Methods for enhancing antibody-induced cell lysis and treating cancer

专利摘要:

公开号:AU2009212978A1
申请号:U2009212978
申请日:2009-09-01
公开日:2009-10-01
发明作者:Gunther Hartmann；George Weiner
申请人:Coley Pharmaceutical GmbH；University of Iowa Research Foundation UIRF；
IPC主号:A61K39-395

专利说明:
Australian Patents Act 1990 - Regulation 3.2 ORIGINAL COMPLETE SPECIFICATION STANDARD PATENT Invention Title Methods for enhancing antibody-induced cell lysis and treating cancer" The following statement is a full description of this invention, including the best method of performing it known to me/us: P/00/0 Il WO 01/97843 PCT/USOI/20154 -1 METHODS FOR ENHANCING ANTIBODY-INDUCED CELL LYSIS AND TREATING CANCER PRIORITY 5 This application claims the benefit of U.S. Provisional Application No. 60/213,346, filed June 22, 2000. FIELD OF THE INVENTION The invention relates to the treatment and prevention of cancer using 10 immunostimulatory nucleic acids and antibodies. BACKGROUND OF THE INVENTION Cancer is the second leading cause of death, resulting in one out of every four deaths in the United States. In 1997, the estimated total number of new diagnoses for lung, breast, 15 prostate, colorectal and ovarian cancer was approximately two million. Due to the ever increasing aging population in the United States, it is reasonable to expect that rates of cancer incidence will continue to grow. Cancer is a disease which involves the uncontrolled growth (i.e., division) of cells. Some of the known mechanisms which contribute to the uncontrolled proliferation of cancer cells 20 include growth factor independence, failure to detect genomic mutation, and inappropriate cell signaling. The ability of cancer cells to ignore normal growth controls may result in an increased rate of proliferation. Although the causes of cancer have not been firmly established, there are some factors known to contribute, or at least predispose a subject, to cancer. Such factors include particular genetic mutations (e.g., BRCA gene mutation for 25 breast cancer, APC for colon cancer), exposure to suspected cancer-causing agents, or carcinogens (e.g., asbestos, UV radiation) and familial disposition for particular cancers such as breast cancer. Cancer is currently treated using a variety of modalities including surgery, radiation therapy and chemotherapy. The choice of treatment modality will depend upon the type, 30 location and dissemination of the cancer. For example, surgery and radiation therapy may be more appropriate in the case of solid well-defined tumor masses and less practical in the case of non-solid tumor cancers such as leukemia and lymphoma. One of the advantages of WO 01/97843 PCT/USO1/20154 -2 surgery and radiation therapy is the ability to control to some extent the impact of the therapy, and thus to limit the toxicity to normal tissues in the body. However, surgery and radiation therapy are often followed by chemotherapy to guard against any remaining or radio-resistant cancer cells. Chemotherapy is also the most appropriate treatment for 5 disseminated cancers such as leukemia and lymphoma as well as metastases. More recently, the use of CpG containing nucleic acids has been proposed for the treatment and prevention of cancer. We have found that unmethylated CG-dinucleotides within certain sequence contexts (CpG DNA) are recognized by the vertebrate immune system as foreign DNA (bacterial or viral). CpG DNA activates a coordinated set of immune 10 responses that include innate immunity (macrophages, dendritic cells, and natural killer cells), humoral immunity, and cellular immunity. Krieg AM et al., Pharmacol Ther 84:113 20 (1999); Krieg AM et al., Curr Top Microbiol Immunol 247:1-21 (2000); Wagner H, Adv Immunol 73:329-68 (1999). As a vaccine adjuvant, CpG DNA is at least as effective as the gold standard complete Freund's adjuvant (CFA), but induces higher Th1 activity and 15 demonstrates less toxicity. Chu RS et al., JExp Med 186:1623-31 (1997); Weiner GJ et al., Proc Natl Acad Sci USA 94:10833-7 (1997); Roman M et al., Nat Med 3:849-54 (1997); Lipford GB et al., Eur JImmunol 27:2340-4 (1997); Davis HL et al., JImmunol 160:870-6 (1998). Recently, we identified a human CpG motif which triggers proliferation and activation of primary human B cells. Hartmann G et al., JImmunol 164:944-53 (2000). 20 SUMMARY OF THE INVENTION The invention relates in some aspects to methods for treating and preventing cancer using immunostimulatory nucleic acids and antibodies. Thus in one aspect the invention is a method for treating or preventing cancer. The method involves administering to a subject 25 having or at risk of developing cancer an effective amount to upregulate CD20 expression of a nucleic acid, and an anti-CD20 antibody. The cancer, in some embodiments, is B-cell lymphoma associated with low levels of CD20 expression. The B-cell lymphoma in other embodiments is B-cell chronic lymphocytic leukemia (B-CLL) or a marginal zone lymphoma. In some embodiments the CD20 antibody is C2B8 or Rituximab. 30 The invention in other aspects relates to a method for diagnosing lymphoma by isolating a B cell from a subject and identifying a change in ccll surface markers when the B cell is contacted with an immunostimulatory nucleic acid, wherein the cell surface marker WO 01/97843 PCT/USOI/20154 -3 induced on the B cell is indicative of the type of lymphoma. In some embodiments the subject has a type of lymphoma. In some embodiments the subject is suspected of having a type of lymphoma. The method may optionally include a method for treating cancer by administering to the subject an immunostimulatory nucleic acid and an antibody specific for 5 the cell surface antigens induced on the B cell in order to treat the cancer. In another aspect the invention is a method for treating or preventing cancer by administering to a subject having or at risk of developing cancer an effective amount to induce expression of a surface antigen on a cancer cell surface, of a nucleic acid, and administering to the subject an antibody selected from the group consisting of an anti-CD22 10 antibody and an anti-CD19 antibody. According to another aspect of the invention, a method for treating lymphoma is provided. The method includes the steps of isolating a B cell from a subject having lymphoma, identifying a surface antigen which is not expressed or which is expressed on the surface of the B cell in an amount lower than that of a control B cell, administering to the 15 subject an antibody specific for the identified surface antigen and an immunostimulatory nucleic acid in order to treat the lymphoma, wherein the nucleic acid is administered in an effective amount to upregulate expression of the surface antigen on the lymphoma cell surface. A method for treating a lymphoma resistant to antibody therapy is provided according 20 to another aspect of the invention. The method includes administering to a subject having a lymphoma resistant to therapy with an antibody specific for a surface antigen, an antibody specific for the surface antigen to which the lymphoma is resistant and a nucleic acid in order to treat the lymphoma, wherein the nucleic acid is administered in an effective amount to upregulate expression of the surface antigen on the lymphoma cell surface. 25 The surface antigen maybe any type of surface antigen which is capable of being expressed on the surface of a cancer cell and which is induced by stimulation with immunostimulatory nucleic acids. In some embodiments the surface antigen is CD20, CD40, CD22, or CD19. In other embodiments the lymphoma is B-CLL or marginal zone lymphoma. In some embodiments the antibody is an anti-CD20 antibody. In some 30 embodiments the anti-CD20 antibody is C2B8. In another embodiment the anti-CD20 antibody is Rituximab.
WO 01/97843 PCT/US01/20154 -4 In some preferred embodiments the antibody is a human IgG1 antibody. In some preferred embodiments the antibody is a murine IgG2a antibody. In some embodiments the methods also include administering an anti-cancer therapy to the subject. 5 The invention also includes a method for treating cancer in a human by administering to a human an immunostimulatory nucleic acid and an antibody of IgGI isotype (an IgG1 isotype antibody as used herein refers to a human or humanized IgG1 unless otherwise specified), which binds to a cell surface antigen of a cancer cell and wherein the nucleic acid and the antibody are administered in an effective amount for killing the cancer cell. 10 Optionally the nucleic acid and the antibody are administered together. Alternatively the nucleic acid and the antibody may be administered separately. In some embodiments the method includes the step of administering a cancer therapy. As used herein the term "a cancer therapy" is meant to embrace a single medicament, a plurality of medicaments of a particular class and a plurality of medicaments of different 15 classes, and includes but is not limited to chemotherapeutic agents, cancer vaccines, biological response modifiers, and hormone therapies. A chemotherapeutic agent may be selected from the group consisting of methotrexate, vincristine, adriamycin, cisplatin, non-sugar containing chloroethylnitrosoureas, 5 fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol, fragyline, Meglamine 20 GLA, valrubicin, carmustaine and poliferposan, MMI270, BAY 12-9566, RAS famesyl transferase inhibitor, famesyl transferase inhibitor, MMP, MTA/LY231514, LY264618/Lometexol, Glamolec, CI-994, TNP-470, Hycamtin/Topotecan, PKC412, Valspodar/PSC833, Novantrone/Mitroxantrone, Metaret/Suramin, Batimastat, E7070, BCH 4556, CS-682, 9-AC, AG3340, AG3433, Incel/VX-710, VX-853, ZD0101, ISI641, ODN 25 698, TA 2516/Marmistat, BB2516/Marmistat, CDP 845, D2163, PD183805, DX8951f, Lemonal DP 2202, FK 317, Picibanil/OK-432, AD 32/Valrubicin, Metastron/strontium derivative, Temodal/Temozolomide, Evacet/liposomal doxorubicin, Yewtaxan/Paclitaxel, Taxol/Paclitaxel, Xeload/Capecitabine, Furtulon/Doxifluridine, Cyclopax/oral paclitaxel, Oral Taxoid, SPU-077/Cisplatin, HMR 1275/Flavopiridol, CP-358 (774)/EGFR, CP-609 30 (754)/RAS oncogene inhibitor, BMS-1 82751/oral platinum, UFT(Tegafur/Uracil), Ergamisol/Levamisole, Eniluracil/776C85/5FU enhancer, Campto/Levamisole, Camptosar/Irinotecan, Tumodex/Ralitrexed, Leustatin/Cladribine, Paxex/Paclitaxel, WO 01/97843 PCT/USO1I/20154 -5 Doxil/liposomal doxorubicin, Caelyx/liposomal doxorubicin, Fludara/Fludarabine, Pharmarubicin/Epirubicin, DepoCyt, ZD 1839, LU 79553/Bis-Naphtalimide, LU 103793/Dolastain, Caetyx/liposomal doxorubicin, Genzar/Gemcitabine, ZD 0473/Anormed, YM 116, Iodine seeds, CDK4 and CDK2 inhibitors, PARP inhibitors, D4809/Dexifosamide, 5 Ifes/Mesnex/Ifosamide, Vumon/Teniposide, Paraplatin/Carboplatin, Plantinol/cisplatin, Vepeside/Etoposide, ZD 9331, Taxotere/Docetaxel, prodrug of guanine arabinoside, Taxane Analog, nitrosoureas, alkylating agents such as Melphalan, Cyclophosphamide, Aminoglutethimide, Asparaginase, Busulfan, Carboplatin, Chlorombucil, Cytarabine HCl, Dactinomycin, Daunorubicin HCl, Estramustine phosphate sodium, Etoposide (VP16-213), 10 Floxuridine, Fluorouracil (5-FU), Flutarnide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Interferon Alfa-2b, Leuprolide acetate (LHRH-releasing factor analogue), Lomustine (CCNU), Mechlorethamine HCl (nitrogen mustard), Mercaptopurine, Mesna, Mitotane (o,p'-DDD), Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HC, Streptozocin, Tanoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Amsacrine (m 15 AMSA), Azacitidine, Erythropoietin, Hexamethylmelamine (H1MM), Interleukin 2, Mitoguazone (methyl-GAG; methyl glyoxal bis-guanylhydrazone; MGBG), Pentostatin (2'deoxycoformycin), Semustine (methyl-CCNU), Teniposide (VM-26) and Vindesine sulfate. In some preferred embodiments the chemotherapeutic agent may be selected from the 20 group consisting of methotrexate, vincristine, adriamycin, cisplatin, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol, valrubicin, Novantrone/Mitroxantrone, Evacet/liposomal doxorubicin, Yewtaxan/Paclitaxel, Taxol/Paclitaxel, SPU-077/Cisplatin, HMR 1275/Flavopiridol, BMS-182751/oral platinum, Leustatin/Cladribine, Paxex/Paclitaxel, Doxil/liposomal doxorubicin, Caelyx/liposomal doxorubicin, Fludara/Fludarabine, 25 Pharmarubicin/Epirubicin, DepoCyt, Caetyx/liposomal doxorubicin, Gemzar/Gemcitabine, Ifes/Mesnex/Ifosamide, Vumon/Teniposide, Paraplatin/Carboplatin, Plantinol/cisplatin, Vepeside/Etoposide, Taxotere/Docetaxel, prodrug of guanine arabinoside, nitrosoureas, alkylating agents such as melphalan and cyclophosphamide, Asparaginase, Busulfan, Carboplatin, Chlorombucil, Cytarabine HCI, Daunorubicin HCl, Etoposide (VP16-213), 30 Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Interferon Alfa-2b, Lomustine (CCNU), Mechlorethaminc HCl (nitrogen mustard), Mercaptopurine, Mitoxantrone HC1, Procarbazine HCI, Thioguanine, Thiotepa, Vinblastine sulfate, WO 01/97843 PCT/US01/20154 -6 Azacitidine, Interleukin 2, Pentostatin (2'deoxycoformycin), Teniposide (VM-26), GM-CSF, and Vindesine sulfate. A cancer vaccine may be selected from the group consisting of EGF, Anti-idiotypic cancer vaccines, Gp75 antigen, GMK melanoma vaccine, MGV ganglioside conjugate 5 vaccine, Her2/neu, Ovarex, M-Vax, O-Vax, L-Vax, STn-KHL theratope, BLP25 (MUC-1), liposomal idiotypic vaccine, Melacine, peptide antigen vaccines, toxin/antigen vaccines, MVA-based vaccine, PACIS, BCG vaccine, TA--HPV, TA-CIN, DISC-virus and InmuCyst/TheraCys. Biological response modifiers include interferon, and lymphokines such as IL-2. Hormone replacement therapy includes tamoxifen alone or in combination with 10 progesterone. In a further embodiment, the cancer therapy is interferon-c: (e.g., INTRON@ A, Schering). The cancer may be selected from the group consisting of basal cell carcinoma, bladder cancer, bone cancer, brain and central nervous system (CNS) cancer, breast cancer, cervical cancer, colon and rectum cancer, connective tissue cancer, esophageal cancer, eye cancer, 15 kidney cancer, larynx cancer, leukemia, liver cancer, lung cancer, Hodgkin's lymphoma, non Hodgkin's lymphoma, melanoma, myeloma, oral cavity cancer (e.g., lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, rhabdomyosarcoma, skin cancer, stomach cancer, testicular cancer, and uterine cancer. In preferred embodiments, the cancer to be treated may be selected from the group consisting of bone cancer, brain and CNS 20 cancer, connective tissue cancer, esophageal cancer, eye cancer, Hodgkin's lymphoma, larynx cancer, oral cavity cancer (e.g., lip, tongue, mouth, and pharynx), skin cancer, and testicular cancer. In another aspect the invention encompasses a kit. The kit includes a package including at least two containers, the first container housing an immunostimulatory nucleic 25 acid, the second container housing an antibody specific for a cell surface antigen, and instructions for screening a cell to determine whether the immunostimulatory nucleic acid upregulates expression of the cell surface antigen. In one embodiment the antibody is selected from the group consisting of an anti-CD20 antibody, an anti-CD19 antibody, and an anti-CD22 antibody. 30 The nucleic acids useful according to the invention are immunostimulatory nucleic acids and in some embodiments are immunostimulatory CpG nucleic acids having an WO 01/97843 PCT/US01/20154 -7 unmethylated CpG motif, immunostimulatory T-rich nucleic acids, immunostimulatory poly G nucleic acids, bacterial DNA, yeast DNA, or eukaryotic DNA. In some embodiments the nucleic acid does not hybridize with genomic DNA or RNA under stringent conditions. In other embodiments the nucleic acid does hybridize with 5 genomic DNA or RNA under stringent conditions. The nucleic acid may have natural linkages or may include at least one modified backbone intemucleotide linkage. In some embodiments the modified backbone is a phosphate backbone modification. In other embodiments the modified backbone is a peptide modified oligonucleotide backbone. The nucleic acid may also include native bases or 10 modified bases. The nucleotide backbone may be chimeric, or the nucleotide backbone is entirely modified. The immunostimulatory nucleic acid can have any length greater than 6 nucleotides, but in some embodiments is between 8 and 100 nucleotide residues in length. In other embodiments the nucleic acid comprises at least 20 nucleotides, at least 24 nucleotides, at 15 least 27, nucleotides, or at least 30 nucleotides. The nucleic acid may be single-stranded or double-stranded. In some embodiments the nucleic acid is isolated and in other embodiments the nucleic acid may be a synthetic nucleic acid. The CpG nucleic acid in one embodiment contains at least one unmethylated CpG dinucleotide having a sequence including at least the following formula: 5' XIX 2
CGX
3
X
4 3' 20 wherein C is unmethylated, wherein X 1 , X 2 , X 3 , and X 4 are nucleotides. In one embodiment the 5' X 1
X
2
CGX
3
X
4 3' sequence of the CpG nucleic acid is a non-palindromic sequence, and in other embodiments it is a palindromic sequence. In some embodiments XiX 2 are nucleotides selected from the group consisting of: GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT, and TpG; and X 3
X
4 are 25 nucleotides selected from the group consisting of: TpT, CpT, ApT, TpG, ApG, CpG, TpC, ApC, CpC, TpA, ApA, and CpA. In other embodiments X 1
X
2 are GpA or GpT and X 3
X
4 are TpT. In yet other embodiments Xi or X 2 or both are purines and X 3 or X4 or both are pyrimidines or XIX 2 are GpA and X 3 or X 4 or both are pyrimidines. In one embodiment X 2 is a T and X 3 is a pyrimidine. 30 In other embodiments the CpG nucleic acid has a sequence selected from the group consisting of SEQ ID NOs: 19, 35-37, 39-42, 91, 92, 101, 108, 111, 135, 141, 151, 274, 277, 280, 286, 319, 350, 363, 368, 375, 495-498, 517, 518, 524, 529, 545, 548, 549, 555, 557, WO 01/97843 PCT/USO1/20154 -8 560-563, 566, 585, 590, 591, 595, 599, 603, 605, 611, 614-616, 650, 676, 679, 682, 684, 702, 703, 707-710,717-720, 729-732, 752, 755, 770, and 801-803. In some embodiments the T-rich immunostimulatory nucleic acid is a poly-T nucleic acid comprising 5' TTTT 3'. In yet other embodiments the poly-T nucleic acid comprises 5 5' XIX 2
TTTTX
3
X
4 3' wherein X 1 , X 2 , X 3 , and X 4 are nucleotides. In some embodiments
XIX
2 is TT and/or X 3
X
4 is TT. In other embodiments X 1
X
2 is selected from the group consisting of TA, TG, TC, AT, AA, AG, AC, CT, CC, CA, CG, GT, GG, GA, and GC; and/or X 3
X
4 is selected from the group consisting of TA, TG, TC, AT, AA, AG, AC, CT, CC, CA, CG, GT, GG, GA, and GC. 10 The T-rich immunostimulatory nucleic acid may have only a single poly-T motif or it may have a plurality of poly-T nucleic acid motifs. In some embodiments the T-rich immunostimulatory nucleic acid comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 T motifs. In other embodiments it comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 CpG motifs. In some embodiments the 15 plurality of CpG motifs and poly-T motifs are interspersed. In yet other embodiments at least one of the plurality of poly-T motifs comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 contiguous T nucleotide residues. In other embodiments the plurality of poly-T motifs is at least 3 motifs and wherein at least 3 motifs each comprises at least 3 contiguous T nucleotide residues or 20 the plurality of poly-T motifs is at least 4 motifs and wherein the at least 4 motifs each comprises at least 3 contiguous T nucleotide residues. The T-rich immunostimulatory nucleic acid may include one or more CpG motifs. In other embodiments the T-rich immunostimulatory nucleic acid is free of one or more CpG dinucleotides. 25 In other embodiments the T-rich immunostimulatory nucleic acid has poly A, poly-G, and/or poly C motifs. In other embodiments the T-rich immunostimulatory nucleic acid is free of two poly C sequences of at least 3 contiguous C nucleotide residues. Preferably the T rich immunostimulatory nucleic acid is free of two poly A sequences of at least 3 contiguous A nucleotide residues. In other embodiments the T-rich immunostimulatory nucleic acid 30 comprises a nucleotide composition of greater than 25% C or greater than 25% A. In yet other embodiments the T-rich immunostimulatory nucleic acid is free of poly-C sequences, poly-G sequences or poly-A sequences.
WO 01/97843 PCT/USOI/20154 -9 In some cases the T-rich immunostimulatory nucleic acid may be free of poly-T motifs, but rather, comprises a nucleotide composition of greater than 25% T. In other embodiments the T-rich immunostimulatory nucleic acid may have poly-T motifs and also comprise a nucleotide composition of greater than 25% T. In some embodiments the T-rich 5 immunostimulatory nucleic acid comprises a nucleotide composition of greater than 25% T, greater than 30% T, greater than 40% T, greater than 50% T, greater than 60% T, greater than 80% T, or greater than 90% T nucleotide residues. In some embodiments the poly-G nucleic acid comprises: 5' XIX 2
GGGX
3 X4 3' wherein XI, X 2 , X 3 , and X 4 are nucleotides. In embodiments at least one of X 3 and X 4 are a 10 G or both of X 3 and X 4 are a G. In other embodiments the poly-G nucleic acid comprises the following formula: 5' GGGNGGG 3' wherein N represents between 0 and 20 nucleotides. In yet other embodiments the poly-G nucleic acid comprises the following formula: 5' GGGNGGGNGGG 3' wherein N represents between 0 and 20 nucleotides. The poly-G immunostimulatory nucleic acid may include one or more CpG motifs or 1s T-rich motifs. In other embodiments the poly-G nucleic acid is free of one or more CpG dinucleotides or poly-T motifs. Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. 20 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts data from flow cytometry which demonstrates the induction of a morphologic change in marginal zone lymphoma cells upon CpG oligonucleotide stimulation. Malignant B cells from a patient with marginal zone lymphoma were stimulated 25 with no oligonucleotide (A and D), control oligonucleotide (ODN 2017, SEQ ID NO: 168, B and B) or CpG oligonucleotide (ODN 2006, SEQ ID NO: 729, C and F) and analyzed by flow cytometry. A, B, and C illustrate forward scatter (FSC; x-axis) vs. side scatter (SSC; y-axis). D, E and F illustrate CD1 9 expression (x-axis) against FSC (y-axis). Figure 2 depicts data from flow cytometry which demonstrates the change in 30 expression of surface antigens on marginal zone lymphoma cells upon CpG oligodoexynucleotide (ODN) treatment. Flow cytometric analysis of surface antigen expression on malignant B cells from a patient with marginal zone lymphoma was performed WO 01/97843 PCT/USO1/20154 -10 using either CpG or non-CpG oligonucleotide. Thin curves indicate incubation with medium alone, dotted curves indicate incubation with control oligonucleotide, and bold curves indicate incubation with CpG oligonucleotide. Figure 3 is a set of bar graphs depicting changes in expression of surface antigens on 5 primary cells representing different B-cell malignancies and cells of a benign follicular hyperplasia upon treatment with, from left to right in each panel: negative control, no oligonucleotide, control oligonucleotide (ODN 2017, SEQ ID NO: 168), or CpG oligonucleotide (ODN 2006, SEQ ID NO: 729). Each panel represents one experiment. Figure 4 is a set of graphs depicting the observation that the effect of CpG 10 oligonucleotide on CD20 (top) and CD40 (bottom) is dependent on the baseline level of expression of CD20 and CD40. Cells from lymph node biopsies, peripheral blood or pleural fluid from patients with different B-cell malignancies were incubated with or without CpG oligonucleotide, and expression of CD20 and CD40 was measured by flow cytometry. Figure 5 depicts data from flow cytometry which demonstrates the effect of CpG 15 oligonucleotide-induced proliferation of malignant and normal B cells. Peripheral blood mononuclear cells from patients with B-CLL (left) or marginal zone lymphoma with circulating malignant cells (right), were incubated with CpG oligonucleotide (bottom) or medium alone (top) and evaluated by two-color flow cytometry. CFSE fluorescence (x-axis) and expression of CD5 (B-CLL) or CD 19 (marginal zone lymphoma) (y-axis) were 20 evaluated. Figure 6 is a graph depicting the survival ofmice injected on Day 0 with tumor cells in response to CpG simulation in combination with murine IgG2a and murine IgG1 anti tumor antibodies. Treatments are shown as filled squares, untreated controls; filled circles, murine IgGI; filled triangles, murine IgGI plus CpG; filled diamonds, murine IgG2a; and 25 open squares, murine IgG2a plus CpG. DETAILED DESCRIPTION Present cancer treatments are often ineffective as well as being associated with a high degree of patient morbidity. The invention provides methods and products for the more 30 effective treatment of cancer using a combination of immunostimulatory nucleic acids, antibodies, and optionally cancer therapies.
WO 01/97843 PCT/USOI/20154 The invention is based, in part, on the surprising discovery that administration to a subject of immunostimulatory nucleic acids induces the expression of cell surface antigens including CD20, CD 19, and CD22 on the surface of a cancer cell and that the induction of these antigens leads to enhanced antibody-dependent cellular cytotoxicity (ADCC). It was 5 previously believed that CpG oligonucleotides enhanced ADCC by influencing the effector cell (e.g., by activating natural killer (NK) cells). Now it has been discovered according to the invention that immunostimulatory nucleic acids actually cause the induction of specific antigens CD20, CD19, and CD22, each of which can be targeted by specific antibody therapies. The discovery that immunostimulatory nucleic acids are capable of upregulating 10 expression of certain target antigens on the surface of cancer cells, supports the development of therapies using immunostimulatory nucleic acids in combination with specific antibodies which interact with these cell surface antigens. Thus, in one aspect, the invention provides a method for treating or preventing cancer which involves the administration to a subject of a combination of an immunostimulatory nucleic acid and an antibody which specifically 15 interacts with CD20, CD 19, and CD22 in an effective amount to prevent or treat the cancer. Additionally, it was discovered that the increased expression of these and other cell surface antigens varies widely depending upon the histological state of the tumor cell studied. The effect of immunostimulatory nucleic acids on different types of primary malignant B cells and reactive follicular hyperplasia was extensively examined. All B-cell lymphoma 20 cells tested increased in size and granularity, upregulated activation markers (CD80, CD86, CD40, CD54, CD69), and upregulated antigen presentation molecules (class I major histocompatibility complex (MHC 1), class II major histocompatibilty complex (MHC II)) in response to immunostimulatory nucleic acids. A control poly-C oligodeoxynucleotide (ODN) showed only minor effects. The extent of phenotypic change induced by 25 immunostimulatory nucleic acids differed from sample to sample. Immunostimulatory nucleic acids, but not control nucleic acids, increased the expression of co-stimulatory molecules (e.g., CD40, CD80, CD86, CD54) on malignant B cells without altering the phenotype of B cells derived from reactive follicular hyperplasia. Immunostimulatory nucleic acids also enhanced expression of both class I and class II MHC in most samples. 30 CD20 expression was increased in response to immunostimulatory nucleic acids, most notably in B-CLL and marginal zone lymphoma.
WO 01/97843 PCT/US01/20154 -12 Furthermore, an inverse correlation was found between baseline expression of specific cell surface antigens and their expression after exposure to immunostimulatory nucleic acids. Thus the most significant increase in expression of these molecules was found in those samples that had the lowest (or no) baseline levels. These data indicate that 5 immunostimulatory nucleic acids may reverse low expression of co-stimulatory molecules on malignant B cells that correspond to a low level of activation, while their effects on cells that are already in an activated state are less profound. Thus, the invention relates to methods for identifying an appropriate therapy for a lymphoma patient, and for treating the patient using that therapy. The method can be to accomplished by isolating a B cell from a lymphoma patient and comparing the surface antigens expressed on the malignant B cell with those expressed on normal B cells. The antigens which are expressed in low levels or not at all on the malignant B cell can be identified. The subject can then be treated using a combination of an immunostimulating nucleic acid and an antibody which specifically recognizes the antigen(s) which are expressed 15 in low levels or not at all on the malignant B cell. The invention is also useful for treating cancers which are resistant to monoclonal antibody therapy. It has been discovered according to the invention, that immunostimulatory nucleic acids can reverse the resistance of tumor cells and render tumor cells which were previously non-responsive or only weakly responsive, sensitive to therapy. In particular it 20 has been discovered that immunostimulatory nucleic acids can cause a phenotypic change to a resistant tumor cell that renders it sensitive to monoclonal antibody therapy. For instance, the monoclonal anti-CD20 antibody Rituximab has been shown to be effective clinically in several trials and has recently been approved for the therapy of follicular B cell lymphoma. Maloney DG, Semin Oncol 26:74-8 (1999); Foran JM et al., J Clin Oncol 18:317-24 (2000); 25 Witzig TE et al., JClin Oncol 17:3793-803 (1999); Davis TA et al., J Clin Oncol 17:1851-7 (1999); Wiseman GA et al., Clin Cancer Res 5:3281s-3286s (1999); Grillo-Lopez AJ et al, Semin Oncol 26:66-73 (1999). There are reports that with lymphomas a small minority of tumors that re-emerge following Rituximab therapy can lack CD20 expression. Davis TA et al., Clin Cancer Res 5:611-5 (1999); Kinoshita T et al., J Clin Oncol 16:3916 (1998). The 30 immunostimulatory nucleic acids of the invention are useful for treating this set of resistant tumors. Additionally, Rituximab has not been useful for the treatment of all types of B cell malignancies. Expression of CD20 is relatively low on B-CLL cells, which provides an WO 01/97843 PCTIUSOI/20154 -13 explanation for why Rituximab is less effective for CLL than for some other B-cell malignancies. Grinaldi L et al., JClin Pathol 51:364-9 (1998). The immunostimulatory nucleic acids of the invention are also useful for treating these tumors. The humanized monoclonal antibody 1D 10 recognizes an HLA-DR variant antigen. 5 Link BK et al., Blood 81:3343-9 (1993). This antibody is currently being tested in a phase I clinical trial in patients with lymphoma. One limitation to the use of this antibody is that the target antigen is only expressed by approximately 50% ofB-cell lymphomas. Interestingly, its expression was upregulated by immunostimulatory nucleic acids in all lymphoma samples tested. It was discovered according to the invention that immunostimulatory nucleic acids 10 may enhance the efficacy of therapy with these and other antibodies by increasing expression of target antigen. Thus in another aspect the invention includes methods for treating lymphoma by administering to a subject an immunostimulatory nucleic acid and antibodies specific for HLA-DR. One useful antibody is the humanized monoclonal antibody 1D10. It is particularly useful for treating resistant tumors. 