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Duplex microfluidic SERS detection of pathogen antigens with nanoyeast single-chain variable fragments.

PubMed

Wang, Yuling; Rauf, Sakandar; Grewal, Yadveer S; Spadafora, Lauren J; Shiddiky, Muhammad J A; Cangelosi, Gerard A; Schlücker, Sebastian; Trau, Matt

2014-10-07

Quantitative and accurate detection of multiple biomarkers would allow for the rapid diagnosis and treatment of diseases induced by pathogens. Monoclonal antibodies are standard affinity reagents applied for biomarkers detection; however, their production is expensive and labor-intensive. Herein, we report on newly developed nanoyeast single-chain variable fragments (NYscFv) as an attractive alternative to monoclonal antibodies, which offers the unique advantage of a cost-effective production, stability in solution, and target-specificity. By combination of surface-enhanced Raman scattering (SERS) microspectroscopy using glass-coated, highly purified SERS nanoparticle clusters as labels, with a microfluidic device comprising multiple channels, a robust platform for the sensitive duplex detection of pathogen antigens has been developed. Highly sensitive detection for individual Entamoeba histolytica antigen EHI_115350 (limit of detection = 1 pg/mL, corresponding to 58.8 fM) and EHI_182030 (10 pg/mL, corresponding 453 fM) with high specificity has been achieved, employing the newly developed corresponding NYscFv as probe in combination with SERS microspectroscopy at a single laser excitation wavelength. Our first report on SERS-based immunoassays using the novel NYscFv affinity reagent demonstrates the flexibility of NYscFv fragments as viable alternatives to monoclonal antibodies in a range of bioassay platforms and paves the way for further applications.

Screening and identification of human ZnT8-specific single-chain variable fragment (scFv) from type 1 diabetes phage display library.

PubMed

Wu, Qian; Wang, Xiaodong; Gu, Yong; Zhang, Xiao; Qin, Yao; Chen, Heng; Xu, Xinyu; Yang, Tao; Zhang, Mei

2016-07-01

Zinc transporter 8 (ZnT8) is a major autoantigen and a predictive marker in type 1 diabetes (T1D). To investigate ZnT8-specific antibodies, a phage display library from T1D was constructed and single-chain antibodies against ZnT8 were screened and identified. Human T1D single-chain variable fragment (scFv) phage display library consists of approximately 1×10(8) clones. After four rounds of bio-panning, seven unique clones were positive by phage ELISA. Among them, C27 and C22, which demonstrated the highest affinity to ZnT8, were expressed in Escherichia coli Top10F' and then purified by affinity chromatography. C27 and C22 specifically bound ZnT8 N/C fusion protein and ZnT8 C terminal dimer with one Arg325Trp mutation. The specificity to human islet cells of these scFvs were further confirmed by immunohistochemistry. In conclusion, we have successfully constructed a T1D phage display antibody library and identified two ZnT8-specific scFv clones, C27 and C22. These ZnT8-specific scFvs are potential agents in immunodiagnostic and immunotherapy of T1D.

Modeling Antibody Diversity.

ERIC Educational Resources Information Center

Baker, William P.; Moore, Cathy Ronstadt

1998-01-01

Understanding antibody structure and function is difficult for many students. The rearrangement of constant and variable regions during antibody differentiation can be effectively simulated using a paper model. Describes a hands-on laboratory exercise which allows students to model antibody diversity using readily available resources. (PVD)

Protective Effect of Vaccine Promoted Neutralizing Antibodies against the Intracellular Pathogen Chlamydia trachomatis.

PubMed

Olsen, Anja Weinreich; Lorenzen, Emma Kathrine; Rosenkrands, Ida; Follmann, Frank; Andersen, Peter

2017-01-01

There is an unmet need for a vaccine to control Chlamydia trachomatis ( C.t .) infections. We have recently designed a multivalent heterologous immuno-repeat 1 (Hirep1) vaccine construct based on major outer membrane protein variable domain (VD) 4 regions from C.t . serovars (Svs) D-F. Hirep1 administered in the Cationic Adjuvant Formulation no. 1 (CAF01) promoted neutralizing antibodies in concert with CD4 + T cells and protected against genital infection. In the current study, we examined the protective role of the antibody (Ab) response in detail. Mice were vaccinated with either Hirep1 or a vaccine construct based on a homologous multivalent construct of extended VD4's from SvF (extVD4 F *4), adjuvanted in CAF01. Hirep1 and extVD4 F *4 induced similar levels of Ab and cell-mediated immune responses but differed in the fine specificity of the B cell epitopes targeted in the VD4 region. Hirep1 induced a strong response toward a neutralizing epitope (LNPTIAG) and the importance of this epitope for neutralization was demonstrated by competitive inhibition with the corresponding peptide. Immunization with extVD4 F *4 skewed the response to a non-neutralizing epitope slightly upstream in the sequence. Vaccination with Hirep1 as opposed to extVD4 F *4 induced significant protection against infection in mice both in short- and long-term vaccination experiments, signifying a key role for Hirep1 neutralizing antibodies during protection against C.t . Finally, we show that passive immunization of Rag1 knockout mice with Hirep1 antibodies completely prevented the establishment of infection in 48% of the mice, demonstrating an isolated role for neutralizing antibodies in controlling infection. Our data emphasize the role of antibodies in early protection against C.t . and support the inclusion of neutralizing targets in chlamydia vaccines.

Protective Effect of Vaccine Promoted Neutralizing Antibodies against the Intracellular Pathogen Chlamydia trachomatis

PubMed Central

Olsen, Anja Weinreich; Lorenzen, Emma Kathrine; Rosenkrands, Ida; Follmann, Frank; Andersen, Peter

2017-01-01

There is an unmet need for a vaccine to control Chlamydia trachomatis (C.t.) infections. We have recently designed a multivalent heterologous immuno-repeat 1 (Hirep1) vaccine construct based on major outer membrane protein variable domain (VD) 4 regions from C.t. serovars (Svs) D–F. Hirep1 administered in the Cationic Adjuvant Formulation no. 1 (CAF01) promoted neutralizing antibodies in concert with CD4+ T cells and protected against genital infection. In the current study, we examined the protective role of the antibody (Ab) response in detail. Mice were vaccinated with either Hirep1 or a vaccine construct based on a homologous multivalent construct of extended VD4’s from SvF (extVD4F*4), adjuvanted in CAF01. Hirep1 and extVD4F*4 induced similar levels of Ab and cell-mediated immune responses but differed in the fine specificity of the B cell epitopes targeted in the VD4 region. Hirep1 induced a strong response toward a neutralizing epitope (LNPTIAG) and the importance of this epitope for neutralization was demonstrated by competitive inhibition with the corresponding peptide. Immunization with extVD4F*4 skewed the response to a non-neutralizing epitope slightly upstream in the sequence. Vaccination with Hirep1 as opposed to extVD4F*4 induced significant protection against infection in mice both in short- and long-term vaccination experiments, signifying a key role for Hirep1 neutralizing antibodies during protection against C.t. Finally, we show that passive immunization of Rag1 knockout mice with Hirep1 antibodies completely prevented the establishment of infection in 48% of the mice, demonstrating an isolated role for neutralizing antibodies in controlling infection. Our data emphasize the role of antibodies in early protection against C.t. and support the inclusion of neutralizing targets in chlamydia vaccines. PMID:29312283

Structural classification of CDR-H3 revisited: a lesson in antibody modeling.

PubMed

Kuroda, Daisuke; Shirai, Hiroki; Kobori, Masato; Nakamura, Haruki

2008-11-15

Among the six complementarity-determining regions (CDRs) in the variable domains of an antibody, the third CDR of the heavy chain (CDR-H3), which lies in the center of the antigen-binding site, plays a particularly important role in antigen recognition. CDR-H3 shows significant variability in its length, sequence, and structure. Although difficult, model building of this segment is the most critical step in antibody modeling. Since our first proposal of the "H3-rules," which classify CDR-H3 structure based on amino acid sequence, the number of experimentally determined antibody structures has increased. Here, we revise these H3-rules and propose an improved classification scheme for CDR-H3 structure modeling. In addition, we determine the common features of CDR-H3 in antibody drugs as well as discuss the concept of "antibody druggability," which can be applied as an indicator of antibody evaluation during drug discovery.

Community-Based Seroepidemiological Survey of Hepatitis E Virus Infection in Catalonia, Spain▿

PubMed Central

Buti, Maria; Domínguez, Àngela; Plans, Pere; Jardí, Rossend; Schaper, Mélani; Espuñes, Jordi; Cardeñosa, Neus; Rodríguez-Frías, Francisco; Esteban, Rafael; Plasència, Antoni; Salleras, Luis

2006-01-01

The objective of the study was to investigate the prevalence of immunoglobulin G (IgG) antibodies to hepatitis E virus (HEV) infection in a population sample from Catalonia and to analyze the demographic and clinical variables associated with the presence of these antibodies. A total of 1,280 subjects between 15 and 74 years of age were selected randomly from urban and rural areas. Data for sociodemographic and clinical variables were collected by using a questionnaire. IgG antibodies to HEV were determined by an immunoenzymatic method. The odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated for studied variables. Multiple logistic regression analysis was used to determine which variables were independently associated with the prevalence of HEV infection. Anti-HEV antibodies were detected in 96 (7.3%) of the 1,280 samples analyzed. The prevalence of antibodies was greater among males (7.8%) than among women (7%) and increased with age for both sexes, from 3% among subjects 15 to 24 years of age to 12% among subjects ≥65 years of age. Bivariate analysis of the sociodemographic and clinical variables showed an association between the prevalence of hepatitis E virus infection and minor surgery (OR, 1.96; 95% CI, 1.24 to 3.11), abdominal surgery (OR, 1.74; 95% CI, 1.12 to 2.73), and, for women, being uniparous or multiparous (OR, 2.84; 95% CI, 1.19 to 6.79). The multivariate analysis showed an association with minor surgery only (OR, 1.68; 95% CI, 1.03 to 2.70). In conclusion, anti-HEV antibodies were detected in 7.3% of the Catalan population. The seroprevalence of anti-HEV antibodies increased with age and was associated with previous minor surgery. PMID:17050741

An improved, rapid competitive ELISA using a novel conserved 3B epitope for the detection of serum antibodies to foot-and-mouth disease virus.

PubMed

Chung, Chungwon J; Clavijo, Alfonso; Bounpheng, Mangkey A; Uddowla, Sabena; Sayed, Abu; Dancho, Brooke; Olesen, Ian C; Pacheco, Juan; Kamicker, Barbara J; Brake, David A; Bandaranayaka-Mudiyanselage, Carey L; Lee, Stephen S; Rai, Devendra K; Rieder, Elizabeth

2018-06-01

The highly contagious foot-and-mouth disease virus (FMDV) afflicts cloven-hoofed animals, resulting in significant costs because of loss of trade and recovery from disease. We developed a sensitive, specific, and rapid competitive ELISA (cELISA) to detect serum antibodies to FMDV. The cELISA utilized a monoclonal blocking antibody specific for a highly conserved FMDV nonstructural 3B epitope, a recombinant mutant FMDV 3ABC coating protein, and optimized format variables including serum incubation for 90 min at 20-25°C. Samples from 16 animals experimentally infected with one FMDV serotype (A, O, Asia, or SAT-1) demonstrated early detection capacity beginning 7 d post-inoculation. All samples from 55 vesicular stomatitis virus antibody-positive cattle and 44 samples from cloven-hoofed animals affected by non-FMD vesicular diseases were negative in the cELISA, demonstrating 100% analytical specificity. The diagnostic sensitivity was 100% against sera from 128 cattle infected with isolates of all FMDV serotypes, emphasizing serotype-agnostic results. Diagnostic specificities of U.S. cattle ( n = 1135) and swine ( n = 207) sera were 99.4% and 100%, respectively. High repeatability and reproducibility were demonstrated with 3.1% coefficient of variation in percent inhibition data and 100% agreement using 2 kit lots and 400 negative control serum samples, with no difference between bench and biosafety cabinet operation. Negative results from vaccinated, uninfected cattle, pig, and sheep sera confirmed the DIVA (differentiate infected from vaccinated animals) capability. This rapid (<3 h), select agent-free assay with high sensitivity and specificity, DIVA capability, and room temperature processing capability will serve as a useful tool in FMDV surveillance, emergency preparedness, response, and outbreak recovery programs.

Bayesian hierarchical modeling for subject-level response classification in peptide microarray immunoassays

PubMed Central

Imholte, Gregory; Gottardo, Raphael

2017-01-01

Summary The peptide microarray immunoassay simultaneously screens sample serum against thousands of peptides, determining the presence of antibodies bound to array probes. Peptide microarrays tiling immunogenic regions of pathogens (e.g. envelope proteins of a virus) are an important high throughput tool for querying and mapping antibody binding. Because of the assay’s many steps, from probe synthesis to incubation, peptide microarray data can be noisy with extreme outliers. In addition, subjects may produce different antibody profiles in response to an identical vaccine stimulus or infection, due to variability among subjects’ immune systems. We present a robust Bayesian hierarchical model for peptide microarray experiments, pepBayes, to estimate the probability of antibody response for each subject/peptide combination. Heavy-tailed error distributions accommodate outliers and extreme responses, and tailored random effect terms automatically incorporate technical effects prevalent in the assay. We apply our model to two vaccine trial datasets to demonstrate model performance. Our approach enjoys high sensitivity and specificity when detecting vaccine induced antibody responses. A simulation study shows an adaptive thresholding classification method has appropriate false discovery rate control with high sensitivity, and receiver operating characteristics generated on vaccine trial data suggest that pepBayes clearly separates responses from non-responses. PMID:27061097

The antigenic evolution of influenza: drift or thrift?

PubMed Central

Wikramaratna, Paul S.; Sandeman, Michi; Recker, Mario; Gupta, Sunetra

2013-01-01

It is commonly assumed that antibody responses against the influenza virus are polarized in the following manner: strong antibody responses are directed at highly variable antigenic epitopes, which consequently undergo ‘antigenic drift’, while weak antibody responses develop against conserved epitopes. As the highly variable epitopes are in a constant state of flux, current antibody-based vaccine strategies are focused on the conserved epitopes in the expectation that they will provide some level of clinical protection after appropriate boosting. Here, we use a theoretical model to suggest the existence of epitopes of low variability, which elicit a high degree of both clinical and transmission-blocking immunity. We show that several epidemiological features of influenza and its serological and molecular profiles are consistent with this model of ‘antigenic thrift’, and that identifying the protective epitopes of low variability predicted by this model could offer a more viable alternative to regularly update the influenza vaccine than exploiting responses to weakly immunogenic conserved regions. PMID:23382423

Larger differences in utilization of rarely requested tests in primary care in Spain.

PubMed

Salinas, Maria; López-Garrigós, Maite; Flores, Emilio; Uris, Joaquín; Leiva-Salinas, Carlos

2015-01-01

The study was performed to compare and analyze the inter-departmental variability in the request of rarely requested laboratory tests in primary care, as opposed to other more common and highly requested tests. Data from production statistics for the year 2012 from 76 Spanish laboratories was used. The number of antinuclear antibodies, antistreptolysin O, creatinine, cyclic citrullinated peptide antibodies, deaminated peptide gliadine IgA antibodies, glucose, protein electrophoresis, rheumatoid factor, transglutaminase IgA antibodies, urinalysis and uric acid tests requested was collected. The number of test requests per 1000 inhabitants was calculated. In order to explore the variability the coefficient of quartile dispersion was calculated. The smallest variation was seen for creatinine, glucose, uric acid and urinalysis; the most requested tests. The tests that were least requested showed the greatest variability. Our study shows through a very simplified approach, in a population close to twenty million inhabitants, how in primary care, the variability in the request of laboratory tests is inversely proportional to the request rate.

Affinity maturation in an HIV broadly neutralizing B-cell lineage through reorientation of variable domains.

PubMed

Fera, Daniela; Schmidt, Aaron G; Haynes, Barton F; Gao, Feng; Liao, Hua-Xin; Kepler, Thomas B; Harrison, Stephen C

2014-07-15

Rapidly evolving pathogens, such as human immunodeficiency and influenza viruses, escape immune defenses provided by most vaccine-induced antibodies. Proposed strategies to elicit broadly neutralizing antibodies require a deeper understanding of antibody affinity maturation and evolution of the immune response to vaccination or infection. In HIV-infected individuals, viruses and B cells evolve together, creating a virus-antibody "arms race." Analysis of samples from an individual designated CH505 has illustrated the interplay between an antibody lineage, CH103, and autologous viruses at various time points. The CH103 antibodies, relatively broad in their neutralization spectrum, interact with the CD4 binding site of gp120, with a contact dominated by CDRH3. We show by analyzing structures of progenitor and intermediate antibodies and by correlating them with measurements of binding to various gp120s that there was a shift in the relative orientation of the light- and heavy-chain variable domains during evolution of the CH103 lineage. We further show that mutations leading to this conformational shift probably occurred in response to insertions in variable loop 5 (V5) of the HIV envelope. The shift displaced the tips of the light chain away from contact with V5, making room for the inserted residues, which had allowed escape from neutralization by the progenitor antibody. These results, which document the selective mechanism underlying this example of a virus-antibody arms race, illustrate the functional significance of affinity maturation by mutation outside the complementarity determining region surface of the antibody molecule.

Antibody purification-independent microarrays (PIM) by direct bacteria spotting on TiO2-treated slides.

PubMed

De Marni, Marzia L; Monegal, Ana; Venturini, Samuele; Vinati, Simone; Carbone, Roberta; de Marco, Ario

2012-02-01

The preparation of effective conventional antibody microarrays depends on the availability of high quality material and on the correct accessibility of the antibody active moieties following their immobilization on the support slide. We show that spotting bacteria that expose recombinant antibodies on their external surface directly on nanostructured-TiO(2) or epoxy slides (purification-independent microarray - PIM) is a simple and reliable alternative for preparing sensitive and specific microarrays for antigen detection. Variable domains of single heavy-chain antibodies (VHHs) against fibroblast growth factor receptor 1 (FGFR1) were used to capture the antigen diluted in serum or BSA solution. The FGFR1 detection was performed by either direct antigen labeling or using a sandwich system in which FGFR1 was first bound to its antibody and successively identified using a labeled FGF. In both cases the signal distribution within each spot was uniform and spot morphology regular. The signal-to-noise ratio of the signal was extremely elevated and the specificity of the system was proved statistically. The LOD of the system for the antigen was calculated being 0.4ng/mL and the dynamic range between 0.4ng/mL and 10μg/mL. The microarrays prepared with bacteria exposing antibodies remain fully functional for at least 31 days after spotting. We finally demonstrated that the method is suitable for other antigen-antibody pairs and expect that it could be easily adapted to further applications such as the display of scFv and IgG antibodies or the autoantibody detection using protein PIMs. Copyright © 2011. Published by Elsevier Inc.

¹⁵N, ¹³C and ¹H resonance assignments and secondary structure determination of a variable heavy domain of a heavy chain antibody.

PubMed

Prosser, Christine E; Waters, Lorna C; Muskett, Frederick W; Veverka, Vaclav; Addis, Philip W; Griffin, Laura M; Baker, Terry S; Lawson, Alastair D G; Wernery, Ulrich; Kinne, Jorg; Henry, Alistair J; Taylor, Richard J; Carr, Mark D

2014-04-01

Heavy chain antibodies differ in structure to conventional antibodies lacking both the light chain and the first heavy chain constant domain (CH1). Characteristics of the antigen-binding variable heavy domain of the heavy chain antibody (VHH) including the smaller size, high solubility and stability make them an attractive alternative to more traditional antibody fragments for detailed NMR-based structural analysis. Here we report essentially complete backbone and side chain (15)N, (13)C and (1)H assignments for a free VHH. Analysis of the backbone chemical shift data obtained indicates that the VHH is comprised predominantly of β-sheets corresponding to nearly 60% of the protein backbone.

Impact of Uniform Methods on Interlaboratory Antibody Titration Variability: Antibody Titration and Uniform Methods.

PubMed

Bachegowda, Lohith S; Cheng, Yan H; Long, Thomas; Shaz, Beth H

2017-01-01

-Substantial variability between different antibody titration methods prompted development and introduction of uniform methods in 2008. -To determine whether uniform methods consistently decrease interlaboratory variation in proficiency testing. -Proficiency testing data for antibody titration between 2009 and 2013 were obtained from the College of American Pathologists. Each laboratory was supplied plasma and red cells to determine anti-A and anti-D antibody titers by their standard method: gel or tube by uniform or other methods at different testing phases (immediate spin and/or room temperature [anti-A], and/or anti-human globulin [AHG: anti-A and anti-D]) with different additives. Interlaboratory variations were compared by analyzing the distribution of titer results by method and phase. -A median of 574 and 1100 responses were reported for anti-A and anti-D antibody titers, respectively, during a 5-year period. The 3 most frequent (median) methods performed for anti-A antibody were uniform tube room temperature (147.5; range, 119-159), uniform tube AHG (143.5; range, 134-150), and other tube AHG (97; range, 82-116); for anti-D antibody, the methods were other tube (451; range, 431-465), uniform tube (404; range, 382-462), and uniform gel (137; range, 121-153). Of the larger reported methods, uniform gel AHG phase for anti-A and anti-D antibodies had the most participants with the same result (mode). For anti-A antibody, 0 of 8 (uniform versus other tube room temperature) and 1 of 8 (uniform versus other tube AHG), and for anti-D antibody, 0 of 8 (uniform versus other tube) and 0 of 8 (uniform versus other gel) proficiency tests showed significant titer variability reduction. -Uniform methods harmonize laboratory techniques but rarely reduce interlaboratory titer variance in comparison with other methods.

Method for altering antibody light chain interactions

DOEpatents

Stevens, Fred J.; Stevens, Priscilla Wilkins; Raffen, Rosemarie; Schiffer, Marianne

2002-01-01

A method for recombinant antibody subunit dimerization including modifying at least one codon of a nucleic acid sequence to replace an amino acid occurring naturally in the antibody with a charged amino acid at a position in the interface segment of the light polypeptide variable region, the charged amino acid having a first polarity; and modifying at least one codon of the nucleic acid sequence to replace an amino acid occurring naturally in the antibody with a charged amino acid at a position in an interface segment of the heavy polypeptide variable region corresponding to a position in the light polypeptide variable region, the charged amino acid having a second polarity opposite the first polarity. Nucleic acid sequences which code for novel light chain proteins, the latter of which are used in conjunction with the inventive method, are also provided.

IgM anti-myeloperoxidase antibody-secreting lymphocytes are present in the peripheral repertoire of lupus mice but rarely differentiate into IgG-producing cells

PubMed Central

Vittecoq, O; Brard, F; Jovelin, F; Le Loet, X; Tron, F; Gilbert, D

1999-01-01

Two IgM, κ anti-myeloperoxidase (MPO) monoclonal antibodies, 6D6 and 9B5, bound to MPO in a solid-phase enzyme-linked immunosorbent assay were derived from the splenocytes of (NZB × NZW) F1 and MRL/lpr-lpr mice, respectively. 6D6 gave a characteristic perinuclear immunofluorescence staining pattern on ethanol-fixed human neutrophils, bound to the native form of MPO by immunoblotting and had a high constant affinity for MPO as demonstrated by real-time specific interaction. 9B5 produced a cytoplasmic immunofluorescence staining pattern, reacted with the heavy chain of MPO and had a low constant affinity for MPO. The heavy-and light-chain variable region genes of monoclonal antibodies (mAb) 6D6 and 9B5 were sequenced and found to be highly homologous to germline genes and to contain negatively charged amino acids in the complementarity determining regions. IgM MPO-binding activity was observed in most BW and MRL/lpr-lpr mouse sera, which may correspond to polyclonal activation of B cells, whereas IgG anti-MPO antibodies could be rarely detected. Thus, this study indicates that (i) BW and MRL/lpr-lpr mice do not delete IgM anti-MPO secreting B cells, do not maintain these B cells in a state of anergy, but most individuals are not able to spontaneously induce the class-switching of this autoantibody population; (ii) IgM anti-MPO antibodies can recognize different epitopes on MPO and produce different immunofluorescence staining pattern on ethanol-fixed human neutrophils, as demonstrated by the immunochemical properties of the two lupus-mouse derived mAb. PMID:10540169

DISTINCT ANTIBODY SPECIES: STRUCTURAL DIFFERENCES CREATING THERAPEUTIC OPPORTUNITIES

PubMed Central

Muyldermans, Serge; Smider, Vaughn V.

2016-01-01

Antibodies have been a remarkably successful class of molecules for binding a large number of antigens in therapeutic, diagnostic, and research applications. Typical antibodies derived from mouse or human sources use the surface formed by complementarity determining regions (CDRs) on the variable regions of the heavy chain/light chain heterodimer, which typically forms a relatively flat binding surface. Alternative species, particularly camelids and bovines, provide a unique paradigm for antigen recognition through novel domains which form the antigen binding paratope. For camelids, heavy chain antibodies bind antigen with only a single heavy chain variable region, in the absence of light chains. In bovines, ultralong CDR-H3 regions form an independently folding minidomain, which protrudes from the surface of the antibody and is diverse in both its sequence and disulfide patterns. The atypical paratopes of camelids and bovines potentially provide the ability to interact with different epitopes, particularly recessed or concave surfaces, compared to traditional antibodies. PMID:26922135

Length-independent structural similarities enrich the antibody CDR canonical class model.

PubMed

Nowak, Jaroslaw; Baker, Terry; Georges, Guy; Kelm, Sebastian; Klostermann, Stefan; Shi, Jiye; Sridharan, Sudharsan; Deane, Charlotte M

2016-01-01

Complementarity-determining regions (CDRs) are antibody loops that make up the antigen binding site. Here, we show that all CDR types have structurally similar loops of different lengths. Based on these findings, we created length-independent canonical classes for the non-H3 CDRs. Our length variable structural clusters show strong sequence patterns suggesting either that they evolved from the same original structure or result from some form of convergence. We find that our length-independent method not only clusters a larger number of CDRs, but also predicts canonical class from sequence better than the standard length-dependent approach. To demonstrate the usefulness of our findings, we predicted cluster membership of CDR-L3 sequences from 3 next-generation sequencing datasets of the antibody repertoire (over 1,000,000 sequences). Using the length-independent clusters, we can structurally classify an additional 135,000 sequences, which represents a ∼20% improvement over the standard approach. This suggests that our length-independent canonical classes might be a highly prevalent feature of antibody space, and could substantially improve our ability to accurately predict the structure of novel CDRs identified by next-generation sequencing.

Synthetic Fab Fragments that Bind the HIV-1 gp41 Heptad Repeat Regions

PubMed Central

Liu, Yanyun; Regula, Lauren K.; Stewart, Alex; Lai, Jonathan R.

2011-01-01

Recent work has demonstrated that antibody phage display libraries containing restricted diversity in the complementarity determining regions (CDRs) can be used to target a wide variety of antigens with high affinity and specificity. In the most extreme case, antibodies whose combining sites are comprised of only two residues – tyrosine and serine – have been identified against several protein antigens. [F. A. Fellouse, B. Li, D. M. Compaan, A. A. Peden, S. G. Hymowitz, and S. S. Sidhu, J. Mol. Biol., 348 (2005) 1153–1162.] Here, we report the isolation and characterization of antigen-binding fragments (Fabs) from such “minimalist” diversity synthetic antibody libraries that bind the heptad repeat regions of human immunodeficiency virus type 1 (HIV-1) gp41. We show that these Fabs are highly specific for the HIV-1 epitope and comparable in affinity to a single chain variable fragment (scFv) derived from a natural antibody repertoire that targets the same region. Since the heptad repeat regions of HIV-1 gp41 are required for viral entry, these Fabs have potential for use in therapeutic, research, or diagnostic applications. PMID:21925149

Intraocular Penetration of a vNAR: In Vivo and In Vitro VEGF165 Neutralization.

PubMed

Camacho-Villegas, Tanya A; Mata-González, María Teresa; García-Ubbelohd, Walter; Núñez-García, Linda; Elosua, Carolina; Paniagua-Solis, Jorge F; Licea-Navarro, Alexei F

2018-03-31

Variable new antigen receptor domain (vNAR) antibodies are novel, naturally occurring antibodies that can be isolated from naïve, immune or synthetic shark libraries. These molecules are very interesting to the biotechnology and pharmaceutical industries because of their unique characteristics related to size and tissue penetrability. There have been some approved anti-angiogenic therapies for ophthalmic conditions, not related to vNAR. This includes biologics and chimeric proteins that neutralize vascular endothelial growth factor (VEGF) 165 , which are injected intravitreal, causing discomfort and increasing the possibility of infection. In this paper, we present a vNAR antibody against human recombinant VEGF 165 (rhVEGF 165 ) that was isolated from an immunized Heterodontus francisci shark. A vNAR called V13, neutralizes VEGF 165 cytokine starting at 75 μg/mL in an in vitro assay based on co-culture of normal human dermal fibroblasts (NHDFs) and green fluorescence protein (GFP)-labeled human umbilical vein endothelial cells (HUVECs) cells. In the oxygen-induced retinopathy model in C57BL/6:Hsd mice, we demonstrate an endothelial cell count decrease. Further, we demonstrate the intraocular penetration after topical administration of 0.1 μg/mL of vNAR V13 by its detection in aqueous humor in New Zealand rabbits with healthy eyes after 3 h of application. These findings demonstrate the potential of topical application of vNAR V13 as a possible new drug candidate for vascular eye diseases.

Anti-Aβ single-chain variable fragment antibodies exert synergistic neuroprotective activities in Drosophila models of Alzheimer's disease

PubMed Central

Fernandez-Funez, Pedro; Zhang, Yan; Sanchez-Garcia, Jonatan; de Mena, Lorena; Khare, Swati; Golde, Todd E.; Levites, Yona; Rincon-Limas, Diego E.

2015-01-01

Both active and passive immunotherapy protocols decrease insoluble amyloid-ß42 (Aß42) peptide in animal models, suggesting potential therapeutic applications against the main pathological trigger in Alzheimer's disease (AD). However, recent clinical trials have reported no significant benefits from humanized anti-Aß42 antibodies. Engineered single-chain variable fragment antibodies (scFv) are much smaller and can easily penetrate the brain, but identifying the most effective scFvs in murine AD models is slow and costly. We show here that scFvs against the N- and C-terminus of Aß42 (scFv9 and scFV42.2, respectively) that decrease insoluble Aß42 in CRND mice are neuroprotective in Drosophila models of Aß42 and amyloid precursor protein neurotoxicity. Both scFv9 and scFv42.2 suppress eye toxicity, reduce cell death in brain neurons, protect the structural integrity of dendritic terminals in brain neurons and delay locomotor dysfunction. Additionally, we show for the first time that co-expression of both anti-Aß scFvs display synergistic neuroprotective activities, suggesting that combined therapies targeting distinct Aß42 epitopes can be more effective than targeting a single epitope. Overall, we demonstrate the feasibility of using Drosophila as a first step for characterizing neuroprotective anti-Aß scFvs in vivo and identifying scFv combinations with synergistic neuroprotective activities. PMID:26253732

Passive West Nile virus antibody transfer from maternal Eastern Screech-Owls (Megascops asio) to progeny

USGS Publications Warehouse

Hahn, D.C.; Nemeth, N.M.; Edwards, E.; Bright, P.R.; Komar, N.

2006-01-01

Transovarial antibody transfer in owls has not been demonstrated for West Nile virus (WNV). We sampled chicks from captive adult WNV-antibody-positive Eastern Screech-Owls (Megascops asio) to evaluate the prevalence of transovarial maternal antibody transfer, as well as titers and duration of maternal antibodies. Twenty-four owlets aged 1 to 27 days old circulated detectable antibodies with neutralizing antibody titers ranging from 20 to 1600 (median 1:40). Demonstrating that WNV antibodies are passively transferred transovarially is important for accurate interpretation of serologic data from young birds.

Development of anti-bovine IgA single chain variable fragment and its application in diagnosis of foot-and-mouth disease

PubMed Central

Sridevi, N. V.; Shukra, A. M.; Neelakantam, B.; Anilkumar, J.; Madhanmohan, M.; Rajan, S.; Dev Chandran

2014-01-01

Recombinant antibody fragments like single chain variable fragments (scFvs) represent an attractive yet powerful alternative to immunoglobulins and hold great potential in the development of clinical diagnostic/therapeutic reagents. Structurally, scFvs are the smallest antibody fragments capable of retaining the antigen-binding capacity of whole antibodies and are composed of an immunoglobulin (Ig) variable light (VL) and variable heavy (VH) chain joined by a flexible polypeptide linker. In the present study, we constructed a scFv against bovine IgA from a hybridoma cell line IL-A71 that secretes a monoclonal antibody against bovine IgA using recombinant DNA technology. The scFv was expressed in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC). The binding activity and specificity of the scFv was established by its non-reactivity toward other classes of immunoglobulins as determined by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. Kinetic measurement of the scFv indicated that the recombinant antibody fragment had an affinity in picomolar range toward purified IgA. Furthermore, the scFv was used to develop a sensitive ELISA for the detection of foot and mouth disease virus (FMDV) carrier animals. PMID:24678404

Human immunodeficiency virus type 1 gp41 antibodies that mask membrane proximal region epitopes: antibody binding kinetics, induction, and potential for regulation in acute infection.

PubMed

Alam, S Munir; Scearce, Richard M; Parks, Robert J; Plonk, Kelly; Plonk, Steven G; Sutherland, Laura L; Gorny, Miroslaw K; Zolla-Pazner, Susan; Vanleeuwen, Stacie; Moody, M Anthony; Xia, Shi-Mao; Montefiori, David C; Tomaras, Georgia D; Weinhold, Kent J; Karim, Salim Abdool; Hicks, Charles B; Liao, Hua-Xin; Robinson, James; Shaw, George M; Haynes, Barton F

2008-01-01

Two human monoclonal antibodies (MAbs) (2F5 and 4E10) against the human immunodeficiency virus type 1 (HIV-1) envelope g41 cluster II membrane proximal external region (MPER) broadly neutralize HIV-1 primary isolates. However, these antibody specificities are rare, are not induced by Env immunization or HIV-1 infection, and are polyspecific and also react with lipids such as cardiolipin or phosphatidylserine. To probe MPER anti-gp41 antibodies that are produced in HIV-1 infection, we have made two novel murine MAbs, 5A9 and 13H11, against HIV-1 gp41 envelope that partially cross-blocked 2F5 MAb binding to Env but did not neutralize HIV-1 primary isolates or bind host lipids. Competitive inhibition assays using labeled 13H11 MAb and HIV-1-positive patient plasma samples demonstrated that cluster II 13H11-blocking plasma antibodies were made in 83% of chronically HIV-1 infected patients and were acquired between 5 to 10 weeks after acute HIV-1 infection. Both the mouse 13H11 MAb and the three prototypic cluster II human MAbs (98-6, 126-6, and 167-D) blocked 2F5 binding to gp41 epitopes to variable degrees; the combination of 98-6 and 13H11 completely blocked 2F5 binding. These data provide support for the hypothesis that in some patients, B cells make nonneutralizing cluster II antibodies that may mask or otherwise down-modulate B-cell responses to immunogenic regions of gp41 that could be recognized by B cells capable of producing antibodies like 2F5.

Differential recognition of the multiple banded antigen isoforms across Ureaplasma parvum and Ureaplasma urealyticum species by monoclonal antibodies.

PubMed

Aboklaish, Ali F; Ahmed, Shatha; McAllister, Douglas; Cassell, Gail; Zheng, Xiaotian T; Spiller, Owen B

2016-08-01

Two separate species of Ureaplasma have been identified that infect humans: Ureaplasma parvum and Ureaplasma urealyticum. Most notably, these bacteria lack a cell wall and are the leading infectious organism associated with infection-related induction of preterm birth. Fourteen separate representative prototype bacterial strains, called serovars, are largely differentiated by the sequence of repeating units in the C-terminus of the major surface protein: multiple-banded antigen (MBA). Monoclonal antibodies that recognise single or small groups of serovars have been previously reported, but these reagents remain sequestered in individual research laboratories. Here we characterise a panel of commercially available monoclonal antibodies raised against the MBA and describe the first monoclonal antibody that cross-reacts by immunoblot with all serovars of U. parvum and U. urealyticum species. We also describe a recombinant MBA expressed by Escherichia coli which facilitated further characterisation by immunoblot and demonstrate immunohistochemistry of paraffin-embedded antigens. Immunoblot reactivity was validated against well characterised previously published monoclonal antibodies and individual commercial antibodies were found to recognise all U. parvum strains, only serovars 3 and 14 or only serovars 1 and 6, or all strains belonging to U. parvum and U. urealyticum. MBA mass was highly variable between strains, consistent with variation in the number of C-terminal repeats between strains. Antibody characterisation will enable future investigations to correlate severity of pathogenicity to MBA isoform number or mass, in addition to development of antibody-based diagnostics that will detect infection by all Ureaplasma species or alternately be able to differentiate between U. parvum, U. urealyticum or mixed infections. Copyright © 2016 Elsevier B.V. All rights reserved.

Tetravalent anti-CD20/CD3 bispecific antibody for the treatment of B cell lymphoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Chia-Yen; Chen, Gregory J.; Tai, Pei-Han

Bispecific antibodies (bsAbs) are second generation antibodies for therapeutic application in immunotherapy. One of the major strategies of the bsAb platform is the recruitment of immune effector T cells by incorporating an anti-CD3 domain. A bispecific T-cell engager (BiTE), with one end having an affinity for CD3 and the other end with affinity for CD19, has been approved in the US and Europe for the treatment of acute lymphoblastic leukemia. However, due to their small size and lack of Fc region, these single-chain variable fragment (scFv) bsAbs have short half-lives in vivo. Additionally, poor solubility, structural instability, and low production yieldsmore » have also become major challenges in the bulk production process. To overcome these challenges, we have engineered a tetravalent bsAb with bivalent binding specificity for the CD20 and CD3 antigen in an immunoglobulin G (IgG) format. The fusion of the anti-CD3 scFvs to the CD20 antibody via a linker-hinge domain (LHD) results in improved antibody stabilization and properties. Here we demonstrate this antibody's highly efficient cancer cell elimination in a dose-dependent manner in a CD20-expressing B lymphoblastoid cell line in vitro. Our data suggest the potential clinical application of this bsAb for the treatment of CD20-expressing B cell malignancies. - Highlights: • A bispecific antibody (bsAb) can increase immunotherapeutic efficacy. • A tetravalent bsAb with binding specificity for the CD20 and CD3 antigens is proposed. • A linker-hinge domain (LHD) within the bsAb results in improved antibody properties.« less

Identification of inhibitory scFv antibodies targeting fibroblast activation protein utilizing phage display functional screens

PubMed Central

Zhang, Jiping; Valianou, Matthildi; Simmons, Heidi; Robinson, Matthew K.; Lee, Hyung-Ok; Mullins, Stefanie R.; Marasco, Wayne A.; Adams, Gregory P.; Weiner, Louis M.; Cheng, Jonathan D.

2013-01-01

Fibroblast activation protein (FAP) is a serine protease selectively expressed on tumor stromal fibroblasts in epithelial carcinomas and is important in cancer growth, adhesion, and metastases. As FAP enzymatic activity is a potent therapeutic target, we aimed to identify inhibitory antibodies. Using a competitive inhibition strategy, we used phage display techniques to identify 53 single-chain variable fragments (scFvs) after three rounds of panning against FAP. These scFvs were expressed and characterized for binding to FAP by surface plasmon resonance and flow cytometry. Functional assessment of these antibodies yielded an inhibitory scFv antibody, named E3, which could attenuate 35% of FAP cleavage of the fluorescent substrate Ala-Pro-7-amido-4-trifluoromethylcoumarin compared with nonfunctional scFv control. Furthermore, a mutant E3 scFv was identified by yeast affinity maturation. It had higher affinity (4-fold) and enhanced inhibitory effect on FAP enzyme activity (3-fold) than E3. The application of both inhibitory anti-FAP scFvs significantly affected the formation of 3-dimensional FAP-positive cell matrix, as demonstrated by reducing the fibronectin fiber orientation from 41.18% (negative antibody control) to 34.06% (E3) and 36.15% (mutant E3), respectively. Thus, we have identified and affinity-maturated the first scFv antibody capable of inhibiting FAP function. This scFv antibody has the potential to disrupt the role of FAP in tumor invasion and metastasis.—Zhang, J., Valianou, M., Simmons, H., Robinson, M. K., Lee, H.-O., Mullins, S. R., Marasco, W. A., Adams, G. P., Weiner, L. M., Cheng, J. D. Identification of inhibitory ScFv antibodies targeting fibroblast activation protein utilizing phage display functional screens. PMID:23104982

Chimeric recombinant antibody fragments in cardiac troponin I immunoassay.

PubMed

Hyytiä, Heidi; Heikkilä, Taina; Brockmann, Eeva-Christine; Kekki, Henna; Hedberg, Pirjo; Puolakanaho, Tarja; Lövgren, Timo; Pettersson, Kim

2015-03-01

To introduce a novel nanoparticle-based immunoassay for cardiac troponin I (cTnI) utilizing chimeric antibody fragments and to demonstrate that removal of antibody Fc-part and antibody chimerization decrease matrix related interferences. A sandwich-type immunoassay for cTnI based on recombinant chimeric (mouse variable/human constant) antigen binding (cFab) antibodies and intrinsically fluorescent nanoparticles was developed. To test whether using chimeric antibody fragments helps to avoid matrix related interferences, samples (n=39) with known amounts of triglycerides, bilirubin, rheumatoid factor (RF) or human anti-mouse antibodies (HAMAs) were measured with the novel assay, along with a previously published nanoparticle-based research assay with the same antibody epitopes. The limit of detection (LoD) was 3.30ng/L. Within-laboratory precision for 29ng/L and 2819ng/L cTnI were 13.7% and 15.9%, respectively. Regression analysis with Siemens ADVIA Centaur® yielded a slope (95% confidence intervals) of 0.18 (0.17-1.19) and a y-intercept of 1.94 (-1.28-3.91) ng/L. When compared to a previously published nanoparticle-based assay, the novel assay showed substantially reduced interference in the tested interference prone samples, 15.4 vs. 51.3%. A rheumatoid factor containing sample was decreased from 241ng/L to

High Inter-Individual Diversity of Point Mutations, Insertions, and Deletions in Human Influenza Virus Nucleoprotein-Specific Memory B Cells

PubMed Central

Bussmann, Bianca M.; Horn, Susanne; Sieg, Michael; Jassoy, Christian

2015-01-01

The diversity of virus-specific antibodies and of B cells among different individuals is unknown. Using single-cell cloning of antibody genes, we generated recombinant human monoclonal antibodies from influenza nucleoprotein-specific memory B cells in four adult humans with and without preceding influenza vaccination. We examined the diversity of the antibody repertoires and found that NP-specific B cells used numerous immunoglobulin genes. The heavy chains (HCs) originated from 26 and the kappa light chains (LCs) from 19 different germ line genes. Matching HC and LC chains gave rise to 43 genetically distinct antibodies that bound influenza NP. The median lengths of the CDR3 of the HC, kappa and lambda LC were 14, 9 and 11 amino acids, respectively. We identified changes at 13.6% of the amino acid positions in the V gene of the antibody heavy chain, at 8.4 % in the kappa and at 10.6 % in the lambda V gene. We identified somatic insertions or deletions in 8.1% of the variable genes. We also found several small groups of clonal relatives that were highly diversified. Our findings demonstrate broadly diverse memory B cell repertoires for the influenza nucleoprotein. We found extensive variation within individuals with a high number of point mutations, insertions, and deletions, and extensive clonal diversification. Thus, structurally conserved proteins can elicit broadly diverse and highly mutated B-cell responses. PMID:26086076

Targeting CD157 in AML using a novel, Fc-engineered antibody construct

PubMed Central

Krupka, Christina; Lichtenegger, Felix S.; Köhnke, Thomas; Bögeholz, Jan; Bücklein, Veit; Roiss, Michael; Altmann, Torben; Do, To Uyen; Dusek, Rachel; Wilson, Keith; Bisht, Arnima; Terrett, Jon; Aud, Dee; Pombo-Villar, Esteban; Rohlff, Christian; Hiddemann, Wolfgang; Subklewe, Marion

2017-01-01

Antibody-based immunotherapy represents a promising strategy to eliminate chemorefractory leukemic cells in acute myeloid leukemia (AML). In this study, we evaluated a novel Fc-engineered antibody against CD157 (MEN1112) for its suitability as immunotherapy in AML. CD157 was expressed in 97% of primary AML patient samples. A significant, albeit lower expression level of CD157 was observed within the compartment of leukemia-initiating cells, which are supposed to be the major source of relapse. In healthy donor bone marrow, CD157 was expressed on CD34+ cells. In ex vivo assays, MEN1112 triggered natural killer (NK) cell-mediated cytotoxicity against AML cell lines and primary AML cells. Compared to its parental analogue, the Fc-engineered antibody exhibited higher antibody dependent cellular cytotoxicity responses. Using NK cells from AML patients, we observed heterogeneous MEN1112-mediated cytotoxicity against AML cells, most likely due to well-documented defects in AML-NK cells and corresponding inter-patient variations in NK cell function. Cytotoxicity could not be correlated to the time after completion of chemotherapy. In summary, we could demonstrate that CD157 is strongly expressed in AML. MEN1112 is a promising antibody construct that showed high cytotoxicity against AML cells and warrants further clinical testing. Due to variability in NK-cell function of AML patients, the time of application during the course of the disease as well as combinatorial strategies might influence treatment results. PMID:28415689

Production of Recombinant Human scFv Against Tetanus Toxin Heavy Chain by Phage Display Technology.

PubMed

Khalili, Ehsan; Lakzaei, Mostafa; Rasaee, Mohhamad Javad; Aminian, Mahdi

2015-10-01

Tetanus, as a major cause of death in developing countries, is caused by tetanus neurotoxin. Recombinant antibodies against tetanus neurotoxin can be useful in tetanus management. Phage display of antibody fragments from immune human antibody libraries with single chain constructs combining the variable fragments (scFv) has been one of the most prominent technologies in antibody engineering. The aim of this study was the generation of a single chain fragment of variable region (scFv) library and selection of specific antibodies with high affinity against tetanus toxin. Immune human single chain fragment variable (HuscFv) antibody phagemid library was displayed on pIII of filamentous bacteriophage. Selection of scFv clones was performed against tetanus toxin antigens after three rounds of panning. The selected scFv clones were analyzed for inhibition of tetanus toxin binding to ganglioside GT1b. After the third round of panning, over 35 HuscFv phages specific for tetanus toxin were isolated from this library of which 15 clones were found to bind specifically to tetanus toxin. The selected HuscFv phages expressed as a soluble HuscFv peptide and some clones showed positive signals against tetanus toxin. We found that six HuscFv clones inhibit toxin binding to ganglioside GT1b. These selected antibodies can be used in the management of tetanus.

Production and characterization of a single chain variable fragment (scFv) for the mycotoxin deoxynivalenol

USDA-ARS?s Scientific Manuscript database

Deoxynivalenol (DON)is a mycotoxin produced by certain fungi that infest cereal grains worldwide. A hybridoma cell line producing a monoclonal antibody (Mab) recognizing DON was used as the starting point in the development of a recombinant single chain variable fragment (scFv) antibody. The scFv wa...

DARPA Antibody Technology Program Standardized Test Bed for Antibody Characterization: Characterization of an MS2 ScFv Antibody

DTIC Science & Technology

2016-03-01

performance in an enzyme-linked immunosorbent assay ( ELISA ), with little regard for quantification of the full spectrum of variables affecting antibody...Program (ATP) Quality MS2 coat protein (MS2CP) Enzyme-linked immunosorbent assay ( ELISA ) 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT...5 2.7 ELISA ................................................................................................................5

Phage display antibodies against ectromelia virus that neutralize variola virus: Selection and implementation for p35 neutralizing epitope mapping.

PubMed

Khlusevich, Yana; Matveev, Andrey; Baykov, Ivan; Bulychev, Leonid; Bormotov, Nikolai; Ilyichev, Ivan; Shevelev, Georgiy; Morozova, Vera; Pyshnyi, Dmitrii; Tikunova, Nina

2018-04-01

In this study, five phage display antibodies (pdAbs) against ectromelia virus (ECTV) were selected from vaccinia virus (VACV)-immune phage-display library of human single chain variable fragments (scFv). ELISA demonstrated that selected pdAbs could recognize ECTV, VACV, and cowpox virus (CPXV). Atomic force microscopy visualized binding of the pdAbs to VACV. Three of the selected pdAbs neutralized variola virus (VARV) in the plaque reduction neutralization test. Western blot analysis of ECTV, VARV, VACV, and CPXV proteins indicated that neutralizing pdAbs bound orthopoxvirus 35 kDa proteins, which are encoded by the open reading frames orthologous to the ORF H3L in VACV. The fully human antibody fh1A was constructed on the base of the VH and VL domains of pdAb, which demonstrated a dose-dependent inhibition of plaque formation after infection with VARV, VACV, and CPXV. To determine the p35 region responsible for binding to neutralizing pdAbs, a panel of truncated p35 proteins was designed and expressed in Escherichia coli cells, and a minimal p35 fragment recognized by selected neutralizing pdAbs was identified. In addition, peptide phage-display combinatorial libraries were applied to localize the epitope. The obtained data indicated that the epitope responsible for recognition by the neutralizing pdAbs is discontinuous and amino acid residues located within two p35 regions, 15-19 aa and 232-237 aa, are involved in binding with neutralizing anti-p35 antibodies. Copyright © 2018. Published by Elsevier B.V.

Comparing domain interactions within antibody Fabs with kappa and lambda light chains.

PubMed

Toughiri, Raheleh; Wu, Xiufeng; Ruiz, Diana; Huang, Flora; Crissman, John W; Dickey, Mark; Froning, Karen; Conner, Elaine M; Cujec, Thomas P; Demarest, Stephen J

2016-10-01

IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.

Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGFβ1 antibody

PubMed Central

Honey, Denise M.; Best, Annie; Qiu, Huawei

2018-01-01

ABSTRACT Metelimumab (CAT192) is a human IgG4 monoclonal antibody developed as a TGFβ1-specific antagonist. It was tested in clinical trials for the treatment of scleroderma but later terminated due to lack of efficacy. Subsequent characterization of CAT192 indicated that its TGFβ1 binding affinity was reduced by ∼50-fold upon conversion from the parental single-chain variable fragment (scFv) to IgG4. We hypothesized this result was due to decreased conformational flexibility of the IgG that could be altered via engineering. Therefore, we designed insertion mutants in the elbow region and screened for binding and potency. Our results indicated that increasing the elbow region linker length in each chain successfully restored the isoform-specific and high affinity binding of CAT192 to TGFβ1. The crystal structure of the high binding affinity mutant displays large conformational rearrangements of the variable domains compared to the wild-type antigen-binding fragment (Fab) and the low binding affinity mutants. Insertion of two glycines in both the heavy and light chain elbow regions provided sufficient flexibility for the variable domains to extend further apart than the wild-type Fab, and allow the CDR3s to make additional interactions not seen in the wild-type Fab structure. These interactions coupled with the dramatic conformational changes provide a possible explanation of how the scFv and elbow-engineered Fabs bind TGFβ1 with high affinity. This study demonstrates the benefits of re-examining both structure and function when converting scFv to IgG molecules, and highlights the potential of structure-based engineering to produce fully functional antibodies. PMID:29333938

Sequence intrinsic somatic mutation mechanisms contribute to affinity maturation of VRC01-class HIV-1 broadly neutralizing antibodies

PubMed Central

Hwang, Joyce K.; Wang, Chong; Du, Zhou; Meyers, Robin M.; Kepler, Thomas B.; Neuberg, Donna; Kwong, Peter D.; Mascola, John R.; Joyce, M. Gordon; Bonsignori, Mattia; Haynes, Barton F.; Yeap, Leng-Siew; Alt, Frederick W.

2017-01-01

Variable regions of Ig chains provide the antigen recognition portion of B-cell receptors and derivative antibodies. Ig heavy-chain variable region exons are assembled developmentally from V, D, J gene segments. Each variable region contains three antigen-contacting complementarity-determining regions (CDRs), with CDR1 and CDR2 encoded by the V segment and CDR3 encoded by the V(D)J junction region. Antigen-stimulated germinal center (GC) B cells undergo somatic hypermutation (SHM) of V(D)J exons followed by selection for SHMs that increase antigen-binding affinity. Some HIV-1–infected human subjects develop broadly neutralizing antibodies (bnAbs), such as the potent VRC01-class bnAbs, that neutralize diverse HIV-1 strains. Mature VRC01-class bnAbs, including VRC-PG04, accumulate very high SHM levels, a property that hinders development of vaccine strategies to elicit them. Because many VRC01-class bnAb SHMs are not required for broad neutralization, high overall SHM may be required to achieve certain functional SHMs. To elucidate such requirements, we used a V(D)J passenger allele system to assay, in mouse GC B cells, sequence-intrinsic SHM-targeting rates of nucleotides across substrates representing maturation stages of human VRC-PG04. We identify rate-limiting SHM positions for VRC-PG04 maturation, as well as SHM hotspots and intrinsically frequent deletions associated with SHM. We find that mature VRC-PG04 has low SHM capability due to hotspot saturation but also demonstrate that generation of new SHM hotspots and saturation of existing hotspot regions (e.g., CDR3) does not majorly influence intrinsic SHM in unmutated portions of VRC-PG04 progenitor sequences. We discuss implications of our findings for bnAb affinity maturation mechanisms. PMID:28747530

Ultrastructural characteristics of tau filaments in tauopathies: immuno-electron microscopic demonstration of tau filaments in tauopathies.

PubMed

Arima, Kunimasa

2006-10-01

The microtubule-associated protein tau aggregates into filaments in the form of neurofibrillary tangles, neuropil threads and argyrophilic grains in neurons, in the form of variable astrocytic tangles in astrocytes and in the form of coiled bodies and argyrophilic threads in oligodendrocytes. These tau filaments may be classified into two types, straight filaments or tubules with 9-18 nm diameters and "twisted ribbons" composed of two parallel aligned components. In the same disease, the fine structure of tau filaments in glial cells roughly resembles that in neurons. In sporadic tauopathies, individual tau filaments show characteristic sizes, shapes and arrangements, and therefore contribute to neuropathologic differential diagnosis. In frontotemporal dementias caused by tau gene mutations, variable filamentous profiles were observed in association with mutation sites and insoluble tau isoforms, including straight filaments or tubules, paired helical filament-like filaments, and twisted ribbons. Pre-embedding immunoelectron microscopic studies were carried out using anti-3-repeat tau and anti-4-repeat tau specific antibodies, RD3 and RD4. Straight tubules in neuronal and astrocytic Pick bodies were immunolabeled by the anti-3-repeat tau antibody. The anti-4-repeat tau antibody recognized abnormal tubules comprising neurofibrillary tangles, coiled bodies and argyrophilic threads in progressive supranuclear palsy (PSP) and corticobasal degeneration. In the pre-embedding immunoelectron microscopic study using the phosphorylated tau AT8 antibody, tuft-shaped astrocytes of PSP were found to be composed of bundles of abnormal tubules in processes and perikarya of protoplasmic astrocytes. In this study, the 3-repeat tau or 4-repeat tau epitope was detected in situ at the ultrastructural level in abnormal tubules in representative pathological lesions in Pick's disease, PSP and corticobasal degeneration.

B cell epitopes on infliximab identified by oligopeptide microarray with unprocessed patient sera.

PubMed

Homann, Arne; Röckendorf, Niels; Kromminga, Arno; Frey, Andreas; Jappe, Uta

2015-10-29

Autoimmune diseases like rheumatoid arthritis and inflammatory bowel disease are treated with TNF-alpha-blocking antibodies such as infliximab and adalimumab. A common side effect of therapeutic antibodies is the induction of anti-drug antibodies, which may reduce therapeutic efficacy. In order to reveal immunogenic epitopes on infliximab which are responsible for the adverse effects, sera from patients treated with infliximab were screened by ELISA for anti-infliximab antibodies. Sera containing high levels of anti-drug-antibodies (>1.25 µg/ml) were analyzed in an oligopeptide microarray system containing immobilized 15-meric oligopeptides from the infliximab amino acid sequence. Immunogenic infliximab IgG-epitopes were identified by infrared fluorescence scanning and comparison of infliximab-treated patients versus untreated controls. Six relevant epitopes on infliximab were recognized by the majority of all patient sera: 4 in the variable and 2 in the constant region. Three of the epitopes in the variable region are located in the TNF-alpha binding region of infliximab. The fourth epitope of the variable part of infliximab is located close to the TNF-alpha binding region and contains an N-glycosylation sequon. The sera positive for anti-infliximab antibodies do not contain antibodies against adalimumab as determined by ELISA. Thus, there is no infliximab-adalimumab cross-reactivity as determined by these systems. Our data shall contribute to a knowledge-based recommendation for a potentially necessary therapy switch from infliximab to another type of TNF-alpha-blocker. The characterization of immunogenic epitopes on therapeutic monoclonal antibodies using unprocessed patient sera shall lead to direct translational aspects for the development of less immunogenic therapeutic antibodies. Patients benefit from less adverse events and longer lasting drug effects.

Immunological observations following vasectomy.

PubMed

Choi, Y J; Reiner, L; Ney, C

1979-09-15

Lymphocytotoxic antibodies (LCA) against panels of normal lymphocytes and leukemic B-cells were demonstrated in vasectomized men. Since vasectomy is known to induce antibody formation to spermatozoa, the demonstration of these lymphocytotoxic antibodies may be related to antigenic constituents of spermatozoa such as HLA or B-cell alloantigens. Long term follow-up is needed to clarify the clinical significance of these antibodies.

Seroprevalence of Powassan virus in New England deer, 1979-2010.

PubMed

Nofchissey, Robert A; Deardorff, Eleanor R; Blevins, Tia M; Anishchenko, Michael; Bosco-Lauth, Angela; Berl, Erica; Lubelczyk, Charles; Mutebi, John-Paul; Brault, Aaron C; Ebel, Gregory D; Magnarelli, Louis A

2013-06-01

Powassan virus and its subtype, deer tick virus, are closely related tick-borne flaviviruses that circulate in North America. The incidence of human infection by these agents appears to have increased in recent years. To define exposure patterns among white-tailed deer, potentially useful sentinels that are frequently parasitized by ticks, we screened serum samples collected during 1979-2010 in Connecticut, Maine, and Vermont for neutralizing antibody by using a novel recombinant deer tick virus-West Nile virus chimeric virus. Evidence of exposure was detected in all three states. Overall our results demonstrate that seroprevalence is variable in time and space, suggesting that risk of exposure to Powassan virus is similarly variable.

Seroprevalence of Powassan Virus in New England Deer, 1979–2010

PubMed Central

Nofchissey, Robert A.; Deardorff, Eleanor R.; Blevins, Tia M.; Anishchenko, Michael; Bosco-Lauth, Angela; Berl, Erica; Lubelczyk, Charles; Mutebi, John-Paul; Brault, Aaron C.; Ebel, Gregory D.; Magnarelli, Louis A.

2013-01-01

Powassan virus and its subtype, deer tick virus, are closely related tick-borne flaviviruses that circulate in North America. The incidence of human infection by these agents appears to have increased in recent years. To define exposure patterns among white-tailed deer, potentially useful sentinels that are frequently parasitized by ticks, we screened serum samples collected during 1979–2010 in Connecticut, Maine, and Vermont for neutralizing antibody by using a novel recombinant deer tick virus–West Nile virus chimeric virus. Evidence of exposure was detected in all three states. Overall our results demonstrate that seroprevalence is variable in time and space, suggesting that risk of exposure to Powassan virus is similarly variable. PMID:23568288

Development of single chain variable fragment (scFv) antibodies against surface proteins of ‘Ca. Liberibacter asiaticus’

USDA-ARS?s Scientific Manuscript database

‘Ca. Liberibacter asiaticus’ is the causal agent of citrus huanglongbing, the most serious disease of citrus worldwide. We have developed and applied immunization and affinity screening methods to develop a primary library of recombinant single chain variable fragment (scFv) antibodies in an M13 vec...

Creation of a Ligand-Dependent Enzyme by Fusing Circularly Permuted Antibody Variable Region Domains.

PubMed

Iwai, Hiroto; Kojima-Misaizu, Miki; Dong, Jinhua; Ueda, Hiroshi

2016-04-20

Allosteric control of enzyme activity with exogenous substances has been hard to achieve, especially using antibody domains that potentially allow control by any antigens of choice. Here, in order to attain this goal, we developed a novel antibody variable region format introduced with circular permutations, called Clampbody. The two variable-region domains of the antibone Gla protein (BGP) antibody were each circularly permutated to have novel termini at the loops near their domain interface. Through their attachment to the N- and C-termini of a circularly permutated TEM-1 β-lactamase (cpBLA), we created a molecular switch that responds to the antigen peptide. The fusion protein specifically recognized the antigen, and in the presence of some detergent or denaturant, its catalytic activity was enhanced up to 4.7-fold in an antigen-dependent manner, due to increased resistance to these reagents. Hence, Clampbody will be a powerful tool for the allosteric regulation of enzyme and other protein activities and especially useful to design robust biosensors.

Duration of serum antibody responses following vaccination and revaccination of cattle with non-living commercial Pasteurella haemolytica vaccines.

PubMed

Confer, A W; Fulton, R W; Clinkenbeard, K D; Driskel, B A

1998-12-01

This study was designed to determine the duration of serum antibody responses to Pasteurella haemolytica whole cells (WC) and leukotoxin (LKT) in weanling beef cattle vaccinated with various non-living P. haemolytica vaccines. Serum antibodies to P. haemolytica antigens were determined periodically through day 140 by enzyme-linked immunosorbent assays. At day 140, cattle were revaccinated, and antibody responses periodically determined through day 196. Three vaccines were used in two experiments (A and B), OneShot, Presponse HP/tK, and Septimune PH-K. In general, all three vaccines between 7 and 14 days induced antibody responses to WC after vaccination. Antibodies to LKT were induced with OneShot and Presponse. Revaccination at days 28 and 140 usually stimulated anamnestic responses. Serum antibodies to the various antigens remained significantly increased for up to 84 days after vaccination or revaccination. The intensity and duration of antibody responses were variable depending on the experiment and vaccines used. Vaccination with OneShot usually stimulated the greatest responses to WC. Vaccination with OneShot or Presponse resulted in equivalent primary anti-LKT responses. In experiment B, spontaneous seroconversion was found in numerous calves on day 112. Revaccination of those cattle at day 140 resulted in markedly variable antibody responses such that several groups had no increase in antibody responses.

A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties

PubMed Central

Tiller, Thomas; Schuster, Ingrid; Deppe, Dorothée; Siegers, Katja; Strohner, Ralf; Herrmann, Tanja; Berenguer, Marion; Poujol, Dominique; Stehle, Jennifer; Stark, Yvonne; Heßling, Martin; Daubert, Daniela; Felderer, Karin; Kaden, Stefan; Kölln, Johanna; Enzelberger, Markus; Urlinger, Stefanie

2013-01-01

This report describes the design, generation and testing of Ylanthia, a fully synthetic human Fab antibody library with 1.3E+11 clones. Ylanthia comprises 36 fixed immunoglobulin (Ig) variable heavy (VH)/variable light (VL) chain pairs, which cover a broad range of canonical complementarity-determining region (CDR) structures. The variable Ig heavy and Ig light (VH/VL) chain pairs were selected for biophysical characteristics favorable to manufacturing and development. The selection process included multiple parameters, e.g., assessment of protein expression yield, thermal stability and aggregation propensity in fragment antigen binding (Fab) and IgG1 formats, and relative Fab display rate on phage. The framework regions are fixed and the diversified CDRs were designed based on a systematic analysis of a large set of rearranged human antibody sequences. Care was taken to minimize the occurrence of potential posttranslational modification sites within the CDRs. Phage selection was performed against various antigens and unique antibodies with excellent biophysical properties were isolated. Our results confirm that quality can be built into an antibody library by prudent selection of unmodified, fully human VH/VL pairs as scaffolds. PMID:23571156

Dry immunochemical sensor for the detection of PETN vapor

NASA Astrophysics Data System (ADS)

Lukens, Herbert Richard

1992-05-01

Giaever (1973) showed that an indium semi-mirror coated with a monolayer of a substance would undergo reduced optical reflectance after incubation with a solution of the substance's antibody. He was able to see the effect with the naked eye. Lukens and Williams (1977) reversed the process by first attaching the target substance's antibody to the semi-mirror, after which the device would show a decline in optical density when exposed to a solution of the target substance. Lukens and Williams (1982) subsequently found that the device could also be used as an immunochemical film badge (IFB) to detect an airborne target substance. Early efforts to develop the IFB for the detection of airborne substances were plagued by such a high degree of performance variability that there were doubts in some quarters that an airborne target substance could bind to its antibody until Lukens (1990) demonstrated such binding in experiments with radiolabeled cocaine and morphine. Recently improved semi-mirrors and densitometry have been obtained and have led to improved performance of IFB's. As shown in this paper, IFB's can now be constructed that detect PETN vapor in a few seconds.

Polymorphisms in HLA-DPB1 Are Associated With Differences in Rubella Virus–Specific Humoral Immunity After Vaccination

PubMed Central

Lambert, Nathaniel D.; Haralambieva, Iana H.; Kennedy, Richard B.; Ovsyannikova, Inna G.; Pankratz, Vernon Shane; Poland, Gregory A.

2015-01-01

Vaccination with live attenuated rubella virus induces a strong immune response in most individuals. However, small numbers of subjects never reach or maintain protective antibody levels, and there is a high degree of variability in immune response. We have previously described genetic polymorphisms in HLA and other candidate genes that are associated with interindividual differences in humoral immunity to rubella virus. To expand our previous work, we performed a genome-wide association study (GWAS) to discover single-nucleotide polymorphisms (SNPs) associated with rubella virus–specific neutralizing antibodies. We identified rs2064479 in the HLA-DPB1 genetic region as being significantly associated with humoral immune response variations after rubella vaccination (P = 8.62 × 10−8). All other significant SNPs in this GWAS were located near the HLA-DPB1 gene (P ≤ 1 × 10−7). These findings demonstrate that polymorphisms in HLA-DPB1 are strongly associated with interindividual differences in neutralizing antibody levels to rubella vaccination and represent a validation of our previous HLA work. PMID:25293367

HIV-1 VACCINES. Diversion of HIV-1 vaccine-induced immunity by gp41-microbiota cross-reactive antibodies.

PubMed

Williams, Wilton B; Liao, Hua-Xin; Moody, M Anthony; Kepler, Thomas B; Alam, S Munir; Gao, Feng; Wiehe, Kevin; Trama, Ashley M; Jones, Kathryn; Zhang, Ruijun; Song, Hongshuo; Marshall, Dawn J; Whitesides, John F; Sawatzki, Kaitlin; Hua, Axin; Liu, Pinghuang; Tay, Matthew Z; Seaton, Kelly E; Shen, Xiaoying; Foulger, Andrew; Lloyd, Krissey E; Parks, Robert; Pollara, Justin; Ferrari, Guido; Yu, Jae-Sung; Vandergrift, Nathan; Montefiori, David C; Sobieszczyk, Magdalena E; Hammer, Scott; Karuna, Shelly; Gilbert, Peter; Grove, Doug; Grunenberg, Nicole; McElrath, M Juliana; Mascola, John R; Koup, Richard A; Corey, Lawrence; Nabel, Gary J; Morgan, Cecilia; Churchyard, Gavin; Maenza, Janine; Keefer, Michael; Graham, Barney S; Baden, Lindsey R; Tomaras, Georgia D; Haynes, Barton F

2015-08-14

An HIV-1 DNA prime vaccine, with a recombinant adenovirus type 5 (rAd5) boost, failed to protect from HIV-1 acquisition. We studied the nature of the vaccine-induced antibody (Ab) response to HIV-1 envelope (Env). HIV-1-reactive plasma Ab titers were higher to Env gp41 than to gp120, and repertoire analysis demonstrated that 93% of HIV-1-reactive Abs from memory B cells responded to Env gp41. Vaccine-induced gp41-reactive monoclonal antibodies were non-neutralizing and frequently polyreactive with host and environmental antigens, including intestinal microbiota (IM). Next-generation sequencing of an immunoglobulin heavy chain variable region repertoire before vaccination revealed an Env-IM cross-reactive Ab that was clonally related to a subsequent vaccine-induced gp41-reactive Ab. Thus, HIV-1 Env DNA-rAd5 vaccine induced a dominant IM-polyreactive, non-neutralizing gp41-reactive Ab repertoire response that was associated with no vaccine efficacy. Copyright © 2015, American Association for the Advancement of Science.

Detection of bacterial-reactive natural IgM antibodies in desert bighorn sheep populations

PubMed Central

Palmer, Amy L.; Zielke, Ryszard A.; Sikora, Aleksandra E.; Beechler, Brianna R.; Jolles, Anna E.; Epps, Clinton W.

2017-01-01

Ecoimmunology is a burgeoning field of ecology which studies immune responses in wildlife by utilizing general immune assays such as the detection of natural antibody. Unlike adaptive antibodies, natural antibodies are important in innate immune responses and often recognized conserved epitopes present in pathogens. Here, we describe a procedure for measuring natural antibodies reactive to bacterial antigens that may be applicable to a variety of organisms. IgM from desert bighorn sheep plasma samples was tested for reactivity to outer membrane proteins from Vibrio coralliilyticus, a marine bacterium to which sheep would have not been exposed. Immunoblotting demonstrated bighorn sheep IgM could bind to a variety of bacterial cell envelope proteins while ELISA analysis allowed for rapid determination of natural antibody levels in hundreds of individual animals. Natural antibody levels were correlated with the ability of plasma to kill laboratory strains of E. coli bacteria. Finally, we demonstrate that natural antibody levels varied in two distinct populations of desert bighorn sheep. These data demonstrate a novel and specific measure of natural antibody function and show that this varies in ecologically relevant ways. PMID:28662203

Chimeric Anti-Human Podoplanin Antibody NZ-12 of Lambda Light Chain Exerts Higher Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity Compared with NZ-8 of Kappa Light Chain.

PubMed

Kaneko, Mika K; Abe, Shinji; Ogasawara, Satoshi; Fujii, Yuki; Yamada, Shinji; Murata, Takeshi; Uchida, Hiroaki; Tahara, Hideaki; Nishioka, Yasuhiko; Kato, Yukinari

2017-02-01

Podoplanin (PDPN), a type I transmembrane 36-kDa glycoprotein, is expressed not only in normal cells, such as renal epithelial cells (podocytes), lymphatic endothelial cells, and pulmonary type I alveolar cells, but also in cancer cells, including brain tumors and lung squamous cell carcinomas. Podoplanin activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelets, and the podoplanin/CLEC-2 interaction facilitates blood/lymphatic vessel separation. We previously produced neutralizing anti-human podoplanin monoclonal antibody (mAb), clone NZ-1 (rat IgG 2a , lambda), which neutralizes the podoplanin/CLEC-2 interaction and inhibits platelet aggregation and cancer metastasis. Human-rat chimeric antibody, NZ-8, was previously developed using variable regions of NZ-1 and human constant regions of heavy chain (IgG 1 ) and light chain (kappa chain). Although NZ-8 showed high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human podoplanin-expressing cancer cells, the binding affinity of NZ-8 was lower than that of NZ-1. Herein, we produced a novel human-rat chimeric antibody, NZ-12, the constant regions of which consist of IgG 1 heavy chain and lambda light chain. Using flow cytometry, we demonstrated that the binding affinity of NZ-12 was much higher than that of NZ-8. Furthermore, ADCC and CDC activities of NZ-12 were significantly increased against glioblastoma cell lines (LN319 and D397) and lung cancer cell line (PC-10). These results suggested that NZ-12 could become a promising therapeutic antibody against podoplanin-expressing brain tumors and lung cancers.

Binding diversity of antibodies against external and internal epitopes of the multidrug resistance gene product P-glycoprotein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lehne, G.; De Angelis, P.; Clausen, O.P.F.

1995-07-01

P-glycoprotein (Pgp) is a trans-membraneous protein that is associated with multidrug resistance (MDR) in human cancer, including hepatocellular carcinomas and leukemia. There is no consensus regarding methods of choice for analysis of Pgp expression, and development of reliable analytical methods is now essential. We have studied the Pgp expression in human hepatoma and leukemia cell lines using flow cytometry. The aim of the study was to compare binding properties of anti-Pgp antibodies reacting with surface (MRK16, UIC2) and cytoplasmic (C219, JSB-1) epitopes to assess which antibody performed best with respect to fluorescence discrimination. By histogram subtraction the fractions of resistantmore » human hepatoma cells positive for Pgp were 99% (MRK16), 97% (UIC2), 77% (USB-1), and 51% (C219), demonstrating variations in antibody reactivity. The resolution in detecting decreasing levels of Pgp in hepatoma cells was superior for the externally binding antibodies, showing that there is a correlation between antibody reactivity and fluorescence discrimination. Similar results were obtained for parental and resistant KG1a human leukemia cell lines. The Pgp epitopes remained reactive to the anti-Pgp MAbs after methanol fixation and cryopreservation. By dual parameter flow cytometry it was shown that Pgp expression in viable cells may be assessed together with uptake of epirubicin, which was low in cells expressing high levels of Pgp and vice versa. In conclusion, all tested antibodies proved useful for flow cytometric detection of high levels of Pgp, but the externally binding ones were superior in detection of low and variable levels of Pgp. 36 refs., 8 figs., 1 tab.« less

Generation of a Canine Anti-EGFR (ErbB-1) Antibody for Passive Immunotherapy in Dog Cancer Patients

PubMed Central

Wang, Wei; Weichselbaumer, Marlene; Matz, Miroslawa; Mader, Alexander; Steinfellner, Willibald; Meitz, Sarah; Mechtcheriakova, Diana; Sobanov, Yuri; Willmann, Michael; Stockner, Thomas; Spillner, Edzard; Kunert, Renate; Jensen-Jarolim, Erika

2014-01-01

Passive immunotherapy with monoclonal antibodies represents a cornerstone of human anticancer therapies, but has not been established in veterinary medicine yet. As the tumor-associated antigen EGFR (ErbB-1) is highly conserved between humans and dogs, and considering the effectiveness of the anti-EGFR antibody cetuximab in human clinical oncology, we present here a “caninized” version of this antibody, can225IgG, for comparative oncology studies. Variable region genes of 225, the murine precursor of cetuximab, were fused with canine constant heavy gamma and kappa chain genes, respectively, and transfected into Chinese hamster ovary (CHO) DUKX-B11 cells. Of note, 480 clones were screened and the best clones were selected according to productivity and highest specificity in EGFR-coated ELISA. Upon purification with Protein G, the recombinant cetuximab-like canine IgG was tested for integrity, correct assembly, and functionality. Specific binding to the surface of EGFR-overexpressing cells was assessed by flow cytometry and immunofluorescence; moreover, binding to canine mammary tissue was demonstrated by immunohistochemistry. In cell viability and proliferation assays, incubation with can225IgG led to significant tumor cell growth inhibition. Moreover, this antibody mediated significant tumor cell killing via phagocytosis in vitro. We thus present here, for the first time, the generation of a canine IgG antibody and its hypothetical structure. On the basis of its cetuximab-like binding site, on the one hand, and the expression of a 91% homologous EGFR molecule in canine cancer, on the other hand, this antibody may be a promising research compound to establish passive immunotherapy in dog patients with cancer. PMID:24755200

Antibody responses to prime-boost vaccination with an HIV-1 gp145 envelope protein and chimpanzee adenovirus vectors expressing HIV-1 gp140.

PubMed

Emmer, Kristel L; Wieczorek, Lindsay; Tuyishime, Steven; Molnar, Sebastian; Polonis, Victoria R; Ertl, Hildegund C J

2016-10-23

Over 2 million individuals are infected with HIV type 1 (HIV-1) each year, yet an effective vaccine remains elusive. The most successful HIV-1 vaccine to date demonstrated 31% efficacy. Immune correlate analyses associated HIV-1 envelope (Env)-specific antibodies with protection, thus providing a path toward a more effective vaccine. We sought to test the antibody response from novel prime-boost vaccination with a chimpanzee-derived adenovirus (AdC) vector expressing a subtype C Env glycoprotein (gp)140 combined with either a serologically distinct AdC vector expressing gp140 of a different subtype C isolate or an alum-adjuvanted, partially trimeric gp145 from yet another subtype C isolate. Three different prime-boost regimens were tested in mice: AdC prime-protein boost, protein prime-AdC boost, and AdC prime-AdC boost. Each regimen was tested at two different doses of AdC vector in a total of six experimental groups. Sera were collected at various time points and evaluated by ELISA for Env-specific antibody binding, isotype, and avidity. Antibody functionality was assessed by pseudovirus neutralization assay. Priming with AdC followed by a protein boost or sequential immunizations with two AdC vectors induced HIV-1 Env-specific binding antibodies, including those to the variable region 2, whereas priming with protein followed by an AdC boost was relatively ineffective. Antibodies that cross-neutralized tier 1 HIV-1 from different subtypes were elicited with vaccine regimens that included immunizations with protein. Our study warrants further investigation of AdC vector and gp145 protein prime-boost vaccines and their ability to protect against acquisition in animal challenge studies.

Transplanting Sensitized Kidney Transplant Patients With Equivalent Outcomes Utilizing Stringent HLA Crossmatching.

