Targeting CD39 in Cancer Reveals an Extracellular ATP- and Inflammasome-Driven Tumor Immunity
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Published OnlineFirst November 7, 2019; DOI: 10.1158/2159-8290.CD-19-0541 RESEARCH ARTICLE Targeting CD39 in Cancer Reveals an Extracellular ATP- and Inflammasome-Driven Tumor Immunity Xian-Yang Li1, Achim K. Moesta2, Christos Xiao3, Kyohei Nakamura1, Mika Casey1, Haiyan Zhang1, Jason Madore1, Ailin Lepletier1, Amelia Roman Aguilera1, Ashmitha Sundarrajan4, Celia Jacoberger-Foissac3, Clifford Wong2, Tracy dela Cruz2, Megan Welch2, Alana G. Lerner2, Bradley N. Spatola2, Vanessa B. Soros2, John Corbin2, Ana C. Anderson5, Maike Effern6,7,8, Michael Hölzel6,7, Simon C. Robson9, Rebecca L. Johnston10, Nicola Waddell10, Corey Smith11, Tobias Bald4, Nishamol Geetha1, Courtney Beers2, Michele W. L. Teng3, and Mark J. Smyth1 ABSTRACT We explored the mechanism of action of CD39 antibodies that inhibit ectoen- zyme CD39 conversion of extracellular ATP (eATP) to AMP and thus potentially augment eATP–P2-mediated proinflammatory responses. Using syngeneic and humanized tumor models, we contrast the potency and mechanism of anti-CD39 mAbs with other agents targeting the adenosinergic pathway. We demonstrate the critical importance of an eATP–P2X7–ASC–NALP3- inflammasome–IL18 pathway in the antitumor activity mediated by CD39 enzyme blockade, rather than simply reducing adenosine as mechanism of action. Efficacy of anti-CD39 activity was underpinned by CD39 and P2X7 coexpression on intratumor myeloid subsets, an early signature of macrophage depletion, and active IL18 release that facilitated the significant expansion of intratumor effector T cells. More importantly, anti-CD39 facilitated infiltration into T cell–poor tumors and rescued anti–PD-1 resistance. Anti-human CD39 enhanced human T-cell proliferation and Th1 cytokine production and sup- pressed human B-cell lymphoma in the context of autologous Epstein–Barr virus–specific T-cell transfer. SIGNIFICANCE: Overall, these data describe a potent and novel mechanism of action of antibodies that block mouse or human CD39, triggering an eATP–P2X7–inflammasome–IL18 axis that reduces intratumor macrophage number, enhances intratumor T-cell effector function, overcomes anti–PD-1 resistance, and potentially enhances the efficacy of adoptive T-cell transfer. 1Immunology in Cancer and Infection Laboratory, QIMR Berghofer Medical QIMR Berghofer Medical Research Institute, Herston, Queensland, Research Institute, Herston, Queensland, Australia. 2Tizona Therapeutics, Australia. 11Translational and Human Immunology Laboratory, QIMR San Francisco, California. 3Cancer Immunoregulation and Immunother- Berghofer Medical Research Institute, Herston, Queensland, Australia. apy Laboratory, QIMR Berghofer Medical Research Institute, Herston, Note: Supplementary data for this article are available at Cancer Discovery 4 Queensland, Australia. Oncology and Cellular Immunology Laboratory, Online (http://cancerdiscovery.aacrjournals.org/). QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia. 5Harvard Medical School and Brigham and Women’s Hospital, X.-Y. Li and A.K. Moesta contributed equally to this work. Boston, Massachusetts. 6Unit of RNA Biology, Department of Clinical Corresponding Author: Mark J. Smyth, QIMR Berghofer Medical Research Chemistry and Clinical Pharmacology, University Hospital Bonn, Univer- Institute, 300 Herston Road, Herston, Queensland 4006, Australia. Phone: sity of Bonn, Bonn, Germany. 7Institute of Experimental Oncology (IEO), 617-3845-3957; Fax: 617-3362-0111; E-mail: Mark.Smyth@qimrberghofer. University Hospital Bonn, University of Bonn, Bonn, Germany. 8Depart- edu.au ment of Microbiology and Immunology, The University of Melbourne at the Cancer Discov 2019;9:1–20 Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia. 9Departments of Medicine and Anesthesia, Beth Israel Deaconess doi: 10.1158/2159-8290.CD-19-0541 Medical Center, Boston, Massachusetts. 10Medical Genomics Laboratory, ©2019 American Association for Cancer Research. OF1 | CANCER DISCOVERY DECEMBER 2019 www.aacrjournals.org Downloaded from cancerdiscovery.