Rationalizing Enhanced Affinity of Engineered T‐Cell Receptors in Cancer Immunotherapy Through Interaction Energy Calculations and Residue Correlation Analysis

ABSTRACT The advancement of T cell engineering has significantly transformed the field of cancer immunotherapy. In particular, T cells equipped with modified T cell receptors present a promising therapeutic strategy, especially for addressing solid tumors. Nonetheless, critical obstacles, including suboptimal clinical response rates, off‐target toxicity, and the immunosuppressive nature of the tumor microenvironment, have impeded the full clinical implementation of this approach. Understanding the molecular determinants governing the interaction between T‐cell receptors and major histocompatibility complex molecules is pivotal not only for designing TCRs capable of selectively and effectively recognizing MHC on cancer cells but also for minimizing off‐target toxicity, thereby improving the safety profile of TCR‐based therapies. In this study, we used a test case involving a natural TCR (c728) and its affinity‐enhanced variant (c796), which differ by a single conservative mutation in the region. Through molecular dynamics simulations, MM/PBSA binding energy and Free Energy Perturbation calculations, residue‐specific energy decomposition, and correlation analyses, we dissected the molecular basis of the engineered TCR's six‐fold increase in binding affinity for the peptide–MHC complex compared to its parental counterpart. Interestingly, our results indicate that this affinity enhancement is not directly attributable to the mutation itself but rather to the dynamic interplay of both proximal and distal residues that are either directly correlated with the mutation or connected via allosteric pathways. Our findings, which align with experimental data, highlight the nuanced role of structural flexibility and allosteric communication in shaping TCR‐pMHC interactions. By demonstrating the utility of combining computational techniques to unravel these dynamics, this work emphasizes how similar approaches can guide the rational design of engineered TCRs with improved efficacy and specificity, advancing their application in cancer immunotherapy.