CD73: an emerging checkpoint for cancer immunotherapy

Abstract

CD73 is a novel immune checkpoint associated with adenosine metabolism that promotes tumor progression by suppressing antitumor immune response and promoting angiogenesis. The inhibition of CD73, in combination with immune checkpoint blockade, targeted therapy or conventional therapy, improves antitumor effects in numerous preclinical mouse models of cancer. Emerging evidence suggests that the combination of anti-CD73 and immune checkpoint blockade has promising clinical activity in patients with advanced solid tumors. In this review, we will discuss the specific role of CD73 on both tumor cells and nontumor cells in regulating tumor immunity and tumorigenesis and provide an update on the current view of the antitumor activity of targeting CD73 by mAb or small molecule selective inhibitors in preclinical and clinical settings.

CD73, known as ecto-5′-nucleotidase, is a cell surface glycosylphosphatidlinositol-anchored glycoprotein. CD73 is essential for the generation of extracellular adenosine from 5′-adenosine monophosphate (5′-AMP) through the coordinated action of CD39 (ecto-nucleoside triphosphate diphosphohydrolase-1; ecto-NTPDase1) that catalyzes the phosphohydrolysis of ATP and ADP to AMP) [1,2]. Additionally, extracellular adenosine could be generated from the noncanonical pathway of CD38 (NAD⁺ nucleosidase)-CD203a (ecto-nucleotide pyrophosphatase/phosphodiesterase 1)-CD73 that is independent of CD39 [3]. CD73 is found on the surface of a variety of cell types, including endothelial cells, subtypes of lymphocytes, stromal cells and select types of tumor cells. Adenosine induces immunosuppression [4], angiogenesis [5], mucosal hydration [6] and ischemic preconditioning [7]. During pathophysiological conditions, CD73-generated adenosine protects from tissue destruction through inflammation, ischemia and hypoxia [2]. Despite the lack of extracellular adenosine signaling in CD73^−/− mice, extracellular ATP and ADP levels remain almost completely unchanged. Strikingly, CD73^−/− mice are viable, indicating that CD73 is not vital under normal physiologic conditions. However, recent work found that, CD73 insufficiency contributes to arterial calcification in humans with an autosomal recessive disease caused by loss-of-function mutations of Cd73. CD73 knockout (CD73^−/−) mice also recapitulated some of the characteristics associated with arterial calcification cue to deficiency of CD73 (ACDC) [8].

Previous studies have underscored a negative role for extracellular adenosine within the tumor microenvironment by activating four distinct adenosine receptors (G protein-coupled receptors): A₁R, A_2AR, A_2BR and A₃R. Adenosine activation of A_2AR triggers cyclic AMP and protein kinase A signaling, which inhibits T cell receptor signaling [9] and promotes the induction of Foxp3⁺ regulatory T cells (Tregs) [10]. In contrast, adenosine activation of A_2BR decreases the barrier function of vascular endothelium [11] and promotes myeloid cells [12–15] to acquire an anti-inflammatory (M2) phenotype that suppresses immune-mediated tumor cell eradication. Consistent with the results from A2AR^−/− [16] and A2BR^−/− mice [17], we and others have shown that CD73^−/− mice more readily reject tumors as compared with wild-type (WT) mice due to enhanced antitumor immunity [18] and decreased carcinogenesis [19]. Unexpectedly, one recent report indicated a decrease of CD73 expression in poorly differentiated advanced-stage endometrial carcinoma and ovarian high-grade serous carcinoma [20]. Strikingly, CD73-generated adenosine inhibits disease progression in these tumors [20]. CD73-generated adenosine seems to be required to sustain a physiological balance that maintains epithelial integrity in endometrial carcinomas with stable cell–cell adhesions mediated by A₁R. As the epithelial barrier function is essential for the inhibition of endometrial carcinomas with fewer intervening stromal or inflammatory cells in interconnected malignant glands [21], the loss of CD73 promotes adenosine deaminase endometrial tumor progression. This phenomenon is completely contrary to that observed in other tumor types, and results likely from context-dependent functions of the receptor in different tumor types. These findings provide a distinct perspective on the role of CD73-generated adenosine in tumor development. The roles of CD73 on tumor and host cells as well as its prognosis and therapeutic value in human cancers have been recently discussed [22,23]. In this review, we aim at providing a broader view of how CD73 is involved in the immune evasion and tumor progression, thereby highlighting the importance of combination treatment arising from CD73 targeting currently under investigation in early phase clinical trials. The limitations, challenges and the possible future research directions of anti-CD73 immunotherapy are also reviewed.

Effects of CD73 activity by tumor cells

CD73 is expressed by many different types of cancer and promotes tumor outgrowth, metastasis [18,24–31] and drug resistance [32,33] among leukemia, malignant glioma, melanoma, ovarian-, colon-, breast- and bladder-cancer [34–37].

