A cleavable adapter to reduce nonspecific cytotoxicity of recombinant immunotoxins.
One of the problems associated with the administration of immunotoxins is hypersensitivity reaction such as vascular leak syndrome. This may be prevented by decreasing the plasma half-life. To improve immunotoxins with respect to reduced side effects, we have previously described the development of a cleavable adapter. This adapter links the toxic moiety and ligand that are usually directly coupled. In our study, the cytotoxicity of saporin linked either directly or via the adapter to epidermal growth factor (EGF) was evaluated in vitro. The immunotoxins exhibited similar cytotoxic activity towards A-431 and HER14 cells (IC(50) < 10 nM). The supernatant from 6 hr cultures of HER14 cells incubated in the presence of the adapter-containing immunotoxin exhibited a significantly reduced cytotoxicity as compared to the directly coupled immunotoxin. Western blotting revealed that the adapter was cleaved, thus supporting our proposal that cleavable adapters may reduce nonspecific effects. A similar reduced half-life was detected in platelet-poor plasma. In contrast MCF-7 cells remain unaffected by the immunotoxins. This was shown to be due to the absence of detectable EGF-receptor in comparison to A-431 and HER14 cells as determined by Western blotting. Furthermore, we could show that the adapter does not exert an effect on the N-glycosidase activity of saporin. These results suggest that the use of cleavable adapters may be a useful tool in immunotoxins for reducing the killing of surrounding noncancerous cells due to nonspecific binding.
Heisler
Iring
I
Institut für Klinische Chemie und Pathobiochemie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Berlin, Germany.
Keller
Jutta
J
Tauber
Rudolf
R
Sutherland
Mark
M
Fuchs
Hendrik
H
D000225:Adenine / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D017931:DNA Primers / Q000737:chemistry; D004815:Epidermal Growth Factor / Q000633:toxicity; D066246:ErbB Receptors / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006207:Half-Life; D006514:Hepatitis B Surface Antigens / Q000235:genetics / Q000378:metabolism; D006801:Humans; D000922:Immunotoxins / Q000633:toxicity*; D066298:In Vitro Techniques; D009699:N-Glycosyl Hydrolases / Q000633:toxicity*; D010940:Plant Proteins / Q000633:toxicity*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism / Q000633:toxicity; D054789:Ribosome Inactivating Proteins, Type 1; D000076985:Saporins; D014407:Tumor Cells, Cultured / Q000187:drug effects* / Q000378:metabolism
D017931:DNA Primers; D006514:Hepatitis B Surface Antigens; D000922:Immunotoxins; D010940:Plant Proteins; D011994:Recombinant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D006026:Glycoside Hydrolases; D009699:N-Glycosyl Hydrolases; D000076985:Saporins; D000225:Adenine