Study of Antigen-Binding Properties of Bispecific Antibodies

M. B. Smirnova,¹ N. N. Dergunova,² E. A. Kizim,¹ Yu. S. Massino,¹ V. A. Nikulina,² O. L. Segal,¹ E. B. Tereshkina,² G. I. Kolyaskina,² and A. D. Dmitriev^1,3

¹Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, ul. Butlerova 5a, Moscow, 117865 Russia; fax: (7-095) 338-85-00; E-mail: vnd@ihnerv.msk.su

²Mental Health Center, Russian Academy of Medical Sciences, Zagorodnoe Shosse 2/2, Moscow, 113152 Russia; fax: (7-095) 952-89-40; E-mail: kgi@rcmh.msk.ru

³To whom correspondence should be addressed.

Submitted May 21, 1996; revision submitted September 10, 1996.

Antigen-binding properties of bispecific antibodies (bAbs) produced by mouse hybrid hybridomas were studied. One of the bAbs held binding sites for two different antigens with relatively high molecular mass: human IgG (M_r . 160,000) and horseradish peroxidase (HRP, M_r . 40,000). Another bAbs showed specificity to antigens differing in molecular mass by more than an order of magnitude: peptide alpha-endorphin (END, M_r . 1600) and HRP (M_r . 40,000). The studied antibodies also contained different immunoglobulin chains. Both heavy chains of the anti-IgG/HRP bAbs molecule were of mouse subclass IgG1. Anti-END/HRP bAbs was formed by a combination of heavy chains which belong to two subclasses of IgG: IgG2a and IgG1. bAbs were purified from ascitic fluid by a two-step affinity chromatography on columns with Sepharose-4B conjugated with the corresponding antigen. Radioimmune and immunoenzyme assays were used to analyze antigen--antibody binding and equilibrium constants of association (K_a) for each parental antibody and bAbs were determined by the Scatchard method. No significant changes in the affinity of bAbs antigen-binding sites were observed as compared to the corresponding parental antibodies. It was also shown that bAbs interaction with an excess of one of the antigens did not affect binding of the other antigen to the second bAbs site. Two-dimensional gel electrophoresis was used to analyze the composition of bAbs light and heavy chains specific to END/HRP. This analysis corroborated that bAbs molecules contained light and heavy chains from both parental hybridomas. Hence, it was demonstrated that the hybridoma fusion method can provide bispecific IgG molecules fully preserving antigen binding properties of the parental antibodies.

KEY WORDS: tetradoma, bispecific antibody, affinity.

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