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Definitions

• Pancreatic ductal adenocarcinoma is the fourth most common cause of cancer death in the United States, accounting for 30,000 deaths yearly in the United States (Jemal et al. 2009). Pancreatic cancer is characterized by a rapid disease progression and absence of specific symptoms, largely precluding an early diagnosis and meaningful treatment (Stathis & Moore, 2010; Schneider et al. 2005).
• aptamers that specifically bind pancreatic cancer cells are provided.
• Such pancreatic cancer cell aptamers may include an RNA molecule that specifically binds a pancreatic cancer cell surface protein.
• the RNA molecule that is used as an aptamer in accordance with the embodiments described herein may include a nucleotide sequence of GAAUGCCC (SEQ ID NO: 8).
• the RNA molecule may include a nucleotide sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.
• the aptamer may be conjugated to one or more therapeutic agents, one or more diagnostic agents, or a combination thereof.
• the one or more therapeutic agents may be selected from an shRNA molecule, an siRNA molecule, an mRNA molecule, or an miRNA molecule.
• methods for delivering a therapeutic agent to a pancreatic cancer cell may include a step of contacting the pancreatic cancer cell with a pancreatic cancer cell aptamer conjugate.
• the pancreatic cancer cell aptamer conjugate may include a pancreatic cell aptamer component and a therapeutic agent component.
• the pancreatic cell aptamer component includes an RNA molecule that specifically binds a pancreatic cancer cell surface protein, resulting in internalization of the pancreatic cell aptamer conjugate - such as those described herein.
• the therapeutic agent component may include any suitable therapeutic agent that can be conjugated to an mRNA molecule including, but not limited to, an shRNA molecule, an siRNA molecule, an mRNA molecule, or an miRNA molecule.
• Such a method may include a step of administering a therapeutically effective amount of a pancreatic cell aptamer, wherein the pancreatic cell aptamer comprises an RNA molecule that specifically binds a pancreatic cancer cell surface protein, and wherein the pancreatic cell aptamer prevents binding of a pancreatic cell ligand.
• the RNA molecule that is used as an aptamer in accordance with the embodiments described herein may include a nucleotide sequence of GAAUGCCC (SEQ ID NO: 8).
• the RNA molecule may include a nucleotide sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.
• the aptamer may be conjugated to one or more therapeutic agents, one or more diagnostic agents, or a combination thereof.
• the aptamers may be part of a pharmaceutical composition for use in the methods of treating pancreatic cancer. Said pharmaceutical compositions may further comprise one or more additional therapeutic agents (e.g., chemotherapeutics).
• FIG. 1 is a schematic diagram illustrating a selection/counterselection SELEX process for selecting pancreatic cancer cell-specific aptamers according to some embodiments. Briefly, a population of 2'F-Py RNAs was incubated with a nonpancreatic cancer cell line (Huh7 hepatocarcinoma cell line) or a healthy pancreatic cell line for the counterselection step. Unbound oligonucleotides sequences were recovered and incubated with a pancreatic cancer cell line (Panc-1 ) for the selection step. Unbound sequences were discarded and bound sequences were recovered by total RNA extraction. Sequences enriched by the selection step are then amplified before the subsequent cycle of selection. (SELEX method based on an adapted version of Esposito et al. 201 1 ).
• Figure 2 shows the secondary structure of six RNA aptamers selected from randomized N40 RNA libraries according to some embodiments.
• the secondary structures of the six aptamers (A) SEQ ID NO:1 ; (B) SEQ ID NO:2; (C) SEQ ID NO:3; (D) SEQ ID NO:4; (E) SEQ ID NO:5; and (F) SEQ ID NO:6; were predicted using the Mfold program.
• RNA library pool is shown as Lib. PC; Panc-1 , NC; Huh7.
• Figure 4 illustrates cell-internalization of target cells by confocal microscopy.
• Cells were grown in 35mm dishes and incubated with 100nM of Cy3- labeled RNA. After one hour incubation, cells were washed and took images using 40x magnification.
• A Initial RNA library pool was incubated in Panc-1 and Huh7.
• B Each aptamer clones were incubated in Panc-1 . Red; Cy3-labeled RNA, Blue: Hoechest 33342 (Nuclear dye for living cells).
• Figure 5 illustrates cell-internalization in other types of pancreatic cancer cells by confocal microscopy.
• Each RNA aptamer clones labeled with Cy3 were applied to different type of pancreatic cancer cells.
• Cells were grown in 35mm dishes and incubated with 100nM of RNA. After one hour incubation, cells were washed and took images using 40x magnification.
• A AsPC-1 .
• B CFPAC-1 .
• C BxPC-1 .
• Figure 6 illustrates cell-internalization in normal primary pancreatic cells by confocal microscopy.
• Each of the RNA aptamer clones labeled with Cy3 was applied to different type of normal pancreatic cells.
• Cells were grown in 35mm dishes and incubated with 100nM of RNA. After one hour incubation, cells were washed and took images using 40x magnification.
• Red Cy3-labeled RNA (none shown)
• Blue Hoechest 33342 (Nuclear dye for living cells). None of the normal pancreatic cancer cells internalized the RNA aptamers.
• Figure 7 shows the binding affinity of P19, P15 and P1 aptamers.
• the measurement of dissociation constant (K D ) was done by physiology function of Zeiss LSM using various concentrations (15.6-500nM) of Cy3 labeled aptamers.
• the affinity of P19, P15, and P1 were 64.76nM, 70.72nM, and 1 12.2nM, respectively.
