Masuda Kenji, Sakamoto Kenzo, Kojima Miki, Aburatani Takahide, Ueda Takuya, Ueda Hiroshi
Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba, Japan.
FEBS J. 2006 May;273(10):2184-94. doi: 10.1111/j.1742-4658.2006.05232.x.
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.
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)）相互作用很强，但抗溶菌酶IgG HyHEL-10具有相当强的亲和力，然而在没有抗原的情况下，其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，采用了一种可以切换两种展示模式的新型噬菌体展示系统。我们聚焦于HyHEL-10 V(H)和V(L)在结构域界面相互面对的两个框架区2区域，并构建了一个组合文库，其中每个框架区2残基与D1.3的相应残基混合，D1.3具有更强的V(H)/V(L)相互作用。编码V(H)基因7和V(L)琥珀密码子基因9的噬菌粒文库用于转化TG-1（sup+），并筛选展示功能性可变区的噬菌体。然后将筛选出的噬菌体用于感染非抑制菌株，含有展示V(H)的噬菌体和可溶性V(L)片段的培养上清液用于评估V(H)/V(L)相互作用强度。结果清楚地表明存在一个强烈影响V(H)/V(L)相互作用强度的关键框架区2残基（H39），并且抗原结合亲和力与V(H)/V(L)相互作用之间存在显著的正相关，特别是在存在一组特定V(L)残基的情况下。SPR生物传感器也证实了H39突变对野生型可变区的影响，由于结合速率增加，抗原结合亲和力提高了几倍，而单链可变区则略有下降。
