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Sample records for affinity human antibodies

  1. Humanization of high-affinity antibodies targeting glypican-3 in hepatocellular carcinoma

    PubMed Central

    Zhang, Yi-Fan; Ho, Mitchell

    2016-01-01

    Glypican-3 (GPC3) is a cell-surface heparan sulfate proteoglycan highly expressed in hepatocellular carcinoma (HCC). We have generated a group of high-affinity mouse monoclonal antibodies targeting GPC3. Here, we report the humanization and testing of these antibodies for clinical development. We compared the affinity and cytotoxicity of recombinant immunotoxins containing mouse single-chain variable regions fused with a Pseudomonas toxin. To humanize the mouse Fvs, we grafted the combined KABAT/IMGT complementarity determining regions (CDR) into a human IgG germline framework. Interestingly, we found that the proline at position 41, a non-CDR residue in heavy chain variable regions (VH), is important for humanization of mouse antibodies. We also showed that two humanized anti-GPC3 antibodies (hYP7 and hYP9.1b) in the IgG format induced antibody-dependent cell-mediated cytotoxicity and complement-dependent-cytotoxicity in GPC3-positive cancer cells. The hYP7 antibody was tested and showed inhibition of HCC xenograft tumor growth in nude mice. This study successfully humanizes and validates high affinity anti-GPC3 antibodies and sets a foundation for future development of these antibodies in various clinical formats in the treatment of liver cancer. PMID:27667400

  2. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

    PubMed Central

    Noy-Porat, Tal; Rosenfeld, Ronit; Ariel, Naomi; Epstein, Eyal; Alcalay, Ron; Zvi, Anat; Kronman, Chanoch; Ordentlich, Arie; Mazor, Ohad

    2016-01-01

    Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication. PMID:26950154

  3. Purification of antibodies to O antigen of Salmonella Typhimurium from human serum by affinity chromatography.

    PubMed

    O'Shaughnessy, Colette M; Micoli, Francesca; Gavini, Massimiliano; Goodall, Margaret; Cobbold, Mark; Saul, Allan; Maclennan, Calman A

    2013-01-31

    Nontyphoidal Salmonellae (NTS) are a common cause of bacteraemia in children and HIV-infected adults in Sub-Saharan Africa. We have previously shown that antibodies play a key role in both bactericidal and cellular mechanisms of immunity to NTS, but found that high concentrations of antibody to Salmonella Typhimurium O antigen (OAg) in the serum of some HIV-infected African adults is associated with impaired killing of NTS. To further investigate the function of antibodies to the OAg of NTS, we developed a method to purify these antibodies from human serum by affinity chromatography. Purified Salmonella Typhimurium OAg was activated with adipic acid dihydrazide (ADH) via two different chemistries before linking to N-hydroxysuccinamide-Sepharose resin: one ADH molecule was introduced per OAg chain on its terminal 3-deoxy-D-manno-octulosonic acid sugar (OAg-ADH), or multiple ADH molecules were attached along the OAg chain after oxidation with sodium periodate (OAgoxADH). Both resulting columns worked well when tested with commercial polyclonal anti-O:4,5 antibodies from rabbit serum. Over 90% of the applied antibodies bound to the resin and 89% of these antibodies were then eluted as detected by ELISA. OAg-ADH was preferred as the method for OAg derivatisation as it does not modify the saccharide chain and can be applied to OAg from different bacteria. Both columns were able to bind OAg-specific antibodies in human serum, but antibody recovery was initially low. Different elution buffers were tested and different amounts of OAg-ADH were linked to the resin to improve the yield. Optimal recovery (51%) was obtained by loading 1mg of activated OAg per ml of resin and eluting with 0.1M glycine, 0.1M NaCl pH2.4. The column matrix could be regenerated following elution with no detectable loss in performance for over ten uses. This method offers the potential to purify antibodies to Salmonella OAg from polyclonal serum following vaccination or natural exposure to Salmonella

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

    PubMed

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

    2013-02-01

    We showed previously that humanization of 528, a murine anti-epidermal growth factor receptor (EGFR) antibody, causes reduced affinity for its target. Here, to improve the affinity of the humanized antibody for use in cancer immunotherapy, we constructed phage display libraries focused on the complementarity-determining regions (CDRs) of the antibody and carried out affinity selection. Two-step selections using libraries constructed in a stepwise manner enabled a 32-fold affinity enhancement of humanized 528 (h528). Thermodynamic analysis of the interactions between the variable domain fragment of h528 (h528Fv) mutants and the soluble extracellular domain of EGFR indicated that the h528Fv mutants obtained from the first selection showed a large increase in negative enthalpy change due to binding, resulting in affinity enhancement. Furthermore, mutants from the second selection showed a decrease in entropy loss, which led to further affinity maturation. These results suggest that a single mutation in the heavy chain variable domain (i.e. Tyr(52) to Trp) enthalpically contributed for overcoming the energetic barrier to the antigen-antibody interaction, which was a major hurdle for the in vitro affinity maturation of h528. We reported previously that the humanized bispecific diabody hEx3 Db, which targets EGFR and CD3, shows strong anti-tumor activity. hEx3 Db mutants, in which the variable domains of h528 were replaced with those of the affinity-enhanced mutants, were prepared and characterized. In a growth inhibition assay of tumor cells, the hEx3 Db mutants showed stronger anti-tumor activity than that of hEx3 Db, suggesting that affinity enhancement of h528Fv enhances the anti-tumor activity of the bispecific diabody. PMID:23118340

  5. Affinity purification of antibodies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Antibodies are provided in a variety of formats that includes antiserum, hybridoma culture supernatant or ascites. They can all be used successfully in crude form for the detection of target antigens by immunoassay. However, it is advantageous to use purified antibody in defined quantity to facil...

  6. Affinity Purification of Antibodies.

    PubMed

    Hnasko, Robert M; McGarvey, Jeffery A

    2015-01-01

    Antibodies are provided in a variety of formats that include antiserum, hybridoma culture supernatant, or ascites. They can all be used successfully in crude form for the detection of target antigens by immunoassay. However, it is advantageous to use purified antibody in defined quantity to facilitate assay reproducibility, economy, and reduced interference of nonspecific components as well as improved storage, stability, and bio-conjugation. Although not always necessary, the relative simplicity of antibody purification using commercially available protein-A, protein-G, or protein-L resins with basic chromatographic principles warrants purification when antibody source material is available in sufficient quantity. Here, we define three simple methods using immobilized (1) protein-A, (2) protein-G, and (3) protein-L agarose beads to yield highly purified antibody. PMID:26160561

  7. Engineering antibody affinity and specificity.

    PubMed

    Webster, D M; Roberts, S; Cheetham, J C; Griest, R; Rees, A R

    1988-01-01

    A combination of ab initio calculations, "knowledge-based prediction", molecular graphics and site-directed mutagenesis has enabled us to probe the molecular details of antibody:antigen recognition and binding and to alter the affinity and specificity of an antibody for its antigen. The significance of electrostatic hydrogen bonding, hydrophilic/hydrophobic patch matching and van der Waals interactions as well as CDR:CDR interactions are discussed in relation to the results of site-directed mutagenesis experiments on the anti-lysozyme antibody Gloop2. The ability to generate reconstructed antibodies, chimeric antibodies, catalytic antibodies and the use of modelled antibodies for the design of drugs is discussed. PMID:3209295

  8. Germline variable region gene segment derivation of human monoclonal anti-Rh(D) antibodies. Evidence for affinity maturation by somatic hypermutation and repertoire shift.

    PubMed Central

    Bye, J M; Carter, C; Cui, Y; Gorick, B D; Songsivilai, S; Winter, G; Hughes-Jones, N C; Marks, J D

    1992-01-01

    To date, there has been no systematic study of the process of affinity maturation of human antibodies. We therefore sequenced the variable region genes (V genes) of 14 human monoclonal antibodies specific for the erythrocyte Rh(D) alloantigen and determined the germline gene segments of origin and extent of somatic hypermutation. These data were correlated with determinations of antibody affinity. The four IgM antibodies (low affinity) appear to be derived from two germline heavy chain variable region gene segments and one or two germline light chain variable region gene segments and were not extensively mutated. The 10 IgG antibodies (higher affinity) appear to be derived from somatic hypermutation of these V gene segments and by use of new V gene segments or V gene segment combinations (repertoire shift). Affinity generally increased with increasing somatic hypermutation; on average, there were 8.9 point mutations in the V gene segments of the four IgM antibodies (Ka = 1-4 x 10(7)/M-1) compared with 19 point mutations in the V gene segments of the 10 IgG antibodies. The four highest affinity antibodies (Ka = 0.9-3 x 10(9)/M-1) averaged 25.5 point mutations. The use of repertoire shift and somatic hypermutation in affinity maturation of human alloantibodies is similar to data obtained in inbred mice immunized with haptens. PMID:1469099

  9. Affinity chromatography of human leukocyte and diploid cell interferons on sepharose-bound antibodies.

    PubMed

    Berg, K; Ogburn, C A; Paucker, K; Mogensen, K E; Cantell, K

    1975-02-01

    Interferons produced in human peripheral leukocytes (LE) and foreskin fibroblast (FS-4) cells were subjected to affinity chromatography on Sepharose-bound globulins from rabbits immunized with these interferons. Anti-LE interferon sera neutralized both interferons, but titers against FS-4 interferon were consistently lower than those against LE interferon. Anti-FS-4 interferon sera neutralized only FS-4 but not LE interferon. Accordingly, affinity columns constructed with anti-FS-4 globulin excluded LE but not FS-4 interferon, whereas those prepared with anti-LE interferon globulin bound and eluted both LE and FS-4 interferons. Purification of native interferons of both types on anti-LE interferon-Sepharose ranged from 680- to 3,600-fold and recoveries from 72 to 126%. Specific activities of eluate pools varied from 4 to 30 times 10-6 reference (B, 69/19) units per milligram protien.

  10. Generation of a novel high-affinity monoclonal antibody with conformational recognition epitope on human IgM.

    PubMed

    Sarikhani, Sina; Mirshahi, Manouchehr; Gharaati, Mohammad Reza; Mirshahi, Tooran

    2010-11-01

    As IgM is the first isotype of antibody which appears in blood after initial exposure to a foreign antigen in the pattern of primary response, detection, and quantification of this molecule in blood seems invaluable. To approach these goals, generation, and characterization of a highly specific mAb (monoclonal antibody) against human IgM were investigated. Human IgM immunoglobulins were used to immunize Balb/c mice. Spleen cells taken from the immunized animals were fused with SP2/O myeloma cells using PEG (polyethylene glycol, MW 1450) as fusogen. The hybridomas were cultured in HAT containing medium and supernatants from the growing hybrids were screened by enzyme-linked immunosorbent assay (ELISA) using plates coated with pure human IgM and the positive wells were then cloned at limiting dilutions. The best clone designated as MAN-1, was injected intraperitoneally to some Pristane-injected mice. Anti-IgM mAb was purified from the animals' ascitic fluid by protein-G sepharose followed by DEAE-cellulose ion exchange chromatography. MAN-1 interacted with human IgM with a very high specificity and affinity. The purity of the sample was tested by SDS-PAGE and the affinity constant was measured (K(a) = 3.5 x 10(9)M(-1). Immunoblotting and competitive ELISA were done and the results showed that the harvested antibody recognizes a conformational epitope on the mu chain of human IgM and there was no cross-reactivity with other subclasses of immunoglobulins. Furthermore, isotyping test was done and the results showed the subclass of the obtained mAb which was IgG(1)kappa. PMID:20162378

  11. Affinity maturation of a novel antagonistic human monoclonal antibody with a long VH CDR3 targeting the Class A GPCR formyl-peptide receptor 1.

    PubMed

    Douthwaite, Julie A; Sridharan, Sudharsan; Huntington, Catherine; Hammersley, Jayne; Marwood, Rose; Hakulinen, Jonna K; Ek, Margareta; Sjögren, Tove; Rider, David; Privezentzev, Cyril; Seaman, Jonathan C; Cariuk, Peter; Knights, Vikki; Young, Joyce; Wilkinson, Trevor; Sleeman, Matthew; Finch, Donna K; Lowe, David C; Vaughan, Tristan J

    2015-01-01

    Therapeutic monoclonal antibodies targeting G-protein-coupled receptors (GPCRs) are desirable for intervention in a wide range of disease processes. The discovery of such antibodies is challenging due to a lack of stability of many GPCRs as purified proteins. We describe here the generation of Fpro0165, a human anti-formyl peptide receptor 1 (FPR1) antibody generated by variable domain engineering of an antibody derived by immunization of transgenic mice expressing human variable region genes. Antibody isolation and subsequent engineering of affinity, potency and species cross-reactivity using phage display were achieved using FPR1 expressed on HEK cells for immunization and selection, along with calcium release cellular assays for antibody screening. Fpro0165 shows full neutralization of formyl peptide-mediated activation of primary human neutrophils. A crystal structure of the Fpro0165 Fab shows a long, protruding VH CDR3 of 24 amino acids and in silico docking with a homology model of FPR1 suggests that this long VH CDR3 is critical to the predicted binding mode of the antibody. Antibody mutation studies identify the apex of the long VH CDR3 as key to mediating the species cross-reactivity profile of the antibody. This study illustrates an approach for antibody discovery and affinity engineering to typically intractable membrane proteins such as GPCRs.

  12. Generation, affinity maturation, and characterization of a human anti-human NKG2D monoclonal antibody with dual antagonistic and agonistic activity

    PubMed Central

    Kwong, Ka Yin; Baskar, Sivasubramanian; Zhang, Hua; Mackall, Crystal L.; Rader, Christoph

    2008-01-01

    Summary In humans, NKG2D is an activating receptor on NK cells and a costimulatory receptor on certain T cells and plays a central role in mediating immune responses in autoimmune diseases, infectious diseases, and cancer. Monoclonal antibodies that antagonize or agonize immune responses mediated by human NKG2D are considered to be of broad and potent therapeutic utility. Nonetheless, monoclonal antibodies to NKG2D that are suitable for clinical investigations have not been published yet. Here we describe the generation, affinity maturation, and characterization of a fully human monoclonal antibody to human NKG2D. Using phage display technology based on a newly generated naïve human Fab library in phage display vector pC3C followed by a tandem chain shuffling process designed for minimal deviation from natural human antibody sequences, we selected a human Fab, designated KYK-2.0, with high specificity and affinity to human NKG2D. KYK-2.0 Fab blocked the binding of the natural human NKG2D ligands MICA, MICB, and ULBP2 as potently as a commercially available mouse anti-human NKG2D monoclonal antibody in IgG format. Conversion of KYK-2.0 Fab to IgG1 resulted in subnanomolar avidity for human NKG2D. KYK-2.0 IgG1 was found to selectively recognize defined subpopulations of human lymphocytes known to express NKG2D, i.e. the majority of human CD8+, CD16+, and CD56+ cells as well as a small fraction of human CD4+ cells. In solution, KYK-2.0 IgG1 interfered with the cytolytic activity of ex vivo expanded human NK cells. By contrast, immobilized KYK-2.0 IgG1 was found to strongly induce human NK cell activation. The dual antagonistic and agonistic activity promises a wide range of therapeutic applications for KYK-2.0 IgG1 and its derivatives. PMID:18809410

  13. Improving antibody binding affinity and specificity for therapeutic development.

    PubMed

    Bostrom, Jenny; Lee, Chingwei V; Haber, Lauric; Fuh, Germaine

    2009-01-01

    Affinity maturation is an important part of the therapeutic antibody development process as in vivo activity often requires high binding affinity. Here, we describe a targeted approach for affinity improvement of therapeutic antibodies. Sets of CDR residues that are solvent accessible and relatively diverse in natural antibodies are targeted for diversification. Degenerate oligonucleotides are used to generate combinatorial phage-displayed antibody libraries with varying degree of diversity at randomized positions from which high-affinity antibodies can be selected. An advantage of using antibodies for therapy is their exquisite target specificity, which enables selective antigen binding and reduces off-target effects. However, it can be useful, and often it is necessary, to generate cross-reactive antibodies binding to not only the human antigen but also the corresponding non-human primate or rodent orthologs. Such cross-reactive antibodies can be used to validate the therapeutic targeting and examine the safety profile in preclinical animal models before committing to a costly development track. We show how affinity improvement and cross-species binding can be achieved in a one-step process.

  14. AGIA Tag System Based on a High Affinity Rabbit Monoclonal Antibody against Human Dopamine Receptor D1 for Protein Analysis

    PubMed Central

    Yano, Tomoya; Takeda, Hiroyuki; Uematsu, Atsushi; Yamanaka, Satoshi; Nomura, Shunsuke; Nemoto, Keiichirou; Iwasaki, Takahiro; Takahashi, Hirotaka; Sawasaki, Tatsuya

    2016-01-01

    Polypeptide tag technology is widely used for protein detection and affinity purification. It consists of two fundamental elements: a peptide sequence and a binder which specifically binds to the peptide tag. In many tag systems, antibodies have been used as binder due to their high affinity and specificity. Recently, we obtained clone Ra48, a high-affinity rabbit monoclonal antibody (mAb) against dopamine receptor D1 (DRD1). Here, we report a novel tag system composed of Ra48 antibody and its epitope sequence. Using a deletion assay, we identified EEAAGIARP in the C-terminal region of DRD1 as the minimal epitope of Ra48 mAb, and we named this sequence the “AGIA” tag, based on its central sequence. The tag sequence does not include the four amino acids, Ser, Thr, Tyr, or Lys, which are susceptible to post-translational modification. We demonstrated performance of this new tag system in biochemical and cell biology applications. SPR analysis demonstrated that the affinity of the Ra48 mAb to the AGIA tag was 4.90 × 10−9 M. AGIA tag showed remarkably high sensitivity and specificity in immunoblotting. A number of AGIA-fused proteins overexpressed in animal and plant cells were detected by anti-AGIA antibody in immunoblotting and immunostaining with low background, and were immunoprecipitated efficiently. Furthermore, a single amino acid substitution of the second Glu to Asp (AGIA/E2D) enabled competitive dissociation of AGIA/E2D-tagged protein by adding wild-type AGIA peptide. It enabled one-step purification of AGIA/E2D-tagged recombinant proteins by peptide competition under physiological conditions. The sensitivity and specificity of the AGIA system makes it suitable for use in multiple methods for protein analysis. PMID:27271343

  15. Modern affinity reagents: Recombinant antibodies and aptamers.

    PubMed

    Groff, Katherine; Brown, Jeffrey; Clippinger, Amy J

    2015-12-01

    Affinity reagents are essential tools in both basic and applied research; however, there is a growing concern about the reproducibility of animal-derived monoclonal antibodies. The need for higher quality affinity reagents has prompted the development of methods that provide scientific, economic, and time-saving advantages and do not require the use of animals. This review describes two types of affinity reagents, recombinant antibodies and aptamers, which are non-animal technologies that can replace the use of animal-derived monoclonal antibodies. Recombinant antibodies are protein-based reagents, while aptamers are nucleic-acid-based. In light of the scientific advantages of these technologies, this review also discusses ways to gain momentum in the use of modern affinity reagents, including an update to the 1999 National Academy of Sciences monoclonal antibody production report and federal incentives for recombinant antibody and aptamer efforts. In the long-term, these efforts have the potential to improve the overall quality and decrease the cost of scientific research.

  16. Visualizing Antibody Affinity Maturation in Germinal Centers

    PubMed Central

    Tas, Jeroen M.J.; Mesin, Luka; Pasqual, Giulia; Targ, Sasha; Jacobsen, Johanne T.; Mano, Yasuko M.; Chen, Casie S.; Weill, Jean-Claude; Reynaud, Claude-Agnès; Browne, Edward P.; Meyer-Hermann, Michael; Victora, Gabriel D.

    2016-01-01

    Antibodies somatically mutate to attain high affinity in germinal centers (GCs). There, competition between B cell clones and among somatic mutants of each clone drives an increase in average affinity across the population. The extent to which higher-affinity cells eliminating competitors restricts clonal diversity is unknown. By combining multiphoton microscopy and sequencing, we show that tens to hundreds of distinct B cell clones seed each GC, and that GCs lose clonal diversity at widely disparate rates. Furthermore, efficient affinity maturation can occur in the absence of homogenizing selection, ensuring that many clones can mature in parallel within the same GC. Our findings have implications for development of vaccines in which antibodies with non-immunodominant specificities must be elicited, as is the case for HIV-1 and influenza. PMID:26912368

  17. Latest technologies for the enhancement of antibody affinity.

    PubMed

    Wark, Kim L; Hudson, Peter J

    2006-08-01

    High affinity antibodies are crucial both for the discovery and validation of biomarkers for human health and disease and as clinical diagnostic and therapeutic reagents. This review describes some of the latest technologies for the design, mutation and selection of high affinity antibodies that provide a paradigm for molecular evolution of a far wider range of proteins including enzymes. Strategies include both in vivo and in vitro methods and embrace the latest concepts for antibody display and selection. Specifically, affinity enhancement can be tailored to the target-binding surface, typically the complementary determining region (CDR) loops in antibodies, whereas enhanced stability, expression or catalytic properties can be affected by selected changes to the core protein scaffold. Together, these technologies provide a rapid and powerful strategy to drive the next generation of protein-based reagents for numerous clinical, environmental and agribusiness applications.

  18. Antibody-free magnetic cell sorting of genetically modified primary human CD4+ T cells by one-step streptavidin affinity purification.

    PubMed

    Matheson, Nicholas J; Peden, Andrew A; Lehner, Paul J

    2014-01-01

    Existing methods for phenotypic selection of genetically modified mammalian cells suffer disadvantages of time, cost and scalability and, where antibodies are used to bind exogenous cell surface markers for magnetic selection, typically yield cells coated with antibody-antigen complexes and beads. To overcome these limitations we have developed a method termed Antibody-Free Magnetic Cell Sorting in which the 38 amino acid Streptavidin Binding Peptide (SBP) is displayed at the cell surface by the truncated Low Affinity Nerve Growth Receptor (LNGFRF) and used as an affinity tag for one-step selection with streptavidin-conjugated magnetic beads. Cells are released through competition with the naturally occurring vitamin biotin, free of either beads or antibody-antigen complexes and ready for culture or use in downstream applications. Antibody-Free Magnetic Cell Sorting is a rapid, cost-effective, scalable method of magnetic selection applicable to either viral transduction or transient transfection of cell lines or primary cells. We have optimised the system for enrichment of primary human CD4+ T cells expressing shRNAs and exogenous genes of interest to purities of >99%, and used it to isolate cells following Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing.

  19. Humanized Affinity-matured Monoclonal Antibody 8H9 Has Potent Antitumor Activity and Binds to FG Loop of Tumor Antigen B7-H3.

    PubMed

    Ahmed, Mahiuddin; Cheng, Ming; Zhao, Qi; Goldgur, Yehuda; Cheal, Sarah M; Guo, Hong-fen; Larson, Steven M; Cheung, Nai-kong V

    2015-12-11

    B7-H3 (CD276) is both an inhibitory ligand for natural killer cells and T cells and a tumor antigen that is widely expressed among human solid tumors. Anti-B7-H3 mouse monoclonal antibody 8H9 has been successfully used for radioimmunotherapy for patients with B7-H3(+) tumors. We present the humanization, affinity maturation, and epitope mapping of 8H9 based on structure determination, modeling, and yeast display methods. The crystal structure of ch8H9 Fab fragment was solved to 2.5-Å resolution and used as a template for humanization. By displaying the humanized 8H9 single chain Fv (scFv) on the surface of yeast, the affinity was matured by sequential random mutagenesis and fluorescence-activated cell sorting. Six mutations (three in the complementarity-determining region and three in the framework regions) were identified and incorporated into an affinity-matured humanized 8H9 construct (hu8H9-6m) and an affinity-matured chimeric 8H9 construct (ch8H9-6m). The hu8H9-6m scFv had a 160-fold improvement in affinity (0.9 nm KD) compared with parental hu8H9 scFv (144 nm KD). The IgG formats of ch8H9-6m and hu8H9-6m (nanomolar to subnanomolar KD) had 2-9-fold enhancements in affinity compared with their parental forms, potent in vitro antibody-dependent cell-mediated cytotoxicity (0.1-0.3 μg/ml EC50), and high tumor uptake in mouse xenografts. Based on in silico docking studies and experimental validation, the molecular epitope of 8H9 was determined to be dependent on the FG loop of B7-H3, a region critical to its function in immunologic blockade and unique among anti-B7-H3 antibodies published to date. PMID:26487718

  20. FYWHCLDE-based affinity chromatography of IgG: effect of ligand density and purifications of human IgG and monoclonal antibody.

    PubMed

    Zhao, Wei-Wei; Shi, Qing-Hong; Sun, Yan

    2014-08-15

    This work reports the development of an octapeptide-based affinity adsorbent for the purification of human IgG (hIgG) and monoclonal antibody (mAb). The octapeptide was FYWHCLDE selected earlier by the biomimetic design of affinity peptide ligands for hIgG. The ligand was coupled to Sepharose gel at four densities from 10.4 to 31.0μmol/mL, and the effect of peptide density on the adsorption of hIgG and bovine serum albumin (BSA) was first investigated. The binding capacity of hIgG increased from 104.2 to 176.4mg/mL within the ligand density range, and the binding affinity (dissociation constant) kept at 2.4-3.7μM. Batch adsorption revealed that the selectivity of FYWHCLDE-Sepharose for IgG was 30-40 times over BSA. The effective pore diffusivity of IgG decreased somewhat with increasing ligand density, but the dynamic binding capacity at 10% breakthrough, measured by using 10-fold diluted human serum as feedstock, doubled with increasing ligand density from 10.4 to 31.0μmol/mL due to the remarkable increase of static binding capacity. By using the affinity column with a ligand density of 23.9μmol/mL, hIgG and humanized mAb purifications from human serum and cell culture supernatant, respectively, were achieved at high purities and recovery yields. Finally, the robustness of the peptide gel was demonstrated by recycled use of the affinity column in 20 breakthrough cycles. PMID:24947889

  1. Molecular engineering of high affinity single-chain antibody fragment for endothelial targeting of proteins and nanocarriers in rodents and humans.

    PubMed

    Greineder, Colin F; Hood, Elizabeth D; Yao, Anning; Khoshnejad, Makan; Brenner, Jake S; Johnston, Ian H; Poncz, Mortimer; Gottstein, Claudia; Muzykantov, Vladimir R

    2016-03-28

    Endothelial cells (EC) represent an important target for pharmacologic intervention, given their central role in a wide variety of human pathophysiologic processes. Studies in lab animal species have established that conjugation of drugs and carriers with antibodies directed to surface targets like the Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1, a highly expressed endothelial transmembrane protein) help to achieve specific therapeutic interventions in ECs. To translate such "vascular immunotargeting" to clinical practice, it is necessary to replace antibodies by advanced ligands that are more amenable to use in humans. We report the molecular design of a single chain variable antibody fragment (scFv) that binds with high affinity to human PECAM-1 and cross-reacts with its counterpart in rats and other animal species, allowing parallel testing in vivo and in human endothelial cells in microfluidic model. Site-specific modification of the scFv allows conjugation of protein cargo and liposomes, enabling their endothelial targeting in these models. This study provides a template for molecular engineering of ligands, enabling studies of drug targeting in animal species and subsequent use in humans. PMID:26855052

  2. Selective targeting of the IL23 pathway: Generation and characterization of a novel high-affinity humanized anti-IL23A antibody

    PubMed Central

    Singh, Sanjaya; Kroe-Barrett, Rachel R; Canada, Keith A; Zhu, Xiang; Sepulveda, Eliud; Wu, Helen; He, Yaqin; Raymond, Ernest L; Ahlberg, Jennifer; Frego, Lee E; Amodeo, Laura M; Catron, Katrina M; Presky, David H; Hanke, Jeffrey H

    2015-01-01

    Herein, we describe the generation and characterization of BI 655066, a novel, highly potent neutralizing anti-interleukin-23 (IL23) monoclonal antibody in clinical development for autoimmune conditions, including psoriasis and Crohn's disease. IL23 is a key driver of the differentiation, maintenance, and activity of a number of immune cell subsets, including T helper 17 (Th17) cells, which are believed to mediate the pathogenesis of several immune-mediated disorders. Thus, IL23 neutralization is an attractive therapeutic approach. Designing an antibody for clinical activity and convenience for the patient requires certain properties, such as high affinity, specificity, and solubility. These properties were achieved by directed design of the immunization, lead identification, and humanization procedures. Favorable substance and pharmacokinetic properties were established by biophysical assessments and studies in cynomolgus monkeys. PMID:25905918

  3. Strain specificity and binding affinity requirements of neutralizing monoclonal antibodies to the C4 domain of gp120 from human immunodeficiency virus type 1.

    PubMed Central

    Nakamura, G R; Byrn, R; Wilkes, D M; Fox, J A; Hobbs, M R; Hastings, R; Wessling, H C; Norcross, M A; Fendly, B M; Berman, P W

    1993-01-01

    The binding properties of seven CD4-blocking monoclonal antibodies raised against recombinant gp120 of human immunodeficiency virus type 1 strain MN (HIV-1MN) and two CD4-blocking monoclonal antibodies to recombinant envelope glycoproteins gp120 and gp160 of substrain IIIB of HIVLAI were analyzed. With a panel of recombinant gp120s from seven diverse HIV-1 isolates, eight of the nine antibodies were found to be strain specific and one was broadly cross-reactive. Epitope mapping revealed that all nine antibodies bound to epitopes located in the fourth conserved domain (C4) of gp120. Within this region, three distinct epitopes could be identified: two were polymorphic between HIV-1 strains, and one was highly conserved. Studies with synthetic peptides demonstrated that the conserved epitope, recognized by antibody 13H8, was located between residues 431 and 439. Site-directed mutagenesis of gp120 demonstrated that residue 429 and/or 432 was critical for the binding of the seven antibodies to gp120 from HIV-1MN. Similarly, residues 423 and 429 were essential for the binding of monoclonal antibody 5C2 raised against gp120 from HIV-1IIIB. The amino acids located at positions 423 and 429 were found to vary between strains of HIV-1 as well as between molecular clones derived from the MN and LAI isolates of HIV-1. Polymorphism at these positions prevented the binding of virus-neutralizing monoclonal antibodies and raised the possibility that HIV-1 neutralization serotypes may be defined on the basis of C4 domain sequences. Analysis of the binding characteristics of the CD4-blocking antibodies demonstrated that their virus-neutralizing activity was directly proportional to their gp120-binding affinity. These studies account for the strain specificity of antibodies to the C4 domain of gp120 and demonstrate for the first time that antibodies to this region can be as effective as those directed to the principal neutralizing determinant (V3 domain) in neutralizing HIV-1

  4. Affinity-purified antibodies of defined specificity for use in a solid-phase microplate radioimmunoassay of human Tamm-Horsfall glycoprotein in urine.

    PubMed

    Hunt, J S; McGiven, A R; Groufsky, A; Lynn, K L; Taylor, M C

    1985-05-01

    Rabbit antibodies to human Tamm-Horsfall glycoprotein (prepared by salt precipitation from normal urine) were purified by affinity chromatography using columns containing Tamm-Horsfall glycoprotein linked to CNBr-activated Sepharose 4B. The specificity of these antibodies was determined by analysis of their binding characteristics on Western blots of Tamm-Horsfall protein from sodium dodecyl sulphate/polyacrylamide gradient gels and comparison with the reactivity of monoclonal antibodies to this glycoprotein. Optimal conditions of adsorption to poly(vinyl chloride) microtitre plates were established such that these purified antibodies could be used in a solid-phase radioimmunoassay for the determination of urinary Tamm-Horsfall-glycoprotein concentration. The specificity of the immunoassay was confirmed by competitive inhibition of the urinary Tamm-Horsfall glycoprotein by purified freeze-dried material in solution. A standard curve obtained with this material showed the radioimmunoassay to have a sensitivity of at least 5 ng/ml, with linearity between 30 and 600 ng/ml. The mean coefficient of variation over the linear section of the curve was 11.3 +/- 2.2% (n = 13). The effects of dialysis and freezing of urine samples before determination of Tamm-Horsfall-glycoprotein concentrations were investigated and the mean 24 h urinary excretion rate in 60 normal donors was shown to be 84.9 +/- 44.1 mg.

  5. Production and characterization of high-affinity human monoclonal antibodies to human immunodeficiency virus type 1 envelope glycoproteins in a mouse model expressing human immunoglobulins.

    PubMed

    Sheppard, Neil C; Davies, Sarah L; Jeffs, Simon A; Vieira, Sueli M; Sattentau, Quentin J

    2007-02-01

    Human (Hu) monoclonal antibodies (MAbs) against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) are useful tools in the structural and functional analysis of Env, are under development both as potential prophylaxis and as therapy for established HIV-1 infection, and have crucial roles in guiding the design of preventative vaccines. Despite representing more than 50% of infections globally, no MAbs have been generated in any species against C clade HIV-1 Env. To generate HuMAbs to a novel Chinese C clade Env vaccine candidate (primary isolate strain HIV-1(97CN54)), we used BAB5 mice that express a human immunoglobulin (Ig) M antibody repertoire in place of endogenous murine immunoglobulins. When immunized with HIV-1(97CN54) Env, these mice developed antigen-specific IgM antibodies. Hybridoma fusions using splenocytes from these mice enabled the isolation of two Env-specific IgM HuMAbs: N3C5 and N03B11. N3C5 bound to HIV-1 Env from clades A and C, whereas N03B11 bound two geographically distant clade C isolates but not Env from other clades. These HuMAbs bind conformational epitopes within the immunodominant region of the gp41 ectodomain. N3C5 weakly neutralized the autologous isolate in the absence of complement and weakly enhanced infection in the presence of complement. N03B11 has no effect on infectivity in either the presence or the absence of complement. These novel HuMAbs are useful reagents for the study of HIV-1 Env relevant to the global pandemic, and mice producing human immunoglobulin present a tool for the production of such antibodies.

  6. Capillary electrophoresis of affinity complexes between subviral 80S particles of human rhinovirus and monoclonal antibody 2G2.

    PubMed

    Kremser, Leopold; Petsch, Martina; Blaas, Dieter; Kenndler, Ernst

    2006-07-01

    Human rhinoviruses (HRVs), the main etiologic agents of the common cold, transform into subviral B- or 80S particles (they sediment at 80S upon sucrose density gradient centrifugation) during infection and, in vitro, upon exposure to a temperature between 50 and 56 degrees C. With respect to the native virion they lack the genomic RNA and the viral capsid protein VP4. 80S particles are unstable and easily disintegrate into their components, VP1, VP2, and VP3 in buffers containing SDS. However, this detergent was found to be a necessary constituent of the BGE for the analysis of these viruses and their complexes with receptors and antibodies by CE. We here demonstrate that dodecylpoly(ethyleneglycol ether) (D-PEG) a nonionic detergent, is suitable for analysis of subviral particles as it preserves their integrity, in contrast to SDS. Electrophoresis of the 80S particles in borate buffer (pH 8.3, 100 mM) containing 10 mM D-PEG resulted in a well-defined electrophoretic peak. The identity of the peak was confirmed, among other means, by complexation with mAb 2G2, which recognizes a structural epitope exclusively present on subviral particles but not on native virus. Upon incubation of the 80S particles with mAb 2G2 the peak disappeared, but a new peak, attributed to the antibody complex emerged. The separation system allowed following the time course of the transformation of intact HRV serotype 2 into 80S particles upon incubation at temperatures between 40 and 65 degrees C. We also demonstrate that subviral particles derived from HRV2 labeled with the fluorescence dyes FITC or Cy3.5 were stable in the separation system containing D-PEG. Dye-modified particles were still recognized by mAb 2G2, suggesting that the exposed lysines that are derivatized by the reagent do not form part of the epitope of the antibody.

  7. Measuring an antibody affinity distribution molecule by molecule.

    PubMed

    Temirov, Jamshid P; Bradbury, Andrew R M; Werner, James H

    2008-11-15

    Single molecule fluorescence microscopy was used to observe the binding and unbinding of hapten decorated quantum dots to individual surface immobilized antibodies. The fluorescence time history from an individual antibody site can be used to calculate its binding affinity. While quantum dot blinking occurs during these measurements, we describe a simple empirical method to correct the apparent/observed affinity to account for the blinking contribution. The combination of many single molecule affinity measurements from different antibodies yields not only the average affinity, it directly measures the full shape and character of the surface affinity distribution function.

  8. Measuring an antibody affinity distribution molecule by molecule

    SciTech Connect

    Bradbury, Andrew M; Werner, James H; Temirov, Jamshid

    2008-01-01

    Single molecule fluorescence mIcroscopy was used to observe the binding and unbinding of hapten decorated quantum dots with individual surface immobilized antibodies. The fluorescence time history from an individual antibody site can be used to calculate its binding affinity. While quantum dot blinking occurs during these measurements, we describe a simple empirical method to correct the apparent/observed affinity to account for the blinking contribution. The combination of many single molecule affinity measurements from different antibodies yields not only the average affinity, it directly measures the full shape and character of the surface affinity distribution function.

  9. Isoelectric focusing-affinity immunoblot analysis of mouse monoclonal antibodies to the four human IgG subclasses

    NASA Technical Reports Server (NTRS)

    Hamilton, Robert G.; Roebber, Marianne; Rodkey, L. Scott; Reimer, Charles B.

    1987-01-01

    Isoelectric focusing (IEF)/affinity immunoblotting and enzyme-linked immunosorbent assay (ELISA) were used for parallel analysis of murine monoclonal antihuman IgG-subclass antisera (MoAbs). Coomassie Blue-stained protein bands in the pH region 5.5-8.0 were shown to be murine IgG by direct blotting onto nitrocellulose followed by detection with conjugated antimouse IgG. Use of IgG myeloma antigen-coated nitrocellulose in the IEF-affinity immunoblot allowed detection of the charge microheterogeneity of MoAbs. The MoAb group contained one to five major dense bands flanked by up to four minor fainter bands, all with pIs ranging from 6.1 to 7.8. Semiquantitative estimates of binding specificity in the IEF-affinity blot compared well with cross-reactivity data obtained from a quantitative ELISA.

  10. Antibody response and antibody affinity maturation in cats with experimental proliferative immune complex glomerulonephritis.

    PubMed

    Bishop, S A; Bailey, M; Lucke, V M; Stokes, C R

    1992-07-01

    An experimental model of proliferative glomerulonephritis (GN) in the cat, which closely resembles human proliferative forms of GN, has been used to study the role of antibody and antibody affinity in the development of immune complex-mediated renal disease. The serum IgG and IgM antibody response to antigen, average antibody affinity (avidity) and affinity heterogeneity of the IgG and IgM populations was assessed at varying times after commencement of chronic immunization with the antigen, human serum albumin (HSA), by enzyme immunoassay. Cats could be classified according to whether they were "low", "intermediate" or "high" IgG responders, by quantification of serum IgG values. Cats with the lowest serum IgG values failed to develop glomerulonephritis. However, there was no relationship between actual IgG values and the severity of the induced disease. In contrast to IgG, there was no division of cats into low or high IgM anti-HSA responders. Again, cats with the lowest IgM values failed to develop GN, but, more interestingly, a late, marked increase in serum IgM anti-HSA occurred only in cats that developed clinical signs of GN (anterior uveitis and nephrotic syndrome). Maturation of average, functional IgG affinity (avidity) for HSA following chronic immunization was clearly demonstrated for all cats. At the end of the experiment, all cats had IgG of high affinity for HSA and the average affinity heterogeneity of the IgG populations was less than in measurements taken earlier. Values of IgG affinity at the end of the experiment were very similar both in cats which developed GN and in those which remained clinically, biochemically and pathologically normal. In contrast to IgG antibody, some cats developed IgM of increased affinity, whilst others produced antibody of reduced affinity, following chronic immunization. There was no correlation between the development of disease and the production of either low or high affinity IgM antibody. Data indicated that an

  11. Calculation of antibody affinity in homogeneous and heterogeneous systems.

    PubMed

    Chalquest, R R

    1988-12-01

    Antibody affinity is an important determinant of all antibody-antigen reactions. A new computer program, AFCRV, was developed to calculate binding constants with data from a radioimmunoassay on most microcomputers in the laboratory by using constant-ratio dilution curves. Evaluation of a homogeneous or heterogeneous antibody in the presence of a single antigen can be accomplished.

  12. Antibody-based affinity cryo-EM grid.

    PubMed

    Yu, Guimei; Li, Kunpeng; Jiang, Wen

    2016-05-01

    The Affinity Grid technique combines sample purification and cryo-Electron Microscopy (cryo-EM) grid preparation into a single step. Several types of affinity surfaces, including functionalized lipids monolayers, streptavidin 2D crystals, and covalently functionalized carbon surfaces have been reported. More recently, we presented a new affinity cryo-EM approach, cryo-SPIEM, which applies the traditional Solid Phase Immune Electron Microscopy (SPIEM) technique to cryo-EM. This approach significantly simplifies the preparation of affinity grids and directly works with native macromolecular complexes without need of target modifications. With wide availability of high affinity and high specificity antibodies, the antibody-based affinity grid would enable cryo-EM studies of the native samples directly from cell cultures, targets of low abundance, and unstable or short-lived intermediate states.

  13. Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display.

    PubMed

    Hanes, J; Schaffitzel, C; Knappik, A; Plückthun, A

    2000-12-01

    Here we applied ribosome display to in vitro selection and evolution of single-chain antibody fragments (scFvs) from a large synthetic library (Human Combinatorial Antibody Library; HuCAL) against bovine insulin. In three independent ribosome display experiments different clusters of closely related scFvs were selected, all of which bound the antigen with high affinity and specificity. All selected scFvs had affinity-matured up to 40-fold compared to their HuCAL progenitors, by accumulating point mutations during the ribosome display cycles. The dissociation constants of the isolated scFvs were as low as 82 pM, which validates the design of the naïve library and the power of this evolutionary method. We have thus mimicked the process of antibody generation and affinity maturation with a synthetic library in a cell-free system in just a few days, obtaining molecules with higher affinities than most natural antibodies.

  14. Measurement of affinity of viral monoclonal antibodies by ELISA titration of free antibody in equilibrium mixtures.

    PubMed

    Azimzadeh, A; Van Regenmortel, M H

    1991-08-01

    The binding affinity of a monoclonal antibody (Mab) to tobacco mosaic virus (TMV) was determined by measuring, in an enzyme-linked immunosorbent assay, the amount of free antibody present after ultracentrifugation of virus-antibody complexes at equilibrium. In antibody excess, univalent binding of Mabs was observed and the affinity constant was K = 3.2 +/- 0.4 10(8) l/mol; in antigen excess, bivalent antibody binding was observed and the antibody avidity was about 15 times higher. In antigen excess, it was imperative to correct experimental data for the presence of 0.55% inactive molecules in the immunopurified antibody preparation. Modelling studies suggest that in the case of antibodies of increasing affinity, it becomes increasingly important to correct for the presence of inactive antibody in the binding assay.

  15. Antibody Affinity Maturation in Fishes—Our Current Understanding

    PubMed Central

    Magor, Brad G.

    2015-01-01

    It has long been believed that fish lack antibody affinity maturation, in part because they were thought to lack germinal centers. Recent research done on sharks and bony fishes indicates that these early vertebrates are able to affinity mature their antibodies. This article reviews the functionality of the fish homologue of the immunoglobulin (Ig) mutator enzyme activation-induced cytidine deaminase (AID). We also consider the protein and molecular evidence for Ig somatic hypermutation and antibody affinity maturation. In the context of recent evidence for a putative proto-germinal center in fishes we propose some possible reasons that observed affinity maturation in fishes often seems lacking and propose future work that might shed further light on this process in fishes. PMID:26264036

  16. Three Recombinant Engineered Antibodies against Recombinant Tags with High Affinity and Specificity.

    PubMed

    Zhao, Hongyu; Shen, Ao; Xiang, Yang K; Corey, David P

    2016-01-01

    We describe three recombinant engineered antibodies against three recombinant epitope tags, constructed with divalent binding arms to recognize divalent epitopes and so achieve high affinity and specificity. In two versions, an epitope is inserted in tandem into a protein of interest, and a homodimeric antibody is constructed by fusing a high-affinity epitope-binding domain to a human or mouse Fc domain. In a third, a heterodimeric antibody is constructed by fusing two different epitope-binding domains which target two different binding sites in GFP, to polarized Fc fragments. These antibody/epitope pairs have affinities in the low picomolar range and are useful tools for many antibody-based applications.

  17. Three Recombinant Engineered Antibodies against Recombinant Tags with High Affinity and Specificity

    PubMed Central

    Zhao, Hongyu; Shen, Ao; Xiang, Yang K.; Corey, David P.

    2016-01-01

    We describe three recombinant engineered antibodies against three recombinant epitope tags, constructed with divalent binding arms to recognize divalent epitopes and so achieve high affinity and specificity. In two versions, an epitope is inserted in tandem into a protein of interest, and a homodimeric antibody is constructed by fusing a high-affinity epitope-binding domain to a human or mouse Fc domain. In a third, a heterodimeric antibody is constructed by fusing two different epitope-binding domains which target two different binding sites in GFP, to polarized Fc fragments. These antibody/epitope pairs have affinities in the low picomolar range and are useful tools for many antibody-based applications. PMID:26943906

  18. Comparison of affinity chromatography and adsorption to vaccinia virus recombinant infected cells for depletion of antibodies directed against respiratory syncytial virus glycoproteins present in a human immunoglobulin preparation.

    PubMed

    Sastre, Patricia; Melero, José A; García-Barreno, Blanca; Palomo, Concepción

    2005-06-01

    Antibodies directed against human respiratory syncytial virus (HRSV) glycoproteins were depleted from a commercial immunoglobulin preparation (RespiGam) by two different methods. The first method consisted of repeated adsorption of RespiGam to Sepharose beads with covalently bound soluble forms of the two major viral glycoproteins (F or G). The second method consisted of adsorption of immunoglobulins to live cells expressing F or G glycoproteins on their surfaces after infection with vaccinia virus recombinants. While the first method removed efficiently antibodies that reacted with F and/or G glycoproteins by ELISA, it was inefficient in the elimination of anti-HRSV neutralizing antibodies. In contrast, the second method removed efficiently anti-HRSV antibodies that both reacted by ELISA and neutralized virus infectivity. These results confirm that human neutralizing antibodies are directed exclusively against HRSV F and G glycoproteins, and, they raise the possibility that F and G glycoproteins inserted into cell membranes differ antigenically from their soluble forms linked covalently to Sepharose beads.

  19. Production Of Human Antibodies

    NASA Technical Reports Server (NTRS)

    Sammons, David W.; Neil, Garry A.

    1993-01-01

    Process for making human monoclonal antibodies based on combination of techniques. Antibodies made active against specific antigen. Process involves in vivo immunization of human B lymphocyte cells in mice. B cells of interest enriched in vitro before fusion. Method potentially applicable to any antigen. Does not rely on use of Epstein-Barr virus at any step. Human lymphocytes taken from any source.

  20. Novel Human Three-Domain Antibody Fragments Against sTNFα as Well as tmTNFα with High Affinity Generated by the Combination of Ribosome Display and E. coli Expression System.

    PubMed

    Zhao, X-L; Tian, L-F; Zhang, S-J; Li, J-M; Feng, H; Wang, L-M; Wang, S; Wang, J; Wang, T; Chen, W-Q

    2016-04-01

    Human tumour necrosis factor α (hTNFα) has been proved to be a validated therapeutic target in a number of immune-mediated inflammatory diseases (IMIDs). Fully human monoclonal antibodies (mAbs) that can neutralize soluble hTNFα (sTNFα) as well as transmembrane hTNFα (tmTNFα) are more desirable hTNFα antagonists. Here, we report that novel anti-hTNFα human low-molecular-weight MAbs have been selected and identified using both sTNFα and tmTNFα as target antigens by the combination of ribosome display and E. coli expression system for the first time. As a newly born engineering small molecular antibody, three-domain antibody fragment (VH /κ) provides an alternative promising molecular principle to generate biological agents for TNFα-dependent IMIDs. In this study, a panel of novel human VH /κs (F09, F21, F49 and F409) with high affinity (10(-10) -10(-9) mol/l) to neutralize sTNFα as well as tmTNFα was generated by the combination of ribosome display and E. coli expression system. Among the four clones, F21 and F409 could reduce cytotoxicity on L929 cells induced by sTNFα as well as tmTNFα effectively, and both of them had great potential to inhibit hTNFα-mediated NF-κB activation. Soluble F21 and F409 were also able to inhibit the binding of hTNFα to TNFR1 and TNFR2. The new human antibodies described here have desirable capability to neutralize sTNFα as well as tmTNFα effectively with high affinity and reasonable stability; this may provide an alternative approach for patients who are not responding adequately to currently available anti-TNFα agents. PMID:26860639

  1. Mining human antibody repertoires

    PubMed Central

    2010-01-01

    Human monoclonal antibodies (mAbs) have become drugs of choice for the management of an increasing number of human diseases. Human antibody repertoires provide a rich source for human mAbs. Here we review the characteristics of natural and non-natural human antibody repertoires and their mining with non-combinatorial and combinatorial strategies. In particular, we discuss the selection of human mAbs from naïve, immune, transgenic and synthetic human antibody repertoires using methods based on hybridoma technology, clonal expansion of peripheral B cells, single-cell PCR, phage display, yeast display and mammalian cell display. Our reliance on different strategies is shifting as we gain experience and refine methods to the efficient generation of human mAbs with superior pharmacokinetic and pharmacodynamic properties. PMID:20505349

  2. Molecular modeling of the affinity chromatography of monoclonal antibodies.

    PubMed

    Paloni, Matteo; Cavallotti, Carlo

    2015-01-01

    Molecular modeling is a methodology that offers the possibility of studying complex systems such as protein-ligand complexes from an atomistic point of view, making available information that can be difficultly obtained from experimental studies. Here, a protocol for the construction of molecular models of the interaction between antibodies and ligands that can be used for an affinity chromatography process is presented. The outlined methodology focuses mostly on the description of a procedure that may be adopted to determine the structure and free energy of interaction between the antibody and the affinity ligand. A procedure to extend the proposed methodology to include the effect of the environment (buffer solution, spacer, support matrix) is also briefly outlined. PMID:25749965

  3. 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.

  4. Effects of whole-body irradiation on antibody affinity.

    PubMed Central

    Gorini, G; Adorini, L; Boraschi, D; Di Michele, A; Doria, G

    1977-01-01

    Mice exposed to a sublethal dose of X-rays were immunized with alum-precipitated DNP-KLH (dinitrophenyl-keyhole limpet haemocyanin) and B. pertussis either before or after irradiation. The primary anti-DNP antibody response was evaluated during 8 weeks after immunization by the equilibrium dialysis technique using ammonium sulphate- precipitated serum globulins and the ligand 3H-labelled xi-DNP-L-Lysine. The serum concentrations of antibody sites in mice immunized 1-5 days before or 2 h-8 weeks after 450 rad were below the values in unirradiated controls at all bleeding times. Antibody affinity, however, was found to be up to 20 fold higher in irradiated mice than in control mice when antigen was injected before, or 3-8 weeks after, irradiation. Spleen cells from mice exposed to 450 rad 1-9 weeks before killing were stimulated in vitro with PHA, ConA, or LPS. Recovery profiles of mitotic responsiveness suggest that enhancement of antibody affinity in irradiated mice could result from relative lack of suppressor T Cells. PMID:198358

  5. Binding Affinity, Specificity and Comparative Biodistribution of the Parental Murine Monoclonal Antibody MX35 (Anti-NaPi2b) and Its Humanized Version Rebmab200.

    PubMed

    Lindegren, Sture; Andrade, Luciana N S; Bäck, Tom; Machado, Camila Maria L; Horta, Bruno Brasil; Buchpiguel, Carlos; Moro, Ana Maria; Okamoto, Oswaldo Keith; Jacobsson, Lars; Cederkrantz, Elin; Washiyama, Kohshin; Aneheim, Emma; Palm, Stig; Jensen, Holger; Tuma, Maria Carolina B; Chammas, Roger; Hultborn, Ragnar; Albertsson, Per

    2015-01-01

    The aim of this preclinical study was to evaluate the characteristics of the monoclonal antibody Rebmab200, which is a humanized version of the ovarian-specific murine antibody MX35. This investigation contributes to the foundation for future clinical α-radioimmunotherapy of minimal residual ovarian cancer with 211At-Rebmab200. Here, the biodistribution of 211At-Rebmab200 was evaluated, as was the utility of 99mTc-Rebmab200 for bioimaging. Rebmab200 was directly compared with its murine counterpart MX35 in terms of its in-vitro capacity for binding the immobilized NaPi2B epitope and live cells; we also assessed its biodistribution in nude mice carrying subcutaneous OVCAR-3 tumors. Tumor antigen and cell binding were similar between Rebmab200 and murine MX35, as was biodistribution, including normal tissue uptake and in-vivo tumor binding. We also demonstrated that 99mTc-Rebmab200 can be used for single-photon emission computed tomography of subcutaneous ovarian carcinomas in tumor-bearing mice. Taken together, our data support the further development of Rebmab200 for radioimmunotherapy and diagnostics. PMID:25970341

  6. Binding Affinity, Specificity and Comparative Biodistribution of the Parental Murine Monoclonal Antibody MX35 (Anti-NaPi2b) and Its Humanized Version Rebmab200

    PubMed Central

    Lindegren, Sture; Andrade, Luciana N. S.; Bäck, Tom; Machado, Camila Maria L.; Horta, Bruno Brasil; Buchpiguel, Carlos; Moro, Ana Maria; Okamoto, Oswaldo Keith; Jacobsson, Lars; Cederkrantz, Elin; Washiyama, Kohshin; Aneheim, Emma; Palm, Stig; Jensen, Holger; Tuma, Maria Carolina B.; Chammas, Roger; Hultborn, Ragnar; Albertsson, Per

    2015-01-01

    The aim of this preclinical study was to evaluate the characteristics of the monoclonal antibody Rebmab200, which is a humanized version of the ovarian-specific murine antibody MX35. This investigation contributes to the foundation for future clinical α-radioimmunotherapy of minimal residual ovarian cancer with 211At-Rebmab200. Here, the biodistribution of 211At-Rebmab200 was evaluated, as was the utility of 99mTc-Rebmab200 for bioimaging. Rebmab200 was directly compared with its murine counterpart MX35 in terms of its in-vitro capacity for binding the immobilized NaPi2B epitope and live cells; we also assessed its biodistribution in nude mice carrying subcutaneous OVCAR-3 tumors. Tumor antigen and cell binding were similar between Rebmab200 and murine MX35, as was biodistribution, including normal tissue uptake and in-vivo tumor binding. We also demonstrated that 99mTc-Rebmab200 can be used for single-photon emission computed tomography of subcutaneous ovarian carcinomas in tumor-bearing mice. Taken together, our data support the further development of Rebmab200 for radioimmunotherapy and diagnostics. PMID:25970341

  7. Binding Affinity, Specificity and Comparative Biodistribution of the Parental Murine Monoclonal Antibody MX35 (Anti-NaPi2b) and Its Humanized Version Rebmab200.

    PubMed

    Lindegren, Sture; Andrade, Luciana N S; Bäck, Tom; Machado, Camila Maria L; Horta, Bruno Brasil; Buchpiguel, Carlos; Moro, Ana Maria; Okamoto, Oswaldo Keith; Jacobsson, Lars; Cederkrantz, Elin; Washiyama, Kohshin; Aneheim, Emma; Palm, Stig; Jensen, Holger; Tuma, Maria Carolina B; Chammas, Roger; Hultborn, Ragnar; Albertsson, Per

    2015-01-01

    The aim of this preclinical study was to evaluate the characteristics of the monoclonal antibody Rebmab200, which is a humanized version of the ovarian-specific murine antibody MX35. This investigation contributes to the foundation for future clinical α-radioimmunotherapy of minimal residual ovarian cancer with 211At-Rebmab200. Here, the biodistribution of 211At-Rebmab200 was evaluated, as was the utility of 99mTc-Rebmab200 for bioimaging. Rebmab200 was directly compared with its murine counterpart MX35 in terms of its in-vitro capacity for binding the immobilized NaPi2B epitope and live cells; we also assessed its biodistribution in nude mice carrying subcutaneous OVCAR-3 tumors. Tumor antigen and cell binding were similar between Rebmab200 and murine MX35, as was biodistribution, including normal tissue uptake and in-vivo tumor binding. We also demonstrated that 99mTc-Rebmab200 can be used for single-photon emission computed tomography of subcutaneous ovarian carcinomas in tumor-bearing mice. Taken together, our data support the further development of Rebmab200 for radioimmunotherapy and diagnostics.

  8. Affinity enhancement of antibodies: how low-affinity antibodies produced early in immune responses are followed by high-affinity antibodies later and in memory B-cell responses.

    PubMed

    Eisen, Herman N

    2014-05-01

    The antibodies produced initially in response to most antigens are high molecular weight (MW) immunoglobulins (IgM) with low affinity for the antigen, while the antibodies produced later are lower MW classes (e.g., IgG and IgA) with, on average, orders of magnitude higher affinity for that antigen. These changes, often termed affinity maturation, take place largely in small B-cell clusters (germinal center; GC) in lymphoid tissues in which proliferating antigen-stimulated B cells express the highly mutagenic cytidine deaminase that mediates immunoglobulin class-switching and sequence diversification of the immunoglobulin variable domains of antigen-binding receptors on B cells (BCR). Of the large library of BCR-mutated B cells thus rapidly generated, a small minority with affinity-enhancing mutations are selected to survive and differentiate into long-lived antibody-secreting plasma cells and memory B cells. BCRs are also endocytic receptors; they internalize and cleave BCR-bound antigen, yielding peptide-MHC complexes that are recognized by follicular helper T cells. Imperfect correlation between BCR affinity for antigen and cognate T-cell engagement may account for the increasing affinity heterogeneity that accompanies the increasing average affinity of antibodies. Conservation of mechanisms underlying mutation and selection of high-affinity antibodies over the ≈200 million years of evolution separating bird and mammal lineages points to the crucial role of antibody affinity enhancement in adaptive immunity.

  9. Influence of affinity on antibody determination in microtiter ELISA systems

    SciTech Connect

    Peterman, J.H.; Voss, E.W. Jr.; Butler, J.E.

    1986-03-01

    Theoretically, all immunoassays are affinity (Ka) dependent when the product of the antibody (Ab) Ka and the free epitope concentration is less than 10. Thus, the degree of dependence on Ka depends on the concentration of available antigen in the system. The authors examined the binding of /sup 125/I-anti-fluorescein (a-FLU) monoclonal antibodies of different affinities to FLU-gelatin adsorbed on Immunlon 2 microtiter plates. Data obtained were in general agreement with our theoretical predictions; the percent of /sup 125/I-a-FLU which bound correlated with Ka, as did the shape of the titration curves. Measurement of 5 a-FLU monoclonals by the ELISA showed that the determination of Ab concentrations depends on the FLU-gelatin concentration, epitope density, and on the relationship between the Kas of test samples and the reference standard Ab preparation. Thus the ELISA is Ka dependent and should not be used routinely to estimate the absolute amount to Ab in unknown samples. However, the Ka dependency of the ELISA might provide a convenient assay for the estimation of the relative functional Ka (rfKa) of antibody preparations.

  10. [The effect of blood serum polyreactive immunoglobulins on serum antibody affinity determination].

    PubMed

    Bobrovnik, S A; Demchenko, M A; Komisarenko, S V

    2010-01-01

    The presence of polyreactive immunoglobulins in sera may substantially influence on the accuracy of antibody affinity determination. In order to obtain precise values of antibody affinity one should apply one of two following ways. First, one should block polyreactive immunoglobulins with high concentration of Twin 20 and high concentration of any antigen which does not interact with studying antibody. After this antibody affinity may be determined by traditional methods. Another way is the application of the method suggested by us earlier, which allow determining affinity of two antibodies in a mixture and the relation of their concentrations.

  11. Enhanced antigen-antibody binding affinity mediated by an anti-idiotypic antibody

    SciTech Connect

    Sawutz, D.G.; Koury, R.; Homcy, C.J.

    1987-08-25

    The authors previously described the production of four monoclonal antibodies to the ..beta..-adrenergic receptor antagonist alprenolol. One of these antibodies, 5B7 (IgG/sub 2a/, kappa), was used to raise anti-idiotypic antisera in rabbits. In contrast to the expected results, one of the anti-idiotypic antisera (R9) promotes (/sup 125/I)iodocyanopinodolol (ICYP) binding to antibody 5B7. In the presence of R9, the dissociation constant decreases 100-fold from 20 to 0.3 nM. This increase in binding affinity of antibody 5B7 for ICYP is not observed in the presence of preimmune, rabbit anti-mouse or anti-idiotypic antisera generated to a monoclonal antibody of a different specificity. Furthermore, R9 in the absence of 5B7 does not bind ICYP. The F(ab) fragments of 5B7 and T9 behaved in a similar manner, and the soluble complex responsible for the high-affinity interaction with ICYP can be identified by gel filtration chromatography. The elution position of the complex is consistent with a 5B7 F(ab)-R9 F(ab) dimer, indicating that polyvalency is not responsible for the enhanced ligand binding. Kinetic analysis of ICYP-5B7 binding revealed that the rate of ICYP dissociation from 5B7 in the presence of R9 is approximately 100 times slower than in the absence of R9, consistent with the 100-fold change in binding affinity of 5B7 for ICYP. The available data best fit a model in which an anti-idiotypic antibody binds at or near the binding site of the idiotype participating in the formation of a hybrid ligand binding site. This would allow increased contact of the ligand with the idiotype-anti-idiotype complex and result in an enhanced affinity of the ligand interaction.

  12. Phase 1/2 study of ocaratuzumab, an Fc-engineered humanized anti-CD20 monoclonal antibody, in low-affinity FcγRIIIa patients with previously treated follicular lymphoma.

    PubMed

    Ganjoo, Kristen N; de Vos, Sven; Pohlman, Brad L; Flinn, Ian W; Forero-Torres, Andres; Enas, Nathan H; Cronier, Damien M; Dang, Nam H; Foon, Kenneth A; Carpenter, Susan P; Slapak, Christopher A; Link, Brian K; Smith, Mitchell R; Mapara, Markus Y; Wooldridge, James E

    2015-01-01

    This phase 2 study assessed the safety and efficacy of ocaratuzumab, a humanized anti-CD20 monoclonal antibody. Fifty patients with previously treated follicular lymphoma (FL) and a low-affinity genotype of FcγRIIIa received ocaratuzumab 375 mg/m(2) weekly for 4 weeks. Grade 3/4/5 adverse events (AEs) were reported in 11/1/1 patients, respectively. Serious AEs were reported by 11/50 patients, and three discontinued due to AEs. One patient died from aspiration pneumonia due to possibly drug-related nausea and vomiting. Investigator-assessed response rate was 30% (15/50), including four complete responses (CR), three CR unconfirmed (CRu) and eight partial responses (PR). Investigator-assessed median Progression-free survivial (PFS) was 38.3 weeks. Ocaratuzumab's pharmacokinetic profile was similar to that reported for rituximab. Lymphocyte subset analysis showed significant, selective reduction of B-cells during and after ocaratuzumab treatment. Ocaratuzumab at this dose and schedule is active and well tolerated in patients with previously treated FL with low affinity FcγRIIIa genotypes. ClinTrials registry number: NCT00354926.

  13. Humanized Antibodies for Antiviral Therapy

    NASA Astrophysics Data System (ADS)

    Co, Man Sung; Deschamps, Marguerite; Whitley, Richard J.; Queen, Cary

    1991-04-01

    Antibody therapy holds great promise for the treatment of cancer, autoimmune disorders, and viral infections. Murine monoclonal antibodies are relatively easy to produce but are severely restricted for therapeutic use by their immunogenicity in humans. Production of human monoclonal antibodies has been problematic. Humanized antibodies can be generated by introducing the six hypervariable regions from the heavy and light chains of a murine antibody into a human framework sequence and combining it with human constant regions. We humanized, with the aid of computer modeling, two murine monoclonal antibodies against herpes simplex virus gB and gD glycoproteins. The binding, virus neutralization, and cell protection results all indicate that both humanized antibodies have retained the binding activities and the biological properties of the murine monoclonal antibodies.

  14. Affinity Maturation of Monoclonal Antibody 1E11 by Targeted Randomization in CDR3 Regions Optimizes Therapeutic Antibody Targeting of HER2-Positive Gastric Cancer.

    PubMed

    Ko, Bong-Kook; Choi, Soyoung; Cui, Lei Guang; Lee, Young-Ha; Hwang, In-Sik; Kim, Kyu-Tae; Shim, Hyunbo; Lee, Jong-Seo

    2015-01-01

    Anti-HER2 murine monoclonal antibody 1E11 has strong and synergistic anti-tumor activity in HER2-overexpressing gastric cancer cells when used in combination with trastuzumab. We presently optimized this antibody for human therapeutics. First, the complementarity determining regions (CDRs) of the murine antibody were grafted onto human germline immunoglobulin variable genes. No difference in affinity and biological activity was observed between chimeric 1E11 (ch1E11) and humanized 1E11 (hz1E11). Next, affinity maturation of hz1E11 was performed by the randomization of CDR-L3 and H3 residues followed by stringent biopanning selection. Milder selection pressure favored the selection of more diverse clones, whereas higher selection stringency resulted in the convergence of the panning output to a smaller number of clones with improved affinity. Clone 1A12 had four amino acid substitutions in CDR-L3, and showed a 10-fold increase in affinity compared to the parental clone and increased potency in an in vitro anti-proliferative activity assay with HER2-overepxressing gastric cancer cells. Clone 1A12 inhibited tumor growth of NCI-N87 xenograft model with similar efficacy to trastuzumab alone, and the combination treatment of 1A12 and trastuzumab completely removed the established tumors. These results suggest that humanized and affinity matured monoclonal antibody 1A12 is a highly optimized molecule for future therapeutic development against HER2-positive tumors.

  15. Optimal fusion of antibody binding domains resulted in higher affinity and wider specificity.

    PubMed

    Dong, Jinhua; Kojima, Tomoki; Ohashi, Hiroyuki; Ueda, Hiroshi

    2015-11-01

    Antibody is a very important protein in biotechnological and biomedical fields because of its high affinity and specificity to various antigens. Due to the rise of human antibody therapeutics, its cost-effective purification is an urgent issue for bio-industry. In this study, we made novel fusion proteins PAxPG with a flexible (DDAKK)n linker between the two Ig binding domains derived from Staphylococcus protein A and Streptococcus protein G. The fusion proteins bound human and mouse IgGs and their fragments with up to 58-times higher affinity and wider specificity than the parental binding domains. Interestingly, the optimal linker for human Fab fragment was n = 4, which was close to the modeled distance between the termini of domains bound to heavy chain, implying increased avidity as a possible mechanism. For binding to Fc, the longest n=6 linker gave the highest affinity, implying longer interchain distance between the two binding sites. The novel fusion protein with optimized interdomain linker length will be a useful tool for the purification and detection of various IgGs including mouse IgG1 that binds only weakly to natural protein A. PMID:25910963

  16. Combining somatic mutations present in different in vivo affinity-matured antibodies isolated from immunized Lama glama yields ultra-potent antibody therapeutics.

    PubMed

    Klarenbeek, Alex; Blanchetot, Christophe; Schragel, Georg; Sadi, Ava S; Ongenae, Nico; Hemrika, Wieger; Wijdenes, John; Spinelli, Silvia; Desmyter, Aline; Cambillau, Christian; Hultberg, Anna; Kretz-Rommel, Anke; Dreier, Torsten; De Haard, Hans J W; Roovers, Rob C

    2016-04-01

    Highly potent human antibodies are required to therapeutically neutralize cytokines such as interleukin-6 (IL-6) that is involved in many inflammatory diseases and malignancies. Although a number of mutagenesis approaches exist to perform antibody affinity maturation, these may cause antibody instability and production issues. Thus, a robust and easy antibody affinity maturation strategy to increase antibody potency remains highly desirable. By immunizing llama, cloning the 'immune' antibody repertoire and using phage display, we selected a diverse set of IL-6 antagonistic Fabs. Heavy chain shuffling was performed on the Fab with lowest off-rate, resulting in a panel of variants with even lower off-rate. Structural analysis of the Fab:IL-6 complex suggests that the increased affinity was partly due to a serine to tyrosine switch in HCDR2. This translated into neutralizing capacity in an in vivo model of IL-6 induced SAA production. Finally, a novel Fab library was designed, encoding all variations found in the natural repertoire of VH genes identified after heavy chain shuffling. High stringency selections resulted in identification of a Fab with 250-fold increased potency when re-formatted into IgG1. Compared with a heavily engineered anti-IL-6 monoclonal antibody currently in clinical development, this IgG was at least equally potent, showing the engineering process to have had led to a highly potent anti-IL-6 antibody.

  17. Affinity improvement of a therapeutic antibody to methamphetamine and amphetamine through structure-based antibody engineering.

    PubMed

    Thakkar, Shraddha; Nanaware-Kharade, Nisha; Celikel, Reha; Peterson, Eric C; Varughese, Kottayil I

    2014-01-14

    Methamphetamine (METH) abuse is a worldwide threat, without any FDA approved medications. Anti-METH IgGs and single chain fragments (scFvs) have shown efficacy in preclinical studies. Here we report affinity enhancement of an anti-METH scFv for METH and its active metabolite amphetamine (AMP), through the introduction of point mutations, rationally designed to optimize the shape and hydrophobicity of the antibody binding pocket. The binding affinity was measured using saturation binding technique. The mutant scFv-S93T showed 3.1 fold enhancement in affinity for METH and 26 fold for AMP. The scFv-I37M and scFv-Y34M mutants showed enhancement of 94, and 8 fold for AMP, respectively. Structural analysis of scFv-S93T:METH revealed that the substitution of Ser residue by Thr caused the expulsion of a water molecule from the cavity, creating a more hydrophobic environment for the binding that dramatically increases the affinities for METH and AMP.

  18. Affinity improvement of a therapeutic antibody to methamphetamine and amphetamine through structure-based antibody engineering

    PubMed Central

    Thakkar, Shraddha; Nanaware-Kharade, Nisha; Celikel, Reha; Peterson, Eric C.; Varughese, Kottayil I.

    2014-01-01

    Methamphetamine (METH) abuse is a worldwide threat, without any FDA approved medications. Anti-METH IgGs and single chain fragments (scFvs) have shown efficacy in preclinical studies. Here we report affinity enhancement of an anti-METH scFv for METH and its active metabolite amphetamine (AMP), through the introduction of point mutations, rationally designed to optimize the shape and hydrophobicity of the antibody binding pocket. The binding affinity was measured using saturation binding technique. The mutant scFv-S93T showed 3.1 fold enhancement in affinity for METH and 26 fold for AMP. The scFv-I37M and scFv-Y34M mutants showed enhancement of 94, and 8 fold for AMP, respectively. Structural analysis of scFv-S93T:METH revealed that the substitution of Ser residue by Thr caused the expulsion of a water molecule from the cavity, creating a more hydrophobic environment for the binding that dramatically increases the affinities for METH and AMP. PMID:24419156

  19. Human antibody production in transgenic animals.

    PubMed

    Brüggemann, Marianne; Osborn, Michael J; Ma, Biao; Hayre, Jasvinder; Avis, Suzanne; Lundstrom, Brian; Buelow, Roland

    2015-04-01

    Fully human antibodies from transgenic animals account for an increasing number of new therapeutics. After immunization, diverse human monoclonal antibodies of high affinity can be obtained from transgenic rodents, while large animals, such as transchromosomic cattle, have produced respectable amounts of specific human immunoglobulin (Ig) in serum. Several strategies to derive animals expressing human antibody repertoires have been successful. In rodents, gene loci on bacterial artificial chromosomes or yeast artificial chromosomes were integrated by oocyte microinjection or transfection of embryonic stem (ES) cells, while ruminants were derived from manipulated fibroblasts with integrated human chromosome fragments or human artificial chromosomes. In all strains, the endogenous Ig loci have been silenced by gene targeting, either in ES or fibroblast cells, or by zinc finger technology via DNA microinjection; this was essential for optimal production. However, comparisons showed that fully human antibodies were not as efficiently produced as wild-type Ig. This suboptimal performance, with respect to immune response and antibody yield, was attributed to imperfect interaction of the human constant region with endogenous signaling components such as the Igα/β in mouse, rat or cattle. Significant improvements were obtained when the human V-region genes were linked to the endogenous CH-region, either on large constructs or, separately, by site-specific integration, which could also silence the endogenous Ig locus by gene replacement or inversion. In animals with knocked-out endogenous Ig loci and integrated large IgH loci, containing many human Vs, all D and all J segments linked to endogenous C genes, highly diverse human antibody production similar to normal animals was obtained.

