Efficiency landscape of bioorthogonal click reactions producing bispecific antibody conjugates

  • Cell Rep Methods. 2026 Jun 15;6(6):101416. doi: 10.1016/j.crmeth.2026.101416.
László Petri  1 ,  Írisz K Kovács  2 ,  Boglárka Szabó  2 ,  Tímea Imre  3 ,  Anita Rácz  4 ,  Vijay Chudasama  5 ,  Peter A Szijj  6 ,  György M Keserű  7
Affiliations
  • 1. Medicinal Chemistry Research Group, HUN-REN Research Centre for Natural Sciences, 2 Magyar tudósok krt, 1117 Budapest, Hungary; National Drug Discovery and Development Laboratory, HUN-REN Research Centre for Natural Sciences, 2 Magyar tudósok krt, 1117 Budapest, Hungary.
  • 2. Medicinal Chemistry Research Group, HUN-REN Research Centre for Natural Sciences, 2 Magyar tudósok krt, 1117 Budapest, Hungary; National Drug Discovery and Development Laboratory, HUN-REN Research Centre for Natural Sciences, 2 Magyar tudósok krt, 1117 Budapest, Hungary; Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, 4 Szt. Gellért tér, 1111 Budapest, Hungary.
  • 3. Medicinal Chemistry Research Group, HUN-REN Research Centre for Natural Sciences, 2 Magyar tudósok krt, 1117 Budapest, Hungary; National Drug Discovery and Development Laboratory, HUN-REN Research Centre for Natural Sciences, 2 Magyar tudósok krt, 1117 Budapest, Hungary; MS Metabolomics Research Laboratory, HUN-REN Research Centre for Natural Sciences, 2 Magyar tudósok krt, 1117 Budapest, Hungary.
  • 4. National Drug Discovery and Development Laboratory, HUN-REN Research Centre for Natural Sciences, 2 Magyar tudósok krt, 1117 Budapest, Hungary; Plasma Chemistry Research Group, HUN-REN Research Centre for Natural Sciences, 2 Magyar tudósok krt, 1117 Budapest, Hungary.
  • 5. Department of Chemistry, University College London, 20 Gordon Street, London WC1H OAJ, UK.
  • 6. Department of Chemistry, University College London, 20 Gordon Street, London WC1H OAJ, UK; Stanford Sarafan ChEM-H and Department of Chemistry, Stanford University, Stanford, CA 94305, USA. Electronic address: [email protected].
  • 7. Medicinal Chemistry Research Group, HUN-REN Research Centre for Natural Sciences, 2 Magyar tudósok krt, 1117 Budapest, Hungary; National Drug Discovery and Development Laboratory, HUN-REN Research Centre for Natural Sciences, 2 Magyar tudósok krt, 1117 Budapest, Hungary; Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, 4 Szt. Gellért tér, 1111 Budapest, Hungary. Electronic address: [email protected].
PMID: 42019503 DOI: 10.1016/j.crmeth.2026.101416
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.

Keywords
CP: biotechnology; antibody; bioconjugation; bioorthogonal chemistry; bispecific antibody; click chemistry; reaction optimization.
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