• FIG. 8 illustrates cell internalization competition assays by confocal microscopy.
• Panc-1 cells were incubated with fluorescently labeled P19 RNA (200 nM) and increasing amounts (1 ⁇ ) of unlabeled each clone aptamers as competitors against the labeled RNA.
• the fluorescence intensity was quantified in the presence of increasing amounts of competitors using confocal microscopy and analyzed statistically.
• FIG. 9 shows cell proliferation assays for cells treated with P1 and P19 aptamers.
• Cell proliferation was quantified by WST-1 reagent following the manufacturer's guidelines.
• Panc-1 2.5x10 5 cells
• WST-1 reagent was used at a 1 :100 dilution to plates and incubated for one hour.
• the enzymatic reaction was measured at 450 ⁇ using Bio-Tek ELISA reader
• FIG. 10 illustrates results of in vivo experiments.
• Panc-1 pancreatic cancer cells were injected subcutaneously (s.c.) on the flank in five NOD/SCID mice. After 2 weeks, mice were divided into two groups. One group served as untreated controls and the others injected 10ug with P1 combined with P19. Aptamers were injected via tail vein. A total of 4 times was injected per animal. Student t-test was used for statistical analysis. * TTEST: P value ⁇ 0.05.
• FIG 1 1 illustrates results of gemcitabine-resistant tumor animal experiments.
• Gemcitabine-resistant ASPC-1 (2.8x10 6 ) cells were injected subcutaneously (s.c.) on the flank in twelve 5-weeks-old female NOD/SCID mice. After 3 weeks, mice were divided into four groups. One group served as untreated controls and the others injected with P1 , P19, and P1 combined with P19 (P1 +P19). Aptamers were injected via tail vein. A total of 4 times was injected per animal every two days and sacrificed at day 9. When compared to the control, all three treatment groups showed a significant anti-tumor effect ( * P ⁇ 0.05)
• pancreatic cancer cell aptamers systems for cell specific delivery and methods for their use are provided herein. According to the embodiments described herein, the pancreatic cancer cell aptamers may be used alone or in combination with therapeutic or diagnostic agents and molecules for treatment, diagnosis and monitoring of pancreatic cancer.
• aptamers for targeting pancreatic cancer cells are provided. Said aptamers may be used for treating pancreatic cell cancer, malignancies or for any other disease or condition related to pancreatic cells.
• An "aptamer” is any suitable small molecule, such as a nucleic acid or a peptide molecule that binds specifically to a target, such as a small molecule, protein, nucleic acid, cell, tissue or organism. Aptamers that target specific cell surface proteins can be employed as delivery molecules to target a distinct cell type, thereby reducing off-target effects or other unwanted side effects. Further, by binding a specific cell surface protein, the aptamers may also be used as a therapeutic agent on their own.
• the aptamer (or aptamer component) is a nucleic acid aptamer.
• aptamers with binding affinities in nanomolar range have been utilized for flexible applications ranging from diagnostic to therapeutic assay formats (Zhou & Rossi 2009).
• aptamers that target specific cell surface proteins may be employed as delivery molecules to target a distinct cell type, hence reducing off- target effects or other unwanted side effects (Zhou et al. 2008).
• the nucleic acid aptamer is an RNA aptamer.
• RNA aptamer may be any suitable RNA molecule that can be used on its own as a stand-alone molecule, or may be integrated as part of a larger RNA molecule having multiple functions, such as an RNA interference molecule in accordance with some embodiments.
• the pancreatic cell aptamer may be located in an exposed region of an shRNA molecule (e.g., the loop region of the shRNA molecule) to allow the shRNA or miRNA molecule to bind a surface receptor on the target cell, then after it is internalized, is processed by the target cell's RNA interference pathways.
• the nucleic acid that forms the nucleic acid aptamer may comprise naturally occurring nucleosides, modified nucleosides, naturally occurring nucleosides with hydrocarbon linkers (e.g., an alkylene) or a polyether linker (e.g., a PEG linker) inserted between one or more nucleosides, modified nucleosides with hydrocarbon or PEG linkers inserted between one or more nucleosides, or a combination of thereof.
• hydrocarbon linkers e.g., an alkylene
• a polyether linker e.g., a PEG linker
• nucleotides or modified nucleotides of the nucleic acid aptamer can be replaced with a hydrocarbon linker or a polyether linker provided that the binding affinity and selectivity of the nucleic acid aptamer is not substantially reduced by the substitution.
• aptamers that target and selectively bind pancreatic cancer cells are generated and selected.
• Selection of aptamers may be accomplished by any suitable method known in the art, including an optimized protocol for in vitro selection, known as SELEX (Systemic Evolution of Ligands by Exponential enrichment).
• SELEX Systemic Evolution of Ligands by Exponential enrichment
• SELEX process has been established as a general technique for aptamer selection, it is not predictable nor is it standardized for use with any target. Instead, the SELEX process must be optimized and customized for each particular target molecule.
• Each SELEX experiment includes its own challenges and is not guaranteed to work for all targets.
• the target molecule should be stable and easily reproduced for each round of SELEX, because the SELEX process involves multiple rounds of binding, selection, and amplification to enrich the nucleic acid molecules.
• the nucleic acids that exhibit specific binding to the target molecule have to be present in the initial library.
• the SELEX process for a single target may need to be repeated with different starting libraries. Aptamer selection using SELEX is unpredictable. Even when all of the factors are optimized for successful aptamer selection, the SELEX process does not always yield viable aptamers for every target molecule.
