Rational protein engineering to enhance MHC-independent T cell receptors

Chimeric antigen receptor (CAR)-based therapies have pioneered synthetic cellular immunity against cancer, however remain limited in their scope and long-term efficacy. Emerging data suggest that dysregulated CAR-driven T cell activation causes T cell dysfunction and therapeutic failure. To re-engage the endogenous T cell response, we designed hybrid MHC-independent T cell receptors (miTCRs) by linking antibody variable domains to TCR constant domains. While functional, we observed stark differences in miTCR-driven T cell function that were dependent on receptor orientation. Using predictive structural modeling, we observed significant biochemical conflicts at the hybrid variable-constant domain interface. To overcome this, we performed iterative sequence modifications and structural modeling to design a panel of miTCR variants predicted to have improved interface stability. Functional screening nominated a variant with superior efficacy to all other miTCRs as well as a standard CAR against high burdens of leukemia. ### Competing Interest Statement J.-F.C. and N.S. have submitted patent applications related to this work. N.S. is an inventor on patents related to adoptive cell therapies, held by Washington University and the University of Pennsylvania (some licensed to Novartis). Unrelated to this work, N.S. has served as a consultant for several companies involved in cell therapies and is a board member for Phoreus Biotech. J.F.D. receives research funding from Amphivena Therapeutics, NeoImmuneTech, Macrogenics, Incyte, Bioline Rx, Wugen; has equity ownership in Magenta Therapeutics, Wugen; consults for Incyte, RiverVest Venture Partners, hC Bioscience, Inc.; and is a board member for RiverVest Venture Partners, Magenta Therapeutics.