Targeting Adenosinergic Signaling in Cancer Immunotherapy
Adenosine confers potent immunosuppressive as well as direct tumor-promoting effects in the TME. Thus, approaches to both blocking its generation and hindering binding to its receptors have become important areas of research (Figure 3). Indeed, extensive pre-clinical experimentation has firmly established that targeting the adenosinergic signaling on its own (Table 1) or in combination with emerging IMTs or established cancer treatments (Table 2) shows important promise and soundly supports the clinical evaluation (Table 3) of these concepts. Here we present an overview of such pre-clinical and clinical studies.
Figure 3  Approaches for blocking adenosinergic signaling in the TME. The inhibitory effects of adenosine in the TME can be circumvented by administration of mAbs or small molecules that target enzymes involved in the catabolism of ATP and NAD, such as CD39,CD73 and CD38, as well as by pharmacologic antagonists of A2AR and A2BR to block adenosine-mediated signaling. Whereas multiple such mAbs and pharmacologic inhibitors/antagonists display antitumor activity within murine models of solid tumors (Tables 1, 2), depicted are only those currently evaluated in patients with solid tumor malignancies (Table 3). Finally, treatments that reduce the extracellular export of ATP, such as oxygenation to reverse hypoxia, can attenuate adenosinergic signaling.
Table 1  Evaluation of adenosine-axis blockade in murine models of solid malignancies.
Target   Treatment   Tumor model   Outcome of adenosine axis blockade depends on presence/unhindered function of   Impact on the TME
CD73  ➣ mAbs:
TY/23 (368, 378–384)
2C5 IgG2a (384)
Oleclumab (385)
AD2 (386)
➣ Pharmacologic inhibitor:
APCP (378, 379, 381, 383, 384, 387–389)  ➣ Breast:
4T1.2 (368, 382, 383)
E0771 (368)
LM3a (386)
MDA-MB-231a (386)
➣ Melanoma:
B16-SIY (378)
B16-F10 (379, 381, 384, 387, 389)
K1735 (381)
LWT1 (384)
➣ Ovarian:
ID8 (378, 388)
➣ Prostate:
TRAMP-C1 (380)
RM-1 (382)
➣ Colon:
CT26 (385)
MC38-OVA (382)
➣ Fibrosarcoma: MCA-induced (380, 382)
➣ Lymphoma:
EG7 (379)  ➣ Primary tumor expansionrate ↓ (368, 378–382, 385, 387, 389)
Host CD73 (387)
A2AR on hematopoietic cells (368)
T cells, NK cells or B cells (368)
T cells (381)
CD8 T cells (380)
IFN-γ (382)
IL-17A (381)
Partially retained inmice depleted of B cells (381)
Retained in perforin KO mice (382)
➣ Metastasis formation ↓ (368, 379, 380, 384, 386)
Retained against tumor cells with significantly reduced CD73 (386)
Host CD73 (380)
MDSCs (384)
Retained in mice depleted of T cells or NK cells (380)
Retained in SCID mice lacking T cells, NK cells and functional B cells (386)
Host FcRIV (384)
FcR binding capacity (384)
Independent of the capacity to suppress CD73 catalytic activity (384, 386)
➣ Survival↑ (378, 382, 384, 388)  ↑ CD8+ T cells (381, 385, 389)↑ tumor-specific CD8+ T cells (382)↓ CD73 on CD4+/CD8+ T cells (382, 384)↑ B cells (381)↓ Tregs (389)↑ MDSCs (384)↓ CD73 on MDSCs (384)↑ IFN-γ, TNF-α, IL-17A (381)↓ Ki67+ cells (381)↓ Bcl-2+ cells (381)↓ Microvessel density (383)↓ VEGF (383)
CD39  ➣ mAb:
9-8B (390)
➣ Pharmacologic inhibitor:
POM-1 (391)  ➣ Melanoma: B16-F10 (391)
➣ Colon: MCA38 (391)
➣ Sarcoma: IGN-SRC- 004a (390)  ➣ Primary tumor expansion rate ↓ (391)
