Abstract
A recombinant single-chain molecule, scFv-K12G0, containing the variable domains of the monoclonal antibody MA-15C5, specific for fragment D-dimer of human cross-linked fibrin, was constructed and expressed in Spodoptera frugiperda, Sf9, insect cells. The Arg108 carboxyl-terminal amino acid of the variable domain of the light-chain of the antibody was connected through a synthetic Ala-Gly-Gln-Gly-Ser-Ser-Val peptide linker with the Gln1 amino-terminal amino acid of the variable domain of its heavy chain. scFv-K12G0 was secreted by the infected Sf9 cells at a rate of 10 micrograms/10(6) cells within 48 h, resulting in conditioned medium with a maximal concentration of 15 mg of scFv-K12G0/liter. The molecule, purified to homogeneity by ion exchange chromatography and gel filtration, migrated as a single Mr band on reduced sodium dodecyl sulfate-gel electrophoresis. It bound to immobilized fragment D-dimer with an affinity constant of 4.0 x 10(9) M-1 (2.0 x 10(10) M-1 for intact MA-15C5). Clearing of scFv-K12G0 from the circulation in rabbits occurred with an initial half-life (t1/2 alpha) of 10 min and a clearance of 5.1 ml min-1, as compared to 90 min and 210 ml min-1 for intact MA-15C5. Nephrectomy resulted in a prolongation of t1/2 alpha to 110 min, suggesting that the rapid clearance of scFv-K12G0 occurs primarily via the kidney, presumably by glomerular filtration. The results indicate that the single-chain recombinant molecule scFv-K12G0 is secreted in functionally intact form and suggest that it may be useful for targeting of radioisotopes or plasminogen activators to blood clots in vivo.
Highlights
A recombinantsingle-chain molecule, scFv-KlzGo, The murine monoclonal antibody MA-15C5l directed against containing the variable domaionfsthe monoclonal an- fragment D-dimerof cross-linked human fibrind,eveloped by tibody MA-15C5, specific forfragmentD-dimer of Holvoet et al [3], has shown promise for thrombus imaging human cross-linked fibrin, was constructed and ex- in animal models [3, 4]
Ser-Val peptide linker with the Gln' amino-terminal intact murine monoclonal antibodies conjugated amino acid of the variable domainof its heavy chain. to isotopes or toxins have been utilized for targeting agents scFv-KlzGowas secretedby the infected Sf9 cells at a for the diagnosis and treatmenotf malignant ocrardiovascular rate of 10 wg/106cells within 40 h, resulting incondi- diseases [18],the immunogenicity of murine monoclonal antioned medium with a maximal concentrationof 15mg of scFv-KlzGo/liter
The molecule, purified to homogeneity by ion exchange chromatography and gel filtration, migrated as a single M, band on reduced sodium dodecyl sulfate-gel electrophoresis. It bound to immobilized fragment D-dimer with an affinity constant of 4.0 X los M"
Summary
Ser-Val peptide linker with the Gln' amino-terminal intact murine monoclonal antibodies conjugated amino acid of the variable domainof its heavy chain. 15C5 was connected througha synthetic peptide linker to thededuced that the Glu-Ser dipeptidecould be replaced by the original amino-terminal aminoacid of its heavy chain variable domain Ly~'"~-Arg'V~L*carboxyl-terminal amino acids and that the Tyr-Val (VH).The fibrin fragment D-dimer binding capacity and the dipeptide could be replaced by the original Gln1-Va12VH aminopharmacokinetic propertiesof scFv-KIPGowere determined.
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