IL-12 is a potent immunostimulatory cytokine, but its impact as an antitumor drug in clinical practice is limited. Upsurge of regulatory T cells (Treg) in the tumor milieu has been proposed to limit the efficacy of the treatment. In this paper, two drugs (cyclophosphamide [CPA] and anti-CD25 mAb) widely used to eliminate Treg were used in an attempt to enhance the antitumor effect of IL-12 gene therapy. Both anti-CD25 and CPA combined with IL-12 were able to deplete intratumoral Treg and myeloid-derived suppressor cells (MDSC), but only IL-12 plus CPA achieved significant antitumor activity in mice with large established s.c. colon carcinoma. This therapeutic effect was associated with the emergence of a heterogeneous population of myeloid cells within the tumor, termed inflammatory myeloid cells (IMC), composed of Ly6ChighLy6Glow inflammatory monocytes and Ly6GhighLy6C+ neutrophils. IMC showed a distinctive pattern of cytokine/chemokine production, and in contrast to MDSC, they did not induce conversion of naive CD4+ T cells into Treg. The appearance of IMC coincided with intense tumor infiltration by effector T cells, which was abrogated by elimination of IMC by anti-Gr1 mAb, a maneuver that abolished the antitumor effect of the therapy. Therefore, the combination of IL-12 and CPA eliminates intratumoral Treg and MDSC, while it induces the appearance of IMC within the tumor microenvironment. The latter effect is essential to facilitate effector T cell infiltration and subsequent tumor elimination.
Interferon alpha (IFNα) is widely used for the treatment of viral hepatitis but substantial toxicity hampers its clinical use. In this work, we aimed at improving the efficacy of IFNα therapy by increasing the IFNα half‐life and providing liver tropism. We selected apolipoprotein A‐I (ApoA‐I) as the stabilizing and targeting moiety. We generated plasmids encoding IFNα, albumin bound to IFNα (ALF), or IFNα linked to ApoA‐I (IA) and mice were treated either by hydrodynamic administration of the plasmids or by injection of the corresponding recombinant proteins or high‐density lipoproteins containing IA. The plasma half‐life of IA was intermediate between IFNα and ALF. IA was targeted to the liver and induced higher hepatic expression of interferon‐stimulated genes than IFNα or even ALF. IA exhibits stronger in vivo antiviral activity than IFNα and the hematologic cytopenic effects of IA are milder than those observed when using IFNα or ALF. In contrast to IFNα, IA does not cause activation‐dependent cell death of lymphocytes in vitro. Accordingly, in vivo studies showed that IA boosts T‐cell immune responses more efficiently than IFNα or ALF. The difference in immunostimulatory activity between IFNα and IA disappears in scavenger receptor class B type I (SR‐BI) knockout mice, suggesting that crosstalk between SR‐BI and IFNα receptor is essential for enhanced induction of cytotoxic T cells by IA. Conclusion: Anchoring IFNα to ApoA‐I prolongs the half‐life of IFNα and promotes targeting to the liver. Importantly, the fusion protein shows increased immunostimulatory properties and lower hematological toxicity. (HEPATOLOGY 2011;)
Background & Aims Current HBV management is challenging as treatment with nucleos(t)ide analogs needs to be maintained indefinitely and because IFNα therapy is associated with considerable toxicity. Previously we showed that linking IFNα to apolipoprotein A-I generates a molecule (IA) with distinct antiviral and immunostimulatory activities which lacks the hematological toxicity of IFNα. Methods Here, we analyze the antiviral potential of an adeno-associated vector encoding interferon alpha fused to apolipoprotein A-I (AAV-IA) in comparison to a vector encoding only IFNα (AAV-IFN) in two animal models of chronic hepadnavirus infection. Results In HBV transgenic mice, we found that both vectors induced marked reductions in serum and liver HBV DNA and in hepatic HBV RNA but AAV-IFN caused lethal pancytopenia. Woodchucks with chronic WHV infection that were treated by intrahepatic injection of vectors encoding the woodchuck sequences (AAV-wIFN or AAV-wIA) experienced only a slight reduction of viremia which was associated with hematological toxicity and high mortality when using AAV-wIFN while AAV-wIA was well tolerated. However, when we tested AAV-wIA or a control vector encoding woodchuck apolipoprotein A-I (AAV-wApo) in combination with entecavir, we found that AAV-wApo-treated animals exhibited an immediate rebound of viral load upon entecavir withdrawal while, in AAV-wIA-treated woodchuks, viremia and antigenemia remained at low levels for several weeks following entecavir interruption. Conclusions Treatment with AAV-IA is safe and elicits antiviral effects in animal models with difficult-to-treat chronic hepadnavirus infection. AAV-IA in combination with nucleos(t)ide analogs represents a promising approach for the treatment of HBV infection in highly viremic patients.
