Combinatorial Treatment Approaches
Since multiple ecto-enzymes with redundant functions contribute toward extracellular adenosine production and both A2AR and A2BR triggering mediate the majority of adenosine's pro-tumoral effects, monotherapies may not be sufficient to block the adenosine-signaling axis. In addition, there is strong rationale for combination with IMTs, such as ICB of PD-1/PDL-1 or CTLA-4, as well as ACT, radiotherapy and chemotherapy, to further unleash the cytotoxic capacity of T cells, which, as will be discussed, can become highly sensitized to adenosine-mediated immunosuppression.

Combinations of Adenosine-Axis Blockade Agents
Concurrent mAb-mediated (418) or pharmacologic (47) inhibition of CD39 and CD73 failed to potentiate CD73-blockade-induced suppression of adenosine production by Tregs and ovarian cancer cell lines. These findings are corroborated by the observation that skin biopsies derived from CD39−/−CD73−/− mice have identical capacity to produce adenosine upon injury induction with counterpart biopsies derived from CD73−/− mice (424).
Alone the same lines, others addressed whether simultaneous blockade of CD73 and of A2AR would result in higher anti-tumor efficacy. Of note, CD73−/−A2AR−/− mice present superior tumor control as compared to single knockout mice (384). Moreover, tumors in A2AR-null mice express twice as much CD73 at their core when compared to tumors formed in wild-type mice (384). Indeed, dual therapy with an anti-CD73 mAb and an A2AR agonist confers superior tumor protection as compared to either one as a monotherapy (384). However, this additive effect is lost when CD73 is targeted with a pharmacologic inhibitor, thus underscoring the capacity of CD73 to promote tumor progression in a catalytic activity-independent manner (384). In light of these studies, Evotec and Exscientia have partnered to develop A2AR/CD73 bi-specific inhibitory molecules (425), whereas NCT03454451, NCT03549000 as well as the Phase Ib/II clinical trial NCT03381274 sponsored by MedImmune all include solid tumor-bearing patient cohorts scheduled to be treated with combinations of an anti-CD73 mAb along with a pharmacologic A2AR antagonist.

Adenosine-Axis and PD-1 Blockade
Briefly, PD-1 is an immunosuppressive receptor that upon binding to its ligands, PDL-1 and PDL-2, dampens T-cell activity thereby enabling tumors to evade immune-destruction. Blockade of the PD-1-PDL-1/2 signaling axis results in durable complete responses in the clinic for a fraction of treated patients (1), and many pre-clinical and clinical studies have explored concomitant inhibition of adenosine production, or antagonism of A2AR and A2BR, to improve response rates.
It has been demonstrated that CD73+ tumor cells are resistant to PD-1 ICB (401) and that simultaneous mAb-mediated blockade of CD73 and PD-1 synergistically enhances tumor control and survival in mice (382, 385). Mechanistically, the dual therapy augments intra-tumoral CD8+ tumor-specific T cells (382, 385) and IFNγ mRNA levels (382) as compared to single-agent treatments. Several clinical trials assessing anti-CD73 mAb treatment along with anti-PD-1 mAb (NCT03454451, NCT03549000) or anti-PDL-1 mAb (NCT02503774, NCT03773666, NCT03267589, NCT03334617) of advanced solid tumors are recruiting or underway. Intra-tumoral upregulation of CD38 and subsequent adenosine production was recently identified as a mechanism of acquired resistance to PD-1/PD-L1 blockade and mAb-mediated or pharmacologic inhibition of CD38 was shown to significantly improve the anti-tumor efficacy of an anti-PDL-1 mAb (96). In terms of mechanisms, tumors from mice receiving the combinatorial therapy displayed higher accumulation of CD8+ T cells, effector memory CD8+ T cells, ICOS+ CD4+ T cells and lower levels of MDSCs and Tregs as compared to tumors from single-agent treated mice (96).
The potential for synergy between the co-administration of A2R antagonists with anti-PD-1 mAb is underscored by the observations that PD-1 blockade enhances A2AR expression on tumor-infiltrating CD8+ T cells (401), as well as that PD-1 blockade is more efficacious, in terms of increasing the survival of tumor-bearing mice, when these mice lack the A2AR (400). Vice versa, A2AR triggering on the surface of CD8+ T cells derived from tumor tissue (382), tumor draining lymph nodes or spleen (396) promotes PD-1 expression suggesting that simultaneous PD-1 blockade would boost the anti-tumor efficacy of A2A antagonism. Indeed, several groups demonstrated that concurrent provision of PD-1 checkpoint inhibitors along with A2AR antagonists is more effective than single-agent treatments at reducing tumor growth rate (96, 396, 400, 401) and metastasis formation (394, 401), as well as at improving survival (394, 396, 401). Moreover, the combination enables increased production of IFNγ and GzB by CD8+ tumor infiltrating T cells (401) while augmenting the intra-tumoral presence of NK cells (394). Five clinical trials for the treatment of solid-tumor patient cohorts with A2AR antagonists along with anti-PD-1 Ab (NCT02403193, NCT03207867) or anti-PD-L1 Ab (NCT02655822, NCT03337698, NCT02740985) are ongoing. Finally, dual therapy comprising A2BR antagonism and PD-1 blockade is superior to either monotherapy at decreasing metastasis and improving survival of tumor-bearing mice (359). However, no clinical trials have been launched to date to explore this combination in human cancer patients.

