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Design and production of novel tetravalent bispecific antibodies

Nature Biotechnology volume 15, pages 159–163 (1997)Cite this article

Abstract

We have produced novel bispecific antibodies by fusing the DNA encoding a single chain antibody (ScFv) after the C terminus (CH3-ScFv) or after the hinge (Hinge-ScFv) with an antibody of a different specificity. The fusion protein is expressed by gene transfection in the context of a murine variable region. Transfectomas secrete a homogeneous population of the recombinant antibody with two different specificities, one at the N terminus (anti-dextran) and one at the C terminus (anti-dansyl). The CH3-ScFv antibody, which maintains the constant region of human lgG3, has some of the associated effector functions such as long half-life and Fc receptor binding. The Hinge-ScFv antibody which lacks the CH2 and CH3 domains has no known effector functions.

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References

  1. Flavell, D.J., Cooper, S., Morland, B. and Flavell, S.U. 1991. Characteristics and performance of a bispecific F (ab′gamma)2 antibody for delivering saporin to a CD7+ human acute T-cell leukaemia cell line. Br. J. Cancer 64: 274–280.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Runge, M.S., Bode, C., Savard, C.E., Matsueda, G.R. and Haber, E. 1990. Antibody-directed fibrinolysis: a bispecific (Fab′)2 that binds to fibrin and tissue plasminogen activator. Bioconjug. Chem. 1: 274–277.

    Article  CAS  PubMed  Google Scholar 

  3. Raso, V. and Griffin, T. 1981. Hybrid antibodies with dual specificity for the delivery of ricin to immunoglobulin-bearing target cells. Cancer Res. 41: 2073–2078.

    CAS  PubMed  Google Scholar 

  4. Glennie, M.J., McBride, H.M., Worth, A.T. and Stevenson, G.T. 1987. Preparation and performance of bispecific F(ab′ gamma)2 antibody containing thioether-linked Fab′ gamma fragments. J. Immunol. 139: 2367–2375.

    CAS  PubMed  Google Scholar 

  5. Glennie, M.J., Brennand, D.M., Bryden, F., McBride, H.M., Stirpe, F., Worth, A.T. and Stevenson, G.T., 1988. F(ab′ gamma)2 antibody for the delivery of saporin in the treatment of lymphoma. J. Immunol. 141: 3662–3670.

    CAS  PubMed  Google Scholar 

  6. Reading, C., 1983. Bifunctional Antibodies, pp. 235 in Hybridomas and Cellular Immortality. Press, P. (ed.). Plenum Press: New York.

    Google Scholar 

  7. Martinis, J., Franz, K.J.F. and Bartholomew, R. 1982. Proceedings of the Thirteenth Colloquium on Protides of the Biological Fluids. Peter, H.E. (ed.). New York: Pergamon Press. 311.

    Google Scholar 

  8. Milstein, C. and Cuello, A.C. 1983. Hybrid hybridomas and their use in immuno-histochemistry. Nature 305: 537–540.

    Article  CAS  PubMed  Google Scholar 

  9. Kontsekova, E., Kolcunova, A. and Kontsek, P. 1992. Quadroma-secreted bi(interferon alpha 2—peroxidase) specific antibody suitable for one-step immunoassay. Hybridoma 11: 461–468.

    Article  CAS  PubMed  Google Scholar 

  10. Lloyd, F.J. and Goldrosen, M. 1991. The production of a bispecific anti-CEA, anti-hapten (4-amino-phthalate) hybrid-hybridoma. J. Natl. Med. Assoc. 83: 901–904.

    PubMed  PubMed Central  Google Scholar 

  11. Tada, H., Kurokawa, T., Seita, T., Watanabe, T. and Iwasa, S. 1994. Expression and characterization of a chimeric bispecific antibody against fibrin and against urokinase-type plasminogen activator. J. Biotechnol. 33: 157–174.

    Article  CAS  PubMed  Google Scholar 

  12. Pack, P. and Pluckthun, A. 1992. Miniantibodies: use of amphipathic helices to produce functional, flexibly linked dimeric FV fragments with high avidity in Escherichia coli. Biochemistry 31: 1579–1584.

