Abstract
Chemokine CC motif receptor (CCR) 5 is a major drug target for both inflammation and virology indications. The primary function of CCR5 is to mediate the trafficking of CCR5-expressing lymphocytes to any of the CCR5 ligands, which are often increased during inflammatory responses. In addition, CCR5 is a coreceptor for HIV, mediating R5 tropic HIV infection of CCR5-expressing CD4 T cells. We report the use of a novel method to assay the pharmacodynamic (PD) properties of small-molecule and antibody inhibitors of CCR5 ligand-induced activation by measuring phosphorylation of serine residue 349 in the cytoplasmic tail of human CCR5 using phosphoflow cytometry in whole blood. This assay is highly specific and measures CCR5 phosphorylation in both CD8(+) and CD4(+) T cells and allows the calculation of inhibitor IC(50) values from both lymphocyte subsets in the presence of CCR5 antagonists. In addition, this assay is cross-reactive to nonhuman primates and allows PD analysis in whole blood from rhesus and cynomolgus macaque. Using this assay, we identified different ligand-dependent response properties between CD8(+) and CD4(+) T cells, although CCR5 antagonists behave with similar properties against both cell types. The use of this assay may be of particular benefit to monitor PD effects of CCR5 inhibitors during drug development, preclinical in vivo studies, and in patients currently being treated for HIV or CCR5-mediated inflammatory diseases with CCR5 inhibitors. Similar phosphoflow approaches to other GPCR targets on circulating lymphocytes may prove to be the most reliable PD assay for preclinical and potentially clinical development.