  20. In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-Domain Antibodies (VHHs) and a VHH-Fc Antibody

    PubMed Central

    Richard, Gabrielle; Meyers, Ashley J.; McLean, Michael D.; Arbabi-Ghahroudi, Mehdi; MacKenzie, Roger; Hall, J. Christopher

    2013-01-01

    Small recombinant antibody fragments (e.g. scFvs and VHHs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab’)2 antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama VHHs were isolated from an immune VHH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α–Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity VHH (C2) was fused to a human Fc fragment to create a VHH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to α–Cbtx. Mouse α–Cbtx challenge studies showed that our highest affinity VHHs (C2 and C20) and the VHH2-Fc antibody effectively neutralized lethality induced by α–Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx VHHs and VHH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy. PMID:23894495

  1. Tailored Immunogens Direct Affinity Maturation toward HIV Neutralizing Antibodies.

    PubMed

    Briney, Bryan; Sok, Devin; Jardine, Joseph G; Kulp, Daniel W; Skog, Patrick; Menis, Sergey; Jacak, Ronald; Kalyuzhniy, Oleksandr; de Val, Natalia; Sesterhenn, Fabian; Le, Khoa M; Ramos, Alejandra; Jones, Meaghan; Saye-Francisco, Karen L; Blane, Tanya R; Spencer, Skye; Georgeson, Erik; Hu, Xiaozhen; Ozorowski, Gabriel; Adachi, Yumiko; Kubitz, Michael; Sarkar, Anita; Wilson, Ian A; Ward, Andrew B; Nemazee, David; Burton, Dennis R; Schief, William R

    2016-09-01

    Induction of broadly neutralizing antibodies (bnAbs) is a primary goal of HIV vaccine development. VRC01-class bnAbs are important vaccine leads because their precursor B cells targeted by an engineered priming immunogen are relatively common among humans. This priming immunogen has demonstrated the ability to initiate a bnAb response in animal models, but recall and maturation toward bnAb development has not been shown. Here, we report the development of boosting immunogens designed to guide the genetic and functional maturation of previously primed VRC01-class precursors. Boosting a transgenic mouse model expressing germline VRC01 heavy chains produced broad neutralization of near-native isolates (N276A) and weak neutralization of fully native HIV. Functional and genetic characteristics indicate that the boosted mAbs are consistent with partially mature VRC01-class antibodies and place them on a maturation trajectory that leads toward mature VRC01-class bnAbs. The results show how reductionist sequential immunization can guide maturation of HIV bnAb responses.

  2. Tailored Immunogens Direct Affinity Maturation toward HIV Neutralizing Antibodies.

    PubMed

    Briney, Bryan; Sok, Devin; Jardine, Joseph G; Kulp, Daniel W; Skog, Patrick; Menis, Sergey; Jacak, Ronald; Kalyuzhniy, Oleksandr; de Val, Natalia; Sesterhenn, Fabian; Le, Khoa M; Ramos, Alejandra; Jones, Meaghan; Saye-Francisco, Karen L; Blane, Tanya R; Spencer, Skye; Georgeson, Erik; Hu, Xiaozhen; Ozorowski, Gabriel; Adachi, Yumiko; Kubitz, Michael; Sarkar, Anita; Wilson, Ian A; Ward, Andrew B; Nemazee, David; Burton, Dennis R; Schief, William R

    2016-09-01

    Induction of broadly neutralizing antibodies (bnAbs) is a primary goal of HIV vaccine development. VRC01-class bnAbs are important vaccine leads because their precursor B cells targeted by an engineered priming immunogen are relatively common among humans. This priming immunogen has demonstrated the ability to initiate a bnAb response in animal models, but recall and maturation toward bnAb development has not been shown. Here, we report the development of boosting immunogens designed to guide the genetic and functional maturation of previously primed VRC01-class precursors. Boosting a transgenic mouse model expressing germline VRC01 heavy chains produced broad neutralization of near-native isolates (N276A) and weak neutralization of fully native HIV. Functional and genetic characteristics indicate that the boosted mAbs are consistent with partially mature VRC01-class antibodies and place them on a maturation trajectory that leads toward mature VRC01-class bnAbs. The results show how reductionist sequential immunization can guide maturation of HIV bnAb responses. PMID:27610570

  3. New High Affinity Monoclonal Antibodies Recognize Non-Overlapping Epitopes On Mesothelin For Monitoring And Treating Mesothelioma

    PubMed Central

    Zhang, Yi-Fan; Phung, Yen; Gao, Wei; Kawa, Seiji; Hassan, Raffit; Pastan, Ira; Ho, Mitchell

    2015-01-01

    Mesothelin is an emerging cell surface target in mesothelioma and other solid tumors. Most antibody drug candidates recognize highly immunogenic Region I (296–390) on mesothelin. Here, we report a group of high-affinity non-Region I rabbit monoclonal antibodies. These antibodies do not compete for mesothelin binding with the immunotoxin SS1P that binds Region I of mesothelin. One pair of antibodies (YP218 and YP223) is suitable to detect soluble mesothelin in a sandwich ELISA with high sensitivity. The new assay can also be used to measure serum mesothelin concentration in mesothelioma patients, indicating its potential use for monitoring patients treated with current antibody therapies targeting Region I. The antibodies are highly specific and sensitive in immunostaining of mesothelioma. To explore their use in tumor therapy, we have generated the immunotoxins based on the Fv of these antibodies. One immunotoxin (YP218 Fv-PE38) exhibits potent anti-tumor cytotoxicity towards primary mesothelioma cell lines in vitro and an NCI-H226 xenograft tumor in mice. Furthermore, we have engineered a humanized YP218 Fv that retains full binding affinity for mesothelin-expressing cancer cells. In conclusion, with their unique binding properties, these antibodies may be promising candidates for monitoring and treating mesothelioma and other mesothelin-expressing cancers. PMID:25996440

  4. Sequential class switching is required for the generation of high affinity IgE antibodies

    PubMed Central

    Xiong, Huizhong; Dolpady, Jayashree; Wabl, Matthias; Curotto de Lafaille, Maria A.

    2012-01-01

    IgE antibodies with high affinity for their antigens can be stably cross-linked at low concentrations by trace amounts of antigen, whereas IgE antibodies with low affinity bind their antigens weakly. In this study, we find that there are two distinct pathways to generate high and low affinity IgE. High affinity IgE is generated through sequential class switching (μ→γ→ε) in which an intermediary IgG phase is necessary for the affinity maturation of the IgE response, where the IgE inherits somatic hypermutations and high affinity from the IgG1 phase. In contrast, low affinity IgE is generated through direct class switching (μ→ε) and is much less mutated. Mice deficient in IgG1 production cannot produce high affinity IgE, even after repeated immunizations. We demonstrate that a small amount of high affinity IgE can cause anaphylaxis and is pathogenic. Low affinity IgE competes with high affinity IgE for binding to Fcε receptors and prevents anaphylaxis and is thus beneficial. PMID:22249450

  5. Design, synthesis and application of benzyl-sulfonate biomimetic affinity adsorbents for monoclonal antibody purification from transgenic corn.

    PubMed

    Maltezos, Anastasios; Platis, Dimitris; Vlachakis, Dimitrios; Kossida, Sophia; Marinou, Marigianna; Labrou, Nikolaos E

    2014-01-01

    The human anti-human immunodeficiency virus (HIV) antibody 2G12 (mAb 2G12) is one of the most broadly neutralizing antibodies against HIV that recognizes a unique epitope on the surface glycoprotein gp120. In the present work, a limited affinity-ligand library was synthesized and evaluated for its ability to bind and purify recombinant mAb 2G12 expressed in transgenic corn. The affinity ligands were structural fragments of polysulfonate triazine dye Cibacron Blue 3GA (CB3GA) and represent novel lead scaffolds for designing synthetic affinity ligands. Solid phase chemistry was used to synthesize variants of CB3GA lead ligand. One immobilized ligand, bearing 4-aminobenzyl sulfonic acid (4ABS) linked on two chlorine atoms of the triazine ring (4ABS-Trz-4ABS), displayed high affinity for mAb 2G12. Absorption equilibrium, 3D molecular modelling and molecular dynamics simulation studies were carried out to provide a detailed picture of the 4ABS-Trz-4ABS interaction with mAb 2G12. This biomimetic affinity ligand was exploited for the development of a facile two-step purification protocol for mAb 2G12. In the first step of the procedure, mAb 2G12 was purified on an S-Sepharose FF cation exchanger, and in the second step, mAb 2G12 was purified using affinity chromatography on 4ABS-Trz-4ABS affinity adsorbent. Analysis of the antibody preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay showed that the mAb 2G12 was fully active and of sufficient purity suitable for analytical applications.

  6. Design, synthesis and application of benzyl-sulfonate biomimetic affinity adsorbents for monoclonal antibody purification from transgenic corn.

    PubMed

    Maltezos, Anastasios; Platis, Dimitris; Vlachakis, Dimitrios; Kossida, Sophia; Marinou, Marigianna; Labrou, Nikolaos E

    2014-01-01

    The human anti-human immunodeficiency virus (HIV) antibody 2G12 (mAb 2G12) is one of the most broadly neutralizing antibodies against HIV that recognizes a unique epitope on the surface glycoprotein gp120. In the present work, a limited affinity-ligand library was synthesized and evaluated for its ability to bind and purify recombinant mAb 2G12 expressed in transgenic corn. The affinity ligands were structural fragments of polysulfonate triazine dye Cibacron Blue 3GA (CB3GA) and represent novel lead scaffolds for designing synthetic affinity ligands. Solid phase chemistry was used to synthesize variants of CB3GA lead ligand. One immobilized ligand, bearing 4-aminobenzyl sulfonic acid (4ABS) linked on two chlorine atoms of the triazine ring (4ABS-Trz-4ABS), displayed high affinity for mAb 2G12. Absorption equilibrium, 3D molecular modelling and molecular dynamics simulation studies were carried out to provide a detailed picture of the 4ABS-Trz-4ABS interaction with mAb 2G12. This biomimetic affinity ligand was exploited for the development of a facile two-step purification protocol for mAb 2G12. In the first step of the procedure, mAb 2G12 was purified on an S-Sepharose FF cation exchanger, and in the second step, mAb 2G12 was purified using affinity chromatography on 4ABS-Trz-4ABS affinity adsorbent. Analysis of the antibody preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay showed that the mAb 2G12 was fully active and of sufficient purity suitable for analytical applications. PMID:24375581

  7. Assessment of Solvated Interaction Energy Function for Ranking Antibody-Antigen Binding Affinities.

    PubMed

    Sulea, Traian; Vivcharuk, Victor; Corbeil, Christopher R; Deprez, Christophe; Purisima, Enrico O

    2016-07-25

    Affinity modulation of antibodies and antibody fragments of therapeutic value is often required in order to improve their clinical efficacies. Virtual affinity maturation has the potential to quickly focus on the critical hotspot residues without the combinatorial explosion problem of conventional display and library approaches. However, this requires a binding affinity scoring function that is capable of ranking single-point mutations of a starting antibody. We focus here on assessing the solvated interaction energy (SIE) function that was originally developed for and is widely applied to scoring of protein-ligand binding affinities. To this end, we assembled a structure-function data set called Single-Point Mutant Antibody Binding (SiPMAB) comprising several antibody-antigen systems suitable for this assessment, i.e., based on high-resolution crystal structures for the parent antibodies and coupled with high-quality binding affinity measurements for sets of single-point antibody mutants in each system. Using this data set, we tested the SIE function with several mutation protocols based on the popular methods SCWRL, Rosetta, and FoldX. We found that the SIE function coupled with a protocol limited to sampling only the mutated side chain can reasonably predict relative binding affinities with a Spearman rank-order correlation coefficient of about 0.6, outperforming more aggressive sampling protocols. Importantly, this performance is maintained for each of the seven system-specific component subsets as well as for other relevant subsets including non-alanine and charge-altering mutations. The transferability and enrichment in affinity-improving mutants can be further enhanced using consensus ranking over multiple methods, including the SIE, Talaris, and FOLDEF energy functions. The knowledge gained from this study can lead to successful prospective applications of virtual affinity maturation. PMID:27367467

  8. Assessment of Solvated Interaction Energy Function for Ranking Antibody-Antigen Binding Affinities.

    PubMed

    Sulea, Traian; Vivcharuk, Victor; Corbeil, Christopher R; Deprez, Christophe; Purisima, Enrico O

    2016-07-25

    Affinity modulation of antibodies and antibody fragments of therapeutic value is often required in order to improve their clinical efficacies. Virtual affinity maturation has the potential to quickly focus on the critical hotspot residues without the combinatorial explosion problem of conventional display and library approaches. However, this requires a binding affinity scoring function that is capable of ranking single-point mutations of a starting antibody. We focus here on assessing the solvated interaction energy (SIE) function that was originally developed for and is widely applied to scoring of protein-ligand binding affinities. To this end, we assembled a structure-function data set called Single-Point Mutant Antibody Binding (SiPMAB) comprising several antibody-antigen systems suitable for this assessment, i.e., based on high-resolution crystal structures for the parent antibodies and coupled with high-quality binding affinity measurements for sets of single-point antibody mutants in each system. Using this data set, we tested the SIE function with several mutation protocols based on the popular methods SCWRL, Rosetta, and FoldX. We found that the SIE function coupled with a protocol limited to sampling only the mutated side chain can reasonably predict relative binding affinities with a Spearman rank-order correlation coefficient of about 0.6, outperforming more aggressive sampling protocols. Importantly, this performance is maintained for each of the seven system-specific component subsets as well as for other relevant subsets including non-alanine and charge-altering mutations. The transferability and enrichment in affinity-improving mutants can be further enhanced using consensus ranking over multiple methods, including the SIE, Talaris, and FOLDEF energy functions. The knowledge gained from this study can lead to successful prospective applications of virtual affinity maturation.

  9. Defining the human gallbladder proteome by transcriptomics and affinity proteomics.

    PubMed

    Kampf, Caroline; Mardinoglu, Adil; Fagerberg, Linn; Hallström, Björn M; Danielsson, Angelika; Nielsen, Jens; Pontén, Fredrik; Uhlen, Mathias

    2014-11-01

    Global protein analysis of human gallbladder tissue is vital for identification of molecular regulators and effectors of its physiological activity. Here, we employed a genome-wide deep RNA sequencing analysis in 28 human tissues to identify the genes overrepresented in the gallbladder and complemented it with antibody-based immunohistochemistry in 48 human tissues. We characterized human gallbladder proteins and identified 140 gallbladder-specific proteins with an elevated expression in the gallbladder as compared to the other analyzed tissues. Five genes were categorized as enriched, with at least fivefold higher levels in gallbladder, 60 genes were categorized as group enriched with elevated transcript levels in gallbladder shared with at least one other tissue and 75 genes were categorized as enhanced with higher expression than the average expression in other tissues. We explored the localization of the genes within the gallbladder through cell-type specific antibody-based protein profiling and the subcellular localization of the genes through immunofluorescent-based profiling. Finally, we revealed the biological processes and metabolic functions carried out by these genes through the use of GO, KEGG Pathway, and HMR2.0 that is compilation of the human metabolic reactions. We demonstrated the results of the combined analysis of the transcriptomics and affinity proteomics.

  10. Maturation of functional antibody affinity in animals immunised with synthetic foot-and-mouth disease virus.

    PubMed

    Mulcahy, G; Reid, E; Dimarchi, R D; Gale, C; Doel, T R

    1992-03-01

    A good correlation exists between specific neutralising antibody titre and protection against challenge with foot-and-mouth disease virus (FMDV) in infected or virus-vaccinated cattle, but not in the case of animals immunised with synthetic FMDV peptides. Therefore, mechanisms other than simple neutralisation are likely to be important in vivo. Antibody affinity may influence the protective capacity of sera from immunised animals and experiments were carried out to measure the functional affinity for synthetic FMDV peptide of sera from guinea pigs and cattle given various synthetic vaccines. In guinea pigs given a single dose of synthetic vaccine, antibody affinity increased with time after immunisation. In cattle, however, administration of a second dose of peptide 21 days after the first markedly retarded the process of affinity maturation. For guinea pig sera of equivalent neutralising activity, those of higher functional affinity had higher protective indices than those of lower functional affinity. Knowledge of the importance of antibody affinity in protection against FMD is important for an improved understanding of the mechanisms of protection and for the design of novel vaccines. PMID:1316628

  11. Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity.

    PubMed

    Boder, E T; Midelfort, K S; Wittrup, K D

    2000-09-26

    Single-chain antibody mutants have been evolved in vitro with antigen-binding equilibrium dissociation constant K(d) = 48 fM and slower dissociation kinetics (half-time > 5 days) than those for the streptavidin-biotin complex. These mutants possess the highest monovalent ligand-binding affinity yet reported for an engineered protein by over two orders of magnitude. Optimal kinetic screening of randomly mutagenized libraries of 10(5)-10(7) yeast surface-displayed antibodies enabled a >1,000-fold decrease in the rate of dissociation after four cycles of affinity mutagenesis and screening. The consensus mutations are generally nonconservative by comparison with naturally occurring mouse Fv sequences and with residues that do not contact the fluorescein antigen in the wild-type complex. The existence of these mutants demonstrates that the antibody Fv architecture is not intrinsically responsible for an antigen-binding affinity ceiling during in vivo affinity maturation.

  12. Yeast Display-Based Antibody Affinity Maturation Using Detergent-Solubilized Cell Lysates.

    PubMed

    Tillotson, Benjamin J; Lajoie, Jason M; Shusta, Eric V

    2015-01-01

    It is often desired to identify or engineer antibodies that target membrane proteins (MPs). However, due to their inherent insolubility in aqueous solutions, MPs are often incompatible with in vitro antibody discovery and optimization platforms. Recently, we adapted yeast display technology to accommodate detergent-solubilized cell lysates as sources of MP antigens. The following protocol details the incorporation of cell lysates into a kinetic screen designed to obtain antibodies with improved affinity via slowed dissociation from an MP antigen. PMID:26060070

  13. Yeast display-based antibody affinity maturation using detergent-solubilized cell lysates

    PubMed Central

    Tillotson, Benjamin J.; Lajoie, Jason M.; Shusta, Eric V.

    2016-01-01

    Summary It is often desired to identify or engineer antibodies that target membrane proteins (MPs). However, due to their inherent insolubility in aqueous solutions, MPs are often incompatible with in vitro antibody discovery and optimization platforms. Recently, we adapted yeast display technology to accommodate detergent-solubilized cell lysates as sources of MP antigens. The following protocol details the incorporation of cell lysates into a kinetic screen designed to obtain antibodies with improved affinity via slowed dissociation from an MP antigen. PMID:26060070

  14. Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies

    PubMed Central

    Schlothauer, Tilman; Rueger, Petra; Stracke, Jan Olaf; Hertenberger, Hubert; Fingas, Felix; Kling, Lothar; Emrich, Thomas; Drabner, Georg; Seeber, Stefan; Auer, Johannes; Koch, Stefan; Papadimitriou, Apollon

    2013-01-01

    The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity. PMID:23765230

  15. Co-evolution of affinity and stability of grafted amyloid-motif domain antibodies.

    PubMed

    Julian, Mark C; Lee, Christine C; Tiller, Kathryn E; Rabia, Lilia A; Day, Evan K; Schick, Arthur J; Tessier, Peter M

    2015-10-01

    An attractive approach for designing lead antibody candidates is to mimic natural protein interactions by grafting peptide recognition motifs into the complementarity-determining regions (CDRs). We are using this approach to generate single-domain (VH) antibodies specific for amyloid-forming proteins such as the Alzheimer's Aβ peptide. Here, we use random mutagenesis and yeast surface display to improve the binding affinity of a lead VH domain grafted with Aβ residues 33-42 in CDR3. Interestingly, co-selection for improved Aβ binding and VH display on the surface of yeast yields antibody domains with improved affinity and reduced stability. The highest affinity VH domains were strongly destabilized on the surface of yeast as well as unfolded when isolated as autonomous domains. In contrast, stable VH domains with improved affinity were reliably identified using yeast surface display by replacing the display antibody that recognizes a linear epitope tag at the terminus of both folded and unfolded VH domains with a conformational ligand (Protein A) that recognizes a discontinuous epitope on the framework of folded VH domains. Importantly, we find that selection for improved stability using Protein A without simultaneous co-selection for improved Aβ binding leads to strong enrichment for stabilizing mutations that reduce antigen binding. Our findings highlight the importance of simultaneously optimizing affinity and stability to improve the rapid isolation of well-folded and specific antibody fragments.

  16. Co-evolution of affinity and stability of grafted amyloid-motif domain antibodies.

    PubMed

    Julian, Mark C; Lee, Christine C; Tiller, Kathryn E; Rabia, Lilia A; Day, Evan K; Schick, Arthur J; Tessier, Peter M

    2015-10-01

    An attractive approach for designing lead antibody candidates is to mimic natural protein interactions by grafting peptide recognition motifs into the complementarity-determining regions (CDRs). We are using this approach to generate single-domain (VH) antibodies specific for amyloid-forming proteins such as the Alzheimer's Aβ peptide. Here, we use random mutagenesis and yeast surface display to improve the binding affinity of a lead VH domain grafted with Aβ residues 33-42 in CDR3. Interestingly, co-selection for improved Aβ binding and VH display on the surface of yeast yields antibody domains with improved affinity and reduced stability. The highest affinity VH domains were strongly destabilized on the surface of yeast as well as unfolded when isolated as autonomous domains. In contrast, stable VH domains with improved affinity were reliably identified using yeast surface display by replacing the display antibody that recognizes a linear epitope tag at the terminus of both folded and unfolded VH domains with a conformational ligand (Protein A) that recognizes a discontinuous epitope on the framework of folded VH domains. Importantly, we find that selection for improved stability using Protein A without simultaneous co-selection for improved Aβ binding leads to strong enrichment for stabilizing mutations that reduce antigen binding. Our findings highlight the importance of simultaneously optimizing affinity and stability to improve the rapid isolation of well-folded and specific antibody fragments. PMID:26386257

  17. Co-evolution of affinity and stability of grafted amyloid-motif domain antibodies

    PubMed Central

    Julian, Mark C.; Lee, Christine C.; Tiller, Kathryn E.; Rabia, Lilia A.; Day, Evan K.; Schick, Arthur J.; Tessier, Peter M.

    2015-01-01

    An attractive approach for designing lead antibody candidates is to mimic natural protein interactions by grafting peptide recognition motifs into the complementarity-determining regions (CDRs). We are using this approach to generate single-domain (VH) antibodies specific for amyloid-forming proteins such as the Alzheimer's Aβ peptide. Here, we use random mutagenesis and yeast surface display to improve the binding affinity of a lead VH domain grafted with Aβ residues 33–42 in CDR3. Interestingly, co-selection for improved Aβ binding and VH display on the surface of yeast yields antibody domains with improved affinity and reduced stability. The highest affinity VH domains were strongly destabilized on the surface of yeast as well as unfolded when isolated as autonomous domains. In contrast, stable VH domains with improved affinity were reliably identified using yeast surface display by replacing the display antibody that recognizes a linear epitope tag at the terminus of both folded and unfolded VH domains with a conformational ligand (Protein A) that recognizes a discontinuous epitope on the framework of folded VH domains. Importantly, we find that selection for improved stability using Protein A without simultaneous co-selection for improved Aβ binding leads to strong enrichment for stabilizing mutations that reduce antigen binding. Our findings highlight the importance of simultaneously optimizing affinity and stability to improve the rapid isolation of well-folded and specific antibody fragments. PMID:26386257

  18. The Purification of Natural and Recombinant Peptide Antibodies by Affinity Chromatographic Strategies.

    PubMed

    Ma, Hui; O'Kennedy, Richard

    2015-01-01

    The purification of peptide antibodies (e.g., IgG, IgY, scFv, and Fab) are described in this chapter. Affinity chromatographic purification, a very convenient and effective antibody purification strategy, is used to isolate peptide antibodies based on specific binding, i.e., binding of the antibody to a column on which its specific ligand is immobilized with subsequent elution of the purified antibody. In addition, the application of purification methods based on the use of proteins A, G, and L, each of which bind to specific domains on an antibody/fragment, or the use of specific tags (e.g., histidine and biotin) attached to antibodies or antigens are also described.

  19. Quality control of murine monoclonal antibodies using isoelectric focusing affinity immunoblot analysis

    NASA Technical Reports Server (NTRS)

    Hamilton, Robert G.; Rodkey, L. Scott; Reimer, Charles B.

    1987-01-01

    The quality control of murine hybridoma secretory products has been performed using two approaches for isoelectric focusing affinity immunoblot analysis: (1) a method in which antigen-coated nitrocellulose is placed on top of an acrylamide gel containing isoelectrically focused ascites to bind the antigen specific monoclonal antibody; and (2) a method in which focused ascite proteins were passively blotted onto nitrocellulose and specific monoclonal antibodies were detected with enzyme-conjugated antigen. Analysis by both methods of batches of ascites containing antihuman IgG antibodies that were produced by six hybridomas permitted effective monitoring of immunoreactive antibodies for pI microheterogeneity.

  20. Affinity immunoblotting - High resolution isoelectric focusing analysis of antibody clonotype distribution

    NASA Technical Reports Server (NTRS)

    Knisley, Keith A.; Rodkey, L. Scott

    1986-01-01

    A sensitive and specific method is proposed for the analysis of specific antibody clonotype changes occurring during an immune response and for comparing multiple sera for antibody clonotype similarities. Polyclonal serum antibodies separated by isoelectric focusing (IEF) were analyzed by an affinity immunoblotting method using antigen-coated nitrocellulose membranes. Antibodies present on the surface of the acrylamide gels following IEF bind the antigen on the nitrocellulose when the coated nitrocellulose is laid over the gels. The technique has been used to analyze Ig clonotypes specific for five protein antigens and two carbohydrate antigens. Optimal antigen concentrations for coating the nitrocellulose membranes were found to range from 10-100 microgram/ml.

  1. Human antibody technology and the development of antibodies against cytomegalovirus.

    PubMed

    Ohlin, Mats; Söderberg-Nauclér, Cecilia

    2015-10-01

    Cytomegalovirus (CMV) is a virus that causes chronic infections in a large set of the population. It may cause severe disease in immunocompromised individuals, is linked to immunosenescence and implied to play an important role in the pathogenesis of cardiovascular diseases and cancer. Modulation of the immune system's abilities to manage the virus represent a highly viable therapeutic option and passive immunotherapy with polyclonal antibody preparations is already in clinical use. Defined monoclonal antibodies offer many advantages over polyclonal antibodies purified from serum. Human CMV-specific monoclonal antibodies have consequently been thoroughly investigated with respect to their potential in the treatment of diseases caused by CMV. Recent advances in human antibody technology have substantially expanded the breadth of antibodies for such applications. This review summarizes the fundamental basis for treating CMV disease by use of antibodies, the basic technologies to be used to develop such antibodies, and relevant human antibody specificities available to target this virus.

  2. Topography of the high-affinity lysine binding site of plasminogen as defined with a specific antibody probe

    SciTech Connect

    Miles, L.A.; Plow, E.F.

    1986-11-04

    An antibody population that reacted with the high-affinity lysine binding site of human plasminogen was elicited by immunizing rabbits with an elastase degradation product containing kringles 1-3 (EDP I). This antibody was immunopurified by affinity chromatography on plasminogen-Sepharose and elution with 0.2 M 6-aminohexanoic acid. The eluted antibodies bound (/sup 125/I)EDP I, (/sup 125/I)Glu-plasminogen, and (/sup 125/I)Lys-plasminogen in radioimmunoassays, and binding of each ligand was at least 99% inhibited by 0.2 M 6-aminohexanoic acid. The concentrations for 50% inhibition of (/sup 125/I)EDP I binding by tranexamic acid, 6-aminohexanoic acid, and lysine were 2.6, 46, and l730 ..mu..M, respectively. Similar values were obtained with plasminogen and suggested that an unoccupied high-affinity lysine binding site was required for antibody recognition. The antiserum reacted exclusively with plasminogen derivatives containing the EDP I region and did not react with those lacking an EDP I region, or with tissue plasminogen activator or prothrombin, which also contains kringles. By immunoblotting analyses, a chymotryptic degradation product of M/sub r/ 20,000 was derived from EDP I that retained reactivity with the antibody. ..cap alpha../sub 2/-Antiplasmin inhibited the binding of radiolabeled EDP I, Glu-plasminogen, or Lys-plasminogen by the antiserum, suggesting that the recognized site is involved in the noncovalent interaction of the inhibitor with plasminogen. The binding of (/sup 125/I)EDP I to fibrin was also inhibited by the antiserum. The observations provide independent evidence for the role of the high-affinity lysine binding site in the functional interactions of plasminogen with its primary substrate and inhibitor.

  3. Affinity maturation of T-cell receptor-like antibodies for Wilms tumor 1 peptide greatly enhances therapeutic potential

    PubMed Central

    Zhao, Qi; Ahmed, Mahiuddin; Tassev, Dimiter V.; Hasan, Aisha; Kuo, Tzu-Yun; Guo, Hong-fen; O’Reilly, Richard J.; Cheung, Nai-Kong V.

    2016-01-01

    WT1126 (RMFPNAPYL) is a human leukocyte antigen-A2 (HLA-A2) restricted peptide derived from Wilms tumor protein (WT1), which is widely expressed in a broad spectrum of leukemias, lymphomas and solid tumors. A novel T-cell-receptor (TCR)-like single chain variable fragment (scFv) antibody specific for the T cell epitope consisting of the WT1/HLA-A2 complex was isolated from a human scFv phage library. This scFv was affinity-matured by mutagenesis combined with yeast display, and structurally analyzed using a homology model. This monovalent scFv showed a 100-fold affinity improvement (dissociation constant [KD]= 3nM) and exquisite specificity towards its targeted epitope or HLA-A2+/WT1+ tumor cells. Bivalent scFv-huIgG1-Fc fusion protein demonstrated an even higher avidity (KD = 2pM) binding to the T cell epitope and to tumor targets, and was capable of mediating antibody-dependent cell-mediated cytotoxicity or tumor lysis by chimeric antigen receptor (CAR)-expressing human T or NK-92-MI transfected cells. This antibody demonstrated specific and potent cytotoxicity in vivo towards WT1-positive leukemia xenograft that was HLA-A2 restricted. In summary, T cell epitopes can provide novel targets for antibody-based therapeutics. By combining phage and yeast displays and scFv-Fc fusion platforms, a strategy for developing high affinity TCR-like antibodies could be rapidly explored for potential clinical development. PMID:25987253

  4. A strategy of designing the ligand of antibody affinity chromatography based on molecular dynamics simulation.

    PubMed

    Dai, Lu; Li, Weikang; Sun, Fei; Li, Baizhi; Li, Hongrui; Zhang, Hongxing; Zheng, Qingchuan; Liang, Chongyang

    2016-09-01

    Designing affinity ligands has always been the development focus of affinity chromatography. Previous antibody affinity ligand designs were mostly based on the crystal structure of protein A (UniProt code number: P38507), and the antibody-binding domains were modified according to the properties of amino acid residues. Currently, more effective bioinformatic prediction and experimental validation has been used to improve the design of antibody affinity ligands. In the present study, the complex crystal structure (the domain D of protein A and the Fab segment of IgM, PDB code: 1DEE) was used as the model. The vital site that inhibits the binding between domain D and IgM was estimated by means of molecular dynamics (MD) simulation, then MM-GBSA calculations were used to design a mutant of domain D (K46E) for improving affinity on the above vital site. The binding analysis using Biacore showed the association and dissociation parameters of K46E mutant that were optimized with IgM. The affinity increase of K46E mutant preferred for IgM, the affinity order is K46E tetramer (KD=6.02×10(-9)M)>K46E mutant (KD=6.66×10(-8)M)>domain D (KD=2.17×10(-7)M). Similar results were obtained when the optimized ligands were immobilized to the chromatography medium. A complete designing strategy was validated in this study, which will provide a novel insight into designing new ligands of antibody affinity chromatography media.

  5. A strategy of designing the ligand of antibody affinity chromatography based on molecular dynamics simulation.

    PubMed

    Dai, Lu; Li, Weikang; Sun, Fei; Li, Baizhi; Li, Hongrui; Zhang, Hongxing; Zheng, Qingchuan; Liang, Chongyang

    2016-09-01

    Designing affinity ligands has always been the development focus of affinity chromatography. Previous antibody affinity ligand designs were mostly based on the crystal structure of protein A (UniProt code number: P38507), and the antibody-binding domains were modified according to the properties of amino acid residues. Currently, more effective bioinformatic prediction and experimental validation has been used to improve the design of antibody affinity ligands. In the present study, the complex crystal structure (the domain D of protein A and the Fab segment of IgM, PDB code: 1DEE) was used as the model. The vital site that inhibits the binding between domain D and IgM was estimated by means of molecular dynamics (MD) simulation, then MM-GBSA calculations were used to design a mutant of domain D (K46E) for improving affinity on the above vital site. The binding analysis using Biacore showed the association and dissociation parameters of K46E mutant that were optimized with IgM. The affinity increase of K46E mutant preferred for IgM, the affinity order is K46E tetramer (KD=6.02×10(-9)M)>K46E mutant (KD=6.66×10(-8)M)>domain D (KD=2.17×10(-7)M). Similar results were obtained when the optimized ligands were immobilized to the chromatography medium. A complete designing strategy was validated in this study, which will provide a novel insight into designing new ligands of antibody affinity chromatography media. PMID:27524303

  6. Development of new versions of anti-human CD34 monoclonal antibodies with potentially reduced immunogenicity

    SciTech Connect

    Qian Weizhu; Wang Ling; Li Bohua; Wang Hao; Hou Sheng; Hong Xueyu; Zhang Dapeng; Guo Yajun

    2008-03-07

    Despite the widespread clinical use of CD34 antibodies for the purification of human hematopoietic stem/progenitor cells, all the current anti-human CD34 monoclonal antibodies (mAbs) are murine, which have the potential to elicit human antimouse antibody (HAMA) immune response. In the present study, we developed three new mouse anti-human CD34 mAbs which, respectively, belonged to class I, class II and class III CD34 epitope antibodies. In an attempt to reduce the immunogenicity of these three murine mAbs, their chimeric antibodies, which consisted of mouse antibody variable regions fused genetically to human antibody constant regions, were constructed and characterized. The anti-CD34 chimeric antibodies were shown to possess affinity and specificity similar to that of their respective parental murine antibodies. Due to the potentially better safety profiles, these chimeric antibodies might become alternatives to mouse anti-CD34 antibodies routinely used for clinical application.

  7. Human germline antibody gene segments encode polyspecific antibodies.

    PubMed

    Willis, Jordan R; Briney, Bryan S; DeLuca, Samuel L; Crowe, James E; Meiler, Jens

    2013-04-01

    Structural flexibility in germline gene-encoded antibodies allows promiscuous binding to diverse antigens. The binding affinity and specificity for a particular epitope typically increase as antibody genes acquire somatic mutations in antigen-stimulated B cells. In this work, we investigated whether germline gene-encoded antibodies are optimal for polyspecificity by determining the basis for recognition of diverse antigens by antibodies encoded by three VH gene segments. Panels of somatically mutated antibodies encoded by a common VH gene, but each binding to a different antigen, were computationally redesigned to predict antibodies that could engage multiple antigens at once. The Rosetta multi-state design process predicted antibody sequences for the entire heavy chain variable region, including framework, CDR1, and CDR2 mutations. The predicted sequences matched the germline gene sequences to a remarkable degree, revealing by computational design the residues that are predicted to enable polyspecificity, i.e., binding of many unrelated antigens with a common sequence. The process thereby reverses antibody maturation in silico. In contrast, when designing antibodies to bind a single antigen, a sequence similar to that of the mature antibody sequence was returned, mimicking natural antibody maturation in silico. We demonstrated that the Rosetta computational design algorithm captures important aspects of antibody/antigen recognition. While the hypervariable region CDR3 often mediates much of the specificity of mature antibodies, we identified key positions in the VH gene encoding CDR1, CDR2, and the immunoglobulin framework that are critical contributors for polyspecificity in germline antibodies. Computational design of antibodies capable of binding multiple antigens may allow the rational design of antibodies that retain polyspecificity for diverse epitope binding.

  8. Heterophile antibodies in human transplantation

    PubMed Central

    Rapaport, F. T.; Kano, K.; Milgrom, F.

    1968-01-01

    Sensitization of human recipients with transplantation antigens (leucocytes, skin, or kidney allografts) has resulted in the appearance of serum hemagglutinins directed against sheep, guinea pig, and rat erythrocytes. Such hemagglutinins have been identified as IgG and IgM antibodies. Their appearance was not related to AB0 erythrocyte group incompatibility between donors and recipients, and the antibodies were not of the Forssman or Paul-Bunnel type. The antibody responses appeared to be primarily directed against antigen(s) present on rat erythrocytes, but shared to varying extents by other species. The peak antibody titers occurred in association with allograft rejection. In this regard, they may be of interest as a possible early warning system for the diagnosis and prompt management of rejection crises in clinical organ transplantation. Images PMID:4866325

  9. Affinity-based methodologies and ligands for antibody purification: advances and perspectives.

    PubMed

    Roque, Ana C A; Silva, Cláudia S O; Taipa, M Angela

    2007-08-10

    Many successful, recent therapies for life-threatening diseases such as cancer and rheumatoid arthritis are based on the recognition between native or genetically engineered antibodies and cell-surface receptors. Although naturally produced by the immune system, the need for antibodies with unique specificities and designed for single application, has encouraged the search for novel antibody purification strategies. The availability of these products to the end-consumer is strictly related to manufacture costs, particularly those attributed to downstream processing. Over the last decades, academia and industry have developed different types of interactions and separation techniques for antibody purification, affinity-based strategies being the most common and efficient methodologies. The affinity ligands utilized range from biological to synthetic designed molecules with enhanced resistance and stability. Despite the successes achieved, the purification "paradigm" still moves interests and efforts in the continuous demand for improved separation performances. This review will focus on recent advances and perspectives in antibody purification by affinity interactions using different techniques, with particular emphasis on affinity chromatography.

  10. Affinity-based methodologies and ligands for antibody purification: advances and perspectives.

    PubMed

    Roque, Ana C A; Silva, Cláudia S O; Taipa, M Angela

    2007-08-10

    Many successful, recent therapies for life-threatening diseases such as cancer and rheumatoid arthritis are based on the recognition between native or genetically engineered antibodies and cell-surface receptors. Although naturally produced by the immune system, the need for antibodies with unique specificities and designed for single application, has encouraged the search for novel antibody purification strategies. The availability of these products to the end-consumer is strictly related to manufacture costs, particularly those attributed to downstream processing. Over the last decades, academia and industry have developed different types of interactions and separation techniques for antibody purification, affinity-based strategies being the most common and efficient methodologies. The affinity ligands utilized range from biological to synthetic designed molecules with enhanced resistance and stability. Despite the successes achieved, the purification "paradigm" still moves interests and efforts in the continuous demand for improved separation performances. This review will focus on recent advances and perspectives in antibody purification by affinity interactions using different techniques, with particular emphasis on affinity chromatography. PMID:17618635

  11. Antibody Binding Selectivity: Alternative Sets of Antigen Residues Entail High-Affinity Recognition

    PubMed Central

    Nominé, Yves; Choulier, Laurence; Travé, Gilles; Vernet, Thierry; Altschuh, Danièle

    2015-01-01

    Understanding the relationship between protein sequence and molecular recognition selectivity remains a major challenge. The antibody fragment scFv1F4 recognizes with sub nM affinity a decapeptide (sequence 6TAMFQDPQER15) derived from the N-terminal end of human papilloma virus E6 oncoprotein. Using this decapeptide as antigen, we had previously shown that only the wild type amino-acid or conservative replacements were allowed at positions 9 to 12 and 15 of the peptide, indicating a strong binding selectivity. Nevertheless phenylalanine (F) was equally well tolerated as the wild type glutamine (Q) at position 13, while all other amino acids led to weaker scFv binding. The interfaces of complexes involving either Q or F are expected to diverge, due to the different physico-chemistry of these residues. This would imply that high-affinity binding can be achieved through distinct interfacial geometries. In order to investigate this point, we disrupted the scFv–peptide interface by modifying one or several peptide positions. We then analyzed the effect on binding of amino acid changes at the remaining positions, an altered susceptibility being indicative of an altered role in complex formation. The 23 starting variants analyzed contained replacements whose effects on scFv1F4 binding ranged from minor to drastic. A permutation analysis (effect of replacing each peptide position by all other amino acids except cysteine) was carried out on the 23 variants using the PEPperCHIP® Platform technology. A comparison of their permutation patterns with that of the wild type peptide indicated that starting replacements at position 11, 12 or 13 modified the tolerance to amino-acid changes at the other two positions. The interdependence between the three positions was confirmed by SPR (Biacore® technology). Our data demonstrate that binding selectivity does not preclude the existence of alternative high-affinity recognition modes. PMID:26629896

  12. Maximum-Entropy Models of Sequenced Immune Repertoires Predict Antigen-Antibody Affinity

    PubMed Central

    Marcatili, Paolo; Pagnani, Andrea

    2016-01-01

    The immune system has developed a number of distinct complex mechanisms to shape and control the antibody repertoire. One of these mechanisms, the affinity maturation process, works in an evolutionary-like fashion: after binding to a foreign molecule, the antibody-producing B-cells exhibit a high-frequency mutation rate in the genome region that codes for the antibody active site. Eventually, cells that produce antibodies with higher affinity for their cognate antigen are selected and clonally expanded. Here, we propose a new statistical approach based on maximum entropy modeling in which a scoring function related to the binding affinity of antibodies against a specific antigen is inferred from a sample of sequences of the immune repertoire of an individual. We use our inference strategy to infer a statistical model on a data set obtained by sequencing a fairly large portion of the immune repertoire of an HIV-1 infected patient. The Pearson correlation coefficient between our scoring function and the IC50 neutralization titer measured on 30 different antibodies of known sequence is as high as 0.77 (p-value 10−6), outperforming other sequence- and structure-based models. PMID:27074145

  13. A humanized monoclonal antibody targeting Staphylococcus aureus.

    PubMed

    Patti, Joseph M

    2004-12-01

    This current presentation describes the in vitro and in vivo characterization of Aurexis (tefibazumab), a humanized monoclonal antibody that exhibits a high affinity and specificity and for the Staphylococcus aureus MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecules) protein ClfA. Aurexis inhibited ClfA binding to human fibrinogen, and enhanced the opsonophagocytic uptake of ClfA-coated beads. Preclinical in vivo testing revealed that a single administration of Aurexis significantly protected against an IV challenge with a methicillin resistant S. aureus (MRSA) strain in murine septicemia and rabbit infective endocarditis (IE) models. Safety and pharmacokinetic data from a 19-patient phase I study support continued evaluation of Aurexis in phase II studies. PMID:15576200

  14. Characterization of monoclonal antibodies against human lactoferrin.

    PubMed

    van Berkel, Patrick H C; van Veen, Harrie A; Geerts, Marlieke E J; Nuijens, Jan H

    2002-09-15

    The iron-binding glycoprotein human lactoferrin (hLF) is involved in the host defense against infection and is a modulator of inflammatory reactions. We generated monoclonal antibodies (mAbs) to hLF as tools to assist both structure-function studies and the development of recombinant human lactoferrin for applications in human health care. Binding experiments with ten distinct anti-hLF mAbs to tryptic and recombinant hLF fragments in ELISA and/or on immunoblots revealed that five mAbs bound to conformational epitopes residing in the N-lobe (residues 1 to 334), whereas the other five bound to C-lobe conformational epitopes (residues 335 to 692). None of the mAbs bound to hLF denatured upon reduction. Monoclonal antibody E11 appeared to bind to the arginine-rich N-terminus of hLF, which is the binding site for heparin, bacterial lipopolysaccharide, human lysozyme, DNA and receptors. The dissociation constant of the distinct mAbs for hLF ranged from 0.5 to 18 nM, without differences in affinity for unsaturated or iron-saturated hLF, indicating that the conformational changes subject to incorporation of iron do not seem to affect the exposure and/or conformation of the antibody epitopes. The mAbs did not bind to human transferrin, a protein closely related to hLF in size, primary amino acid sequence and structure. Two C-lobe specific mAbs, E2 and E8, cross-reacted with bovine and/or porcine lactoferrin, indicating that human, bovine and porcine lactoferrin share antigenic determinants. This panel of mAbs will be used to develop quantitative and qualitative immunoassays for hLF and to delineate which regions of hLF are relevant to its anti-infective and anti-inflammatory properties. PMID:12165435

  15. CD4+ T Cells Promote Antibody Production but Not Sustained Affinity Maturation during Borrelia burgdorferi Infection

    PubMed Central

    Elsner, Rebecca A.; Hastey, Christine J.

    2014-01-01

    CD4 T cells are crucial for enhancing B cell-mediated immunity, supporting the induction of high-affinity, class-switched antibody responses, long-lived plasma cells, and memory B cells. Previous studies showed that the immune response to Borrelia burgdorferi appears to lack robust T-dependent B cell responses, as neither long-lived plasma cells nor memory B cells form for months after infection, and nonswitched IgM antibodies are produced continuously during this chronic disease. These data prompted us to evaluate the induction and functionality of B. burgdorferi infection-induced CD4 TFH cells. We report that CD4 T cells were effectively primed and TFH cells induced after B. burgdorferi infection. These CD4 T cells contributed to the control of B. burgdorferi burden and supported the induction of B. burgdorferi-specific IgG responses. However, while affinity maturation of antibodies against a prototypic T-dependent B. burgdorferi protein, Arthritis-related protein (Arp), were initiated, these increases were reversed later, coinciding with the previously observed involution of germinal centers. The cessation of affinity maturation was not due to the appearance of inhibitory or exhausted CD4 T cells or a strong induction of regulatory T cells. In vitro T-B cocultures demonstrated that T cells isolated from B. burgdorferi-infected but not B. burgdorferi-immunized mice supported the rapid differentiation of B cells into antibody-secreting plasma cells rather than continued proliferation, mirroring the induction of rapid short-lived instead of long-lived T-dependent antibody responses in vivo. The data further suggest that B. burgdorferi infection drives the humoral response away from protective, high-affinity, and long-lived antibody responses and toward the rapid induction of strongly induced, short-lived antibodies of limited efficacy. PMID:25312948

  16. Identification and subcellular localization of a 21-kilodalton molecule using affinity-purified antibodies against. cap alpha. -transforming growth factor

    SciTech Connect

    Hazarika, P.; Pardue, R.L.; Earls, R.; Dedman, J.R.

    1987-04-07

    Monospecific antibodies were generated against each of six different peptide sequences derived from rat and human ..cap alpha..-transforming growth factor (..cap alpha..-TGF). The affinity-purified antibody to the 17 amino acid carboxyl-terminal portion of the molecule proved most useful in detecting ..cap alpha..-TGF. When used in a peptide-based radioimmunoassay, it was possible to measure nanogram quantities of native ..cap alpha..-TGF in conditioned cell culture media. When used to analyze cell lysate, these antibodies specifically recognized a 21-kilodalton protein species. Indirect immunofluorescence localization procedures revealed a high concentration of ..cap alpha..-TCF in a perinuclear ring with a diffuse cytoplasmic distribution. These results suggest that a precursor form of ..cap alpha..-TGF has a cellular role beyond that of an autocrine growth factor.

  17. Intra-clonal competition inhibits the formation of high affinity antibody secreting cells1

    PubMed Central

    Le, Thuc-vy L.; Kim, Tea Hyun; Chaplin, David D.

    2010-01-01

    Protective immunity requires a diverse, polyclonal B cell repertoire. We demonstrate that affinity maturation of the humoral response to a hapten is impaired when pre-existing clonally restricted cells recognizing the hapten are dominant in the B cell repertoire. B1- 8i+/− mice, which feature a high frequency of B cells with nitrophenyl (NP) binding specificity, respond to NP-haptenated proteins with the production of NP-specific antibodies, but affinity maturation is impaired due to insufficient generation of high affinity antibody producing cells. We manipulated the frequency of NP-specific B cells by adoptive transfer of B1-8 B cells into naïve, wild-type recipients. Remarkably, when 104 B1-8 B cells were transferred, these cells supported efficient affinity maturation and plasma cell differentiation. In contrast, when 106 B1-8 cells were transferred, affinity maturation did not occur. These data indicate that restricting the frequency of clonally related B cells is required to support affinity maturation. PMID:18941192

  18. Affinity chromatography for purification of two urokinases from human urine.

    PubMed

    Takahashi, R; Akiba, K; Koike, M; Noguchi, T; Ezure, Y

    2000-05-26

    A new affinity chromatography (hydrophobic-mediated affinity chromatography), which was characterized by the matrix having both affinity site to urokinase and hydrophobic site, was established for the purification of urokinase from human urine. The hydrophobic affinity matrix (tentatively named PAS in the text) was prepared by immobilizing 6-aminocaproic acid on Sepharose CL-6B, followed by a coupling p-aminobenzamidine to a part of the hydrophobic site on the matrix. The PAS matrix was applied to the purification of urokinase from human urine, and high- and low-molecular weight pure urokinases were efficiently obtained in high yield by the present method. PMID:10892585

  19. Deconvolution of antibody affinities and concentrations by non-linear regression analysis of competitive ELISA data.

    SciTech Connect

    Stevens, F. J.; Bobrovnik, S. A.; Biosciences Division; Palladin Inst. Biochemistry

    2007-12-01

    Physiological responses of the adaptive immune system are polyclonal in nature whether induced by a naturally occurring infection, by vaccination to prevent infection or, in the case of animals, by challenge with antigen to generate reagents of research or commercial significance. The composition of the polyclonal responses is distinct to each individual or animal and changes over time. Differences exist in the affinities of the constituents and their relative proportion of the responsive population. In addition, some of the antibodies bind to different sites on the antigen, whereas other pairs of antibodies are sterically restricted from concurrent interaction with the antigen. Even if generation of a monoclonal antibody is the ultimate goal of a project, the quality of the resulting reagent is ultimately related to the characteristics of the initial immune response. It is probably impossible to quantitatively parse the composition of a polyclonal response to antigen. However, molecular regression allows further parameterization of a polyclonal antiserum in the context of certain simplifying assumptions. The antiserum is described as consisting of two competing populations of high- and low-affinity and unknown relative proportions. This simple model allows the quantitative determination of representative affinities and proportions. These parameters may be of use in evaluating responses to vaccines, to evaluating continuity of antibody production whether in vaccine recipients or animals used for the production of antisera, or in optimizing selection of donors for the production of monoclonal antibodies.

  20. Affinity Chromatography of Native and Recombinant Proteins from Receptors for Insulin and IGF-I to Recombinant Single Chain Antibodies.

    PubMed

    Fujita-Yamaguchi, Yoko

    2015-01-01

    Affinity chromatography is an efficient method to isolate proteins by taking advantage of their affinities for specific molecules such as substrates, inhibitors, antigens, ligands, antibodies, and other interacting molecules, including subunits. Nowadays, we take the effectiveness and excellence of this technology for granted. This essay will mainly cover the use of affinity chromatography based on my experience. PMID:26579073

  1. Affinity Chromatography of Native and Recombinant Proteins from Receptors for Insulin and IGF-I to Recombinant Single Chain Antibodies

    PubMed Central

    Fujita-Yamaguchi, Yoko

    2015-01-01

    Affinity chromatography is an efficient method to isolate proteins by taking advantage of their affinities for specific molecules such as substrates, inhibitors, antigens, ligands, antibodies, and other interacting molecules, including subunits. Nowadays, we take the effectiveness and excellence of this technology for granted. This essay will mainly cover the use of affinity chromatography based on my experience. PMID:26579073

  2. Monoclonal antibodies to human glycophorin A and cell lines for the production thereof

    DOEpatents

    Vanderlaan, Martin; Bigbee, William L.; Jensen, Ronald H.; Fong, Stella S. N.; Langlois, Richard G.

    1988-01-01

    Cloned mouse hybridoma cell lines have been established which continuously produce antibodies that are highly specific to and exhibit high affinity for glycophorin A.sup.N and differentiate between the M and N forms of human glycophorin A.

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

    PubMed Central

    Schmidt, Michael M.; Thurber, Greg M.

    2010-01-01

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

  4. Antibody humanization by structure-based computational protein design

    PubMed Central

    Choi, Yoonjoo; Hua, Casey; Sentman, Charles L; Ackerman, Margaret E; Bailey-Kellogg, Chris

    2015-01-01

    Antibodies derived from non-human sources must be modified for therapeutic use so as to mitigate undesirable immune responses. While complementarity-determining region (CDR) grafting-based humanization techniques have been successfully applied in many cases, it remains challenging to maintain the desired stability and antigen binding affinity upon grafting. We developed an alternative humanization approach called CoDAH (“Computationally-Driven Antibody Humanization”) in which computational protein design methods directly select sets of amino acids to incorporate from human germline sequences to increase humanness while maintaining structural stability. Retrospective studies show that CoDAH is able to identify variants deemed beneficial according to both humanness and structural stability criteria, even for targets lacking crystal structures. Prospective application to TZ47, a murine anti-human B7H6 antibody, demonstrates the approach. Four diverse humanized variants were designed, and all possible unique VH/VL combinations were produced as full-length IgG1 antibodies. Soluble and cell surface expressed antigen binding assays showed that 75% (6 of 8) of the computationally designed VH/VL variants were successfully expressed and competed with the murine TZ47 for binding to B7H6 antigen. Furthermore, 4 of the 6 bound with an estimated KD within an order of magnitude of the original TZ47 antibody. In contrast, a traditional CDR-grafted variant could not be expressed. These results suggest that the computational protein design approach described here can be used to efficiently generate functional humanized antibodies and provide humanized templates for further affinity maturation. PMID:26252731

  5. 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. PMID:27391930

  6. Affinity binding of inclusion bodies on supermacroporous monolithic cryogels using labeling with specific antibodies.

    PubMed

    Ahlqvist, Josefin; Kumar, Ashok; Sundström, Heléne; Ledung, Erika; Hörnsten, E Gunnar; Enfors, Sven-Olof; Mattiasson, Bo

    2006-03-23

    A new chromatographic method based on affinity supermacroporous monolithic cryogels is developed for binding and analyzing inclusion bodies during fermentation. The work demonstrated that it is possible to bind specific IgG and IgY antibodies to the 15 and 17 amino acids at the terminus ends of a 33 kDa target protein aggregated as inclusion bodies. The antibody treated inclusion bodies from lysed fermentation broth can be specifically retained in protein A and pseudo-biospecific ligand sulfamethazine modified supermacroporous cryogels. The degree of binding of IgG and IgY treated inclusion bodies to the Protein A and sulfamethazine gels are investigated, as well as the influence of pH on the sulfamethazine ligand. Optimum binding of 78 and 72% was observed on both protein A and sulfamethazine modified cryogel columns, respectively, using IgG labeling of the inclusion bodies. The antibody treated inclusion bodies pass through unretained in the sulfamethazine supermacroporous gel at pH that does not favour the binding between the ligand on the gel and the antibodies on the surface of inclusion bodies. Also the unlabeled inclusion bodies went through the gel unretained, showing no non-specific binding or trapping within the gel. These findings may very well be the foundation for the building of a powerful analytical tool during fermentation of inclusion bodies as well as a convenient way to purify them from fermentation broth. These results also support our earlier findings [Kumar, A., Plieva, F.M., Galaev, I.Yu., Mattiasson, B., 2003. Affinity fractionation of lymphocytes using a monolithic cyogel. J. Immunol. Methods 283, 185-194] with mammalian cells that were surface labeled with specific antibodies and recognized on protein A supermacroporous gels. A general binding and separation system can be established on antibody binding cryogel affinity matrices.

  7. AB-Bind: Antibody binding mutational database for computational affinity predictions.

    PubMed

    Sirin, Sarah; Apgar, James R; Bennett, Eric M; Keating, Amy E

    2016-02-01

    Antibodies (Abs) are a crucial component of the immune system and are often used as diagnostic and therapeutic agents. The need for high-affinity and high-specificity antibodies in research and medicine is driving the development of computational tools for accelerating antibody design and discovery. We report a diverse set of antibody binding data with accompanying structures that can be used to evaluate methods for modeling antibody interactions. Our Antibody-Bind (AB-Bind) database includes 1101 mutants with experimentally determined changes in binding free energies (ΔΔG) across 32 complexes. Using the AB-Bind data set, we evaluated the performance of protein scoring potentials in their ability to predict changes in binding free energies upon mutagenesis. Numerical correlations between computed and observed ΔΔG values were low (r = 0.16-0.45), but the potentials exhibited predictive power for classifying variants as improved vs weakened binders. Performance was evaluated using the area under the curve (AUC) for receiver operator characteristic (ROC) curves; the highest AUC values for 527 mutants with |ΔΔG| > 1.0 kcal/mol were 0.81, 0.87, and 0.88 using STATIUM, FoldX, and Discovery Studio scoring potentials, respectively. Some methods could also enrich for variants with improved binding affinity; FoldX and Discovery Studio were able to correctly rank 42% and 30%, respectively, of the 80 most improved binders (those with ΔΔG < -1.0 kcal/mol) in the top 5% of the database. This modest predictive performance has value but demonstrates the continuing need to develop and improve protein energy functions for affinity prediction. PMID:26473627

  8. High Affinity Antibodies against Influenza Characterize the Plasmablast Response in SLE Patients After Vaccination

    PubMed Central

    Kaur, Kaval; Zheng, Nai-Ying; Smith, Kenneth; Huang, Min; Li, Lie; Pauli, Noel T.; Henry Dunand, Carole J.; Lee, Jane-Hwei; Morrissey, Michael; Wu, Yixuan; Joachims, Michelle L.; Munroe, Melissa E.; Lau, Denise; Qu, Xinyan; Krammer, Florian; Wrammert, Jens; Palese, Peter; Ahmed, Rafi; James, Judith A.; Wilson, Patrick C.

    2015-01-01

    Breakdown of B cell tolerance is a cardinal feature of systemic lupus erythematosus (SLE). Increased numbers of autoreactive mature naïve B cells have been described in SLE patients and autoantibodies have been shown to arise from autoreactive and non-autoreactive precursors. How these defects, in the regulation of B cell tolerance and selection, influence germinal center (GC) reactions that are directed towards foreign antigens has yet to be investigated. Here, we examined the characteristics of post-GC foreign antigen-specific B cells from SLE patients and healthy controls by analyzing monoclonal antibodies generated from plasmablasts induced specifically by influenza vaccination. We report that many of the SLE patients had anti-influenza antibodies with higher binding affinity and neutralization capacity than those from controls. Although overall frequencies of autoreactivity in the influenza-specific plasmablasts were similar for SLE patients and controls, the variable gene repertoire of influenza-specific plasmablasts from SLE patients was altered, with increased usage of JH6 and long heavy chain CDR3 segments. We found that high affinity anti-influenza antibodies generally characterize the plasmablast responses of SLE patients with low levels of autoreactivity; however, certain exceptions were noted. The high-avidity antibody responses in SLE patients may also be correlated with cytokines that are abnormally expressed in lupus. These findings provide insights into the effects of dysregulated immunity on the quality of antibody responses following influenza vaccination and further our understanding of the underlying abnormalities of lupus. PMID:25951191

  9. Purification of infective bluetongue virus particles by immuno-affinity chromatography using anti-core antibody.

    PubMed

    Chand, Karam; Biswas, Sanchay K; Mondal, Bimalendu

    2016-03-01

    An immuno-affinity chromatography technique for purification of infective bluetongue virus (BTV) has been descried using anti-core antibodies. BTV anti-core antibodies (prepared in guinea pig) were mixed with cell culture-grown BTV-1 and then the mixture was added to the cyanogens bromide-activated protein-A Sepharose column. Protein A binds to the antibody which in turn binds to the antigen (i.e. BTV). After thorough washing, antigen-antibody and antibody-protein A couplings were dissociated with 4M MgCl2, pH6.5. Antibody molecules were removed by dialysis and virus particles were concentrated by spin column ultrafiltration. Dialyzed and concentrated material was tested positive for BTV antigen by a sandwich ELISA and the infectivity of the chromatography-purified virus was demonstrated in cell culture. This method was applied for selective capture of BTV from a mixture of other viruses. As group-specific antibodies (against BTV core) were used to capture the virus, it is expected that virus of all BTV serotypes could be purified by this method. This method will be helpful for selective capture and enrichment of BTV from concurrently infected blood or tissue samples for efficient isolation in cell culture. Further, this method can be used for small scale purification of BTV avoiding ultracentrifugation. PMID:26925450

  10. Purification of anti-bromelain antibodies by affinity precipitation using pNIPAm-linked bromelain.

    PubMed

    Mahmood, Rubab

    2016-01-01

    Affinity precipitation has emerged as a very useful technique for the purification of proteins. Here it has been employed for the purification of anti-bromelain antibodies from rabbit serum. A system has been developed for reversibly binding and thermoprecipitating antibodies. Anti-bromelain antibodies were raised in rabbit by immunizing it with bromelain. Poly-N-isopropylacrylamide (pNIPAm)-bromelain conjugate was prepared and incubated with rabbit serum. After that the temperature was raised for thermal precipitation of the polymer. Antibodies were then eluted from the complex by incubating it with a small volume of buffer, pH 3.0. This method is very effective in concentrating the antibodies. Purity and specificity of the antibodies were checked by gel electrophoresis and enzyme-linked immunosorbent assay (ELISA), respectively. The study of the effect of pH and temperature on the binding of the antibodies to the conjugate showed that the optimum binding occurred at pH 8.0 and 25°C.The polymer enzyme conjugate was further used for another cycle.

  11. A general method for greatly improving the affinity of antibodies by using combinatorial libraries

    PubMed Central

    Rajpal, Arvind; Beyaz, Nurten; Haber, Lauric; Cappuccilli, Guido; Yee, Helena; Bhatt, Ramesh R.; Takeuchi, Toshihiko; Lerner, Richard A.; Crea, Roberto

    2005-01-01

    Look-through mutagenesis (LTM) is a multidimensional mutagenesis method that simultaneously assesses and optimizes combinatorial mutations of selected amino acids. The process focuses on a precise distribution within one or more complementarity determining region (CDR) domains and explores the synergistic contribution of amino acid side-chain chemistry. LTM was applied to an anti-TNF-α antibody, D2E7, which is a challenging test case, because D2E7 was highly optimized (Kd = 1 nM) by others. We selected and incorporated nine amino acids, representative of the major chemical functionalities, individually at every position in each CDR and across all six CDRs (57 aa). Synthetic oligonucleotides, each introducing one amino acid mutation throughout the six CDRs, were pooled to generate segregated libraries containing single mutations in one, two, and/or three CDRs for each VH and VL domain. Corresponding antibody libraries were displayed on the cell surface of yeast. After positive binding selection, 38 substitutions in 21 CDR positions were identified that resulted in higher affinity binding to TNF-α. These beneficial mutations in both VH and VL were represented in two combinatorial beneficial mutagenesis libraries and selected by FACS to produce a convergence of variants that exhibit between 500- and 870-fold higher affinities. Importantly, these enhanced affinities translate to a 15- to 30-fold improvement in in vitro TNF-α neutralization in an L929 bioassay. Thus, this LTM/combinatorial beneficial mutagenesis strategy generates a comprehensive energetic map of the antibody-binding site in a facile and rapid manner and should be broadly applicable to the affinity maturation of antibodies and other proteins. PMID:15939870

  12. Probing Cocaine-Antibody Interactions in Buffer and Human Serum

    PubMed Central

    Ramakrishnan, Muthu; Alves De Melo, Fernando; Kinsey, Berma M.; Ladbury, John E.; Kosten, Thomas R.; Orson, Frank M.

    2012-01-01

    Background Despite progress in cocaine immunotherapy, the kinetic and thermodynamic properties of antibodies which bind to cocaine and its metabolites are not well understood. It is also not clear how the interactions between them differ in a complex matrix such as the serum present in the human body. In the present study, we have used microscale thermophoresis (MST), isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR) we have evaluated the affinity properties of a representative mouse monoclonal (mAb08) as well as those of polyclonal antibodies purified from vaccinated mouse and human patient serum. Results MST analysis of fluorescently tagged mAb08 binding to cocaine reveals an approximately 15 fold decrease in its equilibrium dissociation constant in 20–50% human serum compared with that in saline buffer. A similar trend was also found using enriched polyclonal antibodies purified from vaccinated mice and patient serum, for which we have used fluorescently tagged bovine serum albumin conjugated to succinyl norcocaine (BSA-SNC). This conjugate closely mimics both cocaine and the hapten used to raise these antibodies. The ITC data also revealed that cocaine has a moderate affinity of about 2 µM to 20% human serum and very little interaction with human serum albumin or nonspecific human IgG at that concentration range. In a SPR inhibition experiment, the binding of mAb08 to immobilized BSA-SNC was inhibited by cocaine and benzoylecgonine in a highly competitive manner, whereas the purified polyclonal antibodies from vaccinated humans and mice, revealed preferential selectivity to pharmacologically active cocaine but not to the inactive metabolite benzoylecgonine. We have also developed a simple binding model to simulate the challenges associated with cocaine immunotherapy using the variable quantitative and kinetic properties of the antibodies. Conclusions High sensitivity calorimetric determination of antibody binding to cocaine and its

  13. High-affinity antibodies to the 1,4-dihydropyridine Ca2+-channel blockers

    SciTech Connect

    Campbell, K.P.; Sharp, A.; Strom, M.; Kahl, S.D.

    1986-05-01

    Antibodies with high affinity and specificity for the 1,4-dihydropyridine Ca2+-channel blockers have been produced in rabbits by immunization with dihydropyridine-protein conjugates. Anti-dihydropyridine antibodies were found to specifically bind (/sup 3/H)nitrendipine, (/sup 3/H)-nimodipine, (/sup 3/H)nisoldipine, and (/sup 3/H)PN 200-110 (all 1,4-dihydropyridine Ca2+-channel blockers) with high affinity, while (/sup 3/H)verapamil, (/sup 3/H)diltiazem, and (/sup 3/H)trifluoperazine were not recognized. The average dissociation constant of the (/sup 3/H)nitrendipine-antibody complex was 0.06 (+/- 0.02) X 10(-9) M for an antiserum studied in detail and ranged from 0.01 to 0.24 X 10(-9) M for all antisera. Inhibition of (/sup 3/H)nitrendipine binding was specific for the 1,4-dihydropyridine Ca2+-channel modifiers and the concentrations required for half-maximal inhibition ranged between 0.25 and 0.90 nM. Structurally unrelated Ca2+-channel blockers, calmodulin antagonists, inactive metabolites of nitrendipine, and UV-inactivated nisoldipine did not modify (/sup 3/H)nitrendipine binding to the anti-dihydropyridine antibodies. Dihydropyridines without a bulky substituent in the 4-position of the heterocycle were able to displace (/sup 3/H)nitrendipine binding, but the concentrations required for half-maximal inhibition were greater than 800 nM. In summary, anti-dihydropyridine antibodies have been shown to have high affinity and specificity for the 1,4-dihydropyridine Ca2+-channel blockers and to exhibit dihydropyridine binding properties similar to the membrane receptor for the 1,4-dihydropyridine Ca2+-channel blockers.

  14. Design, expression and characterization of a single chain anti-CD20 antibody; a germline humanized antibody derived from Rituximab.

    PubMed

    Ahmadzadeh, Vahideh; Farajnia, Safar; Hosseinpour Feizi, Mohammad Ali; Khavarinejad, Ramazan Ali

    2014-10-01

    CD20 is a B cell lineage specific surface antigen involved in various B cell malignancies. So far, several murine and chimeric antibodies have been produced against this antigen among which Rituximab is a commercially approved antibody widely used in treatment of cancers associated with CD20 overexpression. The current study reports the production and characterization of a humanized single chain version of Rituximab through CDR grafting method. For either heavy or light chain variable domains, a human antibody with the highest sequence homology to Rituximab was selected from human germline sequences and used as framework donors. Vernier zone residues in framework regions were replaced with those of Rituximab to retain the antigen binding affinity of parental antibody. The reactivity of humanized single chain antibody with CD20 was examined by ELISA and dot blot assays. The ability of antibody to suppress the growth of CD20 overexpressing Raji cells was tested by MTT assay. Analysis of reactivity with CD20 antigen revealed that the humanized single chain antibody reacted to the target antigen with high affinity. Proliferation inhibition assay showed that humanized scFv could suppress the proliferation of Raji cells efficiently in a dose-dependent manner. This successful production of a humanized scFv with the ability to inhibit growth of CD20-expressing cancer cell may provide a promising alternative strategy for CD20 targeted therapy.

  15. Affinity maturation by targeted diversification of the CDR-H2 loop of a monoclonal Fab derived from a synthetic naïve human antibody library and directed against the internal trimeric coiled-coil of gp41 yields a set of Fabs with improved HIV-1 neutralization potency and breadth

    PubMed Central

    Gustchina, Elena; Louis, John M.; Frisch, Christian; Ylera, Francisco; Lechner, Annette; Bewley, Carole A.; Clore, G. Marius

    2009-01-01

    Previously we reported a broadly HIV-1 neutralizing mini-antibody (Fab 3674) of modest potency that was derived from a human non-immune phage library by panning against the chimeric gp41-derived construct NCCG-gp41. This construct presents the N-heptad repeat of the gp41 ectodomain as a stable, helical, disulfide-linked trimer that extends in helical phase from the six-helix bundle of gp41. In this paper, Fab 3674 was subjected to affinity maturation against the NCCG-gp41 antigen by targeted diversification of the CDR-H2 loop to generate a panel of Fabs with diverse neutralization activity. Three affinity-matured Fabs selected for further study, Fabs 8060, 8066 and 8068, showed significant increases in both potency and breadth of neutralization against HIV-1 pseudotyped with envelopes of primary isolates from the standard subtypes B and C HIV-1 reference panels. The parental Fab 3674 is 10-20 fold less potent in monovalent than bivalent format over the entire B and C panels of HIV-1 pseudotypes. Of note is that the improved neutralization activity of the affinity-matured Fabs relative to the parental Fab 3674 was, on average, significantly greater for the Fabs in monovalent than bivalent format. This suggests that the increased avidity of the Fabs for the target antigen in bivalent format can be partially offset by kinetic and/or steric advantages afforded by the smaller monovalent Fabs. Indeed, the best affinity-matured Fab (8066) in monovalent format (∼50 kDa) was comparable in HIV-1 neutralization potency to the parental Fab 3674 in bivalent format (∼120 kDa) across the subtypes B and C reference panels. PMID:19695655

  16. Affinity improvement of a therapeutic antibody by structure-based computational design: generation of electrostatic interactions in the transition state stabilizes the antibody-antigen complex.

    PubMed

    Kiyoshi, Masato; Caaveiro, Jose M M; Miura, Eri; Nagatoishi, Satoru; Nakakido, Makoto; Soga, Shinji; Shirai, Hiroki; Kawabata, Shigeki; Tsumoto, Kouhei

    2014-01-01

    The optimization of antibodies is a desirable goal towards the development of better therapeutic strategies. The antibody 11K2 was previously developed as a therapeutic tool for inflammatory diseases, and displays very high affinity (4.6 pM) for its antigen the chemokine MCP-1 (monocyte chemo-attractant protein-1). We have employed a virtual library of mutations of 11K2 to identify antibody variants of potentially higher affinity, and to establish benchmarks in the engineering of a mature therapeutic antibody. The most promising candidates identified in the virtual screening were examined by surface plasmon resonance to validate the computational predictions, and to characterize their binding affinity and key thermodynamic properties in detail. Only mutations in the light-chain of the antibody are effective at enhancing its affinity for the antigen in vitro, suggesting that the interaction surface of the heavy-chain (dominated by the hot-spot residue Phe101) is not amenable to optimization. The single-mutation with the highest affinity is L-N31R (4.6-fold higher affinity than wild-type antibody). Importantly, all the single-mutations showing increase affinity incorporate a charged residue (Arg, Asp, or Glu). The characterization of the relevant thermodynamic parameters clarifies the energetic mechanism. Essentially, the formation of new electrostatic interactions early in the binding reaction coordinate (transition state or earlier) benefits the durability of the antibody-antigen complex. The combination of in silico calculations and thermodynamic analysis is an effective strategy to improve the affinity of a matured therapeutic antibody. PMID:24475232

  17. Autoimmunity and antibody affinity maturation are modulated by genetic variants on mouse chromosome 12.

    PubMed

    Collin, Roxanne; Dugas, Véronique; Chabot-Roy, Geneviève; Salem, David; Zahn, Astrid; Di Noia, Javier M; Rauch, Joyce; Lesage, Sylvie

    2015-04-01

    Autoimmune diseases result from a break in immune tolerance leading to an attack on self-antigens. Autoantibody levels serve as a predictive tool for the early diagnosis of many autoimmune diseases, including type 1 diabetes. We find that a genetic locus on mouse chromosome 12 influences the affinity maturation of antibodies as well as autoantibody production. Thus, we generated a NOD.H2(k) congenic strain bearing B10 alleles at the locus comprised within the D12Mit184 and D12Mit12 markers, which we named NOD.H2(k)-Chr12. We determined the biological relevance of the Chr12 locus on the autoimmune process using an antigen-specific TCR transgenic autoimmune mouse model. Specifically, the 3A9 TCR transgene, which recognizes a peptide from hen egg lysozyme (HEL) in the context of I-A(k), and the HEL transgene, which is expressed under the rat-insulin promoter (iHEL), were bred into the NOD.H2(k)-Chr12 congenic strain. In the resulting 3A9 TCR:iHEL NOD.H2(k)-Chr12 mice, we observed a significant decrease in diabetes incidence as well as a decrease in both the quantity and affinity of HEL-specific IgG autoantibodies relative to 3A9 TCR:iHEL NOD.H2(k) mice. Notably, the decrease in autoantibodies due to the Chr12 locus was not restricted to the TCR transgenic model, as it was also observed in the non-transgenic NOD.H2(k) setting. Of importance, antibody affinity maturation upon immunization and re-challenge was also impeded in NOD.H2(k)-Chr12 congenic mice relative to NOD.H2(k) mice. Together, these results demonstrate that a genetic variant(s) present within the Chr12 locus plays a global role in modulating antibody affinity maturation.

