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Journal of Biochemistry Advance Access originally published online on January 23, 2008
Journal of Biochemistry 2008 143(5):593-601; doi:10.1093/jb/mvn009
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© 2008 The Japanese Biochemical Society.

Ig L-chain Shuffling for Affinity Maturation of Phage Library-derived Human Anti-human MCP-1 Antibody Blocking its Chemotactic Activity

Keisuke Yoshinaga1, Miyuki Matsumoto2, Masaharu Torikai2, Kazuki Sugyo1, Saori Kuroki1, Kentaro Nogami1, Ryo Matsumoto1, Shuhei Hashiguchi1, Yuji Ito1, Toshihiro Nakashima2 and Kazuhisa Sugimura1,*

1Department of Bioengineering, Faculty of Engineering, Kagoshima University, Korimoto 1-21-40, Kagoshima, Kagoshima 890-0065; and 2The Chemo-Sero-Therapeutic Research Institute, Kyokushi Kikuchi, Kumamoto 869-1298, Japan

*To whom correspondence should be addressed. Tel: +81-99-285-8345, Fax: +81-99-258-4706, E-mail: kazu{at}be.kagoshima-u.ac.jp

Received November 13, 2007; Accepted January 7, 2008


  Abstract

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 koff value with no influence of the antigen specificity. MC32 variants #22 or #56 showed ~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.

Key Words: affinity maturation, human antibody, L-chain shuffling, MCP-1, phage display library

Abbreviations: AP, alkaline-phosphatase; BSA, bovine serum albumin; CDR, complementary determining region; FR, framework; HPLC, high performance liquid chromatography; HRP, horseradish peroxidase; HAS, human serum albumin; IC50, 50% inhibitory concentration; IgG, immunoglobulin G; Ig L-chain, immunoglobulin light chain; IPTG, isopropyl-thio-β-D-galactopyranoside; MCP-1, monocyte chemotactic protein-1; MIP-1{alpha}, macrophage inflammatory protein-1{alpha}; PBMC, peripheral blood mononuclear cell; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PEG, poly(ethylene glycol); RU, response unit; scFv, single-chain variable fragment; TU, transforming unit


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