4.1 CD73 and Adenosine Pathway
The adenosine pathway, involving the enzymes CD73 and CD39, plays a crucial role in shaping the immunosuppressive TME. These enzymes are highly expressed in various cell types within the TME, including tumor cells, endothelial cells, and infiltrating immune cells such as Tregs and stromal cells [359]. Notably, CD73 and CD39 are upregulated in response to adenosine signaling and the hypoxic conditions commonly found in tumors. The generation of adenosine through the CD39/CD73 pathway is a key mechanism underlying the immunosuppressive function of Tregs [359, 360].
The extracellular degradation of ATP by CD39 and CD73 contributes significantly to immunosuppression. This process reduces ATP-dependent immune activation and results in the production of adenosine (ADO) [359, 360]. CD73 activity on the cell surface is the rate-limiting step in the production of extracellular adenosine, a process that hinders antitumor immunity and supports tumor progression [361]. Adenosine, a nucleoside molecule, exerts its potent immunosuppressive effects by binding to specific receptors on immune cells, primarily the A2A and A2B adenosine receptors. This receptor engagement triggers a cascade of intracellular signaling events that inhibit immune cell activation and effector functions, thus enabling tumors to evade immune detection and destruction [359, 360, 362, 363].
CD73, also known as ecto-5’-nucleotidase , is an is a glycosyl-phosphatidylinositol-linked cell membrane-bound ecto-enzyme, encoded by the gene NT5E [364]. Its primary function involves catalyzing the conversion of extracellular AMP (adenosine monophosphate) to adenosine. This enzymatic activity leads to the accumulation of adenosine in the tumor milieu, particularly heightened under conditions of tissue hypoxia and inflammation typical of solid tumors [359]. Adenosine, upon binding to A2A and A2B receptors (A2AR and A2BR) expressed on T cells and natural killer T (NKT) cells, monocytes, macrophages, dendritic cells and NK cells, exerts potent immunosuppressive effects. These effects include the suppression of cytotoxic T cell responses, inhibition of dendritic cell maturation and antigen presentation, promotion of Treg cell differentiation and function, and reduction in pro-inflammatory cytokine production such as IFN-γ and TNF-α. [365]. A2AR is up-regulated in macrophages in response to NF-κB, STAT1 and PPARγ as well as adenosine signaling, and A2AR activation inhibits the secretion of neutrophil chemokines, thereby reducing the inflammatory response [359].  Thus, CD73-mediated adenosine production plays a critical role in fostering an immunosuppressive environment within tumors, contributing to immune evasion and supporting tumor progression. CD73 has been shown to play a role in various cancer processes, such as metastasis [366], tumor invasion [367], and increased cell proliferation [368].
CD39, also known as ectonucleoside triphosphate diphosphohydrolase-1 or NTPDase 1, is an ectoenzyme prominently expressed on immune cells, including Tregs and subsets of activated T cells [369, 370]. Its primary function involves the hydrolysis of ATP (adenosine triphosphate) and ADP (adenosine diphosphate) into AMP (adenosine monophosphate). This enzymatic activity serves as a critical step in the production of adenosine within the TME. By generating AMP, CD39 acts upstream of CD73, facilitating the subsequent conversion of AMP into adenosine [371].
A significant proportion of cancer patients fail to respond to immunotherapies such as PD-1/PD-L1 and CTLA-4 blockade, indicating that other immunosuppressive pathways may contribute to immune evasion in these non-responding tumors [372]. The adenosinergic pathway, presents a promising new therapeutic approach in cancer immunotherapy, though still in its early stages [370]. Preclinical studies and clinical trial data have shown that targeting this pathway is a viable therapeutic strategy for the future. Small-molecule inhibitors and monoclonal antibodies targeting CD39, CD73 and A2AR have been developed for cancer therapy [359, 373]. As small molecules could cross physiologic barriers in TME, they are better than monoclonal antibodies (mAb) which are macromolecules. Bastid et al. in their study showed that, administering a CD39 inhibitor or blocking antibody reduced the tumor-induced suppression of CD4 and CD8 T-cell proliferation and enhanced the cytotoxic activity of CTLs and NK cells [374]. In a lung cancer model study, an anti-CD39 monoclonal antibody, which inhibits the mouse ectoenzyme CD39, was found to increase CD107a expression in infiltrating NK cells and stimulate IFN-γ release, leading to enhanced cancer cell killing and anti-metastatic effects cells [375]. Simmilarly, in mouse model of melanoma, the administration of anti-CD39 monoclonal antibody (mAb) stimulated the release of IFN-γ , resulting in the eradication of cancer cells [375]. A study by Lu et al. demonstrated that the bifunctional antibody-ligand trap, ES014 (targeting CD39/TGF-β), effectively inhibited CD39, preventing the degradation of extracellular ATP, while also neutralizing autocrine/paracrine TGF-β near target cells leading to restoration of anti-tumor immunity [376]. Anti-CD73 monoclonal antibody (3F7), in a mouse model of TNBC, suppressed tumor growth and metastasis [376]. An experimental study by Jin et al. found that the combination of tumor CD73 knockdown with tumor-specific T-cell transfer successfully cured all tumor-bearing mice. Notably, adoptive T-cell immunotherapy alone provided no therapeutic benefit in mice with tumors that did not undergo CD73 knockdown [377]. Another study in tumor-bearing mouse models, showed that anti-CD73 antibodies can amplify the anticancer effects of both anti-CTLA-4 and anti-PD-1 immunotherapies in MC38-OVA (colon) and RM-1 (prostate) subcutaneous tumors and established metastatic 4T1.2 breast cancer. The activity of anti-PD-1 mAb was also enhanced by anti-CD73 mAb, against 3-methylcholanthrene (MCA)-induced fibrosarcomas [378]. In a study conducted by Perrot et al., it was observed that the antibodies IPH5201 and IPH5301, which target the human membrane-associated and soluble forms of CD39 and CD73 respectively, effectively inhibited the hydrolysis of immunogenic ATP into immunosuppressive adenosine. The mechanism of action involved stimulating dendritic cells and macrophages, as well as restoring the activation of T cells isolated from cancer patients [379]. Drugs that block the A2AR-mediated adenosinergic pathway could boost antitumor immunity by counteracting the effects of extracellular adenosine generated by both tissue cells and Tregs. Pharmacological treatment of mice with A2AR antagonists enhanced antitumor T-cell activity, leading to greater inhibition of tumor growth, destruction of metastases, and reduced neovascularization of cancerous tissues [380].
