A Generic Approach for Miniaturized Unbiased High-Throughput Screens of Bispecific Antibodies and Biparatopic Antibody-Drug Conjugates.

The toolbox of modern antibody engineering allows the design of versatile novel functionalities exceeding nature's repertoire. Many bispecific antibodies comprise heterodimeric Fc portions recently validated through the approval of several bispecific biotherapeutics. While heterodimerization methodologies have been established for low-throughput large-scale production, few approaches exist to overcome the bottleneck of large combinatorial screening efforts that are essential for the identification of the best possible bispecific antibody.

Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities.

Controlled Fab arm exchange (cFAE) has proven to be a generic and versatile technology for the efficient generation of IgG-like bispecific antibodies (DuoBodies or DBs), with several in clinical development and one product, amivantamab, approved by the Food and Drug Administration. In this study, we expand the cFAE-toolbox by incorporating VHH-modules at the C-termini of DB-IgGs, termed DB-VHHs. This approach enables the combinatorial generation of tri- and tetraspecific molecules with flexible valencies in a straightforward fashion.

Site-Specific Conjugation of Native Antibodies Using Engineered Microbial Transglutaminases.

Site-specific bioconjugation technologies are frequently employed to generate homogeneous antibody-drug conjugates (ADCs) and are generally considered superior to stochastic approaches like lysine coupling. However, most of the technologies developed so far require undesired manipulation of the antibody sequence or its glycan structures. Herein, we report the successful engineering of microbial transglutaminase enabling efficient, site-specific conjugation of drug-linker constructs to position HC-Q295 of native, fully glycosylated IgG-type antibodies.