• selection of an aptamer may be accomplished by applying a SELEX process against whole living/intact cells in culture to obtain aptamers that selectively target an antigen that is specifically expressed on a target cell.
• a whole cell SELEX process may include an approach that includes both counterselection and selection, which is specifically designed for enrichment of aptamers against cell surface tumor-specific targets ( Figure 1 ).
• a SELEX process was used to generate a panel of RNA aptamers that are able to bind pancreatic cancer cells, but do not bind unrelated cancer cell or healthy cell types.
• the pancreatic cancer cell aptamers have a sequence that may include SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, which are described in detail in the Example below and in Figures 2A-2F.
• aptamers As described in the Examples below, at least two aptamers have been determined to be effective in reducing tumor size. These aptamers share a common nucleotide motif GAAUGCCC (SEQ ID NO: 8). As such, a pancreatic cell aptamer used in accordance with the embodiments described herein may include a nucleotide sequence of GAAUGCCC (SEQ ID NO: 8).
• the aptamers described herein target a cell surface molecule or an endocytotic membrane associated protein (e.g., a membrane receptor or a glycoprotein) that is overexpressed on pancreatic cancer cells or is specifically expressed only on pancreatic cancer cells.
• an endocytotic membrane associated protein e.g., a membrane receptor or a glycoprotein
• the aptamer selection process described above may be used to develop aptamers that bind known cell surface molecules and endocytotic membrane associated proteins, or may also be used to discover new cell surface molecules that act as pancreatic cell biomarkers and are specific to pancreatic cells.
• the pancreatic cancer cell aptamers can act as a cell-specific delivery vehicle, a therapeutic agent, or both. Further, these aptamers are likely able to inhibit or suppress proliferation of pancreatic cancer cells or otherwise interfere with a cancerous pathway by blocking a receptor or other membrane associated protein, preventing a ligand from binding. Therefore, the pancreatic cancer cell aptamers may be used for at least two functions: inhibition of proliferation and survival of pancreatic cancer cells and as a delivery vehicle for therapeutic and/or diagnostic agents. As described below, the pancreatic cancer cell aptamers can deliver therapeutic or diagnostic agents efficiently to pancreatic cancer cell lines.
• the aptamers described herein may be conjugated to a therapeutic agent, forming a therapeutic aptamer conjugate.
• conjugated to or “conjugate” refers to two or more entities or the state of two or more entities which are linked by a direct or indirect covalent or non-covalent interaction.
• an association is covalent.
• a covalent association is mediated by a linker moiety.
• an association is non-covalent (e.g.
• pancreatic cancer cell aptamers described herein may be used as a cell-specific delivery vehicle to deliver a therapeutic or diagnostic payload to pancreatic cancer cells.
• the pancreatic cancer cell aptamers described herein may be conjugated to one or more therapeutic agents to form a therapeutic aptamer conjugate.
• a "therapeutic agent” as used herein is an atom, molecule, or compound that is useful in the treatment of cancer or other conditions described herein.
• therapeutic agents that may be conjugated to a pancreatic cell aptamer include, but are not limited to, drugs, chemotherapeutic agents, therapeutic antibodies and fragments thereof, toxins, radioisotopes, enzymes (e.g., enzymes to cleave prodrugs to a cytotoxic agent at the site of the tumor), nucleases, hormones, immunomodulators, antisense oligonucleotides, nucleic acid molecules (e.g., mRNA molecules, cDNA molecules or RNAi molecules such as siRNA or shRNA), chelators, boron compounds, photoactive agents and dyes.
• drugs chemotherapeutic agents, therapeutic antibodies and fragments thereof, toxins, radioisotopes, enzymes (e.g., enzymes to cleave prodrugs to a cytotoxic agent at the site of the tumor), nucleases, hormones, immunomodulators, antisense oligonucleotides, nucleic acid molecules (e.g., mRNA
• the therapeutic agent may also include a metal, metal alloy, intermetallic or core-shell nanoparticle bound to a chelator that acts as a radiosensitizer to render the targeted cells more sensitive to radiation therapy as compared to healthy cells.
• the therapeutic agent may include paramagnetic nanoparticles for MRI contrast agents (e.g., magnetite or Fe3O 4 ) and may be used with other types of therapies (e.g., photodynamic and hyperthermal therapies) and imaging (e.g., fluorescent imaging (Au and CdSe)).
• the pancreatic cell aptamer is conjugated to a nucleic acid molecule which acts as the therapeutic agent.
• the nucleic acid molecule that is conjugated to the aptamer is an RNA molecule.
• RNA molecules that may be conjugated to the aptamer in accordance with the embodiments described herein may include, but are not limited to, ribosomal RNA (rRNA), messenger RNA (mRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), small cytoplasmic RNA (scRNA), micro RNA (miRNA), small interfering RNA (siRNA), and short hairpin RNA (shRNA).
• the nucleic acid molecule is an RNA interference molecule (e.g., an siRNA or shRNA molecule) that, when delivered to a target cell by the aptamer, is internalized by the cell and acts to suppress or silence the expression of one or more oncogenes or of any protein or peptide that is associated with cancer by targeting an mRNA molecule.
• RNA interference molecule e.g., an siRNA or shRNA molecule
• the RNA interference molecule is (i) an siRNA, shRNA, miRNA or other RNA molecule which targets an mRNA molecule which encodes K-ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) and/or SHH (Sonic Hedgehog), (ii) an mRNA molecule which encodes an anti-apoptotic protein (e.g., Bcl-xL, Bcl-2, survivin, Hax-1 , AKT2, Mcl-1 ), or (iii) any other RNA molecule that inhibits or enhances expression of a protein that is associated with cancer.
