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 glucagonlike peptide-1 levels. Thus, the anti-GCGR mAb could represent an effective new therapeutic for the treatment of type 2 diabetes.

Uncontrolled hepatic glucose output (HGO) contributes significantly to the pathological hyperglycemic state of patients with type 2 diabetes. Glucagon, through action on its receptor, stimulates HGO, thereby leading to increased glycemia. Antagonizing the glucagon signaling pathway represents an attractive therapeutic approach for the treatment of type 2 diabetes. We previously reported the generation and characterization of several high-affinity monoclonal antibodies (mAbs) targeting the glucagon receptor (GCGR). In the present study, we demonstrate that a 5-week treatment of diet-induced obese mice with mAb effectively normalized nonfasting blood glucose. Similar treatment also reduced fasting blood glucose without inducing hypoglycemia or other undesirable metabolic perturbations. In addition, no hypoglycemia was found in db/db mice that were treated with a combination of insulin and mAb. Long-term treatment with the mAb caused dose-dependent hyperglucagonemia and minimal to mild ␣-cell hyperplasia in lean mice. There was no evidence of pancreatic ␣-cell neoplastic transformation in mice treated with mAb for as long as 18 weeks. Treatment-induced hyperglucagonemia and ␣-cell hyperplasia were reversible after treatment withdrawal for periods of 4 and 10 weeks, respectively. It is noteworthy that pancreatic ␤-cell function was preserved, as demonstrated by improved glucose tolerance throughout the 18-week treatment period. Our studies further support the concept that long-term inhibition of GCGR signaling by a mAb could be an effective approach for controlling diabetic hyperglycemia.

No abstract

SummaryWe determined the expression of ORAI1 protein in rodent and non-rodent tissues using a monoclonal antibody directed against an extracellular loop of the protein. Previous reports using antibodies directed at the C-terminus of ORAI1 have not detected central nervous system (CNS) expression. Our results demonstrate broad tissue expression that includes the CNS using a unique monoclonal antibody specific to an extracellular loop of ORAI1. In addition, we present in situ hybridization (ISH) results using a probe within the middle of the mouse coding region showing CNS expression of Orai1 RNA. We contrast the patterns of rodent and human tissue expression and conclude that rodents have similar expression of ORAI1 in most tissue types when compared to primates, with an important exception being the male reproductive system, where human-specific expression is observed. (J Histochem Cytochem 62:864-878, 2014)

The transient receptor potential ankyrin 1 (TRPA1) channel has been implicated in different pathophysiologies that include asthma, cough, itch, and inflammatory pain. Agonists of TRPA1 such as mustard oil and its key component allyl isothiocyanate (AITC) cause pain and neurogenic inflammation in humans and pain behaviors in rodents. Hence, TRPA1 antagonists are being pursued as potential therapeutics. With the goal of generating monoclonal antibodies (mAbs) to human TRPA1 that could act as selective antagonists, we immunized mice with a variety of antigens expressing TRPA1 channels. After generation of hybridomas, the hybridoma conditioned media were screened to identify the mAbs that bind TRPA1 channels by a flow cytometry assay utilizing U2OS or Chinese hamster ovary (CHO) cells stably expressing TRPA1. The purified IgGs from the hybridomas that showed selective binding to TRPA1 were evaluated for antagonism in agonist-induced 45 Ca 21 uptake assays using CHO-TRPA1 cells. Several of the mAbs showed concentration-dependent inhibition of AITC and cold (4°C) activation of TRPA1. The most potent mAb, 2B10, had IC 50 values of approximately 260 and 90 nM in the two assays, respectively. These antagonist mAbs also blocked osmotically activated TRPA1 as well as activation by an endogenous agonist (4-oxo-2-nonenal). In summary, we generated mouse mAbs against TRPA1 that act as antagonists of multiple modes of TRPA1 activation.