A number of factors contribute to the modulation of CD73 expression on tumor cells. Estrogen receptors (ERs) have been shown to dominantly and negatively regulate CD73 expression, and its ability to generate adenosine in breast cancer [37]. As a result, the increased expression of CD73 may relate to breast cancer progression in a subset of ER-negative tumor cells. CD73 expression is also often induced under hypoxic conditions in the tumor microenvironment, as the cd73 gene transcription is directly mediated by HIF-1α [38–41]. In addition, many proinflammatory factors promote induction of CD73 expression, including TGF-β, IFNs, TNF, IL-1β and prostaglandin E2 [42,43]. Furthermore, tumor cell CD73 expression is regulated through the Wnt and cAMP pathways [44,45]. CD73 expression is also induced epigenetically, as CD73 expression is downregulated via methylation-dependent transcriptional silencing in human melanoma cell lines [46]. Particularly, melanomas lacking CD73 methylation are more likely to relapse. In addition, activated MAPK pathway in cooperation with the proinflammatory cytokines such as TNFα, promotes CD73 expression on melanoma cells [47,48]. Emerging evidence also suggests aberrant CD73 regulation at the transcriptional and post-transcriptional (e.g., miRNA) level in a variety of different cancer subtypes [49]. Together, these observations collectively support the potential for therapeutically targeting CD73 in melanoma and beyond.

The extracellular adenosine generated by CD73-expressing tumor cells [24,25] negatively regulates the activation and effector phases of the antitumor T cell response, while also promoting T cell apoptosis. CD73 is also required for cancer cell proliferation independent of immune regulation. For example, silencing CD73 expression with specific shRNAs inhibits the proliferation of breast cancer cells (MB-MDA-231), leading to increased cell-cycle arrest and apoptosis [31]. Likewise, treatment with APCP (α, β-methylene adenosine-5-disphosphate), a selective CD73 enzyme inhibitor, inhibits cancer cell proliferation in a dose-dependent manner [31,50,51]. Conversely, CD73 overexpression in breast cancer cells (MCF-7) increases cell viability and promotes cell-cycle progression. Similarly, CD73 overexpressing MCF-7 cells grow more rapidly than parental MCF-7 cells, while suppressing CD73 mRNA with siRNA suppresses tumor growth in mouse xenograft models [28,31]. In glioma cells, APCP treatment causes a 30% reduction of cell proliferation, while the addition of adenosine increases cell proliferation by 35%. Taken together, CD73-generated adenosine may promote cancer cell growth via its enzyme activity [29]. However, this effect is not universal, as adenosine induces apoptosis in gastric carcinoma cells [52], and ovarian cancer cells through the pro-apoptotic molecules Bax and caspase-3 [53]. Tumor cell CD73 expression also promotes tumor metastasis in mouse models, likely depending on the autocrine activation of A_2BR [24]. Tumor cell CD73 expression [28–30], or the activation of other adenosine receptors [54,55], promotes chemotaxis and invasiveness. Strikingly, CD73 activity by tumor cells also involves tumor angiogenesis by facilitating VEGF production in a mouse breast cancer model [56]. CD73 is also overexpressed on cancer stem cells [57,58] or cancer-initiating cells [59], and CD73 inhibition attenuates sphere formation and tumor initiation [57,59] highlighting the druggability of CD73 in the context of cancer stem cell/cancer-initiating cell-directed therapies. These results indicate a complex and contextual role for CD73 in regulating cancer cell viability, stemness and immune suppression, warranting further investigation in vivo.

Effects of CD73 activity by nontumor cells

Endothelial cells

A subset of endothelial cells expresses CD73 [60] and may contribute to angiogenesis within the tumor microenvironment. Indeed, WT mice formed more capillary-like structures in pulmonary microvascular endothelial cells than CD73^−/− hosts, and this difference was more evident when pulmonary microvascular endothelial cells were in vitro cultured with cancer cell-conditioned medium. The extent and density of tumor angiogenesis was greater in WT mice as compared with CD73^−/− deficient mice in vivo [61]. Additionally, the treatment of anti-CD73 monoclonal antibody (mAb) or APCP led to impaired angiogenesis and decreased tumor growth in several murine tumor models [56,62]. There was also evidence showing that the in vitro formation of capillary-like tubes by human umbilical vein endothelial cells is affected by CD73 expression but independent of its associated enzyme activity (i.e., extracellular adenosine) [56]. Furthermore, tumor cell CD73 promotes metastasis through adenosine-independent attachment to endothelium [63]. Taken together, current studies demonstrate that both tumor and endothelial cell CD73 synergistically contribute to tumor angiogenesis. However, the exact role of adenosine-independent function of CD73 demands additional investigation.

In an experimental lung metastasis model, CD73^−/− mice were found to be resistant to tumor metastasis after the intravenous injection of B16F10 melanoma cells or TRAMP-C1 prostate cancer cells [19,26]. Notably, the pro-metastatic effects of host CD73 were dependent on its expression by nonhematopoietic cells; most likely attributable to endothelial cells. On the other hand, we found that endothelial cell CD73 expression was associated with limited T cell infiltration of tumors [18] and an enhancement of tumor growth. Despite remaining not fully understood, the bulk of the existing evidence points to CD73-expressing endothelium as a contributor to tumor growth and metastasis.

