Small molecule AZD4635 inhibitor of A2AR signaling rescues immune cell function including CD103+ dendritic cells enhancing anti-tumor immunity.

Accumulation of extracellular adenosine within the microenvironment is a strategy exploited by tumors to escape detection by the immune system. Adenosine signaling through the adenosine 2A receptor (A2AR) on immune cells elicits a range of immunosuppressive effects which promote tumor growth and limit the efficacy of immune checkpoint inhibitors. Preclinical data with A2AR inhibitors have demonstrated tumor regressions in mouse models by rescuing T cell function; however, the mechanism and role on other immune cells has not been fully elucidated. METHODS: We report here the development of a small molecule A2AR inhibitor including characterization of binding and inhibition of A2AR function with varying amounts of a stable version of adenosine. Functional activity was tested in both mouse and human T cells and dendritic cells (DCs) in in vitro assays to understand the intrinsic role on each cell type. The role of adenosine and A2AR inhibition was tested in DC differentiation assays as well as co-culture assays to access the cross-priming function of DCs. Syngeneic models were used to assess tumor growth alone and in combination with alphaprogrammed death-ligand 1 (αPD-L1). Immunophenotyping by flow cytometry was performed to examine global immune cell changes upon A2AR inhibition. RESULTS: We provide the first report of AZD4635, a novel small molecule A2AR antagonist which inhibits downstream signaling and increases T cell function as well as a novel mechanism of enhancing antigen presentation by CD103+ DCs. The role of antigen presentation by DCs, particularly CD103+ DCs, is critical to drive antitumor immunity providing rational to combine a priming agent AZD4635 with check point blockade. We find adenosine impairs the maturation and antigen presentation function of CD103+ DCs. We show in multiple syngeneic mouse tumor models that treatment of AZD4635 alone and in combination with αPD-L1 led to decreased tumor volume correlating with enhanced CD103+ function and T cell response. We extend these studies into human DCs to show that adenosine promotes a tolerogenic phenotype that can be reversed with AZD4635 restoring antigen-specific T cell activation. Our results support the novel role of adenosine signaling as an intrinsic negative regulator of CD103+ DCs maturation and priming. We show that potent inhibition of A2AR with AZD4635 reduces tumor burden and enhances antitumor immunity. This unique mechanism of action in CD103+ DCs may contribute to clinical responses as AZD4635 is being evaluated in clinical trials with IMFINZI (durvalumab, αPD-L1) in patients with solid malignancies. CONCLUSION: We provide evidence implicating suppression of adaptive and innate immunity by adenosine as a mechanism for immune evasion by tumors. Inhibition of adenosine signaling through selective small molecule inhibition of A2AR using AZD4635 restores T cell function via an internal mechanism as well as tumor antigen cross-presentation by CD103+ DCs resulting in antitumor immunity.

In vitro investigations into the roles of drug transporters and metabolizing enzymes in the disposition and drug interactions of dolutegravir, a HIV integrase inhibitor.

Dolutegravir (DTG; S/GSK1349572) is a potent HIV-1 integrase inhibitor with a distinct resistance profile and a once-daily dose regimen that does not require pharmacokinetic boosting. This work investigated the in vitro drug transport and metabolism of DTG and assessed the potential for clinical drug-drug interactions. DTG is a substrate for the efflux transporters P-glycoprotein (Pgp) and human breast cancer resistance protein (BCRP). Its high intrinsic membrane permeability limits the impact these transporters have on DTG's intestinal absorption. UDP-glucuronosyltransferase (UGT) 1A1 is the main enzyme responsible for the metabolism of DTG in vivo, with cytochrome P450 (P450) 3A4 being a notable pathway and UGT1A3 and UGT1A9 being only minor pathways. DTG demonstrated little or no inhibition (IC(50) values > 30 µM) in vitro of the transporters Pgp, BCRP, multidrug resistance protein 2, organic anion transporting polypeptide 1B1/3, organic cation transporter (OCT) 1, or the drug metabolizing enzymes CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, UGT1A1, or 2B7. Further, DTG did not induce CYP1A2, 2B6, or 3A4 mRNA in vitro using human hepatocytes. DTG does inhibit the renal OCT2 (IC(50) = 1.9 µM) transporter, which provides a mechanistic basis for the mild increases in serum creatinine observed in clinical studies. These in vitro studies demonstrate a low propensity for DTG to be a perpetrator of clinical drug interactions and provide a basis for predicting when other drugs could result in a drug interaction with DTG.