CCR5 Inhibition Research Articles

Abstract 2350: Vaccination enhances anti-tumor immunity in ovarian cancer following repolarization of the tumor microenvironment with CCR2 blockade

Abstract Introduction: Ovarian cancer (OC) expresses the tumor associated antigen mesothelin and contains a relative abundance of T-cells. However, ovarian cancer is also infiltrated by immunosuppressive tumor associated macrophages (TAM) that dominate the tumor microenvironment (TME). The CCL2/CCR2 chemokine axis is co-opted by various human malignancies to facilitate the recruitment of bone marrow (BM) derived inflammatory monocytes (IM) to the TME where they become immunosuppressive TAMs. Herein, we explore the rationale for combination of a CCR2 inhibitor (CCR2i) with a mesothelin peptide vaccine. Methods: Monocyte counts were obtained from preoperative CBCs under IRB approval. Mice were vaccinated with a dual eight-mer peptide (50 nM/vaccination) on days 0 and 7 with an irradiated peptide pulsed dendritic cell boost on day 14. Mice were challenged with 4 million syngeneic OC cells (ID8) on day 15. CCR2 inhibitor (Tocris) and CCR2 KO mice were used. Results: Preoperative monocyte counts of human ovarian cancer patients were stratified into low (>1 SD below mean), mid (within 1 SD of mean), and high (>1 SD above the mean) groups. Patients with a high monocyte count (n = 15) had a significantly decreased median survival of 1.2 years compared to 4.8 years in the low monocyte group (n = 15). The mid group (n = 69) had a median survival of 3.5 years (p 0.001). The hazard ratio between the low and high groups was 0.24 (0.05-0.39). Flow cytometry of peripheral blood from these patients demonstrated that the majority of these monocytes were CCR2+ inflammatory monocytes. Human OC overexpresses CCL2 compared to normal ovarian tissue and analysis of the TME from resected human OC patients revealed an abundance of CCR2+ TAM, which greatly outnumbered tumor infiltrating lymphocytes (TIL). In a murine ID8 tumor model, which recapitulates features of human OC, CCR2i prevented IM egress from the bone marrow with a resultant decrease in TAM at the primary tumor site. Furthermore, there was an increase in TIL infiltrate following CCR2 blockade. Addition of vaccine to CCR2i caused an improvement of effector to suppressor ratio and prolonged survival compared to vaccine (p = 0.02) or CCR2i alone (p = 0.02) and control (p<0.0001). Conclusion: Thus far vaccination has not provided durable patient responses in OC. Therapies targeting the immunosuppressive TME are an attractive treatment modality to enhance vaccination and facilitate anti-tumor immunity in OC. Citation Format: Darren Cullinan, Pratibha Binder, Timothy Nywening, Ivy Wilkinson-Ryan, Brian Belt, Peter Goedegebuure, David Linehan, Matthew Powell, William Hawkins. Vaccination enhances anti-tumor immunity in ovarian cancer following repolarization of the tumor microenvironment with CCR2 blockade. [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 2350.

Abstract 564: Combination therapy of chemokine receptor inhibition plus PD-L1 blockade potentiates antitumor effects in a murine model of breast cancer

Abstract 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.

Abstract 1449: In vivo targeted silencing of CCR1 and CCR5 repolarizes pro-tumoral myeloid cells in retinoblastoma positive neutrophils with a strong anti-tumor activity