T cells

Regulatory T cells (Tregs; CD4⁺CD25⁺FoxP3⁺) mediate immunotolerance and help tumor cells evade immunosurveillance by suppressing the immune response. One of the main mechanisms for Treg-mediated tumor immunosuppression is dependent on the extracellular adenosine generated by CD73 [18]. CD73 is abundantly expressed by Tregs and is frequently coexpressed with CD39. CD73, in combination with CD39, renders an enzymatically driven accumulation of immunosuppressive adenosine by Tregs. Accordingly, Tregs derived from either CD73^−/− or CD39^−/− mice have impaired suppressive functions [64,65]. Unlike WT murine Tregs, CD73^−/− Tregs fail to promote tumor growth [18,26]. Similarly, human Treg CD73 inhibition decreases their immunosuppressive capability, in vitro [66]. Coincidently, CD73 expression is observed on circulating Tregs isolated from head and neck cancer patients as compared with healthy donors [67]. In contrast to murine Tregs, natural human Tregs are often CD39-positive, but express little or no cell surface CD73. Inducible CD39⁺CD73⁺ Tregs significantly increase in peripheral blood, sentinel lymph node and tumor infiltrating lymphocytes of melanoma patients [68]; especially in response to high-dose IL-2 therapy [69]. Although the full role of CD73 on human Tregs is still being characterized, it is generally accepted that they generate adenosine through paracrine interaction(s) with local CD73-expressing cells and/or tumor-derived exosomes [70,71].

Aside from Tregs, murine CD4⁺Foxp3⁻ conventional T and CD8⁺ T cells express CD73 when exposed to TGF-β. In healthy donors, peripheral human CD8⁺ T cells express CD73 [72] and the activation of CD8⁺ T cells downregulate CD73 expression [73]. Within tumors, CD73 is highly expressed by memory CD8⁺ T cells and negligible on terminally differentiated effector CD8⁺ T cells [74]. CD73-mediated adenosine signaling through A_2AR is associated with the transition from naive/memory CD8⁺ T cells into effector cells by the inhibition of Wnt-signaling [74,75]. CD73 expression is also associated with a subset of CD4⁺ effector T cells enriched in polyfunctional Th1/17 cells [76]. In resected human breast and ovarian tumors, multicolor immunofluorescence confirms the codetection of CD73⁺CD4⁺ effector T cells and CD39⁺ Tregs within the immune infiltrates [76]. Increased CD73 levels are associated with increased adenosine generation and immunosuppressive Th17 functions, and increased tumor growth in mouse tumor models [77]. In contrast, genetic ablation of CD73 or treating with IL-1β rather than TGF-β to program Th17 cells, ex vivo, induces stemness and improves the antitumor effects of adoptive Th17 cell therapy [78].

Natural killer cells

Within the tumor microenvironment, natural killer (NK) cells highly express CD73, which is found at negligible levels in healthy individuals [79]. NK cell CD73 expression endows them with suppressive functions via adenosine generation. A_2AR is primarily expressed by NK cells and upregulated further under pathological conditions, such as lung injury and inflammation [80]. A_2AR activation suppresses the activation and cytotoxic function of NK cells [81–84]. Accordingly, CD73 adenosine and A_2AR activation causes NK cell suppression of antitumor functions while simultaneously promoting tumor metastasis [81,85,86]. Therefore, blockade of the CD73-A_2AR axis may potentiate the antitumor effects of NK cell-based immunotherapy. However, different from the effect of A_2AR activation, A_3AR agonists can stimulate NK cytotoxicity and promote the antitumor function [87]. Furthermore, the presence of adenosine renders NK cells hyporesponsive to IL-12 and IL-15 stimulation ex vivo, exhibiting enhanced IFN-γ expression from CD56⁺ subsets [84]. These results argue against the adenosine-mediated general NK cell inhibition and suggest instead the cellular contexture dependent effect of adenosine on NK cells.

Macrophages

In healthy individuals, circulating CD14⁺ monocytes express low levels of CD73, while tissue-resident macrophages express both CD39 and CD73 [88]. Activation of macrophages through TLR activation induces the release of ATP, which is rapidly hydrolyzed by cell surface CD39 [89]. The expression levels of CD39 and CD73 regulates adenosine generation [90]. Macrophages are divided into two primary subsets, the pro-inflammatory M1 population and the anti-inflammatory M2 population. M1 macrophages express lower levels of CD39 and CD73 as compared with the M2 subset [90]. Changes in the ectoenzyme activities of CD39 and CD73 may fine-tune their functions in the inflammatory setting. In a murine model of myocardial infection, CD73 blockade by APCP augmented the predominance of the M1 subset [91]. Similarly, ablating CD73 activity in tumor-bearing mice decreases M2 macrophages and increases the M1 subset [27]. However, LPS and TNF-α induced CD73 on human macrophages, and the addition of exogenous AMP or CD73 inhibitor has no effect on their polarization [92]. To better identify the effects of CD73 on macrophages during tumor outgrowth, additional studies are required to evaluate the importance of CD73 for differentiation and function of tumor-associated macrophages isolated, especially from human tumors.