• an anti-apoptotic protein e.g., Bcl-xL, Bcl-2, survivin, Hax-1 , AKT2, Mcl-1
• any other RNA molecule that inhibits or enhances expression of a protein that is associated with cancer.
• the nucleic acid molecule is an mRNA molecule that is expressed intracellular ⁇ as part of a therapeutic or diagnostic payload.
• the mRNA component may include a cDNA molecule.
• the mRNA component may express a full wild type protein or peptide in a target cell, or may express at least the biologically active portion of the protein or peptide.
• the mRNA molecule acts as a therapeutic agent by expressing a protein or peptide that is missing or altered in the target cell, a cytotoxic protein or peptide to kill the target cell, an apoptotic triggering protein or peptide, or any other anti-cancer protein or peptide.
• Chemotherapeutic agents that may be used in accordance with the embodiments described herein are often cytotoxic or cytostatic in nature and may include, but are not limited to, alkylating agents, antimetabolites, anti-tumor antibiotics, topoisomerase inhibitors, mitotic inhibitors hormone therapy, targeted therapeutics and immunotherapeutics.
• the chemotherapeutic agents that may be used as therapeutic agents in accordance with the embodiments of the disclosure include, but are not limited to,13-cis-Retinoic Acid, 2-Chlorodeoxyadenosine, 5- Azacitidine, 5-Fluorouracil, 6-Mercaptopurine, 6-Thioguanine, actinomycin-D, adriamycin, aldesleukin, alemtuzumab, alitretinoin, all-transretinoic acid, alpha interferon, altretamine, amethopterin, amifostine, anagrelide, anastrozole, arabinosylcytosine, arsenic trioxide, amsacrine, aminocamptothecin, aminoglutethimide, asparaginase, azacytidine, bacillus calmette-guerin (BCG), bendamustine, bevacizumab, bexarotene, bicalut
• Therapeutic antibodies and functional fragments thereof, that may be used as therapeutic agents in accordance with the embodiments of the disclosure include, but are not limited to, alemtuzumab, bevacizumab, cetuximab, edrecolomab, gemtuzumab, ibritumomab tiuxetan, panitumumab, rituximab, tositumomab, and trastuzumab and other antibodies associated with specific diseases listed herein.
• Toxins that may be used as therapeutic agents in accordance with the embodiments of the disclosure include, but are not limited to, ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.
• Radioisotopes that may be used as therapeutic agents in accordance with the embodiments of the disclosure include, but are not limited to, 32 P, 89 Sr, 90 Y, 99m Tc, "Mo, 131 1, 153 Sm, 177 Lu, 186 Re, 213 Bi, 223 Ra and 225 Ac.
• the pancreatic cell aptamers described herein may be conjugated to one or more diagnostic agents (or "imaging agents"), forming a diagnostic aptamer conjugate.
• the diagnostic aptamer conjugate may to target and visualize pancreatic cells in vivo via an imaging method (e.g., positron emission tomography (PET), computer assisted tomography (CAT), single photon emission computerized tomography, x-ray, fluoroscopy, and magnetic resonance imaging (MRI)).
• PET positron emission tomography
• CAT computer assisted tomography
• single photon emission computerized tomography x-ray, fluoroscopy
• MRI magnetic resonance imaging
• a diagnostic or imaging agent may include, but is not limited to a fluorescent, luminescent, or magnetic protein, peptide or derivatives thereof (e.g., genetically engineered variants).
• Fluorescent proteins that may be used include, but are not limited to, green fluorescent protein (GFP), enhanced GFP (EGFP), red, blue, yellow, cyan, and sapphire fluorescent proteins, and reef coral fluorescent protein.
• Luminescent proteins that may be used include, but are not limited to, luciferase, aequorin and derivatives thereof. Numerous fluorescent and luminescent dyes and proteins are known in the art (see, e.g., U.S.
• Patent Application Publication 2004/0067503 Valeur, B., "Molecular Fluorescence: Principles and Applications,” John Wiley and Sons, 2002; Handbook of Fluorescent Probes and Research Products, Molecular Probes, 9.sup.th edition, 2002; and The Handbook-A Guide to Fluorescent Probes and Labeling Technologies, Invitrogen, 10th edition, available at the Invitrogen web site; both of which are hereby incorporated by reference as if fully set forth herein.
• a pancreatic cell aptamer may be further conjugated to or otherwise associated with a non-protein diagnostic agent or a delivery vehicle such as a nanoparticle, radioactive substances (e.g., radioisotopes, radionuclides, radiolabels or radiotracers), dyes, contrast agents, fluorescent compounds or molecules, bioluminescent compounds or molecules, enzymes and enhancing agents (e.g., paramagnetic ions).
• a nanoparticle for example quantum dots and metal nanoparticles (described below) may also be suitable for use as a diagnostic agent or a therapeutic agent (e.g., using hyperthermal and photodynamic therapies as well as diagnostic agents through fluorescence and or MRI contrast).
• Fluorescent and luminescent substances that may be used as an additional diagnostic agent in accordance with the embodiments of the disclosure include, but are not limited to, a variety of organic or inorganic small molecules commonly referred to as "dyes,” “labels,” or “indicators.” Examples include fluorescein, rhodamine, acridine dyes, Alexa dyes, and cyanine dyes.