    Article  CAS  PubMed  Google Scholar 

  13. George, A.J., Titus, J.A., Jost, C.R., Kurucz, I., Perez, R., Andrew, S.M., Nicholls, P.J., Huston, J.S. and Segal, D.M. 1994. Redirection of T cell-mediated cyto-toxicity by a recombinant single-chain Fv molecule. J. Immunol. 152: 1802–1811.

    CAS  PubMed  Google Scholar 

  14. Chaudhary, V.K., Batra, J.K., Gallo, M.G., Willingham, M.C., FitzGerald, D.J. and Pastan, I. 1990. A rapid method of cloning functional variable-region antibody genes in Escherichia coli as single-chain immunotoxins [published erratum appears in Proc .Nat. Acad. Sci. USA 1990 Apr;87(8):3253]. Proc. Natl. Acad. Sci. USA 87: 1066–1070.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Ayala, M., Duenas, M., Santos, A., Vazquez, J., Menendez, A., Silva, A. and Gavilondo, J.V. 1992. Bacterial single-chain antibody fragments, specific for carcinoembryonic antigen. Biotechniques 13: 790–799.

    CAS  PubMed  Google Scholar 

  16. Tao, M.H., Canfield, S.M. and Morrison, S.L. 1991. The differential ability of human lgG1 and lgG4 to activate complement is determined by the COOH-terminal sequence of the CH2 domain. J. Exp. Med. 173: 1025–1028.

    Article  CAS  PubMed  Google Scholar 

  17. Huston, J.S., Levinson, D., Mudgett, H.M., Tai, M.S., Novotny, J., Margolies, M.N., Ridge, R.J., Bruccoleri, R.E., Haber, E., Crea, R. et al. 1989. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Gene 77: 51–59.

    Article  Google Scholar 

  18. Ayala, M., Balint, R.F., Fernandez, de, Cossio, L., Canaan, H.J., Larrick, J.W., and Gavilondo, J.V. 1995. Variable region sequence modulates periplasmic export of a single-chain Fv antibody fragment in Escherichia coli. Biotechniques 18: 835–838.

    Google Scholar 

  19. Wallick, S.C., Kabat, E.A. and Morrison, S.L. 1988. Glycosylation of a VH residue of a monoclonal antibody against alpha (1—>;6) dextran increases its affinity for antigen. J. Immunol. 141: 3516–3522.

    Google Scholar 

  20. Tao, M.H. and Morrison, S.L. 1989. Studies of aglycosylated chimeric mouse-human IgG. Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region. J. Immunol. 143: 2595–2601.

    CAS  PubMed  Google Scholar 

  21. Kirn, J.K., Tsen, M.F., Ghetie, V. and Ward, E.S. 1994. Identifying amino acid residues that influence plasma clearance of murine lgG1 fragments by site-directed mutagenesis. Eur. J. Immunol. 24: 542–548.

    Article  Google Scholar 

  22. Kim, J.K., Tsen, M.F., Ghetie, V. and Ward, E.S. 1994. Catabolism of the murine lgG1 molecule: evidence that both CH2-CH3 domain interfaces are required for persistence of lgG1 in the circulation of mice. Scand. J. Immunol. 40: 457–465.

    CAS  Google Scholar 

  23. Kim, J.K., Tsen, M.F., Ghetie, V. and Ward, E.S. 1995. Evidence that the hinge region plays a role in maintaining serum levels of the murine lgG1 molecule. Mol. Immunol. 32: 467–475.

    Article  CAS  PubMed  Google Scholar 

  24. Duncan, A.R. and Winter, G. 1988. The binding site for C1q on IgG. Nature 332: 738–740.

    Article  CAS  PubMed  Google Scholar 

  25. Tao, M.H., Smith, R.I. and Morrison, S.L. 1993. Structural features of human immunoglobulin G that determine isotype-specific differences in complement activation. J. Exp. Med. 178: 661–667.

