MMP14 is a novel target of PTH signaling in osteocytes that controls resorption by regulating soluble RANKL production.

Parathyroid hormone (PTH) affects the skeleton by acting on osteocytes (Ots) in bone through yet unclear mechanisms. We report that matrix metalloproteinase 14 (MMP14) expression/activity are increased in bones from mice with genetic constitutive activation (ca) of the PTH receptor 1 (PTH1R) in Ots (caPTH1ROt) and in bones from mice exposed to elevated PTH levels but not in mice lacking [conditional knockout (cKO)] the PTH1R in Ots (cKOPTH1ROt). Furthermore, PTH upregulates MMP14 in human bone cultures and in Ot-enriched bones from floxed control mice but not from cKOPTH1ROt mice. MMP14 activity increases soluble receptor activator of NF-κΒ ligand production, which in turn, stimulates osteoclast differentiation and resorption. Pharmacologic inhibition of MMP14 activity reduced the high bone remodeling exhibited by caPTH1ROt mice or induced by chronic PTH elevation and decreased bone resorption but allowed full stimulation of bone formation induced by PTH injections, thereby potentiating bone gain. Thus, MMP14 is a new member of the intricate gene network activated in Ots by PTH1R signaling that can be targeted to adjust the skeletal responses to PTH in favor of bone preservation.-Delgado-Calle, J., Hancock, B., Likine, E. F., Sato, A. Y., McAndrews, K., Sanudo, C., Bruzzaniti, A., Riancho, J. A., Tonra, J. R., Bellido, T. MMP14 is a novel target of PTH signaling in osteocytes that controls resorption by regulating soluble RANKL production.

Structural model of a2-subunit N-terminus and its binding interface for Arf-GEF CTH2: Implication for regulation of V-ATPase, CTH2 function and rational drug design.

We have previously identified the interaction between mammalian V-ATPase a2-subunit isoform and cytohesin-2 (CTH2) and studied molecular details of binding between these proteins. In particular, we found that six peptides derived from the N-terminal cytosolic domain of a2 subunit (a2N1-402) are involved in interaction with CTH2 (Merkulova, Bakulina, Thaker, Grüber, & Marshansky, 2010). However, the actual 3D binding interface was not determined in that study due to the lack of high-resolution structural information about a-subunits of V-ATPase. Here, using a combination of homology modeling and NMR analysis, we generated the structural model of complete a2N1-402 and uncovered the CTH2-binding interface. First, using the crystal-structure of the bacterial M. rubber Icyt-subunit of A-ATPase as a template (Srinivasan, Vyas, Baker, & Quiocho, 2011), we built a homology model of mammalian a2N1-352 fragment. Next, we combined it with the determined NMR structures of peptides a2N368-395 and a2N386-402 of the C-terminal section of a2N1-402. The complete molecular model of a2N1-402 revealed that six CTH2 interacting peptides are clustered in the distal and proximal lobe sub-domains of a2N1-402. Our data indicate that the proximal lobe sub-domain is the major interacting site with the Sec7 domain of first CTH2 protein, while the distal lobe sub-domain of a2N1-402 interacts with the PH-domain of second CTH2. Indeed, using Sec7/Arf-GEF activity assay we experimentally confirmed our model. The interface formed by peptides a2N1-17 and a2N35-49 is involved in specific interaction with Sec7 domain and regulation of GEF activity. These data are critical for understanding of the cross-talk between V-ATPase and CTH2 as well as for the rational drug design to regulate their function.

Selective oral ROCK2 inhibitor down-regulates IL-21 and IL-17 secretion in human T cells via STAT3-dependent mechanism.

Rho-associated kinase 2 (ROCK2) regulates the secretion of proinflammatory cytokines and the development of autoimmunity in mice. Data from a phase 1 clinical trial demonstrate that oral administration of KD025, a selective ROCK2 inhibitor, to healthy human subjects down-regulates the ability of T cells to secrete IL-21 and IL-17 by 90% and 60%, respectively, but not IFN-γ in response to T-cell receptor stimulation in vitro. Pharmacological inhibition with KD025 or siRNA-mediated inhibition of ROCK2, but not ROCK1, significantly diminished STAT3 phosphorylation and binding to IL-17 and IL-21 promoters and reduced IFN regulatory factor 4 and nuclear hormone RAR-related orphan receptor γt protein levels in T cells derived from healthy subjects or rheumatoid arthritis patients. Simultaneously, treatment with KD025 also promotes the suppressive function of regulatory T cells through up-regulation of STAT5 phosphorylation and positive regulation of forkhead box p3 expression. The administration of KD025 in vivo down-regulates the progression of collagen-induced arthritis in mice via targeting of the Th17-mediated pathway. Thus, ROCK2 signaling appears to be instrumental in regulating the balance between proinflammatory and regulatory T-cell subsets. Targeting of ROCK2 in man may therefore restore disrupted immune homeostasis and have a role in the treatment of autoimmunity.