  18. Affinity purification of egg yolk immunoglobulins (IgY) using a human mycoplasma protein.

    PubMed

    Jiang, Xuemei; Diraviyam, Thirumalai; Zhang, Xiaoying

    2016-02-15

    Egg yolk immunoglobulin (IgY) is a superior functional equivalent to mammalian IgG. However, the preparation of refined and highly purified IgY is still attributed as difficult task. Protein M (a transmembrane protein from human mycoplasma) has been newly demonstrated as an ideal affinity regent for mammalian antibody purification. This study aimed to evaluate the interaction between protein M and IgY. The results showed protein M could be a superior affinity reagent for IgY, scFv as well as IgYΔFc, based on pull down and western blot investigations; in addition, it was found that ∼125 times increase of effective IgY in the elutent was obtained using protein M affinity chromatography column compared with traditional IgY extraction methods. This indicates, the purification strategy of protein M is entirely different to traditional IBPs and the salient purification feature of protein M would be a breakthrough for purifying not only non-mammalian antibodies, but also monoclonal antibodies and engineered antibodies based on variable region.

  19. BIOINTERACTION ANALYSIS BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY: KINETIC STUDIES OF IMMOBILIZED ANTIBODIES

    PubMed Central

    Nelson, Mary Anne; Moser, Annette; Hage, David S.

    2009-01-01

    A system based on high-performance affinity chromatography was developed for characterizing the binding, elution and regeneration kinetics of immobilized antibodies and immunoaffinity supports. This information was provided by using a combination of frontal analysis, split-peak analysis and peak decay analysis to determine the rate constants for antibody-antigen interactions under typical sample application and elution conditions. This technique was tested using immunoaffinity supports that contained monoclonal antibodies for 2,4-dichlorophenoxyacetic acid (2,4-D). Association equilibrium constants measured by frontal analysis for 2,4-D and related compounds with the immobilized antibodies were 1.7–12 × 106 M−1 at pH 7.0 and 25°C. Split-peak analysis gave association rate constants of 1.4–12 × 105 M−1s−1 and calculated dissociation rate constants of 0.01–0.4 s−1 under the application conditions. Elution at pH 2.5 for the analytes from the antibodies was examined by peak decay analysis and gave dissociation rate constants of 0.056–0.17 s−1. A comparison of frontal analysis results after various periods of column regeneration allowed the rate of antibody regeneration to be examined, with the results giving a first-order regeneration rate constant of 2.4 × 10−4 s−1. This combined approach and the information it provides should be useful in the design and optimization of immunoaffinity chromatography and other analytical methods that employ immobilized antibodies. The methods described are not limited to the particular analytes and antibodies employed in this study but should be useful in characterizing other targets, ligands and supports. PMID:19394281

  20. Affine differential geometry analysis of human arm movements.

    PubMed

    Flash, Tamar; Handzel, Amir A

    2007-06-01

    Humans interact with their environment through sensory information and motor actions. These interactions may be understood via the underlying geometry of both perception and action. While the motor space is typically considered by default to be Euclidean, persistent behavioral observations point to a different underlying geometric structure. These observed regularities include the "two-thirds power law", which connects path curvature with velocity, and "local isochrony", which prescribes the relation between movement time and its extent. Starting with these empirical observations, we have developed a mathematical framework based on differential geometry, Lie group theory and Cartan's moving frame method for the analysis of human hand trajectories. We also use this method to identify possible motion primitives, i.e., elementary building blocks from which more complicated movements are constructed. We show that a natural geometric description of continuous repetitive hand trajectories is not Euclidean but equi-affine. Specifically, equi-affine velocity is piecewise constant along movement segments, and movement execution time for a given segment is proportional to its equi-affine arc-length. Using this mathematical framework, we then analyze experimentally recorded drawing movements. To examine movement segmentation and classification, the two fundamental equi-affine differential invariants-equi-affine arc-length and curvature are calculated for the recorded movements. We also discuss the possible role of conic sections, i.e., curves with constant equi-affine curvature, as motor primitives and focus in more detail on parabolas, the equi-affine geodesics. Finally, we explore possible schemes for the internal neural coding of motor commands by showing that the equi-affine framework is compatible with the common model of population coding of the hand velocity vector when combined with a simple assumption on its dynamics. We then discuss several alternative explanations

  1. Monoclonal IgM antibody exhibiting high-affinity binding and cryoglobulin properties.

    PubMed Central

    Ballard, D W; Kranz, D M; Voss, E W

    1983-01-01

    A monoclonal IgM antibody (18-2-3) derived from cell fusion of (NZB X NZW) F1 splenocytes following secondary immunization with fluorescein-conjugated keyhole limpet hemocyanin was shown to exhibit high intrinsic binding affinity and cryoinsolubility. Affinity-purified preparations were determined to be IgM by immunochemical, electrophoretic, and chromatographic analyses. An intrinsic association constant (Ka) of 2.9 X 10(10) M-1 (at 2 degrees C) was measured by first-order dissociation-rate analysis. Antibody solubility at low concentration (approximately equal to 50 micrograms/ml) was shown, by absorption spectroscopy, to be temperature dependent between 4 degrees C and 32 degrees C. Insolubility at low temperature (4 degrees C) was reversible in the presence of homologous fluorescyl hapten, indicative of active site involvement in the mechanism of cryoglobulin-18-2-3 complex formation. Characteristics of clone 18-2-3 are discussed in terms of (i) its potential use as a model for examining the mechanism of cryoprecipitation and (ii) the proposed relationship between affinity maturation and the IgM to IgG class switch. Images PMID:6348779

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

    PubMed Central

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

    2012-01-01

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

  3. Water channel in the binding site of a high affinity anti-methotrexate antibody.

    PubMed

    Gayda, Susan; Longenecker, Kenton L; Manoj, Sharmila; Judge, Russell A; Saldana, Sylvia C; Ruan, Qiaoqiao; Swift, Kerry M; Tetin, Sergey Y

    2014-06-17

    In the present study, we report the structure of the free and drug-bound Fab fragment of a high affinity anti-methotrexate antibody and perform a thermodynamic analysis of the binding process. The anti-methotrexate Fab fragment features a remarkably rigid tunnel-like binding site that extends into a water channel serving as a specialized route to move solvent out and into the site upon ligand binding and dissociation. This new finding in antibody structure-function relationships directly relates to the fast association (1 × 10⁷ M⁻¹ s⁻¹) and slow dissociation (4 × 10⁻⁵ s⁻¹) rates determined for mAb ADD056, resulting in a very strong binding with a K(D) ~ 3.6 pM at 20 °C. As follows from the X-ray data analysis, the methotrexate-antibody complex is stabilized by an extended network of hydrogen bonds and stacking interactions. The analysis also shows structural involvement of the CDR H3 in formation of the water channel revealing another important role of this hypervariable region. This suggests a new direction in natural affinity maturation and opens a new possibility in antibody engineering. Methotrexate is a widely used therapeutic agent for many malignant diseases and inflammatory disorders. Unfortunately, it may also interfere with central aspects of metabolism and thereby cause inevitable side effects. Therefore, methotrexate therapy requires careful monitoring of drug blood levels, which is traditionally done by immunoassays. An understanding of the structure-function properties of antibodies selected for drug monitoring substantiates the performance and robustness of such tests.

  4. Water channel in the binding site of a high affinity anti-methotrexate antibody.

    PubMed

    Gayda, Susan; Longenecker, Kenton L; Manoj, Sharmila; Judge, Russell A; Saldana, Sylvia C; Ruan, Qiaoqiao; Swift, Kerry M; Tetin, Sergey Y

    2014-06-17

    In the present study, we report the structure of the free and drug-bound Fab fragment of a high affinity anti-methotrexate antibody and perform a thermodynamic analysis of the binding process. The anti-methotrexate Fab fragment features a remarkably rigid tunnel-like binding site that extends into a water channel serving as a specialized route to move solvent out and into the site upon ligand binding and dissociation. This new finding in antibody structure-function relationships directly relates to the fast association (1 × 10⁷ M⁻¹ s⁻¹) and slow dissociation (4 × 10⁻⁵ s⁻¹) rates determined for mAb ADD056, resulting in a very strong binding with a K(D) ~ 3.6 pM at 20 °C. As follows from the X-ray data analysis, the methotrexate-antibody complex is stabilized by an extended network of hydrogen bonds and stacking interactions. The analysis also shows structural involvement of the CDR H3 in formation of the water channel revealing another important role of this hypervariable region. This suggests a new direction in natural affinity maturation and opens a new possibility in antibody engineering. Methotrexate is a widely used therapeutic agent for many malignant diseases and inflammatory disorders. Unfortunately, it may also interfere with central aspects of metabolism and thereby cause inevitable side effects. Therefore, methotrexate therapy requires careful monitoring of drug blood levels, which is traditionally done by immunoassays. An understanding of the structure-function properties of antibodies selected for drug monitoring substantiates the performance and robustness of such tests. PMID:24832237

  5. Design of Cyclic Peptides That Bind Protein Surfaces with Antibody-Like Affinity

    PubMed Central

    Millward, Steven W.; Fiacco, Stephen; Austin, Ryan J.; Roberts, Richard W.

    2012-01-01

    There is a pressing need for new molecular tools to target protein surfaces with high affinity and specificity. Here, we describe cyclic messenger RNA display with a trillion-member covalent peptide macrocycle library. Using this library, we have designed a number of high-affinity, redox-insensitive, cyclic peptides that target the signaling protein Gαi1. In addition to cyclization, our library construction took advantage of an expanded genetic code, utilizing nonsense suppression to insert N-methylphenylalanine as a 21st amino acid. The designed macrocycles exhibit several intriguing features. First, the core motif seen in all of the selected variants is the same and shares an identical context with respect to the macrocyclic scaffold, consistent with the idea that selection simultaneously optimizes both the cyclization chemistry and the structural placement of the binding epitope. Second, detailed characterization of one molecule, cyclic Gαi binding peptide (cycGiBP), demonstrates substantially enhanced proteolytic stability relative to that of the parent linear molecule. Third and perhaps most important, the cycGiBP peptide binds the target with very high affinity (Ki ≈ 2.1 nM), similar to those of many of the best monoclonal antibodies and higher than that of the βγ heterodimer, an endogenous Gαi1 ligand. Overall the work provides a general route to design novel, low-molecular-weight, high-affinity ligands that target protein surfaces. PMID:17894440

  6. Design of cyclic peptides that bind protein surfaces with antibody-like affinity.

    PubMed

    Millward, Steven W; Fiacco, Stephen; Austin, Ryan J; Roberts, Richard W

    2007-09-21

    There is a pressing need for new molecular tools to target protein surfaces with high affinity and specificity. Here, we describe cyclic messenger RNA display with a trillion-member covalent peptide macrocycle library. Using this library, we have designed a number of high-affinity, redox-insensitive, cyclic peptides that target the signaling protein G alpha i1. In addition to cyclization, our library construction took advantage of an expanded genetic code, utilizing nonsense suppression to insert N-methylphenylalanine as a 21st amino acid. The designed macrocycles exhibit several intriguing features. First, the core motif seen in all of the selected variants is the same and shares an identical context with respect to the macrocyclic scaffold, consistent with the idea that selection simultaneously optimizes both the cyclization chemistry and the structural placement of the binding epitope. Second, detailed characterization of one molecule, cyclic G alpha i binding peptide (cycGiBP), demonstrates substantially enhanced proteolytic stability relative to that of the parent linear molecule. Third and perhaps most important, the cycGiBP peptide binds the target with very high affinity ( K i approximately 2.1 nM), similar to those of many of the best monoclonal antibodies and higher than that of the betagamma heterodimer, an endogenous G alpha i1 ligand. Overall the work provides a general route to design novel, low-molecular-weight, high-affinity ligands that target protein surfaces.

  7. Affitins as robust tailored reagents for affinity chromatography purification of antibodies and non-immunoglobulin proteins.

    PubMed

    Béhar, Ghislaine; Renodon-Cornière, Axelle; Mouratou, Barbara; Pecorari, Frédéric

    2016-04-01

    Affinity chromatography is a convenient way of purifying proteins, as a high degree of purity can be reached in one step. The use of tags has greatly contributed to the popularity of this technique. However, the addition of tags may not be desirable or possible for the production of biopharmaceuticals. There is thus a need for tailored artificial affinity ligands. We have developed the use of archaeal extremophilic proteins as scaffolds to generate affinity proteins (Affitins). Here, we explored the potential of Affitins as ligand to design affinity columns. Affitins specific for human immunoglobulin G (hIgG), bacterial PulD protein, and chicken egg lysozyme were immobilized on an agarose matrix. The columns obtained were functional and highly selective for their cognate target, even in the presence of exogenous proteins as found in cell culture media, ascites and bacterial lysates, which result in a high degree of purity (∼95%) and recovery (∼100%) in a single step. Anti-hIgG Affitin columns withstand repetitive cycles of purification and cleaning-in-place treatments with 0.25 M NaOH as well as Protein A does. High levels of Affitin productions in Escherichia coli makes it possible to produce these affinity columns at low cost. Our results validate Affitins as a new class of tailored ligands for the affinity chromatography purification of potentially any proteins of interest including biopharmaceuticals.

  8. Affitins as robust tailored reagents for affinity chromatography purification of antibodies and non-immunoglobulin proteins.

    PubMed

    Béhar, Ghislaine; Renodon-Cornière, Axelle; Mouratou, Barbara; Pecorari, Frédéric

    2016-04-01

    Affinity chromatography is a convenient way of purifying proteins, as a high degree of purity can be reached in one step. The use of tags has greatly contributed to the popularity of this technique. However, the addition of tags may not be desirable or possible for the production of biopharmaceuticals. There is thus a need for tailored artificial affinity ligands. We have developed the use of archaeal extremophilic proteins as scaffolds to generate affinity proteins (Affitins). Here, we explored the potential of Affitins as ligand to design affinity columns. Affitins specific for human immunoglobulin G (hIgG), bacterial PulD protein, and chicken egg lysozyme were immobilized on an agarose matrix. The columns obtained were functional and highly selective for their cognate target, even in the presence of exogenous proteins as found in cell culture media, ascites and bacterial lysates, which result in a high degree of purity (∼95%) and recovery (∼100%) in a single step. Anti-hIgG Affitin columns withstand repetitive cycles of purification and cleaning-in-place treatments with 0.25 M NaOH as well as Protein A does. High levels of Affitin productions in Escherichia coli makes it possible to produce these affinity columns at low cost. Our results validate Affitins as a new class of tailored ligands for the affinity chromatography purification of potentially any proteins of interest including biopharmaceuticals. PMID:26952369

  9. Affinity binding of antibodies to supermacroporous cryogel adsorbents with immobilized protein A for removal of anthrax toxin protective antigen.

    PubMed

    Ingavle, Ganesh C; Baillie, Les W J; Zheng, Yishan; Lis, Elzbieta K; Savina, Irina N; Howell, Carol A; Mikhalovsky, Sergey V; Sandeman, Susan R

    2015-05-01

    Polymeric cryogels are efficient carriers for the immobilization of biomolecules because of their unique macroporous structure, permeability, mechanical stability and different surface chemical functionalities. The aim of the study was to demonstrate the potential use of macroporous monolithic cryogels for biotoxin removal using anthrax toxin protective antigen (PA), the central cell-binding component of the anthrax exotoxins, and covalent immobilization of monoclonal antibodies. The affinity ligand (protein A) was chemically coupled to the reactive hydroxyl and epoxy-derivatized monolithic cryogels and the binding efficiencies of protein A, monoclonal antibodies to the cryogel column were determined. Our results show differences in the binding capacity of protein A as well as monoclonal antibodies to the cryogel adsorbents caused by ligand concentrations, physical properties and morphology of surface matrices. The cytotoxicity potential of the cryogels was determined by an in vitro viability assay using V79 lung fibroblast as a model cell and the results reveal that the cryogels are non-cytotoxic. Finally, the adsorptive capacities of PA from phosphate buffered saline (PBS) were evaluated towards a non-glycosylated, plant-derived human monoclonal antibody (PANG) and a glycosylated human monoclonal antibody (Valortim(®)), both of which were covalently attached via protein A immobilization. Optimal binding capacities of 108 and 117 mg/g of antibody to the adsorbent were observed for PANG attached poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] and Valortim(®) attached poly(AAm-AGE) cryogels, respectively, This indicated that glycosylation status of Valortim(®) antibody could significantly increase (8%) its binding capacity relative to the PANG antibody on poly(AAm-AGE)-protien-A column (p < 0.05). The amounts of PA which remained in the solution after passing PA spiked PBS through PANG or Valortim bound poly(AAm-AGE) cryogel were significantly (p < 0

  10. Affinity binding of antibodies to supermacroporous cryogel adsorbents with immobilized protein A for removal of anthrax toxin protective antigen.

    PubMed

    Ingavle, Ganesh C; Baillie, Les W J; Zheng, Yishan; Lis, Elzbieta K; Savina, Irina N; Howell, Carol A; Mikhalovsky, Sergey V; Sandeman, Susan R

    2015-05-01

    Polymeric cryogels are efficient carriers for the immobilization of biomolecules because of their unique macroporous structure, permeability, mechanical stability and different surface chemical functionalities. The aim of the study was to demonstrate the potential use of macroporous monolithic cryogels for biotoxin removal using anthrax toxin protective antigen (PA), the central cell-binding component of the anthrax exotoxins, and covalent immobilization of monoclonal antibodies. The affinity ligand (protein A) was chemically coupled to the reactive hydroxyl and epoxy-derivatized monolithic cryogels and the binding efficiencies of protein A, monoclonal antibodies to the cryogel column were determined. Our results show differences in the binding capacity of protein A as well as monoclonal antibodies to the cryogel adsorbents caused by ligand concentrations, physical properties and morphology of surface matrices. The cytotoxicity potential of the cryogels was determined by an in vitro viability assay using V79 lung fibroblast as a model cell and the results reveal that the cryogels are non-cytotoxic. Finally, the adsorptive capacities of PA from phosphate buffered saline (PBS) were evaluated towards a non-glycosylated, plant-derived human monoclonal antibody (PANG) and a glycosylated human monoclonal antibody (Valortim(®)), both of which were covalently attached via protein A immobilization. Optimal binding capacities of 108 and 117 mg/g of antibody to the adsorbent were observed for PANG attached poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] and Valortim(®) attached poly(AAm-AGE) cryogels, respectively, This indicated that glycosylation status of Valortim(®) antibody could significantly increase (8%) its binding capacity relative to the PANG antibody on poly(AAm-AGE)-protien-A column (p < 0.05). The amounts of PA which remained in the solution after passing PA spiked PBS through PANG or Valortim bound poly(AAm-AGE) cryogel were significantly (p < 0

  11. TRIM21 Immune Signaling Is More Sensitive to Antibody Affinity Than Its Neutralization Activity.

    PubMed

    Foss, Stian; Watkinson, Ruth E; Grevys, Algirdas; McAdam, Martin B; Bern, Malin; Høydahl, Lene Stokken; Dalhus, Bjørn; Michaelsen, Terje E; Sandlie, Inger; James, Leo C; Andersen, Jan Terje

    2016-04-15

    Ab-coated viruses can be detected in the cytosol by the FcR tripartite motif-containing 21 (TRIM21), which rapidly recruits the proteasomal machinery and triggers induction of immune signaling. As such, TRIM21 plays a key role in intracellular protection by targeting invading viruses for destruction and alerting the immune system. A hallmark of immunity is elicitation of a balanced response that is proportionate to the threat, to avoid unnecessary inflammation. In this article, we show how Ab affinity modulates TRIM21 immune function. We constructed a humanized monoclonal IgG1 against human adenovirus type 5 (AdV5) and a panel of Fc-engineered variants with a wide range of affinities for TRIM21. We found that IgG1-coated viral particles were neutralized via TRIM21, even when affinity was reduced by as much as 100-fold. In contrast, induction of NF-κB signaling was more sensitive to reduced affinity between TRIM21 and the Ab variants. Thus, TRIM21 mediates neutralization under suboptimal conditions, whereas induction of immune signaling is balanced according to the functional affinity for the incoming immune stimuli. Our findings have implications for engineering of antiviral IgG therapeutics with tailored effector functions. PMID:26962230

  12. Alteration of Electrostatic Surface Potential Enhances Affinity and Tumor Killing Properties of Anti-ganglioside GD2 Monoclonal Antibody hu3F8.

    PubMed

    Zhao, Qi; Ahmed, Mahiuddin; Guo, Hong-fen; Cheung, Irene Y; Cheung, Nai-Kong V

    2015-05-22

    Ganglioside GD2 is highly expressed on neuroectodermal tumors and an attractive therapeutic target for antibodies that have already shown some clinical efficacy. To further improve the current antibodies, which have modest affinity, we sought to improve affinity by using a combined method of random mutagenesis and in silico assisted design to affinity-mature the anti-GD2 monoclonal antibody hu3F8. Using yeast display, mutants in the Fv with enhanced binding over the parental clone were FACS-sorted and cloned. In silico modeling identified the minimal key interacting residues involved in the important charged interactions with the sialic acid groups of GD2. Two mutations, D32H (L-CDR1) and E1K (L-FR1) altered the electrostatic surface potential of the antigen binding site, allowing for an increase in positive charge to enhance the interaction with the negatively charged GD2-pentasaccharide headgroup. Purified scFv and IgG mutant forms were then tested for antigen specificity by ELISA, for tissue specificity by immunohistochemistry, for affinity by BIACORE, for antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity in vitro, and for anti-tumor efficacy in xenografted humanized mice. The nearly 7-fold improvement in affinity of hu3F8 with a single D32H (L-CDR1) mutation translated into a ∼12-fold improvement in NK92MI-transfected CD16-mediated ADCC, a 6-fold improvement in CD32-mediated ADCC, and a 2.5-fold improvement in complement-mediated cytotoxicity while maintaining restricted normal tissue cross-reactivity and achieving substantial improvement in tumor ablation in vivo. Despite increasing GD2 affinity, the double mutation D32H (L-CDR1) and E1K (L-FR1) did not further improve anti-tumor efficacy.

  13. Alteration of Electrostatic Surface Potential Enhances Affinity and Tumor Killing Properties of Anti-ganglioside GD2 Monoclonal Antibody hu3F8.

    PubMed

    Zhao, Qi; Ahmed, Mahiuddin; Guo, Hong-fen; Cheung, Irene Y; Cheung, Nai-Kong V

    2015-05-22

    Ganglioside GD2 is highly expressed on neuroectodermal tumors and an attractive therapeutic target for antibodies that have already shown some clinical efficacy. To further improve the current antibodies, which have modest affinity, we sought to improve affinity by using a combined method of random mutagenesis and in silico assisted design to affinity-mature the anti-GD2 monoclonal antibody hu3F8. Using yeast display, mutants in the Fv with enhanced binding over the parental clone were FACS-sorted and cloned. In silico modeling identified the minimal key interacting residues involved in the important charged interactions with the sialic acid groups of GD2. Two mutations, D32H (L-CDR1) and E1K (L-FR1) altered the electrostatic surface potential of the antigen binding site, allowing for an increase in positive charge to enhance the interaction with the negatively charged GD2-pentasaccharide headgroup. Purified scFv and IgG mutant forms were then tested for antigen specificity by ELISA, for tissue specificity by immunohistochemistry, for affinity by BIACORE, for antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity in vitro, and for anti-tumor efficacy in xenografted humanized mice. The nearly 7-fold improvement in affinity of hu3F8 with a single D32H (L-CDR1) mutation translated into a ∼12-fold improvement in NK92MI-transfected CD16-mediated ADCC, a 6-fold improvement in CD32-mediated ADCC, and a 2.5-fold improvement in complement-mediated cytotoxicity while maintaining restricted normal tissue cross-reactivity and achieving substantial improvement in tumor ablation in vivo. Despite increasing GD2 affinity, the double mutation D32H (L-CDR1) and E1K (L-FR1) did not further improve anti-tumor efficacy. PMID:25851904

  14. Alteration of Electrostatic Surface Potential Enhances Affinity and Tumor Killing Properties of Anti-ganglioside GD2 Monoclonal Antibody hu3F8*

    PubMed Central

    Zhao, Qi; Ahmed, Mahiuddin; Guo, Hong-fen; Cheung, Irene Y.; Cheung, Nai-Kong V.

    2015-01-01

    Ganglioside GD2 is highly expressed on neuroectodermal tumors and an attractive therapeutic target for antibodies that have already shown some clinical efficacy. To further improve the current antibodies, which have modest affinity, we sought to improve affinity by using a combined method of random mutagenesis and in silico assisted design to affinity-mature the anti-GD2 monoclonal antibody hu3F8. Using yeast display, mutants in the Fv with enhanced binding over the parental clone were FACS-sorted and cloned. In silico modeling identified the minimal key interacting residues involved in the important charged interactions with the sialic acid groups of GD2. Two mutations, D32H (L-CDR1) and E1K (L-FR1) altered the electrostatic surface potential of the antigen binding site, allowing for an increase in positive charge to enhance the interaction with the negatively charged GD2-pentasaccharide headgroup. Purified scFv and IgG mutant forms were then tested for antigen specificity by ELISA, for tissue specificity by immunohistochemistry, for affinity by BIACORE, for antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity in vitro, and for anti-tumor efficacy in xenografted humanized mice. The nearly 7-fold improvement in affinity of hu3F8 with a single D32H (L-CDR1) mutation translated into a ∼12-fold improvement in NK92MI-transfected CD16-mediated ADCC, a 6-fold improvement in CD32-mediated ADCC, and a 2.5-fold improvement in complement-mediated cytotoxicity while maintaining restricted normal tissue cross-reactivity and achieving substantial improvement in tumor ablation in vivo. Despite increasing GD2 affinity, the double mutation D32H (L-CDR1) and E1K (L-FR1) did not further improve anti-tumor efficacy. PMID:25851904

  15. Monoclonal Antibody That Defines Human Myoepithelium

    NASA Astrophysics Data System (ADS)

    Dairkee, Shahnaz Hashmi; Blayney, Carlene; Smith, Helene S.; Hackett, Adeline J.

    1985-11-01

    We have isolated a mouse monoclonal antibody that, upon immunohistochemical localization in frozen sections, displays specificity for human myoepithelial cells in the resting mammary gland, sweat glands, and salivary glands. Furthermore, this antibody was strongly and homogeneously reactive with frozen sections of 3 of 60 breast carcinoma specimens. Using immunolocalization techniques in conjunction with polyacrylamide gel electrophoresis, we have determined that the reactivity of this monoclonal antibody is directed toward a 51,000-dalton keratin polypeptide. The potential uses of this antibody in the prognosis of human mammary carcinoma and in understanding the role of the myoepithelium in development and differentiation are discussed.

  16. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

    PubMed Central

    Boulet-Audet, Maxime; Kazarian, Sergei G.; Byrne, Bernadette

    2016-01-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan. PMID:27470880

  17. Quantitation of tyrosine hydroxylase, protein levels: Spot immunolabeling with an affinity-purified antibody

    SciTech Connect

    Haycock, J.W. )

    1989-09-01

    Tyrosine hydroxylase was purified from bovine adrenal chromaffin cells and rat pheochromocytoma using a rapid (less than 2 days) procedure performed at room temperature. Rabbits were immunized with purified enzyme that was denatured with sodium dodecylsulfate, and antibodies to tyrosine hydroxylase were affinity-purified from immune sera. A Western blot procedure using the affinity-purified antibodies and {sup 125}I-protein A demonstrated a selective labeling of a single Mr approximately 62,000 band in samples from a number of different tissues. The relative lack of background {sup 125}I-protein A binding permitted the development of a quantitative spot immunolabeling procedure for tyrosine hydroxylase protein. The sensitivity of the assay is 1-2 ng of enzyme. Essentially identical standard curves were obtained with tyrosine hydroxylase purified from rat pheochromocytoma, rat corpus striatum, and bovine adrenal medulla. An extract of PC 12 cells (clonal rat pheochromocytoma cells) was calibrated against purified rat pheochromocytoma tyrosine hydroxylase and used as an external standard against which levels of tyrosine hydroxylase in PC12 cells and other tissue were quantified. With this procedure, qualitative assessment of tyrosine hydroxylase protein levels can be obtained in a few hours and quantitative assessment can be obtained in less than a day.

  18. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies.

    PubMed

    Boulet-Audet, Maxime; Kazarian, Sergei G; Byrne, Bernadette

    2016-01-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan. PMID:27470880

  19. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

    NASA Astrophysics Data System (ADS)

    Boulet-Audet, Maxime; Kazarian, Sergei G.; Byrne, Bernadette

    2016-07-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan.

  20. Transgenic mouse strains as platforms for the successful discovery and development of human therapeutic monoclonal antibodies.

    PubMed

    Green, Larry L

    2014-03-01

    Transgenic mice have yielded seven of the ten currently-approved human antibody drugs, making them the most successful platform for the discovery of fully human antibody therapeutics. The use of the in vivo immune system helps drive this success by taking advantage of the natural selection process that produces antibodies with desirable characteristics. Appropriately genetically-engineered mice act as robust engines for the generation of diverse repertoires of affinity- matured fully human variable regions with intrinsic properties necessary for successful antibody drug development including high potency, specificity, manufacturability, solubility and low risk of immunogenicity. A broad range of mAb drug targets are addressable in these mice, comprising both secreted and transmembrane targets, including membrane multi-spanning targets, as well as human target antigens that share high sequence identity with their mouse orthologue. Transgenic mice can routinely yield antibodies with sub-nanomolar binding affinity for their antigen, with lead candidate mAbs frequently possessing affinities for binding to their target of less than 100 picomolar, without requiring any ex vivo affinity optimization. While the originator transgenic mice platforms are no longer broadly available, a new generation of transgenic platforms is in development for discovery of the next wave of human therapeutic antibodies.

  1. High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors

    PubMed Central

    Batsuli, Glaivy; Deng, Wei; Healey, John F.; Parker, Ernest T.; Baldwin, W. Hunter; Cox, Courtney; Nguyen, Brenda; Kahle, Joerg; Königs, Christoph; Li, Renhao; Lollar, Pete

    2016-01-01

    Inhibitor formation in hemophilia A is the most feared treatment-related complication of factor VIII (fVIII) therapy. Most inhibitor patients with hemophilia A develop antibodies against the fVIII A2 and C2 domains. Recent evidence demonstrates that the C1 domain contributes to the inhibitor response. Inhibitory anti-C1 monoclonal antibodies (mAbs) have been identified that bind to putative phospholipid and von Willebrand factor (VWF) binding epitopes and block endocytosis of fVIII by antigen presenting cells. We now demonstrate by competitive enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry that 7 of 9 anti-human C1 mAbs tested recognize an epitope distinct from the C1 phospholipid binding site. These mAbs, designated group A, display high binding affinities for fVIII, weakly inhibit fVIII procoagulant activity, poorly inhibit fVIII binding to phospholipid, and exhibit heterogeneity with respect to blocking fVIII binding to VWF. Another mAb, designated group B, inhibits fVIII procoagulant activity, fVIII binding to VWF and phospholipid, fVIIIa incorporation into the intrinsic Xase complex, thrombin generation in plasma, and fVIII uptake by dendritic cells. Group A and B epitopes are distinct from the epitope recognized by the canonical, human-derived inhibitory anti-C1 mAb, KM33, whose epitope overlaps both groups A and B. Antibodies recognizing group A and B epitopes are present in inhibitor plasmas from patients with hemophilia A. Additionally, group A and B mAbs increase fVIII clearance and are pathogenic in a hemophilia A mouse tail snip bleeding model. Group A anti-C1 mAbs represent the first identification of pathogenic, weakly inhibitory antibodies that increase fVIII clearance. PMID:27381905

  2. Antilymphocytic antibodies and marrow transplantation. VIII. Recipient conditioning with Clq-affine monoclonal anti-pan T antibodies prevents GVHD in homozygous fully mismatched mice

    SciTech Connect

    Thierfelder, S.; Kummer, U.; Schuh, R.; Mysliwietz, J.

    1986-10-01

    An approach to suppressing secondary disease with antibodies was studied that differed from conventional antibody treatment of donor marrow in vitro. It consisted of the selection of anti-Thy-1 antibodies with high affinity for Clq, the first subunit of the complement cascade, and a single injection of such antibodies into prospective irradiated marrow recipients. Monoclonal mouse IgM and rat IgG 2c antibodies of high titers in complement-dependent test systems but with low affinity for Clq caused little immunosuppression. Monoclonal rat IgG2b or mouse IgG2a anti-Thy-1 antibodies with high affinity for Clq prevented acute and chronic mortality of graft-v-host disease (GVHD), however, when injected in irradiated CBA or AKR mice prior to C57BL/6 spleen and/or bone marrow cell transfusion. This treatment simultaneously suppressed residual host-v-graft reactivity of the irradiated mice, so that permanent hematopoietic engraftment ensued even at 5 or 6 Gy. Full chimerism and specific tolerance were obtained. Primary immune response to SRBC was clearly depressed in the chimeras; secondary immune response was not. Clearance of T cell antibody activity (greater than 6 days), timing, and dose of injected antibody, as well as other modalities of the conditioning treatment that may have contributed to the remarkable immunosuppression, are discussed.

  3. Profiling human antibody responses by integrated single-cell analysis.

    PubMed

    Ogunniyi, Adebola O; Thomas, Brittany A; Politano, Timothy J; Varadarajan, Navin; Landais, Elise; Poignard, Pascal; Walker, Bruce D; Kwon, Douglas S; Love, J Christopher

    2014-05-19

    Comprehensive characterization of the antigen-specific B cells induced during infections or following vaccination would facilitate the discovery of novel antibodies and inform how interventions shape protective humoral responses. The analysis of human B cells and their antibodies has been performed using flow cytometry to evaluate memory B cells and expanded plasmablasts, while microtechnologies have also provided a useful tool to examine plasmablasts/plasma cells after vaccination. Here we present an integrated analytical platform, using arrays of subnanoliter wells (nanowells), for constructing detailed profiles for human B cells comprising the immunophenotypes of these cells, the distribution of isotypes of the secreted antibodies, the specificity and relative affinity for defined antigens, and for a subset of cells, the genes encoding the heavy and light chains. The approach combines on-chip image cytometry, microengraving, and single-cell RT-PCR. Using clinical samples from HIV-infected subjects, we demonstrate that the method can identify antigen-specific neutralizing antibodies, is compatible with both plasmablasts/plasma cells and activated memory B cells, and is well-suited for characterizing the limited numbers of B cells isolated from tissue biopsies (e.g., colon biopsies). The technology should facilitate detailed analyses of human humoral responses for evaluating vaccines and their ability to raise protective antibody responses across multiple anatomical compartments. PMID:24602776

  4. Natural human antibodies to amyloid beta peptide.

    PubMed

    Szabo, Paul; Relkin, Norman; Weksler, Marc E

    2008-06-01

    Properties of human, natural anti-Abeta antibodies and commercially available intravenous immunoglobulin (IVIg) have been examined in light of the beneficial effects of passive immunotherapy with IVIg for patients with mild to moderate Alzheimer's disease (AD). Anti-Abeta antibodies in IVIg recognize conformation-specific epitopes as well as linear epitopes from different regions of the Abeta peptide. Anti-Abeta antibodies in circulation, especially those with high avidity, are often masked by ligands and the avidity of these antibodies increases upon dissociation of the bound ligands from the antibodies. Such natural anti-Abeta antibodies have the capacity to prevent Abeta oligomer-induced neurotoxicity in N2A neuroblastoma cells. This neuro-protective effect may reflect the therapeutic potential of the natural anti-Abeta antibodies found in IVIg for the treatment of patients with AD.

  5. Kinetic analysis of a high-affinity antibody/antigen interaction performed by multiple Biacore users.

    PubMed

    Katsamba, Phinikoula S; Navratilova, Iva; Calderon-Cacia, Maria; Fan, Linsey; Thornton, Kevin; Zhu, Mingde; Bos, Tim Vanden; Forte, Carla; Friend, Della; Laird-Offringa, Ite; Tavares, Gisele; Whatley, John; Shi, Ergang; Widom, Angela; Lindquist, Kevin C; Klakamp, Scott; Drake, Andrew; Bohmann, David; Roell, Marina; Rose, Larry; Dorocke, Jill; Roth, Bruce; Luginbühl, Béatrice; Myszka, David G

    2006-05-15

    To explore the reliability of Biacore-based assays, 22 study participants measured the binding of prostate-specific antigen (PSA) to a monoclonal antibody (mAb). Each participant was provided with the same reagents and a detailed experimental protocol. The mAb was immobilized on the sensor chip at three different densities and a two-step assay was used to determine the kinetic and affinity parameters of the PSA/mAb complex. First, PSA was tested over a concentration range of 2.5-600 nM to obtain k(a) information. Second, to define the k(d) of this stable antigen/antibody complex accurately, the highest PSA concentration was retested with the dissociation phase of each binding cycle monitored for 1h. All participants collected data that could be analyzed to obtain kinetic parameters for the interaction. The association and the extended-dissociation data derived from the three antibody surfaces were globally fit using a simple 1:1 interaction model. The average k(a) and k(d) for the PSA/mAb interaction as calculated from the 22 analyses were (4.1+/-0.6) x 10(4) M(-1) s(-1) and (4.5+/-0.6) x 10(-5) s(-1), respectively. Overall, the experimental standard errors in the rate constants were only approximately 14%. Based on the kinetic rate constants, the affinity (K(D)) of the PSA/mAb interaction was 1.1+/-0.2 nM.

  6. Engineered Cystine-Knot Peptides That Bind αvβ3 Integrin With Antibody-Like Affinities

    PubMed Central

    Silverman, Adam P.; Levin, Aron M.; Lahti, Jennifer L.; Cochran, Jennifer R.

    2010-01-01

    The αvβ3 integrin receptor is an important cancer target due to its overexpression on many solid tumors and the tumor neovasculature, and its role in metastasis and angiogenesis. We used a truncated form of the Agouti-related protein (AgRP), a 4 kDa cystine-knot peptide with four disulfide bonds and four solvent-exposed loops, as a scaffold for engineering peptides that bound to αvβ3 integrins with high affinity and specificity. A yeast-displayed cystine-knot peptide library was generated by substituting a 6-amino acid loop of AgRP with a 9-amino acid loop containing the Arg-Gly-Asp (RGD) integrin recognition motif and randomized flanking residues. Mutant cystine-knot peptides were screened in a high-throughput manner by fluorescence-activated cell sorting (FACS) to identify clones with high affinity to detergent-solubilized αvβ3 integrin receptor. Select integrin-binding peptides were expressed recombinantly in Pichia pastoris and were tested for their ability to bind to human cancer cells expressing various integrin receptors. These studies showed that the engineered AgRP peptides bound to cells expressing αvβ3 integrins with affinities ranging from 15 nM to 780 pM. Furthermore, the engineered peptides were shown bind specifically to αvβ3 integrins, and had only minimal or no binding to αvβ5, α5β1, and αiibβ3 integrins. The engineered AgRP peptides were also shown to inhibit cell adhesion to the extracellular matrix protein vitronectin, which is a naturally-occurring ligand for αvβ3 and other integrins. Next, to evaluate whether the other three loops of AgRP could modulate integrin specificity, we made second generation libraries by individually randomizing these loops in one of the high affinity integrin-binding variants. Screening of these loop-randomized libraries against αvβ3 integrins resulted in peptides that retained high affinities for αvβ3 and had increased specificities for αvβ3 over αiibβ3 integrins. Collectively, these data

  7. Potent dengue virus neutralization by a therapeutic antibody with low monovalent affinity requires bivalent engagement.

    PubMed

    Edeling, Melissa A; Austin, S Kyle; Shrestha, Bimmi; Dowd, Kimberly A; Mukherjee, Swati; Nelson, Christopher A; Johnson, Syd; Mabila, Manu N; Christian, Elizabeth A; Rucker, Joseph; Pierson, Theodore C; Diamond, Michael S; Fremont, Daved H

    2014-04-01

    We recently described our most potently neutralizing monoclonal antibody, E106, which protected against lethal Dengue virus type 1 (DENV-1) infection in mice. To further understand its functional properties, we determined the crystal structure of E106 Fab in complex with domain III (DIII) of DENV-1 envelope (E) protein to 2.45 Å resolution. Analysis of the complex revealed a small antibody-antigen interface with the epitope on DIII composed of nine residues along the lateral ridge and A-strand regions. Despite strong virus neutralizing activity of E106 IgG at picomolar concentrations, E106 Fab exhibited a ∼20,000-fold decrease in virus neutralization and bound isolated DIII, E, or viral particles with only a micromolar monovalent affinity. In comparison, E106 IgG bound DENV-1 virions with nanomolar avidity. The E106 epitope appears readily accessible on virions, as neutralization was largely temperature-independent. Collectively, our data suggest that E106 neutralizes DENV-1 infection through bivalent engagement of adjacent DIII subunits on a single virion. The isolation of anti-flavivirus antibodies that require bivalent binding to inhibit infection efficiently may be a rare event due to the unique icosahedral arrangement of envelope proteins on the virion surface. PMID:24743696

  8. Direct binding of radioiodinated monoclonal antibody to tumor cells: significance of antibody purity and affinity for drug targeting or tumor imaging

    SciTech Connect

    Kennel, S.J.; Foote, L.J.; Lankford, P.K.; Johnson, M.; Mitchell, T.; Braslawsky, G.R.

    1983-01-01

    For MoAb to be used efficiently for drug targeting and tumor imaging, the fraction of antibody binding to tumor cells must be maximized. We have studied the binding of 125I MoAb in three different tumor systems. The fraction of antibody that could be bound to the cell surface was directly proportional to the antibody purity. The affinity constant also limits the fraction of antibody that can bind to cells at a given antigen concentration. Rearrangement of the standard expression for univalent equilibrium binding between two reactants shows that in antigen excess, the maximum fraction of antibody that can bind (formula; see text). Binding data using four different MoAb with three cell systems confirm this relationship. Estimates for reasonable concentrations of tumor antigens in vivo indicate that antibodies with binding constants less than 10(8) M-1 are not likely to be useful for drug targeting or tumor imaging.

  9. Purification of infectious canine parvovirus from cell culture by affinity chromatography with monoclonal antibodies.

    PubMed

    Rimmelzwaan, G F; Groen, J; Juntti, N; Teppema, J S; UytdeHaag, F G; Osterhaus, A D

    1987-03-01

    Immuno affinity chromatography with virus neutralizing monoclonal antibodies, directed to the haemagglutinating protein of canine parvovirus (CPV) was used to purify and concentrate CPV from infected cell culture. The procedure was monitored by testing the respective fractions in an infectivity titration system, in an ELISA, in a haemagglutination assay and by negative contrast electron microscopy to quantify CPV or CPV antigen. The degree of purification was further estimated by testing the fractions for total protein content in a colorimetric method, for bovine serum albumin content in an ELISA and by SDS-PAGE. Over 99% of the contaminating proteins proved to be removed, and 20% or 70-90% of infectious CPV or CPV antigen, respectively, was recovered.

  10. Discovery of high affinity anti-ricin antibodies by B cell receptor sequencing and by yeast display of combinatorial VH:VL libraries from immunized animals.

    PubMed

    Wang, Bo; Lee, Chang-Han; Johnson, Erik L; Kluwe, Christien A; Cunningham, Josephine C; Tanno, Hidetaka; Crooks, Richard M; Georgiou, George; Ellington, Andrew D

    2016-01-01

    Ricin is a toxin that could potentially be used as a bioweapon. We identified anti-ricin A chain antibodies by sequencing the antibody repertoire from immunized mice and by selecting high affinity antibodies using yeast surface display. These methods led to the isolation of multiple antibodies with high (sub-nanomolar) affinity. Interestingly, the antibodies identified by the 2 independent approaches are from the same clonal lineages, indicating for the first time that yeast surface display can identify native antibodies. The new antibodies represent well-characterized reagents for biodefense diagnostics and therapeutics development.

  11. Discovery of high affinity anti-ricin antibodies by B cell receptor sequencing and by yeast display of combinatorial VH:VL libraries from immunized animals.

    PubMed

    Wang, Bo; Lee, Chang-Han; Johnson, Erik L; Kluwe, Christien A; Cunningham, Josephine C; Tanno, Hidetaka; Crooks, Richard M; Georgiou, George; Ellington, Andrew D

    2016-01-01

    Ricin is a toxin that could potentially be used as a bioweapon. We identified anti-ricin A chain antibodies by sequencing the antibody repertoire from immunized mice and by selecting high affinity antibodies using yeast surface display. These methods led to the isolation of multiple antibodies with high (sub-nanomolar) affinity. Interestingly, the antibodies identified by the 2 independent approaches are from the same clonal lineages, indicating for the first time that yeast surface display can identify native antibodies. The new antibodies represent well-characterized reagents for biodefense diagnostics and therapeutics development. PMID:27224530

  12. Aflatoxin metabolism in humans: detection of metabolites and nucleic acid adducts in urine by affinity chromatography

    SciTech Connect

    Groopman, J.D.; Donahue, P.R.; Zhu, J.Q.; Chen, J.S.; Wogan, G.N.

    1985-10-01

    A high-affinity IgM monoclonal antibody specific for aflatoxins was covalently bound to Sepharose 4B and used as a preparative column to isolate aflatoxin derivatives from the urine of people and experimental animals who had been exposed to the carcinogen environmentally or under laboratory conditions. Aflatoxin levels were quantified by radioimmunoassay and high-performance liquid chromatography after elution from the affinity column. In studies on rats injected with ( UC)aflatoxin B1, the authors identified the major aflatoxin-DNA adduct, 2,3-dihydro-2-(N7-guanyl)-3-hydroxy-aflatoxin B1 (AFB1-N7-Gua), and the oxidative metabolites M1 and P1 as the major aflatoxin species present in the urine. When this methodology was applied to human urine samples obtained from people from the Guangxi Province of China exposed to aflatoxin B1 through dietary contamination, the aflatoxin metabolites detected were also AFB1-N7-Gua and aflatoxins M1 and P1. Therefore, affinity chromatography using a monoclonal antibody represents a useful and rapid technique with which to isolate this carcinogen and its metabolites in biochemical epidemiology and for subsequent quantitative measurements, providing exposure information that can be used for risk assessment.

  13. Efficient generation of human IgA monoclonal antibodies.

    PubMed

    Lorin, Valérie; Mouquet, Hugo

    2015-07-01

    Immunoglobulin A (IgA) is the most abundant antibody isotype produced in humans. IgA antibodies primarily ensure immune protection of mucosal surfaces against invading pathogens, but also circulate and are present in large quantities in blood. IgAs are heterogeneous at a molecular level, with two IgA subtypes and the capacity to form multimers by interacting with the joining (J) chain. Here, we have developed an efficient strategy to rapidly generate human IgA1 and IgA2 monoclonal antibodies in their monomeric and dimeric forms. Recombinant monomeric and dimeric IgA1/IgA2 counterparts of a prototypical IgG1 monoclonal antibody, 10-1074, targeting the HIV-1 envelope protein, were produced in large amounts after expression cloning and transient transfection of 293-F cells. 10-1074 IgAs were FPLC-purified using a novel affinity-based resin engrafted with anti-IgA chimeric Fabs, followed by a monomers/multimers separation using size exclusion-based FPLC. ELISA binding experiments confirmed that the artificial IgA class switching of 10-1074 did not alter its antigen recognition. In summary, our technical approach allows the very efficient production of various forms of purified recombinant human IgA molecules, which are precious tools in dissecting IgA B-cell responses in physiological and pathophysiological conditions, and studying the biology, function and therapeutic potential of IgAs.

  14. Octapeptide-based affinity chromatography of human immunoglobulin G: comparisons of three different ligands.

    PubMed

    Zhao, Wei-Wei; Liu, Fu-Feng; Shi, Qing-Hong; Sun, Yan

    2014-09-12

    In an earlier work, we have developed a biomimetic design strategy based on the human IgG (hIgG)-Protein A interactions and identified an affinity ligand for hIgG, FYWHCLDE, which ranked top one in a pool of 14 potential candidates. Herein, two more octapeptides, FYCHWALE and FYCHTIDE, were identified, and the binding and purification of hIgG on the affinity columns packed with the three octapeptide-modified Sepharose gels were extensively studied and compared to find more effective octapeptide-based affinity ligands. It was found that all the three ligands bound hIgG and Fc fragment but barely bound Fab fragment, and the binding to hIgG and Fc was mainly by electrostatic interactions. The optimum binding pH values for the three ligands were different from each other, but kept in the range of 5.0-6.0. Ligand binding competition revealed that the binding sites on hIgG for the three octapeptides were similar to those for Protein A. Adsorption isotherms revealed that hIgG binding capacity was in the range of 64-104mg/mL drained gel in the order of FYWHCLDE>FYCHWALE>FYCHTIDE. Then, purifications of hIgG and human monoclonal antibody from human serum and cell culture supernatant, respectively, were achieved with the three affinity columns at high purities and recovery yields. Finally, the molecular basis for the binding affinity of the peptides for the Fc fragment of hIgG was elucidated by molecular dynamics simulations. PMID:25064536

  15. Conformation-Dependent High-Affinity Potent Ricin-Neutralizing Monoclonal Antibodies

    PubMed Central

    Hu, Wei-Gang; Yin, Junfei; Chau, Damon; Hu, Charles Chen; Lillico, Dustin; Yu, Justin; Negrych, Laurel M.; Cherwonogrodzky, John W.

    2013-01-01

    Ricin is a potential biothreat agent with no approved antidote available for ricin poisoning. The aim of this study was to develop potent antibody-based antiricin antidotes. Four strong ricin resistant hybridoma clones secreting antiricin monoclonal antibodies (mAbs) were developed. All four mAbs are bound to conformational epitopes of ricin toxin B (RTB) with high affinity (KD values from 2.55 to 36.27 nM). RTB not only triggers cellular uptake of ricin, but also facilitates transport of the ricin toxin A (RTA) from the endoplasmic reticulum to the cytosol, where RTA exerts its toxic activity. The four mAbs were found to have potent ricin-neutralizing capacities and synergistic effects among them as determined by an in vitro neutralization assay. In vivo protection assay demonstrated that all four mAbs had strong efficacy against ricin challenges. D9 was found to be exceptionally effective. Intraperitoneal (i.p.) administration of D9, at a dose of 5 μg, 6 weeks before or 6 hours after an i.p. challenge with 5 × LD50 of ricin was able to protect or rescue 100% of the mice, indicating that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin poisoning for both prophylactic and therapeutic purposes. PMID:23484120

  16. Conformation-dependent high-affinity potent ricin-neutralizing monoclonal antibodies.

    PubMed

    Hu, Wei-Gang; Yin, Junfei; Chau, Damon; Hu, Charles Chen; Lillico, Dustin; Yu, Justin; Negrych, Laurel M; Cherwonogrodzky, John W

    2013-01-01

    Ricin is a potential biothreat agent with no approved antidote available for ricin poisoning. The aim of this study was to develop potent antibody-based antiricin antidotes. Four strong ricin resistant hybridoma clones secreting antiricin monoclonal antibodies (mAbs) were developed. All four mAbs are bound to conformational epitopes of ricin toxin B (RTB) with high affinity (KD values from 2.55 to 36.27 nM). RTB not only triggers cellular uptake of ricin, but also facilitates transport of the ricin toxin A (RTA) from the endoplasmic reticulum to the cytosol, where RTA exerts its toxic activity. The four mAbs were found to have potent ricin-neutralizing capacities and synergistic effects among them as determined by an in vitro neutralization assay. In vivo protection assay demonstrated that all four mAbs had strong efficacy against ricin challenges. D9 was found to be exceptionally effective. Intraperitoneal (i.p.) administration of D9, at a dose of 5 μ g, 6 weeks before or 6 hours after an i.p. challenge with 5 × LD50 of ricin was able to protect or rescue 100% of the mice, indicating that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin poisoning for both prophylactic and therapeutic purposes. PMID:23484120

  17. Prospective Design of Anti-Transferrin Receptor Bispecific Antibodies for Optimal Delivery into the Human Brain.

    PubMed

    Kanodia, J S; Gadkar, K; Bumbaca, D; Zhang, Y; Tong, R K; Luk, W; Hoyte, K; Lu, Y; Wildsmith, K R; Couch, J A; Watts, R J; Dennis, M S; Ernst, J A; Scearce-Levie, K; Atwal, J K; Ramanujan, S; Joseph, S

    2016-05-01

    Anti-transferrin receptor (TfR)-based bispecific antibodies have shown promise for boosting antibody uptake in the brain. Nevertheless, there are limited data on the molecular properties, including affinity required for successful development of TfR-based therapeutics. A complex nonmonotonic relationship exists between affinity of the anti-TfR arm and brain uptake at therapeutically relevant doses. However, the quantitative nature of this relationship and its translatability to humans is heretofore unexplored. Therefore, we developed a mechanistic pharmacokinetic-pharmacodynamic (PK-PD) model for bispecific anti-TfR/BACE1 antibodies that accounts for antibody-TfR interactions at the blood-brain barrier (BBB) as well as the pharmacodynamic (PD) effect of anti-BACE1 arm. The calibrated model correctly predicted the optimal anti-TfR affinity required to maximize brain exposure of therapeutic antibodies in the cynomolgus monkey and was scaled to predict the optimal affinity of anti-TfR bispecifics in humans. Thus, this model provides a framework for testing critical translational predictions for anti-TfR bispecific antibodies, including choice of candidate molecule for clinical development. PMID:27299941

  18. Isolation of human lactate dehydrogenase isoenzyme X by affinity chromatography.

    PubMed Central

    Kolk, A H; van Kuyk, L; Boettcher, B

    1978-01-01

    Human isoenzyme LDH-X (lactate dehydrogenase isoenzyme X) was isolated from seminal fluid of frozen semen samples by affinity chromatography by using oxamate-Sepharose and AMP-Sepharose. In the presence of 1.6 mM-NAD+, isoenzyme LDH-X does not bind to AMP-Sepharose, whereas the other lactate dehydrogenase isoenzymes do. This is the crucial point in the isolation of isoenzyme LDH-X from the other isoenzymes. The purified human isoenzyme LDH-X had a specific activity of 146 units/mg of protein. Images Fig. 2. Fig. 3. PMID:213050

  19. Glycosylation of plant produced human antibodies.

    PubMed

    Kallolimath, Somanath; Steinkellner, Herta

    2015-12-23

    Human immunoglobulins circulate as highly heterogeneously glycosylated mixture of otherwise homogeneous protein backbones. A series of studies, mainly on IgG, have unequivocally proven that antibodies modulate their effector function through sugars present in the Fc domain. However, our limited technology in producing complex proteins such as antibodies, with defined glycan structures hamper in depths studies. This review introduces a plant based expression platform enabling engineering of antibody glycans. The procedure is based on the simultaneous delivery of appropriate constructs, carrying cDNAs of target proteins (e.g. heavy and light chain of antibodies) in combination with human glycosylation enzymes into plant leaves. Harvesting of recombinant proteins one week post construct delivery allows high speed and flexibility. Major achievements include the production of functional active slialylated pentameric IgMs in tobacco leaves. The system provides a viable approach to the generation of antibodies with defined glycoforms on demand, contributing to studies on antibody glycans and the development of novel antibody based drugs. PMID:27472861

  20. Use of Human Hybridoma Technology To Isolate Human Monoclonal Antibodies.

    PubMed

    Smith, Scott A; Crowe, James E

    2015-02-01

    The human hybridoma technique offers an important approach for isolation of human monoclonal antibodies. A diversity of approaches can be used with varying success. Recent technical advances in expanding the starting number of human antigen-specific B cells, improving fusion efficiency, and isolating new myeloma partners and new cell cloning methods have enabled the development of protocols that make the isolation of human monoclonal antibodies from blood samples feasible. Undoubtedly, additional innovations that could improve efficiency are possible.

  1. Orthobunyavirus Antibodies in Humans, Yucatan Peninsula, Mexico

    PubMed Central

    Saiyasombat, Rungrat; Talavera-Aguilar, Lourdes G.; Garcia-Rejon, Julian E.; Farfan-Ale, Jose A.; Machain-Williams, Carlos; Loroño-Pino, Maria A.

    2012-01-01

    We performed a serologic investigation to determine whether orthobunyaviruses commonly infect humans in the Yucatan Peninsula of Mexico. Orthobunyavirus-specific antibodies were detected by plaque reduction neutralization test in 146 (18%) of 823 persons tested. Further studies are needed to determine health risks for humans from this potentially deadly group of viruses. PMID:23017592

  2. XGFR*, a novel affinity-matured bispecific antibody targeting IGF-1R and EGFR with combined signaling inhibition and enhanced immune activation for the treatment of pancreatic cancer.

    PubMed

    Schanzer, Juergen M; Wartha, Katharina; Moessner, Ekkehard; Hosse, Ralf J; Moser, Samuel; Croasdale, Rebecca; Trochanowska, Halina; Shao, Cuiying; Wang, Peng; Shi, Lei; Weinzierl, Tina; Rieder, Natascha; Bacac, Marina; Ries, Carola H; Kettenberger, Hubert; Schlothauer, Tilman; Friess, Thomas; Umana, Pablo; Klein, Christian

    2016-01-01

    The epidermal growth factor receptor (EGFR) and the insulin-like growth factor-1 receptor (IGF-1R) play critical roles in tumor growth, providing a strong rationale for the combined inhibition of IGF-1R and EGFR signaling in cancer therapy. We describe the design, affinity maturation, in vitro and in vivo characterization of the bispecific anti-IGF-1R/EGFR antibody XGFR*. XGFR* is based on the bispecific IgG antibody XGFR, which enabled heterodimerization of an IGF-1R binding scFab heavy chain with an EGFR-binding light and heavy chain by the "knobs-into-holes" technology. XGFR* is optimized for monovalent binding of human EGFR and IGF-1R with increased binding affinity for IGF-1R due to affinity maturation and highly improved protein stability to oxidative and thermal stress. It bears an afucosylated Fc-portion for optimal induction of antibody-dependent cell-mediated cytotoxicity (ADCC). Stable Chinese hamster ovary cell clones with production yields of 2-3 g/L were generated, allowing for large scale production of the bispecific antibody. XGFR* potently inhibits EGFR- and IGF-1R-dependent receptor phosphorylation, reduces tumor cell proliferation in cells with heterogeneous levels of IGF-1R and EGFR receptor expression and induces strong ADCC in vitro. A comparison of pancreatic and colorectal cancer lines demonstrated superior responsiveness to XGFR*-mediated signaling and tumor growth inhibition in pancreatic cancers that frequently show a high degree of IGF-1R/EGFR co-expression. XGFR* showed potent anti-tumoral efficacy in the orthotopic MiaPaCa-2 pancreatic xenograft model, resulting in nearly complete tumor growth inhibition with significant number of tumor remissions. In summary, the bispecific anti-IGF-1R/EGFR antibody XGFR* combines potent signaling and tumor growth inhibition with enhanced ADCC induction and represents a clinical development candidate for the treatment of pancreatic cancer. PMID:26984378

  3. XGFR*, a novel affinity-matured bispecific antibody targeting IGF-1R and EGFR with combined signaling inhibition and enhanced immune activation for the treatment of pancreatic cancer

    PubMed Central

    Schanzer, Juergen M.; Wartha, Katharina; Moessner, Ekkehard; Hosse, Ralf J.; Moser, Samuel; Croasdale, Rebecca; Trochanowska, Halina; Shao, Cuiying; Wang, Peng; Shi, Lei; Weinzierl, Tina; Rieder, Natascha; Bacac, Marina; Ries, Carola H.; Kettenberger, Hubert; Schlothauer, Tilman; Friess, Thomas; Umana, Pablo; Klein, Christian

    2016-01-01

    ABSTRACT The epidermal growth factor receptor (EGFR) and the insulin-like growth factor-1 receptor (IGF-1R) play critical roles in tumor growth, providing a strong rationale for the combined inhibition of IGF-1R and EGFR signaling in cancer therapy. We describe the design, affinity maturation, in vitro and in vivo characterization of the bispecific anti-IGF-1R/EGFR antibody XGFR*. XGFR* is based on the bispecific IgG antibody XGFR, which enabled heterodimerization of an IGF-1R binding scFab heavy chain with an EGFR-binding light and heavy chain by the “knobs-into-holes” technology. XGFR* is optimized for monovalent binding of human EGFR and IGF-1R with increased binding affinity for IGF-1R due to affinity maturation and highly improved protein stability to oxidative and thermal stress. It bears an afucosylated Fc-portion for optimal induction of antibody-dependent cell-mediated cytotoxicity (ADCC). Stable Chinese hamster ovary cell clones with production yields of 2–3 g/L were generated, allowing for large scale production of the bispecific antibody. XGFR* potently inhibits EGFR- and IGF-1R-dependent receptor phosphorylation, reduces tumor cell proliferation in cells with heterogeneous levels of IGF-1R and EGFR receptor expression and induces strong ADCC in vitro. A comparison of pancreatic and colorectal cancer lines demonstrated superior responsiveness to XGFR*-mediated signaling and tumor growth inhibition in pancreatic cancers that frequently show a high degree of IGF-1R/EGFR co-expression. XGFR* showed potent anti-tumoral efficacy in the orthotopic MiaPaCa-2 pancreatic xenograft model, resulting in nearly complete tumor growth inhibition with significant number of tumor remissions. In summary, the bispecific anti-IGF-1R/EGFR antibody XGFR* combines potent signaling and tumor growth inhibition with enhanced ADCC induction and represents a clinical development candidate for the treatment of pancreatic cancer. PMID:26984378

  4. Mannosylerythritol lipid, a yeast extracellular glycolipid, shows high binding affinity towards human immunoglobulin G

    PubMed Central

    Im, Jae Hong; Nakane, Takashi; Yanagishita, Hiroshi; Ikegami, Toru; Kitamoto, Dai

    2001-01-01

    Background There have been many attempts to develop new materials with stability and high affinity towards immunoglobulins. Some of glycolipids such as gangliosides exhibit a high affinity toward immunoglobulins. However, it is considerably difficult to develop these glycolipids into the practical separation ligand due to their limited amounts. We thus focused our attention on the feasible use of "mannosylerythritol lipid A", a yeast glycolipid biosurfactant, as an alternative ligand for immunoglobulins, and undertook the investigation on the binding between mannosylerythritol lipid A (MEL-A) and human immunoglobulin G (HIgG). Results In ELISA assay, MEL-A showed nearly the same binding affinity towards HIgG as that of bovine ganglioside GM1. Fab of human IgG was considered to play a more important role than Fc in the binding of HIgG by MEL-A. The bound amount of HIgG increased depending on the attached amount of MEL-A onto poly (2-hydroxyethyl methacrylate) (polyHEMA) beads, whereas the amount of human serum albumin slightly decreased. Binding-amount and -selectivity of HIgG towards MEL-A were influenced by salt species, salt concentration and pH in the buffer solution. The composite of MEL-A and polyHEMA, exhibited a significant binding constant of 1.43 × 106 (M-1) for HIgG, which is approximately 4-fold greater than that of protein A reported. Conclusions MEL-A shows high binding-affinity towards HIgG, and this is considered to be due to "multivalent effect" based on the binding molar ratio. This is the first report on the binding of a natural human antibody towards a yeast glycolipid. PMID:11604104

  5. Molecular Insights into Fully Human and Humanized Monoclonal Antibodies

    PubMed Central

    Davies, Julian; Glasebrook, Andrew; Tang, Ying; Glaesner, Wolfgang; Nickoloff, Brian J.

    2016-01-01

    In recent years, a large number of therapeutic monoclonal antibodies have come to market to treat a variety of conditions including patients with immune-mediated chronic inflammation. Distinguishing the relative clinical efficacy and safety profiles of one monoclonal antibody relative to another can be difficult and complex due to different clinical designs and paucity of head-to-head comparator studies. One distinguishing feature in interpreting clinical trial data by dermatologists may begin by determining whether a monoclonal antibody is fully human or humanized, which can be discerned by the generic name of the drug. Herein, this commentary highlights the distinctions and similarities of fully human and humanized monoclonal antibodies in their nomenclature, engineering, and clinical profiles. While there are a number of differences between these types of monoclonal antibodies, current evidence indicates that this designation does not impart any measurable impact on overall clinical efficacy and safety profiles of a given drug. Based on molecular insights provided in this commentary, it is clear that each monoclonal antibody, irrespective of being fully human or humanized, should be individually assessed for its clinical impact regarding safety and efficacy. Going beyond the type of generic name ascribed to a monoclonal antibody will be an ever-increasing theme for dermatologists as more therapeutic monoclonal antibodies emerge to potentially treat a wider scope of diseases with cutaneous manifestations. PMID:27672407

  6. Optimization of pore structure and particle morphology of mesoporous silica for antibody adsorption for use in affinity chromatography

    NASA Astrophysics Data System (ADS)

    Hikosaka, Ryouichi; Nagata, Fukue; Tomita, Masahiro; Kato, Katsuya

    2016-10-01

    Antibodies have received significant attention for use as antibody drugs, because they bind the objective protein (antigen) via antigen-antibody reactions. Recently, many reports have appeared on various monoclonal antibodies that recognize a single antigen. In this study, monoclonal antibodies are used as adsorbates on mesoporous silica (MPS) for affinity chromatography. MPS has high surface area and large pore volume; moreover, pore diameter, pore structure, and particle morphology are relatively easy to tune by adjusting the conditions of synthesis. The pore structure (two-dimensional (2D) hexagonal and three-dimensional cubic) and particle morphology (spherical and polyhedral) of MPS are optimized for use in a monoclonal antibody/MPS composite. When anti-IgG (one of the monoclonal antibodies) adsorbs on the MPS material and IgG (antigen) binds to anti-IgG/MPS composites, MCM-41p with a 2D-hexagonal pore structure and polyhedral particle morphology has the highest IgG binding efficiency. In addition, the antibody/MPS composites remain stable in chaotropic and low-pH solutions and can be cycled at least five times without decreasing IgG elution. In purification and removal tests, the use of the antibody/MPS composites allows only the objective protein from protein mixtures to be bound and eluted.

  7. 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.

  8. Calcium affinity of human α-actinin 1.

    PubMed

    Backman, Lars

    2015-01-01

    Due to alternative splicing, the human ACTN1 gene codes for three different transcripts of α-actinin; one isoform that is expressed only in the brain and two with a more general expression pattern. The sequence difference is located to the C-terminal domains and the EF-hand motifs. Therefore, any functional or structural distinction should involve this part of the protein. To investigate this further, the calcium affinities of these three isoforms of α-actinin 1 have been determined by isothermal calorimetry. PMID:26020004

  9. Prospective Design of Anti‐Transferrin Receptor Bispecific Antibodies for Optimal Delivery into the Human Brain

    PubMed Central

    Kanodia, JS; Gadkar, K; Bumbaca, D; Zhang, Y; Tong, RK; Luk, W; Hoyte, K; Lu, Y; Wildsmith, KR; Couch, JA; Watts, RJ; Dennis, MS; Ernst, JA; Scearce‐Levie, K; Atwal, JK; Joseph, S

    2016-01-01

    Anti‐transferrin receptor (TfR)‐based bispecific antibodies have shown promise for boosting antibody uptake in the brain. Nevertheless, there are limited data on the molecular properties, including affinity required for successful development of TfR‐based therapeutics. A complex nonmonotonic relationship exists between affinity of the anti‐TfR arm and brain uptake at therapeutically relevant doses. However, the quantitative nature of this relationship and its translatability to humans is heretofore unexplored. Therefore, we developed a mechanistic pharmacokinetic‐pharmacodynamic (PK‐PD) model for bispecific anti‐TfR/BACE1 antibodies that accounts for antibody‐TfR interactions at the blood‐brain barrier (BBB) as well as the pharmacodynamic (PD) effect of anti‐BACE1 arm. The calibrated model correctly predicted the optimal anti‐TfR affinity required to maximize brain exposure of therapeutic antibodies in the cynomolgus monkey and was scaled to predict the optimal affinity of anti‐TfR bispecifics in humans. Thus, this model provides a framework for testing critical translational predictions for anti‐TfR bispecific antibodies, including choice of candidate molecule for clinical development. PMID:27299941

  10. Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcgammaRIII and antibodies lacking core fucose.

    PubMed

    Ferrara, Claudia; Grau, Sandra; Jäger, Christiane; Sondermann, Peter; Brünker, Peter; Waldhauer, Inja; Hennig, Michael; Ruf, Armin; Rufer, Arne Christian; Stihle, Martine; Umaña, Pablo; Benz, Jörg

    2011-08-01

    Antibody-mediated cellular cytotoxicity (ADCC), a key immune effector mechanism, relies on the binding of antigen-antibody complexes to Fcγ receptors expressed on immune cells. Antibodies lacking core fucosylation show a large increase in affinity for FcγRIIIa leading to an improved receptor-mediated effector function. Although afucosylated IgGs exist naturally, a next generation of recombinant therapeutic, glycoenginereed antibodies is currently being developed to exploit this finding. In this study, the crystal structures of a glycosylated Fcγ receptor complexed with either afucosylated or fucosylated Fc were determined allowing a detailed, molecular understanding of the regulatory role of Fc-oligosaccharide core fucosylation in improving ADCC. The structures reveal a unique type of interface consisting of carbohydrate-carbohydrate interactions between glycans of the receptor and the afucosylated Fc. In contrast, in the complex structure with fucosylated Fc, these contacts are weakened or nonexistent, explaining the decreased affinity for the receptor. These findings allow us to understand the higher efficacy of therapeutic antibodies lacking the core fucose and also suggest a unique mechanism by which the immune system can regulate antibody-mediated effector functions.

  11. The Use of Monoclonal Antibodies in Human Prion Disease

    NASA Astrophysics Data System (ADS)

    Bodemer, Walter

    Detection of PrP and its pathological isoform(s) is the key to understanding the etiology and pathogenesis of transmissible spongiform encephalopathy. There is ample evidence that PrP isoforms constitute a major component of an unknown and perhaps unconventional infectious agent. An etiological relationship between human and zoonotic transmissible spongiform encephalopathies may be revealed with monoclonal antibodies. Knowledge of the conformational transition rendering a nonpathogenic, almost ubiquitous cellular protein into a pathogenic one is crucial to defining pathomechanisms. The stepwise or even continuous formation of pathogenic molecules can be monitored. Any improvement in the early diagnosis could help to conceive new therapeutic measures which are not currently available. Determination of PrP isoforms in tissue, cells, or body fluids may be of prognostic value. Many experimental approaches in molecular medicine and molecular biology of the prion protein already rely on monoclonal antibodies. Recombinant antibodies such as the single-chain Fv may soon replace traditional hybridoma techniques. Binding affinity can easily be manipulated by a number of techniques, including in vitro mutagenesis - a step which could never be carried out using the traditional hybridoma technology. Monoclonal antibodies are and will remain an essential support for ongoing research on the prion protein in general and on the unconventional infectious prions.

  12. Characterization of the Native and Denatured Herceptin by ELISA and QCM using a High-Affinity Single Chain Fragment Variable (scFv) Recombinant Antibody

    PubMed Central

    Shang, Yuqin; Mernaugh, Ray

    2012-01-01

    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 an scFv (designated 2B4) to a linear synthetic peptide representing Herceptin’s heavy chain CDR3. 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 amount 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 × 1013 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 non-specific binding events in assays. The potential theoretical and clinical implications of these results and the advantages of using QCM to characterize human therapeutic antibodies in samples are also discussed. PMID:22934911

  13. Fully Human Antagonistic Antibodies against CCR4 Potently Inhibit Cell Signaling and Chemotaxis

    PubMed Central

    Géraudie, Solène; Scheffler, Ulrike; Griep, Remko A.; Reiersen, Herald; Duncan, Alexander R.; Kiprijanov, Sergej M.

    2014-01-01

    Background CC chemokine receptor 4 (CCR4) represents a potentially important target for cancer immunotherapy due to its expression on tumor infiltrating immune cells including regulatory T cells (Tregs) and on tumor cells in several cancer types and its role in metastasis. Methodology Using phage display, human antibody library, affinity maturation and a cell-based antibody selection strategy, the antibody variants against human CCR4 were generated. These antibodies effectively competed with ligand binding, were able to block ligand-induced signaling and cell migration, and demonstrated efficient killing of CCR4-positive tumor cells via ADCC and phagocytosis. In a mouse model of human T-cell lymphoma, significant survival benefit was demonstrated for animals treated with the newly selected anti-CCR4 antibodies. Significance For the first time, successful generation of anti- G-protein coupled chemokine receptor (GPCR) antibodies using human non-immune library and phage display on GPCR-expressing cells was demonstrated. The generated anti-CCR4 antibodies possess a dual mode of action (inhibition of ligand-induced signaling and antibody-directed tumor cell killing). The data demonstrate that the anti-tumor activity in vivo is mediated, at least in part, through Fc-receptor dependent effector mechanisms, such as ADCC and phagocytosis. Anti-CC chemokine receptor 4 antibodies inhibiting receptor signaling have potential as immunomodulatory antibodies for cancer. PMID:25080123

  14. Comparison of biosensor platforms in the evaluation of high affinity antibody-antigen binding kinetics.

    PubMed

    Yang, Danlin; Singh, Ajit; Wu, Helen; Kroe-Barrett, Rachel

    2016-09-01

    The acquisition of reliable kinetic parameters for the characterization of biomolecular interactions is an important component of the drug discovery and development process. While several benchmark studies have explored the variability of kinetic rate constants obtained from multiple laboratories and biosensors, a direct comparison of these instruments' performance has not been undertaken, and systematic factors contributing to data variability from these systems have not been discussed. To address these questions, a panel of ten high-affinity monoclonal antibodies was simultaneously evaluated for their binding kinetics against the same antigen on four biosensor platforms: GE Healthcare's Biacore T100, Bio-Rad's ProteOn XPR36, ForteBio's Octet RED384, and Wasatch Microfluidics's IBIS MX96. We compared the strengths and weaknesses of these systems and found that despite certain inherent systematic limitations in instrumentation, the rank orders of both the association and dissociation rate constants were highly correlated between these instruments. Our results also revealed a trade-off between data reliability and sample throughput. Biacore T100, followed by ProteOn XPR36, exhibited excellent data quality and consistency, whereas Octet RED384 and IBIS MX96 demonstrated high flexibility and throughput with compromises in data accuracy and reproducibility. Our results support the need for a "fit-for-purpose" approach in instrument selection for biosensor studies. PMID:27365220

  15. Antibody VH and VL recombination using phage and ribosome display technologies reveals distinct structural routes to affinity improvements with VH-VL interface residues providing important structural diversity.

