Engineering a single-chain Fv antibody to αvβ6 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-beta 1 and transforming growth factor-beta 3, interact with the specific extracellular matrix proteins and promote migration and invasion of tumor cells. The viral protein (VP1) coat protein of the O(1) British field strain serotype of foot-and-mouth disease virus is 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 a5 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. (C) 2008 Elsevier Ltd. All rights reserved.