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Definitions

• the present invention relates to an oligonucleotide composition for the treatment of cancer.
• BACKGROUND OF THE INVENTION Cancer is an aberrant net accumulation of atypical cells, which can result from an excess of proliferation, an insufficiency of cell death, or a combination of the two.
• Proliferation is the culmination of a cell's progression through the cell cycle resulting in the division of one cell into two cells.
• the 5 major phases of the cell cycle are Go, G ls S, G 2 and M.
• Go phase cells are quiescent. Most cells in the body, at any one time, are in this stage.
• Gi phase cells, responding to signals to divide, produce the RNA and the proteins necessary for DNA synthesis.
• SE early S-phase
• SM middle S-phase
• SL late S-phase
• the cells replicate their DNA.
• G 2 phase proteins are elaborated in preparation for cell division.
• mitotic (M) phase the cell divides into two daughter cells.
• Synthetic oligonucleotides are polyanionic sequences that are internalized in cells (Vlassov et al. Biochim. Biophys. Acta 1197:95, 1994). Synthetic oligonucleotides are reported that bind selectively to nucleic acids (Wagner, R. Nature: 372:333, 1994), to specific cellular proteins (Bates et al. J. Biol. Chem. 274:26369, 1999) and to specific nuclear proteins Scaggiante et al. Eur. J. Biochem. 252:207, 1998) to inhibit proliferation of cancer cells.
• Synthetic 27 base sequences containing guanine (G) and variable amounts of thymine (T) (oligonucleotide GTn), wherein n is > 1 or ⁇ 7 and wherein the number of bases is >20 are reported to inhibit growth of cancer cell lines by sequence specific binding to a 45 kDa nuclear protein, whereas GTn, wherein the number of bases is ⁇ 20, are reported to be inactive against cancer cell lines (Morassutti et al. Nucleosides and Nucleotides 18:1711, 1999).
• Apoptosis is an active cellular death process characterized by distinctive morphological changes that include condensation of nuclear chromatin, cell shrinkage, nuclear disintegration, plasma membrane blebbing, and the formation of membrane-bound apoptotic bodies (Wyllie et al. Int. Rev. Cytol. 68:251,
• a molecular hallmark of apoptosis is degradation of the cell's nuclear
• Caspases (cysteine-aspartyl-specific proteases) have been implicated as key enzymes in the execution of the late stage of apoptosis.
• the caspase family consists of at least fourteen related cysteine aspartyl proteases. All the caspases contain a conserved QACXG (where X is R, Q or G) pentapeptide active-site motif (Cohen G. Biochim. Biophys. Acta 1366:139, 1997).
• a number of caspases are synthesized as inactive proenzymes that are activated following cleavage at caspase specific cleavage sites (Cohen G. Biochim. Biophys. Acta 1366:139, 1997) or as inactive enzymes that require association with regulatory molecules for activation (Stennicke et al. J. Biol. Chem. 274:8359, 1999).
• caspases are involved in activation and proliferation of B and T lymphocytes, in cytokine maturation during inflammation, in differentiation of progenitor cells during erythropoiesis and in development of lens fiber (Fadeel et al. Leukemia 14:1514, 2000).
• caspase 3 is processed during activation of B lymphocytes and of CD4 (+), CD8 (+), CD45RA(+) and CD45RO (+) subsets of T lymphocytes (Alam et al. J. Exp. Med. 190:1879, 1999).
• T lymphocytes stimulation of T lymphocytes by mitogens and by interleukin-2 is associated with activation of the caspase pathway and with cleavage of PARP (Wilheim et al. Eur. J. Immunol. 28:891, 1998).
• caspase 3 activity is necessary for the release of IL-2 by activated T lymphocytes (Posmantur et al. Exp. Cell Res. 244:302, 1998) and for the processing and maturation of the pro-inflammatory cytokine interleukin-16 (Zhang et al. J. Biol. Chem. 273:1144, 1998).
• Cytolysis is the complete or partial destruction of a cell and is mediated by the immune system.
