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In cancers with tumor-infiltrating lymphocytes (TILs), monoclonal antibodies (mAbs) that block immune checkpoints such as CTLA-4 and PD-1/PD-L1 promote antitumor T-cell immunity. Unfortunately, most cancers fail to respond to single-agent immunotherapies. T regulatory cells, myeloid derived suppressor cells (MDSCs), and extensive stromal networks within the tumor microenvironment (TME) dampen antitumor immune responses by preventing T-cell infiltration and/or activation. Few studies have explored combinations of immune-checkpoint antibodies that target multiple suppressive cell populations within the TME, and fewer have studied the combinations of both agonist and antagonist mAbs on changes within the TME. Here, we test the hypothesis that combining a T-cell–inducing vaccine with both a PD-1 antagonist and CD40 agonist mAbs (triple therapy) will induce T-cell priming and TIL activation in mouse models of nonimmunogenic solid malignancies. In an orthotopic breast cancer model and both subcutaneous and metastatic pancreatic cancer mouse models, only triple therapy was able to eradicate most tumors. The survival benefit was accompanied by significant tumor infiltration of IFNγ-, Granzyme B-, and TNFα-secreting effector T cells. Further characterization of immune populations was carried out by high-dimensional flow-cytometric clustering analysis and visualized by t-distributed stochastic neighbor embedding (t-SNE). Triple therapy also resulted in increased infiltration of dendritic cells, maturation of antigen-presenting cells, and a significant decrease in granulocytic MDSCs. These studies reveal that combination CD40 agonist and PD-1 antagonist mAbs reprogram immune resistant tumors in favor of antitumor immunity.

Supplementary Table S1, Supplementary Figures S1-7

Understanding how novel therapeutic combinations alter solid tumor microenvironments (TME) in immunosuppressive tumors such as breast cancer is essential to improve their responses to immune checkpoint inhibitors (ICIs). Entinostat, an oral histone deacetylase inhibitor (HDACi), has been shown to improve responses to ICIs in various tumor models with immunosuppressive TMEs, but the precise alterations induced by entinostat and mechanisms of synergy with ICIs remain unknown. Here, we employ single-cell RNA-sequencing on HER2 overexpressing breast tumors from mice treated with entinostat + ICIs to characterize these changes across cell types in the TME. This analysis demonstrates that treatment with entinostat induces a shift from a pro-tumor to an anti-tumor TME signature characterized predominantly by changes in the myeloid cells. Notably, myeloid-derived suppressor cells (MDSCs) are shifted toward the less suppressive granulocytic phenotype in association with reduced signaling through the STAT3 pathway. In addition, tumor-associated macrophages are shifted toward an anti-tumor M1 phenotype by epigenetic reprogramming. Overall, these entinostat-induced TME changes reduce immunosuppression and increase mechanisms of tumor cell killing to improve ICI responses and broaden the population of patients who could potentially benefit from immunotherapy.

