Susan C. Low

Monomeric Fc Fusions

The delivery of therapeutic proteins by noninvasive routes of administration has been a challenging goal, hence current modes of delivery generally require injections. However, we have recently shown that a naturally occurring receptor, the neonatal Fc receptor (FcRn) can be utilized to carry aerosolized therapeutic proteins conjugated to a portion of its respective ligand (Fc domain of immunoglobulin G) across epithelial cells of the lung to effectively deliver biologically active molecules to the bloodstream. First-generation dimeric Fc fusion molecules were successfully transported by the pulmonary route and biologic activity was demonstrated in both non-human primates and human volunteers. Continuing efforts to improve transport efficiency have led to the development of an alternate configuration of Fc fusion proteins with improved characteristics. These second generation Fc fusion molecules are monomeric with respect to the therapeutic protein and dimeric with respect to the Fc region, and have been termed Fc fusion ‘monomers’. Several different Fc fusion monomers have demonstrated improved transport efficiency, achieving high bioavailabilities for pulmonary delivery in nonhuman primates. While the traditional dimeric Fc fusion molecule generally increases the half-life compared with the unconjugated effector molecule, the monomer configuration has been shown to result in an even greater extension of the circulating half-life, which improves pharmacokinetic parameters for protein therapeutics, whether administered by pulmonary delivery or injection. Finally, many of the Fc monomer fusions have enhanced biologic activity compared with the dimeric configuration. Because of these many advantages, the monomer configuration promises to be an enabling advance to achieve clinically relevant, noninvasive delivery with potentially less frequent administration regimens for a broad range of protein therapeutics. In addition, molecules that are comprised of heterodimeric subunits or multi-subunit complexes can also be constructed as Fc fusions that result in a molecule with enhanced pharmacokinetics and greater bioactivity. Several examples of novel Fc fusion proteins, both monomer and heterodimer are described herein.

Reduction of IgG in nonhuman primates by a peptide antagonist of the neonatal Fc receptor FcRn

The neonatal Fc receptor FcRn provides IgG molecules with their characteristically long half-lives in vivo by protecting them from intracellular catabolism and then returning them to the extracellular space. Other investigators have demonstrated that mice lacking FcRn are protected from induction of various autoimmune diseases, presumably because of the accelerated catabolism of pathogenic IgGs in the animals. Therefore, targeting FcRn with a specific inhibitor may represent a unique approach for the treatment of autoimmune disease or other diseases where the reduction of pathogenic IgG will have a therapeutic benefit. Using phage display peptide libraries, we screened for ligands that bound to human FcRn (hFcRn) and discovered a consensus peptide sequence that binds to hFcRn and inhibits the binding of human IgG (hIgG) in vitro. Chemical optimization of the phage-identified sequences yielded the 26-amino acid peptide dimer SYN1436, which is capable of potent in vitro inhibition of the hIgG–hFcRn interaction. Administration of SYN1436 to mice transgenic for hFcRn induced an increase in the rate of catabolism of hIgG in a dose-dependent manner. Treatment of cynomolgus monkeys with SYN1436 led to a reduction of IgG by up to 80% without reducing serum albumin levels that also binds to FcRn. SYN1436 and related peptides thus represent a previously uncharacterized family of potential therapeutic agents for the treatment of humorally mediated autoimmune and other diseases.

Inhibitors of the FcRn:IgG Protein–Protein Interaction

The neonatal Fc receptor, FcRn, is responsible for controlling the half-life of IgG antibodies. As a result, inhibitors of FcRn have been investigated as a possible way to modulate IgG half-lives. Such inhibitors could have possible applications in reducing autoantibody levels in autoimmune disease states. To date, monoclonal antibodies, engineered Fc domains, and short peptides have been reported to inhibit FcRn function and modulate IgG half-lives in vivo.

PEGylation enhances the therapeutic potential of peptide antagonists of the neonatal Fc receptor, FcRn.

Peptides targeting the human neonatal Fc receptor (FcRn) were conjugated to poly(ethylene glycol) (PEG) polymers to study their effect on inhibition of the IgG:FcRn protein-protein interaction both in vitro and in mice. Both linear (5-40kDa) and branched (20, 40kDa) PEG aldehydes were conjugated to an amine-containing linker of a homodimeric anti-FcRn peptide using reductive alkylation chemistry. It was found that conjugation of PEG to the peptide compromised the in vitro activity, with larger and branched PEGs causing the most dramatic losses in activity. The conjugates were evaluated in transgenic mice for their ability to accelerate the catabolism of human IgG. Optimal pharmacodynamic properties were observed with PEG-peptide conjugates that contained 20-40kDa linear PEGs and a 20kDa branched PEG. The optimal PEG-peptide conjugates were more effective in vivo than the unconjugated peptide control on a mole:mole and mg/kg basis, and represent potential new longer-acting peptide therapeutics for the treatment of humorally-mediated autoimmune disease.

Synthesis and Structure—Activity Relationships of Dimeric Peptide Antagonists of the Human Immunoglobulin G-Human Neonatal Fc Receptor (IgG-FcRn) Interaction

The neonatal Fc receptor, FcRn, regulates the half-life of IgG in vivo and may be a target in the treatment of autoimmune disease. Monomeric peptide antagonists of the human IgG-human FcRn interaction were dimerized using three different synthetic methodologies: thiol/alkyl halide coupling of unprotected peptides, reductive alkylation of unprotected peptides, and on-resin amide bond formation with protected peptides. It was found that dimerization of monomeric peptides increased the in vitro activity of the peptide monomers more than 200-fold. Human IgG catabolism experiments in human FcRn transgenic mice were used to assess the in vivo activity of peptide dimers that possessed different linkers, cyclizations, and affinities for FcRn. Overall, it was found that the linker joining two monomeric peptides had only a minor effect on the in vitro potency but that in vitro potency was predictive of in vivo activity.