U.S. patent application number 12/960030 was filed with the patent office on 2011-06-09 for synthetic agonists of tlr9.
This patent application is currently assigned to IDERA PHARMACEUTICALS, INC.. Invention is credited to SUDHIR AGRAWAL, LAKSHMI BHAGAT, EKAMBAR KANDIMALLA, MALLIKARJUNA PUTTA, DAQING WANG, DONG YU.
Application Number | 20110135669 12/960030 |
Document ID | / |
Family ID | 39512457 |
Filed Date | 2011-06-09 |
United States Patent
Application |
20110135669 |
Kind Code |
A1 |
KANDIMALLA; EKAMBAR ; et
al. |
June 9, 2011 |
SYNTHETIC AGONISTS OF TLR9
Abstract
The invention provides novel oligonucleotide-based compounds
that individually provide distinct immune response profiles through
their interactions as agonists with TLR9. The TLR9 agonists
according to the invention are characterized by specific and unique
chemical modifications, which provide their distinctive immune
response activation profiles.
Inventors: |
KANDIMALLA; EKAMBAR;
(US) ; PUTTA; MALLIKARJUNA; (US) ; YU;
DONG; (WESTBORO, MA) ; BHAGAT; LAKSHMI;
(FRAMINGHAM, MA) ; WANG; DAQING; (BEDFORD, MA)
; AGRAWAL; SUDHIR; (SHREWSBURY, MA) |
Assignee: |
IDERA PHARMACEUTICALS, INC.
|
Family ID: |
39512457 |
Appl. No.: |
12/960030 |
Filed: |
December 3, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11954726 |
Dec 12, 2007 |
7884197 |
|
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12960030 |
|
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60869604 |
Dec 12, 2006 |
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Current U.S.
Class: |
424/184.1 ;
514/44R; 536/23.1 |
Current CPC
Class: |
A61P 11/06 20180101;
C12N 2310/317 20130101; A61P 43/00 20180101; A61P 31/00 20180101;
A61P 37/00 20180101; A61P 35/00 20180101; A61P 37/04 20180101; C12N
2310/3183 20130101; C12N 2310/336 20130101; C12N 2310/321 20130101;
A61P 37/08 20180101; C12N 15/117 20130101; A61P 29/00 20180101;
C12N 2310/17 20130101; C12N 2310/315 20130101; C12N 2310/321
20130101; C12N 2310/3521 20130101 |
Class at
Publication: |
424/184.1 ;
536/23.1; 514/44.R |
International
Class: |
A61K 39/00 20060101
A61K039/00; C07H 21/02 20060101 C07H021/02; A61K 31/712 20060101
A61K031/712; A61K 31/7125 20060101 A61K031/7125; A61P 37/04
20060101 A61P037/04; A61P 35/00 20060101 A61P035/00; A61P 29/00
20060101 A61P029/00; A61P 31/00 20060101 A61P031/00; A61P 37/08
20060101 A61P037/08; A61P 11/06 20060101 A61P011/06 |
Claims
1. A TLR9 agonist comprising a structure selected from the group
consisting of 5'-TAGTCG.sub.1TTCTC-X-CTCTTG.sub.1CTGAT-5',
5'-TCG.sub.1TACG.sub.1TACG.sub.1-X-G.sub.1CATG.sub.1CATG.sub.1CT-5',
5'-TCG.sub.1ATCG.sub.1ATCG.sub.1-X-G.sub.1CTAG.sub.1CTAG.sub.1CT-5',
5'-TCTGTCG.sub.2TTTT-X-TTTTG.sub.2CTGTCT-5',
5'-TCG.sub.2ATCG.sub.2ATCG.sub.2-X-G.sub.2CTAG.sub.2CTAG.sub.2CT-5',
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-M-TCTTG.sub.1CTGTCT-5',
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-L-GACAG.sub.1CTGTCT-5',
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-L-GACAG.sub.1CTGTCT-5',
5'-TCG.sub.1TCG.sub.1ACG.sub.1AT-S-TAG.sub.1CAG.sub.1CTG.sub.1CT-5',
and 5'-TCAGToCG.sub.2TTAC-S-CATTG.sub.2CoTGACT-5', wherein G.sub.1
is 7-deaza-dG; G.sub.2 is AraG; G/A is 2'-O-methylribonucleotides;
o is phosphodiester linkage; X=Glycerol; L is 1,3-Propanediol; M is
1,5-Pentanediol; S is 3-Me-1,3,5-pentanetriol, wherein
internucleoside linkages are selected from the group consisting of
phosphodiester linkages, phosphorothioate linkages and mixtures
thereof.
2. A composition comprising the TLR9 agonist according to claim 1,
and a physiologically acceptable carrier.
3. A vaccine comprising the composition according to claim 2, and
an antigen.
4. A method for generating a TLR9-mediated immune response in a
vertebrate, comprising administering to the vertebrate an effective
amount of the TLR9 agonist according to claim 1.
5. A method for generating a TLR9-mediated immune response in a
vertebrate, comprising administering to the vertebrate an effective
amount of the composition according to claim 2.
6. A method for therapeutically treating a patient having a disease
or disorder, comprising administering to the patient a
therapeutically effective amount of the TLR9 agonist according to
claim 1.
7. A method for therapeutically treating a patient having a disease
or disorder, comprising administering to the patient a
therapeutically effective amount of the composition according to
claim 2.
8. A method for therapeutically treating a patient having a disease
or disorder, comprising administering to the patient a
therapeutically effective amount of the vaccine according to claim
3.
9. A method for preventing a disease or disorder in a patient that
is susceptible to the disease or disorder, comprising administering
to the patient a prophylactically effective amount of the TLR9
agonist according to claim 1.
10. A method for preventing a disease or disorder in a patient that
is susceptible to the disease or disorder, comprising administering
to the patient a prophylactically effective amount of the
composition according to claim 2.
11. A method for preventing a disease or disorder in a patient that
is susceptible to the disease or disorder, comprising administering
to the patient a prophylactically effective amount of the vaccine
according to claim 3.
12. The method according to any of claims 4-11, wherein the disease
or disorder is selected from the group consisting of cancer, an
autoimmune disorder, airway inflammation, inflammatory disorders,
infectious disease, allergy, asthma and a disease caused by a
pathogen.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/954,726, filed Dec. 12, 2007, and claims the benefit of U.S.
Provisional Application Ser. No. 60/869,604, filed on Dec. 12,
2006, the contents of which are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to synthetic chemical compositions
that are useful for modulation of Toll-Like Receptor (TLR)-mediated
immune responses. In particular, the invention relates to agonists
of Toll-Like Receptor 9 (TLR9) that generate unique cytokine and
chemokine profiles.
[0004] 2. Summary of the Related Art
[0005] Toll-like receptors (TLRs) are present on many cells of the
immune system and have been shown to be involved in the innate
immune response (Hornung, V. et a.l, (2002) J. Immunol.
168:4531-4537). In vertebrates, this family consists of eleven
proteins called TLR1 to TLR11, which are known to recognize
pathogen associated molecular patterns from bacteria, fungi,
parasites, and viruses (Poltorak, a. et al. (1998) Science
282:2085-2088; Underhill, D. M., et al. (1999) Nature 401:811-815;
Hayashi, F. et. al (2001) Nature 410:1099-1103; Zhang, D. et al.
(2004) Science 303:1522-1526; Meier, A. et al. (2003) Cell.
Microbiol. 5:561-570; Campos, M. A. et al. (2001) J. Immunol. 167:
416-423; Hoebe, K. et al. (2003) Nature 424: 743-748; Lund, J.
(2003) J. Exp. Med. 198:513-520; Heil, F. et al. (2004) Science
303:1526-1529; Diebold, S. S., et al. (2004) Science 303:1529-1531;
Hornung, V. et al. (2004) J. Immunol. 173:5935-5943).
[0006] TLRs are a key means by which vertebrates recognize and
mount an immune response to foreign molecules and also provide a
means by which the innate and adaptive immune responses are linked
(Akira, S. et al. (2001) Nature Immunol. 2:675-680; Medzhitov, R.
(2001) Nature Rev. Immunol. 1:135-145). Some TLRs are located on
the cell surface to detect and initiate a response to extracellular
pathogens and other TLRs are located inside the cell to detect and
initiate a response to intracellular pathogens.
[0007] TLR9 is known to recognize unmethylated CpG motifs in
bacterial DNA and in synthetic oligonucleotides. (Hemmi, H. et al.
(2000) Nature 408:740-745). Other modifications of CpG-containing
phosphorothioate oligonucleotides can also affect their ability to
act as modulators of immune response through TLR9 (see, e.g., Zhao
et al., Biochem. Pharmacol. (1996) 51:173-182; Zhao et al. (1996)
Biochem Pharmacol. 52:1537-1544; Zhao et al. (1997) Antisense
Nucleic Acid Drug Dev. 7:495-502; Zhao et al (1999) Bioorg. Med.
Chem. Lett. 9:3453-3458; Zhao et al. (2000) Bioorg. Med. Chem.
Lett. 10:1051-1054; Yu, D. et al. (2000) Bioorg. Med. Chem. Lett.
10:2585-2588; Yu, D. et al. (2001) Bioorg. Med. Chem. Lett.
11:2263-2267; and Kandimalla, E. et al. (2001) Bioorg. Med. Chem.
9:807-813). Naturally occurring agonists of TLR9 have been shown to
produce anti-tumor activity (e.g. tumor growth and angiogenesis)
resulting in an effective anti-cancer response (e.g. anti-leukemia)
(Smith, J. B. and Wickstrom, E. (1998) J. Natl. Cancer Inst.
90:1146-1154). In addition, TLR9 agonists have been shown to work
synergistically with other known anti-tumor compounds (e.g.
cetuximab, irinotecan) (Vincenzo, D., et al. (2006) Clin. Cancer
Res. 12(2):577-583).
[0008] Certain TLR9 agonists are comprised of 3'-3' linked DNA
structures containing a core CpR dinucleotide, wherein the R is a
modified guanosine (U.S. patent application Ser. No. 10/279,684).
In addition, specific chemical modifications have allowed the
preparation of specific oligonucleotide analogs that generate
distinct modulations of the immune response. In particular,
structure activity relationship studies have allowed identification
of synthetic motifs and novel DNA-based compounds that generate
specific modulations of the immune response and these modulations
are distinct from those generated by unmethylated CpG
dinucleotides. (Kandimalla, E. et al. (2005) Proc. Natl. Acad. Sci.
USA 102:6925-6930. Kandimalla, E. et al. (2003) Proc. Nat. Acad.
Sci. USA 100:14303-14308; Cong, Y. et al. (2003) Biochem Biophys
Res. Commun. 310:1133-1139; Kandimalla, E. et al. (2003) Biochem.
Biophys. Res. Commun. 306:948-953; Kandimalla, E. et al. (2003)
Nucleic Acids Res. 31:2393-2400; Yu, D. et al. (2003) Bioorg. Med.
Chem. 11:459-464; Bhagat, L. et al. (2003) Biochem. Biophys. Res.
Commun. 300:853-861; Yu, D. et al. (2002) Nucleic Acids Res.
30:4460-4469; Yu, D. et al. (2002) J. Med. Chem. 45:4540-4548. Yu,
D. et al. (2002) Biochem. Biophys. Res. Commun. 297:83-90;
Kandimalla. E. et al. (2002) Bioconjug. Chem. 13:966-974; Yu, D. et
al. (2002) Nucleic Acids Res. 30:1613-1619; Yu, D. et al. (2001)
Bioorg. Med. Chem. 9:2803-2808; Yu, D. et al. (2001) Bioorg. Med.
Chem. Lett. 11:2263-2267; Kandimalla, E. et al. (2001) Bioorg. Med.
Chem. 9:807-813; Yu, D. et al. (2000) Bioorg. Med. Chem. Lett.
10:2585-2588; Putta, M. et al. (2006) Nucleic Acids Res.
34:3231-3238).
[0009] The inventors have surprisingly discovered that unique
modifications to the sequence flanking the core CpR dinucleotide
produce novel agonists of TLR9 that generate distinct cytokine and
chemokine profiles in vitro and in vivo. This ability to
"custom-tune" the cytokine and chemokine response to a CpR
containing oligonucleotide promises to provide the ability to
prevent and/or treat various disease conditions in a
disease-specific and even a patient-specific manner. Thus, there is
a need for new oligonucleotide analog compounds to provide such
custom-tuned responses.
BRIEF SUMMARY OF THE INVENTION
[0010] The invention provides novel oligonucleotide-based compounds
that individually provide distinct immune response profiles through
their interactions as agonists with TLR9. The TLR9 agonists
according to the invention are characterized by specific and unique
chemical modifications, which provide their distinctive immune
response activation profiles.
[0011] The TLR9 agonists according to the invention induce immune
responses in various cell types and in various in vitro and in vivo
experimental models, with each agonist providing a distinct immune
response profile. As such, they are useful as tools to study the
immune system, as well as to compare the immune systems of various
animal species, such as humans and mice. The TLR9 agonists
according to the invention are also useful in the prevention and/or
treatment of various diseases, either alone, in combination with
other drugs, or as adjuvants for antigens used as vaccines.
[0012] Thus, in a first aspect, the invention provides
oligonuceotide-based agonists of TLR9.
[0013] In a second aspect, the invention provides a composition
comprising an oligonucleotide-based TLR9 agonist ("a compound")
according to the invention and a physiologically acceptable
carrier.
[0014] In a third aspect, the invention provides a vaccine.
Vaccines according to this aspect comprise a composition according
to the invention, and further comprise an antigen.
[0015] In a fourth aspect, the invention provides methods for
generating a TLR9-mediated immune response in a vertebrate, such
methods comprising administering to the vertebrate a compound,
composition or vaccine according to the invention.
[0016] In a fifth aspect, the invention provides methods for
therapeutically treating a patient having a disease or disorder,
such methods comprising administering to the patient a compound,
composition or vaccine according to the invention.
[0017] In a sixth aspect, the invention provides methods for
preventing a disease or disorder, such methods comprising
administering to the patient a compound, composition or vaccine
according to the invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0018] The invention provides novel oligonucleotide-based compounds
that individually provide distinct immune response profiles through
their interactions as agonists with TLR9. The TLR9 agonists
according to the invention are characterized by unique chemical
modifications, which provide their distinctive immune response
activation profiles. All publications cited herein reflect the
level of skill in the art and are hereby incorporated by reference
in their entirety. Any conflict between the teachings of these
references and this specification shall be resolved in favor of the
latter.
[0019] The TLR9 agonists according to the invention induce immune
responses in various cell types and in various in vivo and in vitro
experimental models, with each agonist providing a distinct immune
response profile. As such, they are useful as tools to study the
immune system, as well as to compare the immune systems of various
animal species, such as humans and mice. The TLR9 agonists
according to the invention are also useful in the prevention and/or
treatment of various diseases, either alone, in combination with
other drugs, or as adjuvants for antigens used as vaccines.
[0020] Certain TLR9 agonists according to the invention are shown
in Table I below. In this table, the oligonucleotide-based TLR9
agonists have all phosphorothioate (PS) linkages, except where
indicated. Except where indicated, all nucleotides are
deoxyribonucleotides. Those skilled in the art will recognize,
however, that phosphodiester (PO) linkages, or a mixture of PS and
PO linkages can be used.
