anti-CXCR4 Antibody Research Articles

Phase I dose-escalation study of F50067, a humanized anti-CXCR4 monoclonal antibody alone and in combination with lenalidomide and low-dose dexamethasone, in relapsed or refractory multiple myeloma.

PurposeMultiple myeloma (MM) remains an incurable disease as tumor cells ultimately resist to all available drugs. Homing of tumor cells to the bone marrow microenvironment, involving especially the CXCR4/SDF-1 axis, allows them to survive, proliferate and resist to therapy. F50067, a humanized anti-CXCR4 IgG1 antibody, has promising preclinical activity in MM.We present a phase I multicenter escalation study in relapsed/refractory MM (RRMM) to determine the maximum tolerated dose (MTD) for F50067 alone and in combination with lenalidomide and low dose dexamethasone (Len-Dex).Experimental design14 end-stage RRMM patients received F50067 single agent (n = 10) or in combination with Len-Dex (n = 4).ResultsOne dose-limiting toxicity was observed, a grade 4 neutropenia lasting more than 7 days in combination arm. MTD could not be established. Thrombocytopenia was observed in 100% and neutropenia in 92.9% of patients with no cases of febrile neutropenia and no severe bleeding or hematoma. Non-hematological adverse events were of mild to moderate severity.Nine patients (6 in single arm and 3 in combination arm) were evaluable for response, with 66.7% overall response rate (≥PR) in combination arm, and 33.3% of disease control (≥SD) in single agent arm. At the time of study termination, 55.6% had progressed.ConclusionThis study suggests that egression of tumor cells to the blood stream can represent a novel therapeutic strategy for MM. However, because of significant hematological toxicity, this study had to be discontinued. Further studies are needed to validate the feasibility of this approach in clinical practice.

Disruption of CXCR3 function impedes the development of Sjögren's syndrome-like xerostomia in non-obese diabetic mice

The chemokine receptor CXCR3 plays an important role in T cell recruitment in various immune responses and autoimmune diseases. Expression of CXCR3 ligands, including CXCL9, CXCL10, and CXCL11, is elevated in the salivary glands of patients with Sjögren's syndrome (SS). To elucidate whether interaction between CXCR3 and its ligands is required for the development of SS, we administrated an anti-CXCR3 blocking antibody (CXCR3-173) to the non-obese diabetic (NOD) mice, a well-defined model of SS, during the stage prior to disease onset. Treatment with this anti-CXCR3 antibody significantly improved salivary secretion, indicating a remission of SS clinical manifestation. Anti-CXCR3 treatment did not affect the gross leukocyte infiltration of the submandibular glands (SMGs) as assessed by hematoxylin and eosin staining. However, flow cytometric analysis showed that anti-CXCR3 treatment markedly reduced the percentage of CXCR3+CD8 T and CXCR3+CD44+CD8 T cells, without affecting that of CXCR3+CD4 T and CXCR3+CD44+CD4 T cells in the SMGs and submandibular lymph nodes, suggesting a preferential effect of this anti-CXCR3 treatment on CXCR3-expressing effector CD8 T cells. Meanwhile, SMG expression of inflammatory factor TNF-α was markedly diminished by anti-CXCR3 treatment. In accordance, anti-CXCR3 significantly enhanced SMG expression of tight junction protein claudin-1 and water channel protein aquaporin 5, two molecules that are crucial for normal salivary secretion and can be down-regulated by TNF-α. Taken together, these findings demonstrated that the interaction between the endogenous CXCR3 and its ligands plays a pro-inflammatory and pathogenic role in the development of SS-like xerostomia in the NOD mouse model.

Screening of cancer tissue arrays identifies CXCR4 on adrenocortical carcinoma: correlates with expression and quantification on metastases using 64Cu-plerixafor PET.

Expression of the chemokine receptor CXCR4 by many cancers correlates with aggressive clinical behavior. As part of the initial studies in a project whose goal was to quantify CXCR4 expression on cancers non-invasively, we examined CXCR4 expression in cancer samples by immunohistochemistry using a validated anti-CXCR4 antibody. Among solid tumors, we found expression of CXCR4 on significant percentages of major types of kidney, lung, and pancreatic adenocarcinomas, and, notably, on metastases of clear cell renal cell carcinoma and squamous cell carcinoma of the lung. We found particularly high expression of CXCR4 on adrenocortical cancer (ACC) metastases. Microarrays of ACC metastases revealed correlations between expression of CXCR4 and other chemokine system genes, particularly CXCR7/ACKR3, which encodes an atypical chemokine receptor that shares a ligand, CXCL12, with CXCR4. A first-in-human study using 64Cu-plerixafor for PET in an ACC patient prior to resection of metastases showed heterogeneity among metastatic nodules and good correlations among PET SUVs, CXCR4 staining, and CXCR4 mRNA. Additionally, we were able to show that CXCR4 expression correlated with the rates of growth of the pulmonary lesions in this patient. Further studies are needed to understand better the role of CXCR4 in ACC and whether targeting it may be beneficial. In this regard, non-invasive methods for assessing CXCR4 expression, such as PET using 64Cu-plerixafor, should be important investigative tools.

