Immunotherapies targeting programmed cell death protein-1 (PD-1) and programmed cell death ligand-1 (PD-L1) checkpoints elicit durable antitumoral effects in multiple cancer indications. Objective response rates of 20–30% in urothelial cancer and head and neck squamous cell carcinoma and 50–60% in melanoma and Merkel cell carcinoma have been achieved in treatment-naïve advanced-disease patients but were generally lower in pretreated patients [1, 2]. To explain the wide variation in responses to checkpoint inhibition, pharmacodynamic data obtained in various checkpoint inhibitor trials were analyzed, and the concept of a tumor immunity continuum was developed, differentiating between inflamed and noninflamed tumors [3]. Inflamed tumors are characterized by the presence of tumor-infiltrating CD8⁺ T cells, increased IFN-γ signaling, expression of PD-L1, and high tumor mutational burden (TMB). Noninflamed tumors are immunologically ignorant, are poorly infiltrated by lymphocytes, and rarely express PD-L1. In-between, there are tumors that, although immunogenic, show increased influence of immunosuppressive stroma, myeloid-derived suppressor cells (MDSCs) or M2 macrophages, each of which suppresses T cell activation within the tumor immune microenvironment (TIME) or prevents infiltration of T cells into the tumor (reviewed in [3, 4]).

Clinical responses to PD-(L)1 blockade correlate with the presence of intratumoral T cells [5, 6]. Accordingly, tumors nonresponsive to anti-PD-(L)1 therapy are either deficient of T cells or, if T cell-inflamed, comprise mainly T cells of an exhausted phenotype, experience immunosuppression by myeloid cells, or evade cytotoxic T cell recognition by downregulating antigen presentation [7–9]. Administration of anti-PD-(L)1 monotherapy to these patients appears to be ineffective, indicating the need to combine PD-(L)1 blockade with additional immunomodulating drugs.

HDAC inhibitors (HDACis) are epigenetic modifiers known to have pleiotropic effects that increase immune responses by enhancing expression of cancer-germline antigens (CGA), MHC class I and II molecules (MHC-I and -II), components of the antigen-processing machinery (APM), and T cell-recruiting chemokines [10–15]. Inhibition of class I HDACs is associated with reduced number and immunosuppressive function of MDSCs and regulatory T cells (Tregs) [16, 17]. In murine models treated with combinations of HDACi with PD-(L)1 blockade, antitumor activity was superior to single-agent therapy [14, 18–20]; however, the mechanisms associated with these effects, particularly in tumors of different immunophenotypes, have not been fully elucidated. Although epigenetic drugs are being evaluated in combination with immunotherapy in several clinical trials, translational data on the immunomodulatory effects of class I-selective HDACis are scarce. In breast cancer patients, the number of peripheral MDSCs was significantly reduced upon treatment with the HDACi entinostat and the aromatase inhibitor exemestane [21].

Here, we studied the immunomodulatory effects of the class I-selective oral HDACi domatinostat (4SC-202). Domatinostat was previously tested in 24 patients with advanced hematological malignancies (phase I trial NCT01344707). Signs of anticancer activity were observed, including one patient with a complete response, one patient with a partial response and 18 patients with disease stabilization as the best overall response. Domatinostat was well tolerated, showing an acceptable safety profile [22].

To characterize the immune-related effects of domatinostat, two mouse syngeneic tumor models with low intrinsic response to checkpoint therapy and different levels of T cell infiltration were analyzed for immunologic changes in the TIME and antitumor activity. In both tumor models, domatinostat increased the number of intratumoral cytotoxic CD8⁺ T cells (cytotoxic T lymphocytes, CTLs), with the relative effect being more pronounced in tumors with low levels of pre-existing CTLs. In CTL-low tumors, domatinostat substantially induced the expression of Ifng, IFN-γ response genes and the PD-1 blockade response signature. In CTL-high tumors, domatinostat significantly increased CTLs expressing activation and proliferation markers, even within the PD-1/LAG3-double-positive CTL subpopulation.

