David Maussang

Human cytomegalovirus-encoded chemokine receptor US28 promotes tumorigenesis.

Human cytomegalovirus (HCMV) is a widely spread herpesvirus, suggested to play a role in tumor progression. US28, a chemokine receptor encoded by HCMV, binds a broad spectrum of chemokines and constitutively activates various pathways linked to proliferation. Our studies reveal that expression of US28 induces a proangiogenic and transformed phenotype by up-regulating the expression of vascular endothelial growth factor and enhancing cell growth and cell cycle progression. US28-expressing cells promote tumorigenesis when injected into nude mice. The G protein-uncoupled constitutively inactive mutant of US28, induces delayed and attenuated tumor formation, indicating the importance of constitutive receptor activity in the early onset of tumor development. Importantly, also in glioblastoma cells infected with the newly isolated clinical HCMV strain Titan, US28 was shown to be involved in the HCMV-induced angiogenic phenotype. Hence, the constitutively activated chemokine receptor US28 might act as a viral oncogene and enhance and/or promote HCMV-associated tumor progression.

HCMV-Encoded Chemokine Receptor US28 Mediates Proliferative Signaling Through the IL-6–STAT3 Axis

A viral G protein–coupled receptor may initiate a positive feedback loop to promote tumor proliferation and vascularization. A Viral Pathway to Tumor Development Human cytomegalovirus (HCMV), a widespread human herpesvirus that persists in a latent form, is associated with pathological processes in immunocompromised hosts and has been implicated in the development of several forms of cancer, including glioblastoma. HCMV encodes a G protein–coupled receptor, US28, that resembles a chemokine receptor and constitutively activates signaling pathways associated with cell proliferation. Slinger et al. expressed US28 in cultured cells to explore the mechanisms through which it could promote tumor development. They found that US28 stimulated the production and secretion of both vascular endothelial growth factor (VEGF) and the cytokine interleukin-6 (IL-6) and defined a signaling pathway whereby US28 increased cell proliferation through IL-6–dependent activation of the JAK1-STAT3 axis. IL-6 is itself a target of STAT3, leading the authors to propose that US28-dependent production and secretion of IL-6 and consequent autocrine and paracrine STAT3 activation lead to establishment of a positive feedback loop that promotes proliferation of both infected and neighboring cells. Analyses of human glioblastoma tissue revealed US28 and activated STAT3 in cells lining blood vessels, suggesting that US28 may play a role in tumor vascularization. US28 is a viral G protein (heterotrimeric guanosine triphosphate–binding protein)–coupled receptor encoded by the human cytomegalovirus (HCMV). In addition to binding and internalizing chemokines, US28 constitutively activates signaling pathways linked to cell proliferation. Here, we show increased concentrations of vascular endothelial growth factor and interleukin-6 (IL-6) in supernatants of US28-expressing NIH 3T3 cells. Increased IL-6 was associated with increased activation of the signal transducer and activator of transcription 3 (STAT3) through upstream activation of the Janus-activated kinase JAK1. We used conditioned growth medium, IL-6–neutralizing antibodies, an inhibitor of the IL-6 receptor, and short hairpin RNA targeting IL-6 to show that US28 activates the IL-6–JAK1–STAT3 signaling axis through activation of the transcription factor nuclear factor κB and the consequent production of IL-6. Treatment of cells with a specific inhibitor of STAT3 inhibited US28-dependent [3H]thymidine incorporation and foci formation, suggesting a key role for STAT3 in the US28-mediated proliferative phenotype. US28 also elicited STAT3 activation and IL-6 secretion in HCMV-infected cells. Analyses of tumor specimens from glioblastoma patients demonstrated colocalization of US28 and phosphorylated STAT3 in the vascular niche of these tumors. Moreover, increased phospho-STAT3 abundance correlated with poor patient outcome. We propose that US28 induces proliferation in HCMV-infected tumors by establishing a positive feedback loop through activation of the IL-6–STAT3 signaling axis.