15 The invention also relates to the discovery of a specific subclass, or isotype, of antibody which when combined with immunostimulatory nucleic acids produces a synergistic immune response. Another subclass, or isotype, does not even provide an additive response when combined with immunostimulatory nucleic acids. It was discovered according to the invention that the combination of immunostimulatory nucleic acids and human antibodies of 20 the IgG1 isotype results in an increased (synergistic) survival rate. When immunostimulatory nucleic acids are combined with human antibodies of the IgG2 isotype, no increase in survival rate is observed over the use of the IgG2 antibody alone. The IgG2 isotype (which correlates with the murine IgG1 isotype) is believed to be recognized by the Fc receptor designated CD16 that is expressed largely by NK cells. Immunostimulatory nucleic acids are 25 known to activate NK cells, and thus, it is surprising that immunostimulatory nucleic acids do not enhance the therapeutic effect of human IgG2 or murine IgGl antibodies. Since NK cells are believed to be involved in ADCC and are activated by immunostimulatory nucleic acids, it was surprising that antibodies of the human IgG2 (or murine IgG1) isotype do not produce a synergistic or even additive response when administered with immunostimulatory nucleic 30 acids. A cancer cell is a cell that divides and reproduces abnormally due to a loss of normal growth control. Cancer cells almost always arise from at least one genetic mutation. In some WO 01/97843 PCTIUSOI/20154 -14 instances, it is possible to distinguish cancer cells from their normal counterparts based on profiles of expressed genes and proteins, as well as to the level of their expression. Genes commonly affected in cancer cells include oncogenes, such as ras, neu/HER2/erbB, myb, myc and abl, as well as tumor suppressor genes such as p53, Rb, DCC, RET and WT. 5 Cancer-related mutations in some of these genes leads to a decrease in their expression or a complete deletion. In others, mutations cause an increase in expression or the expression of an activated variant of the normal counterpart. The term "tumor" is usually equated with neoplasm, which literally means "new growth" and is used interchangeably with "cancer." A "neoplastic disorder" is any disorder 10 associated with cell proliferation, specifically with a neoplasm. A "neoplasm" is an abnormal mass of tissue that persists and proliferates after withdrawal of the carcinogenic factor that initiated its appearance. There are two types of neoplasms, benign and malignant. Nearly all benign tumors are encapsulated and are noninvasive; in contrast, malignant tumors are almost never encapsulated but invade adjacent tissue by infiltrative destructive growth. This 15 infiltrative growth can be followed by tumor cells implanting at sites discontinuous with the original tumor. The method of the invention can be used to treat neoplastic disorders in humans, including but not limited to: sarcoma, carcinoma, fibroma, glioma, leukemia, lymphoma, melanoma, myeloma, neuroblastoma, retinoblastoma, and rhabdomyosarcoma, as well as each of the other tumors described herein. 20 "Cancer" as used herein refers to an uncontrolled growth of cells which interferes with the normal functioning of the bodily organs and systems. Cancers which migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs. Hemopoietic cancers, such as leukemia, are able to out-compete the normal hemopoietic compartments in a subject, thereby 25 leading to hemopoietic failure (in the form of anemia, thrombocytopenia and neutropenia), ultimately causing death. A metastasis is a region of cancer cells, distinct from the primary tumor location, resulting from the dissemination of cancer cells from the primary tumor to other parts of the body. At the time of diagnosis of the primary tumor mass, the subject may be monitored for 30 the presence of metastases. Metastases are most often detected through the sole or combined use of magnetic resonance imaging (MRJ) scans, computed tomography (CT) scans, blood WO 01/97843 PCT/USOI/20154 - 15 and platelet counts, liver function studies, chest X-rays and bone scans in addition to the monitoring of specific symptoms. Cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cervical cancer; 5 choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal caricer; eye cancer; cancer of the head and neck; gastric cancer; intra-epithelial neoplasm; kidney cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small cell and non-small cell); lymphoma including Hodgkin's and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, 1o tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; renal cancer; cancer of the respiratory system; sarcoma; skin cancer; stomach cancer; testicular cancer; thyroid cancer; uterine cancer; cancer of the urinary system, as well as other carcinomas and sarcomas. The immunostimulatory nucleic acids and antibodies are useful for treating or i5 preventing cancer in a subject. A "subject" unless otherwise specified shall mean a human or vertebrate mammal including but not limited to a dog, cat, horse, cow, pig, sheep, goat, or primate, e.g., monkey. Thus the invention can be used to treat cancer and tumors in human and non human subjects. Cancer is one of the leading causes of death in companion animals (i.e., cats and dogs). Cancer usually strikes older animals which, in the case of house pets, 20 have become integrated into the family. Forty-five percent of dogs older than 10 years of age are likely to succumb to the disease. The most common treatment options include surgery, chemotherapy and radiation therapy. Other treatment modalities which have been used with some success are laser therapy, cryotherapy, hyperthermia and immunotherapy. The choice of treatment depends on the type of cancer and degree of dissemination. Unless the 25 malignant growth is confined to a discrete area in the body, it is difficult to remove only malignant tissue without also affecting normal cells. Malignant disorders commonly diagnosed in dogs and cats include but are not limited to lymphosarcoma, osteosarcoma, mammary tumors, mastocytoma, brain tumor, melanoma, adenosquamous carcinoma, carcinoid lung tumor, bronchial gland tumor, bronchiolar 30 adenocarcinoma, fibroma, myxochondroma, pulmonary sarcoma, neurosarcoma, osteoma, papilloma, retinoblastoma, Ewing's sarcoma, Wilms' tumor, Burkitt's lymphoma, microglioma, neuroblastoma, osteoclastoma, oral neoplasia, fibrosarcoma, osteosarcoma and WO 01/97843 PCT/USO1/20154 -16 rhabdomyosarcoma. Other neoplasias in dogs include genital squamous cell carcinoma, transmissable venereal tumor, testicular tumor, seminoma, Sertoli cell tumor, hemangiopericytoma, histiocytoma, chloroma (granulocytic sarcoma), corneal papilloma, corneal squamous cell carcinoma, hemangiosarcoma, pleural mesothelioma, basal cell tumor, 5 thymoma, stomach tumor, adrenal gland carcinoma, oral papillomatosis, hemangioendothelioma and cystadenoma. Additional malignancies diagnosed in cats include follicular lymphoma, intestinal lymphosarcoma, fibrosarcoma and pulmonary squamous cell carcinoma. The ferret, an ever-more popular house pet, is known to develop insulinoma, lymphoma, sarcoma, neuroma, pancreatic islet cell tumor, gastric MALT lymphoma and 10 gastric adenocarcinoma. Neoplasias affecting agricultural livestock include leukemia, hemangiopericytoma and bovine ocular neoplasia (in cattle); preputial fibrosarcoma, ulcerative squamous cell carcinoma, preputial carcinoma, connective tissue neoplasia and mastocytoma (in horses); hepatocellular carcinoma (in swine); lymphoma and pulmonary adenomatosis (in sheep); 15 pulmonary sarcoma, lymphoma, Rous sarcoma, reticuloendotheliosis, fibrosarcoma, nephroblastoma, B-cell lymphoma and lymphoid leukosis (in avian species); retinoblastoma, hepatic neoplasia, lymphosarcoma lymphoblasticc lymphoma), plasmacytoid leukemia and swimbladder sarcoma (in fish), caseous lymphadenitis (CLA): chronic, infectious, contagious disease of sheep and goats caused by the bacterium Corynebacterium pseudotuberculosis, 20 and contagious lung tumor of sheep caused byjaagsiekte. In one aspect, a method for treating cancer is provided which involves administering the compositions of the invention to a subject having cancer. A "subject having cancer" is a subject that has been diagnosed with a cancer. In some embodiments, the subject has a cancer type characterized by a solid mass tumor. The solid tumor mass, if present, may be a 25 primary tumor mass. A primary tumor mass refers to a growth of cancer cells in a tissue resulting from the transformation of a normal cell of that tissue. In most cases, the primary tumor mass is identified by the presence of a cyst, which can be found through visual inspection or palpation methods, or by irregularity in shape, texture or weight of the tissue. However, some primary tumors are not palpable and can be detected only through 30 medical imaging techniques such as X-rays (e.g., mammography), or by needle aspirations. The use of these latter techniques is more common in early detection. Molecular and WO 01/97843 PCT/USOI/20154 -17 phenotypic analysis of cancer cells within a tissue will usually confirm if the cancer is endogenous to the tissue or if the lesion is due to metastasis from another site. With respect to the prophylactic treatment methods, the invention is aimed at administering the compositions of the invention to a subject at risk of developing cancer. A 5 subject at risk of developing a cancer is one who has a high probability of developing cancer. These subjects include, for instance, subjects having a genetic abnormality, the presence of which has been demonstrated to have a correlative relation to a higher likelihood of developing a cancer. Subjects exposed to cancer-causing agents such as tobacco, asbestos, or other chemical toxins are also subjects at risk of developing cancers used herein. When a 10 subject at risk of developing a cancer is treated with an immunostimulatory nucleic acid, an antibody and optionally a cancer therapy, on a regular basis, such as monthly, the cancer growth will be prevented from initiating. This aspect of the invention is particularly advantageous when the subjects employed in certain trades which are exposed to cancer causing agents on an ongoing basis. For example, many airborne, or inhaled, carcinogens 15 such as tobacco smoke and asbestos have been associated with lung cancer. A carcinogen is an agent capable of initiating development of malignant cancers. Exposure to carcinogens generally increases the risk of neoplasms in subjects, usually by affecting DNA directly. Carcinogens may take one of several forms such as chemical, electromagnetic radiation, or may be an inert solid body. 20 Substances for which there is sufficient evidence to establish a causal relationship in cancer in humans are referred to as confirmed human carcinogens. Included in this category are the following substances: Aflatoxins, Alcoholic beverages, Aluminium production, 4 aminobiphenyl, Arsenic and arsenic compounds, Asbestos, Manufacture of auramine, Azathioprine, Benzene, Benzidine, Beryllium and beryllium compounds, Betel quid with 25 tobacco, Bis(chloromethyl)ether and chloromethyl methyl ether (technical grade), Boot and shoe manufacture and repair (occupational exposure), 1,4 Butanediol dimethanesulphonate (Myleran), Cadmium and cadmium compounds, Chlorambucil, Chlornaphazine, 1-(2 Chlorocthyl)-3-(4-methylcyclohexyl)-l nitrosourea, Chloromethyl methyl ether (technical), Chromium compounds (hexavalent), Coal gasification, Coal tar pitches, Coal tars, Coke 30 production, Cyclophosphamide, Cyclosporin, Erionite, Ethylene oxide, Furniture and cabinet making, Underground haematite mining with exposure to radon, Iron and steel founding, Isopropyl alcohol manufacture (strong acid process), Manufacture of magenta, Melphalan, 8- WO 01/97843 PCT/USOI/20154 - 18 Methoxypsoralen (Methoxsalen) plus ultraviolet radiation, Mineral oils-untreated and mildly treated oils, MOPP and other combined chemotherapy for cancer, Mustard gas (sulphur mustard), 2-Naphthylamine, Nickel and nickel compounds (essentially sulphate and sulphide), Nonsteroidal estrogens (not necessarily all in group) includes diethylstilbestrol, 5 Estrogen replacement therapy, and Combined oral contraceptives and sequential oral contraceptives, Steroidal estrogens (not all in group), Painter (occupational exposure as a painter), Phenacetin (analgesic mixtures containing), Rubber industry, Salted fish (Chinese style), Solar radiation, Shale oils, Soots, Sulphuric acid (occupational exposures to strong inorganic-acid mists of sulphuric acid), Talc containing asbestiform fibres, Thiotepa, 10 Tobacco products (smokeless), Tobacco smoke, Treosulphan, and Vinyl chloride. Substances for which there is a lesser degree of evidence in humans but sufficient evidence in animal studies, or degrees of evidence considered unequivocal of mutagenicity in mammalian cells, are referred to as probable human carcinogens. This category of substances includes: Acrylamide, Acrylonitrile, Adriamycin, Anabolic steroids, Azacitidine, 15 Benzanthracene, Benzidine-based dyes (technical grade), Direct Black 38, Direct Blue 6, Direct Brown 95, Benzopyrenel,3-Butadiene, Captafol, Bischloroethyl nitrosourea (BCNU), 1-(2-Chloroethyl)-3-cyclohexyl-l-nitrosourea (CCNU), Chloramphenicolpara-Chloro-ortho toluidine and its strong acid salts, Chlorozotocin, Cisplatin, Creosotes, Dibenzanthracene, Diesel engine exhaust, Diethyl sulphate, Dimethylcarbamoyl chloride, Dimethyl sulphate, 20 Epichlorohydrin, Ethylene dibromide, N-ethyl-N-nitrosourea, Formaldehyde, Glass manufacturing industry (occupational exposure), Art glass (glass containers and pressed ware), Hairdresser or barber (occupational exposure, probably dyes), Insecticide use (occupational), IQ (2-Amino-3-methylimidazo[4,5-f]quinoline), Mate drinking (hot), 5 Methoxypsoralen, 4,4'-Methylenebis(2-chloroaniline) (MOCA), N-Methyl-N-nitro-N 25 nitrosoguanidine (MNNG), N-Methyl-N-nitrosourea, Nitrogen mustard, N Nitrosodiethylamine, N-Nitrosodiinethylamine, Petroleum refining (occupational refining exposures), Phenacetin, Polychlorinated biphenyls, Procarbazine hydrochloride, Silica (crystalline), Styrene-7,8-oxide, Tris(1-azaridinyl)phosphine sulphide (Thiotepa), Tris(2,3 dibromopropyl) phosphate, Ultraviolet radiation: A, B and C including sunlamps and 30 sunbeds, and Vinyl bromide. Substances for which there is sufficient evidence in animal tests are referred to as possible human carcinogens. This category of substances includes: A-C(2-Amino-9H- WO 01/97843 PCT/US01/20154 -19 pyrido[2,3-b]indole), Acetaldehyde, Acetamide, AF-2[2-(2-Furyl)-3-(5-nitro-2 furyl)acrylamide, para-Aminoazobenzene, ortho-Aminoazobenzene, 2-Amino-5-(5-nitro-2 furyl)-1,3,4-thiadiazole, Amitrole, ortho-Anisidine, Antimony trioxide, Aramite, Atrazine, Attapulgite, Azaserine, Benzo[b]fluoranthene, Benzo[j]fluoranthene, Benzo[k]fluoranthene, 5 Benzyl violet, Bitumens (extracts of steam-refined and air-refined bitumens), Bleomycins, Bracken ferns, Bromodichloromethane, Butylated hydroxyanisole (BHA), i-Butyrolactone, Caffeic acid, Carbon black extract, Carbon tetrachloride, Carrageenan (degraded), Ceramic fibres, Chloramphenicol, Chlordane, Chlordecone, Chlorendic acid, Chlorinated paraffins of average carbon-chain length C12 and average degree of chlorination approx 60%, alpha 10 Chlorinated toluenes (not necessarily all in group), Benzotrichloride, para-Chloroaniline, Chloroform, Chlorophenols, Pentachlorophenol, 2,4,6-Trichlorophenol, Chlorophenoxy herbicides (not necessarily all in group), 4-Chloro-ortho-phenylenediamine, CI Acid Red 114, CI Basic Red 9, CI Direct Blue 15, Citrus Red No.2, Cobalt and cobalt compounds, Coffee (bladder), para-Cresidine, Cycasin, Dacarbazine, Dantron (1,8 15 dihydroxyanthraquinone), Daunomycin, DDT, N,N'-Diacetylbenzidine, 4,4' Diaminodiphenyl ether, 2,4-Diaminotoluene, Dibenz[a,hlacridine, Dibenz[aj]acridine, 7H Dibenzo[c,g]carbazole, Dibenzo[a,e]pyrene, Dibenzo[a,h]pyrene, Dibenzo[a,i]pyrene, Dibenzo[a,1]pyrene, 1,2-Dibromo-3-chloropropane, para-Dichlorobenzene, 3,3' Dichlorobenzene, 3,3'-Dichloro-4,4'-diaminodiphenyl ether, 1,2-Dichloroethane, 20 Dichloromethane, 1,3-Dichloropropene (technical grade), Dichlorvos, Diepoxybutane, Diesel fuel (marine), Di(2-ethylhexyl)phthalate, 1,2-Diethylhydrazine, Diglycidyl resorcinol ether, Dihydrosafrole, Diisopropyl sulfate, 3,3'-Dimethoxybenzidine, para Dimethylaminoazobenzene, trans-2-[(Dimethylamino)methylimino]-5-[2-(5-nitro-2 firy1[vinyll-1,3,4-oxidiazole, 2,6-Dimethylaniline (2,6-Xylidene), 3,3'-Dimethylbenzidine 25 (ortho-tolidine), Dimethylformamide, 1,1-Dimethyihydrazine, 1,2-Dimethylhydrazine, 1,6 Dinitropyrene, 1,8-Dinitropyrene, 1,4-Dioxane, Disperse Blue, lEthyl acrylate, Ethylene thiourea, Ethyl methanesulphonate, 2-(2-Formylhydrazino)-4-(5-nitro-2-furyl)thiazole, Fuel oils (residual, heavy), Fusarium moniliforme (toxins derived from), Fumonisin BI; Fumonisin B2; Fusarin C, Gasoline, Gasoline engine exhausts, Glasswool, Glu-P-1 (2 30 Amino-6-methyldipyrido[1,2-a:3'2'-d]imidazole), Glu-P-2(-Aminodipyrido[1,2-a:3'2' d]imidazole), Glycidaldehyde, Griseofulvin, HC Blue No 1, Heptachlor, Hexachlorobenzene, Hexachlorocyclohexanes Technical grades alpha isomer gamma isomer (lindane), WO 01/97843 PCT/USO1/20154 -20 Hexamethylphosphoramide, Hydrazine, Indeno[1,2,3-cd]pyrene, Iron-dextran complex, Isoprene, Lasiocarpine, Lead and lead compounds (inorganic), Magenta (containing CI Basic Red 9), Man-made mineral fibres (see glasswool, rockwool, slagwool, and ceramic fibres), MeA-a-C (2-Amino-3-methyl-9H-pyrido[2,3-b]indoie), MeIQ (2-Amino-3,4 5 dimethylimidazo[4,5-f]-quinolone), MeIQx (2-Amino-3,8-dimethylamidazo[4,5 f]quinoxaline), Methylmercury compounds (methylmercuric chloride), Melphalan, 2 Methylaziridine, Methylazoxymethanol and its acetate, 5-Methylchrysene, 4,4' Methylenebis(2-methylaniline), 4,4'-Methylenedianiline, Methylmethanesulphonate, 2 methyl-1-nitroanthraquinone (uncertain purity), N-methyl-N-nitrosourethane, 10 Methylthiouracil, Metronidazole, Mirex, Mitomycin, Monocrotaline 5-(Morpholinomethyl) 3-[(5-nitrofurfurylidene)amino]-2-oxazolidinone, Nafenopin, Niridazole, 5 Nitroacenaphthene, 6-Nitrochrysene, Nitrofen (technical grade), 2-Nitrofluorenel-[(5 Nitrofurfurylidene)amino]-2-imidazolidinone, N-[4-(5-Nitro-2-furyl)-2-thiazolylJacetamide, Nitrogen mustard, N-oxide, Nitrolotriacetic acid and its salts, 2-Nitropropanel-Nitropyrene, 15 4-Nitropyrene, N-Nitrosodi-n-butylamine, N-Nitrosodiethanolamine, N-Nitrosodi-n propylamine, 3-(N-Nitrosomethylamino)propionitrile, 4-(N-Nitrosomethylamino)-1-(3 pyri dyl)-l-butanone (NNK), N-Nitrosomethylethylamine, N-Nitrosonethylvinylamine, N Nitrosomorpholine, N-Nitrosonomicotine, N-Nitrosopiperidene, N-Nitrosopyrrolidine, N Nitrososarcosine, Ochratoxin A, Oil Orange, Panfuran S (containing 20 dihydroxymethylfuratzine), Phenazopyridine hydrochloride, Phenobarbital, Phenoxybenzamine hydrochloride, Phenyl glycidyl ether, PhenytoinPhlP (2-Amino-i methyl-6-phenylimidazo[4,5-b]pyridine, Pickled vegetables, traditional Asian, Polybrominated biphenyls, Ponceau MXPonceau 3R, Potassium bromate, 1,3-Propane sultone, Propylene oxide, Progestins, Medroxyprogesterone acetate, A-Propiolactone, 25 Propylthiouracil, Rockwool, Saccharin, Safrole, Slagwool, Sodium ortho-phenylphenate, Sterigmatocystin, Streptozotocin, Styrene, Sulfallate, 2,3,7,8-Tetrachlorodibenzo-para-dioxin (TCDD), Tetrachloroethylene, Textile manufacturing (occupational exposures), Thiocetamide, 4,4'-Thiodianiline, Thiourea, Toluene, diisocyanatcsortho-Toluidine, Toxaphene (polychlorinated camphenes), Trichlormethine (trimustine hydrochloride), Trp-P 30 1 (3-Amino-1,4-dimethyl-5-H-pyrido[4,3-b]indole), Trp-P-2 (3-Amino-i -methyl-5H pyrido[4,3-b]indole), Trypan blue, Uracil mustard, Urethane, 4-Vinylcyclohexene, 4 Vinylcyclohexene diepoxide, Welding fumes, Wood industries and Carpentry and joinery.
WO 01/97843 PCTIUSOI/20154 -21 Subjects at risk of developing cancer also include those who have a genetic predisposition to cancer. In many cases, genetic predisposition to cancer can be identified by studying the occurrence of cancer in family members. Examples of genetic predisposition to common forms of cancer include, but are not limited to, mutation of BRCA1 and BRCA2 in 5 familial breast cancer, mutation of APC in familial colony cancer (familial polyposis coli), mutation of MSH12 and MLHI in hereditary nonpolyposis colon cancer (HNPCC), mutation of p53 in Li-Fraumeni syndrome, mutation of Rb1 in retinoblastoma, mutation of RET in multiple endocrine neoplasia type 2 (MEN2), mutation of VHL in renal cancer and mutation of WTI in Wilms' tumor. Other cancers for which a familial predisposition has been 10 identified include ovarian, prostate, melanoma and lung cancer. It has been estimated that almost half of all currently diagnosed cancers will be treated with some form of cancer medicament. However, many forms of cancer, including melanoma, colorectal, prostate, endometrial, cervical and bladder cancer, do not respond well to treatment with cancer medicaments. In fact, only about 5-10 percent of cancers can be 15 cured using cancer medicaments alone. These include some forms of leukemias and lymphomas, testicular cancer, choriocarcinoma, Wilms' tumor, Ewing's sarcoma, neuroblastoma, small-cell lung cancer and ovarian cancer. Treatment of still other cancers, including breast cancer, requires a combination therapy of surgery or radiotherapy in conjunction with a cancer medicament. 20 The immunostimulatory nucleic acids are administered in combination with antibodies which specifically bind to cancer cell surface antigens. These antibodies include but are not limited to anti-CD20 antibodies, anti-CD40 antibodies, anti-CD19 antibodies, anti-CD22 antibodies, anti-HLA-DR antibodies, anti-CD80 antibodies, anti-CD86 antibodies, anti-CD54 antibodies, and anti-CD69 antibodies. These antibodies are available from 25 commercial sources or may be synthesized de novo. Commercially available anti-CD20 antibodies include but are not limited to those presented in Table 1 below. Table 1. Commercially Available Anti-CD20 Antibodies. Product/Supplicr Catalog # Monoclonal Antibody to CD20, Human, ANC-169-020 Purified, 100 pg Alexis Corp.
WO 01/97843 PCT/USOI/20154 -22 Product/Supplier Catalog # CD20, B-Cell Bab Mouse: anti-Human V6021 Clone: L26 Isotype: IgG2a, Kappa; Concentrated Biomeda Corporation CD20, B-Cell Mab Mouse: anti-Human V1018 Clone: L26 Isotype: IgG2a, Kappa; Concentrated Biomeda Corporation CD20, B-Cell MAb Mouse: anti-Human K026 Clone; L26 Isotype: IgG2a, Kappa; Dehydrated Biomeda Corporation. CD20, B-Cell Mab Mouse: anti-Human 058D Clone: L26 Isotype: IgG2a, Kappa; Prediluted Biomeda Corporation Mouse anti-Human CD20 AHS2022 BioSource International Mouse anti-Hurmn CD20 AHS2001 BioSource International Mouse anti-Human CD20 AHS2028 BioSource Intemnational Mouse ant-Human CD20 AHS2002 BioSource Inernational Mouse anti-Humnan CD20 AHS2021 BioSource International Mouse Anti-CD20, B-Cell, Human IgG2a MOB004 Antibody, Kappa, Supernatant, Clone L26,1 mL BIOTREND Chemikalien GmbH AnTesti-CD20, Human, Mouse, 100 ptg 217670 Calbiochem Mouse Monoclonal Anti-(Human CD20) MHCD2000 IgG3 Antibody, Clone 1I47, 0.5 mL Caltag Laboratories Mouse Monoclonal Anti-(Human CD20) MHCD2000-4 IgG3 Antibody, Clone B-ly 1, 1 mL Caltag Laboratories Mouse Monoclonal Anti-(Human CD20), MON1111 Mature B-cell) igG1 Antibody, Clone MEM-97, I mL Caltag Laboratories CD20, B-cell, Mouse Anti-Human, Clone: N150230 L26, Isotype: IgG2a, kappa, Ready-to-Use, LSAB2, EnVision & EnVision Doublestain, Monoclonal Antibody, 12 mL DAKO Corp. CD20, B-cell, Mouse Anti-Human, Clone: N150289 L26, Isotype: IgG2a, kappa, Ready-to-Use, LSAB2, EnVision & EnVision Doublestain, Monoclonal Antibody, Packaged for DAKO Autostainer, 33 mL DAKO Corp. CD20, L26 B-cell Marker, Mouse Anti-Human, M075501 Human, Monoclonal Antibody, 1 mL DAKO Corp. CD20, L26 B-cell Marker, Mouse Anti-Human M077401 Monoclonal Antibody, 1 mL DAKO Corp. MxH B cell, CD20 RTU, 12 mL L185030 DAKO Corp.
WO 01/97843 PCTIUS01/20154 - 23 Product/Supplier Catalog # Monoclonal Anti-B-Cell, CD20 IgG2a Mob 004 Antibody, Clone L26, concentrated, 1 mL Diagnostic BioSystems Monoclonal Anti-CD20, B-Cell IgG1 Mob 241 Antibody, Clone 7D1, concentrated, I mL Diagnostic BioSystems Monoclonal Anti-CD20, B-Cell IgG2a Mob 004-01 Antibody, Clone L26, Concentrated, I mL Diagnostic BioSystems Rabbit Polyclonal Anti-CD20, B-cell RP 041 Antibody, Concentrated, I mL Diagnostic Biosystems Coulter* Antibodies to Human CDs::CD20 COIM 1455 Fisher Scientific Co. Coulter* Antibodies to Human CDs::CD20 C06603858 Fisher Scientific Co. Coulter* Antibodies to Human CDs::CD20 COIM 1342 Fisher Scientific Co. Coulter* Antibodies to Human CDs::CD20 COIM 1565 Fisher Scientific Co. Coulter* Antibodies to Human CDs::CD20 COIM 1454 Fisher Scientific Co. Coulter* Antibodies to Human CDs::CD20 C06604106 Fisher Scientific Co. Coulter* Antibodies to Human CDs::CD20 C06603446 Fisher Scientific Co. Coulter* Antibodies to Human CDs::CD20 COLM 1456 Fisher Scientific Co. Coulter* Antibodies to Human CDs::CD20 COIM 1451 Fisher Scientific Co. Coulter* Antibodies to Human CDs::CD20 C06602381 Fisher Scientific Co. Coulter* Antibodies to Human CDs::CD20 COIM1925 Fisher Scientific Co. Coulter* Antibodies to Human CDs::CD20 C06602140 Fisher Scientific Co. CD20, Pan B-cell marker, Mouse Anti- M077401 Human, Monoclonal Antibody, I mL DAKO Corp. MxH B Cell, CD20 RTU, 12 mL L185030 DAKO Corp. Monoclonal Anti-B-Cell, CD20 IgG2a Mob 004 Antibody, Clone L26, Concentrated, 1 mL Diagnostic BioSystems Monoclonal Anti-CD20, B-Cell IgG I Mob 241 Antibody, Clone 7D 1, Concentrated,. I mL Diagnostic BioSystems Monoclonal Anti-CD20, B-Cell IgG2a Mob 004-01 Antibody, Clone L26, Concentrated, I mL Diagnostic BioSystems Rabbit Polyclonal Anti-CD20, B-cell RP 041 Antibody, Concentrated, 1 mL Diagnostic BioSystems Coulter* Antibodies to Human CDs::CD20 COIM 1455 Fisher Scientific Co.
WO 01/97843 PCT/USOI/20154 -24 Product/Supplier Catalog # Coulter* Antibodies to Human CDs::CD20 C06603858 Fisher Scientific Co. Coulter* Antibodies to Human CDs::CD20 COIM 1342 Fisher Scientific Co. Coulter* Antibodies to Human CDs::CD20 COIM 1565 Fisher Scientific Co. Coulter* Antibodies to Human CDs::CD20 COIM 1454 Fisher Scientific Co. Coulter* Antibodies to Human CDs::CD20 C06604106 Fisher Scientific Co. Coulter* Antibodies to Human CDs::CD20 C06603446 Fisher Scientific Co. Coulter* Antibodies to Human CDs::CD20 COIM 1456 Fisher Scientific Co. Coulter* Antibodies to Human CDs::CD20 COIM 1451 Fisher Scientific Co. Coulter* Antibodies to Human CDs::CD20 C06602381 Fisher Scientific Co. Coulter* Antibodies to Human CDs::CD20 COIM 1925 Fisher Scientific Co. Coulter* Antibodies to Human CDs::CD20 C06602140 Fisher Scientific Co. Coulter* Antibodies to Human CDs::CD20 C06602471 Fisher Scientific Co. CD20 (B Cell) AM-1 165-11 InnoGenex Coulter* Antibodies to Human CDs::CD20 C06602471 Fisher Scientific Co. CD20 (B Cell) AM-1165-11 InnoGenex CD20 (B Cell), Unpurified AM-1165-11 (0.1 mg/O.1 mL), Clone: B1, Isotype: InnoGenex Mouse Monoclonal Anti-CD20 Ab-1 (B- MS-340-SO Cell Marker) IgGh/K Antibody, Clone: L26, Workshop, 0.1 mL Lab Vision Corp. Mouse Monoclonal Anti-CD20 Ab-1 (B- MS-340-S1 Cell Marker) IgG2a/K Antibody, Clone: L26, Workshop, 0.5 mL Lab Vision Corp. Mouse Monoclonal Anti-CD20 Ab-1 (B- MS-340-S Cell Marker) IgG 2 1 /K Antibody, Clone: L26, Workshop, 1.0 mL Lab Vision Corp. Mouse Monoclonal Anti-CD20 Ab-I (B- MS-340-R7 Cell Marker) IgG2./K Antibody, Clone: L26, Workshop, 7.0 mL Lab Vision Corp. Mouse Monoclonal Anti-CD20 Ab-1 (B- MS-431-P1 Cell Marker) IgG2'/K Antibody, Clone: B9E9, Workshop V; 100 pg Lab Vision Corp.
WO 01/97843 PCT/USOI/20154 - 25 Product/Supplier Catalog # Mouse Monoclonal Anti-CD20 Ab-1 (B- MS-431-P Cell Marker) IgG 2 /K Antibody, Clone: 139E9, Workshop V; 200 pg Lab Vision Corp. Mouse Monoclonal Anti-CD20 (Ab- I (B- MS.431-PO Cell Marker) lgGa/K Antibody, Clone: B9E9, Workshop V; 20 jig Lab Vision Corp. Mouse Monoclonal Anti-CD20 Ab- I (B- MS-758-P1 Cell Marker) IgGI/K Antibody, Clone: 93-11B3, Workshop V; Code: CD20.4, 100 Mg Lab Vision Corp. Mouse Monoclonal Anti-CD20 Ab-3 (B- MS-758-P Cell Marker) IgGic Antibody, Clone: 93-1B3, Workshop V; Code: CD20.4, 200 pg Lab Vision Corp. Mouse Monoclonal Anti-CD20 Ab-3 (B- MS-758-PO Cell Marker) IgG/ Antibody, Clone: 93-1B33, Workshop V; Code: CD20.4 Lab Vision Corp. Human CD20, B Cell, 6 mL MAB-0020 Maxim Biotech Inc. Mouse Monoclonal Anti-B Cell, CD20 A9004C IgG2. K Antibody, Concentrate, 1 mL Scytek Mouse Monoclonal Anti-B Cell, CD20 A20003 IgG2. K Antibody, Ready-to-Use, I mL Scytek Mouse Monoclonal Anti-CD20, B Cell A9001C IgGu. K Antibody, Concentrate, mL (Clone: L26) Scytek Mouse Monoclonal Anti-CD20, B Cell A00003 IgGz. K Antibody, Ready-to-Use, 6 mL Serytec Mouse Monoclonal Anti-(Human CD20 MCA 1807 IgGI Antibody, Clone 7D1, 1. mL Serotec, Inc. Mouse Monoclonal Anti-(Human CD20 MCA 1822 IgG1b Antibody, Clone AT80, 0.2 mg Serotec, Inc. Mvous~eMonoclonal Anti-(Human CD20 MCA 1710 IgG2b Antibody, Clone 2H7, 0.2 mg Serotec, Inc. Antibody Panels, Hematopoietic 324-01 Markers, Lymphocyte Related Antigens, CD20, B Cell, Clone 1.26, Concentrated, 1 mL, Ab Source Mouse, Ab# 324 Signet Pathology Systems, Inc. Antibody Panels, Hematopoietic 324-13 Markers, Lymphocyte Related Antigens, CD20, B Cell, Clone L26, Level 1, 3 mL, Ab 324 Signet Pathology Systems, Inc.
WO 01/97843 PCT/USO1I/20154 -26 Product/Supplier Catalog # Antibody Panels, Hematopoietic 324-16 Markers, Lymphocyte Related Antigens, CD20, B Cell, Clone L26, level 1, 6 mL, Ab Source Mouse, Ab# 324 Signet Pathology Systems, Inc. Antibody Panels, Hematopoictic 324-26 Markers, Lymphocyte Related Antigens, CD20, B Cell, Clone L26, Level 2, 6 mL, Ab Source Mouse, Ab# 324 Signet Pathology Systems, Inc. Monoclonal Mouse anti-CD20, B9E9, Epitope- 07-2003 Affinity Purified-Unconjugated, IgG2,-K, 200 p±g Zymed Laboratories, Inc. Antibodies are well known to those of ordinary skill in the science of immunology. As used herein, the term "antibody" means not only intact antibody molecules but also fragments of antibody molecules retaining specific binding ability. Such fragments are also 5 well known in the art and are regularly employed both in vitro and in vivo. In particular, as used herein, the term "antibody" means not only intact immunoglobulin molecules but also the well-known active fragments F(ab') 2 , and Fab. F(ab') 2 , and Fab fragments which lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody. Wahl RL et al., JNucl Med 24:316-25 10 (1983). Antibody fragments which are particularly useful according to the methods of the invention are those which are bispecific and constructed to enhance FcR binding, e.g., include an Fc portion. These include, but are not limited to Medarex antibodies (MDX-21 0, 220, 22, 447, and 260). Other non-Fc containing fragments which interact with the antigens induced on the cell surface are also useful. These are particularly useful in combination with 15 immunotoxins and/or radioactivity. The fragments can be delivered separately from the immunotoxins or radioactivity or conjugated thereto (e.g., radiolabled antibodies or antibody fragments). Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity-determining regions (CDRs), which directly interact with the epitope of 20 the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity-determining regions (CDRI through CDR3). The CDRs, and in particular the CDR3 regions, and more particularly the heavy 25 chain CDR3, are largely responsible for antibody specificity.
WO 01/97843 PCT/USOI/20154 -27 It is now well-established in the art that the non-CDR regions of a marrimalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies in which non-human 5 CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody. Thus, for example, PCT Intemational Publication Number WO 92/04381 teaches the production and use of humanized murine RSV antibodies in which at least a portion of the murine FR regions have been replaced by FR regions of human origin. Such antibodies, including fragments of intact antibodies with antigen-binding ability, are often referred to as 10 "chimeric" antibodies. A "humanized monoclonal antibody" as used herein is a human monoclonal antibody or functionally active fragment thereof having human constant regions and a binding CDR3 region from a mammal of a species other than a human. Humanized monoclonal antibodies may be made by any method known in the art. Humanized monoclonal antibodies, for example, may be constructed by replacing the non-CDR regions 15 of a non-human mammalian antibody with similar regions of human antibodies while retaining the epitopic specificity of the original antibody. For example, non-human CDRs and optionally some of the framework regions may be covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody. There are entities in the United States which will synthesize humanized antibodies from specific murine antibody regions 20 commercially, such as Protein Design Labs (Mountain View California). European Patent Application 0239400, the entire contents of which is hereby incorporated by reference, provides an exemplary teaching of the production and use of humanized monoclonal antibodies in which at least the CDR portion of a murine (or other non-human mammal) antibody is included in the humanized antibody. Briefly, the following 25 methods are useful for constructing a humanized CDR monoclonal antibody including at least a portion of a mouse CDR. A first replicable expression vector including a suitable promoter operably linked to a DNA sequence encoding at least a variable domain of an Ig heavy or light chain and the variable domain comprising framework regions from a human antibody and a CDR region of a murine antibody is prepared. Optionally a second replicable 30 expression vector is prepared which includes a suitable promoter operably linked to a DNA sequence encoding at least the variable domain of a complementary human Ig light or heavy chain respectively. A cell line is then transformed with the vectors. Preferably the cell line is WO 01/97843 PCT/US01/20154 -28 an immortalized mammalian cell line of lymphoid origin, such as a myeloma, hybridoma, trioma, or quadroma cell line, or is a normal lymphoid cell which has been immortalized by transformation with a virus. The transformed cell line is then cultured under conditions known to those of skill in the art to produce the humanized antibody. 5 As set forth in European Patent Application 0239400 several techniques are well known in the art for creating the particular antibody domains to be inserted into the replicable vector. (Preferred vectors and recombinant techniques are discussed in greater detail below.) For example, the DNA sequence encoding the domain may be prepared by oligonucleotide synthesis. Alternatively a synthetic gene lacking the CDR regions in which four framework 10 regions are fused together with suitable restriction sites at the junctions, such that double stranded synthetic or restricted subcloned CDR cassettes with sticky ends could be ligated at the junctions of the framework regions. Another method involves the preparation of the DNA sequence encoding the variable CDR containing domain by oligonucleotide site directed mutagenesis. Each of these methods is well known in the art. Therefore, those 15 skilled in the art may construct humanized antibodies containing a murine CDR region without destroying the specificity of the antibody for its epitope. Human monoclonal antibodies may be made by any of the methods known in the art, such as those disclosed in U.S. Patent No. 5,567,610, issued to Borrebaeck et al., U.S. Patent No. 5,565,354, issued to Ostberg, U.S. Patent No. 5,571,893, issued to Baker et al, Kozbor D 20 et al., Jlmmunol 133:3001-5 (1984), Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc, New York, 1987), and Boerner Petal., Jlmniunol 147:86-95 (1991). In addition to the conventional methods for preparing human monoclonal antibodies, such antibodies may also be prepared by immunizing transgenic animals that are capable of producing human antibodies (e.g., Jakobovits A et al., 25 Proc Natl Acad Sci USA 90:2551-5 (1993); Jakobovits A et al., Nature 362:255-8 (1993); Bruggermann et al., Year in Immunology 7:33 (1993); and U.S. Patent No. 5,569,825 issued to Lonberg). Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in 30 general, Clark, W.R. (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Imnimunology, 7th Ed., Blackwcll Scientific Publications, Oxford). The pFc' and Fc regions, for example, are effectors of the WO 01/97843 PCT/USOI/20154 -29 complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F(ab') 2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, 5 or which has been produced without the Fe region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody 10 specificity) and Fd fragments retain epitope-binding ability in isolation. Other antibodies useful according to the invention are antibodies of the IgG1 isotype. As mentioned above, anti-IgG1 isotype antibody as used herein refers to a human or humanized anti-IgG1 unless otherwise specified. IgGI isotype antibodies are well known in the art and include at least the antibodies listed in Table 2 below. 15 Table 2: Cancer Immunotherapies In Development Or On The Market. Marketer Brand Name (Generic Name) Indication IDEC/Genentech, Rituxanm (rituximab, Mabthera) (IDEC- non-Hodgkin's lymphoma Inc./Hoffmann-LaRoche (first C2B8, chirneric murine/human anti-CD20 monoclonal antibody licensed for MAb) the treatment of cancer in the U.S.) GenentecfHoffmann-La Roche Herceptin, anti-Her2 hMAb Breast/ovarian Cytogen Corp. Quadramet (CYT-424) radiotherapeutic Bone metastases agent Centocor/Glaxo/Ajinomoto Panorex@ (17-lA) marinee monoclonal Adjuvant therapy for antibody) colorectal (Dukes-C) Centocor/Ajinomoto Panorex@ (17-lA) (chimeric murine Pancreatic, lung, breast, monoclonal antibody) ovary IDEC IDEC-Y2B8 (murine, anti-CD20 MAb non-Hodgkin's lymphoma labeled with Yttrium-90) ImClone Systems BEC2 (anti-idiotypic MAb, mimics the GD 3 Small cell lung epitope) (with BCG) ImClone Systems C225 (chimeric monoclonal antibody to Renal cell epidermal growth factor receptor (EGFr)) Techniclone International/Alpha Oncolym (Lym-1 monoclonal antibody non-Hodgkin's lymphoma Therapeutics linked to 131 iodine) Protein Design Labs SMART M195 Ab, humanized Acute myleoid leukemia WO 01/97843 PCT/USOl/20154 -30 Marketer Brand Name (Generic Name) Indication Techniclone I LYM-1 (Oncolymvm) non-Hodgkin's lymphoma Corporation/Cambridge Antibody Technology Aronex Pharmaceuticals, Inc. ATRAGEN@ Acute promyelocytic leukemia 1mClone Systems C225 (chimeric anti-EGFr monoclonal Head & neck, non-small antibody) + cisplatin or radiation cell lung cancer Altarex, Canada Ovarex (B43.13, anti-idiotypic CA125, Ovarian mouse MAb) Coulter Pharma (Clinical results Bexxar (anti-CD20 Mab labeled with I) non-Hodgkin's lymphoma have been positive, but the drug has been associated with significant bone marrow toxicity) Aronex Pharmaceuticals, Inc. ATRAGEN@ Kaposi's sarcoma IDEC Pharmaceuticals Rituxanm (MAb against CD20) pan-B Ab in B cell lymphoma CorpJGenentech combo. with chemotherapy LeukoSite/flex Oncology LDP-03, huMAb to the leukocyte antigen Chronic lymphocytic CAMPATH leukemia (CLL) Center of Molecular Immunology ior t6 (anti CD6, murine MAb) CTCL Cancer Medarex/Novartis MDX-210 (humanized anti-HER-2 bispecific Breast, ovarian antibody) Medarex/Novartis MDX-2 10 (humanized anti-HER-2 bispecific Prostate, non-small cell antibody) lung, pancreatic, breast Medarex MDX-1 I (complement activating receptor Acute myelogenous (CAR) monoclonal antibody) leukemia (AML) Medarex/Novartis MDX-2 10 (humanized anti-HER-2 bispecific Renal and colon antibody) Medarex MDX- II (complement activating receptor Ex vivo bone marrow (CAR) monoclonal antibody) purging in acute myelogenous leukemia (AML) Medarex MDX-22 (humanized bispecific antibody, Acute myleoid leukemia MAb-conjugates) (complement cascade activators) Cytogen OV103 (Yttrium-90 labeUed antibody) Ovarian Cytogen OV103 (Yttrium-90 labelled antibody) Prostate Aronex Pharmaceuticals, Inc. ATRAGEN@ non-Hodgkin's lymphoma Glaxo Wellcome plc 3622W94 MAb that binds to EGP40 (17-A) non-small cell lung, pancarcinoma antigen on adenocarcinomas prostate (adjuvant) Genentech Anti-VEGF, RhuMAb (inhibits Lung, breast, prostate, angiogenesis) colorectal Protein Design Labs Zenapax (SMART Anti-Tac (IL-2 receptor) Leukemia, lymphoma Ab, humanized) Protein Design Labs SMART M195 Ab, humanized Acute promyelocytic leukemia WO 01/97843 PCT/USO1/20154 -31 Marketer Brand Name (Generic Name) Indication ImClone Systems C225 (chimeric anti-EGFr monoclonal Breast antibody ) + taxol ImClone Systems (licensed from C225 (chimeric anti-EGFr monoclonal prostate RPR) antibody ) + doxorubicin ImClone Systems C225 (chimeric anti-EGFr monoclonal prostate antibody ) + adriamycin ImClone Systems BEC2 (anti-idiotypic MAb, mimics the GD 3 Melanoma epitope) Medarex MDX-210 (humanized anti-HER-2 bispecific Cancer antibody) Medarex MDX-220 (bispecific for tumors that express Lung, colon, prostate, TAG-72) ovarian, endometrial, pancreatic and gastric Medarex/Novartis MDX-210 (humanized anti-HER-2 bispecific Prostate antibody) Medarex/Merck KgaA MDX-447 (humanized anti-EGF receptor EGF receptor cancers bispecific antibody) (head & neck, prostate, lung, bladder, cervical, ovarian) Medarex/Novartis MDX-2 10 (humanized anti-HER-2 bispecific Comb. Therapy with G antibody) CSF for various cancers, esp. breast IDEC MELIMMUNE-2 (murine monoclonal Melanoma antibody therapeutic vaccine) IDEC MELIMMUNE-l (murine monoclonal Melanoma antibody therapeutic vaccine) Immunomedics, Inc. CEACIDETM (1-131) Colorectal and other NeoRx PretargetTM radioactive antibodies non-Hodgkin's B cell lymphoma Novopharm Biotech, Inc. NovoMAb-G2 (pancarcinoma specific Ab) Cancer Techniclone Corporation/ TNT (chimeric MAb to histone antigens) Brain Cambridge Antibody Technology Techniclone TNT (chimeric MAb to histone antigens) Brain International/Cambridge Antibody Technology Novopharm Gliomab-H (Monoclonals - Humanized Abs) Brain, melanomas, neuroblastomas Genetics Institute/AHP GNI-250 Mab Colorectal Merck KgaA EMD-72000 (chimeric-EGF antagonist) Cancer Immunomedics LymphoCide (humanized LL2 antibody) non-Hodgkin's B-cell lymphoma Immunex/AHP CMA 676 (monoclonal antibody conjugate) Acute myelogenous leukemia Novopharm Biotech, Inc. Monopharm-C Colon, lung, pancreatic Novopharm Biotech, Inc. 4B5 anti-idiotype Ab Melanoma, small-cell lung WO 01/97843 PCT/US01/20154 -32 Marketer Brand Name (Generic Name) Indication Center of Molecular Immunology ior egf/r3 (anti EGF-R humanized Ab) Radioimmunotherapy Center of Molecular Immunology ior c5 (murine MAb colorectal) for Colorectal radioimmunotherapy Creative BioMolecules/ BABS (biosynthetic antibody binding site) Breast cancer Chiron Proteins ImClone Systems/Chugai FLK-2 (monoclonal antibody to fetal liver Tumor-associated kinase-2 (FLK-2)) angiogenesis ImmunoGen, Inc. Humanized MAb/small-drug conjugate Small-cell lung Medarex, Inc. MDX-260 bispecific, targets GD-2 Melanoma, glioma, neuroblastoma Procyon Biopharma, Inc. ANA Ab Cancer Protein Design Labs SMART IDI0 Ab B-cell lymphoma Protein Design Labs/Novartis SMART ABL 364 Ab Breast, lung, colon Immunomedics, Inc. ImmuRAIT-CEA Colorectal In some embodiments the nucleic acid and antibody are administered in combination with a cancer therapy. As used herein, a "cancer therapy" refers to an agent which prevents growth of a cancer cell by decreasing or slowing the rate of growth, by inhibiting growth 5 altogether, or by killing or inducing apoptosis of the cancer cell. Thus, as used herein, "treating cancer" includes preventing the development of a cancer, reducing the symptoms of cancer, and/or inhibiting the growth of an established cancer. In other aspects, the cancer therapy is administered to a subject at risk of developing a cancer for the purpose of reducing the risk of developing the cancer. Various types of medicaments for the treatment of cancer lo are described herein. For the purpose of this specification, cancer therapies are classified as chemotherapeutic agents, cancer vaccines, hormone therapy, biological response modifiers, surgical procedures, and radiotherapy aimed at treating cancer. Additionally, the methods of the invention are intended to embrace the use of more than one cancer therapy along with the immunostimulatory nucleic acids and antibody. As an example, where appropriate, the i5 immunostimulatory nucleic acids may be administered with a both a chemotherapeutic agent and a radiotherapy. Cancer therapies function in a variety of ways. Some cancer therapies work by targeting physiological mechanisms that are specific to tumor cells. Examples include the targeting of specific genes and their gene products (i.e., proteins primarily) which are mutated 20 in cancers. Such genes include but are not limited to oncogenes (e.g., Ras, Her2, bcl-2), tumor suppressor genes (e.g., EGF, p53, Rb), and cell cycle targets (e.g., CDK4, p21, WO 01/97843 PCT/USOI/20154 -33 telomerase). Cancer therapies can alternately target signal transduction pathways and molecular mechanisms which are altered in cancer cells. Other cancer therapies target cells other than cancer cells. For example, some medicaments prime the immune system to attack tumor cells (i.e., cancer vaccines). Still 5 other medicaments, called angiogenesis inhibitors, function by attacking the blood supply of solid tumors. Since the most malignant cancers are able to metastasize (i.e., exit the primary tumor site and seed a distal tissue, thereby forming a secondary tumor), medicaments that impede this metastasis are also useful in the treatment of cancer. Angiogenic mediators include basic FGF, VEGF, angiopoietins, angiostatin, endostatin, TNF-a, TNP-470, 10 thrombospondin-1, platelet factor 4, CAI, and certain members of the integrin family of proteins. One category of this type of medicament is a metalloproteinase inhibitor, which inhibits the enzymes used by the cancer cells to exit the primary tumor site and extravasate into another tissue. As used herein, chemotherapeutic agents encompass both chemical and biological 15 agents. These agents function to inhibit a cellular activity which the cancer cell is dependent upon for continued survival. Categories of chemotherapeutic agents include alkylating/alkaloid agents, antimetabolites, hormones or hormone analogs, and miscellaneous antineoplastic drugs. Most if not all of these agents are directly toxic to cancer cells and do not require immune stimulation. Chemotherapeutic agents which are currently in 20 development or in use in a clinical setting are shown in Table 3 below. Table 3: Cancer Drugs In Development Or On The Market. Marketer Brand Name Generic Name Indication Abbott TNP 470/AGM 1470 Fragyline Anti-Angiogenesis in Cancer Takeda TNP 470/AGM 1470 1 Fragyline Anti-Angiogenesis in Cancer Scotia Meglamine GIA Meglamine GLA Bladder Cancer Medeva Valstar Valrubicin Bladder Cancer -Refractory in situ carcinoma Medeva Valstar Valrubicin Bladder Cancer - Papillary Cancer Rhone Poulenc Gliadel Wafer Carmustaine + Polifepr Osan Brain Tumor Warner Lambert Undisclosed Cancer (b) Undisclosed Cancer (1,) Cancer Bristol-Myers RAS Famesyl Transferase RAS FamesylTransferase Cancer Squibb Inhibitor Inhibitor Novartis MMI 270 MMI 270 Cancer Bayer BAY 12-9566 BAY 12-9566 Cancer Merck Famesyl Transferase Inhibitor Famesyl Transferase Cancer (Solid tumors Inhibitor pancreas, colo lung, breast) Pfizer PFE MMAP Cancer, angiogenesis WO 01/97843 PCT/USOI/20154 -34 Marketer Brand Name Generic Name Indication Pfizer PFE Tyrosine Kinase Cancer, angiogenesis Lilly MTAILY 231514 MTA/LY 231514 Cancer Solid Tumors Lilly LY 264618/Lometexol Lometexol Cancer Solid Tumors Scotia Glamolec LiGLA (lithium-gamma Cancer, pancreatic, breast, linolenate) colon Warner Lambert CI-994 CI-994 Cancer, Solid Tumors I Leukemia Schering AG Angiogenesis inhibitor Angiogenesis Inhibitor Cancer / Cardio Takeda TNP-470 n/k Malignant Tumor Smithkline lycamtin Topotecan Metastatic Ovarian Cancer Beecham Novartis PKC 412 PKC 412 Multi-Drug Resistant Cancer Novartis Valspodar PSC 833 Myeloid Leukemia/Ovarian Cancer Immune Novantrone Mitoxantrone Pain related to hormone refractory prostate cancer. Warner Lambert Metaret Suramin Prostate Genentech Anti-VEGF Anti-VEGF Prostate / Breast / Colorectal / NSCL Cancer British Biotech Batimastat Batimastat (BB94) Pterygium Eisai E 7070 E 7070 Solid Tumors Biochem BCH-4556 BCH-4556 Solid Tumors Pharma Sankyo CS-682 CS-682 Solid Tumors Agouron AG2037 AG2037 Solid Tumors ]DEC Pharma 9-AC 9-AC Solid Tumors Agouron VEGF/b-FGF Inhibitors VEGF/b-FGF Inhibitors Solid Tumors Agouron AG3340 AG3340 Solid Tumors / Macular Degeneration Vertex Incel VX-710 Solid Tumors -IV Vertex VX-853 VX-853 Solid Tumors -Oral Zeneca ZD 0101 (inj) ZD 0101 Solid Tumors Novartis IS1 641 IS1 641 Solid Tumors Novartis ODN 698 ODN 698 Solid Tumors Tanube Seiyaku TA 2516 Marimistat Solid Tumors British Biotech Marimastat Marimastat (BB 2516) Solid Tumors Celltech CDP 845 Aggrecanase Inhibitor Solid Tumors / Breast Cancer Chiroscience D2163 D2163 Solid Tumors / Metastases Warner Lambert PD 183805 PD 183805 Daiichi DX8951f DX8951f Anti-Cancer Daiichi Lemonal DP 2202 Lemonal DP 2202 Anti-Cancer Fujisawa FK 317 FK 317 Anticancer Antibiotic Chugai Picibanil OK-432 Antimalignant Tumor Nycomed AD 32/valrubicin Valrubicin Bladder Cancer-Refractory Arnersham In situ Carcinoma Nycomed Metastron Strontium Derivative Bone Cancer (adjunct Amershan therapy, Pain) Schering Plough Temodal Temozolomide Brain Tumors Schering Plough _ Temodal Tenozolonide Brain Tumors Liposome Evacet Doxorubicin, Liposonmal Breast Cancer Nycomed Yewtaxan Paclitaxel Breast Cancer Advanced, Amersham- Ovarian Cancer Advanced WO 01/97843 PCT/USOl/20154 -35 Marketer Brand Name Generic Name Indication Bristol-Myers Taxol Paclitaxel Breast Cancer Advanced, Squibb Ovarian Cancer Advanced, NSCLC Roche Xeloda Capecitabine Breast Cancer, Colorectal Cancer Roche Furtulon Doxifluridine Breast Cancer, Colorectal Cancer, Gastric Cancer Pharmacia & Adriamycin Doxorubicin Breast Cancer, Leukemia Upjohn Ivax Cyclopax Paclitaxel, Oral Breast/Ovarian Cancer Rhone Poulenc Oral Taxoid Oral Taxoid Broad Cancer AHP Novantrone Mitoxantrone Cancer Sequus SPI-077 Cisplatin, Stealth Cancer Hoechst HMR 1275 Flavopiridol Cancer Pfizer CP-358, 774 EGFR Cancer Pfizer CP-609, 754 RAS Oncogene Inhibitor Cancer Bristol-Myers BMS-182751 Oral Platinum Cancer (Lung, Ovarian) Squibb Bristol-Myers UFT (Tegafur/Uracil) UFT (Tegafur/Uracil) Cancer Oral Squibb Johnson & Ergamisol Levamisole Cancer Therapy Johnson Glaxo Wellcome Eniluracil/776C85 5FU Enhancer Cancer, Refractory Solid & Colorectal Cancer Johnson & Ergamisol Levamisole Colon Cancer Johnson Rhone Poulenc Campto Irinotecan Colorectal Cancer, Cervical Cancer Pharmacia & Camptosar Irinotecan Colorectal Cancer, Cervical Upjohn Cancer Zeneca Tomudex Ralitrexed Colorectal Cancer, Lung Cancer, Breast Cancer Johnson & Leustain Cladribine Hairy Cell Leukaemia Johnson Ivax Paxene Pachtaxel Kaposi Sarcoma Sequus Doxil Doxorubicin, Liposomal KS/Cancer Sequus Caelyx Doxorubicin, Liposomal KS/Cancer Schering AG Fludara Fludarabine Leukaemia Pharmacia & Pharmorubicin Epirubicin Lung/Breast Cancer Upjohn Chiron DepoCyt DepoCyt Neoplastic Meningitis Zeneca ZD1839 ZD 1839 Non Small Cell Lung Cancer, Pancreatic Cancer BASF LU 79553 Bis-Naphtalimide Oncology BASF LU 103793 Dolastain Oncology Schering Plough Caetyx Doxorubicin-Liposome Ovarian/Breast Cancer Lilly Germzar Gemcitabine Pancreatic Cancer, Non Small Cell Lung Cancer, Breast, Bladder and Ovarian Zeneca ZD 0473/Anormed ZD 0473/Anormed Platinum based NSCL, ovarian etc. Yarnanouchi YM 116 YM 116 Prostate Cancer Nycomed Sceds/I- 125 Rapid St Iodine Seeds Prostate Cancer Amersham_ A gouron Cdk4/cdk2 inhibitors cdk4/cdk2 inhibitors Solid Tumors WO 01/97843 PCTUSO1/20154 -36 Marketer Brand Name Generic Name Indication Agouron PARP inhibitors PARP Inhibitors Solid Tumors Chiroscience D4809 Dexifosamide Solid Tumors Bristol-Myers UFT (Tegafur/Uracil) UFT (Tegafur/Uracil) Solid Tumors Squibb Sankyo Krestin Krestin Solid Tumors Asta Medica Ifex/Mesnex Ifosamide Solid Tumors Bristol-Myers Ifex/Mesnex Ifosamide Solid Tumors Squibb Bristol-Myers Vumon Teniposide Solid Tumors Squibb Bristol-Myers Paraplatin Carboplatin Solid Tumors Squibb Bristol-Myers Plantinol Cisplatin, Stealth Solid Tumors Squibb Bristol-Myers Plantinol Cisplatin Solid Tumors Squibb Bristol-Myers Vepeside Etoposide Solid Tumors Melanoma Squibb Zeneca ZD 9331 ZD 9331 Solid Tumors, Advanced Colorectal Chugai Taxotere Docetaxel Solid Tumors, Breast Cancer Rhone Poulenc Taxotere Docetaxel Solid Tumors, Breast Cancer Glaxo Wellcome Prodrug of guanine arabinside prodrug of arabinside T Cell Leukemia/Lymphoma & B Cell Neoplasm Bristol-Myers Taxane Analog Taxane Analog Taxol follow up Squibb Another useful anti-cancer therapy is Interferon-a (e.g., INTRON@ A, Schering). The compounds useful according to the invention are nucleic acids. The nucleic acids may be double-stranded or single-stranded. Generally, double-stranded molecules may be 5 more stable in vivo, while single-stranded molecules may have increased activity. The terms "nucleic acid" and "oligonucleotide" refer to multiple nucleotides (i.e., molecules comprising a sugar (e.g., ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g., cytosine (C), thymine (T) or uracil (U)) or a substituted purine (e.g., adenine (A) or guanine (G)) or a modified base. As 10 used herein, the terms refer to oligoribonucleotides as well as oligodeoxyribonucleotides. The terms shall also include polynucleosides (i.e., a polynucleotide minus the phosphate) and any other organic base-containing polymer. The terms "nucleic acid" and "oligonucleotide" also encompass nucleic acids or oligonucleotides with a covalently modified base and/or sugar. For example, they include nucleic acids having backbone sugars which are covalently 15 attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position. Thus modified nucleic acids may include a 2'-O-alkylated ribose group. In addition, modified nucleic acids may include WO 01/97843 PCT/USO1/20154 -37 sugars such as arabinose instead of ribose. Thus the nucleic acids may be heterogeneous in backbone composition thereby containing any possible combination of polymer units linked together such as peptide-nucleic acids (which have amino acid backbone with nucleic acid bases). In some embodiments the nucleic acids are homogeneous in backbone composition. 5 Nucleic acids also can include base analogs such as C-5 propyne modified bases. Wagner RW et al., Nature Biotechnol 14:840-4 (1996). Purines and pyrimidines include but are not limited to adenine, cytosine, guanine, thymine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-dianinopurine, hypoxanthine, and other naturally and non-naturally occurring nucleobases, substituted and unsubstituted aromatic moieties. 10 The nucleic acid is a linked polymer of bases or nucleotides. As used herein with respect to linked units of a nucleic acid, "linked" or "linkage" means two entities are bound to one another by any physicochemical means. Any linkage known to those of ordinary skill in the art, covalent or non-covalent, is embraced. Such linkages are well known to those of ordinary skill in the art. Natural linkages, which are those ordinarily found in nature 15 connecting the individual units of a nucleic acid , are most common. The individual units of a nucleic acid may be linked, however, by synthetic or modified linkages. Whenever a nucleic acid is represented by a sequence of letters it will be understood that the nucleotides are in 5' - 3' order from left to right and that "A" denotes adenosine, "C" denotes cytosine, "G" denotes guanosine, "T" denotes thynidine, and "U" denotes uracil 20 unless otherwise noted. Nucleic acid molecules useful according to the invention can be obtained from natural nucleic acid sources (e.g., genomic nuclear or mitochondrial DNA or cDNA), or are synthetic (e.g., produced by oligonucleotide synthesis). Nucleic acids isolated from existing nucleic acid sources are referred to herein as native, natural, or isolated nucleic acids. The nucleic 25 acids useful according to the invention may be isolated from any source, including eukaryotic sources, prokaryotic sources, nuclear DNA, mitochondrial DNA, etc. Thus, the term nucleic acid encompasses both synthetic and isolated nucleic acids. The term "isolated" as used herein refers to a nuclcic acid which is substantially free of other nucleic acids, proteins, lipids, carbohydrates or other materials with which it is naturally associated. The nucleic 30 acids can be produced on a large scale in plasmids, (see Sambrook T et al., "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press, New York, 1989) and separated into smaller pieces or administered whole. After being administered to a WO 01/97843 PCT/US01/20154 -38 subject the plasmid can be degraded into oligonucleotides. One skilled in the art can purify viral, bacterial, eukaryotic, etc., nucleic acids using standard techniques, such as those employing restriction enzymes, exonucleases or endonucleases. For use in the instant invention, the nucleic acids can be synthesized de novo using 5 any of a number of procedures well known in the art. For example, the b-cyanoethyl phosphoramidite method (Beaucage SL et al., Tetrahedron Lett 22:1859, 1981); nucleoside H-phosphonate method (Garegg et al., Tetrahedron Lett 27:4051-4, 1986; Froehler et al., Nucl Acid Res 14:5399-407, 1986; Garegg et al., Tetrahedron Lett 27:4055-8, 1986; Gaffney et al., Tetrahedron Lett 29:2619-22, 1988). These chemistries can be performed by a variety 10 of automated oligonucleotide synthesizers available in the market. In some embodiments, the nucleic acids useful according to the invention are immunostimulatory nucleic acids. An immunostimulatory nucleic acid is any nucleic acid, as described above, which is capable of modulating an immune response. A nucleic acid which modulates an immune response is one which produces any form of immune stimulation, 15 including, but not limited to, induction of cytokines, B-cell activation, T-cell activation, monocyte activation. Immunostimulatory nucleic acids include, but are not limited to, CpG nucleic acids, methylated CpG nucleic acids, T-rich nucleic acids, poly-G nucleic acids, and nucleic acids having phosphate modified backbones, such as phosphorothioate backbones. A "CpG nucleic acid" or a "CpG immunostimulatory nucleic acid" as used herein is a 20 nucleic acid containing at least one unmethylated CpG dinucleotide (cytosine-guanine dinucleotide sequence, i.e., "CpG DNA" or DNA containing a 5' cytosine followed by 3' guanosine and linked by a phosphate bond) and activates a component of the immune system. The entire CpG nucleic acid can be unmethylated or portions may be unmethylated but at least the C of the 5' CG 3' must be unmethylated. 25 In one embodiment the invention provides a CpG nucleic acid represented by at least the formula: 5' NiXICGX 2
N
2 3' wherein X, and X 2 are nucleotides and N is any nucleotide and Ni and N 2 are nucleic acid sequences composed of from about 0-25 N's each. In some embodiments Xi is adenine, 30 guanine, or thymine and X 2 is cytosine, adenine, or thymine. In other embodiments Xi is cytosine and/or X 2 is guanine. In other embodiments the CpG nucleic acid is represented by at least the formula: WO 01/97843 PCT/USO1/20154 -39 5' NiX 1
X
2
CGX
3
X
4
N
2 3' wherein X 1 , X 2 , X 3 , and X 4 are nucleotides. In some embodiments, XIX2 are nucleotides selected from the group consisting of: GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT, and TpG; and X 3
X
4 are nucleotides selected from the group consisting of: TpT, 5 CpT, ApT, TpG, ApG, CpG, TpC, ApC, CpC, TpA, ApA, and CpA; N is any nucleotide and Ni and N 2 are nucleic acid sequences composed of from about 0-25 N's each. In some embodiments, X 1
X
2 are GpA or GpT and X 3
X
4 are TpT. In other embodiments X1 or X 2 or both are purines and X 3 or X 4 or both are pyrimidines or X 1
X
2 are GpA and X3 or X 4 or both are pyrimidines. 10 In some embodiments Ni and N 2 of the nucleic acid do not contain a CCGG or CGCG quadmer or more than one CCG or CGG trimer. The effect of a CCGG or CGCG quadmer or more than one CCG or CGG trimer depends in part on the status of the nucleic acid backbone. For instance, if the nucleic acid has a phosphodiester backbone or a chimeric backbone the inclusion of these sequences in the nucleic acid will only have minimal if any 15 affect on the biological activity of the nucleic acid. If the backbone is completely phosphorothioate or significantly phosphorothioate then the inclusion of these sequences may have more influence on the biological activity or the kinetics of the biological activity, but compounds containing these sequences are still useful. In another embodiment the CpG nucleic acid has the sequence 5' TCNITXIX 2
CGX
3
X
4 3'. 20 A "T-rich nucleic acid" or "T-rich innunostimulatory nucleic acid" is a nucleic acid which includes at least one poly-T sequence and/or which has a nucleotide composition of greater than 25% T nucleotide residues and which activates a component of the immune system. A nucleic acid having a poly-T sequence includes at least four Ts in a row, such as 5' TTTT 3'. Preferably the T-rich nucleic acid includes more than one poly-T sequence. In 25 preferred embodiments the T-rich nucleic acid may have 2, 3, 4, etc., poly-T sequences, such as oligonucleotide #2006 (5' TCGTCGT'TTGTCGTTTTGTCGTT 3', SEQ ID NO: 729). One of the most highly immunostimulatory T-rich oligonucleotides discovered according to the invention is a nucleic acid composed entirely of T nucleotide residues, e.g., oligonucleotide #2183 (5'TTTTTTITTTI1fiTTTT 3', SEQ ID NO: 841). Other 30 T-rich nucleic acids have a nucleotide composition of greater than 25% T nucleotide residues, but do not necessarily include a poly-T sequence. In these T-rich nucleic acids the T nucleotide resides may be separated from one another by other types of nucleotide residues, WO 01/97843 PCT/US01/20154 -40 i.e., G, C, and A. In some embodiments the T-rich nucleic acids have a nucleotide composition of greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 99%, T nucleotide residues and every integer % in between. Preferably the T-rich nucleic acids have at least one poly-T sequence and a nucleotide composition of greater than 25% T nucleotide residues. In one embodiment the T-rich nucleic acid is represented by at least the formula: 5' XIX 2 TTTTX3X 4 3' wherein X 1 , X 2 , X3, and X 4 are nucleotides. In one embodiment X 1
X
2 is TT and/or X 3
X
4 is IT. In another embodiment XiX 2 are any one of the following nucleotides TA, TG, TC, AT, AA, AG, AC, CT, CC, CA, CG, GT, GG, GA, and GC; and X 3
X
4 are any one of the 10 following nucleotides TA, TG, TC, AT, AA, AG, AC, CT, CC, CA, CG, GT, GG, GA, and GC. In some embodiments it is preferred that the T-rich nucleic acid does not contain poly-C (CCCC), poly-A (AAAA), poly-G (GGGG), CpG motifs, or multiple GGs. In other embodiments the T-rich nucleic acid includes these motifs. Thus in some embodiments of 15 the invention the T-rich nucleic acids include CpG dinucleotides and in other embodiments the T-rich nucleic acids are free of CpG dinucleotides. The CpG dinucleotides may be methylated or unmethylated. Poly-G containing nucleic acids are also immunostimulatory. A variety of references, including Pisetsky DS et al., Mol Biol Rep 18:217-21 (1993); Krieger M et al., Annu Rev 20 Biochem 63:601-37 (1994); Macaya RF et al., Proc Nall Acad Sci USA 90:3745-9 (1993); Wyatt JR et al., Proc Natl Acad Sci USA 91:1356-60 (1994); Rando and Hogan, 1998, In: Applied Antisense Oligonucleotide Technology, eds. Krieg AM and Stein C, pp. 335-352; and Kimura Y et al., JBiochem (Tokyo) 116:991-4 (1994) also describe the immunostimulatory properties of poly-G nucleic acids. 25 Poly G nucleic acids preferably are nucleic acids having the following formulas: 5' XIX 2
GGGX
3
X
4 3' wherein Xi, X 2 , X 3 , and X 4 are nucleotides. In preferred embodiments at least one of X 3 and
X
