Tuning antibody stability and function by rational designs of framework mutations

Artificial intelligence and machine learning models have been developed to engineer antibodies for specific recognition of antigens, however these approaches often focus on the antibody complementarity determining region (CDR) whilst ignoring the immunoglobulin framework (FW) which provides structural rigidity and support for the flexible CDR loops. Here we present an integrated computational-experimental workflow, combining static structure analyses, molecular dynamics simulations and in vitro physicochemical and functional assays to generate rational designs of FW mutations for modulating antibody stability and activity. We first showed that recent antibody-specific language models lacked insights in FW mutagenesis, in comparison to approaches which utilised antibody structure information. Using the widely-used breast cancer therapeutic trastuzumab as a use case, we designed stabilising mutants which were distal to the CDR and preserved the antibody's functionality to engage its cognate antigen (HER2) and induce antibody-dependent cellular cytotoxicity (ADCC). Interestingly, guided by local backbone motions predicted using molecular dynamics simulations, we designed a FW mutation on the trastuzumab light chain which retained antigen-binding effects but lost Fab-mediated and Fc-mediated effector functions. This highlighted effects of FW on immunological functions engendered in distal areas of the antibody, and the importance to consider attributes other than binding affinity when assessing antibody function. Our approach incorporates interdomain dynamics and distal effects between FW and the Fc domains, expands the scope of antibody engineering beyond the CDR, and underscores the importance of a holistic perspective that considers the entire antibody structure as a whole in optimising antibody stability, developability and function.