TABLE-US-00001 TABLE I Seq. ID. No./ Oligo No. Sequence and
Modifications 1 5'-TCAGTC TTAC-X-CATT CTGACT-5' 2 5'-TCTGTC
TTAG-X-GATT CTGTCT-5' 3 5'-CAGTC TTCAG-X-GACTT CTGAC-5' 4 5'-TCTGTC
TTTT-X-TTTT CTGTCT-5' 5 5'-TCTGTC TTGT-X-TGTT CTGTCT-5' 6 5'-TAGTC
TTTTT-X-TTTTT CGTAT-5' 7 5'-TGGTC TTCTT-X-TTCTT CTGGT-5' 8 5'-TAGTC
TTGTA-X-ATGTT CTGAT-5' 9 5'-TAGTC TTCTC-X-CTCTT CTGAT-5' 10 5'-TC
TC TTCTT-X-TTCTT CTGCT-5' 11 5'-TC TAC TAC -X- CAT CAT CT-5' 12
5'-TC TC AC AT-X-TA CA CT CT-5' 13 5'-TC ATC ATC -X- CTA CTA CT-5'
14 5'-TCAGAC TTAC-X-CATT CAGACT-5' 15 5'-TCTGAC TTAG-X-GATT
CAGTCT-5' 16 5'-CAGAC TTCAG-X-GACTT CAGAC-5' 17 5'-TCTGAC
TTTT-X-TTTT CAGTCT-5' 18 5'-TCTGACGTTGT-X-TGTT CAGTCT-5' 19
5'-TAGAC TTTTT-X-TTTTT CAGAT-5' 20 5'-TGGAC TTCTT-X-TTCTT CAGGT-5'
21 5'-TAGACGTTGTA-X-ATGTT CAGAT-5' 22 5'-TAGAC TTCTC-X-CTCTT
CAGAT-5' 23 5'-TC TC TTCTT-X-TTCTT CT CT-5' 24 5'-TCAGTC
TTAC-X-CATT CTGACT-5' 25 5'-TCTGTC TTAG-X-GATT CTGTCT-5' 26
5'-CAGTC TTCAG-X-GACTT CTGAC-5' 27 5'-TCTGTC TTTT-X-TTTT CTGTCT-5'
28 5'-TCTGTC TTGT-X-TGTT CTGTCT-5' 29 5'-TAGTC TTTTT-X-TTTTT
CGTAT-5' 30 5'-TGGTC TTCTT-X-TTCTT CTGGT-5' 31 5'-TAGTC
TGTA-X-ATGTT CTGAT-5' 32 5'-TAGTC TTCTC-X-CTCTT CTGAT-5' 33 5'-TC
TC TTCTT-X-TTCTT CT CT-5' 34 5'-TC TAC TAC -X- CAT CAT CT-5' 35
5'-TC TC AC AT-X-TA CA CT CT-5' 36 5'-TC ATC ATC -X- CTA CTA CT-5'
37 5'-TCTGTCGTTCT-Y-TCTTGCTGTCT-5' 38 5'-TCTGAC TTCT-Y-TCTT
CAGTCT-5' 39 5'-TC AAC TTC -Y- CTT CAA CT-5' 40 5'-TC TC
TTCTG-Y-GTCTT CT CT-5' 41 5'-TCAGT GTTAG-Y-GATTG TGACT-5' 42
5'-TCTGT GTTCT-Y-TCTTG TGTCT-5' 43 5'-TCGTTGL-Y-LGTTGCT-5' 44
5'-TCGTTGM-Y-MGTTGCT-5' 45 5'-TC TTGM-Y-MGTT CT-5' 46
5'-TCGTTGM-X-MGTTGCT-5' 47 & 93 5'-TC AAC TTC -M-TCTT CTGTCT-5'
48 & 94 5'-TC AAC TTC -M-GACA CTGTCT-5' 49 [(5'-TCTGAC
TTCT).sub.2Y].sub.2Y 50 5'-TCTGTC TTCT-Y-TCTT CTGTCT-5' 51
5'-TCTGAC TTCT-Y-TCTT CAGTCT-5' 52 5'-TCTGT TCT-Y-TCTT TGTCT-5' 53
5'-TCTGACGTTCT-Z-TCTTGCAGTCT-5' 54 5'-TCTGAC TTCT-Z-TCTT CAGTCT-5'
55 5'-TCTGTC TTCT-Z-TCTT CTGTCT-5' 56 5'-TCTGAC TTCT-S-TCTT
CAGTCT-5' 57 5'-TCTGTC TTCT-S-TCTT CTGTCT-5' 58 5'-TC AAC TTC -S-
CTT CAA CT-5' 59 5'-TCAGTC TTAG-S-GATT CTGACT-5' 60 5'-TCTGTC
TTCUo-X-oUCTT CTGTCT-5' 61 5'-TCTGTC TTCoUo-X-oUoCTT CTGTCT-5' 62
5'-TCTGTC TTCU-X-UCTT CTGTCT-5' 63 5'-CTGTC TTCUC-X-CUCTT CTGTC-5'
64 5'-TC AAC TTCG-X-GCTT CAA CT-5' 65 & 95 5'-TC AAC TTC
-L-GACA CTGTCT-5' 66 5'-TCTGTC TTCUo-X-oUCTT CTGCTC-5' 67 5'-TCTGTC
TTCoUo-X-oUoCTT CTGCTC-5' 68 5'-TCTGTC TTCU-X-UCTT CTGCTC-5' 69
5'-CTGTC TTCUC-X-CUCTT CTGCTC-5' 70 5'-TC AAC TTCG-X-GCTT CAA CT-5'
71 & 95 5'-TC AAC TTC -L-GACA CTGTCT-5' 72 5'-TCTGTC
TTAG-S-GATT CTGTCT-5' 73 5'-CAGTC TTCAG-Z-GACTT CTGAC-5' 74 5'-TC
TC AC AT-S-TA CA CT CT-5' 75 5'-TCAGTC TTAC-X-CATT CTGACT-5' 76
5'-TCAoGToC TTAC-X-CATT CoTGoACT-5' 77 5'-UCAGTC TTAC-X-CATT
CTGACU-5' 78 5'-TCAGTC TTAoC-X-CoATT CTGACT-5' 79 5'-TAGToC
TTTTT-X-TTTTT CoTGTAT-5' 80 5'-TCTGToC TTGT-X-TGTT CoTGTCT-5' 81
5'-TAGoToC TTTTT-X-TTTTT CoToGTAT-5' 82 5'-TC oToC AoC AT-X-TA CoA
CoTo CT-5' 83 5'-TC AoToC oAoTC -X- ToAo CoToA CT-5' 84 5'-TCAGToC
TTAC-S-CATT CoTGACT-5' 85 & 96 5'-TCTGoToC TAG-Z-GATT
CoToGTCT-5' 86 5'-TC TC TTTL-S-LTTT CT CT-5' 87 5'-LTC TC
TTTL-S-LTTT CT CTL-5' 88 5'-TC C TTTL-Z-LTTT CT CT-5' 89 5'-LTC TC
TTTL-Z-LTTT CT CTL-5' 90 5'-TC TC TTTL-X-LTTT CT CT-5' 91 & 97
5'-LTC TC TTL-X-LTTT CT CTL-5' 92 5'-TCAGTC TTAC-X-CATT CTGACT-5' =
7-deaza-dG; = AraG; = N.sup.1-Me-dG; =
1-(2'-deoxy-.beta.-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine;
U/C/G/A = 2'-O-methylribonucleotides; o = phosphodiester linkage; X
= Glycerol; Y = 1,3,5-Pentanetriol; L = 1,3-Propanediol; M =
1,5-Pentanediol; Z = cis,cis-1,3,5-Cyclohexanetriol; S =
3-Me-1,3,5-pentanetriol
[0021] Exemplar TLR9 agonists from Table I were tested for immune
stimulatory activity in HEK293 cells expressing TLR9, as described
in Example 2. The results shown in Table II(a), II(b), II(c), and
II(d) below demonstrate that specific chemical modifications to
3'-3' linked oligonucleotides will alter their TLR9 mediated NF-kB
activation profile 24 hours after administration. More generally,
these data demonstrate that specific chemical modifications to
3'-3' linked oligonucleotides can be used to increase or decrease
NF-kB activation.
TABLE-US-00002 TABLE II(a) NF-kB Activation Profiles in HEK293
Cells Expressing TLR9 Fold Changes in NF-kB Seq. ID. No./ Activity
.+-. SD Oligo No. Sequences and Modification (5'-3') at 10 .mu.g/ml
1 5'-TCAGTC TTAC-X-CATT CTGACT-5' 13.94 .+-. 0.33 2 5'TCTGTC
TTAG-X-GATT CTGTCT-5' 6.76 .+-. 0.12 3 5'-CAGTC TTCAG-X-GACTT
CTGAC-5' 10.45 .+-. 0.13 4 5'-TCTGTC TTTT-X-TTTT CTGTCT-5' 7.84
.+-. 0.02 5 5'-TCTGTC TTGT-X-TGTT CTGTCT-5' 9.45 .+-. 1.31 6
5'-TAGTC TTTTT-X-TTTTT CGTAT-5' 6.95 .+-. 0.05 7 5'-TGGTC
TTCTT-X-TTCTT CTGGT-5' 5.02 .+-. 0.13 8 5'-TAGTC TTGTA-X-ATGTT
CTGAT-5' 2.75 .+-. 0.46 9 5'-TAGTC TTCTC-X-CTCTT CTGAT-5' 12.59
.+-. 0.26 10 5'-TC TC TTCTT-X-TTCTT CT CT-5' 13.24 .+-. 1.58 14
5'-TCAGAC TTAC-X-CATT CAGACT-5' 14.32 .+-. 0.19 15 5'-TCTGAC
TTAG-X-GATT CAGTCT-5' 12.19 .+-. 0.94 16 5'-CAGAC TTCAG-X-GACTT
CAGAC-5' 16.42 .+-. 0.44 17 5'-TCTGAC TTTT-X-TTTT CAGTCT-5' 16.49
.+-. 1.13 18 5'-TCTGAC TTGT-X-TGTT CAGTCT-5' 14.63 .+-. 0.03 19
5'-TAGAC TTTTT-X-TTTTT CAGAT-5' 16.06 .+-. 0.71 20 5'-TGGAC
TTCTT-X-TTCTT CAGGT-5' 13.28 .+-. 0.42 21 5'-TAGAC TTGTA-X-ATGTT
CAGAT-5' 11.75 .+-. 0.42 22 5'-TAGAC TTCTC-X-CTCTT CAGAT-5' 13.70
.+-. 0.21 Media 1 .+-. 0.07
TABLE-US-00003 TABLE II(b) NF-kB Activation Profiles in HEK293
Cells Expressing TLR9 Fold Changes Seq. ID. in NF-kB No./ Activity
Oligo Sequences and Modification at No. (5'-3') 10 .mu.g/ml 37
5'-TCTGTCGTTCT-Y-TCTTGCTGTCT-5' 11.35 38 5'-TCTGAC TTCT-Y-TCTT
CAGTCT-5' 11.51 50 5'-TCTGTC TTCT-Y-TCTT CTGTCT-5' 12.32 52
5'-TCTGTC TTCT-Y-TCTT CTGTCT-5' 9.40 57 5'-TCTGTC TTCT-S-TCTT
CTGTCT-5' 11.05 PBS 1.00
TABLE-US-00004 TABLE II(c) NF-kB Activation Profiles in HEK293
Cells Expressing TLR9 Fold Changes in Seq. ID. No./ NF-.kappa.B
Activity .+-. Oligo No. Sequences and Modification (5'-3') SD at 10
.mu.g/ml 60 5'-TCTGTC TTCUo-X-oUCTT CTGTCT-5' 11.2 .+-. 0.06 61
5'-TCTGTC TTCoUo-X-oUoCTT CTGTCT-5' 9.5 .+-. 0.06 62 5'-TCTGTC
TTCU-X-UCTT CTGTCT-5' 6.6 .+-. 0.04 63 5'-CTGTC TTCUC-X-CUCTT
CTGTC-5' 14.6 .+-. 0.09 64 5'-TC AAC TTCG-X-GCTT CAA CT-5' 32.1
.+-. 0.6 65 & 95 5'-TC AAC TTC -L-GACA CTGTCT-5' 23.7 .+-. 0.2
PBS 1 .+-. 0.03
TABLE-US-00005 TABLE II(d) NF-kB Activation Profiles in HEK293
Cells Expressing TLR9 Fold Changes in Seq. ID. No./ NF-.kappa.B
Activity .+-. Oligo No. Sequences and Modification (5'-3') SD at 10
.mu.g/ml 72 5'-TCTGTC TTAG-S-GATT CTGTCT-5' 3.81 .+-. 0.14 73
5'-CAGTC TTCAG-Z-GACTT CTGAC-5' 5.47 .+-. 0.17 74 5'-TC TC AC
AT-S-TA CA CT CT-5' 9.46 .+-. 1.35; 9.18 .+-. 0.09 75 5'-TCAGTC
TTAC-X-CATT CTGACT-5' 5.08 .+-. 0.58; 6.91 .+-. 1.52 77 5'-UCAGTC
TTAC-X-CATT CTGACU-5' 5.33 .+-. 0.25; 4.85 .+-. 0.46 78 5'-TCAGTC
TTAoC-X-CoATT CTGACT-5' 7.63 .+-. 0.54; 10.79 .+-. 0.9 79 5'-TAGToC
TTTTT-X-TTTTT CoTGTAT-5' 5.63 .+-. 1.46; 5.75 .+-. 0.45 80
5'-TCTGToC TTGT-X-TGTT CoTGTCT-5' 9.60 .+-. 1.39; 9.75 .+-. 0.25 81
5'-TAGoToC TTTTT-X-TTTTT CoToGTAT-5' 5.63 .+-. 0.46; 6.22 .+-. 0.12
82 5'-TC oToC AoC AT-X-TA CoA CoTo CT-5' 9.71 .+-. 0.75; 12.59 .+-.
0.3 83 5'-TC AoToC oAoTC -X- CToAo CoToA CT-5' 7.24 .+-. 0.42 84
5'-TCAGToC TTAC-S-CATT CoTGACT-5' 8.92 .+-. 0.88; 10.33 .+-. 0.2;
12.16 .+-. 1.5 85 & 96 5'-TCTGoToC TAG-Z-GATT CoToGTCT-5' 9.13
.+-. 1.25; 8.05 .+-. 0.39; 11.34 .+-. 0.3 86 5'-TC TC TTTL-S-LTTT
CT CT-5' 11.61 .+-. 0.6 88 5'-TC C TTTL-Z-LTTT CT CT-5' 9.61 .+-.
0.14; 9.32 .+-. 0.20 89 5'-LTC TC TTTL-Z-LTTT CT CTL-5' 2.57 .+-.
0.28 90 5'-TC TC TTTL-X-LTTT CT CT-5' 9.65 .+-. 1.78; 9.57 .+-.
0.18 92 5'-TCAGTC TTAC-X-CATT CTGACT-5' 5.67 .+-. 0.25; 7.25 .+-.
1.23 Media 1.0 .+-. 0.17; 1.0 .+-. 0.25; 1.0 .+-. 0.02; 1.0 .+-.
0.11
[0022] Exemplar TLR9 agonists from Table I were tested for immune
stimulatory activity in the C57BL/6 mouse spleenocyte IL-12 assay,
as described in Example 3. The results shown in Table III(a),
III(b) and III(c) below demonstrate that specific chemical
modifications to 3'-3' linked oligonucleotides will alter their
TLR9 mediated IL-12 activation profile in spleen cells 24 hours
after administration and that this activation profile may be dose
dependent depending on the chemical modification. More generally,
these data demonstrate that specific chemical modifications to
3'-3' linked oligonucleotides can be used to increase or decrease
IL-12 activation.
TABLE-US-00006 TABLE III(a) Induction of IL-12 Secretion in C57BL/6
Mouse Spleen Cell Cultures IL-12 Seq. ID No./ (pg/m .+-. SD1) Oligo
No. Sequences and Modification (5'-3') at 1 .mu.g/ml at 3 .mu.g/ml
1 5'-TCAGTC TTAC-X-CATT CTGACT-5' 733 .+-. 5 638 .+-. 14 2
5'-TCTGTC TTAG-X-GATT CTGTCT-5' 919 .+-. 8 660 .+-. 8 3 5'-CAGTC
TTCAG-X-GACTT CTGAC-5' 500 .+-. 26 634 .+-. 49 4 5'-TCTGTC
TTTT-X-TTTT CTGTCT-5' 822 .+-. 09 516 .+-. 07 5 5'-TCTGTC
TTGT-X-TGTT CTGTCT-5' 636 .+-. 6 369 .+-. 4 6 5'-TAGTC
TTTTT-X-TTTTT CGTAT-5' 857 .+-. 0 115 .+-. 0 7 5'-TGGTC
TTCTT-X-TTCTT CTGGT-5' 61 .+-. 0 357 .+-. 16 8 5'-TAGTC
TTGTA-X-ATGTT CTGAT-5' 253 .+-. 10 120 .+-. 13 9 5'-TAGTC
TTCTC-X-CTCTT CTGAT-5' 743 .+-. 33 553 .+-. 12 10 5'-TC TC
TTCTT-X-TTCTT CT CT-5' 714 .+-. 0 913 .+-. 12 14 5'-TCAGAC
TTAC-X-CATT CAGACT-5' 1654 .+-. 64 1592 .+-. 27 15 5'-TCTGAC
TTAG-X-GATT CAGTCT-5' 1299 .+-. 2 1257 .+-. 8 16 5'-CAGAC
TTCAG-X-GACTT CAGAC-5' 1152 .+-. 11 1134 .+-. 0 17 5'-TCTGAC
TTTT-X-TTTT CAGTCT-5' 1370 .+-. 4 1015 .+-. 7 18 5'-TCTGAC
TTGT-X-TGTT CAGTCT-5' 1140 .+-. 16 816 .+-. 4 19 5'-TAGAC
TTTTT-X-TTTTT CAGAT-5' 1215 .+-. 32 719 .+-. 3 20 5'-TGGAC
TTCTT-X-TTCTT CAGGT-5' 814 .+-. 9 645 .+-. 40 21 5'-TAGAC
TTGTA-X-ATGTT CAGAT-5' 835 .+-. 34 750 .+-. 16 22 5'-TAGAC
TTCTC-X-CTCTT CAGAT-5' 1211 .+-. 26 898 .+-. 24 Media 154 .+-. 1
154 .+-. 1
TABLE-US-00007 TABLE III(b) Induction of IL-12 Secretion in C57BL/6
Mouse Spleen Cell Cultures Seq. ID. No./ IL-12 Oligo No. Sequences
and Modification (5'-3') (pg/ml .+-. SD) 24 5'-TCAGTC TTAC-X-CATT
CTGACT-5' 932 .+-. 18 892 .+-. 2 25 5'-TCTGTC TTAG-X-GATT CTGTCT-5'
771 .+-. 6 604 .+-. 6 26 5'-CAGTC TTCAG-X-GACTT CTGAC-5' 835 .+-. 4
905 .+-. 4 27 5'-TCTGTC TTTT-X-TTTT CTGTCT-5' 571 .+-. 11 502 .+-.
