Engineering disulfide-linked single-chain Fv dimers [(sFv')₂] with improved solution and targeting properties: anti-digoxin 26–10 (sFv')₂ and anti-c-erbB-2 741F8 (sFv')₂ made by protein folding and bonded through C-terminal cysteinyl peptides

Abstract

Single-chain Fv fusions with C-terminal cysteinyl peptides (sFv') have been engineered using model sFv proteins based upon the 26-10 anti-digoxin IgG and 741F8 anti-c-erbB-2 IgG monoclonal antibodies. As part of the 741F8 sFv construction process, the PCR-amplified 741F8 VH gene was modified in an effort to correct possible primer-induced errors. Genetic replacement of the N-terminal beta-strand sequence of 741F8 VH with that from the FR1 of anti-c-erbB-2 520C9 VH resulted in a dramatic improvement of sFv folding yields. Folding in urea-glutathione redox buffers produced active sFv' with a protected C-terminal sulfhydryl, presumably as the mixed disulfide with glutathione. Disulfide-bonded (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-association. The 741F8 sFv binds to the extracellular domain (ECD) of the c-erbB-2 oncogene protein, which is often overexpressed in breast cancer and other adenocarcinomas. The recombinant ECD was prepared to facilitate the analysis of 741F8 binding site properties; the cloned ECD gene, modified to encode a C-terminal Ser-Gly-His6 peptide, was transfected into Chinese hamster ovary cells using a vector that also expressed dihydrofolate reductase to facilitate methotrexate amplification. Optimized cell lines expressed ECD-His6 at high levels in a cell bioreactor; after isolation by immobilized metal affinity chromatography, final ECD yields were as high as 47 mg/l. An animal tumor model complemented physicochemical studies of 741F8 species and indicated increased tumor localization of the targeted 741F8 (sFv')2 over other monovalent 741F8 species.

Avidity-Mediated Enhancement of In vivo Tumor Targeting by Single-Chain Fv Dimers

“…The 741F8-1 sFvV, which is specific for the tumor-associated antigen c-erbB-2 (HER2/neu), and the 26-10-1 sFvV, which recognizes digoxin and related cardiac glycosides, such as ouabain, were produced with a COOH-terminal Gly 4 -Cys peptide as described (6,17,20). The sFv constructs employed in this study are described in Table 1.…”

“…The sFv constructs employed in this study are described in Table 1. These proteins were expressed in Escherichia coli as cytoplasmic inclusion bodies that were denatured in guanidine HCl, refolded, and purified as described (20). 741F8-1 (sFvV) 2 homodimers, 741F8-1/26-10-1 (sFvV) 2 heterodimers, and 26-10-1 (sFvV) 2 homodimers were produced as described (6).…”

Prolonged in vivo tumour retention of a human diabody targeting the extracellular domain of human HER2/neu

“…In a BlAcore flow cell, approximately 1400 RU (for the scFv) or 600 RU (for the diabody) of HER2/neu ECD (90 kDa;McCartney, 1994) were coupled to a CM5 sensor chip (J0nsson et al, 1991). Association rates were measured under continuous flow of 5 pl min-1 using concentrations ranging from 5.0 x 10-8 to 8.0 x 10-7 M. k1on was determined from a plot of [In (dR/dt)]/t vs concentration (Karlson et al, 1991).…”

Radiometal labeling of recombinant proteins by a genetically engineered minimal chelation site: technetium-99m coordination by single-chain Fv antibody fusion proteins through a C-terminal cysteinyl peptide.

“…Our primary model was the 26-10 sFv, which recognizes digoxin and related cardiac glycosides such as ouabain (1). For tumor localization studies, we used the 741F8 sFv, which targets the tumorassociated antigen c-erbB-2 (7,16). The 26-10-1 sFv' and 741F8-1 sFv' were fusions with a C-terminal Gly4Cys peptide; the 26-10-1 sFv' had a Ser between the light chain variable region and Gly4Cys.…”