Characterization of a recombinant single-chain molecule comprising the variable domains of a monoclonal antibody specific for human fibrin fragment D-dimer.
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.
Laroche
Y
Y
Corvas International NV, Ghent, Belgium.
Demaeyer
M
M
Stassen
J M
JM
Gansemans
Y
Y
Demarsin
E
E
Matthyssens
G
G
Collen
D
D
Holvoet
P
P
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology* / Q000493:pharmacokinetics; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000276:immunology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000276:immunology; D008969:Molecular Sequence Data; D009036:Moths; D009693:Nucleic Acid Hybridization; D011994:Recombinant Proteins / Q000276:immunology; D014162:Transfection
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D005337:Fibrin; D004247:DNA