Pooled screening for CAR function identifies novel IL13Rα2-targeted CARs for treatment of glioblastoma

Chimeric antigen receptor therapies have demonstrated potent efficacy in treating B cell malignancies, but have yet to meaningfully translate to solid tumors. Here, we utilize our pooled screening platform, CARPOOL, to expedite the discovery of CARs with anti-tumor functions necessary for solid tumor efficacy. We performed selections in primary human T cells expressing a library of 1.3×106 3rd generation CARs targeting IL13Rα2, a cancer testis antigen commonly expressed in glioblastoma. Selections were performed for cytotoxicity, proliferation, memory formation, and persistence upon repeated antigen challenge. Each enriched CAR robustly produced the phenotype for which it was selected, and one enriched CAR triggered potent cytotoxicity and long-term proliferation upon in vitro tumor rechallenge. It also showed significantly improved persistence and comparable antigen-specific tumor control in a microphysiological human in vitro model and a xenograft model of human glioblastoma. Taken together, this work demonstrates the utility of extending CARPOOL to diseases beyond hematological malignancies and represents the largest exploration of signaling combinations in human primary cells to date. ### Competing Interest Statement The library approach described in this paper is the subject of a US patent application (PCT/US2020/017794) with M.E.B. as an inventor. A.P. is a member of the scientific advisory board and equity holder of AIM Biotech Pte. Ltd. M.E.B. is a founder, consultant, and equity holder of Kelonia Therapeutics and Abata Therapeutics. Khloe Gordon is currently employed at Ginkgo Bioworks, Inc. The other authors declare no competing interests.