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Revealing the impact of CD70 expression on the manufacture and functions of CAR-70 T-cells based on single-cell transcriptomics

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Cancer Immunology, Immunotherapy Aims and scope Submit manuscript

Abstract

Background

Chimeric antigen receptor-modified T cells (CAR T-cells) have shown exhilarative clinical efficacy for hematological malignancies. However, a shared antigen pool between healthy and malignant T-cells remains a concept to be technically and clinically explored for CAR T-cell therapy in T-cell cancers. No guidelines for engineering CAR T-cells targeting self-expressed antigens are currently available.

Method

Based on anti-CD70 CAR (CAR-70) T-cells, we constructed CD70 knock-out and wild-type CAR (CAR-70KO and CAR-70WT) T-cells and evaluated their manufacturing and anti-tumor capability. Single-cell RNA sequencing and TCR sequencing were performed to further reveal the underlying differences between the two groups of CAR T-cells.

Results

Our data showed that the disruption of target genes in T-cells before CAR transduction advantaged the expansion and cell viability of CAR T-cells during manufacturing periods, as well as the degranulation, anti-tumor efficacy, and proliferation potency in response to tumor cells. Meanwhile, more naïve and central memory phenotype CAR+ T-cells, with higher TCR clonal diversity, remained in the final products in KO samples. Gene expression profiles revealed a higher activation and exhaustion level of CAR-70WT T-cells, while signaling transduction pathway analysis identified a higher level of the phosphorylation-related pathway in CAR-70KO T-cells.

Conclusion

This study evidenced that CD70 stimulation during manufacturing process induced early exhaustion of CAR-70 T-cells. Knocking-out CD70 in T-cells prevented the exhaustion and led to a better-quality CAR-70 T-cell product. Our research will contribute to good engineering CAR T-cells targeting self-expressed antigens.

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Availability of data and materials

Data were contained in the manuscript, and raw data for the scRNA-seq and scTCR-seq are available via HRA002418 at the National Genome Data Center (NGDC) (https://ngdc.cncb.ac.cn/).

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Acknowledgements

We gratefully thank Wei Mu for the instructions for manufacturing the CAR T-cells.

Funding

This work was supported by the National Key R&D Program of China (2021YFF0703704), National Natural Science Foundation of China (82270183, 32100527, and 82100241), Natural Sciences Foundation of Hubei Province of China (2020CFB790), and the Excellent Young Science Foundation Project of Tongji Hospital (No.2020YQ0012).

Author information

Author notes

  1. Jiali Cheng and Yuyan Zhao have contributed equally to this work.

Authors and Affiliations

  1. Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China

    Jiali Cheng, Xinyue Deng, Shengnan Ding & Xiaojian Zhu

  2. Center for Artificial Intelligence Biology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, 430074, Hubei, China

    Yuyan Zhao, Hui Hu & An-Yuan Guo

  3. Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China

    Ling Tang

  4. Department of Internal Medicine, Cleveland Clinic, Akron General, Akron, OH, 44307, USA

    Yuhao Zeng

  5. Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China

    Hui Hu

  6. Department of Hematology, Wuhan No.1 Hospital, Wuhan, 430030, China

    Qing Li

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  1. Jiali Cheng
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Contributions

A-YG, QL and XZ proposed the ideas, oversaw the whole project, and revised the manuscript. JC conducted the functional experiments and analyzed the related data. Y. Zhao and HH analyzed the data of scRNA and scTCR sequencing. LT and SD helped with the experiments and participated in the discussion. JC, Y. Zhao and Y. Zeng wrote the manuscript.

Corresponding authors

Correspondence to An-Yuan Guo, Qing Li or Xiaojian Zhu.

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Cheng, J., Zhao, Y., Hu, H. et al. Revealing the impact of CD70 expression on the manufacture and functions of CAR-70 T-cells based on single-cell transcriptomics. Cancer Immunol Immunother 72, 3163–3174 (2023). https://doi.org/10.1007/s00262-023-03475-7

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