Toll-Like Receptor Signaling in Small Intestinal Epithelium Promotes B-Cell Recruitment and IgA Production in Lamina Propria

BACKGROUND & AIMS Several lines of evidence support a role for Toll-like receptor (TLR) signaling to protect the intestine from pathogenic infection. We hypothesized that TLR signaling at the level of the intestinal epithelium is critical for mucosal immune responses. METHODS We generated transgenic mice that express a constitutively active form of TLR4 in the intestinal epithelium (V-TLR4 mice). Lamina propria cellularity was evaluated by immunostaining and flow cytometry. Immunoglobulin (Ig) A levels in the stool and serum were measured by enzyme-linked immunosorbent assay. Chemokine and cytokine expression were analyzed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS V-TLR4 transgenic mice reproduced normally and had a normal life span. Constitutive activity of TLR4 in the intestinal epithelium promoted recruitment of B cells and an increase in fecal IgA levels. Intestinal epithelial cells of V-TLR4 mice expressed higher levels of CCL20 and CCL28, chemokines known to be involved in B-cell recruitment, and of a proliferation-inducing ligand (APRIL), a cytokine that promotes T-cell-independent class switching of B cells to IgA. The changes in B-cell numbers and IgA levels were blocked by simultaneous expression in intestinal epithelial cells of M3, a herpes virus protein that binds and inhibits multiple chemokines. CONCLUSIONS TLR signaling in the intestinal epithelial cells significantly elevated the production of IgA in the intestine. This effect was mediated by TLR-induced expression of a specific set of chemokines and cytokines that promoted both recruitment of B cells into the lamina propria and IgA class switching of B cells.

CXCR4 nanobodies (VHH-based single variable domains) potently inhibit chemotaxis and HIV-1 replication and mobilize stem cells

The important family of G protein-coupled receptors has so far not been targeted very successfully with conventional monoclonal antibodies. Here we report the isolation and characterization of functional VHH-based immunoglobulin single variable domains (or nanobodies) against the chemokine receptor CXCR4. Two highly selective monovalent nanobodies, 238D2 and 238D4, were obtained using a time-efficient whole cell immunization, phage display, and counterselection method. The highly selective VHH-based immunoglobulin single variable domains competitively inhibited the CXCR4-mediated signaling and antagonized the chemoattractant effect of the CXCR4 ligand CXCL12. Epitope mapping showed that the two nanobodies bind to distinct but partially overlapping sites in the extracellular loops. Short peptide linkage of 238D2 with 238D4 resulted in significantly increased affinity for CXCR4 and picomolar activity in antichemotactic assays. Interestingly, the monovalent nanobodies behaved as neutral antagonists, whereas the biparatopic nanobodies acted as inverse agonists at the constitutively active CXCR4-N3.35A. The CXCR4 nanobodies displayed strong antiretroviral activity against T cell-tropic and dual-tropic HIV-1 strains. Moreover, the biparatopic nanobody effectively mobilized CD34-positive stem cells in cynomolgus monkeys. Thus, the nanobody platform may be highly effective at generating extremely potent and selective G protein-coupled receptor modulators.

Pharmacological modulation of chemokine receptor function

G protein‐coupled chemokine receptors and their peptidergic ligands are interesting therapeutic targets due to their involvement in various immune‐related diseases, including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, chronic obstructive pulmonary disease, HIV‐1 infection and cancer. To tackle these diseases, a lot of effort has been focused on discovery and development of small‐molecule chemokine receptor antagonists. This has been rewarded by the market approval of two novel chemokine receptor inhibitors, AMD3100 (CXCR4) and Maraviroc (CCR5) for stem cell mobilization and treatment of HIV‐1 infection respectively. The recent GPCR crystal structures together with mutagenesis and pharmacological studies have aided in understanding how small‐molecule ligands interact with chemokine receptors. Many of these ligands display behaviour deviating from simple competition and do not interact with the chemokine binding site, providing evidence for an allosteric mode of action. This review aims to give an overview of the evidence supporting modulation of this intriguing receptor family by a range of ligands, including small molecules, peptides and antibodies. Moreover, the computer‐assisted modelling of chemokine receptor–ligand interactions is discussed in view of GPCR crystal structures. Finally, the implications of concepts such as functional selectivity and chemokine receptor dimerization are considered.