Host CD39 (391)
➣ Survival ↑ (390)
Retained in NOG mice lacking T cells, B cells, NK cells and functional macrophages (390)
CD38  ➣ mAb:
NIMR-5 (96)
➣ Pharmacologic inhibitor:
Rhein (96)  ➣ Lung:
344SQ (96)
LLC-JSP (96)
531LN3 (96)  ➣ Primary tumor expansion rate ↓ (96)
CD8+ T cells (96)  ↑ CD8+ T cells (96)↑ CD44hiCD62Llo CD8+ T cells (96)↓ PD-1+TIME3+ CD8+ T cells (96)↓ Tregs (96)↓ MDSCs (96)
Intratumoral hypoxia  Respiratory hyperoxia (60% O2) (9, 293)  ➣ Breast: 4T1 (293)
➣ Melanoma:
B16 (9)
B16-F10 (293)
CL8-1 (9)
➣ Fibrosarcoma: MCA205 (9, 293)  ➣ Primary tumor expansion rate ↓ (9)
➣ Metastasis formation ↓ (293)
CD4 > CD8 > NK cells (293)
Host A2AR (293)
Independent of 60%O2-induced ROS production (293)
➣ Survival ↑ (9, 293)   ↓ Hypoxia (9, 293)↓ HIF- 1α (9)↑ FHL-1, FIH-1, VHL (HIF- 1α inhibitors) (9)↓ CD39, CD73, A2AR, A2BR, COX-2 mRNA (9)↓ extracellular adenosine (9)↑ CD8+, CD69+, CD44+ cells (293)↓ Tregs (293)↑ IL-2, IL-12, CXCL9, CXCL10, CXCL11 mRNA (293)↓ TGF-β (293)↓ FOXP3 in Tregs (293)↓ CD39, CD73, CTLA-4 on Tregs (293)↑ MHC class I on tumor cells (9)↓ VEGF, VEGF mRNA (9)↓ Microvessel density (9)
A2AR  ➣ Antagonists:
ZM241385 (38, 54)
ZM241365 (389)
SCH58261 (54, 292, 384, 392–394)
FSPTP (395)
CPI-444 (396)
PBF-509 (397)  ➣ Breast:
4T1.2 (292)
➣ Melanoma:
B16-F10 (292, 384, 389, 394, 395, 397)
CL8-1 (38)
BRAFV600E-PTEN-deficient mice (393)
LWT1 (393)
➣ Colon:
CT26 (396)
MC38 (396)
➣ NSCLC: PC9a (54)
➣ Bladder: MB49 (395)
➣ HNSCC: Tgfbr1/Pten double KO (392)
➣ Fibrosarcoma: MCA205 (397)  ➣ Primary tumor expansion rate ↓ (38, 54, 389, 392, 393, 396)
T cells (38)
Retained in NUDE micelackingT cells (54)
➣ Metastasis formation ↓ (292, 384, 393, 394, 397)
Tumor CD73 (292, 394)
Host A2AR (292)
T cells, B cells or NK cells (292)
Perforin (292)
➣ Survival↑ (384, 396)
CD8 T cells > NK cells (394)  ↓ CD8+, CD4+ T cells (395)↑ CD8+ T cells (389, 392, 393)↑ CD69 on CD8+ T cells (393)↓ A2AR+ CD8+ T cells (392)↑ IFN-γ+ CD8+ T cells (392)↑ T-bet, 41-BB in/on CD44+CD8+ T cells (396)↑ IFN-γ, TNF-α production by CD8+ T cells (392)↑ stimulation-induced IFN-γ/TNF-αproduction by CD8+ T cells (396)↑ NK cells (393, 395)↑ GzB+ NK cells (292)↓ Tregs (389, 392, 393)↓ PD-1, LAG3, FOXP3 on Tregs (396)↓ A2AR+ Tregs (392)↓ FOXP3 (392)
A2BR  ➣ Antagonists:
PSB1115 (292, 315, 359, 398)
ATL-801 (399)  ➣ Breast:
4T1.2 (292, 359, 368, 399)
E0771 (359)
➣ Melanoma:
B16-F10 (292, 315, 359, 398)
LWT1 (359)
➣ Bladder: MB49 (399)  ➣ Primary tumor expansion rate ↓ (315, 398, 399)
Mature T cells (399)
T cells (398)
Host A2BR (399)
Host CXCR3 (399)
Retained in A2AR−/− mice (399)
Retained in mice depleted of MDSCs but lost upon adoptive transfer of MDSCs (398)
➣ Metastasis formation ↓ (292, 359, 368)
Tumor CD73 (292)
Retained in RAG−/−cγ−/− mice lacking T cells, B cells and NK cells (292)
Retained in mice depleted of T cells, NK cells D11c+ DCs or macrophages (359)
➣ Survival↑ (359)
Tumor A2BR (359)
Retained in mice depleted of T cells or NK cells (359)  ↑ CD8+ T cells (315, 398)↑ CXCR3+ T cells (399)↑ NKT cells (315, 398)↓ MDSCs (315, 398)↑IFN-γ, CXCL10 mRNA (399)↑ IFN-γ, TNF-α, GzB (398)↓ MCP-1, IL-10 (398)↓ VEGF (315)↓ Microvessel density (315)
a  Patient-derived tumor cell lines, NSCLC, Non-Small-Cell LungCancer. HNSCC, Head and neck squamous cellcarcinoma.
