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Integration of humanized ROBO1 CAR in PD-1 locus in natural killer cells delivers synergistic tumor-killing effect against non-small cell lung cancer

Cancer Gene Therapy (2025)Cite this article

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Abstract

Lung cancer is the most common cancer and one of the leading causes of cancer-related deaths in the world, however, the treatment of non-small cell lung cancer (NSCLC) is still limited, and it is a clinically urgent problem. ROBO1 is an important surface receptor on tumor cells, but the role of humanized chimeric antigen receptor (CAR) modified natural killer (NK) cells targeting ROBO1 in NSCLC is rarely explored. Furthermore, the role of PD-1 in NK cell killing tumor cells remains controversial. In this study, we identified the expression pattern of ROBO1 in lung squamous cell carcinoma (LUSC) by searching biological information databases. We constructed hROBO1-CAR-NK-92 cells and performed functional identification.We inserted the hROBO1-CAR at the PD-1 locus and performed functional detection in vitro and in vivo. The results showed that ROBO1 expression was significantly increased in LUSC. After inserting the hROBO1-CAR sequence at the PD-1 locus, the PD-1-KO-hROBO1-CAR-NK-92 cells had the best long-term killing ability and cytokine secretion ability, and had a significant inhibitory effect on tumor growth in the mouse xenograft model. We also observed that the long-term killing ability of PD-1-KO-hROBO1-CAR-NK-92 cells was achieved by inhibiting cell senescence via knocking out PD-1. These studies proposed ROBO1 as a key target for CAR-NK therapy in NSCLC and integrated hROBO1 CAR in PD-1 locus in NK cells, resulting in synergistic tumor killing effects in NSCLC, presenting a new treatment strategy for solid tumor treatment.

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Fig. 1: Humanization and specificity detection of ROBO1 antibody.
Fig. 2: Construction, identification, and characterization of hROBO1-CAR-NK cells.
Fig. 3: hROBO1-CAR-NK-92 cells induce cell lysis of human solid tumor cell lines in vitro.
Fig. 4: Knocking in the CAR at the PD-1 locus enhanced long-term lysis and cytokine release of hROBO1-CAR-NK-92.
Fig. 5: PD-1-KO-hROBO1-CAR-NK-92 cells inhibited tumor growth in vivo in the Xenotransplantation model of lung cancer cells.
Fig. 6: PD-1-KO reduces the H1299 cells-induced cell senescence of hROBO1-CAR-NK-92 cells.
Fig. 7: PD-1 KO reduces SASP components, but not apoptosis of hROBO1-CAR-NK-92 cells induced by PMA.
Fig. 8: Inhibition of PD-1 enhances the killing effect of hROBO1 on NSCLC cells.

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Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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Funding

This study was supported by the National Natural Science Foundation of China (82241053), Fundamental Research Funds for the Central Universities of Central South University (2020zzts772), Hunan Provincial Innovation Foundation for Postgraduate (CX20220321), and Postgraduate Innovative Project of Central South University (2021XQLX51).

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Authors and Affiliations

  1. Department of Physiology, Xiangya School of Basic Medical Science, Central South University, Changsha, Hunan, China

    Jia-Hao Tao, Wen-Jing Zhong, Chen-Yu Zhang, Yu-Biao Liu, Jin Ling, Yong Zhou & Cha-Xiang Guan

  2. Ascle Therapeutics, Suzhou, Jiangsu, China

    Jia-Hao Tao, Jun Zhang & Hua-Shun Li

  3. Changsha Jinyu Medical Laboratory, Changsha, Hunan, China

    Chun-Yan Tang

  4. Department of Geriatrics, Respiratory Medicine, Xiangya Hospital, Central South University, Changsha, Hunan, China

    Jia-Xi Duan

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  1. Jia-Hao Tao
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  2. Jun Zhang
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  4. Jia-Xi Duan
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  5. Wen-Jing Zhong
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Contributions

JHT, JZ, HSL, YZ and CXG conceptualized and designed this study. JHT performed most of the experiments, and J.Z performed partial experiments. JZ, CYT, JXD, CYZ, and YBL executed the acquisition and analysis of data. JHT, JZ and LJ prepared figures, performed statistical analysis, and wrote the manuscript. JHT, JZ, HSL,YZ, CXG, JXD, CYZ, YBL and LJ provided administrative, technical, or material support. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Jun Zhang, Hua-Shun Li, Yong Zhou or Cha-Xiang Guan.

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The experimental use of mice in the present study was approved by the Ethics Committee of the Institute of Clinical Pharmacology of Central South University (No. 2021-XMSB-0194) to keep with the guidelines of the National Institutes of Health.

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Tao, JH., Zhang, J., Tang, CY. et al. Integration of humanized ROBO1 CAR in PD-1 locus in natural killer cells delivers synergistic tumor-killing effect against non-small cell lung cancer. Cancer Gene Ther (2025). https://doi.org/10.1038/s41417-025-00957-x

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