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BackgroundPoorly immunogenic tumors are hardly responsive to immunotherapies such as immune checkpoint blockade (ICB) and are, therefore, a therapeutic challenge. Combination with other immunotherapies and/or immunogenic therapies, such as radiotherapy (RT), could make these tumors more immune responsive. We have previously shown that the immunocytokine L19–IL2 combined with single-dose RT resulted in 75% tumor remission and a 20% curative abscopal effect in the T cell-inflamed C51 colon carcinoma model. This treatment schedule was associated with the upregulation of inhibitory immune checkpoint (IC) molecules on tumor-infiltrating T cells, leading to only tumor growth delay in the poorly immunogenic Lewis lung carcinoma (LLC) model.MethodsWe aimed to trigger curative therapeutic responses in three tumor models (LLC, C51 and CT26) by “pushing the accelerator” of tumor immunity with L19–IL2 and/or “releasing the brakes” with ICB, such as antibodies directed against cytotoxic T lymphocyte associated protein 4 (CTLA-4), programmed death 1 (PD-1) or its ligand (PD-L1), combined with single-dose RT (10 Gy or 5 Gy). Primary tumor endpoint was defined as time to reach four times the size of tumor volume at start of treatment (4T×SV). Multivariate analysis of 4T×SV was performed using the Cox proportional hazards model comparing each treatment group with controls. Causal involvement of T and natural killer (NK) cells in the anti-tumor effect was assessed by in vivo depletion of T, NK or both cell populations. Immune profiling was performed using flow cytometry on single cell suspensions from spleens, bone marrow, tumors and blood.ResultsCombining RT, anti-PD-L1 and L19–IL2 cured 38% of LLC tumors, which was both CD8+ T and NK cell dependent. LLC tumors were resistant to RT +anti-PD-L1 likely explained by the upregulation of other IC molecules and increased T regulatory cell tumor infiltration. RT+L19–IL2 outperformed RT+ICB in C51 tumors; effects were comparable in CT26 tumors. Triple combinations were not superior to RT+L19–IL2 in both these models.ConclusionsThis study demonstrated that combinatorial strategies rationally designed on biological effects can turn immunotherapy-resistant tumors into immunologically responsive tumors. This hypothesis is currently being tested in the international multicentric randomized phase 2 trial: ImmunoSABR (NCT03705403).

Background While significant strides in the treatment of metastatic bladder cancer have been made with immune checkpoint inhibitors, the treatment of carcinoma in situ and non-muscle invasive, non-metastatic (superficial) human urothelial carcinoma, also termed non-muscle invasive bladder cancer (NMIBC), remains intractable with bacillus Calmette-Guerin (BCG) employed as the standard of care. In this study, an immunocytokine, NHS-muIL12, which consists of two molecules of murine IL-12 fused to NHS76, a tumor necrosis-targeting human IgG1, was examined as an immunotherapeutic in an orthotopic MB49luc bladder tumor model. Methods The antitumor activity of systemic administration of NHS-muIL12 was investigated on MB49luc tumors, an aggressive, bioluminescent orthotopic bladder cancer model. Temporal studies were carried out on MB49luc bladder tumors harvested during various time points during NHS-muIL12 treatment and cellular changes associated with the reduction in tumor burden following NHS-muIL12 were determined by flow cytometry. Effects of those changes on the proliferation/activation of lymphoid cells were also determined. Results Studies revealed a significant reduction in MB49luc bladder tumor burden occurring between days 3 and 6 after the third and final systemic administration of NHS-muIL12. Temporal analyses of the MB49luc bladder tumor microenvironment (TME) initially revealed a large accumulation of myeloid-derived suppressor cells (MDSCs) and macrophages that elicited potent immunosuppression. Immunosuppression was characterized by the inability of CD4+ and CD8+ T cells to respond to broad-based immune stimulants. NHS-muIL12 administration resulted in temporal-dependent reductions in the number of MDSCs, macrophages and tumor-associated TGF-β, which culminated in a re-ignition of CD4+ and CD8+ T cells to elicit potent antitumor responses against MB49luc bladder tumors. Conclusions These findings provide strong evidence that the systemic administration of an immunocytokine consisting of a tumor-targeting Ig through recognition of DNA and DNA-histone complexes coupled to muIL-12 can effectively target the bladder TME; this significantly reduces the myeloid cellular compartment and reverts an immunosuppressive to an immunopermissive TME, ultimately resulting in antitumor effects. These studies provide further rationale for the employment of NHS-IL12 as an immunomodulator and clinical immunotherapeutic for NMIBC.

