Vinay Bhaskar

Monoclonal antibodies targeting IL-1 beta reduce biomarkers of atherosclerosis in vitro and inhibit atherosclerotic plaque formation in Apolipoprotein E-deficient mice

OBJECTIVE Atherosclerosis is a condition that is increasingly contributing to worldwide mortality through complications such as stroke and myocardial infarction. IL-1β plays multiple direct, local roles in the formation and stability of the atheroma by eliciting the production of additional cytokines and proteolytic enzymes from macrophages, endothelial cells (EC) and smooth muscle cells (SMC). We therefore tested whether an anti-IL-1β antibody, XOMA 052, might inhibit the secretion of pro-atherogenic cytokines from macrophages in vitro and affect a positive outcome in the Apolipoprotein E-deficient mouse (ApoE(-/-)) model of atherosclerosis in vivo. METHODS AND RESULTS In an in vitro co-culture model, XOMA 052 inhibited macrophage-induced secretion of key atherogenic cytokines from EC and SMC, including IL-6, IL-8, MCP-1 and TNFα. The release of degradative enzymes, such as the matrix metalloproteinases MMP-3 and MMP-9, was also decreased by XOMA 052. In addition, XOMA 052 inhibited the secretion of IL-7 from EC and IL-4 from SMC, cytokines not previously reported to be driven by IL-1β in this context. In vivo, XMA052 MG1K, a chimeric murine version of XOMA 052, inhibited the formation of atherosclerotic lesions in the ApoE(-/-) model at all three doses tested. This effect was comparable to that reported for complete genetic ablation of IL-1β or IL-1R1 on an ApoE(-/-) background and was associated with decreases in plasma non-HDL/HDL cholesterol ratio and plaque lipid content and macrophage infiltration. CONCLUSIONS These results demonstrate for the first time that an antibody targeting IL-1β can inhibit the progression of atherosclerosis in vivo, highlighting the importance of this key cytokine in cardiovascular disease.

A Fully Human, Allosteric Monoclonal Antibody That Activates the Insulin Receptor and Improves Glycemic Control

Many patients with diabetes mellitus (both type 1 and type 2) require therapy to maintain normal fasting glucose levels. To develop a novel treatment for these individuals, we used phage display technology to target the insulin receptor (INSR) complexed with insulin and identified a high affinity, allosteric, human monoclonal antibody, XMetA, which mimicked the glucoregulatory, but not the mitogenic, actions of insulin. Biophysical studies with cultured cells expressing human INSR demonstrated that XMetA acted allosterically and did not compete with insulin for binding to its receptor. XMetA was found to function as a specific partial agonist of INSR, eliciting tyrosine phosphorylation of INSR but not the IGF-IR. Although this antibody activated metabolic signaling, leading to enhanced glucose uptake, it neither activated Erk nor induced proliferation of cancer cells. In an insulin resistant, insulinopenic model of diabetes, XMetA markedly reduced elevated fasting blood glucose and normalized glucose tolerance. After 6 weeks, significant improvements in HbA1c, dyslipidemia, and other manifestations of diabetes were observed. It is noteworthy that hypoglycemia and weight gain were not observed during these studies. These studies indicate, therefore, that allosteric monoclonal antibodies have the potential to be novel, ultra-long acting, agents for the regulation of hyperglycemia in diabetes.

XMetA, an allosteric monoclonal antibody to the insulin receptor, improves glycaemic control in mice with diet-induced obesity.

XMetA, a high‐affinity, fully human monoclonal antibody, allosterically binds to and activates the insulin receptor (INSR). Previously, we found that XMetA normalized fasting glucose and glucose tolerance in insulinopenic mice. To determine whether XMetA is also beneficial for reducing hyperglycaemia due to the insulin resistance of obesity, we have now evaluated XMetA in hyperinsulinemic mice with diet‐induced obesity. XMetA treatment of these mice normalized fasting glucose for 4 weeks without contributing to weight gain. XMetA also corrected glucose tolerance and improved non‐high density lipoprotein cholesterol. These studies indicate, therefore, that monoclonal antibodies that allosterically activate the INSR, such as XMetA, have the potential to be novel agents for the treatment of hyperglycaemia in conditions associated with the insulin resistance of obesity.