Fluorescent CXCR4 targeting peptide as alternative for antibody staining in Ewing sarcoma

BackgroundEwing sarcoma is an aggressive, highly metastatic primary bone and soft tissue tumor most frequently occurring in the bone of young adolescents. Patients, especially those diagnosed with a metastatic disease, have a poor overall survival. Chemokine receptor CXCR4 has a key pro-tumorigenic role in the tumor microenvironment of Ewing sarcoma and has been suggested to be involved in the increased metastatic propensity. Earlier studies on CXCR4 protein expression in Ewing sarcoma yielded contradictory results when compared to CXCR4 RNA expression studies. Previously, we demonstrated that CXCR4 expression could be detected in vivo using the fluorescently tagged CXCR4-specific peptide MSAP-Ac-TZ14011. Therefore, we studied the membranous CXCR4 expression in Ewing sarcoma cell lines using MSAP-Ac-TZ14011.MethodsThe CXCR4 membrane expression levels were studied in EWS cell lines by flow cytometry using the hybrid peptide MSAP-Ac-TZ14011 and were correlated to CXCR4 RNA expression levels. The measurements were compared to levels detected using the CXCR4 antibody ab2074 under various cell preparation conditions. In addition, the staining patterns were analyzed by confocal fluorescence microscopy over time.ResultsThe hybrid peptide MSAP-Ac-TZ14011 levels showed a strong and better correlation of CXCR4 membrane expression with the CXCR4 RNA expression levels than observed with the anti-CXCR4 antibody ab2074. With the hybrid peptide MSAP-Ac-TZ14011 using live cell confocal microscopy CXCR4 membrane staining and internalization was detected and the signal intensity correlated well with CXCR4 mRNA expression levels.ConclusionsThe fluorescently labeled CXCR4 targeting peptide-based method provides a reliable alternative to antibody staining to study the CXCR4 membrane expression in live cells using either flow cytometry or live cell fluorescence microscopy. The fluorescently tagged CXCR4 targeting peptide could enable in vivo detection of CXCR4 expression in Ewing sarcoma which may help to stratify cases for anti-CXCR4 therapy.

Targeting the CXCR4 pathway using a novel anti-CXCR4 IgG1 antibody (PF-06747143) in chronic lymphocytic leukemia

BackgroundThe CXCR4-CXCL12 axis plays an important role in the chronic lymphocytic leukemia (CLL)-microenvironment interaction. Overexpression of CXCR4 has been reported in different hematological malignancies including CLL. Binding of the pro-survival chemokine CXCL12 with its cognate receptor CXCR4 induces cell migration. CXCL12/CXCR4 signaling axis promotes cell survival and proliferation and may contribute to the tropism of leukemia cells towards lymphoid tissues and bone marrow. Therefore, we hypothesized that targeting CXCR4 with an IgG1 antibody, PF-06747143, may constitute an effective therapeutic approach for CLL.MethodsPatient-derived primary CLL-B cells were assessed for cytotoxicity in an in vitro model of CLL microenvironment. PF-06747143 was analyzed for cell death induction and for its potential to interfere with the chemokine CXCL12-induced mechanisms, including migration and F-actin polymerization. PF-06747143 in vivo efficacy was determined in a CLL murine xenograft tumor model.ResultsPF-06747143, a novel-humanized IgG1 CXCR4 antagonist antibody, induced cell death of patient-derived primary CLL-B cells, in presence or absence of stromal cells. Moreover, cell death induction by the antibody was independent of CLL high-risk prognostic markers. The cell death mechanism was dependent on CXCR4 expression, required antibody bivalency, involved reactive oxygen species production, and did not require caspase activation, all characteristics reminiscent of programmed cell death (PCD). PF-06747143 also induced potent B-CLL cytotoxicity via Fc-driven antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity activity (CDC). PF-06747143 had significant combinatorial effect with standard of care (SOC) agents in B-CLL treatment, including rituximab, fludarabine (F-ara-A), ibrutinib, and bendamustine. In a CLL xenograft model, PF-06747143 decreased tumor burden and improved survival as a monotherapy, and in combination with bendamustine.ConclusionsWe show evidence that PF-06747143 has biological activity in CLL primary cells, supporting a rationale for evaluation of PF-06747143 for the treatment of CLL patients.

