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High-Level Expression of a Recombinant Antibody from Myeloma Cells Using a Glutamine Synthetase Gene as an Amplifiable Selectable Marker

Bio/Technology volume 10, pages 169–175 (1992)Cite this article

Abstract

We report a method for introducing a glutamine synthetase (GS) selectable marker into myeloma cells in which transfectants are selected by growth in a glutamine-free medium. Vector amplification can subsequently be selected using the specffic inhibitor of GS, methionine sulphoximine (MSX). Using this system, DNA sequences encoding a chimeric B72.3 IgG4 antibody were expressed from hCMV-MIE promoters in NSO myeloma cells. A cell line was isolated after a single round of selection for vector amplification which contains approximately 4 copies of the vector, secretes 10–15 pg/cell/day cB72.3 antibody during exponential growth and can accumulate 560 mg/l antibody in a fed-batch air-lift fermentation system. Productivity is stable in the absence of MSX selection.

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References

  1. Sahagan, B.G., Dorai, H., Saltzgaber-Muller, J., Toneguzzo, F., Guindon, C.A., Lilly, S.P., McDonald, K.W., Morrissey, D.V., Stone, B.A., and Davis, G.L. 1986. A genetically engineered murine/human chimeric antibody retains specificity for human tumor-associated antigen. J. Immunol. 137: 1066–1074.

    CAS  PubMed  Google Scholar 

  2. Nishimura, Y., Yokoyama, M., Araki, K., Ueda, R., Kudo, A., and Watanabe, T. 1987. Recombinant human-mouse chimeric monoclonal antibody specific for common acute lymphocytic leukemia antigen. Cancer Res 47: 999–1005.

    CAS  Google Scholar 

  3. Sun, L.K., Curtis, P., Rakowicz-Szulczynska, E., Ghrayeb, J., Chang, N., Morrison, S.L., and Koprowski, H. 1987. Chimeric antibody with human constant regions and mouse variable regions directed against carcinoma-associated antigen 17-lA. Proc. Natl. Acad. Sci. USA 84: 214–218.

    Article  CAS  Google Scholar 

  4. Liu, A.Y., Robinson, R.R., Murray, E.D., Ledbetter, J.A., Hellstrom, I., and Hellstrom, K.E. 1987. Production of a mouse-human chimeric monoclonal antibody to CD20 with potent Fc-dependent biologic activity. J. Immunol. 139: 3521–3526.

    CAS  PubMed  Google Scholar 

  5. Beidler, C.B., Ludwig, J.R., Cardenas, J., Phelps, J., Papworth, C.G., Melcher, E., Sierzega, M., Myers, L.J., Unger, B.W., and Fisher, M. 1988. Cloning and high level expression of a chimeric antibody with specificity for human carcinoembryonic antigen. J. Immunol. 141: 4053–4060.

    CAS  PubMed  Google Scholar 

  6. Morrison, S.L. and Oi, V.T. 1984. Transfectomas provide novel chimeric antibodies. Science 229: 1202–1210.

    Article  Google Scholar 

  7. Neumaier, M., Shively, L., Chen, F.S., Gaida, F.J., Ilgen, C., Paxton, R.J., Shively, J.E., and Riggs, A.D. 1990. Cloning of the genes for T84.66, an antibody that has a high specificity and affinity for carcinoembryonic antigen, and expression of chimeric human/mouse T84.66 genes in myeloma and Chinese hamster ovary cells. Cancer Res. 50: 2128–2134.

    CAS  PubMed  Google Scholar 

  8. Bebbington, C.R. 1991. Expression of recombinant antibody genes in non-lymphoid mammalian cells. Methods 2: 136–145.

    Article  CAS  Google Scholar 

  9. Wood, C.R., Dorner, A.J., Morris, G.E., Alderman, E.M., Wilson, D., O'Hara, R.M., and Kaufman, R.J. 1990. High level synthesis of immunoglobulins in Chinese hamster ovary cells. J. Immunol. 145: 3011–3016.

    CAS  PubMed  Google Scholar 

  10. Page, M.J. and Sydenham, M.A. 1991. High level expression of the humanized monoclonal antibody Campath-1H in Chinese hamster ovary cells. Bio/Technology 9: 64–68.

    CAS  PubMed  Google Scholar 

  11. Dorai, H. and Moore, G.P. 1987. The effect of dihydrofolate reduc tase-mediated gene amplification on the expression of transfected immunoglobulin genes. J. Immunol. 139: 4232–4241.

    CAS  Google Scholar 

  12. Gillies, S.D., Dorai, H., Wesolowski, J., Majeau, G., Young, D., Boyd, J., and James, K. 1989. Expression of human anti-tetanus toxoid antibody in transfected murine myeloma cells. Bio/Technology 7: 799–804.

