title: Production and binding analyses of a humanised scFv against a cryptic epitope on tumour-associated fibronectin.
abstract: Tumour-associated splice variants of fibronectin are a major source of tumour-matrix associated targets and are proving very successful in the development of clinical agents to treat cancer. One of the first monoclonal antibodies to be produced to this target, murine BC-1, recognises a cryptic epitope in domain 7 of the B-form splice variant (EDB-FN). Antibody fragments based on this immunoglobulin (IgG) were unstable, but BC-1 humanisation provided an opportunity to produce a more stable single-chain Fv (scFv). The variable domains of the humanized BC-1 IgG were sub-cloned and constructed into a scFv (HuBC-1 scFv) which was successfully expressed in Escherichia coli. The scFv retained its conformationally-sensitive epitope recognition and demonstrated a good affinity to the target of around 50 nM as measured by ELISA, Surface Plasmon Resonance and Flow Cytometry. Furthermore, the scFv was thermostable and stable in serum allowing substantial localisation to human tumours grown in mouse xenograft models. This scFv could form the basis of future tumour-specific biopharmaceuticals.
authors.affiliation: Imperial College London, Exhibition Road, London, SW7 2AZ, United Kingdom.
authors.forename: Nor Adzimah
authors.initials: NA
authors.lastname: Johdi
authors.forename: Ruth
authors.initials: R
authors.lastname: Harman
authors.forename: Irene
authors.initials: I
authors.lastname: Sanjuan
authors.forename: Christina
authors.initials: C
authors.lastname: Kousparou
authors.forename: Nigel
authors.initials: N
authors.lastname: Courtenay-Luck
authors.forename: Mahendra P
authors.initials: MP
authors.lastname: Deonarain
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D000939:Epitopes / Q000235:genetics / Q000276:immunology*; D005353:Fibronectins / Q000276:immunology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D051379:Mice; D020033:Protein Isoforms / Q000235:genetics / Q000276:immunology; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D020349:Surface Plasmon Resonance
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D005353:Fibronectins; D007128:Immunoglobulin Fragments; D020033:Protein Isoforms; D057127:Single-Chain Antibodies