Engineering a single-chain Fv antibody to alpha v beta 6 integrin using the specificity-determining loop of a foot-and-mouth disease virus.
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.
Kogelberg
Heide
H
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
Tolner
Berend
B
Thomas
Gareth J
GJ
Di Cara
Danielle
D
Minogue
Shane
S
Ramesh
Bala
B
Sodha
Serena
S
Marsh
Dan
D
Lowdell
Mark W
MW
Meyer
Tim
T
Begent
Richard H J
RH
Hart
Ian
I
Marshall
John F
JF
Chester
Kerry
K
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
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