• Activated macrophages and monocytes produce bioactive molecules that include, but are not limited to cytokines.
• Cytokines include, but are not limited to, interleukin (IL)-l, IL-1 beta, IL-6,
• IL-10 IL-10
• IL-12 IL-12
• TNF-alpha TNF-alpha
• IL-6 induces B cell differentiation, stimulates IgG secretion (Taga et al. J. Exp. Med. 166:967, 1987), induces cytotoxic T cell differentiation (Lee et al. Vaccine 17:490, 1999), promotes megakaryocyte maturation (Ishibashi et al. Proc. Natl. Acad. Sci. USA 86:8953, 1989) and functions both as an anti-proliferative factor (Mori et al. Biochem. Biophys. Res. Comm. 257:609,
• IL-12 alone and in combination with other cytokines, promotes the maturation of leukocytes and induces the secretion of interferon-gamma.
• TNF-alpha causes necrosis of solid tumors (Porter et al. Trends in
• compositions and methods that induce cell cycle arrest in cancer cells, inhibit proliferation of cancer cells, activate caspases in cancer cells, induce apoptosis in cancer cells and stimulate cytokine production by immune system cells.
• the present invention fulfills this need by providing a composition and method comprising a 2 to 20 base 3'-OH, 5'-OH synthetic oligonucleotide (hereinafter "sequence") selected from the group consisting of (G x T y ) n; (T y G x ) n, a(G x T y ) n, a(T y G x ) n, (G x T y ) complicatb, (T y G x ) n b, a(G x T y ) complicatb, a(T y G x ) n b, wherein x and y is an integer between 1 and 7, n is an integer between 1 and 12, a and b are one or more As, Cs, Gs or Ts, and wherein the sequence induces a response selected from the group consisting of induction of cell cycle arrest, inhibition of proliferation, activation of caspases and induction
• sequence induces a response
• a composition comprising a sequence and a pharmaceutically acceptable carrier is administered to an animal, including a human, having cancer in an amount effective to treat the cancer in the animal.
• the unexpected and surprising ability of the sequence to induce cell cycle arrest, inhibit proliferation, activate caspases and induce apoptosis and in cancer cells and to stimulate cyotkine production by immune system cells addresses a long felt unfulfilled need in the medical arts and provides an important benefit for animals, including humans.
• Another object of the present invention is to provide a composition and method that induces senescence in cells.
• Another object of the present invention is to provide a composition and method that induces apoptosis in cells.
• Another object of the present invention is to provide a composition and method that induces apoptosis in cancer cells independent of p21/waf-l/CIP.
• Another object of the present invention is to provide a composition and method that induces apoptosis in cancer cells independent of pi 5 ⁇ nk4B .
• Another object of the present invention is to provide a composition and method that induces apoptosis in cancer cells independent of pl6 ⁇ nk4 .
• Another object of the present invention is to provide a composition and method that induces apoptosis in cancer cells independent of hormone dependence.
• Another object of the present invention is to provide a composition and method that induces apoptosis in cancer cells independent of drug resistance.
• Another object of the present invention is to provide a composition and method to treat an autoimmune disease. Another object of the present invention is to provide a composition and method to treat a lymphoproliferative disease.
• Another object of the present invention is to provide a composition and method to modulate T- or B-cell activation.
• Another object of the present invention is to provide a composition and method to modulate progenitor cell maturation.
• Another object of the present invention is to provide a composition and method to modulate erythropoiesis.
• Another object of the present invention is to provide a composition and method to modulate transcription factors in cells. Another object of the present invention is to provide a composition and method that potentiates the effect of other therapeutic agents on cells.
• Another object of the present invention is to provide a composition and method that potentiates the effect of chemotherapeutic agents on cancer cells.
• Another object of the present invention is to provide a composition and method that potentiates the anti-neoplastic effect of radiation.