PubMed

Rohan, Vinayak S; Taber, David J; Moussa, Omar; Pilch, Nicole A; Denmark, Signe; Meadows, Holly B; McGillicuddy, John W; Chavin, Kenneth D; Baliga, Prabhakar K; Bratton, Charles F

2017-02-01

Elevated panel reactive antibody levels have been traditionally associated with increased acute rejection rate and decreased long-term graft survival after kidney transplant. In this study, our objective was to determine patient and allograft outcomes in sensitized kidney transplant recipients with advanced HLA antibody detection and stringent protein sequence epitope analyses. This was a subanalysis of a prospective, risk-stratified randomized controlled trial that compared interleukin 2 receptor antagonist to rabbit antithymocyte globulin induction in 200 kidney transplant recipients, examining outcomes based on panel reactive antibody levels of < 20% (low) versus ≥ 20% (high, sensitized). The study was conducted between February 2009 and July 2011. All patients underwent solid-phase single antigen bead assays to detect HLA antibodies and stringent HLA epitope analyses with protein sequence alignment for virtual crossmatching. Delayed graft function, acute rejection rates, and graft loss were the main outcomes measured. Both the low (134 patients) and high (66 patients) panel reactive antibody level cohorts had equivalent induction and maintenance immunosuppression. Patients in the high-level group were more likely to be female (P < .001), African American (P < .001), and received a kidney from a deceased donor (P = .004). Acute rejection rates were similar between the low (rate of 8%) and high (rate of 9%) panel reactive antibody groups (P = .783). Delayed graft function, borderline rejection, graft loss, and death were not different between groups. Multivariate analyses demonstrated delayed graft function to be the strongest predictor of acute rejection (odds ratio, 5.7; P = .005); panel reactive antibody level, as a continuous variable, had no significant correlation with acute rejection (C statistic, 0.48; P = .771). Appropriate biologic matching with single antigen bead assays and stringent epitope analyses provided excellent outcomes in sensitized patients regardless of the induction therapy choice.

[Construction of dengue virus-specific full-length fully human antibody libraries by mammalian display technology].

PubMed

Wen, Yangming; Lan, Kaijian; Wang, Junjie; Yu, Jingyi; Qu, Yarong; Zhao, Wei; Zhang, Fuchun; Tan, Wanlong; Cao, Hong; Zhou, Chen

2013-06-01

To construct dengue virus-specific full-length fully human antibody libraries using mammalian cell surface display technique. Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) from convalescent patients with dengue fever. The reservoirs of the light chain and heavy chain variable regions (LCκ and VH) of the antibody genes were amplified by RT-PCR and inserted into the vector pDGB-HC-TM separately to construct the light chain and heavy chain libraries. The library DNAs were transfected into CHO cells and the expression of full-length fully human antibodies on the surface of CHO cells was analyzed by flow cytometry. Using 1.2 µg of the total RNA isolated from the PBMCs as the template, the LCκ and VH were amplified and the full-length fully human antibody mammalian display libraries were constructed. The kappa light chain gene library had a size of 1.45×10(4) and the heavy chain gene library had a size of 1.8×10(5). Sequence analysis showed that 8 out of the 10 light chain clones and 7 out of the 10 heavy chain clones randomly picked up from the constructed libraries contained correct open reading frames. FACS analysis demonstrated that all the 15 clones with correct open reading frames expressed full-length antibodies, which could be detected on CHO cell surfaces. After co-transfection of the heavy chain and light chain gene libraries into CHO cells, the expression of full-length antibodies on CHO cell surfaces could be detected by FACS analysis with an expressible diversity of the antibody library reaching 1.46×10(9) [(1.45×10(4)×80%)×(1.8×10(5)×70%)]. Using 1.2 µg of total RNA as template, the LCκ and VH full-length fully human antibody libraries against dengue virus have been successfully constructed with an expressible diversity of 10(9).

Application of two different kinds of sera against the Proteus penneri lipopolysaccharide core region in search of epitopes determining cross-reactions with antibodies.

PubMed

Palusiak, Agata; Dzieciatkowska, Monika; Sidorczyk, Zygmunt

2008-01-01

Proteus penneri lipopolysaccharide (LPS) core regions are characterized by a greater structural variability than that observed in other Enterobacteriaceae. This fact and the small amount of published data concerning the serological activity of this part of P. penneri LPS prompted an examination of which fragment might determine cross-reactions with antibodies. To date, such epitopes have been found in the LPS core regions of P. mirabilis and P. vulgaris strains. Proteus sp. LPSs were tested with unabsorbed rabbit antisera by enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot, and once again by ELISA or passive immunohemolysis after the absorption of these antisera with selected LPSs. The serological studies of P. penneri 8 LPS demonstrated antibodies in the tested antisera recognizing a common epitope located in the core regions of six of the LPSs, i.e. P. penneri 8, 34, 133, 7, 14, and 15. Additionally, another type of antibody directed against some fragment of P. penneri 13 and the core regions of other LPSs investigated was observed in one antiserum. A distal, trisaccharide fragment of the P. penneri 8 LPS core region is suggested to determine the cross-reactions of the tested antisera with the six P. penneri LPSs.

The effect of freeze-dried antibody concentrations on its stability in the presence of trehalose and hydroxypropyl-β-cyclodextrin: a Box-Behnken statistical design.

PubMed

Faghihi, Homa; Khalili, Fatemeh; Amini, Mohsen; Vatanara, Alireza

2017-09-01

The present study aimed at preparation and optimization of stable freeze-dried immunoglobulin G (IgG) applying proper amount of antibody with efficient combination of trehalose and hydroxypropyl-β-cyclodextrin (HPβCD). Response surface methodology was employed through a three-factor, three-level Box-Behnken design. Amounts of IgG (X 1 ), trehalose (X 2 ) and HPβCD (X 3 ) were independent variables. Aggregation following process (Y 1 ), after one month at 45 °C (Y 2 ), upon two month at 45 °C (Y 3 ) and beta-sheet content of IgG (Y 4 ) were determined as dependent variables. Results were fitted to quadratic models (except for beta-sheet content), describing the inherent relationship between main factors. Optimized formulation composed of 55.85 mg IgG, 52.51 mg trehalose and 16.01 mg HPβCD was prepared. The calculated responses of the optimized formulation were as follows: Y 1  = 0.19%, Y 2  = 0.78%, Y 3  = 1.88% and Y 4  = 68.60%, respectively. The thermal analysis confirmed the amorphous nature of optimum formulation and the integrity of IgG was shown to be favorably preserved. Validation of the optimization study demonstrated high degree of prognostic ability. The DOE study successfully predicted the optimum values of antibody as well as stabilizers for desirable process and storage stabilization of freeze-dried IgG.

Insulin aspart pharmacokinetics: an assessment of its variability and underlying mechanisms.

PubMed

Rasmussen, Christian Hove; Røge, Rikke Meldgaard; Ma, Zhulin; Thomsen, Maria; Thorisdottir, Rannveig Linda; Chen, Jian-Wen; Mosekilde, Erik; Colding-Jørgensen, Morten

2014-10-01

Insulin aspart (IAsp) is used by many diabetics as a meal-time insulin to control post-prandial glucose levels. As is the case with many other insulin types, the pharmacokinetics (PK), and consequently the pharmacodynamics (PD), is associated with clinical variability, both between and within individuals. The present article identifies the main physiological mechanisms that govern the PK of IAsp following subcutaneous administration and quantifies them in terms of their contribution to the overall variability. CT scanning data from Thomsen et al. (2012) are used to investigate and quantify the properties of the subcutaneous depot. Data from Brange et al. (1990) are used to determine the effects of insulin chemistry in subcutis on the absorption rate. Intravenous (i.v.) bolus and infusion PK data for human insulin are used to understand and quantify the systemic distribution and elimination (Pørksen et al., 1997; Sjöstrand et al., 2002). PK and PD profiles for type 1 diabetics from Chen et al. (2005) are analyzed to demonstrate the effects of IAsp antibodies in terms of bound and unbound insulin. PK profiles from Thorisdottir et al. (2009) and Ma et al. (2012b) are analyzed in the nonlinear mixed effects software Monolix® to determine the presence and effects of the mechanisms described in this article. The distribution of IAsp in the subcutaneous depot show an initial dilution of approximately a factor of two in a single experiment. Injected insulin hexamers exist in a chemical equilibrium with monomers and dimers, which depends strongly on the degree of dilution in subcutis, the presence of auxiliary substances, and a variety of other factors. Sensitivity to the initial dilution in subcutis can thus be a cause of some of the variability. Temporal variations in the PK are explained by variations in the subcutaneous blood flow. IAsp antibodies are found to be a large contributor to the variability of total insulin PK in a study by Chen et al. (2005), since only the free fraction is eliminated via the receptors. The contribution of these and other sources of variability to the total variability is quantified via a population PK analysis and two recent clinical studies (Thorisdottir et al., 2009; Ma et al., 2012b), which support the presence and significance of the identified mechanisms. IAsp antibody binding, oligomeric transitions in subcutis, and blood flow dependent variations in absorption rate seem to dominate the PK variability of IAsp. It may be possible via e.g. formulation design to reduce some of these variability factors. Copyright © 2014 Elsevier B.V. All rights reserved.

Very Few Substitutions in a Germ Line Antibody Are Required To Initiate Significant Domain Exchange ▿

PubMed Central

Huber, Michael; Le, Khoa M.; Doores, Katie J.; Fulton, Zara; Stanfield, Robyn L.; Wilson, Ian A.; Burton, Dennis R.

2010-01-01

2G12 is a broadly neutralizing anti-HIV-1 monoclonal human IgG1 antibody reactive with a high-mannose glycan cluster on the surface of glycoprotein gp120. A key feature of this very highly mutated antibody is domain exchange of the heavy-chain variable region (VH) with the VH of the adjacent Fab of the same immunoglobulin, which assembles a multivalent binding interface composed of two primary binding sites in close proximity. A non-germ line-encoded proline in the elbow between VH and CH1 and an extensive network of hydrophobic interactions in the VH/VH′ interface have been proposed to be crucial for domain exchange. To investigate the origins of domain exchange, a germ line version of 2G12 that behaves as a conventional antibody was engineered. Substitution of 5 to 7 residues for those of the wild type produced a significant fraction of domain-exchanged molecules, with no evidence of equilibrium between domain-exchanged and conventional forms. Two substitutions not previously implicated, AH14 and EH75, are the most crucial for domain exchange, together with IH19 at the VH/VH′ interface and PH113 in the elbow region. Structural modeling gave clues as to why these residues are essential for domain exchange. The demonstration that domain exchange can be initiated by a small number of substitutions in a germ line antibody suggests that the evolution of a domain-exchanged antibody response in vivo may be more readily achieved than considered to date. PMID:20702640

Antibody-Dependent Cell-Mediated Cytotoxicity Effector-Enhanced EphA2 Agonist Monoclonal Antibody Demonstrates Potent Activity against Human Tumors1

PubMed Central

Bruckheimer, Elizabeth M; Fazenbaker, Christine A; Gallagher, Sandra; Mulgrew, Kathy; Fuhrmann, Stacy; Coffman, Karen T; Walsh, William; Ready, Shannon; Cook, Kim; Damschroder, Melissa; Kinch, Michael; Kiener, Peter A; Woods, Rob; Gao, Changshou; Dall'Acqua, William; Wu, Herren; Coats, Steven

2009-01-01

EphA2 is a receptor tyrosine kinase that has been shown to be overexpressed in a variety of human tumor types. Previous studies demonstrated that agonist monoclonal antibodies targeting EphA2 induced the internalization and degradation of the receptor, thereby abolishing its oncogenic effects. In this study, the in vitro and in vivo antibody-dependent cell-mediated cytotoxicity (ADCC) activity of EphA2 effector-enhanced agonist monoclonal antibodies was evaluated. With tumor cell lines and healthy human peripheral blood monocytes, the EphA2 antibodies demonstrated ∼80% tumor cell killing. In a dose-dependent manner, natural killer (NK) cells were required for the in vitro ADCC activity and became activated as demonstrated by the induction of cell surface expression of CD107a. To assess the role of NK cells on antitumor efficacy in vivo, the EphA2 antibodies were evaluated in xenograft models in severe compromised immunodeficient (SCID) mice (which have functional NK cells and monocytes) and SCID nonobese diabetic (NOD) mice (which largely lack functional NK cells and monocytes). Dosing of EphA2 antibody in the SCID murine tumor model resulted in a 6.2-fold reduction in tumor volume, whereas the SCID/nonobese diabetic model showed a 1.6-fold reduction over the isotype controls. Together, these results demonstrate that the anti-EphA2 monoclonal antibodies may function through at least two mechanisms of action: EphA2 receptor activation and ADCC-mediated activity. These novel EphA2 monoclonal antibodies provide additional means by which host effector mechanisms can be activated for selective destruction of EphA2-expressing tumor cells. PMID:19484140

Clinical Utility of Acetylcholine Receptor Antibody Testing in Ocular Myasthenia Gravis.

PubMed

Peeler, Crandall E; De Lott, Lindsey B; Nagia, Lina; Lemos, Joao; Eggenberger, Eric R; Cornblath, Wayne T

2015-10-01

The sensitivity of acetylcholine receptor (AChR) antibody testing is thought to be lower in ocular myasthenia gravis (OMG) compared with generalized disease, although estimates in small-scale studies vary. There is little information in the literature about the implications of AChR antibody levels and progression from OMG to generalized myasthenia gravis. To test the hypothesis that serum AChR antibody testing is more sensitive in OMG than previously reported and to examine the association between AChR antibody levels and progression from OMG to generalized myasthenia gravis. A retrospective, observational cohort study was conducted of 223 patients (mean [SD] age, 59.2 [16.4] years; 139 [62.3%] male) diagnosed with OMG between July 1, 1986, and May 31, 2013, at 2 large, academic medical centers. Baseline characteristics, OMG symptoms, results of AChR antibody testing, and progression time to generalized myasthenia gravis (if this occurred) were recorded for each patient. Multiple logistic regression was used to measure the association between all clinical variables and antibody result. Kaplan-Meier survival analysis was performed to examine time to generalization. Among the 223 participants, AChR antibody testing results were positive in 158 participants (70.9%). In an adjusted model, increased age at diagnosis (odds ratio [OR], 1.03; 95% CI, 1.01-1.04; P = .007) and progression to generalized myasthenia gravis (OR, 2.92; 95% CI, 1.18-7.26; P = .02) were significantly associated with positive antibody test results. Women were less likely to have a positive antibody test result (OR, 0.36; 95% CI, 0.19-0.68; P = .002). Patients who developed symptoms of generalized myasthenia gravis had a significantly higher mean (SD) antibody level than those who did not develop symptoms of generalized myasthenia gravis (12.7 [16.5] nmol/L vs 4.2 [7.9] nmol/L; P = .002). We demonstrate a higher sensitivity of AChR antibody testing than previously reported in the largest cohort of patients with OMG available to date. Older age, male sex, and progression to generalized myasthenia gravis were significantly associated with a positive antibody test result. In addition, to our knowledge, this is the first report of an association between high AChR antibody levels and progression from OMG to generalized disease.

Design and Characterization of a Human Monoclonal Antibody that Modulates Mutant Connexin 26 Hemichannels Implicated in Deafness and Skin Disorders

PubMed Central

Xu, Liang; Carrer, Andrea; Zonta, Francesco; Qu, Zhihu; Ma, Peixiang; Li, Sheng; Ceriani, Federico; Buratto, Damiano; Crispino, Giulia; Zorzi, Veronica; Ziraldo, Gaia; Bruno, Francesca; Nardin, Chiara; Peres, Chiara; Mazzarda, Flavia; Salvatore, Anna M.; Raspa, Marcello; Scavizzi, Ferdinando; Chu, Youjun; Xie, Sichun; Yang, Xuemei; Liao, Jun; Liu, Xiao; Wang, Wei; Wang, Shanshan; Yang, Guang; Lerner, Richard A.; Mammano, Fabio

2017-01-01

Background: Mutations leading to changes in properties, regulation, or expression of connexin-made channels have been implicated in 28 distinct human hereditary diseases. Eight of these result from variants of connexin 26 (Cx26), a protein critically involved in cell-cell signaling in the inner ear and skin. Lack of non-toxic drugs with defined mechanisms of action poses a serious obstacle to therapeutic interventions for diseases caused by mutant connexins. In particular, molecules that specifically modulate connexin hemichannel function without affecting gap junction channels are considered of primary importance for the study of connexin hemichannel role in physiological as well as pathological conditions. Monoclonal antibodies developed in the last three decades have become the most important class of therapeutic biologicals. Recombinant methods permit rapid selection and improvement of monoclonal antibodies from libraries with large diversity. Methods: By screening a combinatorial library of human single-chain fragment variable (scFv) antibodies expressed in phage, we identified a candidate that binds an extracellular epitope of Cx26. We characterized antibody action using a variety of biochemical and biophysical assays in HeLa cells, organotypic cultures of mouse cochlea and human keratinocyte-derived cells. Results: We determined that the antibody is a remarkably efficient, non-toxic, and completely reversible inhibitor of hemichannels formed by connexin 26 and does not affect direct cell-cell communication via gap junction channels. Importantly, we also demonstrate that the antibody efficiently inhibits hyperative mutant Cx26 hemichannels implicated in autosomal dominant non-syndromic hearing impairment accompanied by keratitis and hystrix-like ichthyosis-deafness (KID/HID) syndrome. We solved the crystal structure of the antibody, identified residues that are critical for binding and used molecular dynamics to uncover its mechanism of action. Conclusions: Although further studies will be necessary to validate the effect of the antibody in vivo, the methodology described here can be extended to select antibodies against hemichannels composed by other connexin isoforms and, consequently, to target other pathologies associated with hyperactive hemichannels. Our study highlights the potential of this approach and identifies connexins as therapeutic targets addressable by screening phage display libraries expressing human randomized antibodies. PMID:29018324

Isolation and characterization of anti c-met single chain fragment variable (scFv) antibodies.

PubMed

Qamsari, Elmira Safaie; Sharifzadeh, Zahra; Bagheri, Salman; Riazi-Rad, Farhad; Younesi, Vahid; Abolhassani, Mohsen; Ghaderi, Sepideh Safaei; Baradaran, Behzad; Somi, Mohammad Hossein; Yousefi, Mehdi

2017-12-01

The receptor tyrosine kinase (RTK) Met is the cell surface receptor for hepatocyte growth factor (HGF) involved in invasive growth programs during embryogenesis and tumorgenesis. There is compelling evidence suggesting important roles for c-Met in colorectal cancer proliferation, migration, invasion, angiogenesis, and survival. Hence, a molecular inhibitor of an extracellular domain of c-Met receptor that blocks c-Met-cell surface interactions could be of great thera-peutic importance. In an attempt to develop molecular inhibitors of c-Met, single chain variable fragment (scFv) phage display libraries Tomlinson I + J against a specific synthetic oligopeptide from the extracellular domain of c-Met receptor were screened; selected scFv were then characterized using various immune techniques. Three c-Met specific scFv (ES1, ES2, and ES3) were selected following five rounds of panning procedures. The scFv showed specific binding to c-Met receptor, and significantly inhibited proliferation responses of a human colorectal carcinoma cell line (HCT-116). Moreover, anti- apoptotic effects of selected scFv antibodies on the HCT-116 cell line were also evaluated using Annexin V/PI assays. The results demonstrated rates of apoptotic cell death of 46.0, 25.5, and 37.8% among these cells were induced by use of ES1, ES2, and ES3, respectively. The results demonstrated ability to successfully isolate/char-acterize specific c-Met scFv that could ultimately have a great therapeutic potential in immuno-therapies against (colorectal) cancers.

Myeloma-Derived Light Chain Paired with a Diagnostic Monoclonal Antibody Hinders Immunoassay Performance.

PubMed

Tu, Bailin; Tieman, Bryan; Moore, Jeffrey; Pan, You; Muerhoff, A Scott

2017-06-01

Monoclonal antibodies are widely used as the capture and detection reagents in diagnostic immunoassays. In the past, myeloma fusion partners expressing endogenous heavy and/or light chains were often used to generate hybridoma cell lines. As a result, mixed populations of antibodies were produced that can cause inaccurate test results, poor antibody stability, and significant lot-to-lot variability. We describe one such scenario where the P3U1 (P3X63Ag8U.1) myeloma fusion partner was used in the generation of a hybridoma producing protein induced vitamin K absence/antagonist-II (PIVKA II) antibody. The hybridoma produces three subpopulations of immunoglobulin as determined by ion exchange (IEx) chromatography that exhibit varying degrees of immunoreactivity (0%, 50%, or 100%) to the target antigen as determined by Surface Plasmon Resonance. To produce an antibody with the highest possible sensitivity and specificity, the antigen-specific heavy and light chain variable domains (VH and VL) were cloned from the hybridoma and tethered to murine IgG1 and kappa scaffolds. The resulting recombinant antibody was expressed in Chinese hamster ovary cells and is compatible for use in a diagnostic immunoassay.

Enzyme-linked immunosorbent assay with monoclonal and single-chain variable fragment antibodies selective to coplanar polychlorinated biphenyls.

PubMed

Inui, Hideyuki; Takeuchi, Tetsuya; Uesugi, Akari; Doi, Fumito; Takai, Mikio; Nishi, Kosuke; Miyake, Shiro; Ohkawa, Hideo

2012-02-22

Coplanar polychlorinated biphenyls (Co-PCBs) consisting of non-ortho and mono-ortho-chlorinated PCBs are dioxin-like compounds and cause wide contamination in the environment. To monitor Co-PCB residues, it was attempted to establish an enzyme-linked immunosorbent assay (ELISA) with monoclonal and recombinant antibodies selective to Co-PCBs. When 3,3',5,5'-tetrachlorobiphenoxybutyric acid (PCBH)-keyhole limpet hemocyanin conjugate was immunized into mice, two monoclonal antibodies, Mab-0217 and Mab-4444, were obtained. 3,3',5,5'-Tetrachlorobiphenyl (PCB80) was determined with an IC(50) value of 2.6 and 0.46 ng mL(-1) in ELISA based on Mab-0217 and Mab-4444, respectively. Mab-4444 cross-reacted with Co-PCB congeners, except for PCB77 and PCB81. Mab-0217 reacted with PCB80 and cross-reacted with PCB111. A single-chain variable fragment (scFv) antibody derived from Mab-4444 was produced in recombinant Escherichia coli cells. The scFv antibody showed nearly the same sensitivity toward PCBH as the parent monoclonal antibody in ELISA. These results clearly suggested that Mab-4444 and its scFv antibodies were suitable for monitoring the representative congeners of Co-PCBs.

Heterodimeric bispecific single chain variable fragments (scFv) killer engagers (BiKEs) enhance NK-cell activity against CD133+ colorectal cancer cells

PubMed Central

JU, Schmohl; MK, Gleason; PR, Dougherty; JS, Miller; DA, Vallera

2015-01-01

Background Natural killer (NK) cells are potent cytotoxic lymphocytes that play a critical role in tumor immunosurveillance and control. Cancer stem cells (CSC) initiate and sustain tumor cell growth, mediate drug refractory cancer relapse and express the well-known surface marker CD133. Methods DNA fragments from two fully humanized single chain fragment variable (scFv) antibody recognizing CD16 on NK-cells and CD133 on CSC were genetically spliced forming a novel drug, 16 × 133 BiKE that simultaneously recognizes these antigen to facilitate an immunologic synapse. The anti-CD133 was created using a fusion protein prepared by fusing DNA fragments encoding the two extracellular domains of CD133. Immunization of mice with the resulting fusion protein generated an unique antibody that recognized the molecular framework and was species cross-reactive. Results In vitro 51chromium release cytotoxicity assays at both high and low effector:target ratios demonstrated the ability of the heterodimeric biological drug to greatly enhance NK-cell killing of human Caco-2 colorectal carcinoma cells known to overexpress CD133. The tumor associated antigen specificity of the drug for CD133 even enhanced NK-cell cytotoxicity against the NK-resistant human Burkitt's lymphoma Daudi cell line, which has less than 5% CD133 surface expression. Flow cytometry analysis revealed increases in NK-cell degranulation and Interferon-γ production upon co-culture with Caco-2 targets in the presence of the drug. Conclusion These studies demonstrate that the innate immune system can be effectively recruited to kill CSC using bispecific antibodies targeting CD133, and that this anti-CD133 scFv may be useful in this bispecific platform or, perhaps, in the design of more complex trispecific molecules for carcinoma therapy. PMID:26566946

Mice with megabase humanization of their immunoglobulin genes generate antibodies as efficiently as normal mice.

PubMed

Murphy, Andrew J; Macdonald, Lynn E; Stevens, Sean; Karow, Margaret; Dore, Anthony T; Pobursky, Kevin; Huang, Tammy T; Poueymirou, William T; Esau, Lakeisha; Meola, Melissa; Mikulka, Warren; Krueger, Pamela; Fairhurst, Jeanette; Valenzuela, David M; Papadopoulos, Nicholas; Yancopoulos, George D

2014-04-08

Mice genetically engineered to be humanized for their Ig genes allow for human antibody responses within a mouse background (HumAb mice), providing a valuable platform for the generation of fully human therapeutic antibodies. Unfortunately, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which their genetic humanization was carried out. Heretofore, HumAb mice have been generated by disrupting the endogenous mouse Ig genes and simultaneously introducing human Ig transgenes at a different and random location; KO-plus-transgenic humanization. As we describe in the companion paper, we attempted to make mice that more efficiently use human variable region segments in their humoral responses by precisely replacing 6 Mb of mouse Ig heavy and kappa light variable region germ-line gene segments with their human counterparts while leaving the mouse constant regions intact, using a unique in situ humanization approach. We reasoned the introduced human variable region gene segments would function indistinguishably in their new genetic location, whereas the retained mouse constant regions would allow for optimal interactions and selection of the resulting antibodies within the mouse environment. We show that these mice, termed VelocImmune mice because they were generated using VelociGene technology, efficiently produce human:mouse hybrid antibodies (that are rapidly convertible to fully human antibodies) and have fully functional humoral immune systems indistinguishable from those of WT mice. The efficiency of the VelocImmune approach is confirmed by the rapid progression of 10 different fully human antibodies into human clinical trials.

Sensitivity of HER-2/neu antibodies in archival tissue samples: potential source of error in immunohistochemical studies of oncogene expression.

PubMed

Press, M F; Hung, G; Godolphin, W; Slamon, D J

1994-05-15

HER-2/neu oncogene amplification and overexpression of breast cancer tissue has been correlated with poor prognosis in women with both node-positive and node-negative disease. However, several studies have not confirmed this association. Review of these studies reveals the presence of considerable methodological variability including differences in study size, follow-up time, techniques and reagents. The majority of papers with clinical follow-up information are immunohistochemical studies using archival, paraffin-embedded breast cancers, and a variety of HER-2/neu antibodies have been used in these studies. Very little information, however, is available about the ability of the antibodies to detect overexpression following tissue processing for paraffin-embedding. Therefore, a series of antibodies, reported in the literature or commercially available, were evaluated to assess their sensitivity and specificity as immunohistochemical reagents. Paraffin-embedded samples of 187 breast cancers, previously characterized as frozen specimens for HER-2/neu amplification by Southern blot and for overexpression by Northern blot, Western blot, and immunohistochemistry, were used. Two multitumor paraffin-embedded tissue blocks were prepared from the previously analyzed breast cancers as a panel of cases to test a series of previously studied and/or commercially available anti-HER-2/neu antibodies. Immunohistochemical staining results obtained with 7 polyclonal and 21 monoclonal antibodies in sections from paraffin-embedded blocks of these breast cancers were compared. The ability of these antibodies to detect overexpression was extremely variable, providing an important explantation for the variable overexpression rate reported in the literature.

Young Age at Diagnosis of Type 1 Diabetes Is Associated with the Development of Celiac Disease-Associated Antibodies in Children Living in Newfoundland and Labrador, Canada.

PubMed

Pall, Harpreet; Newhook, Leigh A; Aaron, Hillary; Curtis, Joseph; Randell, Ed

2015-10-14

The objectives of this study were to establish the prevalence of positive antibodies to endomysium (EMA) and tissue transglutaminase (tTG) in children with type 1 diabetes living in Newfoundland and Labrador (NL), and to examine clinical features associated with positive antibodies. Patients were recruited from the pediatric diabetes clinic. One hundred sixty-seven children with type 1 diabetes from the 280 children followed at the clinic were prospectively screened for celiac disease using EMA and tTG. The variables of Irish descent, age at onset of diabetes, duration of diabetes, sex, family history of celiac disease, hemoglobin A1C (A1C), ferritin, gastrointestinal symptoms, and body mass index were compiled for all patients. The group of patients with positive antibodies to EMA and/or tTG was compared to the group with negative antibodies. The prevalence of patients with positive antibodies to EMA and/or tTG was 16.8% (n = 28). One patient had also been previously diagnosed with symptomatic celiac disease. The two statistically significant variables with positive antibodies were an earlier age at onset of diabetes (Mann-Whitney U two-tailed test: mean difference 3.2 years, 95% CI 1.7-4.8 years, p < 0.0001) and longer duration of diabetes (Mann-Whitney U two-tailed test: mean difference 2.9 years, 95% CI 1.3-4.4 years, p < 0.0001). Irish descent was associated with positive antibodies but did not reach statistical significance. On logistic regression analysis performed with these three variables together, only age at onset of diabetes remained significant. There is a high prevalence of celiac disease-associated antibodies in children living in NL with type 1 diabetes. Unlike other clinical features, an earlier age at onset of diabetes was predictive for positive antibodies. As the majority of children with positive antibodies did not have signs or symptoms of celiac disease, routine screening for celiac disease in type 1 diabetes is recommended.

The catalytic activity of a recombinant single chain variable fragment nucleic acid-hydrolysing antibody varies with fusion tag and expression host.

PubMed

Lee, Joungmin; Kim, Minjae; Seo, Youngsil; Lee, Yeonjin; Park, Hyunjoon; Byun, Sung June; Kwon, Myung-Hee

2017-11-01

The antigen-binding properties of single chain Fv antibodies (scFvs) can vary depending on the position and type of fusion tag used, as well as the host cells used for expression. The issue is even more complicated with a catalytic scFv antibody that binds and hydrolyses a specific antigen. Herein, we investigated the antigen-binding and -hydrolysing activities of the catalytic anti-nucleic acid antibody 3D8 scFv expressed in Escherichia coli or HEK293f cells with or without additional amino acid residues at the N- and C-termini. DNA-binding activity was retained in all recombinant forms. However, the DNA-hydrolysing activity varied drastically between forms. The DNA-hydrolysing activity of E. coli-derived 3D8 scFvs was not affected by the presence of a C-terminal human influenza haemagglutinin (HA) or His tag. By contrast, the activity of HEK293f-derived 3D8 scFvs was completely lost when additional residues were included at the N-terminus and/or when a His tag was incorporated at the C-terminus, whereas a HA tag at the C-terminus did not diminish activity. Thus, we demonstrate that the antigen-binding and catalytic activities of a catalytic antibody can be separately affected by the presence of additional residues at the N- and C-termini, and by the host cell type. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Customizing monoclonal antibodies for the treatment of methamphetamine abuse: current and future applications.

PubMed

Peterson, Eric C; Gentry, W Brooks; Owens, S Michael

2014-01-01

Monoclonal antibody-based medications designed to bind (+)-methamphetamine (METH) with high affinity are among the newest approaches to the treatment of METH abuse and the associated medical complications. The potential clinical indications for these medications include treatment of overdose, reduction of drug dependence, and protection of vulnerable populations from METH-related complications. Research designed to discover and conduct preclinical and clinical testing of these antibodies suggests a scientific vision for how intact monoclonal antibody (mAb) (singular and plural) or small antigen-binding fragments of mAb could be engineered to optimize the proteins for specific therapeutic applications. In this review, we discuss keys to success in this development process including choosing predictors of specificity, efficacy, duration of action, and safety of the medications in disease models of acute and chronic drug abuse. We consider important aspects of METH-like hapten design and how hapten structural features influence specificity and affinity, with an example of a high-resolution X-ray crystal structure of a high-affinity antibody to demonstrate this structural relationship. Additionally, several prototype anti-METH mAb forms such as antigen-binding fragments and single-chain variable fragments are under development. Unique, customizable aspects of these fragments are presented with specific possible clinical indications. Finally, we discuss clinical trial progress of the first in kind anti-METH mAb, for which METH is the disease target instead of vulnerable central nervous system networks of receptors, binding sites, and neuronal connections. © 2014 Elsevier Inc. All rights reserved.

Production and characterization of single-chain antibody (scFv) against 3ABC non-structural protein in Escherichia coli for sero-diagnosis of Foot and Mouth Disease virus.

PubMed

Sharma, Gaurav K; Mahajan, Sonalika; Matura, Rakesh; Subramaniam, Saravanan; Mohapatra, Jajati K; Pattnaik, Bramhadev

2014-11-01

Differentiation of Foot-and-Mouth Disease infected from vaccinated animals is essential for effective implementation of vaccination based control programme. Detection of antibodies against 3ABC non-structural protein of FMD virus by immunodiagnostic assays provides reliable indication of FMD infection. Sero-monitoring of FMD in the large country like India is a big task where thousands of serum samples are annually screened. Currently, monoclonal or polyclonal antibodies are widely used in these immunodiagnostic assays. Considering the large population of livestock in the country, an economical and replenishable alternative of these antibodies was required. In this study, specific short chain variable fragment (scFv) antibody against 3B region of 3ABC poly-protein was developed. High level of scFv expression in Escherichia coli system was obtained by careful optimization in four different strains. Two formats of enzyme immunoassays (sandwich and competitive ELISAs) were optimized using scFv with objective to differentiate FMD infected among the vaccinated population. The assays were statistically validated by testing 2150 serum samples. Diagnostic sensitivity/specificity of sandwich and competitive ELISAs were determined by ROC method as 92.2%/95.5% and 89.5%/93.5%, respectively. This study demonstrated that scFv is a suitable alternate for immunodiagnosis of FMD on large scale. Copyright © 2014 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

In vivo analysis of replication and immunogenicity of proviral clones of human T-lymphotropic virus type 1 with selective envelope surface-unit mutations

PubMed Central

Silverman, Lee R.; Phipps, Andrew J.; Montgomery, Andy; Fernandez, Soledad; Tsukahara, Tomonori; Ratner, Lee; Lairmore, Michael D.

2005-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell lymphoma/leukemia (ATL). The HTLV-1 envelope gene exhibits limited variability when examined from infected individuals, but has not been tested using infectious clones of the virus in animal models. In vitro assays indicate that HTLV-1 envelope (Env) Ser75Ile, Asn95Asp, and Asn195Asp surface unit (SU) mutants are able to replicate in and immortalize lymphocytes. Herein, we examined the effects of these Env mutants in rabbits inoculated with HTLV-1 immortalized ACH.75, ACH.95, or ACH.195 cell lines (expressing full-length molecular clones with the SU mutations) or the ACH.1 cell line (expressing wild-type SU). All rabbits became infected, and the fidelity of the mutations was maintained throughout the 8-week study. However, SU point mutations resulted in decreased antibody responses to viral group-associated antigen (Gag) and Env antigens. ACH.195 rabbits had a selective decreased antibody response to SU, and one ACH.195 rabbit had an antibody response to both HTLV-1 and HTLV-2 SUs. Some mutant inoculation groups had altered proviral loads. However, peripheral-blood mononuclear cell (PBMC) proviral loads did not correlate with antibody responses. Our data are the first to demonstrate that mutations in critical determinants of HTLV-1 Env SU altered antibody responses and proviral loads, but do not prevent viral replication in vivo. PMID:16046523

Biochemical and Structural Characterization of Lysophosphatidic Acid Binding by a Humanized Monoclonal Antibody

DOE Office of Scientific and Technical Information (OSTI.GOV)

J Fleming; J Wojciak; M Campbell

Lysophosphatidic acid (LPA) is a common product of glycerophospholipid metabolism and an important mediator of signal transduction. Aberrantly high LPA concentrations accompany multiple disease states. One potential approach for treatment of these diseases, therefore, is the therapeutic application of antibodies that recognize and bind LPA as their antigen. We have determined the X-ray crystal structure of an anti-LPA antibody (LT3015) Fab fragment in its antigen-free form to 2.15 {angstrom} resolution and in complex with two LPA isotypes (14:0 and 18:2) to resolutions of 1.98 and 2.51 {angstrom}, respectively. The variable CDR (complementarity-determining region) loops at the antigen binding site adoptmore » nearly identical conformations in the free and antigen-bound crystal structures. The crystallographic models reveal that the LT3015 antibody employs both heavy- and light-chain CDR loops to create a network of eight hydrogen bonds with the glycerophosphate head group of its LPA antigen. The head group is almost completely excluded from contact with solvent, while the hydrocarbon tail is partially solvent-exposed. In general, mutation of amino acid residues at the antigen binding site disrupts LPA binding. However, the introduction of particular mutations chosen strategically on the basis of the structures can positively influence LPA binding affinity. Finally, these structures elucidate the exquisite specificity demonstrated by an anti-lipid antibody for binding a structurally simple and seemingly unconstrained target molecule.« less

Neutralisation and binding of VHS virus by monovalent antibody fragments.

PubMed

Cupit, P M; Lorenzen, N; Strachan, G; Kemp, G J; Secombes, C J; Cunningham, C

2001-12-04

We have previously reported the cloning and characterisation of the heavy and light chain variable domain genes encoding three monoclonal antibodies (Mabs) that bind viral haemorrhagic septicaemia virus (VHSV). Two of these antibodies, 3F1H10 and 3F1A2 both neutralised the virus though 3F1A2 appeared to recognise a broader range of virus isolates. The variable domains of these two antibodies differ by only four residues (Lorenzen et al., 2000a. Fish Shellfish Immunol. 10, 129-142). To further study the mechanism of neutralisation, Fab fragments as well as a series of recombinant bacterial single chain antibody (scAb) fragments were generated from the three anti-VHSV Mabs and their variable domain genes, respectively. Fabs and scAbs derived from the neutralising Mabs were both able to neutralise the VHSV type 1 isolate DK-F1. In addition, a series of scAb fragments were produced using the 3F1H10 variable heavy (VH) chain and variable light (Vkappa) chain domains but containing, either alone or in dual combination, each of the four different residues present in 3F1A2. The dissociation constants of Mabs 3F1H10 and 3F1A2 and their respective Fab and scAb fragments were measured by BIAcore analysis and found to correlate with the capacity of each molecule to neutralise DK-F1. These investigations, together with computer assisted molecular analysis of the theoretical influence of each mutation on antigen binding, led to the identification of a single mutation at position 35a in the VH domain as having the most marked impact on viral neutralisation.

A novel redox method for rapid production of functional bi-specific antibodies for use in early pilot studies.

PubMed

Carlring, Jennifer; De Leenheer, Evy; Heath, Andrew William

2011-01-01

We demonstrate here a rapid alternative method for the production of functional bi-specific antibodies using the mild reducing agent 2-mercaptoethanesulfonic acid sodium salt (MESNA). Following reduction of a mixture of two monoclonal antibodies with MESNA to break inter heavy chain bonds, this solution is dialysed under oxidising conditions and antibodies are allowed to reform. During this reaction a mixture of antibodies is formed, including parental antibodies and bi-specific antibody. Bi-specific antibodies are purified over two sequential affinity columns. Following purification, bi-specificity of antibodies is determined in enzyme-linked immunosorbent assays and by flow cytometry. Using this redox method we have been successful in producing hybrid and same-species bi-specific antibodies in a time frame of 6-10 working days, making this production method a time saving alternative to the time-consuming traditional heterohybridoma technology for the production of bi-specific antibodies for use in early pilot studies. The use of both rat and mouse IgG antibodies forming a rat/mouse bi-specific antibody as well as producing a pure mouse bi-specific antibody and a pure rat bi-specific antibody demonstrates the flexibility of this production method.

Efficient generation of monoclonal antibodies from single rhesus macaque antibody secreting cells.

PubMed

Meng, Weixu; Li, Leike; Xiong, Wei; Fan, Xuejun; Deng, Hui; Bett, Andrew J; Chen, Zhifeng; Tang, Aimin; Cox, Kara S; Joyce, Joseph G; Freed, Daniel C; Thoryk, Elizabeth; Fu, Tong-Ming; Casimiro, Danilo R; Zhang, Ningyan; A Vora, Kalpit; An, Zhiqiang

2015-01-01

Nonhuman primates (NHPs) are used as a preclinical model for vaccine development, and the antibody profiles to experimental vaccines in NHPs can provide critical information for both vaccine design and translation to clinical efficacy. However, an efficient protocol for generating monoclonal antibodies from single antibody secreting cells of NHPs is currently lacking. In this study we established a robust protocol for cloning immunoglobulin (IG) variable domain genes from single rhesus macaque (Macaca mulatta) antibody secreting cells. A sorting strategy was developed using a panel of molecular markers (CD3, CD19, CD20, surface IgG, intracellular IgG, CD27, Ki67 and CD38) to identify the kinetics of B cell response after vaccination. Specific primers for the rhesus macaque IG genes were designed and validated using cDNA isolated from macaque peripheral blood mononuclear cells. Cloning efficiency was averaged at 90% for variable heavy (VH) and light (VL) domains, and 78.5% of the clones (n = 335) were matched VH and VL pairs. Sequence analysis revealed that diverse IGHV subgroups (for VH) and IGKV and IGLV subgroups (for VL) were represented in the cloned antibodies. The protocol was tested in a study using an experimental dengue vaccine candidate. About 26.6% of the monoclonal antibodies cloned from the vaccinated rhesus macaques react with the dengue vaccine antigens. These results validate the protocol for cloning monoclonal antibodies in response to vaccination from single macaque antibody secreting cells, which have general applicability for determining monoclonal antibody profiles in response to other immunogens or vaccine studies of interest in NHPs.

Monoclonal Antibody Analysis of Keratin Expression in the Central Nervous System

NASA Astrophysics Data System (ADS)

Franko, Maryellen C.; Gibbs, Clarence J.; Rhoades, Dorothy A.; Carleton Gajdusek, D.

1987-05-01

A monoclonal antibody directed against a 65-kDa brain protein demonstrates an epitope found in keratin from human epidermis. By indirect immunofluorescence, the antibody decorates intracytoplasmic filaments in a subclass of astrocytes and Purkinje cells of adult hamster brain. Double-label immunofluorescence study using antibody to glial fibrillary acidic protein and this antibody reveals the 65-kDa protein to be closely associated with glial filaments in astrocytes of fetal mouse brain cultures. Immunoblot analysis of purified human epidermal keratin and hamster brain homogenate confirms the reactivity of this antibody to epidermal keratin polypeptides. All the major epidermal keratins were recognized by this antibody. It did not bind to the remaining major intermediate filament proteins. These findings suggest that monoclonal antibody 34C9 recognizes a cytoskeletal structure connected with intermediate filaments. In addition, the monoclonal antibody demonstrates that epidermal keratins share an epitope not only among themselves but also with a ``neural keratin.''

Characterization of antibodies directed against the immunoglobulin light kappa chain variable chain region (VK) of hepatitis C virus-related type-II mixed cryoglobulinemia and B-cell proliferations.

PubMed

de Re, Valli; Simula, Maria Paola; Pavan, Alessandro; Garziera, Marica; Marin, Dolores; Dolcetti, Riccardo; de Vita, Salvatore; Sansonno, Domenico; Geremia, Silvano; Toffoli, Giuseppe

2009-09-01

Autoimmune type-II cryoglobulinemia (II-MC) is sustained by hepatitis C virus (HCV) infection and B-cell (oligo)clones. This is the reason why the disease may be considered an "indolent B-cell lymphoma (NHL)." B clones show a restricted use of immunoglobulin variable genes (BCR), in particular in the use of the variable kappa (VK)3-20/15 light chain, and show a homology between their BCR functional regions and those of autoimmune rheumatoid factors. We underlined the BCR unique repertoire with frequent rheumatoid factor activity also observed in other autoimmune disorders associated with NHL. The immunoglobulin idiotype is a clonal B-cell marker and an ideal target for immunotherapy. Five monoclonal antibodies were produced in our laboratory toward the VK3-20 of a subject with HCV infection and a II-MC-associated NHL. Epitope determination was performed using the epitope excision approach. Monoclonal antibody reactivity was tested in vitro in ELISA, Western blot, and cytofluorimetry. Data confirmed that a panel of antibodies, reactive against shared idiotypes, can be produced from patients with HCV-associated B-cell lymphoproliferative diseases, thus obviating the need to produce an anti-idiotype antibody for each patient.

Human non-neutralizing HIV-1 envelope monoclonal antibodies limit the number of founder viruses during SHIV mucosal infection in rhesus macaques

DOE PAGES

Santra, Sampa; Tomaras, Georgia D.; Warrier, Ranjit; ...

2015-08-03

HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4⁺ T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant regionmore » of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses.« less

Human Non-neutralizing HIV-1 Envelope Monoclonal Antibodies Limit the Number of Founder Viruses during SHIV Mucosal Infection in Rhesus Macaques

PubMed Central

Liao, Hua-Xin; Pollara, Justin; Liu, Pinghuang; Alam, S. Munir; Zhang, Ruijun; Cocklin, Sarah L.; Shen, Xiaoying; Duffy, Ryan; Xia, Shi-Mao; Schutte, Robert J.; Pemble IV, Charles W.; Dennison, S. Moses; Li, Hui; Chao, Andrew; Vidnovic, Kora; Evans, Abbey; Klein, Katja; Kumar, Amit; Robinson, James; Landucci, Gary; Forthal, Donald N.; Montefiori, David C.; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Rerks-Ngarm, Supachai; Robb, Merlin L.; Michael, Nelson L.; Kim, Jerome H.; Soderberg, Kelly A.; Giorgi, Elena E.; Blair, Lily; Korber, Bette T.; Moog, Christiane; Shattock, Robin J.; Schmitz, Joern E.; Moody, M. A.; Gao, Feng; Ferrari, Guido; Shaw, George M.; Haynes, Barton F.

2015-01-01

HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4+ T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant region of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Thus, some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses. PMID:26237403

Human non-neutralizing HIV-1 envelope monoclonal antibodies limit the number of founder viruses during SHIV mucosal infection in rhesus macaques

DOE Office of Scientific and Technical Information (OSTI.GOV)

Santra, Sampa; Tomaras, Georgia D.; Warrier, Ranjit

HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4⁺ T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant regionmore » of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses.« less

Long-term patterns of immune investment by wild deer mice infected with Sin Nombre virus.

PubMed

Lehmer, Erin M; Jones, Jeremy D; Bego, Mariana G; Varner, Johanna M; Jeor, Stephen St; Clay, Christine A; Dearing, M Denise

2010-01-01

Immunocompetence of animals fluctuates seasonally, However, there is little consensus on the cause of these fluctuations. Some studies have suggested that these patterns are influenced by changes in reproductive condition, whereas others have suggested that differences result from seasonal variations in energy expenditures. The objective of our study was to examine these contrasting views of immunity by evaluating seasonal patterns of immune response and reproduction in wild populations of deer mice Peromyscus maniculatus exposed to Sin Nombre virus (SNV). Over three consecutive fall (September, October, November) and three consecutive spring (March, April, May) sampling periods, we used titration enzyme-linked immunosorbent assay (ELISA) to quantify virus-specific antibody production in 48 deer mice infected with SNV. Levels of reproductive hormones were quantified using ELISA. SNV antibody titers reached their lowest level during November (geometric mean titer [GMT] = 420) and their highest levels during September (GMT = 5,545) and May (GMT = 3,582), suggesting that the immune response of deer mice to SNV has seasonal patterns. The seeming decrease in antibody titer over winter coupled with the consistency in body masses suggests that during winter, immunocompetence may be compromised to offset the energetic costs of maintenance functions, including those associated with maintaining body mass. Deer mice showed distinct sex-based differences in SNV antibody production, with males producing higher antibody titers (GMT = 3,333) than females (GMT = 1,477). Levels of reproductive hormones do not appear to influence antibody production in either males or females, as there was no correlation between estradiol concentrations and SNV antibody titer in female deer mice (r² = 0.26), nor was there a significant relationship between levels of testosterone and SNV antibody titers in males (r² = 0.28). Collectively, this study demonstrates that immunocompetence of wild deer mice is seasonally variable; however, reproduction is not the primary stressor responsible for this variation. Rather, the data suggest that deer mice may compromise immunocompetence during winter to offset other maintenance costs during this period.

Identification of antigen-specific human monoclonal antibodies using high-throughput sequencing of the antibody repertoire.

PubMed

Liu, Ju; Li, Ruihua; Liu, Kun; Li, Liangliang; Zai, Xiaodong; Chi, Xiangyang; Fu, Ling; Xu, Junjie; Chen, Wei

2016-04-22

High-throughput sequencing of the antibody repertoire provides a large number of antibody variable region sequences that can be used to generate human monoclonal antibodies. However, current screening methods for identifying antigen-specific antibodies are inefficient. In the present study, we developed an antibody clone screening strategy based on clone dynamics and relative frequency, and used it to identify antigen-specific human monoclonal antibodies. Enzyme-linked immunosorbent assay showed that at least 52% of putative positive immunoglobulin heavy chains composed antigen-specific antibodies. Combining information on dynamics and relative frequency improved identification of positive clones and elimination of negative clones. and increase the credibility of putative positive clones. Therefore the screening strategy could simplify the subsequent experimental screening and may facilitate the generation of antigen-specific antibodies. Copyright © 2016 Elsevier Inc. All rights reserved.

Enrichment of high affinity subclasses and glycoforms from serum-derived IgG using FcγRs as affinity ligands.

PubMed

Boesch, Austin W; Kappel, James H; Mahan, Alison E; Chu, Thach H; Crowley, Andrew R; Osei-Owusu, Nana Y; Alter, Galit; Ackerman, Margaret E

2018-05-01

As antibodies continue to gain predominance in drug discovery and development pipelines, efforts to control and optimize their activity in vivo have matured to incorporate sophisticated abilities to manipulate engagement of specific Fc binding partners. Such efforts to promote diverse functional outcomes include modulating IgG-Fc affinity for FcγRs to alternatively potentiate or reduce effector functions, such as antibody-dependent cellular cytotoxicity and phagocytosis. While a number of natural and engineered Fc features capable of eliciting variable effector functions have been demonstrated in vitro and in vivo, elucidation of these important functional relationships has taken significant effort through use of diverse genetic, cellular and enzymatic techniques. As an orthogonal approach, we demonstrate use of FcγR as chromatographic affinity ligands to enrich and therefore simultaneously identify favored binding species from a complex mixture of serum-derived pooled polycloncal human IgG, a load material that contains the natural repertoire of Fc variants and post-translational modifications. The FcγR-enriched IgG was characterized for subclass and glycoform composition and the impact of this bioseparation step on antibody activity was measured in cell-based effector function assays including Natural Killer cell activation and monocyte phagocytosis. This work demonstrates a tractable means to rapidly distinguish complex functional relationships between two or more interacting biological agents by leveraging affinity chromatography followed by secondary analysis with high-resolution biophysical and functional assays and emphasizes a platform capable of surveying diverse natural post-translational modifications that may not be easily produced with high purity or easily accessible with recombinant expression techniques. © 2018 Wiley Periodicals, Inc.

Idiopathic Inflammatory Myopathies

PubMed Central

Dimachkie, Mazen M.; Barohn, Richard J.

2012-01-01

The idiopathic inflammatory myopathies are a group of rare disorders including polymyositis (PM), dermatomyositis (DM), and autoimmune necrotizing myopathies (NMs). The idiopathic inflammatory myopathies share many similarities. They present acutely, subacutely, or chronically with marked proximal and symmetric muscle weakness, except for associated distal and asymmetric weakness in inclusion body myositis. The idiopathic inflammatory myopathies also share a variable degree of creatine kinase (CK) elevation and a nonspecifically abnormal electromyogram demonstrating an irritative myopathy. The muscle pathology demonstrates inflammatory exudates of variable distribution within the muscle fascicle. Despite these similarities, the idiopathic inflammatory myopathies are a heterogeneous group. The overlap syndrome (OS) refers to the association of PM, DM, or NM with connective tissue disease, such as scleroderma or systemic lupus erythematosus. In addition to elevated antinuclear antibodies (ANA), patients with OS may be weaker in the proximal arms than the legs mimicking the pattern seen in some muscular dystrophies. In this review, we focus on DM, PM, and NM and examine current and promising therapies. PMID:23117947

RAPID CLONING OF HIGH AFFINITY HUMAN MONOCLONAL ANTIBODIES AGAINST INFLUENZA VIRUS

PubMed Central

Wrammert, Jens; Smith, Kenneth; Miller, Joe; Langley, Trey; Kokko, Kenneth; Larsen, Christian; Zheng, Nai-Ying; Mays, Israel; Garman, Lori; Helms, Christina; James, Judith; Air, Gillian M.; Capra, J. Donald; Ahmed, Rafi; Wilson, Patrick C.

2008-01-01

Pre-existing neutralizing antibody provides the first line of defense against pathogens in general. For influenza virus, annual vaccinations are given to maintain protective levels of antibody against the currently circulating strains. Here we report that after booster vaccination there was a rapid and robust influenza-specific IgG+ antibody-secreting plasma cell (ASC) response that peaked at approximately day 7 and accounted for up to 6% of peripheral blood B cells. These ASCs could be distinguished from influenza-specific IgG+ memory B cells that peaked 14 to 21 days after vaccination and averaged 1% of all B cells. Importantly, as much as 80% of ASCs purified at the peak of the response were influenza specific. This ASC response was characterized by a highly restricted B cell receptor (BCR) repertoire that in some donors were dominated by only a few B cell clones. This pauci-clonal response, however, showed extensive intraclonal diversification from accumulated somatic mutations. We used the immunoglobulin variable regions isolated from sorted single ASCs to produce over fifty human monoclonal antibodies (mAbs) that bound to the three influenza vaccine strains with high affinity. This strategy demonstrates that we can generate multiple high affinity mAbs from humans within a month after vaccination. The panel of influenza virus specific human mAbs allowed us to address the issue of original antigenic sin (OAS) - the phenomenon where the induced antibody shows higher affinity to a previously encountered influenza virus strain compared to the virus strain present in the vaccine1. However, we found that the vast majority of the influenza virus specific mAbs showed the highest affinity for the current vaccine strain. Thus, OAS does not seem to be a common occurrence in normal healthy adults receiving influenza vaccination. PMID:18449194

Rapid cloning of high-affinity human monoclonal antibodies against influenza virus.

PubMed

Wrammert, Jens; Smith, Kenneth; Miller, Joe; Langley, William A; Kokko, Kenneth; Larsen, Christian; Zheng, Nai-Ying; Mays, Israel; Garman, Lori; Helms, Christina; James, Judith; Air, Gillian M; Capra, J Donald; Ahmed, Rafi; Wilson, Patrick C

2008-05-29

Pre-existing neutralizing antibody provides the first line of defence against pathogens in general. For influenza virus, annual vaccinations are given to maintain protective levels of antibody against the currently circulating strains. Here we report that after booster vaccination there was a rapid and robust influenza-specific IgG+ antibody-secreting plasma cell (ASC) response that peaked at approximately day 7 and accounted for up to 6% of peripheral blood B cells. These ASCs could be distinguished from influenza-specific IgG+ memory B cells that peaked 14-21 days after vaccination and averaged 1% of all B cells. Importantly, as much as 80% of ASCs purified at the peak of the response were influenza specific. This ASC response was characterized by a highly restricted B-cell receptor (BCR) repertoire that in some donors was dominated by only a few B-cell clones. This pauci-clonal response, however, showed extensive intraclonal diversification from accumulated somatic mutations. We used the immunoglobulin variable regions isolated from sorted single ASCs to produce over 50 human monoclonal antibodies (mAbs) that bound to the three influenza vaccine strains with high affinity. This strategy demonstrates that we can generate multiple high-affinity mAbs from humans within a month after vaccination. The panel of influenza-virus-specific human mAbs allowed us to address the issue of original antigenic sin (OAS): the phenomenon where the induced antibody shows higher affinity to a previously encountered influenza virus strain compared with the virus strain present in the vaccine. However, we found that most of the influenza-virus-specific mAbs showed the highest affinity for the current vaccine strain. Thus, OAS does not seem to be a common occurrence in normal, healthy adults receiving influenza vaccination.

Synergistic capture of Clostridium botulinum Type A neurotoxin by scFv antibodies to novel epitopes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gray, Sean A.; Barr, John R.; Kalb, Suzanne R.

2011-10-01

A non-immune library of human single chain fragment variable (scFv) antibodies displayed on Saccharomyces cerevisiae was screened for binding to the Clostridium botulinum neurotoxin serotype A binding domain [BoNT/A (Hc)] with the goal of identifying scFv to novel epitopes. To do this, an antibody-mediated labeling strategy was used in which antigen-binding yeast clones were selected after labeling with previously characterized monoclonal antibodies (MAbs) specific to the Hc. Twenty unique scFv clones were isolated that bound Hc. Of these, three also bound to full-length BoNT/A toxin complex with affinities ranging from 5 nM to 170 nM. Epitope binning showed that themore » three unique clones recognized at least two epitopes that were distinct from one another and from the detection MAbs. After production in E. coli, the scFv were coupled to magnetic particles and tested for their ability to capture BoNT/A holotoxin using an Endopep-MS assay. In this assay, toxin captured by scFv coated magnetic particles was detected by incubation of the complex with a peptide containing a BoNT/A-specific cleavage sequence. Mass spectrometry was used to detect the ratio of intact peptide to cleavage products as evidence for toxin capture. When tested individually, each of the scFv showed a weak positive Endopep-MS result. However, when the particles were coated with all three scFv simultaneously, they exhibited significantly higher Endopep-MS activity, consistent with synergistic binding. These results demonstrate novel approaches toward the isolation and characterization of scFv antibodies specific to unlabeled antigen. They also provide evidence that distinct scFv antibodies can work synergistically to increase the efficiency of antigen capture onto a solid support.« less

Construction and characterization of a highly reactive chicken-derived single-chain variable fragment (scFv) antibody against Staphylococcus aureus developed with the T7 phage display system.

PubMed

Li, Jingquan; Xu, Yongping; Wang, Xitao; Li, Yuan; Wang, Lili; Li, Xiaoyu

2016-06-01

The purpose of this study was to construct a single-chain variable fragment (scFv) antibody from chicken egg yolk immunoglobulin (IgY) by means of genetic engineering and subsequent panning for a specific antibody against Staphylococcus aureus. We amplified the scFv using blood and spleen obtained from 100-day-old Roman chickens immunized with inactivated S. aureus and subsequently constructed a T7 phage display antibody library using phage display technology. Four non-repeated blood scFv and 6 spleen scFv were obtained following 3 rounds of panning of the T7 phage display antibody library, enzyme-linked immunosorbent assay and sequencing. These 10 scFv were cloned into the prokaryotic expression vector pCold I with expression induced at a low temperature. Four soluble proteins were obtained. Among them, soluble protein SFV6 derived from the spleen showed good reactivity against S. aureus using indirect ELISA and produced a particularly strong antibacterial effect in vitro. We were successful in isolating a highly specific scFv antibody against S. aureus from the spleen phage display library. This study provides a simple and rapid method for the quick preparation of a large number of antibodies against S. aureus and provides the foundation for the positioning of antibodies in the organism and the study of the antibacterial mechanism through which the antibody functions. Copyright © 2016 Elsevier B.V. All rights reserved.

Cloning, bacterial expression and crystallization of Fv antibody fragments

NASA Astrophysics Data System (ADS)

E´, Jean-Luc; Boulot, Ginette; Chitarra, V´ronique; Riottot, Marie-Madeleine; Souchon, H´le`ne; Houdusse, Anne; Bentley, Graham A.; Narayana Bhat, T.; Spinelli, Silvia; Poljak, Roberto J.

1992-08-01

The variable Fv fragments of antibodies, cloned in recombinant plasmids, can be expressed in bacteria as functional proteins having immunochemical properties which are very similar or identical with those of the corresponding parts of the parent eukaryotic antibodies. They offer new possibilities for the study of antibody-antigen interactions since the crystals of Fv fragments and of their complexes with antigen reported here diffract X-rays to a higher resolution that those obtained with the cognate Fab fragments. The Fv approach should facilitate the structural study of the combining site of antibodies and the further characterization of antigen-antibody interactions by site-directed mutagenesis experiments.

Expression of enhancing-activity-free neutralizing antibody against dengue type 1 virus in plasmid-inoculated mice.

PubMed

Yamanaka, Atsushi; Pitaksajjakul, Pannamthip; Ramasoota, Pongrama; Konishi, Eiji

2015-11-09

Most candidate dengue vaccines currently under development induce neutralizing antibodies, which are considered important for immunoprotection. However, the concomitant induction of infection-enhancing antibodies is an unavoidable concern. In contrast, a neutralizing antibody developed for passive immunotherapy has been engineered to eliminate its enhancing activity. Therefore, a strategy for the long-term expression of enhancing-activity-free neutralizing antibodies may resolve this concern. A mouse monoclonal antibody, 7F4, of the IgG3 subclass and with no detectable enhancing activity, was selected as the model neutralizing antibody to evaluate the potential of this strategy. Equal amounts of commercial vector (pFUSE)-based plasmids containing 7F4 heavy (H)- or light (L)-chain variable region genes were mixed and used for the cotransfection of 293T cells and co-delivery into ICR and BALB/c mice. The recombinant plasmids were designed to express IgG2b or IgG3 subclass antibodies (p7F4G2b or p7F4G3, respectively). 293T cells transfected with 2 μg of p7F4G2b or p7F4G3 produced approximately 15,000 or 800 ng/ml IgG in the culture fluids, respectively. The dose is expressed as the total amount of H- and L-chain plasmids. Neutralizing antibody was detected dose-dependently in ICR mice inoculated with 50-200 μg of p7F4G2b. A 1:2 dilution of sera from ICR and BALB/c mice inoculated with 100 μg of p7F4G3 showed average plaque reduction levels of >70% on day 3 and >90% on days 5-9. BALB/c mice maintained detectable neutralizing antibody for at least 3 months. The neutralizing antibody expressed by p7F4G3 in mice showed no enhancing activity. Although the expression of neutralizing antibodies from immunoglobulin genes is a type of passive immunization, its durability can be utilized as a dengue vaccine strategy. This "proof-of-concept" study using a mouse model demonstrates that the enhancing-activity-free characteristic of this strategy augurs well for dengue vaccine development, although further improvement is required. Copyright © 2015 Elsevier Ltd. All rights reserved.

Expression and assembly of a fully active antibody in algae

NASA Astrophysics Data System (ADS)

Mayfield, Stephen P.; Franklin, Scott E.; Lerner, Richard A.

2003-01-01

Although combinatorial antibody libraries have solved the problem of access to large immunological repertoires, efficient production of these complex molecules remains a problem. Here we demonstrate the efficient expression of a unique large single-chain (lsc) antibody in the chloroplast of the unicellular, green alga, Chlamydomonas reinhardtii. We achieved high levels of protein accumulation by synthesizing the lsc gene in chloroplast codon bias and by driving expression of the chimeric gene using either of two C. reinhardtii chloroplast promoters and 5' and 3' RNA elements. This lsc antibody, directed against glycoprotein D of the herpes simplex virus, is produced in a soluble form by the alga and assembles into higher order complexes in vivo. Aside from dimerization by disulfide bond formation, the antibody undergoes no detectable posttranslational modification. We further demonstrate that accumulation of the antibody can be modulated by the specific growth regime used to culture the alga, and by the choice of 5' and 3' elements used to drive expression of the antibody gene. These results demonstrate the utility of alga as an expression platform for recombinant proteins, and describe a new type of single chain antibody containing the entire heavy chain protein, including the Fc domain.

CD22 expression on blastic plasmacytoid dendritic cell neoplasms and reactivity of anti-CD22 antibodies to peripheral blood dendritic cells.

PubMed

Reineks, Edmunds Z; Osei, Ebenezer S; Rosenberg, Arlene; Auletta, Jeffrey; Meyerson, Howard J

2009-07-01

We identified CD22 expression on a blastic plasmacytoid dendritic cell (pDC) neoplasm presenting as a leukemia in a child. CD22 expression, as determined by the antibody s-HCL-1, was also noted on the neoplastic cells from three additional patients with blastic pDC tumors identified at our institution. Subsequently we determined that peripheral blood pDCs react with the s-HCL-1 antibody demonstrating that normal pDCs express CD22. Evaluation of five additional anti-CD22 antibodies indicated that staining of pDCs with these reagents was poor except for s-HCL-1. Therefore, the detection of CD22 on pDCs is best demonstrated with the use of this specific antibody clone. All anti-CD22 antibodies stained conventional DCs. We also evaluated the reactivity of the anti-CD22 antibodies with basophils and noted that the pattern of staining was similar to that seen with pDCs. The studies demonstrate that normal DCs and pDC neoplasms express CD22, and highlight clone specific differences in anti-CD22 antibody reactivity patterns on pDCs and basophils. (c) 2009 Clinical Cytometry Society.

Increased Antibody Affinity Confers Broad In Vitro Protection against Escape Mutants of Severe Acute Respiratory Syndrome Coronavirus

PubMed Central

Rani, Mridula; Bolles, Meagan; Donaldson, Eric F.; Van Blarcom, Thomas; Baric, Ralph; Iverson, Brent

2012-01-01

Even though the effect of antibody affinity on neutralization potency is well documented, surprisingly, its impact on neutralization breadth and escape has not been systematically determined. Here, random mutagenesis and DNA shuffling of the single-chain variable fragment of the neutralizing antibody 80R followed by bacterial display screening using anchored periplasmic expression (APEx) were used to generate a number of higher-affinity variants of the severe acute respiratory syndrome coronavirus (SARS-CoV)-neutralizing antibody 80R with equilibrium dissociation constants (KD) as low as 37 pM, a >270-fold improvement relative to that of the parental 80R single-chain variable fragment (scFv). As expected, antigen affinity was shown to correlate directly with neutralization potency toward the icUrbani strain of SARS-CoV. Additionally, the highest-affinity antibody fragment displayed 10-fold-increased broad neutralization in vitro and completely protected against several SARS-CoV strains containing substitutions associated with antibody escape. Importantly, higher affinity also led to the suppression of viral escape mutants in vitro. Escape from the highest-affinity variant required reduced selective pressure and multiple substitutions in the binding epitope. Collectively, these results support the hypothesis that engineered antibodies with picomolar dissociation constants for a neutralizing epitope can confer escape-resistant protection. PMID:22696652

Detection of Pituitary Antibodies by Immunofluorescence: Approach and Results in Patients With Pituitary Diseases

PubMed Central

Ricciuti, Adriana; De Remigis, Alessandra; Landek-Salgado, Melissa A.; De Vincentiis, Ludovica; Guaraldi, Federica; Lupi, Isabella; Iwama, Shintaro; Wand, Gary S.; Salvatori, Roberto

2014-01-01

Context: Pituitary antibodies have been measured mainly to identify patients whose disease is caused or sustained by pituitary-specific autoimmunity. Although reported in over 100 publications, they have yielded variable results and are thus considered of limited clinical utility. Objectives: Our objectives were to analyze all publications reporting pituitary antibodies by immunofluorescence for detecting the major sources of variability, to experimentally test these sources and devise an optimized immunofluorescence protocol, and to assess prevalence and significance of pituitary antibodies in patients with pituitary diseases. Study Design and Outcome Measures: We first evaluated the effect of pituitary gland species, section fixation, autofluorescence quenching, blockade of unwanted antibody binding, and use of purified IgG on the performance of this antibody assay. We then measured cross-sectionally the prevalence of pituitary antibodies in 390 pituitary cases and 60 healthy controls, expressing results as present or absent and according to the (granular, diffuse, perinuclear, or mixed) staining pattern. Results: Human pituitary was the best substrate to detect pituitary antibodies and yielded an optimal signal-to-noise ratio when treated with Sudan black B to reduce autofluorescence. Pituitary antibodies were more common in cases (95 of 390, 24%) than controls (3 of 60, 5%, P = .001) but did not discriminate among pituitary diseases when reported dichotomously. However, when expressed according to their cytosolic staining, a granular pattern was highly predictive of pituitary autoimmunity (P < .0001). Conclusion: We report a comprehensive study of pituitary antibodies by immunofluorescence and provide a method and an interpretation scheme that should be useful for identifying and monitoring patients with pituitary autoimmunity. PMID:24606106

Idiotypic Cascades in Cancer Patients Treated with Monoclonal Antibody CO17-1A

NASA Astrophysics Data System (ADS)

Wettendorff, Martine; Iliopoulos, Dimitrios; Tempero, Margaret; Kay, David; Defreitas, Elaine; Koprowski, Hilary; Herlyn, Dorothee

1989-05-01

We have previously shown that gastrointestinal cancer patients treated with monoclonal antibody CO17-1A (Ab1) developed anti-idiotypic antibodies (Ab2) to the Ab1. We now demonstrate that patients produce anti-anti-idiotypic antibodies (Ab3) to their autologous Ab2. Ab3 were demonstrated in culture supernatants of peripheral blood mononuclear cells from five Ab1-treated patients after stimulation of the cells with heterologous Ab2 that functionally mimicked the tumor antigen (Ag) defined by Ab1 and immunologically cross reacted with the patients' Ab2. Ab3 shared idiotopes with Ab1 and were Ab1-like in their binding specificities to tumor cells, Ag, and Ab2. Such antibodies were also elicited by stimulating cells with Ag. However, they were not produced by stimulating posttreatment mononuclear cells with control proteins or by stimulating pretreatment cells with either Ag or Ab2. Our results demonstrate idiotypic cascades in cancer patients treated with monoclonal antibody. Ag-specific Ab3 responses may underlie delayed clinical responses often observed in cancer patients treated with monoclonal antibodies of various specificities.

An anti-hapten camelid antibody reveals a cryptic binding site with significant energetic contributions from a nonhypervariable loop

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fanning, Sean W.; Horn, James R.

2014-03-05

Conventional anti-hapten antibodies typically bind low-molecular weight compounds (haptens) in the crevice between the variable heavy and light chains. Conversely, heavy chain-only camelid antibodies, which lack a light chain, must rely entirely on a single variable domain to recognize haptens. While several anti-hapten VHHs have been generated, little is known regarding the underlying structural and thermodynamic basis for hapten recognition. Here, an anti-methotrexate VHH (anti-MTX VHH) was generated using grafting methods whereby the three complementarity determining regions (CDRs) were inserted onto an existing VHH framework. Thermodynamic analysis of the anti-MTX VHH CDR1-3 Graft revealed a micromolar binding affinity, while themore » crystal structure of the complex revealed a somewhat surprising noncanonical binding site which involved MTX tunneling under the CDR1 loop. Due to the close proximity of MTX to CDR4, a nonhypervariable loop, the CDR4 loop sequence was subsequently introduced into the CDR1-3 graft, which resulted in a dramatic 1000-fold increase in the binding affinity. Crystal structure analysis of both the free and complex anti-MTX CDR1-4 graft revealed CDR4 plays a significant role in both intermolecular contacts and binding site conformation that appear to contribute toward high affinity binding. Additionally, the anti-MTX VHH possessed relatively high specificity for MTX over closely related compounds aminopterin and folate, demonstrating that VHH domains are capable of binding low-molecular weight ligands with high affinity and specificity, despite their reduced interface.« less

Computational Prediction of Neutralization Epitopes Targeted by Human Anti-V3 HIV Monoclonal Antibodies

PubMed Central

Shmelkov, Evgeny; Krachmarov, Chavdar; Grigoryan, Arsen V.; Pinter, Abraham; Statnikov, Alexander; Cardozo, Timothy

2014-01-01

The extreme diversity of HIV-1 strains presents a formidable challenge for HIV-1 vaccine design. Although antibodies (Abs) can neutralize HIV-1 and potentially protect against infection, antibodies that target the immunogenic viral surface protein gp120 have widely variable and poorly predictable cross-strain reactivity. Here, we developed a novel computational approach, the Method of Dynamic Epitopes, for identification of neutralization epitopes targeted by anti-HIV-1 monoclonal antibodies (mAbs). Our data demonstrate that this approach, based purely on calculated energetics and 3D structural information, accurately predicts the presence of neutralization epitopes targeted by V3-specific mAbs 2219 and 447-52D in any HIV-1 strain. The method was used to calculate the range of conservation of these specific epitopes across all circulating HIV-1 viruses. Accurately identifying an Ab-targeted neutralization epitope in a virus by computational means enables easy prediction of the breadth of reactivity of specific mAbs across the diversity of thousands of different circulating HIV-1 variants and facilitates rational design and selection of immunogens mimicking specific mAb-targeted epitopes in a multivalent HIV-1 vaccine. The defined epitopes can also be used for the purpose of epitope-specific analyses of breakthrough sequences recorded in vaccine clinical trials. Thus, our study is a prototype for a valuable tool for rational HIV-1 vaccine design. PMID:24587168

Biophysical characterization and structure of the Fab fragment from the NIST reference antibody, RM 8671.

PubMed

Karageorgos, Ioannis; Gallagher, Elyssia S; Galvin, Connor; Gallagher, D Travis; Hudgens, Jeffrey W

2017-11-01

Monoclonal antibody pharmaceuticals are the fastest-growing class of therapeutics, with a wide range of clinical applications. To assure their safety, these protein drugs must demonstrate highly consistent purity and stability. Key to these objectives is higher order structure measurements validated by calibration to reference materials. We describe preparation, characterization, and crystal structure of the Fab fragment prepared from the NIST Reference Antibody RM 8671 (NISTmAb). NISTmAb is a humanized IgG1κ antibody, produced in murine cell culture and purified by standard biopharmaceutical production methods, developed at the National Institute of Standards and Technology (NIST) to serve as a reference material. The Fab fragment was derived from NISTmAb through papain cleavage followed by protein A based purification. The purified Fab fragment was characterized by SDS-PAGE, capillary gel electrophoresis, multi-angle light scattering, size exclusion chromatography, mass spectrometry, and x-ray crystallography. The crystal structure at 0.2 nm resolution includes four independent Fab molecules with complete light chains and heavy chains through Cys 223, enabling assessment of conformational variability and providing a well-characterized reference structure for research and engineering applications. This nonproprietary, publically available reference material of known higher-order structure can support metrology in biopharmaceutical applications, and it is a suitable platform for validation of molecular modeling studies. Published by Elsevier Ltd.

Studies of thermostability in Camelus bactrianus (Bactrian camel) single-domain antibody specific for the mutant epidermal-growth-factor receptor expressed by Pichia.

PubMed

Omidfar, Kobra; Rasaee, Mohhamad Javad; Kashanian, Soheila; Paknejad, Malieheh; Bathaie, Zahra

2007-01-01

Camelids have a unique immune system capable of producing heavy-chain antibodies lacking the light chains and CH1 (constant heavy-chain domain 1). It has been shown that, in contrast with conventional antibody fragments, the variable domains of these heavy-chain antibodies are functional at or after exposure to high temperatures. In the present study, the VHH (variable domain of heavy-chain antibody) camel antibody was subcloned into vector Ppiczc and expressed in Pichia pastoris. ORB1-83 VHH antibody recognizes the external domain of the mutant EGFR [EGF (epidermal growth factor) receptor], EGFR VIII. This tumour-specific antigen is ligand-independent, contains a constitutively active tyrosine kinase domain and has been shown to be present in a number of human malignancies. We report here that, although expression from P. pastoris resulted in a significantly increased level of expression of the anti-EGFR VIII VHH antibodies compared with Escherichia coli [Omidfar, Rasaee, Modjtahedi, Forouzandeh, Taghikhani, Bakhtiari, Paknejad and Kashanian (2004) Tumor Biol. 25, 179-187; Omidfar, Rasaee, Modjtahedi, Forouzandeh, Taghikhani and Golmakany (2004) Tumor Biol. 25, 296-305], this antibody selectively bound to the EGFR VIII peptide and reacted specifically with the immunoaffinity-purified antigen from non-small-cell lung cancer. Furthermore, thermal denaturation stability and CD spectra analysis of the Camelus bactrianus (Bactrian camel) VHH and heavy-chain antibodies at different temperature proved reversibility and binding activity after heat denaturation. Our results indicate that the P. pastoris expression system may be useful for the expression of camel single domain antibody and the ability of the expressed protein to reversibly melt without aggregation, allowing it to regain binding activity after heat denaturation.

In vitro antibody-enzyme conjugates with specific bactericidal activity.

PubMed

Knowles, D M; Sulivan, T J; Parker, C W; Williams, R C

1973-06-01

IgG with antibacterial antibody opsonic activity was isolated from rabbit antisera produced by intravenous hyperimmunization with several test strains of pneumococci, Group A beta-hemolytic streptococci, Staphylococcus aureus, Proteus mirabilis, Pseudomonas aeruginosa, and Escherichia coli. Antibody-enzyme conjugates were prepared, using diethylmalonimidate to couple glucose oxidase to IgG antibacterial antibody preparations. Opsonic human IgG obtained from serum of patients with subacute bacterial endocarditis was also conjugated to glucose oxidase. Antibody-enzyme conjugates retained combining specificity for test bacteria as demonstrated by indirect immunofluorescence. In vitro test for bactericidal activity of antibody-enzyme conjugates utilized potassium iodide, lactoperoxidase, and glucose as cofactors. Under these conditions glucose oxidase conjugated to antibody generates hydrogen peroxide, and lactoperoxidase enzyme catalyzes the reduction of hydrogen peroxide with simultaneous oxidation of I(-) and halogenation and killing of test bacteria. Potent in vitro bactericidal activity of this system was repeatedly demonstrated for antibody-enzyme conjugates against pneumococci, streptococci, S. aureus, P. mirabilis, and E. coli. However, no bactericidal effect was demonstrable with antibody-enzyme conjugates and two test strains of P. aeruginosa. Bactericidal activity of antibody-enzyme conjugates appeared to parallel original opsonic potency of unconjugated IgG preparations. Antibody-enzyme conjugates at concentrations as low as 0.01 mg/ml were capable of intense bactericidal activity producing substantial drops in surviving bacterial counts within 30-60 min after initiation of assay. These in vitro bactericidal systems indicate that the concept of antibacterial antibody-enzyme conjugates may possibly be adaptable as a mechanism for treatment of patients with leukocyte dysfunction or fulminant bacteremia.

