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Generating potent and persistent antitumor immunity via affinity-tuned CAR-T cells targeting mesothelin

Acta Pharmacologica Sinica volume 46, pages 2849–2862 (2025) Cite this article

Abstract

Chimeric antigen receptor (CAR)-T cell therapy for solid tumors faces challenges of insufficient efficacy and a high recurrence rate. Mesothelin (MSLN) is a membrane glycoprotein highly expressed in various solid tumors that has restricted low expression in normal tissues such as the pleura, peritoneum, and pericardium. We previously performed affinity maturation based on the parental antibody M912, and constructed the phage display library. In this study we identified four novel human anti-MSLN antibodies (LP12, HP4-11, HP4-41/LP6, and HP4-44/LP2) with varying degrees of enhanced affinity. These third-generation CARs targeting MSLN were packaged into lentiviral vectors to generate stable CAR-T cells. The CAR-T variants induced robust cytolytic activity, significant cytokine production, and activation-induced clonal proliferation against various MSLN-positive tumors in vitro, and effectively cleared disseminated tumors in mice. A single administration of the CAR-T variant LP12 potently eradicated various types of MSLN-positive solid tumors, achieved long-term persistence in vivo, effectively prevented tumor recurrence, and exhibited no non-specific toxicity. Therefore, optimizing the affinity of antigen-binding domain in CAR represents a promising strategy for advancing the development of safe and effective CAR-T cell therapies. The LP12 CAR-T cells developed in this study have potential applications in patients with MSLN-positive solid tumors.

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Schematic illustration of the generation and antitumor mechanism of affinity-tuned MSLN-targeted CAR-T cells. The expression plasmids carrying different anti-MSLN CAR genes were packaged into lentiviral vectors. Lentiviral transduction of human CD3+ T cells was performed to generate CAR-T cells, which were then expanded. After injection of moderately affinity-tuned MSLN-targeted CAR-T cells into mice, they enter the bloodstream, recognize, and infiltrate the solid tumors. They specifically recognize and bind to MSLN on the surface of tumor cells, and upon activation, release IFN-γ, IL-2, and TNF-α to exert cytolytic activity. Subsequently, they undergo clonal proliferation and primarily differentiate into effector memory CAR-T cells, maintaining long-term antitumor immunity and effectively preventing recurrence.

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Fig. 1: Generation and characterization of MSLN-targeted CAR-T cells and expression profile of human MSLN.
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Fig. 2: MSLN-targeted CAR-T variants exhibit significantly enhanced antigen-specific cytotoxicity and activation.
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Fig. 3: MSLN-targeted CAR-T variants effectively eradicate peritoneal disseminated tumors and prevent recurrence.
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Fig. 4: CAR-T variants LP12 and HP4-11 proliferate and persist in vivo to counter tumor recurrence.
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Fig. 5: CAR-T variants LP12 and HP4-11 did not exhibit non-specific toxicity during treatment.
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Fig. 6: CAR-T variant LP12 outperforms HP4-11 in eradicating large MSLN-positive solid tumors and preventing recurrence.
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Fig. 7: LP12 CAR-T cells effectively infiltrate and eradicate MSLN-positive orthotopic pancreatic solid tumors.
The alternative text for this image may have been generated using AI.

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Acknowledgements

This research was supported by the National Natural Science Foundation of China (Grant Nos. 81773621 and 82073751 to JW Zhu), and the National Science and Technology Major Project “Key New Drug Creation and Manufacturing Program” of China (Grant No. 2019ZX09732001-019 to JW Zhu). We thank Jecho Laboratories and Jecho Biopharmaceuticals for their valuable technical support.

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Authors and Affiliations

  1. School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China

    Ya-li Yue, Jun-jun Liu, Hang Ma, Lei Wang, Jia-wei Zhang, Yan-lin Bian, Ming-yuan Wu, Yun-sheng Yuan, Bao-hong Zhang & Jian-wei Zhu

  2. Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, Shanghai, 200240, China

    Ya-li Yue, Jun-jun Liu, Hang Ma, Lei Wang, Jia-wei Zhang, Yan-lin Bian, Ming-yuan Wu, Yun-sheng Yuan, Bao-hong Zhang & Jian-wei Zhu

  3. Jecho Institute Co., Ltd, Shanghai, 200240, China

    Zhi-di Pan & Jian-wei Zhu

  4. Jecho Laboratories, Inc, Frederick, MD, 21704, USA

    Shu-sheng Wang, Yue-qing Xie, Hua Jiang, Xiao-dong Xiao & Jian-wei Zhu

  5. Jecho Biopharmaceuticals Co., Ltd, Tianjin, 300467, China

    Jian-wei Zhu

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Contributions

JWZhu, YLY, and HM conceptualized and designed the study. YLY and HM performed molecular cloning and plasmid construction. YLY, JJL, ZDP, and JWZhang performed in vitro assays. YLY, JJL, and LW carried out animal experiments. YLY, JJL, and ZDP analyzed the experimental data. YLY, JWZhu, XDX, HJ, YQX, SSW, YLB, BHZ, MYW, and YSY contributed to writing, original draft, review, and editing. All authors critically reviewed the manuscript.

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Correspondence to Bao-hong Zhang, Xiao-dong Xiao or Jian-wei Zhu.

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Competing interests

ZDP is an employee of Jecho Institute Co., Ltd. (Shanghai, China). SSW, YQX, HJ, and XDX are employees of Jecho Laboratories, Inc. (Frederick, MD, USA). No potential conflicts of interest were disclosed by the other authors.

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Yue, Yl., Liu, Jj., Ma, H. et al. Generating potent and persistent antitumor immunity via affinity-tuned CAR-T cells targeting mesothelin. Acta Pharmacol Sin 46, 2849–2862 (2025). https://doi.org/10.1038/s41401-025-01572-0

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