2 28 5'-TCTGTC TTGT-X-TGTT CTGTCT-5' 567 .+-. 0 698 .+-. 77 29
5'-TAGTC TTTTT-X-TTTTT CGTAT-5' 975 .+-. 24 656 .+-. 33 30 5'-TGGTC
TTCTT-X-TTCTT CTGGT-5' 426 .+-. 16 393 .+-. 1 31 5'-TAGTC
TTGTA-X-ATGTT CTGAT-5' 568 .+-. 23 575 .+-. 14 32 5'-TAGTC
TTCTC-X-CTCTT CTGAT-5' 960 .+-. 2 647 .+-. 13 33 5'-TC TC
TTCTT-X-TTCTT CT CT-5' 659 .+-. 10 1014 .+-. 1 34 5'-TC TAC TAC -X-
CAT CAT CT-5' 1044 .+-. 66 1109 .+-. 32 35 5'-TC TC AC AT-X-TA CA
CT CT-5' 1406 .+-. 36 968 .+-. 4 36 5'-TC ATC ATC -X- CTA CTA CT-5'
912 .+-. 3 1035 .+-. 11 media 190 .+-. 4
TABLE-US-00008 TABLE III(c) Induction of IL-12 Secretion in C57BL/6
Mouse Spleen Cell Cultures Seq. ID No./ IL-12 Oligo No. Sequences
and Modification (5'-3') (pg/ml .+-. SD) 72 5'-TCTGTC TTAG-S-GATT
CTGTCT-5' 988 .+-. 224 73 5'-CAGTC TTCAG-Z-GACTT CTGAC-5' 504 .+-.
76 74 5'-TC TC AC AT-S-TA CA CT CT-5' 906 .+-. 47 75 5'-TCAGTC
TTAC-X-CATT CTGACT-5' 473 .+-. 67 77 5'-UCAGTC TTAC-X-CATT
CTGACU-5' 265 .+-. 19 78 5'-TCAGTC TTAoC-X-CoATT CTGACT-5' 833 .+-.
63 79 5'-TAGToC TTTTT-X-TTTTT CoTGTAT-5' 380 .+-. 54 80 5'-TCTGToC
TTGT-X-TGTT CoTGTCT-5' 1502 .+-. 162 81 5'-TAGoToC TTTTT-X-TTTTT
CoToGTAT-5' 370 .+-. 47 82 5'-TC oToC AoC AT-X-TA CoA CoTo CT-5'
1599 .+-. 156 83 5'-TC AoToC oAoTC -X- CToAo CoToA CT-5' 1203 .+-.
109 84 5'-TCAGToC TTAC-S-CATT CoTGACT-5' 838 .+-. 61 85 & 96
5'-TCTGoToC TAG-Z-GATT CoToGTCT-5' 589 .+-. 45 86 5'-TC TC
TTTL-S-LTTT CT CT-5' 1603 .+-. 167 88 5'-TC C TTTL-Z-LTTT CT CT-5'
1643 .+-. 40 89 5'-LTC TC TTTL-Z-LTTT CT CTL-5' 450 .+-. 50 90
5'-TC TC TTTL-X-LTTT CT CT-5' 1393 .+-. 9 92 5'-TCAGTC TTAC-X-CATT
CTGACT-5' 383 .+-. 23 Media 82 .+-. 4; 168 .+-. 15
[0023] Exemplar TLR9 agonists from Table I were tested for immune
stimulatory activity in the C57BL/6 mouse spleenocyte IL-6 assay,
as described in Example 3. The results shown in Table IV(a), IV(b),
and IV(c) below demonstrate that specific chemical modifications to
3'-3' linked oligonucleotides will alter their TLR9 mediated IL-6
activation profile in spleen cells 24 hours after administration
and that this activation profile may be dose dependent depending on
the chemical modification. More generally, these data demonstrate
that specific chemical modifications to 3'-3' linked
oligonucleotides can be used to increase or decrease IL-6
activation.
TABLE-US-00009 TABLE IV(a) Induction of IL-6 Secretion in C57BL/6
Mouse Spleen Cell Cultures Seq. ID. No./ IL-6 (pg/ml .+-. SD) Oligo
No. Sequences and Modification (5'-3') at 1 .mu.g/ml at 3 .mu.g/ml
1 5'-TCAGTC TTAC-X-CATT CTGACT-5' 2436 .+-. 93 6282 .+-. 138 2
5'-TCTGTC TTAG-X-GATT CTGTCT-5' 1812 .+-. 95 5758 .+-. 55 3
5'-CAGTC TTCAG-X-GACTT CTGAC-5' 1650 .+-. 63 3349 .+-. 46 4
5'-TCTGTC TTTT-X-TTTT CTGTCT-5' 707 .+-. 59 7018 .+-. 3 5 5'-TCTGTC
TTGT-X-TGTT CTGTCT-5' 1302 .+-. 56 5874 .+-. 83 6 5'-TAGTC
TTTTT-X-TTTTT CGTAT-5' 1025 .+-. 93 1677 .+-. 12 7 5'-TGGTC
TTCTT-X-TTCTT CTGGT-5' 453 .+-. 8 3068 .+-. 3 8 5'-TAGTC
TTGTA-X-ATGTT CTGAT-5' 914 .+-. 74 1147 .+-. 30 9 5'-TAGTC
TTCTC-X-CTCTT CTGAT-5' 3570 .+-. 21 12114 .+-. 86 10 5'-TC TC
TTCTT-X-TTCTT CT CT-5' 77 .+-. 0 1657 .+-. 17 14 5'-TCAGAC
TTAC-X-CATT CAGACT-5' 2605 .+-. 15 7206 .+-. 16 15 5'-TCTGAC
TTAG-X-GATT CAGTCT-5' 1705 .+-. 28 6538 .+-. 63 16 5'-CAGAC
TTCAG-X-GACTT CAGAC-5' 1081 .+-. 20 3765 .+-. 18 17 5'-TCTGAC
TTTT-X-TTTT CAGTCT-5' 1711 .+-. 32 8386 .+-. 33 18 5'-TCTGAC
TTGT-X-TGTT CAGTCT-5' 1725 .+-. 0 7340 .+-. 142 19 5'-TAGAC
TTTTT-X-TTTTT CAGAT-5' 984 .+-. 16 3312 .+-. 22 20 5'-TGGAC
TTCTT-X-TTCTT CAGGT-5' 515 .+-. 77 1828 .+-. 22 21 5'-TAGAC
TTGTA-X-ATGTT CAGAT-5' 221 .+-. 5 1539 .+-. 9 22 5'-TAGAC
TTCTC-X-CTCTT CAGAT-5' 1593 .+-. 19 6960 .+-. 81 Media 0 .+-. 0 0
.+-. 0
TABLE-US-00010 TABLE IV(b) Induction of IL-6 Secretion in C57BL/6
Mouse Spleen Cell Cultures (24 hours) Seq. ID. No./ Oligo No.
Sequences and Modification (5'-3') IL-6 (pg/ml .+-. SD) 24
5'-TCAGTC TTAC-X-CATT CTGACT-5' 8276 .+-. 35 11634 .+-. 83 25
5'-TCTGTC TTAG-X-GATT CTGTCT-5' 5428 .+-. 106 11860 .+-. 154 26
5'-CAGTC TTCAG-X-GACTT CTGAC-5' 6389 .+-. 15 12402 .+-. 77 27
5'-TCTGTC TTTT-X-TTTT CTGTCT-5' 3977 .+-. 89 8058 .+-. 46 28
5'-TCTGTC TTGT-X-TGTT CTGTCT-5' 4333 .+-. 59 10555 .+-. 49 29
5'-TAGTC TTTTT-X-TTTTT CGTAT-5' 3380 .+-. 24 9348 .+-. 90 30
5'-TGGTC TTCTT-X-TTCTT CTGGT-5' 2452 .+-. 45 4028 .+-. 15 31
5'-TAGTC TTGTA-X-ATGTT CTGAT-5' 2574 .+-. 45 6426 .+-. 40 32
5'-TAGTC TTCTC-X-CTCTT CTGAT-5' 6432 .+-. 4 10872 .+-. 413 33 5'-TC
TC TTCTT-X-TTCTT CT CT-5' 6136 .+-. 24 10408 .+-. 7 34 5'-TC TAC
TAC -X- CAT CAT CT-5' 7840 .+-. 61 15642 .+-. 56 35 5'-TC TC AC
AT-X-TA CA CT CT-5' 8004 .+-. 141 15174 .+-. 54 36 5'-TC ATC ATC
-X- CTA CTA CT-5' 5590 .+-. 259 14788 .+-. 441 media 346 .+-. 0
TABLE-US-00011 TABLE IV(c) Induction of IL-6 Secretion in C57BL/6
Mouse Spleen Cell Cultures (24 hours) Seq. ID. No./ IL-6 Oligo No.
Sequences and Modification (5'-3') (pg/ml .+-. SD) 72 5'-TCTGTC
TTAG-S-GATT CTGTCT-5' 8388 .+-. 1609 73 5'-CAGTC TTCAG-Z-GACTT
CTGAC-5' 4198 .+-. 1602 74 5'-TC TC AC AT-S-TA CA CT CT-5' 18828
.+-. 1448 75 5'-TCAGTC TTAC-X-CATT CTGACT-5' 3689 .+-. 109 77
5'-UCAGTC TTAC-X-CATT CTGACU-5' 91 .+-. 12 78 5'-TCAGTC
TTAoC-X-CoATT CTGACT-5' 22047 .+-. 8443 79 5'-TAGToC TTTTT-X-TTTTT
CoTGTAT-5' 1234 .+-. 508 80 5'-TCTGToC TTGT-X-TGTT CoTGTCT-5' 14025
.+-. 369 81 5'-TAGoToC TTTTT-X-TTTTT CoToGTAT-5' 373 .+-. 61 82
5'-TC oToC AoC AT-X-TA CoA CoTo CT-5' 86355 .+-. 4638 83 5'-TC
AoToC oAoTC -X- CToAo CoToA CT-5' 10871 .+-. 1996 84 5'-TCAGToC
TTAC-S-CATT CoTGACT-5' 30346 .+-. 1670 85 & 96 5'-TCTGoToC
TAG-Z-GATT CoToGTCT-5' 113 .+-. 11 86 5'-TC TC TTTL-S-LTTT CT CT-5'
15654 .+-. 470 88 5'-TC C TTTL-Z-LTTT CT CT-5' 16317 .+-. 659 89
5'-LTC TC TTTL-Z-LTTT CT CTL-5' 1259 .+-. 215 90 5'-TC TC
TTTL-X-LTTT CT CT-5' 13864 .+-. 344 92 5'-TCAGTC TTAC-X-CATT
CTGACT-5' 2171 .+-. 186 Media 51 .+-. 3; 60 .+-. 1
[0024] Exemplar TLR9 agonists from Table I were tested for immune
stimulatory activity in the human B-cell proliferation assay, as
described in Example 4. The results shown in Table V(a), V(b),
V(c), V(d) and V(e) below demonstrate that specific chemical
modifications to 3'-3' linked oligonucleotides will alter their
TLR9 mediated B-cell proliferation activity and that this
activation profile may be dose dependent depending on the chemical
modification. More generally, these data demonstrate that specific
chemical modifications to 3'-3' linked oligonucleotides can be used
to regulate B-cell proliferation.
TABLE-US-00012 TABLE V(a) Human B-Cell Proliferation Assay Seq. ID.
No./ [.sup.3H]-T (cpm .+-. SD) Oligo No. Sequences and Modification
(5'-3') at 1 .mu.g/ml at 3 .mu.g/ml 1 5'-TCAGTC TTAC-X-CATT
CTGACT-5' 9170 .+-. 5038 7556 .+-. 3260 2 5'-TCTGTC TTAG-X-GATT
CTGTCT-5' 9907 .+-. 4299 9405 .+-. 3319 3 5'-CAGTC TTCAG-X-GACTT
CTGAC-5' 7594 .+-. 4088 7094 .+-. 1526 4 5'-TCTGTC TTTT-X-TTTT
CTGTCT-5' 13130 .+-. 6721 12343 .+-. 4336 5 5'-TCTGTC TTGT-X-TGTT
CTGTCT-5' 11990 .+-. 5511 12102 .+-. 5618 6 5'-TAGTC TTTTT-X-TTTTT
CGTAT-5' 13676 .+-. 3676 14223 .+-. 6073 7 5'-TGGTC TTCTT-X-TTCTT
CTGGT-5' 7286 .+-. 2800 7007 .+-. 424 8 5'-TAGTC TTGTA-X-ATGTT
CTGAT-5' 7858 .+-. 2877 8757 .+-. 3733 9 5'-TAGTC TTCTC-X-CTCTT
CTGAT-5' 7834 .+-. 2397 6840 .+-. 2158 Media 559 .+-. 355 559 .+-.
355
TABLE-US-00013 TABLE V(b) Human B-Cell Proliferation Assay Seq. ID.
No./ Oligo No. Sequences and Modification (5'-3') [.sup.3H]-T (cpm
.+-. SD) 24 5'-TCAGTC TTAC-X-CATT CTGACT-5' 12015 .+-. 2721 22634
.+-. 7474 25 5'-TCTGTC TTAG-X-GATT CTGTCT-5' 12033 .+-. 1502 28048
.+-. 14380 26 5'-CAGTC TTCAG-X-GACTT CTGAC-5' 8738 .+-. 2957 23675
.+-. 11455 27 5'-TCTGTC TTTT-X-TTTT CTGTCT-5' 17623 .+-. 4158 24309
.+-. 7340 28 5'-TCTGTC TTGT-X-TGTT CTGTCT-5' 13631 .+-. 01735 18438
.+-. 3212 29 5'-TAGTC TTTTT-X-TTTTT CGTAT-5' 12051 .+-. 5367 19867
.+-. 9831 30 5'-TGGTC TTCTT-X-TTCTT CTGGT-5' 17206 .+-. 8474 18061
.+-. 9703 31 5'-TAGTC TTGTA-X-ATGTT CTGAT-5' 21600 .+-. 10694 22746
.+-. 13411 32 5'-TAGTC TTCTC-X-CTCTT CTGAT-5' 15827 .+-. 8603 26722
.+-. 16455 33 5'-TC TC TTCTT-X-TTCTT CT CT-5' 19269 .+-. 14059
21945 .+-. 11281 34 5'-TC TAC TAC -X- CAT CAT CT-5' 11228 .+-. 4499
17990 .+-. 7547 35 5'-TC TC AC AT-X-TA CA CT CT-5' 13364 .+-. 2570
22787 .+-. 3265 36 5'-TC ATC ATC -X- CTA CTA CT-5' 14071 .+-. 3313
31519 .+-. 2373 media 634 .+-. 166
TABLE-US-00014 TABLE V(c) Human B-Cell Proliferation Assay Seq. ID.
No./ Oligo No. Sequences and Modification (5'-3') [.sup.3H]-T (cpm
.+-. SD) 38 5'-TCTGAC TTCT-Y-TCTT CAGTCT-5' 4714 .+-. 1043 4535
.+-. 1269 39 5'-TC AAC TTC -Y- CTT CAA CT-5' 3664 .+-. 219 7556
.+-. 1615 40 5'-TC TC TTCTG-Y-GTCTT CT CT-5' 4346 .+-. 453 6093
.+-. 2052 41 5'-TCAGTCGTTAG-Y-GATTGCTGACT-5' 3585 .+-. 495 4371
.+-. 1380 42 5'-TCTGTCGTTCT-Y-TCTTGCTGTCT-5' 5607 .+-. 1163 5202
.+-. 1980 43 5'-TCGTTGL-Y-LGTTGCT-5' 3302 .+-. 359 4767 .+-. 737 47
& 93 5'-TC AAC TTC -M-TCTT CTGTCT-5' 6010 .+-. 1951 6469 .+-.