Myeloid-derived suppressor cells

MDSCs (myeloid-derived suppressor cells) are a heterogeneous population consisting of different immature myeloid cell subsets that accumulate during tumor development. MDSCs infiltrate human tumors and their presence is associated with decreased efficacy of immune checkpoint blockade and other immunotherapies [93]. In tumor-bearing mice, CD39 and CD73 are coexpressed on MDSCs [14,94,95] and subsequent A_2BR activation promote their expansion [14]. Furthermore, A_2BR blockade reduces tumor-infiltrating MDSCs in a mouse melanoma model [96]. Conversely, treatment with an A_2BR agonist enhances MDSC tumor infiltration [97]. These results are supported by human studies showing higher CD39 and CD73 levels on MDSCs isolated from cancer patients as compared with healthy donors [98–100]. CD39 and CD73 coexpression also identifies a distinct inflammatory subpopulation among activated MDSCs with enriched suppressive molecular signatures in non-small-cell lung cancer patients [99]. Specifically, tumor-derived TGF-β induces CD39/CD73 coexpression on MDSCs through activation of the mTOR-HIF-1α pathway. Moreover, MDSC CD39/CD73 contributes to their immunosuppressive function and predicts the response to chemotherapy [99]. Consistently, metformin treatment decreases the expression and ectoenzyme activity of CD73 on monocytic and polymononuclear MDSC subsets in ovarian cancer (OC) patients, which is associated with improved T cell-mediated antitumor immunity [100]. OC patients treated with metformin have a downregulation of CD39/CD73 expression on MDSCs through activation of AMP-activated protein kinase α and subsequent suppression of HIF-1α. Metformin treatment induces a simultaneous increase in the antitumor activities of circulating CD8⁺ T cells and correlates with a longer overall survival in diabetic patients with OC. Collectively, these data suggest that the inhibition of CD39/CD73-dependent MDSCs may provide for new opportunities to improve future cancer immunotherapeutic strategies.

Other immune cells & stromal cells

CD73 has long been recognized to be a marker for B cell differentiation [101]. Several studies demonstrated that, CD73 is expressed on a subset of memory B cells [102,103]. In vitro blockade of B cell CD73 activity from healthy controls results in an impairment of IgG class switching. Although there is a paucity of data for B cell CD73 expression and its function in cancer, a recent study reported a critical role for B cells in providing antitumor activity while using APCP in a mouse melanoma model [104]. We have shown that CD73 is expressed on type 2 innate lymphoid cells (ILC2s) and can be significantly elevated on tumoral ST2⁺ ILC2s in the IL-33-rich tumor microenvironment [90]. Importantly, the ILC2-derived CD73 is likely involved in suppressing NK cell-mediated antitumor activities [90].

Cancer associated fibroblasts (CAFs) are an important stromal component in a majority of tumor microenvironments. High levels of CAF CD73 expression is associated with poor prognosis in patients with OC [105] and nonmuscle invasive bladder cancer [106]. CD73-expressing CAFs are also detected in human triple negative breast cancer [107] and OC [108]. In these two studies, CAFs enhanced the survival, as well as the content of Tregs; partly dependent on CD73 activity. In a mouse model of OC, CAF CD73 expression inhibits T cell-mediated antitumor immunity [105], similar to the immunosuppressive effect of tumor cell CD73 [25]. Furthermore, CD73 expression in prostate epithelium is associated with immune suppression and progression of prostate cancer, while its expression in the stroma is shown to be associated with a good disease prognosis [109].

CD73 blockade in cancer therapy

In 2006, the Sitkovsky group presented seminal findings on how A₂AR protect tumors from antitumor-armed effector T cells [16]. Based on the first report, accumulating evidence has supported the therapeutic potential of targeting the CD73-A₂AR axis in numerous types of cancer over the past decade. Stagg et al. originally showed that, CD73 mAb treatment inhibited breast tumor growth and metastasis [24], while we reported that CD73 small molecule inhibitor treatment with APCP decreased the progression of OC and increased animal subject survival [25]. These two independent groups both subsequently demonstrated that host and tumor cell CD73 contribute to tumor growth and metastasis using multiple mouse models, highlighting the requirement for optimal antitumor activity to target both tissue compartments. The different experimental methods for inhibiting CD73 expression and its activity in the preclinical setting is summarized in Table 1.

Table 1. . The strategies to block CD73 in tumor cells and tumor-bearing animals.

