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Purpose: Agonist antibodies targeting the T-cell costimulatory receptor 4-1BB (CD137) are among the most effective immunotherapeutic agents across preclinical cancer models. In the clinic, however, development of these agents has been hampered by dose-limiting liver toxicity. Lack of knowledge of the mechanisms underlying this toxicity has limited the potential to separate 4-1BB agonist–driven tumor immunity from hepatotoxicity.Experimental Design: The capacity of 4-1BB agonist antibodies to induce liver toxicity was investigated in immunocompetent mice, with or without coadministration of checkpoint blockade, via (i) measurement of serum transaminase levels, (ii) imaging of liver immune infiltrates, and (iii) qualitative and quantitative assessment of liver myeloid and T cells via flow cytometry. Knockout mice were used to clarify the contribution of specific cell subsets, cytokines, and chemokines.Results: We find that activation of 4-1BB on liver myeloid cells is essential to initiate hepatitis. Once activated, these cells produce interleukin-27 that is required for liver toxicity. CD8 T cells infiltrate the liver in response to this myeloid activation and mediate tissue damage, triggering transaminase elevation. FoxP3+ regulatory T cells limit liver damage, and their removal dramatically exacerbates 4-1BB agonist–induced hepatitis. Coadministration of CTLA-4 blockade ameliorates transaminase elevation, whereas PD-1 blockade exacerbates it. Loss of the chemokine receptor CCR2 blocks 4-1BB agonist hepatitis without diminishing tumor-specific immunity against B16 melanoma.Conclusions: 4-1BB agonist antibodies trigger hepatitis via activation and expansion of interleukin-27–producing liver Kupffer cells and monocytes. Coadministration of CTLA-4 and/or CCR2 blockade may minimize hepatitis, but yield equal or greater antitumor immunity. Clin Cancer Res; 24(5); 1138–51. ©2018 AACR.

Supplemental Figure 1: Peak of 4-1BB mediate liver transaminase level and gating strategy for flow cytometry analysis of liver immune infiltrates. Supplemental Figure 2: Representative flow cytometry analysis of liver immune infiltrates. Supplemental Figure 3: Administration of 4-1BB agonist antibodies initiates liver pathology through activation of liver-resident myeloid cells. Supplemental Figure 4: Effects of IL-27 pathway inactivation on CD4 T cells. Supplemental Figure 5: Representative flow cytometry analysis of liver immune infiltrates.

BackgroundPancreatic ductal adenocarcinoma (PDAC) is resistant to many available therapies including immunotherapy because of its highly complex tumor microenvironment (TME). PDAC TME consists of a significant proportion of stromal cells, such as endothelial cells, perivascular cells, and cancer-associated fibroblasts (CAFs). Recent work indicates how CAFs can orchestrate the crosstalk cancer and immune cells, and contribute to many aspects of tumor progression, including angiogenesis, senescence, and inflammation. Recent studies based on scRNA-seq have increased understanding of CAF heterogeneity in PDAC in both human and genetically engineered mouse models (GEMMs) is of high interest. To understand the translatability of GEMMs in the setting of PDAC, we conducted a thorough scRNA-seq meta-analysis on CAFs across GEMMs and PDAC human samples. Hereafter, we characterized CAFs multi-dimensionally based on transcriptional, chromatin accessibility, and spatial profiles. Finally, we suggested certain transcription factors may be regulatory drivers of heterogeneous CAF phenotypes in both human and GEMMs.MethodsWe collected publicly available and internally generated scRNA-seq data of PDAC CAFs from human and mouse. After dataset alignment and label transfer, we conducted differential expression analysis across CAF subsets to characterize myofibroblasts (myCAFs) and other CAF subsets of interest. Bioinformatically, we further interrogated CAF heterogeneity in terms of regulatory potential of transcription factors, gene set enrichment, and functional state transition. Complemented by epigenomic assessment, we investigated chromatin accessibility and transcription factor binding availability on the single-cell level. Finally, to investigate the TME organization and spatial neighborhood of cell-to-cell interaction, we explored potential functional differences across location and transcriptional changes of CAF subsets by spatial transcriptomics.ResultsWe found that myofibroblasts (myCAFs) make up a substantial proportion of the CAF population, in both human and mouse TME. In a combination of transcriptional profiling, chromatin accessibility assessment, and spatial transcriptomics, we elucidated potential functional and phenotypic differences within myCAF population and compared to other CAF subsets in the TME. While myofibroblasts are traditionally described as matrix remodeling related, heterogeneity in myofibroblasts may suggest additional roles played by this specific subset. In addition, CAFs in human and mouse share similarities, in terms of transcriptional profiles and phenotypes. The use of GEMMs facilitates our understanding of CAF heterogenous behavior and phenotypes in the PDAC TME.ConclusionsHere, we presented a comprehensive overview of CAF heterogeneity in mouse PDAC models and human datasets. Our observations highlight molecular differences in CAFs, which facilitates our understanding on PDAC stromal microenvironment and translatability in GEMMs in imitating human TME.