An allosteric inhibitor of sirtuin 2 deacetylase activity exhibits broad-spectrum antiviral activity.

Most drugs used to treat viral disease target a virus-coded product. They inhibit a single virus or virus family, and the pathogen can readily evolve resistance. Host-targeted antivirals can overcome these limitations. The broad-spectrum activity achieved by host targeting can be especially useful in combating emerging viruses and for treatment of diseases caused by multiple viral pathogens, such as opportunistic agents in immunosuppressed patients. We have developed a family of compounds that modulate sirtuin 2, an NAD+-dependent deacylase, and now report the properties of a member of that family, FLS-359. Biochemical and x-ray structural studies show that the drug binds to sirtuin 2 and allosterically inhibits its deacetylase activity. FLS-359 inhibits the growth of RNA and DNA viruses, including members of the coronavirus, orthomyxovirus, flavivirus, hepadnavirus, and herpesvirus families. FLS-359 acts at multiple levels to antagonize cytomegalovirus replication in fibroblasts, causing modest reductions in viral RNAs and DNA, together with a much greater reduction in infectious progeny, and it exhibits antiviral activity in humanized mouse models of infection. Our results highlight the potential of sirtuin 2 inhibitors as broad-spectrum antivirals and set the stage for further understanding of how host epigenetic mechanisms impact the growth and spread of viral pathogens.

Plinabulin, a Distinct Microtubule-Targeting Chemotherapy, Promotes M1-Like Macrophage Polarization and Anti-tumor Immunity.

Reprogramming tumor infiltrating myeloid cells to elicit pro-inflammatory responses is an exciting therapeutic maneouver to improve anti-tumor responses. We recently demonstrated that a distinct microtubule-targeting drug, plinabulin-a clinical-stage novel agent-modulates dendritic cell maturation and enhances anti-tumor immunity. Here, we investigated the effects of plinabulin on macrophage polarization in vitro and in vivo. Plinabulin monotherapy induced significant tumor growth inhibition in mice bearing subcutaneous MC38 colon cancer. Importantly, the regressing tumors were characterized by an increase in M1-like/M2-like tumor-associated macrophages (TAM) ratio. The efficacy of plinabulin remained unaltered in T cell-deficient Rag2-/- mice, suggesting an important role of macrophages in driving the drug's anti-tumor effect. Exposure of murine and healthy human macrophages to plinabulin induced polarization toward the M1 phenotype, including increased expression of co-stimulatory molecules CD80, CD86 and pro-inflammatory cytokines IL-1ß, IL-6, and IL-12. M2-associated immunosuppressive cytokines IL-10 and IL-4 were reduced. This pro-inflammatory M1-like skewing of TAMs in response to plinabulin was dependent on the JNK pathway. Functionally, plinabulin-polarized human M1 macrophages directly killed HuT 78 tumor cells in vitro. Importantly, plinabulin induced a functional M1-like polarization of tumor infiltrating macrophages in murine tumors as well as in tumor samples from ovarian cancer patients, by preferentially triggering M1 proliferation. Our study uncovers a novel immunomodulatory effect of plinabulin in directly triggering M1 polarization and proliferation as well as promoting TAM anti-tumoral effector functions.

Plinabulin ameliorates neutropenia induced by multiple chemotherapies through a mechanism distinct from G-CSF therapies.

