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

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. and Chester, Kerry (2008) Engineering a single chain Fv antibody to ?v?6 integrin using the specificity-determining loop of a foot-and-mouth disease virus. Journal of Molecular Biology, 382 (2), 385-401. (doi:10.1016/j.jmb.2008.07.013).

Abstract

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.

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