Abstract Introduction: Depending on their polarization, myeloid cells can either promote or restrain tumor progression. By secreting myelopoietic factors tumours promote myeloid cells polarization toward a phenotype that provides immune protection and that stimulate neoplastic cells growth, invasiveness, and metastasis. Chemokines and their cognate receptors are thought to play a key role in myeloid cell trafficking, however, their study in vivo is hindered by their signal redundancy and integration. Methods: By taking advantage of a newly developed nanoplatform that allows the in vivo preferential transfection of tumor infiltrating myeloid cells (TIMC, aka Myeloid derived suppressor cells, MDSC, and tumor associated macrophages, TAM), we evaluated the effect of CCR-1, CCR-2, CCR-4, CCR-5 and/or CCR-7 silencing on TAMC trafficking, phenotype, and function. Results: While CCR-4 and -7 targeted silencing did not affect tumor progression, simultaneous silencing of CCR-1 and CCR-5 significantly restrained tumor growth and greatly modifies tumor micro-environment. Mechanistic in vivo and in vitro analysis surprisingly indicate that myeloid cell trafficking was not altered whereas myeloid cell polarization was. In particular, CCR-1 and CCR-5 blockade restrained MDSC differentiation and promote the accumulation of retinoblastoma positive, neutrophil-like cells with a strong tumoricidal activity. Discussion: Our data indicate that CCR-1 and CCR-5 engagement (most likely via CCL-3 and CCL-4) is necessary for the pro-tumoral polarization of myeloid cells in cancer. Our findings not only improve our understanding of myeloid cells differentiation in cancer but, also, strongly suggest that targeted inhibition of CCR1 and CCR5 on TIMC can be a new and effective anti-tumor immune-therapeutic strategy that convert pro-tumoral MDSCs into tumoricidal neutrophils. Citation Format: Serena Zilio, Jennifer Vella, Adriana De La Fuente, Vincenzo Bronte, Emilia Mazza, Silvio Bicciato, Paolo Serafini. In vivo targeted silencing of CCR1 and CCR5 repolarizes pro-tumoral myeloid cells in retinoblastoma positive neutrophils with a strong anti-tumor activity. [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 1449.

A Subset of Patients with Acute Myeloid Leukemia Has Leukemia Cells Characterized by Chemokine Responsiveness and Altered Expression of Transcriptional as well as Angiogenic Regulators.

Acute myeloid leukemia (AML) is an aggressive and heterogeneous bone marrow malignancy, the only curative treatment being intensive chemotherapy eventually in combination with allogeneic stem cell transplantation. Both the AML and their neighboring stromal cells show constitutive chemokine release, but chemokines seem to function as regulators of AML cell proliferation only for a subset of patients. Chemokine targeting is therefore considered not only for immunosuppression in allotransplanted patients but also as a possible antileukemic strategy in combination with intensive chemotherapy or as part of disease-stabilizing treatment at least for the subset of patients with chemokine-responsive AML cells. In this study, we characterized more in detail the leukemia cell phenotype of the chemokine-responsive patients. We investigated primary AML cells derived from 79 unselected patients. Standardized in vitro suspension cultures were used to investigate AML cell proliferation, and global gene expression profiles were compared for chemokine responders and non-responders identified through the proliferation assays. CCL28-induced growth modulation was used as marker of chemokine responsiveness, and 38 patients were then classified as chemokine-responsive. The effects of exogenous CCL28 (growth inhibition/enhancement/no effect) thus differed among patients and was also dependent on the presence of exogenous hematopoietic growth factors as well as constitutive AML cell cytokine release. The effect of CCR1 inhibition in the presence of chemokine-secreting mesenchymal stem cells also differed among patients. Chemokine-responsive AML cells showed altered expression of genes important for (i) epigenetic transcriptional regulation, particularly lysine acetylation; (ii) helicase activity, especially DExD/H RNA helicases; and (iii) angioregulatory proteins important for integrin binding. Thus, chemokine responsiveness is part of a complex AML cell phenotype with regard to extracellular communication and transcriptional regulation. Chemokine targeting in chemokine-responsive patients may thereby alter AML cell trafficking and increase their susceptibility toward antileukemic treatment, e.g., conventional chemotherapy or targeting of other phenotypic characteristics of the chemokine-responsive cells.

Centrality of bone marrow in the severity of gadolinium-based contrast-induced systemic fibrosis.