Polymorphisms in HLA-DPB1 are associated with differences in rubella virus-specific humoral immunity after vaccination.

PubMed

Lambert, Nathaniel D; Haralambieva, Iana H; Kennedy, Richard B; Ovsyannikova, Inna G; Pankratz, Vernon Shane; Poland, Gregory A

2015-03-15

Vaccination with live attenuated rubella virus induces a strong immune response in most individuals. However, small numbers of subjects never reach or maintain protective antibody levels, and there is a high degree of variability in immune response. We have previously described genetic polymorphisms in HLA and other candidate genes that are associated with interindividual differences in humoral immunity to rubella virus. To expand our previous work, we performed a genome-wide association study (GWAS) to discover single-nucleotide polymorphisms (SNPs) associated with rubella virus-specific neutralizing antibodies. We identified rs2064479 in the HLA-DPB1 genetic region as being significantly associated with humoral immune response variations after rubella vaccination (P = 8.62 × 10(-8)). All other significant SNPs in this GWAS were located near the HLA-DPB1 gene (P ≤ 1 × 10(-7)). These findings demonstrate that polymorphisms in HLA-DPB1 are strongly associated with interindividual differences in neutralizing antibody levels to rubella vaccination and represent a validation of our previous HLA work. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Engineering of a novel zipFv using leucine zipper motif against rabies virus glycoprotein G with improved protection potency in vivo.

PubMed

Xi, Hualong; Zhang, Kaixin; Yin, Yanchun; Gu, Tiejun; Sun, Qing; Li, Zhuang; Cheng, Yue; Jiang, Chunlai; Kong, Wei; Wu, Yongge

2017-06-01

Rabies is an acute zoonotic infectious disease with a high fatality rate but is preventable with vaccination and rabies immunoglobulin (RIG). The single-chain Fv fragment (scFv), a small engineered antigen-binding protein derived from antibody variable heavy (V H ) and light (V L ) chains connected by a peptide linker, can potentially be used to replace RIG. Here, we produced two peptides V H -JUN-HIS and V L -FOS-HA separately in Escherichia coli and assembled them to form zipFv successfully in vitro. The new zipFv utilizes FOS and JUN leucine zippers to form an antibody structure similar to the IgG counterpart with two free N-terminal ends of V H and V L . The zipFv protein showed notable improvement in binding ability and affinity over its corresponding scFv. The zipFv also demonstrated greater stability in serum and the same protective rate as RIG against challenge with a standard rabies virus (CVS-24) in mice. Our results indicated zipFv as a novel and efficient antibody form with enhanced neutralizing potency. Copyright © 2017. Published by Elsevier B.V.

Insertion of scFv into the hinge domain of full-length IgG1 monoclonal antibody results in tetravalent bispecific molecule with robust properties.

PubMed

Bezabeh, Binyam; Fleming, Ryan; Fazenbaker, Christine; Zhong, Haihong; Coffman, Karen; Yu, Xiang-Qing; Leow, Ching Ching; Gibson, Nerea; Wilson, Susan; Stover, C Kendall; Wu, Herren; Gao, Changshou; Dimasi, Nazzareno

By simultaneous binding two disease mediators, bispecific antibodies offer the opportunity to broaden the utility of antibody-based therapies. Herein, we describe the design and characterization of Bs4Ab, an innovative and generic bispecific tetravalent antibody platform. The Bs4Ab format comprises a full-length IgG1 monoclonal antibody with a scFv inserted into the hinge domain. The Bs4Ab design demonstrates robust manufacturability as evidenced by MEDI3902, which is currently in clinical development. To further demonstrate the applicability of the Bs4Ab technology, we describe the molecular engineering, biochemical, biophysical, and in vivo characterization of a bispecific tetravalent Bs4Ab that, by simultaneously binding vascular endothelial growth factor and angiopoietin-2, inhibits their function. We also demonstrate that the Bs4Ab platform allows Fc-engineering similar to that achieved with IgG1 antibodies, such as mutations to extend half-life or modulate effector functions.

Stereophysicochemical variability plots highlight conserved antigenic areas in Flaviviruses

PubMed Central

Schein, Catherine H; Zhou, Bin; Braun, Werner

2005-01-01

Background Flaviviruses, which include Dengue (DV) and West Nile (WN), mutate in response to immune system pressure. Identifying escape mutants, variant progeny that replicate in the presence of neutralizing antibodies, is a common way to identify functionally important residues of viral proteins. However, the mutations typically occur at variable positions on the viral surface that are not essential for viral replication. Methods are needed to determine the true targets of the neutralizing antibodies. Results Stereophysicochemical variability plots (SVPs), 3-D images of protein structures colored according to variability, as determined by our PCPMer program, were used to visualize residues conserved in their physical chemical properties (PCPs) near escape mutant positions. The analysis showed 1) that escape mutations in the flavivirus envelope protein are variable residues by our criteria and 2) two escape mutants found at the same position in many flaviviruses sit above clusters of conserved residues from different regions of the linear sequence. Conservation patterns in T-cell epitopes in the NS3- protease suggest a similar mechanism of immune system evasion. Conclusion The SVPs add another dimension to structurally defining the binding sites of neutralizing antibodies. They provide a useful aid for determining antigenically important regions and designing vaccines. PMID:15845145

The short-term culture of lymphoid tissue from immunized guinea-pigs

PubMed Central

Dresser, Ann M.

1965-01-01

Lymph node tissue from guinea-pigs immunized against one of several antigens was cultured in different media in order to determine conditions under which maximal amounts of antibody are formed in vitro. The amounts of antibody formed were variable and bore no relation to the level of serum antibody in the tissue donor. Heterologous sera of several origins, when included in the culture medium, varied very markedly in their capacity to support antibody production in vitro. PMID:5847427

Antiviral Activity of HIV gp120 Targeting Bispecific T Cell Engager (BiTE®) Antibody Constructs.

PubMed

Brozy, Johannes; Schlaepfer, Erika; Mueller, Christina K S; Rochat, Mary-Aude; Rampini, Silvana K; Myburgh, Renier; Raum, Tobias; Kufer, Peter; Baeuerle, Patrick A; Muenz, Markus; Speck, Roberto F

2018-05-02

Today's gold standard in HIV therapy is the combined antiretroviral therapy (cART). It requires strict adherence by patients and life-long medication, which can lower the viral load below detection limits and prevent HIV-associated immunodeficiency, but cannot cure patients. The bispecific T cell engaging (BiTE®) antibody technology has demonstrated long-term relapse-free outcomes in patients with relapsed and refractory acute lymphocytic leukemia. We here generated BiTE® antibody constructs that target the HIV-1 envelope protein gp120 (HIV gp120) using either the scFv B12 or VRC01, the first two extracellular domains (1+2) of human CD4 alone or joined to the single chain variable fragment (scFv) of the antibody 17b fused to an anti-human CD3ϵ scFv. These engineered human BiTE® antibody constructs showed engagement of T cells for redirected lysis of HIV gp120-transfected CHO cells. Furthermore, they substantially inhibited HIV-1 replication in PBMCs as well as in macrophages co-cultured with autologous CD8+ T-cells, the most potent being the human CD4(1+2) BiTE® antibody construct and the CD4(1+2)L17b BiTE® antibody construct. The CD4(1+2) h BiTE® antibody construct promoted HIV infection of human CD4-/CD8+ T cells. In contrast, the neutralizing B12 and the VRC01 BiTE® antibody constructs as well as the CD4(1+2)L17b BiTE® antibody construct did not. Thus, BiTE® antibody constructs targeting HIV gp120 are very promising for constraining HIV and warrant further development as novel antiviral therapy with curative potential. Importance HIV is a chronic infection well controlled with the current cART. However, we lack cure of HIV, and the HIV pandemic goes on. Here we showed in vitro and ex vivo t hat a bispecific T-cell engaging (BiTE®) antibody construct targeting HIV gp120 resulted in substantially reduced HIV replication. In addition, these BiTE® antibody constructs display efficient killing of gp120 expressing cells and inhibited replication in ex vivo HIV-infected PBMCs or macrophages. We believe that BiTE® antibody constructs recognizing HIV gp120 could be a very valuable strategy for a cure of HIV in combination with cART and compounds, which reverse latency. Copyright © 2018 American Society for Microbiology.

Generation of human Fab antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences.

PubMed

Andris-Widhopf, Jennifer; Steinberger, Peter; Fuller, Roberta; Rader, Christoph; Barbas, Carlos F

2011-09-01

The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human Fab (fragment antigen binding) antibody libraries. In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final Fab products that are used for cloning.

Development of β-lactoglobulin-specific chimeric human IgEκ monoclonal antibodies for in vitro safety assessment of whey hydrolysates.

PubMed

Knipping, Karen; Simons, Peter J; Buelens-Sleumer, Laura S; Cox, Linda; den Hartog, Marcel; de Jong, Niels; Teshima, Reiko; Garssen, Johan; Boon, Louis; Knippels, Léon M J

2014-01-01

Cow's milk-derived whey hydrolysates are nutritional substitutes for allergic infants. Safety or residual allergenicity assessment of these whey hydrolysates is crucial. Currently, rat basophilic leukemia RBL-2H3 cells expressing the human IgE receptor α-chain (huFcεRIα-RBL-2H3), sensitized with serum IgE from cow's milk allergic children, are being employed to assess in vitro residual allergenicity of these whey hydrolysates. However, limited availability and inter-lot variation of these allergic sera impede standardization of whey hydrolysate safety testing in degranulation assays. An oligoclonal pool of chimeric human (chu)IgE antibodies against bovine β-lactoglobulin (a major allergen in whey) was generated to increase sensitivity, specificity, and reproducibility of existing degranulation assays. Mice were immunized with bovine β-lactoglobulin, and subsequently the variable domains of dissimilar anti-β-lactoglobulin mouse IgG antibodies were cloned and sequenced. Six chimeric antibodies were generated comprising mouse variable domains and human constant IgE/κ domains. After sensitization with this pool of anti-β-lactoglobulin chuIgEs, huFcεRIα-expressing RBL-2H3 cells demonstrated degranulation upon cross-linking with whey, native 18 kDa β-lactoglobulin, and 5-10 kDa whey hydrolysates, whereas a 3 kDa whey hydrolysate and cow's milk powder (mainly casein) showed no degranulation. In parallel, allergic serum IgEs were less sensitive. In addition, our pool anti-β-lactoglobulin chuIgEs recognized multiple allergenic immunodominant regions on β-lactoglobulin, which were also recognized by serum IgEs from cow's milk allergic children. Usage of our 'unlimited' source and well-defined pool of β-lactoglobulin-specific recombinant chuIgEs to sensitize huFcεRIα on RBL-2H3 cells showed to be a relevant and sensitive alternative for serum IgEs from cow's milk allergic patients to assess safety of whey-based non-allergic hydrolyzed formula.

Determination of allergen specificity by heavy chains in grass pollen allergen-specific IgE antibodies.

PubMed

Gadermaier, Elisabeth; Flicker, Sabine; Lupinek, Christian; Steinberger, Peter; Valenta, Rudolf

2013-04-01

Affinity and clonality of allergen-specific IgE antibodies are important determinants for the magnitude of IgE-mediated allergic inflammation. We sought to analyze the contribution of heavy and light chains of human allergen-specific IgE antibodies for allergen specificity and to test whether promiscuous pairing of heavy and light chains with different allergen specificity allows binding and might affect affinity. Ten IgE Fabs specific for 3 non-cross-reactive major timothy grass pollen allergens (Phl p 1, Phl p 2, and Phl p 5) obtained by means of combinatorial cloning from patients with grass pollen allergy were used to construct stable recombinant single chain variable fragments (ScFvs) representing the original Fabs and shuffled ScFvs in which heavy chains were recombined with light chains from IgE Fabs with specificity for other allergens by using the pCANTAB 5 E expression system. Possible ancestor genes for the heavy chain and light chain variable region-encoding genes were determined by using sequence comparison with the ImMunoGeneTics database, and their chromosomal locations were determined. Recombinant ScFvs were tested for allergen specificity and epitope recognition by means of direct and sandwich ELISA, and affinity by using surface plasmon resonance experiments. The shuffling experiments demonstrate that promiscuous pairing of heavy and light chains is possible and maintains allergen specificity, which is mainly determined by the heavy chains. ScFvs consisting of different heavy and light chains exhibited different affinities and even epitope specificity for the corresponding allergen. Our results indicate that allergen specificity of allergen-specific IgE is mainly determined by the heavy chains. Different heavy and light chain pairings in allergen-specific IgE antibodies affect affinity and epitope specificity and thus might influence clinical reactivity to allergens. Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

How can we reduce costs of solid-phase multiplex-bead assays used to determine anti-HLA antibodies?

PubMed

Kamburova, E G; Wisse, B W; Joosten, I; Allebes, W A; van der Meer, A; Hilbrands, L B; Baas, M C; Spierings, E; Hack, C E; van Reekum, F E; van Zuilen, A D; Verhaar, M; Bots, M L; Drop, A C A D; Plaisier, L; Seelen, M A J; Sanders, J S F; Hepkema, B G; Lambeck, A J; Bungener, L B; Roozendaal, C; Tilanus, M G J; Vanderlocht, J; Voorter, C E; Wieten, L; van Duijnhoven, E M; Gelens, M; Christiaans, M H L; van Ittersum, F J; Nurmohamed, A; Lardy, N M; Swelsen, W; van der Pant, K A; van der Weerd, N C; Ten Berge, I J M; Bemelman, F J; Hoitsma, A; van der Boog, P J M; de Fijter, J W; Betjes, M G H; Heidt, S; Roelen, D L; Claas, F H; Otten, H G

2016-09-01

Solid-phase multiplex-bead assays are widely used in transplantation to detect anti-human leukocyte antigen (HLA) antibodies. These assays enable high resolution detection of low levels of HLA antibodies. However, multiplex-bead assays are costly and yield variable measurements that limit the comparison of results between laboratories. In the context of a Dutch national Consortium study we aimed to determine the inter-assay and inter-machine variability of multiplex-bead assays, and we assessed how to reduce the assay reagents costs. Fifteen sera containing a variety of HLA antibodies were used yielding in total 7092 median fluorescence intensities (MFI) values. The inter-assay and inter-machine mean absolute relative differences (MARD) of the screening assay were 12% and 13%, respectively. The single antigen bead (SAB) inter-assay MARD was comparable, but showed a higher lot-to-lot variability. Reduction of screening assay reagents to 50% or 40% of manufacturers' recommendations resulted in MFI values comparable to 100% of the reagents, with an MARD of 12% or 14%, respectively. The MARD of the 50% and 40% SAB assay reagent reductions were 11% and 22%, respectively. From this study, we conclude that the reagents can be reliably reduced at least to 50% of manufacturers' recommendations with virtually no differences in HLA antibody assignments. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The binding of an anti-PD-1 antibody to FcγRΙ has a profound impact on its biological functions.

PubMed

Zhang, Tong; Song, Xiaomin; Xu, Lanlan; Ma, Jie; Zhang, Yanjuan; Gong, Wenfeng; Zhang, Yilu; Zhou, Xiaosui; Wang, Zuobai; Wang, Yali; Shi, Yingdi; Bai, Huichen; Liu, Ning; Yang, Xiaolong; Cui, Xinxin; Cao, Yanping; Liu, Qi; Song, Jing; Li, Yucheng; Tang, Zhiyu; Guo, Mingming; Wang, Lai; Li, Kang

2018-04-23

Antibodies targeting PD-1 have been demonstrated durable anti-cancer activity in certain cancer types. However, the anti-PD-1 antibodies are less or not efficacious in many situations, which might be attributed to co-expression of multiple inhibitory receptors or presence of immunosuppressive cells in the tumor microenvironment. Most of the anti-PD-1 antibodies used in clinical studies are of IgG4 isotype with the S228P mutation (IgG4 S228P ). The functional impact by the interaction of anti-PD-1 IgG4 S228P antibody with Fc gamma receptors (FcγRs) is poorly understood. To assess the effects, we generated a pair of anti-PD-1 antibodies: BGB-A317/IgG4 S228P and BGB-A317/IgG4-variant (abbreviated as BGB-A317), with the same variable regions but two different IgG4 Fc-hinge sequences. There was no significant difference between these two antibodies in binding to PD-1. However, BGB-A317/IgG4 S228P binds to human FcγRI with high affinity and mediates crosslinking between PD-1 and FcγRI. In contrast, BGB-A317 does neither. Further cell-based assays showed that such crosslinking could reverse the function of an anti-PD-1 antibody from blocking to activating. More importantly, the crosslinking induces FcγRI + macrophages to phagocytose PD-1 + T cells. In a mouse model transplanted with allogeneic human cancer cells and PBMCs, BGB-A317 showed significant tumor growth inhibition, whereas BGB-A317/IgG4 S228P had no such inhibition. Immunohistochemistry study revealed an inverse correlation between FcγRI + murine macrophage infiltration and the density of CD8 + PD-1 + human T cells within tumors in the BGB-A317/IgG4 S228P -treated group. These evidences suggested that FcγRI + binding and crosslinking had negative impact on the anti-PD-1 antibody-mediated anti-cancer activity.

A preclinical study comparing approaches for augmenting the immunogenicity of a heptavalent KLH-conjugate vaccine against epithelial cancers.

PubMed

Ragupathi, Govind; Koide, Fusataka; Sathyan, Natarajan; Kagan, Ella; Spassova, Maria; Bornmann, William; Gregor, Polly; Reis, Celso A; Clausen, Henrik; Danishefsky, Samuel J; Livingston, Philip O

2003-10-01

Previously using a series of monovalent vaccines, we demonstrated that the optimal method for inducing an antibody response against cancer cell-surface antigens is covalent conjugation of the antigens to keyhole limpet hemocyanin (KLH) and the use of a saponin adjuvant. We have prepared a heptavalent-KLH conjugate vaccine containing the seven epithelial cancer antigens GM2, Globo H, Lewis(y), TF(c), Tn(c), STn(c), and glycosylated MUC1. In preparation for testing this vaccine in the clinic, we tested the impact on antibody induction of administering the individual conjugates plus adjuvant compared with a mixture of the seven conjugates plus adjuvant, and of several variables thought to augment immunogenicity. These include approaches for decreasing suppressor cell activity or increasing helper T-lymphocyte activity (low dose cyclophosphamide or anti-CTLA-4 MAb), different saponin adjuvants at various doses (QS-21 and GPI-0100), and different methods of formulation (lyophilization and use of polysorbate 80). We find that: (1). Immunization with the heptavalent-KLH conjugate plus GPI-0100 vaccine induces antibodies against the seven antigens of comparable titer to those induced by the individual-KLH conjugate vaccines, high titers of antibodies against Tn (median ELISA titer IgM/IgG 320/10240), STn (640/5120), TF (320/10240), MUC1 (80/20480), and globo H (640/40); while lower titers of antibodies against Lewis(y)()(160/0) and only occasional antibodies against GM2 are induced. (2). These antibodies reacted with the purified synthetic antigens by ELISA, and with naturally expressed antigens on the cancer cell surface by FACS. (3). None of the approaches for further altering the suppressor cell/helper T-cell balance nor changes to the standard formulation by lyophilization or use of polysorbate 80 had any impact on antibody titers. (4). An optimal dose of saponin adjuvant, QS-21 (50 microg) or GPI-0100 (1000 microg), is required for optimal antibody titers. This heptavalent vaccine is sufficiently optimized for testing in the clinic.

A polyclonal antibody against extracellular loops 1 of chNHE1 blocks avian leukosis virus subgroup J infection.

PubMed

Meng, Wei; Zhou, Defang; Li, Chengui; Wang, Guihua; Huang, Libo; Cheng, Ziqiang

2018-05-02

Avian leukosis virus subgroup J (ALV-J), an oncogenic retrovirus, induces myelocytomas and other various tumors, leading to great economical losses in poultry industry. It is a great challenge to develop effective preventive methods for ALV-J control due to its antigenic variations in the variable regions of envelope. In present study, we generated a mouse polyclonal antibody targeting the first extracellular loop (ECL1) of chicken Na + /H + exchanger isoform 1 (chNHE1), the receptor of ALV-J, to block ALV-J infection in vitro and in vivo. In ALV-J infected DF-1 cells, chNHE1 expression and the intracellular pH (pHi) were up-regulated with "wave" pattern, indicating that the disequilibrium of ALV-J infected cells associated with chNHE1. Next, we validated that ALV-J infection was significantly blocked with time dependent after treating with anti-ECL1 antibody and accordingly the pHi value were recovered, indicating the blockage of ALV-J infection did not affect Na + /H + exchange. Furthermore, in anti-ECL1 antibody treatment chickens that infected by ALV-J, weight gain and immune organs were recovered, and viral loads were significantly decreased, and the tissue injury and inflammation were reduced significantly from 21 to 35 days of age. The study demonstrated that anti-ECL1 antibody effectively blocks ALV-J infection without affecting Na + /H + exchange, and sheds light on a novel strategy for retroviruses control. Copyright © 2018 Elsevier Ltd. All rights reserved.

A strategy to identify linker-based modules for the allosteric regulation of antibody-antigen binding affinities of different scFvs

PubMed Central

Thie, Holger

2017-01-01

ABSTRACT Antibody single-chain variable fragments (scFvs) are used in a variety of applications, such as for research, diagnosis and therapy. Essential for these applications is the extraordinary specificity, selectivity and affinity of antibody paratopes, which can also be used for efficient protein purification. However, this use is hampered by the high affinity for the protein to be purified because harsh elution conditions, which may impair folding, integrity or viability of the eluted biomaterials, are typically required. In this study, we developed a strategy to obtain structural elements that provide allosteric modulation of the affinities of different antibody scFvs for their antigen. To identify suitable allosteric modules, a complete set of cyclic permutations of calmodulin variants was generated and tested for modulation of the affinity when substituting the linker between VH and VL. Modulation of affinity induced by addition of different calmodulin-binding peptides at physiologic conditions was demonstrated for 5 of 6 tested scFvs of different specificities and antigens ranging from cell surface proteins to haptens. In addition, a variety of different modulator peptides were tested. Different structural solutions were found in respect of the optimal calmodulin permutation, the optimal peptide and the allosteric effect for scFvs binding to different antigen structures. Significantly, effective linker modules were identified for scFvs with both VH-VL and VL-VH architecture. The results suggest that this approach may offer a rapid, paratope-independent strategy to provide allosteric regulation of affinity for many other antibody scFvs. PMID:28055297

Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain

PubMed Central

Liu, Rui; Liang, Xiao; Xiang, Dandan; Guo, Yirong; Liu, Yihua; Zhu, Guonian

2016-01-01

Triazophos is a widely used organophosphorous insecticide that has potentially adverse effects to organisms. In the present study, a high-affinity single-chain variable fragment (scFv) antibody with specific lambda light chain was developed for residue monitoring. First, the specific variable regions were correctly amplified from a hybridoma cell line 8C10 that secreted monoclonal antibody (mAb) against triazophos. The regions were then assembled as scFv via splicing by overlap extension polymerase chain reaction. Subsequently, the recombinant anti-triazophos scFv-8C10 was successfully expressed in Escherichia coli strain HB2151 in soluble form, purified through immobilized metal ion affinity chromatography, and verified via Western blot and peptide mass fingerprinting analyses. Afterward, an indirect competitive enzyme-linked immunosorbent assay was established based on the purified anti-triazophos scFv-8C10 antibody. The assay exhibited properties similar to those based on the parent mAb, with a high sensitivity (IC50 of 1.73 ng/mL) to triazophos and no cross reaction for other organophosphorus pesticides; it was reliable in detecting triazophos residues in spiked water samples. Moreover, kinetic measurement using a surface plasmon resonance biosensor indicated that the purified scFv-8C10 antibody had a high affinity of 1.8 × 10−10 M and exhibited good binding stability. Results indicated that the recombinant high-affinity scFv-8C10 antibody was an effective detection material that would be promising for monitoring triazophos residues in environment samples. PMID:27338340

Serosurvey of selected viruses in captive giant pandas (Ailuropoda melanoleuca) in China.

PubMed

Qin, Qin; Li, Desheng; Zhang, Hemin; Hou, Rong; Zhang, Zhihe; Zhang, Chenglin; Zhang, Jinguo; Wei, Fuwen

2010-05-19

Serum samples from 92 giant pandas in three captive facilities were tested for antibodies against five viruses of carnivores. Antibody titers against canine distemper virus (CDV) in two facilities in which giant pandas were vaccinated were variable. The canine adenovirus (CAV-1) and canine parvovirus (CPV) titers in vaccinated group were both positive, but titers were not high and varied among individual except one vaccinated panda had extremely high CAV-1 titer, indicating infection with the field virus following vaccination. Our results suggest that the vaccines used for these giant pandas do not elicit consistent antibody titers. Antibody titers against CDV, CPV and CAV-1 in unvaccinated giant pandas were highly variable, especially CPV titer. Almost half of sera were CPV antibody positive, and CPV titers were high enough to suggest infection with the virus. Canine coronavirus (CCV) and canine parainfluenza virus (CPIV) titers were not detected in all serum samples. The results of this study emphasize the need for research on infectious diseases of giant pandas and development of suitable vaccines for the species. Copyright 2009 Elsevier B.V. All rights reserved.

Heterologous Coproduction of Enterocin A and Pediocin PA-1 by Lactococcus lactis: Detection by Specific Peptide-Directed Antibodies

PubMed Central

Martínez, José M.; Kok, Jan; Sanders, Jan W.; Hernández, Pablo E.

2000-01-01

Antibodies against enterocin A were obtained by immunization of rabbits with synthetic peptides PH4 and PH5 designed, respectively, on the N- and C-terminal amino acid sequences of enterocin A and conjugated to the carrier protein KLH. Anti-PH4-KLH antibodies not only recognized enterocin A but also pediocin PA-1, enterocin P, and sakacin A, three bacteriocins which share the N-terminal class IIa consensus motif (YGNGVXC) that is contained in the sequence of the peptide PH4. In contrast, anti-PH5-KLH antibodies only reacted with enterocin A because the amino acid sequences of the C-terminal parts of class IIa bacteriocins are highly variable. Enterocin A and/or pediocin PA-1 structural and immunity genes were introduced in Lactococcus lactis IL1403 to achieve (co)production of the bacteriocins. The level of production of the two bacteriocins was significantly lower than that obtained by the wild-type producers, a fact that suggests a low efficiency of transport and/or maturation of these bacteriocins by the chromosomally encoded bacteriocin translocation machinery of IL1403. Despite the low production levels, both bacteriocins could be specifically detected and quantified with the anti-PH5-KLH (anti-enterocin A) antibodies isolated in this study and the anti-PH2-KLH (anti-pediocin PA-1) antibodies previously generated (J. M. Martínez, M. I. Martínez, A. M. Suárez, C. Herranz, P. Casaus, L. M. Cintas, J. M. Rodríguez, and P. E. Hernández, Appl. Environ. Microbiol. 64:4536–4545, 1998). In this work, the availability of antibodies for the specific detection and quantification of enterocin A and pediocin PA-1 was crucial to demonstrate coproduction of both bacteriocins by L. lactis IL1403(pJM04), because indicator strains that are selectively inhibited by each bacteriocin are not available. PMID:10919819

Antibody expressing pea seeds as fodder for prevention of gastrointestinal parasitic infections in chickens.

PubMed

Zimmermann, Jana; Saalbach, Isolde; Jahn, Doreen; Giersberg, Martin; Haehnel, Sigrun; Wedel, Julia; Macek, Jeanette; Zoufal, Karen; Glünder, Gerhard; Falkenburg, Dieter; Kipriyanov, Sergey M

2009-09-11

Coccidiosis caused by protozoans of genus Eimeria is a chicken parasitic disease of great economical importance. Conventional disease control strategies depend on vaccination and prophylactic use of anticoccidial drugs. Alternative solution to prevent and treat coccidiosis could be provided by passive immunization using orally delivered neutralizing antibodies. We investigated the possibility to mitigate the parasitic infection by feeding poultry with antibody expressing transgenic crop seeds. Using the phage display antibody library, we generated a panel of anti-Eimeria scFv antibody fragments with high sporozoite-neutralizing activity. These antibodies were expressed either transiently in agrobacteria-infiltrated tobacco leaves or stably in seeds of transgenic pea plants. Comparison of the scFv antibodies purified either from tobacco leaves or from the pea seeds demonstrated no difference in their antigen-binding activity and molecular form compositions. Force-feeding experiments demonstrated that oral delivery of flour prepared from the transgenic pea seeds had higher parasite neutralizing activity in vivo than the purified antibody fragments isolated from tobacco. The pea seed content was found to protect antibodies against degradation by gastrointestinal proteases (>100-fold gain in stability). Ad libitum feeding of chickens demonstrated that the transgenic seeds were well consumed and not shunned. Furthermore, feeding poultry with shred prepared from the antibody expressing pea seeds led to significant mitigation of infection caused both by high and low challenge doses of Eimeria oocysts. The results suggest that our strategy offers a general approach to control parasitic infections in production animals using cost-effective antibody expression in crop seeds affordable for the animal health market.

Antibody expressing pea seeds as fodder for prevention of gastrointestinal parasitic infections in chickens

PubMed Central

Zimmermann, Jana; Saalbach, Isolde; Jahn, Doreen; Giersberg, Martin; Haehnel, Sigrun; Wedel, Julia; Macek, Jeanette; Zoufal, Karen; Glünder, Gerhard; Falkenburg, Dieter; Kipriyanov, Sergey M

2009-01-01

Background Coccidiosis caused by protozoans of genus Eimeria is a chicken parasitic disease of great economical importance. Conventional disease control strategies depend on vaccination and prophylactic use of anticoccidial drugs. Alternative solution to prevent and treat coccidiosis could be provided by passive immunization using orally delivered neutralizing antibodies. We investigated the possibility to mitigate the parasitic infection by feeding poultry with antibody expressing transgenic crop seeds. Results Using the phage display antibody library, we generated a panel of anti-Eimeria scFv antibody fragments with high sporozoite-neutralizing activity. These antibodies were expressed either transiently in agrobacteria-infiltrated tobacco leaves or stably in seeds of transgenic pea plants. Comparison of the scFv antibodies purified either from tobacco leaves or from the pea seeds demonstrated no difference in their antigen-binding activity and molecular form compositions. Force-feeding experiments demonstrated that oral delivery of flour prepared from the transgenic pea seeds had higher parasite neutralizing activity in vivo than the purified antibody fragments isolated from tobacco. The pea seed content was found to protect antibodies against degradation by gastrointestinal proteases (>100-fold gain in stability). Ad libitum feeding of chickens demonstrated that the transgenic seeds were well consumed and not shunned. Furthermore, feeding poultry with shred prepared from the antibody expressing pea seeds led to significant mitigation of infection caused both by high and low challenge doses of Eimeria oocysts. Conclusion The results suggest that our strategy offers a general approach to control parasitic infections in production animals using cost-effective antibody expression in crop seeds affordable for the animal health market. PMID:19747368

Generation, Diversity Determination, and Application to Antibody Selection of a Human Naïve Fab Library

PubMed Central

Kim, Sangkyu; Park, Insoo; Park, Seung Gu; Cho, Seulki; Kim, Jin Hong; Ipper, Nagesh S.; Choi, Sun Shim; Lee, Eung Suk; Hong, Hyo Jeong

2017-01-01

We constructed a large naïve human Fab library (3 × 1010 colonies) from the lymphocytes of 809 human donors, assessed available diversities of the heavy-chain variable (VH) and κ light-chain variable (VK) domain repertoires, and validated the library by selecting Fabs against 10 therapeutically relevant antigens by phage display. We obtained a database of unique 7,373 VH and 41,804 VK sequences by 454 pyrosequencing, and analyzed the repertoires. The distribution of VH and VK subfamilies and germline genes in our antibody repertoires slightly differed from those in earlier published natural antibody libraries. The frequency of somatic hypermutations (SHMs) in heavy-chain complementarity determining region (HCDR)1 and HCDR2 are higher compared with the natural IgM repertoire. Analysis of position-specific SHMs in CDRs indicates that asparagine, threonine, arginine, aspartate and phenylalanine are the most frequent non-germline residues on the antibody-antigen interface and are converted mostly from the germline residues, which are highly represented in germline SHM hotspots. The amino acid composition and length-dependent changes in amino acid frequencies of HCDR3 are similar to those in previous reports, except that frequencies of aspartate and phenylalanine are a little higher in our repertoire. Taken together, the results show that this antibody library shares common features of natural antibody repertoires and also has unique features. The antibody library will be useful in the generation of human antibodies against diverse antigens, and the information about the diversity of natural antibody repertoires will be valuable in the future design of synthetic human antibody libraries with high functional diversity. PMID:28927259

Generation, Diversity Determination, and Application to Antibody Selection of a Human Naïve Fab Library.

PubMed

Kim, Sangkyu; Park, Insoo; Park, Seung Gu; Cho, Seulki; Kim, Jin Hong; Ipper, Nagesh S; Choi, Sun Shim; Lee, Eung Suk; Hong, Hyo Jeong

2017-09-30

We constructed a large naïve human Fab library (3 × 10 10 colonies) from the lymphocytes of 809 human donors, assessed available diversities of the heavy-chain variable (VH) and κ light-chain variable (VK) domain repertoires, and validated the library by selecting Fabs against 10 therapeutically relevant antigens by phage display. We obtained a database of unique 7,373 VH and 41,804 VK sequences by 454 pyrosequencing, and analyzed the repertoires. The distribution of VH and VK subfamilies and germline genes in our antibody repertoires slightly differed from those in earlier published natural antibody libraries. The frequency of somatic hypermutations (SHMs) in heavy-chain complementarity determining region (HCDR)1 and HCDR2 are higher compared with the natural IgM repertoire. Analysis of position-specific SHMs in CDRs indicates that asparagine, threonine, arginine, aspartate and phenylalanine are the most frequent non-germline residues on the antibody-antigen interface and are converted mostly from the germline residues, which are highly represented in germline SHM hotspots. The amino acid composition and length-dependent changes in amino acid frequencies of HCDR3 are similar to those in previous reports, except that frequencies of aspartate and phenylalanine are a little higher in our repertoire. Taken together, the results show that this antibody library shares common features of natural antibody repertoires and also has unique features. The antibody library will be useful in the generation of human antibodies against diverse antigens, and the information about the diversity of natural antibody repertoires will be valuable in the future design of synthetic human antibody libraries with high functional diversity.

Structural models of antibody variable fragments: A method for investigating binding mechanisms

NASA Astrophysics Data System (ADS)

Petit, Samuel; Brard, Frédéric; Coquerel, Gérard; Perez, Guy; Tron, François

1998-03-01

The value of comparative molecular modeling for elucidating structure-function relationships was demonstrated by analyzing six anti-nucleosome autoantibody variable fragments. Structural models were built using the automated procedure developed in the COMPOSER software, subsequently minimized with the AMBER force field, and validated according to several standard geometric and chemical criteria. Canonical class assignment from Chothia and Lesk's [Chottin and Lesk, J. Mol. Biol., 196 (1987) 901; Chothia et al., Nature, 342 (1989) 877] work was used as a supplementary validation tool for five of the six hypervariable loops. The analysis, based on the hypothesis that antigen binding could occur through electrostatic interactions, reveals a diversity of possible binding mechanisms of anti-nucleosome or anti-histone antibodies to their cognate antigen. These results lead us to postulate that anti-nucleosome autoantibodies could have different origins. Since both anti-DNA and anti-nculeosome autoantibodies are produced during the course of systemic lupus erythematosus, a non-organ specific autoimmune disease, a comparative structural and electrostatic analysis of the two populations of autoantibodies may constitute a way to elucidate their origin and the role of the antigen in tolerance breakdown. The present study illustrates some interests, advantages and limits of a methodology based on the use of comparative modeling and analysis of molecular surface properties.

Tumour homing and therapeutic effect of colloidal nanoparticles depend on the number of attached antibodies

PubMed Central

Colombo, Miriam; Fiandra, Luisa; Alessio, Giulia; Mazzucchelli, Serena; Nebuloni, Manuela; De Palma, Clara; Kantner, Karsten; Pelaz, Beatriz; Rotem, Rany; Corsi, Fabio; Parak, Wolfgang J.; Prosperi, Davide

2016-01-01

Active targeting of nanoparticles to tumours can be achieved by conjugation with specific antibodies. Specific active targeting of the HER2 receptor is demonstrated in vitro and in vivo with a subcutaneous MCF-7 breast cancer mouse model with trastuzumab-functionalized gold nanoparticles. The number of attached antibodies per nanoparticle was precisely controlled in a way that each nanoparticle was conjugated with either exactly one or exactly two antibodies. As expected, in vitro we found a moderate increase in targeting efficiency of nanoparticles with two instead of just one antibody attached per nanoparticle. However, the in vivo data demonstrate that best effect is obtained for nanoparticles with only exactly one antibody. There is indication that this is based on a size-related effect. These results highlight the importance of precisely controlling the ligand density on the nanoparticle surface for optimizing active targeting, and that less antibodies can exhibit more effect. PMID:27991503

An evaluation of an aptamer for use as an affinity reagent with MS: PCSK9 as an example protein.

PubMed

Gupta, Vinita; Lassman, Michael E; McAvoy, Thomas; Lee, Anita Yh; Chappell, Derek L; Laterza, Omar F

2016-08-01

For quantitative immunoaffinity IA-LC-MS, the utility of antibodies has been demonstrated many times but the utility of aptamers as affinity reagents is unproven. Immunoaffinity reagents including a monoclonal antibody and an aptamer were coupled to magnetic beads and used as part of an enrichment strategy for PCSK9 quantitation in plasma. With limited method development, we have established a comparison of an anti-PCSK9 aptamer with an anti-PCSK9 monoclonal antibody. The background that results from a tryptic digest of affinity enrichment in plasma was demonstrated for each reagent using high-resolution full scan MS. The assay recovery was demonstrated for multiple concentrations of aptamer in plasma with different concentrations of PCSK9 protein. The aptamer achieved comparable enrichment to the antibody, but with lower peptide background, thus demonstrating the potential use of aptamers for IA-LC-MS.

A comparative study of monoclonal antibodies. 1. Phase behavior and protein-protein interactions

PubMed Central

Lewus, Rachael A.; Levy, Nicholas E.; Lenhoff, Abraham M.; Sandler, Stanley I.

2018-01-01

Protein phase behavior is involved in numerous aspects of downstream processing, either by design as in crystallization or precipitation processes, or as an undesired effect, such as aggregation. This work explores the phase behavior of eight monoclonal antibodies (mAbs) that exhibit liquid-liquid separation, aggregation, gelation, and crystallization. The phase behavior has been studied systematically as a function of a number of factors, including solution composition and pH, in order to explore the degree of variability among different antibodies. Comparisons of the locations of phase boundaries show consistent trends as a function of solution composition; however, changing the solution pH has different effects on each of the antibodies studied. Furthermore, the types of dense phases formed varied among the antibodies. Protein-protein interactions, as reflected by values of the osmotic second virial coefficient, are used to correlate the phase behavior. The primary findings are that values of the osmotic second virial coefficient are useful for correlating phase boundary locations, though there is appreciable variability among the antibodies in the apparent strengths of the intrinsic protein-protein attraction manifested. However, the osmotic second virial coefficient does not provide a clear basis to predict the type of dense phase likely to result under a given set of solution conditions. PMID:25378269

Evaluation of Preexisting Anti-Hemagglutinin Stalk Antibody as a Correlate of Protection in a Healthy Volunteer Challenge with Influenza A/H1N1pdm Virus.

PubMed

Park, Jae-Keun; Han, Alison; Czajkowski, Lindsay; Reed, Susan; Athota, Rani; Bristol, Tyler; Rosas, Luz Angela; Cervantes-Medina, Adriana; Taubenberger, Jeffery K; Memoli, Matthew J

2018-01-23

Influenza virus hemagglutinin (HA) surface glycoprotein is currently the primary target of licensed influenza vaccines. Recently, broadly reactive antibodies that target the stalk region of the HA have become a major focus of current novel vaccine development. These antibodies have been observed in humans after natural infection with influenza A virus, but the data are limited. Using samples and data from the uniquely controlled setting of an influenza A/H1N1 virus human challenge study of healthy volunteers, we performed a secondary analysis that for the first time explores the role of anti-HA stalk antibody as a human correlate of protection. An anti-HA stalk antibody enzyme-linked immunosorbent assay (ELISA) was performed on samples from 65 participants challenged with a 2009 H1N1pdm virus. Pre- and postchallenge anti-HA stalk titers were then correlated with multiple outcome measures to evaluate anti-HA stalk antibody titer as a correlate of protection. Anti-HA stalk antibody titers were present before challenge and rose in response to challenge in 64% of individuals. Those individuals with higher titers at baseline were less likely to develop shedding, but not less likely to develop symptoms. Similar to the hemagglutination inhibition (HAI) titer, the baseline anti-HA stalk antibody titer did not independently predict a decrease in the severity of influenza disease, while the antineuraminidase (neuraminidase inhibition [NAI]) titer did. As a correlate of protection, the naturally occurring anti-HA stalk antibody titer is predictive of a reduction of certain aspects of disease similar to HAI titer, but the NAI titer is the only identified correlate that is an independent predictor of a reduction of all assessed influenza clinical outcome measures. IMPORTANCE This is the first study to evaluate preexisting anti-HA stalk antibodies as a predictor of protection. We use a healthy volunteer influenza challenge trial for an examination of the role such antibodies play in protection. This study demonstrates that anti-HA stalk antibodies are naturally generated in response to an infection, but there is significant variability in response. Similar to antibodies that target the HA head, baseline anti-HA stalk antibody titer is a correlate of protection in terms of reduced shedding, but it is not a predictor of reduced clinical disease or an independent predictor of disease severity. These results, in the context of the limited data available in humans, suggest that vaccines that induce anti-HA stalk antibodies could play a role in future vaccine strategies, but alone, this target may be insufficient to induce a fully protective vaccine and overcome some of the issues identified with current vaccines.

Avian Diagnostic and Therapeutic Antibodies to Viral Emerging Pathogens

DOE Office of Scientific and Technical Information (OSTI.GOV)

David Bradley

2011-03-31

During the current period the following key objectives were achieved: demonstration of high titer antibody production by geese following immunization with inactived H1N1 virus; completion of the epitope mapping of West Nile Virus-specific goose antibodies and initiation of epitope mapping of H1N1 flu-specific goose antibodies; advancement in scalable purification of goose antibodies.

Another Piece of the Antibody Puzzle: Observations from the HALT study\\.

PubMed

Snyder, Laurie D; Tinckam, Kathryn J

2018-06-04

In the rapidly evolving domain of clinical transplantation immunobiology, the interrogation and interpretation of HLA antibodies and their associated clinical consequences are in the spotlight. In lung transplant, HLA antibodies, in particular donor specific antibodies (DSA), are a determining component of the lung transplant antibody mediated rejection (AMR) definition (1). DSA after lung transplant are widely regarded as poor prognosticator, though sparse data to date necessitate ongoing discourse and continued investigation into incidence, timing and treatment. Prior studies reported a wide range of DSA incidence with differing consequences on a background of highly variable timing, methods, antibody analytic strategies and clinical definitions. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Macrophages are critical effectors of antibody therapies for cancer.

PubMed

Weiskopf, Kipp; Weissman, Irving L

2015-01-01

Macrophages are innate immune cells that derive from circulating monocytes, reside in all tissues, and participate in many states of pathology. Macrophages play a dichotomous role in cancer, where they promote tumor growth but also serve as critical immune effectors of therapeutic antibodies. Macrophages express all classes of Fcγ receptors, and they have immense potential to destroy tumors via the process of antibody-dependent phagocytosis. A number of studies have demonstrated that macrophage phagocytosis is a major mechanism of action of many antibodies approved to treat cancer. Consequently, a number of approaches to augment macrophage responses to therapeutic antibodies are under investigation, including the exploration of new targets and development of antibodies with enhanced functions. For example, the interaction of CD47 with signal-regulatory protein α (SIRPα) serves as a myeloid-specific immune checkpoint that limits the response of macrophages to antibody therapies, and CD47-blocking agents overcome this barrier to augment phagocytosis. The response of macrophages to antibody therapies can also be enhanced with engineered Fc variants, bispecific antibodies, or antibody-drug conjugates. Macrophages have demonstrated success as effectors of cancer immunotherapy, and further investigation will unlock their full potential for the benefit of patients.

Phase I study of obinutuzumab (GA101) in Japanese patients with relapsed or refractory B-cell non-Hodgkin lymphoma.

PubMed

Ogura, Michinori; Tobinai, Kensei; Hatake, Kiyohiko; Uchida, Toshiki; Suzuki, Tatsuya; Kobayashi, Yukio; Mori, Masakazu; Terui, Yasuhito; Yokoyama, Masahiro; Hotta, Tomomitsu

2013-01-01

As CD20 has become an established target for treating B-cell malignancies, there is interest in developing anti-CD20 antibodies with different functional activity from rituximab that might translate into improved efficacy. Obinutuzumab (GA101) is a glycoengineered, humanized type II anti-CD20 monoclonal antibody that has demonstrated superior activity to type I antibodies in preclinical studies and is currently being investigated in phase III trials. In this phase I dose-escalating study in Japanese patients with relapsed/refractory B-cell non-Hodgkin lymphoma, the primary endpoint was to characterize the safety of GA101; secondary endpoints were efficacy, pharmacokinetics and pharmacodynamics. Patients received up to nine doses of GA101 with up to 52 weeks' follow up. Most adverse events were grade 1 or 2 infusion-related reactions, and 10 grade 3/4 adverse events occurred. No dose-limiting toxicities were observed and the maximum tolerated dose was not identified. Out of 12 patients, 7 responded (end-of-treatment response rate 58%), with 2 complete responses and 5 partial responses. Responses were observed from low to high doses, and no dose-efficacy relationship was observed. B-cell depletion occurred in all patients after the first infusion and was maintained for the duration of treatment. Serum levels of GA101 increased in a dose-dependent fashion, although there was inter-patient variability. This phase I study demonstrated that GA101 has an acceptable safety profile and offers encouraging activity to Japanese patients with relapsed/refractory B-cell non-Hodgkin lymphoma. © 2012 Japanese Cancer Association.

Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system.

PubMed

Krebber, A; Bornhauser, S; Burmester, J; Honegger, A; Willuda, J; Bosshard, H R; Plückthun, A

1997-02-14

A prerequisite for the use of recombinant antibody technologies starting from hybridomas or immune repertoires is the reliable cloning of functional immunoglobulin genes. For this purpose, a standard phage display system was optimized for robustness, vector stability, tight control of scFv-delta geneIII expression, primer usage for PCR amplification of variable region genes, scFv assembly strategy and subsequent directional cloning using a single rare cutting restriction enzyme. This integrated cloning, screening and selection system allowed us to rapidly obtain antigen binding scFvs derived from spleen-cell repertoires of mice immunized with ampicillin as well as from all hybridoma cell lines tested to date. As representative examples, cloning of monoclonal antibodies against a his tag, leucine zippers, the tumor marker EGP-2 and the insecticide DDT is presented. Several hybridomas whose genes could not be cloned in previous experimental setups, but were successfully obtained with the present system, expressed high amounts of aberrant heavy and light chain mRNAs, which were amplified by PCR and greatly exceeded the amount of binding antibody sequences. These contaminating variable region genes were successfully eliminated by employing the optimized phage display system, thus avoiding time consuming sequencing of non-binding scFv genes. To maximize soluble expression of functional scFvs subsequent to cloning, a compatible vector series to simplify modification, detection, multimerization and rapid purification of recombinant antibody fragments was constructed.

Loss of Asparagine-Linked Glycosylation Sites in Variable Region 5 of Human Immunodeficiency Virus Type 1 Envelope Is Associated with Resistance to CD4 Antibody Ibalizumab ▿

PubMed Central

Toma, Jonathan; Weinheimer, Steven P.; Stawiski, Eric; Whitcomb, Jeannette M.; Lewis, Stanley T.; Petropoulos, Christos J.; Huang, Wei

2011-01-01

Ibalizumab (formerly TNX-355) is a first-in-class, monoclonal antibody inhibitor of CD4-mediated human immunodeficiency type 1 (HIV-1) entry. Multiple clinical trials with HIV-infected patients have demonstrated the antiviral activity, safety, and tolerability of ibalizumab treatment. A 9-week phase Ib study adding ibalizumab monotherapy to failing drug regimens led to transient reductions in HIV viral loads and the evolution of HIV-1 variants with reduced susceptibility to ibalizumab. This report characterizes these variants by comparing the phenotypic susceptibilities and envelope (env) sequences of (i) paired baseline and on-treatment virus populations, (ii) individual env clones from selected paired samples, and (iii) env clones containing site-directed mutations. Viruses with reduced susceptibility to ibalizumab were found to exhibit reduced susceptibility to the anti-CD4 antibody RPA-T4. Conversely, susceptibility to soluble CD4, which targets the HIV-1 gp120 envelope protein, was enhanced. No changes in susceptibility to the fusion inhibitor enfuvirtide or the CCR5 antagonist maraviroc were observed. Functionally, viruses with reduced ibalizumab susceptibility also displayed high levels of infectivity relative to those of paired baseline viruses. Individual env clones exhibiting reduced ibalizumab susceptibility contained multiple amino acid changes in different regions relative to the paired baseline clones. In particular, clones with reduced susceptibility to ibalizumab contained fewer potential asparagine-linked glycosylation sites (PNGSs) in variable region 5 (V5) than did paired ibalizumab-susceptible clones. The reduction in ibalizumab susceptibility due to the loss of V5 PNGSs was confirmed by site-directed mutagenesis. Taken together, these findings provide important insights into resistance to this new class of antiretroviral drug. PMID:21289125

High efficient expression of a functional humanized single-chain variable fragment (scFv) antibody against CD22 in Pichia pastoris.

PubMed

Zarei, Najmeh; Vaziri, Behrouz; Shokrgozar, Mohammad Ali; Mahdian, Reza; Fazel, Ramin; Khalaj, Vahid

2014-12-01

Single-chain variable fragments (scFvs) have recently emerged as attractive candidates in targeted immunotherapy of various malignancies. The anti-CD22 scFv is able to target CD22, on B cell surface and is being considered as a promising molecule in targeted immunotherapy of B cell malignancies. The recombinant anti-CD22 scFv has been successfully expressed in Escherichia coli; however, the insufficient production yield has been a major bottleneck for its therapeutic application. The methylotrophic yeast Pichia pastoris has become a highly popular expression host for the production of a wide variety of recombinant proteins such as antibody fragments. In this study, we used the Pichia expression system to express a humanized scFv antibody against CD22. The full-length humanized scFv gene was codon optimized, cloned into the pPICZαA and expressed in GS115 strain. The maximum production level of the scFv (25 mg/L) were achieved at methanol concentration, 1 %; pH 6.0; inoculum density, OD600 = 3 and the induction time of 72 h. The correlation between scFv gene dosage and expression level was also investigated by real-time PCR, and the results confirmed the presence of such correlation up to five gene copies. Immunofluorescence and flow cytometry studies and Biacore analysis demonstrated binding to CD22 on the surface of human lymphoid cell line Raji and recombinant soluble CD22, respectively. Taken together, the presented data suggest that the Pichia pastoris can be considered as an efficient host for the large-scale production of anti-CD22 scFv as a promising carrier for targeted drug delivery in treatment of CD22(+) B cell malignancies.

A single-chain fragment against prostate specific membrane antigen as a tool to build theranostic reagents for prostate cancer.

PubMed

Frigerio, B; Fracasso, G; Luison, E; Cingarlini, S; Mortarino, M; Coliva, A; Seregni, E; Bombardieri, E; Zuccolotto, G; Rosato, A; Colombatti, M; Canevari, S; Figini, M

2013-06-01

Prostate carcinoma is the most common non-cutaneous cancer in developed countries and represents the second leading cause of death. Early stage androgen dependent prostate carcinoma responds well to conventional therapies, but relatively few treatment options exist for patients with hormone-refractory prostate cancer. One of the most suitable targets for antibody-mediated approaches is prostate specific membrane antigen (PSMA) which is a well known tumour associated antigen. PSMA is a type II integral cell-surface membrane protein that is not secreted, and its expression density and enzymatic activity are increased progressively in prostate cancer compared to normal prostate epithelium, thereby making PSMA an ideal target for monoclonal antibody imaging and therapy. To obtain a small protein that can better penetrate tissue, we have engineered a single-chain variable fragment (scFv) starting from the variable heavy and light domains of the murine anti-PSMA monoclonal antibody D2B. scFvD2B was analysed in vitro for activity, stability, internalisation ability and in vivo for targeting specificity. Maintenance of function and immunoreactivity as well as extremely high radiolabelling efficiency and radiochemical purity were demonstrated by in vitro assays and under different experimental conditions. Despite its monovalent binding, scFvD2B retained a good strength of binding and was able to internalise around 40% of bound antigen. In vivo we showed its ability to specifically target only PSMA expressing prostate cancer xenografts. Due to these advantageous properties, scFvD2B has the potential to become a good theranostic reagent for early detection and therapy of prostate cancers. Published by Elsevier Ltd.

Cross-neutralizing human anti-poliovirus antibodies bind the recognition site for cellular receptor

PubMed Central

Chen, Zhaochun; Fischer, Elizabeth R.; Kouiavskaia, Diana; Hansen, Bryan T.; Ludtke, Steven J.; Bidzhieva, Bella; Makiya, Michelle; Agulto, Liane; Purcell, Robert H.; Chumakov, Konstantin

2013-01-01

Most structural information about poliovirus interaction with neutralizing antibodies was obtained in the 1980s in studies of mouse monoclonal antibodies. Recently we have isolated a number of human/chimpanzee anti-poliovirus antibodies and demonstrated that one of them, MAb A12, could neutralize polioviruses of both serotypes 1 and 2. This communication presents data on isolation of an additional cross-neutralizing antibody (F12) and identification of a previously unknown epitope on the surface of poliovirus virions. Epitope mapping was performed by sequencing of antibody-resistant mutants and by cryo-EM of complexes of virions with Fab fragments. The results have demonstrated that both cross-neutralizing antibodies bind the site located at the bottom of the canyon surrounding the fivefold axis of symmetry that was previously shown to interact with cellular poliovirus receptor CD155. However, the same antibody binds to serotypes 1 and 2 through different specific interactions. It was also shown to interact with type 3 poliovirus, albeit with about 10-fold lower affinity, insufficient for effective neutralization. Antibody interaction with the binding site of the cellular receptor may explain its broad reactivity and suggest that further screening or antibody engineering could lead to a universal antibody capable of neutralizing all three serotypes of poliovirus. PMID:24277851

A large population-based association study between HLA and KIR genotypes and measles vaccine antibody responses.

PubMed

Ovsyannikova, Inna G; Schaid, Daniel J; Larrabee, Beth R; Haralambieva, Iana H; Kennedy, Richard B; Poland, Gregory A

2017-01-01

Human antibody response to measles vaccine is highly variable in the population. Host genes contribute to inter-individual antibody response variation. The killer cell immunoglobulin-like receptors (KIR) are recognized to interact with HLA molecules and possibly influence humoral immune response to viral antigens. To expand on and improve our previous work with HLA genes, and to explore the genetic contribution of KIR genes to the inter-individual variability in measles vaccine-induced antibody responses, we performed a large population-based study in 2,506 healthy immunized subjects (ages 11 to 41 years) to identify HLA and KIR associations with measles vaccine-induced neutralizing antibodies. After correcting for the large number of statistical tests of allele effects on measles-specific neutralizing antibody titers, no statistically significant associations were found for either HLA or KIR loci. However, suggestive associations worthy of follow-up in other cohorts include B*57:01, DQB1*06:02, and DRB1*15:05 alleles. Specifically, the B*57:01 allele (1,040 mIU/mL; p = 0.0002) was suggestive of an association with lower measles antibody titer. In contrast, the DQB1*06:02 (1,349 mIU/mL; p = 0.0004) and DRB1*15:05 (2,547 mIU/mL; p = 0.0004) alleles were suggestive of an association with higher measles antibodies. Notably, the associations with KIR genotypes were strongly nonsignificant, suggesting that KIR loci in terms of copy number and haplotypes are not likely to play a major role in antibody response to measles vaccination. These findings refine our knowledge of the role of HLA and KIR alleles in measles vaccine-induced immunity.

Development of cell-penetrating bispecific antibodies targeting the N-terminal domain of androgen receptor for prostate cancer therapy.

PubMed

Goicochea, Nancy L; Garnovskaya, Maria; Blanton, Mary G; Chan, Grace; Weisbart, Richard; Lilly, Michael B

2017-12-01

Castration-resistant prostate cancer cells exhibit continued androgen receptor signaling in spite of low levels of ligand. Current therapies to block androgen receptor signaling act by inhibiting ligand production or binding. We developed bispecific antibodies capable of penetrating cells and binding androgen receptor outside of the ligand-binding domain. Half of the bispecific antibody molecule consists of a single-chain variable fragment of 3E10, an anti-DNA antibody that enters cells. The other half is a single-chain variable fragment version of AR441, an anti-AR antibody. The resulting 3E10-AR441 bispecific antibody enters human LNCaP prostate cells and accumulates in the nucleus. The antibody binds to wild-type, mutant and splice variant androgen receptor. Binding affinity of 3E10-AR441 to androgen receptor (284 nM) was lower than that of the parental AR441 mAb (4.6 nM), but could be improved (45 nM) through alternative placement of the affinity tags, and ordering of the VH and VK domains. The 3E10-AR441 bispecific antibody blocked genomic signaling by wild-type or splice variant androgen receptor in LNCaP cells. It also blocked non-genomic signaling by the wild-type receptor. Furthermore, bispecific antibody inhibited the growth of C4-2 prostate cancer cells under androgen-stimulated conditions. The 3E10-AR441 biAb can enter prostate cancer cells and inhibits androgen receptor function in a ligand-independent manner. It may be an attractive prototype agent for prostate cancer therapy. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Hypothyroidism after x irradiation to the neck: three case reports and a brief review of the literature

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adler, R.A.; Corrigan, D.F.; Wartofsky, L.

1976-05-01

Three patients who developed hypothyroidism after x irradiation to the neck are presented. The first two cases demonstrate that patients can develop clinical and chemical hypothyroidism after a very short interval following radiotherapy. Hypothyroidism developed in the first patient in the absence of surgical manipulation of the neck, or a large iodine load 4 months after receiving 6800 rad of x-ray therapy to his neck for carcinoma of the larynx. The second patient developed hypothyroidism approximately 6 months after his radiotherapy for carcinoma of the esophagus. Both of these patients demonstrated high titers of serum antithyroid antibodies. A third patientmore » with Hodgkin's disease did not manifest clinical symptoms and signs of hypothyroidism until 6 years after radiation therapy. These cases demonstrate the variability of onset of hypothyroidism after radiotherapy and emphasize the need for careful evaluation of thyroid function before and after neck irradiation.« less

Integration of lyoplate based flow cytometry and computational analysis for standardized immunological biomarker discovery.

PubMed

Villanova, Federica; Di Meglio, Paola; Inokuma, Margaret; Aghaeepour, Nima; Perucha, Esperanza; Mollon, Jennifer; Nomura, Laurel; Hernandez-Fuentes, Maria; Cope, Andrew; Prevost, A Toby; Heck, Susanne; Maino, Vernon; Lord, Graham; Brinkman, Ryan R; Nestle, Frank O

2013-01-01

Discovery of novel immune biomarkers for monitoring of disease prognosis and response to therapy in immune-mediated inflammatory diseases is an important unmet clinical need. Here, we establish a novel framework for immunological biomarker discovery, comparing a conventional (liquid) flow cytometry platform (CFP) and a unique lyoplate-based flow cytometry platform (LFP) in combination with advanced computational data analysis. We demonstrate that LFP had higher sensitivity compared to CFP, with increased detection of cytokines (IFN-γ and IL-10) and activation markers (Foxp3 and CD25). Fluorescent intensity of cells stained with lyophilized antibodies was increased compared to cells stained with liquid antibodies. LFP, using a plate loader, allowed medium-throughput processing of samples with comparable intra- and inter-assay variability between platforms. Automated computational analysis identified novel immunophenotypes that were not detected with manual analysis. Our results establish a new flow cytometry platform for standardized and rapid immunological biomarker discovery with wide application to immune-mediated diseases.

Integration of Lyoplate Based Flow Cytometry and Computational Analysis for Standardized Immunological Biomarker Discovery

PubMed Central

Villanova, Federica; Di Meglio, Paola; Inokuma, Margaret; Aghaeepour, Nima; Perucha, Esperanza; Mollon, Jennifer; Nomura, Laurel; Hernandez-Fuentes, Maria; Cope, Andrew; Prevost, A. Toby; Heck, Susanne; Maino, Vernon; Lord, Graham; Brinkman, Ryan R.; Nestle, Frank O.

2013-01-01

Discovery of novel immune biomarkers for monitoring of disease prognosis and response to therapy in immune-mediated inflammatory diseases is an important unmet clinical need. Here, we establish a novel framework for immunological biomarker discovery, comparing a conventional (liquid) flow cytometry platform (CFP) and a unique lyoplate-based flow cytometry platform (LFP) in combination with advanced computational data analysis. We demonstrate that LFP had higher sensitivity compared to CFP, with increased detection of cytokines (IFN-γ and IL-10) and activation markers (Foxp3 and CD25). Fluorescent intensity of cells stained with lyophilized antibodies was increased compared to cells stained with liquid antibodies. LFP, using a plate loader, allowed medium-throughput processing of samples with comparable intra- and inter-assay variability between platforms. Automated computational analysis identified novel immunophenotypes that were not detected with manual analysis. Our results establish a new flow cytometry platform for standardized and rapid immunological biomarker discovery with wide application to immune-mediated diseases. PMID:23843942

Use of modified live feline panleukopenia virus vaccine to immunize dogs against canine parvovirus.

PubMed

Pollock, R V; Carmichael, L E

1983-02-01

Modified live feline panleukopenia virus (FPLV) vaccine protected dogs against canine parvovirus (CPV) infection. However, unlike the long-lived (greater than or equal to 20-month) immunity engendered by CPV infection, the response of dogs to living FPLV was variable. Doses of FPLV (snow leopard strain) in excess of 10(5.7) TCID50 were necessary for uniform immunization; smaller inocula resulted in decreased success rates. The duration of immunity, as measured by the persistence of hemagglutination-inhibiting antibody, was related to the magnitude of the initial response to vaccination; dogs with vigorous initial responses resisted oronasal CPV challenge exposure 6 months after vaccination, and hemagglutination-inhibiting antibodies persisted in such dogs for greater than 1 year. Limited replication of FPLV in dogs was demonstrated, but unlike CPV, the feline virus did not spread to contact dogs or cats. Adverse reactions were not associated with living FPLV vaccination, and FPLV did not interfere with simultaneous response to attenuated canine distemper virus.

A structural basis for antigen recognition by the T cell-like lymphocytes of sea lamprey

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng, Lu; Velikovsky, C. Alejandro; Xu, Gang

Adaptive immunity in jawless vertebrates is mediated by leucine-rich repeat proteins called 'variable lymphocyte receptors' (VLRs). Two types of VLR (A and B) are expressed by mutually exclusive lymphocyte populations in lamprey. VLRB lymphocytes resemble the B cells of jawed vertebrates; VLRA lymphocytes are similar to T cells. We determined the structure of a high-affinity VLRA isolated from lamprey immunized with hen egg white lysozyme (HEL) in unbound and antigen-bound forms. The VLRA-HEL complex demonstrates that certain VLRAs, like {gamma}{delta} T-cell receptors (TCRs) but unlike {alpha}{beta} TCRs, can recognize antigens directly, without a requirement for processing or antigen-presenting molecules. Thus,more » these VLRAs feature the nanomolar affinities of antibodies, the direct recognition of unprocessed antigens of both antibodies and {gamma}{delta} TCRs, and the exclusive expression on the lymphocyte surface that is unique to {alpha}{beta} and {gamma}{delta} TCRs.« less

[Prevalence of antibodies to hepatitis A virus in healthcare workers and associated variables].

PubMed

Carreras-Valls, Rosa; Valverde-Lozano, Judit; Benito-Carreras, Dolors; Inglés-Torruella, Joan; Vilardell-Ynaraja, Miquel; Garre-Olmo, Josep; Gil-Soto, Rosa; Escalé-Roca, Margarita

2013-01-01

Vaccination against hepatitis A is recommended in risk groups, including healthcare workers. The objective of this study was to determine the prevalence of antibodies to HAV (IgG) among workers in the healthcare setting in order to establish criteria for vaccination. A cross-sectional, analytic, observational study of 4,864 employees was undertaken in four healthcare companies in Catalonia (Spain). The variables gathered included personal data, professional category, location of employment, and serology. The overall prevalence of antibodies to HAV was 52.7%. The prevalence significantly increased with greater age. The mean age of seropositive workers was 41.5 years compared with 34.3 in workers with negative serology. The highest prevalence of antibodies was found in cleaning employees (74.2%) and catering staff (75.3%). Given the high prevalence of seronegative adults susceptible to infection and the characteristics of their professional activities, vaccination of all staff working in health institutions should be considered. Copyright © 2012 SESPAS. Published by Elsevier Espana. All rights reserved.

PET imaging of osteosarcoma in dogs using a fluorine-18-labeled monoclonal antibody fab fragment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Page, R.L.; Garg, P.K.; Gard, S.

Four dogs with histologically confirmed osteogenic sarcoma were studied with PET following intravenous injection of the {sup 18}F-labeled Fab fragment of TP-3, a monoclonal antibody specific for human and canine osteosarcomas. The antibody fragment was labeled using the N-succinimidyl (8-(4{prime}-({sup 18}F)fluorobenzyl)amino)suberate acylation agent. Blood clearance of activity was biphasic in all dogs but half-times were variable (T{sub 1/2{beta}} = 2-13 hr). Catabolism of labeled Fab was reflected by the decrease in protein-associated activity in serum from more than 90% at 1 min to 60%-80% at 4 hr. PET images demonstrated increased accumulation of {sup 18}F at the primary tumor sitemore » relative to normal contralateral bone in one dog as early as 15 min after injection. Biopsies obtained after euthanasia indicated higher uptake at the edges of the tumor as observed on the PET scans. Tumor uptake was 1-3 x 10{sup -3}% injected dose/g, a level similar to that reported for other Fab fragments in human tumors. In the three dogs with metastatic disease, early PET images reflected activity in the blood pool but later uptake was observed in suspected metastatic sites. These results, although preliminary, suggest that PET imaging of {sup 18}F-labeled antibody fragments is feasible and that dogs with spontaneous tumors could be a valuable model for preclinical research with radioimmunoconjugates. 34 refs., 6 figs., 2 tabs.« less

Customizing Monoclonal Antibodies for the Treatment of Methamphetamine Abuse: Current and Future Applications

PubMed Central

Peterson, Eric C.; Gentry, W. Brooks

2015-01-01

Monoclonal antibody-based medications designed to bind (+)-methamphetamine (METH) with high affinity are among the newest approaches to the treatment of METH abuse, and the associated medical complications. The potential clinical indications for these medications include treatment of overdose, reduction of drug dependence, and protection of vulnerable populations from METH-related complications. Research designed to discover and conduct preclinical and clinical testing of these antibodies suggest a scientific vision for how intact mAb (singular and plural) or small antigen binding fragments of mAb could be engineered to optimize the proteins for specific therapeutic applications. In this review we discuss keys to success in this development process including choosing predictors of specificity, efficacy, duration of action, and safety of the medications in disease models of acute and chronic drug abuse. We consider important aspects of METH-like hapten design and how hapten structural features influence specificity and affinity, with an example of a high-resolution x-ray crystal structure of a high affinity antibody to demonstrate this structural relationship. Additionally, several prototype anti-METH mAb forms such as antigen binding fragments (Fab) and single chain variable fragments (scFv) are under development. Unique, customizable aspects of these fragments are presented with specific possible clinical indications. Finally, we discuss clinical trial progress of the first in kind anti-METH mAb, for which the METH is the disease target instead of vulnerable central nervous system networks of receptors, binding sites and neuronal connections. PMID:24484976

Single-Domain Antibodies and the Promise of Modular Targeting in Cancer Imaging and Treatment.

PubMed

Iezzi, María Elena; Policastro, Lucía; Werbajh, Santiago; Podhajcer, Osvaldo; Canziani, Gabriela Alicia

2018-01-01

Monoclonal antibodies and their fragments have significantly changed the outcome of cancer in the clinic, effectively inhibiting tumor cell proliferation, triggering antibody-dependent immune effector cell activation and complement mediated cell death. Along with a continued expansion in number, diversity, and complexity of validated tumor targets there is an increasing focus on engineering recombinant antibody fragments for lead development. Single-domain antibodies (sdAbs), in particular those engineered from the variable heavy-chain fragment (VHH gene) found in Camelidae heavy-chain antibodies (or IgG2 and IgG3), are the smallest fragments that retain the full antigen-binding capacity of the antibody with advantageous properties as drugs. For similar reasons, growing attention is being paid to the yet smaller variable heavy chain new antigen receptor (VNAR) fragments found in Squalidae. sdAbs have been selected, mostly from immune VHH libraries, to inhibit or modulate enzyme activity, bind soluble factors, internalize cell membrane receptors, or block cytoplasmic targets. This succinct review is a compilation of recent data documenting the application of engineered, recombinant sdAb in the clinic as epitope recognition "modules" to build monomeric, dimeric and multimeric ligands that target, tag and stall solid tumor growth in vivo . Size, affinity, specificity, and the development profile of sdAbs drugs are seemingly consistent with desirable clinical efficacy and safety requirements. But the hepatotoxicity of the tetrameric anti-DR5-VHH drug in patients with pre-existing anti-drug antibodies halted the phase I clinical trial and called for a thorough pre-screening of the immune and poly-specific reactivities of the sdAb leads.

Generation of a Highly Reactive Chicken-Derived Single-Chain Variable Fragment against Fusarium verticillioides by Phage Display

PubMed Central

Hu, Zu-Quan; Liu, Jin-Long; Li, He-Ping; Xing, Shu; Xue, Sheng; Zhang, Jing-Bo; Wang, Jian-Hua; Nölke, Greta; Liao, Yu-Cai

2012-01-01

Fusarium verticillioides is the primary causal agent of Fusarium ear and kernel rot in maize, producing fumonisin mycotoxins that are toxic to humans and domestic animals. Rapid detection and monitoring of fumonisin-producing fungi are pivotally important for the prevention of mycotoxins from entering into food/feed products. Chicken-derived single-chain variable fragments (scFvs) against cell wall-bound proteins from F. verticillioides were isolated from an immunocompetent phage display library. Comparative phage enzyme-linked immunosorbant assays (ELISAs) and sequencing analyses identified four different scFv antibodies with high sensitivity. Soluble antibody ELISAs identified two highly sensitive scFv antibodies, FvCA3 and FvCA4, with the latter being slightly more sensitive. Three-dimensional modeling revealed that the FvCA4 may hold a better overall structure with CDRH3, CDRL1 and CDRL3 centered in the core region of antibody surface compared with that of other scFvs. Immunofluorescence labeling revealed that the binding of FvCA4 antibody was localized to the cell walls of conidiospores and hyphae of F. verticillioides, confirming the specificity of this antibody for a surface target. This scFv antibody was able to detect the fungal mycelium as low as 10−2 μg/mL and contaminating mycelium at a quantity of 10−2 mg/g maize. This is the first report that scFv antibodies derived from phage display have a wide application for rapid and accurate detection and monitoring of fumonisin-producing pathogens in agricultural samples. PMID:22837678

Production of anti-amoxicillin ScFv antibody and simulation studying its molecular recognition mechanism for penicillins.

PubMed

Liu, Jing; Zhang, Hui C; Duan, Chang F; Dong, Jun; Zhao, Guo X; Wang, Jian P; Li, Nan; Liu, Jin Z; Li, Yu W

2016-11-01

The molecular recognition mechanism of an antibody for its hapten is very interesting. The objective of this research was to study the intermolecular interactions of an anti-amoxicillin antibody with penicillin drugs. The single chain variable fragment (ScFv) antibody was generated from a hybridoma cell strain excreting the monoclonal antibody for amoxicillin. The recombinant ScFv antibody showed similar recognition ability for penicillins to its parental monoclonal antibody: simultaneous recognizing 11 penicillins with cross-reactivities of 18-107%. The three-dimensional structure of the ScFv antibody was simulated by using homology modeling, and its intermolecular interactions with 11 penicillins were studied by using molecular docking. Results showed that three CDRs are involved in antibody recognition; CDR L3 Arg 100, CDR H3 Tyr226, and CDR H3 Arg 228 were the key contact amino acid residues; hydrogen bonding was the main antibody-drug intermolecular force; and the core structure of penicillin drugs was the main antibody binding position. These results could explain the recognition mechanism of anti-amoxicillin antibody for amoxicillin and its analogs. This is the first study reporting the production of ScFv antibody for penicillins and stimulation studying its recognition mechanism.

Recombinant scFv antibodies against infectious pancreatic necrosis virus isolated by flow cytometry.

PubMed

Xu, Li-Ming; Zhao, Jing-Zhuang; Liu, Miao; Cao, Yong-Sheng; Yin, Jia-Sheng; Liu, Hong-Bai; Lu, Tongyan

2016-11-01

Infectious pancreatic necrosis is a significant disease of farmed salmonids in China. In this study, a single chain variable fragment (scFv) antibody library derived from rainbow trout (Oncorhynchus mykiss) and viral protein VP2 of a Chinese infectious pancreatic necrosis virus (IPNV) isolate ChRtm213 were co-expressed by a bacterial display technology. The library was subjected to three rounds of screening by flow cytometry (FCM) to select IPNV specific antibodies. Six antibody clones with different mean fluorescence intensities (MFI) were obtained by picking colonies at random. The antibody clones were expressed and purified. The purified IPNV-specific scFv antibodies were used successfully in Western blotting, enzyme linked immunosorbent assay (ELISA) and an immunofluorescence antibody test (IFAT). This method provides a high throughput means to screen an antibody library by flow cytometry, and isolate a panel of antibody that can be used as potential reagents for the detection and study of IPNV that are prevalent in China. Copyright © 2016 Elsevier B.V. All rights reserved.

Near-Atomic Resolution Structure of a Highly Neutralizing Fab Bound to Canine Parvovirus.

PubMed

Organtini, Lindsey J; Lee, Hyunwook; Iketani, Sho; Huang, Kai; Ashley, Robert E; Makhov, Alexander M; Conway, James F; Parrish, Colin R; Hafenstein, Susan

2016-11-01

Canine parvovirus (CPV) is a highly contagious pathogen that causes severe disease in dogs and wildlife. Previously, a panel of neutralizing monoclonal antibodies (MAb) raised against CPV was characterized. An antibody fragment (Fab) of MAb E was found to neutralize the virus at low molar ratios. Using recent advances in cryo-electron microscopy (cryo-EM), we determined the structure of CPV in complex with Fab E to 4.1 Å resolution, which allowed de novo building of the Fab structure. The footprint identified was significantly different from the footprint obtained previously from models fitted into lower-resolution maps. Using single-chain variable fragments, we tested antibody residues that control capsid binding. The near-atomic structure also revealed that Fab binding had caused capsid destabilization in regions containing key residues conferring receptor binding and tropism, which suggests a mechanism for efficient virus neutralization by antibody. Furthermore, a general technical approach to solving the structures of small molecules is demonstrated, as binding the Fab to the capsid allowed us to determine the 50-kDa Fab structure by cryo-EM. Using cryo-electron microscopy and new direct electron detector technology, we have solved the 4 Å resolution structure of a Fab molecule bound to a picornavirus capsid. The Fab induced conformational changes in regions of the virus capsid that control receptor binding. The antibody footprint is markedly different from the previous one identified by using a 12 Å structure. This work emphasizes the need for a high-resolution structure to guide mutational analysis and cautions against relying on older low-resolution structures even though they were interpreted with the best methodology available at the time. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Near-Atomic Resolution Structure of a Highly Neutralizing Fab Bound to Canine Parvovirus

PubMed Central

Organtini, Lindsey J.; Lee, Hyunwook; Iketani, Sho; Huang, Kai; Ashley, Robert E.; Makhov, Alexander M.; Conway, James F.

2016-01-01

ABSTRACT Canine parvovirus (CPV) is a highly contagious pathogen that causes severe disease in dogs and wildlife. Previously, a panel of neutralizing monoclonal antibodies (MAb) raised against CPV was characterized. An antibody fragment (Fab) of MAb E was found to neutralize the virus at low molar ratios. Using recent advances in cryo-electron microscopy (cryo-EM), we determined the structure of CPV in complex with Fab E to 4.1 Å resolution, which allowed de novo building of the Fab structure. The footprint identified was significantly different from the footprint obtained previously from models fitted into lower-resolution maps. Using single-chain variable fragments, we tested antibody residues that control capsid binding. The near-atomic structure also revealed that Fab binding had caused capsid destabilization in regions containing key residues conferring receptor binding and tropism, which suggests a mechanism for efficient virus neutralization by antibody. Furthermore, a general technical approach to solving the structures of small molecules is demonstrated, as binding the Fab to the capsid allowed us to determine the 50-kDa Fab structure by cryo-EM. IMPORTANCE Using cryo-electron microscopy and new direct electron detector technology, we have solved the 4 Å resolution structure of a Fab molecule bound to a picornavirus capsid. The Fab induced conformational changes in regions of the virus capsid that control receptor binding. The antibody footprint is markedly different from the previous one identified by using a 12 Å structure. This work emphasizes the need for a high-resolution structure to guide mutational analysis and cautions against relying on older low-resolution structures even though they were interpreted with the best methodology available at the time. PMID:27535057

Protection against canine parvovirus type 2 infection in puppies by colostrum-derived antibodies.

