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Several types of psoriasiform dermatitis are associated with increased IL-36 cytokine activity in the skin. A rare, but severe, psoriasis-like disorder, generalized pustular psoriasis (GPP), is linked to loss-of-function mutations in the gene encoding IL-36RA, an important negative regulator of IL-36 signaling. To understand the effects of IL-36 dysregulation in a mouse model, we studied skin inflammation induced by intradermal injections of preactivated IL-36α. We found the immune cells infiltrating IL-36α–injected mouse skin to be of dramatically different composition than those infiltrating imiquimod-treated skin. The IL-36α–induced leukocyte population comprised nearly equal numbers of CD4+ αβ T cells, neutrophils, and inflammatory dendritic cells, whereas the imiquimod-induced population comprised γδ T cells and neutrophils. Ligands for chemokine receptors CCR6 and CXCR2 are increased in both GPP and IL-36α–treated skin, which led us to test an optimized small-molecule antagonist (CCX624) targeting CCR6 and CXCR2 in the IL-36α model. CCX624 significantly reduced the T cell, neutrophil, and inflammatory dendritic cell infiltrates and was more effective than saturating levels of an anti–IL-17RA mAb at reducing inflammatory symptoms. These findings put CCR6 and CXCR2 forward as novel targets for a mechanistically distinct therapeutic approach for inflammatory skin diseases involving dysregulated IL-36 signaling, such as GPP.

Background and Aims Diabetic nephropathy (DN) is a syndrome characterized by pathological levels of proteinuria, glomerular lesions and reduction in the glomerular filtration rate (GFR) in diabetic patients. Several lines of evidence support a role of CCR2 in the pathogenesis of DN. Recent studies have demonstrated that existing small molecule CCR2 antagonists can be placed in two classes, distinguished by their interaction with one of two distinct binding sites on CCR2: an extracellular site for which antagonists compete directly with native ligands (the orthosteric site), or an intracellular allosteric binding site. To better understand the functional differences between these antagonists we compared an example of each class in a murine model of DN. Our results revealed that the allosteric, but not the orthosteric CCR2 inhibitors ameliorated the proteinuria and improved glomerular histopathology in this model of DN. Method We used db/db mice to model DN. Several structurally distinct CCR2 inhibitors with that bind to either the allosteric (CCR2-RA-[R] and CCX872) or orthosteric (MK-0812 and CCX598) sites were compared. Pharmacokinetic (PK) properties and renal functional parameters were assessed, including trough drug levels and proteinuria (urinary albumin excretion rate UAER; urine albumin creatinine ratio UACR). Histopathology and electron microscopy (EM) were performed to assess any potential tissue-protective effects of the antagonists. Results Both the allosteric inhibitors (CCR2-RA-[R] and CCX872) and the orthosteric inhibitors (MK-0812 and CCX598) were potent CCR2 antagonists with desirable PK in mouse in so far as both classes effectively blocked CCR2-mediated monocyte migration into the peritoneal cavity in the thioglycollate-induced peritonitis model. At comparable drug coverage levels, CCR2-RA-[R] and CCX872 rapidly and significantly reduced UAER/UACR (CCX872: 70 % at day 7 vs vehicle, p Conclusion Allosteric antagonists of CCR2 provide significant and rapid renal protection in the db/db murine model of DN, as evidenced by improvements in renal function and histological parameters, while potent orthosteric CCR2 antagonists were not effective in the model. Our data suggest that targeting the effectiveness of CCR2 antagonists in DN are directly dependent on binding to the allosteric site of CCR2.

Background and Aims We have recently demonstrated that small molecule inhibitors of CCR2 rapidly reduced proteinuria and preserved renal structure in animal models of diabetic nephropathy (DN) and focal segmental glomerular sclerosis (FSGS). Similar beneficial effects of CCR2 inhibition were also observed in DN patients in a large diabetic nephropathy Phase 2 clinical trial. Despite its rapid and robust effects in these kidney diseases, the mechanism by which CCR2 inhibition affords such renal protection remains unclear. CCR2 is well-known to be expressed on cells of the immune system, where it mediates inflammation and immunological functions by controlling the trafficking of monocytes and T cells. However, immune cell expression of CCR2 may not adequately explain the rapid pharmacologic benefits of CCR2 inhibitors in DN and FSGS because these diseases do not exhibit appreciable kidney inflammation. To better understand the protective mechanism(s) of CCR2 inhibition in renal diseases, we have used a variety of in vivo nephropathy models to determine if CCR2 is expressed on kidney parenchymal cells. Our results suggest that a subset of CD45 negative, renal parietal epithelial cells express CCR2. Method To characterize CCR2 expression in the kidney we used CCR2 RFP heterozygous mice (CCR2RFP/+) in which one copy of CCR2 was replaced by red fluorescent protein (RFP), whose expression is under the control of the endogenous CCR2 promoter. This approach provided a highly sensitive method for CCR2 detection by both flow cytometry and immunofluorescence, and compensated for the lack of validated, commercially available anti-CCR2 antibodies. Enriched glomerular cells were obtained by a non-enzymatic disruption of kidneys, followed by centrifugation at low speed (80gs), and sieving through 300µm and 150µm meshes. The Adriamycin (ADR)-nephropathy model with CCR2 RFP heterozygous mice was used to determine CCR2 expression on renal cells. 5/6 nephrectomy models of FSGS, as well as the db/db diabetic nephropathy model were used to access the change of activated parietal epithelial cells and proteinuria, both in the presence and absence of a CCR2 inhibitor in the setting of kidney injury. Results We observed dramatic increases in the number of activated parietal epithelial cells (PECs, identified as CD133+CD24+CD44hi cells by flow cytometry) in all three renal disease models. In the ADR nephropathy model, we found that in 30-40% of activated PEC cells within an enriched population of glomerular cells expressed CCR2/RFP. CD44hi RFP+ increased much more dramatically than CD44hi RFP- cells in response to ADR, and this was highly correlated with proteinuria, suggesting that CCR2 plays an important role in PEC cell activation and kidney pathology in ADR induced kidney injury. Furthermore, we found that blocking CCR2 with the CCR2-specific antagonist CCX872 significantly reduced both proteinuria and the number of activated PECs in the 5/6 nephropathy and db/db diabetic nephropathy models. Conclusion This is the first report that CCR2 is present on a subset of activated renal PECs. This population is markedly upregulated during kidney injury in a variety of murine models of nephropathy. Changes in the abundance CCR2+ activated PECs in the glomerular enriched preparations in response to CCR2 inhibition suggests that CCR2 antagonism may represent a novel approach for the treatment of chronic kidney disease.

