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Next-generation immunotherapy - Chimeric antigen receptor-modified T cells targeting cancer and immune checkpoint blockade: therapeutic efficacy and molecular mechanisms of T cell exhaustion.


T cells containing chimeric receptors composed of antibody binding motifs (CAR T cells, chimeric antigen receptor T cells), capable to be directly activated when in contact with a specific antigen, are a potent new generation of cancer immunotherapy, unexploited by Brazilian researchers. Extraordinary results have been obtained using CAR T cells for the treatment of hematological tumors. However CAR T cells therapy has some barriers to be overcome for the treatment of solid tumors, such as the CAR T cells exhaustion induced by the immunosuppressive tumor microenvironment. This project aims to produce CAR T cells able to efficiently migrate to tumor sites to block T cell exhaustion and kill tumor cells. These CAR T cells will be also applied as model of exhaustion induction/reversal to study the effect of extracellular matrix compounds in T cell exhaustion, with focus in the role of glycosaminoglycans (GAGs). GAGs are heteropolysaccharides involved in several cellular processes and can work as co-receptors during T cell stimulation. The carbonic anhydrase IX (CAIX) is an important antigen overexpressed in solid tumors in hypoxia, with remarkable expression in clear cell renal cell carcinoma (ccRCC). In this project, an anti-CAIX CAR lentiviral vector will be adapted in its second cassette to express an anti-programmed cell death ligand-1 (PD-L1) antibody with high affinity to be released to the medium, blocking T cell exhaustion. In addition, CAR structure will be modified by insertion of 4-1BB as a co-stimulatory domain, to replace CD28. 4-1BB has shown superior results to extend CAR T cells lifespan and decrease T cell exhaustion, when compared to CD28. T cells will be selected, activated and transduced with the lentiviral constructs and transduction efficiency will be confirmed. The cytotoxic effect of anti-CAIX CAR T cells will be evaluated in vitro in ccRCC cells and the expression of exhaustion markers will be also analyzed. A model of ccRCC in NSG mice will be used to evaluate the activity of CAR T cells in vivo. The tumor growth will be measured by bioluminescence. The expression levels of Ki67 and PD-L1 will be used to confirm the anti-tumor effect of CAR T cells in situ by immunohistochemistry (IHC). The tumor infiltrating lymphocytes will be analyzed by IHC detection of granzyme B and CD3. T cells will be activated, expanded and treated with inhibitors of GAGs synthesis/ sulfation. These cells will be also co-cultured with CHO cells with or without GAG production. After these different incubations, T cells will be evaluated for exhaustion markers, cytokine secretion and cytotoxicity. The GAG profile before and after exhaustion will be evaluated and we will verify if immune checkpoint blockade by anti-PD-L1 antibodies can change GAGs synthesis by T cells or ccRCC cells, influencing tumor malignancy. This innovative project will achieve the production of a next generation immunotherapy, with great antitumor potential. Besides, the role of tumor microenvironment molecules in T cell exhaustion will provide valuable information to improve the immune system function against cancer. (AU)