Systemic fibrosis can be induced in humans with gadolinium-based contrast, and cumulative doses correlate with severity. Bone marrow-derived fibrocytes accumulate in the dermis. Whether target organs liberate chemokines to recruit these fibrocytes or whether fibrocytes are stimulated to home to the affected tissue is unknown. Transgenic (tagged) donor rats were treated with gadolinium-based contrast. Bone marrow was obtained from diseased animals and age-matched controls. Rats with subtotal nephrectomies were lethally irradiated and underwent salvage transplantation with either the contrast-naïve or contrast-exposed bone marrow. Groups were randomly assigned to control or contrast treatment. Contrast treatment led to dermal fibrosis, and this was exacerbated in recipients of contrast-exposed marrow. Fibronectin, C-C chemokine receptors (CCRs)2 and 7, and oxidative stress were all increased in skin from contrast-treated animals-all parameters more severe in recipients of contrast-treated animals. The respective ligands, monocyte chemoattractant protein and C-C motif ligand 19, were both elevated in skin from contrast-treated animals. Coadministration of gadolinium-based contrast and a CCR2 inhibitor reduced the severity of skin disease as well as dermal cellularity. The functional role of chemokines in the effects of gadolinium-based contrast was further confirmed in in situ coculture studies using neutralizing CCR2 antibodies. These data implicate dermal liberation of specific chemokines in the recruitment of circulating bone marrow-derived cells. The disease is augmented by bone marrow exposure to contrast, which explains why multiple exposures correlate with severity.-Drel, V. R., Tan, C., Barnes, J. L., Gorin, Y., Lee, D.-Y., Wagner, B. Centrality of bone marrow in the severity of gadolinium-based contrast-induced systemic fibrosis.

The inhibition of CCR2 to modify the microenvironment in pancreatic cancer mouse model and to support the profiling of the CCR2 inhibitor CCX872-B in patients.

e15743Background: The CCL2/CCR2 axis regulates the recruitment of monocytes to sites of inflammation. We implanted a murine K-Ras+ pancreatic cancer cell line into mice to determine if inhibition o...

Targeting tumour-associated macrophages with CCR2 inhibition in combination with FOLFIRINOX in patients with borderline resectable and locally advanced pancreatic cancer: a single-centre, open-label, dose-finding, non-randomised, phase 1b trial

In pancreatic ductal adenocarcinoma, the CCL2-CCR2 chemokine axis is used to recruit tumour-associated macrophages for construction of an immunosuppressive tumour microenvironment. This pathway has prognostic implications in pancreatic cancer, and blockade of CCR2 restores anti-tumour immunity in preclinical models. We aimed to establish the safety, tolerability, and recommended phase 2 oral dose of the CCR2 inhibitor PF-04136309 in combination with FOLFIRINOX chemotherapy (oxaliplatin and irinotecan plus leucovorin and fluorouracil). We did this open-label, dose-finding, non-randomised, phase 1b study at one centre in the USA. We enrolled treatment-naive patients aged 18 years or older with borderline resectable or locally advanced biopsy-proven pancreatic ductal adenocarcinoma, an Eastern Cooperative Oncology Group performance status of 1 or less, measurable disease as defined by Response Evaluation Criteria in Solid Tumors version 1.1, and normal end-organ function. Patients were allocated to receive either FOLFIRINOX alone (oxaliplatin 85 mg/m(2), irinotecan 180 mg/m(2), leucovorin 400 mg/m(2), and bolus fluorouracil 400 mg/m(2), followed by 2400 mg/m(2) 46-h continuous infusion), administered every 2 weeks for a total of six treatment cycles, or in combination with oral PF-04136309, administered at a starting dose of 500 mg twice daily in a standard 3 + 3 dose de-escalation design. Both FOLFIRINOX and PF-04136309 were simultaneously initiated with a total treatment duration of 12 weeks. The primary endpoints were the safety, tolerability, and recommended phase 2 dose of PF-04136309 plus FOLFIRINOX, with an expansion phase planned at the recommended dose. We analysed the primary outcome by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT01413022. Between April 19, 2012, and Nov 12, 2014, we treated 47 patients with FOLFIRINOX alone (n=8) or with FOLFIRINOX plus PF-04136309 (n=39). One patient had a dose-limiting toxic effect in the dose de-escalation group receiving FOLFIRINOX plus PF-04136309 at 500 mg twice daily (n=6); this dose was established as the recommended phase 2 dose. We pooled patients in the expansion-phase group (n=33) with those in the dose de-escalation group that received PF-04136309 at the recommended phase 2 dose for assessment of treatment-related toxicity. Six (75%) of the eight patients receiving FOLFIRINOX alone were assessed for treatment toxicity, after exclusion of two (25%) patients due to insurance coverage issues. The median duration of follow-up for treatment toxicity was 72·0 days (IQR 49·5-89·0) in the FOLFIRINOX alone group and 77·0 days (70·0-90·5) in the FOLFIRINOX plus PF-04136309 group. No treatment-related deaths occurred. Two (5%) patients in the FOLFIRINOX plus PF-04136309 group stopped treatment earlier than planned due to treatment-related toxic effects. Grade 3 or higher adverse events reported in at least 10% of the patients receiving PF-04136309 included neutropenia (n=27), febrile neutropenia (n=7), lymphopenia (n=4), diarrhoea (n=6), and hypokalaemia (n=7). Grade 3 or higher adverse events reported in at least 10% of patients receiving FOLFIRINOX alone were neutropenia (n=6), febrile neutropenia (n=1), anaemia (n=2), lymphopenia (n=1), diarrhoea (n=2), hypoalbuminaemia (n=1), and hypokalaemia (n=3). Therapy was terminated because of treatment-related toxicity in one (17%) of the six patients receiving FOLFIRINOX alone. 16 (49%) of 33 patients receiving FOLFIRINOX plus PF-04136309 who had undergone repeat imaging achieved an objective tumour response, with local tumour control achieved in 32 (97%) patients. In the FOLFIRINOX alone group, none of the five patients with repeat imaging achieved an objective response, although four (80%) of those patients achieved stable disease. CCR2-targeted therapy with PF-04136309 in combination with FOLFIRINOX is safe and tolerable. Washington University-Pfizer Biomedical Collaborative.