4 are a G. In other embodiments both of X 3 and X 4 are a G. In yct other embodiments the preferred formula is 5' GGGNGGG 3', or 5' GGGNGGGNGGG 3' wherein N represents 30 between 0 and 20 nucleotides. In other embodiments the poly-G nucleic acid is free of unmethylated CG dinucleotides, such as, for example, the nucleic acids listed in Table 4 below as SEQ .ID NOs: 12-14, 23, 56, 100, 155, 163, 182, 227, 237, 246, 400, 407, 429, 430, WO 01/97843 PCT/US01/20154 -41 432, 435, 438, 439, 446,450, 451, 480, 487, 493, 522, 661, 662, 671-673, 807, 808, 821, 823, and 834. In other embodiments the poly-G nucleic acid includes at least one unmethylated CG dinucleotide, such as, for example, the nucleic acids listed in Table 4 below as SEQ ID NOs: 6, 7, 22, 26, 28-30, 87, 115, 141, 177, 191, 209, 254, 258, 267, 303, 317, 329, 335, 344, 5 345, 395, 414, 417, 418,423-426, 428, 431,433, 434, 436, 437, 440, 442-445, 447-449, 458, 460, 463, 467-469, 474, 515, 516, 594, 638-640, 663, 664, 727, 752, 776, 795, 799, 817, 818, 831, and 832. Nucleic acids having modified backbones, such as phosphorothioate backbones, also fall within the class of immunostimulatory nucleic acids. U.S. Patents Nos. 5,723,335 and 10 5,663,153 issued to Hutcherson, et al. and related PCT publication W095/26204 describe immune stimulation using phosphorothioate oligonucleotide analogues. These patents describe the ability of the phosphorothioate backbone to stimulate an immune response in a non-sequence specific manner. The immunostimulatory nucleic acids may be any size but in some embodiments are 15 in the range of between 6 and 100 or in some embodiments between 8 and 35 nucleotides in size. Immunostimulatory nucleic acids can be produced on a large scale in plasmids. These may be administered in plasmid form or alternatively they can be degraded into oligonucleotides. "Palindromic sequence" shall mean an inverted repeat (i.e., a sequence such as 20 ABCDEE'D'C'B'A' in which A and A' are bases capable of forming the usual Watson-Crick base pairs and which includes at least 6 nucleotides in the palindrome. In vivo, such sequences may form double-stranded structures. In one embodiment the nucleic acid contains a palindromic sequence. In some embodiments when the nucleic acid is a CpG nucleic acid, a palindromic sequence used in this context refers to a palindrome in which the 25 CpG is part of the palindrome, and optionally is the center of the palindrome. In another embodiment the nucleic acid is free of a palindrome. A nucleic acid that is free of a palindrome does not have any regions of 6 nucleotides or greater in length which are palindromic. A nucleic acid that is free of a palindrome can include a region of less than 6 nucleotides which are palindromic. 30 A "stabilized nucleic acid molecule" shall mean a nucleic acid molecule that is relatively resistant to in vivo degradation (e.g., via an exonuclease or endonuclease). Stabilization can be a function of length or secondary structure. Nucleic acids that are tens to WO 01/97843 PCT/US01/20154 -42 hundreds of kbs long are relatively resistant to in vivo degradation. For shorter nucleic acids, secondary structure can stabilize and increase their effect. For example, if the 3' end of an oligonucleotide has self-complementarity to an upstream region, so that it can fold back and form a sort of stem loop structure, then the oligonucleotide becomes stabilized and therefore 5 exhibits more activity. Some stabilized oligonucleotides of the instant invention have a modified backbone. It has been demonstrated that modification of the oligonucleotide backbone provides enhanced activity of the nucleic acids when administered in vivo. Nucleic acids, including at least two phosphorothioate linkages at the 5' end of the oligonucleotide and multiple 10 phosphorothioate linkages at the 3' end, preferably 5, may provide maximal activity and protect the oligonucleotide from degradation by intracellular exo- and endo-nucleases. Other modified oligonucleotides include phosphodiester modified oligonucleotide, combinations of phosphodiester and phosphorothioate oligonucleotide, methylphosphonate, methylphosphorothioate, phosphorodithioate, and combinations thereof. Each of these 15 combinations and their particular effects on immune cells is discussed in more detail in PCT Published Patent Application W098/18810 claiming priority to U.S. Serial Nos. 08/738,652 (now issued as U.S. Patent No. 6,207,646 B1) and 08/960,774 (now issued as U.S. Patent No. 6,239,116 B1), filed on October 30, 1996 and October 30, 1997 respectively, the entire contents of which is hereby incorporated by reference. It is believed that these modified 20 oligonucleotides may show more stimulatory activity due to enhanced nuclease resistance, increased cellular uptake, increased protein binding, and/or altered intracellular localization. Both phosphorothioate and phosphodiester nucleic acids are active in immune cells. Other stabilized oligonucleotides include: nonionic DNA analogs, such as alkyl- and aryl-phosphates (in which the charged phosphonate oxygen is replaced by an alkyl or aryl 25 group), phosphodiester and alkylphosphotriesters, in which the charged oxygen moiety is alkylated. Oligonucleotides which contain diol, such as tetraethyleneglycol or hexaethyleneglycol, at either or both termini have also been shown to be substantially resistant to nuclease degradation. For use in vivo, nucleic acids are preferably relatively resistant to degradation (e.g., 30 via endonucleases and exonucleases). Secondary structures, such as stem loops, can stabilize nucleic acids against degradation. Alternatively, nucleic acid stabilization can be accomplished via phosphate backbone modifications. One type of stabilized nucleic acid has WO 01/97843 PCT/USO1/20154 -43 at least a partial phosphorothioate modified backbone. Phosphorothioates may be synthesized using automated techniques employing either phosphoramidate or H-phosphonate chemistries. Aryl- and alkyl-phosphonates can be made, e.g., as described in U.S. Patent No. 4,469,863; and alkylphosphotriesters (in which the charged oxygen moiety is 5 alkylated as described in U.S. Patent No. 5,023,243 and European Patent No. 092,574) can be prepared by automated solid phase synthesis using commercially available reagents. Methods for making other DNA backbone modifications and substitutions have been described. Uhlmann E et al., Chem Rev 90:544-84 (1990); Goodchild J, Bioconjugate Chem 1:165-87 (1990). 10 The immunostimulatory nucleic acids having backbone modifications useful according to the invention in some embodiments are S- or R-chiral immunostimulatory nucleic acids. An "S chiral immunostimulatory nucleic acid" as used herein is an immunostimulatory nucleic acid wherein at least two nucleotides have a backbone modification forming a chiral center and wherein a plurality of the chiral centers have S 15 chirality. An "R chiral immunostimulatory nucleic acid" as used herein is an immunostimulatory nucleic acid wherein at least two nucleotides have a backbone modification forming a chiral center and wherein a plurality of the chiral centers have R chirality. The backbone modification may be any type of modification that forms a chiral center. The modifications include but are not limited to phosphorothioate, 20 methylphosphonate, methylphosphorothioate, phosphorodithioate, 2'-OMe and combinations thereof. In other embodiments they are non-chiral. A non-chiral nucleic acid is any nucleic acid which does not have at least two chiral centers. The chiral immunostimulatory nucleic acids must have at least two nucleotides within the nucleic acid that have a backbone modification. All or less than all of the nucleotides in 25 the nucleic acid, however, may have a modified backbone. Of the nucleotides having a modified backbone (referred to as chiral centers), a plurality have a single chirality, S or R. A "plurality" as used herein refers to an amount greater than or equal to 75%. Thus, less than all of the chiral centers may have S or R chirality as long as a plurality of the chiral centers have S or R chirality. In some embodiments at least 75%, 80%, 85%, 90%, 95%, or 100% of 30 the chiral centers have S or R chirality. In other embodiments at least 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotides have backbone modifications.
WO 01/97843 PCT/USOI/20154 -44 The S- and R- chiral immunostimulatory nucleic acids may be prepared by any method known in the art for producing chirally pure oligonucleotides. Stec et al. teach methods for producing stereopure phosphorothioate oligodeoxynucleotides using an oxathiaphospholane. Stec WJ et al.,JAm Chem Soc 117:12019 (1995). Other methods for 5 making chirally pure oligonucleotides have been described by companies such as ISIS Pharmaceuticals. U.S. Patents which disclose methods for generating stereopure oligonucleotides include 5,212,295, 5,359,052, 5,506,212, 5,512,668, 5,521,302, 5,599,797, 5,837,856, 5,856,465, and 5,883,237, each of which is hereby incorporated by reference in its entirety. 10 Other sources of nucleic acids useful according to the invention include standard viral and bacterial vectors, many of which are commercially available. In its broadest sense, a "vector" is any nucleic acid material which is ordinarily used to deliver and facilitate the transfer of nucleic acids to cells. The vector as used herein may be an empty vector or a vector carrying a gene which can be expressed. In the case when the vector is carrying a 15 gene the vector generally transports the gene to the target cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In this case the vector optionally includes gene expression sequences to enhance expression of the gene in target cells such as immune cells, but it is not required that the gene be expressed in the cell. 20 In general, vectors include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources. Viral vectors are one type of vector and include, but are not limited to, nucleic acid sequences from the following viruses: retrovirus, such as Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, and Rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; 25 polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art. Some viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with a nucleic acid to be delivered. Non-cytopathic viruses include retroviruses, the life cycle of which involves reverse 30 transcription of genomic viral RNA into DNA. Standard protocols for producing empty vectors or vectors carrying genes (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a WO 01/97843 PCT/USO1/20154 -45 packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and/or infection of the target cells with viral particles) are provided in Kriegler M, "Gene Transfer and Expression, A Laboratory Manual," W.H. Freeman Co., New York (1990) and Murry EJ, Ed., "Methods in 5 Molecular Biology," vol. 7, Humana Press, Inc., Cliffton, New Jersey (1991). Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well-known to those of skill in the art. See e.g., Sambrook et al., "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been found to be particularly 10 advantageous for delivering genes to cells in vivo because of their inability to replicate within and integrate into a host genome. Some plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pcDNA3.1, pSV40, and pBlueScript. Other plasmids are well-known to those of ordinary skill in the art. 15 Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. It has recently been discovered that plasmids (empty or gene-carrying) can be delivered to the immune system using bacteria. Modified forms of bacteria such as Salmonella can be transfected with the plasmid and used as delivery vehicles. The bacterial 20 delivery vehicles can be administcred to a host subject orally or by other administration means. The bacteria deliver the plasmid to immune cells, e.g., dendritic cells, probably by passing through the gut barrier. High levels of immune protection have been established using this methodology. Such methods of delivery are useful for the aspects of the invention utilizing systemic delivery of nucleic acid. 25 As used herein, administration of an immunostimulatory nucleic acid is intended to embrace the administration of one or more immunostimulatory nucleic acids which may or may not differ in terms of their profile, sequence, backbone modifications and biological effect. As an example, CpG nucleic acids and T-rich nucleic acids may be administered to a single subject along with an antibody and optionally a cancer therapy. In another example, a 30 plurality of CpG nucleic acids which differ in nucleotide sequence may also be administered to a subject.
WO 01/97843 PCT/USO1/20154 - 46 Some of the nucleic acids useful according to the invention and described herein are presented in Table 4 below. Table 4: Exemplary Nucleic Acids. SEQUENCE BACKBONE SE MNO aaaaaa 8 1 aaaaaaaaaaaaaaaaaaaa 0 2 aaaaaccccccccccaaaaa 0 3 aaaacatgacgttcaaa~aa a o0 4 aaaacatgacgttcaaaaaa s2 5 aaaacatgacgttc ggg 808 6 aaaacatgacgttcgqqggg s2 7 aaaacgtt 0 8 aaaatcaacgttgaaaaaaa BOB 9 aaaatctqtgcttttaaaaaa BOB 10 aaaattgacgttttaaaaaa SOS 11 aaacattctgggggaattttaagaagtaaacat 0 12 aaacattctgggggaattttaagaagttcCcCttcccc 0 13 aaacattctgggggaattttgtctagtaaacat 0 14 aacgctcgaccttcqat 0 15 Oagtgctca 16 aagtgc caqcSOS 17 Oagt 18 oagtc 19 aagt 20 Oagtg 21 Oaqtaggqa 22 aaggtggggcagtctcaggga 23 aatagtcgccataacaaaac 0 24 aatagtcgccatccccccc 0 25 aatagtcgccatcccgggac 0 26 aataqtcgccatcgcgcgac 0 27 aatagtcgccatggcgqggc 0 28 aattctctatcggggcttctgtgtctgttgctggttccgctttat 0 29 acaaccacqagaacgqgaac 30 acaacgtt 0 31 acaacgttga 0 32 accacaacgagaggaacgca 33 accatcctgaqqccattcgg 34 accatgqacgaactgtttcccctc a 35 accatg acgaCctgtttcccctc 8 36 accatqqacgagctgtttCCCCtC B 37 accatggacgaqctgtttcccctc 38 accatggacgatctgtttcccctc a 39 accatgqacggtctgtttcccctc S 40 accatggacqtactgtttcccctc 41 accatggacgttctgtttcccctc S 42 acccatcaatagctctqtgc 1a 43 acccgtcgtaattatagtaaaaccc ___44_ accgcatggattctaggcca ____45__ accttattaagattgtgcaatgtgacgtcctttagca cgcaaga 0 46 acgctggaccttccat 47______ aCgtc"gttCCCCCCCCCCCC 0 48 WO 01/97843 PCT/USO1/20154 - 47 SEQENC BACKBONE SEQ ID NO: acgtgt a 49 actagacgttagtgtga 0 50 actagacgttagtgtga 8 51 actggacgttagcgtga 0 52 acttctcatagtccctttggtccag 0 53 agaacgtt 0 54 agacagacacgaaacgaccg ________ 55 agactcatgggaaaatcccacatttga 0 56 agatagcaaatcggctgacg 0 57 agatggttctcagataaagcggaa ________ 58 agcaccgaacgtgagagg 0 59 agcacggtagccttccta ________ 60 agcagctttagagctttagagctt a 61 agcatcaggaacgacatgga 0 62 agcatcaggaccgacatgga 0 63 agcgctga 0 64 agctcaacgtcatgc 0 65 agctccatggtgctcactg s 66 aggatatc 0 67 aggtacagccagactac~a ________ 68 agicccgigaacgiattcac 0 69 agtgactctccagcgttctc 0 70 agtgcgattcgagatcg 0 71_____ agtgcgattgcagatcg 0 72_____ agtgct a 73 agttgcaact 0 75_____ ataaagcgaaactagcagcagtttc 0 76 ataacgtt 0 77 ataatagagcttcaagcaag a 78 ataatccagcttgaaccaag a 79 ataatcgacgttcaagcaag 80 ataatcgacgttcccCcccc 8 81 ataatcgtcgtt caagcaag 8 82 ataatcgtgcgttcaagaaag a 83 atagacaaaaattccctccccggagCc 0 84 atatatatatatatatat a 85 atatctaatcaaaa-cattaacaaa 0 86 atcaggaacgtcatgggaagc 0 87 atc4_acctacgtgcgttctc 0 8e atcgacctacgtgcgttztc 0 89 atcgactcgagccgttctc 0 90 atcgactctcgagcqttctc 0 91 atcgactctcgagcgttctc Boa 92 atcgactctcgagtgttctc 0 93 atcgactctcgagzgttctc 0 94 atcgactctctcgagcgttctc 0 9s atcgacttcgagcglttctc 0 96 atcgatcgagcgttctc 0 97 atcgatgt 0 98 atcggaggactggcgcgccg 99 atctggtgagggcaagctatg s 100 atgacgttcctgacgtt s 0 atgcactctgcagcgttctc 0 102 atgcatgt 0 103 WO 01/97843 PCTIUSOI/20154 -48 SEQUENCE BACKBONE SEQ ID NO: atgcccctcaacgtt 0 104 atgctaaaggacgtc acattgca o 105 atggaaggtccacgttctc o 106 atggaaggtccagcgttct 0 107 atggaaggtccagcgttctc 0 108 atggaaggtccagtgttctc o 109 atggaaggtcgagcgttctc o 110 atggactctccagcgttctc o 111 atgtcctcggtcctgatgct o 112 atgtttactagacaaaattcccccagaatgttt o 113 atgtttacttcttaaaattcccccagaatgttt o 114 attca tcggggcgggcgag o 115 atzgacctacgtgcgttctc 0 116 atzgactctzgagzgttctc 0 117 batggaaggtccagcgttctc o [ ie bgagaacgctccagcactgat o 119 bgagaacgctcgaccttcgat 0 120 bgagaazgctccagcactgat o 121 bgagaazgctcgaccttcgat o 122 bgagcaagctggaccttccat 0 123 bgagcaagztggaccttccat 0 124 bgctagacgttagcgtga o 125 btcaacgtt o 126 btccatgacgttcctgatgct o 127 btccatgagcttcctgatget o 128 btccattccatgacgttcctgatgcttcca os 129 btccattccattctaggcctgagtcttccat os 130 btcgtcgttttgtcgttttgtcgttttttt os 131 btttttccatgtcgttcctgatgcttttt os 132 btttttcgtcgttcccccccccccc os 133 caaacgtt o 134 caacgtOt 135 caogagatgctaacaatgca 5 136 caatcaatctgaggagaccc 137 cacaccttggtcaatgtcacgt 0 138 caccaccttggtcaatgtcacqt o 139 catggt agcctttccta 140 eacgttgaggggcat S 141 cactgtccttcgtcga SOS 142 cagacacagaagcccgatagacg 143 cagattgtgcaatgtctcga 0 144 cataacataggaatatttactcctcgc o 145 cataggatctcgagctcggaaagtc ccctac 146 catgagctcatctggaggaagcgg o 147 catttccacgatttccca 0 148 cattttacgggcgggcgggc 149 ccaaatatcgaggtcaagcac 156 ccaacgtt a 151 ccacgtcgaccctcaggcga a 152 ccacgtggac ct ctagc 0 153 ccactcacatctgctgctccacaag o 154 ccagatgage tcatgggt tt etcc o 155 ccaggttaapaggaaatgacttcggg 0 156 ccaggttgtatagaggc 157 ccagtgctgatcaccgatatcctgttcggIcagtcg 158 WO 01/97843 PCT/USO1/20154 -49 SEQUENCE BACKBONE SEQ ID NO: ccatcgat 0 159 ccatgcat 0 160 ccatgctaacctctagc 0 161 ccatgtcggtcctgatgct 0 162 ccccaaagggatgagaagtt 0 163 cccccaaaaaaaaaaccccc 0 164 cccccc a 165 cccccccc a 166 cccccccccccc B 167 cccccccccccccccccccc a 168 ccccccCCcccccccc BOB 169 cccccccccccccccccccccccc a 170 ccccccccccccccccccCccCccCCc s 171 CccccccccccccccccccccccccCcccCcc B 172 ccccttgacgttttcccccc BoB 173 cccgaagtcatttcctcttaacctgg 0 174 ccgaacaggatatcggtgatcagcac 175 ccgcttcotccagatgagctcatg o 176 ccgcttcctccagatgagctcatgggtttctccaccaag 0 177 ccggccggccggccggccgg o 178 ccgtcgttcccccccccccc 0 179 cctacgttgtatgcgcccagct o 180 cctccaaatgaaagaccccc 181 cctctatacaacctgggac 182 ccttccatgtcggtcctgat sos 183 ccttcgat 0 184 cgaacgtt 0 185 cgacga o 186 cgacgt a 187 cgactctcgagcgttctc 0 1BB cgactgccgaacaggatatcggtgatcagcactgg 189 cgccgtcgcggcggttgg o 190 cgcctggggctggtctgg o 191 cgcgcgcgcgcgcgcgcgcg s 192 cgcgcgcgcgcgcgcgcgcg o 193 cgcgta B 194 cgctagaggttagcgtga 0 195 cgctggaccttccat 0 196 cgctggaccttccatgtcgg Boa 197 cggctgacgtcatcaa B 198 cgggcgactcagtctatcgg 199 cgggcttacggcggatgctg 200 cggtagccttccta 201 cgtaccttacggtga o 202 cgtacg B 203 cgtcga a 204 cgtcga o 205 cgtcgt B 206 cgtcgtcgt 0 207 cgtcgtcgtcgtcgtcgtcgt 8 208 cgtctatcgggcttctgtgtctg 209 cgttcg B 210 ctaacgtt 0 211 ctaatctttctaatttttttctaa 6 212 ctagataaagcggaaccagcaacagacacagaagccccgatagag o 213 WO 01/97843 PCT/USOI/20154 -50 SEQUENCE BACKBONE SEQ ID NO: ctagcgct 0 214 ctagcggctgacgtcataaagctagc s 215 ctagcggctgacgtcatcaagctag _ 216 ctagcggctgacgtcatcaatctag 0 217 ctagcggctgagctcataaagctagc 8 218 ctagcttgatgacgtcagccgctag 0 219 ctagcttgatgagctcagccgctag 0 220 ctagetttatgacgtcagcegetage 8 221 ctaggctgacgtcatcaagctagt o 222 ctagtgtgacgtcatcaagctag | 223 ctatcggaggactggcgcgcc 224 ctatcggaggactggcgcgccg 225 ctcaacgctggaccttccat o 226 ccatgggtttctccaccaag 0 227 ctccagctccaagaaaggacg 0 228 ctcgccccgccccgatcgaat o 229 ctctccaagctcacttacag 230 ctctctgtaggcccgcttgg s 231 ctcttgcgacctggaaggta 232 ctgacgtcat 0 233 ctgacgtg o 234 ctgattgctctctcgtga SOS 235 ctgattgctctctcgtga 0 236 ctgcagcctgggac 0 237 ctgcgttagcaatttaactgtg 0 238 ctgctgagactggag a 239 ctgctgctgctgctgctgctg S 240 ctggaccttccatgtc sos 241 ctggaccttccatgtcgg sos 242 ctggtctttctggtttttttctgg | 243 ctggtctttctggtttttttctgg o 244 etgtaagtgagettggagag 245 ctgtatgaaacaaattttcctctttgggca 0 246 Ctgtca 8 247 ctgtcaggaactgcaggtaagg o 248 ctgtcccatatttttagaca 249 ctgtcg a 250 otgtcg a 251 ctgtcgttcccccccccccc 0 252 ctgtgctttctgtgtttttctgtg a 253 cttggagggcctcccggcgg 254 cttggtggagaaacccatgag 0 255 cttggtggagaaacccatgagctcatctggaggaagcgg 0 256 ctttccgttggacccctggg s 257 czggczggczgggczccgg o 258 faacgttga 0 259 fcgcgaattcgcg 0 260 ftcaacgtt 0 261 gaaacgtt 0 262 gaaactgctgctagtttcgctttat 0 263 gaaccttccatgctgtt 264 gaaccttccatgctgttccg 265 gaacgctggaccttccat 266 gaagttcacgttgaggggcat 0 267 gaagtttctggtaagtcttcg 0 268 WO 0 1/97843 PCT/USOI/20154 - 51 SEQUENCE BACKBONE SEQ ID NO: gaccttccat _ 269 gaccttccatgtcggtcctgat 270 gaccttctatgtcggtcctg 271 gacgtcat 0 272 gactgacgtcagcgt 0 273 gagaacgatggacc~tccat 0 274 gagaacgctagaccttctat 0 275 gagaacgctccaccttccat 0 276 gagaacgctccagcactgat 0 277 gagaacgctccagcttcgat 0 278 gagaacgctccgacct tcgat 8279 gagaacgctcgaccttccat 0 280 gagaacgctcgaccttcgatb a 281 gagaacgctggacctatccat 0 282 gagaacgctggacctcatcatccat 0 283 gagaacqctggacctcatccat 0 284 gagaacgctggaccttcc 285 gagaacgctggacCtt ccat 286 gagaacgctggaccttccat 13 287 gagaacgctggaccttccatgt ______ _ 2B8 gagaacgctggacctt cgat 0 289 gagaacgctggaccttcgta 0 290 gagaacgctggaccttgcat 0 291 gagaacgctggacgctcatccat 0 292 gagaacgctggacttccat 0 293 gagaacgctggaczttccat 0 294 gaga acgctggatccat 0 295 gagaatgctggaccttccat 0 296 gagaaz ctg accttccat 0 297 gagaccgctcgaccttcgat 298 gagcaagctggaccttccat a 299 gagcaagctggacc tccatb 8 300 gaggaacgtcatggagaggaacgtcaty9aglaggaacgtcatgga 0 301 gaggaaggigiggaigacgt 0 302 gagqggaccattttacqggc 303 gatccagattctgccaggtcactgtgactggat 0 304 gatccagattctgctgagtcactgtgactggat 0- 305 gatccagtcacagtgacctggcagaatctggat 0306 gatccagtcacagtgactcagcagaatctggat 0 307 gatccggctgactcatcactagatc 0 308 atcgctgatctaatgctcg BOB 309 gatcggaggactgccgcccg ______ 310 gatctagtqatgaqtcagccqqatc 0 311 gattcaacttgcgctcatcttaggc 0 312 gcaacgtt 0 313 gcaatattgcb 0 314 gcaatattgcf 0 315 gcacatcgtcccgcagccga a 316 gcaqcctctatacaacctqgacqgga __ 317 gcatagcgttgagct BOB 318 gcatgacgttgjagct s 319 qcatgacqttgaqct BOB 320 gcatgacgttgagct 0 321 gcatgacgttgagct s 322 gcatgagcttgagctga 0 323 WO001/97843 PCTIUSOII2O154 - 52 SEQUENCE BACKBONE SEQ ID NO: gcatgatgttgagct 0 324 gcatgazgttgagct 0 325 gcatggcgttgaqct BOB 326 gcatgtagctgagct 0 327 gcatgtcgttgagct BOB 328 gcattcatcaggcgggqcaagaat 0 329 gcattgcgttgagct BOB 330 gcatttcgaggagct 0 331 gccaccaaaacttgtccatg ________ 332 gccagatgttagctgga 0 333 gccatggacgaactgttccccctc B 334 gcgacgggcggcgcgcgccc B 335 gcgacggtcggcgcgcgcc s 336 gcgacgtgcggcgcgcgccc B 337 gcgacgttcggcgcgcgcec 13 338 gcgatgtcgttcctgatgcg 0 339 gcgatgtcgttcctgatgct 0 340 gcgccagtcctccgatagac 341 gcgcgcgcgcgcgcgcgcg 0 342 gcgctaccggtagcctgagt ________ 343 gcggcgggcggcgCgcgCtC 0 344 gcggcgggcggcgcgcgccc 8 345 9 9 cggtcgscgcgcgccc -B 346 gcggcgtgcggcgcgcgccc B 347 gcggcgttcggcgcgcgccc B 348 gcgtcgttcccccccccccc 0 349 gcgtgcgttgtcgttgtcgtt s 350 gcgtttttttttgcg a 351 gctaaacgttagcgt 0 352 gctaacgttagcgtga 0 353 gctatagcgta 0___354_ gctagcttagcgg 0 362 gctaacttagcgt 0 356 gctagacgatagcgtga 364 gctagacgttagcta 0 358 qctagacgttac~ - O 366 gctagacgtaaggtga 0 360 gct agacgtctagg 0 368 Sctagacgttagcg 0 369 gctagacgttagc 0 370 gctagacgttagcgtga 0371 gctagaggttagctga 0 372 gctagacgttagctga BO 373 gctagacgttaggctg 0 367 gctagacgttagtgt 0 375 gctagacgttagct 0 370 gctagagttagcgtga 0 377 gctagatgttagcgt 0375 WO 01/97843 PCT/USOI/201S4 - 53 SEQUENCE BACKBONESQD NO: gctagazgttagtgt 0 379 gctagctttagagctttagaqctt 0 380 gctaggcgttagcgt 0 381 gctagtcgatagc 0 382 gctagtcgatagcgt 0 383 gctagtcgctagc 0 384 gctandcghhagc 0 385 gctatgacgttccaaggg s 386 gctcga 8 387 gctcgttcagcgcgtct 80os 388 gctgaaccttccatgctgtt 389 5ctgagctcatgccgtctgc SOS 390 gctggaccttccat 392 gctggaccttccat 0 392 gctggccagcttacctcccg ________ 393 qcgaatgatgg ___________ 394____ gcttgacgtctagc 0 402 ggattagtatgtag agag 0 43 gcgagcttcgagCat 40539 gcatgaggcttacggg 406 ggcaactttc 0ggagCt 407 ggcagcataagaaa0 408 ggaattataatagatatagaagt 0 409 ggagtatcctgactcac g 0 411 ggactttcgaCagccat 402 ggcatgtcagtcaggg 413 ggcccgggtttca tgt tct 414 ggccgatcccatgatgCCC 415 gggaatgaagatt tattaaa 0 416 ggggactttccgctggggactttccagggggactttcc 5os 417 99 gagggaggaacttcttaaaattcccccagaatgttt 0 418 ggagga 419 c5gagg 420 gggatagtcaaa 421 gggagagtaaaaSOS 422 ggggatgagttcgggg s2423 ggggcatgacgttcgggggg SOS 424 ggggcatgagct tcgggggg S 425 ggggcatgagcttcgqqggg BOB 426 ggggcctctatacaacctggg 427 gqqqqacgttgggg 0 428 gggggsg~ggggSOB - ~ 429 999999999999999-9999R 0 430 ggggggttgfggaaaacccggacttcctgca 0 431 gggggttttttttttggggg 0 432 ggggtaatcgatcaggggg BOS 433 WO 011/97843 PCT/USOI/20154 - 54 SEQUENCE BACKBONE SEQ ID NO: ggggtaatcgatgagggggg 0 434 ggggtaatgcatcagggggg 008 435 gqgtcaacgttgaqgggg BOB 436 ggggtcaacgttgagqgggg S 437 ggtcaagcttqa~jg999g GOB 438 ggggtcaagtctgag gggg BOB 439 ggggtccagcgtgcgccatggggg 808 440 ggggtccctgagactgcc 441 ggggtcgaccttggagggggg BOB 442 ggggtcgacgtcgag gggg 443 ggggtcgtcgttttgggggg 444 ggggtctgtcgttttggggg -SOS- 445 ggggtctgtgcttttgggggg BOB 446 gqggtgacgt tcagggggg SOS 447 ggggtgtcgttcagggggg SOB 448 ggggttgacgttttgggggg --- OB 449 ggggt tgggggt t S 450 ggtacctgtggggacattgtg 0 451 ggtgaggtg a 452 ggtggtgtaggttttgg 0 453 ggttacggtctgtccsatat 454 ggttcacgtgctcatggctg 0 455 gtaacgtt 0 456 gtagccttccta _________ 457 gtaggggactttccgagctcgagatcctatg 0 458 gtcactcgtggtacctcga 8 459 gtccatggcgtgcgggatga ________ 460 gtcccaggttgtatagaggctgc ________ 461 qtccccatttcccagaqgaqgaaat 0 462 gtccgggccaggccaaagtc a 463 gtcggtcctgatgctgttcc Gas 464 gtctatcggaggactggcgc 465 gtctgtcccatgatctcgaa 466 gtgaaticgttcicgggict_ 0 467 gtgccggggtctccgggc a 468 gtgccggggtctccgggc 0 469 gtgcgcgcgagcccgaaatc s 470 gtgctgatcaccgatatcctgttcgg ________ 471 gtgcttgaccaccgatatttgg ________ 472 gtggttacggtcgtgccat ________ 473 gtgtcggggtctccgggc 0_______ 474 gttctcagataaagcggaaccagcaacagacacagaa ________ 475 gttgaaacccgagaacatcat ________ 476 gttggat acaggccagactttgttg 0 477 gtttttatataatttggg 0 478 gzaatattgcb 0___479___ ________________cc 480 taaactt 5483. tagaaacagcattcttcttttagggcagcaca ________ 487 tagacgtc 0___488__ WO 01/97843 PCT/USO 1120154 - 55 SEQUENCE BACKBONE SEQ ID NO: tagacgttagcgtga 0489 tatagtccctgagactgcccaccttctcaacaacc 490 tatcggaggactggcgcgccg ________ 491 tatgccgcgcccggacttat SOS 492 tcaaatgtgggattttccatgagtct 0 493 tcaacgt 8 494 tcaacgtc 0 495 tcaacgtt p-ethoxy 496 tcaacgtt a 497 tcaacgtt 0 498 tcaacgttaacgttaacgtt -0 499 tcaacgttaacgttaacgttaacgttaacgttb S 500 tcaacgttga S 5 01 tcaacgttga 0 502 tcaacgttgab053 tcactp-ethoxy 505 tcaagctaacctg 0___506___ tcaatgctafctta 0____507__ [tcaacgtatc0508 tcaczgtta 50 teaaccagtgtg ttCta B___510___ tcagcgctggcttaggggtcg 0_______ 511_____ tcacgcta__________0 _512 tcagctacgtctg051 tcacgcttgtctt 0_______ 519 tcagacacccgatcaggtgtctga 0 520 tcaqgacgaccagctggtttcctga 0 516 tcagzgct 0 523 tcagcgtgc 8 524 tccagctctctgtt 525 tccagagttgcagttgtcg 0 520 tcagcaccgatcgctggcg 0 527 tcaccagtggtgctt -- SOS-____ 522 tcazcacttct 0 529 tcacgagt a 0 530 tccaagacgttaat 0 531 tccaagagttaagt 0 52 tccac tggcat c O 527 tccacgacgtgtctagct S 534 tccacgacgttttcgacgtt 8 535 tccagacgttgaa gt 0o 530 icca gactttccta 0t 537 tccagagctttgcagt 0 538 tccaggacgttcctagttct 0 539 tccaggatttctagttt ___________ 540 tccaggact gcctagtt 0 541 tccagttrctt~ctcagtct -0 542 tccagttcgagcctagttct 0 543 WO 01/97843 PCTIUSOI/20154 -56 SEQUENCE -BACKBONE_ SEQ ID) NO: tccataacgttcctg gtct GOB 544 tccataacgttcctgatgct 0 545 -tccatagcgatcctagcgat 0 546 tccatagcggtcctagcggt 0 547 tccatagcgttcctagcgtt a 548 tccatagcgttcctagcgtt 0 549 tccatcacgtgcctgagtct 6013 550 tccatgacattcctgatgct 0 55) tccatgacggtcctgacggt 8 552 tccatgacggtcctgacggt 0 553 tccatgacggtcctgagtct BOB 554 tccatgacggtcctgatgct 0 555 tccatgacgtccctgagtct 6016 556 tccatgacgtccctgatgct 0 557 tccatgacgttcctagttct -0 558 tccatgacgttcct ctccatgacgt tcctctccatgacgttcctc 0 559 tccatgacgttcctgacgtt 8 560 tccatgacgttcctgacgtt ________ 561 tccatgacgttcctgacgtt 808 562 tccatgacgttcctgacgtt 0 563 tccatgacgttcctgagtct SOS 564 t ccatgacgttcctgat cc ________ 565 tccatgacgttcctgatgct 0 566 tccatgacgttcctgatgct S 567 tccatgacgttcctgcagttcctgacgtLt a 568 tccatgacgttcctgccgtt 13 569 tccatgacgttcctgcgttt 8 570 tccatgacgttcctggcggg 8 571 tccatgacgttcztgatgct 0 572 tccatgagcttcctgagctt 8 573 tccatgagcttcctgagtct 0 574 tccatgagcttcctgagtct p-ethoxy 575 tccatgagcttcctgagtct s 576 tccatgagcttcctgatgct a2 577 tccatgagcttccttgagtct ________ 578 tccatgaigttcctgaigtt S 579 tccatgatgttcctagttct 0 580 tccatgazgttcctagttct 0 581. tccatgazgttcctgatgct 0 582 tccatgazgttcctgazgtt a 583 tccatgccggtcctgagtct BOB 584 tacatgccggtcctgatgct 0 585 tccatgccggtcctgccggt 0 586 tccatgccgttcctgccgtt 6 587 tccatgccgttcctgccgtt 0 588 tccatglcgcgtcctgcgcgt 0 589 tccatgcgtgcgtgcgt ttt a 590 tccatgcgttgcgttgcgtt a 591 tccatgctggtcctgagtct 808 592 tccatgctggtCCtgatgct 0 593 tccatggcgggcctggcggg a 594 tccat gcggtc~ctgatqct 0 595 tccatgtagttcctagttct 0 596 tccatgtccttcctgatgct I_______ 9 tccatgtcgatcctgagtct BOB 598 WO 01/97843 PCTJIJSO 1120154 - 57 SEQENCE BACKBONE SEQ ID NO: tccatgtcgatcctgatgct 0 599 tccatgtcgctcctgagtct BOB 600 tccatgtcgctcctgatcct 0 601 tccatgtcggtcctgagtct 808 602 tccatgtcggtcctgatgct 603 tccatgtcggtcctgatgct 8 604 tccatgtcggtcctgctgat 0 605 tccatgtcggt zctgatgct 0 606 tccatgtcgttccgcgcgcg 0 607 tccatgtcgttcctagttct 608 tccatgtcgttcctgagtct SOS 609 tccatgtcgttcctgatgcg 0 610 tccatgtcgttcctgatgct 0 611 tccatgtcgttcctgccgct 0 612 tccatgtcgttcctgtagct 0 613 tccatgtcgttcctgtcgtt e 614 tccatgtcgttcctgtcgtt o 615 tccatgtcgtttttgtcgtt S616 tccatgtgcttcctgatgct 0 617 tccatgtzggtcctgagtct SOS 618 tccatgtzggtcctgatgct 0 619 tccatgtzgttcctgatgqt 0 620 tccatgtzgttcctgtzgtt 8 621 tccattgcgttccttgcgtt 0 622 tcccgacggtgaagt 0 623 t cccgc cgttgaagt 0 624 tcccgcgcgttccglcgcgtt S 625 tccctgagactgccccacctt ________ 626 tccgatcg 0 627 tccggacggtgaagt 0 628 tccggccgttgaagt o 629 tccgtacg 0 630 tcctaacgttguagt 0 631 tcctagcgttgaagt 0 632 tcctcacgttgaagt 0 633 tcctga a 634 tcctgaaaaggaagt a 635 tcctgacgatgaagt 0 636 tcctgacgctgaagt 0 637 tcctgacggggaagt 0 638 tcctgacggggaagt a 639 tcctgacggggagt a 640 tcctgacggtgaagt 0 641 tcctgacggtgaagt S 642 tcctgacgtagaagt 0 643 tcctgacgtcgaagt 0 644 tcctgacgtggaagt 0 645 tcctgacgtgaagt ______ 646 tcctgacgttaga 0647 tcctgacgttccc 0 648 tcctgacgttcccctggcggtcccctgtcgct 0 649 tcctgacgttcctgacgtt a 650 tcctgacgttcctggcggtcctgtcgct 0 651 tcctgacgtt cttc 0_______ 652 tcctqacqttcggcgcgcgccc a 653 WO 01/97843 PCTJUSOI/20154 - 58 SEQUENCE BACKBONE SE! ID NO: tcctgacgttgaagt 0- 654 tcctgacgttgaagt a 655 tcctgagcttgaagt 0 656 tcctg3agcttgaagt B 657 tcctgazgttgaagt 0 658 tcctgccgttgaagt 0 659 tcctgccgttgaagt 8 660 tcctggaggggaagt 0 661 tcctggaggggaagt a 662 tcctggcggggaagt 0 663 tcctggcggggaagt a664 tcctggcggtcctggcggtt a665 tcctggcggtgaagt 0 666 tcctggcggtgaagt B 667 tcctggcgtggaagt ____66B___ tcctggcgttgatgt 0 tt tcctggcgttgaagtcgtgga670ag tcctlgcgttgggaaq a~t agacaga 0_671 tcctgtgttccgCgt 0 679 tcctjgtcgigaagt o 680 tcctgtcgtctggtttttt 0 684 tcctgtcgcttttcctg gc a 682 tcctgtcgttcgacg t 0 683 tcctgtcgt tcctgtcgttgagcg a 6774 tccgtc tcttgtcagcg caai 0 678 tctcg tta t gtcCCCCC 0 686 tccgtgttg~aatatg 0 680 tccgcgttgaagttttttC 0 688 tccgtcgtttttgtcgttta- t 8 682 tcg at 0 690 tcggcgtt cccc cccc ccc c 0 691 tcgagcattccactt 0 683 tcgcattcccccCcccccc 0 694 tcgcgtCCcgttgcCCCCCCC g a- 689 tcqga~~CCCCCC 0 696 tcgtcgtttc a 6.92 tcgtcttccccttcttc a 698 tcgtcgctgtctccgcttctt ______98__ tcgtcgctgtcgttt 70 tcgtcgtcgtcgtt70 WO 01/97843 PCTJUS01/20154 -59 SEQUENCE BACKBONE SEQ 1U) NO: tcgtcgtcgtcgtt 92o 709 tcgtcgtcgtcgtt oa2 710 tcgtcgttccccccccC s 711 tcgtcgttcccccccccccc 0 712 tcgtcgttccccccccccccb 0 713 tcgtcgttccccccczcccc 0 714 tg gs ggtgtcgtgg gc 8 715 tctgtgtgtgttgt 716 tctgtt8tgct 717 tctstgcgt6ct 06 718 tctctttgttttct B 719 tctgtgcttttst OB 720 tcgtgttcgtctttgacgt 8721 tctcttgggcttctgt 722 tcgtcgtttgt cgttt tgt cgtt a 723 tcgtcgttttgacgttttgacgtt 8 724 tcgtcgttttgacgttttgtcgtt S 725 tcgtcsttttgcgtgcsttt a 726 tcgtcgt tttgtcgtt ttgggggg, 727 tcgtcgttttgtcgttttgtcgt a2 728 tcgtcgttttgtcgttttgtcgtt 8 729 tcgtcgttttgtcgttttgtcgtt SOB 730 tcgtcgttttgtcgttttgtcgtt 0 731 tcgtcgttttgtcgttttgtcgtt s2 1 732 tcgtcgttttgtcgttttgtcgttb 0 733 tcgtcgttttgtcgttttgtcgttttgtcgtt S 734 tcgtcgt tttgtggttttgtggtt B 735 tcgtcgttttttgtcgttttttgtcgtt S 736 tcgtcgtttttttttttttt I 3 tcgtga a 738 tcgtga 0 739 tcgtgg 13 740 tcgtzgttccccccccccc 0 741 tcntcgtnttntcgtnttntcgtl 8 742 tctaaaaaccatctattcttaaccct 0 743 tctagcgtttttagcgttcc 809 744 tctatcccaggtgttcctgttag 0 745 tctatcgacgttcaagcaag S 746 tctccatcctatggttttatcg 0 747 tctccatgatggttttatcg 748 tctcccagcgagcgagcgccat 8 749 tctcccagcgagcgccat 8 750 tctcccagcgcgcgccat a 751 tctcccagcgggcgcat B 752 tctcccagcgtacgccat 8 753 tctcccagcgtcgccat a 754 tcccggtcca 755 occcggtcca 756 tcccacggc06tt O 757 tcccactggctt OB 758 tcccactcggaca 759 tcccgctcttttSOS 760 tcccggtatt 761. tctcccagcgttgcgccatat SOB 762 tctcccatcgtcgccat 8 763 WO 01/97843 PCTJUSOI/20154 - 60 SEQUENCE BACKBONE SEQ ED NO: tctcccgacgtgcgccat 13 764 tctcccqtcgtgcgccat s 765 tctccctgcgtgcgccatat BOB 766 tctcctagcgtgcgccatat BOB 767 tctgacgtcatctgacgttggctgacgtct 0 768 tctgcgtgcgtgcgccatat soB 76 tcttcgaa 0 770 tcttgcgatgctaaaggacgtcacattgcacaatcttaataaggt 0 77. tctttattagtgactcagcacttggca 0 772 tcztgacgttgaagt 0 773 tgaacgtt 0 774 tgcaatgtgacgtcctttagcat 0 '775 tgcaggaagtccgggttttccccaacccccc 0 776 tgcatcagctct B 777 tgCatca(,Cctt BOB 77B tgcatcccccaggccaccat B 779 tgcatgccgtacacagctct 130B 780 tgcatqccqtacacaqctct B 78. tgcatgccgtacacagctct 0 782 tgcatgccgtgcatccgtacacagctct 6 783 tgccaagtgctgagtcactaataaag-a 0 784 tgcccaaagaggaaaatttgtttcatacag 0 785 tgcgctct B 786 tgctagactqtqcctqtacct 787 tgctagctgtgcctgtacct B 788 tgctgcttccccccccccc 0 789 tgctgcttcccccccCcccc 8 790 tgctgcttttgtgcttttgtgctt 0 791 tgctgcttttgtqcttttgtgctt B 792 tggaccttccat 793 tggaccttctatgtcggtcc 794 tggagggtgagggtggggccagagcgggtggggctgattggaa 0 795 tgg3agqtcccaccgagatcggaq o 796 tggttacggtctgt~ccatg ________ 797 tgtatctctctgaaggact 0 798 tgtccaqccgagqqqaccat ________ 799 tgtcccatgtttttagaagc ________ 800 tgtcgttgtcgtt S 802. tgtcgttgtcgttgtcgttgtcgtt S 802 tgtcgtttgtcgtttgtcgtt 8 803 ttaacggtggtagcggtattggtc 0 804 ttaacgtt 0 805 ttaagaccaataccgctaccaccg 0 806 ttaggacaaggtctagggtg 807 ttagggttagggttagggtt s2 808 ttcagttgtcttgctgcttagctaa089 ttcagcctgcaaatgcg810 ttccagctgtccgctgg812 ttcgggcggactcctccattSO 1 ttcgggcggactcctccatt 0 81.8 WO 01/97843 PCT/USOI/20154 -61 SEQUENCE BACKBONE SEQ ID NO: ttcgtcgttttgtcgttttgtcgtt a 819 ttctgtgtctgttgctggttccgctttatctgagaac 820 ttgaaactgaggtgggac 821 ttgccccatattttagaaac 822 ttggggggggtt a 823 ttgtactctccatgatggtt 824 tttaccttttataaacataactaaaacaaa o 825 tttgaatcctcagcggtctccagtggc 0 826 tttgaattcaggactggtgaggttgag 0 827 tttgaattcctgtacagaagcgagaasc 0 828 tttgagaacgctggaccttc 08 829 tttgcggcc9ctagacttaacctgagagata 0 830 tttgggcccacgagagacagagacacttc o 831 tttgggcccgcttctcgettctgtacacg 0 832 ttttctagdgaggtgcacaatgctctgg 0 833 tttttgggggggggttttt o 834 tttttttttttttf 0 835 tttttttttttttf so 836 tttttttttttttttttt S 837 tttttttttttttttttttt a 838 tttttttttttttttttttt 0 839 ttttttttttttttttttttt a 840 tttttttttttttttttttttttt 841 ttttttttttttttttttttttttttt s842 tzaacgtt o 843 tzgtCgttcccccccccccc 0 844 tzgtCgttttgtCgttttgtCgtt 0 845 tZgtggttaccccccccccc o 846 tzgtzgttttgtzgttttgtzgtt 0 847 tzgtzgttttgtzgttttgtzgtt a 848 In Table 4 with respect to sequences the letter symbols aside from a, c, t, and g are defined as follows: "b" indicates a biotin moiety attached to that end of the oligonucleotide when it is single and is listed on the 5' or 3' end of oligonucleotide; "d" represents a, g, or t; 5 "f' represents fluorescein isothiocyanate (F1TC) moiety attached to the 5' or 3' end of oligonucleotide; "h" represents a, c, or t; "i" represents inosine; "n" represents any nucleotide; "z" represents 5-methylcytosine. Also in Table 4 with respect to backbones the notations are defined as follows: "o" represents phosphodiester; "os" represents phosphorothioate and phosphodiester chimeric 10 with phosphodiester on 5' end; "os2" represents phosphorodithioate and phosphodiester chimeric with phosphodiester on 5' end; "p-ethoxy" represents p-ethoxy backbone (see, e.g., U.S. Patent No. 6,015,886); "po" represents phosphodiester; "s" represents phosphorothioate; "s2" represents phosphorodithioate; "s2o" represents phosphorodithioate and phosphodiester chimeric with phosphodiester on 3' end; "so" represents phosphorothioate and phosphodiestcr WO 01/97843 PCT/USO 1/20154 -62 chimeric with phosphodiester on 3' end; and "sos" represents chimeric phosphorothioate/phosphodiester with phosphorothioate at the 5' and 3' ends. The nucleic acids are delivered in effective amounts. The term "effective amount" of a immunostimulatory nucleic acid refers to the amount necessary or sufficient to realize a 5 desired biologic effect. For example, an effective amount of an immunostimulatory nucleic acid could be that amount necessary to cause activation of the immune system. According to some aspects of the invention, an effective amount is that amount of an immunostimulatory nucleic acid and that amount of an antibody, which when combined or co-administered, results in the prevention or the treatment of the cancer. In some embodiments a synergistic 10 effect is observed. A synergistic amount is that amount which produces an anti-cancer response that is greater than the sum of the individual effects of either the immunostimulatory nucleic acid and the antibody alone. For example, a synergistic combination of an immunostimulatory nucleic acid and an antibody provides a biological effect which is greater than the combined biological effect which could have been achieved using each of the 15 components (i.e., the nucleic acid and the antibody) separately. The biological effect may be the amelioration and or absolute elimination of symptoms resulting from the cancer. In another embodiment, the biological effect is the complete abrogation of the cancer, as evidenced for example, by the absence of a tumor or a biopsy or blood smear which is free of cancer cells. 