PURPOSE: Chemotherapy-induced neutropenia (CIN) increases the risk of infections and mortality in cancer patients. G-CSF therapies are approved for the treatment of CIN, but non-G-CSF therapies are needed to increase efficacy and minimize side effects. Plinabulin is an inhibitor of tubulin polymerization that ameliorates CIN caused in patients by the microtubule stabilizer docetaxel. The present study evaluates the potential of plinabulin to reduce neutropenia induced by chemotherapies of different classes in a manner not dependent on increasing G-CSF. METHODS: The anti-CIN benefits of plinabulin were tested in rodents co-treated with docetaxel, cyclophosphamide or doxorubicin. Effects on G-CSF levels were evaluated in tissues by immunoassay. Flow cytometry was utilized to test treatment effects on femur bone marrow cell counts from immunocompetent mice-bearing orthotopic 4T1 breast cancer tumors. RESULTS: Plinabulin alleviated neutropenia induced by microtubule stabilizing, DNA cross-linking and DNA intercalating chemotherapies, yet did not affect bone marrow or blood G-CSF levels. The number of lineage-/Sca1+/c-Kit+ (LSK) hematopoietic stem/progenitor cells (HSPC) in murine bone marrow collected 2 days after treatment was not affected by docetaxel monotherapy despite increased plasma G-CSF in this group. LSK cell number was, however, increased when plinabulin was combined with docetaxel, without affecting G-CSF. CONCLUSIONS: Results support the clinical testing of plinabulin as a non-G-CSF-based treatment for CIN associated with chemotherapies of different mechanisms. Results also support HSPC as a focal point for future mechanism-of-action work aimed at understanding the ability of plinabulin to reduce this serious side effect of cytotoxic therapy in cancer patients.

Functional significance of VEGFR-2 on ovarian cancer cells.

Vascular endothelial growth factor receptor (VEGFR) has recently been discovered on ovarian cancer cells, but its functional significance is unknown and is the focus of this study. By protein analysis, A2780-par and HeyA8 ovarian cancer cell lines expressed VEGFR-1 and HeyA8 A2774, and SKOV3ip1 expressed VEGFR-2. By in situ hybridization (ISH), 85% of human ovarian cancer specimens showed moderate to high VEGFR-2 expression, whereas only 15% showed moderate to high VEGFR-1 expression. By immunofluorescence, little or no VEGFR-2 was detected in normal ovarian surface epithelial cells, whereas expression was detected in 75% of invasive ovarian cancer specimens. To differentiate between the effects of tumor versus host expression of VEGFR, nude mice were injected with SKOV3ip1 cells and treated with either human VEGFR-2 specific antibody (1121B), murine VEGFR-2 specific antibody (DC101) or the combination. Treatment with 1121B reduced SKOV3ip1 cell migration by 68% (p < 0.01) and invasion by 72% (p < 0.01), but exposure to VEGFR-1 antibody had no effect. Treatment with 1121B effectively blocked VEGF-induced phosphorylation of p130Cas. In vivo treatment with either DC101 or 1121B significantly reduced tumor growth alone and in combination in the SKOV3ip1 and A2774 models. Decreased tumor burden after treatment with DC101 or 1121B correlated with increased tumor cell apoptosis, decreased proliferative index, and decreased microvessel density. These effects were significantly greater in the combination group (p < 0.001). We show functionally active VEGFR-2 is present on most ovarian cancer cells. The observed anti-tumor activity of VEGF-targeted therapies may be mediated by both anti-angiogenic and direct anti-tumor effects.

In vivo method for establishing synergy between antibodies to epidermal growth factor receptor and vascular endothelial growth factor receptor-2.

Targeted therapy for cancer is shifting towards an approach of inhibiting multiple pathways, justified in part by the ability of cancer cells to overcome the inhibition of a single pathway. However the literature is replete with preclinical data supporting the anticancer potential of numerous combinations of targeted agents, making it difficult to select the combination strategies to invest in through clinical development. One characteristic of a combination strategy that can be utilized for prioritization is synergy. Synergy indicates that the effect of the combination is greater than that predicted from the monotherapy potencies. Here we describe a detailed method for establishing synergy between two treatments in vivo. We utilized this method to establish that antibodies targeting the epidermal growth factor receptor and vascular endothelial growth factor receptor-2 are synergistic with regard to antitumor effects, in a BxPC-3 subcutaneous xenograft model for pancreatic cancer.

Prioritization of EGFR/IGF-IR/VEGFR2 combination targeted therapies utilizing cancer models.

