U.S. patent application number 11/056463 was filed with the patent office on 2005-08-04 for methods related to immunostimulatory nucleic acid-induced interferon.
This patent application is currently assigned to Coley Pharmaceutical GmbH. Invention is credited to Bratzler, Robert L., Hartmann, Gunther, Krieg, Arthur M..
Application Number | 20050169888 11/056463 |
Document ID | / |
Family ID | 34810902 |
Filed Date | 2005-08-04 |
United States Patent
Application |
20050169888 |
Kind Code |
A1 |
Hartmann, Gunther ; et
al. |
August 4, 2005 |
Methods related to immunostimulatory nucleic acid-induced
interferon
Abstract
Methods and compositions are provided for extending the clinical
utility of IFN-.alpha. in the treatment of a variety of viral and
proliferative disorders. Among other aspects, the invention
provides methods which increase the efficacy of IFN-.alpha.
treatment and reduce IFN-.alpha. treatment-related side effects. In
addition, methods are provided for supporting the survival and for
activating natural interferon producing cells (IPCs) in vitro
without exogenous IL-3 or GM-CSF. The invention is based on the
discovery that certain CpG and non-CpG ISNAs promote survival and
stimulation of IPCs.
Inventors: |
Hartmann, Gunther; (Munich,
DE) ; Bratzler, Robert L.; (Concord, MA) ;
Krieg, Arthur M.; (Wellesley, MA) |
Correspondence
Address: |
WOLF GREENFIELD & SACKS, PC
FEDERAL RESERVE PLAZA
600 ATLANTIC AVENUE
BOSTON
MA
02210-2211
US
|
Assignee: |
Coley Pharmaceutical GmbH
Langenfeld
MA
Coley Pharmaceutical Group, Inc.
Wellesley
IA
University of Iowa Research Foundation
Iowa City
|
Family ID: |
34810902 |
Appl. No.: |
11/056463 |
Filed: |
February 11, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11056463 |
Feb 11, 2005 |
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09672126 |
Sep 27, 2000 |
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60156147 |
Sep 27, 1999 |
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Current U.S.
Class: |
424/85.7 ;
514/44A |
Current CPC
Class: |
A61K 38/21 20130101;
A61K 2039/5158 20130101; A61P 31/22 20180101; A61K 38/21 20130101;
C12N 2501/056 20130101; A61P 31/20 20180101; A61P 35/00 20180101;
A61P 31/18 20180101; A61K 2300/00 20130101; A61K 2039/55561
20130101; C12N 2501/22 20130101; C12N 2501/23 20130101; C12N 5/0639
20130101; C12N 15/117 20130101; A61P 31/14 20180101; A61K 2039/5154
20130101 |
Class at
Publication: |
424/085.7 ;
514/044 |
International
Class: |
A61K 038/21; A61K
048/00 |
Claims
1-46. (canceled)
47. A method of treating a subject to activate interferon-producing
cells (IPCs) of the subject comprising isolating IPCs from a
subject in need of such treatment, culturing the IPCs in vitro,
contacting the IPCs in vitro with an effective amount of an
isolated immunostimulatory nucleic acid, and returning the
contacted IPCs to the subject.
48. The method of claim 47, further comprising contacting the IPCs
in vitro with a growth factor.
49. The method of claim 47, further comprising contacting the IPCs
in vitro with IL-3.
50. The method of claim 47, further comprising contacting the IPCs
in vitro with GM-CSF.
51. The method of claim 47, wherein the IPCs are cultured in vitro
in the absence of IL-3.
52. The method of claim 47, wherein the IPCs are cultured in vitro
in the absence of GM-CSF.
53. The method of claim 47, wherein the immunostimulatory nucleic
acid is modified.
54. The method of claim 47, wherein the immunostimulatory nucleic
acid comprises a backbone with at least one nuclease-resistant
intemucleotide linkage selected from the group consisting of:
phosphorothioate, phosphorodithioate, methylphosphonate, and
peptide.
55. The method of claim 47, wherein the immunostimulatory nucleic
acid comprises at least one nucleotide analog or derivative.
56. The method of claim 47, wherein the immunostimulatory nucleic
acid is not a palindrome.
57. The method of claim 47, wherein the immunostimulatory nucleic
acid is a CpG nucleic acid.
58. The method of claim 47, wherein the immunostimulatory nucleic
acid is a non-CpG nucleic acid.
59. The method of claim 58, wherein the non-CpG immunostimulatory
nucleic acid is a T-rich nucleic acid.
60. The method of claim 58, wherein the non-CpG immunostimulatory
nucleic acid is a poly-G nucleic acid.
61. The method of claim 47, wherein the immunostimulatory nucleic
acid is any combination of at least two nucleic acids selected from
the group consisting of: CpG nucleic acids, T-rich nucleic acids,
and poly-G nucleic acids.
62. The method of claim 47, wherein the immunostimulatory nucleic
acid is between 8 and 100 nucleotides in length.
63. The method of claim 47, wherein the immunostimulatory nucleic
acid is between 12 and 40 nucleotides in length.
64. The method of claim 47, wherein the immunostimulatory nucleic
acid has a sequence selected from the group consisting of
13 ggGGTCAACGTTGAgggggG ODN 1585 SEQ ID NO:1
tcgtcgttttgtcgttttgtcgtt ODN 2022 SEQ ID NO:2 ggggtcgtcgttttgggggg
ODN 2184 SEQ ID NO:3 tcgtcgttttgtcgttttgggggg ODN 2185 SEQ ID NO:4
ggggtcgacgtcgagggggg ODN 2192 SEQ ID NO:5 ggggtcatcgatgagggggg ODN
2204 SEQ ID NO:6 ggGGGACGATCGTCgggggG ODN 2216 SEQ ID NO:7
gggggtcgtacgacgggggg ODN 2217 SEQ ID NO:8 ggGGGACGATATCGTCgggggG
ODN 2245 SEQ ID NO:9 ggGGGACGACGTCGTCgggggG ODN 2246 SEQ ID NO:10
ggGGGACGAGCTCGTCgggggG ODN 2247 SEQ ID NO:11 ggGGGACGTACGTCgggggG
ODN 2248 SEQ ID NO:12 ggGGGACGATCGTTGggggG ODN 2252 SEQ ID NO:13
ggGGAACGATCGTCgggggG ODN 2253 SEQ ID NO:14 ggGGGGACGATCGTCgggggG
ODN 2254 SEQ ID NO:15 ggGGGACGATCGTCGgggggG ODN 2255 SEQ ID NO:16
ggGGGTCATCGATGAgggggG ODN 2260 SEQ ID NO:17 ggGGTCGTCGACGAgggggG
ODN 2293 SEQ ID NO:18 ggGGTCGTTCGAACGAgggggG ODN 2294 SEQ ID NO:19
ggGGACGTTCGAACGTgggggG ODN 2295 SEQ ID NO:20 ggGGAACGACGTCGTTgggggG
ODN 2297 SEQ ID NO:21 ggGGAACGTACGTCgggggG ODN 2298 SEQ ID NO:22
ggGGAACGTACGTACGTTgggggG ODN 2299 SEQ ID NO:23 ggGGTCACCGGTGAgggggG
ODN 2300 SEQ ID NO:24 ggGGTCGACGTACGTCGAgggggG ODN 2301 SEQ ID
NO:25 ggGGACCGGTACCGGTgggggG ODN 2302 SEQ ID NO:26
ggGTCGACGTCGAgggggG ODN 2303 SEQ ID NO:27 ggGGTCGACGTCGagggg ODN
2304 SEQ ID NO:28 ggGGAACGTTAACGTTgggggG ODN 2305 SEQ ID NO:29
ggGGACGTCGACGTggggG ODN 2306 SEQ ID NO:30 ggGGGTCGTTCGTTgggggG ODN
2311 SEQ ID NO:31 ggGACGATCGTCGgggggG ODN 2328 SEQ ID NO:32
ggGTCGTCGACGAggggggG ODN 2329 SEQ ID NO:33 ggTCGTCGACGAGgggggG ODN
2330 SEQ ID NO:34 ggGGACGATCGTCGgggggG ODN 2332 SEQ ID NO:35
ggGGTCGACGTCGACGTCGAGgggggG, ODN 2334 SEQ ID NO:36 and
ggGGACGACGTCGTGgggggG, ODN 2336 SEQ ID NO:37
wherein each lower case letter represents phosphorothioate linkage
and each upper case letter indicates phosphodiester linkage.
65-121. (canceled)
122. A method of enhancing efficacy of IFN-.alpha. treatment in a
subject in need of such treatment, comprising administering to a
subject in need of such treatment an amount of a pharmaceutical
composition comprising IFN-.alpha. effective for treating a
condition of the subject; isolating natural interferon-producing
cells (IPCs) from a donor; contacting the isolated IPCs ex vivo
with an amount of a pharmaceutical composition comprising an
immunostimulatory nucleic acid effective for inducing the IPCs to
release IFN-.alpha.; and administering the contacted cells to the
subject.
123. The method of claim 122, wherein the donor is the subject.
124. The method of claim 122 further comprising contacting the
isolated IPCs with an antigen.
125. The method of claim 122, wherein the administering the
contacted cells comprises local injection.
126. The method of claim 125, wherein the local injection is via a
blood vessel supplying a target tissue.
127. The method of claim 126, wherein the blood vessel is selected
from the group consisting of a hepatic artery, a portal vein, a
celiac artery, and a splenic artery.
128. The method of claim 122, wherein the immunostimulatory nucleic
acid is modified.
129. The method of claim 122, wherein the immunostimulatory nucleic
acid comprises a backbone with at least one nuclease-resistant
intemucleotide linkage selected from the group consisting of:
phosphorothioate, phosphorodithioate, methylphosphonate, and
peptide.
130. The method of claim 122, wherein the immunostimulatory nucleic
acid comprises at least one nucleotide analog or derivative.
131. The method of claim 122, wherein the immunostimulatory nucleic
acid is not a palindrome.
132. The method of claim 122, wherein the immunostimulatory nucleic
acid is a CpG nucleic acid.
133. The method of claim 122, wherein the immunostimulatory nucleic
acid is a non-CpG nucleic acid.
134. The method of claim 133, wherein the non-CpG immunostimulatory
nucleic acid is a T-rich nucleic acid.
135. The method of claim 133, wherein the non-CpG immunostimulatory
nucleic acid is a poly-G nucleic acid.
136. The method of claim 122, wherein the immunostimulatory nucleic
acid is any combination of at least two nucleic acids selected from
the group consisting of: CpG nucleic acids, T-rich nucleic acids,
and poly-G nucleic acids.
137. The method of claim 122, wherein the immunostimulatory nucleic
acid is between 8 and 100 nucleotides in length.
138. The method of claim 122, wherein the immunostimulatory nucleic
acid is between 12 and 40 nucleotides in length.
139. The method of claim 122, wherein the immunostimulatory nucleic
acid has a sequence selected from the group consisting of
14 ggGGTCAACGTTGAgggggG ODN 1585 SEQ ID NO:1
tcgtcgttttgtcgttttgtcgtt ODN 2022 SEQ ID NO:2 ggggtcgtcgttttgggggg
ODN 2184 SEQ ID NO:3 tcgtcgttttgtcgttttgggggg ODN 2185 SEQ ID NO:4
ggggtcgacgtcgagggggg ODN 2192 SEQ ID NO:5 ggggtcatcgatgagggggg ODN
2204 SEQ ID NO:6 ggGGGACGATCGTCgggggG ODN 2216 SEQ ID NO:7
gggggtcgtacgacgggggg ODN 2217 SEQ ID NO:8 ggGGGACGATATCGTCgggggG
ODN 2245 SEQ ID NO:9 ggGGGACGACGTCGTCgggggG ODN 2246 SEQ ID NO:10
ggGGGACGAGCTCGTCgggggG ODN 2247 SEQ ID NO:11 ggGGGACGTACGTCgggggG
ODN 2248 SEQ ID NO:12 ggGGGACGATCGTTGggggG ODN 2252 SEQ ID NO:13
ggGGAACGATCGTCgggggG ODN 2253 SEQ ID NO:14 ggGGGGACGATCGTCgggggG
ODN 2254 SEQ ID NO:15 ggGGGACGATCGTCGgggggG ODN 2255 SEQ ID NO:16
ggGGGTCATCGATGAgggggG ODN 2260 SEQ ID NO:17 ggGGTCGTCGACGAgggggG
ODN 2293 SEQ ID NO:18 ggGGTCGTTCGAACGAgggggG ODN 2294 SEQ ID NO:19
ggGGACGTTCGAACGTgggggG ODN 2295 SEQ ID NO:20 ggGGAACGACGTCGTTgggggG
ODN 2297 SEQ ID NO:21 ggGGAACGTACGTCgggggG ODN 2298 SEQ ID NO:22
ggGGAACGTACGTACGTTgggggG ODN 2299 SEQ ID NO:23 ggGGTCACCGGTGAgggggG
ODN 2300 SEQ ID NO:24 ggGGTCGACGTACGTCGAgggggG ODN 2301 SEQ ID
NO:25 ggGGACCGGTACCGGTgggggG ODN 2302 SEQ ID NO:26
ggGTCGACGTCGAgggggG ODN 2303 SEQ ID NO:27 ggGGTCGACGTCGagggg ODN
2304 SEQ ID NO:28 ggGGAACGTTAACGTTgggggG ODN 2305 SEQ ID NO:29
ggGGACGTCGACGTggggG ODN 2306 SEQ ID NO:30 ggGGGTCGTTCGTTgggggG ODN
2311 SEQ ID NO:31 ggGACGATCGTCGgggggG ODN 2328 SEQ ID NO:32
ggGTCGTCGACGAggggggG ODN 2329 SEQ ID NO:33 ggTCGTCGACGAGgggggG ODN
2330 SEQ ID NO:34 ggGGACGATCGTCGgggggG ODN 2332 SEQ ID NO:35
ggGGTCGACGTCGACGTCGAGgggggG, ODN 2334 SEQ ID NO:36 and
ggGGACGACGTCGTGgggggG, ODN 2336 SEQ ID NO:37
wherein each lower case letter represents phosphorothioate linkage
and each upper case letter indicates phosphodiester linkage.
140. The method of claim 122, wherein the subject has a condition
selected from the group consisting of a proliferative disorder and
a viral infection.
141. The method of claim 122, wherein the subject has a
proliferative disorder selected from the group consisting of: hairy
cell leukemia, chronic myelogenous leukemia, cutaneous T-cell
leukemia, multiple myeloma, follicular lymphoma, malignant
melanoma, squamous cell carcinoma, AIDS-related Kaposi's sarcoma,
renal cell carcinoma, prostate carcinoma, bladder cell carcinoma,
cervical dysplasia, and colon carcinoma.
142. The method of claim 122, wherein the subject has a viral
infection selected from the group consisting of: hepatitis B,
hepatitis C, condyloma acuminatum, human immunodeficiency virus,
herpes, cytomegalovirus, Epstein-Barr virus, and
papillomavirus.
143-203. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application No. 60/156,147, filed Sep. 27, 1999, the entire
contents of which are hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] Human interferon alpha (IFN-.alpha.), also known as
leukocyte interferon and .alpha. interferon, comprises a family of
extracellular signaling proteins with antiviral, antiproliferative,
and immunomodulatory activities. The first type of interferon to be
identified and commercialized, IFN-.alpha. remains the most widely
used interferon for clinical applications.
[0003] IFN-.alpha. is a member of the family of Type I interferons,
which also includes IFN-.beta., omega (leukocyte (II)) interferon
and tau (trophoblast) interferon. Omega and tau interferons are not
clinically used. IFN-.beta., also known as fibroblast interferon,
is well characterized but less utilized than IFN-.alpha. in the
clinic. Fibroblasts are the predominant cellular producers of
IFN-.beta.. IFN-.beta. has been approved in the United States for
the treatment of relapsing forms of multiple sclerosis. Interferon
gamma (IFN-.gamma.), also known as gamma interferon, is the only
known type II interferon. IFN-.gamma. is produced by activated T
lymphocytes and plays an important role in the establishment of a
Th1 immune response. Its therapeutic use is limited. In the United
States, human IFN-.gamma. has been approved for reducing the
frequency and severity of infections with chronic granulomatous
disease.
[0004] IFN-.alpha. itself represents a family of more than a dozen
related, homologous proteins (isoforms, Table 1), each encoded by a
unique gene and each exhibiting a unique activity profile. The
activities of the different .alpha. interferon species on viruses
can vary as much as twenty-fold or more.
[0005] IFN-.alpha. products in clinical use are recombinant
proteins or highly purified natural proteins of a single isoform.
Recombinant IFN-.alpha. has been approved for use in the treatment
of a variety of tumors and viral diseases (Table 2).
[0006] Until recently, B lymphocytes were believed to be the
predominant producers of IFN-.alpha.. Recently a new cell type has
been identified in the peripheral blood as the major source of Type
I interferon production. These previously unidentified "natural
interferon producing cells" (IPC) had been described for many years
as a rare CD4.sup.+/MHC class II.sup.+ population (1:1000 within
peripheral blood mononuclear cells (PBMC)) capable of synthesizing
extremely large amounts of type I IFN upon viral infection. Cella M
et al. Nat Med 5:919 (1999); Galy A et al. Blood 95:128 (2000);
Siegal F P et al. Science 284:1835 (1999). After isolation of IPCs
from the peripheral blood, IL-3 is required for survival of this
cell type.
1TABLE 1 Family of Human IFN-.alpha. IFN-.alpha.A (IFN-.alpha.2a)
IFN-.alpha.2 (IFN-.alpha.2b) IFN-.alpha.4b (IFN-.alpha.4)
IFN-.alpha.B2 (IFN-.alpha.8) IFN-.alpha.C (IFN-.alpha.10)
IFN-.alpha.D (IFN-.alpha.1) IFN-.alpha.F (IFN-.alpha.21)
IFN-.alpha.G (IFN-.alpha.5) IFN-.alpha.H2 (IFN-.alpha.14)
IFN-.alpha.I (IFN-.alpha.17) IFN-.alpha.J1 (IFN-.alpha.7)
IFN-.alpha.K (IFN-.alpha.6) IFN-.alpha.M1 IFN-.alpha.N
IFN-.alpha.WA (IFN-.alpha.16)
[0007]
2TABLE 2 Current Clinical Approval of IFN-.alpha. Approved Outside
Approved in the United States the United States Chronic hepatitis B
Multiple myeloma Chronic hepatitis C Renal cell carcinoma Hairy
cell leukemia Bladder cell carcinoma Cutaneous T-cell leukemia
Colon carcinoma Chronic myeloid leukemia Cervical dysplasia
Non-Hodgkin's lymphoma Laryngeal papillomatosis Adjuvant therapy
for malignant melanoma Kaposi's Sarcoma (AIDS-related) Condylomata
acuminata (venereal warts)
[0008] Dendritic cells (DC) are thought to play a key role in the
priming of immune responses against neoantigens. Banchereau J et
al., Nature 392:245 (1998). Recent evidence suggests the presence
of several distinct DC subtypes in human peripheral blood. Zhong R
K et al. J Immunol 163:1254 (1999). These subtypes of DC include
myeloid DC (mDC) and plasmacytoid DC (pDC, also known as DC2
cells). Precursor dendritic cells contain two subsets, a
CD11c.sup.+/CD123.sup.+/- population (precursor of mDC) and a
CD11c.sup.-/CD123.sup.++ population (precursor of pDC). The latter
has recently attracted major attention since it was reported to be
identical with the natural type I IFN producing cell (IPC).
O'Doherty U et al. J Exp Med 178:1067 (1993); Grouard G et al. J
Exp Med 185:1101 (1997); Thomas R et al. J Immunol 153:4016 (1994).
Upon maturation this cell type develops characteristic features of
DC. O'Doherty U et al. J Exp Med 178:1067 (1993); Thomas R et al. J
Immunol 153:4016 (1994); Galy A et al. Blood 95:128 (2000); Chehimi
J et al. Immunology 68:488. (1989).
