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.

Immune checkpoint inhibitors against PD-1, PD-L1 and CTLA-4 have altered the treatment paradigm for various types of cancers in the past decade. However, they offer clinical benefits to only a subset of patients. Evaluation and identification of an appropriate therapeutic approach to improve intratumoral immune status are needed for better treatment outcomes. We previously demonstrated that intratumoral expression of IL-7 and IL-12 increased tumor-infiltrating lymphocytes in poorly immunogenic tumors, resulting in a higher tumor regression rate than IL-12 alone. However, the mechanism underlying the difference in efficacy with and without IL-7 remains unclear. Here, we identified a previously unknown effect of IL-7 on the T cell receptor (TCR) repertoire of intratumoral CD8+ T cells, which is induced in the presence of IL-12. While IL-7 alone increased the diversity of intratumoral CD8+ T cells, IL-7 with IL-12 increased a limited number of high-frequency clones, conversely augmenting IL-12 function to increase the clonality. The proportion of mice with multiple high-frequency clones in tumors correlated with that achieving complete tumor regression in efficacy studies. These findings provide a scientific rationale for combining IL-7 and IL-12 in anticancer immunotherapy and unveil a novel IL-7 function on intratumoral TCR repertoire.

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

Immune checkpoint blockade targeting PD-1, PD-L1 and CTLA-4 have demonstrated remarkable clinical responses in patients with advanced cancers; however, a durable benefit is obtained in only a subset of patients. To overcome this therapeutic limitation, a number of immunotherapies and their combinations have been investigated. Among these, in situ vaccination using tumor-selective oncolytic viruses has proved an attractive approach. Viruses not only directly kill cancer cells through their replication, but also activate antitumor immunity triggered by the release of tumor antigens from killed cells. We have explored this approach using tumor-selective oncolytic vaccinia viruses (VVs) carrying immunomodulatory genes. We previously demonstrated that intratumoral treatment with a VV carrying human IL-7 and murine IL-12 genes (hIL-7/mIL-12-VV) enhanced immune responses in treated tumors and induced complete tumor regression in syngeneic mouse models (AACR-NCI-EORTC 2019). Here, using a bilaterally tumor-bearing mouse model, we examined whether unilateral virus injection affected untreated distant tumors. All tumors that had been directly injected with hIL-7/mIL-12-VV completely regressed. At the same time, untreated tumors in three of six mice also disappeared. We further confirmed significant increases in tumor-infiltrating lymphocytes in both treated and untreated tumors after treatment with hIL-7/mIL-12-VV. Unlike the treated tumors, the untreated distant tumors showed no detectable viral DNA by qPCR. PD-L1 expression was also upregulated in this model following treatment with hIL-7/mIL-12-VV in both treated and untreated tumors. This led us to examine the effects of combining this virotherapy with anti-PD-1 antibody. While monotherapies with anti-PD-1 antibody did not show significant antitumor efficacy, hIL-7/mIL-12-VV prior to this agent elicited improved antitumor activity in the virus-untreated distant tumors, suggesting that hIL-7/mIL-12-VV systemically sensitized tumors to anti-PD-1 antibody. Evidence that intratumoral immune status was changed following hIL-7/mIL-12-VV was found in the upregulation of various immune pathways analyzed using the Nanostring Pancancer Immune Profiling panel, and the increased clonality of T-cell repertoire in tumors. These results support the clinical development of VV carrying IL-7 and IL-12 as both monotherapy and as a combination partner with anti-PD-1 antibody for patients with advanced tumors. Citation Format: Shinsuke Nakao, Midori Yamashita, Mamoru Tasaki, Tatsuya Kawase, Motomu Nakatake, Hajime Kurosaki, Masamichi Mori, Masahiro Takeuchi, Takafumi Nakamura. Oncolytic vaccinia virus encoding IL-7 and IL-12 systemically sensitizes tumors to anti-PD-1 antibody in a syngeneic mouse model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4561.

Decreased reactivity in mixed lymphocyte culture (MLC) was observed in patients within 1 yr after allogeneic and autologous bone marrow transplantation. Suppressor activity of peripheral blood mononuclear cells (PBMC) from transplant patients was studied by adding these cells as modulator cells to a bidirectional MLC with cells from normal individuals. PBMC from transplant patients markedly suppressed MLC reactivity in a dose-dependent manner. Suppressor activity was present in cells forming rosettes with sheep erythrocytes. Treatment of modulator cells with monoclonal antibodies against T cell differentiation antigens (OKT8, OKIa1) and complement completely abolished suppression of MLC. Suppressor activity was unaffected by 30 Gy irradiation. Suppressor activity declined gradually after transplantation and was inversely correlated with MLC reactivity of each patient at a significant level (p less than 0.01). These observations suggest that OKT8+ Ia+ radioresistant suppressor T cells play a role in the development of decreased MLC reactivity observed during the early post-transplant period.