Characterization of the Rhesus Cytomegalovirus US28 Locus

Human cytomegalovirus (CMV) US28 (and the related open reading frame [ORF] US27) are G-protein-coupled receptor homologs believed to play a role in viral pathogenesis. In vitro, US28 has been shown to bind and internalize ligands, as well as activate intracellular signaling in response to certain chemokines, and to initiate the migration of smooth muscle cells to chemokine gradients. To assess the role of US28 in vivo, we examined the rhesus model and sequenced and characterized the rhesus CMV US28 locus. We found that rhesus CMV carries five tandem homologs of US28, all widely divergent from US28 and from each other. By reverse transcription-PCR and Northern analysis, we demonstrated expression of these ORFs in infected cells. With stable cell lines expressing these ORFs, we analyzed the homolog's binding and signaling characteristics across a wide range of chemokines and found one (RhUS28.5) to have a ligand binding profile similar to that of US28. In addition, we localized US28 and the rhesus CMV homolog RhUS28.5 to the envelope of infectious virions, suggesting a role in viral entry or cell tropism

Macrophage inflammatory protein-1 beta induces migration and activation of human thymocytes

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CCR6, a CC chemokine receptor that interacts with macrophage inflammatory protein 3alpha and is highly expressed in human dendritic cells.

Dendritic cells initiate immune responses by ferrying antigen from the tissues to the lymphoid organs for presentation to lymphocytes. Little is known about the molecular mechanisms underlying this migratory behavior. We have identified a chemokine receptor which appears to be selectively expressed in human dendritic cells derived from CD34+ cord blood precursors, but not in dendritic cells derived from peripheral blood monocytes. When stably expressed as a recombinant protein in a variety of host cell backgrounds, the receptor shows a strong interaction with only one chemokine among 25 tested: the recently reported CC chemokine macrophage inflammatory protein 3alpha. Thus, we have designated this receptor as the CC chemokine receptor 6. The cloning and characterization of a dendritic cell CC chemokine receptor suggests a role for chemokines in the control of the migration of dendritic cells and the regulation of dendritic cell function in immunity and infection

OP0001 BARICITINIB, TOFACITINIB, UPADACITINIB, FILGOTINIB, AND CYTOKINE SIGNALING IN HUMAN LEUKOCYTE SUBPOPULATIONS: AN UPDATED EX-VIVO COMPARISON

Background:everal JAKi are now used for the treatment of RA; approved doses include baricitinib (bari) 2- and/or 4-mg QD, tofacitinib (tofa) 5-mg BID, upadacitinib (upa) 15-mg QD. The JAK selectivity these agents is proposed to vary across the class.Objectives:In vitro cellular pharmacology of bari to tofa, upa, and filgotinib (filgo) were compared.Methods:PBMCs from 6 healthy donors were incubated with the JAKis over a 7- to 8-point concentration range. Following cytokine stimulation, levels of pSTAT were measured and IC50 calculated in gated leukocyte subpopulations. Therapeutic dose relevance was assessed using calculated mean concentration-time (CT) profiles over 24 hours for bari 2- and 4-mg QD; tofa 5- and 10-mg BID; upa 15- and 30-mg QD; filgo 100- and 200-mg QD. Average daily % inhibition of pSTAT (%SI) was calculated for each JAKi, cytokine, and cell type; filgo %SI integrated parent drug + metabolite.Results:The cytokines did not signal in all cell types (Figure 1). When signaling was detected, IC50 and %SI for a particular JAKi were generally similar across cell types, with dose-dependent inhibition (Figures 1 &amp; 2). Based on IC50s, upa was most and filgo/metabolite least potent across JAK2/2 or JAK2/TYK2-dependent (IL-3, GM-CSF, G-CSF), JAK1/3-dependent (IL-2, 4, 15, 21), and JAK1/2/TYK2 dependent (IL-6 &amp; 10, IFN-α &amp; γ) signaling pathways.Figure 1.IC50 values (nM) for baricitinib, tofacitinib, upadacitinib, filgotinib (parent and metabolite) in cytokine-stimulated human PBMC preparations. *p&lt;0.01, **p&lt;0.001, ***p&lt;0.0001 vs. bari.Incorporating CT profiles, no agent potently or continuously inhibited an individual cytokine signaling pathway throughout the dosing interval. Comparing bari 4-mg to tofa 5-mg BID, upa 15-mg QD, and filgo 100-mg QD, %SI of JAK2/2 or JAK2/TYK2-dependent cytokines was highest with bari 4-mg and upa. Inhibition of JAK1/2/TYK2 cytokines was highest with bari 4-mg. Inhibition of JAK2/2 or JAK2/TYK2, and of JAK1/3-dependent cytokines was lowest for filgo 100-mg QD. For bari 2-mg QD vs. these other JAKi doses, %SI of JAK2/2 or JAK2/TYK2 was highest with upa followed by bari 2-mg. The highest inhibitors of the JAK1/2/TYK2-dependent cytokines varied by cytokine / cell type though consistently included upa. Inhibition of JAK1/3 was highest for upa and tofa. Across comparisons, filgo 100-mg QD showed the least %SI of JAK2/2 or JAK2/TYK2-dependent, and of JAK1/3-dependent cytokines. Filgo reached the highest levels of %SI among agents only for 200-mg QD vs. lower doses of the other JAKi (for selected JAK1/2/TYK2-dependent cytokines).Conclusion:JAKis display different in vitro pharmacologic profiles which, coupled to their in vivo pharmacokinetics, suggest they modulate distinct cytokine pathways to differing degrees and durations over 24 hours. Ex vivo whole cell assays seem distinct from cell free kinase inhibition assays in determining the overall cytokine modulatory potential of members of the JAKi class.References:[1]McInnes IB, et al. Arthritis Res Ther. 2019 Aug 2;21(1):183Figure 2.Baricitinib, tofacitinib, upadacitinib, filgotinib: calculated average percent daily STAT inhibition for selected cytokines. -p&lt;0.01, --p&lt;0.001, ---p&lt;0.0001 significantly lower compared to bari (vs. 2-mg if left of vertical line “|”, vs. 4-mg if right of vertical line “|”). +p&lt;0.01, ++p&lt;0.001, +++p&lt;0.0001 significantly higher compared to bari (vs. 2-mg if left of vertical line “|”, vs. 4-mg if right of vertical line “|”).Disclosure of Interests:Iain McInnes Grant/research support from: Bristol-Myers Squibb, Celgene, Eli Lilly and Company, Janssen, and UCB, Consultant of: AbbVie, Bristol-Myers Squibb, Celgene, Eli Lilly and Company, Gilead, Janssen, Novartis, Pfizer, and UCB, Guilherme Rocha Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Richard E Higgs Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Daniel Dairaghi Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Thomas Wehrman Shareholder of: An insignificant amount in AbbVie as part of a larger portfolio, Consultant of: Primity Bio Inc. works with many pharmaceutical and biotech companies and provides CRO services., Evan Wang Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Zhang Xin Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Jorge Ross Terres Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Terence Rooney Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Peter C. Taylor Grant/research support from: Celgene, Eli Lilly and Company, Galapagos, and Gilead, Consultant of: AbbVie, Biogen, Eli Lilly and Company, Fresenius, Galapagos, Gilead, GlaxoSmithKline, Janssen, Nordic Pharma, Pfizer Roche, and UCB</jats:sec