    PubMed

    Groves, Maria A T; Amanuel, Lily; Campbell, Jamie I; Rees, D Gareth; Sridharan, Sudharsan; Finch, Donna K; Lowe, David C; Vaughan, Tristan J

    2014-01-01

    In vitro selection technologies are an important means of affinity maturing antibodies to generate the optimal therapeutic profile for a particular disease target. Here, we describe the isolation of a parent antibody, KENB061 using phage display and solution phase selections with soluble biotinylated human IL-1R1. KENB061 was affinity matured using phage display and targeted mutagenesis of VH and VL CDR3 using NNS randomization. Affinity matured VHCDR3 and VLCDR3 library blocks were recombined and selected using phage and ribosome display protocol. A direct comparison of the phage and ribosome display antibodies generated was made to determine their functional characteristics.In our analyses, we observed distinct differences in the pattern of beneficial mutations in antibodies derived from phage and ribosome display selections, and discovered the lead antibody Jedi067 had a ~3700-fold improvement in KD over the parent KENB061. We constructed a homology model of the Fv region of Jedi067 to map the specific positions where mutations occurred in the CDR3 loops. For VL CDR3, positions 94 to 97 carry greater diversity in the ribosome display variants compared with the phage display. The positions 95a, 95b and 96 of VLCDR3 form part of the interface with VH in this model. The model shows that positions 96, 98, 100e, 100f, 100 g, 100h, 100i and 101 of the VHCDR3 include residues at the VH and VL interface. Importantly, Leu96 and Tyr98 are conserved at the interface positions in both phage and ribosome display indicating their importance in maintaining the VH-VL interface. For antibodies derived from ribosome display, there is significant diversity at residues 100a to 100f of the VH CDR3 compared with phage display. A unique deletion of isoleucine at position 102 of the lead candidate, Jedi067, also occurs in the VHCDR3.As anticipated, recombining the mutations via ribosome display led to a greater structural diversity, particularly in the heavy chain CDR3, which in turn

  16. Ultrasensitive Analysis of Binding Affinity of HIV Receptor and Neutralizing Antibody Using Solution-Phase Electrochemiluminescence Assay

    PubMed Central

    Xu, Xiao-Hong Nancy; Wen, Zhaoyang; Brownlow, William J.

    2012-01-01

    Binding of a few ligand molecules with its receptors on cell surface can initiate cellular signaling transduction pathways, and trigger viral infection of host cells. HIV-1 infects host T-cells by binding its viral envelope protein (gp120) with its receptor (a glycoprotein, CD4) on T cells. Primary strategies to prevent and treat HIV infection is to develop therapies (e.g., neutralizing antibodies) that can block specific binding of CD4 with gp120. The infection often leads to the lower counts of CD4 cells, which makes it an effective biomarker to monitor the AIDS progression and treatment. Despite research over decades, quantitative assays for effective measurements of binding affinities of protein-protein (ligand-receptor, antigen-antibody) interactions remains highly sought. Solid-phase electrochemiluminescence (ECL) immunoassay has been commonly used to capture analytes from the solution for analysis, which involves immobilization of antibody on solid surfaces (micron-sized beads), but it cannot quantitatively measure binding affinities of molecular interactions. In this study, we have developed solution-phase ECL assay with a wide dynamic range (0–2 nM) and high sensitivity and specificity for quantitative analysis of CD4 at femtomolar level and their binding affinity with gp120 and monoclonal antibodies (MABs). We found that binding affinities of CD4 with gp120 and MAB (Q4120) are 9.5×108 and 1.2×109 M−1, respectively. The results also show that MAB (Q4120) of CD4 can completely block the binding of gp120 with CD4, while MAB (17b) of gp120 can only partially block their interaction. This study demonstrates that the solution-phase ECL assay can be used for ultrasensitive and quantitative analysis of binding affinities of protein-protein interactions in solution for better understating of protein functions and identification of effective therapies to block their interactions. PMID:23565071

  17. Combination of isothermal titration calorimetry and time-resolved luminescence for high affinity antibody-ligand interaction thermodynamics and kinetics.

    PubMed

    Aweda, Tolulope A; Meares, Claude F

    2012-02-01

    For experiments using synthetic ligands as probes for biological experiments, it is useful to determine the specificity and affinity of the ligands for their receptors. As ligands with higher affinities are developed (K(A)>10(8)M(-1); K(D)<10(-8)M), a new challenge arises: to measure these values accurately. Isothermal titration calorimetry measures heat produced or consumed during ligand binding, and also provides the equilibrium binding constant. However, as normally practiced, its range is limited. Displacement titration, where a competing weaker ligand is used to lower the apparent affinity of the stronger ligand, can be used to determine the binding affinity as well as the complete thermodynamic data for ligand-antibody complexes with very high affinity. These equilibrium data have been combined with kinetic measurements to yield the rate constants as well. We describe this methodology, using as an example antibody 2D12.5, which captures yttrium S-2-(4-aminobenzyl)-1, 4, 7, 10-tetraazacyclododecanetetraacetate.

  18. Development of an automated mid-scale parallel protein purification system for antibody purification and affinity chromatography.

    PubMed

    Zhang, Chi; Long, Alexander M; Swalm, Brooke; Charest, Ken; Wang, Yan; Hu, Jiali; Schulz, Craig; Goetzinger, Wolfgang; Hall, Brian E

    2016-12-01

    Protein purification is often a bottleneck during protein generation for large molecule drug discovery. Therapeutic antibody campaigns typically require the purification of hundreds of monoclonal antibodies (mAbs) during the hybridoma process and lead optimization. With the increase in high-throughput cloning, faster DNA sequencing, and the use of parallel protein expression systems, a need for high-throughput purification approaches has evolved, particularly in the midsize range between 20 ml and 100 ml. To address this we modified a four channel Gilson solid phase extraction system (referred to as MG-SPE) with switching valves and sample holding loops to be able to perform standard affinity purification using commercially available columns and micro-titer format deep well blocks. By running 4 samples in parallel, the MG-SPE has the capacity to purify up to 24 samples of greater than 50 ml each using a single-step affinity purification protocol or a two-step protocol consisting of affinity chromatography followed by desalting/buffer exchange overnight (∼12 h run time). Our evaluation of affinity purification using mAbs and Fc-fusion proteins from mammalian cell supernatants demonstrates that the MG-SPE compared favorably with industry standard systems for both protein quality and yield. Overall the system is simple to operate and fills a void in purification processes where a simple, efficient, automated system is needed for affinity purification of midsize research samples. PMID:27498022

  19. Development of an automated mid-scale parallel protein purification system for antibody purification and affinity chromatography.

    PubMed

    Zhang, Chi; Long, Alexander M; Swalm, Brooke; Charest, Ken; Wang, Yan; Hu, Jiali; Schulz, Craig; Goetzinger, Wolfgang; Hall, Brian E

    2016-12-01

    Protein purification is often a bottleneck during protein generation for large molecule drug discovery. Therapeutic antibody campaigns typically require the purification of hundreds of monoclonal antibodies (mAbs) during the hybridoma process and lead optimization. With the increase in high-throughput cloning, faster DNA sequencing, and the use of parallel protein expression systems, a need for high-throughput purification approaches has evolved, particularly in the midsize range between 20 ml and 100 ml. To address this we modified a four channel Gilson solid phase extraction system (referred to as MG-SPE) with switching valves and sample holding loops to be able to perform standard affinity purification using commercially available columns and micro-titer format deep well blocks. By running 4 samples in parallel, the MG-SPE has the capacity to purify up to 24 samples of greater than 50 ml each using a single-step affinity purification protocol or a two-step protocol consisting of affinity chromatography followed by desalting/buffer exchange overnight (∼12 h run time). Our evaluation of affinity purification using mAbs and Fc-fusion proteins from mammalian cell supernatants demonstrates that the MG-SPE compared favorably with industry standard systems for both protein quality and yield. Overall the system is simple to operate and fills a void in purification processes where a simple, efficient, automated system is needed for affinity purification of midsize research samples.

  20. "Velcro" engineering of high affinity CD47 ectodomain as signal regulatory protein α (SIRPα) antagonists that enhance antibody-dependent cellular phagocytosis.

    PubMed

    Ho, Chia Chi M; Guo, Nan; Sockolosky, Jonathan T; Ring, Aaron M; Weiskopf, Kipp; Özkan, Engin; Mori, Yasuo; Weissman, Irving L; Garcia, K Christopher

    2015-05-15

    CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the "don't-eat-me" signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined "Velcro" engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that "Velcro" engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy.

  1. “Velcro” Engineering of High Affinity CD47 Ectodomain as Signal Regulatory Protein α (SIRPα) Antagonists That Enhance Antibody-dependent Cellular Phagocytosis*

    PubMed Central

    Ho, Chia Chi M.; Guo, Nan; Sockolosky, Jonathan T.; Ring, Aaron M.; Weiskopf, Kipp; Özkan, Engin; Mori, Yasuo; Weissman, Irving L.; Garcia, K. Christopher

    2015-01-01

    CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the “don't-eat-me” signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined “Velcro” engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that “Velcro” engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy

  2. Autoreactivity of primary human immunoglobulins ancestral to hypermutated human antibodies that neutralize HCMV.

    PubMed

    McLean, Gary R; Cho, Chin-wen; Schrader, John W

    2006-05-01

    The human antibody response to the AD-2S1 epitope of glycoprotein B (gB) of human cytomegalovirus (HCMV) is dominated by a family of closely related somatically mutated antibodies. These antibodies neutralize viral infectivity and the genes encoding them are derived from two commonly used germ-line variable (V) region genes, IGHV3-30 and IGKV3-11. Recombination of these V genes with the appropriate junctional diversity generates genes that encode primary immunoglobulins that bind to AD-2S1. To further understand the initial primary immunoglobulin response to AD-2S1 we synthesized the germ-line-based ancestor of one such family of antibodies and showed that it bound gB at the AD-2S1 epitope. Here we show that the germ-line ancestor of a second family of antibodies likewise binds to gB. We further show that one of the ancestral primary immunoglobulins, but not the other, also recognized autoantigens. In contrast, the hypermutated derivatives did not demonstrate autoreactivity and minor structural changes in the primary immunoglobulin were sufficient to generate or abolish autoreactivity or to change specificity. Thus, our demonstration that the ancestor of a highly mutated, non-autoreactive antiviral IgG antibody binds nuclear and cell-surface autoantigens indicates for the first time that self-reactivity is not necessarily a barrier to development into a follicular B lymphocyte that undergoes antigen-initiated affinity maturation.

  3. Glycosylation of a VH residue of a monoclonal antibody against alpha (1- ---6) dextran increases its affinity for antigen

    PubMed Central

    1988-01-01

    We have observed that antidextran hybridomas with potential N-linked glycosylation sites in VH have higher affinity for polymeric dextran and for isomaltoheptaose than those lacking potential glycosylation sites. In these studies we have used gene transfection and expression techniques to verify that the carbohydrate addition sites in VH were used. The carbohydrate of the VH region was accessible for binding by the lectin Con A. By ELISA analysis it was demonstrated that the aKa of the antibody for dextran was influenced by the presence of carbohydrate in VH, with the aglycosylated antibody having an aKa 15-fold lower than its untreated counterpart. The aKa for antigen of antibodies that contain carbohydrate only in their constant region was unaffected by lack of carbohydrate. Thus, not only the amino acid sequence of the variable region but also its carbohydrate moieties can determine the magnitude of the antigen-antibody interaction. PMID:2459288

  4. Postbooster Antibodies from Humans as Source of Diphtheria Antitoxin

    PubMed Central

    Avila-Alonso, Ana; González-Rivera, Milagros; Tamayo, Eduardo; Eiros, Jose María; Almansa, Raquel

    2016-01-01

    Diphtheria antitoxin for therapeutic use is in limited supply. A potential source might be affinity-purified antibodies originally derived from plasma of adults who received a booster dose of a vaccine containing diphtheria toxoid. These antibodies might be useful for treating even severe cases of diphtheria. PMID:27314309

  5. The Interplay of Antigen Affinity, Internalization, and Pharmacokinetics on CD44-Positive Tumor Targeting of Monoclonal Antibodies.

    PubMed

    Glatt, Dylan M; Beckford Vera, Denis R; Parrott, Matthew C; Luft, J Christopher; Benhabbour, S Rahima; Mumper, Russell J

    2016-06-01

    Monoclonal antibodies (mAbs) offer promise as effective tumor targeting and drug delivery agents for cancer therapy. However, comparative biological and clinical characteristics of mAbs targeting the same tumor-associated antigen (TAA) often differ widely. This study examined the characteristics of mAbs that impact tumor targeting using a panel of mAb clones specific to the cancer-associated cell-surface receptor and cancer stem cell marker CD44. CD44 mAbs were screened for cell-surface binding, antigen affinity, internalization, and CD44-mediated tumor uptake by CD44-positive A549 cells. It was hypothesized that high-affinity, rapidly internalizing CD44 mAbs would result in high tumor uptake and prolonged tumor retention. Although high-affinity clones rapidly bound and were internalized by A549 cells in vitro, an intermediate-affinity clone demonstrated significantly greater tumor uptake and retention than high-affinity clones in vivo. Systemic exposure, rather than high antigen affinity or rapid internalization, best associated with tumor targeting of CD44 mAbs in A549 tumor-bearing mice. PMID:27079967

  6. Antigen-Antibody Affinity for Dry Eye Biomarkers by Label Free Biosensing. Comparison with the ELISA Technique

    PubMed Central

    Laguna, Maríafe; Holgado, Miguel; Hernandez, Ana L.; Santamaría, Beatriz; Lavín, Alvaro; Soria, Javier; Suarez, Tatiana; Bardina, Carlota; Jara, Mónica; Sanza, Francisco J.; Casquel, Rafael

    2015-01-01

    The specificity and affinity of antibody-antigen interactions is a fundamental way to achieve reliable biosensing responses. Different proteins involved with dry eye dysfunction: ANXA1, ANXA11, CST4, PRDX5, PLAA and S100A6; were validated as biomarkers. In this work several antibodies were tested for ANXA1, ANXA11 and PRDX5 to select the best candidates for each biomarker. The results were obtained by using Biophotonic Sensing Cells (BICELLs) as an efficient methodology for label-free biosensing and compared with the Enzyme-Linked Immuno Sorbent Assay (ELISA) technique. PMID:26287192

  7. Antigen-Antibody Affinity for Dry Eye Biomarkers by Label Free Biosensing. Comparison with the ELISA Technique.

    PubMed

    Laguna, Maríafe; Holgado, Miguel; Hernandez, Ana L; Santamaría, Beatriz; Lavín, Alvaro; Soria, Javier; Suarez, Tatiana; Bardina, Carlota; Jara, Mónica; Sanza, Francisco J; Casquel, Rafael

    2015-08-13

    The specificity and affinity of antibody-antigen interactions is a fundamental way to achieve reliable biosensing responses. Different proteins involved with dry eye dysfunction: ANXA1, ANXA11, CST4, PRDX5, PLAA and S100A6; were validated as biomarkers. In this work several antibodies were tested for ANXA1, ANXA11 and PRDX5 to select the best candidates for each biomarker. The results were obtained by using Biophotonic Sensing Cells (BICELLs) as an efficient methodology for label-free biosensing and compared with the Enzyme-Linked Immuno Sorbent Assay (ELISA) technique.

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

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

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

  9. Purification and characterization of natural antibodies that recognize a human brain lectin.

    PubMed

    Lutomski, D; Caron, M; Bourin, P; Lefebure, C; Bladier, D; Joubert-Caron, R

    1995-03-01

    We have recently identified oligoclonal IgG antibodies that are related to a human brain lectin (HBL14) from serum and cerebrospinal fluid of patients with neurological disorders. They were termed lectin-like IgG (L-IgG) (Joubert-Caron et al., 1994a,b). In this paper, the occurrence of antibodies reactive both towards HBL14 and L-IgG was investigated. Binding of antibodies to HBL14 was demonstrated by solid-phase ELISA and chromatography on immobilized HBL14. Fab fragments of these antibodies were also shown to bind to HBL14. The specificity of the antibodies towards HBL14 was studied using a panel of different antigens. Our data show that individual sera from healthy people as well as a pool of immunoglobulins from 80 blood donors contain an IgG autoreactivity to HBL14, while no IgM autoreactivity was detected. Anti-HBL14 antibodies from sera were purified using affinity chromatography on immobilized HBL14. Affinity chromatography further allowed us to demonstrate that the binding of anti-HB14 antibodies was mediated through their Fab fragments. A higher amount of anti-HBL14 antibodies was purified using a L-IgG-depleted fraction of sera. The binding of anti-HBL14 antibodies to L-IgG was confirmed by ELISA. Finally, anti-HBL14 antibodies were found to be polyreactive. These results indicate the occurrence of a novel class of natural antibodies reactive towards a human brain lectin and suggest that these antibodies may participate in immunoregulatory mechanisms probably though idiotypic/anti-idiotypic interaction.

  10. Human antibody Fc deamidation in vivo.

    PubMed

    Liu, Y Diana; van Enk, Jian Zhang; Flynn, Gregory C

    2009-10-01

    Protein and peptide deamidation occurs spontaneously in vitro under relatively mild conditions. For antibodies and other therapeutic proteins, great effort is placed in manufacturing and storage to minimize this form of degradation. Concern has been especially great in cases where deamidation has been shown to impact protein activity. Here we monitored asparagine deamidation from a recombinant human antibody in humans and found that among the conserved sites, only Asn 384 deamidated at an appreciable rate. Under physiological temperature and pH conditions, in vitro antibody deamidation followed similar kinetics, indicating that simple incubation reactions may be used to model in vivo behavior. Endogenous IgG isolated from human serum possessed 23% deamidation at this site, further demonstrating that this modification is naturally occurring. Thus, deamidation generated in manufacturing and storage does not fully determine the patient exposure to the attribute. Instead, pharmacokinetic data along with the deamidation kinetics are combined to predict patient exposure. The deamidation rate can also be used to estimate the serum lifetime of antibodies. This approach could potentially be used to estimate turnover for other cellular or extracellular proteins.

  11. Ebolavirus Nucleoprotein C-Termini Potently Attract Single Domain Antibodies Enabling Monoclonal Affinity Reagent Sandwich Assay (MARSA) Formulation

    PubMed Central

    Sherwood, Laura J.; Hayhurst, Andrew

    2013-01-01

    Background Antigen detection assays can play an important part in environmental surveillance and diagnostics for emerging threats. We are interested in accelerating assay formulation; targeting the agents themselves to bypass requirements for a priori genome information or surrogates. Previously, using in vitro affinity reagent selection on Marburg virus we rapidly established monoclonal affinity reagent sandwich assay (MARSA) where one recombinant antibody clone was both captor and tracer for polyvalent nucleoprotein (NP). Hypothesizing that the closely related Ebolavirus genus may share the same Achilles' heel, we redirected the scheme to see whether similar assays could be delivered and began to explore their mechanism. Methods and Findings In parallel we selected panels of llama single domain antibodies (sdAb) from a semi-synthetic library against Zaire, Sudan, Ivory Coast, and Reston Ebola viruses. Each could perform as both captor and tracer in the same antigen sandwich capture assay thereby forming MARSAs. All sdAb were specific for NP and those tested required the C-terminal domain for recognition. Several clones were cross-reactive, indicating epitope conservation across the Ebolavirus genus. Analysis of two immune shark sdAb revealed they also targeted the C-terminal domain, and could be similarly employed, yet were less sensitive than a comparable llama sdAb despite stemming from immune selections. Conclusions The C-terminal domain of Ebolavirus NP is a strong attractant for antibodies and enables sensitive sandwich immunoassays to be rapidly generated using a single antibody clone. The polyvalent nature of nucleocapsid borne NP and display of the C-terminal region likely serves as a bountiful affinity sink during selections, and a highly avid target for subsequent immunoassay capture. Combined with the high degree of amino acid conservation through 37 years and across wide geographies, this domain makes an ideal handle for monoclonal affinity reagent

  12. Direct binding of radioiodinated monoclonal antibody to tumor cells: significance of antibody purity and affinity for drug targeting or tumor imaging

    SciTech Connect

    Kennel, S.J.; Foote, L.J.; Lankford, P.K.; Johnson, M.; Mitchell, T.; Braslawsky, G.R.

    1983-01-01

    For MoAb to be used efficiently for drug targeting and tumor imaging, the fraction of antibody binding to tumor cells must be maximized. The authors have studied the binding of /sup 125/I MoAb in three different tumor systems. The fraction of antibody that could be bound to the cell surface was directly proportional to the antibody purity. The affinity constant also limits the fraction of antibody that can bind to cells at a given antigen concentration. Rearrangement of the standard expression for univalent equilibrium binding between two reactants shows that in antigen excess, the maximum fraction of antibody that can bind =Ka(Ag total)/1 + Ka(Ag total). Binding data using four different MoAb with three cell systems confirm this relationship. Estimates for reasonable concentrations of tumor antigens in vivo indicate that antibodies with binding constants less than 10/sup 8/ M/sup -1/ are not likely to be useful for drug targeting or tumor imaging.

  13. Preferential germline usage and VH/VL pairing observed in human antibodies selected by mRNA display.

    PubMed

    Chen, Lei; Kutskova, Yuliya A; Hong, Feng; Memmott, John E; Zhong, Suju; Jenkinson, Megan D; Hsieh, Chung-Ming

    2015-10-01

    Since the invention of phage display, in vitro antibody display technologies have revolutionized the field of antibody discovery. In combination with antibody libraries constructed with sequences of human origin, such technologies enable accelerated therapeutic antibody discovery while bypassing the laborious animal immunization and hybridoma generation processes. Many in vitro display technologies developed since aim to differentiate from phage display by displaying full-length IgG proteins, utilizing eukaryotic translation system and codons, increasing library size or real-time kinetic selection by fluorescent activated cell sorting. We report here the development of an mRNA display technology and an accompanying HCDR3 size spectratyping monitor for human antibody discovery. Importantly, the mRNA display technology maintains a monovalent linkage between the mRNA (genotype) and display binding protein (phenotype), which minimizes avidity effect common in other display systems and allows for a stringent affinity and off-rate selection. The mRNA display technology successfully identified 100 human antibodies in 15 different selections against various targets from naïve human antibody libraries. These antibodies in general have high affinity and diversity. By analyzing the germline usage and combination of antibodies selected by the mRNA display technology, we identified trends and determined the productivity of each germline subgroup in the libraries that could serve as the knowledge base for constructing fully synthetic, next generation antibody libraries.

  14. Antibody diffusion in human cervical mucus.

    PubMed Central

    Saltzman, W M; Radomsky, M L; Whaley, K J; Cone, R A

    1994-01-01

    The mucosal immune system actively transports large quantities of antibodies into all mucus secretions, and these secreted antibodies help prevent infectious entry of many pathogens. Mucus is generally thought to protect epithelial cells by forming a diffusional barrier through which only small molecules can pass. However, electron microscopy indicates that the pore size in mucus is approximately 100 nm, which suggests that antibodies as well as other large molecules might also diffuse through mucus. We measured the diffusion coefficients for antibodies and other proteins within human midcycle cervical mucus using two techniques: fluorescence imaging of concentration profiles and fluorescence photobleaching recovery. The two techniques are complementary, since the rates of diffusion are observed over millimeter distances with fluorescence imaging of concentration profiles and micron distances with fluorescence photobleaching recovery. Both methods yielded essentially the same diffusion coefficients. In contrast to previous reports indicating mucus significantly impedes diffusion of small molecules, antibody diffusion in mucus was relatively unimpeded. In our observations IgG, IgG fragments, IgA, and IgM diffused almost as rapidly in cervical mucus as in water (1.0 > Dmucus/Dwater > 0.7). Simple models for diffusion through water-filled pores suggest that the hydrodynamic pore size for cervical mucus is approximately 100 nm, smaller than the approximately 1000 nm pore size of a collagen gel (at 1 mg/ml) and larger than the approximately 10 nm pore size of gelatin (at 100 mg/ml). This estimated pore size is consistent both with electron micrographs and geometric models of interfiber spacing. Based on these results, we predict that particles as large as viruses can diffuse rapidly through human midcycle cervical mucus, provided the particle forms no adhesive interactions with mucus glycoproteins. Images FIGURE 4 PMID:8161703

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  17. Characterization of golimumab, a human monoclonal antibody specific for human tumor necrosis factor α.

    PubMed

    Shealy, David J; Cai, Ann; Staquet, Kim; Baker, Audrey; Lacy, Eilyn R; Johns, Laura; Vafa, Omid; Gunn, George; Tam, Susan; Sague, Sarah; Wang, Dana; Brigham-Burke, Mike; Dalmonte, Paul; Emmell, Eva; Pikounis, Bill; Bugelski, Peter J; Zhou, Honghui; Scallon, Bernard J; Giles-Komar, Jill

    2010-01-01

    We prepared and characterized golimumab (CNTO148), a human IgG1 tumor necrosis factor alpha (TNFα) antagonist monoclonal antibody chosen for clinical development based on its molecular properties. Golimumab was compared with infliximab, adalimumab and etanercept for affinity and in vitro TNFα neutralization. The affinity of golimumab for soluble human TNFα, as determined by surface plasmon resonance, was similar to that of etanercept (18 pM versus 11 pM), greater than that of infliximab (44 pM) and significantly greater than that of adalimumab (127 pM, p=0.018).  The concentration of golimumab necessary to neutralize TNFα-induced E-selectin expression on human endothelial cells by 50% was significantly less than those for infliximab (3.2 fold; p=0.017) and adalimumab (3.3-fold; p=0.008) and comparable to that for etanercept. The conformational stability of golimumab was greater than that of infliximab (primary melting temperature [Tm] 74.8 °C vs. 69.5 °C) as assessed by differential scanning calorimetry.  In addition, golimumab showed minimal aggregation over the intended shelf life when formulated as a high concentration liquid product (100 mg/mL) for subcutaneous administration.  In vivo, golimumab at doses of 1 and 10 mg/kg significantly delayed disease progression in a mouse model of human TNFα-induced arthritis when compared with untreated mice, while infliximab was effective only at 10 mg/kg. Golimumab also significantly reduced histological scores for arthritis severity and cartilage damage, as well as serum levels of pro-inflammatory cytokines and chemokines associated with arthritis. Thus, we have demonstrated that golimumab is a highly stable human monoclonal antibody with high affinity and capacity to neutralize human TNFα in vitro and in vivo.

  18. Importance of Hypervariable Region 2 for Stability and Affinity of a Shark Single-Domain Antibody Specific for Ebola Virus Nucleoprotein

    PubMed Central

    Anderson, George P.; Teichler, Daniel D.; Zabetakis, Dan; Shriver-Lake, Lisa C.; Liu, Jinny L.; Lonsdale, Stephen G.; Goodchild, Sarah A.; Goldman, Ellen R.

    2016-01-01

    Single-domain antibodies derived from the unique New Antigen Receptor found in sharks have numerous potential applications, ranging from diagnostic reagents to therapeutics. Shark-derived single-domain antibodies possess the same characteristic ability to refold after heat denaturation found in single-domain antibodies derived from camelid heavy-chain-only antibodies. Recently, two shark derived single-domain antibodies specific for the nucleoprotein of Ebola virus were described. Our evaluation confirmed their high affinity for the nucleoprotein, but found their melting temperatures to be low relative to most single-domain antibodies. Our first approach towards improving their stability was grafting antigen-binding regions (complementarity determining regions) of one of these single-domain antibodies onto a high melting temperature shark single-domain antibody. This resulted in two variants: one that displayed excellent affinity with a low melting temperature, while the other had poor affinity but a higher melting temperature. These new proteins, however, differed in only 3 amino acids within the complementarity determining region 2 sequence. In shark single-domain antibodies, the complementarity determining region 2 is often referred to as hypervariable region 2, as this segment of the antibody domain is truncated compared to the sequence in camelid single-domain antibodies and conventional heavy chain variable domains. To elucidate which of the three amino acids or combinations thereof were responsible for the affinity and stability we made the 6 double and single point mutants that covered the intermediates between these two clones. We found a single amino acid change that achieved a 10°C higher melting temperature while maintaining sub nM affinity. This research gives insights into the impact of the shark sdAb hypervariable 2 region on both stability and affinity. PMID:27494523

  19. Importance of Hypervariable Region 2 for Stability and Affinity of a Shark Single-Domain Antibody Specific for Ebola Virus Nucleoprotein.

    PubMed

    Anderson, George P; Teichler, Daniel D; Zabetakis, Dan; Shriver-Lake, Lisa C; Liu, Jinny L; Lonsdale, Stephen G; Goodchild, Sarah A; Goldman, Ellen R

    2016-01-01

    Single-domain antibodies derived from the unique New Antigen Receptor found in sharks have numerous potential applications, ranging from diagnostic reagents to therapeutics. Shark-derived single-domain antibodies possess the same characteristic ability to refold after heat denaturation found in single-domain antibodies derived from camelid heavy-chain-only antibodies. Recently, two shark derived single-domain antibodies specific for the nucleoprotein of Ebola virus were described. Our evaluation confirmed their high affinity for the nucleoprotein, but found their melting temperatures to be low relative to most single-domain antibodies. Our first approach towards improving their stability was grafting antigen-binding regions (complementarity determining regions) of one of these single-domain antibodies onto a high melting temperature shark single-domain antibody. This resulted in two variants: one that displayed excellent affinity with a low melting temperature, while the other had poor affinity but a higher melting temperature. These new proteins, however, differed in only 3 amino acids within the complementarity determining region 2 sequence. In shark single-domain antibodies, the complementarity determining region 2 is often referred to as hypervariable region 2, as this segment of the antibody domain is truncated compared to the sequence in camelid single-domain antibodies and conventional heavy chain variable domains. To elucidate which of the three amino acids or combinations thereof were responsible for the affinity and stability we made the 6 double and single point mutants that covered the intermediates between these two clones. We found a single amino acid change that achieved a 10°C higher melting temperature while maintaining sub nM affinity. This research gives insights into the impact of the shark sdAb hypervariable 2 region on both stability and affinity. PMID:27494523

  20. High affinity nanobodies against human epidermal growth factor receptor selected on cells by E. coli display

    PubMed Central

    Salema, Valencio; Mañas, Carmen; Cerdán, Lidia; Piñero-Lambea, Carlos; Marín, Elvira; Roovers, Rob C.; Van Bergen en Henegouwen, Paul M.P.; Fernández, Luis Ángel

    2016-01-01

    ABSTRACT Most therapeutic antibodies (Abs) target cell surface proteins on tumor and immune cells. Cloning of Ab gene libraries in E. coli and their display on bacteriophages is commonly used to select novel therapeutic Abs binding target antigens, either purified or expressed on cells. However, the sticky nature of bacteriophages renders phage display selections on cells challenging. We previously reported an E. coli display system for expression of VHHs (i.e., nanobodies, Nbs) on the surface of bacteria and selection of high-affinity clones by magnetic cell sorting (MACS). Here, we demonstrate that E. coli display is also an attractive method for isolation of Nbs against cell surface antigens, such as the epidermal growth factor receptor (EGFR), upon direct selection and screening of Ab libraries on live cells. We employ a whole cell-based strategy using a VHH library obtained by immunization with human tumor cells over-expressing EGFR (i.e., A431), and selection of bacterial clones bound to murine fibroblast NIH-3T3 cells transfected with human EGFR, after depletion of non-specific clones on untransfected cells. This strategy resulted in the isolation of high-affinity Nbs binding distinct epitopes of EGFR, including Nbs competing with the ligand, EGF, as characterized by flow cytometry of bacteria displaying the Nbs and binding assays with purified Nbs using surface plasmon resonance. Hence, our study demonstrates that E. coli display of VHH libraries and selection on cells enables efficient isolation and characterization of high-affinity Nbs against cell surface antigens. PMID:27472381

  1. Development of monoclonal antibodies against parathyroid hormone: genetic control of the immune response to human PTH

    SciTech Connect

    Nussbaum, S.R.; Lin, C.S.; Potts, J.T. Jr.; Rosenthal, A.S.; Rosenblatt, M.

    1985-01-01

    Seventeen monocloanl antibodies against the aminoterminal portion of parathyroid hormone (PTH) were generated by using BALB/c mouse for immunization fully biologically active synthetic human PTH-(1-34) and bovine PTH-(1-84) as immunogens, monoclonal antibody methods, and a solid-phase screening assay. Isotypic analysis of these monoclonal antibodies was performed using affinity purified goat antimouse immunoglobulins specific for IgG heavy chains and ..mu..(IgM). All antibodies were IgM as evidenced by 40 times greater than background activity when 25,000 cpm of /sup 125/I-labelled goat anti-mouse IgM was used as second antibody in a radioimmunoassay.

  2. Oriented covalent immobilization of antibodies onto heterofunctional agarose supports: a highly efficient immuno-affinity chromatography platform.

    PubMed

    Batalla, Pilar; Bolívar, Juan M; Lopez-Gallego, Fernando; Guisan, Jose M

    2012-11-01

    The development of new bioconjugates formed by one antibody optimally bound (through its Fc region) to fairly inert solid surfaces is of primary relevance in immuno-affinity chromatography. Immunoglobulins G (IgG) have a Fc region very rich in histidine (His) residues. In this way, immobilization of IgGs on heterofunctional metal chelate-glyoxyl supports (Ag-Me(2+)/G) takes place in two steps: firstly the antibodies are conjugated to the support via His-metal coordination bonds. Secondly, their incubation under alkaline condition promotes an intramolecular covalent attachment between lysine residues at the Fc region and glyoxyl groups on the support surface. The IgG that recognizes as antigen the HRP (antiHRP-IgG) has been conjugated to Ag-Me(2+)/G supports. The resulting bioconjugate is highly inert and able to specifically bind the antigen (HRP) without significant unspecific binding of any other proteins, resulting in an excellent HRP purification platform. The binding activity of this bioconjugate has been optimized by controlling the antibody distribution throughout the bead's surface in order to avoid high antibody densities that led to a low binding activity of the antibodies. The optimal antibody distribution has been achieved when these proteins were slowly immobilized on Ag-Cu(2+)/G in presence of imidazole. This bioconjugate was able to bind up to 1.5 moles of antigen per mole of antibody, only 1.3-fold less than the antibody in solution. Hence, we have been able to develop an optimal protocol to prepare bioconjugated composites in an oriented and irreversible fashion which results in highly efficient and specific surfaces for the exclusive biological recognition. PMID:23021645

  3. Characterization of novel neutralizing monoclonal antibodies specific to human neurturin.

    PubMed

    Hongo, J A; Tsai, S P; Moffat, B; Schroeder, K A; Jung, C; Chuntharapai, A; Lampe, P A; Johnson, E M; de Sauvage, F J; Armanini, M; Phillips, H; Devaux, B

    2000-08-01

    Neurturin (NTN) a structural and functional relative of glial cell line-derived neurotrophic factor, was originally identified based on its ability to support the survival of sympathetic neurons in culture. Similar to glial cell line-derived neurotrophic factor (GDNF), Neurturin has been shown to bind to a high affinity glycosylphosphatidylinositol (GPI)-linked receptor (GFRalpha2) and induce phosphorylation of the tyrosine kinase receptor Ret, resulting in the activation of the mitogen activated protein kinase (MAPK) signalling pathway. A panel of six novel murine monoclonal antibodies (MAbs) specific to human Neurturin has been developed and characterized. Four of the MAbs tested inhibit, to varying degrees, binding of NTN to the GPI-linked GFRalpha2 receptor. Three MAbs cross-react with the murine homolog. These antibodies have been shown to be useful reagents for Western blotting, immunohistochemistry, and also for the development of a sensitive, quantitative enzyme-linked immunosorbent assay (ELISA) for human NTN. Novel, specific MAbs with varying epitope specificities and blocking activity will be valuable tools for both the in vitro and in vivo characterization of NTN and its relationship to the GFRalpha2 and Ret receptors.

  4. Characterization of novel neutralizing monoclonal antibodies specific to human neurturin.

    PubMed

    Hongo, J A; Tsai, S P; Moffat, B; Schroeder, K A; Jung, C; Chuntharapai, A; Lampe, P A; Johnson, E M; de Sauvage, F J; Armanini, M; Phillips, H; Devaux, B

    2000-08-01

    Neurturin (NTN) a structural and functional relative of glial cell line-derived neurotrophic factor, was originally identified based on its ability to support the survival of sympathetic neurons in culture. Similar to glial cell line-derived neurotrophic factor (GDNF), Neurturin has been shown to bind to a high affinity glycosylphosphatidylinositol (GPI)-linked receptor (GFRalpha2) and induce phosphorylation of the tyrosine kinase receptor Ret, resulting in the activation of the mitogen activated protein kinase (MAPK) signalling pathway. A panel of six novel murine monoclonal antibodies (MAbs) specific to human Neurturin has been developed and characterized. Four of the MAbs tested inhibit, to varying degrees, binding of NTN to the GPI-linked GFRalpha2 receptor. Three MAbs cross-react with the murine homolog. These antibodies have been shown to be useful reagents for Western blotting, immunohistochemistry, and also for the development of a sensitive, quantitative enzyme-linked immunosorbent assay (ELISA) for human NTN. Novel, specific MAbs with varying epitope specificities and blocking activity will be valuable tools for both the in vitro and in vivo characterization of NTN and its relationship to the GFRalpha2 and Ret receptors. PMID:11001403

  5. A likelihood-based index of protein protein binding affinities with application to influenza HA escape from antibodies.

    PubMed

    Watabe, Teruaki; Kishino, Hirohisa; de Oliveira Martins, Leonardo; Kitazoe, Yasuhiro

    2007-08-01

    In many biological systems, proteins interact with other organic molecules to produce indispensable functions, in which molecular recognition phenomena are essential. Proteins have kept or gained their functions during molecular evolution. Their functions seem to be flexible, and a few amino acid substitutions sometimes cause drastic changes in function. In order to monitor and predict such drastic changes in the early stages in target populations, we need to identify patterns of structural changes during molecular evolution causing decreases or increases in the binding affinity of protein complexes. In previous work, we developed a likelihood-based index to quantify the degree to which a sequence fits a given structure. This index was named the sequence-structure fitness (SSF) and is calculated empirically based on amino acid preferences and pairwise interactions in the structural environment present in template structures. In the present work, we used the SSF to develop an index to measure the binding affinity of protein-protein complexes defined as the log likelihood ratio, contrasting the fitness of the sequences to the structure of the complex and that of the uncomplexed proteins. We applied the developed index to the complexes formed between influenza A hemagglutinin (HA) and four antibodies. The antibody-antigen binding region of HA is under strong selection pressure by the host immune system. Hence, examination of the long-term adaptation of HA to the four antibodies could reveal the strategy of the molecular evolution of HA. Two antibodies cover the HA receptor-binding region, while the other two bind away from the receptor-binding region. By focusing on branches with a significant decline in binding ability, we could detect key amino acid replacements and investigate the mechanism via conditional probabilities. The contrast between the adaptations to the two types of antibodies suggests that the virus adapts to the immune system at the cost of structural

  6. Surface antigens on cat leukemic cells induced by feline leukemia virus: area density and antibody-binding affinity.

    PubMed

    Boone, C W; Gordin, F; Kawakami, T G

    1973-04-01

    The binding of autologous bovine antibody to feline leukemia virus-induced cell surface antigens (FeCSA) on cat leukemia cells was studied by performing certain titration procedures with a mixture of immune and normal sera labeled with different iodine radioisotopes (paired-label technique). By using plots of titration data which conformed to linear equations derived from the mass action law, we determined the following constants. (i) The density of FeCSA was 2.03 x 10(6) sites per cell, or 5,230 sites per mum(2). (ii) The equilibrium constant of the FeCSA-antibody reaction was 2.67 x 10(7), from which the antibody binding affinity or standard free energy of the FeCSA-antibody bond was determined to be - 10.48 kcal (-43,869.28 J) per mol. The use of the techniques described to measure the concentration of antibody in antiserum, in micrograms per milliliter, is discussed.

  7. Affinity Maturation of a Potent Family of HIV Antibodies Is Primarily Focused on Accommodating or Avoiding Glycans.

    PubMed

    Garces, Fernando; Lee, Jeong Hyun; de Val, Natalia; de la Pena, Alba Torrents; Kong, Leopold; Puchades, Cristina; Hua, Yuanzi; Stanfield, Robyn L; Burton, Dennis R; Moore, John P; Sanders, Rogier W; Ward, Andrew B; Wilson, Ian A

    2015-12-15

    The high-mannose patch on the HIV-1 envelope (Env) glycoprotein is the epicenter for binding of the potent broadly neutralizing PGT121 family of antibodies, but strategies for generating such antibodies by vaccination have not been defined. We generated structures of inferred antibody intermediates by X-ray crystallography and electron microscopy to elucidate the molecular events that occurred during evolution of this family. Binding analyses revealed that affinity maturation was primarily focused on avoiding, accommodating, or binding the N137 glycan. The overall antibody approach angle to Env was defined very early in the maturation process, yet some variation evolved in the PGT121 family branches that led to differences in glycan specificities in their respective epitopes. Furthermore, we determined a crystal structure of the recombinant BG505 SOSIP.664 HIV-1 trimer with a PGT121 family member at 3.0 Å that, in concert with these antibody intermediate structures, provides insights to advance design of HIV vaccine candidates. PMID:26682982

  8. Monoclonal antibodies to human hemoglobin S and cell lines for the production thereof

    DOEpatents

    Jensen, R.H.; Vanderlaan, M.; Bigbee, W.L.; Stanker, L.H.; Branscomb, E.W.; Grabske, R.J.

    1984-11-29

    The present invention provides monoclonal antibodies specific to and distinguishing between hemoglobin S and hemoglobin A and methods for their production and use. These antibodies are capable of distinguishing between two hemoglobin types which differ from each other by only a single amino acid residue. The antibodies produced according to the present method are useful as immunofluorescent markers to enumerate circulating red blood cells which have the property of altered expression of the hemoglobin gene due to somatic mutation in stem cells. Such a measurement is contemplated as an assay for in vivo cellular somatic mutations in humans. Since the monoclonal antibodies produced in accordance with the instant invention exhibit a high degree of specificity to and greater affinity for hemoglobin S, they are suitable for labeling human red blood cells for flow cytometric detection of hemoglobin genotype. 4 figs.

  9. Monoclonal antibodies to human hemoglobin S and cell lines for the production thereof

    DOEpatents

    Jensen, Ronald H.; Vanderlaan, Martin; Bigbee, William L.; Stanker, Larry H.; Branscomb, Elbert W.; Grabske, Robert J.

    1988-01-01

    The present invention provides monoclonal antibodies specific to and distinguish between hemoglobin S and hemoglobin A and methods for their production and use. These antibodies are capable of distinguishing between two hemoglobin types which differ from each other by only a single amino acid residue. The antibodies produced according to the present method are useful as immunofluorescent markers to enumerate circulating red blood cells which have the property of altered expression of the hemoglobin gene due to somatic mutation in stem cells. Such a measurement is contemplated as an assay for in vivo cellular somatic mutations in humans. Since the monoclonal antibodies produced in accordance with the instant invention exhibit a high degree of specificity to and greater affinity for hemoglobin S, they are suitable for labeling human red blood cells for flow cytometric detection of hemoglobin genotype.

  10. Competitive Selection from Single Domain Antibody Libraries Allows Isolation of High-Affinity Antihapten Antibodies That Are Not Favored in the llama Immune Response

    PubMed Central

    Rosa, Sofia Tabares-da; Rossotti, Martin; Carleiza, Carmen; Carrión, Federico; Pritsch, Otto; Ahn, Ki Chang; Last, Jerold A.; Hammock, Bruce D; González-Sapienza, Gualberto

    2011-01-01

    Single-domain antibodies (sdAbs) found in camelids, lack a light chain and their antigen-binding site sits completely in the heavy-chain variable domain (VHH). Their simplicity, thermostability, and ease in expression have made VHHs highly attractive. While this has been successfully exploited for macromolecular antigens, their application to the detection of small molecules is still limited to a very few reports, mostly describing low affinity VHHs. Using triclocarban (TCC) as a model hapten, we found that conventional antibodies, IgG1 fraction, reacted with free TCC with a higher relative affinity (IC50 51.0 ng/mL) than did the sdAbs (IgG2 and IgG3, 497 and 370 ng/mL, respectively). A VHH library was prepared, and by elution of phage with limiting concentrations of TCC and competitive selection of binders, we were able to isolate high-affinity clones, KD 0.98–1.37 nM (SPR) which allowed development of a competitive assay for TCC with an IC50 = 3.5 ng/mL (11 nM). This represents a 100-fold improvement with regard to the performance of the sdAb serum fraction, and it is 100-fold better than the IC50 attained with other anti-hapten VHHs reported thus far. Despite the modest overall anti-hapten sdAbs response in llamas, a small subpopulation of high affinity VHHs are generated that can be isolated by carefully design of the selection process. PMID:21827167

  11. Human Monoclonal Antibodies to Pf 155, a Major Antigen of Malaria Parasite Plasmodium falciparum

    NASA Astrophysics Data System (ADS)

    Udomsangpetch, Rachanee; Lundgren, Katarina; Berzins, Klavs; Wahlin, Birgitta; Perlmann, Hedvig; Troye-Blomberg, Marita; Carlsson, Jan; Wahlgren, Mats; Perlmann, Peter; Bjorkman, Anders

    1986-01-01

    Pf 155, a protein of the human malaria parasite Plasmodium falciparum, is strongly immunogenic in humans and is believed to be a prime candidate for the preparation of a vaccine. Human monoclonal antibodies to Pf 155 were obtained by cloning B cells that had been prepared from an immune donor and transformed with Epstein-Barr virus. When examined by indirect immunofluorescence, these antibodies stained the surface of infected erythrocytes, free merozoites, segmented schizonts, and gametocytes. They bound to a major polypeptide with a relative molecular weight of 155K and to two minor ones (135K and 120K), all having high affinity for human glycophorin. The antibodies strongly inhibited merozoite reinvasion in vitro, suggesting that they might be appropriate reagents for therapeutic administration in vivo.

  12. Variation in antigen-antibody affinity among serotypes of Salmonella O4 serogroup, determined using specific antisera.

    PubMed

    Aribam, Swarmistha Devi; Elsheimer-Matulova, Marta; Matsui, Hidenori; Hirota, Jiro; Shiraiwa, Kazumasa; Ogawa, Yohsuke; Hikono, Hirokazu; Shimoji, Yoshihiro; Eguchi, Masahiro

    2015-11-01

    Serotyping is widely used for typing Salmonella during surveillance, and depends on determining the lipopolysaccharide (LPS) O-antigen and the flagellar protein (H-antigens) components. As the O-antigen is highly variable, and structurally unique to each serotype, we investigated the binding affinities of LPS from Salmonella serotypes of O4 serogroup with specific anti-antigen serum via immunoblot and enzyme-linked immunosorbent assays. Since the serotypes from O4 serogroup also express the O-antigen factor 12, O12 antiserum was also used for the analysis. LPS from the different serotypes showed different binding affinities with the antisera. Therefore, based on the antigen-antibody affinity, a modified agglutination assay was carried out by using O4 and O12 antisera. Although serotypes from O4 serogroup have the common O-antigen factors 4 and 12, the analysis showed that the degree of agglutination reaction is different for each of the serotypes. We suggest that Salmonella serogroup O4 serotypes exhibit different binding affinities with specific antisera despite the presence of common O-antigen factors 4 and 12.

  13. Precolumn affinity capillary electrophoresis for the identification of clinically relevant proteins in human serum: application to human cardiac troponin I.

    PubMed

    Dalluge, J J; Sander, L C

    1998-12-15

    An approach has been developed to the on-line extraction and identification of clinical disease-state marker proteins in human serum. Fabrication of capillaries with integral packed beds for the online determination of human cardiac troponin I (cTnI), a diagnostic marker for myocardial infarction, at clinically relevant levels (2 nmol/L) in serum is demonstrated. The technique, termed precolumn affinity capillary electrophoresis (PA-CE), utilizes a short (approximately 5 mm) packed bed of porous silica containing covalently immobilized monoclonal anti-cTnI antibodies directly integrated within a separation capillary for the selective retention of cTnI from a complex matrix. Following a rinsing step to eliminate nonspecifically bound serum proteins and other impurities from the column, desorption of the antigen into the separation region of the PA-CE capillary for subsequent measurement of femto-molar amounts of cTnI by CE is effected by the injection of an appropriate elution buffer. Advantages of this approach over previously reported affinity preconcentration techniques, related applications for PA-CE technology, and its potential for use in the development of a certified reference material for cTnI in serum are discussed. PMID:9868922

  14. VH-VL orientation prediction for antibody humanization candidate selection: A case study.

    PubMed

    Bujotzek, Alexander; Lipsmeier, Florian; Harris, Seth F; Benz, Jörg; Kuglstatter, Andreas; Georges, Guy

    2016-01-01

    Antibody humanization describes the procedure of grafting a non-human antibody's complementarity-determining regions, i.e., the variable loop regions that mediate specific interactions with the antigen, onto a β-sheet framework that is representative of the human variable region germline repertoire, thus reducing the number of potentially antigenic epitopes that might trigger an anti-antibody response. The selection criterion for the so-called acceptor frameworks (one for the heavy and one for the light chain variable region) is traditionally based on sequence similarity. Here, we propose a novel approach that selects acceptor frameworks such that the relative orientation of the 2 variable domains in 3D space, and thereby the geometry of the antigen-binding site, is conserved throughout the process of humanization. The methodology relies on a machine learning-based predictor of antibody variable domain orientation that has recently been shown to improve the quality of antibody homology models. Using data from 3 humanization campaigns, we demonstrate that preselecting humanization variants based on the predicted difference in variable domain orientation with regard to the original antibody leads to subsets of variants with a significant improvement in binding affinity.

  15. Characterization of specific high affinity receptors for human tumor necrosis factor on mouse fibroblasts

    SciTech Connect

    Hass, P.E.; Hotchkiss, A.; Mohler, M.; Aggarwal, B.B.

    1985-10-05

    Mouse L-929 fibroblasts, an established line of cells, are very sensitive to lysis by human lymphotoxin (hTNF-beta). Specific binding of a highly purified preparation of hTNF-beta to these cells was examined. Recombinant DNA-derived hTNF-beta was radiolabeled with (TH)propionyl succinimidate at the lysine residues of the molecule to a specific activity of 200 microCi/nmol of protein. (TH)hTNF-beta was purified by high performance gel permeation chromatography and the major fraction was found to be monomeric by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The labeled hTNF-beta was fully active in causing lysis of L-929 fibroblasts and bound specifically to high affinity binding sites on these cells. Scatchard analysis of the binding data revealed the presence of a single class of high affinity receptors with an apparent Kd of 6.7 X 10(-11) M and a capacity of 3200 binding sites/cell. Unlabeled recombinant DNA-derived hTNF-beta was found to be approximately 5-fold more effective competitive inhibitor of binding than the natural hTNF-beta. The binding of hTNF-beta to these mouse fibroblasts was also correlated with the ultimate cell lysis. Neutralizing polyclonal antibodies to hTNF-beta efficiently inhibited the binding of (TH)hTNF-beta to the cells. The authors conclude that the specific high affinity binding site is the receptor for hTNF-beta and may be involved in lysis of cells.

  16. Analysis of Aged Human Serum Albumin Affinity for Doxazosin.

    PubMed

    Chudzik, Mariola; Równicka-Zubik, Joanna; Pożycka, Jadwiga; Pawelczak, Bartosz; Sulkowska, Anna

    2016-01-01

    Structural changes of human serum albumin (HSA) caused by old age and coexisting diseases result in differences in the binding of doxazosin (DOX). DOX is a postsynaptic α1- adrenoreceptor antagonist used for treatment of hypertension and benign prostatic hyperplasia. In elderly people suffering from various renal or hepatic diseases the significant portion of N-form of human serum albumin (normal) is converted to A-form (aged). The differences in binding of doxazosin to N- and Aform of albumin are an important factor, which may determines therapeutic dosage and toxicity of the test drug. To indicate these differences, the technique of fluorescence spectroscopy was used. The association constant (Ka) obtained from fluorescence quenching demonstrated that doxazosin has higher affinity for AHSA than for HSA. In order to describe the cooperativity in binding process, the values of the Hill's coefficient has been analysed. For DOX-HSA system (λex 295 nm) Hill's coefficient is close to 1 and it indicates that there is a single class of binding sites. For DOX-HSA (λex 275 nm) and DOX-AHSA (λex 275 nm and λex 295 nm) systems we observed positive cooperativity (nH>1). A greater red shift of fluorescence emission maximum of AHSA than HSA in the presence of DOX was observed. This suggests that the binding of DOX to AHSA was accompanied by a stronger increase in polarity around the fluorophores in comparison to HSA. The binding interaction between DOX and HSA has been also studied by molecular docking simulation.

  17. Framework selection can influence pharmacokinetics of a humanized therapeutic antibody through differences in molecule charge

    PubMed Central

    Li, Bing; Tesar, Devin; Boswell, C Andrew; Cahaya, Hendry S; Wong, Anne; Zhang, Jianhuan; Meng, Y Gloria; Eigenbrot, Charles; Pantua, Homer; Diao, Jinyu; Kapadia, Sharookh B; Deng, Rong; Kelley, Robert F

    2014-01-01

    Pharmacokinetic (PK) testing of a humanized (κI, VH3 framework) and affinity matured anti-hepatitis C virus E2-glycoprotein (HCV-E2) antibody (hu5B3.κ1VH3.v3) in rats revealed unexpected fast clearance (34.9 mL/day/kg). This antibody binds to the rat recycling receptor FcRn as expected for a human IgG1 antibody and does not display non-specific binding to baculovirus particles in an assay that is correlated with fast clearance in cynomolgus monkey. The antigen is not expressed in rat so target-dependent clearance does not contribute to PK. Removal of the affinity maturation changes (hu5B3.κ1VH3.v1) did not restore normal clearance. The antibody was re-humanized on a κ4, VH1 framework and the non-affinity matured version (hu5B3.κ4VH1.v1) was shown to have normal clearance (8.5 mL/day/kg). Since the change in framework results in a lower pI, primarily due to more negative charge on the κ4 template, the effect of additional charge variation on antibody PK was tested by incorporating substitutions obtained through phage display affinity maturation of hu5B3.κ1VH3.v1. A variant having a pI of 8.61 gave very fast clearance (140 mL/day/kg) whereas a molecule with pI of 6.10 gave slow clearance (5.8 mL/kg/day). Both antibodies exhibited comparable binding to rat FcRn, but biodistribution experiments showed that the high pI variant was catabolized in liver and spleen. These results suggest antibody charge can have an effect on PK through alterations in antibody catabolism independent of FcRn-mediated recycling. Furthermore, introduction of affinity maturation changes into the lower pI framework yielded a candidate with PK and virus neutralization properties suitable for clinical development. PMID:25517310

  18. The effects of affinity-purified anti-DNA antibodies from patients with systemic lupus erythematosus on the fluorescent antinuclear antibody assay using HEp-2 cells.

    PubMed

    Suzuki, Kimihiro; Kawamura, Masahide; Mineo, Midori; Shinohara, Tadashi; Kataharada, Koji; Okada, Makoto; Takada, Kunio; Miyawaki, Shoji; Ohsuzu, Fumitaka

    2002-01-01

    The aim of this study was to clarify the effects of anti-dsDNA antibodies on the titer and the nuclear staining pattern(s) in a fluorescent antinuclear antibody (FANA) assay using HEp-2 cells. Anti-dsDNA derived from 14 patients with systemic lupus erythematosus (SLE) was individually affinity-purified. The anti-dsDNA titer of the purified anti-dsDNA solution was measured by radioimmunoassay (RIA) or by enzyme-linked immunosorbent assay (ELISA). In the FANA assay, the anti-dsDNA solution was diluted in a stepwise manner and its titer was expressed by the endpoint dilution. The nuclear staining pattern in the anti-dsDNA solution was examined at the 1:5 and 1:20 dilutions and at the endpoint dilution. The anti-dsDNA titers of the affinity-purified anti-dsDNA solution were high enough (13 to 126 IU/ml) to be measured by RIA. However, the antinuclear antibody (ANA) titers of this solution were relatively low: 1:20 to 1:320. In the study of nuclear staining the peripheral pattern was observed in nine of the 14 cases at a 1:5 dilution. However, at the endpoint dilution, all cases exhibited the homogeneous pattern. These findings indicate that in the FANA assay using HEp-2 cells, 1) although serum samples show high anti-dsDNA titers by RIA or by ELISA, the antibodies' direct contribution to ANA titers is limited, and 2) when samples reveal a homogeneous staining pattern at the endpoint dilution, this suggests the presence of anti-dsDNA.

  19. A novel antibody discovery platform identifies anti-influenza A broadly neutralizing antibodies from human memory B cells.

    PubMed

    Xiao, Xiaodong; Chen, Yan; Varkey, Reena; Kallewaard, Nicole; Koksal, Adem C; Zhu, Qing; Wu, Herren; Chowdhury, Partha S; Dall'Acqua, William F

    2016-07-01

    Monoclonal antibody isolation directly from circulating human B cells is a powerful tool to delineate humoral responses to pathological conditions and discover antibody therapeutics. We have developed a platform aimed at improving the efficiencies of B cell selection and V gene recovery. Here, memory B cells are activated and amplified using Epstein-Barr virus infection, co-cultured with CHO-muCD40L cells, and then assessed by functional screenings. An in vitro transcription and translation (IVTT) approach was used to analyze variable (V) genes recovered from each B cell sample and identify the relevant heavy/light chain pair(s). We achieved efficient amplification and activation of memory B cells, and eliminated the need to: 1) seed B cells at clonal level (≤1 cell/well) or perform limited dilution cloning; 2) immortalize B cells; or 3) assemble V genes into an IgG expression vector to confirm the relevant heavy/light chain pairing. Cross-reactive antibodies targeting a conserved epitope on influenza A hemagglutinin were successfully isolated from a healthy donor. In-depth analysis of the isolated antibodies suggested their potential uses as anti-influenza A antibody therapeutics and uncovered a distinct affinity maturation pathway. Importantly, our results showed that cognate heavy/light chain pairings contributed to both the expression level and binding abilities of our newly isolated VH1-69 family, influenza A neutralizing antibodies, contrasting with previous observations that light chains do not significantly contribute to the function of this group of antibodies. Our results further suggest the potential use of the IVTT as a powerful antibody developability assessment tool. PMID:27049174

  20. Binding affinity of anti-xylitol antibodies to canine hepatic vessels.

    PubMed

    Imai, Akihiro; Nishita, Toshiho; Ichihara, Nobutsune; Shirota, Kinji; Orito, Kensuke

    2012-09-15

    Xylitol is used as a sugar substitute in food products. Dogs have been reported to experience lethal liver injury after accidental ingestion of xylitol. Because liver injury may be a serious consequence of canine immune-mediated reactions, antibodies produced against xylitol may attack the liver. Therefore, in the present study, we evaluated whether binding sites for xylitol antibodies are located at the liver or not. Anti-xylitol antibodies were generated by immunization of rabbits with a xylose-bovine serum albumin conjugate. Immunohistological examination showed that binding sites for the anti-xylitol antibodies were located in the hepatic arteries and the portal veins. Western blotting analyses by using a canine liver homogenate showed 4 protein bands with different molecular weights which reacted with anti-xylitol antibodies. Therefore, binding of anti-xylitol antibodies to the vessels may be the first step in an immune-mediated pathogenic response in xylitol toxicity. Further studies are necessary to determine the effects of anti-xylitol antibodies on the liver in the pathogenesis of xylitol toxicity.

  1. Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration.

    PubMed

    Koenig, Patrick; Lee, Chingwei V; Sanowar, Sarah; Wu, Ping; Stinson, Jeremy; Harris, Seth F; Fuh, Germaine

    2015-09-01

    The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies.

  2. Neutralization of Japanese Encephalitis Virus by heme-induced broadly reactive human monoclonal antibody

    PubMed Central

    Gupta, Nimesh; de Wispelaere, Mélissanne; Lecerf, Maxime; Kalia, Manjula; Scheel, Tobias; Vrati, Sudhanshu; Berek, Claudia; Kaveri, Srinivas V.; Desprès, Philippe; Lacroix-Desmazes, Sébastien; Dimitrov, Jordan D.

    2015-01-01

    Geographical expansion and re-emerging new genotypes of the Japanese encephalitis virus (JEV) require the development of novel therapeutic approaches. Here, we studied a non-conventional approach for antibody therapy and show that, upon exposure to heme, a fraction of natural human immunoglobulins acquires high-affinity reactivity with the antigenic domain-III of JEV E glycoprotein. These JEV-reactive antibodies exhibited neutralizing activity against recently dominant JEV genotypes. This study opens new therapeutic options for Japanese encephalitis. PMID:26542535

  3. Neutralization of Japanese Encephalitis Virus by heme-induced broadly reactive human monoclonal antibody.

    PubMed

    Gupta, Nimesh; de Wispelaere, Mélissanne; Lecerf, Maxime; Kalia, Manjula; Scheel, Tobias; Vrati, Sudhanshu; Berek, Claudia; Kaveri, Srinivas V; Desprès, Philippe; Lacroix-Desmazes, Sébastien; Dimitrov, Jordan D

    2015-01-01

    Geographical expansion and re-emerging new genotypes of the Japanese encephalitis virus (JEV) require the development of novel therapeutic approaches. Here, we studied a non-conventional approach for antibody therapy and show that, upon exposure to heme, a fraction of natural human immunoglobulins acquires high-affinity reactivity with the antigenic domain-III of JEV E glycoprotein. These JEV-reactive antibodies exhibited neutralizing activity against recently dominant JEV genotypes. This study opens new therapeutic options for Japanese encephalitis. PMID:26542535

  4. Serial studies on the affinity and heterogeneity of human autoantibodies to thyroglobulin.

    PubMed Central

    Devey, M E; Bleasdale-Barr, K M; McLachlan, S M; Bradbury, J; Clark, F; Young, E T

    1989-01-01

    The functional affinity and heterogeneity of autoantibodies to thyroglobulin (Tg) were measured by an IgG subclass-specific solid-phase competition ELISA in patients with autoimmune thyroid disease. High-affinity IgG1 and IgG4 antibodies formed the major anti-Tg response. Both titre and affinity of IgG3 and IgG2 anti-Tg were generally low but in some Hashimoto's disease patients high-affinity IgG2 anti-Tg were found and IgG2 anti-Tg, unlike those of other subclasses, showed very restricted heterogeneity. The affinity of IgG4 anti-Tg was similar in patients with thyroid disease and their clinically euthyroid (normal) relatives. In contrast, a progressive increase in IgG1 anti-Tg affinity was seen in clinically euthyroid individuals compared with their relatives with thyroid disease and high titred Hashimoto's disease patients, suggesting that either rising titres of high affinity IgG1 anti-Tg or affinity maturation of IgG1 anti-Tg may be indicative of impending hypothyroidism. PMID:2776357

  5. Human antibody fragments specific for the epidermal growth factor receptor selected from large non-immunised phage display libraries.

    PubMed

    Souriau, Christelle; Rothacker, Julie; Hoogenboom, Hennie R; Nice, Edouard

    2004-09-01

    Antibodies to EGFR have been shown to display anti-tumour effects mediated in part by inhibition of cellular proliferation and angiogenesis, and by enhancement of apoptosis. Humanised antibodies are preferred for clinical use to reduce complications with HAMA and HAHA responses frequently seen with murine and chimaeric antibodies. We have used depletion and subtractive selection strategies on cells expressing the EGFR to sample two large antibody fragment phage display libraries for the presence of human antibodies which are specific for the EGFR. Four Fab fragments and six scFv fragments were identified, with affinities of up to 2.2nM as determined by BIAcore analysis using global fitting of the binding curves to obtain the individual rate constants (ka and kd). This overall approach offers a generic screening method for the identification of growth factor specific antibodies and antibody fragments from large expression libraries and has potential for the rapid development of new therapeutic and diagnostic reagents.

  6. Preclinical evaluation of multistep targeting of diasialoganglioside GD2 using an IgG-scFv bispecific antibody with high affinity for GD2 and DOTA metal complex.

    PubMed

    Cheal, Sarah M; Xu, Hong; Guo, Hong-fen; Zanzonico, Pat B; Larson, Steven M; Cheung, Nai-Kong

    2014-07-01

    Bispecific antibodies (BsAb) have proven to be useful targeting vectors for pretargeted radioimmunotherapy (PRIT). We sought to overcome key PRIT limitations such as high renal radiation exposure and immunogenicity (e.g., of streptavidin-antibody fusions), to advance clinical translation of this PRIT strategy for diasialoganglioside GD2-positive [GD2(+)] tumors. For this purpose, an IgG-scFv BsAb was engineered using the sequences for the anti-GD2 humanized monoclonal antibody hu3F8 and C825, a murine scFv antibody with high affinity for the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) complexed with β-particle-emitting radiometals such as (177)Lu and (90)Y. A three-step regimen, including hu3F8-C825, a dextran-based clearing agent, and p-aminobenzyl-DOTA radiolabeled with (177)Lu (as (177)Lu-DOTA-Bn; t1/2 = 6.71 days), was optimized in immunocompromised mice carrying subcutaneous human GD2(+) neuroblastoma (NB) xenografts. Absorbed doses for tumor and normal tissues were approximately 85 cGy/MBq and ≤3.7 cGy/MBq, respectively, with therapeutic indices (TI) of 142 for blood and 23 for kidney. A therapy study (n = 5/group; tumor volume, 240 ± 160 mm(3)) with three successive PRIT cycles (total (177)Lu: ∼33 MBq; tumor dose ∼3,400 cGy), revealed complete tumor response in 5 of 5 animals, with no recurrence up to 28 days after treatment. Tumor ablation was confirmed histologically in 4 of 5 mice, and normal organs showed minimal overall toxicities. All nontreated mice required sacrifice within 12 days (>1.0-cm(3) tumor volume). We conclude that this novel anti-GD2 PRIT approach has sufficient TI to successfully ablate subcutaneous GD2(+)-NB in mice while sparing kidney and bone marrow.

  7. Fully Human Monoclonal Antibody Inhibitors of the Neonatal Fc Receptor Reduce Circulating IgG in Non-Human Primates

    PubMed Central

    Nixon, Andrew E.; Chen, Jie; Sexton, Daniel J.; Muruganandam, Arumugam; Bitonti, Alan J.; Dumont, Jennifer; Viswanathan, Malini; Martik, Diana; Wassaf, Dina; Mezo, Adam; Wood, Clive R.; Biedenkapp, Joseph C.; TenHoor, Chris

    2015-01-01

    The therapeutic management of antibody-mediated autoimmune disease typically involves immunosuppressant and immunomodulatory strategies. However, perturbing the fundamental role of the neonatal Fc receptor (FcRn) in salvaging IgG from lysosomal degradation provides a novel approach – depleting the body of pathogenic immunoglobulin by preventing IgG binding to FcRn and thereby increasing the rate of IgG catabolism. Herein, we describe the discovery and preclinical evaluation of fully human monoclonal IgG antibody inhibitors of FcRn. Using phage display, we identified several potent inhibitors of human-FcRn in which binding to FcRn is pH-independent, with over 1000-fold higher affinity for human-FcRn than human IgG-Fc at pH 7.4. FcRn antagonism in vivo using a human-FcRn knock-in transgenic mouse model caused enhanced catabolism of exogenously administered human IgG. In non-human primates, we observed reductions in endogenous circulating IgG of >60% with no changes in albumin, IgM, or IgA. FcRn antagonism did not disrupt the ability of non-human primates to mount IgM/IgG primary and secondary immune responses. Interestingly, the therapeutic anti-FcRn antibodies had a short serum half-life but caused a prolonged reduction in IgG levels. This may be explained by the high affinity of the antibodies to FcRn at both acidic and neutral pH. These results provide important preclinical proof of concept data in support of FcRn antagonism as a novel approach to the treatment of antibody-mediated autoimmune diseases. PMID:25954273

  8. Fully human monoclonal antibody inhibitors of the neonatal fc receptor reduce circulating IgG in non-human primates.

    PubMed

    Nixon, Andrew E; Chen, Jie; Sexton, Daniel J; Muruganandam, Arumugam; Bitonti, Alan J; Dumont, Jennifer; Viswanathan, Malini; Martik, Diana; Wassaf, Dina; Mezo, Adam; Wood, Clive R; Biedenkapp, Joseph C; TenHoor, Chris

    2015-01-01

    The therapeutic management of antibody-mediated autoimmune disease typically involves immunosuppressant and immunomodulatory strategies. However, perturbing the fundamental role of the neonatal Fc receptor (FcRn) in salvaging IgG from lysosomal degradation provides a novel approach - depleting the body of pathogenic immunoglobulin by preventing IgG binding to FcRn and thereby increasing the rate of IgG catabolism. Herein, we describe the discovery and preclinical evaluation of fully human monoclonal IgG antibody inhibitors of FcRn. Using phage display, we identified several potent inhibitors of human-FcRn in which binding to FcRn is pH-independent, with over 1000-fold higher affinity for human-FcRn than human IgG-Fc at pH 7.4. FcRn antagonism in vivo using a human-FcRn knock-in transgenic mouse model caused enhanced catabolism of exogenously administered human IgG. In non-human primates, we observed reductions in endogenous circulating IgG of >60% with no changes in albumin, IgM, or IgA. FcRn antagonism did not disrupt the ability of non-human primates to mount IgM/IgG primary and secondary immune responses. Interestingly, the therapeutic anti-FcRn antibodies had a short serum half-life but caused a prolonged reduction in IgG levels. This may be explained by the high affinity of the antibodies to FcRn at both acidic and neutral pH. These results provide important preclinical proof of concept data in support of FcRn antagonism as a novel approach to the treatment of antibody-mediated autoimmune diseases. PMID:25954273

  9. Phase Transitions in Antibody Solutions: from Pharmaceuticals to Human Disease

    NASA Astrophysics Data System (ADS)

    Wang, Ying; Lomakin, Aleksey; Benedek, George; Dana Farber Cancer Institute Collaboration; Amgen Inc. Collaboration

    2014-03-01

    Antibodies are very important proteins. Natural antibodies play essential role in the immune system of human body. Pharmaceutical antibodies are used as drugs. Antibodies are also indispensable tools in biomedical research and diagnostics. Recently, a number of observations of phase transitions of pharmaceutical antibodies have been reported. These phase transitions are undesirable from the perspective of colloid stability of drug solutions in processing and storage, but can be used for protein purification, X-ray crystallography, and improving pharmokinetics of drugs. Phase transitions of antibodies can also take place in human body, particularly in multiple myeloma patients who overproduce monoclonal antibodies. These antibodies, in some cases, crystallize at body temperature and cause severe complications called cryoglobulinemia. I will present the results of our current studies on phase transitions of both pharmaceutical antibodies and cryoglobulinemia-associated antibodies. These studies have shown that different antibodies have different propensity to undergo phase transitions, but their phase behavior has universal features which are remarkably different from those of spherical proteins. I will discuss how studies of phase behavior can be useful in assessing colloid stability of pharmaceutical antibodies and in early diagnostics of cryoglobulinemia, as well as general implications of the fact that some antibodies can precipitate at physiological conditions.

  10. Purification of proteins specifically binding human endogenous retrovirus K long terminal repeat by affinity elution chromatography.

    PubMed

    Trubetskoy, D O; Zavalova, L L; Akopov, S B; Nikolaev, L G

    2002-11-01

    A novel affinity elution procedure for purification of DNA-binding proteins was developed and employed to purify to near homogeneity the proteins recognizing a 21 base pair sequence within the long terminal repeat of human endogenous retroviruses K. The approach involves loading the initial protein mixture on a heparin-agarose column and elution of protein(s) of interest with a solution of double-stranded oligonucleotide containing binding sites of the protein(s). The affinity elution has several advantages over conventional DNA-affinity chromatography: (i) it is easier and faster, permitting to isolate proteins in a 1 day-one stage procedure; (ii) yield of a target protein is severalfold higher than that in DNA-affinity chromatography; (iii) it is not necessary to prepare a special affinity support for each factor to be isolated. Theaffinity elution could be a useful alternative to conventional DNA-affinity chromatography.

  11. Flow-Cytometric Isolation of Human Antibodies from a Nonimmune Saccharomyces cerevisiae Surface Display Library

    SciTech Connect

    Feldhaus, Michael ); Siegel, Robert W. ); Opresko, Lee ); Coleman, James R. ); Feldhaus, Jane M. ); Yeung, Yik A.; Cochran, Jennifer R.; Heinzelman, Peter; Colby, David; Swers, Jeffrey; Graff, Christilyn; Wiley, H Steven ); Wittrup, K D.

    2003-02-28

    A nonimmune library of 109 human antibody scFv fragments has been cloned and expressed on the surface of yeast, and nanomolar-affinity scFvs routinely obtained by magnetic bead screening and flow cytometric sorting. The yeast library can be amplified 1010-fold without measurable loss of clonal diversity, enabling effectively indefinite expansion of the library. The expression, stability, and antigen binding properties of more than 50 isolated scFv clones were assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps and thereby expediting the isolation of novel affinity reagents. The ability to use multiplex library screening demonstrates the utility of this approach for high throughput antibody isolation for proteomics applications.

