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(12) Patent Application Publication (10) Pub. No.: US 2009/0137519 A1 Krieg Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2009/0137519 A1 Krieg Et Al US 20090 137519A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0137519 A1 Krieg et al. (43) Pub. Date: May 28, 2009 (54) SEMI-SOFT C-CLASS Related U.S. Application Data MMUNOSTIMULATORY OLGONUCLEOTDES (62) Division of application No. 1 1/255,100, filed on Oct. 20, 2005. (75) Inventors: Arthur M. Krieg, Wellesley, MA (US); Ulrike Samulowitz (60) Provisional application No. 60/620,759, filed on Oct. Langenfeld (DE); Jorg Vollmer, 20, 2004. Dusseldorf (DE); Eugen Uhlmann, O O Glashuetter (DE) Publication Classification (51) Int. Cl. Correspondence Address: A63L/7088 (2006.01) WOLF GREENFIELD & SACKS, PC. C7H 2L/00 (2006.01) 6OO ATLANTIC AVENUE BOSTON, MA 02210-2206 (US) (52) U.S. Cl. .......................................... 514/44; 536/22.1 (73) Assignees: Coley Pharmaceutical Group, (57) ABSTRACT Inc., New York, NY (US); Coley Pharmaceutical GmbH The invention relates to specific C-Class semi-soft CpG Duesseldorf (DE) s immunostimulatory oligonucleotides that are useful for stimulating an immune response. In particular the oligonucle (21) Appl. No.: 12/336,986 otides are useful for treating allergy, such as allergic rhinitis and asthma, cancer and infectious disease. Such as hepatitis B (22) Filed: Dec. 17, 2008 and hepatitis C. A UNINFECTED UNREATED B VIRUS NFECTED TEST VEHICLE C VIRUS INFECTED SEOD NO:10.03 mg/kg D VIRUS INFECTED SEQID NO:1, 0.3 mg/kg E VIRUSINFECTED SEQID NO:1.3 mg/kg F VIRUSINFECTED SEQID NO2, 0.03 mg/kg G VIRUS INFECTED SEQID NO;2,0.3 mg/kg H VIRUSINFECTED SEQID NO:2, 3 mg/kg I VIRUS INFECTED SEQID NO:3,0.03 mg/kg J VIRUS INFECTED SEQID NO:3,0.3 mg/kg K VIRUS INFECTED SEQID NO:3, 3 mg/kg NFLUENZAWIRUS LOAD (ABSORBANCE UNITS) 2.5- 2.0- 15 1.O- 0.5- n.d. 0.0-1 r. A B C. D. E F G. H J K Patent Application Publication May 28, 2009 Sheet 1 of 52 US 2009/0137519 A1 SEOID NO:1 SEOID NO:2 E 5000 S3000 40003000- E:2000 1500 is 2000: is 1000 aF 1000O. ! 500O O.O. O.1 O.O1 O.1 1 ODNuM) ODNEM) SEOID NO:3 SEOID NO:4 E 2500- E 10000 R 2000- R 8000 - g 1500- 6000 1000- 4000 it e 500- E 2000 o O --- O O.O1 0.1 1 O,O1 O.1 1 ODNuM) ODNuM) SEOID NO:5 SEO ED NO:6 E 2500 E 3000 S. 2000 S. 2500 1500 O 2000 1 OOO - 10001500 D. 500 500 O LE O O.O1 O.1 O.O. O. ODNEuM) ODNuM) N- Ny - Patent Application Publication May 28, 2009 Sheet 2 of 52 US 2009/0137519 A1 NASAL RESESTANCE (cmH2O) mimin1) 150 1.25 OO -& - UNCHALLENGED ... ANTIGENCHALLENGED, TREATED WITH TEST VEHICLE O.75 ANTIGENCHALLENGED, TREATED WITH SEQID NO:71 mg/kg O.50 O 10 20 30 40 TIME AFTER ANTIGEN CHALLENGE (MINUTES) Fig. 2A NASAL RESISTANCE (cmH2O) milmin) "& UNCHALLENGED 150 . ANTIGENCHALLENGED, 1.25 TREATED WITH TEST VEHICLE ANTIGENCHALLENGED, 1.OO TREATED WITH SEQID NO:7 0.03 mg/kg - ANTIGENCHALLENGED, O.75 TREATED WITH SEQID NO:70.1 mg/kg