The role of interface framework residues in determining antibody V(H)/V(L) 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.
Masuda
Kenji
K
Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba, Japan.
Sakamoto
Kenzo
K
Kojima
Miki
M
Aburatani
Takahide
T
Ueda
Takuya
T
Ueda
Hiroshi
H
D000595:Amino Acid Sequence; D000941:Antigens / Q000378:metabolism*; D001666:Binding Sites, Antibody; D002478:Cells, Cultured; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019151:Peptide Library
D000941:Antigens; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library