KRAS is most frequently mutated in pancreatic adenocarcinomas (PDAC) with more than 90% of cases having a KRAS mutation. This leads to constitutive activation of the downstream signaling pathways, driving tumor cell proliferation whilst co-opting the tumor microenvironment (TME) to promote tumorigenesis and immune escape. The mutant specific KRASG12C inhibitor (AMG510) has been shown to suppress tumor growth and reconfigure the immune TME. However, covalent binding KRASG12C inhibitors have no efficacy on other KRAS alleles, such as G12D and V, the majority of KRAS mutations in PDAC (37% and 28%, respectively). Here, we use a SOS1 inhibitor (BI 1701963) in combination with a MEK inhibitor, blocking two non-redundant steps in the KRAS pathway, on a library of congenic tumor clones derived from an autochthonous mouse model of PDAC deemed the KPCY (Pdx-1-Cre, KRASG12D, p53-/-, YFP+). These tumor lines can generate both “hot” and “cold” TMEs regarding T cell infiltration. We observed that combined SOS1 and MEK inhibition suppressed tumor growth of all KPCY lines irrespective of their cold or hot TME status. In some KPCY lines, CD8 T-cells significantly increased in the TME following combination treatment. We found that the CD8 T-cell increase is restricted to the individual tumor site and resulted from both recruitment and intra-tumoral division. This was accompanied by a decrease of tumor associated macrophages and myeloid-derived suppressor cells. Single-cell RNAseq profiling of the TME was performed on three KPCY lines with distinct immune response to the treatment to characterize cellular composition change induced by the SOS1 and MEK inhibitor combination. To take advantage of the treatment-induced immune TME reconfiguration, we examined combination immune-oncology (IO) targeting T cells and antigen-presenting cells and found that the combination of SOS1i and MEKi with PD-1/CTLA4/CD40 enhanced and prolonged tumor growth inhibition mediated by anti-tumor immunity. Collectively, these results reveal that the combination of SOS1 and MEK inhibitors can be used to convert cold to hot tumors and highlights how we can rationalize combination of cancer-targeted therapy and IO therapy to prolong anti-tumor effects. Citation Format: Robert J Norgard, Pratha Budhani, Sarah O'Brian, Jessica Potts, Brianna Flynn, Xavier Salce, Joshua Tagore, Lucinda Thiede, Joseph Seco, Suzanne Segal, Mark Love, Ania Mikucka, Charlie Cote, Ruby Wasti, Mohanapriya Kamalakannan, Astrid Jeschko, Gabriela Melo-Zainzinger, Angela Mcnabola, Varenka Rodriguez Rodriguez DiBlasi, Joseph Tumang, Ulrike Tontsch-Grunt, Melanie Hinkel, Jeanine Pignatelli, Francesca Trapani, Sandra Martinez-Morilla, Kaja Kostyrko, Marco H Hofmann, Abhishek Kashyap, Emilio Flano, Kang Liu. Combination of SOS1::KRAS inhibitor with a MEK inhibitor reconfigures the immune tumor microenvironment of KRASG12D pancreatic ductal adenocarcinomas and sensitizes to immunotherapy [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B23.

