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Opportunities and challenges for anti-CD73 cancer therapy “...CD73 expression on regulatory T cells appears to impair antitumor immunity by directly suppressing antitumor T‑cell responses, whereas endothelium-derived CD73 limits the migration of tumor-specific CD8+ cells to the tumor site, thereby inhibiting antitumor immunity.” KEYWORDS: adenosine n CD73 n ecto‑5´‑nucleotidase n immunosuppression n microenvironment n tumor

The emerging characterization of the roles of CD73 in tumor growth and metastasis has presented opportunities to develop effective and specific immunotherapies for various human cancers. Here, I will provide a concise perspective on the current progress and challenges of anti-CD73 therapy. Elevated levels of extracellular adenosine within the tumor microenvironment have recently been described [1,2] . CD73, or ecto5´-nucleotidase (ecto-5´-NT, EC 3.1.3.5), is a cell surface rate-limiting enzyme that catalyzes the dephosphorylation of extracellular AMP to adenosine [3,4] . This ecto-enzymatic cascade in tandem with CD39 (ecto-ATPase) generates adenosine from ATP that in turn signals through adenosine receptors. It is becoming increasingly clear that CD73-generated adenosine plays a key role in the regulation of inflammatory reactions and immune responses by modulating endothelial adhesion, transmigration and T‑cell activities [5–7] . Because CD73 is constitutively expressed in many types of cancer cells, based on the immunomodulatory property of adenosine [8–10] , the role of CD73 in tumor immunity was initially evaluated on cancer cells. We [11] and Smyth’s group [12] have recently documented that specific targeting of tumorderived CD73 using siRNA potently reduced tumor growth. Importantly, extracellular adenosine generated by tumor CD73 inhibited both the proliferation and cytotoxicity of tumor-specific CD8 + T  cells through activation of the adenosine receptor A 2A R [11] . Thus, CD73 expressed by tumor cells specifically impairs adaptive antitumor immune responses through its ecto-enzymatic activity, suggesting an important mechanism of tumor-induced immune evasion. We each concluded that tumor

CD73 is a novel target for cancer treatment to improve antitumor immunity [11,12] . Proof-ofprinciple studies performed by Smyth’s group reported that anti-CD73 monoclonal antibody (mAb) treatment was effective in reducing the tumorigenesis and metastasis of breast cancer [12] . Likewise, we showed that anti-CD73 smallmolecule inhibitors reduced ovarian cancer progression and increased mice survival [11] . Similar observations have recently been made by Hausler and his colleagues using human primary ovarian cancer cells and cell lines in which CD73 and/or CD39 siRNA or small-molecule inhibitors promoted CD4 + T‑cell proliferation, NK cell-mediated lysis and cytotoxic T‑cell activity [13] . In addition, exosomes secreted by cancer cells express CD39 and CD73, and conversion of 5´-AMP to adenosine by exosome-derived CD73 suppressed T‑cell activation [14] . Because CD73 is expressed on many host cell types, such as subsets of lymphocytes, endothelial cells and dendritic cells, in later studies Stagg and his colleagues [15] , Salmi’s group [16] and ours [17] addressed the crucial role of host CD73 expression and activity in several transplantable tumor models using the same CD73-deficient mice. These studies collectively revealed that: ƒƒ CD73-deficient mice are resistant to the growth and metastasis of immunogenic tumors;

10.2217/IMT.12.83 © 2012 Future Medicine Ltd

Immunotherapy (2012) 4(9), 861–865

Bin Zhang Biotherapy Center of the First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, China and Department of Medicine, Cancer Therapy & Research Center, University of Texas Health Science Center, San Antonio, TX 78229, USA and Robert H Lurie Comprehensive Cancer Center, Department of Medicine – Division of Hematology/Oncology, Northwestern University, Feinberg School of Medicine, 300 E Superior Street-Tarry 13-766, Chicago, IL 60611, USA Tel.: +1 312 503 1104 Fax: +1 312 503 0189 [email protected]

ƒƒ The protective effect of host’s CD73 deficiency on primary tumors is dependent on CD8 + T cells and associated with increased endogenous antitumor T‑cell immunity; ƒƒ CD73 deficiency on both hematopoietic and nonhematopoietic cells is required to limit tumor growth and host CD73 deficiency or blockade increased tumor antigen-specific T‑cell homing to tumors.

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In the hematopoietic compartment, the protumorigenic effect of CD4 + CD25 + regulatory T cells was in part dependent on their expression of CD73, which is in line with previous studies [18] . On the other hand, the prometastatic effect of host-derived CD73 is dependent on CD73 expression on nonhematopoietic cells. To elucidate further the role of CD73 on de novo tumorigenesis, Stagg and his colleagues in a more recent study elegantly demonstrated that CD73 facilitates carcinogen-induced tumor initiation and tumor growth by suppressing immuno­surveillance via IFN‑g, NK cells and CD8 + T cells [19] . They found that CD73 deficiency also reduced de  novo prostate tumorigenesis in TRAMP transgenic mice. Moreover, anti-CD73 mAb treatment effectively inhibited growth of established carcinogen-induced tumors or prostate tumors and suppressed the development of experimental lung metastases of prostate tumors. The findings obtained from the above three independent groups are similar and complementary to one another. Therefore, it is clear now both tumor and host CD73 significantly contributes to tumor growth and metastasis.

