Background:Existing treatments for cholangiocarcinoma have poor efficacy.However,chimeric antigen receptor-T(CAR-T)cells are emerging as a potential therapeutic strategy.Solid tumors possess multiple adverse factors i...Background:Existing treatments for cholangiocarcinoma have poor efficacy.However,chimeric antigen receptor-T(CAR-T)cells are emerging as a potential therapeutic strategy.Solid tumors possess multiple adverse factors in an immunosuppressive microenvironment that impair CAR-T cell infiltration and function.This study aimed to improve the function of CAR-T cells through knock down immune checkpoints and immunosuppressive molecular receptors.Methods:We evaluated the expression of epidermal growth factor receptor(EGFR)and B7 homolog 3 protein(B7H3)antigens in cholangiocarcinoma tissues using immunohistochemistry and screened specific immune checkpoints in the cholangiocarcinoma microenvironment via flow cytometry.Subsequently,we engineered CAR-T cells targeting EGFR and B7H3 antigens.We simultaneously knocked down immune checkpoints and immunosuppressive molecular receptors in CAR-T cells by constructing two clusters of small hairpin RNAs and evaluated the engineered CAR-T cells for antitumor activity both in vitro,using tumor cell lines and cholangiocarcinoma organoid models,and in vivo,using humanized mouse models.Results:We observed high expression of EGFR and B7H3 antigens in cholangiocarcinoma tissues.EGFR-CAR-T and B7H3-CAR-T cells demonstrated specific anti-tumor activity.We found an abundance of programmed cell death protein 1(PD-1),T cell immunoglobulin and mucin domain-containing protein 3(Tim-3),and T cell immunoglobulin and ITIM domain(Tigit)on infiltrated CD8^(+)T cells in the cholangiocarcinoma microenvironment.We then decreased the expression of these 3 proteins on the surface of CAR-T cells,named PTG-scFV-CAR-T cells.Furthermore,we knocked-down the expression of transforming growth factor beta receptor(TGFβR),interleukin-10 receptor(IL-10R),and interleukin-6 receptor(IL-6R)of PTG-scFV-CAR-T cells.Those cells,named PTG-T16R-scFVCAR-T cells,potently killed tumor cells in vitro and promoted apoptosis of tumor cells in a cholangiocarcinoma organoidmodel.Finally,the PTG-T16R-scFv-CART cells showed greater inhibitory effect on tumor growth in vivo,and were superior in prolonging the survival of mice.Conclusions:Our results revealed that PTG-T16R-scFV-CAR-T cells with knockdown of sextuplet inhibitory molecules exhibited strong immunity against cholangiocarcinoma and long-term efficacy both in vitro and in vivo.This strategy provides an effective and personalized immune cell therapy against cholangiocarcinoma.展开更多
基金Guangzhou Science and Technology Innovation development Special fund,Grant/Award Number:202102020386Basic and Applied Basic Research Fund Committee of Guangdong Province,Grant/Award Numbers:2021A1515111209,2022A1515010547+1 种基金Science Foundation of Guangdong Provincial Bureau of traditional Chinese Medicine,Grant/Award Number:20211090National Natural Science Foundation of China,Grant/Award Numbers:82103331,82171825,82202036。
文摘Background:Existing treatments for cholangiocarcinoma have poor efficacy.However,chimeric antigen receptor-T(CAR-T)cells are emerging as a potential therapeutic strategy.Solid tumors possess multiple adverse factors in an immunosuppressive microenvironment that impair CAR-T cell infiltration and function.This study aimed to improve the function of CAR-T cells through knock down immune checkpoints and immunosuppressive molecular receptors.Methods:We evaluated the expression of epidermal growth factor receptor(EGFR)and B7 homolog 3 protein(B7H3)antigens in cholangiocarcinoma tissues using immunohistochemistry and screened specific immune checkpoints in the cholangiocarcinoma microenvironment via flow cytometry.Subsequently,we engineered CAR-T cells targeting EGFR and B7H3 antigens.We simultaneously knocked down immune checkpoints and immunosuppressive molecular receptors in CAR-T cells by constructing two clusters of small hairpin RNAs and evaluated the engineered CAR-T cells for antitumor activity both in vitro,using tumor cell lines and cholangiocarcinoma organoid models,and in vivo,using humanized mouse models.Results:We observed high expression of EGFR and B7H3 antigens in cholangiocarcinoma tissues.EGFR-CAR-T and B7H3-CAR-T cells demonstrated specific anti-tumor activity.We found an abundance of programmed cell death protein 1(PD-1),T cell immunoglobulin and mucin domain-containing protein 3(Tim-3),and T cell immunoglobulin and ITIM domain(Tigit)on infiltrated CD8^(+)T cells in the cholangiocarcinoma microenvironment.We then decreased the expression of these 3 proteins on the surface of CAR-T cells,named PTG-scFV-CAR-T cells.Furthermore,we knocked-down the expression of transforming growth factor beta receptor(TGFβR),interleukin-10 receptor(IL-10R),and interleukin-6 receptor(IL-6R)of PTG-scFV-CAR-T cells.Those cells,named PTG-T16R-scFVCAR-T cells,potently killed tumor cells in vitro and promoted apoptosis of tumor cells in a cholangiocarcinoma organoidmodel.Finally,the PTG-T16R-scFv-CART cells showed greater inhibitory effect on tumor growth in vivo,and were superior in prolonging the survival of mice.Conclusions:Our results revealed that PTG-T16R-scFV-CAR-T cells with knockdown of sextuplet inhibitory molecules exhibited strong immunity against cholangiocarcinoma and long-term efficacy both in vitro and in vivo.This strategy provides an effective and personalized immune cell therapy against cholangiocarcinoma.
