Cooperative Immune-Mediated Mechanisms of the HDAC Inhibitor Entinostat, an IL15 Superagonist, and a Cancer Vaccine Effectively

Published OnlineFirst October 23, 2019; DOI: 10.1158/1078-0432.CCR-19-0727

CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY

Cooperative Immune-Mediated Mechanisms of the HDAC Inhibitor Entinostat, an IL15 Superagonist, and a Cancer Vaccine Effectively Synergize as a Novel Cancer Therapy Kristin C. Hicks1, Karin M. Knudson1, Karin L. Lee1, Duane H. Hamilton1, James W. Hodge1, William D. Figg2, Peter Ordentlich3, Frank R. Jones4, Shahrooz Rabizadeh4,5,6, Patrick Soon-Shiong4,5,6, Jeffrey Schlom1, and Soﬁa R. Gameiro1

ABSTRACT ◥ Purpose: Immunotherapy has demonstrated clinical efficacy in minimal levels of granzyme B. For the first time, we demonstrate subsets of patients with solid carcinomas. Multimodal therapies that the addition of entinostat to N-803 plus vaccine promoted using agents that can affect different arms of the immune system significant tumor control, correlating with increased expression of and/or tumor microenvironment (TME) might increase clinical genes associated with tumor inflammation, enhanced infiltration of þ responses. activated CD8 T cells with maximal granzyme B, T-cell responses Experimental Design: We demonstrate that entinostat, a to multiple tumor-associated antigens, increased serum IFNg, class I histone deacetylase inhibitor, enhances the antitumor reduction of regulatory T cells in the TME, and decreased expression efficacy of the IL15 superagonist N-803 plus vaccine in 4T1 of the checkpoint V-domain Ig suppressor of T-cell activation triple-negative breast and MC38-CEA colon murine carcinoma (VISTA) on multiple immune subsets. models. A comprehensive immune and gene-expression analysis Conclusions: Collectively, these data demonstrate that the was performed in the periphery and/or TME of MC38-CEA synergistic combination of entinostat, N-803, and vaccine elicits tumor–bearing mice. potent antitumor activity by generating a more inflamed TME. þ Results: Although N-803 plus vaccine induced peripheral CD8 These findings thus form the rationale for the use of this T-cell activation and cytokine production, there was no reduction in combination of agents for patients harboring poorly or nonin- þ tumor burden and poor tumor infiltration of CD8 T cells with flamed solid carcinomas.

Introduction nostimulatory cytokine N-803, and the histone deacetylase inhibitor (HDACi) entinostat. Immunotherapy has induced unprecedented long-lasting clinical To promote antigen-specific immune responses, we utilized ade- responses across multiple carcinoma types (1). However, the majority noviral vaccines targeting the TAAs human carcinoembryonic antigen of patients fail to respond or develop resistance. Clinical benefit may be (CEA) or murine Twist1. This novel recombinant adenoviral vaccine achieved by rationally combining immunotherapies to simultaneously platform has previously been described (2, 3). CEA has been shown to target multiple components of the immune system and tumor micro- be highly expressed in multiple human carcinomas, but not murine (4). environment (TME), shifting the balance of immune suppression and In order to target human CEA in mice, we implanted MC38 cells stably activation to promote a more inflamed TME. This encompasses transfected with human CEA into CEA-transgenic mice (CEA-Tg) generating and expanding tumor antigen–specific immune responses, andvaccinatedagainstCEAtoovercomeimmunetolerancetothe increasing their cytolytic potential, and promoting their infiltration now murine self-antigen CEA (5). Vaccines targeting CEA have into the TME. To this end, we utilized the combination of a therapeutic been shown to induce CEA-specific T cells and delay tumor growth cancer vaccine against a tumor-associated antigen (TAA), the immu- in the absence of autoimmunity (3, 6–8). Alternatively, we vaccinated against Twist1, a human and murine transcription factor associated with the metastatic process and poor patient progno- sis (9). We previously demonstrated that vaccinating against Twist1 1 Laboratory of Tumor Immunology and Biology, Center for Cancer Research, reduces tumor growth (10). 2 fi NCI, NIH, Bethesda, Maryland. Clinical Pharmacology Program, Of ce of the To expand antigen-specific T cells generated from vaccination and/ Clinical Director, Center for Cancer Research, NCI, NIH, Bethesda, Maryland. 3Syndax Pharmaceuticals, Inc., Waltham, Massachusetts. 4ImmunityBio, Culver or cascade responses, we utilized N-803, comprising an IL15 super- City, California. 5NantOmics, LLC, Culver City, California. 6NantWorks, Culver agonist (IL-15N72D) bound to an IL15 receptor alpha/IgG1 Fc fusion City, California. protein (formerly ALT-803). N-803 has enhanced biological activity and a substantially longer half-life than native IL15 (11). Additionally, Note: Supplementary data for this article are available at Clinical Cancer þ Research Online (http://clincancerres.aacrjournals.org/). N-803 increases CD8 T-cell and natural killer (NK) cell numbers and fi J. Schlom and S.R. Gameiro contributed equally to this article. function to promote antitumor ef cacy in multiple murine tumor models (11–13). Currently, N-803 is being examined clinically in Corresponding Author: Jeffrey Schlom, NCI, 10 Center Drive, Building 10, Room combination with multiple therapies, including vaccine. 8B09, Bethesda, MD 20892. Phone: 240-858-3463; Fax: 240-541-4558; E-mail: [email protected] Mounting preclinical and clinical evidence suggests that combining HDAC inhibitors with immunotherapy generates potent antitumor Clin Cancer Res 2020;XX:XX–XX efficacy (14, 15). In particular, the class I HDACi entinostat reduces the doi: 10.1158/1078-0432.CCR-19-0727 number and/or function of immunosuppressive regulatory T cells 2019 American Association for Cancer Research. (Treg) or myeloid-derived suppressor cells (MDSC; refs. 16–20) and