[0009] The frequency of IPCs in PBMC in normal individuals varies
between 0.2 and 0.6 percent. They are characterized by the absence
of lineage markers CD3 (T cells), CD14 (monocytes), CD19 (B cells)
and CD56 (NK cells), by the absence of CD11c, and by their
expression of CD4, CD123 (IL-3 receptor .alpha., IL-3R.alpha.) and
MHC class II. Grouard G et al. J Exp Med 185:1101-11 (1997);
Rissoan M-C et al. Science 283:1183-6 (1999); Siegal F P et al.
Science 284:1835-7 (1999); Cella M et al. Nat Med 5:919-23 (1999).
Morphologically IPCs resemble lymphocytes. IPCs can be isolated
from PBMC by a combination of magnetic bead activated cell sorting
(MACS) and fluorescence-activated cell sorting (flow cytometry,
FACS). Without addition of IL-3, most of the IPCs die within 3 days
of cell culture. Infection of IPCs with herpes simplex virus (HSV,
Siegal F P et al. Science 284:1835-7 (1999)) or influenza virus
(Cella M et al. Nat Med 5:919-23 (1999)) leads to production of
large amounts of type I interferons as measured by a bioassay
(protection of fibroblasts against vesicular stomatitis virus).
[0010] Aside from its role in carrying the genetic code, DNA has
recently been shown to function as a signaling molecule (Krieg A M,
1998, Biodrugs). The immune systems of higher eukaryotes appear to
have evolved a mechanism to detect prokaryotic nucleic acids based
on their content of unmethylated CpG dinucleotides in particular
base contexts. Krieg A M et al. Nature 374:546-9 (1995).
Unmethylated CpG dinucleotides are common in bacterial DNA, but are
underrepresented ("CpG suppression") and are methylated in
vertebrate DNA. Bird AP Trends in Genetics 3:342 (1987). DNA
containing these unmethylated CpG dinucleotides in immune
stimulatory base contexts ("CpG motifs") triggers humoral immunity
by inducing B cell activation, resistance to activation-induced
apoptosis, and IL-6 and IgM secretion. Krieg A M et al. Nature
374:546-9 (1995); Yi A K et al. J Immunol 157:5394 (1996); and
Klinman D et al. Proc Natl Acad Sci USA 93:2879 (1996). Such CpG
DNA also directly activates monocytes and macrophage to secrete
Th1-like cytokines. Ballas Z K et al. J Immunol 157:1840 (1996);
Cowdery J S et al. J Immunol 156:4570 (1996); and Halpern M D et
al. Cell Immunol 167:72 (1996). This leads to the activation of
natural killer (NK) cell lytic activity and IFN-.gamma. secretion.
Ballas Z K et al. J Immunol 157:1840 (1996); Cowdery J S et al. J
Immunol 156:4570 (1996); and Chace J H Clin Immunol Immunopath
84:185-93 (1997).
[0011] Yamamoto et al. reported in 1988 their findings that a
nucleic acid fraction, designated MY-1, extracted from
Mycobacterium bovis (BCG) induced type I interferon in vitro.
Yamamoto S et al. Jpn J Cancer Res 79:866-73 (1988). Subsequently
Tokunaga et al. subsequently synthesized a panel of 45-mer
oligonucleotides with sequence present in cDNA encoding three
randomly selected known BCG proteins and found that one sequence,
BCG-A4, was a strong inducer of type I IFN in mouse spleen cell
suspensions. Tokunaga T et al. Microbiol Immunol 36:55-66 (1992). A
5' 30-mer fragment, BCG-A4a, was reported to be as potent an
inducer of IFN as the intact 45-mer BCG-A4.
3 (SEQ ID NO:163) BCG-A4 ACCGATGACGTCGCCGGTGACGGCAC-
CACGACGGCCACCGTGCTG (SEQ ID NO:164) BCG-A4a
ACCGATGACGTCGCCGGTGACGCCACCACG
[0012] These workers went on to report that all oligonucleotides
that induced IFN included a hexamer palindromic sequence GACGTC
(present in BCG-A4 and BCG-A4a), AGCGCT, and AACGTT, but not
ACCGGT. Yamamoto S et al. J Immunol 148:4072-6 (1992). Kimura et
al. then found that among 30-mer phosophodiester
oligodeoxynucleotides (ODNs) containing the hexamer palindrome
AACGTT and oligoA, oligoC, oligoT, and oligoG ends, the latter
(GGGGGGGGGGGGAACGTTGGGGGGGGGGGG; SEQ ID NO:165) was the strongest
inducer of type I IFN in mouse spleen cell suspensions. Kimura Y et
al. J Biochem (Toyo) 116:991-4 (1994).
[0013] Recently it was surprisingly discovered that CpG ODN
sequences with the strongest activity on human B cells did not
induce detectable levels of type I IFN in PBMC. Hartmann G et al. J
Immunol 164:1617-24 (2000).
SUMMARY OF THE INVENTION
[0014] It was discovered according to the invention that certain
immunostimulatory nucleic acids (ISNAs) are especially suited as
single agents to promote both survival and stimulation of IPCs. It
was also discovered according to the invention that certain ISNAs
obviate the requirement of IL-3 for IPC survival and the
requirement of viral infection for IPC activation.
[0015] In addition, it was surprisingly discovered according to the
invention that certain CpG ISNA induce the production of large
amounts of type I IFN but have minimal effects activating B cells,
while certain other CpG ISNA strongly activate human B cells and
IPCs but have minimal effects inducing type I IFN. Surprisingly, it
was discovered that the CpG ISNA that are strong inducers of type I
IFN do not necessarily contain a hexamer palindrome GACGTC, AGCGCT,
or AACGTT described by Yamamoto and colleagues. Yamamoto S et al. J
Immunol 148:4072-6 (1992).
[0016] These discoveries open avenues for the use of ISNA, and
especially certain CpG ISNA, as a therapeutic agent for clinical
applications which call for the use of IFN-.alpha.. Clinical
strategies comprise local and systemic in vivo administration of
ISNA as well as ex vivo strategies in which in vitro ISNA-activated
isolated IPCs are reinfused into the patient locally or
systemically. These therapeutic strategies include the combination
with other growth factors (IL-3, GM-CSF, flt3-ligand, etc.) as well
as with other stimuli (superantigens, viral products). CpG ISNA of
the invention that are inducers of type I IFN also allow the in
vitro production of natural interferons using a permanent cell line
derived from IPCs. Since natural IFN-.alpha. is a family of more
than a dozen separate gene products, the individual products of
which have unique activity profiles, the clinical use of natural
interferon may be preferable compared to recombinant IFN-.alpha.
derived from a single recombinant IFN-.alpha. gene.
[0017] It was also surprisingly discovered according to the
invention that type I IFN activates a subset of T lymphocytes
called .gamma..delta. T cells. In addition, it was further
discovered according to the invention that CpG ODN that are strong
inducers of type I IFN, but not CpG ODN that are strong activators
of B cells and pDCs without being strong inducers of type I IFN,
can activate .gamma..delta. T cells present within a population of
peripheral blood mononuclear cells (PBMCs). Without meaning to be
held to a particular theory, it appears likely that type I
IFN-inducing CpG ODN can activate .gamma..delta. T cells present
within the PBMC by their ability to induce secretion of type I IFN
by IPCs also present in the PBMC.
[0018] In addition to the ability to activate .gamma..delta. T
cells, it was also surprisingly discovered according to the
invention that type I IFN-inducing CpG ODN, but not CpG ODN that
are strong activators of B cells and pDCs without being strong
inducers of type I IFN, can enhance proliferation of
antigen-activated .gamma..delta. T cells present within a
population of PBMCs. In particular, proliferation is enhanced in
connection with the presence of specific nonpeptide antigen, for
example, the phosphoantigen isopentenyl pyrophosphate (IPP).
[0019] It was also surprisingly discovered according to the
invention that certain CpG ODN in combination with IPP
synergistically induce the production of IFN-.gamma. and perforin
in .gamma..delta. T cells.
[0020] In another surprising discovery according to the invention,
it was found that type I IFN-inducing CpG ODN, but not CpG ODN that
are strong activators of B cells and pDCs without being strong
inducers of type I IFN, can block CD40-stimulated IL-12 production
in PBMC. It was surprisingly found, in addition, that CpG ODN that
are strong activators of B cells and pDCs without being strong
inducers of type I IFN had the opposite effect, i.e., these ODN
actually enhanced CD40-stimulated IL-12 production in PBMC.
[0021] It was further discovered according to the invention that
that CpG ODN that are strong activators of B cells and pDCs without
being strong inducers of type I IFN are better promoters of
antigen-specific priming and recall of human cytotoxic T
lymphocytes (CTLs) than are CpG ODN that are potent inducers of
type I IFN.
[0022] According to one aspect of the invention, an improvement is
provided for therapies involving administration of IFN-.alpha. to
subjects. The improvement involves co-administering an effective
amount of an isolated ISNA. In one embodiment, the IFN-.alpha. is
administered at the clinically established effective dose for
IFN-.alpha. alone. In another embodiment, the IFN-.alpha. is
administered at a dosage below the clinically established effective
dose for IFN-.alpha. alone. The IFN-.alpha. also can be
administered at the maximum tolerated dose for IFN-.alpha. in the
absence of the oligonucleotide. In other embodiments, the
IFN-.alpha. is administered at 20 percent below, 30 percent below,
40 percent below, or even 50 percent below the maximum tolerated
dose of IFN-.alpha. or the clinically established effective dose
for IFN-.alpha. alone.
[0023] In some embodiments, the ISNA is a CpG nucleic acid. In
other embodiments the ISNA is a non-CpG nucleic acid, i.e., the
ISNA is not a CpG nucleic acid. The non-CpG nucleic acid in one
embodiment is a T-rich nucleic acid. In another embodiment the
non-CpG nucleic acid is a poly-G nucleic acid. In yet another
embodiment the immunostimulatory nucleic acid is any combination of
at least two nucleic acids selected from the group including CpG
nucleic acids, T-rich nucleic acids, and poly-G nucleic acids.
[0024] In some embodiments, the ISNA is modified. In certain
embodiments, the ISNA has a modified backbone with at least one
nuclease-resistant internucleotide linkage. A nuclease-resistant
internucleotide linkage can be selected from the group which
includes a phosphorothioate linkage, a phosphorodithioate linkage,
a methylphosphonate linkage, and a peptide linkage. In certain
embodiments a modified ISNA includes at least one nucleotide analog
or at least one nucleotide analog. The ISNA is a palindrome in
certain embodiments, while in other embodiments, the ISNA is not a
palindrome. In some preferred embodiments the ISNA is between 8 and
100 nucleotides in length, while in other preferred embodiments the
ISNA is between 12 and 40 nucleotides in length. Preferred sizes,
sequences and modifications are described in greater detail
below.
[0025] In certain preferred embodiments the ISNA is a chimeric CpG
ODN exemplified by formula 5' Y.sub.1N.sub.1CGN.sub.2Y.sub.23'
wherein Y.sub.1 and Y.sub.2 are, independent of one another,
nucleic acid molecules having between 1 and 10 nucleotides, and
wherein Y.sub.1 includes at least one modified internucleotide
linkage; Y.sub.2 includes at least one modified internucleotide
linkage; and N.sub.1 and N.sub.2 are nucleic acid molecules, each
independent of one another, having between 0 and 20 nucleotides and
in some embodiments, between 3 and 8 nucleotides, but wherein
N.sub.1CGN.sub.2 has at least 6 nucleotides in total and wherein
the nucleotides of N.sub.1CGN.sub.2 have a phosphodiester
backbone.
[0026] In certain preferred embodiments the ISNA has a sequence
corresponding to
4 ggGGTCAACGTTGAgggggG ODN 1585 SEQ ID NO:1
tcgtcgttttgtcgttttgtcgtt ODN 2022 SEQ ID NO:2 ggggtcgtcgttttgggggg
ODN 2184 SEQ ID NO:3 tcgtcgttttgtcgttttgggggg ODN 2185 SEQ ID NO:4
ggggtcgacgtcgagggggg ODN 2192 SEQ ID NO:5 ggggtcatcgatgagggggg ODN
2204 SEQ ID NO:6 ggGGGACGATCGTCgggggG ODN 2216 SEQ ID NO:7
gggggtcgtacgacgggggg ODN 2217 SEQ ID NO:8 ggGGGACGATATCGTCgggggG
ODN 2245 SEQ ID NO:9 ggGGGACGACGTCGTCgggggG ODN 2246 SEQ ID NO:10
ggGGGACGAGCTCGTCgggggG ODN 2247 SEQ ID NO:11 ggGGGACGTACGTCgggggG
ODN 2248 SEQ ID NO:12 ggGGGACGATCGTTGggggG ODN 2252 SEQ ID NO:13
ggGGAACGATCGTCgggggG ODN 2253 SEQ ID NO:14 ggGGGGACGATCGTCgggggG
ODN 2254 SEQ ID NO:15 ggGGGACGATCGTCGgggggG ODN 2255 SEQ ID NO:16
ggGGGTCATCGATGAgggggG ODN 2260 SEQ ID NO:17 ggGGTCGTCGACGAgggggG
ODN 2293 SEQ ID NO:18 ggGGTCGTTCGAACGAgggggG ODN 2294 SEQ ID NO:19
ggGGACGTTCGAACGTgggggG ODN 2295 SEQ ID NO:20 ggGGAACGACGTCGTTgggggG
ODN 2297 SEQ ID NO:21 ggGGAACGTACGTCgggggG ODN 2298 SEQ ID NO:22
ggGGAACGTACGTACGTTgggggG ODN 2299 SEQ ID NO:23 ggGGTCACCGGTGAgggggG
ODN 2300 SEQ ID NO:24 ggGGTCGACGTACGTCGAgggggG ODN 2301 SEQ ID
NO:25 ggGGACCGGTACCGGTgggggG ODN 2302 SEQ ID NO:26
ggGTCGACGTCGAgggggG ODN 2303 SEQ ID NO:27 ggGGTCGACGTCGagggg ODN
2304 SEQ ID NO:28 ggGGAACGTTAACGTTgggggG ODN 2305 SEQ ID NO:29
ggGGACGTCGACGTggggG ODN 2306 SEQ ID NO:30 ggGGGTCGTTCGTTgggggG ODN
2311 SEQ ID NO:31 ggGACGATCGTCGgggggG ODN 2328 SEQ ID NO:32
ggGTCGTCGACGAggggggG ODN 2329 SEQ ID NO:33 ggTCGTCGACGAGgggggG ODN
2330 SEQ ID NO:34 ggGGACGATCGTCGgggggG ODN 2332 SEQ ID NO:35
ggGGTCGACGTCGACGTCGAGgggggG, ODN 2334 SEQ ID NO:36 and
ggGGACGACGTCGTGgggggG, ODN 2336 SEQ ID NO:37
[0027] wherein each lower case letter represents phosphorothioate
linkage and each upper case letter indicates phosphodiester
linkage.
[0028] In certain more preferred embodiments the ISNA has a
sequence corresponding to
5 SEQ ID NO:11) ggGGGACGAGCTCGTCgggggG (ODN 2247;, SEQ ID NO:16)
ggGGGACGATCGTCGgggggG (ODN 2255;, SEQ ID NO:20)
ggGGACGTTCGAACGTgggggG (ODN 2295;, SEQ ID NO:36)
ggGGTCGACGTCGACGTCGAGgggggG (ODN 2334;, or SEQ ID NO:37)
ggGGACGACGTCGTGgggggG (ODN 2336;,
[0029] wherein each lower case letter represents phosphorothioate
linkage and each upper case letter indicates phosphodiester
linkage.
[0030] In one embodiment, the improvement further involves
co-administering granulocyte-monocyte colony-stimulating factor
(GM-CSF) to the subject.
[0031] In another embodiment, the subject has a condition selected
from the group consisting of a proliferative disorder and a viral
infection. In one embodiment, the subject has a proliferative
disorder such as hairy cell leukemia, chronic myelogenous leukemia,
cutaneous T-cell leukemia, multiple myeloma, follicular lymphoma,
malignant melanoma, squamous cell carcinoma, AIDS-related Kaposi's
sarcoma, renal cell carcinoma, prostate carcinoma, bladder cell
carcinoma, cervical dysplasia, and colon carcinoma. In another
embodiment the subject has a viral infection such as hepatitis B,
hepatitis C, condyloma acuminatum, human immunodeficiency virus,
herpes, cytomegalovirus, Epstein-Barr virus, and
papillomavirus.
[0032] According to another aspect of the invention, a method is
provided for supplementing IFN-.alpha. treatment of a subject. This
aspect of the invention involves administering to a subject in need
of IFN-.alpha. treatment an effective amount of IFN-.alpha. and an
ISNA of the invention. The IFN-.alpha. doses, ISNAs, concurrent
therapy, and conditions calling for treatment with IFN-.alpha.
according to this aspect of the invention are the same as those
described above.
[0033] According to another aspect of the invention, a method is
provided for treating a subject to activate IPCs of the subject.
The method involves isolating IPCs from the subject in need of such
treatment, culturing the isolated IPCs in vitro, contacting the
IPCs in vitro with an effective amount of an isolated ISNA, and
returning the contacted cells to the subject. The cells can also be
contacted in vitro with a growth factor or with a cytokine. In one
embodiment, the method further involves contacting the IPC cells in
vitro with IL-3 or GM-CSF. In another embodiment, the cells are
cultured in vitro in the absence of IL-3 and/or GM-CSF. The ISNAs
and conditions calling for treatment with IFN-.alpha. according to
this aspect of the invention are as described above.
[0034] According to another aspect of the invention, a method is
provided for increasing the efficacy of IFN-.alpha. treatment of a
subject. The method involves administering to a subject in need of
treatment with IFN-.alpha. a pharmaceutical composition including
IFN-.alpha. and co-administering to the subject a pharmaceutical
composition including an ISNA in an amount which, together with the
administered IFN-.alpha., is an effective IFN-.alpha. treatment.
The efficacy of the IFN-.alpha. treatment is greater than the
efficacy of administering the same amount of IFN-.alpha. in the
absence of co-administering the ISNA. The ISNAs and conditions
calling for treatment with IFN-.alpha. according to this aspect of
the invention are as described above. In one embodiment, the
pharmaceutical compositions are administered locally.
[0035] According to another aspect of the invention, a method is
provided for decreasing the dose of IFN-.alpha. needed for
effective treatment of a subject. The method involves administering
to a subject in need of treatment with IFN-.alpha. a pharmaceutical
composition comprising IFN-.alpha. and co-administering to the
subject a pharmaceutical composition including an ISNA. The amount
of administered IFN-.alpha. is less than an amount of IFN-.alpha.
required to achieve the same therapeutic benefit in the absence of
co-administering the ISNA. In certain embodiments, the amount of
administered IFN-.alpha. is at least 20 percent, at least 30
percent, at least 40 percent, or even at least 50 percent below the
amount of IFN-.alpha. required in the absence of coadministering
the immunostimulatory nucleic acid. The pharmaceutical composition
including the ISNA can be administered locally. The ISNAs and
conditions calling for treatment with IFN-.alpha. according to this
aspect of the invention are as described above.
[0036] According to another aspect of the invention, a method is
provided for preventing an IFN-.alpha. treatment-related side
effect in a subject receiving or in need of treatment with
IFN-.alpha.. The method involves administering to a subject in need
of the treatment an IFN-.alpha. pharmaceutical composition and a
pharmaceutical composition comprising an immunostimulatory nucleic
acid in an amount which, together with the administered
IFN-.alpha., is an effective IFN-.alpha. treatment. The IFN-.alpha.
treatment-related side effect is reduced in comparison to the side
effect when IFN-.alpha. is administered in the absence of
co-administering ISNA. The IFN-.alpha. treatment-related side
effect may be systemic. The IFN-.alpha. treatment-related side
effect prevented by the method can include any one of flu-like
syndrome, fever, headache, chills, myalgia, fatigue, anorexia,
nausea, vomiting, diarrhea, and depression. The pharmaceutical
composition including the ISNA can be administered locally. The
ISNAs and conditions calling for treatment with IFN-.alpha.
according to this aspect of the invention are as described
above.
[0037] According to another aspect of the invention, a method is
provided for enhancing the efficacy of IFN-.alpha. treatment in a
subject in need of such treatment. The method involves
administering to a subject in need of such treatment an effective
amount of a pharmaceutical composition containing IFN-.alpha. for
treating the condition, isolating natural IFN-producing cells from
a donor, contacting the isolated IFN-producing cells ex vivo with
an amount of an ISNA effective for inducing the IFN-producing cells
to release IFN-.alpha., and administering the contacted cells to
the subject. The donor can be, but does not have to be, the
subject. The method further can comprise contacting the isolated
cells with an antigen. The administration of the cells can be
accomplished in any manner suitable for the purposes of the method,
and can include local injection. The local injection can be via a
blood vessel supplying a target tissue. The blood vessel can be
selected from, among others, hepatic artery, portal vein, celiac
artery, and splenic artery. The ISNAs and conditions calling for
treatment with IFN-.alpha. according to this aspect of the
invention are as described above.
[0038] According to another aspect of the invention, a method is
provided for supporting survival of IPCs in vitro. The method
involves isolating such cells from a subject, culturing the cells
in a sterile medium suitable for tissue culture, and contacting the
cells in vitro with an amount of ISNA effective to support the
growth of the cells in the absence of IL-3. In a preferred
embodiment the cells can be precursor type 2 dendritic cells. The
culture conditions also can be selected to be free of IL-3 and/or
free of GM-CSF, or they can include IL-3, GM-CSF, or other growth
factors and cytokines. Preferred ISNAs, including oligonucleotides,
sequences, modifications and the like according to this aspect of
the invention, are as described above.
[0039] According to another aspect of the invention, a method is
provided for stimulating isolated IPCs in vitro. The method
involves isolating such cells from a subject, culturing the cells
in a sterile medium suitable for tissue culture, and contacting the
cells in vitro with an amount of ISNA effective to induce secretion
of at least one type I interferon or the expression of CD 80. The
culture conditions can be in the presence or absence of
interleukin-3, GM-CSF, or other growth factors and cytokines. The
IPCs can be precursor type 2 dendritic cells. Preferred ISNAs,
including oligonucleotides, sequences, modifications and the like
according to this aspect of the invention, are as described
above.
[0040] According to another aspect of the invention, a method is
provided for stimulating the production of an array of at least 3,
4, 5, 6, 7 or even 8 or more interferon sub-types. The method
involves contacting IFN-producing cells with an ISNA. The cells may
or may not be isolated. The contacting may be in vivo or in vitro.
Preferred ISNAs, including oligonucleotides, sequences,
modifications and the like according to this aspect of the
invention, are as described herein.
[0041] According to another aspect of the invention, a method is
provided for inhibiting IL-12 production. The method involves
contacting IL-12-producing cells, in the presence of
interferon-producing cells under conditions in which the
IL-12-producing cells normally produce IL-12, with an
immunostimulatory nucleic acid in an amount effective for inducing
secretion of type I interferon. In certain embodiments the
immunostimulatory nucleic acid includes at least one of SEQ ID
NO:1-37.
[0042] According to yet another aspect of the invention, a method
for activating .gamma..delta. T cells is provided. In one
embodiment the method involves contacting .gamma..delta. T cells
with type I IFN. In another embodiment the method involves
contacting .gamma..delta. T cells within a population of cells that
includes interferon-producing cells with an immunostimulatory
nucleic acid in an amount effective for inducing type I IFN.
[0043] In another aspect the invention provides a method for
promoting the proliferation of .gamma..delta. T cells. The method
involves contacting a .gamma..delta. T cell with an
immunostimulatory nucleic acid and an inducer of .gamma..delta. T
cell proliferation, in an amount effective to induce a greater
proliferative response in the presence of the immunostimulatory
nucleic acid than in its absence. In certain embodiments the
immunostimulatory nucleic acid is a CpG nucleic acid. In preferred
embodiments the immunostimulatory nucleic acid is selected from
among SEQ ID NO:1-37. The inducer of .gamma..delta. T cell
proliferation in some embodiments is a phosphoantigen, preferably
IPP.
[0044] In another aspect the invention provides an isolated nucleic
acid having a sequence selected from the group which includes:
6 tcgtcgttttgtcgttttgtcgtt ODN 2022 SEQ ID NO:2
ggggtcgtcgttttgggggg ODN 2184 SEQ ID NO:3 tcgtcgttttgtcgttttgggggg
ODN 2185 SEQ ID NO:4 ggggtcgacgtcgagggggg ODN 2192 SEQ ID NO:5
ggggtcatcgatgagggggg ODN 2204 SEQ ID NO:6 ggGGGACGATCGTCgggggG ODN
2216 SEQ ID NO:7 gggggtcgtacgacgggggg ODN 2217 SEQ ID NO:8
ggGGGACGATATCGTCgggggG ODN 2245 SEQ ID NO:9 ggGGGACGACGTCGTCgggggG
ODN 2246 SEQ ID NO:10 ggGGGACGAGCTCGTCgggggG ODN 2247 SEQ ID NO:11
ggGGGACGTACGTCgggggG ODN 2248 SEQ ID NO:12 ggGGGACGATCGTTGggggG ODN
2252 SEQ ID NO:13 ggGGAACGATCGTCgggggG ODN 2253 SEQ ID NO:14
ggGGGGACGATCGTCgggggG ODN 2254 SEQ ID NO:15 ggGGGACGATCGTCGgggggG
ODN 2255 SEQ ID NO:16 ggGGGTCATCGATGAgggggG ODN 2260 SEQ ID NO:17
ggGGTCGTCGACGAgggggG ODN 2293 SEQ ID NO:18 ggGGTCGTTCGAACGAgggggG
ODN 2294 SEQ ID NO:19 ggGGACGTTCGAACGTgggggG ODN 2295 SEQ ID NO:20
ggGGAACGACGTCGTTgggggG ODN 2297 SEQ ID NO:21 ggGGAACGTACGTCgggggG
ODN 2298 SEQ ID NO:22 ggGGAACGTACGTACGTTgggggG ODN 2299 SEQ ID
NO:23 ggGGTCACCGGTGAgggggG ODN 2300 SEQ ID NO:24
ggGGTCGACGTACGTCGAgggggG ODN 2301 SEQ ID NO:25
ggGGACCGGTACCGGTgggggG ODN 2302 SEQ ID NO:26 ggGTCGACGTCGAgggggG
ODN 2303 SEQ ID NO:27 ggGGTCGACGTCGagggg ODN 2304 SEQ ID NO:28
ggGGAACGTTAACGTTgggggG ODN 2305 SEQ ID NO:29 ggGGACGTCGACGTggggG
ODN 2306 SEQ ID NO:30 ggGGGTCGTTCGTTgggggG ODN 2311 SEQ ID NO:31
ggGACGATCGTCGgggggG ODN 2328 SEQ ID NO:32 ggGTCGTCGACGAggggggG ODN
2329 SEQ ID NO:33 ggTCGTCGACGAGgggggG ODN 2330 SEQ ID NO:34
ggGGACGATCGTCGgggggG ODN 2332 SEQ ID NO:35
ggGGTCGACGTCGACGTCGAGgggggG, ODN 2334 SEQ ID NO:36 and
ggGGACGACGTCGTGgggggG, ODN 2336 SEQ ID NO:37
[0045] wherein each lower case letter represents phosphorothioate
linkage and each upper case letter indicates phosphodiester
linkage.
[0046] In yet another aspect the invention provides a
pharmaceutical composition containing an isolated nucleic acid
having a sequence selected from the group which includes:
7 tcgtcgttttgtcgttttgtcgtt ODN 2022 SEQ ID NO:2
ggggtcgtcgttttgggggg ODN 2184 SEQ ID NO:3 tcgtcgttttgtcgttttgggggg
ODN 2185 SEQ ID NO:4 ggggtcgacgtcgagggggg ODN 2192 SEQ ID NO:5
ggggtcatcgatgagggggg ODN 2204 SEQ ID NO:6 ggGGGACGATCGTCgggggG ODN
2216 SEQ ID NO:7 gggggtcgtacgacgggggg ODN 2217 SEQ ID NO:8
ggGGGACGATATCGTCgggggG ODN 2245 SEQ ID NO:9 ggGGGACGACGTCGTCgggggG
ODN 2246 SEQ ID NO:10 ggGGGACGAGCTCGTCgggggG ODN 2247 SEQ ID NO:11
ggGGGACGTACGTCgggggG ODN 2248 SEQ ID NO:12 ggGGGACGATCGTTGggggG ODN
2252 SEQ ID NO:13 ggGGAACGATCGTCgggggG ODN 2253 SEQ ID NO:14
ggGGGGACGATCGTCgggggG ODN 2254 SEQ ID NO:15 ggGGGACGATCGTCGgggggG
ODN 2255 SEQ ID NO:16 ggGGGTCATCGATGAgggggG ODN 2260 SEQ ID NO:17
ggGGTCGTCGACGAgggggG ODN 2293 SEQ ID NO:18 ggGGTCGTTCGAACGAgggggG
ODN 2294 SEQ ID NO:19 ggGGACGTTCGAACGTgggggG ODN 2295 SEQ ID NO:20
ggGGAACGACGTCGTTgggggG ODN 2297 SEQ ID NO:21 ggGGAACGTACGTCgggggG
ODN 2298 SEQ ID NO:22 ggGGAACGTACGTACGTTgggggG ODN 2299 SEQ ID
NO:23 ggGGTCACCGGTGAgggggG ODN 2300 SEQ ID NO:24
ggGGTCGACGTACGTCGAgggggG ODN 2301 SEQ ID NO:25
ggGGACCGGTACCGGTgggggG ODN 2302 SEQ ID NO:26 ggGTCGACGTCGAgggggG
ODN 2303 SEQ ID NO:27 ggGGTCGACGTCGagggg ODN 2304 SEQ ID NO:28
ggGGAACGTTAACGTTgggggG ODN 2305 SEQ ID NO:29 ggGGACGTCGACGTggggG
ODN 2306 SEQ ID NO:30 ggGGGTCGTTCGTTgggggG ODN 2311 SEQ ID NO:31
ggGACGATCGTCGgggggG ODN 2328 SEQ ID NO:32 ggGTCGTCGACGAggggggG ODN
2329 SEQ ID NO:33 ggTCGTCGACGAGgggggG ODN 2330 SEQ ID NO:34
ggGGACGATCGTCGgggggG ODN 2332 SEQ ID NO:35
ggGGTCGACGTCGACGTCGAGgggggG, ODN 2334 SEQ ID NO:36 and
ggGGACGACGTCGTGgggggG, ODN 2336 SEQ ID NO:37
[0047] wherein each lower case letter represents phosphorothioate
linkage and each upper case letter indicates phosphodiester
linkage, plus a pharmaceutically acceptable carrier. In some
embodiments the pharmaceutical composition also contains
IFN-.alpha..
[0048] According to another aspect of the invention, an interferon
composition for administration to a subject is provided. The
composition includes interferon in a container for administration
to a subject. The amount of the interferon in the container is at
least about 10 percent less than the maximum tolerated dose (MTD).
Preferably the amount of interferon in the container is at least
about 20 percent below the MTD, at least 30 percent below the MTD,
at least 40 percent below the MTD, or even at least 50 percent
below the MTD. The container also can include an ISNA.
[0049] In still another aspect of the invention, kits for
administration of interferon and an ISNA to a subject are provided.
The kits include a container containing a composition which
includes IFN-.alpha. and instructions for administering the
interferon to a subject in need of such treatment in an amount
which is at least about 10 percent less than the MTD, 20 percent
less than the MTD, 30 percent less than the MTD, 40 percent less
than the MTD, or 50 percent less than the MTD. The kit can include,
in the same container or in a separate container, an ISNA. The kit
also can include instructions for treating a subject with a
condition susceptible to treatment with IFN-.alpha..
[0050] Each of the limitations of the invention can encompass
various embodiments of the invention. It is, therefore, anticipated
that each of the limitations of the invention involving any one
element or combinations of elements can be included in each aspect
of the invention.
[0051] These and other aspects of the invention are described in
greater detail below.
BRIEF DESCRIPTION OF THE FIGURES
[0052] FIG. 1 depicts FACS analyses of cell populations during the
isolation and characterization of IPCs performed with magnetic
beads and flow cytometry. From left to right are shown: selection
of lin-/MHC class II+ cells from PBMCs; further selection of
CD123+/MHC class II+ cells from lin-/CD4+/MHC class II+ cells; and
characterization of freshly isolated lin-/CD4+/MHC class II+/CD123+
IPCs as CD80-.
[0053] FIG. 2 depicts FACS analyses of survival and activation
(CD80) of freshly isolated IPCs after incubation for two days in
the presence of selected growth factors and stimuli. Growth factor
(GM-CSF) and/or stimulus (CpG oligonucleotide or LPS) for each
panel: top left, none; top middle, CpG oligonucleotide; top right,
LPS; bottom left, GM-CSF; and bottom middle, GM-CSF plus CpG
oligonucleotide. The number in the upper right corner of each panel
is the mean fluorescence intensity (MFI) for CD80. Results are
representative of five independent experiments.
[0054] FIG. 3 depicts FACS analyses showing the different effects
CpG and poly IC have on the survival and activation of freshly
isolated IPCs. All cells were cultured for three days in the
presence of IL-3. Cells were then cultured for an additional 24
hours with the addition of: nothing (left panels); CpG (middle
panels); or poly IC (right panels). MFI for CD80 is shown in the
top right of each of the bottom panels. Results are representative
of three independent experiments.
[0055] FIG. 4 is a graph depicting the concentration of IFN-.alpha.
(determined by IFN-.alpha.-specific ELISA) present in the
supernatant of IPCs cultured for two days in the presence of IL-3
and GM-CSF, either with CpG oligonucleotide (solid bar) or without
CpG oligonucleotide (open bar). Results are representative of three
independent experiments.
[0056] FIG. 5 is a graph depicting the concentration of IFN-.alpha.
induced in the supernatants of PBMC from different donors following
incubation for 48 hours in the presence of 3 .mu.M ODN 2006 (n=7),
1585 (n=7), 2197 (n=6), 2198 (n=5), or media without added ODN
(n=7). Error bars indicate SEM.
[0057] FIG. 6 is a graph depicting the dose-response of CpG
ODN-induced IFN-.alpha. synthesis by PBMC cultured for 48 hours in
the presence of ODN 2216, 1585, 2006, and 2243 at concentrations
ranging from 0.2 to 12 .mu.g/ml.
[0058] FIG. 7 is a graph depicting CpG ODN-mediated stimulation of
IFN-.alpha. and IFN-.beta. production in PBMC enriched for
plasmacytoid dendritic cells, with (n=3) and without (n=4) addition
of lipofectin (10 .mu.g/ml). PBMC were cultured for 48 hours in the
presence of IL-3 alone (-) or IL-3 with the addition of ODN 2006,
1585, 2197, or 2216. Results are presented as means of 3 or 4
independent experiments with different donors, each performed in
duplicate. Error bars indicate SEM. *p<0.0018 (Bonferroni-Dunn
correction).
[0059] FIG. 8 is a series of four graphs depicting results of four
FACS experiments examining intracellular IFN-.alpha.. Panel A,
identification of lin+ and lin.sup.- cells. Panel B, identification
of CD123.sup.+/-/HLA DR.sup.++ mDC (gate II) and CD123.sup.++/HLA
DR.sup.+ pDC (gate III) in lin.sup.- cells. Panel C, lack of
staining for intracellular IFN-.alpha. in lin.sup.+ cells. Panel D,
staining for intracellular IFN-.alpha. in lin.sup.- cells.
[0060] FIG. 9 is a series of six graphs depicting results of six
FACS experiments examining intracellular IFN-.alpha. (Panels A) and
TNF-.alpha. (Panels B) in lin.sup.-/HLA DR.sup.+ plasmacytoid
dendritic cell precursor cells after stimulation with different CpG
oligonucleotides (2006, 2216, both at 3 .mu.g/ml). Brefeldin A was
added during incubation for TNF-.alpha.. MFI, mean fluorescence
intensity.
[0061] FIG. 10 is a graph depicting CD86 expression on plasmacytoid
dendritic cells in response to IL-3 alone (-) or to IL-3 with
various CpG ODN (2006, 1585, 2197, or 2216, each at 3 .mu.g/ml).
Results are presented as means of three independent experiments
with cells from different donors. Error bars indicate SEM.
*p<0.0018 (Bonferroni-Dunn correction).
[0062] FIG. 11 depicts a graph representing a FACS purification of
plasmacytoid dendritic cells (Panel A) and the secretion of
IFN-.alpha. and IFN-.beta. by purified plasmacytoid dendritic cells
in response to IL-3 with and without ODN 2216 (Panel B).
[0063] FIG. 12 depicts NK cell-mediated lysis of K562 cells
following exposure of PBMC to ODN 2216, 1585, 2006, 2118, IL-2, or
media alone.
[0064] FIG. 13 is a graph depicting the concentration of IL-8
(determined by an IL-8-specific ELISA) present in the supernatant
of IPCs cultured for two days in the presence of IL-3 alone (left),
IL-3 supplemented with CpG oligonucleotide (middle), or IL-3
supplemented with poly IC (right). Results are representative of
three independent experiments.
[0065] FIG. 14 is a graph depicting IFN-.gamma. production by
.gamma..delta. T cells in response to CpG ODN 2006, 1585, or 2216
in the presence or absence of nonpeptide antigen isopentenyl
pyrophosphate (IPP). Results are shown in terms of mean
fluorescence intensity (MFI) for intracellular staining for
IFN-.gamma., with medium alone as negative control. Data are
presented as mean+SEM; * (p<0,01) and ** (p<0,001) indicate p
values calculated by Student's t-test for paired samples comparing
medium control to CpG ODN and IPP alone to IPP+CpG ODN.
[0066] FIG. 15 is a pair of graphs depicting proliferation of
.gamma..delta. T cells in response to CpG ODN 2006, 1585, or 2216
in the presence or absence of nonpeptide antigen isopentenyl
pyrophosphate (IPP). Panel A depicts the kinetics of .gamma..delta.
T cell expansion, over 10 days, from one representative experiment.
Panel B depicts the expansion of .gamma..delta. T cells 10 days
after stimulation with IPP alone or in combination with different
CpG ODN. Between 9 and 16 donors were analyzed for each ODN. Data
are presented as x-fold increase compared to IPP alone (mean+SEM);
* indicates p<0,05 (IPP versus IPP+CpG ODN.
[0067] FIG. 16 is a graph depicting regulation of CD40-induced
IL-12p70 production by type I IFN and by various CpG ODN. Data are
shown as x-fold of IL-12p70 production by anti-CD40 alone (mean=143
pg/ml) and represent the mean+SEM of three different donors.
[0068] FIG. 17 is a series of graphs depicting the effects of CpG
ODN 2006, 1585, and 2216 on recall and primary peptide-specific
human CTL responses. Panels A and C, peptide-specific IFN-.gamma.
producing CTL as a percentage of all CD8.sup.+ T cells for the
recall antigen influenza-matrix peptide and for the primary antigen
melan-A/mart-1 peptide, respectively.
[0069] Panels B and D, antigen-specific tetramer-positive staining
CD8.sup.+ T cells for the recall antigen influenza-matrix peptide
and for the primary antigen melan-A/mart-1 peptide,
respectively.
[0070] FIG. 18 is a schematic representation of a kit which
includes a container containing a composition which includes
IFN-.alpha. in an amount which is at least about 10 percent less
than the maximum tolerated dose (MTD) and, in the same container or
in a separate container, an ISNA. The kit also can include
instructions for treating a subject with a condition susceptible to
treatment with IFN-.alpha..
DETAILED DESCRIPTION OF THE INVENTION
[0071] The invention involves the discovery that a particular
subset of blood cells, natural IFN-producing cells (IPCs), are
stimulated by ISNAs to produce IFN-.alpha.. This discovery was
surprising because it was previously unknown what component of
UV-irradiated virus or of heat-killed bacteria was responsible for
inducing IFN-.alpha. production by IPCs. Siegal F P et al. Science
284:1835-7 (1999). The discovery was also surprising because mature
DC2s, which arise from the IPCs, are not strong producers of
IFN-.alpha.. Furthermore, it was also known that monocyte-derived
dendritic cells (DC1s) do not produce IFN-.alpha. in response to
CpG nucleic acids. It also was surprising that a broad array of
IFN-.alpha. molecules is stimulated. In addition, the invention
involves the local induction of IFN-.alpha. at the site of ISNA
administration, thus avoiding toxic effects associated with
systemic administration of IFN-.alpha. in doses necessary to
achieve similar local concentration of IFN-.alpha.. The invention
also involves the unexpected discovery that ISNAs can stimulate
IFN-producing cells to activate them to express the costimulatory
molecule CD80 (B7-1). Another unexpected discovery is that ISNAs
can support the survival of IFN-producing cells even in the absence
of interleukin-3. These various discoveries have led to the in
vivo, ex vivo and in vitro inventions described herein.
[0072] An ISNA is a nucleic acid molecule which, upon contacting
cells of the immune system, is itself capable of inducing contacted
cells of the immune system to proliferate and/or to become
activated. The contacting can be direct or indirect, e.g., the ISNA
may directly stimulate a first type of immune cell to express a
product which may in turn stimulate a second type of immune cell
which has not been exposed to, or is not responsive to, the ISNA.
The immunostimulatory effect of the ISNA is separate from any
product that might happen to be encoded by the sequence of the
ISNA. Similarly, the immunostimulatory effect of an ISNA is
distinct from and does not rely upon any antisense mechanism. Only
certain nucleic acids are ISNAs. Originally it was believed that
certain palindromic sequences were immunostimulatory. Tokunaga T et
al. Microbiol Immunol 36:55-66 (1992); Yamamoto T et al. Antisense
Res Dev 4:119-22 (1994). Further work demonstrated that
non-palindromic sequences are also immunostimulatory provided they
contained CpG dinucleotides within particular sequence contexts
(CpG motifs). Krieg A M et al. Nature 374:546-9 (1995). The ISNAs
can be single-stranded or double-stranded. Generally,
double-stranded nucleic acid molecules are more stable in vivo,
while single-stranded nucleic acid molecules have increased immune
activity. Thus in some aspects of the invention it is preferred
that the ISNA be single-stranded and in other aspects it is
preferred that the ISNA be double-stranded.
[0073] The terms "nucleic acid" and "oligonucleotide" are used
interchangeably to mean multiple, covalently linked nucleotides,
wherein each nucleotide comprises a sugar (e.g., ribose or
deoxyribose) linked to a phosphate group and to an exchangeable
organic base, which is either a substituted pyrimidine (e.g.,
cytosine (C), thymine (T) or uracil (U)) or a substituted purine
(e.g., adenine (A) or guanine (G).
[0074] As used herein, the terms "nucleic acid" and
"oligonucleotide" refer to oligoribonucleotides as well as
oligodeoxyribonucleotides. The terms shall also include
polynucleosides (i.e., a polynucleotide minus the phosphate) and
any other organic base-containing polymer. Nucleic acid molecules
can be obtained from existing nucleic acid sources (e.g., genomic
DNA or cDNA), but are preferably synthetic (e.g., produced by
oligonucleotide synthesis).
[0075] The terms "nucleic acid" and "oligonucleotide" also
encompass nucleic acids or oligonucleotides with a covalently
modified base and/or sugar. For example, they include nucleic acids
having backbone sugars which are covalently attached to low
molecular weight organic groups other than a hydroxyl group at the
3' position and other than a phosphate group at the 5' position.
Thus modified nucleic acids may include a 2'-O-alkylated ribose
group. In addition, modified nucleic acids may include sugars such
as arabinose instead of ribose. Thus the nucleic acids may be
heterogeneous in backbone composition thereby containing any
possible combination of polymer units linked together such as
peptide nucleic acids (which have an amino acid backbone with
nucleic acid bases). In some embodiments the nucleic acids are
homogeneous in backbone composition.
[0076] Nucleic acids also can include base analogs such as C-5
propyne modified bases. Wagner et al. Nature Biotechnology
14:840-844 (1996). Purines and pyrimidines include but are not
limited to adenine, cytosine, guanine, thymine, 5-methylcytosine,
2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine,
hypoxanthine, and other naturally and non-naturally occurring
nucleobases, substituted and unsubstituted aromatic moieties.
[0077] The nucleic acid is a linked polymer of bases, nucleobase
analogs, or nucleotides. As used herein with respect to linked
units of a nucleic acid, "linked" or "linkage" means two entities
are bound to one another by any physicochemical means. Any linkage
known to those of ordinary skill in the art, covalent or
non-covalent, is embraced. Such linkages are well known to those of
ordinary skill in the art. Natural linkages, which are those
ordinarily found in nature connecting the individual units of a
nucleic acid, are most common. The individual units of a nucleic
acid may be linked, however, by synthetic or modified linkages.
[0078] A CpG oligonucleotide is an oligonucleotide which includes
at least one unmethylated CpG dinucleotide. An oligonucleotide
containing at least one unmethylated CpG dinucleotide is a nucleic
acid molecule which contains an unmethylated cytosine-guanine
dinucleotide sequence (i.e., "CpG DNA" or DNA containing a 5'
cytosine followed by 3' guanine and linked by a phosphate bond) and
activates the immune system. The entire CpG oligonucleotide can be
unmethylated or portions may be unmethylated but at least the C of
the 5' CG 3' must be unmethylated. The CpG oligonucleotides can be
double-stranded or single-stranded. The terms CpG oligonucleotide
or CpG nucleic acid as used herein refer to an immunostimulatory
CpG oligonucleotide or a nucleic acid unless otherwise
indicated.
[0079] In one preferred embodiment the invention provides a CpG
oligonucleotide represented by at least the formula:
5' X.sub.1X.sub.2CGX.sub.3X.sub.4 3'
[0080] wherein X.sub.1, X.sub.2, X.sub.3, and X.sub.4 are
nucleotides. In one embodiment X.sub.2 is adenine, guanine, or
thymine. In another embodiment X.sub.3 is cytosine, adenine, or
thymine.
[0081] In another embodiment the invention provides an isolated CpG
oligonucleotide represented by at least the formula:
5' N.sub.1X.sub.1X.sub.2CGX.sub.3X.sub.4N.sub.2 3'
[0082] wherein X.sub.1, X.sub.2, X.sub.3, and X.sub.4 are
nucleotides and N is any nucleotide and N.sub.1 and N.sub.2 are
nucleic acid sequences composed of from about 0-25 N's each. In one
embodiment X.sub.1X.sub.2 is a dinucleotide selected from the group
consisting of: GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA,
TpT, and TpG; and X.sub.3X.sub.4 is a dinucleotide selected from
the group consisting of: TpT, ApT, TpG, ApG, CpG, TpC, ApC, CpC,
TpA, ApA, and CpA. Preferably X.sub.1X.sub.2 is GpA or GpT and
X.sub.3X.sub.4 is TpT. In other embodiments X.sub.1 or X.sub.2 or
both are purines and X.sub.3 or X.sub.4 or both are pyrimidines or
X.sub.1X.sub.2 is GpA and X.sub.3 or X.sub.4 or both are
pyrimidines. In another preferred embodiment X.sub.1X.sub.2 is a
dinucleotide selected from the group consisting of: TpA, ApA, ApC,
ApG, and GpG. In yet another embodiment X.sub.3X.sub.4 is a
dinucleotide selected from the group consisting of: TpT, TpA, TpG,
ApA, ApG, GpA, and CpA. X.sub.1X.sub.2 in another embodiment is a
dinucleotide selected from the group consisting of: TpT, TpG, ApT,
GpC, CpC, CpT, TpC, GpT and CpG; X.sub.3 is a nucleotide selected
from the group consisting of A and T and X.sub.4 is a nucleotide,
but wherein when X.sub.1X.sub.2 is TpC, GpT, or CpG, X.sub.3X.sub.4
is not TpC, ApT or ApC.
[0083] In another preferred embodiment the CpG oligonucleotide has
the sequence 5' TCN.sub.1TX.sub.1X.sub.2CGX.sub.3X.sub.4 3'. The
CpG oligonucleotides of the invention in some embodiments include
X.sub.1X.sub.2 selected from the group consisting of GpT, GpG, GpA
and ApA and X.sub.3X.sub.4 is selected from the group consisting of
TpT, CpT and TpC.
[0084] For facilitating uptake into cells, ISNAs, including
CpG-containing oligonucleotides, are preferably in the range of 8
to 100 bases in length. However, nucleic acids of any size greater
than 8 nucleotides (even many kb long) are capable of inducing an
immune response according to the invention if sufficient
immunostimulatory motifs are present, since larger nucleic acids
are degraded into oligonucleotides inside of cells. Preferably the
ISNA is in the range of between 8 and 100 nucleotides in length. In
some preferred embodiments the ISNA is between 12 and 40
nucleotides in length. In more preferred embodiments the ISNA is
between 8 and 30 nucleotides in length. In most preferred
embodiments the ISNA is between 8 and 24 nucleotides in length.
[0085] "Palindromic sequence" shall mean an inverted repeat, i.e.,
a sequence such as ABCDEE'D'C'B'A' in which A and A', B and B', C
and C', D and D', and E and E' are bases capable of forming the
usual Watson-Crick base pairs. In vivo, such palindromic sequences
may form double-stranded structures. In one embodiment the CpG
oligonucleotide contains a palindromic sequence. A palindromic
sequence used in this context refers to a palindrome in which the
CpG is part of the palindrome, and preferably is the center of the
palindrome. In another embodiment the CpG oligonucleotide is free
of a palindrome. A CpG oligonucleotide that is free of a palindrome
is one in which the CpG dinucleotide is not part of a palindrome.
Such an oligonucleotide may include a palindrome in which the CpG
is not the center of the palindrome.
[0086] The CpG nucleic acid sequences of the invention are those
broadly described above as well as disclosed in PCT Published
Patent Applications PCT/US95/01570 and PCT/US97/19791 claiming
priority to U.S. Ser. Nos. 08/386,063 and 08/960,774, filed on Feb.
7, 1995 and Oct. 30, 1997 respectively. Exemplary sequences include
but are not limited to those immunostimulatory sequences shown in
Table 3 and Table 5.
8TABLE 3 Exemplary CpG ISNAs AACGTTCT SEQ ID NO:38
ACCATGGACGAACTGTTTCCCCTC SEQ ID NO:39 ACCATGGACGACCTGTTTCCCCTC SEQ
ID NO:40 ACCATGGACGAGCTGTTTCCCCTC SEQ ID NO:41
ACCATGGACGATCTGTTTCCCCTC SEQ ID NO:42 ACCATGGACGGTCTGTTTCCCCTC SEQ
ID NO:43 ACCATGGACGTACTGTTTCCCCTC SEQ ID NO:44
ACCATGGACGTTCTGTTTCCCCTC SEQ ID NO:45 AGCTATGACGTTCCAAGG SEQ ID
NO:46 ATAGGAGGTCCAACGTTCTC SEQ ID NO:47 ATCGACTCTCGAACGTTCTC SEQ ID
NO:48 ATCGACTCTCGAGCGTTCTC SEQ ID NO:49 ATGACGTTCCTGACGTT SEQ ID
NO:50 ATGGAAGGTCCAACGTTCTC SEQ ID NO:51 ATGGAAGGTCCAGCGTTCTC SEQ ID
NO:52 ATGGACTCTCCAGCGTTCTC SEQ ID NO:53 ATGGAGGCTCCATCGTTCTC SEQ ID
NO:54 CAACGTT SEQ ID NO:55 CACGTTGAGGGGCAT SEQ ID NO:56 CCAACGTT
SEQ ID NO:57 GAGAACGATGGACCTTCCAT SEQ ID NO:58
GAGyAACGCTCCAGCACTGAT SEQ ID NO:59 GAGAACGCTCGACCTTCCAT SEQ ID
NO:60 GAGAACGCTCGACCTTCGAT SEQ ID NO:61 GAGAACGCTGGACCTTCCAT SEQ ID
NO:62 GCATGACGTTGAGCT SEQ ID NO:63 GCGTGCGTTGTCGTTGTCGTT SEQ ID
NO:64 GCTAGACGTTAGCGT SEQ ID NO:65 GCTAGACGTTAGTGT SEQ ID NO:66
GCTAGATGTTAGCGT SEQ ID NO:67 GGGGTCAACGTTGACGGGG SEQ ID NO:68
GGGGTCAGTCGTGACGGGG SEQ ID NO:69 GTCGYT SEQ ID NO:70 TCAACGTC SEQ
ID NO:71 TCAACGTT SEQ ID NO:72 TCAGCGCT SEQ ID NO:73 TCAGCGTGCGCC
SEQ ID NO:74 TCATCGAT SEQ ID NO:75 TCCACGACGTTTTCGACGTT SEQ ID
NO:76 TCCATAACGTTCCTGATGCT SEQ ID NO:77 TCCATAGCGTTCCTAGCGTT SEQ ID
NO:78 TCCATCACGTGCCTGATGCT SEQ ID NO:79 TCCATGACGGTCCTGATGCT SEQ ID
NO:80 TCCATGACGTCCCTGATGCT SEQ ID NO:81 TCCATGACGTGCCTGATGCT SEQ ID
NO:82 TCCATGACGTTCCTGACGTT SEQ ID NO:83 TCCATGACGTFFCCTGATGCT SEQ
ID NO:84 TCCATGCCGGTCCTGATGCT SEQ ID NO:85 TCCATGCGTGCGTGCGTTTT SEQ
ID NO:86 TCCATGCGTTGCGTTGCGTT SEQ ID NO:87 TCCATGGCGGTCCTGATGCT SEQ
ID NO:88 TCCATGTCGATCCTGATGCT SEQ ID NO:89 TCCATGTCGCTCCTGATGCT SEQ
ID NO:90 TCCATGTCGGTCCTGACGCA SEQ ID NO:91 TCCATGTCGGTCCTGATGCT SEQ
ID NO:92 TCCATGTCGGTCCTGCTGAT SEQ ID NO:93 TCCATGTCGTCCCTGATGCT SEQ
ID NO:94 TCCATGTCGTTCCTGTCGTT SEQ ID NO:95 TCCATGTCGTTTTTGTCGTT SEQ
ID NO:96 TCCTGACGTTCCTGACGTT SEQ ID NO:97 TCCTGTCGTTCCTGTCGTT SEQ
ID NO:98 TCCTGTCGTTCCTTGTCGTT SEQ ID NO:99 TCCTGTCGTTTTTTGTCGTT SEQ
ID NO:100 TCCTTGTCGTTCCTGTCGTT SEQ ID NO:101 TCGTCGCTGTCTCCCCTTCTT
SEQ ID NO:102 TCGTCGCTGTCTGCCCTTCTT SEQ ID NO:103
TCGTCGCTGTTGTCGTTTCTT SEQ ID NO:104 TCGTCGTCGTCGTT SEQ ID NO:105
TCGTCGTTGTCGTTGTCGTT SEQ ID NO:106 TCGTCGTTGTCGTTTTGTCGTT SEQ ID
NO:107 TCGTCGTTTTGTCGTTTTGTCGTT SEQ ID NO:108 TCTCCCAGCGGGCGCAT SEQ
ID NO:109 TCTCCCAGCGTGCGCCAT SEQ ID NO:110 TCTTCGAA SEQ ID NO:111
TCTTCGAT SEQ ID NO:112 TGTCGTTGTCGTT SEQ ID NO:113
TGTCGTTGTCGTTGTCGTT SEQ ID NO:114 TGTCGTTGTCGTTGTCGTTGTCGTT SEQ ID
NO:115 TGTCGTTTGTCGTTTGTCGTT SEQ ID NO:116 TGTCGYT SEQ ID
NO:117
[0087] The immunostimulatory nucleic acids of the invention also
include nucleic acids having T-rich motifs. As used herein, a
"T-rich nucleic acid" is a nucleic acid which includes at least one
poly-T sequence and/or which has a nucleotide composition of
greater than 25% T nucleotide residues. A nucleic acid having a
poly-T sequence includes at least four Ts in a row, such as 5' TTTT
3'. Preferably the T-rich nucleic acid includes more than one
poly-T sequence. In preferred embodiments the T-rich nucleic acid
may have 2, 3, 4, etc., poly-T sequences. One of the most highly
immunostimulatory T-rich oligonucleotides is a nucleic acid
composed entirely of T nucleotide residues. Other T-rich nucleic
acids have a nucleotide composition of greater than 25% T
nucleotide residues, but do not necessarily include a poly-T
sequence. In these T-rich nucleic acids the T nucleotide residues
may be separated from one another by other types of nucleotide
residues, i.e., G, C, and A. In some embodiments the T-rich nucleic
acids have a nucleotide composition of greater than 35%, 40%, 50%,
60%, 70%, 80%, 90%, and 99%, T nucleotide residues and every
integer % in between. Preferably the T-rich nucleic acids have at
least one poly-T sequence and a nucleotide composition of greater
than 25% T nucleotide residues.
[0088] T-rich nucleic acids are also described and claimed in U.S.
Ser. No. ______ filed on Sep. 25, 2000, claiming priority to U.S.
Provisional Patent Application No. 60/156,113 filed on Sep. 25,
1999, which is hereby incorporated by reference. Many of the CpG
ODN presented in Table 3 are also T-rich nucleic acids as defined
here.
[0089] A number of references also describe the immunostimulatory
properties of poly-G nucleic acids (defined below). Pisetsky and
Reich (1993) Mol Biol Reports 18:217-221; Krieger and Herz (1994)
Ann Rev Biochem 63:601-637; Macaya et al. (1993) Proc Natl Acad Sci
USA 90:3745-3749; Wyatt et al. (1994) Proc Natl Acad Sci USA
91:1356-1360; Rando and Hogan (1998) In: Applied Antisense
Oligonucleotide Technology, eds. Krieg and Stein, p. 335-352; and
Kimura et al. (1994) J Biochem 116:991-994. Poly-G-containing
oligonucleotides are useful for treating and preventing bacterial
and viral infections.
[0090] It was previously suggested in the prior art that poly-G
rich oligonucleotides inhibit the production of IFN-.beta. by
compounds such as CpG oligonucleotides, concanavalin A, bacterial
DNA, or the combination of phorbol 12-myristate 13-acetate (PMA)
and the calcium ionophore A 23187 (Halperin and Pisetsky (1995)
Immunopharmacol 29:47-52), as well as block the downstream effects
of IFN-.gamma.. For instance, Ramanathan et al. has shown that a
poly-G oligonucleotide inhibits the binding of IFN-.gamma. to its
receptor, which prevents the normal enhancement of MHC class I and
ICAM-1 in response to IFN-.gamma.. Ramanathan et al. (1994)
Transplantation 57:612-615. Poly-G oligonucleotides were also found
to be able to inhibit the secretion of IFN-.gamma. from
lymphocytes. Halperin and Pisetsky (1995) Immunopharmacol
29:47-52.
[0091] Poly-G nucleic acids preferably are nucleic acids having the
following formula:
5' X.sub.1X.sub.2GGGX.sub.3X.sub.4 3'
[0092] wherein X.sub.1, X.sub.2, X.sub.3, and X.sub.4 are
nucleotides. In preferred embodiments at least one of X.sub.3 and
X.sub.4 is a G. In other embodiments both of X.sub.3 and X.sub.4
are G's. In yet other embodiments the preferred formula is 5'
GGGNGGG 3' or 5' GGGNGGGNGGG 3', wherein N represents between 0 and
20 nucleotides. In other embodiments the poly-G nucleic acid is
free of CpG dinucleotides, such as, for example, the nucleic acids
listed in Table 4 as SEQ ID NOs 95-114, 117-121, 123-130, 132, and
133. In other embodiments the poly-G nucleic acid includes at least
one CpG dinucleotide, such as, for example, the nucleic acids
listed in Table 4 as SEQ ID NOs 115, 116, 122, 131, and 134-136.
