Rational Protein Engineering to Enhance MHC-Independent T-cell Receptors

Chimeric antigen receptor (CAR)–based therapies have pioneered synthetic cellular immunity but remain limited in their long-term efficacy. Emerging data sug-gest that dysregulated CAR-driven T-cell activation causes T-cell dysfunction and therapeutic failure. To re-engage the precision of the endogenous T-cell response, we designed MHC-independent T-cell receptors (miTCR) by linking antibody variable domains to T-cell receptor constant chains. Using predictive modeling, we observed that this standard “cut and paste” approach to synthetic protein design resulted in myriad biochemical conflicts at the hybrid variable–constant domain interface. Through iterative modeling and sequence modifications, we developed structure-enhanced miTCRs which significantly improved receptor-driven T-cell function across multiple tumor models. We found that 41BB costimulation specifically prolonged miTCR T-cell persistence and enabled improved leu-kemic control in vivo compared with classic CAR T cells. Collectively, we have identified core features of hybrid receptor structure responsible for regulating function. Significance: Improving the durability of engineered T-cell immunotherapies is critical to enhancing efficacy. We used a structure-informed design to evolve improved miTCR function across several models. This work underscores the central role of synthetic receptor structure in T-cell function and provides a framework for improved receptor engineering.