Final Report Summary - CANCER THERAPIES (Use of adoptive T cell transfer in combination with oncolytic adenoviruses for cancer treatment) Adoptive transfer of large numbers of human T lymphocytes genetically modified to express chimeric antigen receptors (CARs) is an attractive therapy for the treatment of cancer. Recently, some dramatic tumor regressions in patients with B-cell malignancies using CARs targeting CD19 have sparked great interest. CARs consist of an antigen-binding domain from an antibody coupled to an intracellular domain of the CD3-z chain. CAR-mediated T cell responses can be enhanced with the addition of an intracellular domain of a costimulatory receptor, such as CD28 or 4-1BB.Much of the research in tumor immunology has been focused on using CD8+ T cells. However, CD4+ T helper cells can also promote tumor rejection. Preclinical experiments in mice revealed that tumor immunotherapies that use CD4+ cells polarized as Th17 cells are more effective than therapies using non-polarized or Th1-based approaches, and new evidence suggest that CD4+ T cells polarized into Th17 cells can promote long-lived antitumour immunity. However, the therapeutic potential of Th17 cells in enhancing ACT therapy remained unknown.Previous data from Dr. June's lab established that inducible costimulatory (ICOS) protein is critical for the sustained expansion of human Th17 cells after their primary activation. Our hypothesis here was that redirection of Th17 cells with chimeric receptors containing the ICOS intracellular domain in tandem with TCR-z signal-transduction domain could promote and retain the Th17 phenotype after antigen recognition.In order to test this hypothesis, we generated a chimeric receptor containing a single-chain variable fragment that binds mesothelin (SS1 scFv) linked to the signaling domains of ICOS and the z chain of the CD3 complex (SS1-ICOSz). Peripheral blood CD4 T cells were cultured with activating beads coated with anti-CD3 and anti-ICOS antibodies in the presence of Th17 polarizing cytokines. After 24h, cells were transduced with lentiviral vectors encoding SS1-based chimeric receptors containing the TCR-z signal-transduction domain alone (SS1-z) or in combination with the CD28 (SS1-28z), CD137 (SS1-BBz) or ICOS (SS1-ICOSz) intracellular domains. Th17 cells were expanded until they rest down and then used for functional assays.When stimulated with aAPC or tumor cells expressing mesothelin, Th17 cells redirected with the SS1-ICOSz chimeric receptor secreted high amounts of IL17-A, IL17-F and CCL20 but nominal levels of IL-2. Moreover, Th17 activation through ICOS induced the expression of CD161, consistent with a predominant Th17 phenotype. By contrast, Th17 cells redirected with the SS1-28z, secreted higher amounts of IL-2 and IFNg but nominal levels of IL17-A and IL17-F, and had low CD161 expression. Microarray analysis indicated that Th17 cells redirected with the ICOS chimeric receptor maintained a core molecular signature of Th17 cells. When transferred into NSG mice with large vascularized pre-established tumors, Th17/Tc17 cells redirected with SS1-ICOS-z mediated enhanced antitumor responses, with 70% of mice showing complete remission. Our studies indicate that the expression of selected CAR endodomains, like ICOS, can significantly enhance the in vivo persistence of different T cell subsets and can program T cells for their subsequent differentiation fates and effector functions. Based on these results, we believe that the design of novel ICOS-based CARs has the potential to augment antitumor effects in clinical trials. Another possible strategy to increase the anti-tumor effect and persistence of CAR-T cells that we tested here is the combination of CAR-T cells with oncolytic adenoviruses. Oncolytic viruses are designed to selectively target, replicate in and kill cancer cells, while sparing normal cells. By replicating within the tumor, oncolytic adenoviruses cause direct tumor debulking while providing danger signals necessary to awake the immune system and break immunotolerance. The main goal for the returning period of this fellowship was to evaluate the ability of oncolytic adenoviruses to boost and maintain engineered T cells in tumor bearing mice. Our hypothesis here was that a previous administration of an oncolytic adenovirus in tumor bearing mice can reduce tumor burden, revert immunosuppression and enhance stability and function of adoptively transferred engineered T cells.We demonstrated that treatment of tumor cells with oncolytic virus can decrease PD1 and PDL1 expression in CAR-T cells and tumor cells, respectively, while enhancing in vitro CAR-T cell expansion upon antigen recognition. Importantly, the combination of VCN1 and CAR-T cells showed the best antitumor effects when administered into NSG mice bearing mesothelin+ subcutaneous tumors. Tumors treated with the combination therapy had slightly higher percentages of CD45+ T cells than tumors treated only with CAR-T cells, although differences were not significant. Interestingly, we saw a significant inverse correlation between the percentage of CD45+ T cells and the tumor volume in mice treated with CAR-T cells and virus, while no correlation was observed in mice treated with only CAR-T cells. Of note, spleens and tumors treated with the combination of VCN1 and CAR-T cells had an increase in the ratio of CD8+/CD4+ T cells when compared to tumors treated with only CAR-T cells. Also, a significant inverse correlation between the percentage of CD8+ T cells (versus CD45+ T cells) and the tumor volume was observed in mice treated with CAR-T cells and virus, while no correlation was seen in mice treated with only CAR-T cells. More experiments are required to clarify if combination of oncolytic viruses and CAR-T cells has a synergistic effect. We believe that these results warrant further investigation of the in vitro and in vivo effects of combining oncolytic adenoviruses and CAR-T cells.The use of a CAR containing the ICOS intracellular domain to redirect Th17 cells was patented in 2012: “Use of ICOS-Based CARs to Enhance Antitumor Activity and CAR Persistence”. Also, these results were published in the prestigious journal “Blood” in 2014: “ICOS-based chimeric antigen receptors sustain bipolar TH17/TH1 cells” and presented at several top scientific meetings.
