Gary Elliott

Osteoprotegerin Ligand Is a Cytokine that Regulates Osteoclast Differentiation and Activation

The ligand for osteoprotegerin has been identified, and it is a TNF-related cytokine that replaces the requirement for stromal cells, vitamin D3, and glucocorticoids in the coculture model of in vitro osteoclastogenesis. OPG ligand (OPGL) binds to a unique hematopoeitic progenitor cell that is committed to the osteoclast lineage and stimulates the rapid induction of genes that typify osteoclast development. OPGL directly activates isolated mature osteoclasts in vitro, and short-term administration into normal adult mice results in osteoclast activation associated with systemic hypercalcemia. These data suggest that OPGL is an osteoclast differentiation and activation factor. The effects of OPGL are blocked in vitro and in vivo by OPG, suggesting that OPGL and OPG are key extracellular regulators of osteoclast development.

T-cell co-stimulation through B7RP-1 and ICOS.

T-cell activation requires co-stimulation through receptors such as CD28 (refs 1,2,3) and antigen-specific signalling through the T-cell antigen receptor. Here we describe a new murine co-stimulatory receptor–ligand pair. The receptor, which is related to CD28 and is the homologue of the human protein ICOS, is expressed on activated T cells and resting memory T cells. The ligand, which has homology to B7 molecules and is called B7-related protein-1 (B7RP-1), is expressed on B cells and macrophages. ICOS and B7RP-1 do not interact with proteins in the CD28–B7 pathway, and B7RP-1 co-stimulates T cells in vitro independently of CD28. Transgenic mice expressing a B7RP-1–Fc fusion protein show lymphoid hyperplasia in the spleen, lymph nodes and Peyers patches. Presensitized mice treated with B7RP-1–Fc during antigen challenge show enhanced hypersensitivity. Therefore, B7RP-1 exhibits co-stimulatory activities in vitro and in vivo. ICOS and B7RP-1 define a new and distinct receptor–ligand pair that is structurally related to CD28–B7 and is involved in the adaptive immune response.

Bendamustine (Treanda) Displays a Distinct Pattern of Cytotoxicity and Unique Mechanistic Features Compared with Other Alkylating Agents

Purpose: Bendamustine has shown clinical activity in patients with disease refractory to conventional alkylator chemotherapy. The purpose of this study was to characterize the mechanisms of action of bendamustine and to compare it with structurally related compounds. Experimental Design: Bendamustine was profiled in the National Cancer Institute in vitro antitumor screen. Microarray-based gene expression profiling, real-time PCR, immunoblot, cell cycle, and functional DNA damage repair analyses were used to characterize response to bendamustine and compare it with chlorambucil and phosphoramide mustard. Results: Bendamustine displays a distinct pattern of activity unrelated to other DNA-alkylating agents. Its mechanisms of action include activation of DNA-damage stress response and apoptosis, inhibition of mitotic checkpoints, and induction of mitotic catastrophe. In addition, unlike other alkylators, bendamustine activates a base excision DNA repair pathway rather than an alkyltransferase DNA repair mechanism. Conclusion: These results suggest that bendamustine possesses mechanistic features that differentiate it from other alkylating agents and may contribute to its distinct clinical efficacy profile.

Fully Human Monoclonal Antibodies to Hepatocyte Growth Factor with Therapeutic Potential against Hepatocyte Growth Factor/c-Met–Dependent Human Tumors

c-Met is a well-characterized receptor tyrosine kinase for hepatocyte growth factor (HGF). Compelling evidence from studies in human tumors and both cellular and animal tumor models indicates that signaling through the HGF/c-Met pathway mediates a plethora of normal cellular activities, including proliferation, survival, migration, and invasion, that are at the root of cancer cell dysregulation, tumorigenesis, and tumor metastasis. Inhibiting HGF-mediated signaling may provide a novel therapeutic approach for treating patients with a broad spectrum of human tumors. Toward this goal, we generated and characterized five different fully human monoclonal antibodies that bound to and neutralized human HGF. Antibodies with subnanomolar affinities for HGF blocked binding of human HGF to c-Met and inhibited HGF-mediated c-Met phosphorylation, cell proliferation, survival, and invasion. Using a series of human-mouse chimeric HGF proteins, we showed that the neutralizing antibodies bind to a unique epitope in the beta-chain of human HGF. Importantly, these antibodies inhibited HGF-dependent autocrine-driven tumor growth and caused significant regression of established U-87 MG tumor xenografts. Treatment with anti-HGF antibody rapidly inhibited tumor cell proliferation and significantly increased the proportion of apoptotic U-87 MG tumor cells in vivo. These results suggest that an antibody to an epitope in the beta-chain of HGF has potential as a novel therapeutic agent for treating patients with HGF-dependent tumors.