BACKGROUND: Rational strategies utilizing anticancer efficacy and biological principles are needed for the prioritization of specific combination targeted therapy approaches for clinical development, from among the many with experimental support. MATERIALS AND METHODS: Antibodies targeting epidermal growth factor receptor (EGFR) (cetuximab), insulin-like growth factor-1 receptor (IGF-IR) (IMC-A12) or vascular endothelial growth factor receptor 2 (VEGFR2) (DC101), were dosed alone or in combination, in 11 human tumor xenograft models established in mice. Efficacy readouts included the tumor burden and incidence of metastasis, as well as tumor active hypoxia inducible factor-1 (HIF-1), human VEGF and blood vessel density. RESULTS: Cetuximab and DC101 contributed potent and non-overlapping benefits to the combination approach. Moreover, DC101 prevented escape from IMC-A12 + cetuximab in a colorectal cancer model and cetuximab prevented escape from DC101 therapy in a pancreatic cancer model. CONCLUSION: Targeting VEGFR2 + EGFR was prioritized over other treatment strategies utilizing EGFR, IGF-IR and VEGFR2 antibodies. The criteria that proved to be valuable were a non-overlapping spectrum of anticancer activity and the prevention of resistance to another therapy in the combination.

Arylphthalazines as potent, and orally bioavailable inhibitors of VEGFR-2.

A series of arylphthalazine derivatives were synthesized and evaluated as antagonists of VEGF receptor II (VEGFR-2). IM-094482 57, which was prepared in two steps from commercially available starting materials, was found to be a potent inhibitor of VEGFR-2 in enzymatic, cellular and mitogenic assays (comparable activity to ZD-6474). Additionally, 57 inhibited the related receptor, VEGF receptor I (VEGFR-1), and showed excellent exposure when dosed orally to female CD-1 mice.

Vascular endothelial growth factor receptor 3 is involved in tumor angiogenesis and growth.

Vascular endothelial growth factor receptor 3 (VEGFR-3) binds VEGF-C and VEGF-D and is essential for the development of the lymphatic vasculature. Experimental tumors that overexpress VEGFR-3 ligands induce lymphatic vessel sprouting and enlargement and show enhanced metastasis to regional lymph nodes and beyond, whereas a soluble form of VEGFR-3 that blocks receptor signaling inhibits these changes and metastasis. Because VEGFR-3 is also essential for the early blood vessel development in embryos and is up-regulated in tumor angiogenesis, we wanted to determine if an antibody targeting the receptor that interferes with VEGFR-3 ligand binding can inhibit primary tumor growth. Our results show that antibody interference with VEGFR-3 function can inhibit the growth of several human tumor xenografts in immunocompromised mice. Immunohistochemical analysis showed that the blood vessel density of anti-VEGFR-3-treated tumors was significantly decreased and hypoxic and necrotic tumor tissue was increased when compared with tumors treated with control antibody, indicating that blocking of the VEGFR-3 pathway inhibits angiogenesis in these tumors. As expected, the anti-VEGFR-3-treated tumors also lacked lymphatic vessels. These results suggest that the VEGFR-3 pathway contributes to tumor angiogenesis and that effective inhibition of tumor progression may require the inhibition of multiple angiogenic targets.

Tumor growth inhibition with cetuximab and chemotherapy in non-small cell lung cancer xenografts expressing wild-type and mutated epidermal growth factor receptor.

PURPOSE: Targeting the epidermal growth factor receptor (EGFR) is a validated approach to treat cancer. In non-small cell lung cancer (NSCLC), EGFR contains somatic mutations in 10% of patients, which correlates with increased response rates to small molecule inhibitors of EGFR. We analyzed the effects of the monoclonal IgG1 antibody Erbitux (cetuximab) in NSCLC xenografts with wild-type (wt) or mutated EGFR. EXPERIMENTAL DESIGN: NSCLC cell lines were grown s.c. in nude mice. Dose-dependent efficacy was established for cetuximab. To determine whether combination therapy produces tumor regressions, cetuximab was dosed at half-maximal efficacy with chemotherapy used at maximum tolerated dose. RESULTS: Cetuximab showed antitumor activity in wt (A549, NCI-H358, NCI-H292) and mutated [HCC-827 (delE746-A750), NCI-H1975 (L858R, T790M)] EGFR-expressing xenografts. In the H292 model, cetuximab and docetaxel combination therapy was more potent to inhibit tumor growth than cetuximab or docetaxel alone. Cisplatin augmented efficacy of cetuximab to produce 6 of 10 regressions, whereas 1 of 10 regressions was found with cetuximab and no regression was found with cisplatin. Using H1975 xenografts, gemcitabine increased efficacy of cetuximab resulting in 12 of 12 regressions. Docetaxel with cetuximab was more efficacious with seven of nine regressions compared with single treatments. Cetuximab inhibited autophosphorylation of EGFR in both H292 and H1975 tumor lysates. Exploring the underlying mechanism for combination effects in the H1975 xenograft model, docetaxel in combination with cetuximab added to the antiproliferative effects of cetuximab but was the main component in this drug combination to induce apoptosis. CONCLUSIONS: Cetuximab showed antitumor activity in NSCLC models expressing wt and mutated EGFR. Combination treatments increased the efficacy of cetuximab, which may be important for the management of patients with chemorefractory NSCLC.