PubMed

Mila, Hanna; Grellet, Aurélien; Desario, Costantina; Feugier, Alexandre; Decaro, Nicola; Buonavoglia, Canio; Chastant-Maillard, Sylvie

2014-01-01

During the first weeks of life puppies remain protected against canine parvovirus type 2 (CPV2) infection thanks to maternally derived antibodies (MDA) absorbed with colostrum after birth. The objective of the present study was to present the variability in CPV2-specific passive immune transfer and its consequences in puppies naturally exposed to the parvovirus. Seventy-nine puppies from one breeding kennel were included in the study at birth and followed until 56 d of age. Once per week the MDA titre for CPV2 specific antibodies was determined in blood. Viral excretion was also evaluated on a rectal swab by CPV2 PCR assay and puppies were weighed to determine growth rate. At 2 d of age, thirty-four out of seventy-nine puppies (43 %) had MDA ≤1:160 (designed group A) and forty-five puppies (57 %) had greater MDA titres (designed group B). The level of absorbed maternal antibodies was shown to be associated with breed size and growth rate during the first 48 h of life. The MDA level declined with age in all cases; however, the proportion of puppies with the antibody level considered as protective against CPV2 infection was significantly higher in group B compared with A from day 2 until 42. Among all puppies surviving until 56 d of age, sixty-seven out of seventy (95·7 %) underwent CPV2 infection. However, puppies from group A excreted CPV2 significantly earlier than puppies from group B. The present study demonstrates the link between passive immune transfer, in terms of level of specific MDA absorbed, and length of the protection period against parvovirus infection in weaning puppies.

Quantitative Assessment of the Heterogeneity of PD-L1 Expression in Non-Small-Cell Lung Cancer.

PubMed

McLaughlin, Joseph; Han, Gang; Schalper, Kurt A; Carvajal-Hausdorf, Daniel; Pelekanou, Vasiliki; Rehman, Jamaal; Velcheti, Vamsidhar; Herbst, Roy; LoRusso, Patricia; Rimm, David L

2016-01-01

Early-phase trials with monoclonal antibodies targeting PD-1 (programmed cell death protein 1) and PD-L1 (programmed cell death 1 ligand 1) have demonstrated durable clinical responses in patients with non-small-cell lung cancer (NSCLC). However, current assays for the prognostic and/or predictive role of tumor PD-L1 expression are not standardized with respect to either quantity or distribution of expression. To demonstrate PD-L1 protein distribution in NSCLC tumors using both conventional immunohistochemistry (IHC) and quantitative immunofluorescence (QIF) and compare results obtained using 2 different PD-L1 antibodies. PD-L1 was measured using E1L3N and SP142, 2 rabbit monoclonal antibodies, in 49 NSCLC whole-tissue sections and a corresponding tissue microarray with the same 49 cases. Non-small-cell lung cancer biopsy specimens from 2011 to 2012 were collected retrospectively from the Yale Thoracic Oncology Program Tissue Bank. Human melanoma Mel 624 cells stably transfected with PD-L1 as well as Mel 624 parental cells, and human term placenta whole tissue sections were used as controls and for antibody validation. PD-L1 protein expression in tumor and stroma was assessed using chromogenic IHC and the AQUA (Automated Quantitative Analysis) method of QIF. Tumor-infiltrating lymphocytes (TILs) were scored in hematoxylin-eosin slides using current consensus guidelines. The association between PD-L1 protein expression, TILs, and clinicopathological features were determined. PD-L1 expression discordance or heterogeneity using the diaminobenzidine chromogen and QIF was the main outcome measure selected prior to performing the study. Using chromogenic IHC, both antibodies showed fair to poor concordance. The PD-L1 antibodies showed poor concordance (Cohen κ range, 0.124-0.340) using conventional chromogenic IHC and showed intra-assay heterogeneity (E1L3N coefficient of variation [CV], 6.75%-75.24%; SP142 CV, 12.17%-109.61%) and significant interassay discordance using QIF (26.6%). Quantitative immunofluorescence showed that PD-L1 expression using both PD-L1 antibodies was heterogeneous. Using QIF, the scores obtained with E1L3N and SP142 for each tumor were significantly different according to nonparametric paired test (P < .001). Assessment of 588 serial section fields of view from whole tissue showed discordant expression at a frequency of 25%. Expression of PD-L1 was correlated with high TILs using both E1L3N (P = .007) and SP142 (P = .02). Objective determination of PD-L1 protein levels in NSCLC reveals heterogeneity within tumors and prominent interassay variability or discordance. This could be due to different antibody affinities, limited specificity, or distinct target epitopes. Efforts to determine the clinical value of these observations are under way.

Single Domain Antibodies as New Biomarker Detectors

PubMed Central

Fischer, Katja; Leow, Chiuan Yee; Chuah, Candy; McCarthy, James

2017-01-01

Biomarkers are defined as indicators of biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. Biomarkers have been widely used for early detection, prediction of response after treatment, and for monitoring the progression of diseases. Antibodies represent promising tools for recognition of biomarkers, and are widely deployed as analytical tools in clinical settings. For immunodiagnostics, antibodies are now exploited as binders for antigens of interest across a range of platforms. More recently, the discovery of antibody surface display and combinatorial chemistry techniques has allowed the exploration of new binders from a range of animals, for instance variable domains of new antigen receptors (VNAR) from shark and variable heavy chain domains (VHH) or nanobodies from camelids. These single domain antibodies (sdAbs) have some advantages over conventional murine immunoglobulin owing to the lack of a light chain, making them the smallest natural biomarker binders thus far identified. In this review, we will discuss several biomarkers used as a means to validate diseases progress. The potential functionality of modern singe domain antigen binders derived from phylogenetically early animals as new biomarker detectors for current diagnostic and research platforms development will be described. PMID:29039819

Somatic diversification in the heavy chain variable region genes expressed by human autoantibodies bearing a lupus-associated nephritogenic anti-DNA idiotype

DOE Office of Scientific and Technical Information (OSTI.GOV)

Demaison, C.; Chastagner, P.; Theze, J.

1994-01-18

Monoclonal anti-DNA antibodies bearing a lupus nephritis-associated idiotype were derived from five patients with systemic lupus erythematosus (SLE). Genes encoding their heavy (H)-chain variable (V[sub H]) regions were cloned and sequenced. When compared with their closest V[sub h] germ-line gene relatives, these sequences exhibit a number of silent (S) and replacement (R) substitutions. The ratios of R/S mutations were much higher in the complementarity-determining regions (CDRs) of the antibodies than in the framework regions. Molecular amplification of genomic V[sub H] genes and Southern hybridization with somatic CDR2-specific oligonucleotide probes showed that the configuration of the V[sub H] genes corresponding tomore » V[sub H] sequences in the nephritogenic antibodies is not present in the patient's own germ-line DNA, implying that the B-cell clones underwent somatic mutation in vivo. These findings, together with the characteristics of the diversity and junctional gene elements utilized to form the antibody, indicate that these autoantibodies have been driven through somatic selection processes reminiscent of those that govern antibody responses triggered by exogenous stimuli.« less

A Simple Model System to Demonstrate Antibody Structure and Functions.

ERIC Educational Resources Information Center

O'Kennedy, Richard

1991-01-01

A model that can be used to show the arrangement of light and heavy chains, disulfide linkages, domains, and subclass variations in antibodies is given. It can be constructed and modified to illustrate Fab, F(ab')2, and Fc fragments, single domain and bifunctional antibodies, and labeling of antibodies. (Author)

Human antibodies fix complement to inhibit Plasmodium falciparum invasion of erythrocytes and are associated with protection against malaria.

PubMed

Boyle, Michelle J; Reiling, Linda; Feng, Gaoqian; Langer, Christine; Osier, Faith H; Aspeling-Jones, Harvey; Cheng, Yik Sheng; Stubbs, Janine; Tetteh, Kevin K A; Conway, David J; McCarthy, James S; Muller, Ivo; Marsh, Kevin; Anders, Robin F; Beeson, James G

2015-03-17

Antibodies play major roles in immunity to malaria; however, a limited understanding of mechanisms mediating protection is a major barrier to vaccine development. We have demonstrated that acquired human anti-malarial antibodies promote complement deposition on the merozoite to mediate inhibition of erythrocyte invasion through C1q fixation and activation of the classical complement pathway. Antibody-mediated complement-dependent (Ab-C') inhibition was the predominant invasion-inhibitory activity of human antibodies; most antibodies were non-inhibitory without complement. Inhibitory activity was mediated predominately via C1q fixation, and merozoite surface proteins 1 and 2 were identified as major targets. Complement fixation by antibodies was very strongly associated with protection from both clinical malaria and high-density parasitemia in a prospective longitudinal study of children. Ab-C' inhibitory activity could be induced by human immunization with a candidate merozoite surface-protein vaccine. Our findings demonstrate that human anti-malarial antibodies have evolved to function by fixing complement for potent invasion-inhibitory activity and protective immunity. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Generation of human scFv antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences.

PubMed

Andris-Widhopf, Jennifer; Steinberger, Peter; Fuller, Roberta; Rader, Christoph; Barbas, Carlos F

2011-09-01

The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human scFv (single chain antibody fragment) libraries using a short linker (GGSSRSS) or a long linker (GGSSRSSSSGGGGSGGGG). In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final scFv products that are used for cloning.

Lack of association between ectoparasite intensities and rabies virus neutralizing antibody seroprevalence in wild big brown bats (Eptesicus fuscus), Fort Collins, Colorado

USGS Publications Warehouse

Pearce, R.D.; O'Shea, T.J.; Shankar, V.; Rupprecht, C.E.

2007-01-01

Recently, bat ectoparasites have been demonstrated to harbor pathogens of potential importance to humans. We evaluated antirabies antibody seroprevalence and the presence of ectoparasites in big brown bats (Eptesicus fuscus) sampled in 2002 and 2003 in Colorado to investigate if an association existed between ectoparasite intensity and exposure to rabies virus (RV). We used logistic regression and Akaike's Information Criteria adjusted for sample size (AICc) in a post-hoc analysis to investigate the relative importance of three ectoparasite species, as well as bat colony size, year sampled, age class, colony size, and year interaction on the presence of rabies virus neutralizing antibodies (VNA) in serum of wild E. fuscus. We obtained serum samples and ectoparasite counts from big brown bats simultaneously in 2002 and 2003. Although the presence of ectoparasites (Steatonyssus occidentalis and Spinturnix bakeri) were important in elucidating VNA seroprevalence, their intensities were higher in seronegative bats than in seropositive bats, and the presence of a third ectoparasite (Cimex pilosellus) was inconsequential. Colony size and year sampled were the most important variables in these AICc models. These findings suggest that these ectoparasites do not enhance exposure of big brown bats to RV. ?? 2007 Mary Ann Liebert, Inc.

Ultra Q-bodies: quench-based antibody probes that utilize dye-dye interactions with enhanced antigen-dependent fluorescence.

PubMed

Abe, Ryoji; Jeong, Hee-Jin; Arakawa, Dai; Dong, Jinhua; Ohashi, Hiroyuki; Kaigome, Rena; Saiki, Fujio; Yamane, Kyosuke; Takagi, Hiroaki; Ueda, Hiroshi

2014-04-11

Recently, we described a novel reagentless fluorescent biosensor strategy named Quenchbody, which functions via the antigen-dependent removal of the quenching effect on a fluorophore that is attached to a single-chain antibody variable region. To explore the practical utility of Quenchbodies, we prepared antibody Fab fragments that were fluorolabeled at either one or two of the N-terminal regions, using a cell-free translation-mediated position-specific protein labeling system. Unexpectedly, the Fab fragment labeled at the heavy chain N-terminal region demonstrated a deeper quenching and antigen-dependent release compared to that observed using scFv. Moreover, when the Fab was fluorolabeled at the two N-termini with either the same dye or with two different dyes, an improved response due to enhanced quenching via dye-dye interactions was observed. On the basis of this approach, several targets, including peptides, proteins, and haptens, as well as narcotics, were quantified with a higher response up to 50-fold. In addition, differentiation of osteosarcoma to osteoblasts was successfully imaged using a similarly fluorolabeled recombinant Fab protein prepared from E. coli. Due to its versatility, this "Ultra-Quenchbody" is expected to exhibit a range of applications from in vitro diagnostics to the live imaging of various targets in situ.

SAbPred: a structure-based antibody prediction server

PubMed Central

Dunbar, James; Krawczyk, Konrad; Leem, Jinwoo; Marks, Claire; Nowak, Jaroslaw; Regep, Cristian; Georges, Guy; Kelm, Sebastian; Popovic, Bojana; Deane, Charlotte M.

2016-01-01

SAbPred is a server that makes predictions of the properties of antibodies focusing on their structures. Antibody informatics tools can help improve our understanding of immune responses to disease and aid in the design and engineering of therapeutic molecules. SAbPred is a single platform containing multiple applications which can: number and align sequences; automatically generate antibody variable fragment homology models; annotate such models with estimated accuracy alongside sequence and structural properties including potential developability issues; predict paratope residues; and predict epitope patches on protein antigens. The server is available at http://opig.stats.ox.ac.uk/webapps/sabpred. PMID:27131379

Reshaping Human Antibodies: Grafting an Antilysozyme Activity

NASA Astrophysics Data System (ADS)

Verhoeyen, Martine; Milstein, Cesar; Winter, Greg

1988-03-01

The production of therapeutic human monoclonal antibodies by hybridoma technology has proved difficult, and this has prompted the ``humanizing'' of mouse monoclonal antibodies by recombinant DNA techniques. It was shown previously that the binding site for a small hapten could be grafted from the heavy-chain variable domain of a mouse antibody to that of a human myeloma protein by transplanting the hypervariable loops. It is now shown that a large binding site for a protein antigen (lysozyme) can also be transplanted from mouse to human heavy chain. The success of such constructions may be facilitated by an induced-fit mechanism.

Conatumumab (AMG 655) coated nanoparticles for targeted pro-apoptotic drug delivery.

PubMed

Fay, Francois; McLaughlin, Kirsty M; Small, Donna M; Fennell, Dean A; Johnston, Patrick G; Longley, Daniel B; Scott, Christopher J

2011-11-01

Colloidal nanoparticle drug delivery systems have attracted much interest for their ability to enable effective formulation and delivery of therapeutic agents. The selective delivery of these nanoparticles to the disease site can be enhanced by coating the surface of the nanoparticles with targeting moieties, such as antibodies. In this current work, we demonstrate that antibodies on the surface of the particles can also elicit key biological effects. Specifically, we demonstrate the induction of apoptosis in colorectal HCT116 cancer cells using PLGA nanoparticles coated with Conatumumab (AMG 655) death receptor 5-specific antibodies (DR5-NP). We show that DR5-NP preferentially target DR5-expressing cells and present a sufficient density of antibody paratopes to induce apoptosis via DR5, unlike free AMG 655 or non-targeted control nanoparticles. We also demonstrate that DR5-targeted nanoparticles encapsulating the cytotoxic drug camptothecin are effectively targeted to the tumour cells, thereby producing enhanced cytotoxic effects through simultaneous drug delivery and apoptosis induction. These results demonstrate that antibodies on nanoparticulate surfaces can be exploited for dual modes of action to enhance the therapeutic utility of the modality. Copyright © 2011 Elsevier Ltd. All rights reserved.

Heterogeneous distribution of antigens on human platelets demonstrated by fluorescence flow cytometry.

PubMed

Dunstan, R A; Simpson, M B

1985-12-01

We have used fluorescence flow cytometry to analyse cell-to-cell variability in the density of platelet ABH, Ii, Lewis, P, P1A1, Bak,a and HLA class I antigens. Human IgG and IgM antibodies were used in a two-stage assay with goat FITC-conjugated antihuman IgG (H&L) antibody as the label, followed by single cell analysis of 10 000 platelets per sample using a 256-channel fluorescence flow cytometer (Becton-Dickinson FACS Analyser). Computer analysis of fluorescence intensity histograms for mean and peak channel and coefficient of variation shows that the degree of heterogeneity in platelet antigen density varies with each particular blood group. The broad fluorescence distribution curves with oligosaccharide antigens (CVs: A = 53, B = 40, I = 44, Lea = 40, P = 40) indicate that these antigens possess a greater variability in the number of sites per cell compared to the more homogeneous distribution of P1,A1 BaK,a and HLA (CVs: P1A1 = 24, HLA = 30). These findings may partly account for the mechanism by which transfusion of ABO-incompatible platelets results in a biphasic survival curve, with a period of early rapid removal of those platelets with a high density of antigen sites, followed by a relatively normal survival curve for those platelets that possess only a few or no antigen sites. In contrast, P1A1 and HLA sites are less variable in number from one platelet to another in a given donor, and immune-mediated removal would be more likely to approximate a single exponential curve.

An innovative and highly drug-tolerant approach for detecting neutralizing antibodies directed to therapeutic antibodies.

PubMed

Sloan, John H; Conway, Richard G; Pottanat, Thomas G; Troutt, Jason S; Higgs, Richard E; Konrad, Robert J; Qian, Yue-Wei

2016-10-01

Immunogenicity testing of biotherapeutic drugs is a regulatory requirement. Herein, we describe a drug-tolerant assay for detecting neutralizing antibodies against a therapeutic antibody. Excess target of the therapeutic antibody was incorporated into the detection step of an affinity capture elution assay. Signal generated from binding of antidrug antibody (ADA) to the therapeutic antibody was compared with signal from binding of ADA to the therapeutic antibody preincubated with its target. The results demonstrated that the target blocked binding of the therapeutic antibody to neutralizing monkey ADA and to two anti-idiotypic antibodies. This highly drug-tolerant novel approach enables the detection of neutralizing antibodies and allows for one basic assay format to achieve complete characterization of ADA responses.

Affinity-based precipitation via a bivalent peptidic hapten for the purification of monoclonal antibodies.

PubMed

Handlogten, Michael W; Stefanick, Jared F; Deak, Peter E; Bilgicer, Basar

2014-09-07

In a previous study, we demonstrated a non-chromatographic affinity-based precipitation method, using trivalent haptens, for the purification of mAbs. In this study, we significantly improved this process by using a simplified bivalent peptidic hapten (BPH) design, which enables facile and rapid purification of mAbs while overcoming the limitations of the previous trivalent design. The improved affinity-based precipitation method (ABP(BPH)) combines the simplicity of salt-induced precipitation with the selectivity of affinity chromatography for the purification of mAbs. The ABP(BPH) method involves 3 steps: (i) precipitation and separation of protein contaminants larger than immunoglobulins with ammonium sulfate; (ii) selective precipitation of the target-antibody via BPH by inducing antibody-complex formation; (iii) solubilization of the antibody pellet and removal of BPH with membrane filtration resulting in the pure antibody. The ABP(BPH) method was evaluated by purifying the pharmaceutical antibody trastuzumab from common contaminants including CHO cell conditioned media, DNA, ascites fluid, other antibodies, and denatured antibody with >85% yield and >97% purity. Importantly, the purified antibody demonstrated native binding activity to cell lines expressing the target protein, HER2. Combined, the ABP(BPH) method is a rapid and scalable process for the purification of antibodies with the potential to improve product quality while decreasing purification costs.

Structural and functional characterization of a novel scFv anti-HSP60 of Strongyloides sp.

PubMed Central

Levenhagen, Marcelo Arantes; de Almeida Araújo Santos, Fabiana; Fujimura, Patrícia Tiemi; Caneiro, Ana Paula; Costa-Cruz, Julia Maria; Goulart, Luiz Ricardo

2015-01-01

Phage display is a powerful technology that selects specific proteins or peptides to a target. We have used Phage Display to select scFv (single-chain variable fragment) clones from a combinatorial library against total proteins of Strongyloides venezuelensis. After scFv characterization, further analysis demonstrated that this recombinant fragment of antibody was able to bind to an S. venezuelensis antigenic fraction of ~65 kDa, present in the body periphery and digestive system of infective larvae (L3), as demonstrated by immunofluorescence. Mass spectrometry results followed by bioinformatics analysis showed that this antigenic fraction was a heat shock protein 60 (HSP60) of Strongyloides sp. The selected scFv was applied in serodiagnosis by immune complexes detection in serum samples from individuals with strongyloidiasis using a sandwich enzyme-linked immunosorbent assay (ELISA), showing sensitivity of 97.5% (86.84–99.94), specificity of 98.81 (93.54–99.97), positive likelihood ratio of 81.60 and an area under the curve of 0.9993 (0.9973–1.000). Our study provided a novel monoclonal scFv antibody fragment which specifically bound to HSP60 of Strongyloides sp. and was applied in the development of an innovative serodiagnosis method for the human strongyloidiasis. PMID:25994608

The influence of proteasome inhibitor MG132, external radiation and unlabeled antibody on the tumor uptake and biodistribution of 188Re-labeled anti-E6 C1P5 antibody in cervical cancer in mice

PubMed Central

Phaeton, Rébécca; Wang, Xing Guo; Einstein, Mark H.; Goldberg, Gary L.; Casadevall, Arturo; Dadachova, Ekaterina

2009-01-01

Background Human Papillomavirus (HPV) infection is considered a necessary step for the development of cervical cancer and >95% of all cervical cancers have detectable HPV sequences. We have recently demonstrated the efficacy of radioimmunotherapy (RIT) which targeted viral oncoprotein E6 in treatment of experimental cervical cancer We hypothesized that pre-treatment of tumor cells with various agents which cause cell death and/or elevation of E6 levels would increase the accumulation of radiolabeled antibodies to E6 in cervical tumors. Methods HPV-16 positive CasKi cells were treated in vitro with up to 6 Gy of external radiation, or proteasome inhibitor MG-132 or unlabeled anti-E6 antibody C1P5 and cell death was assessed. Biodistribution of 188Rhenium (188Re)-labeled C1P5 antibody was performed in both control and radiation MG-132 treated CasKi tumor-bearing nude mice. Results . 188Re-C1P5 antibody demonstrated tumor specificity and very low uptake and fast clearance from the major organs. The amount of tumor uptake was enhanced by MG-132 but was unaffected by pre-treatment with radiation. In addition, in vitro studies demonstrated an unanticipated effect of unlabeled antibody on the amount of cell death, a finding that was suggested by our previous in vivo studies in CasKi tumor model. Conclusion We demonstrated that pre-treatment of cervical tumors with proteasome inhibitor MG-132 and with unlabeled antibody to E6 can serve as a means to generate non-viable cancer cells and to elevate the levels of target oncoproteins in the cells for increasing the accumulation of targeted radiolabeled antibodies in tumors. These results favor further development of RIT of cervical cancers targeting viral antigens. PMID:20127955

Development of a competitive ELISA using a truncated E2 recombinant protein as antigen for detection of antibodies to classical swine fever virus.

PubMed

Clavijo, A; Lin, M; Riva, J; Mallory, M; Lin, F; Zhou, E M

2001-02-01

The sequence encoding a truncated E2 glycoprotein of the Alfort/187 strain of classical swine fever virus (CSFV) was expressed in Escherichia coli using the pET expression system and the recombinant product purified by Ni-NTA agarose affinity chromatography. The antigenicity of this recombinant protein was demonstrated by immunoblot using anti- CSFV-specific antibodies. A monoclonal antibody was produced against the truncated E2 protein and used as competitor in an ELISA for the detection of antibodies to CSFV. Specific antibodies were demonstrated by competitive ELISA (C-ELISA) as early as 21 days post-infection (dpi) in experimentally infected pigs. Seroconversion was demonstrated by C-ELISA and neutralising peroxidase-linked assay (NPLA) in all infected animals by 4 weeks. No cross-reaction with antibodies to bovine viral diarrhoea virus (BVDV) was seen in the C-ELISA using sera from experimentally infected pigs. The C-ELISA is not intended as a substitute for the NPLA. However, it is expected it will be useful for monitoring and prevalence studies. It will also assist in testing a large number of samples in the event of an outbreak. Copyright 2001 Harcourt Publishers Ltd.

Combinatorial Libraries of Arrayable Single-Chain Antibodies

NASA Astrophysics Data System (ADS)

Benhar, Itai

Antibodies that bind their respective targets with high affinity and specificity have proven to be essential reagents for biological research. Antibody phage display has become the leading tool for the rapid isolation of single-chain variable fragment (scFv) antibodies in vitro for research applications, but there is usually a gap between scFv isolation and its application in an array format suitable for high-throughput proteomics. In this chapter, we present our antibody phage display system where antibody isolation and scFv immobilization are facilitated by the design of the phagemid vector used as platform. In our system, the scFvs are fused at their C-termini to a cellulose-binding domain (CBD) and can be immobilized onto cellulose-based filters. This made it possible to develop a unique filter lift screen that allowed the efficient screen for multiple binding specificities, and to directly apply library-derived scFvs in an antibody spotted microarray.

Molecular Simulation of Receptor Occupancy and Tumor Penetration of an Antibody and Smaller Scaffolds: Application to Molecular Imaging.

PubMed

Orcutt, Kelly D; Adams, Gregory P; Wu, Anna M; Silva, Matthew D; Harwell, Catey; Hoppin, Jack; Matsumura, Manabu; Kotsuma, Masakatsu; Greenberg, Jonathan; Scott, Andrew M; Beckman, Robert A

2017-10-01

Competitive radiolabeled antibody imaging can determine the unlabeled intact antibody dose that fully blocks target binding but may be confounded by heterogeneous tumor penetration. We evaluated the hypothesis that smaller radiolabeled constructs can be used to more accurately evaluate tumor expressed receptors. The Krogh cylinder distributed model, including bivalent binding and variable intervessel distances, simulated distribution of smaller constructs in the presence of increasing doses of labeled antibody forms. Smaller constructs <25 kDa accessed binding sites more uniformly at large distances from blood vessels compared with larger constructs and intact antibody. These observations were consistent for different affinity and internalization characteristics of constructs. As predicted, a higher dose of unlabeled intact antibody was required to block binding to these distant receptor sites. Small radiolabeled constructs provide more accurate information on total receptor expression in tumors and reveal the need for higher antibody doses for target receptor blockade.

Antibody-functionalized porous silicon nanoparticles for vectorization of hydrophobic drugs.

PubMed

Secret, Emilie; Smith, Kevin; Dubljevic, Valentina; Moore, Eli; Macardle, Peter; Delalat, Bahman; Rogers, Mary-Louise; Johns, Terrance G; Durand, Jean-Olivier; Cunin, Frédérique; Voelcker, Nicolas H

2013-05-01

We describe the preparation of biodegradable porous silicon nanoparticles (pSiNP) functionalized with cancer cell targeting antibodies and loaded with the hydrophobic anti-cancer drug camptothecin. Orientated immobilization of the antibody on the pSiNP is achieved using novel semicarbazide based bioconjugate chemistry. To demonstrate the generality of this targeting approach, the three antibodies MLR2, mAb528 and Rituximab are used, which target neuroblastoma, glioblastoma and B lymphoma cells, respectively. Successful targeting is demonstrated by means of flow cytometry and immunocytochemistry both with cell lines and primary cells. Cell viability assays after incubation with pSiNPs show selective killing of cells expressing the receptor corresponding to the antibody attached on the pSiNP. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The challenges of modelling antibody repertoire dynamics in HIV infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Luo, Shishi; Perelson, Alan S.

Antibody affinity maturation by somatic hypermutation of B-cell immunoglobulin variable region genes has been studied for decades in various model systems using well-defined antigens. While much is known about the molecular details of the process, our understanding of the selective forces that generate affinity maturation are less well developed, particularly in the case of a co-evolving pathogen such as HIV. Despite this gap in understanding, high-throughput antibody sequence data are increasingly being collected to investigate the evolutionary trajectories of antibody lineages in HIV-infected individuals. Here, we review what is known in controlled experimental systems about the mechanisms underlying antibody selectionmore » and compare this to the observed temporal patterns of antibody evolution in HIV infection. In addition, we describe how our current understanding of antibody selection mechanisms leaves questions about antibody dynamics in HIV infection unanswered. Without a mechanistic understanding of antibody selection in the context of a co-evolving viral population, modelling and analysis of antibody sequences in HIV-infected individuals will be limited in their interpretation and predictive ability.« less

The challenges of modelling antibody repertoire dynamics in HIV infection

DOE PAGES

Luo, Shishi; Perelson, Alan S.

2015-07-20

Antibody affinity maturation by somatic hypermutation of B-cell immunoglobulin variable region genes has been studied for decades in various model systems using well-defined antigens. While much is known about the molecular details of the process, our understanding of the selective forces that generate affinity maturation are less well developed, particularly in the case of a co-evolving pathogen such as HIV. Despite this gap in understanding, high-throughput antibody sequence data are increasingly being collected to investigate the evolutionary trajectories of antibody lineages in HIV-infected individuals. Here, we review what is known in controlled experimental systems about the mechanisms underlying antibody selectionmore » and compare this to the observed temporal patterns of antibody evolution in HIV infection. In addition, we describe how our current understanding of antibody selection mechanisms leaves questions about antibody dynamics in HIV infection unanswered. Without a mechanistic understanding of antibody selection in the context of a co-evolving viral population, modelling and analysis of antibody sequences in HIV-infected individuals will be limited in their interpretation and predictive ability.« less

Use of the single cell gel electrophoresis (comet assay) for comparing apoptotic effect of conventional antibodies versus nanobodies

PubMed Central

Shaker, Ghada H.; Melake, Nahla A.

2011-01-01

The large molecular size of antibodies is considered one major factor preventing them from becoming more efficient therapeutically. It is well established that all camelids have unique antibodies circulating in their blood called heavy-chain antibodies (HcAbs). Unlike antibodies from other species, these HcAbs contain a single variable domain and two constant domains (CH2 and CH3). HcAbs are a novel type of immunoglobulin-like, antigen binding protein with beneficial pharmacokinetic properties that are ideally suited to targeting cellular antigens for molecular imaging or therapeutic purposes. Since the antigen-binding site of dromedary HcAb is comprised in one single domain, it was referred to as nanobody. In the present work, the different IgG subclasses from immunized camel (Camelus dromedairus) were purified employing their different affinity for protein A column (PA) and protein G column (PG). Characterization of IgG subclasses was done by using 12% SDS–PAGE under reducing conditions. Protein bands were visualized after staining with Coomassie Brilliant Blue, showing two bands at 50 kDa and 30 kDa in case of IgG1 while IgG2 and IgG3 produce only one band at 46 kDa and 43 kDa respectively. The induction of apoptosis by either conventional or nanobodies was evaluated on two different cell lines, Colon and Hepatic cancer cell (HCT116 and HepG2), using the comet assay. Induced apoptosis were confirmed by visualizing DNA fragmentation bands on 2% agarose gel, and the gel was photographed under UV light. This study demonstrates the successful targeting of human cancer colon cell lines by nanobodies in vitro. It may open perspectives for their future use as tumor target vehicle, due to their small size, soluble behavior and they interact with epitopes that are less antigenic for conventional antibodies. PMID:23960797

Antibody responses to the chlamydial heat shock proteins hsp60 and hsp70 are H-2 linked.

PubMed Central

Zhong, G; Brunham, R C

1992-01-01

The effects of both H-2 and non-H-2 genes on antibody responses to two Chlamydia trachomatis heat shock proteins (hsp60 and hsp70) were investigated. These chlamydial proteins are homologs of Escherichia coli GroEL (hsp60) and DnaK (hsp70) and are highly sequence conserved between bacterial and mammalian sources. Antibody responses among 17 different strains of mice immunized with C. trachomatis serovar B and serovar C elementary bodies were evaluated by immunoblot, radioimmunoprecipitation and enzyme-linked immunosorbent assay. Antibody responses to the two proteins displayed host genetic restriction. Of six distinctive H-2 haplotypes, only H-2d generated high antibody responses to hsp70. Five of the six H-2 haplotypes, i.e., H-2a, H-2d, H-2k, H-2q, and H-2s, produced high antibody responses to hsp60. Only the H-2b-bearing strain had low antibody responses to hsp60. By using congenic and H-2 recombinant strains, the genes responsible for regulating antibody responses to hsp70 and hsp60 were mapped to the K-IA region of the H-2 locus. In F1 hybrid crosses between high and low responders, high responses to hsp60 and hsp70 were dominant traits. Other genes outside the H-2 locus also influenced antibody responses to hsp60 and hsp70, since inbred strains of identical H-2 but different background genes displayed variable antibody responses to the proteins. The genetic control of murine immune responses to C. trachomatis hsp60, a putative chlamydial immunopathologic antigen, suggests that a similar genetic mechanism may also exist in humans, and this observation may help to explain the observed variability in the spectrum of chlamydial diseases seen in humans. Images PMID:1639484

Relationship between natural and heme-mediated antibody polyreactivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hadzhieva, Maya; Vassilev, Tchavdar; Bayry, Jagadeesh

Polyreactive antibodies represent a considerable fraction of the immune repertoires. Some antibodies acquire polyreactivity post-translationally after interaction with various redox-active substances, including heme. Recently we have demonstrated that heme binding to a naturally polyreactive antibody (SPE7) results in a considerable broadening of the repertoire of recognized antigens. A question remains whether the presence of certain level of natural polyreactivity of antibodies is a prerequisite for heme-induced further extension of antigen binding potential. Here we used a second monoclonal antibody (Hg32) with unknown specificity and absence of intrinsic polyreactivity as a model to study the potential of heme to induce polyreactivitymore » of antibodies. We demonstrated that exposure to heme greatly extends the antigen binding potential of Hg32, suggesting that the intrinsic binding promiscuity is not a prerequisite for the induction of polyreactivity by heme. In addition we compared the kinetics and thermodynamics of the interaction of heme-exposed antibodies with a panel of unrelated antigens. These analyses revealed that the two heme-sensitive antibodies adopt different mechanisms of binding to the same set of antigens. This study contributes to understanding the phenomenon of induced antibody polyreactivity. The data may also be of importance for understanding of physiological and pathological roles of polyreactive antibodies. - Highlights: • Exposure of certain monoclonal IgE antibodies to heme results in gain of antigen binding polyreactivity. • Natural polyreactivity of antibodies is dispensable for acquisition of polyreactivity through interaction with heme. • Heme-induced monoclonal IgE antibodies differ in their thermodynamic mechanisms of antigen recognition.« less

Facile Affinity Maturation of Antibody Variable Domains Using Natural Diversity Mutagenesis

PubMed Central

Tiller, Kathryn E.; Chowdhury, Ratul; Li, Tong; Ludwig, Seth D.; Sen, Sabyasachi; Maranas, Costas D.; Tessier, Peter M.

2017-01-01

The identification of mutations that enhance antibody affinity while maintaining high antibody specificity and stability is a time-consuming and laborious process. Here, we report an efficient methodology for systematically and rapidly enhancing the affinity of antibody variable domains while maximizing specificity and stability using novel synthetic antibody libraries. Our approach first uses computational and experimental alanine scanning mutagenesis to identify sites in the complementarity-determining regions (CDRs) that are permissive to mutagenesis while maintaining antigen binding. Next, we mutagenize the most permissive CDR positions using degenerate codons to encode wild-type residues and a small number of the most frequently occurring residues at each CDR position based on natural antibody diversity. This mutagenesis approach results in antibody libraries with variants that have a wide range of numbers of CDR mutations, including antibody domains with single mutations and others with tens of mutations. Finally, we sort the modest size libraries (~10 million variants) displayed on the surface of yeast to identify CDR mutations with the greatest increases in affinity. Importantly, we find that single-domain (VHH) antibodies specific for the α-synuclein protein (whose aggregation is associated with Parkinson’s disease) with the greatest gains in affinity (>5-fold) have several (four to six) CDR mutations. This finding highlights the importance of sampling combinations of CDR mutations during the first step of affinity maturation to maximize the efficiency of the process. Interestingly, we find that some natural diversity mutations simultaneously enhance all three key antibody properties (affinity, specificity, and stability) while other mutations enhance some of these properties (e.g., increased specificity) and display trade-offs in others (e.g., reduced affinity and/or stability). Computational modeling reveals that improvements in affinity are generally not due to direct interactions involving CDR mutations but rather due to indirect effects that enhance existing interactions and/or promote new interactions between the antigen and wild-type CDR residues. We expect that natural diversity mutagenesis will be useful for efficient affinity maturation of a wide range of antibody fragments and full-length antibodies. PMID:28928732

An efficient method for variable region assembly in the construction of scFv phage display libraries using independent strand amplification

PubMed Central

Sotelo, Pablo H.; Collazo, Noberto; Zuñiga, Roberto; Gutiérrez-González, Matías; Catalán, Diego; Ribeiro, Carolina Hager; Aguillón, Juan Carlos; Molina, María Carmen

2012-01-01

Phage display library technology is a common method to produce human antibodies. In this technique, the immunoglobulin variable regions are displayed in a bacteriophage in a way that each filamentous virus displays the product of a single antibody gene on its surface. From the collection of different phages, it is possible to isolate the virus that recognizes specific targets. The most common form in which to display antibody variable regions in the phage is the single chain variable fragment format (scFv), which requires assembly of the heavy and light immunoglobulin variable regions in a single gene. In this work, we describe a simple and efficient method for the assembly of immunoglobulin heavy and light chain variable regions in a scFv format. This procedure involves a two-step reaction: (1) DNA amplification to produce the single strand form of the heavy or light chain gene required for the fusion; and (2) mixture of both single strand products followed by an assembly reaction to construct a complete scFv gene. Using this method, we produced 6-fold more scFv encoding DNA than the commonly used splicing by overlap extension PCR (SOE-PCR) approach. The scFv gene produced by this method also proved to be efficient in generating a diverse scFv phage display library. From this scFv library, we obtained phages that bound several non-related antigens, including recombinant proteins and rotavirus particles. PMID:22692130

Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display.

PubMed

Yuan, Qing; Jordan, Ramon; Brlansky, Ronald H; Istomina, Olga; Hartung, John

2015-10-01

Xylella fastidiosa is a member of the gamma proteobacteria. It is fastidious, insect-vectored and xylem-limited and causes a variety of diseases, some severe, on a wide range of economically important perennial crops, including grape and citrus. Antibody based detection assays are commercially available for X. fastidiosa, and are effective at the species, but not at the subspecies level. We have made a library of scFv antibody fragments directed against X. fastidiosa subsp. pauca strain 9a5c (citrus) by using phage display technology. Antibody gene repertoires were PCR-amplified using 23 primers for the heavy chain variable region (V(H)) and 21 primers for the light chain variable region (V(L)). The V(H) and V(L) were joined by overlap extension PCR, and then the genes of the scFv library were ligated into the phage vector pKM19. The library contained 1.2×10(7) independent clones with full-length scFv inserts. In each of 3cycles of affinity-selection with 9a5c, about 1.0×10(12) phage were used for panning with 4.1×10(6), 7.1×10(6), 2.1×10(7) phage recovered after the first, second and third cycles, respectively. Sixty-six percent of clones from the final library bound X. fastidiosa 9a5c in an ELISA. Some of these scFv antibodies recognized strain 9a5c and did not recognize X. fastidiosa strains that cause Pierce's disease of grapevine. Published by Elsevier B.V.

Fab-based bispecific antibody formats with robust biophysical properties and biological activity.

PubMed

Wu, Xiufeng; Sereno, Arlene J; Huang, Flora; Lewis, Steven M; Lieu, Ricky L; Weldon, Caroline; Torres, Carina; Fine, Cody; Batt, Micheal A; Fitchett, Jonathan R; Glasebrook, Andrew L; Kuhlman, Brian; Demarest, Stephen J

2015-01-01

A myriad of innovative bispecific antibody (BsAb) platforms have been reported. Most require significant protein engineering to be viable from a development and manufacturing perspective. Single-chain variable fragments (scFvs) and diabodies that consist only of antibody variable domains have been used as building blocks for making BsAbs for decades. The drawback with Fv-only moieties is that they lack the native-like interactions with CH1/CL domains that make antibody Fab regions stable and soluble. Here, we utilize a redesigned Fab interface to explore 2 novel Fab-based BsAbs platforms. The redesigned Fab interface designs limit heavy and light chain mixing when 2 Fabs are co-expressed simultaneously, thus allowing the use of 2 different Fabs within a BsAb construct without the requirement of one or more scFvs. We describe the stability and activity of a HER2×HER2 IgG-Fab BsAb, and compare its biophysical and activity properties with those of an IgG-scFv that utilizes the variable domains of the same parental antibodies. We also generated an EGFR × CD3 tandem Fab protein with a similar format to a tandem scFv (otherwise known as a bispecific T cell engager or BiTE). We show that the Fab-based BsAbs have superior biophysical properties compared to the scFv-based BsAbs. Additionally, the Fab-based BsAbs do not simply recapitulate the activity of their scFv counterparts, but are shown to possess unique biological activity.

Fab-based bispecific antibody formats with robust biophysical properties and biological activity

PubMed Central

Wu, Xiufeng; Sereno, Arlene J; Huang, Flora; Lewis, Steven M; Lieu, Ricky L; Weldon, Caroline; Torres, Carina; Fine, Cody; Batt, Micheal A; Fitchett, Jonathan R; Glasebrook, Andrew L; Kuhlman, Brian; Demarest, Stephen J

2015-01-01

A myriad of innovative bispecific antibody (BsAb) platforms have been reported. Most require significant protein engineering to be viable from a development and manufacturing perspective. Single-chain variable fragments (scFvs) and diabodies that consist only of antibody variable domains have been used as building blocks for making BsAbs for decades. The drawback with Fv-only moieties is that they lack the native-like interactions with CH1/CL domains that make antibody Fab regions stable and soluble. Here, we utilize a redesigned Fab interface to explore 2 novel Fab-based BsAbs platforms. The redesigned Fab interface designs limit heavy and light chain mixing when 2 Fabs are co-expressed simultaneously, thus allowing the use of 2 different Fabs within a BsAb construct without the requirement of one or more scFvs. We describe the stability and activity of a HER2×HER2 IgG-Fab BsAb, and compare its biophysical and activity properties with those of an IgG-scFv that utilizes the variable domains of the same parental antibodies. We also generated an EGFR × CD3 tandem Fab protein with a similar format to a tandem scFv (otherwise known as a bispecific T cell engager or BiTE). We show that the Fab-based BsAbs have superior biophysical properties compared to the scFv-based BsAbs. Additionally, the Fab-based BsAbs do not simply recapitulate the activity of their scFv counterparts, but are shown to possess unique biological activity. PMID:25774965

Palladium-109 labeled anti-melanoma monoclonal antibodies

DOEpatents

Srivastava, S.C.; Fawwaz, R.A.; Ferrone, S.

1984-04-30

The invention consists of new monoclonal antibodies labelled with Palladium 109, a beta-emitting radionuclide, the method of preparing this material, and its use in the radiotherapy of melanoma. The antibodies are chelate-conjugated and demonstrate a high uptake in melanomas. (ACR)

Single-Domain Antibodies and the Promise of Modular Targeting in Cancer Imaging and Treatment

PubMed Central

Iezzi, María Elena; Policastro, Lucía; Werbajh, Santiago; Podhajcer, Osvaldo; Canziani, Gabriela Alicia

2018-01-01

Monoclonal antibodies and their fragments have significantly changed the outcome of cancer in the clinic, effectively inhibiting tumor cell proliferation, triggering antibody-dependent immune effector cell activation and complement mediated cell death. Along with a continued expansion in number, diversity, and complexity of validated tumor targets there is an increasing focus on engineering recombinant antibody fragments for lead development. Single-domain antibodies (sdAbs), in particular those engineered from the variable heavy-chain fragment (VHH gene) found in Camelidae heavy-chain antibodies (or IgG2 and IgG3), are the smallest fragments that retain the full antigen-binding capacity of the antibody with advantageous properties as drugs. For similar reasons, growing attention is being paid to the yet smaller variable heavy chain new antigen receptor (VNAR) fragments found in Squalidae. sdAbs have been selected, mostly from immune VHH libraries, to inhibit or modulate enzyme activity, bind soluble factors, internalize cell membrane receptors, or block cytoplasmic targets. This succinct review is a compilation of recent data documenting the application of engineered, recombinant sdAb in the clinic as epitope recognition “modules” to build monomeric, dimeric and multimeric ligands that target, tag and stall solid tumor growth in vivo. Size, affinity, specificity, and the development profile of sdAbs drugs are seemingly consistent with desirable clinical efficacy and safety requirements. But the hepatotoxicity of the tetrameric anti-DR5-VHH drug in patients with pre-existing anti-drug antibodies halted the phase I clinical trial and called for a thorough pre-screening of the immune and poly-specific reactivities of the sdAb leads. PMID:29520274

Evaluating the Use of Antibody Variable Region (Fv) Charge as a Risk Assessment Tool for Predicting Typical Cynomolgus Monkey Pharmacokinetics*

PubMed Central

Bumbaca Yadav, Daniela; Sharma, Vikas K.; Boswell, Charles Andrew; Hotzel, Isidro; Tesar, Devin; Shang, Yonglei; Ying, Yong; Fischer, Saloumeh K.; Grogan, Jane L.; Chiang, Eugene Y.; Urban, Konnie; Ulufatu, Sheila; Khawli, Leslie A.; Prabhu, Saileta; Joseph, Sean; Kelley, Robert F.

2015-01-01

The pharmacokinetic (PK) behavior of monoclonal antibodies in cynomolgus monkeys (cynos) is generally translatable to that in humans. Unfortunately, about 39% of the antibodies evaluated for PKs in cynos have fast nonspecific (or non-target-mediated) clearance (in-house data). An empirical model relating variable region (Fv) charge and hydrophobicity to cyno nonspecific clearance was developed to gauge the risk an antibody would have for fast nonspecific clearance in the monkey. The purpose of this study was to evaluate the predictability of this empirical model on cyno nonspecific clearance with antibodies specifically engineered to have either high or low Fv charge. These amino acid changes were made in the Fv region of two test antibodies, humAb4D5-8 and anti-lymphotoxin α. The humAb4D5-8 has a typical nonspecific clearance in cynos, and by making it more positively charged, the antibody acquires fast nonspecific clearance, and making it less positively charged did not impact its clearance. Anti-lymphotoxin α has fast nonspecific clearance in cynos, and making it more positively charged caused it to clear even faster, whereas making it less positively charged caused it to clear slower and within the typical range. These trends in clearance were also observed in two other preclinical species, mice and rats. The effect of modifying Fv charge on subcutaneous bioavailability was also examined, and in general bioavailability was inversely related to the direction of the Fv charge change. Thus, modifying Fv charge appears to impact antibody PKs, and the changes tended to correlate with those predicted by the empirical model. PMID:26491012

A murine monoclonal antibody based enzyme-linked immunosorbent assay for almond (Prunus dulcis L.) detection.

PubMed

Su, Mengna; Venkatachalam, Mahesh; Liu, Changqi; Zhang, Ying; Roux, Kenneth H; Sathe, Shridhar K

2013-11-13

A sandwich enzyme-linked immunosorbent assay (ELISA) using anti-almond soluble protein rabbit polyclonal antibodies as capture antibodies and murine monoclonal antibody 4C10 as the detection antibodies was developed. The assay is specific and sensitive (3-200 ng almond protein/mL) for almond detection. The standardized assay is accurate (<15% CV) and reproducible (intra- and inter assay variability <15% CV). The assay did not register any cross-reactivity with the tested food matrices, suggesting the assay to be almond amandin specific. The assay could detect the presence of declared almond in the tested matched commercial samples. Further, the assay reliably detected the presence of almonds in the laboratory prepared food samples spiked with almond flour.

Immunological aspects of Giardia infections

PubMed Central

Heyworth, Martin F.

2014-01-01

Immunodeficiency, particularly antibody deficiency, predisposes to increased intensity and persistence of Giardia infections. Giardia-infected immunocompetent hosts produce serum and intestinal antibodies against Giardia trophozoites. The number of Giardia muris trophozoites, in mice with G. muris infection, is reduced by intra-duodenal administration of anti-G. muris antibody. Giardia intestinalis antigens that are recognised by human anti-trophozoite antibodies include variable (variant-specific) and invariant proteins. Nitric oxide (NO) appears to contribute to host clearance of Giardia trophozoites. Arginine is a precursor of NO and is metabolised by Giardia trophozoites, possibly reducing its availability for generation of NO by the host. Work with mice suggests that T lymphocytes and interleukin-6 (IL-6) contribute to clearance of Giardia infection via mechanisms independent of antibodies. PMID:25347704

Theoretic criteria for antibody penetration into solid tumors and micrometastases.

PubMed

Thurber, Greg M; Zajic, Stefan C; Wittrup, K Dane

2007-06-01

Targeting tumors with antibody-based therapeutics is a complex task presenting multiple kinetic barriers. Antibody internalization and clearance inhibit uptake both in solid tumors, limited by tumor vascular permeability, and in micrometastases, limited by diffusion. A modeling exercise is used to introduce 2 simple criteria that must be less than unity for saturation of both tumors and micrometastases. The clearance modulus and the Thiele modulus are ratios of the plasma clearance rate and antibody catabolism, respectively, to the tumor tissue penetration rate. Even low rates of antigen internalization from constitutive membrane turnover can significantly retard antibody penetration. Rapid clearance of single-chain variable fragments also hinders uptake, often more than counterbalancing their more rapid extravasation and diffusion. The model illustrates that with the large resistance from the tumor capillary, antibodies may be more suitable for targeting micrometastases than vascularized tumors.

Virus diseases of the salmonidae in the western United States. III. Immunopathological aspects

USGS Publications Warehouse

Klontz, George W.; Yasutake, William T.; Parisot, T.J.

1965-01-01

The immune response among fish, from a phylogenetic standpoint, presents a progressive pattern of increasing development. The cyclostomes have been shown to have only feeble immunologic responsiveness. One of their number, the hagfish, appeared to be totally lacking in the ability to actively acquire antibodies.Among the elasmobranchs, the sharks have received the most study immunologically. This group demonstrated a variable response to antigenic stimulationOf the teleosts, the salmonids and the cyprinids have been the more frequent recipients of experimentally introduced antigens. These fishes, as well as other species of teleosts, are quite active and quite consistent in their response to various antigens.

Investigation of within- and between-herd variability of bovine leukaemia virus bulk tank milk antibody levels over different sampling intervals in the Canadian Maritimes.

PubMed

John, Emily E; Nekouei, Omid; McClure, J T; Cameron, Marguerite; Keefe, Greg; Stryhn, Henrik

2018-06-01

Bulk tank milk (BTM) samples are used to determine the infection status and estimate dairy herd prevalence for bovine leukaemia virus (BLV) using an antibody ELISA assay. BLV ELISA variability between samples from the same herd or from different herds has not been investigated over long time periods. The main objective of this study was to determine the within-herd and between-herd variability of a BTM BLV ELISA assay over 1-month, 3-month, and 3-year sampling intervals. All of the Canadian Maritime region dairy herds (n = 523) that were active in 2013 and 2016 were included (83.9% and 86.9% of total herds in 2013 and 2016, respectively). BLV antibody levels were measured in three BTM samples collected at 1-month intervals in early 2013 as well as two BTM samples collected over a 3-month interval in early 2016. Random-effects models, with fixed effects for sample replicate and province and random effects for herd, were used to estimate the variability between BTM samples from the same herd and between herds for 1-month, 3-month, and 3-year sampling intervals. The majority of variability of BTM BLV ELISA results was seen between herds (1-month, 6.792 ± 0.533; 3-month, 7.806 ± 0.652; 3-year, 6.222 ± 0.528). Unexplained variance between samples from the same herd, on square-root scale, was greatest for the 3-year (0.976 ± 0.104), followed by the 1-month (0.611 ± 0.035) then the 3-month (0.557 ± 0.071) intervals. Variability of BTM antibody levels within the same herd was present but was much smaller than the variability between herds, and was greatest for the 3-year sampling interval. The 3-month sampling interval resulted in the least variability and is appropriate to use for estimating the baseline level of within-herd prevalence for BLV control programs. Knowledge of the baseline variability and within-herd prevalence can help to determine effectiveness of control programs when BTM sampling is repeated at longer intervals. Copyright © 2018 Elsevier B.V. All rights reserved.

Alveolar macrophages are critical for broadly-reactive antibody-mediated protection against influenza A virus in mice.

PubMed

He, Wenqian; Chen, Chi-Jene; Mullarkey, Caitlin E; Hamilton, Jennifer R; Wong, Christine K; Leon, Paul E; Uccellini, Melissa B; Chromikova, Veronika; Henry, Carole; Hoffman, Kevin W; Lim, Jean K; Wilson, Patrick C; Miller, Matthew S; Krammer, Florian; Palese, Peter; Tan, Gene S

2017-10-10

The aim of candidate universal influenza vaccines is to provide broad protection against influenza A and B viruses. Studies have demonstrated that broadly reactive antibodies require Fc-Fc gamma receptor interactions for optimal protection; however, the innate effector cells responsible for mediating this protection remain largely unknown. Here, we examine the roles of alveolar macrophages, natural killer cells, and neutrophils in antibody-mediated protection. We demonstrate that alveolar macrophages play a dominant role in conferring protection provided by both broadly neutralizing and non-neutralizing antibodies in mice. Our data also reveal the potential mechanisms by which alveolar macrophages mediate protection in vivo, namely antibody-induced inflammation and antibody-dependent cellular phagocytosis. This study highlights the importance of innate effector cells in establishing a broad-spectrum antiviral state, as well as providing a better understanding of how multiple arms of the immune system cooperate to achieve an optimal antiviral response following influenza virus infection or immunization.Broadly reactive antibodies that recognize influenza A virus HA can be protective, but the mechanism is not completely understood. Here, He et al. show that the inflammatory response and phagocytosis mediated by the interaction between protective antibodies and macrophages are essential for protection.

Aging and the Immune Response to the Haemophilus influenzae Type b Capsular Polysaccharide: Retention of the Dominant Idiotype and Antibody Function in the Elderly

PubMed Central

Lucas, Alexander H.; Reason, Donald C.

1998-01-01

Anti-Haemophilus influenzae b polysaccharide (Hib PS) antibodies elicited in elderly subjects following conjugate vaccination expressed a light-chain variable-region (VL)-associated idiotype and had functional activities similar to those previously observed in children and younger adults. These findings indicate that advanced age is not accompanied by shifts in the major VL component of the Hib PS-specific repertoire or by diminution of the protective function of antibodies. PMID:9529108

Simplifying complex sequence information: a PCP-consensus protein binds antibodies against all four Dengue serotypes.

PubMed

Bowen, David M; Lewis, Jessica A; Lu, Wenzhe; Schein, Catherine H

2012-09-14

Designing proteins that reflect the natural variability of a pathogen is essential for developing novel vaccines and drugs. Flaviviruses, including Dengue (DENV) and West Nile (WNV), evolve rapidly and can "escape" neutralizing monoclonal antibodies by mutation. Designing antigens that represent many distinct strains is important for DENV, where infection with a strain from one of the four serotypes may lead to severe hemorrhagic disease on subsequent infection with a strain from another serotype. Here, a DENV physicochemical property (PCP)-consensus sequence was derived from 671 unique sequences from the Flavitrack database. PCP-consensus proteins for domain 3 of the envelope protein (EdomIII) were expressed from synthetic genes in Escherichia coli. The ability of the purified consensus proteins to bind polyclonal antibodies generated in response to infection with strains from each of the four DENV serotypes was determined. The initial consensus protein bound antibodies from DENV-1-3 in ELISA and Western blot assays. This sequence was altered in 3 steps to incorporate regions of maximum variability, identified as significant changes in the PCPs, characteristic of DENV-4 strains. The final protein was recognized by antibodies against all four serotypes. Two amino acids essential for efficient binding to all DENV antibodies are part of a discontinuous epitope previously defined for a neutralizing monoclonal antibody. The PCP-consensus method can significantly reduce the number of experiments required to define a multivalent antigen, which is particularly important when dealing with pathogens that must be tested at higher biosafety levels. Copyright © 2012 Elsevier Ltd. All rights reserved.

Combining phage display with de novo protein sequencing for reverse engineering of monoclonal antibodies.

PubMed

Rickert, Keith W; Grinberg, Luba; Woods, Robert M; Wilson, Susan; Bowen, Michael A; Baca, Manuel

2016-01-01

The enormous diversity created by gene recombination and somatic hypermutation makes de novo protein sequencing of monoclonal antibodies a uniquely challenging problem. Modern mass spectrometry-based sequencing will rarely, if ever, provide a single unambiguous sequence for the variable domains. A more likely outcome is computation of an ensemble of highly similar sequences that can satisfy the experimental data. This outcome can result in the need for empirical testing of many candidate sequences, sometimes iteratively, to identity one which can replicate the activity of the parental antibody. Here we describe an improved approach to antibody protein sequencing by using phage display technology to generate a combinatorial library of sequences that satisfy the mass spectrometry data, and selecting for functional candidates that bind antigen. This approach was used to reverse engineer 2 commercially-obtained monoclonal antibodies against murine CD137. Proteomic data enabled us to assign the majority of the variable domain sequences, with the exception of 3-5% of the sequence located within or adjacent to complementarity-determining regions. To efficiently resolve the sequence in these regions, small phage-displayed libraries were generated and subjected to antigen binding selection. Following enrichment of antigen-binding clones, 2 clones were selected for each antibody and recombinantly expressed as antigen-binding fragments (Fabs). In both cases, the reverse-engineered Fabs exhibited identical antigen binding affinity, within error, as Fabs produced from the commercial IgGs. This combination of proteomic and protein engineering techniques provides a useful approach to simplifying the technically challenging process of reverse engineering monoclonal antibodies from protein material.

Combining phage display with de novo protein sequencing for reverse engineering of monoclonal antibodies

PubMed Central

Rickert, Keith W.; Grinberg, Luba; Woods, Robert M.; Wilson, Susan; Bowen, Michael A.; Baca, Manuel

2016-01-01

ABSTRACT The enormous diversity created by gene recombination and somatic hypermutation makes de novo protein sequencing of monoclonal antibodies a uniquely challenging problem. Modern mass spectrometry-based sequencing will rarely, if ever, provide a single unambiguous sequence for the variable domains. A more likely outcome is computation of an ensemble of highly similar sequences that can satisfy the experimental data. This outcome can result in the need for empirical testing of many candidate sequences, sometimes iteratively, to identity one which can replicate the activity of the parental antibody. Here we describe an improved approach to antibody protein sequencing by using phage display technology to generate a combinatorial library of sequences that satisfy the mass spectrometry data, and selecting for functional candidates that bind antigen. This approach was used to reverse engineer 2 commercially-obtained monoclonal antibodies against murine CD137. Proteomic data enabled us to assign the majority of the variable domain sequences, with the exception of 3–5% of the sequence located within or adjacent to complementarity-determining regions. To efficiently resolve the sequence in these regions, small phage-displayed libraries were generated and subjected to antigen binding selection. Following enrichment of antigen-binding clones, 2 clones were selected for each antibody and recombinantly expressed as antigen-binding fragments (Fabs). In both cases, the reverse-engineered Fabs exhibited identical antigen binding affinity, within error, as Fabs produced from the commercial IgGs. This combination of proteomic and protein engineering techniques provides a useful approach to simplifying the technically challenging process of reverse engineering monoclonal antibodies from protein material. PMID:26852694

Proteomic Identification of Monoclonal Antibodies from Serum

PubMed Central

2015-01-01

Characterizing the in vivo dynamics of the polyclonal antibody repertoire in serum, such as that which might arise in response to stimulation with an antigen, is difficult due to the presence of many highly similar immunoglobulin proteins, each specified by distinct B lymphocytes. These challenges have precluded the use of conventional mass spectrometry for antibody identification based on peptide mass spectral matches to a genomic reference database. Recently, progress has been made using bottom-up analysis of serum antibodies by nanoflow liquid chromatography/high-resolution tandem mass spectrometry combined with a sample-specific antibody sequence database generated by high-throughput sequencing of individual B cell immunoglobulin variable domains (V genes). Here, we describe how intrinsic features of antibody primary structure, most notably the interspersed segments of variable and conserved amino acid sequences, generate recurring patterns in the corresponding peptide mass spectra of V gene peptides, greatly complicating the assignment of correct sequences to mass spectral data. We show that the standard method of decoy-based error modeling fails to account for the error introduced by these highly similar sequences, leading to a significant underestimation of the false discovery rate. Because of these effects, antibody-derived peptide mass spectra require increased stringency in their interpretation. The use of filters based on the mean precursor ion mass accuracy of peptide-spectrum matches is shown to be particularly effective in distinguishing between “true” and “false” identifications. These findings highlight important caveats associated with the use of standard database search and error-modeling methods with nonstandard data sets and custom sequence databases. PMID:24684310

Avian Diagnostic and Therapeutic Antibodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bradley, David Sherman

2012-12-31

A number of infectious agents have the potential of causing significant clinical symptomology and even death, but dispite this, the number of incidence remain below the level that supports producing a vaccine. Therapeutic antibodies provide a viable treatment option for many of these diseases. We proposed that antibodies derived from West Nile Virus (WNV) immunized geese would be able to treat WNV infection in mammals and potential humans. We demonstrated that WNV specific goose antibodies are indeed successful in treating WNV infection both prophylactically and therapeutically in a golden hamster model. We demonstrated that the goose derived antibodies are non-reactogenic,more » i.e. do not cause an inflammatory response with multiple exposures in mammals. We also developed both a specific pathogen free facility to house the geese during the antibody production phase and a patent-pending purification process to purify the antibodies to greater than 99% purity. Therefore, the success of these study will allow a cost effective rapidly producible therapeutic toward clinical testing with the necessary infrastructure and processes developed and in place.« less

Macrophage-mediated trogocytosis leads to death of antibody-opsonized tumor cells

PubMed Central

Velmurugan, Ramraj; Challa, Dilip K.; Ram, Sripad; Ober, Raimund J.; Ward, E. Sally

2016-01-01

Understanding the complex behavior of effector cells such as monocytes or macrophages in regulating cancerous growth is of central importance for cancer immunotherapy. Earlier studies using CD20-specific antibodies have demonstrated that the Fcγ receptor (FcγR)-mediated transfer of the targeted receptors from tumor cells to these effector cells through trogocytosis can enable escape from antibody therapy, leading to the viewpoint that this process is pro-tumorigenic. In the current study we demonstrate that persistent trogocytic attack results in the killing of HER2-overexpressing breast cancer cells. Further, antibody engineering to increase FcγR interactions enhances this tumoricidal activity. These studies extend the complex repertoire of activities of macrophages to trogocytic-mediated cell death of HER2-overexpressing target cells and have implications for the development of effective antibody-based therapies. PMID:27226489

Development of β-Lactoglobulin-Specific Chimeric Human IgEκ Monoclonal Antibodies for In Vitro Safety Assessment of Whey Hydrolysates

PubMed Central

Buelens-Sleumer, Laura S.; Cox, Linda; den Hartog, Marcel; de Jong, Niels; Teshima, Reiko; Garssen, Johan; Boon, Louis; Knippels, Léon M. J.

2014-01-01

Background Cow’s milk-derived whey hydrolysates are nutritional substitutes for allergic infants. Safety or residual allergenicity assessment of these whey hydrolysates is crucial. Currently, rat basophilic leukemia RBL-2H3 cells expressing the human IgE receptor α-chain (huFcεRIα-RBL-2H3), sensitized with serum IgE from cow’s milk allergic children, are being employed to assess in vitro residual allergenicity of these whey hydrolysates. However, limited availability and inter-lot variation of these allergic sera impede standardization of whey hydrolysate safety testing in degranulation assays. Objective An oligoclonal pool of chimeric human (chu)IgE antibodies against bovine β-lactoglobulin (a major allergen in whey) was generated to increase sensitivity, specificity, and reproducibility of existing degranulation assays. Methods Mice were immunized with bovine β-lactoglobulin, and subsequently the variable domains of dissimilar anti-β-lactoglobulin mouse IgG antibodies were cloned and sequenced. Six chimeric antibodies were generated comprising mouse variable domains and human constant IgE/κ domains. Results After sensitization with this pool of anti-β-lactoglobulin chuIgEs, huFcεRIα-expressing RBL-2H3 cells demonstrated degranulation upon cross-linking with whey, native 18 kDa β-lactoglobulin, and 5–10 kDa whey hydrolysates, whereas a 3 kDa whey hydrolysate and cow’s milk powder (mainly casein) showed no degranulation. In parallel, allergic serum IgEs were less sensitive. In addition, our pool anti-β-lactoglobulin chuIgEs recognized multiple allergenic immunodominant regions on β-lactoglobulin, which were also recognized by serum IgEs from cow’s milk allergic children. Conclusion Usage of our ‘unlimited’ source and well-defined pool of β-lactoglobulin-specific recombinant chuIgEs to sensitize huFcεRIα on RBL-2H3 cells showed to be a relevant and sensitive alternative for serum IgEs from cow’s milk allergic patients to assess safety of whey-based non-allergic hydrolyzed formula. PMID:25153680

A Diverse Repertoire of Human Immunoglobulin Variable Genes in a Chicken B Cell Line is Generated by Both Gene Conversion and Somatic Hypermutation.

PubMed

Leighton, Philip A; Schusser, Benjamin; Yi, Henry; Glanville, Jacob; Harriman, William

2015-01-01

Chicken immune responses to human proteins are often more robust than rodent responses because of the phylogenetic relationship between the different species. For discovery of a diverse panel of unique therapeutic antibody candidates, chickens therefore represent an attractive host for human-derived targets. Recent advances in monoclonal antibody technology, specifically new methods for the molecular cloning of antibody genes directly from primary B cells, has ushered in a new era of generating monoclonal antibodies from non-traditional host animals that were previously inaccessible through hybridoma technology. However, such monoclonals still require post-discovery humanization in order to be developed as therapeutics. To obviate the need for humanization, a modified strain of chickens could be engineered to express a human-sequence immunoglobulin variable region repertoire. Here, human variable genes introduced into the chicken immunoglobulin loci through gene targeting were evaluated for their ability to be recognized and diversified by the native chicken recombination machinery that is present in the B-lineage cell line DT40. After expansion in culture the DT40 population accumulated genetic mutants that were detected via deep sequencing. Bioinformatic analysis revealed that the human targeted constructs are performing as expected in the cell culture system, and provide a measure of confidence that they will be functional in transgenic animals.

Cloning and molecular characterization of the cDNAs encoding the variable regions of an anti-CD20 monoclonal antibody.

PubMed

Shanehbandi, Dariush; Majidi, Jafar; Kazemi, Tohid; Baradaran, Behzad; Aghebati-Maleki, Leili

2017-01-01

CD20-based targeting of B-cells in hematologic malignancies and autoimmune disorders is associated with outstanding clinical outcomes. Isolation and characterization of VH and VL cDNAs encoding the variable regions of the heavy and light chains of monoclonal antibodies (MAb) is necessary to produce next generation MAbs and their derivatives such as bispecific antibodies (bsAb) and single-chain variable fragments (scFv). This study was aimed at cloning and characterization of the VH and VL cDNAs from a hybridoma cell line producing an anti-CD20 MAb. VH and VL fragments were amplified, cloned and characterized. Furthermore, amino acid sequences of VH, VL and corresponding complementarity-determining regions (CDR) were determined and compared with those of four approved MAbs including Rituximab (RTX), Ibritumomab tiuxetan, Ofatumumab and GA101. The cloned VH and VL cDNAs were found to be functional and follow a consensus pattern. Amino acid sequences corresponding to the VH and VL fragments also indicated noticeable homologies to those of RTX and Ibritumomab. Furthermore, amino acid sequences of the relating CDRs had remarkable similarities to their counterparts in RTX and Ibritumomab. Successful recovery of VH and VL fragments encourages the development of novel CD20 targeting bsAbs, scFvs, antibody conjugates and T-cells armed with chimeric antigen receptors.

Simple Objective Detection of Human Lyme Disease Infection Using Immuno-PCR and a Single Recombinant Hybrid Antigen

PubMed Central

Halpern, Micah D.; Molins, Claudia R.; Schriefer, Martin

2014-01-01

A serology-based tiered approach has, to date, provided the most effective means of laboratory confirmation of clinically suspected cases of Lyme disease, but it lacks sensitivity in the early stages of disease and is often dependent on subjectively scored immunoblots. We recently demonstrated the use of immuno-PCR (iPCR) for detecting Borrelia burgdorferi antibodies in patient serum samples that were positive for Lyme disease. To better understand the performance of the Lyme disease iPCR assay, the repeatability and variability of the background of the assay across samples from a healthy population (n = 36) were analyzed. Both of these parameters were found to have coefficients of variation of <3%. Using eight antigen-specific iPCR assays and positive call thresholds established for each assay, iPCR IgM and/or IgG diagnosis from Lyme disease patient serum samples (n = 12) demonstrated a strong correlation with that of 2-tier testing. Furthermore, a simplified iPCR approach using a single hybrid antigen and detecting only IgG antibodies confirmed the 2-tier diagnosis in the Lyme disease patient serum samples (n = 12). Validation of the hybrid antigen IgG iPCR assay using a blinded panel of Lyme disease and non-Lyme disease patient serum samples (n = 92) resulted in a sensitivity of 69% (95% confidence interval [CI], 50% to 84%), compared to that of the 2-tier analysis at 59% (95% CI, 41% to 76%), and a specificity of 98% (95% CI, 91% to 100%) compared to that of the 2-tier analysis at 97% (95% CI, 88% to 100%). A single-tier hybrid antigen iPCR assay has the potential to be an improved method for detecting host-generated antibodies against B. burgdorferi. PMID:24899074

Structural and functional characterization of an anti-West Nile virus monoclonal antibody and its single-chain variant produced in glycoengineered plants

PubMed Central

Lai, Huafang; He, Junyun; Hurtado, Jonathan; Stahnke, Jake; Fuchs, Anja; Mehlhop, Erin; Gorlatov, Sergey; Loos, Andreas; Diamond, Michael S.; Chen, Qiang

2014-01-01

Previously, our group engineered a plant-derived monoclonal antibody (MAb pE16) that efficiently treated West Nile virus (WNV) infection in mice. In this study, we developed a pE16 variant consisting of a single-chain variable fragment (scFv) fused to the heavy chain constant domains (CH) of human IgG (pE16scFv-CH). pE16 and pE16scFv-CH were expressed and assembled efficiently in Nicotiana benthamiana ΔXF plants, a glycosylation mutant lacking plant specific N-glycan residues. Glycan analysis revealed that ΔXF plant-derived pE16scFv-CH (ΔXFpE16scFv-CH) and pE16 (ΔXFpE16) both displayed a mammalian glycosylation profile. ΔXFpE16 and ΔXFpE16scFv-CH demonstrated equivalent antigen binding affinity and kinetics, and slightly enhanced neutralization of WNV in vitro compared to the parent mammalian cell-produced E16 (mE16). A single dose of ΔXFpE16 or ΔXFpE16scFv-CH protected mice against WNV-induced mortality even 4 days after infection at equivalent rates as mE16. This study provides a detailed tandem comparison of the expression, structure and function of a therapeutic MAb and its single-chain variant produced in glycoengineered plants. Moreover, it demonstrates the development of anti-WNV MAb therapeutic variants that are equivalent in efficacy to pE16, simpler to produce, and likely safer to use as therapeutics due to their mammalian N-glycosylation. This platform may lead to a more robust and cost effective production of antibody-based therapeutics against WNV infection and other infectious, inflammatory, or neoplastic diseases. PMID:24975464

Validation of an enzyme-linked immunosorbent assay for the quantification of citrullinated histone H3 as a marker for neutrophil extracellular traps in human plasma.

PubMed

Thålin, Charlotte; Daleskog, Maud; Göransson, Sophie Paues; Schatzberg, Daphne; Lasselin, Julie; Laska, Ann-Charlotte; Kallner, Anders; Helleday, Thomas; Wallén, Håkan; Demers, Mélanie

2017-06-01

There is an emerging interest in the diverse functions of neutrophil extracellular traps (NETs) in a variety of disease settings. However, data on circulating NETs rely largely upon surrogate NET markers such as cell-free DNA, nucleosomes, and NET-associated enzymes. Citrullination of histone H3 by peptidyl arginine deiminase 4 (PAD4) is central for NET formation, and citrullinated histone H3 (H3Cit) is considered a NET-specific biomarker. We therefore aimed to optimize and validate a new enzyme-linked immunosorbent assay (ELISA) to quantify the levels of H3Cit in human plasma. A standard curve made of in vitro PAD4-citrullinated histones H3 allows for the quantification of H3Cit in plasma using an anti-histone antibody as capture antibody and an anti-histone H3 citrulline antibody for detection. The assay was evaluated for linearity, stability, specificity, and precision on plasma samples obtained from a human model of inflammation before and after lipopolysaccharide injection. The results revealed linearity and high specificity demonstrated by the inability of detecting non-citrullinated histone H3. Coefficients of variation for intra- and inter-assay variability ranged from 2.1 to 5.1% and from 5.8 to 13.5%, respectively, allowing for a high precision. Furthermore, our results support an inflammatory induction of a systemic NET burden by showing, for the first time, clear intra-individual elevations of plasma H3Cit in a human model of lipopolysaccharide-induced inflammation. Taken together, our work demonstrates the development of a new method for the quantification of H3Cit by ELISA that can reliably be used for the detection of NETs in human plasma.

Kinetics of anti-carcinoembryonic antigen antibody internalization: effects of affinity, bivalency, and stability

PubMed Central

Schmidt, Michael M.; Thurber, Greg M.

2010-01-01

Theoretical analyses suggest that the cellular internalization and catabolism of bound antibodies contribute significantly to poor penetration into tumors. Here we quantitatively assess the internalization of antibodies and antibody fragments against the commonly targeted antigen carcinoembryonic antigen (CEA). Although CEA is often referred to as a non-internalizing or shed antigen, anti-CEA antibodies and antibody fragments are shown to be slowly endocytosed by LS174T cells with a half-time of 10–16 h, a time scale consistent with the metabolic turnover rate of CEA in the absence of antibody. Anti-CEA single chain variable fragments (scFvs) with significant differences in affinity, stability against protease digestion, and valency exhibit similar uptake rates of bound antibody. In contrast, one anti-CEA IgG exhibits unique binding and trafficking properties with twice as many molecules bound per cell at saturation and significantly faster cellular internalization after binding. The internalization rates measured herein can be used in simple computational models to predict the microdistribution of these antibodies in tumor spheroids. PMID:18408925

Genetic relationships of antibody response, viremia level and weight gain in pigs experimentally infected with porcine reproductive and respiratory syndrome virus

USDA-ARS?s Scientific Manuscript database

Genetic and antigenic variability between Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) isolates has encumbered the development of effective vaccines. Therefore, the potential of genetic selection on PRRSV antibody response to improve resistance to PRRSV infection was assessed using da...

Autoimmune Encephalitis: Pathophysiology and Imaging Review of an Overlooked Diagnosis.

PubMed

Kelley, B P; Patel, S C; Marin, H L; Corrigan, J J; Mitsias, P D; Griffith, B

2017-06-01

Autoimmune encephalitis is a relatively new category of immune-mediated disease involving the central nervous system that demonstrates a widely variable spectrum of clinical presentations, ranging from the relatively mild or insidious onset of cognitive impairment to more complex forms of encephalopathy with refractory seizure. Due to its diverse clinical features, which can mimic a variety of other pathologic processes, autoimmune encephalitis presents a diagnostic challenge to clinicians. Imaging findings in patients with these disorders can also be quite variable, but recognizing characteristic findings within limbic structures suggestive of autoimmune encephalitis can be a key step in alerting clinicians to the potential diagnosis and ensuring a prompt and appropriate clinical work-up. In this article, we review antibody-mediated encephalitis and its various subtypes with a specific emphasis on the role of neuroimaging in the diagnostic work-up. © 2017 by American Journal of Neuroradiology.

Variability in Immunohistochemical Detection of Programmed Death Ligand 1 (PD-L1) in Cancer Tissue Types

PubMed Central

Scognamiglio, Giosuè; De Chiara, Anna; Di Bonito, Maurizio; Tatangelo, Fabiana; Losito, Nunzia Simona; Anniciello, Annamaria; De Cecio, Rossella; D’Alterio, Crescenzo; Scala, Stefania; Cantile, Monica; Botti, Gerardo

2016-01-01

In normal cell physiology, programmed death 1 (PD-1) and its ligand, PD-L1, play an immunoregulatory role in T-cell activation, tolerance, and immune-mediated tissue damage. The PD-1/PD-L1 pathway also plays a critical role in immune escape of tumor cells and has been demonstrated to correlate with a poor prognosis of patients with several types of cancer. However, recent reports have revealed that the immunohistochemical (IHC) expression of the PD-L1 in tumor cells is not uniform for the use of different antibodies clones, with variable specificity, often doubtful topographical localization, and with a score not uniquely defined. The purpose of this study was to analyze the IHC expression of PD-L1 on a large series of several human tumors to correctly define its staining in different tumor tissues. PMID:27213372

Array-Based Rational Design of Short Peptide Probe-Derived from an Anti-TNT Monoclonal Antibody.

