Recombinant bispecific antibodies for the targeting of adenoviruses to CEA-expressing tumour cells: a comparative analysis of bacterially expressed single-chain diabody and tandem scFv

• Published in: The journal of gene medicine 2004-06, Vol.6 (6), p.642-651
• Main Authors: Korn, Tina, Nettelbeck, Dirk M., Völkel, Tina, Müller, Rolf, Kontermann, Roland E.
• Format: Article
• Language: English
• Subjects: Adenoviridae - genetics; Adenovirus; Antibodies, Bispecific - genetics; Antibodies, Bispecific - metabolism; Bacteria - genetics; Base Sequence; Breast Neoplasms - genetics; Breast Neoplasms - immunology; carcinoembryonic antigen; Carcinoembryonic Antigen - metabolism; Carcinoma - genetics; Carcinoma - immunology; Gene therapy; Gene Transfer Techniques; Humans; Immunoglobulin Variable Region - genetics; Molecular Sequence Data; Peptide Library; phage display; Protein Engineering - methods; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; single-chain diabody; tandem scFv; targeting; Tumor Cells, Cultured
• ISSN: 1099-498X; 1521-2254
• DOI: 10.1002/jgm.555

We have generated two distinct recombinant bispecific antibody molecules for the retargeting of adenoviral vectors to CEA‐expressing tumour cells. These antibody molecules were produced by combining the antigen‐binding sites of a neutralising anti‐fibre knob scFv (S11) and an anti‐CEA antibody either in a single‐chain diabody format (scDb CEA‐S11) or a tandem scFv format (taFv CEA‐S11). In order to facilitate expression of taFv CEA‐S11 in bacteria we selected from a phage display library taFv molecules with an optimised linker that connects the two scFv fragments. ScDb CEA‐S11 and taFv CEA‐S11 were expressed and purified in soluble form from the bacterial periplasm with yields of approximately 100 µg per litre of bacterial culture. In vitro, both bispecific molecules mediated selective and enhanced transduction of CEA‐expressing tumour cells by recombinant adenoviruses. These assays did not reveal any differences in efficiency of adenoviral transduction by the two antibody formats. However, compared with taFv CEA‐S11, scDb CEA‐S11 exhibited a 2‐ to 3‐fold increased stability in human plasma at 37 °C. In summary, we could demonstrate that both formats are suitable for adenovirus targeting to tumour cells with similar biological activity in vitro. Copyright © 2004 John Wiley & Sons, Ltd.