282 Improving solid tumor CAR-T therapies by modulating T cell transcriptional states using combinatorial in vitro CRISPR screening followed by in vivo validation

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Background Chronic antigen stimulation coupled with a suppressive tumor microenvironment can limit CAR-T therapies in solid tumors by causing T cell exhaustion. The epigenetic and transcriptional programs driving this dysfunctional state have been well characterized, opening an opportunity to reprogram T cells towards a more potent therapeutic state. Methods To identify T cell state genetic modulators of exhaustion, we delivered a library of >550 single gain-of-function (GOF), loss-of function (LOF), and combinatorial gene perturbations (G/G, L/L, G/L) to human CAR-T cells followed by two weeks of repetitive in vitro stimulation with cell lines expressing cognate antigens. Readouts measuring T cell proliferation, tumor cell killing, and scRNA-seq allow us to functionally and molecularly characterize T cell activity as a function of each unique perturbation. To bridge our in vitro work with in vivo efficacy, we selected top performing perturbations from our arrayed screen that increased either tumor killing, T cell proliferation, or both, indicative of a reduced exhausted state, and dosed a cohort of tumor-bearing mice with CAR-T cells. We selected stringent in vivo model conditions by using large tumors and small T cells inoculums to provide a stress test of the T cell perturbations evaluated. Results These perturbations markedly increased the anti-tumor activity of CAR-T cells, confirming their performance in the in vitro assay. Conclusions This pre-clinical study demonstrates that our in vitro screening platform is an effective predictor of potent CAR-T cell modulators with strong in vivo anti-tumor activity.

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