Abstract
The immunoglobulin Fc region is a homodimer consisted of two sets of CH2 and CH3 domains and has been exploited to generate two-arm protein fusions with high expression yields, simplified purification processes and extended serum half-life. However, attempts to generate one-arm fusion proteins with monomeric Fc, with one set of CH2 and CH3 domains, are often plagued with challenges such as weakened binding to FcRn or partial monomer formation. Here, we demonstrate the generation of a stable IgG4 Fc monomer with a unique combination of mutations at the CH3-CH3 interface using rational design combined with in vitro evolution methodologies. In addition to size-exclusion chromatography and analytical ultracentrifugation, we used multi-angle light scattering (MALS) to show that the engineered Fc monomer exhibits excellent monodispersity. Furthermore, crystal structure analysis (PDB ID: 5HVW) reveals monomeric properties supported by disrupted interactions at the CH3-CH3 interface. Monomeric Fc fusions with Fab or scFv achieved FcRn binding and serum half-life comparable to wildtype IgG. These results demonstrate that this monomeric IgG4 Fc is a promising therapeutic platform to extend the serum half-life of proteins in a monovalent format.
Highlights
The homodimeric immunoglobulin fragment crystallizable, Fc, has been widely utilized to form fusion proteins with enzymes, growth factors, immune modulators, and target-binding moieties such as single chain fragment variable (scFv) [1,2,3,4] (Fig 1A)
Our results show that a library selection strategy combining thermal selection and rational template designs can lead to monomeric Fc fusion proteins which have the desired biophysical, structural and pharmacokinetics (PK) properties
We used multi-angle light scattering (MALS) and analytical ultracentrifugation analyses, in addition to sizeexclusion chromatography, to characterize the monomeric Fc and used the crystal structure to demonstrate that our IgG4 based monomeric Fc exhibits an Immunoglobulin G (IgG) Fc-like structure
Summary
The homodimeric immunoglobulin fragment crystallizable, Fc, has been widely utilized to form fusion proteins with enzymes, growth factors, immune modulators, and target-binding moieties such as scFv [1,2,3,4] (Fig 1A). Both as research tools and as therapeutic agents, Fcfusion proteins are able to harness FcRn-mediated serum half-life extension provided by the Fc domain. There have been several examples of proteins fused on one arm of the Fc, e.g., erythropoietin, coagulation factor IX, and interferon, that exhibited similar or improved stability and biological activities compared to conventional Fc fusions [5,6,7,8]. There are particular signaling pathways, such as receptor tyrosine kinases, which require
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