Affinity maturation of humanized anti-epidermal growth factor receptor antibody using a modified phage-based open sandwich selection method
| Autor: | Izumi Kumagai, Mitsuo Umetsu, Kazuki Kobayashi, Kenji Oyama, Takamitsu Maru, Hideaki Sanada, Takeshi Nakanishi, Ryutaro Asano |
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| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Phage display Mutant Antibody Affinity Immunoglobulin Variable Region lcsh:Medicine medicine.disease_cause Antibodies Monoclonal Humanized Article Affinity maturation 03 medical and health sciences Peptide Library medicine Humans Peptide library lcsh:Science Mutation Multidisciplinary biology Chemistry lcsh:R Molecular biology In vitro ErbB Receptors 030104 developmental biology biology.protein lcsh:Q Antibody Function (biology) |
| Zdroj: | Scientific Reports Scientific Reports, Vol 8, Iss 1, Pp 1-9 (2018) |
| ISSN: | 2045-2322 |
| Popis: | Affinity maturation is one of the cardinal strategies for improving antibody function using in vitro evolutionary methods; one such well-established method is phage display. To minimise gene deletion, we previously developed an open sandwich (OS) method wherein selection was performed using only phage-displaying VH fragments after mixing with soluble VL fragments. The decrease in anti-EGFR antibody 528 affinity through humanization was successfully recovered by selecting VH mutants using this OS method. However, the affinity was not similar to that of parental 528. For further affinity maturation, we aimed to isolate VL mutants that act in synergy with VH mutants. However, the OS method could not be applied for selecting VL fragments because the preparation of soluble VH fragments was hampered by their instability and insolubility. Therefore, we initially designed a modified OS method based on domain-swapping of VH fragments, from added soluble Fv fragments to phage-displaying VL fragments. Using this novel Fv-added OS selection method, we successfully isolated VL mutants, and one of the Fv comprising VH and VL mutants showed affinity almost equivalent to that of parental 528. This method is applicable for engineering other VL fragments for affinity maturation. |
| Databáze: | OpenAIRE |
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