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Sample records for scfv antibody fragments

  1. Refolded scFv Antibody Fragment against Myoglobin Shows Rapid Reaction Kinetics

    PubMed Central

    Song, Hyung-Nam; Jang, Jun-Hyuck; Kim, Young-Wan; Kim, Dong-Hyung; Park, Sung-Goo; Lee, Myung Kyu; Paek, Se-Hwan; Woo, Eui-Jeon

    2014-01-01

    Myoglobin is one of the early biomarkers for acute myocardial infarction. Recently, we have screened an antibody with unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid reaction kinetics are thought to be an early IgG form produced during early stage of in vivo immunization. We produced a recombinant scFv fragment for the premature antibody from Escherichia coli using refolding technology. The scFv gene was constructed by connection of the VH–VL sequence with a (Gly4Ser)3 linker. The scFv fragment without the pelB leader sequence was expressed at a high level, but the solubility was extremely low. A high concentration of 8 M urea was used for denaturation. The dilution refolding process in the presence of arginine and the redox reagents GSH and GSSH successfully produced a soluble scFv protein. The resultant refolded scFv protein showed association and dissociation values of 9.32 × 10−4 M−1·s−1 and 6.29 × 10−3 s−1, respectively, with an affinity value exceeding 107 M−1 (kon/koff), maintaining the original rapid reaction kinetics of the premature antibody. The refolded scFv could provide a platform for protein engineering for the clinical application for diagnosis of heart disease and the development of a continuous biosensor. PMID:25530617

  2. RECOMBINANT SINGLE CHAIN VARIABLE FRAGMENT ANTIBODIES (scFv) AGAINST Pro144-Leu155 FRAGMENT OF HUMAN PROTEIN C.

    PubMed

    Oliinyk, O S; Palyvoda, K O; Lugovskaya, N E; Kolibo, D V; Lugovskoy, E V; Komisarenko, S V

    2015-01-01

    The aim of this work was to obtain the recombinant single chain variable fragments of antibodies (scFv) against human protein C, the key component of blood anticoagulation system. For this purpose a peptide that mimics a Pro144-Leu155 sequence of protein C was synthesized and the murine immune scFv library against this peptide was constructed. The protein C specific scFv 9E were selected from the constructed library by the phage-display method. The scFv 9E dissociation constant was found to be 2∙10(-9) M. It was shown that scFv 9E were suitable for protein C detection by ELISA and Western blotting. Selected scFv could be further used for protein C investigation and for the development of quantitative methods for protein C detection in human blood.

  3. Radioiodination and biodistribution of the monoclonal antibody TU-20 and its scFv fragment

    NASA Astrophysics Data System (ADS)

    Kubaštová, H.; Kleinova, V.; Seifert, D.; Fišer, M.; Kranda, K.

    2006-01-01

    The ability of the monoclonal antibody TU-20 and its scFv fragment to specifically bind to the C-end of the class III beta-tubulin makes these preparations useful as potential diagnostics for in vivo determination of neurodegenerative diseases that entail degradation of neuronal cytoskeleton. To examine this hypothesis, TU-20 and its scFv were labelled with 125I and their properties were extensively investigated. TU-20 and its scFv were labelled via chloramine-T with the yield 90 95% and 64 78%, respectively. Their quality control, performed by an ELISA and gel electrophoresis, determined adequate properties for further studies. The in vitro experiment, involving autoradiography and immunohistochemistry of mice’ brain slices, enabled confirmation of preserved immunospecificity of the radiolabelled substances. Finally, the in vivo biodistribution proved differences in elimination of either TU-20, scFv TU-20, or iodide from the mice.

  4. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ‘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...

  6. Construction of Recombinant Single Chain Variable Fragment (ScFv) Antibody Against Superantigen for Immunodetection Using Antibody Phage Display Technology.

    PubMed

    Singh, Pawan Kumar; Agrawal, Ranu; Kamboj, D V; Singh, Lokendra

    2016-01-01

    Superantigens are a class of antigens that bind to the major histocompatibility complex class (MHC) II and T-cell receptor (TCR) and cause the nonspecific activation of T cells, resulting in a massive release of pro-inflammatory mediators. They are produced by the gram-positive organisms Staphylococcus aureus and Streptococcus pyogenes, and by a variety of other microbes such as viruses and mycoplasma, and cause toxic shock syndrome (TSS) and even death in some cases. The immunodetection of superantigens is difficult due to the polyclonal activation of T-cells leading to nonspecific antibody production. The production of recombinant monoclonal antibodies against superantigens can solve this problem and are far better than polyclonal antibodies in terms of detection. Here, we describe the construction of recombinant single chain variable fragments (ScFv) antibodies against superantigens with specific reference to SEB (staphylococcal enterotoxin B) using antibody phage display technology.

  7. Identification of scFv antibody fragments that specifically recognise the heroin metabolite 6-monoacetylmorphine but not morphine.

    PubMed

    Moghaddam, Amir; Borgen, Tine; Stacy, John; Kausmally, Louise; Simonsen, Bjørg; Marvik, Ole J; Brekke, Ole Henrik; Braunagel, Michael

    2003-09-01

    Use of phage display of recombinant antibodies and large repertoire naïve antibody libraries for identifying antibodies of high specificity has been extensively reported. Nevertheless, there have been few reported antibodies to haptens that have originated from naïve antibody libraries with potential use in diagnostics. We have used chain shuffling of lead single-chain fragment variable (scFv) antibodies, isolated from a naïve antibody library, to screen for antibodies that specifically recognise the major metabolite of heroin, 6-monoacetylmorphine (6MAM). The antibodies were identified by screening high-density colonies of Escherichia coli expressing soluble scFv antibody fragments without prior expression on bacteriophage (phage display). The antibodies recognise 6MAM with affinities of 1-3x10(-7) M with no crossreactivity to morphine. These antibodies can potentially be used for developing a rapid immunoassay in drug-testing programs. To our knowledge, this is the first report of an antibody that distinguishes 6MAM from its de-acetylated form, morphine.

  8. Transgenic tobacco plants expressing a dimeric single-chain variable fragment (scfv) antibody against Salmonella enterica serotype Paratyphi B.

    PubMed

    Makvandi-Nejad, Shokouh; McLean, Michael D; Hirama, Tomoko; Almquist, Kurt C; Mackenzie, C Roger; Hall, J Christopher

    2005-10-01

    Transgenic tobacco plants were produced that express an anti-Salmonella enterica single-chain variable fragment (scFv) antibody that binds to the lipopolysaccharide (LPS) of S. enterica Paratyphi B. The coding sequence of this scFv was optimized for expression in tobacco, synthesized and subsequently placed behind three different promoters: an enhanced tobacco constitutive ubiquitous promoter (EntCUP4), and single- and double-enhancer versions of the Cauliflower Mosaic Virus 35S promoter (CaMV 35S). These chimeric genes were introduced into Nicotiana tabacum cv. 81V9 by Agrobacterium-mediated transformation and 50 primary transgenic (T(0)) plants per construct were produced. Among these plants, 23 were selected for the ability to express active scFv as determined by enzyme-linked immunosorbent assay (ELISA) using S. enterica LPS as antigen. Expanded bed adsorption-immobilized metal affinity chromatography (EBA-IMAC) was used to purify 41.7 mug of scFv/g from leaf tissue. Gel filtration and surface plasmon resonance (SPR) analyses demonstrated that the purified scFv was active as a dimer or higher-order multimer. In order to identify T(1) plants suitable for development of homozygous lines with heritable scFv expression, kanamycin-resistance segregation analyses were performed to determine the number of T-DNA loci in each T(0) plant, and quantitative ELISA and immunoblot analyses were used to compare expression of active and total anti-Salmonella scFv, respectively, in the T(1) generation. As S. enterica causes millions of enteric fevers and hundreds of thousands of deaths worldwide each year, large-scale production and purification of this scFv will have potential for uses in diagnosis and detection, as a therapeutic agent, and in applications such as water system purification.

  9. NOVEL AMYLOID-BETA SPECIFIC scFv and VH ANTIBODY FRAGMENTS FROM HUMAN AND MOUSE PHAGE DISPLAY ANTIBODY LIBRARIES

    PubMed Central

    Medecigo, M.; Manoutcharian, K.; Vasilevko, V.; Govezensky, T.; Munguia, M. E.; Becerril, B.; Luz-Madrigal, A.; Vaca, L.; Cribbs, D. H.; Gevorkian, G.

    2010-01-01

    Anti-amyloid immunotherapy has been proposed as an appropriate therapeutic approach for Alzheimer’s disease (AD). Significant efforts have been made towards the generation and assessment of antibody-based reagents capable of preventing and clearing amyloid aggregates as well as preventing their synaptotoxic effects. In this study, we selected a novel set of human anti-amyloid-beta peptide 1-42 (Aβ1-42) recombinant monoclonal antibodies in a single chain fragment variable (scFv) and a single domain (VH) formats. We demonstrated that these antibody fragments recognize in a specific manner amyloid beta deposits in APP/Tg mouse brains, inhibit toxicity of oligomeric Aβ1-42 in neuroblastoma cell cultures in a concentration-dependently manner and reduced amyloid deposits in APP/Tg2576 mice after intracranial administration. These antibody fragments recognize epitopes in the middle/C-terminus region of Aβ, which makes them strong therapeutic candidates due to the fact that most of the Aβ species found in the brains of AD patients display extensive N-terminus truncations/modifications. PMID:20451261

  10. Targeted Multiplex Imaging Mass Spectrometry with Single Chain Fragment Variable (scfv) Recombinant Antibodies

    NASA Astrophysics Data System (ADS)

    Thiery, Gwendoline; Mernaugh, Ray L.; Yan, Heping; Spraggins, Jeffrey M.; Yang, Junhai; Parl, Fritz F.; Caprioli, Richard M.

    2012-10-01

    Recombinant scfv antibodies specific for CYP1A1 and CYP1B1 P450 enzymes were combined with targeted imaging mass spectrometry to simultaneously detect the P450 enzymes present in archived, paraffin-embedded, human breast cancer tissue sections. By using CYP1A1 and CYP1B1 specific scfv, each coupled to a unique reporter molecule (i.e., a mass tag) it was possible to simultaneously detect multiple antigens within a single tissue sample with high sensitivity and specificity using mass spectrometry. The capability of imaging multiple antigens at the same time is a significant advance that overcomes technical barriers encountered when using present day approaches to develop assays that can simultaneously detect more than a single antigen in the same tissue sample.

  11. Targeted Multiplex Imaging Mass Spectrometry with Single Chain Fragment Variable (scfv) Recombinant Antibodies

    PubMed Central

    Thiery, Gwendoline; Mernaugh, Ray L.; Yan, Heping; Spraggins, Jeffrey M.; Yang, Junhai; Parl, Fritz F.; Caprioli, Richard M.

    2012-01-01

    Recombinant scfv antibodies specific for CYP1A1 and CYP1B1 P450 enzymes were combined with targeted imaging mass spectrometry to simultaneously detect the P450 enzymes present in archived, paraffin-embedded, human breast cancer tissue sections. By using CYP1A1 and CYP1B1 specific scfv, each coupled to a unique reporter molecule (i.e., a mass tag) it was possible to simultaneously detect multiple antigens within a single tissue sample with high sensitivity and specificity using mass spectrometry. The capability of imaging multiple antigens at the same time is a significant advance that overcomes technical barriers encountered when using present day approaches to develop assays that can simultaneously detect more than a single antigen in the same tissue sample. PMID:22869296

  12. 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.

  13. Construction of a human functional single-chain variable fragment (scFv) antibody recognizing the malaria parasite Plasmodium falciparum.

    PubMed

    Wajanarogana, Sumet; Prasomrothanakul, Teerawat; Udomsangpetch, Rachanee; Tungpradabkul, Sumalee

    2006-04-01

    Falciparum malaria is one of the most deadly and profound human health problems around the tropical world. Antimalarial drugs are now considered to be a powerful treatment; however, there are drugs currently being used that are resistant to Plasmodium falciparum parasites spreading in different parts of the world. Although the protective immune response against intraerythrocytic stages of the falciparum malaria parasite is still not fully understood, immune antibodies have been shown to be associated with reduced parasite prevalence. Therefore antibodies of the right specificity present in adequate concentrations and affinity are reasonably effective in providing protection. In the present study, VH (variable domain of heavy chain) and VL (variable domain of light chain) were isolated from human blood lymphocytes of P. falciparum in one person who had high serum titre to RESA (ring-infected erythrocyte surface antigen). Equal amounts of VH and VL were assembled together with universal linker (G4S)3 to generate scFvs (single-chain variable fragments). The scFv antibodies were expressed with a phage system for the selection process. Exclusively, an expressed scFv against asynchronous culture of P. falciparum-infected erythrocytes was selected and characterized. Sequence analysis of selected scFv revealed that this clone could be classified into a VH family-derived germline gene (VH1) and Vkappa family segment (Vkappa1). Using an indirect immunofluorescence assay, we could show that soluble expressed scFv reacted with falciparum-infected erythrocytes. The results encourage the further study of scFvs for development as a potential immunotherapeutic agent.

  14. Cloning single-chain antibody fragments (ScFv) from hyrbidoma cells.

    PubMed

    Toleikis, Lars; Frenzel, André

    2012-01-01

    Despite the rising impact of the generation of antibodies by phage display and other technologies, hybridoma technology still provides a valuable tool for the generation of high-affinity binders against different targets. But there exist several limitations of using hybridoma-derived antibodies. The source of the hybridoma clones are mostly rat or mouse B-lymphocytes. Therefore a human-anti-mouse or human-anti-rat antibody response may result in immunogenicity of these antibodies. This leads to the necessity of humanization of these antibodies where the knowledge of the amino acid sequence of the proteins is inalienable. Furthermore, additional in vitro modifications, e.g., affinity maturation or fusion to other proteins, are dependent on cloning of the antigen-binding domains.Here we describe the isolation of RNA from hybridoma cells and the primers that can be used for the amplification of VL and VH as well as the cloning of the antibody in scFv format and its expression in Escherichia coli.

  15. Selection of scFv Antibody Fragments Binding to Human Blood versus Lymphatic Endothelial Surface Antigens by Direct Cell Phage Display

    PubMed Central

    Keller, Thomas; Kalt, Romana; Raab, Ingrid; Schachner, Helga; Mayrhofer, Corina; Kerjaschki, Dontscho; Hantusch, Brigitte

    2015-01-01

    The identification of marker molecules specific for blood and lymphatic endothelium may provide new diagnostic tools and identify new targets for therapy of immune, microvascular and cancerous diseases. Here, we used a phage display library expressing human randomized single-chain Fv (scFv) antibodies for direct panning against live cultures of blood (BECs) and lymphatic (LECs) endothelial cells in solution. After six panning rounds, out of 944 sequenced antibody clones, we retrieved 166 unique/diverse scFv fragments, as indicated by the V-region sequences. Specificities of these phage clone antibodies for respective compartments were individually tested by direct cell ELISA, indicating that mainly pan-endothelial cell (EC) binders had been selected, but also revealing a subset of BEC-specific scFv antibodies. The specific staining pattern was recapitulated by twelve phage-independently expressed scFv antibodies. Binding capacity to BECs and LECs and differential staining of BEC versus LEC by a subset of eight scFv antibodies was confirmed by immunofluorescence staining. As one antigen, CD146 was identified by immunoprecipitation with phage-independent scFv fragment. This antibody, B6-11, specifically bound to recombinant CD146, and to native CD146 expressed by BECs, melanoma cells and blood vessels. Further, binding capacity of B6-11 to CD146 was fully retained after fusion to a mouse Fc portion, which enabled eukaryotic cell expression. Beyond visualization and diagnosis, this antibody might be used as a functional tool. Overall, our approach provided a method to select antibodies specific for endothelial surface determinants in their native configuration. We successfully selected antibodies that bind to antigens expressed on the human endothelial cell surfaces in situ, showing that BECs and LECs share a majority of surface antigens, which is complemented by cell-type specific, unique markers. PMID:25993332

  16. Isolation of soluble scFv antibody fragments specific for small biomarker molecule, L-Carnitine, using phage display.

    PubMed

    Abou El-Magd, Rabab M; Vozza, Nicolas F; Tuszynski, Jack A; Wishart, David S

    2016-01-01

    Isolation of single chain antibody fragment (scFv) clones from naïve Tomlinson I+J phage display libraries that specifically bind a small biomarker molecule, L-Carnitine, was performed using iterative affinity selection procedures. L-Carnitine has been described as a conditionally essential nutrient for humans. Abnormally high concentrations of L-Carnitine in urine are related to many health disorders including diabetes mellitus type 2 and lung cancer. ELISA-based affinity characterization results indicate that selectants preferentially bind to L-Carnitine in the presence of key bioselecting component materials and closely related L-Carnitine derivatives. In addition, the affinity results were confirmed using biophysical fluorescence quenching for tyrosine residues in the V segment. Small-scale production of the soluble fragment yielded 1.3mg/L using immunopure-immobilized protein A affinity column. Circular Dichroism data revealed that the antibody fragment (Ab) represents a folded protein that mainly consists of β-sheets. These novel antibody fragments may find utility as molecular affinity interface receptors in various electrochemical biosensor platforms to provide specific L-Carnitine binding capability with potential applications in metabolomic devices for companion diagnostics and personalized medicine applications. It may also be used in any other biomedical application where detection of the L-Carnitine level is important.

  17. Aptamers, antibody scFv, and antibody Fab' fragments: An overview and comparison of three of the most versatile biosensor biorecognition elements.

    PubMed

    Crivianu-Gaita, Victor; Thompson, Michael

    2016-11-15

    The choice of biosensing elements is crucial for the development of the optimal biosensor. Three of the most versatile biosensing elements are antibody single-chain Fv fragments (scFv), antibody fragment-antigen binding (Fab') units, and aptamers. This article provides an overview of these three biorecognition elements with respects to their synthesis/engineering, various immobilization techniques, and examples of their use in biosensors. Furthermore, the final section of the review compares and contrasts their characteristics (time/cost of development, ease and variability of immobilization, affinity, stability) illustrating their advantages and disadvantages. Overall, scFv fragments are found to display the highest customizability (i.e. addition of functional groups, immobilizing peptides, etc.) due to recombinant synthesis techniques. If time and cost are an issue in the development of the biosensor, Fab' fragments should be chosen as they are relatively cheap and can be developed quickly from whole antibodies (several days). However, if there are sufficient funds and time is not a factor, aptamers should be utilized as they display the greatest affinity towards their target analytes and are extremely stable (excellent biosensor regenerability).

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

    PubMed

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

    2016-03-01

    'Candidatus 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 vector, pKM19. The antibody population is enriched for antibodies that bind antigens of 'Ca. Liberibacter asiaticus'. The primary library has more than 10(7) unique antibodies and the genes that encode them. We have screened this library for antibodies that bind to specifically-chosen proteins that are present on the surface of 'Ca. Liberibacter asiaticus'. These proteins were used as targets for affinity-based selection of scFvs that bind to the major outer membrane protein, OmpA; the polysaccharide capsule protein KpsF; a protein component of the type IV pilus (CapF); and, two flagellar proteins FlhA and FlgI. These scFvs have been used in ELISA and dot blot assays against purified protein antigens and 'Ca. Liberibacter asiaticus' infected plant extracts. We have also recloned many of these scFvs into a plasmid expression vector designed for the production of scFvs. Screening of these scFvs was more efficient when phage-bound, rather than soluble scFvs, were used. We have demonstrated a technology to produce antibodies at will and against any protein target encoded by 'Ca. Liberibacter asiaticus'. Applications could include advanced diagnostic methods for huanglongbing and the development of immune labeling reagents for in planta applications.

  19. Cross-Neutralization Activity of Single-Chain Variable Fragment (scFv) Derived from Anti-V3 Monoclonal Antibodies Mediated by Post-Attachment Binding.

    PubMed

    Maruta, Yasuhiro; Kuwata, Takeo; Tanaka, Kazuki; Alam, Muntasir; Valdez, Kristel Paola Ramirez; Egami, Yoshika; Suwa, Yoshiaki; Morioka, Hiroshi; Matsushita, Shuzo

    2016-09-21

    The V3 loop in the envelope (Env) of HIV-1 is one of the major targets of neutralizing antibodies. However, this antigen is hidden inside the Env trimer in most isolates and is fully exposed only during CD4-gp120 interaction. Thus, primary HIV-1 isolates are relatively resistant to anti-V3 antibodies because IgG is too large to access the V3 loop. To overcome this obstacle, we constructed single-chain variable fragments (scFvs) from anti-V3 monoclonal antibodies 0.5γ, 5G2, and 16G6. Enhanced neutralization by 0.5γ and 5G2 scFvs was observed in strains resistant to their IgG counterparts. Neutralization coverage by 0.5γ scFv reached up to 90% of the tested viruses (tier 2 and 3 classes). The temperature-regulated neutralization assay revealed that extensive cross-neutralization of 0.5γ scFv can be explained by post-attachment neutralization. Neutralization assay involving viruses carrying an inter-subunit disulfide bond (SOS virus) showed that the neutralization-susceptible timeframe after attachment was 60 to 120 min. These results indicate that the scFvs efficiently access the V3 loop and subsequently neutralize HIV-1, even after virus attachment to the target cells. Based on its broad and potent neutralizing activity, further development of anti-V3 scFv for therapeutic and preventive strategies is warranted.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  1. Cultivation of Pichia pastoris carrying the scFv anti LDL (-) antibody fragment. Effect of preculture carbon source.

    PubMed

    Arias, Cesar Andres Diaz; Marques, Daniela de Araujo Viana; Malpiedi, Luciana Pellegrini; Maranhão, Andrea Queiroz; Parra, Dulcineia Abdalla Saes; Converti, Attilio; Junior, Adalberto Pessoa

    2017-02-09

    Antibodies and antibody fragments are nowadays among the most important biotechnological products, and Pichia pastoris is one of the most important vectors to produce them as well as other recombinant proteins. The conditions to effectively cultivate a P. pastoris strain previously genetically modified to produce the single-chain variable fragment anti low density lipoprotein (-) under the control of the alcohol oxidase promoter have been investigated in this study. In particular, it was evaluated if, and eventually how, the carbon source (glucose or glycerol) used in the preculture preceding cryopreservation in 20% glycerol influences both cell and antibody fragment productions either in flasks or in bioreactor. Although in flasks the volumetric productivity of the antibody fragment secreted by cells precultured, cryopreserved and reactivated in glycerol was 42.9% higher compared with cells precultured in glucose, the use of glycerol in bioreactor led to a remarkable shortening of the lag phase, thereby increasing it by no less than thrice compared to flasks. These results are quite promising in comparison with those reported in the literature for possible future industrial applications of this cultivation, taking into account that the overall process time was reduced by around 8h.

  2. Molecular characterization of a single-chain antibody variable fragment (scFv) specific for PspA from Streptococcus pneumoniae.

    PubMed

    Jang, ShinA; Kim, Gyuhee; Oh, Jihye; Lee, Seungyeop; Kim, Dongho; Kim, Kook-Han; Kim, Yong Ho; Rhee, Dong-Kwon; Lee, Sangho

    2017-01-01

    Streptococcus pneumoniae is a major infectious agent responsible for pneumonia, otitis media, sepsis and meningitis. Pneumococcal surface protein A (PspA) is a well-characterized virulence factor localized on the surface and a target for vaccine development. In this study, we screened a single-chain antibody variable fragment (scFv) using phage display from a human synthetic library to select a clone 2B11. Affinity (Kd) of 2B11 was measured to be 5 nM using biolayer interferometry. 2B11 exhibited a dose-dependent recognition of recombinant PspA with no cross-reactivity towards pneumococcal antigens. The epitope on PspA was defined to residues 231-242 by mutational analysis. Molecular docking analysis supported the experimentally determined epitope, suggesting that the helix spanning residues 231-242 can bind to 2B11 with residues in the CDR-H3 (complementarity determining region 3 in the heavy chain) actively participating in the molecular contacts. Comparison of 2B11 with a commercial PspA antibody revealed that 2B11 exhibited a better specificity towards recombinant PspA antigen. 2B11 was capable of detecting endogenous PspA from pneumococcal lysates with affinity similar to that of the commercial antibody. Our study provides a molecular tool for biosensors detecting pneumococcal diseases.

  3. Enhancement of hERG channel activity by scFv antibody fragments targeted to the PAS domain

    PubMed Central

    Harley, Carol A.; Starek, Greg; Jones, David K.; Fernandes, Andreia S.; Robertson, Gail A.; Morais-Cabral, João H.

    2016-01-01

    The human human ether-à-go-go–related gene (hERG) potassium channel plays a critical role in the repolarization of the cardiac action potential. Changes in hERG channel function underlie long QT syndrome (LQTS) and are associated with cardiac arrhythmias and sudden death. A striking feature of this channel and KCNH channels in general is the presence of an N-terminal Per-Arnt-Sim (PAS) domain. In other proteins, PAS domains bind ligands and modulate effector domains. However, the PAS domains of KCNH channels are orphan receptors. We have uncovered a family of positive modulators of hERG that specifically bind to the PAS domain. We generated two single-chain variable fragments (scFvs) that recognize different epitopes on the PAS domain. Both antibodies increase the rate of deactivation but have different effects on channel activation and inactivation. Importantly, we show that both antibodies, on binding to the PAS domain, increase the total amount of current that permeates the channel during a ventricular action potential and significantly reduce the action potential duration recorded in human cardiomyocytes. Overall, these molecules constitute a previously unidentified class of positive modulators and establish that allosteric modulation of hERG channel function through ligand binding to the PAS domain can be attained. PMID:27516548

  4. Generation of a Novel Bacteriophage Library Displaying scFv Antibody Fragments from the Natural Buffalo Host to Identify Antigens from Adult Schistosoma japonicum for Diagnostic Development

    PubMed Central

    Hosking, Christopher G.; McWilliam, Hamish E. G.; Driguez, Patrick; Piedrafita, David; Li, Yuesheng; McManus, Donald P.; Ilag, Leodevico L.; Meeusen, Els N. T.; de Veer, Michael J.

    2015-01-01

    The development of effective diagnostic tools will be essential in the continuing fight to reduce schistosome infection; however, the diagnostic tests available to date are generally laborious and difficult to implement in current parasite control strategies. We generated a series of single-chain antibody Fv domain (scFv) phage display libraries from the portal lymph node of field exposed water buffaloes, Bubalus bubalis, 11–12 days post challenge with Schistosoma japonicum cercariae. The selected scFv-phages showed clear enrichment towards adult schistosomes and excretory-secretory (ES) proteins by immunofluorescence, ELISA and western blot analysis. The enriched libraries were used to probe a schistosome specific protein microarray resulting in the recognition of a number of proteins, five of which were specific to schistosomes, with RNA expression predominantly in the adult life-stage based on interrogation of schistosome expressed sequence tags (EST). As the libraries were enriched by panning against ES products, these antigens may be excreted or secreted into the host vasculature and hence may make good targets for a diagnostic assay. Further selection of the scFv library against infected mouse sera identified five soluble scFv clones that could selectively recognise soluble whole adult preparations (SWAP) relative to an irrelevant protein control (ovalbumin). Furthermore, two of the identified scFv clones also selectively recognised SWAP proteins when spiked into naïve mouse sera. These host B-cell derived scFvs that specifically bind to schistosome protein preparations will be valuable reagents for further development of a cost effective point-of-care diagnostic test. PMID:26684756

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

    SciTech Connect

    Gray, Sean A.; Barr, John R.; Kalb, Suzanne R.; Marks, James D.; Baird, Cheryl L.; Cangelosi, Gerard A.; Miller, Keith D.; Feldhaus, Michael J.

    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 the 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.

  6. Fast conversion of scFv to Fab antibodies using type IIs restriction enzymes.

    PubMed

    Sanmark, Hanna; Huovinen, Tuomas; Matikka, Tero; Pettersson, Tiina; Lahti, Maria; Lamminmäki, Urpo

    2015-11-01

    Single chain variable fragment (scFv) antibody libraries are widely used for developing novel bioaffinity reagents, although Fab or IgG molecules are the preferred antibody formats in many final applications. Therefore, rapid conversion methods for combining multiple DNA fragments are needed to attach constant domains to the scFv derived variable domains. In this study we describe a fast and easy cloning method for the conversion of single framework scFv fragments to Fab fragments using type IIS restriction enzymes. All cloning steps excluding plating of the Fab transformants can be done in 96 well plates and the procedure can be completed in one working day. The concept was tested by converting 69 scFv clones into Fab format on 96 well plates, which resulted in 93% success rate. The method is particularly useful as a high-throughput tool for the conversion of the chosen scFv clones into Fab molecules in order to analyze them as early as possible, as the conversion can significantly affect the binding properties of the chosen clones.

  7. Biodistribution of the Radiolabeled Anti III {beta}-Tubulin scFv Fragment in Mice

    SciTech Connect

    Kleinova, Veronika; Svecova, H.; Chaloupkova, H.; Kranda, K.; Fiser, M.

    2007-11-26

    For studies of new potential radiopharmaceutical such as radiolabeled compound, the biodistribution exoeriments are essential to describe behavior of the substance in organism. The specific binding of the scFv fragment of the monoclonal antibody TU-20 to the C-end of the class III {beta}-tubulin makes this substance useful as a potential diagnostics for in vivo neurodegenerative diseases determination. To examine this hypothesis, scFv was radio-labeled with {sup 125}I and {sup 123}I, and its biochemical properties were studied. The in vivo bio-distribution confirmed the expected elimination behavior of the radio-labeled scFv TU-20 in mice. The bi-exponential model for two-phase clearance to determine short phase half-life t{sub 1/2{alpha}} and long phase half-life t{sub 1/2{beta}} values was used to evaluate the experimental data.

  8. Generation of a recombinant single-chain variable fragment (scFv) targeting 5-methyl-2'-deoxycytidine.

    PubMed

    Ohshima, Motohiro; Tadakuma, Tomomi; Hayashi, Hideki; Inoue, Kazuyuki; Itoh, Kunihiko

    2010-01-01

    We generated a single-chain variable fragment (scFv) against 5-methyl-2'-deoxycytidine (m(5)dCyd) using phage display technology. The heavy and light chain variable region genes were amplified by the polymerase chain reaction (PCR) from hybridoma cell line FMC9 and assembled as an scFv fragment with a flexible linker (Gly(4)-Ser)(3). The scFv DNA fragment was then cloned into pCANTAB-5E, and a phage displaying the scFv was produced. Antigen-positive phage clones were successfully selected by enzyme-linked immunosorbent assay (ELISA). The scFv was modified with FLAG and His tags for detection and purification. The scFv reacted strongly with m(5)dCyd and weakly with 5-methylcytidine (m(5)Cyd) but not with cytidine (Cyd) and 1-methyladenosine in a manner similar to the monoclonal antibody (MoAb). Although the specificities of scFv and MoAb were almost identical, the sensitivity of the scFv (IC(50) 0.054 microg/ml) was approximately 80 times higher than that of the parent MoAb (IC(50) 4.27 microg/ml), determined by inhibition ELISA. As a biochemical application of this scFv, we quantified the m(5)dCyd content of genomic DNA by enzymatic hydrolysis using inhibition ELISA. The cancer cell lines HeLa, HeLa S3 and MDA-MB-453 contained approximately 1% of the methylated DNA in total genomic DNA, as did peripheral blood cell genomic DNA from healthy volunteers, but HT29 and T-47D showed hypomethylation compared with the HeLa, HeLa S3 and MDA-MB-453 cell lines. The scFv generated here may be applicable to the assessment of cellular DNA methylation levels and is more sensitive than the MoAb.

  9. Engineering production of functional scFv antibody in E. coli by co-expressing the molecule chaperone Skp

    PubMed Central

    Wang, Rongzhi; Xiang, Shuangshuang; Feng, Youjun; Srinivas, Swaminath; Zhang, Yonghui; Lin, Mingshen; Wang, Shihua

    2013-01-01

    Single-chain variable fragment (scFv) is a class of engineered antibodies generated by the fusion of the heavy (VH) and light chains (VL) of immunoglobulins through a short polypeptide linker. ScFv play a critical role in therapy and diagnosis of human diseases, and may in fact also be developed into a potential diagnostic and/or therapeutic agent. However, the fact that current scFv antibodies have poor stability, low solubility, and affinity, seriously limits their diagnostic and clinical implication. Here we have developed four different expression vectors, and evaluated their abilities to express a soluble scFv protein. The solubility and binding activity of the purified proteins were determined using both SDS-PAGE and ELISA. Amongst the four purified proteins, the Skp co-expressed scFv showed the highest solubility, and the binding activity to antigen TLH was 3-4 fold higher than the other three purified scFv. In fact, this scFv is specific for TLH and does not cross-react with other TLH-associated proteins and could be used to detect TLH directly in real samples. These results suggest that the pACYC-Duet-skp co-expression vector might be a useful tool for the production of soluble and functional scFv antibody. PMID:24224158

  10. Efficient production of anti-fluorescein and anti-lysozyme as single-chain anti-body fragments (scFv) by Brevibacillus expression system.

    PubMed

    Onishi, Hiromasa; Mizukami, Makoto; Hanagata, Hiroshi; Tokunaga, Masao; Arakawa, Tsutomu; Miyauchi, Akira

    2013-10-01

    Expression of scFv in Brevibacillus choshinensis was tested using combinations of three different promoters and four different secretion signals. Two model scFv constructs, i.e., His-scFvFLU and His-scFvHEL, were successfully expressed with some of the combinations. Ni Sepharose column and size exclusion chromatography resulted in fairly pure preparations of these two proteins. The purified His-scFvFLU inhibited fluorescence from fluorescein, while the purified His-scFvHEL inhibited lysozyme activity. Relatively high yield of His-scFvFLU (∼40%) and His-scFvHEL (∼30%) was achieved with the expression and purification system described here.

  11. Binding of HIV-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (Fab) and single chain variable fragment (ScFv) antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations.

    PubMed

    Louis, John M; Aniana, Annie; Lohith, Katheryn; Sayer, Jane M; Roche, Julien; Bewley, Carole A; Clore, G Marius

    2014-01-01

    We previously reported a series of antibodies, in fragment antigen binding domain (Fab) formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066) and non-neutralizing (8062) antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv) formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥ 150-fold) in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

  12. Single-Chain Fragment Variable Antibody Piezoimmunosensors

    PubMed Central

    Shen, Zhihong; Stryker, Gabrielle A.; Mernaugh, Ray L.; Yu, Lei; Yan, Heping; Zeng, Xiangqun

    2008-01-01

    In this paper, we describe a novel nonlabeled biosensor with high diagnostic potential for rapid and sensitive detection of antigens in complex biological samples. The biosensor comprises a piezoimmunosensor (PZ) displaying a specially constructed recombinant antibody on its surface. The recombinant single-chain fragment variable (scFv) antibody contained a cysteine within the linker amino acid sequence used to join the scFv variable heavy and light chains. The presence of cysteine induced the scFv construct to self-assemble as a densely packed rigid monolayer on the gold surface of a quartz crystal microbalance. scFv molecules in this self-assembled mono-layer (SAM) exhibited a defined orientation and high areal densities, with scFv-modified microbalance surfaces displaying 35 times as many variable antigen-binding sites per square centimeter as surfaces modified with whole antibody. Experimental data show that the scFv SAM PZ is superior to Fab fragment, Fab fragment containing a free sulfhydryl group (i.e., Fab-SH), and whole antibody PZs regarding sensitivity and specificity. Because of their small uniform size (MW ≈ 27000) and the ease with which they can be modified using genetic engineering, scFv’s have significant advantages over whole antibodies in microbalance biosensor systems. We demonstrate here that the use of scFv containing a cysteine within the scFv linker sequence (i.e., scFv-cys) for preparation of biosensor surfaces markedly increases the density of available antigen-binding sites, yielding a system that is highly selective, rapid, and capable of detecting low concentrations of antigens in complex samples. PMID:15679346

  13. 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.

  14. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library

    PubMed Central

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

    2016-01-01

    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective. PMID:27626445

  15. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library.

    PubMed

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

    2016-09-11

    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

  16. Development of single-chain variable fragments (scFv) against influenza virus targeting hemagglutinin subunit 2 (HA2).

    PubMed

    Li, Tai-Wei; Cheng, Shu-Fang; Tseng, Yen-Tzu; Yang, Yu-Chih; Liu, Wen-Chun; Wang, Sheng-Cyuan; Chou, Mei-Ju; Lin, Yu-Jen; Wang, Yueh; Hsiao, Pei-Wen; Wu, Suh-Chin; Chang, Ding-Kwo

    2016-01-01

    Influenza A viruses (IAV) are widespread in birds and domestic poultry, occasionally causing severe epidemics in humans and posing health threats. Hence, the need to develop a strategy for prophylaxis or therapy, such as a broadly neutralizing antibody against IAV, is urgent. In this study, single-chain variable fragment (scFv) phage display technology was used to select scFv fragments recognizing influenza envelope proteins. The Tomlinson I and J scFv phage display libraries were screened against the recombinant HA2 protein (rHA2) for three rounds. Only the third-round elution sample of the Tomlinson J library showed high binding affinity to rHA2, from which three clones (3JA18, 3JA62, and 3JA78) were chosen for preparative-scale production as soluble antibody by E. coli. The clone 3JA18 was selected for further tests due to its broad affinity for influenza H1N1, H3N2 and H5N1. Simulations of the scFv 3JA18-HA trimer complex revealed that the complementarity-determining region of the variable heavy chain (VH-CDR2) bound the stem region of HA. Neutralization assays using a peptide derived from VH-CDR2 also supported the simulation model. Both the selected antibody and its derived peptide were shown to suppress infection with H5N1 and H1N1 viruses, but not H3N2 viruses. The results also suggested that the scFvs selected from rHA2 could have neutralizing activity by interfering with the function of the HA stem region during virus entry into target cells.

  17. PVY-resistant transgenic potato plants expressing an anti-NIa protein scFv antibody.

    PubMed

    Gargouri-Bouzid, Radhia; Jaoua, Leïla; Rouis, Souad; Saïdi, Mohamed Najib; Bouaziz, Donia; Ellouz, Radhouane

    2006-06-01

    A synthetic gene encoding a single chain Fv fragment of an antibody directed against the nuclear inclusion a (NIa) protein of potato virus Y (PVY) was used to transform two commercial potato cultivars (Claustar and BF15). The NIa protease forms the nuclear inclusion body A and acts as the major protease in the cleavage of the viral polyprotein into functional proteins. Immunoblot analysis showed that most of the resulting transgenic plants accumulate high levels of the transgenic protein. Furthermore, a majority of the selected transgenic lines showed an efficient and complete protection against the challenge virus after mechanical inoculation with PVYO strain. Two transgenic lines showed an incomplete resistance with delayed appearance of symptoms accompanied by low virus titers, whereas one line developed symptoms during the first days after inoculation but recovered rapidly, leading to a low virus accumulation rate. These results confirm that expression of scFv antibody is able to inhibit a crucial step in the virus multiplication, such as polyprotein cleavage is a powerful strategy for engineered virus resistance. It can lead to a complete resistance that was not obtained previously by expression of scFv directed against the viral coat protein.

  18. Construction of scFv derived from a tumor-associated monoclonal antibody having tumoricidal activity on human hepatocellular carcinoma.

    PubMed

    Tungpradabkul, Sumalee; Sandee, Duanpen; Puthong, Songchan; Laohathai, Kingkarn

    2005-04-01

    A mouse monoclonal antibody (Mab-HepTAA43), classified as an anti-tumor-associated antigen, was raised by immunizing BALB/c mice with the Thai human hepatocellular carcinoma S102 (HCC-S102) cell line cells using hybridoma techniques. The Mab-HepTAA43 reacted with and markedly inhibited the growth of human hepatocellular carcinoma cell lines as well as a tumor mass in an animal model. Human hepatoma transplanted into nude mice did not show metastasis after 20 injections amounting to a total of about 4 mg of the Mab over 1-month period. A single-chain variable fragment (scFv) molecule derived from the Mab was constructed by phage display method. DNA sequence analysis of the active variable regions of both heavy- and light-chains of the cDNA clone was subsequently performed. The scFv43 molecule contains a V(L) kappa type and a unique V(H) sequence having 88% amino acid homology to that of Mab-MAK B raised against tumor-associated antigen. Immunohistochemical staining on frozen sections of paired hepatoma (NCI-I) and normal liver tissue from the same individual showed that both scFv43 and Mab-HepTAA43 antibodies reacted with hepatoma but not with normal liver tissue. The results suggest that scFv43 may be useful in the immunotherapy of hepatocellular carcinoma.

  19. In vivo imaging of prostate cancer using an anti-PSMA scFv fragment as a probe

    PubMed Central

    Mazzocco, Claire; Fracasso, Giulio; Germain-Genevois, Coralie; Dugot-Senant, Nathalie; Figini, Mariangela; Colombatti, Marco; Grenier, Nicolas; Couillaud, Franck

    2016-01-01

    We aimed to evaluate a fluorescent-labeled single chain variable fragment (scFv) of the anti-PSMA antibody as a specific probe for the detection of prostate cancer by in vivo fluorescence imaging. An orthotopic model of prostate cancer was generated by injecting LNCaP cells into the prostate lobe. ScFvD2B, a high affinity anti-PSMA antibody fragment, was labeled using a near-infrared fluorophore to generate a specific imaging probe (X770-scFvD2B). PSMA-unrelated scFv-X770 was used as a control. Probes were injected intravenously into mice with prostate tumors and fluorescence was monitored in vivo by fluorescence molecular tomography (FMT). In vitro assays showed that X770-scFvD2B specifically bound to PSMA and was internalized in PSMA-expressing LNCaP cells. After intravenous injection, X770-scFvD2B was detected in vivo by FMT in the prostate region. On excised prostates the scFv probe co-localized with the cancer cells and was found in PSMA-expressing cells. The PSMA-unrelated scFv used as a control did not label the prostate cancer cells. Our data demonstrate that scFvD2B is a high affinity contrast agent for in vivo detection of PSMA-expressing cells in the prostate. NIR-labeled scFvD2B could thus be further developed as a clinical probe for imaging-guided targeted biopsies. PMID:26996325

  20. A Recombinant Human Anti-Platelet scFv Antibody Produced in Pichia pastoris for Atheroma Targeting

    PubMed Central

    Vallet-Courbin, Amelie; Larivière, Mélusine; Hocquellet, Agnès; Hemadou, Audrey; Parimala, Sarjapura-Nagaraja; Laroche-Traineau, Jeanny; Santarelli, Xavier; Clofent-Sanchez, Gisèle; Jacobin-Valat, Marie-Josée; Noubhani, Abdelmajid

    2017-01-01

    Cells of the innate and adaptive immune system are key factors in the progression of atherosclerotic plaque, leading to plaque instability and rupture, potentially resulting in acute atherothrombotic events such as coronary artery disease, cerebrovascular disease and peripheral arterial disease. Here, we describe the cloning, expression, purification, and immunoreactivity assessment of a recombinant single-chain variable fragment (scFv) derived from a human anti-αIIbβ3 antibody (HuAb) selected to target atheromatous lesions for the presence of platelets. Indeed, platelets within atheroma plaques have been shown to play a role in inflammation, in platelet-leucocyte aggregates and in thrombi formation and might thus be considered relevant biomarkers of atherosclerotic progression. The DNA sequence that encodes the anti-αIIbβ3 TEG4 scFv previously obtained from a phage-display selection on activated platelets, was inserted into the eukaryote vector (pPICZαA) in fusion with a tag sequence encoding 2 cysteines useable for specific probes grafting experiments. The recombinant protein was expressed at high yields in Pichia pastoris (30 mg/L culture). The advantage of P. pastoris as an expression system is the production and secretion of recombinant proteins in the supernatant, ruling out the difficulties encountered when scFv are produced in the cytoplasm of bacteria (low yield, low solubility and reduced affinity). The improved conditions allowed for the recovery of highly purified and biologically active scFv fragments ready to be grafted in a site-directed way to nanoparticles for the imaging of atherosclerotic plaques involving inflammatory processes and thus at high risk of instability. PMID:28125612

  1. Inhibiting angiogenesis with human single-chain variable fragment antibody targeting VEGF.

    PubMed

    Hosseini, Hossien; Rajabibazl, Masoumeh; Ebrahimizadeh, Walead; Dehbidi, Gholamreza Rafiei

    2015-01-01

    Vascular endothelial growth factor (VEGF) is a highly specific angiogenesis factor which has crucial roles in the angiogenesis of tumors. Anti-angiogenesis agents can inhibit growth and metastasis of tumor cells. Single-chain variable fragments (scFv) have the same affinity as whole antibodies and smaller size, thus result in more tissue permeability and higher production yield. In this research we aim to isolate a human scFv antibody against VEGF that inhibits angiogenesis. For that, we have used human scFv phage library to isolate a specific scFv antibody against binding site of VEGF. The human scFv phage library was amplified according to the manufacture protocol and panned against recombinant VEGF. ScFv antibody was isolated after five rounds of panning. Phage ELISA was used for detection of the highest affinity binder (HR6). Soluble HR6 scFv was expressed in non-suppressor strain of Escherichia coli HB2151 and purified using Ni-NTA chromatography. In vivo and in vitro function of the HR6 scFv was analyzed by chorioallantoic membrane assay and endothelial cell proliferation assay on VEGF stimulated HUVECs. Result of the cross reactivity showed that HR6 scFv specifically bounds to VEGF. The affinity was calculated to be 1.8×10(-7)M. HR6 could stop HUVEC proliferation in a dose dependent manner and anti-angiogenesis activity was observed using 10μg of HR6 in chorioallantoic membrane assay. In this work, we demonstrate that a HR6 scFv selected from human library phage display specifically blocks VEGF signaling, furthermore, this scFv has an anti-angiogenesis effect and because of its small size has more tissue diffusion. The HR6 antibody was isolated form a human library thus, it is not immunogenic for humans and could serve as a potential therapeutic agent in cancer.

  2. The change of the scFv into the Fab format improves the stability and in vivo toxin neutralization capacity of recombinant antibodies.

    PubMed

    Quintero-Hernández, Veronica; Juárez-González, Victor R; Ortíz-León, Mauricio; Sánchez, Rosalba; Possani, Lourival D; Becerril, Baltazar

    2007-02-01

    The antigen-binding fragment (Fab) has been considered a more functionally stable version of recombinant antibodies than single chain antibody fragments (scFvs), however this intuitive consideration has not been sufficiently proven in vivo. This communication shows that three out of four specific scFvs against a scorpion toxin, with different affinities and stabilities, become neutralizing in vivo when expressed as Fabs, despite the fact that they are not neutralizing in the scFv format. A scFv fragment previously obtained from a neutralizing mouse antibody (BCF2) was used to produce three derived scFvs by directed evolution. Only one of them was neutralizing, however when expressed as Fab, all of them became neutralizing fragments in vivo. The initial scFvBCF2 (earlier used for directed evolution) was not neutralizing in the scFv format. After expressing it as Fab did not become a neutralizing fragment, but did reduce the intoxication symptoms of experimental mice. The stability of the four Fabs derived from their respective scFvs was improved when tested in the presence of guanidinium chloride. The in vitro stability of the Fab format has been shown earlier, but the physiological consequences of this stability are shown in this communication. The present results indicate that improved functional stability conferred by the Fab format can replace additional maturation steps, when the affinity and stability are close to the minimum necessary to be neutralizing.

  3. Production of a single-chain fragment of the murine anti-idiotypic antibody ACA125 as phage-displayed and soluble antibody by recombinant phage antibody technique.

    PubMed

    Schlebusch, H; Reinartz, S; Kaiser, R; Grünn, U; Wagner, U

    1997-02-01

    The F(ab')2 fragment of the murine monoclonal anti-idiotypic antibody ACA125 mimicking the tumor-associated antigen CA125 is used as a vaccine for the induction of an anti-tumoral immunity in patients with ovarian carcinoma. We tried to generate a single-chain fragment (ScFv) composed of ACA125 heavy- and light-chain variable domains connected by a polypeptide linker as an alternative to the corresponding F(ab')2 fragment. Heavy- and light-chain genes of antibody-producing mouse hybridoma cell line were amplified separately and assembled into a ScFv gene with linker DNA by the polymerase chain reaction (PCR). The ScFv gene was ligated into the phagemid vector pCANTAB5E, which allows the production of both phage-displayed and soluble ScFv. Transformed Escherichia coli TG1 cells were infected with M13K07 helper phage to yield recombinant phage, which display ScFv fragments as a g3p fusion protein on the surface of the filamentous phage M13. Recombinant phages could be selected by binding to the idiotypic antibody OC125 after one round of panning and directly used to reinfect E. coli TG1 cells. The E. coli nonsuppressor strain HB2151 was infected with an antigen-positive phage clone, previously screened by enzyme-linked immunosorbent assay (ELISA), to express soluble ScFv fragments. Functional soluble ScFv binding to the idiotypic antibody OC125 F(ab')2 could be detected in the bacterial periplasm by Western blot and ELISA. The variable heavy- and light-chain genes of the ACA125 ScFv fragment were further sequenced and compared with known antibody sequences.

  4. Penetration of engineered antibody fragments into the eye

    PubMed Central

    THIEL, M A; COSTER, D J; STANDFIELD, S D; BRERETON, H M; MAVRANGELOS, C; ZOLA, H; TAYLOR, S; YUSIM, A; WILLIAMS, K A

    2002-01-01

    Antibodies are powerful immunotherapeutic agents but their use for treating ocular disorders is limited by their poor penetration into the eye. We hypothesized that antibody fragments of relatively small size might penetrate the cornea more readily. Monovalent single chain variable region (scFv) antibody fragments and divalent miniantibodies were engineered from existing monoclonal antibodies, expressed in a bacterial expression system, and purified by metal ion affinity chromatography. Corneoscleral preparations from normal pig and cat eyes were mounted in a corneal perfusion chamber. Intact antibodies and antibody fragments were applied topically to the anterior corneal surface over 12-h periods, and samples were collected from the artificial anterior chamber. Similar experiments were performed with whole enucleated pig and human eyes. Penetration of antibodies and fragments was quantified by high-sensitivity flow cytometry on appropriate target cells. Both monovalent scFv and divalent miniantibody fragments (but not whole immunoglobulin molecules) passed through de-epithelialized and intact corneas after topical administration, and could be detected by antigen binding. Addition of 0·5% sodium caprate facilitated penetration through intact corneas. Topically-applied scFv was found to penetrate into the anterior chamber fluid of rabbit eyes in vivo. The engineered fragments were stable and resistant to ocular proteases. Monovalent and divalent antibody constructs of molecular weight 28 kD and 67 kD, respectively, can penetrate through intact corneas into the anterior chamber, with retention of appropriate antigen-binding activity. Such constructs may form novel therapeutic agents for topical ophthalmic use. PMID:11982592

  5. Antibody Fragments as Probe in Biosensor Development

    PubMed Central

    Saerens, Dirk; Huang, Lieven; Bonroy, Kristien; Muyldermans, Serge

    2008-01-01

    Today's proteomic analyses are generating increasing numbers of biomarkers, making it essential to possess highly specific probes able to recognize those targets. Antibodies are considered to be the first choice as molecular recognition units due to their target specificity and affinity, which make them excellent probes in biosensor development. However several problems such as difficult directional immobilization, unstable behavior, loss of specificity and steric hindrance, may arise from using these large molecules. Luckily, protein engineering techniques offer designed antibody formats suitable for biomarker analysis. Minimization strategies of antibodies into Fab fragments, scFv or even single-domain antibody fragments like VH, VL or VHHs are reviewed. Not only the size of the probe but also other issues like choice of immobilization tag, type of solid support and probe stability are of critical importance in assay development for biosensing. In this respect, multiple approaches to specifically orient and couple antibody fragments in a generic one-step procedure directly on a biosensor substrate are discussed. PMID:27873779

  6. Enzymatic Assembly for scFv Library Construction.

    PubMed

    Kato, Mieko; Hanyu, Yoshiro

    2017-01-01

    Recombinant monoclonal antibodies can be established by displaying single-chain variable fragment (scFv) antibody libraries on phages and then biopanning against the target. For constructing superior scFv libraries, antibody light-chain variable region (VL) and heavy-chain variable region (VH) fragments must be assembled into scFvs without loss of diversity. A high-quality scFv library is a prerequisite for obtaining strong binders from the scFv library. However, the technical challenges associated with the construction of a diverse library have been the bottleneck in the establishment of recombinant antibodies through biopanning. Here, we describe a simple and efficient method for assembling VL and VH fragments through the concerted action of λ-exonuclease and Bst DNA polymerase. We successfully used this method to construct a diverse chicken scFv library.

  7. The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi

    PubMed Central

    Joosten, Vivi; Lokman, Christien; van den Hondel, Cees AMJJ; Punt, Peter J

    2003-01-01

    In this review we will focus on the current status and views concerning the production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. We will focus on single-chain antibody fragment production (scFv and VHH) by these lower eukaryotes and the possible applications of these proteins. Also the coupling of fragments to relevant enzymes or other components will be discussed. As an example of the fusion protein strategy, the 'magic bullet' approach for industrial applications, will be highlighted. PMID:12605725

  8. Expression of recombinant antibody (single chain antibody fragment) in transgenic plant Nicotiana tabacum cv. Xanthi.

    PubMed

    Dobhal, S; Chaudhary, V K; Singh, A; Pandey, D; Kumar, A; Agrawal, S

    2013-12-01

    Plants offer an alternative inexpensive and convenient technology for large scale production of recombinant proteins especially recombinant antibodies (plantibodies). In this paper, we describe the expression of a model single chain antibody fragment (B6scFv) in transgenic tobacco. Four different gene constructs of B6scFv with different target signals for expression in different compartments of a tobacco plant cell with and without endoplasmic reticulum (ER) retention signal were used. Agrobacterium mediated plant transformation of B6scFv gene was performed with tobacco leaf explants and the gene in regenerated plants was detected using histochemical GUS assay and PCR. The expression of B6scFv gene was detected by western blotting and the recombinant protein was purified from putative transgenic tobacco plants using metal affinity chromatography. The expression level of recombinant protein was determined by indirect enzyme-linked immunosorbent assay. The highest accumulation of protein was found up to 3.28 % of the total soluble protein (TSP) in plants expressing B6scFv 1003 targeted to the ER, and subsequently expression of 2.9 % of TSP in plants expressing B6scFv 1004 (with target to apoplast with ER retention signal). In contrast, lower expression of 0.78 and 0.58 % of TSP was found in plants expressing antibody fragment in cytosol and apoplast, without ER retention signal. The described method/system could be used in the future for diverse applications including expression of other recombinant molecules in plants for immunomodulation, obtaining pathogen resistance against plant pathogens, altering metabolic pathways and also for the expression of different antibodies of therapeutic and diagnostic uses.

  9. Fragmentation of monoclonal antibodies

    PubMed Central

    Vlasak, Josef

    2011-01-01

    Fragmentation is a degradation pathway ubiquitously observed in proteins despite the remarkable stability of peptide bond; proteins differ only by how much and where cleavage occurs. The goal of this review is to summarize reports regarding the non-enzymatic fragmentation of the peptide backbone of monoclonal antibodies (mAbs). The sites in the polypeptide chain susceptible to fragmentation are determined by a multitude of factors. Insights are provided on the intimate chemical mechanisms that can make some bonds prone to cleavage due to the presence of specific side-chains. In addition to primary structure, the secondary, tertiary and quaternary structures have a significant impact in modulating the distribution of cleavage sites by altering local flexibility, accessibility to solvent or bringing in close proximity side chains that are remote in sequence. This review focuses on cleavage sites observed in the constant regions of mAbs, with special emphasis on hinge fragmentation. The mechanisms responsible for backbone cleavage are strongly dependent on pH and can be catalyzed by metals or radicals. The distribution of cleavage sites are different under acidic compared to basic conditions, with fragmentation rates exhibiting a minimum in the pH range 5–6; therefore, the overall fragmentation pattern observed for a mAb is a complex result of structural and solvent conditions. A critical review of the techniques used to monitor fragmentation is also presented; usually a compromise has to be made between a highly sensitive method with good fragment separation and the capability to identify the cleavage site. The effect of fragmentation on the function of a mAb must be evaluated on a case-by-case basis depending on whether cleavage sites are observed in the variable or constant regions, and on the mechanism of action of the molecule. PMID:21487244

  10. Expression of anti-tumor necrosis factor alpha (TNFα) single-chain variable fragment (scFv) in Spirodela punctata plants transformed with Agrobacterium tumefaciens.

    PubMed

    Balaji, Parthasarathy; Satheeshkumar, P K; Venkataraman, Krishnan; Vijayalakshmi, M A

    2016-05-01

    Therapeutic antibodies against tumor necrosis factor alpha (TNFα) have been considered effective for some of the autoimmune diseases such as rheumatoid arthritis, Crohn's diseases, and so on. But associated limitations of the current therapeutics in terms of cost, availability, and immunogenicity have necessitated the need for alternative candidates. Single-chain variable fragment (scFv) can negate the limitations tagged with the anti-TNFα therapeutics to a greater extent. In the present study, Spirodela punctata plants were transformed with anti-TNFα through in planta transformation using Agrobacterium tumefaciens strain, EHA105. Instead of cefotaxime, garlic extract (1 mg/mL) was used to remove the agrobacterial cells after cocultivation. To the best of our knowledge, this report shows for the first time the application of plant extracts in transgenic plant development. 95% of the plants survived screening under hygromycin. ScFv cDNA integration in the plant genomic DNA was confirmed at the molecular level by PCR. The transgenic protein expression was followed up to 10 months. Expression of scFv was confirmed by immunodot blot. Protein expression levels of up to 6.3% of total soluble protein were observed. β-Glucuronidase and green fluorescent protein expressions were also detected in the antibiotic resistant plants. The paper shows the generation of transgenic Spirodela punctuata plants through in planta transformation.

  11. Selection of single chain variable fragments (scFv) against the glycoprotein antigen of the rabies virus from a human synthetic scFv phage display library and their fusion with the Fc region of human IgG1

    PubMed Central

    Ray, K; Embleton, M J; Jailkhani, B L; Bhan, M K; Kumar, R

    2001-01-01

    We have prepared human recombinant antibody molecules against the glycoprotein antigen of the rabies virus (GPRV) based on the single chain variable fragment (scFv) format. Anti-GPRV scFvs were selected from a human synthetic scFv phage display library with a repertoire of approximately 109 specificities. After three rounds of selection against the PV11 strain of the virus, 40% of the clones tested recognized the rabies antigen. Of the 20 positive clones that were sequenced, five distinct sequences were identified. These distinct scFvs were cloned into a mammalian expression vector carrying the human IgG1 Fc region. The specificity of the resulting scFv-Fc molecules for GPRV was established by ELISA, dot blot and western blot analyses and membrane immunofluorescence. Two of the scFv-Fc fusion proteins neutralized the PV11 strain in a standard in vivo neutralization assay where the virus was incubated with the scFv-Fc molecules before intracranial inoculation in mice. These anti-GPRV scFv-Fc molecules have the potential to be used as an alternative to the presently available HRIG, for use in post-exposure preventive treatment. PMID:11472431

  12. Isolation of human single chain variable fragment antibodies against specific sperm antigens for immunocontraceptive development

    PubMed Central

    Samuel, A.S.; Naz, R.K.

    2008-01-01

    BACKGROUND Contraceptive vaccines can provide valuable alternatives to current methods of contraception. We describe here the development of sperm-reactive human single chain variable fragment (scFv) antibodies of defined sperm specificity for immunocontraception. METHODS Peripheral blood leukocytes (PBL) from antisperm antibody-positive immunoinfertile and vasectomized men were activated with human sperm antigens in vitro, and the complementary DNA prepared and PCR-amplified using primers based on all the variable regions of heavy and light chains of immunoglobulins. The scFv repertoire was cloned into pCANTAB5E vector to create a human scFv antibody library. RESULTS Panning of the library against specific sperm antigens yielded several clones, and the four strongest reactive were selected for further analysis. These clones had novel sequences with unique complementarity-determining regions. ScFv antibodies were expressed, purified and analyzed for human sperm reactivity and effect on human sperm function. AFA-1 and FAB-7 scFv antibodies both reacted with fertilization antigen-1 antigen, but against different epitopes. YLP20 antibody reacted with the expected human sperm protein of 48 ± 5 kDa. The fourth antibody, AS16, reacted with an 18 kDa sperm protein and seems to be a human homologue of the mouse monoclonal recombinant antisperm antibody that causes sperm agglutination. All these antibodies inhibited human sperm function. CONCLUSIONS This is the first study to report the use of phage display technology to obtain antisperm scFv antibodies of defined antigen specificity. These antibodies will find clinical applications in the development of novel immunocontraceptives, and specific diagnostics for immunoinfertility. PMID:18372255

  13. The Development of a Recombinant scFv Monoclonal Antibody Targeting Canine CD20 for Use in Comparative Medicine.

    PubMed

    Jain, Saurabh; Aresu, Luca; Comazzi, Stefano; Shi, Jianguo; Worrall, Erin; Clayton, John; Humphries, William; Hemmington, Sandra; Davis, Paul; Murray, Euan; Limeneh, Asmare A; Ball, Kathryn; Ruckova, Eva; Muller, Petr; Vojtesek, Borek; Fahraeus, Robin; Argyle, David; Hupp, Ted R

    2016-01-01

    Monoclonal antibodies are leading agents for therapeutic treatment of human diseases, but are limited in use by the paucity of clinically relevant models for validation. Sporadic canine tumours mimic the features of some human equivalents. Developing canine immunotherapeutics can be an approach for modeling human disease responses. Rituximab is a pioneering agent used to treat human hematological malignancies. Biologic mimics that target canine CD20 are just being developed by the biotechnology industry. Towards a comparative canine-human model system, we have developed a novel anti-CD20 monoclonal antibody (NCD1.2) that binds both human and canine CD20. NCD1.2 has a sub-nanomolar Kd as defined by an octet red binding assay. Using FACS, NCD1.2 binds to clinically derived canine cells including B-cells in peripheral blood and in different histotypes of B-cell lymphoma. Immunohistochemical staining of canine tissues indicates that the NCD1.2 binds to membrane localized cells in Diffuse Large B-cell lymphoma, Marginal Zone Lymphoma, and other canine B-cell lymphomas. We cloned the heavy and light chains of NCD1.2 from hybridomas to determine whether active scaffolds can be acquired as future biologics tools. The VH and VL genes from the hybridomas were cloned using degenerate primers and packaged as single chains (scFv) into a phage-display library. Surprisingly, we identified two scFv (scFv-3 and scFv-7) isolated from the hybridoma with bioactivity towards CD20. The two scFv had identical VH genes but different VL genes and identical CDR3s, indicating that at least two light chain mRNAs are encoded by NCD1.2 hybridoma cells. Both scFv-3 and scFv-7 were cloned into mammalian vectors for secretion in CHO cells and the antibodies were bioactive towards recombinant CD20 protein or peptide. The scFv-3 and scFv-7 were cloned into an ADEPT-CPG2 bioconjugate vector where bioactivity was retained when expressed in bacterial systems. These data identify a recombinant anti-CD20

  14. The Development of a Recombinant scFv Monoclonal Antibody Targeting Canine CD20 for Use in Comparative Medicine

    PubMed Central

    Jain, Saurabh; Aresu, Luca; Comazzi, Stefano; Shi, Jianguo; Worrall, Erin; Clayton, John; Humphries, William; Hemmington, Sandra; Davis, Paul; Murray, Euan; Limeneh, Asmare A.; Ball, Kathryn; Ruckova, Eva; Muller, Petr; Vojtesek, Borek; Fahraeus, Robin; Argyle, David; Hupp, Ted R.

    2016-01-01

    Monoclonal antibodies are leading agents for therapeutic treatment of human diseases, but are limited in use by the paucity of clinically relevant models for validation. Sporadic canine tumours mimic the features of some human equivalents. Developing canine immunotherapeutics can be an approach for modeling human disease responses. Rituximab is a pioneering agent used to treat human hematological malignancies. Biologic mimics that target canine CD20 are just being developed by the biotechnology industry. Towards a comparative canine-human model system, we have developed a novel anti-CD20 monoclonal antibody (NCD1.2) that binds both human and canine CD20. NCD1.2 has a sub-nanomolar Kd as defined by an octet red binding assay. Using FACS, NCD1.2 binds to clinically derived canine cells including B-cells in peripheral blood and in different histotypes of B-cell lymphoma. Immunohistochemical staining of canine tissues indicates that the NCD1.2 binds to membrane localized cells in Diffuse Large B-cell lymphoma, Marginal Zone Lymphoma, and other canine B-cell lymphomas. We cloned the heavy and light chains of NCD1.2 from hybridomas to determine whether active scaffolds can be acquired as future biologics tools. The VH and VL genes from the hybridomas were cloned using degenerate primers and packaged as single chains (scFv) into a phage-display library. Surprisingly, we identified two scFv (scFv-3 and scFv-7) isolated from the hybridoma with bioactivity towards CD20. The two scFv had identical VH genes but different VL genes and identical CDR3s, indicating that at least two light chain mRNAs are encoded by NCD1.2 hybridoma cells. Both scFv-3 and scFv-7 were cloned into mammalian vectors for secretion in CHO cells and the antibodies were bioactive towards recombinant CD20 protein or peptide. The scFv-3 and scFv-7 were cloned into an ADEPT-CPG2 bioconjugate vector where bioactivity was retained when expressed in bacterial systems. These data identify a recombinant anti-CD20

  15. 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.

  16. The protective effects and underlying mechanism of an anti-oligomeric Aβ42 single-chain variable fragment antibody.

    PubMed

    Zhang, Yuan; Chen, Xu; Liu, Jinyu; Zhang, Yingjiu

    2015-12-01

    Oligomeric Aβ42 aggregates have been identified as one of the major neurotoxic components of Alzheimer's disease (AD). Immunotherapy targeted against these Aβ42 aggregates has been proposed as an appropriate therapeutic approach for the treatment of AD. Here, we report an anti-oligomeric Aβ42 single-chain variable fragment (scFv) antibody, named MO6, obtained from the human antibody library of a healthy donor. ScFv MO6 specifically recognized and bound to the oligomeric Aβ42 (Aβ42 oligomers and immature protofibrils; 18-37 kDa), and reduced their levels mainly by blocking their formation, although scFv MO6 also induced disaggregation of Aβ42 aggregates. More importantly, scFv MO6 ameliorated or attenuated Aβ42-induced cytotoxicity and increased cell viability by up to 33%. Furthermore, scFv MO6 efficiently passed through an in vitro blood-brain barrier (BBB) model with a delivery efficiency of 66% after 60 min post-administration. ScFv MO6 is a monovalent antibody with an affinity constant (KD) of 5.2×10(-6) M for Aβ42 oligomers. Molecular docking simulations of Aβ42 to scFv MO6 revealed that the approach and specific binding of scFv MO6 to oligomeric Aβ42 aggregates was achieved by conformational recognition and directed induction, which resulted in a more dynamic adaptation of Aβ42 to scFv MO6, occurring mainly in the N-terminal (3-4), middle (12-19) and C-terminal (34-42) regions of Aβ42. This binding mode of scFv MO6 to Aβ42 explains its protective effects against oligomeric Aβ42. Our findings may be applied for the design of a smaller antibody specific for Aβ42 oligermers.

  17. Secretion of an immunoreactive single-chain variable fragment antibody against mouse interleukin 6 by Lactococcus lactis.

    PubMed

    Shigemori, Suguru; Ihara, Masaki; Sato, Takashi; Yamamoto, Yoshinari; Nigar, Shireen; Ogita, Tasuku; Shimosato, Takeshi

    2017-01-01

    Interleukin 6 (IL-6) is an important pathogenic factor in development of various inflammatory and autoimmune diseases and cancer. Blocking antibodies against molecules associated with IL-6/IL-6 receptor signaling are an attractive candidate for the prevention or therapy of these diseases. In this study, we developed a genetically modified strain of Lactococcus lactis secreting a single-chain variable fragment antibody against mouse IL-6 (IL6scFv). An IL6scFv-secretion vector was constructed by cloning an IL6scFv gene fragment into a lactococcal secretion plasmid and was electroporated into L. lactis NZ9000 (NZ-IL6scFv). Secretion of recombinant IL6scFv (rIL6scFv) by nisin-induced NZ-IL6scFv was confirmed by western blotting and was optimized by tuning culture conditions. We found that rIL6scFv could bind to commercial recombinant mouse IL-6. This result clearly demonstrated the immunoreactivity of rIL6scFv. This is the first study to engineer a genetically modified strain of lactic acid bacteria (gmLAB) that produces a functional anti-cytokine scFv. Numerous previous studies suggested that mucosal delivery of biomedical proteins using gmLAB is an effective and low-cost way to treat various disorders. Therefore, NZ-IL6scFv may be an attractive tool for the research and development of new IL-6 targeting agents for various inflammatory and autoimmune diseases as well as for cancer.

  18. Blocking monocyte transmigration in in vitro system by a human antibody scFv anti-CD99. Efficient large scale purification from periplasmic inclusion bodies in E. coli expression system.

    PubMed

    Moricoli, Diego; Muller, William Anthony; Carbonella, Damiano Cosimo; Balducci, Maria Cristina; Dominici, Sabrina; Watson, Richard; Fiori, Valentina; Weber, Evan; Cianfriglia, Maurizio; Scotlandi, Katia; Magnani, Mauro

    2014-06-01

    Migration of leukocytes into 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 and the absence of toxic reagents utilized for solubilization and refolding step 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 periplasmic 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 signaling. This protocol can be useful for the successful purification of other monomeric scFvs which are expressed as periplasmic inclusion bodies in bacterial systems.

  19. Suppression of Aggrus/podoplanin-induced platelet aggregation and pulmonary metastasis by a single-chain antibody variable region fragment.

    PubMed

    Miyata, Kenichi; Takagi, Satoshi; Sato, Shigeo; Morioka, Hiroshi; Shiba, Kiyotaka; Minamisawa, Tamiko; Takami, Miho; Fujita, Naoya

    2014-12-01

    Almost all highly metastatic tumor cells possess high platelet aggregating abilities, thereby form large tumor cell-platelet aggregates in the microvasculature. Embolization of tumor cells in the microvasculature is considered to be the first step in metastasis to distant organs. We previously identified the platelet aggregation-inducing factor expressed on the surfaces of highly metastatic tumor cells and named as Aggrus. Aggrus was observed to be identical to the marker protein podoplanin (alternative names, T1α, OTS-8, and others). Aggrus is frequently overexpressed in several types of tumors and enhances platelet aggregation by interacting with the platelet receptor C-type lectin-like receptor 2 (CLEC-2). Here, we generated a novel single-chain antibody variable region fragment (scFv) by linking the variable regions of heavy and light chains of the neutralizing anti-human Aggrus monoclonal antibody MS-1 with a flexible peptide linker. Unfortunately, the generated KM10 scFv failed to suppress Aggrus-induced platelet aggregation in vitro. Therefore, we performed phage display screening and finally obtained a high-affinity scFv, K-11. K-11 scFv was able to suppress Aggrus-induced platelet aggregation in vitro. Moreover, K-11 scFv prevented the formation of pulmonary metastasis in vivo. These results suggest that K-11 scFv may be useful as metastasis inhibitory scFv and is expected to aid in the development of preclinical and clinical examinations of Aggrus-targeted cancer therapies.

  20. Optimization of a single-chain antibody fragment overexpression in Escherichia coli using response surface methodology

    PubMed Central

    Akbari, V.; Sadeghi, H. Mir Mohammad; Jafarian-Dehkordi, A.; Chou, C. Perry; Abedi, D.

    2015-01-01

    Human epidermal growth factor receptor (HER) family plays an important role in various types of cancers. As a result, antibodies against HER and the mechanism of antigen-antibody binding action are under active investigation. We previously constructed a single-chain variable fragment (ScFv) against HER2, i.e. anti-Her2 ScFv, for expressing in the Escherichia coli. In the present study, we report the optimization of anti-Her2 ScFv expression in an E. coli host of BL21 (DE3) pLysS using response surface methodology based on tuning of three cultivation variables, including isopropyl-beta-D-thiogalactopyranoside (IPTG) concentration, temperature and post-induction time. A model for protein expression according to the Box-Behnken design predicted a maximal anti-Her2 ScFv expression at 37 °C, a post-induction time of 10.45 h and 0.75 mM IPTG. In addition, strategies based on inclusion body isolation and affinity chromatography were applied to purify anti-Her2 ScFv. The purity of the final product for inclusion bodies isolation and purification by Ni-NTA resin were 70 % and 95 %, respectively. The solubilization of the inclusion bodies was carried out using two denaturant agents, guanidine hydrochloride and urea. The present study showed that guanidine hydrochloride was more effective than urea in solubilizing the inclusion bodies. PMID:26430460

  1. A novel human recombinant antibody fragment capable of neutralizing Mexican scorpion toxins.

    PubMed

    Riaño-Umbarila, Lidia; Olamendi-Portugal, Timoteo; Morelos-Juárez, Citlalli; Gurrola, Georgina B; Possani, Lourival D; Becerril, Baltazar

    2013-12-15

    Using phage display and directed evolution, our group has progressed in the construction of a second family of human single chain variable fragments (scFv) which bind to scorpion toxins dangerous to mammals. It was observed that scFv C1 only bound initially to toxin Cn2, which constitutes 6.8% of whole venom from the scorpion Centruroides noxius Hoffman. Only a few amino acid changes were necessary to extend its recognition to other similar toxins and without affecting the recognition for its primary antigen (Cn2 toxin). One variant of scFv C1 (scFv 202F) was selected after two cycles of directed evolution against Cll1 toxin, the second major toxic component from the venom of the Mexican scorpion Centruroides limpidus limpidus Karsh (0.5% of the whole venom). scFv 202F is also capable of recognizing Cn2 toxin. Despite not having the highest affinity for toxins Cll1 (KD = 25.1 × 10(-9) M) or Cn2 (KD = 8.1 × 10(-9) M), this antibody fragment neutralized one LD50 of each one of these toxins. Additionally, scFv 202F moderately recognized Cll2 toxin which constitutes 1.5% of the venom from C. limpidus. Based on our previous experience, we consider that these results are promising; consequently, we continue working on generating new optimized variants from scFv C1 that could be part of a recombinant scorpion anti-venom from human origin, that might reach the market in the near future.