X > Y: X contributes more than Y to the anti-tumor effect of adenosine axis modulation.
Table 2  Evaluation of concomitant adenosine-axis blockade in murine models of solid malignancies.
Combinatorial schemes   Treatments   Tumor model   Outcome of concomitant adenosine axis blockade depends on presence/unhindered function of   Impact on the TME
CD73 inhibition & A2AR antagonism  ➣ anti-CD73mAb:
TY/23 (384)
➣ A2AR pharmacologic antagonist:
SCH58261 (384)  ➣ Breast: 4T1.2 (384)
➣ Melanoma:
B16-F10 (384)
LWT1 (384)  ➣ Metastasis formation ↓ (384)
➣ Survival ↑ (384)
NK cells > CD8+ T cells (384)
IFN-γ (384)
Perforin (partial dependence) (384)
PD-1 ICB & CD73 inhibition  ➣ anti-PD-1mAb:
RMP1-14 (382, 385)
➣ anti-CD73mAbs:
Oleclumab (385)
TY/23 (382)  ➣ Breast: 4T1.2 (382)
➣ Colon:
CT26 (385)
MC38 (382)
➣ Prostate: RM-1 (382)  ➣ Primary tumor expansion rate ↓ (382, 385)
➣ Survival ↑ (382, 385)  ↑ Tumor-specific CD8+ T cells (382, 385)↑ IFN-γ mRNA (382)
PD-1 ICB & CD38 inhibition  ➣ anti-PD-L1mAb:
9G2 (96)
➣ anti-CD38mAb:
NIMR-5 (96)
➣ CD38 pharmacologic inhibitor:
Rhein (96)  ➣ Lung:
344SQ (96)
LLC-JSP (96)  ➣ Primary tumor expansion rate ↓ (96)
➣ Metastasis formation ↓ (96)  ↑ CD8+ T cells (96)↑ CD44hiCD62Llo CD8+ T cells (96)↓ PD-1+TIME3+ CD8+ T cells (96)↑ CD4+ICOS+ T cells (96)↓ Tregs (96)↓ MDSCs (96)
PD-1 ICB & A2AR antagonism  ➣ anti-PD-L1:
9G2 (mAb) (96)
B7-DC/Fc (400)
➣ anti-PD-1mAb:
RMP1-14 (396, 401)
➣ A2AR antagonists:
SCH58261 (96, 394, 401)
ZM241385 (400)
SYN115 (401)
CPI-444 (396)  ➣ Breast:
AT3 (401)
4T1.2 (394, 401)
➣ Melanoma: B16-F10 (394)
➣ Colon:
MC38 (396, 401)
CT26 (396)
➣ Lung:
344SQ (96)
LLC-JSP (96)
➣ Lymphoma: EL4 (400)  ➣ Primary tumor expansion rate ↓ (96, 396, 400, 401)
IFN-γ (401)
Retained in perforin KO mice (401)
➣ Metastasis formation ↓ (394, 401)
Tumor CD73 (394)
NK cells > CD8+ T cells (394)
➣ Survival ↑ (394, 396, 401)
CD8+ T cells > NK cells (394)  ↑ IFN-γ+ CD8+ or tumor-specific T cells (401)↑ GzB+ CD8+ T cells (401)↑ NK cells (394)
PD-1 ICB & A2BR antagonism  ➣ anti-PD-1mAb:
RMP1-14 (359)
➣ A2BR antagonist:
PSB1115 (359)  ➣ Melanoma: B16-F10 (359)
➣ Breast: 4T1.2 (359)  ➣ Metastasis formation ↓ (359)
➣ Survival ↑ (359)
CTLA-4 ICB & CD73 inhibition  ➣ anti-CTLA-4mAbs:
9H10 (389)
UC10-4F10 (382)
➣ CD73 pharmacologic inhibitor:
APCP (389)
➣ anti-CD73mAb:
TY/23 (382)  ➣ Breast: 4T1.2 (382)