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Facility (Frederick, MD). Mice were housed in microisolator cages Translational Relevance under pathogen-free conditions, in accordance with Association for Immunotherapy has demonstrated clinical efficacy in subsets of Assessment and Accreditation of Laboratory Animal Care guidelines. patients with solid carcinomas. Multimodal therapies using agents that can affect different arms of the immune system and/or tumor Murine tumor studies microenvironment might increase clinical responses. Here we MC38-CEA cells (3 105) were implanted subcutaneously (s.c.) demonstrate that entinostat, a class I HDAC inhibitor, synergizes into the right flank of CEA-Tg or NSG mice. 4T1 cells (5 104) with an IL15 superagonist (N-803) and vaccine to promote sig- were orthotopically implanted into the mammary fat pad of Balb/c nificant tumor control in both murine colon and triple-negative mice. Once tumors reached 25 mm3 on average, mice were ran- breast carcinoma models. Although vaccine/N-803 activated domized to receive control (Cntrl) or entinostat (ENT) diet alone, þ CD8 T cells in the periphery, these remained largely excluded or in combination with Ad-CEA or Ad-Twist vaccine (V, 1010 virus from the tumor. The addition of entinostat maximized activation, particles, s.c.). Vaccinated mice received vaccine boosts plus N-803 þ infiltration, and cytolytic function of CD8 T cells in the tumor (1 mg, s.c.) 14 and 21 days after tumor implant. Tumor volume was while reducing immune suppression in both the tumor and measured twice weekly and calculated by (length2 width)/2. For periphery. These data identify novel cooperative immune- tumor rechallenge, tumor cells were implanted as before into cured mediated mechanisms by which entinostat, an IL15 superagonist, mice and na€ve controls at least 1 month after tumor rejection. and a therapeutic vaccine synergize to generate a more inflamed Quantification of 4T1 lung metastasis was performed as previously tumor microenvironment, resulting in potent antitumor activity. described (26). These findings form the rationale for clinical translation for Additional materials and methods are described in the Supplemen- patients with solid carcinomas. tary Data, including Supplementary Table S1, listing the antibodies used for flow cytometry.

Statistical analysis alters the tumor phenotype to be more amenable to immune Statistical analyses were performed in GraphPad Prism 7 (Graph- killing (21–23). Therefore, we hypothesized that entinostat would Pad Software) and listed in figure legends. Statistical significance was increase the antitumor efficacy of N-803 combined with a vaccine. set at P < 0.05. , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001. Here, we demonstrate that this novel combination promotes significant antitumor efficacy in 4T1 triple-negative breast (TNB) and Study approval MC38-CEA colon carcinoma models. We provide a comprehensive All animal studies were conducted under approval of the NIH analysis of immune subsets in the spleen and TME, identifying how the Intramural Animal Care and Use Committee. effects of each therapy cooperate to produce significant antitumor efficacy. Associated pathways were investigated by gene-expression analysis of the TME. Notably, the combination therapy improved Results þ CD8 T-cell function, activation, and tumor infiltration. Overall, these Entinostat synergizes with N-803 and vaccine to control tumor findings provide a rationale for combining entinostat, N-803, and a growth therapeutic cancer vaccine in the clinical setting. As monotherapies, entinostat, N-803, and therapeutic cancer vaccines targeting TAAs have demonstrated significant, but not curative, antitumor efficacy in murine models of solid carcinomas Materials and Methods (3, 7, 10, 12, 16–22, 27–30). In the MC38-CEA colon cancer model, Tumor cell lines administration of N-803 with Ad-CEA vaccination produced sig- 4T1 murine TNB cancer cells from the American Type Culture nificant, although modest, antitumor efficacy (Supplementary Collection were maintained according to supplier recommendations. Fig. S1A). Using antigen-specific 9-mer peptides in an ELISPOT, MC38 murine colon carcinoma cells expressing human CEA (MC38- we examined T-cell responses against the vaccination antigen CEA) were generated and maintained as previously described (24). All CEA and the cascade antigens, janus kinase 1 (Jak1), prostaglandin cell lines were low passage and free of Mycoplasma as determined by F receptor (ptgfr), and the retroviral protein p15e. Jak1 and ptgfr MycoAlert Mycoplasma Detection Kit (Lonza). are two recently identified neoepitopes in this model (31). There was a trend toward N-803 plus vaccine generating superior antigen- Vaccines and reagents specific T-cell responses against the vaccination and cascade antigens The adenoviral Ad5 [E1-, E2b-]-CEA (Ad-CEA) vaccine was con- relative to either agent alone. More importantly, vaccine promoted structed as previously described (3). Under Cooperative Research and greater responses to neoantigens compared with N-803 alone (Sup- Development Agreements with the NCI, entinostat was provided plementary Fig. S1B). Given the immune altering effects of by Syndax, and N-803, Ad-CEA and Ad-Twist vaccines were provided entinostat (16–23), we hypothesized that its addition to N-803 plus by ImmunityBio. Entinostat was formulated in a low-fat diet with vaccine would synergistically enhance tumor control. Given the short 35% sucrose for target murine doses of 3, 6, 9, and 12 mg/kg/day half-life of entinostat in mice, entinostat was administered continu- (Research Diets). A dose of 12 mg/kg/day was used with CEA-Tg and ously in the diet to better mimic the longer half-life observed in cancer nod-scid-gamma (NSG) mice and 3 mg/kg/day for Balb/c mice. patients (32, 33). MC38-CEA tumor–bearing mice fed an entinostat Entinostat was quantified in plasma as previously described (25). diet had plasma levels of entinostat within the therapeutic window achieved in patients (Supplementary Fig. S1C; ref. 33). Animals Next, the antitumor efficacy of entinostat combined with N-803 and Six- to 8-week-old female Balb/c, NSG, and C57BL/6 CEA-Tg Ad-Twist vaccination was examined in the 4T1 TNB cancer murine mice (5) were obtained from the NCI Frederick Cancer Research model. Entinostat, N-803 plus vaccine, and the combination of the