    Article  CAS  PubMed  Google Scholar 

  26. Duncan, A.R. and Winter, G. 1994. The binding site for C1q on IgG. J. Exp. Med. 180: 1087–1096.

    Article  Google Scholar 

  27. Duncan, A.R., Woof, J.M., Partridge, L.J., Burton, D.R. and Winter, G. 1988. Localization of the binding site for the human high-affinity Fc receptor on IgG. Nature 332: 738–740.

    Article  CAS  PubMed  Google Scholar 

  28. Canfield, S.M. and Morrison, S.L. 1991. The binding affinity of human IgG for its high affinity Fc receptor is determined by multiple amino acids in the CH2 domain and is modulated by the hinge region. J. Exp. Med. 173: 1483–1491.

    Article  CAS  PubMed  Google Scholar 

  29. Chappel, M.S., Isenman, D.E., Everett, M., Xu, Y.Y., Dorrington, K.J. and Klein, M.H. 1991. Identification of the Fc gamma receptor class I binding site in human IgG through the use of recombinant lgG1/lgG2 hybrid and point-mutated antibodies. Proc. Natl. Acad. Sci. USA 88: 9036–9040.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Dangl, J.L., Parks, D.R., Oi, V.T. and Herzenberg, L.A. 1982. Rapid isolation of cloned isotype switch variants using fluorescence activated cell sorting. Cytometry 2: 395–401.

    Article  CAS  PubMed  Google Scholar 

  31. Ho, S.N., Hunt, H.D., Norton, R.M., Pullen, J.K. and Pease, L.R. 1989. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77: 51–59.

    Article  CAS  PubMed  Google Scholar 

  32. Horton, R.M., Ho, S.N., Pullen, J.K., Hunt, H.D., Cai, Z. and Pease, L.R. 1993. Gene splicing by overlap extension. Methods Enzymol. 217: 270–279.

    Article  CAS  PubMed  Google Scholar 

  33. Holton, T.A. and Graham, M.W. 1991. A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors. Nucleic Acids Res. 19.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Shin, S.U., Wright, A., Bonagura, V., and Morrison, S.L. 1992. Genetically-engineered antibodies: tools for the study of diverse properties of the antibody molecule. Immunol. Rev. 130: 87–107.

    Article  CAS  PubMed  Google Scholar 

  35. Wright, A., Shin, S.U. and Morrison, S.L. 1992. Genetically engineered antibodies: progress and prospects. Crit. Rev. Immunol. 12: 125–168.

    CAS  PubMed  Google Scholar 

  36. Shin, S.U. and Morrison, S.L. 1989. Production and properties of chimeric antibody molecules. Methods Enzymol. 178: 459–476.

    Article  CAS  PubMed  Google Scholar 

  37. Nieto, A., Gaya, A., Jansa, M., Moreno, C. and Vives, J. 1984. Direct measurement of antibody affinity distribution by hapten-inhibition enzyme immunoassay. Mol. Immunol. 21: 537–543.

    Article  CAS  PubMed  Google Scholar 

  38. Wright, A. and Morrison, S.L. 1994. Effect of altered CH2-associated carbohydrate structure on the functional properties and in vivo fate of chimeric mouse-human immunoglobulin G1. J. Exp. Med. 180: 1087–1096.

    Article  CAS  PubMed  Google Scholar 

  39. Smith, R.I. and Morrison, S.L. 1994. Recombinant polymeric IgG: an approach to engineering more potent antibodies. Bio-Technology 12: 683–688.

    CAS  PubMed  Google Scholar 

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Author notes

  1. Sherie L. Morrison: Corresponding author (e-mail: sheriem@microbio.lifesci.ucla.edu).

Authors and Affiliations

  1. Department of Microbiology and Molecular Genetics and the Molecular Biology Institute, University of California at Los Angeles, 405 HilgardAve., Los Angeles, CA, 90095

    M. Josefina Coloma & Sherie L. Morrison

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  1. M. Josefina Coloma
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Coloma, M., Morrison, S. Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol 15, 159–163 (1997). https://doi.org/10.1038/nbt0297-159

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