Dissemination of HIV-1 from latently infected cells to resting CD4+ T cells requires CD4 and actin remodeling.

Abstract The major barrier to HIV-1 eradication is the replication-competent proviruses in long-lived latently infected CD4+ T cells. Current efforts to purge latently HIV-1-infected cells have focused on developing “shock and kill” approaches to force reactivation of the proviruses in these cells to be killed after reactivation of virus gene expression by viral cytopathic effects, host immune responses, or both. Cell-free virus infection and cell-to-cell transmission via the virological synapses (VS) are the two modes of HIV-1 dissemination between CD4+ T cells. Several studies have demonstrated that cell-to-cell spread, in which HIV-1 disseminates through direct contact of infected cells with surrounding uninfected cells, is a far more efficient process than that of cell-free virus infection. In this work, we studied the mechanism of cell-to-cell transmission between primary CD4+ T cells and the HIV-1 latently infected cell line ACH-2. When primary resting CD4+ T cells were co-cultured with ACH-2 cells under latency reversing agent Romidepsin stimulation, about 20.7% cells had been infected without becoming activated. Additionally, blocking with anti-CD4 or anti-gp120 neutralizing antibodies (VRC01) decreased de novo infection, and neither anti-CXCR4 antibodies nor chemokine inhibitors demonstrated the ability to inhibit cell-to-cell transmission. We also found that co-cultured resting CD4+ T cells carried higher levels of active cofilin, the actin-depolymerizing factor, suggesting actin plays a role in VS formation and viral pathogenesis. These results suggest HIV-1 Env gp120 binds with the CD4 receptor, not chemokine coreceptor to form VS during cell-to-cell transmission.

CXCR3 blockade impedes the development of Sjögren’s syndrome-like xerostomia in non-obese diabetic mice

Abstract The chemokine receptor CXCR3 plays an important role in T cell recruitment in various immune responses and autoimmune diseases. Expression of CXCR3 ligands, including CXCL9, CXCL10 and CXCL11, is elevated in the salivary glands of patients with Sjögren’s syndrome (SS). To elucidate whether interaction between CXCR3 and its ligands is required for the development of SS, we administrated an anti-CXCR3 blocking antibody to non-obese diabetic (NOD) mice, a well-defined model of SS, during the stage prior to disease onset. CXCR3 blockade significantly improved salivary secretion, indicating a remission of SS clinical manifestation. CXCR3 blockade did not affect the gross leukocyte infiltration of the submandibular glands (SMG) as assessed by H&E staining. However, flow cytometric analysis showed that anti-CXCR3 treatment markedly reduced the percentage of CXCR3+CD8 T cells and CD44+CD8 T cells, without affecting that of CXCR3+CD4 T cells and CD44+CD4 T cells in the SMG and submandibular lymph nodes, suggesting a selective effect of CXCR3 blockade on effector T cytotoxic 1 cells. Meanwhile, SMG expression of inflammatory factor TNF-α was markedly diminished by anti-CXCR3 treatment. Consistent with this, CXCR3 blockade significantly enhanced SMG expression of tight junction protein claudin-1 and water channel protein aquaporin-5, two factors that are crucially required for normal salivary secretion and can be down-regulated by TNF-α. Taken together, our findings demonstrated that the interaction between endogenous CXCR3 and its ligands plays a proinflammatory and pathogenic role in the development of SS-like xerostomia in NOD mouse model.

CXCR3 mediates ascites-directed tumor cell migration and predicts poor outcome in ovarian cancer patients

Intraabdominal tumor dissemination is a major hallmark of epithelial ovarian cancer (EOC), but the underlying mechanisms have not been fully elucidated. The CXCR3 chemokine receptor supports migration of tumor cells to metastatic sites, but its role in ovarian cancer metastasis is largely unknown. Herein, we first screened two independent cohorts of high-grade serous ovarian cancers (HGSCs, discovery set n=60, validation set n=117) and 102 metastatic lesions for CXCR3 expression. In primary tumors, CXCR3 was particularly overexpressed by tumor cells at the invasive front. In intraabdominal metastases, tumor cells revealed a strong CXCR3 expression regardless of its expression in the corresponding primary tumor, suggesting a selection of CXCR3-overexpressing cancer cells into peritoneal niches. In support of this, CXCR3 mediated the migration of tumor cell lines OVCAR3 and SKOV3 toward malignant ascites, which was inhibited by a monoclonal anti-CXCR3 antibody in vitro. These results were prospectively validated in ascites-derived tumor cells from EOC patients ex vivo (n=9). Moreover, tumor cell-associated overexpression of CXCR3 in advanced ovarian cancer patients was associated with a reduced progression-free survival (PFS) and overall survival (OS), which remained independent of optimal debulking, age, FIGO stage and lymph node involvement (PFS: hazard ratio (HR) 2.11, 95% confidence interval (CI) 1.30–3.45, P=0.003; OS: HR 2.36, 95% CI 1.50–3.71, P<0.001). These results in ovarian cancer patients identify CXCR3 as a potential new target to confine peritoneal spread in ovarian cancer after primary cytoreductive surgery.