• Administration of an effective amount of a sequence of the present invention to an animal, including a human, is a therapeutic treatment that prevents, treats or eliminates a disease including, but not limited to, cancer, rheumatoid arthritis, lympho-proliferative disorders and asthma.
• Cancers include, but are not limited to, squamous cell carcinoma, fibrosarcoma, sarcoid carcinoma, melanoma, mammary cancer, lung cancer, colorectal cancer, renal cancer, osteosarcoma, cutaneous melanoma, basal cell carcinoma, pancreatic cancer, bladder cancer, brain cancer, ovarian cancer, prostate cancer, leukemia, lymphoma and metastases derived therefrom.
• sequences of the present invention form a new family of structures and that they do not function as antisense RNAs, antisense DNAs, triple helix forming DNAs, telomerase inhibitors, transcription activators or transcription inhibitors.
• control 0 ⁇ g/ml (control) and 100 ⁇ g/ml (treated) of 2 to 45 base sequences containing A, C, G and T for 48 h.
• Jurkat T cells were incubated with 3, 6, 9, 12, 14, 15, 18, 21 and 24 base GT sequences (Table 3).
• Jurkat T cells were incubated with 6 base GT, AG, CG, GG, AGT and CGT sequences (Table 4).
• GT SEQ ID NO:25 inhibited proliferation 60%
• AGT SEQ ID NO:49 inhibited proliferation 45%.
• NO:49-phosphorothioate is reported to inhibit telomerase activity and to induce apoptosis in Burkitt lymphoma cells (Mata et al. Toxicol. Appl.
• telomerase activity To determine telomerase activity, extracts from 2 x 10 5 Jurkat T cells were assayed using the PCR-telomeric repeat amplification protocol (TRAP) (Roche, Laval, Quebec, Canada). At concentrations between 1 and 100 ⁇ g/ml, GT SEQ ID NO:25-phosphodiester showed between 0 and 10% anti- telomerase activity, whereas AGT SEQ ID NO:49-phosphorothioate showed between 30 and 75% anti-telomerase activity. Neither GT SEQ ID NO:25- phosphorothioate nor GT SEQ ID NO:49-phosphodiester showed any anti- telomerase actvity.
• TRAP PCR-telomeric repeat amplification protocol
• HL-60 promyelocytic leukemia cells are a p53 mutated human suspension tumor model. HL-60 cells were incubated with 6 base GT sequences (Table 6).
• MCF-7 human breast cancer cells are a caspase 3 negative, estrogen-dependent human solid tumor model.
• MCF-7 cells (5 x 10 5 cells/ml) were incubated with 3 and 6 base GT sequences (Table 7).
• PC-3 prostate cancer cells are a p53 mutated, androgen-independent human solid tumor model.
• PC-3 cells (5 x 10 5 cells/ml) were incubated with 3 and 6 base GT sequences (Table 8).
• LNCaP prostate cancer cells are a TGF-beta 1 receptor negative, androgen-independent, metastatic human solid tumor model. LNCaP cells (5 x 10 5 cells/ml) were incubated with 6 base GT sequences (Table 9).
• SK-OV-3 ovarian cancer cells are a p53 mutated, p21/waf-l/Cip deleted, pl5 mk4B deleted, pl6 ⁇ nk4 deleted, metastatic human solid tumor model.
• SK-OV-3 cells (5 x 10 5 cells/ml) were incubated with 2, 6 and 18 base GT sequences (Table 12).
• Jurkat human leukemia T cells (Table 13), LNCaP human prostate cancer cells (5 x 10 5 cells/ml) (Table 14) and MCF-7 human breast cancer cells (5 x 10 5 cells/ml) (Table 15) were incubated with 6 base GT sequences, having either oxygen (phosphodiester) or sulfur (phosphorothioate) as a nonbridging atom on the phosphate group.
• Jurkat human leukemia T cells (Table 16) and MCF-7 human breast cancer cells (5 x 10 5 cells/ml) (Table 17) were incubated with the 6 base GT SEQ ID NO:25, wherein either oxygen (phosphodiester) or sulfur (phosphorothioate) was the nonbridging atom on the phosphate group.
