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Chimeric antigen receptor clustering via cysteines enhances T-cell efficacy against tumor

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Abstract

Chimeric antigen receptor (CAR) T-cell therapy achieves great success for hematological malignancies. However, clinical trials have revealed some limitations in both improving the efficacy and reducing the relapse, which calls for innovative strategies to engineer more powerful CAR-T cells. Promoting the formation of CAR clusters provides an alternative approach and potentially improves current CAR T-cell therapy against cancers. Here, we generated CARCys-T cells using a 4-1BB-derived hinge region including 11 cysteines residues. The cysteines in the hinge were found to facilitate CARCys clustering upon antigen stimulation and promote the antitumor activity of CAR-T cells. Compared with most conventionally used CAR-T cells with CD8α-derived hinge (CARconv-T cells), CARCys-T cells exhibited larger diameter of CAR clusters and enhanced antigen-specific tumor lysis both in vitro and in vivo. In addition, the CARCys-mediated enhancement could be applied to HER2, CD19 as well as GPC3-targeted CAR-T cells. More importantly, CARCys-T cells showed potent antitumor efficacy in clinically relevant patient-derived primary tumor cells and organoids. Thus, the novel hinge containing 11 cysteines provides a promising strategy to facilitate CAR clustering and maximize anti-tumor activity of CAR-T cells, which emphasizes the importance of CAR clustering to improve CAR T-cell therapy in the clinic.

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Acknowledgements

The authors thank National Center for Protein Science Shanghai (NCPSS) for STORM system, Prof. Yanhui Xu (Fudan University), Prof. Chenqi Xu (University of Chinese Academy of Sciences) and Ms. Yang Yu (NCPSS) for valuable suggestions, Dr. Yufei Zhang of Olympus (Beijing) sales & service co., Ms. Yao Li of NCPSS, Ms. Shenglan Gu of International Peace Maternity and Child Health Hospital for excellent technical assistance.

Funding

This work was supported by the National Natural Science Foundation of China (82121002, 82130050, 81870375), Shanghai Rising-Star Program (18QA1401000), Open Research Fund of State Key Laboratory of Genetic Engineering, Fudan University (SKLGE1911), and Shenzhen Institute of Synthetic Biology Scientific Research Program (ZTXM20214002). SA is a Senior Clinical Investigator of the Research Fund-Flanders (FWO) (Belgium). GR is supported by a Doctoral Grant Strategic Basic Research of the FWO (Grant 1S72821N) and the MeToYou Foundation (Belgium).

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Authors and Affiliations

  1. Department of Immunology, School of Basic Medical Sciences, Biotherapy Research Center, and Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, China

    Yuedi Wang, Yiyuan Gao, Congyi Niu, Jiawen Qian, Jingbo Qie, Lin Chen, Jie Liu, Feifei Luo & Yiwei Chu

  2. Department of Digestive Diseases, Huashan Hospital, Fudan University, Shanghai, 200040, China

    Yuedi Wang, Lin Chen, Jie Liu & Feifei Luo

  3. Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China

    Yuedi Wang

  4. Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, 100871, China

    Bo Wang

  5. Laboratory of Medical Molecular Virology of the Ministry of Health and Ministry of Education, School of Basic Medical Sciences, Fudan University, Shanghai, 200032, China

    Shushu Zhao

  6. Vaccine & Infectious Disease Institute, Laboratory of Experimental Hematology, University of Antwerp, Antwerp, Belgium

    Gils Roex & Sébastien Anguille

  7. Department of General Surgery, Huashan Hospital, Fudan University, Shanghai, 200040, China

    Chenhe Yi

  8. Division of Hematology and Center for Cell Therapy & Regenerative Medicine, Antwerp University Hospital, Antwerp, Belgium

    Sébastien Anguille

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  1. Yuedi Wang
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  2. Yiyuan Gao
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  3. Congyi Niu
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  9. Lin Chen
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Contributions

YC, FL, and YW designed the experiments. YW and FL wrote the manuscript. YC, FL, JL, SA, GR, and JQ revised the manuscript. YW, YG, CN, SZ, GR, LC, and CY performed the experiments and acquired the data. YW, FL, BW, JQ, and LC analyzed the data and prepared the figures. All authors provided intellectual input and read the manuscript.

Corresponding authors

Correspondence to Jie Liu, Feifei Luo or Yiwei Chu.

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The authors have no relevant financial or non-financial interests to disclose.

Ethical approval and ethical standards.

Human N87, Huh7, Ramos and K562 cell lines were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Science (Shanghai, China). Human peripheral blood mononuclear cells isolated from healthy donors or patients with hepatocellular carcinoma (HCC) were purchased from the Shanghai Blood Center (Shanghai, China), or obtained from Huashan hospital (Shanghai, China), respectively. Fresh human HCC samples were obtained from surgery performed at the Huashan hospital. Informed consent was obtained from HCC patients involved, and the project was approved by the Ethical Committee of Medical Research, Huashan Hospital of Fudan University (No. 2013-005). Six- to eight-week-old female nude mice (BALB/c) were purchased from SLAC (Shanghai, China) and kept under specific pathogen-free conditions in animal facility of Fudan University. The animal experimental protocol was approved by the Ethics Committee of Fudan University (201901003Z) following the Guidelines for the Care and Use of Laboratory Animals (No. 55 issued by Ministry of Health, China on January 25th, 1998).

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Wang, Y., Gao, Y., Niu, C. et al. Chimeric antigen receptor clustering via cysteines enhances T-cell efficacy against tumor. Cancer Immunol Immunother 71, 2801–2814 (2022). https://doi.org/10.1007/s00262-022-03195-4

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