Evidence of CCR2-independent transmigration of Ly6Chi monocytes into the brain after permanent cerebral ischemia in mice

Previously we showed that INCB3344, a CCR2 antagonist, inhibits transmigration of Ly6Chi monocytes into the brain after ischemia-reperfusion. Here we tested the effect of CCR2 inhibition during permanent cerebral ischemia. Mice were administered either vehicle (dimethyl sulfoxide/carboxymethylcellulose) or INCB3344 (30 or 100mg/kg IP) 1h before middle cerebral artery occlusion and at 2 and 6h after the initiation of ischemia. After 24h, we assessed functional outcome, infarct volume and quantified immune cells in blood and brain. The increase in circulating bone marrow-derived Ly6Chi monocytes, but not the infiltration of those cells into the brain, was blocked by the CCR2 antagonist. INCB3344 had no effect on either neurological deficit or infarct volume. Our data confirm that cerebral ischemia triggers a CCR2-dependent increase in circulating Ly6Chi monocytes, but suggest that in the absence of reperfusion these cells may transmigrate into the ischemic brain in a CCR2-independent manner.

AMPK-Activated Protein Kinase Suppresses Ccr2 Expression by Inhibiting the NF-κB Pathway in RAW264.7 Macrophages.

C-C chemokine receptor 2 (Ccr2) is a key pro-inflammatory marker of classic (M1) macrophage activation. Although Ccr2 is known to be expressed both constitutively and inductively, the full regulatory mechanism of its expression remains unclear. AMP-activated protein kinase (AMPK) is not only a master regulator of energy homeostasis but also a central regulator of inflammation. In this study, we sought to assess AMPK’s role in regulating RAW264.7 macrophage Ccr2 protein levels in resting (M0) or LPS-induced M1 states. In both M0 and M1 RAW264.7 macrophages, knockdown of the AMPKα1 subunit by siRNA led to increased Ccr2 levels whereas pharmacologic (A769662) activation of AMPK, attenuated LPS-induced increases in Ccr2 expression in an AMPK dependent fashion. The increases in Ccr2 levels by AMPK downregulation were partially reversed by NF-κB inhibition whereas TNF-a inhibition had minimal effects. Our results indicate that AMPK is a negative regulator of Ccr2 expression in RAW264.7 macrophages, and that the mechanism of action of AMPK inhibition of Ccr2 is mediated, in part, through the NF-κB pathway.