• EL-4 murine lymphoma T cells are a suspension tumor model. EL-4 murine lymphoma T cells were incubated with 6, 18, 27 and 33 base GT sequences and with a 15 base ACG sequence (Table 18).
• A20 murine leukemia B cells are a suspension tumor model. A20 murine leukemia B cells were incubated with 6 base GT sequences (Table 19).
• D-17 canine osteosarcoma cells and CF-51 canine mammary gland cancer cells are solid tumor models.
• D-17 canine osteosarcoma cells (5 x 10 5 cells/ml) (Table 20) and CF-51 canine mammary gland cancer cells (5 x 10 5 cells/ml) (Table 21) were incubated with 6, 9, 17, 27 and 33 base GT- sequences and with a 15 base ACG sequence.
• GT SEQ ID NO:25 and 27 base GT SEQ ID NO:l potentiated the antineoplastic effect of 0.1 and 1.0 ⁇ g/ml of 5- fluorouracil on Jurkat T cell proliferation and GT SEQ ID NO:25 potentiated the antineoplastic effect of 0.1 and 1.0 ⁇ g/ml cisplatin on Jurkat T cell proliferation.
• MCF-7 human breast cancer cells (5 X 10 5 cells/ml) were incubated with 1.0 ⁇ g/ml of 6 base GT SEQ ID NO:25 and of 27 base GT SEQ ID NO:l in the presence of 0, 0.1, 1.0 or 10.0 ⁇ g/ml of 5-fluorouracil or tamoxifen
• Tamoxifen is an estrogen antagonist.
• MCF-7 cell proliferation Twenty-seven base SEQ ID NO:l did not potentiate the antineoplastic activity of 5-fluorouracil or of tamoxifen on MCF-7 cell proliferation.
• BCG derived sequences are reported to inhibit tumor growth in vivo
• BCG derived sequences inhibited Jurkat T cell proliferation ⁇ 24%.
• phosphatidylserine in the plasma membrane during apoptosis was measured by flow cytometry using FITC-conjugated annexin V (BD Pharmingen, San Diego, CA). NuMA release into the supernatant was determined using a commercial ELISA kit
• Jurkat human leukemia T cells were incubated with 50 ⁇ M of 3, 4, 5, 6 and 7 GT base sequences, a 5 base ACGT sequence, 6 base AG, GG, AGT and CGT sequences and a 7 base GG sequence.
• Table 31 shows % of cells in apoptosis (positive for phosphatidyl-serine/annexin V staining (PS/A-V)) and
• Example 24 Increase in intracellular calcium (Ca + ).
• Apoptosis can be initiated by ligands that bind to cell surface receptors including, but not limited to, Fas (CD95) and tumor necrosis factor (TNF).
• ligands that bind to cell surface receptors including, but not limited to, Fas (CD95) and tumor necrosis factor (TNF).
• Caspases initiate the lethal proteolytic cascade of apoptosis execution associated with nuclear DNA-fragmentation, release of nuclear matrix proteins (NuMA) and loss of cell substrate contact.
• Jurkat human leukemia T cells (1 x 10 6 /ml) were incubated with 6 and 27 GT base sequences (Table 32). NuMA was determined as in Example 23.
• % NuMA release with 6 base GT SEQ ID NO:25 was greater than % NuMA release with 27 base GT SEQ ID NOs: 1, 2, 3 and 4.
• Jurkat human cell leukemia T cells were incubated for 24 h with 6 base
• SEQ ID NOs:81, 82 and 83 rich in GT, inhibited proliferation of, induced cell cycle arrest in and induced apoptosis of Jurkat T cells, whereas SEQ ID NO: 15, rich in AC, did not inhibit proliferation of, induce cell cycle arrest in or induce apoptosis of Jurkat T cells.