Tumor cell	Cancer type	Treatment	Efficiency
MDA-MB-231	Human breast cancer	APCP	Xenograft of MDA-MB-231 cells in nude mice with APCP treatment have a lower volume and weight than those of the control group [50]
		Anti-CD73 (AD2)	Anti-CD73 monoclonal antibody inhibits metastasis formation by a mechanism independent of CD73 catalytic activity but fails to inhibit primary tumor growth [110]
		SiRNA-CD73	CD73 siRNA effectively causes MB-MDA-231 tumor growth suppression in vitro and in vitro, prevention of adhesion to extracellular matrix and inhibition of invasion and migration [28,31]
U138MG	Human brain glioblastoma	APCP	In vitro APCP treatment of glioma cells results in a significant reduction of glioma cell proliferation [111]
T24	Human bladder carcinoma	APCP	In vitro APCP treatment results in a significant decrease of T24 cell number as compared with control cells [51]
GBC-SD	Human gallbladder carcinoma	SiRNA-CD73	CD73 knockdown inhibits tumor cell proliferation, migration and motility [112]
Glioblastoma Multiforme	Human brain cancer	SiRNA-CD73	CD73 knockdown reverses multiple-drug resistance of GBM cells [33]
E0771	Murine breast cancer	Anti-CD73 (TY/23)	Anti-CD73 monoclonal antibody treatment significantly delays primary E0771 tumor growth in immune-competent mice [24]
ID8	Murine ovarian cancer	APCP	A survival advantage is observed in ID8-bearing mice with APCP treatment in vivo [25]
		Anti-CD73 (TY/23)	Peritoneal ID8 tumor model showed a median 146-day survival of mice treated with TY/23 as superior to that of mice without treatment [18]
ID8-OVA	Murine ovarian cancer	SiRNA-CD73	CD73 knockdown renders tumor cells more susceptible to T-cell killing [25]
B16-SIY	Murine melanoma	APCP	In vivo APCP treatment effectively suppresses B16-SIY tumor growth [18]
		Anti-CD73 (TY/23)	Delayed tumor growth was observed in B16-SIY bearing mice treated with the anti-CD73 mAb TY/23 as compared with a control group [18]
B16F10	Murine melanoma	APCP	In vivo APCP treatment induces significant tumor regression by promoting the release of Th1- and Th17-associated cytokines in the tumor microenvironment [104]
		CD73 ablation	CD73 ablation significantly suppresses the growth and metastasis of B16F10 [18]
4T1.2	Murine breast cancer	Anti-CD73 (TY/23)	Anti-CD73 monoclonal antibody treatment significantly delays primary 4T1.2 and E0771 tumor growth in immune-competent mice and significantly inhibits the development of spontaneous 4T1.2 lung metastases [24]
4T1	Murine breast cancer	siRNA-CD73 loaded nanoparticle	Decreased expression of tumor cell CD73 contributes to decreased tumor growth and metastasis and improves animal subject survival [113,114]
MCA-induced fibrosarcoma	Murine	Anti-CD73 (TY/23)	Anti-CD73 monoclonal antibody treatment effectively suppresses growth of established 3-methylcholanthrene (MCA)-induced tumors [19]
		CD73 ablation	CD73 deficiency suppresses the development of MCA-induced fibrosarcomas [19]
TRAMP-C1	Murine prostate cancer	Anti-CD73 (TY/23)	Anti-CD73 monoclonal antibody treatment suppresses the growth of TRAMP-C1 prostate tumors and inhibits the development of TRAMP-C1 lung metastases [19]
Prostate cancer	Murine	CD73 ablation	CD73 deficiency also suppresses prostate tumorigenesis in TRAMP transgenic mice [19]
LWT1	Murine melanoma	Anti-CD73 (TY/23)	Anti-CD73 monoclonal antibody treatment significantly decreases metastatic burden as compared with control treatment [115]
		CD73 ablation	CD73 ablation improves metastatic control as compared with wild-type mice [115]
MC38-OVA	Murine colon cancer	CD73 ablation	CD73 ablation significantly suppresses the growth of MC38-OVA [26]
EL4/EG7	Murine lymphoma	CD73 ablation	CD73 ablation significantly suppresses the growth of EL4/EG7 [18,26]
AT-3	Murine mammary tumor	CD73 ablation	CD73 ablation significantly suppresses the growth of AT-3 [26]

While single agent CD73 blockade is not curative, CD73 inhibition improves the antitumor activity of blockade immune checkpoint treatments including CTLA-4- and PD-1-mAb [116–119]. Strikingly, tumor cell CD73 expression attenuates the immune response evoked by anti-PD-1 mAb treatment, while anti-PD-1 mAb treatment augments the expression level of A_2AR, especially on CD8⁺ tumor-infiltrating lymphocytes [117]. As expected, the combination of anti-PD-1 and anti-CD73 mAbs, or an A_2AR antagonist, results in more effective antitumor T cell-mediated immune responses [117]. Interestingly, the activation of A_2AR enhances PD-1 expression on tumor-specific CD8⁺ T cells and CD4⁺ Foxp3⁺ Tregs [116], indicating a reciprocal loop of the CD73-A_2AR axis and how PD-1 contributes to the synergy of combination immunotherapy. Indeed, the removal of hypoxic environment, a primary driver of the CD73-A_2AR axis, promotes antitumor activity of dual PD-1 and CTLA-4 blockade in an A_2AR dependent manner [85]. In addition, blockade of A_2AR profoundly increases the antitumor activity of CAR T cell, particularly when combined with PD-1 blockade [120]. Similarly, anti-CD73 improves the intratumoral homing of CAR-NK in vivo. Furthermore, the combination of anti-CD73 with NKG2D-engineered CAR-NK cells achieves synergistic antitumor effect in CD73⁺ human lung cancer xenograft model [121].

