The development of bone‐rebuilding anabolic agents for potential use in the treatment of bone loss conditions, such as osteoporosis, has been a long‐standing goal. Genetic studies in humans and mice ...have shown that the secreted protein sclerostin is a key negative regulator of bone formation, although the magnitude and extent of sclerostin's role in the control of bone formation in the aging skeleton is still unclear. To study this unexplored area of sclerostin biology and to assess the pharmacologic effects of sclerostin inhibition, we used a cell culture model of bone formation to identify a sclerostin neutralizing monoclonal antibody (Scl‐AbII) for testing in an aged ovariectomized rat model of postmenopausal osteoporosis. Six‐month‐old female rats were ovariectomized and left untreated for 1 yr to allow for significant estrogen deficiency‐induced bone loss, at which point Scl‐AbII was administered for 5 wk. Scl‐AbII treatment in these animals had robust anabolic effects, with marked increases in bone formation on trabecular, periosteal, endocortical, and intracortical surfaces. This not only resulted in complete reversal, at several skeletal sites, of the 1 yr of estrogen deficiency‐induced bone loss, but also further increased bone mass and bone strength to levels greater than those found in non‐ovariectomized control rats. Taken together, these preclinical results establish sclerostin's role as a pivotal negative regulator of bone formation in the aging skeleton and, furthermore, suggest that antibody‐mediated inhibition of sclerostin represents a promising new therapeutic approach for the anabolic treatment of bone‐related disorders, such as postmenopausal osteoporosis.
Peptibodies or peptide-Fc fusions are an attractive alternative therapeutic format to monoclonal antibodies. They consist of biologically active peptides grafted onto an Fc domain. This approach ...retains certain desirable features of antibodies, notably an increased apparent affinity through the avidity conferred by the dimerization of two Fcs and a long plasma residency time. Peptibodies can be made in E. coli using recombinant technology. The manufacturing process involves fermentation and downstream processing, including refolding and multiple column chromatographic steps, that result in overall yields and quality suitable for commercial development. Romiplostim, marketed under the brand name Nplate®, is the first peptibody to be approved by the United States Food and Drug Administration and the European Medicines Agency and is indicated for the treatment of immune thrombocytopenic purpura. AMG 386, a peptibody antagonist to angiopoietins 1 and 2, is being evaluated in Phase 3 clinical testing in combination with chemotherapy in women with ovarian cancer. AMG 819, a peptibody targeting nerve growth factor for pain has also progressed to clinical trials. These peptibodies illustrate the versatility of the modality.
AMG 386 is an investigational first-in-class peptide-Fc fusion protein (peptibody) that inhibits angiogenesis by preventing the interaction of angiopoietin-1 (Ang1) and Ang2 with their receptor, ...Tie2. Although the therapeutic value of blocking Ang2 has been shown in several models of tumorigenesis and angiogenesis, the potential benefit of Ang1 antagonism is less clear. To investigate the consequences of Ang1 neutralization, we have developed potent and selective peptibodies that inhibit the interaction between Ang1 and its receptor, Tie2. Although selective Ang1 antagonism has no independent effect in models of angiogenesis-associated diseases (cancer and diabetic retinopathy), it induces ovarian atrophy in normal juvenile rats and inhibits ovarian follicular angiogenesis in a hormone-induced ovulation model. Surprisingly, the activity of Ang1 inhibitors seems to be unmasked in some disease models when combined with Ang2 inhibitors, even in the context of concurrent vascular endothelial growth factor inhibition. Dual inhibition of Ang1 and Ang2 using AMG 386 or a combination of Ang1- and Ang2-selective peptibodies cooperatively suppresses tumor xenograft growth and ovarian follicular angiogenesis; however, Ang1 inhibition fails to augment the suppressive effect of Ang2 inhibition on tumor endothelial cell proliferation, corneal angiogenesis, and oxygen-induced retinal angiogenesis. In no case was Ang1 inhibition shown to (a) confer superior activity to Ang2 inhibition or dual Ang1/2 inhibition or (b) antagonize the efficacy of Ang2 inhibition. These results imply that Ang1 plays a context-dependent role in promoting postnatal angiogenesis and that dual Ang1/2 inhibition is superior to selective Ang2 inhibition for suppression of angiogenesis in some postnatal settings.