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Figure 1. Entinostat synergizes with N-803 and vaccine to control tumor growth and enhance survival. A–C, 4T1 tumor cells were orthotopically implanted in Balb/c mice (day 0) and treated as depicted in the schematic. A, Treatment schedule and tumor volumes on day 26. B, Number of 4T1 lung colonies (day 27; n ¼ 17–18 mice/group). C, The table depicts the number and percentage of mice within each treatment group with the indicated range of 4T1 lung tumor colonies, the mean number of colonies, and percent reduction from control. D–F, MC38-CEA tumor cells were implanted s.c. in the right ﬂank of CEA-Tg mice and treated as depicted in the schematic. D, Primary tumor growth curves. E, Survival curves of combined data from 2 independent experiments; the table below depicts median OS in days (d) and number of cured mice (n ¼ 18–20 mice/group). F, Cured mice and na€ve CEA-Tg mice were implanted with MC38-CEA tumor cells. Graph shows survival data combined from 3 independent experiments. The table below depicts median OS and number of mice with memory response. G, On day 24, serum from MC38-CEA tumor–bearing mice was analyzed for cytokine levels. Dashed line indicates cytokine levels for na€ve mouse. Graphs show mean SEM from (A–C) 1 experiment or (D–G)2–3 experiments. , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001. A and D, Two-way ANOVA; B and G, one-way ANOVA; and E, Mantel–Cox.

three therapies minimally, but significantly, reduced primary tumor 25 days (control) to 55 days, curing 5% of mice (Fig. 1D and E). growth versus control (Fig. 1A). However, the combination generated This effect was immune mediated as entinostat did not affect tumor a greater reduction in the number of lung metastases (73%) than either growth in immune-deficient NSG mice (Supplementary Fig. S1D). therapy alone. Entinostat or N-803 plus vaccine reduced lung metas- Survival was further improved to 73 days with the combination, tasis by 42% and 46%, respectively (Fig. 1B and C). curing 28% of mice (Fig. 1E). Upon tumor rechallenge, 67% of The efficacy of this combination was assessed in a second tumor cured mice had a protective memory response (Fig. 1F). Overall, model using MC38-CEA. Although N-803 plus vaccine had no our data suggest that combining entinostat, N-803, and a tumor- significant antitumor effects, entinostat alone significantly delayed specific vaccine has significant antitumor efficacy in two distinct tumor growth and enhanced median overall survival (OS) from murine models of solid carcinomas.

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Combination therapy is well tolerated and promotes an Despite reduced Treg numbers, their proliferative capacity (Ki67) was immune-stimulatory cytokine profile increased by entinostat (Fig. 3C). Notably, treatment with entinostat Because combining multiple therapies can lead to adverse effects, alone or in combination reduced V-domain Ig suppressor of T-cell we monitored toxicity of the combination therapy in the MC38- activation (VISTA) levels on Tregs (Fig. 3C). VISTA is an immune CEAmodel.Miceshowednosignificant weight change during the checkpoint expressed on human and murine lymphocytes that course of treatment (Supplementary Fig. S2A). Via organ histopa- negatively regulates T-cell function and promotes conversion of þ thology, fatty changes to the liver were observed in mice treated CD4 T cells into Tregs (35–37). Also, VISTA has been implicated with entinostat alone or in combination, with no other treatment as an immune evasion mechanism for patients on immunothera- abnormalities identified (Supplementary Fig. S2B). Serum chemis- py (38, 39). Together, these data suggest that combination therapy is try analysis identified a modest, albeit significant elevation in reducing splenic Treg numbers and potentially decreasing their chloride and albumin in mice treated with entinostat alone, with immunosuppressive capacity. no significant effects observed with combination therapy (Supple- Entinostat has also been reported to reduce the number and mentary Fig. S2C). Of note, splenic cellularity was reduced with function of MDSCs (17–20). Entinostat treatment reduced the entinostat alone or in combination, which was associated with a number of splenic monocytic-MDSCs (M-MDSC) and granulo- large decrease in B cells (Supplementary Fig. S3A and S3B). This cytic-MDSCs (G-MDSC) due to the decreased splenic cellularity. effect is consistent with transient lymphopenia reported in patients However, the frequency of G-MDSCs, but not M-MDSCs, increased treated with entinostat (32, 33). Overall, the combination of entino- with entinostat treatment (Supplementary Fig. S4A and S4B). stat, N-803, and vaccine was well tolerated. Combination treatment also elevated programmed death-ligand 1 To deduce the mechanism of antitumor activity, we monitored (PD-L1) and decreased VISTA on both MDSC subsets (Supple- the systemic effects of treatment via serum cytokines. For all mentary Fig. S4A and S4B). mechanistic investigations in the MC38-CEA model, mice were Because entinostat reduced VISTA on both Tregs and MDSCs, this sacrificed on day 24 after tumor implant. Combination treatment effect was further interrogated by treating non–tumor-bearing CEA- significantly elevated the proinflammatory cytokines IFNg and Tg and Balb/c mice with different doses of entinostat. In both strains, þ þ TNFa 3-fold and 2-fold, respectively, whereas entinostat or N- entinostat significantly decreased VISTA on CD4 and CD8 T cells, 803 plus vaccine alone had no significant effect (Fig. 1G). The anti- G-MDSCs, and M-MDSCs (Supplementary Fig. S5). These data inflammatory cytokine IL10 was not altered relative to control mice, suggest a generalized mechanism by which entinostat reduces VISTA whereas the immunosuppressive cytokine TGFb1 was decreased levels on immune cells. Overall, these data suggest that entinostat alone 66% with entinostat alone or in combination (Fig. 1G). Entinostat or in combination is reducing the immune suppression in the periph- alone or in combination reduced CXCL1 (KC/GRO) serum levels to ery by reducing Tregs and VISTA expression. that of a na€ve mouse (Fig. 1G). Recently, ablation of CXCL1 was implicated in enhanced T-cell tumor infiltration and response to Combination therapy promotes a more active T-cell phenotype immunotherapy (34). These data suggest that entinostat is syner- in the spleen gizing with N-803 plus vaccine to promote a proinflammatory Because the decrease in immune suppression suggests that cytokine environment. T cells may be more active, we examined the effects on T cells. We observed that entinostat alone or in combination reduced CD4 þ CD8 T cells are required for the antitumor efficacy of the numbers due to decreased splenic cellularity. The proportion of þ combination therapy splenic CD4 T cells increased with entinostat (Supplementary To determine which immune effector subset was required for Fig. S6A). Combination therapy induced a more active phenotype þ antitumor efficacy of the combination therapy, the effect of depleting on CD4 T cells by increasing the activation marker CD25 and each cell type on MC38-CEA tumor growth and survival was exam- decreasing VISTA (Supplementary Fig. S6A and S6B). Combination ined. Depletion efficacy was confirmed in the blood (Fig. 2A–C). therapy produced similar effects in the blood of 4T1 tumor–bearing þ CD8 T cells were required for the antitumor efficacy of the combi- mice, reducing VISTA levels, while the frequency of activated þ nation therapy as OS decreased from 58.5 days with combination (CD44hi)ortotalCD4 T cells remained unchanged (Supplemen- therapy to 29 and 28 days with CD8 or CD8þNK depletion, respec- tary Fig. S6C). tively. Surprisingly, CD4 depletion cured 80% of combination-treated Combination therapy did not significantly alter the frequency of þ mice. NK cell depletion had no effect on tumor growth and survival. CD8 T cells, but a reduction in cell number was observed due to Notably, even with CD8þNK depletion, tumor growth was still decreased spleen cellularity (Fig. 3D). Both N-803 plus vaccine and significantly reduced compared with control mice, suggesting addi- combination therapy promoted a central memory phenotype in þ tional components may be providing minor antitumor benefits with splenic CD8 T cells, an effect previously reported with N-803 combination treatment (Fig. 2D and E). These data demonstrate that (Fig. 3E; refs. 12, 13, 40). Combination therapy promoted an active þ þ CD8 T cells are essential for the antitumor efficacy of the combina- phenotype on CD8 T cells by increasing CD44hi, CD25, and Ki67 and tion therapy. decreasing VISTA (Fig. 3F and G). With the exception of CD25, the activation phenotype was driven by N-803 plus vaccine as these effects In the spleen, entinostat decreased the number and inhibitory were not observed with entinostat alone. In addition, the ratio of þ phenotype of Tregs and reduced VISTA expression splenic CD8 T cells to Tregs was significantly increased with entino- To further elucidate the mechanism of action of the combination stat alone or in combination (Fig. 3H). In the blood of 4T1 tumor– therapy, we examined the immune subsets in the periphery (spleen and bearing mice, similar effects were observed in the frequency of total þ tumor-draining lymph node). Because results were extremely similar and CD44hi CD8 T cells and levels of VISTA with combination in both tissues, only data from the spleen are shown. Concordant with therapy (Supplementary Fig. S7). Altogether, these data suggest that in þ previous reports (16, 29), entinostat treatment alone or in combination the spleen, combination therapy increases CD8 T-cell activation, significantly reduced splenic Tregs and FoxP3 expression (Fig. 3A–C). while reducing Treg numbers and their suppressive phenotype, thus

