LY3298176, a novel dual GIP and GLP-1 receptor agonist for the treatment of type 2 diabetes mellitus: From discovery to clinical proof of concept.

OBJECTIVE: A novel dual GIP and GLP-1 receptor agonist, LY3298176, was developed to determine whether the metabolic action of GIP adds to the established clinical benefits of selective GLP-1 receptor agonists in type 2 diabetes mellitus (T2DM). METHODS: LY3298176 is a fatty acid modified peptide with dual GIP and GLP-1 receptor agonist activity designed for once-weekly subcutaneous administration. LY3298176 was characterised in vitro, using signaling and functional assays in cell lines expressing recombinant or endogenous incretin receptors, and in vivo using body weight, food intake, insulin secretion and glycemic profiles in mice. A Phase 1, randomised, placebo-controlled, double-blind study was comprised of three parts: a single-ascending dose (SAD; doses 0.25-8 mg) and 4-week multiple-ascending dose (MAD; doses 0.5-10 mg) studies in healthy subjects (HS), followed by a 4-week multiple-dose Phase 1 b proof-of-concept (POC; doses 0.5-15 mg) in patients with T2DM (ClinicalTrials.gov no. NCT02759107). Doses higher than 5 mg were attained by titration, dulaglutide (DU) was used as a positive control. The primary objective was to investigate safety and tolerability of LY3298176. RESULTS: LY3298176 activated both GIP and GLP-1 receptor signaling in vitro and showed glucose-dependent insulin secretion and improved glucose tolerance by acting on both GIP and GLP-1 receptors in mice. With chronic administration to mice, LY3298176 potently decreased body weight and food intake; these effects were significantly greater than the effects of a GLP-1 receptor agonist. A total of 142 human subjects received at least 1 dose of LY3298176, dulaglutide, or placebo. The PK profile of LY3298176 was investigated over a wide dose range (0.25-15 mg) and supports once-weekly administration. In the Phase 1 b trial of diabetic subjects, LY3298176 doses of 10 mg and 15 mg significantly reduced fasting serum glucose compared to placebo (least square mean [LSM] difference [95% CI]: -49.12 mg/dL [-78.14, -20.12] and -43.15 mg/dL [-73.06, -13.21], respectively). Reductions in body weight were significantly greater with the LY3298176 1.5 mg, 4.5 mg and 10 mg doses versus placebo in MAD HS (LSM difference [95% CI]: -1.75 kg [-3.38, -0.12], -5.09 kg [-6.72, -3.46] and -4.61 kg [-6.21, -3.01], respectively) and doses of 10 mg and 15 mg had a relevant effect in T2DM patients (LSM difference [95% CI]: -2.62 kg [-3.79, -1.45] and -2.07 kg [-3.25, -0.88], respectively. The most frequent side effects reported with LY3298176 were gastrointestinal (vomiting, nausea, decreased appetite, diarrhoea, and abdominal distension) in both HS and patients with T2DM; all were dose-dependent and considered mild to moderate in severity. CONCLUSIONS: Based on these results, the pharmacology of LY3298176 translates from preclinical to clinical studies. LY3298176 has the potential to deliver clinically meaningful improvement in glycaemic control and body weight. The data warrant further clinical evaluation of LY3298176 for the treatment of T2DM and potentially obesity.

Impact of variable domain glycosylation on antibody clearance: an LC/MS characterization.

Variable (Fv) domain N-glycosylation sites are found in approximately 20% of human immunoglobulin Gs (IgGs) in addition to the conserved N-glycosylation sites in the C(H)2 domains. The carbohydrate structures of the Fv glycans and their impact on in vivo half-life are not well characterized. Oligosaccharide structures in a humanized anti-Abeta IgG1 monoclonal antibody (Mab) with an N-glycosylation site in the complementary determining region (CDR2) of the heavy chain variable region were elucidated by LC/MS analysis following sequential exoglycosidase treatments of the endoproteinase Lys-C digest. Results showed that the major N-linked oligosaccharide structures in the Fv region have three characteristics (core-fucosylated biantennary oligosaccharides with one or two N-glycolylneuraminic acid [NeuGc] residues, zero or one alpha-linked Gal residue, and zero or one beta-linked GalNAc residue), whereas N-linked oligosaccharides in the Fc region contained typical Fc glycans (core-fucosylated, biantennary oligosaccharides with zero to two Gal residues). To elucidate the contribution of Fv glycans to the half-life of the antibody, a method that allows capture of the Mab and determination of its glycan structures at various time points after administration to mice was developed. Anti-Abeta antibody in mouse serum was immunocaptured by immobilized goat anti-human immunoglobulin Fc(gamma) antibody resin, and the captured material was treated with papain to generate Fab and Fc for LC/MS analysis. Different glycans in the Fc region showed the same clearance rate as demonstrated previously. In contrast to many other non-antibody glycosylated therapeutics, there is no strong correlation between oligosaccharide structures in the Fv region and their clearance rates in vivo. Our data indicated that biantennary oligosaccharides lacking galactosylation had slightly faster clearance rates than other structures in the Fv domain.

Glycosylation of erythropoietin affects receptor binding kinetics: role of electrostatic interactions.

Erythropoietin (EPO) is a cytokine produced by the kidney whose function is to stimulate red blood cell production in the bone marrow. Previously, it was shown that the affinity of EPO for its receptor, EPOR, is inversely related to the sialylation of EPO carbohydrate. To better understand the properties of EPO that modulate its receptor affinity, various glycoforms were analyzed using surface plasmon resonance. The system used has been well characterized and is based on previous reports employing an EPOR-Fc chimera captured on a Protein A surface. Using three variants of EPO containing different levels of sialylation, we determined that sialic acid decreased the association rate constant (k(on)) about 3-fold. Furthermore, glycosylated EPO had a 20-fold slower k(on) than nonglycosylated EPO, indicating that the core carbohydrate also negatively impacted k(on). The effect of electrostatic forces on EPO binding was studied by measuring binding kinetics in varying NaCl concentrations. Increasing NaCl concentration resulted in a slower k(on) while having little impact on k(off), suggesting that long-range electrostatic interactions are primarily important in determining the rate of association between EPO and EPOR. Furthermore, the glycosylation content (i.e., nonglycosylated vs glycosylated, sialylated vs desialylated) affected the overall sensitivities of k(on) to [NaCl], indicating that sialic acid and the glycan itself each impact the overall effect of these electrostatic forces.