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.

Absence of retroviral vector-mediated transformation of gene-modified T cells after long-term engraftment in mice

“…The chimeric gene was composed of extracellular singlechain anti-Le Y , [46][47][48] linked to the hinge region of CD8 followed by the transmembrane and cytoplasmic domains of CD28 and the cytoplasmic portion of CD3-z. 49 The gene was incorporated into the Moloney murine leukemia virus-based plasmid, pSAMEN.…”

Production and characterisation of a recombinant scFv reactive with human gastrointestinal carcinomas

“…However, the use of whole antibody for in vivo imaging of cancer targets may have limitations due to their large molecular weight, with subsequent slow tumour uptake and long serum half-life (Power et al, 2001). Recombinant single chain variable fragments (scFvs), in which the two variable domains are covalently joined via a flexible peptide linker have overcome many of these problems.…”

Gene-modified T cells as immunotherapy for multiple myeloma and acute myeloid leukemia expressing the Lewis Y antigen

“…DNA encoding the anti-Le Y chimeric receptor was generated by using standard molecular biology techniques using an scFv 16 generated from the humanized monoclonal antibody hu3S193. 41 The construct used in this study was derived from the one described by Westwood et al 15 by excising the sequences coding for the Neomycin (Neo)-resistance gene and the IRES sequence by cutting at the HindIII/SpHI sites and subsequent religation.…”

“…The vector construct has been described before in detail by Westwood et al 15 An important advantage of this particular construct is that its sequence is fully humanized and therefore less immunogenic than constructs containing murine elements. [16][17][18] Furthermore, besides CD3 zeta, it contains a co-stimulatory signaling motif from the CD28 co-receptor, which has previously been demonstrated to enhance T-cell activation compared with CD3 zeta alone. 19,20 A further advantage of Le Y as a target structure is that anti-Le Y chimeric receptor expressing T cells could potentially target a broad range of malignancies that express Le Y .…”