Despite the success of immune checkpoint blockade against melanoma, many "cold" tumors like prostate cancer remain unresponsive. We found that hypoxic zones were prevalent across preclinical prostate cancer and resisted T cell infiltration even in the context of CTLA-4 and PD-1 blockade. We demonstrated that the hypoxia-activated prodrug TH-302 reduces or eliminates hypoxia in these tumors. Combination therapy with this hypoxia-prodrug and checkpoint blockade cooperated to cure more than 80% of tumors in the transgenic adenocarcinoma of the mouse prostate-derived (TRAMP-derived) TRAMP-C2 model. Immunofluorescence imaging showed that TH-302 drives an influx of T cells into hypoxic zones, which were expanded by checkpoint blockade. Further, combination therapy reduced myeloid-derived suppressor cell density by more than 50%, and durably reduced the capacity of the tumor to replenish the granulocytic subset. Spontaneous prostate tumors in TRAMP transgenic mice, which completely resist checkpoint blockade, showed minimal adenocarcinoma tumor burden at 36 weeks of age and no evidence of neuroendocrine tumors with combination therapy. Survival of Pb-Cre4, Ptenpc-/-Smad4pc-/- mice with aggressive prostate adenocarcinoma was also significantly extended by this combination of hypoxia-prodrug and checkpoint blockade. Hypoxia disruption and T cell checkpoint blockade may sensitize some of the most therapeutically resistant cancers to immunotherapy.

Purpose: Agonist antibodies targeting the T-cell costimulatory receptor 4-1BB (CD137) are among the most effective immunotherapeutic agents across preclinical cancer models. In the clinic, however, development of these agents has been hampered by dose-limiting liver toxicity. Lack of knowledge of the mechanisms underlying this toxicity has limited the potential to separate 4-1BB agonist–driven tumor immunity from hepatotoxicity. Experimental Design: The capacity of 4-1BB agonist antibodies to induce liver toxicity was investigated in immunocompetent mice, with or without coadministration of checkpoint blockade, via (i) measurement of serum transaminase levels, (ii) imaging of liver immune infiltrates, and (iii) qualitative and quantitative assessment of liver myeloid and T cells via flow cytometry. Knockout mice were used to clarify the contribution of specific cell subsets, cytokines, and chemokines. Results: We find that activation of 4-1BB on liver myeloid cells is essential to initiate hepatitis. Once activated, these cells produce interleukin-27 that is required for liver toxicity. CD8 T cells infiltrate the liver in response to this myeloid activation and mediate tissue damage, triggering transaminase elevation. FoxP3+ regulatory T cells limit liver damage, and their removal dramatically exacerbates 4-1BB agonist–induced hepatitis. Coadministration of CTLA-4 blockade ameliorates transaminase elevation, whereas PD-1 blockade exacerbates it. Loss of the chemokine receptor CCR2 blocks 4-1BB agonist hepatitis without diminishing tumor-specific immunity against B16 melanoma. Conclusions: 4-1BB agonist antibodies trigger hepatitis via activation and expansion of interleukin-27–producing liver Kupffer cells and monocytes. Coadministration of CTLA-4 and/or CCR2 blockade may minimize hepatitis, but yield equal or greater antitumor immunity. Clin Cancer Res; 24(5); 1138–51. ©2018 AACR.

Evofosfamide (TH-302) is a clinical-stage hypoxia-activated prodrug of a DNA-crosslinking nitrogen mustard that has potential utility for human papillomavirus (HPV) negative head and neck squamous cell carcinoma (HNSCC), in which tumor hypoxia limits treatment outcome. We report the preclinical efficacy, target engagement, preliminary predictive biomarkers and initial clinical activity of evofosfamide for HPV-negative HNSCC. Evofosfamide was assessed in 22 genomically characterized cell lines and 7 cell line–derived xenograft (CDX), patient-derived xenograft (PDX), orthotopic, and syngeneic tumor models. Biomarker analysis used RNA sequencing, whole-exome sequencing, and whole-genome CRISPR knockout screens. Five advanced/metastatic HNSCC patients received evofosfamide monotherapy (480 mg/m2 qw × 3 each month) in a phase 2 study. Evofosfamide was potent and highly selective for hypoxic HNSCC cells. Proliferative rate was a predominant evofosfamide sensitivity determinant and a proliferation metagene correlated with activity in CDX models. Evofosfamide showed efficacy as monotherapy and with radiotherapy in PDX models, augmented CTLA-4 blockade in syngeneic tumors, and reduced hypoxia in nodes disseminated from an orthotopic model. Of 5 advanced HNSCC patients treated with evofosfamide, 2 showed partial responses while 3 had stable disease. In conclusion, evofosfamide shows promising efficacy in aggressive HPV-negative HNSCC, with predictive biomarkers in development to support further clinical evaluation in this indication.