PubMed

Okochi, Mina; Muto, Masaki; Yanai, Kentaro; Tanaka, Masayoshi; Onodera, Takeshi; Wang, Jin; Ueda, Hiroshi; Toko, Kiyoshi

2017-10-09

Complementarity-determining regions (CDRs) are sites on the variable chains of antibodies responsible for binding to specific antigens. In this study, a short peptide probe for recognition of 2,4,6-trinitrotoluene (TNT), was identified by testing sequences derived from the CDRs of an anti-TNT monoclonal antibody. The major TNT-binding site in this antibody was identified in the heavy chain CDR3 by antigen docking simulation and confirmed by an immunoassay using a spot-synthesis based peptide array comprising amino acid sequences of six CDRs in the variable region. A peptide derived from heavy chain CDR3 (RGYSSFIYWF) bound to TNT with a dissociation constant of 1.3 μM measured by surface plasmon resonance. Substitution of selected amino acids with basic residues increased TNT binding while substitution with acidic amino acids decreased affinity, an isoleucine to arginine change showed the greatest improvement of 1.8-fold. The ability to create simple peptide binders of volatile organic compounds from sequence information provided by the immune system in the creation of an immune response will be beneficial for sensor developments in the future.

Amino acid sequence of the Fv region of a human monoclonal IgM (protein WEA) with antibody activity against 3,4-pyruvylated galactose in Klebsiella polysaccharides K30 and K33.

PubMed Central

Goñi, F; Frangione, B

1983-01-01

We have determined the amino acid sequence of the Fv [variable heavy (VH) and variable light (VL)] region of a human monoclonal IgM-kappa with antibody activity against 3,4-pyruvylated galactose, isolated from the plasma of patient WEA with Waldenström macroglobulinemia. The VH region has 114 residues, belongs to subgroup III, and has a very short third complementarity-determining region (CDR3), probably due to a small D segment/or an unusual D-J rearrangement (D, diversity; J, joining). The VL region has 108 residues and belongs to subgroup V kappa I. Compared to other members of the human VHIII and V kappa I families, WEA Fv does not appear to have significant differences within the framework residues but has unique CDRs that might be responsible for the particular antibody activity. Another IgM-kappa (GAL), which has an as-yet-undetermined antibody activity, shares a striking homology in V kappa with WEA, including an identical CDR1. PMID:6410398

Neutralization of soluble, synaptotoxic amyloid β species by antibodies is epitope specific.

PubMed

Zago, Wagner; Buttini, Manuel; Comery, Thomas A; Nishioka, Christopher; Gardai, Shyra J; Seubert, Peter; Games, Dora; Bard, Frédérique; Schenk, Dale; Kinney, Gene G

2012-02-22

Several anti-amyloid β (Aβ) antibodies are under evaluation for the treatment of Alzheimer's disease (AD). Clinical studies using the N-terminal-directed anti-Aβ antibody bapineuzumab have demonstrated reduced brain PET-Pittsburg-B signals, suggesting the reduction of Aβ plaques, and reduced levels of total and phosphorylated tau protein in the CSF of treated AD patients. Preclinical studies using 3D6 (the murine form of bapineuzumab) have demonstrated resolution of Aβ plaque and vascular burdens, neuritic dystrophy, and preservation of synaptic density in the transgenic APP mouse models. In contrast, few studies have evaluated the direct interaction of this antibody with synaptotoxic soluble Aβ species. In the current report, we demonstrated that 3D6 binds to soluble, synaptotoxic assemblies of Aβ(1-42) and prevents multiple downstream functional consequences in rat hippocampal neurons including changes in glutamate AMPA receptor trafficking, AD-type tau phosphorylation, and loss of dendritic spines. In vivo, we further demonstrated that 3D6 prevents synaptic loss and acutely reverses the behavioral deficit in the contextual fear conditioning task in transgenic mouse models of AD, two endpoints thought to be linked to synaptotoxic soluble Aβ moieties. Importantly C-terminal anti-Aβ antibodies were ineffective on these endpoints. These results, taken with prior studies, suggest that N-terminal anti-Aβ antibodies effectively interact with both soluble and insoluble forms of Aβ and therefore appear particularly well suited for testing the Aβ hypothesis of AD.

Molecular imprint of enzyme active site by camel nanobodies: rapid and efficient approach to produce abzymes with alliinase activity.

PubMed

Li, Jiang-Wei; Xia, Lijie; Su, Youhong; Liu, Hongchun; Xia, Xueqing; Lu, Qinxia; Yang, Chunjin; Reheman, Kalbinur

2012-04-20

Screening of inhibitory Ab1 antibodies is a critical step for producing catalytic antibodies in the anti-idiotypic approach. However, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional antibodies become the limiting step. Because camelid-derived nanobodies possess the potential to preferentially bind to the active site of enzymes due to their small size and long CDR3, we have developed a novel approach to produce antibodies with alliinase activities by exploiting the molecular mimicry of camel nanobodies. By screening the camelid-derived variable region of the heavy chain cDNA phage display library with alliinase, we obtained an inhibitory nanobody VHHA4 that recognizes the active site. Further screening with VHHA4 from the same variable domain of the heavy chain of a heavy-chain antibody library led to a higher incidence of anti-idiotypic Ab2 abzymes with alliinase activities. One of the abzymes, VHHC10, showed the highest activity that can be inhibited by Ab1 VHHA4 and alliinase competitive inhibitor penicillamine and significantly suppressed the B16 tumor cell growth in the presence of alliin in vitro. The results highlight the feasibility of producing abzymes via anti-idiotypic nanobody approach.

Human anti-CD30 recombinant antibodies by guided phage antibody selection using cell panning

PubMed Central

Klimka, A; Matthey, B; Roovers, R C; Barth, S; Arends, J-W; Engert, A; Hoogenboom, H R

2000-01-01

In various clinical studies, Hodgkin’s patients have been treated with anti-CD30 immunotherapeutic agents and have shown promising responses. One of the problems that appeared from these studies is the development of an immune response against the non-human therapeutics, which limits repeated administration and reduces efficacy. We have set out to make a recombinant, human anti-CD30 single-chain variable fragment (scFv) antibody, which may serve as a targeting moiety with reduced immunogenicity and more rapid tumour penetration in similar clinical applications. Rather than selecting a naive phage antibody library on recombinant CD30 antigen, we used guided selection of a murine antibody in combination with panning on the CD30-positive cell line L540. The murine monoclonal antibody Ki-4 was chosen as starting antibody, because it inhibits the shedding of the extracellular part of the CD30 antigen. This makes the antibody better suited for CD30-targeting than most other anti-CD30 antibodies. We have previously isolated the murine Ki-4 scFv by selecting a mini-library of hybridoma-derived phage scFv-antibodies via panning on L540 cells. Here, we report that phage display technology was successfully used to obtain a human Ki-4 scFv version by guided selection. The murine variable heavy (VH) and light (VL) chain genes of the Ki-4 scFv were sequentially replaced by human V gene repertoires, while retaining only the major determinant for epitope-specificity: the heavy-chain complementarity determining region 3 (CDR3) of murine Ki-4. After two rounds of chain shuffling and selection by panning on L540 cells, a fully human anti-CD30 scFv was selected. It competes with the parental monoclonal antibody Ki-4 for binding to CD30, inhibits the shedding of the extracellular part of the CD30 receptor from L540 cells and is thus a promising candidate for the generation of anti-CD30 immunotherapeutics. © 2000 Cancer Research Campaign PMID:10901379

Mining Naïve Rabbit Antibody Repertoires by Phage Display for Monoclonal Antibodies of Therapeutic Utility.

PubMed

Peng, Haiyong; Nerreter, Thomas; Chang, Jing; Qi, Junpeng; Li, Xiuling; Karunadharma, Pabalu; Martinez, Gustavo J; Fallahi, Mohammad; Soden, Jo; Freeth, Jim; Beerli, Roger R; Grawunder, Ulf; Hudecek, Michael; Rader, Christoph

2017-09-15

Owing to their high affinities and specificities, rabbit monoclonal antibodies (mAbs) have demonstrated value and potential primarily as basic research and diagnostic reagents, but, in some cases, also as therapeutics. To accelerate access to rabbit mAbs bypassing immunization, we generated a large naïve rabbit antibody repertoire represented by a phage display library encompassing >10 billion independent antibodies in chimeric rabbit/human Fab format and validated it by next-generation sequencing. Panels of rabbit mAbs selected from this library against two emerging cancer targets, ROR1 and ROR2, revealed high diversity, affinity, and specificity. Moreover, ROR1- and ROR2-targeting rabbit mAbs demonstrated therapeutic utility as components of chimeric antigen receptor-engineered T cells, further corroborating the value of the naïve rabbit antibody library as a rich and virtually unlimited source of rabbit mAbs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates demonstrate tumor specificity and anti-tumor activity.

PubMed

Prendergast, Jillian M; Galvao da Silva, Ana Paula; Eavarone, David A; Ghaderi, Darius; Zhang, Mai; Brady, Dane; Wicks, Joan; DeSander, Julie; Behrens, Jeff; Rueda, Bo R

Targeted therapeutics that can differentiate between normal and malignant tumor cells represent the ideal standard for the development of a successful anti-cancer strategy. The Sialyl-Thomsen-nouveau antigen (STn or Sialyl-Tn, also known as CD175s) is rarely seen in normal adult tissues, but it is abundantly expressed in many types of human epithelial cancers. We have identified novel antibodies that specifically target with high affinity the STn glycan independent of its carrier protein, affording the potential to recognize a wider array of cancer-specific sialylated proteins. A panel of murine monoclonal anti-STn therapeutic antibodies were generated and their binding specificity and efficacy were characterized in vitro and in in vivo murine cancer models. A subset of these antibodies were conjugated to monomethyl auristatin E (MMAE) to generate antibody-drug conjugates (ADCs). These ADCs demonstrated in vitro efficacy in STn-expressing cell lines and significant tumor growth inhibition in STn-expressing tumor xenograft cancer models with no evidence of overt toxicity.

Simultaneous Measurements of Auto-Immune and Infectious Disease Specific Antibodies Using a High Throughput Multiplexing Tool

PubMed Central

Asati, Atul; Kachurina, Olga; Kachurin, Anatoly

2012-01-01

Considering importance of ganglioside antibodies as biomarkers in various immune-mediated neuropathies and neurological disorders, we developed a high throughput multiplexing tool for the assessment of gangliosides-specific antibodies based on Biolpex/Luminex platform. In this report, we demonstrate that the ganglioside high throughput multiplexing tool is robust, highly specific and demonstrating ∼100-fold higher concentration sensitivity for IgG detection than ELISA. In addition to the ganglioside-coated array, the high throughput multiplexing tool contains beads coated with influenza hemagglutinins derived from H1N1 A/Brisbane/59/07 and H1N1 A/California/07/09 strains. Influenza beads provided an added advantage of simultaneous detection of ganglioside- and influenza-specific antibodies, a capacity important for the assay of both infectious antigen-specific and autoimmune antibodies following vaccination or disease. Taken together, these results support the potential adoption of the ganglioside high throughput multiplexing tool for measuring ganglioside antibodies in various neuropathic and neurological disorders. PMID:22952605

Selection of cholera toxin specific IgNAR single-domain antibodies from a naïve shark library.

PubMed

Liu, Jinny L; Anderson, George P; Delehanty, James B; Baumann, Richard; Hayhurst, Andrew; Goldman, Ellen R

2007-03-01

Shark immunoglobulin new antigen receptor (IgNAR, also referred to as NAR) variable domains (Vs) are single-domain antibody (sdAb) fragments containing only two hypervariable loop structures forming 3D topologies for a wide range of antigen recognition and binding. Their small size ( approximately 12kDa) and high solubility, thermostability and binding specificity make IgNARs an exceptional alternative source of engineered antibodies for sensor applications. Here, two new shark NAR V display libraries containing >10(7) unique clones from non-immunized (naïve) adult spiny dogfish (Squalus acanthias) and smooth dogfish (Mustelus canis) sharks were constructed. The most conserved consensus sequences derived from random clone sequence were compared with published nurse shark (Ginglymostoma cirratum) sequences. Cholera toxin (CT) was chosen for panning one of the naïve display libraries due to its severe pathogenicity and commercial availability. Three very similar CT binders were selected and purified soluble monomeric anti-CT sdAbs were characterized using Luminex(100) and traditional ELISA assays. These novel anti-CT sdAbs selected from our newly constructed shark NAR V sdAb library specifically bound to soluble antigen, without cross reacting with other irrelevant antigens. They also showed superior heat stability, exhibiting slow loss of activity over the course of one hour at high temperature (95 degrees C), while conventional antibodies lost all activity in the first 5-10min. The successful isolation of target specific sdAbs from one of our non-biased NAR libraries, demonstrate their ability to provide binders against an unacquainted antigen of interest.

Antiproliferative and Apoptotic Effects of Novel Anti-ROR1 Single-Chain Antibodies in Hematological Malignancies.

PubMed

Aghebati-Maleki, Leili; Younesi, Vahid; Baradaran, Behzad; Abdolalizadeh, Jalal; Motallebnezhad, Morteza; Nickho, Hamid; Shanehbandi, Dariush; Majidi, Jafar; Yousefi, Mehdi

2017-04-01

Receptor tyrosine kinase-like orphan receptor (ROR) proteins are a conserved family of tyrosine kinase receptors that function in developmental processes including cell survival, differentiation, cell migration, cell communication, cell polarity, proliferation, metabolism, and angiogenesis. ROR1 has recently been shown to be expressed in various types of cancer cells but not normal cells. Pharmacokinetics and pharmacodynamics of single-chain Fragment variable (scFv) antibodies provide potential therapeutic advantages over whole antibody molecules. In the present study, scFvs against a specific peptide from the extracellular domain of ROR1 were selected using phage display technology. The selected scFvs were further characterized using polyclonal and monoclonal phage enzyme-linked immunosorbent assay (ELISA), soluble monoclonal ELISA, colony PCR, and sequencing. Antiproliferative and apoptotic effects of selected scFv antibodies were also evaluated in lymphoma and myeloma cancer cell lines using MTT and annexin V/PI assays. The results of ELISA indicated specific reactions of the isolated scFvs against the ROR1 peptide. Colony PCR confirmed the presence of full-length V H and Vκ inserts. The percentages of cell growth after 24 h of treatment of cells with individual scFv revealed that the scFv significantly inhibited the growth of the RPMI8226 and chronic lymphocytic leukemia (CLL) cells in comparison with the untreated cells ( p < 0.05). Interestingly, 24-h treatment with specific scFv induced apoptosis cell death in the RPMI8226 and CLL cells. Taken together, our results demonstrate that targeting of ROR1 using peptide-specific scFv can be an effective immunotherapy strategy in hematological malignancies.

Identification of novel tumor antigens with patient-derived immune-selected antibodies

PubMed Central

Rodriguez-Pinto, Daniel; Sparkowski, Jason; Keough, Martin P.; Phoenix, Kathryn N.; Vumbaca, Frank; Han, David K.; Gundelfinger, Eckart D.; Beesley, Philip

2010-01-01

The identification of tumor antigens capable of eliciting an immune response in vivo may be an effective method to identify therapeutic cancer targets. We have developed a method to identify such antigens using frozen tumor-draining lymph node samples from breast cancer patients. Immune responses in tumor-draining lymph nodes were identified by immunostaining lymph node sections for B-cell markers (CD20&CD23) and Ki67 which revealed cell proliferation in germinal center zones. Antigen-dependent somatic hypermutation (SH) and clonal expansion (CE) were present in heavy chain variable (VH) domain cDNA clones obtained from these germinal centers, but not from Ki67 negative germinal centers. Recombinant VH single-domain antibodies were used to screen tumor proteins and affinity select potential tumor antigens. Neuroplastin (NPTN) was identified as a candidate breast tumor antigen using proteomic identification of affinity selected tumor proteins with a recombinant VH single chain antibody. NPTN was found to be highly expressed in approximately 20% of invasive breast carcinomas and 50% of breast carcinomas with distal metastasis using a breast cancer tissue array. Additionally, NPTN over-expression in a breast cancer cell line resulted in a significant increase in tumor growth and angiogenesis in vivo which was related to increased VEGF production in the transfected cells. These results validate NPTN as a tumor-associated antigen which could promote breast tumor growth and metastasis if aberrantly expressed. These studies also demonstrate that humoral immune responses in tumor-draining lymph nodes can provide antibody reagents useful in identifying tumor antigens with applications for biomarker screening, diagnostics and therapeutic interventions. PMID:18568347

Serum antibody titers following routine rabies vaccination in African elephants.

PubMed

Miller, Michele A; Olea-Popelka, Francisco

2009-10-15

To evaluate serum antibody titers in captive African elephants (Loxodonta africana) following routine vaccination with a commercially available, inactivated rabies vaccine. Seroepidemiologic study. 14 captive African elephants from a single herd. Elephants were vaccinated as part of a routine preventive health program. Initially, elephants were vaccinated annually (2 mL, IM), and blood was collected every 4 or 6 months for measurement of rabies virus-neutralizing antibody titer by means of the rapid fluorescent focus inhibition test. Individual elephants were later switched to an intermittent vaccination schedule to allow duration of the antibody response to be determined. All elephants had detectable antibody responses following rabies vaccination, although there was great variability among individual animals in regard to antibody titers, and antibody titers could be detected as long as 24 months after vaccine administration. Young animals were found to develop an antibody titer following administration of a single dose of the rabies vaccine. Age and time since vaccination had significant effects on measured antibody titers. Results indicated that African elephants developed detectable antibody titers in response to inoculation with a standard large animal dose of a commercially available, inactivated rabies vaccine. The persistence of detectable antibody titers in some animals suggested that vaccination could be performed less frequently than once a year if antibody titers were routinely monitored.

Construction and characterization of VL-VH tail-parallel genetically engineered antibodies against staphylococcal enterotoxins.

PubMed

He, Xianzhi; Zhang, Lei; Liu, Pengchong; Liu, Li; Deng, Hui; Huang, Jinhai

2015-03-01

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus have increasingly given rise to human health and food safety. Genetically engineered small molecular antibody is a useful tool in immuno-detection and treatment for clinical illness caused by SEs. In this study, we constructed the V(L)-V(H) tail-parallel genetically engineered antibody against SEs by using the repertoire of rearranged germ-line immunoglobulin variable region genes. Total RNA were extracted from six hybridoma cell lines that stably express anti-SEs antibodies. The variable region genes of light chain (V(L)) and heavy chain (V(H)) were cloned by reverse transcription PCR, and their classical murine antibody structure and functional V(D)J gene rearrangement were analyzed. To construct the eukaryotic V(H)-V(L) tail-parallel co-expression vectors based on the "5'-V(H)-ivs-IRES-V(L)-3'" mode, the ivs-IRES fragment and V(L) genes were spliced by two-step overlap extension PCR, and then, the recombined gene fragment and V(H) genes were inserted into the pcDNA3.1(+) expression vector sequentially. And then the constructed eukaryotic expression clones termed as p2C2HILO and p5C12HILO were transfected into baby hamster kidney 21 cell line, respectively. Two clonal cell lines stably expressing V(L)-V(H) tail-parallel antibodies against SEs were obtained, and the antibodies that expressed intracytoplasma were evaluated by enzyme-linked immunosorbent assay, immunofluorescence assay, and flow cytometry. SEs can stimulate the expression of some chemokines and chemokine receptors in porcine IPEC-J2 cells; mRNA transcription level of four chemokines and chemokine receptors can be blocked by the recombinant SE antibody prepared in this study. Our results showed that it is possible to get functional V(L)-V(H) tail-parallel genetically engineered antibodies in same vector using eukaryotic expression system.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Conroy, W.G.

Structural relatedness between the variable region of anti-ligand antibodies and opioid binding sites allowed the generation of anti-idiotypic antibodies which recognized opioid receptors. The IgG{sub 3}k antibodies which bound to opioid receptors were obtained when an anti-morphine antiserum was the idiotype. Both antibodies bound to opioid receptors, but only one of these blocked the binding of ({sup 3}H)naloxone. The antibody which did not inhibit the binding of ({sup 3}H)naloxone was itself displaced from the receptor by opioid ligands. The unique binding properties displayed by this antibody indicated that anti-idiotypic antibodies are not always a perfect image of the original ligand,more » and therefore may be more useful than typical ligands as probes for the receptor. An auto-anti-idiotypic technique was successfully used to obtain anti-opioid receptor antibodies. Another IgG{sub 3}k antibody that blocked the binding of ({sup 3}H)naloxone to rat brain opioid receptors was obtained when a mouse was immunized with naloxone conjugated to bovine serum albumin. These data confirmed that an idiotype-anti-idiotype network which can generate an anti-receptor antibody normally functions when an opioid ligand is introduced into an animal in an immunogenic form.« less

Mechanisms of monoclonal antibody stabilization and release from silk biomaterials

PubMed Central

Guziewicz, Nicholas A.; Massetti, Andrew J.; Perez-Ramirez, Bernardo J.; Kaplan, David L.

2013-01-01

The availability of stabilization and sustained delivery systems for antibody therapeutics remains a major clinical challenge, despite the growing development of antibodies for a wide range of therapeutic applications due to their specificity and efficacy. A mechanistic understanding of protein-matrix interactions is critical for the development of such systems and is currently lacking as a mode to guide the field. We report mechanistic insight to address this need by using well-defined matrices based on silk gels, in combination with a monoclonal antibody. Variables including antibody loading, matrix density, charge interactions, hydrophobicity and water access were assessed to clarify mechanisms involved in the release of antibody from the biomaterial matrix. The results indicate that antibody release is primarily governed by hydrophobic interactions and hydration resistance, which are controlled by silk matrix chemistry, peptide domain distribution and protein density. Secondary ionic repulsions are also critical in antibody stabilization and release. Matrix modification by free methionine incorporation was found to be an effective strategy for mitigating encapsulation induced antibody oxidation. Additionally, these studies highlight a characterization approach to improve the understanding and development of other protein sustained delivery systems, with broad applicability to the rapidly developing monoclonal antibody field. PMID:23859659

Striational antibodies in myasthenia gravis: reactivity and possible clinical significance.

PubMed

Romi, Fredrik; Skeie, Geir Olve; Gilhus, Nils Erik; Aarli, Johan Arild

2005-03-01

Myasthenia gravis is an autoimmune disease caused, in most cases, by antibodies attaching to the acetylcholine receptor. Some myasthenia gravis patients have antibodies that bind in a cross-striational pattern to skeletal and heart muscle tissue sections (striational antibodies). These antibodies react with epitopes on the muscle proteins titin and ryanodine receptor, are found mainly in sera of patients with thymoma and late-onset myasthenia gravis, and may correlate with myasthenia gravis severity. Their presence may predict an unsatisfactory outcome after thymectomy. The detection of titin and ryanodine receptor antibodies provides more specific clinical information than the immunofluorescent demonstration of striational antibodies.

Usefulness of thyrogastric immune features as predictors of pernicious anaemia that lacks intrinsic factor antibody.

PubMed

Chan, J; Chan, H Y F

2011-08-01

The study aims to evaluate the diagnostic utility of thyrogastric immune features in the identification of intrinsic factor antibody negative (IFA -ve) pernicious anaemia (PA) patients. Clinico-pathological features of 'intrinsic factor antibody positive (IFA +ve) PA' and 'IFA -ve presumed PA' Chinese patients in a single hospital (2001-2009) were studied. Coefficients of independent variables identified were used as weighted scores. The result was validated by patients (1994-2000) with Schilling test done. Comparison of 127 'IFA +ve PA' and 130 'IFA -ve presumed PA' patients showed four independent variables, namely (+) gastric parietal cell (GPC) antibody (OR, 2.907, 95%; CI, 2.346-3.468; P < 0.001), (+) antithyroid antibodies (OR, 3.098, 95%; CI, 2.496-3.70; P < 0.001), (+) gastric atrophy (OR, 3.827, 95%; CI, 3.041-4.64; P = 0.001), and (-) Helicobacter pylori (HP) organisms (OR, 0.134, 95%; CI, -1.60-1.869; P = 0.023). The respective scores were 1.067, 1.131, 1.342 and -2.012. Total scores for each patient ranged from 3.54 to -2.012. When the cut-off score 1.528 was applied to the validation sample (n = 75), the specificity of identifying IFA -ve PA was 100%, sensitivity 53%, positive predictive value 100%, and negative predictive value 36%. Patients with two out of three features, GPC, antithyroid antibodies, gastric atrophy, but without HP organisms; or three features with HP organisms, can be predicted to have PA. © 2011 Blackwell Publishing Ltd.

Evaluation of the Biological Properties and Cross-Reactive Antibody Response to H10 Influenza Viruses in Ferrets

PubMed Central

Sutton, Troy C.; Lamirande, Elaine W.; Czako, Rita

2017-01-01

ABSTRACT The recent outbreak of avian origin H10N7 influenza among seals in northern Europe and two fatal human infections with an avian H10N8 virus in China have demonstrated that H10 viruses can spread between mammals and cause severe disease in humans. To gain insight into the potential for H10 viruses to cross the species barrier and to identify a candidate vaccine strain, we evaluated the in vitro and in vivo properties and antibody response in ferrets to 20 diverse H10 viruses. H10 virus infection of ferrets caused variable weight loss, and all 20 viruses replicated throughout the respiratory tract; however, replication in the lungs was highly variable. In glycan-binding assays, the H10 viruses preferentially bound “avian-like” α2,3-linked sialic acids. Importantly, several isolates also displayed strong binding to long-chain “human-like” α2,6-linked sialic acids and exhibited comparable or elevated neuraminidase activity relative to human H1N1, H2N2, and H3N2 viruses. In hemagglutination inhibition assays, 12 antisera cross-reacted with ≥14 of 20 H10 viruses, and 7 viruses induced neutralizing activity against ≥15 of the 20 viruses. By combining data on weight loss, viral replication, and the cross-reactive antibody response, we identified A/mallard/Portugal/79906/2009 (H10N7) as a suitable virus for vaccine development. Collectively, our findings suggest that H10 viruses may continue to sporadically infect humans and other mammals, underscoring the importance of developing an H10 vaccine for pandemic preparedness. IMPORTANCE Avian origin H10 influenza viruses sporadically infect humans and other mammals; however, little is known about viruses of this subtype. Thus, we characterized the biological properties of 20 H10 viruses in vitro and in ferrets. Infection caused mild to moderate weight loss (5 to 15%), with robust viral replication in the nasal tissues and variable replication in the lung. H10 viruses preferentially bind “avian-like” sialic acids, although several isolates also displayed binding to “human-like” sialic acid receptors. This is consistent with the ability of H10 viruses to cross the species barrier and warrants selection of an H10 vaccine strain. By evaluating the cross-reactive antibody response to the H10 viruses and combining this analysis with viral replication and weight loss findings, we identified A/mallard/Portugal/79906/2009 (H10N7) as a suitable H10 vaccine strain. PMID:28701401

Clinical and Serological Predictors of Suicide in Schizophrenia and Major Mood Disorders.

PubMed

Dickerson, Faith; Origoni, Andrea; Schweinfurth, Lucy A B; Stallings, Cassie; Savage, Christina L G; Sweeney, Kevin; Katsafanas, Emily; Wilcox, Holly C; Khushalani, Sunil; Yolken, Robert

2018-03-01

Persons with serious mental illness are at high risk for suicide, but this outcome is difficult to predict. Serological markers may help to identify suicide risk. We prospectively assessed 733 persons with a schizophrenia spectrum disorder, 483 with bipolar disorder, and 76 with major depressive disorder for an average of 8.15 years. The initial evaluation consisted of clinical and demographic data as well as a blood samples from which immunoglobulin G antibodies to herpes viruses and Toxoplasma gondii were measured. Suicide was determined using data from the National Death Index. Cox proportional hazard regression models examined the role of baseline variables on suicide outcomes. Suicide was associated with male sex, divorced/separated status, Caucasian race, and elevated levels of antibodies to Cytomegalovirus (CMV). Increasing levels of CMV antibodies were associated with increasing hazard ratios for suicide. The identification of serological variables associated with suicide might provide more personalized methods for suicide prevention.

Conserved Structural Elements in the V3 Crown of HIV-1 gp120

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang, X.; Burke, V; Totrov, M

2010-01-01

Binding of the third variable region (V3) of the HIV-1 envelope glycoprotein gp120 to the cell-surface coreceptors CCR5 or CXCR4 during viral entry suggests that there are conserved structural elements in this sequence-variable region. These conserved elements could serve as epitopes to be targeted by a vaccine against HIV-1. Here we perform a systematic structural analysis of representative human anti-V3 monoclonal antibodies in complex with V3 peptides, revealing that the crown of V3 has four conserved structural elements: an arch, a band, a hydrophobic core and the peptide backbone. These are either unaffected by or are subject to minimal sequencemore » variation. As these regions are targeted by cross-clade neutralizing human antibodies, they provide a blueprint for the design of vaccine immunogens that could elicit broadly cross-reactive protective antibodies.« less

Epigallocatechin-3-gallate preferentially induces aggregation of amyloidogenic immunoglobulin light chains

PubMed Central

Hora, Manuel; Carballo-Pacheco, Martin; Weber, Benedikt; Morris, Vanessa K.; Wittkopf, Antje; Buchner, Johannes; Strodel, Birgit; Reif, Bernd

2017-01-01

Antibody light chain amyloidosis is a rare disease caused by fibril formation of secreted immunoglobulin light chains (LCs). The huge variety of antibody sequences puts a serious challenge to drug discovery. The green tea polyphenol epigallocatechin-3-gallate (EGCG) is known to interfere with fibril formation in general. Here we present solution- and solid-state NMR studies as well as MD simulations to characterise the interaction of EGCG with LC variable domains. We identified two distinct EGCG binding sites, both of which include a proline as an important recognition element. The binding sites were confirmed by site-directed mutagenesis and solid-state NMR analysis. The EGCG-induced protein complexes are unstructured. We propose a general mechanistic model for EGCG binding to a conserved site in LCs. We find that EGCG reacts selectively with amyloidogenic mutants. This makes this compound a promising lead structure, that can handle the immense sequence variability of antibody LCs. PMID:28128355

High Titers ofChlamydia trachomatis Antibodies in Brazilian Women with Tubal Occlusion or Previous Ectopic Pregnancy

PubMed Central

Machado, A. C. S.; Guimarães, E. M. B.; Sakurai, E.; Fioravante, F. C. R.; Amaral, W. N.; Alves, M. F. C.

2007-01-01

Objective. To evaluate serum chlamydia antibody titers (CATs) in tubal occlusion or previous ectopic pregnancy and the associated risk factors.Methods. The study population consisted of 55 women wih tubal damage and 55 parous women. CAT was measured using the whole-cell inclusion immunofluorescence test and cervical chlamydial DNA detected by PCR. Odds ratios were calculated to assess variables associated withC. trachomatis infection.Results. The prevalence of chlamydial antibodies and antibody titers in women with tubal occlusion or previous ectopic pregnancy was significantly higher (P < .01) than in parous women. Stepwise logistic regression analysis showed that chlamydia IgG antibodies were associated with tubal damage and with a larger number of lifetime sexual partners.Conclusions. Chlamydia antibody titers were associated with tubal occlusion, prior ectopic pregnancy, and with sexual behavior, suggesting that a chlamydia infection was the major contributor to the tubal damage in these women. PMID:17541464

Homologous and heterologous antibody responses of mice immunized with purified feline herpesvirus type 1 and canine herpesvirus glycoproteins.

PubMed

Limcumpao, J A; Horimoto, T; Xuan, X N; Tohya, Y; Azetaka, M; Takahashi, E; Mikami, T

1991-06-01

The three glycoproteins each of feline herpesvirus type 1 (FHV-1) and canine herpesvirus (CHV) were purified by affinity chromatography using glycoprotein-specific monoclonal antibodies and used individually or in combination in immunizing mice to determine their relative immunogenicity. All the glycoproteins induced detectable virus neutralizing antibodies to the homologous virus but FHV-1 gp143/108 and its cross-reacting counterpart, CHV gp145/112, elicited the highest titers not only to the homologous virus but to the heterologous virus as well. The production of ELISA antibodies after glycoprotein immunization was variable, while hemagglutination-inhibiting antibodies were produced by only 1 out of 10 FHV-1 gp60-inoculated mice. In general, the antibody titers induced by CHV glycoproteins were lower than those by FHV-1 glycoproteins. These results indicate that these glycoproteins may be useful as subunit vaccines against FHV-1 and CHV infections.

Correlation between sonography and antibody activity in patients with Hashimoto thyroiditis.

PubMed

Willms, Arnulf; Bieler, Dan; Wieler, Helmut; Willms, Diana; Kaiser, Klaus P; Schwab, Robert

2013-11-01

Patients with Hashimoto thyroiditis show structural changes of the thyroid that can be identified by a variety of sonographic criteria. We conducted this study to investigate whether there is a correlation between sonography and antibody activity and to assess the role of sonography in the diagnosis and follow-up of Hashimoto thyroiditis. In addition, we present a new classification system (termed the VESINC system [volume, echogenicity, sonographic texture, pseudonodular hypoechoic infiltration, nodules, and cysts]), which helps improve the clarity of sonographic findings. The study included 223 consecutive patients with previously diagnosed Hashimoto autoimmune thyroiditis who attended the thyroid clinic of the German Armed Forces Central Hospital in Koblenz for follow-up examinations between 2006 and 2008. Laboratory tests were performed to measure the levels of free triiodothyronine, free thyroxine, thyrotropin, anti-thyroglobulin antibodies (TgAbs), and antithyroid peroxidase antibodies (TPOAbs). Sonography was performed according to a strict protocol. We then assessed whether a correlation existed between antibody activity and the 6 sonographic variables of the VESINC system. Hypoechogenicity, heterogeneity, and pseudonodular hypoechoic infiltration were associated with significantly higher TPOAb activity (P < .001). There were no significant correlations between the other sonographic variables examined (cysts, nodules, and volume) or the biometric data with the TPOAb and TgAb levels. In addition, an assessment of TgAb levels did not show significant differences in correlations with any of the sonographic variables. Sonography is a noninvasive diagnostic imaging modality that provides information about the level of inflammatory activity. Markedly decreased echogenicity, heterogeneity, and multifocal pseudoinodular hypoechoic infiltration are indicative of a high level of inflammatory activity. The sonographic classification system presented here (VESINC system) can be a useful tool for comparing sonographic findings in a rapid and objective manner during follow-up of Hashimoto thyroiditis.

Development of an affinity-matured humanized anti-epidermal growth factor receptor antibody for cancer immunotherapy.

PubMed

Nakanishi, Takeshi; Maru, Takamitsu; Tahara, Kazuhiro; Sanada, Hideaki; Umetsu, Mitsuo; Asano, Ryutaro; Kumagai, Izumi

2013-02-01

We showed previously that humanization of 528, a murine anti-epidermal growth factor receptor (EGFR) antibody, causes reduced affinity for its target. Here, to improve the affinity of the humanized antibody for use in cancer immunotherapy, we constructed phage display libraries focused on the complementarity-determining regions (CDRs) of the antibody and carried out affinity selection. Two-step selections using libraries constructed in a stepwise manner enabled a 32-fold affinity enhancement of humanized 528 (h528). Thermodynamic analysis of the interactions between the variable domain fragment of h528 (h528Fv) mutants and the soluble extracellular domain of EGFR indicated that the h528Fv mutants obtained from the first selection showed a large increase in negative enthalpy change due to binding, resulting in affinity enhancement. Furthermore, mutants from the second selection showed a decrease in entropy loss, which led to further affinity maturation. These results suggest that a single mutation in the heavy chain variable domain (i.e. Tyr(52) to Trp) enthalpically contributed for overcoming the energetic barrier to the antigen-antibody interaction, which was a major hurdle for the in vitro affinity maturation of h528. We reported previously that the humanized bispecific diabody hEx3 Db, which targets EGFR and CD3, shows strong anti-tumor activity. hEx3 Db mutants, in which the variable domains of h528 were replaced with those of the affinity-enhanced mutants, were prepared and characterized. In a growth inhibition assay of tumor cells, the hEx3 Db mutants showed stronger anti-tumor activity than that of hEx3 Db, suggesting that affinity enhancement of h528Fv enhances the anti-tumor activity of the bispecific diabody.

High-frequency expression of a conserved kappa light-chain variable-region gene in chronic lymphocytic leukemia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kipps, T.J.; Fong, S.; Tomhave, E.

Malignant B lymphocytes from several patients with chronic lymphocytic leukemia (CLL) were examined for reactivity with murine monoclonal antibody 17.109. This antibody, prepared against the rheumatoid factor (RF) paraprotein Sie, recognizes a cross reactive idiotype on 48% of human IgM RF paraproteins, but does not react with IgM paraproteins without RF activity or substantially with normal pooled immunoglobulin. The 17.109-reactive idiotype is a marker for a kappa III variable-region gene, designated V/sub kappa/RF, that is conserved in outbred human populations. In a limited study of 31 CLL patients, the leukemic cells from 5 of 20 patients with kappa light chain-expressingmore » CLL were recognized by the 17.109 monoclonal antibody. Despite having malignant cells specifically reactive with this antibody, patients with 17.109-positive CLL did not have elevated serum levels of circulating antibody bearing 17.109-reactive determinants. Total RNAs isolated from the CLL B lymphocytes, or from hybridomas produced by fusing the CLL cells with the WI-L2-729-HF/sub 2/ cell line, were fractionated electrophoretically and examined by blot hybridization. Under stringent hybridization conditions capable of discerning a single base-pair mismatch, RNA from the 17.109-idiotype-positive CLL cells hybridized to synthetic oligonucleotide probes corresponding to framework and complementary-determining regions in the V/sub kappa/RF gene. The high frequency of the 17.109-associated idiotype and the V/sub kappa/RF gene in CLL suggests that the disease may arise from B lymphocytes that express a restricted set of inherited immunoglobulin variable-region genes with little or no somatic mutation.« less

INDUCTION OF RABBIT ANTIBODY WITH MOLECULAR UNIFORMITY AFTER IMMUNIZATION WITH GROUP C STREPTOCOCCI

PubMed Central

Eichmann, Klaus; Lackland, Henry; Hood, Leroy; Krause, Richard M.

1970-01-01

Antibodies with uniform properties may occur in rabbits after immunization with Group C streptococci. These precipitating antibodies possess specificity for the group-specific carbohydrate. Not uncommonly, their concentration is between 20 and 40 mg/ml of antiserum. Evidence for molecular uniformity in the case of one of these antibodies, described in detail here, includes: individual antigenic specificity; monodisperse distribution of the light chains by alkaline urea polyacrylamide disc electrophoresis; and a single amino acid in each of the first three N-terminal positions of the light chains. When the amino acid sequence of rabbit antibody b+ light chains (κ type) are aligned against their human κ counterparts, a definite homology is observed between the N-terminus of the human and the rabbit variable region. PMID:5409946

Immunological aspects of Giardia infections.

PubMed

Heyworth, Martin F

2014-01-01

Immunodeficiency, particularly antibody deficiency, predisposes to increased intensity and persistence of Giardia infections. Giardia-infected immunocompetent hosts produce serum and intestinal antibodies against Giardia trophozoites. The number of Giardia muris trophozoites, in mice with G. muris infection, is reduced by intra-duodenal administration of anti-G. muris antibody. Giardia intestinalis antigens that are recognised by human anti-trophozoite antibodies include variable (variant-specific) and invariant proteins. Nitric oxide (NO) appears to contribute to host clearance of Giardia trophozoites. Arginine is a precursor of NO and is metabolised by Giardia trophozoites, possibly reducing its availability for generation of NO by the host. Work with mice suggests that T lymphocytes and interleukin-6 (IL-6) contribute to clearance of Giardia infection via mechanisms independent of antibodies. © M.F. Heyworth, published by EDP Sciences, 2014.

Demonstration of the salmonid humoral response to Renibacterium salmoninarum using a monoclonal antibody against salmonid immunoglobulin

USGS Publications Warehouse

Bartholomew, J.L.; Arkoosh , M.R.; Rohovec, J.S.

1991-01-01

The specificity of the antibody response of salmonids to Renibacterium salmoninarum antigens was demonstrated by western blotting techniques that utilized a monoclonal antibody against salmonid immunoglobulin. In this study, the specificity of the response in immunized chinook salmon Oncorhynchus tshawytschawas compared with the response in naturally infected chinook salmon and coho salmon O. kisutch, and immunized rabbits. The antibody response in immunized salmon and rabbits and the naturally infected fish was primarily against the 57–58kilodalton protein complex. In addition to recognizing these proteins in the extracellular fraction and whole-cell preparations, antibody from the immunized salmon and rabbits detected four proteins with lower molecular masses. Western blotting techniques allow identification of the specific antigens recognized and are a useful tool for comparing the immunogenicity of different R. salmoninarumpreparations. Immunofluorescent techniques with whole bacteria were less sensitive than western blotting in detecting salmonid anti-R. salmoninarumantibody.

[Value of using polyethylene glycol in the indirect antiglobulin test with erythrocytes].

PubMed

Michalewska, B; Walewska, I; Seyfriedowa, H; Kuśnierz-Alejska, G

1991-01-01

A series of comparative investigations was carried out for establishing the value of the indirect antiglobulin test (IAT) in its first phase. The results showed that PEG, in relation to NaCl solution of low ionic strength, increased the reactivity of various antibodies of different specificity present in 72 studied sera. A significant superiority of IAT-PEG to IAT-BISS was noted in 45.8% of these sera. In search for antibodies in sera of 1160 patients IAT-PEG detected 7 antibodies not reacting in the IAT-LISS, and 5 of them had Rh system specificity, one was anti-K and one anti-Jka. Although the IAT-PEG was more sensitive than IAT-LISS in the detection of anti-Rh antibodies, it was less sensitive in this range than the enzymatic LEN test with which not only 5 of these antibodies were demonstrated but which detected also 8 antibodies not demonstrated in IAT-PEG or IAT-LISS. All 5 antibodies not detected by the IAT-PEG but demonstrated in the IAT-LISS were specific for the Lewis system. The per cent of reactions regarded as non-specific in the IAT-PEG was 1.3%. The IAT-PEG modification may be recommended as a sensitive, simple and specific method extending the possibilities of immunohematological diagnosis, especially in cases of post-transfusion reactions and in difficulties of interpretation connected with the occurrence of weakly positive reactions in the IAT-LISS.

A Spectrum of Monoclonal Antibodies Reactive with Human Mammary Tumor Cells

NASA Astrophysics Data System (ADS)

Colcher, D.; Horan Hand, P.; Nuti, M.; Schlom, J.

1981-05-01

Splenic lymphocytes of mice, immunized with membrane-enriched fractions of metastatic human mammary carcinoma tissues, were fused with the NS-1 non-immunoglobulin-secreting murine myeloma cell line. This resulted in the generation of hybridoma cultures secreting immunoglobulins reactive in solid-phase radioimmunoassays with extracts of metastatic mammary carcinoma cells from involved livers, but not with extracts of apparently normal human liver. As a result of further screening of immunoglobulin reactivities and double cloning of cultures, 11 monoclonal antibodies were chosen that demonstrated reactivities with human mammary tumor cells and not with apparently normal human tissues. These monoclonal antibodies could be placed into at least five major groups on the basis of their differential binding to the surface of various live human mammary tumor cells in culture, to extracts of mammary tumor tissues, or to tissue sections of mammary tumor cells studied by the immunoperoxidase technique. Whereas a spectrum of reactivities to mammary tumors was observed with the 11 monoclonal antibodies, no reactivity was observed to apparently normal cells of the following human tissues: breast, lymph node, lung, skin, testis, kidney, thymus, bone marrow, spleen, uterus, thyroid, intestine, liver, bladder, tonsils, stomach, prostate, and salivary gland. Several of the antibodies also demonstrated a ``pancarcinoma'' reactivity, showing binding to selected non-breast carcinomas. None of the monoclonal antibodies showed binding to purified ferritin or carcinoembryonic antigen. Monoclonal antibodies of all five major groups, however, demonstrated binding to human metastatic mammary carcinoma cells both in axillary lymph nodes and at distal sites.

Prevalence, sensitivity and specificity of antibodies against carbamylated proteins in a monocentric cohort of patients with rheumatoid arthritis and other autoimmune rheumatic diseases.

PubMed

Pecani, Arbi; Alessandri, Cristiano; Spinelli, Francesca Romana; Priori, Roberta; Riccieri, Valeria; Di Franco, Manuela; Ceccarelli, Fulvia; Colasanti, Tania; Pendolino, Monica; Mancini, Riccardo; Truglia, Simona; Barbati, Cristiana; Vomero, Marta; Sabatinelli, Danilo; Morello, Francesca; Valesini, Guido; Conti, Fabrizio

2016-11-25

Antibodies against carbamylated proteins (anti-CarP) have been recently identified in the sera of patients with rheumatoid arthritis (RA). The objective of the study was to evaluate the prevalence, sensitivity and specificity of anti-CarP compared to anti-citrullinated peptide antibodies (ACPA) and rheumatoid factor (RF), replicating the existing data in a large cohort of Italian patients with RA and extending the evaluation to other autoimmune rheumatic diseases (AIRDs). Serum samples (n = 607) from 309 patients with RA, 200 disease controls and 98 normal healthy subjects (NHS) were evaluated. Anti-CarP were detected using carbamylated fetal calf serum as the antigen. ACPAs were detected using second-generation ELISA and IgM RF was assessed as part of routine analysis. Anti-CarP antibodies were detected in 117 patients with RA (34.4%), ACPA in 190 patients (61.4%) and RF in 202 patients (65.3%). Two (2.04%) of the NHS were positive for anti-CarP, one NHS (1.02%) was positive for ACPA and three NHS were positive for RF (3.06%). Among disease controls, anti-CarP antibodies were detected in 33 patients (16.5%), ACPA in 29 patients (14.5%) and RF in 64 patients (32%). In particular, 16.8% of patients with systemic lupus erythematosus and 31.1% of patients with Sjögren syndrome were positive for anti-CarP. The sensitivity of anti-CarP, ACPA and RF was 46.8%, 61.8% and 64.4%, respectively and specificity was 91.95%, 89.93% and 76.51%, respectively. The present study extends the knowledge of anti-CarP antibodies, confirming previous data on the diagnostic accuracy of anti-CarP in RA in a large cohort of Italian patients. Anti-CarP antibodies demonstrated relatively low sensitivity and slightly higher specificity compared to ACPA and RF. Even if predominantly present in RA, anti-CarP was detected in a variable percentage of patients with other autoimmune rheumatic diseases and their generation could be attributed to the inflammatory status; the clinical relevance of anti-CarP antibodies in these latter patients should be further determined.

Anti-Yo antibody uptake and interaction with its intracellular target antigen causes Purkinje cell death in rat cerebellar slice cultures: a possible mechanism for paraneoplastic cerebellar degeneration in humans with gynecological or breast cancers.

PubMed

Greenlee, John E; Clawson, Susan A; Hill, Kenneth E; Wood, Blair; Clardy, Stacey L; Tsunoda, Ikuo; Carlson, Noel G

2015-01-01

Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat cerebellar slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat cerebellar slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not initiated by brain mononuclear cells. Purkinje cell death was not simply due to intraneuronal antibody accumulation.

Anti-Yo Antibody Uptake and Interaction with Its Intracellular Target Antigen Causes Purkinje Cell Death in Rat Cerebellar Slice Cultures: A Possible Mechanism for Paraneoplastic Cerebellar Degeneration in Humans with Gynecological or Breast Cancers

PubMed Central

Greenlee, John E.; Clawson, Susan A.; Hill, Kenneth E.; Wood, Blair; Clardy, Stacey L.; Tsunoda, Ikuo; Carlson, Noel G.

2015-01-01

Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat cerebellar slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat cerebellar slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not initiated by brain mononuclear cells. Purkinje cell death was not simply due to intraneuronal antibody accumulation. PMID:25885452

Construction of Rabbit Immune Antibody Libraries.

PubMed

Nguyen, Thi Thu Ha; Lee, Jong Seo; Shim, Hyunbo

2018-01-01

Rabbits have distinct advantages over mice as a source of target-specific antibodies. They produce higher affinity antibodies than mice, and may elicit strong immune response against antigens or epitopes that are poorly immunogenic or tolerated in mice. However, a great majority of currently available monoclonal antibodies are of murine origin because of the wider availability of murine fusion partner cell lines and well-established tools and protocols for fusion and cloning of mouse hybridoma. Phage-display selection of antibody libraries is an alternative method to hybridoma technology for the generation of target-specific monoclonal antibodies. High-affinity monoclonal antibodies from nonmurine species can readily be obtained by constructing immune antibody libraries from B cells of the immunized animal and screening the library by phage display. In this article, we describe the construction of a rabbit immune Fab library for the facile isolation of rabbit monoclonal antibodies. After immunization, B-cell cDNA is obtained from the spleen of the animal, from which antibody variable domain repertoires are amplified and assembled into a Fab repertoire by PCR. The Fab genes are then cloned into a phagemid vector and transformed to E. coli, from which a phage-displayed immune Fab library is rescued. Such a library can be biopanned against the immunization antigen for rapid identification of high-affinity, target-specific rabbit monoclonal antibodies.

Immune TB Antibody Phage Display Library as a Tool To Study B Cell Immunity in TB Infections.

PubMed

Hamidon, Nurul Hamizah; Suraiya, Siti; Sarmiento, Maria E; Acosta, Armando; Norazmi, Mohd Nor; Lim, Theam Soon

2018-03-01

B cells and in particular antibodies has always played second fiddle to cellular immunity in regard to tuberculosis (TB). However, recent studies has helped position humoral immunity especially antibodies back into the foray in relation to TB immunity. Therefore, the ability to correlate the natural antibody responses of infected individuals toward TB antigens would help strengthen this concept. Phage display is an intriguing approach that can be utilized to study antibody-mediated responses against a particular infection via harvesting the B cell repertoire from infected individuals. The development of disease-specific antibody libraries or immune libraries is useful to better understand antibody-mediated immune responses against specific disease antigens. This study describes the generation of an immune single-chain variable fragment (scFv) library derived from TB-infected individuals. The immune library with an estimated diversity of 10 9 independent clones was then applied for the identification of monoclonal antibodies against Mycobacterium tuberculosis α-crystalline as a model antigen. Biopanning of the library isolated three monoclonal antibodies with unique gene usage. This strengthens the role of antibodies in TB immunity in addition to the role played by cellular immunity. The developed library can be applied against other TB antigens and aid antibody-derived TB immunity studies in the future.

Isolation and characterization of an IgNAR variable domain specific for the human mitochondrial translocase receptor Tom70.

PubMed

Nuttall, Stewart D; Krishnan, Usha V; Doughty, Larissa; Pearson, Kylie; Ryan, Michael T; Hoogenraad, Nicholas J; Hattarki, Meghan; Carmichael, Jennifer A; Irving, Robert A; Hudson, Peter J

2003-09-01

The new antigen receptor (IgNAR) from sharks is a disulphide bonded dimer of two protein chains, each containing one variable and five constant domains, and functions as an antibody. In order to assess the antigen-binding capabilities of isolated IgNAR variable domains (VNAR), we have constructed an in vitro library incorporating synthetic CDR3 regions of 15-18 residues in length. Screening of this library against the 60 kDa cytosolic domain of the 70 kDa outer membrane translocase receptor from human mitochondria (Tom70) resulted in one dominant antigen-specific clone (VNAR 12F-11) after four rounds of in vitro selection. VNAR 12F-11 was expressed into the Escherichia coli periplasm and purified by anti-FLAG affinity chromatography at yields of 3 mg x L(-1). Purified protein eluted from gel filtration columns as a single monomeric protein and CD spectrum analysis indicated correct folding into the expected beta-sheet conformation. Specific binding to Tom70 was demonstrated by ELISA and BIAcore (Kd = 2.2 +/- 0.31 x 10(-9) m-1) indicating that these VNAR domains can be efficiently displayed as bacteriophage libraries, and selected against target antigens with an affinity and stability equivalent to that obtained for other single domain antibodies. As an initial step in producing 'intrabody' variants of 12F-11, the impact of modifying or removing the conserved immunoglobulin intradomain disulphide bond was assessed. High affinity binding was only retained in the wild-type protein, which combined with our inability to affinity mature 12F-11, suggests that this particular VNAR is critically dependent upon precise CDR loop conformations for its binding affinity.

Quantitative spatiotemporal analysis of antibody fragment diffusion and endocytic consumption in tumor spheroids.

PubMed

Thurber, Greg M; Wittrup, K Dane

2008-05-01

Antibody-based cancer treatment depends upon distribution of the targeting macromolecule throughout tumor tissue, and spatial heterogeneity could significantly limit efficacy in many cases. Antibody distribution in tumor tissue is a function of drug dosage, antigen concentration, binding affinity, antigen internalization, drug extravasation from blood vessels, diffusion in the tumor extracellular matrix, and systemic clearance rates. We have isolated the effects of a subset of these variables by live-cell microscopic imaging of single-chain antibody fragments against carcinoembryonic antigen in LS174T tumor spheroids. The measured rates of scFv penetration and retention were compared with theoretical predictions based on simple scaling criteria. The theory predicts that antibody dose must be large enough to drive a sufficient diffusive flux of antibody to overcome cellular internalization, and exposure time must be long enough to allow penetration to the spheroid center. The experimental results in spheroids are quantitatively consistent with these predictions. Therefore, simple scaling criteria can be applied to accurately predict antibody and antibody fragment penetration distance in tumor tissue.

Quantitative Spatiotemporal Analysis of Antibody Fragment Diffusion and Endocytic Consumption in Tumor Spheroids

PubMed Central

Thurber, Greg M.; Wittrup, K. Dane

2010-01-01

Antibody-based cancer treatment depends upon distribution of the targeting macromolecule throughout tumor tissue, and spatial heterogeneity could significantly limit efficacy in many cases. Antibody distribution in tumor tissue is a function of drug dosage, antigen concentration, binding affinity, antigen internalization, drug extravasation from blood vessels, diffusion in the tumor extracellular matrix, and systemic clearance rates. We have isolated the effects of a subset of these variables by live-cell microscopic imaging of single-chain antibody fragments against carcinoembryonic antigen in LS174T tumor spheroids. The measured rates of scFv penetration and retention were compared with theoretical predictions based on simple scaling criteria. The theory predicts that antibody dose must be large enough to drive a sufficient diffusive flux of antibody to overcome cellular internalization, and exposure time must be long enough to allow penetration to the spheroid center. The experimental results in spheroids are quantitatively consistent with these predictions. Therefore, simple scaling criteria can be applied to accurately predict antibody and antibody fragment penetration distance in tumor tissue. PMID:18451160

Malaria Prevention by New Technology: Vectored Delivery of Antibody Genes

DTIC Science & Technology

2017-10-01

AWARD NUMBER: W81XWH-15-1-0401 TITLE: Malaria Prevention by New Technology : Vectored Delivery of Antibody Genes PRINCIPAL INVESTIGATOR: Gary...CONTRACT NUMBER Malaria Prevention by New Technology : Vectored Delivery of Antibody Genes 5b. GRANT NUMBER W81XWH-15-1-0401 5c. PROGRAM ELEMENT...whole animals. Using a specific technology originally applied to expression of HIV antibodies, we demonstrated that mice can be protected from

Detection of IgA antibodies and quantification of IgA antibody-producing cells specific for ovalbumin or Trichinella spiralis in the rat.

PubMed

Van Loveren, H; Osterhaus, A D; Nagel, J; Schuurman, H J; Vos, J G

1988-09-01

This report describes procedures to quantify IgA responses in the rat sensitized to ovalbumin or infected with the parasite Trichinella spiralis: an ELISPOT detecting specific IgA antibody-producing cells in lymph nodes, and an ELISA demonstrating IgA antibody in serum and gut mucosal scrapings. For this purpose a mouse monoclonal anti-rat IgA antibody was produced. This IgG1-kappa 1 antibody recognized rat IgA but not rat IgM, IgG, or IgE. It proved very suitable in both assays. Using this reagent we could demonstrate large numbers of IgA anti-ovalbumin-producing cells in the mesenteric lymph nodes 15 days after sensitization to ovalbumin via the Peyer's patches. At 28 days after sensitization the numbers were much lower. IgA antibody titres to ovalbumin in serum were maximal between days 14 and 21 after immunization. Maximal numbers of IgA anti-T. spiralis-producing cells were found in the mesenteric lymph nodes 12 days after infection with muscle larvae, followed by a sharp decrease at 15 days. Maximal IgA anti-T. spiralis antibody titres in serum and mucus scrapings of small intestines were found on days 10 and 12 after oral infection with the parasite.

Investigating interactions between phospholipase B-Like 2 and antibodies during Protein A chromatography.

PubMed

Tran, Benjamin; Grosskopf, Vanessa; Wang, Xiangdan; Yang, Jihong; Walker, Don; Yu, Christopher; McDonald, Paul

2016-03-18

Purification processes for therapeutic antibodies typically exploit multiple and orthogonal chromatography steps in order to remove impurities, such as host-cell proteins. While the majority of host-cell proteins are cleared through purification processes, individual host-cell proteins such as Phospholipase B-like 2 (PLBL2) are more challenging to remove and can persist into the final purification pool even after multiple chromatography steps. With packed-bed chromatography runs using host-cell protein ELISAs and mass spectrometry analysis, we demonstrated that different therapeutic antibodies interact to varying degrees with host-cell proteins in general, and PLBL2 specifically. We then used a high-throughput Protein A chromatography method to further examine the interaction between our antibodies and PLBL2. Our results showed that the co-elution of PLBL2 during Protein A chromatography is highly dependent on the individual antibody and PLBL2 concentration in the chromatographic load. Process parameters such as antibody resin load density and pre-elution wash conditions also influence the levels of PLBL2 in the Protein A eluate. Furthermore, using surface plasmon resonance, we demonstrated that there is a preference for PLBL2 to interact with IgG4 subclass antibodies compared to IgG1 antibodies. Copyright © 2016 Elsevier B.V. All rights reserved.

Caspr2-reactive antibody cloned from a mother of an ASD child mediates an ASD-like phenotype in mice.

PubMed

Brimberg, L; Mader, S; Jeganathan, V; Berlin, R; Coleman, T R; Gregersen, P K; Huerta, P T; Volpe, B T; Diamond, B

2016-12-01

Autism spectrum disorder (ASD) occurs in 1 in 68 births, preferentially affecting males. It encompasses a group of neurodevelopmental abnormalities characterized by impaired social interaction and communication, stereotypic behaviors and motor dysfunction. Although recent advances implicate maternal brain-reactive antibodies in a causative role in ASD, a definitive assessment of their pathogenic potential requires cloning of such antibodies. Here, we describe the isolation and characterization of monoclonal brain-reactive antibodies from blood of women with brain-reactive serology and a child with ASD. We further demonstrate that male but not female mice exposed in utero to the C6 monoclonal antibody, binding to contactin-associated protein-like 2 (Caspr2), display abnormal cortical development, decreased dendritic complexity of excitatory neurons and reduced numbers of inhibitory neurons in the hippocampus, as well as impairments in sociability, flexible learning and repetitive behavior. Anti-Caspr2 antibodies are frequent in women with brain-reactive serology and a child with ASD. Together these studies provide a methodology for obtaining monclonal brain-reactive antibodies from blood B cells, demonstrate that ASD can result from in utero exposure to maternal brain-reactive antibodies of single specificity and point toward the exciting possibility of prognostic and protective strategies.

Technical note: Protozoa-specific antibodies raised in sheep plasma bind to their target protozoa in the rumen.

PubMed

Williams, Y J; Rea, S M; Popovski, S; Skillman, L C; Wright, A-D G

2014-12-01

Binding of IgG antibodies to Entodinium spp. in the rumen of sheep (Ovis aries) was investigated by adding IgG, purified from plasma, directly into the rumen. Plasma IgG was sourced from sheep that had or had not been immunized with a vaccine containing whole fixed Entodinium spp. cells. Ruminal fluid was sampled approximately 2 h after each antibody dosing. Binding of protozoa by a specific antibody was detected using an indirect fluorescent antibody test. An antibody titer in the ruminal fluid was determined by ELISA, and the concentration of ruminal fluid ammonia-N and ruminal pH were also determined. Entodinium spp. and total protozoa from IgG-infused sheep were enumerated by microscopic counts. Two-hourly additions of IgG maintained a low antibody titer in the rumen for 12 h and the binding of the antibody to the rumen protozoa was demonstrated. Increased ammonia-N concentrations and altered ruminal fluid pH patterns indicated that additional fermentation of protein was occurring in the rumen after addition of IgG. No reduction in numbers of Entodinium spp. was observed (P>0.05). Although binding of antibodies to protozoa has been demonstrated in the rumen, it is unclear how much cell death occurred. On the balance of probability, it would appear that the antibody was degraded or partially degraded, and the impact of this on protozoal populations and the measurement of a specific titer is also unclear.

Recombinant DNA technology for melanoma immunotherapy: anti-Id DNA vaccines targeting high molecular weight melanoma-associated antigen.

PubMed

Barucca, A; Capitani, M; Cesca, M; Tomassoni, D; Kazmi, U; Concetti, F; Vincenzetti, L; Concetti, A; Venanzi, F M

2014-11-01

Anti-idiotypic MK2-23 monoclonal antibody (anti-Id MK2-23 mAb), which mimics the high molecular weight melanoma-associated antigen (HMW-MAA), has been used to implement active immunotherapy against melanoma. However, due to safety and standardization issues, this approach never entered extensive clinical trials. In the present study, we investigated the usage of DNA vaccines as an alternative to MK2-23 mAb immunization. MK2-23 DNA plasmids coding for single chain (scFv) MK2-23 antibody were constructed via the insertion of variable heavy (V H) and light (V L) chains of MK2-23 into the pVAC-1mcs plasmids. Two alternative MK2-23 plasmids format V H/V L, and V L/V H were assembled. We demonstrate that both polypeptides expressed by scFv plasmids in vitro retained the ability to mimic HMW-MAA antigen, and to elicit specific anti-HMW-MAA humoral and cellular immunoresponses in immunized mice. Notably, MK2-23 scFv DNA vaccines impaired the onset and growth of transplantable B16 melanoma cells not engineered to express HMW-MAA. This pilot study suggests that optimized MK2-23 scFv DNA vaccines could potentially provide a safer and cost-effective alternative to anti-Id antibody immunization, for melanoma immunotherapy.

IDEPI: rapid prediction of HIV-1 antibody epitopes and other phenotypic features from sequence data using a flexible machine learning platform.

PubMed

Hepler, N Lance; Scheffler, Konrad; Weaver, Steven; Murrell, Ben; Richman, Douglas D; Burton, Dennis R; Poignard, Pascal; Smith, Davey M; Kosakovsky Pond, Sergei L

2014-09-01

Since its identification in 1983, HIV-1 has been the focus of a research effort unprecedented in scope and difficulty, whose ultimate goals--a cure and a vaccine--remain elusive. One of the fundamental challenges in accomplishing these goals is the tremendous genetic variability of the virus, with some genes differing at as many as 40% of nucleotide positions among circulating strains. Because of this, the genetic bases of many viral phenotypes, most notably the susceptibility to neutralization by a particular antibody, are difficult to identify computationally. Drawing upon open-source general-purpose machine learning algorithms and libraries, we have developed a software package IDEPI (IDentify EPItopes) for learning genotype-to-phenotype predictive models from sequences with known phenotypes. IDEPI can apply learned models to classify sequences of unknown phenotypes, and also identify specific sequence features which contribute to a particular phenotype. We demonstrate that IDEPI achieves performance similar to or better than that of previously published approaches on four well-studied problems: finding the epitopes of broadly neutralizing antibodies (bNab), determining coreceptor tropism of the virus, identifying compartment-specific genetic signatures of the virus, and deducing drug-resistance associated mutations. The cross-platform Python source code (released under the GPL 3.0 license), documentation, issue tracking, and a pre-configured virtual machine for IDEPI can be found at https://github.com/veg/idepi.

Recent progress in the development of anthrax vaccines.

PubMed

Kaur, Manpreet; Bhatnagar, Rakesh

2011-12-01

Bacillus anthracis is the etiological agent of anthrax. Although anthrax is primarily an epizootic disease; humans are at risk for contracting anthrax. The potential use of B. anthracis spores as biowarfare agent has led to immense attention. Prolonged vaccination schedule of current anthrax vaccine and variable protection conferred; often leading to failure of therapy. This highlights the need for alternative anthrax countermeasures. A number of approaches are being investigated to substitute or supplement the existing anthrax vaccines. These relied on expression of Protective antigen (PA), the key protective immunogen; in bacterial or plant systems; or utilization of attenuated strains of B. anthracis for immunization. Few studies have established potential of domain IV of PA for immunization. Other targets including the spore, capsule, S-layer and anthrax toxin components have been investigated for imparting protective immunity. It has been shown that co-immunization of PA with domain I of lethal factor that binds PA resulted in higher antibody responses. Of the epitope based vaccines, the loop neutralizing determinant, in particular; elicited robust neutralizing antibody response and conferred 97% protection upon challenge. DNA vaccination resulted in varying degree of protection and seems a promising approach. Additionally, the applicability of monoclonal and therapeutic antibodies in the treatment of anthrax has also been demonstrated. The recent progress in the direction of anthrax prophylaxis has been evaluated in this review.

Cell-penetrating anti-GFAP VHH and corresponding fluorescent fusion protein VHH-GFP spontaneously cross the blood-brain barrier and specifically recognize astrocytes: application to brain imaging.

PubMed

Li, Tengfei; Bourgeois, Jean-Pierre; Celli, Susanna; Glacial, Fabienne; Le Sourd, Anne-Marie; Mecheri, Salah; Weksler, Babette; Romero, Ignacio; Couraud, Pierre-Olivier; Rougeon, François; Lafaye, Pierre

2012-10-01

Antibodies normally do not cross the blood-brain barrier (BBB) and cannot bind an intracellular cerebral antigen. We demonstrate here for the first time that a new class of antibodies can cross the BBB without treatment. Camelids produce native homodimeric heavy-chain antibodies, the paratope being composed of a single-variable domain called VHH. Here, we used recombinant VHH directed against human glial fibrillary acidic protein (GFAP), a specific marker of astrocytes. Only basic VHHs (e.g., pI=9.4) were able to cross the BBB in vitro (7.8 vs. 0% for VHH with pI=7.7). By intracarotid and intravenous injections into live mice, we showed that these basic VHHs are able to cross the BBB in vivo, diffuse into the brain tissue, penetrate into astrocytes, and specifically label GFAP. To analyze their ability to be used as a specific transporter, we then expressed a recombinant fusion protein VHH-green fluorescent protein (GFP). These "fluobodies" specifically labeled GFAP on murine brain sections, and a basic variant (pI=9.3) of the fusion protein VHH-GFP was able to cross the BBB and to label astrocytes in vivo. The potential of VHHs as diagnostic or therapeutic agents in the central nervous system now deserves attention.

Human immunoglobulin allotypes

PubMed Central

Lefranc, Marie-Paule

2009-01-01

More than twenty recombinant monoclonal antibodies are approved as therapeutics. Almost all of these are based on the whole IgG isotype format, but vary in the origin of the variable regions between mouse (chimeric), humanized mouse and fully human sequences; all of those with whole IgG format employ human constant region sequences. Currently, the opposing merits of the four IgG subclasses are considered with respect to the in vivo biological activities considered to be appropriate to the disease indication being treated. Human heavy chain genes also exhibit extensive structural polymorphism(s) and, being closely linked, are inherited as a haplotype. Polymorphisms (allotypes) within the IgG isotype were originally discovered and described using serological reagents derived from humans; demonstrating that allotypic variants can be immunogenic and provoke antibody responses as a result of allo-immunization. The serologically defined allotypes differ widely within and between population groups; therefore, a mAb of a given allotype will, inevitably, be delivered to a cohort of patients homozygous for the alternative allotype. This publication reviews the serologically defined human IgG allotypes and considers the potential for allotype differences to contribute to or potentiate immunogenicity. PMID:20073133

Differential Expression of Immunogenic Proteins on Virulent Mycobacterium tuberculosis Clinical Isolates

PubMed Central

Klepp, Laura; Vazquez, Camila; Rocha, Roxana Valeria; Blanco, Federico Carlos; López, Beatriz; Bigi, Fabiana; Sasiain, María del Carmen

2014-01-01

Molecular epidemiology has revealed that Mycobacterium tuberculosis (Mtb), formerly regarded as highly conserved species, displays a considerable degree of genetic variability that can influence the outcome of the disease as well as the innate and adaptive immune response. Recent studies have demonstrated that Mtb families found worldwide today differ in pathology, transmissibility, virulence, and development of immune response. By proteomic approaches seven proteins that were differentially expressed between a local clinical isolate from Latin-American-Mediterranean (LAM) and from Haarlem (H) lineages were identified. In order to analyze the immunogenic ability, recombinant Rv2241, Rv0009, Rv0407, and Rv2624c proteins were produced for testing specific antibody responses. We found that these proteins induced humoral immune responses in patients with drug-sensitive and drug-resistant tuberculosis with substantial cross-reactivity among the four proteins. Moreover, such reactivity was also correlated with anti-Mtb-cell surface IgM, but not with anti-ManLAM, anti-PPD, or anti-Mtb-surface IgG antibodies. Therefore, the present results describe new Mtb antigens with potential application as biomarkers of TB. PMID:25105140

Efficacy of a therapeutic cocaine vaccine in rodent models.

PubMed

Fox, B S; Kantak, K M; Edwards, M A; Black, K M; Bollinger, B K; Botka, A J; French, T L; Thompson, T L; Schad, V C; Greenstein, J L; Gefter, M L; Exley, M A; Swain, P A; Briner, T J

1996-10-01

Cocaine abuse is a major medical and public health concern in the United States, with approximately 2.1 million people dependent on cocaine. Pharmacological approaches to the treatment of cocaine addiction have thus far been disappointing, and new therapies are urgently needed. This paper describes an immunological approach to cocaine addiction. Antibody therapy for neutralization of abused drugs has been described previously, including a recent paper demonstrating the induction of anti-cocaine antibodies. However, both the rapidity of entry of cocaine into the brain and the high doses of cocaine frequently encountered have created challenges for an antibody-based therapy. Here we demonstrate that antibodies are efficacious in an animal model of addiction. Intravenous cocaine self-administration in rats was inhibited by passive transfer of an anti-cocaine monoclonal antibody. To actively induce anti-cocaine antibodies, a cocaine vaccine was developed that generated a high-titer, long-lasting antibody response in mice. Immunized mice displayed a significant change in cocaine pharmacokinetics, with decreased levels of cocaine measured in the brain of immunized mice only 30 seconds after intravenous (i.v.) administration of cocaine. These data establish the feasibility of a therapeutic cocaine vaccine for the treatment of cocaine addiction.

SAbDab: the structural antibody database

PubMed Central

Dunbar, James; Krawczyk, Konrad; Leem, Jinwoo; Baker, Terry; Fuchs, Angelika; Georges, Guy; Shi, Jiye; Deane, Charlotte M.

2014-01-01

Structural antibody database (SAbDab; http://opig.stats.ox.ac.uk/webapps/sabdab) is an online resource containing all the publicly available antibody structures annotated and presented in a consistent fashion. The data are annotated with several properties including experimental information, gene details, correct heavy and light chain pairings, antigen details and, where available, antibody–antigen binding affinity. The user can select structures, according to these attributes as well as structural properties such as complementarity determining region loop conformation and variable domain orientation. Individual structures, datasets and the complete database can be downloaded. PMID:24214988

Eradication of Human Hepatic and Pulmonary Melanoma Metastases in SCID Mice by Antibody--Interleukin 2 Fusion Proteins

NASA Astrophysics Data System (ADS)

Becker, Jurgen C.; Pancook, James D.; Gillies, Stephen D.; Mendelsohn, John; Reisfeld, Ralph A.

1996-04-01

Antibody--cytokine fusion proteins combine the unique targeting ability of antibodies with the multifunctional activity of cytokines. Here, we demonstrate the therapeutic efficacy of such constructs for the treatment of hepatic and pulmonary metastases of different melanoma cell lines. Two antibody--interleukin 2 (IL-2) fusion proteins, ch225-IL2 and ch14.18-IL2, constructed by fusion of a synthetic sequence coding for human IL-2 to the carboxyl end of the Cγ 1 gene of the corresponding antibodies, were tested for their therapeutic efficacy against xenografted human melanoma in vivo. Tumorspecific fusion proteins completely inhibited the growth of hepatic and pulmonary metastases in C.B-17 scid/scid mice previously reconstituted with human lymphokine-activated killer cells, whereas treatment with combinations of the corresponding antibodies plus recombinant IL-2 only reduced the tumor load. Even when treatment with fusion proteins was delayed up to 8 days after inoculation of tumor cells, it still resulted in complete eradication of micrometastases that were established at that time point. Selection of tumor cell lines expressing or lacking the targeted antigen of the administered fusion protein proved the specificity of the observed antitumor effect. Biodistribution analysis demonstrated that the tumorspecific fusion protein accumulated not only in subcutaneous tumors but also in lungs and livers affected with micrometastases. Survival times of animals treated with the fusion protein were more than doubled as compared to those treated with the combination of the corresponding antibody plus IL-2. Our data demonstrate that an immunotherapeutic approach using cytokines targeted by antibodies to tumor sites has potent effects against disseminated human melanoma.

Oxidation in the complementarity-determining regions differentially influences the properties of therapeutic antibodies

PubMed Central

Dashivets, Tetyana; Stracke, Jan; Dengl, Stefan; Knaupp, Alexander; Pollmann, Jan; Buchner, Johannes; Schlothauer, Tilman

2016-01-01

ABSTRACT Therapeutic antibodies can undergo a variety of chemical modification reactions in vitro. Depending on the site of modification, either antigen binding or Fc-mediated functions can be affected. Oxidation of tryptophan residues is one of the post-translational modifications leading to altered antibody functionality. In this study, we examined the structural and functional properties of a therapeutic antibody construct and 2 affinity matured variants thereof. Two of the 3 antibodies carry an oxidation-prone tryptophan residue in the complementarity-determining region of the VL domain. We demonstrate the differences in the stability and bioactivity of the 3 antibodies, and reveal differential degradation pathways for the antibodies susceptible to oxidation. PMID:27612038

Dual-color Proteomic Profiling of Complex Samples with a Microarray of 810 Cancer-related Antibodies*

PubMed Central

Schröder, Christoph; Jacob, Anette; Tonack, Sarah; Radon, Tomasz P.; Sill, Martin; Zucknick, Manuela; Rüffer, Sven; Costello, Eithne; Neoptolemos, John P.; Crnogorac-Jurcevic, Tatjana; Bauer, Andrea; Fellenberg, Kurt; Hoheisel, Jörg D.

2010-01-01

Antibody microarrays have the potential to enable comprehensive proteomic analysis of small amounts of sample material. Here, protocols are presented for the production, quality assessment, and reproducible application of antibody microarrays in a two-color mode with an array of 1,800 features, representing 810 antibodies that were directed at 741 cancer-related proteins. In addition to measures of array quality, we implemented indicators for the accuracy and significance of dual-color detection. Dual-color measurements outperform a single-color approach concerning assay reproducibility and discriminative power. In the analysis of serum samples, depletion of high-abundance proteins did not improve technical assay quality. On the contrary, depletion introduced a strong bias in protein representation. In an initial study, we demonstrated the applicability of the protocols to proteins derived from urine samples. We identified differences between urine samples from pancreatic cancer patients and healthy subjects and between sexes. This study demonstrates that biomedically relevant data can be produced. As demonstrated by the thorough quality analysis, the dual-color antibody array approach proved to be competitive with other proteomic techniques and comparable in performance to transcriptional microarray analyses. PMID:20164060

Clonal expansion and somatic hypermutation of V(H) genes of B cells from cerebrospinal fluid in multiple sclerosis.

PubMed Central

Qin, Y; Duquette, P; Zhang, Y; Talbot, P; Poole, R; Antel, J

1998-01-01

The cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients is characterized by increased concentrations of immunoglobulin (Ig), which on electrophoretic analysis shows restricted heterogeneity (oligoclonal bands). CSF Ig is composed of both serum and intrathecally produced components. To examine the properties of intrathecal antibody-producing B cells, we analyzed Ig heavy-chain variable (V(H)) region genes of B cells recovered from the CSF of 12 MS patients and 15 patients with other neurological diseases (OND). Using a PCR technique, we could detect rearrangements of Ig V(H) genes in all samples. Sequence analysis of complementarity-determining region 3 (CDR3) of rearranged VDJ genes revealed expansion of a dominant clone or clones in 10 of the 12 MS patients. B cell clonal expansion was identified in 3 of 15 OND. The nucleotide sequences of V(H) genes from clonally expanded CSF B cells in MS patients demonstrated the preferential usage of the V(H) IV family. There were numerous somatic mutations, mainly in the CDRs, with a high replacement-to-silent ratio; the mutations were distributed in a way suggesting that these B cells had been positively selected through their antigen receptor. Our results demonstrate that in MS CSF, there is a high frequency of clonally expanded B cells that have properties of postgerminal center memory or antibody-forming lymphocytes. PMID:9727074

Red colored IgG4 caused by vitamin B12 from cell culture media combined with disulfide reduction at harvest

PubMed Central

Derfus, Gayle E; Dizon-Maspat, Jemelle; Broddrick, Jared T; Velayo, Arleene C; Toschi, Josh D; Santuray, Rodell T; Hsu, Stephen K; Winter, Charles M; Krishnan, Rajesh; Amanullah, Ashraf

2014-01-01

While many antibody therapeutics are formulated at low concentration (~10–20 mg/mL) for intravenous administration, high concentration (> 100 mg/mL) formulations may be required for subcutaneous delivery in certain clinical indications. For such high concentration formulations, product color is more apparent due to the higher molecular density across a given path-length. Color is therefore a product quality attribute that must be well-understood and controlled, to demonstrate process consistency and enable clinical trial blinding. Upon concentration of an IgG4 product at the 2000 L manufacturing scale, variability in product color, ranging from yellow to red, was observed. A small-scale experimental model was developed to assess the effect of processing conditions (medium composition and harvest conditions) on final bulk drug substance (BDS) color. The model was used to demonstrate that, for two distinct IgG4 products, red coloration occurred only in the presence of disulfide reduction-mediated antibody dissociation. The red color-causing component was identified as vitamin B12, in the hydroxocobalamin form, and the extent of red color was correlated with the cobalt (vitamin B12) concentration in the final pools. The intensity of redness in the final BDS was modulated by changing the concentration of vitamin B12 in the cell culture media. PMID:24552690

An unusual cysteine VL87 affects the antibody fragment conformations without interfering with the disulfide bond formation.

