TNF-alpha-"armed" TCR vectors to enhance adoptive cell therapy for solid tumors

PROJECT SUMMARY/ABSTRACT Cancer immunotherapy with T-cells expressing a transgenic T-cell receptor (TCR) or chimeric antigen receptor (CAR) can generate dramatic clinical responses in a variety of solid tumors. However, a significant number of patients do not respond to therapy. We have previously shown that increased secretion activity of tumor necrosis factor alpha (TNF-alpha) is associated with superior clinical responses to TCR-T cell therapy. Furthermore, we have shown that increased TNF-alpha activity reduces immunosuppressive T helper 2 (Th2) cell activity, which is associated with inferior response to therapy due to their anti-effector functionality. We have generated novel lentiviral vectors which simultaneously encode for TNF-alpha along with a tumor antigen-specific TCR or CAR (which we have termed a TNF-alpha-“armed” TCR/CAR vector). Treatment with TNF-alpha-armed TCR-T cells results in enhanced anti-tumor activity in vitro and in vivo in preliminary humanized mouse xenograft studies. Importantly, the improved antitumor functionality was not associated with increased systemic toxicity, due to the antigen-dependent nature of the increased TNF-alpha secretion. We hypothesize that utilizing TNF-alpha-armed TCR/CAR-T cells will result in superior antitumor efficacy of the transgenic T-cells compared to conventional TCR/CAR-T cells. We will explore our hypothesis in a wide variety of TCRs and CARs and associated tumor types via two related but independent aims. Aim 1 will evaluate the impact of transgenic TNF-alpha co-delivery with a transgenic TCR or CAR on T-cell anti- tumor functionality and toxicology in humanized murine xenografts, as well as T-cell infiltration into the tumor microenvironment and gene expression patterns of the tumor tissue in response to the increased TNF-alpha activity via spatial transcriptomics/proteomics. We hypothesize that TNF-alpha-armed TCR/CARs will result in superior anti-tumor functionality in vivo compared to conventional TCR/CAR-T cells without increased toxicity. Aim 2 will explore the T-cell intrinsic impact of increased TNF-alpha activity on T-cell functional phenotype and the underlying signaling mechanisms in the face of chronic antigen stimulation. Tumor-infiltrating lymphocytes obtained from the same humanized mouse xenografts obtained in Aim 1 will be interrogated with mass cytometry, single-cell cytokinetic polyfunctionality assays, and single-cell phospho-proteomics. Repetitive tumor cell stimulation assays in vitro, as well as TNF-receptor-2 knockouts will serve as confirmatory experiments. We hypothesize that the addition of transgenic TNF-alpha to TCR/CAR-T cell therapeutics will augment TNF- alpha/TNF-receptor 2 signaling, leading to inhibition of Th2 immunosuppressive cytokinetic activity. This project represents an innovative approach to improve transgenic T-cell therapeutics and further elucidate the role of TNF-alpha in T-cell biology. Our findings will set the stage for the translation of TNF-alpha-armed TCR/CAR T-cells into the next generation of adoptive T-cell therapies for solid tumors.