  12. Preparation of factor IX deficient human plasma by immunoaffinity chromatography using a monoclonal antibody.

    PubMed

    Goodall, A H; Kemble, G; O'Brien, D P; Rawlings, E; Rotblat, F; Russell, G C; Janossy, G; Tuddenham, E G

    1982-03-01

    A murine hybridoma clone is described that grows continuously in culture and produces a monoclonal antibody we have called Royal Free Monoclonal Antibody to factor IX No. 1 (RFF-IX/1). This has high affinity for a coagulation site on factor IX. RFF-IX/1 immobilised on sepharose can be used to deplete factor IX from normal human plasma. This immunoaffinity depleted plasma is indistinguishable from severe Christmas disease plasma and can be used as the substrate in a one stage coagulation assay for factor IX. The affinity column has high capacity and can be regenerated so that large scale production from normal plasma of factor IX deficient plasma as a diagnostic reagent is now feasible.

  13. STRUCTURE OF A HIGH-AFFINITY “MIMOTOPE” PEPTIDE BOUND TO HIV-1-NEUTRALIZING ANTIBODY b12 EXPLAINS ITS INABILITY TO ELICIT gp120 CROSS-REACTIVE ANTIBODIES

    PubMed Central

    Saphire, Erica Ollmann; Montero, Marinieve; Menendez, Alfredo; van Houten, Nienke E.; Irving, Melita B.; Pantophlet, Ralph; Zwick, Michael B.; Parren, Paul W. H. I.; Burton, Dennis R.; Scott, Jamie K.; Wilson, Ian A.

    2007-01-01

    The human antibody b12 recognizes a discontinuous epitope on gp120 and is one of the rare monoclonal antibodies that neutralize a broad range of primary HIV-1 isolates. We previously reported the isolation of B2.1, a dimeric peptide that binds with high specificity to b12 and competes with gp120 for b12 antibody binding. Here, we show that the affinity of B2.1 was improved 60-fold over its synthetic-peptide counterpart by fusing it to the N-terminus of a soluble protein. This affinity, which is within an order of magnitude of that of gp120, probably more closely reflects the affinity of the phage-borne peptide. The crystal structure of a complex between Fab of b12 and B2.1 was determined at 1.8 Å resolution. The structural data allowed the differentiation of residues that form critical contacts with b12 from those required for maintenance of the antigenic structure of the peptide, and revealed that three contiguous residues mediate B2.1's critical contacts with b12. This single region of critical contact between the B2.1 peptide and the b12 paratope is unlikely to mimic the discontinuous key binding residues involved in the full b12 epitope for gp120, as previously identified by alanine scanning substitutions on the gp120 surface. These structural observations are supported by experiments that demonstrate that B2.1 is an ineffective immunogenic mimic of the b12 epitope on gp120. Indeed, an extensive series of immunizations with B2.1 in various forms failed to produce gp120 cross-reactive sera. The functional and structural data presented here, however, suggest that the mechanism by which b12 recognizes the two antigens is very different. Here, we present the first crystal structure of peptide bound to an antibody that was originally raised against a discontinuous protein epitope. Our results highlight the challenge of producing immunogens that mimic discontinuous protein epitopes, and the necessity of combining complementary experimental approaches in analyzing

  14. Analysis of free drug fractions in human serum by ultrafast affinity extraction and two-dimensional affinity chromatography.

    PubMed

    Zheng, Xiwei; Podariu, Maria; Matsuda, Ryan; Hage, David S

    2016-01-01

    Ultrafast affinity extraction and a two-dimensional high performance affinity chromatographic system were used to measure the free fractions for various drugs in serum and at typical therapeutic concentrations. Pooled samples of normal serum or serum from diabetic patients were utilized in this work. Several drug models (i.e., quinidine, diazepam, gliclazide, tolbutamide, and acetohexamide) were examined that represented a relatively wide range of therapeutic concentrations and affinities for human serum albumin (HSA). The two-dimensional system consisted of an HSA microcolumn for the extraction of a free drug fraction, followed by a larger HSA analytical column for the further separation and measurement of this fraction. Factors that were optimized in this method included the flow rates, column sizes, and column switching times that were employed. The final extraction times used for isolating the free drug fractions were 333-665 ms or less. The dissociation rate constants for several of the drugs with soluble HSA were measured during system optimization, giving results that agreed with reference values. In the final system, free drug fractions in the range of 0.7-9.5% were measured and gave good agreement with values that were determined by ultrafiltration. Association equilibrium constants or global affinities were also estimated by this approach for the drugs with soluble HSA. The results for the two-dimensional system were obtained in 5-10 min or less and required only 1-5 μL of serum per injection. The same approach could be adapted for work with other drugs and proteins in clinical samples or for biomedical research. PMID:26462924

  15. Computationally driven antibody engineering enables simultaneous humanization and thermostabilization.

    PubMed

    Choi, Yoonjoo; Ndong, Christian; Griswold, Karl E; Bailey-Kellogg, Chris

    2016-10-01

    Humanization reduces the immunogenicity risk of therapeutic antibodies of non-human origin. Thermostabilization can be critical for clinical development and application of therapeutic antibodies. Here, we show that the computational antibody redesign method Computationally Driven Antibody Humanization (CoDAH) enables these two goals to be accomplished simultaneously and seamlessly. A panel of CoDAH designs for the murine parent of cetuximab, a chimeric anti-EGFR antibody, exhibited both substantially improved thermostabilities and substantially higher levels of humanness, while retaining binding activity near the parental level. The consistently high quality of the turnkey CoDAH designs, over a whole panel of variants, suggests that the computationally directed approach encapsulates key determinants of antibody structure and function.

  16. Expression of high-affinity IL-4 receptors on human melanoma, ovarian and breast carcinoma cells.

    PubMed Central

    Obiri, N I; Siegel, J P; Varricchio, F; Puri, R K

    1994-01-01

    It has previously been shown that murine sarcoma cells express high-affinity IL-4 receptors (IL-4R) which are internalized after binding to the ligand (Puri et al., Cancer Res 1991; 51:3011-7). We have also reported that human renal cell carcinoma cells express high-affinity IL-4R, and IL-4 inhibits tumour growth in vitro (Obiri et al., J Clin Invest 1993; 91:88). In this study we investigated the expression and function of IL-4R on other human solid tumours. Human melanoma, ovarian carcinoma and breast carcinoma cell lines were assessed for the cell surface expression of IL-4R by radio-ligand receptor binding and for IL-4R gene expression by Northern blot analysis. Primary cultures of mesothelioma and neurofibrosarcoma cells were similarly investigated. Human melanoma, ovarian carcinoma and breast carcinoma cell lines expressed IL-4R on their cell surface with a dissociation constant (Kd) of 140-549 pM. These tumour lines expressed a single 4 kb species of mRNA for IL-4R. Similarly, primary cultures of mesothelioma and neurofibrosarcoma cells were positive for the IL-4R mRNA by Northern blot analysis. Fresh, non-cultured mesothelioma and neurofibrosarcoma tumour sections were also positive for the presence of IL-4R as determined by immunohistochemistry of frozen sections using anti-IL-4R antibody. In order to study possible functions of IL-4R, we evaluated the effects of IL-4 on cell growth and its effect on MHC antigen expression in the presence or absence of interferon-gamma (IFN-gamma). In tissue culture, IL-4 reduced the growth of tumour cell lines and primary cell cultures studied. IL-4 had very little effect on MHC class I antigen expression on ovarian, breast and melanoma cell lines; however, MHC class II (HLA-DR) expression was enhanced on melanoma and breast carcinoma cells. IL-4 also enhanced the IFN-gamma-induced class II expression on melanoma and breast carcinoma cells. Taken together, our observations indicate that IL-4R are expressed on a variety of

  17. Tools to therapeutically harness the human antibody response.

    PubMed

    Wilson, Patrick C; Andrews, Sarah F

    2012-10-01

    The natural human antibody response is a rich source of highly specific, neutralizing and self-tolerant therapeutic reagents. Recent advances have been made in isolating and characterizing monoclonal antibodies that are generated in response to natural infection or vaccination. Studies of the human antibody response have led to the discovery of crucial epitopes that could serve as new targets in vaccine design and in the creation of potentially powerful immunotherapies. With a focus on influenza virus and HIV, herein we summarize the technological tools used to identify and characterize human monoclonal antibodies and describe how these tools might be used to fight infectious diseases.

  18. Affinity purification of antibodies using immobilized FB domain of protein A.

    PubMed

    Solomon, B; Raviv, O; Leibman, E; Fleminger, G

    1992-04-24

    A continuous method for the efficient digestion of protein A into active fragments (FB, Mr = 7000) using immobilized trypsin was developed. These fragments originate from almost identical five-repeated monovalent Fc-binding units of 58 residues each. The fragments obtained were found to be similar to the recently described genetically engineered fragment B. Antibody-binding characteristics of the FB domain and also of intact protein A, immobilized on to adipic dihydrazide-modified Eupergit CB6200 beads, were investigated. Based on the experimental data obtained, a high-performance liquid chromatographic column containing C30N Eupergit C-immobilized FB domain was prepared and its performance in antibody purification was compared with that of Eupergit C-immobilized intact protein A. PMID:1517325

  19. Idiotypes of pre-existing human anti-carcinoma anti-T and anti-Tn antibodies.

    PubMed

    Zanetti, M; Lenert, G; Springer, G F

    1993-02-01

    All humans normally possess antibodies, predominantly IgM, that react specifically with the Thomsen-Friedenreich (T) and the Tn antigens which are present in immunoreactive form on > 85% of all human carcinomas, but not in healthy and otherwise diseased tissues. We report here a serological study of idiotype expression and antigen reactivity of the anti-T and anti-Tn antibodies. Idiotypy was analyzed with rabbit antibodies raised against, and made specific for, affinity-purified polyclonal anti-T and anti-Tn antibodies from blood group A1B healthy adult donors. Anti-T and anti-Tn antibodies cross-reacted idiotypically in spite of their distinct epitope specificities. By adsorbing anti-T antibodies on insolubilized synthetic T carbohydrate we could firmly link idiotype expression with antigen reactivity. The relation of idiotype expression to the antigen-binding site of plant seed lectins was also studied; one originated from Arachis hypogaea [peanut agglutinin (PNA)], the other from Artocarpus integrifolia (Jacalin). PNA inhibited only anti-T antibodies. Jacalin inhibited both anti-T and anti-Tn antibodies in a dose-dependent manner. Neither idiotypic nor anti-idiotypic antibodies diminished the binding of lectins to T and Tn epitopes. The shared idiotypes on natural anti-T and anti-Tn antibodies permit consideration of application of their anti-idiotypes in treatment and/or prevention of human carcinoma.

  20. The influence of orientation and number of copies of T and B cell epitopes on the specificity and affinity of antibodies induced by chimeric peptides.

    PubMed

    Partidos, C; Stanley, C; Steward, M

    1992-10-01

    CBA and TO mice were immunized with chimeric peptide immunogens consisting of B cell (residues 404-414) and T cell (residues 288-302) epitopes from the F protein of measles virus. The chimeras were co-linearly synthesized to contain one or two copies of the T cell epitope linked to one or two copies of the B cell epitope via a glycine.glycine spacer. Two orientations were synthesized such that the T cell epitope(s) were located at either the amino or carboxyl terminus of the B cell epitope(s). The levels of antibody induced following immunization with the chimeras were assessed by enzyme-linked immunosorbent assay using microtiter plates coated with either the homologous chimera or the B cell epitope sequence. The affinities of the anti-chimera antibodies for the B cell epitope were assessed by a fluid-phase double-isotope radioimmunoassay. All the chimeras induced good antibody responses in both strains of mice with specificity for the B cell epitope. Chimeras containing two copies of the T cell epitope induced antibodies with higher affinity for the B cell epitope than did chimeras containing one copy of the T cell epitope or two copies of the B cell epitope. Furthermore, the amino terminal location of the T cell epitope in relation to the B cell epitope in the chimera induced higher affinity anti-B cell antibody than did the reverse orientation. These results suggest that orientation of epitopes and amino acid composition of chimeric peptides affect antigen processing and presentation to T cells which govern both the specificity and affinity of antibody produced. Thus, for the production of synthetic peptide immunogens with vaccine potential, attention needs to be given to the number and orientation of the component epitopes required to produce highest affinity antibody.

  1. Immune complex relay by subcapsular sinus macrophages and non-cognate B cells drives antibody affinity maturation

    PubMed Central

    Phan, Tri Giang; Green, Jesse A.; Gray, Elizabeth E.; Xu, Ying; Cyster, Jason G.

    2009-01-01

    Subcapsular sinus (SCS) macrophages capture antigens from lymph and present them intact for B cell encounter and follicular delivery. However, the properties of SCS macrophages are poorly defined. Here we show SCS macrophage development depended on lymphotoxin-α1β2 and the cells had low lysosomal enzyme expression and retained opsonized antigens on their surface. Intravital imaging revealed immune complexes moving along macrophage processes into the follicle. Moreover, non-cognate B cells relayed antigen opsonized by newly produced antibodies from the subcapsular sinus to the germinal center and affinity maturation was impaired when this transport process was disrupted. Thus, we characterize SCS macrophages as specialized antigen-presenting cells functioning at the apex of an antigen transport chain that promotes humoral immunity. PMID:19503106

  2. Arginine as an eluent overcomes the hindrance of monoclonal antibody quantification by dextran sulfate in protein A affinity chromatography.

    PubMed

    Kim, Bong Gyun; Park, Hong Woo

    2015-01-01

    Analytical chromatography using protein A affinity columns was employed for the fast and simple quantitative analysis of monoclonal antibodies (mAb) from suspension cultures of recombinant Chinese hamster ovary (rCHO) cells. Reliable results could not be obtained from analysis of rCHO cell culture supernatants containing dextran sulfate using elution buffers such as phosphate, glycine, or MgCl2 . These problems increased as the number of analysis and the concentration of dextran sulfate in samples increased. Arginine was identified as an alternative eluent to overcome the hindrance by dextran sulfate. When the samples contain dextran sulfate up to 100 mg/L, the elution buffer containing 0.6-1.0 M arginine at pH 3.0-3.8 is useful for the effective analysis. Reproducible results in the mAb quantification could be obtained by this developed arginine elution buffer from rCHO cell culture supernatants containing dextran sulfate.

  3. Arginine as an eluent overcomes the hindrance of monoclonal antibody quantification by dextran sulfate in protein A affinity chromatography.

    PubMed

    Kim, Bong Gyun; Park, Hong Woo

    2015-01-01

    Analytical chromatography using protein A affinity columns was employed for the fast and simple quantitative analysis of monoclonal antibodies (mAb) from suspension cultures of recombinant Chinese hamster ovary (rCHO) cells. Reliable results could not be obtained from analysis of rCHO cell culture supernatants containing dextran sulfate using elution buffers such as phosphate, glycine, or MgCl2 . These problems increased as the number of analysis and the concentration of dextran sulfate in samples increased. Arginine was identified as an alternative eluent to overcome the hindrance by dextran sulfate. When the samples contain dextran sulfate up to 100 mg/L, the elution buffer containing 0.6-1.0 M arginine at pH 3.0-3.8 is useful for the effective analysis. Reproducible results in the mAb quantification could be obtained by this developed arginine elution buffer from rCHO cell culture supernatants containing dextran sulfate. PMID:26363185

  4. Isolation of human anti-serum albumin Fab antibodies with an extended serum-half life.

    PubMed

    Kang, Hyeon-Ju; Kim, Hye-Jin; Cha, Sang-Hoon

    2016-01-01

    The serum albumin (SA) has been exploited to generate long-acting biotherapeutics by taking advantage of the FcRn-mediated recycling mechanism in a direct or an indirect way. Since Fab fragments have been proven to be clinically safe for human usage, we assumed that human anti-SA Fab antibodies could have a great potential as a carrier molecule to extend the serum half-life of therapeutic proteins. We, herein, had attempted to isolate anti-SA Fab antibodies from HuDVFab-8L antibody library via a phage display technology, and identified eight discrete human Fab antibodies. One of the Fab antibodies, SL335, showed the strongest binding reactivity to human SA with nM range of affinity at both pH 6 and pH 7.4, and cross-reacted to SAs from various species including rat, mouse, canine and monkey. The in vivo pharmacokinetic assay using a rat model indicated that SL335 has approximately 10 fold longer serum half-life and 26 to 44-fold increase in AUC0 → ∞ compared to the negative control Fab molecule in both intravenous and subcutaneous administrations. Knowing that Fabs have proven to be safe in clinics for a long time, SL335 seems to have a great potential in generating long-acting protein drugs by tagging effector molecules with either chemical conjugation or genetic fusion.

  5. Improvement in affinity and HIV-1 neutralization by somatic mutation in the heavy chain first complementarity-determining region of antibodies triggered by HIV-1 infection.

    PubMed

    Torán, J L; Sánchez-Pulido, L; Kremer, L; del Real, G; Valencia, A; Martínez-A, C

    2001-01-01

    We assessed the impact of somatic hypermutation in the framework region 1 (FR1) and complementarity-determining region 1 (CDR1) of three clonally-related heavy chains from the human monovalent antigen-binding fragments Fab S19, S8 and S20 on gp120 binding and HIV-1 neutralization capacity. Nucleotide changes were introduced in the heavy chains to revert single and multiple amino acid residues, and two Fab libraries were constructed with the same light chain to express equivalent amounts of parental and reverted phage Fab. We studied the contribution of each amino acid replacement to antigen binding by calculating the frequency of phage Fab retrieval after competitive library selection on gp120. Whereas mutations in FR1 had no effect on antigen binding, somatic replacements in the CDR1 of the heavy chain (HCDR1) appeared to produce significant changes. In S19 HCDR1, somatic mutation of residue 32 reduced gp120 binding. In Fab S20, the Arg(30) and Asp(31) somatically replaced residues in HCDR1 improved antigen binding. Both of these residues are necessary to increase Fab binding to gp120; reversion of either residue alone results in a decrease in binding. The impact of these two replacements was confirmed by the greater neutralization capacity of S20 compared to the other Fab. Molecular modeling of S20 HCDR1 suggests that Arg(30) and Asp(31) are the main interaction sites for gp120, increasing antibody affinity and promoting the enhanced neutralization ability of S20. These findings are consistent with a gp120-driven process, supporting a role for affinity maturation and intraclonal evolution of HIV-1 neutralizing antibodies.

  6. Label-free Fab and Fc affinity/avidity profiling of the antibody complex half-life for polyclonal and monoclonal efficacy screening.

    PubMed

    Read, Thomas; Olkhov, Rouslan V; Williamson, E Diane; Shaw, Andrew M

    2015-09-01

    A unified approach to affinity screening for Fab and Fc interactions of an antibody for its antigen and FcγR receptor has been developed. An antigen array is used for the Fab affinity and cross-reactivity screening and protein A/G proxy is the FcγR receptor. The affinities are derived using a simple 1:1 binding model with a consistent error analysis. The association and dissociation kinetics are measured over optimised times for accurate determination. The Fab/Fc affinities are derived for ten antibodies: mAb-actin (mouse), pAb-BSA (sheep), pAb-collagen V (rabbit), pAb-CRP (goat), mAb-F1 (mouse), mAbs (mouse) 7.3, 12.3, 29.3, 36.3 and 46.3 raised against LcrV in Yersinia pestis. The rate of the dissociation of antigen-antibody complexes relates directly to their immunological function as does the Fc-FcγR complex and a new half-life plot has been defined with a Fab/Fc half-life range of 17-470 min. The upper half-life value points to surface avidity. Two antibodies that are protective as an immunotherapy define a Fab half-life >250 min and an Fc half-life >50 min as characteristics of ideal interactions which can form the basis of an antibody screen for immunotherapy.

  7. The natural antibody repertoire of sharks and humans recognizes the potential universe of antigens.

    PubMed

    Adelman, Miranda K; Schluter, Samuel F; Marchalonis, John J

    2004-02-01

    In ancestral sharks, a rapid emergence in the evolution of the immune system occurred, giving jawed-vertebrates the necessary components for the combinatorial immune response (CIR). To compare the natural antibody (NAb) repertoires of the most divergent vertebrates with the capacity to produce antibodies, we isolated NAbs to the same set of antigens by affinity chromatography from two species of Carcharhine sharks and from human polyclonal IgG and IgM antibody preparations. The activities of the affinity-purified anti-T-cell receptor (anti-TCR) NAbs were compared with those of monoclonal anti-TCR NAbs that were generated from a systemic lupus erythematosus patient. We report that sharks and humans, representing the evolutionary extremes of vertebrate species sharing the CIR, have NAbs to human TCRs, Igs, the human senescent cell antigen, and to numerous retroviral antigens, indicating that essential features of the combinatorial repertoire and the capacity to recognize the potential universe of antigens is shared among all jawed-vertebrates.

  8. Co-administration of CpG oligonucleotides enhances the late affinity maturation process of human anti-hepatitis B vaccine response.

    PubMed

    Siegrist, Claire-Anne; Pihlgren, Maria; Tougne, Chantal; Efler, Sue M; Morris, Mary Lou; AlAdhami, Mohammed J; Cameron, D William; Cooper, Curtis L; Heathcote, Jenny; Davis, Heather L; Lambert, Paul-Henri

    2004-12-16

    We assessed the avidity maturation process elicited by human immunization with alum-adsorbed HBsAg alone or with a novel adjuvant containing CpG motifs (CpG 7909). Mean avidity indexes and distribution of low- and high-avidity anti-HBs indicated that avidity maturation essentially takes place late after priming. CpG 7909 markedly enhanced this affinity maturation process, increasing the pool of high-avidity antibodies. The influence of CpG 7909 was antigen-specific, isotype-specific and distinct from the influence on anti-HBs production, as avidity did not correlate with anti-HBs IgG titers. This is the first demonstration that a novel human adjuvant may induce antibodies with higher antigen-binding affinity. PMID:15542181

  9. Synthetic antibodies with a human framework that protect mice from lethal Sudan ebolavirus challenge.

    PubMed

    Chen, Gang; Koellhoffer, Jayne F; Zak, Samantha E; Frei, Julia C; Liu, Nina; Long, Hua; Ye, Wei; Nagar, Kaajal; Pan, Guohua; Chandran, Kartik; Dye, John M; Sidhu, Sachdev S; Lai, Jonathan R

    2014-10-17

    The ebolaviruses cause severe and rapidly progressing hemorrhagic fever. There are five ebolavirus species; although much is known about Zaire ebolavirus (EBOV) and its neutralization by antibodies, little is known about Sudan ebolavirus (SUDV), which is emerging with increasing frequency. Here we describe monoclonal antibodies containing a human framework that potently inhibit infection by SUDV and protect mice from lethal challenge. The murine antibody 16F6, which binds the SUDV envelope glycoprotein (GP), served as the starting point for design. Sequence and structural alignment revealed similarities between 16F6 and YADS1, a synthetic antibody with a humanized scaffold. A focused phage library was constructed and screened to impart 16F6-like recognition properties onto the YADS1 scaffold. A panel of 17 antibodies were characterized and found to have a range of neutralization potentials against a pseudotype virus infection model. Neutralization correlated with GP binding as determined by ELISA. Two of these clones, E10 and F4, potently inhibited authentic SUDV and conferred protection and memory immunity in mice from lethal SUDV challenge. E10 and F4 were further shown to bind to the same epitope on GP as 16F6 with comparable affinities. These antibodies represent strong immunotherapeutic candidates for treatment of SUDV infection. PMID:25140871

  10. Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies.

    PubMed

    Yu, Guimei; Vago, Frank; Zhang, Dongsheng; Snyder, Jonathan E; Yan, Rui; Zhang, Ci; Benjamin, Christopher; Jiang, Xi; Kuhn, Richard J; Serwer, Philip; Thompson, David H; Jiang, Wen

    2014-07-01

    Single particle cryo-electron microscopy (cryo-EM) is an emerging powerful tool for structural studies of macromolecular assemblies (i.e., protein complexes and viruses). Although single particle cryo-EM requires less concentrated and smaller amounts of samples than X-ray crystallography, it remains challenging to study specimens that are low-abundance, low-yield, or short-lived. The recent development of affinity grid techniques can potentially further extend single particle cryo-EM to these challenging samples by combining sample purification and cryo-EM grid preparation into a single step. Here we report a new design of affinity cryo-EM approach, cryo-SPIEM, that applies a traditional pathogen diagnosis tool Solid Phase Immune Electron Microscopy (SPIEM) to the single particle cryo-EM method. This approach provides an alternative, largely simplified and easier to use affinity grid that directly works with most native macromolecular complexes with established antibodies, and enables cryo-EM studies of native samples directly from cell cultures. In the present work, we extensively tested the feasibility of cryo-SPIEM with multiple samples including those of high or low molecular weight, macromolecules with low or high symmetry, His-tagged or native particles, and high- or low-yield macromolecules. Results for all these samples (non-purified His-tagged bacteriophage T7, His-tagged Escherichiacoli ribosomes, native Sindbis virus, and purified but low-concentration native Tulane virus) demonstrated the capability of cryo-SPIEM approach in specifically trapping and concentrating target particles on TEM grids with minimal view constraints for cryo-EM imaging and determination of 3D structures.

  11. Germline humanization of a murine Abeta antibody and crystal structure of the humanized recombinant Fab fragment.

    PubMed

    Robert, Remy; Streltsov, Victor A; Newman, Janet; Pearce, Lesley A; Wark, Kim L; Dolezal, Olan

    2010-02-01

    Alzheimer's disease is the most common form of dementia, affecting 26 million people worldwide. The Abeta peptide (39-43 amino acids) derived from the proteolytic cleavage of the amyloid precursor protein is one of the main constituents of amyloid plaques associated with disease pathogenesis and therefore a validated target for therapy. Recently, we characterized antibody fragments (Fab and scFvs) derived from the murine monoclonal antibody WO-2, which bind the immunodominant epitope ((3)EFRH(6)) in the Abeta peptide at the N-terminus. In vitro, these fragments are able to inhibit fibril formation, disaggregate preformed amyloid fibrils, and protect neuroblastoma cells against oligomer-mediated toxicity. In this study, we describe the humanization of WO-2 using complementary determining region loop grafting onto the human germline gene and the determination of the three-dimensional structure by X-ray crystallography. This humanized version retains a high affinity for the Abeta peptide and therefore is a potential candidate for passive immunotherapy of Alzheimer's disease.

  12. Detection of Hepatitis C core antibody by dual-affinity yeast chimera and smartphone-based electrochemical sensing.

    PubMed

    Aronoff-Spencer, Eliah; Venkatesh, A G; Sun, Alex; Brickner, Howard; Looney, David; Hall, Drew A

    2016-12-15

    Yeast cell lines were genetically engineered to display Hepatitis C virus (HCV) core antigen linked to gold binding peptide (GBP) as a dual-affinity biobrick chimera. These multifunctional yeast cells adhere to the gold sensor surface while simultaneously acting as a "renewable" capture reagent for anti-HCV core antibody. This streamlined functionalization and detection strategy removes the need for traditional purification and immobilization techniques. With this biobrick construct, both optical and electrochemical immunoassays were developed. The optical immunoassays demonstrated detection of anti-HCV core antibody down to 12.3pM concentrations while the electrochemical assay demonstrated higher binding constants and dynamic range. The electrochemical format and a custom, low-cost smartphone-based potentiostat ($20 USD) yielded comparable results to assays performed on a state-of-the-art electrochemical workstation. We propose this combination of synthetic biology and scalable, point-of-care sensing has potential to provide low-cost, cutting edge diagnostic capability for many pathogens in a variety of settings.

  13. Detection of Hepatitis C core antibody by dual-affinity yeast chimera and smartphone-based electrochemical sensing.

    PubMed

    Aronoff-Spencer, Eliah; Venkatesh, A G; Sun, Alex; Brickner, Howard; Looney, David; Hall, Drew A

    2016-12-15

    Yeast cell lines were genetically engineered to display Hepatitis C virus (HCV) core antigen linked to gold binding peptide (GBP) as a dual-affinity biobrick chimera. These multifunctional yeast cells adhere to the gold sensor surface while simultaneously acting as a "renewable" capture reagent for anti-HCV core antibody. This streamlined functionalization and detection strategy removes the need for traditional purification and immobilization techniques. With this biobrick construct, both optical and electrochemical immunoassays were developed. The optical immunoassays demonstrated detection of anti-HCV core antibody down to 12.3pM concentrations while the electrochemical assay demonstrated higher binding constants and dynamic range. The electrochemical format and a custom, low-cost smartphone-based potentiostat ($20 USD) yielded comparable results to assays performed on a state-of-the-art electrochemical workstation. We propose this combination of synthetic biology and scalable, point-of-care sensing has potential to provide low-cost, cutting edge diagnostic capability for many pathogens in a variety of settings. PMID:27472403

  14. RNA recognition by a human antibody against brain cytoplasmic 200 RNA.

    PubMed

    Jung, Euihan; Lee, Jungmin; Hong, Hyo Jeong; Park, Insoo; Lee, Younghoon

    2014-06-01

    Diverse functional RNAs participate in a wide range of cellular processes. The RNA structure is critical for function, either on its own or as a complex form with proteins and other ligands. Therefore, analysis of the RNA conformation in cells is essential for understanding their functional mechanisms. However, no appropriate methods have been established as yet. Here, we developed an efficient strategy for panning and affinity maturation of anti-RNA human monoclonal antibodies from a naïve antigen binding fragment (Fab) combinatorial phage library. Brain cytoplasmic 200 (BC200) RNA, which is also highly expressed in some tumors, was used as an RNA antigen. We identified MabBC200-A3 as the optimal binding antibody. Mutagenesis and SELEX experiments showed that the antibody recognized a domain of BC200 in a structure- and sequence-dependent manner. Various breast cancer cell lines were further examined for BC200 RNA expression using conventional hybridization and immunoanalysis with MabBC200-A3 to see whether the antibody specifically recognizes BC200 RNA among the total purified RNAs. The amounts of antibody-recognizable BC200 RNA were consistent with hybridization signals among the cell lines. Furthermore, the antibody was able to discriminate BC200 RNA from other RNAs, supporting the utility of this antibody as a specific RNA structure-recognizing probe. Intriguingly, however, when permeabilized cells were subjected to immunoanalysis instead of purified total RNA, the amount of antibody-recognizable RNA was not correlated with the cellular level of BC200 RNA, indicating that BC200 RNA exists as two distinct forms (antibody-recognizable and nonrecognizable) in breast cancer cells and that their distribution depends on the cell type. Our results clearly demonstrate that anti-RNA antibodies provide an effective novel tool for detecting and analyzing RNA conformation.

  15. Single-chain Fv antibody fragments retain binding properties of the monoclonal antibody raised against peptide P1 of the human prion protein.

    PubMed

    Skrlj, Nives; Serbec, Vladka Curin; Dolinar, Marko

    2010-03-01

    Prion diseases are incurable neurodegenerative diseases that affect both humans and animals. The infectious agent is a pathogenic form of the prion protein that accumulates in brain as amyloids. Currently, there is neither cure nor reliable preclinical diagnostics on the market available. The growing number of reports shows that passive immunisation is one of the most promising strategies for prion disease therapy, where antibodies against prions may prevent and even cure the infection. Since antibodies are large molecules and, thus, might not be suitable for the therapy, different antibody fragments are a good alternative. Therefore, we have designed and prepared single-chain antibody fragments (scFvs) derived from the PrP(Sc)-specific murine monoclonal antibody V5B2. Using a new expression vector pMD204, we produced scFvs in two opposing chain orientations in the periplasm of Escherichia coli. Both recombinant antibody fragments retained the specificity of the parent antibody and one of these exhibited binding properties comparable to the corresponding murine Fab fragments with the affinity in nM range. Our monovalent antibody fragments are of special interest in view of possible therapeutic reagents for prion diseases as well as for development of a new generation of diagnostics. PMID:19597999

  16. Mechanism of human antibody-mediated neutralization of Marburg virus.

    PubMed

    Flyak, Andrew I; Ilinykh, Philipp A; Murin, Charles D; Garron, Tania; Shen, Xiaoli; Fusco, Marnie L; Hashiguchi, Takao; Bornholdt, Zachary A; Slaughter, James C; Sapparapu, Gopal; Klages, Curtis; Ksiazek, Thomas G; Ward, Andrew B; Saphire, Erica Ollmann; Bukreyev, Alexander; Crowe, James E

    2015-02-26

    The mechanisms by which neutralizing antibodies inhibit Marburg virus (MARV) are not known. We isolated a panel of neutralizing antibodies from a human MARV survivor that bind to MARV glycoprotein (GP) and compete for binding to a single major antigenic site. Remarkably, several of the antibodies also bind to Ebola virus (EBOV) GP. Single-particle EM structures of antibody-GP complexes reveal that all of the neutralizing antibodies bind to MARV GP at or near the predicted region of the receptor-binding site. The presence of the glycan cap or mucin-like domain blocks binding of neutralizing antibodies to EBOV GP, but not to MARV GP. The data suggest that MARV-neutralizing antibodies inhibit virus by binding to infectious virions at the exposed MARV receptor-binding site, revealing a mechanism of filovirus inhibition. PMID:25723164

  17. A humanized anti-DLL4 antibody promotes dysfunctional angiogenesis and inhibits breast tumor growth

    PubMed Central

    Jia, Xuelian; Wang, Wenyi; Xu, Zhuobin; Wang, Shijing; Wang, Tong; Wang, Min; Wu, Min

    2016-01-01

    Blockage of Delta-like 4 (DLL4)-directed Notch signaling induces excessive tip cell formation and endothelial proliferation resulting in dysfunctional angiogenesis in tumors. MMGZ01, as a murine anti-human DLL4 monoclonal antibody, specifically binds to human DLL4 and blocks Notch pathway. Here, the structure of MMGZ01 variable fragment (Fv) was established and framework region (FR) residues which supported complementarily determining region (CDR) loop conformation were identified. Important residues interactions were also identified through docking MMGZ01 Fv with antigen epitope in DLL4. To humanize the murine antibody, we modified MMGZ01 Fv through CDR grafting and the reconstructed antibody (H3L2) maintained similar structure and binding affinity to parental MMGZ01 after back mutation of 12 canonical murine residues in the FRs. Meanwhile, H3L2 promoted human umbilical vein endothelial cell (HUVEC) proliferation through inhibiting DLL4-directed Notch pathway. Moreover, in MDA-MB-231-bearing nude mice, H3L2 induced dysfunctional angiogenesis and tumor cell apoptosis and showed superior anti-tumor activity. In conclusion, H3L2 is an ideal humanized antibody that inhibits tumor growth through targeting DLL4-Notch pathway and has attracting potentials for clinical applications. PMID:27301650

  18. An improved Protein G with higher affinity for human/rabbit IgG Fc domains exploiting a computationally designed polar network

    PubMed Central

    Jha, Ramesh K.; Gaiotto, Tiziano; Bradbury, Andrew R.M.; Strauss, Charlie E.M.

    2014-01-01

    Protein G is an IgG binding protein that has been widely exploited for biotechnological purposes. Rosetta protein modeling identified a set of favorable polar mutations in Protein G, at its binding interface with the Fc domain of Immunoglobulin G, that were predicted to increase the stability and tighten the binding relative to native Protein G, with only a minor perturbation of the binding mode seen in the crystal structure. This triple mutant was synthesized and evaluated experimentally. Relative to the native protein G, the mutant showed a 3.5-fold enhancement in display level on the surface of yeast and a 5-fold tighter molar affinity for rabbit and human IgG. We attribute the improved affinity to a network of hydrogen bonds exploiting specific polar groups on human and rabbit Fc. The relative specificity increased as well since there was little affinity enhancement for goat and mouse Fc, while the affinity for rat Fc was poorer by half. This designed Protein G will be useful in biotechnological applications as a recombinant protein, where its improved affinity, display and specificity will increase antibody capture sensitivity and capacity. Furthermore, the display of this protein on the surface of yeast introduces the concept of the use of yeast as an affinity matrix. PMID:24632761

  19. Growth factors with heparin binding affinity in human synovial fluid

    SciTech Connect

    Hamerman, D.; Taylor, S.; Kirschenbaum, I.; Klagsbrun, M.; Raines, E.W.; Ross, R.; Thomas, K.A.

    1987-12-01

    Synovial effusions were obtained from the knees of 15 subjects with joint trauma, menisceal or ligamentous injury, or osteoarthritis. Heparin-Sepharose affinity chromatography of these synovial fluids revealed, in general, three major peaks of mitogenic activity as measured by incorporation of /sup 3/H-thymidine into 3T3 cells. Gradient elution patterns showed activities at 0.5M NaCl, which is characteristic of platelet derived growth factor, and at 1.1 M NaCl and 1.6M NaCl, indicative of acidic and basic fibroblast growth factors, respectively. The identities of these mitogenic fractions were confirmed by specific immunologic and receptor-binding assays. The presence of platelet derived, acidic and basic fibroblast growth factors in the synovial fluid may contribute to wound healing in the arthritic joint.

  20. A cocktail of humanized anti-pertussis toxin antibodies limits disease in murine and baboon models of whooping cough.

    PubMed

    Nguyen, Annalee W; Wagner, Ellen K; Laber, Joshua R; Goodfield, Laura L; Smallridge, William E; Harvill, Eric T; Papin, James F; Wolf, Roman F; Padlan, Eduardo A; Bristol, Andy; Kaleko, Michael; Maynard, Jennifer A

    2015-12-01

    Despite widespread vaccination, pertussis rates are rising in industrialized countries and remain high worldwide. With no specific therapeutics to treat disease, pertussis continues to cause considerable infant morbidity and mortality. The pertussis toxin is a major contributor to disease, responsible for local and systemic effects including leukocytosis and immunosuppression. We humanized two murine monoclonal antibodies that neutralize pertussis toxin and expressed them as human immunoglobulin G1 molecules with no loss of affinity or in vitro neutralization activity. When administered prophylactically to mice as a binary cocktail, antibody treatment completely mitigated the Bordetella pertussis-induced rise in white blood cell counts and decreased bacterial colonization. When administered therapeutically to baboons, antibody-treated, but not untreated control animals, experienced a blunted rise in white blood cell counts and accelerated bacterial clearance rates. These preliminary findings support further investigation into the use of these antibodies to treat human neonatal pertussis in conjunction with antibiotics and supportive care. PMID:26631634

  1. A cocktail of humanized anti-pertussis toxin antibodies limits disease in murine and baboon models of whooping cough.

    PubMed

    Nguyen, Annalee W; Wagner, Ellen K; Laber, Joshua R; Goodfield, Laura L; Smallridge, William E; Harvill, Eric T; Papin, James F; Wolf, Roman F; Padlan, Eduardo A; Bristol, Andy; Kaleko, Michael; Maynard, Jennifer A

    2015-12-01

    Despite widespread vaccination, pertussis rates are rising in industrialized countries and remain high worldwide. With no specific therapeutics to treat disease, pertussis continues to cause considerable infant morbidity and mortality. The pertussis toxin is a major contributor to disease, responsible for local and systemic effects including leukocytosis and immunosuppression. We humanized two murine monoclonal antibodies that neutralize pertussis toxin and expressed them as human immunoglobulin G1 molecules with no loss of affinity or in vitro neutralization activity. When administered prophylactically to mice as a binary cocktail, antibody treatment completely mitigated the Bordetella pertussis-induced rise in white blood cell counts and decreased bacterial colonization. When administered therapeutically to baboons, antibody-treated, but not untreated control animals, experienced a blunted rise in white blood cell counts and accelerated bacterial clearance rates. These preliminary findings support further investigation into the use of these antibodies to treat human neonatal pertussis in conjunction with antibiotics and supportive care.

  2. Purification of human copper, zinc superoxide dismutase by copper chelate affinity chromatography

    SciTech Connect

    Weslake, R.J.; Chesney, S.L.; Petkau, A.; Friesen, A.D.

    1986-05-15

    Copper, zinc superoxide dismutase was isolated from human red blood cell hemolysate by DEAE-Sepharose and copper chelate affinity chromatography. Enzyme preparations had specific activities ranging from 3400 to 3800 U/mg and recoveries were approximately 60% of the enzyme activity in the lysate. Copper chelate affinity chromatography resulted in a purification factor of about 60-fold. The homogeneity of the superoxide dismutase preparation was analyzed by sodium dodecyl sulfate-gel electrophoresis, analytical gel filtration chromatography, and isoelectric focusing.

  3. Oral priming with replicating adenovirus serotype 4 followed by subunit H5N1 vaccine boost promotes antibody affinity maturation and expands H5N1 cross-clade neutralization.

    PubMed

    Khurana, Surender; Coyle, Elizabeth M; Manischewitz, Jody; King, Lisa R; Ishioka, Glenn; Alexander, Jeff; Smith, Jon; Gurwith, Marc; Golding, Hana

    2015-01-01

    A Phase I trial conducted in 2009-2010 demonstrated that oral vaccination with a replication competent Ad4-H5 (A/Vietnam) vector with dosages ranging from 107-1011 viral particles was well tolerated. HA-specific T-cell responses were efficiently induced, but very limited hemagglutination-inhibiting (HI) humoral responses were measured. However, a single boost of Ad4-H5-Vtn vaccinated individuals with a unadjuvanted licensed H5N1 (A/Vietnam) subunit vaccine resulted in superior HI titers compared with unprimed subjects. In the current study, the impact of Ad4-H5 priming on the quality of the polyclonal humoral immune response was evaluated using a real-time kinetics assay by surface plasmon resonance (SPR). Total binding of serum polyclonal antibodies from the Ad4-H5-Vtn primed groups against both homologous H5N1-A/Vietnam/1194/2004 (clade 1) and heterologous A/Indonesia-5/2005 (clade 2.1) HA1 head domain was significantly higher compared with sera from individuals that received subunit H5N1 vaccination alone. SPR measurements also demonstrated that the antigen-antibody complex dissociation rates (a surrogate for antibody affinity) of serum antibodies against the HA1 of H5N1-A/Vietnam were significantly higher in the Ad4-H5 primed groups compared with those from the unprimed group. Furthermore, strong correlations were observed between the antibody affinities for HA1 (but not HA2) and the virus neutralization titers against the homologous strain and a panel of heterologous clade 2 H5N1 strains. These findings support the concept of oral prime-boost vaccine approaches against pandemic influenza to elicit long-term memory B cells with high affinity capable of rapid response to variant pandemic viruses likely to emerge and adapt to human transmissions.

  4. The remarkable flexibility of the human antibody repertoire; isolation of over one thousand different antibodies to a single protein, BLyS.

    PubMed

    Edwards, Bryan M; Barash, Steven C; Main, Sarah H; Choi, Gil H; Minter, Ralph; Ullrich, Stephen; Williams, Elizabeth; Du Fou, Leila; Wilton, Jane; Albert, Vivian R; Ruben, Steve M; Vaughan, Tristan J

    2003-11-14

    It is well established that the humoral immune response can generate antibodies to many different antigens. The antibody diversity required to achieve this is believed to be substantial. However, the extent to which the immune repertoire can generate structural diversity against a single target antigen has never been addressed. Here, we have used phage display to demonstrate the extraordinary capacity of the human antibody repertoire. Over 1000 antibodies, all different in amino acid sequence, were generated to a single protein, B-lymphocyte stimulator (BLyS protein). This is a highly diverse panel of antibodies as exemplified by the extensive heavy and light chain germline usage: 42/49 functional heavy chain germlines and 19/33 V(lambda) and 13/35 V(kappa) light chain germlines were all represented in the panel of antibodies. Moreover, a high level of sequence diversity was observed in the V(H) CDR3 domains of these antibodies, with 568 different amino acid sequences identified. Thus we have demonstrated that specific recognition of a single antigen can be achieved from many different VDJ combinations, illustrating the remarkable problem-solving ability of the human immune repertoire. When studied in a biochemical assay, around 500 (40%) of these antibodies inhibited the binding of BLyS to its receptors on B-cell lines. The most potent antibodies inhibited BLyS binding with sub-nanomolar IC(50) values and with sub-nanomolar affinities. Such antibodies provide excellent choices as candidates for the treatment of BLyS-associated autoimmune diseases.

  5. Immunoglobulin gene insertions and deletions in the affinity maturation of HIV-1 broadly reactive neutralizing antibodies.

    PubMed

    Kepler, Thomas B; Liao, Hua-Xin; Alam, S Munir; Bhaskarabhatla, Rekha; Zhang, Ruijun; Yandava, Chandri; Stewart, Shelley; Anasti, Kara; Kelsoe, Garnett; Parks, Robert; Lloyd, Krissey E; Stolarchuk, Christina; Pritchett, Jamie; Solomon, Erika; Friberg, Emma; Morris, Lynn; Karim, Salim S Abdool; Cohen, Myron S; Walter, Emmanuel; Moody, M Anthony; Wu, Xueling; Altae-Tran, Han R; Georgiev, Ivelin S; Kwong, Peter D; Boyd, Scott D; Fire, Andrew Z; Mascola, John R; Haynes, Barton F

    2014-09-10

    Induction of HIV-1 broad neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development but has remained challenging partially due to unusual traits of bnAbs, including high somatic hypermutation (SHM) frequencies and in-frame insertions and deletions (indels). Here we examined the propensity and functional requirement for indels within HIV-1 bnAbs. High-throughput sequencing of the immunoglobulin (Ig) VHDJH genes in HIV-1 infected and uninfected individuals revealed that the indel frequency was elevated among HIV-1-infected subjects, with no unique properties attributable to bnAb-producing individuals. This increased indel occurrence depended only on the frequency of SHM point mutations. Indel-encoded regions were generally proximal to antigen binding sites. Additionally, reconstruction of a HIV-1 CD4-binding site bnAb clonal lineage revealed that a large compound VHDJH indel was required for bnAb activity. Thus, vaccine development should focus on designing regimens targeted at sustained activation of bnAb lineages to achieve the required SHM and indel events.

  6. Relative binding affinities of bisphosphonates for human bone and relationship to antiresorptive efficacy.

    PubMed

    Leu, Chih-Tai; Luegmayr, Eva; Freedman, Leonard P; Rodan, Gideon A; Reszka, Alfred A

    2006-05-01

    Potent bisphosphonates (BPs) preferentially bind bone at sites of active osteoclastic bone resorption, where they are taken up by the osteoclast and inhibit resorption. We tested the hypothesis that BP affinity to human bone affects antiresorptive potency. [(1)(4)C]-Alendronate binding to human bone was saturable and reversible with an apparent Kd of 72 microM by Scatchard analysis. In competition binding assays, unlabeled alendronate (Ki: 61 microM) was slightly more potent than pyrophosphate (Ki = 156 microM) in blocking [(1)(4)C]-alendronate binding. Likewise, most tested BPs, including etidronate (Ki: 91 microM), ibandronate (116 microM), pamidronate (83 microM), risedronate (85 microM) and zoledronate (81 microM), showed comparable affinities. Interestingly, tiludronate (173 microM; P < 0.05 vs. all other BPs) and especially clodronate (806 microM; P > 0.0001 vs. all other BPs) displayed significantly weaker affinity for bone. The weak affinity of clodronate translated into a requirement for 10-fold higher dosing in in vitro bone resorption assays when bone was pretreated with BP and subsequently washed prior to adding osteoclasts. In stark contrast, neither alendronate nor risedronate lost any efficacy after washing the bone surface. These findings suggest that most clinically tested BPs may have similar affinities for human bone. For those with reduced affinity, this may translate into lower potency that necessitates higher dosing.

  7. An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Iuraşcu, Marius-Ionuţ; Marroquin Belaunzanar, Osiris; Cozma, Claudia; Petrausch, Ulf; Renner, Christoph; Przybylski, Michael

    2016-06-01

    HLA-B27 homodimer formation is believed to be a hallmark of HLA-B27 associated spondyloarthritides. Recently, we have generated a homodimer-specific monoclonal antibody (HD6) and have demonstrated that HLA-B27 homodimer complexes are present on monocytes of healthy HLA-B27 gene carriers at low levels, with significantly increased levels at active disease. The capability of the HD6 antibody to discriminate between correctly formed HLA-B27 heterotrimers and pathology-associated homodimers is striking and cannot be explained by the primary structure of HLA-B27. We hypothesized that HD6 accesses a unique epitope and used affinity-mass spectrometry for its identification. The HD6 antibody was immobilized on an activated sepharose affinity column, and HLA-B27 homodimer characterized for affinity. The epitope was identified by proteolytic epitope excision and MALDI mass spectrometry, and shown to comprise a discontinuous Cys-203- 257-Cys mixed-disulfide peptide structure that is not accessible in HLA-B27 heterotrimers due to protection by noncovalently linked β2-microglobulin. The epitope peptides were synthesized by solid phase peptide synthesis, and the two monomeric peptide components, HLA-B27(203-219) and HLA-B27(257-273), as well as the homo- and hetero-dimeric disulfide linked combinations prepared. The affinity binding constants KD towards the antibodies were determined using a surface acoustic wave (SAW) biosensor, and showed the highest affinity with a KD of approximately 40 nM to the HD6 antibody for the (203-219)-SS-(257-273) mixed disulfide epitope.

  8. Preclinical evaluation of multistep targeting of diasialoganglioside GD2 using a IgG-scFv bispecific antibody with high affinity for GD2 and DOTA metal complex

    PubMed Central

    Cheal, Sarah M.; Xu, Hong; Guo, Hong-fen; Zanzonico, Pat B.; Larson, Steven M.; Cheung, Nai-Kong

    2014-01-01

    Bispecific antibodies (BsAb) have proven to be useful targeting vectors for pretargeted radioimmunotherapy (PRIT). We sought to overcome key PRIT limitations such as high renal radiation exposure and immunogenicity (e.g. of streptavidin-antibody fusions), to advance clinical translation of this PRIT strategy for diasialoganglioside GD2-positive (GD2(+)) tumors. For this purpose, a IgG-scFv BsAb was engineered using the sequences for the anti-GD2 humanized monoclonal antibody hu3F8 (1) and C825, a murine scFv antibody with high affinity for the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) complexed with beta-particle emitting radiometals such as 177Lu and 90Y (2, 3). A three-step regimen including hu3F8-C825, a dextran-based clearing agent, and p-aminobenzyl-DOTA radiolabeled with 177Lu (as 177Lu-DOTA-Bn; t1/2 = 6.71 days (d)) was optimized in immunocompromised mice carrying subcutaneous (s.c.) human GD2(+) neuroblastoma (NB) xenografts. Absorbed doses for tumor and normal tissues were ∼85 cGy/MBq and ≤3.7 cGy/MBq, respectively, with therapeutic indicies (TI) of 142 for blood and 23 for kidney. A therapy study (n = 5 per group; tumor volume: 240 ± 160 mm3) with three successive PRIT cycles (total 177Lu: ∼33 MBq; tumor dose ∼3400 cGy), revealed complete tumor response in 5/5 animals, with no recurrence up to 28 d post-treatment. Tumor ablation was confirmed histologically in 4/5 mice, and normal organs showed minimal overall toxicities. All non-treated mice required sacrifice within 12 d (>1.0 cm3 tumor volume). We conclude that this novel anti-GD2 PRIT approach has sufficient TI to successfully ablate s.c. GD2(+)–NB in mice while sparing kidney and bone marrow. PMID:24944121

  9. Combining Phage and Yeast Cell Surface Antibody Display to Identify Novel Cell Type-Selective Internalizing Human Monoclonal Antibodies.

    PubMed

    Bidlingmaier, Scott; Su, Yang; Liu, Bin

    2015-01-01

    Using phage antibody display, large libraries can be generated and screened to identify monoclonal antibodies with affinity for target antigens. However, while library size and diversity is an advantage of the phage display method, there is limited ability to quantitatively enrich for specific binding properties such as affinity. One way of overcoming this limitation is to combine the scale of phage display selections with the flexibility and quantitativeness of FACS-based yeast surface display selections. In this chapter we describe protocols for generating yeast surface antibody display libraries using phage antibody display selection outputs as starting material and FACS-based enrichment of target antigen-binding clones from these libraries. These methods should be widely applicable for the identification of monoclonal antibodies with specific binding properties. PMID:26060069

  10. Prevalence of antibodies to human herpesviruses 6 and 7 in early infancy and age at primary infection.

    PubMed

    Cermelli, C; Fabio, G; Montorsi, M; Sabbatini, A M; Portolani, M

    1996-01-01

    Sera from a sample of children aged 3 months to 6 years and from cord blood were tested in an indirect immunofluorescence assay (IFA) for reactivity to human herpesviruses 6 (HHV-6) and 7 (HHV-7). HHV-6 seropositivity values rose from 19% to 79.3% in the first 18 months of life, while HHV-7 seroprevalence reached a similar value (75.9%) in children aged 3-6 years. These results show that HHV-7, like HHV-6, is a prevalent virus in infancy. In cord blood sera, assayed to study infant humoral situation at birth, similar values for the two viruses (78.9% for HHV-6 and 76.3% for HHV-7) were found. HHV-6 and HHV-7 IgG antibody affinity to the corresponding antigens was assessed by the end point antibody titration in the presence and absence of urea 8M. This test distinguishes antibodies of recent (low affinity) or past (high affinity) production. Together, the data on seroprevalence and antibody affinity suggest that HHV-6 primary infection generally precedes that by HHV-7. These results are discussed in the light of a different pathogenetic role of the two viruses.

  11. Computational protein design suggests that human PCNA-partner interactions are not optimized for affinity.

    PubMed

    Fridman, Yearit; Gur, Eyal; Fleishman, Sarel J; Aharoni, Amir

    2013-02-01

    Increasing the affinity of binding proteins is invaluable for basic and applied biological research. Currently, directed protein evolution experiments are the main approach for generating such proteins through the construction and screening of large mutant libraries. Proliferating cell nuclear antigen (PCNA) is an essential hub protein that interacts with many different partners to tightly regulate DNA replication and repair in all eukaryotes. Here, we used computational design to generate human PCNA mutants with enhanced affinity for several different partners. We identified double mutations in PCNA, outside the main partner binding site, that were predicted to increase PCNA-partner binding affinities compared to the wild-type protein by forming additional hydrophobic interactions with conserved residues in the PCNA partners. Affinity increases were experimentally validated with four different PCNA partners, demonstrating that computational design can reveal unexpected regions where affinity enhancements in natural systems are possible. The designed PCNA mutants can be used as a valuable tool for further examination of the regulation of PCNA-partner interactions during DNA replication and repair both in vitro and in vivo. More broadly, the ability to engineer affinity increases toward several PCNA partners suggests that interaction affinity is not an evolutionarily optimized trait of this system. PMID:23011891

  12. Antibody Response to Cryptococcus neoformans Proteins in Rodents and Humans

    PubMed Central

    Chen, Lin-Chi; Goldman, David L.; Doering, Tamara L.; Pirofski, Liise-anne; Casadevall, Arturo

    1999-01-01

    The prevalence and specificity of serum antibodies to Cryptococcus neoformans proteins was studied in mice and rats with experimental infection, in individuals with or without a history of potential laboratory exposure to C. neoformans, human immunodeficiency virus (HIV)-positive individuals who developed cryptococcosis, in matched samples from HIV-positive individuals who did not develop cryptococcosis, and in HIV-negative individuals. Rodents had little or no serum antibody reactive with C. neoformans proteins prior to infection. The intensity and specificity of the rodent antibody response were dependent on the species, the mouse strain, and the viability of the inoculum. All humans had serum antibodies reactive with C. neoformans proteins regardless of the potential exposure, the HIV infection status, or the subsequent development of cryptococcosis. Our results indicate (i) a high prevalence of antibodies reactive with C. neoformans proteins in the sera of rodents after cryptococcal infection and in humans with or without HIV infection; (ii) qualitative and quantitative differences in the antibody profiles of HIV-positive individuals; and (iii) similarities and differences between humans, mice, and rats with respect to the specificity of the antibodies reactive with C. neoformans proteins. The results are consistent with the view that C. neoformans infections are common in human populations, and the results have implications for the development of vaccination strategies against cryptococcosis. PMID:10225877

  13. Autoimmune anti-androgen-receptor antibodies in human serum.

    PubMed Central

    Liao, S; Witte, D

    1985-01-01

    Circulating autoantibodies to human and rat androgen receptors are present at high titers in the blood sera of some patients with prostate diseases. The antibodies from some serum samples were associated with a purified IgG fraction and interacted with the 3.8S cytosolic androgen-receptor complexes of rat ventral prostate to form 9- to 12S units. Other serum samples, however, formed 14- to 19S units, suggesting that other immunoglobulins might be involved. In the presence of an anti-human immunoglobulin as a second antibody, the androgen-receptor-antibody complexes could be immunoprecipitated. The antibodies interacted with the nuclear and the cytosolic androgen-receptor complexes, either the DNA-binding or the nonbinding form, but not with receptors for estradiol, progestin, or dexamethasone from a variety of sources. Human testosterone/estradiol-binding globulin, rat epididymal androgen-binding protein, or rat prostate alpha-protein (a nonreceptor steroid-binding protein) also did not interact with the antibodies to form immunoprecipitates. About 37% of male and 3% of female serum samples screened had significant antibody titer. The chance of finding serum with a high titer is much better in males older than 66 years than in the younger males or females at all ages. The presence of the high-titer antibodies may make it possible to prepare monoclonal antibodies to androgen receptors without purification of the receptors for immunization. PMID:3866227

  14. Crystal structure of human prostate-specific antigen in a sandwich antibody complex.

    PubMed

    Stura, Enrico A; Muller, Bruno H; Bossus, Marc; Michel, Sandrine; Jolivet-Reynaud, Colette; Ducancel, Frédéric

    2011-12-01

    Human prostate-specific antigen (PSA or human kallikrein-related peptidase 3) present in small quantities in the sera of healthy men becomes elevated in prostate cancer (PCa) and other prostate disorders. The ability to identify the free PSA fraction associated with PCa could increase the reliability of the PSA diagnostic test. Here we present the crystal structure of human PSA from seminal fluid in a sandwich complex with two monoclonal antibodies (mAbs). MAb 5D5A5 captures total PSA with exceptionally high affinity, and mAb 5D3D11 selectively discriminates between free PSA subforms that are more abundant in sera from patients with PCa. Although the antigen is not of seric origin, several insights into cancer diagnosis can be discerned from this complex. MAb 5D3D11 recognizes a PSA conformation different from that previously reported. Interacting with the kallikrein loop, the PSA N-linked glycan attached to asparagine 61 is an uncommonly complex sialated triantennary chain. O-linked glycosylation is observed at threonine 125. The description of how PSA subforms in prostatic fluid can be discriminated using pairs of antibodies is a first step in the design of new strategies that are capable of real discrimination among PSA subforms, which will lead to the formulation of more reliable diagnostic tests. In a companion article [Muller, B. H., Savatier, A., L'Hostis, G., Costa, N., Bossus, M., Michel, S., et al. (2011). In vitro affinity maturation of an anti-PSA antibody for prostate cancer diagnostic assay. J. Mol. Biol.], we describe engineering efforts to improve the affinity of mAb 5D3D11, a first step towards such goal. PMID:22037582

  15. Purification of human immunoglobulins A, G and M from Cohn fraction II/III by small peptide affinity chromatography.

    PubMed

    Liu, Zhuo; Gurgel, Patrick V; Carbonell, Ruben G

    2012-11-01

    This work describes attempts to purify human IgG, IgA and IgM from Cohn fraction II/III using HWRGWV affinity peptide resin. The effects of peptide density and different elution additives on recovery of the three antibodies were investigated. At low peptide density, salting-in salts such as magnesium chloride and calcium chloride facilitated antibody elution. Ethylene glycol, urea and arginine also facilitated elution because of their ability to decrease hydrophobic interactions, hydrogen bonding and electrostatic interactions. However, at high peptide density, no recovery improvements were observed because of increased non-specific hydrophobic interactions. The final elution conditions for each antibody were chosen based on the resulting yields and purities when a 10:2:1mg/mL mixture of human IgG, IgA and IgM was used as starting material. Different pretreatment methods were employed in order to improve the purity of antibodies from Cohn fraction II/III. After pretreatment with caprylic acid precipitation or combination of caprylic acid and polyethylene glycol precipitation, purities over 95% and yields of about 60% were obtained for hIgG, which are comparable to current chromatographic purification methods involving two chromatography steps when hIgG is isolated from plasma fractions. A hIgA-enriched fraction with 42% hIgA and 56% hIgG, as well as a hIgM enriched fraction with 46% hIgM, 28% hIgA and 24% hIgG, were obtained as the by-products. PMID:23026261

  16. Human plasma contains cross-reactive Abeta conformer-specific IgG antibodies.

    PubMed

    O'Nuallain, Brian; Acero, Luis; Williams, Angela D; Koeppen, Helen P McWilliams; Weber, Alfred; Schwarz, Hans P; Wall, Jonathan S; Weiss, Deborah T; Solomon, Alan

    2008-11-25

    Two conformers of aggregated Abeta, i.e., fibrils and oligomers, have been deemed important in the pathogenesis of Alzheimer's disease. We now report that intravenous immune globulin (IVIG) derived from pools of human plasma contains IgGs that recognize conformational epitopes present on fibrils and oligomers, but not their soluble monomeric precursor. We have used affinity chromatography to isolate these antibodies and have shown that they cross-reacted with comparable nanomolar avidity with both types of Abeta aggregates; notably, binding was not inhibited by soluble Abeta monomers. Our studies provide further support for investigating the therapeutic use of IVIG in Alzheimer's disease.

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

    PubMed

    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

  18. 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

  19. A simple nonradioactive method for the determination of the binding affinities of antibodies induced by hapten bioconjugates for drugs of abuse.

    PubMed

    Torres, Oscar B; Antoline, Joshua F G; Li, Fuying; Jalah, Rashmi; Jacobson, Arthur E; Rice, Kenner C; Alving, Carl R; Matyas, Gary R

    2016-02-01

    The accurate analytical measurement of binding affinities of polyclonal antibody in sera to heroin, 6-acetylmorphine (6-AM), and morphine has been a challenging task. A simple nonradioactive method that uses deuterium-labeled drug tracers and equilibrium dialysis (ED) combined with ultra performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) to measure the apparent dissociation constant (K d) of antibodies to 6-AM and morphine is described. The method can readily detect antibodies with K d in the low nanomolar range. Since heroin is rapidly degraded in sera, esterase inhibitors were included in the assay, greatly reducing heroin hydrolysis. MS/MS detection directly measured the heroin in the assay after overnight ED, thereby allowing the quantitation of % bound heroin in lieu of K d as an alternative measurement to assess heroin binding to polyclonal antibody sera. This is the first report that utilizes a solution-based assay to quantify heroin-antibody binding without being confounded by the presence of 6-AM and morphine and to measure K d of polyclonal antibody to 6-AM. Hapten surrogates 6-AcMorHap, 6-PrOxyHap, MorHap, DiAmHap, and DiPrOxyHap coupled to tetanus toxoid (TT) were used to generate high affinity antibodies to heroin, 6-AM, and morphine. In comparison to competition ED-UPLC/MS/MS which gave K d values in the nanomolar range, the commonly used competition enzyme-linked immunosorbent assay (ELISA) measured the 50% inhibition concentration (IC50) values in the micromolar range. Despite the differences in K d and IC50 values, similar trends in affinities of hapten antibodies to heroin, 6-AM, and morphine were observed by both methods. Competition ED-UPLC/MS/MS revealed that among the five TT-hapten bioconjugates, TT-6-AcMorHap and TT-6-PrOxyHap induced antibodies that bound heroin, 6-AM, and morphine. In contrast, TT-MorHap induced antibodies that poorly bound heroin, while TT-DiAmHap and TT-DiPrOxyHap induced antibodies either did not

  20. The binding affinity of a soluble TCR-Fc fusion protein is significantly improved by crosslinkage with an anti-C{beta} antibody

    SciTech Connect

    Ozawa, Tatsuhiko; Horii, Masae; Kobayashi, Eiji; Jin, Aishun; Kishi, Hiroyuki; Muraguchi, Atsushi

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer A novel soluble TCR composed of TCR V and C regions with Ig Fc region is generated. Black-Right-Pointing-Pointer TCR-Fc protein immobilized by an anti-C{beta} antibody bound to a p/MHC tetramer. Black-Right-Pointing-Pointer Binding affinity of TCR-Fc was markedly increased by binding with anti-C{beta} antibody. -- Abstract: The identification and cloning of tumor antigen-specific T cell receptors (TCRs) and the production of the soluble form of the TCR (sTCR) contributed to the development of diagnostic and therapeutic tools for cancer. Recently, several groups have reported the development of technologies for the production of sTCRs. The native sTCR has a very low binding affinity for the antigenic peptide/MHC (p/MHC) complex. In this study, we established a technology to produce high affinity, functional sTCRs. We generated a novel sTCR-Fc fusion protein composed of the TCR V and C regions of the TCR linked to the immunoglobulin (Ig) Fc region. A Western blot analysis revealed that the molecular weight of the fusion protein was approximately 60 kDa under reducing conditions and approximately 100-200 kDa under non-reducing conditions. ELISAs using various antibodies showed that the structure of each domain of the TCR-Fc protein was intact. The TCR-Fc protein immobilized by an anti-C{beta} antibody effectively bound to a p/MHC tetramer. An SPR analysis showed that the TCR-Fc protein had a low binding affinity (KD; 1.1 Multiplication-Sign 10{sup -5} M) to the p/MHC monomer. Interestingly, when the TCR-Fc protein was pre-incubated with an anti-C{beta} antibody, its binding affinity for p/MHC increased by 5-fold (2.2 Multiplication-Sign 10{sup -6} M). We demonstrated a novel method for constructing a functional soluble TCR using the Ig Fc region and showed that the binding affinity of the functional sTCR-Fc was markedly increased by an anti-C{beta} antibody, which is probably due to the stabilization of the V

  1. Selection of high affinity peptide ligands for detection of circulating antibodies in neurocysticercosis.

    PubMed

    da Silva Ribeiro, Vanessa; Manhani, Marianna Nascimento; Cardoso, Rone; Vieira, Carlos Ueira; Goulart, Luiz Ricardo; Costa-Cruz, Julia Maria

    2010-04-01

    Neurocysticercosis (NC), caused by Taenia solium, is the most common infection caused by helminthes of the human central nervous system. In this study, a random peptide phage display library was used to isolate peptide ligands as potential markers for neurocysticercosis diagnosis, because occurrence of cross-reactions with other helminthes species in the current used markers. We selected different peptides using IgG purified from pooled sera of neurocysticercosis patients. To investigate the diagnostic potential of recombinant peptides, we have tested different panels of serum samples by Phage-ELISA, and 10 phage clones strongly bound to the anti-T. solium IgGs in NC sera, with an accuracy range from 84.2% to 95%. The phage clones, NC(4)1 and NC(2)8, presented the highest sensitivity and specificity (100%), respectively, and most important, some phage clones did not react with patients' sera from Echinococcus granulosus infected patients. The validation with a competitive ELISA assay demonstrated that the selected phages could mimic T. solium epitopes and bind specifically to the pool of NC sera. Finally, the two recombinant antigens may become potential biomarkers for serodiagnosis of NC, and the Phage-ELISA demonstrated to be a very good assay, being reproducible, simple, fast, and low-cost due to its production through Escherichia coli culture, allowing a high throughput screening of NC.

  2. Development and Characterization of a New Antipeptide Monoclonal Antibody Directed to Human CD20 Antigen.

    PubMed

    Habibi-Anbouhi, Mahdi; Azadmanesh, Kayhan; Behdani, Mahdi; Hajizadeh-Saffar, Ensiyeh; Vahabpour, Rouhollah; Shokrgozar, Mohammad Ali

    2015-09-01

    The rapid expansion of immunotherapeutic approaches for treatment of various diseases, including cancers, has been greatly facilitated by the invention of new generation of antibodies. Clinical studies have indicated that anti-CD20 mAb-based therapies represent an effective treatment for various diseases with overexpression of CD20 on their cell surface, such as non-Hodgkin's lymphoma, hemolytic anemia, as well as autoimmune diseases like rheumatoid arthritis. Technically, due to a short extra membrane domain, the recombinant CD20 protein is a difficult antigen to raise immune responses. In search for new monoclonal antibodies, the authors used an antigenic polypeptide, which yielded numbers of new binders that may lead to production of anti-CD20 antibodies, with improved diagnostic or clinical attributes. Mice were immunized with extra membrane loop of human CD20 (exCD20) polypeptide. The exCD20 antigen showed a desired immune response and was able to develop a monoclonal antibody, 3B4C10, which reacted well with peptide antigen as well as native antigen on the surface of Raji B-cell line. The antibody 3B4C10 with a balanced K(on) and K(off) may be applicable in the construction of affinity columns or beads for isolation and purification of CD20-positive cells and cancer stem cells. PMID:26352927

  3. Development of Human Monoclonal Antibodies Against Respiratory Syncytial Virus Using a High Efficiency Human Hybridoma Technique.

    PubMed

    Alvarado, Gabriela; Crowe, James E

    2016-01-01

    Human monoclonal antibodies against RSV have high potential for use as prophylaxis or therapeutic molecules, and they also can be used to define the structure of protective epitopes for rational vaccine design. In the past, however, isolation of human monoclonal antibodies was difficult and inefficient. Here, we describe contemporary methods for activation and proliferation of primary human memory B cells followed by cytofusion to non-secreting myeloma cells by dielectrophoresis to generate human hybridomas secreting RSV-specific monoclonal antibodies. We also provide experimental methods for screening human B cell lines to obtain RSV-specific lines, especially lines secreting neutralizing antibodies. PMID:27464688

  4. 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

    2016-01-01

    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

  5. 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

  6. Two monoclonal antibodies selective for human mammary carcinoma.

    PubMed

    White, C A; Dulbecco, R; Allen, R; Bowman, M; Armstrong, B

    1985-03-01

    Mouse myeloma cells were fused with spleen cells from BALB/c mice immunized with the MCF-7 human mammary carcinoma cell line. Among hybridomas, two (3B18 and 15A8) were selected and cloned. Hybridoma 3B18 produces kappa-IgG1 antibodies that react with a cytoplasmic component of MCF-7 cells. In immunoperoxidase assays, 3B18 reacts with 27 of 31 specimens of human mammary carcinoma. It reacts most consistently with poorly differentiated and infiltrating ductal breast cancers, but it also reacts with isolated cells in 3 of 5 benign mammary pathological lesions with a variable distribution. The antibody does not react with normal mammary epithelium. It does not react with any normal human tissues, and it reacts with only one of 19 other cancers tested. Hybridoma 15A8 produces kappa-IgG1 antibodies that react with the surface membranes of the cells of two human breast cancer cell lines but not with a human fibroblast cell line. In immunoperoxidase assays, the antibody reacted with 28 out of 31 human mammary carcinomas. The antibody also reacts more weakly with normal human epithelial cells of breast, renal proximal tubule, skin, esophagus, and salivary gland, but no other normal tissue. The antibody was unreactive with 14 of 18 other malignant tissues tested. Since 3B18 and 15A8 detect antigens found predominantly in human mammary carcinomas and, possibly, distinguish overlapping categories of human mammary carcinomas, they may prove useful in determining the cellular lineage from which human mammary carcinomas arise, or they may have other clinical applications in breast cancer.

  7. Adjuvant dependence of APS pathology-related low-affinity antibodies during secondary immune response to tetanus toxoid in BALB/c mice.

    PubMed

    Zivković, Irena; Petrušić, Vladimir; Dimitrijević, Rajna; Stojanović, Marijana; Dimitrijević, Ljiljana

    2013-05-01

    One of the established animal models for autoimmune disease antiphospholipid syndrome (APS) is TTd hyperimmunization of mice. Tetanus toxoid (TTd) and plasma protein β2GPI share structural homology so that immunization with TTd induces appearance of cross-reactive antibodies. In this paper, we have investigated the presence and dynamic of fluctuation of specific (anti-TTd) and auto (anti-β2GPI) antibodies induced in BALB/c mice during secondary immune response after TTd immunization with alhydrogel or glycerol as adjuvants. In addition, we followed the induced reproductive pathology as a sign of autoimmune outcome. We show undoubtedly adjuvant dependance of (1) level of induced anti-TTd IgG antibodies, (2) changes in levels of low-affinity anti-β2GPI IgG antibodies, and (3) change in fecundity and fertility during secondary immune response. These findings once more indicate the importance of chosen adjuvants used for successful immunization and eventual autoantibody outcome, this time associated with the processes involving low affinity, natural antibodies.

  8. 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.

  9. Isolation of a Trypanosoma cruzi antigen by affinity chromatography with a monoclonal antibody. Preliminary evaluation of its possible applications in serological tests.