Astrocyte-Derived CCL2 is Associated with M1 Activation and Recruitment of Cultured Microglial Cells

Background/Aims: Microglia are an essential player in central nervous system inflammation. Recent studies have demonstrated that the astrocytic chemokine, CCL2, is associated with microglial activation in vivo. However, CCL2-induced microglial activation has not yet been studied in vitro. The purpose of the current study was to understand the role of astrocyte-derived CCL2 in microglial activation and to elucidate the underlying mechanism(s). Methods: Primary astrocytes were pre-treated with CCL2 siRNA and stimulated with TNF-α. The culture medium (CM) was collected and added to cultures of microglia, which were incubated with and without CCR2 inhibitor. Microglial cells were analyzed by quantitative RT-PCR to determine whether they polarized to the M1 or M2 state. Microglial migratory ability was assessed by transwell migration assay. Results: TNF-α stimulated the release of CCL2 from astrocytes, even if the culture media containing TNF-α was replaced with fresh media after 3 h. CM from TNF-α-stimulated astrocytes successfully induced microglial activation, which was ascertained by increased activation of M1 and enhanced migration ability. In contrast, CM from astrocytes pretreated with CCL2 siRNA showed no effect on microglial activation, compared to controls. Additionally, microglia pre-treated with RS102895, a CCR2 inhibitor, were resistant to activation by CM from TNF-α-stimulated astrocytes. Conclusion: This study demonstrates that the CCL2/CCR2 pathway of astrocyte-induced microglial activation is associated with M1 polarization and enhanced migration ability, indicating that this pathway could be a useful target to ameliorate inflammation in the central nervous system.

Abstract A90: Combination therapy of chemokine receptor inhibition plus PDL-1 blockade potentiates anti-tumor effects in a murine model of breast cancer

Abstract 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 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 an anti-PDL1 antibody significantly reduced tumor burden in an orthotopic breast cancer mouse model. Primary tumor growth and lung metastasis were modestly reduced by either agent (anti-PDL-1 or CCR1 inhibitor) alone, but the combination of CCR1 inhibitor plus anti-PDL1 antibody resulted in significantly reduced tumor burden as compared to either of the single agents. Orthotropic breast cancer cell engraftment induces 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 in primary tumors, showing that CCR1 blockade of MDSC trafficking translates into reduced tumor burden. 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. Furthermore, CCR1 and its ligands, as well as PDL-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 the most aggressive forms of breast cancer. Citation Format: Heiyoun Jung. Combination therapy of chemokine receptor inhibition plus PDL-1 blockade potentiates anti-tumor effects in a murine model of breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A90.

Inhibition of chemokine receptor CCR2 reduces sarcoma cell transendothelial migration and metastasis to the lungs.

Inhibition of chemokine receptor CCR2 reduces sarcoma cell transendothelial migration and metastasis to the lungs.

Targeted delivery of CCR2 antagonist to activated pulmonary endothelium prevents metastasis

Enhanced levels of the inflammatory chemokine CCL2 are known to correlate with increased tumorigenesis and metastases, and thereby poor prognosis for cancer patients. The CCL2-CCR2 chemokine axis was shown to facilitate the metastatic initiation through the recruitment of inflammatory monocytes and the activation of endothelial cells at metastatic sites. Both steps are required for efficient cancer cell trans-endothelial migration and seeding in the targeted tissue. The translation of preclinical evidence proved to be challenging due to systemic effects of chemokine inhibition and limited target specificity. Here we tested an approach of a targeted delivery of the CCR2 antagonist Teijin Compound 1 to metastatic sites. VCAM-1 binding peptide tagged liposomes carrying the CCR2 antagonist enabled a specific delivery to cancer cell-activated endothelium. The subsequent binding of target-sensitive liposomes triggered the release of the Teijin Compound 1 and thereby local inhibition of CCR2 in the lungs. Blocking of CCR2 resulted in reduced induction of the lungs vascular permeability, and thereby reduced tumor cell extravasation. However, the recruitment of inflammatory monocytes to the pre-metastatic lungs remained unaltered. Endothelial VCAM-1 targeted delivery of the CCR2 antagonist resulted in inhibition of pulmonary metastases both in a murine (MC-38GFP cells) and a human xenograft (patient-derived cells) model. Thus, timely- and spatially-defined inhibition of CCR2 signaling represents a potential therapeutic approach for treatment of metastasis without affecting homeostatic functions.

Reduction in disease progression by inhibition of transforming growth factor α-CCL2 signaling in experimental posttraumatic osteoarthritis.

