Tuning cell fate with a toolbox of DoubleCatcher bispecific binder scaffolds

Thesis

Molecular interactions underpin the regulatory networks of cells and biological systems. At the cell surface, differences in receptor-receptor interactions have profound consequences on cell behaviour and disease progression. Molecular bridges can be designed to replicate spatially sensitive biomolecular interactions, or to explore synergy arising from new interactions, and are emerging as promising tools in therapeutic discovery and synthetic biology. Bispecific binders, which bind simultaneously to two different targets, are an example of such a tool. Standard approaches for bispecific synthesis are limited by specialised binder formats or lack geometric tunability. Instead, I designed SpyMask: a platform for the modular, stepwise dimerisation of diverse ligands using SpyTag/SpyCatcher peptide-protein superglue. Mixing of SpyCatcher and SpyTag in solution results in spontaneous isopeptide bond formation between the pair. As such, SpyTag-bearing binders are covalently conjugated onto a tandem-SpyCatcher scaffold, here termed DoubleCatcher. To impart control into the platform, I engineered DoubleCatcher to possess one native, reactive Catcher and one whose reactivity is activated by a site-specific protease. I further designed a panel of DoubleCatcher scaffolds that are locked in distinct three-dimensional architectures, from which SpyTag-conjugated modules will project in different orientations. I assembled a matrix of anti-HER2 bispecific binders in parallel by coupling a panel of anti-HER2-binders to the DoubleCatcher toolbox in all possible permutations, using a simple and scalable protocol. Different modular combinations revealed anti-proliferative or pro-proliferative activity on HER2-addicted cancer cells. Crucially, activity depended on both the order of the binders within the assembly and the geometry of DoubleCatcher scaffolds. These findings emphasise the need for assembly and screening tools, such as SpyMask, for the combinatorial dimerisation of molecules in distinct geometries. Potential applications of SpyMask-discovered bispecifics range from interrogating cancer cell-surface receptor networks to fine-tuning chimeric antigen receptor-T cell responses.

Authors: Driscoll, CL

• DOI: 10.5287/ora-kzaq6vegm
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: bioconjugation; bispecific antibodies; antibody engineering; spycatcher/spytag; engineering biology; synthetic biology; protein biochemistry; protein engineering
• Subjects: Biochemistry; Chemical biology; Bispecific antibodies; Synthetic biology