Aaron George Winters

Sclerostin Antibody Treatment Increases Bone Formation, Bone Mass, and Bone Strength in a Rat Model of Postmenopausal Osteoporosis

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 sclerostins 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 sclerostins 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.

Antihepcidin antibody treatment modulates iron metabolism and is effective in a mouse model of inflammation-induced anemia

Iron maldistribution has been implicated in multiple diseases, including the anemia of inflammation (AI), atherosclerosis, diabetes, and neurodegenerative disorders. Iron metabolism is controlled by hepcidin, a 25-amino acid peptide. Hepcidin is induced by inflammation, causes iron to be sequestered, and thus, potentially contributes to AI. Human hepcidin (hHepc) overexpression in mice caused an iron-deficient phenotype, including stunted growth, hair loss, and iron-deficient erythropoiesis. It also caused resistance to supraphysiologic levels of erythropoiesis-stimulating agent, supporting the hypothesis that hepcidin may influence response to treatment in AI. To explore the role of hepcidin in inflammatory anemia, a mouse AI model was developed with heat-killed Brucella abortus treatment. Suppression of hepcidin mRNA was a successful anemia treatment in this model. High-affinity antibodies specific for hHepc were generated, and hHepc knock-in mice were produced to enable antibody testing. Antibody treatment neutralized hHepc in vitro and in vivo and facilitated anemia treatment in hHepc knock-in mice with AI. These data indicate that antihepcidin antibodies may be an effective treatment for patients with inflammatory anemia. The ability to manipulate iron metabolism in vivo may also allow investigation of the role of iron in a number of other pathologic conditions.

A fully human anti-hepcidin antibody modulates iron metabolism in both mice and nonhuman primates

Iron maldistribution has been implicated in the etiology of many diseases including the anemia of inflammation (AI), atherosclerosis, diabetes, and neurodegenerative disorders. Iron metabolism is controlled by hepcidin, a 25-amino-acid peptide. Hepcidin is induced by inflammation and causes iron to be sequestered within cells of the reticuloendothelial system, suppressing erythropoiesis and blunting the activity of erythropoiesis stimulating agents (ESAs). For this reason, neutralization of hepcidin has been proposed as a therapeutic treatment of AI. The aim of the current work was to generate fully human anti-hepcidin antibodies (Abs) as a potential human therapeutic for the treatment of AI and other iron maldistribution disorders. An enzyme-linked immunosorbent assay was established using these Abs to identify patients likely to benefit from either ESAs or anti-hepcidin agents. Using human hepcidin knock-in mice, the mechanism of action of the Abs was shown to be due to an increase in available serum iron leading to enhanced red cell hemoglobinization. One of the Abs, 12B9m, was validated in a mouse model of AI and demonstrated to modulate serum iron in cynomolgus monkeys. The 12B9m Ab was deemed to be an appropriate candidate for use as a potential therapeutic to treat AI in patients with kidney disease or cancer.

Dickkopf-1 regulates bone formation in young growing rodents and upon traumatic injury.

The physiological role of Dickkopf‐1 (Dkk1) during postnatal bone growth in rodents and in adult rodents was examined utilizing an antibody to Dkk1 (Dkk1‐Ab) that blocked Dkk1 binding to both low density lipoprotein receptor‐related protein 6 (LRP6) and Kremen2, thereby preventing the Wnt inhibitory activity of Dkk1. Treatment of growing mice and rats with Dkk1‐Ab resulted in a significant increase in bone mineral density because of increased bone formation. In contrast, treatment of adult ovariectomized rats did not appreciably impact bone, an effect that was associated with decreased Dkk1 expression in the serum and bone of older rats. Finally, we showed that Dkk1 plays a prominent role in adult bone by mediating fracture healing in adult rodents. These data suggest that, whereas Dkk1 significantly regulates bone formation in younger animals, its role in older animals is limited to pathologies that lead to the induction of Dkk1 expression in bone and/or serum, such as traumatic injury.

An antibody to IP-10 is a potent antagonist of cell migration in vitro and in vivo and does not affect disease in several animal models of inflammation

IP-10 secretion is induced by pro-inflammatory cytokines and mediates the migration of CXCR3+ cells. Its elevation in clinical samples has been associated with multiple inflammatory diseases and its antagonism has been reported to be effective in several animal models of inflammatory disease. We generated a mouse anti-mouse IP-10 monoclonal antibody (mAb; Clone 20A9) that specifically bound murine IP-10 with high affinity and inhibited in vitro IP-10 induced BaF3/mCXCR3 cell migration with an IC50 of ∼4 nM. The 20A9 mAb was completely absorbed in vivo and had dose proportional pharmacokinetic exposure with a serum half life of 2.4–6 days. The 20A9 mAb inhibited IP-10 mediated T-cell recruitment to the airways, indicating that it is effective in vivo. However, administration of the 20A9 mAb had no significant effect on disease in mouse models of delayed type hypersensitivity, collagen induced arthritis, cardiac allograft transplantation tolerance, EAE or CD4+ CD45RBHi T-cell transfer-induced IBD. These data suggest that the 20A9 mAb can antagonize IP-10 mediated chemotaxis in vitro and in vivo and that this is insufficient to cause a therapeutic benefit in multiple mouse models of inflammatory disease.