Immune checkpoint blockade is effective in “hot” tumors like melanoma with pre-existing immune infiltrates; however, “cold” tumors like prostate cancer fail to respond. We found that prostate cancers harbor regions of hypoxia that resist T cell infiltration even in the context of anti-CTLA-4 (cytotoxic T lymphocyte associated protein-4) and anti-PD-1 (programmed cell death protein 1) blockade. These hypoxic zones serve as islands of immune privilege through the recruitment and suppressive polarization of immature myeloid cells into myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM). We found that targeted hypoxia ablation using TH-302, a hypoxia-activated prodrug, sensitized both transplantable and spontaneous models of prostate cancer to checkpoint blockade, coincident with enhanced T cell infiltration and effector function and loss of MDSC recruitment and suppressive function. Tumors treated with the combination of TH-302 and checkpoint blockade showed a reduced capacity to suppressively polarize new myeloid immigrants, implying a durable reconditioning of the tumor microenvironment (TME) into an immune-infiltrated, pro-inflammatory milieu. T cells infiltrating combination-treated tumors exhibited increased mitochondrial respiration, consistent with creation of a metabolically favorable milieu for T cell function. Based on these findings, we hypothesized that other approaches capable of metabolically rewiring the TME should promote anti-tumor immunity and sensitize checkpoint blockade-resistant tumors to immunotherapy. With this in mind, we performed a longitudinal study comparing a panel of different mitochondrial respiration inhibitors and a glutaminase inhibitor for their efficacy in reducing hypoxia, improving T cell infiltration and decreasing myeloid cell recruitment and suppressive polarization using immunofluorescence staining and confocal microscopy. Our preliminary data suggests that inhibitors targeting mitochondrial respiration, rather than those targeting glutamine metabolism synergize with checkpoint blockade and exhibit the highest efficacy in increasing T cell recruitment. We continue to characterize the dynamics of hypoxia reduction, duration of normalization following drug withdrawal, and impact on the immune microenvironment of these diverse approaches to metabolic reconditioning. Citation Format: Priyamvada Jayaprakash, Midan Ai, Arthur Liu, Pratha Budhani, Todd Bartkowiak, Jie Sheng, Casey Ager, Courtney Nicholas, Ashvin Jaiswal, Yanqiu Sun, Krishna Shah, Sadhana Balasubramanyam, Nan Li, Guocan Wang, Jing Ning, Anna Zal, Tomasz Zal, Michael Curran. Targeting hypoxia-induced immune suppression to overcome immunotherapy resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5011.

Immune checkpoint blockade has revolutionized cancer immunotherapy, and combination treatment with antibodies against multiple inhibitory receptors promises to improve efficacy substantially. However, the spatiotemporal and combinatorial expression profile of various checkpoint receptors on effector T cells and the effect of combined blockade on immune function remain poorly defined. In this study we set out to determine the co-expression kinetics and functional impact of multiple checkpoint receptors (PD-1, LAG-3, TIM-3, and BTLA) on tumor antigen-specific effector T cells in controlled in vitro culture systems and in the tumor environment. To elicit tumor antigen-specific responses we immunized mice with irradiated MC38 tumor cells engineered to express chicken ovalbumin (OVA; MC38.OVA) and used pentamer reagents to identify OVA257-264-specific CD8 T cells. Repeated stimulation with irradiated MC38.OVA cells or irradiated OVA-pulsed splenocytes ex vivo resulted in robust proliferation and activation of antigen-specific T cells as determined by BrdU incorporation and production of IFN-γ. Most OVA-specific cells expressed PD-1 ex vivo and maintained PD-1 expression during repeated cycles of in vitro stimulation. In contrast, antigen-specific T cells did not express TIM-3 or LAG-3 ex vivo, yet both receptors were induced on the majority of PD1 positive, but not PD-1 negative, cells upon repeated stimulation ex vivo, resulting in double and triple positive cells. The frequency of BTLA expressing cells remained low under all conditions. The addition of PD-1 and TIM-3 blocking antibodies during ex vivo restimulation enhanced T cell activation, demonstrating that these receptors mediate inhibitory function. Consistent with our in vitro data demonstrating that repeated antigen stimulation promotes the coordinated expression of checkpoint receptors, we found that the vast majority of MC38.OVA or Colon26 tumor infiltrating T cells (TILs) express PD-1, and most PD-1 positive cells also co-express TIM-3 and LAG-3, whereas these receptors are largely absent from T cells in secondary lymphoid organs. Importantly, combination treatment of tumor-bearing mice with anti-PD-1 and anti-TIM-3, or anti-PD-1 and anti-LAG-3 antibodies enhanced the immune control of tumor growth in both models as compared to the respective monotherapies. Finally, in vitro activation of human PBMC-derived T cells resulted in a similar sequential upregulation of checkpoint inhibitors ultimately resulting in their co-expression. Here, we have established a culture system to delineate the hierarchical expression of inhibitor checkpoint receptors on tumor antigen-specific murine T cells during repeated antigen stimulation. Defining the inhibitory checkpoint receptor landscape will guide the process of identifying the most promising combinations of checkpoint blockers in cancer immunotherapy. Citation Format: Robert J. Durso, Jerry Pei, Michelle Russell, Pratha Budhani, Elena Burova, Chandrika Taduriyasas, Ella Ioffe, Markus Mohrs, Gavin Thurston, Jie Dai. Multiple immune checkpoint receptors are co-expressed on tumor antigen-specific T cells and contribute to tumor immune evasion [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B129.