ANTIGENCHALLENGED, O.50 TREATED WITH SEQID NO:70.3 mg/kg O O 20 30 40 TFMEAFTERANTIGEN CHALLENGE (MINUTES) Fig.2B Patent Application Publication May 28, 2009 Sheet 3 of 52 US 2009/0137519 A1 NUMBER OFSNEEZES (10 MINUTES) 60 50 40 BO 2O 1 O O UNCHALLENGED ANTIGENCHALLENGED SEOD NO:7 BUDESONIDE O 3 30 300 300 ug/kg ug/kg Fig. 3A NUMBER OF RUBS (10 MINUTES) 16 14 12 1O UNCHALLENGED ANTIGEN CHALLENGED SEQID NO:7 BUDESONIDE O 3 30 300 BOO lg/kg ug/kg Fig. 3B Patent Application Publication May 28, 2009 Sheet 4 of 52 US 2009/0137519 A1 TOTAL LEUKOCYTES (x103/mi) 2OOO WRUSDOSE 1500 -o- 50 EED50 - A - 500 ED50 SOO O O 1 3 6 9 14 DAYS AFTER INFECTION Fig. 4 Patent Application Publication May 28, 2009 Sheet 5 of 52 US 2009/0137519 A1 A UNNFECTED UNTREATED B VIRUS INFECTED TEST VEHICLE C VIRUS INFECTED SEQID NO:10.03 mg/kg D VIRUS INFECTED SEQID NO;1, 0.3 mg/kg E VIRUSINFECTED SEQID NO:1, 3 mg/kg F VIRUS INFECTED SEQID NO2, 0.03 mg/kg G VIRUS INFECTED SEQID NO:2, 0.3 mg/kg H VIRUSINFECTED SEQED NO:2, 3 mg/kg I VIRUS INFECTED SEQID NO:3,0.03 mg/kg J VIRUS INFECTED SEC ID NO:3,0.3 mg/kg K VIRUS INFECTED SEQID NO:3, 3 mg/kg NFLUENZAWIRUS LOAD (ABSORBANCE UNITS) 2.5 2.0 1.5 1.O O.5 n.d. 0.0-1 A B CD E F. G. H J K Fig.5 Patent Application Publication May 28, 2009 Sheet 6 of 52 US 2009/0137519 A1 A UNINFECTED UNTREATED B VIRUS INFECTED TEST VEHICLE TOTAL LEUKOCYTES (x103/ml) C VIRUSINFECTED SEQLD NO:10.03 mg/kg 1 OOO D VIRUS INFECTED SEQID NO:1, 0.3 mg/kg E VIRUS INFECTED SEQID NO:1, 3 mg/kg 750 F VIRUSINFECTED SEQID NO2, 0.03 mg/kg G VIRUSINFECTED SEQID NO2, 0.3 mg/kg 500 H VIRUSINFECTED SEQID NO:2, 3 mg/kg 250 I VIRUSINFECTED SEQID NO:3,0.03 mg/kg J VIRUS INFECTED SEOID NO:3,0.3 mg/kg O K VIRUSINFECTED SEQID NO:3, 3 mg/kg N Y - Fig.6A A UNENFECTED UNTREATED B VIRUS INFECTED ES WEHICLE NEUTROPHILS (x10.3/ml) C VIRUSINFECTED SEQID NO:10.03 mg/kg 700 D VERUSINFECTED SEQID NO;1, 0.3 mg/kg 600 E VIRUSINFECTED SEQID NO:1, 3 mg/kg 5 OO F VIRUS INFECTED SEQID NO2, 0.03 mg/kg 40 G VIRUSINFECTED SEQID NO:2,0.3 mg/kg 300 H VIRUSINFECTED SEOID NO:2, 3 mg/kg 200 I VIRUS INFECTED SEQID NO:3,0.03 mg/kg 100 J VIRUS INFECTED SEQED NO:3,0.3 mg/kg K VIRUS INFECTED SEQID NO:3, 3 mg/kg Patent Application Publication May 28, 2009 Sheet 7 of 52 US 2009/0137519 A1 3 A UNINFECTED UNTREATED MONONUCLEAR CELLS (x10/ml) B WRUS INFECTED TEST WEHICLE 600 C VIRUS INFECTED SEQID NO:10.03 mg/kg 500 D VIRUSINFECTED SEQID NO:1, 0.3 mg/kg 400 E VIRUSINFECTED SEQDNO;1.3 mg/kg 300 F VIRUSINFECTED SEQ DNO2, 0.03 mg/kg G VIRUSINFECTED SEQID NO2, 0.3 mg/kg 200 H VIRUSINFECTED SEQID NO2, 3 mg/kg 100 I VIRUS INFECTED SEQID NO:3, 0.03 mg/kg J VIRUSINFECTED SEQED NO:3,0.3 mg/kg K VIRUS INFECTED SEQID NO:3, 3 mg/kg N - - - - Fig.6C Patent Application Publication May 28, 2009 Sheet 8 of 52 US 2009/0137519 A1 A UNCHALLENGED UNTREATED B ANTIGENCHALLENGED UNTREATED C ANTIGENCHALLENGED WEHICLE D ANTIGENCHALLENGED SEQED NO:7, 1.g/kg E ANTIGENCHALLENGED SEQDN0:7, 10 pg/kg EOSINOPHILS (x103/ml) F