Supplementary Figure 7 from Aptamer:Toxin Conjugates that Specifically Target Prostate Tumor Cells

Supplementary Figures 1-6 from Aptamer:Toxin Conjugates that Specifically Target Prostate Tumor Cells

Nucleic acids that bind to cells and are subsequently internalized could prove to be novel delivery reagents. An anti-prostate specific membrane antigen aptamer that has previously been shown to bind to prostate tumor cells was coupled to siRNAs via a modular streptavidin bridge. The resulting conjugates could be simply added onto cells without any further preparation, and were taken up within 30 min. The siRNA-mediated inhibition of gene expression was as efficient as observed with conventional lipid-based reagents, and was dependent upon conjugation to the aptamer. These results suggest new venues for the therapeutic delivery of siRNAs and for the development of reagents that can be used to probe cellular physiology.

B19 Prostate cancer is the second leading cause of death amongst males in America. Diagnosis and prognosis of prostate cancer is heavily based upon cell morphology and the levels of prostate specific antigen (PSA), two factors that are prone to false negatives and misdiagnosis. The identification of molecular alternatives to the detection of all tumors in general as well as cell specific markers to specific tumor types can be very useful. Nucleic acid aptamers serve as excellent detection tools due to their affinity for the target they are selected against as well their versatile nature that allows them to be coupled to molecules such as siRNA, DNA tags, quantum dots, peptides etc. We have now directly selected aptamers against prostate cancer biomarker molecules on the surfaces of cultured prostate cancer cell lines, LNCaP and PC3. These cell lines differ in the expression of the tumor marker prostate specific membrane antigen (PSMA), a very promising cell surface biomarker. LNCaP cells over-express this surface marker while PC3 have very little PSMA on their surface. Two aptamers, LN101 and LN104, selected against LNCaP cells and two aptamers, PC301 and PC304, selected against PC3 specifically were generated from negative selection while four aptamers, LP702, LP705, LP719 and LP720 were generated through positive selections. The sensitive nature of these aptamers as cancer diagnostic tools was demonstrated by FACs, confocal microscopy and a novel technique called the Proximity Ligation Assay (PLA). The principle of PLA involves the use of two affinity tags on the surface of the target, which when in proximity to one another can ligate in the presence of a connector nucleotide (splint) and the amplicon generated is detected via real-time PCR. In order to adapt aptamers to PLA, we constructed a five-piece PLA system where aptamers were annealed to specific 39and 59 DNA probes via specific RNA linkers or extensions that had been chosen based on their ability to leave the aptamer structure intact. This construct was used in target specific detection of cognate cancer cells in a heterogeneous mixture of cells, the sensitivity of detection being as efficient as 10 LNCaP and 10 PC3 cells in a mixture of 105 HeLA cells, with little to no cross-reactivity. In conclusion, aptamer-mediated PLA is extremely sensitive and target specific presenting itself as a potential molecular sensor for early cancer detection.

B21 Functional nucleic acids, known as aptamers, can be selected from random sequence populations. Aptamers can potentially be applied in conventional roles for identifying and typing tumors, essentially as substitutes for antibodies or other staining reagents. Fluorescent aptamers have been used to label cells in flow cytometry. A 2â™-fluoropyrimidine modified RNA pool that contains a random sequence core of 30 nucleotides flanked by primer binding sites has been generated and selected against three lung tumor cell lines that differ in their Epidermal Growth Factor Receptor (EGFR) expression were chosen as targets for selection. NCI-H358 is from a non-small cell lung cancer and expresses wild type EGFR. NCI-1650 is from a bronchoalveolar carcinoma and expresses a mutant form of EGFR (in-frame deletion delE746-A750). NCIH526 is from a small cell lung cancer (SCLC) cell lines and expresses low to undetectable levels of EGFR. Selection of aptamers against these three lines serves several purposes: first, it will potentially demonstrate that aptamers against cell surfaces can be used to differentiate different types of lung tumor cells as well as typing tumor cells from normal cells. These aptamers can further be modified for early detection of tumorigenic cells. Second, aptamers may be derived that recognize particular types or levels of EGFR, an important tumor marker. Finally, selection methods that target both adherent and suspension cells will be developed.