Particularly preferred ISNAs are SEQ ID NOs 134, 135, and 136.
9TABLE 4 Poly-G ISNAs ATGGAAGGTCCAAGGGGCTC SEQ ID NO:118
ATGGAAGGTCCAGGGGGCTC SEQ ID NO:119 ATGGAAGGTCCGGGGTTCTC SEQ ID
NO:120 ATGGACTCTCCGGGGTTCTC SEQ ID NO:121 ATGGACTCTGGAGGGGGCTC SEQ
ID NO:122 ATGGACTCTGGAGGGGTCTC SEQ ID NO:123 ATGGACTCTGGGGGGTTCTC
SEQ ID NO:124 ATGGAGGCTCCATGGGGCTC SEQ ID NO:125
GAGAAGGGGCCAGCACTGAT SEQ ID NO:126 GAGAAGGGGGGACCTTCCAT SEQ ID
NO:127 GAGAAGGGGGGACCTTGGAT SEQ ID NO:128 GCATGAGGGGGAGCT SEQ ID
NO:129 GCTAGAGGGAGTGT SEQ ID NO:130 GCTAGAGGGGAGGGT SEQ ID NO:131
GCTAGATGTTAGGGG SEQ ID NO:132 GGGGGACGATCGTCGGGGGG SEQ ID NO:133
GGGGGGGGGGGGGGGGGGGG SEQ ID NO:134 GGGGTCAACGTTGAGGGGGG SEQ ID
NO:135 GGGGTCGACGTCGAGGGGGG SEQ ID NO:136 TCCATCGGGGGCCTGATGCT SEQ
ID NO:137 TCCATGAGGGGCCTGATGCT SEQ ID NO:138 TCCATGCGGGTGGGGATGCT
SEQ ID NO:139 TCCATGGGGGTCCTGATGCT SEQ ID NO:140
TCCATGGGGTCCCTGATGCT SEQ ID NO:141 TCCATGGGGTGCCTGATGCT SEQ ID
NO:142 TCCATGGGGTTCCTGATGCT SEQ ID NO:143 TCCATGTGGGGCCTGATGCT SEQ
ID NO:144 TCCATGTGGGGCCTGCTGAT SEQ ID NO:145 TCCATGTGGGTGGGGATGCT
SEQ ID NO:146
[0093] More generally, ISNAs of the invention can include any
combination of at least two types of ISNAs, including CpG nucleic
acids, T-rich nucleic acids, and poly-G nucleic acids. Such
combinations can occur in the form of chimeric nucleic acids, in
which the at least two types of ISNA are represented in a single
nucleic acid molecule.
[0094] In addition, at least two individual nucleic acid molecules
with different sequences and/or different types of ISNA, can be
used together. The at least two individual nucleic acid molecules
used together can represent a single type or at least two types of
ISNA.
[0095] A preferred composition for inducing IFN-.alpha. is a
composition including an oligonucleotide having a phosphate
modification at the 3' and 5' ends of the molecule with a
phosphodiester central region. This preferred molecule is
exemplified by the following formula:
5' Y.sub.1N.sub.1CGN.sub.2Y.sub.2 3'
[0096] wherein Y.sub.1 and Y.sub.2 are, independent of one another,
nucleic acid molecules having between 1 and 10 nucleotides, and
wherein Y.sub.1 includes at least one modified intemucleotide
linkage and Y.sub.2 includes at least one modified internucleotide
linkage and wherein N.sub.1 and N.sub.2 are nucleic acid molecules,
each independent of one another having between 0 and 20 nucleotides
and in some embodiments, between 3 and 8 nucleotides, but wherein
N.sub.1CGN.sub.2 has at least 6 nucleotides in total and wherein
the nucleotides of N.sub.1CGN.sub.2 have a phosphodiester backbone.
Oligonucleotides having one or more phosphorothioate-modified
intemucleotide linkages with a central region having one or more
phosphodiester intemucleotide linkages demonstrated unexpectedly
high ability to induce IFN-.alpha.. The activity of these
oligonucleotides was particularly high when the first two and last
five intemucleotide linkages include phosphate modifications and/or
the oligonucleotide included poly-G ends.
[0097] Y.sub.1 and Y.sub.2 are considered independent of one
another. This means that each of Y.sub.1 and Y.sub.2 may or may not
have different sequences and different backbone linkages from one
another in the same molecule. The sequences vary, but in some cases
Y.sub.1 and Y.sub.2 have a poly-G sequence. A poly-G sequence
refers to at least 3 Gs in a row. In other embodiments the poly-G
sequence refers to at least 4, 5, 6, 7, or 8 Gs in a row.
[0098] In some embodiments Y.sub.1 and Y.sub.2 have between 3 and 8
or between 4 and 7 nucleotides. At least one of these nucleotides
includes a modified intemucleotide linkage. In some embodiments
Y.sub.1 and Y.sub.2 include at least two modified intemucleotide
linkages, and in other embodiments Y.sub.1 and Y.sub.2 include
between two and five modified intemucleotide linkages. In yet other
embodiments Y.sub.1 has two modified intemucleotide linkages and
Y.sub.2 has five modified intemucleotide linkages. In other
embodiments Y.sub.1 has five modified intemucleotide linkages and
Y.sub.2 has two modified intemucleotide linkages.
[0099] Exemplary preferred ISNAs of the invention for inducing
secretion of type I IFN are shown in Table 5 below with lower case
letters indicating phosphorothioate linkages and upper case letters
indicating phosphodiester linkages.
10TABLE 5 Exemplary Preferred ISNAs for Inducing Type I IFN
ggGGTCAACGTTGAgggggG ODN 1585 SEQ ID NO:1 tcgtcgttttgtcgttttgtcgtt
ODN 2022 SEQ ID NO:2 ggggtcgtcgttttgggggg ODN 2184 SEQ ID NO:3
tcgtcgttttgtcgttttgggggg ODN 2185 SEQ ID NO:4 ggggtcgacgtcgagggggg
ODN 2192 SEQ ID NO:5 ggggtcatcgatgagggggg ODN 2204 SEQ ID NO:6
ggGGGACGATCGTCgggggG ODN 2216 SEQ ID NO:7 gggggtcgtacgacgggggg ODN
2217 SEQ ID NO:8 ggGGGACGATATCGTCgggggG ODN 2245 SEQ ID NO:9
ggGGGACGACGTCGTCgggggG ODN 2246 SEQ ID NO:10 ggGGGACGAGCTCGTCgggggG
ODN 2247 SEQ ID NO:11 ggGGGACGTACGTCgggggG ODN 2248 SEQ ID NO:12
ggGGGACGATCGTTGggggG ODN 2252 SEQ ID NO:13 ggGGAACGATCGTCgggggG ODN
2253 SEQ ID NO:14 ggGGGGACGATCGTCgggggG ODN 2254 SEQ ID NO:15
ggGGGACGATCGTCGgggggG ODN 2255 SEQ ID NO:16 ggGGGTCATCGATGAgggggG
ODN 2260 SEQ ID NO:17 ggGGTCGTCGACGAgggggG ODN 2293 SEQ ID NO:18
ggGGTCGTTCGAACGAgggggG ODN 2294 SEQ ID NO:19 ggGGACGTTCGAACGTgggggG
ODN 2295 SEQ ID NO:20 ggGGAACGACGTCGTTgggggG ODN 2297 SEQ ID NO:21
ggGGAACGTACGTCgggggG ODN 2298 SEQ ID NO:22 ggGGAACGTACGTACGTTgggggG
ODN 2299 SEQ ID NO:23 ggGGTCACCGGTGAgggggG ODN 2300 SEQ ID NO:24
ggGGTCGACGTACGTCGAgggggG ODN 2301 SEQ ID NO:25
ggGGACCGGTACCGGTgggggG ODN 2302 SEQ ID NO:26 ggGTCGACGTCGAgggggG
ODN 2303 SEQ ID NO:27 ggGGTCGACGTCGagggg ODN 2304 SEQ ID NO:28
ggGGAACGTTAACGTTgggggG ODN 2305 SEQ ID NO:29 ggGGACGTCGACGTggggG
ODN 2306 SEQ ID NO:30 ggGGGTCGTTCGTTgggggG ODN 2311 SEQ ID NO:31
ggGACGATCGTCGgggggG ODN 2328 SEQ ID NO:32 ggGTCGTCGACGAggggggG ODN
2329 SEQ ID NO:33 ggTCGTCGACGAGgggggG ODN 2330 SEQ ID NO:34
ggGGACGATCGTCGgggggG ODN 2332 SEQ ID NO:35
ggGGTCGACGTCGACGTCGAGgggggG, ODN 2334 SEQ ID NO:36 and
ggGGACGACGTCGTGgggggG. ODN 2336 SEQ ID NO:37
[0100] For use in the instant invention, the nucleic acids can be
synthesized de novo using any of a number of procedures well known
in the art. For example, the nucleic acids can be synthesized using
the .beta.-cyanoethyl phosphoramidite method (Beaucage SL and
Caruthers M H Tetrahedron Lett 22:1859 (1981)) or the nucleoside
H-phosphonate method (Garegg et al. Tetrahedron Lett 27:4051
(1986); Froehler et al. Nucl Acid Res 14:5399 (1986); Garegg et al.
Tetrahedron Lett 27:4055 (1986); Gaffney et al. Tetrahedron Lett
29:2619 (1988)). These chemistries can be performed by a variety of
automated oligonucleotide synthesizers available in the market.
These oligonucleotides are referred to as synthetic
oligonucleotides. Alternatively, ISNAs can be produced on a large
scale in plasmids, (see Sambrook, T., et al., "Molecular Cloning: A
Laboratory Manual", Cold Spring Harbor Laboratory Press, New York,
1989) and separated into smaller pieces or administered whole.
Oligonucleotides can be prepared from existing nucleic acid
sequences (e.g., genomic DNA or cDNA) using known techniques, such
as those employing restriction enzymes, exonucleases or
endonucleases. Oligonucleotides prepared in this manner are
referred to as isolated oligonucleotides. The term ISNA encompasses
both synthetic and isolated immunostimulatory nucleic acids.
[0101] For use in vivo, ISNAs are preferably relatively resistant
to degradation (e.g., are stabilized). A "stabilized nucleic acid
molecule" shall mean a nucleic acid molecule that is relatively
resistant to in vivo degradation (e.g., via an exo- or
endo-nuclease). Stabilization can be a function of length or
secondary structure. For example, ISNAs that are tens to hundreds
of kbs long are relatively resistant to in vivo degradation. For
shorter ISNAs, secondary structure can stabilize and increase their
effect. For example, if the 3' end of an oligonucleotide has
self-complementarity to an upstream region, so that it can fold
back and form a sort of stem loop structure, then the
oligonucleotide becomes stabilized and therefore exhibits more
activity.
[0102] Alternatively, nucleic acid stabilization can be
accomplished via phosphate backbone modifications. Preferred
stabilized oligonucleotides of the instant invention have a
modified backbone. It has been demonstrated that modification of
the oligonucleotide backbone provides enhanced activity of the
ISNAs when administered in vivo. These stabilized structures are
preferred because the ISNAs of the invention have at least a
partial modified backbone. For example, CpG oligonucleotides of a
given sequence which include at least two phosphorothioate linkages
at the 5' end of the oligonucleotide and multiple phosphorothioate
linkages at the 3' end, preferably five, provide maximal activity
and protect the oligonucleotide from degradation by intracellular
exo- and endo-nucleases. Other modified oligonucleotides include
phosphodiester modified oligonucleotides, combinations of
phosphodiester and phosphorothioate oligonucleotide,
methylphosphonate, methylphosphorothioate, phosphorodithioate, and
combinations thereof. Each of these combinations and their
particular effects on immune cells is discussed in more detail in
PCT Published Patent Applications PCT/US95/01570 and PCT/US97/19791
claiming priority to U.S. Ser. Nos. 08/386,063 and 08/960,774,
filed on Feb. 7, 1995 and Oct. 30, 1997, respectively, the entire
contents of which is hereby incorporated by reference. It is
believed that these modified backbone oligonucleotides may show
more stimulatory activity due to enhanced nuclease resistance,
increased cellular uptake, increased protein binding, and/or
altered intracellular localization.
[0103] Modified backbones such as phosphorothioates may be
synthesized using automated techniques employing either
phosphoramidate or H-phosphonate chemistries. Aryl- and
alkyl-phosphonates can be made, e.g., as described in U.S. Pat. No.
4,469,863; and alkylphosphotriesters (in which the charged oxygen
moiety is alkylated as described in U.S. Pat. No. 5,023,243 and
European Patent No. 092,574) can be prepared by automated solid
phase synthesis using commercially available reagents. Methods for
making other DNA backbone modifications and substitutions have been
described. Uhlmann E and Peyman A Chem Rev 90:544 (1990); Goodchild
J Bioconjugate Chem 1:165 (1990).
[0104] Other stabilized oligonucleotides include: nonionic DNA
analogs, such as alkyl- and aryl-phosphates (in which the charged
phosphonate oxygen is replaced by an alkyl or aryl group), and
alkylphosphodiester and alkylphosphotriesters (in which the charged
oxygen moiety is alkylated). Oligonucleotides which contain diol,
such as tetraethyleneglycol or hexaethyleneglycol, at either or
both termini have also been shown to be substantially resistant to
nuclease degradation.
[0105] In some embodiments the ISNAs useful according to the
invention are S and R chiral ISNAs. An "S chiral ISNA" as used
herein is an ISNA wherein at least two nucleotides have a backbone
modification forming a chiral center and wherein a plurality of the
chiral centers have S chirality. An "R chiral ISNA" as used herein
is an ISNA wherein at least two nucleotides have a backbone
modification forming a chiral center and wherein a plurality of the
chiral centers have R chirality. The backbone modification may be
any type of modification that forms a chiral center. The
modifications include but are not limited to phosphorothioate,
phosphorodithioate, methylphosphonate, methylphosphorothioate, and
combinations thereof.
[0106] The chiral ISNAs must have at least two nucleotides within
the oligonucleotide that have a backbone modification. All or less
than all of the nucleotides in the oligonucleotides, however, may
have a modified backbone. Of the nucleotides having a modified
backbone (referred to as chiral centers), a plurality have a single
chirality, S or R. A "plurality" as used herein refers to an amount
greater than 50 percent. Thus, less than all of the chiral centers
may have S or R chirality as long as a plurality of the chiral
centers have S or R chirality. In some embodiments at least 55
percent, 60 percent, 65 percent, 70 percent, 75 percent, 80
percent, 85 percent, 90 percent, 95 percent, or 100 percent of the
chiral centers have S or R chirality. In other embodiments at least
55 percent, 60 percent, 65 percent, 70 percent, 75 percent, 80
percent, 85 percent, 90 percent, 95 percent, or 100 percent of the
nucleotides have backbone modifications.
[0107] The S and R chiral ISNAs may be prepared by any method known
in the art for producing chirally pure oligonucleotides. Many
references teach methods for producing stereopure phosphorothioate
oligodeoxynucleotides using an oxathiaphospholane method have been
published. Stec W J et al. J Am Chem Soc 117:12019 (1995). Other
methods for making chirally pure oligonucleotides have been
described by companies such as ISIS Pharmaceuticals. U.S. Patents
have also described these methods. For instance, U.S. Pat. Nos.
5,883,237; 5,837,856; 5,599,797; 5,512,668; 5,856,465; 5,359,052;
5,506,212; 5,521,302; and 5,212,295, each of which is hereby
incorporated by reference in its entirety, disclose methods for
generating stereopure oligonucleotides.
[0108] A "subject" shall mean a human or vertebrate animal
including but not limited to a dog, cat, horse, cow, pig, sheep,
goat, chicken, non-human primate (e.g., monkey), fish (aquaculture
species, e.g., salmon), rabbit, rat, and mouse.
[0109] A "subject having a proliferative disorder" is a subject
that has detectable and unwanted proliferating cells. The unwanted
proliferating cell can be cancerous cells in a subject with cancer.
The cancer may be a malignant or non-malignant cancer. Cancers or
tumors include but are not limited to biliary tract cancer; bladder
cancer; brain cancer; breast cancer; cervical cancer;
choriocarcinoma; colon cancer; endometrial cancer; esophageal
cancer; gastric cancer; intraepithelial neoplasms; leukemia; liver
cancer; lung cancer (e.g., small cell and non-small cell);
lymphoma; melanoma; multiple myeloma; neuroblastomas; oral cancer;
ovarian cancer; pancreas cancer; prostate cancer; rectal cancer;
renal cancer, sarcomas; skin cancer; stomach cancer; testicular
cancer; and thyroid cancer; as well as other carcinomas and
sarcomas. In other embodiments the unwanted proliferating cells can
be non-cancerous, e.g., cells associated with an autoimmune
condition or inflammatory condition.
[0110] A "subject having a viral infection" is a subject that has
been exposed to a virus and has acute or chronic manifestations or
detectable levels of the virus in the body.
[0111] Examples of viruses that have been found in humans include
but are not limited to: Retroviridae (e.g., human immunodeficiency
viruses, such as HIV-1 (also referred to as HTLV-III, LAV or
HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP;
Picornaviridae (e.g., polio viruses, hepatitis A virus;
enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses);
Calciviridae (e.g., strains that cause gastroenteritis);
Togaviridae (e.g., equine encephalitis viruses, rubella viruses);
Flaviridae (e.g., hepatitis C virus (HCV), dengue viruses,
encephalitis viruses, yellow fever viruses); Coronoviridae (e.g.,
coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses,
rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae
(e.g., parainfluenza viruses, mumps virus, measles virus,
respiratory syncytial virus); Orthomyxoviridae (e.g., influenza
viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses,
phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever
viruses); Reoviridae (e.g., reoviruses, orbiviurses and
rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus);
Parvovirida (parvoviruses); Papovaviridae (papilloma viruses,
polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae
(herpes simplex virus (HSV) 1 and 2, varicella zoster virus,
cytomegalovirus (CMV), herpes virus; Poxviridae (variola viruses,
vaccinia viruses, pox viruses); Iridoviridae (e.g., African swine
fever virus); and unclassified viruses (e.g., the etiological
agents of spongiform encephalopathies, the agent of delta hepatitis
(thought to be a defective satellite of hepatitis B virus), the
unclassified agents of non-A, non-B hepatitis (class 1=internally
transmitted; class 2=parenterally transmitted); Norwalk and related
viruses, and astroviruses).
[0112] Although many of the viruses described above relate to human
disorders, the invention is also useful for treating nonhuman
vertebrates. Nonhuman vertebrates are also capable of developing
infections which can be prevented or treated with the ISNAs
disclosed herein. For instance, in addition to the treatment of
infectious human diseases, the methods of the invention are useful
for treating infections of animals.
[0113] Infectious virus of both human and non-human vertebrates
include retroviruses, RNA viruses and DNA viruses. This group of
retroviruses includes both simple retroviruses and complex
retroviruses. The simple retroviruses include the subgroups of
B-type retroviruses, C-type retroviruses and D-type retroviruses.
