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

The ?v?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-?1 and transforming growth factor-?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 O1 British field strain serotype of foot-and-mouth disease virus is a high-affinity ligand for ?v?6, and we recently reported that a peptide derived from VP1 exhibited ?v?6-specific binding in vitro and in vivo. We hypothesized that this peptide could confer binding specificity of an antibody to ?v?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 ?v?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 ?v?6 but not with ?5?1, ?v?3, ?v?5, ?v?8 or CEA. B6-2 was internalized into ?v?6-expressing cells and inhibited ?v?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 ?v?6-mediated cell adhesion. Thus, we have used a rational stepwise approach to create a humanized scFv with therapeutic potential to block ?v?6-mediated cancer cell invasion or to deliver and internalize toxins specifically to ?v?6-expressing tumors