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Generation and Characterization of an IgG4 Monomeric Fc Platform

Journal Article · · PLoS ONE
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  1. MedImmune, Gaithersburg, MD (United States)
  2. AstraZeneca Pharmaceuticals, Waltham, MA (United States)
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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.
Research Organization:
Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Organization:
MedImmune, subsidiary of AstraZeneca Plc.
OSTI ID:
1438919
Journal Information:
PLoS ONE, Journal Name: PLoS ONE Journal Issue: 8 Vol. 11; ISSN 1932-6203
Publisher:
Public Library of ScienceCopyright Statement
Country of Publication:
United States
Language:
ENGLISH

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Bacteriophages
Cell fusion
Cloning
Crystal structure
Crystallization
Enzyme-linked immunoassays
Serum proteins
Ultracentrifugation