Retraction Note to: Nuclear Protein C23 on the Cell Surface Plays an Important Role in Activation of CXCR4 Signaling in Glioblastoma.

The chemokine receptor CXCR4 and its ligand stromal cell-derived factor 1 (SDF-1) plays an important role in tumor progression and are associated with angiogenesis. Meanwhile, the implications of C23 in multiple signaling pathways have been also investigated. However, the effects of C23 on CXCR4 pathway in glioblastoma are not fully characterized. In the present study, C23 and CXCR4 of U87 cell line were inhibited by anti-C23 and anti-CXCR4 antibodies, respectively; and then C23 and CXCR4 siRNAs were used to knock down endogenous C23 and CXCR4, respectively. In addition, MTT assay was also introduced. Our data showed that either anti-C23 or anti-CXCR4 antibodies efficaciously repressed the phosphorylation levels of ERK (p < 0.000) and AKT (p < 0.000) compared with SDF-1 alone and control. As expected, either C23 or CXCR4 siRNAs indeed resulted in C23 and CXCR4 knockdown and further suppressed the expression of p-ERK and p-AKT. Most importantly, immunoprecipitation revealed C23 interacted with CXCR4 once U87 was exposed to SDF-1 treatment. In addition, MTT assay identified that C23 or CXCR4 siRNAs could obviously decreased cell proliferation capacity (p = 0.002). In conclusion, our results suggest that C23 plays a crucial role in activation of SDF-1-induced ERK and PI3K/AKT pathways via interacting with CXCR4. Furthermore, C23 could be recommended as an important element in glioblastoma development and a new target for glioblastoma treatment.

Elucidation of Distinct Roles of Guinea Pig CXCR1 and CXCR2 in Neutrophil Migration toward IL-8 and GROα by Specific Antibodies.

Chemokine receptors CXCR1 and CXCR2 are conserved between guinea pigs and humans, but the distinct role of each receptor in chemotactic responses of neutrophils against chemokine ligands has not been elucidated due in part to the lack of specific inhibitors against these receptors in guinea pigs. In this study, we investigated the roles of guinea pig CXCR1 and CXCR2 on neutrophils in chemotactic responses to guinea pig interleukin (IL)-8 and growth-regulated oncogene (GRO)α by using specific inhibitory antibodies against these receptors. Neutrophil migration induced by IL-8 was partially inhibited by either anti-CXCR1 antibody or anti-CXCR2 antibody. In addition, the migration was inhibited completely when both anti-CXCR1 and anti-CXCR2 antibodies were combined. On the other hand, neutrophil migration induced by GROα was not inhibited by anti-CXCR1 antibody while inhibited profoundly by anti-CXCR2 antibody. These results indicated that CXCR1 and CXCR2 mediated migration induced by the IL-8 synergistically and only CXCR2 mediated migration induced by GROα in guinea pig neutrophils. Our findings on ligand selectivity of CXCR1 and CXCR2 in guinea pigs are consistent with those in humans.

CCR7 and CXCR4 Expression in Primary Head and Neck Squamous Cell Carcinomas and Nodal Metastases – a Clinical and Immunohistochemical Study

Background:Squamous cell carcinomas (SCCs) are common head and neck malignancies demonstrating lymph node LN involvement. Recently chemokine receptor overxpression has been reported in many cancers. Of particular interest, CCR7 appears to be a strong mediator of LN metastases, while CXCR4 may mediate distant metastases. Any relations between their expression in primary HNSCCs and metastatic lymph nodes need to be clarified.Aims:To investigate CCR7 andCXCR4 expression in primary HNSCCs of all tumor sizes, clinical stages and histological grades, as well as involved lymph nodes, then make comparisons, also with control normal oral epithelium.Materials and Methods:The sample consisted of 60 formalin-fixed, paraffin-embedded specimens of primary HNSCCs, 77 others of metastasi-positive lymph nodes, and 10 of control normal oral epithelial tissues. Sections were conventionally stained with H&E and immunohistochemically with monoclonal anti-CCR7 and monoclonal anti-CXCR4 antibodies. Positive cells were counted under microscopic assessment in four fields (X40) per case.Results:There was no variation among primary HNSCC tumors staining positive for CCR7 and CXCR4 with tumor size of for CCR7 with lymph node involvement. However, a difference was noted between primary HNSCC tumors stained by CXCR4 with a single as compared to more numerous node involvement. CXCR4 appear to vary with the clinical stagebut no links were noted with histological grades. Staining for primary HNSCC tumors and metastatic lymph nodes correlated.