// Jonathan Fallon 1,* , Robert Tighe 2,* , Giorgio Kradjian 2 , Wilson Guzman 2 , Anna Bernhardt 2 , Berend Neuteboom 2 , Yan Lan 2 , Helen Sabzevari 2 , Jeffrey Schlom 1 , John W. Greiner 1 1 Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland USA; 2 EMD Serono Research and Development Institute, Billerica, Massachusetts USA * These authors contributed equally to this work. Correspondence: Jeffrey Schlom, email: // Keywords : immunocytokine, interleukin-12, tumor necrosis therapy, T cells, immunotherapy Received : February 19, 2014 Accepted : March 23, 2014 Published : March 24, 2014 Abstract Targeted delivery of IL-12 might turn this cytokine into a safer, more effective cancer therapeutic. Here we describe a novel immunocytokine, NHS-IL12, consisting of two molecules of IL-12 fused to a tumor necrosis-targeting human IgG1 (NHS76). The addition of the human IgG1 moiety resulted in a longer plasma half-life of NHS-IL12 than recombinant IL-12, and a selective targeting to murine tumors in vivo . Data from both in vitro assays using human PBMCs and in vivo primate studies showed that IFN-gamma production by immune cells is attenuated following treatment with the immunocytokine, suggesting an improved toxicity profile than seen with recombinant IL-12 alone. NHS-IL12 was superior to recombinant IL-12 when evaluated as an anti-tumor agent in three murine tumor models. Mechanistic studies utilizing immune cell subset-depleting antibodies, flow cytometric methods, and in vitro cytotoxicity and ELISA assays all indicated that the anti-tumor effects of NHS-IL12 were primarily CD8+ T cell-dependent and likely IL-12-mediated. Combining NHS-IL12 treatment with a cancer vaccine, radiation, or chemotherapy resulted in greater anti-tumor effects than each individual therapy alone. These preclinical findings provide a rationale for the clinical testing of this immunocytokine, both as a single agent and in combination with vaccines, radiation and chemotherapy.

Targeted delivery of IL-12 might make this cytokine a safer, more effective cancer therapeutic. Here we evaluated whether fusing IL-12 to a tumor necrosis-targeting human IgG1 (NHS76) could improve its anti-tumor efficacy in vivo. In vivo imaging and immunohistochemistry staining were used to assess the ability of this novel immunocytokine (NHS-IL12) to target murine tumors. The anti-tumor efficacy of NHS-IL12 was examined at various doses and treatment schedules. Immune cell subset-depleting antibodies, flow cytometric methods, and in vitro cytotoxicity and ELISA assays were used to investigate the mechanisms of action of NHS-IL12. This immunocytokine was investigated in several models and in all cases NHS-IL12 outperformed recombinant IL-12 in anti-tumor activity. Depletion of immune cell subsets revealed that the anti-tumor activity depends primarily on CD8+ cells; mice that had complete tumor regression following NHS-IL12 treatment displayed durable anti-tumor memory responses. Combining NHS-IL12 treatment with a cancer vaccine, radiation, or chemotherapy resulted in greater anti-tumor effects than each individual therapy alone. These preclinical findings provide a rationale for the clinical testing of this immunocytokine, both as a single agent and in combination with vaccines, radiation, and chemotherapy. NHS-IL12 is currently being evaluated in a Phase 1 clinical trial (NCT01417546). Citation Format: Jonathan K. Fallon, Robert Tighe, Amanda J. Lyon, Helen Sabzevari, Jeffrey Schlom, John W. Greiner. The immunocytokine NHS-IL12 as a potential cancer therapeutic. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2573. doi:10.1158/1538-7445.AM2014-2573