Background: Checkpoint inhibition is a very successful treatment strategy in cancers that are naturally immunogenic by attracting T cells into the tumor microenvironment (TME) and promoting cytotoxic signaling pathways. While this strategy has shown some efficacy in metastatic breast cancer, most breast cancers are not highly immunogenic likely due to an immunosuppressive microenvironment and a lack of tumor antigen expression and recognition. One strategy to transform the breast TME is to use epigenetic modulation to affect activation and trafficking of myeloid derived suppressor cells (MDSCs), known to alter the immunogenicity of the TME and sensitize tumors to checkpoint modulation. We hypothesize that combinatorial therapy primes the TME by altering infiltration and function of MDSCs leading to a more robust T cell response. Methods: We are using a HER-2/neu transgenic mouse model with tumor challenge of syngeneic cell lines to test the efficacy of different combinations of an epigenetic agent, the histone deacetylase inhibitor entinostat (ENT), checkpoint inhibitors anti -programmed cell death protein (a-PD-1) and anti-cytotoxic T-lymphocyte-associated protein 4 (a-CTLA-4) antibodies, with and without anti-HER2 antibodies. We will examine treatment effects on tumor growth, and hope to identify co-stimulatory and inhibitory factors regulating T cell and MDSC responses. Characterization of tumor infiltrating lymphocytes and their functional capabilities are being investigated in primary tumors using fluorescence-activated cell sorting, nanostring gene expression profiling, and immunohistochemistry. Results: We found significant improvement in survival and delay in tumor growth in mice treated with ENT in combination with a-PD-1 and/or a-CTLA-4. Addition of anti-HER2 therapy to ENT and a-CTLA4 or a-PD1 also significantly improves survival and delay in tumor growth. We also found addition of ENT to checkpoint inhibition leads to significantly increased infiltration of granulocytic-MDSCs into the TME. We demonstrate an increase in CD8+ T effector cells in mice treated with combination therapy. Flow cytometric evaluation of markers of T cell activation, exhaustion, and MDSC function demonstrate significantly increased T cell activation, exhaustion, and myeloid function however it is unclear how this directly effects the phenotype we have observed in these mice. Gene expression profiling of both MDSCs and lymphocytes infiltrating tumors is underway to help determine significant changes in immune related pathways that lead to our observed outcomes. Conclusions: Addition of ENT to checkpoint inhibition significantly increases infiltration of innate and adaptive immune cells into the highly tolerant neu-N breast tumors and leads to improved survival and decreased tumor burden. Functional assays are underway and future studies will further delineate changes in immune infiltration as well as genetic alterations responsible for these observations. It is our hope that these novel findings will provide further rationale for combination therapy and improve the response rate of these immune therapies in patients with breast cancer. Citation Format: Roussos Torres ET, Ma H, Christmas B, Armstrong T, Jaffee EM. Combination checkpoint inhibition and epigenetic modulation promotes tumor suppression and improves survival in Her2+ models of breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-08-01.

Background: Immune checkpoint inhibitors (ICIs) provide benefit for immunogenic cancers that naturally attract T cell infiltration, but have limited benefit for patients with tumors that lack natural T cells in the tumor microenvironment (TME) including many breast cancers. Suboptimal and inconsistent immune responsiveness in many cancers is likely the result of a lack of tumor antigen expression and/or recognition together with multiple suppressive signals within the TME that provide a formidable barrier to T cell infiltration. Emerging data suggest that these factors can be overcome by combining epigenetic modulation that reprograms myeloid-derived suppressor cells (MDSCs) and upregulate T cell-attracting signals within the TME, with ICIs. Our paralleled preclinical and clinical trial investigates the mechanisms and efficacy of the epigenetic modulator, entinostat (ENT) to reprogram the immune suppressive cells within the TME. Methods: We conducted a multicenter phase 1 clinical trial (NCT02453620) of entinostat combined with nivolumab +/- ipilimumab in patients with advanced cancers including hormone-receptor positive or triple-negative breast cancer (TNBC). Mandatory tumor samples are being obtained pre-treatment, post-treatment with 2 weeks of entinostat, and 8 weeks post combination therapy with ICIs. We used immunohistochemical (IHC) for CD8 and FoxP3 to determine a ratio representative of immune infiltration in response to therapy. Preclinical studies utilize mouse models of breast cancer (NeuN and 4T1) and mirror the treatment scheme used in the clinical trial. We employed multiparameter flow cytometry, single cell RNA sequencing, bulk RNA sequencing of tumor specimens and patient samples to specifically interrogate the role of ENT in modulating STAT3 as a master regulator of downstream inflammatory pathways. Results: With regard to the clinical trial, out of 35 patients enrolled, 11 were diagnosed with breast cancer. We observed 4 partial responses, 2 responses in patients with breast cancer, with an ORR of 12%. We have almost completed accrual of our expansion cohort of 15 patients with advanced HER2 negative breast cancer. The primary outcomes of the clinical trial will be reported elsewhere, here we are focusing on important correlative findings which suggest a combination of ENT with ICIs alters CD8/FoxP3 ratios in certain patients. Bulk-RNA sequencing or patient samples is underway. Preliminary data from animal models obtained using scRNA-seq has begun to elucidate altered MDSC signaling pathways, and to identify gene expression changes in immune, stromal, and tumor cells following treatment with ENT. These data are also expected to identify new targets for ICI sensitization. Conclusions: Preliminary results with IHC staining suggests increased immune infiltration with combination therapy. Sequencing analysis will likely provide deeper insight into mechanisms driving response. The mechanisms of response to ICIs in patients with breast cancer have yet to be elucidated. These studies will provide the necessary scientific evidence to uncover mechanisms behind the transformation of the immunosuppressive TME and provide new targets that could improve clinical response to ICIs to maximize patient survival. The core biological themes explored will likely have a broad impact in the field of breast cancer and affect the paradigm of therapies available to patients in the clinic. These findings will help determine how treatment with ICIs will be successful to obtain durable responses for breast cancer patients. Citation Format: Evanthia T Roussos Torres, Luciane Tsukamoto Kagohara, Emily Davis, Christine Rafie, Brian Christmas, Qingfeng Zhu, Chenguang Wang, David Lim, Robert Anders, Elana Fertig, Vincent Chung, Patricia Lorusso, Adam Brufsky, Elizabeth M Jaffee, Vered Stearns, Roisin Connolly. Reprogramming the suppressive tumor microenvironment of breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-06.

