CAR-T therapies to treat T-cell malignancies face unique hurdles. Normal and malignant T cells usually express the same target for CAR, leading to fratricide. CAR-T cells targeting CD7, which is expressed in various malignant T cells, have limited expansion due to fratricide. Using CRISPR/Cas9 to knockout CD7 can reduce the fratricide. Here we developed a 2-in-1 strategy to insert EF1α-driven CD7-specific CAR at the disrupted CD7 locus and compared it to two other known strategies: one was random integration of CAR by a retrovirus and the other was site-specific integration at T-cell receptor alpha constant (TRAC) locus, both in the context of CD7 disruption. All three types of CD7 CAR-T cells with reduced fratricide could expand well and displayed potent cytotoxicity to both CD7+ tumor cell lines and patient-derived primary tumors. Moreover, EF1α-driven CAR expressed at the CD7 locus enhances tumor rejection in a mouse xenograft model of T-cell acute lymphoblastic leukemia (T-ALL), suggesting great clinical application potential. Additionally, this 2-in-1 strategy was adopted to generate CD7-specific CAR-NK cells as NK also expresses CD7, which would prevent contamination from malignant cells. Thus, our synchronized antigen-knockout CAR-knockin strategy could reduce the fratricide and enhance anti-tumor activity, advancing clinical CAR-T treatment of T-cell malignancies.
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The authors thank the support of Zhejiang Provincial Key Laboratory of Immunity and Inflammatory Diseases, and thank Yueting Xin, Chun Guo, Jiajia Wang, Yingying Huang from the core facilities, Zhejiang University School of Medicine for their technical support.
This research was funded by the National Key R&D Program of China 2021YFA0909900 (JS), 2018YFA0800102 (YG), the National Natural Science Foundation of China grants 31971324 (JS), 31970555 (YG), 82161138028 (JS), 81973993 (XG), Zhejiang Provincial Natural Science Foundation grant LR20H160003 (JS), LR20C070001 (XG) and the Leading Innovative and Entrepreneur Team Introduction Program of Zhejiang grant 2020R01006 (JS, YG).
The authors declare no competing interests.
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Jiang, J., Chen, J., Liao, C. et al. Inserting EF1α-driven CD7-specific CAR at CD7 locus reduces fratricide and enhances tumor rejection. Leukemia 37, 1660–1670 (2023). https://doi.org/10.1038/s41375-023-01948-3