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Therapeutic peptides or proteins include clotting factors, hormones, growth factors, cytokines, enzymes, and antibodies. The half-life of peptides and proteins in human serum is dictated by several factors, including size, charge, proteolytic sensitivity, nature of their biology, turnover rate of proteins they bind, and other factors. However, many therapeutic peptides and proteins exhibit short plasma half-lives, typically from a few minutes to a few hours, leading to the necessity for frequent or continuous injections. Three types of proteins human IgGs, HSA, and transferrin persist for much longer in human serum than would be predicted just by their size. The exaggerated persistence of human IgGs and HSA has been determined to be due to their binding to the neonatal Fc receptor.

Fig.1 FcRn-dependent recycling and protection of monomeric IgG versus degradation and potentially presentation of complexed IgG. (Mancuso, 2014)

Principle of Fc-fusion Based Half-Life Extension

FcRn is a heterodimeric receptor, closely related to major histocompatibility complex (MHC) class I receptors, which is widely expressed in vascular epithelial cells, endothelial cells, intestinal epithelial cells, mammary epithelial cells, placental membranes, monocytes, macrophages, dendritic cells, and polymorphonuclear (PMN) leukocytes. Upon pinocytosis of serum proteins by cells of the reticuloendothelial system, human IgG1, IgG2, and IgG4 isotypes and HSA bind FcRn in a pH-dependent manner. As the vesicles are acidified, the IgGs and HSA bind FcRn, which allows them to be translocated back to the cell surface for recycling back into the circulation, while non-FcRn-bound proteins are targeted for lysosomal degradation. Upon exposure to the neutral pH at the cell surface, the IgGs and HSA are released back into the circulation. This recycling mechanism confers a nominal 14- to 21-day half-life on human IgG1, IgG2, and IgG4, and a ~19-day half-life on HSA.

Table.1 Nominal half-life values of human proteins in human serum. (Strohl, 2015)

Most of the Fc-fusion proteins are produced by genetic fusion of the C-terminus of a biological moiety to the N-terminus of the IgG-Fc domain. The strong interaction of the IgG-CH3 domains creates a stable Fc-structure and allows more complex structures to be fused to the flexible hinge regions. In the hinge region, the disulfide bonds reside at the base of either monomeric, homodimer, or heterodimer structures. The Fc-fusion partners can be subdivided into four major groups: the extracellular domains (ECD) of natural receptors and novel binding domains, such as functionally active peptides, genetically engineered binding constructs acting as cytokine traps, or recombinant enzymes.

Fig.2 Schematic representation of selected Fc-fusion proteins classified based on their ligand-binding domain that can be derived from a receptor ECD. (Duivelshof, 2021)

This binding occurs via specific residues in the Fc of the antibody, giving these IgG isotypes a nominal 2- to 3-week half-life in human serum. The concept of using IgG Fc as a fusion partner to significantly increase the half-life of a therapeutic peptide or protein has been around since the late 1980s. Many Fc fusion proteins of various types have been made over the past few decades, virtually all of which were intended to prolong the half-life of a protein or peptide.

References

1. Mancuso, M. E. and Mannucci, P. M. Fc-fusion technology and recombinant FVIII and FIX in the management of the hemophilias. Drug Des Devel Ther. 2014, 8: 365-71.
2. Strohl, W. R. Fusion Proteins for Half-Life Extension of Biologics as a Strategy to Make Biobetters. BioDrugs. 2015, 29(4): 215-39.
3. Duivelshof, B. L.; et al. Therapeutic Fc-fusion proteins: Current analytical strategies. J Sep Sci. 2021, 44(1): 35-62.

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