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.

Moderate expression of CD39 in GPC3-CAR-T cells shows high efficacy against hepatocellular carcinoma

CD39 serves as a crucial biomarker for neoantigen-specific CD8^(+)T cells and is associated with antitumor activity and exhaustion.However,the relationship between CD39 expression levels and the function of chimeric antigen receptor T(CAR-T)cells remains controversial.This study aimed to investigate the role of CD39 in the functional performance of CAR-T cells against hepatocellular carcinoma(HCC)and explore the therapeutic potential of CD39 modulators,such as mitochondrial division inhibitor-1(mdivi-1),or knockdown CD39 through short hairpin RNA.Our findings demonstrated that glypican-3-CAR-T cells with moderate CD39 expression exhibited a strong antitumor activity,while high and low levels of CD39 led to an impaired cellular function.Methods modulating the proportion of CD39 intermediate(CD39^(int))-phenotype CAR-T cells such as mdivi-1 and CD39 knockdown enhanced and impaired T cell function,respectively.The combination of mdivi-1 and CD39 knockdown in CAR-T cells yielded the highest proportion of infiltrated CD39^(int)CAR-T cells and demonstrated a robust antitumor activity in vivo.In conclusion,this study revealed the crucial role of CD39 in CAR-T cell function,demonstrated the potential therapeutic efficacy of combining mdivi-1 with CD39 knockdown in HCC,and provided a novel treatment strategy for HCC patients in the field of cellular immunotherapy.

Anti-PD-L1 antibody enhances curative effect of cryoablation via antibody-dependent cell-mediated cytotoxicity mediating PD-L1^(high)CD11b^(+)cells elimination in hepatocellular carcinoma

Cryoablation(CRA)and microwave ablation(MWA)are two main local treatments for hepatocellular carcinoma(HCC).However,which one is more curative and suitable for combining with immunotherapy is still controversial.Herein,CRA induced higher tumoral PD-L1 expression and more T cells infiltration,but less PD-L1^(high)CD11b^(+)myeloid cells infiltration than MWA in HCC.Furthermore,CRA had better curative effect than MWA for anti-PD-L1 combination therapy in mouse models.Mechanistically,anti-PD-L1 antibody facilitated infiltration of CD8^(+)T cells by enhancing the secretion of CXCL9 from cDC1 cells after CRA therapy.On the other hand,anti-PD-L1 antibody promoted the infiltration of NK cells to eliminate PD-L1^(high)CD11b^(+)myeloid cells by antibody-dependent cell-mediated cytotoxicity(ADCC)effect after CRA therapy.Both aspects relieved the immunosuppressive microenvironment after CRA therapy.Notably,the wild-type PD-L1 Avelumab(Bavencio),compared to the mutant PD-L1 atezolizumab(Tecentriq),was better at inducing the ADCC effect to target PD-L1^(high)CD11b^(+)myeloid cells.Collectively,our study uncovered the novel insights that CRA showed superior curative effect than MWA in combining with anti-PD-L1 antibody by strengthening CTL/NK cell immune responses,which provided a strong rationale for combining CRA and PD-L1 blockade in the clinical treatment for HCC.

Coating with flexible DNA network enhanced T-cell activation and tumor killing for adoptive cell therapy

Adoptive cell therapy(ACT)is an emerging powerful cancer immunotherapy,which includes a complex process of genetic modification,stimulation and expansion.During these in vitro or ex vivo manipulation,sensitive cells are inescapability subjected to harmful external stimuli.Although a variety of cytoprotection strategies have been developed,their application on ACT remains challenging.Herein,a DNA network is constructed on cell surface by rolling circle amplification(RCA),and T cell-targeted trivalent tetrahedral DNA nanostructure is used as a rigid scaffold to achieve high-efficient and selective coating for T cells.The cytoprotective DNA network on T-cell surface makes them aggregate over time to form cell clusters,which exhibit more resistance to external stimuli and enhanced activities in human peripheral blood mononuclear cells and liver cancer organoid killing model.Overall,this work provides a novel strategy for in vitro T cell-selective protection,which has a great potential for application in ACT.