In combination with PD-(L)1 blockade, domatinostat was able to significantly increase tumor response rates and survival of animals, particularly in tumors with high CTL levels. Likewise, double blockade of the inhibitory immune checkpoint receptors PD-1 and LAG3 or agonistic targeting of the costimulatory receptor 4-1BB augmented the antitumor effects of domatinostat in the CTL-high in vivo model.

Gene expression analysis of patient-derived melanoma biopsies after 14 days of domatinostat treatment (ongoing phase I/II trial NCT03278665) demonstrated increased expression of IFNG, the 10-gene IFN-γ signature, the pembrolizumab response signature, and APM/MHC genes and increased immune cytolytic activity scores in individual samples compared with baseline.

In summary, our data provide mechanistic insights into the immunomodulatory effects of domatinostat in cancer, supporting further clinical development of domatinostat in combination with immunotherapy.

HDACis are known to upregulate the expression of CGA, MHC-I and -II, APM and chemokine genes, which are associated with enhanced immunogenicity and improved recognition of tumor cells by T cells [10–15]. In addition, some HDACis were shown to reduce the number and function of immunosuppressive cells [16, 17]. Overall, HDACis induce changes in the TIME that support antitumoral immune responses [18–20] and may thus be ideal candidates for combination with cancer immunotherapies.

Domatinostat is a class I-selective HDACi currently in clinical development for the treatment of advanced cutaneous melanoma (NCT03278665) and gastrointestinal cancer (NCT03812796). In these trials, domatinostat is administered in combination with PD-1 and PD-L1 blockade, respectively. To characterize potential antitumor and immunologic effects, we performed a series of in vivo experiments with domatinostat alone and in combination with immunotherapies using mouse syngeneic tumor models of low intrinsic response to PD-(L)1 blockade and different levels of T cell infiltration.

T cell immunity requires the recognition of antigens. Tumor escape mechanisms thus comprise impaired tumor antigen expression, malfunctioning of the APM and/or surface presentation of peptides by MHC-I molecules, preventing recognition and binding of CTLs and subsequent destruction of tumor cells [35]. Domatinostat increased the expression of CGA, APM and MHC-I genes both in vitro and in vivo, addressing these particular resistance mechanisms. Interestingly, domatinostat also increased MHC-II molecules on tumor and immune cells in vivo. The expression of MHC-II and costimulatory molecules on tumor cells, particularly within a proinflammatory TIME, was shown to augment tumor-specific CTL and T-helper responses, leading to tumor rejection and protective long-term and memory immune responses [36]. In triple-negative breast cancer and colorectal carcinoma, MHC-II expression has correlated with a favorable prognosis of patients [37, 38]. In anti-PD-1-treated melanoma patients, MHC-II positivity in addition to ubiquitous MHC-I expression has been associated with CD4⁺ and CD8⁺ T cell infiltrates and has been predictive for the response to PD-1 blockade and overall survival [39]. Upregulation of MHC-II molecules by domatinostat may thus enhance antitumoral immune responses in vivo.

IFN-γ signaling supports antitumor immune responses in several ways. It can upregulate the expression of MHC molecules and APM in both tumor and immune cells [40], promote tumoricidal activity of macrophages [41], and be crucial for T and NK cell trafficking into tumors through induction of the chemokines CXCL10 and − 11 [42]. In melanoma patients, a 10-gene IFN-γ-related gene signature has been associated with response to PD-1 blockade and clinical benefit [5]. In CT26 tumors, treatment with domatinostat increased the expression of Ifng and IFN-γ response genes, which are known to enhance inflammation and support immune responses against tumor cells. While domatinostat directly affected APM/MHC genes, the upregulation of IFN-γ did not seem to be an immediate effect of domatinostat on Ifng gene expression. Although domatinostat increased intratumoral expression of Ifng and IFN-γ target genes in vivo, it neither upregulated their expression in the CT26 cell line nor induced IFN-γ in isolated peripheral blood mononuclear cells (PBMCs) in vitro (Additional file 2: Figure S7), suggesting an indirect effect. In CT26 tumors, elevated Ifng gene expression was associated with increased intratumoral CTLs, which may be the source of IFN-γ in vivo. The combination of domatinostat with IFN-γ in vitro resulted in a stronger upregulation of MHC-I than either agent alone (Additional file 2: Figure S1c,d), indicating a possible synergistic effect of domatinostat and IFN-γ on MHC expression in the TIME.

