Cytotoxic and antitumor activity of a recombinant immunotoxin composed of disulfide-stabilized anti-Tac Fv fragment and truncated Pseudomonas exotoxin.
Disulfide-stabilized Fv (dsFv)-immunotoxins are recombinant immunotoxins in which the inherently unstable Fv moiety, composed of the VH-VL heterodimer, is stabilized by a disulfide bond engineered between structurally conserved framework positions of VH and VL. Anti-Tac(dsFv)-PE38KDEL is composed of such a dsFv, directed to the alpha subunit of the IL2 receptor (IL2R), and containing a truncated form of Pseudomonas exotoxin (PE38KDEL). We have found this new type of immunotoxin to be indistinguishable in its in vitro activity and specificity from its single-chain immunotoxin counterpart, anti-Tac(Fv)-PE38KDEL. We have now examined the therapeutically relevant factors, including stability, pharmacokinetics, and antitumor activity of this new disulfide-stabilized Fv-immunotoxin. We found that anti-Tac(dsFv)-PE38KDEL was specifically cytotoxic to human activated T-lymphocytes in addition to IL2R bearing cell lines. Anti-Tac(dsFv)-PE38KDEL was considerably more stable at 37 degrees C in human serum and in buffered saline than the single-chain immunotoxin, anti-Tac(Fv)-PE38KDEL. The half-life in blood was similar for both immunotoxins (approx. 20 min). The therapeutic potential of the disulfide-stabilized immunotoxin was evaluated using an animal model of immunodeficient mice bearing subcutaneous tumor xenografts of human IL2R-bearing cells. Anti-Tac(dsFv)-PE38KDEL caused complete regression of tumors with no toxic effects in mice. Because dsFv-immunotoxins are more stable and can be produced with significantly improved yields compared to scFv-immunotoxins, dsFv-immunotoxin may be more useful for therapeutic applications.
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Y
Y
Reiter
R J
RJ
Kreitman
U
U
Brinkmann
I
I
Pastan
D036002:ADP Ribose Transferases; D000818:Animals; D000970:Antineoplastic Agents / Q000493:pharmacokinetics* / Q000633:toxicity*; D001427:Bacterial Toxins; D004220:Disulfides / Q000494:pharmacology*; D004355:Drug Stability; D005098:Exotoxins / Q000493:pharmacokinetics* / Q000633:toxicity*; D005260:Female; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000633:toxicity; D000922:Immunotoxins / Q000378:metabolism* / Q000633:toxicity*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D009374:Neoplasms, Experimental / Q000188:drug therapy / Q000276:immunology; D010446:Peptide Fragments / Q000276:immunology / Q000493:pharmacokinetics / Q000633:toxicity; D010957:Plasmids; D015375:Receptors, Interleukin-2 / Q000276:immunology*; D011994:Recombinant Proteins / Q000633:toxicity; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000970:Antineoplastic Agents; D001427:Bacterial Toxins; D004220:Disulfides; D005098:Exotoxins; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D010446:Peptide Fragments; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C055966:immunoglobulin Fv