CRISPR/Cas9 mediated deletion of the adenosine A2A receptor enhances CAR T cell efficacy.

Adenosine is an immunosuppressive factor that limits anti-tumor immunity through the suppression of multiple immune subsets including T cells via activation of the adenosine A2A receptor (A2AR). Using both murine and human chimeric antigen receptor (CAR) T cells, here we show that targeting A2AR with a clinically relevant CRISPR/Cas9 strategy significantly enhances their in vivo efficacy, leading to improved survival of mice. Effects evoked by CRISPR/Cas9 mediated gene deletion of A2AR are superior to shRNA mediated knockdown or pharmacological blockade of A2AR. Mechanistically, human A2AR-edited CAR T cells are significantly resistant to adenosine-mediated transcriptional changes, resulting in enhanced production of cytokines including IFNγ and TNF, and increased expression of JAK-STAT signaling pathway associated genes. A2AR deficient CAR T cells are well tolerated and do not induce overt pathologies in mice, supporting the use of CRISPR/Cas9 to target A2AR for the improvement of CAR T cell function in the clinic.

Differential GLP-1R Binding and Activation by Peptide and Non-peptide Agonists.

Peptide drugs targeting class B1 G-protein-coupled receptors (GPCRs) can treat multiple diseases; however, there remains substantial interest in the development of orally delivered non-peptide drugs. Here, we reveal unexpected overlap between signaling and regulation of the glucagon-like peptide-1 (GLP-1) receptor by the non-peptide agonist PF 06882961 and GLP-1 that was not observed for another compound, CHU-128. Compounds from these patent series, including PF 06882961, are currently in clinical trials for treatment of type 2 diabetes. High-resolution cryoelectron microscopy (cryo-EM) structures reveal that the binding sites for PF 06882961 and GLP-1 substantially overlap, whereas CHU-128 adopts a unique binding mode with a more open receptor conformation at the extracellular face. Structural differences involving extensive water-mediated hydrogen bond networks could be correlated to functional data to understand how PF 06882961, but not CHU-128, can closely mimic the pharmacological properties of GLP-1. These findings will facilitate rational structure-based discovery of non-peptide agonists targeting class B GPCRs.

Targeting Adenosine Receptor Signaling in Cancer Immunotherapy.

The immune system plays a major role in the surveillance and control of malignant cells, with the presence of tumor infiltrating lymphocytes (TILs) correlating with better patient prognosis in multiple tumor types. The development of 'checkpoint blockade' and adoptive cellular therapy has revolutionized the landscape of cancer treatment and highlights the potential of utilizing the patient's own immune system to eradicate cancer. One mechanism of tumor-mediated immunosuppression that has gained attention as a potential therapeutic target is the purinergic signaling axis, whereby the production of the purine nucleoside adenosine in the tumor microenvironment can potently suppress T and NK cell function. The production of extracellular adenosine is mediated by the cell surface ectoenzymes CD73, CD39, and CD38 and therapeutic agents have been developed to target these as well as the downstream adenosine receptors (A1R, A2AR, A2BR, A3R) to enhance anti-tumor immune responses. This review will discuss the role of adenosine and adenosine receptor signaling in tumor and immune cells with a focus on their cell-specific function and their potential as targets in cancer immunotherapy.

Determination of adenosine A1 receptor agonist and antagonist pharmacology using Saccharomyces cerevisiae: implications for ligand screening and functional selectivity.

The budding yeast, Saccharomyces cerevisiae, is a convenient system for coupling heterologous G protein-coupled receptors (GPCRs) to the pheromone response pathway to facilitate empirical ligand screening and/or GPCR mutagenesis studies. However, few studies have applied this system to define GPCR-G protein-coupling preferences and furnish information on ligand affinities, efficacies, and functional selectivity. We thus used different S. cerevisiae strains, each expressing a specific human Galpha/yeast Gpa1 protein chimera, and determined the pharmacology of various ligands of the coexpressed human adenosine A(1) receptor. These assays, in conjunction with the application of quantitative models of agonism and antagonism, revealed that (-)-N(6)-(2-phenylisopropyl)adenosine was a high-efficacy agonist that selectively coupled to Gpa/1Galpha(o), Gpa1/Galpha(i1/2), and Gpa1/Galpha(i3), whereas the novel compound, 5'-deoxy-N(6)-(endo-norborn-2-yl)-5'-(2-fluorophenylthio)adenosine (VCP-189), was a lower-efficacy agonist that selectively coupled to Gpa1/Galpha(i) proteins; the latter finding suggested that VCP-189 might be functionally selective. The affinity of the antagonist, 8-cyclopentyl-1,3-dipropylxanthine, was also determined at the various strains. Subsequent experiments performed in mammalian Chinese hamster ovary cells monitoring cAMP formation/inhibition, intracellular calcium mobilization, phosphorylation of extracellular signal-regulated kinase 1 and 2 or (35)S-labeled guanosine 5'-(gamma-thio)triphosphate binding, were in general agreement with the yeast data regarding agonist efficacy estimation and antagonist affinity estimation, but revealed that the apparent functional selectivity of VCP-189 could be explained by differences in stimulus-response coupling between yeast and mammalian cells. Our results suggest that this yeast system is a useful tool for quantifying ligand affinity and relative efficacy, but it may lack the sensitivity required to detect functional selectivity of low-efficacy agonists.