Targeting novel negative regulators to improve CAR T cell therapy responses in solid tumors

While CAR T cell therapy has achieved unequivocal success in treating hematologic malignancies, solid tumors have consistently resisted this treatment. This highlights a crucial need to identify strategies that improve CAR T cell efficacy in solid tumors. To address this challenge, we performed in vivo CRISPR screens, targeting one quarter of the murine genome, to identify regulators of CD8+ T cell abundance within solid tumors. These screens led to the prioritization of 11 targets whose knockout (KO), increased CD8+ T cell abundance in tumors. Notably, KO of these genes in adoptively transferred CD8+ T cells improved tumor control in solid tumor models. Based on these findings, we hypothesize that targeting these genes may similarly augment the efficacy of CAR T cells against solid tumors. We will test this hypothesis using two human CAR T cell-solid tumor in vivo models (colorectal cancer and lung cancer). In Aim 1, we will assess KO CAR T cells targeting each of the 11 prioritized genes, along with paired controls, by evaluating their proliferation following repeat antigen stimulation and their serial killing capacity in vitro. In Aim 2, we will select the 4 top candidate targets for in vivo evaluation, assessing

their effects on anti-tumor immunity, expansion, and effector phenotypes. Together, these studies will prioritize genetic targets that enhance CAR T cell performance in our models, guiding selection of a lead “armored” CAR for translation to the clinic.