US007 271156 B2 (12) United States Patent (10) Patent No.: US 7,271,156 B2 Krieg et al. (45) Date of Patent: Sep. 18, 2007 (54) IMMUNOSTIMULATORY NUCLEIC ACIDS 6,544,518 B1 * 4/2003 Friede et al. ............ 424,184.1 6,653.292 B1* 11/2003 Krieg et al. .................. 514,44 (75) Inventors: Arthur M. Krieg, Wellesley, MA (US); 6,727,230 B1 4/2004 Hutcherson et al. Jörg Vollmer, Düsseldorf (DE): 6.821,957 B2 11/2004 Krieg et al. 6,943,240 B2 9, 2005 Bauer et al. Christian Schetter, Hilden (DE) 6,949,520 B1 9, 2005 Hartmann et al. Assignees: University of Iowa Research 2001/0044416 A1 11/2001 McCluskie et al. (73) 2002/009 1097 A1 7/2002 Bratzler et al. Foundation, Iowa City, IA (US); Coley 2002/0156033 A1 10, 2002 Bratzler et al. Pharmaceutical GmbH, Dusseldorf 2002/0164341 A1 11/2002 Davis et al. (DE) 2002fO165178 A1 11/2002 Schetter et al. Notice: 2002fO1981.65 A1 12/2002 Bratzler et al. (*) Subject to any disclaimer, the term of this 2003/0026782 A1 2/2003 Krieg et al. patent is extended or adjusted under 35 2003/0026801 A1 2/2003 Weiner et al. U.S.C. 154(b) by 0 days. 2003/0050261 A1 3/2003 Krieg et al. 2003/0050268 A1* 3/2003 Krieg et al. .................. 514,44 (21) Appl. No.: 10/314,578 2003/0055014 A1 3/2003 Bratzler 2003/009 1599 A1 5/2003 Davis et al. (22) Filed: Dec. 9, 2002 2003/010.0527 A1 5/2003 Krieg et al. 2003. O139364 A1 7/2003 Krieg et al. (65) Prior Publication Data 2003. O14831.6 A1 8/2003 Lipford et al. US 2003/0212026 A1 Nov. 13, 2003 2003/0148976 A1* 8/2003 Krieg et al. .................. 514,44 2003. O166001 A1 9/2003 Lipford Related U.S. Application Data 2003/0181406 A1* 9, 2003 Schetter et al. ............... 514,44 2003/019 1079 A1 10/2003 Krieg et al. (63) Continuation of application No. 09/669,187, filed on 2003/0212026 A1* 11/2003 Krieg et al. .................. 514,44 Sep. 25, 2000. 2003/022401.0 A1 12/2003 Davis et al. (60) Provisional application No. 60/227,436, filed on Aug. 23, 2000, provisional application No. 60/156,135, (Continued) filed on Sep. 27, 1999, provisional application No. 60/156,113, filed on Sep. 25, 1999. FOREIGN PATENT DOCUMENTS WO WO96.O2555 A1 2, 1996 (51) Int. C. WO WO98.37919 A1 * 9, 1998 (2006.01) WO WO98,401.00 9, 1998 (2006.01) WO WO99,56755 A1 11, 1999 (2006.01) WO WO99,58118 11, 1999 (2006.01) A 6LX 9/52 (2006.01) (Continued) AOIN 25/08 (2006.01) AOIN 43/04 (2006.01) OTHER PUBLICATIONS (52) U.S. Cl. ...................... 514/44; 536/23.1536/25.3: Krieg et al. Immunopharmacology, 2000, 48:303-305.* 536/25.6; 424/408; 424/409: 424/422; 424/430; Tighe et al. J. Allergy Clin. Immunol. 2000, 106:124-134.* 424/434; 424/450; 424/457; 424/468; 424/490 McCluskie et al. Vaccine, 2001, 19:2657-2660.* (58) Field of Classification Search .................. 514/44; Jones et al. Vaccine, 1999, 17:3065-3071.