Abstract
Bispecific antibodies (BsAb) are promising therapeutic tools in tomorrow's medicine. Expression systems favoring efficient heterodimerization of intermediate-sized bispecific antibodies will significantly improve existing production methods. By C-terminal fusion of scFv molecules to the Fd- and the L-chains efficient heterodimerization in mammalian cells was obtained and a novel intermediate sized, disulfide stabilized BsAb could be efficiently produced. This type of antibody derivative easily allows for the production of trispecific antibodies, BsAb with bivalent binding for one antigen, or immunoconjugates.
Similar content being viewed by others
References
Brissinck J, Demanet C, Moser M, Leo O and Thielemans K. (1991) Treatment of mice bearing BCL1 lymphoma with bispecific antibodies. J Immunol 147: 4019-4026
De Jonge J, Brissinck J, Heirman C, Demanet C, Leo O, Moser M and Thielemans K. (1995) Production and characterization of bispecific single-chain antibody fragments. Mol Immunol 32: 1405-1412
De Sutter K, Feys V, Van de Voorde A and Fiers W. (1992) Production of functionally active murine and murine::human chimeric F(ab')2 fragments in COS-1 cells. Gene 113: 223-230
DuBridge RB, Tang P, Hsia HC, Leong PM, Miller JH and Calos MP. (1987) Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system. Mol Cell Biol 7: 379-87
Grooten J and Fiers W. (1989) Acquisition by the murine host of responsiveness toward various neoplastic cell lines, but not toward self, through adoptive transfer of a helper T-lymphocyte clone with antiself specificity. Cancer Res 49: 3872-8
Hendershot L, Bole D, Kohler G and Kearney JF. (1987) Assembly and secretion of heavy chains that do not associate posttranslationally with immunoglobulin heavy chain-binding protein. J Cell Biol 104: 761-7
Hendrix PG, Dauwe SE, Van de Voorde A, Nouwen EJ, Hoylaerts MF and De Broe ME. (1991) Radiolocalisation and imaging of stably HPLAP-transfected MO4 tumours with monoclonal antibodies and fragments. Br J Cancer 64: 1060-1068
Holliger P, Prospero T and Winter G. (1993) “Diabodies”: small bivalent and bispecific antibody fragments. Proc Natl Acad Sci USA 90: 6444-6448
Horn U, Strittmatter W, Krebber A, Knupfer U, Kujau M, Wenderoth R, Muller K, Matzku S, Pluckthun A and Riesenberg D. (1996) High volumetric yields of functional dimeric miniantibodies in Escherichia coli, using an optimized expression vector and high-cell-density fermentation under non-limited growth conditions. Appl Microbiol Biotechnol 46: 524-532
Hu S, Shively L, Raubitschek A, Sherman M, Williams LE, Wong JY, Shively JE and Wu AM. (1996) Minibody: A novel engineered anti-carcinoembryonic antigen antibody fragment (single-chain Fv-CH3) which exhibits rapid, high-level targeting of xenografts. Cancer Res 56: 3055-61
Luo D, Geng M, Noujaim AA and Madiyalakan R. (1997) An engineered bivalent single-chain antibody fragment that increases antigen binding activity. J Biochem (Tokyo) 121: 831-4
Mack M, Riethmuller G and Kufer P. (1995) A small bispecific antibody construct expressed as a functional single-chain molecule with high tumor cell cytotoxicity. Proc Natl Acad Sci USA 92: 7021-7025
Milenic DE, Yokota T, Filpula DR, Finkelman MA, Dodd SW, Wood JF, Whitlow M, Snoy P and Schlom J. (1991) Construction, binding properties, metabolism, and tumor targeting of a single-chain Fv derived from the pancarcinoma monoclonal antibody CC49. Cancer Res 51: 6363-71
Muller KM, Arndt KM, Strittmatter W and Pluckthun A. (1998) The first constant domain (C(H)1 and C(L)) of an antibody used as heterodimerization domain for bispecific miniantibodies. FEBS Lett 422: 259-64
Niwa H, Yamamura K and Miyazaki J. (1991) Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108: 193-9
O'Mahoney JV and Adams TE. (1994) Optimization of experimental variables influencing reporter gene expression in hepatoma cells following calcium phosphate transfection. DNA Cell Biol 13: 1227-32
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
Ridgway JB, Presta LG and Carter P. (1996) 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng 9: 617-621
Smans KA, Hoylaerts MF, Narisawa S, Millan JL and De Broe ME. (1995) Bispecific antibody-mediated lysis of placental and germ cell alkaline phosphatase targeted solid tumors in immunocompetent mice. Cancer Res 55: 4383-4390
Zhu Z, Zapata G, Shalaby R, Snedecor B, Chen H and Carter P. (1996) High level secretion of a humanized bispecific diabody from Escherichia coli. Biotechnology (N Y) 14: 192-6
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Schoonjans, R., Willems, A., Grooten, J. et al. Efficient heterodimerization of recombinant bi- and trispecific antibodies. Bioseparation 9, 179–183 (2000). https://doi.org/10.1023/A:1008120203269
Issue Date:
DOI: https://doi.org/10.1023/A:1008120203269