20 The effective amount of immunostimulatory nucleic acid necessary to treat a cancer or in the reduction of the risk of developing a cancer may vary depending upon the sequence of the immunostimulatory nucleic acid, the backbone constituents of the nucleic acid, and the mode of delivery of the nucleic acid. The effective amount for any particular application can also vary depending on such factors as the cancer being treated, the particular 25 immunostimulatory nucleic acid being administered (e.g., the nature, number or location of immunostimulatory motifs in the nucleic acid), the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular immunostimulatory nucleic acid and antibody combination without necessitating undue experimentation. Combined with the teachings provided herein, by 30 choosing among the various active compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and preferred mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned WO 01/97843 PCT/USO1/20154 -63 which does not cause substantial toxicity and yet is entirely effective to treat the particular subject. Therapeutic doses of cancer therapies are well known in the field of medicine for the treatment of cancer. These dosages have been extensively described in references such as 5 Remington's Pharmaceutical Sciences, 18th ed., 1990; as well as many other medical references relied upon by the medical profession as guidance for the treatment of cancer. Therapeutic dosages of immunostimulatory nucleic acids have also been described in the art and methods for identifying therapeutic dosages in subjects are described in more detail herein. 10 Subject doses of the compounds described herein typically range from about 0.1 pig to 10 mg per administration, which depending on the application could be given daily, weekly, or monthly and any other amount of time therebetween. More typically mucosal or local doses range from about 10 ptg to 5 mg per administration, and most typically from about 100 tg to 1 mg, with 2 - 4 administrations being spaced hours, days or weeks apart. More 15 typically, immune stimulant doses range from 1 pg to 10 mg per administration, and most typically 10 pg to I mg, with daily or weekly administrations. Subject doses of the compounds described herein for parenteral delivery, wherein the compounds are delivered without another therapeutic agent are typically 5 to 10,000 times higher than the effective mucosal dose or for immune stimulant applications, and more typically 10 to 1,000 times 20 higher, and most typically 20 to 100 times higher. More typically parenteral doses for these purposes range from about 10 j.g to 5 mg per administration, and most typically from about 100 ig to I mg, with 2 - 4 administrations being spaced hours, days or weeks apart. In some embodiments, however, parenteral doses for these purposes may be used in a range of 5 to 10,000 times higher than the typical doses described above. 25 For any compound described herein the therapeutically effective amount can be initially determined from animal models, e.g., the animal models described herein. A therapeutically effective dose can also be determined from human data for CpG nucleic acids which have been tested in humans (human clinical trials have been initiated and the results publicly disseminated) and for compounds which are known to exhibit similar 30 pharmacological activities. Higher doses may be required for parenteral administration, as described above. The applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy WO 01/97843 PCT/USOI/20154 -64 based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan. The formulations of the invention are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, 5 buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients. For use in therapy, an effective amount of the nucleic acid can be administered to a subject by any mode that delivers the nucleic acid to a subject. "Administering" the pharmaceutical composition of the present invention may be accomplished by any means 10 known to the skilled artisan. Some routes of administration include but are not limited to oral, intranasal, intratracheal, inhalation, ocular, vaginal, rectal, parenteral (e.g., intramuscular, intradermal, intravenous or subcutaneous injection) and direct injection. For oral administration, the compounds (i.e., nucleic acids and antibodies) can be delivered alone without any pharmaceutical carriers or formulated readily by combining the 15 active compound(s) with pharmaceutically acceptable carriers well known in the art. The tenn "pharmaceutically-acceptable carrier" means one or more compatible solid or liquid filler, dilutants or encapsulating substances which are suitable for administration to a human or other vertebrate animal. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. 20 The components of the pharmaceutical compositions also are capable of being commingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion 25 by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, 30 potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or WO 01/97843 PCT/US01/20154 -65 alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers for neutralizing internal acid conditions. Dragee cores may be provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl 5 pyrrolidone, carbopol gel, polycthylenc glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. Pharmaceutical preparations which can be used orally include push-fit capsules made 10 of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene 15 glycols. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. 20 For administration by inhalation, the compounds for use according to the present invention may be conveniently delivered in the form of an aerosol spray, from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlkrodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to 25 deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. 30 Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as WO 01/97843 PCT/US01/20154 -66 suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active 5 compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable 10 stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. The compounds may also be formulated in rectal or vaginal compositions such as 15 suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable 20 oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, 25 and polymers such as polyethylene glycols. Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical 30 compositions may also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries WO 01/97843 PCT/US01/20154 -67 such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of present methods for drug delivery, see Langer R, Science 249:1527-33 (1990), which is 5 incorporated herein by reference. The nucleic acids and/or antibodies may be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not 10 limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group. 15 Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v). The nucleic acids or other therapeutics useful in the invention may be delivered in 20 mixtures with additional antibodies. A mixture may consist of several antibodies in addition to the nucleic acid. A variety of administration routes are available. The particular mode selected will depend, of course, upon the particular nucleic acids or antibodies selected, the particular condition being treated and the dosage required for therapeutic efficacy. The methods of this 25 invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of an immune response without causing clinically unacceptable adverse effects. Preferred modes of administration are discussed above. The compositions may conveniently be presented in unit dosage form and may be 30 prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the compounds into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately WO 01/97843 PCT/USOI/20154 -68 bringing the compounds into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. Liquid dose units are vials or ampoules. Solid dose units are tablets, capsules and suppositories. Other delivery systems can include time-release, delayed release or sustained release 5 delivery systems. Such systems can avoid repeated administrations of the compounds, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. 10 Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent No. 5,075,109. Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di-, and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; 15 partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which an agent of the invention is contained in a form within a matrix such as those described in U.S. Patent Nos. 4,452,775, 4,675,189, and 5,736,152, and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Patent Nos. 3,854,480, 5,133,974 and 5,407,686. In 20 addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation. The nucleic acid may be directly administered to the subject or may be administered in conjunction with a pharmaceutically acceptable carrier or a delivery vehicle. The nucleic acid and optionally other therapeutic agents may be administered alone (e.g., in saline or 25 buffer) or using any delivery vehicles known in the art. One type of delivery vehicle is referred to herein as a nucleic acid delivery complex. A "nucleic acid delivery complex" shall mean a nucleic acid molecule associated with (e.g., ionically or covalently bound to; or encapsulated within) a targeting means (e.g., a molecule that results in higher affinity binding to target cell (e.g., dendritic cell surfaces and/or increased cellular uptake by target cells). 30 Examples of nucleic acid delivery complexes include nucleic acids associated with: a sterol (e.g., cholesterol), a lipid (e.g., a cationic lipid, virosome or Liposome), or a target cell specific binding agent (e.g., a ligand recognized by target cell specific receptor). Preferred WO 01/97843 PCT/US01/20154 - 69 complexes may be sufficiently stable in vivo to reduce significant uncoupling prior to internalization by the target cell. However, the complex may be cleavable under appropriate conditions within the cell so that the nucleic acid may be released in a functional form. The nucleic acids may be delivered by non-invasive methods as described above. 5 Non-invasive delivery of compounds is desirable for treatment of children, elderly, animals, and even adults and also to avoid the risk of needle-stick injury. Delivery vehicles for delivering compounds to mucosal surfaces have been described and include but are not limited to: Cochleates (Gould-Fogerite et al., 1994, 1996); Emulsomes (Vancott et al., 1998, Lowell et al., 1997); ISCOMs (Mowat et al., 1993, Carlsson et al., 1991, Hu et al., 1998, 10 Morein et al., 1999); Liposomes (Childers et al., 1999, Michalek et al., 1989, 1992, de Haan 1995a, 1995b); Live bacterial vectors (e.g., Salmonella, Escherichia coli, Bacillus Calmetne Gudrin, Shigella, Lactobacillus) (Hone et al., 1996, Pouwels et al., 1998, Chatfield et al., 1993, Stover et al., 1991, Nugent et al., 1998); Live viral vectors (e.g., Vaccinia, adenovirus, Herpes Simplex) (Gallichan et al., 1993, 1995, Moss et al., 1996, Nugent et al., 1998, Flexner 15 et al., 1988, Morrow et al., 1999); Microspheres (Gupta et al., 1998, Jones et al., 1996, Maloy et al., 1994, Moore et al., 1995, O'Hagan et al., 1994, Eldridge et al., 1989); nucleic acid vaccines (Fynan et al., 1993, Kuklin et al., 1997, Sasaki et al., 1998, Okada et al., 1997, Ishii et al., 1997); Polymers (e.g., carboxymethylcellulose, chitosan) (Hamajima et al., 1998, Jabbal-Gill et al., 1998); Polymer rings (Wyatt et al., 1998); Proteosomes (Vancott et al., 20 1998, Lowell et al., 1988, 1996, 1997); Sodium Fluoride (Hashi et al., 1998); Transgenic plants (Tacket et al., 1998, Mason et al., 1998, Haq et al., 1995); Virosomes (Gluck et al., 1992, Mengiardi et al., 1995, Cryz et al., 1998); Virus-like particles (Jiang et al., 1999, Leibl et al., 1998). The invention also includes kits. The kits generally include a package with a plurality 25 of containers housing active agents and instructions for carrying out the methods of the invention. The active agents include but are not limited to immunostimulatory nucleic acids, antibodies such as antibodies specific for a cell surface antigen, and anti-cancer therapies. The following examples are provided to illustrate specific instances of the practice of the present invention and are not to be construed as limiting the present invention to these 30 examples. As will be apparent to one of ordinary skill in the art, the present invention will find application in a variety of compositions and methods.
WO 01/97843 PCT/US01/20154 -70 Examples Introduction: Extensive cross-talk exists between healthy B cells and T cells. There is evidence that malignant B cells also communicate with T cells. However, malignant cells appear to differ 5 from their normal counterparts in a number of ways, including a decreased tendency to undergo apoptosis in response to normal signals, altered expression of a variety of surface markers, and altered ability to function as effective antigen presenting cells. Lagneaux L et al., Blood 91:2387-96 (1998); Gordon J et al., Leukemia 7 Suppl 2:S5-9 (1993); Gordon J et al., Adv Exp Med Biol 406:139-44 (1996); Chaperot L et al., Exp Hematol 27:479-88 (1999). 10 Immunotherapeutic approaches have recently become part of our therapy of some subtypes of B-cell malignancy. Improved immunotherapy of B-cell malignancy will need to be designed based on the growing understanding of the cellular immunology of this disease. Schultze JL et al., JMol Med 77:322-32 (1999). A variety of cellular receptors and antigens are involved in growth, differentiation and 15 apoptosis of B-cell malignancies. Antibodies or ligands against a variety of antigens can cause growth inhibition or even apoptosis including CD20, surface immunoglobulins, MHC II, CD80, CD86 and CD40. Maloney DG, Semin Oncol 26:74-8 (1999); McLaughlin P et al., Semin Oncol 26:79-87 (1999); Shan D et al., Blood 91:1644-52 (1998); Coiffier B et al., Blood 92:1927-32 (1998); McLaughlin P et al., Oncology (Huntingt) 12:1763-70, 1775-7 20 (1998); Tutt AL et al., JInmunol 161:3176-85 (1998); Funakoshi S et al., Blood 83:2787-94 (1994); Mayumi M et al., JAllergy Clin Immunol 98:S238-47 (1996); Higaki Y et al., Immunol Cell Biol 72:205-14 (1994); Elsasser D et al., Blood 87:3803-12 (1996); Link BK et al., Blood 81:3343-9 (1993); Link BK et al., Int J Cancer 77:251-6 (1998). The relative contribution of antibody dependent cellular cytotoxicity (ADCC) versus trans-membrane 25 signaling mediated by anti-B cell antibodies remains unclear. In the present study, we examined how CpG-DNA impacts on the phenotype, apoptosis and proliferation of different types of B-cell malignancy including follicular B-cell lymphoma and B-CLL. Materials and Methods: Cell culture: Fresh lymph node samples were obtained from the operating suite and 30 were minced with a scalpel under aseptic conditions. The resulting suspension was passed sequentially through a sterilized sieve-tissue grinder containing a nylon mesh screen, a 150 Am mesh screen and a 60 pm mesh screen. Alternatively, mononuclear cells were obtained WO 01/97843 PCT/USOI/20154 -71 from peripheral blood or pleural fluid as described. Hartmann G et al., JPharmacol Exp Other 285:920-8 (1998). Red blood cells were removed by resuspending the cells in 5 ml ACK lysis buffer according to standard procedures. Cells were frozen slowly and stored in liquid nitrogen. For analysis, cells were thawed and resuspended in 10 % (v/v) heat 5 inactivated (56*C, I h) FCS (HyClone, Logan, UT), 1.5 mM L-glutamine (all from Gibco BRL, Grand Island, NY) and incubated on a 96-well-plate (1 x 106 cells/ml) in the presence of ODN as indicated below. Not all assays were performed for all samples because of the limited number of cells available for some samples. Oligonucleotides: Nuclease-resistant phosphorothioate-modified 10 oligodeoxynucleotide (ODN) were purchased from Operon Technologies (Alameda, CA) and Hybridon Specialty Products (Milford, MA). Sequences were as follows: CpG ODN 2006: 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3'(SEQ ID NO: 729), and control ODN 2017: 5'-CCCCCCCCCCCCCCCCCCCC-3' (SEQ ID NO: 168). ODN was diluted in TE (10 mM Tris-HCI, 1 mM EDTA, pH 8) using pyrogen-free reagents. ODN was added at a final 15 concentration of 5 gg/ml. Flow cytometry: Cells were washed and resuspended in ice-cold PBS or Annexin V binding buffer (10 mM HEPES/NaOH, 140 mM NaCl, 2.5 mM CaC 2 , pH 7.4). Murine or human serum was added (final concentration 1%) to block non-specific binding of antibodies. Surface antigen staining was performed as described. Hartmann G et al., JPharmacol Exp 20 Ther 285:920-8 (1998). In brief, I x 105 cells per sample were stained with CyChrome labeled anti-CD19 and FITC- or PE-labeled antibodies as indicated for 20 min on ice. They were then washed and analyzed by flow cytometry. Monoclonal antibodies to CD40 (5C3), CD69 (FN50), CD80 (L307.4), CD86 (IT2.2), CD54 (HA58), MHC I (G46-2.6) and MHC II (TU39) as well as isotype controls (IgGI, MOPC-21 and IgG2a, G155-178) were purchased 25 from PharMingen, San Diego, CA. FITC-labeled polyclonal anti-human Ig was purchased from Southern Biotech, Birmingham, AL. ID10, a monoclonal humanized antibody directed against a variant of HLA-DR was produced in our laboratory as described earlier. Link BK et al., Blood 81:3343-9 (1993). C2B8, a monoclonal humanized anti-CD20 antibody, was purchased from IDEC Pharmaceuticals, San Diego, CA. ID10 and C2B8 were labeled with 30 FITC according to standard protocols. The analysis gate was set on viable cells identified according to FSC/SSC characteristics and Annexin V staining (> 97 % viable cells within analysis gate). Spectral overlap was corrected by appropriate compensation. Flow WO 01/97843 PCTIUS01/20154 -72 cytometric data from I x 104 cells per sample were acquired on a FACScan (Beckton Dickinson Immunocytometry Systems, San Jose, CA). Data were analyzed using the computer program FlowJo (version 2.5.1, Tree Star, Inc., Stanford, CA). CFSE staining: CFSE 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester, 5 Molecular Probes, USA, is a fluorescein-derived intracellular fluorescent label which is divided equally between daughter cells upon cell division. Staining of cells with CFSE allows both quantification and immunophenotyping of proliferating cells in a mixed cell suspension. Interference between oligonucleotide degradation products and thymidine uptake (standard proliferation assay) is avoided by using this method. The technique has described 10 in detail previously. Lyons AB et al., JImmunol Methods 171:131-7 (1994). Briefly, cells were washed twice in PBS, resuspended in PBS (1 x 107 cells/ml) containing CFSE at a final concentration of 1 FM, and incubated at 37*C for 10 minutes. Cells were washed three times with PBS. TUNEL assay: A two-color DNA strand break labeling assay, based on a 15 modification of the assay described by Li et al. (Li X et al., ExpCell Res 222:28-37 (1996)) was used to assess B-cell proliferation in response to CpG ODN. This assay involved terminal transferase-mediated dUTP nick end labeling (TUNEL) before and after induction of DNA strand breaks in BrdU-labeled cells. Briefly, cells were cultured for 3 days with and without ODN. They were then incubated for 16 hours in 10p±M BrdU and placed onto slides 20 by cytospin. Cells were then in 1% paraformaldehyde in PBS for 15 minutes followed by 20 minutes in 70% ethanol. DNA cleavage indicative of apoptosis cells was detected by labeling the 3'-DNA end of nicked strands with FITC-ddUTP (Boehringer-Mannheim). The use of dideoxy-dUT prevented further elongation of the 3'-ends in subsequent steps. Slides were then placed face-down on a 2mm support at both ends on a UV transilluminator and 25 exposed for 5 minutes. The new DNA strand breaks induced by photolysis at sites of BrdU incorporation (i.e., proliferating cells) were detected by a second TUNEL labeling using tetramethylrhodamine-dUTP (TMR-dUTP, Boehringer-Mannheim). Both TUNEL staining steps included incubating slides in 50p of TdT mix (3 4 pl distilled water, 10p.l of 5X TdT buffer, 5p1 of 25mM cobalt chloride, 12.5 units terminal transferase and 0.5nmol 30 fluorochrome-conjugated-dUTP) (Boehringer-Mannheim) under a coverslip for one hour at 37*C in a humidified chamber. The slides were then washed in 5 quick changes of distilled water followed by 3 changes of 2XSSC containing 30% formamide for 5 minutes each at WO 01/97843 PCT/USOI/20154 -73 room temperature. After the second TUNEL labeling step, cells were counterstained for CD19, and also stained with Wright solution for blood cell differentiation and mounted in Vectashield media containing DAPI counterstain (Vector Laboratories, Burlingame, CA). The morphology and staining of cells were assessed using both visible light and fluorescence 5 microscopy. Apoptotic cells were identified by green fluorescence (FITC label), and proliferating cells by red fluorescence (TMR label). The percentage of apoptotic and proliferating cells was determined by counting at least 200 cells per sample by three observers blinded to whether cells were treated with ODN. Mean and standard error were determined for each sample based on these three readings. 10 Example 1: Immunostimulatory nucleic acids Induce morphological and phenotypic changes in malignant B cells. Our prior studies demonstrated that activation of naive human B cells by CpG ODN results in increased cell size (FSC) and granularity (SSC). Hartmann G et al., JImmunol 15 164:944-53 (2000). We therefore first determined whether such changes also occur in malignant B cells. Primary malignant B cells were obtained from lymph node biopsies, peripheral blood, or pleural fluid of patients with various types of B-cell malignancy. In addition, cells from the lymph node of a patient with benign reactive follicular hyperplasia were studied. Nine samples in total were evaluated (see Table 5). Cells were incubated for 20 72 hours in media containing CpG ODN 2006 (5 pig/ml) or control ODN 2017. FSC and SSC were examined with gating on CD19+ viable cells (Figure 1). Varying degrees of change in FSC and SSC were noted in response to CpG ODN 2006 when compared to control ODN 2017 or medium alone. Comparable changes were not found in the cells from the patient with benign reactive follicular hyperplasia. 25 Figure 1 depicts the morphologic changes of marginal zone lymphoma cells upon CpG ODN stimulation. Malignant B cells from a patient with marginal zone lymphoma were stimulated with 5 pg/ml of no ODN (A and D), control ODN (B and E) or CpG ODN (C and F) for 72 hours and analyzed by flow cytometry. A, B, and C illustrate FSC (x-axis) vs. SSC (y-axis). D, E and F illustrate CD19 expression (x-axis) against FSC (y-axis), allowing for 30 separation of B cells from other leukocyte subpopulations. Upon stimulation with CpG ODN, B cells shifted up and to the right, indicating an increase in granularity and size. No changes could be detected without stimulation or on stimulation with the non-CpG ODN.
WO 01/97843 PCT/USOI/20154 -74 Expression of CD20, CD40, CD69, CD80, CD86, surface Ig, CD54, MHC I, MHC II, and an HLA-DR variant antigen (moAb IDIO) were examined on viable CD19+ cells after incubation of cells with CpG ODN for 72 hours. Each of these markers was upregulated to varying extents in response to the CpG ODN 2006 compared to the control ODN 2017 (Fig. 5 2, Fig. 3). Figure 2 depicts the expression of surface antigens on marginal zone lymphoma cells upon CpG ODN treatment. Flow cytometric analysis of surface antigen expression on malignant B cells from a patient with marginal zone lymphoma was performed 72 hours after stimulation with 5 sg/ml of either CpG ODN or non-CpG ODN. On stimulation with CpG 10 ODN, median fluorescence intensity for all markers tested shifted to the right, indicating an increase in surface expression. Thin curves indicate incubation with medium alone, dotted curves incubation with control ODN, and bold curves incubation with CpG ODN. Figure 3 depicts the expression of surface antigens on primary cells representing different B-cell malignancies and cells of a benign follicular hyperplasia upon CpG ODN 15 treatment. Cells from lymph node biopsies, peripheral blood or pleural fluid from patients with different B-cell malignancies were incubated for 72 hours with either media alone, control ODN or CpG ODN. Each panel represents one experiment. CD20 was expressed to varying degrees in all samples tested. As is well known, baseline CD20 expression was lower in the B-CLL samples when compared to the B-cell 20 malignancies of other histologies. CpG-ODN 2006 but not the control ODN 2017 increased CD20 expression in both B-CLLs and both marginal zone lymphomas. No or only little upregulation was seen in the other lymphoma samples. Non-malignant CD1 9+ cells derived from the reactive follicular hyperplasia decreased CD20 expression in response to CpG (Fig. 3). This data demonstrated a reverse correlation between the baseline expression of CD20 25 and CD40, and expression of these markers after incubation with CpG ODN; thus the lower the baseline level of CD20 and CD40, the higher was the responsiveness to CpG ODN (r: 0.6; -0.4) (Fig. 4). This correlation was less clear for the other markers. CD19+ cells derived from the reactive follicular hyperplasia showed high baseline expression of activation markers which was not further upregulated by CpG. 30 Figure 4 shows the CpG ODN effect on CD20 and CD40 is dependent on the baseline level of expression. Cells from lymph node biopsies, peripheral blood or pleural fluid from patients with different B-cell malignancies (see Table 5) were incubated with or WO 01/97843 PCT/USOI/20154 -75 without CpG ODN for 72 hours. Expression of CD20 and CD40 was measured by flow cytometry. Baseline expression of CD20 and CD40 with medium alone was compared to the expression of CD20 and CD40 in the presence of CpG ODN. The coefficients of correlation are indicated. 5 Table 5: Percentage Of CD19+ Cells In Samples Tested. Number Histology Source % CD19+ Cells 1 Chronic Lymphocytic Leukemia 1 Peripheral > 98 % ____ ___ ____ ____ ____ ____ ____ ____ Blood _ _ _ _ _ _ _ Peripheral 7 2 Chronic Lymphocytic Leukemia 2 70% 3 Large Cell Lymphoma 1 Pleural Fluid 55 % 4 Large Cell Lymphoma 2 Lymph Node 75 % 5 Mantle Cell Lymphoma Lymph Node 98 % 6 Diffuse Mixed Small and Large Cell Lymph Node 50 % Lymphoma 7 Marginal Zone Lymphoma 1 Lymph Node 80 % 8 Marginal Zone Lymphoma 2 Peripheral >94 % ____ ___ ____ ____ ____ ____ ____ ____ Blood _ _ _ _ _ _ _ 9 Reactive Follicular Hyperplasia Lymph Node 35 % Example 2: Immunostimulatory nucleic acids induce proliferation and apoptosis of malignant B cells. 10 CpG induces a strong proliferative response of primary human B cells. Hartmann G et al., Jlmmunol 164:944-53 (2000). Two techniques were used to assess whether CpG ODN is capable of inducing proliferation of B-CLL cells. For select samples, cells were stained with CFSE and incubated for four days. Proliferation of cells is indicated by a loss of CFSE stain with every cell division. In B-CLL, CD5 can be used to identify malignant B cells 15 among CD19+ cells. Proliferation of malignant B cells (CD5+ and CD19+) was lower than proliferation of normal B cells (CD5- and CD19+) (Fig. 5). For the marginal zone lymphoma, CpG ODN 2006 induced proliferation of the CD19+ cell population (Fig. 5). Figure 5 shows a comparison of CpG ODN induced proliferation of malignant and normal B cells. Peripheral blood mononuclear cells from two patients, one with B-CLL and 20 one with marginal zone lymphoma with circulating malignant cells, were incubated for 72 hours with CpG ODN or medium alone and evaluated by two-color flow cytometry. CFSE fluorescence (x-axis) and expression of CD5 (CLL) or CD 19 (marginal zone lymphoma) (y- WO 01/97843 PCT/US01120154 -76 axis) were evaluated. In CLL, CpG ODN enhanced proliferation of both CD5+ and the CD5 cells. However the relative number of proliferating cells and the number of divisions is lower in the CD5- subset than in the CD5+ subset. In marginal zone lymphoma CpG ODN enhanced proliferation in the CD19+ cell subset. 5 No consistent pattern was apparent related to determining whether CpG ODN altered the percent of dead cells as determined by morphological criteria (see Table 6). Table 6: Percent Apoptotic Cells Based On Morphologic Criteria. Numbr Histology Media CpG ODN 2006 N Chronic Lymphocytic 25.9 21.5 1____ Leukemia 1 2 Chronic Lymphocytic 32.6 45.3 Leukemia 2 3 Large Cell Lymphoma 1 33.9 26.2 4 Large Cell Lymphorna 2 16.0 9.8 5 Mantle Cell Lymphoma 55.1 60.0 6 Diffuse Mixed Small and 27.6 26.6 Large Cell Lymphoma 7 Marginal Zone 32.9 32.8 Lymphoma 1 8 Marginal Zone 38.8 56.0 Lymphoma 2 9 Reactive Follicular 8.6 18.0 SHyperplasiaI 10 A TUNEL assay was utilized to assess the effect of CpG ODN on both proliferation and apoptosis. The results are shown in Table 7. Table 7: Apoptosis And Proliferation As Determined By TUNEL. Sample Baseline CpG ODN Control ODN Apop I Prolif Apop Prolif Apop Prolif 1663141 15 8 11 10 12 5 12142812 3 <1 1 10 2 12 12141811 <1 <1 <1 11 15 Example 3: CpG ODN enhance the therapeutic effect of murine IgGZa (which relates to human IgGI) but not murlne IgGI (which relates to human IgG2) anti-tumor antibody.
-77 CpG ODN when combined with antibody of murine subtype IgG2a dramatically promotes survival in mice having tumors. Mice were injected i.p. with 5000 T3C cells on day 0. They were then given 100 pg anti-idiotype monoclonal antibody as either IgGI (MS5A10) or IgG2a (MS1 1G6) on days 5, 7, and 10. In this model, the target antigen is the idiotype 5 expressed by the lymphoma cells. Therefore, the anti-tumor antibodies are also "anti idiotype." These antibodies (MS5AI0 and MS11G6) are simultaneously both anti-tumor antibodies and anti-idiotype antibodies. Twenty micrograms of CpG ODN 1826 (5'TCCATGACGTTCCTGACGTT3'; SEQ ID NO:560) was given at the same time. Results are shown in Figure 6. Untreated controls had a median survival time (MST) of 17 10 days after inoculation with tumor. Mice treated with murine IgGl antibody plus CpG ODN had survival that was similar to those treated with murine IgGl antibody alone (MST 28 days and 27 days, respectively). In contrast, mice treated with murine IgG2a plus CpG ODN had survival that was significantly improved when compared to mice treated with murine IgG2a alone (MST 45 days and 37 days, respectively). 15 The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope 20 of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention. 25 All references, patents and patent publications that are recited in this application are incorporated in their entirety herein by reference. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and 30 "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the 35 common general knowledge in Australia.