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Figure 2. CD8þ T cells are required for the antitumor efﬁcacy of combination therapy. MC38-CEA tumor–bearing mice treated as in Fig. 1D with PBS or combination therapy underwent depletion of CD4, CD8, NK, or CD8þNK cells. Graphs show the percentage of (A) CD8þ,(B) CD4þ,(C) NK cells remaining in blood after depletion as mean SEM of 3 mice/group. D, Primary tumor growth graphed as mean SEM. # indicates Cntrl group was signiﬁcantly different (P < 0.0001) from all other groups; , P < 0.0001, two-way ANOVA. E, Survival data graphed; the table below depicts median OS in days (d), and number of cured mice. Data represent 1 independent experiment (n ¼ 9–10 mice/group). ENT, entinostat; V, vaccine.

shifting the immune balance toward a more activated, less suppressed Tregs (Fig. 4C and D). A previous study suggested that Tregs utilize þ peripheral environment. granzyme B and perforin to inhibit CD8 T cells in the TME (41). Therefore, although the enhanced granzyme B levels suggest Tregs Combination of entinostat with N-803 plus vaccine significantly may be more suppressive on a per cell basis, overall immune reduced Treg tumor infiltration suppression by Tregs may be reduced due to markedly decreased Next, we investigated the effect on immune subsets in the TME. tumor infiltration. Although the reduction of Tregsinthespleenwasdrivenby Tumor infiltration of either G-MDSCs or M-MDSCs was unaf- entinostat, in the TME, both N-803 plus vaccine and combination fected by any treatment (Supplementary Fig. S8A and S8C). therapy reduced the number of infiltrated Tregs (Fig. 4A). The Combination therapy decreased VISTA expression and enhanced proliferative capacity(Ki67)oftumor-infiltrating Tregs was aug- PD-L1 levels on M-MDSCs with no effect on G-MDSCs (Supple- mented with entinostat and combination therapy (Fig. 4B). Of note, mentary Fig. S8B and S8D). Altogether, these data suggest that the VISTA expression was reduced, whereas granzyme B levels were combination therapy has mixed effects on the MDSC inhibitory significantly elevated with combination therapy in tumor-infiltrated phenotype.

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Figure 3. In the spleen, entinostat reduces Tregs and N-803 and vaccine drive a CD8 activation phenotype. MC38-CEA tumor–bearing mice were treated as described in Fig. 1D and sacriﬁced on day 24. Tregs and CD8þ T cells in spleen were analyzed by ﬂow cytometry. All gating strategies are listed in Supplementary Table S2. A, Representative dot plots showing gates for FoxP3þ Tregs. B, Treg frequency and numbers. C, Expression of FoxP3 (gMFI), Ki67, and VISTA on Tregs. D, CD8þ T-cell þ hi hi hi hi þ frequency and numbers. E, Frequency of CD8 T cells with central memory phenotype CD44 CD62L CD127 (TCM). F, Frequency of CD44 CD8 T cells. G, Expression levels of CD25, Ki67, and VISTA on CD8þ T cells. H, Ratio of CD8þ T cells to Tregs. Graphs show mean SEM of combined data from 2 independent experiments; n ¼ 11–12 mice/group. , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001, one-way ANOVA. ENT, entinostat; V, vaccine.