PubMed

Attallah, Carolina; Aguilar, María Fernanda; Garay, A Sergio; Herrera, Fernando E; Etcheverrigaray, Marina; Oggero, Marcos; Rodrigues, Daniel E

2017-10-01

The Cys residues are almost perfectly conserved in all antibodies. They contribute significantly to the antibody fragment stability. The relevance of two natural contiguous Cys residues of an anti-recombinant human-follicle stimulation hormone (rhFSH) in a format of single-chain variable fragment (scFv) was studied. This scFv contains 5 Cys residues: V H 22 and V H 92 in the variable heavy chain (V H ) and V L 23, V L 87 and V L 88 in the variable light chain (V L ). The influence of two unusual contiguous Cys at positions V L 87 and V L 88 was studied by considering the wild type fragment and mutant variants: V L -C88S, V L -C87S, V L -C87Y. The analysis was carried out using antigen-binding ability measurement by indirect specific ELISA and a detailed molecular modeling that comprises homology methods, long molecular dynamics simulations and docking. We found that V L -C87 affected the antibody fragment stability without interfering with the disulfide bond formation. The effect of mutating the V L -C87 by a usual residue at this position like Tyr caused distant structural changes at the V H region that confers a higher mobility to the V H -CDR2 and V H -CDR3 loops improving the scFv binding to the antigen. Copyright © 2017 Elsevier Ltd. All rights reserved.

Recognition of Naegleriae ameba surface protein epitopes by anti-human CD45 antibodies.

PubMed

Ravine, Terrence J; Polski, Jacek M; Jenkins, James

2010-04-01

Phagocytosis is a highly conserved mechanism exhibited by both free-living amebas and mammalian blood cells. Similarities demonstrated by either cell type during engulfment of the same bacterial species may imply analogous surface proteins involved in receptor-mediated endocytosis. The increased availability of anti-human leukocyte antibodies or clusters of differentiation (CD) markers used in conjunction with flow cytometric (FCM) and/or immunohistochemical (IHC) analysis provides investigators with a relatively easy method to screen different cell populations for comparable plasma membrane proteins. In this study, we incubated Naegleria and Acanthamoeba amebas with several directly conjugated anti-human leukocyte monoclonal antibodies (mAb) for similarly recognized amebic epitopes. CD marker selection was based upon a recognized role of each mAb in phagocyte activation and/or uptake of bacteria. These included CD14, CD45, and CD206. In FCM, only one CD45 antibody demonstrated strong reactivity with both Naegleria fowleri and Naegleria gruberi that was not expressed in similarly tested Acanthamoeba species. Additional testing of N. gruberi by IHC demonstrated reactivity to a different CD45 antibody. Our results suggest a possible utility of using anti-human leukocyte antibodies to screen amebic cells for similarly expressed protein epitopes. In doing so, several important items must be considered when selecting potential mAbs for testing to increase the probability of a positive result.

Purification of monoclonal antibodies from clarified cell culture fluid using Protein A capture continuous countercurrent tangential chromatography

PubMed Central

Dutta, Amit K.; Tran, Travis; Napadensky, Boris; Teella, Achyuta; Brookhart, Gary; Ropp, Philip A.; Zhang, Ada W.; Tustian, Andrew D.; Zydney, Andrew L.; Shinkazh, Oleg

2015-01-01

Recent studies using simple model systems have demonstrated that Continuous Countercurrent Tangential Chromatography (CCTC) has the potential to overcome many of the limitations of conventional Protein A chromatography using packed columns. The objective of this work was to optimize and implement a CCTC system for monoclonal antibody purification from clarified Chinese Hamster Ovary (CHO) cell culture fluid using a commercial Protein A resin. Several improvements were introduced to the previous CCTC system including the use of retentate pumps to maintain stable resin concentrations in the flowing slurry, the elimination of a slurry holding tank to improve productivity, and the introduction of an “after binder” to the binding step to increase antibody recovery. A kinetic binding model was developed to estimate the required residence times in the multi-stage binding step to optimize yield and productivity. Data were obtained by purifying two commercial antibodies from two different manufactures, one with low titer (~0.67 g/L) and one with high titer (~6.9 g/L), demonstrating the versatility of the CCTC system. Host cell protein removal, antibody yields and purities were similar to that obtained with conventional column chromatography; however, the CCTC system showed much higher productivity. These results clearly demonstrate the capabilities of continuous countercurrent tangential chromatography for the commercial purification of monoclonal antibody products. PMID:25747172

Purification of monoclonal antibodies from clarified cell culture fluid using Protein A capture continuous countercurrent tangential chromatography.

PubMed

Dutta, Amit K; Tran, Travis; Napadensky, Boris; Teella, Achyuta; Brookhart, Gary; Ropp, Philip A; Zhang, Ada W; Tustian, Andrew D; Zydney, Andrew L; Shinkazh, Oleg

2015-11-10

Recent studies using simple model systems have demonstrated that continuous countercurrent tangential chromatography (CCTC) has the potential to overcome many of the limitations of conventional Protein A chromatography using packed columns. The objective of this work was to optimize and implement a CCTC system for monoclonal antibody purification from clarified Chinese Hamster Ovary (CHO) cell culture fluid using a commercial Protein A resin. Several improvements were introduced to the previous CCTC system including the use of retentate pumps to maintain stable resin concentrations in the flowing slurry, the elimination of a slurry holding tank to improve productivity, and the introduction of an "after binder" to the binding step to increase antibody recovery. A kinetic binding model was developed to estimate the required residence times in the multi-stage binding step to optimize yield and productivity. Data were obtained by purifying two commercial antibodies from two different manufactures, one with low titer (∼ 0.67 g/L) and one with high titer (∼ 6.9 g/L), demonstrating the versatility of the CCTC system. Host cell protein removal, antibody yields and purities were similar to those obtained with conventional column chromatography; however, the CCTC system showed much higher productivity. These results clearly demonstrate the capabilities of continuous countercurrent tangential chromatography for the commercial purification of monoclonal antibody products. Copyright © 2015 Elsevier B.V. All rights reserved.

Defect in IgV gene somatic hypermutation in common variable immuno-deficiency syndrome.

PubMed

Levy, Y; Gupta, N; Le Deist, F; Garcia, C; Fischer, A; Weill, J C; Reynaud, C A

1998-10-27

Common Variable Immuno-Deficiency (CVID) is the most common symptomatic primary antibody-deficiency syndrome, but the basic immunologic defects underlying this syndrome are not well defined. We report here that among eight patients studied (six CVID and two hypogammaglobulinemic patients with recurrent infections), there is in two CVID patients a dramatic reduction in Ig V gene somatic hypermutation with 40-75% of IgG transcripts totally devoid of mutations in the circulating memory B cell compartment. Functional assays of the T cell compartment point to an intrinsic B cell defect in the process of antibody affinity maturation in these two cases.

Detection of Chlamydophila psittaci antibodies from captive birds at the Ninoy Aquino Parks and Wildlife Nature Center, Quezon City, Philippines.

PubMed

Maluping, Ramon P; Oronan, Rey B; Toledo, Steven U

2007-01-01

The present study was undertaken to detect the presence of Chlamydophila psittaci antibodies in captive birds at the Wildlife Rescue Center, Ninoy Aquino Parks and Wildlife Nature Center, Quezon City, Philippines. Blood was collected from 36 birds of different species and the presence of antibodies against C. psittaci was detected using an ELISA-based test kit. 25% of the samples demonstrated antibodies against C. psittaci. The results of this study confirmed the presence of C. psittaci antibodies among the captive birds examined.

[Immunodetection of bacteriophages by a piezoelectric resonator with lateral electric field].

PubMed

Gulii, O I; Zaitsev, B D; Shikhabudinov, A M; Teplykh, A A; Borodina, I A; Pavlii, S A; Larionova, O S; Fomin, A S; Staroverov, S A; Dykman, L A; Ignatov, O V

2016-01-01

It has been demonstrated that electroacoustic analysis with polyclonal antibodies can be used for bacteriophage detection. The frequency dependences of the real and imaginary parts of electrical impedance of a resonator with a viral suspension with antibodies were shown to be essentially different from the dependences of a resonator with control viral suspension without antibodies. It was shown that ΦAl-Sp59b bacteriophages were detected with the use of antibodies in the presence of foreign virus particles. The ΦAl-Sp59b bacteriophage content in the analyzed suspension was ~1010–106 phages/mL; the time of analysis was no more than 5 min. The optimally informative parameter for obtaining reliable information was the change in the real or imaginary part of electrical impedance at a fixed frequency near the resonance upon the addition of specific antibodies to the analyzed suspension. It was demonstrated that the interaction between bacteriophages and antibodies can be recorded, offering good prospects for the development of a biological sensor for liquid-phase identification and virus detection.

Balancing Selectivity and Efficacy of Bispecific Epidermal Growth Factor Receptor (EGFR) × c-MET Antibodies and Antibody-Drug Conjugates*

PubMed Central

Sellmann, Carolin; Doerner, Achim; Knuehl, Christine; Rasche, Nicolas; Sood, Vanita; Krah, Simon; Rhiel, Laura; Messemer, Annika; Wesolowski, John; Schuette, Mark; Becker, Stefan; Toleikis, Lars; Kolmar, Harald; Hock, Bjoern

2016-01-01

Bispecific antibodies (bsAbs) and antibody-drug conjugates (ADCs) have already demonstrated benefits for the treatment of cancer in several clinical studies, showing improved drug selectivity and efficacy. In particular, simultaneous targeting of prominent cancer antigens, such as EGF receptor (EGFR) and c-MET, by bsAbs has raised increasing interest for potentially circumventing receptor cross-talk and c-MET-mediated acquired resistance during anti-EGFR monotherapy. In this study, we combined the selectivity of EGFR × c-MET bsAbs with the potency of cytotoxic agents via bispecific antibody-toxin conjugation. Affinity-attenuated bispecific EGFR × c-MET antibody-drug conjugates demonstrated high in vitro selectivity toward tumor cells overexpressing both antigens and potent anti-tumor efficacy. Due to basal EGFR expression in the skin, ADCs targeting EGFR in general warrant early safety assessments. Reduction in EGFR affinity led to decreased toxicity in keratinocytes. Thus, the combination of bsAb affinity engineering with the concept of toxin conjugation may be a viable route to improve the safety profile of ADCs targeting ubiquitously expressed antigens. PMID:27694443

Seroprevalence of Toxoplasma gondii antibodies in humans from rural Western Amazon, Brazil.

PubMed

Cavalcante, G T; Aguilar, D M; Camargo, L M A; Labruna, M B; de Andrade, H F; Meireles, L R; Dubey, J P; Thulliez, P; Dias, R A; Gennari, S M

2006-06-01

Antibodies to Toxoplasma gondii were assayed in sera of 266 humans from 71 farms located at Rondônia State, Western Amazon, Brazil, by the modified agglutination test (MAT) and the indirect immunofluorescent antibody test (IFAT). Antibodies were found in 195 humans (73.3%), with MAT titers of 1:25 in 11, 1:50 in 11, 1:100 in 16, 1:200 in 27, 1:400 in 38, 1:800 in 37, 1:1,600 in 22, and 1:3,200 or higher in 33. From the 71 farms visited, 69 had seropositive humans. Prevalence of anti-T. gondii antibodies increased with age of the people (P < 0.05), and no difference was observed in the occurrence by gender (P > 0.05). A sanitary questionnaire was applied in each farm, and statistical association between the serologic status and several variables were analyzed. Home-grown vegetable consumption and origin of drinking water (well or river) were the independent variables that displayed significant association (P = 0.002 and 0.02, respectively). Higher values of occurrence were found in people with consumption of home-grown vegetables (76.1%) and people that drink well water (75.4%) compared with people that did not consume this type of food (61.9%) and drink river water (55.2%). By IFAT (> or = 1:16), 194 of 266 (73%) humans were seropositive and there was a good correlation between MAT and IFAT.

Effects of Darwinian Selection and Mutability on Rate of Broadly Neutralizing Antibody Evolution during HIV-1 Infection

PubMed Central

Sheng, Zizhang; Schramm, Chaim A.; Connors, Mark; Morris, Lynn; Mascola, John R.; Kwong, Peter D.; Shapiro, Lawrence

2016-01-01

Accumulation of somatic mutations in antibody variable regions is critical for antibody affinity maturation, with HIV-1 broadly neutralizing antibodies (bnAbs) generally requiring years to develop. We recently found that the rate at which mutations accumulate decreases over time, but the mechanism governing this slowing is unclear. In this study, we investigated whether natural selection and/or mutability of the antibody variable region contributed significantly to observed decrease in rate. We used longitudinally sampled sequences of immunoglobulin transcripts of single lineages from each of 3 donors, as determined by next generation sequencing. We estimated the evolutionary rates of the complementarity determining regions (CDRs), which are most significant for functional selection, and found they evolved about 1.5- to 2- fold faster than the framework regions. We also analyzed the presence of AID hotspots and coldspots at different points in lineage development and observed an average decrease in mutability of less than 10 percent over time. Altogether, the correlation between Darwinian selection strength and evolutionary rate trended toward significance, especially for CDRs, but cannot fully explain the observed changes in evolutionary rate. The mutability modulated by AID hotspots and coldspots changes correlated only weakly with evolutionary rates. The combined effects of Darwinian selection and mutability contribute substantially to, but do not fully explain, evolutionary rate change for HIV-1-targeting bnAb lineages. PMID:27191167

Bayesian analysis and classification of two Enzyme-Linked Immunosorbent Assay (ELISA) tests without a gold standard

PubMed Central

Zhang, Jingyang; Chaloner, Kathryn; McLinden, James H.; Stapleton, Jack T.

2013-01-01

Reconciling two quantitative ELISA tests for an antibody to an RNA virus, in a situation without a gold standard and where false negatives may occur, is the motivation for this work. False negatives occur when access of the antibody to the binding site is blocked. Based on the mechanism of the assay, a mixture of four bivariate normal distributions is proposed with the mixture probabilities depending on a two-stage latent variable model including the prevalence of the antibody in the population and the probabilities of blocking on each test. There is prior information on the prevalence of the antibody, and also on the probability of false negatives, and so a Bayesian analysis is used. The dependence between the two tests is modeled to be consistent with the biological mechanism. Bayesian decision theory is utilized for classification. The proposed method is applied to the motivating data set to classify the data into two groups: those with and those without the antibody. Simulation studies describe the properties of the estimation and the classification. Sensitivity to the choice of the prior distribution is also addressed by simulation. The same model with two levels of latent variables is applicable in other testing procedures such as quantitative polymerase chain reaction tests where false negatives occur when there is a mutation in the primer sequence. PMID:23592433

A droplet-based heterogeneous immunoassay for screening single cells secreting antigen-specific antibodies.

PubMed

Akbari, Samin; Pirbodaghi, Tohid

2014-09-07

High throughput heterogeneous immunoassays that screen antigen-specific antibody secreting cells are essential to accelerate monoclonal antibody discovery for therapeutic applications. Here, we introduce a heterogeneous single cell immunoassay based on alginate microparticles as permeable cell culture chambers. Using a microfluidic device, we encapsulated single antibody secreting cells in 35-40 μm diameter alginate microbeads. We functionalized the alginate to capture the secreted antibodies inside the microparticles, enabling single cell analysis and preventing the cross-talk between the neighboring encapsulated cells. We demonstrated non-covalent functionalization of alginate microparticles by adding three secondary antibodies to the alginate solution to form high molecular weight complexes that become trapped in the porous nanostructure of alginate and capture the secreted antibodies. We screened anti-TNF-alpha antibody-secreting cells from a mixture of antibody-secreting cells.

De novo sequencing and resurrection of a human astrovirus-neutralizing antibody

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bogdanoff, Walter A.; Morgenstern, David; Bern, Marshall

Monoclonal antibody (mAb) therapeutics targeting cancer, autoimmune diseases, inflammatory diseases, and infectious diseases are growing exponentially. Although numerous panels of mAbs targeting infectious disease agents have been developed, their progression into clinically useful mAbs is often hindered by the lack of sequence information and/or loss of hybridoma cells that produce them. Here we combine the power of crystallography and mass spectrometry to determine the amino acid sequence and glycosylation modification of the Fab fragment of a potent human astrovirus-neutralizing mAb. We used this information to engineer a recombinant antibody single-chain variable fragment that has the same specificity as the parentmore » monoclonal antibody to bind to the astrovirus capsid protein. Furthermore, this antibody can now potentially be developed as a therapeutic and diagnostic agent.« less

De novo sequencing and resurrection of a human astrovirus-neutralizing antibody

DOE PAGES

Bogdanoff, Walter A.; Morgenstern, David; Bern, Marshall; ...

2016-03-14

Monoclonal antibody (mAb) therapeutics targeting cancer, autoimmune diseases, inflammatory diseases, and infectious diseases are growing exponentially. Although numerous panels of mAbs targeting infectious disease agents have been developed, their progression into clinically useful mAbs is often hindered by the lack of sequence information and/or loss of hybridoma cells that produce them. Here we combine the power of crystallography and mass spectrometry to determine the amino acid sequence and glycosylation modification of the Fab fragment of a potent human astrovirus-neutralizing mAb. We used this information to engineer a recombinant antibody single-chain variable fragment that has the same specificity as the parentmore » monoclonal antibody to bind to the astrovirus capsid protein. Furthermore, this antibody can now potentially be developed as a therapeutic and diagnostic agent.« less

A residue-specific shift in stability and amyloidogenicity of antibody variable domains.

PubMed

Nokwe, Cardine N; Zacharias, Martin; Yagi, Hisashi; Hora, Manuel; Reif, Bernd; Goto, Yuji; Buchner, Johannes

2014-09-26

Variable (V) domains of antibodies are essential for antigen recognition by our adaptive immune system. However, some variants of the light chain V domains (VL) form pathogenic amyloid fibrils in patients. It is so far unclear which residues play a key role in governing these processes. Here, we show that the conserved residue 2 of VL domains is crucial for controlling its thermodynamic stability and fibril formation. Hydrophobic side chains at position 2 stabilize the domain, whereas charged residues destabilize and lead to amyloid fibril formation. NMR experiments identified several segments within the core of the VL domain to be affected by changes in residue 2. Furthermore, molecular dynamic simulations showed that hydrophobic side chains at position 2 remain buried in a hydrophobic pocket, and charged side chains show a high flexibility. This results in a predicted difference in the dissociation free energy of ∼10 kJ mol(-1), which is in excellent agreement with our experimental values. Interestingly, this switch point is found only in VL domains of the κ family and not in VLλ or in VH domains, despite a highly similar domain architecture. Our results reveal novel insight into the architecture of variable domains and the prerequisites for formation of amyloid fibrils. This might also contribute to the rational design of stable variable antibody domains. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Nanoparticles for the delivery of therapeutic antibodies: Dogma or promising strategy?

PubMed

Sousa, Flávia; Castro, Pedro; Fonte, Pedro; Kennedy, Patrick J; Neves-Petersen, Maria Teresa; Sarmento, Bruno

2017-10-01

Over the past two decades, therapeutic antibodies have demonstrated promising results in the treatment of a wide array of diseases. However, the application of antibody-based therapy implies multiple administrations and a high cost of antibody production, resulting in costly therapy. Another disadvantage inherent to antibody-based therapy is the limited stability of antibodies and the low level of tissue penetration. The use of nanoparticles as delivery systems for antibodies allows for a reduction in antibody dosing and may represent a suitable alternative to increase antibody stability Areas covered: We discuss different nanocarriers intended for the delivery of antibodies as well as the corresponding encapsulation methods. Recent developments in antibody nanoencapsulation, particularly the possible toxicity issues that may arise from entrapment of antibodies into nanocarriers, are also assessed. In addition, this review will discuss the alterations in antibody structure and bioactivity that occur with nanoencapsulation. Expert opinion: Nanocarriers can protect antibodies from degradation, ensuring superior bioavailability. Encapsulation of therapeutic antibodies may offer some advantages, including potential targeting, reduced immunogenicity and controlled release. Furthermore, antibody nanoencapsulation may aid in the incorporation of the antibodies into the cells, if intracellular components (e.g. intracellular enzymes, oncogenic proteins, transcription factors) are to be targeted.

Thermodynamics of antibody-antigen interaction revealed by mutation analysis of antibody variable regions.

PubMed

Akiba, Hiroki; Tsumoto, Kouhei

2015-07-01

Antibodies (immunoglobulins) bind specific molecules (i.e. antigens) with high affinity and specificity. In order to understand their mechanisms of recognition, interaction analysis based on thermodynamic and kinetic parameters, as well as structure determination is crucial. In this review, we focus on mutational analysis which gives information about the role of each amino acid residue in antibody-antigen interaction. Taking anti-hen egg lysozyme antibodies and several anti-small molecule antibodies, the energetic contribution of hot-spot and non-hot-spot residues is discussed in terms of thermodynamics. Here, thermodynamics of the contribution from aromatic, charged and hydrogen bond-forming amino acids are discussed, and their different characteristics have been elucidated. The information gives fundamental understanding of the antibody-antigen interaction. Furthermore, the consequences of antibody engineering are analysed from thermodynamic viewpoints: humanization to reduce immunogenicity and rational design to improve affinity. Amino acid residues outside hot-spots in the interface play important roles in these cases, and thus thermodynamic and kinetic parameters give much information about the antigen recognition. Thermodynamic analysis of mutant antibodies thus should lead to advanced strategies to design and select antibodies with high affinity. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Investigation of a panel of monoclonal antibodies and polyclonal sera against anthrax toxins resulted in identification of an anti-lethal factor antibody with disease-enhancing characteristics.

PubMed

Kulshreshtha, Parul; Tiwari, Ashutosh; Priyanka; Joon, Shikha; Sinha, Subrata; Bhatnagar, Rakesh

2015-12-01

Hybridomas were created using spleen of mice that were actively immunized with rLFn (recombinant N-terminal domain of lethal factor). Later on, separate group of mice were immunized with rLFn to obtain a polyclonal control for passive immunization studies of monoclonal antibodies. This led to the identification of one cohort of rLFn-immnized mice that harboured disease-enhancing polyclonal antibodies. At the same time, the monoclonal antibodies secreted by all the hybridomas were being tested. Two hybridomas secreted monoclonal antibodies (H10 and H8) that were cross-reactive with EF (edema factor) and LF (lethal factor), while the other two hybridomas secreted LF-specific antibodies (H7 and H11). Single chain variable fragment (LETscFv) was derived from H10 hybridoma. H11 was found to have disease-enhancing property. Combination of H11 with protective monoclonal antibodies (H8 and H10) reduced its disease enhancing nature. This in vitro abrogation of disease-enhancement provides the proof of concept that in polyclonal sera the disease enhancing character of a fraction of antibodies is overshadowed by the protective nature of the rest of the antibodies generated on active immunization. Copyright © 2015. Published by Elsevier Ltd.

Single chain variable fragment antibodies block aggregation and toxicity induced by familial ALS-linked mutant forms of SOD1.

PubMed

Ghadge, Ghanashyam D; Pavlovic, John D; Koduvayur, Sujatha P; Kay, Brian K; Roos, Raymond P

2013-08-01

Approximately 10% of amyotrophic lateral sclerosis (ALS) cases are familial (known as FALS) with an autosomal dominant inheritance pattern, and ~25% of FALS cases are caused by mutations in Cu/Zn superoxide dismutase (SOD1). There is convincing evidence that mutant SOD1 (mtSOD1) kills motor neurons (MNs) because of a gain-of-function toxicity, most likely related to aggregation of mtSOD1. A number of recent reports have suggested that antibodies can be used to treat mtSOD1-induced FALS. To follow up on the use of antibodies as potential therapeutics, we generated single chain fragments of variable region antibodies (scFvs) against SOD1, and then expressed them as 'intrabodies' within a motor neuron cell line. In the present study, we describe isolation of human scFvs that interfere with mtSOD1 in vitro aggregation and toxicity. These scFvs may have therapeutic potential in sporadic ALS, as well as FALS, given that sporadic ALS may also involve abnormalities in the SOD1 protein or activity. Copyright © 2013 Elsevier Inc. All rights reserved.

Blocking monocyte transmigration in in vitro system by an anti-CD99 human antibody in single chain fragment variable (scFv) format. Efficient large scale purification of biological active scFv from inclusion bodies in E. coli expression system

PubMed Central

Moricoli, Diego; Muller, William A.; Carbonella, Damiano Cosimo; Balducci, Maria Cristina; Dominici, Sabrina; Fiori, Valentina; Watson, Richard; Weber, Evan; Cianfriglia, Maurizio; Scotlandi, Katia; Magnani, Mauro

2015-01-01

Migration of leukocytes into a site of inflammation involves several steps mediated by various families of adhesion molecules. CD99 play a significant role in transendothelial migration (TEM) of leukocytes. Inhibition of TEM by specific monoclonal antibody (mAb) can provide a potent therapeutic approach to treating inflammatory conditions. However, the therapeutic utilization of whole IgG can lead to an inappropriate activation of Fc receptor-expressing cells inducing serious adverse side effects due to cytokine release. In this regard, specific recombinant antibody in single chain variable fragments (scFvs) originated by phage library may offer a solution by affecting TEM function in a safe clinical context. However, this consideration requires large scale production of functional scFv antibodies under GMP conditions and hence, the absence of toxic reagents utilized for the solubilization and refolding steps of inclusion bodies that may discourage industrial application of these antibody fragments. In order to apply the scFv anti-CD99 named C7A in a clinical setting we herein describe an efficient and large scale production of the antibody fragments expressed in E.coli as insoluble protein avoiding gel filtration chromatography approach, and laborious refolding step pre- and post-purification. Using differential salt elution which is a simple, reproducible and effective procedure we are able to separate scFv in monomer format from aggregates. The purified scFv antibody C7A exhibits inhibitory activity comparable to an antagonistic conventional mAb, thus providing an excellent agent for blocking CD99 signalling. Thanks to the original purification protocol that can be extended to other scFvs that are expressed as inclusion bodies in bacterial systems, the scFv anti-CD99 C7A herein described represents the first step towards the construction of new antibody therapeutic. PMID:24798881

Screening for single-chain variable fragment antibodies against multiple Cry1 toxins from an immunized mouse phage display antibody library.

PubMed

Dong, Sa; Bo, Zongyi; Zhang, Cunzheng; Feng, Jianguo; Liu, Xianjin

2018-04-01

Single-chain variable fragment (scFv) is a kind of antibody that possess only one chain of the complete antibody while maintaining the antigen-specific binding abilities and can be expressed in prokaryotic system. In this study, scFvs against Cry1 toxins were screened out from an immunized mouse phage displayed antibody library, which was successfully constructed with capacity of 6.25 × 10 7  CFU/mL. Using the mixed and alternative antigen coating strategy and after four rounds of affinity screening, seven positive phage-scFvs against Cry1 toxins were selected and characterized. Among them, clone scFv-3H9 (MG214869) showing relative stable and high binding abilities to six Cry1 toxins was selected for expression and purification. SDS-PAGE indicated that the scFv-3H9 fragments approximately 27 kDa were successfully expressed in Escherichia coli HB2151 strain. The purified scFv-3H9 was used to establish the double antibody sandwich enzyme-linked immunosorbent assay method (DAS-ELISA) for detecting six Cry1 toxins, of which the lowest detectable limits (LOD) and the lowest quantitative limits (LOQ) were 3.14-11.07 and 8.22-39.44 ng mL -1 , respectively, with the correlation coefficient higher than 0.997. The average recoveries of Cry1 toxins from spiked rice leaf samples were ranged from 84 to 95%, with coefficient of variation (CV) less than 8.2%, showing good accuracy for the multi-residue determination of six Cry1 toxins in agricultural samples. This research suggested that the constructed phage display antibody library based on the animal which was immunized with the mixture of several antigens under the same category can be used for the quick and effective screening of generic antibodies.

Anti-H antibody of unusually high titer showing variable reactivities against group A red cells and broad thermal amplitude in a patient with lymphoma.

PubMed

Choi, Seung Jun; Kim, Hoon Seok; Lee, Jaewoong; Song, Young-Sun; Lim, Jihyang

2018-02-01

We report a case of a patient with high titer anti-H antibody showing broad thermal amplitude and variable reactivities against group A red cells. A 62-year-old Korean female was diagnosed with diffuse large B cell lymphoma involving multiple organs. Her ABO/RhD type was A+ and her genotype was ABO*A.01.01/ABO*O.01.02. Antibody screening test (AST) and antibody identification test (IDT) were strongly positive for all reagent cells. Anti-human globulin (AHG) test revealed an antibody titer of 1:256 for 37 °C phase and trace positivity for poly- and mono-specific C3d. Reactivity was stronger for O+ red cells than that for A+ red cells across all temperatures tested (4 °C, room temperature (RT) and 37 °C). This was also found for AHG phase. Anti-IH was ruled out based on agglutination of O+ cord cells (CCs). Antibody was determined as IgM anti-H after DTT treatment. Three batches of 10 A+ red cells from random donors were tested with three consecutive serums for crossmatching using tube method. Interestingly, out of thirty A+ red cells tested, 20 cells at RT, 11 cells at 37 °C and 11 cells in the AHG phase showed reactivity of greater than 2+. The patient was transfused with 6 units of packed RBCs subsequently. Chemotherapy (R-CHOP regimen) and Helicobacter pylori eradication were then started. Her antibody titer gradually decreased following such treatment. In conclusion, we identified a case of patient with high titer anti-H with broad thermal amplitude, suggesting that anti-H antibodies might need to be considered for cases with pan-agglutination in AST and IDT. Copyright © 2018 Elsevier Ltd. All rights reserved.

Polyclonal and monoclonal antibodies in clinic.

PubMed

Wootla, Bharath; Denic, Aleksandar; Rodriguez, Moses

2014-01-01

Immunoglobulins (Ig) or antibodies are heavy plasma proteins, with sugar chains added to amino-acid residues by N-linked glycosylation and occasionally by O-linked glycosylation. The versatility of antibodies is demonstrated by the various functions that they mediate such as neutralization, agglutination, fixation with activation of complement and activation of effector cells. Naturally occurring antibodies protect the organism against harmful pathogens, viruses and infections. In addition, almost any organic chemical induces antibody production of antibodies that would bind specifically to the chemical. These antibodies are often produced from multiple B cell clones and referred to as polyclonal antibodies. In recent years, scientists have exploited the highly evolved machinery of the immune system to produce structurally and functionally complex molecules such as antibodies from a single B clone, heralding the era of monoclonal antibodies. Most of the antibodies currently in the clinic, target components of the immune system, are not curative and seek to alleviate symptoms rather than cure disease. Our group used a novel strategy to identify reparative human monoclonal antibodies distinct from conventional antibodies. In this chapter, we discuss the therapeutic relevance of both polyclonal and monoclonal antibodies in clinic.

Growth inhibition of tumor cells in vitro by using monoclonal antibodies against gonadotropin-releasing hormone receptor.

PubMed

Lee, Gregory; Ge, Bixia

2010-07-01

As the continuation of a previous study, synthetic peptides corresponding to the extracellular domains of human gonadotropin-releasing hormone (GnRH) receptor were used to generate additional monoclonal antibodies which were further characterized biochemically and immunologically. Among those identified to recognize GnRH receptor, monoclonal antibodies designated as GHR-103, GHR-106 and GHR-114 were found to exhibit high affinity (Kd < or = 1 x 10(-8) M) and specificity to GnRH receptor as judged by the whole cell binding immunoassay and Western blot assay. Both anti-GnRH receptor monoclonal antibodies and GnRH were shown to compete for the same binding site of GnRH receptor on the surface of cultured cancer cells. Growth inhibitions of cancer cells cultured in vitro were demonstrated by cellular apoptosis experiments (TUNEL and MTT assays) under different conditions of treatment with GHR-106 monoclonal antibody or GnRH analogs. It was generally observed that both GnRH I and GHR-106 effectively induce the apoptosis of cultured cancer cells as determined by TUNEL and MTT assays. Consistently, suppressions of gene expressions at mRNA levels were demonstrated with several ribosomal proteins (P0, P1, P2 and L37), when cancer cells were incubated with GnRH or GHR-106. The widespread expressions of GnRH receptor in almost all of the studied human cancer cell lines were also demonstrated by RT-PCR and Western blot assay, as well as indirect immunofluorescence assay with either of these monoclonal antibodies as the primary antibody. In view of the longer half life of antibodies as compared to that of GnRH or its analogs, anti-GnRH receptor monoclonal antibodies in humanized forms could function as GnRH analogs and serve as an ideal candidate of anti-cancer drugs for therapeutic treatments of various cancers in humans as well as for fertility regulations.

Silk micrococoons for protein stabilisation and molecular encapsulation

NASA Astrophysics Data System (ADS)

Shimanovich, Ulyana; Ruggeri, Francesco S.; de Genst, Erwin; Adamcik, Jozef; Barros, Teresa P.; Porter, David; Müller, Thomas; Mezzenga, Raffaele; Dobson, Christopher M.; Vollrath, Fritz; Holland, Chris; Knowles, Tuomas P. J.

2017-07-01

Naturally spun silks generate fibres with unique properties, including strength, elasticity and biocompatibility. Here we describe a microfluidics-based strategy to spin liquid native silk, obtained directly from the silk gland of Bombyx mori silkworms, into micron-scale capsules with controllable geometry and variable levels of intermolecular β-sheet content in their protein shells. We demonstrate that such micrococoons can store internally the otherwise highly unstable liquid native silk for several months and without apparent effect on its functionality. We further demonstrate that these native silk micrococoons enable the effective encapsulation, storage and release of other aggregation-prone proteins, such as functional antibodies. These results show that native silk micrococoons are capable of preserving the full activity of sensitive cargo proteins that can aggregate and lose function under conditions of bulk storage, and thus represent an attractive class of materials for the storage and release of active biomolecules.

Monoclonal Antibodies Attached to Carbon Nanotube Transistors for Paclitaxel Detection

NASA Astrophysics Data System (ADS)

Lee, Wonbae; Lau, Calvin; Richardson, Mark; Rajapakse, Arith; Weiss, Gregory; Collins, Philip; UCI, Molecular Biology; Biochemistry Collaboration; UCI, Departments of Physics; Astronomy Collaboration

Paclitaxel is a naturally-occurring pharmaceutical used in numerous cancer treatments, despite its toxic side effects. Partial inhibition of this toxicity has been demonstrated using weakly interacting monoclonal antibodies (3C6 and 8A10), but accurate monitoring of antibody and paclitaxel concentrations remains challenging. Here, single-molecule studies of the kinetics of antibody-paclitaxel interactions have been performed using single-walled carbon nanotube field-effect transistors. The devices were sensitized with single antibody attachments to record the single-molecule binding dynamics of paclitaxel. This label-free technique recorded a range of dynamic interactions between the antibody and paclitaxel, and it provided sensitive paclitaxel detection for pM to nM concentrations. Measurements with two different antibodies suggest ways of extending this working range and uncovering the mechanistic differences among different antibodies.

Dengue virus specific IgY provides protection following lethal dengue virus challenge and is neutralizing in the absence of inducing antibody dependent enhancement

PubMed Central

Williams, Katherine L.; Harris, Eva; Alvine, Travis D.; Henderson, Thomas; Schiltz, James; Nilles, Matthew L.; Bradley, David S.

2017-01-01

Dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are severe disease manifestations that can occur following sequential infection with different dengue virus serotypes (DENV1-4). At present, there are no licensed therapies to treat DENV-induced disease. DHF and DSS are thought to be mediated by serotype cross-reactive antibodies that facilitate antibody-dependent enhancement (ADE) by binding to viral antigens and then Fcγ receptors (FcγR) on target myeloid cells. Using genetically engineered DENV-specific antibodies, it has been shown that the interaction between the Fc portion of serotype cross-reactive antibodies and FcγR is required to induce ADE. Additionally, it was demonstrated that these antibodies were as neutralizing as their non-modified variants, were incapable of inducing ADE, and were therapeutic following a lethal, antibody-enhanced infection. Therefore, we hypothesized that avian IgY, which do not interact with mammalian FcγR, would provide a novel therapy for DENV-induced disease. We demonstrate here that goose-derived anti-DENV2 IgY neutralized DENV2 and did not induce ADE in vitro. Anti-DENV2 IgY was also protective in vivo when administered 24 hours following a lethal DENV2 infection. We were also able to demonstrate via epitope mapping that both full-length and alternatively spliced anti-DENV2 IgY recognized different epitopes, including epitopes that have not been previously identified. These observations provide evidence for the potential therapeutic applications of goose-derived anti-DENV2 IgY. PMID:28686617

Dengue virus specific IgY provides protection following lethal dengue virus challenge and is neutralizing in the absence of inducing antibody dependent enhancement.

PubMed

Fink, Ashley L; Williams, Katherine L; Harris, Eva; Alvine, Travis D; Henderson, Thomas; Schiltz, James; Nilles, Matthew L; Bradley, David S

2017-07-01

Dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are severe disease manifestations that can occur following sequential infection with different dengue virus serotypes (DENV1-4). At present, there are no licensed therapies to treat DENV-induced disease. DHF and DSS are thought to be mediated by serotype cross-reactive antibodies that facilitate antibody-dependent enhancement (ADE) by binding to viral antigens and then Fcγ receptors (FcγR) on target myeloid cells. Using genetically engineered DENV-specific antibodies, it has been shown that the interaction between the Fc portion of serotype cross-reactive antibodies and FcγR is required to induce ADE. Additionally, it was demonstrated that these antibodies were as neutralizing as their non-modified variants, were incapable of inducing ADE, and were therapeutic following a lethal, antibody-enhanced infection. Therefore, we hypothesized that avian IgY, which do not interact with mammalian FcγR, would provide a novel therapy for DENV-induced disease. We demonstrate here that goose-derived anti-DENV2 IgY neutralized DENV2 and did not induce ADE in vitro. Anti-DENV2 IgY was also protective in vivo when administered 24 hours following a lethal DENV2 infection. We were also able to demonstrate via epitope mapping that both full-length and alternatively spliced anti-DENV2 IgY recognized different epitopes, including epitopes that have not been previously identified. These observations provide evidence for the potential therapeutic applications of goose-derived anti-DENV2 IgY.

Immune response and histology of humoral rejection in kidney transplantation.

PubMed

González-Molina, Miguel; Ruiz-Esteban, Pedro; Caballero, Abelardo; Burgos, Dolores; Cabello, Mercedes; Leon, Miriam; Fuentes, Laura; Hernandez, Domingo

2016-01-01

The adaptive immune response forms the basis of allograft rejection. Its weapons are direct cellular cytotoxicity, identified from the beginning of organ transplantation, and/or antibodies, limited to hyperacute rejection by preformed antibodies and not as an allogenic response. This resulted in allogenic response being thought for decades to have just a cellular origin. But the experimental studies by Gorer demonstrating tissue damage in allografts due to antibodies secreted by B lymphocytes activated against polymorphic molecules were disregarded. The special coexistence of binding and unbinding between antibodies and antigens of the endothelial cell membranes has been the cause of the delay in demonstrating the humoral allogenic response. The endothelium, the target tissue of antibodies, has a high turnover, and antigen-antibody binding is non-covalent. If endothelial cells are attacked by the humoral response, immunoglobulins are rapidly removed from their surface by shedding and/or internalization, as well as degrading the components of the complement system by the action of MCP, DAF and CD59. Thus, the presence of complement proteins in the membrane of endothelial cells is transient. In fact, the acute form of antibody-mediated rejection was not demonstrated until C4d complement fragment deposition was identified, which is the only component that binds covalently to endothelial cells. This review examines the relationship between humoral immune response and the types of acute and chronic histological lesion shown on biopsy of the transplanted organ. Copyright © 2016 Sociedad Española de Nefrología. Published by Elsevier España, S.L.U. All rights reserved.

The INNs and outs of antibody nonproprietary names

PubMed Central

Jones, Tim D.; Carter, Paul J.; Plückthun, Andreas; Vásquez, Max; Holgate, Robert G.E.; Hötzel, Isidro; Popplewell, Andrew G.; Parren, Paul W.H.I.; Enzelberger, Markus; Rademaker, Hendrik J.; Clark, Michael R.; Lowe, David C.; Dahiyat, Bassil I.; Smith, Victoria; Lambert, John M.; Wu, Herren; Reilly, Mary; Haurum, John S.; Dübel, Stefan; Huston, James S.; Schirrmann, Thomas; Janssen, Richard A.J.; Steegmaier, Martin; Gross, Jane A.; Bradbury, Andrew R.M.; Burton, Dennis R.; Dimitrov, Dimiter S.; Chester, Kerry A.; Glennie, Martin J.; Davies, Julian; Walker, Adam; Martin, Steve; McCafferty, John; Baker, Matthew P.

2016-01-01

An important step in drug development is the assignment of an International Nonproprietary Name (INN) by the World Health Organization (WHO) that provides healthcare professionals with a unique and universally available designated name to identify each pharmaceutical substance. Monoclonal antibody INNs comprise a –mab suffix preceded by a substem indicating the antibody type, e.g., chimeric (-xi-), humanized (-zu-), or human (-u-). The WHO publishes INN definitions that specify how new monoclonal antibody therapeutics are categorized and adapts the definitions to new technologies. However, rapid progress in antibody technologies has blurred the boundaries between existing antibody categories and created a burgeoning array of new antibody formats. Thus, revising the INN system for antibodies is akin to aiming for a rapidly moving target. The WHO recently revised INN definitions for antibodies now to be based on amino acid sequence identity. These new definitions, however, are critically flawed as they are ambiguous and go against decades of scientific literature. A key concern is the imposition of an arbitrary threshold for identity against human germline antibody variable region sequences. This leads to inconsistent classification of somatically mutated human antibodies, humanized antibodies as well as antibodies derived from semi-synthetic/synthetic libraries and transgenic animals. Such sequence-based classification implies clear functional distinction between categories (e.g., immunogenicity). However, there is no scientific evidence to support this. Dialog between the WHO INN Expert Group and key stakeholders is needed to develop a new INN system for antibodies and to avoid confusion and miscommunication between researchers and clinicians prescribing antibodies. PMID:26716992

Peptide docking of HIV-1 p24 with single chain fragment variable (scFv) by CDOCKER algorithm

NASA Astrophysics Data System (ADS)

Karim, Hana Atiqah Abdul; Tayapiwatana, Chatchai; Nimmanpipug, Piyarat; Zain, Sharifuddin M.; Rahman, Noorsaadah Abdul; Lee, Vannajan Sanghiran

2014-10-01

In search for the important residues that might have involve in the binding interaction between the p24 caspid protein of HIV-1 fragment (MET68 - PRO90) with the single chain fragment variable (scFv) of FAB23.5, modern computational chemistry approach has been conducted and applied. The p24 fragment was initially taken out from the 1AFV protein molecule consisting of both light (VL) and heavy (VH) chains of FAB23.5 as well as the HIV-1 caspid protein. From there, the p24 (antigen) fragment was made to dock back into the protein pocket receptor (antibody) by using the CDOCKER algorithm to conduct the molecular docking process. The score calculated from the CDOCKER gave 15 possible docked poses with various docked ligand's positions, the interaction energy as well as the binding energy. The best docked pose that imitates the original antigen's position was determined and further processed to the In Situ minimization to obtain the residues interaction energy as well as to observe the hydrogen bonds interaction in the protein-peptide complex. Based on the results demonstrated, the specific residues in the complex that have shown immense lower interaction energies in the 5Å vicinity region from the peptide are from the heavy chain (VH:TYR105) and light chain (VL: ASN31, TYR32, and GLU97). Those residues play vital roles in the binding mechanism of Antibody-Antigen (Ab-Ag) complex of p24 with FAB23.5.

[Research of Human-mouse Chimeric Antibodies Against Ebola Virus Nucleoprotein].

PubMed

Zhou, Rongping; Sun, Lina; Liu, Yang; Wu, Wei; Li, Chuan; Liang, Mifang; Qiu, Peihong

2016-01-01

The Ebola virus is highly infectious and can result in death in ≤ 90% of infected subjects. Detection of the Ebola virus and diagnosis of infection are extremely important for epidemic control. Presently, Chinese laboratories detect the nucleic acids of the Ebola virus by real-time reverse transcription-polymerase chain reaction (RT-PCR). However, such detection takes a relatively long time and necessitates skilled personnel and expensive equipment. Enzyme-linked immunosorbent assay (ELISA) of serum is simple, easy to operate, and can be used to ascertain if a patient is infected with the Ebola virus as well as the degree of infection. Hence, ELISA can be used in epidemiological investigations and is a strong complement to detection of nucleic acids. Cases of Ebola hemorrhagic fever have not been documented in China, so quality-control material for positive serology is needed. Construction and expression of human-mouse chimeric antibodies against the nucleoprotein of the Ebola virus was carried out. Genes encoding variable heavy (VH) and variable light (VL) chains were extracted and amplified from murine hybridoma cells. Genes encoding the VH and VL chains of monoclonal antibodies were amplified by RT-PCR. According to sequence analyses, a primer was designed to amplify functional sequences relative to VH and VL chain. The eukaryotic expression vector HL51-14 carrying some human antibody heavy chain- and light chain-constant regions was used. IgG antibodies were obtained by transient transfection of 293T cells. Subsequently, immunological detection and immunological identification were identified by ELISA, immunofluorescence assay, and western blotting. These results showed that we constructed and purified two human- mouse chimeric antibodies.

Immunoadsorption of anti-HLA antibodies for highly sensitized patients awaiting renal transplantation.

PubMed

Hiesse, C; Kriaa, F; Rousseau, P; Farahmand, H; Bismuth, A; Fries, D; Charpentier, B

1992-01-01

Fifteen end-stage renal disease patients with high titres of panel reactive (PRA) antibodies were treated with immunoadsorption (IA) on sepharose-bound protein A columns in order to remove anti-HLA antibodies and facilitate transplantation. Infectious complications were not observed after IA and transplantation, and the procedure was well tolerated. In spite of the use of adjunctive immunosuppressive treatment with cyclophosphamide and prednisolone, this method produced only variable effects in lowering panel reaction antibodies, and was hampered by high de novo resynthesis of anti-HLA antibodies. Patients whose pre-IA antibody titre was greater than or equal to 1:64 clearly did not benefit from the procedure, but other immunological criteria were not predictive of efficacy. Twelve patients were transplanted on the basis of a negative cross-match with current serum, historical sera being retrospectively tested. Surprisingly, seven patients received a well-matched graft with both pre- and post-IA negative cross-matching. Graft survival was 86% in this group. Conversely, in the group of five transplants which were performed in recipients having a positive historical cross-match with the donor, graft survival was only 40%. One patient died with a functional graft, and two grafts failed due to hyperacute humoral rejection. Humoral rejection in a third patient was successfully treated by a second IA course and administration of polyclonal IgG. We conclude that IA is a safe procedure for managing hyperimmunized transplant candidates. However, its efficacy remains variable, and a better definition of patients who should benefit from IA needs to be found.

Maternally derived egg hormones, antibodies and antimicrobial proteins: common and different pathways of maternal effects in Japanese quail.

PubMed

Okuliarova, Monika; Kankova, Zuzana; Bertin, Aline; Leterrier, Christine; Mostl, Erich; Zeman, Michal

2014-01-01

Avian eggs contain a variety of maternally-derived substances that can influence the development and performance of offspring. The levels of these egg compounds vary in relation to environmental and genetic factors, but little is known about whether there are correlative links between maternal substances in the egg underlying common and different pathways of maternal effects. In the present study, we investigated genetically determined variability and mutually adjusted deposition of sex hormones (testosterone-T, androstenedione-A4 and progesterone-P4), antibodies (IgY) and antimicrobial proteins (lysozyme) in eggs of Japanese quail (Coturnix japonica). We used different genetic lines that were independently selected for yolk T concentrations, duration of tonic immobility and social reinstatement behaviour, since both selections for behavioural traits (fearfulness and social motivation, respectively) produced considerable correlative responses in yolk androgen levels. A higher selection potential was found for increased rather than decreased yolk T concentrations, suggesting that there is a physiological minimum in egg T levels. Line differences in yolk IgY concentrations were manifested within each selection experiment, but no consistent inter-line pattern between yolk IgY and T was revealed. On the other hand, a consistent inverse inter-line pattern was recorded between yolk IgY and P4 in both selections for behavioural traits. In addition, selections for contrasting fearfulness and social motivation were associated with changes in albumen lysozyme concentrations and an inverse inter-line pattern between the deposition of yolk IgY and albumen lysozyme was found in lines selected for the level of social motivation. Thus, our results demonstrate genetically-driven changes in deposition of yolk T, P4, antibodies and albumen lysozyme in the egg. This genetic variability can partially explain mutually adjusted maternal deposition of sex hormones and immune-competent molecules but the inconsistent pattern of inter-line differences across all selections indicates that there are other underlying mechanisms, which require further studies.

Structure-based design of broadly protective group a streptococcal M protein-based vaccines.

PubMed

Dale, James B; Smeesters, Pierre R; Courtney, Harry S; Penfound, Thomas A; Hohn, Claudia M; Smith, Jeremy C; Baudry, Jerome Y

2017-01-03

A major obstacle to the development of broadly protective M protein-based group A streptococcal (GAS) vaccines is the variability within the N-terminal epitopes that evoke potent bactericidal antibodies. The concept of M type-specific protective immune responses has recently been challenged based on the observation that multivalent M protein vaccines elicited cross-reactive bactericidal antibodies against a number of non-vaccine M types of GAS. Additionally, a new "cluster-based" typing system of 175M proteins identified a limited number of clusters containing closely related M proteins. In the current study, we used the emm cluster typing system, in combination with computational structure-based peptide modeling, as a novel approach to the design of potentially broadly protective M protein-based vaccines. M protein sequences (AA 16-50) from the E4 cluster containing 17 emm types of GAS were analyzed using de novo 3-D structure prediction tools and the resulting structures subjected to chemical diversity analysis to identify sequences that were the most representative of the 3-D physicochemical properties of the M peptides in the cluster. Five peptides that spanned the range of physicochemical attributes of all 17 peptides were used to formulate synthetic and recombinant vaccines. Rabbit antisera were assayed for antibodies that cross-reacted with E4 peptides and whole bacteria by ELISA and for bactericidal activity against all E4GAS. The synthetic vaccine rabbit antisera reacted with all 17 E4M peptides and demonstrated bactericidal activity against 15/17 E4GAS. A recombinant hybrid vaccine containing the same E4 peptides also elicited antibodies that cross-reacted with all E4M peptides. Comprehensive studies using structure-based design may result in a broadly protective M peptide vaccine that will elicit cluster-specific and emm type-specific antibody responses against the majority of clinically relevant emm types of GAS. Copyright © 2016 Elsevier Ltd. All rights reserved.

Anaphylactic Reactions to Oligosaccharides in Red Meat: a Syndrome in Evolution

PubMed Central

2012-01-01

Objective While most allergic responses to food are directed against protein epitopes and occur within 30 minutes of ingesting the allergen, recent studies suggest that delayed reactions may occur, sometimes mediated by IgE antibodies directed against carbohydrate moieties. The objective of this review is to summarize the clinical features and management of delayed hypersensitivity reactions to mammalian meat mediated by IgE antibodies to galactose-alpha 1,3-galactose (alpha-gal), an oligosaccharide. Methods A PubMed search was conducted with MeSH terms: galactosyl-(1,3) galactose, oligosaccharides, cetuximab, allergy/hypersensitivity, and anaphylaxis. Reported cases with alpha-gal-mediated reactions were reviewed. This research study was approved by the Institutional Review Board of East Tennessee State University. Results Thirty-two cases of adults presenting with red-meat induced allergy thought to be related to oligosaccharides have been reported in the literature so far, making this a rare and evolving syndrome. Most of these patients demonstrated delayed reactions to beef, as was seen in the case reported by us in this manuscript. IgE specific to alpha-gal was identified in most patients with variable response to skin testing with beef and pork. Inhibition studies in some cases showed that the IgE antibodies to beef were directed towards alpha-gal in the meat rather than the protein. The patients often reported history of tick bites, the significance of which is unclear at present. Reactions to cetuximab, a monoclonal antibody, are mediated by a similar mechanism, with IgE antibodies directed against an alpha-gal moiety incorporated in the drug structure. Conclusion Alpha-gal is an oligosaccharide recently incriminated in delayed anaphylactic reactions to mammalian meats such as to beef, pork, and lamb. It appears that anaphylactic reactions to the anti-cancer biological agent, cetuximab, may be linked mechanistically to the same process. More studies are required to understand the underlying molecular basis for these delayed reactions in specific, and their broader implications for host defense in general. PMID:22397506

Determination of royal jelly freshness by ELISA with a highly specific anti-apalbumin 1, major royal jelly protein 1 antibody*

PubMed Central

Shen, Li-rong; Wang, Yi-ran; Zhai, Liang; Zhou, Wen-xiu; Tan, Liang-liang; Li, Mei-lu; Liu, Dan-dan; Xiao, Fa

2015-01-01

Major royal jelly protein 1 (MRJP1), designated apalbumin 1, has been regarded as a freshness marker of royal jelly (RJ). A MRJP1-specific peptide (IKEALPHVPIFD) identified by bioinformatics analysis of homologous members of the major royal protein family was synthesized and used to raise polyclonal anti-MRJP1 antibody (anti-SP-MRJP1 antibody). Western blot analysis showed that anti-SP-MRJP1 antibody only reacted with MRJP1 in RJ. In contrast, the previously reported antibody against recombinant MRJP1 (anti-R-MRJP1 antibody) reacted with other members of MRJP family in RJ. Enzyme-linked immunosorbent assay (ELISA) using anti-SP-MRJP1 antibody demonstrated that MRJP1 content in RJ stored at 40 °C significantly degraded by 37.3%, 55.9%, 58.0%, 60.6%, 65.7%, 72.7%, and 73.1% at 7, 14, 21, 28, 35, 42, and 49 d, respectively, when compared with MRJP1 content in fresh RJ (0 d). Optical density analysis of MRJP bands from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles demonstrated that the degradation of MRJP1, MRJP2, MRJP3, and MRJP5 in RJ was strongly and positively correlated with the period of storage (P<0.0001). Our results indicated anti-SP-MRJP1 antibody was highly specific for MRJP1, and ELISA using the antibody is a sensitive and easy-to-use method to determine the freshness and authenticity of RJ. PMID:25644470

Long Term Maintenance of Polysaccharide-specific Antibodies by IgM Secreting Cells1

PubMed Central

Foote, Jeremy B.; Mahmoud, Tamer I.; Vale, Andre M.; Kearney, John F.

2011-01-01

Many bacteria-associated polysaccharides induce long-lived antibody responses that protect against pathogenic microorganisms. The maintenance of polysaccharide-specific antibody titers may be due to long-lived plasma cells or ongoing antigen-driven B cell activation due to polysaccharide persistence. BALB/c and VHJ558.3 transgenic (TG) mice respond to α 1→3-dextran (DEX) by generating a peak anti-DEX response at 7 days, followed by maintenance of serum antibody levels for up to 150 days. Analysis of the cellular response to DEX identified a population of short-lived, cyclophosphamide sensitive DEX-specific plasmablasts in the spleen, and a quiescent, cyclophosphamide resistant DEX-specific antibody-secreting population in the bone marrow. BrdU pulse-chase experiments demonstrated the longevity of the DEX-specific antibody-secreting population in the bone marrow. Splenic DEX-specific plasmablasts were located in the red pulp with persisting DEX-associated CD11c+ dendritic cells 90 days after immunization, whereas DEX was not detected in the bone marrow after 28 days. Selective depletion of short-lived DEX-specific plasmablasts and memory B1b B cells using cyclophosphamide and anti-CD20 treatment had a minimal impact on the maintenance of serum anti-DEX antibodies. Collectively, these findings demonstrate that the maintenance of serum polysaccharide-specific antibodies is the result of continuous antigen-driven formation of short-lived plasmablasts in the spleen and a quiescent population of antibody-secreting cells maintained in the bone marrow for a long duration. PMID:22116821

Determination of royal jelly freshness by ELISA with a highly specific anti-apalbumin 1, major royal jelly protein 1 antibody.

PubMed

Shen, Li-rong; Wang, Yi-ran; Zhai, Liang; Zhou, Wen-xiu; Tan, Liang-liang; Li, Mei-lu; Liu, Dan-dan; Xiao, Fa

2015-02-01

Major royal jelly protein 1 (MRJP1), designated apalbumin 1, has been regarded as a freshness marker of royal jelly (RJ). A MRJP1-specific peptide (IKEALPHVPIFD) identified by bioinformatics analysis of homologous members of the major royal protein family was synthesized and used to raise polyclonal anti-MRJP1 antibody (anti-SP-MRJP1 antibody). Western blot analysis showed that anti-SP-MRJP1 antibody only reacted with MRJP1 in RJ. In contrast, the previously reported antibody against recombinant MRJP1 (anti-R-MRJP1 antibody) reacted with other members of MRJP family in RJ. Enzyme-linked immunosorbent assay (ELISA) using anti-SP-MRJP1 antibody demonstrated that MRJP1 content in RJ stored at 40 °C significantly degraded by 37.3%, 55.9%, 58.0%, 60.6%, 65.7%, 72.7%, and 73.1% at 7, 14, 21, 28, 35, 42, and 49 d, respectively, when compared with MRJP1 content in fresh RJ (0 d). Optical density analysis of MRJP bands from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles demonstrated that the degradation of MRJP1, MRJP2, MRJP3, and MRJP5 in RJ was strongly and positively correlated with the period of storage (P<0.0001). Our results indicated anti-SP-MRJP1 antibody was highly specific for MRJP1, and ELISA using the antibody is a sensitive and easy-to-use method to determine the freshness and authenticity of RJ.

Quantitative measurement of IgE antibodies to purified allergens using streptavidin linked to a high-capacity solid phase.

PubMed

Erwin, Elizabeth A; Custis, Natalie J; Satinover, Shama M; Perzanowski, Matthew S; Woodfolk, Judith A; Crane, Julian; Wickens, Kristin; Platts-Mills, Thomas A E

2005-05-01

Commercially available assays for IgE antibody provide results in international units per milliliter for many allergen extracts, but this is not easily achieved with purified or novel allergens. To develop assays for IgE antibody suitable for purified or novel allergens by using a commercially available immunosorbent. Streptavidin coupled to a high-capacity immunosorbent (CAP) was used to bind biotinylated purified allergens from mite (Der p 1 and Der p 2), cat (Fel d 1), and dog (Can f 1). Assays for IgE antibody to these allergens were performed on sera from children (asthma and control) as well as adults with atopic dermatitis. The results were validated by serial dilution of sera with high and low levels of IgE antibody and were quantitated in international units per milliliter by using a standard curve. Values for IgE antibody to Der p 1, Der p 2, and Fel d 1 correlated with values obtained with the allergen extracts (r2 = 0.80, 0.84, and 0.95, respectively; P < .001 in each case). Furthermore, the values for IgE antibody in sera from children with high exposure to mite and cat allergens demonstrated 10-fold higher levels of IgE antibody to Der p 1 and Der p 2 than to Fel d 1 (P < .001). The streptavidin immunosorbent technique provides a new method for quantifying IgE antibody to purified proteins. The results provide evidence about the high quantities of IgE antibody to purified inhalant allergens in patients with atopic dermatitis. In addition, the results demonstrate major differences in IgE antibodies specific for mite and cat allergens among children with high exposure to both allergens.

Screening of a ScFv Antibody With High Affinity for Application in Human IFN-γ Immunoassay

PubMed Central

Yang, Hang; Zhong, Yanfang; Wang, Juncheng; Zhang, Qinghong; Li, Xiulan; Ling, Sumei; Wang, Shihua; Wang, Rongzhi

2018-01-01

Interferon gamma (IFN-γ), a signal proinflammatory cytokine secreted by immune cell, and plays a critical role in the pathogenesis and progression of many diseases. It has been regarded as an important marker for determination of disease-specific immune responses. Therefore, it is urgent to develop a feasible and accurate method to detect IFN-γ in clinic real blood samples. Until now, the immunoassay based on singe chain variable fragment (scFv) antibody for human IFN-γ is still not reported. In the present study, an scFv antibody named scFv-A8 with high specificity was obtained by phage display and biopanning, with the affinity 2.6 × 109 L/mol. Maltose binding protein (MBP) was used to improve the solubility of scFv by inserting an linker DNA between scFv and MBP tag, and the resulted fusion protein (MBP-LK-scFv) has high solubility and antigen biding activity. The expressed and purified MBP-LK-scFv antibody was used to develop the indirect competitive enzyme-linked immunosorbent assay (ELISA) (ic-ELISA) for detection of human IFN-γ, and the result indicated that the linear range to detect IFN-γ was 6–60 pg/mL with IC50 of 25 pg/mL. The limit of detection was 2 pg/mL (1.3 fm), and the average recovery was 85.05%, further demonstrating that the detection method based on scFv has higher recovery and accuracy. Hence, the developed ic-ELISA can be used to detect IFN-γ in real samples, and it may be further provided a scientific basis for disease diagnosis. PMID:29563896

Seroepidemiology of Toxoplasma gondii in pregnant women in Aguascalientes City, Mexico: a cross-sectional study.

PubMed

Alvarado-Esquivel, Cosme; Terrones-Saldívar, María Del Carmen; Hernández-Tinoco, Jesús; Muñoz-Terrones, María Daniela Enriqueta; Gallegos-González, Roberto Oswaldo; Sánchez-Anguiano, Luis Francisco; Reyes-Robles, Martha Elena; Jaramillo-Juárez, Fernando; Liesenfeld, Oliver; Estrada-Martínez, Sergio

2016-07-01

We determined the seroprevalence and correlates of Toxoplasma gondii infection in pregnant women in Aguascalientes City, Mexico. A cross-sectional survey. Pregnant women were enrolled in the central Mexican city of Aguascalientes. We studied 338 pregnant women who attended prenatal care in 3 public health centres. Women were examined for IgG/IgM antibodies to T. gondii by using commercially available enzyme immunoassays, and an avidity test. Multiple analyses were used to determine the association of T. gondii seropositivity with the characteristics of the pregnant women. Of the 338 pregnant women studied, 21 (6.2%) had IgG antibodies to T. gondii, and 1 (4.8%) of them was also positive for IgM antibodies to T. gondii. Avidity of IgG antibodies to T. gondii was high in the IgM-positive sample. Logistic regression analysis of sociodemographic, behavioural and housing variables showed that T. gondii seropositivity was associated with white ethnicity (OR=149.4; 95% CI 10.8 to 2054.1; p<0.01), not washing hands before eating (OR=6.41; 95% CI 1.73 to 23.6; p=0.005) and use of latrine (OR=37.6; 95% CI 4.63 to 306.31; p=0.001). Results demonstrate that pregnant women in Aguascalientes City have a low seroprevalence of T. gondii infection. However, this low prevalence indicates that most pregnant women are at risk for a primary infection. Factors associated with T. gondii exposure found in this study, including food hygiene, may be useful to determine preventive measures against T. gondii infection and its sequelae. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Human scFv antibodies (Afribumabs) against Africanized bee venom: Advances in melittin recognition.

PubMed

Pessenda, Gabriela; Silva, Luciano C; Campos, Lucas B; Pacello, Elenice M; Pucca, Manuela B; Martinez, Edson Z; Barbosa, José E

2016-03-15

Africanized Apis mellifera bees, also known as killer bees, have an exceptional defensive instinct, characterized by mass attacks that may cause envenomation or death. From the years 2000-2013, 77,066 bee accidents occurred in Brazil. Bee venom comprises several substances, including melittin and phospholipase A2 (PLA2). Due to the lack of antivenom for bee envenomation, this study aimed to produce human monoclonal antibody fragments (single chain fragment variable; scFv), by using phage display technology. These fragments targeted melittin and PLA2, the two major components of bee venom, to minimize their toxic effects in cases of mass envenomation. Two phage antibody selections were performed using purified melittin. As the commercial melittin is contaminated with PLA2, phages specific to PLA2 were also obtained during one of the selections. Specific clones for melittin and PLA2 were selected for the production of soluble scFvs, named here Afribumabs: prefix: afrib- (from Africanized bee); stem/suffix: -umab (fully human antibody). Afribumabs 1 and 2 were tested in in vitro and in vivo assays to assess their ability to inhibit the toxic actions of purified melittin, PLA2, and crude bee venom. Afribumabs reduced hemolysis caused by purified melittin and PLA2 and by crude venom in vitro and reduced edema formation in the paws of mice and prolonged the survival of venom-injected animals in vivo. These results demonstrate that Afribumabs may contribute to the production of the first non-heterologous antivenom treatment against bee envenomation. Such a treatment may overcome some of the difficulties associated with conventional immunotherapy techniques. Copyright © 2016 Elsevier Ltd. All rights reserved.

Detection of anthrax protective antigen (PA) using europium labeled anti-PA monoclonal antibody and time-resolved fluorescence ◊

PubMed Central

Stoddard, Robyn A.; Quinn, Conrad P.; Schiffer, Jarad M.; Boyer, Anne E.; Goldstein, Jason; Bagarozzi, Dennis A.; Soroka, Stephen D.; Dauphin, Leslie A.; Hoffmaster, Alex R.

2015-01-01

Inhalation anthrax is a rare but acute infectious disease following adsorption of Bacillus anthracis spores through the lungs. The disease has a high fatality rate if untreated, but early and correct diagnosis has a significant impact on case patient recovery. The early symptoms of inhalation anthrax are, however, non-specific and current anthrax diagnostics are primarily dependent upon culture and confirmatory real-time PCR. Consequently, there may be a significant delay in diagnosis and targeted treatment. Rapid, culture-independent diagnostic tests are therefore needed, particularly in the context of a large scale emergency response. The aim of this study was to evaluate the ability of monoclonal antibodies to detect anthrax toxin proteins that are secreted early in the course of B. anthracis infection using a time-resolved fluorescence (TRF) immunoassay. We selected monoclonal antibodies that could detect protective antigen (PA), as PA83 and also PA63 and LF in the lethal toxin complex. The assay reliable detection limit (RDL) was 6.63 × 10−6 μM (0.551 ng/ml) for PA83 and 2.51 × 10−5 μM (1.58 ng/ml) for PA63. Despite variable precision and accuracy of the assay, PA was detected in 9 out of 10 sera samples from anthrax confirmed case patients with cutaneous (n=7), inhalation (n=2), and gastrointestinal (n=1) disease. Anthrax Immune Globulin (AIG), which has been used in treatment of clinical anthrax, interfered with detection of PA. This study demonstrates a culture-independent method of diagnosing anthrax through use of monoclonal antibodies to detect PA and LF in the lethal toxin complex. PMID:24857756

Closely Related Antibody Receptors Exploit Fundamentally Different Strategies for Steroid Recognition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Verdino, P.; Aldag, C.; Hilvert, D.

2009-05-26

Molecular recognition by the adaptive immune system relies on specific high-affinity antibody receptors that are generated from a restricted set of starting sequences through homologous recombination and somatic mutation. The steroid binding antibody DB3 and the catalytic Diels-Alderase antibody 1E9 derive from the same germ line sequences but exhibit very distinct specificities and functions. However, mutation of only two of the 36 sequence differences in the variable domains, Leu{sup H47}Trp and Arg{sup H100}Trp, converts 1E9 into a high-affinity steroid receptor with a ligand recognition profile similar to DB3. To understand how these changes switch binding specificity and function, we determinedmore » the crystal structures of the 1E9 Leu{sup H47}Trp/Arg{sup H100}Trp double mutant (1E9dm) as an unliganded Fab at 2.05 {angstrom} resolution and in complex with two configurationally distinct steroids at 2.40 and 2.85 {angstrom}. Surprisingly, despite the functional mimicry of DB3, 1E9dm employs a distinct steroid binding mechanism. Extensive structural rearrangements occur in the combining site, where residue H47 acts as a specificity switch and H100 adapts to different ligands. Unlike DB3, 1E9dm does not use alternative binding pockets or different sets of hydrogen-bonding interactions to bind configurationally distinct steroids. Rather, the different steroids are inserted more deeply into the 1E9dm combining site, creating more hydrophobic contacts that energetically compensate for the lack of hydrogen bonds. These findings demonstrate how subtle mutations within an existing molecular scaffold can dramatically modulate the function of immune receptors by inducing unanticipated, but compensating, mechanisms of ligand interaction.« less

Expression of full-length HER2 protein in Sf9 insect cells and its presentation on the surface of budded virus-like particles.

PubMed

Nika, Lisa; Wallner, Jakob; Palmberger, Dieter; Koczka, Krisztina; Vorauer-Uhl, Karola; Grabherr, Reingard

2017-08-01

Biomarkers of cancer are often glycosylated membrane receptor proteins present on the cellular surface. In order to develop new antibodies for cancer diagnostics or treatment, it is a main pre-requisite that these target proteins are available in a native conformation. However, membrane receptor proteins are notoriously difficult to produce due to their hydrophobic nature and complex architecture. Here, we used the baculovirus-insect cell expression system to produce budded virus-like particles (VLPs) as the scaffold for the presentation of complex membrane proteins. Since the human epidermal growth factor receptor 2 (HER2) is known to be overexpressed in a number of cancers it was chosen as model for a tumor antigen. VLPs displaying full-length HER2 on the surface were produced in Spodoptera frugiperda 9 (Sf9) insect cells and purified by sucrose gradient ultracentrifugation. The number of secreted particles was quantified by nanoparticle tracking analysis. To confirm the presence of HER2 protein on the surface, VLPs were labeled with gold-conjugated antibodies and analyzed by transmission electron microscopy. Functionality of displayed HER2 was investigated by ELISA and a newly established biolayer interferometry based technique. Detection was accomplished using the specific monoclonal antibody Herceptin and filamentous phages displaying a single-chain variable fragment of an anti-HER2 antibody. Significant stronger binding of Herceptin and anti-HER2 phages to HER2-displaying VLPs as compared to control VLPs was demonstrated. Thus, we suggest that Sf9 insect cells are highly feasible for the fast and easy production of various budded VLPs that serve as a platform for full-length membrane receptor presentation. Copyright © 2017. Published by Elsevier Inc.

Chronic Toxoplasma gondii in Nurr1-Null Heterozygous Mice Exacerbates Elevated Open Field Activity

PubMed Central

Eells, Jeffrey B.; Varela-Stokes, Andrea; Guo-Ross, Shirley X.; Kummari, Evangel; Smith, Holly M.; Cox, Erin; Lindsay, David S.

2015-01-01

Latent infection with Toxoplasma gondii is common in humans (approximately 30% of the global population) and is a significant risk factor for schizophrenia. Since prevalence of T. gondii infection is far greater than prevalence of schizophrenia (0.5-1%), genetic risk factors are likely also necessary to contribute to schizophrenia. To test this concept in an animal model, Nurr1-null heterozygous (+/-) mice and wild-type (+/+) mice were evaluate using an emergence test, activity in an open field and with a novel object, response to bobcat urine and prepulse inhibition of the acoustic startle response (PPI) prior to and 6 weeks after infection with T. gondii. In the emergence test, T. gondii infection significantly decreased the amount of time spent in the cylinder. Toxoplasma gondii infection significantly elevated open field activity in both +/+ and +/- mice but this increase was significantly exacerbated in +/- mice. T. gondii infection reduced PPI in male +/- mice but this was not statistically significant. Aversion to bobcat urine was abolished by T. gondii infection in +/+ mice. In female +/- mice, aversion to bobcat urine remained after T. gondii infection while the male +/- mice showed no aversion to bobcat urine. Antibody titers of infected mice were a critical variable associated with changes in open field activity, such that an inverted U shaped relationship existed between antibody titers and the percent change in open field activity with a significant increase in activity at low and medium antibody titers but no effect at high antibody titers. These data demonstrate that the Nurr1 +/- genotype predisposes mice to T. gondii-induced alterations in behaviors that involve dopamine neurotransmission and are associated with symptoms of schizophrenia. We propose that these alterations in murine behavior were due to further exacerbation of the altered dopamine neurotransmission in Nurr1 +/- mice. PMID:25855987

Chronic Toxoplasma gondii in Nurr1-null heterozygous mice exacerbates elevated open field activity.

PubMed

Eells, Jeffrey B; Varela-Stokes, Andrea; Guo-Ross, Shirley X; Kummari, Evangel; Smith, Holly M; Cox, Erin; Lindsay, David S

2015-01-01

Latent infection with Toxoplasma gondii is common in humans (approximately 30% of the global population) and is a significant risk factor for schizophrenia. Since prevalence of T. gondii infection is far greater than prevalence of schizophrenia (0.5-1%), genetic risk factors are likely also necessary to contribute to schizophrenia. To test this concept in an animal model, Nurr1-null heterozygous (+/-) mice and wild-type (+/+) mice were evaluate using an emergence test, activity in an open field and with a novel object, response to bobcat urine and prepulse inhibition of the acoustic startle response (PPI) prior to and 6 weeks after infection with T. gondii. In the emergence test, T. gondii infection significantly decreased the amount of time spent in the cylinder. Toxoplasma gondii infection significantly elevated open field activity in both +/+ and +/- mice but this increase was significantly exacerbated in +/- mice. T. gondii infection reduced PPI in male +/- mice but this was not statistically significant. Aversion to bobcat urine was abolished by T. gondii infection in +/+ mice. In female +/- mice, aversion to bobcat urine remained after T. gondii infection while the male +/- mice showed no aversion to bobcat urine. Antibody titers of infected mice were a critical variable associated with changes in open field activity, such that an inverted U shaped relationship existed between antibody titers and the percent change in open field activity with a significant increase in activity at low and medium antibody titers but no effect at high antibody titers. These data demonstrate that the Nurr1 +/- genotype predisposes mice to T. gondii-induced alterations in behaviors that involve dopamine neurotransmission and are associated with symptoms of schizophrenia. We propose that these alterations in murine behavior were due to further exacerbation of the altered dopamine neurotransmission in Nurr1 +/- mice.

Escherichia coli surface display of single-chain antibody VRC01 against HIV-1 infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Lin-Xu; School of Biological Sciences, University of Nebraska—Lincoln, Lincoln, NE 68583; Mellon, Michael

Human immunodeficiency virus type 1 (HIV-1) transmission and infection occur mainly via the mucosal surfaces. The commensal bacteria residing in these surfaces can potentially be employed as a vehicle for delivering inhibitors to prevent HIV-1 infection. In this study, we have employed a bacteria-based strategy to display a broadly neutralizing antibody VRC01, which could potentially be used to prevent HIV-1 infection. The VRC01 antibody mimics CD4-binding to gp120 and has broadly neutralization activities against HIV-1. We have designed a construct that can express the fusion peptide of the scFv-VRC01 antibody together with the autotransporter β-barrel domain of IgAP gene frommore » Neisseria gonorrhoeae, which enabled surface display of the antibody molecule. Our results indicate that the scFv-VRC01 antibody molecule was displayed on the surface of the bacteria as demonstrated by flow cytometry and immunofluorescence microscopy. The engineered bacteria can capture HIV-1 particles via surface-binding and inhibit HIV-1 infection in cell culture. - Highlights: • Designed single-chain VRC01 antibody was demonstrated to bind HIV-1 envelope gp120. • Single-chain VRC01 antibody was successfully displayed on the surface of E. coli. • Engineered bacteria can absorb HIV-1 particles and prevent HIV-1 infection in cell culture.« less

Comparative Assessment of Clinically Utilized CD20-directed Antibodies in Chronic Lymphocytic Leukemia Cells Reveals Divergent NK cell, Monocyte and Macrophage Properties

PubMed Central

Rafiq, Sarwish; Butchar, Jonathon P.; Cheney, Carolyn; Mo, Xiaokui; Trotta, Rossana; Caligiuri, Michael; Jarjoura, David; Tridandapani, Susheela; Muthusamy, Natarajan; Byrd, John C.

2013-01-01

CD20 is a widely validated, B cell specific target for therapy in B cell malignancies. Rituximab is an anti-CD20 antibody that when combined with chemotherapy prolongs survival of CLL patients. Ofatumumab and GA101 (obinutuzumab) are CD20-directed antibodies now being developed as alternative agents to rituximab in CLL based upon different properties of enhanced direct cell death (DCD), NK cell-mediated antibody dependent cellular cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). Despite wide spread study, ofatumumab and GA101 have not been directly compared to one another, nor studied for interaction with monocytes and macrophages that are critical to CD20-mediated antibody efficacy in murine models. In CLL cells, we show that DCD is greatest with GA101 and CDC with ofatumumab. GA101 promotes enhanced NK cell activation and ADCC at high antibody concentrations. Ofatumumab has superior antibody dependent cellular phagocytosis (ADCP) with monocyte derived macrophages (MDM). GA101 demonstrated reduced activation of monocytes with diminished pERK, TNF-α release, and FcγRIIa recruitment to lipid rafts. These data demonstrate GA101 and ofatumumab are superior to rituximab against CLL cells via different mechanisms of potential tumor elimination. These findings bear relevance to potential combination strategies with each of these anti-CD20 antibodies in the treatment of CLL. PMID:23418626

Comprehensive Cross-Clade Characterization of Antibody-Mediated Recognition, Complement-Mediated Lysis, and Cell-Mediated Cytotoxicity of HIV-1 Envelope-Specific Antibodies toward Eradication of the HIV-1 Reservoir.