ANTIGENCHALLENGED SEQIDN0:7, 100 g/kg 700 G ANTIGENCHALLENGED SEQDN0:1, 1ug/kg 600 H ANTIGENCHALLENGED SEQID NO:1, 10 g/kg 500 ANTIGENCHALLENGED SEQID NO;1,100 g/kg J ANTIGENCHALLENGED SEQID NO2, 1 g/kg 400 K ANTIGENCHALLENGED SEQID NO2, 10 g/kg 300 L ANTIGENCHALLENGED SEQID NO2,100 g/kg MANTIGENCHALLENGED SEQIDNC3, 1g/kg N ANTIGENCHALLENGED SEQID NO:3, 10 g/kg O ANTIGENCHALLENGED SEQIDNO3,100 g/kg A UNCHALLENGED UNTREATED B AMTGEN CHALLENGED UNTREATED C ANTIGENCHALLENGED WEHICLE D ANTIGENCHALLENGED SEQID NO:7, 19/kg E ANTIGENCHALLENGED SEQID NO7, 10 pg/kg NEUTROPHILS (x103/ml) F ANTIGENCHALLENGED SEQIDN07, 100 g/kg 1 OO G ANTIGENCHALLENGED SEQID NO:1, 1 g/kg H ANTIGENCHALLENGED SEQID NO:1, 10 g/kg | ANTIGENCHALLENGED SEQID NO:1, 100 g/kg J ANTIGENCHALLENGED SEQDNO2, 1 g/kg K ANTIGENCHALLENGED SEQED NO2, 10 pg/kg L ANTIGENCHALLENGED SEQID NO:2, 100 g/kg Patent Application Publication May 28, 2009 Sheet 9 of 52 US 2009/0137519 A1 A UNCHAENGED UNTREATED E ANTEGEN CHALLENGED UNTREATED C ANTIGENCHALENGED WEHICLE D ANTIGENCHALLENGED SEQID NO:7, 1 g/kg TOTALT CELS E ANTIGENCHALLENGED SEQID NO:7, 10 g/kg (CD3t CELLS, x10.3/mi) F ANTIGENCHALLENGED SEQIDNO:7, 100 g/kg 600 G ANTIGENCHALLENGED SEQID NO:1, 1 g/kg H ANTIGENCHALLENGED SEQID NO:1, 10 g/kg 500 | ANTIGENCHALLENGED SEQID NO:1, 100 g/kg 400 J ANTIGENCHALLENGED SEQID NO2, 1 g/kg BOO | | || K ANTIGENCHALLENGED SEQID NO2, 10 g/kg L ANTIGENCHALLENGED SEQIDNO2, 10Cug/kg 2OO MANTIGENCHALLENGED SEQDNC3, 1 g/kg 1 OO i N ANTIGENCHALLENGED SEQDNO3, 10 pg/kg O-I O ANTIGENCHALLENGED SEQIDNO:3, 100 g/kg A B C D E F GH J K L MNO Y- - - Fig.8A A UNCHALLENGED UNTREATED B ANTIGENCHALLENGED UNTREATED C ANTIGENCHALENGED WEHICLE D ANTIGENCHALLENGED SEQIDN0:7, 1 g/kg CD4+ T CELLS E ANTIGENCHALLENGED SEQIDN07, 10ug/kg (CD3t CD4+CELLS, x103/ml) F ANTIGENCHALLENGED SEQID NO7, 100 pg/kg 4OO G ANTIGENCHALLENGED SEQDNO:1, 1 g/kg H ANTIGENCHALLENGED SEQDN0:1, 10 pg/kg 300 | ANTIGENCHALLENGED SEQIDNO:1,100 g/kg J ANTIGENCHALLENGED SEQID NO2, lug/kg 300 K ANTIGENCHALLENGED SEQID NO2, 10 g/kg L ANTIGENCHALLENGED SEQID NO2, 100 g/kg OO MANTIGENCHALLENGED SEQID NO3, 1 g/kg N ANTIGENCHALLENGED SEQID NO3, 10 g/kg O O ANTIGENCHALLENGED SEQID NO3, 100 g/kg A B C D EF GH KL Y -- - - Fig.8B Patent Application Publication May 28, 2009 Sheet 10 of 52 US 2009/0137519 A1 8 HOURS AFTER DOSNG 15 HOURS AFTER DOSING WITH 0.1 mg/kg WITH 0.1 mg/kg TOTAL LEUKOCYTES (x103/ml) TOTAL LEUKOCYTES (x10.3/mi) 400 400 3OO 300 200 200 1OO 1 OO O'CONTROL'7' ' ' ' O'CONTROL" 7" ' " ' " 's SEOID NO SEO D NO Fig 9A Fig 9B 8 HOURS AFTER DOSING 15 HOURS AFTER DOSING WITH 1 mg/kg WITH 1 mg/kg TOTAL LEUKOCYTES(x10.3/ml) TOTAL LEUKOCYTES (x103/ml) 400 400 300 300 200 200 1OO 100 O CONTROL 7 1 2 3 O CONTROL"7" 1 2 3 SEOD NO SEC ID NO Fig 9C Fig 9D Patent Application Publication May 28, 2009 Sheet 11 of 52 US 2009/0137519 A1 8 HOURS AFTER DOSING 15 HOURS AFTER DOSNG WITH 0.1 mg/kg WITH 0.1 mg/kg IFNo.
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