• Enzymes that may be used as an additional diagnostic agent in accordance with the embodiments of the disclosure include, but are not limited to, horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, ⁇ - galactosidase, ⁇ -glucoronidase or ⁇ -lactamase. Such enzymes may be used in combination with a chromogen, a fluorogenic compound or a luminogenic compound to generate a detectable signal.
• Radioactive substances that may be used as an additional diagnostic agent in accordance with the embodiments of the disclosure include, but are not limited to, 18 F, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99m Tc, "Mo, 105 Pd, 105 Rh, 111 Ag, 11 1 ln, 123 l, 124 l, 125 l, 131 l, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154"1581 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 lr, 198 Au, 199 Au, 21 1 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 2
• Paramagnetic ions that may be used as an additional diagnostic agent in accordance with the embodiments of the disclosure include, but are not limited to, ions of transition and lanthanide metals (e.g., metals having atomic numbers of 21 -29, 42, 43, 44, or 57-71 ). These metals include ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.
• transition and lanthanide metals e.g., metals having atomic numbers of 21 -29, 42, 43, 44, or 57-71 .
• These metals include ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.
• the agent when the diagnostic agent is a radioactive metal or paramagnetic ion, the agent may be reacted with another long-tailed reagent having a long tail with one or more chelating groups attached to the long tail for binding these ions.
• the long tail may be a polymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chain having pendant groups to which may be added for binding to the metals or ions.
• chelating groups examples include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), DOTA, NOTA, NETA, porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and like groups.
• EDTA ethylenediaminetetraacetic acid
• DTPA diethylenetriaminepentaacetic acid
• DOTA DOTA
• NOTA NOTA
• NETA NETA
• porphyrins polyamines
• crown ethers bis-thiosemicarbazones
• polyoximes and like groups.
• the chelate is normally linked to the PSMA antibody or functional antibody fragment by a group which enables the formation of a bond to the molecule with minimal loss of immunoreactivity and minimal aggregation and/or internal cross-linking.
• chelates when complexed with non-radioactive metals, such as manganese, iron and gadolinium are useful for MRI, when used along with the antibodies and carriers described herein.
• Macrocyclic chelates such as NOTA, DOTA, and TETA are of use with a variety of metals and radiometals including, but not limited to, radionuclides of gallium, yttrium and copper, respectively.
• Other ring-type chelates such as macrocyclic polyethers, which are of interest for stably binding nuclides, such as 223 Ra for RAIT may be used.
• chelating moieties may be used to attach a PET diagnostic agent, such as an AI- 18 F complex, to a targeting molecule for use in PET analysis.
• the aptamers may be conjugated to both a therapeutic and a diagnostic agent. Therefore, any of the above diagnostic and therapeutic agents may be used in combination to form an aptamer conjugate that targets pancreatic cells to deliver both a diagnostic and a therapeutic payload with a single dose.
• pancreatic cancer cell aptamers Therapeutic uses of pancreatic cancer cell aptamers
• the aptamers and the aptamer-therapeutic agent conjugates described herein have at least a dual function that provides a basis for treating pancreatic cancer.
• the pancreatic cell aptamers may be used on their own to inhibit or suppress proliferation and survival of pancreatic cancer cells, and may also be used to eradicate existing primary or metastatic tumors.
• Pancreatic cancers and tumors that may be treated using the methods described herein include, but are not limited to acinar cell carcinoma, adenocarcinoma, adenosquamous carcinoma, giant cell tumor, intraductal papillary- mucinous neoplasm (IPMN), musinous cystadenocarcinoma, pancreatoblastoma, serous cystadenocarcinoma, solid and pseudopapillary tumors, gastrinoma (Zollinger- Ellison Syndrome), glucagonoma, insulinoma, nonfunctional islet cell tumors, somatostatinoma, secondary tumors derived from multiple endocrine neoplasia Type-1 , or vasoactive intestinal peptide-releasing tumor (IPMN), musinous cystadenocarcinoma, pancreatoblastoma, serous cystadenocarcinoma, solid and pseudopapillary tumors, gastrinoma (Zollinger- Ellis
• Treating" or “treatment” of a condition may refer to preventing the condition, slowing the onset or rate of development of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or ending symptoms associated with the condition, generating a complete or partial regression of the condition, or some combination thereof.
• an aptamer or an aptamer conjugate such as those described herein may be used to treat pancreatic cancer, wherein the treatment refers to suppression of pancreatic cancer cell proliferation rate, an increase in pancreatic cancer cell death, or a decreased tumor size resulting in regression or eradication of a tumor.
• the treatments described herein may be used in any suitable subject, including a human subject or any mammalian or avian subject that needs treatment in accordance with the methods described herein (e.g., dogs, cats, horses, rabbits, mice, rats, pigs, cows).
• the methods for treating the pancreatic cancer include administering a therapeutically effective amount of a therapeutic composition.
• An "effective amount,” “therapeutically effective amount” or “effective dose” is an amount of a composition (e.g., a therapeutic composition or agent) that produces a desired therapeutic effect in a subject, such as preventing or treating a target condition or alleviating symptoms associated with the condition.
• the precise therapeutically effective amount is an amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject.
• This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration.
• One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, namely by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy 21 st Edition, Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005.
• the therapeutic composition may include, among other things, an aptamer, a therapeutic agent, an aptamer-therapeutic agent or a combination thereof.
• Aptamers, therapeutic agents, and aptamer-therapeutic agents suitable for use according to the embodiments described herein may include, but are not limited to, those described above and in the Examples below.
