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A novel safer CD19CAR with shRNA interference of IFN-γ can reduce multiple cytokine levels without significantly compromising its killing efficacy

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Abstract

Cytokine release syndrome (CRS) is a great challenge for the application of anti-CD19 CAR-T cell therapy. The aim of this study was to investigate the effect of knocking down interferon gamma (IFN-γ) by shRNA as a potential strategy to reduce the cytokine storms. A newly designed short hairpin interference RNA of IFN-γ (shIFN-γ) in CD19CAR gene was constructed. Several cellular model systems of approach using Nalm-6 cell lines including Nalm-6CD19pos and Nalm-6CD19neg with or without monocytes and endothelial cells were used to analyze the different levels of cytokines after shIFN-γ-anti-CD19CAR-T cell targeted therapy. The activity of this novel CD19CAR-T was evaluated both in vitro and in NSG mouse model. The killing efficacy of shIFN-γ-anti-CD19CAR-T at the E:T ratio of 2:1 was similar to that of regular anti-CD19CAR-T at the E:T ratio of 1:1. The IFN-γ level in the shIFN-γ-anti-CD19CAR-T cell group was (2673.1 ± 307.4) pg/ml at the E:T ratio of 2:1 which was significantly lower than that ((8261.5 ± 345.5) pg/ml) in the regular anti-CD19CAR-T group at the E:T ratio of 1:1. Cytotoxicity experiments in vitro showed significantly reduced concentrations of IFN-γ, IL-6 and TNFα in the shIFN-γ-anti-CD19CAR-T cell group compared to regular anti-CD19CAR-T cell group. Both regular anti-CD19CAR and shIFN-γ-CD19CAR-T exerted bystander killing effect in vitro. We conclude that shIFN-γ-anti-CD19CAR-T cells can reduce the generation of cytokine storms without significantly compromising their therapeutic efficacy in the preclinical setting. In mouse model, 3 × 106 shIFN-γ-anti-CD19CAR-T cells/mouse generated the similar killing efficacy to that with 2 × 106 regular anti-CD19CAR-T cells/mouse.

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Data Availability

Data will be made available on request.

Abbreviations

CRS:

Cytokine release syndrome

IFN-γ:

Interferon‐gamma

shIFN-γ:

short hairpin interference RNA of IFN-γ

CAR-T:

Chimeric antigen receptor T cells

R/R B-ALL:

relapsed/refractory B-lineage acute lymphoblastic leukemia

IL-6:

interleukin 6

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Acknowledgements

Authors would like to thank all the staffs from the department/center of hematology-oncology and laboratory for their technical support.

Funding

This work was supported in part by grants from the National Natural Science Foundation of China (grant no. 81770202 and 81470304), Zhejiang Medical and Health Science and Technology Plan Project (No. 2022KY868), the Key Project from Science and Technology Department of Zhejiang Province (No. 2019C03032), Pediatric Leukemia Diagnostic and Therapeutic Technology Research Center of Zhejiang Province (No. JBZX-201904), Zhejiang Medical and Health Science and Technology Plan Project (No. 2022KY1051) and the Scientific Research Foundation of Zhejiang University City College (J-202103).

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Author notes

  1. Ping Zhang and Peiting Ying are contributed equally to this work.

Authors and Affiliations

  1. Department/Center of Hematology-oncology, Children’s Hospital of Zhejiang University School of Medicine, Hangzhou, PR China

    Ping Zhang, Peiting Ying, Hongzhe Li, Ning Zhao, Rongrong Liu, Weiqun Xu & Yongmin Tang

  2. Pediatric Leukemia Diagnostic and Therapeutic Technology Research Center of Zhejiang Province, National Clinical Research Center for Child Health, #57 Zhuganxiang Road, Yan-an Street, Hangzhou, 310003, PR China

    Ping Zhang, Peiting Ying, Hongzhe Li, Ning Zhao, Rongrong Liu, Weiqun Xu & Yongmin Tang

  3. Department of Clinical Medicine, School of Medicine, Hangzhou City University, No. 50, Huzhou Street, Gongshu District, Hangzhou, Zhejiang Province, 310015, PR China

    Sisi Li

  4. Department of Colorectal Surgery and Oncology (Key Laboratory of Cancer Prevetion and Intervention, China National Ministry of Education, Key Laboratory of Molecular Biology in Medical Sciences), The Second Affiliated Hospital of Zhejiang University School of Medicine, No. 88 Jiefang Road, Hangzhou, Zhejiang Province, 310009, PR China

    Yang Tang

  5. Zhejiang Provincial Clinical Research Center for CANCER, No. 88 Jiefang Road, Hangzhou, 310009, China

    Yang Tang

  6. Cancer Center of Zhejiang University, No. 88 Jiefang Road, Hangzhou, 310009, China

    Yang Tang

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  1. Ping Zhang
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Contributions

P.Z. and P.Y. are co-first authors of this article who did experiments and performed data analysis. P.Z. wrote the manuscript draft. Y.T., H.L. and N.Z. did part of experiments. R.L .and S.L. assisted in completing the statistical analysis. Y.T. designed the work and amended the manuscript. Y.T. assisted in amending the manuscript.

Corresponding authors

Correspondence to Yang Tang or Yongmin Tang.

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This study was approved by the Institutional of animal care and use committee, ZJCLA. (ID :ZJCLA-IACUC-20030115)

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The authors declare no competing interests.

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Zhang, P., Ying, P., Li, H. et al. A novel safer CD19CAR with shRNA interference of IFN-γ can reduce multiple cytokine levels without significantly compromising its killing efficacy. Apoptosis 29, 556–567 (2024). https://doi.org/10.1007/s10495-023-01925-2

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