In vivo, domatinostat induced 8- and 1.6-fold increases in cytotoxic T cells, resulting in CTL levels of ~ 1 and 22% of the total tumor cells, in CTL-low CT26 and CTL-high C38 tumors, respectively. Despite high baseline CTL levels, C38 tumors have shown limited responses to PD-(L)1 blockade, suggesting impaired functionality of CTLs. T cells persistently exposed to inflammatory signals or antigenic stimulation are known to become exhausted over time [43]. T cell exhaustion is characterized by the coexpression of several inhibitory checkpoints, including PD-1 and LAG3. Indeed, a proportion of CTLs expressed PD-1, LAG3 or both, indicating previous antigen-specific activation and emerging exhaustion of T cells in our study. Since C38 fragments are used for passaging the tumor between animals, the cells might have reached a “mature” immune phenotype promoting T cell exhaustion in this model.

Although ~ 20% of CTLs were proliferating (3.2% Ki67⁺ within the 13.5% of total cells that were CTLs) in vehicle-treated C38 tumors, no tumor control was achieved. Domatinostat strongly increased the percentage of activated and proliferating CTLs without affecting the absolute number of nonactivated CTLs (non-EM, CD69⁻, GITR⁻, PD-1⁻, LAG3⁻) or the expression levels of the inhibitory receptors PD-1 and LAG3 on CTLs positive for these markers (Additional file 2: Figure S5a,b). While PD-1 blockade had no effects (Additional file 2: Figure S5c), domatinostat significantly increased the number of proliferating CTLs within the PD-1⁺/LAG3⁺ subpopulation, indicating a beneficial effect of domatinostat on the functionality of CTLs coexpressing these exhaustion markers.

Overall, domatinostat increased not only the overall number of CTLs but also the number of activated and proliferating CTLs of the EM phenotype. Domatinostat thus induced the generation of functional, tumor-specific T cells necessary for efficacious antitumor immune responses. Indeed, mean tumor volumes decreased significantly on domatinostat monotherapy compared with vehicle in both syngeneic mouse models.

The observed actions of domatinostat on the TIME together with the known mechanisms of resistance to immunotherapy indicate a high potential value of its combination with immune checkpoint blockade. This was further supported by the intratumoral upregulation of genes associated with responses to pembrolizumab [5] and nivolumab [27] in CT26 tumors treated with domatinostat. Therefore, different regimens were tested in vivo for their antitumor effects. The combination of domatinostat and PD-(L)1 blockade significantly prolonged survival in animals with CT26 and C38 tumors, with 10 and 56% of the animals being completely tumor-free at the end of the study, respectively. Combination therapy was thus efficacious in both tumor immunophenotypes, with a higher benefit in tumors with pre-existing CTLs.

In contrast to other cancer indications, the expression of MHC-II is correlated with poor prognosis in melanoma [44]. MHC-II is a ligand of the inhibitory checkpoint receptor LAG3, which is substantially expressed on melanoma-infiltrating T cells [45]. Domatinostat increased the expression of MHC-II on both tumor and immune cells, which is beneficial for CD4⁺ T cell priming in principle; however, interactions with the LAG3 receptor could subvert the CD4⁺ T cell response against the tumor [46]. Hence, we hypothesized that blocking LAG3 in addition to PD-1 blockade may increase tumor-specific T cell responses promoted by domatinostat. Indeed, the triple combination of domatinostat, anti-PD-1 and anti-LAG3 resulted in an increased response rate compared with the corresponding mono- or double therapies in the C38 tumor model.

