title: Relative stabilities of IgG1 and IgG4 Fab domains: influence of the light-heavy interchain disulfide bond architecture.
abstract: The stability of therapeutic antibodies is a prime pharmaceutical concern. In this work we examined thermal stability differences between human IgG1 and IgG4 Fab domains containing the same variable regions using the thermofluor assay. It was found that the IgG1 Fab domain is up to 11°C more stable than the IgG4 Fab domain containing the same variable region. We investigated the cause of this difference with the aim of developing a molecule with the enhanced stability of the IgG1 Fab and the biological properties of an IgG4 Fc. We found that replacing the seven residues, which differ between IgG1 C(H) 1 and IgG4 C(H) 1 domains, while retaining the native IgG1 light-heavy interchain disulfide (L-H) bond, did not affect thermal stability. Introducing the IgG1 type L-H interchain disulfide bond (DSB) into the IgG4 Fab resulted in an increase in thermal stability to levels observed in the IgG1 Fab with the same variable region. Conversely, replacement of the IgG1 L-H interchain DSB with the IgG4 type L-H interchain DSB reduced the thermal stability. We utilized the increased stability of the IgG1 Fab and designed a hybrid antibody with an IgG1 C(H) 1 linked to an IgG4 Fc via an IgG1 hinge. This construct has the expected biophysical properties of both the IgG4 Fc and IgG1 Fab domains and may therefore be a pharmaceutically relevant format.
authors: UCB Pharma, Slough, Berkshire SL1 3WE, United Kingdom. james.heads@UCB.com
authors: James T
authors: JT
authors: Heads
authors: Ralph
authors: R
authors: Adams
authors: Lena E
authors: LE
authors: D'Hooghe
authors: Matt J T
authors: MJ
authors: Page
authors: David P
authors: DP
authors: Humphreys
authors: Andrew G
authors: AG
authors: Popplewell
authors: Alastair D
authors: AD
authors: Lawson
authors: Alistair J
authors: AJ
authors: Henry
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D004220:Disulfides / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D013696:Temperature
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D004220:Disulfides; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains