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

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

doi:10.1038/s41598-018-23796-3

Hideaki Sanada, Takeshi Nakanishi + Show 6 more

Open Access

https://doi.org/10.1038/s41598-018-23796-3

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Abstract

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.

Highlights

Hybridoma[1] and humanization[2,3] remain the major practical techniques used for obtaining specific antibodies and for their application as therapeutics, respectively
For VL selection, this open sandwich (OS) method could not be applied because the preparation of soluble variable heavy domain (VH) fragments was not possible owing to their instability
A mixture of these two fragments showed obvious binding activity, analysis was performed immediately after mixing. These results indicate that domain swapping of h528 VH occurs quickly, and that this Fv-added OS selection method can be applied for the affinity maturation of VH fragments

Summary

Introduction

Hybridoma[1] and humanization[2,3] remain the major practical techniques used for obtaining specific antibodies and for their application as therapeutics, respectively. To minimise the size of the loaded fragment antibody on the phage for preventing gene deletion, we previously developed the open sandwich (OS) selection method, in which selection was performed using a phage displaying only VH fragments, after mixing with soluble VL fragments[15,16]. This method has resulted in successful antibody engineering, such as isolation of antibodies with specific conversion and affinity maturation[17,18,19]. This method may be useful for engineering antibody VL fragments and integrating isolated high-affinity VL mutants into engineered antibodies previously constructed by us based on h528 Fv19,27,28 for increasing their affinity and tumour-inhibitory effects

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