Nonalcoholic fatty liver disease (NAFLD) is a common disease, closely associated with obesity and insulin resistance. We investigated the presence of a subset of myeloid cells associated with metabolic disturbance in the liver of patients with NAFLD and a murine model of obesity-induced liver disease. Gene and protein expression in liver and serum was investigated with RT-PCR or ELISA and correlated to clinical disease. Liver-infiltrating immune cells were isolated from normal or diseased human liver for flow cytometric analysis. In animal experiments, mice were fed a high-fat diet (60% of calories from fat) for 16 wk, or high-fat diet with 30% fructose for 32 wk to induce steatohepatitis and fibrosis. A small molecule inhibitor of CC chemokine receptor 2 (CCR2), CCX872, was administered to some mice. A subset of CD11c+CD206+ immune cells was enriched in human liver tissue, and greater infiltration was observed in NAFLD. The presence of CD11c+CD206+ myeloid cells correlated with systemic insulin resistance. CD11c+CD206+ cells expressed high levels of CCR2, and liver CC chemokine ligand 2 (CCL2) expression was increased in nonalcoholic steatohepatitis and correlated with disease activity. In mice, CCR2 inhibition reduced infiltration of liver CD11b+CD11c+F4/80+ monocytes, which are functional homologs of human CD11c+CD206+ cells, and improved liver injury and glycemic control. A role for CCR2/CCL2 in human NAFLD has long been postulated. These data confirm a role for this chemokine/receptor axis, through mediating adipose and hepatic infiltration of myeloid cells. Inhibition of CCR2 improved hepatic inflammation and fibrosis in murine models of NAFLD. These data confirm the rationale for targeting CCR2 to treat NAFLD. NEW & NOTEWORTHY These data show for the first time that CD11c+CD206+ myeloid cells, previously associated with human adipose tissue inflammation, infiltrate into liver tissue in nonalcoholic fatty liver disease. These cells express CCR2. Inhibition of CCR2 in mice inhibits hepatic inflammation caused by a murine homolog of these myeloid cells and improves experimental liver disease.

Introduction: Cancer cells can escape tumor-specific T cell responses via engagement of inhibitory immune checkpoints. PD-L1/PD-1 interaction is one of the major checkpoints that limit effector T cell function against cancer cells, and monoclonal antibodies that block this interaction have been approved as therapies in multiple tumor indications. As a next generation therapy, small molecule inhibitors of PD-L1 potentially have advantageous properties compared to approved monoclonal antibodies, such as better penetration into solid tumors, reduced immunogenicity, lack of Fc-mediated side effects, convenience of oral administration, and lower cost of goods. We therefore embarked on an effort to identify and develop an orally available small molecule capable of targeting PD-L1/PD-1 interactions. Methods: Inhibition of the PD-L1/PD-1 interaction was measured using a binding assay, followed by a cell culture system assessing PD-1 inhibition of T cell receptor (TCR) activation. Human T cell responses were assessed in vitro using the mixed lymphocyte reaction (MLR) assay, and a human peripheral blood mononuclear cell (PBMC)-mediated tumor cell killing assay. For in vivo studies CCX559 was dosed orally in a syngeneic tumor model and in a human tumor cell/PBMC co-implantation model in immune deficient mice. Results: Using structural information and focused medicinal chemistry, we identified CCX559 as a potent inhibitor of PD-L1 interaction with PD-1. CCX559 prevented PD-L1/PD-1 inhibition of TCR signaling in a cell-based reporter assay, increased IFNγ secretion in allogeneic MLR assays, and increased tumor cell killing by human PBMCs. We demonstrated that CCX559 potentially employs multiple mechanisms to inhibit PD-L1, which are distinct from those published for human anti-PD-L1 antibodies. In murine tumor models, orally administered CCX559 reduced tumor growth similarly to a clinically-approved anti-human PD-L1 antibody. Summary: CCX559 is a highly potent, small molecule PD-L1 inhibitor that can be orally administered. CCX559 enhanced primary human T cell activity in vitro and demonstrated anti-tumor efficacy in two murine tumor models. Based on its unique mechanism of PD-L1 inhibition, strong anti-tumor activity, desirable drug properties, and good safety profile, we plan to advance CCX559 into clinical development in the first half of 2021. Citation Format: Chris Li, Marta Vilalta, Linda S. Ertl, Yu Wang, Yibin Zeng, Pingchen Fan, Christopher Lange, Darren McMurtrie, Ju Yang, Rebecca Lui, Ryan Scamp, Vicky Chhina, Alice Kumamoto, Ryan Ong, Ton Dang, Ashton Easterday, Niky Zhao, Shirley Liu, Rajinder Singh, Israel Charo, Kathleen Sullivan, Thomas J. Schall, Penglie Zhang. CCX559 is a potent orally-administered small molecule PD-L1 inhibitor that induces anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1274.

