Enhancement of CAR-T cell activity against cholangiocarcinoma by simultaneous knockdown of six inhibitory membrane proteins

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.

Infection of wild-type mice by SARS-CoV-2 B.1.351 variant indicates a possible novel cross-species transmission route

COVID-19 is identified as a zoonotic disease caused by SARS-CoV-2,which also can cross・transmit to many animals but not mice.Genetic modifications of SARS-CoV-2 or mice enable the mice susceptible to viral infection.Although neither is the natural situation,they are currently utilized to establish mouse infection models.Here we report a direct contact transmission of SARS-CoV-2 variant B.1.351 in wild-type mice.The SARS-CoV-2(B.1.351)re plicated efficiently and induced significant pathological changes in lungs and tracheas,accompanied by elevated proinflammatory cytokines in the lungs and sera.Mechanistically,the receptor-binding domain(RBD)of SARS-CoV-2(B.1.351)spike protein turned to a high binding affinity to mouse angiotensin-converting enzyme 2(mACE2),allowing the mice highly susceptible to SARS-CoV-2(B.1.351)infection.Our work suggests that SARS-CoV-2(B.1.351)expands the host range and therefore increases its transmission route without adapted mutation.As the wild house mice live with human populations quite closely,this possible transmission route could be potentially risky.In addition,because SARS-CoV-2(B.1.351)is one of the major epidemic strains and the mACE2 in laboratory-used mice is naturally expressed and regulated,the SARS-CoV-2(B.1.351)/mice could be a much convenient animal model system to study COVID-19 pathogenesis and evaluate antiviral inhibitors and vaccines.