Affinity maturation of humanized anti-epidermal growth factor receptor antibody using a modified phage-based open sandwich selection method.
Affinity maturation of humanized anti-epidermal growth factor receptor antibody using a modified phage-based open sandwich selection method.
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DOI：
10.1038/s41598-018-23796-3
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发表时间：
2018-04-03
影响因子：
4.6
通讯作者：
Kumagai I
中科院分区：
文献类型：
--
作者：
Sanada H;Kobayashi K;Oyama K;Maru T;Nakanishi T;Umetsu M;Asano R;Kumagai I
亲和成熟是利用体外进化方法改善抗体功能的主要策略之一；其中一种行之有效的方法是噬菌体展示。为了最大限度地减少基因缺失，我们之前开发了一种开放三明治（OS）方法，在与可溶性VL片段混合后，仅使用显示噬菌体的VH片段进行选择。通过这种OS方法选择VH突变体，成功地恢复了人源化过程中抗egfr抗体528亲和力的降低。亲本528的亲和度与亲本528的亲和度不相似。为了进一步的亲和成熟，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片段的制备受其不稳定性和不溶性的限制，OS方法不能用于VL片段的选择。因此，我们初步设计了一种基于VH片段域交换的改进OS方法，从添加的可溶性Fv片段到显示噬菌体的VL片段。利用这种新的添加Fv的OS选择方法，我们成功分离出了VL突变体，其中一个由VH和VL突变体组成的Fv具有与亲本528几乎相当的亲和力。该方法同样适用于其他VL片段的亲和成熟工程。
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.
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影响因子：
3.9
作者：
Asano, Ryutaro;Kawaguchi, Hiroko;Kumagai, Izumi
通讯作者：
Kumagai, Izumi
影响因子：
4.8
作者：
Nishimiya, Y;Tsumoto, K;Kumagai, I
通讯作者：
Kumagai, I
影响因子：
5.6
作者：
Kipriyanov, SM;Moldenhauer, G;Little, M
通讯作者：
Little, M
影响因子：
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作者：
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通讯作者：
Vaughan TJ
影响因子：
5.3
作者：
Asano R;Shimomura I;Konno S;Ito A;Masakari Y;Orimo R;Taki S;Arai K;Ogata H;Okada M;Furumoto S;Onitsuka M;Omasa T;Hayashi H;Katayose Y;Unno M;Kudo T;Umetsu M;Kumagai I
通讯作者：
Kumagai I