The role of interface framework residues in determining antibody VH/VL interaction strength and antigen-binding affinity
The role of interface framework residues in determining antibody VH/VL interaction strength and antigen-binding affinity
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DOI：
10.1111/j.1742-4658.2006.05232.x
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发表时间：
2006-05-01
期刊：
影响因子：
5.4
通讯作者：
Ueda, H
中科院分区：
文献类型：
--
作者：
Masuda, K;Sakamoto, K;Ueda, H
虽然许多具有强抗原结合亲和力的抗体具有稳定的可变区，其具有强抗体重链可变区片段（V-H）/抗体轻链可变区片段（V-L）相互作用，但抗溶菌酶IgG HyHEL-10在不存在抗原的情况下具有相当强的亲和力，但V-H/V-L相互作用强度非常弱。为了研究抗原结合亲和力与V-H/V-L相互作用强度之间的可能关系，采用了一种可切换两种展示模式的新型噬菌体展示系统。我们关注HyHEL-10的两个框架区2区域V-H和V-L，在结构域界面处彼此面对，并且制备组合文库，其中每个框架区2残基与D1.3残基混合，D1.3具有更强的V-H/V-L相互作用。用编码V-H基因7和V-L琥珀密码子基因9的噬菌粒文库转化TG-1（sup（+）），筛选出具有功能可变区的噬菌粒。将筛选出的重组质粒感染非抑制菌株，用含有V-H展示质粒和可溶性V-L片段的培养上清检测V-H/V-L相互作用强度。结果清楚地表明，存在一个关键的框架区2残基（H39），强烈影响V-H/V-L相互作用的强度，和抗原结合亲和力和V-H/V-L相互作用之间的显着的正相关性，特别是在一组特定的V-L残基的存在下。H39突变对野生型可变区的影响也被SPR生物传感器证实为由于缔合速率增加而使抗原结合亲和力增加数倍，而对于单链可变区观察到略微降低。
While many antibodies with strong antigen-binding affinity have stable variable regions with a strong antibody heavy chain variable region fragment (V-H)/antibody light chain variable region fragment (V-L) interaction, the anti-lysozyme IgG HyHEL-10 has a fairly strong affinity, yet a very weak V-H/V-L interaction strength, in the absence of antigen. To investigate the possible relationship between antigen-binding affinity and V-H/V-L interaction strength, a novel phage display system that can switch two display modes was employed. We focused on the two framework region 2 regions of the HyHEL-10 V-H and V-L, facing each other at the domain interface, and a combinatorial library was made in which each framework region 2 residue was mixed with that of D1.3, which has a far stronger V-H/V-L interaction. The phagemid library, encoding V-H gene 7 and V-L amber codon gene 9, was used to transform TG-1 (sup(+)), and the phages displaying functional variable regions were selected. The selected phages were then used to infect a nonsuppressing strain, and the culture supernatant containing V-H-displaying phages and soluble V-L fragment was used to evaluate the V-H/V-L interaction strength. The results clearly showed the existence of a key framework region 2 residue (H39) that strongly affects V-H/V-L interaction strength, and a marked positive correlation between the antigen-binding affinity and the V-H/V-L interaction, especially in the presence of a set of particular V-L residues. The effect of the H39 mutation on the wild-type variable region was also confirmed by a SPR biosensor as a several-fold increase in antigen-binding affinity owing to an increased association rate, while a slight decrease was observed for the single-chain variable region.