CCR6, a CC chemokine receptor that interacts with macrophage inflammatory protein 3alpha and is highly expressed in human dendritic cells.

Dendritic cells initiate immune responses by ferrying antigen from the tissues to the lymphoid organs for presentation to lymphocytes. Little is known about the molecular mechanisms underlying this migratory behavior. We have identified a chemokine receptor which appears to be selectively expressed in human dendritic cells derived from CD34+ cord blood precursors, but not in dendritic cells derived from peripheral blood monocytes. When stably expressed as a recombinant protein in a variety of host cell backgrounds, the receptor shows a strong interaction with only one chemokine among 25 tested: the recently reported CC chemokine macrophage inflammatory protein 3alpha. Thus, we have designated this receptor as the CC chemokine receptor 6. The cloning and characterization of a dendritic cell CC chemokine receptor suggests a role for chemokines in the control of the migration of dendritic cells and the regulation of dendritic cell function in immunity and infection

Characterization of Pharmacologic and Pharmacokinetic Properties of CCX168, a Potent and Selective Orally Administered Complement 5a Receptor Inhibitor, Based on Preclinical Evaluation and Randomized Phase 1 Clinical Study

The complement 5a receptor has been an attractive therapeutic target for many autoimmune and inflammatory disorders. However, development of a selective and potent C5aR antagonist has been challenging. Here we describe the characterization of CCX168 (avacopan), an orally administered selective and potent C5aR inhibitor. CCX168 blocked the C5a binding, C5a-mediated migration, calcium mobilization, and CD11b upregulation in U937 cells as well as in freshly isolated human neutrophils. CCX168 retains high potency when present in human blood. A transgenic human C5aR knock-in mouse model allowed comparison of the in vitro and in vivo efficacy of the molecule. CCX168 effectively blocked migration in in vitro and ex vivo chemotaxis assays, and it blocked the C5a-mediated neutrophil vascular endothelial margination. CCX168 was effective in migration and neutrophil margination assays in cynomolgus monkeys. This thorough in vitro and preclinical characterization enabled progression of CCX168 into the clinic and testing of its safety, tolerability, pharmacokinetic, and pharmacodynamic profiles in a Phase 1 clinical trial in 48 healthy volunteers. CCX168 was shown to be well tolerated across a broad dose range (1 to 100 mg) and it showed dose-dependent pharmacokinetics. An oral dose of 30 mg CCX168 given twice daily blocked the C5a-induced upregulation of CD11b in circulating neutrophils by 94% or greater throughout the entire day, demonstrating essentially complete target coverage. This dose regimen is being tested in clinical trials in patients with anti-neutrophil cytoplasmic antibody-associated vasculitis. Trial Registration ISRCTN registry with trial ID ISRCTN13564773