title: Construction, expression and characterisation of a single-chain diabody derived from a humanised anti-Lewis Y cancer targeting antibody using a heat-inducible bacterial secretion vector.
abstract: A single-chain antibody fragment (scFv) of the humanised monoclonal antibody, hu3S193, that reacts specifically with Le(y) antigen expressed in numerous human epithelial carcinomas was constructed. A five-residue linker joined the C-terminus of the V(H) and the N-terminus of the V(L), which prevented V-domain association into a monomeric scFv and instead directed non-covalent association of two scFvs into a dimer or diabody. The diabody was secreted into the E. coli periplasm using a heat-inducible vector, pPOW3, and recovered as a soluble, correctly processed protein, following osmotic shock or solubilised with 4 M urea from the insoluble fraction. The diabody from both fractions was isolated by a rapid batch affinity chromatography procedure, using the FLAG affinity tag to minimise degradation and aggregation. The purified diabody has an Mr of approximately 54 kDa, was stable and demonstrated similar binding activity as the parent monoclonal antibody, as measured by FACS and BIAcore analyses. The radiolabelled diabody showed a rapid tumour uptake, with fast blood clearance, proving it to be an excellent potential candidate as a tumour-imaging agent.
authors.affiliation: CSIRO Health Sciences and Nutrition, Parkville, Victoria, Australia. barbara.power@hsn.csiro.au
authors.forename: B E
authors.initials: BE
authors.lastname: Power
authors.forename: J M
authors.initials: JM
authors.lastname: Caine
authors.forename: J E
authors.initials: JE
authors.lastname: Burns
authors.forename: D R
authors.initials: DR
authors.lastname: Shapira
authors.forename: M K
authors.initials: MK
authors.lastname: Hattarki
authors.forename: K
authors.initials: K
authors.lastname: Tahtis
authors.forename: F T
authors.initials: FT
authors.lastname: Lee
authors.forename: F E
authors.initials: FE
authors.lastname: Smyth
authors.forename: A M
authors.initials: AM
authors.lastname: Scott
authors.forename: A A
authors.initials: AA
authors.lastname: Kortt
authors.forename: P J
authors.initials: PJ
authors.lastname: Hudson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology* / Q000493:pharmacokinetics; D000951:Antigens, Neoplasm / Q000276:immunology; D015374:Biosensing Techniques; D002277:Carcinoma / Q000378:metabolism / Q000628:therapy*; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D005822:Genetic Vectors; D006358:Hot Temperature; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D007700:Kinetics; D007983:Lewis Blood Group Antigens / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008969:Molecular Sequence Data; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D007135:Immunoglobulin Variable Region; D007983:Lewis Blood Group Antigens; C094162:Lewis Y antigen