Efficient Construction of a Diabody Using a Refolding System: Anti- Carcinoembryonic Antigen Recombinant Antibody Fragment

Abstract

Recombinant fragments of the variable region of antibodies are useful in many experimental and clinical applications. However, it can be difficult to obtain these materials in soluble form after their expression in bacteria. Here, we report an efficient procedure for preparing several variable-domain fragments (Fv), single-chain Fv (scFv), and a diabody (the smallest functional bispecific antibody) of anti-carcinoembryonic antigen (CEA) antibody by overexpression in Escherichia coli in inclusion bodies, using a refolding system to obtain renatured proteins. Two types of refolded Fv were prepared: (i) Heavy and light chains of the immunoglobulin variable regions (VH and VL, respectively) were coexpressed with a dicistronic expression vector (designated Fv(co)); (ii) VH and VL were expressed separately, mixed stoichiometrically, and refolded (designated Fv(mix)). All samples refolded with high efficiency; Fv(co), Fv(mix), scFv, and the bispecific diabody bound to several CEA-positive cell lines, exactly as did soluble Fv fragments secreted by E. coli (Fv(sol)) and the parent IgG. The refolded fragments inhibited binding of the parent IgG to CEA-positive cell lines, indicating that their epitope is identical to that of IgG. The bispecific diabody, which combined variable-region fragments of anti-CEA antibody with variable-region fragments of anti-CD3 antibody, was also prepared using the refolding system. This refolded diabody could bind to lymphokine-activated killer cells. In addition, its cytotoxicity toward human bile duct carcinoma TFK-1 and other several other CEA-positive cell lines was concentration-dependent. Taken together, our results suggest that a refolding procedure can be used to prepare various functional antibody fragments (Fv, scFv, and diabody).

Thermodynamic Consequences of Mutations in Vernier Zone Residues of a Humanized Anti-human Epidermal Growth Factor Receptor Murine Antibody, 528

“…For functional characterization of the Fvs, c-Myc peptide tag was fused to the C terminus of both chains. Flow cytometric analyses using some EGFR-positive cells and anti-c-Myc peptide tag antibody 9E10 indicated that both Fv fragments had binding profiles identical to that of IgG, and specific inhibition of the binding of the Fvs to the cell surface antigen by the IgG was confirmed (data not shown) (37)(38)(39)41). These results suggest that m528Fv and h528Fv recognized the same epitope structure of EGFR as the parental 528 IgG.…”

“…1 shows the amino acid sequences of the variable domain fragment of anti-human EGFR murine antibody 528 (m528Fv) and of humanized 528Fv (h528Fv). The genes encoding h528Fv were chemically synthesized by a method based on overlap extension PCR as described previously (38,42). The NcoI ϩ SacII-digested PCR products were cloned into pUC19-based plasmid pRA (43).…”

Humanization of the Bispecific Epidermal Growth Factor Receptor × CD3 Diabody and Its Efficacy as a Potential Clinical Reagent

“…Preparation of the diabodies from an E. coli intracellular insoluble fraction by refolding has been described previously (19,21,36). In brief, vector-transformed cultures of E. coli strain BL21 (DE3) were incubated at 37jC in Luria-Bertani broth.…”

Highly Effective Recombinant Format of a Humanized IgG-like Bispecific Antibody for Cancer Immunotherapy with Retargeting of Lymphocytes to Tumor Cells

“…Preparation of hEx3 Diabody and hEx3-scFv-Fc MoleculehEx3 diabody was prepared from inclusion bodies expressed in Escherichia coli as described previously (14,15,21,22). For hEx3-scFv-Fc, CHO cells were cotransfected with equal amounts of DNA from vector pcDNA-hOHL-Fc and pcDNAh5HL-CL.…”