• an RNA aptamer that may be used as part of the therapeutic composition may include a sequence of SEQ ID NO:8.
• the RNA aptamer may include a sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6 ( Figures 2A-2F).
• the therapeutic composition may also include one or more pharmaceutically acceptable carriers.
• a "pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body.
• the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or some combination thereof.
• Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It also must be suitable for contact with any tissue, organ, or portion of the body that it may encounter, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.
• the therapeutic compositions described herein may be administered by any suitable route of administration.
• a route of administration may refer to any administration pathway known in the art, including but not limited to aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, transdermal (e.g., topical cream or ointment, patch), or vaginal.
• transdermal administration may be accomplished using a topical cream or ointment or by means of a transdermal patch.
• Parenter refers to a route of administration that is generally associated with injection, including infraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal.
• the pharmaceutical composition may optionally include, in addition to the one or more aptamer or aptamer conjugates, one or more additional therapeutic agents, such as an anti-cancer agent, antibiotic, anti-viral agent, anti-HIV agent, anti-parasite agent, anti-protozoal agent, anesthetic, anticoagulant, inhibitor of an enzyme, steroidal agent, steroidal or nonsteroidal anti-inflammatory agent, antihistamine, immunosuppressant agent, antineoplastic agent, antigen, vaccine, antibody, decongestant, sedative, opioid, analgesic, anti-pyretic, birth control agent, hormone, prostaglandin, progestational agent, anti- glaucoma agent, ophthalmic agent, anti-cholinergic, analgesic, anti-depressant, antipsychotic, neurotoxin, hypnotic, tranquilizer, anti-convulsant, muscle relaxant, anti- Parkinson agent, anti-spasmodic, muscle contractant, channel
• additional therapeutic agents such as an anti-cancer
• pancreatic cell aptamers may also serve as a pancreatic cell specific targeting delivery vehicle to deliver a therapeutic or diagnostic payload to a particular cell. Therefore, according to some embodiments, methods for delivering a therapeutic payload (or a therapeutic agent) to a pancreatic cancer cell are provided. Such methods may include a step of contacting the pancreatic cancer cell with a pancreatic cancer cell aptamer conjugate, wherein the pancreatic cell aptamer conjugate comprises a pancreatic cell aptamer component and a therapeutic agent component (i.e., the therapeutic payload).
• the pancreatic cell aptamer component may be any suitable aptamer, for example, a nucleic acid aptamer.
• the nucleic acid aptamer is an RNA molecule that specifically binds a pancreatic cancer cell surface protein or other molecule, resulting in internalization of the pancreatic cell aptamer conjugate by the pancreatic cancer cell.
• the therapeutic agent component may be an siRNA molecule, an miRNA molecule, an shRNA molecule, or an mRNA molecule as described with respect to aptamer-RNA chimeras described herein.
• the pancreatic cell aptamer or aptamer conjugates may be used to deliver a diagnostic payload to pancreatic cancer cells or a pancreatic tumor cell.
• the pancreatic cell aptamer or aptamer conjugate may be used in methods of diagnosing pancreatic cancer.
• the methods for diagnosing a pancreatic cancer or pancreatic malignancy may include a step of administering to a subject suspected of having a pancreatic cancer or a pancreatic cancer malignancy, an effective amount of a pancreatic cancer cell aptamer that is conjugated to a diagnostic agent.
• the diagnostic agent may include one or more diagnostic agents, such as those described above.
• the method may further include a step of subjecting the subject to a diagnostic imaging technique (e.g., MRI, PET, CT, SPECT, PET/CT, PET/MRI, or other suitable imaging method) to visualize any diagnostic agent that is delivered to pancreatic cancer cells.
• a diagnostic imaging technique e.g., MRI, PET, CT, SPECT, PET/CT, PET/MRI, or other suitable imaging method
• Visualization of a diagnostic agent localized to an organ that is susceptible to pancreatic cancer (primary or metastatic cancer) such as the pancreas or liver, by the diagnostic imaging technique indicate that the subject has or likely has a form of pancreatic cancer such as those described above.
• aptamers that are identified using systematic evolution of ligands by exponential enrichment (SELEX) as an in vitro selection strategy can adopt complex structures to bind target proteins with high affinities and specificities (Ellington & Szostak 1990; Tuerk 1997).
• aptamers may be selected to recognize a wide variety of targets from small molecules to proteins and nucleic acids in cultured cells and whole organisms (Ulrich et al. 2002; Wang et al. 2000; Blank et al. 2001 ; Daniels et al. 2003; Hicke et al. 2001 ; Wilson & Szostak 1999).
• the Example below describes a cell-based SELEX assay for the identification of pancreatic cancer cell surface biomarkers and the therapeutic delivery of siRNAs into pancreatic cancer cells.
• a 2'-fluropyrimidine-RNA (2'F-RNA) combinatorial library was used to isolate 2'F RNA aptamers against a Panc-1 cell line, which is an aggressive pancreatic cancer cell type.
• aptamers selectively internalized into pancreatic cancer cells and the selected aptamers are candidates for targeted delivery of therapeutic siRNAs and other agents into these cells.
• Example 1 Generation of pancreatic cell aptamers for use in therapeutic
• Panc-1 CTL-1469
• Capan-1 HTB-79
• CFPAC-1 CL-1918
• MIA PaCa-2 CL-1420
• BxPC-3 CL-1687
• AsPC-1 CL-1682
• CCCAGAGGUGAUGGAUCCCCC-3' (SEQ ID NO:7) was constructed by in vitro transcription of synthetic DNA templates with NTPs (2'F UTP, 2'F CTP, GTP, ATP, Epicentre Biotechnologies, Madision, Wl) and T7 RNA polymerase.
