Influence of FcRn binding properties on the gastrointestinal absorption and exposure profile of Fc molecules.

The neonatal Fc receptor (FcRn) represents a transport system with the potential to facilitate absorption of biologics across the gastrointestinal barrier. How biologics interact with FcRn to enable their gastrointestinal absorption, and how these interactions might be optimized in a biological therapeutic are not well understood. Thus, we studied the absorption of Fc molecules from the intestine using three IgG4-derived Fc variants with different, pH-dependent FcRn binding and release profiles. Using several different intestinal models, we consistently observed that FcRn binding affinity correlated with transcytosis. Our findings support targeting FcRn to enable intestinal absorption of biologics and highlight additional strategic considerations for future work.

Disposition of Basal Insulin Peglispro Compared with 20-kDa Polyethylene Glycol in Rats Following a Single Intravenous or Subcutaneous Dose.

Basal insulin peglispro (BIL) comprises insulin lispro covalently bound to a 20-kDa polyethylene glycol (PEG) at lysine B28. The biologic fate of BIL and unconjugated PEG were examined in rats given a single 0.5-mg/kg i.v. or 2-mg/kg s.c. dose of BIL with (14)C label in 20-kDa PEG or (125)I label in lispro. Unconjugated (14)C-labeled 20-kDa PEG was dosed at 10 mg/kg i.v. or s.c. Blood, urine, and feces were collected up to 336 hours after dosing. Radioactivity was measured by scintillation spectrometry, and BIL was quantitated by enzyme-linked immunosorbent assay. Area under the curve and half-life for immunoreactive BIL were lower than those for both (14)C and (125)I after subcutaneous and intravenous administration. The half-lives of (14)C after BIL and PEG dosing were similar. The clearance of immunoreactive BIL was 2.4-fold faster than that of (14)C and 1.6-fold faster than (125)I. After a subcutaneous dose of BIL, immunoreactive BIL accounted for 31% of the circulating (125)I and 16% of the circulating (14)C, indicating extensive catabolism of BIL. Subcutaneous bioavailability of BIL was 23%-29%; bioavailability for unconjugated PEG was 78%. For unconjugated PEG, most of the (14)C dose was recovered in urine. For BIL, ≥86% of (125)I was eliminated in urine and (14)C was eliminated about equally in urine and feces. The major (14)C-labeled catabolism product of BIL in urine was 20-kDa PEG with lysine attached. The attachment of 20-kDa PEG to lispro in BIL led to a different elimination pathway for PEG compared with unconjugated 20-kDa PEG.

Generation and activity of a humanized monoclonal antibody that selectively neutralizes the epidermal growth factor receptor ligands transforming growth factor-α and epiregulin.

At least seven distinct epidermal growth factor (EGF) ligands bind to and activate the EGF receptor (EGFR). This activation plays an important role in the embryo and in the maintenance of adult tissues. Importantly, pharmacologic EGFR inhibition also plays a critical role in the pathophysiology of diverse disease states, especially cancer. The roles of specific EGFR ligands are poorly defined in these disease states. Accumulating evidence suggests a role for transforming growth factor α (TGFα) in skin, lung, and kidney disease. To explore the role of Tgfa, we generated a monoclonal antibody (mAb41) that binds to and neutralizes human Tgfa with high affinity (KD = 36.5 pM). The antibody also binds human epiregulin (Ereg) (KD = 346.6 pM) and inhibits ligand induced myofibroblast cell proliferation (IC50 values of 0.52 and 1.12 nM for human Tgfa and Ereg, respectively). In vivo, a single administration of the antibody to pregnant mice (30 mg/kg s.c. at day 14 after plug) or weekly administration to neonate mice (20 mg/kg s.c. for 4 weeks) phenocopy Tgfa knockout mice with curly whiskers, stunted growth, and expansion of the hypertrophic zone of growth plate cartilage. Humanization of this monoclonal antibody to a human IgG4 antibody (LY3016859) enables clinical development. Importantly, administration of the humanized antibody to cynomolgus monkeys is absent of the skin toxicity observed with current EGFR inhibitors used clinically and no other pathologies were noted, indicating that neutralization of Tgfa could provide a relatively safe profile as it advances in clinical development.

Engineering and characterization of the long-acting glucagon-like peptide-1 analogue LY2189265, an Fc fusion protein.

BACKGROUND: Glucagon-like peptide-1 (GLP-1) receptor agonists are novel agents for type 2 diabetes treatment, offering glucose-dependent insulinotropic effects, reduced glucagonemia and a neutral bodyweight or weight-reducing profile. However, a short half-life (minutes), secondary to rapid inactivation by dipeptidyl peptidase-IV (DPP-IV) and excretion, limits the therapeutic potential of the native GLP-1 hormone. Recently, the GLP-1 receptor agonist exenatide injected subcutaneously twice daily established a novel therapy class. Developing long-acting and efficacious GLP-1 analogues represents a pivotal research goal. We developed a GLP-1 immunoglobulin G (IgG4) Fc fusion protein (LY2189265) with extended pharmacokinetics and activity. METHODS: In vitro and in vivo activity of LY2189265 was characterized in rodent and primate cell systems and animal models. RESULTS: LY2189265 retained full receptor activity in vitro and elicited insulinotropic activity in islets similar to native peptide. Half-life in rats and cynomolgus monkeys was 1.5-2 days, and serum immunoreactivity representing active compound persisted > 6 days. In rats, LY2189265 enhanced insulin responses during graded glucose infusion 24 h after one dose. LY2189265 increased glucose tolerance in diabetic mice after one dose and lowered weight and delayed hyperglycaemia when administered twice weekly for 4 weeks. In monkeys, LY2189265 significantly increased glucose-dependent insulin secretion for up to a week after one dose, retained efficacy when administered subchronically (once weekly for 4 weeks) and was well tolerated. CONCLUSIONS: LY2189265 retains the effects of GLP-1 with increased half-life and efficacy, supporting further evaluation as a once-weekly treatment of type 2 diabetes.

Engineered FcRn Binding Fusion Peptides Significantly Enhance the Half-Life of a Fab Domain in Cynomolgus Monkeys.

There is a rapidly growing reinvigoration of the investigation of small proteins, cyclic peptides, and mAb derived domains as biotherapies. The drugability of these structures are challenged by fast peripheral clearance properties that can reduce their potential to be realized as medicines. Engineering strategies have been of limited value because mechanistically the half-life benefit is manifested by increasing the molecular weight and/or the hydrodyanimc radius which slows the molecule's renal elimination, but can result in the inherent loss of activity and target accessibility. The present work evaluated an alternative approach using smaller peptide sequences which bind to the neonatal Fc receptor (FcRn). Results revealed, small linear and cyclic FcRn binding peptides (FcRnBPs) fused to a combination of the N- and C-termini of a Fab can significantly improve the pharmacokinetics of the protein in cynomolgus monkeys relative to the parental Fab. The linear and cyclic conformations, as well as, the number of FcRnBPs fused to the Fab both influence the clearance and the extent of pharmacokinetic benefit. FcRnBP fusion protein kinetics were also affected by a combination of post-translation modifications and non-specific binding properties. The results in this report lay some foundation in fostering the advent of newer technologies toward successfully improving the pharmacokinetics of proteins, peptides, and mAb-derived domains. Additional work in the integration of a variety of factors including the intended site of action, tissue disposition, metabolism, toxicity and pharmacokinetic, and pharmacodynamics relationship of the intended therapeutic modality are key areas for advancement of these approaches.