Development and preclinical validation of a TRBC1-targeted CAR-T therapy for T-cell malignancies

T-cell malignancies are among the most challenging areas in hematological oncology, as the close immunophenotypic similarity between malignant and healthy T cells has long hindered the development of safe and specific targeted therapies. Chimeric antigen receptor (CAR) T-cell approaches face similar limitations, since targeting pan-T-cell antigens leads to fratricide during manufacturing and life-threatening T-cell aplasia in vivo. The mutually exclusive expression of the T-cell receptor β-chain constant regions TRBC1 and TRBC2 offers a unique solution. By selectively targeting one variant, it is theoretically possible to eliminate malignant clones while preserving half of the normal T-cell repertoire. This thesis describes the preclinical development and functional validation of CAR-T-One, a second-generation CAR-T therapy targeting TRBC1⁺ malignancies. In vitro studies demonstrated potent, antigen-dependent cytotoxicity against TRBC1⁺ tumor models (Jurkat T-ALL and H9 TCL), sustained serial killing, and complete resistance of TRBC2⁺ lines (SUDHL-1, SUP-T1). Importantly, CAR-T-One maintained strict antigen selectivity, sparing TRBC2⁺ cells in mixed cultures without evidence of bystander toxicity. Single-cell functional imaging confirmed efficient killing of malignant TRBC1⁺ targets with preservation of TRBC2⁺ cells, while healthy TRBC1⁺ cells showed context-dependent susceptibility, also observed in non-transduced controls. Analysis of CAR dynamics revealed stable expression and no evidence of cis/trans masking or functional impairment due to trogocytosis. Notably, CAR-transduced tumor cells remained susceptible to killing, addressing concerns about malignant transduction during manufacturing. In vivo xenograft models further supported therapeutic efficacy. In minimal residual disease settings, CAR-T-One induced deep tumor clearance and prolonged survival in most animals, while in high-tumor-burden models it achieved consistent, though temporary, complete remissions. Overall, these findings establish TRBC1-targeted CAR-T-One cells as a selective and effective immunotherapeutic strategy, providing a strong preclinical foundation for clinical translation in T-cell malignancies.