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

Abstract

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 micro g 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 degrees C. In summary, we could demonstrate that both formats are suitable for adenovirus targeting to tumour cells with similar biological activity in vitro.

Cytotoxic Enhancement of a Bispecific Diabody by Format Conversion to Tandem Single-chain Variable Fragment (taFv)

“…A previous comparative study of scDb and scFv formats (15), for example, showed that both have similar biological activities in vitro, and these results differ from our results. This difference between results may suggest that the most suitable format of small BsAbs depends on the Fv used and/or their combination, but it seems clear, at least, that converting formats has the potential to increase functionality.…”

“…Hence, refolding techniques or mammalian expression systems are routinely used to produce soluble taFv (25,26). Researchers have engineered linker length (27,28) and optimized linkers using phage display technology (15) to promote the expression of soluble taFv; however, whether these linker sequences are applicable to other taFv is unclear. Additionally, the peptide tag usually required for purification may affect the function of small BsAbs in samples prepared using bacterial or mammalian expression systems.…”

“…To date, several studies have compared various small BsAb formats, including multimeric formats, and their differences in function; however, no consensus had been established on the most appropriate format (15,33,34). A previous comparative study of scDb and scFv formats (15), for example, showed that both have similar biological activities in vitro, and these results differ from our results.…”

“…To date, however, there have been few reports presenting comparative analyses of bispecific Dbs and taFv consisting of identical valuable fragments (15) and no reports that discuss differences in binding kinetics and cross-linking ability. There have also been no reports on the influence of format on the cytotoxicity of small BsAbs that retarget immune cells against tumor cells.…”

Novel antibodies as anticancer agents

“…The main antigens that have been used as targets for bispecific antibodies are overexpressed by tumor cells, including EGFR (Witlox et al, 2002;Reusch et al, 2006), HER2 (Zhu et al, 1996;Maletz et al, 2001a), CD19 (Haagen et al, 1994;Csoka et al, 1996;Chen et al, 1998a, b;Kipriyanov et al, 1998;Loffler et al, 2000b;Manzke et al, 2001a, b;Dreier et al, 2003c), CD20 (Brandl et al, 1999;Xiong et al, 2002;Yu et al, 2005), EpCAM (Maletz et al, 2001b;Wimberger et al, 2003), MUC-1 (Katayose et al, 1996) and CEA (FitzGerald et al, 1997;Holliger et al, 1999;Korn et al, 2004).…”