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 |
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Authors: | Power B E Caine J M Burns J E Shapira D R Hattarki M K Tahtis K Lee F T Smyth F E Scott A M Kortt A A Hudson P J |
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Institution: | (1) CSIRO Health Sciences and Nutrition, 343 Royal Parade, Parkville, Victoria 3052, Australia e-mail: barbara.power@hsn.csiro.au Tel.: +61-3-96627100; Fax: +61-3-96627313, AU;(2) CRC for Diagnostic Technologies, 343 Royal Parade, Parkville, Victoria 3052, Australia, AU;(3) Tumor Targeting Program, Ludwig Institute for Cancer Research, Austin and Repatriation Medical Centre, Heidelberg, Victoria 3084, Australia, AU |
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Abstract: | A single-chain antibody fragment (scFv) of the humanised monoclonal antibody, hu3S193, that reacts specifically with Ley antigen expressed in numerous human epithelial carcinomas was constructed. A five-residue linker joined the C-terminus of
the VH and the N-terminus of the VL, 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 4M 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 ˜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.
Received: 16 November 2000 / Accepted: 8 March 2001 |
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Keywords: | Diabodies Single-chain antibodies Bacterial expression Ley Cancer targeting |
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