SpyMask enables combinatorial assembly of bispecific binders

Journal article

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

Authors: Driscoll, CL; Keeble, AH; Howarth, MR

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Nature Research
• Journal: Nature Communications
• Volume: 15
• Issue: 1
• Pages: 2403
• Article number: 2403
• Publication date: 2024-03-16
• DOI: 10.1038/s41467-024-46599-9
• EISSN: 2041-1723
• ISSN: 2041-1723
• Language: English
• Keywords: Immunology; Scaffold; Disulfide bond; Modular design; Computational biology; Chemistry; Computer science; Antibody; Protease; Nanotechnology; Monoclonal antibody; Antigen; Enzyme; Tandem; Bispecific antibody; Combinatorial chemistry; Materials science; Epitope; Biochemistry; Biology; Database