Bispecific antibodies may bind to two or more different antigens or targets. Currently, bispecific antibodies are produced by a number of different recombinant strategies. Such strategies include single chain variable fragment (scFv)-derived formats such as diabodies, tandem diabodies, BiTes and DARTs, as well as others. The production of products using these methods can be time consuming and costly, especially for upstream screening of multiple antibody pairs. In addition, a number of chemical approaches have been developed which primarily exploit the reactivity of lysine or cysteine residues within the antibody. Drawbacks of these approaches include the production of highly cross-linked unwanted species, difficult and/or time-consuming characterization and purification of the desired products with poor yield, and the lack of site-specificity of the conjugation sites can impair or destroy antibody binding affinities. We previously introduced a highly-efficient method for the site-specific, directional, crosslinking of essentially any two off-the-shelf antibodies resulting in a high yield of the desired tetravalent bispecific pairs. The site-specific method, targeting the heavy chain glycans, preserves antigen binding activity and ensures the directionality of conjugation. The method involves four main steps, 1) Fc glycan cleavage with endoglycosidase S2 (EndoS2) leaving 2 core GlcNAc residues (one per heavy chain), 2) azide-activation of the core GlcNAcs with a mutant GalT(Y289L) enzyme, 3) conjugation of azide-activated antibodies with one of two Diels Alder orthogonal reactive-pair functional groups, transcyclooctene (TCO) or methytetrazine (Tzn) and 4) combining equimolar amounts of each TCO/Tzn mAb pairs to yield site-specific, directionally conjugated, tetrameric bispecific antibodies. The four-step method allows for production of the bispecific pairs over a 4 day period requiring at least 2 overnight incubations. To streamline this process, we have employed an automated workflow that reduces bispecific antibody production time by more than 50%. Our results demonstrate that completion of the first 3 steps (TCO/Tzn activations) can be accomplished in a single overnight run, while the final pair conjugation step can be completed within a few hours on day 2. Mass spec and enzymatic analyses of the labeled antibodies confirm site specific crosslinking sites are limited to the antibody Fc domain glycosylation sites. Gel analyses and spectroscopic studies show greater than 80% yields of the bispecific pairs without any purification. Furthermore, mass spectrometric analyses show that the antibodies contain only a single crosslink between the heavy chain glycan sites. Our results demonstrate that multiple tetravalent bispecific antibody pairs can be rapidly prepared and screened in less than a 1 week time frame using any off-the-shelf IgG's.
展开▼