A New Anti-CXCR4 Antibody That Blocks the CXCR4/SDF-1 Axis and Mobilizes Effector Cells.

The type IV C-X-C-motif chemokine receptor (CXCR4) is expressed in a large variety of human cancers, including hematologic malignancies, and this receptor and its ligand, stromal cell-derived factor-1 (SDF-1), play a crucial role in cancer progression. We generated a humanized immunoglobulin G1 mAb, hz515H7, which binds human CXCR4, efficiently competes for SDF-1 binding, and induces a conformational change in CXCR4 homodimers. Furthermore, it inhibits both CXCR4 receptor-mediated G-protein activation and β-arrestin-2 recruitment following CXCR4 activation. The binding of the hz515H7 antibody to CXCR4 inhibits the SDF-1-induced signaling pathway, resulting in reduced phosphorylation of downstream effectors, such as Akt, Erk1/2, p38, and GSK3β. Hz515H7 also strongly inhibits cell migration and proliferation and, while preserving normal blood cells, induces both antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against neoplastic cells. In mouse xenograft models, hz515H7 displays antitumor activities with multiple hematologic tumor cell lines, with its Fc-mediated effector functions proving essential in this context. Furthermore, hz515H7 binds to primary tumor cells from acute myeloid leukemia and multiple myeloma patients. Collectively, our results demonstrate two major mechanisms of action, making hz515H7 unique in this regard. Its potential as a best-in-class molecule is currently under investigation in a phase I clinical trial. Mol Cancer Ther; 15(8); 1890-9. ©2016 AACR.

Abstract 1729: Withaferin A blocks formation of IFN-γ-induced metastatic cancer stem cells through inhibition of the CXCR4/CXCL12 pathway in the UP-LN1 carcinoma cell model

Abstract As our understanding of cancer stem cell (CSC) biology improves, there is an urgent need to search for inhibitory agents of CSCs and metastatic CSCs (mCSCs) positive for CXCR4. Withaferin A (WA), a withanolide extracted from the medicinal plant Withania somnifera, has been shown to exhibit anti-cancer effects through multiple mechanisms. Whether WA could selectively target CSCs, mCSCs, or non-CSCs of a gastrointestinal (GI) carcinoma tumor remains unclear. Side population analysis, flow cytometric phenotyping and sorting, non-invasive imaging in conjunction with xenotransplantation, and immunohistology were used in this investigation. Using the lymph node metastatic GI cancer cell line UP-LN1, consisting of CD44high/CD24low grape-like floating (F) and CD44low/CD24high adherent (A) cell subsets, this study demonstrated that, as compared with parental UP-LN1 cells or A cells, WA preferentially reduced F-cell proliferation, tumor sphere formation, and side population cells in vitro in greater efficiencies by apoptosis. This action was mechanistically mediated via the down-regulation of CXCR4/CXCL12 and STAT3/IL-6 pathways, both of which are instrumental in the acquisition of metastatic ability. Attenuation of IFN-γ-induced CXCR4 expression in F cells by knockdown with siRNA or blocking with anti-CXCR4 antibody, followed by Western blot analysis, showed significantly reduced metastatic potential in vitro. The extent of in vitro anti-invasive effect of WA on the IFN-γ-treated F cells was significantly greater than on the F cells without WA treatment, or F cells treated with control siRNA or with control IgG antibody. The observed in vitro effects of WA on the CSC and mCSC targeting were validated by data obtained with non-invasive imaging in NOD/SCID mouse xenotransplantation. We conclude that WA could efficiently block the formation of both CSCs and mCSCs in the UP-LN1 cell line, suggesting that WA may be considered an effective therapeutic agent for GI malignancies exhibiting grape-like tumor sphere. Citation Format: Andy Shau-Bin Chou, Li-Lei Ting, Chin-Hsuan Hsieh, See-Tong Pang, Shuen-Kuei Liao. Withaferin A blocks formation of IFN-γ-induced metastatic cancer stem cells through inhibition of the CXCR4/CXCL12 pathway in the UP-LN1 carcinoma cell model. [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 1729.