Transforming growth factor α (TGFα) is increased in osteoarthritic (OA) cartilage in rats and humans and modifies chondrocyte phenotype. CCL2 is increased in OA cartilage and stimulates proteoglycan loss. This study was undertaken to test whether TGFα and CCL2 cooperate to promote cartilage degradation and whether inhibiting either reduces disease progression in a rat model of posttraumatic OA. Microarray analysis was used to profile expression of messenger RNA (mRNA) for Tgfa, Ccl2, and related genes in a rat model of posttraumatic OA. Rat primary chondrocytes and articular cartilage explants were treated with TGFα in the presence or absence of MEK-1/2, p38, phosphatidylinositol 3-kinase, Rho-associated protein kinase, or CCR2 inhibitors and immunostained for markers of cartilage degradation. The rat model was used to administer pharmacologic inhibitors of TGFα (AG1478) and CCL2 (RS504393) signaling for up to 10 weeks and assess histopathology and serum biomarkers of cartilage synthesis (C-propeptide of type II collagen [CPII]) and breakdown (C2C). Tgfa and Ccl2 mRNA were simultaneously up-regulated in articular cartilage in the rat model of posttraumatic OA. TGFα induced expression of CCL2, Mmp3, and Tnf in primary chondrocytes. Cleavage of type II collagen and aggrecan (by matrix metalloproteinases and ADAMTS-4/5, respectively) induced by TGFα was blocked by pharmacologic inhibition of CCL2 in cartilage explants. In vivo pharmacologic inhibition of TGFα or CCL2 signaling reduced Osteoarthritis Research Society International cartilage histopathology scores and increased serum CPII levels, but only TGFα inhibition reduced C2C levels intreated versus untreated rat OA cartilage. TGFα signaling stimulates cartilage degradation via a CCL2-dependent mechanism, but pharmacologic inhibition of the TGFα-CCL2 axis reduces experimental posttraumatic OA progression in vivo.

CCR2 inhibition: a panacea for diabetic kidney disease?

CCR2 inhibition: a panacea for diabetic kidney disease?

Abstract CT223: CCX872: Pharmacodynamic study of a potent and selective CCR2 antagonist in human volunteers and plans for phase Ib trial in patients with pancreatic cancer

Abstract Recent clinical results have highlighted the importance of myeloid-derived suppressor cells in enhancing the ability of the host immune system to limit the growth of tumors. Chemokine receptors control the directed trafficking and persistence of peripheral blood mononuclear cells, including effector and suppressor cells, to the tumor microenvironment. Myeloid derived suppressor cells in particular express high levels of the chemokine receptor CCR2, and pre-clinical data suggests that CCR2 plays an important role in the recruitment of these suppressor cells to tumors. Here we report pharmacodynamic details of a potent and selective small molecule antagonist of CCR2, CCX872, and our plans for a phase Ib trial in patients with pancreatic cancer. CCX872 has been successfully evaluated in a phase I single and multiple ascending dose study (3 to 300 mg) in 40 healthy volunteers. All dose levels were well tolerated and safe. CCX872-B showed a dose-linear PK profile. Ex vivo CCR2 occupancy and internalization assays revealed that blood monocytes from CCX872, but not placebo-treated subjects, were impaired in their ability to bind to or internalize exogenously added CCL2, indicating that CCX872 blocked CCR2 in the treated subjects. The 300 mg daily dose of CCX872 exhibited 104 ± 3% blockade at 2 hours and 93 ± 7% blockade at 24 hours. We calculated that 150 mg twice a day would provide over 90% CCR2 inhibition at all times. Analysis of data from the Tumor Cancer Genome Atlas revealed high levels of expression of CCL2 in pancreatic tumors, and co-expression of CCR2 with monocyte/macrophage markers such as CD68 and CSF1R. In pre-clinical studies, we found that a CCR2 antagonist reduced the growth of the pancreatic tumor cell line, PancO2, in rodent xenograft models. Based in part on these data we have initiated a phase Ib single arm, open label, multicenter study of CCX872 in patients with un-resectable pancreatic cancer. In Part A, subjects will receive a single dose of CCX872 for pharmacokinetic and pharmacodynamics analyses. In Part B, up to 50 subjects will be treated for at least 12 weeks with CCX872 in addition to FOLFIRINOX (5-fluorouracil, leucovorin, irinotecan, and oxaliplatin). Responders and those with stable disease after 12 weeks will continue treatment until disease progression, unacceptable toxicity or death. The primary endpoints are safety/tolerability and efficacy (progression-free rate at 6 months based on RECIST criteria). Other endpoints include overall survival, and effect on the tumor microenvironment and immunological biomarkers in the blood. This trial will test the hypothesis that CCR2 plays an important role in enabling the myeloid suppressor cell response to pancreatic cancer, and will determine if addition of a CCR2 antagonist can enhance the standard of care for these patients Citation Format: Anne-Marie Duliege, Stefan Sleijfer, Ashley Bischof, Joanne Tan, Penglie Zhang, Lisa Seitz, Daniel Dairaghi, Pirow Bekker, Israel Charo, Thomas Schall. CCX872: Pharmacodynamic study of a potent and selective CCR2 antagonist in human volunteers and plans for phase Ib trial in patients with pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT223. doi:10.1158/1538-7445.AM2015-CT223

