6 Abscopal effect after intratumoral ISF35 with anti-CTLA-4 and anti-PD-1 antibody blockade. brain. Therapeutic efficacy is associated with increases in the systemic level of tumor-specific CD8+ T cells, and an increased ratio of intratumoral CD8+ T cells to CD4+ Tregs. These results provide a proof of concept of systemic antitumor activity after intratumoral CD40 triggering with ISF35 in combination with checkpoint blockade for multifocal cancer, including the brain. Introduction Although surgical resection is a reliable treatment for localized melanoma, treatment options for metastatic melanoma are limited. Brain metastasis is a major clinical problem in patients with advanced melanoma1, and the incidence of brain metastasis is increasing yearly. Immunotherapies, like T cell checkpoint blockade with anti-CTLA-4 and anti-PD-1antibodies, have improved the median survival of patients with metastatic melanoma and brain melanoma. However, the majority of patients are still not cured by these therapies2, leaving Leupeptin hemisulfate a need for more effective melanoma therapy. One strategy to enhance the efficacy of checkpoint blockade therapy is to increase the frequency of tumor-specific T cells, for example, by antitumor vaccination. A critical aspect for successful tumor vaccines is the selection of suitable tumor antigens. However, it can be difficult to find antigens with high, tumor-restricted expression in a sufficiently large fraction of patients to allow for development of a commercially viable vaccine therapy3. An alternative strategy is to use immunomodulators that directly activate innate and adaptive immune cells in the tumor microenvironment, facilitating the generation of T cells against often-unique neoantigens encoded by tumor-specific mutations. We and others have reported that intratumoral Leupeptin hemisulfate immunotherapies induce systemic, tumor-specific T cell responses that can target metastases, distant from the initially treated tumor mass, making this a promising approach for the treatment of metastatic cancers4C6. Activation of tumor-specific T cell responses has been shown to require activation of the CD40 receptor on antigen-presenting cells7. In this regard, agonist CD40 antibody and the cognate CD40 ligand (CD40L) are candidates for tumor immunotherapy. Preclinical and clinical studies with agonist CD40 antibody have shown induction of antitumor immune responses and evidence of efficacy7, 8. However, systemically delivered CD40 agonists have resulted in various adverse effects during clinical testing, such as cytokine release syndrome and organ-specific toxicities9. ISF35 is a nonreplicating adenovirus encoding a humanCmouse chimeric, optimized form of CD40L that is a potent CD40 agonist10. ISF35 is delivered by intratumoral injection, resulting in membrane-bound CD40L expression that targets the CD40 receptor on antigen-presenting cells at the site of injection. Cell-surface trimeric CD40L, such as ISF35, results in enhanced CD40 receptor clustering, critical for optimal immune activation. This localized intratumoral ISF35 expression has not caused off-target adverse events such as cytokine release syndrome observed with systemic CD40 mAbs treatment, likely due to its induction of membrane-bound CD40L that is not cleaved Leupeptin hemisulfate into the circulation11. ISF35 has previously been tested as a monotherapy and in combination with chemo-immunotherapy for chronic lymphocytic leukemia patients, with signs of clinical efficacy and transient, mild adverse events consisting primarily of flu-like symptoms12. This makes intratumoral ISF35 a candidate for the induction of tumor-specific T cells, which could possibly synergize with checkpoint blockade therapy in melanoma and other cancers. To test this hypothesis, we examined the antitumor activity and mechanism of action of intratumoral ISF35 against injected and distant, uninjected tumors. We also examined if intratumoral ISF35 could overcome primary resistance to PD-1 and CTLA-4 checkpoint blockade therapy, including against tumors in the brain. Results Induction of antitumor immunity after ISF35 therapy Since melanoma tumors contain antigen-presenting cells which constitutively express CD40 on their surface, we hypothesized that the activation of these APCs with IFITM2 the CD40 agonist ISF35 might induce tumor-specific CD8 T cell immunity and effective treatment of local and disseminated tumors. Intratumoral injection of ISF35 into s.c. B16.F10 melanomas significantly suppressed tumor growth and prolonged the survival of mice compared to either control-recombinant adenovirus or saline control-treated mice (test, Fig.?1aCc). ISF35 also inhibited tumor growth and prolonged mouse survival significantly better than the intratumoral-injected agonist CD40 mAb (test; Fig.?1c). To ensure that antitumor activity of ISF35 was not limited to B16 melanoma, we also used ISF35 to treat established BP melanomas derived from the test or one-way ANOVA. *test or one-way ANOVA (test or one-way ANOVA. *test. *test; Supplementary Fig.?2b); however, in contrast to triple combination, dual combination was unable to cure the mice from tumor (Fig.?6c Leupeptin hemisulfate and Supplementary Fig.?2b). Open in a separate window Fig. 6 Abscopal effect after intratumoral ISF35 with anti-CTLA-4 and anti-PD-1 antibody blockade. a Treatment strategy. b Growth of injected and distant, uninjected B16.F10 tumors (test or one-way ANOVA. *test, and.