Abstract We have used RNA aptamer:gelonin conjugates to target and specifically destroy cells overexpressing the known cancer biomarker prostate-specific membrane antigen (PSMA). Aptamer:toxin conjugates have an IC50 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)

Aptamers that bind to prostate specific membrane antigen (PSMA) were conjugated to luminescent CdSe and CdTe nanocrystals for cell-labeling studies. The aptamer-nanocrystal conjugates showed specific targeting of both fixed and live cells that overexpressed PSMA. More importantly, aptamers were able to label cells dispersed in a collagen gel matrix simulating tissue. The specific binding abilities and synthetic accessibility of aptamers combined with the photostability and small size of semiconductor nanocrystals offers a powerful and general tool for cellular imaging.

Proc Amer Assoc Cancer Res, Volume 46, 2005 6183 PSMA is a transmembrane receptor whose expression increases on prostatic tumor cells during disease progression. This protein is also expressed on the endotheluim of tumor vasculature but not normal vasculature. Aptamers are small nucleic acids selected to bind proteins such as cell-surface tumor antigens with high affinity and specificity. Aptamers have the potential to serve as replacements for cell-targeting antibodies or other cell-targeting ligands. We utilized a modified RNA aptamer (designated PSMAA) known to bind the external domain of PSMA with high affinity (previously reported Ki = 2 nM). The 22 kDa PSMAA molecule was conjugated to recombinant gelonin(rGel) toxin using the heterobifunctional cross-linking agent SPDP. The PSMAA/rGel conjugate was then purified by ion exchange and gel permeation chromatography. The final product was uncontaminated by free aptamer or free rGel and migrated as a single species (∼50 kDa) by SDS-PAGE. Analysis of the construct demonstrated that the rGel component was enzymatically active compared to free rGel. In addition, the conjugate was found to bind specifically to PSMA-expressing LNCAP cells. Cytotoxicity studies of the PSMAA/rGel conjugate demonstrated an I.C.50 of 32 nM on antigen-positive LNCAP cells compared to an I.C.50 of 350,000 nM on PC3 cells, which contain much less antigen; a targeting index of approximately 10,000 fold. Internalization studies should reveal the details of toxin conjugate entry, and animal model studies are ongoing. These data suggest that aptamers can be used to successfully deliver protein molecules such as toxins to tumor cells and may provide a novel approach to development of targeted therapeutic agents. The fact that aptamers can be chemically synthesized and thereby site-specifically conjugated makes them especially interesting as targeting ligands. The PSMAA-rGel construct may prove useful to target PSMA on prostate cells and on tumor vasculature. Research conducted, in part, by the Clayton Foundation for Research.

Cancer of the prostate affects approximately 1 in 11 men. Current early screening for prostate cancer utilizes digital rectal examinations to detect anomalies in the prostate gland and blood test screenings for upregulated levels of prostate specific antigen (PSA). Many of these tests are invasive and can often be inconclusive as PSA levels may be heightened due to benign factors. Prostate specific membrane antigen (PSMA), a well-characterized integral membrane protein, is expressed in virtually all prostate cancers and often correlates with cancer aggressiveness. Therefore, it may be used as an indicator of cancer growth and metastases. PSMA-specific antibodies have been identified and conjugated to fluorescent markers for cancer cell targeting; however, both the antibodies and markers possess significant limitations in their pharmaceutical and diagnostic value. Here we report the use of semiconductor nanocrystals bioconjugated to PSMA-specific aptamer recognition molecules for prostate carcinoma cell targeting. The nanocrystal/aptamer bioconjugates are small biocompatible probes with the potential for color-tunability for multicolor imaging. Ongoing in vitro and in vivo research seeks to introduce these nanoparticle bioconjugates into medical diagnostics.