PubMed

Mujib, Shariq; Liu, Jun; Rahman, A K M Nur-Ur; Schwartz, Jordan A; Bonner, Phil; Yue, Feng Yun; Ostrowski, Mario A

2017-08-15

Immunotherapy with passive administration of broadly neutralizing HIV-1 envelope-specific antibodies (bnAbs) in the setting of established infection in vivo has yielded mixed results. The contribution of different antibodies toward the direct elimination of infected cells is poorly understood. In this study, we determined the ability of 12 well-characterized anti-HIV-1 neutralizing antibodies to recognize and eliminate primary CD4 T cells infected with HIV-1 belonging to clades A, B, C, and D, via antibody-dependent complement-mediated lysis (ADCML) and antibody-dependent cell-mediated cytotoxicity (ADCC), in vitro We further tested unique combinations of these antibodies to determine the optimal antibody cocktails to be tested in future clinical trials. We report that antibody binding to infected CD4 T cells is highly variable and correlates with ADCML and ADCC processes. Particularly, antibodies targeting the envelope glycan shield (2G12) and V1/V2 site (PG9, PG16, and PGT145) are best at recognizing HIV-1-infected CD4 T cells. However, only PG9 and PG16 and their combinations with other bnAbs sufficiently induced the elimination of HIV-1-infected CD4 T cells by ADCML, ADCC, or both. Notably, CD4 binding site antibodies VRC01, 3BNC117, and NIH45-46 G54W did not exhibit recognition of infected cells and were unable to induce their killing. Future trials geared toward the development of a cure for HIV/AIDS should incorporate V1/V2 antibodies for maximal clearance of infected cells. With the use of only primary immune cells, we conducted a comprehensive cross-clade physiological analysis to aid the direction of antibodies as therapeutics toward the development of a cure for HIV/AIDS. IMPORTANCE Several antibodies capable of neutralizing the majority of circulating HIV-1 strains have been identified to date and have been shown to prevent infection in animal models. However, the use of combinations of such broadly neutralizing antibodies (bnAbs) for the treatment and eradication of HIV-1 in infected humans remains uncertain. In this study, we tested the ability of bnAbs to directly recognize and eliminate primary human CD4 T cells infected with diverse HIV-1 strains representative of the global epidemic by antibody-dependent pathways. We also tested several combinations of bnAbs in our assays in order to maximize the clearance of infected cells. We show that the ability of bnAbs to identify and kill infected cells is highly variable and that only a few of them are able to exert this function. Our data will help guide the formulation of bnAbs to test in future human trials aimed at the development of a cure. Copyright © 2017 American Society for Microbiology.

Biomarkers in the evolution of multiple sclerosis.

PubMed

Berger, Thomas

2017-11-01

Nonimaging biomarkers can be applied in differential diagnosis, evaluation of disease progression and therapy monitoring of multiple sclerosis (MS). Presence of oligoclonal IgG bands in cerebrospinal fluid is a diagnostic element and a negative predictor of MS evolution. AQP4 antibodies are pathogenic and diagnostic for neuromyelitis optica spectrum disorder. Antibodies to myelin oligodendrocyte glycoprotein develop in about 50% of predominantly pediatric patients with acute disseminated encephalomyelitis, but their possible role in pathogenesis is unknown. Currently, there are no individualized biomarkers suitable to track disease progression. Neutralizing antibodies against IFN-β, natalizumab and daclizumab arise with variable frequency and reduce treatment efficacy. The anti-John Cunningham virus antibody index has potential as a biomarker for risk of progressive multifocal leukoencephalopathy.

Developmental pathway for potent V1V2-directed HIV-neutralizing antibodies

PubMed Central

Doria-Rose, Nicole A.; Schramm, Chaim A.; Gorman, Jason; Moore, Penny L.; Bhiman, Jinal N.; DeKosky, Brandon J.; Ernandes, Michael J.; Georgiev, Ivelin S.; Kim, Helen J.; Pancera, Marie; Staupe, Ryan P.; Altae-Tran, Han R.; Bailer, Robert T.; Crooks, Ema T.; Cupo, Albert; Druz, Aliaksandr; Garrett, Nigel J.; Hoi, Kam H.; Kong, Rui; Louder, Mark K.; Longo, Nancy S.; McKee, Krisha; Nonyane, Molati; O’Dell, Sijy; Roark, Ryan S.; Rudicell, Rebecca S.; Schmidt, Stephen D.; Sheward, Daniel J.; Soto, Cinque; Wibmer, Constantinos Kurt; Yang, Yongping; Zhang, Zhenhai; Mullikin, James C.; Binley, James M.; Sanders, Rogier W.; Wilson, Ian A.; Moore, John P.; Ward, Andrew B.; Georgiou, George; Williamson, Carolyn; Abdool Karim, Salim S.; Morris, Lynn; Kwong, Peter D.; Shapiro, Lawrence; Mascola, John R.

2015-01-01

Summary Antibodies capable of neutralizing HIV-1 often target variable regions 1 and 2 (V1V2) of the HIV-1 envelope, but the mechanism of their elicitation has been unclear. Here we define the developmental pathway by which such antibodies are generated and acquire the requisite molecular characteristics for neutralization. Twelve somatically related neutralizing antibodies (CAP256-VRC26.01-12) were isolated from CAPRISA-donor CAP256; each antibody contained the protruding tyrosine-sulfated, anionic antigen-binding loop (CDR H3) characteristic of this category of antibodies. Their unmutated ancestor emerged between weeks 30–38 post-infection with a 35-residue CDR H3, and neutralized the virus that superinfected this individual 15 weeks after initial infection. Improved neutralization breadth occurred by week 59 with modest affinity maturation, and was preceded by extensive diversification of the virus population. HIV-1 V1V2-directed neutralizing antibodies can thus develop relatively rapidly through initial selection of B cells with a long CDR H3, and limited subsequent somatic hypermutation, an important vaccine insight. PMID:24590074

An integrated top-down and bottom-up proteomic approach to characterize the antigen-binding fragment of antibodies.

PubMed

Dekker, Lennard; Wu, Si; Vanduijn, Martijn; Tolić, Nikolai; Stingl, Christoph; Zhao, Rui; Luider, Theo; Paša-Tolić, Ljiljana

2014-05-01

We have previously shown that different individuals exposed to the same antigen produce antibodies with identical mutations in their complementarity determining regions (CDR), suggesting that CDR tryptic peptides can serve as biomarkers for disease diagnosis and prognosis. Complete Fabs derived from disease specific antibodies have even higher potential; they could potentially be used for disease treatment and are required to identify the antigens toward which the antibodies are directed. However, complete Fab sequence characterization via LC-MS analysis of tryptic peptides (i.e. bottom-up) has proven to be impractical for mixtures of antibodies. To tackle this challenge, we have developed an integrated bottom-up and top-down MS approach, employing 2D chromatography coupled with Fourier transform mass spectrometry (FTMS), and applied this approach for full characterization of the variable parts of two pharmaceutical monoclonal antibodies with sensitivity comparable to the bottom-up standard. These efforts represent an essential step toward the identification of disease specific antibodies in patient samples with potentially significant clinical impact. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Recent Trends in Antibody-based Oncologic Imaging

PubMed Central

Kaur, Sukhwinder; Venktaraman, Ganesh; Jain, Maneesh; Senapati, Shantibhusan; Garg, Pradeep K.; Batra, Surinder K.

2011-01-01

Antibodies, with their unmatched ability for selective binding to any target, are considered as potentially the most specific probes for imaging. Their clinical utility, however, has been limited chiefly due to their slow clearance from the circulation, longer retention in non-targeted tissues and the extensive optimization required for each antibody-tracer. The development of newer contrast agents, combined with improved conjugation strategies and novel engineered forms of antibodies (diabodies, minibodies, single chain variable fragments, and nanobodies), have triggered a new wave of antibody-based imaging approaches. Apart from their conventional use with nuclear imaging probes, antibodies and their modified forms are increasingly being employed with non-radioisotopic contrast agents (MRI and ultrasound) as well as newer imaging modalities, such as quantum dots, near infra red (NIR) probes, nanoshells and surface enhanced Raman spectroscopy (SERS). The review article provides new developments in the usage of antibodies and their modified forms in conjunction with probes of various imaging modalities such as nuclear imaging, optical imaging, ultrasound, MRI, SERS and nanoshells in preclinical and clinical studies on the diagnosis, prognosis and therapeutic responses of cancer. PMID:22104729

Somatic Hypermutation-Induced Changes in the Structure and Dynamics of HIV-1 Broadly Neutralizing Antibodies.

PubMed

Davenport, Thaddeus M; Gorman, Jason; Joyce, M Gordon; Zhou, Tongqing; Soto, Cinque; Guttman, Miklos; Moquin, Stephanie; Yang, Yongping; Zhang, Baoshan; Doria-Rose, Nicole A; Hu, Shiu-Lok; Mascola, John R; Kwong, Peter D; Lee, Kelly K

2016-08-02

Antibody somatic hypermutation (SHM) and affinity maturation enhance antigen recognition by modifying antibody paratope structure to improve its complementarity with the target epitope. SHM-induced changes in paratope dynamics may also contribute to antibody maturation, but direct evidence of this is limited. Here, we examine two classes of HIV-1 broadly neutralizing antibodies (bNAbs) for SHM-induced changes in structure and dynamics, and delineate the effects of these changes on interactions with the HIV-1 envelope glycoprotein (Env). In combination with new and existing structures of unmutated and affinity matured antibody Fab fragments, we used hydrogen/deuterium exchange with mass spectrometry to directly measure Fab structural dynamics. Changes in antibody structure and dynamics were positioned to improve complementarity with Env, with changes in dynamics primarily observed at the paratope peripheries. We conclude that SHM optimizes paratope complementarity to conserved HIV-1 epitopes and restricts the mobility of paratope-peripheral residues to minimize clashes with variable features on HIV-1 Env. Copyright © 2016 Elsevier Ltd. All rights reserved.

Recent Developments in Antibody-Based Assays for the Detection of Bacterial Toxins

PubMed Central

Zhu, Kui; Dietrich, Richard; Didier, Andrea; Doyscher, Dominik; Märtlbauer, Erwin

2014-01-01

Considering the urgent demand for rapid and accurate determination of bacterial toxins and the recent promising developments in nanotechnology and microfluidics, this review summarizes new achievements of the past five years. Firstly, bacterial toxins will be categorized according to their antibody binding properties into low and high molecular weight compounds. Secondly, the types of antibodies and new techniques for producing antibodies are discussed, including poly- and mono-clonal antibodies, single-chain variable fragments (scFv), as well as heavy-chain and recombinant antibodies. Thirdly, the use of different nanomaterials, such as gold nanoparticles (AuNPs), magnetic nanoparticles (MNPs), quantum dots (QDs) and carbon nanomaterials (graphene and carbon nanotube), for labeling antibodies and toxins or for readout techniques will be summarized. Fourthly, microscale analysis or minimized devices, for example microfluidics or lab-on-a-chip (LOC), which have attracted increasing attention in combination with immunoassays for the robust detection or point-of-care testing (POCT), will be reviewed. Finally, some new materials and analytical strategies, which might be promising for analyzing toxins in the near future, will be shortly introduced. PMID:24732203

An integrated top-down and bottom-up proteomic approach to characterize the antigen binding fragment of antibodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dekker, Leendert J.; Wu, Si; vanDuijn, Martijn M.

2014-05-31

We have previously shown that different individuals exposed to the same antigen produce antibodies with identical mutations in their complementarity determining regions (CDR), suggesting that CDR tryptic peptides can serve as biomarkers for disease diagnosis and prognosis. Complete Fabs derived from disease specific antibodies have even higher potential; they could potentially be used for disease treatment and are required to identify the antigens towards which the antibodies are directed. However, complete Fab sequence characterization via LC-MS analysis of tryptic peptides (i.e. bottom-up) has proven to be impractical for mixtures of antibodies. To tackle this challenge, we have developed an integratedmore » bottom-up and top-down MS approach, employing 2D chromatography coupled with Fourier transform mass spectrometry (FTMS), and applied this approach for full characterization of the variable parts of two pharmaceutical monoclonal antibodies with sensitivity comparable to the bottom-up standard. These efforts represent an essential step towards the identification of disease specific antibodies in patient samples with potentially significant clinical impact.« less

Neuronal Surface Antibody-Mediated Autoimmune Encephalitis

PubMed Central

Linnoila, Jenny J.; Rosenfeld, Myrna R.; Dalmau, Josep

2016-01-01

In the past few years, many autoimmune encephalitides have been identified, with specific clinical syndromes and associated antibodies against neuronal surface antigens. There is compelling evidence that many of these antibodies are pathogenic and most of these encephalitides are highly responsive to immunotherapies. The clinical spectra of some of these antibody-mediated syndromes, especially those reported in only a few patients, are evolving. Others, such as anti-N-methyl-D-aspartate (NMDA) receptor encephalitis, are well characterized. Diagnosis involves recognizing the specific syndromes and identifying the antibody in a patient’s cerebrospinal fluid (CSF) and/or serum. These syndromes are associated with variable abnormalities in CSF, magnetic resonance imaging, and electroencephalography. Treatment is often multidisciplinary and should be focused upon neutralizing the effects of antibodies and eliminating their source. Overlapping disorders have been noted, with some patients having more than one neurologic autoimmune disease. In other patients, viral infections such as herpes simplex virus encephalitis trigger robust antineuronal autoimmune responses. PMID:25369441

Development and characterization of novel chimeric monoclonal antibodies for broad spectrum neutralization of rabies virus.

PubMed

Kim, Pan Kyeom; Keum, Sun Ju; Osinubi, Modupe O V; Franka, Richard; Shin, Ji Young; Park, Sang Tae; Kim, Man Su; Park, Mi Jung; Lee, Soo Young; Carson, William; Greenberg, Lauren; Yu, Pengcheng; Tao, Xiaoyan; Lihua, Wang; Tang, Qing; Liang, Guodong; Shampur, Madhusdana; Rupprecht, Charles E; Chang, Shin Jae

2017-01-01

Current post-exposure prophylaxis for rabies virus infection has several limitations in terms of supply, cost, safety, and efficacy. Attempts to replace human or equine rabies immune globulins (HRIG or ERIG) have been made by several companies and institutes. We developed potent monoclonal antibodies to neutralize a broad spectrum of rabies viruses by screening hybridomas received from the U.S. Centers for Disease Control and Prevention (CDC). Two kinds of chimeric human antibodies (chimeric #7 and #17) were constructed by cloning the variable regions from selected hybridomas and the constant region of a human antibody. Two antibodies were bound to antigenic site III and I/IV, respectively, and were able to neutralize 51 field isolates of rabies virus that were isolated at different times and places such as Asia, Africa, North America, South America, and Australia. These two antibodies neutralize rabies viruses with high efficacy in an in vivo test using Syrian hamster and mouse models and show low risk for adverse immunogenicity.

Antibody derivatization and conjugation strategies: application in preparation of stealth immunoliposome to target chemotherapeutics to tumor.

PubMed

Manjappa, Arehalli S; Chaudhari, Kiran R; Venkataraju, Makam P; Dantuluri, Prudhviraju; Nanda, Biswarup; Sidda, Chennakesavulu; Sawant, Krutika K; Murthy, Rayasa S Ramachandra

2011-02-28

A great deal of effort has been made over the years to develop liposomes that have targeting vectors (oligosaccharides, peptides, proteins and vitamins) attached to the bilayer surface. Most studies have focused on antibody conjugates since procedures for producing highly specific monoclonal antibodies are well established. Antibody conjugated liposomes have recently attracted a great deal of interest, principally because of their potential use as targeted drug delivery systems and in diagnostic applications. A number of methods have been reported for coupling antibodies to the surface of stealth liposomes. The objective of this review is to enumerate various strategies which are employed in the modification and conjugation of antibodies to the surface of stealth liposomes. This review also describes various derivatization techniques of lipids prior and after their use in the preparation of liposomes. The use of single chain variable fragments and affibodies as targeting ligands in the preparation of immunoliposomes is also discussed. Copyright © 2010 Elsevier B.V. All rights reserved.

Progress in overcoming the chain association issue in bispecific heterodimeric IgG antibodies

PubMed Central

Klein, Christian; Sustmann, Claudio; Thomas, Markus; Stubenrauch, Kay; Croasdale, Rebecca; Schanzer, Jürgen; Brinkmann, Ulrich; Kettenberger, Hubert; Regula, Jörg T.; Schaefer, Wolfgang

2012-01-01

The development of bispecific antibodies has attracted substantial interest, and many different formats have been described. Those specifically containing an Fc part are mostly tetravalent, such as stabilized IgG-scFv fusions or dual-variable domain (DVD) IgGs. However, although they exhibit IgG-like properties and technical developability, these formats differ in size and geometry from classical IgG antibodies. Thus, considerable efforts focus on bispecific heterodimeric IgG antibodies that more closely mimic natural IgG molecules. The inherent chain association problem encountered when producing bispecific heterodimeric IgG antibodies can be overcome by several methods. While technologies like knobs-into-holes (KiH) combined with a common light chain or the CrossMab technology enforce the correct chain association, other approaches, e.g., the dual-acting Fab (DAF) IgGs, do not rely on a heterodimeric Fc part. This review discusses the state of the art in bispecific heterodimeric IgG antibodies, with an emphasis on recent progress. PMID:22925968

Antibody-controlled actuation of DNA-based molecular circuits.

PubMed

Engelen, Wouter; Meijer, Lenny H H; Somers, Bram; de Greef, Tom F A; Merkx, Maarten

2017-02-17

DNA-based molecular circuits allow autonomous signal processing, but their actuation has relied mostly on RNA/DNA-based inputs, limiting their application in synthetic biology, biomedicine and molecular diagnostics. Here we introduce a generic method to translate the presence of an antibody into a unique DNA strand, enabling the use of antibodies as specific inputs for DNA-based molecular computing. Our approach, antibody-templated strand exchange (ATSE), uses the characteristic bivalent architecture of antibodies to promote DNA-strand exchange reactions both thermodynamically and kinetically. Detailed characterization of the ATSE reaction allowed the establishment of a comprehensive model that describes the kinetics and thermodynamics of ATSE as a function of toehold length, antibody-epitope affinity and concentration. ATSE enables the introduction of complex signal processing in antibody-based diagnostics, as demonstrated here by constructing molecular circuits for multiplex antibody detection, integration of multiple antibody inputs using logic gates and actuation of enzymes and DNAzymes for signal amplification.

Antibody-controlled actuation of DNA-based molecular circuits

NASA Astrophysics Data System (ADS)

Engelen, Wouter; Meijer, Lenny H. H.; Somers, Bram; de Greef, Tom F. A.; Merkx, Maarten

2017-02-01

DNA-based molecular circuits allow autonomous signal processing, but their actuation has relied mostly on RNA/DNA-based inputs, limiting their application in synthetic biology, biomedicine and molecular diagnostics. Here we introduce a generic method to translate the presence of an antibody into a unique DNA strand, enabling the use of antibodies as specific inputs for DNA-based molecular computing. Our approach, antibody-templated strand exchange (ATSE), uses the characteristic bivalent architecture of antibodies to promote DNA-strand exchange reactions both thermodynamically and kinetically. Detailed characterization of the ATSE reaction allowed the establishment of a comprehensive model that describes the kinetics and thermodynamics of ATSE as a function of toehold length, antibody-epitope affinity and concentration. ATSE enables the introduction of complex signal processing in antibody-based diagnostics, as demonstrated here by constructing molecular circuits for multiplex antibody detection, integration of multiple antibody inputs using logic gates and actuation of enzymes and DNAzymes for signal amplification.

Two-color SERS microscopy for protein co-localization in prostate tissue with primary antibody-protein A/G-gold nanocluster conjugates

NASA Astrophysics Data System (ADS)

Salehi, Mohammad; Schneider, Lilli; Ströbel, Philipp; Marx, Alexander; Packeisen, Jens; Schlücker, Sebastian

2014-01-01

SERS microscopy is a novel staining technique in immunohistochemistry, which is based on antibodies labeled with functionalized noble metal colloids called SERS labels or nanotags for optical detection. Conventional covalent bioconjugation of these SERS labels cannot prevent blocking of the antigen recognition sites of the antibody. We present a rational chemical design for SERS label-antibody conjugates which addresses this issue. Highly sensitive, silica-coated gold nanoparticle clusters as SERS labels are non-covalently conjugated to primary antibodies by using the chimeric protein A/G, which selectively recognizes the Fc part of antibodies and therefore prevents blocking of the antigen recognition sites. In proof-of-concept two-color imaging experiments for the co-localization of p63 and PSA on non-neoplastic prostate tissue FFPE specimens, we demonstrate the specificity and signal brightness of these rationally designed primary antibody-protein A/G-gold nanocluster conjugates.SERS microscopy is a novel staining technique in immunohistochemistry, which is based on antibodies labeled with functionalized noble metal colloids called SERS labels or nanotags for optical detection. Conventional covalent bioconjugation of these SERS labels cannot prevent blocking of the antigen recognition sites of the antibody. We present a rational chemical design for SERS label-antibody conjugates which addresses this issue. Highly sensitive, silica-coated gold nanoparticle clusters as SERS labels are non-covalently conjugated to primary antibodies by using the chimeric protein A/G, which selectively recognizes the Fc part of antibodies and therefore prevents blocking of the antigen recognition sites. In proof-of-concept two-color imaging experiments for the co-localization of p63 and PSA on non-neoplastic prostate tissue FFPE specimens, we demonstrate the specificity and signal brightness of these rationally designed primary antibody-protein A/G-gold nanocluster conjugates. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr05890e

Development of a homogeneous assay format for p53 antibodies using fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Neuweiler, Hannes; Scheffler, Silvia; Sauer, Markus

2005-08-01

The development of reliable methods for the detection of minute amounts of antibodies directly in homogeneous solution represents one of the major tasks in the current research field of molecular diagnostics. We demonstrate the potential of fluorescence correlation spectroscopy (FCS) in combination with quenched peptide-based fluorescence probes for sensitive detection of p53 antibodies directly in homogeneous solution. Single tryptophan (Trp) residues in the sequences of short, synthetic peptide epitopes of the human p53 protein efficiently quench the fluorescence of an oxazine fluorophore attached to the amino terminal ends of the peptides. The fluorescence quenching mechanism is thought to be a photoinduced electron transfer reaction from Trp to the dye enabled by the formation of intramolecular complexes between dye and Trp. Specific recognition of the epitope by the antibody confines the conformational flexibility of the peptide. Consequently, complex formation between dye and Trp is abolished and fluorescence is recovered. Using fluorescence correlation spectroscopy (FCS), antibody binding can be monitored observing two parameters simultaneously: the diffusional mobility of the peptide as well as the quenching amplitude induced by the conformational flexibility of the peptide change significantly upon antibody binding. Our data demonstrate that FCS in combination with fluorescence-quenched peptide epitopes opens new possibilities for the reliable detection of antibody binding events in homogeneous solution.

The importance of antineuraminidase antibodies in resistance to influenza A and immunologic memory for their synthesis.

PubMed Central

Naikhin, A. N.; Tsaritsina, I. M.; Oleinikova, E. V.; Syrodoeva, L. G.; Korchanova, N. L.; Denisov, G. M.; Shvartsman YaS

1983-01-01

Eight hundred and seventy-seven sera from 360 adults aged 18-50 who were under permanent observation from October 1980 to March 1981 have been studied by haemagglutination-inhibition (HI) and erythrocyte elution-inhibition (EI) tests--a simplified method of antineuraminidase antibody titration. It was demonstrated in some subjects infected with influenza A H1N1 and H3N2 viruses that the antibody rise was to one of the surface antigens only--haemagglutinin or neuraminidase. These subjects made up 5.2-25.8% of all examinees. The protective effect of antibodies to neuraminidase was similar to that of antihaemagglutinins. Interaction of both types of antibodies was observed in protection against the disease. Data have been obtained on the influence of antineuraminidase antibodies in decreasing the severity of natural infection with influenza A. A study of heterologous immunologic responses to haemagglutinin and neuraminidase among persons immunized with live influenza A H1N1 and H3N2 vaccines and among children naturally infected with influenza A H3N2 demonstrated the presence of immunologic memory for antineuraminidase antibody synthesis. Thus, the suggestion of a common antigenic structure for neuraminidase N1 and N2 is made. PMID:6886409

Antibodies against toluene diisocyanate protein conjugates. Three methods of measurement.

PubMed

Patterson, R; Harris, K E; Zeiss, C R

1983-12-01

With the use of canine antisera against toluene diisocyanate (TDI)-dog serum albumin (DSA), techniques for measuring antibody against TDI-DSA were evaluated. The use of an ammonium sulfate precipitation assay showed suggestive evidence of antibody binding but high levels of TDI-DSA precipitation in the absence of antibody limit any usefulness of this technique. Double-antibody co-precipitation techniques will measure total antibody or Ig class antibody against 125I-TDI-DSA. These techniques are quantitative. The polystyrene tube radioimmunoassay is a highly sensitive method of detecting and quantitatively estimating IgG antibody. The enzyme linked immunosorbent assay is a rapidly adaptable method for the quantitative estimation of IgG, IgA, and IgM against TDI-homologous proteins. All these techniques were compared and results are demonstrated by using the same serum sample for analysis.

Influenza A(H7N9) Virus Antibody Responses in Survivors 1 Year after Infection, China, 2017

PubMed Central

Ma, Mai-Juan; Liu, Cheng; Wu, Meng-Na; Zhao, Teng; Wang, Guo-Lin; Yang, Yang; Gu, Hong-Jing; Cui, Peng-Wei; Pang, Yuan-Yuan; Tan, Ya-Yun; Hang, Hui; Lin, Bao; Qin, Jiang-Chun; Cheng, Li-Ling

2018-01-01

Avian influenza A(H7N9) virus has caused 5 epidemic waves in China since its emergence in 2013. We investigated the dynamic changes of antibody response to this virus over 1 year postinfection in 25 patients in Suzhou City, Jiangsu Province, China, who had laboratory-confirmed infections during the fifth epidemic wave, October 1, 2016–February 14, 2017. Most survivors had relatively robust antibody responses that decreased but remained detectable at 1 year. Antibody response was variable; several survivors had low or undetectable antibody titers. Hemagglutination inhibition titer was >1:40 for <40% of the survivors. Measured in vitro in infected mice, hemagglutination inhibition titer predicted serum protective ability. Our findings provide a helpful serologic guideline for identifying subclinical infections and for developing effective vaccines and therapeutics to counter H7N9 virus infections. PMID:29432091

Influenza A(H7N9) Virus Antibody Responses in Survivors 1 Year after Infection, China, 2017.

PubMed

Ma, Mai-Juan; Liu, Cheng; Wu, Meng-Na; Zhao, Teng; Wang, Guo-Lin; Yang, Yang; Gu, Hong-Jing; Cui, Peng-Wei; Pang, Yuan-Yuan; Tan, Ya-Yun; Hang, Hui; Lin, Bao; Qin, Jiang-Chun; Fang, Li-Qun; Cao, Wu-Chun; Cheng, Li-Ling

2018-04-01

Avian influenza A(H7N9) virus has caused 5 epidemic waves in China since its emergence in 2013. We investigated the dynamic changes of antibody response to this virus over 1 year postinfection in 25 patients in Suzhou City, Jiangsu Province, China, who had laboratory-confirmed infections during the fifth epidemic wave, October 1, 2016-February 14, 2017. Most survivors had relatively robust antibody responses that decreased but remained detectable at 1 year. Antibody response was variable; several survivors had low or undetectable antibody titers. Hemagglutination inhibition titer was >1:40 for <40% of the survivors. Measured in vitro in infected mice, hemagglutination inhibition titer predicted serum protective ability. Our findings provide a helpful serologic guideline for identifying subclinical infections and for developing effective vaccines and therapeutics to counter H7N9 virus infections.

[Immunologic and transplantation biology studies in patients with terminal renal failure].

PubMed

Lanzer, G

1985-01-01

Best possible tissue compatibility is a fundamental precondition for a successful organ transplantation. The desirable tissue compatibility is mainly defined by transplantation-antigens of the donor and the recipient and therefore--beside ABO antigens--the characteristics of HLA-system are the focal point of the preoperative immunological diagnosis. Based on the results of comperative examinations it is demonstrated, that HLA-antisera have such a widespread biological variability, that they lead sometimes to a faulty diagnosis which in turn causes the biological importance of the HLA-system to be doubted. The preoperative immunologic diagnosis should include an estimation of the risk factors in the patient. The consideration of the crossmatch between donor and recipient is a decisive factor in organ transplantation. The starting point is the preoperative antibody monitoring which checks the patients serum reactions against a panel of blood donors to see, whether the patient is a "high" or "low"-- responder to allogenetic stimuli. A positive reaction in the crossmatch is brought about by different kinds of antibodies whereby only in the presence of auto-antibody or cold-reactive B-cell antibody a transplantation may take place. The antibody characterisation in preoperative diagnosis is supported by the results of the immunologic antibody monitoring, whereby--because of the results here presented-- it can be confirmed, that through the knowledge of the antibody specifities which have been checked in a positive crossmatch transplantation on highly sensitized patients can take place with a prospect of success. Supplementing the "Eurotransplant" results the HLA-DRw6 antigen is shown not only to be an indication of risk in transplantation, but may also be an aetiopathogenetic factor. In the presentated statistics it can be shown, that in patients suffering from glomerulonephritis or pyelonephritis and requiring dialysis treatment the HLA - DRw6 antigen occur more frequently than in the control group of healthy blood donors. In glomerulonephritis patients there is additionally a significant change in distribution favourable to HLA - DRw10 shown. The determination of genetically caused risk factors is appreciably supported by characterisation of lymphocyte subpopulations and diagnosis of changes in the Complement-system. Changes in T-lymphocyte subpopulations pointing to proceedings of immunostimulation and conditions of the activated Complement-system represent warning signals in organ transplantation.

[Study of nucleic acid structure by immunochemical methods. I. Antibodies specific for 6-sulfo-5,6-dihydro-4-methoxyaminopyrimidinone-2].

PubMed

Poverennyĭ, A M; Podgorodnichenko, V K; Monastyrskaia, G S; Bryskina, L E; Sverdlov, E D

1978-01-01

Immunization of animals with DNA modified by a mixture of bisulphite and O-methylhydroxylamine and methylated bovine serum albumin results in production of antibodies mainly reacting with modified DNA. Antibodies that react with denatured DNA were produced in minute quantity. It was shown that elicited antibodies possess a high specificity and have the ability to recognize only nucleotides with a double modification. The immune sera were fractionated by Sephadex G-200 column chromatography and the antibody activity was demonstrable in the 19S and 7S fractions. The attempts to induce synthesis of antibodies by injection of DNA modified by O-methylhydroxylamine failed.

[Regression analysis to select native-like structures from decoys of antigen-antibody docking].

PubMed

Chen, Zhengshan; Chi, Xiangyang; Fan, Pengfei; Zhang, Guanying; Wang, Meirong; Yu, Changming; Chen, Wei

2018-06-25

Given the increasing exploitation of antibodies in different contexts such as molecular diagnostics and therapeutics, it would be beneficial to unravel properties of antigen-antibody interaction with modeling of computational protein-protein docking, especially, in the absence of a cocrystal structure. However, obtaining a native-like antigen-antibody structure remains challenging due in part to failing to reliably discriminate accurate from inaccurate structures among tens of thousands of decoys after computational docking with existing scoring function. We hypothesized that some important physicochemical and energetic features could be used to describe antigen-antibody interfaces and identify native-like antigen-antibody structure. We prepared a dataset, a subset of Protein-Protein Docking Benchmark Version 4.0, comprising 37 nonredundant 3D structures of antigen-antibody complexes, and used it to train and test multivariate logistic regression equation which took several important physicochemical and energetic features of decoys as dependent variables. Our results indicate that the ability to identify native-like structures of our method is superior to ZRANK and ZDOCK score for the subset of antigen-antibody complexes. And then, we use our method in workflow of predicting epitope of anti-Ebola glycoprotein monoclonal antibody-4G7 and identify three accurate residues in its epitope.

CCL22-specific Antibodies Reveal That Engagement of Two Distinct Binding Domains on CCL22 Is Required for CCR4-mediated Function.

PubMed

Santulli-Marotto, Sandra; Wheeler, John; Lacy, Eilyn R; Boakye, Ken; Luongo, Jennifer; Wu, Sheng-Jiun; Ryan, Mary

2015-12-01

CCL22 inactivation in vivo occurs by cleavage at the N-terminus; however, it is unclear whether this encompasses the entire site of CCR4 interaction. CCL17 also binds CCR4 and its function requires binding via two discrete binding sites. Using monoclonal antibodies (MAbs), we report that there are two separate sites on CCL22 that are required for CCR4-mediated function. The CCL22-specific antibodies bind with affinities of 632 ± 297 pM (MC2B7) and 308 ± 43 pM (MAB4391) and neither exhibited detectable binding to CCL17. Both antibodies are comparable in their ability to inhibit CCL22-mediated calcium mobilization; however, competition binding studies demonstrate that MC2B7 and MAB4391 bind to distinct epitopes on CCL22. Both antibodies inhibit function through CCR4, which is demonstrated by loss of β-arrestin recruitment in a reporter cell line. In both assays, blocking either site independently abolished CCL22 function, suggesting that concurrent engagement of both sites with CCR4 is necessary for function. This is the first demonstration that CCL22 has two distinct binding sites that are required for CCR4 function. These antibodies are valuable tools for better understanding the interaction and function of CCL22 and CCR4 and will potentially help further understanding of the differential outcomes of CCL17 and CCL22 interaction with CCR4.

[Comparison of several methods for detecting anti-erythrocyte alloantibodies].

PubMed

Bencomo, A A

1990-08-01

The efficacy of different methods for anti-red cell antibodies detection was assessed, variations being found in accordance with the specificity of the alloantibodies. The usefulness of enzyme tests in anti-Rh antibody detection was demonstrated, as well as that of low ionic strength saline solutions in detecting anti-Kell, anti-Duffy and anti-Kidd antibodies. Serum precipitation with 15% polyethyleneglycol 8000 previously to indirect antiglobulin test was found the most sensitive method, providing the best results in all the antibodies studied.

ACTION OF TRITIATED TETANUS TOXIN AND TOXOID UPON THE ANTIBODY FORMING MECHANISM. Period Covered: April 1, 1962 to March 31, 1963

DOE Office of Scientific and Technical Information (OSTI.GOV)

Speirs, R.

1962-12-01

Progress is reported in studies of hypersensitivity and antibody formation in mice in which tritiated antigens were used as tracers. Data are included from a study on the initiation of antibody production in irradiated mice and studies on hemopoiesis and antibody production as demonstrated in radioautograms of spleen, peritoneal fluid, bone marrow, and thymus from mice injected with antigen foliowed by tritiated thymidine and autopsied at specific times. (C.H.)

How-to-do-it: Immunological Assays for the Classroom 1. Enzyme Linked Immunosorbent Assay (ELISA): A Laboratory Tool for Demonstration of Antibody-Antigen Interaction.

ERIC Educational Resources Information Center

Russo, A. J.; And Others

1984-01-01

Background information, list of required materials, and procedures are provided for an immunological assay which has been modified for use as a classroom/laboratory demonstration of antigen-antibody reaction. The assay is designed for a two and one-half hour laboratory period but may be modified for one hour laboratories. (JN)

Varicella-zoster virus immunity among health care workers in Catalonia.

PubMed

Urbiztondo, L; Bayas, J M; Broner, S; Costa, J; Esteve, M; Campins, M; Borrás, E; Domínguez, A

2014-10-14

To determine varicella-zoster virus (VZV) immunity among healthcare workers (HCWs). Cross-sectional study. HCWs attending voluntary periodic health examinations between June 2008 and December 2010. Six public hospitals and five primary care areas in Catalonia, Spain. A self-administered questionnaire was given to eligible HCWs. Variables including age, sex, professional category, type of centre, history of varicella infection, and VZV vaccination were collected. The study was carried out using a convenience sample. The prevalence of antibodies and positive and negative predictive values (PPV and NPV) of the history of clinical VZV infection or vaccination were calculated. Crude and adjusted odds ratios (OR and ORa) and their 95% confidence intervals (CI) were calculated to determine the variables associated with antibody prevalence. Of 705 HCWs who agreed to participate, 644 were finally included. The overall prevalence of antibodies to varicella was 94.9% (95% CI: 92.9-96.4). Of the variables studied, only age was associated with serological susceptibility to VZV. HCWs aged 25-35 years had the highest serological susceptibility (8.1%, 95% CI: 4.6-13.0). The prevalence of antibodies was 96% in subjects reporting previous VZV infection or vaccination, compared with 93% in subjects who did not report these states or did not know. The high proportion of serologically-susceptible HCWs found in this study indicates the need to develop for screening and vaccination strategies in Catalonia. Due to the high capacity of propagation of the VZV in health settings and its consequences, VZV vaccination programmes in HCWs should be reinforced. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparison of mHTT Antibodies in Huntington’s Disease Mouse Models Reveal Specific Binding Profiles and Steady-State Ubiquitin Levels with Disease Development

PubMed Central

Bayram-Weston, Zubeyde; Jones, Lesley; Dunnett, Stephen B.; Brooks, Simon P.

2016-01-01

Huntington’s disease (HD) cellular pathology is characterised by the aggregation of mutant huntingtin (mHTT) protein into inclusion bodies. The present paper compared the sensitivity of five widely used mHTT antibodies (S830; MW8; EM48; 1C2; ubiquitin) against mice from five commonly used HD mouse models (R6/1; YAC128; HdhQ92; B6 HdhQ150; B6 x129/Ola HdhQ150) at two ages to determine: the most sensitive antibodies for each model; whether mHTT antibody binding differed depending on aggregation stage (diffuse versus frank inclusion); the role of ubiquitin during aggregation as the ubiquitin proteosome system has been implicated in disease development. The models demonstrated unique profiles of antibody binding even when the models varied only by background strain (HdhQ150). MW8 was highly sensitive for detecting frank inclusions in all lines whereas EM48, ubiquitin and 1C2 demonstrated consistent staining in all models irrespective of age or form of mHTT. MW8 and S830 were the most sensitive antibodies with 1C2 the least. Ubiquitin levels were stable for each model regardless of age. Ubiquitin was particularly sensitive in young YAC128 mice that demonstrate an absence of inclusions until ~12 months of age suggesting high affinity to mHTT in its diffuse form. The data indicate that generalisations across models regarding the quantification of aggregations may not be valid and that mHTT antibody binding is unique to the mouse model and sensitive to changes in inclusion development. PMID:27196694

Species-Specific Immunodetection of an Entamoeba histolytica Cyst Wall Protein

PubMed Central

Kearney, Moira R.; Siddique, Abdullah; Ali, Ibne K.; Gilchrist, Carol A.; Arju, Tuhinur; Hoffstrom, Benjamin; Nguyen, Felicia K.; Petri, William A.; Haque, Rashidul; Cangelosi, Gerard A.

2016-01-01

Entamoeba histolytica causes intestinal disease in endemic settings throughout the world. Diagnosis of E. histolytica infection would be improved by the identification of biomarkers that are expressed by cysts of E. histolytica, but not by cysts of closely related commensal species of Entamoeba. Herein, we describe two novel monoclonal antibodies (1A4 and 1D3) produced against a spacer region of the E. histolytica Jacob2 lectin, an outer cyst wall protein. These reagents demonstrated no cross-reaction to E. dispar recombinant antigen and low picomolar molecular detection limits when paired in ELISA sandwich assays. In an immunofluorescence microscopy assay, the α-Jacob2 murine antibodies labeled cysts of three xenically cultured E. histolytica isolates but did not label cysts of three E. bangladeshi isolates. Monoclonal antibody 1A4 did not cross-react with xenic cultures of three E. dispar isolates, demonstrating specificity to E. histolytica, while monoclonal antibody 1D3 cross-reacted with two out of three E. dispar isolates. Both antibodies labeled cysts in formalin-fixed slides, a potential logistical advantage in some settings. The monoclonal antibody 1A4 was also used in an immunofluorescence microscopy assay with formalin-fixed stool specimens. Seven out of ten ELISA-positive stool specimens exhibited 1A4-labeled cyst-like objects, compared to one out of seven ELISA-negative specimens. These results demonstrate that antibodies generated against the flexible spacer of E. histolytica Jacob2 lectin recognize and bind to Jacob2 protein in whole cysts and are capable of differentiating Entamoeba species in fixed specimens. Thus, Jacob2 is a promising biomarker for use in diagnosing E. histolytica infection. PMID:27152855

Increasing the sensitivity of reverse phase protein arrays by antibody-mediated signal amplification

PubMed Central

2010-01-01

Background Reverse phase protein arrays (RPPA) emerged as a useful experimental platform to analyze biological samples in a high-throughput format. Different signal detection methods have been described to generate a quantitative readout on RPPA including the use of fluorescently labeled antibodies. Increasing the sensitivity of RPPA approaches is important since many signaling proteins or posttranslational modifications are present at a low level. Results A new antibody-mediated signal amplification (AMSA) strategy relying on sequential incubation steps with fluorescently-labeled secondary antibodies reactive against each other is introduced here. The signal quantification is performed in the near-infrared range. The RPPA-based analysis of 14 endogenous proteins in seven different cell lines demonstrated a strong correlation (r = 0.89) between AMSA and standard NIR detection. Probing serial dilutions of human cancer cell lines with different primary antibodies demonstrated that the new amplification approach improved the limit of detection especially for low abundant target proteins. Conclusions Antibody-mediated signal amplification is a convenient and cost-effective approach for the robust and specific quantification of low abundant proteins on RPPAs. Contrasting other amplification approaches it allows target protein detection over a large linear range. PMID:20569466

Anti-GM1 antibodies as a model of the immune response to self-glycans.

PubMed

Nores, Gustavo A; Lardone, Ricardo D; Comín, Romina; Alaniz, María E; Moyano, Ana L; Irazoqui, Fernando J

2008-03-01

Glycans are a class of molecules with high structural variability, frequently found in the plasma membrane facing the extracellular space. Because of these characteristics, glycans are often considered as recognition molecules involved in cell social functions, and as targets of pathogenic factors. Induction of anti-glycan antibodies is one of the early events in immunological defense against bacteria that colonize the body. Because of this natural infection, antibodies recognizing a variety of bacterial glycans are found in sera of adult humans and animals. The immune response to glycans is restricted by self-tolerance, and no antibodies to self-glycans should exist in normal subjects. However, antibodies recognizing structures closely related to self-glycans do exist, and can lead to production of harmful anti-self antibodies. Normal human sera contain low-affinity anti-GM1 IgM-antibodies. Similar antibodies with higher affinity or different isotype are found in some neuropathy patients. Two hypotheses have been developed to explain the origin of disease-associated anti-GM1 antibodies. According to the "molecular mimicry" hypothesis, similarity between GM1 and Campylobacter jejuni lipopolysaccharide carrying a GM1-like glycan is the cause of Guillain-Barré syndrome associated with anti-GM1 IgG-antibodies. According to the "binding site drift" hypothesis, IgM-antibodies associated with disease originate through changes in the binding site of normally occurring anti-GM1 antibodies. We now present an "integrated" hypothesis, combining the "mimicry" and "drift" concepts, which satisfactorily explains most of the published data on anti-GM1 antibodies.

Reconciling the Structural Attributes of Avian Antibodies*

PubMed Central

Conroy, Paul J.; Law, Ruby H. P.; Gilgunn, Sarah; Hearty, Stephen; Caradoc-Davies, Tom T.; Lloyd, Gordon; O'Kennedy, Richard J.; Whisstock, James C.

2014-01-01

Antibodies are high value therapeutic, diagnostic, biotechnological, and research tools. Combinatorial approaches to antibody discovery have facilitated access to unique antibodies by surpassing the diversity limitations of the natural repertoire, exploitation of immune repertoires from multiple species, and tailoring selections to isolate antibodies with desirable biophysical attributes. The V-gene repertoire of the chicken does not utilize highly diverse sequence and structures, which is in stark contrast to the mechanism employed by humans, mice, and primates. Recent exploitation of the avian immune system has generated high quality, high affinity antibodies to a wide range of antigens for a number of therapeutic, diagnostic and biotechnological applications. Furthermore, extensive examination of the amino acid characteristics of the chicken repertoire has provided significant insight into mechanisms employed by the avian immune system. A paucity of avian antibody crystal structures has limited our understanding of the structural consequences of these uniquely chicken features. This paper presents the crystal structure of two chicken single chain fragment variable (scFv) antibodies generated from large libraries by phage display against important human antigen targets, which capture two unique CDRL1 canonical classes in the presence and absence of a non-canonical disulfide constrained CDRH3. These structures cast light on the unique structural features of chicken antibodies and contribute further to our collective understanding of the unique mechanisms of diversity and biochemical attributes that render the chicken repertoire of particular value for antibody generation. PMID:24737329

Antibody engineering of a cytotoxic monoclonal antibody 84 against human embryonic stem cells: Investigating the effects of multivalency on cytotoxicity.

PubMed

Klement, Maximilian; Zheng, Jiyun; Liu, Chengcheng; Tan, Heng-Liang; Wong, Victor Vai Tak; Choo, Andre Boon-Hwa; Lee, Dong-Yup; Ow, Dave Siak-Wei

2017-02-10

Antibody fragments have shown targeted specificity to their antigens, but only modest tissue retention times in vivo and in vitro. Multimerization has been used as a protein engineering tool to increase the number of binding units and thereby enhance the efficacy and retention time of antibody fragments. In this work, we explored the effects of valency using a series of self-assembling polypeptides based on the GCN4 leucine zipper multimerization domain fused to a single-chain variable fragment via an antibody upper hinge sequence. Four engineered antibody fragments with a valency from one to four antigen-binding units of a cytotoxic monoclonal antibody 84 against human embryonic stem cells (hESC) were constructed. We hypothesized that higher cytotoxicity would be observed for fragments with increased valency. Flow cytometry analysis revealed that the trimeric and tetrameric engineered antibody fragments resulted in the highest degree of cytotoxicity to the undifferentiated hESC, while the engineered antibody fragments were observed to have improved tissue penetration into cell clusters. Thus, a trade off was made for the trimeric versus tetrameric fragment due to improved tissue penetration. These results have direct implications for antibody-mediated removal of undifferentiated hESC during regenerative medicine and cell therapy. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

Maternal immunity enhances systemic recall immune responses upon oral immunization of piglets with F4 fimbriae.

PubMed

Nguyen, Ut V; Melkebeek, Vesna; Devriendt, Bert; Goetstouwers, Tiphanie; Van Poucke, Mario; Peelman, Luc; Goddeeris, Bruno M; Cox, Eric

2015-06-23

F4 enterotoxigenic Escherichia coli (ETEC) cause diarrhoea and mortality in piglets leading to severe economic losses. Oral immunization of piglets with F4 fimbriae induces a protective intestinal immune response evidenced by an F4-specific serum and intestinal IgA response. However, successful oral immunization of pigs with F4 fimbriae in the presence of maternal immunity has not been demonstrated yet. In the present study we aimed to evaluate the effect of maternal immunity on the induction of a systemic immune response upon oral immunization of piglets. Whereas F4-specific IgG and IgA could be induced by oral immunization of pigs without maternal antibodies and by intramuscular immunization of pigs with maternal antibodies, no such response was seen in the orally immunized animals with maternal antibodies. Since maternal antibodies can mask an antibody response, we also looked by ELIspot assays for circulating F4-specific antibody secreting cells (ASCs). Enumerating the F4-specific ASCs within the circulating peripheral blood mononuclear cells, and the number of F4-specific IgA ASCs within the circulating IgA(+) B-cells revealed an F4-specific immune response in the orally immunized animals with maternal antibodies. Interestingly, results suggest a more robust IgA booster response by oral immunization of pigs with than without maternal antibodies. These results demonstrate that oral immunization of piglets with F4-specific maternal antibodies is feasible and that these maternal antibodies seem to enhance the secondary systemic immune response. Furthermore, our ELIspot assay on enriched IgA(+) B-cells could be used as a screening procedure to optimize mucosal immunization protocols in pigs with maternal immunity.

EphA2 Targeted Chemotherapy Using an Antibody Drug Conjugate in Endometrial Carcinoma

PubMed Central

Lee, Jeong-Won; Stone, Rebecca L.; Lee, Sun Joo; Nam, Eun Ji; Roh, Ju-Won; Nick, Alpa M.; Han, Hee-Dong; Shahzad, Mian M.K.; Kim, Hye-Sun; Mangala, Lingegowda S.; Jennings, Nicholas B.; Mao, Shenlan; Gooya, John; Jackson, Dowdy; Coleman, Robert L.; Sood, Anil K.

2013-01-01

Purpose EphA2 overexpression is frequently observed in endometrial cancers, and is predictive of poor clinical outcome. Here, we utilize an antibody drug conjugate (MEDI-547) composed of a fully human monoclonal antibody against both human and murine EphA2 (1C1) and the tubulin polymerization inhibitor, monomethylauristatin F (MMAF). Experimental design EphA2 expression was examined in endometrial cancer cell lines by Western Blot. Specificity of MEDI-547 was examined by antibody degradation and internalization assays. Viability and apoptosis were investigated in endometrial cancer cell lines and orthotopic tumor models. Results EphA2 was expressed in the Hec-1A and Ishikawa cells, but was absent in the SPEC-2 cells. Antibody degradation and internalization assays showed that the antibody drug conjugate decreased EphA2 protein levels and was internalized in EphA2 positive cells (Hec-1A and Ishikawa). Moreover, in vitro cytotoxicity and apoptosis assays demonstrated that the antibody drug conjugate decreased viability and increased apoptosis of Hec-1A and Ishikawa cells. In vivo therapy experiments in mouse orthotopic models with this antibody drug conjugate resulted in 86 to 88% growth inhibition (P < 0.001) in the orthotopic Hec-1A and Ishikawa models compared to controls. Moreover, the mice treated with this antibody drug conjugate had a lower incidence of distant metastasis compared with controls. The anti-tumor effects of the therapy were related to decreased proliferation and increased apoptosis of tumor and associated endothelial cells. Conclusions The preclinical data for endometrial cancer treatment using MEDI-547 demonstrate substantial anti-tumor activity. PMID:20388851

A high-throughput platform for population reformatting and mammalian expression of phage display libraries to enable functional screening as full-length IgG.

PubMed

Xiao, Xiaodong; Douthwaite, Julie A; Chen, Yan; Kemp, Ben; Kidd, Sara; Percival-Alwyn, Jennifer; Smith, Alison; Goode, Kate; Swerdlow, Bonnie; Lowe, David; Wu, Herren; Dall'Acqua, William F; Chowdhury, Partha S

Phage display antibody libraries are a rich resource for discovery of potential therapeutic antibodies. Single-chain variable fragment (scFv) libraries are the most common format due to the efficient display of scFv by phage particles and the ease by which soluble scFv antibodies can be expressed for high-throughput screening. Typically, a cascade of screening and triaging activities are performed, beginning with the assessment of large numbers of E. coli-expressed scFv, and progressing through additional assays with individual reformatting of the most promising scFv to full-length IgG. However, use of high-throughput screening of scFv for the discovery of full-length IgG is not ideal because of the differences between these molecules. Furthermore, the reformatting step represents a bottle neck in the process because each antibody has to be handled individually to preserve the unique VH and VL pairing. These problems could be resolved if populations of scFv could be reformatted to full-length IgG before screening without disrupting the variable region pairing. Here, we describe a novel strategy that allows the reformatting of diverse populations of scFv from phage selections to full-length IgG in a batch format. The reformatting process maintains the diversity and variable region pairing with high fidelity, and the resulted IgG pool enables high-throughput expression of IgG in mammalian cells and cell-based functional screening. The improved process led to the discovery of potent candidates that are comparable or better than those obtained by traditional methods. This strategy should also be readily applicable to Fab-based phage libraries. Our approach, Screening in Product Format (SiPF), represents a substantial improvement in the field of antibody discovery using phage display.

Next-Generation DNA Sequencing of VH/VL Repertoires: A Primer and Guide to Applications in Single-Domain Antibody Discovery.

PubMed

Henry, Kevin A

2018-01-01

Immunogenetic analyses of expressed antibody repertoires are becoming increasingly common experimental investigations and are critical to furthering our understanding of autoimmunity, infectious disease, and cancer. Next-generation DNA sequencing (NGS) technologies have now made it possible to interrogate antibody repertoires to unprecedented depths, typically by sequencing of cDNAs encoding immunoglobulin variable domains. In this chapter, we describe simple, fast, and reliable methods for producing and sequencing multiplex PCR amplicons derived from the variable regions (V H , V H H or V L ) of rearranged immunoglobulin heavy and light chain genes using the Illumina MiSeq platform. We include complete protocols and primer sets for amplicon sequencing of V H /V H H/V L repertoires directly from human, mouse, and llama lymphocytes as well as from phage-displayed V H /V H H/V L libraries; these can be easily be adapted to other types of amplicons with little modification. The resulting amplicons are diverse and representative, even using as few as 10 3 input B cells, and their generation is relatively inexpensive, requiring no special equipment and only a limited set of primers. In the absence of heavy-light chain pairing, single-domain antibodies are uniquely amenable to NGS analyses. We present a number of applications of NGS technology useful in discovery of single-domain antibodies from phage display libraries, including: (i) assessment of library functionality; (ii) confirmation of desired library randomization; (iii) estimation of library diversity; and (iv) monitoring the progress of panning experiments. While the case studies presented here are of phage-displayed single-domain antibody libraries, the principles extend to other types of in vitro display libraries.

A potent human anti-eotaxin1 antibody, CAT-213: isolation by phage display and in vitro and in vivo efficacy.

PubMed

Main, Sarah; Handy, Rachel; Wilton, Jane; Smith, Stephen; Williams, Liz; Fou, Leila Du; Andrews, John; Conroy, Louise A; May, Richard; Anderson, Ian; Vaughan, Tristan J

2006-12-01

The CC chemokine, eotaxin1 (CCL11) is an important regulator of eosinophil function. A marked accumulation of eosinophils in tissues has been correlated with the up-regulation of eotaxin1 expression in several diseases. The potential therapeutic value of neutralizing the effects of eotaxin1 in inflammatory conditions (including asthma) is under investigation. A human single-chain fragment variable antibody that neutralizes human eotaxin1 (CAT-212) was produced using antibody phage display and converted to whole antibody IgG4 format (CAT-213). A novel approach to lead optimization in which the length of the variable heavy chain complementarity-determining region 3 was reduced by one amino acid resulted in an increase in potency of >1000-fold compared with the parent anti-eotaxin1 antibody. The optimized antibody binds eotaxin1 with high affinity (80.4 pM) and specificity. CAT-213 and CAT-212 do not bind or neutralize a range of other human proteins including human monocyte chemoattractant protein-1, a structurally similar chemokine. CAT-213 neutralizes the ability of eotaxin1 to cause an increase in intracellular calcium signaling (with an IC(50) value of 2.86 nM), migration of CCR3-expressing L1.2 cells (with an IC(50) value of 0.48 nM), and inhibition of the eotaxin1-evoked shape change of human eosinophils in vitro (with an IC(50) of 0.71 nM). Local administration of CAT-213 to mice (1-100 microg kg(-1)) attenuates dermal eosinophilia induced by human eotaxin1, achieving >90% inhibition of eosinophil influx. CAT-213 may therefore be of therapeutic value in inhibiting diseases in which eotaxin1 and eosinophils play a major role, for example, severe asthma.

Primary antibody deficiencies at Queen Rania Children Hospital in Jordan: single center experience.

PubMed

Habahbeh, Zeyad M; Abu-Shukair, Mohammad E; Almutereen, Mohammad A; Alzyoud, Raed M; Wahadneh, Adel M

2014-03-01

Primary antibody deficiency, the most common primary immunodeficiency disorder, represents a heterogeneous spectrum of conditions caused by a defect in any critical stage of B cell development and is characterized by impaired production of normal amounts of antigen-specific antibodies. This retrospective study aimed at description and analysis of demographic, clinical, immunological features and complications of subjects diagnosed with primary antibody deficiency at a referral center in Jordan. The medical records of pediatric patients who were diagnosed as primary antibody deficiency (PAD) during the period from January 2006 to June 2013 were reviewed. Patients were diagnosed as PADs based on the Pan-American Group for Immunodeficiency (PAGID) and the European Society for Immunodeficiency (ESID) diagnostic criteria. A total number of 53 patients with PAD were identified; 37(70%) males and 16(30%) females, 16(30%) patients with congenital agammaglobulinemia, 16(30%) patients with common variable immunodeficiency, 4(7.5%) patients with IgG subclass deficiency, 10(19%) cases with transient hypogammaglobulinemia of infancy and 7(13.5%) patients as undefined PAD. The most common infection among patients was pneumonia (62%); followed by suppurative otitis media in 49% of patients. Cytopenia was the most noted autoimmune association and was found at prevalence of 22 %, other autoimmune associations (17%) including inflammatory arthritis, discoid lupus, inflammatory bowel disease, vasculitis and celiac disease. The prevalence of long-term complications was 58%, the most frequent ones were; stunted growth in 13%, bronchiectasis and lymphoproliferation in 11% for each. Our results indicated that congenital agammaglobulinemia and common variable immunodeficiency are the most frequent primary antibody deficiency in our patients. The awareness of families, general population as well as primary health physicians is crucial in the establishment of early diagnosis and prompt commencement of appropriate therapy for PADs.

Highly sensitive and unbiased approach for elucidating antibody repertoires

PubMed Central

Lin, Sherry G.; Ba, Zhaoqing; Du, Zhou; Zhang, Yu; Hu, Jiazhi; Alt, Frederick W.

2016-01-01

Developing B lymphocytes undergo V(D)J recombination to assemble germ-line V, D, and J gene segments into exons that encode the antigen-binding variable region of Ig heavy (H) and light (L) chains. IgH and IgL chains associate to form the B-cell receptor (BCR), which, upon antigen binding, activates B cells to secrete BCR as an antibody. Each of the huge number of clonally independent B cells expresses a unique set of IgH and IgL variable regions. The ability of V(D)J recombination to generate vast primary B-cell repertoires results from a combinatorial assortment of large numbers of different V, D, and J segments, coupled with diversification of the junctions between them to generate the complementary determining region 3 (CDR3) for antigen contact. Approaches to evaluate in depth the content of primary antibody repertoires and, ultimately, to study how they are further molded by secondary mutation and affinity maturation processes are of great importance to the B-cell development, vaccine, and antibody fields. We now describe an unbiased, sensitive, and readily accessible assay, referred to as high-throughput genome-wide translocation sequencing-adapted repertoire sequencing (HTGTS-Rep-seq), to quantify antibody repertoires. HTGTS-Rep-seq quantitatively identifies the vast majority of IgH and IgL V(D)J exons, including their unique CDR3 sequences, from progenitor and mature mouse B lineage cells via the use of specific J primers. HTGTS-Rep-seq also accurately quantifies DJH intermediates and V(D)J exons in either productive or nonproductive configurations. HTGTS-Rep-seq should be useful for studies of human samples, including clonal B-cell expansions, and also for following antibody affinity maturation processes. PMID:27354528

Cross-talk between clinical and host-response parameters of periodontitis in smokers.

PubMed

Nagarajan, R; Miller, C S; Dawson, D; Al-Sabbagh, M; Ebersole, J L

2017-06-01

Periodontal diseases are a major public health concern leading to tooth loss and have also been shown to be associated with several chronic systemic diseases. Smoking is a major risk factor for the development of numerous systemic diseases, as well as periodontitis. While it is clear that smokers have a significantly enhanced risk for developing periodontitis leading to tooth loss, the population varies regarding susceptibility to disease associated with smoking. This investigation focused on identifying differences in four broad sets of variables, consisting of: (i) host-response molecules; (ii) periodontal clinical parameters; (iii) antibody responses to periodontal pathogens and oral commensal bacteria; and (iv) other variables of interest, in a population of smokers with (n = 171) and without (n = 117) periodontitis. Bayesian network structured learning (BNSL) techniques were used to investigate potential associations and cross-talk between the four broad sets of variables. BNSL revealed two broad communities with markedly different topology between the populations of smokers, with and without periodontitis. Confidence of the edges in the resulting network also showed marked variations within and between the periodontitis and nonperiodontitis groups. The results presented validated known associations and discovered new ones with minimal precedence that may warrant further investigation and novel hypothesis generation. Cross-talk between the clinical variables and antibody profiles of bacteria were especially pronounced in the case of periodontitis and were mediated by the antibody response profile to Porphyromonas gingivalis. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Cross-talk between Clinical and Host Response Parameters of Periodontitis in Smokers

PubMed Central

Nagarajan, R.; Miller, C.S.; Dawson, D.; Al-Sabbagh, M.; Ebersole, J.L.

2016-01-01

Periodontal diseases are a major public health concern leading to tooth loss and also shown to be associated with several chronic systemic diseases. Smoking is a major risk factor for developing numerous systemic diseases, as well as periodontitis. While it is clear that smokers have a significantly enhanced risk for developing periodontitis leading to tooth loss, the population varies with regards to susceptibility to disease associated with smoking. This investigation focuses on identifying differences in four broad sets of variables consisting of: (a) host response molecules, (b) periodontal clinical parameters, (c) antibody measures for periodontal pathogens and oral commensal bacteria challenge, and (d) other variables of interest in a smoking population with (n = 171) and without periodontitis (n = 117). Subsequently, Bayesian network structured learning techniques (BNSL) techniques were used to investigate potential associations and cross-talk between the four broad sets of variables. BNSL revealed two broad communities with markedly different topology between the non-periodontitis and periodontitis smoking population. Confidence of the edges in the resulting network also showed marked variations within and between the periodontitis and non-periodontitis groups. The results presented validated known associations, as well as discovered new ones with minimal precedence that may warrant further investigation and novel hypothesis generation. Cross-talk between the clinical variables and antibody profiles of bacteria were especially pronounced in the case of periodontitis and mediated by the antibody response profile to P. gingivalis. PMID:27431617

Laboratory data supporting the clinical trial of antirabies serum in persons bitten by a rabid wolf

PubMed Central

Habel, Karl; Koprowski, Hilary

1955-01-01

Five individuals severely exposed to rabies by wolf bite and treated with a course of phenolized vaccine alone showed no demonstrable antibodies in their sera until the nineteenth day following the start of treatment. Three of these five individuals died of rabies. On the other hand, twelve individuals similarly exposed, who received antirabies serum plus a course of phenolized vaccine, had demonstrable antibodies early in and throughout the period of observation. One individual who received one dose of serum plus a course of vaccine died of rabies. In view of these results, it is apparent that antibody demonstrable early in and throughout the treatment period, and obtained by the combined use of serum and vaccine, is more effective in preventing rabies after severe exposure than is a course of vaccine alone. PMID:13284557

Bioreactor process parameter screening utilizing a Plackett-Burman design for a model monoclonal antibody.

PubMed

Agarabi, Cyrus D; Schiel, John E; Lute, Scott C; Chavez, Brittany K; Boyne, Michael T; Brorson, Kurt A; Khan, Mansoora; Read, Erik K

2015-06-01

Consistent high-quality antibody yield is a key goal for cell culture bioprocessing. This endpoint is typically achieved in commercial settings through product and process engineering of bioreactor parameters during development. When the process is complex and not optimized, small changes in composition and control may yield a finished product of less desirable quality. Therefore, changes proposed to currently validated processes usually require justification and are reported to the US FDA for approval. Recently, design-of-experiments-based approaches have been explored to rapidly and efficiently achieve this goal of optimized yield with a better understanding of product and process variables that affect a product's critical quality attributes. Here, we present a laboratory-scale model culture where we apply a Plackett-Burman screening design to parallel cultures to study the main effects of 11 process variables. This exercise allowed us to determine the relative importance of these variables and identify the most important factors to be further optimized in order to control both desirable and undesirable glycan profiles. We found engineering changes relating to culture temperature and nonessential amino acid supplementation significantly impacted glycan profiles associated with fucosylation, β-galactosylation, and sialylation. All of these are important for monoclonal antibody product quality. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Large-scale sequence and structural comparisons of human naive and antigen-experienced antibody repertoires.

PubMed

DeKosky, Brandon J; Lungu, Oana I; Park, Daechan; Johnson, Erik L; Charab, Wissam; Chrysostomou, Constantine; Kuroda, Daisuke; Ellington, Andrew D; Ippolito, Gregory C; Gray, Jeffrey J; Georgiou, George

2016-05-10

Elucidating how antigen exposure and selection shape the human antibody repertoire is fundamental to our understanding of B-cell immunity. We sequenced the paired heavy- and light-chain variable regions (VH and VL, respectively) from large populations of single B cells combined with computational modeling of antibody structures to evaluate sequence and structural features of human antibody repertoires at unprecedented depth. Analysis of a dataset comprising 55,000 antibody clusters from CD19(+)CD20(+)CD27(-) IgM-naive B cells, >120,000 antibody clusters from CD19(+)CD20(+)CD27(+) antigen-experienced B cells, and >2,000 RosettaAntibody-predicted structural models across three healthy donors led to a number of key findings: (i) VH and VL gene sequences pair in a combinatorial fashion without detectable pairing restrictions at the population level; (ii) certain VH:VL gene pairs were significantly enriched or depleted in the antigen-experienced repertoire relative to the naive repertoire; (iii) antigen selection increased antibody paratope net charge and solvent-accessible surface area; and (iv) public heavy-chain third complementarity-determining region (CDR-H3) antibodies in the antigen-experienced repertoire showed signs of convergent paired light-chain genetic signatures, including shared light-chain third complementarity-determining region (CDR-L3) amino acid sequences and/or Vκ,λ-Jκ,λ genes. The data reported here address several longstanding questions regarding antibody repertoire selection and development and provide a benchmark for future repertoire-scale analyses of antibody responses to vaccination and disease.

Designed Ankyrin Repeat Proteins: A New Approach to Mimic Complex Antigens for Diagnostic Purposes?

PubMed Central

Hausammann, Stefanie; Vogel, Monique; Kremer Hovinga, Johanna A.; Lacroix-Desmazes, Sebastien; Stadler, Beda M.; Horn, Michael P.

2013-01-01

Inhibitory antibodies directed against coagulation factor VIII (FVIII) can be found in patients with acquired and congenital hemophilia A. Such FVIII-inhibiting antibodies are routinely detected by the functional Bethesda Assay. However, this assay has a low sensitivity and shows a high inter-laboratory variability. Another method to detect antibodies recognizing FVIII is ELISA, but this test does not allow the distinction between inhibitory and non-inhibitory antibodies. Therefore, we aimed at replacing the intricate antigen FVIII by Designed Ankyrin Repeat Proteins (DARPins) mimicking the epitopes of FVIII inhibitors. As a model we used the well-described inhibitory human monoclonal anti-FVIII antibody, Bo2C11, for the selection on DARPin libraries. Two DARPins were selected binding to the antigen-binding site of Bo2C11, which mimic thus a functional epitope on FVIII. These DARPins inhibited the binding of the antibody to its antigen and restored FVIII activity as determined in the Bethesda assay. Furthermore, the specific DARPins were able to recognize the target antibody in human plasma and could therefore be used to test for the presence of Bo2C11-like antibodies in a large set of hemophilia A patients. These data suggest, that our approach might be used to isolate epitopes from different sets of anti-FVIII antibodies in order to develop an ELISA-based screening assay allowing the distinction of inhibitory and non-inhibitory anti-FVIII antibodies according to their antibody signatures. PMID:23626669

Anti-P1: the most common unexpected antibodies in northeastern-Thais.

PubMed

Romphruk, A V; Wanhagij, C; Akahat, J; Tantanapornkul, P; Anuphan, T; Pattayaso, P; Puapairoj, C

1999-08-01

The prevalence of unexpected antibodies in the Northeastern-Thai population was studied. Sera were collected from 25,673 blood donors including 18,209 males and 7,464 females. The sera were screened for unexpected antibodies by saline and enzyme techniques. The sera which gave a positive antibody screening test were identified for specificity of antibody. The result demonstrated that 3,928 from 25,673 samples (15.30%) were positive for the antibody screening test and only 3,883 samples could be identified for specificity of antibody. The most common unexpected antibodies were anti-P1, anti-lewis and anti-P1 + anti-lewis with the frequency of 70.8, 18.6 and 10.1 per cent, respectively. The prevalence of anti-P1 in this study was higher than that reported in Central Thailand and Southeast Asia which may due to the high prevalence of liver fluke infection in the Northeastern-Thai population.

Generalized platform for antibody detection using the antibody catalyzed water oxidation pathway.

PubMed

Welch, M Elizabeth; Ritzert, Nicole L; Chen, Hongjun; Smith, Norah L; Tague, Michele E; Xu, Youyong; Baird, Barbara A; Abruña, Héctor D; Ober, Christopher K

2014-02-05

Infectious diseases, such as influenza, present a prominent global problem including the constant threat of pandemics that initiate in avian or other species and then pass to humans. We report a new sensor that can be specifically functionalized to detect antibodies associated with a wide range of infectious diseases in multiple species. This biosensor is based on electrochemical detection of hydrogen peroxide generated through the intrinsic catalytic activity of all antibodies: the antibody catalyzed water oxidation pathway (ACWOP). Our platform includes a polymer brush-modified surface where specific antibodies bind to conjugated haptens with high affinity and specificity. Hydrogen peroxide provides an electrochemical signal that is mediated by Resorufin/Amplex Red. We characterize the biosensor platform, using model anti-DNP antibodies, with the ultimate goal of designing a versatile device that is inexpensive, portable, reliable, and fast. We demonstrate detection of antibodies at concentrations that fall well within clinically relevant levels.

New Coxsackievirus 2Apro and 3Cpro protease antibodies for virus detection and discovery of pathogenic mechanisms.

PubMed

Laitinen, Olli H; Svedin, Emma; Kapell, Sebastian; Hankaniemi, Minna M; Larsson, Pär G; Domsgen, Erna; Stone, Virginia M; Määttä, Juha A E; Hyöty, Heikki; Hytönen, Vesa P; Flodström-Tullberg, Malin

2018-05-01

Enteroviruses (EVs), such as the Coxsackie B-viruses (CVBs), are common human pathogens, which can cause severe diseases including meningitis, myocarditis and neonatal sepsis. EVs encode two proteases (2A pro and 3C pro ), which perform the proteolytic cleavage of the CVB polyprotein and also cleave host cell proteins to facilitate viral replication. The 2A pro cause direct damage to the infected heart and tools to investigate 2A pro and 3C pro expression may contribute new knowledge on virus-induced pathologies. Here, we developed new antibodies to CVB-encoded 2A pro and 3C pro ; Two monoclonal 2A pro antibodies and one 3C pro antibody were produced. Using cells infected with selected viruses belonging to the EV A, B and C species and immunocytochemistry, we demonstrate that the 3C pro antibody detects all of the EV species B (EV-B) viruses tested and that the 2A pro antibody detects all EV-B viruses apart from Echovirus 9. We furthermore show that the new antibodies work in Western blotting, immunocyto- and immunohistochemistry, and flow cytometry to detect CVBs. Confocal microscopy demonstrated the expression kinetics of 2A pro and 3C pro , and revealed a preferential cytosolic localization of the proteases in CVB3 infected cells. In summary, the new antibodies detect proteases that belong to EV species B in cells and tissue using multiple applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Generation of anti-idiotype scFv for pharmacokinetic measurement in lymphoma patients treated with chimera anti-CD22 antibody SM03.

PubMed

Zhao, Qi; Wong, Pui-Fan; Lee, Susanna S T; Leung, Shui-On; Cheung, Wing-Tai; Wang, Jun-Zhi

2014-01-01

Pre-clinical and clinical studies of therapeutic antibodies require highly specific reagents to examine their immune responses, bio-distributions, immunogenicity, and pharmacodynamics in patients. Selective antigen-mimicking anti-idiotype antibody facilitates the assessment of therapeutic antibody in the detection, quantitation and characterization of antibody immune responses. Using mouse specific degenerate primer pairs and splenocytic RNA, we generated an idiotype antibody-immunized phage-displayed scFv library in which an anti-idiotype antibody against the therapeutic chimera anti-CD22 antibody SM03 was isolated. The anti-idiotype scFv recognized the idiotype of anti-CD22 antibody and inhibited binding of SM03 to CD22 on Raji cell surface. The anti-idiotype scFv was subsequently classified as Ab2γ type. Moreover, our results also demonstrated firstly that the anti-idiotype scFv could be used for pharmacokinetic measurement of circulating residual antibody in lymphoma patients treated with chimera anti-CD22 monoclonal antibody SM03. Of important, the present approach could be easily adopted to generate anti-idiotype antibodies for therapeutic antibodies targeting membrane proteins, saving the cost and time for producing a soluble antigen.

Generation of Anti-Idiotype scFv for Pharmacokinetic Measurement in Lymphoma Patients Treated with Chimera Anti-CD22 Antibody SM03

PubMed Central

Zhao, Qi; Wong, Pui-Fan; Lee, Susanna S. T.; Leung, Shui-On; Cheung, Wing-Tai; Wang, Jun-Zhi

2014-01-01

Pre-clinical and clinical studies of therapeutic antibodies require highly specific reagents to examine their immune responses, bio-distributions, immunogenicity, and pharmacodynamics in patients. Selective antigen-mimicking anti-idiotype antibody facilitates the assessment of therapeutic antibody in the detection, quantitation and characterization of antibody immune responses. Using mouse specific degenerate primer pairs and splenocytic RNA, we generated an idiotype antibody-immunized phage-displayed scFv library in which an anti-idiotype antibody against the therapeutic chimera anti-CD22 antibody SM03 was isolated. The anti-idiotype scFv recognized the idiotype of anti-CD22 antibody and inhibited binding of SM03 to CD22 on Raji cell surface. The anti-idiotype scFv was subsequently classified as Ab2γ type. Moreover, our results also demonstrated firstly that the anti-idiotype scFv could be used for pharmacokinetic measurement of circulating residual antibody in lymphoma patients treated with chimera anti-CD22 monoclonal antibody SM03. Of important, the present approach could be easily adopted to generate anti-idiotype antibodies for therapeutic antibodies targeting membrane proteins, saving the cost and time for producing a soluble antigen. PMID:24816427

Localized gene delivery using antibody tethered adenovirus from polyurethane heart valve cusps and intra-aortic implants.

PubMed

Stachelek, S J; Song, C; Alferiev, I; Defelice, S; Cui, X; Connolly, J M; Bianco, R W; Levy, R J

2004-01-01

The present study investigated a novel approach for gene therapy of heart valve disease and vascular disorders. We formulated and characterized implantable polyurethane films that could also function as gene delivery systems through the surface attachment of replication defective adenoviruses using an anti-adenovirus antibody tethering mechanism. Our hypothesis was that we could achieve site-specific gene delivery to cells interacting with these polyurethane implants, and thereby demonstrate the potential for intravascular devices that could also function as gene delivery platforms for therapeutic vectors. Previous research by our group has demonstrated that polyurethane elastomers can be derivatized post-polymerization through a series of chemical reactions activating the hard segment amide groups with alkyl bromine residues, which can enable a wide variety of subsequent chemical modifications. Furthermore, prior research by our group investigating gene delivery intravascular stents has shown that collagen-coated balloon expandable stents can be configured with anti-adenovirus antibodies via thiol-based chemistry, and can then tether adenoviral vectors at doses that lead to high levels of localized arterial neointima expression, but with virtually no distal spread of vector. Thus, we sought to create two-device configurations for our investigations building on this previous research. (1) Polyurethane films coated with Type I collagen were thiol activated to permit covalent attachment of anti-adenovirus antibodies to enable gene delivery via vector tethering. (2) We also formulated polyurethane films with direct covalent attachment of anti-adenovirus antibodies to polyurethane hard segments derivatized with alkyl-thiol groups, thereby also enabling tethering of replication-defective adenoviruses. Both formulations demonstrated highly localized and efficient transduction in cell culture studies with rat arterial smooth muscle cells. In vivo experiments with collagen-coated polyurethane films investigated an abdominal aorta implant model in pigs using a button configuration that simulated the blood contacting environment of a vascular graft. One week explants of the collagen-coated polyurethane films demonstrated 14.3+/-2.5% of neointimal cells on the surface of the implant transduced with green fluorescent protein - adenovirus (AdGFP) vector loadings of 1 x 10(8) PFU. PCR studies demonstrated no detectable vector DNA in blood or distal organs. Similarly, polyurethane films with direct attachment of antivector antibodies to the surface were used in sheep pulmonary valve leaflet replacement studies, simulating the blood contacting environment of a prosthetic heart valve cusp. Polyurethane films with antibody tethered AdGFP vector (10(8) PFU) demonstrated 25.1+/-5.7% of attached cells transduced in these 1 week studies, with no detectable vector DNA in blood or distal organs. In vivo GFP expression was confirmed with immunohistochemistry. It is concluded that site-specific intravascular delivery of adenoviral vectors for gene therapy can be achieved with polyurethane implants utilizing the antivector antibody tethering mechanism.

Response of puppies to canine-origin parvovirus vaccines.

