Аннотация: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.
