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RANTES (regulated on activation, normal T cell expressed and secreted) is a member of the C-C superfamily of chemokines and is reported to function as a potent chemoattractant for monocytes, eosinophils, and a subpopulation of CD4+ T cells. Using a recombinant human type 5 adenovirus containing the murine RANTES cDNA (Ad5E3 mRANTES), which is capable of expressing biologically active cytokine upon infection, we initiated a study to characterize the biologic functions of RANTES cytokine in vivo. Intratracheal administration of Ad5E3 mRANTES targeted transient RANTES expression to the bronchial epithelium of the lung in Sprague-Dawley rats. Bronchoalveolar lavage fluids (BAL) collected at 24 h had increased chemotactic activity vs controls as measured in a murine CD4+ T cell Boyden chamber microchemotaxis assay. There was a dramatic increase in the number of cells (macrophage, monocytes, and neutrophils) recovered from BAL samples taken from Ad5E3 mRANTES-treated animals at 24 h, with a >50-fold increase in monocytes, indicating a proinflammatory effect for this cytokine in vivo. This effect on monocytes was transient, decreasing by 7 days, with evidence of increased eosinophils and lymphocytes at this time. Histologic examination of lung sections at 24 h revealed greatly increased numbers of mononuclear cells, primarily monocytes, within the lungs of Ad5E3 mRANTES-treated animals, with increased extravasation of monocytes around blood vessels, indicating an ongoing process of peripheral blood monocyte recruitment. This study provides further evidence for RANTES to be a monocyte chemoattractant in vivo.

Summary Background Selected cytokines produced by allergen specific CD4+ T cells from atopic individuals contribute to both the specific and non-specific effector mechanisms of the allergic itnmune response. The chemokine family of cytokines and tumour necrosis factor (TNF)-α are leucocyte regulatory and proinflanimatory molecules. The chemokines include interleukin (IL)-8 and the RANTES/SIS cytokines. Objective There has been no systematic survey of chemokine production in T-cell subtypes. Because of their wide range of biological properties, it might be expected that they would be closely regulated by T cells. This paper illustrates one way (through the characterization of T-cell clones) these questions might be addressed. Methods Northern blot analysis was used to quantitate steady state transcription of selected cytokine genes and enzyme linked imtiiunosorbent assay (ELISA) was used to quantitate soluble product. Results mRNA expression of the chemokines (IL-8, HuMIP-1α and HuMIP-1β) and TNFα is upregulated in TH2-like cloned house dust mite reactive human CD4+ T cells under conditions of activation and during the induction phase of anergy. Although the development of anergy superinduces mRNA for both IL-8 and TNFα. protein production is low compared with that released during activation. In contrast. RANTES, a chemoattractant for CD4+/CD45RO+ memory T cells, eosinophils and basophils, is constitutively expressed at the RNA level by the T cells and not modulated by signals of activation and anergy induction. The production of IL-2, IL-4 and IL-5 mRNA and proteins during the induction of anergy peaks at 2h after stimulation, whereas the kinetics following activation of the T cells is delayed in comparison. Conclusion These data show that the induction of the anergic state coincides with post-transcriptional regulation of selected cytokine genes. Further study of these phenomena will impact on our understanding of the mechanisms of induction of anergy and the regulation of allergic immune responses in desensitization.

The chemokine RANTES is a chemoattractant and activating factor for T lymphocytes. Investigation of the signal transduction mechanisms induced by RANTES in T cells revealed tyrosine phosphorylation of multiple protein species with prominent bands at 70-85 and 120-130 kD. Immunoprecipitation and Western analyses revealed that a protein of 125 kD was identical to the focal adhesion kinase (FAK) pp125FAK. RANTES stimulated phosphorylation of FAK as early as 30 seconds and immunoblots using antiphosphotyrosine monoclonal antibodies revealed that there was consistent phosphorylation of a 68-70 kD species in the pp125FAK immunoprecipitates. Immunoblotting and kinase assays showed this to be two separate proteins, the tyrosine kinase zeta-associated protein (ZAP) 70, and the focal adhesion protein paxillin. These results indicate a potentially important role for RANTES in the generation of T cell focal adhesions and subsequent cell activation via a molecular complex containing FAK, ZAP-70, and paxillin.

Chemokines play an important role in immune and inflammatory responses by inducing migration and adhesion of leukocytes. We have isolated a novel chemokine cDNA, designated CCF18, from a cDNA library of an IL-3-dependent murine pro-B cell line, Ba/F3. The cDNA encodes a protein structurally related to the C-C chemokine members. Among this family, C10 shows the highest homology to CCF18, and MIP-1 alpha also has a significant homology but to a lesser extent. CCF18 produced from COS cells induced chemotaxis and Ca2+ flux in CD4+ T cell clones. Moreover, prior administration of MIP-1 alpha desensitized the cells to CCF18. The CCF18 gene (Scya10) was mapped to a middle region of murine chromosome 11, where other genes for several C-C chemokine members are localized. These results clearly indicate that CCF18 is a new member of the C-C chemokine family. Since a high level of CCF18 mRNA is constitutively expressed in macrophage and myeloid cell lines, CCF18 may play a role in inflammatory processes.