C-C chemokine receptor 2 (CCR2) is a key driver of monocyte/macrophage trafficking to sites of inflammation and has long been considered a target for intervention in autoimmune disease. However, systemic administration of CCR2 antagonists is associated with marked increases in CCL2, a CCR2 ligand, in the blood. This heretofore unexplained phenomenon complicates interpretation of in vivo responses to CCR2 antagonism. We report that CCL2 elevation after pharmacological CCR2 blockade is due to interruption in a balance between CCL2 secretion by a variety of cells and its uptake by constitutive internalization and recycling of CCR2. We observed this phenomenon in response to structurally diverse CCR2 antagonists in wild-type mice, and also found substantially higher CCL2 plasma levels in mice lacking the CCR2 gene. Our findings suggest that CCL2 is cleared from blood in a CCR2-dependent but G protein (Gαi, Gαs or Gαq/11)–independent manner. This constitutive internalization is rapid: on a given monocyte, the entire cell surface CCR2 population is turned over in

Introduction: Antibody-based therapies targeting the Programmed cell Death-1/ Programmed Death-Ligand 1 (PD-1/PD-L1) immune checkpoint axis have achieved great success in cancer immunotherapy in recent years. As a next generation therapy, small molecule inhibitors of PD-1/PD-L1 offer the potential for increased tumor penetration, shorter half-life (to better manage immune related adverse events), and lower cost. We therefore embarked on an effort to identify and develop orally available small molecules capable of targeting PD-L1. Methods: We designed and optimized a number of small molecule PD-L1 inhibitors which potently disrupted the interaction of PD-1 with PD-L1. Active compounds were first profiled by an ELISA assay measuring inhibition of the PD-1/PD-L1 interaction, followed by a functional cell-based reporter assay, mixed lymphocyte reaction (MLR) assay, and human peripheral blood mononuclear cell (PBMC)-mediated tumor cell killing assay. Targeted medicinal chemistry efforts were employed to improve potency and oral bioavailability, and candidate compounds were then evaluated in murine tumor models. Due to the specific reactivity of these compounds to human PD-L1 (but not murine PD-L1), a syngeneic tumor model with murine MC-38 colon tumor cells expressing human PD-L1 (MC38-hPD-L1 tumor model) was used. Results: The optimized PD-L1 inhibitors were highly potent in cell-based reporter assay, the MLR assay and PBMC-mediated tumor cell killing assays. These compounds also possess high oral bioavailability and desirable safety profiles. In the MC38-hPD-L1 tumor model, Lead compounds potently reduced tumor growth similarly to an anti-human PD-L1 antibody, which was used as a positive control for the experiments. The tumor microenvironment analysis by flow cytometry demonstrated that these compounds almost completely occupied human PD-L1 on the tumor cells in vivo, and thus could potently block the interaction of PD-1/PD-L1 and enhance the immune responses against tumor. Summary: We have identified and advanced unique small molecule inhibitors of human PD-L1 by rational design and optimization. Molecules resulting from these efforts exhibited marked inhibition of the PD-1/PD-L1 interaction and signaling in vitro, and potent anti-tumor effects in an animal model. Citation Format: Shijie "Chris" Li, Marta Vilalta, Linda S. Ertl, Yu Wang, Yibin Zeng, Pingchen Fan, Christopher Lange, Darren McMurtrie, Ju Yang, Rebecca Lui, Ryan Ong, Vicky Chhina, Alice Kumamoto, Simon Yau, Ton Dang, Ashton Easterday, Shirley Liu, Rajinder Singh, Israel Charo, Thomas J. Schall, Penglie Zhang. Anti-tumor effect of orally available small molecule PD-L1 inhibitors in a murine model of colon adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5693.

The development of anti-TNFα immunotherapies has led to great advances in quality of life for Crohn’s and colitis patients, and has allowed a reduction in steroid use for these diseases. However, a significant proportion of IBD patients is resistant to anti-TNFα agents, or becomes resistant over time. Preclinical research and clinical trials suggest that CCR9 antagonism can effectively reduce colon inflammation. CCR9 is expressed on a subset of T cells dedicated to intestinal trafficking. Antagonism of this receptor inhibits the accumulation of T cells within inflamed intestinal epithelium and lamina propria. Since anti-TNFα and CCR9 antagonists act through entirely different mechanisms, we asked whether these two approaches might synergize and expand the effectiveness and scope of each therapy. We used a piroxicam-accelerated version of the MDR1a−/− spontaneous colitis model in mice. We have found that combined dosing of piroxicam-fed MDR1a−/− mice with a CCR9 antagonist and an anti-TNFα MAb significantly reverses loss of body weight, diarrhea score and colon inflammation. The combined dosing was more effective than anti-TNFα or CCR9 antagonist alone, as measured by colon length/weight ratio, neutrophil accumulation in the colonic epithelium and reversal of the loss in body weight characteristic of this model. These data suggest that combining a CCR9 antagonist with existing anti-TNFα treatment regimens may extend the benefits of anti-TNFα immunotherapy to a larger class of patients suffering from IBD, and may prolong the effectiveness of anti-TNFα immunotherapy for those patients already undergoing such treatments.