Tumors established with cell lines selected for oxaliplatin resistance respond to oxaliplatin if combined with cetuximab.

PURPOSE: To establish whether cetuximab, a chimeric IgG1 antibody targeting epidermal growth factor receptor, has the potential to restore responsiveness to oxaliplatin in preclinical cancer models, as has been shown with irinotecan in irinotecan refractory metastatic colorectal cancer patients. EXPERIMENTAL DESIGN: The effects of cetuximab and oxaliplatin, alone or in combination, were tested in vitro and in vivo using human colorectal cancer cell lines selected for oxaliplatin resistance, as well as parental control cell lines. Evaluations were made of subcutaneous xenograft tumor growth in nu/nu athymic mice, as well as activation of mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2) and AKT, expression of DNA repair genes, density of apurinic/apyrimidinic DNA damage, and accumulation of platinum-DNA adducts in vitro. RESULTS: Oxaliplatin + cetuximab efficacy in murine subcutaneous xenograft models was greater than that of monotherapies and independent of the responsiveness to oxaliplatin monotherapy. In vitro, cetuximab reduced expression of excision repair cross-complementation group 1 and XPF, which are key components of the nucleotide excision repair pathway involved in the excision of platinum-DNA adducts. In addition, cetuximab reduced expression of XRCC1, a component of the base excision repair pathway responsible for the repair of apurinic/apyrimidinic sites. Effects of cetuximab on DNA repair protein levels were downstream to effects on mitogen-activated protein kinase and AKT pathway activation. In line with effects on DNA repair protein expression, cetuximab increased the accumulation of platinum and apurinic/apyrimidinic sites on DNA during oxaliplatin treatment. CONCLUSIONS: Cetuximab has the potential to salvage the benefits of oxaliplatin in oxaliplatin-resistant colorectal cancer patients by reducing DNA repair capacity.

IMC-A12, a human IgG1 monoclonal antibody to the insulin-like growth factor I receptor.

Targeted monoclonal antibody therapy is an important strategy in cancer therapeutics. Among the most promising characteristics of therapeutic targets are those that modulate the growth and survival of malignant neoplasms and their sensitivity to anticancer therapies. The insulin-like growth factor-I receptor (IGF-IR) is overexpressed in many types of solid and hematopoietic malignancies, and has been implicated as a principal cause of heightened proliferative and survival signaling. IGF-IR has also been shown to confer resistance to cytotoxic, hormonal, and targeted therapies, suggesting that therapeutics targeting IGF-IR may be effective against a broad range of malignancies. IMC-A12 (ImClone Systems Incorporated), a fully human monoclonal IgG1 antibody that binds with high affinity to the IGF-IR, inhibits ligand-dependent receptor activation and downstream signaling. IMC-A12 also mediates robust internalization and degradation of the IGF-IR. In human tumor xenograft models, IGF-IR blockade by IMC-A12 results in rapid and profound growth inhibition of cancers of the breast, lung, colon, and pancreas, and many other neoplasms. Although promising single-agent activity has been observed, the most impressive effects of targeting the IGF-IR with IMC-A12 have been noted when this agent was combined with cytotoxic agents or other targeted therapeutics. The results with IMC-A12 to date suggest that it may be an effective therapeutic in a diverse array of oncologic indications.

Synergistic antitumor effects of combined epidermal growth factor receptor and vascular endothelial growth factor receptor-2 targeted therapy.

