Keyvan Fallah-Mehrjardi

CAR T cell qualities, such as persistence and functionality play important roles in determining the outcome of cancer immunotherapy. In spite of full functionality, it has been shown that poor persistence of CAR T cells can limit an effective antitumor immune response. Here, we outline specific strategies that can be employed to overcome intrinsic and extrinsic barriers to CAR T cell persistence. We also offer our viewpoint on how growing use of CAR T cells in various cancers may require modifications in the intrinsic and extrinsic survival signals of CAR T cells. We anticipate these amendments will additionally provide the rationales for generation of more persistent, and thereby, more effective CAR T cell treatments. CAR T cell qualities, such as persistence and functionality play important roles in determining the outcome of cancer immunotherapy. In spite of full functionality, it has been shown that poor persistence of CAR T cells can limit an effective antitumor immune response. Here, we outline specific strategies that can be employed to overcome intrinsic and extrinsic barriers to CAR T cell persistence. We also offer our viewpoint on how growing use of CAR T cells in various cancers may require modifications in the intrinsic and extrinsic survival signals of CAR T cells. We anticipate these amendments will additionally provide the rationales for generation of more persistent, and thereby, more effective CAR T cell treatments.

Abstract Background CAR T cell-based therapies have shown promising results in hematological malignancies. Results of CAR T cell projects in solid tumors have been less impressive, and factors including lack of targetable antigens and immunosuppressive tumor microenvironment (TME) have been suggested as culprits. Adenosine, a metabolite which is highly produced in TME, is known to mediate the suppression of anti-tumor T cell responses via binding and signaling through adenosine 2a receptor (A2aR). Methods In this study, the expression of A2aR and the effects of its activation on the function of fully human anti-mesothelin CAR T cells (MSLN-CAR T), were analyzed. Afterwards, the molecular and pharmacological means to overcome the inhibitory effects of A2aR signaling on CAR T cell function were used. This was performed by targeting A2aR expression in MSLN-CAR T cells using various anti-A2aR shRNA sequences embedded in the CAR vector and an A2aR pharmacological antagonist, SCH-58261. Statistical analyses were performed Prism 7 software. Results Our experiments showed significant A2aR upregulation on T cells during the CAR T cell production procedure (cell activation and transduction). Activation of adenosine signaling using adenosine analog led to the suppression of all major anti-tumor functions in MSLN-CAR T cells. Interestingly, CAR T cells that carried the anti-A2aR shRNA sequences were resistant to the inhibitory effects of adenosine signaling. Pharmacological inhibition of A2aR reversed the reduction in CAR T cell proliferation and cytokine response caused by the adenosine analog; however, it failed to rescue the cytotoxic function of the cells. Conclusion Altogether, our results indicate that mitigating A2aR signaling by genetic targeting of the receptor might be a promising approach in improving CAR T cell function in an unreceptive microenvironment and could potentially improve the outcome of treatment in clinical settings.

BACKGROUND: Lactic acid produced by tumors has been shown to overcome immune surveillance, by suppressing the activation and function of T cells in the tumor microenvironment. The strategies employed to impair tumor cell glycolysis could improve immunosurveillance and tumor growth regulation. Dichloroacetate (DCA) limits the tumor-derived lactic acid by altering the cancer cell metabolism. In this study, the effects of lactic acid on the activation and function of T cells, were analyzed by assessing T cell proliferation, cytokine production and the cellular redox state of T cells. We examined the redox system in T cells by analyzing the intracellular level of reactive oxygen species (ROS), superoxide and glutathione and gene expression of some proteins that have a role in the redox system. Then we co-cultured DCA-treated tumor cells with T cells to examine the effect of reduced tumor-derived lactic acid on proliferative response, cytokine secretion and viability of T cells. RESULT: We found that lactic acid could dampen T cell function through suppression of T cell proliferation and cytokine production as well as restrain the redox system of T cells by decreasing the production of oxidant and antioxidant molecules. DCA decreased the concentration of tumor lactic acid by manipulating glucose metabolism in tumor cells. This led to increases in T cell proliferation and cytokine production and also rescued the T cells from apoptosis. CONCLUSION: Taken together, our results suggest accumulation of lactic acid in the tumor microenvironment restricts T cell responses and could prevent the success of T cell therapy. DCA supports anti-tumor responses of T cells by metabolic reprogramming of tumor cells.

Abstract Although remarkable results have been attained by adoptively transferring T cells expressing fully murine and/or humanized anti‐CD19 chimeric antigen receptors (CARs) to treat B cell malignancies, evidence of human anti‐mouse immune responses against CARs provides a rationale for the development of less immunogenic CARs. By developing a fully human CAR (huCAR), these human anti‐mouse immune responses are likely eliminated. This, perhaps, not only increases the persistence of anti‐CD19 CAR T cells—thereby reducing the risk of tumor relapse—but also facilitates administration of multiple, temporally separated doses of CAR T cells to the same recipient. To these ends, we have designed and constructed a second‐generation fully human anti‐CD19 CAR (or huCAR19) containing a fully human single‐chain variable fragment (ScFv) fused with a CD8a hinge, a 4‐1BB transmembrane domain and intracellular T cell signaling domains of 4‐1BB and CD3z. T cells expressing this CAR specifically recognized and lysed CD19 + target cells produced cytokines and proliferated in vitro. Moreover, cell volume data revealed that our huCAR construct cannot induce antigen‐independent tonic signaling in the absence of cognate antigen. Considering our results, our anti‐CD19 huCAR may overcome issues of transgene immunogenicity that plague trials utilizing CARs containing mouse‐derived ScFvs. These results suggest that this huCAR19 be safely and effectively applied for adaptive T cell immunotherapy in clinical practice.