Checkpoint inhibition has revolutionized treatment in cancers that are naturally immunogenic by enabling infiltration of T cells into the tumor microenvironment (TME) and promoting cytotoxic signaling pathways. While this strategy has shown some efficacy in metastatic breast cancer, most breast cancers lack immunogenic potential due to a strictly sustained immunosuppressive microenvironment and a lack of tumor antigen expression and recognition. One strategy to transform the breast TME is to use epigenetic modulation to affect activation and trafficking of myeloid derived suppressor cells (MDSCs), known to alter the immunogenicity of the TME and sensitize tumors to checkpoint modulation. We show that combinations of an epigenetic agent, the histone deacetylase inhibitor entinostat (ENT), checkpoint inhibitors anti -programmed cell death protein (a-PD-1) and anti-cytotoxic T-lymphocyte-associated protein 4 (a-CTLA-4) antibodies, with and without anti-HER2 antibodies significantly improves survival and anti-tumor effects in HER-2/neu transgenic mouse models. Using fluorescence-activated cell sorting, nanostring gene expression profiling, and other functional assays, we have characterized the various types of tumor infiltrating lymphocytes and myeloid cells and their functional capabilities within primary tumors. We show that addition of ENT to checkpoint inhibition leads to significantly decreased suppressive ability of granulocytic-MDSCs into the TME. We also demonstrate an increase in CD8+ T effector cells in mice treated with combination therapy. Flow cytometric evaluation of markers of T cell activation, exhaustion, and MDSC function demonstrate significantly increased T cell activation, exhaustion, and decreased myeloid function. Nanostring gene expression profiling of both MDSCs and lymphocytes infiltrating tumors determined significant changes in immune related pathways that likely lead to our observed outcomes. In summary, addition of ENT to checkpoint inhibition significantly increases infiltration of innate and adaptive immune cells into the highly tolerant neu-N breast tumors and leads to improved survival and decreased tumor burden. Functional assays demonstrate that this drug combination promotes a less suppressive TME and allows a more robust anti-tumor effect of infiltrating cells. It is our hope that these novel findings will provide further rationale for combination therapy and improve the response rate of these immune therapies in patients with breast cancer. Citation Format: Evanthia T. Roussos Torres, Christine Rafie, Hayley Ma, Brian Christmas, Todd Armstrong, Elizabeth M. Jaffee. Entinostat transforms the suppressive tumor microenvironment of breast cancer and promotes survival and anti-responses when combined with checkpoint inhibition [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 4965.