3332 48 & 94 5'-TC AAC TTC -M-GACA CTGTCT-5' 3507 .+-. 768 4351
.+-. 2101 PBS 545 .+-. 237
TABLE-US-00015 TABLE V(d) Human B-Cell Proliferation Assay
Prolifer- Seq. ID. ation No./ Sequences and Modification Index at
Oligo No. (5'-3') 1 .mu.g/ml 60 5'-TCTGTC TTCUo-X-oUCTT 28.4
CTGTCT-5' 61 5'-TCTGTC TTCoUo-X-oUoCTT 31.3 CTGTCT-5' 62 5'-TCTGTC
TTCU-X-UCTT 42.6 CTGTCT-5' 63 5'-CTGTC TTCUC-X-CUCTT 41.5 CTGTC-5'
64 5'-TC AAC TTCG-X-GCTT 45.6 CAA CT-5' 65 & 95 5'-TC AAC TTC
-L-GACA 23.8 CTGTCT-5' Medium 1
TABLE-US-00016 TABLE V(e) Human B-Cell Proliferation Assay Seq. ID.
Proliferation No./ Index Oligo No. Sequences and Modification
(5'-3') at 1 .mu.g/ml 66 5'-TCTGTC TTCUo-X-oUCTT CTGCTC-5' 13582
.+-. 1296 67 5'-TCTGTC TTCoUo-X-oUoCTT CTGCTC-5' 19250 .+-. 1860 68
5'-TCTGTC TTCU-X-UCTT CTGCTC-5' 24809 .+-. 3983 69 5'-CTGTC
TTCUC-X-CUCTT CTGCTC-5' 21125 .+-. 2056 70 5'-TC AAC TTCG-X-GCTT
CAA CT-5' 20306 .+-. 6796 71 & 95 5'-TC AAC TTC -L-GACA
CTGTCT-5' 11547 .+-. 631 72 5'-TCTGTC TTAG-S-GATT CTGTCT-5' 16603
.+-. 2124 73 5'-CAGTC TTCAG-Z-GACTT CTGAC-5' 9787 .+-. 1290 74
5'-TC TC AC AT-S-TA CA CT CT-5' 16934 .+-. 2628 80 5'-TCTGToC
TTGT-X-TGTT CoTGTCT-5' 16347 .+-. 980; 18093 .+-. 03142 84
5'-TCAGToC TTAC-S-CATT CoTGACT-5' 14546 .+-. 2616 85 & 96
5'-TCTGoToC TAG-Z-GATT CoToGTCT-5' 10051 .+-. 1376 86 5'-TC TC
TTTL-S-LTTT CT CT-5' 18297 .+-. 1246 88 5'-TC C TTTL-Z-LTTT CT
CT-5' 12128 .+-. 2106; 16534 .+-. 1037 89 5'-LTC TC TTTL-Z-LTTT CT
CTL-5' 10749 .+-. 1191 90 5'-TC TC TTTL-X-LTTT CT CT-5' 11357 .+-.
692; 22666 .+-. 54 91 & 97 5'-LTC TC TTL-X-LTTT CT CTL-5' 92
5'-TCAGTC TTAC-X-CATT CTGACT-5' Media 621 .+-. 215
[0025] Exemplar TLR9 agonists from Table I were tested for immune
stimulatory activity in the human PBMC and B-Cell assays for
IL-1Ra, IL-6, IL-10, and IL-12, as described in Example 3. The
results shown in Table VI(a), VI(b), VI(c), VI(d), VI(e), VI(f),
VI(g), and VI(h) below demonstrate that specific chemical
modifications to 3'-3' linked oligonucleotides will alter their
TLR9 mediated IL-1Ra, IL-6, IL-10, and/or IL-12 activation profile
in human PBMCs. More generally, these data demonstrate that
specific chemical modifications to 3'-3' linked oligonucleotides
can be used to increase or decrease IL-1R.alpha., IL-6, IL-10, and
IL-12 activation.
TABLE-US-00017 TABLE VI(a) Human PBMC Assay for IL-1R.alpha., IL-6
and IL-12 IL-1R.alpha. IL-6 IL-12 Seq. ID. (pg/ml) (pg/ml) (pg/ml)
No./ at 10 at 10 at 10 Oligo No. Sequences and Modification (5'-3')
.mu.g/ml .mu.g/ml .mu.g/ml 1 5'-TCAGTC TTAC-X-CATT CTGACT-5' 1331
1035 116.5 2 5'-TCTGTC TTAG-X-GATT CTGTCT-5' 599.5 630 56.5 3
5'-CAGTC TTCAG-X-GACTT CTGAC-5' 1133 867.5 76 4 5'-TCTGTC
TTTT-X-TTTT CTGTCT-5' 926 994 81 5 5'-TCTGTC TTGT-X-TGTT CTGTCT-5'
874.5 813.5 69 6 5'-TAGTC TTTTT-X-TTTTT CGTAT-5' 921 958 90.5 7
5'-TGGTC TTCTT-X-TTCTT CTGGT-5' 1365 1110.5 101.5 8 5'-TAGTC
TTGTA-X-ATGTT CTGAT-5' 744 1048.5 75.5 9 5'-TAGTC TTCTC-X-CTCTT
CTGAT-5' 1239 1084.5 104.5 10 5'-TC TC TTCTT-X-TTCTT CT CT-5' 1727
1415.5 145 11 5'-TC TAC TAC -X- CAT CAT CT-5' 2905.5 2099.5 197.5
12 5'-TC TC AC AT-X-TA CA CT CT-5' 2965 1985.3 192.7 13 5'-TC ATC
ATC -X- CTA CTA CT-5' 3584.5 2543.5 300.5 Media 187 36 21
TABLE-US-00018 TABLE VI(b) Human PBMC Assay for IL-1R.alpha., IL-6
and IL-12 IL-1R.alpha. IL-6 IL-12 Seq. ID. (pg/ml) (pg/ml) (pg/ml)
No./ at 10 at 10 at 10 Oligo No. Sequences and Modification (5'-3')
.mu.g/ml .mu.g/ml .mu.g/ml 24 5'-TCAGTC TTAC-X-CATT CTGACT-5'
10537.8 340.8 350.7 25 5'-TCTGTC TTAG-X-GATT CTGTCT-5' 14850.4
413.6 456.1 26 5'-CAGTC TTCAG-X-GACTT CTGAC-5' 9440.6 335.0 304.4
27 5'-TCTGTC TTTT-X-TTTT CTGTCT-5' 13014.5 499.0 421.9 28 5'-TCTGTC
TTGT-X-TGTT CTGTCT-5' 10270.2 363.2 323.0 29 5'-TAGTC TTTTT-X-TTTTT
CGTAT-5' 11644.7 421.2 362.3 30 5'-TGGTC TTCTT-X-TTCTT CTGGT-5'
10528.9 465.7 339.2 31 5'-TAGTC TTGTA-X-ATGTT CTGAT-5' 19086.4
554.6 551.3 32 5'-TAGTC TTCTC-X-CTCTT CTGAT-5' 15514.6 434.8 462.9
33 5'-TC TC TTCTT-X-TTCTT CT CT-5' 22655.8 551.9 598.6 34 5'-TC TAC
TAC -X- CAT CAT CT-5' 20375.5 456.3 596.3 35 5'-TC TC AC AT-X-TA CA
CT CT-5' 17750.6 383.5 521.9 36 5'-TC ATC ATC -X- CTA CTA CT-5'
23576.8 428.0 706.3 media 799.5 15.7 47.7
TABLE-US-00019 TABLE VI(c) Human PBMC Assay for IL-6 (24 hours)
IL-6 Seq. ID. (pg/ml .+-. No./ Sequences and Modification SD) at
Oligo No. (5'-3') 10 ug/ml 60 5'-TCTGTC TTCUo-X-oUCTT 423 .+-. 1
CTGTCT-5' 61 5'-TCTGTC TTCoUo-X-oUoCTT 938 .+-. 14 CTGTCT-5' 62
5'-TCTGTC TTCU-X-UCTT 497 .+-. 4 CTGTCT-5' 63 5'-CTGTC
TTCUC-X-CUCTT 409 .+-. 2 CTGTC-5' 64 5'-TC AAC TTCG-X-GCTT 474 .+-.
0 CAA CT-5' 65 & 95 5'-TC AAC TTC -L-GACA 626 .+-. 3 CTGTCT-5'
Medium 0 .+-. 0
TABLE-US-00020 TABLE VI(d) Human PBMC Assay for IL-6 (24 hours)
Seq. ID. IL-6 No./ (pg/ml .+-. SD) Oligo No. Sequences and
Modification (5'-3') at 10 .mu.g/ml 66 5'-TCTGTC TTCUo-X-oUCTT
CTGCTC-5' 135.63 67 5'-TCTGTC TTCoUo-X-oUoCTT CTGCTC-5' 117.98 68
5'-TCTGTC TTCU-X-UCTT CTGCTC-5' 300.79 69 5'-CTGTC TTCUC-X-CUCTT
CTGCTC-5' 151.84 70 5'-TC AAC TTCG-X-GCTT CAA CT-5' 268.71 71 &
95 5'-TC AAC TTC -L-GACA CTGTCT-5' 364.23 75 5'-TCAGTC TTAC-X-CATT
CTGACT-5' 722.58 77 5'-UCAGTC TTAC-X-CATT CTGACU-5' 615.21 78
5'-TCAGTC TTAoC-X-CoATT CTGACT-5' 449.96 79 5'-TAGToC TTTTT-X-TTTTT
CoTGTAT-5' 658.10 80 5'-TCTGToC TTGT-X-TGTT CoTGTCT-5' 490.37 81
5'-TAGoToC TTTTT-X-TTTTT CoToGTAT-5' 668.52 82 5'-TC oToC AoC
AT-X-TA CoA CoTo CT-5' 614.15 84 5'-TCAGToC TTAC-S-CATT CoTGACT-5'
603.68; 351.00 85 & 96 5'-TCTGoToC TAG-Z-GATT CoToGTCT-5'
387.97; 464.58 88 5'-TC C TTTL-Z-LTTT CT CT-5' 440.25 90 5'-TC TC
TTTL-X-LTTT CT CT-5' 446.67 92 5'-TCAGTC TTAC-X-CATT CTGACT-5'
605.79 Media 7.12; 3.59
TABLE-US-00021 TABLE VI(e) Human PBMC Assay for IL-10 (24 hours)
IL-10 Seq. ID. No./ (pg/ml .+-. SD) Oligo No. Sequences and
Modification (5'-3') at 10 .mu.g/ml 60 5'-TCTGTC TTCUo-X-oUCTT
CTGTCT-5' 44 .+-. 6 61 5'-TCTGTC TTCoUo-X-oUoCTT CTGTCT-5' 50 .+-.
6 62 5'-TCTGTC TTCU-X-UCTT CTGTCT-5' 42 .+-. 2 63 5'-CTGTC
TTCUC-X-CUCTT CTGTC-5' 55 .+-. 2 64 5'-TC AAC TTCG-X-GCTT CAA CT-5'
11 .+-. 2 65 & 95 5'-TC AAC TTC -L-GACA CTGTCT-5' 26 .+-. 2
Medium 18 .+-. 0
TABLE-US-00022 TABLE VI(f) Human PBMC Assay for IL-12 (24 hours)
Seq. ID. IL-12 No./ (pg/ml .+-. SD) Oligo No. Sequences and
Modification (5'-3') at 10 .mu.g/ml 66 5'-TCTGTC TTCUo-X-oUCTT
CTGCTC-5' 284.03 67 5'-TCTGTC TTCoUo-X-oUoCTT CTGCTC-5' 296.62 68
5'-TCTGTC TTCU-X-UCTT CTGCTC-5' 502.12 69 5'-CTGTC TTCUC-X-CUCTT
CTGCTC-5' 531.48 70 5'-TC AAC TTCG-X-GCTT CAA CT-5' 729.32 71 &
95 5'-TC AAC TTC -L-GACA CTGTCT-5' 810.12 75 5'-TCAGTC TTAC-X-CATT
CTGACT-5' 1678.61 77 5'-UCAGTC TTAC-X-CATT CTGACU-5' 1500.97 78
5'-TCAGTC TTAoC-X-CoATT CTGACT-5' 927.15 79 5'-TAGToC TTTTT-X-TTTTT
CoTGTAT-5' 1013.11 80 5'-TCTGToC TTGT-X-TGTT CoTGTCT-5' 1498.64 81
5'-TAGoToC TTTTT-X-TTTTT CoToGTAT-5' 1019.68 82 5'-TC oToC AoC
AT-X-TA CoA CoTo CT-5' 1220.94 84 5'-TCAGToC TTAC-S-CATT CoTGACT-5'
1450.24; 1604.88 85 & 96 5'-TCTGoToC TAG-Z-GATT CoToGTCT-5'
879.09; 1498.64 88 5'-TC C TTTL-Z-LTTT CT CT-5' 1463.20 90 5'-TC TC
TTTL-X-LTTT CT CT-5' 1417.50 92 5'-TCAGTC TTAC-X-CATT CTGACT-5'
1602.41 Media 54.36; 196.06; 511.18
TABLE-US-00023 TABLE VI(g) Induction of IL-6 in human B cell
cultures (24 hours) IL-6 Seq. ID. No./ (pg/ml .+-. SD) Oligo No.
Sequences and Modification (5'-3') at 10 .mu.g/ml 60 5'-TCTGTC
TTCUo-X-oUCTT CTGTCT-5' 359 .+-. 7 61 5'-TCTGTC TTCoUo-X-oUoCTT
CTGTCT-5' 570 .+-. 37 62 5'-TCTGTC TTCU-X-UCTT CTGTCT-5' 333 .+-. 3
63 5'-CTGTC TTCUC-X-CUCTT CTGTC-5' 593 .+-. 8 64 5'-TC AAC
TTCG-X-GCTT CAA CT-5' 503 .+-. 28 65 & 95 5'-TC AAC TTC -L-GACA
CTGTCT-5' 481 .+-. 13 Medium 86 .+-. 5
TABLE-US-00024 TABLE VI(h) Induction of IL-10 in human B cell
cultures (24 hours) Seq. ID. No./ IL-10 (pg/ml .+-. SD) Oligo No.
Sequences and Modification (5'-3') at 10 .mu.g/ml 60 5'-TCTGTC
TTCUo-X-oUCTT CTGTCT-5' 188 .+-. 0 61 5'-TCTGTC TTCoUo-X-oUoCTT
CTGTCT-5' 286 .+-. 3 62 5'-TCTGTC TTCU-X-UCTT CTGTCT-5' 223 .+-. 10
63 5'-CTGTC TTCUC-X-CUCTT CTGTC-5' 201 .+-. 2 64 5'-TC AAC
TTCG-X-GCTT CAA CT-5' 268 .+-. 0 65 5'-TC AAC TTC -L-GACA CTGTCT-5'
212 .+-. 1 Medium 86 .+-. 5
[0026] Exemplar TLR9 agonists from Table I were tested for immune
stimulatory activity in the human PBMC assay for IFN-.gamma.,
MIP-1.alpha. and MIP-.beta., as described in Example 3. The results
shown in Table VII(a) and VII(b) below demonstrate that specific
chemical modifications to 3'-3' linked oligonucleotides will alter
their TLR9 mediated IFN-.gamma., MIP-1.alpha., and/or MIP-.beta.
activation profile in human PBMCs. More generally, these data
demonstrate that specific chemical modifications to 3'-3' linked
oligonucleotides can be used to increase or decrease IFN-.gamma.,
MIP-1.alpha., and MIP-.beta. activation.
TABLE-US-00025 TABLE VII(a) Human PBMC Assay for IFN-.gamma.,
MIP-1.alpha. and MIP-.beta. IFN-.gamma. MIP-1.alpha. MIP-.beta.
Seq. ID. No./ (pg/ml) (pg/ml) (pg/ml) Oligo No. Sequences and
Modification (5'-3') at 10 ug/ml at 10 ug/ml at 10 ug/ml 1
5'-TCAGTC TTAC-X-CATT CTGACT-5' 86 28 1108 2 5'-TCTGTCG1TTAG-X-GATT
CTGTCT-5' 38.5 11 568.5 3 5'-CAGTC TTCAG-X-GACTT CTGAC-5' 29 8.5
465.5 4 5'-TCTGTC TTTT-X-TTTT CTGTCT-5' 31.5 14 648.5 5 5'-TCTGTC
TTGT-X-TGTT CTGTCT-5' 52.5 12 679 6 5'-TAGTC TTTTT-X-TTTTT CGTAT-5'
66.5 15.5 799 7 5'-TGGTC TTCTT-X-TTCTT CTGGT-5' 68.5 17 889.5 8
5'-TAGTC TTGTA-X-ATGTT CTGAT-5' 77 20 1174 9 5'-TAGTC TTCTC-X-CTCTT
CTGAT-5' 93.5 26.5 1240.5 10 5'-TC TC TTCTT-X-TTCTT CT CT-5' 59.5
29.5 1007 11 5'-TC TAC TAC -X- CAT CAT CT-5' 5237.5 83 2931.5 12
5'-TC TC AC AT-X-TA CA CT CT-5' 2199.7 24.7 2363 13 5'-TC ATC ATC
-X- CTA CTA CT-5' 5619.5 173 2479 Media 40 6 187
TABLE-US-00026 TABLE VII(b) Human PBMC Assay for IFN-.gamma.,
MIP-1.alpha. and MIP-.beta. IFN-.gamma. MIP-1.alpha. MIP-.beta.