    PubMed Central

    Carbonetto, C H; Malchiodi, E L; Chiaramonte, M; Durante de Isola, E; Fossati, C A; Margni, R A

    1990-01-01

    By affinity chromatography with a monoclonal antibody (163B6), obtained in our laboratory, we have isolated a T. cruzi antigen which could be useful for differential diagnosis of Chagas' disease from leishmaniasis. This antigen, a 52-kD protein, reacted with all sera from Chagas' disease patients tested but not with sera from patients with leishmania, in ELISA. The 52-kD antigen is widely distributed in the Trypanosoma genus since the 163B6 monoclonal antibody reacts with T. rangeli and 8 strains and a clone of T. cruzi epimastigotes. Images Fig. 1 Fig. 2 PMID:2119921

  10. Monoclonal antibodies to the cell surface and a soluble form of the human nerve growth factor receptor

    SciTech Connect

    Clagett-Dame, M.; Chung, C.; Chao, M.V.; DiStefano, P.S. )

    1990-12-01

    Monoclonal antibodies (designated IIIG5, VIID1, VIIIC8, and XIF1) have been produced that bind to the human nerve growth factor receptor (NGF-R) as well as to a soluble, truncated form of the receptor (NGF-Rt). The antibodies were generated against partially purified NGF-Rt from the conditioned medium of E9b cells, a transfected mouse fibroblast cell line (Ltk-) that expresses large numbers of the low affinity form of the human NGF-R on its cell surface. Hybridomas were screened by radiometric immunosorbent assay (RISA) and by immunoprecipitation of solubilized cell surface receptor covalently cross-linked to {sup 125}I-NGF. Four positive lines were cloned by limiting dilution and were found to secrete monoclonal antibodies of the IgGl,k subclass. All monoclonal antibodies bound to both NGF-R and NGF-Rt. Two monoclonal antibodies (VIID1, XIF1) immunoblotted the NGF-R from E9b cell preparations resolved on non-reducing sodium dodecyl sulfate (SDS)-polyacrylamide gels. The antibodies immunoprecipitated NGF-R from both E9b cells and from SH-SY5Y human neuroblastoma cells. The monoclonal antibodies bound to monkey (rhesis and cynomolgus) NGF-Rt, but did not cross-react with NGF-R from chick or rat. Results of antibody competition studies demonstrated that three antibodies bound to a similar or overlapping epitope on the NGF-Rt and one monoclonal antibody (IIIG5) recognized a distinct receptor epitope. Antibodies that bound to different sites on the receptor were used to develop a sensitive 2-site RISA. The 2-site RISA can be used to rapidly quantitate NGF-R and NGF-Rt in large numbers of biological samples in the absence of added {sup 125}I-labeled NGF.

  11. Germline V-genes sculpt the binding site of a family of antibodies neutralizing human cytomegalovirus

    SciTech Connect

    Thomson, Christy A.; Bryson, Steve; McLean, Gary R.; Creagh, A. Louise; Pai, Emil F.; Schrader, John W.

    2008-10-17

    Immunoglobulin genes are generated somatically through specialized mechanisms resulting in a vast repertoire of antigen-binding sites. Despite the stochastic nature of these processes, the V-genes that encode most of the antigen-combining site are under positive evolutionary selection, raising the possibility that V-genes have been selected to encode key structural features of binding sites of protective antibodies against certain pathogens. Human, neutralizing antibodies to human cytomegalovirus that bind the AD-2S1 epitope on its gB envelope protein repeatedly use a pair of well-conserved, germline V-genes IGHV3-30 and IGKV3-11. Here, we present crystallographic, kinetic and thermodynamic analyses of the binding site of such an antibody and that of its primary immunoglobulin ancestor. These show that these germline V-genes encode key side chain contacts with the viral antigen and thereby dictate key structural features of the hypermutated, high-affinity neutralizing antibody. V-genes may thus encode an innate, protective immunological memory that targets vulnerable, invariant sites on multiple pathogens.

  12. Analysis of heavy and light chain pairings indicates that receptor editing shapes the human antibody repertoire.

    PubMed

    de Wildt, R M; Hoet, R M; van Venrooij, W J; Tomlinson, I M; Winter, G

    1999-01-22

    In the bone marrow, diversity in the primary antibody repertoire is created by the combinatorial rearrangement of different gene segments and by the association of different heavy and light chains. During the secondary response in the germinal centres, antibodies are diversified by somatic mutation and possibly by further rearrangements, or "receptor editing". Here, we have analysed the pairings of heavy and light chain variable domains (VH and VL) in 365 human IgG+ B cells from peripheral blood, and established that these pairings are largely random. The repertoire is dominated by a limited number of pairings of segments and folds. Among these pairings we identified two identical mutated heavy chains in combination with two different mutated light chains (one kappa and one lambda). This shows that receptor editing occurs in the human periphery and that the same antibody lineage can be subjected to both receptor editing and somatic hypermutation. This suggests that receptor editing may be used together with somatic mutation for the affinity maturation of antibodies. We also propose that receptor editing has shaped variable gene segment use and the evolution of V gene families.

  13. Paratope diversity in the human antibody response to Bacillus anthracis protective antigen.

    PubMed

    Zhou, Jianhui; Ullal, Anuska; Liberato, Justine; Sun, Jinying; Keitel, Wendy; Reason, Donald C

    2008-01-01

    The active component of the licensed human anthrax vaccine (BioThrax, or AVA) is a Bacillus anthracis toxin known as protective antigen (PA). Second generation anthrax vaccines currently under development are also based on a recombinant form of PA. Since the current and future anthrax vaccines are based on this toxin, it is important that the immunobiology of this protein in vaccinated humans be understood in detail. We have isolated and analyzed the PA-specific antibody repertoire from an AVA-vaccinated individual. When examined at the clonal level, we find an antibody response that is complex in terms of the combinatorial elements and immunoglobulin variable genes employed. All PA-specific antibodies had undergone somatic hypermutation and class switch recombination, both signs of affinity maturation. Although the antigenic epitopes recognized by the response were distributed throughout the PA monomer, the majority of antibodies arising in this individual following vaccination recognize determinants located on the amino-terminal (PA20) sub-domain of the molecule. This latter finding may have implications for the rational design of future PA-based anthrax vaccines.

  14. Single-domain antibody-based ligands for immunoaffinity separation of recombinant human lactoferrin from the goat lactoferrin of transgenic goat milk.

    PubMed

    Tillib, S V; Privezentseva, M E; Ivanova, T I; Vasilev, L F; Efimov, G A; Gursky, Y G; Georgiev, G P; Goldman, I L; Sadchikova, E R

    2014-02-15

    Single-domain antibody generation technology was applied to make new Sepharose-bound ligands for affinity separation of closely related proteins, such as human and goat lactoferrin. We generated recombinant antibodies that can selectively bind/recognize only lactoferrins having amino acid sequences identical to that of human natural lactoferrin (anti-hLF Ab). Selected and purified histidine-tagged single-domain antibodies were used as ligands, and different lactoferrins were used as analytes in the kinetics analysis of lactoferrin binding to captured anti-hLF Abs using the Bio-Rad ProteOn XPR36 protein interaction array system. The data obtained were consistent with a 1:1 binding model with very high affinity, practically equal in the case of hLF and rec-hLF (calculated KD varied from 0.43nM to 3.7nM). Interaction of captured fsdAbs with goat LF was significantly weaker and not detectable under the same analysis conditions. We demonstrated the high efficiency of the recombinant human lactoferrin purification from goat lactoferrin and other proteins using the obtained single domain antibody-based affinity ligands. We believe this approach can be used for the generation of single-domain antibody-based affinity media for the efficient separation/purification of a wide spectrum of other highly homologous proteins.

  15. Construction of a Semisynthetic Human VH Single-Domain Antibody Library and Selection of Domain Antibodies against α-Crystalline of Mycobacterium tuberculosis.

    PubMed

    Hairul Bahara, Nur Hidayah; Chin, Siang Tean; Choong, Yee Siew; Lim, Theam Soon

    2016-01-01

    The use of human variable heavy (VH) domain antibodies has been on the rise due to their small scaffold size and simple folding mechanism. A highly diverse library is largely dependent on the diversity introduced within the complementarity-determining region (CDR) cassettes. Here we introduced diversity with the use of a single framework diversifying all three CDRs using tailored codons consisting of degenerate trinucleotides (NNK). The length of the degeneracy in the CDRs was also taken into consideration based on the most frequently occurring length of CDRs and the canonical confirmation for each antibody subfamily. The semisynthetic human VH domain genes were assembled in a single pot using a temperature cascading process. The affinity selection process with Mycobacterium tuberculosis (MTb) α-crystalline was done using a semiautomated process. Enrichment of target-specific clones was observed with successful identification of monoclonal VH domain antibodies for MTb α-crystalline. In short, the semisynthetic library generated was able to select monoclonal VH domain antibodies against full MTb α-crystalline protein with complete semisynthetic CDRs displayed on a single scaffold. The library has the potential to be applied for the isolation of antibodies against other pathogenic proteins.

  16. Heterologous prime-boost vaccination with MF59-adjuvanted H5 vaccines promotes antibody affinity maturation towards the hemagglutinin HA1 domain and broad H5N1 cross-clade neutralization.

    PubMed

    Khurana, Surender; Coyle, Elizabeth M; Dimitrova, Milena; Castellino, Flora; Nicholson, Karl; Del Giudice, Giuseppe; Golding, Hana

    2014-01-01

    In an open label clinical study (2007), MF59-adjuvanted hemagglutinin (HA) vaccine from H5N1-A/Vietnam/1194/2004 (clade 1) was administered to subjects previously vaccinated (primed) with clade 0 H5N3 (A/duck/Singapore/97) vaccine at least 6 years earlier (in 1999 or 2001). The primed individuals responded rapidly and generated high neutralizing antibody titers against the H5N1-Vietnam strain within 7 days of a single booster vaccination. Furthermore, significant cross-neutralization titers were measured against H5N1 clade 0, 1, and 2 viruses. In the current study, the impact of MF59 adjuvant during heterologous priming on the quality of humoral polyclonal immune response in different vaccine arms were further evaluated using real time kinetics assay by surface plasmon resonance (SPR). Total anti-H5N1 HA1 polyclonal sera antibody binding from the heterologous prime-boost groups after a single MF59-H5N1 boost was significantly higher compared with sera from unprimed individuals that received two MF59-H5N1 vaccinations. The antigen-antibody complex dissociation rates (surrogate for antibody affinity) of the polyclonal sera against HA1 of H5N1-A/Vietnam/1194/2004 from the MF59-H5N3 primed groups were significantly higher compared to sera from unadjuvanted primed groups or unprimed individuals that received two MF59-H5N1 vaccines. Furthermore, strong inverse correlations were observed between the antibody dissociation off-rates of the immune sera against HA1 (but not HA2) and the virus neutralization titers against H5 vaccine strains and heterologous H5N1 strains. These findings supports the use of oil-in-water-adjuvanted pandemic influenza vaccines to elicit long term memory B cells with high affinity BCR capable of responding to potential variant pandemic viruses likely to emerge and adapt to human transmissions.

  17. Separation of human breast cancer cells from blood by differential dielectric affinity.

    PubMed Central

    Becker, F F; Wang, X B; Huang, Y; Pethig, R; Vykoukal, J; Gascoyne, P R

    1995-01-01

    Electrorotation measurements were used to demonstrate that the dielectric properties of the metastatic human breast cancer cell line MDA231 were significantly different from those of erythrocytes and T lymphocytes. These dielectric differences were exploited to separate the cancer cells from normal blood cells by appropriately balancing the hydrodynamic and dielectrophoretic forces acting on the cells within a dielectric affinity column containing a microelectrode array. The operational criteria for successful particle separation in such a column are analyzed and our findings indicate that the dielectric affinity technique may prove useful in a wide variety of cell separation and characterization applications. Images Fig. 3 PMID:7846067

  18. Novel asymmetrically engineered antibody Fc variant with superior FcγR binding affinity and specificity compared with afucosylated Fc variant

    PubMed Central

    Mimoto, Futa; Igawa, Tomoyuki; Kuramochi, Taichi; Katada, Hitoshi; Kadono, Shojiro; Kamikawa, Takayuki; Shida-Kawazoe, Meiri; Hattori, Kunihiro

    2013-01-01

    Fc engineering is a promising approach to enhance the antitumor efficacy of monoclonal antibodies (mAbs) through antibody-dependent cell-mediated cytotoxicity (ADCC). Glyco- and protein-Fc engineering have been employed to enhance FcγR binding and ADCC activity of mAbs; the drawbacks of previous approaches lie in their binding affinity to both FcγRIIIa allotypes, the ratio of activating FcγR binding to inhibitory FcγR binding (A/I ratio) or the melting temperature (TM) of the CH2 domain. To date, no engineered Fc variant has been reported that satisfies all these points. Herein, we present a novel Fc engineering approach that introduces different substitutions in each Fc domain asymmetrically, conferring optimal binding affinity to FcγR and specificity to the activating FcγR without impairing the stability. We successfully designed an asymmetric Fc variant with the highest binding affinity for both FcγRIIIa allotypes and the highest A/I ratio compared with previously reported symmetrically engineered Fc variants, and superior or at least comparable in vitro ADCC activity compared with afucosylated Fc variants. In addition, the asymmetric Fc engineering approach offered higher stability by minimizing the use of substitutions that reduce the TM of the CH2 domain compared with the symmetric approach. These results demonstrate that the asymmetric Fc engineering platform provides best-in-class effector function for therapeutic antibodies against tumor antigens. PMID:23406628

  19. Single Cycle Structure-Based Humanization of an Anti-Nerve Growth Factor Therapeutic Antibody

    PubMed Central

    Covaceuszach, Sonia; Marinelli, Sara; Krastanova, Ivet; Ugolini, Gabriele; Pavone, Flaminia; Lamba, Doriano; Cattaneo, Antonino

    2012-01-01

    Most forms of chronic pain are inadequately treated by present therapeutic options. Compelling evidence has accumulated, demonstrating that Nerve Growth Factor (NGF) is a key modulator of inflammatory and nociceptive responses, and is a promising target for the treatment of human pathologies linked to chronic and inflammatory pain. There is therefore a growing interest in the development of therapeutic molecules antagonising the NGF pathway and its nociceptor sensitization actions, among which function-blocking anti-NGF antibodies are particularly relevant candidates. In this respect, the rat anti-NGF αD11 monoclonal antibody (mAb) is a potent antagonist, able to effectively antagonize rodent and human NGF in a variety of in vitro and in vivo systems. Here we show that mAb αD11 displays a significant analgesic effect in two different models of persistent pain in mice, with a remarkable long-lasting activity. In order to advance αD11 mAb towards its clinical application in man, anti-NGF αD11 mAb was humanized by applying a novel single cycle strategy based on the a priori experimental determination of the crystal and molecular structure of the parental Fragment antigen-binding (Fab). The humanized antibody (hum-αD11) was tested in vitro and in vivo, showing that the binding mode and the NGF neutralizing biological activities of the parental antibody are fully preserved, with even a significant affinity improvement. The results firmly establish hum-αD11 as a lead candidate for clinical applications in a therapeutic area with a severe unmet medical need. More generally, the single-cycle structure-based humanization method represents a considerable improvement over the standard humanization methods, which are intrinsically empirical and require several refinement cycles. PMID:22403636

  20. Mechanism of high affinity inhibition of the human urate transporter URAT1

    PubMed Central

    Tan, Philip K.; Ostertag, Traci M.; Miner, Jeffrey N.

    2016-01-01

    Gout is caused by elevated serum urate levels, which can be treated using inhibitors of the uric acid transporter, URAT1. We exploited affinity differences between the human and rat transporters to map inhibitor binding sites in URAT1. Human-rat transporter chimeras revealed that human URAT1 serine-35, phenylalanine-365 and isoleucine-481 are necessary and sufficient to provide up to a 100-fold increase in affinity for inhibitors. Moreover, serine-35 and phenylalanine-365 are important for high-affinity interaction with the substrate urate. A novel URAT1 binding assay provides support for direct interaction with these amino acids; thus, current clinically important URAT1 inhibitors likely bind the same site in URAT1. A structural model suggests that these three URAT1 residues are in close proximity potentially projecting within the channel. Our results indicate that amino acids from several transmembrane segments functionally cooperate to form a high-affinity URAT1 inhibitor binding site that, when occupied, prevents substrate interactions. PMID:27713539

  1. Evaluation of capillary chromatographic supports for immobilized human purine nucleoside phosphorylase in frontal affinity chromatography studies.

    PubMed

    de Moraes, Marcela Cristina; Temporini, Caterina; Calleri, Enrica; Bruni, Giovanna; Ducati, Rodrigo Gay; Santos, Diógenes Santiago; Cardoso, Carmen Lucia; Cass, Quezia Bezerra; Massolini, Gabriella

    2014-04-18

    The aim of this work was to optimize the preparation of a capillary human purine nucleoside phosphorylase (HsPNP) immobilized enzyme reactor (IMER) for characterization and affinity screening studies of new inhibitors by frontal affinity chromatography coupled to mass spectrometry (FAC-MS). For this purpose two monolithic supports, a Chromolith Speed Rod (0.1mm I.D.×5cm) and a methacrylate-based monolithic epoxy polymeric capillary column (0.25mm I.D.×5cm) with epoxy reactive groups were considered and compared to an IMER previously developed using an open fused silica capillary. Each HsPNP-IMER was characterized in terms of catalytic activity using Inosine as standard substrate. Furthermore, they were also explored for affinity ranking experiments. Kd determination was carried out with the based fused silica HsPNP-IMER and the results are herein discussed.

  2. Influence of the galloyl moiety in tea catechins on binding affinity for human serum albumin.

    PubMed

    Minoda, Kanako; Ichikawa, Tatsuya; Katsumata, Tomoharu; Onobori, Ken-ichi; Mori, Taiki; Suzuki, Yukiko; Ishii, Takeshi; Nakayama, Tsutomu

    2010-01-01

    The major catechins of green tea extract are (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg). Recent research has indicated that catechins form complexes with human serum albumin (HSA) in blood, and differences in their binding affinity toward HSA are believed to modulate their bioavailability. In this study, we kinetically investigated the interaction between the catechins and HSA immobilized on a quartz-crystal microbalance (QCM). The association constants obtained from the frequency changes of QCM revealed interactions of ECg and EGCg with HSA that are 100 times stronger than those of EC and EGC. Furthermore, comparisons of these catechins by native-gel electrophoresis/blotting with redox-cycling staining revealed that, in a phosphate buffer, ECg and EGCg have a higher binding affinity toward HSA than EC and EGC. These observations indicate that catechins with a galloyl moiety have higher binding affinities toward HSA than catechins lacking a galloyl moiety.

  3. Broadly neutralizing human monoclonal JC polyomavirus VP1-specific antibodies as candidate therapeutics for progressive multifocal leukoencephalopathy.

    PubMed

    Jelcic, Ivan; Combaluzier, Benoit; Jelcic, Ilijas; Faigle, Wolfgang; Senn, Luzia; Reinhart, Brenda J; Ströh, Luisa; Nitsch, Roger M; Stehle, Thilo; Sospedra, Mireia; Grimm, Jan; Martin, Roland

    2015-09-23

    In immunocompromised individuals, JC polyomavirus (JCPyV) may mutate and gain access to the central nervous system resulting in progressive multifocal leukoencephalopathy (PML), an often fatal opportunistic infection for which no treatments are currently available. Despite recent progress, the contribution of JCPyV-specific humoral immunity to controlling asymptomatic infection throughout life and to eliminating JCPyV from the brain is poorly understood. We examined antibody responses against JCPyV major capsid protein VP1 (viral protein 1) variants in the serum and cerebrospinal fluid (CSF) of healthy donors (HDs), JCPyV-positive multiple sclerosis patients treated with the anti-VLA-4 monoclonal antibody natalizumab (NAT), and patients with NAT-associated PML. Before and during PML, CSF antibody responses against JCPyV VP1 variants show "recognition holes"; however, upon immune reconstitution, CSF antibody titers rise, then recognize PML-associated JCPyV VP1 variants, and may be involved in elimination of the virus. We therefore reasoned that the memory B cell repertoire of individuals who recovered from PML could be a source for the molecular cloning of broadly neutralizing antibodies for passive immunization. We generated a series of memory B cell-derived JCPyV VP1-specific human monoclonal antibodies from HDs and a patient with NAT-associated PML-immune reconstitution inflammatory syndrome (IRIS). These antibodies exhibited diverse binding affinity, cross-reactivity with the closely related BK polyomavirus, recognition of PML-causing VP1 variants, and JCPyV neutralization. Almost all antibodies with exquisite specificity for JCPyV, neutralizing activity, recognition of all tested JCPyV PML variants, and high affinity were derived from one patient who had recovered from PML. These antibodies are promising drug candidates for the development of a treatment of PML. PMID:26400911

  4. Hybridization of an Aβ-specific antibody fragment with aminopyrazole-based β-sheet ligands displays striking enhancement of target affinity.

    PubMed

    Hellmert, Marco; Müller-Schiffmann, Andreas; Peters, Max Sena; Korth, Carsten; Schrader, Thomas

    2015-03-14

    Determining Aβ levels in body fluids remains a powerful tool in the diagnostics of Alzheimer's disease. This report delineates a new supramolecular strategy which increases the affinity of antibodies towards Aβ to make diagnostic procedures more sensitive. A monoclonal antibody IC16 was generated to an N-terminal epitope of Aβ and the variable regions of the heavy and light chains were cloned as a recombinant protein (scFv). A 6 × histidine tag was fused to the C-terminus of IC16-scFv allowing hybridization with a small organic β-sheet binder via Ni-NTA complexation. On the other hand, a multivalent nitrilotriacetic acid (NTA)-equipped trimeric aminopyrazole (AP) derivative was synthesized based on a cyclam platform; and experimental evidence was obtained for efficient Ni(2+)-mediated complex formation with the histidine-tagged antibody species. In a proof of principle experiment the hybrid molecule showed a strong increase in affinity towards Aβ. Thus, the specific binding power of recombinant antibody fragments to their β-sheet rich targets can be conveniently enhanced by non-covalent hybridization with small organic β-sheet binders.

  5. Current status of cancer immunodetection with radiolabeled human monoclonal antibodies.

    PubMed

    De Jager, R; Abdel-Nabi, H; Serafini, A; Pecking, A; Klein, J L; Hanna, M G

    1993-04-01

    The use of radiolabeled murine monoclonal antibodies (MoAbs) for cancer immunodetection has been limited by the development of human antimouse antibodies (HAMA). Human monoclonal antibodies do not elicit a significant human antihuman (HAHA) response. The generation and production of human monoclonal antibodies met with technical difficulties that resulted in delaying their clinical testing. Human monoclonal antibodies of all isotypes have been obtained. Most were immunoglobulin (Ig) M directed against intracellular antigens. Two antibodies, 16.88 (IgM) and 88BV59 (IgG3k), recognize different epitopes on a tumor-associated antigen, CTA 16.88, homologous to cytokeratins 8, 18, and 19. CTA 16.88 is expressed by most epithelial-derived tumors including carcinomas of the colon, pancreas, breast, ovary, and lung. The in vivo targeting by these antibodies is related to their localization in nonnecrotic areas of tumors. Repeated administration of 16.88 over 5 weeks to a cumulative dose of 1,000 mg did not elicit a HAHA response. Two of 53 patients developed a low titer of HAHA 1 to 3 months after a single administration of 88BV59. Planar imaging of colorectal cancer with Iodine-131 (131I)-16.88 was positive in two studies in 9 of 12 and 16 of 20 patients preselected by immunohistochemistry. Tumors less than 2 cm in diameter are usually not detected. The lack of immunogenicity and long tumor residence time (average = 17 days) makes 16.88 a good candidate for therapy. Radioimmunlymphoscintigraphy with indium-111 (111In)-LiLo-16.88 administered by an intramammary route was used in the presurgical staging of primary breast cancer. The negative predictive value of lymph node metastases for tumors less than 3 cm was 90.5%. Planar and single photon emission computed tomography imaging of colorectal carcinoma with technetium-99m (99mTc) 88BV59 was compared with computed tomography (CT) scan in 36 surgical patients. The antibody scan was more sensitive than the CT scan in detecting

  6. Characterization of a low molecular weight protein of the ATP synthetase complex from beef heart and rat liver mitochondria with a high affinity monoclonal antibody.

    PubMed

    Woldegiorgis, G; Contreras, L; Shrago, E

    1990-06-15

    A monoclonal antibody raised against beef heart mitochondria elicited a strong reaction on Western Blot with a 16 kD protein in preparations of beef heart mitochondria, ammonia particles, oligomycin sensitive ATPase and Complex V, in addition to showing a lesser affinity for the partially purified 30 kD ADP/ATP carrier. The antibody also reacted with a 17 kD protein in rat liver mitochondria and an enriched membrane vesicle fraction. The N-terminal sequence of the first twenty amino acids of both the beef heart and rat liver proteins contained significant homology. Comparison with results in the literature indicate that the proteins represent the delta subunit of the ATP synthetase complex. Further evidence suggests that the epitope for the antibody may reside at the C-terminal 30-40 amino acid residues of both proteins.

  7. Development of neutralizing monoclonal antibodies for oncogenic human papillomavirus types 31, 33, 45, 52, and 58.

    PubMed

    Brown, Martha J; Seitz, Hanna; Towne, Victoria; Müller, Martin; Finnefrock, Adam C

    2014-04-01

    Human papillomavirus (HPV) is the etiological agent for all cervical cancers, a significant number of other anogenital cancers, and a growing number of head and neck cancers. Two licensed vaccines offer protection against the most prevalent oncogenic types, 16 and 18, responsible for approximately 70% of cervical cancer cases worldwide and one of these also offers protection against types 6 and 11, responsible for 90% of genital warts. The vaccines are comprised of recombinantly expressed major capsid proteins that self-assemble into virus-like particles (VLPs) and prevent infection by eliciting neutralizing antibodies. Adding the other frequently identified oncogenic types 31, 33, 45, 52, and 58 to a vaccine would increase the coverage against HPV-induced cancers to approximately 90%. We describe the generation and characterization of panels of monoclonal antibodies to these five additional oncogenic HPV types, and the selection of antibody pairs that were high affinity and type specific and recognized conformation-dependent neutralizing epitopes. Such characteristics make these antibodies useful tools for monitoring the production and potency of a prototype vaccine as well as monitoring vaccine-induced immune responses in the clinic. PMID:24574536

  8. Detection of RSV Antibodies in Human Plasma by Enzyme Immunoassays.

    PubMed

    Jadhao, Samadhan J; Anderson, Larry J

    2016-01-01

    Enzyme immunoassays (EIAs) to detect and quantify antibodies against respiratory syncytial virus (RSV) and RSV proteins in human plasma or sera are described. The first EIA uses RSV lysate antigens produced in HEp-2 cell line. The second EIA uses RSV F or G gene-expressed antigen in HEp-2 cells. The third EIA uses 30-amino acid synthetic peptides from central conserved region of G protein of RSV A2 or RSV B1 virus and a peptide from the SARS CoV nucleoprotein as a negative control peptide. All three EIAs have been evaluated for detecting and quantifying the respective antibodies in human sera or plasma. PMID:27464686

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

    PubMed

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

    2016-04-22

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

  10. The detection of circulating antibody in human toxocara infections using the indirect fluorescent antibody test

    PubMed Central

    Bisseru, B.; Woodruff, A. W.

    1968-01-01

    The indirect fluorescent antibody test has been used to detect Toxocara antibodies quantitatively in human sera. The results obtained correlate well with those obtained with the toxocara skin test. Cross reactions have been found with Ascaris lumbricoides and about one in five normal sera give a significant fluorescent reaction. The specificity of the test has been established by Ascaris adsorption studies and by a lack of cross reaction with sera from other helminthic infections. The test may be especially easy to use and therefore particularly valuable in regions where A. lumbricoides infection is relatively rare. Images PMID:4880409

  11. Rescue and expression of human immunoglobulin genes to generate functional human monoclonal antibodies.

    PubMed

    Lewis, A P; Parry, N; Peakman, T C; Crowe, J S

    1992-07-01

    Human monoclonal antibody production has been hampered for many years by the instability of cell lines and low levels of expression of the antibodies. We describe here the rescue of human immunoglobulin genes utilizing micro-mRNA preparation from a small number of human hybridoma cells and conventional cDNA cloning. This allows cloning and immediate high-level expression from full-length human heavy and light chain cDNA molecules and provides a mechanism to rescue whole human monoclonal antibodies of proven efficacy.

  12. Dissection of the Antibody Response against Herpes Simplex Virus Glycoproteins in Naturally Infected Humans

    PubMed Central

    Huang, Zhen-Yu; Whitbeck, J. Charles; Ponce de Leon, Manuel; Lou, Huan; Wald, Anna; Krummenacher, Claude; Eisenberg, Roselyn J.; Cohen, Gary H.

    2014-01-01

    ABSTRACT Relatively little is known about the extent of the polyclonal antibody (PAb) repertoire elicited by herpes simplex virus (HSV) glycoproteins during natural infection and how these antibodies affect virus neutralization. Here, we examined IgGs from 10 HSV-seropositive individuals originally classified as high or low virus shedders. All PAbs neutralized virus to various extents. We determined which HSV entry glycoproteins these PAbs were directed against: glycoproteins gB, gD, and gC were recognized by all sera, but fewer sera reacted against gH/gL. We previously characterized multiple mouse monoclonal antibodies (MAbs) and mapped those with high neutralizing activity to the crystal structures of gD, gB, and gH/gL. We used a biosensor competition assay to determine whether there were corresponding human antibodies to those epitopes. All 10 samples had neutralizing IgGs to gD epitopes, but there were variations in which epitopes were seen in individual samples. Surprisingly, only three samples contained neutralizing IgGs to gB epitopes. To further dissect the nature of these IgGs, we developed a method to select out gD- and gB-specific IgGs from four representative sera via affinity chromatography, allowing us to determine the contribution of antibodies against each glycoprotein to the overall neutralization capacity of the serum. In two cases, gD and gB accounted for all of the neutralizing activity against HSV-2, with a modest amount of HSV-1 neutralization directed against gC. In the other two samples, the dominant response was to gD. IMPORTANCE Antibodies targeting functional epitopes on HSV entry glycoproteins mediate HSV neutralization. Virus-neutralizing epitopes have been defined and characterized using murine monoclonal antibodies. However, it is largely unknown whether these same epitopes are targeted by the humoral response to HSV infection in humans. We have shown that during natural infection, virus-neutralizing antibodies are principally

  13. Inhibition of fibroblast growth factor receptor 3-dependent lung adenocarcinoma with a human monoclonal antibody

    PubMed Central

    Yin, Yongjun; Ren, Xiaodi; Smith, Craig; Guo, Qianxu; Malabunga, Maria; Guernah, Ilhem; Zhang, Yiwei; Shen, Juqun; Sun, Haijun; Chehab, Nabil; Loizos, Nick; Ludwig, Dale L.; Ornitz, David M.

    2016-01-01

    ABSTRACT Activating mutations in fibroblast growth factor receptor 3 (FGFR3) have been identified in multiple types of human cancer and in congenital birth defects. In human lung cancer, fibroblast growth factor 9 (FGF9), a high-affinity ligand for FGFR3, is overexpressed in 10% of primary resected non-small cell lung cancer (NSCLC) specimens. Furthermore, in a mouse model where FGF9 can be induced in lung epithelial cells, epithelial proliferation and ensuing tumorigenesis is dependent on FGFR3. To develop new customized therapies for cancers that are dependent on FGFR3 activation, we have used this mouse model to evaluate a human monoclonal antibody (D11) with specificity for the extracellular ligand-binding domain of FGFR3, that recognizes both human and mouse forms of the receptor. Here, we show that D11 effectively inhibits signaling through FGFR3 in vitro, inhibits the growth of FGFR3-dependent FGF9-induced lung adenocarcinoma in mice, and reduces tumor-associated morbidity. Given the potency of FGF9 in this mouse model and the absolute requirement for signaling through FGFR3, this study validates the D11 antibody as a potentially useful and effective reagent for treating human cancers or other pathologies that are dependent on activation of FGFR3. PMID:27056048

  14. 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.

  15. Human cysticercosis: antigens, antibodies and non-responders.

    PubMed Central

    Flisser, A; Woodhouse, E; Larralde, C

    1980-01-01

    Immunoelectrophoresis of sera from patients with brain cysticercosis against a crude antigenic extract from Cysticercus cellulosae indicates that nearly 50% of the patients do not make sufficient antibodies to ostensively precipitate. The other 50% of the patients who do make precipitating antibodies show a very heterogeneous response in the number of antigens they recognize as well as in the type of antigen--as classified by their electrophoretic mobilities. The most favoured, called antigen B, is recognized by 84% of positive sera and corresponds to one or a limited number of antigens isoelectric at pH 8.6. Indirect immunofluorescence with monospecific anti-human immunoglobulins, performed upon the immunoelectrophoretic preparations, reveal that all cysticercus antigens induced the synthesis of antibodies in the immunoglobulin classes in the order G greater than M greater than E greater than A greater than D. Finally, antigen H (an anodic component) seems to favour IgE relative to its ability to induce IgG. Thus, although in natural infection a good proportion of cysticercotic patients do not seem to mount an energetic antibody response against the parasite, giving rise to some speculations about immunosuppression, the fact that 50% do synthesize antibodies allows for some optimistic expectations from vaccination of humans--in view of the good results of vaccination in experimental animals mediated by IgG antibodies. A likely prospect for a human vaccine would be antigen B because it is the most frequently detected by humans, although its immunizing and toxic properties remain to be properly studied. Images FIG. 1 FIG. 3 FIG. 6 PMID:7389197

  16. Human cysticercosis: antigens, antibodies and non-responders.

    PubMed

    Flisser, A; Woodhouse, E; Larralde, C

    1980-01-01

    Immunoelectrophoresis of sera from patients with brain cysticercosis against a crude antigenic extract from Cysticercus cellulosae indicates that nearly 50% of the patients do not make sufficient antibodies to ostensively precipitate. The other 50% of the patients who do make precipitating antibodies show a very heterogeneous response in the number of antigens they recognize as well as in the type of antigen--as classified by their electrophoretic mobilities. The most favoured, called antigen B, is recognized by 84% of positive sera and corresponds to one or a limited number of antigens isoelectric at pH 8.6. Indirect immunofluorescence with monospecific anti-human immunoglobulins, performed upon the immunoelectrophoretic preparations, reveal that all cysticercus antigens induced the synthesis of antibodies in the immunoglobulin classes in the order G greater than M greater than E greater than A greater than D. Finally, antigen H (an anodic component) seems to favour IgE relative to its ability to induce IgG. Thus, although in natural infection a good proportion of cysticercotic patients do not seem to mount an energetic antibody response against the parasite, giving rise to some speculations about immunosuppression, the fact that 50% do synthesize antibodies allows for some optimistic expectations from vaccination of humans--in view of the good results of vaccination in experimental animals mediated by IgG antibodies. A likely prospect for a human vaccine would be antigen B because it is the most frequently detected by humans, although its immunizing and toxic properties remain to be properly studied.

  17. The high-affinity receptor for IgG, FcγRI, of humans and non-human primates.

    PubMed

    Chenoweth, Alicia M; Trist, Halina M; Tan, Peck-Szee; Wines, Bruce D; Hogarth, P Mark

    2015-11-01

    Non-human primate (NHP) models, especially involving macaques, are considered important models of human immunity and have been essential in preclinical testing for vaccines and therapeutics. Despite this, much less characterization of macaque Fc receptors has occurred compared to humans or mice. Much of the characterization of macaque Fc receptors so far has focused on the low-affinity Fc receptors, particularly FcγRIIIa. From these studies, it is clear that there are distinct differences between the human and macaque low-affinity receptors and their interaction with human IgG. Relatively little work has been performed on the high-affinity IgG receptor, FcγRI, especially in NHPs. This review will focus on what is currently known of how FcγRI interacts with IgG, from mutation studies and recent crystallographic studies of human FcγRI, and how amino acid sequence differences in the macaque FcγRI may affect this interaction. Additionally, this review will look at the functional consequences of differences in the amino acid sequences between humans and macaques.

  18. Binding affinity and agonist activity of putative endogenous cannabinoids at the human neocortical CB1 receptor.

    PubMed

    Steffens, Marc; Zentner, Josef; Honegger, Jürgen; Feuerstein, Thomas J

    2005-01-01

    We investigated the affinity of putative endocannabinoids (2-arachidonylglycerol, 2-AG; noladin ether, virodhamine) for the human neocortical CB1 receptor. Functional activity of these compounds (including anandamide, AEA) was determined by examining basal and forskolin-stimulated cAMP formation. Assays were performed with synaptosomes, prepared from fresh human neocortical tissue. Receptor affinity was assessed from competition binding experiments with the CB1/2 agonist [3H]-CP55.940 in absence or presence of a protease inhibitor to assess enzymatic stability. Noladin ether and virodhamine inhibited [3H]-CP55.940 binding (Ki: 98, 1740 nM, respectively). Protease inhibition decreased the Ki value of virodhamine (Ki: 912 nM), but left that of noladin ether unchanged. 2-AG almost lacked affinity (Ki lymphoblasic )10 microM). Basal cAMP formation was unaffected by AEA and noladin ether, but strongly enhanced by 2-AG and virodhamine. Forskolin-stimulated cAMP formation was inhibited by AEA and noladin ether (IC50: 69, 427 nM, respectively) to the same extent as by CP55.940 (Imax each approximately 30%). Inhibitions by AEA or noladin ether were blocked by the CB1 receptor antagonist AM251. Virodhamine increased forskolin-stimulated cAMP formation, also in presence of AM251, by approximately 20%. 2-AG had no effect; in presence of AM251, however, 10 microM 2-AG stimulated cAMP formation by approximately 15%. Our results suggest, that AEA and noladin ether are full CB1 receptor agonists in human neocortex, whereas virodhamine may act as a CB1 receptor antagonist/inverse agonist. Particularly the (patho)physiological role of 2-AG should be further investigated, since its CB1 receptor affinity and agonist activity especially in humans might be lower than generally assumed. PMID:15588725

  19. Human rotavirus detection by agglutination of antibody-coated erythrocytes.

    PubMed

    Sanekata, T; Okada, H

    1983-06-01

    We sensitized sheep erythrocytes (SRBC) with antibodies against human rotavirus strain Wa (SRBC-antiWa) and antibodies against calf rotavirus strain NCDV (SRBC-antiNCDV). These were readily agglutinated in the presence of homologous antigens, i.e., human rotavirus and calf rotavirus. By the hemagglutination of SRBC-antiWa and SRBC-antiNCDV (reverse passive hemagglutination [RPHA]), titration of rotavirus in extracts from feces of children suffering from diarrhea (61 specimens) was carried out. We found that the ratio of titers determined with SRBC-antiWa and SRBC-antiNCDV varied remarkably from specimen to specimen. This indicated that the antigenic determinants on human rotavirus in patients feces cross-react with antibodies against NCDV to varying extents. To express the cross-reactivity of human rotavirus with antibodies to NCDV, we propose a Wa/NCDV rotavirus index which can be calculated from the RPHA titer with SRBC-antiWa and SRBC-antiNCDV as follows: Wa/NCDV rotavirus index = (antiWa-RPHA titer of specimen/antiWa-RPHA titer of NCDV)/(antiNCDV-RPHA titer of specimen/antiNCDV-RPHA titer of NCDV).

  20. IgY antibodies in human nutrition for disease prevention.

    PubMed

    Müller, Sandra; Schubert, Andreas; Zajac, Julia; Dyck, Terry; Oelkrug, Christopher

    2015-10-20

    Oral administration of preformed specific antibodies is an attractive approach against infections of the digestive system in humans and animals in times of increasing antibiotic resistances. Previous studies showed a positive effect of egg yolk IgY antibodies on bacterial intoxications in animals and humans. Immunization of chickens with specific antigens offers the possibility to create various forms of antibodies. Research shows that orally applied IgY's isolated from egg yolks can passively cure or prevent diseases of the digestive system. The use of these alternative therapeutic drugs provides further advantages: (1) The production of IgY's is a non-invasive alternative to current methods; (2) The keeping of chickens is inexpensive; (3) The animals are easy to handle; (4) It avoids repetitive bleeding of laboratory animals; (5) It is also very cost effective regarding the high IgY concentration within the egg yolk. Novel targets of these antigen specific antibodies are Helicobacter pylori and also molecules involved in signaling pathways in gastric cancer. Furthermore, also dental caries causing bacteria like Streptococcus mutans or opportunistic Pseudomonas aeruginosa in cystic fibrosis patients are possible targets. Therefore, IgY's included in food for human consumption may be able to prevent or cure human diseases.

  1. Facile fabrication and instant application of miniaturized antibody-decorated affinity columns for higher-order structure and functional characterization of TRIM21 epitope peptides.

    PubMed

    Al-Majdoub, M; Opuni, K F M; Koy, C; Glocker, M O

    2013-11-01

    Both epitope excision and epitope extraction methods, combined with mass spectrometry, generate precise informations on binding surfaces of full-length proteins, identifying sequential (linear) or assembled (conformational) epitopes, respectively. Here, we describe the one-step fabrication and application of affinity columns using reversibly immobilized antibodies with highest flexibility with respect to antibody sources and lowest sample amount requirements (fmol range). Depending on the antibody source, we made use of protein G- or protein A-coated resins as support materials. These materials are packed in pipet tips and in combination with a programmable multichannel pipet form a highly efficient epitope mapping system. In addition to epitope identification, the influence of epitope structure modifications on antibody binding specificities could be studied in detail with synthetic peptides. Elution of epitope peptides was optimized such that mass spectrometric analysis was feasible after a single desalting step. Epitope peptides were identified by accurate molecular mass determinations or by partial amino acid sequence analysis. In addition, charge state comparison or ion mobility analysis of eluted epitope peptides enabled investigation of higher-order structures. The epitope peptide of the TRIM21 (TRIM: tripartite motif) autoantigen that is recognized by a polyclonal antibody was determined as assembling an "L-E-Q-L" motif on an α-helix. Secondary structure determination by circular dichroism spectroscopy and structure modeling are in accordance with the mass spectrometric results and the antigenic behavior of the 17-mer epitope peptide variants from the full-length autoantigen. PMID:24094071

  2. Disialoganglioside GD2 on human neuroblastoma cells: target antigen for monoclonal antibody-mediated cytolysis and suppression of tumor growth.

    PubMed

    Mujoo, K; Cheresh, D A; Yang, H M; Reisfeld, R A

    1987-02-15

    A murine monoclonal antibody 14.18 specifically recognizes disialoganglioside GD2, the major ganglioside expressed on the surface of human neuroblastoma cells. This monoclonal antibody (Mab) is of immunoglobulin G3 isotype, has an affinity constant (KA) of 3.5 X 10(8) M-1, and reacts preferentially with tumor cells and fresh frozen tumor tissues of neuroectodermal origin in enzyme-linked immunosorbent assay and immunoperoxidase assays, respectively. Mab 14.18 effectively lyses a number of human neuroblastoma cell lines by two distinct mechanisms, i.e., antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. There is a good correlation between the average number of antibody-binding sites per neuroblastoma cell and the amount of cell lysis observed in complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity. In addition, Mab 14.18 suppresses establishment as well as growth of progressively growing, established human neuroblastoma tumors in nude mice when injected 24 h and 9 days, respectively, after the initial s.c. inoculation of tumor cells. These data suggest that Mab 14.18 can mediate tumor cell killing in vivo and in vitro and may thereby prove useful for immunotherapy of human neuroblastoma.

  3. Humanization of a phosphothreonine peptide-specific chicken antibody by combinatorial library optimization of the phosphoepitope-binding motif.

    PubMed

    Baek, Du-San; Kim, Yong-Sung

    2015-07-31

    Detection of protein phosphorylation at a specific residue has been achieved by using antibodies, which have usually been raised by animal immunization. However, there have been no reports of the humanization of phosphospecific non-human antibodies. Here, we report the humanization of a chicken pT231 antibody specific to a tau protein-derived peptide carrying the phosphorylated threonine at residue 231 (pT231 peptide) as a model for better understanding the phosphoepitope recognition mechanism. In the chicken antibody, the phosphate group of the pT231-peptide antigen is exclusively recognized by complementarity determining region 2 of the heavy chain variable domain (VH-CDR2). Simple grafting of six CDRs of the chicken antibody into a homologous human framework (FR) template resulted in the complete loss of pT231-peptide binding. Using a yeast surface-displayed combinatorial library with permutations of 11 FR residues potentially affecting CDR loop conformations, we identified 5 critical FR residues. The back mutation of these residues to the corresponding chicken residues completely recovered the pT231-peptide binding affinity and specificity of the humanized antibody. Importantly, the back mutation of the FR 76 residue of VH (H76) (Asn to Ser) was critical in preserving the pT231-binding motif conformation via allosteric regulation of ArgH71, which closely interacts with ThrH52 and SerH52a residues on VH-CDR2 to induce the unique phosphate-binding bowl-like conformation. Our humanization approach of CDR grafting plus permutations of FR residues by combinatorial library screening can be applied to other animal antibodies containing unique binding motifs on CDRs specific to posttranslationally modified epitopes. PMID:26036575

  4. Characterization of human sperm surface antigens with monoclonal antibodies.

    PubMed

    Wolf, D P; Sokoloski, J E; Dandekar, P; Bechtol, K B

    1983-10-01

    Monoclonal antibodies (McAb) against human ejaculated sperm were developed from mice immunized with sperm membrane preparations. A solid-phase radioimmunoassay, with dried sperm as antigen, was employed in McAb screening. The tissue and species specificity of monoclonal antibodies HS 2, 4 and 6 were evaluated after absorption of antibody preparations with heterologous sperm, human serum or seminal plasma or cells from other human organs. The sensitivity of HS 2, 4 and 6 antigens to trypsin exposure was determined: HS 4 antigen was highly sensitive while HS 2 and 6 were not. The regional distribution of McAb 4 on intact sperm cells was determined by immunofluorescence staining. HS 4 may be a sperm-coating antigen based on its presence on sperm and in seminal plasma. This possibility led to an investigation of its role in sperm capacitation. HS 4 antibody binding was reduced when capacitated sperm were compared with noncapacitated cells. HS 4 antibody, when present during capacitation and insemination, was without effect on sperm motility or fusion with zona-free hamster eggs. Trypsin removal of as much as 60% of HS 4 antigen from the cell population also did not impact on sperm function. To identify the molecular correlate of HS 4 antigen, membrane components were extracted from washed sperm with Nonidet P-40, concentrated by acetone precipitation and analyzed electrophoretically in SDS-urea on 10% polyacrylamide slab gels. Immunoassays on protein blots with peroxidase-coupled second antibody identified a single reactive species in the molecular weight range of 130,000. Multiple reactive components were detected in blot transfers of seminal plasma.

  5. Human IgG Subclasses against Enterovirus Type 71: Neutralization versus Antibody Dependent Enhancement of Infection

    PubMed Central

    Han, Jian-Feng; Wang, Guang-Chuan; Zhao, Hui; Li, Xiao-Feng; Deng, Yong-Qiang; Zhu, Shun-Ya; Wang, Xiao-Yu; Lin, Fang; Zhang, Fu-Jun; Chen, Wei; Qin, E-De; Qin, Cheng-Feng

    2013-01-01

    The emerging human enterovirus 71 (EV71) represents a growing threat to public health, and no vaccine or specific antiviral is currently available. Human intravenous immunoglobulin (IVIG) is clinical used in treating severe EV71 infections. However, the discovery of antibody dependent enhancement (ADE) of EV71 infection illustrates the complex roles of antibody in controlling EV71 infection. In this study, to identify the distinct role of each IgG subclass on neutralization and enhancement of EV71 infection, different lots of pharmaceutical IVIG preparations manufactured from Chinese donors were used for IgG subclass fractionation by pH gradient elution with the protein A-conjugated affinity column. The neutralization and ADE capacities on EV71 infection of each purified IgG subclass were then assayed, respectively. The neutralizing activity of human IVIG is mainly mediated by IgG1 subclass and to less extent by IgG2 subclass. Interestingly, IgG3 fraction did not have neutralizing activity but enhanced EV71 infection in vitro. These results revealed the different roles of human IgG subclasses on EV71 infection, which is of critical importance for the rational design of immunotherapy and vaccines against severe EV71 diseases. PMID:23700449

  6. Immuno-column for on-line quantification of human serum IgG antibodies to Helicobacter pylori in human serum samples.

    PubMed

    Molina, Luis; Messina, Germán A; Stege, Patrícia W; Salinas, Eloy; Raba, Julio

    2008-09-15

    This study report an human serum IgG antibodies to Helicobacter pylori quantitation procedure based on the multiple use of an immobilized H. pylori antigen on an immuno-column incorporated into an a flow-injection (FI) analytical system. The immuno-adsorbent column was prepared by packing 3-aminopropyl-modified controlled-pore glass (APCPG) covalently linking H. pylori antigens in a 3-cm of Teflon tubing (0.5 i.d.). Antibodies in the serum sample are allowed to react immunologically with the immobilized H. pylori antigen, and the bound antibodies are quantified by alkaline phosphatase (AP) enzyme-labeled second antibodies specific to human IgG. p-Aminophenyl phosphate (pAPP) was converted to p-aminophenol (pAP) by AP and an electroactive product was quantified on glassy carbon electrode (GCE) modified with multiwall carbon nanotubes (MWCNT) (GCE-CNTs) at 0.30 V. The total assay time was 25 min. The calculated detection limits for amperometric detection and the ELISA procedure are 0.62 and 1.8 UmL(-1), respectively. Reproducibility assays were made using repetitive standards of H. pylori-specific antibody and the intra- and inter-assay coefficients of variation were below 5%. The immuno-affinity method showed higher sensitivity and lower time-consumed, demonstrate its potential usefulness for early assessment of human serum immunoglobulin G (IgG) antibodies to H. pylori. PMID:18761158

  7. Human antibody-Fc receptor interactions illuminated by crystal structures.

    PubMed

    Woof, Jenny M; Burton, Dennis R

    2004-02-01

    Immunoglobulins couple the recognition of invading pathogens with the triggering of potent effector mechanisms for pathogen elimination. Different immunoglobulin classes trigger different effector mechanisms through interaction of immunoglobulin Fc regions with specific Fc receptors (FcRs) on immune cells. Here, we review the structural information that is emerging on three human immunoglobulin classes and their FcRs. New insights are provided, including an understanding of the antibody conformational adjustments that are required to bring effector cell and target cell membranes sufficiently close for efficient killing and signal transduction to occur. The results might also open up new possibilities for the design of therapeutic antibodies.

  8. Lineage tracing of human B cells reveals the in vivo landscape of human antibody class switching.

    PubMed

    Horns, Felix; Vollmers, Christopher; Croote, Derek; Mackey, Sally F; Swan, Gary E; Dekker, Cornelia L; Davis, Mark M; Quake, Stephen R

    2016-01-01

    Antibody class switching is a feature of the adaptive immune system which enables diversification of the effector properties of antibodies. Even though class switching is essential for mounting a protective response to pathogens, the in vivo patterns and lineage characteristics of antibody class switching have remained uncharacterized in living humans. Here we comprehensively measured the landscape of antibody class switching in human adult twins using antibody repertoire sequencing. The map identifies how antibodies of every class are created and delineates a two-tiered hierarchy of class switch pathways. Using somatic hypermutations as a molecular clock, we discovered that closely related B cells often switch to the same class, but lose coherence as somatic mutations accumulate. Such correlations between closely related cells exist when purified B cells class switch in vitro, suggesting that class switch recombination is directed toward specific isotypes by a cell-autonomous imprinted state. PMID:27481325

  9. Preparation and characterization of human monoclonal antibodies directed against the hepatitis B virus surface antigen.

    PubMed

    Colucci, G; Kohtz, D S; Waksal, S D

    1986-06-01

    The hepatitis B surface antigen (HBsAg) is highly immunogenic and induces an antibody response which is protective in vivo against hepatitis B virus (HBV) infection. Human monoclonal antibodies specific for HBsAg were produced, which could have potential therapeutic applications. Lymphocytes obtained from a vaccinated donor were stimulated in vitro and fused with the human myeloma cell line GM 4672, and eight hybridomas were obtained. Three of these clones, which reacted in an ELISA against the HBsAg vaccine, were expanded, subcloned and further analyzed. The subclones E7C2, C4C10, and D5B2 were able to bind to different HBsAg preparations, which express various subtypes, and recognized the major HBsAg peptides in Western blot analysis. Cross-inhibition experiments showed that E7C2, C4C10 and D5B2 are directed against the same epitope and have an affinity constant ranging from 5 X 10(7) to 3.3 X 10(9) M. Furthermore, these antibodies stained the surface and cytoplasm of the HBsAg-secreting cell lines PLC/PRF/5 and 4.10. The production of immunoglobulins varies from 0.3 to 1.3 micrograms/ml/10(6) and has remained stable over a period of 8 months. These human monoclonal antibodies, which appear to be directed against an antigenic determinant common to all HBsAg subtypes, could be useful in the study of HBV-related liver diseases as well as in their diagnosis and experimental therapy.

  10. Serum or breast milk immunoglobulins mask the self-reactivity of human natural IgG antibodies.

    PubMed

    Djoumerska-Alexieva, Iglika; Manoylov, Iliyan; Dimitrov, Jordan D; Tchorbanov, Andrey

    2014-04-01

    B cells producing IgG antibodies specific to a variety of self- or foreign antigens are a normal constituent of the immune system of all healthy individuals. These naturally occurring IgG antibodies are found in the serum, external secretions, and pooled human immunoglobulin preparations. They bind with low affinity to antigens, which can also be targets for pathologic autoantibodies. An enhancement of naturally occurring IgG autoantibody activity was observed after treatment of human IgG molecules with protein-destabilizing agents. We have investigated the interactions of human immunoglobulins that were obtained from serum or from breast milk of healthy individuals or IVIg with human liver antigens. Proteins from an individual serum or milk were isolated by two methods, one of which included exposure to low pH and the other did not. Purified serum, mucosal IgM, IgA, and the fraction containing immunoglobulin G F(ab')2 fragments each inhibited the binding of a single donor or pooled IgG to human liver antigens. Our study presents findings regarding the role of the breast milk or serum antibodies in blocking the self-reactivity of IgG antibodies. It supports the suggestion that not IVIg only, but also the pooled human IgM and IgA might possess a potent beneficial immunomodulatory activity in autoimmune patients.

  11. Novel asymmetrically engineered antibody Fc variant with superior FcγR binding affinity and specificity compared with afucosylated Fc variant.

    PubMed

    Mimoto, Futa; Igawa, Tomoyuki; Kuramochi, Taichi; Katada, Hitoshi; Kadono, Shojiro; Kamikawa, Takayuki; Shida-Kawazoe, Meiri; Hattori, Kunihiro

    2013-01-01

    Fc engineering is a promising approach to enhance the antitumor efficacy of monoclonal antibodies (mAbs) through antibody-dependent cell-mediated cytotoxicity (ADCC). Glyco- and protein-Fc engineering have been employed to enhance FcγR binding and ADCC activity of mAbs; the drawbacks of previous approaches lie in their binding affinity to both FcγRIIIa allotypes, the ratio of activating FcγR binding to inhibitory FcγR binding (A/I ratio) or the melting temperature (T(M)) of the C(H)2 domain. To date, no engineered Fc variant has been reported that satisfies all these points. Herein, we present a novel Fc engineering approach that introduces different substitutions in each Fc domain asymmetrically, conferring optimal binding affinity to FcγR and specificity to the activating FcγR without impairing the stability. We successfully designed an asymmetric Fc variant with the highest binding affinity for both FcγRIIIa allotypes and the highest A/I ratio compared with previously reported symmetrically engineered Fc variants, and superior or at least comparable in vitro ADCC activity compared with afucosylated Fc variants. In addition, the asymmetric Fc engineering approach offered higher stability by minimizing the use of substitutions that reduce the T(M) of the C(H)2 domain compared with the symmetric approach. These results demonstrate that the asymmetric Fc engineering platform provides best-in-class effector function for therapeutic antibodies against tumor antigens.

  12. A plasmid containing the human metallothionein II gene can function as an antibody-assisted electrophoretic biosensor for heavy metals.

    PubMed

    Wooten, Dennis C; Starr, Clarise R; Lyon, Wanda J

    2016-01-01

    Different forms of heavy metals affect biochemical systems in characteristic ways that cannot be detected with typical metal analysis methods like atomic absorption spectrometry. Further, using living systems to analyze interaction of heavy metals with biochemical systems can be laborious and unreliable. To generate a reliable easy-to-use biologically-based biosensor system, the entire human metallothionein-II (MT-II) gene was incorporated into a plasmid (pUC57-MT) easily replicated in Escherichia coli. In this system, a commercial polyclonal antibody raised against human metal-responsive transcription factor-1 protein (MTF-1 protein) could modify the electrophoretic migration patterns (i.e. cause specific decreases in agarose gel electrophoretic mobility) of the plasmid in the presence or absence of heavy metals other than zinc (Zn). In the study here, heavy metals, MTF-1 protein, and polyclonal anti-MTF-1 antibody were used to assess pUC57-MT plasmid antibody-assisted electrophoretic mobility. Anti-MTF-1 antibody bound both MTF-1 protein and pUC57-MT plasmid in a non-competitive fashion such that it could be used to differentiate specific heavy metal binding. The results showed that antibody-inhibited plasmid migration was heavy metal level-dependent. Zinc caused a unique mobility shift pattern opposite to that of other metals tested, i.e. Zn blocked the antibody ability to inhibit plasmid migration, despite a greatly increased affinity for DNA by the antibody when Zn was present. The Zn effect was reversed/modified by adding MTF-1 protein. Additionally, antibody inhibition of plasmid mobility was resistant to heat pre-treatment and trypsinization, indicating absence of residual DNA extraction-resistant bacterial DNA binding proteins. DNA binding by anti-DNA antibodies may be commonly enhanced by xenobiotic heavy metals and elevated levels of Zn, thus making them potentially effective tools for assessment of heavy metal bioavailability in aqueous solutions and

  13. An Insertion Mutation That Distorts Antibody Binding Site Architecture Enhances Function of a Human Antibody

    SciTech Connect

    Krause, Jens C.; Ekiert, Damian C.; Tumpey, Terrence M.; Smith, Patricia B.; Wilson, Ian A.; Crowe, Jr., James E.

    2011-09-02

    The structural and functional significance of somatic insertions and deletions in antibody chains is unclear. Here, we demonstrate that a naturally occurring three-amino-acid insertion within the influenza virus-specific human monoclonal antibody 2D1 heavy-chain variable region reconfigures the antibody-combining site and contributes to its high potency against the 1918 and 2009 pandemic H1N1 influenza viruses. The insertion arose through a series of events, including a somatic point mutation in a predicted hot-spot motif, introduction of a new hot-spot motif, a molecular duplication due to polymerase slippage, a deletion due to misalignment, and additional somatic point mutations. Atomic resolution structures of the wild-type antibody and a variant in which the insertion was removed revealed that the three-amino-acid insertion near the base of heavy-chain complementarity-determining region (CDR) H2 resulted in a bulge in that loop. This enlarged CDR H2 loop impinges on adjacent regions, causing distortion of the CDR H1 architecture and its displacement away from the antigen-combining site. Removal of the insertion restores the canonical structure of CDR H1 and CDR H2, but binding, neutralization activity, and in vivo activity were reduced markedly because of steric conflict of CDR H1 with the hemagglutinin antigen.

  14. Pre-Clinical Development of a Humanized Anti-CD47 Antibody with Anti-Cancer Therapeutic Potential

    PubMed Central

    Liu, Jie; Wang, Lijuan; Zhao, Feifei; Tseng, Serena; Narayanan, Cyndhavi; Shura, Lei; Willingham, Stephen; Howard, Maureen; Prohaska, Susan; Volkmer, Jens; Chao, Mark; Weissman, Irving L.; Majeti, Ravindra

    2015-01-01

    CD47 is a widely expressed cell surface protein that functions as a regulator of phagocytosis mediated by cells of the innate immune system, such as macrophages and dendritic cells. CD47 serves as the ligand for a receptor on these innate immune cells, SIRP-alpha, which in turn delivers an inhibitory signal for phagocytosis. We previously found increased expression of CD47 on primary human acute myeloid leukemia (AML) stem cells, and demonstrated that blocking monoclonal antibodies directed against CD47 enabled the phagocytosis and elimination of AML, non-Hodgkin’s lymphoma (NHL), and many solid tumors in xenograft models. Here, we report the development of a humanized anti-CD47 antibody with potent efficacy and favorable toxicokinetic properties as a candidate therapeutic. A novel monoclonal anti-human CD47 antibody, 5F9, was generated, and antibody humanization was carried out by grafting its complementarity determining regions (CDRs) onto a human IgG4 format. The resulting humanized 5F9 antibody (Hu5F9-G4) bound monomeric human CD47 with an 8 nM affinity. Hu5F9-G4 induced potent macrophage-mediated phagocytosis of primary human AML cells in vitro and completely eradicated human AML in vivo, leading to long-term disease-free survival of patient-derived xenografts. Moreover, Hu5F9-G4 synergized with rituximab to eliminate NHL engraftment and cure xenografted mice. Finally, toxicokinetic studies in non-human primates showed that Hu5F9-G4 could be safely administered intravenously at doses able to achieve potentially therapeutic serum levels. Thus, Hu5F9-G4 is actively being developed for and has been entered into clinical trials in patients with AML and solid tumors (ClinicalTrials.gov identifier: NCT02216409). PMID:26390038

  15. 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. PMID:25692880

  16. 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

  17. 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.

  18. Recognition of N-Glycoforms in Human Chorionic Gonadotropin by Monoclonal Antibodies and Their Interaction Motifs*

    PubMed Central

    Li, Daoyuan; Zhang, Ping; Li, Fei; Chi, Lequan; Zhu, Deyu; Zhang, Qunye; Chi, Lianli

    2015-01-01

    The glycosylation of human chorionic gonadotropin (hCG) plays an important role in reproductive tumors. Detecting hCG N-glycosylation alteration may significantly improve the diagnostic accuracy and sensitivity of related cancers. However, developing an immunoassay directly against the N-linked oligosaccharides is unlikely because of the heterogeneity and low immunogenicity of carbohydrates. Here, we report a hydrogen/deuterium exchange and MS approach to investigate the effect of N-glycosylation on the binding of antibodies against different hCG glycoforms. Hyperglycosylated hCG was purified from the urine of invasive mole patients, and the structure of its N-linked oligosaccharides was confirmed to be more branched by MS. The binding kinetics of the anti-hCG antibodies MCA329 and MCA1024 against hCG and hyperglycosylated hCG were compared using biolayer interferometry. The binding affinity of MCA1024 changed significantly in response to the alteration of hCG N-linked oligosaccharides. Hydrogen/deuterium exchange-MS reveals that the peptide β65–83 of the hCG β subunit is the epitope for MCA1024. Site-specific N-glycosylation analysis suggests that N-linked oligosaccharides at Asn-13 and Asn-30 on the β subunit affect the binding affinity of MCA1024. These results prove that some antibodies are sensitive to the structural change of N-linked oligosaccharides, whereas others are not affected by N-glycosylation. It is promising to improve glycoprotein biomarker-based cancer diagnostics by developing combined immunoassays that can determine the level of protein and measure the degree of N-glycosylation simultaneously. PMID:26240146

  19. Production and characterisation of monoclonal antibodies against RAI3 and its expression in human breast cancer

    PubMed Central

    2009-01-01

    Background RAI3 is an orphan G-protein coupled receptor (GPCR) that has been associated with malignancy and may play a role in the proliferation of breast cancer cells. Although its exact function in normal and malignant cells remains unclear and evidence supporting its role in oncogenesis is controversial, its abundant expression on the surface of cancer cells would make it an interesting target for the development of antibody-based therapeutics. To investigate the link with cancer and provide more evidence for its role, we carried out a systematic analysis of RAI3 expression in a large set of human breast cancer specimens. Methods We expressed recombinant human RAI3 in bacteria and reconstituted the purified protein in liposomes to raise monoclonal antibodies using classical hybridoma techniques. The specific binding activity of the antibodies was confirmed by enzyme-linked immunosorbent assay (ELISA), western blot and immunocytochemistry. We carried out a systematic immunohistochemical analysis of RAI3 expression in human invasive breast carcinomas (n = 147) and normal breast tissues (n = 44) using a tissue microarray. In addition, a cDNA dot blot hybridisation assay was used to investigate a set of matched normal and cancerous breast tissue specimens (n = 50) as well as lymph node metastases (n = 3) for RAI3 mRNA expression. Results The anti-RAI3 monoclonal antibodies bound to recombinant human RAI3 protein with high specificity and affinity, as shown by ELISA, western blot and ICC. The cDNA dot blot and immunohistochemical experiments showed that both RAI3 mRNA and RAI3 protein were abundantly expressed in human breast carcinoma. However, there was no association between RAI3 protein expression and prognosis based on overall and recurrence-free survival. Conclusion We have generated a novel, highly-specific monoclonal antibody that detects RAI3 in formaldehyde-fixed paraffin-embedded tissue. This is the first study to report a systematic analysis of RAI3

  20. The Human Antibody Response to the Surface of Mycobacterium tuberculosis

    PubMed Central

    Perley, Casey C.; Frahm, Marc; Click, Eva M.; Dobos, Karen M.; Ferrari, Guido; Stout, Jason E.; Frothingham, Richard

    2014-01-01

    Background Vaccine-induced human antibodies to surface components of Haemophilus influenzae and Streptococcus pneumonia are correlated with protection. Monoclonal antibodies to surface components of Mycobacterium tuberculosis are also protective in animal models. We have characterized human antibodies that bind to the surface of live M. tuberculosis. Methods Plasma from humans with latent tuberculosis (TB) infection (n = 23), active TB disease (n = 40), and uninfected controls (n = 9) were assayed by ELISA for reactivity to the live M. tuberculosis surface and to inactivated M. tuberculosis fractions (whole cell lysate, lipoarabinomannan, cell wall, and secreted proteins). Results When compared to uninfected controls, patients with active TB disease had higher antibody titers to the surface of live M. tuberculosis (Δ = 0.72 log10), whole cell lysate (Δ = 0.82 log10), and secreted proteins (Δ = 0.62 log10), though there was substantial overlap between the two groups. Individuals with active disease had higher relative IgG avidity (Δ = 1.4 to 2.6) to all inactivated fractions. Surprisingly, the relative IgG avidity to the live M. tuberculosis surface was lower in the active disease group than in uninfected controls (Δ = –1.53, p = 0.004). Patients with active disease had higher IgG than IgM titers for all inactivated fractions (ratios, 2.8 to 10.1), but equal IgG and IgM titers to the live M. tuberculosis surface (ratio, 1.1). Higher antibody titers to the M. tuberculosis surface were observed in active disease patients who were BCG-vaccinated (Δ = 0.55 log10, p = 0.008), foreign-born (Δ = 0.61 log10, p = 0.004), or HIV-seronegative (Δ = 0.60 log10, p = 0.04). Higher relative IgG avidity scores to the M. tuberculosis surface were also observed in active disease patients who were BCG-vaccinated (Δ = 1.12, p<0.001) and foreign-born (Δ = 0.87, p = 0.01). Conclusions/Significance Humans

  1. An Integrated Peptide-Antigen Microarray on Plasmonic Gold Films for Sensitive Human Antibody Profiling

    PubMed Central

    Price, Jordan V.; Tabakman, Scott M.; Li, Yanguang; Gong, Ming; Hong, Guosong; Feng, Ju; Utz, Paul J.; Dai, Hongjie

    2013-01-01

    High-throughput screening for interactions of peptides with a variety of antibody targets could greatly facilitate proteomic analysis for epitope mapping, enzyme profiling, drug discovery and biomarker identification. Peptide microarrays are suited for such undertaking because of their high-throughput capability. However, existing peptide microarrays lack the sensitivity needed for detecting low abundance proteins or low affinity peptide-protein interactions. This work presents a new peptide microarray platform constructed on nanostructured plasmonic gold substrates capable of metal enhanced NIR fluorescence enhancement (NIR-FE) by hundreds of folds for screening peptide-antibody interactions with ultrahigh sensitivity. Further, an integrated histone peptide and whole antigen array is developed on the same plasmonic gold chip for profiling human antibodies in the sera of systemic lupus erythematosus (SLE) patients, revealing that collectively a panel of biomarkers against unmodified and post-translationally modified histone peptides and several whole antigens allow more accurate differentiation of SLE patients from healthy individuals than profiling biomarkers against peptides or whole antigens alone. PMID:23923050

  2. Extending the half-life of a fab fragment through generation of a humanized anti-human serum albumin Fv domain: An investigation into the correlation between affinity and serum half-life

    PubMed Central

    Adams, Ralph; Griffin, Laura; Compson, Joanne E.; Jairaj, Mark; Baker, Terry; Ceska, Tom; West, Shauna; Zaccheo, Oliver; Davé, Emma; Lawson, Alastair DG.; Humphreys, David P.; Heywood, Sam

    2016-01-01

    ABSTRACT We generated an anti-albumin antibody, CA645, to link its Fv domain to an antigen-binding fragment (Fab), thereby extending the serum half-life of the Fab. CA645 was demonstrated to bind human, cynomolgus, and mouse serum albumin with similar affinity (1–7 nM), and to bind human serum albumin (HSA) when it is in complex with common known ligands. Importantly for half-life extension, CA645 binds HSA with similar affinity within the physiologically relevant range of pH 5.0 – pH 7.4, and does not have a deleterious effect on the binding of HSA to neonatal Fc receptor (FcRn). A crystal structure of humanized CA645 Fab in complex with HSA was solved and showed that CA645 Fab binds to domain II of HSA. Superimposition with the crystal structure of FcRn bound to HSA confirmed that CA645 does not block HSA binding to FcRn. In mice, the serum half-life of humanized CA645 Fab is 84.2 h. This is a significant extension in comparison with < 1 h for a non-HSA binding CA645 Fab variant. The Fab-HSA structure was used to design a series of mutants with reduced affinity to investigate the correlation between the affinity for albumin and serum half-life. Reduction in the affinity for MSA by 144-fold from 2.2 nM to 316 nM had no effect on serum half-life. Strikingly, despite a reduction in affinity to 62 µM, an extension in serum half-life of 26.4 h was still obtained. CA645 Fab and the CA645 Fab-HSA complex have been deposited in the Protein Data Bank (PDB) with accession codes, 5FUZ and 5FUO, respectively. PMID:27315033

  3. Immunosuppressive human anti-CD83 monoclonal antibody depletion of activated dendritic cells in transplantation.

    PubMed

    Seldon, T A; Pryor, R; Palkova, A; Jones, M L; Verma, N D; Findova, M; Braet, K; Sheng, Y; Fan, Y; Zhou, E Y; Marks, J D; Munro, T; Mahler, S M; Barnard, R T; Fromm, P D; Silveira, P A; Elgundi, Z; Ju, X; Clark, G J; Bradstock, K F; Munster, D J; Hart, D N J

    2016-03-01

    Current immunosuppressive/anti-inflammatory agents target the responding effector arm of the immune response and their nonspecific action increases the risk of infection and malignancy. These effects impact on their use in allogeneic haematopoietic cell transplantation and other forms of transplantation. Interventions that target activated dendritic cells (DCs) have the potential to suppress the induction of undesired immune responses (for example, graft versus host disease (GVHD) or transplant rejection) and to leave protective T-cell immune responses intact (for example, cytomegalovirus (CMV) immunity). We developed a human IgG1 monoclonal antibody (mAb), 3C12, specific for CD83, which is expressed on activated but not resting DC. The 3C12 mAb and an affinity improved version, 3C12C, depleted CD83(+) cells by CD16(+) NK cell-mediated antibody-dependent cellular cytotoxicity, and inhibited allogeneic T-cell proliferation in vitro. A single dose of 3C12C prevented human peripheral blood mononuclear cell-induced acute GVHD in SCID mouse recipients. The mAb 3C12C depleted CMRF-44(+)CD83(bright) activated DC but spared CD83(dim/-) DC in vivo. It reduced human T-cell activation in vivo and maintained the proportion of CD4(+) FoxP3(+) CD25(+) Treg cells and also viral-specific CD8(+) T cells. The anti-CD83 mAb, 3C12C, merits further evaluation as a new immunosuppressive agent in transplantation.

  4. Recognition determinants of broadly neutralizing human antibodies against dengue viruses.

    PubMed

    Rouvinski, Alexander; Guardado-Calvo, Pablo; Barba-Spaeth, Giovanna; Duquerroy, Stéphane; Vaney, Marie-Christine; Kikuti, Carlos M; Navarro Sanchez, M Erika; Dejnirattisai, Wanwisa; Wongwiwat, Wiyada; Haouz, Ahmed; Girard-Blanc, Christine; Petres, Stéphane; Shepard, William E; Desprès, Philippe; Arenzana-Seisdedos, Fernando; Dussart, Philippe; Mongkolsapaya, Juthathip; Screaton, Gavin R; Rey, Félix A

    2015-04-01

    Dengue disease is caused by four different flavivirus serotypes, which infect 390 million people yearly with 25% symptomatic cases and for which no licensed vaccine is available. Recent phase III vaccine trials showed partial protection, and in particular no protection for dengue virus serotype 2 (refs 3, 4). Structural studies so far have characterized only epitopes recognized by serotype-specific human antibodies. We recently isolated human antibodies potently neutralizing all four dengue virus serotypes. Here we describe the X-ray structures of four of these broadly neutralizing antibodies in complex with the envelope glycoprotein E from dengue virus serotype 2, revealing that the recognition determinants are at a serotype-invariant site at the E-dimer interface, including the exposed main chain of the E fusion loop and the two conserved glycan chains. This 'E-dimer-dependent epitope' is also the binding site for the viral glycoprotein prM during virus maturation in the secretory pathway of the infected cell, explaining its conservation across serotypes and highlighting an Achilles' heel of the virus with respect to antibody neutralization. These findings will be instrumental for devising novel immunogens to protect simultaneously against all four serotypes of dengue virus. PMID:25581790

  5. Enzyme-linked immunosorbent assay for quantification of rabies antibodies in human sera.

    PubMed

    Kavaklova, L; Eskenazy, M; Gancheva, T; Vacheva, V

    1984-09-01

    A double antibody enzyme linked immunosorbent assay (ELISA) was elaborated for detection of rabies antibodies in human sera. The procedure consisted of coating polyvinylchloride plates with rabbit antirabies serum followed by attachment of partially purified fixed virus and human rabies antibodies. The rabies-specific antibodies in human antisera were quantified by means of antihuman peroxidase conjugate. Titration of antisera from human volunteers immunized with the "Fermi" vaccine revealed excellent correlation of the virus neutralization test and ELISA.