Pharmacokinetics, brain delivery, and efficacy in brain tumor-bearing mice of glutathione pegylated liposomal doxorubicin (2B3-101).

Brain cancer is a devastating disease affecting many people worldwide. Effective treatment with chemotherapeutics is limited due to the presence of the blood-brain barrier (BBB) that tightly regulates the diffusion of endogenous molecules but also xenobiotics. Glutathione pegylated liposomal doxorubicin (2B3-101) is being developed as a new treatment option for patients with brain cancer. It is based on already marketed pegylated liposomal doxorubicin (Doxil®/Caelyx®), with an additional glutathione coating that safely enhances drug delivery across the BBB. Uptake of 2B3-101 by human brain capillary endothelial cells in vitro was time-, concentration- and temperature-dependent, while pegylated liposomal doxorubicin mainly remained bound to the cells. In vivo, 2B3-101 and pegylated liposomal doxorubicin had a comparable plasma exposure in mice, yet brain retention 4 days after administration was higher for 2B3-101. 2B3-101 was overall well tolerated by athymic FVB mice with experimental human glioblastoma (luciferase transfected U87MG). In 2 independent experiments a strong inhibition of brain tumor growth was observed for 2B3-101 as measured by bioluminescence intensity. The effect of weekly administration of 5 mg/kg 2B3-101 was more pronounced compared to pegylated liposomal doxorubicin (p<0.05) and saline (p<0.01). Two out of 9 animals receiving 2B3-101 showed a complete tumor regression. Twice-weekly injections of 5 mg/kg 2B3-101 again had a significant effect in inhibiting brain tumor growth (p<0.001) compared to pegylated liposomal doxorubicin and saline, and a complete regression was observed in 1 animal treated with 2B3-101. In addition, twice-weekly dosing of 2B3-101 significantly increased the median survival time by 38.5% (p<0.001) and 16.1% (p<0.05) compared to saline and pegylated liposomal doxorubicin, respectively. Overall, these data demonstrate that glutathione pegylated liposomal doxorubicin enhances the effective delivery of doxorubicin to brain tumors and could become a promising new therapeutic option for the treatment of brain malignancies.

The cytomegalovirus-encoded chemokine receptor US28 promotes intestinal neoplasia in transgenic mice.

US28 is a constitutively active chemokine receptor encoded by CMV (also referred to as human herpesvirus 5), a highly prevalent human virus that infects a broad spectrum of cells, including intestinal epithelial cells (IECs). To study the role of US28 in vivo, we created transgenic mice (VS28 mice) in which US28 expression was targeted to IECs. Expression of US28 was detected in all IECs of the small and large intestine, including in cells expressing leucine rich repeat containing GPCR5 (Lgr5), a marker gene of intestinal epithelial stem cells. US28 expression in IECs inhibited glycogen synthase 3β (GSK-3β) function, promoted accumulation of β-catenin protein, and increased expression of Wnt target genes involved in the control of the cell proliferation. VS28 mice showed a hyperplastic intestinal epithelium and, strikingly, developed adenomas and adenocarcinomas by 40 weeks of age. When exposed to an inflammation-driven tumor model (azoxymethane/dextran sodium sulfate), VS28 mice developed a significantly higher tumor burden than control littermates. Transgenic coexpression of the US28 ligand CCL2 (an inflammatory chemokine) increased IEC proliferation as well as tumor burden, suggesting that the oncogenic activity of US28 can be modulated by inflammatory factors. Together, these results indicate that expression of US28 promotes development of intestinal dysplasia and cancer in transgenic mice and suggest that CMV infection may facilitate development of intestinal neoplasia in humans.