Evidence of an interaction between TGF-β1 and the SDF-1/CXCR4/CXCR7 axis in human platelets

BackgroundTGF-β1, SDF-1 and its cognate receptors CXCR4 and CXCR7 are expressed on the surface of human platelets and their expression levels are differently regulated in symptomatic coronary artery disease (CAD). All these proteins and receptors influence outcome in patients with symptomatic CAD. There might be a crosstalk between TGF-β1 and the SDF-1/CXCR4/CXCR7 axis. Interrelations in CAD, especially in the context of platelets, are poorly understood. Therefore, we aimed to provide clinical and experimental evidence of interactions between TGF-β1 and the SDF-1/CXCR4/CXCR7 axis in human platelets. Methods and resultsBlood samples of the complete cohort (n=284) were analysed for platelet surface expression levels of TGF-β1, SDF-1, CXCR4 and CXCR7 by flow cytometry. For stimulation assays platelet rich plasma was treated with TGF-β1 or SDF-1 and then analysed by flow cytometry. Multiple regression analyses were run to show independent associations of TGF-β1 with SDF-1, CXCR4, CXCR7 and clinical cofactors. Both, CXCR4 and CXCR7 significantly predicted TGF-β1 (p<0.001 and p<0.001, respectively). After stimulation with SDF-1, surface expression of TGF-β1 increased significantly when compared to resting platelets [mean TGF-β1 MFI 19.01 vs. mean TGF-β1 MFI 14.01, p<0.001]. Upon receptor blocking with either anti-CXCR4 or anti-CXCR7 monoclonal antibodies the enhancing effect of SDF-1 on TGF-β1 surface expression was significantly blunted. Stimulation with TGF-β1 did not alter SDF-1, CXCR4 or CXCR7 expression significantly. ConclusionsWe provide first clinical and experimental data suggesting a cross-talk between TGF-β and the SDF-1/CXCR4/CXCR7 axis in platelets which does not involve transcriptional modulation as shown previously for other cellular systems.

Inhibition of CXCR4 by LY2624587, a Fully Humanized Anti-CXCR4 Antibody Induces Apoptosis of Hematologic Malignancies.

SDF-1 and CXCR4 are a chemokine and chemokine receptor pair playing critical roles in tumorigenesis. Overexpression of CXCR4 is a hallmark of many hematological malignancies including acute myeloid leukemia, chronic lymphocytic leukemia and non-Hodgkin’s lymphoma, and generally correlates with a poor prognosis. In this study, we developed a humanized anti-CXCR4 monoclonal antibody, LY2624587 as a potent CXCR4 antagonist that was advanced into clinical study for cancer. LY2624587 blocked SDF-1 binding to CXCR4 with an IC50 of 0.26 nM, and inhibited SDF-1-induced GTP binding with a Kb of 0.66 nM. In human lymphoma U937 and leukemia CCRF-CEM cells expressing endogenous CXCR4, LY2624587 inhibited SDF-1-induced cell migration with IC50 values of 3.7 and 0.26 nM, respectively. This antibody also inhibited CXCR4 and SDF-1 mediated cell signaling including activation of MAPK and AKT in tumor cells expressing CXCR4. Bifocal microscopic and flow cytometry analyses revealed that LY2624587 mediated receptor internalization and caused CXCR4 down-regulation on the cell surface. In human hematologic cancer cells, LY2624587 caused dose dependent apoptosis in vitro and in vivo. In mouse xenograft models developed with human leukemia and lymphoma cells expressing high levels of CXCR4, LY2624587 exhibited dose-dependent tumor growth inhibition and provided significant survival benefit in a disseminated lymphoma model. Collectively, we have demonstrated that CXCR4 inhibition by LY2624587 has the potential for the treatment of human hematological malignancies.

