Recombinant immunotoxin containing a disulfide-stabilized Fv directed at erbB2 that does not require proteolytic activation.
PE35/e23(dsFv)KDEL is a recombinant immunotoxin composed of a recombinant form of Pseudomonas exotoxin that does not need proteolytic activation and a disulfide-stabilized Fv fragment of the anti-erbB2 monoclonal antibody e23. In this molecule, the variable heavy (V H) domain is inserted near the carboxyl terminus of PE at position 607 and the variable light (V L) domain is connected to the V H domain by a disulfide bond engineered into the framework region. The disulfide bond forms between cysteines introduced at position 44 of V H and position 99 of V L [Reiter et al. (1994) J. Biol. Chem. 269, 18327-18331]. In contrast to other PE-derived Fv fusion proteins, this type of recombinant toxin does not need proteolytic activation of the toxin domain. PE35/e23(dsFv)KDEL is very cytotoxic toward erbB2 antigen-expressing N87 cells (IC50 = 0.8 ng/mL) despite the fact that it binds to the erbB2 protein only 25% as well as e23(dsFv)PE38KDEL, in which the dsFv moiety is located at the amino terminus of the toxin. The lower binding affinity is probably due to interference by domain III of PE with the amino terminus of e23(V H), possibly where the antigen binding sites are located. Nevertheless, the specificity of immunotoxin is still retained, and it is very stable at 37 degrees C. Because of its small size, stability, and activity without proteolytic processing, this immunotoxin may be advantageous for tumor treatment. PE35/e23(dsFv)KDEL was also used to gain information about whether reduction of the disulfide bonds connecting V H and V L occur in the endoplasmic reticulum (ER) or in a proximal compartment. To do this, we switched the ER retention sequence KDEL from the toxin--V H subunit to the V L subunit. Our results suggest that reduction of the disulfide bond connecting the dsFv heterodimer occurs before the immunotoxin reaches the ER, where translocation to the cytosol appears to occur.
Kuan
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Laboratory of Molecular Biology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
Pastan
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D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D001667:Binding, Competitive; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D017931:DNA Primers; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D005098:Exotoxins / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D006801:Humans; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011500:Protein Synthesis Inhibitors / Q000633:toxicity; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D017931:DNA Primers; D004220:Disulfides; D010450:Endopeptidases; D005098:Exotoxins; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D011500:Protein Synthesis Inhibitors; D018719:Erb-b2 Receptor Tyrosine Kinases; D011994:Recombinant Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A