Augmenting CAR T Cell Function with LIGHT

Chimeric antigen receptor (CAR) T cell therapy has shown remarkable clinical success in the treatment of B-cell acute lymphoblastic leukemia (B-ALL), non-Hodgkin lymphoma (NHL), and multiple myeloma (MM), but its clinical efficacy in other hematologic tumors and solid tumors has been modest. Some of the major challenges that diminish the efficacy of CAR T cells against solid tumors are tumor-associated antigen heterogeneity and the immunosuppressive tumor microenvironment (TME). To overcome these problems, we developed CAR T cells overexpress with LIGHT (TNFSF14), which is a ligand of both LtßR (lymphotoxin beta receptor) on cancer cells and HVEM (herpes virus entry mediator) on immune cells. In addition to the cancer cell-directed cytotoxicity mediated by the CAR T cells themselves, the interaction of LIGHT with LTßR on tumor cells led to antigen-independent killing; moreover, LIGHT also provided immunostimulatory properties to the T cells. Hence, LIGHT-CAR T cells promoted, through multimodal and orthogonal mechanisms, an improved antitumor response through enhanced tumor killing and costimulatory potential. The antigen-independent killing of heterogeneous cancer cells was widely applicable across various cancer cell lines and was mediated by LIGHT CAR T cells in an LTßR-dependent manner. In addition, LIGHT-CAR T cells demonstrated higher proliferative capacity and proinflammatory cytokine secretion than non-LIGHT expressing CAR T cells. Furthermore, LIGHT-CAR T cells conferred significant tumor control and concomitant survival benefit as compared to non-LIGHT CAR T cells in xenograft models of solid tumors with a heterogeneous expression of the target antigen. The application of LIGHT-CAR T cell therapies may provide a novel therapeutic approach to the treatment of solid tumors in the context of antigen-heterogeneous disease thereby preventing outgrowth of antigen-negative populations leading to disease relapse. Overall design: To further elucidate mechanisms of the enhanced cytotoxicity of LIGHT CAR T cells compared to standard second-generation CAR T cells, we performed Cellular Indexing of Transcriptomes and Epitopes (CITE-seq) to evaluate the transcriptional state and surface protein expression of individual CAR T cells before and after co-culture with cancer cells. We generated mesothelin-directed CAR T cells with and without LIGHT (Meso_28z-LT and Meso_28z, respectively) from four healthy human donors (A-D) and sequenced them post-sorting at baseline (t=0) and 48 hours after co-culture with cancer cells (t=48h). We performed cell hashing (32) to combine samples of the same condition (construct + timepoint) from different donors and used an extensive panel of 130 antibody-derived tags (ADTs) to stain cell surface proteins