PURPOSE: Combination therapies that target the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) pathways, are being actively tested for the treatment of cancer. In evaluating combination strategies, the ideal combination would be one in which the treatments interact in a way that is synergistic with regard to antitumor effects. Here, we have evaluated the interaction between anti-EGFR antibody Erbitux (cetuximab) and anti-VEGFR2 antibody, DC101, in preclinical models of pancreatic (BxPC-3) and colon (GEO) cancer. EXPERIMENTAL DESIGN: Analysis of the interaction between cetuximab and DC101 in vivo used a novel method for establishing the upper 95% confidence limits for the combination index (CI) of isobologram analyses, where CI < 1 indicates synergy. Assessment of tumor cell proliferation, apoptosis, VEGF production, and hypoxia, as well as tumor vascularization, was performed to gain insights into the mechanistic basis for synergy between agents targeting different tumor compartments. RESULTS: Monotherapy ED(50) values for tumor growth inhibition ranged from 1.8 to 2.3 mg/kg and 10.5 to 16.6 mg/kg for cetuximab and DC101, respectively. From the dose response of the combination treatment, it was determined that cetuximab and DC101 are synergistic in the BxPC-3 (CI = 0.1, P < 0.01) and GEO (CI = 0.1, P < 0.01) models. Overlapping effects on the tumor cell and vascular compartments form a basis for the interaction, with VEGF production and hypoxia-inducible factor 1alpha potentially acting as molecular links between EGFR and VEGFR2 inhibition. CONCLUSIONS: Results show antitumor synergy for combined EGFR and VEGFR2 targeted therapy, supporting the significant therapeutic potential of this combination strategy.

Anti-vascular endothelial growth factor receptor-1 antagonist antibody as a therapeutic agent for cancer.

PURPOSE: Vascular endothelial growth factor receptor-1 (VEGFR-1) plays important roles in promotion of tumor growth by mediating cellular functions in tumor vascular endothelium and cancer cells. Blockade of VEGFR-1 activation has been shown to inhibit pathologic angiogenesis and tumor growth, implicating VEGFR-1 as a potential therapeutic target for the treatment of cancer. We have thus developed a VEGFR-1 antagonist human monoclonal antibody designated as IMC-18F1 and evaluated its antitumor activity in preclinical experimental models to show the therapeutic potential of the antibody for cancer treatment in clinic. EXPERIMENTAL DESIGN: Human IgG transgenic mice were used for generation of anti-VEGFR-1 antibodies. Anti-VEGFR-1-specific blocking antibodies were identified using solid-phase binding and blocking assays. Inhibitory antitumor cell activity of IMC-18F1 was assessed in cell-based kinase and growth assays. Pharmacokinetic/pharmacodynamic studies were done to determine the association of antibody blood level with antitumor efficacy of the antibody in vivo. Antitumor efficacy of the anti-VEGFR-1 antibodies as monotherapy and in combination with cytotoxic agents was evaluated in human breast cancer xenograft models. RESULTS: A fully human neutralizing antibody, IMC-18F1, was shown to be a high-affinity (KD=54 pmol) inhibitor of VEGFR-1 ligand binding (VEGF-A, VEGF-B, and placental growth factor). IMC-18F1 inhibited ligand-induced intracellular activation of VEGFR-1 and mitogen-activated protein kinase signaling and prevented ligand-stimulated in vitro growth of breast cancer cells. In vivo, IMC-18F1 suppressed the growth of human breast tumor xenografts in association with reduced mitogen-activated protein kinase and Akt activation, reduced tumor cell proliferation, and increased tumor cell apoptosis. Pharmacokinetic/pharmacodynamic studies established a plasma elimination half-life of 5 days for IMC-18F1 and a steady-state trough plasma therapeutic threshold of 88 microg/mL. Importantly, inhibition of mouse and human VEGFR-1 with MF1 and IMC-18F1, respectively, enhanced the antitumor efficacy of cytotoxic agents commonly used to treat breast cancer. CONCLUSIONS: Based on preclinical validation studies, IMC-18F1 anti-VEGFR-1 has potential to provide clinical benefit to cancer patients.

Targeting the platelet-derived growth factor receptor alpha with a neutralizing human monoclonal antibody inhibits the growth of tumor xenografts: implications as a potential therapeutic target.

