Ig L-chain shuffling for affinity maturation of phage library-derived human anti-human MCP-1 antibody blocking its chemotactic activity.
Monocyte chemotactic protein-1 (MCP-1, CC-chemokine ligand 2; CCL2) is involved in the development of various forms of chronic inflammations. Employing the naive human single-chain Fv displaying phage library, we established seven MCP-1-specific scFvs. The MC8 and MC32 clones exhibited blocking activity for the MCP-1-induced chemotaxis of THP-1 cells, in spite of their monovalency. The analysis of V gene usage showed that all clones bore the identical Vh1 gene, IGHV1-24*01, with variable DJ joining sequences, while their Vl usage was relatively varied, suggesting the preferential contribution of the Vh gene. Based on these findings, to minimize the deteriorative influences on the MCP-1 specificity of MC32, we aimed to achieve the affinity maturation of MC32 using MC32 L-chain shuffling library and select MC32 variants. Most MC32 variants increased their affinity by reducing the k(off) value with no influence of the antigen specificity. MC32 variants #22 or #56 showed approximately 15-fold higher affinity than MC32, indicating that the L-chain shuffling library is useful if the Vh is dominantly involved in the determination of the antigen specificity.
Yoshinaga
Keisuke
K
Department of Bioengineering, Faculty of Engineering, Kagoshima University, Korimoto 1-21-40, Kagoshima, Kagoshima 890-0065, Japan.
Matsumoto
Miyuki
M
Torikai
Masaharu
M
Sugyo
Kazuki
K
Kuroki
Saori
S
Nogami
Kentaro
K
Matsumoto
Ryo
R
Hashiguchi
Shuhei
S
Ito
Yuji
Y
Nakashima
Toshihiro
T
Sugimura
Kazuhisa
K
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology / Q000494:pharmacology*; D000915:Antibody Affinity; D018932:Chemokine CCL2 / Q000037:antagonists & inhibitors* / Q000276:immunology*; D002633:Chemotaxis / Q000187:drug effects*; D032621:DNA Shuffling; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D019151:Peptide Library; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D018932:Chemokine CCL2; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D019151:Peptide Library