Abstract 1817: Development of claudin-3 and claudin-4-targeted antiprostate cancer prodrug
Authors: Romanov Victor , Whyard Terry , Waltzer Wayne C. , Gabig Theodore 
Published: May 2015

Abstract
Prostate cancer is the second leading cause of non-cutaneous cancer-related death in males, and effective strategies for treatment of metastatic disease are currently limited to androgen ablation for temporary benefit.
The tight junction proteins, claudin 3 [CLDN3] and claudin 4 [CLDN4], serve as cell-surface receptors for the Clostridium perfringens enterotoxin [CPE]. The toxin is especially lethal for cells expressing large amounts of claudin-3 or -4, which includes many cancer cells. Most prostate cancer cells overexpress CLDN3 and CLDN4, and the over-expressed CLDNs are aberrantly distributed to the basolateral plasma membrane, making these cells particularly sensitive to cytolysis by CPE.
To overcome potential systemic toxicity of native CPE, a modified protoxin was constructed with a tethered ligand attached to the C-terminus connected by a flexible linker containing a PSA-specific protease cleavage site (construct 1). Prostate cancer cells secrete PSA locally that is proteolytically active; however, circulating PSA is inactivated via binding to plasma protease inhibitors. Therefore, CLDN-binding site of the construct 1 become available after PSA cleavage. This engineered protoxin selectively and efficiently lyses PSA-producing prostate cancer cells whereas CLDN3 and CLDN4 positive cells that do not express PSA were resistant to cytolysis. Combination of CLDN3, 4 targeting with PSA activation provides selectivity to CLDN3 and 4 expressing prostate cancer cells that also produce PSA.
Although native CPE has potential usefulness for treating several cancers including prostate where CLDN3 and 4 CPE receptors are overexpressed some challenges with immunogenicity, toxicity, and (possibly) the development of resistance may need to be overcome. An alternative approach is to utilize C-CPE, which corresponds approximately to receptor binding domain I for generation C-CPE fusion compounds with cytotoxic motifs that may be superior to use of native CPE for cancer treatment.
Therefore, we replaced the active part of CPE in construct 1 by thapsigargin (TG) (construct 2). TG is a non-selective toxin that kills cells independently of their proliferative status. Using TG as a cytotoxic part of the drug has appealing prospects with respect to prostate cancer which is characterized by slow proliferation rates of the cancer cells.
For primary investigation TG was conjugated directly to C-CPE (construct 3). This compound was tested against a panel of prostate cancer cells expressing variable amount of CLDN3-4 and PSA. Construct 3 was more effective than TG against C4-2B cells that produced high amount of CLDN3, 4. It shows low efficiency against PC-3 cells that produced lower than C4-2B amount of CLDN3 and 4.
Therefore, PSA-claudin-based dual targeted protoxin construct is a feasible model for the development of clinically useful agents to treat hormone-refractory prostate cancer.
Citation Format: Victor Romanov, Terry Whyard, Wayne C. Waltzer, Theodore Gabig. Development of claudin-3 and claudin-4-targeted antiprostate cancer prodrug. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1817. doi:10.1158/1538-7445.AM2014-1817