  2. Construction of single-chain variable fragment antibodies against MCF-7 breast cancer cells.

    PubMed

    Zuhaida, A A; Ali, A M; Tamilselvan, S; Alitheen, N B; Hamid, M; Noor, A M; Yeap, S K

    2013-11-18

    A phage display library of single chain variable fragment (scFv) against MCF-7 breast cancer cells was constructed from C3A8 hybridoma cells. RNA from the C3A8 was isolated, cDNA was constructed, and variable heavy and light immunoglobulin chain gene region were amplified using PCR. The variable heavy and light chain gene regions were combined with flexible linker, linked to a pCANTAB 5E phagemid vector and electrophoresed into supE strain of Escherichia coli TG1 cells. Forty-eight clones demonstrated positive binding activity to MCF-7 breast cancer cell membrane fragments and the strongest of 48 clones was selected for analysis. The anti-MCF-7 library evaluated by SfiI and NotI digests demonstrated that anti-MCF-7 scFv antibodies possess individual patterns that should be able to recognize distinct human breast cancer cells. The C3A8 scFv, with an apparent molecular weight of 32 kDa, showed high homology (99%) with single chain antibody against rice stripe virus protein P20. In summary, the anti MCF-7 scFv antibody can be used for pretargeting breast cancer for clinical diagnosis of patients; it also has potential for therapeutic applications.

  3. Isolation and characterization of recombinant single chain fragment variable anti-idiotypic antibody specific to Aspergillus fumigatus membrane protein.

    PubMed

    Krishnaswamy, Senthilkumar; Kabir, M Enamul; Rahman, M Mamunur; Miyamoto, Masahiko; Furuichi, Yasuhiro; Komiyama, Tadazumi

    2011-03-07

    Aspergillus fumigatus causes the highly lethal form of invasive aspergillosis (IA). In the present study to develop a novel anti-fungal drug for protection against invasive disease, we identified a single chain fragment variable (scFv) antibody (scFv AF1) by panning against A. fumigatus membrane fraction (AMF) or HM-1 killer toxin (HM-1) neutralizing monoclonal antibody (nmAb-KT) as antigen. The key step was elution of bound phages with phosphate buffered saline (PBS) at pH 7.0 containing AMF. The specificity of soluble scFv AF1 antibody to antigens was verified by ELISA, which specifically binds to both AMF and nmAb-KT. After nucleotide sequencing, clone expression and purification by HisTrap HP affinity column, scFv AF1 showed in vitro anti-fungal activity against A. fumigatus. By SPR analysis it showed high binding affinity to nmAb-KT (K(d)=5.22×10(-11) M). The method used to isolate scFv AF1 was a new method and we believe that it will be applicable to isolate the specific scFv against any kind of membrane protein of yeast or fungus.

  4. Flow cytometry-based methods for assessing soluble scFv activities and detecting pathogen antigens in solution

    SciTech Connect

    Gray, Sean; Weigel, Kris M.; Miller, Keith D.; Ndung'u, Joseph; Buscher, Philippe; Tran, Thao N.; Baird, Cheryl L.; Cangelosi, Gerard A.

    2010-04-01

    Novel methods are reported for evaluating and utilizing single chain fragment variable (scFv) antibodies derived from yeast-display libraries. Yeast-display was used to select scFv specific to invariant surface glycoproteins (ISG) of Trypanosoma brucei. A limiting step in the isolation of scFv from nonimmune libraries is the conversion of highly active yeast-displayed scFv into soluble antibodies that can be used in standard immunoassays. Challenges include limited solubility or activity following secretion and purification of scFv. For this reason, few scFv derived from yeast-display platforms have moved into development and implementation as diagnostic reagents. To address this problem, assays were developed that employ both yeastdisplayed and secreted scFv as analytical reagents. The first is a competitive inhibition flow cytometry (CIFC) assay that detects secreted scFv by virtue of its ability to competitively inhibit the binding of biotinylated antigen to yeast-displayed scFv. The second is an epitope binning assay that uses secreted scFv toidentify additional yeast-displayed scFv that bind nonoverlapping or noncompeting epitopes on an antigen. The epitope binning assay was used not only to identify sandwich assay pairs with yeast-displayed scFv, but also to identify active soluble scFv present in low concentration in a crude expression extract. Finally, a CIFC assay was developed that bypasses entirely the need for soluble scFv expression, by using yeast displayed scFv to detect unlabeled antigen in samples. These methods will facilitate the continued development and practical implementation of scFv derived from yeast-display libraries.

  5. Approach for Half-Life Extension of Small Antibody Fragments That Does Not Affect Tissue Uptake.

    PubMed

    Schneider, Eric L; Hearn, Brian R; Pfaff, Samuel J; Fontaine, Shaun D; Reid, Ralph; Ashley, Gary W; Grabulovski, Stefanie; Strassberger, Verena; Vogt, Lorenz; Jung, Thomas; Santi, Daniel V

    2016-10-07

    The utility of antigen-binding antibody fragments is often limited by their short half-lives. Half-life extension of such fragments is usually accomplished by attachment or binding to high-molecular-weight carriers that reduce the renal elimination rate. However, the higher hydrodynamic radius results in greater confinement in the vascular compartment and, thus, lower tissue distribution. We have developed a chemically controlled drug delivery system in which the drug is covalently attached to hydrogel microspheres by a self-cleaving β-eliminative linker; upon subcutaneous injection, the t1/2,β of the released drug acquires the t1/2 of linker cleavage. In the present work, we compared the pharmacokinetics of an anti-TNFα scFv, the same scFv attached to 40 kDa PEG by a stable linker, and the scFv attached to hydrogel microspheres by a self-cleaving linker. We also developed a general approach for the selective attachment of β-eliminative linkers to the N-termini of proteins. In rats, the scFv had a t1/2,β of 4 h and a high volume of distribution at steady state (Vd,SS), suggesting extensive tissue distribution. The PEG-scFv conjugate had an increased t1/2,β of about 2 days but showed a reduced Vd,SS that was similar to the plasma volume. In contrast, the tissue-penetrable scFv released from the hydrogel system had a t1/2,β of about 2 weeks. Thus, the cleavable microsphere-scFv conjugate releases its protein cargo with a prolonged half-life comparable to that of most full-length mAbs and in a form that has the high tissue distribution characteristic of smaller mAb fragments. Other antigen-binding antibody fragments should be amenable to the half-life extension approach described here.

  6. Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV)

    PubMed Central

    Kirsch, Martina Inga; Hülseweh, Birgit; Nacke, Christoph; Rülker, Torsten; Schirrmann, Thomas; Marschall, Hans-Jürgen; Hust, Michael; Dübel, Stefan

    2008-01-01

    Background Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus group. Several species of this family are also pathogenic to humans and are recognized as potential agents of biological warfare and terrorism. The objective of this work was the generation of recombinant antibodies for the detection of VEEV after a potential bioterrorism assault or an natural outbreak of VEEV. Results In this work, human anti-VEEV single chain Fragments variable (scFv) were isolated for the first time from a human naïve antibody gene library using optimized selection processes. In total eleven different scFvs were identified and their immunological specificity was assessed. The specific detection of the VEEV strains TC83, H12/93 and 230 by the selected antibody fragments was proved. Active as well as formalin inactivated virus particles were recognized by the selected antibody fragments which could be also used for Western blot analysis of VEEV proteins and immunohistochemistry of VEEV infected cells. The anti-VEEV scFv phage clones did not show any cross-reactivity with Alphavirus species of the Western equine encephalitis virus (WEEV) and Eastern equine encephalitis virus (EEEV) antigenic complex, nor did they react with Chikungunya virus (CHIKV), if they were used as detection reagent. Conclusion For the first time, this study describes the selection of antibodies against a human pathogenic virus from a human naïve scFv antibody gene library using complete, active virus particles as antigen. The broad and sensitive applicability of scFv-presenting phage for the immunological detection and diagnosis of Alphavirus species was demonstrated. The selected antibody fragments will improve the fast identification of VEEV in case of a biological warfare or terroristic attack or a natural outbreak. PMID:18764933

  7. Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system

    PubMed Central

    Yin, Gang; Garces, Eudean D; Yang, Junhao; Zhang, Juan; Tran, Cuong; Steiner, Alexander R; Roos, Christine; Bajad, Sunil; Hudak, Susan; Penta, Kalyani; Zawada, James; Pollitt, Sonia

    2012-01-01

    We describe protein synthesis, folding and assembly of antibody fragments and full-length aglycosylated antibodies using an Escherichia coli-based open cell-free synthesis (OCFS) system. We use DNA template design and high throughput screening at microliter scale to rapidly optimize production of single-chain Fv (scFv) and Fab antibody fragments that bind to human IL-23 and IL-13α1R, respectively. In addition we demonstrate production of aglycosylated immunoglobulin G (IgG1) trastuzumab. These antibodies are produced rapidly over several hours in batch mode in standard bioreactors with linear scalable yields of hundreds of milligrams/L over a 1 million-fold change in scales up to pilot scale production. We demonstrate protein expression optimization of translation initiation region (TIR) libraries from gene synthesized linear DNA templates, optimization of the temporal assembly of a Fab from independent heavy chain and light chain plasmids and optimized expression of fully assembled trastuzumab that is equivalent to mammalian expressed material in biophysical and affinity based assays. These results illustrate how the open nature of the cell-free system can be used as a seamless antibody engineering platform from discovery to preclinical development of aglycosylated monoclonal antibodies and antibody fragments as potential therapeutics. PMID:22377750

  8. Ligation-based assembly for constructing mouse synthetic scFv libraries by chain shuffling with in vivo-amplified VH and VL fragments.

    PubMed

    Nishi, Michiru; Jian, Nan; Yamamoto, Keiko; Seto, Haruyo; Nishida, Yuichi; Tonoyama, Yasuhiro; Shimizu, Nobuyoshi; Nishi, Yoshisuke

    2014-10-01

    In vitro assembly of two or three PCR fragments using primers is a common method of constructing scFv fragments for display on the surface of phage. However, mismatch annealing often occurs during in this step, leading to cloning and display of incomplete Fab or scFv fragments. To overcome this limitation, we developed a ligation-based two-fragment assembly (LTFA) protocol that involved separately cloning VH and Vκ fragments into the high-copy-number plasmid pUC18. The VH and Vκ fragments had randomized complementarity-determining region 3 (CDR3) and were joined with a peptidyl linker composed of (G4S)3. Using this approach, complete sequences of scFv fragments were successfully constructed, and the sequencing of 83 scFv clones revealed that none of the sequences, including the linker region, contained deletions or mutations. In contrast, linker sequences generated using a conventional two-fragment PCR assembly (TFPA) protocol often contained sequence anomalies, including large truncations. Using the LTFA protocol, a final library size of 1.0×10(8)cfu was achieved. Examination of the amino acid profiles of the generated scFv fragments within the randomized regions introduced using degenerate codons did not detect any bias from that expected based on stochastic distribution. After several cycles of panning with this library, antigen-specific scFvs against two reference antigens, hen egg lysozyme and streptavidin were detected. In addition, scFvs with specificity against peptidyl antigens in the loop region of the Medaka ortholog of human C6orf89, which encodes a histone deacetylase enhancer that interacts with the bombesin receptor, were also obtained. The LTFA protocol developed here is robust and allows for the easy construction of integral scFv fragments compared with conventional TFPA. Utilizing LTFA, other CDRs can be readily combined. This approach also allows for the in vitro maturation of scFv fragments by separately introducing randomization in CDRs or

  9. Expression and purification of a novel therapeutic single-chain variable fragment antibody against BNP from inclusion bodies of Escherichia coli.

    PubMed

    Bu, Dawei; Zhou, Yuwei; Tang, Jian; Jing, Fang; Zhang, Wei

    2013-12-01

    Abnormal brain natriuretic peptide (BNP) secretion is regarded as the dominating mechanism of cerebral salt wasting syndrome (CSW), which results from a renal loss of sodium and water during intracranial disease leading to hyponatremia. Scale preparation of therapeutic single-chain variable fragment (scFv) that can neutralize elevated circulating BNP may have potential value for clinical use. In this report, we used a recently isolated humanized anti-BNP scFv fragment (3C1) as model antibody (Ab) to evaluate the potential of scale production of this therapeutic protein. The truncated gene encoding for scFv fragment cloned in pET22b (+) was mainly overexpressed as inclusion bodies in Escherichia coli (E. coli) Rosetta (DE3) pLysS cells. The insoluble fragment was solubilized and purified by Ni-NTA agarose resin under denaturation conditions, and recovered via an effective refolding buffer containing 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM EDTA, 0.5 M arginine, 2 mM GSH, 1 mM GSSG, and 5% glycerol. The refolded scFv fragment was concentrated by PEG20000, and dialyzed in PBS (containing 5% glycerol, pH 7.4). The final yield was approximately 10.2 mg active scFv fragment per liter of culture (3.4 g wet weight cells). The scFv fragment was more than 95% pure assessed by SDS-PAGE assay. Recombinant scFv fragment with His tag displayed its immunoreactivity with anti-His tag Ab by western blotting. ELISA showed the scFv fragment specifically bound to BNP, and it displayed similar activity as the traditional anti-BNP monoclonal Ab (mAb). Thus, the current strategy allows convenient small-scale production of this therapeutic protein.

  10. Neutralizing human recombinant antibodies against herpes simplex virus type 1 glycoproteins B from a phage-displayed scFv antibody library.

    PubMed

    Bagheri, Vahid; Nejatollahi, Foroogh; Esmaeili, Seyed Alireza; Momtazi, Amir Abbas; Motamedifar, Mohamad; Sahebkar, Amirhossein

    2017-01-15

    The HSV-1 envelope glycoprotein B (gB) plays a critical role in virus entry into host cells. Neutralizing antibodies can therefore potentially prevent virus entry into target cells and cell-to-cell spread of infection. Our present study focused on the selection of neutralizing single-chain Fv (scFv) antibodies of a phage-displayed nonimmune human scFv antibody library against gB of HSV-1. To enrich specific scFvs, two phage antibodies were isolated against amino acid residues 31-43 derived from the N-terminal part of gB using panning technique. Two scFvs, scFv-gB1 and scFv-gB2, with frequencies of 45% and 20% were obtained from scFv clones after performing PCR and MvaI fingerprinting. In phage ELISA analysis, both gB1 and gB2 scFvs demonstrated high reactivity with the gB peptide. In the neutralization assay, scFv-gB1 and scFv-gB2 represented neutralizing effects of 55% and 59%, respectively. Upon further enhancement of the neutralizing effects of these antibodies, they can be considered as new potential alternatives in the treatment and prophylaxis of HSV-1 infections.

  11. Human monomeric antibody fragments to TRAIL-R1 and TRAIL-R2 that display potent in vitro agonism

    PubMed Central

    Main, Sarah; Newton, Philip; Chodorge, Matthieu; Cadwallader, Karen; Humphreys, Robin; Albert, Vivian; Vaughan, Tristan J; Minter, Ralph R; Edwards, Bryan M

    2009-01-01

    Apoptosis through the TRAIL receptor pathway can be induced via agonistic IgG to either TRAIL-R1 or TRAIL-R2. Here we describe the use of phage display to isolate a substantive panel of fully human anti-TRAIL receptor single chain Fv fragments (scFvs); 234 and 269 different scFvs specific for TRAIL-R1 and TRAIL-R2 respectively. In addition, 134 different scFvs that were cross-reactive for both receptors were isolated. To facilitate screening of all 637 scFvs for potential agonistic activity in vitro, a novel high-throughput surrogate apoptosis assay was developed. Ten TRAIL-R1 specific scFv and 6 TRAIL-R2 specific scFv were shown to inhibit growth of tumor cells in vitro in the absence of any cross-linking agents. These scFv were all highly specific for either TRAIL-R1 or TRAIL-R2, potently inhibited tumor cell proliferation, and were antagonists of TRAIL binding. Moreover, further characterization of TRAIL-R1 agonistic scFv demonstrated significant anti-tumor activity when expressed and purified as a monomeric Fab fragment. Thus, scFv and Fab fragments, in addition to whole IgG, can be agonistic and induce tumor cell death through specific binding to either TRAIL-R1 or TRAIL-R2. These potent agonistic scFv were all isolated directly from the starting phage antibody library and demonstrated significant tumor cell killing properties without any requirement for affinity maturation. Some of these selected scFv have been converted to IgG format and are being studied extensively in clinical trials to investigate their potential utility as human monoclonal antibody therapeutics for the treatment of human cancer. PMID:20068388

  12. Stability engineering of anti-EGFR scFv antibodies by rational design of a lambda-to-kappa swap of the VL framework using a structure-guided approach.

    PubMed

    Lehmann, Andreas; Wixted, Josephine H F; Shapovalov, Maxim V; Roder, Heinrich; Dunbrack, Roland L; Robinson, Matthew K

    2015-01-01

    Phage-display technology facilitates rapid selection of antigen-specific single-chain variable fragment (scFv) antibodies from large recombinant libraries. ScFv antibodies, composed of a VH and VL domain, are readily engineered into multimeric formats for the development of diagnostics and targeted therapies. However, the recombinant nature of the selection strategy can result in VH and VL domains with sub-optimal biophysical properties, such as reduced thermodynamic stability and enhanced aggregation propensity, which lead to poor production and limited application. We found that the C10 anti-epidermal growth factor receptor (EGFR) scFv, and its affinity mutant, P2224, exhibit weak production from E. coli. Interestingly, these scFv contain a fusion of lambda3 and lambda1 V-region (LV3 and LV1) genes, most likely the result of a PCR aberration during library construction. To enhance the biophysical properties of these scFvs, we utilized a structure-based approach to replace and redesign the pre-existing framework of the VL domain to one that best pairs with the existing VH. We describe a method to exchange lambda sequences with a more stable kappa3 framework (KV3) within the VL domain that incorporates the original lambda DE-loop. The resulting scFvs, C10KV3_LV1DE and P2224KV3_LV1DE, are more thermodynamically stable and easier to produce from bacterial culture. Additionally, C10KV3_LV1DE and P2224KV3_LV1DE retain binding affinity to EGFR, suggesting that such a dramatic framework swap does not significantly affect scFv binding. We provide here a novel strategy for redesigning the light chain of problematic scFvs to enhance their stability and therapeutic applicability.

  13. Secretory production of single-chain antibody (scFv) in Brevibacillus choshinensis using novel fusion partner.

    PubMed

    Tokunaga, Masao; Mizukami, Makoto; Yamasaki, Koji; Tokunaga, Hiroko; Onishi, Hiromasa; Hanagata, Hiroshi; Ishibashi, Matsujiro; Miyauchi, Akira; Tsumoto, Kouhei; Arakawa, Tsutomu

    2013-10-01

    Halophilic β-lactamase (BLA) has been successfully used as a novel fusion partner for soluble expression of aggregation-prone foreign proteins in Escherichia coli cytoplasm (Appl Microbiol Biotechnol 86:649-658, 2010b). This halophilic BLA fusion technology was applied here for secretory expression in Brevibacillus. The "Brevibacillus in vivo cloning" method, recently developed by Higeta Shoyu group, for the construction and transformation of Brevibacillus expression vectors facilitates efficient screening of the production conditions of Brevibacillus expression system. Two single-chain antibodies (scFv), HyHEL-10 single chain scFv (scFvHEL) and anti-fluorescein single chain scFv (scFvFLU), were successfully secreted to culture supernatant as a fusion protein with halophilic BLA. The scFvHEL-His, purified after cleavage of BLA portion with thrombin, was fully active: it formed a stoichiometric complex with the antigen, lysozyme, and inhibited the enzymatic activity. The scFvFLU-His, similarly expressed and purified, stoichiometrically inhibited fluorescence intensity of fluorescein. The molecular mass of scFvHEL-His was determined to be 27,800 Da by light scattering measurements, indicating its monomeric structure in solution.

  14. Phage display library selection of a hypoxia-binding scFv antibody for liver cancer metabolic marker discovery

    PubMed Central

    Chen, Hang; Gao, Zhihui; Li, Yao; Sun, Zhongyuan; Xiang, Rong; Zhang, Sihe

    2016-01-01

    Hypoxia, which is frequently observed in liver cancer and metastasis, influences tumor progression and resistance to therapy. Although hypoxia-associated biomarkers are of use in other cancers, none is recognized as a surrogate for hypoxia in liver cancer. In this study, we generated seven unique human single-chain Fv (scFv) antibodies (Abs) specific to hypoxic liver cancer cells, using normoxia-depleted vs hypoxia-selected phage library panning technology. By developing the scFv immunoprecipitation-based mass spectrometry method, the antigen that bound with one of the Abs (H103) was identified as the M2 splice isoform of pyruvate kinase (PKM2), an enzyme that is a key regulator of aerobic glycolysis in cancer cells. Increased expression of PKM2 was induced by hypoxia in liver cancer cell lines. Immunohistochemical (IHC) staining showed that PKM2 was highly expressed in moderately and well differentiated hepatocellular carcinoma (HCC) tissues with a hypovascular staining pattern. High expression of PKM2 was also localized in the perinecrotic area of intrahepatic cholangiocarcinoma (ICC) tissues. The percentage of the HCC or ICC tumor expressing PKM2 was significantly higher with more tumor necrosis, low microvessel density, and advanced stage. Moreover, the H103 scFv Ab was efficiently internalized into hypoxic liver cancer cells and could have potential for targeted drug delivery. Conclusion: our study, for the first time, developed hypoxia-specific scFv Ab H103 to liver cancer cells, and revealed that PKM2 is a promising biomarker for hypoxia in HCC and ICC tissues. These allow further exploration of this valuable Ab and PKM2 antigen for hypoxia targeting in liver cancer. PMID:27203546

  15. Site-specific scFv labelling with invertase via Sortase A mechanism as a platform for antibody-antigen detection using the personal glucose meter

    PubMed Central

    Ismail, Nur Faezee; Lim, Theam Soon

    2016-01-01

    Antibody labelling to reporter molecules is gaining popularity due to its many potential applications for diagnostics and therapeutics. However, non-directional bioconjugation methods which are commonly used often results in the loss of target binding capabilities. Therefore, a site-specific enzymatic based bioconjugation such as sortase-mediated transpeptidation allows for a more rapid and efficient method of antibody conjugation for diagnostic applications. Here we describe the utilization of sortase A bioconjugation to conjugate a single chain fragment variable (scFv) to the extracellular invertase (invB) from Zymomonas mobilis with the aim of developing an invertase based immunoassay. In addition, conjugation to enhanced green fluorescent protein (eGFP) was also validated to show the flexibility of the method. The invertase conjugated complex was successfully applied for the detection of antibody-antigen interaction using a personal glucose meter (PGM) for assay readout. The setup was used in both a direct and competitive assay highlighting the robustness of the conjugate for assay development. The method provides an alternative conjugation process to allow easy exchange of antibodies to facilitate rapid development of diagnostic assays for various diseases on the PGM platform. PMID:26782912

  16. One-step expression and purification of single-chain variable antibody fragment using an improved hexahistidine tag phagemid vector.

    PubMed

    Zhao, Qi; Chan, Yin-Wah; Lee, Susanna Sau-Tuen; Cheung, Wing-Tai

    2009-12-01

    Millions of candidate clones are commonly obtained following rounds of phage-displayed antibody library panning, and expression of those selected single-chain variable fragment (scFv) is required for secondary functional screening to identify positive clones. Large scale functional screening is often hampered by the time-consuming and labor-intensive subcloning of those candidate scFv clones into a bacterial expression vector carrying an affinity tag for scFv purification and detection. To overcome the limitations and to develop a multiplex approach, an improved hexahistidine tag phagemid vector was constructed for one-step scFv expression and purification. By using hexahistidine as an affinity tag, soluble scFvs can be rapidly and cost-effectively captured from Escherichia coli periplasmic extracts. For proof-of-concept, feasibility of the improved phagemid vector was examined against two scFvs, L17E4d targeting a cell surface antigen and L18Hh5 recognizing a monoclonal antibody (mAb). Using 1 ml of Ni-NTA agarose, 0.2-0.5 mg of soluble scFv was obtained from 1 L of bacteria culture, and the purified scFvs bound specifically to their target antigens with high affinity. Moreover, using two randomly selected hapten-specific scFv phage clones, it was demonstrated that the display of scFvs on phage surface was not affected by the hexahistidine affinity tag. These results suggest the improved phagemid vector allows the shuttle of phage-displayed antibody library panning and functional scFv production. Importantly, the improved phagemid vector can be easily adapted for multiplex screening.

  17. Antibody Fragments for On-Site Testing of Cannabinoids Generated via in Vitro Affinity Maturation.

    PubMed

    Morita, Izumi; Oyama, Hiroyuki; Yasuo, Mayumi; Matsuda, Kazuhisa; Katagi, Kengo; Ito, Aya; Tatsuda, Hiroka; Tanaka, Hiroyuki; Morimoto, Satoshi; Kobayashi, Norihiro

    2017-01-01

    Law enforcement against illicit use of cannabis and related substances requires rapid, feasible, and reliable tools for on-site testing of cannabinoids. Notably, methods based on cannabinoid-specific antibodies enable efficient screening of multiple specimens. Antibody engineering may accelerate development of modern and robust testing systems. Here, we used in vitro affinity maturation to generate a single-chain Fv fragment (scFv) that recognizes with high affinity the psychoactive cannabinoid, Δ(9)-tetrahydrocannabinol (THC). A mouse monoclonal antibody against THC, Ab-THC#33, with Ka 6.2×10(7) M(-1) (as Fab fragment) was established by the hybridoma technique. Then, a "wild-type" scFv (wt-scFv) with Ka, 1.1×10(7) M(-1) was prepared by bacterial expression of a fusion gene combining the VH and VL genes for Ab-THC#33. Subsequently, random point mutations in VH and VL were generated separately, and the resulting products were assembled into mutant scFv genes, which were then phage-displayed. Repeated panning identified a mutant scFv (scFv#m1-36) with 10-fold enhanced affinity (Ka 1.1×10(8) M(-1)) for THC, in which only a single conservative substitution (Ser50Thr) was present at the N-terminus of the VH-complementarity-determining region 2 (CDR2) sequence. In competitive enzyme-linked immunosorbent assay (ELISA), the mutant scFv generated dose-response curves with midpoint 0.27 ng/assay THC, which was 3-fold lower than that of wt-scFv. Even higher reactivity with a major THC metabolite, 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol, indicated that the mutant scFv will be useful for testing not only THC in confiscated materials, but also the metabolite in urine. Indeed, the antibody fragment is potentially suitable for use in advanced on-site testing platforms for cannabinoids.

  18. Stainless steel surface functionalization for immobilization of antibody fragments for cardiovascular applications.

    PubMed

    Foerster, A; Hołowacz, I; Sunil Kumar, G B; Anandakumar, S; Wall, J G; Wawrzyńska, M; Paprocka, M; Kantor, A; Kraskiewicz, H; Olsztyńska-Janus, S; Hinder, S J; Bialy, D; Podbielska, H; Kopaczyńska, M

    2016-04-01

    Stainless steel 316 L material is commonly used for the production of coronary and peripheral vessel stents. Effective biofunctionalization is a key to improving the performance and safety of the stents after implantation. This paper reports the method for the immobilization of recombinant antibody fragments (scFv) on stainless steel 316 L to facilitate human endothelial progenitor cell (EPC) growth and thus improve cell viability of the implanted stents for cardiovascular applications. The modification of stent surface was conducted in three steps. First the stent surface was coated with titania based coating to increase the density of hydroxyl groups for successful silanization. Then silanization with 3 aminopropyltriethoxysilane (APTS) was performed to provide the surface with amine groups which presence was verified using FTIR, XPS, and fluorescence microscopy. The maximum density of amine groups (4.8*10(-5) mol/cm(2)) on the surface was reached after reaction taking place in ethanol for 1 h at 60 °C and 0.04M APTS. On such prepared surface the glycosylated scFv were subsequently successfully immobilized. The influence of oxidation of scFv glycan moieties and the temperature on scFv coating were investigated. The fluorescence and confocal microscopy study indicated that the densest and most uniformly coated surface with scFv was obtained at 37 °C after oxidation of glycan chain. The results demonstrate that the scFv cannot be efficiently immobilized without prior aminosilanization of the surface. The effect of the chemical modification on the cell viability of EPC line 55.1 (HucPEC-55.1) was performed indicating that the modifications to the 316 L stainless steel are non-toxic to EPCs.

  19. Femtosecond spectroscopy probes the folding quality of antibody fragments expressed as GFP fusions in the cytoplasm

    SciTech Connect

    Didier, P.; Weiss, E.; Sibler, A.-P.; Philibert, P.; Martineau, P.; Bigot, J.-Y.; Guidoni, L.

    2008-02-22

    Time-resolved femtosecond spectroscopy can improve the application of green fluorescent proteins (GFPs) as protein-folding reporters. The study of ultrafast excited-state dynamics (ESD) of GFP fused to single chain variable fragment (scFv) antibody fragments, allowed us to define and measure an empirical parameter that only depends on the folding quality (FQ) of the fusion. This method has been applied to the analysis of genetic fusions expressed in the bacterial cytoplasm and allowed us to distinguish folded and thus functional antibody fragments (high FQ) with respect to misfolded antibody fragments. Moreover, these findings were strongly correlated to the behavior of the same scFvs expressed in animal cells. This method is based on the sensitivity of the ESD to the modifications in the tertiary structure of the GFP induced by the aggregation state of the fusion partner. This approach may be applicable to the study of the FQ of polypeptides over-expressed under reducing conditions.

  20. Exploiting Cross-reactivity to Neutralize Two Different Scorpion Venoms with One Single Chain Antibody Fragment*

    PubMed Central

    Riaño-Umbarila, Lidia; Contreras-Ferrat, Gabriel; Olamendi-Portugal, Timoteo; Morelos-Juárez, Citlalli; Corzo, Gerardo; Possani, Lourival D.; Becerril, Baltazar

    2011-01-01

    We report the optimization of a family of human single chain antibody fragments (scFv) for neutralizing two scorpion venoms. The parental scFv 3F recognizes the main toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2), albeit with low affinity. This scFv was subjected to independent processes of directed evolution to improve its recognition toward Cn2 (Riaño-Umbarila, L., Juárez-González, V. R., Olamendi-Portugal, T., Ortíz-León, M., Possani, L. D., and Becerril, B. (2005) FEBS J. 272, 2591–2601) and Css2 (this work). Each evolved variant showed strong cross-reactivity against several toxins, and was capable of neutralizing Cn2 and Css2. Furthermore, each variant neutralized the whole venoms of the above species. As far as we know, this is the first report of antibodies with such characteristics. Maturation processes revealed key residue changes to attain expression, stability, and affinity improvements as compared with the parental scFv. Combination of these changes resulted in the scFv LR, which is capable of rescuing mice from severe envenomation by 3 LD50 of freshly prepared whole venom of C. noxius (7.5 μg/20 g of mouse) and C. suffusus (26.25 μg/20 g of mouse), with surviving rates between 90 and 100%. Our research is leading to the formulation of an antivenom consisting of a discrete number of human scFvs endowed with strong cross-reactivity and low immunogenicity. PMID:21156801

  1. Exploiting cross-reactivity to neutralize two different scorpion venoms with one single chain antibody fragment.

    PubMed

    Riaño-Umbarila, Lidia; Contreras-Ferrat, Gabriel; Olamendi-Portugal, Timoteo; Morelos-Juárez, Citlalli; Corzo, Gerardo; Possani, Lourival D; Becerril, Baltazar

    2011-02-25

    We report the optimization of a family of human single chain antibody fragments (scFv) for neutralizing two scorpion venoms. The parental scFv 3F recognizes the main toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2), albeit with low affinity. This scFv was subjected to independent processes of directed evolution to improve its recognition toward Cn2 (Riaño-Umbarila, L., Juárez-González, V. R., Olamendi-Portugal, T., Ortíz-León, M., Possani, L. D., and Becerril, B. (2005) FEBS J. 272, 2591-2601) and Css2 (this work). Each evolved variant showed strong cross-reactivity against several toxins, and was capable of neutralizing Cn2 and Css2. Furthermore, each variant neutralized the whole venoms of the above species. As far as we know, this is the first report of antibodies with such characteristics. Maturation processes revealed key residue changes to attain expression, stability, and affinity improvements as compared with the parental scFv. Combination of these changes resulted in the scFv LR, which is capable of rescuing mice from severe envenomation by 3 LD(50) of freshly prepared whole venom of C. noxius (7.5 μg/20 g of mouse) and C. suffusus (26.25 μg/20 g of mouse), with surviving rates between 90 and 100%. Our research is leading to the formulation of an antivenom consisting of a discrete number of human scFvs endowed with strong cross-reactivity and low immunogenicity.

  2. [Construction and panning of scFv phage display library against recombinant interleukin 4 receptor].

    PubMed

    Yang, Guangyong; Guo, Haitao; Liu, Ximing; He, Guangzhi; Tian, Weiyi; Cai, Kun; Wang, Ping; Wang, Wenjia

    2016-06-01

    Objective To construct the recombinant human interleukin 4 receptor (rhIL-4R) single-chain Fv (scFv) antibody library by phage display technique to obtain the anti-IL-4R scFv clones selected from the library. Methods Total RNA was extracted from splenocytes of the BALB/c mice immunized with rhIL-4R. Complementary DNA fragments of variable heavy (VH) and variable light (VL) chains of the antibodies were prepared by reverse transcription PCR and assembled into scFv by splice overlap extension PCR (SOE-PCR). Both scFv and the pCANTAB5E vector were respectively double-digested with restriction endonuclease Sfi I and Not I, connected with T4 ligase, and then transformed into the competent cells E.coli TG1; it was cultured in medium to obtain the phage scFv antibody library; after three rounds of enrichment and panning, the specific antigen scFv with high affinity was selected for the sequencing. Results After three rounds of panning, we obtained a diversity of approximately 2×10(8) anti-rhIL-4R scFv antibody library. Sequencing analysis of one positive clone showed that the anti-rhIL-4R scFv was 741 bp and coded 247 amino acids. The analysis of VBASE2 database indicated that VH and VL gene sequences of anti-rhIL-4R protein all had three complementarity determining regions and four backbone areas.Conclusion The anti-rhIL-4R scFv was obtained from the scFv antibody library.

  3. Expression of a single-chain variable-fragment antibody against a Fusarium virguliforme toxin peptide enhances tolerance to sudden death syndrome in transgenic soybean plants.

    PubMed

    Brar, Hargeet K; Bhattacharyya, Madan K

    2012-06-01

    Plants do not produce antibodies. However, plants can correctly assemble functional antibody molecules encoded by mammalian antibody genes. Many plant diseases are caused by pathogen toxins. One such disease is the soybean sudden death syndrome (SDS). SDS is a serious disease caused by the fungal pathogen Fusarium virguliforme. The pathogen, however, has never been isolated from diseased foliar tissues. Thus, one or more toxins produced by the pathogen have been considered to cause foliar SDS. One of these possible toxins, FvTox1, was recently identified. We investigated whether expression of anti-FvTox1 single-chain variable-fragment (scFv) antibody in transgenic soybean can confer resistance to foliar SDS. We have created two scFv antibody genes, Anti-FvTox1-1 and Anti-FvTox1-2, encoding anti-FvTox1 scFv antibodies from RNAs of a hybridoma cell line that expresses mouse monoclonal anti-FvTox1 7E8 antibody. Both anti-FvTox1 scFv antibodies interacted with an antigenic site of FvTox1 that binds to mouse monoclonal anti-FvTox1 7E8 antibody. Binding of FvTox1 by the anti-FvTox1 scFv antibodies, expressed in either Escherichia coli or transgenic soybean roots, was initially verified on nitrocellulose membranes. Expression of anti-FvTox1-1 in stable transgenic soybean plants resulted in enhanced foliar SDS resistance compared with that in nontransgenic control plants. Our results suggest that i) FvTox1 is an important pathogenicity factor for foliar SDS development and ii) expression of scFv antibodies against pathogen toxins could be a suitable biotechnology approach for protecting crop plants from toxin-induced diseases.

  4. Delineation of BmSXP antibody V-gene usage from a lymphatic filariasis based immune scFv antibody library.

    PubMed

    Rahumatullah, Anizah; Ahmad, Azimah; Noordin, Rahmah; Lim, Theam Soon

    2015-10-01

    Phage display technology is an important tool for antibody generation or selection. This study describes the development of a scFv library and the subsequent analysis of identified monoclonal antibodies against BmSXP, a recombinant antigen for lymphatic filariasis. The immune library was generated from blood of lymphatic filariasis infected individuals. A TA based intermediary cloning approach was used to increase cloning efficiency for the library construction process. A diverse immune scFv library of 10(8) was generated. Six unique monoclonal antibodies were identified from the 50 isolated clones against BmSXP. Analysis of the clones showed a bias for the IgHV3 and Vκ1 (45.5%) and IgHV2 and Vκ3 (27.3%) gene family. The most favored J segment for light chain is IgKJ1 (45.5%). The most favored D and J segment for heavy chain are IgHD6-13 (75%) and IgHJ3 (47.7%). The information may suggest a predisposition of certain V genes in antibody responses against lymphatic filariasis.

  5. Optimal Neutralization of Centruroides noxius Venom Is Understood through a Structural Complex between Two Antibody Fragments and the Cn2 Toxin.

    PubMed

    Riaño-Umbarila, Lidia; Ledezma-Candanoza, Luis M; Serrano-Posada, Hugo; Fernández-Taboada, Guillermo; Olamendi-Portugal, Timoteo; Rojas-Trejo, Sonia; Gómez-Ramírez, Ilse V; Rudiño-Piñera, Enrique; Possani, Lourival D; Becerril, Baltazar

    2016-01-22

    The current trend of using recombinant antibody fragments in research to develop novel antidotes against scorpion stings has achieved excellent results. The polyclonal character of commercial antivenoms, obtained through the immunization of animals and which contain several neutralizing antibodies that recognize different epitopes on the toxins, guarantees the neutralization of the venoms. To avoid the use of animals, we aimed to develop an equivalent recombinant antivenom composed of a few neutralizing single chain antibody fragments (scFvs) that bind to two different epitopes on the scorpion toxins. In this study, we obtained scFv RU1 derived from scFv C1. RU1 showed a good capacity to neutralize the Cn2 toxin and whole venom of the scorpion Centruroides noxius. Previously, we had produced scFv LR, obtained from a different parental fragment (scFv 3F). LR also showed a similar neutralizing capacity. The simultaneous administration of both scFvs resulted in improved protection, which was translated as a rapid recovery of previously poisoned animals. The crystallographic structure of the ternary complex scFv LR-Cn2-scFv RU1 allowed us to identify the areas of interaction of both scFvs with the toxin, which correspond to non-overlapping sites. The epitope recognized by scFv RU1 seems to be related to a greater efficiency in the neutralization of the whole venom. In addition, the structural analysis of the complex helped us to explain the cross-reactivity of these scFvs and how they neutralize the venom.

  6. Optimal Neutralization of Centruroides noxius Venom Is Understood through a Structural Complex between Two Antibody Fragments and the Cn2 Toxin*

    PubMed Central

    Riaño-Umbarila, Lidia; Ledezma-Candanoza, Luis M.; Serrano-Posada, Hugo; Fernández-Taboada, Guillermo; Olamendi-Portugal, Timoteo; Rojas-Trejo, Sonia; Gómez-Ramírez, Ilse V.; Rudiño-Piñera, Enrique; Possani, Lourival D.; Becerril, Baltazar

    2016-01-01

    The current trend of using recombinant antibody fragments in research to develop novel antidotes against scorpion stings has achieved excellent results. The polyclonal character of commercial antivenoms, obtained through the immunization of animals and which contain several neutralizing antibodies that recognize different epitopes on the toxins, guarantees the neutralization of the venoms. To avoid the use of animals, we aimed to develop an equivalent recombinant antivenom composed of a few neutralizing single chain antibody fragments (scFvs) that bind to two different epitopes on the scorpion toxins. In this study, we obtained scFv RU1 derived from scFv C1. RU1 showed a good capacity to neutralize the Cn2 toxin and whole venom of the scorpion Centruroides noxius. Previously, we had produced scFv LR, obtained from a different parental fragment (scFv 3F). LR also showed a similar neutralizing capacity. The simultaneous administration of both scFvs resulted in improved protection, which was translated as a rapid recovery of previously poisoned animals. The crystallographic structure of the ternary complex scFv LR-Cn2-scFv RU1 allowed us to identify the areas of interaction of both scFvs with the toxin, which correspond to non-overlapping sites. The epitope recognized by scFv RU1 seems to be related to a greater efficiency in the neutralization of the whole venom. In addition, the structural analysis of the complex helped us to explain the cross-reactivity of these scFvs and how they neutralize the venom. PMID:26589800

  7. Baculovirus display of functional antibody Fab fragments.

    PubMed

    Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

    2015-08-01

    The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

  8. Comprehensive optimization of a single-chain variable domain antibody fragment as a targeting ligand for a cytotoxic nanoparticle.

    PubMed

    Zhang, Kathy; Geddie, Melissa L; Kohli, Neeraj; Kornaga, Tad; Kirpotin, Dmitri B; Jiao, Yang; Rennard, Rachel; Drummond, Daryl C; Nielsen, Ulrik B; Xu, Lihui; Lugovskoy, Alexey A

    2015-01-01

    Antibody-targeted nanoparticles have the potential to significantly increase the therapeutic index of cytotoxic anti-cancer therapies by directing them to tumor cells. Using antibodies or their fragments requires careful engineering because multiple parameters, including affinity, internalization rate and stability, all need to be optimized. Here, we present a case study of the iterative engineering of a single chain variable fragment (scFv) for use as a targeting arm of a liposomal cytotoxic nanoparticle. We describe the effect of the orientation of variable domains, the length and composition of the interdomain protein linker that connects VH and VL, and stabilizing mutations in both the framework and complementarity-determining regions (CDRs) on the molecular properties of the scFv. We show that variable domain orientation can alter cross-reactivity to murine antigen while maintaining affinity to the human antigen. We demonstrate that tyrosine residues in the CDRs make diverse contributions to the binding affinity and biophysical properties, and that replacement of non-essential tyrosines can improve the stability and bioactivity of the scFv. Our studies demonstrate that a comprehensive engineering strategy may be required to identify a scFv with optimal characteristics for nanoparticle targeting.

  9. Optimized extraction of a single-chain variable fragment of antibody by using aqueous micellar two-phase systems.

    PubMed

    Malpiedi, Luciana P; Nerli, Bibiana B; Taqueda, Maria E S; Abdalla, Dulcineia S P; Pessoa, Adalberto

    2015-07-01

    In this work, the purification of a single-chain variable fragment (scFv) of an antibody by using liquid-liquid extraction in aqueous micellar two-phase systems was optimized by means of central composite design. Protein partitioning assays were performed by using the selected system composition in previous works: Triton X-114 at 4% wt/wt, yeast fermentation supernatant at 60% wt/wt, McIlvaine buffer pH 7.00. The other system component concentrations, Cibacron Blue F3GA (CB), Fabsorbent™ F1P HF (HF) and NaCl, were selected as independent variables. ScFv recovery percentage (%R) and purification factor (PF) were selected as the responses. According to the optimization process both, scFv recovery percentage and purification factor were favored with the addition of HF and NaCl in a range of concentrations around the central point of the second central composite design (HF 0.0120% w/w, CB 0.0200% w/w, NaCl 0.200% w/w). These experimental conditions allowed the concentration and pre-purification of scFv in the micelle-rich bottom phase of the systems with a recovery percentage superior to 88% and a purification factor of approximately 3.5. These results improved the previously presented works and demonstrated the convenience of using aqueous micellar two-phase systems as a first step in the purification of scFv molecules.

  10. Expression and characterization of single-chain variable fragment antibody against staphylococcal enterotoxin A in Escherichia coli.

    PubMed

    Chen, Weifeng; Hu, Li; Liu, Aiping; Li, Jinquan; Chen, Fusheng; Wang, Xiaohong

    2014-11-01

    The staphylococcal enterotoxins (SEs) are potent gastrointestinal exotoxins synthesized by Staphylococcus aureus, which is responsible for various diseases including septicemia, food poisoning, and toxic shock syndrome, as well as bovine mastitis. Among them, staphylococcal enterotoxin A (SEA) is one of the most commonly present serotypes in staphylococcal food poisoning cases. In this study, the stable hybridoma 3C12 producing anti-SEA monoclonal antibody was established with an equilibrium dissociation constant (KD) of 1.48 × 10(-8) mol·L(-1), its ScFv-coding genes were obtained and then the anti-SEA single chain variable fragment (ScFv) protein was expressed in Escherichia coli. Characterization of the expressed target ScFv protein was analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis, Western blot, and enzyme-linked immunosorbent assay. The results demonstrated that the recombinant anti-SEA ScFv protein retained a specific binding activity for SEA, and the KD value of the soluble ScFv was about 3.75 × 10(-7) mol·L(-1). The overall yield of bioactive anti-SEA ScFv in E. coli flask culture was more than 10 mg·L(-1).

  11. Gladiolus plants transformed with single-chain variable fragment antibodies to Cucumber mosaic virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic plants of Gladiolus ‘Peter Pears’ or ‘Jenny Lee’ were developed that contain single-chain variable fragments (scFv) to Cucumber mosaic virus (CMV) subgroup I or II. The CMV subgroup I heavy and light chain scFv fragments were placed under control of either the duplicated CaMV 35S or suga...

  12. Broadening the neutralizing capacity of a family of antibody fragments against different toxins from Mexican scorpions.

    PubMed

    Rodríguez-Rodríguez, Everardo Remi; Olamendi-Portugal, Timoteo; Serrano-Posada, Hugo; Arredondo-López, Jonathan Noé; Gómez-Ramírez, Ilse; Fernández-Taboada, Guillermo; Possani, Lourival D; Anguiano-Vega, Gerardo Alfonso; Riaño-Umbarila, Lidia; Becerril, Baltazar

    2016-09-01

    New approaches aimed at neutralizing the primary toxic components present in scorpion venoms, represent a promising alternative to the use of antivenoms of equine origin in humans. New potential therapeutics developed by these approaches correspond to neutralizing antibody fragments obtained by selection and maturation processes from libraries of human origin. The high sequence identity shared among scorpion toxins is associated with an important level of cross reactivity exhibited by these antibody fragments. We have exploited the cross reactivity showed by single chain variable antibody fragments (scFvs) of human origin to re-direct the neutralizing capacity toward various other scorpion toxins. As expected, during these evolving processes several variants derived from a parental scFv exhibited the capacity to simultaneously recognize and neutralize different toxins from Centruroides scorpion venoms. A sequence analyses of the cross reacting scFvs revealed that specific mutations are responsible for broadening their neutralizing capacity. In this work, we generated a set of new scFvs that resulted from the combinatorial insertion of these point mutations. These scFvs are potential candidates to be part of a novel recombinant antivenom of human origin that could confer protection against scorpion stings. A remarkable property of one of these new scFvs (ER-5) is its capacity to neutralize at least three different toxins and its complementary capacity to neutralize the whole venom from Centruroides suffusus in combination with a second scFv (LR), which binds to a different epitope shared by Centruroides scorpion toxins.

  13. A novel variable antibody fragment dimerized by leucine zippers with enhanced neutralizing potency against rabies virus G protein compared to its corresponding single-chain variable antibody fragment.

    PubMed

    Li, Zhuang; Cheng, Yue; Xi, Hualong; Gu, Tiejun; Yuan, Ruosen; Chen, Xiaoxu; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2015-12-01

    Fatal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragments (scFv), which are composed of a variable heavy chain (VH) and a variable light chain (VL) connected by a peptide linker, can potentially be used to replace RIG. However, in our previous study, a scFv (scFV57S) specific for the rabies virus (RV) G protein showed a lower neutralizing potency than that of its parent IgG due to lower stability and altered peptide assembly pattern. In monoclonal antibodies, the VH and VL interact non-covalently, while in scFvs the VH is connected covalently with the VL by the artificial linker. In this study, we constructed and expressed two peptides 57VL-JUN-HIS and 57VH-FOS-HA in Escherichia coli. The well-known Fos and Jun leucine zippers were utilized to dimerize VH and VL similarly to the IgG counterpart. The two peptides assembled to form zipFv57S in vitro. Due to the greater similarity in structure with IgG, the zipFv57S protein showed a higher binding ability and affinity resulting in notable improvement of in vitro neutralizing activity over its corresponding scFv. The zipFv57S protein was also found to be more stable and showed similar protective rate as RIG in mice challenged with a lethal dose of RV. Our results not only indicated zipFv57S as an ideal alternative for RIG in PEP but also offered a novel and efficient hetero-dimerization pattern of VH and VL leading to enhanced neutralizing potency.

  14. Microbials for the production of monoclonal antibodies and antibody fragments

    PubMed Central

    Spadiut, Oliver; Capone, Simona; Krainer, Florian; Glieder, Anton; Herwig, Christoph

    2014-01-01

    Monoclonal antibodies (mAbs) and antibody fragments represent the most important biopharmaceutical products today. Because full length antibodies are glycosylated, mammalian cells, which allow human-like N-glycosylation, are currently used for their production. However, mammalian cells have several drawbacks when it comes to bioprocessing and scale-up, resulting in long processing times and elevated costs. By contrast, antibody fragments, that are not glycosylated but still exhibit antigen binding properties, can be produced in microbial organisms, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression systems, strain engineering, and production processes for the three main microbials used in antibody and antibody fragment production, namely Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. PMID:24183828

  15. A scFv antibody targeting common oligomeric epitope has potential for treating several amyloidoses

    PubMed Central

    Zha, Jun; Liu, Xiang-meng; Zhu, Jie; Liu, Shu-ying; Lu, Shuai; Xu, Peng-xin; Yu, Xiao-lin; Liu, Rui-tian

    2016-01-01

    Overproduction or poor clearance of amyloids lead to amyloid aggregation and even amyloidosis development. Different amyloids may interact synergistically to promote their aggregation and accelerate pathology in amyloidoses. Amyloid oligomers assembled from different amyloids share common structures and epitopes, and are considered the most toxic species in the pathologic processes of amyloidoses, which suggests that an agent targeting the common epitope of toxic oligomers could provide benefit to several amyloidoses. In this study, we firstly showed that an oligomer-specific single-chain variable fragment antibody, W20 simultaneously improved motor and cognitive function in Parkinson’s disease and Huntington’s disease mouse models, and attenuated a number of neuropathological features by reducing α-synuclein and mutant huntingtin protein aggregate load and preventing synaptic degeneration. Neuroinflammation and oxidative stress in vivo were also markedly inhibited. The proposed strategy targeting the common epitopes of amyloid oligomers presents promising potential for treating Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, and other amyloidoses. PMID:27824125

  16. Phage display-based generation of novel internalizing antibody fragments for immunotoxin-based treatment of acute myeloid leukemia

    PubMed Central

    Fitting, Jenny; Blume, Tobias; ten Haaf, Andre; Blau, Wolfgang; Gattenlöhner, Stefan; Tur, Mehmet Kemal; Barth, Stefan

    2015-01-01

    The current standard treatment for acute myeloid leukemia (AML) is chemotherapy based on cytarabine and daunorubicine (7 + 3), but it discriminates poorly between malignant and benign cells. Dose-limiting off‑target effects and intrinsic drug resistance result in the inefficient eradication of leukemic blast cells and their survival beyond remission. This minimal residual disease is the major cause of relapse and is responsible for a 5-year survival rate of only 24%. More specific and efficient approaches are therefore required to eradicate malignant cells while leaving healthy cells unaffected. In this study, we generated scFv antibodies that bind specifically to the surface of AML blast cells and AML bone marrow biopsy specimens. We isolated the antibodies by phage display, using subtractive whole-cell panning with AML M2‑derived Kasumi‑1 cells. By selecting for internalizing scFv antibody fragments, we focused on potentially novel agents for intracellular drug delivery and tumor modulation. Two independent methods showed that 4 binders were internalized by Kasumi-1 cells. Furthermore, we observed the AML‑selective inhibition of cell proliferation and the induction of apoptosis by a recombinant immunotoxin comprising one scFv fused to a truncated form of Pseudomonas exotoxin A (ETA'). This method may therefore be useful for the selection of novel disease-specific internalizing antibody fragments, providing a novel immunotherapeutic strategy for the treatment of AML patients. PMID:25760770

  17. Use of antibody fragments (Fv) in immunocytochemistry.

    PubMed

    Kleymann, G; Ostermeier, C; Heitmann, K; Haase, W; Michel, H

    1995-06-01

    We developed a novel antibody fragment (Fv) technique for localization and determination of the surface topology of membrane protein complexes by immunogold electron microscopy. Several hybridoma cell lines producing murine monoclonal antibodies (MAbs) raised against bacterial membrane proteins were established. The cDNAs coding for the variable domains of the MAbs were cloned and expressed in Escherichia coli. The engineered Fv fragments served as trifunctional adapter molecules. The Fv fragment binds to the epitope of the membrane protein. The Strep tag fused to the VH chain was used for one-step affinity purification of the Fv fragments. Immunological detection of the membrane protein-bound Fv fragments in electron microscopy was accomplished either via the Strep tag with colloidal gold-labeled streptavidin or via the c-myc tag, which was fused to the VL chain, in combination with the c-myc tag-specific antibody 9E10 and a colloidal gold-labeled secondary antibody. We examined four Fv fragments directed against the cytochrome c oxidase or the ubiquinol-cytochrome c oxidoreductase of Paracoccus denitrificans and bacteriorhodopsin of Halobacterium halobium to show that this method is generally applicable. In all cases the Fv fragments showed the same results as their corresponding parent antibodies in electron microscopic immunostaining and other applications.

  18. Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments.

    PubMed

    Moghaddam-Taaheri, Parisa; Ikonomova, Svetlana P; Gong, Zifan; Wisniewski, Janna Q; Karlsson, Amy J

    2016-10-15

    Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications.

  19. Isolation and characterisation of Ebolavirus-specific recombinant antibody fragments from murine and shark immune libraries.

    PubMed

    Goodchild, Sarah A; Dooley, Helen; Schoepp, Randal J; Flajnik, Martin; Lonsdale, Stephen G

    2011-09-01

    Members of the genus Ebolavirus cause fulminating outbreaks of disease in human and non-human primate populations with a mortality rate up to 90%. To facilitate rapid detection of these pathogens in clinical and environmental samples, robust reagents capable of providing sensitive and specific detection are required. In this work recombinant antibody libraries were generated from murine (single chain variable domain fragment; scFv) and nurse shark, Ginglymostoma cirratum (IgNAR V) hosts immunised with Zaire ebolavirus. This provides the first recorded IgNAR V response against a particulate antigen in the nurse shark. Both murine scFv and shark IgNAR V libraries were panned by phage display technology to identify useful antibodies for the generation of immunological detection reagents. Two murine scFv were shown to have specificity to the Zaire ebolavirus viral matrix protein VP40. Two isolated IgNAR V were shown to bind to the viral nucleoprotein (NP) and to capture viable Zaire ebolavirus with a high degree of sensitivity. Assays developed with IgNAR V cross-reacted to Reston ebolavirus, Sudan ebolavirus and Bundibugyo ebolavirus. Despite this broad reactivity, neither of IgNAR V showed reactivity to Côte d'Ivoire ebolavirus. IgNAR V was substantially more resistant to irreversible thermal denaturation than murine scFv and monoclonal IgG in a comparative test. The demonstrable robustness of the IgNAR V domains may offer enhanced utility as immunological detection reagents in fieldable biosensor applications for use in tropical or subtropical countries where outbreaks of Ebolavirus haemorrhagic fever occur.

  20. 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

  1. Engineering Peptide Linkers for scFv Immunosensors

    PubMed Central

    Shen, Zhihong; Yan, Heping; Zhang, Ying; Mernaugh, Raymond L.; Zeng, Xiangqun

    2008-01-01

    Using A10B single-chain fragment variable (scFv) as a model system, we demonstrated that the flexibility of scFv linker engineering can be combined with the inherent quick and adaptable characters of surface coupling chemistry (e.g., electrostatic, hydrogen bonding, or covalent attachment) to attach scFv to preformed functionalized self-assembled monolayers (SAMs). Six arginines, which were separated by glycine or serine as spacer, were incorporated in the peptide linker to form a 15-mer peptide linker (RGRGRGRGRSRGGGS). The polycationic arginine peptide was engineered into the A10B scFv-RG3 to favor its adsorption at anionic charged template surface (11-mercaptoundecanoic acid (MUA) and poly(sodium 4-styrenesulfonate (PSS))). This new approach was compared with the other engineered scFv constructs. Our results demonstrated that the anionic charged SAM template facilitated the oriented immobilization of scFvs on the SAM template surface as well as reduced the possibility of protein denaturation when directly immobilized on the solid surface. A 42-fold improvement of detection limits using MUA/A10B scFv-RG3 (less than 0.2 nM experimentally determined) was achieved compared to A10B Fab antibody and a 5-fold improvement was observed compared to A10B scFv that was engineered with a cysteine in the linker sequence. Using protein A-coated gold nanoparticles, a picomolar experimental detection limit was achieved. With 20 amino acids to choose from, engineered recombinant scFv in combination with SAM technology and nanoparticle mass amplification provide an emerging strategy for the development of highly sensitive and specific scFv immunosensors. PMID:18290668

  2. Structural characterization of a therapeutic anti-methamphetamine antibody fragment: oligomerization and binding of active metabolites.

    PubMed

    Peterson, Eric C; Celikel, Reha; Gokulan, Kuppan; Varughese, Kottayil I

    2013-01-01

    Vaccines and monoclonal antibodies (mAb) for treatment of (+)-methamphetamine (METH) abuse are in late stage preclinical and early clinical trial phases, respectively. These immunotherapies work as pharmacokinetic antagonists, sequestering METH and its metabolites away from sites of action in the brain and reduce the rewarding and toxic effects of the drug. A key aspect of these immunotherapy strategies is the understanding of the subtle molecular interactions important for generating antibodies with high affinity and specificity for METH. We previously determined crystal structures of a high affinity anti-METH therapeutic single chain antibody fragment (scFv6H4, K(D) = 10 nM) in complex with METH and the (+) stereoisomer of 3,4-methylenedioxymethamphetamine (MDMA, or "ecstasy"). Here we report the crystal structure of scFv6H4 in homo-trimeric unbound (apo) form (2.60Å), as well as monomeric forms in complex with two active metabolites; (+)-amphetamine (AMP, 2.38Å) and (+)-4-hydroxy methamphetamine (p-OH-METH, 2.33Å). The apo structure forms a trimer in the crystal lattice and it results in the formation of an intermolecular composite beta-sheet with a three-fold symmetry. We were also able to structurally characterize the coordination of the His-tags with Ni(2+). Two of the histidine residues of each C-terminal His-tag interact with Ni(2+) in an octahedral geometry. In the apo state the CDR loops of scFv6H4 form an open conformation of the binding pocket. Upon ligand binding, the CDR loops adopt a closed formation, encasing the drug almost completely. The structural information reported here elucidates key molecular interactions important in anti-methamphetamine abuse immunotherapy.

  3. Effects of a brain-engraftable microglial cell line expressing anti-prion scFv antibodies on survival times of mice infected with scrapie prions.

    PubMed

    Fujita, Koji; Yamaguchi, Yoshitaka; Mori, Tsuyoshi; Muramatsu, Naomi; Miyamoto, Takahito; Yano, Masashi; Miyata, Hironori; Ootsuyama, Akira; Sawada, Makoto; Matsuda, Haruo; Kaji, Ryuji; Sakaguchi, Suehiro

    2011-10-01

    We first verified that a single chain Fv fragment against prion protein (anti-PrP scFv) was secreted by HEK293T cells and prevented prion replication in infected cells. We then stably expressed anti-PrP scFv in brain-engraftable murine microglial cells and intracerebrally injected these cells into mice before or after infection with prions. Interestingly, the injection before or at an early time point after infection attenuated the infection marginally but significantly prolonged survival times of the mice. These suggest that the ex vivo gene transfer of anti-PrP scFvs using brain-engraftable cells could be a possible immunotherapeutic approach against prion diseases.

  4. Development of a hyperimmune anti-MUC-1 single chain antibody fragments phage display library for targeting breast cancer.

    PubMed

    Winthrop, M D; DeNardo, S J; DeNardo, G L

    1999-10-01

    Radioimmunotherapy (RIT) has demonstrated potential for improving clinical cancer therapy. Optimizing the approach has proven difficult thus far. Antibody phage display libraries provide unique molecules that could improve RIT. A phage display library of single chain antibody fragments (scFv) against the MUC-1 mucin molecule, which is expressed on 90% of human breast cancers, was produced from the spleen cells of MUC-1 hyperimmunized BALB/c mice. Increased serum IgG levels, 15 times baseline, were detected following the third immunization. RNA from the spleen cells was isolated, cDNA was made, and variable heavy and variable light immunoglobulin chain gene regions were amplified using PCR technology. The variable heavy and variable light chain gene regions were combined with a flexible linker, ligated into the pCANTAB 5E phagemid vector, and electroporated into TG1 Escherichia coli cells. A library of 10(7) initial colonies was compiled. Forty-six of 288 colonies screened for reactivity demonstrated binding to MUC-1-expressing MCF-7 breast cancer cell membrane fragments. Anti-MUC-1 library diversity evaluated by BstNI digest demonstrated that 52% of the anti-MUC-1 scFv binding MCF-7 possessed individual banding patterns representative of approximately 5 x 10(5) colonies likely able to recognize distinct epitopes present on MUC-1 positive human breast cancers. In summary, the anti-MUC-1 scFv antibody phage library contains diverse scFv molecules, which should provide unique characteristics and epitope recognition. These molecules will be used in the development of pretargeting RIT strategies designed to improve the clinical outcome of patients with breast cancer.

  5. High throughput cytotoxicity screening of anti-HER2 immunotoxins conjugated with antibody fragments from phage-displayed synthetic antibody libraries

    PubMed Central

    Hou, Shin-Chen; Chen, Hong-Sen; Lin, Hung-Wei; Chao, Wei-Ting; Chen, Yao-Sheng; Fu, Chi-Yu; Yu, Chung-Ming; Huang, Kai-Fa; Wang, Andrew H.-J.; Yang, An-Suei

    2016-01-01

    Immunotoxins are an important class of antibody-based therapeutics. The potency of the immunotoxins depends on the antibody fragments as the guiding modules targeting designated molecules on cell surfaces. Phage-displayed synthetic antibody scFv libraries provide abundant antibody fragment candidates as targeting modules for the immunoconjugates, but the discovery of optimally functional immunoconjugates is limited by the scFv-payload conjugation procedure. In this work, cytotoxicity screening of non-covalently assembled immunotoxins was developed in high throughput format to discover highly functional synthetic antibody fragments for delivering toxin payloads. The principles governing the efficiency of the antibodies as targeting modules have been elucidated from large volume of cytotoxicity data: (a) epitope and paratope of the antibody-based targeting module are major determinants for the potency of the immunotoxins; (b) immunotoxins with bivalent antibody-based targeting modules are generally superior in cytotoxic potency to those with corresponding monovalent targeting module; and (c) the potency of the immunotoxins is positively correlated with the densities of the cell surface antigen. These findings suggest that screening against the target cells with a large pool of antibodies from synthetic antibody libraries without the limitations of natural antibody responses can lead to optimal potency and minimal off-target toxicity of the immunoconjugates. PMID:27550798

  6. Generation of “LYmph Node Derived Antibody Libraries” (LYNDAL) for selecting fully human antibody fragments with therapeutic potential

    PubMed Central

    Diebolder, Philipp; Keller, Armin; Haase, Stephanie; Schlegelmilch, Anne; Kiefer, Jonathan D; Karimi, Tamana; Weber, Tobias; Moldenhauer, Gerhard; Kehm, Roland; Eis-Hübinger, Anna M; Jäger, Dirk; Federspil, Philippe A; Herold-Mende, Christel; Dyckhoff, Gerhard; Kontermann, Roland E; Arndt, Michaela AE; Krauss, Jürgen

    2014-01-01

    The development of efficient strategies for generating fully human monoclonal antibodies with unique functional properties that are exploitable for tailored therapeutic interventions remains a major challenge in the antibody technology field. Here, we present a methodology for recovering such antibodies from antigen-encountered human B cell repertoires. As the source for variable antibody genes, we cloned immunoglobulin G (IgG)-derived B cell repertoires from lymph nodes of 20 individuals undergoing surgery for head and neck cancer. Sequence analysis of unselected “LYmph Node Derived Antibody Libraries” (LYNDAL) revealed a naturally occurring distribution pattern of rearranged antibody sequences, representing all known variable gene families and most functional germline sequences. To demonstrate the feasibility for selecting antibodies with therapeutic potential from these repertoires, seven LYNDAL from donors with high serum titers against herpes simplex virus (HSV) were panned on recombinant glycoprotein B of HSV-1. Screening for specific binders delivered 34 single-chain variable fragments (scFvs) with unique sequences. Sequence analysis revealed extensive somatic hypermutation of enriched clones as a result of affinity maturation. Binding of scFvs to common glycoprotein B variants from HSV-1 and HSV-2 strains was highly specific, and the majority of analyzed antibody fragments bound to the target antigen with nanomolar affinity. From eight scFvs with HSV-neutralizing capacity in vitro, the most potent antibody neutralized 50% HSV-2 at 4.5 nM as a dimeric (scFv)2. We anticipate our approach to be useful for recovering fully human antibodies with therapeutic potential. PMID:24256717

  7. SNAP-Tag Technology: A Useful Tool To Determine Affinity Constants and Other Functional Parameters of Novel Antibody Fragments.

    PubMed

    Niesen, Judith; Sack, Markus; Seidel, Melanie; Fendel, Rolf; Barth, Stefan; Fischer, Rainer; Stein, Christoph

    2016-08-17

    Antibody derivatives, such as the single chain fragment variable (scFv), can be developed as diagnostic and therapeutic tools in cancer research, especially in the form of fusion proteins. Such derivatives are easier to produce and modify than monoclonal antibodies (mAbs) and achieve better tissue/tumor penetration. The genetic modification of scFvs is also much more straightforward than the challenging chemical modification of mAbs. Therefore, we constructed two scFvs derived from the approved monoclonal antibodies cetuximab (scFv2112) and panitumumab (scFv1711), both of which are specific for the epidermal growth factor receptor (EGFR), a well-characterized solid tumor antigen. Both scFvs were genetically fused to the SNAP-tag, an engineered version of the human DNA repair enzyme O(6)-alkylguanine DNA alkyltransferase that allows the covalent coupling of benzylguanine (BG)-modified substrates such as fluorescent dyes. The SNAP-tag achieves controllable and irreversible protein modification and is an important tool for experimental studies in vitro and in vivo. The affinity constant of a scFv is a key functional parameter, especially in the context of a fusion protein. Therefore, we developed a method to define the affinity constants of scFv-SNAP fusion proteins by surface plasmon resonance (SPR) spectroscopy. We could confirm that both scFvs retained their functionality after fusion to the SNAP-tag in a variety of procedures and assays, including ELISA, flow cytometry, and confocal microscopy. The experimental procedures described herein, and the new protocol for affinity determination by SPR spectroscopy, are suitable for the preclinical evaluation of diverse antibody formats and derivatives.

  8. Establishment of hapten-specific monoclonal avian IgY by conversion of antibody fragments obtained from combinatorial libraries.

    PubMed

    Deckers, Susanne; Braren, Ingke; Greunke, Kerstin; Meyer, Nadine; Rühl, Dana; Bredehorst, Reinhard; Spillner, Edzard

    2009-01-01

    Nowadays, recombinant antibody and phage display technology enable the efficient generation of immunotools and a subsequent manipulation for optimized affinity, specificity or overall performance. Such advantages are of particular interest for haptenic target structures, such as TNT (2,4,6-trinitrotoluene). The toxicity of TNT and its breakdown products makes a reliable and fast detection of low levels in aqueous samples highly important. In the present study, we aimed for the generation of scFvs (single-chain antibody fragments) specific for the TNT-surrogate TNP (2,4,6-trinitrophenyl) and their subsequent production as monoclonal avian IgY immunoglobulins providing improved assay performance. Therefore we subjected a human synthetic scFv library to selection following different strategies. TNP-specific human antibody fragments could be identified, characterized for their primary structure and evaluated for production as soluble scFv in Escherichia coli. Additionally, a murine TNP-specific antibody fragment was obtained from the hybridoma 11B3; however, the prokaryotic expression level was found to be limited. To generate and evaluate immunoglobulin formats with superior characteristics, all recombinant antibody fragments then were converted into two different chimaeric bivalent IgY antibody formats. After expression in mammalian cells, the IgY antibodies were assessed for their reactivity towards TNT. The IgY antibodies generated on the basis of the combinatorial library proved to be useful for detection of TNT, thereby emphasizing the high potential of this approach for the development of detection devices for immunoassay-based techniques.

  9. Production of a soluble single-chain variable fragment antibody against okadaic acid and exploration of its specific binding.

    PubMed

    He, Kuo; Zhang, Xiuyuan; Wang, Lixia; Du, Xinjun; Wei, Dong

    2016-06-15

    Okadaic acid is a lipophilic marine algal toxin commonly responsible for diarrhetic shellfish poisoning (DSP). Outbreaks of DSP have been increasing and are of worldwide public health concern; therefore, there is a growing demand for more rapid, reliable, and economical analytical methods for the detection of this toxin. In this study, anti-okadaic acid single-chain variable fragment (scFv) genes were prepared by cloning heavy and light chain genes from hybridoma cells, followed by fusion of the chains via a linker peptide. An scFv-pLIP6/GN recombinant plasmid was constructed and transformed into Escherichia coli for expression, and the target scFv was identified with IC-CLEIA (chemiluminescent enzyme immunoassay). The IC15 was 0.012 ± 0.02 μg/L, and the IC50 was 0.25 ± 0.03 μg/L. The three-dimensional structure of the scFv was simulated with computer modeling, and okadaic acid was docked to the scFv model to obtain a putative structure of the binding complex. Two predicted critical amino acids, Ser32 and Thr187, were then mutated to verify this theoretical model. Both mutants exhibited significant loss of binding activity. These results help us to understand this specific scFv-antigen binding mechanism and provide guidance for affinity maturation of the antibody in vitro. The high-affinity scFv developed here also has potential for okadaic acid toxin detection.

  10. In silico experiments of single-chain antibody fragment against drugs of abuse.

    PubMed

    Hu, Guodong; Chen, L Y

    2010-12-01

    Three sets of in silico experiments have been conducted to elucidate the binding mechanics of two drugs, (+)-methamphetamine (METH) and amphetamine (AMP) to the single-chain variable fragment (scFv) recently engineered from anti-METH monoclonal antibody mAb6H4 (IgG, κlight chain, K(d)=11nM). The first set of in silico experiments are long time equilibration runs of scFv:drug complexes and of drug-free scFv both in the solution. They demonstrate how the solution structures of scFv deviate from its crystallographic form with or without drug molecules bound to it. They lead to the prediction that the Arrhenius activation barrier is nearly zero for transitions from the dissociated state to the bound state. The second set of in silico experiments are nonequilibrium dynamics of pulling the drug molecules out of the binding pocket of scFv and the equilibration runs for drugs to fall back into the binding pocket. They demonstrate that extra water molecules (in addition to the two crystallographic waters) exist inside the binding pocket, underneath the drug molecules. These extra waters must have been evaporated from the binding pockets during the crystallization process of the in vitro experiments of structural determination. The third set of in silico experiments are nonequilibrium steered molecular dynamics simulations to determine the absolute binding free energies of METH and AMP to scFv. The center of mass of a drug molecule (METH or AMP) is steered (pulled) towards (forward) and away from (reverse) the binding site, sampling forward and reverse pulling paths. Mechanic work is measured along the pulling paths. The work measurements are averaged through the Brownian dynamics fluctuation dissipation theorem to produce the free-energy profiles of the scFv:drug complexes as a function of the drug-scFv separation. These experiments lead to the theoretical prediction of absolute binding energies of METH and AMP that are in agreement with the in vitro experimental results.

  11. Characterization of the native and denatured herceptin by enzyme linked immunosorbent assay and quartz crystal microbalance using a high-affinity single chain fragment variable recombinant antibody.