The CXCL10/CXCR3 axis promotes cardiac microvascular endothelial cell migration via the p38/FAK pathway in a proliferation-independent manner

CXCL10 is a chemokine with potent chemotactic activity for immune and non-immune cells expressing its receptor CXCR3. Previous studies have demonstrated that CXCL10 is involved in myocardial infarction. However, the role of CXCL10 in cardiac microvascular endothelial cell (CMEC) regulation and related mechanisms remains unclear. In this study, we investigated the effects of CXCL10 on the CMEC migration and explored its potential molecular mechanism by wound healing, cell proliferation and viability analysis. Furthermore, migration-related signaling pathways, including FAK, Erk, p38 and Smad, were examined by Western blotting. We found that CXCL10 significantly promotes CMEC migration under normal conditions and during hypoxia/ischemia. However, no significant differences in CMEC proliferation and viability were observed with or without CXCL10 treatment. CXCL10-mediated CMEC migration was greatly blocked by treatment with an anti-CXCR3 antibody. Although CXCL10 treatment promoted phosphorylation and activation of the FAK, Erk, and p38 pathways during hypoxia/ischemia, CXCL10-mediated CMEC migration was significantly blocked by p38 and FAK inhibitors, but not by an Erk inhibitor. Furthermore, CXCL10-mediated FAK activation was suppressed by the p38 inhibitor. These findings indicated that the CXCL10/CXCR3 pathway promotes the migration of CMECs under normal conditions and during hypoxia/ischemia in a proliferation-independent manner, at least in part, through regulation of the p38/FAK pathways.

Stromal Derived Factor-1/CXCR4 Axis Involved in Bone Marrow Mesenchymal Stem Cells Recruitment to Injured Liver.

The molecular mechanism of bone marrow mesenchymal stromal stem cells (BMSCs) mobilization and migration to the liver was poorly understood. Stromal cell-derived factor-1 (SDF-1) participates in BMSCs homing and migration into injury organs. We try to investigate the role of SDF-1 signaling in BMSCs migration towards injured liver. The expression of CXCR4 in BMSCs at mRNA level and protein level was confirmed by RT-PCR, flow cytometry, and immunocytochemistry. The SDF-1 or liver lysates induced BMSCs migration was detected by transwell inserts. CXCR4 antagonist, AMD3100, and anti-CXCR4 antibody were used to inhibit the migration. The Sprague-Dawley rat liver injury model was established by intraperitoneal injection of thioacetamide. The concentration of SDF-1 increased as modeling time extended, which was determined by ELISA method. The Dir-labeled BMSCs were injected into the liver of the rats through portal vein. The cell migration in the liver was tracked by in vivo imaging system and the fluorescent intensity was measured. In vivo, BMSCs migrated into injured liver which was partially blocked by AMD3100 or anti-CXCR4 antibody. Taken together, the results demonstrated that the migration of BMSCs was regulated by SDF-1/CXCR4 signaling which involved in BMSCs recruitment to injured liver.

Macrophage Densities Correlated with CXC Chemokine Receptor 4 Expression and Related with Poor Survival in Anaplastic Thyroid Cancer.

BackgroundTumor associated macrophages (TAMs) and CXC chemokine receptor 4 (CXCR4) have emerged as potential biomarkers in various human cancers. The aims of this study were to investigate the clinical characteristics of anaplastic thyroid cancer (ATC) patients according to the TAM numbers in the tumor tissue, and to evaluate the associations between CXCR4 expressions and macrophage densities in ATC tumor microenvironment.MethodsTotal 14 ATC samples from thyroid tissue microarray were used. Immunohistochemical staining was performed using anti-CD163 and anti-CXCR4 antibodies. According to the immunoreactivity of CD163, all subjects were divided into two groups: low-CD163 (n=8) and high-CD163 (n=6) groups.ResultsThe mean diagnostic age was 65±7 years and the median tumor size was 4.3 cm, ranging 2.5 to 15 cm. Clinicopathological characteristics were not significantly different between low-CD163 and high-CD163 groups, while age of diagnosis was younger in high-CD163 group than that of low-CD163 group with marginal significance (56.9±5.5 years vs. 67.5±6.8 years, P=0.09). However, overall survival was significantly reduced in high-CD163 group (5.5 months [range, 1 to 10]) compared with low-CD163 groups (8.8 months [range, 6 to 121); log-rank test, P=0.0443). Moreover, high-CD163 group showed strong CXCR4 expressions in both cancer and stromal compartments, while low-CD163 group showed relatively weak, stromal-dominant CXCR4 expressions. Additionally, CD163 and CXCR4 expressions showed a strong positive correlation (γ2=0.432, P=0.013).ConclusionIncreased number of TAMs showed poor overall survival in ATC, suggesting TAMs are potentially a prognostic biomarker for ATC. CXCR4 expression was significantly correlated with CD163-positive TAM densities, which suggest the possible role of CXCR4 in TAM recruitments.

Ulocuplumab (BMS-936564 / MDX1338): a fully human anti-CXCR4 antibody induces cell death in chronic lymphocytic leukemia mediated through a reactive oxygen species-dependent pathway.

