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IMMUNOTHERAPY

Inserting EF1α-driven CD7-specific CAR at CD7 locus reduces fratricide and enhances tumor rejection

Leukemia volume 37, pages 1660–1670 (2023)Cite this article

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Abstract

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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Fig. 1: Potent cytotoxicity coincided with fratricide of CD7 CAR-T cells.
Fig. 2: A 2-in-1 strategy to produce expandable and functional CD7KO CAR-T cells.
Fig. 3: Anti-tumor activity of CD7KO CAR-T cells against primary T-ALL blasts.
Fig. 4: CD7KO CAR-T cells control the progression of T-ALL in the mouse xenograft model.
Fig. 5: The 2-in-1 strategy generated CAR-NK cells with anti-tumor activity.

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Acknowledgements

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.

Funding

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).

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Authors and Affiliations

  1. Bone Marrow Transplantation Center of the First Affiliated Hospital and Department of Cell Biology, Zhejiang University School of Medicine, 866 Yuhangtang Road, Hangzhou, 310058, China

    Jie Jiang, Jiangqing Chen, Yanting Duan, Yajie Wang, Kai Shang & Jie Sun

  2. Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121, China

    Jie Jiang, Jiangqing Chen, Yanting Duan, Yajie Wang, Kai Shang, Ying Gu & Jie Sun

  3. Institute of Hematology, Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, 310058, Zhejiang, China

    Jie Jiang, Jiangqing Chen, Yanting Duan, Yajie Wang, Kai Shang & Jie Sun

  4. Department of Hematology-oncology, Children’s Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China

    Chan Liao & Yongming Tang

  5. Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, 310058, China

    Yanjie Huang & Xiaofei Gao

  6. Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education), the Second Affiliated Hospital, Zhejiang University, School of Medicine, Hangzhou, Zhejiang, 310009, China

    Ying Gu

  7. Institute of Genetics, Zhejiang University and Department of Genetics, Zhejiang University, School of Medicine, Hangzhou, 310058, China

    Ying Gu

  8. Zhejiang Provincial Key Lab of Genetic and Developmental Disorder, Hangzhou, Zhejiang, 310058, China

    Ying Gu

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Contributions

JJ and JS designed the studies and conceived the experiments. JJ, JC, and YW performed most of the experiments; CL, DY, KS conducted data analysis; YH, YT, and XG contributed reagents. JJ, YG, and JS wrote the manuscript. YG and JS supervised the study. All authors read and approved the final manuscript.

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Correspondence to Ying Gu or Jie Sun.

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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

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