Aptamer:Toxin Conjugates that Specifically Target Prostate Tumor Cells Ted C. Chu, 1 John W. Marks III, 2 Laura A. Lavery, 1 Sarah Faulkner, 1 Michael G. Rosenblum, 2 Andrew D. Ellington, 1 and Matthew Levy 1 1 Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas and 2 Department of Bioimmunotherapy, Section of Immunopharmacology and Targeted Therapy, The University of Texas M.D. Anderson Cancer Center, Houston, Texas Abstract We have used RNA aptamer:gelonin conjugates to target and specifically destroy cells overexpressing the known cancer biomarker prostate-specific membrane antigen (PSMA). Apta- mer:toxin conjugates have an IC 50 of 27 nmol/L and display an increased potency of at least 600-fold relative to cells that do not express PSMA. The aptamer not only promotes uptake into target cells but also decreases the toxicity of gelonin in non-target cells. These results validate the notion that ‘‘escort aptamers’’ may be useful for the treatment of specific tumors expressing unique antigen targets. (Cancer Res 2006; 66(12): 5989-92) Introduction The prostate-specific membrane antigen (PSMA) is a type 2 integral membrane glycoprotein expressed on the surface of prostate carcinoma and the neovasculature of most other solid tumors. The antigen is abundantly expressed at all stages of the cancer (1) and is therefore an attractive target for cancer immunotherapy and imaging. Previous efforts to selectively destroy cancer cells have generally focused on the use of antibodies to deliver toxic payloads (2). In particular, the ribosomal toxin gelonin, a small N-glycosidase protein with a molecular weight of 28 kDa, causes cell death by cleaving a specific glycosidic bond in rRNA and thereby disrupting protein synthesis. However, unlike other ribosomal toxins, such as ricin and abrin, gelonin lacks a translocation domain and thus does not efficiently get into cells at significant concentrations (IC 50 f 2 Amol/L; ref. 3). Gelonin is also useful because it can be expressed as a recombinant protein (rGel) in bacteria. Like gelonin, rGel is not efficiently internalized into cells and has very little nascent toxicity (3). rGel can be chemically conjugated or genetically fused to targeting and delivery moieties at either its NH 2 - or COOH-terminal ends or via introduced cysteine residues (4, 5). For example, cytokine vascular endothelial growth factor (VEGF) has been coupled to gelonin, and the conjugate has been shown to specifically kill cancer cells overexpressing the VEGF receptor FLT-1 (6). Aptamers have previously been selected to bind a variety of targets ranging from small molecules (7) to proteins (8) and whole cells (9, 10). The nucleic acid binding species offer a number of significant advantages over antibodies, including greater stability, ease of synthesis, and lower production costs, making them attractive alternatives for use in both diagnostic and therapeutic applications (8, 11). In the current study, we have used a RNA aptamer that specifically binds PSMA to escort gelonin into prostate tumor cells that express PSMA on their surfaces. The toxin conjugate specifically destroys PSMA-positive prostate cancer cells with an IC 50 of 27 nmol/L and displays an increase in toxicity of at least 600-fold when compared with cells that do not express PSMA. Materials and Methods RNA synthesis. The selection and characterization of anti-PSMA aptamers has previously been described (12). All RNAs were synthesized by runoff transcription from double-stranded DNA templates bearing a T7 RNA polymerase promoter. The sequence of the RNA aptamer (A9) used was 5¶-GGGAGGACGAUGCGGACCGAAAAAGACCUGACUUCUAUACUAA- GUCUACGUUCCCAGACGACUCGCCCGA. All transcription reactions were carried out using the Y639F mutant T7 RNA polymerase. Typical reactions (20 AL) were carried out for 8 hours at 37jC and contained 1 transcription buffer [40 mmol/L Tris (pH 8), 30 mmol/L MgCl 2 , and 5 mmol/L spermidine], 1 mmol/L ATP and GTP, 2 mmol/L of 2¶ F dCTP and 2¶ F dUTP (TriLink Biotech, CA), and 2 Ag double-stranded DNA (dsDNA) template. Following transcription, samples were treated with DNase for 10 minutes at 37jC, and the RNA was purified on a denaturing (7 mol/L urea) 8% polyacrylamide gel. Gel slices containing the RNA product of the appropriate size were eluted overnight in H 2 O, and the RNA was recovered by ethanol precipitation. Toxin conjugation. To a 1.6 mL solution of PBS containing (129 Amol/L) anti-PSMA aptamer, a 5-fold molar excess of the cross-linker N -succini- midyl-3-(2-pyridylodithio)propionate (SPDP; Pierce, Rockford, IL) was added and allowed to react for 30 minutes at room temperature. Excess, unreacted SPDP was removed by gel filtration using Sephadex G-25 (Amersham Biosciences, Uppsala, Sweden) gel chromatography. Recombinant gelonin (5-fold molar excess versus aptamer) was reduced by adding 2 mmol/L DTT (Sigma, St. Louis, MO) and stirring for 30 minutes at room temperature. Excess, unreacted DTT was removed by Sephadex G-25 gel chromatography. Aptamer-SPDP was slowly added to the rGelonin-DTT, with stirring, and the conjugation was allowed to proceed overnight (20 hours) at 4jC, under N 2 gas. Iodoacetamide (Sigma) was then added to a concentration of 2 mmol/L to block any remaining, unconjugated aptamer. The concentration of NaCl was reduced to <10 mmol/L by dilution, and the conjugate was applied to a Blue Sepharose (Amersham) column. Unconju- gated aptamer was eluted by washing with PBS [10 mmol/L sodium phosphate, 150 mmol/L NaCl (pH 7.2)], and the conjugate was eluted with 10 mmol/L sodium phosphate, 1,000 mmol/L NaCl. Unconjugated rGelonin was removed by passage through a Superose S-75 fast protein liquid chromatography column (Amersham). Purified conjugate was concentrated using an Amicon Ultra filter (Millipore, Billerica, MA). Although conjugation using SPDP typically proceeds via a primary amine, we found that the unmodified anti-PSMA aptamer still conjugated to rGel. The link between the aptamer and rGel was confirmed by electrophoretic analysis (see Supplementary Data). Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Matthew Levy, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712. Phone: 512-471-6445; Fax: 512-471- 7014; E-mail: mattlevy@mail.utexas.edu. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-4583 www.aacrjournals.org 5989 Cancer Res 2006; 66: (12). June 15, 2006 Priority Report Research. on January 10, 2016. © 2006 American Association for Cancer cancerres.aacrjournals.org Downloaded from