Background: The efficacy of PD-(L)1 blockade depends on the composition of the tumor immune microenvironment (TIME) and is generally higher in tumors with pre-existing cytotoxic T cells (CTL) than in those with low CTL numbers. Nonetheless, a significant proportion of patients with pre-existing immunity fail to respond, indicating a therapeutic potential for combining PD-(L)1 blockade with additional immunomodulatory agents in both CTL-high and-low immune phenotypes. Here, we evaluated domatinostat (4SC-202), a class I-selective histone deacetylase (HDAC) inhibitor, for its effect on the TIME and its antitumoral efficacy using syngeneic mouse models with CTL-high or CTL-low tumors. Methods: Domatinostat was evaluated in PD-1 blockade-insensitive CTL-low (CT26) and CTL-high (C38) syngeneic models alone and in combination with different immune-inhibitory and-stimulatory approaches. Effects on the immunophenotype were assessed via flow cytometry and RNA-seq analyses. The changes in RNA-seq-based immune signatures determined in a murine setting were investigated in patient samples from the first-dose cohort of the SENSITIZE trial (NCT03278665) evaluating domatinostat combined with pembrolizumab in advanced-stage melanoma patients refractory/nonresponding to PD-1 blockade. Results: Domatinostat increased the expression of antigen-presenting machinery (APM) genes and MHC class I and II molecules, along with CTL infiltration, in tumors of both immune phenotypes. In combination with PD-(L)1 blockade, domatinostat augmented antitumor effects substantially above the effects of single-agent therapies, displaying greater benefit in tumors with pre-existing CTLs. In this setting, the combination of domatinostat with agonistic anti-4-1BB or both PD-1 and LAG3 blockade further increased the antitumor efficacy. In CTL-low tumors, domatinostat enhanced the expression of genes known to reinforce immune responses against tumors. Specifically, domatinostat increased the expression of Ifng and genes associated with responses to pembrolizumab and nivolumab. Clinically, these findings were confirmed in patients with advanced melanoma treated with domatinostat for 14 days, who demonstrated elevated expression of APM and MHC genes, the IFNG gene, and the IFN-γ and pembrolizumab response signatures in individual tumor samples. Conclusion: In summary, these data suggest a promising potential of domatinostat in combination with immunotherapy to improve the outcome of refractory cancer patients.

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Various histone deacetylases (HDAC) inhibitors were described as beneficially affecting anti-tumoral immune response. Although different HDAC inhibitors were investigated in syngeneic tumor models, their mode of anti-tumoral action (MOA) is not yet fully understood. Here, we analyzed the anti-tumoral MOA of 4SC-202, an orally available clinical stage small molecule inhibitor targeting HDAC class I. To ensure the relevance for the clinical situation we used a clinically equivalent dosage regimen. 4SC-202's effect on expression of tumor-associated antigen (TAA) and MHC molecules was analyzed in vitro and in vivo. Anti-tumoral efficacy and the impact on tumor microenvironment (TME) were analyzed in syngeneic CT26 and C38 models. Transcriptome analysis was performed by RNA-Seq, and the composition of immune cell subpopulations was determined by flow cytometry and immunohistochemistry. 4SC-202 increased expression of TAA and MHC molecules on tumor cells in vitro and in vivo suggesting a beneficial effect on immunogenicity of tumor cells. 4SC-202 significantly inhibited growth of syngeneic tumors at a dose that was inefficacious in immunocompromised mice. IFN-γ and chemokine expression was increased, and pro-inflammatory IL-1β and IL-23 decreased in the TME of CT26 tumors following 4SC-202 treatment. Detailed analysis revealed that 4SC-202 increased the number of cytotoxic CD8+ T cells (CTLs) in the tumor core without affecting their number in blood. Since the abundance of T cells in the tumor is pre-requisite for the efficacy of immune checkpoint blockade as well as the agonistic 4-1BB antibody, combinations of 4SC-202 with anti-PD-1, and anti-4-1BB antibodies were tested in the C38 model. The response rate to the antibodies alone was low in this model reflecting the refractory clinical situation. 4SC-202 was able to control the tumor growth, but did not induce tumor regression, whereas combination therapies resulted in significantly longer survival and durable complete responses in up to 83% of animals for the combination with the anti-PD-1 antibody. 4SC-202 already demonstrated a favorable safety profile with a low rate of > grade 3 treatment-related adverse events (17%) in a phase I study in heavily pretreated hematological cancer patients. With the daily dosing for 14 consecutive days a disease control rate of 83%, 1 partial and 1 complete response could be achieved. Complimentary to these clinical data, 4SC-202's immune priming capacity offers further options for development in combination with various cancer immunotherapy approaches. Combination of 4SC-202 with PD-1 blockade is now under evaluation in a phase Ib/II clinical trial in advanced cutaneous melanoma patients refractory/non-responding to treatment with anti-PD-1 antibodies (‘SENSITIZE', NCT03278665). Citation Format: Svetlana Hamm, Tanja Wulff, Kerstin Kronthaler, Sabine Schrepfer, Ulrike Parnitzke, Anne Catherine Bretz, René Bartz. 4SC-202 increases immunogenicity of tumor cells, induces infiltration of tumor microenvironment with cytotoxic T cells, and primes tumors for combinations with different cancer immunotherapy approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4722.