Affinity maturation of humanized anti-epidermal growth factor receptor antibody using a modified phage-based open sandwich selection method

Hideaki Sanada, Kazuki Kobayashi, Kenji Oyama, Takamitsu Maru, Takeshi Nakanishi, Mitsuo Umetsu, Ryutaro Asano, Izumi Kumagai

研究成果: Article

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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.

元の言語English
記事番号5414
ジャーナルScientific reports
8
発行部数1
DOI
出版物ステータスPublished - 2018 12 1

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