权利要求:
Claims (78)
[1] 1. A method for treating or preventing cancer, comprising: administering to a subject having or at risk of developing cancer an effective amount 5 to upregulate CD20 expression of a nucleic acid, and an anti-CD20 antibody.
[2] 2. The method of claim 1, wherein the nucleic acid is an immunostimulatory CpG nucleic acid having an unmethylated CpG motif. 10
[3] 3. The method of claim 1, wherein the nucleic acid is an immunostimulatory T-rich nucleic acid.
[4] 4. The method of claim 1, wherein the nucleic acid is an immunostimulatory p6ly-G is nucleic acid.
[5] 5. The method of claim 1, wherein the nucleic acid is bacterial DNA.
[6] 6. The method of claim 1, wherein the nucleic acid is eukaryotic DNA. 20
[7] 7. The method of claim 1, wherein the cancer is B-cell lymphoma associated with low levels of CD20 expression.
[8] 8. The method of claim 7, wherein the B-cell lymphoma is B-cell chronic lymphocytic 25 leukemia (B-CLL).
[9] 9. The method of claim 7, wherein the B-cell lymphoma is a marginal zone lymphoma.
[10] 10. The method of claim 1, wherein the anti-CD20 antibody is C2B8. 30
[11] 11. The method of claim 1, wherein the anti-CD20 antibody is Rituximab. WO 01/97843 PCT/USOI/20154 -79
[12] 12. The method of claim 1, wherein the nucleic acid does not hybridize with genomic DNA or RNA under stringent conditions.
[13] 13. The method of claim 1, wherein the nucleic acid has a modified backbone. 5
[14] 14. The method of claim 13, wherein the modified backbone is a phosphate backbone modification.
[15] 15. The method of claim 13, wherein the modified backbone is a peptide modified 1o oligonucleotide backbone.
[16] 16. The method of claim 1, wherein the nucleic acid is an immunostimulatory nucleic acid. 15
[17] 17. The method of claim 1, wherein the nucleic acid is 8 to 40 nucleotides in length.
[18] 18. The method of claim 1, wherein the nucleic acid is isolated.
[19] 19. The method of claim 1, wherein the nucleic acid is a synthetic nucleic acid. 20
[20] 20. The method of claim 1, wherein the nucleic acid and the anti-CD20 antibody are administered together.
[21] 21. The method of claim 1, wherein the nucleic acid and the anti-CD20 antibody are 25 administered separately.
[22] 22. A method for diagnosing lymphoma, comprising: isolating a B cell from a subject having or suspected of having a type of lymphoma and identifying a change in a cell surface marker when the B cell is contacted with an 30 immunostimulatory nucleic acid, wherein the cell surface marker induced on the B cell is indicative of the type of lymphoma. WO 01/97843 PCT/USOI/20154 -80
[23] 23. The method of claim 22, further comprising a method for treating cancer by administering to the subject an immunostimulatory nucleic acid and an antibody specific for the cell surface marker induced on the B cell in order to treat the cancer. 5
[24] 24. A method for treating or preventing cancer, comprising: administering to a subject having or at risk of developing cancer an effective amount to induce expression of a surface antigen on a cancer cell surface, of a nucleic acid, and administering to the subject an antibody selected from the group consisting of an anti CD22 antibody and an anti-CD19 antibody. 10
[25] 25. The method of claim 24, wherein the nucleic acid is an immunostimulatory CpG nucleic acid having an unmethylated CpG motif.
[26] 26. The method of claim 24, wherein the nucleic acid is an immunostimulatory T-rich 15 nucleic acid.
[27] 27. The method of claim 24, wherein the nucleic acid is an immunostimulatory poly-G nucleic acid. 20
[28] 28. The method of claim 24, wherein the nucleic acid is bacterial DNA.
[29] 29. The method of claim 24, wherein the nucleic acid is eukaryotic DNA.
[30] 30. The method of claim 24, wherein the anti-CD22 antibody is a human IgG1 antibody. 25
[31] 31. The method of claim 24, wherein the anti-CD22 antibody is a murine IgG2a antibody.
[32] 32. The method of claim 24, wherein the anti-CD19 antibody is a human IgGI antibody. 30
[33] 33. The method of claim 24, wherein the anti-CD19 antibody is a murine IgG2a antibody.
[34] 34. A method for treating lymphoma, comprising: WO 01/97843 PCT/USO1/20154 -81 isolating a B cell from a subject having lymphoma, identifying a surface antigen which is not expressed or which is expressed on the surface of the B cell in an amount lower than that of a control B cell, administering to the subject an antibody specific for the identified surface antigen and 5 an immunostimulatory nucleic acid in order to treat the cancer, wherein the immunostimulatory nucleic acid is administered in an effective amount to upregulate expression of the surface antigen on the cancer cell surface.
[35] 35. The method of claim 34, wherein the surface antigen is CD20. 10
[36] 36. The method of claim 34, wherein the surface antigen is CD40.
[37] 37. The method of claim 34, wherein surface antigen is CD22. 15
[38] 38. The method of claim 34, wherein surface antigen is CD19.
[39] 39. The method of claim 34, wherein the lymphoma is B-CLL.
[40] 40. The method of claim 34, wherein the lymphoma is marginal zone lymphoma. 20
[41] 41. The method of claim 34, wherein the antibody is a human IgGI antibody.
[42] 42. The method of claim 34, wherein the antibody is a urine IgG2a antibody. 25
[43] 43. A method for treating a lymphoma resistant to antibody therapy, comprising: administering to a subject having a lymphoma resistant to therapy with an antibody specific for a surface antigen, an antibody specific for the surface antigen to which the lymphoma is resistant and a nucleic acid in order to treat the lymphoma, wherein the nucleic acid is administered in an effective amount to upregulate expression of the surface antigen on 30 the lymphoma cell surface.
[44] 44. The method of claim 43, wherein the surface antigen is CD20. WO 01/97843 PCT/USOI/20154 -82
[45] 45. The method of claim 44, wherein the antibody is Rituximab.
[46] 46. The method of claim 43, wherein the surface antigen is CD40. 5
[47] 47. The method of claim 43, wherein the surface antigen is CD22.
[48] 48. The method of claim 43, wherein the surface antigen is CD19. 10
[49] 49. The method of claim 43, wherein the antibody is a human IgG1 antibody.
[50] 50. The method of claim 43, wherein the antibody is a murine IgG2a antibody.
[51] 51. The method of claim 43, further comprising administering an anti-cancer therapy. 15
[52] 52. The method of claim 51, wherein the anti-cancer therapy is selected from the group consisting of a chemotherapeutic agent or a cancer vaccine.
[53] 53. The method of claim 52, wherein the chemotherapeutic agent is selected from the 20 group consisting of methotrexate, vincristine, adriamycin, cisplatin, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol, valrubicin, Novantrone/Mitroxantrone, Evacet/liposomal doxorubicin, Yewtaxan/Paclitaxel, Taxol/Paclitaxel, Furtulon/Doxifluridine, Cyclopax/oral paclitaxel, SPU-077/Cisplatin, HMR 1275/Flavopiridol, BMS-182751/oral platinum, Leustatin/Cladribine, Paxex/Paclitaxel, Doxil/liposomal doxorubicin, Caelyx/liposomal 25 doxorubicin, Fludara/Fludarabine, Pharmarubicin/Epirubicin, DepoCyt, Caetyx/liposomal doxorubicin, Gemzar/Gemcitabine, Ifes[Mesnex/Ifosamide, Vumon/Teniposide, Paraplatin/Carboplatin, Plantinol/cisplatin, Vepeside/Etoposide, Taxotere/Docetaxel, prodrug of guanine arabinoside, nitrosoureas, Asparaginase, Busulfan, Carboplatin, Chlorombucil, Cytarabine HCl, Daunorubicin HCI, Etoposide (VP 16-213), Hydroxyurea 30 (hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Interferon Alfa-2b, Lomustine (CCNU), Mechlorethamine HCl (nitrogen mustard), Mercaptopurine, Mesna, Mitoxantrone HCI, WO 01/97843 PCT/USO1/20154 -83 Procarbazine HCl, Thioguanine, Thiotepa, Vinblastine sulfate, Azacitidine, Interleukin 2, Pentostatin (2'deoxycoformycin), Teniposide (VM-26), GM-CSF, and Vindesine sulfate.
[54] 54. The method of claim 52, wherein the chemotherapeutic agent is selected from the 5 group consisting of methotrexate, vincristine, adriamycin, cisplatin, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol, valrubicin, Novantrone/Mitroxantrone, Evacet/liposomal doxorubicin, Yewtaxan/Paclitaxel, Taxol/Paclitaxel, SPU-077/Cisplatin, HMR 1275/Flavopiridol, BMS-182751/oral platinum, Leustatin/Cladribine, Paxex/Paclitaxel, Doxilliposomal doxorubicin, Caelyx/liposomal doxorubicin, Fludara/Fludarabine, 10 Pharmarubicin/Epirubicin, DepoCyt, Caetyx/liposomal doxorubicin, Gemzar/Gemcitabine, Ifes/Mesnex/Ifosamide, Vumon/Teniposide, Paraplatin/Carboplatin, Plantinol/cisplatin, Vepeside/Etoposide, Taxotere/Docetaxel, prodrug of guanine arabinoside, nitrosoureas, alkylating agents such as melphalan and cyclophosphamide, Asparaginase, Busulfan, Carboplatin, Chlorombucil, Cytarabine HCl, Daunorubicin HCl, Etoposide (VP16-213), 15 Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Interferon Alfa-2b, Lomustine (CCNU), Mechlorethamine HCI (nitrogen mustard), Mercaptopurine, Mitoxantrone HC1, Procarbazine HCl, Thioguanine, Thiotepa, Vinblastine sulfate, Azacitidine, Interleukin 2, Pentostatin (2'deoxycoformycin), Teniposide (VM-26), GM-CSF, and Vindesine sulfate. 20
[55] 55. The method of claim 52, wherein the cancer vaccine is selected from the group consisting of EGF, Anti-idiotypic cancer vaccines, Gp75 antigen, GMK melanoma vaccine, MGV ganglioside conjugate vaccine, Her2/neu, Ovarex, M-Vax, 0-Vax, L-Vax, STn-KHL theratope, BLP25 (MUC-1), liposomal idiotypic vaccine, Melacine, peptide antigen vaccines, 25 toxin/antigen vaccines, MVA-based vaccine, PACIS, BCG vaccine, TA-HPV, TA-CIN, DISC-virus and ImmuCyst/TheraCys.
[56] 56. A method for treating cancer in a human, comprising: administering to a human an immunostimulatory nucleic acid and an antibody of IgG 1 30 isotype, which binds to a cell surface antigen of a cancer cell and wherein the nucleic acid and the antibody are administered in an effective amount for killing the cancer cell. WO 01/97843 PCT/USOI/20154 -84
[57] 57. The method of claim 56, wherein the nucleic acid is an immunostimulatory CpG nucleic acid having an unmethylated CpG motif.
[58] 58. The method of claim 56, wherein the nucleic acid is an immunostimulatory T-rich 5 nucleic acid.
[59] 59. The method of claim 56, wherein the nucleic acid is an immunostimulatory poly-G nucleic acid. 10
[60] 60. The method of claim 56, wherein the nucleic acid is bacterial DNA.
[61] 61. The method of claim 56, wherein the nucleic acid is eukaryotic DNA.
[62] 62. The method of claim 56, wherein the nucleic acid has a modified backbone. 15
[63] 63. The method of claim 62, wherein the modified backbone is a phosphate backbone modification.
[64] 64. The method of claim 62, wherein the modified backbone is a peptide modified 20 oligonucleotide backbone.
[65] 65. The method of claim 56, wherein the nucleic acid is an immunostimulatory nucleic acid. 25
[66] 66. The method of claim 56, wherein the nucleic acid is 8 to 40 nucleotides in length.
[67] 67. The method of claim 56, wherein the nucleic acid is isolated.
[68] 68. The method of claim 56, wherein the nucleic acid is a synthetic nucleic acid. 30
[69] 69. The method of claim 56, wherein the nucleic acid and the antibody are administered together. WO 01/97843 PCT/USO1/20154 -85
[70] 70. The method of claim 56, wherein the nucleic acid and the antibody are administered separately. 5
[71] 71. The method of claim 56, further comprising administering an anti-cancer therapy.
[72] 72. The method of claim 71, wherein the anti-cancer therapy is selected from the group consisting of a chemotherapeutic agent and a cancer vaccine. 10
[73] 73. The method of claim 72, wherein the chemotherapeutic agent is selected from the group consisting of methotrexate, vincristine, adriamycin, cisplatin, non-sugar containing chloroethylnitrosoureas, 5-fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol, fragyline, Meglamine GLA, valrubicin, carmustaine and poliferposan, MM1270, BAY 12-9566, RAS famesyl transferase inhibitor, famesyl transferase inhibitor, MMP, 15 MTA/LY231514, LY264618/Lometexol, Glamolec, CI-994, TNP-470, Hycamtin/Topotecan, PKC412, Valspodar/PSC833, Novantrone/Mitroxantrone, Metaret/Suramin, Batimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340, AG3433, IncelVX-710, VX-853, ZD0101, IS1641, ODN 698, TA 2516/Marmistat, BB2516/Marmistat, CDP 845, D2163, PD 183805, DX895 If, Lemonal DP 2202, FK 317, Picibanil/OK-432, AD 32/Valrubicin, 20 Metastron/strontium derivative, Temodal/Temozolomide, Evacet/liposomal doxorubicin, Yewtaxan/Paclitaxel, Taxol/Paclitaxel, Xeload/Capecitabine, Furtulon/Doxifluridine, Cyclopax/oral paclitaxel, Oral Taxoid, SPU-077/Cisplatin, HMR 1275/Flavopiridol, CP-358 (774)/EGFR, CP-609 (754)/RAS oncogcnc inhibitor, BMS-182751/oral platinum, UFT(Tegafur/Uracil), Ergamisol/Levamisole, Eniluracil/776C85/5FU enhancer, 25 Campto/Levamisole, Camptosar/Irinotecan, Tumodex/Ralitrexed, Leustatin/Cladribine, Paxex/Paclitaxel, Doxil/liposomal doxorubicin, Caelyx/liposomal doxorubicin, Fludara/Fludarabine, Pharmarubicin/Epirubicin, DepoCyt, ZDI 839, LU 79553/Bis Naphtalimide, LU 103793/Dolastain, Cactyx/liposomal doxorubicin, Gemzar/Gemcitabine, ZD 0473/Anormed, YM 116, Iodine seeds, CDK4 and CDK2 inhibitors, PARP inhibitors, 30 D4809/Dexifosamide, Ifes/Mesnex/Ifosamide, Vumon/Teniposide, Paraplatin/Carboplatin, Plantinol/cisplatin, Vepeside/Etoposide, ZD 9331, Taxotere/Docetaxel, prodrug of guanine arabinoside, Taxane Analog, nitrosoureas, alkylating agents such as melphalan and WO 01/97843 PCT/USO1/20154 -86 cyclophosphamide, Aminoglutethimide, Asparaginase, Busulfan, Carboplatin, Chlorombucil, Cytarabine HCl, Dactinomycin, Daunorubicin HCI, Estramustine phosphate sodium, Etoposide (VP 16-213), Floxuridine, Fluorouracil (5-FU), Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Interferon Alfa-2b, Leuprolide acetate 5 (LHRH-releasing factor analogue), Lomustine (CCNU), Mechlorethamine HCl (nitrogen mustard), Mercaptopurine, Mesna, Mitotane (o,p'-DDD), Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Amsacrine (m-AMSA), Azacitidine, Erythropoietin, Hexamethylmelamine (HMM), Interleukin 2, Mitoguazone (methyl-GAG; methyl glyoxal 10 bis-guanylhydrazone; MGBG), Pentostatin (2'deoxycoformycin), Semustine (methyl CCNU), Teniposide (VM-26), GM-CSF, and Vindesine sulfate.
[74] 74. The method of claim 72, wherein the chemotherapeutic agent is selected from the group consisting of methotrexate, vincristine, adriamycin, cisplatin, mitomycin C, bleomycin, 15 doxorubicin, dacarbazine, taxol, valrubicin, Novantrone/Mitroxantrone, Evacet/liposonal doxorubicin, Yewtaxan/Paclitaxel, TaxoUPaclitaxel, SPU-077/Cisplatin, HMvR 1275/Flavopiridol, BMS-182751/oral platinum, Leustatin/Cladribine, Paxex/Paclitaxel, Doxil/liposomal doxorubicin, Caelyx/liposomal doxorubicin, Fludara/Fludarabine, Pharmarubicin/Epirubicin, DepoCyt, Caetyx/liposomal doxorubicin, Gemzar/Gemcitabine, 20 Ifes/Mesnex/Ifosamide, Vumon/Teniposide, Paraplatin/Carboplatin, Plantinol/cisplatin, Vepeside/Etoposide, Taxotere/Docetaxel, prodrug of guanine arabinoside, nitrosoureas, alkylating agents such as melphalan and cyclophosphamide, Asparaginase, Busulfan, Carboplatin, Chlorombucil, Cytarabinc HCl, Daunorubicin HCl, Etoposide (VP16-213), Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Interferon Alfa-2b, 25 Lomustine (CCNU), Mechlorethamine HCl (nitrogen mustard), Mercaptopurine, Mitoxantrone HCl, Procarbazine HCI, Thioguanine, Thiotepa, Vinblastine sulfate, Azacitidine, Interleukin 2, Pentostatin (2'deoxycoformycin), Teniposide (VM-26), GM-CSF, and Vindesine sulfate. 30
[75] 75. The method of claim 72, wherein the cancer vaccine is selected from the group consisting of EGF, Anti-idiotypic cancer vaccines, Gp75 antigen, GMK melanoma vaccine, MGV ganglioside conjugate vaccine, Her2/neu, Ovarex, M-Vax, O-Vax, L-Vax, STn-KHL -87 theratope, BLP25(MUC-1), liposomal idiotypic vaccine, Melacine, peptide antigen vaccines, toxin/antigen vaccines, MVA-based vaccine, PACIS, BCG vaccine, TA-HPV, TA-CIN, DISC-virus andhnmuCyst/TheraCys.
[76] 76. A kit when used for screening for the expression of a cell surface antigen, comprising: a package including at least two containers, the first container housing an immunostimulatory nucleic acid, the second container housing an antibody specific for a cell surface antigen, and instructions for screening a cell to determine whether the immunostimulatory nucleic acid upregulates expression of the cell surface antigen.
[77] 77. The kit of claim 76, wherein the antibody is selected from the group consisting of an anti-CD20 antibody, an anti-CD 19 antibody, and an anti-CD22 antibody.
[78] 78. A method according to claims 1 to 75 substantially as hereinbefore described with reference to the Figures and Examples and/or a kit according to claims 76 and 77 substantially as hereinbefore described with reference to the Figures and Examples.