• Caspases recognize 3 major peptide substrate sequences: 1) Tyr-Val- Ala-Asp (YVAD, caspase-1, -4 and -5) (SEQ ID NO:84); 2) Asp-Glu-Val- Asp (DEVD, caspase-2, -3 and -7) (SEQ ID NO:85); and, 3) Ile-(Leu)-Glu-X- Asp (I(L)EXD; caspase-8 and -10) (SEQ ID NO: 86) (Thornberry et al. J. Biol. Chem. 272:17907, 1997).
• Sequence recognition in a protein target results in a limited and specific proteolysis of the target as, in a first example, the modulation of caspase 7 activation by caspase 3 or, as in a second example, the degradation of structural protein targets including, but not limited, to lamins or, as in a third example, the activation of enzymes including, but not limited to, PARP.
• caspase activation is determined colorimetrically using an assay based on the cleavage of a caspase-specific peptide conjugated to the color reporter molecule p- nitroanilide, which can be quantitated spectrophotometrically at a wavelength of 405 nm.
• SEQ ID NOs:81, 82 and 83 each induced processing of inactive pro-caspase 3 to active caspase 3, whereas 15 base ACG SEQ ID NO: 67 did not induce processing of inactive pro-caspase 3 to active caspase 3.
• Example 28 As As shown in Figure 2B, caspase 3 activity in 33 base GT
• SEQ ID NO:66 and 11 base GT SEQ ID NOs:81, 82 and 83 treated cells was 105%, 77%, 100% and 60% greater than that in control cells, whereas in 15 base ACG SEQ ID NO:67 treated cells caspase 3 activation was approximately the same as in control cells.
• Example 28 As shown in Figure 3 A, caspase 3 activation in 6 base GT SEQ
• ID NO:25 treated cells was 323% greater than in control cells.
• HEPES pH 7.5 containing 5 mM MgCl 2 , 1 mM dithiothreitol, 1.5 nM aprotinin, 10 mM leupeptin and 2.5 ⁇ m Na orthovanadate, and the protein content of the lysate was determined (Bradford J. Anal. Biochem. 72:248, 1976).
• Activated caspase 7 and PARP cleavage were detected by Western blot analysis.
• Lysate was mixed with Laemmli buffer (Laemmli U. Nature 15:680, 1970), shaken, and heated at 100°C for 4 min.
• Fifty ⁇ g of protein was added to each lane and the proteins were separated by electrophoresis in a 10% (caspase) or a 17% (PARP) sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) at a constant voltage of 100 V for about 1.5 h.
• the separated proteins were electroblotted onto a PVDF membrane. Equal protein loading was monitored by Ponceau red staining of the membrane. The membrane was blocked overnight at 4° C with a buffer containing
• IgG bound to caspase 7 or to PARP was detected with sheep anti-mouse IgG conjugated to horseradish peroxidase (diluted 1:1000 in TBST+5% non-fat dry milk) (Pharmingen). Blots were developed using an enhanced chemiluminescence detection system (ECL, Amersham, Corp., Amersham, UK).
• Example 32 Positive feedback amplification of caspase activation Jurkat human leukemia T cells are incubated for 72 h with NH 2 -
• NH 2 -YVAD-CO-GT, NH 2 -DEVD-CO-GT and NH 2 -IEGD-COO-GT each induce processing of inactive caspase 3 to active caspase 3 and of inactive caspase 7 to active caspase 7, whereas NH 2 -YVAD-CO-ACG, NH 2 -
• DVED-CO-ACG and NH 2 -IEGD-CO-ACG do not induce processing of inactive pro-caspase 3 to active caspase 3 or of inactive caspase 7 to active caspase 7.
• caspase activity within caspase containing cells mediates the release of caspase activating GT sequences from NH 2 -XXXD-COO-GT constructs by proteolysis/hydrolysis. This results in positive amplification of caspase activity (increased levels of caspase 3 and caspase 7) within the cells by the released caspase-activating GT sequences.