    PubMed

    Shang, Yuqin; Mernaugh, Ray; Zeng, Xiangqun

    2012-10-02

    Herceptin/Trastuzumab is a humanized IgG1κ light chain antibody used to treat some forms of breast cancer. A phage-displayed recombinant antibody library was used to obtain a single chain fragment variable (scFv, designated 2B4) to a linear synthetic peptide representing Herceptin's heavy chain CDR3. Enzyme linked immunosorbent assays (ELISAs) and piezoimmunosensor/quartz crystal microbalance (QCM) assays were used to characterize 2B4-binding activity to both native and heat denatured Herceptin. The 2B4 scFv specifically bound to heat denatured Herceptin in a concentration dependent manner over a wide (35-220.5 nM) dynamic range. Herceptin denatures and forms significant amounts of aggregates when heated. UV-vis characterization confirms that Herceptin forms aggregates as the temperature used to heat Herceptin increases. QCM affinity assay shows that binding stoichiometry between 2B4 scFv and Herceptin follows a 1:2 relationship proving that 2B4 scFv binds strongly to the dimers of heat denatured Herceptin aggregates and exhibits an affinity constant of 7.17 × 10(13) M(-2). The 2B4-based QCM assay was more sensitive than the corresponding ELISA. Combining QCM with ELISA can be used to more fully characterize nonspecific binding events in assays. The potential theoretical and clinical implications of these results and the advantages of the use of QCM to characterize human therapeutic antibodies in samples are also discussed.

  12. Probing the soybean Bowman-Birk inhibitor using recombinant antibody fragments.

    PubMed

    Muzard, Julien; Fields, Conor; O'Mahony, James John; Lee, Gil U

    2012-06-20

    The nutritional and health benefits of soy protein have been extensively studied over recent decades. The Bowman-Birk inhibitor (BBI), derived from soybeans, is a double-headed inhibitor of chymotrypsin and trypsin with anticarcinogenic and anti-inflammatory properties, which have been demonstrated in vitro and in vivo. However, the lack of analytical and purification methodologies complicates its potential for further functional and clinical investigations. This paper reports the construction of anti-BBI antibody fragments based on the principle of protein design. Recombinant antibody (scFv and diabody) molecules targeting soybean BBI were produced and characterized in vitro (K(D)~1.10(-9) M), and the antibody-binding site (epitope) was identified as part of the trypsin-specific reactive loop. Finally, an extremely fast purification strategy for BBI from soybean extracts, based on superparamagnetic particles coated with antibody fragments, was developed. To the best of the authors' knowledge, this is the first report on the design and characterization of recombinant anti-BBI antibodies and their potential application in soybean processing.

  13. Phage display of ScFv peptides recognizing the thymidine(6–4)thymidine photoproduct

    PubMed Central

    Zavala, Anamaria G.; Lancaster, Thaddeus; Groopman, John D.; Strickland, Paul T.; Chandrasegaran, Srinivasan

    2000-01-01

    Solar ultraviolet (UV) radiation induces DNA photoproducts in skin cells and is the predominant cause of human skin cancers. To understand human susceptibility to skin cancer and to facilitate the development of prevention measures, highly specific reagents to detect and quantitate UV-induced DNA adducts in human skin will be needed. One approach towards this end is the use of monoclonal antibody-based molecular dosimetry methods. To facilitate the development of photoproduct-specific antibody reagents we have: (i) cloned and sequenced a single chain variable fragment (ScFv) gene coding for one such high affinity monoclonal antibody, αUVssDNA-1 (mAb C3B6), recognizing the thymidine(6–4)thymidine photoproduct; (ii) expressed and displayed the cloned ScFv gene on the surface of phage; (iii) selected functional recombinant phage by panning; (iv) purified the ScFv peptide; (v) shown that the purified ScFv peptide binds to UV-irradiated polythymidylic acid but not unirradiated polythymidylic acid. This is the first demonstration of the use of phage display to select a ScFv recognizing DNA damage. In addition, this is the initial step towards immortalizing the antibody gene for genetic manipulation, structure–function studies and application to human investigations. PMID:10710441

  14. Development of an Immunoassay for Chloramphenicol Based on the Preparation of a Specific Single-Chain Variable Fragment Antibody.

    PubMed

    Du, Xin-jun; Zhou, Xiao-nan; Li, Ping; Sheng, Wei; Ducancel, Frédéric; Wang, Shuo

    2016-04-13

    Specific antibodies are essential for the immune detection of small molecule contaminants. In the present study, the heavy and light variable regions (V(H )and V(L)) of the immunoglobulin genes from a hybridoma secreting a chloramphenicol (CAP)-specific monoclonal antibody (mAb) were cloned and sequenced. In addition, the light and heavy chains obtained from the monoclonal antibody were separated using SDS-PAGE and analyzed using Orbitrap mass spectrometry. The results of DNA sequencing and mass spectrometry analysis were compared, and the V(H) and V(L) chains specific for CAP were determined and used to construct a single-chain variable fragment (scFv). This fragment was recombinantly expressed as a soluble scFv-alkaline phosphatase fusion protein and used to develop a direct competitive ELISA. Compared with the parent mAb, scFv exhibits lower sensitivity but better food matrix resistance. This work highlights the application of engineered antibodies for CAP detection.

  15. Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

    PubMed Central

    Moghaddam-Taaheri, Parisa; Ikonomova, Svetlana P.; Gong, Zifan; Wisniewski, Janna Q.; Karlsson, Amy J.

    2016-01-01

    Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications. PMID:27805609

  16. Vectorization in an oncolytic vaccinia virus of an antibody, a Fab and a scFv against programmed cell death -1 (PD-1) allows their intratumoral delivery and an improved tumor-growth inhibition

    PubMed Central

    Kleinpeter, Patricia; Fend, Laetitia; Thioudellet, Christine; Geist, Michel; Sfrontato, Nathalie; Koerper, Véronique; Fahrner, Catherine; Schmitt, Doris; Gantzer, Murielle; Remy-Ziller, Christelle; Brandely, Renée; Villeval, Dominique; Rittner, Karola; Silvestre, Nathalie; Erbs, Philippe; Zitvogel, Laurence; Quéméneur, Eric; Préville, Xavier; Marchand, Jean-Baptiste

    2016-01-01

    ABSTRACT We report here the successful vectorization of a hamster monoclonal IgG (namely J43) recognizing the murine Programmed cell death-1 (mPD-1) in Western Reserve (WR) oncolytic vaccinia virus. Three forms of mPD-1 binders have been inserted into the virus: whole antibody (mAb), Fragment antigen-binding (Fab) or single-chain variable fragment (scFv). MAb, Fab and scFv were produced and assembled with the expected patterns in supernatants of cells infected by the recombinant viruses. The three purified mPD-1 binders were able to block the binding of mPD-1 ligand to mPD-1 in vitro. Moreover, mAb was detected in tumor and in serum of C57BL/6 mice when the recombinant WR-mAb was injected intratumorally (IT) in B16F10 and MCA 205 tumors. The concentration of circulating mAb detected after IT injection was up to 1,900-fold higher than the level obtained after a subcutaneous (SC) injection (i.e., without tumor) confirming the virus tropism for tumoral cells and/or microenvironment. Moreover, the overall tumoral accumulation of the mAb was higher and lasted longer after IT injection of WR-mAb1, than after IT administration of 10 µg of J43. The IT injection of viruses induced a massive infiltration of immune cells including activated lymphocytes (CD8+ and CD4+). Interestingly, in the MCA 205 tumor model, WR-mAb1 and WR-scFv induced a therapeutic control of tumor growth similar to unarmed WR combined to systemically administered J43 and superior to that obtained with an unarmed WR. These results pave the way for next generation of oncolytic vaccinia armed with immunomodulatory therapeutic proteins such as mAbs. PMID:27853644

  17. Influence of the hydromechanical stress and temperature on growth and antibody fragment production with Bacillus megaterium.

    PubMed

    Lüders, Svenja; David, Florian; Steinwand, Miriam; Jordan, Eva; Hust, Michael; Dübel, Stefan; Franco-Lara, Ezequiel

    2011-07-01

    Bacillus megaterium was used for production of the lysozyme-specific recombinant scFv D1.3 antibody fragment. Key process parameters like the temperature and the hydromechanical stress play a very important role for significant product formation during process development or scale-up. In this study, the influence of these two variables on growth and recombinant antibody fragment production in a 2-L lab-scale bioreactor system was investigated using a central composite design. Especially a significant influence of the hydromechanical stress on antibody fragment production was detected in batch cultivations. While volumetric power inputs of about 0.5 kW/m(3) (agitation rates around 500 min(-1)) are usually employed in batch cultivations, in this work maximal product concentration was found at a volumetric power input of about 0.06 kW/m(3) (agitation rate around 250 min(-1)) and at a high cultivation temperature of 41 °C. The influence of the two process variables at single-cell level was estimated using flow cytometry too. The characterization was done by estimating the membrane potential giving a hint on bioprocess productivity and secretion capability: the best production was obtained through big cells with low specific membrane potential, which grew at low volumetric power inputs and high cultivation temperatures.

  18. Rigidity Emerges during Antibody Evolution in Three Distinct Antibody Systems: Evidence from QSFR Analysis of Fab Fragments

    PubMed Central

    Li, Tong; Tracka, Malgorzata B.; Uddin, Shahid; Casas-Finet, Jose; Jacobs, Donald J.; Livesay, Dennis R.

    2015-01-01

    The effects of somatic mutations that transform polyspecific germline (GL) antibodies to affinity mature (AM) antibodies with monospecificity are compared among three GL-AM Fab pairs. In particular, changes in conformational flexibility are assessed using a Distance Constraint Model (DCM). We have previously established that the DCM can be robustly applied across a series of antibody fragments (VL to Fab), and subsequently, the DCM was combined with molecular dynamics (MD) simulations to similarly characterize five thermostabilizing scFv mutants. The DCM is an ensemble based statistical mechanical approach that accounts for enthalpy/entropy compensation due to network rigidity, which has been quite successful in elucidating conformational flexibility and Quantitative Stability/Flexibility Relationships (QSFR) in proteins. Applied to three disparate antibody systems changes in QSFR quantities indicate that the VH domain is typically rigidified, whereas the VL domain and CDR L2 loop become more flexible during affinity maturation. The increase in CDR H3 loop rigidity is consistent with other studies in the literature. The redistribution of conformational flexibility is largely controlled by nonspecific changes in the H-bond network, although certain Arg to Asp salt bridges create highly localized rigidity increases. Taken together, these results reveal an intricate flexibility/rigidity response that accompanies affinity maturation. PMID:26132144

  19. Rigidity Emerges during Antibody Evolution in Three Distinct Antibody Systems: Evidence from QSFR Analysis of Fab Fragments.

    PubMed

    Li, Tong; Tracka, Malgorzata B; Uddin, Shahid; Casas-Finet, Jose; Jacobs, Donald J; Livesay, Dennis R

    2015-07-01

    The effects of somatic mutations that transform polyspecific germline (GL) antibodies to affinity mature (AM) antibodies with monospecificity are compared among three GL-AM Fab pairs. In particular, changes in conformational flexibility are assessed using a Distance Constraint Model (DCM). We have previously established that the DCM can be robustly applied across a series of antibody fragments (VL to Fab), and subsequently, the DCM was combined with molecular dynamics (MD) simulations to similarly characterize five thermostabilizing scFv mutants. The DCM is an ensemble based statistical mechanical approach that accounts for enthalpy/entropy compensation due to network rigidity, which has been quite successful in elucidating conformational flexibility and Quantitative Stability/Flexibility Relationships (QSFR) in proteins. Applied to three disparate antibody systems changes in QSFR quantities indicate that the VH domain is typically rigidified, whereas the VL domain and CDR L2 loop become more flexible during affinity maturation. The increase in CDR H3 loop rigidity is consistent with other studies in the literature. The redistribution of conformational flexibility is largely controlled by nonspecific changes in the H-bond network, although certain Arg to Asp salt bridges create highly localized rigidity increases. Taken together, these results reveal an intricate flexibility/rigidity response that accompanies affinity maturation.

  20. Dimerisation strategies for shark IgNAR single domain antibody fragments.

    PubMed

    Simmons, David P; Abregu, Fiona A; Krishnan, Usha V; Proll, David F; Streltsov, Victor A; Doughty, Larissa; Hattarki, Meghan K; Nuttall, Stewart D

    2006-08-31

    Immunoglobulin new antigen receptors (IgNARs) are unique single domain antibodies found in the serum of sharks. The individual variable (VNAR) domains bind antigen independently and are candidates for the smallest antibody-based immune recognition units (approximately 13 kDa). Here, we first isolated and sequenced the cDNA of a mature IgNAR antibody from the spotted wobbegong shark (Orectolobus maculatus) and confirmed the independent nature of the VNAR domains by dynamic light scattering. Second, we asked which of the reported antibody fragment dimerisation strategies could be applied to VNAR domains to produce small bivalent proteins with high functional affinity (avidity). In contrast to single chain Fv (scFv) fragments, separate IgNARs could not be linked into a tandem single chain format, with the resulting proteins exhibited only monovalent binding due solely to interaction of the N-terminal domain with antigen. Similarly, incorporation of C-terminal helix-turn-helix (dhlx) motifs, while resulting in efficiently dimerised protein, resulted in only a modest enhancement of affinity, probably due to an insufficiently long hinge region linking the antibody to the dhlx motif. Finally, generation of mutants containing half-cystine residues at the VNAR C-terminus produced dimeric recombinant proteins exhibiting high functional affinity for the target antigens, but at the cost of 50-fold decreased protein expression levels. This study demonstrates the potential for construction of bivalent or bispecific IgNAR-based binding reagents of relatively small size (approximately 26 kDa), equivalent to a monovalent antibody Fv fragment, for formulation into future diagnostic and therapeutic formats.

  1. Site-specific immobilization of recombinant antibody fragments through material-binding peptides for the sensitive detection of antigens in enzyme immunoassays.

    PubMed

    Kumada, Yoichi

    2014-11-01

    The immobilization of an antibody is one of the key technologies that are used to enhance the sensitivity and efficiency of the detection of target molecules in immunodiagnosis and immunoseparation. Recombinant antibody fragments such as VHH, scFv and Fabs produced by microorganisms are the next generation of ligand antibodies as an alternative to conventional whole Abs due to a smaller size and the possibility of site-directed immobilization with uniform orientation and higher antigen-binding activity in the adsorptive state. For the achievement of site-directed immobilization, affinity peptides for a certain ligand molecule or solid support must be introduced to the recombinant antibody fragments. In this mini-review, immobilization technologies for the whole antibodies (whole Abs) and recombinant antibody fragments onto the surfaces of plastics are introduced. In particular, the focus here is on immobilization technologies of recombinant antibody fragments utilizing affinity peptide tags, which possesses strong binding affinity towards the ligand molecules. Furthermore, I introduced the material-binding peptides that are capable of direct recognition of the target materials. Preparation and immobilization strategies for recombinant antibody fragments linked to material-binding peptides (polystyrene-binding peptides (PS-tags) and poly (methyl methacrylate)-binding peptide (PMMA-tag)) are the focus here, and are based on the enhancement of sensitivity and a reduction in the production costs of ligand antibodies. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.

  2. Suppression of human cytochrome P450 aromatase activity by monoclonal and recombinant antibody fragments and identification of a stable antigenic complex.

    PubMed

    Lala, Puloma; Higashiyama, Tadayoshi; Erman, Mary; Griswold, Jennifer; Wagner, Traci; Osawa, Yoshio; Ghosh, Debashis

    2004-03-01

    Human cytochrome P450 aromatase (P450arom) is responsible for biosynthesis of estrogens from androgens. Monoclonal antibody MAb3-2C2 to P450arom specifically binds to a conformational epitope and suppresses the enzyme activity in a dose-dependent manner. The crystal structure of the Fab fragment of MAb3-2C2 has been used to engineer a recombinant single chain antibody fragment (scFv) and a homodimeric variable domain of the light chain (VL(2)). These recombinant antibody fragments have been expressed in Escherichia coli and purified. Here, we show that the recombinant scFv suppresses P450arom activity with an IC(50) value similar to that of natural MAb3-2C2 F(ab')(2). The recombinant VL(2) also exhibits dose-dependent suppression of the P450arom activity, but at a reduced level, demonstrating that the homodimer is unable to fully mimic the complementarity determining region (CDR) of a variable heavy chain (VH)-VL heterodimer. We prepare and purify a stable complex of P450arom with MAb3-2C2 F(ab')(2) and show that the complex migrates and precipitates as a single molecular assembly. Efforts to crystallize P450arom for structure-function studies have yielded small single crystals. Our results suggest that formation of stable complexes with fragments of the monoclonal antibody could provide an alternative method for crystallization of P450arom.

  3. Co-expression of Dsb proteins enables soluble expression of a single-chain variable fragment (scFv) against human type 1 insulin-like growth factor receptor (IGF-1R) in E. coli.

    PubMed

    Sun, Xue-Wen; Wang, Xiao-Hua; Yao, Yan-Bing

    2014-12-01

    Type 1 insulin-like growth factor receptor (IGF-1R) is a promising therapeutic target for cancer treatment. A single-chain variable fragment (scFv) against human IGF-1R forms inclusion body when expressed in periplasmic space of E. coli routinely. Here, we described that co-expression of appropriate disulfide bonds (Dsb) proteins known to catalyze the formation and isomerization of Dsb can markedly recover the soluble expression of target scFv in E. coli. A 50 % recovery in solubility of the scFv was observed upon co-expression of DsbC alone, and a maximum solubility (80 %) was obtained when DsbA and DsbC were co-expressed in combination. Furthermore, the soluble scFv present full antigen-binding activity with IGF-1R, suggesting its correct folding. This study also suggested that the selection of Dsb proteins should be tested case-by-case if the approach of co-expression of Dsb system is adopted to address the problem of insoluble expression of proteins carrying Dsb.

  4. Protection against anthrax toxin by recombinant antibody fragments correlates with antigen affinity.

    PubMed

    Maynard, Jennifer A; Maassen, Catharina B M; Leppla, Stephen H; Brasky, Kathleen; Patterson, Jean L; Iverson, Brent L; Georgiou, George

    2002-06-01

    The tripartite toxin produced by Bacillus anthracis is the key determinant in the etiology of anthrax. We have engineered a panel of toxin-neutralizing antibodies, including single-chain variable fragments (scFvs) and scFvs fused to a human constant kappa domain (scAbs), that bind to the protective antigen subunit of the toxin with equilibrium dissociation constants (K(d)) between 63 nM and 0.25 nM. The entire antibody panel showed high serum, thermal, and denaturant stability. In vitro, post-challenge protection of macrophages from the action of the holotoxin correlated with the K(d) of the scFv variants. Strong correlations among antibody construct affinity, serum half-life, and protection were also observed in a rat model of toxin challenge. High-affinity toxin-neutralizing antibodies may be of therapeutic value for alleviating the symptoms of anthrax toxin in infected individuals and for medium-term prophylaxis to infection.

  5. Production of recombinant scFv against p24 of human immunodeficiency virus type 1 by phage display technology.

    PubMed

    Mohammadzadeh, Sara; Rajabibazl, Masoumeh; Fourozandeh, Mehdi; Rasaee, Mohammad Javad; Rahbarizadeh, Fatemeh; Mohammadi, Mohammad

    2014-02-01

    Phage display has a fundamental role in protein isolation and engineering. Isolated proteins produced with this method can be modified for specific binding and affinity. P24 is the most produced protein during human immune deficiency virus (HIV) replication; especially in the early steps of HIV-1 infection, its evaluation may have diagnostic values. To test the HIV-1 infection, p24 antigen assay appears to be a very promising alternative to RNA assays. In this study, we have generated a recombinant mouse single chain antibody fragment against p24 of the HIV-1 with the use of phage display technology. After isolation of antibody variable-region (V) gene of B cells extracted from the spleen of an immunized mouse, a library of single chain Fv fragments (scFv) was constructed. The library was used in a series of bio-panning processes against recombinant p24 protein expressed from Escherichia coli. The isolated scFv antibody specifically recognizes the HIV-1 capsid protein p24. The affinity constant of the isolated scFv antibody (MF85) was found to be 2×10(-9) M. Our studies showed that the MF85 scFV antibody has similar properties as that of monoclonal antibodies produced by the hybridoma technology.

  6. A chimera of green fluorescent protein with single chain variable fragment antibody against ginsenosides for fluorescence-linked immunosorbent assay.

    PubMed

    Sakamoto, Seiichi; Tanizaki, Yusuke; Pongkitwitoon, Benyakan; Tanaka, Hiroyuki; Morimoto, Satoshi

    2011-05-01

    A chimera of green fluorescent protein extracted from Aequorea coerulescens (AcGFP), a mutant that has been codon optimized for mammalian expression, with single-chain variable fragment (scFv) antibody against ginsenoside Re (GRe-scFv), named fluobody, has been successfully expressed in Escherichia coli (E. coli) to develop simple, speedy, and sensitive fluorescence-linked immunosorbent assay (FLISA). Two chimera proteins were constructed to contain GRe-scFv at the C-terminus of AcGFP (C-fluobody) and at the N-terminus of AcGFP (N-fluobody). These fluobodies were then purified by ion metal affinity chromatography and refolded by stepwise dialysis. The characterization of both fluobodies revealed that C-fluobody was found to be appropriate probe for FLISA as compare with N-fluobody. Furthermore, improvement of limit of detection (LOD) was observed in FLISA using C-fluobody (10 ng/mL) due to its strong fluorescence intensity of AcGFP compared with conventional enzyme-linked immunosorbent assay (ELISA) using parental monoclonal antibody against ginsenoside Re (G-Re), MAb-4G10 (100 ng/mL). Since some steps required in ELISA can be avoided in this present FLISA, speedy and sensitive immunoassay also could be performed using fluobody instead of monoclonal antibody and scFv.

  7. scFv from Antibody That Mimics gp43 Modulates the Cellular and Humoral Immune Responses during Experimental Paracoccidioidomycosis

    PubMed Central

    Jannuzzi, Grasielle Pereira; Tavares, Aldo Henrique F. P.; Kaihami, Gilberto Hideo; de Almeida, José Roberto Fogaça; de Almeida, Sandro Rogério; Ferreira, Karen Spadari

    2015-01-01

    Paracoccidioidomycosis (PCM), caused by Paracoccidioides species is a prevalent systemic and progressive mycosis that occurs in Latin America. It is caused by Paracoccidioides species. Immunization with dendritic cells transfected with a plasmid encoding the scFv (pMAC/PS-scFv) that mimics the main antigen of P. brasiliensis (gp43) confers protection in experimental PCM. DCs link innate and adaptive immunity by recognizing invading pathogens and selecting the type of effector T cell to mediate the immune response. Here, we showed that DC-pMAC/PS-scFv induces the activation of CD4+ and CD8+ T cells. Moreover, our results demonstrated that BALB/c mice infected with P. brasiliensis and treated with DC-pMAC/PS-scFv showed the induction of specific IgG production against gp43 and IFN-γ, IL-12 and IL-4 cytokines. Analysis of regional lymph nodes revealed increases in the expression of clec7a, myd88, tlr2, gata3 and tbx21, which are involved in the immune response. Taken together, our results indicate that the scFv modulates the humoral and cellular immune responses and presents epitopes to CD4+ and CD8+ T cells. PMID:26091522

  8. Construction of human single-chain variable fragment antibodies of medullary thyroid carcinoma and single photon emission computed tomography/computed tomography imaging in tumor-bearing nude mice.

    PubMed

    Liu, Qiong; Pang, Hua; Hu, Xiaoli; Li, Wenbo; Xi, Jimei; Xu, Lu; Zhou, Jing

    2016-01-01

    Medullary thyroid carcinoma (MTC) is a rare tumor of the endocrine system with poor prognosis as it exhibits high resistance against conventional therapy. Recent studies have shown that monoclonal antibodies labeled with radionuclide have become important agents for diagnosing tumors. To elucidate whether single-chain fragment of variable (scFv) antibody labeled with 131I isotope is a potential imaging agent for diagnosing MTC. A human scFv antibody library of MTC using phage display technique was constructed with a capacity of 3x10(5). The library was panned with thyroid epithelial cell lines and MTC cell lines (TT). Western blotting and enzyme-linked immunosorbent assay (ELISA) were used to identify the biological characteristics of the panned scFv. Methyl thiazolyl tetrazolium (MTT) assay was also used to explore the optimal concentration of the TT cell proliferation inhibition rate. They were categorized into TT, SW480 and control groups using phosphate-buffered saline. Western blotting showed that molecular weight of scFv was 28 kDa, cell ELISA showed that the absorbance of TT cell group was significantly increased (P=0.000??) vs. the other three groups, and MTT assay showed that the inhibition rate between the two cell lines was statistically significantly different (P<0.05) when the concentration of scFv was 0.1, 1 and 10 µmol/l. The tumor uptake of 131I-scFv was visible at 12 h and clear image was obtained at 48 h using the single photon emission computed tomography. scFv rapidly and specifically target MTC cells, suggesting the potential of this antibody as an imaging agent for diagnosing MTC.

  9. Considerations in producing preferentially reduced half-antibody fragments.

    PubMed

    Makaraviciute, Asta; Jackson, Carolyn D; Millner, Paul A; Ramanaviciene, Almira

    2016-02-01

    Half-antibody fragments are a promising reagent for biosensing, drug-delivery and labeling applications, since exposure of the free thiol group in the Fc hinge region allows oriented reaction. Despite the structural variations among the molecules of different IgG subclasses and those obtained from different hosts, only generalized preferential antibody reduction protocols are currently available. Preferential reduction of polyclonal sheep anti-digoxin, rabbit anti-Escherichia coli and anti-myoglobin class IgG antibodies to half-antibody fragments has been investigated. A mild reductant 2-mercaptoethylamine (2-MEA) and a slightly stronger reductant tris(2-carboxyethyl)phosphine (TCEP) were used and the fragments obtained were quantitatively determined by SDS-PAGE analysis. It has been shown that the yields of half-antibody fragments could be increased by lowering the pH of the reduction mixtures. However, antibody susceptibility to the reductants varied. At pH4.5 the highest yield of sheep anti-digoxin IgG half-antibody fragments was obtained with 1M 2-MEA. Conversely, rabbit IgG half-antibody fragments could only be obtained with the stronger reductant TCEP. Preferential reduction of rabbit anti-myoglobin IgG antibodies was optimized and the highest half-antibody yield was obtained with 35 mM TCEP. Finally, it has been demonstrated that produced anti-myoglobin half-IgG fragments retained their binding activity.

  10. A strategy for the generation of specific human antibodies by directed evolution and phage display. An example of a single-chain antibody fragment that neutralizes a major component of scorpion venom.

    PubMed

    Riaño-Umbarila, Lidia; Juárez-González, Victor Rivelino; Olamendi-Portugal, Timoteo; Ortíz-León, Mauricio; Possani, Lourival Domingos; Becerril, Baltazar

    2005-05-01

    This study describes the construction of a library of single-chain antibody fragments (scFvs) from a single human donor by individual amplification of all heavy and light variable domains (1.1 x 10(8) recombinants). The library was panned using the phage display technique, which allowed selection of specific scFvs (3F and C1) capable of recognizing Cn2, the major toxic component of Centruroides noxius scorpion venom. The scFv 3F was matured in vitro by three cycles of directed evolution. The use of stringent conditions in the third cycle allowed the selection of several improved clones. The best scFv obtained (6009F) was improved in terms of its affinity by 446-fold, from 183 nm (3F) to 410 pm. This scFv 6009F was able to neutralize 2 LD(50) of Cn2 toxin when a 1 : 10 molar ratio of toxin-to-antibody fragment was used. It was also able to neutralize 2 LD(50) of the whole venom. These results pave the way for the future generation of recombinant human antivenoms.

  11. Crystal structure of a 3B3 variant - A broadly neutralizing HIV-1 scFv antibody

    SciTech Connect

    Clark, K. Reed; Walsh, Scott T.R.

    2009-12-10

    We present the crystal structure determination of an anti-HIV-1 gp120 single-chain variable fragment antibody variant, 3B3, at 2.5 {angstrom} resolution. This 3B3 variant was derived from the b12 antibody, using phage display and site-directed mutagenesis of the variable heavy chain (V{sub H}) complementary-determining regions (CDRs). 3B3 exhibits enhanced binding affinity and neutralization activity against several cross-clade primary isolates of HIV-1 by interaction with the recessed CD4-binding site on the gp120 envelope protein. Comparison with the structures of the unbound and bound forms of b12, the 3B3 structure closely resembles these structures with minimal differences with two notable exceptions. First, there is a reorientation of the CDR-H3 of the V{sub H} domain where the primary sequences evolved from b12 to 3B3. The structural changes in CDR-H3 of 3B3, in light of the b12-gp120 complex structure, allow for positioning an additional Trp side chain in the binding interface with gp120. Finally, the second region of structural change involves two peptide bond flips in CDR-L3 of the variable light (VL) domain triggered by a point mutation in CDR-H3 of Q100eY resulting in changes in the intramolecular hydrogen bonding patterning between the VL and VH domains. Thus, the enhanced binding affinities and neutralization capabilities of 3B3 relative to b12 probably result from higher hydrophobic driving potential by burying more aromatic residues at the 3B3-gp120 interface and by indirect stabilization of intramolecular contacts of the core framework residues between the VL and VH domains possibly through more favorable entropic effect through the expulsion of water.

  12. Noninvasive Imaging of PSMA in Prostate Tumors with 89Zr-Labeled huJ591 Engineered Antibody Fragments: The Faster Alternatives

    PubMed Central

    2015-01-01

    Engineered antibody fragments offer faster delivery with retained tumor specificity and rapid clearance from nontumor tissues. Here, we demonstrate that positron emission tomography (PET) based detection of prostate specific membrane antigen (PSMA) in prostatic tumor models using engineered bivalent antibodies built on single chain fragments (scFv) derived from the intact antibody, huJ591, offers similar tumor delineating properties but with the advantage of rapid targeting and imaging. 89Zr-radiolabeled huJ591 scFv (dimeric scFv-CH3; 89Zr-Mb) and cysteine diabodies (dimeric scFv; 89Zr-Cys-Db) demonstrated internalization and similar Kds (∼2 nM) compared to 89Zr-huJ591 in PSMA(+) cells. Tissue distribution assays established the specificities of both 89Zr-Mb and 89Zr-Cys-Db for PSMA(+) xenografts (6.2 ± 2.5% ID/g and 10.2 ± 3.4% ID/g at 12 h p.i. respectively), while minimal accumulation in PSMA(−) tumors was observed. From the PET images, 89Zr-Mb and 89Zr-Cys-Db exhibited faster blood clearance than the parent huJ591 while tumor-to-muscle ratios for all probes show comparable values across all time points. Ex vivo autoradiography and histology assessed the distribution of the probes within the tumor. Imaging PSMA-expressing prostate tumors with smaller antibody fragments offers rapid tumor accumulation and accelerated clearance; hence, shortened wait periods between tracer administration and high-contrast tumor imaging and lower dose-related toxicity are potentially realized. PMID:24779727

  13. DARPA Antibody Technology Program. Standardized Test Bed for Antibody Characterization: Characterization of an MS2 ScFv Antibody Produced by Illumina

    DTIC Science & Technology

    2016-08-01

    interactions. The Joint Product Management Office for Biosurveillance (JPMO BSV) Critical Reagents Program has recently instituted a quality program...provision of consistent, high- quality assays and reagents for current biodetection platforms as well as the development and validation of future systems...platforms. 15. SUBJECT TERMS Antibody Antibody Technology Program (ATP) Quality Enzyme-linked immunosorbent assay (ELISA) Biosurveillance Single-chain

  14. In situ magnetic separation of antibody fragments from Escherichia coli in complex media

    PubMed Central

    2013-01-01

    Background In situ magnetic separation (ISMS) has emerged as a powerful tool to overcome process constraints such as product degradation or inhibition of target production. In the present work, an integrated ISMS process was established for the production of his-tagged single chain fragment variable (scFv) D1.3 antibodies (“D1.3”) produced by E. coli in complex media. This study investigates the impact of ISMS on the overall product yield as well as its biocompatibility with the bioprocess when metal-chelate and triazine-functionalized magnetic beads were used. Results Both particle systems are well suited for separation of D1.3 during cultivation. While the triazine beads did not negatively impact the bioprocess, the application of metal-chelate particles caused leakage of divalent copper ions in the medium. After the ISMS step, elevated copper concentrations above 120 mg/L in the medium negatively influenced D1.3 production. Due to the stable nature of the model protein scFv D1.3 in the biosuspension, the application of ISMS could not increase the overall D1.3 yield as was shown by simulation and experiments. Conclusions We could demonstrate that triazine-functionalized beads are a suitable low-cost alternative to selectively adsorb D1.3 fragments, and measured maximum loads of 0.08 g D1.3 per g of beads. Although copper-loaded metal-chelate beads did adsorb his-tagged D1.3 well during cultivation, this particle system must be optimized by minimizing metal leakage from the beads in order to avoid negative inhibitory effects on growth of the microorganisms and target production. Hereby, other types of metal chelate complexes should be tested to demonstrate biocompatibility. Such optimized particle systems can be regarded as ISMS platform technology, especially for the production of antibodies and their fragments with low stability in the medium. The proposed model can be applied to design future ISMS experiments in order to maximize the overall product yield

  15. Structural Basis of Neutralization of the Major Toxic Component from the Scorpion Centruroides noxius Hoffmann by a Human-derived Single-chain Antibody Fragment*

    PubMed Central

    Canul-Tec, Juan Carlos; Riaño-Umbarila, Lidia; Rudiño-Piñera, Enrique; Becerril, Baltazar; Possani, Lourival D.; Torres-Larios, Alfredo

    2011-01-01

    It has previously been reported that several single-chain antibody fragments of human origin (scFv) neutralize the effects of two different scorpion venoms through interactions with the primary toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2). Here we present the crystal structure of the complex formed between one scFv (9004G) and the Cn2 toxin, determined in two crystal forms at 2.5 and 1.9 Å resolution. A 15-residue span of the toxin is recognized by the antibody through a cleft formed by residues from five of the complementarity-determining regions of the scFv. Analysis of the interface of the complex reveals three features. First, the epitope of toxin Cn2 overlaps with essential residues for the binding of β-toxins to its Na+ channel receptor site. Second, the putative recognition of Css2 involves mainly residues that are present in both Cn2 and Css2 toxins. Finally, the effect on the increase of affinity of previously reported key residues during the maturation process of different scFvs can be inferred from the structure. Taken together, these results provide the structural basis that explain the mechanism of the 9004G neutralizing activity and give insight into the process of directed evolution that gave rise to this family of neutralizing scFvs. PMID:21489992

  16. Structural Basis of Neutralization of the Major Toxic Component from the Scorpion Centruroides noxius Hoffmann by a Human-derived Single-chain Antibody Fragment

    SciTech Connect

    Canul-Tec, Juan Carlos; Riaño-Umbarila, Lidia; Rudiño-Piñera, Enrique; Becerril, Baltazar; Possani, Lourival D.; Torres-Larios, Alfredo

    2011-08-09

    It has previously been reported that several single-chain antibody fragments of human origin (scFv) neutralize the effects of two different scorpion venoms through interactions with the primary toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2). Here we present the crystal structure of the complex formed between one scFv (9004G) and the Cn2 toxin, determined in two crystal forms at 2.5 and 1.9 {angstrom} resolution. A 15-residue span of the toxin is recognized by the antibody through a cleft formed by residues from five of the complementarity-determining regions of the scFv. Analysis of the interface of the complex reveals three features. First, the epitope of toxin Cn2 overlaps with essential residues for the binding of {beta}-toxins to its Na+ channel receptor site. Second, the putative recognition of Css2 involves mainly residues that are present in both Cn2 and Css2 toxins. Finally, the effect on the increase of affinity of previously reported key residues during the maturation process of different scFvs can be inferred from the structure. Taken together, these results provide the structural basis that explain the mechanism of the 9004G neutralizing activity and give insight into the process of directed evolution that gave rise to this family of neutralizing scFvs.

  17. Structural basis of neutralization of the major toxic component from the scorpion Centruroides noxius Hoffmann by a human-derived single-chain antibody fragment.

    PubMed

    Canul-Tec, Juan Carlos; Riaño-Umbarila, Lidia; Rudiño-Piñera, Enrique; Becerril, Baltazar; Possani, Lourival D; Torres-Larios, Alfredo

    2011-06-10

    It has previously been reported that several single-chain antibody fragments of human origin (scFv) neutralize the effects of two different scorpion venoms through interactions with the primary toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2). Here we present the crystal structure of the complex formed between one scFv (9004G) and the Cn2 toxin, determined in two crystal forms at 2.5 and 1.9 Å resolution. A 15-residue span of the toxin is recognized by the antibody through a cleft formed by residues from five of the complementarity-determining regions of the scFv. Analysis of the interface of the complex reveals three features. First, the epitope of toxin Cn2 overlaps with essential residues for the binding of β-toxins to its Na(+) channel receptor site. Second, the putative recognition of Css2 involves mainly residues that are present in both Cn2 and Css2 toxins. Finally, the effect on the increase of affinity of previously reported key residues during the maturation process of different scFvs can be inferred from the structure. Taken together, these results provide the structural basis that explain the mechanism of the 9004G neutralizing activity and give insight into the process of directed evolution that gave rise to this family of neutralizing scFvs.

  18. HER2-specific immunoligands engaging NKp30 or NKp80 trigger NK-cell-mediated lysis of tumor cells and enhance antibody-dependent cell-mediated cytotoxicity.

    PubMed

    Peipp, Matthias; Derer, Stefanie; Lohse, Stefan; Staudinger, Matthias; Klausz, Katja; Valerius, Thomas; Gramatzki, Martin; Kellner, Christian

    2015-10-13

    NK cells detect tumors through activating surface receptors, which bind self-antigens that are frequently expressed upon malignant transformation. To increase the recognition of tumor cells, the extracellular domains of ligands of the activating NK cell receptors NKp30, NKp80 and DNAM-1 (i.e. B7-H6, AICL and PVR, respectively) were fused to a single-chain fragment variable (scFv) targeting the human epidermal growth factor receptor 2 (HER2), which is displayed by various solid tumors. The resulting immunoligands, designated B7-H6:HER2-scFv, AICL:HER2-scFv, and PVR:HER2-scFv, respectively, bound HER2 and the addressed NK cell receptor. However, whereas B7-H6:HER2-scFv and AICL:HER2-scFv triggered NK cells to kill HER2-positive breast cancer cells at nanomolar concentrations, PVR:HER2-scFv was not efficacious. Moreover, NK cell cytotoxicity was enhanced synergistically when B7-H6:HER2-scFv or AICL:HER2-scFv were applied in combination with another HER2-specific immunoligand engaging the stimulatory receptor NKG2D. In contrast, no improvements were achieved by combining B7-H6:HER2-scFv with AICL:HER2-scFv. Additionally, B7-H6:HER2-scFv and AICL:HER2-scFv enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) by the therapeutic antibodies trastuzumab and cetuximab synergistically, with B7-H6:HER2-scFv exhibiting a higher efficacy. In summary, antibody-derived proteins engaging NKp30 or NKp80 may represent attractive biologics to further enhance anti-tumor NK cell responses and may provide an innovative approach to sensitize tumor cells for antibody-based immunotherapy.

  19. Enhancement of antibody fragment secretion into the Escherichia coli periplasm by co-expression with the peptidyl prolyl isomerase, FkpA, in the cytoplasm.

    PubMed

    Levy, Raphael; Ahluwalia, Kiran; Bohmann, David J; Giang, Hoa M; Schwimmer, Lauren J; Issafras, Hassan; Reddy, Nithin B; Chan, Chung; Horwitz, Arnold H; Takeuchi, Toshihiko

    2013-08-30

    Improper protein folding or aggregation can frequently be responsible for low expression and poor functional activity of antibody fragments secreted into the Escherichia coli periplasm. Expression issues also can affect selection of antibody candidates from phage libraries, since antibody fragments displayed on phage also are secreted into the E. coli periplasm. To improve secretion of properly folded antibody fragments into the periplasm, we have developed a novel approach that involves co-expressing the antibody fragments with the peptidyl prolyl cis-trans isomerase, FkpA, lacking its signal sequence (cytFkpA) which consequently is expressed in the E. coli cytosol. Cytoplasmic expression of cytFkpA improved secretion of functional Fab fragments into the periplasm, exceeding even the benefits from co-expressing Fab fragments with native, FkpA localized in the periplasm. In addition, panning and subsequent screening of large Fab and scFv naïve phage libraries in the presence of cytFkpA significantly increased the number of unique clones selected, as well as their functional expression levels and diversity.

  20. Production and characterization of a single-chain variable fragment linked alkaline phosphatase fusion protein for detection of O,O-diethyl organophosphorus pesticides in a one-step enzyme-linked immunosorbent assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A single-chain variable fragment (scFv) and alkaline phosphatase (AP) fusion protein for detection of O, O-diethyl organophosphorus pesticides (OPs) was produced and characterized. The scFv gene was prepared by cloning VL and VH genes from a hybridoma cell secreting monoclonal antibody with broad-s...

  1. 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

  2. The novel anti-CD19 chimeric antigen receptors with humanized scFv (single-chain variable fragment) trigger leukemia cell killing.

    PubMed

    Qian, Liren; Li, Dan; Ma, Lie; He, Ting; Qi, Feifei; Shen, Jianliang; Lu, Xin-An

    2016-01-01

    The molecular design of CARs (Chimeric Antigen Receptors), especially the scFv, has been a major part to use of CAR-T cells for targeted adoptive immunotherapy. To address this issue, we chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv-based CAR. Next, we generated a panel of humanized scFvs and tested in vitro for their ability to direct CAR-T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. Furthermore, in a xenograft model of lymphoma, human T cells expressing humanized scFvs exhibited the same anti-tumor efficacy as those expressing murine scFv and prolonged survival compared with cells expressing control CAR. Therefore, we uncovered CARs expressing humanized scFv domain that contribute the similar enhanced antileukemic efficacy and survival in tumor bearing mice. These results provide the basis for the future clinical studies of CAR-T cells transduced with humanized scFv directed to CD19.

  3. Bicistronic expression plasmid encoding allergen and anti-IgE single chain variable fragment antibody as a novel DNA vaccine for allergy therapy and prevention.

    PubMed

    Bandbon Balenga, Nariman Aghaei; Thalhamer, Josef; Weiss, Richard

    2006-01-01

    Several approaches have been applied in order to alleviate the difficulties allergic patients are suffering from. Among them DNA vaccination and anti-IgE antibody have shown promising results. Herewith, a combination of both strategies is proposed to minimize IgE production while inducing high levels of blocking IgG and strong Th1 immune responses. A bicistronic expression plasmid including an internal ribosomal entry site (IRES) can express both, allergen and a single chain variable fragment (scFv) antibody against human IgE within antigen presenting cells (APCs) including B cells. Presentation of allergen derived peptides via MHC I and MHC II stimulates specific Th1 responses resulting in high levels of IFN-gamma and IgG. Anti-IgE scFv antibody binds to newly synthesized IgE molecules within B cell cytoplasm and also to free serum IgE, thereby inhibiting attachment of IgE to its receptors on basophils and mast cells. Also, IgE-anti-IgE complex functions as blocking antibody and neutralizes allergens entering the body. Additionally, anti-IgE scFv antibody binds to membrane bound IgE (mIgE) on B cells and interferes with IgE expression. Using assays, such as enzyme linked immunosorbent assay (ELISA), IgG and IgE production in response to this expression system can be evaluated. Also, rat basophil leukemia cell assay (using RBL-2H3 cells) can show the amount of functional IgE in sera as basophil mediator release is regarded as an indicator of the allergic hypersensitive reactions. The proposed approach may result in high levels of blocking IgG and low levels of IgE secretion from B cells. Additionally, it can inhibit activity of IgE in degranulation of basophils and mast cells.

  4. Crystal structure of anti-polysialic acid antibody single chain Fv fragment complexed with octasialic acid: insight into the binding preference for polysialic acid.

    PubMed

    Nagae, Masamichi; Ikeda, Akemi; Hane, Masaya; Hanashima, Shinya; Kitajima, Ken; Sato, Chihiro; Yamaguchi, Yoshiki

    2013-11-22

    Polysialic acid is a linear homopolymer of α2-8-linked sialic acids attached mainly onto glycoproteins. Cell surface polysialic acid plays roles in cell adhesion and differentiation events in a manner that is often dependent on the degree of polymerization (DP). Anti-oligo/polysialic acid antibodies have DP-dependent antigenic specificity, and such antibodies are widely utilized in biological studies for detecting and distinguishing between different oligo/polysialic acids. A murine monoclonal antibody mAb735 has a unique preference for longer polymers of polysialic acid (DP >10), yet the mechanism of recognition at the atomic level remains unclear. Here, we report the crystal structure of mAb735 single chain variable fragment (scFv735) in complex with octasialic acid at 1.8 Å resolution. In the asymmetric unit, two scFv735 molecules associate with one octasialic acid. In both complexes of the unit, all the complementarity-determining regions except for L3 interact with three consecutive sialic acid residues out of the eight. A striking feature of the complex is that 11 ordered water molecules bridge the gap between antibody and ligand, whereas the direct antibody-ligand interaction is less extensive. The dihedral angles of the trisialic acid unit directly interacting with scFv735 are not uniform, indicating that mAb735 does not strictly favor the previously proposed helical conformation. Importantly, both reducing and nonreducing ends of the bound ligand are completely exposed to solvent. We suggest that mAb735 gains its apparent high affinity for a longer polysialic acid chain by recognizing every three sialic acid units in a paired manner.

  5. Crystallization of antibody fragments and their complexes with antigen

    NASA Astrophysics Data System (ADS)

    Boulot, G.; Guillon, V.; Mariuzza, R. A.; Poljak, R. J.; Riottot, M.-M.; Souchon, H.; Spinelli, S.; Tello, D.

    1988-07-01

    Immunoglobulins, myeloma light chains and their fragments, and Fab fragments from monoclonal antibodies of predefined specificity have been crystallized as single components or complexed with their specific antigens. The intersegmental flexibility of antibody molecules has imposed the strategy of attempting to crystallize their Fab and Fc fragments separately. Intrasegmental mobility in Fabs has not been an obstacle to their crystallization, although this has been a low frequency event, occuring in about 1 in 25 to 1 in 50 trials with different Fabs. However, the immune system provides a large functional and structural diversity of antibody molecules so that an active search may eventually reveal antibodies of the desired specificity suitable for crystallization and X-ray diffraction studies.

  6. DNA immunization combined with scFv phage display identifies antagonistic GCGR specific antibodies and reveals new epitopes on the small extracellular loops.

    PubMed

    van der Woning, Bas; De Boeck, Gitte; Blanchetot, Christophe; Bobkov, Vladimir; Klarenbeek, Alex; Saunders, Michael; Waelbroeck, Magali; Laeremans, Toon; Steyaert, Jan; Hultberg, Anna; De Haard, Hans

    2016-01-01

    The identification of functional monoclonal antibodies directed against G-protein coupled receptors (GPCRs) is challenging because of the membrane-embedded topology of these molecules. Here, we report the successful combination of llama DNA immunization with scFv-phage display and selections using virus-like particles (VLP) and the recombinant extracellular domain of the GPCR glucagon receptor (GCGR), resulting in glucagon receptor-specific antagonistic antibodies. By immunizing outbred llamas with plasmid DNA containing the human GCGR gene, we sought to provoke their immune system, which generated a high IgG1 response. Phage selections on VLPs allowed the identification of mAbs against the extracellular loop regions (ECL) of GCGR, in addition to multiple VH families interacting with the extracellular domain (ECD) of GCGR. Identifying mAbs binding to the ECL regions of GCGR is challenging because the large ECD covers the small ECLs in the energetically most favorable 'closed conformation' of GCGR. Comparison of Fab with scFv-phage display demonstrated that the multivalent nature of scFv display is essential for the identification of GCGR specific clones by selections on VLPs because of avid interaction. Ten different VH families that bound 5 different epitopes on the ECD of GCGR were derived from only 2 DNA-immunized llamas. Seven VH families demonstrated interference with glucagon-mediated cAMP increase. This combination of technologies proved applicable in identifying multiple functional binders in the class B GPCR context, suggesting it is a robust approach for tackling difficult membrane proteins.

  7. A VL-linker-VH Orientation Dependent Single Chain Variable Antibody Fragment Against Rabies Virus G Protein with Enhanced Neutralizing Potency in vivo.

    PubMed

    Cheng, Yue; Li, Zhuang; Xi, Hualong; Gu, Tiejun; Yuan, Ruosen; Chen, Xiaoxu; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2016-01-01

    Lethal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragment (scFv), which is composed of a variable heavy chain (VH) and variable light chain (VL) connected by a peptide linker, may be developed as alternative to RIG for neutralizing rabies virus (RV). However, our previously constructed scFv (FV57S) with the (NH2) VH-linker-VL (COOH) orientation showed a lower neutralizing potency than its parent RIG. This orientation may inhibit FV57S from refolding into an intact and correct conformation. Therefore, the RFV57S protein with a VL-linker-VH orientation was constructed based on FV57S. A HIS tag was incorporated to aid in purification and detection of RFV57S and FV57S. However, abilities of RFV57S and FV57S to bind with the anti-HIS tag mAb were different. Therefore, a novel direct ELISA was established by utilizing a biotin-labeled truncated glycoprotein of RV. Although with similar stability and in vitro neutralizing potency as FV57S, RFV57S showed enhanced binding ability, affinity and in vivo protective efficacy against lethal dose of RV. Our studies support the feasibility of developing a scFv with reversed orientation and provide a novel method for evaluating the binding ability, stability and affinity of engineered antibodies recognizing linear epitope.

  8. Conversion of scFv peptide-binding specificity for crystal chaperone development

    SciTech Connect

    Pai, Jennifer C.; Culver, Jeffrey A.; Drury, Jason E.; Motani, Rakesh S.; Lieberman, Raquel L.; Maynard, Jennifer A.

    2012-02-07

    In spite of advances in protein expression and purification over the last decade, many proteins remain recalcitrant to structure determination by X-ray crystallography. One emerging tactic to obtain high-quality protein crystals for structure determination, particularly in the case of membrane proteins, involves co-crystallization with a protein-specific antibody fragment. Here, we report the development of new recombinant single-chain antibody fragments (scFv) capable of binding a specific epitope that can be introduced into internal loops of client proteins. The previously crystallized hexa-histidine-specific 3D5 scFv antibody was modified in the complementary determining region and by random mutagenesis, in conjunction with phage display, to yield scFvs with new biochemical characteristics and binding specificity. Selected variants include those specific for the hexa-histidine peptide with increased expression, solubility (up to 16.6 mg/ml) and sub-micromolar affinity, and those with new specificity for the EE hexa-peptide (EYMPME) and nanomolar affinity. Complexes of one such chaperone with model proteins harboring either an internal or a terminal EE tag were isolated by gel filtration. The 3.1 {angstrom} resolution structure of this chaperone reveals a binding surface complementary to the EE peptide and a {approx}52 {angstrom} channel in the crystal lattice. Notably, in spite of 85% sequence identity, and nearly identical crystallization conditions, the engineered scFv crystallizes in a different space group than the parent 3D5 scFv, and utilizes two new crystal contacts. These engineered scFvs represent a new class of chaperones that may eliminate the need for de novo identification of candidate chaperones from large antibody libraries.

  9. Effects of protein engineering and rational mutagenesis on crystal lattice of single chain antibody fragments.

    PubMed

    Kalyoncu, Sibel; Hyun, Jeongmin; Pai, Jennifer C; Johnson, Jennifer L; Entzminger, Kevin; Jain, Avni; Heaner, David P; Morales, Ivan A; Truskett, Thomas M; Maynard, Jennifer A; Lieberman, Raquel L

    2014-09-01

    Protein crystallization is dependent upon, and sensitive to, the intermolecular contacts that assist in ordering proteins into a three-dimensional lattice. Here we used protein engineering and mutagenesis to affect the crystallization of single chain antibody fragments (scFvs) that recognize the EE epitope (EYMPME) with high affinity. These hypercrystallizable scFvs are under development to assist difficult proteins, such as membrane proteins, in forming crystals, by acting as crystallization chaperones. Guided by analyses of intermolecular crystal lattice contacts, two second-generation anti-EE scFvs were produced, which bind to proteins with installed EE tags. Surprisingly, although noncomplementarity determining region (CDR) lattice residues from the parent scFv framework remained unchanged through the processes of protein engineering and rational design, crystal lattices of the derivative scFvs differ. Comparison of energy calculations and the experimentally-determined lattice interactions for this basis set provides insight into the complexity of the forces driving crystal lattice choice and demonstrates the availability of multiple well-ordered surface features in our scFvs capable of forming versatile crystal contacts.

  10. Effects of protein engineering and rational mutagenesis on crystal lattice of single chain antibody fragments

    PubMed Central

    Kalyoncu, Sibel; Hyun, Jeongmin; Pai, Jennifer C.; Johnson, Jennifer L.; Entzminger, Kevin; Jain, Avni; Heaner, David P.; Morales, Ivan A.; Truskett, Thomas M.; Maynard, Jennifer A.; Lieberman, Raquel L.

    2014-01-01

    Protein crystallization is dependent upon, and sensitive to, the intermolecular contacts that assist in ordering proteins into a three dimensional lattice. Here we used protein engineering and mutagenesis to affect the crystallization of single chain antibody fragments (scFvs) that recognize the EE epitope (EYMPME) with high affinity. These hypercrystallizable scFvs are under development to assist difficult proteins, such as membrane proteins, in forming crystals, by acting as crystallization chaperones. Guided by analyses of intermolecular crystal lattice contacts, two second-generation anti-EE scFvs were produced, which bind to proteins with installed EE tags. Surprisingly, although non-complementarity determining region (CDR) lattice residues from the parent scFv framework remained unchanged through the processes of protein engineering and rational design, crystal lattices of the derivative scFvs differ. Comparison of energy calculations and the experimentally-determined lattice interactions for this basis set provides insight into the complexity of the forces driving crystal lattice choice and demonstrates the availability of multiple well-ordered surface features in our scFvs capable of forming versatile crystal contacts. PMID:24615866

  11. Development and Characterization of Recombinant Antibody Fragments That Recognize and Neutralize In Vitro Stx2 Toxin from Shiga Toxin-Producing Escherichia coli

    PubMed Central

    Luz, Daniela; Chen, Gang; Maranhão, Andrea Q.; Rocha, Leticia B.; Sidhu, Sachdev; Piazza, Roxane M. F.

    2015-01-01

    Background Stx toxin is a member of the AB5 family of bacterial toxins: the active A subunit has N-glycosidase activity against 28S rRNA, resulting in inhibition of protein synthesis in eukaryotic cells, and the pentamer ligand B subunits (StxB) bind to globotria(tetra)osylceramide receptors (Gb3/Gb4) on the cell membrane. Shiga toxin-producing Escherichia coli strains (STEC) may produce Stx1 and/or Stx2 and variants. Strains carrying Stx2 are considered more virulent and related to the majority of outbreaks, besides being usually associated with hemolytic uremic syndrome in humans. The development of tools for the detection and/or neutralization of these toxins is a turning point for early diagnosis and therapeutics. Antibodies are an excellent paradigm for the design of high-affinity, protein-based binding reagents used for these purposes. Methods and Findings In this work, we developed two recombinant antibodies; scFv fragments from mouse hybridomas and Fab fragments by phage display technology using a human synthetic antibody library. Both fragments showed high binding affinity to Stx2, and they were able to bind specifically to the GKIEFSKYNEDDTF region of the Stx2 B subunit and to neutralize in vitro the cytotoxicity of the toxin up to 80%. Furthermore, the scFv fragments showed 79% sensitivity and 100% specificity in detecting STEC strains by ELISA. Conclusion In this work, we developed and characterized two recombinant antibodies against Stx2, as promising tools to be used in diagnosis or therapeutic approaches against STEC, and for the first time, we showed a human monovalent molecule, produced in bacteria, able to neutralize the cytotoxicity of Stx2 in vitro. PMID:25790467

  12. Comparison of the efficiency of antibody selection from semi-synthetic scFv and non-immune Fab phage display libraries against protein targets for rapid development of diagnostic immunoassays.

    PubMed

    Chan, Conrad E Z; Chan, Annie H Y; Lim, Angeline P C; Hanson, Brendon J

    2011-10-28

    Rapid development of diagnostic immunoassays against novel emerging or genetically modified pathogens in an emergency situation is dependent on the timely isolation of specific antibodies. Non-immune antibody phage display libraries are an efficient in vitro method for selecting monoclonal antibodies and hence ideal in these circumstances. Such libraries can be constructed from a variety of sources e.g. B cell cDNA or synthetically generated, and use a variety of antibody formats, typically scFv or Fab. However, antibody source and format can impact on the quality of antibodies generated and hence the effectiveness of this methodology for the timely production of antibodies. We have carried out a comparative screening of two antibody libraries, a semi-synthetic scFv library and a human-derived Fab library against the protective antigen toxin component of Bacillus anthracis and the epsilon toxin of Clostridium botulinum. We have shown that while the synthetic library produced a diverse collection of specific scFv-phage, these contained a high frequency of unnatural amber stops and glycosylation sites which limited their conversion to IgG, and also a high number which lost specificity when expressed as IgG. In contrast, these limitations were overcome by the use of a natural human library. Antibodies from both libraries could be used to develop sandwich ELISA assays with similar sensitivity. However, the ease and speed with which full-length IgG could be generated from the human-derived Fab library makes screening this type of library the preferable method for rapid antibody generation for diagnostic assay development.

  13. Biotechnological applications of recombinant single-domain antibody fragments

    PubMed Central

    2011-01-01

    Background Single-domain antibody fragments possess structural features, such as a small dimension, an elevated stability, and the singularity of recognizing epitopes non-accessible for conventional antibodies that make them interesting for several research and biotechnological applications. Results The discovery of the single-domain antibody's potentials has stimulated their use in an increasing variety of fields. The rapid accumulation of articles describing new applications and further developments of established approaches has made it, therefore, necessary to update the previous reviews with a new and more complete summary of the topic. Conclusions Beside the necessary task of updating, this work analyses in detail some applicative aspects of the single-domain antibodies that have been overseen in the past, such as their efficacy in affinity chromatography, as co-crystallization chaperones, protein aggregation controllers, enzyme activity tuners, and the specificities of the unconventional single-domain fragments. PMID:21658216

  14. The Human Antibody Fragment DIATHIS1 Specific for CEACAM1 Enhances Natural Killer Cell Cytotoxicity Against Melanoma Cell Lines In Vitro

    PubMed Central

    Dupuis, Maria L.; Soriani, Alessandra; Ricci, Biancamaria; Dominici, Sabrina; Moricoli, Diego; Ascione, Alessandro; Santoni, Angela; Magnani, Mauro; Cianfriglia, Maurizio

    2015-01-01

    Several lines of evidence show that de novo expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is strongly associated with reduced disease-free survival of patients affected by metastatic melanoma. Previously published investigations report that homophilic interactions between CEACAM1 expressed on natural killer (NK) cells and tumors inhibit the NK cell-mediated killing independently of major histocompatibility complex class I recognition. This biological property can be physiologically relevant in metastatic melanoma because of the increased CEACAM1 expression observed on NK cells from some patients. Moreover, this inhibitory mechanism in many cases might hinder the efficacy of immunotherapeutic treatments of CEACAM1+ malignancies because of tumor evasion by activated effector cells. In the present study, we designed an in vitro experimental model showing that the human single-chain variable fragment (scFv) DIATHIS1 specific for CEACAM1 is able to enhance the lytic machinery of NK cells against CEACAM1+ melanoma cells. The coincubation of the scFv DIATHIS1 with CEACAM1+ melanoma cells and NK-92 cell line significantly increases the cell-mediated cytotoxicity. Moreover, pretreatment of melanoma cells with scFv DIATHIS1 promotes the activation and the degranulation capacity of in vitro–expanded NK cells from healthy donors. It is interesting to note that the melanoma cell line MelC and the primary melanoma cells STA that respond better to DIATHIS1 treatment, express higher relative levels of CEACAM1-3L and CEACAM1-3S splice variants isoforms compared with Mel501 cells that are less responsive to DIATHIS1-induced NK cell–mediated cytotoxicity. Taken together, our results suggest that the fully human antibody fragment DIATHIS1 originated by biopanning approach from a phage antibody library may represent a relevant biotechnological platform to design and develop completely human antimelanoma therapeutics of biological origin. PMID

  15. Production of human antibody fragments binding to melittin and phospholipase A2 in Africanised bee venom: minimising venom toxicity.

    PubMed

    Funayama, Jaqueline C; Pucca, Manuela B; Roncolato, Eduardo C; Bertolini, Thaís B; Campos, Lucas B; Barbosa, José E

    2012-03-01

    The hybrid created from the crossbreeding of European and African bees, known as the Africanised bee, has provided numerous advantages for current beekeeping. However, this new species exhibits undesirable behaviours, such as colony defence instinct and a propensity to attack en masse, which can result in serious accidents. To date, there is no effective treatment for cases of Africanised bee envenomation. One promising technique for developing an efficient antivenom is the use of phage display technology, which enables the production of human antibodies, thus avoiding the complications of serum therapy, such as anaphylaxis and serum sickness. The aim of this study was to produce human monoclonal single-chain Fv (scFv) antibody fragments capable of inhibiting the toxic effects of Africanised bee venom. We conducted four rounds of selection of antibodies against the venom and three rounds of selection of antibodies against purified melittin. Three clones were selected and tested by enzyme-linked immunosorbent assay to verify their specificity for melittin and phospholipase A2. Two clones (C5 and C12) were specific for melittin, and one (A7) was specific for phospholipase A2. In a kinetic haemolytic assay, these clones were evaluated individually and in pairs. The A7-C12 combination had the best synergistic effect and was chosen to be used in the assays of myotoxicity inhibition and lethality. The A7-C12 combination inhibited the in vivo myotoxic effect of the venom and increased the survival of treated animals.

  16. Selection of Human Antibody Fragments Which Bind Novel Breast Tumor Antigens

    DTIC Science & Technology

    1998-09-01

    cell type specific scFv for tumor targeting and as tools for identifying novel tumor antigens ... tumor specific antigens ). Subsequently, the cells were washed extensively with PBS to remove unbound phage and then incubated at 37°C for 15 minutes... detection and isolation of a tumor cell surface antigen using antibody phage display. J. Immunol. Meth. 203: 11-24. 46. Watters, J.M., Telleman, P.,

  17. Factors affecting the production of a single-chain antibody fragment by Aspergillus awamori in a stirred tank reactor.

    PubMed

    Sotiriadis, A; Keshavarz, T; Keshavarz-Moore, E

    2001-01-01

    A recombinant strain of Aspergillus awamori expressing anti-lysozyme single chain antibody fragments (scFv), under the control of a xylanase promoter, was studied in order to investigate the impact of medium, induction regime and protease production on the expression of the product. Experiments with the time of induction showed that the optimum results are achieved when induction is started in the late exponential phase (21 h after inoculation) improving the titer of the product from 14.5 mg L(-1), obtained in the early exponential phase (7 h after inoculation), to 16.2 mg L(-1). A 100% increase of the carbon (fructose) and nitrogen (ammonium sulfate) sources in the growth medium resulted in an increase in product concentration from 16.2 to 108.9 mg L(-1) and an increase in maximum dry cell weight from 7.5 to 11.5 g L(-1). A 50% reduction in the concentration of the inducer resulted in an increase in the product yield from 10 mg g(-1) dry cell weight to 12 mg g(-1). Proteolytic enzymes were produced during the fermentation up to concentrations equivalent to 1.4 g L(-1) trypsin, but they had no detrimental effect on the concentration of the antibody fragment.

  18. Fast antibody fragment motion: flexible linkers act as entropic spring.

    PubMed

    Stingaciu, Laura R; Ivanova, Oxana; Ohl, Michael; Biehl, Ralf; Richter, Dieter

    2016-03-29

    A flexible linker region between three fragments allows antibodies to adjust their binding sites to an antigen or receptor. Using Neutron Spin Echo Spectroscopy we observed fragment motion on a timescale of 7 ns with motional amplitudes of about 1 nm relative to each other. The mechanistic complexity of the linker region can be described by a spring model with Brownian motion of the fragments in a harmonic potential. Displacements, timescale, friction and force constant of the underlying dynamics are accessed. The force constant exhibits a similar strength to an entropic spring, with friction of the fragment matching the unbound state. The observed fast motions are fluctuations in pre-existing equilibrium configurations. The Brownian motion of domains in a harmonic potential is the appropriate model to examine functional hinge motions dependent on the structural topology and highlights the role of internal forces and friction to function.

  19. Fast antibody fragment motion: flexible linkers act as entropic spring

    SciTech Connect

    Stingaciu, Laura R.; Ivanova, Oxana; Ohl, Michael; Biehl, Ralf; Richter, Dieter

    2016-03-29

    A flexible linker region between three fragments allows antibodies to adjust their binding sites to an antigen or receptor. Using Neutron Spin Echo Spectroscopy we observed fragment motion on a timescale of 7 ns with motional amplitudes of about 1 nm relative to each other. The mechanistic complexity of the linker region can be described by a spring model with Brownian motion of the fragments in a harmonic potential. Displacements, timescale, friction and force constant of the underlying dynamics are accessed. The force constant exhibits a similar strength to an entropic spring, with friction of the fragment matching the unbound state. The observed fast motions are fluctuations in pre-existing equilibrium configurations. In conclusion, the Brownian motion of domains in a harmonic potential is the appropriate model to examine functional hinge motions dependent on the structural topology and highlights the role of internal forces and friction to function.

  20. Fast antibody fragment motion: flexible linkers act as entropic spring

    DOE PAGES

    Stingaciu, Laura R.; Ivanova, Oxana; Ohl, Michael; ...

    2016-03-29

    A flexible linker region between three fragments allows antibodies to adjust their binding sites to an antigen or receptor. Using Neutron Spin Echo Spectroscopy we observed fragment motion on a timescale of 7 ns with motional amplitudes of about 1 nm relative to each other. The mechanistic complexity of the linker region can be described by a spring model with Brownian motion of the fragments in a harmonic potential. Displacements, timescale, friction and force constant of the underlying dynamics are accessed. The force constant exhibits a similar strength to an entropic spring, with friction of the fragment matching the unboundmore » state. The observed fast motions are fluctuations in pre-existing equilibrium configurations. In conclusion, the Brownian motion of domains in a harmonic potential is the appropriate model to examine functional hinge motions dependent on the structural topology and highlights the role of internal forces and friction to function.« less

  1. Fast antibody fragment motion: flexible linkers act as entropic spring

    PubMed Central

    Stingaciu, Laura R.; Ivanova, Oxana; Ohl, Michael; Biehl, Ralf; Richter, Dieter

    2016-01-01

    A flexible linker region between three fragments allows antibodies to adjust their binding sites to an antigen or receptor. Using Neutron Spin Echo Spectroscopy we observed fragment motion on a timescale of 7 ns with motional amplitudes of about 1 nm relative to each other. The mechanistic complexity of the linker region can be described by a spring model with Brownian motion of the fragments in a harmonic potential. Displacements, timescale, friction and force constant of the underlying dynamics are accessed. The force constant exhibits a similar strength to an entropic spring, with friction of the fragment matching the unbound state. The observed fast motions are fluctuations in pre-existing equilibrium configurations. The Brownian motion of domains in a harmonic potential is the appropriate model to examine functional hinge motions dependent on the structural topology and highlights the role of internal forces and friction to function. PMID:27020739

  2. Influence of molecular size on tissue distribution of antibody fragments

    PubMed Central

    Li, Zhe; Krippendorff, Ben-Fillippo; Sharma, Sharad; Walz, Antje C.; Lavé, Thierry; Shah, Dhaval K.

    2016-01-01

    Biodistribution coefficients (BC) allow estimation of the tissue concentrations of proteins based on the plasma pharmacokinetics. We have previously established the BC values for monoclonal antibodies. Here, this concept is extended by development of a relationship between protein size and BC values. The relationship was built by deriving the BC values for various antibody fragments of known molecular weight from published biodistribution studies. We found that there exists a simple exponential relationship between molecular weight and BC values that allows the prediction of tissue distribution of proteins based on molecular weight alone. The relationship was validated by a priori predicting BC values of 4 antibody fragments that were not used in building the relationship. The relationship was also used to derive BC50 values for all the tissues, which is the molecular weight increase that would result in 50% reduction in tissue uptake of a protein. The BC50 values for most tissues were found to be ~35 kDa. An ability to estimate tissue distribution of antibody fragments based on the BC vs. molecular size relationship established here may allow better understanding of the biologics concentrations in tissues responsible for efficacy or toxicity. This relationship can also be applied for rational development of new biotherapeutic modalities with optimal biodistribution properties to target (or avoid) specific tissues. PMID:26496429

  3. Influence of molecular size on tissue distribution of antibody fragments.

    PubMed

    Li, Zhe; Krippendorff, Ben-Fillippo; Sharma, Sharad; Walz, Antje C; Lavé, Thierry; Shah, Dhaval K

    2016-01-01

    Biodistribution coefficients (BC) allow estimation of the tissue concentrations of proteins based on the plasma pharmacokinetics. We have previously established the BC values for monoclonal antibodies. Here, this concept is extended by development of a relationship between protein size and BC values. The relationship was built by deriving the BC values for various antibody fragments of known molecular weight from published biodistribution studies. We found that there exists a simple exponential relationship between molecular weight and BC values that allows the prediction of tissue distribution of proteins based on molecular weight alone. The relationship was validated by a priori predicting BC values of 4 antibody fragments that were not used in building the relationship. The relationship was also used to derive BC50 values for all the tissues, which is the molecular weight increase that would result in 50% reduction in tissue uptake of a protein. The BC50 values for most tissues were found to be ~35 kDa. An ability to estimate tissue distribution of antibody fragments based on the BC vs. molecular size relationship established here may allow better understanding of the biologics concentrations in tissues responsible for efficacy or toxicity. This relationship can also be applied for rational development of new biotherapeutic modalities with optimal biodistribution properties to target (or avoid) specific tissues.

  4. Expression and characterization of recombinant interleukin-21 receptor and its targeting single-chain variable fragment antibodies selected from a human phage display library.

    PubMed

    Wu, Qinhang; Zhang, Juan; Luo, Chen; Zhang, Tao; Wang, Tong; Wang, Min

    2012-10-01

    Interleukin-21 receptor (IL-21R) is widely expressed in lymphocytes, and plays an important role in immunological cell proliferation and cytokine production. The present study aims to express a recombinant extracellular domain of human IL-21R (rhIL-21R-ECD) with high yield, and to screen the anti-IL-21R single-chain variable fragments (scFvs) from a synthetic human phage display library. The rhIL-21R-ECD, being expressed mainly as insoluble inclusion bodies in Escherichia coli BL21 (DE3), was purified and refolded. ELISA analysis showed that the refolded rhIL-21R-ECD bound to its ligand IL-21 in a concentration-dependent manner. Using a phage display technique, anti-IL-21R scFvs were screened from a naïve human phage display library by biopanning. After four rounds of panning, positive clones were isolated, sequenced, and characterized. The clone with highest activity was designated as C2. Flow cytometry analysis showed that the scFv C2 could recognize IL-21R on Jurkat cells. Furthermore, proliferation assay revealed a concentration-dependent inhibitory effect of C2 on the Jurkat cell, with fifty percent inhibitory concentration (IC(50)) of 78 nM. A human scFv antibody C2 with a high binding specificity to IL-21R was isolated and characterized. The antibody showed a concentration-dependent inhibitory effect on Jurkat cell proliferation.

  5. Negative effects of a disulfide bond mismatch in anti-rabies G protein single-chain antibody variable fragment FV57.

    PubMed

    Duan, Ye; Gu, Tiejun; Zhang, Xizhen; Jiang, Chunlai; Yuan, Ruosen; Li, Zhuang; Wang, Dandan; Chen, Xiaoxu; Wu, Chunlai; Chen, Yan; Wu, Yongge; Kong, Wei

    2014-06-01

    Rabies virus (RV) causes a fatal infectious disease requiring efficient post-exposure prophylaxis (PEP), which includes a rabies vaccine and rabies immunoglobulin (RIG). The single-chain antibody variable fragment (scFv), a small engineered antibody fragment derived from an antibody variable heavy chain and light chain, has the potential to replace the current application of RIG. In previous studies, we constructed and evaluated an anti-rabies virus G protein scFv (FV57) based on the monoclonal antibody CR57. Of the five cysteines in FV57, four are linked in intra-chain disulfide bonds (Cys-VH28/Cys-VH98 and Cys-VL16/Cys-VL84), and one is free (Cys-VL85). However, the thiol in Cys-VL85 neighboring Cys-VL84 in the CDR3 of the light chain is likely to mismatch with the thiol in Cys-VL16 during the renaturing process. In order to study effects of the mismatched disulfide bond, Cys-VL85 and Cys-VL84 of FV57 were mutated to serine to construct mutants FV57(VL85S) and FV57(VL84S). Furthermore, the disulfide bonds in the light chain of FV57, FV57(VL85S) and FV57(VL84S) were deleted by mutating Cys-VL16 to serine. All mutants were prepared and evaluated along with the original FV57. The results indicated that the mismatched disulfide bond of FV57 linking the light chain FR1 and CDR3 would confer deleterious negative effects on its activity against RV, likely due to spatial hindrance in the light chain CDR3. Moreover, avoidance of the disulfide bond mismatch provided an additional 30% protective efficacy against RV infection in the mouse RV challenge model. Thus, modifications of FV57 to eliminate the disulfide bond mismatch may provide a candidate therapeutic agent for effective PEP against rabies.

