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Rapid Assay of Phage-Derived Recombinant Human Fabs As Bispecific Antibodies

Bio/Technology volume 13, pages 1221–1224 (1995)Cite this article

Abstract

Specific anti-tumor and anti-viral activities can be conferred on lymphocytic and myeloid effector cells by retargeting them with bispecific antibodies. These are antibodies which possess an anti-target binding region and a region capable of binding specific effector cell surface markers. For the rapid evaluation of recombinant human Fabs as bispecific antibodies, we have constructed a vector that allows for the conversion of Fabs into protein A fusion proteins. These can be used to generate bispecific antibodies when complexed to appropriate anti-effector cell immunoglobulins. As a model system, a protein A fusion derivative of a human recombinant anti-herpes simplex virus (HSV) Fab was constructed and complexed to OKT3, a T cell-activating antibody specific for CD3. This complex reduced HSV-2 yields in infected cells by about three logs relative to controls when incubated on HSV-2-infected cell monolayers in the presence of IL-2-actiyated lymphocytes. The system described allows for the rapid evaluation of recombinant human Fabs as bispecific antibodies for therapeutic applications. In addition, Fab-protein A fusion proteins can be used in ELISA and other immuno-assays with increased sensitivity.

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References

  1. Bolhuis, R.L., Sturm, E., Gratama, J.W. and Braakman, E. 1991. Engineering T lymphocyte antigen specificity. J. Cell. Biochem. 47: 306–310.

    Article  CAS  PubMed  Google Scholar 

  2. Fanger, M.W., Morganelli, P.M. and Guyre, P.M. 1992. Bispecific antibodies. Critical Rev. Immunol. 12: 101–124.

    CAS  Google Scholar 

  3. Staeiz, U.D. and Bevan, M.J. 1986. Hybrid hybridoma producing a bispecific monoclonal antibody that can focus effector T cell activity. Proc. Natl. Acad. Sci. USA 83: 1453–1457.

    Article  Google Scholar 

  4. Gruber, M., Schodin, B.A., Wilson, E.R. and Kranz, D.M. 1994. Efficient tumor cell lysis mediated by a bispecinc single chain antibody expressed in Escherichia coli. J. Immunol. 152: 5368–74.

    CAS  PubMed  Google Scholar 

  5. Holliger, P., Prospero, T. and Winter, C. 1993. “Diabodies”: small bivalent and bispecific antibody fragments. Proc. Natl. Acad. Sci. USA 90: 6444–6448.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Shalaby, M.R., Shepard, H.M., Presta, L., Rodrigues, M.L., Beverley, PC., Feldmann, M. and Carter, P. 1992. Development of humanized bispecinc antibodies reactive with cytotoxic lymphocytes and tumor cells overexpressing the HER2 protooncogene. J. Exp. Med. 175: 217–25.

    Article  CAS  PubMed  Google Scholar 

  7. Jung, G., Ledbetter, J.A. and Muller–Eberhard, H.J. 1987. Induction of cytotoxicity in resting human T lymphocytes bound to tumor cells by antibody heteroconjugates. Proc. Natl. Acad. Sci. USA 84: 4611–4615.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Titus, J.A., Perez, P., Kaubisch, A., Garrido, M.A. and Segal, D.M. 1987. Human K/natural killer cells targeted with hetero-cross-linked antibodies specifically lyse tumor cells in vitro and prevent tumor growth in vivo. J. Immunol 139: 3153–3158.

    CAS  PubMed  Google Scholar 

  9. Bolhuis, R.L., Lamers, C.H., Goey, S.H., Eggennont, A.M., Trimbos, J.B., Stoter, G., Lanzayecchia, A., di Re, E., Miotti, S., Raspagliesi, F., et al. 1992. Adoptive immunotherapy of ovarian carcinoma with bs–MAb–targeted lymphocytes: a multicenter study. Internatl. J. Cancer. 7: 78–81.

    CAS  Google Scholar 

  10. Paya, C.V., McKean, D.J., Segal, D.M., Schoon, R.A., Showalter, S.D. and Leibson, P.J. 1989. Heteroconjigate antibodies enhance cell-mediated anti-Herper simplex virus immunity. J. Immunol. 142: 666–671.