  6. Fibulin-1 purification from human plasma using affinity chromatography on Factor H-Sepharose.

    PubMed

    DiScipio, Richard G; Liddington, Robert C; Schraufstatter, Ingrid U

    2016-05-01

    A method is reported to purify Fibulin-1 from human plasma resulting in a 36% recovery. The steps involve removal of the cryoglobulin and the vitamin K dependent proteins followed by polyethylene glycol and ammonium sulfate precipitations, DEAE-Sephadex column chromatography and finally Factor H-Sepharose affinity purification. The procedure is designed to be integrated into an overall scheme for the isolation of over 30 plasma proteins from a single batch of human plasma. Results from mass spectroscopy, SDS-PAGE, and Western blotting indicate that human plasma Fibulin-1 is a single chain of the largest isotype. Functional binding assays demonstrated calcium ion dependent interaction of Fibulin-1 for fibrinogen, fibronectin, and Factor H. The procedure described is the first to our knowledge that enables a large scale purification of Fibulin-1 from human plasma. PMID:26826315

  7. Quantum dot immunoassays in renewable surface column and 96-well plate formats for the fluorescence detection of Botulinum neurotoxin using high-affinity antibodies

    SciTech Connect

    Warner, Marvin G.; Grate, Jay W.; Tyler, Abby J.; Ozanich, Richard M.; Miller, Keith D.; Lou, Jianlong; Marks, James D.; Bruckner-Lea, Cindy J.

    2009-09-01

    A fluorescence sandwich immunoassay using high affinity antibodies and quantum dot (QD) reporters has been developed for detection of botulinum toxin serotype A (BoNT/A). For the development of the assay, a nontoxic recombinant fragment of the holotoxin (BoNT/A-HC-fragment) has been used as a structurally valid simulant for the full toxin molecule. The antibodies used, AR4 and RAZ1, bind to nonoverlapping epitopes present on both the full toxin and on the recombinant fragment. In one format, the immunoassay is carried out in a 96-well plate with detection in a standard plate reader. Detection down to 31 pM of the BoNT/Hc-fragment was demonstrated with a total incubation time of 3 hours, using AR4 as the capture antibody and QD-coupled RAZ1 as the reporter. In a second format, the AR4 capture antibody was coupled to Sepharose beads, and the immunochemical reactions were carried out in microcentrifuge tubes with an incubation time of 1 hour. These beads were subsequently captured and concentrated in a rotating rod “renewable surface” flow cell as part of a sequential injection fluidic system. This flow cell was equipped with a fiber optic system for fluorescence measurements. In PBS buffer solution matrix, the BoNT/A-HC-fragment was detected to concentrations as low as 5 pM using the fluidic measurement approach.

  8. Method for removal of human antibodies to native DNA from serum

    SciTech Connect

    Diamond, B.A.

    1987-09-01

    A method is described for removing human anti-native DNA antibody from a liquid sample comprising coupling monoclonal, antiidiotypic antibodies capable of binding to a shared idiotype on human anti-native DNA antibody to a medium. The idiotype shares between genetically nonidentical individuals, contacting a liquid sample to the medium to permit binding of human anti-native DNA antibody in the sample to the anti-idiotypic antibodies and separating the sample from the medium to remove the human anti-native DNA antibodies therefrom.

  9. An SF1 affinity model to identify branch point sequences in human introns

    PubMed Central

    Pastuszak, Alexander W.; Joachimiak, Marcin P.; Blanchette, Marco; Rio, Donald C.; Brenner, Steven E.; Frankel, Alan D.

    2011-01-01

    Splicing factor 1 (SF1) binds to the branch point sequence (BPS) of mammalian introns and is believed to be important for the splicing of some, but not all, introns. To help identify BPSs, particularly those that depend on SF1, we generated a BPS profile model in which SF1 binding affinity data, validated by branch point mapping, were iteratively incorporated into computational models. We searched a data set of 117 499 human introns for best matches to the SF1 Affinity Model above a threshold, and counted the number of matches at each intronic position. After subtracting a background value, we found that 87.9% of remaining high-scoring matches identified were located in a region upstream of 3′-splice sites where BPSs are typically found. Since U2AF65 recognizes the polypyrimidine tract (PPT) and forms a cooperative RNA complex with SF1, we combined the SF1 model with a PPT model computed from high affinity binding sequences for U2AF65. The combined model, together with binding site location constraints, accurately identified introns bound by SF1 that are candidates for SF1-dependent splicing. PMID:21071404

  10. 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 ...

  11. Peripherin-Reactive Antibodies in Mouse, Rabbit, and Human Blood

    PubMed Central

    Strom, Alexander; Sonier, Brigitte; Chapman, Harold D.; Mojibian, Majid; Wang, Gen-Sheng; Slatculescu, Cristina R.; Serreze, David V.; Scott, Fraser W.

    2011-01-01

    Type 1 diabetes (T1D) is an autoimmune disorder that results from the destruction of insulin-producing β-cells in the islets of Langerhans. To date, autoimmune T-cell response and antibody reactivity to more than 20 autoantigens have been linked to this disease. Some studies have described the intermediate filament protein peripherin (PRPH) as an autoantigen associated with T1D in non-obese diabetic (NOD) mice. We evaluated immune reactivity of mouse and rabbit sera and human plasma to a 58 kDa protein expressed in RIN-m5F rat insulinoma cells. The protein was isolated using 2-DE and identified by mass spectrometry as PRPH. Antibodies from healthy humans and T1D patients, CD-1 mice, C57BL/6 mice, NOR (non-obese diabetes resistant) mice, and NOD mice reacted with PRPH on Western blots. However, antibody response to PRPH was stronger in NOD than non-autoimmune prone C57BL/6 mice. We conclude that immune reactivity to PRPH is not exclusively associated with NOD mice or human patients with T1D. Furthermore, the frequent occurrence of PRPH-reactive antibodies in mouse and human blood suggests that binding may be non-specific or could reflect the presence of natural autoantibodies against PRPH. These findings point to the need for a re-evaluation of PRPH as a T1D autoantigen in NOD mice and raise the question of the physiological relevance of such widespread immune reactivity against this peripheral nervous system protein. PMID:20113007

  12. Genetically engineered humanized mouse models for preclinical antibody studies.

    PubMed

    Proetzel, Gabriele; Wiles, Michael V; Roopenian, Derry C

    2014-04-01

    The use of genetic engineering has vastly improved our capabilities to create animal models relevant in preclinical research. With the recent advances in gene-editing technologies, it is now possible to very rapidly create highly tunable mouse models as needs arise. Here, we provide an overview of genetic engineering methods, as well as the development of humanized neonatal Fc receptor (FcRn) models and their use for monoclonal antibody in vivo studies.

  13. Human P-glycoprotein exhibits reduced affinity for substrates during a catalytic transition state.

    PubMed

    Ramachandra, M; Ambudkar, S V; Chen, D; Hrycyna, C A; Dey, S; Gottesman, M M; Pastan, I

    1998-04-01

    Human P-glycoprotein (Pgp), a plasma membrane protein that confers multidrug resistance, functions as an ATP-dependent drug efflux pump. Pgp contains two ATP binding/utilization sites and exhibits ATPase activity that is stimulated in the presence of substrates and modulating agents. The mechanism of coupling of ATP hydrolysis to drug transport is not known. To understand the role of ATP hydrolysis in drug binding, it is necessary to develop methods for purifying and reconstituting Pgp that retains properties including stimulation of ATPase activity by known substrates to an extent similar to that in the native membrane. In this study, (His)6-tagged Pgp was expressed in Trichoplusia ni (High Five) cells using the recombinant baculovirus system and purified by metal affinity chromatography. Upon reconstitution into phospholipid vesicles, purified Pgp exhibited specific binding to analogues of substrates and ATP in affinity labeling experiments and displayed a high level of drug-stimulated ATPase activity (specific activity ranging from 4.5 to 6.5 micromol min-1 mg-1). The ATPase activity was inhibited by ADP in a competitive manner, and by vanadate and N-ethylmaleimide at low concentrations. Vanadate which is known to inhibit ATPase activity by trapping MgADP at the catalytic site inhibited photoaffinity labeling of Pgp with substrate analogues, [125I]iodoarylazidoprazosin and [3H]azidopine, only under ATP hydrolysis conditions. Because vanadate-trapped Pgp is known to resemble the ADP and phosphate-bound catalytic transition state, our findings indicate that ATP hydrolysis results in a conformation with reduced affinity for substrates. A catalytic transition conformation with reduced affinity would essentially result in substrate dissociation and supports a model for drug transport in which an ATP hydrolysis-induced conformational change leads to drug release toward the extracellular medium.

  14. Induction of human complement activation without cytolysis by mouse monoclonal antibodies to human leukocyte antigens.

    PubMed

    Sugita, K; Majdic, O; Stockinger, H; Holter, W; Burger, R; Knapp, W

    1987-04-01

    Ten monoclonal antibodies to human leukocyte subsets that had previously been shown to lyse their respective target cells in the presence of rabbit serum as complement source were evaluated for their cytolytic capacity with human complement. Four of the ten were lytic with human complement. All were of IgM type. Antibodies were also evaluated for their capacity to induce C3 binding to target cells. With this method we could demonstrate that, indeed, 3 of the 6 noncytolytic antibodies had the capacity to initiate the human complement activation process and to induce C3 binding. Two of these 3 antibodies were of IgM class (VIT3 and VIM13), one of IgG3 (562). From the practical point of view the most interesting of these 3 antibodies is the nonmitogenic anti-CD3 pan-T cell antibody VIT3. Therefore, this antibody was analyzed in more detail. VIT3 antibody concentrations needed to induce detectable C3 binding to human T cells are very low (down to 1 ng VIT3/ml). Human serum as complement source can also be considerably (100X) diluted before C3 binding becomes undetectable. Activation of C3 is a prerequesite for VIT3-induced C3 binding, and bound C3 seems to lack the C3a fragment. Bound C3, in contrast to the quickly occuring antigenic modulation of the CD3 complex and the simultaneous disappearance of the antibody coat, remains expressed also after prolonged incubation at 37 degrees C. C3 fragments bound to T cells after activation with VIT3 are also recognized by cells bearing C3 receptors of types CR1 and CR2. PMID:3576673

  15. Anti-Lipid IgG Antibodies Are Produced via Germinal Centers in a Murine Model Resembling Human Lupus

    PubMed Central

    Wong-Baeza, Carlos; Reséndiz-Mora, Albany; Donis-Maturano, Luis; Wong-Baeza, Isabel; Zárate-Neira, Luz; Yam-Puc, Juan Carlos; Calderón-Amador, Juana; Medina, Yolanda; Wong, Carlos; Baeza, Isabel; Flores-Romo, Leopoldo

    2016-01-01

    Anti-lipid IgG antibodies are produced in some mycobacterial infections and in certain autoimmune diseases [such as anti-phospholipid syndrome, systemic lupus erythematosus (SLE)]. However, few studies have addressed the B cell responses underlying the production of these immunoglobulins. Anti-lipid IgG antibodies are consistently found in a murine model resembling human lupus induced by chlorpromazine-stabilized non-bilayer phospholipid arrangements (NPA). NPA are transitory lipid associations found in the membranes of most cells; when NPA are stabilized they can become immunogenic and induce specific IgG antibodies, which appear to be involved in the development of the mouse model of lupus. Of note, anti-NPA antibodies are also detected in patients with SLE and leprosy. We used this model of lupus to investigate in vivo the cellular mechanisms that lead to the production of anti-lipid, class-switched IgG antibodies. In this murine lupus model, we found plasma cells (Gr1−, CD19−, CD138+) producing NPA-specific IgGs in the draining lymph nodes, the spleen, and the bone marrow. We also found a significant number of germinal center B cells (IgD−, CD19+, PNA+) specific for NPA in the draining lymph nodes and the spleen, and we identified in situ the presence of NPA in these germinal centers. By contrast, very few NPA-specific, extrafollicular reaction B cells (B220+, Blimp1+) were found. Moreover, when assessing the anti-NPA IgG antibodies produced during the experimental protocol, we found that the affinity of these antibodies progressively increased over time. Altogether, our data indicate that, in this murine model resembling human lupus, B cells produce anti-NPA IgG antibodies mainly via germinal centers. PMID:27746783

  16. Serodiagnosis of human neurocysticercosis using antigenic components of Taenia solium metacestodes derived from the unbound fraction from jacalin affinity chromatography

    PubMed Central

    Machado, Gleyce Alves; de Oliveira, Heliana Batista; Gennari-Cardoso, Margareth Leitão; Mineo, José Roberto; Costa-Cruz, Julia Maria

    2013-01-01

    The aim of the present study was to analyse Taenia solium metacestode antigens that were derived from the unbound fraction of jacalin affinity chromatography and subsequent tert-octylphenoxy poly (oxyethylene) ethanol Triton X-114 (TX-114) partitioning in the diagnosis of human neurocysticercosis (NCC). Immunoassays were designed to detect T. solium-specific IgG antibodies by ELISA and immunoblot. Serum samples were collected from 132 individuals who were categorised as follows: 40 had NCC, 62 presented Taenia spp or other parasitic diseases and 30 were healthy individuals. The jacalin-unbound (Junbound) fraction presented higher sensitivity and specificity rates than the jacalin-bound fraction and only this fraction was subjected to subsequent TX-114 partitioning, resulting in detergent (DJunbound) and aqueous (AJunbound) fractions. The ELISA sensitivity and specificity were 85% and 84.8% for Junbound, 92.5% and 93.5% for DJunboundand 82.5% and 82.6% for AJunbound. By immunoblot, the DJunboundfraction showed 100% sensitivity and specificity and only serum samples from patients with NCC recognised the 50-70 kDa T. solium-specific components. We conclude that the DJunboundfraction can serve as a useful tool for the differential immunodiagnosis of NCC by immunoblot. PMID:23778661

  17. Evidence that the Echinococcus granulosus G6 genotype has an affinity for the brain in humans.

    PubMed

    Sadjjadi, S M; Mikaeili, F; Karamian, M; Maraghi, S; Sadjjadi, F S; Shariat-Torbaghan, S; Kia, E B

    2013-10-01

    The present study investigates the molecular characteristics of cerebral Echinococcus cysts. A total of 10 specimens of cerebral Echinococcus cysts, including six formalin-fixed paraffin blocks and four intact cerebral cysts, were used for this study. The target DNA was successfully amplified from eight samples and sequenced. BLAST analysis indicated that sequenced isolates belong to the Echinococcus granulosus (G6) genotype. All of the eight sampled brain cysts belonged to the G6 genotype, while all of the eight liver cysts belonged to G1. This is a strong indication that G6 has a higher affinity for the human brain than G1.

  18. A versatile bifunctional chelate for radiolabeling humanized anti-CEA antibody with In-111 and Cu-64 at either thiol or amino groups: PET imaging of CEA-positive tumors with whole antibodies.

    PubMed

    Li, Lin; Bading, James; Yazaki, Paul J; Ahuja, Amitkumar H; Crow, Desiree; Colcher, David; Williams, Lawrence E; Wong, Jeffrey Y C; Raubitschek, Andrew; Shively, John E

    2008-01-01

    Radiolabeled anti-carcinoembryonic antigen (CEA) antibodies have the potential to give excellent images of a wide variety of human tumors, including tumors of the colon, breast, lung, and medullar thyroid. In order to realize the goals of routine and repetitive clinical imaging with anti-CEA antibodies, it is necessary that the antibodies have a high affinity for CEA, low cross reactivity and uptake in normal tissues, and low immunogenicity. The humanized anti-CEA antibody hT84.66-M5A (M5A) fulfills these criteria with an affinity constant of >10 (10) M (-1), no reactivity with CEA cross-reacting antigens found in normal tissues, and >90% human protein sequence. A further requirement for routine clinical use of radiolabeled antibodies is a versatile method of radiolabeling that allows the use of multiple radionuclides that differ in their radioemissions and half-lives. We describe a versatile bifunctional chelator, DO3A-VS (1,4,7-tris(carboxymethyl)-10-(vinylsulfone)-1,4,7,10-tetraazacyclododecane) that binds a range of radiometals including 111 In for gamma-ray imaging and 64Cu for positron emission tomography (PET), and which can be conjugated with negligible loss of immunoreactivity either to sulfhydryls (SH) in the hinge region of lightly reduced immunoglobulins or surface lysines (NH) of immunoglobulins. Based on our correlative studies comparing the kinetics of radiolabeled anti-CEA antibodies in murine models with those in man, we predict that 64Cu-labeled intact, humanized antibodies can be used to image CEA positive tumors in the clinic.

  19. Specificity of human anti-carbohydrate IgG antibodies as probed with polyacrylamide-based glycoconjugates.

    PubMed

    Smorodin, E P; Kurtenkov, O A; Sergeyev, B L; Pazynina, G V; Bovin, N V

    2004-01-01

    The TF, Tn, and SiaTn glycotopes are frequently expressed in cancer-associated mucins. Antibodies to these glycotopes were found in human serum. A set of polyacrylamide (PAA)--based glycoconjugates was applied to the direct and competitive enzyme-linked immunosorbent assays (ELISA) to characterize the specificity of serum IgG antibodies. The anti-TF, -Tn and -SiaTn IgG were affinity purified from serum of cancer patients and characterized using PAA-conjugates and free saccharides. The anti-TF and -Tn antibodies were shown to be specific. The anti-TF IgG bound both Galbeta1-3GalNAcalpha- and Galbeta1-3GalNAcbeta-PAA, the latter was three-four times more effective inhibitor of antibody binding. The anti-Tn IgG reacted only with GalNAcalpha-PAA. The anti-SiaTn IgG cross-reacted with Tn-PAA but SiaTn-PAA was five-six times more effective inhibitor in a competitive assay. The IC50 values for PAA-conjugates with the corresponding antibodies typically ranged from 2 to 5 x 10(-8) M. The antibodies display a low specificity to mucin-type glycoconjugates in comparison with PAA-conjugates as was shown for mucins isolated from human malignant tumor tissues, ovine submaxillary mucin (OSM) and asialo-OSM. The unusual IgG-antibody specificity to GalNAcbeta and GalNAcbeta1-3GalNAcbeta ligands was found in human serum.

  20. Specificity of human anti-carbohydrate IgG antibodies as probed with polyacrylamide-based glycoconjugates.

    PubMed

    Smorodin, E P; Kurtenkov, O A; Sergeyev, B L; Pazynina, G V; Bovin, N V

    2004-01-01

    The TF, Tn, and SiaTn glycotopes are frequently expressed in cancer-associated mucins. Antibodies to these glycotopes were found in human serum. A set of polyacrylamide (PAA)--based glycoconjugates was applied to the direct and competitive enzyme-linked immunosorbent assays (ELISA) to characterize the specificity of serum IgG antibodies. The anti-TF, -Tn and -SiaTn IgG were affinity purified from serum of cancer patients and characterized using PAA-conjugates and free saccharides. The anti-TF and -Tn antibodies were shown to be specific. The anti-TF IgG bound both Galbeta1-3GalNAcalpha- and Galbeta1-3GalNAcbeta-PAA, the latter was three-four times more effective inhibitor of antibody binding. The anti-Tn IgG reacted only with GalNAcalpha-PAA. The anti-SiaTn IgG cross-reacted with Tn-PAA but SiaTn-PAA was five-six times more effective inhibitor in a competitive assay. The IC50 values for PAA-conjugates with the corresponding antibodies typically ranged from 2 to 5 x 10(-8) M. The antibodies display a low specificity to mucin-type glycoconjugates in comparison with PAA-conjugates as was shown for mucins isolated from human malignant tumor tissues, ovine submaxillary mucin (OSM) and asialo-OSM. The unusual IgG-antibody specificity to GalNAcbeta and GalNAcbeta1-3GalNAcbeta ligands was found in human serum. PMID:15001840

  1. Characterization of the rabbit homolog of human MUC1 glycoprotein isolated from bladder by affinity chromatography on immobilized jacalin.

    PubMed

    Higuchi, T; Xin, P; Buckley, M S; Erickson, D R; Bhavanandan, V P

    2000-07-01

    The urinary bladder is lined by transitional epithelium, the glycocalyx on the luminal surface has interesting properties and is implicated in protective functions. Glycoconjugates are major components of the glycocalyx, but their biochemical nature is not well understood. Previous studies on rabbit bladder indicated the presence of significant levels of sialoglycoproteins compared to glycosaminoglycans in the epithelium. In this study, rabbit explant cultures were radiolabeled by precursor sugars or amino acids and a major lectin-reactive glycoprotein of rabbit bladder mucosa was isolated by affinity chromatography on jacalin-agarose. The radiolabeled glycoprotein was purified to homogeneity by a second cycle on the lectin column, followed by gel filtration and density gradient centrifugation. The average molecular mass of the glycoprotein was estimated to be 245 kDa and 210 kDa by gel filtration and SDS-PAGE, respectively. Its buoyant density was 1.40 g/ml, suggesting a carbohydrate content of approximately 50%. The percent distribution of glucosamine-derived tritium label in sialic acid, galactosamine, and glucosamine was 30, 52, and 18, respectively. The glycoprotein consisted entirely of small sialylated and neutral oligosaccharides O-glycosidically linked to serine and threonine residues. The same glycoprotein could be immunoprecipitated with an antibody against the carboxy terminal 17 amino acid peptide of human MUC1 mucin glycoprotein. This suggests that this mucin glycoprotein is the rabbit homolog of MUC1 glycoprotein, which has been previously established to be a component of human bladder urothelium and has been purified from human urine and biochemically characterized.

  2. Recombinant human tumor necrosis factor-alpha. Regulation of N-formylmethionylleucylphenylalanine receptor affinity and function on human neutrophils.

    PubMed Central

    Atkinson, Y H; Marasco, W A; Lopez, A F; Vadas, M A

    1988-01-01

    Preincubation of neutrophils with recombinant human tumor necrosis factor-alpha (rH TNF-alpha) enhanced the subsequent release of superoxide anion in response to various concentrations of N-formylmethionylleucylphenylalanine (FMLP). Enhanced superoxide anion production was evident by 5 min and had reached a plateau by 15 min. Not only was the total amount of superoxide anion released greater, but the rate of release was also enhanced threefold by rH TNF-alpha. In contrast, rH TNF-alpha reduced or abolished neutrophil locomotion under agarose in response to a gradient of FMLP. Binding studies of f-Met-Leu-[3H]Phe to purified human neutrophils revealed a heterogeneous binding to unstimulated cells. The high affinity component consisted of approximately 2,000 sites per cell and had an average Kd of 2 +/- 0.7 nM (n = 4). The low affinity component consisted of approximately 40,000 sites per cell and had an average Kd of 180 +/- 50 nM (n = 4). rH TNF-alpha caused conversion to a linear Scatchard plot showing no significant change in total binding sites but a single Kd of 40 +/- 10 nM (n = 4). These data indicate that rH TNF-alpha may influence neutrophil responses to FMLP by regulating the affinity of FMLP receptors. PMID:2830314

  3. Clonal analysis of a human antibody response. Quantitation of precursors of antibody-producing cells and generation and characterization of monoclonal IgM, IgG, and IgA to rabies virus

    PubMed Central

    1990-01-01

    We quantitated and characterized the changes in the human B cell repertoire, at the clonal level, before and after immunization with rabies virus. Moreover, we generated 10 monoclonal cell lines producing IgM, IgG, and IgA antibodies to the virus. We found that in healthy subjects, not previously exposed to the virus, nearly 2% of the circulating B lymphocytes were committed to the production of antibodies that bound the virus. These B cells expressed the surface CD5 molecule. The antibodies they produced were polyreactive IgM that displayed a relatively low affinity for the virus components (Kd, 1.0- 2.4 x 10(-6) g/microliters). After immunization, different anti-virus (IgG and IgA) antibody-producing cells consistently appeared in the circulation and increased from less than 0.005% to greater than 10% of the total B cells committed to the production of IgG and IgA, respectively. Most of such B cells do not express CD5 and produce monoreactive antibodies of high affinity for rabies virus (Kd, 6.5 x 10(-9) to 1.2 x 10(-10) g/microliters). One of these IgG mAbs efficiently neutralized rabies virus in vitro and in vivo, as detailed elsewhere (Dietzschold, B., P. Casali, Y. Ueki, M. Gore, C. E. Rupprecht, A. L. Notkins, and H. Koprowski, manuscript submitted for publication). Hybridization experiments using probes specific for the different human V gene segment families revealed that cell precursors producing low affinity IgM binding to rabies virus utilized a restricted number of VH gene segments (i.e., only members of the VHIIIb subfamily), whereas cell precursors producing high affinity IgG and IgA to rabies virus utilized an assortment of different VH gene segments (i.e., members of the VHI, VHIII, VHIV, and VHVI families and VHIIIb subfamily). In conclusion, our studies show that EBV transformation in conjunction with limiting dilution technology and somatic cell hybridization techniques are useful methods for quantitating, at the B cell clonal level, the human

  4. A model-based approach to predicting the human pharmacokinetics of a monoclonal antibody exhibiting target-mediated drug disposition.

    PubMed

    Luu, Kenneth T; Bergqvist, Simon; Chen, Enhong; Hu-Lowe, Dana; Kraynov, Eugenia

    2012-06-01

    In the drug discovery and development setting, the ability to accurately predict the human pharmacokinetics (PK) of a candidate compound from preclinical data is critical for informing the effective design of the first-in-human trial. PK prediction is especially challenging for monoclonal antibodies exhibiting nonlinear PK attributed to target-mediated drug disposition (TMDD). Here, we present a model-based method for predicting the PK of PF-03446962, an IgG2 antibody directed against human ALK1 (activin receptor-like kinase 1) receptor. Systems parameters as determined experimentally or obtained from the literature, such as binding affinity (k(on) and k(off)), internalization of the drug-target complex (k(int)), target degradation rate (k(deg)), and target abundance (R(0)), were directly integrated into the modeling and prediction. NONMEM 7 was used to model monkey PK data and simulate human PK profiles based on the construct of a TMDD model using a population-based approach. As validated by actual patient data from a phase I study, the human PK of PF-03446962 were predicted within 1- to 2-fold of observations. Whereas traditional approaches fail, this approach successfully predicted the human PK of a monoclonal antibody exhibiting nonlinearity because of TMDD. PMID:22414855

  5. A model-based approach to predicting the human pharmacokinetics of a monoclonal antibody exhibiting target-mediated drug disposition.

    PubMed

    Luu, Kenneth T; Bergqvist, Simon; Chen, Enhong; Hu-Lowe, Dana; Kraynov, Eugenia

    2012-06-01

    In the drug discovery and development setting, the ability to accurately predict the human pharmacokinetics (PK) of a candidate compound from preclinical data is critical for informing the effective design of the first-in-human trial. PK prediction is especially challenging for monoclonal antibodies exhibiting nonlinear PK attributed to target-mediated drug disposition (TMDD). Here, we present a model-based method for predicting the PK of PF-03446962, an IgG2 antibody directed against human ALK1 (activin receptor-like kinase 1) receptor. Systems parameters as determined experimentally or obtained from the literature, such as binding affinity (k(on) and k(off)), internalization of the drug-target complex (k(int)), target degradation rate (k(deg)), and target abundance (R(0)), were directly integrated into the modeling and prediction. NONMEM 7 was used to model monkey PK data and simulate human PK profiles based on the construct of a TMDD model using a population-based approach. As validated by actual patient data from a phase I study, the human PK of PF-03446962 were predicted within 1- to 2-fold of observations. Whereas traditional approaches fail, this approach successfully predicted the human PK of a monoclonal antibody exhibiting nonlinearity because of TMDD.

  6. Selection and application of broad-specificity human domain antibody for simultaneous detection of Bt Cry toxins.

    PubMed

    Xu, Chongxin; Zhang, Xiao; Liu, Xiaoqin; Liu, Yuan; Hu, Xiaodan; Zhong, Jianfeng; Zhang, Cunzheng; Liu, Xianjin

    2016-11-01

    Bt Cry toxin is a kind of bio-toxins that used for genetically modified crops (GMC) transformation widely. In this study, total 15 positive clones could bind the Bt Cry toxins which isolated from a human domain antibody library by 5 rounds affinity selection. According to analyzing of PCR amplification and enzyme-linked immunosorbent assay (ELISA), the most positive phage domain antibody (named F5) gene was cloned into the pET26b vector and expressed in E. coli BL21. The purified antibody was used to develop an indirect competitive ELISA (IC-ELISA) for Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F toxins, respectively. The working range of detection for standard curves in IC-ELISA were 0.258-1.407 μg/mL, the medium inhibition concentration (IC50) were 0.727-0.892 μg/mL and detection limit (IC10) were 0.029-0.074 μg/mL for those Bt Cry toxins. The affinity of F5 domain antibody with Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F toxins were 1.21-5.94 × 10(7) M(-1). The average recoveries of the 5 kinds of Bt Cry toxins from spiked wheat samples were ranged from 81.2%-100.8% with a CV at 2.5%-9.4%. The results showed that we successfully obtained the broad-specificity human domain antibody for simultaneous detection of Bt Cry toxins in agricultural product samples. PMID:27544649

  7. Selection and application of broad-specificity human domain antibody for simultaneous detection of Bt Cry toxins.

    PubMed

    Xu, Chongxin; Zhang, Xiao; Liu, Xiaoqin; Liu, Yuan; Hu, Xiaodan; Zhong, Jianfeng; Zhang, Cunzheng; Liu, Xianjin

    2016-11-01

    Bt Cry toxin is a kind of bio-toxins that used for genetically modified crops (GMC) transformation widely. In this study, total 15 positive clones could bind the Bt Cry toxins which isolated from a human domain antibody library by 5 rounds affinity selection. According to analyzing of PCR amplification and enzyme-linked immunosorbent assay (ELISA), the most positive phage domain antibody (named F5) gene was cloned into the pET26b vector and expressed in E. coli BL21. The purified antibody was used to develop an indirect competitive ELISA (IC-ELISA) for Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F toxins, respectively. The working range of detection for standard curves in IC-ELISA were 0.258-1.407 μg/mL, the medium inhibition concentration (IC50) were 0.727-0.892 μg/mL and detection limit (IC10) were 0.029-0.074 μg/mL for those Bt Cry toxins. The affinity of F5 domain antibody with Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F toxins were 1.21-5.94 × 10(7) M(-1). The average recoveries of the 5 kinds of Bt Cry toxins from spiked wheat samples were ranged from 81.2%-100.8% with a CV at 2.5%-9.4%. The results showed that we successfully obtained the broad-specificity human domain antibody for simultaneous detection of Bt Cry toxins in agricultural product samples.

  8. Net charge and oxygen affinity of human hemoglobin are independent of hemoglobin concentration

    PubMed Central

    1978-01-01

    The dependence of net charge and oxygen affinity of human hemoglobin upon hemoglobin concentration was reinvestigated. In contrast to earlier reports from various laboratories, both functional properties of hemoglobin were found to be independent of hemoglobin concentration. Two findings indicate a concentration-independent net charge of carbonmonoxy hemoglobin at pH 6.6: (A) The pH value of a given carbonmonoty hemoglobin solution remains constant at 6.6 when the hemoglobin concentration is raised from 10 to 40 g/dl, indicating that there is no change in protonation of titratable groups of hemoglobin: (b) the net charge of carbonmonoxy hemoglobin as estimated from the Donnan distribution of 22Na+ shows no dependence on hemoglobin concentration in this concentration range. The oxygen affinity of human hemoglobin was determined from measurements of oxygen concentrations in equilibrated samples using a Lex-O2-Con apparatus (Lexington Instruments, Waltham, Mass.). P50 averaged 11.4 mm Hg at 37 degrees C, pH = 7.2, and ionic strength approximately 0.15. Neither P50 nor Hill's n showed any variation with hemoglobin concentrations increasing from 10 to 40 g/dl. PMID:32221

  9. NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies.

    PubMed

    Moutel, Sandrine; Bery, Nicolas; Bernard, Virginie; Keller, Laura; Lemesre, Emilie; de Marco, Ario; Ligat, Laetitia; Rain, Jean-Christophe; Favre, Gilles; Olichon, Aurélien; Perez, Franck

    2016-01-01

    In vitro selection of antibodies allows to obtain highly functional binders, rapidly and at lower cost. Here, we describe the first fully synthetic phage display library of humanized llama single domain antibody (NaLi-H1: Nanobody Library Humanized 1). Based on a humanized synthetic single domain antibody (hs2dAb) scaffold optimized for intracellular stability, the highly diverse library provides high affinity binders without animal immunization. NaLi-H1 was screened following several selection schemes against various targets (Fluorescent proteins, actin, tubulin, p53, HP1). Conformation antibodies against active RHO GTPase were also obtained. Selected hs2dAb were used in various immunoassays and were often found to be functional intrabodies, enabling tracking or inhibition of endogenous targets. Functionalization of intrabodies allowed specific protein knockdown in living cells. Finally, direct selection against the surface of tumor cells produced hs2dAb directed against tumor-specific antigens further highlighting the potential use of this library for therapeutic applications. PMID:27434673

  10. NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies

    PubMed Central

    Moutel, Sandrine; Bery, Nicolas; Bernard, Virginie; Keller, Laura; Lemesre, Emilie; de Marco, Ario; Ligat, Laetitia; Rain, Jean-Christophe; Favre, Gilles; Olichon, Aurélien; Perez, Franck

    2016-01-01

    In vitro selection of antibodies allows to obtain highly functional binders, rapidly and at lower cost. Here, we describe the first fully synthetic phage display library of humanized llama single domain antibody (NaLi-H1: Nanobody Library Humanized 1). Based on a humanized synthetic single domain antibody (hs2dAb) scaffold optimized for intracellular stability, the highly diverse library provides high affinity binders without animal immunization. NaLi-H1 was screened following several selection schemes against various targets (Fluorescent proteins, actin, tubulin, p53, HP1). Conformation antibodies against active RHO GTPase were also obtained. Selected hs2dAb were used in various immunoassays and were often found to be functional intrabodies, enabling tracking or inhibition of endogenous targets. Functionalization of intrabodies allowed specific protein knockdown in living cells. Finally, direct selection against the surface of tumor cells produced hs2dAb directed against tumor-specific antigens further highlighting the potential use of this library for therapeutic applications. DOI: http://dx.doi.org/10.7554/eLife.16228.001 PMID:27434673

  11. A cocktail of humanized anti-pertussis toxin antibodies limits disease in murine and baboon models of whooping cough

    PubMed Central

    Nguyen, Annalee W.; Wagner, Ellen K.; Laber, Joshua R.; Goodfield, Laura L.; Smallridge, William E.; Harvill, Eric T.; Papin, James F.; Wolf, Roman F.; Padlan, Eduardo A.; Bristol, Andy; Kaleko, Michael; Maynard, Jennifer A.

    2016-01-01

    In spite of wide-spread vaccination, pertussis rates are rising in industrialized countries and remain high world-wide. With no specific therapeutics to treat disease, pertussis continues to cause considerable infant morbidity and mortality. The pertussis toxin is a major contributor to disease, responsible for local and systemic effects including leukocytosis and immunosuppression. Here, we humanized two murine monoclonal antibodies that neutralize pertussis toxin and expressed them as human IgG1 molecules with no loss of affinity or in vitro neutralization activity. When administered prophylactically to mice as a binary cocktail, antibody treatment completely mitigated the B. pertussis-induced rise in white blood cell count and decreased bacterial colonization. When administered therapeutically to baboons, antibody-treated but not control animals experienced a blunted rise in white blood cell count and accelerated bacterial clearance rates. These preliminary findings support further investigation into the use of these antibodies to treat human neonatal pertussis in conjunction with antibiotics and supportive care. PMID:26631634

  12. Estimation of interaction between oriented immobilized green fluorescent protein and its antibody by high performance affinity chromatography and molecular docking.

    PubMed

    Li, Qian; Wang, Jing; Yang, Lingjian; Gao, Xiaokang; Chen, Hongwei; Zhao, Xinfeng; Bian, Liujiao; Zheng, Xiaohui

    2015-07-01

    Although green fluorescence protein (GFP) and its antibody are widely used to track a protein or a cell in life sciences, the binding behavior between them remains unclear. In this work, diazo coupling method that synthesized a new stationary GFP was oriented immobilized on the surface of macro-porous silica gel by a phase. The stationary phase was utilized to confirm the validation of injection amount-dependent analysis in exploring protein-protein interaction that use GFP antibody as a probe. GFP antibody was proved to have one type of binding site on immobilized GFP. The number of binding site and association constant were calculated to be (6.41 ± 0.76) × 10(-10) M and (1.39 ± 0.12) × 10(9) M(-1). Further analysis by molecular docking showed that the binding of GFP to its antibody is mainly driven by hydrogen bonds and salt bridges. These results indicated that injection amount-dependent analysis is capable of exploring the protein-protein interactions with the advantages of ligand and time saving. It is a valuable methodology for the ligands, which are expensive or difficult to obtain. PMID:25727342

  13. Phage display and hybridoma generation of antibodies to human CXCR2 yields antibodies with distinct mechanisms and epitopes.

    PubMed

    Rossant, Christine J; Carroll, Danielle; Huang, Ling; Elvin, John; Neal, Frances; Walker, Edward; Benschop, Joris J; Kim, Eldar E; Barry, Simon T; Vaughan, Tristan J

    2014-01-01

    Generation of functional antibodies against integral membrane proteins such as the G-protein coupled receptor CXCR2 is technically challenging for several reasons, including limited epitope accessibility, the requirement for a lipid environment to maintain structure and their existence in dynamic conformational states. Antibodies to human CXCR2 were generated by immunization in vivo and by in vitro selection methods. Whole cell immunization of transgenic mice and screening of phage display libraries using CXCR2 magnetic proteoliposomes resulted in the isolation of antibodies with distinct modes of action. The hybridoma-derived antibody fully inhibited IL-8 and Gro-α responses in calcium flux and β-arrestin recruitment assays. The phage-display derived antibodies were allosteric antagonists that showed ligand dependent differences in functional assays. The hybridoma and phage display antibodies did not cross-compete in epitope competition assays and mapping using linear and CLIPS peptides confirmed that they recognized distinct epitopes of human CXCR2. This illustrates the benefits of using parallel antibody isolation approaches with different antigen presentation methods to successfully generate functionally and mechanistically diverse antagonistic antibodies to human CXCR2. The method is likely to be broadly applicable to other complex membrane proteins.

  14. Affinities of brompheniramine, chlorpheniramine, and terfenadine at the five human muscarinic cholinergic receptor subtypes.

    PubMed

    Yasuda, S U; Yasuda, R P

    1999-04-01

    Anticholinergic effects are presumed to be the mechanism for the efficacy of chlorpheniramine in symptomatic relief of the common cold. Terfenadine, a second-generation antihistamine, reportedly lacks anticholinergic side effects. We evaluated affinities of two commonly used over-the-counter antihistamines, brompheniramine and chlorpheniramine, as well as terfenadine in comparison with atropine at the five human muscarinic cholinergic receptor subtypes using CHO cells stably transfected with the individual subtypes. Atropine was more potent than all three drugs at m1-m5 (p<0.01). No significant difference was observed between chlorpheniramine and brompheniramine. Atropine, brompheniramine, and chlorpheniramine could not discriminate between m1-m5. Terfenadine demonstrated subtype selectivity at m3. In vitro comparisons in human muscarinic receptor subtypes could potentially be used to predict clinical anticholinergic effects of antihistamines and to target receptor-specific effects of such agents.

  15. Yawn contagion in humans and bonobos: emotional affinity matters more than species.

    PubMed

    Palagi, Elisabetta; Norscia, Ivan; Demuru, Elisa

    2014-01-01

    In humans and apes, yawn contagion echoes emotional contagion, the basal layer of empathy. Hence, yawn contagion is a unique tool to compare empathy across species. If humans are the most empathic animal species, they should show the highest empathic response also at the level of emotional contagion. We gathered data on yawn contagion in humans (Homo sapiens) and bonobos (Pan paniscus) by applying the same observational paradigm and identical operational definitions. We selected a naturalistic approach because experimental management practices can produce different psychological and behavioural biases in the two species, and differential attention to artificial stimuli. Within species, yawn contagion was highest between strongly bonded subjects. Between species, sensitivity to others' yawns was higher in humans than in bonobos when involving kin and friends but was similar when considering weakly-bonded subjects. Thus, emotional contagion is not always highest in humans. The cognitive components concur in empowering emotional affinity between individuals. Yet, when they are not in play, humans climb down from the empathic podium to return to the "understory", which our species shares with apes. PMID:25165630

  16. Yawn contagion in humans and bonobos: emotional affinity matters more than species

    PubMed Central

    Norscia, Ivan; Demuru, Elisa

    2014-01-01

    In humans and apes, yawn contagion echoes emotional contagion, the basal layer of empathy. Hence, yawn contagion is a unique tool to compare empathy across species. If humans are the most empathic animal species, they should show the highest empathic response also at the level of emotional contagion. We gathered data on yawn contagion in humans (Homo sapiens) and bonobos (Pan paniscus) by applying the same observational paradigm and identical operational definitions. We selected a naturalistic approach because experimental management practices can produce different psychological and behavioural biases in the two species, and differential attention to artificial stimuli. Within species, yawn contagion was highest between strongly bonded subjects. Between species, sensitivity to others’ yawns was higher in humans than in bonobos when involving kin and friends but was similar when considering weakly-bonded subjects. Thus, emotional contagion is not always highest in humans. The cognitive components concur in empowering emotional affinity between individuals. Yet, when they are not in play, humans climb down from the empathic podium to return to the “understory”, which our species shares with apes. PMID:25165630

  17. Unique Biological Properties of Catalytic Domain Directed Human Anti-CAIX Antibodies Discovered through Phage-Display Technology

    PubMed Central

    Xu, Chen; Lo, Agnes; Yammanuru, Anuradha; Tallarico, Aimee St. Clair; Brady, Kristen; Murakami, Akikazu; Barteneva, Natasha; Zhu, Quan; Marasco, Wayne A.

    2010-01-01

    Carbonic anhydrase IX (CAIX, gene G250/MN-encoded transmembrane protein) is highly expressed in various human epithelial tumors such as renal clear cell carcinoma (RCC), but absent from the corresponding normal tissues. Besides the CA signal transduction activity, CAIX may serve as a biomarker in early stages of oncogenesis and also as a reliable marker of hypoxia, which is associated with tumor resistance to chemotherapy and radiotherapy. Although results from preclinical and clinical studies have shown CAIX as a promising target for detection and therapy for RCC, only a limited number of murine monoclonal antibodies (mAbs) and one humanized mAb are available for clinical testing and development. In this study, paramagnetic proteoliposomes of CAIX (CAIX-PMPLs) were constructed and used for anti-CAIX antibody selection from our 27 billion human single-chain antibody (scFv) phage display libraries. A panel of thirteen human scFvs that specifically recognize CAIX expressed on cell surface was identified, epitope mapped primarily to the CA domain, and affinity-binding constants (KD) determined. These human anti-CAIX mAbs are diverse in their functions including induction of surface CAIX internalization into endosomes and inhibition of the carbonic anhydrase activity, the latter being a unique feature that has not been previously reported for anti-CAIX antibodies. These human anti-CAIX antibodies are important reagents for development of new immunotherapies and diagnostic tools for RCC treatment as well as extending our knowledge on the basic structure-function relationships of the CAIX molecule. PMID:20224781

  18. Development of purification processes for fully human bispecific antibodies based upon modification of protein A binding avidity

    PubMed Central

    Tustian, Andrew D.; Endicott, Christine; Adams, Benjamin; Mattila, John; Bak, Hanne

    2016-01-01

    ABSTRACT There is strong interest in the design of bispecific monoclonal antibodies (bsAbs) that can simultaneously bind 2 distinct targets or epitopes to achieve novel mechanisms of action and efficacy. Multiple bispecific formats have been proposed and are currently under development. Regeneron's bispecific technology is based upon a standard fully human IgG antibody in order to minimize immunogenicity and improve the pharmacokinetic profile. A single common light chain and 2 distinct heavy chains combine to form the bispecific molecule. One of the heavy chains contains a chimeric Fc sequence form (called Fc*) that ablates binding to Protein A via the constant region. As a result of co-expression of the 2 heavy chains and the common light chain, 3 products are created, 2 of which are homodimeric for the heavy chains and one that is the desired heterodimeric bispecific product. The Fc* sequence allows selective purification of the FcFc* bispecific product on commercially available affinity columns, due to intermediate binding affinity for Protein A compared to the high avidity FcFc heavy chain homodimer, or the weakly binding Fc*Fc* homodimer. This platform requires the use of Protein A chromatography in both a capture and polishing modality. Several challenges, including variable region Protein A binding, resin selection, selective elution optimization, and impacts upon subsequent non-affinity downstream unit operations, were addressed to create a robust and selective manufacturing process. PMID:26963837

  19. Polyreactive Antibodies: Function and Quantification.

    PubMed

    Gunti, Sreenivasulu; Notkins, Abner Louis

    2015-07-15

    Polyreactive antibodies, a major component of the natural antibody repertoire, bind with low affinity to a variety of structurally unrelated antigens. Many of these antibodies are germline or near germline in sequence. Little is known, however, about the function of these antibodies. In the present mini-review we show: (1) that the broad antibacterial activity of the natural antibody repertoire is largely due to polyreactive antibodies, which in the presence of complement lyse bacteria and enhance phagocytosis; (2) that polyreactive antibodies bind to UV- or human immunodeficiency virus-induced apoptotic cells and with complement enhance the phagocytosis of these cells by macrophages; and (3) that dinitrophenol can be used as a surrogate for quantitating the level of polyreactive antibodies in serum. We conclude that polyreactive antibodies protect the host against both foreign invaders and its own damaged/apoptotic cells.

  20. Comparison of nonhuman primate antibodies against Haemophilus influenzae type b polysaccharide with human antibodies in oligoclonality and in vivo protective potency.

    PubMed

    Kim, K H; Park, M K; Peeters, C C; Poolman, J T; Shearer, M H; Kennedy, R C; Nahm, M H

    1994-06-01

    Nonhuman primates are often used as a model for studying vaccines for humans. However, it is not always clear how closely the antibody responses in these species mimic human responses. Recent studies have characterized the human antibody response to Haemophilus influenzae type b (Hib) in great detail. In this study, we have compared the antibody response to Hib of humans with those of other primates. Studies of isoelectric points and V kappa subgroup usage show that, like humans, nonhuman primates produce oligoclonal antibodies. Also, monkey antibodies to the Hib polysaccharide are as protective as human antibodies in an in vivo model of Hib infection. Thus, we conclude that nonhuman primates produce antibodies to Hib polysaccharide that are structurally and functionally similar to human antibodies and are a good model for testing human vaccines.

  1. Design of protease-resistant peptide ligands for the purification of antibodies from human plasma.

    PubMed

    Menegatti, Stefano; Bobay, Benjamin G; Ward, Kevin L; Islam, Tuhidul; Kish, William S; Naik, Amith D; Carbonell, Ruben G

    2016-05-01

    A strategy is presented for developing variants of peptide ligands with enhanced biochemical stability for the purification of antibodies from animal sera. Antibody-binding sequences HWRGWV, HYFKFD, and HFRRHL, previously discovered by our group, were modified with non-natural amino acids to gain resistance to proteolysis, while maintaining target affinity and selectivity. As trypsin and α-chymotrypsin were chosen as models of natural proteolytic enzymes, the basic (arginine and lysine) and aromatic (tryptophan, phenylalanine, and tyrosine) amino acids were replaced with non-natural analogs. Using the docking software HADDOCK, a virtual library of peptide variants was designed and screened in-silico against the known HWRGWV binding site on the pFc fragment of IgG. A pool of selected sequences with the highest predicted free energy of binding was synthesized on chromatographic resin, and the resulting adsorbents were tested for IgG binding and resistance to proteases. The ligand variants exhibited binding capacities and specificities comparable to the original sequences, yet with much higher proteolytic resistances. The sequences HWMetCitGWMetV and HFMetCitCitHL was used for purifying polyclonal IgG from IgG-rich fractions of human plasma, with yields and purity above 90%. Notably, due to electrical neutrality, the variant showed higher selectivity than the original sequence. Binding isotherms were also constructed, which confirmed the docking predictions. This method represents a general strategy for enhancing the biochemical stability as well as the affinity and selectivity of natural or synthetic peptide ligands for bioseparations.

  2. 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

  3. Highly specific off-target binding identified and eliminated during the humanization of an antibody against FGF receptor 4

    PubMed Central

    Reyes, Arthur E; Lin, Benjamin C; Stephan, Jean-Philippe; Desnoyers, Luc; Shen, Ben-Quan

    2011-01-01

    Off-target binding can significantly affect the pharmacokinetics (PK), tissue distribution, efficacy and toxicity of a therapeutic antibody. Herein we describe the development of a humanized anti-fibroblast growth factor receptor 4 (FGFR4) antibody as a potential therapeutic for hepatocellular carcinoma (HCC). A chimeric anti-FGFR4 monoclonal antibody (chLD1) was previously shown to block ligand binding and to inhibit FGFR4-mediated signaling as well as tumor growth in vivo. A humanized version of chLD1, hLD1.vB, had similar binding affinity and in vitro blocking activity, but it exhibited rapid clearance, poor target tissue biodistribution and limited efficacy when compared to chLD1 in a HUH7 human HCC xenograft mouse model. These problems were traced to instability of the molecule in rodent serum. Size exclusion high performance liquid chromatography, immunoprecipitation and mass spectral sequencing identified a specific interaction between hLD1.vB and mouse complement component 3 (C3). A PK study in C3 knock-out mice further confirmed this specific interaction. Subsequently, an affinity-matured variant derived from hLD1.vB (hLD1.v22), specifically selected for its lack of binding to mouse C3 was demonstrated to have a PK profile and in vivo efficacy similar to that of chLD1 in mice. Although reports of non-specific off-target binding have been observed for other antibodies, this represents the first report identifying a specific off-target interaction that affected disposition and biological activity. Screens developed to identify general non-specific interactions are likely to miss the rare and highly specific cross-reactivity identified in this study, thus highlighting the importance of animal models as a proxy for avoiding unexpected clinical outcomes. PMID:21540647

  4. Phage display-derived human antibodies in clinical development and therapy

    PubMed Central

    Frenzel, André; Schirrmann, Thomas; Hust, Michael

    2016-01-01

    ABSTRACT Over the last 3 decades, monoclonal antibodies have become the most important class of therapeutic biologicals on the market. Development of therapeutic antibodies was accelerated by recombinant DNA technologies, which allowed the humanization of murine monoclonal antibodies to make them more similar to those of the human body and suitable for a broad range of chronic diseases like cancer and autoimmune diseases. In the early 1990s in vitro antibody selection technologies were developed that enabled the discovery of “fully” human antibodies with potentially superior clinical efficacy and lowest immunogenicity. Antibody phage display is the first and most widely used of the in vitro selection technologies. It has proven to be a robust, versatile platform technology for the discovery of human antibodies and a powerful engineering tool to improve antibody properties. As of the beginning of 2016, 6 human antibodies discovered or further developed by phage display were approved for therapy. In 2002, adalimumab (Humira®) became the first phage display-derived antibody granted a marketing approval. Humira® was also the first approved human antibody, and it is currently the best-selling antibody drug on the market. Numerous phage display-derived antibodies are currently under advanced clinical investigation, and, despite the availability of other technologies such as human antibody-producing transgenic mice, phage display has not lost its importance for the discovery and engineering of therapeutic antibodies. Here, we provide a comprehensive overview about phage display-derived antibodies that are approved for therapy or in clinical development. A selection of these antibodies is described in more detail to demonstrate different aspects of the phage display technology and its development over the last 25 years. PMID:27416017

  5. Antigen-specific human polyclonal antibodies from hyperimmunized cattle.

    PubMed

    Kuroiwa, Yoshimi; Kasinathan, Poothappillai; Sathiyaseelan, Thillainayagen; Jiao, Jin-an; Matsushita, Hiroaki; Sathiyaseelan, Janaki; Wu, Hua; Mellquist, Jenny; Hammitt, Melissa; Koster, Julie; Kamoda, Satoru; Tachibana, Katsumi; Ishida, Isao; Robl, James M

    2009-02-01

    Antigen-specific human polyclonal antibodies (hpAbs), produced by hyperimmunization, could be useful for treating many human diseases. However, yields from available transgenic mice and transchromosomic (Tc) cattle carrying human immunoglobulin loci are too low for therapeutic applications. We report a Tc bovine system that produces large yields of hpAbs. Tc cattle were generated by transferring a human artificial chromosome vector carrying the entire unrearranged, human immunoglobulin heavy (hIGH) and kappa-light (hIGK) chain loci to bovine fibroblasts in which two endogenous bovine IgH chain loci were inactivated. Plasma from the oldest animal contained >2 g/l of hIgG, paired with either human kappa-light chain (up to approximately 650 microg/ml, fully human) or with bovine kappa- or lambda-light chain (chimeric), with a normal hIgG subclass distribution. Hyperimmunization with anthrax protective antigen triggered a hIgG-mediated humoral immune response comprising a high proportion of antigen-specific hIgG. Purified, fully human and chimeric hIgGs were highly active in an in vitro toxin neutralization assay and protective in an in vivo mouse challenge assay.

  6. Antibody Binding Specificity for Kappa (Vκ) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study

    PubMed Central

    Watzlawik, Jens O.; Kahoud, Robert J.; Wootla, Bharath; Painter, Meghan M.; Warrington, Arthur E.; Carey, William A.; Rodriguez, Moses

    2016-01-01

    Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents in standard laboratory techniques. In contrast, several human IgMs demonstrated proof of efficacy in cancer models and models of CNS disorders including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Reasons for their lack of consideration include difficulties to express, purify and stabilize IgM antibodies, challenge to identify (non-protein) antigens, low affinity binding and fundamental knowledge gaps in carbohydrate and lipid research. This manuscript uses HIgM12 as an example to provide a detailed protocol to detect antigens by Western blotting, immunoprecipitations and immunocytochemistry. HIgM12 targets polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM). Early postnatal mouse brain tissue from wild type (WT) and NCAM knockout (KO) mice lacking the three major central nervous system (CNS) splice variants NCAM180, 140 and 120 was used to evaluate the importance of NCAM for binding to HIgM12. Further enzymatic digestion of CNS tissue and cultured CNS cells using endoneuraminidases led us to identify PSA as the specific binding epitope for HIgM12. PMID:27404858

  7. RA8, A human anti-CD25 antibody against human treg cells

    SciTech Connect

    Arias, Robyn; Flanagan, Meg; Miller, Keith D.; Nien, Yu-Chih; Hu, Peisheng; Gray, Dixon; Khawli, Leslie A.; Epstein, Alan L.

    2007-06-01

    Although anti-CD25 antibodies exist for clinical use in patients, there is a need for the development of a human Treg antibody that will abrogate the immunosuppressive function of this small but critical T cell subtype. Based upon mounting evidence that the level of Treg cells in the tumor microenvironment correlates with clinical prognosis and stage in man, it appears that Treg cells play an important role in the tumor's ability to overcome host immune responses. In mice, the rat anti-mouse CD25 antibody PC61 causes depletion of CD25-bearing Treg cells both peripherally in lymphatic tissues and in the tumor microenvironment, without inducing symptoms of autoimmunity. A similar antibody, though with the ability to delete Treg cells specifically, would be an important new tool for reversing tumor escape associated with Treg immunosuppression in man. To begin to generate such a reagent, we now describe the development of a human anti-CD25 antibody using a novel yeast display library. The target antigen CD25-Fc was constructed and used for five rounds of selection using a non-immune yeast display library that contained as many as 109 single chain variable fragments (scFv). Two unique clones with low KD values (RA4 and RA8) were then selected to construct fully human anti-CD25 antibodies (IgG1/kappa) for stable expression. One antibody, RA8, showed excellent binding to human CD25+ cell lines and to human Treg cells and appears to be an excellent candidate for the generation of a human reagent that may be used in man for the immunotherapy of cancer.

  8. Activation of human complement by immunoglobulin G antigranulocyte antibody.

    PubMed Central

    Rustagi, P K; Currie, M S; Logue, G L

    1982-01-01

    The ability of antigranulocyte antibody to fix the third component of complement (C3) to the granulocyte surface was investigated by an assay that quantitates the binding of monoclonal anti-C3 antibody to paraformaldehyde-fixed cells preincubated with Felty's syndrome serum in the presence of human complement. The sera from 7 of 13 patients with Felty's syndrome bound two to three times as much C3 to granulocytes as sera from patients with uncomplicated rheumatoid arthritis. The complement-activating ability of Felty's syndrome serum seemed to reside in the monomeric IgG-containing serum fraction. For those sera capable of activating complement, the amount of C3 fixed to granulocytes was proportional to the amount of granulocyte-binding IgG present in the serum. Thus, complement fixation appeared to be a consequence of the binding of antigranulocyte antibody to the cell surface. These studies suggest a role for complement-mediated injury in the pathophysiology of immune granulocytopenia, as has been demonstrated for immune hemolytic anemia and immune thrombocytopenia. PMID:7174786

  9. The function and affinity maturation of HIV-1 gp120-specific monoclonal antibodies derived from colostral B cells.

    PubMed

    Jeffries, T L; Sacha, C R; Pollara, J; Himes, J; Jaeger, F H; Dennison, S M; McGuire, E; Kunz, E; Eudailey, J A; Trama, A M; LaBranche, C; Fouda, G G; Wiehe, K; Montefiori, D C; Haynes, B F; Liao, H-X; Ferrari, G; Alam, S M; Moody, M A; Permar, S R

    2016-03-01

    Despite the risk of transmitting HIV-1, mothers in resource-poor areas are encouraged to breastfeed their infants because of beneficial immunologic and nutritional factors in milk. Interestingly, in the absence of antiretroviral prophylaxis, the overwhelming majority of HIV-1-exposed, breastfeeding infants are naturally protected from infection. To understand the role of HIV-1 envelope (Env)-specific antibodies in breast milk in natural protection against infant virus transmission, we produced 19 HIV-1 Env-specific monoclonal antibodies (mAbs) isolated from colostrum B cells of HIV-1-infected mothers and investigated their specificity, evolution, and anti-HIV-1 functions. Despite the previously reported genetic compartmentalization and gp120-specific bias of colostrum HIV Env-specific B cells, the colostrum Env-specific mAbs described here demonstrated a broad range of gp120 epitope specificities and functions, including inhibition of epithelial cell binding and dendritic cell-mediated virus transfer, neutralization, and antibody-dependent cellular cytotoxicity. We also identified divergent patterns of colostrum Env-specific B-cell lineage evolution with respect to crossreactivity to gastrointestinal commensal bacteria, indicating that commensal bacterial antigens play a role in shaping the local breast milk immunoglobulin G (IgG) repertoire. Maternal vaccine strategies to specifically target this breast milk B-cell population may be necessary to achieve safe breastfeeding for all HIV-1-exposed infants. PMID:26242599

  10. The function and affinity maturation of HIV-1 gp120-specific monoclonal antibodies derived from colostral B cells

    PubMed Central

    Jeffries, Thomas L; Sacha, CR; Pollara, Justin; Himes, Jon; Jaeger, Frederick H; Dennison, S Moses; McGuire, Erin; Kunz, Erika; Eudailey, Joshua A; Trama, Ashley M; LaBranche, Celia; Fouda, Genevieve G; Wiehe, Kevin; Montefiori, David C; Haynes, Barton F; Liao, Hua-Xin; Ferrari, Guido; Alam, S Munir; Moody, M Anthony; Permar, Sallie R

    2015-01-01

    Despite the risk of transmitting HIV-1, mothers in resource-poor areas are encouraged to breastfeed their infants due to beneficial immunologic and nutritional factors in milk. Interestingly, in the absence of antiretroviral prophylaxis, the overwhelming majority of HIV-1-exposed, breastfeeding infants are naturally protected from infection. To understand the role of HIV-1 Envelope (Env)-specific antibodies in breast milk in natural protection against infant virus transmission, we produced 19 HIV-1 Env-specific monoclonal antibodies (mAbs) isolated from colostrum B cells of HIV-1-infected mothers and investigated their specificity, evolution and anti-HIV-1 functions. Despite the previously reported genetic compartmentalization and gp120-specific bias of colostrum HIV Env-specific B cells, the colostrum Env-specific mAbs described here demonstrated a broad range of gp120 epitope specificities and functions, including inhibition of epithelial cell binding and dendritic cell mediated virus transfer, neutralization, and antibody-dependent cellular cytotoxicity. Interestingly, we also identified divergent patterns of colostrum Env-specific B cell lineage evolution with respect to cross-reactivity to gastrointestinal commensal bacteria, indicating that commensal bacterial antigens play a role in shaping the local breast milk IgG repertoire. Maternal vaccine strategies to specifically target this breast milk B cell population may be necessary to achieve safe breastfeeding for all HIV-1-exposed infants. PMID:26242599

  11. Influence of IgG Subclass on Human Antimannan Antibody-Mediated Resistance to Hematogenously Disseminated Candidiasis in Mice.

    PubMed

    Nishiya, Casey T; Boxx, Gayle M; Robison, Kerry; Itatani, Carol; Kozel, Thomas R; Zhang, Mason X

    2015-11-16

    Candida albicans is a yeast-like pathogen and can cause life-threatening systemic candidiasis. Its cell surface is enriched with mannan that is resistant to complement activation. Previously, we developed the recombinant human IgG1 antimannan antibody M1g1. M1g1 was found to promote complement activation and phagocytosis and protect mice from systemic candidiasis. Here, we evaluate the influence of IgG subclass on antimannan antibody-mediated protection. Three IgG subclass variants of M1g1 were constructed: M1g2, M1g3, and M1g4. The IgG subclass identity for each variant was confirmed with DNA sequence and subclass-specific antibodies. These variants contain identical M1 Fabs and exhibited similar binding affinities for C. albicans yeast and purified mannan. Yeast cells and hyphae recovered from the kidney of antibody-treated mice with systemic candidiasis showed uniform binding of each variant, indicating constitutive expression of the M1 epitope and antibody opsonization in the kidney. All variants promoted deposition of both murine and human C3 onto the yeast cell surface, with M1g4 showing delayed activation, as determined by flow cytometry and immunofluorescence microscopy. M1g4-mediated complement activation was found to be associated with its M1 Fab that activates the alternative pathway in an Fc-independent manner. Treatment with each subclass variant extended the survival of mice with systemic candidiasis (P < 0.001). However, treatment with M1g1, M1g3, or M1g4, but not with M1g2, also reduced the kidney fungal burden (P < 0.001). Thus, the role of human antimannan antibody in host resistance to systemic candidiasis is influenced by its IgG subclass.

  12. Affinity of neuroleptics for D1 receptor of human brain striatum.

    PubMed Central

    Kanba, S; Suzuki, E; Nomura, S; Nakaki, T; Yagi, G; Asai, M; Richelson, E

    1994-01-01

    We determined the inhibition-dissociation constant (Ki) of a number of neuroleptics for D1 receptors of normal human brain tissue using [3H]SCH23390 [R-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3[benzazepine-7- ol]. SCH23390 had the highest affinity with a Ki of 0.76 nM. Among clinically used drugs, propericiazine showed the highest affinity with a Ki of 10 nM. When neuroleptics were classified according to chemical structures, the Ki values were as follows. Phenothiazines ranged from 10 nM to 250 nM. Butyrophenones ranged from 45 nM to 250 nM. Thioxanthenes ranged from 12 nM to 340 nM. Orthopramines were more than 10,000 nM. The Ki values for the binding site of this study were significantly correlated with those reported in studies using animal brain. The possible relationship between D1 receptors and negative symptoms is discussed. PMID:7918347

  13. The human organic cation transporter OCT1 mediates high affinity uptake of the anticancer drug daunorubicin

    PubMed Central

    Andreev, Emil; Brosseau, Nicolas; Carmona, Euridice; Mes-Masson, Anne-Marie; Ramotar, Dindial

    2016-01-01

    Anthracyclines such as daunorubicin are anticancer agents that are transported into cells, and exert cytotoxicity by blocking DNA metabolism. Although there is evidence for active uptake of anthracyclines into cells, the specific transporter involved in this process has not been identified. Using the high-grade serous ovarian cancer cell line TOV2223G, we show that OCT1 mediated the high affinity (Km ~ 5 μM) uptake of daunorubicin into the cells, and that micromolar amounts of choline completely abolished the drug entry. OCT1 downregulation by shRNA impaired daunorubicin uptake into the TOV2223G cells, and these cells were significantly more resistant to the drug in comparison to the control shRNA. Transfection of HEK293T cells, which accommodated the ectopic expression of OCT1, with a plasmid expressing OCT1-EYFP showed that the transporter was predominantly localized to the plasma membrane. These transfected cells exhibited an increase in the uptake of daunorubicin in comparison to control cells transfected with an empty EYFP vector. Furthermore, a variant of OCT1, OCT1-D474C-EYFP, failed to enhance daunorubicin uptake. This is the first report demonstrating that human OCT1 is involved in the high affinity transport of anthracyclines. We postulate that OCT1 defects may contribute to the resistance of cancer cells treated with anthracyclines. PMID:26861753

  14. ANALYSIS OF DRUG-PROTEIN BINDING BY ULTRAFAST AFFINITY CHROMATOGRAPHY USING IMMOBILIZED HUMAN SERUM ALBUMIN

    PubMed Central

    Mallik, Rangan; Yoo, Michelle J.; Briscoe, Chad J.; Hage, David S.

    2010-01-01

    Human serum albumin (HSA) was explored for use as a stationary phase and ligand in affinity microcolumns for the ultrafast extraction of free drug fractions and the use of this information for the analysis of drug-protein binding. Warfarin, imipramine, and ibuprofen were used as model analytes in this study. It was found that greater than 95% extraction of all these drugs could be achieved in as little as 250 ms on HSA microcolumns. The retained drug fraction was then eluted from the same column under isocratic conditions, giving elution in less than 40 s when working at 4.5 mL/min. The chromatographic behavior of this system gave a good fit with that predicted by computer simulations based on a reversible, saturable model for the binding of an injected drug with immobilized HSA. The free fractions measured by this method were found to be comparable to those determined by ultrafiltration, and equilibrium constants estimated by this approach gave good agreement with literature values. Advantages of this method include its speed and the relatively low cost of microcolumns that contain HSA. The ability of HSA to bind many types of drugs also creates the possibility of using the same affinity microcolumn to study and measure the free fractions for a variety of pharmaceutical agents. These properties make this technique appealing for use in drug binding studies and in the high-throughput screening of new drug candidates. PMID:20227701

  15. Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins.

    PubMed

    Zhong, Nan; Loppnau, Peter; Seitova, Alma; Ravichandran, Mani; Fenner, Maria; Jain, Harshika; Bhattacharya, Anandi; Hutchinson, Ashley; Paduch, Marcin; Lu, Vincent; Olszewski, Michal; Kossiakoff, Anthony A; Dowdell, Evan; Koide, Akiko; Koide, Shohei; Huang, Haiming; Nadeem, Vincent; Sidhu, Sachdev S; Greenblatt, Jack F; Marcon, Edyta; Arrowsmith, Cheryl H; Edwards, Aled M; Gräslund, Susanne

    2015-01-01

    We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols. PMID:26437229

  16. High-resolution crystal structure of the therapeutic antibody pembrolizumab bound to the human PD-1

    PubMed Central

    Horita, Shoichiro; Nomura, Yayoi; Sato, Yumi; Shimamura, Tatsuro; Iwata, So; Nomura, Norimichi

    2016-01-01

    Pembrolizumab is an FDA-approved therapeutic antibody that targets the programmed cell death-1 (PD-1) to block the immune checkpoint pathway for the treatment of various types of cancer. It receives remarkable attention due to the high degree of efficacy. Very recently, the crystal structure of the Fab fragment of pembrolizumab (PemFab) in complex with the extracellular domain of human PD-1 (PD-1ECD) was reported at a resolution of 2.9 Å. However, this relatively low-resolution structural data fails to provide sufficient information on interfacial water molecules at the binding interface that substantially contribute to affinity and specificity between the therapeutic antibody and target. Here, we present the independently determined crystal structure of the Fv fragment of pembrolizumab (PemFv) in complex with the PD-1ECD at a resolution of 2.15 Å. This high-resolution structure allows the accurate mapping of the interaction including water-mediated hydrogen bonds and provides, for the first time, a coherent explanation of PD-1 antagonism by pembrolizumab. Our structural data also provides new insights into the rational design of improved anti-PD-1 therapeutics. PMID:27734966

  17. Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins

    PubMed Central

    Zhong, Nan; Loppnau, Peter; Seitova, Alma; Ravichandran, Mani; Fenner, Maria; Jain, Harshika; Bhattacharya, Anandi; Hutchinson, Ashley; Paduch, Marcin; Lu, Vincent; Olszewski, Michal; Kossiakoff, Anthony A.; Dowdell, Evan; Koide, Akiko; Koide, Shohei; Huang, Haiming; Nadeem, Vincent; Sidhu, Sachdev S.; Greenblatt, Jack F.; Marcon, Edyta; Arrowsmith, Cheryl H.; Edwards, Aled M.; Gräslund, Susanne

    2015-01-01

    We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols. PMID:26437229

  18. Development of human-like scFv-Fc antibodies neutralizing Botulinum toxin serotype B.

    PubMed

    Rasetti-Escargueil, Christine; Avril, Arnaud; Chahboun, Siham; Tierney, Rob; Bak, Nicola; Miethe, Sebastian; Mazuet, Christelle; Popoff, Michel R; Thullier, Philippe; Hust, Michael; Pelat, Thibaut; Sesardic, Dorothea

    2015-01-01

    Botulinum neurotoxins (BoNTs) are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNTs have been classified as category A agents by the Centers for Disease Control and Prevention. To date, 7 subtypes of BoNT/B were identified showing that subtypes B1 (16 strains) and B2 (32 strains) constitute the vast majority of BoNT/B strains. Neutralizing antibodies are required for the development of anti-botulism drugs to deal with the potential risk. In this study, macaques (Macaca fascicularis) were immunized with recombinant light chain (LC) or heavy chain (HC) of BoNT/B2, followed by the construction of 2 hyper-immune phage display libraries. The best single-chain variable fragments (scFvs) isolated from each library were selected according to their affinities and cross reactivity with BoNT/B1 toxin subtype. These scFvs against LC and HC were further analyzed by assessing the inhibition of in vitro endopeptidase activity of BoNT/B1 and B2 and neutralization of BoNT/B1 and B2 toxin-induced paralysis in the mouse ex vivo phrenic nerve assay. The antibodies B2-7 (against HC) and BLC3 (against LC) were produced as scFv-Fc, and, when tested individually, neutralized BoNT/B1 and BoNT/B2 in a mouse ex vivo phrenic nerve assay. Whereas only scFv-Fc BLC3 alone protected mice against BoNT/B2-induced paralysis in vivo, when B2-7 and BLC3 were combined they exhibited potent synergistic protection. The present study provided an opportunity to assess the extent of antibody-mediated neutralization of BoNT/B1 and BoNT/B2 subtypes in ex vivo and in vitro assays, and to confirm the benefit of the synergistic effect of antibodies targeting the 2 distinct functional domains of the toxin in vivo. Notably, the framework regions of the most promising antibodies (B2-7 and BLC3) are close to the human germline sequences, which

  19. Development of human-like scFv-Fc antibodies neutralizing Botulinum toxin serotype B

    PubMed Central

    Rasetti-Escargueil, Christine; Avril, Arnaud; Chahboun, Siham; Tierney, Rob; Bak, Nicola; Miethe, Sebastian; Mazuet, Christelle; Popoff, Michel R; Thullier, Philippe; Hust, Michael; Pelat, Thibaut; Sesardic, Dorothea

    2015-01-01

    Botulinum neurotoxins (BoNTs) are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNTs have been classified as category A agents by the Centers for Disease Control and Prevention. To date, 7 subtypes of BoNT/B were identified showing that subtypes B1 (16 strains) and B2 (32 strains) constitute the vast majority of BoNT/B strains. Neutralizing antibodies are required for the development of anti-botulism drugs to deal with the potential risk. In this study, macaques (Macaca fascicularis) were immunized with recombinant light chain (LC) or heavy chain (HC) of BoNT/B2, followed by the construction of 2 hyper-immune phage display libraries. The best single-chain variable fragments (scFvs) isolated from each library were selected according to their affinities and cross reactivity with BoNT/B1 toxin subtype. These scFvs against LC and HC were further analyzed by assessing the inhibition of in vitro endopeptidase activity of BoNT/B1 and B2 and neutralization of BoNT/B1 and B2 toxin-induced paralysis in the mouse ex vivo phrenic nerve assay. The antibodies B2–7 (against HC) and BLC3 (against LC) were produced as scFv-Fc, and, when tested individually, neutralized BoNT/B1 and BoNT/B2 in a mouse ex vivo phrenic nerve assay. Whereas only scFv-Fc BLC3 alone protected mice against BoNT/B2-induced paralysis in vivo, when B2–7 and BLC3 were combined they exhibited potent synergistic protection. The present study provided an opportunity to assess the extent of antibody-mediated neutralization of BoNT/B1 and BoNT/B2 subtypes in ex vivo and in vitro assays, and to confirm the benefit of the synergistic effect of antibodies targeting the 2 distinct functional domains of the toxin in vivo. Notably, the framework regions of the most promising antibodies (B2–7 and BLC3) are close to the human germline sequences

  20. Development of human-like scFv-Fc antibodies neutralizing Botulinum toxin serotype B.

    PubMed

    Rasetti-Escargueil, Christine; Avril, Arnaud; Chahboun, Siham; Tierney, Rob; Bak, Nicola; Miethe, Sebastian; Mazuet, Christelle; Popoff, Michel R; Thullier, Philippe; Hust, Michael; Pelat, Thibaut; Sesardic, Dorothea

    2015-01-01

    Botulinum neurotoxins (BoNTs) are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNTs have been classified as category A agents by the Centers for Disease Control and Prevention. To date, 7 subtypes of BoNT/B were identified showing that subtypes B1 (16 strains) and B2 (32 strains) constitute the vast majority of BoNT/B strains. Neutralizing antibodies are required for the development of anti-botulism drugs to deal with the potential risk. In this study, macaques (Macaca fascicularis) were immunized with recombinant light chain (LC) or heavy chain (HC) of BoNT/B2, followed by the construction of 2 hyper-immune phage display libraries. The best single-chain variable fragments (scFvs) isolated from each library were selected according to their affinities and cross reactivity with BoNT/B1 toxin subtype. These scFvs against LC and HC were further analyzed by assessing the inhibition of in vitro endopeptidase activity of BoNT/B1 and B2 and neutralization of BoNT/B1 and B2 toxin-induced paralysis in the mouse ex vivo phrenic nerve assay. The antibodies B2-7 (against HC) and BLC3 (against LC) were produced as scFv-Fc, and, when tested individually, neutralized BoNT/B1 and BoNT/B2 in a mouse ex vivo phrenic nerve assay. Whereas only scFv-Fc BLC3 alone protected mice against BoNT/B2-induced paralysis in vivo, when B2-7 and BLC3 were combined they exhibited potent synergistic protection. The present study provided an opportunity to assess the extent of antibody-mediated neutralization of BoNT/B1 and BoNT/B2 subtypes in ex vivo and in vitro assays, and to confirm the benefit of the synergistic effect of antibodies targeting the 2 distinct functional domains of the toxin in vivo. Notably, the framework regions of the most promising antibodies (B2-7 and BLC3) are close to the human germline sequences, which

  1. Peptides and Anti-peptide Antibodies for Small and Medium Scale Peptide and Anti-peptide Affinity Microarrays: Antigenic Peptide Selection, Immobilization, and Processing.