* 536/23.1, 23.6, 23.4, 23.5, 23.51, 23.52, 536/24.3, 25.3, 25.6; 424/408,409, 422, (Continued) 424/430, 434, 450, 457, 468, 490 Primary Examiner Nita Minnifield See application file for complete search history. (74) Attorney, Agent, or Firm Wolf Greenfield & Sacks, (56) References Cited P.C. U.S. PATENT DOCUMENTS (57) ABSTRACT 5,663,153 9, 1997 Hutcherson et al. 5,723,335 3, 1998 Hutcherson et al. The invention relates to immunostimulatory nucleic acid 6,194,388 2, 2001 Krieg et al. compositions and methods of using the compositions. The 6,207,646 3, 2001 Krieg et al. T-rich nucleic acids contain poly T sequences and/or have 6,214,806 4, 2001 Krieg et al. .................. 514,44 greater than 25% T nucleotide residues. The TG nucleic 6,218,371 4, 2001 Krieg et al. .................. 514,44 acids have TG dinucleotides. The C-rich nucleic acids have 6,221,882 4, 2001 Macfarlane at least one poly-C region and/ore greater than 50% c 6,239,116 5, 2001 Krieg et al. .................. 514,44 nucleotides. These immunostimulatory nucleic acids func 6,339,068 1, 2002 Krieg et al. .................. 514,44 tion in a similar manner to nucleic acids containing CpG 6,399,630 6, 2002 Macfarlane 6,406,705 6, 2002 Davis et al. ............. 424,278.1 motifs. The invention also encompasses preferred CpG 6,429,199 8, 2002 Krieg et al. .................. 514,44 nucleic acids. 6.479,504 11, 2002 Macfarlane et al. 6,521,637 2, 2003 Macfarlane 33 Claims, 12 Drawing Sheets US 7,271,156 B2 Page 2 U.S. PATENT DOCUMENTS 2007/0037767 A1 2/2007 Bratzler et al. ............... 514,44 2003/0232074 12, 2003 Lipford et al. FOREIGN PATENT DOCUMENTS 2003/0232856 12, 2003 Macfarlane 2004/OOO9949 1, 2004 Krieg WO WO99, 61056 12/1999 2004.?003.0118 2, 2004 Wagner et al. WO WOOOO6588 A1 2, 2000 2004.0053880 3, 2004 Krieg ............ - - - - - - - - - - - 514,44 WO WOOO.75304 A1 12/2000 2004, OO67902 4, 2004 Bratzler et al. - - - - - - - - - - - - - - - 514,44 WO WO O1/22972 A2 * 4, 2001 2004, OO67905 4, 2004 Krieg WO WO O1/62909 A1 8, 2001 2004/OO87534 5, 2004 Krieg et al. WO WO O2/O69369 A2 * 9, 2002 2004/OO87538 5, 2004 Krieg et al. WO WO O3,O15711 A2 * 2/2003 2004/00924.72 5, 2004 Krieg .......................... 514,44 WO WO 2004/OO7743 A2 1, 2004 2004/0106568 6, 2004 Krieg et al. ... 514/44 WO WO 2004/026888 A2 4/2004 2004/O131628 T/2004 Bratzler et al. 424,184.1 WO WO 2004/094671 A2 11/2004 2004/O132685 T/2004 Krieg et al. .................. 514,44 2004/0142469 T/2004 Krieg et al. OTHER PUBLICATIONS 2004/O143112 T/2004 Krieg et al. 2004/O147468 T/2004 Krieg et al. Askenase, J. Allergy Clin. Immunol., 2000, 106:37-40.* 2004/O152649 8, 2004 Krieg .......................... 514,44 Vollmer et al. Antisense and Nucleic Acid Drug Development, 2002, 2004/O152656 8, 2004 Krieg et al. 12:165-175.* 2004/O152657 8, 2004 Krieg et al. Weiner, J. Leukocyte Biology, 200, 68:455-463.* 2004O162258 8, 2004 Krieg et al. Agrawal et al. TRENDS in Molecular Medicine, 2002. 8/3:114 2004O162262 8, 2004 Krieg et al. 121.* 2004/O167089 8, 2004 Krieg et al. Gelfand, J. Allergy Clin. Immunol., 2004, 114:S135-S138.* 2004/0171150 9, 2004 Krieg et al. Saito et al. Immunology, 2001, 104:226-234.