Molecular Engineering of Efficacious Mono-Valent Ultra-Long Acting Two-Chain Insulin-Fc Conjugates

Abstract

Here, we describe molecular engineering of monovalent ultra-long acting two-chain insulin-Fc conjugates. Insulin-Fc conjugates were synthesized using trifunctional linkers with one amino reactive group for reaction with a lysine residue of insulin and two thiol reactive groups used for re-bridging of a disulfide bond within the Fc molecule. The ultra-long pharmacokinetic profile of the insulin-Fc conjugates was the result of concertedly slowing insulin receptor-mediated clearance by (1) introduction of amino acid substitutions that lowered the insulin receptor affinity and (2) conjugating insulin to the Fc element. Fc conjugation leads to recycling by the neonatal Fc receptor and increase in the molecular size, both contributing to the ultra-long pharmacokinetic and pharmacodynamic profiles.

Chemical Mobilization-Based Capillary Isoelectric Focusing–Mass Spectrometry Using the nanoCEasy Interface for Pharmaceutical Protein Analysis

“…It has already been shown that conjugation of the Fc fragment of an antibody to peptides enables to increase half-life . Similarly, Fc has been used as the conjugator and chemically linked to insulin in the ratio 1:1 to have a long PK profile . Some of the impurities normally relatively easily detected by analysis on insulin (∼6 kDa) can be challenging as soon as the Fc fragment and linker (∼56 kDa) are attached.…”

Toward once-monthly insulin therapy via synergy in two pharmacokinetic protractors: Fc-conjugation and fatty acid acylation

“…The Fc component has several mutations that biophysically stabilize the protein to improve recombinant yields: 227Ala, 234Ala, 235Lys, 297Glu, 315Gln, 384Gln, des447 hIgG4 Fc(227–447). 40 Insulin receptor in vitro phosphorylation analysis indicate that both analogs possess diminished potency compared to human insulin, with Fc-Ins1 at approximately 7% and Fc/FA-Ins1 near 3%. This reduced potency is consistent with previous observations involving Fc-conjugated insulins and it is a consequence of A14 and B25 mutations.…”

“…This reduced potency is consistent with previous observations involving Fcconjugated insulins and it is a consequence of A14 and B25 mutations. 40 The analogs were assessed for their glucose-lowering efficacy in STZ-treated mice following a single subcutaneous injection at a dose of 90 nmol kg À1 (Fig. 1A).…”

“…Mean residence times (MRTs) of 43 hours, 60 hours, and 88 hours in STZinduced Sprague-Dawley rats were reported for Fc-Ins2, Fc-Ins3, and Fc-Ins4, respectively. 40 In a direct comparison focused on glucose-lowering in STZtreated mice (Fig. 2), the dosing for the compounds Fc-Ins2 and Fc-Ins3 was adjusted to 45 nmol kg À1 to accommodate their higher potency.…”

“…Quantitative determination of insulin plasma concentrations was obtained using Luminescence Oxygen Channeling Immunoassay (LOCI)/Alpha-LISA 52,53 and suitable antibodies against the Fc and insulin parts of the molecule as previously described. 40 Briefly, donor beads (Alpha-LISA donor beads, PerkinElmer, Waltham, Massachusetts, USA) were coated with streptavidin, while acceptor beads (Alpha-LISA acceptor beads, PerkinElmer, Waltham, Massachusetts, USA) were conjugated with a mAb specific for the Insulin part of the module. A second pAb, recognizing the Fc part, was biotinylated.…”