Differential subcellular localization, expression and biological toxicity of BRCA1 and the splice variant BRCA1-Δ11b

The mechanism of BRCA1 tumor suppression in human breast and ovarian cells is the focus of intense investigation. In this report, full length BRCA1 (230 kDa) introduced into cells with CMV promoter constructs was nuclear when transgene expression was low whereas high expression resulted in cytoplasmic accumulation, aberrant nuclear and cell morphology. A nuclear localization signal (NLS) was mapped to BRCA1 amino acid positions 262 – 570. We describe a splice variant, BRCA1-Δ11b, missing the majority of exon 11 including the NLS. Exogenous BRCA1-Δ11b (110 kDa) was cytoplasmic and, unlike the full-length protein, overexpression of the protein encoded by the variant did not appear to be toxic. RNA probe titrations and RT – PCR demonstrated that BRCA1 and Δ11b transcripts are coexpressed in a wide variety of cells and tissues. Interestingly, BRCA1-Δ11b message was greatly reduced or absent in several breast and ovarian tumor lines relative to exon 11 transcripts and a Δ9,10 splice variant. Taken together our results suggest that full-length BRCA1 and BRCA1-Δ11b may have distinct roles in cell growth regulation and tumorigenesis.

CSMD1 Is a Novel Multiple Domain Complement-Regulatory Protein Highly Expressed in the Central Nervous System and Epithelial Tissues

In this study, we describe the identification and in vitro functional activity of a novel multiple domain complement regulatory protein discovered based on its homology to short consensus repeat (SCR)-containing proteins of the regulators of complement activation (RCA) gene family. The rat cDNA encodes a predicted 388-kDa protein consisting of 14 N-terminal CUB domains that are separated from each other by a SCR followed by 15 tandem SCR domains, a transmembrane domain, and a short cytoplasmic tail. This protein is the homolog of the human protein of unknown function called the CUB and sushi multiple domains 1 (CSMD1) protein. A cloning strategy that incorporates the two C-terminal CUB-SCR domains and 12 of the tandem SCR repeats was used to produce a soluble rat CSMD1 protein. This protein blocked classical complement pathway activation in a comparable fashion with rat Crry but did not block alternative pathway activation. Analysis of CSMD1 mRNA expression by in situ hybridization and immunolabeling of neurons indicates that the primary sites of synthesis are the developing CNS and epithelial tissues. Of particular significance is the enrichment of CSMD1 in the nerve growth cone, the amoeboid-leading edge of the growing neuron. These results suggest that CSMD1 may be an important regulator of complement activation and inflammation in the developing CNS, and that it may also play a role in the context of growth cone function.

Activation of the Erythropoietin (EPO) Receptor by Bivalent Anti-EPO Receptor Antibodies

Oligomerization of cytokine receptors including the erythropoietin (EPO) receptor has been advanced as a model for activation. If homodimerization of the EPO receptor activates it, then bivalent antibodies raised to the extracellular domain of the EPO receptor should also homodimerize and activate. Mouse monoclonal antibodies (IgG) raised to the soluble, extracellular domain of the human EPO receptor (EPOR) were found that would stimulate thymidine uptake of an human EPO-dependent cell line, UT-7/EPO. Dose response curves showed bell shapes where activity was low at low and high concentrations. Monovalent (Fab) fragments bound to the receptor but did not stimulate thymidine uptake, which indicates that two antibody binding sites are required for activation. The anti-EPOR antibodies stimulated the formation of burst forming unit erythroid colonies from human CD34+ cells purified from peripheral blood. This indicates that homodimerization of the EPO receptor by anti-EPOR antibodies is sufficient for both proliferation and differentiation of erythroid progenitor cells and that the constraints on dimerization necessary for activation are rather loose.

