# SpyMask enables combinatorial assembly of bispecific binders

## TL;DR

SpyMask is a new method to assemble bispecific antibodies using a modular system, allowing for precise control over their structure and activity.

## Contribution

SpyMask introduces a modular and scalable system for bispecific antibody assembly using SpyTag/SpyCatcher chemistry.

## Key findings

- SpyMask enables one-pot assembly and purification of bispecifics in 96-well plates.
- Anti-HER2 bispecifics assembled with SpyMask show activity dependent on binder orientation and geometry.
- Different DoubleCatcher scaffolds produce bispecifics with anti- or pro-proliferative effects.

## 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.

Bispecific antibody architecture is often important for function but rarely optimized. Here, authors present a modular approach to assemble bispecifics in varied formats using a SpyTag/SpyCatcher approach called SpyMask, and build anti-HER2 bispecifics whose activities depend on binder orientation and bispecific geometry.