• N 0 represents 40 nucleotide (nt) sequences formed by equimolar incorporation of A, G, C, and U at each position.
• 2'F-Py RNAs were used.
• RNA molecules that bound to target cells were recovered, amplified by RT-PCR and in vitro transcription, and used in the following selection rounds. In subsequent rounds, the RNA concentration was reduced by 10-fold and incubation time was reduced to create a more stringent condition. To remove RNAs that non-specifically bind the target cells, the counter-selection was carried out at every 3rd round using Huh7 cells.
• cDNAs were amplified.
• the amplified DNA was cloned and individual clones were identified by DNA sequencing. Structures of aptamers were predicted using MFOLD (Zuker 2003), available at http://www.bioinfo.rpi.edu/applications/mfold/using a salt correction algorithm and temperature correction for 25 C.
• RNA aptamers In vitro selection of RNA aptamers to the intact target cells.
• the human pancreatic carcinoma cells (Panc-1 ) were used as target cells for the aptamer selection and the human hepatoma cell line (Huh7) was used for the counter-selection steps to remove non-pancreatic cancer cell specific aptamers.
• a library of 2' Fluroro pyrimidines RNAs (2'F RNA) were used to increase nuclease-resistance and enhance aptamer folding.
• a library of approximately 4 40 different 2'F RNA molecules, containing a 40-nt-long random sequence flanked by defined sequences was screened by SELEX. After 14 cycles of selection, the highly enriched aptamer pools were cloned. The nucleotide sequences of 47 clones were determined.
• sequences P19, P1 and P7 contained a common motif, GAAUGCCC (SEQ ID NO: 8).
• Sequence P15 was found nine times and the length of the random region was 40 nucleotides (nt).
• Sequences P19 and P1 were found six times and the length of the random regions was 40 nt.
• Sequence P1 1 was found four times and P7 was found three times. Both also have 40 nt in the randomized region.
• Sequence P6 was found two times and the length of the randomized region length is 24 nt.
• Example 2 Cell-specific aptamer delivery to pancreatic cancer for use in
• RNA aptamers in the target cells and other types of pancreatic cancer cells
• the cells were grown in 35mm glass bottom dishes (MatTek, Ashland, MA, USA) with seeding at 1 ⁇ 10 6 cells in medium for 24hrs.
• the RNAs were labeled with Cy3 using the Cy3 Silencer siRNA labeling kit (Ambion, TX, USA) following the manufacturer's instructions. Cy3-labeled RNAs at 100nM were added to the cells and incubated for 1 hour. Following the incubation, the cells were stained with 5ug/ml Hoechst 33342 (Molecular Probes, CA, USA) for live cell nuclear staining.
• the images were taken using a Zeiss LSM 510 Meta Inverted 2 photon confocal microscope system under water immersion at 40 magnification.
• Binding assay by flow cytometric analysis was also assessed by flow cytometry.
• cells were detached using a non- enzymatic cell dissociation solution, washed with PBS and suspended in binding buffer. Next, Cy3-labeled aptamers were added and incubated for 1 hours at 37 ° C. The binding of individual aptamers or the starting pool as a control to pancreatic cancer cells was performed in triplicate.
• Flow cytometry was performed on a Guava (Millipore, Billerica, MA, USA) flow cytometer and the data were analyzed with FlowJo software.
• Binding affinity and K D Determination To determine the binding affinity of aptamers to Panc-1 , Kd function of physiology macro provided by a Zeiss LSM 510 Meta Inverted 2 photon confocal microscope system was used. The cells were grown in 35mm glass bottom dishes (MatTek, Ashland, MA, USA) with seeding at 1 ⁇ 10 6 cells in medium for 24hrs. The RNAs were labeled with Cy3 using the Cy3 Silencer siRNA labeling kit (Ambion, TX, USA) following the manufacturer's instructions. Various concentrations of Cy3-labeled RNAs were added to the cells and incubated for 1 hour. After extensive washing, 20 images of each condition of a titration curve were taken. The dissociation constants were calculated using one site binding non-linear curve regression with a Graph Pad Prism.
• Panc-1 cells were prepared as detailed above for the confocal microscopy. 200 nM of Cy3 labeled P19 aptamer was used to compete with either unlabeled clones (1 ⁇ ) in 1 ⁇ Binding buffer prewarmed at 37 °C. Cells were washed three times and took images by confocal microscopy.
• WST-1 assay Cell proliferation was quantified following four treatments of P1 and P19 at 9ug per treatment in Panc-1 (2.5x10 5 cells), using WST-1 reagent following the manufacturer's guidelines (Roche, UK). Briefly, the WST-1 reagent was used at a 1 :100 dilution to plates and incubated for one hour. The enzymatic reaction was measured at 450 ⁇ using Bio-Tek ELISA reader.
• mice Five NOD/SCID mice were injected subcutaneously (s.c.) on the flank with Panc-1 pancreatic cancer cells in 0.05 ml PBS with 0.15 ml Matrigel. After 2 weeks, mice were divided into two groups. One group served as untreated controls and the others injected 10ug with P1 combined with P19. Aptamers were injected via tail vein (i.v.), for a total of 4 times per animal. Animals were sacrificed before tumour disappeared.
