The goal of these studies was to develop a prodrug strategy to selectively deliver an analog of the highly potent, non–cell type–specific cytotoxin TG to human tumors while minimizing toxicity to normal tissues. G202, the lead PSMA-activated prodrug identified here, was efficiently hydrolyzed by PSMA and was ~60-fold more toxic in vitro to PSMA-positive cells compared to PSMA-negative cells. Whereas TG has no therapeutic index and a lethal dose for 100% of the test population (LD100) equals to 0.2 mg/kg in BALB/c mice, two to three doses of G202 (5.6 to 56 mg/kg) produced tumor regression and growth inhibition in vivo in six separate tumor xenograft models with T/C ratios ranging from 0.05 to 0.32. Thus, the PSMA-activated prodrug approach allowed for the systemic delivery of >150-fold more TG molar equivalents compared to TG alone without significant host toxicity.
The choice of PSMA as the activating enzyme was based on its unique enzymatic activity as both a NAALADase and a folate hydrolase, which could allow for the identification and incorporation of specific peptide substrates into a prodrug strategy that are not hydrolyzed by other proteases present in nonprostatic tissues (). In addition, besides almost universal expression by malignant prostate epithelial cells, PSMA is selectively expressed by tumor ECs within a variety of tumor types but is not expressed by normal ECs or by most normal tissues. The results from our staining of 340 human cancers in a tumor tissue array format are consistent with earlier results in which immunohistochemical staining was used to document PSMA expression in tumor ECs.
Our findings document that PSMA is more highly expressed by certain tumor types, with >60% of samples from hepatocellular, ovarian, renal, and breast cancers demonstrating PSMA expression, whereas a lower percentage of PSMA-expressing samples were observed for melanoma, bladder cancer, and mesothelioma. Consistent with earlier studies, we detected no PSMA expression by ECs from 32 normal tissue samples. However, other studies have documented PSMA expression in the proximal tubule of the kidney and in the brain (28
). In this regard, we detected 12ADTβAsp in the kidney in biodistribution studies in the mouse, although 12ADTβAsp concentrations in tumors were 3- to 15-fold higher than that in normal kidney tissue. In addition, transient reversible renal toxicity was observed in animal toxicological studies in the rat and monkey (table S3
). However, pathological analyses of the brains of rats and monkeys treated with three consecutive doses of G202 (10 mg/kg) showed no evidence of G202-related toxicity. This lack of toxicity most likely stems from the inability of the highly charged prodrug to cross the blood-brain barrier, as evidenced by the low amounts of G202 detected in the brain of mice in the biodistribution studies. Finally, unlike traditional cytotoxic chemotherapies, G202 caused no bone marrow toxicity in mouse, rat, or monkey toxicology studies. These results suggest that G202 could be used clinically without a requirement for blood-product support.
Besides PSMA targeting, a further advantage of this approach lies in the use of a TG analog as the targeted cytotoxin. The 12ADTβAsp analog is highly lipophilic and rapidly partitions into cell membranes after release from the peptide carrier, thus minimizing systemic exposure that can result from leakage of an active toxin from a tumor. Unlike the heterogeneous expression of most current anticancer therapeutic targets, the SERCA pump is expressed homogeneously by all cell types and its continued expression is critically important for cell viability. Thus, resistance to 12ADTβAsp via down-regulation of SERCA expression is unlikely to occur. We validated this hypothesis in experiments in which cancer cells were repeatedly exposed to a cytotoxic dose of TG in an attempt to reproduce in vitro the type of exposure that would be achieved in vivo with repeat doses of therapeutic amounts of a TG analog. Here, no change in clonal survival was observed after 11 cycles of repeat exposure and clonal expansion (fig. S4
). In addition, unlike other commonly used chemotherapeutic agents, which are typically cell proliferation–dependent cytotoxins, TG and its analogs can kill both rapidly proliferating and nonproliferating cells with equal potency. The ability to kill nonproliferating cells makes a TG-based approach a particularly suitable choice of agent for the treatment of prostate cancer, because we have demonstrated that within sites of metastatic prostate cancer >95% of the cancer cells are in a proliferatively quiescent G0
). Furthermore, like human prostate cancer cells, ECs and fibroblasts within the tumor stromal compartment also have a low rate of cell proliferation (49
). These observations suggest that TG also represents a favorable choice for a tumor EC–targeted approach.
Finally, a series of mechanistic studies have demonstrated that TG inhibition of the SERCA pump has catastrophic consequences for the cell. Function of the SERCA pump is critical to the maintenance of intracellular calcium homeostasis after calcium-mediated signaling events and to keep endoplasmic reticulum calcium at the high levels required for the maturation of nascent proteins. Perturbation of the endoplasmic reticulum environment by TG results in elevation of cytoplasmic calcium and depletion of endoplasmic reticulum calcium, thus activating the endoplasmic reticulum stress response. Sustained inhibition of the SERCA pump by TG produces an elevation in cytoplasmic calcium to micromolar concentrations that results in the subsequent activation of the apoptotic caspase cascade, release of pro-apoptotic factors from the mitochondria, and direct stimulation of Ca2+
-activated endonucleases, with the end result being the induction of apoptosis in all cell lines tested (6
). Although these mechanistic studies have highlighted the profound intracellular sequelae that follow TG inhibition of the SERCA pump in all cell types, they also underscore the requirement for a targeting strategy if TG analogs are to be considered as potential therapeutic agents for cancer.
The preclinical results presented here demonstrate that the PSMA-activated tumor EC–targeted prodrug strategy produces a sufficient therapeutic index and tumor targeting to support clinical development of this approach. Results from our dose-finding phase 1 study and from the PSMA tumor tissue array staining will guide the design of future studies that test the effectiveness of this unique targeted approach in the treatment of human cancer.