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

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