An example of a B-type retrovirus is mouse mammary tumor virus
(MMTV). The C-type retroviruses include subgroups C-type group A
(including Rous sarcoma virus (RSV), avian leukemia virus (ALV),
and avian myeloblastosis virus (AMV)) and C-type group B (including
murine leukemia virus (MLV), feline leukemia virus (FeLV), murine
sarcoma virus (MSV), gibbon ape leukemia virus (GALV), spleen
necrosis virus (SNV), reticuloendotheliosis virus (RV) and simian
sarcoma virus (SSV)). The D-type retroviruses include Mason-Pfizer
monkey virus (MPMV) and simian retrovirus type 1 (SRV-1). The
complex retroviruses include the subgroups of lentiviruses, T-cell
leukemia viruses and the foamy viruses. Lentiviruses include HIV-1,
but also include HIV-2, SIV, Visna virus, feline immunodeficiency
virus (FIV), and equine infectious anemia virus (EIAV). The T-cell
leukemia viruses include HTLV-1, HTLV-2, simian T-cell leukemia
virus (STLV), and bovine leukemia virus (BLV). The foamy viruses
include human foamy virus (HFV), simian foamy virus (SFV) and
bovine foamy virus (BFV).
[0114] Examples of other RNA viruses that are antigens in
vertebrate animals include, but are not limited to, the following:
members of the family Reoviridae, including the genus Orthoreovirus
(multiple serotypes of both mammalian and avian retroviruses), the
genus Orbivirus (Bluetongue virus, Eugenangee virus, Kemerovo
virus, African horse sickness virus, and Colorado Tick Fever
virus), the genus Rotavirus (human rotavirus, Nebraska calf
diarrhea virus, murine rotavirus, simian rotavirus, bovine or ovine
rotavirus, avian rotavirus); the family Picornaviridae, including
the genus Enterovirus (poliovirus, Coxsackie virus A and B, enteric
cytopathic human orphan (ECHO) viruses, hepatitis A virus, Simian
enteroviruses, Murine encephalomyelitis (ME) viruses, Poliovirus
muris, Bovine enteroviruses, Porcine enteroviruses, the genus
Cardiovirus (encephalomyocarditis virus (EMC), Mengovirus), the
genus Rhinovirus (Human rhinoviruses including at least 113
subtypes; other rhinoviruses), the genus Apthovirus (Foot and Mouth
disease (FMDV); the family Calciviridae, including Vesicular
exanthema of swine virus, San Miguel sea lion virus, Feline
picornavirus and Norwalk virus; the family Togaviridae, including
the genus Alphavirus (Eastern equine encephalitis virus, Semliki
forest virus, Sindbis virus, Chikungunya virus, O'Nyong-Nyong
virus, Ross river virus, Venezuelan equine encephalitis virus,
Western equine encephalitis virus), the genus Flavirius (Mosquito
borne yellow fever virus, Dengue virus, Japanese encephalitis
virus, St. Louis encephalitis virus, Murray Valley encephalitis
virus, West Nile virus, Kunjin virus, Central European tick borne
virus, Far Eastern tick borne virus, Kyasanur forest virus, Louping
III virus, Powassan virus, Omsk hemorrhagic fever virus), the genus
Rubivirus (Rubella virus), the genus Pestivirus (Mucosal disease
virus, Hog cholera virus, Border disease virus); the family
Bunyaviridae, including the genus Bunyvirus (Bunyamwera and related
viruses, California encephalitis group viruses), the genus
Phlebovirus (Sandfly fever Sicilian virus, Rift Valley fever
virus), the genus Nairovirus (Crimean-Congo hemorrhagic fever
virus, Nairobi sheep disease virus), and the genus Uukuvirus
(Uukuniemi and related viruses); the family Orthomyxoviridae,
including the genus Influenza virus (Influenza virus type A, many
human subtypes); Swine influenza virus, and Avian and Equine
Influenza viruses; influenza type B (many human subtypes), and
influenza type C (possible separate genus); the family
paramyxoviridae, including the genus Paramyxovirus (Parainfluenza
virus type 1, Sendai virus, Hemadsorption virus, Parainfluenza
viruses types 2 to 5, Newcastle Disease Virus, Mumps virus), the
genus Morbillivirus (measles virus, subacute sclerosing
panencephalitis virus, distemper virus, Rinderpest virus), the
genus Pneumovirus (respiratory syncytial virus (RSV), Bovine
respiratory syncytial virus and Pneumonia virus of mice); forest
virus, Sindbis virus, Chikungunya virus, O'Nyong-Nyong virus, Ross
river virus, Venezuelan equine encephalitis virus, Western equine
encephalitis virus), the genus Flavirius (Mosquito borne yellow
fever virus, Dengue virus, Japanese encephalitis virus, St. Louis
encephalitis virus, Murray Valley encephalitis virus, West Nile
virus, Kunjin virus, Central European tick borne virus, Far Eastern
tick borne virus, Kyasanur forest virus, Louping III virus,
Powassan virus, Omsk hemorrhagic fever virus), the genus Rubivirus
(Rubella virus), the genus Pestivirus (Mucosal disease virus, Hog
cholera virus, Border disease virus); the family Bunyaviridae,
including the genus Bunyvirus (Bunyamwera and related viruses,
California encephalitis group viruses), the genus Phlebovirus
(Sandfly fever Sicilian virus, Rift Valley fever virus), the genus
Nairovirus (Crimean-Congo hemorrhagic fever virus, Nairobi sheep
disease virus), and the genus Uukuvirus (Uukuniemi and related
viruses); the family Orthomyxoviridae, including the genus
Influenza virus (Influenza virus type A, many human subtypes);
Swine influenza virus, and Avian and Equine Influenza viruses;
influenza type B (many human subtypes), and influenza type C
(possible separate genus); the family paramyxoviridae, including
the genus Paramyxovirus (Parainfluenza virus type 1, Sendai virus,
Hemadsorption virus, Parainfluenza viruses types 2 to 5, Newcastle
Disease Virus, Mumps virus), the genus Morbillivirus (Measles
virus, subacute sclerosing panencephalitis virus, distemper virus,
Rinderpest virus), the genus Pneumovirus (respiratory syncytial
virus (RSV), Bovine respiratory syncytial virus and Pneumonia virus
of mice); the family Rhabdoviridae, including the genus
Vesiculovirus (VSV), Chandipura virus, Flanders-Hart Park virus),
the genus Lyssavirus (Rabies virus), fish Rhabdoviruses, and two
probable Rhabdoviruses (Marburg virus and Ebola virus); the family
Arenaviridae, including Lymphocytic choriomeningitis virus (LCM),
Tacaribe virus complex, and Lassa virus; the family Coronoaviridae,
including Infectious Bronchitis Virus (IBV), Mouse Hepatitis virus,
Human enteric corona virus, and Feline infectious peritonitis
(Feline coronavirus).
[0115] Illustrative DNA viruses that are antigens in vertebrate
animals include, but are not limited to: the family Poxviridae,
including the genus Orthopoxvirus (Variola major, Variola minor,
Monkey pox Vaccinia, Cowpox, Buffalopox, Rabbitpox, Ectromelia),
the genus Leporipoxvirus (Myxoma, Fibroma), the genus Avipoxvirus
(Fowlpox, other avian poxvirus), the genus Capripoxvirus (sheeppox,
goatpox), the genus Suipoxvirus (Swinepox), the genus Parapoxvirus
(contagious postular dermatitis virus, pseudocowpox, bovine papular
stomatitis virus); the family Iridoviridae (African swine fever
virus, Frog viruses 2 and 3, Lymphocystis virus of fish); the
family Herpesviridae, including the .alpha.-Herpesviruses (Herpes
Simplex Types 1 and 2, Varicella-Zoster, Equine abortion virus,
Equine herpes virus 2 and 3, pseudorabies virus, infectious bovine
keratoconjunctivitis virus, infectious bovine rhinotracheitis
virus, feline rhinotracheitis virus, infectious laryngotracheitis
virus) the Beta-herpesviruses (Human cytomegalovirus and
cytomegaloviruses of swine, monkeys and rodents); the
gamma-herpesviruses (Epstein-Barr virus (EBV), Marek's disease
virus, Herpes saimiri, Herpesvirus ateles, Herpesvirus sylvilagus,
guinea pig herpes virus, Lucke tumor virus); the family
Adenoviridae, including the genus Mastadenovirus (Human subgroups
A,B,C,D,E and ungrouped; simian adenoviruses (at least 23
serotypes), infectious canine hepatitis, and adenoviruses of
cattle, pigs, sheep, frogs and many other species, the genus
Aviadenovirus (Avian adenoviruses); and non-cultivatable
adenoviruses; the family Papoviridae, including the genus
Papillomavirus (Human papilloma viruses, bovine papilloma viruses,
Shope rabbit papilloma virus, and various pathogenic papilloma
viruses of other species), the genus Polyomavirus (polyomavirus,
Simian vacuolating agent (SV-40), Rabbit vacuolating agent (RKV), K
virus, BK virus, JC virus, and other primate polyoma viruses such
as Lymphotrophic papilloma virus); the family Parvoviridae
including the genus Adeno-associated viruses, the genus Parvovirus
(Feline panleukopenia virus, bovine parvovirus, canine parvovirus,
Aleutian mink disease virus, etc.). Finally, DNA viruses may
include viruses which do not fit into the above families such as
Kuru and Creutzfeldt-Jacob disease viruses and chronic infectious
neuropathic agents (CHINA virus).
[0116] Each of the foregoing lists is illustrative, and is not
intended to be limiting. In addition, these viruses, either in
intact form or as fragments thereof, can be used as antigens in
immunization procedures. An antigen is a substance recognized by
the immune system as foreign and which induces specific immunity.
Antigens can be carbohydrates (including, e.g., polysaccharides,
glycolipids, and glycoproteins), proteins and polypeptides, as well
as other oligomers, polymers, and small molecules which can bind to
antigen receptors on immune cells. Specific immunity to an antigen
can involve antigen recognition by T cells and/or B cells.
[0117] Nucleic acids containing an appropriate ISNA can be
effective in any vertebrate. Different nucleic acids containing an
ISNA can cause optimal immune stimulation depending on the
mammalian species. Thus an oligonucleotide causing optimal
stimulation or inhibition in humans may not cause optimal
stimulation or inhibition in a mouse, and vice versa. One of skill
in the art can identify the optimal oligonucleotides useful for a
particular mammalian species of interest using routine assays
described herein and/or known in the art, using the guidance
supplied herein.
[0118] The ISNA may be directly administered to the subject or may
be administered in conjunction with a nucleic acid delivery
complex. A "nucleic acid delivery complex" shall mean a nucleic
acid molecule associated with (e.g., ionically or covalently bound
to; or encapsulated within) a targeting means (e.g., a molecule
that results in higher affinity binding to target cell (e.g., B
cell surfaces and/or increased cellular uptake by target cells).
Examples of nucleic acid delivery complexes include nucleic acids
associated with: a sterol (e.g., cholesterol), a lipid (e.g., a
cationic lipid, virosome or liposome), or a target cell specific
binding agent (e.g., a ligand recognized by target cell specific
receptor). Preferred complexes may be sufficiently stable in vivo
to prevent significant uncoupling prior to internalization by the
target cell. However, the complex can be cleavable under
appropriate conditions within the cell so that the nucleic acid is
released in a functional form.
[0119] The ISNA or other therapeutics may be administered alone
(e.g., in saline or buffer) or using any delivery vehicles known in
the art. For instance the following delivery vehicles have been
described: cochleates (Gould-Fogerite et al., 1994, 1996);
emulsomes (Vancott et al., 1998, Lowell et al., 1997); ISCOMs
(Mowat et al., 1993, Carlsson et al., 1991, Hu et., 1998, Morein et
al., 1999); liposomes (Childers et al., 1999, Michalek et al.,
1989, 1992, de Haan 1995a, 1995b); live bacterial vectors (e.g.,
Salmonella, Escherichia coli, Bacillus Calmette-Guerin, Shigella,
Lactobacillus) (Hone et al., 1996, Pouwels et al., 1998, Chatfield
et al., 1993, Stover et al., 1991, Nugent et al., 1998); live viral
vectors (e.g., Vaccinia, adenovirus, Herpes Simplex) (Gallichan et
al., 1993, 1995, Moss et al., 1996, Nugent et al., 1998, Flexner et
al., 1988, Morrow et al., 1999); microspheres (Gupta et al., 1998,
Jones et al., 1996, Maloy et al., 1994, Moore et al., 1995, O'Hagan
et al., 1994, Eldridge et al., 1989); nucleic acid vaccines (Fynan
et al., 1993, Kuklin et al., 1997, Sasaki et al., 1998, Okada et
al., 1997, Ishii et al., 1997); polymers (e.g.,
carboxymethylcellulose, chitosan) (Hamajima et al., 1998,
Jabbal-Gill et al., 1998); polymer rings (Wyatt et al., 1998);
Proteosomes (Vancott et al., 1998, Lowell et al., 1988, 1996,
1997); sodium fluoride (Hashi et al., 1998); transgenic plants
(Tacket et al., 1998, Mason et al., 1998, Haq et al., 1995);
virosomes (Gluck et al., 1992, Mengiardi et al., 1995, Cryz et al.,
1998); virus-like particles (Jiang et al., 1999, Leibl et al.,
1998). Those skilled in the art will recognize that other delivery
vehicles that are known in the art may also be used.
[0120] Combined with the teachings provided herein, by choosing
among the various active compounds and weighing factors such as
potency, relative bioavailability, patient body weight, severity of
adverse side-effects and preferred mode of administration, an
effective prophylactic or therapeutic treatment regimen can be
planned which does not cause substantial toxicity and yet is
entirely effective to treat the particular subject. The effective
amount for any particular application can vary depending on such
factors as the disease or condition being treated, the particular
ISNA being administered (e.g., the number of unmethylated CpG
motifs or their location in the nucleic acid, the degree of
chirality to the oligonucleotide), the antigen, the size of the
subject, or the severity of the disease or condition. One of
ordinary skill in the art can empirically determine the effective
amount of a particular ISNA and/or antigen and/or other therapeutic
agent without necessitating undue experimentation.
[0121] For adult human subjects, doses of the ISNA compounds
described herein typically range from about 50 .mu.g/dose to 20
mg/dose, more typically from about 80 .mu.g/dose to 8 mg/dose, and
most typically from about 800 .mu.g/dose to 4 mg/dose. Stated in
terms of subject body weight, typical dosages range from about 0.5
to 500 .mu.g/kg/dose, more typically from about 1 to 100
.mu.g/kg/dose, and most typically from about 10 to 50
.mu.g/kg/dose. Doses will depend on factors including the route of
administration, e.g., oral administration may require a
substantially larger dose than subcutaneous administration.
[0122] The formulations of the invention are administered in
pharmaceutically acceptable solutions, which may routinely contain
pharmaceutically acceptable concentrations of salt, buffering
agents, preservatives, compatible carriers, adjuvants, and
optionally other therapeutic ingredients.
[0123] The ISNA can be given in conjunction with other agents known
in the art to be useful in combination with IFN-.alpha. to treat
viral and proliferative disorders. Examples of such other agents
currently used or under investigation for use in combination with
IFN-.alpha. include ribavirin, amantadine, chemotherapeutic agents
(e.g., 5-fluorouracil and BCNU), radiation therapy, phototherapy,
and cytokines, including IL-2, IL-12, and IFN-.gamma..
[0124] For use in therapy, an effective amount of the ISNA can be
administered to a subject by any mode that delivers the ISNA to the
desired site, e.g., mucosal, systemic. "Administering" the
pharmaceutical composition of the present invention may be
accomplished by any means known to the skilled artisan. Preferred
routes of administration include but are not limited to oral,
parenteral, intralesional, topical, transdermal, intramuscular,
intranasal, intratracheal, inhalational, ocular, vaginal, and
rectal.
[0125] For oral administration, the compounds (i.e., ISNA, antigen,
other therapeutic agent) can be formulated readily by combining the
active compound(s) with pharmaceutically acceptable carriers well
known in the art. Such carriers enable the compounds of the
invention to be formulated as tablets, pills, dragees, capsules,
liquids, gels, syrups, slurries, suspensions and the like, for oral
ingestion by a subject to be treated. Pharmaceutical preparations
for oral use can be obtained as solid excipient, optionally
grinding a resulting mixture, and processing the mixture of
granules, after adding suitable auxiliaries, if desired, to obtain
tablets or dragee cores. Suitable excipients are, in particular,
fillers such as sugars, including lactose, sucrose, mannitol, or
sorbitol; cellulose preparations such as, for example, maize
starch, wheat starch, rice starch, potato starch, gelatin, gum
tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If
desired, disintegrating agents may be added, such as the
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate. Optionally the oral formulations
may also be formulated in saline or buffers for neutralizing
internal acid conditions or may be administered without any
carriers.
[0126] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0127] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. Microspheres formulated for oral
administration may also be used. Such microspheres have been well
defined in the art. All formulations for oral administration should
be in dosages suitable for such administration.
[0128] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0129] For administration by inhalation, the compounds for use
according to the present invention may be conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g., gelatin for use in an inhaler or insufflator
may be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0130] The compounds, when it is desirable to deliver them
systemically, may be formulated for parenteral administration by
injection, e.g., by bolus injection or continuous infusion.
Formulations for injection may be presented in unit dosage form,
e.g., in ampoules or in multi-dose containers, with an added
preservative. The compositions may take such forms as suspensions,
solutions or emulsions in oily or aqueous vehicles, and may contain
formulatory agents such as suspending, stabilizing and/or
dispersing agents.
[0131] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents which increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
[0132] Alternatively, the active compounds may be in powder form
for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use.
[0133] The compounds may also be formulated in rectal or vaginal
compositions such as suppositories or retention enemas, e.g.,
containing conventional suppository bases such as cocoa butter or
other glycerides.
[0134] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be formulated with suitable polymeric or
hydrophobic materials (for example as an emulsion in an acceptable
oil) or ion exchange resins, or as sparingly soluble derivatives,
for example, as a sparingly soluble salt.
[0135] The pharmaceutical compositions also may comprise suitable
solid or gel phase carriers or excipients. Examples of such
carriers or excipients include but are not limited to calcium
carbonate, calcium phosphate, various sugars, starches, cellulose
derivatives, gelatin, and polymers such as polyethylene
glycols.
[0136] Suitable liquid or solid pharmaceutical preparation forms
are, for example, aqueous or saline solutions for inhalation,
microencapsulated, encochleated, coated onto microscopic gold
particles, contained in liposomes, nebulized, aerosols, pellets for
implantation into the skin, or dried onto a sharp object to be
scratched into the skin. The pharmaceutical compositions also
include granules, powders, tablets, coated tablets,
(micro)capsules, suppositories, syrups, emulsions, suspensions,
creams, drops or preparations with protracted release of active
compounds, in whose preparation excipients and additives and/or
auxiliaries such as disintegrants, binders, coating agents,
swelling agents, lubricants, flavorings, sweeteners or solubilizers
are customarily used as described above. The pharmaceutical
compositions are suitable for use in a variety of drug delivery
systems. For a brief review of methods for drug delivery, see
Langer Science 249:1527 (1990), which is incorporated herein by
reference.
[0137] The ISNAs may be administered per se (neat) or in the form
of a pharmaceutically acceptable salt. When used in medicine the
salts should be pharmaceutically acceptable, but
non-pharmaceutically acceptable salts may conveniently be used to
prepare pharmaceutically acceptable salts thereof. Such salts
include, but are not limited to, those prepared from the following
acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric,
maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric,
methane sulphonic, formic, malonic, succinic,
naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts
can be prepared as alkaline metal or alkaline earth salts, such as
sodium, potassium or calcium salts of the carboxylic acid
group.
[0138] Suitable buffering agents include: acetic acid and a salt
(1-2 percent w/v); citric acid and a salt (1-3 percent w/v); boric
acid and a salt (0.5-2.5 percent w/v); and phosphoric acid and a
salt (0.8-2 percent w/v). Suitable preservatives include
benzalkonium chloride (0.003-0.03 percent w/v); chlorobutanol
(0.3-0.9 percent w/v); parabens (0.01-0.25 percent w/v) and
thimerosal (0.004-0.02 percent w/v).