➣ Melanoma: B16F10 (389)
➣ Colon: MC38 (382)
➣ Prostate: RM-1 (382)  ➣ Primary tumor expansion rate ↓ (382, 389)
CD8+ >> CD4+ T cells (382)
➣ Survival ↑ (382)  ↑ CD8+, CD4+ T cells (389)↑ Tumor-specific CD8+ T cells (382)↑ IFN-γ, T-bet mRNA (382)↑ IFN-γ (389)
CTLA-4 ICB & A2AR antagonism  ➣ anti-CTLA-4mAb:
9H10 (389)
➣ A2AR antagonist:
ZM241365 (389)  ➣ Melanoma: B16F10 (389)  ➣ Primary tumor expansion rate ↓ (389)  ↑ CD8+ T cells (389)↑ IFN-γ, GzB (389)
CTLA-4 ICB & A2BR antagonism  ➣ anti-CTLA-4mAb:
UC10-4F10 (359)
➣ A2BR antagonist:
PSB1115 (359)  ➣ Breast: 4T1.2 (359)
➣ Melanoma: B16-F10 (359)  ➣ Metastasis formation ↓ (359)
➣ Survival ↑ (359)
ACT & CD73 inhibition  ➣ T cells:
2C (SIY-specific) (378)
Reactive to ID8 (378)
OT-I (OVA-specific) (378)
➣ CD73 pharmacologic inhibitor:
APCP (378)
➣ anti-CD73mAb:
TY/23 (378)  ➣ Melanoma: B16-SIY (378)
➣ Ovarian: ID8 (378)
➣ Lymphoma:
EG7 (EL4-OVA) (378)  ➣ Primary tumor expansion rate ↓ (378)
➣ Survival ↑ (378)  ↑ Adoptively transferred T cells (378)
ACT & A2AR antagonism  ➣ T cells:
anti-HER2 CAR+ (135)
OT-I (OVA-specific) (388, 396)
TDLN-derived (402)
Reactive to CMS4 (38)
➣ A2AR antagonists:
CPI-444 (396)
ZM241385 (38, 135, 402)
KW6002 (402)
SCH58261 (135, 388)  ➣ Breast: E0771- HER2 (135)
➣ Melanoma: B16-OVA (396)
➣ Ovarian: ID8-OVA (388)
➣ Fibrosarcoma:
MCA205 (402)
24JK-HER2 (135)
➣ Sarcoma CMS4 (38)  ➣ Primary tumor expansion rate ↓ (135, 396)
PD-1 ICB (135)
IFN-γ (135)
➣ Metastasis formation ↓ (38, 402)
Non-myeloablative pretreatment (402)
➣ Survival ↑ (135, 388, 396, 402) PD-1 ICB (135)  ↑ Adoptively transferred T cells (396)↑ Tbet, 41BB, CD69 in/on adoptively transferred CD8+ cells (396)↑ IFN-γ+ adoptively transferred T cells (135)↑ Stimulation-induced IFN-γ, TNF-α production by adoptively transferred CD8+ T cells (396)↑ Stimulation-induced IFN-γ production by adoptively transferred CD8+ or CD4+ T cells (402)↑ Tbet, 41BB in/on endogenous CD44+ CD8+ cells (396)↑ Stimulation-induced IL-2, IFN-γ, TNF-α production by endogenous CD8+CD44+ T cells (396)
ACT & intratumoral hypoxia aversion  ➣ Respiratory hyperoxia (60%O2)
➣ T cells:
TDLN-derived (293)  ➣ Melanoma: B16-F10 (293)
➣ Fibrosarcoma: MCA205 (293)  ➣ Primary tumor expansion rate ↓ (293)
Host A2AR (293)
➣ Metastasis formation ↓ (293)  ↑ Adoptively transferred T cells (293)↑ IFN-γ+ endogenous/adoptively transferred CD8+ T cells (293)
