使用IF-BETTER门控CAR T细胞增强实体瘤排斥以治疗肝细胞癌

CAR T cell therapy has demonstrated remarkable success in treating hematological malignancies but has been, to date, far less effective against solid tumors. Several common obstacles limiting the activity of CAR T cells in solid tumors have been identified, including low and/or heterogeneous target expression, insufficient T cell functional persistence, T cell exclusion and microenvironmental suppression. We address here the former, focusing on the targeting of glypican‑3 (GPC3) in hepatocellular carcinoma (HCC). GPC3 is a GPI-anchored protein that is commonly but heterogeneously expressed in HCC, ranging from of 10 4 to 10 2 molecules per cell in a panel of HCC tumor cell lines. Whereas conventional CARs are efficacious against tumors expressing several thousand GPC3 molecules per cell, they consistently fail in the lower range of GPC3 expression. To overcome this limitation, we adopted an IF-BETTER gating strategy by co-targeting a second antigen that is broadly expressed in order to increase CAR T cell avidity for the tumor and CAR T cell persistence, depending on the structure of the associated chimeric costimulatory receptor (CCR). In contrast to an OR gate, wherein two antigens are targeted by either two CARs or a dual-specific CAR, the second antigen in an IF-BETTER gate does not on its own elicit cytotoxicity and therefore does not have to be as restricted to tumor cells as the CAR target. We identified IL1RAP as a potential CCR target and paired the GPC3 CAR, incorporating the GC33 scFv and the 1XX signaling module to extend CAR T cell persistence, with CCRs providing different costimulatory signals. We investigated the paired GPC3-1XX CAR/IL1RAP CCRs in vitro and in vivo, targeting tumor cell lines with a range of GPC3 expression levels. The GPC3-1XX + IL1RAP‑CCR configuration preserved on‑target GPC3-restricted cytotoxicity while increasing CAR T cell proliferation and maintaining cytotoxicity after repetitive stimulations, in contrast to control CAR T cells lacking the CCR. In vivo, GPC3-1XX CAR + IL1RAP CCR T cells showed significantly improved tumor control, intratumoral infiltration and survival, most pronounced in the setting of low GPC3 expression. In summary, combining calibrated 1XX CAR signaling with IL1RAP‑gated costimulation increases GPC3 CAR T cell persistence and reduces antigen escape. These data support the clinical translation of an IF-BETTER gated strategy targeting GPC3 in hepatocellular carcinoma.