Adenosine-Axis and CLTA-4 Blockade
The blockade of CTLA-4, an immune checkpoint receptor predominantly expressed by T cells and which competes with the co-stimulatory receptor CD28 for binding to CD80/CD86 on the surface of antigen presenting cells (APCs), has also generated durable clinical responses in advanced cancer patients (1). Tumor-bearing mice receiving CTLA-4 blockade and pharmacologic (389) or Ab-mediated (382) inhibition of CD73 display superior tumor control (382, 389) and overall survival (382) than counterparts receiving single agent treatments. Mechanistically, these dual therapies are more effective than corresponding monotherapies at increasing the intra-tumoral presence of tumor-specific CD8+ T cells (382), CD4+FoxP3neg T cells (389) as well as the levels of IFNγ (389) and of mRNA coding for IFNγ and T-bet (382). Likewise, concomitant provision of CTLA-4 ICB and antagonists of either A2AR (389) or A2BR (359) leads to decreased tumor growth (389) and metastasis formation (359), as well as to higher survival of tumor-bearing mice (359) when compared to single treatments. In terms of mechanisms, combining CTLA-4 ICB with an A2AR antagonist augments intratumoral CD8+ T cell presence as well as IFNγ and GzmB levels (389).

Adenosine-Axis Blockade and Adoptive T Cell Therapy
There are two main approaches to ACT. Either autologous tumor-reactive T cells are expanded from tumor biopsies prior to patient re-infusion [i.e., tumor infiltrating lymphocyte (TIL) therapy], or peripheral blood T cells are gene-engineered to express a tumor-specific T cell receptor (TCR), or a so-called chimeric antigen receptor (CAR; a fusion protein that links scFv-mediated tumor antigen-binding with intracellular endo-domains associated with T cell activation). Cancer patients are typically lymphodepleted prior to ACT, and following infusion they receive high doses of IL-2, both of which support T cell engraftment (426). TIL therapy has achieved robust and durable responses in advanced melanoma patients, while CAR therapy targeting CD19 has yielded unprecedented clinical responses against a variety of advanced, treatment-refractory B cell malignancies (118, 427, 428).
Synergy has been demonstrated between strategies limiting adenosine production blockade and ACT within tumor-bearing mice. Indeed, ACT confers increased control of tumors lacking CD73 expression (388) and dual therapy of ACT and pharmacologic or mAb-mediated inhibition of CD73 was more robust than single treatments at augmenting tumor control and overall survival (378). Mechanistically, pharmacologic inhibition of CD73 potentiated the anti-tumor efficacy of ACT at least by boosting the homing of the adoptively transferred tumor-specific T cells at the tumor sites (378). Likewise, respiratory hyperoxia in mice increased the ability of adoptively transferred T cells to curb primary tumor expansion and metastasis formation by augmenting their capacity to accumulate in the TME and produce IFNγ (293).
Similarly, A2AR deficiency (402) or siRNA-mediated suppression of A2AR and A2BR expression (38) on the surface of adoptively transferred T cells leads to enhanced prevention of metastatic spreading (38, 402) and improved survival of tumor-bearing mice (38). Several groups have validated these observations by demonstrating that ACT and concomitant administration of A2AR antagonists is superior to single treatments in terms of decreasing tumor growth (135, 396), hindering metastasis formation (38, 402) and ultimately improving survival (135, 388, 396, 402). Interestingly, others claim that A2AR antagonism improves the efficacy of adoptively transferred CAR+ T cells only if PD1 ICB is co-administered (135). In terms of mechanisms, concomitant A2AR antagonism not only increases intra-tumoral presence of adoptively transferred T cells (396) but also elevates their activation status. In particular, when A2AR antagonists were co-administered, tumor-derived, adoptively transferred or endogenous CD44+ CD8+ T cells, exhibit heightened expression levels of T-bet, 4-1BB, and CD69 (396) while demonstrating increased capacity to produce IFNγ and TNFα (135, 396, 402).

