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

A single-chain antibody fragment (scFv) of the humanised monoclonal antibod y, 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-do main association into a monomeric scFv and instead directed non-covalent as sociation of two scFvs into a dimer or diabody. The diabody was secreted in to the E. coli periplasm using a heat-inducible vector, pPOW3, and recovere d as a soluble, correctly processed protein, following osmotic shock or sol ubilised with 4M urea from the insoluble fraction. The diabody from both fr actions was isolated by a rapid batch affinity chromatography procedure, us ing the FLAG affinity tag to minimise degradation and aggregation. The puri fied diabody has an M-r of similar to 54 kDa, was stable and demonstrated s imilar binding activity as the parent monoclonal antibody, as measured by F ACS and BIAcore analyses. The radiolabelled diabody showed a rapid tumour u ptake, with fast blood clearance, proving it to be an excellent potential c andidate as a tumour-imaging agent.