þ þ Combination therapy significantly enhanced CD8 T-cell tumor promoted an 18-fold increase in the ratio of CD8 T cells to Tregs infiltration in the TME (Fig. 4I). þ We were particularly interested in investigating the impact of Contrary to the CD8 results, we observed no change in CD4 TILs þ combination therapy on tumor-infiltrating T lymphocytes (TIL) (Supplementary Fig. S8E). CD4 T cells displayed a mixed activation given the depletion results. Combination therapy significantly profile with increased Ki67 and reduced VISTA (inhibitory check- þ enhanced CD8 TILs 4.8-fold over control (Fig. 4E and F). These point), but decreased CD25 (activation; Supplementary Fig. S8F and infiltrated cells had primarily an effector (Teff) or effector memory S8G). Altogether, these data suggest that combination therapy is þ (Tem) phenotype, driven by entinostat (Fig. 4G). Additionally, promoting the infiltration and activation of CD8 T cells with a þ CD8 TILs had an activated phenotype denoted by increased CD25 reduction in Treg tumor infiltration, resulting in a significant increase and Ki67 expression (Fig. 4H). Overall, combination therapy in the CD8/Treg ratio in the TME.

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Figure 4. Combination therapy significantly enhanced CD8þ T-cell tumor infiltration while reducing Tregs in the TME. MC38-CEA tumor–bearing mice were treated as described in Fig. 1D and sacrificed on day 24. Tumor-infiltrated Tregs and CD8þ T cells were analyzed by flow cytometry. All gating strategies are listed in Supplementary Table S2. A, Treg number per mg of tumor and frequency. B and C, Expression of Ki67 and VISTA on Tregs. D, Frequency and gMFI of granzyme B on Tregs. E, CD8þ T-cell number per mg of tumor and frequency. F, Frequency of CD8þ T cells with effector (Teff) and effector memory (Tem) phenotypes. G, Expression of CD25 and Ki67 on CD44hi CD8þ T cells. H, Ratio of tumor-infiltrated CD8þ T cells to Tregs. A–I, Graphs show mean SEM of combined data from 2 independent experiments; n ¼ 12 mice/group. J, Pathway score from the NanoString nCounter advanced analysis software. K, Heat map of the genes increased or decreased 1.5- fold by the combination treatment versus PBS. L, Chemokine levels measured from tumor lysate on day 24. Graphs show mean SEM from 1 independent experiment; n ¼ 4–6 mice/group. Fold change over control is indicated in the table below graph. , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001, one-way ANOVA.

þ þ Increased CD8 TILs are associated with enhanced expression T cells in the spleen. CXCR3 was minimally expressed on CD8 TILs of the chemokine receptor CXCR3 and modulation of (Supplementary Fig. S9A). chemokines Both combination therapy and N-803 plus vaccine alone increased þ þ þ The striking increase in CD8 TILs induced by combination splenic CXCR3 CD8 T cells, yet the superior TME infiltration was therapy led us to interrogate possible mechanisms of tumor infiltration observed only with combination therapy, suggesting that the addition such as chemokines and chemokine receptors. In particular, CXCR3 is of entinostat induces CD8 tumor infiltration. Thus, we postulated that þ one of the main chemokine receptors involved in trafficking of CD8 T entinostat could be modulating chemokines and/or chemokine recep- cells into the TME (42, 43). Combination therapy or N-803 plus tors in the TME, as previously reported (44). NanoString analysis of þ þ vaccine alone significantly increased the frequency of CXCR3 CD8 total tumor RNA revealed a significant upregulation in cytokine and

