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CRISPR/Cas9-mediated PD-1 disruption enhances human mesothelin-targeted CAR T cell effector functions

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Abstract

The interaction between programmed cell death protein 1 (PD-1) on activated T cells and its ligands on a target tumour may limit the capacity of chimeric antigen receptor (CAR) T cells to eradicate solid tumours. PD-1 blockade could potentially enhance CAR T cell function. Here, we show that mesothelin is overexpressed in human triple-negative breast cancer cells and can be targeted by CAR T cells. To overcome the suppressive effect of PD-1 on CAR T cells, we utilized CRISPR/Cas9 ribonucleoprotein-mediated editing to disrupt the programmed cell death-1 (PD-1) gene locus in human primary T cells, resulting in a significantly reduced PD-1hi population. This reduction had little effect on CAR T cell proliferation but strongly augmented CAR T cell cytokine production and cytotoxicity towards PD-L1-expressing cancer cells in vitro. CAR T cells with PD-1 disruption show enhanced tumour control and relapse prevention in vivo when compared with CAR T cells with or without αPD-1 antibody blockade. Our study demonstrates a potential advantage of integrated immune checkpoint blockade with CAR T cells in controlling solid tumours and provides an alternative CAR T cell strategy for adoptive transfer therapy.

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Abbreviations

4-1BB TNF:

Receptor superfamily member 9

CRISPR/Cas9:

sgRNA-guided clustered regularly interspaced short palindrome repeats-associated nuclease Cas9

Meso:

Mesothelin

RFP:

Red fluorescence protein

RNP:

Ribonucleoprotein

T7E1:

T7 endonuclease I

Tim-3:

T cell immunoglobulin and mucin-domain-containing-3

TNBC:

Triple-negative breast cancer

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Acknowledgements

We are grateful to the blood donors for their contribution; Zhouluo Ou for providing the breast cancer cell lines used in this study; Ilya Vinnikov for proofreading the manuscript; and Wei Zhang and Huan Wang for their helpful suggestions.

Funding

This work was supported by the National Natural Science Foundation of China (81402542) and the scholarship of Pujiang Talents in Shanghai to Fang Wei (14PJ1405600).

Author information

Author notes

  1. Wanghong Hu, Zhenguo Zi and Yanling Jin contributed equally.

Authors and Affiliations

  1. Sheng Yushou Center of Cell Biology and Immunology, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Minghang, Shanghai, 200240, China

    Wanghong Hu, Zhenguo Zi, Yanling Jin, Gaoxin Li, Xiaojing Ma & Fang Wei

  2. Affiliated Renji Hospital, Shanghai Jiao Tong University, Shanghai, China

    Kang Shao

  3. MOE and MOH Key Laboratory of Medical Molecular Virology, School of Basic Medicine, Shanghai Medical College, Fudan University, Shanghai, China

    Qiliang Cai

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  1. Wanghong Hu
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Contributions

All authors made substantial contributions to the conception and design of this work. WH, ZZ and YJ performed the experiments, analysed the data and wrote the manuscript. KS contributed to blood donor communication and sample collection. GL contributed to the molecular biology experiments. QC and XM proofread the manuscript and gave intellectual suggestions. FW contributed to the conception and design, data analysis and interpretation, and manuscript writing and provided financial support. All authors viewed and approved the final version of the manuscript.

Corresponding author

Correspondence to Fang Wei.

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Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical approval

For the use of blood samples from healthy donors, written informed consent was obtained in accordance with the of the Shanghai Jiao Tong Human Sample Committee on March 1, 2014. The mouse study was carried out in accordance with the recommendations of East China Normal University Animal Care guidelines from the East China Normal University Animal Care Committee. All experimental protocols were approved on August 1, 2016, by the East China Normal University Animal Care Committee.

Animal source

Six-week-old female NOD-Prkdcscid IL2rgnull (NSG) mice were purchased from Vitalstar, China, and housed in ventilated cages in our pathogen-free facility.

Cell line authentication

The human cell lines MCF7, MDA-MB-231, BT-549, Hs 578T and HeLa were kindly provided by Zouluo Ou (Fudan University, Shanghai, China). The cell lines were authenticated by the Chinese Academy of Sciences Committee Type Culture Collection Cell Bank.

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Hu, W., Zi, Z., Jin, Y. et al. CRISPR/Cas9-mediated PD-1 disruption enhances human mesothelin-targeted CAR T cell effector functions. Cancer Immunol Immunother 68, 365–377 (2019). https://doi.org/10.1007/s00262-018-2281-2

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  • DOI: https://doi.org/10.1007/s00262-018-2281-2

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