Seq. ID. No./ (pg/ml) (pg/ml) (pg/ml) Oligo No. Sequences and
Modification (5'-3') at 10 ug/ml at 10 ug/ml at 10 ug/ml 24
5'-TCAGTC TTAC-X-CATT CTGACT-5' 11.29 84.49 1373.07 25 5'-TCTGTC
TTAG-X-GATT CTGTCT-5' 14.44 90.61 1557.81 26 5'-CAGTC TTCAG-X-GACTT
CTGAC-5' 11.29 84.49 1337.00 27 5'-TCTGTC TTTT-X-TTTT CTGTCT-5'
13.66 109.05 1746.19 28 5'-TCTGTC TTGT-X-TGTT CTGTCT-5' 12.08 87.58
1337.01 29 5'-TAGTC TTTTT-X-TTTTT CGTAT-5' 12.87 82.12 1428.54 30
5'-TGGTC TTCTT-X-TTCTT CTGGT-5' 11.29 105.04 1839.64 31 5'-TAGTC
TTGTA-X-ATGTT CTGAT-5' 13.66 113.18 1995.04 32 5'-TAGTC
TTCTC-X-CTCTT CTGAT-5' 12.08 107.78 1603.54 33 5'-TC TC
TTCTT-X-TTCTT CT CT-5' 13.66 150.26 2785.79 34 5'-TC TAC TAC -X-
CAT CAT CT-5' 13.66 195.82 3966.88 35 5'-TC TC AC AT-X-TA CA CT
CT-5' 10.50 134.83 2878.33 36 5'-TC ATC ATC -X- CTA CTA CT-5' 8.11
107.78 2343.94 media 3.25 6.11 149.2
[0027] Exemplar TLR9 agonists from Table I were tested for immune
stimulatory activity in the human PBMC assay for MCP-1 and
IFN-.alpha., as described in Example 3. The results shown in Table
VIII(a) and VIII(b) below demonstrate that specific chemical
modifications to 3'-3' linked oligonucleotides will alter their
TLR9 mediated MCP-1 and/or IFN-.alpha. activation profile in human
PBMCs. More generally, these data demonstrate that specific
chemical modifications to 3'-3' linked oligonucleotides can be used
to increase or decrease MCP-1 and IFN-.alpha.activation.
TABLE-US-00027 TABLE VIII(a) Human PBMC Assay for MCP-1 and
IFN-.alpha. MCP-1 IFN-.alpha. Seq. ID. No./ (pg/ml) (pg/ml) Oligo
No. Sequences and Modification (5'-3') at 10 ug/ml at 10 ug/ml 1
5'-TCAGTC TTAC-X-CATT CTGACT-5' 3774.5 86 2 5'-TCTGTC TTAG-X-GATT
CTGTCT-5' 841 38.5 3 5'-CAGTC TTCAG-X-GACTT CTGAC-5' 3503 29 4
5'-TCTGTC TTTT-X-TTTT CTGTCT-5' 2514 31.5 5 5'-TCTGTC TTGT-X-TGTT
CTGTCT-5' 2134.5 52.5 6 5'-TAGTC TTTTT-X-TTTTT CGTAT-5' 2154 66.5 7
5'-TGGTC TTCTT-X-TTCTT CTGGT-5' 4201.5 68.5 8 5'-TAGTC
TTGTA-X-ATGTT CTGAT-5' 3620 77 9 5'-TAGTCTTCTC-X-CTCTT CTGAT-5'
4885 935 10 5'-TC TC TTCTT-X-TTCTT CT CT-5' 2672 59.5 11 5'-TC TAC
TAC -X- CAT CAT CT-5' 6793 5237.5 12 5'-TC TC AC AT-X-TA CA CT
CT-5' 6251 2199.7 13 5'-TC ATC ATC -X- CTA CTA CT-5' 6686 5619.5
Media 534 40
TABLE-US-00028 TABLE VIII(b) Human PBMC Assay for MCP-1 and
IFN-.alpha. MCP-1 IFN-.alpha. Seq. ID. No./ (pg/ml) (pg/ml) Oligo
No. Sequences and Modification (5'-3') at 10 ug/ml at 10 ug/ml 24
5'-TCAGTC TTAC-X-CATT CTGACT-5' 12480.44 62.03 25 5'-TCTGTC
TTAG-X-GATT CTGTCT-5' 50410.30 18.07 26 5'-CAGTC TTCAG-X-GACTT
CTGAC-5' 3982.45 42.09 27 5'-TCTGTC TTTT-X-TTTT CTGTCT-5' 53685.37
13.28 28 5'-TCTGTC TTGT-X-TGTT CTGTCT-5' 6116.14 46.03 29 5'-TAGTC
TTTTT-X-TTTTT CGTAT-5' 26435.31 38.08 30 5'-TGGTC TTCTT-X-TTCTT
CTGGT-5' 12012.62 30.38 31 5'-TAGTC TTGTA-X-ATGTT CTGAT-5' 53166.41
26.16 32 5'-TAGTC TTCTC-X-CTCTT CTGAT-5' 36199.26 67.03 33 5'-TC TC
TTCTT-X-TTCTT CT CT-5' 42397.84 31.4 34 5'-TC TAC TAC -X- CAT CAT
CT-5' 62106.60 83.69 35 5'-TC TC AC AT-X-TA CA CT CT-5' 41760.82
29.94 36 5'-TC ATC ATC -X- CTA CTA CT-5' 25530.72 942.37 media
267.37 0
[0028] Exemplar TLR9 agonists from Table I were tested for immune
stimulatory activity in the human PBMC and pDC assays for
IFN-.alpha., IL6, and IL-12, as described in Example 3. The results
shown in Table IX(a), IX(b), IX(c), IX(d), IX(e), IX(f), and IX(g)
below demonstrate that specific chemical modifications to 3'-3'
linked oligonucleotides will alter their TLR9 mediated IFN-.alpha.
activation profile in human PBMCs and pDCs. More generally, these
data demonstrate that specific chemical modifications to 3'-3'
linked oligonucleotides can be used to increase or decrease
IFN-.alpha., IL-6, and IL-12 activation.
TABLE-US-00029 TABLE IX(a) Human PBMC Assay for IFN-.alpha. Seq.
ID. No./ IFN-.alpha. (pg/ml) Oligo No. Sequences and Modification
(5'-3') at 10 ug/ml 10 5'-TC TC TTCTT-X-TTCTT CT CT-5' 142.5 11
5'-TC TAC TAC -X- CAT CAT CT-5' 8065.5 12 5'-TC TC AC AT-X-TA CA CT
CT-5' 7270.5 13 5'-TC ATC ATC -X- CTA CTA CT-5' 8437 Media
109.5
TABLE-US-00030 TABLE IX(b) Human PBMC Assay for IFN-.alpha. (24
hours).sup.a IFN-.alpha. Seq. ID. No./ (pg/ml .+-. SD) Oligo No.
Sequences and Modification (5'-3') at 10 ug/ml 60 5'-TCTGTC
TTCUo-X-oUCTT CTGTCT-5' 257.5 .+-. 539.4 61 5'-TCTGTC
TTCoUo-X-oUoCTT CTGTCT-5' 39.0 .+-. 88.5 62 5'-TCTGTC TTCU-X-UCTT
CTGTCT-5' 84.4 .+-. 143.2 63 5'-CTGTC TTCUC-X-CUCTT CTGTC-5' 34.0
.+-. 37.1 64 5'-TC AAC TTCG-X-GCTT CAA CT-5' 1165 .+-. 704.7 65
& 95 5'-TC AAC TTC -L-GACA CTGTCT-5' 2494.3 .+-. 1880.1 Medium
22.2 .+-. 39.8 .sup.aData are mean of 10 donors
TABLE-US-00031 TABLE IX(c) Human PBMC Assay for IFN-.alpha. (24
hours) IFN-.alpha. Seq. ID. No./ (pg/ml .+-. SD) Oligo No.
Sequences and Modification (5'-3') at 10 ug/ml 68 5'-TCTGTC
TTCU-X-UCTT CTGCTC-5' 53.42 69 5'-CTGTC TTCUC-X-CUCTT CTGCTC-5'
56.30 70 5'-TC AAC TTCG-X-GCTT CAA CT-5' 622.25 71 5'-TC AAC TTC
-L-GACA CTGTCT-5' 1823.24 75 5'-TCAGTC TTAC-X-CATT CTGACT-5' 97.42
77 5'-UCAGTC TTAC-X-CATT CTGACU-5' 92.64 78 5'-TCAGTC TTAoC-X-CoATT
CTGACT-5' 86.38 79 5'-TAGToC TTTTT-X-TTTTT CoTGTAT-5' 86.38 80
5'-TCTGToC TTGT-X-TGTT CoTGTCT-5' 66.13 81 5'-TAGoToC TTTTT-X-TTTTT
CoToGTAT-5' 92.10 82 5'-TC oToC AoC AT-X-TA CoA CoTo CT-5' 65.86 84
5'-TCAGToC TTAC-S-CATT CoTGACT-5' 86.38; 761.92 85 & 96
5'-TCTGoToC TAG-Z-GATT CoToGTCT-5' 98.37; 592.17 88 5'-TC C
TTTL-Z-LTTT CT CT-5' 958.92 90 5'-TC TC TTTL-X-LTTT CT CT-5' 710.90
92 5'-TCAGTC TTAC-X-CATT CTGACT-5' 92.64 Media 32.9; 64.8; 14.5
TABLE-US-00032 TABLE IX(d) Human pDC Assay for IFN-.alpha. (24
hours).sup.a IFN-.alpha. Seq. ID. No./ (pg/ml .+-. SD) Oligo No.
Sequences and Modification (5'-3') at 10 ug/ml 60 5'-TCTGTC
TTCUo-X-oUCTT CTGTCT-5' 1509.5 .+-. 2612.7 61 5'-TCTGTC
TTCoUo-X-oUoCTT CTGTCT-5' 1598.9 .+-. 3727.2 62 5'-TCTGTC
TTCU-X-UCTT CTGTCT-5' 3415.6 .+-. 3903.6 63 5'-CTGTC TTCUC-X-CUCTT
CTGTC-5' 2180.1 .+-. 3882.0 64 5'-TC AAC TTCG-X-GCTT CAA CT-5'
32956.8 .+-. 2639.9 65 & 95 5'-TC AAC TTC -L-GACA CTGTCT-5'
47746.2 .+-. 53192.1 Medium 106.6 .+-. 156.5 .sup.aData are mean of
10 donors
TABLE-US-00033 TABLE IX(e) Human pDC Assay for IFN-.alpha. (24
hours) IFN-.alpha. Seq. ID. No./ (pg/ml .+-. SD) Oligo No.
Sequences and Modification (5'-3') at 10 ug/ml 67 5'-TCTGTC
TTCoUo-X-oUoCTT CTGCTC-5' 174.81 68 5'-TCTGTC TTCU-X-UCTT CTGCTC-5'
900.94 69 5'-CTGTC TTCUC-X-CUCTT CTGCTC-5' 475.64 70 5'-TC AAC
TTCG-X-GCTT CAA CT-5' 4813.76 71 & 95 5'-TC AAC TTC -L-GACA
CTGTCT-5' 15494.15 72 5'-TCTGTC TTAG-S-GATT CTGTCT-5' 1934.09 73
5'-CAGTC TTCAG-Z-GACTT CTGAC-5' 2978.9 74 5'-TC TC AC AT-S-TA CA CT
CT-5' 57697.2 75 5'-TCAGTC TTAC-X-CATT CTGACT-5' 8171 77 5'-UCAGTC
TTAC-X-CATT CTGACU-5' 598.6 78 5'-TCAGTC TTAoC-X-CoATT CTGACT-5'
4245.4 79 5'-TAGToC TTTTT-X-TTTTT CoTGTAT-5' 2807.2 80 5'-TCTGToC
TTGT-X-TGTT CoTGTCT-5' 6133.96 81 5'-TAGoToC TTTTT-X-TTTTT
CoToGTAT-5' 1028.1 82 5'-TC oToC AoC AT-X-TA CoA CoTo CT-5' 17873.5
84 5'-TCAGToC TTAC-S-CATT CoTGACT-5' 190.42.1 85 & 96
5'-TCTGoToC TAG-Z-GATT CoToGTCT-5' 11673.4 86 5'-TC TC TTTL-Z-LTTT
CT CT-5' 10408.2 88 5'-TC C TTTL-Z-LTTT CT CT-5' 14783.3 89 5'-LTC
TC TTTL-Z-LTTT CT CTL-5' 6819.14 90 5'-TC TC TTTL-X-LTTT CT CT-5'
14515.1 92 5'-TCAGTC TTAC-X-CATT CTGACT-5' 307.8 Media 32.9; 0;
0.105
TABLE-US-00034 TABLE IX(f) Human pDC Assay for IL-6 (24 hours) IL-6
Seq. ID. No./ (pg/ml) Oligo No. Sequences and Modification (5'-3')
10 ug/ml 66 5'-TCTGTC TTCUo-X-oUCTT CTGCTC-5' 2384.36 67 5'-TCTGTC
TTCoUo-X-oUoCTT CTGCTC-5' 1405.14 68 5'-TCTGTC TTCU-X-UCTT
CTGCTC-5' 2851.75 69 5'-CTGTC TTCUC-X-CUCTT CTGCTC-5' 1598.06 70
5'-TC AAC TTCG-X-GCTT CAA CT-5' 1625.8 71 & 95 5'-TC AAC TTC
-L-GACA CTGTCT-5' 1648.17 80 5'-TCTGToC TTGT-X-TGTT CoTGTCT-5'
13805.44 84 5'-TCAGToC TTAC-S-CATT CoTGACT-5' 19405.66 85 & 96
5'-TCTGoToC TAG-Z-GATT CoToGTCT-5' 17253.59 88 5'-TC C TTTL-Z-LTTT
CT CT-5' 9277.99 90 5'-TC TC TTTL-X-LTTT CT CT-5' 5092.34 Media
1847.09; 1918.47
TABLE-US-00035 TABLE IX(g) Human pDC Assay for IL-12 (24 hours)
IL-6 Seq. ID. No./ (pg/ml) Oligo No. Sequences and Modification
(5'-3') 10 ug/ml 66 5'-TCTGTC TTCUo-X-oUCTT CTGCTC-5' 624.29 67
5'-TCTGTC TTCoUo-X-oUoCTT CTGCTC-5' 492.13 68 5'-TCTGTC TTCU-X-UCTT
CTGCTC-5' 724.53 69 5'-CTGTC TTCUC-X-CUCTT CTGCTC-5' 895.07 70
5'-TC AAC TTCG-X-GCTT CAA CT-5' 369.77 71 & 95 5'-TC AAC TTC
-L-GACA CTGTCT-5' 567.95 80 5'-TCTGToC TTGT-X-TGTT CoTGTCT-5'
1498.64 84 5'-TCAGToC TTAC-S-CATT CoTGACT-5' 1604.88 85 & 96
5'-TCTGoToC TAG-Z-GATT CoToGTCT-5' 1498.64 88 5'-TC C TTTL-Z-LTTT
CT CT-5' 1463.20 90 5'-TC TC TTTL-X-LTTT CT CT-5' 1417.50 Media
349.26; 397.1
[0029] Exemplar TLR9 agonists from Table I were tested for their
induction of IL-12 and IL-6 in mouse spleen cell cultures, as
described in Example 3. The results shown in Table X(a) and X(b)
below demonstrate that specific chemical modifications to 3'-3'
linked oligonucleotides will alter their TLR9 mediated IL-6 and/or
IL-12 activation profile in spleen cells and that this activation
profile may be dose dependent depending on the chemical
modification. More generally, these data demonstrate that specific
chemical modifications to 3'-3' linked oligonucleotides can be used
to increase or decrease IL-6 and IL-12 activation.
TABLE-US-00036 TABLE X(a) Induction of IL-12 and IL-6 Secretion in
Mouse Spleen Cell Cultures Seq. IL-6 (pg/ml .+-. SD) IL-12 (pg/ml
.+-. SD) ID. No./ at 1 at 3 at 1 at 3 Oligo No Sequence ug/ml ug/ml
ug/ml ug/ml 38 5'-TCTGAC TTCT-Y-TCTT CAGTCT-5' 8354 .+-. 32 24508
.+-. 86 909 .+-. 17 876 .+-. 89 39 5'-TC AAC TTC -Y- CTT CAA CT-5'
3371 .+-. 102 15012 .+-. 25 621 .+-. 12 517 .+-. 19 40 5'-TC TC
TTCTG-Y-GTCTT CT CT-5' 361 .+-. 4 4072 .+-. 1 451 .+-. 13 279 .+-.