    PubMed

    Zhang, Fan; Briones, Andrea; Soloviev, Mikhail

    2016-01-01

    This chapter describes the principles of selection of antigenic peptides for the development of anti-peptide antibodies for use in microarray-based multiplex affinity assays and also with mass-spectrometry detection. The methods described here are mostly applicable to small to medium scale arrays. Although the same principles of peptide selection would be suitable for larger scale arrays (with 100+ features) the actual informatics software and printing methods may well be different. Because of the sheer number of proteins/peptides to be processed and analyzed dedicated software capable of processing all the proteins and an enterprise level array robotics may be necessary for larger scale efforts. This report aims to provide practical advice to those who develop or use arrays with up to ~100 different peptide or protein features.

  2. Monoclonal antibodies specific for human monocytes, granulocytes and endothelium.

    PubMed Central

    Hogg, N; MacDonald, S; Slusarenko, M; Beverley, P C

    1984-01-01

    Four monoclonal antibodies against antigens of human myeloid cells have been produced and thoroughly characterized in terms of their reactions with peripheral blood cells, cell lines, nine lymphoid and non-lymphoid tissues and the polypeptides with which they react. UCHM1 and SmO identify antigens present on the majority of blood monocytes and a variable, but lower, proportion of tissue macrophages. From their morphology and location in tissues, these cells appear to be recirculating monocytes. SMO antigen is also present on platelets. In addition, both antibodies stained endothelial cells, SMO in all tissues examined and UCHM1 variably. Biochemical investigation indicated that the UCHM1 antigen is a protein of 52,000 MW while the SMO antigen could not be indentified. The antibodies TG1 and 28 identify antigens mainly present on granulocytes. While mAb 28 reacted with neutrophils, TG1 also stained eosinophils and stained strongly a proportion of monocytes. TG1 also reacted variably with some non-haemopoietic cell lines. Both antibodies reacted predominantly with granulocytes in tissue sections. MAb TG1 precipitated a single polypeptide of 156,000 MW from monocytes and granulocytes, while mAb 28 precipitated non-convalently associated polypeptides of 83,000 and 155,000 MW from granulocytes but only a single molecule from monocytes, corresponding to the lower MW chain of 83,000. The epitope with which mAb 28 reacts appears not to be exposed on the surface of intact monocytes. This suggests that a similar or identical 83,000 MW molecule is made by both neutrophils and monocytes, but that its expression differs according to cell type. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:6389324

  3. Detection of the human endogenous retrovirus ERV3-encoded Env-protein in human tissues using antibody-based proteomics

    PubMed Central

    Fei, Chen; Atterby, Christina; Edqvist, Per-Henrik; Pontén, Fredrik; Zhang, Wei Wei; Larsson, Erik; Ryan, Frank P

    2014-01-01

    Objectives There is growing evidence to suggest that human endogenous retroviruses (HERVs) have contributed to human evolution, being expressed in development, normal physiology and disease. A key difficulty in the scientific evaluation of this potential viral contribution is the accurate demonstration of virally expressed protein in specific human cells and tissues. In this study, we have adopted the endogenous retrovirus, ERV3, as our test model in developing a reliable high-capacity methodology for the expression of such endogenous retrovirus-coded protein. Design Two affinity-purified polyclonal antibodies to ERV3 Env-encoded protein were generated to detect the corresponding protein expression pattern in specific human cells, tissues and organs. Participants Sampling included normal tissues from 144 individuals ranging from childhood to old age. This included more than forty different tissues and organs and some 216 different cancer tissues representing the twenty commonest forms of human cancer. Setting The Rudbeck Laboratory, Uppsala University and Uppsala University Hospital, Uppsala, Sweden. Main Outcome Measures The potential expression at likely physiological level of the ERV3Env encoded protein in a wide range of human cells, tissues and organs. Results We found that ERV3 encoded Env protein is expressed at substantive levels in placenta, testis, adrenal gland, corpus luteum, Fallopian tubes, sebaceous glands, astrocytes, bronchial epithelium and the ducts of the salivary glands. Substantive expression was also seen in a variety of epithelial cells as well as cells known to undergo fusion in inflammation and in normal physiology, including fused macrophages, myocardium and striated muscle. This contrasted strongly with the low levels expressed in other tissues types. These findings suggest that this virus plays a significant role in human physiology and may also play a possible role in disease. Conclusion This technique can now be extended to the study

  4. Substitution of Heavy Complementarity Determining Region 3 (CDR-H3) Residues Can Synergistically Enhance Functional Activity of Antibody and Its Binding Affinity to HER2 Antigen

    PubMed Central

    Moon, Seung Kee; Park, So Ra; Park, Ami; Oh, Hyun Mi; Shin, Hyun Jung; Jeon, Eun Ju; Kim, Seiwhan; Park, Hyun June; Yeon, Young Joo; Yoo, Young Je

    2016-01-01

    To generate a biobetter that has improved therapeutic activity, we constructed scFv libraries via random mutagenesis of several residues of CDR-H3 and -L3 of hu4D5. The scFv clones were isolated from the phage display libraries by stringent panning, and their anti-proliferative activity against HER2-positive cancer cells was evaluated as a primary selection criterion. Consequently, we selected AH06 as a biobetter antibody that had a 7.2-fold increase in anti-proliferative activity (IC50: 0.81 nM) against the gastric cancer cell line NCI-N87 and a 7.4-fold increase in binding affinity (KD: 60 pM) to HER2 compared to hu4D5. The binding energy calculation and molecular modeling suggest that the substitution of residues of CDR-H3 to W98, F100c, A101 and L102 could stabilize binding of the antibody to HER2 and there could be direct hydrophobic interactions between the aromatic ring of W98 and the aliphatic group of I613 within HER2 domain IV as well as the heavy and light chain hydrophobic interactions by residues F100c, A101 and L102 of CDR-H3. Therefore, we speculate that two such interactions were exerted by the residues W98 and F100c. A101 and L102 may have a synergistic effect on the increase in the binding affinity to HER2. AH06 specifically binds to domain IV of HER2, and it decreased the phosphorylation level of HER2 and AKT. Above all, it highly increased the overall level of p27 compared to hu4D5 in the gastric cancer cell line NCI-N82, suggesting that AH06 could potentially be a more efficient therapeutic agent than hu4D5. PMID:26743905

  5. Substitution of Heavy Complementarity Determining Region 3 (CDR-H3) Residues Can Synergistically Enhance Functional Activity of Antibody and Its Binding Affinity to HER2 Antigen.

    PubMed

    Moon, Seung Kee; Park, So Ra; Park, Ami; Oh, Hyun Mi; Shin, Hyun Jung; Jeon, Eun Ju; Kim, Seiwhan; Park, Hyun June; Yeon, Young Joo; Yoo, Young Je

    2016-03-01

    To generate a biobetter that has improved therapeutic activity, we constructed scFv libraries via random mutagenesis of several residues of CDR-H3 and -L3 of hu4D5. The scFv clones were isolated from the phage display libraries by stringent panning, and their anti-proliferative activity against HER2-positive cancer cells was evaluated as a primary selection criterion. Consequently, we selected AH06 as a biobetter antibody that had a 7.2-fold increase in anti-proliferative activity (IC50: 0.81 nM) against the gastric cancer cell line NCI-N87 and a 7.4-fold increase in binding affinity (KD: 60 pM) to HER2 compared to hu4D5. The binding energy calculation and molecular modeling suggest that the substitution of residues of CDR-H3 to W98, F100c, A101 and L102 could stabilize binding of the antibody to HER2 and there could be direct hydrophobic interactions between the aromatic ring of W98 and the aliphatic group of I613 within HER2 domain IV as well as the heavy and light chain hydrophobic interactions by residues F100c, A101 and L102 of CDR-H3. Therefore, we speculate that two such interactions were exerted by the residues W98 and F100c. A101 and L102 may have a synergistic effect on the increase in the binding affinity to HER2. AH06 specifically binds to domain IV of HER2, and it decreased the phosphorylation level of HER2 and AKT. Above all, it highly increased the overall level of p27 compared to hu4D5 in the gastric cancer cell line NCI-N82, suggesting that AH06 could potentially be a more efficient therapeutic agent than hu4D5.

  6. Isolation of Fully Human Antagonistic RON Antibodies Showing Efficient Block of Downstream Signaling and Cell Migration1

    PubMed Central

    Gunes, Zeynep; Zucconi, Adriana; Cioce, Mario; Meola, Annalisa; Pezzanera, Monica; Acali, Stefano; Zampaglione, Immacolata; De Pratti, Valeria; Bova, Luca; Talamo, Fabio; Demartis, Anna; Monaci, Paolo; La Monica, Nicola; Ciliberto, Gennaro; Vitelli, Alessandra

    2011-01-01

    RON belongs to the c-MET family of receptor tyrosine kinases. As its well-known family member MET, RON and its ligand macrophage-stimulating protein have been implicated in the progression and metastasis of tumors and have been shown to be overexpressed in cancer. We generated and tested a large number of human monoclonal antibodies (mAbs) against human RON. Our screening yielded three high-affinity antibodies that efficiently block ligand-dependent intracellular AKT and MAPK signaling. This effect correlates with the strong reduction of ligand-activated migration of T47D breast cancer cell line. By cross-competition experiments, we showed that the antagonistic antibodies fall into three distinct epitope regions of the RON extracellular Sema domain. Notably, no inhibition of tumor growth was observed in different epithelial tumor xenografts in nude mice with any of the antibodies. These results suggest that distinct properties beside ligand antagonism are required for anti-RON mAbs to exert antitumor effects in vivo. PMID:21286376

  7. The effect of polyamines on the binding of anti-DNA antibodies from patients with SLE and normal human subjects.

    PubMed

    Wang, Xiao; Stearns, Nancy A; Li, Xingfu; Pisetsky, David S

    2014-07-01

    Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus (SLE). To elucidate specificity further, the effect of polyamines on the binding of anti-DNA antibodies from patients with lupus was tested by ELISA to calf thymus (CT) DNA; we also assessed the binding of plasmas of patients and normal human subjects (NHS) to Micrococcus luteus (MC) DNA. As these studies showed, spermine can dose-dependently inhibit SLE anti-DNA binding to CT DNA and can promote dissociation of preformed immune complexes. With MC DNA as antigen, spermine failed to inhibit the NHS anti-DNA binding. Studies using plasmas adsorbed to a CT DNA cellulose affinity indicated that SLE plasmas are mixtures of anti-DNA that differ in inhibition by spermine and binding to conserved and non-conserved determinants. Together, these studies demonstrate that spermine can influence the binding of anti-DNA autoantibodies and may contribute to the antigenicity of DNA.

  8. The binding modes and binding affinities of epipodophyllotoxin derivatives with human topoisomerase IIα.

    PubMed

    Naik, Pradeep Kumar; Dubey, Abhishek; Soni, Komal; Kumar, Rishay; Singh, Harvinder

    2010-12-01

    Epipodophyllotoxin derivatives have important therapeutic value in the treatment of human cancers. These drugs kill cells by inhibiting the ability of topoisomerase II (TP II) to ligate nucleic acids that it cleaves during the double-stranded DNA passage reaction. The 3D structure of human TP IIα was modeled by homology modeling. A virtual library consisting of 143 epipodophyllotoxin derivatives has been developed. Their molecular interactions and binding affinities with modeled human TP IIα have been studied using the docking and Bimolecular Association with Energetics (eMBrAcE) developed by Schrödinger. Structure activity relationship models were developed between the experimental activity expressed in terms of percentage of intracellular covalent TP II-DNA complexes (log PCPDCF) of these compounds and molecular descriptors like docking score and free energy of binding. For both the cases the r2 was in the range of 0.624-0.800 indicating good data fit and r2(cv) was in the range of 0.606-774 indicating that the predictive capabilities of the models were acceptable. Low levels of root mean square error for the majority of inhibitors establish the docking and eMBrAcE based prediction model as an efficient tool for generating more potent and specific inhibitors of human TP IIα by testing rationally designed lead compounds based on epipodophyllotoxin derivatization. PMID:21075653

  9. Specificity and affinity of natural product cyclopentapeptide inhibitors against A. fumigatus, human, and bacterial chitinases.

    PubMed

    Rao, Francesco V; Houston, Douglas R; Boot, Rolf G; Aerts, Johannes M F G; Hodkinson, Michael; Adams, David J; Shiomi, Kazuro; Omura, Satoshi; van Aalten, Daan M F

    2005-01-01

    Family 18 chitinases play key roles in organisms ranging from bacteria to man. There is a need for specific, potent inhibitors to probe the function of these chitinases in different organisms. Such molecules could also provide leads for the development of chemotherapeuticals with fungicidal, insecticidal, or anti-inflammatory potential. Recently, two natural product peptides, argifin and argadin, have been characterized, which structurally mimic chitinase-chitooligosaccharide interactions and inhibit a bacterial chitinase in the nM-mM range. Here, we show that these inhibitors also act on human and Aspergillus fumigatus chitinases. The structures of these enzymes in complex with argifin and argadin, together with mutagenesis, fluorescence, and enzymology, reveal that subtle changes in the binding site dramatically affect affinity and selectivity. The data show that it may be possible to develop specific chitinase inhibitors based on the argifin/argadin scaffolds.

  10. Affinity of rosmarinic acid to human serum albumin and its effect on protein conformation stability.

    PubMed

    Peng, Xin; Wang, Xiangchao; Qi, Wei; Su, Rongxin; He, Zhimin

    2016-02-01

    Rosmarinic acid (RA) is a natural polyphenol contained in many aromatic plants with promising biological activities. The interaction between RA and human serum albumin (HSA) was investigated by multi-spectroscopic, electrochemistry, molecular docking and molecular dynamics simulation methods. The fluorescence emission of HSA was quenched by RA through a combined static and dynamic quenching mechanism, but the static quenching was the major constituent. Fluorescence experiments suggested that RA was bound to HSA with moderately strong binding affinity through hydrophobic interaction. The probable binding location of RA was located near site I of HSA. Additionally, as shown by the Fourier transform infrared (FT-IR) and circular dichroism (CD) spectra, RA can result in conformational and structural alterations of HSA. Furthermore, the molecular dynamics studies were used to investigate the stability of the HSA and HSA-RA system. Altogether, the results can provide an important insight for the applications of RA in the food industry.

  11. Affinity of rosmarinic acid to human serum albumin and its effect on protein conformation stability.

    PubMed

    Peng, Xin; Wang, Xiangchao; Qi, Wei; Su, Rongxin; He, Zhimin

    2016-02-01

    Rosmarinic acid (RA) is a natural polyphenol contained in many aromatic plants with promising biological activities. The interaction between RA and human serum albumin (HSA) was investigated by multi-spectroscopic, electrochemistry, molecular docking and molecular dynamics simulation methods. The fluorescence emission of HSA was quenched by RA through a combined static and dynamic quenching mechanism, but the static quenching was the major constituent. Fluorescence experiments suggested that RA was bound to HSA with moderately strong binding affinity through hydrophobic interaction. The probable binding location of RA was located near site I of HSA. Additionally, as shown by the Fourier transform infrared (FT-IR) and circular dichroism (CD) spectra, RA can result in conformational and structural alterations of HSA. Furthermore, the molecular dynamics studies were used to investigate the stability of the HSA and HSA-RA system. Altogether, the results can provide an important insight for the applications of RA in the food industry. PMID:26304336

  12. Intracellular and circulating neuronal antinuclear antibodies in human epilepsy.

    PubMed

    Iffland, Philip H; Carvalho-Tavares, Juliana; Trigunaite, Abhishek; Man, Shumei; Rasmussen, Peter; Alexopoulos, Andreas; Ghosh, Chaitali; Jørgensen, Trine N; Janigro, Damir

    2013-11-01

    There are overwhelming data supporting the inflammatory origin of some epilepsies (e.g., Rasmussen's encephalitis and limbic encephalitis). Inflammatory epilepsies with an autoimmune component are characterized by autoantibodies against membrane-bound, intracellular or secreted proteins (e.g., voltage gated potassium channels). Comparably, little is known regarding autoantibodies targeting nuclear antigen. We tested the hypothesis that in addition to known epilepsy-related autoantigens, the human brain tissue and serum from patients with epilepsy contain autoantibodies recognizing nuclear targets. We also determined the specific nuclear proteins acting as autoantigen in patients with epilepsy. Brain tissue samples were obtained from patients undergoing brain resections to treat refractory seizures, from the brain with arteriovenous malformations or from post-mortem multiple sclerosis brain. Patients with epilepsy had no known history of autoimmune disease and were not diagnosed with autoimmune epilepsy. Tissue was processed for immunohistochemical staining. We also obtained subcellular fractions to extract intracellular IgGs. After separating nuclear antibody-antigen complexes, the purified autoantigen was analyzed by mass spectrometry. Western blots using autoantigen or total histones were probed to detect the presence of antinuclear antibodies in the serum of patients with epilepsy. Additionally, HEp-2 assays and antinuclear antibody ELISA were used to detect the staining pattern and specific presence of antinuclear antibodies in the serum of patients with epilepsy. Brain regions from patients with epilepsy characterized by blood-brain barrier disruption (visualized by extravasated albumin) contained extravasated IgGs. Intracellular antibodies were found in epilepsy (n=13/13) but not in multiple sclerosis brain (n=4/4). In the brain from patients with epilepsy, neurons displayed higher levels of nuclear IgGs compared to glia. IgG colocalized with extravasated

  13. Affinity and folding properties both influence the selection of antibodies with the selectively infective phage (SIP) methodology.

    PubMed

    Pedrazzi, G; Schwesinger, F; Honegger, A; Krebber, C; Plückthun, A

    1997-10-01

    We investigated which molecules are selected from a model library by the selectively infective phage (SIP) methodology. As a model system, we used the fluorescein binding single-chain Fv fragment FITC-E2, and from a 3D-model, we identified 11 residues potentially involved in hapten binding and mutated them individually to alanines. The binding constant of each mutant was determined by fluorescence titration, and each mutant was tested individually as well as in competitive SIP experiments for infectivity. After three rounds of SIP, only molecules with KD values within a factor of 2 of the tightest binder remain, and among those, a mutant no longer carrying an unnecessary exposed tryptophan residue is preferentially selected. SIP is shown to select for the best overall properties of the displayed molecules, including folding behavior, stability and affinity.

  14. Production of human anti-HLA monoclonal antibodies

    SciTech Connect

    Walker, M.C.; Mercier, F.; Roger, J.; Varin, M.

    1986-03-01

    Only 40% of the several hundred anti-HLA murine monoclonal antibodies (MAbs) that have been made detect HLA-A,B,C or DR specificities previously defined by human alloantisera, the range of recognized specificities is very narrow, and few of the MAbs have proven useful as tissue typing reagents. In hopes of obtaining HLA typing reagents, the authors are developing a protocol for the production of human anti-HLA MAbs from HLA-antigen (Ag) immunized peripheral blood B cells of volunteering renal patients, immunized to one or more HLA Ags through therapeutic blood transfusions. A simple enrichment of the donor B cells has not been sufficient for anti-HLA MAb production, the authors are currently delineating the conditions necessary for increasing the number of HLA-specific donor B cells by in vitro stimulation with cells expressing the HLA Ag to which the B cell donor is immunized. For the production of MAbs, the stimulated B cells are transformed with Epstein-Barr virus and subsequently fused with KR-4 lymphoblastoid cells. Hybridomas are selected by HAT and Ouabain. Supernatants are screened for anti-HLA activity against lymphocyte targets expressing the original immunizing HLA Ag by complement mediated /sup 51/Cr release assay. Antibody specificity is determined by the complement-dependent microcytotoxicity test used for HLA typing.

  15. Separation and characterization of anti-benzylpenicilloyl (BPO) antibodies. I. Biochemical and biophysical properties of anti-BPO-IgG obtained by affinity and subsequent ion-exchange chromatography.

    PubMed

    Scheiner, O; Stemberger, H; Kraft, D; Wiedermann, G

    1978-01-01

    Anti-BPO antibodies were purified by means of affinity chromatography using AH-Sepharose 4B coated with covalently bound BPO groups. Specific elution was achieved by the hapten analogue BPO-epsilon-aminocaproic acid (BPO-EACA); desorption of the remaining antibody was performed thereafter by 0.1 M acetic acid. The resulting antibody fractions--hapten-eluted antibody (H-Ab) and acid eluted antibody (A-Ab), respectively--were further separated by ion-exchange chromatography which led to the appearance of 3 subfractions in the case of H-Ab (H1, H2, H3) and 2 subfractions in the case of A-Ab (A1 and A2). In liquid isoelectrofocusing an inhomogeneous pattern resulted. The bulk of antibodies focused between pH 6.5 and 7.0. The average avidity of H-Ab was found to be higher than that of A-Ab suggesting that avidity may influence the elution pattern in affinity chromatography. The hydrophobic influence of the "spacer" and/or interactions of antibodies directed against the hydrophobic regions of the BPO group may explain why a considerable part of the antibodies could be recovered from the immunosorbent only by acid elution.

  16. Gonadotropin-releasing hormone/human chorionic gonadotropin beta based recombinant antibodies and vaccines.

    PubMed

    Talwar, G P; Vyas, Hemant K; Purswani, Shilpi; Gupta, Jagdish C

    2009-12-01

    Gonadotropin-releasing hormone (GnRH) and human chorionic gonadotropin (hCG) are unique targets for the control of fertility. Immunological approaches to neutralizing these hormones have additional utility in cancer treatment. Vaccines have been developed against both GnRH and hCG and these have undergone Phase I/II clinical trials documenting their safety, reversibility and efficacy. The heterospecies dimer hCG vaccine prevented pregnancy in women of proven fertility without impairment of ovulation or derangement of menstrual regularity and bleeding profiles. The protective threshold of antibody titers to achieve efficacy was determined in these first-ever trials. Recently, a recombinant vaccine against the beta subunit of hCG linked to the B subunit of heat labile enterotoxin has been made and expressed as a glycosylated conjugate in Pichia pastoris. Experiments indicate its ability to generate antibodies above the protective threshold in all immunized Balb/c mice. Ectopic expression of hCG/hCGbeta is observed in many advanced stage cancers of various origins. A chimeric high affinity and specific recombinant antibody against hCGbeta linked to curcumin kills hCGbeta expressing T lymphoblastic leukemia cells without any deleterious effect. Several synthetic and recombinant vaccines have been developed against GnRH. These reduce serum testosterone to castration levels causing atrophy of the prostate. Three Phase I/II clinical trials conducted in India and Austria have shown that these vaccines elicit non-surgical reduction of testosterone, a fall in prostate specific antigen and clinical improvement of prostate carcinoma patients. A multimer recombinant vaccine against GnRH has high efficacy for sterilization of pigs and other animals. PMID:19854518

  17. Lineage tracing of human B cells reveals the in vivo landscape of human antibody class switching

    PubMed Central

    Horns, Felix; Vollmers, Christopher; Croote, Derek; Mackey, Sally F; Swan, Gary E; Dekker, Cornelia L; Davis, Mark M; Quake, Stephen R

    2016-01-01

    Antibody class switching is a feature of the adaptive immune system which enables diversification of the effector properties of antibodies. Even though class switching is essential for mounting a protective response to pathogens, the in vivo patterns and lineage characteristics of antibody class switching have remained uncharacterized in living humans. Here we comprehensively measured the landscape of antibody class switching in human adult twins using antibody repertoire sequencing. The map identifies how antibodies of every class are created and delineates a two-tiered hierarchy of class switch pathways. Using somatic hypermutations as a molecular clock, we discovered that closely related B cells often switch to the same class, but lose coherence as somatic mutations accumulate. Such correlations between closely related cells exist when purified B cells class switch in vitro, suggesting that class switch recombination is directed toward specific isotypes by a cell-autonomous imprinted state. DOI: http://dx.doi.org/10.7554/eLife.16578.001 PMID:27481325

  18. TTAC-0001, a human monoclonal antibody targeting VEGFR-2/KDR, blocks tumor angiogenesis

    PubMed Central

    Lee, Weon Sup; Pyun, Bo-Jeong; Kim, Sung-Woo; Shim, Sang Ryeol; Nam, Ju Ryoung; Yoo, Ji Young; Jin, Younggeon; Jin, Juyoun; Kwon, Young-Guen; Yun, Chae-Ok; Nam, Do-Hyun; Oh, Keunhee; Lee, Dong-Sup; Lee, Sang Hoon; Yoo, Jin-San

    2015-01-01

    Angiogenesis is one of the most important processes for cancer cell survival, tumor growth and metastasis. Vascular endothelial growth factor (VEGF) and its receptor, particularly VEGF receptor-2 (VEGFR-2, or kinase insert domain-containing receptor, KDR), play critical roles in tumor-associated angiogenesis. We developed TTAC-0001, a human monoclonal antibody against VEGFR-2/KDR from a fully human naïve single-chain variable fragment phage library. TTAC-0001 was selected as a lead candidate based on its affinity, ligand binding inhibition and inhibition of VEGFR-2 signal in human umbilical vein endothelial cells (HUVEC). TTAC-0001 inhibited binding of VEGF-C and VEGF-D to VEGFR-2 in addition to VEGF-A. It binds on the N-terminal regions of domain 2 and domain 3 of VEGFR-2. It could inhibit the phosphorylation of VEGFR-2/KDR and ERK induced by VEGF in HUVEC. TTAC-0001 also inhibited VEGF-mediated endothelial cell proliferation, migration and tube formation in vitro, as well as ex vivo vessel sprouting from rat aortic rings and neovascularization in mouse matrigel model in vivo. Our data indicates that TTAC-0001 blocks the binding of VEGFs to VEGFR-2/KDR and inhibits VEGFR-induced signaling pathways and angiogenesis. Therefore, these data strongly support the further development of TTAC-0001 as an anti-cancer agent in the clinic. PMID:25942475

  19. Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits

    PubMed Central

    Robinson, James E.; Hastie, Kathryn M.; Cross, Robert W.; Yenni, Rachael E.; Elliott, Deborah H.; Rouelle, Julie A.; Kannadka, Chandrika B.; Smira, Ashley A.; Garry, Courtney E.; Bradley, Benjamin T.; Yu, Haini; Shaffer, Jeffrey G.; Boisen, Matt L.; Hartnett, Jessica N.; Zandonatti, Michelle A.; Rowland, Megan M.; Heinrich, Megan L.; Martínez-Sobrido, Luis; Cheng, Benson; de la Torre, Juan C.; Andersen, Kristian G.; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Gbakie, Michael; Kanneh, Lansana; Koroma, Veronica J.; Fonnie, Richard; Jalloh, Simbirie C.; Kargbo, Brima; Vandi, Mohamed A.; Gbetuwa, Momoh; Ikponmwosa, Odia; Asogun, Danny A.; Okokhere, Peter O.; Follarin, Onikepe A.; Schieffelin, John S.; Pitts, Kelly R.; Geisbert, Joan B.; Kulakoski, Peter C.; Wilson, Russell B.; Happi, Christian T.; Sabeti, Pardis C.; Gevao, Sahr M.; Khan, S. Humarr; Grant, Donald S.; Geisbert, Thomas W.; Saphire, Erica Ollmann; Branco, Luis M.; Garry, Robert F.

    2016-01-01

    Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design. PMID:27161536

  20. Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits.

    PubMed

    Robinson, James E; Hastie, Kathryn M; Cross, Robert W; Yenni, Rachael E; Elliott, Deborah H; Rouelle, Julie A; Kannadka, Chandrika B; Smira, Ashley A; Garry, Courtney E; Bradley, Benjamin T; Yu, Haini; Shaffer, Jeffrey G; Boisen, Matt L; Hartnett, Jessica N; Zandonatti, Michelle A; Rowland, Megan M; Heinrich, Megan L; Martínez-Sobrido, Luis; Cheng, Benson; de la Torre, Juan C; Andersen, Kristian G; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Gbakie, Michael; Kanneh, Lansana; Koroma, Veronica J; Fonnie, Richard; Jalloh, Simbirie C; Kargbo, Brima; Vandi, Mohamed A; Gbetuwa, Momoh; Ikponmwosa, Odia; Asogun, Danny A; Okokhere, Peter O; Follarin, Onikepe A; Schieffelin, John S; Pitts, Kelly R; Geisbert, Joan B; Kulakoski, Peter C; Wilson, Russell B; Happi, Christian T; Sabeti, Pardis C; Gevao, Sahr M; Khan, S Humarr; Grant, Donald S; Geisbert, Thomas W; Saphire, Erica Ollmann; Branco, Luis M; Garry, Robert F

    2016-01-01

    Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design. PMID:27161536

  1. Preclinical development of AMG 139, a human antibody specifically targeting IL-23

    PubMed Central

    Köck, K; Pan, W J; Gow, J M; Horner, M J; Gibbs, J P; Colbert, A; Goletz, T J; Newhall, K J; Rees, W A; Sun, Y; Zhang, Y; O'Neill, J C; Umble-Romero, A N; Prokop, S P; Krill, C D; Som, L; Buntich, S A; Trimble, M W; Tsuji, W H; Towne, J E

    2015-01-01

    BACKGROUND AND PURPOSE AMG 139 is a human anti-IL-23 antibody currently in a phase II trial for treating Crohn's disease. To support its clinical development in humans, in vitro assays and in vivo studies were conducted in cynomolgus monkeys to determine the pharmacology, preclinical characteristics and safety of this monoclonal antibody. EXPERIMENTAL APPROACH The in vitro pharmacology, pharmacokinetics (PK), pharmacodynamics and toxicology of AMG 139, after single or weekly i.v. or s.c. administration for up to 26 weeks, were evaluated in cynomolgus monkeys. KEY RESULTS AMG 139 bound with high affinity to both human and cynomolgus monkey IL-23 and specifically neutralized the biological activity of IL-23 without binding or blocking IL-12. After a single dose, linear PK with s.c. bioavailability of 81% and mean half-life of 8.4–13 days were observed. After weekly s.c. dosing for 3 or 6 months, AMG 139 exposure increased approximately dose-proportionally from 30 to 300 mg·kg−1 and mean accumulation between the first and last dose ranged from 2- to 3.5-fold. Peripheral blood immunophenotyping, T-cell-dependent antigen responses and bone formation markers were not different between AMG 139 and vehicle treatment. No adverse clinical signs, effects on body weight, vital signs, ophthalmic parameters, clinical pathology, ECG, organ weights or histopathology were observed in the monkeys with the highest dose of AMG 139 tested (300 mg·kg−1 s.c. or i.v.). CONCLUSIONS AND IMPLICATIONS The in vitro pharmacology, PK, immunogenicity and safety characteristics of AMG 139 in cynomolgus monkeys support its continued clinical development for the treatment of various inflammatory diseases. PMID:25205227

  2. Human Monoclonal Antibodies Against a Plethora of Viral Pathogens From Single Combinatorial Libraries

    NASA Astrophysics Data System (ADS)

    Williamson, R. Anthony; Burioni, Roberto; Sanna, Pietro P.; Partridge, Lynda J.; Barbas, Carlos F., III; Burton, Dennis R.

    1993-05-01

    Conventional antibody generation usually requires active immunization with antigen immediately prior to the preparation procedure. Combinatorial antibody library technology offers the possibility of cloning a range of antibody specificities at a single point in time and then accessing these specificities at will. Here we show that human monoclonal antibody Fab fragments against a plethora of infectious agents can be readily derived from a single library. Further examination of a number of libraries shows that whenever antibody against a pathogen can be detected in the serum of the donor, then specific antibodies can be derived from the corresponding library. We describe the generation of human Fab fragments against herpes simplex virus types 1 and 2, human cytomegalovirus, varicella zoster virus, rubella, human immunodeficiency virus type 1, and respiratory syncytial virus. The antibodies are shown to be highly specific and a number are effective in neutralizing virus in vitro.

  3. Characterization of membrane antigens on human cytomegalovirus-infected fibroblasts recognized by human antibodies

    SciTech Connect

    van der Voort, L.H.M.; de Leij, L.F.M.H.; The T.H.

    1989-03-01

    The antigens on the surface of human cytomegalovirus (HCMV)-infected fibroblasts which are recognized by human HCMV antibody-positive sera were characterized. Three HCMV-induced polypeptides, with apparent molecular masses of 53 to 63, 94, and 94 to 120 kilodaltons, were precipitated from /sup 125/I-surface-labeled cell extracts with different sera obtained from healthy individuals. Renal transplant recipients who were suffering from active HCMV infections recognized the same set of antigens. By the use of monoclonal antibodies, these antigens were identified as polypeptides belonging to the gcI and gcIII families of HCMV glycoproteins.

  4. 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.

  5. Method and cell lines for the production of monoclonal antibodies to human glycophorin A

    DOEpatents

    Bigbee, W.L.; Fong, S.S.N.; Jensen, R.H.; Vanderlaan, M.

    Cloned mouse hybridoma cell lines have been established which continuously produce antibodies that differentiate between the M and N forms of human glycophorin A. These antibodies have potential application as human blood group reagents, as markers for terminally differentiated erythroid cells and as immunofluorescent labels of somatically variant human erythrocytes.

  6. Cell lines for the production of monoclonal antibodies to human glycophorin A

    DOEpatents

    Bigbee, William L.; Fong, Stella S. N.; Jensen, Ronald H.; Vanderlaan, Martin; Langlois, Richard G.

    1988-01-01

    Cloned mouse hybridoma cell lines have been established which continuously produce antibodies that differentiate between the M and N forms of human glycophorin A. These antibodies have potential application as human blood group reagents, as markers for terminally differentiated erythroid cells and as immunofluorescent labels of somatically variant human erythrocytes.

  7. Generation, characterization and preclinical studies of a human anti-L1CAM monoclonal antibody that cross-reacts with rodent L1CAM.

    PubMed

    Cho, Seulki; Park, Insoo; Kim, Haejung; Jeong, Mun Sik; Lim, Mooney; Lee, Eung Suk; Kim, Jin Hong; Kim, Semi; Hong, Hyo Jeong

    2016-01-01

    L1 cell adhesion molecule (L1CAM) is aberrantly expressed in malignant tumors and plays important roles in tumor progression. Thus, L1CAM could serve as a therapeutic target and anti-L1CAM antibodies may have potential as anticancer agents. However, L1CAM is expressed in neural cells and the druggability of anti-L1AM antibody must be validated at the earliest stages of preclinical study. Here, we generated a human monoclonal antibody that is cross-reactive with mouse L1CAM and evaluated its pharmacokinetic properties and anti-tumor efficacy in rodent models. First, we selected an antibody (Ab4) that binds human and mouse L1CAM from the human naïve Fab library using phage display, then increased its affinity 45-fold through mutation of 3 residues in the complementarity-determining regions (CDRs) to generate Ab4M. Next, the affinity of Ab4M was increased 1.8-fold by yeast display of single-chain variable fragment containing randomly mutated light chain CDR3 to generate Ab417. The affinities (KD) of Ab417 for human and mouse L1CAM were 0.24 nM and 79.16 pM, respectively. Ab417 specifically bound the Ig5 domain of L1CAM and did not exhibit off-target activity, but bound to the peripheral nerves embedded in normal human tissues as expected in immunohistochemical analysis. In a pharmacokinetics study, the mean half-life of Ab417 was 114.49 h when a single dose (10 mg/kg) was intravenously injected into SD rats. Ab417 significantly inhibited tumor growth in a human cholangiocarcinoma xenograft nude mouse model and did not induce any adverse effect in in vivo studies. Thus, Ab417 may have potential as an anticancer agent. PMID:26785809

  8. The role of human cytochrome P450 enzymes in the formation of 2-hydroxymetronidazole: CYP2A6 is the high affinity (low Km) catalyst.

    PubMed

    Pearce, Robin E; Cohen-Wolkowiez, Michael; Sampson, Mario R; Kearns, Gregory L

    2013-09-01

    Despite metronidazole's widespread clinical use since the 1960s, the specific enzymes involved in its biotransformation have not been previously identified. Hence, in vitro studies were conducted to identify and characterize the cytochrome P450 enzymes involved in the formation of the major metabolite, 2-hydroxymetronidazole. Formation of 2-hydroxymetronidazole in human liver microsomes was consistent with biphasic, Michaelis-Menten kinetics. Although several cDNA-expressed P450 enzymes catalyzed 2-hydroxymetronidazole formation at a supratherapeutic concentration of metronidazole (2000 μM), at a "therapeutic concentration" of 100 μM only CYPs 2A6, 3A4, 3A5, and 3A7 catalyzed metronidazole 2-hydroxylation at rates substantially greater than control vector, and CYP2A6 catalyzed 2-hydroxymetronidazole formation at rates 6-fold higher than the next most active enzyme. Kinetic studies with these recombinant enzymes revealed that CYP2A6 has a Km = 289 μM which is comparable to the Km for the high-affinity (low-Km) enzyme in human liver microsomes, whereas the Km values for the CYP3A enzymes corresponded with the low-affinity (high-Km) component. The sample-to-sample variation in 2-hydroxymetronidazole formation correlated significantly with CYP2A6 activity (r ≥ 0.970, P < 0.001) at substrate concentrations of 100 and 300 μM. Selective chemical inhibitors of CYP2A6 inhibited metronidazole 2-hydroxylation in a concentration-dependent manner and inhibitory antibodies against CYP2A6 virtually eliminated metronidazole 2-hydroxylation (>99%). Chemical and antibody inhibitors of other P450 enzymes had little or no effect on metronidazole 2-hydroxylation. These results suggest that CYP2A6 is the primary catalyst responsible for the 2-hydroxylation of metronidazole, a reaction that may function as a marker of CYP2A6 activity both in vitro and in vivo.

  9. Anti-synthetic peptide antibody reacting at the fusion junction of deletion-mutant epidermal growth factor receptors in human glioblastoma

    SciTech Connect

    Humphrey, P.A.; Zalutsky, M.R.; Fuller, G.N.; Archer, G.E.; Friedman, H.S.; Kwatra, M.M.; Bigner, S.H.; Bigner, D.D. ); Wong, A.J. ); Vogelstein, B. )

    1990-06-01

    The authors have investigated human gliomas that amplify and rearrange the epidermal growth factor receptor gene, with generation of an in-frame deletion mutation of 802 nucleotides in the external domain. This in-frame deletion mutation generates a local amino acid sequence at the fusion junction of what normally were distant polypeptide sequences in the intact epidermal growth factor receptor. This 14-amino acid peptide was chemically synthesized, coupled to keyhole limpet hemocyanin, and used as an immunogen in rabbits. The elicited antibody reacted specifically with the fusion peptide in ELISA. The anti-fusion junction peptide antibody was purified by passage of the antiserum over a peptide affinity column with acidic elution. The purified antibody selectively bound the glioma deletion mutant as compared to the intact epidermal growth factor receptor as assessed by immunocytochemistry, immunofluorescence, immunoprecipitation with gel electrophoresis, and binding experiments using radioiodinated antibody. These data indicate that it is feasible to generate site-specific anti-peptide antibodies that are highly selective for mutant proteins in human tumors. The anti-peptide antibody described here, and other mutation site-specific antibodies, should be ideal candidates for tumor immunoimaging and immunotherapy.

  10. A human antibody recognizing a conserved epitope of H5 hemagglutinin broadly neutralizes highly pathogenic avian influenza H5N1 viruses.

    PubMed

    Hu, Hongxing; Voss, Jarrod; Zhang, Guoliang; Buchy, Philippi; Zuo, Teng; Wang, Lulan; Wang, Feng; Zhou, Fan; Wang, Guiqing; Tsai, Cheguo; Calder, Lesley; Gamblin, Steve J; Zhang, Linqi; Deubel, Vincent; Zhou, Boping; Skehel, John J; Zhou, Paul

    2012-03-01

    Influenza A virus infection is a persistent threat to public health worldwide due to its ability to evade immune surveillance through rapid genetic drift and shift. Current vaccines against influenza A virus provide immunity to viral isolates that are similar to vaccine strains. High-affinity neutralizing antibodies against conserved epitopes could provide immunity to diverse influenza virus strains and protection against future pandemic viruses. In this study, by using a highly sensitive H5N1 pseudotype-based neutralization assay to screen human monoclonal antibodies produced by memory B cells from an H5N1-infected individual and molecular cloning techniques, we developed three fully human monoclonal antibodies. Among them, antibody 65C6 exhibited potent neutralization activity against all H5 clades and subclades except for subclade 7.2 and prophylactic and therapeutic efficacy against highly pathogenic avian influenza H5N1 viruses in mice. Studies on hemagglutinin (HA)-antibody complexes by electron microscopy and epitope mapping indicate that antibody 65C6 binds to a conformational epitope comprising amino acid residues at positions 118, 121, 161, 164, and 167 (according to mature H5 numbering) on the tip of the membrane-distal globular domain of HA. Thus, we conclude that antibody 65C6 recognizes a neutralization epitope in the globular head of HA that is conserved among almost all divergent H5N1 influenza stains. PMID:22238297

  11. 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

  12. Interactions between natural killer cells and antibody Fc result in enhanced antibody neutralization of human immunodeficiency virus type 1.

    PubMed

    Forthal, Donald N; Landucci, Gary; Phan, Tran B; Becerra, Juan

    2005-02-01

    Antibodies can prevent lentivirus infections in animals and may play a role in controlling viral burden in established infection. In preventing and particularly in controlling infection, antibodies likely function in the presence of large quantities of virus. In this study, we explored the mechanisms by which antibodies neutralize large inocula of human immunodeficiency virus type 1 (HIV-1) on different target cells. Immunoglobulin G (IgG) from HIV-infected patients was tested for neutralizing activity against primary R5 strains of HIV-1 at inocula ranging from 100 to 20,000 50% tissue culture infective doses. At all virus inocula, inhibition by antibody was enhanced when target cells for virus growth were monocyte-depleted, peripheral blood mononuclear cells (PBMCs) rather than CD4(+) lymphocytes. However, enhanced inhibition on PBMCs was greatest with larger amounts of virus. Depleting PBMCs of natural killer (NK) cells, which express Fc receptors for IgG (FcgammaRs), abrogated the enhanced antibody inhibition, whereas adding NK cells to CD4(+) lymphocytes restored inhibition. There was no enhanced inhibition on PBMCs when F(ab')(2) was used. Further experiments demonstrated that the release of beta-chemokines, most likely through FcgammaR triggering of NK cells, contributed modestly to the antiviral activity of antibody on PBMCs and that antibody-coated virus adsorbed to uninfected cells provided a target for NK cell-mediated inhibition of HIV-1. These results indicate that Fc-FcgammaR interactions enhance the ability of antibody to neutralize HIV-1. Since FcgammaR-bearing cells are always present in vivo, FcgammaR-mediated antibody function may play a role in the ability of antibody to control lentivirus infection.

  13. Interactions between Natural Killer Cells and Antibody Fc Result in Enhanced Antibody Neutralization of Human Immunodeficiency Virus Type 1

    PubMed Central

    Forthal, Donald N.; Landucci, Gary; Phan, Tran B.; Becerra, Juan

    2005-01-01

    Antibodies can prevent lentivirus infections in animals and may play a role in controlling viral burden in established infection. In preventing and particularly in controlling infection, antibodies likely function in the presence of large quantities of virus. In this study, we explored the mechanisms by which antibodies neutralize large inocula of human immunodeficiency virus type 1 (HIV-1) on different target cells. Immunoglobulin G (IgG) from HIV-infected patients was tested for neutralizing activity against primary R5 strains of HIV-1 at inocula ranging from 100 to 20,000 50% tissue culture infective doses. At all virus inocula, inhibition by antibody was enhanced when target cells for virus growth were monocyte-depleted, peripheral blood mononuclear cells (PBMCs) rather than CD4+ lymphocytes. However, enhanced inhibition on PBMCs was greatest with larger amounts of virus. Depleting PBMCs of natural killer (NK) cells, which express Fc receptors for IgG (FcγRs), abrogated the enhanced antibody inhibition, whereas adding NK cells to CD4+ lymphocytes restored inhibition. There was no enhanced inhibition on PBMCs when F(ab′)2 was used. Further experiments demonstrated that the release of β-chemokines, most likely through FcγR triggering of NK cells, contributed modestly to the antiviral activity of antibody on PBMCs and that antibody-coated virus adsorbed to uninfected cells provided a target for NK cell-mediated inhibition of HIV-1. These results indicate that Fc-FcγR interactions enhance the ability of antibody to neutralize HIV-1. Since FcγR-bearing cells are always present in vivo, FcγR-mediated antibody function may play a role in the ability of antibody to control lentivirus infection. PMID:15681406

  14. A catecholamine transporter from the human parasite Schistosoma mansoni with low affinity for psychostimulants.

    PubMed

    Larsen, Mads B; Fontana, Andréia C K; Magalhães, Lizandra G; Rodrigues, Vanderlei; Mortensen, Ole V

    2011-05-01

    The trematode Schistosoma mansoni is the primary cause of schistosomiasis, a devastating neglected tropical disease that affects 200 million individuals. Identifying novel therapeutic targets for the treatment of schistosomiasis is therefore of great public interest. The catecholamines norepinephrine (NE) and dopamine (DA) are essential for the survival of the parasite as they cause muscular relaxation and a lengthening in the parasite and thereby control movement. Here we characterize a novel dopamine/norepinephrine transporter (SmDAT) gene transcript, from S. mansoni. The SmDAT is expressed in the adult form and in the sporocyst form (infected snails) of the parasite, and also in the egg and miracidium stage. It is absent in the cercariae stage but curiously a transcript missing the exon encoding transmembrane domain 8 was identified in this stage. Heterologous expression of the cDNA in mammalian cells resulted in saturable, dopamine transport activity with an apparent affinity for dopamine comparable to that of the human dopamine transporter. Efflux experiments reveal notably higher substrate selectivity compared with its mammalian counterparts as amphetamine is a much less potent efflux elicitor against SmDAT compared to the human DAT. Pharmacological characterization of the SmDAT revealed that most human DAT inhibitors including psychostimulants such as cocaine were significantly less potent in inhibiting SmDAT. Like DATs from other simpler organisms the pharmacology for SmDAT was more similar to the human norepinephrine transporter. We were not able to identify other dopamine transporting carriers within the completed parasite genome and we hypothesize that the SmDAT is the only catecholamine transporter in the parasite and could be responsible for not only clearing DA but also NE. PMID:21251927

  15. A catecholamine transporter from the human parasite Schistosoma mansoni with low affinity for psychostimulants

    PubMed Central

    Larsen, Mads B.; Fontana, Andréia C. K.; Magalhães, Lizandra G.; Rodrigues, Vanderlei; Mortensen, Ole V.

    2011-01-01

    The trematode Schistosoma mansoni is the primary cause of schistosomiasis, a devastating neglected tropical disease that affects 200 million individuals. Identifying novel therapeutic targets for the treatment of schistosomiasis is therefore of great public interest. The catecholamines norepinephrine (NE) and dopamine (DA) are essential for the survival of the parasite as they cause muscular relaxation and a lengthening in the parasite and thereby control movement. Here we characterize a novel dopamine/norepinephrine transporter (SmDAT) gene transcript, from Schistosoma mansoni. The SmDAT is expressed in the adult form and in the sporocyst form (infected snails) of the parasite, and also in the egg and miracidium stage. It is absent in the cercaria stage but curiously a transcript missing the exon encoding transmembrane domain 8 was identified in this stage. Heterologous expression of the cDNA in mammalian cells resulted in saturable, dopamine transport activity with an apparent affinity for dopamine comparable to that of the human dopamine transporter. Efflux experiments reveal notably higher substrate selectivity compared with its mammalian counterparts as amphetamine is a much less potent efflux elicitor against SmDAT compared to the human DAT. Pharmacological characterization of the SmDAT revealed that most human DAT inhibitors including psychostimulants such as cocaine were significantly less potent in inhibiting SmDAT. Like DATs from other simpler organisms the pharmacology for SmDAT was more similar to the human norepinephrine transporter. We were not able to identify other dopamine transporting carriers within the completed parasite genome and we hypothesize that the SmDAT is the only catecholamine transporter in the parasite and could be responsible for not only clearing DA but also NE. PMID:21251927

  16. Neanderthal and Denisova genetic affinities with contemporary humans: introgression versus common ancestral polymorphisms.

    PubMed

    Lowery, Robert K; Uribe, Gabriel; Jimenez, Eric B; Weiss, Mark A; Herrera, Kristian J; Regueiro, Maria; Herrera, Rene J

    2013-11-01

    Analyses of the genetic relationships among modern humans, Neanderthals and Denisovans have suggested that 1-4% of the non-Sub-Saharan African gene pool may be Neanderthal derived, while 6-8% of the Melanesian gene pool may be the product of admixture between the Denisovans and the direct ancestors of Melanesians. In the present study, we analyzed single nucleotide polymorphism (SNP) diversity among a worldwide collection of contemporary human populations with respect to the genetic constitution of these two archaic hominins and Pan troglodytes (chimpanzee). We partitioned SNPs into subsets, including those that are derived in both archaic lineages, those that are ancestral in both archaic lineages and those that are only derived in one archaic lineage. By doing this, we have conducted separate examinations of subsets of mutations with higher probabilities of divergent phylogenetic origins. While previous investigations have excluded SNPs from common ancestors in principal component analyses, we included common ancestral SNPs in our analyses to visualize the relative placement of the Neanderthal and Denisova among human populations. To assess the genetic similarities among the various hominin lineages, we performed genetic structure analyses to provide a comparison of genetic patterns found within contemporary human genomes that may have archaic or common ancestral roots. Our results indicate that 3.6% of the Neanderthal genome is shared with roughly 65.4% of the average European gene pool, which clinally diminishes with distance from Europe. Our results suggest that Neanderthal genetic associations with contemporary non-Sub-Saharan African populations, as well as the genetic affinities observed between Denisovans and Melanesians most likely result from the retention of ancient mutations in these populations. PMID:23872234

  17. Neutralization of Botulinum Neurotoxin Type E by a Humanized Antibody.

    PubMed

    Derman, Yağmur; Selby, Katja; Miethe, Sebastian; Frenzel, André; Liu, Yvonne; Rasetti-Escargueil, Christine; Avril, Arnaud; Pelat, Thibaut; Urbain, Remi; Fontayne, Alexandre; Thullier, Philippe; Sesardic, Dorothea; Lindström, Miia; Hust, Michael; Korkeala, Hannu

    2016-01-01

    Botulinum neurotoxins (BoNTs) cause botulism and are the deadliest naturally-occurring substances known to humans. BoNTs have been classified as one of the category A agents by the Centers for Disease Control and Prevention, indicating their potential use as bioweapons. To counter bio-threat and naturally-occurring botulism cases, well-tolerated antibodies by humans that neutralize BoNTs are relevant. In our previous work, we showed the neutralizing potential of macaque (Macaca fascicularis)-derived scFv-Fc (scFv-Fc ELC18) by in vitro endopeptidase immunoassay and ex vivo mouse phrenic nerve-hemidiaphragm assay by targeting the light chain of the botulinum neurotoxin type E (BoNT/E). In the present study, we germline-humanized scFv-Fc ELC18 into a full IgG hu8ELC18 to increase its immunotolerance by humans. We demonstrated the protection and prophylaxis capacity of hu8ELC18 against BoNT/E in a mouse model. A concentration of 2.5 ng/mouse of hu8ELC18 protected against 5 mouse lethal dose (MLD) in a mouse protection assay and complete neutralization of 1 LD50 of pure BoNT/E toxin was achieved with 8 ng of hu8ELC18 in mouse paralysis assay. Furthermore, hu8ELC18 protected mice from 5 MLD if injected up to 14 days prior to intraperitoneal BoNT/E administration. This newly-developed humanized IgG is expected to have high tolerance in humans. PMID:27626446

  18. Neutralization of Botulinum Neurotoxin Type E by a Humanized Antibody

    PubMed Central

    Derman, Yağmur; Selby, Katja; Miethe, Sebastian; Frenzel, André; Liu, Yvonne; Rasetti-Escargueil, Christine; Avril, Arnaud; Pelat, Thibaut; Urbain, Remi; Fontayne, Alexandre; Thullier, Philippe; Sesardic, Dorothea; Lindström, Miia; Hust, Michael; Korkeala, Hannu

    2016-01-01

    Botulinum neurotoxins (BoNTs) cause botulism and are the deadliest naturally-occurring substances known to humans. BoNTs have been classified as one of the category A agents by the Centers for Disease Control and Prevention, indicating their potential use as bioweapons. To counter bio-threat and naturally-occurring botulism cases, well-tolerated antibodies by humans that neutralize BoNTs are relevant. In our previous work, we showed the neutralizing potential of macaque (Macaca fascicularis)-derived scFv-Fc (scFv-Fc ELC18) by in vitro endopeptidase immunoassay and ex vivo mouse phrenic nerve-hemidiaphragm assay by targeting the light chain of the botulinum neurotoxin type E (BoNT/E). In the present study, we germline-humanized scFv-Fc ELC18 into a full IgG hu8ELC18 to increase its immunotolerance by humans. We demonstrated the protection and prophylaxis capacity of hu8ELC18 against BoNT/E in a mouse model. A concentration of 2.5 ng/mouse of hu8ELC18 protected against 5 mouse lethal dose (MLD) in a mouse protection assay and complete neutralization of 1 LD50 of pure BoNT/E toxin was achieved with 8 ng of hu8ELC18 in mouse paralysis assay. Furthermore, hu8ELC18 protected mice from 5 MLD if injected up to 14 days prior to intraperitoneal BoNT/E administration. This newly-developed humanized IgG is expected to have high tolerance in humans. PMID:27626446

  19. Human immunodeficiency virus antibody test and seroprevalence in psychiatric patients.

    PubMed

    Naber, D; Pajonk, F G; Perro, C; Löhmer, B

    1994-05-01

    Psychiatric inpatients are at risk for human immunodeficiency virus (HIV) infection. Investigations in the United States revealed seroprevalence rates of 5.5-8.9%. Therefore, inclusion of HIV antibody testing in routine laboratory screening is sometimes suggested. To investigate this issue for inpatients in the Department of Psychiatry, University of Munich, the incidence, reason for HIV testing and results were analyzed. Of 12,603 patients, hospitalized from 1985 to 1993, 4.9% (623 patients, 265 in risk groups) underwent the HIV test after informed consent. Thirty patients (4.8% of those tested) were found to be positive, but only in 5 cases (all of risk groups) was infection newly detected. Data indicate that, in psychiatry, HIV testing is reasonable only in patients in risk groups or if clinical variables suggest HIV infection.

  20. Human immunodeficiency virus antibody test and seroprevalence in psychiatric patients.

    PubMed

    Naber, D; Pajonk, F G; Perro, C; Löhmer, B

    1994-05-01

    Psychiatric inpatients are at risk for human immunodeficiency virus (HIV) infection. Investigations in the United States revealed seroprevalence rates of 5.5-8.9%. Therefore, inclusion of HIV antibody testing in routine laboratory screening is sometimes suggested. To investigate this issue for inpatients in the Department of Psychiatry, University of Munich, the incidence, reason for HIV testing and results were analyzed. Of 12,603 patients, hospitalized from 1985 to 1993, 4.9% (623 patients, 265 in risk groups) underwent the HIV test after informed consent. Thirty patients (4.8% of those tested) were found to be positive, but only in 5 cases (all of risk groups) was infection newly detected. Data indicate that, in psychiatry, HIV testing is reasonable only in patients in risk groups or if clinical variables suggest HIV infection. PMID:8067276

  1. Structure of a human IgA1 Fab fragment at 1.55 Å resolution: potential effect of the constant domains on antigen-affinity modulation.

    PubMed

    Correa, Agustin; Trajtenberg, Felipe; Obal, Gonzalo; Pritsch, Otto; Dighiero, Guillermo; Oppezzo, Pablo; Buschiazzo, Alejandro

    2013-03-01

    Despite being the most abundant class of immunoglobulins in humans and playing central roles in the adaptive immune response, high-resolution structural data are still lacking for the antigen-binding region of human isotype A antibodies (IgAs). The crystal structures of a human Fab fragment of IgA1 in three different crystal forms are now reported. The three-dimensional organization is similar to those of other Fab classes, but FabA1 seems to be more rigid, being constrained by a hydrophobic core in the interface between the variable and constant domains of the heavy chain (VH-CH1) as well as by a disulfide bridge that connects the light and heavy chains, influencing the relative heavy/light-chain orientation. The crystal structure of the same antibody but with a G-isotype CH1 which is reported to display different antigen affinity has also been solved. The differential structural features reveal plausible mechanisms for constant/variable-domain long-distance effects whereby antibody class switching could alter antigen affinity.

  2. Development of recombinant human IgA for anticardiolipin antibodies assay standardization.

    PubMed

    Knappik, Achim; Capuano, Francesco; Frisch, Christian; Ylera, Francisco; Bonelli, Fabrizio

    2009-09-01

    Controls and calibrators in autoimmune assays are typically developed from patient sera. However, the use of sera is accompanied by a number of disadvantages, such as lack of monospecificity, lack of assay comparability, and supply limitations. Ideally, the control reagent would be an antigen-specific human monoclonal antibody preparation that is defined and pure, easy to produce without any supply limitations, and of defined isotype (IgG, IgM, or IgA). The generation of antigen-specific human monoclonal antibodies has been complicated, but recent advances in development of fully human antibodies by means of in vitro antibody gene library selection has opened a way for the isolation of human antibodies to virtually any antigen, including self-antigens. Such antibodies can be converted to any isotype by gene cloning. Here we developed a set of human monoclonal IgA antibodies specific for the cardiolipin-beta2-glycoprotein 1 complex, using the HuCAL technology. We evaluated the IgA variants of those antibodies for their use as standards in IgA anticardiolipin antibody assays and compared these reagents with serum controls. Such recombinant antibodies may ultimately replace patient sera as assay control and calibration reagents. PMID:19758150

  3. Monoclonal Antibody-Directed Effector Cells Selectively Lyse Human Melanoma Cells in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Schulz, Gregor; Bumol, Thomas F.; Reisfeld, Ralph A.

    1983-09-01

    Monoclonal antibody 9.2.27 (mAb 9.2.27) directed to a chondroitin sulfate proteoglycan on human melanoma cells was able to suppress tumor growth in athymic (nu/nu) mice more effectively when bound with polyethylene glycol to murine effector cells than when injected alone. These ``armed'' effector cells also proved more effective than the monoclonal antibody in eliciting antibody-dependent cellular cytotoxicity against human melanoma target cells in vitro.

  4. Reduction of human blood O2 affinity using dihydroxyacetone, phosphate, and pyruvate.

    PubMed

    Kerr, H H; Pantely, G A; Metcalfe, J; Welch, J E

    1979-09-01

    Human blood oxygen affinity (BOA) was measured after blood from six normal donors was incubated with 4 concentrations of dihydroxyacetone (0.022, 0.044, 0.088, and 0.175 M) plus equimolar disodium phosphate and pyruvate (sodium salt) (0.013, 0.025, 0.05 and 0.1 M) in solutions labeled DDP X 1, DDP X 2, DDP X 4, and DDP X 8, respectively. Blood P50 rose (BOA was reduced) from a control value of 26.0 +/- 0.4 Torr (mean +/- SD) to 29.4 +/- 0.6, 30.6 +/- 0.4, 31.9 +/- 0.15 and 33.3 +/- 1.4 Torr after 2 h of incubation at 37 degrees C with solutions DDP X 1, DDP X 2, DDP X 4, and DDP X 8, respectively. P50 changes at 2 h were 75% complete within 30 min. During these incubations, erythrocyte 2,3-diphosphoglycerate (2,3-DPG) concentration rose from 0.76 +/- 0.09 mol/mol Hb (control) to 1.09 +/- 0.17, 1.14 +/- 0.10, 1.33 +/- 0.15, and 1.45 +/- 0.25 mol/mol Hb with increasing solution concentration. BOA is decreased by an increase in erythrocyte 2,3-DPG. Reduced BOA may improve oxygen delivery to ischemic tissues.

  5. Both LCCL-domains of human CRISPLD2 have high affinity for lipid A.

    PubMed

    Vásárhelyi, Viktor; Trexler, Mária; Patthy, László

    2014-02-01

    The LCCL-domain is a recently defined protein module present in diverse extracellular multidomain proteins. Practically nothing is known about the molecular function of these domains; based on functional features of proteins harboring LCCL-domains it has been suggested that these domains might function as lipopolysaccharide-binding domains. Here we show that the two LCCL-domains of human CRISPLD2 protein, a lipopolysaccharide-binding serum protein involved in defense against endotoxin shock, have higher affinity for the lipid A, the toxic moiety of lipopolysaccharides than for ipopolysaccharide. Our observation that the LCCL-domains of CRISPLD2 are specific for the toxic lipid A moiety of the endotoxin suggests that it may block the interaction between endotoxins and the host endotoxin receptors without interfering with the development of antibacterial immunity against the polysaccharide moiety of LPS. We suggest that the anti-inflammatory function of CRISPLD2 protein may account for its role in various pathological and developmental processes. PMID:24090571

  6. Studies of drug interactions with glycated human serum albumin by high-performance affinity chromatography

    PubMed Central

    Matsuda, Ryan; Kye, So-Hwang; Anguizola, Jeanethe; Hage, David S.

    2015-01-01

    Diabetes is a health condition associated with elevated levels of glucose in the bloodstream and affects 366 million people worldwide. Type II diabetes is often treated with sulfonylurea drugs, which are known to bind tightly in blood to the transport protein human serum albumin (HSA). One consequence of the elevated levels of glucose in diabetes is the non-enzymatic glycation of proteins such as HSA. Several areas of HSA are now known to be affected by glycation-related modifications, which may in turn affect the binding of sulfonylurea drugs and other solutes to this protein. This review discusses some recent studies that have examined these changes in drug-protein binding by employing high-performance affinity chromatography (HPAC). A description of the theoretical and experimental techniques that were used in these studies is given. The information on drug interactions with glycated HSA, as obtained through this method, is also summarized. In addition, the potential advantages of this approach in the areas of biointeraction analysis and personalized medicine are considered. PMID:26526139

  7. Studies of drug interactions with glycated human serum albumin by high-performance affinity chromatography.

    PubMed

    Matsuda, Ryan; Kye, So-Hwang; Anguizola, Jeanethe; Hage, David S

    2014-08-01

    Diabetes is a health condition associated with elevated levels of glucose in the bloodstream and affects 366 million people worldwide. Type II diabetes is often treated with sulfonylurea drugs, which are known to bind tightly in blood to the transport protein human serum albumin (HSA). One consequence of the elevated levels of glucose in diabetes is the non-enzymatic glycation of proteins such as HSA. Several areas of HSA are now known to be affected by glycation-related modifications, which may in turn affect the binding of sulfonylurea drugs and other solutes to this protein. This review discusses some recent studies that have examined these changes in drug-protein binding by employing high-performance affinity chromatography (HPAC). A description of the theoretical and experimental techniques that were used in these studies is given. The information on drug interactions with glycated HSA, as obtained through this method, is also summarized. In addition, the potential advantages of this approach in the areas of biointeraction analysis and personalized medicine are considered. PMID:26526139

  8. Targeting human CD27 with an agonist antibody stimulates T-cell activation and antitumor immunity.

    PubMed

    Thomas, Lawrence J; He, Li-Zhen; Marsh, Henry; Keler, Tibor

    2014-01-01

    CD27 is an important co-stimulatory receptor of T cells that can potentially be exploited for immunotherapy. We developed a human IgG1 antibody that targets human CD27, and demonstrated its immunostimulatory and antineoplastic activity in various preclinical models. Currently, the antibody (1F5, CDX-1127) is being tested in patients affected by advanced malignancies. PMID:24605266

  9. Current based antibodies detection from human serum enhanced by secondary antibodies labelled with gold nanoparticles immobilized in a nanogap.

    PubMed

    Marcon, Lionel; Melnyk, Oleg; Stiévenard, Didier

    2008-02-28

    We present the electrical detection of immunoglobulin G (IgGs) from human serum using a nanogap-based biosensor. The detection method is based on the capture of IgGs by a probe immobilized between gold nanoelectrodes of 30-90nm spacing. The captured IgGs are further reacted with secondary antibodies labelled with gold nanoparticles (GNPs). Insertion of GNPs into the nanogap resulted in increasing the conductance through the nanogap. The use of a chip with 90 nanogaps enabled the calculation of a quality factor for the detection which, coupled with a non-linear regression analysis of the data, easily discriminated specific and differential capture of human antibodies by arrayed probes. We obtained a 500-fold higher quality factor with protein A compared to goat anti-murine antibodies. This method can be applied, through these proof-of-concept experiments, to the detection of protein-protein interactions in biological samples.

  10. Sensitive Targeted Quantification of ERK Phosphorylation Dynamics and Stoichiometry in Human Cells without Affinity Enrichment

    SciTech Connect

    Shi, Tujin; Gao, Yuqian; Gaffrey, Matthew J.; Nicora, Carrie D.; Fillmore, Thomas L.; Chrisler, William B.; Gritsenko, Marina A.; Wu, Chaochao; He, Jintang; Bloodsworth, Kent J.; Zhao, Rui; Camp II, David G.; Liu, Tao; Rodland, Karin D.; Smith, Richard D.; Wiley, H. Steven; Qian, Weijun

    2014-12-17

    Mass spectrometry-based targeted quantification is a promising technology for site-specific quantification of posttranslational modifications (PTMs). However, a major constraint of most targeted MS approaches is the limited sensitivity for quantifying low-abundance PTMs, requiring the use of affinity reagents to enrich specific PTMs. Herein, we demonstrate the direct site-specific quantification of ERK phosphorylation isoforms (pT, pY, pTpY) and their relative stoichiometries using a highly sensitive targeted MS approach termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM). PRISM provides effective enrichment of target peptides within a given fraction from complex biological matrix with minimal sample losses, followed by selected reaction monitoring (SRM) quantification. The PRISM-SRM approach enabled direct quantification of ERK phosphorylation in human mammary epithelial cells (HMEC) from as little as 25 µg tryptic peptides from whole cell lysates. Compared to immobilized metal-ion affinity chromatography, PRISM provided >10-fold improvement in signal intensities, presumably due to the better peptide recovery of PRISM for handling small size samples. This approach was applied to quantify ERK phosphorylation dynamics in HMEC treated by different doses of EGF at both the peak activation (10 min) and steady state (2 h). At 10 min, the maximal ERK activation was observed with 0.3 ng/mL dose, whereas the maximal steady state level of ERK activation at 2 h was at 3 ng/ml dose, corresponding to 1200 and 9000 occupied receptors, respectively. At 10 min, the maximally activated pTpY isoform represented ~40% of total ERK, falling to less than 10% at 2 h. The time course and dose-response profiles of individual phosphorylated ERK isoforms indicated that singly phosphorylated pT-ERK never increases significantly, while the increase of pY-ERK paralleled that of pTpY-ERK. This data supports for a processive, rather than

  11. Sensitive Targeted Quantification of ERK Phosphorylation Dynamics and Stoichiometry in Human Cells without Affinity Enrichment

    DOE PAGES

    Shi, Tujin; Gao, Yuqian; Gaffrey, Matthew J.; Nicora, Carrie D.; Fillmore, Thomas L.; Chrisler, William B.; Gritsenko, Marina A.; Wu, Chaochao; He, Jintang; Bloodsworth, Kent J.; et al

    2014-12-17

    Mass spectrometry-based targeted quantification is a promising technology for site-specific quantification of posttranslational modifications (PTMs). However, a major constraint of most targeted MS approaches is the limited sensitivity for quantifying low-abundance PTMs, requiring the use of affinity reagents to enrich specific PTMs. Herein, we demonstrate the direct site-specific quantification of ERK phosphorylation isoforms (pT, pY, pTpY) and their relative stoichiometries using a highly sensitive targeted MS approach termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM). PRISM provides effective enrichment of target peptides within a given fraction from complex biological matrix with minimal sample losses, followed by selected reactionmore » monitoring (SRM) quantification. The PRISM-SRM approach enabled direct quantification of ERK phosphorylation in human mammary epithelial cells (HMEC) from as little as 25 µg tryptic peptides from whole cell lysates. Compared to immobilized metal-ion affinity chromatography, PRISM provided >10-fold improvement in signal intensities, presumably due to the better peptide recovery of PRISM for handling small size samples. This approach was applied to quantify ERK phosphorylation dynamics in HMEC treated by different doses of EGF at both the peak activation (10 min) and steady state (2 h). At 10 min, the maximal ERK activation was observed with 0.3 ng/mL dose, whereas the maximal steady state level of ERK activation at 2 h was at 3 ng/ml dose, corresponding to 1200 and 9000 occupied receptors, respectively. At 10 min, the maximally activated pTpY isoform represented ~40% of total ERK, falling to less than 10% at 2 h. The time course and dose-response profiles of individual phosphorylated ERK isoforms indicated that singly phosphorylated pT-ERK never increases significantly, while the increase of pY-ERK paralleled that of pTpY-ERK. This data supports for a processive, rather than

  12. Human Monoclonal Antibodies Broadly Neutralizing against Influenza B Virus

    PubMed Central

    Yasugi, Mayo; Kubota-Koketsu, Ritsuko; Yamashita, Akifumi; Kawashita, Norihito; Du, Anariwa; Sasaki, Tadahiro; Nishimura, Mitsuhiro; Misaki, Ryo; Kuhara, Motoki; Boonsathorn, Naphatsawan; Fujiyama, Kazuhito; Okuno, Yoshinobu; Nakaya, Takaaki; Ikuta, Kazuyoshi

    2013-01-01

    Influenza virus has the ability to evade host immune surveillance through rapid viral genetic drift and reassortment; therefore, it remains a continuous public health threat. The development of vaccines producing broadly reactive antibodies, as well as therapeutic strategies using human neutralizing monoclonal antibodies (HuMAbs) with global reactivity, has been gathering great interest recently. Here, three hybridoma clones producing HuMAbs against influenza B virus, designated 5A7, 3A2 and 10C4, were prepared using peripheral lymphocytes from vaccinated volunteers, and were investigated for broad cross-reactive neutralizing activity. Of these HuMAbs, 3A2 and 10C4, which recognize the readily mutable 190-helix region near the receptor binding site in the hemagglutinin (HA) protein, react only with the Yamagata lineage of influenza B virus. By contrast, HuMAb 5A7 broadly neutralizes influenza B strains that were isolated from 1985 to 2006, belonging to both Yamagata and Victoria lineages. Epitope mapping revealed that 5A7 recognizes 316G, 318C and 321W near the C terminal of HA1, a highly conserved region in influenza B virus. Indeed, no mutations in the amino acid residues of the epitope region were induced, even after the virus was passaged ten times in the presence of HuMAb 5A7. Moreover, 5A7 showed significant therapeutic efficacy in mice, even when it was administered 72 hours post-infection. These results indicate that 5A7 is a promising candidate for developing therapeutics, and provide insight for the development of a universal vaccine against influenza B virus. PMID:23408886

  13. Separation of recombinant human protein C from transgenic animal milk using immobilized metal affinity chromatography.

    PubMed

    Dalton, J C; Bruley, D F; Kang, K A; Drohan, W N

    1997-01-01

    Protein C is an important serine protease due to its ability to proteolytically cleave activated Factors V and VIII. Excess coagulation and blood agglutination can lead to plugged capillaries, thereby reducing oxygen transport to interstitial tissues. To treat patients with hereditary and acquired protein C deficiency would require a greater amount of Protein C than that available from human plasma. However, the potential demand for this protein could be met by the production of human protein C from transgenic animal mammary glands. Thus, research into inexpensive, efficient methods to purify proteins from transgenic animal milk will be a criti