    CAS  Google Scholar 

  13. Gillies, S.D., Lo, K.M., and Wesolowski, J. 1989. High-level expres sion of chimeric antibodies using adapted cDNA variable region cassettes. J. Immunol. Methods 125: 191–202.

    Article  CAS  Google Scholar 

  14. Broad, D., Boraston, R., and Rhodes, M. 1991. Production of recom binant proteins in serum-free media. Cytotechnology 5: 47–55.

    Article  CAS  Google Scholar 

  15. Cockett, M.I., Bebbington, C.R., and Yarranton, G.T. 1990. High level expression of tissue inhibitor of metalloproteinases in Chinese hamster ovary cells using glutamine synthetase gene amplification. Bio/Technology 8: 662–667.

    CAS  PubMed  Google Scholar 

  16. Meister, A., In: Glutamine Metabolism. Enzymology and Regulation, p. 1–40 Mora, J. and Palacios, R. Eds. . Academic Press, NY.

  17. Kuo, C.F. and Darnell, J.E. 1989. Mouse glutamine synthetase is encoded by a single gene that can be expressed in a localised fashion. J. Mol. Biol. 208: 45–56.

    Article  CAS  Google Scholar 

  18. Feng, B., Shiber, S.K., and Max, S.R. 1990. Glutamine regulates glutamine synthetase expression in skeletal muscle cells in culture. J. Cell. Physiol. 145: 376–380.

    Article  CAS  Google Scholar 

  19. Eagle, H., Oyama, V.I., Levy, M., Horton, C.L., and Fleischman, R. 1956. The growth response of mammalian cells in tissue culture to L-glutamine and L-glutamic acid. J. Biol. Chem. 218: 607–616.

    CAS  PubMed  Google Scholar 

  20. DeMars, R. 1958. The inhibition by glutamine of glutamyl transferase formation in cultures of human cells. Biochem. Biophys. Acta. 27: 435–436.

    Article  CAS  Google Scholar 

  21. Paul, J. and Fottrell, P.F. 1963. The mechanism of D-glutamyltransferase repression in mammalian cells. Biochem. Biophys. Acta. 67: 334–336.

    Article  CAS  Google Scholar 

  22. Griffiths, J.B. 1973. The effects of adapting human diploid cells to grow in glutamic acid media on cell morphology, growth and metabolism. J. Cell Sci. 12: 617–629.

    CAS  PubMed  Google Scholar 

  23. Nagle, S.C. and Brown, B.L. 1971. An improved heat-stable glutamine-free chemically defined medium for growth of mammalian cells. J. Cell. Physiol. 77: 259–264.

    Article  CAS  Google Scholar 

  24. Whittle, N., Adair, J., Lloyd, C., Jenkins, L., Devine, J., Schlom, J., Raubitschek, A., Colcher, D., and Bodmer, M. 1987. Expression in COS cells of a mouse-human chimaeric B72 3 antibody. Protein Eng. 1: 499–505.

    Article  CAS  Google Scholar 

  25. Sanders, P.G. and Wilson, R.H. 1984. Amplification and cloning of the Chinese hamster glutamine synthetase gene. EMBO J. 3: 65–71.

    Article  CAS  Google Scholar 

  26. Emtage, J.S., Angal, S., Doel, M., Harris, T.J.R., Jenkins, B., Lilley, G., and Lowe, P.A. 1983. Synthesis of calf prochymosin in Escherichia coli . Proc. Natl. Acad. Sci. USA 80: 3671–3675.

    Article  CAS  Google Scholar 

  27. Hayward, B.E., Hussain, A., Wilson, R.H., Lyons, A., Woodcock, V., McIntosh, B., and Harris, T.J.R. 1986. The cloning and nucleotide sequence of cDNA for and amplified glutamine synthetase gene from the Chinese hamster. Nucleic Acids Res. 14: 999–1008.

    Article  CAS  Google Scholar 

  28. Subramani, S., Mulligan, R., and Berg, P. 1981. Expression of the mouse dihydrofolate reductase complementary deoxyribonucleic acid in Simian Virus 40 vectors. Mol. Cell. Biol. 1: 854–864.

    Article  CAS  Google Scholar 

  29. Cockett, M.I., Bebbington, C.R., and Yarranton, G.T. 1991. The use of engineered E1A genes to transactivate the hCMV-MIE promoter in permanent CHO cell lines. Nucleic Acids Res. 19: 319–325.

    Article  CAS  Google Scholar 

  30. Galfre, G. and Milstein, C. 1981. Preparation of monoclonal antibodies: strategies and procedures. Methods. Enzymol. 73(B): 3–46.