Radiotherapy & CD73 inhibition  ➣ Radiotherapy:
Single local dose of 20Gy (403, 404)
➣ anti-CD73mAb:
Unspecified (403)
TY/23 (404)  ➣ Breast: TSA (403, 404)  ➣ Primary tumor expansion rate ↓ (403, 404)
BATF3 (403)  ↑ CD103+DCs (403)↑ CD8a+ DCs (404)↑ CD40 on CD8a+ DCs (404)↑ CD8+T cells (404)↑ CD69 on CD8+T cells (404)↑ CD8+T cell/Treg ratio (403)↓ Tregs (404)
Chemotherapy & CD73 inhibition  ➣ Chemotherapy:
Doxorubicin (405)
Paclitaxel (405)
➣ anti-CD73mAb:
TY/23(405)  ➣ Breast:
4T1.2 (405)
AT3 (405)  ➣ Primary tumor expansion rate ↓ (405)
Partially retained in SCID mice lacking T cells, NK cells and functional B cells (405)
CD8+ T cells (405)
➣ Survival↑ (405)  ↑ Tumor-specific CD8+ T cells (405)↑ IFN-γ (405)
Chemotherapy & CD39 inhibition  ➣ Chemotherapy:
Mitoxantrone (406)
Oxaliplatin (406)
➣ CD39 pharmacologic inhibitor:
ARL67156 (406)  ➣ Colon: CT26 (406)
➣ Fibrosarcoma: MCA205 (406)  ➣ Primary tumor expansion rate ↓ (406)
T cells (406)
Knockdown of tumor Atg5 (406)  ↑ Extracellular ATP (406)↑ DCs (406)↑ IFN-γ+ CD4+, CD8+ T cells (406)↑ IL-17A+ γδ T cells (406)↑ IFN-γ (406)
Chemotherapy & A2R antagonism  ➣ Chemotherapy:
Doxorubicin (359, 405, 407)
Dacarbazine (398)
Oxaliplatin (398, 407)
➣ A2R antagonists:
SCH58261(A2AR) (405)
PSB1115 (A2BR) (359, 398)
AB928 (A2AR&A2BR) (407)  ➣ Breast:
4T1.2 (405)
AT3 (359, 405, 407)
➣ Melanoma: B16-F10 (398)  ➣ Primary tumor expansion rate ↓ (398, 405, 407)
Tumor CD73 (405)
➣ Survival ↑ (359)  ↑ CD8+ T cells (398)↑ Tumor-specific CD8+ T cells (407)↑ NKT cells (398)↑ GzB (398)
Targeted therapy & CD73 inhibition  ➣ anti-ErbB2 mAb
7.16.4 (408)
➣ anti-CD73 mAb
TY/23 (408)  ➣ Breast:
H2N100 (408)
TUBO (408)
ErbB2-overexpressing mice (408)  ➣ Primary tumor expansion rate ↓ (408)
Tumor CD73 (408)
B cells, T cells or NK cells (408)
➣ Spontaneous tumor formation ↓ (408)
➣ Metastasis formation ↓ (408)
➣ Survival ↑ (408)  ↑ CD8+ T cells (408)↑ CD4+ FOXP3− T cells (408)↓ MDSCs (408)
Targeted therapy & A2AR antagonism  ➣ BRAF inhibitor:
PLX4720 (393)
➣ MEK inhibitor:
Trametinib (393)
➣ A2AR antagonist:
SCH58261 (393)  ➣ Melanoma:
BRAFV600E-PTEN-deficient mice (393)
BRAFV600E LWT1 (393)  ➣ Primary tumor expansion rate ↓ (393)
➣ Metastasis formation ↓ (393)
TDLN, tumor-draining lymphnode.
Table 3  Clinical evaluation of adenosine-axis targeting in patients with solid tumors.
