Most of the targeting moieties, such as antibody fragments or growth factor domains, used to construct targeted toxins for anticancer therapy derive from secretory proteins. These normally fold in the oxidative environment of the endoplasmic reticulum, and hence their folding in bacterial cells can be quite inefficient. For instance, only low amounts of properly folded antimetastatic chimera constituted by the amino-terminal fragment of human urokinase (ATF) fused to the plant ribosome-inactivating protein saporin could be recovered. ATF-saporin was instead secreted efficiently when expressed in eukaryotic cells protected from autointoxication with neutralizing anti-saporin antibodies. Pichia pastoris is a microbial eukaryotic host where these domains can fold into a transport-competent conformation and reach the extracellular medium. We show here that despite some host toxicity codon-usage optimization greatly increased the expression levels of active saporin but not those of an active-site mutant SAP-KQ in GS115 (his4) strain. The lack of any toxicity associated with expression of the latter confirmed that toxicity is due to saporin catalytic activity. Nevertheless, GS115 (his4) cells in flask culture secreted 3.5 mg/L of a histidine-tagged ATF-saporin chimera showing an IC(50) of 6 x 10(-11) M against U937 cells, thus demonstrating the suitability of this expression platform for secretion of toxic saporin-based chimeras.

Pseudomonas exotoxin (PE)-based immunotoxins (antibody-toxin fusion proteins) have achieved frequent complete remissions in patients with hairy cell leukemia but far fewer objective responses in other cancers. To address possible mechanisms of resistance, we investigated immunotoxin activity in a model system using the colon cancer cell line, DLD1. Despite causing complete inhibition of protein synthesis, there was no evidence that an immunotoxin targeted to the transferrin receptor caused apoptosis in these cells. To address a possible protective role of prosurvival Bcl-2 proteins, the BH3-only mimetic, ABT-737, was tested alone or in combination with immunotoxins. Neither the immunotoxin nor ABT-737 alone activated caspase 3, whereas the combination exhibited substantial activation. In other epithelial cell lines, ABT-737 enhanced the cytotoxicity of PE-related immunotoxins by as much as 20-fold, but did not enhance diphtheria toxin or cycloheximide. Because PE translocates to the cytosol via the endoplasmic reticulum (ER) and the other toxins do not, ABT-737-mediated effects on the ER were investigated. ABT-737 treatment stimulated increased levels of ER stress response factor, ATF4. Because of its activity in the ER, ABT-737 might be particularly well suited for enhancing the activity of immunotoxins that translocate from the ER to the cell cytosol.

SUMMARYThe fate of the type I ribosome-inactivating protein (RIP) saporin when initially targeted to the endoplasmic reticulum (ER) in tobacco protoplasts has been examined. We find that saporin expression causes a marked decrease in protein synthesis, indicating that a fraction of the toxin reaches the cytosol and inactivates tobacco ribosomes. We determined that saporin is largely secreted but some is retained intracellularly, most likely in a vacuolar compartment, thus behaving very differently from the prototype RIP ricin A chain. We also find that the signal peptide can interfere with the catalytic activity of saporin when the protein fails to be targeted to the ER membrane, and that saporin toxicity undergoes signal sequence-specific regulation when the host cell is subjected to ER stress. Replacement of the saporin signal peptide with that of the ER chaperone BiP reduces saporin toxicity and makes it independent of cell stress. We propose that this stress-induced toxicity may have a role in pathogen defence.