Efficiency landscape of bioorthogonal click reactions producing bispecific antibody conjugates

Bioorthogonal conjugation techniques offer a powerful and flexible approach for the modular construction of multifunctional biomolecules, such as bispecific antibodies. In this study, we systematically compared two inverse electron demand Diels-Alder (IEDDA) reactions, between tetrazine and either trans-cyclooctene (TCO) or bicyclo[6.1.0]nonyne (BCN), to generate chemically conjugated bispecific antibody constructs. We applied a design of experiments (DoE) framework to explore how various reaction parameters influence conjugation efficiency. The two systems exhibited distinct reactivity patterns: the BCN-tetrazine reaction proved to be more robust, while the TCO-tetrazine ligation showed a more complex dependency on reaction time and temperature. To assess biological functionality, the bispecific constructs were evaluated by ELISA, confirming preserved antigen-recognition after both conjugation strategies. Both strategies consistently yielded bispecific constructs with comparable physicochemical and functional profiles. These insights support the application of this chemical conjugation strategy as a rapid, tunable, and modular platform for early-stage multispecific antibody development.