“...ectonucleotidase expression by a variety

of stromal components in the tumor microenvironment might be a general immunosuppressive feature associated with cancer progression.” Notably, CD73 expression on regulatory T cells appears to impair antitumor immunity by directly suppressing antitumor T‑cell responses [15,17] , whereas endothelium-derived CD73 limits the migration of tumor-specific CD8 + cells to the tumor site, thereby inhibiting antitumor immunity [17] . Interestingly, tumor-infiltrating Th17 cells also express CD39/CD73 [20] , probably driven by TGF-b [21] . CD39+CD73 + Th17 cells inhibited T‑cell effector functions, and adoptive transfer of those Th17 cells promoted tumor growth through their expression of ectonucleotidases in several different transplantable tumor models [21] . Because those Th17 cells failed to facilitate tumor growth in nude mice upon adoptive transfer, the regulatory activity of CD39 + CD73 + Th17 cells is probably mediated through the regulation of antitumor T‑cell immunity [21] . Furthermore, recent studies suggest that CD73 expressed at high levels on bone marrow-derived granulocytic myeloid-derived suppressor cells has a dual effect on their expansion and suppressive activity [22] , thus contributing to myeloid-derived suppressor cells’ abilities 862

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to limit antitumor immunity. Similarly, it was also reported that inhibition of T‑cell proliferation by multipotent mesenchymal stromal cells was mediated by CD73/CD39 expression and adenosine generation [23,24] . Whether the expression of CD73/CD39 by those cells dictates their immunosuppressive function in the tumor microenvironment has yet to be determined. It is possible that CD73 on other immune cell populations (i.e., certain subsets of B cells, NKT cells and dendritic cells [25–27]) contributes to tumor immunity. In simple terms, ectonucleotidase expression by a variety of stromal components in the tumor microenvironment might be a general immunosuppressive feature associated with cancer progression. Complicating matters further, in addition to the immunoregulatory roles of CD73, CD73derived adenosine can also directly enhance tumor angiogenesis [10] , tumor cell growth, invasion, migration and adhesion [28–30] and/or chemotaxis [15] . Thus, the actual in vivo effect of targeted CD73 cancer treatment might far exceed the expectations raised by published experimental data. Yet furthermore, there are probable functions of CD73 that are independent of its enzymatic activity. For example, CD73 could also possibly function as a novel and specific receptor for the extracellular matrix protein tenascin C and the molecular interaction between these two proteins might influence tumor cell adhesion and migration [29] . CD73 linked via glycosylphosphatidylinositol to the surface of leukemia cells protects them against TRAIL-induced apoptosis by inter­action with death receptor 5 [31] . CD73 engagement triggers a rapid shedding of surface CD73 and results in clustering of CD11a/CD18 integrin and thereby increases the integrin-mediated binding of lymphocytes to endothelium [32,33] . On the other hand, direct engagement of CD73 with a specific mAb exerts profound effects on T‑cell activation by triggering an intracellular signaling cascade [34] . It is possible that the small-molecule inhibitors and mAb against CD73 used in the studies [11,12,15,17,19] have additional effects on nucleotide metabolism, which might independently affect both cancer cell and stromal cell behavior. Thus, the role of CD73 in tumor growth and metastasis independent of ecto-5´-nucleotidase enzymatic activity, if any, will have to be separately evaluated. Given the current availability of CD73 blockade for anticancer treatment for preclinical investigations, it is an appropriate time to ask, what is the true therapeutic potential for targeted CD73 future science group

