SpyMask enables combinatorial assembly of bispecific binders

Claudia L. Driscoll, Anthony H. Keeble and Mark R. Howarth ()
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Claudia L. Driscoll: University of Oxford
Anthony H. Keeble: University of Oxford
Mark R. Howarth: University of Oxford

Nature Communications, 2024, vol. 15, issue 1, 1-17

Abstract: 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.

Date: 2024
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DOI: 10.1038/s41467-024-46599-9

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