Aside from the immune checkpoint-targeted treatments, combination of agonistic anti-4-1BB therapy and CD73 blockade can achieve tumor regression in several mouse models of cancer [122]. Interestingly, anti-4-1BB treatment preferentially induces CD73-negative effector T cell response for tumor inhibition. However, the efficacy of agonistic anti-4-1BB therapy is diminished in TGF-β-rich tumors that sustain the expression of CD73 on intratumoral CD8⁺ T cells. Mechanistically, the efficacy of anti-4-1BB therapy is determined by the activation of STAT3 and mutual regulation between TGF-β and 4-1BB ligation in concomitant CD8⁺ T cell activity. Similar to anti-4-1BB treatment, either anti-GITR or anti-OX40 treatment preferentially drives CD73-negative CD8⁺ T cell immunity. Accordingly, tumor regression has been achieved by combination therapy of CD73 blockade and anti-GITR [122]. Nevertheless, the contribution of CD73-mediated adenosinergic effect to the resistant mechanism of immunostimulatory agonistic cancer therapy remains to be further defined.

NAMPT inhibitors has been combined with anti-CD73 mAbs to improve immune-mediated antitumor effector function(s). NAMPT is a key enzyme in the regulation of intracellular NAD⁺ pool. NAMPT inhibitors suppress tumor cell growth by reducing NAD⁺ and ATP levels [123,124], and have entered the clinical trials. In human cells, CD73 is capable of converting extracellular NAD⁺/nicotinamide mononucleotide to nicotinamide riboside as a precursor for intracellular NAD⁺ biosynthesis [125,126]. Therefore, antitumor functions of combined NAMPT (FK866) and CD73 (APCP) inhibition leads to a significant increase in survival of tumor-bearing mice compared as compared with single treatment in a human OC model [127].

In immunocompetent mouse models of HER2/ErbB2-driven breast cancer, anti-CD73 mAb treatment improves the efficacy of anti-ErbB2 mAb for treating engrafted or spontaneous primary tumors and lung metastases [128]. A recent study reported the increased CD73 expression in melanoma patients with advanced BRAF mutation⁺ disease [129]. Administration of BRAF inhibitor, with or without MEK inhibitor, downregulates CD73 expression, and combination anti-BRAF, MEK and A2A receptor inhibitor treatment results in a significant suppression of tumor initiation and metastasis in a mouse melanoma model [129]. It is becoming increasingly appreciated that a synergy occurs during concurrent treatment with CD73 blockade and conventional antineoplastic agents against cancer [130]. CD73 inhibition enhances doxorubicin-mediated antitumor effects in mice with established metastatic breast tumors [131]. In contrast, CD73 blockade triggers a beneficial effect in the tumor microenvironment of irradiated tumors, reflected by an increased infiltration and activity of CD8⁺ T cells and dendritic cells, with an accompanying decrease of Treg accumulation [132], associated with complete tumor regression during dual CD73 and CTLA-4 blockade in combination with irradiation.

CD73 in clinic

In different types of human cancer, including melanoma, breast cancer, acute lymphocytic leukemia, CLL, glioblastoma, head and neck cancers, high-grade serous OC, endometrial cancer, colorectal, prostate, bladder cancer, gastric cancer, kidney cancer, and pancreatic carcinomas [133,134], the high expression level of CD73 within tumor microenvironment is often associated with worse clinical outcomes. Recently, 122 samples from the BIG 02-98 adjuvant Phase III clinical trial further supports the association of a worse prognosis with higher CD73 expression in the resected tumor. A reduced anti-tumor immune response also correlates with CD73 expression on those samples [135]. CD73 upregulation is also detected in melanoma patients progressing under adoptive T-cell transfer or immune checkpoint blockade [136], indicating a potential adaptive resistance mechanism.