Inhibition of insulin receptor function by a human, allosteric monoclonal antibody: A potential new approach for the treatment of hyperinsulinemic hypoglycemia

Novel therapies are needed for the treatment of hypoglycemia resulting from both endogenous and exogenous hyperinsulinema. To provide a potential new treatment option, we identified XMetD, an allosteric monoclonal antibody to the insulin receptor (INSR) that was isolated from a human antibody phage display library. To selectively obtain antibodies directed at allosteric sites, panning of the phage display library was conducted using the insulin-INSR complex. Studies indicated that XMetD bound to the INSR with nanomolar affinity. Addition of insulin reduced the affinity of XMetD to the INSR by 3-fold, and XMetD reduced the affinity of the INSR for insulin 3-fold. In addition to inhibiting INSR binding, XMetD also inhibited insulin-induced INSR signaling by 20- to 100-fold. These signaling functions included INSR autophosphorylation, Akt activation and glucose transport. These data indicated that XMetD was an allosteric antagonist of the INSR because, in addition to inhibiting the INSR via modulation of binding affinity, it also inhibited the INSR via modulation of signaling efficacy. Intraperitoneal injection of XMetD at 10 mg/kg twice weekly into normal mice induced insulin resistance. When sustained-release insulin implants were placed into normal mice, they developed fasting hypoglycemia in the range of 50 mg/dl. This hypoglycemia was reversed by XMetD treatment. These studies demonstrate that allosteric monoclonal antibodies, such as XMetD, can antagonize INSR signaling both in vitro and in vivo. They also suggest that this class of allosteric monoclonal antibodies has the potential to treat hyperinsulinemic hypoglycemia resulting from conditions such as insulinoma, congenital hyperinsulinism and insulin overdose.

Improved Glucose Metabolism In Vitro and In Vivo by an Allosteric Monoclonal Antibody That Increases Insulin Receptor Binding Affinity

Previously we reported studies of XMetA, an agonist antibody to the insulin receptor (INSR). We have now utilized phage display to identify XMetS, a novel monoclonal antibody to the INSR. Biophysical studies demonstrated that XMetS bound to the human and mouse INSR with picomolar affinity. Unlike monoclonal antibody XMetA, XMetS alone had little or no agonist effect on the INSR. However, XMetS was a strong positive allosteric modulator of the INSR that increased the binding affinity for insulin nearly 20-fold. XMetS potentiated insulin-stimulated INSR signaling ∼15-fold or greater including; autophosphorylation of the INSR, phosphorylation of Akt, a major enzyme in the metabolic pathway, and phosphorylation of Erk, a major enzyme in the growth pathway. The enhanced signaling effects of XMetS were more pronounced with Akt than with Erk. In cultured cells, XMetS also enhanced insulin-stimulated glucose transport. In contrast to its effects on the INSR, XMetS did not potentiate IGF-1 activation of the IGF-1 receptor. We studied the effect of XMetS treatment in two mouse models of insulin resistance and diabetes. The first was the diet induced obesity mouse, a hyperinsulinemic, insulin resistant animal, and the second was the multi-low dose streptozotocin/high-fat diet mouse, an insulinopenic, insulin resistant animal. In both models, XMetS normalized fasting blood glucose levels and glucose tolerance. In concert with its ability to potentiate insulin action at the INSR, XMetS reduced insulin and C-peptide levels in both mouse models. XMetS improved the response to exogenous insulin without causing hypoglycemia. These data indicate that an allosteric monoclonal antibody can be generated that markedly enhances the binding affinity of insulin to the INSR. These data also suggest that an INSR monoclonal antibody with these characteristics may have the potential to both improve glucose metabolism in insulinopenic type 2 diabetes mellitus and correct compensatory hyperinsulinism in insulin resistant conditions.

Selective Allosteric Antibodies to the Insulin Receptor for the Treatment of Hyperglycemic and Hypoglycemic Disorders

Many therapeutic monoclonal antibodies act as antagonists to receptors by targeting and blocking the natural ligand binding site (orthosteric site). In contrast, the use of antibodies to target receptors at allosteric sites (distinct from the orthosteric site) has not been extensively studied. This approach is especially important in metabolic diseases in which endogenous ligand levels are dysregulated. Herein, we review our investigations of 3 categories of human monoclonal antibodies that bind allosterically to the insulin receptor (INSR) and affect its activity: XMetA, XMetS and XMetD. XMetA directly activates the INSR either alone or in combination with insulin. XMetS, in contrast, does not directly activate the INSR but markedly enhances the receptor’s ability to bind insulin and potentiate insulin signaling. Both XMetA and XMetS are effective in controlling hyperglycemia in mouse models of diabetes. A third allosteric antibody, XMetD, is an inhibitor of INSR signaling. This antibody reverses insulin-induced hypoglycemia in a mouse model of hyperinsulinemia. These studies indicate, therefore, that allosteric antibodies to INSR can modulate its signaling and correct conditions of glucose dysregulation. These studies also raise the possibility that the use of allosteric antibodies can be expanded to other receptors for the treatment of metabolic disorders.

An Allosteric Antibody to the Leptin Receptor Reduces Body Weight and Reverses the Diabetic Phenotype in the Lepob/Lepob Mouse

Leptin (LEP) deficiency results in major metabolic perturbations, including obesity, dyslipidemia, and diabetes. Although LEP deficiency can be treated with daily injections of a recombinant LEP, generation of an antibody activating the LEP receptor (LEPR) that has both an intrinsically long half‐life and low immunogenicity could be useful in the treatment of this condition.