Tuning KITv CAR T cells for the solid tumor environment

PROJECT ABSTRACT We developed and translated CD28 costimulated chimeric antigen receptor (M28z CARs) T cells that target mesothelin

(MSLN), a cancer-associated antigen that is overexpressed in a majority of solid tumors; 65 patients have been treated in

phase I/II trials, with no on-target, off-tumor toxicity. Having demonstrated that regionally administered CAR T cells avoid pulmonary sequestration and are enhanced by early antigen-activated CD4 CAR T-cell helper function, we delivered M28z CAR T cells intrapleurally in patients with malignant pleural mesothelioma (MPM), promoting tumor infiltration. To

overcome antigen stress–induced T-cell exhaustion, we treated 27 patients with MPM with an anti-PD1 agent after a single dose of intrapleural M28zCAR T cells (median survival 18 months vs. 10 months following second line pembrolizumab).

To facilitate tumor-specific checkpoint blockade and to achieve functional persistence without repeated administration of anti-PD1 agent, we translated a CAR T-cell–intrinsic PD1 dominant negative receptor (PD1DNR); 8 patients have been

treated, with no CAR- or PD1DNR-related toxicities. Recognizing the necessity of the IFNγ pathway for solid tumor killing by CAR T cells, we exploited a c-KIT mutation, D816V (KITv), as a costimulatory domain (Nat Cancer 2023). A single infusion of M28zKITv CAR T cells was safe, resisted TGFβ-mediated suppression, prolonged survival, and demonstrated

functional persistence in multiple solid tumor models including in low-antigen expressing tumors; clinically available kinase

inhibitors are effective as on/off, tunable safety switch. Herein, we hypothesize that, whereas IFNγ-mediated cytotoxicity is the predominant mechanism of KITv CAR T cells against high-antigen-expressing targets, for low- or non-antigen- expressing targets, IFNγ-mediated upregulation of ICAM1 on tumor cells facilitating LFA1(CAR T-cell)-ICAM1 synapse

(increased avidity) and apoptosis of IFNγ receptor–expressing target cells are the respective biologic mechanisms (Aim 1), KITv-induced IFNγ potentiates the cytotoxicity of CD4 41BBz KITv CAR T cells against low-antigen-expressing targets and that CD28 costimulation promotes the helper function of CD4 28zKITv CAR T cells; however, PD1DNR is essential

for functional persistence (Aim 2), and that PD1DNR-mediated checkpoint blockade extends beyond PDL1-expressing tumor cells and counteracts PDL1-overexpressing M2 macrophages (Aim 3). The significance of our approach lies in its effective combination of a solid tumor–specific scFv that is on target and safe (MSLN), costimulatory domains (KITv,

CD28), checkpoint blockade (PD1DNR), and tumor infiltration (regional delivery). The impact of our proposal extends beyond pleural cancers; the majority of aggressive solid tumors express MSLN. The proposal’s innovations include— exploiting IFNγ-mediated cytotoxic mechanisms in a heterogenous antigen-expressing solid tumor, increased CAR T-cell

avidity through a second synapse, potentiating CD4 CAR T-cell helper function to augment CD8 CAR T cells and counteracting PDL1-expressing M2 macrophages by the CAR T-cell intrinsic PD1DNR.