Preparation of Bispecific Antibodies by Chemical Recombination of Monoclonal Immunoglobulin G ₁ Fragments

Abstract

Preparation of bispecific antibodies by the chemical reassociation of monovalent fragments derived from monoclonal mouse immunoglobulin G1 is inefficient because of side reactions during reoxidation of the multiple disulfide bonds linking the heavy chains. These side reactions can be avoided by using specific dithiol complexing agents such as arsenite and effecting disulfide formation with a thiol activating agent such as 5,5'-dithiobis(2-nitrobenzoic acid). In this way bispecific antibodies were obtained in high yield and free of monospecific contaminants from monoclonal mouse immunoglobulin G1 fragments. The bispecific antibodies were used as agents for the selective immobilization of enzymes.

bisFabs: Tools for rapidly screening hybridoma IgGs for their activities as bispecific antibodies

“…Although methods to produce bispecific F(ab') 2 from recombinant Fab' molecules or from human and murine full-length IgG1 have been described previously, 16,8,[17][18][19] no comprehensive method to produce bispecific antibodies directly from all murine IgG isotype antibodies has been published. We described previously a method to produce bispecific F(ab)' 2 from human IgG1 using a bismaleimide crosslinker.…”

Characterization and analysis of scFv-IgG bispecific antibody size variants

“…In recent years, novel molecular formats, such as antibody-drug conjugates and bispecific antibodies, have entered clinical studies and received FDA approval. Although bispecific antibodies were first identified in the 1960s, 2–6 their therapeutic application was not feasible until the 2000s due to technical challenges in expressing and purifying these formats. 7–15 As of 2017, there were approximately 60 bispecific antibodies in clinical studies 1 and two bispecific antibodies with FDA approval: Blincyto® (blinatumomab, Amgen/Micromet; approved in 2014), and Hemlibra® (emicizumab-kxwh, Chugai/Genentech; approved in 2017).…”

EFab domain substitution as a solution to the light-chain pairing problem of bispecific antibodies

“…1,2 Therefore, efficient methods of generating bispecific antibodies are being pursued. Initial attempts to produce bsAbs as protein therapeutics involved chemical conjugation of monospecific antibodies and fusion of monoclonal antibody (mAb)-expressing cells, 3,4 but low efficiency and the necessity of purification from abundant side products limited the widespread use of these strategies. Advancements in protein engineering and molecular biology have enabled the generation of a variety of new bsAb formats, and more than 60 such formats have been described in the literature.…”