PubMed

Carmichael, L E; Pollock, R V; Joubert, J C

1984-02-01

Pups 9-18 1/2 weeks old were given a single dose of 1 of 4 commercial, live, canine-origin parvovirus vaccines. All 4 vaccines evoked high levels of antibody in seronegative pups, but variable response in those with low levels of maternally derived antibodies. Vaccinal virus spread to unvaccinated contact controls and elicited essentially equivalent titers. No clinical signs of parvovirus infection were observed in vaccinates or controls.

Related Mechanisms of Antibody Somatic Hypermutation and Class Switch Recombination

PubMed Central

HWANG, JOYCE K.; ALT, FREDERICK W.; YEAP, LENG-SIEW

2015-01-01

The primary antibody repertoire is generated by mechanisms involving the assembly of the exons that encode the antigen-binding variable regions of immunoglobulin heavy (IgH) and light (IgL) chains during the early development of B lymphocytes. After antigen-dependent activation, mature B lymphocytes can further alter their IgH and IgL variable region exons by the process of somatic hypermutation (SHM), which allows the selection of B cells in which SHMs resulted in the production of antibodies with increased antigen affinity. In addition, during antigen-dependent activation, B cells can also change the constant region of their IgH chain through a DNA double-strand-break (DSB) dependent process referred to as IgH class switch recombination (CSR), which generates B cell progeny that produce antibodies with different IgH constant region effector functions that are best suited for a elimination of a particular pathogen or in a particular setting. Both the mutations that underlie SHM and the DSBs that underlie CSR are initiated in target genes by activation-induced cytidine deaminase (AID). This review describes in depth the processes of SHM and CSR with a focus on mechanisms that direct AID cytidine deamination in activated B cells and mechanisms that promote the differential outcomes of such cytidine deamination. PMID:26104555

Using llama derived single domain antibodies to target botulinum neurotoxins

NASA Astrophysics Data System (ADS)

Swain, Marla D.; Anderson, George P.; Bernstein, Rachael D.; Liu, Jinny L.; Goldman, Ellen R.

2010-04-01

Llama serum contains both conventional IgG as well as unique forms of antibody that contain only heavy chains where antigen binding is mediated through a single variable domain. These variable domains can be expressed recombinantly and are referred to as single domain antibodies (sdAb). SdAb are among the smallest known naturally derived antigen binding fragments, possess good solubility, thermal stability, and can refold after heat and chemical denaturation. Llamas were immunized with either BoNT A or B toxoid and phage display libraries prepared. Single domain antibodies (sdAb) that were able to detect botulinum neurotoxin (BoNT) serotypes A and B were selected from their respective libraries. Here, the binders obtained by panning the BoNT B library on either BoNT B toxoid or BoNT B complex toxoid coated plates or BoNT B toxin coupled microspheres are described. Using these panning methods, we selected for binders that showed specificity for BoNT B. Phage displayed binders were screened, moved to a protein expression vector and soluble sdAb was produced. Using a Luminex flow cytometer binders were evaluated in direct binding assays. We have exploited the unique properties of sdAb and used them as biological recognition elements in immuno-based sensors that can detect BoNT B.

GLUT-3 expression in human skeletal muscle

NASA Technical Reports Server (NTRS)

Stuart, C. A.; Wen, G.; Peng, B. H.; Popov, V. L.; Hudnall, S. D.; Campbell, G. A.

2000-01-01

Muscle biopsy homogenates contain GLUT-3 mRNA and protein. Before these studies, it was unclear where GLUT-3 was located in muscle tissue. In situ hybridization using a midmolecule probe demonstrated GLUT-3 within all muscle fibers. Fluorescent-tagged antibody reacting with affinity-purified antibody directed at the carboxy-terminus demonstrated GLUT-3 protein in all fibers. Slow-twitch muscle fibers, identified by NADH-tetrazolium reductase staining, possessed more GLUT-3 protein than fast-twitch fibers. Electron microscopy using affinity-purified primary antibody and gold particle-tagged second antibody showed that the majority of GLUT-3 was in association with triads and transverse tubules inside the fiber. Strong GLUT-3 signals were seen in association with the few nerves that traversed muscle sections. Electron microscopic evaluation of human peripheral nerve demonstrated GLUT-3 within the axon, with many of the particles related to mitochondria. GLUT-3 protein was found in myelin but not in Schwann cells. GLUT-1 protein was not present in nerve cells, axons, myelin, or Schwann cells but was seen at the surface of the peripheral nerve in the perineurium. These studies demonstrated that GLUT-3 mRNA and protein are expressed throughout normal human skeletal muscle, but the protein is predominantly found in the triads of slow-twitch muscle fibers.

Rapid isolation of IgNAR variable single-domain antibody fragments from a shark synthetic library.

PubMed

Shao, Cui-Ying; Secombes, Chris J; Porter, Andrew J

2007-01-01

The immunoglobulin isotype IgNAR (Novel Antigen Receptor) was discovered in the serum of the nurse shark (Ginglymostoma cirratum) and wobbegong shark (Orectolobus maculates) as a homodimer of two protein chains, each composed of a single variable domain (V) domain and five constant domains. The IgNAR variable domain contains an intact antigen-binding site and functions as an independent domain able to react to antigen with both high specificity and affinity. Here we describe the successful construction of a synthetic phage-displayed library based upon a single anti-lysozyme clone HEL-5A7 scaffold, which was previously selected from an immune IgNAR variable domain library. The complementarity-determining region 3 (CDR3) loop of this clone was varied in both length and composition and the derived library was used to pan against two model proteins, lysozyme and leptin. A single anti-lysozyme clone (Ly-X20) and anti-leptin clone (Lep-12E1) were selected for further study. Both clones were shown to be functionally expressed in Escherichia coli, extremely thermostable and bind to corresponding antigens specifically. The results here demonstrate that a synthetic IgNAR variable domain library based on a single framework scaffold can be used as a route to generate antigen binders quickly, easily and without the need of immunization.

Chimpanzees Immunized with Recombinant Soluble CD4 Develop Anti-Self CD4 Antibody Responses with Anti-Human Immunodeficiency Virus Activity

NASA Astrophysics Data System (ADS)

Watanabe, Mamoru; Boyson, Jonathan E.; Lord, Carol I.; Letvin, Norman L.

1992-06-01

In view of the efficiency with which human immunodeficiency virus replication can be blocked in vitro with anti-CD4 antibodies, the elicitation of an anti-CD4 antibody response through active immunization might represent a useful therapeutic strategy for AIDS. Here we demonstrate that immunization of chimpanzees with recombinant soluble human CD4 elicited an anti-CD4 antibody response. The elicited antibody bound self CD4 on digitonin-treated but not freshly isolated lymphocytes. Nevertheless, this antibody blocked human immunodeficiency virus replication in chimpanzee and human lymphocytes. These observations suggest that immunization with recombinant soluble CD4 from human immunodeficiency virus-infected humans may be feasible and therapeutically beneficial.

Antibody to intermediate filaments of the cytoskeleton.

PubMed Central

Osung, O A; Chandra, M; Holborow, E J

1982-01-01

IgM antibodies against cultures of intermediate filaments (IMF) of the cytoskeleton were demonstrated by immunofluorescence in the sera of 94 (80%) of 118 patients with seropositive rheumatoid arthritis. These antibodies reacted with IMF in cultures of both human fetal fibroblasts and laryngeal carcinoma (HEp2) cells. Of 10 patients from whom paired synovial fluids were also available 8 had anti-IMF antibodies in both serum and fluid. In seronegative RA the incidence of anti-IMF was 40%, in ankylosing spondylitis 25%, in osteoarthrosis 16%, and in normal subjects 14%. Only a minority of RA sera positive for anti-IMF antibodies were also positive for smooth muscle antibody. Absorption experiments suggest that in RA anti-IMF is directed at the intermediate filament protein, vimentin. Images PMID:7039524

Immunity War: A Novel Therapy for Lymphoma Using T-cell Bispecific Antibodies.

PubMed

Prakash, Ajay; Diefenbach, Catherine S

2018-06-08

The activity of T cell mediated immunotherapies in B cell lymphoma has been limited to date. The novel bi-specific antibody CD20-TCB, has a 2:1 antibody design to maximize T cell engagement, and demonstrates activity in preclinical models. This may represent a novel therapeutic approach for patients with relapsed/refractory NHL. Copyright ©2018, American Association for Cancer Research.

Scintigraphy of normal mouse ovaries with monoclonal antibodies to ZP-2, the major zona pellucida protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

East, I.J.; Keenan, A.M.; Larson, S.M.

1984-08-31

The zona pellucida is an extracellular glycocalyx, made of three sulfated glycoproteins, that surrounds mammalian oocytes. Parenterally administered monoclonal antibodies specific for ZP-2, the most abundant zona protein, localize in the zona pellucida. When labeled with iodine-125, these monoclonal antibodies demonstrate a remarkably high target-to-nontarget tissue ratio and provide clear external radioimaging of ovarian tissue.

Development and Evaluation of a Fluorescent Antibody-Drug Conjugate for Molecular Imaging and Targeted Therapy of Pancreatic Cancer

PubMed Central

Knutson, Steve; Raja, Erum; Bomgarden, Ryan; Nlend, Marie; Chen, Aoshuang; Kalyanasundaram, Ramaswamy; Desai, Surbhi

2016-01-01

Antibodies are widely available and cost-effective research tools in life science, and antibody conjugates are now extensively used for targeted therapy, immunohistochemical staining, or in vivo diagnostic imaging of cancer. Significant advances in site-specific antibody labeling technologies have enabled the production of highly characterized and homogenous conjugates for biomedical purposes, and some recent studies have utilized site-specific labeling to synthesize bifunctional antibody conjugates with both imaging and drug delivery properties. While these advances are important for the clinical safety and efficacy of such biologics, these techniques can also be difficult, expensive, and time-consuming. Furthermore, antibody-drug conjugates (ADCs) used for tumor treatment generally remain distinct from conjugates used for diagnosis. Thus, there exists a need to develop simple dual-labeling methods for efficient therapeutic and diagnostic evaluation of antibody conjugates in pre-clinical model systems. Here, we present a rapid and simple method utilizing commercially available reagents for synthesizing a dual-labeled fluorescent ADC. Further, we demonstrate the fluorescent ADC’s utility for simultaneous targeted therapy and molecular imaging of cancer both in vitro and in vivo. Employing non-site-specific, amine-reactive chemistry, our novel biopharmaceutical theranostic is a monoclonal antibody specific for a carcinoembryonic antigen (CEA) biomarker conjugated to both paclitaxel and a near-infrared (NIR), polyethylene glycol modified (PEGylated) fluorophore (DyLight™ 680-4xPEG). Using in vitro systems, we demonstrate that this fluorescent ADC selectively binds a CEA-positive pancreatic cancer cell line (BxPC-3) in immunofluorescent staining and flow cytometry, exhibits efficient internalization kinetics, and is cytotoxic. Model studies using a xenograft of BxPC-3 cells in athymic mice also show the fluorescent ADC’s efficacy in detecting tumors in vivo and inhibiting tumor growth more effectively than equimolar amounts of unconjugated drug. Overall, our results demonstrate that non-selective, amine-targeting chemistry is an effective dual-labeling method for synthesizing and evaluating a bifunctional fluorescent antibody-drug conjugate, allowing concurrent detection, monitoring and treatment of cancer. PMID:27336622

[Effective combination therapy of plasma exchange and subsequent cyclophosphamide pulses for catastrophic antiphospholipid antibody syndrome: a case report].

PubMed

Miyamae, T; Imagawa, T; Ito, S; Katakura, S; Mori, M; Ibe, M; Mitsuda, T; Aihara, Y; Nakanishi, S; Kohri, T; Yokota, S

1999-06-01

A 7-year-old girl with catastrophic antiphospholipid antibody syndrome was described. She firstly admitted to the local hospital with the complaints of persistent fever and abdominal pain, and was diagnosed as systemic lupus erythematosus with the laboratory findings as follows; positive for antinuclear antibody, anti-DNA antibody, and platelet-associated IgG, thrombocytopenia, and hypocomplementemia. 10 days after the initiation of oral prednisolone, she suddenly manifested tonic convulsion and unconsciousness accompanied by high fever. Because of the unresponsiveness to the methylprednisolone pulse therapy for supposed CNS lupus, she was transferred to our hospital. Her unconsciousness persisted, and pulsation on dorsalis pedis was not palpable on admission. Laboratory investigation revealed the falsely positive VDRL, a prolonged aPTT, positive for lupus-anticoagulant and antiphospholipid antibody. The magnetic resonance image demonstrated multiple spotty hyperintensity (T2) in the brain consistent with multiple hemorrhagic infarcts. Arteriogram demonstrated the infarct of dorsalis pedis, and coronary aneurysms. These findings were compatible with the criteria of catastrophic antiphospholipid antibody syndrome, she was diagnosed as catastrophic antiphospholipid antibody syndrome. The plasma exchange and subsequent cyclophosphamide-pulse therapy, which was given once a month for first 6 months, and later, at 3 months intervals, was effectively administered. This combination and oral anti-thrombotic therapy revealed effective for this kind of fatal disorder.

Humanization of Antibodies Using Heavy Chain Complementarity-determining Region 3 Grafting Coupled with in Vitro Somatic Hypermutation*

PubMed Central

Bowers, Peter M.; Neben, Tamlyn Y.; Tomlinson, Geoffery L.; Dalton, Jennifer L.; Altobell, Larry; Zhang, Xue; Macomber, John L.; Wu, Betty F.; Toobian, Rachelle M.; McConnell, Audrey D.; Verdino, Petra; Chau, Betty; Horlick, Robert A.; King, David J.

2013-01-01

A method for simultaneous humanization and affinity maturation of monoclonal antibodies has been developed using heavy chain complementarity-determining region (CDR) 3 grafting combined with somatic hypermutation in vitro. To minimize the amount of murine antibody-derived antibody sequence used during humanization, only the CDR3 region from a murine antibody that recognizes the cytokine hβNGF was grafted into a nonhomologous human germ line V region. The resulting CDR3-grafted HC was paired with a CDR-grafted light chain, displayed on the surface of HEK293 cells, and matured using in vitro somatic hypermutation. A high affinity humanized antibody was derived that was considerably more potent than the parental antibody, possessed a low pm dissociation constant, and demonstrated potent inhibition of hβNGF activity in vitro. The resulting antibody contained half the heavy chain murine donor sequence compared with the same antibody humanized using traditional methods. PMID:23355464

Automated Antibody De Novo Sequencing and Its Utility in Biopharmaceutical Discovery

NASA Astrophysics Data System (ADS)

Sen, K. Ilker; Tang, Wilfred H.; Nayak, Shruti; Kil, Yong J.; Bern, Marshall; Ozoglu, Berk; Ueberheide, Beatrix; Davis, Darryl; Becker, Christopher

2017-05-01

Applications of antibody de novo sequencing in the biopharmaceutical industry range from the discovery of new antibody drug candidates to identifying reagents for research and determining the primary structure of innovator products for biosimilar development. When murine, phage display, or patient-derived monoclonal antibodies against a target of interest are available, but the cDNA or the original cell line is not, de novo protein sequencing is required to humanize and recombinantly express these antibodies, followed by in vitro and in vivo testing for functional validation. Availability of fully automated software tools for monoclonal antibody de novo sequencing enables efficient and routine analysis. Here, we present a novel method to automatically de novo sequence antibodies using mass spectrometry and the Supernovo software. The robustness of the algorithm is demonstrated through a series of stress tests.

Scale-Dependent Manganese Leaching from Stainless Steel Impacts Terminal Galactosylation in Monoclonal Antibodies.

PubMed

Williamson, Jenna; Miller, Jennifer; McLaughlin, Joseph; Combs, Rodney; Chu, Chia

2018-06-08

N-linked glycosylation profiles are routinely characterized on mammalian-derived protein therapeutic products and achieving consistency in the product-associated glycan attributes is an important indicator that the manufacturing process is under control. More importantly, meeting target glycan profile is a common criterion for ensuring product efficacy. During laboratory process development and subsequent scale up for pilot demonstration for a monoclonal antibody program, discrepancies in the molecule's terminal galactosylation level at 2-L, 100-L, and 6000-L scales were observed. Results from extensive investigations revealed the root cause as manganese leaching from the stainless steel components and that this leaching is dependent on exposed surface area and cultivation time. Although this metal impurity is only present at nanomolar concentrations and difficult to detect, a spike-in study demonstrated that this low level was sufficient to impact the protein glycosylation profiles. Surprisingly, the 2-L glass bioreactor setup exhibited the highest amount of exposure to stainless steel and resulted in both a greater degree of variability and higher overall levels of terminal galactosylation. The use of disposable vessels to minimize stainless steel surface exposure to the cell culture resulted in comparable terminal galactosylation levels to those measured in pilot and commercial bioreactors. The discovery of this leachable effect on the cell culture production process was an essential step in implementing appropriate process control. This article is protected by copyright. All rights reserved. © 2018 American Institute of Chemical Engineers.

Improving the pharmacokinetic properties of biologics by fusion to an anti-HSA shark VNAR domain

PubMed Central

Müller, Mischa R.; Saunders, Kenneth; Grace, Christopher; Jin, Macy; Piche-Nicholas, Nicole; Steven, John; O’Dwyer, Ronan; Wu, Leeying; Khetemenee, Lam; Vugmeyster, Yulia; Hickling, Timothy P.; Tchistiakova, Lioudmila; Olland, Stephane; Gill, Davinder; Jensen, Allan; Barelle, Caroline J.

2012-01-01

Advances in recombinant antibody technology and protein engineering have provided the opportunity to reduce antibodies to their smallest binding domain components and have concomitantly driven the requirement for devising strategies to increase serum half-life to optimise drug exposure, thereby increasing therapeutic efficacy. In this study, we adopted an immunization route to raise picomolar affinity shark immunoglobulin new antigen receptors (IgNARs) to target human serum albumin (HSA). From our model shark species, Squalus acanthias, a phage display library encompassing the variable binding domain of IgNAR (VNAR) was constructed, screened against target, and positive clones were characterized for affinity and specificity. N-terminal and C-terminal molecular fusions of our lead hit in complex with a naïve VNAR domain were expressed, purified and exhibited the retention of high affinity binding to HSA, but also cross-selectivity to mouse, rat and monkey serum albumin both in vitro and in vivo. Furthermore, the naïve VNAR had enhanced pharmacokinetic (PK) characteristics in both N- and C-terminal orientations and when tested as a three domain construct with naïve VNAR flanking the HSA binding domain at both the N and C termini. Molecules derived from this platform technology also demonstrated the potential for clinical utility by being available via the subcutaneous route of delivery. This study thus demonstrates the first in vivo functional efficacy of a VNAR binding domain with the ability to enhance PK properties and support delivery of multifunctional therapies. PMID:23676205

Improving the pharmacokinetic properties of biologics by fusion to an anti-HSA shark VNAR domain.

PubMed

Müller, Mischa R; Saunders, Kenneth; Grace, Christopher; Jin, Macy; Piche-Nicholas, Nicole; Steven, John; O'Dwyer, Ronan; Wu, Leeying; Khetemenee, Lam; Vugmeyster, Yulia; Hickling, Timothy P; Tchistiakova, Lioudmila; Olland, Stephane; Gill, Davinder; Jensen, Allan; Barelle, Caroline J

2012-01-01

Advances in recombinant antibody technology and protein engineering have provided the opportunity to reduce antibodies to their smallest binding domain components and have concomitantly driven the requirement for devising strategies to increase serum half-life to optimise drug exposure, thereby increasing therapeutic efficacy. In this study, we adopted an immunization route to raise picomolar affinity shark immunoglobulin new antigen receptors (IgNARs) to target human serum albumin (HSA). From our model shark species, Squalus acanthias, a phage display library encompassing the variable binding domain of IgNAR (VNAR) was constructed, screened against target, and positive clones were characterized for affinity and specificity. N-terminal and C-terminal molecular fusions of our lead hit in complex with a naïve VNAR domain were expressed, purified and exhibited the retention of high affinity binding to HSA, but also cross-selectivity to mouse, rat and monkey serum albumin both in vitro and in vivo. Furthermore, the naïve VNAR had enhanced pharmacokinetic (PK) characteristics in both N- and C-terminal orientations and when tested as a three domain construct with naïve VNAR flanking the HSA binding domain at both the N and C termini. Molecules derived from this platform technology also demonstrated the potential for clinical utility by being available via the subcutaneous route of delivery. This study thus demonstrates the first in vivo functional efficacy of a VNAR binding domain with the ability to enhance PK properties and support delivery of multifunctional therapies.

Drug of the year: programmed death-1 receptor/programmed death-1 ligand-1 receptor monoclonal antibodies.

PubMed

Robert, Caroline; Soria, Jean-Charles; Eggermont, Alexander M M

2013-09-01

Programmed death-1 receptor (PD-1)/its ligand (PD-L1) antibodies have changed the landscape in oncology in 2013. The most mature results have been obtained in advanced melanoma patients. They indicate important response rates and high quality responses or prolonged duration. Also in renal cancer and in lung cancer remarkable activity has been demonstrated. Thus it is clear that these antibodies have a very broad potential and trials in many tumour types are being initiated. Breaking tolerance at the tumour site is a potent phenomenon and the potential for synergy with other checkpoint inhibitors such as ipilimumab have also been demonstrated in 2013. Long term tumour control now seems achievable and thus the concept of a clinical cure is emerging by modulation of the immune system. These antibodies bring immunotherapy to the forefront and indicate that immune-modulation will be a key component of therapeutic strategies from now on. Because of all these reasons PD-1/PD-L1 antibodies are considered 'drug of the year'. Copyright © 2013 Elsevier Ltd. All rights reserved.

Regulation of monoclonal immunoglobulin G synthesis by antiidiotypic antibody in a patient with hypogammaglobulinemia.

PubMed Central

Mudawwar, F; Awdeh, Z; Ault, K; Geha, R S

1980-01-01

The regulation of in vitro antibody synthesis by antiidiotypic antibodies was studied in a child with hypogammaglobulinemia and a serum immunoglobulin (Ig)G1 kappa M component. A rabbit antiserum was raised against the purified M component and was rendered idiotype specific by extensive absorption with Cohn fraction II and with IgG derived from the patient's parents. Hemagglutination-inhibition studies demonstrated that less than 1 in 300,000 molecules of pooled human IgG carried M component-related idiotypic determinants. 12% of the patient's B cells, but none of her T cells, expressed idiotypic determinants on their surface. Spontaneous de novo synthesis of the M component by the patient's peripheral blood lymphocytes was demonstrated in vitro and was shown to proceed independently of the polyclonal activator pokeweed mitogen. Antiidiotypic rabbit IgG, but not its F(ab')2, fragments, profoundly inhibited the synthesis of M component by the patient's peripheral blood lymphocytes. We concluded that antiidiotypic antibodies may play a role in the regulation of antibody synthesis in man. PMID:6767740

A novel computer algorithm improves antibody epitope prediction using affinity-selected mimotopes: a case study using monoclonal antibodies against the West Nile virus E protein.

PubMed

Denisova, Galina F; Denisov, Dimitri A; Yeung, Jeffrey; Loeb, Mark B; Diamond, Michael S; Bramson, Jonathan L

2008-11-01

Understanding antibody function is often enhanced by knowledge of the specific binding epitope. Here, we describe a computer algorithm that permits epitope prediction based on a collection of random peptide epitopes (mimotopes) isolated by antibody affinity purification. We applied this methodology to the prediction of epitopes for five monoclonal antibodies against the West Nile virus (WNV) E protein, two of which exhibit therapeutic activity in vivo. This strategy was validated by comparison of our results with existing F(ab)-E protein crystal structures and mutational analysis by yeast surface display. We demonstrate that by combining the results of the mimotope method with our data from mutational analysis, epitopes could be predicted with greater certainty. The two methods displayed great complementarity as the mutational analysis facilitated epitope prediction when the results with the mimotope method were equivocal and the mimotope method revealed a broader number of residues within the epitope than the mutational analysis. Our results demonstrate that the combination of these two prediction strategies provides a robust platform for epitope characterization.

A constant threat for HIV: Fc-engineering to enhance broadly neutralizing antibody activity for immunotherapy of the acquired immunodeficiency syndrome.

PubMed

Nimmerjahn, Falk

2015-08-01

Passive immunotherapy with polyclonal or hyperimmune serum immunoglobulin G (IgG) preparations provides an efficient means of protecting immunocompromised patients from microbial infections. More recently, the use of passive immunotherapy to prevent or to treat established infections with the human immunodeficiency virus (HIV) has gained much attention, due to promising preclinical data obtained in monkey and humanized mouse in vivo model systems, demonstrating that the transfer of HIV-specific antibodies can not only prevent HIV infection, but also diminish virus load during chronic infection. Furthermore, an array of broadly neutralizing HIV-specific antibodies has become available and the importance of the IgG constant region as a critical modulator of broadly neutralizing activity has been demonstrated. The aim of this review is to summarize the most recent findings with regard to the molecular and cellular mechanisms responsible for antibody-mediated clearance of HIV infection, and to discuss how this may help to improve HIV therapy via optimizing Fcγ-receptor-dependent activities of HIV-specific antibodies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Viral antibody dynamics in a chiropteran host

PubMed Central

Baker, Kate S; Suu-Ire, Richard; Barr, Jennifer; Hayman, David T S; Broder, Christopher C; Horton, Daniel L; Durrant, Christopher; Murcia, Pablo R; Cunningham, Andrew A; Wood, James L N

2014-01-01

Bats host many viruses that are significant for human and domestic animal health, but the dynamics of these infections in their natural reservoir hosts remain poorly elucidated. In these, and other, systems, there is evidence that seasonal life-cycle events drive infection dynamics, directly impacting the risk of exposure to spillover hosts. Understanding these dynamics improves our ability to predict zoonotic spillover from the reservoir hosts. To this end, we followed henipavirus antibody levels of >100 individual E. helvum in a closed, captive, breeding population over a 30-month period, using a powerful novel antibody quantitation method. We demonstrate the presence of maternal antibodies in this system and accurately determine their longevity. We also present evidence of population-level persistence of viral infection and demonstrate periods of increased horizontal virus transmission associated with the pregnancy/lactation period. The novel findings of infection persistence and the effect of pregnancy on viral transmission, as well as an accurate quantitation of chiropteran maternal antiviral antibody half-life, provide fundamental baseline data for the continued study of viral infections in these important reservoir hosts. PMID:24111634

Delayed LGI1 seropositivity in voltage-gated potassium channel (VGKC)-complex antibody limbic encephalitis

PubMed Central

Sweeney, Michael; Galli, Jonathan; McNally, Scott; Tebo, Anne; Haven, Thomas; Thulin, Perla; Clardy, Stacey L

2017-01-01

We utilise a clinical case to highlight why exclusion of voltage-gated potassium channel (VGKC)-complex autoantibody testing in serological evaluation of patients may delay or miss the diagnosis. A 68-year-old man presented with increasing involuntary movements consistent with faciobrachial dystonic seizures (FBDS). Initial evaluation demonstrated VGKC antibody seropositivity with leucine-rich glioma-inactivated 1 (LGI1) and contactin-associated protein-like 2 (CASPR2) seronegativity. Aggressive immunotherapy with methylprednisolone and plasmapheresis was started early in the course of his presentation. Following treatment with immunotherapy, the patient demonstrated clinical improvement. Repeat serum evaluation 4 months posthospitalisation remained seropositive for VGKC-complex antibodies, with development of LGI1 autoantibody seropositivity. VGKC-complex and LGI1 antibodies remained positive 12 months posthospitalisation. Our findings suggest that clinical symptoms can predate the detection of the antibody. We conclude that when suspicion for autoimmune encephalitis is high in the setting of VGKC autoantibody positivity, regardless of LGI1 or CASPR2 seropositivity, early immunotherapy and repeat testing should be considered. PMID:28432047

Delayed LGI1 seropositivity in voltage-gated potassium channel (VGKC)-complex antibody limbic encephalitis.

PubMed

Sweeney, Michael; Galli, Jonathan; McNally, Scott; Tebo, Anne; Haven, Thomas; Thulin, Perla; Clardy, Stacey L

2017-04-20

We utilise a clinical case to highlight why exclusion of voltage-gated potassium channel (VGKC)-complex autoantibody testing in serological evaluation of patients may delay or miss the diagnosis. A 68-year-old man presented with increasing involuntary movements consistent with faciobrachial dystonic seizures (FBDS). Initial evaluation demonstrated VGKC antibody seropositivity with leucine-rich glioma-inactivated 1 (LGI1) and contactin-associated protein-like 2 (CASPR2) seronegativity. Aggressive immunotherapy with methylprednisolone and plasmapheresis was started early in the course of his presentation. Following treatment with immunotherapy, the patient demonstrated clinical improvement. Repeat serum evaluation 4 months posthospitalisation remained seropositive for VGKC-complex antibodies, with development of LGI1 autoantibody seropositivity. VGKC-complex and LGI1 antibodies remained positive 12 months posthospitalisation. Our findings suggest that clinical symptoms can predate the detection of the antibody. We conclude that when suspicion for autoimmune encephalitis is high in the setting of VGKC autoantibody positivity, regardless of LGI1 or CASPR2 seropositivity, early immunotherapy and repeat testing should be considered. 2017 BMJ Publishing Group Ltd.

Treatment of VGKC complex antibody-associated limbic encephalitis: a systematic review.

PubMed

Radja, Guirindhra Koumar; Cavanna, Andrea Eugenio

2013-01-01

Limbic encephalitis is an autoimmune neuropsychiatric condition characterized by subacute cognitive symptoms, seizures, and affective changes. Although limbic encephalitis is usually caused by an immune reaction secondary to neoplasms, different types of potentially treatable non-paraneoplastic limbic encephalitis (nPLE) have recently been described. In particular, published studies have reported variable responses to immunosuppressive therapy in Voltage-Gated Potassium Channel (VGKC) complex antibody-associated nPLE. This systematic literature review found that the most significant improvements were reported by patients presenting with affective symptoms and consistent neuroradiological changes. In these patients, improved clinical outcomes correlated with the largest decreases in antibody titers.

When monoclonal antibodies are not monospecific: Hybridomas frequently express additional functional variable regions

PubMed Central

Bradbury, Andrew R. M.; Trinklein, Nathan D.; Wilkinson, Ian C.; Tandon, Atul K.; Anderson, Stephen; Bladen, Catherine L.; Jones, Brittany; Aldred, Shelley Force; Bestagno, Marco; Burrone, Oscar; Maynard, Jennifer; Ferrara, Fortunato; Görnemann, Janina; Glanville, Jacob; Wolf, Philipp; Frenzel, Andre; Wong, Julin; Koh, Xin Yu; Eng, Hui-Yan; Lane, David; Lefranc, Marie-Paule; Clark, Mike

2018-01-01

ABSTRACT Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. The most abundant mRNA transcripts found in a hybridoma cell line did not necessarily encode the antibody chains providing the correct specificity. Consequently, when cloning antibody genes, functional validation of all possible VH and VL combinations is required to identify those with the highest affinity and lowest cross-reactivity. These findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use. PMID:29485921

Lipid contribution to the affinity of antigen association with specific antibodies conjugated to liposomes.

PubMed

Klegerman, Melvin E; Huang, Shaoling; Parikh, Devang; Martinez, Janet; Demos, Sasha M; Onyuksel, Hayat A; McPherson, David D

2007-07-01

Immunoliposomes, directed to clinically relevant cell-surface molecules with antibodies, antibody fragments or peptides, are used for site-specific diagnostic evaluation or delivery of therapeutic agents. We have developed intrinsically echogenic liposomes (ELIP) covalently linked to fibrin(ogen)-specific antibodies and Fab fragments for ultrasonic imaging of atherosclerotic plaques. In order to determine the effect of liposomal conjugation on the molecular dynamics of fibrinogen binding, we studied the thermodynamic characteristics of unconjugated and ELIP-conjugated antibody molecules. Utilizing radioimmunoassay and enzyme-linked immunosorbent assay protocols, binding affinities were derived from data obtained at three temperatures. The thermodynamic functions DeltaH(o) , DeltaG(o) and DeltaS(o) were determined from van't Hoff plots and equations of state. The resultant functions indicated that both specific and nonspecific associations of antibody molecules with fibrinogen occurred through a variety of molecular interactions, including hydrophophic, ionic and hydrogen bonding mechanisms. ELIP conjugation of antibodies and Fab fragments introduced a characteristic change in both DeltaH(o) and DeltaS(o) of association, which corresponded to a variable contribution to binding by phospholipid gel-liquid crystal phase transitions. These observations suggest that a reciprocal energy transduction, affecting the strength of antibody-antigen binding, may be a singular characteristic of immunoliposomes, having utility for optimization and further development of the technology.

Prevalence of antibodies to canine parvovirus and reaction to vaccination in client-owned, healthy dogs.

PubMed

Riedl, M; Truyen, U; Reese, S; Hartmann, K

2015-12-12

The purpose of this population-based cohort study was to assess current prevalence of antibodies to canine parvovirus (CPV) in adult, healthy dogs, including risk factors associated with lack of antibodies, and reaction to revaccination with a modified live vaccine (MLV). One hundred dogs routinely presented for vaccination were included in the study and vaccinated with a single dose of a combined MLV. Information was collected on signalment, origin, environment, vaccination history and side effects. Prevaccination and postvaccination antibodies were detected by haemagglutination inhibition. Univariate analysis, followed by multivariate logistic regression, was used to investigate association between different variables and presence of antibodies as well as titre increase. Protective CPV antibodies were present in 86.0 per cent of dogs. Intervals of more than four years since the last vaccination and rare contacts with other dogs were determined as main risk factors for the absence of antibodies. An increase in titres only occurred in 17.0 per cent of dogs. Dogs without protective titres before vaccination or with bodyweight <10 kg were more likely to have an adequate titre increase. Based on these findings, antibody status should be determined instead of periodic vaccinations to ensure reliable protection without unnecessary vaccinations in adult dogs. British Veterinary Association.

Beyond CDR-grafting: Structure-guided humanization of framework and CDR regions of an anti-myostatin antibody.

PubMed

Apgar, James R; Mader, Michelle; Agostinelli, Rita; Benard, Susan; Bialek, Peter; Johnson, Mark; Gao, Yijie; Krebs, Mark; Owens, Jane; Parris, Kevin; St Andre, Michael; Svenson, Kris; Morris, Carl; Tchistiakova, Lioudmila

2016-10-01

Antibodies are an important class of biotherapeutics that offer specificity to their antigen, long half-life, effector function interaction and good manufacturability. The immunogenicity of non-human-derived antibodies, which can be a major limitation to development, has been partially overcome by humanization through complementarity-determining region (CDR) grafting onto human acceptor frameworks. The retention of foreign content in the CDR regions, however, is still a potential immunogenic liability. Here, we describe the humanization of an anti-myostatin antibody utilizing a 2-step process of traditional CDR-grafting onto a human acceptor framework, followed by a structure-guided approach to further reduce the murine content of CDR-grafted antibodies. To accomplish this, we solved the co-crystal structures of myostatin with the chimeric (Protein Databank (PDB) id 5F3B) and CDR-grafted anti-myostatin antibody (PDB id 5F3H), allowing us to computationally predict the structurally important CDR residues as well as those making significant contacts with the antigen. Structure-based rational design enabled further germlining of the CDR-grafted antibody, reducing the murine content of the antibody without affecting antigen binding. The overall "humanness" was increased for both the light and heavy chain variable regions.

Beyond CDR-grafting: Structure-guided humanization of framework and CDR regions of an anti-myostatin antibody

PubMed Central

Apgar, James R.; Mader, Michelle; Agostinelli, Rita; Benard, Susan; Bialek, Peter; Johnson, Mark; Gao, Yijie; Krebs, Mark; Owens, Jane; Parris, Kevin; St. Andre, Michael; Svenson, Kris; Morris, Carl; Tchistiakova, Lioudmila

2016-01-01

ABSTRACT Antibodies are an important class of biotherapeutics that offer specificity to their antigen, long half-life, effector function interaction and good manufacturability. The immunogenicity of non-human-derived antibodies, which can be a major limitation to development, has been partially overcome by humanization through complementarity-determining region (CDR) grafting onto human acceptor frameworks. The retention of foreign content in the CDR regions, however, is still a potential immunogenic liability. Here, we describe the humanization of an anti-myostatin antibody utilizing a 2-step process of traditional CDR-grafting onto a human acceptor framework, followed by a structure-guided approach to further reduce the murine content of CDR-grafted antibodies. To accomplish this, we solved the co-crystal structures of myostatin with the chimeric (Protein Databank (PDB) id 5F3B) and CDR-grafted anti-myostatin antibody (PDB id 5F3H), allowing us to computationally predict the structurally important CDR residues as well as those making significant contacts with the antigen. Structure-based rational design enabled further germlining of the CDR-grafted antibody, reducing the murine content of the antibody without affecting antigen binding. The overall “humanness” was increased for both the light and heavy chain variable regions. PMID:27625211

A fully automated primary screening system for the discovery of therapeutic antibodies directly from B cells.

PubMed

Tickle, Simon; Howells, Louise; O'Dowd, Victoria; Starkie, Dale; Whale, Kevin; Saunders, Mark; Lee, David; Lightwood, Daniel

2015-04-01

For a therapeutic antibody to succeed, it must meet a range of potency, stability, and specificity criteria. Many of these characteristics are conferred by the amino acid sequence of the heavy and light chain variable regions and, for this reason, can be screened for during antibody selection. However, it is important to consider that antibodies satisfying all these criteria may be of low frequency in an immunized animal; for this reason, it is essential to have a mechanism that allows for efficient sampling of the immune repertoire. UCB's core antibody discovery platform combines high-throughput B cell culture screening and the identification and isolation of single, antigen-specific IgG-secreting B cells through a proprietary technique called the "fluorescent foci" method. Using state-of-the-art automation to facilitate primary screening, extremely efficient interrogation of the natural antibody repertoire is made possible; more than 1 billion immune B cells can now be screened to provide a useful starting point from which to identify the rare therapeutic antibody. This article will describe the design, construction, and commissioning of a bespoke automated screening platform and two examples of how it was used to screen for antibodies against two targets. © 2014 Society for Laboratory Automation and Screening.

Application of cyclodextrins in antibody microparticles: potentials for antibody protection in spray drying.

PubMed

Ramezani, Vahid; Vatanara, Alireza; Seyedabadi, Mohammad; Nabi Meibodi, Mohsen; Fanaei, Hamed

2017-07-01

Dry powder formulations are extensively used to improve the stability of antibodies. Spray drying is one of important methods for protein drying. This study investigated the effects of trehalose, hydroxypropyl beta cyclodextrin (HPBCD) and beta cyclodextrin (BCD) on the stability and particle properties of spray-dried IgG. D-optimal design was employed for both experimental design and analysis and optimization of the variables. The size and aerodynamic behavior of particles were determined using laser light scattering and glass twin impinger, respectively. In addition, stability, ratio of beta sheets and morphology of antibody were analyzed using size exclusion chromatography, IR spectroscopy and electron microscopy, respectively. Particle properties and antibody stability were significantly improved in the presence of HPBCD. In addition, particle aerodynamic behavior, in terms of fine-particle fraction (FPF), enhanced up to 52.23%. Furthermore, antibody was better preserved not only during spray drying, but also during long-term storage. In contrast, application of BCD resulted in the formation of larger particles. Although trehalose caused inappropriate aerodynamic property, it efficiently decreased antibody aggregation. HPBCD is an efficient excipient for the development of inhalable protein formulations. In this regard, optimal particle property and antibody stability was obtained with proper combination of cyclodextrins and simple sugars, such as trehalose.

Cbl-independent degradation of Met: ways to avoid agonism of bivalent Met-targeting antibody.

PubMed

Lee, J M; Kim, B; Lee, S B; Jeong, Y; Oh, Y M; Song, Y-J; Jung, S; Choi, J; Lee, S; Cheong, K H; Kim, D U; Park, H W; Han, Y K; Kim, G W; Choi, H; Song, P H; Kim, K A

2014-01-02

The Met receptor tyrosine kinase, found to be constitutively activated in many tumors, has become a leading target for cancer therapy. Disruptions in Met downregulation have been associated with aggressive tumor progression with several therapeutic strategies addressing this aspect of Met biology. Castias B-lineage lymphoma (Cbl) E3 ligase-mediated degradation, which attenuates Met signaling via ligand-dependent Met internalization, is a major negative regulator of Met expression. It is believed that one of the mechanisms by which the therapeutic anti-Met antibodies induce cancer cell death in Met overexpressing tumors is via internalization and subsequent degradation of Met from the cell surface. However, a previously reported Met-targeting antibody demonstrated intrinsic agonistic activity while being capable of inducing Cbl-mediated degradation of Met, suggesting that Cbl-mediated degradation requires receptor activation and impedes therapeutic application. We have developed a potent and selective bivalent Met-targeting antibody (SAIT301) that invokes Met degradation using an alternative regulator LRIG1. In this report, we demonstrate that LRIG1 mediates degradation of Met by SAIT301 and this degradation does not require Met activation. Furthermore, SAIT301 was able to downregulate Met and dramatically inhibit growth of tumors with low or no Cbl expression, as well as tumors with Met exon 14 deletion that prevents Met binding to Cbl. In summary, we demonstrate the enhanced therapeutic potential of a novel tumor-inhibiting anti-Met antibody, SAIT301, which utilizes a Cbl-independent, LRIG1-mediated Met degradation pathway and thereby avoids the agonism that limits the effectiveness of previously reported anti-Met antibodies.

A molecular model for cocaine binding by the immunotherapeutic human/mouse chimeric monoclonal antibody 2E2.

PubMed

Lape, Michael; Paula, Stefan; Ball, William J

2010-06-01

Immunotherapy by cocaine-binding monoclonal antibodies (mAbs) has emerged as a promising strategy for the treatment of cocaine addiction. The human (gamma1 heavy chain)/murine (lambda light chain) chimeric mAb 2E2 has excellent affinity and specificity for cocaine and recent animal studies have demonstrated 2E2's ability in vivo to reduce cocaine levels in the brain as well as alter cocaine self-administration behavior in rats. In this study, we used mAb 2E2 amino acid sequence information to create a homology model for the 3-D structure of its Fv fragment. Subsequent computational docking studies revealed the intermolecular interactions potentially responsible for mAb 2E2's cocaine binding properties. The driving force of cocaine binding was identified as a combination of hydrophobic interactions and a single hydrogen bond between a light chain tyrosine residue and a carbonyl oxygen atom of cocaine. The model also allowed for an in silico evaluation of single/double residue mutations in the heavy and light chain variable regions that might further enhance mAb 2E2's cocaine binding properties. Copyright (c) 2010 Elsevier Masson SAS. All rights reserved.

A Molecular Model for Cocaine Binding by the Immunotherapeutic Human/Mouse Chimeric Monoclonal Antibody 2E2

PubMed Central

Lape, Michael; Paula, Stefan; Ball, William J.

2010-01-01

Immunotherapy by cocaine-binding monoclonal antibodies (mAbs) has emerged as a promising strategy for the treatment of cocaine addiction. The human (γ1 heavy chain)/murine (λ light chain) chimeric mAb 2E2 has excellent affinity and specificity for cocaine and recent animal studies have demonstrated 2E2’s ability in vivo to reduce cocaine levels in the brain as well as alter cocaine self-administration behavior in rats. In this study, we used mAb 2E2 amino acid sequence information to create a homology model for the 3-D structure of its Fv fragment. Subsequent computational docking studies revealed the intermolecular interactions potentially responsible for mAb 2E2’s cocaine binding properties. The driving force of cocaine binding was identified as a combination of hydrophobic interactions and a single hydrogen bond between a light chain tyrosine residue and a carbonyl oxygen atom of cocaine. The model also allowed for an in silico evaluation of single/double residue mutations in the heavy and light chain variable regions that might further enhance mAb 2E2’s cocaine binding properties. PMID:20185210

Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen

PubMed Central

Pallesen, Jesper; Wang, Nianshuang; Corbett, Kizzmekia S.; Wrapp, Daniel; Kirchdoerfer, Robert N.; Turner, Hannah L.; Cottrell, Christopher A.; Becker, Michelle M.; Wang, Lingshu; Shi, Wei; Kong, Wing-Pui; Andres, Erica L.; Kettenbach, Arminja N.; Denison, Mark R.; Chappell, James D.; Graham, Barney S.; Ward, Andrew B.

2017-01-01

Middle East respiratory syndrome coronavirus (MERS-CoV) is a lineage C betacoronavirus that since its emergence in 2012 has caused outbreaks in human populations with case-fatality rates of ∼36%. As in other coronaviruses, the spike (S) glycoprotein of MERS-CoV mediates receptor recognition and membrane fusion and is the primary target of the humoral immune response during infection. Here we use structure-based design to develop a generalizable strategy for retaining coronavirus S proteins in the antigenically optimal prefusion conformation and demonstrate that our engineered immunogen is able to elicit high neutralizing antibody titers against MERS-CoV. We also determined high-resolution structures of the trimeric MERS-CoV S ectodomain in complex with G4, a stem-directed neutralizing antibody. The structures reveal that G4 recognizes a glycosylated loop that is variable among coronaviruses and they define four conformational states of the trimer wherein each receptor-binding domain is either tightly packed at the membrane-distal apex or rotated into a receptor-accessible conformation. Our studies suggest a potential mechanism for fusion initiation through sequential receptor-binding events and provide a foundation for the structure-based design of coronavirus vaccines. PMID:28807998

Proof of the quantitative potential of immunofluorescence by mass spectrometry.

PubMed

Toki, Maria I; Cecchi, Fabiola; Hembrough, Todd; Syrigos, Konstantinos N; Rimm, David L

2017-03-01

Protein expression in formalin-fixed, paraffin-embedded patient tissue is routinely measured by Immunohistochemistry (IHC). However, IHC has been shown to be subject to variability in sensitivity, specificity and reproducibility, and is generally, at best, considered semi-quantitative. Mass spectrometry (MS) is considered by many to be the criterion standard for protein measurement, offering high sensitivity, specificity, and objective molecular quantification. Here, we seek to show that quantitative immunofluorescence (QIF) with standardization can achieve quantitative results comparable to MS. Epidermal growth factor receptor (EGFR) was measured by quantitative immunofluorescence in 15 cell lines with a wide range of EGFR expression, using different primary antibody concentrations, including the optimal signal-to-noise concentration after quantitative titration. QIF target measurement was then compared to the absolute EGFR concentration measured by Liquid Tissue-selected reaction monitoring mass spectrometry. The best agreement between the two assays was found when the EGFR primary antibody was used at the optimal signal-to-noise concentration, revealing a strong linear regression (R 2 =0.88). This demonstrates that quantitative optimization of titration by calculation of signal-to-noise ratio allows QIF to be standardized to MS and can therefore be used to assess absolute protein concentration in a linear and reproducible manner.

Advances in phage display technology for drug discovery.

PubMed

Omidfar, Kobra; Daneshpour, Maryam

2015-06-01

Over the past decade, several library-based methods have been developed to discover ligands with strong binding affinities for their targets. These methods mimic the natural evolution for screening and identifying ligand-target interactions with specific functional properties. Phage display technology is a well-established method that has been applied to many technological challenges including novel drug discovery. This review describes the recent advances in the use of phage display technology for discovering novel bioactive compounds. Furthermore, it discusses the application of this technology to produce proteins and peptides as well as minimize the use of antibodies, such as antigen-binding fragment, single-chain fragment variable or single-domain antibody fragments like VHHs. Advances in screening, manufacturing and humanization technologies demonstrate that phage display derived products can play a significant role in the diagnosis and treatment of disease. The effects of this technology are inevitable in the development pipeline for bringing therapeutics into the market, and this number is expected to rise significantly in the future as new advances continue to take place in display methods. Furthermore, a widespread application of this methodology is predicted in different medical technological areas, including biosensing, monitoring, molecular imaging, gene therapy, vaccine development and nanotechnology.

CCR 20th anniversary commentary: Radioactive Drones for B-cell lymphoma.

PubMed

Knox, Susan J; Levy, Ronald

2015-02-01

In a study published in the March 1, 1996, issue of Clinical Cancer Research, Knox and colleagues (1) demonstrated the safety and efficacy of Yttirium-90 ((90)Y)-anti-CD20 monoclonal antibody therapy, as well as the benefit of preinfusion of unlabeled antibody on radiolabeled antibody biodistribution. Subsequent clinical trials with this radiolabeled antibody led to regulatory approval of this treatment for B-cell lymphoma. See related article by Knox et al., Clin Cancer Res 1996;2(3) Mar 1996; 457-70. ©2015 American Association for Cancer Research.

Design of a single-step immunoassay principle based on the combination of an enzyme-labeled antibody release coating and a hydrogel copolymerized with a fluorescent enzyme substrate in a microfluidic capillary device.

PubMed

Wakayama, Hideki; Henares, Terence G; Jigawa, Kaede; Funano, Shun-ichi; Sueyoshi, Kenji; Endo, Tatsuro; Hisamoto, Hideaki

2013-11-21

A combination of an enzyme-labeled antibody release coating and a novel fluorescent enzyme substrate-copolymerized hydrogel in a microchannel for a single-step, no-wash microfluidic immunoassay is demonstrated. This hydrogel discriminates the free enzyme-conjugated antibody from an antigen-enzyme-conjugated antibody immunocomplex based on the difference in molecular size. A selective and sensitive immunoassay, with 10-1000 ng mL(-1) linear range, is reported.

Improving Antibody-Based Cancer Therapeutics Through Glycan Engineering.

PubMed

Yu, Xiaojie; Marshall, Michael J E; Cragg, Mark S; Crispin, Max

2017-06-01

Antibody-based therapeutics has emerged as a major tool in cancer treatment. Guided by the superb specificity of the antibody variable domain, it allows the precise targeting of tumour markers. Recently, eliciting cellular effector functions, mediated by the Fc domain, has gained traction as a means by which to generate more potent antibody therapeutics. Extensive mutagenesis studies of the Fc protein backbone has enabled the generation of Fc variants that more optimally engage the Fcγ receptors known to mediate cellular effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and cellular phagocytosis. In addition to the protein backbone, the homodimeric Fc domain contains two opposing N-linked glycans, which represent a further point of potential immunomodulation, independent of the Fc protein backbone. For example, a lack of core fucose usually attached to the IgG Fc glycan leads to enhanced ADCC activity, whereas a high level of terminal sialylation is associated with reduced inflammation. Significant growth in knowledge of Fc glycosylation over the last decade, combined with advancement in genetic engineering, has empowered glyco-engineering to fine-tune antibody therapeutics. This has culminated in the approval of two glyco-engineered antibodies for cancer therapy: the anti-CCR4 mogamulizumab approved in 2012 and the anti-CD20 obinutuzumab in 2013. We discuss here the technological platforms for antibody glyco-engineering and review the current clinical landscape of glyco-engineered antibodies.

From VGKC to LGI1 and Caspr2 encephalitis: The evolution of a disease entity over time.

PubMed

van Sonderen, A; Schreurs, M W J; Wirtz, P W; Sillevis Smitt, P A E; Titulaer, M J

2016-10-01

A wide variety of clinical syndromes has been associated with antibodies to voltage-gated potassium channels (VGKCs). Six years ago, it was discovered that patients do not truly have antibodies to potassium channels, but to associated proteins. This enabled the distinction of three VGKC-positive subgroups: anti-LGI1 patients, anti-Caspr2 patients and VGKC-positive patients lacking both antibodies. Patients with LGI1-antibodies have a limbic encephalitis, often with hyponatremia, and about half of the patients have typical faciobrachial dystonic seizures. Caspr2-antibodies cause a more variable syndrome of peripheral or central nervous system symptoms, almost exclusively affecting older males. Immunotherapy seems to be beneficial in patients with antibodies to LGI1 or Caspr2, stressing the need for early diagnosis. Half of the VGKC-positive patients lack antibodies to both LGI1 and Caspr2. This is a heterogeneous group of patients with a wide variety of clinical syndromes, raising the question whether VGKC-positivity is truly a marker of disease in these patients. Data regarding this issue are limited, but a recent study did not show any clinical relevance of VGKC-positivity in the absence of antibodies to LGI1 and Caspr2. The three VGKC-positive subgroups are essentially different, therefore, the lumping term 'VGKC-complex antibodies' should be abolished. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

A Monoclonal Antibody to Cryptococcus neoformans Glucuronoxylomannan Manifests Hydrolytic Activity for Both Peptides and Polysaccharides.

PubMed

Bowen, Anthony; Wear, Maggie P; Cordero, Radames J B; Oscarson, Stefan; Casadevall, Arturo

2017-01-13

Studies in the 1980s first showed that some natural antibodies were "catalytic" and able to hydrolyze peptide or phosphodiester bonds in antigens. Many naturally occurring catalytic antibodies have since been isolated from human sera and associated with positive and negative outcomes in autoimmune disease and infection. The function and prevalence of these antibodies, however, remain unclear. A previous study suggested that the 18B7 monoclonal antibody against glucuronoxylomannan (GXM), the major component of the Cryptococcus neoformans polysaccharide capsule, hydrolyzed a peptide antigen mimetic. Using mass spectrometry and Förster resonance energy transfer techniques, we confirm and characterize the hydrolytic activity of 18B7 against peptide mimetics and show that 18B7 is able to hydrolyze an oligosaccharide substrate, providing the first example of a naturally occurring catalytic antibody for polysaccharides. Additionally, we show that the catalytic 18B7 antibody increases release of capsular polysaccharide from fungal cells. A serine protease inhibitor blocked peptide and oligosaccharide hydrolysis by 18B7, and a putative serine protease-like active site was identified in the light chain variable region of the antibody. An algorithm was developed to detect similar sites present in unique antibody structures in the Protein Data Bank. The putative site was found in 14 of 63 (22.2%) catalytic antibody structures and 119 of 1602 (7.4%) antibodies with no annotation of catalytic activity. The ability of many antibodies to cleave antigen, albeit slowly, supports the notion that this activity is an important immunoglobulin function in host defense. The discovery of GXM hydrolytic activity suggests new therapeutic possibilities for polysaccharide-binding antibodies. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The influence of antibody fragment format on phage display based affinity maturation of IgG

PubMed Central

Steinwand, Miriam; Droste, Patrick; Frenzel, Andrè; Hust, Michael; Dübel, Stefan; Schirrmann, Thomas

2014-01-01

Today, most approved therapeutic antibodies are provided as immunoglobulin G (IgG), whereas small recombinant antibody formats are required for in vitro antibody generation and engineering during drug development. Particularly, single chain (sc) antibody fragments like scFv or scFab are well suited for phage display and bacterial expression, but some have been found to lose affinity during conversion into IgG.   In this study, we compared the influence of the antibody format on affinity maturation of the CD30-specific scFv antibody fragment SH313-F9, with the overall objective being improvement of the IgG. The variable genes of SH313-F9 were randomly mutated and then cloned into libraries encoding different recombinant antibody formats, including scFv, Fab, scFabΔC, and FabΔC. All tested antibody formats except Fab allowed functional phage display of the parental antibody SH313-F9, and the corresponding mutated antibody gene libraries allowed isolation of candidates with enhanced CD30 binding. Moreover, scFv and scFabΔC antibody variants retained improved antigen binding after subcloning into the single gene encoded IgG-like formats scFv-Fc or scIgG, but lost affinity after conversion into IgGs. Only affinity maturation using the Fab-like FabΔC format, which does not contain the carboxy terminal cysteines, allowed successful selection of molecules with improved binding that was retained after conversion to IgG. Thus, affinity maturation of IgGs is dependent on the antibody format employed for selection and screening. In this study, only FabΔC resulted in the efficient selection of IgG candidates with higher affinity by combination of Fab-like conformation and improved phage display compared with Fab. PMID:24262918

A Monoclonal Antibody to Cryptococcus neoformans Glucuronoxylomannan Manifests Hydrolytic Activity for Both Peptides and Polysaccharides*

PubMed Central

Wear, Maggie P.; Cordero, Radames J. B.; Oscarson, Stefan

2017-01-01

Studies in the 1980s first showed that some natural antibodies were “catalytic” and able to hydrolyze peptide or phosphodiester bonds in antigens. Many naturally occurring catalytic antibodies have since been isolated from human sera and associated with positive and negative outcomes in autoimmune disease and infection. The function and prevalence of these antibodies, however, remain unclear. A previous study suggested that the 18B7 monoclonal antibody against glucuronoxylomannan (GXM), the major component of the Cryptococcus neoformans polysaccharide capsule, hydrolyzed a peptide antigen mimetic. Using mass spectrometry and Förster resonance energy transfer techniques, we confirm and characterize the hydrolytic activity of 18B7 against peptide mimetics and show that 18B7 is able to hydrolyze an oligosaccharide substrate, providing the first example of a naturally occurring catalytic antibody for polysaccharides. Additionally, we show that the catalytic 18B7 antibody increases release of capsular polysaccharide from fungal cells. A serine protease inhibitor blocked peptide and oligosaccharide hydrolysis by 18B7, and a putative serine protease-like active site was identified in the light chain variable region of the antibody. An algorithm was developed to detect similar sites present in unique antibody structures in the Protein Data Bank. The putative site was found in 14 of 63 (22.2%) catalytic antibody structures and 119 of 1602 (7.4%) antibodies with no annotation of catalytic activity. The ability of many antibodies to cleave antigen, albeit slowly, supports the notion that this activity is an important immunoglobulin function in host defense. The discovery of GXM hydrolytic activity suggests new therapeutic possibilities for polysaccharide-binding antibodies. PMID:27872188

Heterogeneity of Polyneuropathy Associated with Anti-MAG Antibodies

PubMed Central

Magy, Laurent; Kaboré, Raphaël; Mathis, Stéphane; Lebeau, Prisca; Ghorab, Karima; Caudie, Christiane; Vallat, Jean-Michel

2015-01-01

Polyneuropathy associated with IgM monoclonal gammopathy and anti-myelin associated glycoprotein (MAG) antibodies is an immune-mediated demyelinating neuropathy. The pathophysiology of this condition is likely to involve anti-MAG antibody deposition on myelin sheaths of the peripheral nerves and it is supposed to be distinct from chronic inflammatory demyelinating neuropathy (CIDP), another immune-mediated demyelinating peripheral neuropathy. In this series, we have retrospectively reviewed clinical and laboratory findings from 60 patients with polyneuropathy, IgM gammopathy, and anti-MAG antibodies. We found that the clinical picture in these patients is highly variable suggesting a direct link between the monoclonal gammopathy and the neuropathy. Conversely, one-third of patients had a CIDP-like phenotype on electrodiagnostic testing and this was correlated with a low titer of anti-MAG antibodies and the absence of widening of myelin lamellae. Our data suggest that polyneuropathy associated with anti-MAG antibodies is less homogeneous than previously said and that the pathophysiology of the condition is likely to be heterogeneous as well with the self-antigen being MAG in most of the patients but possibly being another component of myelin in the others. PMID:26065001

Antibodies elicited by the first non-viral prophylactic cancer vaccine show tumor-specificity and immunotherapeutic potential

PubMed Central

Lohmueller, Jason J.; Sato, Shuji; Popova, Lana; Chu, Isabel M.; Tucker, Meghan A.; Barberena, Roberto; Innocenti, Gregory M.; Cudic, Mare; Ham, James D.; Cheung, Wan Cheung; Polakiewicz, Roberto D.; Finn, Olivera J.

2016-01-01

MUC1 is a shared tumor antigen expressed on >80% of human cancers. We completed the first prophylactic cancer vaccine clinical trial based on a non-viral antigen, MUC1, in healthy individuals at-risk for colon cancer. This trial provided a unique source of potentially effective and safe immunotherapeutic drugs, fully-human antibodies affinity-matured in a healthy host to a tumor antigen. We purified, cloned, and characterized 13 IgGs specific for several tumor-associated MUC1 epitopes with a wide range of binding affinities. These antibodies bind hypoglycosylated MUC1 on human cancer cell lines and tumor tissues but show no reactivity against fully-glycosylated MUC1 on normal cells and tissues. We found that several antibodies activate complement-mediated cytotoxicity and that T cells carrying chimeric antigen receptors with the antibody variable regions kill MUC1+ target cells, express activation markers, and produce interferon gamma. Fully-human and tumor-specific, these antibodies are candidates for further testing and development as immunotherapeutic drugs. PMID:27545199

PREVALENCE OF ANTIBODIES FOR SELECTED CANINE PATHOGENS AMONG WOLVES (CANIS LUPUS) FROM THE ALASKA PENINSULA, USA.

PubMed

Watts, Dominique E; Benson, Anna-Marie

2016-07-01

We collected blood samples from wolves ( Canis lupus ) on the Alaska Peninsula, southwest Alaska, US, 2006-11 and tested sera for antibodies to canine adenovirus (CAV), canine coronavirus (CCV), canine distemper virus (CDV), canine herpesvirus (CHV), canine parainfluenza (CPI), canine parvovirus (CPV), Neospora caninum , and Toxoplasma gondii . Detected antibody prevalence was 90% for CAV, 28% for CCV, 12% for CDV, 93% for CHV, 0% for CPI, 20% for CPV, 0% for N. caninum, and 86% for T. gondii . Prevalence of CCV antibodies suggested a seasonal pattern with higher prevalence during spring (43%) than in fall (11%). Prevalence of CCV antibodies also declined during the 6-yr study with high prevalence during spring 2006-08 (80%, n=24) and low prevalence during spring 2009-11 (4%, n=24). Prevalence of N. caninum and T. gondii antibodies were highly variable in the study area during 2006-11. Results suggested that some pathogens might be enzootic on the Alaska Peninsula (e.g., CAV and CHV) while others may be epizootic (e.g., CCV, N. caninum , T. gondii ).

[Histocompatibility tests in a transplantation program].

PubMed

de-Leo-Cervantes, Claudia

2005-01-01

The importance of the role of the histocompatibility laboratory in solid organ transplantation is to perform HLA typing and determine the degree of HLA matching between recipient/donor. It is a useful tool to increase graft survival and decrease chronic rejection. HLA matching has a positive effect on kidney transplants and it has variable impact on other organ transplants. The crossmatch procedure is the most important test in a solid organ transplantation to evaluate the presence of recipient antibodies to antigens expressed on donor white cells. This test decreases the risk of hyperacute humoral rejection or early graft loss. Positive crossmatch is a contraindication for transplantation because it represents the existence of IgG recipient antibodies that will reath againts donor antigens. Antibody evaluation is important in donor-recipient selection and the responsability of the histocompatibility laboratory is to identify clinically relevant anti-donor HLA antibodies. This detection is useful to determine the degree of humoral alloimmunization, expressed as a percent panel reactive antibody (%PRA). This test also provides information about the antibody specificity and can be used for evaluate a patient's immune status providing a significant correlation in selecting donors.

A novel anti-alpha-fetoprotein single-chain variable fragment displays anti-tumor effects in HepG2 cells as a single agent or in combination with paclitaxel.

PubMed

Ji, Xiaonan; Shen, Yanli; Sun, Hao; Gao, Xiangdong

2016-08-01

Human hepatocellular carcinoma (HCC) has a high rate of tumor recurrence and metastasis, resulting in shortened survival time. The function of alpha-fetoprotein (AFP) as a regulatory factor in the growth of HCC cells has been well defined. The aim of this study was to investigate the use of a novel AFP-specific single-chain variable fragment that blocked AFP and inhibited HCC cell growth. The results indicated that the anti-AFP single-chain variable fragment (scFv) induced growth inhibition of AFP-expressing HCC cell lines in vitro through induction of G1 cell cycle arrest and apoptosis. The mechanism of apoptosis probably involved with blocking AFP internalization and regulation of the PTEN/PI3K/Akt signaling network. Moreover, the anti-AFP-scFv also effectively sensitized the HepG2 cells to paclitaxel (PTX) at a lower concentration. The combination effect of PTX and anti-AFP-scFv displayed a synergistic effect on HepG2 cells both in vitro and in vivo. Our results demonstrated that targeting AFP by specific antibodies has potential immunotherapeutic efficacy in human HCC.

Functional Transplant of a Dengue Virus Serotype 3 (DENV3)-Specific Human Monoclonal Antibody Epitope into DENV1.

PubMed

Messer, William B; Yount, Boyd L; Royal, Scott R; de Alwis, Ruklanthi; Widman, Douglas G; Smith, Scott A; Crowe, James E; Pfaff, Jennifer M; Kahle, Kristen M; Doranz, Benjamin J; Ibarra, Kristie D; Harris, Eva; de Silva, Aravinda M; Baric, Ralph S

2016-05-15

The four dengue virus (DENV) serotypes, DENV1 through 4, are endemic throughout tropical and subtropical regions of the world. While first infection confers long-term protective immunity against viruses of the infecting serotype, a second infection with virus of a different serotype carries a greater risk of severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. Recent studies demonstrate that humans exposed to DENV infections develop neutralizing antibodies that bind to quaternary epitopes formed by the viral envelope (E) protein dimers or higher-order assemblies required for the formation of the icosahedral viral envelope. Here we show that the quaternary epitope target of the human DENV3-specific neutralizing monoclonal antibody (MAb) 5J7 can be partially transplanted into a DENV1 strain by changing the core residues of the epitope contained within a single monomeric E molecule. MAb 5J7 neutralized the recombinant DENV1/3 strain in cell culture and was protective in a mouse model of infection with the DENV1/3 strain. However, the 5J7 epitope was only partially recreated by transplantation of the core residues because MAb 5J7 bound and neutralized wild-type (WT) DENV3 better than the DENV1/3 recombinant. Our studies demonstrate that it is possible to transplant a large number of discontinuous residues between DENV serotypes and partially recreate a complex antibody epitope, while retaining virus viability. Further refinement of this approach may lead to new tools for measuring epitope-specific antibody responses and new vaccine platforms. Dengue virus is the most important mosquito-borne pathogen of humans worldwide, with approximately one-half the world's population living in regions where dengue is endemic. Dengue immunity following infection is robust and thought to be conferred by antibodies raised against the infecting virus. However, the specific viral components that these antibodies recognize and how they neutralize the virus have been incompletely described. Here we map a region on dengue virus serotype 3 recognized by the human neutralizing antibody 5J7 and then test the functional significance of this region by transplanting it into a serotype 1 virus. Our studies demonstrate a region on dengue virus necessary for 5J7 binding and neutralization. Our work also demonstrates the technical feasibility of engineering dengue viruses to display targets of protective antibodies. This technology can be used to develop new dengue vaccines and diagnostic assays. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Targeting nanodisks via a single chain variable antibody--apolipoprotein chimera.

PubMed

Iovannisci, David M; Beckstead, Jennifer A; Ryan, Robert O

2009-02-06

Nanodisks (ND) are nanometer scale complexes of phospholipid and apolipoprotein that have been shown to function as drug delivery vehicles. ND harboring significant quantities of the antifungal agent, amphotericin B, or the bioactive isoprenoid, all trans retinoic acid, have been generated and characterized. As currently formulated, ND possess limited targeting capability. In this study, we constructed a single chain variable antibody (scFv).apolipoprotein chimera and assessed the ability of this fusion protein to form ND and recognize the antigen to which the scFv is directed. Data obtained revealed that alpha-vimentin scFv.apolipoprotein A-I is functional in ND formation and antigen recognition, opening the door to the use of such chimeras in targeting drug-enriched ND to specific tissues.

Cloning the Antibody Response in Humans with Chronic Inflammatory Disease: Immunopanning of Subacute Sclerosing Panencephalitis (SSPE) Brain Sections with Antibody Phage Libraries Prepared from SSPE Brain Enriches for Antibody Recognizing Measles Virus Antigens In Situ

PubMed Central

Owens, Gregory P.; Williamson, R. Anthony; Burgoon, Mark P.; Ghausi, Omar; Burton, Dennis R.; Gilden, Donald H.

2000-01-01

In central nervous system (CNS) infectious and inflammatory diseases of known cause, oligoclonal bands represent antibody directed against the causative agent. To determine whether disease-relevant antibodies can be cloned from diseased brain, we prepared an antibody phage display library from the brain of a human with subacute sclerosing panencephalitis (SSPE), a chronic encephalitis caused by measles virus, and selected the library against SSPE brain sections. Antibodies that were retrieved reacted strongly with measles virus cell extracts by enzyme-linked immunosorbent assay and were specific for the measles virus nucleocapsid protein. These antibodies immunostained cells in different SSPE brains but not in control brain. Our data provide the first demonstration that diseased brain can be used to select in situ for antibodies directed against the causative agent of disease and point to the potential usefulness of this approach in identifying relevant antibodies in chronic CNS or systemic inflammatory diseases of unknown cause. PMID:10627565

Fibronectin non-amyloid glomerulopathy.

PubMed

Yong, Jim L; Killingsworth, Murray C; Spicer, S Timothy; Wu, Xiao-Juan

2009-11-20

A 41-year-old Burmese man presented with nephrotic syndrome, a creatinine level of 150 micromol/L and limited clinical history. His renal biopsy demonstrated glomerulopathy with large eosinophilic deposits in the mesangium and capillary loops that were negative for Congo red, slightly positive for periodic acid-Schiff and blue with Masson trichrome stain. Immunofluorescence microscopy with a routine antibody panel was unhelpful. Electron microscopy demonstrated extensive, moderately electron-dense deposits in the subendothelial space, subepithelial space and mesangium that could be differentiated from adjacent basement membrane by their increased electron density. The deposits contained finely granular material and occasional filaments with variable diameter ranging from 9-16 nm. Fibronectin glomerulopathy was suspected from anti-fibronectin immunohistochemistry that showed positive staining of thickened capillary loops. This staining was subsequently confirmed by immunoelectron microscopy demonstrating the presence of cellular fibronectin (cFN) in deposits. Significantly, deposition of fibronectin appeared to have occurred in the absence of thickening or folding of the adjacent basement membrane. The prominent mesangial location of deposits containing a cFN isotype may indicate that retention of local fibronectin produced in the mesangium has contributed to this pathology.

Fibronectin non-amyloid glomerulopathy

PubMed Central

Yong, Jim L; Killingsworth, Murray C; Spicer, S Timothy; Wu, Xiao-Juan

2010-01-01

A 41-year-old Burmese man presented with nephrotic syndrome, a creatinine level of 150 µmol/L and limited clinical history. His renal biopsy demonstrated glomerulopathy with large eosinophilic deposits in the mesangium and capillary loops that were negative for Congo red, slightly positive for periodic acid-Schiff and blue with Masson trichrome stain. Immunofluorescence microscopy with a routine antibody panel was unhelpful. Electron microscopy demonstrated extensive, moderately electron-dense deposits in the subendothelial space, subepithelial space and mesangium that could be differentiated from adjacent basement membrane by their increased electron density. The deposits contained finely granular material and occasional filaments with variable diameter ranging from 9-16 nm. Fibronectin glomerulopathy was suspected from anti-fibronectin immunohistochemistry that showed positive staining of thickened capillary loops. This staining was subsequently confirmed by immunoelectron microscopy demonstrating the presence of cellular fibronectin (cFN) in deposits. Significantly, deposition of fibronectin appeared to have occurred in the absence of thickening or folding of the adjacent basement membrane. The prominent mesangial location of deposits containing a cFN isotype may indicate that retention of local fibronectin produced in the mesangium has contributed to this pathology. PMID:20126589

GABARAPL1 antibodies: target one protein, get one free!

PubMed

Le Grand, Jaclyn Nicole; Chakrama, Fatima Zahra; Seguin-Py, Stéphanie; Fraichard, Annick; Delage-Mourroux, Régis; Jouvenot, Michèle; Risold, Pierre-Yves; Boyer-Guittaut, Michaël

2011-11-01

Atg8 is a yeast protein involved in the autophagic process and in particular in the elongation of autophagosomes. In mammals, several orthologs have been identified and are classed into two subfamilies: the LC3 subfamily and the GABARAP subfamily, referred to simply as the LC3 or GABARAP families. GABARAPL1 (GABARAP-like protein 1), one of the proteins belonging to the GABARAP (GABA(A) receptor-associated protein) family, is highly expressed in the central nervous system and implicated in processes such as receptor and vesicle transport as well as autophagy. The proteins that make up the GABARAP family demonstrate conservation of their amino acid sequences and protein structures. In humans, GABARAPL1 shares 86% identity with GABARAP and 61% with GABARAPL2 (GATE-16). The identification of the individual proteins is thus very limited when working in vivo due to a lack of unique peptide sequences from which specific antibodies can be developed. Actually, and to our knowledge, there are no available antibodies on the market that are entirely specific to GABARAPL1 and the same may be true of the anti-GABARAP antibodies. In this study, we sought to examine the specificity of three antibodies targeted against different peptide sequences within GABARAPL1: CHEM-CENT (an antibody raised against a short peptide sequence within the center of the protein), PTG-NTER (an antibody raised against the N-terminus of the protein) and PTG-FL (an antibody raised against the full-length protein). The results described in this article demonstrate the importance of testing antibody specificity under the conditions for which it will be used experimentally, a caution that should be taken when studying the expression of the GABARAP family proteins.

Anti-GK1 antibodies damage Taenia crassiceps cysticerci through complement activation.

PubMed

Núñez, Guadalupe; Villalobos, Nelly; Herrera, Cinthia P; Navarrete-Perea, José; Méndez, Adriana; Martinez-Maya, José J; Bobes, Raúl J; Fragoso, Gladis; Sciutto, Edda; Aguilar, Laura; Del Arenal, Irene P

2018-06-06

Taeniasis-cysticercosis, a zoonosis caused by Taenia solium, is prevalent in underdeveloped countries, where marginalization promotes its continued transmission. Pig cysticercosis, an essential stage for transmission, is preventable by vaccination. An efficient multiepitope vaccine against pig cysticercosis, S3Pvac, was developed. Previous studies showed that antibodies against one of the S3Pvac components, GK-1, are capable of damaging T. solium cysticerci, inhibiting their ability to transform into the adult stage in golden hamster gut. This study is aimed to evaluate one of the mechanisms that could mediate anti-GK-1 antibody-dependent protection. To this end, pig anti-GK-1 antibodies were produced and purified by using protein A. Proteomic analysis showed that the induced antibodies recognized the respective native cysticercal protein KE7 (Bobes et al. Infect Immun 85:e00395-17, 2017) and two additional T. solium proteins (endophilin B1 and Gp50). A new procedure to evaluate cysticercus viability, based on quantifying the cytochrome c released after parasite damage, was developed. Taenia crassiceps cysticerci were cultured in the presence of differing amounts of anti-GK-1 antibody and complement in a saturating concentration, along with the respective controls. Cysticercus viability was assessed by recording parasite motility, trypan blue exclusion, and cytochrome c levels in cysticercal soluble extract. Anti-GK-1 antibody significantly increased cysticercus damage as measured by all three methods. Parasite evaluation by electron microscopy after treatment with anti-GK-1 antibody plus complement demonstrated cysticercus damage as shorter, capsule-severed microtrichia; a decrease in glycocalyx length with respect to untreated cysts; and disaggregated desmosomes. These results demonstrate that anti-GK-1 antibodies damage cysticerci through classic complement activation.

Antibodies Targeting EMT

DTIC Science & Technology

2017-10-01

impact substrate usage in AKRs . The blue residues are variable positions between AKR1C1-4. These residues are near the active site and may play a ...with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS...and diagnostic biomarkers. We have developed a new technique allowing for discovery of new antibodies that disrupt a key process in cancer progression

Generation of high-affinity, internalizing anti-FGFR2 single-chain variable antibody fragment fused with Fc for targeting gastrointestinal cancers.

PubMed

Borek, Aleksandra; Sokolowska-Wedzina, Aleksandra; Chodaczek, Grzegorz; Otlewski, Jacek

2018-01-01

Fibroblast growth factor receptors (FGFRs) are promising targets for antibody-based cancer therapies, as their substantial overexpression has been found in various tumor cells. Aberrant activation of FGF receptor 2 (FGFR2) signaling through overexpression of FGFR2 and/or its ligands, mutations, or receptor amplification has been reported in multiple cancer types, including gastric, colorectal, endometrial, ovarian, breast and lung cancer. In this paper, we describe application of the phage display technology to produce a panel of high affinity single chain variable antibody fragments (scFvs) against the extracellular ligand-binding domain of FGFR2 (ECD_FGFR2). The binders were selected from the human single chain variable fragment scFv phage display libraries Tomlinson I + J and showed high specificity and binding affinity towards human FGFR2 with nanomolar KD values. To improve the affinity of the best binder selected, scFvF7, we reformatted it to a bivalent diabody format, or fused it with the Fc region (scFvF7-Fc). The scFvF7-Fc antibody construct presented the highest affinity for FGFR2, with a KD of 0.76 nM, and was selectively internalized into cancer cells overexpressing FGFR2, Snu-16 and NCI-H716. Finally, we prepared a conjugate of scFvF7-Fc with the cytotoxic drug monomethyl-auristatin E (MMAE) and evaluated its cytotoxicity. The conjugate delivered MMAE selectively to FGFR2-positive tumor cells. These results indicate that scFvF7-Fc-vcMMAE is a highly potent molecule for the treatment of cancers with FGFR2 overexpression.

In vivo Therapy with Monoclonal Anti-I-A Antibody Suppresses Immune Responses to Acetylcholine Receptor

NASA Astrophysics Data System (ADS)

Waldor, Matthew K.; Sriram, Subramaniam; McDevitt, Hugh O.; Steinman, Lawrence

1983-05-01

A monoclonal antibody to I-A gene products of the immune response gene complex attenuates both humoral and cellular responses to acetylcholine receptor and appears to suppress clinical manifestations of experimental autoimmune myasthenia gravis. This demonstrates that use of antibodies against immune response gene products that are associated with susceptibility to disease may be feasible for therapy in autoimmune conditions such as myasthenia gravis.