0 41 5'-TCAGTCGTTAG-Y-GATTGCTGACT-5' 2496 .+-. 69 17796 .+-. 3 856
.+-. 12 626 .+-. 6 42 5'-TCTGTCGTTCT-Y-TCTTGCTGTCT-5' 8034 .+-. 95
22124 .+-. 57 659 .+-. 7 455 .+-. 18 43 5'-TCGTTGL-Y-LGTTGCT-5'
3127 .+-. 22 14412 .+-. 32 532 .+-. 11 536 .+-. 27 47 & 93
5'-TC AAC TTC -M-TCTT CTGTCT-5' 2685 .+-. 29 15663 .+-. 35 957 .+-.
2 566 .+-. 18 48 & 94 5'-TC AAC TTC -M-GACA CTGTCT-5' 3199 .+-.
69 17016 .+-. 11 792 .+-. 3 528 .+-. 2 PBS 0.00 0.00 87 .+-. 16 87
.+-. 16
TABLE-US-00037 TABLE X(b) Induction of IL-12 and IL-6 Secretion in
Mouse Spleen Cell Cultures IL-12 IL-6 Seq. ID. No./ (pg/ml .+-. SD)
(pg/ml .+-. SD) Oligo No. Sequences and Modification (5'-3') at 1
ug/ml at 1 ug/ml 60 5'-TCTGTC TTCUo-X-oUCTT CTGTCT-5' 4066 .+-. 47
78 .+-. 14 61 5'-TCTGTC TTCoUo-X-oUoCTT CTGTCT-5' 2438 .+-. 81 164
.+-. 21 62 5'-TCTGTC TTCU-X-UCTT CTGTCT-5' 1782 .+-. 67 120 .+-. 36
63 5'-CTGTC TTCUC-X-CUCTT CTGTC-5' 2496 .+-. 105 215 .+-. 19 64
5'-TC AAC TTCG-X-GCTT CAA CT-5' 64796 .+-. 60 3776 .+-. 25 65 &
95 5'-TC AAC TTC -L-GACA CTGTCT-5' 8245 .+-. 244 3776 .+-. 46
Medium 921 .+-. 60 38 .+-. 0
[0030] Exemplar TLR9 agonists from Table I were tested for their
induction of IL-1Ra, IL-6 and IL-12p40p70 in human PBMC cultures,
as described in Example 3. The results shown in Table XI below
demonstrate that specific chemical modifications to 3'-3' linked
oligonucleotides will alter their TLR9 mediated IL-1R.alpha., IL-6,
and IL-12p40p70 activation profile in human PBMCs. More generally,
these data demonstrate that specific chemical modifications to
3'-3' linked oligonucleotides can be used to increase or decrease
IL-1R.alpha., IL-6, and IL-12p40p70 activation.
TABLE-US-00038 TABLE XI IL-1R.alpha., IL-6 and IL-12p40p70 in human
PBMC Seq. ID. No./ Oligo No. Sequence IL-1R.alpha. IL-6 IL-12p40p70
43 5'-TCGTTGL-Y-LGTTGCT-5' 1595.5 1079.5 160.5 44
5'-TCGTTGM-Y-MGTTGCT-5' 1775.0 931.5 148.0 45 5'-TC TTGM-Y-MGTT
CT-5' 954.0 1235.5 71.0 46 5'-TCGTTGM-X-MGTTGCT-5' 1550.0 800.0
127.0 PBS 187.0 36.0 21.0
[0031] As described above, the invention provides, in a first
aspect, oligonucleotide-based synthetic agonists of TLR9. Based
upon certain chemical modifications to the base, sugar, linkage, or
linker, the agonists of TLR9 may possess increased stability when
associated, duplexed, with other of the TLR9 agonist molecules,
while retaining an accessible 5'-end.
[0032] In a second aspect, the invention provides a composition
comprising an oligonucleotide-based TLR9 agonist ("a compound")
according to the invention and a physiologically acceptable
carrier. The term "physiologically acceptable" generally refers to
a material that does not interfere with the effectiveness of the
compound and that is compatible with a biological system such as a
cell, cell culture, tissue, or organism.
[0033] As used herein, the term "carrier" encompasses any
excipient, diluent, filler, salt, buffer, stabilizer, solubilizer,
oil, lipid, lipid containing vesicle, microspheres, liposomal
encapsulation, or other material well known in the art for use in
physiologically acceptable formulations. It will be understood that
the characteristics of the carrier, excipient, or diluent will
depend on the route of administration for a particular application.
The preparation of physiologically acceptable formulations
containing these materials is described in, e.g., Remington's
Pharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack
Publishing Co., Easton, Pa., 1990.
[0034] The active compound is included in the physiologically
acceptable carrier or diluent in an amount sufficient to deliver to
a patient a prophylactically or therapeutically effective amount
without causing serious toxic effects in the patient treated. The
term an "effective amount" or a "sufficient amount" generally
refers to an amount sufficient to affect a desired biological
effect, such as beneficial results. Thus, an "effective amount" or
"sufficient amount" will depend upon the context in which it is
being administered. The effective dosage range of the
physiologically acceptable derivatives can be calculated based on
the weight of the parent compound to be delivered. If the
derivative exhibits activity in itself, the effective dosage can be
estimated as above using the weight of the derivative, or by other
means known to those skilled in the art.
[0035] In a third aspect, the invention provides a vaccine.
Vaccines according to this aspect comprise a composition according
to the invention, and further comprise an antigen. An antigen is a
molecule that elicits a specific immune response. Such antigens
include, without limitation, proteins, peptides, nucleic acids,
carbohydrates and complexes or combinations of any of the same.
Antigens may be natural or synthetic and generally induce an immune
response that is specific for that antigen. Any such antigen may
optionally be linked to an immunogenic protein, such as keyhole
limpet hemocyanin (KLH), cholera toxin B subunit, or any other
immunogenic carrier protein.
[0036] Vaccines according to the invention may further include any
of the plethora of known adjuvants, including, without limitation,
Freund's complete adjuvant, KLH, monophosphoryl lipid A (MPL),
alum, and saponins, including QS-21, imiquimod, R848, or
combinations thereof.
[0037] In a fourth aspect, the invention provides methods for
generating a TLR9-- mediated immune response in a vertebrate, such
methods comprising administering to the vertebrate a compound,
composition or vaccine according to the invention. In some
embodiments, the vertebrate is a mammal. For purposes of this
invention, the term "mammal" is expressly intended to include
humans and animals. In preferred embodiments, the compound,
composition or vaccine is administered to a vertebrate in need of
immune stimulation.
[0038] In the methods according to this aspect of the invention,
administration of a compound, composition or vaccine according to
the invention can be by any suitable route, including, without
limitation, parenteral, oral, intratumoral, sublingual,
transdermal, topical, intranasal, aerosol, intraocular,
intratracheal, intrarectal, mucosal, vaginal, by gene gun, dermal
patch or in eye drop or mouthwash form. Administration of the
compound, composition or vaccine can be carried out using known
procedures at dosages and for periods of time effective to reduce
symptoms or surrogate markers of the disease. When administered
systemically, the compound, composition or vaccine is preferably
administered at a sufficient dosage to attain a blood level of a
compound according to the invention from about 0.0001 micromolar to
about 10 micromolar. For localized administration, much lower
concentrations than this may be effective, and much higher
concentrations may be tolerated without serious toxic effects.
Preferably, a total dosage of a compound according to the invention
ranges from about 0.001 mg per patient per day to about 200 mg per
kg body weight per day. It may be desirable to administer
simultaneously, or sequentially a therapeutically effective amount
of one or more of the therapeutic compositions of the invention to
an individual as a single treatment episode.
[0039] In certain preferred embodiments, a compound, composition or
vaccine according to the invention is administered in combination
with another agent, including without limitation antibodies,
cytotoxic agents, allergens, antibiotics, antisense
oligonucleotides, SiRNA, aptamers, ribozymes, targeted therapies,
peptides, proteins, gene therapy vectors, DNA vaccines, and/or
adjuvants to enhance the specificity or magnitude of the immune
response.
[0040] For purposes of this aspect of the invention, the term "in
combination with" means in the course of treating a disease or
disorder in a patient, administering the compound, composition or
vaccine according to the invention and/or the other agent in any
order, including simultaneous administration, as well as temporally
spaced order of up to several hours, days or weeks apart. Such
combination treatment may also include more than a single
administration of the compound, composition or vaccine according to
the invention, and/or the other agent. The administration of the
compound, composition or vaccine according to the invention and/or
the other agent may be by the same or different routes.
[0041] The methods according to this aspect of the invention are
useful for the prophylactic or therapeutic treatment of human or
animal disease. For example, the methods are useful for pediatric
and veterinary vaccine applications. The methods are also useful
for model studies of the immune system.
[0042] In a fifth aspect, the invention provides methods for
therapeutically treating a patient having a disease or disorder,
such methods comprising administering to the patient a compound,
composition or vaccine according to the invention. In various
embodiments, the disease or disorder to be treated is cancer, an
autoimmune disorder, infectious disease, airway inflammation,
inflammatory disorders, allergy, asthma or a disease caused by a
pathogen or allergen. Pathogens include for example bacteria,
parasites, fungi, viruses, viroids, and prions. Administration is
carried out as described for the fourth aspect of the
invention.
[0043] The term "treatment" generally refers to an approach
intended to obtain a beneficial or desired results, which may
include alleviation of symptoms, or delaying or ameliorating a
disease progression.
[0044] For purposes of the invention, the term "allergy" generally
refers to an inappropriate immune response characterized by
inflammation and includes, without limitation, food allergies and
respiratory allergies. The term "airway inflammation" includes,
without limitation, asthma. As used herein, the term "autoimmune
disorder" refers to disorders in which "self" components (e.g.,
proteins) undergo attack by the immune system. Such term includes
autoimmune asthma. The term "cancer" includes, without limitation,
any malignant growth or tumor caused by abnormal or uncontrolled
cell proliferation and/or division. Cancers may occur in humans
and/or animals and may arise in any and all tissues. Treating a
patient having cancer with the invention may include administration
of a compound, composition or vaccine according to the invention
such that the abnormal or uncontrolled cell proliferation and/or
division is affected.
[0045] In a sixth aspect, the invention provides methods for
preventing a disease or disorder, such methods comprising
administering to the patient a compound, composition or vaccine
according to the invention. In various embodiments, the disease or
disorder to be prevented is cancer, an autoimmune disorder, airway
inflammation, inflammatory disorders, infectious disease, allergy,
asthma or a disease caused by a pathogen. Pathogens include,
without limitation, bacteria, parasites, fungi, viruses, viroids,
and prions. Administration is carried out as described for the
fourth aspect of the invention.
[0046] In any of the methods according to the invention, the
compound, composition or vaccine according to the invention can be
administered in combination with any other agent useful for
preventing or treating the disease or condition that does not
diminish the immune stimulatory effect of the compound, composition
or vaccine according to the invention. In any of the methods
according to the invention, the agent useful for preventing or
treating the disease or condition includes, but is not limited to,
vaccines, antigens, antibodies, cytotoxic agents, allergens,
antibiotics, antisense oligonucleotides, TLR agonist, peptides,
proteins, gene therapy vectors, DNA vaccines and/or adjuvants to
enhance the specificity or magnitude of the immune response, or
co-stimulatory molecules such as cytokines, chemokines, protein
ligands, trans-activating factors, peptides and peptides comprising
modified amino acids. For example, in the prevention and/or
treatment of cancer, it is contemplated that the compound,
composition or vaccine according to the invention may be
administered in combination with a chemotherapeutic compound or a
monoclonal antibody. Alternatively, the agent can include DNA
vectors encoding for antigen or allergen. In these embodiments, the
compound, composition or vaccine according to the invention can
variously act as adjuvants and/or produce direct immunomodulatory
effects.
[0047] The following examples are intended to further illustrate
certain preferred embodiments of the invention and are not intended
to limit the scope of the invention in any way.
Example 1
Synthesis of Oligonucleotides, Pentane-1,3,5-triol,
Pentane-1,5-diol and cis-1,3,5-Cyclohexanetriol Linkers and
Functionalization of CPG and OligoPrep Solid Supports
[0048] Control pore glass-derivatized 3-methyl-1,3,5-pentanetriol
linker (5) was achieved from commercially available
3-methyl-1,3,5-triol 1 as shown in Scheme 1. Initially, bis-DMT
protected alcohol 2 was prepared in good yield from 1 by treating
with DMTC1 in the presence of DMAP. The conventional method of
derivatization of CPG was not possible due to the low yields of the
succinylation product at 3-hydroxyl of 2, possibly due to steric
effects. However, the linker derivatized CPG 5 was prepared by
following the alternate approach which eliminates the need for
making succinate 3 (Scheme 1). In this route, initially, the CPG
beads were activated by treating with 3% trichloroacetic acid (TCA)
in dichloromethane (DCM) at room temperature (r.t.) to liberate
maximum number of reactive amino groups on the surface of CPG. The
activated CPG beads were then derivatized with succinic anhydride
in the presence of DMAP to provide CPG beads 4. Finally, CPG
derivatized linker 5 was obtained by condensation of 2 with
carboxylic groups of CPG 4 in the presence of
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride
(DEC)/DMAP. After derivatization, the residual carboxylic groups
were eliminated by capping reaction with pentachlorophenol.
##STR00001##
[0049] 1,3,5-Pentanetriol linker derivatized CPG 10 and OligoPrep
11 were prepared starting from the commercially available diethyl
3-hydroxy glutarate 6 (Scheme 2). Reduction of 6 with LiAlH.sub.4
yielded 1,3,5-pentanetriol 7 in quantitative yield. The triol 7 was
then selectively protected with DMTC1 in the presence of DMAP to
afford bis-DMT protected alcohol 8, which was then successfully
converted into succinate 9, which is ready to load on to the solid
support, by treating with succinic anhydride in the presence of
DMAP. Attachment of 9 to CPG was accomplished in quantitative
loading yield in the presence of DIC/DMAP in pyridine/acetonitrile
mixture (10). Whereas, the above protocol gave very poor loading
yield in the case of OligoPrep250, a PVA solid support,
functionalization. However, quantitative loading yield was achieved
in the presence of TBTU/DMAP in acetonitrile (11). The loading was
maintained at about .about.40 .mu.mol/g on CPG (10) and .about.125
.mu.mol/g on OligoPrep250 (11) supports, respectively, which
increases the nucleotide coupling efficiencies and final
yields.
##STR00002##
[0050] The C5 linker functionalized supports 10 and 11 are ideal
for making immunomers with identical sequences. Immunomers with
unidentical sequences also exhibited potent immune stimulatory
activity in our studies. Appropriately protected C5 linker, such as
14 (Scheme 3), is required in order to make immunomers with
unidentical sequences. One of the hydroxyl groups of commercially
available 1,5-pentanediol was selectively protected with DMT
followed by phosphitylation with 2-cyanoethyl
N,N-diisopropylchlorophos-phosphoramidite afforded the required C5
linker 14 (Scheme 31
##STR00003##
[0051] We have also focused our attention on the design and
development of CpG DNA dendrimers as potent synthetic immune
modulatory motifs. In order to make CpG DNA dendrimers,
appropriately protected linker phosphoramidites are essential. The
C5 linker phosphramidites 15 and 16 were prepared from di-DMT
alcohols 2 and 8, respectively, by phosphitylation with
2-cyanoethyl N,N-diisopropylchlorophosphosphoramidite as shown in
Scheme 4.
##STR00004##
[0052] cis-Cyclohexanetriol linker derivatized with CPG 20 was
accomplished as shown in Scheme 5. bis-DMT protected
cis-1,3,5-cyclohexanetriol 18 was achieved from commercially
available cis-1,3,5-cyclohexanetriol (17). The subsequent
succinylation of the unprotected hydroxyl of 18 with succinic
anhydride in the presence of DMAP afforded desired bis-DMT
succinate 19 in 78% yield. Derivatization of CPG with succinate 19
was accomplished in quantitative loading yield (20, 40 .mu.mol/g)
in the presence of DIC/DMAP in pyridine/acetonitrile mixture.
##STR00005##
[0053] Reagents such as diethyl 3-hydroxy glutarate, lithium
aluminum hydride (LiAlH.sub.4), 4,4-dimethoxytrityl chloride
(DMTC1), 4-dimethylaminopyridine (DMAP), succinic anhydride,
O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (TBTU), N,N'-diisopropylcarbodiimide (DIC),
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (DEC),
trichloroacetic acid (TCA), N-methylimidazole (NMI), triethylamine
(TEA), diisopropylethylamine (DIPEA) and solvents such as pyridine,
dichloromethane (DCM) and tetrahydrofuran (THF) were obtained from
Sigma-Aldrich (St. Louis, Mo.) and used without further
purification unless mentioned otherwise. Long chain alkyl amine
controlled pore glass (CPG; 120-200 mesh, 500 .ANG., 90-120
.mu.mol/g NH.sub.2 groups) was obtained from CPG Inc. (Lincoln
Park, N.J.) and OligoPrep250 was obtained from Merck KGaA
(Germany). Cap A (acetic anhydride/2,6-lutidine/THF 1:1:8) and Cap
B (N-methylimidazole/THF 16:84) reagents were obtained from Applied
Biosystems (Foster City, Calif.). All reactions were performed in
glassware which had been oven dried at 120.degree. C. for at least
3 hrs prior to use. TLCs were run on silica gel 60 F.sub.254 coated
on aluminum sheets, and visualized by UV light or by a 5%
phosphomolybdic acid (PMA) solution from Sigma-Aldrich (St. Louis,
Mo.). Solvents such as ethyl acetate (EtOAc), hexanes, DCM,
methanol, t-butyl methyl ether for chromatography were obtained
from J. T. Baker and used without purification. Flash column
chromatography was performed using silica gel 60 (mesh size
0.040-0.063 mm & 230-400 mesh ASTM) which was obtained from EMD
Chemicals (Gibbstown, N.J.). NMR spectra were performed on Varian
400 MHz Unity Inova instrument. Chemical shifts (.delta.) are in
ppm relative to TMS and all coupling constants (J) are in Hz.