We describe the isolation of a cDNA that encodes human lymphotactin (Ltn), a new class of lymphocyte-specific chemokine. Human Ltn shows similarity to some members of the C-C chemokine family but has lost the first and third cysteine residues that are characteristic of the C-C and C-X-C chemokines. Ltn is chemotactic for lymphocytes but not for monocytes, a characteristic that makes it unique among chemokines. In addition, calcium flux desensitization studies indicate that Ltn uses a unique receptor. The human Ltn gene maps to a different chromosome than do the C-C and C-X-C chemokine families. Taken together, these characteristics indicate that Ltn is the first example of a new class of human chemokines with preferential effects on lymphocytes.

We have characterized the IL-8-induced signal transduction processes in T lymphocytes. A basal level of IL-8 receptor expression was shown on mixed PBL, as identified by using phycoerythrin (PE)-coupled IL-8, and this expression was increased following IL-2 stimulation. Scatchard analysis of T cells revealed competitive binding of IL-8 with a Kd of 0.55 nM, with approximately 1200 receptors per cell, on freshly isolated T cells. After 24 h in culture following purification, reverse transcriptase PCR (RT-PCR) analyses show the mRNA for only the type B IL-8R on these cultured T lymphocytes and the cell line MOLT-4. Stimulation of T lymphocytes or T cell clones with IL-8 led to generation of inositol trisphosphate and calcium flux. In addition, when T cells were prelabeled with [3H]oleic acid, IL-8 caused a long lasting, time- and dose-related increase in [3H]phosphatidylethanol (PtE), indicating activation of phospholipase D (PLD). By contrast, this IL-8-dependent PLD activity was undetectable in IL-8-stimulated neutrophils. PLD activation appeared to be downstream of protein kinase C, because several inhibitors abrogated the increase in [3H]PtE, whereas guanosine-5'-O-(3-thiotriphosphate (GTP(gamma)S) and inositol trisphosphorothioate (IP3S3) both increased the generation of [3H]PtE. Together, these results demonstrate that the IL-8RB receptor is sufficient to mediate phospholipase C (PLC) and PLD activation in T lymphocytes, but not in neutrophils, and indicate an important difference in receptor usage and signal transduction pathways between IL-8-stimulated lymphocytes and neutrophils.

The C-C chemokines MIP-1 alpha, MCP-1, and RANTES, but not MIP-1 beta, induce the chemotaxis of NK and IL-2-activated NK (IANK) cells, as determined in microchemotaxis assay. Only RANTES and MCP-1, but not MIP-1 alpha were able to induce the chemokinesis of NK cells. In contrast, none of the C-C chemokines tested was able to induce the chemokinesis of IANK cells. IANK cell chemotaxis in response to MCP-1 or RANTES but not MIP-1 alpha, was inhibited by pertussis toxin (PT). In contrast, cholera toxin (CT) inhibited the ability of all three chemokines to induce the chemotaxis of IANK cells. IANK cells intoxicated with PT lost their ability to migrate in response to RANTES and MCP-1 but not MIP-1 alpha, whereas those intoxicated with CT lost their ability to migrate in response to the three C-C chemokines tested. These results suggest that guanine nucleotide binding (G) proteins are coupled to C-C chemokine receptors in IANK cells. Subsequently, we observed that MIP-1 alpha, MCP-1, and RANTES, but not MIP-1 beta, enhance the binding of guanosine 5'-O-(thiotriphosphate), and increase the hydrolysis of [32P]GTP in IANK cell membranes. Further analysis showed that MIP-1 alpha, RANTES, or MCP-1 did not enhance GTP binding in membranes prepared from IANK cells intoxicated with CT, whereas only RANTES and MCP-1 but not MIP-1 alpha lost their ability to enhance GTP binding to IANK cell membranes prepared from PT-intoxicated cells. The differential inhibitory activity of CT and PT suggests that C-C chemokine receptors are coupled to different G proteins in IANK cells.