Semliki Forest virus (SFV) represents a promising gene therapy vector for tumor treatment, because it produces high levels of recombinant therapeutic proteins while inducing apoptosis in infected cells. In this study, we constructed a SFV vector expressing murine interferon alpha (IFNa). IFNa displays antitumor activity mainly by enhancing an antitumor immune response, as well as by a direct antiproliferative effect. In spite of the antiviral activity of IFNa, SFV-IFN could be produced in BHK cells at high titers. This vector was able to infect TC-1 cells, a tumor cell line expressing E6 and E7 proteins of human papillomavirus, leading to high production of IFNa both in vitro and in vivo. When injected into subcutaneous TC-1 tumors implanted in mice, SFV-IFN was able to induce an E7-specific cytotoxic T lymphocyte response, and to modify tumor infiltrating immune cells, reducing the percentage of T regulatory cells and activating myeloid cells. As a consequence, SFV-IFN was able to eradicate 58% of established tumors treated 21 days after implantation with long-term tumor-free survival and very low toxicity. SFV-IFN was also able to induce significant antitumor responses in a subcutaneous tumor model of murine colon adenocarcimoma. These data suggest that local production of IFNa by intratumoral injection of recombinant SFV-IFN could represent a potent new strategy to treat tumors in patients.
BackgroundModified vaccinia virus Ankara (MVA) are genetically engineered non-replicating viral vectors. Intratumoral administration of MVA induces a cyclic GMP-AMP synthase-mediated type I interferon (IFN) response and the production of high levels of the transgenes engineered into the viral genome such as tumor antigens to construct cancer vaccines. Although type I IFNs are essential for establishing CD8-mediated antitumor responses, this cytokine family may also give rise to immunosuppressive mechanisms.MethodsIn vitro assays were performed to evaluate the activity of simvastatin and atorvastatin on type I IFN signaling and on antigen presentation. Surface levels of IFN α/β receptor 1, endocytosis of bovine serum albumin-fluorescein 5 (6)-isothiocyanate, signal transducer and activator of transcription (STAT) phosphorylation, and real-time PCR of IFN-stimulated genes were assessed in the murine fibroblast cell line L929. In vivo experiments were performed to characterize the effect of simvastatin on the MVA-induced innate immune response and on the antitumor effect of MVA-based antitumor vaccines in B16 melanoma expressing ovalbumin (OVA) and Lewis lung carcinoma (LLC)-OVA tumor models. RNAseq analysis, depleting monoclonal antibodies, and flow cytometry were used to evaluate the MVA-mediated immune response.ResultsIn this work, we identified commonly prescribed statins as potent IFNα pharmacological inhibitors due to their ability to reduce surface expression levels of IFN-α/β receptor 1 and to reduce clathrin-mediated endocytosis. Simvastatin and atorvastatin efficiently abrogated for 8 hours the transcriptomic response to IFNα and enhanced the number of dendritic cells presenting an OVA-derived peptide bound to major histocompatibility complex (MHC) class I. In vivo, intraperitoneal or intramuscular administration of simvastatin reduced the inflammatory response mediated by peritumoral administration of MVA and enhanced the antitumor activity of MVA encoding tumor-associated antigens. The synergistic antitumor effects critically depend on CD8+ cells, whereas they were markedly improved by depletion of CD4+ lymphocytes, T regulatory cells, or NK cells. Either MVA-OVA alone or combined with simvastatin augmented B cells, CD4+ lymphocytes, CD8+ lymphocytes, and tumor-specific CD8+ in the tumor-draining lymph nodes. However, only the treatment combination increased the numbers of these lymphocyte populations in the tumor microenvironment and in the spleen.ConclusionIn conclusion, blockade of IFNα functions by simvastatin markedly enhances lymphocyte infiltration and the antitumor activity of MVA, prompting a feasible drug repurposing.
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