Inhibition of WISE Preserves Renal Allograft Function

Wnt-modulator in surface ectoderm (WISE) is a secreted modulator of Wnt signaling expressed in the adult kidney. Activation of Wnt signaling has been observed in renal transplants developing interstitial fibrosis and tubular atrophy; however, whether WISE contributes to chronic changes is not well understood. Here, we found moderate to high expression of WISE mRNA in a rat model of renal transplantation and in kidneys from normal rats. Treatment with a neutralizing antibody against WISE improved proteinuria and graft function, which correlated with higher levels of β-catenin protein in kidney allografts. In addition, treatment with the anti-WISE antibody reduced infiltration of CD68(+) macrophages and CD8(+) T cells, attenuated glomerular and interstitial injury, and decreased biomarkers of renal injury. This treatment reduced expression of genes involved in immune responses and in fibrogenic pathways. In summary, WISE contributes to renal dysfunction by promoting tubular atrophy and interstitial fibrosis.

Identification of Antibody and Small Molecule Antagonists of Ferroportin-Hepcidin Interaction

The iron exporter ferroportin and its ligand, the hormone hepcidin, control fluxes of stored and recycled iron for use in a variety of essential biochemical processes. Inflammatory disorders and malignancies are often associated with high hepcidin levels, leading to ferroportin down-regulation, iron sequestration in tissue macrophages and subsequent anemia. The objective of this research was to develop reagents to characterize the expression of ferroportin, the interaction between ferroportin and hepcidin, as well as to identify novel ferroportin antagonists capable of maintaining iron export in the presence of hepcidin. Development of investigative tools that enabled cell-based screening assays is described in detail, including specific and sensitive monoclonal antibodies that detect endogenously-expressed human and mouse ferroportin and fluorescently-labeled chemically-synthesized human hepcidin. Large and small molecule antagonists inhibiting hepcidin-mediated ferroportin internalization were identified, and unique insights into the requirements for interaction between these two key iron homeostasis molecules are provided.

A multifactorial screening strategy to identify anti-idiotypic reagents for bioanalytical support of antibody therapeutics.

Antibodies are critical tools for protein bioanalysis; their quality and performance dictate the caliber and robustness of ligand binding assays. After immunization, polyclonal B cells generate a diverse antibody repertoire against constant and variable regions of the therapeutic antibody immunogen. Herein we describe a comprehensive and multifactorial screening strategy to eliminate undesirable constant region-specific antibodies and select for anti-idiotypic antibodies with specificity for the unique variable region. Application of this strategy is described for the therapeutic antibody Mab-A case study. Five different factors were evaluated to select a final antibody pair for the quantification of therapeutics in biological matrices: (i) matrix effect in preclinical and clinical matrices, (ii) assay sensitivity with lower limit of quantification goal of single-digit ng/ml (low pM) at a signal-to-background ratio greater than 5, (iii) epitope distinction or nonbridging antibody pair, (iv) competition with target and inhibitory capacity enabling measurement of free drug, and (v) neutralizing bioactivity using bioassay. The selected antibody pair demonstrated superior assay sensitivity with no or minimal matrix effect in common biological samples, recognized two distinct binding epitopes on the therapeutic antibody variable region, and featured inhibitory and neutralizing effects with respect to quantification of free drug levels.

Novel Anti-c-Mpl Monoclonal Antibodies Identified Multiple Differentially Glycosylated Human c-Mpl Proteins in Megakaryocytic Cells But Not in Human Solid Tumors

Thrombopoietin and its cognate receptor, c-Mpl, are the primary molecular regulators of megakaryocytopoiesis and platelet production. To date the pattern of c-Mpl expression in human solid tumors and the distribution and biochemical properties of c-Mpl proteins in hematopoietic tissues are largely unknown. We have recently developed highly specific mouse monoclonal antibodies (MAb) against human c-Mpl. In this study we used these antibodies to demonstrate the presence of full-length and truncated human c-Mpl proteins in various megakaryocytic cell types, and their absence in over 100 solid tumor cell lines and in the 12 most common primary human tumor types. Quantitative assays showed a cell context-dependent distribution of full-length and truncated c-Mpl proteins. All forms of human c-Mpl protein were found to be modified with extensive N-linked glycosylation but different degrees of sialylation and O-linked glycosylation. Of note, different variants of full-length c-Mpl protein exhibiting differential glycosylation were expressed in erythromegakaryocytic leukemic cell lines and in platelets from healthy human donors. This work provides a comprehensive analysis of human c-Mpl mRNA and protein expression on normal and malignant hematopoietic and non-hematopoietic cells and demonstrates the multiple applications of several novel anti-c-Mpl antibodies.