• mice For the gemcitabine resistant tumour test, twelve 5-week-old female NOD/SCID mice were injected subcutaneously (S.C.) on the flank with 2.8x10 6 ASPC-1 pancreatic cancer cells in 0.05 ml PBS with 0.15 ml Matrigel. After 3 weeks, mice were divided into four groups. One group served as untreated controls and the others injected with P1 (10ug per injection), P19 (10ug per injection) and P1 combined with P19 (5ug of P1 with 5ug of P19 per injection). Aptamers were injected through tail vein (i.v.) for a total of 4 times per animal (at day 1 , 3, 5, and 7). Animals were sacrificed at day 9.
• RNA aptamers specifically bind to and are internalized in pancreatic cancer cells. Flow cytometric analyses of the individual clones revealed the aptamers bound to the target cells ( Figure 3). In order to determine that the selected six different aptamers were internalized in the pancreatic cancer cells, the live-cell confocal microscopy with the Cy3-labeled RNA transcripts was carried out. The RNA aptamers were internalized specifically in target cells Panc-1 ( Figure 4B), but not the Huh7 control cells ( Figure 4A). Non-specific weak binding was observed when initial the RNA library pool was incubated withPanc-1 .
• Figure 4B shows that the aptamers aggregate within the cytoplasm, suggesting that the RNA aptamers enter into cells via receptor-mediated endocytosis.
• the aptamers recognize different type of pancreatic cancer cells, five different pancreatic cancer cell lines were tested for aptamer uptake. All six of the tested aptamers internalized in all the pancreatic cancer cells ( Figures 5A, 5B, 5C, 5D, 5E).
• RNA aptamers that target pancreatic cancer cells were developed and the selected RNA aptamers were demonstrated to internalize within the cells, indicating that the RNA aptamers described herein may be used as targeting agents to deliver therapeutic agents to pancreatic cancer cells, such as siRNAs or chemotherapy agents.
• the aptamers are not internalized by normal pancreatic cells.
• primary epithelial pancreatic cells were incubated with Cy3 labeled aptamers as described above.
• Cy3 labeled aptamers As shown in Figures 6A and 6B, none of the Cy3 labeled aptamers were internalized by the normal pancreatic cells, indicating that the aptamers bind specifically to a cell surface molecule (e.g., a cell surface protein) that is expressed on pancreatic cancer cells, but is not expressed on normal pancreatic cells.
• a cell surface molecule e.g., a cell surface protein
• RNA aptamer binding affinity of target cells To estimate the affinity of the RNA aptamers, Physiology function of confocal microscopy was utilized. The measured dissociation constants (K D ) of P19, P15, and P1 were 64.76nM, 70.72nM, and 1 12.2nM, respectively ( Figure 7). To determine whether each aptamer binds to the pancreatic cancer cells via the same or different cell surface proteins, Panc-1 cells were incubated with fluorescently labeled P19 RNA (100 nM) and increasing amounts (1 ⁇ ) of each unlabeled aptamer as a competitor against the labeled chimeras (Figure 8).
• the fluorescence intensity of labeled RNAs was measured in the presence of increasing amounts of competitors using confocal microscopy.
• the intensity of P19 competed with unlabeled P19 was significantly decreased (indicating competition for the same target); while others showed insignificant changes, indicating that each RNA aptamer has a different binding site on the same target or binds different targets.
• RNA aptamers The anti-tumor effect of selected RNA aptamers.
• three aptamer clones P19, P15 and P1 ) were injected into SCID mice intravenously (i.v.).
• P19 and P1 inhibited cell proliferation in vitro ( Figure 9).
• P19 and P1 also significantly reduced the tumor size in Panc-1 engrafted mice when administered via i.v. injection ( Figure 10), even in gemcitabine resistant pancreatic cancer ( Figure 1 1 ). Based on these results, the selected P19 and P1 aptamers may be used as an effective for pancreatic cancer on their own due to their anti-cancer, tumor regressing effects.
• the P19 and P1 aptamers effectively target and are internalized by pancreatic cancer cells, they also have a dual function in that they can deliver a payload (e.g., a therapeutic agent) to the pancreatic cancer cells, resulting in an additional anti-cancer effect.
• a payload e.g., a therapeutic agent
• the P19 and P1 aptamers may be used as delivery agents for delivery of one or more therapeutic agents that target K-ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) and SHH (Sonic Hedgehog) according to some embodiments.
• K-ras V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog
• SHH Sonic Hedgehog
• pancreatic cancer cells may spread to the liver even at the pre-neoplastic stage (Rhim et al. 2012).
• intravenous administration of the aptamers for use as a systemic therapy is particularly important in that the vast majority of patients with pancreatic cancer, since most likely, the patients have distant tumour spread at the time of diagnosis.
• the specific RNA aptamers against pancreatic cancer may be used as part of a drug or a pharmaceutical composition for systemic therapy, and may also be used for diagnosis and staging of pancreatic cancer.
• RNA aptamers that target pancreatic cancer cells.
• RNA aptamers themselves were shown to have anti-tumor effect on their own, and also specifically target pancreatic cancer cells - not normal pancreatic cells.
• the RNA aptamers described herein may be used as targeting agents to deliver therapeutic agents (e.g., siRNA or chemotherapeutics) or diagnostic agents to pancreatic cancer cells.
• Neoptolemos J. P., et al. A randomized trial of chemoradiotherapy and chemotherapy after resection of pancreatic cancer. The New England journal of medicine 350, 1200-1210 (2004).
• Pancreatic cancer molecular pathogenesis and new therapeutic targets. Nat Rev Gastroenterol Hepatol, 6, 412-422.

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