Claudin 3 and 4 are of particular interest as targets for the delivery of protein toxins because they are both consistently over-expressed on some types of tumors and, once bound to their ligand, are rapidly internalized by an endocytotic process. A fusion toxin in which the protein synthesis inhibitory factor (PSIF) was attached to a C-terminal fragment of CPE (C-CPE) was capable of inducing cytolysis in CLDN3/4-expressing MCF-7 human breast cancer cells [
10] implying that C-CPE-PSIF must have entered the cytosol. However, the work reported here has exposed some limitations to this approach primarily related to the challenge of getting toxic proteins out of endosomal compartments. We opted to explore CPE-based targeting using the protein toxin gelonin since the requirement for gelonin to escape endosomal/lysosomal compartments to reach the ribosome is well-established, and this toxin has been successfully used to make a variety of immunotoxins in the past [
2,
14,
23,
24].
The various fusion proteins tested turned out to be quite stable in the presence of cells and tissue culture media, and those that did get into the 2008 cells were not extensively degraded intracellularly. We have previously documented that the 2008 human ovarian cancer cells express claudin 3 and 4 at levels that are readily detectable by Western blot analysis [
12]. Despite the fact that the addition of CPE did not enhance the cellular accumulation of rGel as measured by Western blot analysis, enhanced uptake relative to native rGel was clearly detected by immunofluorescent staining with an anti-gelonin antibody. The pattern of dispersed punctuate staining is consistent with accumulation in the endosomal/lysosomal compartment. However, this degree of enhancement failed to increase the cytotoxicity of the rGel, suggesting that the rGel could not escape from the compartment into which CPE delivered it. The fact that the treatment with a low concentration of chloroquine, which permeabilizes endosomes, enhanced the toxicity of CPE-E
9-G
4S-R
9-rGel provides a second line of evidence that CPE delivers rGel into a subcellular compartment from which it cannot readily escape. It is noteworthy that chloroquine, the anti-malaria drug clinically used in humans, can also be applied to achieve endosome disruption
in vivo without severe side effects [
25], thus holding promise for further optimization and development as a combined therapy with CPE-based toxins.
Arginine-rich peptides have been extensively used to enhance cellular uptake of various types of cargo, and there is a reasonable presumption that they can deliver cargo out of subcellular compartments as well although there is little information on this point. When R
9 was added to either rGel alone or the CPE-G
4S-rGel fusion protein it markedly increased uptake and cytotoxicity. However, it also obliterated the selectivity afforded by the CPE component (data not shown). In an attempt to mask the R
9 sequence until the CPE had successfully delivered the rGel into the cell, we utilized the strategy of neutralizing the charge on R
9 with an E
9 sequence previously demonstrated by Tsien and colleagues [
18,
26]. In this approach the tumor selectivity is achieved by including a sequence between the R
9 and E
9 that is cleaved by a matrix metalloproteinase to remove the negatively charged E
9 glutamate sequence allowing subsequent tumor penetration of a cargo by R
9. Our design differs in that the recruitment of the fusion toxin to tumor cells is mediated through ligand-receptor interaction, R
9 provides a mechanism for getting out of intracellular compartment and E9 offers an approach to masking the non-specific toxicity of R
9-rGel. The addition of E
9, attached via a flexible G
4S linker, did indeed substantially reduce the toxicity of R
9-rGel and CPE-R
9-rGel confirming in this model the observation that the electrostatic interaction between the E
9 and R
9 sequences when they are tethered together disables the ability of R
9 to translocate cargo across lipid membranes [
18,
26]. However, despite the fact that the R
9 sequence itself is a substrate for the endosomal/lysosomal protease furin, it appears that R
9-rGel was not released from CPE-E
9-G
4S-R
9-rGel after cellular uptake. Given the great potency of R
9-rGel, one would expect even a modest release to be detected as an increase in cytotoxicity. Interestingly, neither a modification of the R
9 sequence directed at enhancing its cleavage by furin nor the use of a GFLG linker increased the cytotoxicity of rGel delivered into the intracellular compartment to which it is directed by CPE. Thus, if the use of R
9 to enhance release of rGel or other protein toxins from intracellular compartment is to be successful, a sequence that is more readily cleaved needs to be found or another approach to linking the E
9 to the R
9 developed. Importantly, the CPE-E
9-linker-R
9-rGel proteins provide an excellent model system with which to test other types of linkers.