In addition to the inhibitory receptors PD-1 and LAG3, the costimulatory receptor 4-1BB (CD137) is also highly expressed on exhausted T cells [32]. Stimulation of 4-1BB was shown to increase T cell responses and improve the antitumor effects of PD-1 blockade in vivo by improving T cell metabolic and respiratory capacities [47, 48]. In the C38 tumor model, the combination of domatinostat with an agonistic 4-1BB antibody doubled the tumor response rate over anti-4-1BB alone, further emphasizing the ability of domatinostat to increase the functional activity of CTLs.

Translational data on the immunologic effects of HDACis are scarce so far. Entinostat, like domatinostat a class I-selective HDACi, was shown to reduce the number and function of MDSCs in murine models in which combination therapies with checkpoint inhibitors were evaluated [48]. Based on this mode of action, peripheral MDSCs were evaluated in advanced breast cancer patients treated with a combination of entinostat and exemestane [21]. Blood samples collected after two weeks of therapy revealed significantly decreased MDSCs. These findings were confirmed in an ongoing trial administering entinostat to patients with advanced solid tumors [49]. Of note, following domatinostat treatment, we observed a slight reduction in MDSCs in CT26 tumors, but not in blood (Additional file 2: Figure S2c,d). To the best of our knowledge, there are no published data describing immunological changes in tumors of patients treated with class I-selective HDACis as monotherapy.

To characterize the effects of domatinostat in humans, biopsies from 6 patients with advanced melanoma (ongoing phase I/II trial SENSITIZE) were analyzed before and after 14 days of domatinostat therapy. Immune cell composition at baseline illustrated considerable heterogeneity between the patients. Nevertheless, clinical gene expression data of baseline versus treated tumor lesions confirmed our previous murine in vivo findings. Domatinostat increased IFNG expression in 5/6 patients, the scores for the 10-gene IFN-γ-related and pembrolizumab response signatures in 4/6 patients, and APM/MHC and cytolytic activity expression in 3/6 patients each. Of note, the patient with the lowest presence of immune cells in the pretreatment biopsy showed the highest upregulation of gene expression in all scores applied. The low number of samples does not allow statistically reliable interpretations. Nonetheless, although preliminary, the data obtained from patients treated with 100 mg domatinostat once daily in the first-dose cohort of the trial further support the development of domatinostat in combination with cancer immunotherapy.

In summary, we have demonstrated that domatinostat increased both the overall number of CTLs and the number of functional cytotoxic T cells, which may effectively target tumor cells and ensure the induction of antitumor immune responses. In addition, domatinostat enhanced the expression of CGA, APM and MHC-I and -II genes both in vitro and in vivo. These effects may increase the immunogenicity of tumor cells and support the recognition of tumor cells by CD4⁺ and CD8⁺ T cells. By inducing Ifng and IFN-γ-related genes, domatinostat established a proinflammatory TIME, which is known to reinforce immune responses against tumor cells. Specifically, domatinostat induced the expression of genes associated with responses to pembrolizumab and nivolumab, further supporting the suitability of domatinostat for combination therapy with PD-1 blockade.

Domatinostat monotherapy exhibited antitumor activity in all in vivo systems analyzed. In combination with PD-(L)1 blockade, domatinostat augmented the antitumor effects substantially above the effects observed for single-agent therapies, with a greater benefit in tumors with pre-existing CTLs. In this setting, combination of domatinostat with the agonistic 4-1BB antibody or with both PD-1 and LAG3 blockade further increased the antitumor efficacy.

Based on the observed preliminary translational immunomodulatory effects of domatinostat, synergy with immune checkpoint inhibition may also be expected in patients. Since there is an urgent need to increase response rates and improve survival in patients on immunotherapy, combination with domatinostat is a potential treatment option for cancer patients.