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.
In 2018, the United States Food and Drug Administration (FDA) approved Aimovig (erenumab) for the prevention of migraine. Erenumab is the first FDA approved antibody therapeutic against a ...G-protein-coupled receptor, the canonical receptor of calcitonin gene related peptide (CGRP-R). A novel, epitope-focused antigen was created to reconstruct the extracellular domains of the CGRP-R in a stable conformation. Successful inoculation of XenoMouse animals and careful screening yielded multiple candidate molecules for high potency and exquisite selectivity toward the CGRP-R over related receptors. These efforts led to the discovery of erenumab which has demonstrated the desired efficacy and safety profiles in multiple clinical studies for the prevention of migraine. The innovation developed in the discovery of erenumab furthers the ability to target G-coupled protein receptors using antibody approaches.
We and others recently reported tumor necrosis factor (TNF) and apoptosis ligand-related leukocyte-expressed ligand 1 (TALL-1) as a novel member of the TNF ligand family that is functionally involved ...in B cell proliferation. Transgenic mice overexpressing TALL-1 have severe B cell hyperplasia and lupus-like autoimmune disease. Here, we describe expression cloning of a cell surface receptor for TALL-1 from a human Burkitt's lymphoma RAJI cell library. The cloned receptor is identical to the previously reported TNF receptor (TNFR) homologue transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI). Murine TACI was subsequently isolated from the mouse B lymphoma A20 cells. Human and murine TACI share 54% identity overall. Human TACI exhibits high binding affinities to both human and murine TALL-1. Soluble TACI extracellular domain protein specifically blocks TALL-1-mediated B cell proliferation without affecting CD40- or lipopolysaccharide-mediated B cell proliferation in vitro. In addition, when injected into mice, soluble TACI inhibits antibody production to both T cell-dependent and -independent antigens. By yeast two-hybrid screening of a B cell library with TACI intracellular domain, we identified that, like many other TNFR family members, TACI intracellular domain interacts with TNFR-associated factor (TRAF)2, 5, and 6. Correspondingly, TACI activation in a B cell line results in nuclear factor kappaB and c-Jun NH(2)-terminal kinase activation. The identification and characterization of the receptor for TALL-1 provides useful information for the development of a treatment for B cell-mediated autoimmune diseases such as systemic lupus erythematosus.
IL-18-binding protein (IL-18BP) is a natural IL-18 inhibitor. Human IL-18BP isoform a was produced as fusion construct with human IgG1 Fc and assessed for binding and neutralizing IL-18. IL-18BP-Fc ...binds human, mouse, and rat IL-18 with high affinity (K(D) 0.3-5 nM) in a BIAcore-based assay. In vitro, IL-18BP-Fc blocks IL-18 (100 ng/ml)-induced IFN-gamma production by KG1 cells (EC(50) = 0.3 microg/ml). In mice challenged with an LD(90) of LPS (15 mg/kg), IL-18BP-Fc (5 mg/kg) administered 10 min before LPS blocks IFN-gamma production and protects against lethality. IL-18BP-Fc administered 10 min before LPS blocks IFN-gamma production induced by LPS (5 mg/kg) with ED(50) of 0.005 mg/kg. Furthermore, IL-18BP-Fc (5 mg/kg) abrogates LPS (5 mg/kg)-induced IFN-gamma production even when administered 6 days before LPS but shows no effect when administered 9 or 12 days before LPS. Given 10 min before LPS challenge to mice primed 12 days in advance with heat-killed Propionibacterium acnes, IL-18BP-Fc prevents LPS-induced liver damage and IFN-gamma and Fas ligand expression. Given at the moment of priming with P. acnes, IL-18BP-Fc decreases P. acnes-induced granuloma formation, macrophage-inflammatory protein-1alpha and macrophage-inflammatory protein-2 production and prevents sensitization to LPS. IL-18BP-Fc also prevents Con A-induced liver damage and IFN-gamma and Fas ligand expression as well as liver damage induced by Pseudomonas aeruginosa exotoxin A or by anti-Fas agonistic Ab. In conclusion, IL-18BP can be engineered and produced in recombinant form to generate an IL-18 inhibitor, IL-18BP-Fc, endowed with remarkable in vitro and in vivo properties of binding and neutralizing IL-18.