Introduction: Chemokines and their receptors influence many of the hallmark processes in cancer: they act not only on infiltrating leukocytes, but also on fibroblasts, endothelial cells, and directly on some types of tumor cells. C-C chemokine receptor 4 (CCR4) and its ligands have been found to be highly expressed in multiple types of human tumors and are associated with poor prognosis. CCR4 antagonism has been demonstrated to reduce tumor growth in various mouse tumor models. Here we assess small-molecule inhibition of CCR4 as a therapeutic agent to potentiate the effects of checkpoint inhibitors in the CT26 and KCM tumor models. Methods: The subcutaneous CT26 colon cancer model and the orthotopic KCM pancreatic cancer model were used to assess the effects of a CCR4 inhibitor, CCX6239, in combination with anti-CTLA-4 antibody. CT26 cells were implanted into the flanks of 9wk female Balb/c mice. Mice were randomized into study groups based on the tumor sizes on day 6, and dosing of both CCX6239 and anti-CTLA-4 started on day 7. For the orthotopic pancreatic cancer model, KCM cells were implanted directly into the pancreas, and dosing of CCX6239 and anti-CTLA-4 also began on day 7. CCX6239 was dosed orally twice daily at 30mg/kg, and anti-CTLA-4 was dosed IP on days 7, 11 and 15 at 100μg/mouse. Results: Blockade of CCR4 appreciably enhanced the therapeutic effects of anti-CTLA-4 in both models. Combined anti-CTLA-4/CCR4 inhibitor significantly decreased tumor size and increased the proportion of long-term survivors in the CT26 model. The antitumor response was CT26-specific; long-term survivors were resistant to reinoculation with CT26 cells (without further dosing of either drug), but 4T1 breast tumors grew well upon challenge of CT26 survivors. Mice with tumor regression exhibited a high proportion of CD8 T cells that recognized a CT26-specific neoantigen, as illustrated by AH1 peptide-MHC tetramer staining. Although long-term survival has not yet been examined in the KCM model, CCX6239 alone significantly reduced tumor burden in 3 independent studies. Anti-CTLA-4 alone provided substantial inhibition of tumor growth in this model, which was further enhanced by CCX6239. Summary: A specific CCR4 small-molecule inhibitor enhanced the effectiveness of anti-CTLA-4 in 2 preclinical models, suggesting that targeting CCR4 may be a viable clinical approach for combinatorial treatment with checkpoint blockers. Citation Format: Shijie “Chris” Li, James J. Campbell, Linda S. Ertl, Zhenhua Miao, Vicky Chhina, Alice Kumamoto, Simon Yau, Ton Dang, Penglie Zhang, Thomas J. Schall, Rajinder Singh. C-C chemokine receptor 4 (CCR4) antagonism enhances the effectiveness of checkpoint Inhibition in mouse tumor models [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B08.

mAbs that neutralize IL-17 or its receptor have proven efficacious in treating moderate-to-severe psoriasis, confirming IL-17 as an important driver of this disease. In mice, a rare population of T cells, γδT17 cells, appears to be a dominant source of IL-17 in experimental psoriasis. These cells traffic between lymph nodes and the skin, and are identified by their coexpression of the TCR variable regions γ4 and δ4. These cells are homologous to the Vγ9Vδ2 T cell population identified in human psoriatic plaques. In this study we report that a potent and specific small molecule antagonist of the CCR6 chemokine receptor, CCX2553, was efficacious in reducing multiple aspects of psoriasis in two different murine models of the disease. Administration of CCX2553 ameliorated skin inflammation in both the IL-23–induced ear swelling model and the topical imiquimod model, and significantly reduced the number of γδT17 cells in inflamed skin. γδT17 cells were greatly reduced in imiquimod-treated skin of CCR6−/− mice, but adoptively transferred wild-type (CCR6+/+) γδT17 cells homed normally to the skin of imiquimod-treated CCR6−/− mice. Our data suggest that γδT17 cells are completely dependent on CCR6 for homing to psoriasiform skin. Thus, CCR6 may constitute a novel target for a mechanistically distinct therapeutic approach to treating psoriasis.

Necrotizing and crescentic GN (NCGN) with a paucity of glomerular immunoglobulin deposits is associated with ANCA. The most common ANCA target antigens are myeloperoxidase (MPO) and proteinase 3. In a manner that requires activation of the alternative complement pathway, passive transfer of antibodies to mouse MPO (anti-MPO) induces a mouse model of ANCA NCGN that closely mimics human disease. Here, we confirm the importance of C5aR/CD88 in the mediation of anti-MPO–induced NCGN and report that C6 is not required. We further demonstrate that deficiency of C5a-like receptor (C5L2) has the reverse effect of C5aR/CD88 deficiency and results in more severe disease, indicating that C5aR/CD88 engagement enhances inflammation and C5L2 engagement suppresses inflammation. Oral administration of CCX168, a small molecule antagonist of human C5aR/CD88, ameliorated anti-MPO–induced NCGN in mice expressing human C5aR/CD88. These observations suggest that blockade of C5aR/CD88 might have therapeutic benefit in patients with ANCA-associated vasculitis and GN.

