新型 CD4 特异性 CAR-NK 细胞疗法治疗 T 细胞恶性肿瘤的临床前评价

T-cell leukemias and lymphomas (TCL) are a rare and heterogeneous group of cancers with limited treatment options and an overall poor prognosis. In contrast to B-cell malignancies, the development of cellular therapies for TCL has not yet resulted in an approved product. The development of autologous CAR T cell approaches is challenging because of the shared antigens between the malignant T lymphocytes and healthy T cells, that can lead to fratricide, product contamination, manufacturing limitations, and T-cell aplasia.

To address these limitations, we developed CD4-specific chimeric antigen receptor (CAR) natural killer (NK) cells as an off-the-shelf immunotherapeutic strategy. CD4 CAR NK cells were generated from peripheral blood via retroviral transduction, and cytotoxicity was tested against 17 TCL cell lines representing six TCL subtypes. Additionally, RNA sequencing was performed to profile NK cell ligand expression across these cell lines. We further characterized CD4 CAR NK cells, cultivated in gas-permeable G-Rex culture plates, and assessed their killing capacities against primary patient-derived TCL samples.

CD4 CAR NK cells showed CAR expression levels of up to 60% and demonstrated natural and specific cytotoxicity across T-cell malignancy cell lines. NK ligand analysis of the cell lines highlights how the balance of inhibitory versus activating signals shapes both natural and CAR-mediated responses. Using gas-permeable G-Rex culture plates, we developed a standardized manufacturing protocol, achieving over 15-fold expansion by day 14 and over 50-fold by day 21 post-isolation. Cells maintained high viability, transduction efficiency, and cytotoxic functionality, and exhibited potent activity against primary patient-derived TCL samples.

Together, these findings demonstrate the feasibility, scalability, and therapeutic potential of CD4-directed CAR NK cells in T-cell malignancies, supporting their further development towards clinical application.