A cleavable adapter to reduce nonspecific cytotoxicity of recombinant immunotoxins

Abstract

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.

Saporin as a novel suicide gene in anticancer gene therapy

“…11 and in anticancer therapy, to construct immunotoxins or ligand toxins [18][19][20][21] and SAP complementary DNA (cDNA) has been used for expressing targeted recombinant chimeras. 17,[22][23][24][25] When a SAP-based secretory chimera was expressed in Xenopus laevis oocytes, total inhibition of cellular protein synthesis was observed that could be restored only by supplementing neutralizing anti-SAP antibodies into the oocyte cytosol, 26 indicating that, although almost all of the chimera was secreted, the few molecules escaping from the secretory pathway were sufficient to inactivate protein synthesis in the host cells.…”

Saponins modulate the intracellular trafficking of protein toxins

“…Saporin and saporin-EGF were cloned previously [14]. All proteins were expressed in Escherichia coli Rosetta DE3 pLys (Novabiochem, Schwalbach, Germany) and isolated by Nickelnitrilotriacetate chromatography as described elsewhere [15].…”

Diving through Membranes: Molecular Cunning to Enforce the Endosomal Escape of Antibody-Targeted Anti-Tumor Toxins

“…Regardless of the endosomal escape mechanism, drugs that exert their effect inside the cytosol or in organelles should accumulate suitably within the cell to achieve maximum toxicity. A promising strategy to achieve intracellular trapping is the inclusion of a cleavable peptide that, once cleaved inside the cytosol, converts the drug to a membrane-impermeable protein by removing any motif or domain used for membrane translocation [46,47]. An analogous procedure describes the insertion of a natural organelle-specific cleavage recognition site of the mitochondrial malate dehydrogenase signal sequence.…”