CCR5 Targeted Cell Therapy for HIV and Prevention of Viral Escape.

Allogeneic transplantation with CCR5-delta 32 (CCR5-d32) homozygous stem cells in an HIV infected individual in 2008, led to a sustained virus control and probably eradication of HIV. Since then there has been a high degree of interest to translate this approach to a wider population. There are two cellular ways to do this. The first one is to use a CCR5 negative cell source e.g., hematopoietic stem cells (HSC) to copy the initial finding. However, a recent case of a second allogeneic transplantation with CCR5-d32 homozygous stem cells suffered from viral escape of CXCR4 quasi-species. The second way is to knock down CCR5 expression by gene therapy. Currently, there are five promising techniques, three of which are presently being tested clinically. These techniques include zinc finger nucleases (ZFN), clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 nuclease (CRISPR/Cas9), transcription activator-like effectors nuclease (TALEN), short hairpin RNA (shRNA), and a ribozyme. While there are multiple gene therapy strategies being tested, in this review we reflect on our current knowledge of inhibition of CCR5 specifically and whether this approach allows for consequent viral escape.

CCR2 inhibition sequesters multiple subsets of leukocytes in the bone marrow.

Chemokine receptor CCR2 mediates monocyte mobilization from the bone marrow (BM) and subsequent migration into target tissues. The degree to which CCR2 is differentially expressed in leukocyte subsets, and the contribution of CCR2 to these leukocyte mobilization from the BM are poorly understood. Using red fluorescence protein CCR2 reporter mice, we found heterogeneity in CCR2 expression among leukocyte subsets in varying tissues. CCR2 was highly expressed by inflammatory monocytes, dendritic cells, plasmacytoid dendritic cells and NK cells in all tissues. Unexpectedly, more than 60% of neutrophils expressed CCR2, albeit at low levels. CCR2 expression in T cells, B cells and NK T cells was greatest in the BM compared to other tissues. Genetic CCR2 deficiency markedly sequestered all leukocyte subsets in the BM, with reciprocal reduction noted in the peripheral blood and spleen. CCR2 inhibition via treatment with CCR2 signaling inhibitor propagermanium produced similar effects. Propagermanium also mitigated lipopolysaccharide-induced BM leukocyte egress. Consistent with its functional significance, CCR2 antibody staining revealed surface CCR2 expression within a subset of BM neutrophils. These results demonstrate the central role CCR2 plays in mediating leukocyte mobilization from the BM, and suggest a role for CCR2 inhibition in managing monocytes/macrophages-mediated chronic inflammatory conditions.

Pancreatic cancer: optimizing treatment options, new, and emerging targeted therapies.