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst November 7, 2019; DOI: 10.1158/2159-8290.CD-19-0541 INTRODUCTION by targeting tumor metabolic processes, such as the ATP–adeno- Immune-checkpoint blockade (ICB) using antagonistic sine axis, is a new and promising avenue for therapeutic invention. antibodies to CTLA4, PD-1, and PD-L1 has revolutionized the Purinergic signaling in the TME plays a key role in the regula- cancer treatment paradigm (1). However, despite the unprec- tion of immune responses. In solid tumors, ATP is abundantly edented responses achieved among select “immunogenically released in the extracellular space owing to cell death in the hot” tumor types with these therapies, the majority of patients tumor core, metabolic and/or hypoxic stress, and proinflam- still fail to achieve clinically relevant responses in those indi- matory signals that stimulate active export of ATP, leading cations, and several tumor types show profound resistance to an accumulation of extracellular ATP (eATP) levels far in to ICB (2). In addition, a significant proportion of patients excess of those found in healthy tissues (4, 5). eATP acts as a who initially demonstrate antitumor responses following ICB proinflammatory stimulus by agonizing P2 purinergic recep- therapy eventually become refractory and experience tumor tors (e.g., P2X7) on immune cells (6). However, tumors are pro- relapse (3). Taken together, these observations reveal the need ficient at scavenging eATP, converting it to immunosuppressive for additional immunotherapeutics and suggest that addi- adenosine by means of two ectonucleotidases, CD39 and CD73, tional immune-escape mechanisms remain to be uncovered. expressed on malignant cells, regulatory immune cells, and Although a multitude of clinical agents have entered the the vasculature (7). Adenosine exerts its suppressive function clinic as single agents or combination therapies with established directly by binding to A2A receptors on multiple immune cells ICBs, the majority of these fall into two categories: antagonists such as phagocytes, dendritic cells (DC), natural killer (NK) of additional immune checkpoints (e.g., LAG3, TIM3, and cells, T cells, and B cells (8–14). By controlling the initial steps TIGIT) and agonists of costimulatory molecules (e.g., GITR, in the phosphohydrolytic cascade, CD39 acts as the master OX40, and 4-1BB). Altering the tumor microenvironment (TME) regulator of this dynamic balance between proinflammatory December 2019 CANCER DISCOVERY | OF2 Downloaded from cancerdiscovery.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst November 7, 2019; DOI: 10.1158/2159-8290.CD-19-0541 RESEARCH ARTICLE Li et al. eATP and immunosuppressive adenosine within the TME, and inhibitors of A2AR and antagonistic antibodies against CD73. thereby fosters a broadly immunosuppressive milieu (6). An outstanding question has been whether targeting CD39 offers In addition to elevated expression levels of CD39 in blood any therapeutic advantage by targeting a different mechanism of neoplasias and multiple solid-tumor settings (15–17), CD39 is action than these other approaches. Here we report the use of broadly expressed on the vasculature and specifically found on novel antibodies that selectively block the enzymatic function certain immune subsets, including B cells, NK cells, DCs, mono- of CD39 in mouse and human. We use these to determine that cytes, macrophages, and regulatory T cells (Treg; ref. 18). Within targeting CD39 may be a more potent approach by virtue of the the TME, CD39 expression on Tregs (19, 20) and myeloid-derived unique eATP–P2X7–inflammasome–IL18 mechanism of action suppressor cells (21, 22) has been shown to be directly correlated that reduces intratumor macrophages and enhances CD8+ T-cell with the ability of these professional immunoregulatory cells to effector function in the TME. In particular, we provide differ- suppress T-cell function. CD8+ T cells, which show little detect- entiation between therapeutic antibodies targeting CD39 and able CD39 in peripheral blood, express significantly elevated agents targeting other components of the adenosinergic system. CD39 levels across multiple human tumor types, including gas- Finally, we demonstrate the therapeutic potential of targeting tric cancer, renal cell carcinoma, non–small cell lung carcinoma CD39 in solid tumors, either as monotherapy or in potential (NSCLC), head and neck squamous cell carcinoma, breast cancer, ICB combinations, where anti–PD-1 alone was not effective. and melanoma (23, 24). This apparent upregulation is accompa- nied by reduced polyfunctionality and induction of T-cell exhaus- RESULTS tion signatures (24, 25). Recent reports also suggest that CD39 is a marker of tumor-reactive effector T-cell subsets (25, 26) and is Efficacy of Anti-Mouse CD39 mAb Monotherapy increasingly appreciated as a regulatory marker (27). Despite the fact that pharmacologic inhibitors of CD39, such The impact of CD39 on tumor growth and antitumor as POM1, have previously been described to have potent antimet- immunity has mostly been delineated using global CD39 gene– astatic activity (30, 31), questions around specificity, therapeutic targeted mice; published data suggested that growth of multiple half-life, pharmacokinetics, and toxicity have always left doubts syngeneic tumors was reduced in these mice (28,