  6. Polyreactivity of natural antibodies: exchange by HL-fragments.

    PubMed

    Sedykh, M A; Buneva, V N; Nevinsky, G A

    2013-12-01

    The polyreactivity of binding (formation of antibody (AB) complexes not only with specific but also with foreign antigens) is a widespread phenomenon that in some cases can be caused by a conformational lability of the antigen-binding sites of antibodies (which increases upon treatment with various destabilizing agents) and leads to AB binding with very different antigens. Some ABs exist as dimers of the initial ABs and their idiotypes (or anti-idiotypes) capable of producing intramolecular cyclic complexes with features of polyreactants. Another mechanism of binding polyreactivity is an exchange in blood by halves of IgG4 molecules (HL-fragments) against various antigens. Also, for the first time catalytic polyfunctionality of human milk ABs has been detected, which is caused by an exchange by HL-fragments between molecules of λ- and κ-IgG (IgG1-IgG4) and also by λ- and κ-sIgA against different antigens with formation of very different chimeric antibodies. This review considers all possible pathways of formation of polyspecific immunoglobulins and their biological functions described in the literature, as well as mechanisms of binding polyreactivity and catalytic polyfunctionality of natural antibodies.

  7. Large-Scale Purification of r28M: A Bispecific scFv Antibody Targeting Human Melanoma Produced in Transgenic Cattle

    PubMed Central

    Spiesberger, Katrin; Paulfranz, Florian; Egger, Anton; Reiser, Judith; Vogl, Claus; Rudolf-Scholik, Judith; Mayrhofer, Corina; Grosse-Hovest, Ludger; Brem, Gottfried

    2015-01-01

    Background 30 years ago, the potential of bispecific antibodies to engage cytotoxic T cells for the lysis of cancer cells was discovered. Today a variety of bispecific antibodies against diverse cell surface structures have been developed, the majority of them produced in mammalian cell culture systems. Beside the r28M, described here, no such bispecific antibody is known to be expressed by transgenic livestock, although various biologicals for medical needs are already harvested—mostly from the milk—of these transgenics. In this study we investigated the large-scale purification and biological activity of the bispecific antibody r28M, expressed in the blood of transgenic cattle. This tandem single-chain variable fragment antibody is designed to target human CD28 and the melanoma/glioblastoma-associated cell surface chondroitin sulfate proteoglycan 4 (CSPG4). Results With the described optimized purification protocol an average yield of 30 mg enriched r28M fraction out of 2 liters bovine plasma could be obtained. Separation of this enriched fraction by size exclusion chromatography into monomers, dimers and aggregates and further testing regarding the biological activity revealed the monomer fraction as being the most appropriate one to continue working with. The detailed characterization of the antibody’s activity confirmed its high specificity to induce the killing of CSPG4 positive cells. In addition, first insights into tumor cell death pathways mediated by r28M-activated peripheral blood mononuclear cells were gained. In consideration of possible applications in vivo we also tested the effect of the addition of different excipients to r28M. Conclusion Summing up, we managed to purify monomeric r28M from bovine plasma in a large-scale preparation and could prove that its biological activity is unaffected and still highly specific and thus, might be applicable for the treatment of melanoma. PMID:26469402

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. Xylella fastidiosa subsp pauca causes citrus variegat...

  9. ScFv anti-heparan sulfate antibodies unexpectedly activate endothelial and cancer cells through p38 MAPK: implications for antibody-based targeting of heparan sulfate proteoglycans in cancer.

    PubMed

    Christianson, Helena C; van Kuppevelt, Toin H; Belting, Mattias

    2012-01-01

    Tumor development requires angiogenesis and anti-angiogenic therapies have been introduced in the treatment of cancer. In this context, heparan sulfate proteoglycans (HSPGs) emerge as interesting targets, owing to their function as co-receptors of major, pro-angiogenic factors. Accordingly, previous studies have suggested anti-tumor effects of heparin, i.e. over-sulfated HS, and various heparin mimetics; however, a significant drawback is their unspecific mechanism of action and potentially serious side-effects related to their anticoagulant properties. Here, we have explored the use of human ScFv anti-HS antibodies (αHS) as a more rational approach to target HSPG function in endothelial cells (ECs). αHS were initially selected for their recognition of HS epitopes localized preferentially to the vasculature of patient glioblastoma tumors, i.e. highly angiogenic brain tumors. Unexpectedly, we found that these αHS exhibited potent pro-angiogenic effects in primary human ECs. αHS were shown to stimulate EC differentiation, which was associated with increased EC tube formation and proliferation. Moreover, αHS supported EC survival under hypoxia and starvation, i.e. conditions typical of the tumor microenvironment. Importantly, αHS-mediated proliferation was efficiently counter-acted by heparin and was absent in HSPG-deficient mutant cells, confirming HS-specific effects. On a mechanistic level, binding of αHS to HSPGs of ECs as well as glioblastoma cells was found to trigger p38 MAPK-dependent signaling resulting in increased proliferation. We conclude that several αHS that recognize HS epitopes abundant in the tumor vasculature may elicit a pro-angiogenic response, which has implications for the development of antibody-based targeting of HSPGs in cancer.

  10. Construction of an antimyoglobin single-chain variable fragment with rapid reaction kinetics.

    PubMed

    Jang, Jun-Hyuck; Kim, Dong-Hyung; Paek, Se-Hwan; Woo, Eui-Jeon; Kim, Young-Wan

    2016-01-01

    Antibodies with rapid reaction kinetics (high association and dissociation rates), named reversible antibodies, are used to perform continuous monitoring of sensitive disease biomarkers. In cases of acute myocardial infarction (AMI), continuous monitoring and early diagnosis are important. Human myoglobin (Myo) is a useful biomarker for AMI during the early stage after the onset of symptoms. In this study, a single-chain variable fragment (scFv) specific to Myo was derived from an IgG antibody that has rapid reaction kinetics. Enzyme-linked immunosorbent assay revealed that recombinant scFv exhibited 3.8-fold reduced affinity compared with the parent IgG antibody based on the antibody concentration necessary for 50% of the maximum signal. The scFv retained the rapid reaction kinetic mode with average kon and koff of 2.63 × 10(5) M(-1) Sec(-1) and 3.25 × 10(-3) Sec(-1) , respectively, which were reduced to 10- and 2.3-fold compared with those of the parent antibody. The equilibrium constant for the association of the scFv (KA = 8.09 × 10(7) M(-1) ) was 4.6-fold lower than that of its parent IgG antibody. This scFv may be a starting point for further mutagenesis/kinetic and structural analyses providing valuable insight into the mechanism of reversible antibodies.

  11. Generation of high-affinity fully human anti-interleukin-8 antibodies from its cDNA by two-hybrid screening and affinity maturation in yeast.

    PubMed

    Ding, Ling; Azam, Mark; Lin, Yu-Huei; Sheridan, James; Wei, Shuanghong; Gupta, Gigi; Singh, Rakesh K; Pauling, Michelle H; Chu, Waihei; Tran, Antares; Yu, Nai-Xuan; Hu, Jiefeng; Wang, Wei; Long, Hao; Xiang, Dong; Zhu, Li; Hua, Shao-Bing

    2010-10-01

    We have developed a technology for rapidly generating novel and fully human antibodies by simply using the antigen DNA. A human single-chain variable fragment (scFv) antibody library was constructed in a yeast two-hybrid vector with high complexity. After cloning cDNA encoding the mature sequence of human interleukin-8 (hIL8) into the yeast two-hybrid system vector, we have screened the human scFv antibody library and obtained three distinct scFv clones that could specifically bind to hIL8. One clone was chosen for further improvement by a novel affinity maturation process using the error-prone PCR of the scFv sequence followed by additional rounds of yeast two-hybrid screening. The scFv antibodies of both primary and affinity-matured scFv clones were expressed in E. coli. All purified scFvs showed specific binding to hIL8 in reciprocal coimmunoprecipitation and ELISA assays. All scFvs, as well as a fully human IgG antibody converted from one of the scFv clones and expressed in the mammalian cells, were able to effectively inhibit hIL8 in neutrophil chemotaxis assays. The technology described can generate fully human antibodies with high efficiency and low cost.

  12. Purification of human monoclonal antibodies and their fragments.

    PubMed

    Müller-Späth, Thomas; Morbidelli, Massimo

    2014-01-01

    This chapter summarizes the most common chromatographic mAb and mAb fragment purification methods, starting by elucidating the relevant properties of the compounds and introducing the various chromatography modes that are available and useful for this application. A focus is put on the capture step affinity and ion exchange chromatography. Aspects of scalability play an important role in judging the suitability of the methods. The chapter introduces also analytical chromatographic methods that can be utilized for quantification and purity control of the product. In the case of mAbs, for most purposes the purity obtained using an affinity capture step is sufficient. Polishing steps are required if material of particularly high purity needs to be generated. For mAb fragments, affinity chromatography is not yet fully established, and the capture step potentially may not provide material of high purity. Therefore, the available polishing techniques are touched upon briefly. In the case of mAb isoform and bispecific antibody purification, countercurrent chromatography techniques have been proven to be very useful and a part of this chapter has been dedicated to them, paying tribute to the rising interest in these antibody formats in research and industry.

  13. A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses

    PubMed Central

    Liu, Han-Lin; Lin, Wei-Fang; Hu, Wen-Chi; Lee, Yung-An

    2015-01-01

    Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (VH) and light (VL) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (VL-VH) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli. PMID:26209665

  14. A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses.

    PubMed

    Liu, Han-Lin; Lin, Wei-Fang; Hu, Wen-Chi; Lee, Yung-An; Chang, Ya-Chun

    2015-10-01

    Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (VH) and light (VL) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (VL-VH) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli.

  15. Synthesis and pre-clinical evaluation of an 18F-labeled single-chain antibody fragment for PET imaging of epithelial ovarian cancer

    PubMed Central

    Sharma, Sai Kiran; Wuest, Melinda; Way, Jenilee D; Bouvet, Vincent R; Wang, Monica; Wuest, Frank R

    2016-01-01

    Anti-CA125 antibodies have been used in immunoassays to quantify levels of shed antigen in the serum of patients who are under surveillance for epithelial ovarian cancer (EOC). However, there is currently no molecular imaging probe in the clinic for the assessment of CA125 expression in vivo. The present study describes the development of an 18F-labeled single-chain variable fragment (scFv) for PET imaging of CA125 in preclinical EOC models. Anti-CA125 scFv was derived from MAb-B43.13 by recombinant expression of the fragment in E.coli. Fragment scFv-B43.13 was purified via immobilized metal affinity chromatography and characterized for antigen binding via immuno-staining and flow cytometry. Prosthetic group N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) was used for radiolabeling of scFv-B43.13. Preclinical ovarian cancer models were developed based on ovarian cancer cell lines OVCAR3 (CA125-positive) and SKOV3 (CA125-negative) in NIH-III mice. The radiopharmacological profile of 18F-labeled scFv-B43.13 ([18F]FBz-scFv-B43.13) was studied with PET. [18F]FBz-scFv-B43.13 was prepared in radiochemical yields of 3.7 ± 1.8% (n = 5) at an effective specific activity of 3.88 ± 0.76 GBq/µmol (n = 5). The radiotracer demonstrated selective uptake in CA125-positive OVCAR3 cells and virtually no uptake in CA125-negative SKOV3 cells. Standardized uptake values (SUV) of radioactivity uptake in OVCAR3 tumors was 0.5 (n = 3) and 0.3 (n = 2) in SKOV3 tumors after 60 min post injection (p.i.). PMID:27508105

  16. Exploitation of rolling circle amplification for the construction of large phage-display antibody libraries.

    PubMed

    Shahsavarian, Melody A; Le Minoux, Damien; Matti, Kalyankumar M; Kaveri, Srini; Lacroix-Desmazes, Sébastien; Boquet, Didier; Friboulet, Alain; Avalle, Bérangère; Padiolleau-Lefèvre, Séverine

    2014-05-01

    Phage display antibody libraries have proven to have a significant role in the discovery of therapeutic antibodies and polypeptides with desired biological and physicochemical properties. Obtaining a large and diverse phage display antibody library, however, is always a challenging task. Various steps of this technique can still undergo optimization in order to obtain an efficient library. In the construction of a single chain fragment variable (scFv) phage display library, the cloning of the scFv fragments into a phagemid vector is of crucial importance. An efficient restriction enzyme digestion of the scFv DNA leads to its proper ligation with the phagemid followed by its successful cloning and expression. Here, we are reporting a different approach to enhance the efficiency of the restriction enzyme digestion step. We have exploited rolling circle amplification (RCA) to produce a long strand of DNA with tandem repeats of scFv sequences, which is found to be highly susceptible to restriction digestion. With this important modification, we are able to construct a large phage display antibody library of naive SJL/J mice. The size of the library is estimated as ~10(8) clones. The number of clones containing a scFv fragment is estimated at 90%. Hence, the present results could considerably aid the utilization of the phage-display technique in order to get an efficiently large antibody library.

  17. Expression, purification, and characterization of anti-plumbagin single-chain variable fragment antibody in Sf9 insect cell.

    PubMed

    Sakamoto, Seiichi; Taura, Futoshi; Tsuchihashi, Ryota; Putalun, Waraporn; Kinjo, Junei; Tanaka, Hiroyuki; Morimoto, Satoshi

    2010-12-01

    Plumbagin (PL; 5-hydroxy-2-methyl-1, 4-naphthoquinone) is an important secondary metabolite, mainly produced in the Plumbago zeylanica L. (Plumbaginaceae). A single-chain variable fragment (scFv) antibody, fusion of the variable regions of the heavy chain and light chain of immunoglobulin against PL (PL-scFv) was expressed by Bac-to-Bac Baculovirus Expression System using Spodoptera frugiperda (Sf9) insect cells and characterized to investigate potential use of PL-scFv as a tool for plant immunomodulation. Functional PL-scFv expressed in the Sf9 insect cells were purified using cation exchange chromatography followed by immobilized metal ion affinity chromatography (IMAC). The yields of the purified PL-scFv in the culture supernatant and Sf9 insect cells were 2.0 mg and 5.2 mg per 1 liter of Sf9 culture medium, respectively. Recombinant purified PL-scFv was then characterized by the indirect competitive enzyme-linked immunosorbent assay (ELISA). The cross-reactivity and sensitivity of PL-scFv expressed in Sf9 insect cells were compared with PL-scFv expressed in Escherichia coli and its parental anti-plumbagin monoclonal antibody (MAb 3A3) secreted from hybridoma cells. Intriguingly, the specificity of the PL-scFv expressed in Sf9 insect cells was found to be different from that expressed in E. coli and parental MAb 3A3, although the detectable level (0.2-25 μg/mL) was the same in ELISA using each antibody. Even more interestingly, the characteristics of PL-scFv, which have wide cross-reactivity against 1,4-napththoquinone, suggest its potential use as a tool for plant immunomodulation not only for breeding Plumbaginacea family containing PL but also for breeding other medicinal plants containing bioactive naphthoquinones.

  18. High-level iodination of monoclonal antibody fragments for radiotherapy

    SciTech Connect

    Ferens, J.M.; Krohn, K.A.; Beaumier, P.L.; Brown, J.P.; Hellstroem, I.; Hellstroem, K.E.; Carrasquillo, J.A.; Larson, S.M.

    1984-03-01

    Two different murine monoclonal antibody Fab fragments specific for p97, a melanoma-associated antigen, were labeled with I-131 at high activity levels without excessive chemical damage. Up to 20 mg of Fab were labeled with up to 300 mCi of I-131 using the chloramine-T method and large working volumes at room temperature. As much as 90% of the initial activity was recovered as labeled product. The labeled Fabs varied in their sensitivity to radioiodination damage, as measured by an in vitro cell-binding assay. Radioiodination was performed safely using a remote iodination apparatus. The final product was of radiopharmaceutical quality suitable for clinical diagnosis and experimental radiotherapy in humans.

  19. A Nanotechnology-Based Platform for Extending the Pharmacokinetic and Binding Properties of Anti-methamphetamine Antibody Fragments

    PubMed Central

    Nanaware-Kharade, Nisha; Thakkar, Shraddha; Gonzalez III, Guillermo A.; Peterson, Eric C.

    2015-01-01

    To address the need for effective medications to aid in the treatment of methamphetamine (METH) abuse, we used a nanotechnology approach to customize the in vivo behavior of an anti-METH single chain antibody (scFv7F9Cys). Anti-METH scFv7F9Cys was conjugated to dendrimer nanoparticles via a polyethylene glycol (PEG) linker to generate high-order conjugates termed dendribodies. We found that the high affinity (KD = 6.2 nM) and specificity for METH was unchanged after nanoparticle conjugation. The dendribodies were administered in an i.v. bolus to male Sprague Dawley rats after starting a s.c. infusion of METH. The PCKN values for clearance and volume of distribution of scFv7F9Cys after conjugation to dendrimers decreased 45 and 1.6-fold respectively, and the terminal elimination half-life increased 20-fold. Organ distribution of scFv7F9Cys and dendribody in blood and urine agreed well with the PCKN data. Renal clearance appeared to be the major route of elimination for both experimental medications. We have thus successfully developed a novel multivalent METH-binding nanomedicine by conjugating multiple anti-METH scFvs to dendrimer nanoparticles, extending the scFv half-life from 1.3 (±0.3) to 26 (±2.6) hr. These data suggest that the dendribody design could be a feasible platform for generating multivalent antibodies with customizable PCKN profiles. PMID:26159352

  20. A Nanotechnology-Based Platform for Extending the Pharmacokinetic and Binding Properties of Anti-methamphetamine Antibody Fragments.

    PubMed

    Nanaware-Kharade, Nisha; Thakkar, Shraddha; Gonzalez, Guillermo A; Peterson, Eric C

    2015-07-10

    To address the need for effective medications to aid in the treatment of methamphetamine (METH) abuse, we used a nanotechnology approach to customize the in vivo behavior of an anti-METH single chain antibody (scFv7F9Cys). Anti-METH scFv7F9Cys was conjugated to dendrimer nanoparticles via a polyethylene glycol (PEG) linker to generate high-order conjugates termed dendribodies. We found that the high affinity (KD = 6.2 nM) and specificity for METH was unchanged after nanoparticle conjugation. The dendribodies were administered in an i.v. bolus to male Sprague Dawley rats after starting a s.c. infusion of METH. The PCKN values for clearance and volume of distribution of scFv7F9Cys after conjugation to dendrimers decreased 45 and 1.6-fold respectively, and the terminal elimination half-life increased 20-fold. Organ distribution of scFv7F9Cys and dendribody in blood and urine agreed well with the PCKN data. Renal clearance appeared to be the major route of elimination for both experimental medications. We have thus successfully developed a novel multivalent METH-binding nanomedicine by conjugating multiple anti-METH scFvs to dendrimer nanoparticles, extending the scFv half-life from 1.3 (± 0.3) to 26 (± 2.6) hr. These data suggest that the dendribody design could be a feasible platform for generating multivalent antibodies with customizable PCKN profiles.

  1. Production and characterization of recombinant scFv against digoxin by phage display technology.

    PubMed

    Alirezapour, Behruz; Rajabibazl, Masoumeh; Rasaee, Mohhamad Javad; Omidfar, Kobra

    2013-06-01

    The cardiac glycoside digoxin is widely used for the treatment of congestive heart failure and cardiac arrhythmias. Digoxin is a highly toxic drug and consequently is routinely measured in sera of treated patients. In such cases, antibodies are required against digoxin for detection as well as detoxification purposes. To obtain recombinant single chain antibody against digoxin, RNA was extracted from spleen of BALB/c mice immunized with digoxin-BSA and converted to cDNA. The gene fragment corresponding to the variable regions of the repertoire of antibody genes were amplified by PCR. ScFv construct was generated by randomly joining individual heavy- and light-chain variable domains through gene splicing by overlapping extension PCR. Recombinant phage library expressing scFv polypeptides were produced. Phages with higher affinity toward digoxin were selected in the biopanning process. Sensitivity of produced recombinant MAb (AR85) was determined to be about 100 pg/well, while intact MAb (BBA) produced by hybridoma technology (data not shown) was reported to be around 100 pg/well too. The saturation value for recombinant scFv MAb was found to be 1000 ng/well while that for hybridoma MAb was reported to be 10 ng/well. The affinity constant of recombinant MAb (AR85) towards digoxin was also found to be around ka=3.8×10(7) M(-1) while that for hybridoma MAb (BBA) was reported to be ka=2.6×10(8) M(-1).

  2. Anti-CD20 single chain variable antibody fragment-apolipoprotein A-I chimera containing nanodisks promote targeted bioactive agent delivery to CD20-positive lymphomas.

    PubMed

    Crosby, Natasha M; Ghosh, Mistuni; Su, Betty; Beckstead, Jennifer A; Kamei, Ayako; Simonsen, Jens B; Luo, Bing; Gordon, Leo I; Forte, Trudy M; Ryan, Robert O

    2015-08-01

    A fusion protein comprising an α-CD20 single chain variable fragment (scFv) antibody, a spacer peptide, and human apolipoprotein (apo) A-I was constructed and expressed in Escherichia coli. The lipid interaction properties intrinsic to apoA-I as well as the antigen recognition properties of the scFv were retained by the chimera. scFv•apoA-I was formulated into nanoscale reconstituted high-density lipoprotein particles (termed nanodisks; ND) and incubated with cultured cells. α-CD20 scFv•apoA-I ND bound to CD20-positive non-Hodgkins lymphoma (NHL) cells (Ramos and Granta) but not to CD20-negative T lymphocytes (i.e., Jurkat). Binding to NHL cells was partially inhibited by pre-incubation with rituximab, a monoclonal antibody directed against CD20. Confocal fluorescence microscopy analysis of Granta cells following incubation with α-CD20 scFv•apoA-I ND formulated with the intrinsically fluorescent hydrophobic polyphenol, curcumin, revealed α-CD20 scFv•apoA-I localizes to the cell surface, while curcumin off-loads and gains entry to the cell. Compared to control incubations, viability of cultured NHL cells was decreased upon incubation with α-CD20 scFv•apoA-I ND harboring curcumin. Thus, formulation of curcumin ND with α-CD20 scFv•apoA-I as the scaffold component confers cell targeting and enhanced bioactive agent delivery, providing a strategy to minimize toxicity associated with chemotherapeutic agents.

  3. Holliday junctions generate super-bright antibodies and antibody fragments in sortase-catalyzed reactions

    PubMed Central

    Li, Zeyang; Theile, Christopher S.; Chen, Guan-Yu; Bilate, Angelina M.; Duarte, Joao N.; Avalos, Ana M.; Fang, Tao; Barberena, Roberto; Sato, Shuji; Ploegh, Hidde L.

    2015-01-01

    Site-specific modification of proteins with fluorophores can render a protein fluorescent without compromising its function. To avoid self-quenching of multiple fluorophores installed in close proximity, we used Holliday junctions to label proteins site-specifically. Holliday junctions enable modification with multiple fluorophores at reasonably precise spacing. We designed a Holliday junction with three of its four arms modified with a fluorophore of choice and the remaining arm equipped with a dibenzocyclooctyne substituent to render it reactive with an azide-modified, fluorescent single domain antibody fragment or an intact immunoglobulin, produced in a sortase-catalyzed reaction. We conclude that fluorescent Holliday junctions improve fluorescence yields for both single domain and full-sized antibodies, without deleterious effects on antigen binding. PMID:26252716

  4. Characterization of Tumor-Avid Antibody Fragments Genetically Engineered for Mono-Specific Radionuclide Chelation

    SciTech Connect

    Quinn, T.P.

    2003-12-31

    The successful clinical application of targeted-radiopharmaceuticals depends on the development of molecules that optimize tumor specific radionuclide deposition and minimize non-specific organ irradiation. To this end, this proposal outlines a research effort to identify and evaluate novel antibodies and antibody fragments that bind breast tumors. The tumor-avid antibodies will be investigated for as imaging and therapeutic agents and to gain a better understanding of the pharmacokinetics and metabolism of radiolabeled tumor-avid antibody fragments through the use of site-specifically labeled molecules. Antibodies or antibody fragments, that bind breast carcinoma carbohydrate antigens, will be obtained from hybridoma or bacteriophage library screening. More specifically, antibody fragments that bind the carcinoma-associated Thomsen-Friedenreich (T) antigen will be radiolabeled with {sup 99m}Tc and {sup 188}Re at a natural amino acid chelation site and will be investigated in vivo for their abilities to target human breast tumors. In addition, site-specific radiolabeled antibody fragments will be biosynthesized using misacylated suppressor tRNAs. Homogeneously radiolabeled populations of antibody fragments will be used to investigate the effects of radionuclide location and chelation chemistries on their biodistribution and metabolism. It is hypothesized that site-specifically radiolabeled antibody fragments will possess enhanced tumor imaging and therapeutic properties due to optimal label location and conjugation chemistries. New insights into the factors that govern antibody metabolism in vivo are also expected from this work. Results from these studies should enhance our ability to design and synthesize radiolabeled antibody fragments that have improved pharmacokinetic properties. The studies in this proposal involve basic research into the development of antibody-based radiopharmaceuticals, with the ultimate goal of application in humans. This type of basic

  5. Solution Equilibrium Titration for High-Throughput Affinity Estimation of Unpurified Antibodies and Antibody Fragments.

    PubMed

    Della Ducata, Daniela; Jaehrling, Jan; Hänel, Cornelia; Satzger, Marion; Wolber, Meike; Ostendorp, Ralf; Pabst, Stefan; Brocks, Bodo

    2015-12-01

    The generation of therapeutic antibodies with extremely high affinities down to the low picomolar range is today feasible with state-of-the art recombinant technologies. However, reliable and efficient identification of lead candidates with the desired affinity from a pool of thousands of antibody clones remains a challenge. Here, we describe a high-throughput procedure that allows reliable affinity screening of unpurified immunoglobulin G or antibody fragments. The method is based on the principle of solution equilibrium titration (SET) using highly sensitive electrochemiluminescence as a readout system. Because the binding partners are not labeled, the resulting KD represents a sound approximation of the real affinity. For screening, diluted bacterial lysates or cell culture supernatants are equilibrated with four different concentrations of a soluble target molecule, and unbound antibodies are subsequently quantified on 384-well Meso Scale Discovery (MSD) plates coated with the respective antigen. For determination of KD values from the resulting titration curves, fit models deduced from the law of mass action for 1:1 and 2:1 binding modes are applied to assess hundreds of interactions simultaneously. The accuracy of the method is demonstrated by comparing results from different screening campaigns from affinity optimization projects with results from detailed affinity characterization.

  6. Single chain Fab (scFab) fragment

    PubMed Central

    Hust, Michael; Jostock, Thomas; Menzel, Christian; Voedisch, Bernd; Mohr, Anja; Brenneis, Mariam; Kirsch, Martina I; Meier, Doris; Dübel, Stefan

    2007-01-01

    Background The connection of the variable part of the heavy chain (VH) and and the variable part of the light chain (VL) by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv) was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv) fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab) with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display. Results Here, we demonstrate that the introduction of a polypeptide linker between the fragment difficult (Fd) and the light chain (LC), resulting in the formation of a single chain Fab fragment (scFab), can lead to improved production of functional molecules. We tested the impact of various linker designs and modifications of the constant regions on both phage display efficiency and the yield of soluble antibody fragments. A scFab variant without cysteins (scFabΔC) connecting the constant part 1 of the heavy chain (CH1) and the constant part of the light chain (CL) were best suited for phage display and production of soluble antibody fragments. Beside the expression system E. coli, the new antibody format was also expressed in Pichia pastoris. Monovalent and divalent fragments (DiFabodies) as well as multimers were characterised. Conclusion A new antibody design offers the generation of bivalent Fab derivates for antibody phage display and production of soluble antibody fragments. This antibody format is of particular value for high throughput proteome binder generation projects, due to the avidity effect and the possible use of common standard sera

  7. Cloning and expression of an anti-LDL(-) single-chain variable fragment, and its inhibitory effect on experimental atherosclerosis.

    PubMed

    Kazuma, Soraya M; Cavalcante, Marcela F; Telles, Andréia E R; Maranhão, Andrea Queiroz; Abdalla, Dulcineia S P

    2013-01-01

    The in vivo modified forms of low-density lipoprotein (LDL) are important for the formation of foam cells and as mediators of the immuno-inflammatory process involved in the progression of atherosclerosis. Electronegative LDL, LDL(-), is a LDL subfraction with pro-inflammatory properties that is present in human blood. To investigate possible atheroprotective effects, an anti-LDL(-) single-chain variable fragment (scFv) was expressed in the methylotrophic yeast Pichia pastoris and its activity was evaluated in vitro against macrophages and in experimental atherosclerosis in Ldlr(-/-) mice. The recombinant 2C7 scFv was produced in a yield of 9.5 mg of protein/L. The specificity and affinity of purified 2C7 scFv against LDL(-) was confirmed by ELISA. To assess the activity of 2C7 scFv on foam cell formation, RAW 264.7 macrophages were exposed to LDL(-) in the presence or absence of 2C7 scFv. The 2C7 scFv inhibited the uptake of LDL(-) by macrophages in a dose-dependent manner, and internalization of LDL(-) by these cells was found to be mediated by the CD36 and CD14 receptor. In addition, compared with untreated cells, lipid accumulation in macrophages was decreased, and the expression of Cd36, Tlr-4 and Cox-2 was downregulated in macrophages treated with 2C7 scFv. Importantly, compared with untreated mice, the treatment of Ldlr(-/-) mice with 2C7 scFv decreased the atherosclerotic lesion area at the aortic sinus. In conclusion, our data show that 2C7 scFv inhibits foam cell formation and atherosclerotic plaque development by modulating the expression of genes relevant to atherogenesis. These results encourage further use of this antibody fragment in the development of new therapeutic strategies that neutralize the pro-atherogenic effects of LDL(-).

  8. Digoxin-Specific Antibody Fragment Dosing: A Case Series.

    PubMed

    Chhabra, Neeraj; Valento, Matthew; Bryant, Sean M; Aks, Steven E

    Digoxin-specific antibody fragments (DSFab) are used for the treatment of poisoning by cardiac glycosides, such as pharmaceutical digoxin. Dosing of this therapy for chronic and acute poisonings is based on the steady-state serum concentrations of digoxin, historical data in acute ingestions, or empiric regimens purportedly based on the average requirements. Empiric dosing for adult patients involves utilization of 3-6 vials for chronic poisoning and 10-20 vials for acute poisoning. The aim of this study was to describe the average dosing requirements based on the steady-state serum concentration of digoxin or historical data and compare this with the empiric dosing regimens. We performed a retrospective analysis of cases over an 11-year period presented to the Illinois Poison Center where administration of DSFab was recommended. We identified 140 cases of chronic digoxin poisoning and 26 cases or acute digoxin poisoning for analysis. The average dose of DSFab recommended in the cases of chronic digoxin poisoning was 3.05 vials (SD ± 1.31). The average dose of DSFab recommended in the cases of acute digoxin poisoning was 6.33 vials (SD ± 5.26). These values suggest that empiric dosing regimens may overestimate the need for DSFab in cases of both chronic and acute poisonings of pharmaceutical digoxin.

  9. A Combinatory Antibody–Antigen Microarray Assay for High-Content Screening of Single-Chain Fragment Variable Clones from Recombinant Libraries

    PubMed Central

    Jansson, Bo; Stuhr-Hansen, Nicolai; Kovács, András; Welinder, Charlotte

    2016-01-01

    We have developed a combinatory antibody–antigen microarray for direct screening of multiple single-chain fragment variable (scFv) clones with no need for pre-purification or enrichment before screening. The straightforward workflow allows for early selection of binders to predefined peptide and glycopeptide targets. A capture antibody is contact printed on microarray slides, side by side with the antigens of interest. A large number of scFv clones, in supernatants, are printed on top of the capture antibody and the antigen in a “spot-on-spot” print. The printed scFv clones, which bind to the capture antibody, are detected using biotinylated antigen, while the binding of scFv clones to the printed antigen is detected through a mouse anti-tag antibody. Two different analyses are thus performed on the same slide, generating two kinds of information: one on the ability of an individual scFv clone to bind to the soluble form of the antigen, which may favour selection for higher affinity rather than avidity, while the other allows the identification of large numbers of clones, simultaneously, due to the binding of scFv clones to densely presented antigens, thus providing an overall increased hit rate. The functionality of the new screening approach was illustrated through the generation of antibodies against peptides from the chaperone complex Ku70/Ku80 and the GalNAcα-serine/threonine epitope on the IgA1 alpha chain hinge region. In total, 659 scFv clones were screened with a hit rate of approximately 20%. This approach allowed the identification of functional antibodies in both cases, illustrating the usefulness and capacity of this combinatory microarray screening technique for efficient analysis and validation of antibodies at an early stage of antibody generation. PMID:28002485

  10. Pharmacological efficacy of anti-IL-1β scFv, Fab and full-length antibodies in treatment of rheumatoid arthritis.

    PubMed

    Qi, Jianying; Ye, Xianlong; Ren, Guiping; Kan, Fangming; Zhang, Yu; Guo, Mo; Zhang, Zhiyi; Li, Deshan

    2014-02-01

    Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that mainly causes the synovial joint inflammation and cartilage destruction. Interleukin-1β (IL-1β) is an important proinflammatory cytokine involved in the pathogenesis of RA. In this study, we constructed and expressed anti-IL-1β-full-length antibody in CHO-K1-SV, anti-IL-1β-Fab and anti-IL-1β-scFv in Rosetta. We compared the therapeutic efficacy of three anti-IL-1β antibodies for CIA mice. Mice with CIA were subcutaneously injected with humanized anti-IL-1β-scFv, anti-IL-1β-Fab or anti-IL-1β-full-length antibody. The effects of treatment were determined by arthritis severity score, autoreactive humoral, cellular immune responses, histological lesion and cytokines production. Compared with anti-IL-1β-scFv treatments, anti-IL-1β-Fab and anti-IL-1β-full-length antibody therapy resulted in more significant effect in alleviating the severity of arthritis by preventing bone damage and cartilage destruction, reducing humoral and cellular immune responses, and down-regulating the expression of IL-1β, IL-6, IL-2, IFN-γ, TNF-α and MMP-3 in inflammatory tissue. The therapeutic effects of anti-IL-1β-Fab and anti-IL-1β-full-length antibodies on CIA mice had no significant difference. However, production of anti-IL-1β-full-length antibody in eukaryotic system is, in general, time-consuming and more expensive than that of anti-IL-1β-Fab in prokaryotic systems. In conclusion, as a small molecule antibody, anti-IL-1β-Fab is an ideal candidate for RA therapy.

  11. Selection of a human butyrylcholinesterase-like antibody single-chain variable fragment resistant to AChE inhibitors from a phage library expressed in E. coli

    PubMed Central

    Podestà, Adriano; Rossi, Serena; Massarelli, Ilaria; Carpi, Sara; Adinolfi, Barbara; Fogli, Stefano; Bianucci, Anna Maria; Nieri, Paola

    2014-01-01

    Organophosphates are potent poisoning agents that cause severe cholinergic toxicity. Current treatment has been reported to be unsatisfactory and novel antidotes are needed. In this study, we used a single-chain variable fragment (scFv) library to select a recombinant antibody fragment (WZ1–14.2.1) with butyrylcholinesterase-like catalytic activity by using an innovative method integrating genetic selection and the bait-and-switch strategy. Ellman assay demonstrated that WZ1–14.2.1 has Michaelis-Menten kinetics in the hydrolysis of all the three substrates used, acetylthiocholine, propionylthiocholine and butyrylthiocholine. Notably, the catalytic activity was resistant to the following acetylcholinesterase inhibitors: neostigmine, iso-OMPA, chlorpyrifos oxon, dichlorvos, and paraoxon ethyl. Otherwise, the enzymatic activity of WZ1–14.2.1 was inhibited by the selective butyrylcholinesterase inhibitor, ethopropazine, and by the Ser-blocking agent phenylmethanesuphonyl fluoride. A hypothetical 3D structure of the WZ1–14.2.1 catalytic site, compatible with functional results, is proposed on the basis of a molecular modeling analysis. PMID:24675419

  12. Selection of a human butyrylcholinesterase-like antibody single-chain variable fragment resistant to AChE inhibitors from a phage library expressed in E. coli.

    PubMed

    Podestà, Adriano; Rossi, Serena; Massarelli, Ilaria; Carpi, Sara; Adinolfi, Barbara; Fogli, Stefano; Bianucci, Anna Maria; Nieri, Paola

    2014-01-01

    Organophosphates are potent poisoning agents that cause severe cholinergic toxicity. Current treatment has been reported to be unsatisfactory and novel antidotes are needed. In this study, we used a single-chain variable fragment (scFv) library to select a recombinant antibody fragment (WZ1-14.2.1) with butyrylcholinesterase-like catalytic activity by using an innovative method integrating genetic selection and the bait-and-switch strategy. Ellman assay demonstrated that WZ1-14.2.1 has Michaelis-Menten kinetics in the hydrolysis of all the three substrates used, acetylthiocholine, propionylthiocholine and butyrylthiocholine. Notably, the catalytic activity was resistant to the following acetylcholinesterase inhibitors: neostigmine, iso-OMPA, chlorpyrifos oxon, dichlorvos, and paraoxon ethyl. Otherwise, the enzymatic activity of WZ1-14.2.1 was inhibited by the selective butyrylcholinesterase inhibitor, ethopropazine, and by the Ser-blocking agent phenylmethanesuphonyl fluoride. A hypothetical 3D structure of the WZ1-14.2.1 catalytic site, compatible with functional results, is proposed on the basis of a molecular modeling analysis.

  13. Photoluminescence detection of 2,4,6-trinitrotoluene (TNT) binding on diatom frustule biosilica functionalized with an anti-TNT monoclonal antibody fragment.

    PubMed

    Zhen, Le; Ford, Nicole; Gale, Debra K; Roesijadi, Guritno; Rorrer, Gregory L

    2016-05-15

    A selective and label-free biosensor for detection of the explosive compound 2,4,6-trinitrotoluene (TNT) in aqueous solution was developed based on the principle of photoluminescence quenching of upon immunocomplex formation with antibody-functionalized diatom frustule biosilica. The diatom frustule is an intricately nanostructured, highly porous biogenic silica material derived from the shells of microscopic algae called diatoms. This material emits strong visible blue photoluminescence (PL) upon UV excitation. PL-active frustule biosilica was isolated from cultured cells of the marine diatom Pinnularia sp. and functionalized with a single chain variable fragment (scFv) derived from an anti-TNT monoclonal antibody. When TNT was bound to the anti-TNT scFv-functionalized diatom frustule biosilica, the PL emission from the biosilica was partially quenched due to the electrophilic nature of the nitro (-NO2) groups on the TNT molecule. The dose-response curve for immunocomplex formation of TNT on the scFv-functionalized diatom frustule biosilica had a half-saturation binding constant of 6.4 ± 2.4·10(-8)M and statistically-significant measured detection limit of 3.5·10(-8)M. The binding and detection were selective for TNT and TNB (trinitrobenzene) but not RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) or 2,6-DNT (2,6-dinitrotoluene).

  14. Site-directed immobilisation of antibody fragments for detection of C-reactive protein.

    PubMed

    Vikholm-Lundin, Inger; Albers, Willem M

    2006-01-15

    C-reactive protein, CRP antibody Fab'-fragments have been attached on pre-cleaned gold slides and protein repellent polymers have been used to block the remaining free space in between the antibody fragments. At optimal conditions the antibody fragments are site-directly immobilised on the surface and non-specific binding is reduced. The amount of Fab'-fragments in the polymer host monolayer has been optimised for various buffers. Binding of CRP to Fab'-fragment/polymer layers produced in phosphate buffered saline decreased with NaCl salt concentration. In a 1M NaCl phosphate buffer, the antibodies seem to be randomly oriented on the surface with a similar response to CRP as that of an antibody F(ab)(2)-fragment layer. In a 150 mM NaCl phosphate buffer, on the other hand, the fragments seem to be site-directly oriented and the response to CRP was fivefold. The highest response to CRP was obtained to a layer with a Fab'-fragment concentration of 60 microg/ml. CRP could be detected in a concentration range of 1 ng/ml to 50 microg/ml from a standard solution in phosphate buffer and in a range of 4 ng/ml to 50 microg/ml from serum/PBS. CRP was, moreover, successfully detected in patient samples with good reproducibility. The layer would thus be sensitive enough to analyse the CRP concentration in human serum for predicting cardiovascular disease.

  15. Effect of radiochemical modification on biodistribution of scFvD2B antibody fragment recognising prostate specific membrane antigen.

    PubMed

    Frigerio, Barbara; Benigni, Fabio; Luison, Elena; Seregni, Ettore; Pascali, Claudio; Fracasso, Giulio; Morlino, Sara; Valdagni, Riccardo; Mezzanzanica, Delia; Canevari, Silvana; Figini, Mariangela

    2015-11-01

    Antibody-based reagents represent a promising strategy as clinical diagnostic tools. Prostate cancer (PCa) is the second-leading cause of death in males in the Western population. There is a presently unmet need for accurate diagnostic tool to localize and define the extent of both primary PCa and occult recurrent disease. One of the most suitable targets for PCa is the prostate-specific membrane antigen (PSMA) recognised by the monoclonal antibody D2B that we re-shaped into the single chain Fv (scFv format). Aim of this study was to evaluate in preclinical in vivo models the target specificity of scFvD2B after labelling with different radionuclides. (111)In radiolabelling was performed via the chelator Bz-NOTA, and (131)I radioiodination was performed using iodogen. The potential for molecular imaging and the biological behaviour of the radiolabelled scFvD2B were evaluated in mice bearing two subcutaneous PCa isogenic cell lines that differed only in PSMA expression. Biodistribution studies were performed at 3, 9, 15 and 24h after injection to determine the optimal imaging time point. A significant kidney accumulation, as percentage of injected dose of tissue (%ID/g), was observed for (111)In-scFvD2B at 3h after injection (45%ID/g) and it was maintained up to 24h (26%ID/g). By contrast, kidney accumulation of (131)I-scFvD2B was only marginally (0.3%ID/g at 24h). At the optimal time point defined between 15h and 24h, regardless of the radionuclide used, the scFvD2B was able to localize significantly better in the PSMA expressing tumours compared to the negative control; with (131)I-scFvD2B yielding a significantly better target/background ratio compared to (111)In-scFvD2B. These data suggest that, besides antigen specificity, chemical modification may affect antibody fragment biodistribution.

  16. Specific recognition of a tetrahedral phosphonamidate transition state analogue group by a recombinant antibody Fab fragment.

    PubMed

    Hua, T D; Lamaty, F; Souriau, C; Rolland-Fulcrand, V; Lazaro, R; Viallefont, P; Lefranc, M P; Weill, M

    1996-06-01

    In order to obtain antibodies able to catalyse a peptide synthesis, a naive combinatorial library of human Fab antibody fragments was screened with the phosphonamidate transition state analogue of the reaction. Several Fab fragments were able to bind the analogue. Competitive binding studies performed with molecules containing representative parts of the hapten showed that two Fabs were able to recognize specifically the tetrahedral phosphorus present in the hapten.

  17. Single-chain antibody-fragment M6P-1 possesses a mannose 6-phosphate monosaccharide-specific binding pocket that distinguishes N-glycan phosphorylation in a branch-specific manner†

    PubMed Central

    Blackler, Ryan J; Evans, Dylan W; Smith, David F; Cummings, Richard D; Brooks, Cory L; Braulke, Thomas; Liu, Xinyu; Evans, Stephen V; Müller-Loennies, Sven

    2016-01-01

    The acquisition of mannose 6-phosphate (Man6P) on N-linked glycans of lysosomal enzymes is a structural requirement for their transport from the Golgi apparatus to lysosomes mediated by the mannose 6-phosphate receptors, 300 kDa cation-independent mannose 6-phosphate receptor (MPR300) and 46 kDa cation-dependent mannose 6-phosphate receptor (MPR46). Here we report that the single-chain variable domain (scFv) M6P-1 is a unique antibody fragment with specificity for Man6P monosaccharide that, through an array-screening approach against a number of phosphorylated N-glycans, is shown to bind mono- and diphosphorylated Man6 and Man7 glycans that contain terminal αMan6P(1 → 2)αMan(1 → 3)αMan. In contrast to MPR300, scFv M6P-1 does not bind phosphodiesters, monophosphorylated Man8 or mono- or diphosphorylated Man9 structures. Single crystal X-ray diffraction analysis to 2.7 Å resolution of Fv M6P-1 in complex with Man6P reveals that specificity and affinity is achieved via multiple hydrogen bonds to the mannose ring and two salt bridges to the phosphate moiety. In common with both MPRs, loss of binding was observed for scFv M6P-1 at pH values below the second pKa of Man6P (pKa = 6.1). The structures of Fv M6P-1 and the MPRs suggest that the change of the ionization state of Man6P is the main driving force for the loss of binding at acidic lysosomal pH (e.g. lysosome pH ∼ 4.6), which provides justification for the evolution of a lysosomal enzyme transport pathway based on Man6P recognition. PMID:26503547

  18. Conversion of a Mouse Fab into a Whole Humanized IgG Antibody for Detecting Botulinum Toxin

    DTIC Science & Technology

    2006-04-01

    pentavalent toxoid; Fab, antibody fragment; HRP, horseradish peroxidase; LCκ, kappa light chain ; scFv, single- chain antibody fragments; VL, variable light ...The variable regions from an anti-botulinum Fab were cloned into human IgG heavy and light chain vectors and produced in myeloma cells. Purified...from an anti-botulinum Fab were cloned into human IgG heavy and light chain vectors and produced in myeloma cells. Purified humanized IgG demonstrated

  19. Expression of Human Immunodeficiency Virus Type 1 Neutralizing Antibody Fragments Using Human Vaginal Lactobacillus

    PubMed Central

    Marcobal, Angela; Liu, Xiaowen; Zhang, Wenlei; Dimitrov, Antony S.; Jia, Letong; Lee, Peter P.; Fouts, Timothy R.; Parks, Thomas P.

    2016-01-01

    Abstract Eradication of human immunodeficiency virus type 1 (HIV-1) by vaccination with epitopes that produce broadly neutralizing antibodies is the ultimate goal for HIV prevention. However, generating appropriate immune responses has proven difficult. Expression of broadly neutralizing antibodies by vaginal colonizing lactobacilli provides an approach to passively target these antibodies to the mucosa. We tested the feasibility of expressing single-chain and single-domain antibodies (dAbs) in Lactobacillus to be used as a topical microbicide/live biotherapeutic. Lactobacilli provide an excellent platform to express anti-HIV proteins. Broadly neutralizing antibodies have been identified against epitopes on the HIV-1 envelope and have been made into active antibody fragments. We tested single-chain variable fragment m9 and dAb-m36 and its derivative m36.4 as prototype antibodies. We cloned and expressed the antibody fragments m9, m36, and m36.4 in Lactobacillus jensenii-1153 and tested the expression levels and functionality. We made a recombinant L. jensenii 1153-1128 that expresses dAb-m36.4. All antibody fragments m9, m36, and m36.4 were expressed by lactobacilli. However, we noted the smaller m36/m36.4 were expressed to higher levels, ≥3 μg/ml. All L. jensenii-expressed antibody fragments bound to gp120/CD4 complex; Lactobacillus-produced m36.4 inhibited HIV-1BaL in a neutralization assay. Using a TZM-bl assay, we characterized the breadth of neutralization of the m36.4. Delivery of dAbs by Lactobacillus could provide passive transfer of these antibodies to the mucosa and longevity at the site of HIV-1 transmission. PMID:26950606

  20. Engineering of a recombinant trivalent single-chain variable fragment antibody directed against rabies virus glycoprotein G with improved neutralizing potency.

    PubMed

    Turki, Imène; Hammami, Akil; Kharmachi, Habib; Mousli, Mohamed

    2014-02-01

    Human and equine rabies immunoglobulins are currently available for passive immunization against rabies. However, these are hampered by the limited supply and some drawbacks. Advances in antibody engineering have led to overcome issues of clinical applications and to improve the protective efficacy. In the present study, we report the generation of a trivalent single-chain Fv (scFv50AD1-Fd), that recognizes the rabies virus glycoprotein, genetically fused to the trimerization domain of the bacteriophage T4 fibritin, termed 'foldon' (Fd). scFv50AD1-Fd was expressed as soluble recombinant protein in bacterial periplasmic space and purified through affinity chromatography. The molecular integrity and stability were analyzed by polyacrylamide gradient-gel electrophoresis, size-exclusion chromatography and incubation in human sera. The antigen-binding properties of the trimeric scFv were analyzed by direct and competitive-ELISA. Its apparent affinity constant was estimated at 1.4 ± 0.25 × 10(9)M(-1) and was 75-fold higher than its monovalent scFv (1.9 ± 0.68 × 10(7)M(-1)). The scFv50AD1-Fd neutralized rabies virus in a standard in vitro and in vivo neutralization assay. We showed a high neutralization activity up to 75-fold compared with monovalent format and the WHO standard serum. The gain in avidity resulting from multivalency along with an improved biological activity makes the trivalent scFv50AD1-Fd construct an important reagent for rabies protection. The antibody engineering approach presented here may serve as a strategy for designing a new generation of anti-rabies for passive immunotherapy.

  1. The biochemical properties of antibodies and their fragments

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Immunoglobulins (Ig) or antibodies are a powerful molecular recognition tools that can be used to identify minute quantities of a given target analyte. Their antigen binding properties define both the sensitivity and selectivity of an immunoassay. Understanding the biochemical properties of this c...

  2. Construction of a scFv Library with Synthetic, Non-combinatorial CDR Diversity.

    PubMed

    Bai, Xuelian; Shim, Hyunbo

    2017-01-01

    Many large synthetic antibody libraries have been designed, constructed, and successfully generated high-quality antibodies suitable for various demanding applications. While synthetic antibody libraries have many advantages such as optimized framework sequences and a broader sequence landscape than natural antibodies, their sequence diversities typically are generated by random combinatorial synthetic processes which cause the incorporation of many undesired CDR sequences. Here, we describe the construction of a synthetic scFv library using oligonucleotide mixtures that contain predefined, non-combinatorially synthesized CDR sequences. Each CDR is first inserted to a master scFv framework sequence and the resulting single-CDR libraries are subjected to a round of proofread panning. The proofread CDR sequences are assembled to produce the final scFv library with six diversified CDRs.

  3. Selection and characterization of single-chain recombinant antibodies against phosphoprotein of Newcastle disease virus.

    PubMed

    Li, Benqiang; Ye, Jiaxin; Lin, Yuan; Wang, Man; Jia, Rui; Zhu, Jianguo

    2014-09-01

    Phosphoprotein (P), involved in virus RNA replication and transcription, had become a new target for the research on treating Newcastle disease virus (NDV). Here we described the cloning and expression of phosphoprotein from NDV, and then screened the anti-P antibodies from the chicken single chain fragment variable (scFv) library, which were generated from chickens immunized with the ND vaccines. As a first step, the recombinant expression vector pET28a-P was successfully constructed. In a following step, two anti-P positive scFv clones from the scFv library were selected by indirect enzyme-linked immunosorbent assay (ELISA) method. The sequence analysis of two positive clones showed that there were more variation in complementary determine region (CDR) of VH and VL, and the CDR3 in VH exhibited a significant change in amino acid number and type. In another experiment, the purified scFv antibodies used in the assay was shown to be specific for NDV-P by western blot. The results indicated that the strategy we used in this experiment proved to be convenient way for screening scFv antibody, which paved a new way for the immunization diagnosis and the exploration of integrated control of NDV.

  4. Isolation and characterization of anti ROR1 single chain fragment variable antibodies using phage display technique.

    PubMed

    Aghebati-Maleki, Leili; Younesi, Vahid; Jadidi-Niaragh, Farhad; Baradaran, Behzad; Majidi, Jafar; Yousefi, Mehdi

    2017-01-01

    Receptor tyrosine kinase-like orphan receptor (ROR1) belongs to one of the families of receptor tyrosine kinases (RTKs). RTKs are involved in the various physiologic cellular functions including proliferation, migration, survival, signaling and differentiation. Several RTKs are deregulated in various cancers implying the targeting potential of these molecules in cancer therapy. ROR1 has recently been shown to be expressed in various types of cancer cells but not in normal adult cells. Hence a molecular inhibitor of extracellular domain of ROR1 that inhibits ROR1-cell surface interaction is of great therapeutic importance. In an attempt to develop molecular inhibitors of ROR1, we screened single chain variable fragment (scFv) phage display libraries, Tomlinson I + J, against one specific synthetic oligopeptide from extracellular domain of ROR1 and selected scFvs were characterized using various immunological techniques. Several ROR1 specific scFvs were selected following five rounds of panning procedure. The scFvs showed specific binding to ROR1 using immunological techniques. Our results demonstrate successful isolation and characterization of specific ROR1 scFvs that may have great therapeutic potential in cancer immunotherapy.

  5. Preparation of Recombinant Human Monoclonal Antibody Fab Fragments Specific for Entamoeba histolytica

    PubMed Central

    Tachibana, Hiroshi; Cheng, Xun-Jia; Watanabe, Katsuomi; Takekoshi, Masataka; Maeda, Fumiko; Aotsuka, Satoshi; Kaneda, Yoshimasa; Takeuchi, Tsutomu; Ihara, Seiji

    1999-01-01

    Genes coding for human antibody Fab fragments specific for Entamoeba histolytica were cloned and expressed in Escherichia coli. Lymphocytes were separated from the peripheral blood of a patient with an amebic liver abscess. Poly(A)+ RNA was isolated from the lymphocytes, and then genes coding for the light chain and Fd region of the heavy chain were amplified by a reverse transcriptase PCR. The amplified DNA fragments were ligated with a plasmid vector and were introduced into Escherichia coli. Three thousand colonies were screened for the production of antibodies to E. histolytica HM-1:IMSS by an indirect fluorescence-antibody (IFA) test. Lysates from five Escherichia coli clones were positive. Analysis of the DNA sequences of the five clones showed that three of the five heavy-chain sequences and four of the five light-chain sequences differed from each other. When the reactivities of the Escherichia coli lysates to nine reference strains of E. histolytica were examined by the IFA test, three Fab fragments with different DNA sequences were found to react with all nine strains and another Fab fragment was found to react with seven strains. None of the four human monoclonal antibody Fab fragments reacted with Entamoeba dispar reference strains or with other enteric protozoan parasites. These results indicate that the bacterial expression system reported here is effective for the production of human monoclonal antibodies specific for E. histolytica. The recombinant human monoclonal antibody Fab fragments may be applicable for distinguishing E. histolytica from E. dispar and for use in the serodiagnosis of amebiasis. PMID:10225840

  6. Affinity separation using an Fv antibody fragment-"smart" polymer conjugate.

    PubMed

    Fong, Robin B; Ding, Zhongli; Hoffman, Allan S; Stayton, Patrick S

    2002-08-05

    Poly(N-isopropylacrylamide), or PNIPAAm, is considered a "smart" polymer because it sharply precipitates when heated above a critical temperature, about 32 degrees C in water, and redissolves when cooled. Conjugates made of PNIPAAm and IgG antibodies also exhibit the same critical temperature behavior. Interestingly, antigens that are complexed with these conjugates can also be phase-separated along with the conjugates. In this work, we conjugated PNIPAAm for the first time to the immunoglobulin Fv fragment, the smallest fragment of an antibody that still retains the antigenic affinity of the whole antibody. For our studies, we used an Fv fragment that strongly binds hen egg white lysozyme (HEL). The purified Fv fragment-polymer conjugate precipitated at the same temperature as did the pure polymer. After addition of the conjugate to a mixture containing HEL and after thermal separation of the conjugate at 37 degrees C, the amount of HEL in solution was reduced by as much as 80%. We were able to demonstrate the reversibility of the separation through three cycles of precipitation and dissolution. It was also possible to recover free HEL by thermal separation of the conjugate in the presence of an eluant, 50 mM diethylamine. The conjugate can then be recycled for second use. In conclusion, immunoseparations can be performed using smart polymer conjugates made with just the variable domains of an antibody. Unlike whole antibodies, fragments of antibodies can be produced in Escherichia coli, allowing easier genetic engineering of the antibody and tailoring of the conjugate.

  7. Fragmentation, labeling and biodistribution studies of KS1/4, a monoclonal antibody

    SciTech Connect

    Mohd, S.B.

    1987-01-01

    In this study, an IgG2a (KS1/4), a monoclonal antibody (MoAb) specific against a human lung adenocarcinoma (UCLA P-3) was successfully fragmented enzymatically to yield F(ab')/sub 2/ and Fab by using pepsin and papain, respectively. The kinetic of fragmentation of the MoAb was compared to that of human immunoglobulin G (IgG). A similar pattern of fragmentation was observed with both antibodies with a higher percentage yield of the F(ab')/sub 2/ and Fab obtained upon the fragmentation of the IgG by the enzymes. The KS1/4 and the two fragments were labeled with three different radionuclides, namely iodine-131, indium-111 and selenium-75. The radioiodination of the MoAb and the fragments was carried out by using a modified chloramine-T method. Radiometal labeling of the MoAb and the fragments with indium-111 was performed by using DTPA as a bifunctional chelating agent, while intrinsic labeling of the MoAb was done by culturing the hybridoma in the presence of /sup 75/Se-methionine. The biodistribution of the radiolabeled MoAb, F(ab')/sub 2/ and Fab fragments were performed by injecting the preparations intravenously into nude mice bearing human lung adenocarcinoma.

  8. Dendritic Cells Transfected with scFv from Mab 7.B12 Mimicking Original Antigen gp43 Induces Protection against Experimental Paracoccidioidomycosis

    PubMed Central

    Ferreira, Karen S.; Maranhão, Andrea Q.; Garcia, Maria C. C.; Brígido, Marcelo M.; Santos, Suelen S.; Lopes, José D.; Almeida, Sandro R.

    2011-01-01

    Paracoccidioidomycosis (PCM), endemic in Latin America, is a progressive systemic mycosis caused by Paracoccidioides brasiliensis (P. brasiliensis), which primarily attacks lung tissue. Dendritic cells (DCs) are able to initiate a response in naïve T cells, and they also participate in Th-cell education. Furthermore, these cells have been used for therapy in several disease models. Here we transfected DCs with a plasmid (pMAC/PS-scFv) encoding a single chain variable fragment (scFv) of an anti-Id antibody that is capable of mimicking gp43, the main antigenic component of P. brasiliensis. First, Balb/c mice were immunized subcutaneously with pMAC/PS-scFv and, after seven days, scFv protein was presented to the regional lymph nodes cells. Moreover, we showed that the DCs transfected with scFv were capable of efficiently activating proliferation of total lymph node cells and inducing a decrease in lung infection. Therefore, our results suggested that the use of scFv-transfected DCs may be a promising therapy in the paracoccidioidomycosis (PCM) model. PMID:21249212

  9. Composition and method for detecting cancer with technetium labeled antibody fragments

    SciTech Connect

    Burchiel, S. W.; Crockford, D. R.; Rhodes, B. A.

    1984-10-23

    F(ab')/sub 2/ or Fab fragments of antibodies to: (a) human chorionic gonadotropin (hCG), hCG alpha subunit, hCG beta subunit, or an hCG-like material; or (b) other tumor specific or tumor associated molecules, to include carcinoembryonic antigen (CEA), alpha fetoprotein (AFP), human melanoma associated antigens, human sarcoma associated antigens or other antigens, are radiolabeled with technetium-99m (Tc-99m). When the F(ab')/sub 2/ or Fab fragments of antibody to such tumor associated antigens are injected intravenously into a patient, the radiolabeled composition accumulates at tumor sites. The accumulation of the cancer seeking radiopharmaceutical at tumor sites permits detection by external gamma scintigraphy. Thus, the composition is useful in the monitoring, localization and detection of cancer in the body. In an alternative composition, a double antibody approach to tumor localization using radiolabeled F(ab')/sub 2/ or Fab fragments is utilized. In this approach, a tumor specific antibody in the form of IgG, F(ab')/sub 2/ or Fab is first administered to a patient intravenously. Following a sufficient period of time, a second antibody in the form of F(ab')/sub 2/ or Fab is administered. The second antibody is radiolabeled with Tc-99m and has the property that it is reactive with the first antibody. This double antibody method has the advantage over a single antibody approach in that smaller tumors can be localized and detected and that the total amount of radioactive trace localized at the cancer site is increased.

  10. Anti-sulfotyrosine antibodies

    DOEpatents

    Bertozzi, Carolyn R.; Kehoe, John; Bradbury, Andrew M.

    2009-09-15

    The invention provides anti-sulfotyrosine specific antibodies capable of detecting and isolating polypeptides that are tyrosine-sulfated. The sulfotyrosine antibodies and antibody fragments of the invention may be used to discriminate between the non-sulfated and sulfated forms of such proteins, using any number of immunological assays, such ELISAs, immunoblots, Western Blots, immunoprecipitations, and the like. Using a phage-display system, single chain antibodies (scFvs) were generated and screened against tyrosine-sulfated synthetic peptide antigens, resulting in the isolation of scFvs that specifically recognize sulfotyrosine-containing peptides and/or demonstrate sulfotyrosine-specific binding in tyrosine sulfated proteins. The VH and VL genes from one such sulfotyrosine-specific scFv were employed to generate a full length, sulfotyrosine-specific immunoglobulin.

  11. Characterization of an anti-Bla g 1 scFv: epitope mapping and cross-reactivity.

    PubMed

    Mueller, Geoffrey A; Ankney, John A; Glesner, Jill; Khurana, Taruna; Edwards, Lori L; Pedersen, Lars C; Perera, Lalith; Slater, Jay E; Pomés, Anna; London, Robert E

    2014-06-01

    Bla g 1 is a major allergen from Blatella germanica and one of the primary allergens used to assess cockroach allergen exposure. The epitope of an anti-Bla g 1 scFv was mapped in order to better understand cross reactivity with other group 1 cockroach allergens and patient IgE epitopes. X-ray crystallography was used to determine the structure of the scFv. The scFv epitope on Bla g 1 was located by alanine scanning site-directed mutagenesis and ELISA. Twenty-six rBla g 1-GST alanine mutants were evaluated for variations in binding to the scFv compared to the wild type allergen. Six mutants showed a significant difference in scFv binding affinity. These mutations clustered to form a discontinuous epitope mainly comprising two helices of Bla g 1. The allergen-scFv complex was modeled based on the results, and the epitope region was found to have low sequence similarity with Per a 1, especially among the residues identified as functionally important for the scFv binding to Bla g 1. Indeed, the scFv failed to bind Per a 1 in American cockroach extract. The scFv was unable to inhibit the binding of IgE antibodies from a highly cockroach allergic patient to Bla g 1. Based on the surface area of Bla g 1 occluded by the scFv, putative regions of patient IgE-Bla g 1 interactions can be inferred. This scFv could be best utilized as a capture antibody in an IgE detection ELISA, or to differentiate Bla g 1 from Per a 1 in environmental exposure assays.

  12. Engineering Venom’s Toxin-Neutralizing Antibody Fragments and Its Therapeutic Potential

    PubMed Central

    Alvarenga, Larissa M.; Zahid, Muhammad; di Tommaso, Anne; Juste, Matthieu O.; Aubrey, Nicolas; Billiald, Philippe; Muzard, Julien

    2014-01-01

    Serum therapy remains the only specific treatment against envenoming, but anti-venoms are still prepared by fragmentation of polyclonal antibodies isolated from hyper-immunized horse serum. Most of these anti-venoms are considered to be efficient, but their production is tedious, and their use may be associated with adverse effects. Recombinant antibodies and smaller functional units are now emerging as credible alternatives and constitute a source of still unexploited biomolecules capable of neutralizing venoms. This review will be a walk through the technologies that have recently been applied leading to novel antibody formats with better properties in terms of homogeneity, specific activity and possible safety. PMID:25153256

  13. Selective disulfide reduction for labeling and enhancement of Fab antibody fragments.

    PubMed

    Kirley, Terence L; Greis, Kenneth D; Norman, Andrew B

    2016-11-25

    Many methods have been developed for chemical labeling and enhancement of the properties of antibodies and their common fragments, including the Fab and F(ab')2 fragments. Somewhat selective reduction of some antibody disulfide bonds has been previously achieved, yielding antibodies and antibody fragments that can be labeled at defined sites, enhancing their utility and properties. Selective reduction of the two hinge disulfide bonds present in F(ab')2 fragments using mild reduction has been useful. However, such reduction is often not quantitative and results in the reduction of multiple disulfide bonds, and therefore subsequent multiple labeling or conjugation sites are neither homogenous nor stoichiometric. Here, a simple and efficient selective reduction of the single disulfide bond linking the partial heavy chain and the intact light chain which compose the Fab fragment is accomplished utilizing tris(2-carboxyethyl)phosphine (TCEP) immobilized on agarose beads. The resultant reduced cysteine residues were labeled with several cysteine-selective fluorescent reagents, as well as by cysteine-directed PEGylation. These two cysteine residues can also be re-ligated by means of a bifunctional cysteine cross-linking agent, dibromobimane, thereby both restoring a covalent linkage between the heavy and light chains at this site, far removed from the antigen binding site, and also introducing a fluorescent probe. There are many other research and clinical uses for these selectively partially reduced Fab fragments, including biotinylation, toxin and drug conjugation, and incorporation of radioisotopes, and this technique enables simple generation of very useful Fab fragment derivatives with many potential applications.

  14. A novel anti-PSMA human scFv has the potential to be used as a diagnostic tool in prostate cancer

    PubMed Central

    Han, Yueheng; Wei, Ming; Han, Sen; Lin, Ruihe; Sun, Ziyong; Yang, Fa; Jiao, Dian; Xie, Pin; Zhang, Lingling; Yang, An-Gang; Zhao, Aizhi; Wen, Weihong; Qin, Weijun

    2016-01-01

    Prostate cancer (PCa) is the most commonly diagnosed malignancy and the second leading cause of cancer related death in men. The early diagnosis and treatment of PCa are still challenging due to the lack of efficient tumor targeting agents in traditional managements. Prostate specific membrane antigen (PSMA) is highly expressed in PCa, while only has limited expression in other organs, providing an ideal target for the diagnosis and therapy of PCa. The antibody library technique has opened the avenue for the discovery of novel antibodies to be used in the diagnosis and therapy of cancer. In this paper, by screening a large yeast display naive human single chain antibody fragment (scFv) library, we obtained a high affinity scFv targeting PSMA, called gy1. The gy1 scFv was expressed in E.coli and purified via a C terminal 6His tag. The binding affinity of gy1 was shown to be at the nanomolar level and gy1 can specifically bind with PSMA positive cancer cells, and binding triggers its rapid internalization through the endosome-lysosome pathway. The specific targeting of gy1 to PSMA positive tumor tissues was also evaluated in vivo. We showed that the IRDye800CW labeled gy1 can efficiently target and specifically distribute in PSMA positive tumor tissues after being injected into xenograft nude mice. This study indicated that the novel antibody gy1 could be used as a great tool for the development of PSMA targeted imaging and therapy agents for PCa. PMID:27448970

  15. Novel multispecific heterodimeric antibody format allowing modular assembly of variable domain fragments.

    PubMed

    Egan, Timothy J; Diem, Dania; Weldon, Richard; Neumann, Tessa; Meyer, Sebastian; Urech, David M

    2017-01-01

    Multispecific antibody formats provide a promising platform for the development of novel therapeutic concepts that could facilitate the generation of safer, more effective pharmaceuticals. However, the production and use of such antibody-based multispecifics is often made complicated by: 1) the instability of the antibody fragments of which they consist, 2) undesired inter-subunit associations, and 3) the need to include recombinant heterodimerization domains that confer distribution-impairing bulk or enhance immunogenicity. In this paper, we describe a broadly-applicable method for the stabilization of human or humanized antibody Fv fragments that entails replacing framework region IV of a Vκ1/VH3-consensus Fv framework with the corresponding germ-line sequence of a λ-type VL chain. We then used this stable Fv framework to generate a novel heterodimeric multispecific antibody format that assembles by cognate VL/VH associations between 2 split variable domains in the core of the complex. This format, termed multispecific antibody-based therapeutics by cognate heterodimerization (MATCH), can be applied to produce homogeneous and highly stable antibody-derived molecules that simultaneously bind 4 distinct antigens. The heterodimeric design of the MATCH format allows efficient in-format screening of binding domain combinations that result in maximal cooperative activity.

  16. Novel multispecific heterodimeric antibody format allowing modular assembly of variable domain fragments

    PubMed Central

    Egan, Timothy J.; Diem, Dania; Weldon, Richard; Neumann, Tessa; Meyer, Sebastian; Urech, David M.

    2017-01-01

    ABSTRACT Multispecific antibody formats provide a promising platform for the development of novel therapeutic concepts that could facilitate the generation of safer, more effective pharmaceuticals. However, the production and use of such antibody-based multispecifics is often made complicated by: 1) the instability of the antibody fragments of which they consist, 2) undesired inter-subunit associations, and 3) the need to include recombinant heterodimerization domains that confer distribution-impairing bulk or enhance immunogenicity. In this paper, we describe a broadly-applicable method for the stabilization of human or humanized antibody Fv fragments that entails replacing framework region IV of a Vκ1/VH3-consensus Fv framework with the corresponding germ-line sequence of a λ-type VL chain. We then used this stable Fv framework to generate a novel heterodimeric multispecific antibody format that assembles by cognate VL/VH associations between 2 split variable domains in the core of the complex. This format, termed multispecific antibody-based therapeutics by cognate heterodimerization (MATCH), can be applied to produce homogeneous and highly stable antibody-derived molecules that simultaneously bind 4 distinct antigens. The heterodimeric design of the MATCH format allows efficient in-format screening of binding domain combinations that result in maximal cooperative activity. PMID:27786600

  17. [Construction of combinatorial immune library of single chain human antibodies to orthopoxviruses and selection from this library antibodies to recombinant protein prA30L of variola virus].

    PubMed

    Dubrovskaia, V V; Ulitin, A B; Laman, A G; Gileva, I P; Bormotov, N I; Il'ichev, A A; Brovko, F A; Shchelkunov, S N; Belanov, E F; Tikunova, N V

    2007-01-01

    A combinatorial immune library of human single-chain antibody fragments (scFv) was constructed on the base of genes encoding variable domains of heavy and light chains of immunoglobulins cloned from the lymphocytes of four vaccinia virus (VACV) vaccinated donors. The size of the library was 3 x 10(7) independent clones. After the library was enriched with the clones producing scFv against recombinant analogue of variola virus surface protein prA30L, a panel of unique antibodies specific to both prA30L and VACV was selected from the library. A plaque reduction neutralization test was performed for all selected antibodies and two antibodies were shown to be able to neutralize plaque formation of VACV in Vero E6 cells monolayer. Binding specificities of these antibodies were confirmed using ELISA and Western blot analysis. To determine the amino acid sequences of neutralizing antibodies their genes were sequenced.

  18. Association of selenocysteine transfer RNA fragments with serum antibody response to Mycoplasma spp. in beef cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective was to identify transfer RNA fragments (tRFs) associated with a serum antibody response to Mycoplasma spp. in beef cattle. Serum from sixteen beef calves was collected at three points: in summer after calves were born, in fall at weaning, and in the following spring. All sera collected...

  19. Effects of sheep digoxin-specific antibodies and their Fab fragments on digoxin pharmacokinetics in dogs.

    PubMed Central

    Butler, V P; Schmidt, D H; Smith, T W; Haber, E; Raynor, B D; Demartini, P

    1977-01-01

    Intact sheep antidigoxin antibodies and their Fab fragments have both been found to exert profound effects on digoxin pharmacokinetics in [3H] digoxin-treated dogs. Both classes of molecule remove digoxin from the extravascular space and sequester it in the circulation in protein-bound form, a form in which the digoxin is presumably inactive. These two classes of molecule differ, however, in that the intact antibody molecules interfere with digoxin excretion, thereby promoting the retention of the glycoside; this retained digoxin is eventually released in free, active form when the administered antibody is metabolically degraded. In contrast, urinary excretion of digoxin continues in Fab-treated dogs, with significant quantities of digoxin being excreted promptly in the urine in complex with Fab fragments. These differences in urinary excretion, together with the probable decreased immunogenicity of sheep antidigoxin Fab fragments, suggest that such fragments possess potential advantages over intact antibody molecules for use in the therapy of life-threatening digoxin intoxication in man. PMID:299860

  20. Antibody fragments for stabilization and crystallization of G protein-coupled receptors and their signaling complexes.

    PubMed

    Shukla, Arun K; Gupta, Charu; Srivastava, Ashish; Jaiman, Deepika

    2015-01-01

    G protein-coupled receptors (GPCRs) are one of the key players in extracellular signal recognition and their subsequent communications with cellular signaling machinery. Crystallization and high-resolution structure determination of GPCRs has been one of the major advances in the area of GPCR biology over the last 7-8 years. There have primarily been three approaches to GPCR crystallization till date. These are fusion protein strategy, thermostabilization, and antibody fragment-mediated crystallization. Of these, antibody fragment-mediated crystallization has not only provided the first breakthrough in structure determination of a non-rhodopsin GPCR but it has also assisted in obtaining structures of fully active conformations of GPCRs. Antibody fragment approach has also been crucial in obtaining structural information on GPCR signaling complexes. Here, we highlight the specific examples of GPCR crystal structures that have utilized antibody fragments for promoting crystallogenesis and structure solution. We also discuss emerging powerful technologies such as the nanobody technology and the synthetic phage display libraries in the context of GPCR crystallization and underline how these tools are likely to propel key GPCR structural studies in future.