Pancreatic ductal adenocarcinoma (PDAC) cells (PCC) have an exceptional propensity to metastasize early into intratumoral, chemokine-secreting nerves. However, we hypothesized the opposite process, that precancerous pancreatic cells secrete chemokines that chemoattract Schwann cells (SC) of nerves and thus induce ready-to-use routes of dissemination in early carcinogenesis. Here we show a peculiar role for the chemokine CXCL12 secreted in early PDAC and for its receptors CXCR4/CXCR7 on SC in the initiation of neural invasion in the cancer precursor stage and the resulting delay in the onset of PDAC-associated pain. SC exhibited cancer- or hypoxia-induced CXCR4/CXCR7 expression in vivo and in vitro and migrated toward CXCL12-expressing PCC. Glia-specific depletion of CXCR4/CXCR7 in mice abrogated the chemoattraction of SC to PCC. PDAC mice with pancreas-specific CXCL12 depletion exhibited diminished SC chemoattraction to pancreatic intraepithelial neoplasia and increased abdominal hypersensitivity caused by augmented spinal astroglial and microglial activity. In PDAC patients, reduced CXCR4/CXCR7 expression in nerves correlated with increased pain. Mechanistically, upon CXCL12 exposure, SC down-regulated the expression of several pain-associated targets. Therefore, PDAC-derived CXCL12 seems to induce tumor infiltration by SC during early carcinogenesis and to attenuate pain, possibly resulting in delayed diagnosis in PDAC.

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.

Mouse CT26 colorectal tumors are heavily infiltrated by tumor-specific CD8 T cells, but nevertheless grow rapidly in Balb/c mice. These tumors are partially responsive to anti-PD-1 (α-PD-1) monoclonal antibody therapy, which suggests that an active suppression of the tumor-specific cytotoxic T cells exists in the untreated tumor. As chemokine receptor 2 (CCR2) is expressed by a potentially suppressive leukocyte subset within these tumors (monocytic myeloid derived suppressor cells aka M-MDSCs), we aimed to test whether CCR2 blockade could enhance the anti-tumor effects of α-PD-1. We have found that the therapeutic effects of α-PD-1 therapy are appreciably enhanced by specific blockade of CCR2 via a small molecule antagonist. This combined α-PD-1/CCR2i approach significantly decreases overall tumor size and increases the proportion of long-term survivors, with more than 50% of the mice (up to 73%) showing complete regression of a previously established tumor. The effects of this combined therapy are dependent on the presence of CD8+ T cells, as tumors do not respond to the therapy in CD8-depleted mice. The anti-CT26 tumor response is specific: long term survivors are resistant to re-inoculation with the CT26 tumor (even without further dosing of either drug) but are not resistant to the 4T1 breast tumor. CCR2 antagonism alters the tumor microenvironment by reducing the number of M-MDSC per gram of tumor (a CCR2hi population phenotypically defined as CD11b+/Ly6G-/Ly6Chi). Reduction in tumor size is inversely proportional to the ratio of CD8 T cells to M-MDSC. These data are consistent with a hypothesis that CCR2 antagonism enhances α-PD-1 therapy by preventing M-MDSC from accumulating within the tumor, thus reducing their suppressive effects on cytotoxic T cells. Citation Format: James J. Campbell, Christine Janson, Linda Ertl, Chris Li, Zhenhua Miao, Antoni Krasinski, Rebecca Lui, Venkat Mali, Jeffrey McMahon, Yibin Zheng, Yu Wang, Xiaoping Zang, Vicky Chhina, Marta Vilalta, Alice Kumamoto, Ton Dang, Shirley Liu, Simon Yao, Penglie Zhang, Thomas J. Schall, Rajinder Singh. Inhibition of chemokine receptor 2 (CCR2) with a small molecule antagonist enhances the effectiveness of checkpoint inhibition by altering the tumor microenvironment in mouse colorectal tumors: Reducing tumor size and increasing long term survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1745.

Generalized pustular psoriasis (GPP) is a rare inflammatory skin disorder with an etiology distinct from common plaque psoriasis. GPP patients often do not respond to therapeutic agents used routinely to treat plaque psoriasis. Genetic evidence suggests that GPP arises from dysfunction in the IL36/IL36Ra/IL36R signaling axis, and many aspects of GPP can be re-created in the mouse by intradermal (ID) injection of pre-activated IL36. We have used this ID-IL36 model to study the leukocyte populations that accumulate within GPP skin. In a previous study, we reported that a small molecule CCR6 antagonist, CCX2553, ameliorates inflammation in an imiquimod-induced model of plaque psoriasis, and we now find this is to be the case in the GPP model as well. However, in contrast to the imiquimod model, a conventional CD4+ αβ T cell population accumulates in the ID-IL36-treated skin instead of the characteristic γδT17 population found in imiquimod-treated skin. CCX2553 reduces IL36-induced inflammation while preventing skin accumulation of this CD4+ αβT cell population. Thus, although disparate T cell populations are associated with each model, inflammation is ameliorated by CCR6 antagonism in both models. These findings suggest that CCR6 may constitute a novel target for a mechanistically distinct approach towards GPP therapy.

Pancreatic cancer is an aggressive malignancy with a 5 year survival rate of less than five percent. The predominant immune cells infiltrating the tumor microenvironment are monocytes/macrophages, which are reported to support tumor growth by suppressing host immune responses to the tumor. Recruitment of monocytes to various tissues, including tumors, is dependent upon activation of the chemokine receptor CCR2 by one or more of the chemokines CCL2, CCL8 and CCL13. In preclinical and clinical studies, inhibition of CCR2 in pancreatic cancer has shown to decrease tumor progression by blocking recruitment and accumulation of monocytes/macrophages in the tumor microenvironment. Analysis of human pancreatic tumors revealed elevation of both CCL2 and CSF1, which recruit monocytes, as well as the monocyte marker CD14, in advanced pancreatic cancers. Current immunotherapy using checkpoint inhibitors are effective in some tumors, but lack efficacy in immune insensitive cancers, including pancreatic cancer. Here, we report that the inhibition of CCR2 using a small molecule antagonist potentiates anti-PD-1 immunotherapy in a syngeneic, orthotropic mouse model of pancreatic cancer. Our data reveal that blocking CCR2 decreases tumor burden by blocking monocyte infiltration and creating a microenvironment more favorable for CD8 T cells activity, and provide a mechanistic rationale for investigating the combination of a CCR2 antagonist and an immune checkpoint inhibitor in pancreatic cancer. Citation Format: Christine Janson, Heiyoun Jung, Linda Ertl, Shirley Liu, Ton Dang, Yibin Zeng, Antoni Krasinski, Jeff McMahon, Penglie Zhang, Israel Charo, Rajinder Singh, Thomas J. Schall. Inhibition of CCR2 potentiates checkpoint inhibitor immunotherapy in murine model of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5655. doi:10.1158/1538-7445.AM2017-5655