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chemokine signaling with entinostat alone or in combination, includ- versus spleen, with combination therapy significantly enhancing CD4 ing many receptor and ligand combinations implicated in CD8 tumor IFNg and TNFa production in the TME (Supplementary Fig. S11A– infiltration (refs. 42, 43, 45–47; Fig. 4J and K). Specifically, entinostat S11F). Moreover, the percentage of bifunctional cells in the TME that elevated both the receptor CXCR3 and its ligands CXCL9, CXCL10, produce both cytokines was increased with combination therapy þ and CXCL11, as well as CCR5 and its ligands CCL3, CCL4, and CCL5 (Supplementary Fig. S11F). These data suggest that CD4 T cells (Fig. 4K). Chemokine protein levels were also examined in the tumor provide cytokine support in the TME. þ lysate using a bead-based immunoassay. Despite the transcripts for Combination therapy enhanced the frequency of splenic CD8 CXCL9 (MIG) and CXCL10 (IP-10) being elevated with combination T cells that produce IFNg or TNFa and the amount produced per cell, therapy, protein levels in the tumor lysate were not enhanced, with as well as the frequency of bifunctional cells (Fig. 5C–E). This effect CXCL10 being significantly reduced (Supplementary Fig. S9B). How- was driven by N-803 plus vaccine as entinostat did not elevate ever, consistent with the transcript levels, CXCL13 and CCL4 (MIP- cytokine production. In the TME, combination therapy increased 1B) proteins were significantly elevated with entinostat alone or in production of TNFa [% and geometric mean fluorescence intensity combination. CCL2 (MCP-1), CCL3 (MIP-1A), and CCL5 (RANTES) (gMFI)], but not IFNg (Fig. 5F and G). Only N-803 plus vaccine proteins were increased 1.6- to 2-fold with combination therapy versus alone increased the frequency of bifunctional cells (Fig. 5H). control, albeit not significantly (Fig. 4L). Of note, chemokines were Overall, these data suggest that combination therapy is enhancing examined on day 24 when significant CD8 tumor infiltration was T-cell function in the spleen and TME. This is further supported by already present. Nonetheless, these data suggest a possible mechanism NanoString analysis of the TME where entinostat alone or in for the enhanced CD8 tumor infiltration whereby entinostat is mod- combination enhanced genes involved in costimulatory and inter- ulating tumor chemokines to attract T cells, whereas N-803 plus feron signaling (Fig. 5I and J). þ vaccine is elevating the expression of the receptor CXCR3 on CD8 T cells in the periphery. Entinostat and N-803 plus vaccine synergize to elevate granzyme B levels in both murine and human CD8þ T cells þ Entinostat elevates the expression of MHC class I on tumor cells Although N-803 plus vaccine generated more bifunctional CD8 HDAC inhibitors have been shown to modulate MHC class I and TILs, that did not translate into significant antitumor efficacy. Thus, antigen-processing machinery in vitro in human carcinoma cells, cytokine production may be not the sole indicator of the ability of þ increasing their recognition and lysis by antigen-specific cytotoxic CD8 T cells to exert tumor control. The granular exocytosis pathway þ CD8 T cells (21–23). Interestingly, NanoString analysis of TME is one of the major components of the cytolytic ability of a T cell, which showed that entinostat alone or in combination elevated genes utilizes perforin and granzymes A and B to induce cell death (48). Our involved in antigen processing and presentation, including several NanoString analysis of MC38-CEA tumors indicated entinostat alone MHC genes (Supplementary Fig. S10A). This elevation in MHC gene or in combination increased cytotoxicity genes (Fig. 6A). Specifically, expression was also observed at the protein level in the nonimmune, genes for granzymes and perforin were elevated, with granzyme B CD45-negative cells, with entinostat treatment alone or in combina- being increased the most (Fig. 6B). Therefore, we measured granzyme tion increasing MHC class I (H-2Kb, H-2Db) expression on a per cell B levels by flow cytometry. Combination therapy significantly elevated þ þ basis (Supplementary Fig. S10B and S10C). granzyme B levels in splenic CD8 and CD4 T cells versus all other To assess the direct effect of entinostat on tumor cells, we exposed therapies (Supplementary Fig. S12A). In the TME, N-803 plus vaccine þ MC38-CEA and 4T1 cells in vitro to a clinically relevant concentration enhanced the percentage of CD8 TILs containing granzyme B relative ¼ (Cmax 0.5 mmol/L) of entinostat. Entinostat exposure increased the to control but not the amount of granzyme B per cell (gMFI; Fig. 6C expression of MHC class I on MC38-CEA cells and 4T1 cells, and D). Notably, combination therapy increased granzyme B levels suggesting these tumor cells would be more amenable to T-cell 2.8-fold versus control (% and gMFI), an effect driven by entinostat recognition and killing (Supplementary Fig. S10E, S10F, S10H, and (Fig. 6C and D). Entinostat alone or in combination also elevated þ S10I). Of note, both in vivo and in vitro exposure to entinostat elevated granzyme B levels in CD4 TILs (% and gMFI; Supplementary the expression of PD-L1 on MC38-CEA or 4T1 cells (Supplementary Fig. S12B). The promotion of granzyme B in the TME by entinostat Fig. S10D, S10G, and S10J). Although increased PD-L1 expression may alone or in combination correlated with augmented tumor necrosis by be decreasing the antitumor efficacy of the combination therapy by hematoxylin and eosin (H&E) staining (Supplementary Fig. S13A– impeding T cells, the combination therapy is still producing significant S13C). þ tumor control. CD8 TILs and granzyme B levels were also examined in the TME via immunofluorescence (Fig. 6E–G). Combination therapy increased þ þ þ N-803 plus vaccine promotes bifunctional CD4 and CD8 CD8 TILs 7-fold and total granzyme B puncta 7.9-fold in the TME T cells versus control (Fig. 6F and G). These data are similar to what was þ Given the necessity of CD8 T cells for the antitumor efficacy determined by flow cytometry (Figs. 4E and 6C). Of note, the of combination therapy, the effect on T-cell function was investigated. quantification of total granzyme B puncta is a summation of granzyme Previously, we demonstrated that the combination of vaccine plus N- in cytotoxic cells and any that has been recently released. These data are þ 803 enhanced antigen-specific T-cell responses (Supplementary comparable to monitoring granzyme B levels in all CD45 cells by flow Fig. S1B). The addition of entinostat to the duet further improved cytometry (Supplementary Fig. S12C). the number of IFNg-producing cells specific for CEA, p15e, and The effect of entinostat and N-803 on granzyme B levels was neoepitopes relative to either therapy alone (Fig. 5A and B). These assessed in healthy donor (HD) peripheral blood mononuclear cells data indicate that the combination of all three therapies is required for (PBMC) exposed in vitro to entinostat followed by N-803 and then significant induction of CEA and cascade immune responses. TCR stimulated. Granzyme B levels were elevated in T cells upon Next, we investigated the ability of T cells to produce IFNg and stimulation regardless of treatment. Furthermore, exposure to both N- þ TNFa upon T-cell receptor (TCR) stimulation. The treatment effects 803 and entinostat elevated granzyme B levels in human CD8 and þ on CD4 cytokine production were substantially larger in the TME CD4 T cells regardless of stimulation (Fig. 6H; Supplementary

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Figure 5. Effect of combination therapy on CD8þ T-cell function. MC38-CEA tumor–bearing mice were treated as described in Fig. 1D and sacriﬁced on day 24. A and B, ELISPOT assay performed as in Materials and Methods with indicated peptides. IFNg spot-forming cells (SFC) calculated from the number of spots with peptide of interest minus the number of spots with null peptide. Results expressed per 500,000 splenocytes. Cells from the spleen (C–E)ortumor(F–H)were restimulated ex vivo with anti-CD3/anti-CD28 for 4 hours. Intracellular levels of IFNg (C and F)andTNFa (D and G)wereexaminedbyﬂow cytometry. E and H, Frequency of IFNg/TNFa double producers (DP) of CD8þ CD44hi T cells. Graphs show mean SEM from 1 experiment, 4–6mice/group(F–H)or2combined independent experiments, 10–11 mice/group (A–E). I and J, Pathway score via the NanoString nCounter advanced analysis software. Heat maps of the genes increased or decreased 3-fold by the combination treatment versus PBS. , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001, one-way ANOVA. ENT, entinostat; V, vaccine.

