Background Adenosine signaling pathway has emerged as a key metabolic pathway that regulates tumor immunity.1 Adenosine-mediated immune suppression within the tumor microenvironment has been shown to be one of the mechanisms for resistance to immune checkpoint blockade (ICB) therapies.2 3 Strategies targeting adenosine receptors or adenosine-generating enzymes CD38, CD39 and CD73, which convert NAD or ATP to AMP, and AMP to adenosine, are under active clinical trials or preclinical development.4 6 Currently small molecule inhibitor (SMI) is the only modality that is under clinical investigation for targeting adenosine receptor 2a (A2aR).4–6
Methods Humanized anti-A2aR antibodies were generated from mouse antibodies discovered by hybridoma technology. Antibody specificity and cross-reactivity were determined by flow cytometry. Antibody was compared with SMIs CPI-444 and AB-928 for its potency in 1) inhibiting cAMP generation in A2aR-overexpressing HEK293 cells in the presence of agonists adenosine or adenosine analog NECA using cAMP release assay, and 2) restoring NECA-suppressed effector cell activation and inflammatory cytokine IFN-gamma and IL-2 production using human PBMC cells stimulated with anti-CD3/anti-CD28. To validate its anti-cancer effect, anti-A2aR antibody was administered alone or in combination with Ipilimumab, Opdivo or Avastin to determine the inhibition of human lung cancer or melanoma tumor CDX growth in human PBMC-reconstituted NCG or MHC I&II dKO NSG mice.
Results The humanized antibody specifically binds to human A2aR and cross-reacts to cynomolgus monkey homologue. It induced internalization of A2aR and displayed 10-fold higher potency than clinical stage SMIs CPI-444 and AB928 in in vitro assay. Moreover, it restored the function of adenosine-induced suppression of cell activation and inflammatory cytokine production by immune effector cells, e.g., T and NK cells. Pilot thermal stress study and a single dose mouse PK analysis supported favorable developability of the humanized antibody. Anti-A2aR antibody alone inhibited the growth of solid tumor, e.g. melanoma and lung cancer, in human PBMC grafted mice. Furthermore, it showed significant synergistic effect on inhibiting tumor growth when combined with Ipilimumab, Opdivo or Avastin, through multiple mechanisms including reduced ratios of Tregs:CD8+ T cells within the tumor microenvironment.
Conclusions The antagonist A2aR antibody represents a promising novel therapeutic candidate to potentiate combinatorial approaches for cancer immunotherapy via enhancing anti-tumor responses in solid tumors that only partially respond or are completely resistance to ICB therapies due to elevated adenosine level in tumor microenvironment.
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Ethics Approval All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) and performed in accordance with current regulations and standards of the United States Department of Agriculture and the National Institute of Health.
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