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SpyMask Enables Combinatorial Assembly of Bispecific Binders

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Driscoll, Claudia L  ORCID logo
Keeble, Anthony 


Bispecific antibodies are a successful and expanding therapeutic class, bridging two cell-types or engaging two different molecules on the same cell. 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. Assembly on DoubleCatcher is efficient in phosphate-buffered saline at 37 °C, with half-times less than 5 min for both SpyCatcher arms and over 98% bispecific purity. 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 plate format. 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 the geometry of DoubleCatcher scaffolds. 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.



Epitopes, Antibodies, Bispecific, Peptide Hydrolases

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Nature Communications

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Nature Portfolio
Biotechnology and Biological Sciences Research Council (BB/S007369/1)
Biotechnology and Biological Sciences Research Council (BB/R000344/1)
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