The CXCR4 receptor (Chemokine C-X-C motif receptor 4) is highly expressed in different hematological malignancies including chronic lymphocytic leukemia (CLL). The CXCR4 ligand (CXCL12) stimulates CXCR4 promoting cell survival and proliferation, and may contribute to the tropism of leukemia cells towards lymphoid tissues. Therefore, strategies targeting CXCR4 may constitute an effective therapeutic approach for CLL. To address that question, we studied the effect of Ulocuplumab (BMS-936564), a fully human IgG4 anti-CXCR4 antibody, using a stroma – CLL cells co-culture model. We found that Ulocuplumab (BMS-936564) inhibited CXCL12 mediated CXCR4 activation-migration of CLL cells at nanomolar concentrations. This effect was comparable to AMD3100 (Plerixafor - Mozobil), a small molecule CXCR4 inhibitor. However, Ulocuplumab (BMS-936564) but not AMD3100 induced apoptosis in CLL at nanomolar concentrations in the presence or absence of stromal cell support. This pro-apoptotic effect was independent of CLL high-risk prognostic markers, was associated with production of reactive oxygen species and did not require caspase activation. Overall, these findings are evidence that Ulocuplumab (BMS-936564) has biological activity in CLL, highlight the relevance of the CXCR4-CXCL12 pathway as a therapeutic target in CLL, and provide biological rationale for ongoing clinical trials in CLL and other hematological malignancies.

Targeting the CXCR4-CXCL12 Pathway Using an Anti-CXCR4 IgG1 Antibody (PF-06747143) in Chronic Lymphocytic Leukemia

Abstract Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in adults in the Western world. This leukemia is not curable and resistance to therapy is promoted by factors present in the tumor microenvironment including the chemokine CXCL12 (SDF-1), which interacts with its receptor CXCR4 and is thought to promote cancer cell survival. Here we explored the therapeutic potential of blocking CXCL12-CXCR4 interactions using PF-06747143, a humanized IgG1 antibody specific for CXCR4, which is expressed at high levels by CLL cells. Using primary leukemia cells from CLL patients, we found that PF-06747143 inhibited CXCL12-induced cell migration and blocked cytoskeletal changes via F-actin polymerization similar to AMD-3100 (Mozobil, a small molecule inhibitor of CXCR4). In addition, PF-06747143 induced apoptosis on CLL cells cultured alone or in the presence of human bone marrow-derived stromal cells (stroma-NK-tert). The pro-apoptotic activity of PF-06747143 was independent of high-risk prognostic factors including IGHV mutation status, ZAP-70 expression or TP53 mutation / 17p-deletion. Interestingly, AMD-3100, which binds and inhibits signaling through CXCR4, did not induce cell death in CLL or any of the cell lines tested. PF-06747143 did not induce apoptosis on normal B and T cells, and the ability of this anti-CXCR4 antibody to induce cell death on CLL cells appeared to be dependent on the crosslinking of CXCR4. This was supported by the fact that a Fab only fragment derived from PF-06747143 did not induce apoptosis despite of its high binding affinity for CXCR4. We observed that PF-06747143 induced complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in CLL cells. However, this antibody did not induce caspase activation but rather its cell death activity appeared to be dependent on the production of reactive oxygen species (ROS) in leukemia cells. This effect was similar to that observed with other ROS dependent antibodies such as obinutuzumab (Gazyva). ROS induction was observed with PF-06747143, but not its Fab derived fragment and preceded apoptosis suggesting that this is critical component of its mechanism of action. We evaluated synergism of PF-06747143 with other CLL therapeutic agents and observed that this antibody synergized with fludarabine, bendamustine, ibrutinib and rituximab in the majority of CLL patient samples tested. In summary, our studies showed that PF-06747143, a CXCR4 IgG1 antibody is a potent inhibitor of the CXCR4-CXCL12 pathway and induces cell death primarily in CLL cells but not in normal lymphocytes. The cytotoxic effect of PF-06747143 was similar in CLL cells cultured alone or with stromal cells, suggesting that this antibody has the potential to overcome the protective effect of the tumor microenvironment. We also showed that PF-06747143 induced programmed cell death on CLL cells was dependent on ROS production and that this antibody synergized with agents currently used for the treatment of CLL patients. Overall, these findings highlight the biological relevance of the CXCR4-CXCL12 pathway in CLL, and provide rationale for clinical evaluation of PF-06747143 in CLL and other cancers. Disclosures Choi: Gilead: Consultancy, Other: Advisory Board, Speakers Bureau; AbbVie: Consultancy, Other: Advisory Board, Research Funding. Kipps:Pharmacyclics Abbvie Celgene Genentech Astra Zeneca Gilead Sciences: Other: Advisor.