* 2004/0171571 9, 2004 Krieg et al. - - - - - - - - - - - - - - - - - - 514,44 2004/O181045 9, 2004 Krieg et al. Taube et al. Int. Arch. Allergy Immunol., 2004, 135:173-186.* 2004/O198680 10, 2004 Krieg .......................... 514,44 Asakura et al. International Archives of Allergy and Immunology, 2004/O198688 10, 2004 Krieg et al. 1998, 116:49-52.* 2004/02298.35 11, 2004 Krieg et al. Takeda et al., J. Exp. Med., Aug. 4, 1997, 186/3:449-454.* 2004/0234512 11, 2004 Wagner et al. Kataoka, et al., “Antitumor activity of synthetic oligonucleotides 2004/O235770 11, 2004 Davis et al. .................. 514,44 with sequences from cDNA encoding proteins of Mycobacterium 2004/O235774 11, 2004 Bratzler et al. ............... 514,44 bovis BCG”, Jpn. J. Cancer Res., 83:244-247 (1992). 2004/0235777 11/2004 Wagner et al. Krieg, et al., “Editorial:Phosphorothioate oligodeoxynucleotides: 2004/O235778 11, 2004 Wagner et al. antisense or anti-protein’?”. Anitsense Res. Devlp., 5:241 (1995). 2004/0266719 12, 2004 McCluskie et al. Krieg, A.M., "Leukocyte stimulation by oligodeoxynucleotides'. 2005, 0004061 1/2005 Krieg et al. Applied Antisense Oligonucleotide Technology, 24:431-448 (1998). 2005, 0004062 1/2005 Krieg et al. Sun, et al., “Mitogenicity of DNA from different organisms for 2005/OOO9774 1/2005 Krieg et al. murine B cells”. J. Immunol. 159:3119-3125 (1997(. 2005/OO32734 2, 2005 Davis et al. Krieg, et al., “Brief Communication: Oligodeoxynucleotide modi 2005/OO32736 2, 2005 Krieg et al. fications determine the magnitude of B cell stimulation by CpG 2005/OO37403 2, 2005 Krieg et al. motifs'. Antisense Nucleic Acid. Drug Developmen, 6:133-139 2005, 0037985 2, 2005 Krieg et al. (1996). 2005.0043529 2, 2005 Davis et al. Messina, et al., “Stimulation of in vitro murine lymphocyte prolif 2005.00492.15 3, 2005 Krieg et al. eration by bacterial DNA”, J. of Immunol., 147(6): 1759-1764 2005.0049216 3, 2005 Krieg et al. (1991). 2005.00546O1 3, 2005 Wagner et al. McIntyre, et al., “A sense phosphorothioate oligonucleotide directed 2005.00546O2 3, 2005 Krieg et al. to the intiation codon of transcription factor NF-kB p65 causes 2005/0059619 3, 2005 Krieg et al. sequence-specific immune stimulation'. Antisense Res. and 2005/00596.25 3, 2005 Krieg et al. Devlop., 3:309-322 (1993). 2005/0070491 3, 2005 Krieg et al. Hartmann, et al., “Mechanism and function of a newly identified 2005.00753O2 4, 2005 Hutcherson et al. CpG DNA motif in human primary b cells'. J. of Immunol. 2005. O080034 4, 2005 Standring et al. ............. 514.46 164:944-952 (2000). 2005, 0100983 5/2005 Bauer et al. Yi, et al., “CpG DNA rescues B cells from apoptosis by activating 2005/0101554 5/2005 Krieg et al. NFKB and preventing mitochondrial membrane potential disruption 2005/0101557 5/2005 Krieg et al. via a chloroquine-sensitive pathway. International Immunol. 2005/O119273 6, 2005 Lipford et al. 11(2):2015-2024 (1999). 2005/O123523 6, 2005 Krieg et al.