Efficiency landscape of bioorthogonal click reactions producing bispecific antibody conjugates

Petri, László and Kovács, Írisz Karolina and Szabó, Boglárka and Imre, Tímea and Rácz, Anita and Chudasama, Vijay and Szijj, Peter A. and Keserű, György Miklós (2026) Efficiency landscape of bioorthogonal click reactions producing bispecific antibody conjugates. CELL REPORTS METHODS, 6 (6). No. 101416. ISSN 2667-2375

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Abstract

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.

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