title: Engineering a single-chain Fv antibody to alpha v beta 6 integrin using the specificity-determining loop of a foot-and-mouth disease virus.
abstract: 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.
authors.affiliation: Cancer Research UK Targeting and Imaging Group, Department of Oncology, Royal Free & University College Medical School, Hampstead Campus, London NW3 2PF, UK. h.kogelberg@ucl.ac.uk
authors.forename: Heide
authors.initials: H
authors.lastname: Kogelberg
authors.forename: Berend
authors.initials: B
authors.lastname: Tolner
authors.forename: Gareth J
authors.initials: GJ
authors.lastname: Thomas
authors.forename: Danielle
authors.initials: D
authors.lastname: Di Cara
authors.forename: Shane
authors.initials: S
authors.lastname: Minogue
authors.forename: Bala
authors.initials: B
authors.lastname: Ramesh
authors.forename: Serena
authors.initials: S
authors.lastname: Sodha
authors.forename: Dan
authors.initials: D
authors.lastname: Marsh
authors.forename: Mark W
authors.initials: MW
authors.lastname: Lowdell
authors.forename: Tim
authors.initials: T
authors.lastname: Meyer
authors.forename: Richard H J
authors.initials: RH
authors.lastname: Begent
authors.forename: Ian
authors.initials: I
authors.lastname: Hart
authors.forename: John F
authors.initials: JF
authors.lastname: Marshall
authors.forename: Kerry
authors.initials: K
authors.lastname: Chester
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000951:Antigens, Neoplasm / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D002272:Carcinoembryonic Antigen / Q000737:chemistry / Q000235:genetics; D002460:Cell Line; D002465:Cell Movement; D022801:Complementarity Determining Regions / Q000737:chemistry*; D029544:Foot-and-Mouth Disease Virus; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D016023:Integrins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering
chemical_list: D000951:Antigens, Neoplasm; D036022:Capsid Proteins; D002272:Carcinoembryonic Antigen; D022801:Complementarity Determining Regions; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D016023:Integrins; D009842:Oligopeptides; C442501:VP1 protein, Foot-and-mouth disease virus; C073878:integrin alphavbeta6; C047981:arginyl-glycyl-aspartic acid