While it has long been established that the chemokine receptor CCR9 and its ligand CCL25 are essential for the movement of leukocytes into the small intestine and the development of small-intestinal inflammation, the role of this chemokine-receptor pair in colonic inflammation is not clear. Toward this end, we compared colonic CCL25 protein levels in healthy individuals to those in patients with ulcerative colitis. In addition, we determined the effect of CCR9 pharmacological inhibition in themdr1a−/−mouse model of ulcerative colitis. Colon samples from patients with ulcerative colitis had significantly higher levels of CCL25 protein compared to healthy controls, a finding mirrored in themdr1a−/−mice. In themdr1a−/−mice, CCR9 antagonists significantly decreased the extent of wasting and colonic remodeling and reduced the levels of inflammatory cytokines in the colon. These findings indicate that the CCR9:CCL25 pair plays a causative role in ulcerative colitis and suggest that CCR9 antagonists will provide a therapeutic benefit in patients with colonic inflammation.

Chemokine (C-C motif) receptor 2 (CCR2) is central for the migration of monocytes into inflamed tissues. The novel CCR2 antagonist CCX140-B, which is currently in two separate phase 2 clinical trials in diabetic nephropathy, has recently been shown to reduce hemoglobin A1c and fasting blood glucose levels in type 2 diabetics. In this report, we describe the effects of this compound on glycemic and renal function parameters in diabetic mice. Since CCX140-B has a low affinity for mouse CCR2, transgenic human CCR2 knockin mice were generated and rendered diabetic with either a high-fat diet (diet-induced obesity) or by deletion of the leptin receptor gene ( db/ db). CCX140-B treatment in both models resulted in decreased albuminuria, which was associated with decreased glomerular hypertrophy and increased podocyte density. Moreover, treatment of diet-induced obese mice with CCX140-B resulted in decreased levels of fasting blood glucose and insulin, normalization of homeostatic model assessment of insulin resistance values, and decreased numbers of adipose tissue inflammatory macrophages. Unlike other CCR2 antagonists, CCX140-B had no effect on plasma levels of the CCR2 ligand CCL2 or on the numbers of blood monocytes. These results support the ongoing evaluation of this molecule in diabetic subjects with impaired renal function.

Objective CCR2 inhibition has produced promising experimental and clinical anti-hyperglycemic effects. These results support the thesis that insulin resistance and Type 2 diabetes (T2D) are associated with chronic unresolved inflammation. The aim of this study was to provide a broad analysis of the various physiological changes occurring in mouse models of T2D in connection with pharmacological CCR2 inhibition. Materials/Methods A mouse-active chemical analogue of the clinical candidate CCX140-B was tested in diet-induced obese (DIO) mice and db/db mice. Measurements included: adipose tissue inflammatory macrophage counts; peripheral blood glucose levels at steady-state and after glucose and insulin challenges; peripheral blood insulin and adiponectin levels; 24-h urine output and urinary glucose levels; pancreatic islet number and size; hepatic triglyceride and glycogen content; and hepatic glucose-6-phosphatase levels. Results In DIO mice, the CCR2 antagonist completely blocked the recruitment of inflammatory macrophages to visceral adipose tissue. The mice exhibited reduced hyperglycemia and insulinemia, improved insulin sensitivity, increased circulating adiponectin levels, decreased pancreatic islet size and increased islet number. It also reduced urine output, glucose excretion, hepatic glycogen and triglyceride content and glucose 6-phosphatase levels. Similar effects were observed in the db/db diabetic mice. Conclusions These data indicate that pharmacological inhibition of CCR2 in models of T2D can reduce inflammation in adipose tissue, alter hepatic metabolism and ameliorate multiple diabetic parameters. These mechanisms may contribute to the promising anti-diabetic effects seen in humans with at least one CCR2 antagonist.