The murine beta-chemokine TCA3 was purified to homogeneity. The biologic activities of the purified glycoprotein were evaluated in vivo and in vitro. Mice injected i.p. with 1- to 100-ng purified rTCA3 exhibited a rapid influx of neutrophils and macrophages. Increased numbers of neutrophils and monocytes were observed in peripheral blood within 15 min and peak at 45 min. After 45 min neutrophil and macrophage levels were increased in the peritoneal exudate with peak levels occurring at 2 h, followed by a subsequent decline by 24 h. Inflammatory responses were induced in a dose-dependent fashion. The in vivo inflammatory responses were mirrored by the pattern of TCA3-induced chemotaxis in vitro. Neutrophils and macrophages responded to similar concentrations of TCA3 (3 x 10(-9) to 10(-8) M). Lymph node cells responded to other chemokines but did not migrate to TCA3. We also demonstrated that rTCA3 stimulates a transient increase in cytoplasmic free calcium in monocytic cells through a PTX-sensitive pathway. Cross-desensitization studies indicate that TCA3 acts independently of other beta-chemokines (MIP-1 alpha and RANTES) and the alpha-chemokine IL-8. Furthermore, TCA3 does not induce a Ca2 lux in cells transfected with cDNA for the C-C CKR-1 chemokine receptor, supporting the conclusion that there are distinct receptors for TCA3.

Macrophage inflammatory proteins-1 (MIP-1) alpha and beta are members of the C-C branch of the platelet factor 4 superfamily of cytokines, recently designated the "chemokine" superfamily. It has been suggested that the major cellular targets for the biologic activities of the C-C chemokines are the mononuclear leukocytes. However, the original designation of murine MIP-1 proteins as inflammatory mediators was based on suggestions that they activated neutrophil functions such as chemotaxis, the respiratory burst, and degranulation. In this study, we have evaluated the ability of human (Hu) MIP-1 alpha and beta to affect purified human neutrophil function. Although both rHuMIP-1 alpha and -1 beta stimulated significant calcium mobilization in human monocytes, only HuMIP-1 alpha exerted a detectable effect on neutrophils. HuMIP-1 alpha stimulated a small, dose-dependent increase in intracellular calcium, which was accompanied by a simultaneous change in right-angle light scatter, the latter indicating induction of shape change. While the effect of HuMIP-1 alpha on calcium mobilization in neutrophils was small when compared with that elicited by IL-8 or Gro alpha, it had similar characteristics to that by other receptor-dependent neutrophil agonists in that it was dependent on pertussis toxin-sensitive G proteins and on both mobilization of calcium from intracellular sources as well as influx from the extracellular environment. In addition, stimulation of neutrophils with HuMIP-1 alpha led to desensitization to subsequent additions of HuMIP-1 alpha. The stimulatory effect of HuMIP-1 alpha on neutrophil calcium mobilization and shape change was not coupled to other standard measures of neutrophil effector function. For instance, neither HuMIP-1 alpha nor -1 beta had any detectable stimulatory effect on the Na+/H+ antiport, degranulation, actin polymerization, or chemotaxis. Moreover, although HuMIP-1 alpha binding could easily be measured on monocytes or monocytic cell lines, the number of sites were too few to characterize on neutrophils by the same technique. Taken together, these results show that neither HuMIP-1 alpha nor -1 beta stimulate significant neutrophil activation and support the concept that the biologic effects of members of the C-C branch of the platelet factor 4 superfamily are not primarily directed toward neutrophils.

A chronic inflammatory disease may be characterized by an accumulation of activated leukocytes at the site of inflammation. Since the chemokine RANTES may play an active role in recruiting leukocytes into inflammatory sites, we investigated the ability of cultured human synovial fibroblasts isolated from patients suffering from rheumatoid arthritis to produce this chemokine and compared its regulation to that of the closely related chemokine gene, interleukin-8 (IL-8). In unstimulated synovial fibroblasts, the expression of mRNA for both chemokines was undetectable, but was increased in both a time- and dose-dependent manner upon stimulation with the monokines tumor necrosis factor alpha (TNF alpha) and interleukin-1 beta (IL-1 beta). Preincubation of the cells with cycloheximide "superinduced" the level of IL-8 mRNA stimulated by TNF alpha and IL-1 beta and RANTES mRNA stimulated by IL-1 beta, but decreased the expression of RANTES mRNA in response to TNF alpha. In addition, differential regulation of these genes was noted when synovial fibroblasts were stimulated with a combination of cytokines. IL-4 down-regulated and IFN gamma enhanced the TNF alpha- and IL-1 beta-induced increase in RANTES mRNA, whereas the induction of IL-8 mRNA by TNF alpha or IL-1 beta was inhibited by IFN gamma and augmented by IL-4. Moreover, a combination of TNF alpha and IL-1 beta synergistically induced IL-8 mRNA expression, whereas under the same conditions, the level of expression of RANTES mRNA was less than that induced by TNF alpha alone. These observations were also reflected at the level of chemokine secretion. These studies demonstrate that by expressing the chemokines RANTES and IL-8, synovial fibroblasts may participate in the ongoing inflammatory process in rheumatoid arthritis. In addition, the observation that these chemokine genes are differentially regulated, depending upon the presence of different cytokines, indicates that the type of cellular infiltrate and the progress of the inflammatory disease is likely to depend on the relative levels of stimulatory and inhibitory cytokines.