• Example 33 Induction of cytokine production Unless stated otherwise, 1 x 10 6 cells were incubated with 100 ⁇ g/ml of each of the sequences tested for 48 h at 37°C in 5% CO 2 . Production of cytokines IL-6, IL-10, IL-12, IL-1 beta and TNF-alpha was determined in pg/ml in 100 ⁇ l of culture supernatant using the appropriate commercial ELISA (BioSource, Camarillo CA). The IL-12 ELISA measures both IL-12 p70 complex and free p40 subunit. Results are expressed as the "fold" (x) increase in cytokine production by treated cells compared to control cells.
• THP-1 human acute monocytic leukemia cells were incubated with 2, 3, 6, 9, 12, 14, 15 and 18 base GT sequences and production of the cyotkine IL-6 was determined (Table 35). Table 35 Cytokine production by THP-1 human acute monocytic leukemia cells
• THP-1 human acute monocytic leukemia cells were incubated with 6 base GT, AG, CG, GG, AGT and CGT sequences and production of the cytokines IL-12 and IL-6 was determined (Table 36).
• BCG derived sequences A-3 (SEQ ID NO:73), A-4 (SEQ ID NO:74), A-6 (SEQ ID NO:75), A-7 (SEQ ID NO:76), M3 (SEQ ID NO:77) and Alpha 1 (SEQ ID NO:78) are reported to activate NK cells in vivo (Kataoka et al.
• THP-1 human acute monocytic leukemia cells were incubated with 6 base GT sequences, having either an oxygen (phosphodiester) or a sulfur (phosphorothioate) as the nonbridging atom on the phosphate groups and production of the cytokine IL-12 was determined (Table 39).
• THP-1 human acute monocytic leukemia cells were incubated with 6 base GT SEQ ID NO:25, having either an oxygen (phosphodiester) or a sulfur (phosphorothioate) as the nonbridging atom on the phosphate groups and production of the cytokine IL-12 was determined (Table 40).
• PBMCs were isolated from 4 healthy rhesus monkeys as in Example 35. PBMCs were incubated with 6 base GT, AGT, CGT, AG, CG ands GG sequences and production of the cytokines IL-6, IL-12 and TNF-alpha was determined (Table 42).
• LNCaP human prostate cancer cells are implanted subcutaneously, as xenografts, in male nude nu nu mice.
• the mice are divided into 5 groups of 10 mice.
• group 1 mice receive saline
• group 2 mice receive 3 base SEQ ID NO:8
• group 3 mice receive 6 base GT SEQ ID NO:25
• group 4 mice receive 6 base AG
• SEQ ID NO:45 and group 5 mice receive 45 base BCG A-3 SEQ ID NO:69.
• the mice are sacrificed and tumor mass is determined.
• Group 1 mice have the most tumor mass
• group 5 mice have less tumor mass than group 1 mice and groups 2, 3 and 4 mice have less tumor mass than groups 1 and 5 mice.
• Example 41 Effect of 3, 6, 8 and 27 base sequences on EL-4 murine T lymphomas EL-4 murine T lymphoma cells are implanted into C57/BL6 mice.
• mice are divided into 6 groups of 10 mice. On day 0, group 1 mice receive saline, group 2 mice receive 3 base GT SEQ ID NO: 8, group 3 mice receive 6 base SEQ ID NO:25, group 4 mice receive 6 base AG SEQ ID NO:45, group 5 mice receive 18 base GT SEQ ID NO: 18 and group 6 mice receive 27 base GT SEQ ID NO:l. After 4 weeks of treatment, the mice are sacrificed and tumor mass is determined. Group 1 mice have the most tumor mass, groups 2, 3, 4, 5 and 6 mice have less tumor mass than group 1 mice.
• Human colon cancer cell lines are maintained as adherent cell cultures.
• Cells in the exponential growth phase are treated with 2-20 base GT, GA, GC or GG sequences in the dose range 0 ⁇ g/ml to 100 ⁇ l/ml for 24-72 hours.
• GA, GC or GG sequences inhibit proliferation, induce cell cycle arrest and induce apoptosis in the colon cancer cell lines.

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