Current clinical trials are evaluating adenosinergic pathway targets either as monotherapy or in combination therapy:
  1. CD39 inhibitors like, anti-CD39 monoclonal antibody [NCT05508373, JS019] [NCT05234853, PUR001,], CD39 antagonist [NCT04261075, IPH5201] [NCT04336098, SRF617] [NCT05075564, ES002023], anti-CD39/TGF-β bispecific antibody [NCT05381935, ES014], CD39 antagonist with chemotherapy [NCT04336098, Combination SRF617 with pembrolizumab, gemcitabine, albuminbound paclitaxel], Anti-CD39 antibody with immunotherapy [NCT04306900, Combination TTX-030 with immunotherapy and/or chemotherapy]
  2. CD373 inhibitors like, Anti-CD73 monoclonal antibody [NCT05431270, PT199] [NCT05174585, JAB-BX102]; CD73 antagonist [NCT04797468, HLX23] [NCT04104672, AB680] [NCT03736473, MEDI9447 (oleclumab)] [NCT04148937, LY3475070] [NCT03549000, NZV930] [NCT02754141, BMS-986179] [NCT05227144, ORIC-533]; Anti-CD73 antibody with immunotherapy [NCT05174585, JAB-BX102 with pembrolizumab] [NCT05431270, PT199 with an anti-PD-1 monoclonal antibody] [NCT03549000, NZV930 with PDR001] [NCT04672434, Sym024 with Sym021]; Anti-CD73 antibody with chemotherapy [NCT05143970, IPH5301 with chemotherapy and trastuzumab] [NCT04572152, AK119 with AK104] [NCT05119998, IBI325 with sintilimab] [NCT04940286, oleclumab with gemcitabine, nab-paclitaxel, durvalumab]; Anti-CD73-TGFβ-Trap antibody with chemotherapy [ NCT03954704, dalutrafusp (GS-1423) with mFOLFOX6 regimen]; CD73 antagonist with immunotherapy [NCT04148937, LY3475070 with pembrolizumab] [NCT02754141, BMS-986179 with nivolumab (BMS-936558)] [NCT04989387, INCA00186 with INCB106385 and/or retifanlimab]
  3. A2AR antagonist [NCT05501054, Ciforadenant (CPI-444)] [NCT04969315, TT-10] [NCT03207867, Taminadenant (NIR178)] [NCT02403193, PBF-509] [NCT05117177, Inupadenant (EOS100850)] [NCT04580485, INCB106385] [NCT04478513, AZD4635]; A2AR and A2BR antagonist [NCT04262856, Etrumadenant (AB928)];Anti-CD39 antibody with A2AR and A2BR [NCT05177770, SRF617 with AB928 (Etrumadenent) and AB122 (zimberelimab)];Anti-CD73 antibody with A2AR antagonist [NCT04089553, AZD4635 with durvalumab or oleclumab (MEDI9447)] [NCT03454451, CPI-006 with ciforadenant or pembrolizumab] [NCT03381274, oleclumab (MEDI9447) with AZD4635]; A2AR antagonist with chemotherapy [NCT05403385, inupadenant (EOS100850) with Chemotherapy]; A2AR antagonist with immunotherapy [NCT04580485, INCB106385 with immunotherapy] [NCT05501054, lpilimumab, nivolumab with ciforadenant (CPI-444)] [NCT03549000, NZV930 with PDR001 and /or NIR178] [NCT02403193, taminadenant with PDR001] NCT03207867, NIR178 with PDR001] [NCT04895748, DFF332, spartalizumab with taminadenant].