Project description: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 LtβR (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 LTβR 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 LTβR-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.
Project description:Chimeric antigen receptor (CAR)-T cell-based immunotherapy for cancer and immunological diseases has made great strides, but it still faces multiple hurdles. Finding the right molecular targets to engineer T cells toward a desired function has broad implications for the armamentarium of T cell-centered therapies. Here, we developed a dead-guide RNA (dgRNA)-based CRISPR activation screen in primary CD8+ T cells, and identified gain-of-function (GOF) targets for CAR-T engineering. Targeted knock-in or overexpression of a lead target, PRODH2, enhanced CAR-T-based killing and in vivo efficacy in multiple cancer models. Transcriptomics and metabolomics in CAR-T cells revealed that augmenting PRODH2 expression re-shaped broad and distinct gene expression and metabolic programs. Mitochondrial, metabolic and immunological analyses showed that PRODH2 engineering enhances the metabolic and immune functions of CAR-T cells against cancer. Together these findings provide a system for identification of GOF immune boosters, and demonstrate PRODH2 as a target to enhance CAR-T efficacy.
Project description:Chimeric antigen receptor (CAR)-T cell-based immunotherapy for cancer and immunological diseases has made great strides, but it still faces multiple hurdles. Finding the right molecular targets to engineer T cells toward a desired function has broad implications for the armamentarium of T cell-centered therapies. Here, we developed a dead-guide RNA (dgRNA)-based CRISPR activation screen in primary CD8+ T cells, and identified gain-of-function (GOF) targets for CAR-T engineering. Targeted knock-in or overexpression of a lead target, PRODH2, enhanced CAR-T-based killing and in vivo efficacy in multiple cancer models. Transcriptomics and metabolomics in CAR-T cells revealed that augmenting PRODH2 expression re-shaped broad and distinct gene expression and metabolic programs. Mitochondrial, metabolic and immunological analyses showed that PRODH2 engineering enhances the metabolic and immune functions of CAR-T cells against cancer. Together these findings provide a system for identification of GOF immune boosters, and demonstrate PRODH2 as a target to enhance CAR-T efficacy.
Project description:Chimeric antigen receptor (CAR) T-cell therapy targeting CD19 (CART-19) represents a significant advance in the treatment of patients with relapsed or refractory CD19+ B-cell lymphomas. However, a significant portion of patients either relapse or fail to respond. Moreover, many patients have symptomatic disease, requiring bridging radiation therapy (RT) during the period of CAR T-cell manufacturing. To investigate the impact of 1 to 2 fractions of low-dose RT on CART-19 treatment response, we developed a mouse model using A20 lymphoma cells for CART-19 therapy. We found that low-dose fractionated RT had a positive effect on generating abscopal systemic antitumor responses beyond the irradiated site. The combination of RT with CART-19 therapy resulted in additive effects on tumor growth in irradiated masses. Notably, a significant additional increase in antitumor effect was observed in nonirradiated tumors. Mechanistically, our results validate activation of the cyclic guanosine adenosine synthetase/stimulator of interferon genes pathway, tumor-associated antigen crosspriming, and elicitation of epitope spreading. Collectively, our findings suggest that RT may serve as an optimal priming and bridging modality for CAR T-cell therapy, overcoming treatment resistance and improving clinical outcomes in patients with CD19+ hematologic malignancies.
Project description:We performed transcriptome sequencing on Neo-2/15 stimulated CAR NK cells,to shed light on the function and phenotype changes of CAR-NK cells stimulated by IL-2 and Neo-2/15.
Project description:Adoptive cell therapy, a subset of cancer immunotherapy, is collection of therapeutic approaches which aim to redirect the immune system by reprogramming patient T-cells to target antigenic molecules differentially and specifically expressed in certain cancers. One promising immunotherapy technique is CAR T-cell therapy, where cancer cells are targeted through the expression a chimeric antigen receptor (CAR), a synthetic trans- membrane receptor that functionally compensates for the T-cell receptor (TCR) but targets a tumor associated antigen on the cancer cell surface. While CAR T-cell therapy is promising with two clinically approved second-generation CARs (Kymriah and Yescarta), few studies have investigated the mechanism of signal propagation in T-cells and no studies have investigated the potential signaling response in the target cells. To gain further insight to CAR-based signaling, we stimulated third generation CD19 CAR-expressing Jurkat T-cells by co-culture with SILAC labeled CD19HI Raji B-cells and used two phosphoenrichment strategies coupled with liquid chromatography-tandem mass spec- trometry (LC-MS/MS) to detect and analyze global phosphorylation changes in both cell populations. Analysis of the phosphopeptides originating from the CD19-CAR T cells revealed an increase in many phosphorylation events necessary for canonical TCR signaling. We also observed for the first time a significant decrease in B-cell receptor- related phosphopeptide abundance in CD19HI Raji B-cells after co-culture with CD19-targetted CAR T-cells.