SpyMask Enables Combinatorial Assembly of Bispecific Binders

Bispecific antibodies are a successful and expanding therapeutic class, bridging two cell-types or engaging two different molecules on the same cell. Bispecifics unlock avenues towards synergy, resistance evasion, and signaling bias. Standard approaches to generate bispecifics are complicated by the need for disulfide reduction/oxidation or cloning of each binder molecule in two different formats. Here we present a modular approach to bispecifics using SpyTag/SpyCatcher spontaneous amidation, where all binders are cloned in the same format, bearing a SpyTag. Two SpyTag-fused antigen-binding modules can be precisely conjugated onto DoubleCatcher, a tandem SpyCatcher where the second Catcher is unreactive until unveiling of reactivity using site-specific protease. Assembly on DoubleCatcher is efficient in phosphate-buffered saline at 37 {degrees}C, with half-times less than 5 min for both SpyCatcher arms and over 97% bispecific homogeneity. We engineer a panel of DoubleCatchers, locked through disulfide bonds to direct binders to project in different directions from the hub. We establish a generalized methodology for one-pot assembly and purification of bispecifics in 96-well plate format. A panel of Fab, affibody or nanobody binders recognizing different sites on HER2 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 the order of the binders within the assembly and the geometry of DoubleCatcher scaffolds. These findings support the need for straightforward assembly in different formats. SpyMask provides a simple and scalable tool to discover synergy in bispecific activity, through modulating receptor organization and geometry.