The role of interface framework residues in determining antibody VH/VL interaction strength and antigen-binding affinity

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.