Agonist antibodies against the T cell costimulatory receptor 4-1BB have proven extraordinarily efficacious in the treatment of a multitude of pre-clinical murine tumors both in mono- and combination therapy settings. Although administration of α4-1BB in Phase I and II clinical trials has led to disease stabilization and partial remission in some cases of melanoma; high grade, dose limiting liver inflammation has slowed the progress of these agents toward FDA approval. We sought to uncover the mechanisms by which 4-1BB agonist antibodies trigger hepatotoxicity in hopes of discovering approaches by which the anti-tumor and hepatotoxic effects could be separated. We show that α4-1BB mediated liver damage initiates through stimulation of myeloid cells, followed by subsequent recruitment and activation of CD8 and CD4 T cells in the liver. Moreover, we show that the inflammatory cytokine IL-27 is essential in myeloid conversion of T cells into mediators of liver damage. Conversely, we show that Foxp3+ regulatory T cells are potent suppressors of 4-1BB agonist mediated hepatotoxicity; as depletion of Tregs exacerbates liver pathology. Our results go on to support the use of combination therapies in the treatment of tumor malignancies, in particular CTLA-4 blockade which may expand Tregs in the liver to ameliorate α4-1BB mediated toxicities while also amplifying anti-tumor immune responses.

Targeting immune inhibitory (CTLA-4, PD-1) or stimulatory (OX-40, 4-1BB) co-receptors has proven to be a potent anti-cancer therapeutic strategy, eliciting robust anti-tumor T cell responses and yielding compelling pre-clinical and clinical data in a variety of cancers. We have previously shown in pre-clinical models that 4-1BB agonist antibodies engender strong anti-tumor T cell responses capable of driving regression of established tumors and durable tumor-free survival. This impressive anti-tumor effect is due, in part, to the unique ability of 4-1BB co-stimulation to differentiate a novel subset of T cells that acquires a highly cytotoxic effector profile typically only seen in NK cells. This phenotype- known as ThEO (CD4+) or TcEO (CD8+)-is driven by the T-box transcription factor Eomesodermin, and is further characterized by expression of the inhibitory receptor KLRG1. We show here that, despite high level effector function and expression of several co-inhibitory receptors (PD-1, TIM-3, KLRG1), these ThEO/TcEO phenotype T cells efficiently establish immunologic memory and recall with high frequency upon secondary tumor challenge. Further, they maintain high effector function on recall and can traffic to tissue sites far removed from the original malignancy. These findings provide mechanistic insight into the process of memory formation and give evidence for the capacity of ThEO/TcEO cells to prevent tumor recurrence.