Fig. S12D). Overall, combination therapy elevates the cytolytic poten- immunotherapy. Here we provide the rationale for combining the tial of T cells in the periphery and TME, which was recapitulated in HD HDACi entinostat with the IL15 superagonist N-803 plus a ther- PBMCs in vitro. apeutic cancer vaccine. Although the combination therapy was well tolerated, entinostat-induced lymphopenia was observed. This is concordant with reports of transient, non–dose-limiting leukopenia Discussion observed in some patients treated with entinostat (32, 33). Impor- Combining therapies to alleviate immunosuppression while pro- tantly, despite lymphopenia, the combination therapy had signif- moting immune effector function may improve clinical responses to icant antitumor effects in both murine models.

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Figure 6. Combination therapy enhances granzyme B levels in immune effectors. MC38-CEA tumor–bearing mice were treated as described in Fig. 1D and sacrificed on day 24. A, Pathway score via the NanoString nCounter advanced analysis software. B, Key genes from the cytotoxicity pathway upregulated. C, Representative histograms of granzyme B staining on CD44hi CD8þ T cells in the tumor. D, Frequency and geometric mean fluorescence intensity (gMFI) of granzyme B on CD44hi CD8þ TILs. Graphs show mean SEM from 2 combined experiments (n ¼ 11–12 mice/group). E, Representative confocal images of tumors stained for DAPI, CD8, and granzyme B at 40 magnification. Scale bar, 20 mm. White arrows indicate CD8þ TIL positive for granzyme B. Quantification of the number of CD8þ T cells (F) or granzyme B puncta per field (G). F and G, 6 fields quantified per tumor. Graphs show mean SEM from 1 experiment; n ¼ 3 mice/group. H, Granzyme B gMFI quantified from human CD8þ T cells from 5 healthy donor PBMCs treated in vitro with indicated treatment with and without CD3/CD28 stimulation. Graphs show mean SEM from 2 combined experiments. , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001, one-way ANOVA. ENT, entinostat; V, vaccine.

Comprehensive studies in the laboratory have demonstrated that N-803 increases antigen cascade to p15e and neoepitope antigens, N-803 in combination with vaccine hassuperiorantitumoreffects an effect not observed with N-803 alone. Antigen cascade has been relative to either agent alone (K.P. Fabian; manuscript in prepara- repeatedly observed both preclinically and clinically with therapeu- tion). Ergo, in this study, we utilized N-803 plus vaccine as the tic cancer vaccines and linked to the generation of durable long- “vaccine component.” Nonetheless, the contribution of the vaccine term antitumor activity and superior clinical outcomes (49, 50). is evident in the ELISPOT data where the addition of a vaccine to Importantly, we also demonstrated that the addition of entinostat to

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the vaccine/N-803 duet further enhances antigen cascade, which is (CXCL13, CCL2, CCL3, CCL4, and CCL5) may be playing a role given þ not observed with entinostat alone. Given the long duration of their implication in increasing CD8 TILs (45–47). One study showed tumor control in the MC38-CEA model by the combination ther- there was synergy between CXCR3 chemokines and CCL5 in attracting apy, one could speculate this is duetotheepitopespreadingwe T cells into melanoma tumors (45). Although we did not investigate þ observed in CD8 T-cell responses, including to neoantigens. the expression of other chemokine receptors, this study suggests that Although we have not identified antigen-specific T cells in the TME, CCL3, CCL4, and CCL5 may be contributing to CD8 tumor it appears that we are inducing an antigen cascade as evident from the infiltration. ELISPOT data. In addition, the loss of antitumor activity with the CD8 A key finding of our studies and a final instance of N-803 and þ depletion demonstrates the role of antigen-specificCD8 T cells in vaccine synergizing with entinostat was the enhanced cytolytic poten- þ the antitumor effect. Interestingly, we observed that depletion of tial of CD8 TILs observed at both the gene and protein levels. For the þ CD4 T cells cured 80% of the combination-treated mice. Others first time, we demonstrate that entinostat synergizes with N-803 plus þ þ have reported similar findings, with one study demonstrating that vaccine to increase granzyme B levels in CD4 and CD8 T cells in the þ CD8 T-cell responses were enhanced in CD4-depleted mice periphery and TME. This elevation in granzyme B in T cells was resulting in tumor control (51, 52). Hence, it is possible that recapitulated in human PBMCs after in vitro exposure to N-803 and/or þ þ depletion of CD4 T cells may have enhanced CD8 T-cell entinostat. Previous research has shown that granzyme B levels are responses elicited by combination therapy. In addition, the inherent elevated in human PBMCs treated with N-803 (13). Here, we dem- þ deletion of Tregs encompassed by CD4 T-cell depletion could onstrated that exposure to entinostat and N-803 elevated granzyme B further contribute to CD8-mediated tumor resolution. Although levels in T cells to a greater extent than either agent alone, suggesting a our data indicated that after 17 days of entinostat treatment Tregs cooperative mechanism that further enhances the cytolytic potential of were reduced, we have never determined how rapidly this effect T cells. occurs.ItisfeasiblethatentinostatdoesnotalterTregsatthe For the first time, we show that a therapy reduces the inhibitory initiation of treatment. Therefore, Treg depletion in the early stages checkpoint VISTA on CD8, CD4, Tregs, and MDSCs in tumor- of tumor growth may be sufficient to resolve tumors in conjunction bearing and non–tumor-bearing mice. Although the precise role of þ with combination therapy. It is unclear if depletion of CD4 T cells VISTA in antitumor immune responsesisstillunderinvestigation, later in tumor growth would have produced a different result, VISTA has been shown to negatively affect T-cell function (35, 36). especially given that combination therapy increased cytokine pro- Thus, it is possible that the decrease in VISTA is contributing to the þ duction by CD4 T cells in the TME. improved antitumor activity observed with combination therapy. Our comprehensive immune analysis in the spleen and TME Additionally, it has been suggested that inhibiting/reducing mul- unveiled several instances where N-803 plus vaccine synergized tiple checkpoints may improve the antitumor efficacy of checkpoint with entinostat. For example, entinostat increased genes associated blockade. Therefore, it is tempting to speculate that the reduction of with antigen processing and presentation as well as protein and VISTA via entinostat could, in part, explain why entinostat transcript levels of MHC on tumor cells. This would result in enhances the antitumor effects of an antiprogrammed cell death- increased CD8 recognition of tumor cells. To this end, we observed 1(aPD-1) treatment, although VISTA levels were not reported in an increase in antigen-specific T cells only in combination-treated those studies (17, 18). It has been reported that entinostat reduces mice. This could be a consequence of entinostat enhancing the the suppressive ability of MDSCs through multiple mecha- expression of transcripts involved in antigen presentation, costi- nisms (17, 20). The reduction of VISTA on MDSCs may provide mulatory signaling, and interferon, in combination with CD8 an additional mechanism as to how entinostat reduces the sup- activation induced by N-803 and vaccine (Supplementary pressive capacity of MDSCs. Fig. S14, A's). Moreover, entinostat alone or in combination pro- To conclude, the antitumor efficacy of the combination therapy þ moted effector memory CD8 TILs. Because memory T cells have stems from simultaneously targeting multiple aspects of the immune an enhanced proliferative capacity and better longevity than effector system and the tumor, thereby shifting the balance of immune T cells (53), one could speculate that entinostat promoting effector suppression and activation in the TME toward a more activated and memory formation in the tumor contributes to the antitumor inflamed microenvironment. Combination therapy enhanced tumor efficacy of the combination therapy. infiltration of CD8 TILs with increased cytolytic potential able to The superior CD8 tumor infiltration elicited by combination ther- efficiently target tumor cells whose phenotype had been altered by apy further illustrates the three therapies synergizing. We demon- entinostat to render them more amenable to recognition and killing strated that N-803 plus vaccine increased the expression of CXCR3 on (Supplementary Fig. S14, A's and B's). Further, these CD8 TILs would þ peripheral CD8 T cells, a receptor shown to be required for efficient also display increased functionality given the reduction in immune T-cell tumor infiltration (42, 43). NanoString analysis revealed an suppression arising from a decrease in tumor Tregs and VISTA levels entinostat-induced increase in cytokine and chemokine signaling, (Supplementary Fig. S14, C's). including augmented transcripts for the CXCR3 ligands, CXCL9, Although combination therapy tripled survival of MC38-CEA CXCL10, and CXCL11. This is concordant with previous reports tumor–bearing mice and induced various immune effects associated showing that HDAC inhibitors, including entinostat, elevate these with antitumor efficacy, it did not resolve all the tumors. It is feasible chemokines (44, 54). The protein levels of these chemokines at day 24 that T-cell exhaustion or immune regulatory elements were induced at did not follow this trend. Nonetheless, one could speculate that the a later time point. Alternatively, through our comprehensive studies, entinostat-induced increase in CXCL9, CXCL10, and CXCL11,in we observed that PD-L1 was elevated on both tumor cells and MDSCs þ combination with N-803 plus vaccine-induced elevation in CXCR3 after entinostat treatment, suggesting that PD-1/PD-L1 blockade þ CD8 T cells, would lead to increased CD8 tumor infiltration (Sup- could further enhance tumor control. To this end, ongoing studies plementary Fig. S14, B's). Additionally, due to the pleiotropic and have been initiated combining entinostat with M7824, a bifunctional promiscuous nature of chemokines and chemokine receptors, it is molecule comprising an anti–PD-L1 fused to a TGFb trap. Impor- possible that other chemokines upregulated with combination therapy tantly, all of the agents used in this study are currently being examined