[0139] The pharmaceutical compositions of the invention contain an
effective amount of an ISNA and optionally antigens and/or other
therapeutic agents optionally included in a
pharmaceutically-acceptable carrier. The term
"pharmaceutically-acceptable carrier" means one or more compatible
solid or liquid filler, diluants or encapsulating substances which
are suitable for administration to a human or other vertebrate
animal. The term "carrier" denotes an organic or inorganic
ingredient, natural or synthetic, with which the active ingredient
is combined to facilitate the application. The components of the
pharmaceutical compositions also are capable of being commingled
with the compounds of the present invention, and with each other,
in a manner such that there is no interaction which would
substantially impair the desired pharmaceutical efficiency.
[0140] A variety of administration routes are available. The
particular mode selected will depend, of course, upon the
particular adjuvants or antigen selected, the particular condition
being treated and the dosage required for therapeutic efficacy. The
methods of this invention, generally speaking, may be practiced
using any mode of administration that is medically acceptable,
meaning any mode that produces effective levels of an immune
response without causing clinically unacceptable adverse effects.
Preferred modes of administration are discussed above.
[0141] The compositions may conveniently be presented in unit
dosage form and may be prepared by any of the methods well known in
the art of pharmacy. All methods include the step of bringing the
compounds into association with a carrier which constitutes one or
more accessory ingredients. In general, the compositions are
prepared by uniformly and intimately bringing the compounds into
association with a liquid carrier, a finely divided solid carrier,
or both, and then, if necessary, shaping the product. Liquid dose
units are vials or ampoules. Solid dose units are tablets, capsules
and suppositories. For treatment of a patient, depending on
activity of the compound, manner of administration, purpose of the
immunization (i.e., prophylactic or therapeutic), nature and
severity of the disorder, age and body weight of the patient,
different doses may be necessary. The administration of a given
dose can be carried out both by single administration in the form
of an individual dose unit or else several smaller dose units.
[0142] Other delivery systems can include time-release, delayed
release or sustained release delivery systems. Such systems can
avoid repeated administrations of the compounds, increasing
convenience to the subject and the physician. Many types of release
delivery systems are available and known to those of ordinary skill
in the art. They include polymer-based systems such as
poly(lactide-glycolide), copolyoxalates, polycaprolactones,
polyesteramides, polyorthoesters, polyhydroxybutyric acid, and
polyanhydrides. Microcapsules of the foregoing polymers containing
drugs are described in, for example, U.S. Pat. No. 5,075,109.
Delivery systems also include non-polymer systems that are: lipids
including sterols such as cholesterol, cholesterol esters and fatty
acids or neutral fats such as mono-, di- and tri-glycerides;
hydrogel release systems; silastic systems; peptide based systems;
wax coatings; compressed tablets using conventional binders and
excipients; partially fused implants; and the like. Specific
examples include, but are not limited to: (a) erosional systems in
which an agent of the invention is contained in a form within a
matrix such as those described in U.S. Pat. Nos. 4,452,775,
4,675,189, and 5,736,152, and (b) diffusional systems in which an
active component permeates at a controlled rate from a polymer such
as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686.
In addition, pump-based hardware delivery systems can be used, some
of which are adapted for implantation.
[0143] As used herein, a method which calls for administration of
IFN-.alpha. refers to a clinical method for treating a subject by
administration of IFN-.alpha. with the aim of achieving a desired
therapeutic result. There are a number of clinical indications for
which IFN-.alpha. is an established therapeutic agent. A subject in
need of IFN-.alpha. treatment has a clinical indication for which
IFN-.alpha. is an established therapeutic agent. These clinical
indications include certain viral infections and certain
proliferative disorders, notably cancers and pre-cancerous
conditions. The viral infections for which IFN-.alpha. has current
approval for use in the United States are hepatitis B, hepatitis C,
and condylomata acuminata (venereal or anogenital warts). The
neoplasms for which IFN-.alpha. has current approval for use in the
United States are hairy cell leukemia, cutaneous T-cell leukemia,
chronic myelogenous leukemia (CML), non-Hodgkin's lymphoma,
malignant melanoma, and AIDS-related Kaposi's sarcoma. Outside of
the United States, IFN-.alpha. is also in clinical use for bladder
cell carcinoma, colon carcinoma, renal cell carcinoma, multiple
myeloma, cervical dysplasia, and laryngeal papillomatosis. Other
indications under investigation for IFN-.alpha. treatment include
other viral infections and other cancers, including, for example,
Behcet's disease, HIV, prostate cancer, small cell lung cancer,
pancreatic cancer, squamous cell carcinoma, glioma, and malignant
pleural mesothelioma, among others.
[0144] As used herein, a pharmaceutical composition comprising
IFN-.alpha. refers to a preparation of recombinant or natural
IFN-.alpha. suitable for pharmaceutical use. IFN-.alpha. may be
derived from natural material (e.g., leukocytes, myeloblasts,
lymphocytes) or material derived therefrom (e.g., cell lines), or
those prepared with recombinant DNA technology. Details of the
cloning of IFN-.alpha. and the direct expression thereof,
especially in Escherichia coli, have been the subject of many
publications. The preparation of recombinant IFNs is known, for
example, from Gray et al. Nature 295:503-8 (1982), Goeddel et al.
Nature 284:316-20 (1980), Goeddel et al. Nature 290:20-26 (1981),
and EP 174143. In the United States IFN-.alpha. is available as
recombinant human IFN-.alpha.2a (ROFERON-A), recombinant human
IFN-.alpha.2b (INTRON A), and as purified natural IFN-.alpha.n3
(ALFERON N). Outside the United States, IFN-.alpha. is also
available as purified natural IFN-.alpha.n1 (WELLFERON).
[0145] As used herein, a clinically established effective dose for
IFN-.alpha. alone refers to a dose of recombinant or natural
IFN-.alpha., administered in the absence of another agent that
increases IFN-.alpha. bioavailability, that is the standard
recommended dose for a particular clinical indication. A clinically
established effective dose for IFN-.alpha. alone can encompass,
however, the use of IFN-.alpha. in combination with other agents
and treatment modalities, such as conventional chemotherapy,
radiation therapy, antiviral agents, and surgery. In a majority of
subjects the standard recommended dose of IFN-.alpha. for a
particular clinical indication would be expected to exert a desired
clinical effect. In a given clinical application in a given
subject, a clinically established effective dose for IFN-.alpha.
alone can also refer to a dose of IFN that is, or has been, or
would be expected to be effective in that subject for treating a
condition of the subject. For example, a subject might be
responsive to a dose of IFN-.alpha. alone that is less than the
standard recommended dose. Conversely, a subject might be unable to
tolerate a clinically established effective dose due to actual or
anticipated side effects of the IFN-.alpha. treatment.
[0146] The maximum tolerated dose (MTD) for any therapeutic
compound is identified as part of its clinical evaluation. For
example, phase I trials can include a determination of the maximum
tolerated dose, dose-limiting toxicities (DLT) and pharmacokinetics
of a test compound. Thus, the MTD for any Food and Drug
Administration (FDA) approved therapeutic compound is known to
those of ordinary skill in the art as a matter of the public
record. The MTD for any particular therapeutic compound may vary
according to its formulation (e.g., injectable formulation,
implantable bioerodible polymer formulation, oral formulation),
route of delivery (e.g., intravenous, oral, intratumoral), manner
of delivery (e.g., infusion, bolus injection), dosing schedule
(e.g., hourly, daily, weekly) and the like. MTD frequently is
defined as the highest dose level at which 50 percent of subjects
administered with the drug develop a dose-limiting toxicity. Other
definitions which are clinically relevant and generally accepted
will be known to one of ordinary skill in the art.
[0147] Examples of MTD for various types of IFN-.alpha. have been
published in studies involving various routes of administration,
indications, combinations with other agents, and clinical settings.
In one study the MTD of recombinant IFN-.alpha.2a was 18 million
international units (IU) when given intramuscularly three times
weekly in combination with phototherapy for treatment of cutaneous
T-cell lymphoma (mycosis fungoides and Sezary syndrome). Kuzel T M
et al. J Natl Cancer Inst 82:203-7 (1990). In an independent study,
the MTD of IFN-.alpha.2b for the treatment of cutaneous T-cell
lymphoma was found to be 18 million IU given intramuscularly three
times a week. Qiu B and Chen M Chin Med J (Engl) 109:404-6 (1996).
The MTD for IFN-.alpha.2a was lower, 3 million IU subcutaneously
three times a week, for patients undergoing high dose pelvic
radiation for rectal cancer. Perera F et al. Int J Radiat Oncol
Biol Phys 37:297-303 (1997). The MTD for IFN-.alpha.2b was 10
million IU daily for patients with AIDS-related Kaposi's sarcoma
following cytotoxic chemotherapy. Gill P S et al. J Biol Response
Mod 9:512-6 (1990). In yet another study the MTD of IFN-.alpha.2b
was 18 million IU/m.sup.2 given weekly as a 24-hour infusion in
combination with 5-fluorouracil and leucovorin to patients with
metastatic colorectal cancer. Cascinu S et al. Anticancer Drugs
7:520-4 (1996).
[0148] Measurement of maximum tolerated dose may be expressed as
weight of drug per weight of subject, weight of drug per body
surface area, etc. The MTD of anticancer compounds is frequently
expressed as weight per square meters (mg/m.sup.2) of body surface
area. MTD also may be expressed as a dose relative to a time
component, such as weight of drug per body surface area per
day.
[0149] For therapeutics which have not yet been subjected to human
clinical trials, or subjected to any determination of the MTD in
humans (e.g., experimental or highly toxic compounds), one of skill
in the art can estimate the MTD by using animal models. Calculation
of MTD in animals may be based on a number of physiological
parameters, such as death, particular toxicities, and drug-induced
weight loss. Using death as an endpoint, the MTD may be the highest
dose given test animals in which each member of the test group
survived. Using toxicity as an endpoint, the MTD may be the dose at
which moderate but not severe toxicity is observed. Using weight
loss as an endpoint, the MTD may be the dose above which a certain
percent change in body weight is induced. Other methods for
determining MTDs using animal models and various endpoints are
known to one of ordinary skill in the art. Correlation of animal
MTDs to human MTDs for a therapeutic compound is an accepted
practice in the pharmaceutical arts.
[0150] Thus the invention in one aspect provides compositions and
formulations for administration to a subject, preferably a human
subject, containing an amount of interferon which is below the
maximum tolerated dose for interferon.
[0151] In one aspect of the invention, an improved method is
provided for treating a subject in need of treatment with
IFN-.alpha. which entails coadministration of an effective amount
of an isolated ISNA in conjunction with administration of
IFN-.alpha.. As used herein, an effective amount of an isolated
ISNA refers to that amount of an isolated ISNA that causes cells to
produce IFN-.alpha.. In one preferred embodiment an effective
amount of an isolated ISNA refers to that amount of an isolated
ISNA that causes cells in vivo to produce IFN-.alpha.. In another
preferred embodiment an effective amount of an isolated ISNA refers
to that amount of an isolated ISNA corresponding to an amount that
causes cells in vitro to produce IFN-.alpha.. In another preferred
embodiment an effective amount of an isolated ISNA refers to that
amount of an isolated ISNA that causes an increase in the local or
circulating amount of IFN-.alpha. above the corresponding level
which would occur only from the administration of exogenous
IFN-.alpha.. In another preferred embodiment an effective amount of
an isolated ISNA refers to that amount of an isolated ISNA that
increases the therapeutic benefit of exogenous IFN-.alpha. above
that which would be obtained without the ISNA. In yet another
embodiment an effective amount of an isolated ISNA refers to that
amount of an isolated ISNA that would allow the same therapeutic
effect to be achieved for a given amount of IFN-.alpha. when the
oligonucleotide is coadministered with a lower dose of
IFN-.alpha..
[0152] As used herein, the term coadminister refers to
administering at least two agents in clinical association with one
another. Coadministration can include administering the at least
two agents together or sequentially. In a preferred embodiment,
ISNA is administered either before, at the same time as, or after
administering IFN-.alpha., provided the local or systemic
concentration of IFN-.alpha. is increased over the corresponding
concentration of IFN-.alpha. that would be achieved by
administering the same amount of IFN-.alpha. alone. Coadminister
means the administration of interferon-.alpha. close enough in time
to the administration of the ISNA such that their effects are more
than the effects that would be achieved if administering either one
on its own at the same dose. Preferably, the effects are at least
additive. They also may be administered via different modes, for
example such as administering the interferon systemically and
administering the ISNA locally.
[0153] In certain embodiments involving simultaneous
coadministration, the IFN-.alpha. and the ISNA may be prepared as a
single formulation. In other embodiments involving simultaneous
coadministration, the IFN-.alpha. and the ISNA may be prepared and
administered separately. In this latter instance individual
IFN-.alpha. and ISNA formulations may be packaged together as a kit
with instructions for simultaneous administration. Simlarly, in
embodiments calling for consecutive coadministration, individual
IFN-.alpha. and ISNA formulations may be packaged together as a kit
with instructions for their sequential administration.
[0154] As used herein, administered locally refers to
administration by a route that achieves a local concentration of
IFN-.alpha. that exceeds the systemic concentration of IFN-.alpha..
For example, local administration to a particular lesion or organ
could be accomplished by direct injection into the lesion or organ
or by direct injection into an afferent blood vessel associated
with and supplying the lesion or organ to be treated. In the
example local administration to the liver, local administration can
be accomplished by injection or infusion into the hepatic artery,
the celiac artery, or the portal vein.
[0155] In another aspect, the invention provides a method of
supplementing IFN-.alpha. treatment of a subject in need of
IFN-.alpha. treatment wherein an effective amount of IFN-.alpha.
and an isolated ISNA are both administered to the subject. The
IFN-.alpha. induced by the ISNA supplements the IFN-.alpha.
directly administered to the subject, thus extending the clinical
efficacy of a given dose of IFN-.alpha.. Furthermore, because the
ISNA-induced IFN-.alpha. typically includes a plurality of
subtypes, while the directly administered IFN-.alpha. typically
includes only a single subtype, the range of biological effects
afforded by IFN-.alpha. treatment is also expanded by the
coadministration of the ISNA and the IFN-.alpha..
[0156] The invention also provides a method of increasing the
efficacy of IFN-.alpha. treatment of a subject. The method
according to this aspect of the invention involves administering to
a subject in need of treatment with IFN-.alpha. a pharmaceutical
composition comprising IFN-.alpha. and coadministering to the
subject in need of such treatment a pharmaceutical composition
comprising an ISNA in an amount which, together with the
administered IFN-.alpha., is an effective IFN-.alpha. treatment,
wherein the efficacy of the IFN-.alpha. treatment is greater than
the efficacy of administering the same amount of IFN-.alpha. in the
absence of coadministering the ISNA.
[0157] As used herein, a method of enhancing efficacy or increasing
efficacy of IFN-.alpha. treatment of a subject refers to a method
in which the effect of administering a given dose of IFN-.alpha. to
a subject results in a greater clinical effect than expected or
previously observed when using that same dose of IFN-.alpha.. In a
preferred embodiment the method entails coadministering an amount
of ISNA in an amount effective for inducing the production of
IFN-.alpha. by IPCs. The amount of IFN-.alpha. achieved locally or
systemically in this method reflects contributions both from
administered IFN-.alpha. and from induced IFN-.alpha., thereby
achieving an enhanced efficacy of IFN-.alpha. treatment for a given
dose of administered IFN-.alpha.. The increased efficacy could be
manifested as, for example, a greater degree of response to
treatment, a more rapid course of response to treatment, or
improved compliance with the treatment regimen.
[0158] According to another aspect of the invention, a method is
provided for decreasing a dose of IFN-.alpha. effective for
treating a subject in need of treatment with IFN-.alpha.. The
method involves administering to a subject in need of treatment
with IFN-.alpha. a pharmaceutical composition comprising
IFN-.alpha., and coadministering to the subject in need of such
treatment a pharmaceutical composition comprising an
immunostimulatory nucleic acid in an amount which, together with
the administered IFN-.alpha., is an effective IFN-.alpha.
treatment, and wherein the amount of administered IFN-.alpha. is
less than an amount of IFN-.alpha. required in the absence of
coadministering the immunostimulatory nucleic acid.
[0159] As used herein, a method of decreasing a dose of IFN-.alpha.
effective for treating a subject refers to a method in which
IFN-.alpha. is administered to a subject in an amount or with a
frequency that is reduced compared to a previously established
amount or frequency, while achieving a desired clinical effect in
treating a condition of the subject. In a preferred embodiment the
dose amount can be reduced by a clinically determined extent to an
amount that is, for example, at least 10 percent below the
customary or maximum tolerated dose of IFN-.alpha. alone. In other
more preferred embodiments, the IFN-.alpha. dose amount can be
reduced by a clinically determined extent to an amount that is at
least 20 percent, at least 30 percent, or at least 40 percent below
the customary or maximum tolerated dose of IFN-.alpha. alone. In a
most preferred embodiment, the IFN-.alpha. dose amount can be
reduced by a clinically determined extent to an amount that is at
least 50 percent below the customary or maximum tolerated dose of
IFN-.alpha. alone. In another preferred embodiment the dose
frequency can be reduced by a clinically determined extent to a
frequency that is, for example, at least 10 percent below the
customary or maximum tolerated dose of IFN-.alpha. alone. In other
more preferred embodiments, the IFN-.alpha. dose frequency can be
reduced by a clinically determined extent to a frequency that is at
least 20 percent, at least 30 percent, or at least 40 percent below
the customary or maximum tolerated dose of IFN-.alpha. alone. In a
most preferred embodiment, the IFN-.alpha. dose frequency can be
reduced by a clinically determined extent to a frequency that is at
least 50 percent below the customary or maximum tolerated dose of
IFN-.alpha. alone.
[0160] Yet another aspect of the invention is a method of
preventing an IFN-.alpha. treatment-related side effect in a
subject receiving or in need of treatment with IFN-.alpha.. The
method entails administering to a subject in need of treatment with
IFN-.alpha. a pharmaceutical composition comprising IFN-.alpha. and
coadministering to the subject in need of such treatment a
pharmaceutical composition comprising an immunostimulatory nucleic
acid in an amount which, together with the administered
IFN-.alpha., is an effective IFN-.alpha. treatment, and wherein an
IFN-.alpha. treatment-related side effect is reduced in comparison
to the side effect when IFN-.alpha. is administered in the absence
of coadministering the immunostimulatory nucleic acid.
[0161] Assays for IFN-.alpha. are well known in the art. These
include direct tests, e.g., enzyme-linked immunosorbent assay
(ELISA) specific for at least one IFN-.alpha., and indirect tests,
e.g., functional tests including NK cell activation/cytotoxicity
(Trinchieri G Adv Immunol 47:187-376 (1989) and phenotyping by
fluorescence-activated cell sorting (FACS) analysis for class I
MHC. Additional specific assay methods well known in the art can be
particularly useful in settings where local concentration or local
presence of IFN-.alpha. is of interest; these methods include, for
example, immunohistochemistry, nucleic acid hybridization (e.g.,
Northern blotting), Western blotting, reverse
transcriptase/polymerase chain reaction (RT/PCR), and in situ
RT/PCR. A further method, involving detection of intracellular
IFN-.alpha. by flow cytometry, is disclosed below in Example 6.
[0162] As used herein, a method of preventing an IFN-.alpha.
treatment-related side effect in a subject refers to a method of
reducing the incidence or severity of an IFN-.alpha.
treatment-related side effect experienced by a subject receiving
IFN-.alpha. treatment. As used herein, an IFN-.alpha.
treatment-related side effect is a clinical side effect that is
induced in a subject as a result of administration of IFN-.alpha.
to the subject. A number of such side effects have been well
documented through clinical experience and clinical trials. Such
side effects are frequently dose-limiting in a subject. Systemic
IFN-.alpha. treatment-related side effects most commonly
encountered include: influenza-like syndrome, fever, headache,
chills, myalgia, fatigue, anorexia, nausea, vomiting, diarrhea,
depression, hypothyroidism, neutropenia, and anemia. In a preferred
embodiment the IFN-.alpha. treatment-related side effect is reduced
sufficiently to promote greater compliance with IFN-.alpha.
treatment. In another preferred embodiment the IFN-.alpha.
treatment-related side effect is reduced sufficiently to permit
resumption of IFN-.alpha. treatment otherwise precluded by the side
effect. In another preferred embodiment the IFN-.alpha.
treatment-related side effect is reduced sufficiently to permit an
intensification of IFN-.alpha. treatment.