Cells in the rheumatoid synovium express high levels of major histocompatibility complex (MHC) class II molecules in vivo. We have therefore examined the ability of engagement of MHC class II molecules by the superantigen Staphylococcal enterotoxin A (SEA) to activate interleukin 6 (IL-6) and IL-8 gene expression in type B synoviocytes isolated from patients with rheumatoid arthritis. SEA had a minimal or undetectable effect on the expression of either gene in resting synoviocytes, as determined by Northern blot and specific enzyme-linked immunosorbent assay. However, induction of MHC class II molecule expression after treatment of synoviocytes with interferon gamma (IFN-gamma) enabled the cells to respond to SEA in a dose-dependent manner, resulting in an increase in both the level of steady-state mRNA for IL-6 and IL-8, and the release of these cytokines into the supernatant. IFN-gamma by itself had no effect on the expression of either cytokine. Pretreatment of the cells with the transcription inhibitor actinomycin D prevented the increase in cytokine mRNA induced by SEA, whereas cycloheximide superinduced mRNA for both cytokines after stimulation by SEA. Taken together, these results indicate that signaling through MHC class II molecules may represent a novel mechanism by which inflammatory cytokine production is regulated in type B rheumatoid synoviocytes, and potentially provides insight into the manner by which superantigens may initiate and/or propagate autoimmune diseases.

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.

Background In ANCA vasculitis, the anaphylatoxin C5a amplifies neutrophil influx and activation through C5aR. CCX168, an oral specific C5aR antagonist, is in clinical development for ANCA-associated renal vasculitis (AARV). Objectives A Phase 2 clinical trial was conducted to investigate the potential of CCX168 to contribute to disease remission and permit glucocorticoid reduction or avoidance in patients with active AARV receiving cyclophosphamide (CYC). Methods This randomized, double-blind, placebo-controlled Phase 2 trial was performed in a stepwise manner. In Step 1 (N=12), CCX168+CYC+low dose prednisone (20 mg/day starting dose) was compared to standard-of-care (SOC) (CYC+high dose prednisone, 60 mg/day starting dose). In Step 2 (N=14), CCX168+CYC and no prednisone was compared to SOC. Eligible patients had GPA, MPA, or renal limited vasculitis, were PR3 or MPO-ANCA positive, and had active renal vasculitis with a GFR >30ml/min. The dose of CCX168 was 30 mg BID PO for 12 weeks and the dose of CYC was 15 mg/kg IV q2-3 wks. Results Baseline characteristics and efficacy results at Week 12 are shown in the table. The number of steroid rescue events was not higher in the CCX168 groups compared to SOC. The incidence of renal remission was higher in the CCX168 groups compared to the SOC control group. The percent decrease from baseline in BVAS, urinary ACR and urinary MCP-1/creatinine was higher in the CCX168 groups compared to SOC. Renal function, as measured by eGFR, increased in all 3 groups, with the largest increase (6.8 mL/min/1.73 m2) in the CCX168+low dose steroids group. CCX168 appeared to be well tolerated. No unexpected serious adverse reactions were observed with CCX168 use. There was one early withdrawal from study, in the control, SOC, group. Conclusions CCX168 plus CYC appear to be at least as effective, if not more effective, as full dose steroids plus CYC in treatment of patients with an ANCA associated renal vasculitis flare. Disclosure of Interest D. Jayne Consultant for: ChemoCentryx, Inc., A. Bruchfeld Consultant for: ChemoCentryx, Inc., M. Schaier: None declared, K. Ciechanowski: None declared, L. Harper Consultant for: ChemoCentryx, Inc., M. Jadoul: None declared, M. Segelmark Consultant for: ChemoCentryx, Inc., D. Selga: None declared, I. Szombati: None declared, M. Venning Consultant for: ChemoCentryx, Inc., C. Hugo: None declared, P. Van Daele: None declared, O. Viklicky: None declared, A. Potarca Consultant for: ChemoCentryx, Inc., T. Schall Shareholder of: ChemoCentryx, Inc., Employee of: ChemoCentryx, Inc., P. Bekker Shareholder of: ChemoCentryx, Inc., Employee of: ChemoCentryx, Inc. DOI 10.1136/annrheumdis-2014-eular.3728

