CAR T-cell intrinsic PD1 Dominant Negative Receptor (PD1DNR) functions by both cell-intrinsic and extrinsic mechanisms

Abstract

Abstract Objectives: We have shown that chimeric antigen receptor (CAR) T cells with T-cell intrinsic PD1 dominant negative receptor (PD1DNR) prolong functional persistence. Herein, we further define T-cell intrinsic and extrinsic mechanistic functions of PD1DNR. Methods: Human T cells were retrovirally transduced with mesothelin targeted, CD28 co-stimulated CARs– either M28z (M) or M28z with PD1DNR (MDNR). Cytotoxicity, proliferation, phenotype, cytokine and effector and inhibitory protein expression were assessed by chromium release, cell count, flow cytometry, olink and mass spectrometry, respectively. MDNR extrinsic activity was measured by the ELISA for soluble PD1 and assessing cytotoxic functions of M cells following co-culture with MDNR supernatant. CAR T-cell functional persistence was assessed in vitro by repeated antigen stimulation and in vivo by tumor treadmill test and mice survival. Results: Following repeat antigen stimulation, MDNR cells demonstrate retained cytotoxicity, proliferation, accumulation, and enrichment of TNF-related cytokines (≥1× Log2), and overexpression of T-cell activation and proliferation proteins (GZMA, GNLY, IFNAR2, MFAP4). Soluble PD1 was 10-fold higher in MDNR supernatants. Co-culture of M cells with MDNR supernatant demonstrated higher cytotoxicity (p<0.01). Mice treated with a single dose of MDNR show enhanced survival compared to M (median survival 68.5 vs. 27 days, P<0.01). MDNR displayed also superior in-vivo functionality against advanced stage disease (median survival 62 vs. 12 days, P<0.01). Conclusions: PD1DNR expression in CAR T cells functions by both cell-intrinsic and extrinsic functions that are beneficial in solid tumors adoptive cell therapy.