    CAS  PubMed  Google Scholar 

  11. Tutt, A., Stevenson, G.T. and Glennie, M.J. 1991. Trispecific F(ab′)3 derivatives that use cooperative signaling via the TCR/CD3 complex and CD2 to activate and redirect resting cytotoxic T cells. J. Immunol. 147: 60–69.

    CAS  PubMed  Google Scholar 

  12. Voss, L.M., David, C.S., Showalter, S.D., Paya, C.V. and Leibson, P.J. 1992. Heteroconjugate antibodies enhance cell-mediates anti-herpes simplex virus immunity in vivo. Internatl. Immunol. 4: 417–420.

    Article  CAS  Google Scholar 

  13. Woodle, E.S., Thistlethwaite, J.R., Jolliffe, L.K., Zivin, R.A., Collins, A., Adair, J.R., Bodmer, M., Athwal, D., Alegre, M.L. and Bluestone, J.A. 1992. Humanized OKT3 antibodies: successful transfer of immune modulating properties and idiotype expression. J. Immunol. 148: 2756–2763.

    CAS  PubMed  Google Scholar 

  14. Burton, D.R. and Barbas, C.F. 1994. Human antibodies from combinatorial libraries. Adv. Immunol. 57: 191–280.

    Article  CAS  PubMed  Google Scholar 

  15. Sjödahl, J. 1977. Structural studies on the four repetitive Fc binding regions in protein A from Staphylococcus aureus. European J. Biochem. 78: 471–479.

    Article  Google Scholar 

  16. Inganas, M. 1981. Comparison of mechanisms of interaction between protein A from Staphylococcus aureus and human monoclonal IgG, IgA and IgM in relation to the classical Fc gamma and the alternative F(ab′)2 epsilon protein A interactions. Scand. J. Immunol. 13: 343–352.

    Article  CAS  PubMed  Google Scholar 

  17. Harlow, E. and Lane, D. 1988. Antibodies: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 616–619.

    Google Scholar 

  18. Nilsson, B., Abrahmsen, L. and Uhlen, M. 1985. Immobilization and purification of enzymes with staphylococcal protein A Gene fusion vectors. EMBO J. 4: 1075–1080.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Nilsson, B. and Abrahmsen, L. 1990. Fusions to staphylococcal protein A. Methods in Enzymol. 185: 144–161.

    Article  CAS  Google Scholar 

  20. Biguzzi, S. 1982. Fc gamma-like determinants on immunoglobulin variable regions: identification by staphylococcal protein A. Scand. J. Immunol. 15:(6)605–18.

    Article  CAS  PubMed  Google Scholar 

  21. Sasso, E.H., Silverman, G.J. and Mannik, M. 1989. Human IgM molecules that bind staphylococcal protein A contain VHIII H chains. J. Immunol. 142(8):2778–83.

    CAS  PubMed  Google Scholar 

  22. Barbas, C.F. 3d, Kang, A.S., Lerner, R.A. and Benkovic, S.J. 1991. Assembly of combinatorial antibody libraries on phage surfaces: the Gene in site. Proc. Natl. Acad. Sci. USA 88: 7978–7982.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Burton, D.R. and Barbas, C.F. III 1993. Human antibodies to HIV-1 by recombinant DNA methods. Chemical Immunology 56: 112–1126.

    Article  CAS  PubMed  Google Scholar 

  24. Ito, W. and Kurosawa, Y. 1993. Development of an artificial antibody system with multiple valency using an Fv fragment fused to a fragment of protein A. J. Biol. Chem. 268: 20668–20675.

    CAS  PubMed  Google Scholar 

  25. Tai, M.S., Mudgett–Hunter, M., Levinson, D., Wu, G.M., Haber, E., Oppermann, H. and Huston, J.S. 1990. A bifunctional fusion protein containing Fc-binding fragment B of staphylococcal protein A amino terminal to antidigoxin single-chain Fv. Biochem. 29: 8024–8030.

    Article  CAS  Google Scholar 

  26. Gandecha, A.R., Owen, M.R., Cockburn, B., Wbitelam, G.C. 1992. Production and secretion of a bifunctional staphylococcal protein A:antiphytochrome single-chain Fv fusion protein in Escherichia coli. Gene 122: 361–365.

