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SpyMask enables combinatorial assembly of bispecific binders

Author

Listed:
  • Claudia L. Driscoll

    (University of Oxford
    University of Cambridge)

  • Anthony H. Keeble

    (University of Oxford
    University of Cambridge)

  • Mark R. Howarth

    (University of Oxford
    University of Cambridge)

Abstract

Bispecific antibodies are a successful and expanding therapeutic class. Standard approaches to generate bispecifics are complicated by the need for disulfide reduction/oxidation or specialized formats. Here we present SpyMask, a modular approach to bispecifics using SpyTag/SpyCatcher spontaneous amidation. Two SpyTag-fused antigen-binding modules can be precisely conjugated onto DoubleCatcher, a tandem SpyCatcher where the second SpyCatcher is protease-activatable. We engineer a panel of structurally-distinct DoubleCatchers, from which binders project in different directions. We establish a generalized methodology for one-pot assembly and purification of bispecifics in 96-well plates. A panel of binders recognizing different HER2 epitopes were coupled to DoubleCatcher, revealing unexpected combinations with anti-proliferative or pro-proliferative activity on HER2-addicted cancer cells. Bispecific activity depended sensitively on both binder orientation and DoubleCatcher scaffold geometry. These findings support the need for straightforward assembly in different formats. SpyMask provides a scalable tool to discover synergy in bispecific activity, through modulating receptor organization and geometry.

Suggested Citation

  • Claudia L. Driscoll & Anthony H. Keeble & Mark R. Howarth, 2024. "SpyMask enables combinatorial assembly of bispecific binders," Nature Communications, Nature, vol. 15(1), pages 1-17, December.
  • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-46599-9
    DOI: 10.1038/s41467-024-46599-9
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