Molecular target   Clinical Trial identifier   Agents   Phase   Design overview*   Solid tumor indications   Sponsor   Launched on
CD73  NCT02503774  Oleclumab  I  ➣ Single agent  Advanced solid malignancies  MedImmune  2015
➣ In combination with durvalumab (anti-PD-L1)
NCT03736473  Oleclumab  I  ➣ Single agent  Advanced solid malignancies  AstraZeneca  2018
NCT03773666  Oleclumab  I  ➣ In combination with durvalumab (anti-PD-L1)  Muscle-invasive Bladder Cancer  Dana-Farber Cancer Institute  2018
NCT03267589  Oleclumab  II  ➣ In combination with durvalumab (anti-PD-L1)  Relapsed ovarian cancer  Nordic Society for Gynecologic Oncology  2018
NCT03334617  Oleclumab  II  ➣ In combination with durvalumab (anti-PD-L1)  PD-1/PD-L1 inhibition-resistant NSCLC  AstraZeneca  2018
NCT03742102  Oleclumab  Ib/II  ➣ In combination with durvalumab (anti-PD-L1) and paclitaxel (chemotherapy)  Metastatic Triple Negative Breast Cancer  AstraZeneca  2018
NCT03611556  Oleclumab  Ib/II  ➣ In combination with gemcitabine (chemotherapy) and nab-paclitaxel (chemotherapy)  Metastatic pancreatic cancer  MedImmune  2018
➣ In combination with gemcitabine and nab-paclitaxel and durvalumab (anti-PD-L1)
➣ In combination with mFOLFOX (chemotherapy regimen comprising oxaliplatin, leucovorin, 5-FU)
NCT03381274  Oleclumab  Ib/II  ➣ In combination with osimertinib (EGFRT790Minhibitor)  Advanced NSCLC  MedImmune  2018
➣ In combination with AZD4635 (A2Aantagonist)
NCT02754141  BMS-986179  I/IIa  ➣ Single agent  Advanced solid malignancies  Bristol-Myers Squibb  2016
➣ In combination with nivolumab (anti-PD-1)
➣ In combination with rHuPH20 (drug deliveryenzyme)
NCT03454451  CPI-006  I/Ib  ➣ Single agent  Advanced solid malignancies  Corvus Pharmaceuticals  2018
➣ In combination with CPI-444 (A2Aantagonist)
➣ In combination with pembrolizumab (anti-PD-1)
NCT03549000  NZV930  I/Ib  ➣ Single agent  Advanced solid malignancies  Novartis  2018
➣ In combination with spartalizumab (anti-PD-1)
➣ In combination with NIR178 (A2Aantagonist)
➣ In combination with NIR178 andspartalizumab
CD38  NCT03473730  Daratumumab  I  ➣ Single agent  Metastatic Renal Cell Carcinoma or Muscle Invasive Bladder Cancer  M.D. Anderson Cancer Center  2017
A2A  NCT02403193  NIR178  I/Ib  ➣ Single agent  Advanced NSCLC  Palobiofarma  2015
➣ In combination with spartalizumab (anti-PD-1)
NCT03207867  NIR178  II  ➣ Single agent  Advanced solid malignancies  Novartis  2017
➣ In combination with spartalizumab (anti-PD-1)
NCT03742349  NIR178  Ib  ➣ In combination with spartalizumab (anti-PD-1) and LAG525(anti-LAG3)  Triple-negative Breast Cancer  Novartis  2018
NCT02655822  CPI-444  I/Ib  ➣ Single agent  Advanced solid malignancies  Corvus Pharmaceuticals  2016
➣ In combination with atezolizumab (anti-PD-L1)
NCT03337698  CPI-444  Ib/II  ➣ Single agent  Metastatic NSCLC  Hoffmann-La Roche  2017
➣ In combination with atezolizumab (anti-PD-L1)
NCT02740985  AZD4635  I  ➣ Single agent  Advanced solid malignancies  AstraZeneca  2016
➣ In combination with durvalumab (anti-PD-L1)
A2B  NCT03274479  PBF-1129  I  ➣ Single agent  Advanced NSCLC  Palobiofarma  2018
NSCLC, non-small-cell lung cancer.
*  Mentioned are schemes comprising at least one adenosine-axis modulator.

Blockade of Adenosine Generation
As previously described, CD73 is an nucleotidase that converts AMP, generated from CD39- or CD38/CD203-mediated catabolism of ATP or NAD respectively, to adenosine. Its central role in adenosine generation is underscored by the fact that CD73-deficient mice display drastically decreased interstitial levels of adenosine, not only at steady state, but also upon induction of trauma or hypoxia (409, 410). CD73 knock-out mice exhibit hindered tumor growth and metastatic spreading (378–380, 387) and mice inoculated with tumor cells lacking CD73 survive longer than mice inoculated with tumor cells expressing this ecto-enzyme (378, 388). Indeed, administration of anti-CD73 monoclonal antibodies (mAb) (368, 378–386) or of a CD73-specific pharmacologic inhibitor (378, 379, 381, 383, 384, 387–389) impairs tumor growth (368, 378–382, 385, 387, 389) and metastasis (368, 379, 380, 384, 386) while increasing survival (378, 382, 384, 388). Of note, CD73 can also act as an adhesion/signaling molecule to promote metastasis in a catalytic-activity independent manner (386, 411, 412). Mechanistically, the aforementioned treatments have been shown to promote intra-tumoral accumulation of CD8+ T cells (381, 382, 385, 389), B cells (381) as well as of Th1- and Th17-associated cytokines (381) while decreasing the levels of intra-tumoral VEGF (383) and the presence of Tregs (389). Of note, even though metastasis can be modestly inhibited by anti-CD73 therapy in an immune-system independent fashion (368, 386), most of the antitumor effect of CD73 blockade is due to alleviation of A2AR-mediated immunosuppression (368).