Background Chemokines are key regulators of leukocyte activation and recruitment to sites of inflammation. Of particular relevance in rheumatoid arthritis (RA), the chemokine receptors CCR1 and CCR6 are thought to drive monocyte/macrophage and Th17 cell recruitment, respectively, into RA joints. We recently demonstrated the clinical benefit of our first-generation CCR1 antagonist (CCX354) in a Phase 2 trial in RA patients (Tak et al., 2012). To date, no therapeutic agents targeting CCR6 have progressed into clinical evaluation. Objectives To identify orally active small molecules that selectively target either CCR1 or CCR6 and possess overall profiles suitable for clinical development. Key selection criteria include their potency on CCR1 or CCR6-expressing primary human cells, their selectivity, and their ability to prevent chemotaxis of freshly isolated human blood leukocytes towards RA synovial fluid samples. Methods The in vitro potency of CCX486 (CCR1 antagonist) was assessed by inhibition of CCL15-mediated chemotaxis of THP-1 cells and human blood monocytes. The in vitro potency of C0339589 (CCR6 antagonist) was assessed by inhibition of CCL20-mediated chemotaxis and binding using a human NK cell line and CCR6+ enriched human PBMC. CCX486 and C0339589 were assessed for their ability to block the CCR1 and CCR6-mediated chemotaxis, respectively, of human blood leukocytes towards RA synovial fluids. Results CCX486 potently blocks CCL15-mediated chemotaxis with an IC50 of 0.61 nM (THP-1 cells). In 100% human serum, CCX486 blocks CCL15-mediated chemotaxis with an IC50 of 1.0 nM (THP-1 cells) and 1.8 nM (human monocytes). CCX486 also completely blocks the monocyte-chemotactic activity displayed by human RA synovial fluid samples. C0339589 potently blocks CCL20-mediated chemotaxis of CCR6+ enriched human PBMC (IC50: 38 nM). C0339589 also blocks the lymphocyte-chemotactic activity displayed by human RA synovial fluid samples (IC50 ~ 10-30 nM). Both CCX486 and C0339589 are highly selective for CCR1 and CCR6, respectively, when tested against a wide screen of chemokine and chemoattractant receptors. Conclusions CCR1: A second-generation CCR1 antagonist (CCX486) was designed de novo using existing knowledge from several other CCR1 programs that have previously advanced into clinical evaluation. This compound, which shares very little structural similarity to first-generation molecule CCX354, is nevertheless equally selective for CCR1 and orally bioavailable in preclinical species (Dairaghi et al., 2011). CCX486 is about 20 times more potent on human CCR1 than CCX354. CCX486 was shown to block CCR1-mediated chemotaxis of human blood monocytes towards RA synovial fluid samples. CCR6: Several chemical lead series were identified using a proprietary high-throughput screening format. Optimization of one of these series resulted in advanced molecules such as C0339589. This molecule is highly potent on human and mouse CCR6 and displays properties conducive to its use for the treatment of diseases such as RA, psoriasis and multiple sclerosis. References (a) Dairaghi, D. et al. (2011) Clin. Pharmacol. Ther., 89(5), 726; (b) Tak, P.P. et al. (2012) Ann. Rheum. Dis., online May 15, 2012. Disclosure of Interest J. Jaen Shareholder of: ChemoCentryx, Employee of: ChemoCentryx, D. Dairaghi Shareholder of: ChemoCentryx, Employee of: ChemoCentryx, M. Leleti Shareholder of: ChemoCentryx, Employee of: ChemoCentryx, J. Powers Shareholder of: ChemoCentryx, Employee of: ChemoCentryx, Y. Wang Shareholder of: ChemoCentryx, Employee of: ChemoCentryx, P. Zhang Shareholder of: ChemoCentryx, Employee of: ChemoCentryx, T. Schall Shareholder of: ChemoCentryx, Employee of: ChemoCentryx

