CD28-KITv CAR T cells with PD-1 dominant negative receptor - PROJECT ABSTRACT The success of chimeric antigen receptor (CAR) T-cell therapy in solid tumors requires antigen targets with no on-target, off-tumor toxicity, effective tumor infiltration, cytotoxicity and proliferation in an immunosuppressive environment, and revival of antigen stress-induced exhausted CAR T cells. We translated CD28-costimulated CARs (M28z) that target mesothelin (MSLN), a cancer-associated antigen that we have documented expression in majority of solid tumors; 64 patients have been treated to date, with no on-target, off-tumor toxicity. Having demonstrated that regionally administered CAR T cells avoid pulmonary sequestration and benefit from early antigen-activated CD4 helper CAR T-cell function, we delivered CAR T cells intrapleurally in patients with malignant pleural mesothelioma (MPM), promoting tumor infiltration. To address T-cell exhaustion, we either treated patients with anti-PD1 agent after CAR T cells or employed tumor-specific checkpoint blockade by CAR T-cell intrinsic PD1 dominant negative receptor (PD1DNR); 34 patients have been treated to date, with no CAR- or PD1DNR-related toxicities and with responses by imaging, and increased survival. To promote IFNγ-mediated cytotoxicity shown to be essential for solid tumor killing, we exploited a c-KIT mutation, D816V (KITv), as a costimulatory domain. KITv CAR T cells show antigen- activation induced IFNγ signaling, enhanced cytotoxicity, and when added as signal 3 to CD28 (signal 2), provide a synergistic function, resist TGFβ-mediated suppression, and prolong functional persistence. Clinically available kinase inhibitors provide an on/off, tunable safety switch for KITv CAR T cells. To effectively deliver these next-generation CAR T cells to solid tumors, we developed a translational strategy of non- ablative, tumor-targeted radiation therapy (RT) to generate a chemokine gradient that facilitates systemically administered CAR T-cell chemotaxis, tumor infiltration, proliferation, and persistence. Herein, we seek to translate the M28zKITv-PD1DNR CAR T cells to address key limitations in solid tumor cell therapy. In UG3 phase, we will explore the hypothesis that PD1DNR checkpoint blockade extends beyond tumor cells and counteracts PDL1-expressing M2 macrophages with immune suppressor function (Aim 1). We will define optimal regimen of non-ablative, tumor-targeted RT to promote tumor infiltration of systemically administered CAR T cells, achieving efficacy similar to that with regional delivery. In Aim 3, we will submit an IND application, a process with which we are familiar and have track record of success. In UH3 phase, we will conduct a phase I study to investigate the safety, functional activity and efficacy, and markers of response in patients with MPM. The significance of our approach lies in its effective combination of solid tumor-specific– scFv that is on-target and safe (MSLN), costimulatory domains (CD28, KITv), checkpoint blockade (PD1DNR), and a strategy of promoting solid tumor-infiltration (RT) of CAR T cells. The impact of our proposal extends beyond MPM (>150,000/year pleural cancers in the U.S.); majority of aggressive solid tumors express MSLN.
