The immune status of the tumor microenvironment is a key indicator in determining the antitumor effectiveness of immunotherapies. Data support the role of activation and expansion of tumor-infiltrating lymphocytes (TILs) in increasing the benefit of immunotherapies in patients with solid tumors. We found that intratumoral injection of a tumor-selective oncolytic vaccinia virus encoding interleukin-7 (IL-7) and IL-12 into tumor-bearing immunocompetent mice activated the inflammatory immune status of previously poorly immunogenic tumors and resulted in complete tumor regression, even in distant tumor deposits. Mice achieving complete tumor regression resisted rechallenge with the same tumor cells, suggesting establishment of long-term tumor-specific immune memory. Combining this virotherapy with anti–programmed cell death-1 (PD-1) or anti–cytotoxic T lymphocyte antigen 4 (CTLA4) antibody further increased the antitumor activity as compared to virotherapy alone, in tumor models unresponsive to either of the checkpoint inhibitor monotherapies. These findings suggest that administration of an oncolytic vaccinia virus carrying genes encoding for IL-7 and IL-12 has antitumor activity in both directly injected and distant noninjected tumors through immune status changes rendering tumors sensitive to immune checkpoint blockade. The benefit of intratumoral IL-7 and IL-12 expression was also observed in humanized mice bearing human cancer cells. These data support further investigation in patients with non-inflamed solid tumors.

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Characterization of the intratumoral immune status is important for developing immunotherapies and evaluating their antitumor effectiveness. CD8 + T cells are one of the most important cell types that directly and indirectly contribute to antitumor efficacy by releasing cytolytic molecules and inflammatory cytokines in the tumor microenvironment. Previously, we engineered a tumor-selective oncolytic vaccinia virus that encodes interleukin-7 (IL-7) and IL-12 and demonstrated its usefulness as an agent for in situ vaccination against tumors, with data showing that antitumor efficacy was reliant upon CD8 + T cells recruited by viral treatment. Here, we investigated the phenotypic changes in intratumoral CD8 + T cells caused by this oncolytic virus and found increased expression of inducible co-stimulator (ICOS) in PD-1 − CD8 + T cells. Unlike previously reported ICOS + CD8 + T cells, a subset of ICOS + PD-1 − CD8 + T cells showed effector function characterized by granzyme B expression. ICOS expression was induced by the backbone virus, which did not encode any immune transgenes and was independent of upregulation of the type I interferon pathway. Not only did we identify a novel effector cell subset characterized by ICOS expression, but our findings also shed light on a potential unknown aspect of the mechanism of oncolytic vaccinia virotherapy.

While immune checkpoint inhibitors have markedly improved therapeutic outcomes for cancer patients, only a subset of patients exhibit a long-term durable response. A number of approaches have been trialed in preclinical and clinical studies to overcome this, including in situ vaccination using tumor-selective oncolytic viruses. Oncolytic vaccinia virus (VV), which is highly cytolytic across a wide range of tumor types, kills cancer cells and induces antitumor immune responses via DAMPs, PAMPs and tumor antigen release. To maximize the potential of oncolytic virus therapy, we examined methods to further upregulate a tumor’s inflammatory status based on the intratumoral IL-7 or IL-12 expression. IL-7 is crucial for T cell homeostasis and is known to increase tumor-reactive T cells. Meanwhile, IL-12 is known to activate both innate and adaptive immunity via IFN-γ produced by NK cells, cytotoxic T cells and CD4-positive T cells. We engineered and administered VVs carrying human IL-7 (hIL-7-VV) and murine IL-12 (mIL-12-VV) and evaluated antitumor responses in immunocompetent mice with poorly immunogenic Lewis lung carcinoma (LLC) tumors. Monotherapy with hIL-7-VV or mIL-12-VV induced complete tumor regression (CR) in 0 or 1 of 7 mice, respectively, whereas combination treatment with hIL-7-VV and mIL-12-VV induced CR in 4 of 7 mice with no body weight loss. Intratumoral injection of hIL-7-VV in combination with mIL-12-VV significantly increased tumor-infiltrating lymphocytes and induced murine IFN-γ production. Mice that had previously achieved CR following treatment with a VV carrying both human IL-7 and murine IL-12 (hIL-7/mIL-12-VV) rejected rechallenged tumors, suggesting that hIL-7/mIL-12-VV induced the establishment of antitumor immune memory. Next, to assess the effect of oncolytic activity and immune activation potential against human tumors, we engineered a VV carrying human IL-7 and human IL-12 genes (hIL-7/hIL-12-VV) and evaluated its effect in humanized mice bearing human cancer cells. Intratumoral administration of hIL-7/hIL-12-VV altered the immune status of tumors and induced tumor regression, consistent with the activity of hIL-7/mIL-12-VV observed in immunocompetent mouse models. Therefore, oncolytic VV-induced intratumoral IL-7 and IL-12 expression enhanced the immune response in the tumor microenvironment and improved antitumor efficacy. These results support the clinical development of VV carrying IL-7 and IL-12 for patients with non-inflamed cold tumors. Citation Format: Shinsuke Nakao, Yukinori Arai, Mamoru Tasaki, Midori Yamashita, Nobuaki Amino, Ryuji Murakami, Tatsuya Kawase, Motomu Nakatake, Hajime Kurosaki, Masamichi Mori, Masahiro Takeuchi, Takafumi Nakamura. Effect of oncolytic vaccinia virus-induced IL-7 and IL-12 expression on tumor immune microenvironment in poorly immunogenic murine cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B019. doi:10.1158/1535-7163.TARG-19-B019

Our previous studies showed that dietary nucleotides fed to mice enhanced the secretion of interleukin 7 (IL-7) and transforming growth factor beta (TGF-beta) from intestinal epithelial cells (IECs). To explore whether nucleotides influence IECs directly to enhance the secretion of the cytokines or not, the effects of nucleotides added in vitro on the cytokine secretion from primary-cultured murine IECs were examined. When the mixture of nucleotide 5'-monophosphates (CMP, GMP, IMP, and UMP) or individual nucleotide 5'-monophosphates were added to the primary culture of IECs derived from BALB/c mice, the secretion of IL-7, but not that of TGF-beta, was increased significantly. Addition of nucleotides to the culture did not alter the number of the IECs. Secretion of IL-6 and granulocyte-macrophage colony-stimulating factor, which are known to be secreted from IECs, was not enhanced by the addition of nucleotides. These results demonstrate that nucleotides can affect IECs directly to enhance the secretion of IL-7, and suggest that the increased secretion of TGF-beta from IECs by dietary nucleotides was due to indirect effects of the nucleotides, which may affect intestinal microflora or cells other than IECs that in turn influence the cytokine secretion of IECs.