A hallmark of many non-immunogenic cancers is the lack of tumor infiltrating lymphocytes (TIL) and/or failure to mount a robust anti-tumor T cell response via multiple mechanisms. The presence of T regulatory cells and myeloid derived suppressor cells (MDSCs) serve to dampen the immune response, and furthermore, tumor antigen-specific T cell tolerance limits the efficacy of therapeutic cancer vaccines. CD40 signaling is critical to the decision of whether cytotoxic T lymphocytes become primed or tolerized. Administration of monoclonal CD40 agonistic antibody (Ab) has been shown to promote CD8 activation in vivo, and likely alters the myeloid component of the tumor microenvironment. Our study asks the question of whether combining a T cell inducing vaccine and PD1 inhibition with CD40 agonistic Ab can induce T cell priming and TIL activation in non-immunogenic solid malignancies. We utilized mouse models of pancreatic ductal adenocarcinoma (PDAC) and breast cancer to assess the effects of drug combinations on intratumoral immune responses. Tumor-bearing mice were treated with a GM-CSF secreting vaccine (GVAX) + anti-PD1 Ab alone or in combination with CD40 Ab, or isotype control Ab, and monitored for survival. A separate cohort of mice were analyzed by immunohistochemistry and multi-color flow cytometry to assess T cell infiltration/activation and myeloid maturation. In a hemisplenectomy model of PDAC in which tumor cells were surgically implanted into wild-type recipients, mice treated with isotype control Abs succumbed to disease with extensive liver and peritoneal metastases at 35-70 days. GVAX + anti-PD1 Ab treatment displayed some efficacy, although 70% of mice eventually developed fatal liver metastases. In contrast, CD40 Ab was highly active, with 90% long-term survival afforded by a single administration of Ab, and mice treated with GVAX + anti-PD1 Ab + CD40 Ab had 100% survival. Similar trends in treatment efficacy were observed following subcutaneous tumor implantation of PDAC tumor cells in the lower limb. In an orthotopic model in which HER2/neu-expressing breast tumor cells were implanted into the mammary fat pad of syngeneic neu-N mice, we demonstrated delayed tumor progression and increased median survival in mice treated with GVAX + anti-PD1 Ab + CD40 Ab relative to either therapy alone. Further characterization of immune populations was carried out by high dimensional flow cytometric analysis utilizing PhenoGraph clustering and visualized by t-SNE. Changes were observed in monocytic and dendritic cell infiltration and maturation in the tumors of combination-treated mice. A significant decrease in granulocytic MDSCs was associated with response, as well as an increase in mature antigen presenting cells. In conclusion, GVAX, anti-PD1 and CD40 agonist Ab have potential synergy in modulating anti-tumor immunity in non-immunogenic cancers. Citation Format: Hayley S. Ma, Evanthia Roussos Torres, Bibhav Poudel, Tara Robinson, Brian Christmas, Kayla Cruz, Skylar Woolman, Christine Rafie, Blake Scott, Valerie Wall, Todd Armstrong, Elizabeth Jaffee. Combination CD40 agonist and PD-1 antagonist antibody therapy enhances vaccine induced T cell responses in non-immunogenic cancers [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 4936.

TPS4151Background: The use of antibody therapy targeting immune checkpoints has become a major focus of cancer immunotherapy, with the most compelling activity seen in the minority of patients with...