  1. Utility of recombinant Fragment C for assessment of anti-tetanus antibodies in plasma

    PubMed Central

    Ramakrishnan, Girija; Pedersen, Karl; Guenette, Denis; Sink, Joyce; Haque, Rashidul; Petri, William A.; Herbein, Joel; Gilchrist, Carol A.

    2016-01-01

    Anti-tetanus antibodies in biological samples are typically detected using an ELISA based on toxoided tetanus neurotoxin as antigen. We demonstrate that recombinantly produced Fragment C of the toxin heavy chain (rFragC) is an effective alternative antigen for assessment of tetanus- immune status in plasma samples. PMID:25749462

  2. Integrative Expression System for Delivery of Antibody Fragments by Lactobacilli▿ †

    PubMed Central

    Martín, M. Cruz; Pant, Neha; Ladero, Victor; Günaydın, Gökçe; Andersen, Kasper Krogh; Álvarez, Beatriz; Martínez, Noelia; Alvarez, Miguel A.; Hammarström, Lennart; Marcotte, Harold

    2011-01-01

    A series of expression cassettes which mediate secretion or surface display of antibody fragments was stably integrated in the chromosome of Lactobacillus paracasei. L. paracasei producing surface-anchored variable domain of llama heavy chain (VHH) (ARP1) directed against rotavirus showed efficient binding to rotavirus and protection in the mouse model of rotavirus infection. PMID:21257814

  3. Targeting of viral interleukin-10 with an antibody fragment specific to damaged arthritic cartilage improves its therapeutic potency

    PubMed Central

    2014-01-01

    Introduction We previously demonstrated that a single-chain fragment variable (scFv) specific to collagen type II (CII) posttranslationally modified by reactive oxygen species (ROS) can be used to target anti-inflammatory therapeutics specifically to inflamed arthritic joints. The objective of the present study was to demonstrate the superior efficacy of anti-inflammatory cytokines when targeted to inflamed arthritic joints by the anti-ROS modified CII (anti-ROS-CII) scFv in a mouse model of arthritis. Methods Viral interleukin-10 (vIL-10) was fused to anti-ROS-CII scFv (1-11E) with a matrix-metalloproteinase (MMP) cleavable linker to create 1-11E/vIL-10 fusion. Binding of 1-11E/vIL-10 to ROS-CII was determined by enzyme-linked immunosorbent assay (ELISA), Western blotting, and immune-staining of arthritic cartilage, whereas vIL-10 bioactivity was evaluated in vitro by using an MC-9 cell-proliferation assay. Specific in vivo localization and therapeutic efficacy of 1-11E/vIL-10 was tested in the mouse model of antigen-induced arthritis. Results 1-11E/vIL-10 bound specifically to ROS-CII and to damaged arthritic cartilage. Interestingly, the in vitro vIL-10 activity in the fusion protein was observed only after cleavage with MMP-1. When systemically administered to arthritic mice, 1-11E/vIL-10 localized specifically to the arthritic knee, with peak accumulation observed after 3 days. Moreover, 1-11E/vIL-10 reduced inflammation significantly quicker than vIL-10 fused to the control anti-hen egg lysozyme scFv (C7/vIL10). Conclusions Targeted delivery of anti-inflammatory cytokines potentiates their anti-arthritic action in a mouse model of arthritis. Our results further support the hypothesis that targeting biotherapeutics to arthritic joints may be extended to include anti-inflammatory cytokines that lack efficacy when administered systemically. PMID:25029910

  4. Directed immobilization of reduced antibody fragments onto a novel SAM on gold for myoglobin impedance immunosensing.

    PubMed

    Billah, Md Morsaline; Hodges, Christopher S; Hays, Henry C W; Millner, P A

    2010-11-01

    The successful construction of an immunosensor depends on having an effective procedure for immobilising the bio-recognition element to the transducer surface. In the present study, an amino-terminated 4-aminothiophenol (ATP) self-assembled monolayer (SAM) was modified with heterobifunctional crosslinker sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate to couple reduced anti-myoglobin half-antibody fragments. The disulphide groups present in the hinge region of IgG molecules were selectively cleaved by 2-mercaptoethylamine to produce reduced half-antibody fragments with free sulphydryl groups. The maleimide terminated 4-ATP SAM modified surface was coupled to these reduced antibody fragments to produce highly oriented immobilization of the half-antibody via its Fc domain and to allow free access to the Fv bindings sites. This represents an improvement by comparison with biotin/avidin mediated IgG attachment which is essentially randomly oriented. Functional immunosensors were able to detect myoglobin in both phosphate buffered saline and whole serum over the range of concentrations from 10(-13)M to 10(-6)M, and order of magnitude better than avidin/biotin linked immunosensors. In addition, atomic force microscopy (AFM) was carried out to elucidate the nanotopology of the immunosensor surface at different stages of fabrication; the images demonstrate that half antibodies bind as described and show structural changes on subsequent antigen binding.

  5. Effect of single-chain antibody targeting of the ligand-binding domain in the anaplastic lymphoma kinase receptor

    PubMed Central

    Stylianou, DC; Auf der Maur, A; Kodack, DP; Henke, RT; Hohn, S; Toretsky, JA; Riegel, AT; Wellstein, A

    2013-01-01

    The tyrosine kinase receptor anaplastic lymphoma kinase (ALK) and its ligand, the growth factor pleiotrophin (PTN), are highly expressed during the development of the nervous system and have been implicated in the malignant progression of different tumor types. Here, we describe human single-chain variable fragment (scFv) antibodies that target the ligand-binding domain (LBD) in ALK and show the effect in vitro and in vivo. The ALK LBD was used as a bait in a yeast two-hybdrid system to select human scFv from a library with randomized complementarity-determining region 3 domains. Surface plasmon resonance showed high-affinity binding of the selected scFv. The anti-ALK scFv competed for binding of PTN to ALK in intact cells and inhibited PTN-dependent signal transduction through endogenous ALK. Invasion of an intact endothelial cell monolayer by U87MG human glioblastoma cells was inhibited by the anti-ALK scFv. In addition, the growth of established tumor xenografts in mice was reversed after the induction of the conditional expression of the anti-ALK scFv. In archival malignant brain tumors expression levels of ALK and PTN were found elevated and appear correlated with poor patient survival. This suggests a rate-limiting function of the PTN/ALK interaction that may be exploited therapeutically. PMID:19633684

  6. Strategies to stabilize compact folding and minimize aggregation of antibody-based fragments

    PubMed Central

    Schrum, Adam G.

    2015-01-01

    Monoclonal antibodies (mAbs) have proven to be useful for development of new therapeutic drugs and diagnostic techniques. To overcome the difficulties posed by their complex structure and folding, reduce undesired immunogenicity, and improve pharmacokinetic properties, a plethora of different Ab fragments have been developed. These include recombinant Fab and Fv segments that can display improved properties over those of the original mAbs upon which they are based. Antibody (Ab) fragments such as Fabs, scFvs, diabodies, and nanobodies, all contain the variable Ig domains responsible for binding to specific antigenic epitopes, allowing for specific targeting of pathological cells and/or molecules. These fragments can be easier to produce, purify and refold than a full Ab, and due to their smaller size they can be well absorbed and distributed into target tissues. However, the physicochemical and structural properties of the immunoglobulin (Ig) domain, upon which the folding and conformation of all these Ab fragments is based, can limit the stability of Ab-based drugs. The Ig domain is fairly sensitive to unfolding and aggregation when produced out of the structural context of an intact Ab molecule. When unfolded, Ab fragments may lose their specificity as well as establish non-native interactions leading to protein aggregation. Aggregated antibody fragments display altered pharmacokinetic and immunogenic properties that can augment their toxicity. Therefore, much effort has been placed in understanding the factors impacting the stability of Ig folding at two different levels: 1) intrinsically, by studying the effects of the amino acid sequence on Ig folding; 2) extrinsically, by determining the environmental conditions that may influence the stability of Ig folding. In this review we will describe the structure of the Ig domain, and the factors that impact its stability, to set the context for the different approaches currently used to achieve stable recombinant Ig

  7. 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.

  8. Efficient expression of single chain variable fragment antibody against paclitaxel using the Bombyx mori nucleopolyhedrovirus bacmid DNA system and its characterizations.

    PubMed

    Yusakul, Gorawit; Sakamoto, Seiichi; Tanaka, Hiroyuki; Morimoto, Satoshi

    2016-07-01

    A single chain variable fragment (scFv), the smallest unit of functional recombinant antibody, is an attractive format of recombinant antibodies for various applications due to its small fragment and possibility of genetic engineering. Hybridoma clone 3A3 secreting anti-paclitaxel monoclonal antibody was used to construct genes encoding its variable domains of heavy (VH) and light (VL) chains. The VH and VL domains were linked to be the PT-scFv3A3 using flexible peptide linker in a format of VH-(GGGGS)5-VL. The PT-scFv3A3 was primarily expressed using the pET28a(+) vector in the Escherichia coli system, and was then further expressed by using the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Interestingly, the reactivity of PT-scFv3A3 expressed in the hemolymph of B. mori using the BmNPV bacmid DNA system was much higher than that expressed in the E. coli system. Using indirect competitive enzyme-linked immunosorbent assay (icELISA), the PT-scFv3A3 (B. mori) reacted not only with immobilized paclitaxel, but also with free paclitaxel in a concentration-dependent manner, with the linear range of free paclitaxel between 0.156 and 5.00 µg/ml. The PT-scFv3A3 (B. mori) exhibited less cross-reactivity (%) than its parental MAb clone 3A3 against paclitaxel-related compounds, including docetaxel (31.1 %), 7-xylosyltaxol (22.1 %), baccatin III (<0.68 %), 10-deacetylbaccatin III (<0.68 %), 1-hydroxybaccatin I (<0.68 %), and 1-acetoxy-5-deacetylbaccatin I (<0.68 %). With the exception of cephalomannine, the cross-reactivity was slightly increased to 8.50 %. The BmNPV bacmid DNA system was a highly efficient expression system of active PT-scFv3A3, which is applicable for PT-scFv3A3-based immunoassay of paclitaxel. In addition, the PT-scFv3A3 can be applied to evaluate its neutralizing property of paclitaxel or docetaxel toxicity.

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

    SciTech Connect

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

    1994-09-01

    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 site 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.

  10. Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment

    PubMed Central

    Kirley, Terence L; Norman, Andrew B

    2015-01-01

    Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region. PMID:25692880

  11. Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment.

    PubMed

    Kirley, Terence L; Norman, Andrew B

    2015-01-01

    Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region.

  12. The effect of internalizing human single chain antibody fragment on liposome targeting to epithelioid and sarcomatoid mesothelioma.

    PubMed

    Iyer, Arun K; Su, Yang; Feng, Jinjin; Lan, Xiaoli; Zhu, Xiaodong; Liu, Yue; Gao, Dongwei; Seo, Youngho; Vanbrocklin, Henry F; Courtney Broaddus, V; Liu, Bin; He, Jiang

    2011-04-01

    Immunoliposomes (ILs) anchored with internalizing human antibodies capable of targeting all subtypes of mesothelioma can be useful for targeted imaging and therapy of this malignant disease. The objectives of this study were to evaluate both the in vitro and in vivo tumor targeted internalization of novel internalizing human single chain antibody (scFv) anchored ILs on both epithelioid (M28) and sarcomatoid (VAMT-1) subtypes of human mesothelioma. ILs were prepared by post-insertion of mesothelioma-targeting human scFv (M1) onto preformed liposomes and radiolabeled with (111)In ((111)In-IL-M1), along with control non-targeted liposomes ((111)In-CL). Incubation of (111)In-IL-M1 with M28, VAMT-1, and a control non-tumorigenic cell line (BPH-1) at 37 °C for 24 h revealed efficient binding and rapid internalization of ILs into both subtypes of tumor cells but not into the BPH-1 cells; internalization accounted for approximately 81-94% of total cell accumulation in mesothelioma cells compared to 37-55% in control cells. In tumor-bearing mice intravenous (i.v.) injection of (111)In-IL-M1 led to remarkable tumor accumulation: 4% and 4.7% injected dose per gram (% ID/g) for M28 and VAMT-1 tumors, respectively, 48 h after injection. Furthermore, tumor uptake of (111)In-IL-M1 in live xenograft animal models was verified by single photon emission computed tomography (SPECT/CT). In contrast, i.v. injection of (111)In-CL in tumor-bearing mice revealed very low uptake in both subtypes of mesothelioma, 48 h after injection. In conclusion, M1 scFv-anchored ILs showed selective tumor targeting and rapid internalization into both epithelioid and sarcomatoid subtypes of human mesothelioma, demonstrating its potential as a promising vector for enhanced tumor drug targeting.

  13. Purification of molecular machines and nanomotors using phage-derived monoclonal antibody fragments.

    PubMed

    Esteban, Olga; Christ, Daniel; Stock, Daniela

    2013-01-01

    Molecular machines and nanomotors are sophisticated biological assemblies that convert potential energy stored either in transmembrane ion gradients or in ATP into kinetic energy. Studying these highly dynamic biological devices by X-ray crystallography is challenging, as they are difficult to produce, purify, and crystallize. Phage display technology allows us to put a handle on these molecules in the form of highly specific antibody fragments that can also stabilize conformations and allow versatile labelling for electron microscopy, immunohistochemistry, and biophysics experiments.Here, we describe a widely applicable protocol for selecting high-affinity monoclonal antibody fragments against a complex molecular machine, the A-type ATPase from T. thermophilus that allows fast and simple purification of this transmembrane rotary motor from its wild-type source. The approach can be readily extended to other integral membrane proteins and protein complexes as well as to soluble molecular machines and nanomotors.

  14. Affinity maturation of single-chain variable fragment specific for aflatoxin B(1) using yeast surface display.

    PubMed

    Min, Won-Ki; Kim, Sung-Gun; Seo, Jin-Ho

    2015-12-01

    As aflatoxin B1 is one of the most toxic mycotoxins, it is important to detect and to quantify aflatoxin B1 accurately by immunological methods. To enhance aflatoxin B1-binding affinity of the single-chain variable fragment, yeast surface display technique combined with fluorescence-activated cell sorting was applied. A randomly mutated scFv library was subjected to 4 rounds of fluorescence-activated cell sorting, resulting in isolation of 5 scFv variants showing an affinity improvement compared to the parental wild type scFv. The best scFv with a 9-fold improvement in affinity for aflatoxin B1 exhibited similar specificity to the monoclonal antibody. Most of the mutations in scFv-M37 were located outside of the canonical antigen-contact loops, suggesting that its affinity improvement might be driven by an allosteric effect inducing scFv-M37 to form a more favorable binding pocket for aflatoxin B1 than the wild type scFv.

  15. Integrating scFv into xMAP Assays for the Detection of Marine Toxins

    PubMed Central

    Shriver-Lake, Lisa C.; Liu, Jinny L.; Brozozog Lee, P. Audrey; Goldman, Ellen R.; Dietrich, Richard; Märtlbauer, Erwin; Anderson, George P.

    2016-01-01

    Marine toxins, such as saxitoxin and domoic acid are associated with algae blooms and can bioaccumulate in shell fish which present both health and economic concerns. The ability to detect the presence of toxin is paramount for the administration of the correct supportive care in case of intoxication; environmental monitoring to detect the presence of toxin is also important for prevention of intoxication. Immunoassays are one tool that has successfully been applied to the detection of marine toxins. Herein, we had the variable regions of two saxitoxin binding monoclonal antibodies sequenced and used the information to produce recombinant constructs that consist of linked heavy and light variable domains that make up the binding domains of the antibodies (scFv). Recombinantly produced binding elements such as scFv provide an alternative to traditional antibodies and serve to “preserve” monoclonal antibodies as they can be easily recreated from their sequence data. In this paper, we combined the anti-saxitoxin scFv developed here with a previously developed anti-domoic acid scFv and demonstrated their utility in a microsphere-based competitive immunoassay format. In addition to detection in buffer, we demonstrated equivalent sensitivity in oyster and scallop matrices. The potential for multiplexed detection using scFvs in this immunoassay format is demonstrated. PMID:27879646

  16. Effect of chloramine-T labeling conditions on the stability of monoclonal antibodies and their fragments

    SciTech Connect

    DeNardo, G.L.; DeNardo, S.J.; Miyao, N.P.; Peng, J.S.; Epstein, A.L.; Cardiff, R.D.

    1985-05-01

    Rapid in vivo degradation of radioiodinated monoclonal antibodies (MAb) has been reported. Conditions for radioiodination have varied. The purposes of this study were to compare the stability of MAb and their fragments when iodinated with chloramine-T (CT) under different conditions, and to compare methods for quality assessment of the radioiodinated molecules. A B-cell lymphoma MAb (Lym-1, IgG2a) and its FAb fragment, and a mammary cancer MAb(B6.01, IgG1) and its F(Ab')/sub 2/ fragment were iodinated with I-125 at CT:AB and I:Ab ratios of 1:1 and 1:10. Molecular sieving (TSK-3000) high performance liquid chromatography (HPLC), cellulose acetate electrophoresis (CAE) at 11 and 45 minutes and solid phase immunoreactivity (IRA) were used to observe stability of the molecules when stored at 4/sup 0/C. Radiochemical yield was greater than 95% in all instances. Iodination at CT:Ab and I:Ab ratios of 1:1 induced progressive degradation in all species which was most marked for the fragments. Iodination at CT:Ab and I:Ab ratios of 1:10 resulted in no observable degradation over 21 days. There was no significant difference in degradation between the IgG2a and IgG1 antibody when iodinated under identical circumstances. HPLC, CAE for 11 minutes and IRA, but not CAE for 45 minutes, revealed comparable changes. The authors conclude that lesser amounts of chloramine-T can be used to iodinate MAb and their fragments without loss of radiochemical efficiency and with improved stability of the species. MAb fragments are more vulnerable to chloramine-T. These observations may explain, at least in part, rapid in vivo degradation of radioiodinated MAb.

  17. Understanding the molecular recognition between antibody fragments and protein A biomimetic ligand.

    PubMed

    Branco, Ricardo J F; Dias, Ana M G C; Roque, Ana C A

    2012-06-29

    Affinity chromatography with protein A from Staphylococcus aureus (SpA) is the most widespread and accepted methodology for antibody capture during the downstream process of antibody manufacturing. A triazine based ligand (ligand 22/8) was previously developed as an inexpensive and robust alternative to SpA chromatography (Li et al. and Teng et al.). Despite the experimental success, there is no structural information on the binding modes of ligand 22/8 to antibodies, namely to Immunoglobulin G (IgG) molecules and fragments. In this work, we addressed this issue by a molecular docking approach allied to molecular dynamics simulations. Theoretical results confirmed the preference of the synthetic ligand to bind IgG through the binding site found in the crystallographic structure of the natural complex between SpA and the Fc fragment of IgG. Our studies also suggested other unknown "hot-spots" for specific binding of the affinity ligand at the hinge between V(H) and C(H)1 domains of Fab fragment. The best docking poses were further analysed by molecular dynamics studies at three different protonation states (pH 3, 7 and 11). The main interactions between ligand 22/8 and the IgG fragments found at pH 7 were weaker at pH 3 and pH 11 and in these conditions the ligand start losing tight contact with the binding site, corroborating the experimental evidence for protein elution from the chromatographic adsorbents at these pH conditions.

  18. A Tat-grafted anti-nucleic acid antibody acquires nuclear-localization property and a preference for TAR RNA

    SciTech Connect

    Jeong, Jong-Geun; Kim, Dong-Sik; Kim, Yong-Sung; Kwon, Myung-Hee

    2011-03-18

    Highlights: {yields} We generate '{sub H3}Tat-3D8' by grafting Tat{sub 48-60} peptide to VH CDR of 3D8 scFv antibody. {yields} {sub H3}Tat-3D8 antibody retains nucleic acid binding and hydrolyzing activities. {yields} {sub H3}Tat-3D8 acquires a preference for TAR RNA structure. {yields} Properties of Tat{sub 48-60} is transferred to an antibody via Tat-grafting into a CDR. -- Abstract: The 3D8 single chain variable fragment (3D8 scFv) is an anti-nucleic acid antibody that can hydrolyze nucleic acids and enter the cytosol of cells without reaching the nucleus. The Tat peptide, derived from the basic region of the HIV-1 Tat protein, translocates to cell nuclei and has TAR RNA binding activity. In this study, we generated a Tat-grafted antibody ({sub H3}Tat-3D8) by replacing complementarity-determining region 3 (CDR3) within the VH domain of the 3D8 scFv with a Tat{sub 48-60} peptide (GRKKRRQRRRPPQ). {sub H3}Tat-3D8 retained the DNA-binding and DNA-hydrolyzing activity of the scFv, and translocated to the nuclei of HeLa cells and preferentially recognized TAR RNA. Thus, the properties associated with the Tat peptide were transferred to the antibody via Tat-grafting without loss of the intrinsic DNA-binding and hydrolyzing activities of the 3D8 scFv antibody.

  19. Antibody fragment-conjugated polymeric micelles incorporating platinum drugs for targeted therapy of pancreatic cancer.

    PubMed

    Ahn, Jooyeon; Miura, Yutaka; Yamada, Naoki; Chida, Tsukasa; Liu, Xueying; Kim, Ahram; Sato, Ryuta; Tsumura, Ryo; Koga, Yoshikatsu; Yasunaga, Masahiro; Nishiyama, Nobuhiro; Matsumura, Yasuhiro; Cabral, Horacio; Kataoka, Kazunori

    2015-01-01

    Antibody-mediated therapies including antibody-drug conjugates (ADCs) have shown much potential in cancer treatment by tumor-targeted delivery of cytotoxic drugs. However, there is a limitation of payloads that can be delivered by ADCs. Integration of antibodies to drug-loaded nanocarriers broadens the applicability of antibodies to a wide range of therapeutics. Herein, we developed antibody fragment-installed polymeric micelles via maleimide-thiol conjugation for selectively delivering platinum drugs to pancreatic tumors. By tailoring the surface density of maleimide on the micelles, one tissue factor (TF)-targeting Fab' was conjugated to each carrier. Fab'-installed platinum-loaded micelles exhibited more than 15-fold increased cellular binding within 1 h and rapid cellular internalization compared to non-targeted micelles, leading to superior in vitro cytotoxicity. In vivo, Fab'-installed micelles significantly suppressed the growth of pancreatic tumor xenografts for more than 40 days, outperforming non-targeted micelles and free drugs. These results indicate the potential of Fab'-installed polymeric micelles for efficient drug delivery to solid tumors.

  20. Human single-chain variable fragment antibody inhibits macrophage migration inhibitory factor tautomerase activity.

    PubMed

    Tarasuk, Mayuri; Poungpair, Ornnuthchar; Ungsupravate, Duangporn; Bangphoomi, Kunan; Chaicumpa, Wanpen; Yenchitsomanus, Pa-Thai

    2014-03-01

    Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine, secreted from a variety of immune cells, that regulates innate and adaptive immune responses. Elevation of MIF levels in plasma correlates with the severity of inflammatory diseases in humans. Inhibition of MIF or its tautomerase activity ameliorates disease severity by reducing inflammatory responses. In this study, the human single-chain variable fragment (HuScFv) antibody specific to MIF was selected from the human antibody phage display library by using purified recombinant full-length human MIF (rMIF) as the target antigen. Monoclonal HuScFv was produced from phage-transformed bacteria and tested for their binding activities to rMIF by indirect enzyme-linked immunosorbent assay as well as to native MIF by western blot analysis and immunofluorescence assay. The HuScFv with highest binding signal to rMIF also inhibited the tautomerase activities of both rMIF and native MIF in human monoblastic leukemia (U937) cells in a dose-dependent manner. Mimotope searching and molecular docking concordantly demonstrated that the HuScFv interacted with Lys32 and Ile64 in the MIF tautomerase active site. To the best of our knowledge, this is the first study to focus on MIF-specific fully-human antibody fragment with a tautomerase-inhibitory effect that has potential to be developed as anti-inflammatory biomolecules for human use.

  1. Production, purification and biological characterization of mono-PEGylated anti-IL-17A antibody fragments.

    PubMed

    Koussoroplis, Salome-Juliette; Heywood, Sam; Uyttenhove, Catherine; Barilly, Céline; Van Snick, Jacques; Vanbever, Rita

    2013-09-15

    The aim of this study was to maximize the yield of the production of mono-PEGylated anti-interleukin-17A (anti-IL-17A) antibody fragments using large (≥ 20 kDa) polyethylene glycol (PEG) chains. Particular attention was paid to selectively yield mono-PEGylated species to maintain the maximum possible functionality and to simplify the purification. Neutralization of IL-17A by antibody constructs might find application for the treatment of bronchial hyperreactivity. Amino-directed and sulfhydryl-directed PEGylation of the native antibody fragments were compared. The former was selected as it produced the most interesting construct in terms of yield and preservation of biological activity. In particular, the F(ab')2-PEG conjugate with one 40 kDa branched PEG prepared in this study was produced at a 42% yield. The conjugate presented only a slight decrease in its binding activity and in its in vitro inhibitory potency offering interesting perspectives for in vivo studies.

  2. Functional expression of a single-chain antibody to ErbB-2 in plants and cell-free systems

    PubMed Central

    Galeffi, Patrizia; Lombardi, Alessio; Pietraforte, Immacolata; Novelli, Flavia; Di Donato, Monica; Sperandei, Maria; Tornambé, Andrea; Fraioli, Rocco; Martayan, Aline; Natali, Pier Giorgio; Benevolo, Maria; Mottolese, Marcella; Ylera, Francisco; Cantale, Cristina; Giacomini, Patrizio

    2006-01-01

    Background Aberrant signaling by ErbB-2 (HER 2, Neu), a member of the human Epidermal Growth Factor (EGF) receptor family, is associated with an aggressive clinical behaviour of carcinomas, particularly breast tumors. Antibodies targeting the ErbB-2 pathway are a preferred therapeutic option for patients with advanced breast cancer, but a worldwide deficit in the manufacturing capacities of mammalian cell bioreactors is foreseen. Methods Herein, we describe a multi-platform approach for the production of recombinant Single chain Fragments of antibody variable regions (ScFvs) to ErbB-2 that involves their functional expression in (a) bacteria, (b) transient as well as stable transgenic tobacco plants, and (c) a newly developed cell-free transcription-translation system. Results An ScFv (ScFv800E6) was selected by cloning immunoglobulin sequences from murine hybridomas, and was expressed and fully functional in all the expression platforms, thereby representing the first ScFv to ErbB-2 produced in hosts other than bacteria and yeast. ScFv800E6 was optimized with respect to redox synthesis conditions. Different tags were introduced flanking the ScFv800E6 backbone, with and without spacer arms, including a novel Strep II tag that outperforms conventional streptavidin-based detection systems. ScFv800E6 was resistant to standard chemical radiolabeling procedures (i.e. Chloramine T), displayed a binding ability extremely similar to that of the parental monovalent Fab' fragment, as well as a flow cytometry performance and an equilibrium binding affinity (Ka approximately 2 × 108 M-1) only slightly lower than those of the parental bivalent antibody, suggesting that its binding site is conserved as compared to that of the parental antibody molecule. ScFv800E6 was found to be compatible with routine reagents for immunohistochemical staining. Conclusion ScFv800E6 is a useful reagent for in vitro biochemical and immunodiagnostic applications in oncology, and a candidate for

  3. A Naturally Occurring Antibody Fragment Neutralizes Infectivity of Diverse Infectious Agents

    PubMed Central

    Polonelli, Luciano; Ciociola, Tecla; Elviri, Lisa; Zanello, Pier Paolo; Giovati, Laura; Arruda, Denise C.; Muñoz, Julián E.; Mortara, Renato A.; Morace, Giulia; Borghi, Elisa; Galati, Serena; Marin, Oriano; Casoli, Claudio; Pilotti, Elisabetta; Ronzi, Paola; Travassos, Luiz R.; Magliani, Walter; Conti, Stefania

    2016-01-01

    A phosphorylated peptide, named K40H, derived from the constant region of IgMs was detected in human serum by liquid chromatography coupled to high-resolution mass spectrometry. Synthetic K40H proved to exert a potent in vitro activity against fungal pathogens, and to inhibit HIV-1 replication in vitro and ex vivo. It also showed a therapeutic effect against an experimental infection by Candida albicans in the invertebrate model Galleria mellonella. K40H represents the proof of concept of the innate role that naturally occurring antibody fragments may exert against infectious agents, shedding a new light upon the posthumous role of antibodies and opening a new scenario on the multifaceted functionality of humoral immunity. PMID:27725769

  4. Generation, use, and validation of receptor-selective antibodies.

    PubMed

    Mackrill, John J

    2004-01-01

    Antibodies have proved invaluable in the study of G-protein-coupled receptors (GPCRs). The utility of these immunoglobulin probes for investigation of protein structures and functions arises from their selectivity as well as their versatility. Antibodies can be used to analyze GPCR size, abundance, distribution, turnover, modification, interaction with other proteins, and functional properties. In this chapter, techniques for the generation and characterization of receptor-selective antibodies are described. Two protocols are given for the generation of antibodies: (1) development of polyclonal antibodies (PAbs) against synthetic peptides corresponding to a specific site within a GPCR and (2) selection of synthetic single-chain fragment variable (scFv) monoclonal antibodies (MAbs) from libraries expressed on the surface of bacteriophage. Immunoblot and enzyme-linked immunosorbent assays for characterization of the selectivity and affinity of such antibodies are described. Finally, methods are given for improvement of the titer and specificity of PAbs.

  5. Role of Fc fragments in antibody-mediated recovery from ocular and subcutaneous herpes simplex virus infections.

    PubMed Central

    Oakes, J E; Lausch, R N

    1981-01-01

    The contributions of the Fc fragment of virus-specific antibody in the resistance of mice to peripheral herpes simplex virus infection were investigated. Rabbit anti-herpes simplex virus-specific F(ab')2 fragments prepared by pepsin digestion of immune immunoglobulin G (IgG) were found to be inactive in complement-mediated cytolysis while retaining their capacity to neutralize virus infectivity in vitro. When F(ab')2 fragments were passively transferred either before or simultaneously with virus inoculation, they were as efficient as intact IgG was in protecting animals from virus challenge. However, if passive transfer was delayed until 8 h after herpes simplex virus infection, only IgG antibody was protective. The loss of protective activity could not be attributed to a rapid disappearance of F(ab')2 fragments, because comparable levels of F(ab')2 fragments and IgG antibody were maintained in the blood of recipients during the time that antibody mediated its protective effects. The inability of F(ab')2 subunits to activate complement was also not a factor, because complement-deficient A/J mice and complement-sufficient SJL/J mice recovered from herpes simplex virus infection after the passive transfer of IgG. We concluded that the Fc component of the antibody molecule is needed to resolve intracellular infection and that the mechanism by which antibody mediates recovery remains undefined but does not appear to involve virus neutralization or complement activation. PMID:6266961

  6. Production of single chain Fab (scFab) fragments in Bacillus megaterium

    PubMed Central

    Jordan, Eva; Al-Halabi, Laila; Schirrmann, Thomas; Hust, Michael; Dübel, Stefan

    2007-01-01

    Background The demand on antigen binding reagents in research, diagnostics and therapy raises questions for novel antibody formats as well as appropriate production systems. Recently, the novel single chain Fab (scFab) antibody format combining properties of single chain Fv (scFv) and Fab fragments was produced in the Gram-negative bacterium Escherichia coli. In this study we evaluated the Gram-positive bacterium Bacillus megaterium for the recombinant production of scFab and scFvs in comparison to E. coli. Results The lysozyme specific D1.3 scFab was produced in B. megaterium and E. coli. The total yield of the scFab after purification obtained from the periplasmic fraction and culture supernatant of E. coli was slightly higher than that obtained from culture supernatant of B. megaterium. However, the yield of functional scFab determined by analyzing the antigen binding activity was equally in both production systems. Furthermore, a scFv fragment with specificity for the human C reactive protein was produced in B. megaterium. The total yield of the anti-CRP scFv produced in B. megaterium was slightly lower compared to E. coli, whereas the specific activity of the purified scFvs produced in B. megaterium was higher compared to E. coli. Conclusion B. megaterium allows the secretory production of antibody fragments including the novel scFab antibody format. The yield and quality of functional antibody fragment is comparable to the periplasmic production in E. coli. PMID:18042285

  7. Immobilization and functional reconstitution of antibody Fab fragment by solid-phase refolding.

    PubMed

    Kumada, Yoichi; Hamasaki, Kyoto; Nakagawa, Aya; Sasaki, Eiju; Shirai, Tatsunori; Okumura, Masahiro; Inoue, Manami; Kishimoto, Michimasa

    2013-12-31

    In this study, we demonstrated the successful preparation of a Fab antibody-immobilized hydrophilic polystyrene (phi-PS) plate via one- and two-step solid-phase refolding methods. Both polystyrene-binding peptide (PS-tag)-fused Fd fragment of heavy chain (Fab H-PS) and full-length of light-chain (Fab L-PS) were individually produced in insoluble fractions of Escherichia coli cells, and they were highly purified in the presence of 8M of urea. Antigen-binding activities of Fab antibody immobilized were correctly recovered by the one-step solid-phase refolding method that a mixture of Fab H-PS and Fab L-PS was immobilized in the presence of 0.5-2M urea, followed by surface washing of the phi-PS plate with PBST. These results indicate that by genetic fusion of a PS-tag, a complex between Fab H and Fab L was efficiently immobilized on the surface of a phi-PS plate even in the presence of a low concentration of urea, and was then correctly refolded to retain its high antigen-binding activity via removal of the urea. A two-step solid-phase refolding method whereby Fab H-PS and Fab L-PS were successively refolded on the surface of a phi-PS plate also resulted in Fab antibody formation on the plate. Furthermore, both the binding affinity and the specificity of the Fab antibody produced by the two-step method were highly maintained, according to the results of sandwich ELISA and competitive ELISA using Fab antibody-immobilized plate via two-step solid-phase refolding. Thus, the solid-phase refolding method demonstrated in this study should be quite useful for the preparation of a Fab antibody-immobilized PS surface with high efficiency from individually produced Fab H-PS and Fab L-PS. This method will be applicable to the preparation of a large Fab antibody library on the surface of a PS plate for use in antibody screening.

  8. Expression of an anti-botulinum toxin A neutralizing single-chain Fv recombinant antibody in transgenic tobacco.

    PubMed

    Almquist, Kurt C; McLean, Michael D; Niu, Yongqing; Byrne, Greg; Olea-Popelka, Fernando C; Murrant, Coral; Barclay, Jack; Hall, J Christopher

    2006-03-15

    Botulinum neurotoxins (BoNTs) are the most poisonous substances known and are thus classified as high-risk threats for use as bioterror agents. To examine the potential of transgenic plants as bioreactors for the production of BoNT antidotes, we transformed tobacco with an optimized, synthetic gene encoding a botulinum neurotoxin A (BoNT/A) neutralizing single-chain Fv (scFv) recombinant antibody fragment. In vitro mouse muscle twitch assays demonstrated the functional utility of this scFv extracted from tobacco for neutralizing the paralytic effects of BoNT/A at neuromuscular junctions. Based on the efficiency of the scFv capture process and the dose required to antidote a human being, 1-2 ha of this tobacco could yield up to 4 kg of scFv, which would be enough to contribute to the manufacture of 1,000,000 therapeutic doses of a monoclonal antibody (mAb) cocktail capable of neutralizing the effects of BoNT poisoning. Transgenic plants could provide an inexpensive production platform for expression of multiple mAbs toward the creation of polyclonal therapies (i.e. pooled mAbs) as the next improvement in recombinant antibody therapy.

  9. Evaluation of selectivity in homologous multimodal chromatographic systems using in silico designed antibody fragment libraries.

    PubMed

    Karkov, Hanne Sophie; Woo, James; Krogh, Berit Olsen; Ahmadian, Haleh; Cramer, Steven M

    2015-12-24

    This study describes the in silico design, surface property analyses, production and chromatographic evaluations of a diverse set of antibody Fab fragment variants. Based on previous findings, we hypothesized that the complementarity-determining regions (CDRs) constitute important binding sites for multimodal chromatographic ligands. Given that antibodies are highly diversified molecules and in particular the CDRs, we set out to examine the generality of this result. For this purpose, four different Fab fragments with different CDRs and/or framework regions of the variable domains were identified and related variants were designed in silico. The four Fab variant libraries were subsequently generated by site-directed mutagenesis and produced by recombinant expression and affinity purification to enable examination of their chromatographic retention behavior. The effects of geometric re-arrangement of the functional moieties on the multimodal resin ligands were also investigated with respect to Fab variant retention profiles by comparing two commercially available multimodal cation-exchange ligands, Capto MMC and Nuvia cPrime, and two novel multimodal ligand prototypes. Interestingly, the chromatographic data demonstrated distinct selectivity trends between the four Fab variant libraries. For three of the Fab libraries, the CDR regions appeared as major binding sites for all multimodal ligands. In contrast, the fourth Fab library displayed a distinctly different chromatographic behavior, where Nuvia cPrime and related multimodal ligand prototypes provided markedly improved selectivity over Capto MMC. Clearly, the results illustrate that the discriminating power of multimodal ligands differs between different Fab fragments. The results are promising indications that multimodal chromatography using the appropriate multimodal ligands can be employed in downstream bioprocessing for challenging selective separation of product related variants.

  10. The identification of affinity peptide ligands specific to the variable region of human antibodies.

    PubMed

    Akiyama, Yasuto; Miyata, Haruo; Komiyama, Masaru; Nogami, Masahiro; Ozawa, Kazumichi; Oshita, Chie; Kume, Akiko; Ashizawa, Tadashi; Sakura, Naoki; Mochizuki, Tohru; Yamaguchi, Ken

    2014-01-01

    Of all potential biological therapeutics, monoclonal antibody (mAb)-based therapies are becoming the dominant focus of clinical research. In particular, smaller recombinant antibody fragments such as single-chain variable fragments (scFv) have become the subject of intense focus. However, an efficient affinity ligand for antibody fragment purification has not been developed. In the present study, we designed a consensus sequence for the human antibody heavy or light chain-variable regions (Fv) based on the antibody sequences available in the ImMunoGeneTics information system (IMGT), and synthesized these consensus sequences as template Fv antibodies. We then screened peptide ligands that specifically bind to the repertoire-derived human Fv consensus antibody using a 12-mer-peptide library expressed-phage display method. Subsequently, 1 peptide for the VH template and 8 peptides for the VK template were selected as the candidate ligands after 4 rounds of panning the phage display. Using peptide-bead-based immunoprecipitation, the code-4 and code-13 peptides showed recovery rates of the VH and VK templates that were 20-30% and 40-50%, respectively. Both peptides exhibited better recovery rates for trastuzumab scFv (approximately 40%). If it were possible to identify the best combination of VH and VK-binding peptides among the ligand peptides suitable for the human mAb Fv sequence, the result could be a promising purification tool that might greatly improve the cost efficiencies of the purification process.

  11. Engineering an antibody with picomolar affinity to DOTA chelates of multiple radionuclides for pretargeted radioimmunotherapy and imaging

    PubMed Central

    Orcutt, Kelly Davis; Slusarczyk, Adrian L; Cieslewicz, Maryelise; Ruiz-Yi, Benjamin; Bhushan, Kumar R; Frangioni, John V; Wittrup, K Dane

    2014-01-01

    Introduction In pretargeted radioimmunotherapy (PRIT), a bifunctional antibody is administered and allowed to pre-localize to tumor cells. Subsequently, a chelated radionuclide is administered and captured by cell-bound antibody while unbound hapten clears rapidly from the body. We aim to engineer high-affinity binders to DOTA chelates for use in PRIT applications. Methods We mathematically modeled antibody and hapten pharmacokinetics to analyze hapten tumor retention as a function of hapten binding affinity. Motivated by model predictions, we used directed evolution and yeast surface display to affinity mature the 2D12.5 antibody to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), reformatted as a single chain variable fragment (scFv). Results Modeling predicts that for high antigen density and saturating bsAb dose, a hapten binding affinity of 100 picomolar (pM) is needed for near-maximal hapten retention. We affinity matured 2D12.5 with an initial binding constant of about 10 nanomolar (nM) to DOTA-yttrium chelates. Affinity maturation resulted in a 1000-fold affinity improvement to biotinylated DOTA-yttrium, yielding an 8.2 ± 1.9 picomolar binder. The high-affinity scFv binds DOTA complexes of lutetium and gadolinium with similar picomolar affinity and indium chelates with low nanomolar affinity. When engineered into a bispecific antibody construct targeting carcinoembryonic antigen (CEA), pretargeted high-affinity scFv results in significantly higher tumor retention of a 111In-DOTA hapten compared to pretargeted wild-type scFv in a xenograft mouse model. Conclusions We have engineered a versatile, high-affinity DOTA-chelate-binding scFv. We anticipate it will prove useful in developing pretargeted imaging and therapy protocols to exploit the potential of a variety of radiometals. PMID:21315278

  12. Expression and structural characterization of anti-T-antigen single-chain antibodies (scFvs) and analysis of their binding to T-antigen by surface plasmon resonance and NMR spectroscopy.

    PubMed

    Yuasa, Noriyuki; Koyama, Tsubasa; Subedi, Ganesh P; Yamaguchi, Yoshiki; Matsushita, Misao; Fujita-Yamaguchi, Yoko

    2013-12-01

    T-antigen (Galβ1-3GalNAcα-1-Ser/Thr), also known as Thomsen-Friedenreich antigen (TF antigen), is an oncofetal antigen commonly found in cancerous tissues. Availability of anti-T-antigen human antibodies could lead to the development of cancer diagnostics and therapeutics. Four groups of single-chain variable fragment (scFv) genes were previously isolated from a phage library (Matsumoto-Takasaki et al. (2009) Isolation and characterization of anti-T-antigen single chain antibodies from a phage library. BioSci Trends 3:87-95.). Here, four anti-T-antigen scFv genes belonging to Group 1-4 were expressed and produced in a Drosophila S2 cell expression system. ELISA and surface plasmon resonance (SPR) analyses confirmed the binding activity of 1E8 scFv protein to various T-antigen presenting conjugates. NMR experiments provided evidence of the folded nature of the 1E8 scFv protein. ScFv-ligand contact was identified by STD NMR, indicating that the galactose unit of T-antigen at the non-reducing end was primarily recognized by 1E8 scFv. This thus provides direct evidence of T-antigen specificity.

  13. Single-Chain Antibody Library

    DOE Data Explorer

    Baird, Cheryl

    Researchers at Pacific Northwest National Laboratory (PNNL) have constructed a nonimmune library consisting of 109 human antibody scFv fragments, which have been cloned and expressed on the surface of yeast. Nanomolar-affinity scFvs are routinely obtained by magnetic bead screening and flow cytometric sorting. The yeast library can be amplified 1010 fold without measurable loss of clonal diversity. This allows for indefinite expansion of the library. All scFv clones can be assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps. The ability to use multiplex library screening demonstrates the utility of this approach for high-throughput antibody isolation for proteomic applications. The yeast library may be used for research projects or teaching performed for U.S. Government purposes only. If you would like to request an aliquot of the single-chain antibody library for your research, please print and fill out the Materials Transfer Agreement (MTA) [PDF, 20K]. The website provides the contact information for mailing the MTA. [copied from http://www.sysbio.org/dataresources/singlechain.stm

  14. Screening, expression, and characterization of an anti-human oxidized low-density lipoprotein single-chain variable fragment.

    PubMed

    Kumano-Kuramochi, Miyuki; Fujimura, Takashi; Komba, Shiro; Maeda-Yamamoto, Mari; Machida, Sachiko

    2016-09-01

    Increased levels of oxidized low-density lipoprotein (OxLDL) in the blood circulation are correlated with atherosclerosis. Monoclonal antibody-based detection systems have been reported for OxLDL. We identified novel single-chain variable fragments (scFvs) having affinity for human OxLDL and related ligands. We constructed an scFv library from nonimmunized human spleen mRNA. Two types (γ+κ and μ+λ) of scFv phage libraries were enriched by biopanning, and five scFv clones with affinity for OxLDL were identified. The γκ5 scFv, which showed the highest affinity for OxLDL, was cloned into pET-22b(+) and expressed in Escherichia coli BL21(DE3). γκ5, expressed as an inclusion body in BL21(DE3), was refolded and purified. The specificity and sensitivity of γκ5 were analyzed using enzyme-linked immunosorbent assays (ELISAs). The γκ5 scFv showed affinity for OxLDL and acetylated LDL. The sensitivity of γκ5 to low concentrations (1-2 μg/mL) of OxLDL was higher than that to AcLDL and LDL. Finally, we developed a sandwich ELISA using γκ5 and CTLD14 (a lectin-like OxLDL receptor-1 ligand recognition region), which allowed specific detection of OxLDL at a level below 0.1 μg/mL. Our results indicated that the γκ5 scFv was a promising molecule for the detection of modified LDL at very low concentrations.

  15. [Molecular dynamics of immune complex of photoadduct-containing DNA with Fab-Anti-DNA antibody fragment].

    PubMed

    Akberova, N I; Zhmurov, A A; Nevzorova, T A; Litvinov, R I

    2016-01-01

    Antibodies to DNA play an important role in the pathogenesis of autoimmune diseases. The elucidation of structural mechanisms of both the antigen recognition and the interaction of anti-DNA antibodies with DNA will help to understand the role of DNA-containing immune complexes in various pathologies and can provide a basis for new treatment modalities. Moreover, the DNA-antibody complex is an analog of specific intracellular DNA-protein interactions. In this work, we used in silico molecular dynamic simulations of bimolecular complexes of the dsDNA segment containing the Fab fragment of an anti-DNA antibody to obtain the detailed thermodynamic and structural characteristics of dynamic intermolecular interactions. Using computationally modified crystal structure of the Fab-DNA complex (PDB ID: 3VW3), we studied the equilibrium molecular dynamics of the 64M-5 antibody Fab fragment associated with the dsDNA fragment containing the thymine dimer, the product of DNA photodamage. Amino acid residues that constitute paratopes and the complementary nucleotide epitopes for the Fab-DNA construct were identified. Stacking and electrostatic interactions were found to play the main role in mediating the most specific antibody-dsDNA contacts, while hydrogen bonds were less significant. These findings may shed light on the formation and properties of pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus associated with skin photosensitivity and DNA photodamage.

  16. A single mutation in framework 2 of the heavy variable domain improves the properties of a diabody and a related single-chain antibody.

    PubMed

    Rodríguez-Rodríguez, Everardo Remi; Ledezma-Candanoza, Luis M; Contreras-Ferrat, Luis Gabriel; Olamendi-Portugal, Timoteo; Possani, Lourival D; Becerril, Baltazar; Riaño-Umbarila, Lidia

    2012-10-26

    Excellent results regarding improved therapeutic properties have been often obtained through the conversion of a single-chain variable fragment (scFv) into a noncovalent dimeric antibody (diabody) via peptide linker shortening. We utilized this approach to obtain a dimeric version of the human scFv 6009F, which was originally engineered to neutralize the Cn2 toxin of Centruroides noxius scorpion venom. However, some envenoming symptoms remained with diabody 6009F. Diabody 6009F was subjected to directed evolution to obtain a variant capable of eliminating envenoming symptoms. After two rounds of biopanning, diabody D4 was isolated. It exhibited a single mutation (E43G) in framework 2 of the heavy-chain variable domain. Diabody D4 displayed an increase in T(m) (thermal transition midpoint temperature) of 6.3°C compared with its dimeric precursor. The importance of the E43G mutation was tested in the context of the human scFv LR, a highly efficient antibody against Cn2, which was previously generated by our group [Riaño-Umbarila, L., Contreras-Ferrat, G., Olamendi-Portugal, T., Morelos-Juárez, C., Corzo, G., Possani, L. D. and Becerril, B. (2011). J. Biol. Chem.286, 6143-6151]. The new variant, scFv LER, displayed an increase in T(m) of 3.4°C and was capable of neutralizing 2 LD(50) of Cn2 toxin with no detectable symptoms when injected into mice at a 1:1 toxin-to-antibody molar ratio. These results showed that the E43G mutation might increase the therapeutic properties of these antibody fragments. Molecular modeling and dynamics results suggest that the rearrangement of the hydrogen-bonding network near the E43G mutation could explain the improved functional stability and neutralization properties of both the diabody D4 and scFv LER.

  17. Polynucleotides encoding anti-sulfotyrosine antibodies

    DOEpatents

    Bertozzi, Carolyn R [Berkeley, CA; Kehoe, John [Saint Davids, PA; Bradbury, Andrew M [Santa Fe, NM

    2011-01-11

    The invention provides anti-sulfotyrosine specific antibodies capable of detecting and isolating polypeptides that are tyrosine-sulfated. The sulfotyrosine antibodies and antibody fragments of the invention may be used to discriminate between the non-sulfated and sulfated forms of such proteins, using any number of immunological assays, such ELISAs, immunoblots, Western Blots, immunoprecipitations, and the like. Using a phage-display system, single chain antibodies (scFvs) were generated and screened against tyrosine-sulfated synthetic peptide antigens, resulting in the isolation of scFvs that specifically recognize sulfotyrosine-containing peptides and/or demonstrate sulfotyrosine-specific binding in tyrosine sulfated proteins. The VH and VL genes from one such sulfotyrosine-specific scFv were employed to generate a full length, sulfotyrosine-specific immunoglobulin.

  18. Preventing phage lysis of Lactococcus lactis in cheese production using a neutralizing heavy-chain antibody fragment from llama.

    PubMed

    Ledeboer, A M; Bezemer, S; de Hiaard, J J W; Schaffers, I M; Verrips, C T; van Vliet, C; Düsterhöft, E M; Zoon, P; Moineau, S; Frenken, L G J

    2002-06-01

    Bacteriophage infection is still a persistent problem in large dairy processes despite extensive studies over the last decades. Consequently, new methods are constantly sought to prevent phage infection. In this paper, we show that phage neutralizing heavy-chain antibody fragments, obtained from Camelidae and produced at a large scale in the generally regarded as safe microorganism Saccharomyces cerevisiae, can effectively be used to impede phage induced lysis during a cheese process. The growth inhibition of the cheese starter culture by 10(5) pfu/ml cheese-milk of the small isometric-headed 936-type phage p2 was prevented by the addition of only 0.1 microg/ml (7 nM) of the neutralizing antibody fragment. The use of such antibody fragments in cheese manufacturing are a realistic and interesting option because of the small amount of antibody fragments that are needed. Moreover the antibodies are produced in a food grade microorganism and can easily be isolated from the fermentation liquid in a pure and DNA free form.

  19. A Nucleic-Acid Hydrolyzing Single Chain Antibody Confers Resistance to DNA Virus Infection in HeLa Cells and C57BL/6 Mice

    PubMed Central

    Lee, Gunsup; Yu, Jaelim; Cho, Seungchan; Byun, Sung-June; Kim, Dae Hyun; Lee, Taek-Kyun; Kwon, Myung-Hee; Lee, Sukchan

    2014-01-01

    Viral protein neutralizing antibodies have been developed but they are limited only to the targeted virus and are often susceptible to antigenic drift. Here, we present an alternative strategy for creating virus-resistant cells and animals by ectopic expression of a nucleic acid hydrolyzing catalytic 3D8 single chain variable fragment (scFv), which has both DNase and RNase activities. HeLa cells (SCH7072) expressing 3D8 scFv acquired significant resistance to DNA viruses. Virus challenging with Herpes simplex virus (HSV) in 3D8 scFv transgenic cells and fluorescence resonance energy transfer (FRET) assay based on direct DNA cleavage analysis revealed that the induced resistance in HeLa cells was acquired by the nucleic acid hydrolyzing catalytic activity of 3D8 scFv. In addition, pseudorabies virus (PRV) infection in WT C57BL/6 mice was lethal, whereas transgenic mice (STG90) that expressed high levels of 3D8 scFv mRNA in liver, muscle, and brain showed a 56% survival rate 5 days after PRV intramuscular infection. The antiviral effects against DNA viruses conferred by 3D8 scFv expression in HeLa cells as well as an in vivo mouse system can be attributed to the nuclease activity that inhibits viral genome DNA replication in the nucleus and/or viral mRNA translation in the cytoplasm. Our results demonstrate that the nucleic-acid hydrolyzing activity of 3D8 scFv confers viral resistance to DNA viruses in vitro in HeLa cells and in an in vivo mouse system. PMID:24968358

  20. Multiparameter optimization method and enhanced production of secreted recombinant single-chain variable fragment against the HIV-1 P17 protein from Escherichia coli by fed-batch fermentation.

    PubMed

    Paopang, Porntip; Kasinrerk, Watchara; Tayapiwatana, Chatchai; Seesuriyachan, Phisit; Butr-Indr, Bordin

    2016-01-01

    The single-chain fragment variable (scFv) was used to produce a completely functional antigen-binding fragment in bacterial systems. The advancements in antibody engineering have simplified the method of producing Fv fragments and made it more efficient and generally relevant. In a previous study, the scFv anti HIV-1 P17 protein was produced by a batch production system, optimized by the sequential simplex optimization method. This study continued that work in order to enhance secreted scFv production by fed-batch cultivation, which supported high volumetric productivity and provided a large amount of scFvs for diagnostic and therapeutic research. The developments in cell culture media and process parameter settings were required to realize the maximum production of cells. This study investigated the combined optimization methods, Plackett-Burman design (PBD) and sequential simplex optimization, with the aim of optimize feed medium. Fed-batch cultivation with an optimal feeding rate was determined. The result demonstrated that a 20-mL/hr feeding rate of the optimized medium can increase cell growth, total protein production, and scFv anti-p17 activity by 4.43, 1.48, and 6.5 times more than batch cultivation, respectively. The combined optimization method demonstrated novel power tools for the optimization strategy of multiparameter experiments.

  1. Generating Recombinant Antibodies against Putative Biomarkers of Retinal Injury

    PubMed Central

    Kierny, Michael R.; Cunningham, Thomas D.; Bouhenni, Rachida A.; Edward, Deepak P.; Kay, Brian K.

    2015-01-01

    Candidate biomarkers, indicative of disease or injury, are beginning to overwhelm the process of validation through immunological means. Recombinant antibodies developed through phage-display offer an alternative means of generating monoclonal antibodies faster than traditional immunization of animals. Peptide segments of putative biomarkers of laser induced injury in the rabbit, discovered through mass spectrometry, were used as targets for a selection against a library of phage-displayed human single-chain variable fragment (scFv) antibodies. Highly specific antibodies were isolated to four of these unique peptide sequences. One antibody against the retinal protein, Guanine Nucleotide-Binding Protein Beta 5 (GBB5), had a dissociation constant ~300 nM and recognized the full-length endogenous protein in retinal homogenates of three different animal species by western blot. Alanine scanning of the peptide target identified three charged and one hydrophobic amino acid as the critical binding residues for two different scFvs. To enhance the utility of the reagent, one scFv was dimerized through a Fragment-crystallizable hinge region (i.e., Fc) and expressed in HEK-293 cells. This dimeric reagent yielded a 25-fold lower detection limit in western blots. PMID:25902199

  2. Generating Recombinant Antibodies against Putative Biomarkers of Retinal Injury.

    PubMed

    Kierny, Michael R; Cunningham, Thomas D; Bouhenni, Rachida A; Edward, Deepak P; Kay, Brian K

    2015-01-01

    Candidate biomarkers, indicative of disease or injury, are beginning to overwhelm the process of validation through immunological means. Recombinant antibodies developed through phage-display offer an alternative means of generating monoclonal antibodies faster than traditional immunization of animals. Peptide segments of putative biomarkers of laser induced injury in the rabbit, discovered through mass spectrometry, were used as targets for a selection against a library of phage-displayed human single-chain variable fragment (scFv) antibodies. Highly specific antibodies were isolated to four of these unique peptide sequences. One antibody against the retinal protein, Guanine Nucleotide-Binding Protein Beta 5 (GBB5), had a dissociation constant ~300 nM and recognized the full-length endogenous protein in retinal homogenates of three different animal species by western blot. Alanine scanning of the peptide target identified three charged and one hydrophobic amino acid as the critical binding residues for two different scFvs. To enhance the utility of the reagent, one scFv was dimerized through a Fragment-crystallizable hinge region (i.e., Fc) and expressed in HEK-293 cells. This dimeric reagent yielded a 25-fold lower detection limit in western blots.

  3. Design and construction of a new human naïve single-chain fragment variable antibody library, IORISS1.

    PubMed

    Pasello, Michela; Zamboni, Silvia; Mallano, Alessandra; Flego, Michela; Picci, Piero; Cianfriglia, Maurizio; Scotlandi, Katia

    2016-04-20

    Human monoclonal antibodies are a powerful tool with increasingly successful exploitations and the single chain fragment variable format can be considered the building block for the implementation of more complex and effective antibody-based constructs. Phage display is one of the best and most efficient methods to isolate human antibodies selected from an efficient and variable phage display library. We report a method for the construction of a human naïve single-chain variable fragment library, termed IORISS1. Many different sets of oligonucleotide primers as well as optimized electroporation and ligation reactions were used to generate this library of 1.2×10(9) individual clones. The key difference is the diversity of variable gene templates, which was derived from only 15 non-immunized human donors. The method described here, was used to make a new human naïve single-chain fragment variable phage display library that represents a valuable source of diverse antibodies that can be used as research reagents or as a starting point for the development of therapeutics. Using biopanning, we determined the ability of IORISS1 to yield antibodies. The results we obtained suggest that, by using an optimized protocol, an efficient phage antibody library can be generated.

  4. Antibody fragment recognition layers for surface plasmon resonance biosensing: a parametric study

    NASA Astrophysics Data System (ADS)

    Magalhães, André; Bordeira, Sandro; Almeida, Ana Cristina; Fontes, Vanessa; Costa, Maria João L.; Fonseca, Luís P.; da Fonseca, João Garcia

    2009-02-01

    A comparative study is reported regarding the use of two different surface plasmon resonance (SPR) biosensors, a homemade SPR grating biosensor and a reference prism coupled biosensor, to perform quantification of C-reactive protein (CRP) in human blood serum. Surface functionalization was conducted using anti-CRP fragments immobilized directly on gold. Adsorption time optimization for the antibody fragments monolayer, non-specific binding (NSB) resistance evaluation and CRP detection were conducted, with better results achieved by the grating biosensor on all topics, namely less functionalization time, higher resistance to NSB and wider CRP dynamic concentration range. A study regarding comparison between continuous flow and surface coating immobilization is also reported in this work. We have shown that surface coating immobilization achieves similar NSB resistance and CRP detection results, allowing a 75% assay cost reduction by lower solution volume requirement. Results suggest that the coating immobilization technique is the best suited to be used in further studies in order to obtain a viable immunosensor for CRP and other biomarkers detection in complex biological fluids.

  5. Anti-neuropilin 1 antibody Fab' fragment conjugated liposomal docetaxel for active targeting of tumours.

    PubMed

    Manjappa, Arehalli S; Goel, Peeyush N; Gude, Rajiv P; Ramachandra Murthy, Rayasa S

    2014-09-01

    Neuropilin-1, a transmembrane receptor entailed in wide range of human tumour cell lines and diverse neoplasms, mediates the effects of VEGF and Semaphorins during the processes of cellular proliferation, survival and migration. In view of this, we had developed and evaluated in vitro and in vivo efficacy of anti-neuropilin-1 immunoliposomes against neuropilin-1 receptor expressing tumours. The PEGylated liposomes loaded with docetaxel were prepared using thin film hydration method. Functionalised PEGylated liposomes were prepared using post-insertion technique. Anti-neuropilin-1 immunoliposomes were prepared by covalently conjugating Fab' fragments of neuropilin-1 antibody to functionalised PEGylated liposomes via thioether linkage. In vivo evaluation of Taxotere and liposomal formulations was performed using intradermal tumour model to demonstrate anti-angiogenic and tumour regression ability. The modified Fab' fragments and immunoliposomes were found to be immunoreactive against A549 cells. Further, docetaxel loaded PEGylated liposomes and PEGylated immunoliposomes demonstrated higher in vitro cytotoxicity than Taxotere formulation at the same drug concentration and exposure time. The live imaging showed distinctive cellular uptake of functional immunoliposomes. Further, significant decrease in micro-blood vessel density and tumour volumes was observed using bio-engineered liposomes. The results clearly highlight the need to seek neuropilin-1 as one of the prime targets in developing an anti-angiogenic therapy.

  6. Identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage. lambda. immunoexpression library

    SciTech Connect

    Mullinax, R.L.; Gross, E.A.; Amberg, J.R.; Hogrefe, H.H.; Kubitz, M.M.; Greener, A.; Alting-Mees, M.; Ardourel, D.; Short, J.M.; Sorge, J.A. ); Hay, B.N.; Shopes, B. )

    1990-10-01

    The authors have applied a molecular biology approach to the identification of human monoclonal antibodies. Human peripheral blood lymphocyte mRNA was converted to cDNA and a select subset was amplified by the polymerase chain reaction. These products, containing coding sequences for numerous immunoglobulin heavy- and {kappa} light-chain variable and constant region domains, were inserted into modified bacteriophase {lambda} expression vectors and introduced into Escherichia coli by infection to yield a combinatorial immunoexpression library. Clones with binding activity to tetanus toxoid were identified by filter hybridization with radiolabeled antigen and appeared at a frequency of 0.2{percent} in the library. These human antigen binding fragments, consisting of a heavy-chain fragment covalently linked to a light chain, displayed high affinity of binding to tetanus toxoid with equilibrium constants in the nanomolar range but did not cross-react with other proteins tested. They estimate that this human immunoexpression library contains 20,000 clones with high affinity and specificity to our chosen antigen.

  7. A system for repertoire cloning and phage display of murine and leporid antibody fragments.

    PubMed

    Schüller, Carolin; Wiebe, Julia C; Pegel, Antje; Kramer, Karl; Skerra, Arne; Hock, Bertold

    2010-01-01

    Even though rabbit antibodies (Abs) are known to exceed murine Abs with respect to specificity, affinity, and stability, cloned leporid immune repertoires have been rarely considered in recombinant Ab preparation for environmental analysis. We have developed a set of four tet(p/o)-based phasmid vectors that allow the efficient cloning of both murine and leporid Ab repertoires. These vectors differ in the design of the cloning sites, choice of signal peptides, and antibiotic selection markers. A set of 39 primer oligodeoxynucleotides has been developed for the PCR amplification of rabbit Ab genes, representing the most exhaustive coverage of the leporid immune repertoire described so far. The atrazine-specific murine Fab fragment K411B and a cloned V-gene repertoire from sulfonamide-immunized rabbits were used to compare these phasmids with respect to expression of Fab fragments, phagemid titers, and number of Fab displaying phagemid particles. Our results show that the ratio of recombinant phagemids could be increased up to 65% of total phage titer by utilizing the appropriate phasmid. Based on this system, the selection of two sulfonamide-specific rabbit Abs, SA2 23 and SA2 90, was accomplished after a single phagemid panning round.

  8. Rice-based oral antibody fragment prophylaxis and therapy against rotavirus infection

    PubMed Central

    Tokuhara, Daisuke; ρlvarez, Beatriz; Mejima, Mio; Hiroiwa, Tomoko; Takahashi, Yuko; Kurokawa, Shiho; Kuroda, Masaharu; Oyama, Masaaki; Kozuka-Hata, Hiroko; Nochi, Tomonori; Sagara, Hiroshi; Aladin, Farah; Marcotte, Harold; Frenken, Leon G.J.; Iturriza-Gómara, Miren; Kiyono, Hiroshi; Hammarström, Lennart; Yuki, Yoshikazu

    2013-01-01

    Rotavirus-induced diarrhea is a life-threatening disease in immunocompromised individuals and in children in developing countries. We have developed a system for prophylaxis and therapy against rotavirus disease using transgenic rice expressing the neutralizing variable domain of a rotavirus-specific llama heavy-chain antibody fragment (MucoRice-ARP1). MucoRice-ARP1 was produced at high levels in rice seeds using an overexpression system and RNAi technology to suppress the production of major rice endogenous storage proteins. Orally administered MucoRice-ARP1 markedly decreased the viral load in immunocompetent and immunodeficient mice. The antibody retained in vitro neutralizing activity after long-term storage (>1 yr) and boiling and conferred protection in mice even after heat treatment at 94°C for 30 minutes. High-yield, water-soluble, and purification-free MucoRice-ARP1 thus forms the basis for orally administered prophylaxis and therapy against rotavirus infections. PMID:23925294

  9. Dual Beneficial Effect of Interloop Disulfide Bond for Single Domain Antibody Fragments*

    PubMed Central

    Govaert, Jochen; Pellis, Mireille; Deschacht, Nick; Vincke, Cécile; Conrath, Katja; Muyldermans, Serge; Saerens, Dirk

    2012-01-01

    The antigen-binding fragment of functional heavy chain antibodies (HCAbs) in camelids comprises a single domain, named the variable domain of heavy chain of HCAbs (VHH). The VHH harbors remarkable amino acid substitutions in the framework region-2 to generate an antigen-binding domain that functions in the absence of a light chain partner. The substitutions provide a more hydrophilic, hence more soluble, character to the VHH but decrease the intrinsic stability of the domain. Here we investigate the functional role of an additional hallmark of dromedary VHHs, i.e. the extra disulfide bond between the first and third antigen-binding loops. After substituting the cysteines forming this interloop cystine by all 20 amino acids, we selected and characterized several VHHs that retain antigen binding capacity. Although VHH domains can function in the absence of an interloop disulfide bond, we demonstrate that its presence constitutes a net advantage. First, the disulfide bond stabilizes the domain and counteracts the destabilization by the framework region-2 hallmark amino acids. Second, the disulfide bond rigidifies the long third antigen-binding loop, leading to a stronger antigen interaction. This dual beneficial effect explains the in vivo antibody maturation process favoring VHH domains with an interloop disulfide bond. PMID:22128183

  10. Nebulized anti-IL-13 monoclonal antibody Fab' fragment reduces allergen-induced asthma.

    PubMed

    Hacha, Jonathan; Tomlinson, Kate; Maertens, Ludovic; Paulissen, Geneviève; Rocks, Natacha; Foidart, Jean-Michel; Noel, Agnès; Palframan, Roger; Gueders, Maud; Cataldo, Didier D

    2012-11-01

    IL-13 is a prototypic T helper type 2 cytokine and a central mediator of the complex cascade of events leading to asthmatic phenotype. Indeed, IL-13 plays key roles in IgE synthesis, bronchial hyperresponsiveness, mucus hypersecretion, subepithelial fibrosis, and eosinophil infiltration. We assessed the potential efficacy of inhaled anti-IL-13 monoclonal antibody Fab' fragment on allergen-induced airway inflammation, hyperresponsiveness, and remodeling in an experimental model of allergic asthma. Anti-IL-13 Fab' was administered to mice as a liquid aerosol generated by inExpose inhalation system in a tower allowing a nose-only exposure. BALB/c mice were treated by PBS, anti-IL-13 Fab', or A33 Fab' fragment and subjected to ovalbumin exposure for 1 and 5 weeks (short-term and long-term protocols). Our data demonstrate a significant antiasthma effect after nebulization of anti-IL-13 Fab' in a model of asthma driven by allergen exposure as compared with saline and nonimmune Fab fragments. In short- and long-term protocols, administration of the anti-IL-13 Fab' by inhalation significantly decreased bronchial responsiveness to methacholine, bronchoalveolar lavage fluid eosinophilia, inflammatory cell infiltration in lung tissue, and many features of airway remodeling. Levels of proinflammatory mediators and matrix metalloprotease were significantly lower in lung parenchyma of mice treated with anti-IL-13 Fab'. These data demonstrate that an inhaled anti-IL-13 Fab' significantly reduces airway inflammation, hyperresponsiveness, and remodeling. Specific neutralization of IL-13 in the lungs using an inhaled anti-IL-13 Fab' could represent a novel and effective therapy for the treatment of asthma.

  11. A humanized anti-M2 scFv shows protective in vitro activity against influenza

    SciTech Connect

    Bradbury, Andrew M; Velappan, Nileena; Schmidt, Jurgen G

    2008-01-01

    M2 is one of the most conserved influenza proteins, and has been widely prospected as a potential universal vaccine target, with protection predominantly mediated by antibodies. In this paper we describe the creation of a humanized single chain Fv from 14C2, a potent monoclonal antibody against M2. We show that the humanized scFv demonstrates similar activity to the parental mAb: it is able to recognize M2 in its native context on cell surfaces and is able to show protective in vitro activity against influenza, and so represents a potential lead antibody candidate for universal prophylactic or therapeutic intervention in influenza.

  12. Expression and production of llama variable heavy-chain antibody fragments (V(HH)s) by Aspergillus awamori.

    PubMed

    Joosten, Vivi; Gouka, Robin J; van den Hondel, Cees A M J J; Verrips, C Theo; Lokman, B Christien

    2005-01-01

    We report the expression and production of llama variable heavy-chain antibody fragments (V(HH)s) by Aspergillus awamori. Fragments encoding V(HH)s were cloned in a suitable Aspergillus expression vector and transformants secreting V(HH) fragments were analysed for integrated gene copy-numbers, mRNA levels and protein production. Functional V(HH)s were detected in the culture medium, indicating the feasibility of producing this type of protein in a fungal expression system. Secreted V(HH)s were subjected to (extracellular) degradation, which could be partially prevented by the addition of BSA to the culture medium.

  13. Early Detection of NSCLC with scFv Selected against IgM Autoantibody

    PubMed Central

    Pedchenko, Tetyana; Mernaugh, Ray; Parekh, Dipti; Li, Ming; Massion, Pierre P.

    2013-01-01

    Survival of patients with lung cancer could be significantly prolonged should the disease be diagnosed early. Growing evidence indicates that the immune response in the form of autoantibodies to developing cancer is present before clinical presentation. We used a phage-displayed antibody library to select for recombinant scFvs that specifically bind to lung cancer-associated IgM autoantibodies. We selected for scFv recombinant antibodies reactive with circulating IgM autoantibodies found in the serum of patients with early stage lung adenocarcinoma but not matched controls. Discriminatory performance of 6 selected scFvs was validated in an independent set of serum from stage 1 adenocarcinoma and matching control groups using two independent novel methods developed for this application. The panel of 6 selected scFvs predicted cancer based on seroreactivity value with sensitivity of 0.8 and specificity of 0.87. Receiver Operative Characteristic curve (ROC) for combined 6 scFv has an AUC of 0.88 (95%CI, 0.76–1.0) as determined by fluorometric microvolume assay technology (FMAT) The ROC curve generated using a homogeneous bridging Mesa Scale Discovery (MSD) assay had an AUC of 0.72 (95% CI, 0.59–0.85). The panel of all 6 antibodies demonstrated better discriminative power than any single scFv alone. The scFv panel also demonstrated the association between a high score - based on seroreactivity - with poor survival. Selected scFvs were able to recognize lung cancer associated IgM autoantibodies in patient serum as early as 21 months before the clinical presentation of disease. The panel of antibodies discovered represents a potential unique non-invasive molecular tool to detect an immune response specific to lung adenocarcinoma at an early stage of disease. PMID:23585862

  14. Immunoscintigraphy of human pancreatic carcinoma in nude mice with I-131-F(ab')/sub 2/-fragments of monoclonal antibodies

    SciTech Connect

    Senekowitsch, R.; Maul, F.D.; Wenisch, H.J.C.; Kriegel, H.; Hor, G.

    1985-05-01

    In the present study radioiodinated F(ab')/sub 2/-fragments of CA19-9 and antibody that reacts specifically with human gastrointestinal cancer were examined for their ability to detect human pancreatic carcinoma hosted in nude mice. Tumor-bearing mice received 80..mu..Ci of I-131-F(ab')/sub 2/ with a specific activity of 1.8..mu..Ci/..mu..g. All mice were imaged after the injection and every 24hr up to 6 days. The retained radioactivity was also registered with a whole-body counter immediately after imaging. As a control F(ab's)/sub 2/ of a nonspecific antibody were administered in parallel to another group of animals bearing the same tumor. Three animals of each group were killed at 1,2,4 and 8 days for determination of the distribution of both labeled antibody-fragments. On scintigraphic images obtained with the CA19-9-F(ab')/sub 2/ the tumors could be visualized 24hr after injection, the best dilineation however was achieved 96hr p.i.. The biodistribution data exhibited a more rapid blood clearance for the specific fragments compared to that for the unspecific ones. Tumors showed an increase in uptake up to 48hr reaching 1.7% of the injected dose per gram, declining to values of 0.08%/g at day 6 p.i.. The highest tumor-to-blood ratios were found after 96h. They were 7 for the CA19-9-fragments compared to 1.5 for the unspecific fragments. The whole body counting revealed a more rapid excretion for the fragments of the specific monoclonal antibodies than for the unspecific ones. In summary the authors were able to show that CA19-9-F(ab')/sub 2/-fragments can be used for immunodetection of human pancreatic carcinoma hosted in nude mice.

  15. Electrochemical detection of vascular endothelial growth factors (VEGFs) using VEGF antibody fragments modified Au NPs/ITO electrode.