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同族专利:

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公开号 | 公开日

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US7534772B2|2009-05-19|

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AU2001270134B2|2006-06-15|

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AU2009203061C1|2012-06-28|

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AU2009203061A1|2009-08-20|

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DK1296714T3|2009-12-07|

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PT1296714E|2009-10-15|

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AU2009203061B2|2012-02-23|

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AU7013401A|2002-01-02|

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CA2410371A1|2001-12-27|

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AU2006216542B2|2009-04-30|

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EP1296714A2|2003-04-02|

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ES2332444T3|2010-02-05|

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AU2006216542A1|2006-10-12|

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JP2003535907A|2003-12-02|

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DE60139689D1|2009-10-08|

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SI1296714T1|2010-01-29|

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WO2001097843A3|2003-01-23|

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CA2410371C|2015-11-17|

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EP1296714B1|2009-08-26|

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US20030026801A1|2003-02-06|

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AT440618T|2009-09-15|

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WO2001097843A2|2001-12-27|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

US3854480A|1969-04-01|1974-12-17|Alza Corp|Drug-delivery system|

---

US3906092A|1971-11-26|1975-09-16|Merck & Co Inc|Stimulation of antibody response|

---

US4469863A|1980-11-12|1984-09-04|Ts O Paul O P|Nonionic nucleic acid alkyl and aryl phosphonates and processes for manufacture and use thereof|

---

US4675189A|1980-11-18|1987-06-23|Syntex Inc.|Microencapsulation of water soluble active polypeptides|

---

US5023243A|1981-10-23|1991-06-11|Molecular Biosystems, Inc.|Oligonucleotide therapeutic agent and method of making same|

---

US4452775A|1982-12-03|1984-06-05|Syntex Inc.|Cholesterol matrix delivery system for sustained release of macromolecules|

---

US5567610A|1986-09-04|1996-10-22|Bioinvent International Ab|Method of producing human monoclonal antibodies and kit therefor|

---

US5565354A|1986-09-05|1996-10-15|Sandoz Ltd.|Production of human monoclonal antibodies specific for hepatitis B surface antigen|

---

US5075109A|1986-10-24|1991-12-24|Southern Research Institute|Method of potentiating an immune response|

---

US6214804B1|1989-03-21|2001-04-10|Vical Incorporated|Induction of a protective immune response in a mammal by injecting a DNA sequence|

---

US5133974A|1989-05-05|1992-07-28|Kv Pharmaceutical Company|Extended release pharmaceutical formulations|

---

US5786189A|1989-11-29|1998-07-28|Smithkline Beecham Biologicals |Vaccine|

---

US5599797A|1991-10-15|1997-02-04|Isis Pharmaceuticals, Inc.|Oligonucleotides having phosphorothioate linkages of high chiral purity|

---

US5212295A|1990-01-11|1993-05-18|Isis Pharmaceuticals|Monomers for preparation of oligonucleotides having chiral phosphorus linkages|

---

US5506212A|1990-01-11|1996-04-09|Isis Pharmaceuticals, Inc.|Oligonucleotides with substantially chirally pure phosphorothioate linkages|

---

US5248670A|1990-02-26|1993-09-28|Isis Pharmaceuticals, Inc.|Antisense oligonucleotides for inhibiting herpesviruses|

---

DE69127627T2|1990-08-29|1998-02-19|Genpharm Int|Production and Use Non-human transgene heterologous antibodies for production|

---

US5208146A|1990-11-05|1993-05-04|The Regents Of The University Of California|Murine monoclonal anti-idiotype antibodies|

---

US5512668A|1991-03-06|1996-04-30|Polish Academy Of Sciences|Solid phase oligonucleotide synthesis using phospholane intermediates|

---

US5359052A|1991-08-05|1994-10-25|Polish Academy Of Sciences|Chalcophospholanes useful in the synthesis of oligonucleoside phosphorothioates, phosphorodithioates and related selenates|

---

US6015886A|1993-05-24|2000-01-18|Chemgenes Corporation|Oligonucleotide phosphate esters|

---

US5407686A|1991-11-27|1995-04-18|Sidmak Laboratories, Inc.|Sustained release composition for oral administration of active ingredient|

---

US5585479A|1992-07-24|1996-12-17|The United States Of America As Represented By The Secretary Of The Navy|Antisense oligonucleotides directed against human ELAM-I RNA|

---

US5679647A|1993-08-26|1997-10-21|The Regents Of The University Of California|Methods and devices for immunizing a host against tumor-associated antigens through administration of naked polynucleotides which encode tumor-associated antigenic peptides|

---

US5849719A|1993-08-26|1998-12-15|The Regents Of The University Of California|Method for treating allergic lung disease|

---

IL111658D0|1993-11-16|1995-01-24|Genta Inc|Chirally enriched synthetic phosphonate oligomers|

---

US6727230B1|1994-03-25|2004-04-27|Coley Pharmaceutical Group, Inc.|Immune stimulation by phosphorothioate oligonucleotide analogs|

---

WO1995026204A1|1994-03-25|1995-10-05|Isis Pharmaceuticals, Inc.|Immune stimulation by phosphorothioate oligonucleotide analogs|

---

US5534615A|1994-04-25|1996-07-09|Genentech, Inc.|Cardiac hypertrophy factor and uses therefor|

---

US20030026782A1|1995-02-07|2003-02-06|Arthur M. Krieg|Immunomodulatory oligonucleotides|

---

EP1167377B2|1994-07-15|2012-08-08|University of Iowa Research Foundation|Immunomodulatory oligonucleotides|

---

US6429199B1|1994-07-15|2002-08-06|University Of Iowa Research Foundation|Immunostimulatory nucleic acid molecules for activating dendritic cells|

---

US6207646B1|1994-07-15|2001-03-27|University Of Iowa Research Foundation|Immunostimulatory nucleic acid molecules|

---

US20030050263A1|1994-07-15|2003-03-13|The University Of Iowa Research Foundation|Methods and products for treating HIV infection|

---

US7935675B1|1994-07-15|2011-05-03|University Of Iowa Research Foundation|Immunostimulatory nucleic acid molecules|

---

US6239116B1|1994-07-15|2001-05-29|University Of Iowa Research Foundation|Immunostimulatory nucleic acid molecules|

---

US5736152A|1995-10-27|1998-04-07|Atrix Laboratories, Inc.|Non-polymeric sustained release delivery system|

---

US5969135A|1995-11-02|1999-10-19|Icn Pharmaceuticals, Inc.|Oligonucleotide analogs with an amino acid or a modified amino alcohol residue|

---

US5780448A|1995-11-07|1998-07-14|Ottawa Civic Hospital Loeb Research|DNA-based vaccination of fish|

---

US5856465A|1996-05-24|1999-01-05|Polska Akademia Nauk Centrum Badan Molekularnych I Makromolekularnych|Compositions and methods for the synthesis of chirally pure organophosphorus nucleoside derivatives|

---

EP0855184A1|1997-01-23|1998-07-29|Grayson B. Dr. Lipford|Pharmaceutical composition comprising a polynucleotide and an antigen especially for vaccination|

---

WO1998037919A1|1997-02-28|1998-09-03|University Of Iowa Research Foundation|USE OF NUCLEIC ACIDS CONTAINING UNMETHYLATED CpG DINUCLEOTIDE IN THE TREATMENT OF LPS-ASSOCIATED DISORDERS|

---

EP1005368B1|1997-03-10|2009-09-02|Ottawa Hospital Research Institute|Use of nucleic acids containing unmethylated CpG dinucleotide in combination with alum asadjuvants|

---

US6406705B1|1997-03-10|2002-06-18|University Of Iowa Research Foundation|Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant|

---

US6306393B1|1997-03-24|2001-10-23|Immunomedics, Inc.|Immunotherapy of B-cell malignancies using anti-CD22 antibodies|

---

US6183744B1|1997-03-24|2001-02-06|Immunomedics, Inc.|Immunotherapy of B-cell malignancies using anti-CD22 antibodies|

---

DE69838294T2|1997-05-20|2009-08-13|Ottawa Health Research Institute, Ottawa|Process for the preparation of nucleic acid constructs|

---

WO1999001154A1|1997-07-03|1999-01-14|University Of Iowa Research Foundation|Method for inhibiting immunostimulatory dna associated responses|

---

CA2323929C|1998-04-03|2004-03-09|University Of Iowa Research Foundation|Methods and products for stimulating the immune system using immunotherapeutic oligonucleotides and cytokines|

---

AT305507T|1998-05-14|2005-10-15|Coley Pharm Gmbh|METHOD FOR REGULATING HEMATOPOIESE BY USING CPG OLIGONUCLEOTIDE|

---

NZ508927A|1998-05-22|2003-12-19|Ottawa Health Research Inst|Methods and products for inducing mucosal immunity|

---

JP2002522511A|1998-08-11|2002-07-23|アイデックファーマスーティカルズコーポレイション|Combination therapy of B cell lymphoma including administration of anti-CD20 antibody|

---

CA2350058C|1998-11-09|2015-10-13|Idec Pharmaceuticals Corporation|Treatment of hematologic malignancies associated with circulating tumor cells using chimeric anti-cd20 antibody|

---

AU5937100A|1999-06-23|2001-01-09|Datum, Inc.|System and method for providing a trusted third party clock and trusted local clock|

---

DE60022665T2|1999-09-25|2006-06-22|Coley Pharmaceutical Gmbh|IMMUNOSTIMULATING NUCLEIC ACIDS|

---

US6949520B1|1999-09-27|2005-09-27|Coley Pharmaceutical Group, Inc.|Methods related to immunostimulatory nucleic acid-induced interferon|

---

US20010044416A1|2000-01-20|2001-11-22|Mccluskie Michael J.|Immunostimulatory nucleic acids for inducing a Th2 immune response|

---

US7585847B2|2000-02-03|2009-09-08|Coley Pharmaceutical Group, Inc.|Immunostimulatory nucleic acids for the treatment of asthma and allergy|

---

US20020156033A1|2000-03-03|2002-10-24|Bratzler Robert L.|Immunostimulatory nucleic acids and cancer medicament combination therapy for the treatment of cancer|

---

US20040131628A1|2000-03-08|2004-07-08|Bratzler Robert L.|Nucleic acids for the treatment of disorders associated with microorganisms|

---

US20020091097A1|2000-09-07|2002-07-11|Bratzler Robert L.|Nucleic acids for the prevention and treatment of sexually transmitted diseases|

---

AT510850T|2000-09-15|2011-06-15|Coley Pharm Gmbh|PROCESS FOR HIGH-SPEED SCREENING OF CPG-BASED IMMUNOGONISTS AND ANTAGONISTS|

---

WO2002069369A2|2000-12-08|2002-09-06|Coley Pharmaceutical Gmbh|Cpg-like nucleic acids and methods of use thereof|

---

WO2002053141A2|2000-12-14|2002-07-11|Coley Pharmaceutical Group, Inc.|Inhibition of angiogenesis by nucleic acids|

---

US20030050268A1|2001-03-29|2003-03-13|Krieg Arthur M.|Immunostimulatory nucleic acid for treatment of non-allergic inflammatory diseases|

---

AU2002318944A1|2001-08-01|2003-02-17|Coley Pharmaceutical Gmbh|Methods and compositions relating to plasmacytoid dendritic cells|

---

NZ552377A|2001-08-17|2008-08-29|Coley Pharm Gmbh|Combination motif immune stimulatory oligonucleotides with improved activity|

---

IL160837D0|2001-10-05|2004-08-31|Coley Pharm Gmbh|Toll-like receptor 3 signaling agonists and antagonists|

---

AU2002360278A1|2001-10-12|2003-11-11|Coley Pharmaceutical Gmbh|Methods and products for enhancing immune responses using imidazoquinoline compounds|

---

JP4846200B2|2002-04-04|2011-12-28|コーリーファーマシューティカルゲーエムベーハー|Immunostimulatory G and U-containing oligoribonucleotides|

---

WO2003103586A2|2002-06-05|2003-12-18|Coley Pharmaceutical Group, Inc.|Method for treating autoimmune or inflammatory diseases with combinations of inhibitory oligonucleotides and small molecule antagonists of immunostimulatory cpg nucleic acids|

---

US7605138B2|2002-07-03|2009-10-20|Coley Pharmaceutical Group, Inc.|Nucleic acid compositions for stimulating immune responses|

---

US7807803B2|2002-07-03|2010-10-05|Coley Pharmaceutical Group, Inc.|Nucleic acid compositions for stimulating immune responses|

---

US7569553B2|2002-07-03|2009-08-04|Coley Pharmaceutical Group, Inc.|Nucleic acid compositions for stimulating immune responses|

---

US20040053880A1|2002-07-03|2004-03-18|Coley Pharmaceutical Group, Inc.|Nucleic acid compositions for stimulating immune responses|

---

US7576066B2|2002-07-03|2009-08-18|Coley Pharmaceutical Group, Inc.|Nucleic acid compositions for stimulating immune responses|

---

AR040996A1|2002-08-19|2005-04-27|Coley Pharm Group Inc|IMMUNE STIMULATING NUCLEIC ACIDS|

---

CA2503693A1|2002-10-29|2004-05-13|Coley Pharmaceutical Group, Ltd.|Use of cpg oligonucleotides in the treatment of hepatitis c virus infection|

---

WO2004053104A2|2002-12-11|2004-06-24|Coley Pharmaceutical Group, Inc.|5’ cpg nucleic acids and methods of use|

---

WO2004087203A2|2003-04-02|2004-10-14|Coley Pharmaceutical Group, Ltd.|Immunostimulatory nucleic acid oil-in-water formulations for topical application|

---

EA200600069A1|2003-06-20|2006-08-25|Коли Фармасьютикал Гмбх|LOW-MOLECULAR ANTAGONISTS OF TOLL-SIMILAR RECEPTORS |

---

EP1663316A2|2003-09-25|2006-06-07|Coley Pharmaceutical Group, Inc.|Nucleic acid lipophilic conjugates|

---

US20050215501A1|2003-10-24|2005-09-29|Coley Pharmaceutical Group, Inc.|Methods and products for enhancing epitope spreading|

---

US20050239733A1|2003-10-31|2005-10-27|Coley Pharmaceutical Gmbh|Sequence requirements for inhibitory oligonucleotides|

---

US20050100983A1|2003-11-06|2005-05-12|Coley Pharmaceutical Gmbh|Cell-free methods for identifying compounds that affect toll-like receptor 9 signaling|

---

AU2005243250A1|2004-04-02|2005-11-24|Coley Pharmaceutical Gmbh|Immunostimulatory nucleic acids for inducing IL-10 responses|

---

JP2008506683A|2004-07-18|2008-03-06|コーリーファーマシューティカルグループ，リミテッド|Methods and compositions for inducing innate immune responses|

---

MY159370A|2004-10-20|2016-12-30|Coley Pharm Group Inc|Semi-soft-class immunostimulatory oligonucleotides|

---

EP1851314A2|2005-02-24|2007-11-07|Coley Pharmaceutical Group, Inc.|Immunostimulatory oligonucleotides|

---

EP1957647B1|2005-11-25|2015-03-04|Zoetis Belgium S.A.|Immunostimulatory oligoribonucleotides|US6727230B1|1994-03-25|2004-04-27|Coley Pharmaceutical Group, Inc.|Immune stimulation by phosphorothioate oligonucleotide analogs|

---

US6429199B1|1994-07-15|2002-08-06|University Of Iowa Research Foundation|Immunostimulatory nucleic acid molecules for activating dendritic cells|

---

US20030026782A1|1995-02-07|2003-02-06|Arthur M. Krieg|Immunomodulatory oligonucleotides|

---

US6207646B1|1994-07-15|2001-03-27|University Of Iowa Research Foundation|Immunostimulatory nucleic acid molecules|

---

US6239116B1|1994-07-15|2001-05-29|University Of Iowa Research Foundation|Immunostimulatory nucleic acid molecules|