Adenosine-Axis Blockade Combined With Radiotherapy, Chemotherapy or Targeted Therapies
It is well documented that radiotherapy (RT) as well as several chemotherapeutic (CT) drugs have the capacity to induce ATP release (406, 429–433). Since such regimens also elevate the expression levels of CD39 (405, 407, 434) and CD73 (405, 407, 435–437), it is possible that the concentration of interstitial adenosine in the TME rises sharply upon application of these treatments. Therefore, several investigators have explored whether concomitant provision of agents targeting the adenosine axis increase the anti-tumor efficacy of RT or of various CT agents.
Indeed, mAb-mediated inhibition of CD73 increased the anti-tumor efficacy of RT (403, 404) and this synergistic effect was even more apparent upon concurrent CTLA-4-blockade (404). Mechanistically, CD73 inhibition increases the presence of CD8+ T cells as well as of CD8α+ or CD103+ DCs within irradiated tumors while decreasing Tregs (403, 404). Moreover, concomitant CD73 blockade was shown to increase the activation status of CD8+ T cells and CD8α+ DCs within irradiated tumors as evidenced by the elevated expression levels of CD69 and CD40, respectively (404). Likewise, concurrent mAb-mediated inhibition of CD73 (405) or pharmacologic blockade of CD39 activity (406) boosted the tumor control (405, 406) and survival (405) of mice treated with the CT drugs Doxorubicin (405), Paclitaxel (405), and Mitoxantrone (406). Of note, such dual therapies were shown to not only augment intra-tumoral presence of DCs (406) and tumor-specific CD8+ T cells (405) but also the fraction of intra-tumoral CD4+ or CD8+ T cells producing IFNγ (406) as well as the levels of IFNγ in the TME (405, 406). In light of such observations, the clinical trials NCT03611556 and NCT03742102 are set to decipher the potency of CT regimens when provided in combination with the CD73-blocking Ab Oleclumab, supplemented or not by PD-1 blockade.
Along the same lines, others explored if direct antagonism of A2AR and A2BR would augment the antitumor effects of CT agents. Indeed, tumor-bearing mice treated with Doxorubicin (359, 405, 407), Dacarbazine (398), or Oxaliplatin (398, 407) in combination with A2AR (405), A2BR (359, 398), or dual A2AR/A2BR antagonists (407) displayed superior tumor control (398, 405, 407) or survived longer (359). Of note, tumors derived from mice treated with the combination of Dacarbazine and PSB1115, an A2BR antagonist, were more heavily infiltrated by CD8+ T cells as well as NKT cells and contained higher levels of GzB than tumors derived from counterpart mice subjected to Dacarbazine monotherapy (398). Likewise, concomitant administration of AB928, a dual A2AR and A2BR antagonist, along with Doxorubicin or Oxaliplatin increased the intra-tumoral detection of tumor-specific CD8+ T cells (407).
Finally, others have sought to decipher whether adenosine axis blockade enhances the anti-tumor efficacy of particular targeted therapies. For instance, it has been recently demonstrated that high expression levels of CD73 in tumors derived from breast cancer patients are associated with resistance to Trastuzumab, an anti-HER2/ErbB2 mAb, and that artificial CD73 overexpression promotes resistance to Trastuzumab-like therapy in immunocompetent murine models of breast cancer (408). Subsequently, the authors moved on to show that when such mice receive dual therapy comprising anti-CD73 and anti-ERB2 mAbs they exhibit inferior tumor expansion rate as well as reduced metastatic spreading and survive longer than counterpart mice treated with either single agent treatments (408). In terms of mechanisms, the combinatorial therapy significantly increases the intra-tumoral presence of CD8+ and CD4+FoxP3neg T cells while decreasing MDSCs (408). In addition, melanoma patients harboring BRAF-mutant tumors exhibit a trend for elevated expression of CD73 whereas co-administration of an A2AR antagonist in mice bearing BRAF-mutant tumors increased the therapeutic benefit achieved either by BRAF inhibition or by the combination of BRAF and MEK inhibitors (393). Finally, CD73 and A2AR are overexpressed in NSCLCs harboring EGFR mutations (438) and even though preclinical studies demonstrating increased efficacy of concomitant inhibition of EGFR and A2AR are not currently publicly available, the clinical trial NCT03381274 includes a cohort of patients with advanced NSCLC that will receive both an EGFR inhibitor and an A2AR antagonist.