Molecular Tailoring of Bispecific Antibody–BottlebrushConjugates - Bispecific antibodies (bsAbs) have emerged as a transformative class of biologic therapeutics that enable targeting of multiple antigens or pathways, generating potent and highly selective therapeutic responses. Despite significant advances in bsAb formats, the overall tunability of bsAb structure remains limited and the impact of spatial configuration between binding sites, e.g., distance and orientation, on efficacy remains underexplored. We hypothesize that optimal targeting of different antigens within the same or multiple cell types will depend on the inter-binding-domain distances between antibody components of bsAbs. Testing this hypothesis requires a new synthetic route to bsABC-like molecules with controlled, rigid spacing between two antibody binding domains, which could be achieved by linking together two antibodies with a rigid macromolecular “ruler.” This project aims to develop synthetic methods to access such systems, using bottlebrush polymer s(BPs) as the rulers for constructing bsAbs with defined lengths and access to length scales not possible with traditional bsAbs. By leveraging single-chain variable fragment (scFv) technology, precise polymerization, and advanced conjugation techniques, a family of bispecific “Antibody–Bottlebrush Conjugates (bsABCs) will be synthesized, incorporating adjustable bottlebrush scaffolds and scFv species to investigate the effects of inter-antigen distance and targeting specificity on cell targeting and immune recruitment. Variables such as ABC length and aspect ratio will be tuned to identify their impacts on cell binding and activation using a suite of in vitro assays. Additionally, synthetic methods for installing small molecule imaging fluorophore into the bottlebrush domains of bsABCs will be developed; these fluorophores will enable imagining of bsABC cellular uptake and their syntheses will provide guidelines for future drug- carrying bsABCs. Finally, building on these learnings, a synthetic strategy for accessing trispecific antibody– bottlebrush conjugates (tsABCs) is proposed, wherein three unique binding domains from antibodies are linked together through a macromolecular core. Comprehensive in vitro studies will evaluate the impacts of molecular structure on the function of bsABCs and tsABCs, deepening the understanding of the importance of spatial configuration in multi-functional antibody–conjugate design and providing design principles for bsAb formats in future clinical applications. It is expected that precise control over antibody architecture and structure will improve pharmacokinetics, reduce dosing requirements, and enhance T cell-tumor cell interactions, ultimately leading to safer and more effective disease treatments.
