Abstact

The development of anti-CD3 antibody-based T cell engager therapeutics has improved the treatment of various malignancies, yet the challenge of achieving tumor-specific targeting while minimizing on-target off-tumor effects in normal tissues remains a substantial hurdle. One promising strategy to address this issue involves engineering antibodies with conditional pH-dependent binding affinities, capitalizing on the acidic microenvironment characteristics of tumors (pH ~ 6.5-6.8) compared to the neutral pH of healthy tissues (pH ~ 7.4). In this study, we focus on the pH-engineering of antibody binders against the human CD3 antigen, a critical component of T cell activation, to achieve preferential binding at acidic pH. Using molecular dynamics (MD) simulations on the reported CD3varepsilon antibody binder 40G5c, we shed light on possible molecular mechanisms of the pH-responsiveness of key mutations and their impact on the overall binder structure at physiological or acidic pH. Our study highlights how MD has emerged as a powerful tool to guide and explain intrinsic pH-dependent molecular mechanisms in antibody engineering. Lastly, we report that our engineered CD3 binders preferentially bind and activate T cells under acidic pH conditions and display favorable affinity and pH-window profiles.