A recombinant immunotoxin containing a disulfide-stabilized Fv fragment.
B3(dsFv)-PE38KDEL is a recombinant immunotoxin composed of the Fv region of monoclonal antibody B3 connected to a truncated form of Pseudomonas exotoxin (PE38KDEL), in which the unstable Fv heterodimer (composed of heavy- and light-chain variable regions) is held together and stabilized by a disulfide bond [termed disulfide-stabilized Fv (dsFV)]. A computer modeled structure of the B3(Fv), made by mutating and energy minimizing the amino acid sequence and structure of McPC603, enabled us to identify positions in conserved framework regions that "hypothetically" could be used for disulfide stabilization without changing the structure or affecting antigen binding. This prediction was evaluated experimentally by constructing a disulfide-linked two-chain dsFv-immunotoxin that was produced in Escherichia coli. The activity and specificity of this immunotoxin was indistinguishable from its single-chain Fv (scFv) counterpart, indicating that, as in B3(scFv), the structure of the binding region is retained in B3(dsFv). Because we introduced the stabilizing disulfide bond in between two framework residues in a position that is conserved in most Fv molecules, this method of linkage between the heavy- and light-chain variable regions should be generally applicable to construct immunotoxins and dsFv molecules using other antibodies. Furthermore, the finding that B3(dsFv) was much more stable at 37 degrees C in human plasma than B3(scFv) indicates that dsFvs are possibly more versatile for therapeutic application than scFvs.
Brinkmann
U
U
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Reiter
Y
Y
Jung
S H
SH
Lee
B
B
Pastan
I
I
D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry* / Q000633:toxicity; D001427:Bacterial Toxins; D001483:Base Sequence; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D003198:Computer Simulation; D004220:Disulfides / Q000032:analysis; D004355:Drug Stability; D005098:Exotoxins / Q000096:biosynthesis / Q000737:chemistry* / Q000633:toxicity; D006801:Humans; D000922:Immunotoxins / Q000096:biosynthesis / Q000737:chemistry* / Q000633:toxicity; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000633:toxicity; D015183:Restriction Mapping; D014407:Tumor Cells, Cultured; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D004220:Disulfides; D005098:Exotoxins; D000922:Immunotoxins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A