Platelet-derived growth factor receptor alpha (PDGFRalpha) is a type III receptor tyrosine kinase that is expressed on a variety of tumor types. A neutralizing monoclonal antibody to human PDGFRalpha, which did not cross-react with the beta form of the receptor, was generated. The fully human antibody, termed 3G3, has a Kd of 40 pmol/L and blocks both PDGF-AA and PDGF-BB ligands from binding to PDGFRalpha. In addition to blocking ligand-induced cell mitogenesis and receptor autophosphorylation, 3G3 inhibited phosphorylation of the downstream signaling molecules Akt and mitogen-activated protein kinase. This inhibition was seen in both transfected and tumor cell lines expressing PDGFRalpha. The in vivo antitumor activity of 3G3 was tested in human glioblastoma (U118) and leiomyosarcoma (SKLMS-1) xenograft tumor models in athymic nude mice. Antibody 3G3 significantly inhibited the growth of U118 (P=0.0004) and SKLMS-1 (P <0.0001) tumors relative to control. These data suggest that 3G3 may be useful for the treatment of tumors that express PDGFRalpha.

ROCK2 signaling is required to induce a subset of T follicular helper cells through opposing effects on STATs in autoimmune settings.

Rho-associated kinase 2 (ROCK2) determines the balance between human T helper 17 (TH17) cells and regulatory T (Treg) cells. We investigated its role in the generation of T follicular helper (TFH) cells, which help to generate antibody-producing B cells under normal and autoimmune conditions. Inhibiting ROCK2 in normal human T cells or peripheral blood mononuclear cells from patients with active systemic lupus erythematosus (SLE) decreased the number and function of TFH cells induced by activation ex vivo. Moreover, inhibition of ROCK2 activity decreased the abundance of the transcriptional regulator Bcl6 (B cell lymphoma 6) and increased that of Blimp1 by reducing the binding of signal transducer and activator of transcription 3 (STAT3) and increasing that of STAT5 to the promoters of the genes Bcl6 and PRDM1, respectively. In the MRL/lpr murine model of SLE, oral administration of the selective ROCK2 inhibitor KD025 resulted in a twofold reduction in the numbers of TFH cells and antibody-producing plasma cells in the spleen, as well as a decrease in the size of splenic germinal centers, which are the sites of interaction between TFH cells and B cells. KD025-treated mice showed a substantial improvement in both histological and clinical scores compared to those of untreated mice and had reduced amounts of Bcl6 and phosphorylated STAT3, as well as increased STAT5 phosphorylation. Together, these data suggest that ROCK2 signaling plays a critical role in controlling the development of TFH cells induced by autoimmune conditions through reciprocal regulation of STAT3 and STAT5 activation.

Mice deficient in fractalkine are less susceptible to cerebral ischemia-reperfusion injury.

Fractalkine (FKN), also known as neurotactin, is a CX(3)C chemokine that exists in both secreted and neuronal membrane-bound forms and is upregulated during brain inflammation. There is accumulating evidence that FKN induces chemotaxis by binding to its receptor CX(3)CR1 on leukocytes and microglia. We generated FKN-deficient mice to study the role of FKN in postischemic brain injury. After transient focal cerebral ischemia, FKN-deficient mice had a 28% reduction in infarction size and lower mortality rate, when compared to wild-type littermates. The findings of this study indicate a possible role for FKN in augmenting postischemic injury and mortality after transient focal cerebral ischemia.

Anti GPVI human antibodies neutralizing collagen-induced platelet aggregation isolated from a combinatorial phage display library.

Glycoprotein VI is a type I membrane protein identified as a key platelet receptor for collagen. In vitro binding of the GPVI receptor with collagen leads to activation and ultimately to aggregation of platelets. In vivo, GPVI-collagen interactions could cause formation of occlusive thrombi within vessels with damaged endothelial barriers. GPVI antagonists are therefore important therapeutics in patients suffering from collagen-mediated ischemic disorders such as myocardial infarction or stroke. Polyclonal antibodies to GPVI prepared from one patient serum have previously been described. However, only their monovalent Fab fragments, incapable of receptor crosslinking, were found to protect platelets from collagen-mediated aggregation. Here we describe GPVI-neutralizing human antibodies derived from a combinatorial phage display library of single-chain antibodies. By selecting phage on GPVI-expressing U937 cells, we isolated five specific antibodies - A4, A9, A10, C3 and C9. Of the set A10 and C3 specifically blocked GPVI binding to collagen-rich adventitial layers in aorta sections. The higher affinity antibody A10 inhibited binding of snake-venom convulxin to GPVI. It also specifically protected human platelets from collagen-induced aggregation in vitro. A10-bound platelets could still be activated by ADP or thrombin suggesting that this human scFv may represent an original anti-platelet agent for the treatment of collagen-mediated thrombotic diseases.