Biochemical Characterization of AMG 102: A Neutralizing, Fully Human Monoclonal Antibody to Human and Nonhuman Primate Hepatocyte Growth Factor

AMG 102 is a fully human monoclonal antibody that selectively targets and neutralizes hepatocyte growth factor/scatter factor (HGF/SF). A detailed biochemical and functional characterization of AMG 102 was done to support its clinical development for the treatment of cancers dependent on signaling through the HGF/SF:c-Met pathway. In competitive equilibrium binding experiments, AMG 102 bound to human and cynomolgus monkey HGF with affinities of approximately 19 pmol/L and 41 pmol/L, respectively. However, AMG 102 did not detect mouse or rabbit HGF on immunoblots. Immunoprecipitation experiments showed that AMG 102 preferentially bound to the mature, active form of HGF, and incubation of AMG 102/HGF complexes with kallikrein protease indicated that AMG 102 had no apparent effect on proteolytic processing of the inactive HGF precursor. AMG 102 inhibited human and cynomolgus monkey HGF-induced c-Met autophosphorylation in PC3 cells with IC50 values of 0.12 nmol/L and 0.24 nmol/L, respectively. AMG 102 also inhibited cynomolgus monkey HGF-induced migration of human MDA-MB-435 cells but not rat HGF-induced migration of mouse 4T1 cells. Epitope-mapping studies of recombinant HGF molecules comprising human/mouse chimeras and human-to-mouse amino acid substitutions showed that amino acid residues near the NH2-terminus of the β-chain are critical for AMG 102 binding. Bound AMG 102 protected one trypsin protease cleavage site near the NH2-terminus of the β-chain of human HGF, further substantiating the importance of this region for AMG 102 binding. Currently, AMG 102 is in phase II clinical trials in a variety of solid tumor indications. Mol Cancer Ther; 9(2); 400–9

Fully Human Monoclonal Antibodies Antagonizing the Glucagon Receptor Improve Glucose Homeostasis in Mice and Monkeys

Antagonizing the glucagon signaling pathway represents an attractive therapeutic approach for reducing excess hepatic glucose production in patients with type 2 diabetes. Despite extensive efforts, there is currently no human therapeutic that directly inhibits the glucagon/glucagon receptor pathway. We undertook a novel approach by generating high-affinity human monoclonal antibodies (mAbs) to the human glucagon receptor (GCGR) that display potent antagonistic activity in vitro and in vivo. A single injection of a lead antibody, mAb B, at 3 mg/kg, normalized blood glucose levels in ob/ob mice for 8 days. In addition, a single injection of mAb B dose-dependently lowered fasting blood glucose levels without inducing hypoglycemia and improved glucose tolerance in normal C57BL/6 mice. In normal cynomolgus monkeys, a single injection improved glucose tolerance while increasing glucagon and active glucagon-like peptide-1 levels. Thus, the anti-GCGR mAb could represent an effective new therapeutic for the treatment of type 2 diabetes.

Characterization of cells and gene-targeted mice deficient for the p53-binding kinase homeodomain-interacting protein kinase 1 (HIPK1)

The tumor suppressor p53 is regulated in part by binding to cellular proteins. We used p53 as bait in the yeast two-hybrid system and isolated homeodomain-interacting protein kinase 1 (HIPK1) as a p53-binding protein. Deletion analysis showed that amino acids 100–370 of p53 and amino acids 885-1093 of HIPK1 were sufficient for HIPK1–p53 interaction. HIPK1 was capable of autophosphorylation and specific serine phosphorylation of p53. The HIPK1 gene was highly expressed in human breast cancer cell lines and oncogenically transformed mouse embryonic fibroblasts. HIPK1 was localized to human chromosome band 1p13, a site frequently altered in cancers. Gene-targeted HIPK1−/− mice were grossly normal but oncogenically transformed HIPK1 −/− mouse embryonic fibroblasts exhibited reduced transcription of Mdm2 and were more susceptible than transformed HIPK1+/+ cells to apoptosis induced by DNA damage. Carcinogen-treated HIPK1 −/− mice developed fewer and smaller skin tumors than HIPK1+/+ mice. HIPK1 may thus play a role in tumorigenesis, perhaps by means of the regulation of p53 and/or Mdm2.