[0054] 1,5-Bis-dimethoxytrityloxy-3-methyl-pentan-3-ol (2). DMTC1
(3.6 g, 10.5 mmol, 2.1 equiv) in pyridine (25 mL) was added drop
wise to an ice cold (0.degree. C.) and stirring solution of
3-methyl-1,3,5-pentanetiol (1, 0.7 g, 5 mmol) and DMAP (0.24 g) in
dry pyridine (25 mL) under nitrogen atmosphere. The reaction
mixture was allowed to slowly reach room temperature (-4 h) and
continued stirring for overnight. TLC (hexanes/t-butyl methyl ether
2:1 containing 0.5% TEA) indicated the completion of the reaction.
Pyridine rotoevaporated to dryness, residue was dissolved in ethyl
acetate (250 mL) and washed successively with water (2.times.100
mL), saturated NH.sub.4Cl solution (2.times.100 mL), brine
(2.times.100 mL) and water (2.times.100 mL). Ethyl acetate layer
dried over anhydrous MgSO.sub.4 and rotoevaporated to dryness. The
residue was purified on silica gel flash column chromatography
using hexane/t-butyl methyl ether (3:1) containing 0.5%
triethylamine to give bis-DMT product 2 as a white foam (2.9 g,
78%). .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta. 1.56 (s,
3H--CH.sub.3), 1.74-1.79 (m, 4H, --CH.sub.2CHCH.sub.2--), 3.19-3.26
(m, 4H, 1 & 5-CH.sub.2--), 3.78 (d, 12H, --OCH.sub.3), 6.82
(dd, J=8.8, 8H, Ar--H), 7.16-7.40 (m, 18H, Ar--H).
[0055] Preparation of bis-DMT-3-methyl-pentanetriol derivatized CPG
(5): LCAA-CPG (5 g) was added to 3% TCA (50 mL) and slowly agitated
at room temperature for 3 h. The CPG was filtered and washed with
9:1 mixture of TEA/DIPEA (50 mL) followed by DCM (5.times.100 ml).
The activated CPG dried under high vacuum for 1 h. A solution of
succinic anhydride (1 g, 10 mmol) and DMAP (0.2 g) in pyridine (30
mL) was added to the above CPG (5 g) and the slurry was shaken at
room temperature for 24 hrs. Solutions filtered off and CPG was
washed with pyridine (2.times.25 mL) followed by DCM (5.times.25
mL) and dried under high vacuum for 2 h to obtain the succinic acid
derivative of CPG 4. A solution of di-DMT-3-methyl-pentanetriol 2
(87 mg), DMAP (30 mg), TEA (100 .mu.L) and DEC (0.4 g) in dry
pyridine (10 mL) was added to CPG 4 and shaken at room temperature
for 15 h. Pentachlorophenol (0.14 g) added to the above mixture and
shaken for additional 15 hrs. Solutions filtered off, CPG was
thoroughly washed with pyridine (2.times.25 mL) followed by DCM
(5.times.25 mL) and dried under high vacuum in a desiccator for
over night to get Di-DMT-3-methyl-pentanetriol derivatized CPG 5.
Loading was determined by treating small portion of CPG with 3% TCA
in DCM and assayed DMT content 37 .mu.mol/g) by measuring
absorbance at 498 mm.
[0056] Synthesis of pentane-1,3,5-triol (7): Diethyl 3-hydroxy
glutarate (6, 25 g, 122.4 mmol) in THF (100 mL) was added dropwise
to a 1M solution of LiAlH.sub.4 in THF (400 mL) at 0.degree. C.
under argon atmosphere with vigorous stirring. After addition,
reaction mixture was allowed to reach r. t. (.about.4 h) and
stirred for overnight. The reaction mixture was cooled to
-78.degree. C. (acetone/dry ice bath) and quenched by dropwise
addition of saturated NH.sub.4Cl solution (50 mL) giving white
precipitate. The reaction mixture was diluted with another 500 mL
of THF and white precipitate was filtered through Celite. The
precipitate was treated with boiling THF (250 mL) and filtered. The
combined organic solution was dried over anhydrous MgSO.sub.4 and
solvent removed by rotoevaporation. The residue was purified on a
silica gel flash column chromatography using DCM/EtOAc/methanol
(6:3:1) affording triol 7 (11.4 g, 95.5 mmol, 78%) as a colorless
oil. %). .sup.1H-NMR (DMSO-d.sub.6, 400 MHz): .delta. 1.39-1.54 (m,
4H, --CH.sub.2CH(OH)CH.sub.2--), 3.47 (t, J=6.6, 4H, 1 &
5-CH.sub.2--), 3.61-3.68 (m, 1H, --CH(HO)--) and 4.23 (bs, 3H, 1, 3
& 5-OH).
[0057] 1,5-Bis-[(4,4-dimethoxyphenyl)-phenylmethoxy]-pentan-3-ol
(8). DMTC1 (16.6 g, 49 mmol, 2.1 equiv) in pyridine (100 mL) was
added dropwise to an ice cold (0.degree. C.) and stirring solution
of pentanetriol 7 (2.8 g, 23 mmol) and DMAP (2.85 g, 1 equiv) in
dry pyridine (50 mL) under argon atmosphere. The reaction mixture
was allowed to slowly reach r. t. (.about.4 h) and continued
stirring for overnight. TLC (hexanes/EtOAc 3:1 containing 0.5% TEA)
indicated the completion of the reaction. Pyridine rotoevaporated
to dryness, residue was dissolved in DCM (500 mL) and washed
successively with water (500 mL), saturated NH.sub.4Cl solution
(500 mL), brine (500 mL) and water (2.times.500 mL). DCM layer
dried over anhydrous MgSO.sub.4 and rotoevaporated to dryness. The
residue was purified on silica gel flash column chromatography
using hexane/EtOAc (3:1) containing 0.5% TEA to give bis-DMT
alcohol 8 as a white foam (12.1 g, 16.7 mmol, 72%). .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta. 1.65-1.81 (m, 4H,
--CH.sub.2CHCH.sub.2--), 3.16-3.31 (m, 4H, DMTO--CH.sub.2--), 3.78
(s, 12H, --OCH.sub.3), 3.95-4.01 (m, 1H, --CH--), 6.81 (d, J=8.8,
8H, Ar--H), 7.17-7.42 (m, 18H, Ar--H). .sup.13C NMR (CDCl.sub.3,
75.5 MHz): 37.22, 55.38, 61.81, 69.58, 86.54, 113.27, 126.88,
128.01, 128.26, 130.15, 136.44, 145.16, 158.55.
[0058] 3(1,5-O-Dimethoxytrityl pentanetriol)succinic acid (9):
bis-DMT alcohol 8 (12 g, 16.6 mmol) and DMAP (4.04 g, 33.2 mmol)
were dissolved in dry pyridine (150 mL) and succinic anhydride
(3.31 g, 33.2 mmol) was added portion wise at r.t. with vigorous
stirring. The reaction mixture was stirred for over night and
pyridine rotoevaporated to dryness. Residue was dissolved in DCM
(500 mL) and successively washed with ice cold 10% citric acid
solution (2.times.500 mL) and water (2.times.500 mL). DCM layer was
dried over anhydrous MgSO.sub.4, concentrated to 50 mL volume using
rotoevaporator and purified by silica gel flash column
chromatography using 0.fwdarw.2% methanol in DCM containing 0.5%
TEA to get pure triethylammnium salt of succinate 9 as white foam
(11.4 g, 13.8 mmol, 83%). .sup.1H-NMR (CDCl3, 400 MHz): .delta.
1.20 (t, J=7.6, 9H, --N(CH.sub.2CH.sub.3).sub.3), 1.80-1.85 (m, 4H,
--CH.sub.2CHCH.sub.2--), 2.40 (s, 4H, --COCH.sub.2CH.sub.2CO--),
2.90 (q, 6H, --N(CH.sub.2CH.sub.3).sub.3), 3.03-3.11 (m, 4H, 1
& 5-CH.sub.2--), 3.78 (s, 12H, --OCH.sub.3), 5.20-5.26 (m, 1H,
--CH--), 6.81 (d, J=8.8, 8H, Ar--H), 7.17-7.42 (m, 18H, Ar--H).
.sup.13C NMR (CDCl.sub.3, 75.5 MHz): 9.09, 30.94, 31.77, 34.45,
45.08, 52.92, 55.32, 59.73, 69.73, 86.00, 113.12, 126.71, 127.85,
128.32, 130.11, 136.54, 145.22, 158.40, 173.07 and 177.71.
[0059] Preparation of bis-DMT pentanetriol loaded CPG (10): A
solution of succinate 9 (0.83 g, 1 mmol), DMAP (0.4 g, 3.3 mmol)
and DIC (5 mL) in 1:6 mixture of pyridine/acetonitrile (105 mL) was
added to CPG (25 g) and the slurry was shaken for 24 hrs. Solutions
filtered off and CPG was washed with acetonitrile containing 5%
pyridine (100 mL) and acetonitrile (250 mL). Cap A (ABI, 89 mL) and
Cap B (ABI, 100 mL) solutions were added to CPG support and shaken
for 4 h. Solutions filtered off, CPG washed with acetonitrile
containing 5% pyridine (2.times.100 mL) followed by acetonitrile
(2.times.250 mL) and dried under high vacuum for 30 min. A solution
of TBDMSCl (5.6 g) and imidazole (1.4 g) in acetonitrile containing
5% pyridine (150 mL) was added to CPG and shaken for 4 h. Solution
filtered off, CPG was successively washed with acetonitrile
containing 5% pyridine (3.times.100 mL) and DCM (4.times.250 mL)
and dried under high vacuum in a desiccator for over night to get
dry CPG support 10. Loading was determined by treating small
portion of CPG with 3% TCA in DCM and assayed DMT content (40
.mu.mol/g) by measuring absorbance at 498 mm.
[0060] Preparation of bis-DMT pentanetriol loaded OligoPrep250
(11): OligoPrep250 (100 g preswollen in acetonitrile) was taken in
a peptide synthesis vessel and washed with anhydrous acetonitrile
(3.times.100 mL). Succinate 9 (1.752 g, 2.125 mmol), DMAP (1.82 g,
14.87 mmol), TBTU (3.41 g, 10.62 mmol) and acetonitrile (100 mL)
were added to OligoPrep250 and the slurry was shaken for 4 hrs.
Solution filtered off and OligoPrep was washed with acetonitrile
containing 1% TEA (2.times.100 mL) and acetonitrile (5.times.100
mL). Cap A (50 mL: NMI/pyridine/acetonitrile=2:3:5) and Cap B (50
mL: acetic anhydride/acetonitrile=1:4) solutions were added to
solid support and shaken for 6 hrs. Solutions filtered off, solid
support washed with acetonitrile (2.times.100 mL) and repeated the
capping reaction one more time. Solutions filtered off, solid
support washed with acetonitrile containing 1% TEA (3.times.100 mL)
followed by acetonitrile (5.times.100 mL) and dried under high
vacuum in a desiccator for 24 hrs to get dry OligoPrep250 support
11 (26.4 g). Loading was determined by treating small portion of
OligoPrep with 3% TCA in DCM and assayed DMT content (138
.mu.mol/g) by measuring absorbance at 498 mm.
[0061] 5-Dimethoxytrityloxy-Pentane-1-ol (13): Pentanediol 12 (12.5
g, 120 mmol) and DMAP (14.6 g, 120 mmol) were dissolved in dry
pyridine (100 mL), cooled to -10.degree. C. and maintained under
argon atmosphere. DMTCl (37.3 g, 110 mmol, 0.92 equiv) in pyridine
(150 mL) was added drop wise with vigorous stirring. The reaction
mixture was allowed to slowly reach r.t. (.about.4 h) and continued
stirring for overnight. Pyridine rotoevaporated to dryness, residue
dissolved in DCM (500 mL) and successively washed with water (250
mL), saturated NH.sub.4Cl solution (2.times.250 mL), brine (250 mL)
and water (2.times.250 mL). DCM layer dried over anhydrous
MgSO.sub.4 and rotoevaporated to dryness. The residue was purified
by silica gel flash column chromatography using hexanes/EtOAc (3:1)
containing 0.5% TEA to give mono-DMT protected alcohol 13 as a
colorless syrup (28.2 g, 58%). .sup.1H-NMR (CDCl.sub.3, 400 MHz):
.delta. 1.39-1.47 (m, 2H, --CH.sub.2CH.sub.2CH.sub.2OH),
1.49-1.1.56 (m, 2H, DMTO--CH.sub.2CH.sub.2--), 1.60-1.68 (m, 2H,
--CH.sub.2CH.sub.2OH), 3.06 (t, 2H, J=6.2, DMTO--CH.sub.2--), 3.60
(t, 2H, J=6.3, --CH.sub.2OH), 3.77 (s, 12H, --OCH.sub.3), 6.82 (d,
J=8.8, 8H, Ar--H), 7.17-7.45 (m, 18H, Ar--H). .sup.13C NMR
(CDCl.sub.3, 75.5 MHz): 22.84, 30.14, 32.92, 55.51, 63.21, 63.57,
85.98, 113.26, 126.87, 128.00, 128.46, 130.30, 136.96, 145.65 and
158.57.
[0062]
5-Dimethoxytrityloxy-Pentane-1-O-(2-cyanoethyl-N,N-diisopropyl)phos-
phoramidite (14): To an ice cold solution of 13 (20.32 g, 50 mmol)
in anhydrous DCM (500 mL) under nitrogen atmosphere was added DIPEA
(26.12 mL, 150 mmol) with vigorous stirring.
2-Cyanoethyl-N,N-diisopropylchlorophosphoramidite (14.2 g, 60 mmol)
was then added dropwise followed by NMI (4 mL, 50 mmol). The
reaction mixture allowed to slowly reach r.t. in .about.4 h and
continued stirring for overnight. TLC in 3:1 hexanes/EtOAc
containing 0.5% TEA exhibited the completion of the reaction. The
reaction mixture was diluted with another 500 mL of DCM and washed
sequentially with saturated aqueous NaHCO.sub.3 (1.times.500 mL),
brine (2.times.500 mL) and water (1.times.500 mL). The organic
layer dried over anhydrous MgSO.sub.4, filtered and rotoevaporated
to dryness. The residue was purified on silica gel flash column
chromatography using 3:1 hexane/EtOAc mixture containing 0.5% TEA
to get 13 as a colorless viscous liquid (22.3 g, 74%). .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta.1.15 (t, 12H, J=7.6,
(Me.sub.2CH).sub.2N--), 1.37-1.45 (m, 2H,
--CH.sub.2CH.sub.2CH.sub.2OP--), 1.52-1.64 (m, 4H,
DMTO--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), 2.55 (t, 2H, J=6.2,
--CH.sub.2CN), 3.01 (t, 2H, J=6.5, --CH.sub.2CH.sub.2CN), 3.49-3.64
(m, 4H, DMTO--CH.sub.2-- and --CH.sub.2OP--), 3.73 (s, 12H,
--OCH.sub.3), 3.71-3.81 (m, 2H, (Me.sub.2CH).sub.2N--), 6.77 (d,
J=8.8, 8H, Ar--H), 7.13-7.41 (m, 18H, Ar--H). .sup.13C NMR
(CDCl.sub.3, 75.5 MHz): 20.56, 20.63, 23.08, 24.82, 24.89, 24.96,
30.05, 31.40, 43.15, 43.27, 55.44, 58.47, 58.66, 63.52, 63.92,
85.89, 113.20, 117.97, 126.81, 127.94, 128.42, 130.25, 136.92,
145.65 and 158.53. .sup.31P-NMR: .delta.145.07.
