ENGINEERING DISULFIDE-LINKED SINGLE-CHAIN FV DIMERS [(SFV')(2)] WITH IMPROVED SOLUTION AND TARGETING PROPERTIES - ANTIDIGOXIN-26-10 (SFV')(2) AND ANTI-C-ERBB-2 741F8 (SFV')(2) MADE BY PROTEIN-FOLDING AND BONDED THROUGH C-TERMINAL CYSTEINYL PEPTIDES

Single-chain Fv fusions with C-terminal cysteinyl peptides (sFv') have been engineered using model sFv proteins based upon the 26-10 anti-di goxin IgG and 741F8 anti-c-erbB-2 IgG monoclonal antibodies, As part o f the 741F8 sFv construction process, the PCR-amplified 741F8 V-H gene was modified in an effort to correct possible primer-induced errors, Genetic replacement of the N-terminal beta-strand sequence of 741F8 V- H With that from the FR1 of anti-c-erbB-2 520C9 V-H resulted in a dram atic improvement of sFv folding yields, Folding in urea-glutathione re dox buffers produced active sFv' with a protected C-terminal sulfhydry l, presumably as the mixed disulfide with glutathione, Disulfide-bonde d (sFv')(2) homodimers were made by disulfide interchange or oxidation after reductive elimination of the blocking group, Both 26-10 (sFv')( 2) and 741F8 (sFv')(2) existed as stable dimers that were well behaved in solution, whereas 741F8 sFv and sFv' exhibited considerable self-a ssociation. The 741F8 sFv binds to the extracellular domain (ECD) of t he c-erbB-2 oncogene protein, which is often overexpressed in breast c ancer and other adenocarcinomas. The recombinant ECD was prepared to f acilitate the analysis of 741F8 binding site properties; the cloned EC D gene, modified to encode a C-terminal Ser-Gly-His(6) peptide, was tr ansfected into Chinese hamster ovary cells using a vector that also ex pressed dihydrofolate reductase to facilitate methotrexate amplificati on, Optimized cell lines expressed ECD-His(6) at high levels in a cell bioreactor; after isolation by immobilized metal affinity chromatogra phy, final ECD yields were as high as 47 mg/l, An animal tumor model c omplemented physicochemical studies of 741F8 species and indicated inc reased tumor localization of the targeted 741F8 (sFv')(2) over other m onovalent 741F8 species.