Study of CAR T cells and mechanisms of resistance in pediatric brain tumors

Division Of Basic Sciences - Nci

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Abstract

I have demonstrated that there is significantly increased infiltration of CD11b+ mouse myeloid cells into the EPN microenvironment after HER2 CAR T cell treatment compared to control CD19 CAR T cells seen by immunohistochemistry. Flow cytometry evaluation revealed a significantly higher population of CD11b+, Ly6C hi cells, defined as monocytic MDSCs, after HER2 CAR treatment in the tumor compared to control CAR T cells. We have demonstrated consistency of this finding in two EPN models and have also used another highly prevalent childhood brain tumor, medulloblastoma, as a comparison model that is much more sensitive to HER2 CAR T cell killing in vivo. Chemokine pathways are co-opted by various tumors to recruit myeloid cells from the bone marrow to establish an immunosuppressive tumor microenvironment. Posterior fossa EPN (811 and 928) release significantly higher amounts of CXCL1, CXCL8, CCL2, and CX3CL1 compared to other cancers in response to CAR T cell therapy in vitro. Each of these chemokines, CXCL1, CXCL8, CCL2, and CX3CL1, alone can potently attract myeloid cells to the TME. This observed chemokine elevation in EPN treated with HER2 CAR T cell therapy implicated the CXCR2, CCR2, and CX3CR1 chemokine pathways in the infiltration of this immunosuppressive population. Further study of ependymoma tumor samples harvested from mice showed a significantly higher percent of monocytic MDSCs with CCR2 surface expression after HER2 CAR T cell treatment compared to control treatment. There was no statistically significant difference in CXCR2 or CX3CR1 expressing myeloid cells after HER2 CAR T cell treatment compared to control treatment. In vivo experiments have demonstrated significant release of the chemokine CCL2 in response to HER2 CAR T cell treatment by EPN tumor cells and this chemokine can mediate infiltration of the immunosuppressive population identified within the tumor via the CCR2 receptor. I hypothesize that infiltrating monocytic MDSCs contribute to the limited effectiveness of CAR T cell therapy in posterior fossa ependymoma. We have recently demonstrated through In vitro studies that TNF-a activates the canonical NF-kB pathway and subsequent secretion of CCL2 that drives monocyte infiltration in PFA Ependymoma after CAR T treatment. We have also performed in vivo evaluation of a CCR2 inhibitor plus HER2 CAR T cells in 2 patient derived xenograft models in conjunction with the University of Colorado and have demonstrated synergy in this treatment approach. We are completing analysis of multi-color immunofluorescence imaging of PFA ependymoma xenografts after treatment to demonstrate changes in immune cell infiltration with CCR2 inhibition. Manuscript preparation is underway for a planned submission in Fall, 2022.