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Cytotoxic activity of anti-mucin 1 chimeric antigen receptor T cells expressing PD-1-CD28 switch receptor against cholangiocarcinoma cells

  • Kamonlapat Supimon
    Affiliations
    Siriraj Center of Research Excellence for Cancer Immunotherapy (SiCORE-CIT), Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

    Division of Molecular Medicine, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
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  • Thanich Sangsuwannukul
    Affiliations
    Siriraj Center of Research Excellence for Cancer Immunotherapy (SiCORE-CIT), Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

    Division of Molecular Medicine, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
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  • Jatuporn Sujjitjoon
    Affiliations
    Siriraj Center of Research Excellence for Cancer Immunotherapy (SiCORE-CIT), Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

    Division of Molecular Medicine, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
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  • Thaweesak Chieochansin
    Affiliations
    Siriraj Center of Research Excellence for Cancer Immunotherapy (SiCORE-CIT), Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

    Division of Molecular Medicine, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
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  • Mutita Junking
    Correspondence
    Correspondence: Asst. Prof. Mutita Junking, Mahidol University, Research Department, Siriraj Center of Research Excellence for Cancer Immunotherapy (SiCORE-CIT) and Division of Molecular Medicine, Faculty of Medicine Siriraj Hospital, Bangkok 10700, Thailand.
    Affiliations
    Siriraj Center of Research Excellence for Cancer Immunotherapy (SiCORE-CIT), Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

    Division of Molecular Medicine, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
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  • Pa-thai Yenchitsomanus
    Correspondence
    Correspondence: Asst. Prof. Mutita Junking, Mahidol University, Research Department, Siriraj Center of Research Excellence for Cancer Immunotherapy (SiCORE-CIT) and Division of Molecular Medicine, Faculty of Medicine Siriraj Hospital, Bangkok 10700, Thailand.
    Affiliations
    Siriraj Center of Research Excellence for Cancer Immunotherapy (SiCORE-CIT), Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

    Division of Molecular Medicine, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
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Published:November 14, 2022DOI:https://doi.org/10.1016/j.jcyt.2022.10.006

      Abstract

      Background aims

      Cholangiocarcinoma (CCA) is a lethal bile-duct cancer that is difficult to treat by current standard procedures. This drawback has prompted us to develop adoptive T-cell therapy for CCA, which requires an appropriate target antigen for binding of chimeric antigen receptor (CAR) T cells. Mucin 1 (MUC1), an overexpressed protein in CCA cells, is a potential target antigen for the CAR T-cell development. However, MUC1 overexpression also is associated with the upregulation of programmed death-ligand 1 (PD-L1), an immune checkpoint protein that prohibits anti-tumor functions of T cells, probably causing poor overall survival of patients with CCA.

      Methods

      To overcome this problem, we developed anti-MUC1-CAR T cells containing PD-1-CD28 switch receptor (SR), namely αM.CAR/SR T cells, to target MUC1 and switch on the inhibitory signal of PD-1/PD-L1 interaction to activate CD28 signaling. Our lentiviral construct contains the sequences that encode anti-MUC1-single chain variable fragment, CD137 and CD3ζ, linked with P2A, PD-1 and CD28.

      Results

      Initially, the upregulations of MUC1 and PD-L1 proteins were confirmed in CCA cell lines. αM.CAR and SR were co-expressed in 53.53 ± 13.89% of transduced T cells, mainly CD8+ T cells (85.7 ± 0.75%, P<0.0001) with the effector memory phenotype (59.22 ± 16.31%, P < 0.01). αM.CAR/SR T cells produced high levels of intracellular tumor necrosis factor-α and interferon-γ in response to the activation by CCA cells expressing MUC1, including KKU-055 (27.18 ± 4.38% and 27.33 ± 5.55%, respectively, P < 0.05) and KKU-213A (47.37 ± 12.67% and 54.55 ± 8.66%, respectively, P < 0.01). Remarkably, the cytotoxic function of αM.CAR/SR T cells against KKU-213A cells expressing PD-L1 was significantly enhanced compared with the αM.CAR T cells (70.69 ± 14.38% versus 47.15 ± 8.413%, respectively; P = 0.0301), correlated with increased granzyme B production (60.6 ± 9.89% versus 43.2 ± 8.95%, respectively; P = 0.0402). Moreover, the significantly enhanced disruption of KKU-213A spheroids by αM.CAR/SR T cells (P = 0.0027), compared with αM.CAR T cells, was also observed.

      Conclusion

      Taken together, the cytotoxic function of αM.CAR/SR T cells was enhanced over the αM.CAR T cells, which are potential to be further tested for CCA treatment.

      Graphical abstract

      Key Words

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