    PubMed

    Kim, Gang-Il; Kim, Kyung-Woo; Oh, Min-Kyu; Sung, Yun-Mo

    2010-03-15

    A new electrochemical technique for the detection of vascular endothelial growth factors (VEGFs) as a cancer-related biomarker is presented in this paper. Gold nanoparticles (Au NPs) were self-assembled onto an indium tin oxide (ITO) electrode to prepare a modified sandwich type electrochemical immunoassay platform. VEGF antibodies were cleaved into two half-fragments by 2-mercaptoethylamine-HCl (2-MEA) and the fragments were immobilized onto the Au NP substrates by their thiol groups. Through this strategy, randomly oriented attachment of antibodies was prevented which frequently occurs in a general use of whole antibody and reduces the number of available sites for the attachment of target molecules. VEGF target molecules were applied to the immunoelectrodes and they combined with the antibody fragments covering the Au NP electrode, forming antigen-antibody complexes. Then, ferrocene-tagged antibodies, which release electrons under a proper applied potential, were added to the system and they combined with the VEGF molecules pre-attached to the antibody fragments. The redox current of ferrocene measured by the differential pulse voltammetry (DPV) increased almost linearly from 1.27 x 10(-4) to 4.17 x 10(-4)A according to the increase in the concentration of the VEGF target molecules from 100 to 600 pg/ml. The measured current values represent the concentration of the VEGF since they are proportional to the number of ferrocene molecules which is in turn proportional to the concentration of VEGF target molecules. Using this modified sandwich immunoassay with the Au NP/ITO electrode, VEGFs as low as 100 pg/ml were detected with high specificity.

  16. Preparation and characterization of anti-tissue factor single-chain variable fragment antibody for cancer diagnosis.

    PubMed

    Sato, Ryuta; Obonai, Toshifumi; Tsumura, Ryo; Tsumoto, Kouhei; Koga, Yoshikatsu; Yasunaga, Masahiro; Matsumura, Yasuhiro

    2014-12-01

    Tissue factor (TF), which serves as the initiator of the extrinsic blood coagulation cascade, has been found to be overexpressed in various solid tumors, especially brain tumors, pancreatic cancer, and gastric cancer. Overexpression of TF is considered to contribute to the high incidence of thrombotic complications and poor prognosis in patients with such cancers. Therefore, detection or targeting of TF may be a promising approach for the diagnosis and treatment of solid tumors that are known to overexpress the protein. Here, we used the recombinant DNA technology to develop an anti-TF single-chain Fv (scFv) of small size and high affinity for its target. The biochemical characteristics of the anti-TF scFv were evaluated using surface plasmon resonance (SPR) sensing and flow cytometry. The data obtained showed that the affinity of the anti-TF scFv was 2.04 × 10(-8) (KD), and that the protein showed significant binding to the cancer cells. Then, Alexa 647-labeled anti-TF scFv and anti-TF IgG were administered to mice bearing chemically induced spontaneous tumors. The maximum tumor to background ratios of anti-TF scFv and anti-TF IgG were obtained 3 and 24 h after the injections, respectively. This study indicates anti-TF scFv may be suitable as an imaging probe for the diagnosis of solid tumors.

  17. Intracellular interference of tick-borne flavivirus infection by using a single-chain antibody fragment delivered by recombinant Sindbis virus.

    PubMed Central

    Jiang, W; Venugopal, K; Gould, E A

    1995-01-01

    A single-chain antibody fragment that identifies a neutralizing epitope on the envelope protein of louping ill and some other tick-borne flaviviruses was previously expressed in soluble form from bacteria and shown to be functionally active in vitro. To see whether or not the single-chain antibody could bind and inactivate infectious virus in vivo, we have used recombinant Sindbis virus as a delivery vehicle for intracellular expression of the antibody fragment. The variable genes and interchain linker encoding the single-chain antibody were cloned into a double subgenomic Sindbis virus expression vector to generate recombinant Sindbis virus. Infection with this recombinant Sindbis virus provided high-level cytoplasmic expression of the antibody fragment in mammalian cells. We demonstrate (i) that the antibody fragment was antigen binding and (ii) that louping ill virus infectivity was significantly reduced in the presence of intracellular antibody expressed by the superinfecting recombinant Sindbis virus. PMID:7815482

  18. An improved affinity tag based on the FLAG peptide for the detection and purification of recombinant antibody fragments.

    PubMed

    Knappik, A; Plückthun, A

    1994-10-01

    The commercially available monoclonal antibodies M1 and M2 were raised against and bind the FLAG sequence DYKDDDDK with high specificity. Using the calcium-dependent M1 antibody and the FLAG tag attached to the N terminus of various fragments of the antibody McPC603 expressed in Escherichia coli, we found that the M1 antibody binds with almost the same affinity to a much shorter version of this sequence (DYKD). Since most antibody light chains start with an aspartate, the addition of only three additional amino acids to the N terminus is sufficient to detect and quantify the expressed antibody fragments using standard immunological methods. Similarly, the heavy chain can be detected specifically with the sequence DYKD, which requires four additional amino acids since most heavy chains do not start with Asp. The signal sequence of both chains that is necessary for the transport of the chains to the periplasm of E. coli is processed correctly. Furthermore, we investigated the influence of the amino acid at the fifth position of the FLAG sequence on the binding affinity of the M1 antibody and found that a glutamate at this position increased the sensitivity in Western blots sixfold over the original long FLAG sequence containing an aspartate residue at this position. Together, the improved FLAG is a versatile tool for both sensitive detection and one-step purification of recombinant proteins.

  19. Phase analysis in single-chain variable fragment production by recombinant Pichia pastoris based on proteomics combined with multivariate statistics.

    PubMed

    Fujiki, Yuya; Kumada, Yoichi; Kishimoto, Michimasa

    2015-08-01

    The proteomics technique, which consists of two-dimensional gel electrophoresis (2-DE), peptide mass fingerprinting (PMF), gel image analysis, and multivariate statistics, was applied to the phase analysis of a fed-batch culture for the production of a single-chain variable fragment (scFv) of an anti-C-reactive protein (CRP) antibody by Pichia pastoris. The time courses of the fed-batch culture were separated into three distinct phases: the growth phase of the batch process, the growth phase of the fed-batch process, and the production phase of the fed-batch process. Multivariate statistical analysis using 2-DE gel image analysis data clearly showed the change in the culture phase and provided information concerning the protein expression, which suggested a metabolic change related to cell growth and production during the fed-batch culture. Furthermore, specific proteins, such as alcohol oxidase, which is strongly related to scFv expression, and proteinase A, which could biodegrade scFv in the latter phases of production, were identified via the PMF method. The proteomics technique provided valuable information about the effect of the methanol concentration on scFv production.

  20. Development of a high-affinity anti-domoic acid sheep scFv and its use in detection of the toxin in shellfish.

    PubMed

    Shaw, Iain; O'Reilly, Aoife; Charleton, Margaret; Kane, Marian

    2008-05-01

    The potential of immunoassays as high-throughput screening tools for the detection of harmful substances in foods will only be realized when convenient methods are available for production of the high affinity antibodies needed for sensitive assay development. Recombinant antibodies offer advantages over traditional monoclonal antibodies in terms of ease of production, much greater antibody repertoire for selection, and versatility. We describe here the development of recombinant antibodies against the common shellfish toxin, domoic acid (DA), utilizing the sheep immunoglobulin system as an effective method for generating high affinity anti-hapten recombinant antibody fragments. A single-chain antibody fragment (scFv) library was generated from a sheep immunized with DA-bovine serum albumin conjugate, and anti-DA scFvs were isolated by phage-display. Three selected scFvs gave I50s of 2.6 to 58 ng/mL (8.3-186 nM) in competitive enzyme-linked immunosorbent assay (ELISA). Assay optimization with one of these scFvs gave a very reproducible standard curve with a range of 0.3 to 5.6 ng/mL (1.0 to 17.9 nM), a mean limit of quantification (LOQ, defined as the I20) of 0.5 ng/mL (1.6 nM), and a mean I50 of 1.2 ng/mL (3.9 nM). When the assay was used for the analysis of crude methanolic extracts of scallop tissues, results obtained correlated well with standard HPLC assay results (R2, 0.90, n = 40; R2, 0.81, n = 34), although ELISA results were lower than HPLC results. Adjusting the cutoff point for DA concentration accordingly from the regulatory 20 mg/kg, the potential of the sheep scFv-based ELISA for use as a screening assay for DA in shellfish extracts was demonstrated.

  1. Use of 18F-2-Fluorodeoxyglucose to Label Antibody Fragments for Immuno-Positron Emission Tomography of Pancreatic Cancer

    PubMed Central

    2015-01-01

    We generated 18F-labeled antibody fragments for positron emission tomography (PET) imaging using a sortase-mediated reaction to install a trans-cyclooctene-functionalized short peptide onto proteins of interest, followed by reaction with a tetrazine-labeled-18F-2-deoxyfluoroglucose (FDG). The method is rapid, robust, and site-specific (radiochemical yields > 25%, not decay corrected). The availability of 18F-2-deoxyfluoroglucose avoids the need for more complicated chemistries used to generate carbon–fluorine bonds. We demonstrate the utility of the method by detecting heterotopic pancreatic tumors in mice by PET, using anti-Class II MHC single domain antibodies. We correlate macroscopic PET images with microscopic two-photon visualization of the tumor. Our approach provides easy access to 18F-labeled antibodies and their fragments at a level of molecular specificity that complements conventional 18F-FDG imaging. PMID:26955657

  2. Evaluation of three different formats of a neutralizing single chain human antibody against toxin Cn2: neutralization capacity versus thermodynamic stability.

    PubMed

    Quintero-Hernández, Veronica; Del Pozo-Yauner, Luis; Pedraza-Escalona, Martha; Juárez-González, Victor R; Alcántara-Recillas, Israel; Possani, Lourival D; Becerril, Baltazar

    2012-04-30

    The single-chain antibody fragment (scFv) 6009F, obtained by directed evolution, neutralizes the effects of the Cn2 toxin, which is the major toxic component of Centruroides noxius scorpion venom. In this work we compared the neutralization capacity and the thermodynamic stability of scFv 6009F with those of two other derived formats: Fab 6009F and diabody 6009F. Additionally, the affinity constants to Cn2 toxin of the three recombinant antibody fragments were determined by means of BIAcore. We found a correlation between the thermodynamic stability of these antibody fragments with their neutralization capacity. The order of thermodynamic stability determined was Fab≫scFv>diabody. The Fab and scFv were capable of neutralizing the toxic effects of Cn2 and whole venom but the diabody was unable to fully neutralize intoxication. In silico analysis of the diabody format indicates that the reduction of stability and neutralization capacity could be explained by a less cooperative interface between the heavy and the light variable domains.

  3. Antibodies with specificities against a dispase-produced 15-kilodalton hexon fragment neutralize adenovirus type 2 infectivity.

    PubMed Central

    Varga, M J; Bergman, T; Everitt, E

    1990-01-01

    During the entrance of adenovirus type 2 into cells, it has been suggested that the virion undergoes a conformational change. In this investigation, we have further characterized the hypothetical conformational change, which the structural protein hexon undergoes in response to low pH. From pH 5.0 to pH 6.0, the proteolytic enzyme dispase cleaved the hexon into a few distinct fragments with a dominating low-molecular-weight fragment with a molecular weight of 15,000 (15K peptide), whereas between pH 6.5 and pH 8.0, the cleavage of the hexon was negligible. The degradation of the hexon with dispase at low pH was not due to an increased activity or alteration of the active site of dispase at low pH. The 15K fragment was identified as a segment of the N-terminal part of the hexon polypeptide beginning at amino acid residue 5. An immune serum produced in response to acid-treated and glutaraldehyde-fixed hexons contained a small amount of antibodies directed towards the 15K fragment, as judged by Western immunoblotting. An anti-15K antibody fraction was isolated by affinity chromatography by removing antibodies recognizing the hexon in the alkaline configuration. Such antibodies displayed a higher relative titer at pH 5.0 than at pH 7.5 in an enzyme-linked immunosorbent assay. The isolated antibodies showed a specific neutralizing capacity five times higher than that of the corresponding unfractionated polyclonal anti-hexon serum; however, the neutralizing ability was independent of pH. The neutralization of adenovirus type 2 infection by the isolated anti-15K antibodies implies that the N-terminal end of the hexon may play a critical role in the early steps of the virion-cell interaction. Images PMID:1696636

  4. Method for preparation of single chain antibodies

    SciTech Connect

    Cheung, Nai-Kong V; Guo, Hong-fen

    2012-04-03

    This invention provides a method for identifying cells expressing a target single chain antibody (scFv) directed against a target antigen from a collection of cells that includes cells that do not express the target scFv, comprising the step of combining the collection of cells with an anti-idiotype directed to an antibody specific for the target antigen and detecting interaction, if any, of the anti-idiotype with the cells, wherein the occurrence of an interaction identifies the cell as one which expresses the target scFv. This invention also provides a method for making a single chain antibody (scFv) directed against an antigen, wherein the selection of clones is made based upon interaction of those clones with an appropriate anti-idiotype, and heretofore inaccessible scFv so made. This invention provides the above methods or any combination thereof. Finally, this invention provides various uses of these methods.

  5. Characterization of Poliovirus Neutralization Escape Mutants of Single-Domain Antibody Fragments (VHHs)

    PubMed Central

    Schotte, Lise; Thys, Bert; Strauss, Mike; Filman, David J.; Rombaut, Bart

    2015-01-01

    To complete the eradication of poliovirus and to protect unvaccinated people subsequently, the development of one or more antiviral drugs will be necessary. A set of five single-domain antibody fragments (variable parts of the heavy chain of a heavy-chain antibody [VHHs]) with an in vitro neutralizing activity against poliovirus type 1 was developed previously (B. Thys, L. Schotte, S. Muyldermans, U. Wernery, G. Hassanzadeh-Ghassabeh, and B. Rombaut, Antiviral Res 87:257–264, 2010, http://dx.doi.org/10.1016/j.antiviral.2010.05.012), and their mechanisms of action have been studied (L. Schotte, M. Strauss, B. Thys, H. Halewyck, D. J. Filman, M. Bostina, J. M. Hogle, and B. Rombaut, J Virol 88:4403–4413, 2014, http://dx.doi.org/10.1128/JVI.03402-13). In this study, neutralization escape mutants were selected for each VHH. Sequencing of the P1 region of the genome showed that amino acid substitutions are found in the four viral proteins of the capsid and that they are located both in proximity to the binding sites of the VHHs and in regions further away from the canyon and hidden beneath the surface. Characterization of the mutants demonstrated that they have single-cycle replication kinetics that are similar to those of their parental strain and that they are all drug (VHH) independent. Their resistant phenotypes are stable, as they do not regain full susceptibility to the VHH after passage over HeLa cells in the absence of VHH. They are all at least as stable as the parental strain against heat inactivation at 44°C, and three of them are even significantly (P < 0.05) more resistant to heat inactivation. The resistant variants all still can be neutralized by at least two other VHHs and retain full susceptibility to pirodavir and 35-1F4. PMID:26014941

  6. Efficient isolation of soluble intracellular single-chain antibodies using the twin-arginine translocation machinery

    PubMed Central

    Fisher, Adam; DeLisa, Matthew P.

    2008-01-01

    One of the most commonly used recombinant antibody formats is the single-chain variable fragment (scFv) that consists of the antibody variable heavy chain connected to the variable light chain by a flexible linker. Since disulfide bonds are often necessary for scFv folding, it can be challenging to express scFvs in the reducing environment of the cytosol. Thus, we sought to develop a method for antigen-independent selection of scFvs that are stable in the reducing cytosol of bacteria. To this end, we applied a recently developed genetic selection for protein folding and solubility based on the quality control feature of the Escherichia coli twin-arginine translocation (Tat) pathway (Fisher et al., 2006 Protein Sci). This selection employs a tripartite sandwich fusion of a protein-of-interest with an N-terminal Tat-specific signal peptide and C-terminal TEM1 β-lactamase, thereby coupling antibiotic resistance with Tat pathway export. Here, we adapted this assay to develop intrabody selection after Tat export (ISELATE), a high-throughput selection strategy for the identification of solubility-enhanced scFv sequences. Using ISELATE for three rounds of laboratory evolution, it was possible to evolve a soluble scFv from an insoluble parental sequence. We also show that ISELATE enables focusing of an scFv library in soluble sequence space prior to functional screening and thus can be used to increase the likelihood of finding functional intrabodies. Finally, the technique was used to screen a large repertoire of naïve scFvs for clones that conferred significant levels of soluble accumulation. In these ways, we show that the Tat quality control mechanism can be harnessed for molecular evolution of scFvs that are soluble in the reducing cytoplasm of E. coli. PMID:18992254

  7. Construction and Screening of Antigen Targeted Immune Yeast Surface Display Antibody Libraries

    SciTech Connect

    Miller, Keith D.; Pefaur, Noah B.; Baird, Cheryl L.

    2008-07-01

    These protocols describe a yeast surface display-based process for the rapid selection of antibodies from immunized mice, eliminating the need for creating and screening hybridoma fusions. A yeast surface display library of single-chain antibody fragments (scFvs) is created from antigen-binding B cells from the splenocytes of immunized mice. The antigen targeted library is then screened for antigen specific scFv by magnetic activated cell sorting (MACS) and fluorescence activated cell sorting (FACS). Library construction and screening can be accomplished in as little as 2 weeks resulting in a panel of scFvs specific for the target antigen.

  8. Construction and Screening of Antigen Targeted Immune Yeast Surface Display Antibody Libraries

    SciTech Connect

    Miller, Keith D.; Pefaur, Noah B.; Baird, Cheryl L.

    2009-08-02

    These protocols describe a yeast surface display-based process for the rapid selection of antibodies from immunized mice, eliminating the need for creating and screening hybridoma fusions. A yeast surface display library of single-chain antibody fragments (scFvs) is created from antigen-binding B cells from the splenocytes of immunized mice. The antigen targeted library is then screened for antigen specific scFv by magneticactivated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Library construction and screening can be accomplished in as little as 2 weeks, resulting in a panel of scFvs specific for the target antigen.

  9. Intein-mediated one-step purification of Escherichia coli secreted human antibody fragments.

    SciTech Connect

    Wu, Wan-Yi; Miller, Keith D.; Coolbaugh, Michael; Wood, David W.

    2011-02-25

    In this work, we apply self-cleaving affinity tag technology to several target proteins secreted into the Escherichia coli periplasm, including two with disulfide bonds. The target proteins were genetically fused to a self-cleaving chitin-binding domain intein tag for purification via a chitin agarose affinity resin. By attaching the intein-tagged fusion genes to the PelB secretion leader sequence, the tagged target proteins were secreted to the periplasmic space and could be recovered in active form by simple osmotic shock. After chitin-affinity purification, the target proteins were released from the chitin-binding domain tag via intein self-cleaving. This was induced by a small change in pH from 8.5 to 6.5 at room temperature, allowing direct elution of the cleaved target protein from the chitin affinity resin. The target proteins include the E. coli maltose-binding protein and b-lactamase enzyme, as well as two human antibody fragments that contain disulfide bonds. In all cases, the target proteins were purified with good activity and yield, without the need for refolding. Overall, this work demonstrates the compatibility of the DI-CM intein with the PelB secretion system in E. coli, greatly expanding its potential to more complex proteins.

  10. 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.

  11. The Antiviral Mechanism of an Influenza A Virus Nucleoprotein-Specific Single-Domain Antibody Fragment

    PubMed Central

    Hanke, Leo; Knockenhauer, Kevin E.; Brewer, R. Camille; van Diest, Eline; Schmidt, Florian I.; Schwartz, Thomas U.

    2016-01-01

    ABSTRACT Alpaca-derived single-domain antibody fragments (VHHs) that target the influenza A virus nucleoprotein (NP) can protect cells from infection when expressed in the cytosol. We found that one such VHH, αNP-VHH1, exhibits antiviral activity similar to that of Mx proteins by blocking nuclear import of incoming viral ribonucleoproteins (vRNPs) and viral transcription and replication in the nucleus. We determined a 3.2-Å crystal structure of αNP-VHH1 in complex with influenza A virus NP. The VHH binds to a nonconserved region on the body domain of NP, which has been associated with binding to host factors and serves as a determinant of host range. Several of the NP/VHH interface residues determine sensitivity of NP to antiviral Mx GTPases. The structure of the NP/αNP-VHH1 complex affords a plausible explanation for the inhibitory properties of the VHH and suggests a rationale for the antiviral properties of Mx proteins. Such knowledge can be leveraged for much-needed novel antiviral strategies. PMID:27965447

  12. Retargeting of adenovirus vectors through genetic fusion of a single-chain or single-domain antibody to capsid protein IX.

    PubMed

    Poulin, Kathy L; Lanthier, Robert M; Smith, Adam C; Christou, Carin; Risco Quiroz, Milagros; Powell, Karen L; O'Meara, Ryan W; Kothary, Rashmi; Lorimer, Ian A; Parks, Robin J

    2010-10-01

    Adenovirus (Ad) vectors are the most commonly used system for gene therapy applications, due in part to their ability to infect a wide array of cell types and tissues. However, many therapies would benefit from the ability to target the Ad vector only to specific cells, such as tumor cells for cancer gene therapy. In this study, we investigated the utility of capsid protein IX (pIX) as a platform for the presentation of single-chain variable-fragment antibodies (scFv) and single-domain antibodies (sdAb) for virus retargeting. We show that scFv can be displayed on the capsid through genetic fusion to native pIX but that these molecules fail to retarget the virus, due to improper folding of the scFv. Redirecting expression of the fusion protein to the endoplasmic reticulum (ER) results in correct folding of the scFv and allows it to recognize its epitope; however, ER-targeted pIX-scFv was incorporated into the Ad capsid at a very low level which was not sufficient to retarget virus infection. In contrast, a pIX-sdAb construct was efficiently incorporated into the Ad capsid and enhanced virus infection of cells expressing the targeted receptor. Taken together, our data indicate that pIX is an effective platform for presentation of large targeting polypeptides on the surface of the virus capsid, but the nature of the ligand can significantly affect its association with virions.

  13. Retargeting of Adenovirus Vectors through Genetic Fusion of a Single-Chain or Single-Domain Antibody to Capsid Protein IX ▿

    PubMed Central

    Poulin, Kathy L.; Lanthier, Robert M.; Smith, Adam C.; Christou, Carin; Risco Quiroz, Milagros; Powell, Karen L.; O'Meara, Ryan W.; Kothary, Rashmi; Lorimer, Ian A.; Parks, Robin J.

    2010-01-01

    Adenovirus (Ad) vectors are the most commonly used system for gene therapy applications, due in part to their ability to infect a wide array of cell types and tissues. However, many therapies would benefit from the ability to target the Ad vector only to specific cells, such as tumor cells for cancer gene therapy. In this study, we investigated the utility of capsid protein IX (pIX) as a platform for the presentation of single-chain variable-fragment antibodies (scFv) and single-domain antibodies (sdAb) for virus retargeting. We show that scFv can be displayed on the capsid through genetic fusion to native pIX but that these molecules fail to retarget the virus, due to improper folding of the scFv. Redirecting expression of the fusion protein to the endoplasmic reticulum (ER) results in correct folding of the scFv and allows it to recognize its epitope; however, ER-targeted pIX-scFv was incorporated into the Ad capsid at a very low level which was not sufficient to retarget virus infection. In contrast, a pIX-sdAb construct was efficiently incorporated into the Ad capsid and enhanced virus infection of cells expressing the targeted receptor. Taken together, our data indicate that pIX is an effective platform for presentation of large targeting polypeptides on the surface of the virus capsid, but the nature of the ligand can significantly affect its association with virions. PMID:20631131

  14. Selection and characterization of a human neutralizing antibody to human fibroblast growth factor-2

    SciTech Connect

    Tao, Jun; Xiang, Jun-Jian; Li, Dan; Deng, Ning; Wang, Hong; Gong, Yi-Ping

    2010-04-09

    Compelling evidences suggest that fibroblast growth factor-2 (FGF-2) plays important roles in tumor growth, angiogenesis and metastasis. Molecules blocking the FGF-2 signaling have been proposed as anticancer agents. Through screening of a human scFv phage display library, we have isolated several human single-chain Fv fragments (scFvs) that bind to human FGF-2. After expression and purification in bacteria, one scFv, named 1A2, binds to FGF-2 with a high affinity and specificity, and completes with FGF-2 binding to its receptor. This 1A2 scFv was then cloned into the pIgG1 vector and expressed in 293T cells. The purified hIgG1-1A2 antibody showed a high binding affinity of 8 x 10{sup -9} M to rhFGF-2. In a set of vitro assays, it inhibited various biological activities of FGF-2 such as the proliferation, migration and tube formation of human umbilical vein endothelial cells. More importantly, hIgG1-1A2 antibody also efficiently blocked the growth while inducing apoptosis of glioma cells. For the first time, we generated a human anti-FGF-2 antibody with proven in vitro anti-tumor activity. It may therefore present a new therapeutic candidate for the treatment of cancers that are dependent on FGF-2 signaling for growth and survival.

  15. Digoxin-specific antibody fragments and a calcium antagonist for reversal of digoxin-induced mesenteric vasoconstriction.

    PubMed

    Hess, T; Scholtysik, G; Salzmann, R; Riesen, W

    1983-10-01

    The effect of digoxin-specific antibody fragments on glycoside-induced mesenteric vasoconstriction were investigated. Digoxin caused a sustained contraction of strips of isolated feline mesenteric artery lasting for several hours, while in anaesthetized cats it produced a significant decrease in blood flow and increase in resistance in the mesenteric artery. In-vitro, digoxin's contractile effect was inhibited by 'prophylactic' addition of antibody to the organ bath, but the clinical use for prophylaxis is not a practical proposition. When the antibodies were added with the contraction of the arterial strip in response to digoxin already established, the tone of the preparation decreased significantly over 3 h, but the effect of the glycoside was not fully reversible. In-vivo, control animals not treated with antibodies developed arrhythmias, mesenteric blood flow fell by more than 50% and resistance increased by more than 80% relative to the initial values. These animals died of ventricular fibrillation before the end of the experiment. Animals treated with digoxin-specific antibody fragments after receiving digoxin injections showed no further decrease in mesenteric blood flow and 90 min after the last dose of digoxin, the flow was recovering and mesenteric resistance decreasing. Furthermore, all the animals that had received antibodies remained in sinus rhythm to the end of the experiment. In view of the latent period to onset of action of the antibodies, valuable time may be lost in impaired mesenteric blood flow. To bridge the gap or, indeed, as primary treatment, calcium antagonists merit consideration; in our experiments mesenteric vasoconstriction was abolished within a few minutes by application of the dihydropyridine calcium antagonist 4-(2,1,3-benzo-oxadiazol-4-yl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylic aid, diethyl ester (PY 108-068).

  16. Redistribution of flexibility in stabilizing antibody fragment mutants follows Le Châtelier's principle.

    PubMed

    Li, Tong; Tracka, Malgorzata B; Uddin, Shahid; Casas-Finet, Jose; Jacobs, Donald J; Livesay, Dennis R

    2014-01-01

    Le Châtelier's principle is the cornerstone of our understanding of chemical equilibria. When a system at equilibrium undergoes a change in concentration or thermodynamic state (i.e., temperature, pressure, etc.), La Châtelier's principle states that an equilibrium shift will occur to offset the perturbation and a new equilibrium is established. We demonstrate that the effects of stabilizing mutations on the rigidity ⇔ flexibility equilibrium within the native state ensemble manifest themselves through enthalpy-entropy compensation as the protein structure adjusts to restore the global balance between the two. Specifically, we characterize the effects of mutation to single chain fragments of the anti-lymphotoxin-β receptor antibody using a computational Distance Constraint Model. Statistically significant changes in the distribution of both rigidity and flexibility within the molecular structure is typically observed, where the local perturbations often lead to distal shifts in flexibility and rigidity profiles. Nevertheless, the net gain or loss in flexibility of individual mutants can be skewed. Despite all mutants being exclusively stabilizing in this dataset, increased flexibility is slightly more common than increased rigidity. Mechanistically the redistribution of flexibility is largely controlled by changes in the H-bond network. For example, a stabilizing mutation can induce an increase in rigidity locally due to the formation of new H-bonds, and simultaneously break H-bonds elsewhere leading to increased flexibility distant from the mutation site via Le Châtelier. Increased flexibility within the VH β4/β5 loop is a noteworthy illustration of this long-range effect.

  17. Redistribution of Flexibility in Stabilizing Antibody Fragment Mutants Follows Le Châtelier’s Principle

    PubMed Central

    Li, Tong; Tracka, Malgorzata B.; Uddin, Shahid; Casas-Finet, Jose; Jacobs, Donald J.; Livesay, Dennis R.

    2014-01-01

    Le Châtelier’s principle is the cornerstone of our understanding of chemical equilibria. When a system at equilibrium undergoes a change in concentration or thermodynamic state (i.e., temperature, pressure, etc.), La Châtelier’s principle states that an equilibrium shift will occur to offset the perturbation and a new equilibrium is established. We demonstrate that the effects of stabilizing mutations on the rigidity ⇔ flexibility equilibrium within the native state ensemble manifest themselves through enthalpy-entropy compensation as the protein structure adjusts to restore the global balance between the two. Specifically, we characterize the effects of mutation to single chain fragments of the anti-lymphotoxin-β receptor antibody using a computational Distance Constraint Model. Statistically significant changes in the distribution of both rigidity and flexibility within the molecular structure is typically observed, where the local perturbations often lead to distal shifts in flexibility and rigidity profiles. Nevertheless, the net gain or loss in flexibility of individual mutants can be skewed. Despite all mutants being exclusively stabilizing in this dataset, increased flexibility is slightly more common than increased rigidity. Mechanistically the redistribution of flexibility is largely controlled by changes in the H-bond network. For example, a stabilizing mutation can induce an increase in rigidity locally due to the formation of new H-bonds, and simultaneously break H-bonds elsewhere leading to increased flexibility distant from the mutation site via Le Châtelier. Increased flexibility within the VH β4/β5 loop is a noteworthy illustration of this long-range effect. PMID:24671209

  18. Obstruction of dengue virus maturation by Fab fragments of the 2H2 antibody.

    PubMed

    Wang, Zhiqing; Li, Long; Pennington, Janice G; Sheng, Ju; Yap, Moh Lan; Plevka, Pavel; Meng, Geng; Sun, Lei; Jiang, Wen; Rossmann, Michael G

    2013-08-01

    The 2H2 monoclonal antibody recognizes the precursor peptide on immature dengue virus and might therefore be a useful tool for investigating the conformational change that occurs when the immature virus enters an acidic environment. During dengue virus maturation, spiky, immature, noninfectious virions change their structure to form smooth-surfaced particles in the slightly acidic environment of the trans-Golgi network, thereby allowing cellular furin to cleave the precursor-membrane proteins. The dengue virions become fully infectious when they release the cleaved precursor peptide upon reaching the neutral-pH environment of the extracellular space. Here we report on the cryo-electron microscopy structures of the immature virus complexed with the 2H2 antigen binding fragments (Fab) at different concentrations and under various pH conditions. At neutral pH and a high concentration of Fab molecules, three Fab molecules bind to three precursor-membrane proteins on each spike of the immature virus. However, at a low concentration of Fab molecules and pH 7.0, only two Fab molecules bind to each spike. Changing to a slightly acidic pH caused no detectable change of structure for the sample with a high Fab concentration but caused severe structural damage to the low-concentration sample. Therefore, the 2H2 Fab inhibits the maturation process of immature dengue virus when Fab molecules are present at a high concentration, because the three Fab molecules on each spike hold the precursor-membrane molecules together, thereby inhibiting the normal conformational change that occurs during maturation.

  19. Effect of size of radiolabeled antibody and fragments on tumor uptake and distribution in nephrectomized mice

    SciTech Connect

    Halpern, S.E.; Buchegger, F.; Schreyer, M.; Mach, J.P.

    1984-01-01

    The importance of molecular size in tumor (T) uptake of intact monoclonal antibody (MAb) of MAb fragments (frag.) is difficult to assess because frag. are excreted by the kidney. To obviate this problem nephrectomized nude mice (M) bearing carcinoembryonic (CEA) secreting human colon (T) were used in the following experiments. Following nephrectomy 3 groups of M were injected intravenously with intact In-111 anti-CEA MAb and simultaneously with I-125 intact, F(ab')2 or Fab anti-CEA-MAb. Iodination was by chloramine T and In-111 labeling by bifunctional chelation. All M were killed 8 h after injection, T and normal tissues (NT) dissected, weighed, and counted against a standard of the injectate. The distribution of intact I-125 and In-111-MAb were nearly identical allowing In-111-MAb to be used for comparison with the I-125 frag. T concentration (conc.) of I-125-F(ab')2 were 25% greater and L conc. were 63% lower than for intact In-111-MAb. T conc. of I-125-Fab were 86% greater and L conc. 64% lower than for intact In-111-MAb. Large T, which produce higher serum CEA levels, increased the L conc. of intact MAb but not I-125 frag. The authors conclude that when renal excretion is prevented there is (a) an inverse relationship between size of a MAb moiety and its T conc. indicating that the improved T/NT ratios observed with frag. are not due only to renal excretion, and (b) the Fc portion of the MAb appears to be critical for L uptake of MAb or immune complexes.

  20. Pharmacokinetics of /sup 99m/Tc(Sn)- and /sup 131/I-labeled anti-carcinoembryonic antigen monoclonal antibody fragments in nude mice

    SciTech Connect

    Zimmer, A.M.; Kazikiewicz, J.M.; Rosen, S.T.; Spies, S.M.

    1987-03-15

    The biodistribution, radioimmunoimaging, and high pressure liquid chromatography activity profiles of /sup 99m/Tc(Sn) and /sup 131/I-labeled anti-carcinoembryonic antigen monoclonal antibody fragments were compared. Nude mice, bearing specific (colon carcinoma, LS174T) and nonspecific (pancreatic carcinoma, MIA) xenografts were given injections of the respective radiolabeled antibody fragments and also of irrelevant /sup 125/I-labeled antibody fragments (MOPC-21). The animals were imaged at 24 h after being given injections, they were sacrificed, and biodistribution studies were performed. Results of the study showed high kidney uptake (48.6% injected dose (ID)/g +/- 8.1% (SD)) and low tumor uptake (1.5% ID/g +/- 0.6%) for /sup 99m/Tc(Sn)-labeled fragments and higher uptake (4.4% ID/g +/- 0.6%) for /sup 131/I-labeled fragments, resulting in a higher localization index for the radioiodinated monoclonal antibody fragments. Imaging results showed good tumor visualization at 24 h after injection for the /sup 131/I-labeled fragments and poor tumor visualization with predominant kidney uptake for /sup 99m/Tc(Sn)-labeled fragments. After radiolabeling, high pressure liquid chromatography analysis indicated that 131I was primarily associated with F(ab')2 fragments, whereas 99mTc was mostly associated with Fab' fragments.

  1. Reconciling the structural attributes of avian antibodies.

    PubMed

    Conroy, Paul J; Law, Ruby H P; Gilgunn, Sarah; Hearty, Stephen; Caradoc-Davies, Tom T; Lloyd, Gordon; O'Kennedy, Richard J; Whisstock, James C

    2014-05-30

    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.

  2. Generation of a stable anti-human CD44v6 scFv and analysis of its cancer-targeting ability in vitro.

    PubMed

    Chen, Yinting; Huang, Kaihong; Li, Xuexian; Lin, Xiangan; Zhu, Zhaohua; Wu, Ying

    2010-06-01

    CD44v6 is a cancer-associated antigen that mainly expresses in a subset of adenocarcinomas. Therefore, in this study, anti-human CD44v6 single-chain variable fragment (scFv) has been selected and characterized because it is the first step of primary importance towards the construction of a novel cancer-targeted agent for cancer diagnosis and therapy. In our study, anti-human CD44v6 scFv was selected from a human phage-displayed scFv library based on its ability to bind in vitro to CD44v6 antigen. Subsequently, immunofluorescent staining and Western blot analyses were performed to measure the binding characteristics of this scFv. In addition, flow cytometric analysis was done to verify its cancer-targeting ability in vitro. And a flow cytometry-based assay was used to determine its equilibrium dissociation constant (K (D)). Finally, one functional anti-CD44v6 scFv was selected and characterized. Nucleotide sequencing verified that it was an incomplete scFv gene but had a variable heavy chain (V(H)) alone. However, anti-CD44v6 scFv demonstrated cell-binding and antigen-binding activities by immunofluorescent staining and Western blot analyses. Furthermore, flow cytometric analysis proved that this scFv specifically targeted CD44v6-expressing cancer cells other than CD44v6 non-expressing normal cells or tumor cells in vitro. The K (D) of this scFv was calculated to be 7.85 +/- 0.93 x 10(-8) M. In summary, the selected human scFv against CD44v6 has specific binding activity and favorable binding affinity despite lacking a variable light chain (V(L)). Moreover, it can effectively and specifically target CD44v6-expressing cancer cells. All these characteristics make anti-CD44v6 scFv a promising agent for cancer detection and anti-cancer therapy.

  3. Production and radioimmunoimaging of novel fully human phage display recombinant antibodies and growth inhibition of lung adenocarcinoma cell line overexpressing Prx I.

    PubMed

    Luo, Yi; Pang, Hua; Li, Shujie; Cao, Hui; Peng, Zhiping; Fan, Chunbo; Li, Shaolin

    2009-07-01

    The Peroxiredoxin I (Prx I) is a member of the Peroxiredoxin family, which is overexpressed in many diverse tumor types and is an anti-apoptosis protein for tumor cell proliferation and survival. Therapeutic strategies targeting the Prx I may therefore be effective broad-spectrum anticancer agents. We constructed a phage display single-chain variable fragment (scFv) antibody library and sieve out the fully human, lung adenocarcinoma-sepcific monoclonal antibodies. The selection on Prx I was performed using above-mentioned lung adenocarcinoma-sepcific monoclonal antibodies with high affinity to Prx I overexpressing lung adenocarcinoma cells. The candidate scFv sequences, based on enzyme-linked immunosorbent assay (ELISA) screening data, were chosen for soluble expression, and a 30 kDa band was observed on polyacrylamide gel electrophoresis as predicted. The purified antibodies were characterized by immunoblotting and showed high specificity to Prx I-overexpressing lung adenocarcinoma cells A549. Radioimmunoimaging was taken to evaluate specificity and distribution of antibodies in vivo. The radiolocalization index (RI) of tumor/serum and tumor/muscle gradually increased, reaching its peak (4.06 +/- 0.13 and 5.17 +/- 0.97, respectively) at 48 h postadministration. Single photon emission computed tomography (SPECT) imaging showed the radioactivity was aggregated in tumor locations and tumor imaging was clearly observed. The internalized scFv resulted in antibody-mediated cell apoptosis and downregulation of Prx I expression. These results demonstrate that the scFv possesses strong antitumor activity on lung adenocarcinoma and may therefore be an effective therapeutic candidate for the treatment of cancers that are dependent on Prx I for growth and survival.

  4. A Novel Human scFv Library with Non-Combinatorial Synthetic CDR Diversity.

    PubMed

    Bai, Xuelian; Kim, Jihye; Kang, Seungmin; Kim, Wankyu; Shim, Hyunbo

    2015-01-01

    The present work describes the construction and validation of a human scFv library with a novel design approach to synthetic complementarity determining region (CDR) diversification. The advantage of synthetic antibody libraries includes the possibility of exerting fine control over factors like framework sequences, amino acid and codon usage, and CDR diversity. However, random combinatorial synthesis of oligonucleotides for CDR sequence diversity also produces many clones with unnatural sequences and/or undesirable modification motifs. To alleviate these issues, we designed and constructed a novel semi-synthetic human scFv library with non-combinatorial, pre-designed CDR diversity and a single native human framework each for heavy, kappa, and lambda chain variable domains. Next-generation sequencing analysis indicated that the library consists of antibody clones with highly nature-like CDR sequences and the occurrence of the post-translational modification motifs is minimized. Multiple unique clones with nanomolar affinity could be isolated from the library against a number of target antigens, validating the library design strategy. The results demonstrate that it is possible to construct a functional antibody library using low, non-combinatorial synthetic CDR diversity, and provides a new strategy for the design of antibody libraries suitable for demanding applications.

  5. Monoclonal Antibody Fragments for Targeting Therapeutics to Growth Plate Cartilage | NCI Technology Transfer Center | TTC

    Cancer.gov

    The NICHD seeks statements of capability or interest from parties interested in collaborative research to co-develop, evaluate, or commercialize treatment of skeletal disorders using targeting antibodies.

  6. Neonatal Immunization with Respiratory Syncytial Virus Glycoprotein Fragment Induces Protective Immunity in the Presence of Maternal Antibodies in Mice

    PubMed Central

    Noh, Youran; Shim, Byoung-Shik; Cheon, In Su; Rho, Semi; Kim, Hee Joo; Choi, Youngjoo; Kang, Chang-Yuil; Chang, Jun

    2013-01-01

    Abstract Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants and the elderly worldwide. The significant morbidity and mortality associated with this infection underscores the urgent need for development of RSV vaccine. In this study, we first show that intranasal administration of RSV glycoprotein core fragment (Gcf) to neonatal mice can induce systemic humoral immune responses and protective immunity against RSV without causing lung eosinophilia, although antibody response was shifted to a Th2 response. Next, we examined whether the presence of maternal anti-RSV antibodies would affect the responsiveness and protection efficacy of Gcf in newborn mice, since infants can possess RSV-specific maternal antibodies due to frequent RSV re-infections to adults. Intranasal administration of Gcf induced antibody response and increased IFNγ secretion and protected mice against RSV challenge without severe lung eosinophilia, even in the presence of high levels of RSV-specific maternal antibodies. Thus, our findings suggest that Gcf may be an effective and safe RSV vaccine during the neonatal period. PMID:23869549

  7. Generation of recombinant monoclonal antibodies to study structure-function of envelope protein VP28 of white spot syndrome virus from shrimp

    SciTech Connect

    Wang Yuzhen; Zhang Xiaohua; Yuan Li; Xu Tao; Rao Yu; Li Jia; Dai Heping

    2008-08-08

    White spot syndrome virus (WSSV) is a major pathogen in shrimp aquaculture. VP28 is one of the most important envelope proteins of WSSV. In this study, a recombinant antibody library, as single-chain fragment variable (scFv) format, displayed on phage was constructed using mRNA from spleen cells of mice immunized with full-length VP28 expressed in Escherichia coli. After several rounds of panning, six scFv antibodies specifically binding to the epitopes in the N-terminal, middle, and C-terminal regions of VP28, respectively, were isolated from the library. Using these scFv antibodies as tools, the epitopes in VP28 were located on the envelope of the virion by immuno-electron microscopy. Neutralization assay with these antibodies in vitro suggested that these epitopes may not be the attachment site of WSSV to host cell receptor. This study provides a new way to investigate the structure and function of the envelope proteins of WSSV.

  8. Affinity improvement by fine tuning of single-chain variable fragment against aflatoxin B1.

    PubMed

    Min, Won-Ki; Na, Kang-In; Yoon, Jung-Hyun; Heo, Yoon-Jee; Lee, Daesang; Kim, Sung-Gun; Seo, Jin-Ho

    2016-10-15

    Aflatoxin B1 (AFB1) produced in Aspergillus flavus is a major hepatocarcinogen found in foods and feed. For effective immunological detection of AFB1 at low concentrations, the development of high affinity antibody for AFB1 is required. Previously, an affinity-maturated single-chain variable fragment containing 6 mutations (scFv-M37) was isolated from an artificial mutagenic library, which showed a 9-fold higher affinity than its wild type scFv. In this study, the effect of the 6 mutated residues on the affinity improvement was characterized using surface plasmon resonance analysis, which identified a deleterious mutation (VH-A110T) located on a framework region of the scFv-M37. The back mutation of VH-A110T resulted in a 3.2-fold affinity improvement, which was attributed to decrease of dissociation rate constant (kd) in interaction between AFB1 and the back mutant scFv. The biophysical analyses using circular dichroism and gel filtration revealed that the back mutation of VH-A110T caused a subtle conformational change of the scFv toward tighter binding to AFB1.

  9. Gluten-specific antibodies of celiac disease gut plasma cells recognize long proteolytic fragments that typically harbor T-cell epitopes

    PubMed Central

    Dørum, Siri; Steinsbø, Øyvind; Bergseng, Elin; Arntzen, Magnus Ø.; de Souza, Gustavo A.; Sollid, Ludvig M.

    2016-01-01

    This study aimed to identify proteolytic fragments of gluten proteins recognized by recombinant IgG1 monoclonal antibodies generated from single IgA plasma cells of celiac disease lesions. Peptides bound by monoclonal antibodies in complex gut-enzyme digests of gluten treated with the deamidating enzyme transglutaminase 2, were identified by mass spectrometry after antibody pull-down with protein G beads. The antibody bound peptides were long deamidated peptide fragments that contained the substrate recognition sequence of transglutaminase 2. Characteristically, the fragments contained epitopes with the sequence QPEQPFP and variants thereof in multiple copies, and they typically also harbored many different gluten T-cell epitopes. In the pull-down setting where antibodies were immobilized on a solid phase, peptide fragments with multivalent display of epitopes were targeted. This scenario resembles the situation of the B-cell receptor on the surface of B cells. Conceivably, B cells of celiac disease patients select gluten epitopes that are repeated multiple times in long peptide fragments generated by gut digestive enzymes. As the fragments also contain many different T-cell epitopes, this will lead to generation of strong antibody responses by effective presentation of several distinct T-cell epitopes and establishment of T-cell help to B cells. PMID:27146306

  10. Proteoliposome-based Selection of a Recombinant Antibody Fragment Against the Human M2 Muscarinic Acetylcholine Receptor

    PubMed Central

    Suharni; Nomura, Yayoi; Arakawa, Takatoshi; Hino, Tomoya; Abe, Hitomi; Nakada-Nakura, Yoshiko; Sato, Yumi; Iwanari, Hiroko; Shiroishi, Mitsunori; Asada, Hidetsugu; Shimamura, Tatsuro; Murata, Takeshi; Kobayashi, Takuya; Hamakubo, Takao; Iwata, So

    2014-01-01

    The development of antibodies against human G-protein-coupled receptors (GPCRs) has achieved limited success, which has mainly been attributed to their low stability in a detergent-solubilized state. We herein describe a method that can generally be applied to the selection of phage display libraries with human GPCRs reconstituted in liposomes. A key feature of this approach is the production of biotinylated proteoliposomes that can be immobilized on the surface of streptavidin-coupled microplates or paramagnetic beads and used as a binding target for antibodies. As an example, we isolated a single chain Fv fragment from an immune phage library that specifically binds to the human M2 muscarinic acetylcholine receptor with nanomolar affinity. The selected antibody fragment recognized the GPCR in both detergent-solubilized and membrane-embedded forms, which suggests that it may be a potentially valuable tool for structural and functional studies of the GPCR. The use of proteoliposomes as immunogens and screening bait will facilitate the application of phage display to this difficult class of membrane proteins. PMID:25545206

  11. Proteoliposome-based selection of a recombinant antibody fragment against the human M2 muscarinic acetylcholine receptor.

    PubMed

    Suharni; Nomura, Yayoi; Arakawa, Takatoshi; Hino, Tomoya; Abe, Hitomi; Nakada-Nakura, Yoshiko; Sato, Yumi; Iwanari, Hiroko; Shiroishi, Mitsunori; Asada, Hidetsugu; Shimamura, Tatsuro; Murata, Takeshi; Kobayashi, Takuya; Hamakubo, Takao; Iwata, So; Nomura, Norimichi

    2014-12-01

    The development of antibodies against human G-protein-coupled receptors (GPCRs) has achieved limited success, which has mainly been attributed to their low stability in a detergent-solubilized state. We herein describe a method that can generally be applied to the selection of phage display libraries with human GPCRs reconstituted in liposomes. A key feature of this approach is the production of biotinylated proteoliposomes that can be immobilized on the surface of streptavidin-coupled microplates or paramagnetic beads and used as a binding target for antibodies. As an example, we isolated a single chain Fv fragment from an immune phage library that specifically binds to the human M2 muscarinic acetylcholine receptor with nanomolar affinity. The selected antibody fragment recognized the GPCR in both detergent-solubilized and membrane-embedded forms, which suggests that it may be a potentially valuable tool for structural and functional studies of the GPCR. The use of proteoliposomes as immunogens and screening bait will facilitate the application of phage display to this difficult class of membrane proteins.

  12. In Vitro Neutralisation of Rotavirus Infection by Two Broadly Specific Recombinant Monovalent Llama-Derived Antibody Fragments

    PubMed Central

    Aladin, Farah; Einerhand, Alexandra W. C.; Bouma, Janneke; Bezemer, Sandra; Hermans, Pim; Wolvers, Danielle; Bellamy, Kate; Frenken, Leon G. J.; Gray, Jim; Iturriza-Gómara, Miren

    2012-01-01

    Rotavirus is the main cause of viral gastroenteritis in young children. Therefore, the development of inexpensive antiviral products for the prevention and/or treatment of rotavirus disease remains a priority. Previously we have shown that a recombinant monovalent antibody fragment (referred to as Anti-Rotavirus Proteins or ARP1) derived from a heavy chain antibody of a llama immunised with rotavirus was able to neutralise rotavirus infection in a mouse model system. In the present work we investigated the specificity and neutralising activity of two llama antibody fragments, ARP1 and ARP3, against 13 cell culture adapted rotavirus strains of diverse genotypes. In addition, immunocapture electron microscopy (IEM) was performed to determine binding of ARP1 to clinical isolates and cell culture adapted strains. ARP1 and ARP3 were able to neutralise a broad variety of rotavirus serotypes/genotypes in vitro, and in addition, IEM showed specific binding to a variety of cell adapted strains as well as strains from clinical specimens. These results indicated that these molecules could potentially be used as immunoprophylactic and/or immunotherapeutic products for the prevention and/or treatment of infection of a broad range of clinically relevant rotavirus strains. PMID:22403728

  13. Site-specific photobiotinylation of antibodies, light chains, and immunoglobulin fragments.

    PubMed

    Pavlinkova, G; Lou, D; Kohler, H

    2000-09-01

    The high affinity of biotin for avidin has been exploited for many antibody-based assays. This requires that biotin is covalently conjugated to the antibody molecule. Several chemically reactive biotinylation reagents are commercially available. Except for the attachment via sulfhydryl groups in the immunoglobulin (Ig) molecule, these reagents attach biotin randomly to various amino acid side chains. Although non-site-specific modification of antibodies does not interfere in most immunoassays, specific application and sensitive antibodies would benefit from site-specific biotinylation. Here we describe an affinity biotinylation technique based on a photoreactive biotin reagent. The design of this reaction was possible from the discovery of a conserved binding site in the variable Ig domain for nucleotides and nucleosides. The described photoaffinity biotinylation offers the advantages of ease, convenience, and production of a reproducible and defined biotinylated antibody preparation.

  14. Nephritogenic lupus antibodies recognize glomerular basement membrane-associated chromatin fragments released from apoptotic intraglomerular cells.

    PubMed

    Kalaaji, Manar; Mortensen, Elin; Jørgensen, Leif; Olsen, Randi; Rekvig, Ole Petter

    2006-06-01

    Antibodies to dsDNA represent a classification criterion for systemic lupus erythematosus. Subpopulations of these antibodies are involved in lupus nephritis. No known marker separates nephritogenic from non-nephritogenic anti-dsDNA antibodies. It is not clear whether specificity for glomerular target antigens or intrinsic antibody-affinity for dsDNA or nucleosomes is a critical parameter. Furthermore, it is still controversial whether glomerular target antigen(s) is constituted by nucleosomes or by non-nucleosomal glomerular structures. Previously, we have demonstrated that antibodies eluted from murine nephritic kidneys recognize nucleosomes, but not other glomerular antigens. In this study, we determined the structures that bind nephritogenic autoantibodies in vivo by transmission electron microscopy, immune electron microscopy, and colocalization immune electron microscopy using experimental antibodies to dsDNA, to histones and transcription factors, or to laminin. The data obtained are consistent and point at glomerular basement membrane-associated nucleosomes as target structures for the nephritogenic autoantibodies. Terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling or caspase-3 assays demonstrate that lupus nephritis is linked to intraglomerular cell apoptosis. The data suggest that nucleosomes are released by apoptosis and associate with glomerulus basement membranes, which may then be targeted by pathogenic anti-nucleosome antibodies. Thus, apoptotic nucleosomes may represent both inducer and target structures for nephritogenic autoantibodies in systemic lupus erythematosus.

  15. A monoclonal antibody to a synthetic fragment of rabies virus glycoprotein binds ligands of the nicotinic cholinergic receptor.

    PubMed

    Rustici, M; Santucci, A; Lozzi, L; Petreni, S; Spreafico, A; Neri, P; Bracci, L; Soldani, P

    1989-09-01

    Rabies virus glycoprotein and snake venom curaremimetic neurotoxins share a region of high homology (30-45 for neurotoxins and 190-203 for the glycoprotein) in the regions that are believed to be responsible for binding the nicotinic acetylcholine receptor. Monoclonal antibodies raised to the 190-203 synthetic fragment of rabies virus glycoprotein were immobilized on a high performance affinity chromatography column and were able to bind neurotoxins. Toxins were displaced from the affinity column by elution at acidic pH and by affinity competition with acetylcholine at neutral pH. Furthermore, the affinity column proved to be useful for the purification of cholinergic ligands. Overall, these results indicate that the paratope of our monoclonal antibodies could behave as an 'internal image' of the nicotinic cholinergic receptor acetylcholine binding site.

  16. Selection of Human Antibody Fragments which Bind Novel Breast Tumor Antigens

    DTIC Science & Technology

    1996-09-01

    diagnosis of node-negative breast cancer patients, for immunotherapy prior to growth of large tumor mass , and as adjuvant therapy for minimal residual...biosensor based on surface plasmon resonance (35). For this technique, antigen is coupled to a derivatized sensor chip capable of detecting changes in mass ...When antibody is passed over the sensor chip, antibody binds to the antigen resulting in an increase in mass which can be quantitated. Measurement of

  17. Heterodimeric bispecific antibody-derivatives against CD19 and CD16 induce effective antibody-dependent cellular cytotoxicity against B-lymphoid tumor cells.

    PubMed

    Kellner, Christian; Bruenke, Joerg; Horner, Heike; Schubert, Joerg; Schwenkert, Michael; Mentz, Kristin; Barbin, Karin; Stein, Christoph; Peipp, Matthias; Stockmeyer, Bernhard; Fey, Georg H

    2011-04-28

    Bispecific scFv antibody-derivatives (bsscFvs) recruiting natural killer (NK) cells for the lysis of malignant cells have therapeutic potential. However, a bsscFv specific for the B-lymphoid tumor antigen CD19 and the trigger molecule CD16 on NK cells had similar affinities for both antigens (42 and 58nM, respectively) and was not optimal for cytotoxicity. Therefore, a bispecific tribody (bsTb) was constructed with two binding sites for CD19 and one for CD16. This bsTb contained a CD19-specific Fab fragment carrying a CD16-specific scFv fused to its light chain and a CD19-specific scFv fused to its heavy chain. The bsTb was compared with a bispecific bibody (bsBb) lacking the CD19-specific scFv. The bsTb had 3-fold greater avidity for CD19 than the bsBb (8 and 24nM, respectively), while both had equal affinity for CD16 (56nM). Both molecules mediated antibody-dependent cellular cytotoxicity (ADCC) of leukemia-derived SEM cells and primary cells from leukemia patients. The bsTb showed half-maximum effective concentrations (EC(50)) of 55pM and promoted equal lysis as the bsBb and the bsscFv at 6- and 12-fold lower concentrations, respectively. Among these three molecules the bsTb showed the most promising in vitro properties which are anticipated to be displayed also in vivo.

  18. The Structure of Natural and Recombinant Antibodies.

    PubMed

    Ma, Hui; O'Kennedy, Richard

    2015-01-01

    Immunoglobulins (Ig) isotypes A, D, E, G, and M are glycoproteins which are mainly composed of a "Y"-shaped Ig monomer (~150 kDa), consisting of two light and two heavy chains. Both light and heavy chains contain variable (N-terminal) and constant regions (C-terminal). Each light chain consists of one variable domain and one constant domain, whereas each heavy chain has one variable domain and three constant domains. However, heavy-chain antibodies consisting of only heavy chains and lacking the light chains are found in camelids and cartilaginous fishes. Unlike other immunoglobulins, the heavy chain of avian antibody IgY (~180 kDa) consists of four constant domains. The single-chain variable fragment (scFv; ~25 kDa) of an antibody contains variable regions of antibody heavy and light chains. The fragment antigen-binding (Fab; ~50 kDa) region has the full antibody light chain but the heavy chain is composed of a variable region and one constant domain.

  19. Next-generation sequencing enables the discovery of more diverse positive clones from a phage-displayed antibody library

    PubMed Central

    Yang, Wonjun; Yoon, Aerin; Lee, Sanghoon; Kim, Soohyun; Han, Jungwon; Chung, Junho

    2017-01-01

    Phage display technology provides a powerful tool to screen a library for a binding molecule via an enrichment process. It has been adopted as a critical technology in the development of therapeutic antibodies. However, a major drawback of phage display technology is that because the degree of the enrichment cannot be controlled during the bio-panning process, it frequently results in a limited number of clones. In this study, we applied next-generation sequencing (NGS) to screen clones from a library and determine whether a greater number of clones can be identified using NGS than using conventional methods. Three chicken immune single-chain variable fragment (scFv) libraries were subjected to bio-panning on prostate-specific antigen (PSA). Phagemid DNA prepared from the original libraries as well as from the Escherichia coli pool after each round of bio-panning was analyzed using NGS, and the heavy chain complementarity-determining region 3 (HCDR3) sequences of the scFv clones were determined. Subsequently, through two-step linker PCR and cloning, the entire scFv gene was retrieved and analyzed for its reactivity to PSA in a phage enzyme immunoassay. After four rounds of bio-panning, the conventional colony screening method was performed for comparison. The scFv clones retrieved from NGS analysis included all clones identified by the conventional colony screening method as well as many additional clones. The enrichment of the HCDR3 sequence throughout the bio-panning process was a positive predictive factor for the selection of PSA-reactive scFv clones. PMID:28336957

  20. Influence of growth temperature on the production of antibody Fab fragments in different microbes: a host comparative analysis.

    PubMed

    Dragosits, Martin; Frascotti, Gianni; Bernard-Granger, Lise; Vázquez, Felícitas; Giuliani, Maria; Baumann, Kristin; Rodríguez-Carmona, Escarlata; Tokkanen, Jaana; Parrilli, Ermenegilda; Wiebe, Marilyn G; Kunert, Renate; Maurer, Michael; Gasser, Brigitte; Sauer, Michael; Branduardi, Paola; Pakula, Tiina; Saloheimo, Markku; Penttilä, Merja; Ferrer, Pau; Luisa Tutino, Maria; Villaverde, Antonio; Porro, Danilo; Mattanovich, Diethard

    2011-01-01

    Microorganisms encounter diverse stress conditions in their native habitats but also during fermentation processes, which have an impact on industrial process performance. These environmental stresses and the physiological reactions they trigger, including changes in the protein folding/secretion machinery, are highly interrelated. Thus, the investigation of environmental factors, which influence protein expression and secretion is still of great importance. Among all the possible stresses, temperature appears particularly important for bioreactor cultivation of recombinant hosts, as reductions of growth temperature have been reported to increase recombinant protein production in various host organisms. Therefore, the impact of temperature on the secretion of proteins with therapeutic interest, exemplified by a model antibody Fab fragment, was analyzed in five different microbial protein production hosts growing under steady-state conditions in carbon-limited chemostat cultivations. Secretory expression of the heterodimeric antibody Fab fragment was successful in all five microbial host systems, namely Saccharomyces cerevisiae, Pichia pastoris, Trichoderma reesei, Escherichia coli and Pseudoalteromonas haloplanktis. In this comparative analysis we show that a reduction of cultivation temperature during growth at constant growth rate had a positive effect on Fab 3H6 production in three of four analyzed microorganisms, indicating common physiological responses, which favor recombinant protein production in prokaryotic as well as eukaryotic microbes.

  1. Radioiodinated iodobenzoyl conjugates of a monoclonal antibody Fab fragment. In vivo comparisons with chloramine-T-labeled Fab

    SciTech Connect

    Wilbur, D.S.; Hadley, S.W.; Grant, L.M.; Hylarides, M.D. )

    1991-03-01

    A comparative investigation of the biodistributions of radioiodinated p- and m-iodobenzoyl conjugates of a monoclonal antibody Fab fragment, NR-LU-10 Fab, and the same antibody Fab fragment radioiodinated by the chloramine-T (ChT) method has been carried out in mice. Coinjected, dual-isotope studies in athymic mice with tumor xenografts have demonstrated that there are only minor differences in the in vivo distributions of the iodobenzoyl-labeled Fabs, except in the excretory organs, kidneys, and intestines, where major differences were observed. Similarly, coinjection of either the p-iodobenzoyl or m-iodobenzoyl conjugate of NR-LU-10 Fab with the Fab radioiodinated with ChT/radioiodide into BALB/c mice provided additional data that indicated that the two iodobenzoyl conjugates distributed similar in a number of selected tissues. The tissue-distribution differences of the regioisomeric iodobenzoyl conjugates in relation to the ChT-radioiodinated Fab were large for the stomach and neck, consistent with previous studies. The most notable difference between the two iodobenzoyl conjugates was the kidney activity, where the m-iodobenzoyl conjugate was similar to the directly labeled Fab, but the p-iodobenzoyl-conjugated Fab was higher by nearly a factor of 2.

  2. Potent neutralization of VEGF biological activities with a fully human antibody Fab fragment directed against VEGF receptor 2

    SciTech Connect

    Miao, H.-Q. . E-mail: hua-quan.miao@imclone.com; Hu, Kun; Jimenez, Xenia; Navarro, Elizabeth; Zhang, Haifan; Lu Dan; Ludwig, Dale L.; Balderes, Paul; Zhu Zhenping . E-mail: zhenping.zhu@imclone.com

    2006-06-23

    Compelling evidence suggest that vascular endothelial growth factor (VEGF) and its receptors, especially receptor 2 (VEGFR2, or kinase insert domain-containing receptor, KDR), play a critical role in angiogenesis under both physiological and pathological conditions, including cancer and angiogenic retinopathies such as age-related macular degeneration (AMD). To this end, inhibition of angiogenesis with antagonists to either VEGF or KDR has yielded significant therapeutic efficacy both in preclinical studies in animal models and in clinical trials in patients with cancer and AMD. We previously reported the identification of a high affinity, fully human anti-KDR antibody fragment, 1121B Fab, through a highly stringent affinity maturation process with a Fab originally isolated from a naive human antibody phage display library. In this study, we demonstrate that 1121B Fab is able to strongly block KDR/VEGF interaction, resulting in potent inhibition of an array of biological activities of VEGF, including activation of the receptor and its signaling pathway, intracellular calcium mobilization, and migration and proliferation of endothelial cells. Taken together, our data lend strong support to the further development of 1121B Fab fragment as an anti-angiogenesis agent in both cancer and angiogenic retinopathies.

  3. Studies of nontarget-mediated distribution of human full-length IgG1 antibody and its FAb fragment in cardiovascular and metabolic-related tissues.

    PubMed

    Davidsson, Pia; Söderling, Ann-Sofi; Svensson, Lena; Ahnmark, Andrea; Flodin, Christine; Wanag, Ewa; Screpanti-Sundqvist, Valentina; Gennemark, Peter

    2015-05-01

    Tissue distribution and pharmacokinetics (PK) of full-length nontargeted antibody and its antigen-binding fragment (FAb) were evaluated for a range of tissues primarily of interest for cardiovascular and metabolic diseases. Mice were intravenously injected with a dose of 10 mg/kg of either human IgG1or its FAb fragment; perfused tissues were collected at a range of time points over 3 weeks for the human IgG1 antibody and 1 week for the human FAb antibody. Tissues were homogenized and antibody concentrations were measured by specific immunoassays on the Gyros system. Exposure in terms of maximum concentration (Cmax ) and area under the curve was assessed for all nine tissues. Tissue exposure of full-length antibody relative to plasma exposure was found to be between 1% and 10%, except for brain (0.2%). Relative concentrations of FAb antibody were the same, except for kidney tissue, where the antibody concentration was found to be ten times higher than in plasma. However, the absolute tissue uptake of full-length IgG was significantly higher than the absolute tissue uptake of the FAb antibody. This study provides a reference PK state for full-length whole and FAb antibodies in tissues related to cardiovascular and metabolic diseases that do not include antigen or antibody binding.

  4. Dynamic force spectroscopy of parallel individual mucin1-antibody bonds

    SciTech Connect

    Sulchek, T A; Friddle, R W; Langry, K; Lau, E; Albrecht, H; Ratto, T; DeNardo, S; Colvin, M E; Noy, A

    2005-05-02

    We used atomic force microscopy (AFM) to measure the binding forces between Mucin1 (MUC1) peptide and a single chain antibody fragment (scFv) selected from a scFv library screened against MUC1. This binding interaction is central to the design of the molecules for targeted delivery of radioimmunotherapeutic agents for prostate and breast cancer treatment. Our experiments separated the specific binding interaction from non-specific interactions by tethering the antibody and MUC1 molecules to the AFM tip and sample surface with flexible polymer spacers. Rupture force magnitude and elastic characteristics of the spacers allowed identification of the bond rupture events corresponding to different number of interacting proteins. We used dynamic force spectroscopy to estimate the intermolecular potential widths and equivalent thermodynamic off rates for mono-, bi-, and tri-valent interactions. Measured interaction potential parameters agree with the results of molecular docking simulation. Our results demonstrate that an increase of the interaction valency leads to a precipitous decline in the dissociation rate. Binding forces measured for mono and multivalent interactions match the predictions of a Markovian model for the strength of multiple uncorrelated bonds in parallel configuration. Our approach is promising for comparison of the specific effects of molecular modifications as well as for determination of the best configuration of antibody-based multivalent targeting agents.

  5. High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells

    PubMed Central

    2013-01-01

    Background The demand of monospecific high affinity binding reagents, particularly monoclonal antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and purification of recombinant antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable as immunoglobulins (Ig)G in many immunoassays. In contrast, the bivalent scFv-Fc antibody format shares many properties with IgG and has a very high application compatibility. Results In this study transient expression of scFv-Fc antibodies in human embryonic kidney (HEK) 293 cells was optimized. Production levels of 10-20 mg/L scFv-Fc antibody were achieved in adherent HEK293T cells. Employment of HEK293-6E suspension cells expressing a truncated variant of the Epstein Barr virus (EBV) nuclear antigen (EBNA) 1 in combination with production under serum free conditions increased the volumetric yield up to 10-fold to more than 140 mg/L scFv-Fc antibody. After vector optimization and process optimization the yield of an scFv-Fc antibody and a cytotoxic antibody-RNase fusion protein further increased 3-4-fold to more than 450 mg/L. Finally, an entirely new mammalian expression vector was constructed for single step in frame cloning of scFv genes from antibody phage display libraries. Transient expression of more than 20 different scFv-Fc antibodies resulted in volumetric yields of up to 600 mg/L and 400 mg/L in average. Conclusion Transient production of recombinant scFv-Fc antibodies in HEK293-6E in combination with optimized vectors and fed batch shake flasks cultivation is efficient and robust, and integrates well into a high-throughput recombinant antibody generation pipeline. PMID:23802841

  6. Synthesis and application of a N-1' fluorescent biotinyl derivative inducing the specific carboxy-terminal dual labeling of a novel RhoB-selective scFv.

    PubMed

    Chaisemartin, L; Chinestra, P; Favre, G; Blonski, C; Faye, J C

    2009-05-20

    The fluorescent site-specific labeling of protein would provide a new, easy-to-use alternative to biochemical and immunochemical methods. We used an intein-mediated strategy for covalent labeling of the carboxy-terminal amino acid of a RhoB-selective scFv previously isolated from a phage display library (a human synthetic V(H) + V(L) scFv phage library). The scFv fused to the Mxe intein was produced in E. coli and purified and was then labeled with a newly synthesized fluorescent biotinyl cysteine derivative capable of inducing scFv-Mxe intein splicing. In this study, we investigated the splicing and labeling properties of various amino acids in the hinge domain between scFv and Mxe under thiol activation. In this dual labeling system, the fluorescein is used for antibody detection and biotin is used for purification, resulting in a high specific activity for fluorescence. We then checked that the purified biotinylated fluorescent scFv retained its selectivity for RhoB without modification of its affinity.

  7. Selection of recombinant IgE antibodies binding the beta-lactoglobulin allergen in a conformation-dependent manner.

    PubMed

    Jylhä, Sirpa; Mäkinen-Kiljunen, Soili; Haahtela, Tari; Söderlund, Hans; Takkinen, Kristiina; Laukkanen, Marja-Leena

    2009-10-31

    Cow's milk allergy (CMA) is a common food allergy, especially among infants and young children. Approximately 85% of milk-allergic children outgrow their allergy by the age of three but the remaining 15% remain allergic. Bovine beta-lactoglobulin (BLG) is one of the major allergens in cow's milk. There is a definite need for the specific and sensitive detection of allergenic substances. Validated methods are obligatory to demonstrate allergen contamination and even fatal hidden allergens and, thus, to prevent life-threatening conditions of allergic persons. In this study, we constructed human IgE scFv libraries from an adult milk-allergic patient and isolated the first recombinant IgE antibodies specific to a food allergen, BLG. The selection of the IgE antibody libraries with two distinct panning procedures resulted in the enrichment of four clones having different BLG-binding profiles; two of the clones recognize the native BLG whereas the other two recognize only the heat-denatured form of BLG. For further characterization, the scFv fragments were converted to Fab fragments with human IgG1 isotype. The D1 Fab fragment, binding native BLG with nanomolar affinity, also partially inhibited serum IgE binding to BLG. These BLG-specific IgE antibodies can be applied for the detection of both native and denatured BLG in cow's milk products and furthermore, for the optimization of manufacturing processes to develop safe hypoallergenic milk products.

  8. Feasibility study of the Fab fragment of a monoclonal antibody against tissue factor as a diagnostic tool.

    PubMed

    Tsumura, Ryo; Sato, Ryuta; Furuya, Fumiaki; Koga, Yoshikatsu; Yamamoto, Yoshiyuki; Fujiwara, Yuki; Yasunaga, Masahiro; Matsumura, Yasuhiro

    2015-12-01

    Tissue factor (TF) is expressed strongly in various types of cancer, especially cancers that are often refractory to treatment, such as pancreatic cancer. In this study, we compared the differences in the biophysical and pharmacological properties of whole IgG and the Fab fragment of anti-human TF monoclonal antibody (1849 antibodies), in order to determine their suitability for application in the diagnosis and treatment of cancers. In the biophysical examination, we investigated the characteristics of 1849-whole IgG and 1849-Fab by SPR sensing and confocal fluorescence microscopy analysis using recombinant human TF antigen and TF-overexpressing human pancreatic cancer cell line, BxPC3, respectively. After conjugation with Alexa-Flour-647, in vivo imaging was conducted in mice bearing BxPC3 xenograft tumors. Furthermore, the distribution of the conjugates in tumors and major organs was evaluated by ex vivo study. The in vitro experiments showed that 1849 antibodies had high affinity against TF antigen. In addition, 1849-Fab showed a faster dissociation rate from the antigen than 1849-whole IgG. In mice, 1849-Fab-Alexa-Flour-647 showed rapid renal clearance and faster tumor accumulation, achieving a high contrast signal over nearby normal tissues in the early phase and enhanced tumor penetration after administration. On the other hand, 1849-whole IgG-Alexa-Flour-647 showed slow clearance from the blood and sustained high tumor accumulation. These results suggest that 1849-Fab may be a useful tool for pancreatic cancer diagnosis.

  9. Crystal Structure of the Fab Fragment of an Anti-ofloxacin Antibody and Exploration of Its Specific Binding.

    PubMed

    He, Kuo; Du, Xinjun; Sheng, Wei; Zhou, Xiaonan; Wang, Junping; Wang, Shuo

    2016-03-30

    The limited knowledge on the mechanism of interactions between small contaminants and the corresponding antibodies greatly inhibits the development of enzyme-linked immunosorbent assay methods. In this study, the crystal structure of a Fab fragment specific for ofloxacin was obtained. On the basis of the crystal characteristics, the modeling of the interactions between ofloxacin and the Fab revealed that TYR31 and HIS99 of the heavy chain and MET20 and GLN79 of the light chain formed a hydrophobic region and that SER52 and ALA97 of the heavy chain and TYR35 of the light chain formed a salt bridge and two hydrogen bonds for specific binding. The key roles of SER52 and ALA97 were further confirmed by site-directed mutation. A specificity analysis using 14 ofloxacin analogues indicates that the length of the bond formed between the piperazine ring and the antibody plays key roles in specific recognition. This work helps to clarify the mechanisms through which antibodies recognize small molecules and improve immune detection methods.

  10. Kinetic Characterisation of a Single Chain Antibody against the Hormone Abscisic Acid: Comparison with Its Parental Monoclonal

    PubMed Central

    Badescu, George O.; Marsh, Andrew; Smith, Timothy R.; Thompson, Andrew J.; Napier, Richard M.

    2016-01-01

    A single-chain Fv fragment antibody (scFv) specific for the plant hormone abscisic acid (ABA) has been expressed in the bacterium Escherichia coli as a fusion protein. The kinetics of ABA binding have been measured using surface plasmon resonance spectrometry (BIAcore 2000) using surface and solution assays. Care was taken to calculate the concentration of active protein in each sample using initial rate measurements under conditions of partial mass transport limitation. The fusion product, parental monoclonal antibody and the free scFv all have low nanomolar affinity constants, but there is a lower dissociation rate constant for the parental monoclonal resulting in a three-fold greater affinity. Analogue specificity was tested and structure-activity binding preferences measured. The biologically-active (+)-ABA enantiomer is recognised with an affinity three orders of magnitude higher than the inactive (-)-ABA. Metabolites of ABA including phaseic acid, dihydrophaseic acid and deoxy-ABA have affinities over 100-fold lower than that for (+)-ABA. These properties of the scFv make it suitable as a sensor domain in bioreporters specific for the naturally occurring form of ABA. PMID:27023768

  11. Detection of antibodies against synthetic peptides mimicking ureases fragments in sera of rheumatoid arthritis patients.