Tumor hypoxia predicts poor outcomes across all cancers and has long been recognized as a critical source of resistance to both chemotherapy and radiotherapy. Despite the success of T cell immune checkpoint blockade in treating melanoma, aggressive adenocarcinomas of the prostate and pancreas are largely resistant to CTLA-4 and PD-1 antibody therapy in the mouse and in man. We find that hypoxic zones of these tumors resist infiltration by T cells even in the context of robust infiltration of normoxic areas of the same tumor (e.g. in the context of T cell checkpoint blockade). Beyond this lack of accessibility to tumor-specific T cells, hypoxia drives the establishment of a highly interdependent network of immunosuppressive stromal cells. Among these, we find myeloid-derived suppressor cells and myofibroblasts to be the critical populations which act together to suppress T cell responses and mediate immunotherapy resistance. Using the hypoxia-specific prodrug Evofosfamide, we show that disruption of hypoxia in both transplantable and genetically-engineered murine models of prostate cancer sensitizes them to antibody blockade of CTLA-4 and PD-1. Loss of immune resistance is a consequence of re-oxygenation of hypoxia zones which results in 1) loss of myeloid suppressors, 2) reduced capacity to suppressively polarize new myeloid immigrants, and 3) loss of suppressive activation of myofibroblasts. This combination of hypoxia disruption and T cell checkpoint blockade has immense potential to render some of the most therapeutically resistant cancers sensitive to immunotherapy. Citation Format: Midan Ai, Pratha Budhani, Michael A. Curran. Hypoxia is an essential driver of immune suppression in the tumor microenvironment. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr PR003.

Immunotherapy has demonstrated a remarkable potential to cure established cancer, but many tumors either relapse or fail to respond. The molecular mechanisms which drive these tumor relapses remain unknown. We hypothesize that tumors evolve resistance under immunotherapeutic pressure mediated by changes in gene expression which can be selected and propagated through serial passage and then identified and targeted. We are comparing untreated B16 melanoma tumors to those expanding despite treating with combinations checkpoint blockade (CTLA-4, PD-1, and PD-L1), 4-1BB-agonist antibody, adoptive transfer of TRP-1 specific CD4 T-cells, or growth in MHC-mismatched hosts. Through multiple in vivo passages, we have already selected a B16 melanoma tumor line that has evolved almost 100% resistance to combination blockade of CTLA-4, PD-1, and PD-L1, which could cure nearly 90% of tumor-bearing mice in the first cycle. After each passage under selective pressure of combination blockade, we have observed visible signs of increased angiogenesis correlating with enhanced therapy resistance. By determining the pathways engaged by these tumors to develop resistance to tumor-specific immunity, we hope to discover novel characteristics of the mammalian immune system as well as reveal new targets for immunotherapeutic intervention in melanoma patients.

Tumor hypoxia predicts poor outcomes across all cancers and has long been recognized as a critical source of resistance to both chemotherapy and radiotherapy. Despite the success of T cell immune checkpoint blockade in treating melanoma, aggressive adenocarcinomas of the prostate and pancreas are largely resistant to CTLA-4 and PD-1 antibody therapy in the mouse and in man. We find that hypoxic zones of these tumors resist infiltration by T cells even in the context of robust infiltration of T cells in normoxic areas of the same tumor (e.g. in the context of T cell checkpoint blockade). Beyond this lack of accessibility to tumor-specific T cells, hypoxia drives the establishment of a highly interdependent network of immunosuppressive stromal cells. Among these, we find myeloid-derived suppressor cells and myofibroblasts to be the critical populations which act together to suppress T cell responses and mediate immunotherapy resistance. Evofosfamide is a hypoxia-specific chemotherapeutic pro-drug which is activated only in the hypoxic cores of tumors and thus can be co-administered with immunotherapy. We find that Evofosfamide-driven disruption of hypoxia zones sensitizes prostate cancer to antibody blockade of CTLA-4 and PD-1 in both transplantable and genetically-engineered murine models of prostate cancer. Co-administration of Evofosfamide and α-CTLA-4/α-PD-1 promotes tumor rejection in a significantly larger percentage of mice than either therapy alone. Mechanistic studies reveals that loss of immune resistance is a consequence of re-oxygenation of hypoxia zones which results in 1) loss of active myeloid suppressor cells, 2) reduced suppressive capacity of new myeloid immigrants, 3) loss of suppressive activation of myofibroblasts, and 4) enhanced infiltration of effector T cells. Therefore, this combination of hypoxia disruption and T cell checkpoint blockade has immense potential to render some of the most therapeutically resistant cancers sensitive to immunotherapy.