Background Macrophages produce pro-inflammatory cytokines such as TNFα, IL-1, and IL-6, and cause joint destruction. High levels of C-C chemokine receptor 1 (CCR1)-expressing macrophages and CCR1 chemokines have been identified in inflamed synovial fluid and tissue. CCR1 is also involved in osteoclast maturation, mobility, and activation. Orally administered CCX354-C 200 mg QD blocked >90% CCR1 in blood monocytes based on ex vivo assays of Alexa647-MIP-1α binding to CCR1 and was safe and well tolerated in Phase 1. Objectives Primary: Evaluation of safety and tolerability of CCX354-C in RA. Secondary: Evaluation of efficacy based on RA disease response measurements: ACR, DAS28, CRP, and ESR, as well as bone turnover markers. Methods This was a 160-subject randomized, double-blind, placebo-controlled Phase 2 clinical trial. Subjects had moderate to severe RA, were on stable methotrexate treatment for at least 8 weeks, had ≥8 swollen and tender joint counts (SJC, TJC; 66/68 joints), and CRP >5 mg/L. SJC and TJC, and CRP level eligibility were confirmed prior to dosing on Day 1. Eligible subjects were stratified based on prior use of biologic drugs, and current corticosteroid use. Subjects received double-blind placebo (N=54), 100 mg CCX354-C BID (N=53), or 200 mg CCX354-C QD (N=53) orally for 12 weeks. Results Baseline characteristics, mean (SD), were: Age 55 (10) years, 84% female, median RA duration 5 years, DAS28-CRP 5.8 (0.8), median CRP 11 mg/L, median ESR 34 mm/hr. 88% were biologics naive and 55% were current corticosteroid users. CCX354-C was well tolerated by study subjects. No SAEs were reported in the 200 mg QD and placebo groups. Four SAEs not deemed related to CCX354-C (syncope due to blood draw, angina pectoris, MI, temporal lobe epilepsy) were reported in the 100 mg BID group. ACR20 response data were as follows (p-values for 200 mg QD vs. placebo): CRP decrease at Week 12 was statistically significant (p=0.023) in the 200 mg QD group vs. placebo. ACR50, ACR70, DAS28-CRP, ESR, and ACR component results indicated greatest efficacy in the 200 mg QD dose group. C-telopeptide, procollagen type I N-terminal propeptide, and osteocalcin decreases were more pronounced in the CCX354-C groups compared to placebo and reached statistical significance at several time points. Clinical responders had higher plasma CCX354 concentrations than non-responders. Conclusions The novel oral CCR1-specific antagonist CCX354-C at 200 mg QD showed clinical and biologic activity, and appears safe and well tolerated in this study. This is the first demonstration of clinical efficacy in RA with a CCR1 inhibitor, consistent with previous in vitro studies and a synovial biopsy study in humans on the effects of CCR1 blockade. Disclosure of Interest P. P. Tak Consultant for: Abbott Laboratories, Amgen, Arthrogen, AstraZeneca, Bristol Myers Squibb, ChemoCentryx, Johnson & Johnson, Merck, MerckSerono, Novartis, NovImmune, NovoNordisk, Pfizer, Roche/Genentech, Employee of: GlaxoSmithKline, A. Balanescu: None Declared, V. Tseluyko: None Declared, S. Bojin: None Declared, E. Drescher: None Declared, D. Dairaghi Shareholder of: ChemoCentryx, Inc., Employee of: ChemoCentryx, Inc., S. Miao Shareholder of: ChemoCentryx, Inc., Employee of: ChemoCentryx, Inc., V. Marchesin Shareholder of: ChemoCentryx, Inc., Employee of: ChemoCentryx, Inc., J. Jaen Shareholder of: ChemoCentryx, Inc., Employee of: ChemoCentryx, Inc., T. Schall Shareholder of: ChemoCentryx, Inc., Employee of: ChemoCentryx, Inc., P. Bekker Shareholder of: ChemoCentryx, Inc., Employee of: ChemoCentryx, Inc.

Background The C5a receptor (C5aR) is expressed by cells of the innate immune system and is thought to play a key role in numerous autoimmune diseases, such as anti-neutrophil cytoplasmic antibody (ANCA) vasculitis and rheumatoid arthritis (RA). CCX168 is an orally active antagonist of C5aR that has completed Phase 1 evaluation and is currently in a Phase 2 trial for induction of remission in patients with ANCA-associated renal vasculitis (AARV). Objectives To evaluate CCX168 in preclinical and clinical studies to determine safety, pharmacokinetic, and pharmacodynamics properties, and integrate these properties into a PK/PD relationship predictive of receptor coverage required for effective use in treating C5aR-mediated autoimmune diseases in humans. Methods The in vitro potency of CCX168 against C5aR was assessed by [125I]-C5a binding and chemotaxis assays. Inhibition of C5a-induced leukopenia by CCX168 was determined in multiple species. In the Phase 1 program, ex vivo analysis of C5aR coverage on blood neutrophils was performed based on C5a-induced chemotaxis and CD11b expression. A similar PD assay was performed on blood samples from human C5aR knock-in mice dosed orally with CCX168. PK/PD requirements were defined for inhibition of C5aR-mediated neutrophil activation and prevention of anti-MPO IgG induced glomerulonephritis by CCX168 in these mice. Results CCX168 potently blocks [125I]-C5a binding to human C5aR with an IC50 of 0.62 nM, C5a-mediated chemotaxis with an IC50 of 0.25 nM (U937 cells), and C5a-mediated Ca2+ mobilization with an IC50 of 0.2 nM in human neutrophils. In human whole blood, CCX168 blocks C5a-mediated chemotaxis of neutrophils (IC50 1.7 nM) and CD11b upregulation (IC50 4 nM), and also blocks the granulocyte chemotactic activity displayed by human synovial fluid samples (IC50 ∼5 nM). In human C5aR knock-in mice, CCX168 inhibited C5a-induced leukopenia with an ED50 of ∼0.03 mg/kg, and oral dosing to these mice markedly suppressed the induction of glomerulonephritis by anti-MPO IgG. The lowest dose that produced near-maximal therapeutic benefit in this model was 4 mg/kg bid. CCX168 was well tolerated with excellent oral bioavailability and dose-proportional exposure in a Phase 1 study in healthy volunteers, and plasma levels of CCX168 of 197 ng/mL (∼400 nM) were reached after a 100 mg dose, far exceeding the levels required in the anti-MPO mouse model for near-maximal prevention of glomerulonephritis. Twelve hours following a 100 mg single dose of CCX168, there was a 94% reduction in C5a-induced CD11b upregulation on blood neutrophils (ex vivo PD assay). The combined clinical and preclinical PK/PD results indicate a 30 mg bid dosing regimen in humans should result in >90% receptor coverage on human blood leukocytes at all times. Conclusions CCX168 is a highly potent, orally active, antagonist of C5aR which was well tolerated in Phase 1. The PK/PD data from preclinical and human Phase 1 studies indicate that 30 mg CCX168 bid in humans should result in >90% coverage of C5aR in blood at all times, considered optimal for C5aR antagonism in patients with autoimmune diseases such as vasculitis and RA. A Phase 2 trial of CCX168 in patients with AARV is currently underway. Disclosure of Interest J. Powers Employee of: ChemoCentryx, P. Bekker Employee of: ChemoCentryx, D. Dairaghi Employee of: ChemoCentryx, J. Jennette: None Declared, D. Johnson Employee of: ChemoCentryx, M. Leleti Employee of: ChemoCentryx, S. Miao Employee of: ChemoCentryx, L. Seitz Employee of: ChemoCentryx, Y. Wang Employee of: ChemoCentryx, H. Xiao: None Declared, T. Schall Employee of: ChemoCentryx, J. Jaen Employee of: ChemoCentryx

