采用改良的基于噬菌体的开放式三明治筛选方法进行人源化抗表皮生长因子受体抗体的亲和力成熟。

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

机构信息

Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, 980-8579, Japan.

Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, Tokyo, 184-8588, Japan.

出版信息

Sci Rep. 2018 Apr 3;8(1):5414. doi: 10.1038/s41598-018-23796-3.

DOI:10.1038/s41598-018-23796-3

PMID:29615700

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5882652/

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.

摘要

亲和力成熟是利用体外进化方法提高抗体功能的主要策略之一；其中一种成熟的方法是噬菌体展示。为了最小化基因缺失，我们之前开发了一种开放式三明治（OS）方法，其中仅在与可溶性 VL 片段混合后使用展示 VH 片段的噬菌体进行选择。通过使用这种 OS 方法选择 VH 突变体，成功地恢复了人源化抗 EGFR 抗体 528 的亲和力。然而，亲和力与亲本 528 不相似。为了进一步进行亲和力成熟，我们旨在分离与 VH 突变体协同作用的 VL 突变体。然而，由于可溶性 VH 片段的不稳定性和不溶性，OS 方法无法用于选择 VL 片段。因此，我们最初设计了一种基于 VH 片段结构域交换的改良 OS 方法，从添加的可溶性 Fv 片段到噬菌体展示的 VL 片段。使用这种新型 Fv 添加 OS 选择方法，我们成功分离了 VL 突变体，其中一个包含 VH 和 VL 突变体的 Fv 显示出几乎等同于亲本 528 的亲和力。该方法适用于工程其他 VL 片段进行亲和力成熟。