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Dual-function chimeric antigen receptor T cells targeting c-Met and PD-1 exhibit potent anti-tumor efficacy in solid tumors

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Summary

Purpose Programmed cell death 1 (PD-1), which is upregulated under the continuous induction of the tumor microenvironment, causes chimeric antigen receptor (CAR)-T cell hypofunction via interaction with programmed death ligand 1 (PD-L1). This study aimed to construct CAR-T cells that are resistant to PD-1 inhibition to improve the effect of CAR-T cells in solid tumors. Methods We constructed a type of dual-function CAR-T cell that targets tumor-associated antigen c-Met and blocks the binding of PD-1 with PD-L1. The expression of c-Met, PD-L1, and inhibitory receptors was measured using flow cytometry. The cytotoxicity, cytokine release, and differentiation level of CAR-T cells were determined using lactate dehydrogenase release assay, enzyme-linked immunosorbent assay, and flow cytometry, respectively. The levels of p-Akt, p-MAPK, caspase-3, and Bcl2 were detected by western blot. The in vivo anti-tumor effect was evaluated using tumor xenograft models. Results Dual-function CAR-T cells could mediate enhanced active signals upon encountering target antigens and had targeted cytotoxicity to target cells. However, the cytotoxicity of c-Met-CAR-PD-1+ T cells was impaired due to the interaction of PD-1 with PD-L1. By blocking the binding of PD-1 and PD-L1, the novel dual-function CAR-PD-1+ T cells could maintain cytotoxicity to PD-L1+ tumor cells. In tumor tissue, the dual-function CAR-T cells showed lower inhibitory receptor expression and lower differentiation characteristics, which resulted in potent anti-tumor effects and prolonged survival in PD-L1+ tumor xenograft models compared to single-target CAR-T cells. Conclusion These results confirm that the novel dual-function CAR-T cells exhibit stronger anti-tumor activity against solid tumors than traditional single-target CAR-T cells and present a new approach that enhance the activity of CAR-T cells in solid tumors.

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Acknowledgments

We thank Yao Wang from Key Laboratory of Medical Molecular Virology, Ministry of Education and Public Health, School of Basic Medical Sciences, Fudan University for help in flow cytometry.

Funding

This study was funded by the National Key Research Project Bio-safety Key Technology Development Program 2016YFC1201501 and the National Natural Science Foundation of China, No. 31671228.

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Authors and Affiliations

  1. The Key Laboratory of Metabolism and Molecular Medicine, The Ministry of Education and the Department of Biochemistry and Molecular Biology, School of Basic Medicine, Fudan University, P.O. Box #238, No. 138 Yi Xue Yuan Road, Shanghai, China

    Xingxing Yuan, Zujun Sun, Qingyun Yuan, Weihua Hou, Qiaoyan Liang, Yuxiong Wang, Wei Mo & Min Yu

  2. Department of Clinical Laboratory, Shanghai Tongji Hospital, Tongji University School of Medicine, Shanghai, 200065, China

    Zujun Sun

  3. Department of Medical Oncology, Fudan University Shanghai Cancer Center; Department of Oncology, Shanghai Medical College, Fudan University, No. 270 Dong An Road, Shanghai, China

    Huijie Wang

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  1. Xingxing Yuan
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Contributions

All authors contributed to the study conception and design. Min Yu, Wei Mo, Huijie Wang, Zujun Sun and Xingxing Yuan designed the study. Xingxing Yuan performed all the material preparation, experiments and wrote the manuscript. Qingyun Yuan, Qiaoyan Liang, Weihua Hou and Yuxiong Wang assisted with all in vitro experiments. Qingyun Yuan contributed to the in vivo experiments. Xingxing Yuan performed data collection, analysis and interpretation. The first draft of the manuscript was written by Xingxing Yuan and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Huijie Wang or Min Yu.

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All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. All procedures performed in studies involving animals were in accordance with the ethical standards of the department of laboratory animal science of Fudan university and permitted on March 6, 2019.

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ESM 1

Supplementary Fig. 1 The detection of c-Met and PD-L1 in tumor cell lines. (a) The expression of c-Met in each tumor cell line detected by western blot. (b) Flow cytometric analysis of the expression of c-Met in four tumor cell lines. (c) Flow cytometry histogram of PD-L1 expression in four tumor cell lines and PD-L1 up-regulation after stimulation with IFN-γ (40 μg/mL) for 8 h. Supplementary Fig. 2 Representative flow cytometry contour plots of the proportion of CAR-T cells expressing PD-1, LAG-3, and TIM-3 in each group on days 8, 16, and 24 during long-term stimulation. Supplementary Fig. 3 Analysis of tumor-infiltrating CAR-T cells. (a) Quantification of the proportions of perforin+ and granzyme B+ cells within the tumor-infiltrating CAR-T cells of each group 30 days after treatment with CAR-T cells analyzed by flow cytometry (n = 3, mean ± SD, ****P < 0.0001 by one-way ANOVA). (b) Quantification of the inhibitory receptors (PD-1, LAG-3, and TIM-3) on the tumor-infiltrating CAR-T cells 30 days after treatment with CAR-T cells (n = 3, mean ± SD, n.s.: not significant, ***P < 0.001 by one-way ANOVA). (c) Pie charts summarizing the proportion of CD45RA+CD62L+ cells in the tumor-infiltrating CAR-T cells 15 and 30 days after treatment with CAR-T cells (top). Quantification of the proportion of CD62L+CD45RA+ cells in the tumor-infiltrating CAR-T cells (bottom) (n = 3, mean ± SD, ****P < 0.0001 by two-way ANOVA). (d) The frequency of CD8+ cells in tumor-infiltrating CAR-T cells extracted from each group 15 and 30 days after treatment (top). Quantification of the percentages of CD8+ T cells (bottom) (n = 3, mean ± SD, ***P < 0.001, ****P < 0.0001 by two-way ANOVA). (e) The cytotoxicity (left) and IFN-γ secretion (right) of the tumor-infiltrating CAR-T cells in each group were detected in comparison with the original c-Met-PD-1-CAR-T cells (n = 3, mean ± SD, ****P < 0.0001 by two-way ANOVA) (PDF 33429 kb)

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Yuan, X., Sun, Z., Yuan, Q. et al. Dual-function chimeric antigen receptor T cells targeting c-Met and PD-1 exhibit potent anti-tumor efficacy in solid tumors. Invest New Drugs 39, 34–51 (2021). https://doi.org/10.1007/s10637-020-00978-3

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