[0063] Synthesis of phosphoramidites 15 and 16: Synthesized using
general procedure as described for 14. Compound 15--white foam and
yield 69%. .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta. 0.89 (d, 6H,
J=7, (Me.sub.2CH).sub.2N--), 1.05 (d, 6H, J=6.4,
(Me.sub.2CH).sub.2N--), 1.25 (s, 3H, --CH.sub.3), 1.88-1.99 (m, 4H,
--CH.sub.2CHCH.sub.2--), 2.40 (t, 2H, J=6.4, --CH.sub.2CN),
3.10-3.21 (m, 2H, --OCH.sub.2CH.sub.2CN), 3.30-3.40 (m, 2H,
Me.sub.2CH).sub.2N--), 3.43-3.60 (m, 4H, DMTO--CH.sub.2--), 3.77
(d, 12H, --OCH.sub.3), 6.79 (dd, J=8.8, 8H, Ar--H), 7.17-7.42 (m,
18H, Ar--H). .sup.13C NMR (CDCl.sub.3, 75.5 MHz): 20.39, 24.27,
24.72, 43.03, 55.38, 57.73, 60.23, 77.96, 78.04, 86.28, 113.15,
118.01, 126.74, 127.89, 128.31, 129.31, 130.18, 136.80, 139.62,
145.47, 158.44. .sup.31P-NMR: .delta. 135.93. Compound 16--white
foam and yield 79%. .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta. 1.00
(d, 6H, J=6.4, (Me.sub.2CH).sub.2N--), 1.10 (d, 6H, J=6.7,
(Me.sub.2CH).sub.2N--), 1.73-1.99 (m, 4H,
DMTO--CH.sub.2CH.sub.2CHCH.sub.2--), 2.36 (t, 2H, J=6.6,
--CH.sub.2CN), 3.14 (t, 2H, J=6.5, --CH.sub.2CH.sub.2CN), 3.37-3.60
(m, 6H, DMTO--CH.sub.2-- and Me.sub.2CH).sub.2N--), 3.77 (d, 12H,
--OCH.sub.3), 4.14-4.22 (m, 1H, --CHOP--), 6.78 (dd, J=8.8, 8H,
Ar--H), 7.17-7.42 (m, 18H, Ar--H). .sup.13C NMR (CDCl.sub.3, 75.5
MHz): 20.38, 24.56, 24.88, 36.82, 43.08, 55.43, 58.39, 60.45,
69.88, 86.09, 113.18, 117.88, 126.81, 127.93, 128.36, 130.20,
136.77, 137.59, 145.47, 158.49. .sup.31P-NMR: .delta. 145.20.
[0064] cis-3,5-Bis-dimethoxytrityloxy-cyclohexane-1-ol (18).
cis-1,3,5-Cyclohexanetriol dehydrate (5.05 g, 30 mmol) was
dissolved in pyridine (100 mL) and rotoevaporated to dryness and
dried under high vacuum for 48 hrs to obtain anhydrous
cis-1,3,5-cyclohexanetriol (4.05 g, 30.6 mmol). The above anhydrous
cyclohexanetriol and DMAP (7.33 g, 60 mmol) were dissolved in dry
pyridine (100 mL), cooled in ice bath and maintained under nitrogen
atmosphere. DMTC1 (20.4 g, 60 mmol, 2 equiv) in dry pyridine (150
mL) was added drop wise to the above solution with vigorous
stirring. The reaction mixture was allowed to slowly reach r.t.
(.about.4 h) and continued stirring for 24 hrs. TLC in 2:1
hexanes/EtOAc mixture containing 0.5% TEA indicated the presence of
some starting materials. Reaction mixture stirred five more hrs at
60.degree. C. and pyridine rotoevaporated to dryness. The residue
was dissolved in DCM (500 mL) and washed successively with water
(500 mL), saturated NH.sub.4Cl solution (500 mL), brine (500 mL)
and water (2.times.500 mL). DCM layer dried over anhydrous
MgSO.sub.4 and rotoevaporated to dryness. The residue was purified
on silica gel flash column chromatography using 3:1 hexane/EtOAc
mixture containing 0.5% TEA to give bis-DMT product 18 as a white
solid (8.4 g, 38%). .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.
1.04-1.13 (m, 3H, 2, 4 & 6--CH.sub.2--), 1.24-1.28 (m, 3H, 2, 4
& 6--CH.sub.2--), 1.66 (d, 1H, 1-OH), 2.84-2.93 (m, 1H,
--CH--OH), 3.10-3.18 (m, 2H, 3 & 5-CH--), 3.78 (d, 12H,
--OCH.sub.3), 6.78 (d, J=8.8, 8H, Ar--H), 7.16-7.42 (m, 18H,
Ar--H). .sup.13C NMR (CDCl.sub.3, 75.5 MHz): 41.42, 42.67, 55.37,
66.16, 67.91, 86.28, 113.12, 126.83, 127.81, 128.53, 130.41,
137.39, 146.29, 158.53.
[0065] 1(3,5-bis-Dimethoxytrityl-cis-cyclohexanetriol)succinic acid
(19): bis-DMT-cyclohexanetriol 18 (4.05 g, 5.5 mmol) and DMAP (1.34
g, 10.1 mmol) were dissolved in dry pyridine (50 mL) and succinic
anhydride (1.1 g, 10.1 mmol) was added portion wise at r.t. with
vigorous stirring. The reaction mixture was stirred for 48 hrs at
r.t. and TLC in DCM containing 2% methanol and 0.5% TEA indicated
the complete disappearance of starting material. Pyridine
rotoevaporated to dryness, residue dissolved in DCM (250 mL) and
successively washed with ice cold 10% citric acid solution
(2.times.250 mL) and water (2.times.250 mL). DCM layer dried over
anhydrous MgSO.sub.4, concentrated to 50 mL volume using
rotoevaporator and purified by silica gel flash column
chromatography using 0.fwdarw.2% methanol in DCM containing 0.5%
TEA to get pure triethylammonium salt of succinate 19 as white foam
(3.58 g, 78%). .sup.1H-NMR (CDCl.sub.3, 400 MHz .delta. 1.17 (t,
J=7.6, 9H, --N(CH.sub.2CH.sub.3).sub.3), 1.17-1.25 (m, 2H,
4--CH.sub.2--), 1.36-1.51 (m, 4H, 2 & 6-CH.sub.2--), 2.37-2.47
(m, 4H, --OCH.sub.2CH.sub.2CN), 2.86 (q, 6H,
--N(CH.sub.2CH.sub.3).sub.3), 2.99-3.10 (m, 2H, 3 & 5-CH--),
3.78 (d, 12H, --OCH.sub.3), 3.98-4.08 (m, 1H, 1-CH--), 6.76 (dd,
J=8.8, 8H, Ar--H), 7.15-7.37 (m, 18H, Ar--H). .sup.13C NMR
(CDCl.sub.3, 75.5 MHz): 9.38, 31.16, 31.96, 39.02, 41.76, 45.18,
55.37, 67.68, 86.24, 113.12, 126.79, 127.77, 128.42, 130.33,
130.38, 137.15, 137.26, 146.28, 158.54, 172.58 and 178.16.
[0066] Preparation of bis-DMT cyclohexanetriol derivatized CPG 20:
A solution of succinate 19 (0.78 g, 0.93 mmol), DMAP (0.38 g, 2.8
mmol) and DIC (3 mL) in 1:7.5 mixture of pyridine/acetonitrile (85
mL) was added to CPG (22.5 g) and the slurry was agitated for 24
hrs. Solutions filtered off and CPG was washed with acetonitrile
containing 5% pyridine (2.times.100 mL) and acetonitrile
(3.times.100 mL). Cap A (ABI, 80 mL) and Cap B (ABI, 90 mL)
solutions were added to CPG support and shaken for 4 h. Solutions
filtered off, CPG washed with acetonitrile containing 5% pyridine
(2.times.100 mL) followed by acetonitrile (2.times.250 mL) and
dried under high vacuum for 30 min. A solution of TBDMSCl (2.5 g)
and imidazole (0.75 g) in acetonitrile containing 5% pyridine (100
mL) was added to CPG and shaken for 5 h. Solution filtered off, CPG
was successively washed with acetonitrile containing 5% pyridine
(3.times.100 mL) and DCM (4.times.100 mL) and dried under high
vacuum in a desiccator for over night to get dry CPG support 20.
Loading was determined by treating small portion of CPG with 3% TCA
in DCM and assayed DMT content 40 .mu.mol/g) by measuring
absorbance at 498 mm.
Example 2
Cell Culture Conditions and Reagents, Cytokine Induction by
Exemplar Oligos from Table I in HEK293 Cells Expressing Mouse
TLR9
[0067] HEK293 cells stably expressing mouse TLR9 (Invivogen, San
Diego, Calif.) were cultured in 48-well plates in 250 .mu.l/well
DMEM supplemented with 10% heat-inactivated FBS in a 5% CO.sub.2
incubator. At 80% confluence, cultures were transiently transfected
with 400 ng/ml of SEAP (secreted form of human embryonic alkaline
phosphatase) reporter plasmid (pNifty2-Seap) (Invivogen) in the
presence of 4 .mu.l/ml of lipofectamine (Invitrogen, Carlsbad,
Calif.) in culture medium. Plasmid DNA and lipofectamine were
diluted separately in serum-free medium and incubated at room
temperature for 5 minutes. After incubation, the diluted DNA and
lipofectamine were mixed and the mixtures were incubated at room
temperature for 20 minutes. Aliquots of 25 .mu.l of the
DNA/lipofectamine mixture containing 100 ng of plasmid DNA and 1
.mu.l of lipofectamine were added to each well of the cell culture
plate, and the cultures were continued for 4 hours.
[0068] After transfection, medium was replaced with fresh culture
medium, exemplar oligos from Table I were added to the cultures,
and the cultures were continued for 24 hours. At the end of oligo
treatment, 30 .mu.l of culture supernatant was taken from each
treatment and used for SEAP assay following manufacturer's protocol
(Invivogen). Briefly, culture supernatants were incubated with
p-nitrophynyl phosphate substrate and the yellow color generated
was measured by a plate reader at 405 nm (Putta M R et al, Nucleic
Acids Res., 2006, 34:3231-8).
Example 3
Cytokine Induction by Exemplar Oligos from Table I in Human PBMCs,
pDCs, and Mouse Splenocytes
Human PBMC Isolation
[0069] Peripheral blood mononuclear cells (PBMCs) from freshly
drawn healthy volunteer blood (CBR Laboratories, Boston, Mass.)
were isolated by Ficoll density gradient centrifugation method
(Histopaque-1077, Sigma).
Human pDC Isolation
[0070] pDCs were isolated from PBMCs by positive selection using
the BDCA4 cell isolation kits (Miltenyi Biotec) according to the
manufacturer's instructions.
Mouse Splenocyte Isolation
[0071] Spleen cells from 4-8 week old C57BL/6 mice were cultured in
RPMI complete medium as described by Zhao, Q., et al. (Biochem
Pharmacol. 51: 173-182 (1996)) and Branda, R. F., et al. (Biochem.
Pharmacol. 45: 2037-2043 (1993)). All other culture reagents were
purchased from Mediatech (Gaithersburg, Md.).
Cytokine ELISAs
[0072] Human PBMCs or mouse splenocytes were plated in 48-well
plates using 5.times.10.sup.6 cells/ml. Human pDCs were plated in
96-well dishes using 1.times.10.sup.6 cells/ml. The exemplar oligos
from Table I dissolved in DPBS (pH 7.4; Mediatech) were added to
the cell cultures. The cells were then incubated at 37.degree. C.
for 24 hr and the supernatants were collected for luminex multiplex
or ELISA assays. In certain experiments, the levels of IFN-.alpha.,
IL-6, and/or IL-12 were measured by sandwich ELISA. The required
reagents, including cytokine antibodies and standards, were
purchased from PharMingen.
Cytokine Luminex Multiplex
[0073] In certain experiments, the levels of IL-1R.alpha., IL-6,
IL-10, IL-12, IFN-.alpha., IFN-.gamma., MIP-1.alpha., MIP-.beta.,
MCP-1, and IL-12p40p70 in culture supernatants were measured by
Luminex multiplex assays, which were performed using Biosource
human multiplex cytokine assay kits on Luminex 100 instrument and
the data were analyzed using StarStation software supplied by
Applied Cytometry Systems (Sacramento, Calif.).
Example 4
Human B Cell Proliferation Assay in the Presence of Exemplar Oligos
from Table I
[0074] Human B cells were isolated from PBMCs by positive selection
using the CD19 Cell Isolation Kit (Miltenyi Biotec, Auburn, Calif.)
according to the manufacturer's instructions.
[0075] The culture medium used for the assay consisted of RPMI 1640
medium supplemented with 1.5 mM glutamine, 1 mM sodium pyruvate,
0.1 mM non-essential amino acids, 50 .mu.M 2-mercaptoethanol, 100
IU/ml penicillin-streptomycin mix and 10% heat-inactivated fetal
bovine serum.
[0076] A total of 0.5.times.10.sup.6 B cells per ml (i.e.
1.times.10.sup.5/200 .mu.l/well) were stimulated in 96 well flat
bottom plates with different concentrations of exemplar oligos from
Table I in triplicate for a total period of 72 hours. After 66 h,
cells were pulsed with 0.75 .mu.Ci of [.sup.3H]-thymidine (1Ci=37
GBq; Perkin Elmer Life Sciences) in 20 .mu.l RPMI 1640 medium (no
serum) per well and harvested 6-8 h later. The plates were then
harvested using a cell harvester and radioactive incorporation was
determined using standard liquid scintillation technique. In some
cases the corresponding [.sup.3H]-T (cpm) was converted into a
proliferation index and reported as such.
Sequence CWU 1
1
97111DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1tcagtcntta c 11211DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 2tctgtcntta g 11311DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 3cagtcnttca g 11411DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 4tctgtcnttt t 11511DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 5tctgtcnttg t 11611DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 6tagtcntttt t 11711DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 7tggtcnttct t 11811DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 8tagtcnttgt a 11911DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 9tagtcnttct c 111011DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 10tcntcnttct t 111111DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 11tcntacntac n 111211DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 12tcntcnacna t 111311DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 13tcnatcnatc n 111411DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 14tcagacntta c 111511DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 15tctgacntta g 111611DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 16cagacnttca g 111711DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 17tctgacnttt t 111811DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 18tctgacnttg t 111911DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 19tagacntttt t 112011DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 20tggacnttct t 112111DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 21tagacnttgt a 112211DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 22tagacnttct c 112311DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 23tcntcnttct t 112411DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 24tcagtcntta c 112511DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 25tctgtcntta g 112611DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 26cagtcnttca g 112711DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 27tctgtcnttt t 112811DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 28tctgtcnttg t 112911DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 29tagtcntttt t 113011DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 30tggtcnttct t 113111DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 31tagtcnttgta 113211DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 32tagtcnttct c 113311DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 33tcntcnttct t 113411DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 34tcntacntac n 113511DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 35tcntcnacna t 113611DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 36tcnatcnatc n 113711DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 37tctgtcgttc t 113811DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 38tctgacnttc t 113911DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 39tcnaacnttc n 114011DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 40tcntcnttct g 114111DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 41tcagtngtta g 114211DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 42tctgtngttc t 11436DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 43tcgttg 6446DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 44tcgttg
6456DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 45tcnttg 6466DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 46tcgttg 64711DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 47tcnaacnttc n
114811DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 48tcnaacnttc n 114922DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 49tctgacnttc ttctgacntt ct 225011DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 50tctgtcnttc t 115111DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 51tctgacnttc t 115211DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 52tctgtnnttc t 115311DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 53tctgacgttc t 115411DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 54tctgacnttc t 115511DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 55tctgtcnttc t 115611DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 56tctgacnttc t 115711DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 57tctgtcnttc t 115811DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 58tcnaacnttc n 115911DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 59tcagtcntta g 116011DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 60tctgtcnttc u 116111DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 61tctgtcnttc u 116211DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 62tctgtcnttc u 116311DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 63ctgtcnttcu c 116411DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 64tcnaacnttc g 116511DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 65tcnaacnttc n 116611DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 66tctgtcnttc u 116711DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 67tctgtcnttc u 116811DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 68tctgtcnttc u 116911DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 69ctgtcnttcu c 117011DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 70tcnaacnttc g 117111DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 71tcnaacnttc n 117211DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 72tctgtcntta g 117311DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 73cagtcnttca g 117411DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 74tcntcnacna t 117511DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 75tcagtcntta c 117611DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 76tcagtcntta c 117710DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 77cagtcnttac 107811DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 78tcagtcntta c 117911DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 79tagtcntttt t 118011DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 80tctgtcnttg t 118111DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 81tagtcntttt t 118211DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 82tcntcnacna t 118311DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 83tcnatcnatc n 118411DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 84tcagtcntta c 118510DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 85tctgtcntag 10869DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 86tcntcnttt
9879DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 87tcntcnttt 9888DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 88tcncnttt 8899DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 89tcntcnttt
9909DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 90tcntcnttt 9918DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 91tcntcntt 89211DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 92tcagtcntta c
119311DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 93tctgtcnttc t 119411DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 94tctgtcnaca g 119511DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 95tctgtcnaca g 119611DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 96tctgtcntta g 11979DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 97tcntcnttt 9
* * * * *