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in numerous clinical trials. Our proof-of-concept studies indicate that Analysis and interpretation of data (e.g., statistical analysis, biostatistics, the clinical combination of entinostat, N-803, and therapeutic cancer computational analysis): K.C. Hicks, K.L. Lee, D.H. Hamilton, W.D. Figg, vaccines may increase response rates to immunotherapy in patients P. Ordentlich, F.R. Jones, S. Rabizadeh, P. Soon-Shiong, J. Schlom, S.R. Gameiro Writing, review, and/or revision of the manuscript: K.C. Hicks, W.D. Figg, with solid malignancies. F.R. Jones, S. Rabizadeh, P. Soon-Shiong, J. Schlom, S.R. Gameiro Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Schlom Disclosure of Potential Conflicts of Interest Study supervision: J. Schlom, S.R. Gameiro P. Ordentlich is an employee/paid consultant for and holds ownership interest (including patents) in Syndax Pharmaceuticals. F.R. Jones is an employee/paid Acknowledgments consultant for and holds ownership interest (including patents) in Etubics Corpo- The authors thank Curtis Randolph for excellent technical assistance, Debra ration. S. Rabizadeh is an employee/paid consultant for ImmunityBio, and is an Weingarten for her excellent assistance in the preparation of this manuscript, and the advisory board member/unpaid consultant for NantOmics and NantWorks. P. Soon- NCI Collaborative Protein Technology Resource Laboratory and Clinical Support Shiong is an employee/paid consultant for and holds ownership interest (including Laboratory/Frederick National Laboratory for Cancer Research for their excellent fl patents) in NantWorks. No potential con icts of interest were disclosed by the other technical support. This research was supported by the Intramural Research Program authors. of the Center for Cancer Research, NCI, NIH, and by Cooperative Research and Development Agreements (CRADA) between the NCI and Syndax Pharmaceuticals, Authors’ Contributions Inc., and the NCI and NantBioScience, Inc. Conception and design: K.C. Hicks, K.M. Knudson, K.L. Lee, D.H. Hamilton, The costs of publication of this article were defrayed in part by the payment of page W.D. Figg, P. Soon-Shiong, J. Schlom, S.R. Gameiro charges. This article must therefore be hereby marked advertisement in accordance Development of methodology: K.C. Hicks, K.M. Knudson, J.W. Hodge, W.D. Figg, with 18 U.S.C. Section 1734 solely to indicate this fact. P. Soon-Shiong, S.R. Gameiro Acquisition of data (provided animals, acquired and managed patients, provided Received March 1, 2019; revised July 9, 2019; accepted October 18, 2019; facilities, etc.): K.C. Hicks, K.M. Knudson, K.L. Lee, J.W. Hodge, W.D. Figg published first October 23, 2019.

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Kristin C. Hicks, Karin M. Knudson, Karin L. Lee, et al.

Clin Cancer Res Published OnlineFirst October 23, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-19-0727

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