Simultaneous, cell-intrinsic downregulation of PD-1 and TIGIT enhances the effector function of CD19-targeting CAR T cells and promotes an early-memory phenotype

Abstract

CD19 targeting chimeric antigen receptor (CAR) T cells have become an important therapeutic option for patients with relapsed and refractory B cell malignancies. However, recent clinical data indicate that a significant portion of patients still do not benefit from the therapy owing to various resistance mechanisms, including high expression of multiple inhibitory immune checkpoint receptors on activated CAR T cells. Here, we report a lentiviral two in one CAR T approach in which two checkpoint receptors are downregulated simultaneously by a dual short hairpin RNA (shRNA) cassette integrated into a CAR vector. Using this system, we evaluated CD19 targeting CAR T cells in the context of four different checkpoint combinations PD1/TIM3, PD1/LAG3, PD1/CTLA4 and PD1/TIGIT and found that CAR T with PD1/TIGIT downregulation uniquely exerted synergistic antitumor effects in mouse xenograft models compared with PD1 downregulation only, and maintained cytolytic and proliferative capacity upon repeated antigen exposure. Importantly, functional and phenotypic analyses of CAR T cells as well as analyses of transcriptomic profiles suggested that downregulation of PD1 enhances short term effector function, whereas downregulation of TIGIT is primarily responsible for maintaining a less-differentiated/exhausted state, providing a potential mechanism for the observed synergy. The PD1/TIGIT downregulated CAR T cells generated from diffuse large B cell lymphoma patient derived T cells using a clinically applicable manufacturing process also showed robust antitumor activity and significantly improved persistence in vivo compared with conventional CD19 targeting CAR T cells. Overall, our results demonstrate that the cell intrinsic PD1/TIGIT dual downregulation strategy may prove effective in overcoming immune checkpoint mediated resistance in CAR T therapy.