SUMMARY Earlier studies from this laboratory provided evidence for restricted cytokine expression in the T cell population in RA tissues. Specifically, IL-2, IL-4, IL-6 and interferon-gamma (IFN-γ) gene expression levels wore low. The selective chemoattractant and activation effects of chemokines on leucocytes identify them as potentially ideal candidates in mediating selective inflammatory processes in RA. Accordingly, we undertook studies to examine constitutive chemokine gene expression in RA tissues. RANTES, monocyte chemotactic protein-1 (MCP-1) and MIP-1β gene expression was examined in both the T and non-T cell populations in RA peripheral blood (PB), synovial fluid (SF) and synovial tissues (ST). Our results identified elevated levels of both RANTES and MIP-1β gene expression in circulating RA PB and SF T cells. By contrast, MCP-1 expression was virtually absent in RA PB, yet elevated MCP-1 mRNA levels were detected primarily in the non-T cell populations of the SF and ST samples. Histological examination of affected rheumatoid joints revealed extensive RANTES and MIP-1β expression in sites of lymphocyte infiltration and cell proliferation, namely the synovial lining and sublining layers. Fractionation of RA ST patient samples revealed that RANTES expression was restricted to the T cells, whereas MIP-1β expression was detected in both T and non-T fractions. These data suggest that MCP-1, MIP-1β and RANTES may have a central role in the trafficking of reactive molecules involved in immunoregulation and in the inflammatory processes in RA.

We investigated the effect of interleukin-1 (IL-1) activation of human umbilical vein endothelium (HUVE) on human monocyte transendothelial migration induced by chemotactic factors. Monocyte migration across unacdvated endothelium in response to macrophage inflammatory protein-lα (MIP-1α), RANTES, platelet-activating factor (PAF), or monocyte chemoat-tractant protein-1 (MCP-1) was completely inhibited (90%) by monoclonal antibodies (mAbs; 60.3) to CD 18 of the CD11/CD18 complex on the monocyte and partially inhibited (by 75%) in response to C5a. When the HUVE was stimulated with IL-lα (5 h, 0.1 ng/ml), monocyte migration in response to C5a, MIP-lα, RANTES, or PAF was no longer inhibited by mAb to CD18. However, migration was blocked by the combination of mAb to the (α4-integrin (CD49d) chain of very late antigen-4 (CD49d(/CD29) with the mAb to CD18. In contrast to the above stimuli, activation of the HUVE with IL-1α inhibited the transendothelial migration of monocytes in response to MCP-1. mAbs to the adhesion molecules up-regulated on HUVE by IL-1, i.e., E-selectin (CD62E), intercellular adhesion molecule-1 (CD54) or vascular cell adhesion molecule-1 (CD106), did not reverse the inhibitory effect. Transendothelial migration in response to MCP-1 but not to C5a was inhibited by the treatment of monocytes with culture supernatant from IL-1α-stimulated (but not from unstimulated) HUVE. Such supernatant contained chemotactic activity for monocytes, and a mAb to MCP-1 blocked the migration inhibitory effect of IL-1 activation of the HUVE monolayer, as well as the chemotactic activity in the supernatant from IL-1-stimulated HUVE. The inhibitory effect on migration of IL-1-stimulated HUVE was specific for monocytes because polymorphonuclear leukocyte transendothelial migration in response to IL-8 (a related chemokine) was not inhibited by IL-1 activation of HUVE. These results demonstrate that: (1) C5a but not MCP-1, MIP-lα, RANTES, or PAF activates not only a CD 18-dependent but also a VLA-4-dependent mechanism on monocytes, which mediates migration across unstimulated HUVE; (2) IL-1 (tumor necrosis factor-α or lipopolysaccharide) activation of HUVE results in efficient VLA-4 (CD494/CD29)-mediated monocyte transendothelial migration in response to C5a, RANTES, MIP-1α, and PAF, in addition to the CD 18-dependent pathway, but inhibits the response to MCP-1; and (3) this selective inhibition is probably due to MCP-1 production by the activated endothelium monolayer and may be one mechanism of down-regulation by the endothelium of monocyte migration in response to extravascular MCP-1. J. Leukoc. Biol. 58: 71–79,1995.