No doubt encouraged by these pre-clinical studies, four anti-CD73 mAbs are currently being evaluated as monotherapies in small scale trials targeting a variety of solid tumors. In July 2015, MedImmune launched a first in-human trial (NCT02503774) evaluating the human anti-CD73 mAb Oleclumab, which allosterically prevents CD73 from assuming its catalytically active conformation (413). In June 2016, Bristol-Myers Squibb (BMS) launched a Phase I/IIa trial (NCT02754141) to assess the efficacy of BMS-986179, a human IgG2-IgG1 hybrid mAb that not only inhibits CD73-exerted AMP hydrolysis but also induces CD73 internalization (414). In April 2018, Corvus Pharmaceuticals initiated clinical evaluation (NCT03454451) of their humanized anti-CD73 mAb, CPI-006, which directly competes with AMP for the CD73 active site (415). Finally, in July 2018, Novartis listed a Phase I/Ib trial (NCT03549000) evaluating the efficacy of SRF373/NZV930, a human mAb that impedes CD73 activity via a currently undisclosed mechanism, and was pre-clinically developed by Surface Oncology before being exclusively licensed to Novartis for further clinical development.
CD39 also critically contributes to the generation of extracellular adenosine from ATP as evidenced by the fact that deficiency of this enzyme results in significantly decreased adenosine content in tissues, not only at steady state, but also upon ischemia induction (80). Similar to studies with CD73-deficient mice, tumor growth and metastasis are reduced in CD39-null mice (391, 416). In addition, intraperitoneal delivery of a CD39 inhibitor in immunocompetent mice reduces tumor growth rates (391). Administration of an anti-CD39 mAb increased the survival of immuno-deficient mice inoculated with patient-derived tumors (390), indicating that CD39 can also promote tumor growth or metastasis in an immune system independent manner. In terms of mechanisms, several studies have demonstrated that in vitro inhibition of CD39 activity by pharmacologic inhibitors (45, 47, 62) or blocking mAbs (45, 417, 418) results in enhanced functionality of T cells (45, 47, 62, 418) and NK cells (45, 47, 418), as well as decreased Treg-mediated suppression of T cell proliferation (47, 417). Even though restriction of CD39 activity in vitro conclusively alleviates adenosine-induced immunosuppression, a surprisingly small number of studies demonstrate effectiveness of this approach within tumor-bearing mice. Finally, while humanized mAbs targeting CD39, such as IPH52 (Innate Pharma) have been developed, clinical studies exploring CD39 blockade/inhibition have not been launched.
As previously mentioned, the concerted activity of CD38 and CD203a, can functionally replace CD39 toward the generation of extracellular adenosine. Further substantiating the soundness of CD38-blockade as a cancer treatment, immunocompetent CD38-null mice display reduced tumor growth (419) whereas tumors devoid of this ectonucleotidase grow slower both in immuno-competent (96) as well as in immuno-deficient mice (97). Indeed, administration of CD38 mAbs retards tumor growth (96, 420). Interestingly, tumors derived from anti-CD38 mAb-treated mice encompass more CD8+ T cells and less Tregs and MDSCs (96). Moreover, increased fraction of CD8+ T cells infiltrating these tumors display an effector memory phenotype while less of these cells are double positive for the exhaustion markers PD-1 and TIM3 (96). Three anti-CD38 mAbs, Daratumumab (Janssen Biotech), Isatuximab (Sanofi), and MOR202 (Morphosys) are being clinically evaluated. Daratumumab was FDA-approved in 2015 for treating multiple myeloma patients, while to date the most advanced testing of Isatuximab and MOR202 as monotherapies are respectively the Phase II trials NCT01084252, NCT02960555, and NCT02812706, as well as the Phase I/IIa trial NCT01421186. Of note, in addition to modulating the enzymatic activity of CD38, these mAbs also have the capacity to induce cytotoxicity through diverse mechanisms, such as induction of complement activation, Ab-dependent cellular cytotoxicity (ADCC) or phagocytosis, and programmed cell death (420). Albeit extensive clinical experience of utilizing the aforementioned mAbs against CD38-overexpressing hematologic malignancies, the recently launched trial NCT03473730 constitutes the first application of a CD38-specific mAb in patients with solid tumor malignancies.
