R409K mutation prevents acid-induced aggregation of human IgG4
| Autor: | Hideaki Yoshida, Tomoko Haba, Shigeru Iida, Seiji Saito, Keiko Hiraishi, Nobuaki Takahashi, Hiroshi Namisaki, Yoshitaka Tanaka |
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| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Physiology Cell Lines medicine.disease_cause Biochemistry Spectrum Analysis Techniques 0302 clinical medicine Immune Physiology Medicine and Health Sciences Enzyme-Linked Immunoassays Amino Acids skin and connective tissue diseases Receptor chemistry.chemical_classification Mutation Immune System Proteins Multidisciplinary Calorimetry Differential Scanning Protein Stability Organic Compounds Chemistry Antibodies Monoclonal Hydrogen-Ion Concentration Flow Cytometry Isotype Amino acid Spectrophotometry 030220 oncology & carcinogenesis Physical Sciences Amino Acid Analysis Medicine Cytophotometry Biological Cultures Raji Cells Research Article Cell Binding Cell Physiology Substitution Mutation Science Immunology Protein domain Research and Analysis Methods Antibodies Cell Line Protein Aggregates 03 medical and health sciences Protein Domains In vivo parasitic diseases Genetics medicine Humans Immunoassays Molecular Biology Techniques Molecular Biology Molecular Biology Assays and Analysis Techniques Organic Chemistry fungi Chemical Compounds Biology and Life Sciences Proteins Cell Biology In vitro HEK293 Cells 030104 developmental biology Amino Acid Substitution Drug Design Immunoglobulin G Immunologic Techniques Biophysics Cytokine secretion |
| Zdroj: | PLoS ONE PLoS ONE, Vol 15, Iss 3, p e0229027 (2020) |
| ISSN: | 1932-6203 |
| DOI: | 10.1371/journal.pone.0229027 |
| Popis: | Human immunoglobulin G isotype 4 (IgG4) antibodies are suitable for use in either the antagonist or agonist format because their low effector functions prevent target cytotoxicity or unwanted cytokine secretion. However, while manufacturing therapeutic antibodies, they are exposed to low pH during purification, and IgG4 is more susceptible to low-pH-induced aggregation than IgG1. Therefore, we investigated the underlying mechanisms of IgG4 aggregation at low pH and engineered an IgG4 with enhanced stability. By swapping the constant regions of IgG1 and IgG4, we determined that the constant heavy chain (CH3) domain is critical for aggregate formation, but a core-hinge-stabilizing S228P mutation in IgG4 is insufficient for preventing aggregation. To identify the aggregation-prone amino acid, we substituted the CH3 domain of IgG4 with that of IgG1, changing IgG4 Arg409 to a Lys, thereby preventing the aggregation of the IgG4 variant as effectively as in IgG1. A stabilizing effect was also recorded with other variable-region variants. Analysis of thermal stability using differential scanning calorimetry revealed that the R409K substitution increased the Tm value of CH3, suggesting that the R409K mutation contributed to the structural strengthening of the CH3-CH3 interaction. The R409K mutation did not influence the binding to antigens/human Fcγ receptors; whereas, the concurrent S228P and R409K mutations in IgG4 suppressed Fab-arm exchange drastically and as effectively as in IgG1, in both in vitro and in vivo in mice models. Our findings suggest that the IgG4 R409K variant represents a potential therapeutic IgG for use in low-effector-activity format that exhibits increased stability. |
| Databáze: | OpenAIRE |
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