Several inhibitors targeting CD73 or A_2AR are currently being developed and tested in early clinical trials. CD73-targeted mAbs include MEDI9447, BMS-986179, NZV930 and CPI-006 (Table 2). MEDI9447 (Astrazeneca-Medimmune) is a human IgG1λ mAb selectively binding to and inhibiting the ectonucleotidase activity of CD73, which crossreacts with both mouse and human CD73 [119,137]. MEDI9447 removes CD73 from the cell surface by internalization, resulting in the inhibition of AMP conversion into adenosine, relieving AMP-mediated inhibition on T cell proliferation [119]. In immunocompetent mouse tumor models, MEDI9447 decreases immunosuppression and promotes antitumor function(s) [119]. Moreover, the combination of MEDI9447 with an anti-PD-1 antibody, induces greater antitumor effects as a single agent, supporting the initiation of multiple MEDI9447-based Phase I/II trials. Preliminary Phase I data of MEDI9447 (NCT02503774) were recently reported [138]. Treatment with MEDI9447 and durvalumab (anti-PD-L1) had a manageable safety profile and PD consistent with its mechanism of action. There is encouraging clinical activity in pancreatic cancer, and potentially, in colorectal cancer patients following combination therapy. Similar to MEDI9447, BMS-986179 (BMS) is a high-affinity antibody that inhibits CD73 enzyme activity and mediates the internalization of CD73. BMS-986179 treatment enhances antitumor activities of anti-PD-1 mAb in preclinical models [139]. Importantly, preliminary results of the first-in-human Phase I/IIa study of BMS-986179 + nivolumab (anti-PD-1 mAb) in patients with advanced solid tumors (NCT02754141), showed a safety profile similar to that of nivolumab monotherapy and with measurable immunological responsiveness [140]. Aside from anti-CD73 mAbs, small molecule inhibitors targeting CD73 have generated considerable interest. Novel substituted benzothiadiazine derivatives are being developed by GSK to inhibit CD73 activity [141]. Arcus Biosciences have discovered AB680, a highly potent, reversible and selective inhibitor of CD73, with a favorable projected human PK profile suitable for parental administration [142]. Moreover, coadministration of AB680 with an anti-PD-1 mAb, results in a significant reduction of tumor growth associated with increased tumor-infiltrating immune cells [143]. According to a recent global immuno-oncology landscape survey in September 2018 [144], there are 16 active reagents targeting CD73 currently under development. These data collectively support the further evaluation of this approach with other immune checkpoint inhibitors for patients with advanced solid tumors in large cohort clinical studies.

Table 2. . CD73 blockade in the clinic.

Tumor	Drug	Phase	Period	Sponsor	Identifier
Advanced solid tumors	MEDI9447 as monotherapy and in combination with MEDI4736	Phase I	2015–2021	MedImmune LLC	NCT02503774
Advanced solid tumors	BMS-986179 as monotherapy and in combination with nivolumab (BMS-936558)	Phase I/IIa	2016–2020	Bristol-Myers Squibb	NCT02754141
NSCLC, RCC, PDAC, Ovarian cancer, MSS and TNBC	NZV930 as monotherapy and in combination with PDR001 and/or NIR178	Phase I	2018–2022	Novartis Pharmaceuticals	NCT03549000
NSCLC	MEDI9447 in combination with osimertinib or AZD4635	Phase I Phase II	2018–2021	MedImmune LLC	NCT03381274
Ovarian cancer	MEDI9447 in combination with durvalumab (anti-PD-L1), tremelilumab (anti-CTLA-4), and MEDI0562 (anti-OX40)	Phase II	2018–2023	Nordic Society for Gynaecologic Oncology	NCT03267589
TNBC	MEDI9447 in combination with paclitaxel plus carboplatin and durvalumab	Phase I Phase II	2018–2022	Jules Bordet Institute	NCT03616886
NSCLC, RCC, CRC, TNBC, cervical cancer, ovarian cancer, pancreatic cancer, endometrial cancer, sarcoma, HNSCC, bladder cancer and mCRPC	CPI-006 as monotherapy and in combination with CPI-444 or pembrolizumab	Phase I	2018–2023	Corvus Pharmaceuticals, Inc.	NCT03454451

CRC: Colorectal cancer; HNSCC: Squamous cell carcinoma of the head and neck; mCRPC: Metastatic castration-resistant prostate cancer; MSS: Colorectal cancer microsatellite stable; NSCLC: Non-small-cell lung cancer; PDAC: Pancreatic ductal adenocarcinoma; RCC: Renal cell cancer; TNBC: Triple-negative breast cancer.

Interestingly, CD73 and A_2AR functions have recently been shown to be nonredundant in the tumor microenvironment. Antitumor activity of combined inhibition with CD73 and A_2AR coinhibition is more potent than monotherapy with either agent. Thus, targeting these interdependent pathways combinatorially, significantly suppresses tumor initiation, growth and metastasis [115]. Notably, combination AZD4635 (A2AR inhibitor) with MEDI9447 treatment in NSCLC patients is currently undergoing clinical trial (NCT03381274) evaluation.

Importantly, the soluble CD73 (sCD73) enzyme activity is observed in the plasma [145–147]. In patients with metastatic melanoma stage IV, increased basal levels of sCD73 enzyme activity in the blood, before starting nivolumab treatment, are associated with lower response rates to therapy [148], suggesting a potential value of measuring sCD73 activity in the serum from cancer patients to predict the benefit of immune checkpoint therapy [148]. Interestingly, sCD73 and alkaline phosphatase catabolizes AMP to adenosine, whereas soluble ADA (sADA) mediates conversion of adenosine to inosine [145]. There are also significantly higher activities of serum total sADA in patients with breast cancer than healthy subjects [149,150]. However, the expression patterns of these plasma purine-metabolizing enzymes that result in elevated plasma concentrations of anti-inflammatory adenosine in cancer patients remain elusive. Still, more researches are needed to confirm if the sCD73 and/or sADA can be used as serologic prognostic biomarkers in the future.

Conclusion

It has become clear that, CD73 generates adenosine that suppresses antitumor immunity and contributes to tumor outgrowth and/or metastasis. Apart from the immune-dependent effect(s) of CD73, it also promotes tumorigenesis independent of the immune effects. Strikingly, CD73 blockade also suppresses angiogenesis in the tumor [56,61], and both CD73-mediated enzyme and nonenzyme functions contribute to tumor angiogenesis [56]. CD73 also promotes tumor cell adhesion to extracellular matrix proteins [151,152]. These results have led to continued interest in the rapid development of anti-CD73 cancer therapy using mAb and/or small molecule inhibitors in early stage clinical trials. In addition, high levels of CD73 are associated with lymph node metastases and poor prognoses in various types of cancer [153]. CD73 is found to be an independent prognostic factor in cancer patients, including prostate cancer [154] and triple-negative breast cancer (TNBC) [155]. Furthermore, a recent study suggests that sCD73 is a reliable biomarker in patients with metastatic melanoma patients treated with nivolumab [148]. Whether surface CD73 and/or sCD73 serves as a viable biomarker requires further validation.

Future perspective

The field of cancer immunotherapy is evolving rapidly, and CD73-targeted therapies serve as a valuable example of new immune target derived from fundamental new therapeutic approaches. Despite the effective preclinical antitumor activity by CD73 inhibition, in combination with blockade of other immune checkpoints or agonist immunotherapies, the clear therapeutic potential of CD73-targeted therapy remains minimally defined to date in patients with cancer. In addition, manipulating the metabolism of adenosine from CD73 as therapeutic modalities targeting the downstream cascade (such as adenosine deaminase) appears compelling. Deep characterization of CD73 expression, and its function as well as the downstream cascade in the tumor microenvironment during ongoing clinical trials, holds the key to addressing the potential for targeting CD73-mediated adenosinergic axis to substantially benefit human subjects with aggressive malignancies.

Executive summary.

• CD73 is essential for generation of extracellular adenosine through the coordinated action of CD39. Alternatively, extracellular adenosine could be generated from the noncanonical pathway of CD38-CD203a-CD73 that is independent of CD39.

• We aim at providing a broader view of how CD73 is involved in the immune evasion and tumor progression, thereby highlighting the importance of combination treatment arising from CD73 targeting currently under investigation in early phase clinical trials.

Effects of CD73 activity by tumor cells

• CD73 is expressed by many different types of cancer and promotes tumor outgrowth, metastasis and drug resistance.

• There is a complex and contextual role for CD73 in regulating cancer cell viability, stemness and immune suppression.

Effects of CD73 activity by nontumor cells

• CD73-mediated adenosinergic effect has been extensively investigated on a variety of immune cell populations including CD4, CD8, NK, MDSC, macrophage, B cell, ILC2 in the regulation of tumor immunity.

• Both tumor and endothelial cell CD73 synergistically contribute to tumor angiogenesis.

• CAF CD73 expression inhibits T cell-mediated antitumor immunity, similar to the immunosuppressive effect of tumor cell CD73.

CD73 blockade in cancer therapy

• Host and tumor cell CD73 contribute to tumor growth and metastasis in multiple mouse models, highlighting the requirement for optimal antitumor activity to target both tissue compartments.

• While single agent CD73 blockade is not curative, CD73 inhibition improves the antitumor activity of blockade of CTLA-4/PD-1 or agonistic anti-4-1BB/GITR therapy.

• Targeting the CD73-A_2AR axis augments the antitumor activity of CAR-T cells or CAR-NK cells.

• A synergy occurs during concurrent treatment with CD73 blockade and conventional antineoplastic agents, or targeted cancer therapies such as BRAF, MEK, NAMPT and A_2AR inhibitors.

CD73 in clinic

• High expression level of CD73 within tumor microenvironment is often associated with worse clinical outcomes.

• Several inhibitors targeting CD73 or A_2AR with other immune checkpoint inhibitors are currently being developed and tested in early clinical trials.

• Early evidence suggests a potential value of measuring sCD73 activity in the serum/plasma from cancer patients to predict the benefit of immune checkpoint therapy.

Conclusion & future perspective

• Apart from the immune-dependent effect(s) of CD73, it also promotes tumorigenesis independent of the immune effects.

• CD73 blockade is expected to act additively or synergistically with other immune checkpoint inhibitors and agonist immunotherapies.

• Whether surface CD73 and/or soluble CD73 serves as a viable biomarker requires further validation.