    Article  CAS  PubMed  Google Scholar 

  27. Glockshuber, R., Malia, M., Pfitzinger, I. and Pluckthun, A. 1990. A comparison of strategies to stabilize immunoglobulin Fv-fragments. Biochem. 29: 1362–1367.

    Article  CAS  Google Scholar 

  28. Muller, J.D., Nienhaus, G.U., TeUn, S.Y. and Voss, E.W. 1994. Ligand binding to anti-fluorescyl antibodies: stability of the antigen binding site. Biochem. 33: 6221–6227.

    Article  CAS  Google Scholar 

  29. Burioni, R., Williamson, R.A., Sanna, P.P., Bloom, F.E. and Burton, D.R. 1994. Recombinant human Fab to glycoprotein D neutralizes infectivity and prevents cell-to-cell transmission of herpes simplex viruses 1 and 2 in vitro. Proc. Natl. Acad. Sci. USA 91: 355–359.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Kung, P., Goldstein, G., Reinherz, E.L. and Schlossman, S.F. 1979. Monoclonal antibodies denning distinctive human T cell surface antigens. Science 206: 347–349.

    Article  CAS  PubMed  Google Scholar 

  31. Jung, G., Freimann, U., Von MarschaU, Z., Reisfeld, R.A. and WUmarms, W. 1991. Target cell-induced T cell activation with bi- and trispecific antibody fragments. European J. Immunol. 21: 2431–2435.

    Article  CAS  Google Scholar 

  32. Ledbetter, J.A., June, C.H., Martin, P.J., Spooner, C.E., Hansen, J.A. and Meier, K.E. 1986. Valency of CD3 binding and intemalization of the CD3 cell-surface complex control T cell responses to second signals: distinction between effects on protein kinase C, cytoplasmic free calcium, and proliferation. J. Immunol. 136: 3945–3952.

    CAS  PubMed  Google Scholar 

  33. Damle, N.K., Doyle, L.V., Grosmaire, L.S. and Ledbetter, J.A. 1988. Differential regulatory signals delivered by antibody binding to the CD28 (Tp44) molecule during the activation of human T lymphocytes. J. Immunol 140: 1753–1761.

    CAS  PubMed  Google Scholar 

  34. Langone, J.J., Boyle, M.D. and Borsos, T. 1978. Studies on the interaction between protein A and immunoglobulin G.I. Effect of protein A on the functional activity of IgG. J. Immunol. 121: 327–332.

    CAS  PubMed  Google Scholar 

  35. Morelli, D., Villa, E., Tagliabue, E., Perletti, L., Villa, M.L., Menard, S., Balsari, A. and Colnaghi, M.L. 1994. Relevance of antibody valency in EGF receptor modulation. Scandinavian J. Immunol. 39: 453–458.

    Article  CAS  Google Scholar 

  36. Demanet, C., Brissinck, J., Van Mechelen, M., Leo, O. and Thielemans, K. 1991. Treatment of murine B cell lymphoma with bispecific monoclonal antibodies (anti-idiotype x anti-CD3). J. Immunol. 147: 1091–1097

    CAS  PubMed  Google Scholar 

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Author notes

  1. R. Anthony Williamson and Dennis R. Burton: Departments of Immunology, The Scripps Research Institute, 10666 N. Torrey Pines Road, La Jolla, CA 92037.

  2. Dennis R. Burton: Departments of Molecular Biology, The Scripps Research Institute, 10666 N. Torrey Pines Road, La Jolla, CA 92037.

  3. Dario C. Altieri: The Boyer Center for Molecular Medicine, Yale University School of Medicine, 95 Congress Avenue, New Haven, CT 06536.

Authors and Affiliations

  1. Departments of Neuropharmacology, The Scripps Research Institute, 10666 N. Torrey Pines Road, La Jolla, CA, 92037

    Pietro Paolo Sanna, Alessandro De Logu, R. Anthony Williamson, Maria E. Samson & Floyd E. Bloom

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  2. Alessandro De Logu
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Sanna, P., De Logu, A., Williamson, R. et al. Rapid Assay of Phage-Derived Recombinant Human Fabs As Bispecific Antibodies. Nat Biotechnol 13, 1221–1224 (1995). https://doi.org/10.1038/nbt1195-1221

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