Abstract 3627: Targeted delivery of gelonin to claudin-expressing cancer cells and use of activatable cell penetrating peptides to enhance potency

Abstract

Abstract Treatment of tumors with macromolecular toxins directed to cytoplasmic targets requires selective endocytosis followed by release of intact toxin from the endosome/lysosome compartment. The latter step remains a particular challenge. Claudins 3 and 4 are tight junction proteins that are over-expressed in many types of tumors. Although their functional role in cancer progression remains unclear, the differential expression of these proteins between tumor and normal cells, in addition to their membrane localization, makes them prime candidates for targeted cancer therapy. This study utilized the C-terminal 30 amino acid fragment of C. perfringens enterotoxin (CPE), that binds to claudins 3 and 4, to deliver the toxin gelonin (rGel) and examined a new strategy for enhancing delivery to the cytoplasm. CPE was fused to rGel at its N-terminal end via a flexible G4S linker. This molecule was internalized into vesicles from which location it produced little cytotoxicity. 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. To enhance release from the endosomal/lysosomal compartment a poly-arginine sequence (R9) was introduced between the CPE and the rGel. CPE-R9-rGel was 10-fold more cytotoxic but selectivity for claudin-expressing cells was lost. Given that the R9 moiety was so effective at enhancing uptake, we reasoned that it would likely also be good at releasing rGel from the compartment into which CPE delivered it. However, to make use of the R9 sequence in this manner it is necessary to mask the R9 until after CPE-mediated and claudin-dependent endocytotic uptake. To this end an E9-G4S-R9-rGel fusion protein was produced and tested for cytotoxicity. The addition of the E9 through a G4S linker to R9-rGel increased the IC50 by 5.3-fold from 38 to 202 nM indicating that the inhibitory E9 domain can largely neutralize the R9. Addition of CPE to the E9-G4S-R9-rGel fusion protein further reduced the IC50 to 482 nM consistent with the concept that the CPE directs the CPE-E9-G4S-R9-rGel to intracellular vesicles from which it has difficulty in fully escaping. However, addition of endosomolytic reagent chloroquine at a non-toxic concentration increased the inhibition of growth produced by 31.25 nM CPE-E9-G4S-R9-rGel from 10% to 76%. Thus, while CPE provides targeting to claudin-expressing tumor cells, R9 plus chloroquine provide a mechanism for getting out of intracellular compartment, and E9 offers an approach to masking the non-specific toxicity of R9-rGel. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3627. doi:10.1158/1538-7445.AM2011-3627