TARGETING C-ERBB-2 EXPRESSING TUMORS USING SINGLE-CHAIN FV MONOMERS AND DIMERS

Single-chain Fv proteins containing a COOH-terminal cysteine (sFv') we re constructed by using an antidigoxin 26-10 sFv and an anti-c-erbB-2 741F8 sFv, The fully active sFv' proteins were prepared by expression in Escherichia coli as insoluble inclusion bodies, followed by in vitr o refolding using glutathione redox buffers and purification, The COOH -terminal cysteines of the refolded sPv' proteins were protected by a blocking group presumed to be the glutathionyl peptide, which was easi ly and selectively removed by gentle reduction, Air oxidation of the r educed sPv' monomers resulted in the efficient formation of disulfide- linked sFv' homodimers, designated (sFv')(2), which were stable under oxidizing conditions and relatively slow to be disrupted under reducin g conditions. The (26-10-1 sFv')-(741F8-1 sFv') heterodimer,vas prepar ed and possessed dual-antigen specificity; the active bispecific (sFv' )(2) dimerized under native conditions, apparently as a manifestation of self-association by the 741F8 sFv' subunit Biodistribution and imag ing studies that were performed on mice bearing human SK-OV-3 tumor xe nografts that express the c-erbB-2 as a cell surface antigen were revi ewed, Radioiodinated 741F8-2 (sFv')(2) homodimer localized to the tumo rs with high specificity, as evidenced by excellent tumor:normal tissu e ratios, Sagittal section autoradiography of whole animals 24 h after administration of antibody species revealed that 741F8 (sFv')(2) prod uced a stronger tumor image than comparable doses of the 741F8 Fab, mo nomeric sFv', and the 26-10 (sFv')(2) control without the high nonspec ific background distribution of the 741F8 IgG.