Pancreatic cancer is an aggressive malignancy with 5 year survival rate of less than 5 percent. The predominant immune cells infiltrating the tumor microenvironment are monocytes/macrophages, which are reported to support tumor growth by suppressing host immune responses to the tumor. Recruitment of monocytes to various tissues, including tumors, is dependent upon activation of the chemokine receptor CCR2 by one or more of the chemokines CCL2, CCL8 and CCL13. In preclinical and clinical studies, inhibition of CCR2 in pancreatic cancer has shown to decrease tumor progression by blocking recruitment and accumulation of monocytes/macrophages in the tumor microenvironment. Analysis of the TCGA genomic database of human pancreatic tumors revealed elevation of both CCL2 and CSF1, which recruit monocytes, as well as the monocyte marker CD14, in advanced pancreatic cancers. Current immunotherapy using checkpoint inhibitors are effective in some tumors, but lack efficacy in immune insensitive cancers, including pancreatic cancer. Here, we report that the inhibition of CCR2 using small molecule antagonist potentiates anti-PD-1/PD-L1 immunotherapy in a syngeneic, orthotopic mouse model of pancreatic cancer. The KCKO pancreatic cancer cell line, which harbors a K-ras mutation, was implanted into the tail of the pancreas, and a small molecule CCR2 antagonist was administered after a stable tumor mass had formed. Tumor weight correlated well with the per cent of circulating monocytes in the peripheral blood, and the CCR2 antagonist significantly decreased the blood monocyte count. Similar to the human tumor stroma, the KCKO mouse tumors were infiltrated with monocytes and macrophages, and CCR2 antagonist treatment decreased the infiltration of monocyte/ macrophage. PD-L1 expressions are found in the human pancreatic tumor microenvironment, but treatment of patients with anti-PD-1 has not been found to have efficacy. Similarly, anti-PD-1 treatment alone was not effective in the murine KCKO model, but combination treatment with a CCR2 antagonist resulted in significantly smaller tumors. Moreover, this effect was completely reversed by depleting CD8 T cells, suggesting that by blocking monocyte/macrophage recruitment, the CCR2 antagonist relieved suppression of the CD8 T cells. We confirmed this hypothesis by demonstrating that cells from the KCKO tumor microenvironment inhibited CD3/CD28-induced proliferation of CD8 T cells in culture, but that this inhibition was not present when the mice had received the CCR2 antagonist. Taken together, these data reveal that blocking CCR2 decreases tumor burden by blocking monocyte infiltration and creating a microenvironment more favorable for CD8 T cells activity, and provide a mechanistic rationale for investigating the combination of a CCR2 antagonist and an immune checkpoint inhibitor in pancreatic cancer. Citation Format: Heiyoun Jung, Linda Ertl, Christine Janson, Thomas Schall, Israel Charo. Inhibition of CCR2 potentiates the checkpoint inhibitor immunotherapy in pancreatic cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A107.

Trafficking and expansion of myeloid derived suppressor cells (MDSCs) plays a major role in the immune suppression of tumors. MDSCs express chemokine receptors which likely mediate their recruitment to the tumor microenvironment. Suppression of T cells is also mediated by the interaction of programmed death-1 (PD-1) and its ligands which are abundantly expressed in cancer cells and immune infiltrates, including MDSCs. Here, we show that targeting both pathways through administration of a small molecule chemokine receptor antagonist (CCX9588, which blocks CCR1) and a PD-L1 monoclonal antibody (mAb) significantly reduced tumor burden in mice who received orthotopic transplants of 4T1 cells, a cell line used to model triple negative breast cancer. Primary tumor growth was modestly reduced by either agent (PD-L1 mAb or CCR1 inhibitor) alone, but the combination of CCR1 inhibitor plus PD-L1 mAb resulted in significantly reduced tumor progression as compared to either of the single agents. Furthermore, lung metastasis was also significantly reduced by CCR1 antagonist and combination treatment. Orthotropic 4T1 cell engraftment induced robust expansion of CD11b+Ly6Ghi Ly6Chi MDSCs, a subpopulation of which express CCR1. Analysis of the tumor infiltrating cells revealed that CCX9588 significantly reduced the number of MDSCs and increased CD8 T cells infiltration in primary tumors, suggesting that CCR1 blockade of MDSC trafficking in combination with PD-L1 mAb translates into reduced tumor burden, possibly through increased CD8 T cell response against the tumor. Analysis of human breast cancer patient samples from The Cancer Genome Atlas (TCGA) database revealed that the CCR1 ligands, MCP-7 (CCL7) and RANTES (CCL5) are present at significantly higher levels in breast cancers as compared to normal breast tissue. Interestingly, ligands for CCR1 and PD-1 are significantly higher in triple negative breast cancer samples than in the other breast cancer subtypes. These data are the first to show that CCR1 chemokine receptor antagonists can act synergistically with PDL-1 inhibitors, and suggest a novel approach for potentiating the activity of immune cell checkpoint inhibitors in one of the most aggressive forms of breast cancer. Citation Format: Heiyoun Jung, Linda Ertl, Karen Ebsworth, Christine Janson, Penglie Zhang, Punna Sreenivas, Thomas Schall, Israel Charo. Combination therapy of chemokine receptor inhibition plus PD-L1 blockade potentiates antitumor effects in a murine model of breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 564.

The following manuscript was published as a Fast Forward article on February 29, 2012: Sullivan TJ, Dairaghi DJ, Krasinski A, Miao Z, Wang Y, Zhao BN, Baumgart T, Berahovich R, Ertl LS, Pennell A, Seitz L, Miao S, Ungashe S, Wei Z, Johnson D, Boring L, Tsou C-L, Charo IF, Bekker P, Schall TJ, and Jaen JC, Characterization of CCX140-B, an orally bioavailable antagonist of the CCR2 chemokine receptor, for the treatment of type 2 diabetes and associated complications. J Pharmacol Exp Ther jpet.111.190918; doi:10.1124/jpet.111.190918 It was later found that the chemical identity of a compound cited in the article, CCX140-B, was not sufficiently disclosed. The authors are unable, at this time, to provide the chemical identity of CCX140-B in accordance with the editorial policies of The Journal of Pharmacology and Experimental Therapeutics. As a result, the authors have voluntarily withdrawn this manuscript from publication. We apologize for any inconvenience this may cause JPET's readers.

