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Tai MS, McCartney JE, Adams GP, Jin D, Hudziak RM, Oppermann H, Laminet AA, Bookman MA, Wolf EJ, Liu S, Stafford WF, Fand I, Houston LL, Weiner LM, Huston JS
Targeting C-Erbb-2 Expressing Tumors Using Single-Chain Fv Monomers and Dimers
Cancer Research (1995) 55:S5983-S5989.
Abstract
Single-chain Fv proteins containing a COOH-terminal cysteine (sFv') were 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 vitro 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 easily and selectively removed by gentle reduction, Air oxidation of the reduced 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 reducing conditions. The (26-10-1 sFv')-(741F8-1 sFv') heterodimer,vas prepared 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 imaging studies that were performed on mice bearing human SK-OV-3 tumor xenografts that express the c-erbB-2 as a cell surface antigen were reviewed, Radioiodinated 741F8-2 (sFv')(2) homodimer localized to the tumors with high specificity, as evidenced by excellent tumor:normal tissue ratios, Sagittal section autoradiography of whole animals 24 h after administration of antibody species revealed that 741F8 (sFv')(2) produced a stronger tumor image than comparable doses of the 741F8 Fab, monomeric sFv', and the 26-10 (sFv')(2) control without the high nonspecific background distribution of the 741F8 IgG.
Note
Publication Date: 1995-12-01.
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