Engineering a single-chain Fv antibody to alpha v beta 6 integrin using the specificity-determining loop of a foot-and-mouth disease virus

• Published in: Journal of molecular biology Vol. 382; no. 2; pp. 385 - 401
• Main Authors: Kogelberg, Heide, Tolner, Berend, Thomas, Gareth J, Di Cara, Danielle, Minogue, Shane, Ramesh, Bala, Sodha, Serena, Marsh, Dan, Lowdell, Mark W, Meyer, Tim, Begent, Richard H J, Hart, Ian, Marshall, John F, Chester, Kerry
• Format: Journal Article
• Language: English
• Published: England 03.10.2008
• Subjects: Amino Acid Sequence; Animals; Antigens, Neoplasm - chemistry; Antigens, Neoplasm - genetics; Antigens, Neoplasm - immunology; Capsid Proteins - chemistry; Capsid Proteins - genetics; Capsid Proteins - immunology; Carcinoembryonic Antigen - chemistry; Carcinoembryonic Antigen - genetics; Cell Line; Cell Movement; Complementarity Determining Regions - chemistry; Foot-and-Mouth Disease Virus; Humans; Immunoglobulin Fragments - chemistry; Immunoglobulin Fragments - genetics; Immunoglobulin Fragments - immunology; Immunoglobulin Variable Region - chemistry; Immunoglobulin Variable Region - genetics; Immunoglobulin Variable Region - immunology; Integrins - chemistry; Integrins - genetics; Integrins - immunology; Mice; Models, Molecular; Molecular Sequence Data; Oligopeptides - chemistry; Oligopeptides - genetics; Pichia pastoris; Protein Conformation; Protein Engineering
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2008.07.013

The alpha v beta 6 integrin is a promising target for cancer therapy. Its expression is up-regulated de novo on many types of carcinoma where it may activate transforming growth factor-beta1 and transforming growth factor-beta 3, interact with the specific extracellular matrix proteins and promote migration and invasion of tumor cells. The viral protein 1 (VP1) coat protein of the O(1) British field strain serotype of foot-and-mouth disease virus is a high-affinity ligand for alpha v beta 6, and we recently reported that a peptide derived from VP1 exhibited alpha v beta 6-specific binding in vitro and in vivo. We hypothesized that this peptide could confer binding specificity of an antibody to alpha v beta 6. A 17-mer peptide of VP1 was inserted into the complementarity-determining region H3 loop of MFE-23, a murine single-chain Fv (scFv) antibody reactive with carcinoembryonic antigen (CEA). The resultant scFv (B6-1) bound to alpha v beta 6 but retained residual reactivity with CEA. This was eliminated by point mutation (Y100bP) in the variable heavy-chain domain to create an scFv (B6-2) that was as structurally stable as MFE-23 and reacted specifically with alpha v beta 6 but not with alpha 5 beta 1, alpha v beta 3, alpha v beta 5, alpha v beta 8 or CEA. B6-2 was internalized into alpha v beta 6-expressing cells and inhibited alpha v beta 6-dependent migration of carcinoma cells. B6-2 was subsequently humanized. The humanized form (B6-3) was obtained as a non-covalent dimer from secretion in Pichia pastoris (115 mg/l) and was a potent inhibitor of alpha v beta 6-mediated cell adhesion. Thus, we have used a rational stepwise approach to create a humanized scFv with therapeutic potential to block alpha v beta 6-mediated cancer cell invasion or to deliver and internalize toxins specifically to alpha v beta 6-expressing tumors.