The chemokine system represents a diverse group of G protein-coupled receptors responsible for orchestrating cell recruitment under both homeostatic and inflammatory conditions. Chemokine receptor 9 (CCR9) is a chemokine receptor known to be central for migration of immune cells into the intestine. Its only ligand, CCL25, is expressed at the mucosal surface of the intestine and is known to be elevated in intestinal inflammation. To date, there are no reports of small-molecule antagonists targeting CCR9. We report, for the first time, the discovery of a small molecule, CCX282-B, which is an orally bioavailable, selective, and potent antagonist of human CCR9. CCX282-B inhibited CCR9-mediated Ca(2+) mobilization and chemotaxis on Molt-4 cells with IC(50) values of 5.4 and 3.4 nM, respectively. In the presence of 100% human serum, CCX282-B inhibited CCR9-mediated chemotaxis with an IC(50) of 33 nM, and the addition of α1-acid glycoprotein did not affect its potency. CCX282-B inhibited chemotaxis of primary CCR9-expressing cells to CCL25 with an IC(50) of 6.8 nM. CCX282-B was an equipotent inhibitor of CCL25-directed chemotaxis of both splice forms of CCR9 (CCR9A and CCR9B) with IC(50) values of 2.8 and 2.6 nM, respectively. CCX282-B also inhibited mouse and rat CCR9-mediated chemotaxis. Inhibition of CCR9 with CCX282-B results in normalization of Crohn's disease such as histopathology associated with the TNF(ΔARE) mice. Analysis of the plasma level of drug associated with this improvement provides an understanding of the pharmacokinetic/pharmacodynamic relationship for CCR9 antagonists in the treatment of intestinal inflammation.

Background Non-alcoholic fatty liver disease (NAFLD) is a common condition, in which hepatic steatosis is present in up to a third of individuals, whereas the more severe non-alcoholic steatohepatitis (NASH) is seen in about 10%. NAFLD is closely associated with the metabolic syndrome: up to 75% of people with diabetes mellitus, and almost all morbidly obese individuals, have NALFD. Obesity-associated macrophage infiltration of adipose and hepatic tissue is mediated by C-C chemokine receptor type 2 (CCR2), where CCR2+ CD11b+ F4/80+ macrophages contribute to chronic inflammation and insulin resistance. We investigated the efficacy of a small molecule inhibitor of CCR2, CCX872, which is in phase 1 clinical development, for treatment of obesity-associated hepatic steatohepatitis in mice. Methods C57BL/6 mice aged 6 weeks were fed a high-fat diet for 16 weeks. After 8 weeks on this diet, mice were treated with CCX872 (ChemoCentryx, Mountain View, CA, USA) 30 mg/kg once daily or vehicle, both administered subcutaneously. Liver injury was assessed with serum alanine aminotransferase concentration, liver triglyceride content, and flow cytometry of infiltrating cells. Adipose tissue macrophage infiltration was assessed with flow cytometry. Insulin sensitivity was measured by glucose and insulin challenges. Groups were compared with two-tailed Student's t tests. Findings Mice treated with CCX872 had better insulin sensitivity (p vs 532·8 [353·6], p=0·028). Hepatic triglyceride accumulation was significantly lower in CCX872-treated mice (mean 159·9 mg/g [SD 2·6] vs 296·6 [98·5], p vs 16·0 [1·9], p=0·03; mean fluorescence intensity [MFI] 6919 [SD 107·7] vs 7365 [162·9], p=0·04) and in omental adipose tissue (9·2% of CD45+ cells [1·9] vs 21·8 [3·7], p=0·02; MFI 6212 [469·2] vs 7952 [379·5], p=0·02). Interpretation Treatment with a clinical-stage small molecule inhibitor of CCR2 improved steatohepatitis and insulin sensitivity in mice kept on a high-fat diet. Funding UK Medical Research Council.

e13580 Background: Glioblastoma (GBM) is the most common form of malignant brain cancer. Despite aggressive therapy, consisting of radiotherapy, surgical resection, and chemotherapeutic treatment, mean survival from time of diagnosis remains little more than one year. The chemokine receptor CXCR7 is highly expressed on both human glioma cells and tumor-associated vasculature, and may play a key role in tumor growth and survival. Methods: The in vitro activity of the CXCR7 antagonist CCX662 was determined using radioligand binding assays and trans-endothelial migration assays. The therapeutic effects of CCX662 were investigated in the ENU-induced rat model of GBM in combination with radiotherapy. Results: CCX662 is a highly potent and selective small molecule inhibitor of CXCR7. CCX662 inhibits the binding of 125I-CXCL12 to CXCR7 with an IC50 of 9 nM in buffer, and displays minimal serum shift with an IC50 of 18 nM in 100% human serum. CCX662 inhibits the CXCR4-directed trans-endothelial migration of CXCR4+/CXCR7+ NC37 cells towards CXCL12 (SDF1) with an IC50 of 106 nM. CCX662 also potently inhibits binding of 125I-CXCL12 to rat CXCR7 in the presence of 100% rat serum with an IC50 of 14 nM. In vivo inhibition of CXCR7 with CCX662, in concert with radiotherapy, results in a significant extension of survival time in the ENU-induced rat model of GBM. The median survival time for rats (reflective of GBM tumor progression) treated with the combination of irradiation and CCX662 was 234 days compared with 160 days for untreated rats (p 10 and 25-fold, respectively) safety margin relative to highly effective levels of the drug. Conclusions: CCX662 is a highly selective and potent small molecule inhibitor of CXCR7 with profound, therapeutic benefit in an aggressive rodent model of GBM. These data indicate that inhibition of CXCR7, using CCX662, may be a promising strategy for the treatment of glioblastoma.

Meeting abstracts Trafficking and expansion of myeloid derived suppressor cells (MDSCs) plays a major role in immune suppression of tumor. MDSCs express chemokine receptors which likely mediate their recruitment to the tumor microenvironment. Suppression of T cells is also mediated by the