    Article  CAS  Google Scholar 

  31. Kearney, J.F., Radbruch, A., Liesegang, B., and Rajewsky, K. 1979. A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J. Immunol. 123: 1548–1550.

    CAS  PubMed  Google Scholar 

  32. Shulman, M., Wilde, C.D., and Kohler, G. A better cell line for making hybridomas secreting specific antibodies. Nature 276: 269–270.

    Article  CAS  Google Scholar 

  33. Kilmartin, J.V., Wright, B., and Milstein, C. 1982. Rat monoclonal antibodies derived by using a non-secreting rat cell line. J. Cell. Biol. 93: 576–582.

    Article  CAS  Google Scholar 

  34. King, D.J., Adair, J.R., Angal, S., Low, D.C., Proudfoot, K.A., Lloyd, C., Bodmer, M., and Yarranton, G.T. 1992. Expression, purification and characterization of a mouse: human chimeric antibody and chimeric Fab′ fragment. Biochem. J. 281: 317–323.

    Article  CAS  Google Scholar 

  35. Cann, A.J., Koyanagi, Y., and Chen, I.S.Y. 1988. High efficiency transfection of primary human lymphocytes and studies of gene expression. Oncogene 3: 123–128.

    CAS  Google Scholar 

  36. Kadesch, T. and Berg, P. 1986. Effects of the position of the simian virus 40 enhancer on expression of multiple transcription units in a single plasmid. Mol. Cell. Biol. 6: 2593–2601.

    Article  CAS  Google Scholar 

  37. Proudfoot, N.J. 1986. Transcriptional interference and termination between duplicated alpha-globin gene constructs suggests a novel mechanism for gene regulation. Nature 322: 562–565.

    Article  CAS  Google Scholar 

  38. Caulcott, C.A., Boraston, R., Hill, C., Thompson, P.W., and Birch, J.R. 1988. The production and purification of monoclonal antibodies, p. 27–42. In: Complementary Immunoassays. W.P. (Ed.). John Wiley and Sons, NY.

    Google Scholar 

  39. Kohler, G. Immunoglobulin chain loss in hybridoma lines. 1980. Proc. Natl. Acad. Sci. USA 77: 2197–2199.

    Article  CAS  Google Scholar 

  40. Bell, S.L., Bebbington, C.R., Bushell, M.E., Sanders, P.G., Scott, M.F., Spier, R.E., and Wardell, J.N. 1991. Genetic engineering of cellular physiology, p. 304–306. In: Production of Biologicals from Animal Cells in Culture. Spier, R.E., Griffiths,J.B., and Meignier, B. (Eds.). Butterworth-Heinemann, UK.

    Chapter  Google Scholar 

  41. Meilhoc, E., Wittrup, K.D., and Bailey, J.E. 1989. Application of flow cytometric measurement of surface IgG in kinetic analysis of monoclonal antibody synthesis and secretion by murine hybridoma cells. J. Immunol. Methods 121: 167–174.

    Article  CAS  Google Scholar 

  42. Hendershot, L., Bole, D., Kohler, G., and Kearney, J.F. 1987. J. Cell. Biol. 104: 761–767.

    Article  CAS  Google Scholar 

  43. Nakaki, T., Deans, R.J., and Lee, A.S. 1989. Mol. Cell. Biol. 9: 2233–2238.

    Article  CAS  Google Scholar 

  44. Weidle, U.H., Buckel, P., and Wienberg, J. 1988. Amplified expression constructs for human tissue-type plasminogen activator in Chinese hamster ovary cells: instability in the absence of selective pressure. Gene 66: 193–203.

    Article  CAS  Google Scholar 

  45. Stark, G.R. and Wall, G.M. 1984. Gene amplification. Ann. Rev. Biochem. 53: 447–491.

    Article  CAS  Google Scholar 

  46. Pallavacini, M.G., DeTeresa, P.S., Rosette, C., Gray, J., and Wurm, F.M. 1990. Effects of methotrexate on transfected DNA stability in mammalian cells. Mol. Cell. Biol. 10: 401–404.

    Article  Google Scholar 

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Authors and Affiliations

  1. Celltech Ltd., 216, Bath Road, Slough, S11 4EN, U.K.

    C. R. Bebbington, G. Renner, S. Thomson, D. King, D. Abrams & G. T. Yarranton

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Bebbington, C., Renner, G., Thomson, S. et al. High-Level Expression of a Recombinant Antibody from Myeloma Cells Using a Glutamine Synthetase Gene as an Amplifiable Selectable Marker. Nat Biotechnol 10, 169–175 (1992). https://doi.org/10.1038/nbt0292-169

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