Llama-derived Single Variable Domains (Nanobodies) Directed against Chemokine Receptor CXCR7 Reduce Head and Neck Cancer Cell Growth in Vivo

Background: The atypical chemokine receptor CXCR7 is highly expressed in various types of cancer. Results: CXCR7 Nanobodies were generated and show inhibition of β-arrestin2 signaling and secretion of angiogenic CXCL1 in vitro. Anti-CXCR7 Nanobodies reduce tumor growth by inhibiting angiogenesis. Conclusion: CXCR7 inhibition by Nanobodies inhibit head and neck tumor formation. Significance: Anti-CXCR7 therapies are potential novel treatments against head and neck cancer. The chemokine receptor CXCR7, belonging to the membrane-bound G protein-coupled receptor superfamily, is expressed in several tumor types. Inhibition of CXCR7 with either small molecules or small interference (si)RNA has shown promising therapeutic benefits in several tumor models. With the increased interest and effectiveness of biologicals inhibiting membrane-bound receptors we made use of the “Nanobody platform” to target CXCR7. Previously we showed that Nanobodies, i.e. immunoglobulin single variable domains derived from naturally occurring heavy chain-only camelids antibodies, represent new biological tools to efficiently tackle difficult drug targets such as G protein-coupled receptors. In this study we developed and characterized highly selective and potent Nanobodies against CXCR7. Interestingly, the CXCR7-targeting Nanobodies displayed antagonistic properties in contrast with previously reported CXCR7-targeting agents. Several high affinity CXCR7-specific Nanobodies potently inhibited CXCL12-induced β-arrestin2 recruitment in vitro. A wide variety of tumor biopsies was profiled, showing for the first time high expression of CXCR7 in head and neck cancer. Using a patient-derived CXCR7-expressing head and neck cancer xenograft model in nude mice, tumor growth was inhibited by CXCR7-targeting Nanobody therapy. Mechanistically, CXCR7-targeting Nanobodies did not inhibit cell cycle progression but instead reduced secretion of the angiogenic chemokine CXCL1 from head and neck cancer cells in vitro, thus acting here as inverse agonists, and subsequent angiogenesis in vivo. Hence, with this novel class of CXCR7 inhibitors, we further substantiate the therapeutic relevance of targeting CXCR7 in head and neck cancer.

Targeting chemokines and chemokine receptors with antibodies

Chemokines and their receptors are highly interesting therapeutic targets for pharmaceutical and biotechnology companies. In particular, industrial development pipelines are filled with new chemokine-targeting drugs to treat inflammatory diseases and malignancies. In this review, we specifically highlight antibody-based therapeutics and monoclonal antibodies (mAbs) targeting the chemokine system. Besides discussing pitfalls inherently linked to their discovery, we will elaborate on where progress can be made in the development of novel human therapeutic antibodies directed at the chemokine system.

Synthesis, modeling and functional activity of substituted styrene-amides as small-molecule CXCR7 agonists.

The chemokine receptor CXCR7 is an atypical G protein-coupled receptor as it preferentially signals through the β-arrestin pathway rather than through G proteins. CXCR7 is thought to be of importance in cancer and the development of CXCR7-targeting ligands is of huge importance to further elucidate the pharmacology and the therapeutic potential of CXCR7. In the present study, we synthesized 24 derivatives based on a compound scaffold patented by Chemocentryx and obtained CXCR7 ligands with pK(i) values ranging from 5.3 to 8.1. SAR studies were supported by computational 3D Fingerprint studies, revealing several important affinity descriptors. Two key compounds (29 and 30, VUF11207 and VUF11403) were found to be high-potency ligands that induce recruitment of β-arrestin2 and subsequent internalization of CXCR7, making them important tool compounds in future CXCR7 research.