Opportunities & challenges for anti-CD73 cancer therapy

cancer therapy? From a translational perspective, small-molecule inhibitors and mAb against CD73 are effective to treat cancer in multiple mouse tumor models and are well tolerated in mice [15–17] . Moreover, the importance of the CD73-mediated adenosinergic pathway in the process of cell growth and invasion of human cancer cells [30] and Treg-induced immuno­ suppression in various human cancers [35] has been independently confirmed. The development and clinical applications of CD73 blockade using clinical-grade antihuman CD73 mAb and small-molecule inhibitors are thus warranted. Despite these promising results, most published studies so far [11,12,15,17,19] are limited in the size and duration of tumor growth before anti-CD73 treatment. Although these studies have shown strong experimental evidence for the potential application of CD73 blockade in the treatment of small tumors, there is a substantial difference in immune status between an early-stage tumor and a large established tumor [36] . This problem is particularly salient in the clinical setting in which patients harbor an already dysregulated immune system and established cancers. Indeed, inhibiting CD73 alone fails to cure cancer. Moreover, weaker therapeutic effects were observed when large established tumors were treated with CD73 blockade [Wang L, Fan J, Chen S, Zhang B. Unpublished Data] . Nevertheless, the heterogeneous nature of cancer would probably preclude any single cancer immunotherapy from becoming an archetype for therapeutic efficacy [37] . In facing these challenges, investigators have been eager to explore the possibility of achieving the greatest antitumor strategy with CD73 targeting. A few recent studies demonstrate effective therapy against large tumors when using passive antibody or adoptive T‑cell therapy [38] . Given the fact of major modulatory effects of CD73 dependent on adaptive immunity, we consider it likely that CD73 blockade must be complemented by other approaches directed at improving the development and function of antitumor T cells, such as adoptive T‑cell therapy or dendritic cell vaccines. In support, we found that adoptive transfer of antigen-specific T cells cured all mice bearing ovarian tumors in which we knocked down CD73 expression [11] . It is also intriguing to test if synergistic targeting with CD73 and blocking immune checkpoints including CTLA‑4, PD‑1 and B7‑H1 using mAb treatment improves cancer immunotherapy. Finally, CD73 blockade could be effectively combined with chemo­therapy or radiation therapy because tumor CD39/CD73 has potential to convert extracellular ATP released from future science group

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tumor cells induced by chemotherapeutic drugs [39] and radiation [40,41] into adenosine that render tumors more resistant to immune-mediated killing or drug-induced death [42] .

“Despite reinvigorated interest in CD73 expression in the tumor, our understanding is still limited and much more needs to be learned.” Although CD73 has been observed in a broad spectrum of cancer types including leukemia (reviewed in [7]), the clinical importance of CD73 is so far ill-defined. A number of studies indicate that CD73 expression is probably associated with more aggressive, metastatic behaviors, and could serve as a diagnostic/prognostic marker and/or therapeutic target in certain cancer types [5,9] . However, conflicting findings have shown that CD73 expression was correlated with both poor and favorable clinical prognosis, depending on the human cancer type [43–45] . Therefore, it would be interesting to revisit this area and explore the possible correlative ana­lysis of CD73 between the phenotypic characterization of varied immune infiltrates and clinical outcome in a large cohort of different cancer specimens. Despite reinvigorated interest in CD73 expression in the tumor, our understanding is still limited and much more needs to be learned. For example, how is CD73 expression driven in the tumor microenvironment? The in vivo scenario is likely to be much more complex due to the unknown sources regulating CD73 expression in each particular setting during cancer progression. We have demonstrated that the tumor microenvironment contains factors that induce CD73 expression [11] . It is well established that hypoxia upregulates CD73 expression because CD73 has a HIF-1a responsive element in its promoter region [46] . A series of cytokines, such as the type I IFNs, TNF‑a, IL‑1b, prostaglandin E2 and TGF‑b, have also been implicated in the regulation of CD73 expression in various cell types with conflicting results [7] . Although endothelial cells and lymphocytes express structurally similar CD73, the CD73 expression in these two types of cells is differently regulated. For example, IFN‑a upregulates CD73 expression on endothelial cells [47] . Conversely, it fails to regulate CD73 expression on lymphocytes. Although this has been an important observation extrapolated to distinct types of cells, we have not yet identified the cellular and molecular mechanisms underlying the modulation of CD73 expression in the tumor. www.futuremedicine.com

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Possible on-target adverse events have not yet been observed for anti-CD73 therapy in reducing tumor burden in mouse models. However, the potential toxic risk that may be associated with CD73 blockade is still being sought. The exacerbation of a number of inflammatory reactions in CD73-deficient mice has been emphasized [48] . Moreover, human CD73 nonfunctional mutations are involved in increasing ectopic tissue calcification that is associated with an excess risk of cardiovascular events [49] . This has not been observed in CD73-deficient mice, indicating that the expression and/or function of the CD73 murine homolog may differ from that of humans in particular settings. Thus, special caution may be required in the timing and intensity of antiCD73 treatment based upon the specific disease pathogenesis and the stage of inflammation when future studies aim at translating this therapeutic approach to clinical trials in cancer patients. On the whole, we are faced with a good opportunity to develop CD73-targeting immuno­ therapies for treating solid cancers and leukemia [50] based on the knowledge obtained from recent studies. Combined with previous important findings regarding the role of CD39 [51,52] , A 2A R [1] and A 2BR [53,54] in tumor growth and metastasis, the CD39/CD73-mediated adenosinergic effect can now be viewed as one of the most important immunosuppressive regulatory pathways in the tumor microenvironment. The data support the feasibility of potent strategies to harness antitumor immune responses by targeting the References 1

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Financial & competing interests disclosure Financial assistance was provided by NIH grant CA149669, Ovarian Cancer Research Fund (LT/UTHSC/01.2011), CTSA grant (UL1RR025767), the Cancer Therapy and Research Center (2P30 CA054174-17), and the National Natural Science Foundation of China (No. 81171985). The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

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