Pancreatic cancer is the fourth leading cause of cancer death in the US and is expected to become the second leading cause of cancer-related deaths in the next decade. Despite 5-fluorouracil/leucovorin with irinotecan and oxaliplatin (FOLFIRINOX) and gemcitabine/nab-paclitaxel significantly improving outcomes for metastatic cancer, refractory disease still poses significant challenges. Difficulties with early detection and the inherent chemo- and radio-resistant nature of this malignancy led to attempts to define the sequential biology of pancreatic cancer in order to improve survival outcomes. Pancreatic adenocarcinoma is characterized by several germline or acquired genetic mutations, the most common being KRAS (90%), CDK2NA (90%), TP53 (75%–90%), DPC4/SMAD4 (50%). In addition, the tumor microenvironment, chemoresistant cancer stem cells, and the desmoplastic stroma have been the target of some promising clinical investigations. Among the core pathways reproducibly shown to lead the development and progression of this disease, DNA repair, apoptosis, G1/S cell cycle transition, KRAS, Wnt, Notch, Hedgehog, TGF-beta, and other cell invasion pathways, have been the target of “precision therapeutics”. No single molecularly targeted therapeutic though has been uniformly successful, probably due to the tumor heterogeneity, but biomarker research is evolving and it hopes to select more patients likely to benefit. Recent reports note activity with immunotherapies such as CD40 agonists, CCR2 inhibitors, cancer vaccines, and novel combinations against the immunosuppressive tumor milieu are ongoing. While many obstacles still exist, clearly we are making progress in deciphering the heterogeneity within pancreatic cancers. Integrating conventional and immunological targeting will be the key to effective treatment of this deadly disease.

Abstract B60: Role of inflammatory monocyte mobilization in metastatic pancreatic cancer

Abstract Introduction: More than half of patients with Pancreatic Cancer (PC) present with metastasis. Our group and others have shown that PC induces cellular changes in the liver long before metastatic spread. These pre-metastatic changes include a significant recruitment of inflammatory monocytes (IM) by the chemokine CCL2 (produced by pre-metastatic liver) and its receptor CCR2 (expressed on IM). Based on our preliminary data, we hypothesized that IM mobilization to the liver is essential for growth of metastatic PC. We therefore optimized our model of metastatic PC to study the role of CCR2 inhibition on growth of metastatic PC. Methods: We implanted a spontaneously derived murine PC cell line (KCKO) orthotopically in the tail of the pancreas of C57BL/6 (WT) mice. We removed the primary tumor on day 10 and divided the spleen into two hemi-spleens. At this time, mice were injected with luciferase expressing tumor cells in the inferior pole of the spleen which was removed after injection and liver metastases were detected by bioluminescence (BLI). Results: Mice with established liver metastasis were randomized to treatment with CCR2 antagonist (CCR2i), FOLFIRINOX, CCR2i and FOLFIRINOX combination (CCR2i+FOLFIRINOX), or vehicle. Mice were treated for three weeks and were imaged biweekly in order to quantify tumor burden in the liver. Mice were sacrificed after 3 weeks and gene expression and flow cytometry studies were performed on peripheral blood, bone marrow, and liver. Liver metastases were detected by BLI in 70% of mice after 10 days and 100% of mice after 15 days following splenic injection with luciferase expressing tumor cells. Peripheral blood and liver inflammatory monocytes/macrophages were significantly increased in mice bearing liver metastasis. However, CCR2i efficiently blocked the recruitment of inflammatory monocyte/macrophage populations in the liver (p<0.01). Additionally mice treated with CCR2i alone and CCR2i and FOLFIRINOX combination exhibited a shift in the immune gene profile in metastatic liver from a pro-tumor (Type 2) to an anti-tumor (Type 1) immune response. Consequently, metastatic tumor burden was significantly decreased after treatment with CCR2i and FOLFIRINOX. Interestingly, the lowest tumor burden was found in livers of mice treated with combination of CCR2 inhibitor and FOLFIRINOX. This suggests that targeting CCR2 can decrease the growth of liver metastasis in PC. Conclusion: Our murine model of PC metastasis formation recapitulates the human form of the disease. We demonstrate that inflammatory monocyte recruitment to the liver is important in promoting the development of metastasis in liver. CCR2 blockade in combination with FOLFIRINOX decreases macrophages in the liver and impairs growth of liver metastasis. Citation Format: Roheena Z. Panni, Dominic E. Sanford, Brian A. Belt, Timothy M. Nywening, Peter Goedegebuure, David C. Linehan. Role of inflammatory monocyte mobilization in metastatic pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B60.