Skeletal anabolism with PTH is achieved through daily injections that result in brief exposure to the peptide. We hypothesized that similar anabolic effects could be achieved with less frequent but ...more sustained exposures to PTH. A PTH‐Fc fusion protein with a longer half‐life than PTH(1–34) increased cortical and cancellous BMD and bone strength with once‐ or twice‐weekly injections.
Introduction: The anabolic effects of PTH are currently achieved with, and thought to require, daily injections that result in brief exposure to the peptide. We hypothesized that less frequent but more sustained exposures to PTH could also be anabolic for bone, provided that serum levels of PTH were not constant.
Materials and Methods: PTH(1–34) was fused to the Fc fragment of human IgG1 to increase the half‐life of PTH. Skeletal anabolism was examined in mice and rats treated once or twice per week with this PTH‐Fc fusion protein.
Results: PTH‐Fc and PTH(1–34) had similar effects on PTH/PTHrP receptor activation, internalization, and signaling in vitro. However, PTH‐Fc had a 33‐fold longer mean residence time in the circulation of rats compared with that of PTH(1–34). Subcutaneous injection of PTH‐Fc once or twice per week resulted in significant increases in bone volume, density, and strength in osteopenic ovariectomized mice and rats. These anabolic effects occurred in association with hypercalcemia and were significantly greater than those achievable with high concentrations of daily PTH(1–34). PTH‐Fc also significantly improved cortical bone volume and density under conditions where daily PTH(1–34) did not. Antiresorptive co‐therapy with estrogen further enhanced the ability of PTH‐Fc to increase bone mass and strength in ovariectomized rats.
Conclusions: These results challenge the notion that brief daily exposure to PTH is essential for its anabolic effects on cortical and cancellous bone. PTH‐derived molecules with a sustained circulating half‐life may represent a powerful and previously undefined anabolic regimen for cortical and cancellous bone.
Optimal T cell activation requires the interactions of co-stimulatory molecules, such as those in the CD28 and B7 protein families. Recently, we described the co-stimulatory properties of the murine ...ligand to ICOS, which we designated as B7RP-1. Here, we report the co-stimulation of human T cells through the human B7RP-1 and ICOS interaction. This ligand–receptor pair interacts with a KD ~ 33 nM and an off-rate with a t½ > 10 min. Interestingly, tumor necrosis factor (TNF)-α differentially regulates the expression of human B7RP-1 on B cells, monocytes and dendritic cells (DC). TNF-α enhances B7RP-1 expression on B cells and monocytes, while it inhibits it on DC. The human B7RP-1–Fc protein or cells that express membrane-bound B7RP-1 co-stimulate T cell proliferation in vitro. Specific cytokines, such as IFN-γ and IL-10, are induced by B7RP-1 co-stimulation. Although IL-2 levels are not significantly increased, B7RP-1 co-stimulation is dependent on IL-2. These experiments define the human ortholog to murine B7RP-1 and characterize its interaction with human ICOS.
A novel human chemokine STCP-1 (stimulated T cell chemotactic protein) was isolated from an activated macrophage cDNA library. The chemokine has four cysteines positioned in a manner that identifies ...STCP-1 as a member of the CC chemokine family. The amino acid sequence shows 34% identity with RANTES. The gene consists of 3 exons and 2 introns with the position of intron/exon boundaries similar to that of RANTES. The gene is expressed as a 3.4-kilobase transcript on lymph node, thymus, and Appendix. STCP-1 induces Ca2+ mobilization in a small percentage of primary activated T lymphocytes, but on repeated stimulation the percentage of T lymphocytes that respond to STCP-1 increases. The chemokine STCP-1 does not induce Ca2+ mobilization in monocytes, dendritic cells, neutrophils, eosinophils, lipopolysaccharide-activated B lymphocytes, and freshly isolated resting T lymphocytes. Similarly, STCP-1, while acting as a mild chemoattractant for primary activated T lymphocytes, is a potent chemoattractant for chronically activated T lymphocytes but has no chemoattractant activity for monocytes, neutrophils, eosinophils, and resting T lymphocytes. As STCP-1 acts specifically on activated T lymphocytes, it may play a role in the trafficking of activated/effector T lymphocytes to inflammatory sites and other aspects of activated T lymphocyte physiology.
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