---

US7935675B1|1994-07-15|2011-05-03|University Of Iowa Research Foundation|Immunostimulatory nucleic acid molecules|

---

CA2244946C|1996-01-30|2010-04-13|The Regents Of The University Of California|Gene expression vectors which generate an antigen specific immune response and methods of using the same|

---

EP0855184A1|1997-01-23|1998-07-29|Grayson B. Dr. Lipford|Pharmaceutical composition comprising a polynucleotide and an antigen especially for vaccination|

---

US6406705B1|1997-03-10|2002-06-18|University Of Iowa Research Foundation|Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant|

---

US6274591B1|1997-11-03|2001-08-14|Joseph F. Foss|Use of methylnaltrexone and related compounds|

---

US20030158220A1|1997-11-03|2003-08-21|Foss Joseph F.|Use of methylnaltrexone and related compounds to treat chronic opioid use side effects|

---

CA2323929C|1998-04-03|2004-03-09|University Of Iowa Research Foundation|Methods and products for stimulating the immune system using immunotherapeutic oligonucleotides and cytokines|

---

AT305507T|1998-05-14|2005-10-15|Coley Pharm Gmbh|METHOD FOR REGULATING HEMATOPOIESE BY USING CPG OLIGONUCLEOTIDE|

---

NZ508927A|1998-05-22|2003-12-19|Ottawa Health Research Inst|Methods and products for inducing mucosal immunity|

---

US20030022854A1|1998-06-25|2003-01-30|Dow Steven W.|Vaccines using nucleic acid-lipid complexes|

---

US6693086B1|1998-06-25|2004-02-17|National Jewish Medical And Research Center|Systemic immune activation method using nucleic acid-lipid complexes|

---

JP5084984B2|1999-02-17|2012-11-28|シーエスエル、リミテッド|Immunogenic complexes and methods related thereto|

---

US6977245B2|1999-04-12|2005-12-20|The United States Of America As Represented By The Department Of Health And Human Services|Oligodeoxynucleotide and its use to induce an immune response|

---

AU4343700A|1999-04-12|2000-11-14|Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services, The|Oligodeoxynucleotide and its use to induce an immune response|

---

AU777970C|1999-05-07|2006-08-17|F. Hoffman-La Roche Ag|Treatment of autoimmune diseases with antagonists which bind to B cell surface markers|

---

AU778863B2|1999-07-12|2004-12-23|Genentech Inc.|Blocking immune response to a foreign antigen using an antagonist which binds to CD20|

---

KR100917101B1|2000-08-04|2009-09-15|도요 보세키 가부시키가이샤|Flexible metal laminate and production method thereof|

---

DE60022665T2|1999-09-25|2006-06-22|Coley Pharmaceutical Gmbh|IMMUNOSTIMULATING NUCLEIC ACIDS|

---

US6949520B1|1999-09-27|2005-09-27|Coley Pharmaceutical Group, Inc.|Methods related to immunostimulatory nucleic acid-induced interferon|

---

US20010044416A1|2000-01-20|2001-11-22|Mccluskie Michael J.|Immunostimulatory nucleic acids for inducing a Th2 immune response|

---

US7585847B2|2000-02-03|2009-09-08|Coley Pharmaceutical Group, Inc.|Immunostimulatory nucleic acids for the treatment of asthma and allergy|

---

JP5062606B2|2000-02-16|2012-10-31|ジェネンテック，インコーポレイテッド|Use of agonists and antagonists to modulate the activity of TNF-related molecules|

---

US20040131628A1|2000-03-08|2004-07-08|Bratzler Robert L.|Nucleic acids for the treatment of disorders associated with microorganisms|

---

US7030219B2|2000-04-28|2006-04-18|Johns Hopkins University|B7-DC, Dendritic cell co-stimulatory molecules|

---

JP2004530629A|2000-06-07|2004-10-07|バイオシネクサスインコーポレーテッド|Immunostimulatory RNA / DNA hybrid molecule|

---

AT510850T|2000-09-15|2011-06-15|Coley Pharm Gmbh|PROCESS FOR HIGH-SPEED SCREENING OF CPG-BASED IMMUNOGONISTS AND ANTAGONISTS|

---

US20020058029A1|2000-09-18|2002-05-16|Nabil Hanna|Combination therapy for treatment of autoimmune diseases using B cell depleting/immunoregulatory antibody combination|

---

TWI283575B|2000-10-31|2007-07-11|Eisai Co Ltd|Medicinal compositions for concomitant use as anticancer agent|

---

WO2002069369A2|2000-12-08|2002-09-06|Coley Pharmaceutical Gmbh|Cpg-like nucleic acids and methods of use thereof|

---

US7829084B2|2001-01-17|2010-11-09|Trubion Pharmaceuticals, Inc.|Binding constructs and methods for use thereof|

---

US7754208B2|2001-01-17|2010-07-13|Trubion Pharmaceuticals, Inc.|Binding domain-immunoglobulin fusion proteins|

---

US20030133939A1|2001-01-17|2003-07-17|Genecraft, Inc.|Binding domain-immunoglobulin fusion proteins|

---

US7666674B2|2001-07-27|2010-02-23|The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services|Use of sterically stabilized cationic liposomes to efficiently deliver CPG oligonucleotides in vivo|

---

CA2456193A1|2001-08-03|2003-03-27|Medarex, Inc.|Compositions comprising immunostimulatory oligonucleotides and uses thereof to enhance fc receptor-mediated immunotherapies|

---

CN1636067A|2001-08-03|2005-07-06|杰南技术公司|TACIs and BR3 polypeptides and uses thereof|

---

AU2002361468A1|2001-08-14|2003-03-18|The Government Of The United States Of America As Represented By The Secretary Of Health And Human S|Method for rapid generation of mature dendritic cells|

---

NZ552377A|2001-08-17|2008-08-29|Coley Pharm Gmbh|Combination motif immune stimulatory oligonucleotides with improved activity|

---

JP2005501917A|2001-09-07|2005-01-20|ザトラスティーズオブボストンユニバーシティ|Methods and compositions for treating immune complex related diseases|

---

IL160837D0|2001-10-05|2004-08-31|Coley Pharm Gmbh|Toll-like receptor 3 signaling agonists and antagonists|

---

AU2002360278A1|2001-10-12|2003-11-11|Coley Pharmaceutical Gmbh|Methods and products for enhancing immune responses using imidazoquinoline compounds|

---

US8466116B2|2001-12-20|2013-06-18|The Unites States Of America As Represented By The Secretary Of The Department Of Health And Human Services|Use of CpG oligodeoxynucleotides to induce epithelial cell growth|

---

US7615227B2|2001-12-20|2009-11-10|The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services|Use of CpG oligodeoxynucleotides to induce angiogenesis|

---

US7741297B2|2002-02-04|2010-06-22|Oncothyreon Inc.|Immunostimulatory, covalently lipidated oligonucleotides|

---

US8088388B2|2002-02-14|2012-01-03|United Biomedical, Inc.|Stabilized synthetic immunogen delivery system|

---

JP4846200B2|2002-04-04|2011-12-28|コーリーファーマシューティカルゲーエムベーハー|Immunostimulatory G and U-containing oligoribonucleotides|

---

US7968569B2|2002-05-17|2011-06-28|Celgene Corporation|Methods for treatment of multiple myeloma using 3--piperidine-2,6-dione|

---

WO2003103586A2|2002-06-05|2003-12-18|Coley Pharmaceutical Group, Inc.|Method for treating autoimmune or inflammatory diseases with combinations of inhibitory oligonucleotides and small molecule antagonists of immunostimulatory cpg nucleic acids|

---

JP5027387B2|2002-07-01|2012-09-19|タフツ・ユニバーシティ|Methods and compositions for inhibiting multidrug resistance with hyaluronan oligomers|

---

US7576066B2|2002-07-03|2009-08-18|Coley Pharmaceutical Group, Inc.|Nucleic acid compositions for stimulating immune responses|

---

US7569553B2|2002-07-03|2009-08-04|Coley Pharmaceutical Group, Inc.|Nucleic acid compositions for stimulating immune responses|

---

US7605138B2|2002-07-03|2009-10-20|Coley Pharmaceutical Group, Inc.|Nucleic acid compositions for stimulating immune responses|

---

US7807803B2|2002-07-03|2010-10-05|Coley Pharmaceutical Group, Inc.|Nucleic acid compositions for stimulating immune responses|

---

US20040053880A1|2002-07-03|2004-03-18|Coley Pharmaceutical Group, Inc.|Nucleic acid compositions for stimulating immune responses|

---

EP1551877A4|2002-07-25|2006-01-18|Genentech Inc|Taci antibodies and uses thereof|

---

SG166673A1|2002-08-19|2010-12-29|Coley Pharm Group Inc|Immunostimulatory nucleic acids|

---

AR040996A1|2002-08-19|2005-04-27|Coley Pharm Group Inc|IMMUNE STIMULATING NUCLEIC ACIDS|

---

FR2844455B1|2002-09-13|2007-12-14|Lab Francais Du Fractionnement|TREATMENT OF PATHOLOGIES EXCLUDING IMMUNE RESPONSE BY OPTIMIZED ANTIBODIES|

---

US8263091B2|2002-09-18|2012-09-11|The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services|Method of treating and preventing infections in immunocompromised subjects with immunostimulatory CpG oligonucleotides|

---

EP3284753B1|2002-10-17|2019-06-05|Genmab A/S|Human monoclonal antibodies against cd20 for use in the treatment of multiple sclerosis|

---

CA2503693A1|2002-10-29|2004-05-13|Coley Pharmaceutical Group, Ltd.|Use of cpg oligonucleotides in the treatment of hepatitis c virus infection|

---

WO2004053104A2|2002-12-11|2004-06-24|Coley Pharmaceutical Group, Inc.|5’ cpg nucleic acids and methods of use|

---

HU227217B1|2002-12-16|2010-11-29|Genentech Inc|Immunoglobulin variants and uses thereof|

---

US7470714B2|2003-03-04|2008-12-30|Sosei Co., Ltd.|GM-95-containing antitumor effect potentiator, combined antitumor preparation and antitumor agent|

---

WO2004087203A2|2003-04-02|2004-10-14|Coley Pharmaceutical Group, Ltd.|Immunostimulatory nucleic acid oil-in-water formulations for topical application|

---

US20040259899A1|2003-04-08|2004-12-23|Sanghvi Suketu P.|Combination therapy for constipation|

---

WO2004091623A1|2003-04-08|2004-10-28|Progenics Pharmaceuticals. Inc.|Pharmaceutical formulations containing methylnaltrexone|

---

CN1767830A|2003-04-08|2006-05-03|普罗热尼奇制药公司|The use of peripheral opiois antagonists, especially methylnaltrexone to treat irritable bowel syndrome|

---

PL1613350T3|2003-04-09|2009-08-31|Genentech Inc|Therapy of autoimmune disease in a patient with an inadequate response to a tnf-alpha inhibitor|

---

US7569554B2|2003-05-16|2009-08-04|Idera Pharmaceuticals, Inc.|Synergistic treatment of cancer using immunomers in conjunction with therapeutic agents|

---

PL1631313T3|2003-06-05|2015-08-31|Genentech Inc|Combination therapy for b cell disorders|

---

US20050163775A1|2003-06-05|2005-07-28|Genentech, Inc.|Combination therapy for B cell disorders|

---

EA200600069A1|2003-06-20|2006-08-25|Коли Фармасьютикал Гмбх|LOW-MOLECULAR ANTAGONISTS OF TOLL-SIMILAR RECEPTORS |

---

US20050013812A1|2003-07-14|2005-01-20|Dow Steven W.|Vaccines using pattern recognition receptor-ligand:lipid complexes|

---

CA2532556A1|2003-07-29|2005-02-24|Genentech, Inc.|Assay for human anti cd20 antibodies and uses therefor|

---

US20050053602A1|2003-08-29|2005-03-10|Genentech, Inc.|Therapy of ocular disorders|

---

EP1663316A2|2003-09-25|2006-06-07|Coley Pharmaceutical Group, Inc.|Nucleic acid lipophilic conjugates|

---

JP2007509040A|2003-10-11|2007-04-12|イネックスファーマシューティカルズコーポレイション|Methods and compositions for enhancing innate immunity and antibody-dependent cytotoxicity|

---

SG123799A1|2003-10-30|2006-07-26|Coley Pharm Gmbh|C-class oligonucleotide analogs with enchanced immunostimulatory potency|

---

US20050239733A1|2003-10-31|2005-10-27|Coley Pharmaceutical Gmbh|Sequence requirements for inhibitory oligonucleotides|

---

US20050100983A1|2003-11-06|2005-05-12|Coley Pharmaceutical Gmbh|Cell-free methods for identifying compounds that affect toll-like receptor 9signaling|

---

US9050378B2|2003-12-10|2015-06-09|Board Of Regents, The University Of Texas System|N2S2 chelate-targeting ligand conjugates|

---

EP1697419A2|2003-12-19|2006-09-06|Genentech Inc.|Detection of cd20 in transplant rejection|

---

ES2594734T3|2003-12-19|2016-12-22|Autotelic Llc|Combination therapy that associates a TGF-beta antagonist with a chemotherapeutic agent|

---

RU2006126078A|2003-12-19|2008-01-27|Дженентек, Инк. |CD20 DETECTION IN THE THERAPY OF AUTOIMMUNE DISEASES|

---

EP1720568A2|2004-02-19|2006-11-15|Coley Pharmaceutical Group, Inc.|Immunostimulatory viral rna oligonucleotides|

---

TWI235440B|2004-03-31|2005-07-01|Advanced Semiconductor Eng|Method for making leadless semiconductor package|

---

AU2005243250A1|2004-04-02|2005-11-24|Coley Pharmaceutical Gmbh|Immunostimulatory nucleic acids for inducing IL-10 responses|

---

CA2563432A1|2004-04-16|2005-12-01|Genentech, Inc.|Method for augmenting b cell depletion|

---

WO2005103081A2|2004-04-20|2005-11-03|Genmab A/S|Human monoclonal antibodies against cd20|

---

WO2005117972A2|2004-05-05|2005-12-15|Genentech, Inc.|Preventing autoimmune disease by using an anti-cd20 antibody|

---

WO2006007202A2|2004-05-28|2006-01-19|Idexx Laboratories, Inc|Canine cd20 compositions|

---

SV2006002132A|2004-06-04|2006-04-20|Genentech Inc|USE OF AN ANTIBODY FOR THE TREATMENT OF MULTIPLE SCLEROSIS|

---

PA8635501A1|2004-06-04|2006-06-02|Genentech Inc|USE OF AN ANTIBODY FOR THE TREATMENT OF LUPUS|

---

WO2007026190A2|2004-07-18|2007-03-08|Csl Limited|Immuno stimulating complex and oligonucleotide formulations for inducing enhanced interferon-gamma responses|

---

RU2007106722A|2004-07-22|2008-08-27|Дженентек, Инк. |METHOD FOR TREATING SHEHREN'S SYNDROME|

---

CN101087807A|2004-10-05|2007-12-12|健泰科生物技术公司|Method for treating vasculitis|

---

MY159370A|2004-10-20|2016-12-30|Coley Pharm Group Inc|Semi-soft-class immunostimulatory oligonucleotides|

---

CA2586913A1|2004-11-09|2006-05-18|University Of Southern California|Targeted innate immunity|

---

CN101102793A|2005-01-13|2008-01-09|健泰科生物技术公司|Treatment method|

---

EP1845989A1|2005-01-20|2007-10-24|Progenics Pharmaceuticals, Inc.|Use of methylnaltrexone and related compounds to treat post-operative gastrointestinal dysfunction|

---

DOP2006000029A|2005-02-07|2006-08-15|Genentech Inc|ANTIBODY VARIANTS AND USES THEREOF. |

---

US8444973B2|2005-02-15|2013-05-21|Duke University|Anti-CD19 antibodies and uses in B cell disorders|

---

HUE025945T2|2005-02-15|2016-07-28|Univ Duke|Anti-cd19 antibodies and uses in oncology|

---

EP1851314A2|2005-02-24|2007-11-07|Coley Pharmaceutical Group, Inc.|Immunostimulatory oligonucleotides|

---

TW200714289A|2005-02-28|2007-04-16|Genentech Inc|Treatment of bone disorders|

---

KR20070108270A|2005-02-28|2007-11-08|에자이 알앤드디 매니지먼트 가부시키가이샤|Novel combinational use of sulfonamide compound|

---

US8524731B2|2005-03-07|2013-09-03|The University Of Chicago|Use of opioid antagonists to attenuate endothelial cell proliferation and migration|

---

US8518962B2|2005-03-07|2013-08-27|The University Of Chicago|Use of opioid antagonists|

---

EP1861096B1|2005-03-07|2018-12-26|The University of Chicago|Use of opioid antagonists to attenuate endothelial cell proliferation and migration|

---

US20080194611A1|2005-06-03|2008-08-14|Alverdy John C|Modulation of Cell Barrier Dysfunction|

---

US9662325B2|2005-03-07|2017-05-30|The University Of Chicago|Use of opioid antagonists to attenuate endothelial cell proliferation and migration|

---

ZA200708863B|2005-04-08|2009-03-25|Coley Pharm Group Inc|Methods for treating infectious disease exacerbated asthma|

---

AR053579A1|2005-04-15|2007-05-09|Genentech Inc|TREATMENT OF INTESTINAL INFLAMMATORY DISEASE |

---

WO2006116458A2|2005-04-26|2006-11-02|Coley Pharmaceutical Gmbh|Modified oligoribonucleotide analogs with enhances immunostimulatory activity|

---

WO2006121852A2|2005-05-05|2006-11-16|Duke University|Anti-cd19 antibody therapy for autoimmune disease|

---

CN1865275B|2005-05-17|2011-06-15|长春华普生物技术有限公司|Artificial single-chain deoxynucleotide having therapeutic effect to human B cell tumour|

---

WO2006127517A2|2005-05-20|2006-11-30|Genentech, Inc.|Pretreatment of a biological sample from an autoimmune disease subject|

---

AR057035A1|2005-05-25|2007-11-14|Progenics Pharm Inc|SYNTHESIS OF-N-METHYLNTREXONE, PHARMACEUTICAL COMPOSITIONS AND USES|

---

AR057325A1|2005-05-25|2007-11-28|Progenics Pharm Inc|SYNTHESIS OF-N-METHYLNTREXONE, PHARMACEUTICAL COMPOSITIONS AND USES|

---

US8148341B2|2005-07-01|2012-04-03|Index Pharmaceuticals Ab|Method for modulating responsiveness to steroids|

---

DK2269622T3|2005-07-01|2014-03-24|Index Pharmaceuticals Ab|CPG OLIGONUCLEOTIDES USED TO INCREASE STEROID ACTIVITY OF A STEROID DEPENDENT PATIENT|

---

CN101268101A|2005-07-07|2008-09-17|科利制药集团公司|Anti-CTLA-4 antibody and CpG-motif-containing synthetic oligodeoxynucleotide combination therapy for cancer treatment|

---

US20080213273A1|2005-07-25|2008-09-04|Trubion Pharmaceuticals Inc.|Single dose use of CD20-specific binding molecules|

---

PL1912675T3|2005-07-25|2014-10-31|Emergent Product Dev Seattle|B-cell reduction using cd37-specific and cd20-specific binding molecules|

---

US20080279850A1|2005-07-25|2008-11-13|Trubion Pharmaceuticals, Inc.|B-Cell Reduction Using CD37-Specific and CD20-Specific Binding Molecules|

---

EP1924692A2|2005-09-16|2008-05-28|Coley Pharmaceutical GmbH|Modulation of immunostimulatory properties of short interfering ribonucleic acidby nucleotide modification|

---

BRPI0616235A2|2005-09-16|2011-06-14|Coley Pharm Gmbh|phosphodiester main chain immunostimulatory monofilamentated ribonucleic acid|

---

JP2009513629A|2005-10-28|2009-04-02|インデックス・ファーマシューティカルズ・アクチエボラーグ|Compositions and methods for preventing, treating and / or alleviating inflammatory diseases|

---

GB0523041D0|2005-11-11|2005-12-21|Cyclacel Ltd|Combination|

---

MY149159A|2005-11-15|2013-07-31|Hoffmann La Roche|Method for treating joint damage|

---

ES2618543T3|2005-11-23|2017-06-21|Genentech, Inc.|Methods and compositions related to B lymphocyte assays|

---

EP1957647B1|2005-11-25|2015-03-04|Zoetis Belgium S.A.|Immunostimulatory oligoribonucleotides|

---

PT2405002E|2006-02-15|2015-01-05|Adiutide Pharmaceuticals Gmbh|Compositions and methods for oligonucleotide formulations|

---

WO2007146968A2|2006-06-12|2007-12-21|Trubion Pharmaceuticals, Inc.|Single-chain multivalent binding proteins with effector function|

---

US20080031887A1|2006-06-30|2008-02-07|Joseph Lustgarten|Conjugates for inducing targeted immune responses and methods of making and using same|

---

DE102006035618A1|2006-07-31|2008-02-07|Curevac Gmbh|New nucleic acid useful as immuno-stimulating adjuvant for manufacture of a composition for treatment of cancer diseases e.g. colon carcinomas and infectious diseases e.g. influenza and malaria|

---

EP2046954A2|2006-07-31|2009-04-15|Curevac GmbH|NUCLEIC ACID OF FORMULA : GIXmGn, OR : CIXmCn, IN PARTICULAR AS AN IMMUNE-STIMULATING AGENT/ADJUVANT|

---

EP2383297B1|2006-08-14|2013-01-16|Xencor Inc.|Optimized antibodies that target CD19|

---

TW200817048A|2006-09-08|2008-04-16|Wyeth Corp|Dry powder compound formulations and uses thereof|

---

MX2009003398A|2006-09-27|2009-08-12|Coley Pharm Gmbh|Cpg oligonucleotide analogs containing hydrophobic t analogs with enhanced immunostimulatory activity.|

---

US10925977B2|2006-10-05|2021-02-23|Ceil>Point, LLC|Efficient synthesis of chelators for nuclear imaging and radiotherapy: compositions and applications|

---

WO2008057529A2|2006-11-06|2008-05-15|Coley Pharmaceutical Group, Inc.|Peptide-based vaccine compositions to endogenous cholesteryl ester transfer protein |

---

GB0625283D0|2006-12-19|2007-01-24|Cyclacel Ltd|Combination|

---

MX2009010550A|2007-03-29|2009-12-14|Progenics Pharm Inc|Crystal forms of-n-methylnaltrexone bromide and uses thereof.|

---

PE20130480A1|2007-03-29|2013-05-08|Progenics Pharm Inc|HETEROCYCLIC COMPOUNDS AS ANTAGONISTS OF THE PERIPHERAL OPIOID RECEPTOR|

---

MX2009010515A|2007-03-29|2009-10-19|Wyeth Corp|Peripheral opioid receptor and antagonists and uses thereof.|

---

US20100226917A1|2007-04-27|2010-09-09|Astrazeneca Ab|Methods for the treatment of hematologic malignancies|

---

EP2573176B1|2007-05-04|2016-04-06|InDex Pharmaceuticals AB|Tumour growth inhibitory compounds and methods of their use|

---

SG10201406411XA|2007-07-09|2014-11-27|Genentech Inc|Prevention of disulfide bond reduction during recombinant production of polypeptides|

---

WO2009030254A1|2007-09-04|2009-03-12|Curevac Gmbh|Complexes of rna and cationic peptides for transfection and for immunostimulation|

---

CA2699265A1|2007-09-13|2009-03-19|Molecular Insight Pharmaceuticals, Inc.|Infusion and transfer system for use with radioactive agents|

---

EP2233149B1|2007-10-16|2016-02-10|ZymoGenetics, Inc.|Combination of transmembrane activator and calcium modulator and cyclophilin ligand interactorand anti-CD20 agents for treatment of autoimmune disease|

---

EP2077281A1|2008-01-02|2009-07-08|Bergen Teknologioverforing AS|Anti-CD20 antibodies or fragments thereof for the treatment of chronic fatigue syndrome|

---

SG188104A1|2008-01-31|2013-03-28|Curevac Gmbh|Nucleic acids comprising formula a and derivatives thereof as an immunostimulating agents /adjuvants|

---

CA2713568C|2008-02-06|2016-09-20|Progenics Pharmaceuticals, Inc.|Preparation and use of ,-2,2'-bis-methylnaltrexone|

---

US8685995B2|2008-03-21|2014-04-01|The University Of Chicago|Treatment with opioid antagonists and mTOR inhibitors|

---

PL2132228T3|2008-04-11|2012-01-31|Emergent Product Dev Seattle|Cd37 immunotherapeutic and combination with bifunctional chemotherapeutic thereof|

---

US9669057B2|2008-04-25|2017-06-06|Duke University|Regulatory B cells and their uses|

---

EP2307002B1|2008-06-09|2013-01-02|Cyclacel Limited|Combinations of sapacitabine or cndac with dna methyltransferase inhibitors such as decitabine and procaine|

---

AU2009260907A1|2008-06-18|2009-12-23|Index Pharmaceuticals Ab|Combination therapies against cancer|

---

TWI351288B|2008-07-04|2011-11-01|Univ Nat Pingtung Sci & Tech|Cpg dna adjuvant in avian vaccines|

---

DK2350129T3|2008-08-25|2015-08-31|Amplimmune Inc|PREPARATIONS WITH PD-1 ANTAGONISTS AND PROCEDURES FOR USE THEREOF|

---

AR073295A1|2008-09-16|2010-10-28|Genentech Inc|METHODS TO TREAT PROGRESSIVE MULTIPLE SCLEROSIS. MANUFACTURING ARTICLE.|

---

CA2676881C|2008-09-30|2017-04-25|Wyeth|Peripheral opioid receptor antagonists and uses thereof|

---

WO2010037408A1|2008-09-30|2010-04-08|Curevac Gmbh|Composition comprising a complexed rna and a naked mrna for providing or enhancing an immunostimulatory response in a mammal and uses thereof|

---

US20110280934A1|2008-11-04|2011-11-17|Asa Karlsson|Increased Expression of Specific Antigens|

---

WO2010075249A2|2008-12-22|2010-07-01|Genentech, Inc.|A method for treating rheumatoid arthritis with b-cell antagonists|

---

CA3033133C|2009-03-25|2021-11-09|The Board Of Regents Of The University Of Texas System|Compositions for stimulation of mammalian innate immune resistance to pathogens|

---

WO2010132622A2|2009-05-14|2010-11-18|The Regents Of The University Of California|Anticd20-cpg conjugates and methods of treating b cell malignancies|

---

KR20180010324A|2009-08-11|2018-01-30|제넨테크, 인크.|Production of proteins in glutamine-free cell culture media|

---

US20110053829A1|2009-09-03|2011-03-03|Curevac Gmbh|Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids|

---

MX341687B|2010-02-10|2016-08-30|Immunogen Inc|Cd20 antibodies and uses thereof.|

---

WO2012012518A2|2010-07-20|2012-01-26|University Of Miami|Inhibition of nonsense mediated decay pathways|

---

CN107648604A|2010-07-30|2018-02-02|库瑞瓦格股份公司|The purposes of polymer support load compound and polymer support|

---

WO2012019041A2|2010-08-04|2012-02-09|Duke University|Regulatory b cells and their uses|

---

US9044474B2|2010-11-08|2015-06-02|The Ohio State University|Compositions and methods for increasing drug efficacy in cancer|

---

WO2012088272A1|2010-12-21|2012-06-28|Duke University|Methods and compositions combining immunotherapy with monocyte activation|

---

JP5930146B2|2011-03-28|2016-06-08|国立研究開発法人物質・材料研究機構|Immunostimulatory oligonucleotide and therapeutic agent comprising said immunostimulatory oligonucleotide|

---

ES2743691T3|2011-04-14|2020-02-20|Cyclacel Ltd|Dosage regimen for sapacitabine and decitabine in combination to treat acute myeloid leukemia|

---

US20130157261A1|2011-06-01|2013-06-20|The Methodist Hospital Research Institute|Compositions and Methods for Quantitative Histology, Calibration of Images in Fluorescence Microscopy, and ddTUNEL Analyses|

---

US10017739B2|2012-09-06|2018-07-10|Duke University|Methods of expanding and assessing B cells and using expanded B cells to treat disease|

---

US10436680B2|2013-10-15|2019-10-08|Kianoosh Peyvan|Capture, disruption, and extraction apparatus and method|

---

WO2015112749A2|2014-01-22|2015-07-30|The Board Of Trustees Of The Leland Stanford Junior University|Methods and compositions for antibody and antibody-loaded dendritic cell mediated therapy|

---

WO2015149944A2|2014-04-01|2015-10-08|Curevac Gmbh|Polymeric carrier cargo complex for use as an immunostimulating agent or as an adjuvant|

---

US10286065B2|2014-09-19|2019-05-14|Board Of Regents, The University Of Texas System|Compositions and methods for treating viral infections through stimulated innate immunity in combination with antiviral compounds|

---

MX2018003822A|2015-10-02|2018-06-22|Hoffmann La Roche|Bispecific anti-human cd20/human transferrin receptor antibodies and methods of use.|

---

AR106189A1|2015-10-02|2017-12-20|F Hoffmann-La Roche Ag|BIESPECTIFIC ANTIBODIES AGAINST HUMAN A-b AND THE HUMAN TRANSFERRINE RECEIVER AND METHODS OF USE|

---

WO2017070133A1|2015-10-20|2017-04-27|Sorrento Therapeutics, Inc.|Intracellular delivery compounds|

---

US20190185852A1|2016-05-05|2019-06-20|M. Mahmood Hussain|Therapeutically modulating apob and apoai|

---

CN109843327A|2016-07-07|2019-06-04|小利兰·斯坦福大学托管委员会|Antibody adjuvant conjugate|

---

WO2018112407A1|2016-12-15|2018-06-21|Duke University|Antibodies and methods for depleting regulatory b10 cells and use in combination with immune checkpoint inhibitors|

---

AU2018252193A1|2017-04-14|2019-10-17|Tollnine, Inc.|Immunomodulating polynucleotides, antibody conjugates thereof, and methods of their use|

---

CN111826764B|2020-07-28|2021-10-15|吴忠德悦纺织科技有限公司|Preparation method of modified multi-component composite yarn|

法律状态:
2010-02-25| FGA| Letters patent sealed or granted (standard patent)|

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2021-07-15| MK14| Patent ceased section 143(a) (annual fees not paid) or expired|

优先权:

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申请号 | 申请日 | 专利标题

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US21334600P| true| 2000-06-22|2000-06-22||

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US60/213,346||2000-06-22||

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