Checkpoint inhibition is a very successful treatment strategy in cancers that are naturally immunogenic by attracting T cells into the tumor microenvironment (TME) and promoting cytotoxic signaling pathways. While this strategy has shown some efficacy in metastatic breast cancer, most breast cancers are not highly immunogenic likely due to an immunosuppressive microenvironment and a lack of tumor antigen expression and recognition. One strategy to transform the breast TME into one that is immune responsive, is to use epigenetic modulation to expose tumor antigens, promote cytokine production, thus, attracting inflammatory cells into the tumor. Epigenetic modulation also has the potential to affect activation and trafficking of myeloid derived suppressor cells (MDSCs), known to alter the immunogenicity of the TME and thus, sensitizing tumors to checkpoint modulation. We hypothesize that combinatorial therapy primes the TME by increasing immunogenicity via alteration of T cells and MDSCs. We also hypothesize that combination therapy will significantly affect immune modulatory pathways. Alterations in these could serve as a functional read out of combinatorial therapy. We are using a Her-2/neu (neu-N) transgenic mouse model which through the use of various syngeneic cell lines has the capability to inform the response of Her2 and triple negative subtypes of breast cancer to immunotherapy. This model enables us to study the efficacy of different combinations of an epigenetic agent, the histone deacetylase inhibitor entinostat, and checkpoint inhibitors anti -programmed cell death protein (PD1) and anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) antibodies, on tumor growth and metastatic progression, and to help identify co-stimulatory and inhibitory factors regulating T cell and MDSC responses. Characterization of tumor infiltrating lymphocytes and their functional capabilities are being investigated in primary tumors and metastases using fluorescence-activated cell sorting and immunohistochemistry. Thus far, there is a significant improvement in survival and delay in tumor growth in mice inoculated with neu expressing tumor cells treated with combination anti-PD1, anti-CTLA4 and entinostat as well as with anti-PD1 in combination with Entinostat. Preliminary results also suggest that epigenetic modulation with entinostat in combination with checkpoint inhibition improves the immune response within these tumors as evidenced by increased CD8+/T-regulatory cell infiltration, and increased monocytic-MDSCs into the TME. There are ongoing survival studies testing this combination therapy in mice inoculated with triple negative breast tumor cells. Future studies will further delineate the mechanisms by which epigenetic therapy alters the function of the inflammatory response to immunotherapy. Citation Format: Evanthia T. Roussos Torres, Hayley Ma, Brian Christmas, Todd Armstrong, Elizabeth M. Jaffee. Promotion of immunogenicity using epigenetic modulation and immune checkpoint inhibition in mouse models of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3667. doi:10.1158/1538-7445.AM2017-3667

This project aims to test the hypothesis that epigenetic modulatory drugs (EMD) and GVAX, a GM-CSF-secreting whole tumor cell vaccine, are capable of altering the inflammatory environment of pancreatic ductal adenocarcinoma (PDAC) and sensitizing it to checkpoint blocking agents. Recently the use of antibody therapy targeting immune checkpoints, such as CTLA-4 and PD-1, has become a major focus of cancer immunotherapy. In responsive patients, these therapies have resulted in long-term control of chemotherapy-resistant disease. The most compelling activity has been seen in the minority of patients with immunogenic tumors where T cell infiltration naturally occurs. These benefits are not observed in non-immunogenic tumors, such as PDAC, with low expression of tumor-associated antigens (TAA) and a lack of intrinsic T cell infiltrates. Therapies that can alter the tumor microenvironment (TME) and allow infiltration of effector T cells, decrease immunosuppressive cells, and stimulate TAA expression may convert non-immunogenic tumors into cancers sensitive to checkpoint inhibitors. Recent work with EMDs has shown that they are capable of altering the immunogenicity of the TME by inducing the expression of cancer testis antigens as well as increasing tumor cell expression of MHC class II and decreasing Tregs in the TME. Additionally, GVAX has been shown to induce tertiary lymphoid aggregates within the TME of patients with PDAC. We are testing the hypothesis that treatment with EMDs and GVAX can sensitize the inflammatory environment of PDAC to checkpoint blockade inhibition by evaluating changes in immune cell function within the TME via flow cytometry, immunohistochemistry, and gene expression array. We are using a murine model of hepatic metastases of pancreatic cancer which involves injecting syngeneic pancreatic tumor cells into the spleen followed by a hemisplenectomy, resulting in the consistent formation of hepatic metastases that can be monitored by ultrasound. We have evaluated entinostat, a histone deacetylase inhibitor, in combination with GVAX which induces T cell responses, and demonstrated a significant increase in survival when compared with either agent alone. Flow cytometric analysis of the cells infiltrating the TME shows that the combination of entinostat and GVAX causes a significant increase in CD4+ T cell infiltration as well as a shift from an M-MDSC dominant to a more G-MDSC dominant myeloid population. Current studies aim to elucidate the functionality of the MDSC population as well as identify potential changes in the T helper cell subsets via flow cytometry. Additionally, future studies will evaluate changes within checkpoint blockade pathways via flow cytometry and gene expression array to identify pathways that require further modulation to enhance antitumor responses. Citation Format: Brian Christmas, Blake Scott, Todd Armstrong, Nilofer Azad, Elizabeth Jaffee. Epigenetic modulation of the tumor microenvironment enhances vaccine induced T cell responses in a murine model of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1686. doi:10.1158/1538-7445.AM2017-1686