Another approach for limiting the intratumoral interstitial adenosine is the oxygenation of the TME (293). As mentioned, hypoxia promotes build-up of extracellular adenosine at least by inducing upregulation of CD39 and CD73 as well as downregulation of adenosine transporters. Indeed, in pre-clinical models, respiratory hyperoxia (60% oxygen) lowers intra-tumoral adenosine levels (9), tumor growth rates (9), metastasis formation (293) and increases survival of tumor-bearing mice (9, 293). Mechanistically, this treatment boosts MHC-I levels on the tumor-cell surface (9), the presence of CD8+, CD69+, or CD44+ cells within the TME (293) and reduces the presence of Tregs (293) as well as the latter's capacity to express CD39, CD73, CTLA-4, or FoxP3 (293). Moreover, increased oxygenation of tumors not only averts angiogenesis through reduction of VEGF concentration (9), but also dampens expression of molecules associated with immune dysfunction, such as TGF-β, CD39, CD73, A2AR, A2BR and COX-2 (9, 293), the rate-limiting enzyme of PGE2 biosynthesis, while increasing the mRNA levels of pro-inflammatory agents, such as IL-2, and IL-12a (293).

Blockade of Adenosine Receptor Binding
Along with blocking adenosine production with small molecules or mAbs, another approach to inhibit adenosine-induced signaling is to directly block binding to its receptors A2AR and A2BR. Underscoring the potent protumoral effect of A2AR-trigerring, mice devoid of this receptor present reduced rates of tumor growth and metastasis, and in some instances tumors undergo complete rejection (38, 292, 400, 402). In addition, administration of pharmacologic A2AR antagonists recapitulates the anti-tumor effects of A2AR-deletion since it results to reduced primary tumor expansion (38, 54, 389, 392, 393, 396) and metastasis formation (292, 384, 393, 394, 397) ultimately leading to prolonged survival (384, 396). Mechanistically, tumors derived from A2AR-antagonist-treated mice are more heavily infiltrated by CD8+ T cells (389, 392, 393) as well as NK cells (389, 392, 393) and encompass fewer Tregs (389, 392, 393). In addition, in vivo A2AR antagonism leads to increased expression of CD69 (393), T-bet (396), and 4-1BB (396) as well as production of IFNγ and TNFα (392, 396) by intra-tumoral CD8+ T cells. Furthermore, this intervention increases the fraction of intra-tumoral NK cells producing GzB (292) and reduces the expression of PD-1, LAG3, FoxP3 and A2AR by tumor-infiltrating Tregs (392, 396). Interestingly, the A2AR antagonists ZM241385 and SCH58261 exhibit the capacity to curb primary tumor growth even in a T cell-independent manner (54). Notably, A2A antagonism in vivo increases activation induced cell death (AICD) of intra-tumoral T cells (395), a finding corroborating observations that cAMP-accumulation in the T cell cytosol averts terminal effector differentiation and AICD (421, 422). Three A2AR antagonists are currently being evaluated as single agents in Phase I/II trials to treat cancer patients bearing solid tumors. In particular, Corvus Pharmaceuticals, AstraZeneca, and Novartis have undertaken the clinical development of CPI-444 (NCT02655822), AZD4635 (NCT02740985), and NIR178 (NCT02403193, NCT03207867), respectively.
As for A2AR, genetic deletion of A2BR reduces tumor growth rate (399, 423) while A2BR−/− tumor cells display reduced metastatic potential (359, 367). Notably, administration of A2BR antagonists in tumor-bearing mice reduces tumor growth (315, 398, 399) and metastasis (292, 359, 368) eventually prolonging their survival (359). Mechanistically, antagonism of A2BR in vivo augments the intra-tumoral presence of CD8+ T cells (315, 398), NKT (315, 398) as well as the mRNA levels of IFNγ and CXCL10 (399) and the concentration of TNFα, IFNγ, and GzB (398) in the TME. This intervention further results in decreased accumulation of MDSCc (315, 398) and IL-10 (398), as well as reduced levels of VEGF and angiogenesis (315). Based on encouraging preclinical results, Palobiofarma recently launched a dose escalation Phase I study (NCT03274479) administering PBF-1129, a selective A2BR inhibitor, in patients with advanced Non-Small Cell Lung Cancer (NSCLC).

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.