Construction of self-driving anti-αFR CAR-engineered NK cells based on IFN-γ and TNF-α synergistically induced high expression of CXCL10

Abstract

• In this study, we found for the first time that the third-generation αFR-CAR-NK92 effector cells targeting αFR successfully constructed in the previous stage of our research group can produce the chemokine CXCL10 after co-culture with ovarian cancer target cells, and no relevant studies have reported this phenomenon in CAR-T/NK cell therapy. • In ovarian cancer cells, we investigated the signaling pathway that TNF-α or/and IFN-γ induces the secretion of CXCL10, which was not been reported before. • In cancer cells, we studied the mechanism by which TNF-α and IFN-γ synergically induce the increase of CXCL10 secretion in cancer cells, which has not been reported before. • In this study, we used the CXCL10/CXCR3 axis for the first time to construct the fourth-generation CXCR3-αFR-CAR-NK92 cells that co-express the chemokine receptor CXCR3 • We verified the killing function and chemotaxis of CXCR3-αFR-CAR-NK92 cells targeting ovarian cancer cells through in vitro and in vivo experiments. • The CXCR3-αFR-CAR-NK92 cells constructed by us improved the immunotherapy efficacy of αFR-CAR-NK92 cells, providing a new strategy for immunotherapy of ovarian cancer. In particular, it is beneficial to personalized treatment for this patient group of high-grade serous ovarian cancer immune response type (that is, tumor tissue high expression of CXCL10), and to a certain extent, alleviate the difficulty of transport obstruction when CAR-NK92 cells treat solid ovarian cancer. Ovarian cancer is the most malignant gynecological tumor. Previous studies have demonstrated that chimeric antigen receptor (CAR)-engineered NK-92 cells targeting folate receptor α (αFR) (NK-92-αFR-CAR) can specifically kill αFR-positive ovarian cancer cells. However, the migration barrier restricts antitumor effects of CAR-engineered cells. To elucidate the mechanism by which NK-92-αFR-CAR cells induce the secretion of chemokine CXCL10 during killing ovarian cancer cells. It is speculated that NK-92-αFR-CAR-CXCR3A can target αFR and have chemotaxis of CXCL10, and they may have stronger killing effect of ovarian cancer. Study the mechanism of CXCL10 expression strongly induced by TNF-α and IFN-γ combined stimulation in ovarian cancer cells. Construct the fourth generation of NK-92-αFR-CAR-CXCR3A cells, which were co-expressed CXCR3A and αFR-CAR. Evaluate the killing and migration effects of NK-92-αFR-CAR-CXCR3A in vitro and in vivo. RNA sequencing (RNA-seq) first revealed that the expression level of the chemokine CXCL10 was most significantly increased in ovarian cancer cells co-cultured with NK-92-αFR-CAR. Secondly, cytokine stimulation experiments confirmed that IFN-γ and TNF-α secreted by NK-92-αFR-CAR synergistically induced high CXCL10 expression in ovarian cancer cells. Further signaling pathway experiments showed that IFN-γ and TNF-α enhanced the activation level of the IFN-γ-IFNGR-JAK1/2-STAT1-CXCL10 signaling axis. Cytotoxicity experiments showed that NK-92-αFR-CAR-CXCR3A cells could not only efficiently kill αFR-positive ovarian cancer cells in vitro but also secrete IFN-γ and TNF-α. Higher migration than that of NK-92-αFR-CAR was detected in NK-92-αFR-CAR-CXCR3A using transwell assay. NK-92-αFR-CAR-CXCR3A effectively killed tumor cells in different mouse xenograft models of ovarian cancer and increased infiltration into tumor tissue. This study confirmed that IFN-γ and TNF-α secreted by αFR-CAR-engineered NK cells can synergistically induce high expression of CXCL10 in ovarian cancer cells and constructed self-driving αFR-CAR-engineered NK cells that can break through migration barriers based on CXCL10, which may provide a new therapeutic weapon for ovarian cancer.

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