To understand the selective accumulation of memory T helper lymphocytes and of macrophages in delayed-type hypersensitivity (DTH) granulomas, we studied the in situ production of RANTES, a chemokine initially characterized on the basis of its in vitro chemotactic properties for each of these cell populations. RANTES gene expression was studied by in situ hybridization in 15 human lymph nodes presenting typical DTH lesions related to either sarcoidosis or tuberculosis. A positive signal was detected in all cases. Labeling was specific for the DTH lesions, as very few if any positive cells were detected in the normal residual lymphoid tissue surrounding them or in reactive lymph nodes involved in a B lymphocyte response. RANTES gene expression was associated with the production of the protein, which was detected by immunochemistry in DTH lymph nodes. The morphological characteristics and distribution of positive cells in in situ hybridization and immunochemical experiments indicated that macrophages and endothelial cells, two cell populations not previously reported to produce RANTES, contributed to its production in DTH reactions. The ability of macrophages and endothelial cells to produce RANTES was confirmed by in vitro studies with alveolar macrophages and umbilical vein endothelial cells. In view of the chemotactic properties of RANTES for a limited range of cell populations, these results suggest that RANTES production in DTH granulomas may play a role in the selective accumulation of macrophages and memory T helper lymphocytes characterizing this type of cell-mediated immune reaction, and that macrophages and endothelial cells are involved in this production.

Using a rat model of acute lung inflammation induced by intratracheal instillation of lipopolysaccharide (LPS), we investigated the kinetics of mRNA expression and the potential cellular sources of tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2), interleukin (IL)-1 beta, IL-6, RANTES, and transforming growth factor-beta 1 (TGF-beta 1). By Northern blot analysis, TNF-alpha and MIP-2 mRNAs in total lung tissue increased markedly by 30 min and peaked by 1 h after LPS exposure, whereas expression of IL-1 beta and IL-6 was not detected until 1 h and peaked within 6 h. In contrast, neither RANTES nor TGF-beta 1 mRNA was induced by LPS throughout 72 h, although a basal expression was detected in both saline- and LPS-treated lung tissues. At 1 h after LPS, the bronchoalveolar lavage (BAL) fluid contained about 98% alveolar macrophages (AM), whereas by 6 or 12 h, 88% of BAL cells were polymorphonuclear neutrophils (PMN). Upon extraction of total RNA after separation of AM from PMN in BAL, Northern analysis showed that at 1 h, AM expressed pronounced signals for TNF-alpha, MIP-2, IL-1 beta, and IL-6. At 6 and 12 h, however, while cytokine transcripts decreased in AM, PMN exhibited strong signals for these cytokines. A low basal noninducible signal for TGF-beta 1 but not RANTES was detected in both AM and PMN. Finally, by in situ hybridization techniques, PMN in the lung tissue, particularly those located in the vicinity of the bronchiole and vasculature, were demonstrated to localize MIP-2 mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

We have used a cDNA library enriched for T cell-specific sequences to isolate genes expressed by T cells but not by other cell types. We report here one such gene, designated RANTES, which encodes a novel T cell-specific molecule. The RANTES gene product is predicted to be 10 kDa and, after cleavage of the signal peptide, approximately 8 kDa. Of the 68 residues, 4 are cysteines, and there are no sites for N-linked glycosylation. RANTES is expressed by cultured T cell lines that are Ag specific and growth factor dependent. RANTES expression is inducible in PBL by Ag or mitogen. In CTL, expression of RANTES decreases after stimulation with Ag and growth factors. Interestingly, RANTES was not expressed by any T cell tumor line tested. There is significant homology between the RANTES sequence and several other T cell genes, suggesting that they comprise a previously undescribed family of small T cell molecules.