    PubMed

    Konieczna, Iwona; Kwinkowski, Marek; Kolesińska, Beata; Kamiński, Zbigniew; Zarnowiec, Paulina; Kaca, Wiesław

    2012-11-01

    Rheumatoid arthritis (RA) is a chronic disease with an autoimmunological background. RA is mostly characterized by systemic inflammation and injuries of synovial joints. There is a hypothesis that bacterial infections may be connected with development of the disease. It has been suggested that molecular mimicry between bacterial and human antigens may be one of possible mechanisms of RA development. One of potential antigens involved in this mechanism is urease - enzyme with high structural conservatism, occurring in pathogenic and commensal bacteria. We found that the level of antibodies against peptide mimicking urease "flap" region is significantly higher in sera from patients with rheumatoid arthritis in comparison with volunteer blood donor sera. We also observed that antibodies present in RA sera may bind not only specific peptide antigens but also peptides with a slightly different structure.

  12. Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type

    NASA Technical Reports Server (NTRS)

    He, X. M.; Ruker, F.; Casale, E.; Carter, D. C.

    1992-01-01

    The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 degrees. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

  13. Single-Chain Fragment Variable Passive Immunotherapies for Neurodegenerative Diseases

    PubMed Central

    Huang, Liang; Su, Xiaomin; Federoff, Howard J.

    2013-01-01

    Accumulation of misfolded proteins has been implicated in a variety of neurodegenerative diseases including prion diseases, Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD). In the past decade, single-chain fragment variable (scFv) -based immunotherapies have been developed to target abnormal proteins or various forms of protein aggregates including Aβ, SNCA, Htt, and PrP proteins. The scFvs are produced by fusing the variable regions of the antibody heavy and light chains, creating a much smaller protein with unaltered specificity. Because of its small size and relative ease of production, scFvs are promising diagnostic and therapeutic reagents for protein misfolded diseases. Studies have demonstrated the efficacy and safety of scFvs in preventing amyloid protein aggregation in preclinical models. Herein, we discuss recent developments of these immunotherapeutics. We review efforts of our group and others using scFv in neurodegenerative disease models. We illustrate the advantages of scFvs, including engineering to enhance misfolded conformer specificity and subcellular targeting to optimize therapeutic action. PMID:24048248

  14. New directions for half-life extension of protein therapeutics: the rise of antibody Fc domains and fragments.

    PubMed

    Wang, Lili; Ying, Tianlei

    2016-08-23

    Protein-based therapeutics has become one of the most rapidly growing and successful drug class in the clinic. However, there are still a number of key challenges that need to be addressed before the full therapeutic potential of protein drugs can be realized. Of note, many biologically active proteins have very short in vivo half-lives, a fact that has greatly hindered their clinical applications. Consequently, several different strategies including polyethylene glycol modification and fusion with Fc or albumin have been developed and implemented to prolong the serum half-life of protein therapeutics. Here we will focus on the recent advances in the development of Fc-based antibody fragments and domains and their potential use as novel half-life-extending fusion partners for protein therapeutics.

  15. Novel impedimetric immunosensor for the detection and quantitation of Adenovirus using reduced antibody fragments immobilized onto a conducting copolymer surface.

    PubMed

    Caygill, Rebecca L; Hodges, Christopher S; Holmes, Joanne L; Higson, Séamus P J; Blair, G Eric; Millner, Paul A

    2012-02-15

    The number of Adenovirus (Ad) infections detected in immunocompromised people has increased due to the number of patients receiving transplants, as well as the HIV pandemic. Ads cause life-threatening diseases specific to the infected organs of immunocompromised hosts, with discontinuation of immunosuppressive agents necessary to prevent morbidity. The methodology in this paper has been employed to develop a novel impedimetric based assay platform to detect and quantify human Ads, which is comparable in performance to current methods, such as ELISA and PCR, but is also less expensive and faster. Novel immunosensors have been fabricated using polyclonal antibodies raised against a human Ad (Ad5) capsid protein, which were selectively cleaved into antibody fragments by 2-mercaptoethylamine. The fragments were immobilized onto a functionalized conducting copolymer matrix comprising polyaniline and 2-aminobenzylamine. Fully fabricated sensors were incubated with two immunologically distinct serotypes of Ad, Ad5 and Ad3, with between 10 and 10(12)virus particles/mL prior to sensor interrogation. Electrochemical impedance spectroscopy was used to measure the charge transfer resistance of the sensors over a range of frequencies from 25 kHz to 0.1 Hz. Our data demonstrate that the immunosensors specifically detect, and differentiate between, closely related human Ad serotypes with a limit of detection of 10(3)virus particles/mL. In addition, atomic force microscopy was applied to study the sensor surface nanostructure. Future work looks to test virus containing clinical samples but this could be a viable and valuable alternative for point-of-care virus detection and quantification.

  16. Highly efficient production of VHH antibody fragments in Brevibacillus choshinensis expression system.

    PubMed

    Mizukami, Makoto; Tokunaga, Hiroko; Onishi, Hiromasa; Ueno, Yohei; Hanagata, Hiroshi; Miyazaki, Nobuo; Kiyose, Norihiko; Ito, Yuji; Ishibashi, Matsujiro; Hagihara, Yoshihisa; Arakawa, Tsutomu; Miyauchi, Akira; Tokunaga, Masao

    2015-01-01

    Anti-IZUMO1PFF VHH (variable domain of camelid heavy chain antibody) clones, N6 and N15, from immunized alpaca (Lama pacos) phage library were efficiently expressed and their VHH products were secreted into the culture medium of Brevibacillus choshinensis HPD31-SP3, e.g., at a level of 26-95mg in 100ml conventional flask culture. With a 3-L scale fed-batch culture for 65h, the N15 VHH protein with C-terminal His-tag was produced at ∼3g/l culture medium. The N6 and N15 proteins were easily purified to apparent homogeneity by cation exchange and Ni-affinity chromatographies. Both proteins showed specific antigen-binding activity by ELISA and high antigen binding affinity, KD=6.0-8.6nM, by surface plasmon resonance analysis. Size exclusion chromatography-multi-angle laser light scattering analysis revealed that N6 and N15 proteins purified were exclusively monomeric form in phosphate buffered saline. CD spectrum showed beta-sheet rich structure, consistent with a typical antibody structure and also suggested aromatic-aromatic interactions, as indicated by a positive peak at 232nm. Thermal melting analysis of the N15 protein with C-terminal His-tag demonstrated a clear thermal transition with a Tm at 67°C. The heat-denatured sample recovered antigen binding activity upon cooling, indicating a reversible denaturation.

  17. Antibody Drug Conjugate Bioinformatics: Drug Delivery through the Letterbox

    PubMed Central

    Vlachakis, Dimitrios

    2013-01-01

    Antibodies appear to be the first line of defence in the adaptive immune response of vertebrates and thereby are involved in a multitude of biochemical mechanisms, such as regulation of infection, autoimmunity, and cancer. It goes without saying that a full understanding of antibody function is required for the development of novel antibody-interacting drugs. These drugs are the Antibody Drug Conjugates (ADCs), which are a new type of targeted therapy, used for example for cancer. They consist of an antibody (or antibody fragment such as a single-chain variable fragment [scFv]) linked to a payload drug (often cytotoxic). Because of the targeting, the side effects should be lower and give a wider therapeutic window. Overall, the underlying principle of ADCs is to discern the delivery of a drug that is cytotoxic to a target that is cancerous, hoping to increase the antitumoural potency of the original drug by reducing adverse effects and side effects, such as toxicity of the cancer target. This is a pioneering field that employs state-of-the-art computational and molecular biology methods in the fight against cancer using ADCs. PMID:23853668

  18. Specific Conjugation of the Hinge Region for Homogeneous Preparation of Antibody Fragment-Drug Conjugate: A Case Study for Doxorubicin-PEG-anti-CD20 Fab' Synthesis.

    PubMed

    Zhou, Zhan; Zhang, Jing; Zhang, Yan; Ma, Guanghui; Su, Zhiguo

    2016-01-20

    Conventional preparation strategies for antibody-drug conjugates (ADCs) result in heterogeneous products with various molecular sizes and species. In this study, we developed a homogeneous preparation strategy by site-specific conjugation of the anticancer drug with an antibody fragment. The model drug doxorubicin (DOX) was coupled to the Fab' fragment of anti-CD20 IgG at its permissive sites through a heterotelechelic PEG linker, generating an antibody fragment-drug conjugate (AFDC). Anti-CD20 IgG was digested and reduced specifically with β-mercaptoethylamine to generate the Fab' fragment with two free mercapto groups in its hinge region. Meanwhile, DOX was conjugated with α-succinimidylsuccinate ω-maleimide polyethylene glycol (NHS-PEG-MAL) to form MAL-PEG-DOX, which was subsequently linked to the free mercapto containing Fab' fragment to form a Fab'-PEG-DOX conjugate. The dual site-specific bioconjugation was achieved through the combination of highly selective reduction of IgG and introduction of heterotelechelic PEG linker. The resulting AFDC provides an utterly homogeneous product, with a definite ratio of one fragment to two drugs. Laser confocal microscopy and cell ELISA revealed that the AFDC could accumulate in the antigen-positive Daudi tumor cell. In addition, the Fab'-PEG-DOX retained appreciable targeting ability and improved antitumor activity, demonstrating an excellent therapeutic effect on the lymphoma mice model for better cure rate and significantly reduced side effects.

  19. Generation and characterization of chicken-sourced single-chain variable fragments (scFvs) against porcine interferon-gamma (pIFN-γ).

    PubMed

    Chen, Hong-Xiu; He, Fan; Sun, Yuan; Luo, Yuzi; Qiu, Hua-Ji; Zhang, Xiao-Ying; Sutton, Brian J

    2015-01-01

    Development of chicken-sourced antibodies offers an alternative strategy for the development of highly specific antibodies against mammalian proteins with conserved epitopes due to the phylogenetic distance between avian and mammalian species. In this study, the single-chain variable fragments (scFvs) against porcine interferon-gamma was screened and characterized from a hyperimmunized chicken phage display library. The expressed soluble scFvs exhibited highly specific recognition of porcine interferon-gamma in ELISA, Western blot, and immunofluorescence staining assays. Results of the current study indicate that it is possible to develop scFv IgY antibodies to a mammalian interferon by using Biopanning technology. Furthermore, it also confirms that monoclonal avian IgY antibody technique could be applied as a promising tool to produce immunoglobulin molecules with high specificity and affinity towards conserved mammalian epitopes or antigens.

  20. Cross-neutralizing anti-HIV-1 human single chain variable fragments(scFvs) against CD4 binding site and N332 glycan identified from a recombinant phage library

    PubMed Central

    Khan, Lubina; Kumar, Rajesh; Thiruvengadam, Ramachandran; Parray, Hilal Ahmad; Makhdoomi, Muzamil Ashraf; Kumar, Sanjeev; Aggarwal, Heena; Mohata, Madhav; Hussain, Abdul Wahid; Das, Raksha; Varadarajan, Raghavan; Bhattacharya, Jayanta; Vajpayee, Madhu; Murugavel, K. G.; Solomon, Suniti; Sinha, Subrata; Luthra, Kalpana

    2017-01-01

    More than 50% of HIV-1 infection globally is caused by subtype_C viruses. Majority of the broadly neutralizing antibodies (bnAbs) targeting HIV-1 have been isolated from non-subtype_C infected donors. Mapping the epitope specificities of bnAbs provides useful information for vaccine design. Recombinant antibody technology enables generation of a large repertoire of monoclonals with diverse specificities. We constructed a phage recombinant single chain variable fragment (scFv) library with a diversity of 7.8 × 108 clones, using a novel strategy of pooling peripheral blood mononuclear cells (PBMCs) of six select HIV-1 chronically infected Indian donors whose plasma antibodies exhibited potent cross neutralization efficiency. The library was panned and screened by phage ELISA using trimeric recombinant proteins to identify viral envelope specific clones. Three scFv monoclonals D11, C11 and 1F6 selected from the library cross neutralized subtypes A, B and C viruses at concentrations ranging from 0.09 μg/mL to 100 μg/mL. The D11 and 1F6 scFvs competed with mAbs b12 and VRC01 demonstrating CD4bs specificity, while C11 demonstrated N332 specificity. This is the first study to identify cross neutralizing scFv monoclonals with CD4bs and N332 glycan specificities from India. Cross neutralizing anti-HIV-1 human scFv monoclonals can be potential candidates for passive immunotherapy and for guiding immunogen design. PMID:28332627

  1. Neutralization Analysis of a Chicken Single-Chain Variable Fragment Derived from an Immune Antibody Library Against Infectious Bronchitis Virus.

    PubMed

    Lin, Yuan; Li, Benqiang; Ye, Jiaxin; Wang, Man; Wang, Jianhua; Zhang, Ying; Zhu, Jianguo

    2015-09-01

    Avian infectious bronchitis virus (IBV), which is prevalent in many countries causing severe economic loss to the poultry industry, causes infectious bronchitis (IB) in birds. Recombinant single-chain variable fragments (scFvs) have been proven to effectively inhibit many viruses, both in vitro and in vivo, and they could be a potential diagnostic and therapeutic reagent to control IB. In this study, six anti-IBV chicken scFvs, ZL.10, ZL.64, ZL.78, ZL.80, ZL.138, and ZL.256, were obtained by screening random clones from an immune antibody library. An analysis of nucleotide sequences revealed that they represented distinctive genetic sequences and greatly varied in complementarity-determining region three of the heavy chain. Neutralization tests showed that ZL.10, which bound the S1 protein in western blots, inhibited the formation of syncytia in Vero cells 48 h post IBV infection and decreased the transcriptional level of nucleoprotein mRNA to 17.2%, while the other five scFvs, including ZL.78 and ZL.256, that bound the N protein did not. In conclusion, the results suggested that specific and neutralizing chicken scFvs against IBV, which can be safe and economical antibody reagents, can be produced in vitro through prokaryotic expression.

  2. Indications of neutralising anti-idiotypic antibodies and selective proteolytic fragmentation in polyclonal anti-D IgG preparations.

    PubMed

    Gronski, P; Haas, T; Kanzy, E J; Lang, W; Röder, J; Ruhwedel, K; Simshäuser, K

    2003-09-01

    Proteolytic fragmentation is the only suggested cause of potency losses during storage of liquid human polyclonal anti-D Ig. Besides the effect of fragmentation, we have investigated the potential contribution of neutralising anti-idiotypic antibodies (anti-Ids). Potency changes during storage and/or upon pH reduction in anti-D IgG batches with or without addition of plasminogen and urokinase were quantitatively analysed by the autoanalyser (AA) method or by a special procedure of flow cytometry (FC). Moreover, simultaneous changes of the molecular size distribution pattern have been determined by size exclusion chromatography. In contrast to the AA procedure, the particular FC methodology was found to be almost insensitive to proteolysis comprising up to 30% of total IgG. Data interpretation was based on the assumption that both assays cannot detect Ids with neutralised paratopes. In the absence of detectable neutralisation (functional absence of anti-Ids), it could be demonstrated that the anti-D IgG subpopulation is more sensitive to fragmentation by endogenous protease as compared to the unrelated bulk. However, both methods detected batch- and assay-dependently variable potency losses during storage. Moreover, the increase of potency induced by pH reduction correlated with the increase of monomeric IgG, essentially on the expense of dimers. This finding was interpreted to indirectly indicate the neutralising action of anti-Ids known to be the major driving force of dimer formation in polyclonal IgG. A more or less pronounced pH-dependent potency increase was also detectable in three arbitrarily selected batches of two other manufacturers. The data allows to assume that anti-Id-mediated neutralisation can significantly contribute to losses of anti-D potency. In addition, it turned out that anti-D plasma itself can be the source of anti-Ids.

  3. Crystal Structure of Snake Venom Acetylcholinesterase in Complex with Inhibitory Antibody Fragment Fab410 Bound at the Peripheral Site

    PubMed Central

    Bourne, Yves; Renault, Ludovic; Marchot, Pascale

    2015-01-01

    The acetylcholinesterase found in the venom of Bungarus fasciatus (BfAChE) is produced as a soluble, non-amphiphilic monomer with a canonical catalytic domain but a distinct C terminus compared with the other vertebrate enzymes. Moreover, the peripheral anionic site of BfAChE, a surface site located at the active site gorge entrance, bears two substitutions altering sensitivity to cationic inhibitors. Antibody Elec410, generated against Electrophorus electricus acetylcholinesterase (EeAChE), inhibits EeAChE and BfAChE by binding to their peripheral sites. However, both complexes retain significant residual catalytic activity, suggesting incomplete gorge occlusion by bound antibody and/or high frequency back door opening. To explore a novel acetylcholinesterase species, ascertain the molecular bases of inhibition by Elec410, and document the determinants and mechanisms for back door opening, we solved a 2.7-Å resolution crystal structure of natural BfAChE in complex with antibody fragment Fab410. Crystalline BfAChE forms the canonical dimer found in all acetylcholinesterase structures. Equally represented open and closed states of a back door channel, associated with alternate positions of a tyrosine phenol ring at the active site base, coexist in each subunit. At the BfAChE molecular surface, Fab410 is seated on the long Ω-loop between two N-glycan chains and partially occludes the gorge entrance, a position that fully reflects the available mutagenesis and biochemical data. Experimentally based flexible molecular docking supports a similar Fab410 binding mode onto the EeAChE antigen. These data document the molecular and dynamic peculiarities of BfAChE with high frequency back door opening, and the mode of action of Elec410 as one of the largest peptidic inhibitors targeting the acetylcholinesterase peripheral site. PMID:25411244

  4. Intranuclear Delivery of a Novel Antibody-Derived Radiosensitizer Targeting the DNA-Dependent Protein Kinase Catalytic Subunit

    SciTech Connect

    Xiong Hairong; Lee, Robert J.; Haura, Eric B.; Edwards, John G.; Dynan, William S.; Li Shuyi

    2012-07-01

    Purpose: To inhibit DNA double-strand break repair in tumor cells by delivery of a single-chain antibody variable region fragment (ScFv 18-2) to the cell nucleus. ScFv 18-2 binds to a regulatory region of the DNA-dependent protein kinase (DNA-PK), an essential enzyme in the nonhomologous end-joining pathway, and inhibits DNA end-joining in a cell-free system and when microinjected into single cells. Development as a radiosensitizer has been limited by the lack of a method for intranuclear delivery to target cells. We investigated a delivery method based on folate receptor-mediated endocytosis. Methods and Materials: A recombinant ScFv 18-2 derivative was conjugated to folate via a scissile disulfide linker. Folate-ScFv 18-2 was characterized for its ability to be internalized by tumor cells and to influence the behavior of ionizing radiation-induced repair foci. Radiosensitization was measured in a clonogenic survival assay. Survival curves were fitted to a linear-quadratic model, and between-group differences were evaluated by an F test. Sensitization ratios were determined based on mean inhibitory dose. Results: Human KB and NCI-H292 lung cancer cells treated with folate-conjugated ScFv 18-2 showed significant radiosensitization (p < 0.001). Sensitization enhancement ratios were 1.92 {+-} 0.42 for KB cells and 1.63 {+-} 0.13 for NCI-H292 cells. Studies suggest that treatment inhibits repair of radiation-induced DSBs, as evidenced by the persistence of {gamma}-H2AX-stained foci and by inhibition of staining with anti-DNA-PKcs phosphoserine 2056. Conclusions: Folate-mediated endocytosis is an effective method for intranuclear delivery of an antibody-derived DNA repair inhibitor.

  5. Miniaturized antibodies for imaging membrane type-1 matrix metalloproteinase in cancers.

    PubMed

    Kondo, Naoya; Temma, Takashi; Shimizu, Yoichi; Watanabe, Hiroyuki; Higano, Keiichi; Takagi, Yoko; Ono, Masahiro; Saji, Hideo

    2013-04-01

    Since membrane type-1 matrix metalloproteinase (MT1-MMP) plays pivotal roles in tumor progression and metastasis and holds great promise as an early biomarker for malignant tumors, a method of evaluating MT1-MMP expression levels would be valuable for molecular biological and clinical studies. Although we have previously developed a (99m) Tc-labeled anti-MT1-MMP monoclonal IgG ((99m) Tc-MT1-mAb) as an MT1-MMP imaging probe by nuclear medical techniques for this purpose, slow pharmacokinetics were a problem due to its large molecular size. Thus, in this study, our aim was to develop miniaturized antibodies, a single chain antibody fragment (MT1-scFv) and a dimer of two molecules of scFv (MT1-diabody), as the basic structures of MT1-MMP imaging probes followed by in vitro and in vivo evaluation with an (111) In radiolabel. Phage display screening successfully provided MT1-scFv and MT1-diabody, which had sufficiently high affinity for MT1-MMP (KD  = 29.8 and 17.1 nM). Both (111) In labeled miniaturized antibodies showed higher uptake in MT1-MMP expressing HT1080 cells than in non-expressing MCF7 cells. An in vivo biodistribution study showed rapid pharmacokinetics for both probes, which exhibited >20-fold higher tumor to blood radioactivity ratios (T/B ratio), an index for in vivo imaging, than (99m) Tc-MT1-mAb 6 h post-administration, and significantly higher tumor accumulation in HT1080 than MCF7 cells. SPECT images showed heterogeneous distribution and ex vivo autoradiographic analysis revealed that the radioactivity distribution profiles in tumors corresponded to MT1-MMP-positive areas. These findings suggest that the newly developed miniaturized antibodies are promising probes for detection of MT1-MMP in cancer cells.

  6. Utilisation of antibody microarrays for the selection of specific and informative antibodies from recombinant library binders of unknown quality.

    PubMed

    Kibat, Janek; Schirrmann, Thomas; Knape, Matthias J; Helmsing, Saskia; Meier, Doris; Hust, Michael; Schröder, Christoph; Bertinetti, Daniela; Winter, Gerhard; Pardes, Khalid; Funk, Mia; Vala, Andrea; Giese, Nathalia; Herberg, Friedrich W; Dübel, Stefan; Hoheisel, Jörg D

    2016-09-25

    Many diagnostic and therapeutic concepts require antibodies of high specificity. Recombinant binder libraries and related selection approaches allow the efficient isolation of antibodies against almost every target of interest. Nevertheless, it cannot be guaranteed that selected antibodies perform well and interact specifically enough with analytes unless an elaborate characterisation is performed. Here, we present an approach to shorten this process by combining the selection of suitable antibodies with the identification of informative target molecules by means of antibody microarrays, thereby reducing the effort of antibody characterisation by concentrating on relevant molecules. In a pilot scheme, a library of 456 single-chain variable fragment (scFv) binders to 134 antigens was used. They were arranged in a microarray format and incubated with the protein content of clinical tissue samples isolated from pancreatic ductal adenocarcinoma and healthy pancreas, as well as recurrent and non-recurrent bladder tumours. We observed significant variation in the expression of the E3 ubiquitin-protein ligase (CHFR) as well as the glutamate receptor interacting protein 2 (GRIP2), for example, always with more than one of the scFvs binding to these targets. Only the relevant antibodies were then characterised further on antigen microarrays and by surface plasmon resonance experiments so as to select the most specific and highest affinity antibodies. These binders were in turn used to confirm a microarray result by immunohistochemistry analysis.

  7. Methods of preparing and using single chain anti-tumor antibodies

    SciTech Connect

    Cheung, Nai-Kong; Guo, Hong-Fen

    2010-02-23

    This invention provides a method for identifying cells expressing a target single chain antibody (scFv) directed against a target antigen from a collection of cells that includes cells that do not express the target scFv, comprising the step of combining the collection of cells with an anti-idiotype directed to an antibody specific for the target antigen and detecting interaction, if any, of the anti-idiotype with the cells, wherein the occurrence of an interaction identifies the cell as one which expresses the target scFv. This invention also provides a method for making a single chain antibody (scFv) directed against an antigen, wherein the selection of clones is made based upon interaction of those clones with an appropriate anti-idiotype, and heretofore inaccessible scFv so made. This invention provides the above methods or any combination thereof. Finally, this invention provides various uses of these methods.

  8. Crystal structures of a therapeutic single chain antibody in complex with two drugs of abuse-Methamphetamine and 3,4-methylenedioxymethamphetamine.

    PubMed

    Celikel, Reha; Peterson, Eric C; Owens, S Michael; Varughese, Kottayil I

    2009-11-01

    Methamphetamine (METH) is a major drug threat in the United States and worldwide. Monoclonal antibody (mAb) therapy for treating METH abuse is showing exciting promise and the understanding of how mAb structure relates to function will be essential for future development of these important therapies. We have determined crystal structures of a high affinity anti-(+)-METH therapeutic single chain antibody fragment (scFv6H4, K(D)= 10 nM) derived from one of our candidate mAb in complex with METH and the (+) stereoisomer of another abused drug, 3,4-methylenedioxymethamphetamine (MDMA), known by the street name "ecstasy." The crystal structures revealed that scFv6H4 binds to METH and MDMA in a deep pocket that almost completely encases the drugs mostly through aromatic interactions. In addition, the cationic nitrogen of METH and MDMA forms a salt bridge with the carboxylate group of a glutamic acid residue and a hydrogen bond with a histidine side chain. Interestingly, there are two water molecules in the binding pocket and one of them is positioned for a C--H...O interaction with the aromatic ring of METH. These first crystal structures of a high affinity therapeutic antibody fragment against METH and MDMA (resolution = 1.9 A, and 2.4 A, respectively) provide a structural basis for designing the next generation of higher affinity antibodies and also for carrying out rational humanization.

  9. Controlling Rotavirus-associated diarrhea: Could single-domain antibody fragments make the difference?

    PubMed

    Maffey, Lucia; Vega, Celina G; Parreño, Viviana; Garaicoechea, Lorena

    2015-01-01

    Group A Rotavirus (RVA) remains a leading cause of severe diarrhea and child mortality. The variable domain of camelid heavy chain antibodies (VHH) display potent antigen-binding capacity, have low production costs and are suitable for oral therapies. Two sets of anti-RVA VHHs have been developed: ARP1-ARP3; 2KD1-3B2. Here, we explore the potential of both sets as a prevention strategy complementary to vaccination and a treatment option against RVA-associated diarrhea in endangered populations. Both sets have been expressed in multiple production systems, showing extensive neutralizing capacity against strains of RVA in vitro. They were also tested in the neonatal mouse model with various degrees of success in preventing or treating RVA-induced diarrhea. Interestingly, mitigation of the symptoms was also achieved with freeze-dried ARP1, so that it could be applied in areas where cold chains are difficult to maintain. 3B2 was tested in a pre-clinical trial involving gnotobiotic piglets where it conferred complete protection against RVA-induced diarrhea. ARP1 was used in the first clinical trial for anti-RVA VHHs, successfully reducing stool output in infants with RVA diarrhea, with no detected side effects.

  10. STRUCTURE BASED DESIGN OF PROTEIN LIGANDS: A STUDY OF ANTIBODY-LIKE SCAFFOLDS TARGETED AGAINST THE ANTHRAX TOXIN

    SciTech Connect

    P. SHIFLETT; E. HONG-GELLER; ET AL

    2000-12-01

    We have adopted structure-based approaches to enhance the affinities of two single chain antibodies, scFv1 and scFv4, that bind to two different epitopes on the Protective Antigen (PA), a toxin from Bacillus anthracis. In one approach, we have modified scFv4 and re-engineered a novel antibody-like scaffold in which we have placed V{sub L} on the N terminus and V{sub H} on the C-terminus and joined them by a 10 amino-acid-long linker. This scaffold preserves the native V{sub L}-V{sub H} contact interface and the dispositions of the CDR loops. It binds to PA with 10 fold higher affinity than scFv4. In a second approach, we have created a bispecific ligand by covalently joining scFv1 and scFv4 by a flexible linker that supports simultaneous and synergistic binding of the two scFvs to PA. This bispecific scFv1-linker-scFv4 binds to PA with 10 fold higher affinity than the individual scFvs. The newly re-engineered antibody-like scaffold of scFv4 and scFv1-linker-scFv4 are expected to be potent inhibitors of PA binding to the host cells.

  11. Antibody

    MedlinePlus

    An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples ... microorganisms (bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produced when the immune system mistakenly ...

  12. Purification, characterization, and biotinylation of single-chain antibodies.

    PubMed

    Kipriyanov, S M

    1998-01-01

    The variable region (Fv) portion of an antibody is comprised of the antibody V(H) and V(L) domains and is the smallest antibody fragment containing a complete antigen-binding site. To stabilize the association of the recombinant V(H) and V(L) domains, they have been linked in single-chain Fv constructs with a short peptide that bridges the approx 3.5 nm between the carboxy terminus of one domain and the ammo terminus of the other (1-3). An NMR comparison of the unlinked Fv fragment of the antibody McPC603 with the corresponding scFv containing a V(H)-(Gly(4)Ser)(3)-V(L) linker has shown no perturbation of the folding of the variable domains by the linker (4,5). In comparison to the much larger Fab', F(ab')(2), and IgG forms of monoclonal antibody (MAb) from which they are derived, scFvs have lower retention times in nontarget tissues, more rapid blood clearance, and better tumor penetration (6-8). ScFvs, therefore, represent potentially very useful molecules for the targeted delivery of drugs, toxins, or radionuclides to a tumor site.

  13. Effect of protein structure on deamidation rate in the Fc fragment of an IgG1 monoclonal antibody

    PubMed Central

    Sinha, Sandipan; Zhang, Lei; Duan, Shaofeng; Williams, Todd D; Vlasak, Josef; Ionescu, Roxana; Topp, Elizabeth M

    2009-01-01

    The effects of secondary structure on asparagine (N) deamidation in a 22 amino acid sequence (369-GFYPSDIAVEWESNGQPENNYK-390) of the crystallizable (Fc) fragment of a human monoclonal antibody (Fc IgG1) were investigated using high-resolution ultra performance liquid chromatography with tandem mass spectrometry (UPLC/MS). Samples containing either the intact Fc IgG (∼50 kD) (“intact protein”), or corresponding synthetic peptides (“peptide”) were stored in Tris buffer at 37°C and pH 7.5 for up to forty days, then subjected to UPLC/MS analysis with high energy MS1 fragmentation. The peptide deamidated only at N382 to form the isoaspartate (isoD382) and aspartate (D382) products in the ratio of ∼4:1, with a half-life of ∼3.4 days. The succinimide intermediate (Su382) was also detected; deamidation was not observed for the other two sites (N387 and N388) in peptide samples. The intact protein showed a 30-fold slower overall deamidation half-life of ∼108 days to produce the isoD382 and D387 products, together with minor amounts of D382. Surprisingly, the D382 and isoD387 products were not detected in intact protein samples and, as in the peptide samples, deamidation was not detected at N388. The results indicate that higher order structure influences both the rate of N-deamidation and the product distribution. PMID:19544580

  14. A Top-Down Approach to Mechanistic Biological Modeling: Application to the Single-Chain Antibody Folding Pathway

    PubMed Central

    Hildebrandt, Scott; Raden, David; Petzold, Linda; Robinson, Anne Skaja; Doyle, Francis J.

    2008-01-01

    A top-down approach to mechanistic modeling of biological systems is presented and exemplified with the development of a hypothesis-driven mathematical model for single-chain antibody fragment (scFv) folding in Saccharomyces cerevisiae by mediators BiP and PDI. In this approach, model development starts with construction of the most basic mathematical model—typically consisting of predetermined or newly-elucidated biological behavior motifs—capable of reproducing desired biological behaviors. From this point, mechanistic detail is added incrementally and systematically, and the effects of each addition are evaluated. This approach follows the typical progression of experimental data availability in that higher-order, lumped measurements are often more prevalent initially than specific, mechanistic ones. It also necessarily provides the modeler with insight into the structural requirements and performance capabilities of the resulting detailed mechanistic model, which facilitates further analysis. The top-down approach to mechanistic modeling identified three such requirements and a branched dependency-degradation competition motif critical for the scFv folding model to reproduce experimentally observed scFv folding dependencies on BiP and PDI and increased production when both species are overexpressed and promoted straightforward prediction of parameter dependencies. It also prescribed modification of the guiding hypothesis to capture BiP and PDI synergy. PMID:18641066

  15. Site-specific labeling of cysteine-tagged camelid single-domain antibody-fragments for use in molecular imaging.

    PubMed

    Massa, Sam; Xavier, Catarina; De Vos, Jens; Caveliers, Vicky; Lahoutte, Tony; Muyldermans, Serge; Devoogdt, Nick

    2014-05-21

    Site-specific labeling of molecular imaging probes allows the development of a homogeneous tracer population. The resulting batch-to-batch reproducible pharmacokinetic and pharmacodynamic properties are of great importance for clinical translation. Camelid single-domain antibody-fragments (sdAbs)-the recombinantly produced antigen-binding domains of heavy-chain antibodies, also called Nanobodies-are proficient probes for molecular imaging. To safeguard their intrinsically high binding specificity and affinity and to ensure the tracer's homogeneity, we developed a generic strategy for the site-specific labeling of sdAbs via a thio-ether bond. The unpaired cysteine was introduced at the carboxyl-terminal end of the sdAb to eliminate the risk of antigen binding interference. The spontaneous dimerization and capping of the unpaired cysteine required a reduction step prior to conjugation. This was optimized with the mild reducing agent 2-mercaptoethylamine in order to preserve the domain's stability. As a proof-of-concept the reduced probe was subsequently conjugated to maleimide-DTPA, for labeling with indium-111. A single conjugated tracer was obtained and confirmed via mass spectrometry. The specificity and affinity of the new sdAb-based imaging probe was validated in a mouse xenograft tumor model using a modified clinical lead compound targeting the human epidermal growth factor receptor 2 (HER2) cancer biomarker. These data provide a versatile and standardized strategy for the site-specific labeling of sdAbs. The conjugation to the unpaired cysteine results in the production of a homogeneous group of tracers and is a multimodal alternative to the technetium-99m labeling of sdAbs.

  16. A Fully Human Inhibitory Monoclonal Antibody to the Wnt Receptor RYK

    PubMed Central

    Parish, Clare L.; Takano, Elena A.; Fox, Stephen; Layton, Daniel; Nice, Edouard; Stacker, Steven A.

    2013-01-01

    RYK is an unusual member of the receptor tyrosine kinase (RTK) family that is classified as a putative pseudokinase. RYK regulates fundamental biological processes including cell differentiation, migration and target selection, axon outgrowth and pathfinding by transducing signals across the plasma membrane in response to the high affinity binding of Wnt family ligands to its extracellular Wnt inhibitory factor (WIF) domain. Here we report the generation and initial characterization of a fully human inhibitory monoclonal antibody to the human RYK WIF domain. From a naïve human single chain fragment variable (scFv) phage display library, we identified anti-RYK WIF domain–specific scFvs then screened for those that could compete with Wnt3a for binding. Production of a fully human IgG1κ from an inhibitory scFv yielded a monoclonal antibody that inhibits Wnt5a-responsive RYK function in a neurite outgrowth assay. This antibody will have immediate applications for modulating RYK function in a range of settings including development and adult homeostasis, with significant potential for therapeutic use in human pathologies. PMID:24058687

  17. A novel in vivo method for isolating antibodies from a phage display library by neuronal retrograde transport selectively yields antibodies against p75(NTR.).

    PubMed

    Tani, Hiroaki; Osbourn, Jane K; Walker, Edward H; Rush, Robert A; Ferguson, Ian A

    2013-01-01

    The neurotrophin receptor p75(NTR) is utilized by a variety of pathogens to gain entry into the central nervous system (CNS). We tested if this entry portal might be exploited using a phage display library to isolate internalizing antibodies that target the CNS in vivo. By applying a phage library that expressed human single chain variable fragment (scFv) antibodies on their surface to a transected sciatic nerve, we showed that (1) phage conjugated to anti-p75(NTR) antibody or phage scFv library pre-panned against p75(NTR) are internalized by neurons expressing p75(NTR); (2) subsequent retrograde axonal transport separates internalized phage from the applied phage; and, (3) internalized phage can be recovered from a proximal ligature made on a nerve. This approach resulted in 13-fold increase in the number of phage isolated from the injured nerve compared with the starting population, and isolation of 18 unique internalizing p75(NTR) antibodies that were transported from the peripheral nerve into the spinal cord, through the blood-brain barrier. In addition, antibodies recognizing other potentially internalized antigens were identified through in vivo selection using a fully diverse library. Because p75(NTR) expression is upregulated in motor neurons in response to injury and in disease, the p75(NTR) antibodies may have substantial potential for cell-targeted drug/gene delivery. In addition, this novel selection method provides the potential to generate panels of antibodies that could be used to identify further internalization targets, which could aid drug delivery across the blood-brain barrier.

  18. Highly specific PET imaging of prostate tumors in mice with an iodine-124-labeled antibody fragment that targets phosphatidylserine.

    PubMed

    Stafford, Jason H; Hao, Guiyang; Best, Anne M; Sun, Xiankai; Thorpe, Philip E

    2013-01-01

    Phosphatidylserine (PS) is an attractive target for imaging agents that identify tumors and assess their response to therapy. PS is absent from the surface of most cell types, but becomes exposed on tumor cells and tumor vasculature in response to oxidative stresses in the tumor microenvironment and increases in response to therapy. To image exposed PS, we used a fully human PS-targeting antibody fragment, PGN635 F(ab')2, that binds to complexes of PS and β2-glycoprotein I. PGN635 F(ab')2 was labeled with the positron-emitting isotope iodine-124 ((124)I) and the resulting probe was injected into nude mice bearing subcutaneous or orthotopic human PC3 prostate tumors. Biodistribution studies showed that (124)I-PGN635 F(ab')2 localized with remarkable specificity to the tumors with little uptake in other organs, including the liver and kidneys. Clear delineation of the tumors was achieved by PET 48 hours after injection. Radiation of the tumors with 15 Gy or systemic treatment of the mice with 10 mg/kg docetaxel increased localization in the tumors. Tumor-to-normal (T/N) ratios were inversely correlated with tumor growth measured over 28 days. These data indicate that (124)I-PGN635 F(ab')2 is a promising new imaging agent for predicting tumor response to therapy.

  19. Effect of polyethylene glycol conjugation on conformational and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab').

    PubMed

    Roque, Cristopher; Sheung, Anthony; Rahman, Nausheen; Ausar, S Fernando

    2015-02-02

    We have investigated the effects of site specific "hinge" polyethylene glycol conjugation (PEGylation) on thermal, pH, and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab') using a variety of biophysical techniques. The results obtained by circular dichroism (CD), ultraviolet (UV) absorbance, and fluorescence spectroscopy suggested that the physical stability of the Fab' is maximized at pH 6-7 with no apparent differences due to PEGylation. Temperature-induced aggregation experiments revealed that PEGylation was able to increase the transition temperature, as well as prevent the formation of visible and subvisible aggregates. Statistical comparison of the three-index empirical phase diagram (EPD) revealed significant differences in thermal and pH stability signatures between Fab' and PEG-Fab'. Upon mechanical stress, micro-flow imaging (MFI) and measurement of the optical density at 360 nm showed that the PEG-Fab' had significantly higher resistance to surface-induced aggregation compared to the Fab'. Analysis of the interaction parameter, kD, indicated repulsive intermolecular forces for PEG-Fab' and attractive forces for Fab'. In conclusion, PEGylation appears to protect Fab' against thermal and mechanical stress-induced aggregation, likely due to a steric hindrance mechanism.

  20. High production of llama variable heavy-chain antibody fragment (VHH) fused to various reader proteins by Aspergillus oryzae.

    PubMed

    Hisada, Hiromoto; Tsutsumi, Hiroko; Ishida, Hiroki; Hata, Yoji

    2013-01-01

    Llama variable heavy-chain antibody fragment (VHH) fused to four different reader proteins was produced and secreted in culture medium by Aspergillus oryzae. These fusion proteins consisted of N-terminal reader proteins, VHH, and a C-terminal his-tag sequence which facilitated purification using one-step his-tag affinity chromatography. SDS-PAGE analysis of the deglycosylated purified fusion proteins confirmed that the molecular weight of each corresponded to the expected sum of VHH and the respective reader proteins. The apparent high molecular weight reader protein glucoamylase (GlaB) was found to be suitable for efficient VHH production. The GlaB-VHH-His protein bound its antigen, human chorionic gonadotropin, and was detectable by a new ELISA-based method using a coupled assay with glucoamylase, glucose oxidase, peroxidase, maltose, and 3,3',5,5'-tetramethylbenzidine as substrates. Addition of potassium phosphate to the culture medium induced secretion of 0.61 mg GlaB-VHH-His protein/ml culture medium in 5 days.

  1. PEGylation prolongs the pulmonary retention of an anti-IL-17A Fab' antibody fragment after pulmonary delivery in three different species.

    PubMed

    Freches, Danielle; Patil, Harshad P; Machado Franco, Maria; Uyttenhove, Catherine; Heywood, Sam; Vanbever, Rita

    2017-04-15

    The PEGylation of antibody fragments has been shown to greatly prolong their residence time in the lungs in mice. The purpose of this research was to confirm the effect of PEGylation in higher animal species, that is, the rat and the rabbit. An anti-IL-17A Fab' antibody fragment was conjugated to a two-armed 40kDa polyethylene glycol (PEG) via site-selective thiol PEGylation. PEGylation did not significantly alter the binding activity of the Fab' fragment but it largely enhanced its inhibitory potency. PEGylation increased the residence time of the Fab' in the lungs of mice, rats and rabbits. Following intratracheal administration, the unconjugated Fab' was cleared from the lungs within 24h while large quantities of the PEGylated Fab' remained present up to 48h. No significant differences in clearance were noted between the three animal species although there was a tendency of longer residence time in higher species. PEGylation represents a promising approach to sustain the presence of antibody fragments in the lungs and to enhance their therapeutic efficacy in respiratory diseases.

  2. Labeling monoclonal antibodies and F(ab')2 fragments with the alpha-particle-emitting nuclide astatine-211: preservation of immunoreactivity and in vivo localizing capacity.

    PubMed Central

    Zalutsky, M R; Garg, P K; Friedman, H S; Bigner, D D

    1989-01-01

    alpha-Particles such as those emitted by 211At may be advantageous for radioimmunotherapy since they are radiation of high linear energy transfer, depositing high energy over a short distance. Here we describe a strategy for labeling monoclonal antibodies and F(ab')2 fragments with 211At by means of the bifunctional reagent N-succinimidyl 3-(trimethylstannyl)benzoate. An intact antibody, 81C6, and the F(ab')2 fragment of Me1-14 (both reactive with human gliomas) were labeled with 211At in high yield and with a specific activity of up to 4 mCi/mg in a time frame compatible with the 7.2-hr half-life of 211At. Quantitative in vivo binding assays demonstrated that radioastatination was accomplished with maintenance of high specific binding and affinity. Comparison of the biodistribution of 211At-labeled Me1-14 F(ab')2 to that of a nonspecific antibody fragment labeled with 211At and 131I in athymic mice bearing D-54 MG human glioma xenografts demonstrated selective and specific targeting of 211At-labeled antibody in this human tumor model. PMID:2476813

  3. Efficient recovery of the functional IP10-scFv fusion protein from inclusion bodies with an on-column refolding system.

    PubMed

    Guo, Jun-Qing; Li, Qing-Mei; Zhou, Ji-Yong; Zhang, Gai-Ping; Yang, Yan-Yan; Xing, Guang-Xu; Zhao, Dong; You, Shang-You; Zhang, Chu-Yu

    2006-01-01

    A functional IP10-scFv fusion protein retaining the antibody specificity for acidic isoferritin and chemokine function was produced at high level in Esherichia coli (E. coli). IP10-scFv gene from the recombinant plasmid pc3IP104c9 was subcloned into pET28a fused to N-terminal His-tag sequence in frame and overexpressed in E. coli BL21(DE3). With an on-column refolding procedure based on Ni-chelating chromatography, the active fusion protein was recovered efficiently from inclusion bodies with a refolding yield of approximate 45% confirmed by spectrophotometer. The activity of refolded IP10-scFv was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting and enzyme-linked immunosorbent assay. The results showed the fusion protein retains the specific binding activity to AIF with an affinity constant of 4.48x10(-8) M as well as the chemokine function of IP-10. The overall yield of IP10-scFv with bioactivity in E. coli flask culture was more than 40 mg/L.

  4. An improved helper phage system for efficient isolation of specific antibody molecules in phage display.

    PubMed

    Baek, Hyunjung; Suk, Kyoung-ho; Kim, Yong-hwan; Cha, Sanghoon

    2002-03-01

    Phage display technology has been applied in many fields of biological and medical sciences to study molecular interactions and especially in the generation of monoclonal antibodies of human origin. However, extremely low display level of antibody molecules on the surface of phage is an intrinsic problem of a phagemid-based display system resulting in low success rate of isolating specific binding molecules. We show here that display of single-chain antibody fragment (scFv) generated with pIGT3 phagemid can be increased dramatically by using a genetically modified Ex-phage. Ex-phage has a mutant pIII gene that produces a functional wild-type pIII in suppressing Escherichia coli strains but does not make any pIII in non-suppressing E.coli strains. Packaging phagemids encoding antibody-pIII fusion in F+ non-suppressing E.coli strains with Ex-phage enhanced the display level of antibody fragments on the surfaces of recombinant phage particles resulting in an increase of antigen-binding reactivity >100-fold compared to packaging with M13KO7 helper phage. Thus, the Ex-phage and pIGT3 phagemid vector provides a system for the efficient enrichment of specific binding antibodies from a phage display library and, thereby, increases the chance of obtaining more diverse antibodies specific for target antigens.

  5. High-level secretion of recombinant monomeric murine and human single-chain Fv antibodies from Drosophila S2 cells.

    PubMed

    Gilmartin, Allissia A; Lamp, Benjamin; Rümenapf, Till; Persson, Mats A A; Rey, Félix A; Krey, Thomas

    2012-02-01

    Single-chain variable fragment (scFvs) antibodies are small polypeptides (∼26 kD) containing the heavy (V(H)) and light (V(L)) immunoglobulin domains of a parent antibody connected by a flexible linker. In addition to being frequently used in diagnostics and therapy for an increasing number of human diseases, scFvs are important tools for structural biology as crystallization chaperones. Although scFvs can be expressed in many different organisms, the expression level of an scFv strongly depends on its particular amino acid sequence. We report here a system allowing for easy and efficient cloning of (i) scFvs selected by phage display and (ii) individual heavy and light chain sequences from hybridoma cDNA into expression plasmids engineered for secretion of the recombinant fragment produced in Drosophila S2 cells. We validated the method by producing five scFvs derived from human and murine parent antibodies directed against various antigens. The production yields varied between 5 and 12 mg monomeric scFv per liter of supernatant, indicating a relative independence on the individual sequences. The recombinant scFvs bound their cognate antigen with high affinity, comparable with the parent antibodies. The suitability of the produced recombinant fragments for structural studies was demonstrated by crystallization and structure determination of one of the produced scFvs, derived from a broadly neutralizing antibody against the major glycoprotein E2 of the hepatitis C virus. Structural comparison with the Protein Data Bank revealed the typical spatial organization of V(H) and V(L) domains, further validating the here-reported expression system.

  6. High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation

    PubMed Central

    Mendler, Claudia T; Friedrich, Lars; Laitinen, Iina; Schlapschy, Martin; Schwaiger, Markus; Wester, Hans-Jürgen; Skerra, Arne

    2015-01-01

    Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics, their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal filtration. Humanized αHER2 and αCD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli. Cytofluorimetric titration analysis on tumor cell lines confirmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodified αHER2 and αCD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS400. Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodified Fabs were measured in the biodistribution analysis (48 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200, respectively. These findings were confirmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200. Even stronger tumor signals were obtained with the corresponding αCD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS100 vs. 0.24% ID/g for the unmodified Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to significantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions. PMID:25484039

  7. High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation.

    PubMed

    Mendler, Claudia T; Friedrich, Lars; Laitinen, Iina; Schlapschy, Martin; Schwaiger, Markus; Wester, Hans-Jürgen; Skerra, Arne

    2015-01-01

    Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics, their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal filtration. Humanized αHER2 and αCD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli. Cytofluorimetric titration analysis on tumor cell lines confirmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodified αHER2 and αCD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS400. Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodified Fabs were measured in the biodistribution analysis (48 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200, respectively. These findings were confirmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200. Even stronger tumor signals were obtained with the corresponding αCD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS100 vs. 0.24% ID/g for the unmodified Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to significantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions.

  8. [One amino acid mutation in an anti-CD20 antibody fragment that affects the yield bacterial secretion and the affinity].

    PubMed

    Liu, Yin-Xing; Xiong, Dong-Sheng; Fan, Dong-Mei; Shao, Xiao-Feng; Xu, Yuan-Fu; Zhu, Zhen-Ping; Yang, Chun-Zheng

    2003-05-01

    Monoclonal antibodies (mAb) directed against CD20, either unmodified or in radiolabeled forms, have been successfully exploited in clinic as effective therapeutic agents in the management of non-Hodgkin's B-cell lymphoma. The antibody fragment is a potential agent in image and therapy of tumor. To further improve the soluble expression of anti-CD20 antibody Fab' fragment, PCR was used to mutate the anti-CD20 VL and VH genes and its biological activity was identified. The expression vector of chimeric antibody Fab' was constructed and expressed in E. coli. The data of mutant clone DNA sequence showed that the amino acid of light chain gene of the parent anti-CD20 antibody (H47) was successful mutated as Ser (GAG)-Asn (CAG). The soluble expression of mutated anti-CD20 Fab' (CD20-7) was 3.8 mg/g dry cell weight, while the parent (CD20-2) was 1.3 mg/g dry cell weight. The affinity constant Ka of CD20-7 was 2.2 x 10(9) L/mol. The primary results of competitive assays by FACS showed that CD20-7 could partially block the sites through which parent antibody (HI47) bind to Raji cells. There was difference in the Raji cells (CD20+)-binding activity between the mutant CD20-7 and parent CD20-2. The site mutation of anti-CD20 Fab' gene make it possible that the anti-CD20 antibody fragment was succeeded to obtain higher expression. In this thesis, we succeeded in completing mutation and expression of anti-CD20 Fab' genes, distinguishing its biological activity, and obtaining its highly expression. These period results will lay a foundation for development of other kind of anti-CD20 engineering antibody (for instance: Fab' Diabody and miniantibody), and make it possible for anti-CD20 antibody to be applied to tumor therapy in civil in the future.

  9. Selection of Ceratitis capitata (Diptera: Tephritidae) Specific Recombinant Monoclonal Phage Display Antibodies for Prey Detection Analysis

    PubMed Central

    Monzó, César; Urbaneja, Alberto; Ximénez-Embún, Miguel; García-Fernández, Julia; García, José Luis; Castañera, Pedro

    2012-01-01

    Several recombinant antibodies against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), one of the most important pests in agriculture worldwide, were selected for the first time from a commercial phage display library of human scFv antibodies. The specificity and sensitivity of the selected recombinant antibodies were compared with that of a rabbit polyclonal serum raised in parallel using a wide range of arthropod species as controls. The selected recombinant monoclonal antibodies had a similar or greater specificity when compared with classical monoclonal antibodies. The selected recombinant antibodies were successfully used to detect the target antigen in the gut of predators and the scFv antibodies were sequenced and compared. These results demonstrate the potential for recombinant scFv antibodies to be used as an alternative to the classical monoclonal antibodies or even molecular probes in the post-mortem analysis studies of generalist predators. PMID:23272105

  10. Selection of Ceratitis capitata (Diptera: Tephritidae) specific recombinant monoclonal phage display antibodies for prey detection analysis.

    PubMed

    Monzó, César; Urbaneja, Alberto; Ximénez-Embún, Miguel; García-Fernández, Julia; García, José Luis; Castañera, Pedro

    2012-01-01

    Several recombinant antibodies against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), one of the most important pests in agriculture worldwide, were selected for the first time from a commercial phage display library of human scFv antibodies. The specificity and sensitivity of the selected recombinant antibodies were compared with that of a rabbit polyclonal serum raised in parallel using a wide range of arthropod species as controls. The selected recombinant monoclonal antibodies had a similar or greater specificity when compared with classical monoclonal antibodies. The selected recombinant antibodies were successfully used to detect the target antigen in the gut of predators and the scFv antibodies were sequenced and compared. These results demonstrate the potential for recombinant scFv antibodies to be used as an alternative to the classical monoclonal antibodies or even molecular probes in the post-mortem analysis studies of generalist predators.

  11. 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

    2017-03-13

    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 GP1-649 of MARV. Sequencing of 192 selected clones identified 18 clones with distinct VH and VL 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.

  12. Rapid isolation of antibody from a synthetic human antibody library by repeated fluorescence-activated cell sorting (FACS).

    PubMed

    Yim, Sung Sun; Bang, Hyun Bae; Kim, Young Hwan; Lee, Yong Jae; Jeong, Gu Min; Jeong, Ki Jun

    2014-01-01

    Antibodies and their derivatives are the most important agents in therapeutics and diagnostics. Even after the significant progress in the technology for antibody screening from huge libraries, it takes a long time to isolate an antibody, which prevents a prompt action against the spread of a disease. Here, we report a new strategy for isolating desired antibodies from a combinatorial library in one day by repeated fluorescence-activated cell sorting (FACS). First, we constructed a library of synthetic human antibody in which single-chain variable fragment (scFv) was expressed in the periplasm of Escherichia coli. After labeling the cells with fluorescent antigen probes, the highly fluorescent cells were sorted by using a high-speed cell sorter, and these cells were reused without regeneration in the next round of sorting. After repeating this sorting, the positive clones were completely enriched in several hours. Thus, we screened the library against three viral antigens, including the H1N1 influenza virus, Hepatitis B virus, and Foot-and-mouth disease virus. Finally, the potential antibody candidates, which show K(D) values between 10 and 100 nM against the target antigens, could be successfully isolated even though the library was relatively small (∼ 10(6)). These results show that repeated FACS screening without regeneration of the sorted cells can be a powerful method when a rapid response to a spreading disease is required.

  13. Rainbow trout surviving infections of viral haemorrhagic septicemia virus (VHSV) show lasting antibodies to recombinant G protein fragments.

    PubMed

    Encinas, P; Gomez-Casado, E; Fregeneda-Grandes; Olesen, N J; Lorenzen, N; Estepa, A; Coll, J M

    2011-03-01

    Rainbow trout antibodies (Abs) binding to recombinant fragments (frgs) derived from the protein G of the viral haemorrhagic septicemia virus (VHSV)-07.71 strain, could be detected by ELISA (frg-ELISA) in sera from trout surviving laboratory-controlled infections. Abs were detected not only by using sera from trout infected with the homologous VHSV isolate but also with the VHSV-DK-201433 heterologous isolate, which had 13 amino acid changes. Sera from healthy trout and/or from trout surviving infectious haematopoietic necrosis virus (IHNV) infection, were used to calculate cut-off absorbances to differentiate negative from positive sera. Specific anti-VHSV Abs could then be detected by using any of the following frgs: frg11 (56-110), frg15 (65-250), frg16 (252-450) or G21-465. While high correlations were found among the ELISA values obtained with the different frgs, no correlations between any frg-ELISA and complement-dependent 50% plaque neutralization test (PNT) titres could be demonstrated. Between 4 and 10 weeks after VHSV infection, more trout sera were detected as positives by using heterologous frg-ELISA rather than homologous PNT. Furthermore, the percentage of positive sera detected by frg11-ELISA increased with time after infection to reach 100%, while those detected by complement-dependent PNT decreased to 29.4%, thus confirming that the lack of neutralizing Abs does not mean the lack of any anti-VHSV Abs in survivor trout sera. Preliminary results with sera from field samples suggest that further refinements of the frg-ELISA could allow detection of anti-VHSV trout Abs in natural outbreaks caused by different heterologous VHSV isolates. The homologous frg-ELISA method could be useful to follow G immunization attempts during vaccine development and/or to best understand the fish Ab response during VHSV infections. The viral frgs approach might also be used with other fish species and/or viruses.

  14. First successful curative use of digoxin-specific Fab antibody fragments in a life-threatening coconut crab (Birgus latro L.) poisoning.

    PubMed

    Maillaud, C; Barguil, Y; Mikulski, M; Cheze, M; Pivert, C; Deveaux, M; Lapostolle, F

    2012-11-01

    We wish to report the first curative use of digoxin-specific Fab antibody fragments in a coconut crab Birgus latro L. poisoning in New Caledonia. The female patient, aged sixty-three with a previous history of cardiovascular and metabolic dysfunctions, showed marked first-degree atrio-ventricular block and several atrial pauses, and was given 760 mg of digoxin-specific Fab antibody fragments. Shortly after the perfusion her electrocardiogram returned to close to normal with only slight first-degree atrio-ventricular block and no more atrial pauses. Neriifolin LC-MS/MS tests performed on the patient's serum and urine samples confirmed cardenolide poisoning. Another, younger patient, with high neriifolin levels in her serum and urine samples only experienced gastro-intestinal symptoms and was discharged without specific treatment. The consumption of coconut crab in New Caledonia should be avoided even though the first of the two cases reported suggests that digoxin-specific Fab antibody fragments can be effective in the treatment of life-threatening poisoning caused by the ingestion of this crustacean.

  15. A fusogenic dengue virus-derived peptide enhances antitumor efficacy of an antibody-ribonuclease fusion protein targeting the EGF receptor.

    PubMed

    Kiesgen, Stefan; Liebers, Nora; Cremer, Martin; Arnold, Ulrich; Weber, Tobias; Keller, Armin; Herold-Mende, Christel; Dyckhoff, Gerhard; Jäger, Dirk; Kontermann, Roland E; Arndt, Michaela A E; Krauss, Jürgen

    2014-10-01

    Due to its frequent overexpression in a variety of solid tumors the epidermal growth factor receptor (EGFR) is a well-established target for therapeutic interventions in epithelial cancers. In order to target EGFR in head and neck cancer, we have generated a ribonuclease (RNase) fusion protein comprising a humanized anti-EGFR antibody single-chain Fv fragment (scFv) and Ranpirnase, an RNase from Rana pipiens. Fusion of Ranpirnase to the N-terminus of the scFv via a flexible glycine-serine linker (G4S)3 resulted in very poor cytotoxicity of the fusion protein. As endosomal accumulation and lysosomal degradation have been reported to diminish the antitumor efficacy of ribonuclease or toxin-based immunoagents, we explored a fusion peptide from dengue virus that has been reported to be involved in the endosomal escape of the virus. This peptide was introduced as a linker between Ranpirnase and the scFv moiety. The modified immunoRNase exhibited exceptionally high cytotoxicity toward EGFR-expressing head and neck cell lines without affecting specificity. These results indicate that endosomal entrapment needs to be considered for Ranpirnase-based immunoagents and might be overcome by the use of tailored transduction domains from viral proteins.

  16. A stable cytosolic expression of VH antibody fragment directed against PVY NIa protein in transgenic potato plant confers partial protection against the virus.

    PubMed

    Bouaziz, Donia; Ayadi, Malika; Bidani, Amira; Rouis, Souad; Nouri-Ellouz, Oumèma; Jellouli, Raïda; Drira, Noureddine; Gargouri-Bouzid, Radhia

    2009-04-01

    The expression of recombinant antibodies in transgenic plants has been proved to be an efficient approach for large-scale production. However, the stability of these molecules and their accumulation level depend on their molecular properties and cellular targeting. The expression of single-domain antibody fragment (VH) can be advantageous since it offers small length, high expression, solubility and stability. It can therefore be preferred to other antibody derivatives avoiding the expression difficulties related to immunoglobulin domain folding via the formation of disulfide bridge. This report describes the production of transgenic potato plants expressing a VH antibody directed against the NIa protease of potato virus Y. The antibody was driven by the constitutive CaMV 35S RNA promoter. The expression cassette was transferred into potato plants via Agrobacterium tumefaciens mediated transformation. All transgenic lines showed detectable levels of VH protein confirming the efficient translation and stability of this protein. The cellular localisation of the VH antibody was investigated. Transgenic and control plants were transferred in the greenhouse and mechanically inoculated by PVY(o) suspension. Some of the transgenic lines showed delayed symptoms at the first period post inoculation and then displayed a recovery phenomenon while the virions were still detected in the leaves.

  17. Efficient heterologous expression and secretion in Aspergillus oryzae of a llama variable heavy-chain antibody fragment V(HH) against EGFR.

    PubMed

    Okazaki, Fumiyoshi; Aoki, Jun-ichi; Tabuchi, Soichiro; Tanaka, Tsutomu; Ogino, Chiaki; Kondo, Akihiko

    2012-10-01

    We have constructed a filamentous fungus Aspergillus oryzae that secretes a llama variable heavy-chain antibody fragment (V(HH)) that binds specifically to epidermal growth factor receptor (EGFR) in a culture medium. A major improvement in yield was achieved by fusing the V(HH) with a Taka-amylase A signal sequence (sTAA) and a segment of 28 amino acids from the N-terminal region of Rhizopus oryzae lipase (N28). The yields of secreted, immunologically active anti-EGFR V(HH) reached 73.8 mg/1 in a Sakaguchi flask. The V(HH) fragments were released from the sTAA or N28 proteins by an indigenous A. oryzae protease during cultivation. The purified recombinant V(HH) fragment was specifically recognized and could bind to the EGFR with a high affinity.

  18. Effect of linker length between variable domains of single chain variable fragment antibody against daidzin on its reactivity.

    PubMed

    Yusakul, Gorawit; Sakamoto, Seiichi; Pongkitwitoon, Benyakan; Tanaka, Hiroyuki; Morimoto, Satoshi

    2016-07-01

    The peptide linker between variable domains of heavy (VH) and light (VL) chains is one of important factors that influence the characteristics of scFv, including binding activity and specificity against target antigen. The scFvs against daidzin (DZ-scFvs) with different linker lengths were constructed in the format of VH-(GGGGS)n-VL (n = 1, 3, 5, and 7). They were expressed in the hemolymph of silkworm larvae using the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system, and their reactivity against daidzin and related compounds were evaluated using an indirect competitive enzyme-linked immunosorbent assay (icELISA), which is applicable for quantitative analysis of daidzin. The results showed that the reactivity of scFvs against daidzin was increased, whereas specificity slightly decreased when their peptide linker was lengthened. These results suggested that the linker length of DZ-scFvs contributes to its reactivity. In addition, the results emphasize that the linker length could control the reactivity of DZ-scFvs.

  19. [Targeted Delivery of Quantum Dots to HER2-Expressing Tumor Using Recombinant Antibodies].

    PubMed

    Balalaeva, I V; Zdobnova, T A; Sokolova, E A; Deyev, S M

    2015-01-01

    Targeted delivery of semiconductor quantum dots (Q Ds) to tumors overexpressing HER2 cancer marker has been. demonstrated on immunocompromised mice bearing human breast cancer xenografts. To obtain targeted QDs complexes we applied the approach based on the use of protein adaptor system, RNAase barnase and its inhibitor barstar. Specific binding to target cancer marker was achieved through bivalent fusion protein containing two fragments of4D5scFv recombinant antibody and a fragment of barnase. QDs were conjugated to barstar, and final assembly of targeted complexes was obtained through non-covalent specific interaction of barstar, attached to QD, and barnase, that is part of the recombinant targeting protein. The efficient delivery of QDs to HER2-expressing tumor demonstrates the possibilities and prospects of the approach for targeted delivery of nanoparticles to cancer cells in vivo as the way to improve the efficiency of diagnosis and promote development of therapies based on the use of nanoparticles.

  20. Targeting cancer cells by using an antireceptor antibody-photosensitizer fusion protein

    PubMed Central

    Serebrovskaya, Ekaterina O.; Edelweiss, Eveline F.; Stremovskiy, Oleg A.; Lukyanov, Konstantin A.; Chudakov, Dmitry M.; Deyev, Sergey M.

    2009-01-01

    Antibody-photosensitizer chemical conjugates are used successfully to kill cancer cells in photodynamic therapy. However, chemical conjugation of photosensitizers presents several limitations, such as poor reproducibility, aggregation, and free photosensitizer impurities. Here, we report a fully genetically encoded immunophotosensitizer, consisting of a specific anti-p185HER-2-ECD antibody fragment 4D5scFv fused with the phototoxic fluorescent protein KillerRed. Both parts of the recombinant protein preserved their functional properties: high affinity to antigen and light activation of sensitizer. 4D5scFv-KillerRed showed fine targeting properties and efficiently killed p185HER-2-ECD-expressing cancer cells upon light irradiation. It also showed a remarkable additive effect with the commonly used antitumor agent cisplatin, further demonstrating the potential of the approach. PMID:19458251

  1. Whole-body imaging of HER2/neu-overexpressing tumors using scFv-antibody conjugated quantum dots

    NASA Astrophysics Data System (ADS)

    Balalaeva, Irina V.; Zdobnova, Tatiana A.; Brilkina, Anna A.; Krutova, Irina M.; Stremovskiy, Oleg A.; Lebedenko, Elena N.; Vodeneev, Vladimir V.; Turchin, Ilya V.; Deyev, Sergey M.

    2010-02-01

    Semiconductor quantum dots (QDs) are widely used in different fields of bioscience and biotechnology due to their unique optical properties. QDs can be used as fluorescent markers for optical detection and monitoring of deeply located tumors in vivo after specific labeling achieved by conjugating of QDs with targeting molecules. In this work the possibilities of intravital tumor labeling with QDs and subsequent in vivo tumor imaging were estimated. The experiments were run on immunodeficient nu/nu mice bearing human breast carcinoma SKBR-3, overexpressing surface protein HER2/neu. We used quantum dots Qdot 705 ITK (Invitrogen, USA) linked to anti-HER2/neu 4D5 scFv antibody. Antibody scFv fragments as a targeting agent for directed delivery of fluorophores possess significant advantages over full-size antibodies due to their small size, lower cross-reactivity and immunogenicity. QDs were bound to 4D5 scFv by barnase-barstar system (bn-bst) analogous to the streptavidin-biotidin system. Whole-body images were obtained using diffuse fluorescence tomography (DFT) setup with low-frequency modulation and transilluminative configuration of scanning, created at the Institute of Applied Physics of RAS, Russia). DFT-results were confirmed ex vivo by confocal microscopy. We report the results of in vivo whole-body tumor imaging with QDs complexes as contrasting agents. Intravital images of QDs-labeled tumors were obtained using specific tumor cells targeting and fluorescence transilluminative imaging method, while "passive" QD-labeling failed to mark effectively the tumor.

  2. Selection of single chain variable fragments specific for the human-inducible costimulator using ribosome display.

    PubMed

    Pan, Yangbin; Mao, Weiping; Liu, Xuanxuan; Xu, Chong; He, Zhijuan; Wang, Wenqian; Yan, Hao

    2012-11-01

    We applied a ribosome display technique to a mouse single chain variable fragment (scFv) library to select scFvs specific for the inducible costimulator (ICOS). mRNA was isolated from the spleens of BALB/c mice immunized with ICOS protein. Heavy and κ chain genes (VH and κ) were amplified separately by reverse transcriptase polymerase chain reaction, and the anti-ICOS VH/κ chain ribosome display library was constructed with a special flexible linker by overlap extension PCR. The VH/κ chain library was transcribed and translated in vitro using a rabbit reticulocyte lysate system. Then, antibody-ribosome-mRNA complexes were produced and panned against ICOS protein under appropriate conditions. However, in order to isolate specific scFvs for ICOS, negative selection using CD28 was carried out before three rounds of positive selection on ICOS. After three rounds of panning, the selected scFv DNAs were cloned into pET43.1a and detected by SDS-PAGE. Then, enzyme-linked immunosorbent assay showed that we successfully constructed a native ribosome display library, and among seven clones, clone 5 had the highest affinity for the ICOS and low for the CD28. Anti-ICOS scFvs are assessed for binding specificity and affinity and may provide the potential for development of the humanized and acute and chronic allograft rejection.

  3. Reducing heterophilic antibody interference in immunoassays using single chain antibodies

    SciTech Connect

    Baird, Cheryl L.; Tan, Ruimin; Fischer, Christopher J.; Victry, Kristin D.; Zangar, Richard C.; Rodland, Karin D.

    2011-12-15

    Sandwich ELISA microarrays have the potential to simultaneously quantify the levels of multiple diagnostic targets in a biological sample. However, as seen with traditional ELISA diagnostics, heterophilic antibodies (HA) in patient sera have the potential to cause interference in these assays. We demonstrate here that reducing the diagnostic capture antibody to its minimal functional unit, the variable heavy and light domains artificially connected with a short polypeptide linker (scFv), is an effective strategy for reducing the HA assay interference.

  4. ImmunoPET and Near-Infrared Fluorescence Imaging of Pancreatic Cancer with a Dual-Labeled Bispecific Antibody Fragment.

    PubMed

    Luo, Haiming; England, Christopher G; Goel, Shreya; Graves, Stephen A; Ai, Fanrong; Liu, Bai; Theuer, Charles P; Wong, Hing C; Nickles, Robert J; Cai, Weibo

    2017-03-24

    Dual-targeted imaging agents have shown improved targeting efficiencies in comparison to single-targeted entities. The purpose of this study was to quantitatively assess the tumor accumulation of a dual-labeled heterobifunctional imaging agent, targeting two overexpressed biomarkers in pancreatic cancer, using positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging modalities. A bispecific immunoconjugate (heterodimer) of CD105 and tissue factor (TF) Fab' antibody fragments was developed using click chemistry. The heterodimer was dual-labeled with a radionuclide ((64)Cu) and fluorescent dye. PET/NIRF imaging and biodistribution studies were performed in four-to-five week old nude athymic mice bearing BxPC-3 (CD105/TF(+/+)) or PANC-1 (CD105/TF(-/-)) tumor xenografts. A blocking study was conducted to investigate the specificity of the tracer. Ex vivo tissue staining was performed to compare TF/CD105 expression in tissues with PET tracer uptake to validate in vivo results. PET imaging of (64)Cu-NOTA-heterodimer-ZW800 in BxPC-3 tumor xenografts revealed enhanced tumor uptake (21.0 ± 3.4%ID/g; n = 4) compared to the homodimer of TRC-105 (9.6 ± 2.0%ID/g; n = 4; p < 0.01) and ALT-836 (7.6 ± 3.7%ID/g; n = 4; p < 0.01) at 24 h postinjection. Blocking studies revealed that tracer uptake in BxPC-3 tumors could be decreased by 4-fold with TF blocking and 2-fold with CD105 blocking. In the negative model (PANC-1), heterodimer uptake was significantly lower than that found in the BxPC-3 model (3.5 ± 1.1%ID/g; n = 4; p < 0.01). The specificity was confirmed by the successful blocking of CD105 or TF, which demonstrated that the dual targeting with (64)Cu-NOTA-heterodimer-ZW800 provided an improvement in overall tumor accumulation. Also, fluorescence imaging validated the PET imaging, allowing for clear delineation of the xenograft tumors. Dual-labeled heterodimeric imaging agents, like (64)Cu-NOTA-heterodimer-ZW800, may increase the overall tumor

  5. Deep sequencing of phage display libraries to support antibody discovery.

    PubMed

    Ravn, Ulla; Didelot, Gérard; Venet, Sophie; Ng, Kwok-Ting; Gueneau, Franck; Rousseau, François; Calloud, Sébastien; Kosco-Vilbois, Marie; Fischer, Nicolas

    2013-03-15

    The use of next generation sequencing (NGS) for the analysis of antibody sequences both in phage display libraries and during in vitro selection processes has become increasingly popular in the last few years. Here, our methods developed for DNA preparation, sequencing and data analysis are presented. A key parameter has also been to develop new software designed for high throughput antibody sequence analysis that is used in combination with publicly available tools. As an example of our methods, we provide data from the extensive analysis of five scFv libraries generated using different heavy chain CDR3 diversification strategies. The results not only confirm that the library designs were correct but also reveal differences in quality not easily identified by standard DNA sequencing approaches. The very large number of reads permits extensive sequence coverage after the selection process. Furthermore, as samples can be multiplexed, costs decrease and more information is gained per NGS run. Using examples of results obtained post phage display selections against two antigens, frequency and clustering analysis identified novel antibody fragments that were then shown to be specific for the target antigen. In summary, the methods described here demonstrate how NGS analysis enhances quality control of complex antibody libraries as well as facilitates the antibody discovery process.

  6. Single Chain Variable Fragments Produced in Escherichia coli against Heat-Labile and Heat-Stable Toxins from Enterotoxigenic E. coli

    PubMed Central

    Andrade, Fernanda B.; Nepomuceno, Roberto; Silva, Anderson; Munhoz, Danielle D.; Yamamoto, Bruno B.; Luz, Daniela; Abreu, Patrícia A. E.; Horton, Denise S. P. Q.; Elias, Waldir P.; Ramos, Oscar H. P.; Piazza, Roxane M. F.

    2015-01-01

    Background Diarrhea is a prevalent pathological condition frequently associated to the colonization of the small intestine by enterotoxigenic Escherichia coli (ETEC) strains, known to be endemic in developing countries. These strains can produce two enterotoxins associated with the manifestation of clinical symptoms that can be used to detect these pathogens. Although several detection tests have been developed, minimally equipped laboratories are still in need of simple and cost-effective methods. With the aim to contribute to the development of such diagnostic approaches, we describe here two mouse hybridoma-derived single chain fragment variable (scFv) that were produced in E. coli against enterotoxins of ETEC strains. Methods and Findings Recombinant scFv were developed against ETEC heat-labile toxin (LT) and heat-stable toxin (ST), from previously isolated hybridoma clones. This work reports their design, construction, molecular and functional characterization against LT and ST toxins. Both antibody fragments were able to recognize the cell-interacting toxins by immunofluorescence, the purified toxins by ELISA and also LT-, ST- and LT/ST-producing ETEC strains. Conclusion The developed recombinant scFvs against LT and ST constitute promising starting point for simple and cost-effective ETEC diagnosis. PMID:26154103

  7. Single chain Fv fragment specific for human GM-CSF: selection and expression using a bacterial expression library.

    PubMed

    Tapryal, Suman; Pal Khasa, Yogender; Mukherjee, K J

    2010-10-01

    Single chain antibodies (scFvs) are replacing whole antibody molecules since they are easy to produce on large scale and amenable to genetic modifications. Here we report the development of an anti-human granulocyte macrophage colony-stimulating factor (hGM-CSF) scFv as an immunoassay bio-reagent, utilizing an easily scalable bacterial expression system. For this, the V(H) and V(L) gene repertoires were amplified from the immunoglobulin complementary DNA, derived from total RNA of mice splenocytes, pre-sensitized with the antigen. The scFv library was expressed under the strong T7 promoter in BL21 (DE3) Escherichia coli cells. Preliminary screening led to the selection of four potential candidates, which were later subjected to light chain shuffling. Cross-reactivity analysis involving the original and shuffled candidates resulted in the selection of one scFv (scFv196) with no cross-reactivity against E. coli antigens. The binding affinity of the scFv196 for hGM-CSF, measured by surface plasmon resonance, was found to be within the physiological range (K(D) =1.5 μM). The refolded scFv was also shown to recognize and bind the glycosylated antigen, a closer mimic of the physiological GM-CSF, potentiating its use in immunoassays. Expression studies using shake flasks suggested periplasmic export of the scFv196 protein.

  8. Crystallization and preliminary X-ray diffraction analysis of the Fab fragment of WO2, an antibody specific for the Aβ peptides associated with Alzheimer’s disease

    SciTech Connect

    Wun, Kwok S.; Miles, Luke A.; Crespi, Gabriela A. N.; Wycherley, Kaye; Ascher, David B.; Barnham, Kevin J.; Cappai, Roberto; Beyreuther, Konrad; Masters, Colin L.; Parker, Michael W.; McKinstry, William J.

    2008-05-01

    Crystallization and X-ray diffraction data collection of the Fab fragment of the monoclonal antibody WO2 in the absence or presence of amyloid β peptides associated with Alzheimer’s disease are reported. The murine monoclonal antibody WO2 specifically binds the N-terminal region of the amyloid β peptide (Aβ) associated with Alzheimer’s disease. This region of Aβ has been shown to be the immunodominant B-cell epitope of the peptide and hence is considered to be a basis for the development of immunotherapeutic strategies against this prevalent cause of dementia. Structural studies have been undertaken in order to characterize the molecular basis for antibody recognition of this important epitope. Here, details of the crystallization and X-ray analysis of the Fab fragment of the unliganded WO2 antibody in two crystal forms and of the complexes that it forms with the truncated Aβ peptides Aβ{sub 1–16} and Aβ{sub 1–28} are presented. These crystals were all obtained using the hanging-drop vapour-diffusion method at 295 K. Crystals of WO2 Fab were grown in polyethylene glycol solutions containing ZnSO{sub 4}; they belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1} and diffracted to 1.6 Å resolution. The complexes of WO2 Fab with either Aβ{sub 1–@}@{sub 16} or Aβ{sub 1–28} were cocrystallized from polyethylene glycol solutions. These two complex crystals grew in the same space group, P2{sub 1}2{sub 1}2{sub 1}, and diffracted to 1.6 Å resolution. A second crystal form of WO2 Fab was grown in the presence of the sparingly soluble Aβ{sub 1–42} in PEG 550 MME. This second form belonged to space group P2{sub 1} and diffracted to 1.9 Å resolution.

  9. Generation of human single-chain variable fragment antibodies specific to dengue virus non-structural protein 1 that interfere with the virus infectious cycle.

    PubMed

    Poungpair, Ornnuthchar; Bangphoomi, Kunan; Chaowalit, Prapaipit; Sawasdee, Nunghathai; Saokaew, Nichapatr; Choowongkomon, Kiattawee; Chaicumpa, Wanpen; Yenchitsomanus, Pa-thai

    2014-01-01

    Severe forms of dengue virus (DENV) infection frequently cause high case fatality rate. Currently, there is no effective vaccine against the infection. Clinical cases are given only palliative treatment as specific anti-DENV immunotherapy is not available and it is urgently required. In this study, human single-chain variable fragment (HuScFv) antibodies that bound specifically to the conserved non-structural protein-1 (NS1) of DENV and interfered with the virus replication cycle were produced by using phage display technology. Recombinant NS1 (rNS1) of DENV serotype 2 (DENV2) was used as antigen in phage bio-panning to select phage clones that displayed HuScFv from antibody phage display library. HuScFv from two phagemid transformed E. coli clones, i.e., clones 11 and 13, bound to the rNS1 as well as native NS1 in both secreted and intracellular forms. Culture fluids of the HuScFv11/HuScFv13 exposed DENV2 infected cells had significant reduction of the infectious viral particles, implying that the antibody fragments affected the virus morphogenesis or release. HuScFv epitope mapping by phage mimotope searching revealed that HuScFv11 bound to amino acids 1-14 of NS1, while the HuScFv13 bound to conformational epitope at the C-terminal portion of the NS1. Although the functions of the epitopes and the molecular mechanism of the HuScFv11 and HuScFv13 require further investigations, these small antibodies have high potential for development as anti-DENV biomolecules.

  10. An integrated top-down and bottom-up proteomic approach to characterize the antigen binding fragment of antibodies

    SciTech Connect

    Dekker, Leendert J.; Wu, Si; vanDuijn, Martijn M.; Tolic, Nikola; Stingl, Christoph; Zhao, Rui; Luider, Theo N.; Pasa-Tolic, Ljiljana

    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 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 towards the identification of disease specific antibodies in patient samples with potentially significant clinical impact.

  11. Single domain antibodies for the knockdown of cytosolic and nuclear proteins.

    PubMed

    Böldicke, Thomas

    2017-03-08

    Single domain antibodies (sdAbs) from camels or sharks comprise only the variable heavy chain domain. Human sdAbs comprise the variable domain of the heavy chain (VH) or light chain (VL) and can be selected from human antibodies. SdAbs are stable, non aggregating molecules in vitro and in vivo compared to complete antibodies and scFv fragments. They are excellent novel inhibitors of cytosolic/nuclear proteins because they are correctly folded inside the cytosol in contrast to scFv fragments. SdAbs are unique because of their excellent specificity and possibility to target posttranslational modifications such as phosphorylation sites, conformers or interaction regions of proteins that cannot be targeted with genetic knockout techniques and are impossible to knockdown with RNAi. The number of inhibiting cytosolic/nuclear sdAbs is increasing and usage of synthetic single pot single domain libraries will boost the generation of these fascinating molecules without the need of immunization. The most frequently selected antigenic epitopes belong to viral and oncogenic proteins, followed by toxins, proteins of the nervous system as well as plant- and drosophila proteins. It is now possible to select functional sdAbs against virtually every cytosolic/nuclear protein and desired epitope. The development of new endosomal escape protein domains and cell-penetrating peptides for efficient transfection broaden the application of inhibiting sdAbs. Last but not least, the generation of relatively new cell-specific nanoparticles such as polymersomes and polyplexes carrying cytosolic/nuclear sdAb-DNA or -protein will pave the way to apply cytosolic/nuclear sdAbs for inhibition of viral infection and cancer in the clinic. This article is protected by copyright. All rights reserved.

  12. Genetic fusion of single-chain variable fragments to partial spider silk improves target detection in micro- and nanoarrays.

    PubMed

    Thatikonda, Naresh; Delfani, Payam; Jansson, Ronnie; Petersson, Linn; Lindberg, Diana; Wingren, Christer; Hedhammar, My

    2016-03-01

    Immobilizing biomolecules with retained functionality and stability on solid supports is crucial for generation of sensitive immunoassays. However, upon use of conventional immobilization strategies, a major portion of the biomolecules (e.g. antibodies) frequently tends to lose their bioactivity. In this study, we describe a procedure to immobilize human single-chain variable fragment (scFv) via genetic fusion to partial spider silk, which have a high tendency to adhere to solid supports. Two scFvs, directed towards serum proteins, were genetically fused to partial spider silk proteins and expressed as silk fusion proteins in E. coli. Antigen binding ability of scFvs attached to a partial silk protein denoted RC was investigated using microarray analysis, whereas scFvs fused to the NC silk variant were examined using nanoarrays. Results from micro- and nanoarrays confirmed the functionality of scFvs attached to both RC and NC silk, and also for binding of targets in crude serum. Furthermore, the same amount of added scFv gives higher signal intensity when immobilized via partial spider silk compared to when immobilized alone. Together, the results suggest that usage of scFv-silk fusion proteins in immunoassays could improve target detection, in the long run enabling novel biomarkers to be detected in crude serum proteomes.

  13. An Immunosensor Based on Antibody Binding Fragments Attached to Gold Nanoparticles for the Detection of Peptides Derived from Avian Influenza Hemagglutinin H5

    PubMed Central

    Jarocka, Urszula; Sawicka, Róża; Góra-Sochacka, Anna; Sirko, Agnieszka; Zagórski-Ostoja, Włodzimierz; Radecki, Jerzy; Radecka, Hanna

    2014-01-01

    This paper concerns the development of an immunosensor for detection of peptides derived from avian influenza hemagglutinin H5. Its preparation consists of successive gold electrode modification steps: (i) modification with 1,6-hexanedithiol and gold colloidal nanoparticles; (ii) immobilization of antibody-binding fragments (Fab') of anti-hemagglutinin H5 monoclonal antibodies Mab 6-9-1 via S-Au covalent bonds; and (iii) covering the remaining free space on the electrode surfaces with bovine serum albumin. The interactions between Fab' fragments and hemagglutinin (HA) variants have been explored with electrochemical impedance spectroscopy (EIS) in the presence of [Fe(CN)6]3−/4− as an electroactive marker. The immunosensor was able to recognize three different His-tagged variants of recombinant hemagglutinin from H5N1 viruses: H1 subunit (17–340 residues) of A/swan/Poland/305-135V08/2006, the long HA (17–530 residues) A/Bar-headed Goose/Qinghai/12/2005 and H1 subunit (1–345 residues) of A/Vietnam/1194/2004. The strongest response has been observed for the long variant with detection limit of 2.2 pg/mL and dynamic range from 4.0 to 20.0 pg/mL. PMID:25157550

  14. Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354

    SciTech Connect

    Kaufmann, Bärbel; Vogt, Matthew R.; Goudsmit, Jaap; Holdaway, Heather A.; Aksyuk, Anastasia A.; Chipman, Paul R.; Kuhn, Richard J.; Diamond, Michael S.; Rossmann, Michael G.

    2010-11-15

    Many flaviviruses are significant human pathogens, with the humoral immune response playing an essential role in restricting infection and disease. CR4354, a human monoclonal antibody isolated from a patient, neutralizes West Nile virus (WNV) infection at a postattachment stage in the viral life-cycle. Here, we determined the structure of WNV complexed with Fab fragments of CR4354 using cryoelectron microscopy. The outer glycoprotein shell of a mature WNV particle is formed by 30 rafts of three homodimers of the viral surface protein E. CR4354 binds to a discontinuous epitope formed by protein segments from two neighboring E molecules, but does not cause any detectable structural disturbance on the viral surface. The epitope occurs at two independent positions within an icosahedral asymmetric unit, resulting in 120 binding sites on the viral surface. The cross-linking of the six E monomers within one raft by four CR4354 Fab fragments suggests that the antibody neutralizes WNV by blocking the pH-induced rearrangement of the E protein required for virus fusion with the endosomal membrane.

  15. Positron emission tomographic imaging of iodine 124 anti-prostate stem cell antigen-engineered antibody fragments in LAPC-9 tumor-bearing severe combined immunodeficiency mice.

    PubMed

    Fonge, Humphrey; Leyton, Jeffrey V

    2013-05-01

    The humanized antibody (hu1G8) has been shown to localize to prostate stem cell antigen (PSCA) and image PSCA-positive xenografts. We previously constructed hu1G8 anti-PSCA antibody fragments and tested them for tumor targeting and the ability to image prostate cancer at early and late time points postinjection by positron emission tomography (PET). We now then compare the PET imaging and the radioactivity accumulation properties in prostate cancer tumors and nontarget tissues to determine the superior 124I-labeled hu1G8 antibody format. 124I-labeled diabody, minibody, scFv-Fc, scFv-Fc double mutant (DM), and parental IgG were administered into severe combined immunodeficiency (SCID) mice bearing LAPC-9 xenografts and followed by whole-body PET imaging of mice at preselected time points. Regions of interest were manually drawn around tumor and nontarget tissues and evaluated for radioactivity accumulation. The 124I-hu1G8 IgG has its best time point for tumor high-contrast imaging at 168 hours postinjection. The 124I-hu1G8 minibody at 44 hours postinjection results in superior tumor high-contrast imaging compared to the other antibody formats. The 124I-hu1G8 minibody at 44 hours postinjection also has comparable percent tumor radioactivity compared to 124I-hu1G8 IgG at 168 hours postinjection. The 124I-hu1G8 minibody is the best engineered hu1G8 antibody format for imaging prostate cancer.

  16. Bacterial production and structure-functional validation of a recombinant antigen-binding fragment (Fab) of an anti-cancer therapeutic antibody targeting epidermal growth factor receptor.

    PubMed

    Kim, Ji-Hun; Sim, Dae-Won; Park, Dongsun; Jung, Tai-Geun; Lee, Seonghwan; Oh, Taeheun; Ha, Jong-Ryul; Seok, Seung-Hyeon; Seo, Min-Duk; Kang, Ho Chul; Kim, Young Pil; Won, Hyung-Sik

    2016-12-01

    Fragment engineering of monoclonal antibodies (mAbs) has emerged as an excellent paradigm to develop highly efficient therapeutic and/or diagnostic agents. Engineered mAb fragments can be economically produced in bacterial systems using recombinant DNA technologies. In this work, we established recombinant production in Escherichia coli for monovalent antigen-binding fragment (Fab) adopted from a clinically used anticancer mAB drug cetuximab targeting epidermal growth factor receptor (EGFR). Recombinant DNA constructs were designed to express both polypeptide chains comprising Fab in a single vector and to secrete them to bacterial periplasmic space for efficient folding. Particularly, a C-terminal engineering to confer an interchain disulfide bond appeared to be able to enhance its heterodimeric integrity and EGFR-binding activity. Conformational relevance of the purified final product was validated by mass spectrometry and crystal structure at 1.9 Å resolution. Finally, our recombinant cetuximab-Fab was found to have strong binding affinity to EGFR overexpressed in human squamous carcinoma model (A431) cells. Its binding ability was comparable to that of cetuximab. Its EGFR-binding affinity was estimated at approximately 0.7 nM of Kd in vitro, which was quite stronger than the binding affinity of natural ligand EGF. Hence, the results validate that our construction could serve as an efficient platform to produce a recombinant cetuximab-Fab with a retained antigen-binding functionality.

  17. Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells.

    PubMed

    Kaiser, Philipp D; Maier, Julia; Traenkle, Bjoern; Emele, Felix; Rothbauer, Ulrich

    2014-11-01

    In biomedical research there is an ongoing demand for new technologies, which help to elucidate disease mechanisms and provide the basis to develop novel therapeutics. In this context a comprehensive understanding of cellular processes and their pathophysiology based on reliable information on abundance, localization, posttranslational modifications and dynamic interactions of cellular components is indispensable. Besides their significant impact as therapeutic molecules, antibodies are arguably the most powerful research tools to study endogenous proteins and other cellular components. However, for cellular diagnostics their use is restricted to endpoint assays using fixed and permeabilized cells. Alternatively, live cell imaging using fluorescent protein-tagged reporters is widely used to study protein localization and dynamics in living cells. However, only artificially introduced chimeric proteins are visualized, whereas the endogenous proteins, their posttranslational modifications as well as non-protein components of the cell remain invisible and cannot be analyzed. To overcome these limitations, traceable intracellular binding molecules provide new opportunities to perform cellular diagnostics in real time. In this review we summarize recent progress in the generation of intracellular and cell penetrating antibodies and their application to target and trace cellular components in living cells. We highlight recent advances in the structural formulation of recombinant antibody formats, reliable screening protocols and sophisticated cellular targeting technologies and propose that such intrabodies will become versatile research tools for real time cell-based diagnostics including target validation and live cell imaging. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.

  18. Engineered antibody therapies to counteract mutant huntingtin and related toxic intracellular proteins

    PubMed Central

    Butler, David C.; McLear, Julie A.; Messer, Anne

    2013-01-01

    The engineered antibody approach to Huntington's disease (HD) therapeutics is based on the premise that significantly lowering the levels of the primary misfolded mutant protein will reduce abnormal protein interactions and direct toxic effects of the misfolded huntingtin (HTT). This will in turn reduce the pathologic stress on cells, and normalize intrinsic proteostasis. Intracellular antibodies (intrabodies) are single-chain (scFv) and single-domain (dAb; nanobody) variable fragments that can retain the affinity and specificity of full-length antibodies, but can be selected and engineered as genes. Functionally, they represent a protein-based approach to the problem of aberrant mutant protein folding, post-translational modifications, protein-protein interactions, and aggregation. Several intrabodies that bind on either side of the expanded polyglutamine tract of mutant HTT have been reported to improve the mutant phenotype in cell and organotypic cultures, fruit flies, and mice. Further refinements to the difficult challenges of intraneuronal delivery, cytoplasmic folding, and long-term efficacy are in progress. This review covers published studies and emerging approaches on the choice of targets, selection and engineering methods, gene and protein delivery options, and testing of candidates in cell and animal models. The resultant antibody fragments can be used as direct therapeutics and as target validation/drug discovery tools for HD, while the technology is also applicable to a wide range of neurodegenerative and other diseases that are triggered by toxic proteins. PMID:22120646

  19. Crystallization of the receptor-binding domain of parathyroid hormone-related protein in complex with a neutralizing monoclonal antibody Fab fragment

    SciTech Connect

    McKinstry, William J.; Polekhina, Galina; Diefenbach-Jagger, Hannelore; Sato, Koh; Onuma, Etsuro; Gillespie, Matthew T.; Martin, Thomas J.; Parker, Michael W.

    2009-04-01

    Parathyroid hormone-related protein (PTHrP) plays an important role in regulating embryonic skeletal development and is abnormally regulated in the pathogenesis of skeletal complications observed with many cancers and osteoporosis. It exerts its action through binding to a G-protein-coupled seven-transmembrane cell-surface receptor (GPCR). Structurally, GPCRs are very difficult to study by X-ray crystallography. In this study, a monoclonal antibody Fab fragment which recognizes the same region of PTHrP as its receptor, PTH1R, was used to aid in the crystallization of PTHrP. The resultant protein complex was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol as a precipitant. The crystals belonged to the orthorhombic space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 72.6, b = 96.3, c = 88.5 {angstrom}, and diffracted to 2.0 {angstrom} resolution using synchrotron radiation. The crystal structure will shed light on the nature of the key residues of PTHrP that interact with the antibody and will provide insights into how the antibody is able to discriminate between PTHrP and the related molecule parathyroid homone.

  20. Crystallization and preliminary X-ray diffraction analysis of the complex between a human anti-alpha toxin antibody fragment and alpha toxin.

    PubMed

    Oganesyan, Vaheh; Barnes, Arnita; Tkaczyk, Christine; Ferguson, Andrew; Wu, Herren; Dall'Acqua, William F

    2013-03-01

    Staphylococcus aureus alpha toxin (AT) has been crystallized in complex with the Fab fragment of a human antibody (MEDI4893). This constitutes the first reported crystals of AT bound to an antibody. The monoclinic crystals belonged to space group P2₁, with unit-cell parameters a=85.52, b=148.50, c=93.82 Å, β=99.82°. The diffraction of the crystals extended to 2.56 Å resolution. The asymmetric unit contained two MEDI4893 Fab-AT complexes. This corresponds to a crystal volume per protein weight (VM) of 2.3 Å3 Da(-1) and a solvent content of 47%. The three-dimensional structure of this complex will contribute to an understanding of the molecular basis of the interaction of MEDI4893 with AT. It will also shed light on the mechanism of action of this antibody, the current evaluation of which in the field of S. aureus-mediated diseases makes it a particularly interesting case study. Finally, this study will provide the three-dimensional structure of AT in a monomeric state for the first time.

  1. Differential analyses of major allergen proteins in wild-type rice and rice producing a fragment of anti-rotavirus antibody.

    PubMed

    Yuki, Yoshikazu; Kurokawa, Shiho; Kozuka-Hata, Hiroko; Tokuhara, Daisuke; Mejima, Mio; Kuroda, Masaharu; Oyama, Masaaki; Nishimaki-Mogami, Tomoko; Teshima, Reiko; Kiyono, Hiroshi

    2016-04-01

    To develop oral antibody therapy against rotavirus infection, we previously produced a recombinant fragment of llama heavy-chain antibody to rotavirus (ARP1) in rice seeds (MucoRice-ARP1). We intend to use a purification-free rice powder for clinical application but needed to check whether MucoRice-ARP1 had increased levels of known allergen proteins. For this purpose, we used two-dimensional fluorescence difference gel electrophoresis to compare the allergen protein levels in MucoRice-ARP1 and wild-type rice. We detected no notable differences, except in the levels of α-amylase/trypsin inhibitor-like family proteins. Because by this approach we could not completely separate ARP1 from the proteins of this family, we confirmed the absence of changes in the levels of these allergens by using shotgun mass spectrometry as well as immunoblot. By using immunoelectron microscopy, we also showed that RAG2, a member of the α-amylase/trypsin inhibitor-like protein family, was relocated from protein bodies II to the plasma membrane or cell wall in MucoRice-ARP1 seed. The relocation did not affect the level of RAG2. We demonstrated that most of the known rice allergens were not considerably upregulated by the genetic modification in MucoRice-ARP1. Our data suggest that MucoRice-ARP1 is a potentially safe oral antibody for clinical application.

  2. Crystallization of the receptor-binding domain of parathyroid hormone-related protein in complex with a neutralizing monoclonal antibody Fab fragment.

    PubMed

    McKinstry, William J; Polekhina, Galina; Diefenbach-Jagger, Hannelore; Sato, Koh; Onuma, Etsuro; Gillespie, Matthew T; Martin, Thomas J; Parker, Michael W

    2009-04-01

    Parathyroid hormone-related protein (PTHrP) plays an important role in regulating embryonic skeletal development and is abnormally regulated in the pathogenesis of skeletal complications observed with many cancers and osteoporosis. It exerts its action through binding to a G-protein-coupled seven-transmembrane cell-surface receptor (GPCR). Structurally, GPCRs are very difficult to study by X-ray crystallography. In this study, a monoclonal antibody Fab fragment which recognizes the same region of PTHrP as its receptor, PTH1R, was used to aid in the crystallization of PTHrP. The resultant protein complex was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol as a precipitant. The crystals belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 72.6, b = 96.3, c = 88.5 A, and diffracted to 2.0 A resolution using synchrotron radiation. The crystal structure will shed light on the nature of the key residues of PTHrP that interact with the antibody and will provide insights into how the antibody is able to discriminate between PTHrP and the related molecule parathyroid homone.

  3. 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.

  4. Tumour targeting of humanised cross-linked divalent-fab′ antibody fragments: a clinical phase I/II study

    PubMed Central

    Casey, J L; Napier, M P; King, D J; Pedley, R B; Chaplin, L C; Weir, N; Skelton, L; Green, A J; Hope-Stone, L D; Yarranton, G T; Begent, R H J

    2002-01-01

    Antibody engineering has made it possible to design antibodies with optimal characteristics for delivery of radionuclides for tumour imaging and therapy. A humanised divalent-Fab′ cross-linked with a bis-maleimide linker referred to as humanised divalent-Fab′ maleimide was produced as a result of this design process. It is a humanised divalent antibody with no Fc, which can be produced in bacteria and has enhanced stability compared with F(ab′)2. Here we describe a clinical study in patients with colorectal cancer using humanised divalent-Fab′ maleimide generated from the anti-carcinoembryonic antigen antibody A5B7 radiolabelled with iodine-131. Ten patients received an i.v. injection of iodine-131-labelled A5B7 humanised divalent-Fab′ maleimide, and positive tumour images were obtained by gamma camera imaging in eight patients with known lesions, and one previously undetected lesion was identified. True negative results were obtained in two patients without tumour. Area under the curve analysis of serial blood gamma counting and gamma camera images showed a higher tumour to blood ratio compared to A5B7 mF(ab′)2 used previously in the clinic, implying this new molecule may be superior for radioimmunotherapy. MIRD dose calculations showed a relatively high radiation dose to the kidney, which may limit the amount of activity that could be administered in radioimmunotherapy. However the reduction in immunogenicity was also a major advantage for A5B7 humanised divalent-Fab′ maleimide over murine versions of this antibody suggesting that humanised divalent-Fab′ maleimide should be a useful vehicle for repeated therapies. British Journal of Cancer (2002) 86, 1401–1410. DOI: 10.1038/sj/bjc/6600198 www.bjcancer.com © 2002 Cancer Research UK PMID:11986771

  5. Reactivity of German cockroach allergen, Bla g 2, peptide fragments to IgE antibodies in patients' sera.

    PubMed

    Lee, Haeseok; Jeong, Kyoung Yong; Shin, Kwang Hyun; Yi, Myung-hee; Gantulaga, Darambazar; Hong, Chein-Soo; Yong, Tai-Soon

    2008-12-01

    Bla g 2 is a cockroach allergen of great importance. This study was conducted to identify IgE-binding epitope(s) of Bla g 2 using the recombinant protein technique. Approximately 50% of tested sera showed IgE reactivity to Pichia-expressed Bla g 2 (PrBla g 2) and E. coli-expressed Bla g 2 (ErBla g 2). Only 5.3% of serum samples showed stronger reactivity to PrBla g 2 than ErBla g 2, indicating that serum was reactive to conformational or carbohydrate epitopes. The full-length and 5 peptide fragments of Bla g 2 were produced in E. coli. All fragments showed IgE-binding activity to the cockroach-allergy patients' sera. Specifically, peptide fragments of amino acid residue 1-75 and 146-225 appeared to be important for IgE-binding. The information about the IgE-binding epitope of Bla g 2 can aid in the diagnosis and treatment for cockroach allergies.

  6. A dual-targeting PDGFRβ/VEGF-A molecule assembled from stable antibody fragments demonstrates anti-angiogenic activity in vitro and in vivo

    PubMed Central

    Gilbertson, Debra G; Frank, Amanda; Vu, Tuyen; Ardourel, Dan; Ostrander, Craig; Stevens, Brenda; Julien, Susan; Franke, Secil; Meengs, Brent; Brody, Jennifer; Presnell, Scott; Hamacher, Nels B; Lantry, Megan; Wolf, Anitra; Bukowski, Tom; Rosler, Robert; Yen, Cindy; Anderson-Haley, Monica; Brasel, Kenneth; Pan, Qi; Franklin, Hank; Thompson, Penny; Dodds, Mike; Underwood, Sara; Peterson, Scott; Sivakumar, Pallavur V; Snavely, Mark

    2010-01-01

    Targeting angiogenesis is a promising approach to the treatment of solid tumors and age-related macular degeneration (AMD). Inhibition of vascularization has been validated by the successful marketing of monoclonal antibodies (mAbs) that target specific growth factors or their receptors, but there is considerable room for improvement in existing therapies. Combination of mAbs targeting both the VeGF and pDGF pathways has the potential to increase the efficacy of anti-angiogenic therapy without the accompanying toxicities of tyrosine kinase inhibitors and the inability to combine efficiently with traditional chemotherapeutics. However, development costs and regulatory issues have limited the use of combinatorial approaches for the generation of more efficacious treatments. The concept of mediating disease pathology by targeting two antigens with one therapeutic was proposed over two decades ago. While mAbs are particularly suitable candidates for a dual-targeting approach, engineering bispecificity into one molecule can be difficult due to issues with expression and stability, which play a significant role in manufacturability. Here, we address these issues upstream in the process of developing a bispecific antibody (bsAb). Single-chain antibody fragments (scFvs) targeting pDGFRβ and VeGF-A were selected for superior stability. the scFvs were fused to both termini of human Fc to generate a bispecific, tetravalent molecule. resulting molecule displays potent activity, binds both targets simultaneously, and is stable in serum. assembly of a bsAb using stable monomeric units allowed development of an anti-pDGFRB/VeGF-A antibody capable of attenuating angiogenesis through two distinct pathways and represents an efficient method for rapid engineering of dual-targeting molecules. PMID:20065654

  7. Antibody Responses to Recombinant Protein Fragments of the Major Outer Membrane Protein and Polymorphic Outer Membrane Protein POMP90 in Chlamydophila abortus-Infected Pregnant Sheep

    PubMed Central

    Livingstone, Morag; Entrican, Gary; Wattegedera, Sean; Buxton, David; McKendrick, Iain J.; Longbottom, David

    2005-01-01

    Chlamydophila abortus is one of the major causes of infectious abortion in pregnant sheep (enzootic abortion of ewes or EAE) worldwide. Organisms shed in infected placentas and uterine discharges at lambing time are the main sources of environmental contamination, responsible for transmission to susceptible animals and possible human contacts. In the present study, a recently developed test, based on a recombinant fragment of the polymorphic outer membrane protein POMP90 (rOMP90-4 indirect enzyme-linked immunosorbent assay [iELISA]) and one based on the variable segment 2 (VS2) region of the major outer membrane protein (MOMP) (MOMP VS2 iELISA) were compared using sera from C. abortus-infected ewes at different stages throughout pregnancy. The rOMP90 iELISA detected antibody much earlier in pregnancy than the MOMP iELISA, which, like the complement fixation test, detected antibody only at the time of abortion or lambing. No anti-MOMP antibody response could be detected in three of seven experimentally infected ewes. Furthermore, the rOMP90 iELISA detected antibody in an animal that seroconverted during the course of the study, which the MOMP iELISA failed to detect. Overall, the results show that the rOMP90-4 iELISA is considerably more sensitive than the MOMP VS2 iELISA for identifying animals infected with C. abortus. Earlier detection of infection will allow appropriate control measures to be taken to reduce environmental contamination, thus limiting the spread of infection, financial losses, and the possible risks of zoonotic transmission to humans. PMID:15939753

  8. Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type 1

    NASA Technical Reports Server (NTRS)

    He, Xiao M.; Rueker, Florian; Casale, Elena; Carter, Daniel C.

    1992-01-01

    The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 deg. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

  9. Elongation of the C-terminal domain of an anti-amyloid β single-chain variable fragment increases its thermodynamic stability and decreases its aggregation tendency.

    PubMed

    Rivera-Hernández, Geovanny; Marin-Argany, Marta; Blasco-Moreno, Bernat; Bonet, Jaume; Oliva, Baldo; Villegas, Sandra

    2013-01-01

    Amyloid β (Aβ) immunotherapy is considered a promising approach to Alzheimer disease treatment. In contrast to the use of complete antibodies, administration of single-chain variable fragments (scFv) has not been associated with either meningoencephalitis or cerebral hemorrhage. ScFv-h3D6 is known to preclude cytotoxicity of the Aβ 1-42 peptide by removing its oligomers from the amyloid pathway. As is the case for other scFv molecules, the recombinant production of scFv-h3D6 is limited by its folding and stability properties. Here, we show that its urea-induced unfolding pathway is characterized by the presence of an intermediate state composed of the unfolded VL domain and the folded VH domain, which suggests the VL domain as a target for thermodynamic stability redesign. The modeling of the 3D structure revealed that the VL domain, located at the C-terminal of the molecule, was ending before its latest β-strand was completed. Three elongation mutants, beyond VL-K107, showed increased thermodynamic stability and lower aggregation tendency, as determined from urea denaturation experiments and Fourier-transform infrared spectroscopy, respectively. Because the mutants maintained the capability of removing Aβ-oligomers from the amyloid pathway, we expect these traits to increase the half-life of scFv-h3D6 in vivo and, consequently, to decrease the effective doses. Our results led to the improvement of a potential Alzheimer disease treatment and may be extrapolated to other class-I scFv molecules of therapeutic interest.

  10. Epitope Mapping of Antigenic MUC1 Peptides to Breast Cancer Antibody Fragment B27.29: A Heteronuclear NMR Study

    SciTech Connect

    Grinstead, Jeffrey S.; Schuman, Jason T.; Campbell, Ann P.

    2003-11-13

    MUC1 mucin is a breast cancer-associated transmembrane glycoprotein, of which the extracellular domain is formed by the repeating 20-amino acid sequence GVTSAPDTRPAPGSTAPPAH. In neoplastic breast tissue, the highly immunogenic sequence PDTRPAP (in bold above) is exposed. Antibodies raised directly against MUC1-expressing tumors offer unique access to this neoplastic state, as they represent immunologically relevant ''reverse templates'' of the tumor-associated mucin. In a previous study [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], 1H NMR methods were used to correlate the effects of cryptic glycosylation outside of the PDTRPAP core epitope sequence on the recognition and binding of Mab B27.29, a monoclonal antibody raised against breast tumor cells. In the study presented here, isotope-edited NMR methods, including 15N and 13C relaxation measurements, were used to probe the recognition and binding of the PDTRPAP epitope sequence to Fab B27.29. Two peptides were studied: a one-repeat MUC1 16mer peptide of the sequence GVTSAPDTRPAPGSTA and a two-repeat MUC1 40mer peptide of the sequence (VTSAPDTRPAPGSTAPPAHG)2. 15N and 13C NMR relaxation parameters were measured for both peptides free in solution and bound to Fab B27.29. The 13CR T1 values best represent changes in the local correlation time of the peptide epitope upon binding antibody, and demonstrate that the PDTRPAP sequence is immobilized in the antibody-combining site. This result is also reflected in the appearance of the 15N- and 13C-edited HSQC spectra, where line broadening of the same peptide epitope resonances is observed. The PDTRPAP peptide epitope expands upon the peptide epitope identified previously in our group as PDTRP by homonuclear NMR experiments [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], and illustrates the usefulness of the heteronuclear NMR experiments. The implications of these results are discussed within the context of MUC1 breast cancer vaccine design.

  11. An Exopolysaccharide-Deficient Mutant of Lactobacillus rhamnosus GG Efficiently Displays a Protective Llama Antibody Fragment against Rotavirus on Its Surface

    PubMed Central

    Krogh-Andersen, Kasper; Tellgren-Roth, Christian; Martínez, Noelia; Günaydın, Gökçe; Lin, Yin; Martín, M. Cruz; Álvarez, Miguel A.; Hammarström, Lennart

    2015-01-01

    Rotavirus is the leading cause of infantile diarrhea in developing countries, where it causes a high number of deaths among infants. Two vaccines are available, being highly effective in developed countries although markedly less efficient in developing countries. As a complementary treatment to the vaccines, a Lactobacillus strain producing an anti-rotavirus antibody fragment in the gastrointestinal tract could potentially be used. In order to develop such an alternative therapy, the effectiveness of Lactobacillus rhamnosus GG to produce and display a VHH antibody fragment (referred to as anti-rotavirus protein 1 [ARP1]) on the surface was investigated. L. rhamnosus GG is one of the best-characterized probiotic bacteria and has intrinsic antirotavirus activity. Among four L. rhamnosus GG strains [GG (CMC), GG (ATCC 53103), GG (NCC 3003), and GG (UT)] originating from different sources, only GG (UT) was able to display ARP1 on the bacterial surface. The genomic analysis of strain GG (UT) showed that the genes welE and welF of the EPS cluster are inactivated, which causes a defect in exopolysaccharide (EPS) production, allowing efficient display of ARP1 on its surface. Finally, GG (UT) seemed to confer a level of protection against rotavirus-induced diarrhea similar to that of wild-type GG (NCC 3003) in a mouse pup model, indicating that the EPS may not be involved in the intrinsic antirotavirus activity. Most important, GG (EM233), a derivative of GG (UT) producing ARP1, was significantly more protective than the control strain L. casei BL23. PMID:26092449

  12. Recombinant antibodies for specific detection of clostridial [Fe-Fe] hydrogenases.

    PubMed

    Mangayil, Rahul; Karp, Matti; Lamminmäki, Urpo; Santala, Ville

    2016-10-27

    Biological hydrogen production is based on activity of specific enzymes called hydrogenases. Hydrogenases are oxygen sensitive metalloenzymes containing Ni and/or Fe atoms at the active site, catalyzing reversible reduction of protons. Generally, [Fe-Fe] hydrogenases prefer proton reduction to molecular hydrogen, a potential energy carrier molecule that can be produced by bioprocesses in sustainable manner. Thus, monitoring tools have been developed to study the relationship between [Fe-Fe] hydrogenases and biohydrogen production in bioreactors at DNA and RNA levels. In the present study, novel molecular tools are introduced for quantitative monitoring of clostridial [Fe-Fe] hydrogenases at the protein level. Aerobic and anaerobic biopanning (for inactive and active [Fe-Fe] hydrogenase, respectively) of phage displayed single-chain variable fragment (scFv) antibody libraries aided in isolating nine potential scFvs. The enriched antibodies demonstrated high specificity towards Clostridium spp. [Fe-Fe] hydrogenases allowing detection from pure and mixed cultures. Additionally, the antibodies showed different binding characteristics towards hydrogenase catalytic states, providing a possible means for functional detection of clostridial [Fe-Fe] hydrogenases. From hydrogenase-antibody interaction studies we observed that though antibody binding reduced the enzyme catalytic activity, it facilitated to retain hydrogen evolution from oxygen exposed hydrogenases.

  13. Bivalent Brain Shuttle Increases Antibody Uptake by Monovalent Binding to the Transferrin Receptor

    PubMed Central

    Hultqvist, Greta; Syvänen, Stina; Fang, Xiaotian T; Lannfelt, Lars; Sehlin, Dag

    2017-01-01

    The blood-brain barrier (BBB) is an obstacle for antibody passage into the brain, impeding the development of immunotherapy and antibody-based diagnostics for brain disorders. In the present study, we have developed a brain shuttle for active transport of antibodies across the BBB by receptor-mediated transcytosis. We have thus recombinantly fused two single-chain variable fragments (scFv) of the transferrin receptor (TfR) antibody 8D3 to the light chains of mAb158, an antibody selectively binding to Aβ protofibrils, which are involved in the pathogenesis of Alzheimer's disease (AD). Despite the two TfR binders, a monovalent interaction with TfR was achieved due to the short linkers that sterically hinder bivalent binding to the TfR dimer. The design enabled efficient receptor-mediated brain uptake of the fusion protein. Two hours after administration, brain concentrations were 2-3% of the injected dose per gram brain, comparable to small molecular drugs and 80-fold higher than unmodified mAb158. After three days, fusion protein concentrations in AD transgenic mouse brains were 9-fold higher than in wild type mice, demonstrating high in vivo specificity. Thus, our innovative recombinant design markedly increases mAb158 brain uptake, which makes it a strong candidate for improved Aβ immunotherapy and as a PET radioligand for early diagnosis and evaluation of treatment effect in AD. Moreover, this approach could be applied to any target within the brain. PMID:28042336

  14. Recombinant antibodies for specific detection of clostridial [Fe-Fe] hydrogenases

    PubMed Central

    Mangayil, Rahul; Karp, Matti; Lamminmäki, Urpo; Santala, Ville

    2016-01-01

    Biological hydrogen production is based on activity of specific enzymes called hydrogenases. Hydrogenases are oxygen sensitive metalloenzymes containing Ni and/or Fe atoms at the active site, catalyzing reversible reduction of protons. Generally, [Fe-Fe] hydrogenases prefer proton reduction to molecular hydrogen, a potential energy carrier molecule that can be produced by bioprocesses in sustainable manner. Thus, monitoring tools have been developed to study the relationship between [Fe-Fe] hydrogenases and biohydrogen production in bioreactors at DNA and RNA levels. In the present study, novel molecular tools are introduced for quantitative monitoring of clostridial [Fe-Fe] hydrogenases at the protein level. Aerobic and anaerobic biopanning (for inactive and active [Fe-Fe] hydrogenase, respectively) of phage displayed single-chain variable fragment (scFv) antibody libraries aided in isolating nine potential scFvs. The enriched antibodies demonstrated high specificity towards Clostridium spp. [Fe-Fe] hydrogenases allowing detection from pure and mixed cultures. Additionally, the antibodies showed different binding characteristics towards hydrogenase catalytic states, providing a possible means for functional detection of clostridial [Fe-Fe] hydrogenases. From hydrogenase-antibody interaction studies we observed that though antibody binding reduced the enzyme catalytic activity, it facilitated to retain hydrogen evolution from oxygen exposed hydrogenases. PMID:27786270

  15. Human Monoclonal Antibody Fragments Binding to Insulin-like Growth Factors 1 and 2 with Picomolar Affinity

    PubMed Central

    Zhao, Qi; Feng, Yang; Zhu, Zhongyu; Dimitrov, Dimiter S.

    2011-01-01

    The type 1 insulin-like growth factor receptor (IGF1R) and its ligands (IGF1 and IGF2) have been implicated in a variety of physiological processes and in diseases such as cancer. In addition to IGF1R, IGF2 also activates the insulin receptor (IR) isoform A and therefore antibodies against IGF2 can inhibit cell proliferation mediated by the signaling through both IGF1R and IR triggered by IGF2. We identified a new human monoclonal antibody (mAb), m708.2, which bound to IGF1 and IGF2 but not to insulin. m708.2 potently inhibited signal transduction mediated by the interaction of IGF1 or IGF2 with the IGF1R and IGF2 with the IR. It also inhibited the growth of the breast cancer cell line MCF-7. An affinity-matured derivative of m708.2, m708.5, bound to IGF1 with equilibrium dissociation constant, KD = 200 pM and to IGF2 with KD = 60 pM. m708.5 inhibited signal transduction mediated by IGF1 and IGF2 and cancer cell growth more potently than m708.2. These results suggest that m708.5 could have potential as a candidate therapeutic for cancers driven by the IGF1,2 interactions with IGF1R and IR. PMID:21750218

  16. Preparation and activity of conjugate of monoclonal antibody HAb18 against hepatoma F(ab’)2 fragment and staphylococcal enterotoxin A Lian

    PubMed Central

    Yang, Jun; Sui, Yan Fang; Chen, Zhi Nan

    2001-01-01

    AIM: To prepare the conjugate of staphy lococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab’)2 fragment of mAb HAb18 against human hepatocellular carcinoma (HCC), and identify its activity in order to use SAg in the targeting therapy of HCC. METHODS: MAb HAb18 was extracted from the abdominal dropsy of Balb/c mice, and was purified through chromatography column SP 40HR with Fast protein liquid chromatography (FPLC) system. The F(ab’)2 fragment of mAb HAb18 was prepared by papainic digestion method. The conjugate of mAb HAb18 F(ab’)2 fragment and SEA was prepared with chemical conjugating reagent N succinimidyl 3 (2pyridyldithio) propionate (SPDP) and purified through chromatography column Superose 12 with FPLC system. The molecular mass and purity of each collected peak were identified with SDS-PAGE assay. The protein content was assayed by Lowry’s method. The antibody activity of HAb18 F(ab’)2 against HCC in the conjugate was identified by indirect immunocytochemical ABC method, and the activity of SEA in the conjugate to activate peripheral blood mononuclear cells (PBMC) was identified with MTT assay. RESULTS: The IgG mAb HAb18 was extracted, and purified successfully. Immunocytochemical staining demonstrated that it reacted with most of HHCC cells of human HCC cell line. There were two peaks in the process of purification of the prepared HAb18 F(ab’)2 SEA conjugate. SDS-PAGE assay demonstrated that the molecular mass of the first peak was about 130 ku, and the second peak was the mixture of about 45 ku and a little 100 ku proteins. The immunocytochemical staining was similar in HAb18 F(ab’)2 SEA conjugate and HAb18 F(ab’)2, i.e.the cytoplasm and/or cell membranes of most HHCC cells were positively stained. The MTT assay showed that the optical absorbance (A) value at 490 nm of HAb18 F(ab’)2 SEA conjugate was 0.182 ± 0.012, that of negative control was 0.033 ± 0.009, and there was significant difference between them (P

  17. Astatine-211 labeling of an anti-melanoma antibody and its Fab fragment using N-succinimidyl para[{sup 211} At]astatobenzoate : comparisons In Vivo with the para-[{sup 125}1]iodobenzoyl conjugate.

    SciTech Connect

    Hadley, S. W.; Wilbur, D. S.; Gray, M. A.; Atcher, R. W.; Chemistry; NeoRx Corp.; Univ. of Washington Medical Center

    1991-01-01

    Astatine-211 labeling of an anti-melanoma antibody, NR-ML-05, and its Fab fragment using N-succinimidyl para[{sup 211} At]astatobenzoate has been described. Preparation of the astatinated intermediate 2a was accomplished by distilling astatine-211 from an irradiated bismuth target directly into a reaction mixture containing an organometallic compound, N-succinimidyl p-(tri-n-butylstannyl)benzoate (1), and an oxidant, N-chlorosuccinimide, in 5% HOAc/MeOH. Trapping of distilled astatine as 2a was found to be efficient, resulting in 70-90% yields based on the amount of astatine-211 which ranged from 20% to 75%. Conjugation of 2a to NR-ML-05 and its Fab fragment was accomplished in 40-60% yields. The [{sup 211}At]astatobenzoyl-conjugated antibodies were found to be stable in vitro when challenged by strong denaturants and nucleophilic reagents. Coinjected dual-labeled studies of the 2a astatinated antibodies and the same antibodies labeled with N-succinimidyl p-[{sup 125}I]iodobenzoate (2b) in athymic mice bearing the human tumor xenograft A375 Met/Mix demonstrated that both radiolabeled antibodies had equivalent tumor localization. Data from the dual-labeled biodistribution of the intact antibody suggests that the astatine is stably attached. Data from the dual-labeled Fab fragment suggests that a portion of the astatine label is released as astatide, either from the astatinated Fab or from a catabolite.

  18. Antitumor effects of ricin A chain immunotoxins prepared from intact antibodies and Fab' fragments on solid human Hodgkin's disease tumors in mice.

    PubMed

    Engert, A; Martin, G; Pfreundschuh, M; Amlot, P; Hsu, S M; Diehl, V; Thorpe, P

    1990-05-15

    Three monoclonal antibodies which strongly bind to Hodgkin and Reed-Sternberg cells and two corresponding Fab' fragments were linked to deglycosylated ricin A chain (dg A) to evaluate their potential as immunotoxins for the treatment of Hodgkin's disease. Two of the antibodies, Ber-H2 and HRS-3, were shown to bind to the same epitope on the CD30 antigen, whereas the third antibody, IRac, bound to a different antigen. None of the antibodies significantly cross-reacted with normal human tissues as judged by indirect immunofluorescence and immunoperoxidase analyses on frozen sections from 28 normal tissues. All three antibodies formed potent and specific immunotoxins. They inhibited protein synthesis of the L540 Hodgkin's disease cell line in vitro by 50% at concentrations of 1 x 10(-11) M for IRac.dgA, 9 x 10(-11) M for HRS-3.dgA, and 2 x 10(-10) M for Ber-H2.dgA. HRS-3 Fab' and IRac Fab' immunotoxins were 7.8- and 60-fold less cytotoxic, respectively, than their intact counterparts in vitro. In vivo, a single i.v. injection of a dose of Ber-H2.dgA, HRS-3.dgA, or IRac.dgA corresponding to 40% of the LD50 induced lasting complete remissions in 38, 44, and 50%, respectively, of mice with solid s.c. L540 tumors of 60 to 80 mm3 size (0.5-cm diameter). At equivalent dosage (40% of the LD50), the HRS-3 Fab'.dgA and the IRac Fab'.dgA both induced lasting complete remissions in 25% of the mice, although the HRS-3 Fab'.dgA was significantly superior to IRac Fab'.dgA at retarding tumor growth in the remaining animals. The effectiveness of the immunotoxins depended on the size of the tumor at the time of injection, since IRac.dgA treatment induced complete remissions in 100% of mice with small tumors (10 to 20 mm3, approximately 0.3 cm in diameter) but only 13% of mice with larger tumors of 400 to 600 mm3 (approximately 1 cm in diameter). Tumors which regrew after IRac.dgA treatment mainly consisted of antigen-deficient mutants having reduced sensitivity to IRac.dgA but normal

  19. Mapping the Binding Interface of VEGF and a Monoclonal Antibody Fab-1 Fragment with Fast Photochemical Oxidation of Proteins (FPOP) and Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Zhang, Ying; Wecksler, Aaron T.; Molina, Patricia; Deperalta, Galahad; Gross, Michael L.

    2017-03-01

    We previously analyzed the Fab-1:VEGF (vascular endothelial growth factor) system described in this work, with both native top-down mass spectrometry and bottom-up mass spectrometry (carboxyl-group or GEE footprinting) techniques. This work continues bottom-up mass spectrometry analysis using a fast photochemical oxidation of proteins (FPOP) platform to map the solution binding interface of VEGF and a fragment antigen binding region of an antibody (Fab-1). In this study, we use FPOP to compare the changes in solvent accessibility by quantitating the extent of oxidative modification in the unbound versus bound states. Determining the changes in solvent accessibility enables the inference of the protein binding sites (epitope and paratopes) and a comparison to the previously published Fab-1:VEGF crystal structure, adding to the top-down and bottom-up data. Using this method, we investigated peptide-level and residue-level changes in solvent accessibility between the unbound proteins and bound complex. Mapping these data onto the Fab-1:VEGF crystal structure enabled successful characterization of both the binding region and regions of remote conformation changes. These data, coupled with our previous higher order structure (HOS) studies, demonstrate the value of a comprehensive toolbox of methods for identifying the putative epitopes and paratopes for biotherapeutic antibodies.

  20. Genetically engineered multivalent single chain antibody constructs for cancer therapy

    SciTech Connect

    Surinder Batra, Ph D

    2006-02-27

    its tumor: normal tissue ratio for improved therapeutic index, we engineered a variety antibody constructs. These constructs were evaluated using novel approaches like special radionuclides, pretargeting and optimization. Due to the smaller size, the engineered antibody molecules should penetrate better throughout a tumor mass, with less dose heterogeneity, than is the case with intact IgG. Multivalent scFvs with an appropriate radionuclide, therefore, hold promising prospects for cancer therapy and clinical imaging in MAb-based radiopharmaceuticals. In addition, the human anti-mouse antibodies (HAMA) responses in patients against antibody-based therapy are usually directed against the immunoglobulin constant regions; however, anti-idiotypic responses can also be detected. The HAMA responses reduce the efficacy of treatment by removing the circulating antibody molecules, fragments, and possibly scFvs by altering the pharmacokinetic properties of the antibody. HAMA responses against divalent IgG, divalent Ig fragments, and possibly multimeric scFvs could cause immune complex formation with hypersensitivity or allergic reactions that could be harmful to patients. The use of small molecules, such as scFvs (monomeric as well as multimeric), with their shorter biological half-lives and the lack of the constant regions and humanized variable (binding regions) performed in our studies should reduce the development of HAMA. The generation of humanized and fully human scFvs should further reduce the development of HAMA. Specific accomplishments on the project are the production of large amounts of recombinant antibodies as they are required in large amounts for cancer diagnosis and therapy. A variety of single-chain Fv (scFv) constructs were engineered for the desired pharmacokinetic properties. Tetrameric and dimeric scFvs showed a two-fold advantage: (1) there was a considerable gain in avidity as compared to smaller fragments, and (2) the biological half-life was more

  1. A Nanoparticle Platform To Evaluate Bioconjugation and Receptor-Mediated Cell Uptake Using Cross-Linked Polyion Complex Micelles Bearing Antibody Fragments.

    PubMed

    Florinas, Stelios; Liu, Marc; Fleming, Ryan; Van Vlerken-Ysla, Lilian; Ayriss, Joanne; Gilbreth, Ryan; Dimasi, Nazzareno; Gao, Changshou; Wu, Herren; Xu, Ze-Qi; Chen, Shaoyi; Dirisala, Anjaneyulu; Kataoka, Kazunori; Cabral, Horacio; Christie, R James

    2016-05-09

    Targeted nanomedicines are a promising technology for treatment of disease; however, preparation and characterization of well-defined protein-nanoparticle systems remain challenging. Here, we describe a platform technology to prepare antibody binding fragment (Fab)-bearing nanoparticles and an accompanying real-time cell-based assay to determine their cellular uptake compared to monoclonal antibodies (mAbs) and Fabs. The nanoparticle platform was composed of core-cross-linked polyion complex (PIC) micelles prepared from azide-functionalized PEG-b-poly(amino acids), that is, azido-PEG-b-poly(l-lysine) [N3-PEG-b-PLL] and azido-PEG-b-poly(aspartic acid) [N3-PEG-b-PAsp]. These PIC micelles were 30 nm in size and contained approximately 10 polymers per construct. Fabs were derived from an antibody binding the EphA2 receptor expressed on cancer cells and further engineered to contain a reactive cysteine for site-specific attachment and a cleavable His tag for purification from cell culture expression systems. Azide-functionalized micelles and thiol-containing Fab were linked using a heterobifunctional cross-linker (FPM-PEG4-DBCO) that contained a fluorophenyl-maleimide for stable conjugation to Fabs thiols and a strained alkyne (DBCO) group for coupling to micelle azide groups. Analysis of Fab-PIC micelle conjugates by fluorescence correlation spectroscopy, size exclusion chromatography, and UV-vis absorbance determined that each nanoparticle contained 2-3 Fabs. Evaluation of cellular uptake in receptor positive cancer cells by real-time fluorescence microscopy revealed that targeted Fab-PIC micelles achieved higher cell uptake than mAbs and Fabs, demonstrating the utility of this approach to identify targeted nanoparticle constructs with unique cellular internalization properties.

  2. A recombinant mimetics of the HIV-1 gp41 prehairpin fusion intermediate fused with human IgG Fc fragment elicits neutralizing antibody response in the vaccinated mice

    SciTech Connect

    Qi, Zhi; Pan, Chungen; Lu, Hong; Shui, Yuan; Li, Lin; Li, Xiaojuan; Xu, Xueqing; Liu, Shuwen; Jiang, Shibo

    2010-07-30

    Research highlights: {yields} One recombinant mimetics of gp41 prehairpin fusion intermediate (PFI) consisting of gp41 N46 sequence, foldon and IgG Fc, designated N46FdFc, was expressed. {yields} N46FdFc-induced antibodies in mice that neutralized HIV-1 infection, inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. {yields} These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines. -- Abstract: HIV-1 gp41 prehairpin fusion intermediate (PFI) composed of three N-terminal heptad repeats (NHR) plays a crucial role in viral fusion and entry and represents an attractive target for anti-HIV therapeutics (e.g., enfuvirtide) and vaccines. In present study, we constructed and expressed two recombinant gp41 PFI mimetics, designated N46Fd and N46FdFc. N46Fd consists of N46 (residues 536-581) in gp41 NHR and foldon (Fd), a trimerization motif. N46FdFc is composed of N46Fd fused with human IgG Fc fragment as an immunoenhancer. We immunized mice with N46 peptide, N46Fd and N46FdFc, respectively, and found that only N46FdFc elicited neutralizing antibody response in mice against infection by HIV-1 strains IIIB (clade B, X4), 92US657 (clade B, R5), and 94UG103 (clade A, X4R5). Anti-N46FdFc antibodies inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines.

  3. Crystallization and preliminary X-ray diffraction analysis of the Fab fragment of WO2, an antibody specific for the A[beta] peptides associated with Alzheimer's disease

    SciTech Connect

    Wun, Kwok S.; Miles, Luke A.; Crespi, Gabriela A.N.; Wycherley, Kaye; Ascher, David B.; Barnham, Kevin J.; Cappai, Roberto; Beyreuther, Konrad; Masters, Colin L.; Parker, Michael W.; McKinstry, William J.

    2008-05-28

    The murine monoclonal antibody WO2 specifically binds the N-terminal region of the amyloid {beta} peptide (A{beta}) associated with Alzheimer's disease. This region of A{beta} has been shown to be the immunodominant B-cell epitope of the peptide and hence is considered to be a basis for the development of immunotherapeutic strategies against this prevalent cause of dementia. Structural studies have been undertaken in order to characterize the molecular basis for antibody recognition of this important epitope. Here, details of the crystallization and X-ray analysis of the Fab fragment of the unliganded WO2 antibody in two crystal forms and of the complexes that it forms with the truncated Az{beta} peptides A{beta}{sub 1-16} and A{beta}{sub 1-28} are presented. These crystals were all obtained using the hanging-drop vapour-diffusion method at 295 K. Crystals of WO2 Fab were grown in polyethylene glycol solutions containing ZnSO{sub 4}; they belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1} and diffracted to 1.6 {angstrom} resolution. The complexes of WO2 Fab with either A{beta}{sub 1-16} or A{beta}{sub 1-28} were cocrystallized from polyethylene glycol solutions. These two complex crystals grew in the same space group, P2{sub 1}2{sub 1}2{sub 1}, and diffracted to 1.6 {angstrom} resolution. A second crystal form of WO2 Fab was grown in the presence of the sparingly soluble A{beta}{sub 1-42} in PEG 550 MME. This second form belonged to space group P2{sub 1} and diffracted to 1.9 {angstrom} resolution.

  4. Detection and quantification of affinity ligand leaching and specific antibody fragment concentration within chromatographic fractions using surface plasmon resonance.

    PubMed

    Thillaivinayagalingam, Pranavan; Newcombe, Anthony R; O'Donovan, Kieran; Francis, Richard; Keshavarz-Moore, Eli

    2007-12-01

    Rapid analyses of chromatographic steps within a biopharmaceutical manufacturing process are often desirable to evaluate column performance, provide mass balance data and to permit accurate calculations of yields and recoveries. Using SPR (surface plasmon resonance) biosensor (Biacore) technology, we have developed a sandwich immunoassay to quantify polyclonal anti-digoxin Fab fragments used for the production of the FDA (Food and Drug Administration)-approved biotherapeutic DigiFab. The results show that specific Fab may be quantified in all affinity process streams and accurate yield and mass balance data calculated. Control experiments using sheep Fab and Fc indicate that the assay is specific to DigiFab. The quantification of potential leached ligand within chromatographic fractions may also be technically challenging, particularly when low-molecular-mass ligands are covalently coupled with an affinity absorbent. Typical methods to assess ligand leakage such as DDMA (digoxin-dicarboxymethoxylamine; digoxin analogue) often involve the use of labelled ligands and relatively complex and labour-intensive analytical techniques. Using the same analytical methodologies, an assay to detect leached or eluted ligand off the column was developed. The results indicate minimal levels of leached ligand in all chromatographic fractions, with total levels of leached DDMA calculated to be 1.52 microg. This is less than 0.01% of the total amount of DDMA coupled with the laboratory-scale affinity column. The SPR methods described in the present study may be applicable for the rapid in-process analysis of specific polyclonal Fab fragments (within a polyclonal mixture) and to rapidly assess leakage of small molecule ligands covalently attached to chromatographic supports.

  5. Occurrence of anti-Leptospira spp. antibodies in Rhipidomys spp. from a forest fragment of the Brazilian Cerrado.

    PubMed

    Gomes, D O; Ramos, G B; Alves, V B A; Ciuffa, A Z; Cuccato, L P; Dos Reis, T F M; Lima, A M C; Gonçalves, M C; Tolesano, G V; Rodrigues, V S; Szabó, M P J

    2017-03-01

    Leptospirosis is a zoonotic disease of world importance, and its transmission depends on the interaction between humans and animals. Given the necessity to investigate potential hosts of Leptospira spp., this study verified the prevalence of different serovars in the species of Rhipidomys spp., a widespread sigmodont rodent in Brazil. The studied population originates from a semi-evergreen forest located in the county of Uberlândia, in the state of Minas Gerais. The microscopic agglutination test (MAT) was performed with 14 serovars. Thirteen out of the 43 wild rodents captured showed a positive agglutination reaction, with a greater prevalence of the serovars Pyrogenes, Copenhageni, and Canicola. This study found a prevalence of 30.3% anti-Leptospira spp. antibodies; all positive animals were reactive to more than one serovar.

  6. A single domain antibody fragment that recognizes the adaptor ASC defines the role of ASC domains in inflammasome assembly

    PubMed Central

    Schmidt, Florian I.; Lu, Alvin; Chen, Jeff W.; Ruan, Jianbin; Tang, Catherine

    2016-01-01

    Myeloid cells assemble inflammasomes in response to infection or cell damage; cytosolic sensors activate pro–caspase-1, indirectly for the most part, via the adaptors ASC and NLRC4. This leads to secretion of proinflammatory cytokines and pyroptosis. To explore complex formation under physiological conditions, we generated an alpaca single domain antibody, VHHASC, which specifically recognizes the CARD of human ASC via its type II interface. VHHASC not only impairs ASCCARD interactions in vitro, but also inhibits inflammasome activation in response to NLRP3, AIM2, and NAIP triggers when expressed in living cells, highlighting a role of ASC in all three types of inflammasomes. VHHASC leaves the Pyrin domain of ASC functional and stabilizes a filamentous intermediate of inflammasome activation. Incorporation of VHHASC-EGFP into these structures allowed the visualization of endogenous ASCPYD filaments for the first time. These data revealed that cross-linking of ASCPYD filaments via ASCCARD mediates the assembly of ASC foci. PMID:27069117

  7. Structures of synthetic O-antigen fragments from serotype 2a Shigella flexneri in complex with a protective monoclonal antibody

    PubMed Central

    Vulliez-Le Normand, B.; Saul, F. A.; Phalipon, A.; Bélot, F.; Guerreiro, C.; Mulard, L. A.; Bentley, G. A.

    2008-01-01

    The anti-LPS IgG mAb F22-4, raised against Shigella flexneri serotype 2a bacteria, protects against homologous, but not heterologous, challenge in an experimental animal model. We report the crystal structures of complexes formed between Fab F22-4 and two synthetic oligosaccharides, a decasaccharide and a pentadecasaccharide that were previously shown to be both immunogenic and antigenic mimics of the S. flexneri serotype 2a O-antigen. F22-4 binds to an epitope contained within two consecutive 2a serotype pentasaccharide repeat units (RU). Six sugar residues from a contiguous nine-residue segment make direct contacts with the antibody, including the nonreducing rhamnose and both branching glucosyl residues from the two RUs. The glucosyl residue, whose position of attachment to the tetrasaccharide backbone of the RU defines the serotype 2a O-antigen, is critical for recognition by F22-4. Although the complete decasaccharide is visible in the electron density maps, the last four pentadecasaccharide residues from the reducing end, which do not contact the antibody, could not be traced. Although considerable mobility in the free oligosaccharides can thus be expected, the conformational similarity between the individual RUs, both within and between the two complexes, suggests that short-range transient ordering to a helical conformation might occur in solution. Although the observed epitope includes the terminal nonreducing residue, binding to internal epitopes within the polysaccharide chain is not precluded. Our results have implications for vaccine development because they suggest that a minimum of two RUs of synthetic serotype 2a oligosaccharide is required for optimal mimicry of O-Ag epitopes. PMID:18621718

  8. Novel camelid antibody fragments targeting recombinant nucleoprotein of Araucaria hantavirus: a prototype for an early diagnosis of Hantavirus Pulmonary Syndrome.

    PubMed

    Pereira, Soraya S; Moreira-Dill, Leandro S; Morais, Michelle S S; Prado, Nidiane D R; Barros, Marcos L; Koishi, Andrea C; Mazarrotto, Giovanny A C A; Gonçalves, Giselle M; Zuliani, Juliana P; Calderon, Leonardo A; Soares, Andreimar M; Pereira da Silva, Luiz H; Duarte dos Santos, Claudia N; Fernandes, Carla F C; Stabeli, Rodrigo G

    2014-01-01

    In addition to conventional antibodies, camelids produce immunoglobulins G composed exclusively of heavy chains in which the antigen binding site is formed only by single domains called VHH. Their particular characteristics make VHHs interesting tools for drug-delivery, passive immunotherapy and high-throughput diagnosis. Hantaviruses are rodent-borne viruses of the Bunyaviridae family. Two clinical forms of the infection are known. Hemorrhagic Fever with Renal Syndrome (HFRS) is present in the Old World, while Hantavirus Pulmonary Syndrome (HPS) is found on the American continent. There is no specific treatment for HPS and its diagnosis is carried out by molecular or serological techniques, using mainly monoclonal antibodies or hantavirus nucleoprotein (N) to detect IgM and IgG in patient serum. This study proposes the use of camelid VHHs to develop alternative methods for diagnosing and confirming HPS. Phage display technology was employed to obtain VHHs. After immunizing one Lama glama against the recombinant N protein (prNΔ₈₅) of a Brazilian hantavirus strain, VHH regions were isolated to construct an immune library. VHHs were displayed fused to the M13KO7 phage coat protein III and the selection steps were performed on immobilized prNΔ₈₅. After selection, eighty clones recognized specifically the N protein. These were sequenced, grouped based mainly on the CDRs, and five clones were analyzed by western blot (WB), surface plasmon resonance (SPR) device, and ELISA. Besides the ability to recognize prNΔ85 by WB, all selected clones showed affinity constants in the nanomolar range. Additionaly, the clone KC329705 is able to detect prNΔ₈₅ in solution, as well as the native viral antigen. Findings support the hypothesis that selected VHHs could be a powerful tool in the development of rapid and accurate HPS diagnostic assays, which are essential to provide supportive care to patients and reduce the high mortality rate associated with hantavirus

  9. Selection and characterization of naturally occurring single-domain (IgNAR) antibody fragments from immunized sharks by phage display.

    PubMed

    Dooley, Helen; Flajnik, Martin F; Porter, Andrew J

    2003-09-01

    The novel immunoglobulin isotype novel antigen receptor (IgNAR) is found in cartilaginous fish and is composed of a heavy-chain homodimer that does not associate with light chains. The variable regions of IgNAR function as independent domains similar to those found in the heavy-chain immunoglobulins of Camelids. Here, we describe the successful cloning and generation of a phage-displayed, single-domain library based upon the variable domain of IgNAR. Selection of such a library generated from nurse sharks (Ginglymostoma cirratum) immunized with the model antigen hen egg-white lysozyme (HEL) enabled the successful isolation of intact antigen-specific binders matured in vivo. The selected variable domains were shown to be functionally expressed in Escherichia coli, extremely stable, and bind to antigen specifically with an affinity in the nanomolar range. This approach can therefore be considered as an alternative route for the isolation of minimal antigen-binding fragments with favorable characteristics.

  10. A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior.

    PubMed

    Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H; Muyldermans, Serge; Declerck, Paul J; Huang, Mingdong; Andreasen, Peter A; Ngo, Jacky Chi Ki

    2016-07-15

    A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors.

  11. Single chain fragment variable antibodies developed by using as target the 3rd fibronectin type III homologous repeat fragment of human neural cell adhesion molecule L1 promote cell migration and neuritogenesis.

    PubMed

    Tang, Dan-Yang; Yu, Yang; Zhao, Xuan-Jun; Schachner, Melitta; Zhao, Wei-Jiang

    2015-01-15

    L1CAM plays important roles during ontogeny, including promotion of neuronal cell migration and neuritogenesis, and stimulation of axonal outgrowth, fasciculation and myelination. These functions are at least partially exerted through a 16-mer amino acid sequence in the third fibronectin type III-like repeat of L1, which associates with several interaction partners, including integrins, other adhesion molecules and growth factor receptors. Here, using the Tomlinson I library for phage display, we obtained two single-chain variable fragment antibodies (scFvs) against this peptide sequence of human L1, hereafter called H3 peptide. Both scFvs recognize the H3 peptide and the extracellular domain of L1, as tested by enzyme-linked immunosorbent assay (ELISA), Western blot analysis and immunofluorescence staining of L1 expresssing cells. Furthermore, both scFvs reduce U-87 MG cell adhesion to fibronectin, while stimulating cell migration. Application of scFvs to human neuroblastoma SK-N-SH cells promote process outgrowth. Similar to triggering of endogenous L1 functions at the cell surface, both scFvs activate the signal transducers Erk and Src in these cells. Our results indicate that scFvs against a functionally pivotal domain in L1 trigger its regeneration-beneficial functions in vitro, encouraging thoughts on therapy of neurodegenerative diseases in the hope to ameliorate human nervous system diseases.

  12. Preparation and evaluation of an immunoaffinity sorbent with Fab' antibody fragments for the analysis of opioid peptides by on-line immunoaffinity solid-phase extraction capillary electrophoresis-mass spectrometry.

    PubMed

    Medina-Casanellas, Silvia; Benavente, Fernando; Barbosa, José; Sanz-Nebot, Victoria

    2013-07-30

    An immunoaffinity (IA) sorbent with antibody fragments was prepared for the analysis of opioid peptides by on-line immunoaffinity solid-phase extraction capillary electrophoresis-mass spectrometry (IA-SPE-CE-MS). The antibody fragmentation was evaluated by MALDI-TOF-MS. Fab' fragments obtained from a polyclonal IgG antibody against Endomorphins 1 and 2 (End1 and End2) were covalently attached to succinimidyl silica particles to prepare the IA sorbent. An IA-SPE-CE-MS methodology was established analyzing standard solutions of End1 and End2 and acceptable repeatability, linearity ranges and LODs (0.5 and 5 ng mL(-1), respectively) were obtained. The LOD of End1 was slightly better than that previously obtained using an IA sorbent with intact antibodies (1 ng mL(-1)). In human plasma samples, End1 and End2 could be detected at 1 and 50 ng mL(-1), respectively, which meant an improvement of 100 and 2-fold with regard to the LODs using an IA sorbent with intact antibodies (100 ng mL(-1)).

  13. Single Chain Antibodies Against gp55 of Human Cytomegalovirus (HCMV) for Prophylaxis and Treatment of HCMV Infections

    PubMed Central

    Moazen, Bahareh; Ebrahimi, Elahe; Nejatollahi, Foroogh

    2016-01-01

    Background: Immunotherapy is a promising prospective new treatment for cytomegalovirus (CMV) infections. Neutralizing effects have been reported using monoclonal antibodies. Recombinant single chain antibodies (scFvs) due to their advantages over monoclonal antibodies are potential alternatives and provide valuable clinical agents. Objectives: The aim of this study was to select specific single chain antibodies against gp55 of CMV and to evaluate their neutralizing effects. In the present study, we selected specific single chain antibodies against glycoprotein 55 (gp55) of CMV for their use in treatment and diagnosis. Materials and Methods: Single chain antibodies specific against an epitope located in the C-terminal part of gp55 were selected from a phage antibody display library. After four rounds of panning, twenty clones were amplified by the polymerase chain reaction (PCR) and fingerprinted by MvaI restriction enzyme. The reactivities of the specific clones were tested by the enzyme-linked immunosorbent assay (ELISA) and the neutralizing effects were evaluated by the plaque reduction assay. Results: Fingerprinting of selected clones revealed three specific single chain antibodies (scFv1, scFv2 and scFv3) with frequencies 25%, 20 and 20%. The clones produced positive ELISA with the corresponding peptide. The percentages of plaque reduction for scFv1, scFv2 and scFv3 were 23.7, 68.8 and 11.6, respectively. Conclusions: Gp55 of human CMV is considered as an important candidate for immunotherapy. In this study, we selected three specific clones against gp55. The scFvs reacted only with the corresponding peptide in a positive ELISA. The scFv2 with 68.8% neutralizing effect showed the potential to be considered for prophylaxis and treatment of CMV infections, especially in solid organ transplant recipients, for whom treatment of CMV is urgently needed. The scFv2 with neutralizing effect of 68.8%, has the potential to be considered for treatment of these patients

  14. Optimization of IGF-1R SPECT/CT imaging using 111In-labeled F(ab')2 and Fab fragments of the monoclonal antibody R1507.

    PubMed

    Heskamp, Sandra; van Laarhoven, Hanneke W M; Molkenboer-Kuenen, Janneke D M; Bouwman, Wilbert H; van der Graaf, Winette T A; Oyen, Wim J G; Boerman, Otto C

    2012-08-06

    The insulin-like growth factor 1 receptor (IGF-1R) is a potential new target for the treatment of breast cancer. Patients with breast cancer lesions that express IGF-1R may benefit from treatment with anti-IGF-1R antibodies. IGF-1R expression can be visualized using radiolabeled R1507, a monoclonal antibody directed against IGF-1R. However, antibodies clear slowly from the circulation, resulting in low tumor-to-background ratios early after injection. Therefore, we aimed to accelerate targeting of IGF-1R using radiolabeled R1507 F(ab')2 and Fab fragments. In vitro, immunoreactivity, binding affinity and internalization of R1507 IgG, F(ab')2 and Fab were determined using the triple negative IGF-1R-expressing breast cancer cell line SUM149. In vivo, pharmacokinetics of (111)In-labeled R1507 IgG, F(ab')2 and Fab were studied in mice bearing subcutaneous SUM149