[0163] In another aspect of the invention, a second method is
provided for enhancing efficacy of IFN-.alpha. treatment in a
subject in need of such treatment. The method involves the steps of
administering to a subject in need of such treatment an amount of a
pharmaceutical composition comprising IFN-.alpha. effective for
treating a condition of the subject, isolating natural
interferon-producing cells (IPCs) from a donor, contacting the
isolated IPCs ex vivo with an amount of a pharmaceutical
composition comprising an immunostimulatory nucleic acid effective
for inducing the IPCs to release IFN-.alpha. and administering the
contacted cells to the subject. The donor and the subject can be a
single individual or they can be different individuals. In certain
embodiments the contacted cells are administered to the subject in
a local fashion, e.g., via injection or infusion into a blood
vessel supplying a target tissue to be treated. The method
according to this aspect of the invention can optionally include
contacting the isolated IPCs with an antigen. In certain
embodiments the method may also include contacting the isolated
IPCs with a growth factor which the IPCs do not produce themselves.
Such a growth factor not produced by IPCs can include, for example,
IL-3 or GM-CSF, and would exclude IL-8 and TNF-.alpha..
[0164] As used herein, the term growth factor refers to a soluble
signaling factor that induces a responsive cell type to undergo
maturation and mitosis. Categories of growth factors include a
number of cytokines, growth factors per se, and hormones. Specific
examples of growth factors include, without limitation, IL-1, IL-2,
IL-3, IL-6, GM-CSF, G-CSF, PDGF, TGF-.beta., NGF, IGFs, growth
hormone, erythropoietin, thrombopoietin, and the like. In addition
to naturally occurring growth factors, growth factor analogs and
growth factor derivatives such as fusion proteins can be used for
the purposes of the invention.
[0165] As used herein, the term natural interferon-producing cell
(IPC) refers to a specialized type of leukocyte that is the chief
producer of IFN-.alpha. in response to enveloped viruses, bacteria,
and tumors. IPCs are lineage negative (lin-)/CD4+/MHC class II+
cells that are present in low frequency in peripheral blood
mononuclear cells (PBMCs) and in tonsillar tissue. Siegal F P et
al. Science 284:1835-7 (1999); Grouard G et al. J Exp Med
185:1101-11 (1997). The frequency of IPCs in PBMCs in normal
individuals varies between 0.2 and 0.6 percent. They are
characterized by the absence of lineage markers CD3 (T cells), CD14
(monocytes), CD19 (B cells) and CD56 (NK cells), by the absence of
CD11c, and by their expression of CD4, CD123 (IL-3 receptor
.alpha., IL-3R.alpha.) and MHC class II. Grouard G et al. J Exp Med
185:1101-11 (1997); Rissoan M-C et al. Science 283:1183-86 (1999);
Siegal F P et al. Science 284:1835-7 (1999); Cella M et al. Nat Med
5:919-23 (1999).
[0166] As used herein, isolating IPCs from a subject refers to a
process of removing from the subject a body fluid or tissue
containing IPCs and enriching for the IPCs from the body fluid or
tissue to an extent that at least 1 percent of the cells are IPCs.
In a most preferred embodiment at least 99 percent of cells are
IPCs. In another preferred embodiment at least 95 percent of cells
are IPCs. In another preferred embodiment at least 90 percent of
cells are IPCs. In another preferred embodiment at least 80 percent
of cells are IPCs. In another preferred embodiment at least 70
percent of cells are IPCs. In another preferred embodiment at least
60 percent of cells are IPCs. In another preferred embodiment at
least 50 percent of cells are IPCs. In another preferred embodiment
at least 40 percent of cells are IPCs. In another preferred
embodiment at least 30 percent of cells are IPCs. In another
preferred embodiment at least 20 percent of cells are IPCs. In
another preferred embodiment at least 10 percent of cells are IPCs.
In another preferred embodiment at least 5 percent of cells are
IPCs. The enriching can be achieved by a series of selecting steps
which can include, for example, a negative selection of
lineage-positive cells by contacting cells with magnetic beads
conjugated with antibodies specific for lineage markers (i.e.,
anti-CD3, anti-CD11c, anti-CD14, anti-CD16, anti-CD19, anti-CD56)
and then passing the contacted cells over a depletion column in the
presence of a strong magnetic field; a positive selection step
involving contacting the cells passing through the depletion column
with microbead-conjugated anti-CD4 and passing the contacted cells
over a positive selection column; and further enhancement for IPCs
by fluorescence-activated cell sorting (FACS) using anti-CD123 and
anti-MHC class II. Other methods can be employed to equal effect,
as will be appreciated by those skilled in the art, provided they
result in the isolation or enrichment of (lin-)/CD4+/CD123+/MHC
class II+ interferon producing cells to an extent that at least 1
percent of the viable cells are IPCs.
[0167] In yet another aspect of the invention, a method is provided
for supporting the survival of natural interferon-producing cells
(IPCs) in vitro. The method involves isolating IPCs from a subject
(as described above), culturing the IPCs in a sterile medium
suitable for tissue culture, and contacting the IPCs in vitro with
an amount of immunostimulatory nucleic acid effective to support
the growth of the IPCs in the absence of interleukin 3 (IL-3). In a
preferred embodiment, the IPCs are precursor type 2 dendritic cells
(pDC2s; plasmacytoid monocytes). Siegal F P et al. Science
284:1835-7 (1999). The IPCs can be cultured under suitable tissue
culture conditions either with or, more notably, without exogenous
IL-3 and/or GM-CSF.
[0168] As used herein, a method of supporting survival of
interferon-producing cells (IPCs) in vitro refers to providing a
factor or inducing a signal that promotes the viability of IPCs
placed in in vitro culture. For example, in the absence of IL-3,
normally most IPCs die within three days of being placed into cell
culture. Addition of IL-3 to IPCs will support the survival of IPCs
in culture. According to this aspect of the invention, IL-3 is not
required for IPC survival in vitro if the IPCs are contacted with
an effective amount of ISNA.
[0169] The invention in another aspect provides a method for
stimulating isolated interferon-producing cells (IPCs) in vitro.
The method includes the steps of isolating IPCs from a subject
(described above), culturing the IPCs in a sterile medium suitable
for tissue culture, and contacting the IPCs in vitro with an amount
of immunostimulatory nucleic acid effective to induce secretion of
at least one type I interferon. In a preferred embodiment the type
I interferon induced by the method is IFN-.alpha.. As described
above, preferred IPCs are precursor type 2 dendritic cells (pDC2s;
plasmacytoid monocytes). Siegal F P et al. Science 284:1835-7
(1999). Importantly, the IPCs can be stimulated in culture in the
absence of GM-CSF and without viral infection by, e.g., Sendai
virus, HSV or influenza virus. Activation can be assayed using
methods well known in the art, including FACS analysis of the cell
surface activation marker CD80 and ELISA or bioassay (e.g.,
protection of fibroblasts against vesicular stomatitis virus) for
type I IFN.
[0170] As used herein, a method of stimulating isolated
interferon-producing cells (IPCs) in vitro refers to providing a
factor or inducing a signal that results in a change in IPC size,
morphology, or expression of a cell surface antigen, transcript, or
a secreted product that is not characteristic of IPCs in the
absence of the factor or signal. Freshly isolated IPCs, in the
absence of a signal provided by viral infection, CD40L ligation, or
GM-CSF, display a smooth round lymphoid morphology with a diameter
of 8-10 .mu.m and do not express CD80 or CD86 on their cell
surface. Grouard G et al. J Exp Med 185:1101-11 (1999). Similarly,
freshly isolated IPCs do not secrete IFN-.alpha. in large amounts.
Siegal F P et al. Science 284:1835-7 (1999). In contrast, IPCs
exposed to IL-3 in vitro develop pseudopods and a veiled
morphology, express CD80 and CD86 on their surface, and secrete
large amounts of type I IFN (IFN-.alpha. and IFN-.beta.) when
exposed to ultraviolet-irradiated herpes simplex virus, Sendai
virus, or heat-killed Staphylococcus aureus. Grouard G et al. J Exp
Med 185:1101-11 (1999); Siegal F P et al. Science 284:1835-7
(1999). According to this aspect of the invention, ISNA can be used
in place of viral infection, CD40L ligation, or GM-CSF to induce a
signal effective for stimulating isolated IPCs in vitro.
[0171] The invention further provides a method for treating a
subject to activate interferon-producing cells (IPCs) of the
subject. The method involves isolating IPCs from a subject in need
of such treatment, culturing the IPCs in vitro, contacting the IPCs
in vitro with an effective amount of an isolated immunostimulatory
nucleic acid, and returning the contacted IPCs to the subject. IPCs
are isolated from a subject as described above and placed into
culture under suitable in vitro cell culture conditions. Such
culture conditions may optionally include provision of exogenous
growth factor, including IL-3 or GM-CSF. However, IL-3 or GM-CSF
may not be required for the purposes of the method. According to
this method, the subject may be treated without direct
administration of a pharmaceutical preparation of IFN-.alpha..
Activation of the IPCs can be assayed as described above, with
reference made to IPCs similarly obtained and cultured but not
contacted with ISNA.
[0172] In yet another aspect, the invention provides a method for
stimulating production of a plurality of type I IFN subtypes. The
method involves contacting IPCs with an amount of immunostimulatory
nucleic acid effective to induce secretion of at least two type I
interferons. In one embodiment the IPCs are brought into contact
with ISNA in vivo. In another embodiments the IPCs are isolated
and/or are contacted with ISNA in vitro under suitable cell culture
conditions. Various other embodiments result in the induction of at
least three, at least four, at least five, at least six, at least
seven, and at least eight subtypes of type I IFN. The various
subtypes can be determined using methods well described in the art
and known to those of skill in the art, e.g., subtype-specific
ELISA, amino-terminal sequencing, and mass spectrometry (MS).
[0173] Matrix-assisted laser desorption/ionization time-of-flight
(MALDI TOF)-MS and electrospray ionization (ESI)-MS are now
standard methods used for identifying peptides available in
femtomole quantities. Mann M and Talbo G Curr Opin Biotechnol
7:11-19 (1996); Mann M and Wilm M Trends Biochem Sci 20:219-24
(1995); Mann M et al. Anal Chem 61:1702-8 (1989). Individual bands
were cut out of the polyacrylamide gel, cleaved with trypsin and
then eluted to yield peptide fragments that are subjected to MALDI
TOF-MS or ESI-MS analysis. The combination of mass/charge data from
the MS, cleavage site specificity of the trypsin digest, and
peptide sequence data permitted identification of individual
proteins and peptides. MALDI TOF-MS analysis gives a mass
fingerprint of the cleaved and analyzed proteins. The fingerprint
is useful only for scanning against a database of calculated
peptide masses corresponding to fully sequenced proteins. The
ESI-MS analysis is more difficult, but it permits identification
based on comparison to either complete or partial sequence data.
Mass accuracies for either method can exceed 0.01 percent, i.e., 1
Da per 10 kDa.
[0174] In another aspect the invention relates to the discovery
that type I IFN induces activation and proliferation of
.gamma..delta. T cells. The .gamma..delta. T cells are
antigen-specific T cells in a preactivated stage which respond to
common phosphate-containing non-peptide antigens. Examples of
.gamma..delta. T cell antigens include phosphate-containing
non-peptide molecules from heat-killed mycobacteria; isopentenyl
pyrophosphate (IPP) and related prenyl pyrophosphate derivatives;
monoethyl phosphate; and .gamma.-monoethyl derivatives of
nucleoside and deoxynucleoside triphosphates. Tanaka Y et al.
Nature 375:155-8 (1995). Previous studies showed that
.gamma..delta. T cells can secrete a variety of lymphockines and
mount cytolytic responses. For example, exposure of .gamma..delta.
T cells to these phosphate-containing non-peptide antigens
stimulates IFN-.gamma. production in the absence of APC. While
clearly belonging to the T-cell lineage, human .gamma..delta. T
cells are distinctly different from .alpha./.beta. T cells, and
they share several features with NK cells. The observations that
.gamma..delta. cells accumulate in lesions caused by mycobacterial
infections, respond to virally infected cells in a
virus-nonspecific manner, require neither antigen-processing nor
antigen-presenting cells, and that they are preferentially located
in various epithelia, together suggest that .gamma..delta. cells
may be responsive to pattern recognition and responsible for a
first line of defense.
[0175] It was discovered according to this aspect of the invention
that CpG ODN in combination with IPP synergistically induced
activation of human .gamma..delta. T cells present within PBMC, as
measured by the production of IFN-.gamma. and perforin. In
addition, it was also discovered according to this aspect of the
invention that CpG ODN in combination with IPP synergistically
induced proliferation of human .gamma..delta. T cells present
within PBMC. These effects were abrogated by isolating
.gamma..delta. T cells from PBMC or by addition of neutralizing
antibodies to type I IFN, and they were reproduced by the addition
of recombinant type I IFN. Notably, ODN 2216 and 1585, both strong
inducers of type I IFN, were more potent in their effects on
.gamma..delta. T cells than ODN 2006.
[0176] In humans Th1 responses are driven by IL-12 and/or
IFN-.gamma.. IL-12 and IFN-.alpha./.beta. both promote IFN-.gamma.
synthesis in T cells and NK cells. It was previously known that
IL-12 promotes .gamma..delta. T cells to secrete IFN-.gamma.. Since
IFN-.beta. has been described to downregulate IL-12 production,
experiments were performed to study the effect on IL-12 production
exerted by ISNA which induce type I IFN. The results of these
experiments (Example 13) demonstrate that certain CpG ODN suppress
CD40-dependent IL-12p70 production by an
IFN-.alpha./.beta.-mediated negative feedback mechanism on IL-12p40
mRNA production. Thus the interaction of T cells and
antigen-presenting cells via CD40L leads to a cytokine milieu
dominated by IL-12 or IFN-.alpha./.beta.. Although both promote Th1
responses, CpG ODN which are better inducers of B cell activation
than of type I IFN may be superior for priming naive T cells, and
conversely CpG ODN which are strong inducers of type I IFN may have
higher activity to support preactivated and memory T cells.
[0177] According to another aspect of the invention, an interferon
composition for administration to a subject is provided. The
composition includes recombinant or natural interferon in a
container for administration to a subject. The amount of the
interferon in the container is at least about 10 percent less than
the maximum tolerated dose (MTD). Preferably the amount of
interferon in the container is at least about 20 percent below the
MTD, at least 30 percent below the MTD, at least 40 percent below
the MTD, or even at least 50 percent below the MTD. In other
embodiments, the amount of interferon in the container is at least
about 20 percent below, 30 percent below, 40 percent below, or even
50 percent below the clinically established effective dose. The
container also can include an ISNA.
[0178] In still another aspect of the invention, kits for
administration of interferon and an ISNA to a subject are provided.
Referring to FIG. 18 depicting a kit 11, the kits include a
container 19 containing a composition 17 which includes IFN-.alpha.
and instructions 21 for administering the interferon to a subject
in need of such treatment in an amount which is at least about 10
percent less than the maximum tolerated dose (MTD), 20 percent less
than the MTD, 30 percent less than the MTD, 40 percent less than
the MTD, or 50 percent less than the MTD. The kit 11 can include,
in the same container or in a separate container 19, an ISNA. The
kit also can include instructions 21 for treating a subject with a
condition susceptible to treatment with IFN-.alpha.. Examples of
such conditions, proliferative and viral, are as described above.
Kit 11 also includes a box-like package 15.
[0179] The present invention is further illustrated by the
following Examples, which in no way should be construed as further
limiting. The entire contents of all of the references (including
literature references, issued patents, published patent
applications, and co-pending patent applications) cited throughout
this application are hereby expressly incorporated by
reference.
EXAMPLES
Example 1
Isolation and Characterization of IPCs
[0180] Peripheral blood mononuclear cells (PBMCs) contain a total
of 0.2 to 0.4 percent IPCs, which are characterized by the lack of
lineage markers (CD3, CD14, CD16, CD19, CD20, CD56) and can be
distinguished from other lineage-negative cells by the expression
of CD4, CD123 (IL-3R.alpha.), and MHC class II.
[0181] IPCs were isolated from peripheral blood by using the
VARIOMACS technique (Milteny Biotec, Auburn, Calif.) and the
technique previously described. O'Doherty U et al. J Exp Med
178:1067-76 (1993). PBMCs were obtained from buffy coats of healthy
blood donors by Ficoll-Paque density gradient centrifugation
(Histopaque-1077, Sigma) as previously described. Hartmann G et al.
Antisense Nucleic Acid Drug Dev 6:291-9 (1996). Monoclonal
antibodies directed to CD3 (UCHT1), CD14 (M5E2), and CD19 (B43)
were purchased from PharMingen (San Diego). PBMCs were incubated
with anti-CD3, CD14, CD16, CD19, and CD56 antibodies conjugated to
colloidal superparamagnetic microbeads and passed over a depletion
column in a strong magnetic field. Resulting lineage-negative
(lin-) cells in the flow-through were incubated with a
microbead-conjugated antibody to CD4 and passed over a positive
selection column. Further purification of IPCs to >99 percent
from lin-/CD4+ cells was achieved by fluorescence-activated cell
sorting (FACS) using phycoerythrin (PE)-labeled anti-CD123 and
FITC-labeled anti-MHC class II.
[0182] Surface antigen staining was performed as previously
described. Hartmann G et al. J Pharmacol Exp Ther 285:920-8 (1998).
Monoclonal antibodies to MHC class II (HLA-DR, Immun-357) and CD80
(MAB104) were purchased from Immunotech (Marseilles, France). All
other antibodies were purchased from PharMingen (San Diego): mAbs
to CD3 (UCHT1), CD14 (M5E2), CD19 (B43), and CD86 (2331 (FUN-1)).
FITC-labeled IgG1,.kappa. (MOPC-21) and phycoerythrin-labeled
IgG2b,.kappa. were used to control for specific staining. Lyons A B
and Parish C R J Immunol Methods 171:131-7 (1994).
[0183] Flow cytometric data were acquired on a FACScan (Becton
Dickinson Immunocytometry Systems, San Jose, Calif.). Spectral
overlap was corrected by appropriate compensation. Analysis was
performed on viable cells within a morphologic gate (forward
scattering (FSC), side scattering (SSC), >94 percent of cells
MHC class II positive and lineage marker negative). Data were
analyzed with the computer program FLOWJO (version 2.5.1, Tree
Star, Stanford, Calif.).
[0184] Results. Viability as determined by trypan blue exclusion
was >95 percent. Freshly isolated IPCs are negative for
costimulatory molecules CD80 and CD86. FIG. 1 depicts FACS analyses
of IPCs isolated from PBMCs with magnetic beads and flow cytometry.
From left to right are shown: selection of lin-/MHC class II+ cells
from PBMCs; further selection of CD123+/MHC class II+ cells from
lin-/MHC class II+ cells; and characterization of freshly isolated
lin-/MHC class II+/CD123+ IPCs as CD80-.
Example 2
CpG Oligonucleotide Supports the Survival and Activation of IPCs In
Vitro
[0185] The majority of freshly isolated IPCs die within 3 days if
not incubated in the presence of IL-3 or GM-CSF. Remaining live
cells are not activated or are only weakly activated. If CpG
oligonucleotide but no other growth factors are added to the cell
culture of IPCs, IPCs survive and become highly activated as shown
by their increased expression of costimulatory molecules (e.g.,
CD80, FIG. 2).
[0186] Freshly isolated IPCs (see Example 1) were suspended in RPMI
1640 culture medium supplemented with 10 percent (vol/vol)
heat-inactivated (56.degree. C., 1 h) FCS (HyClone), 1.5 mM
L-glutamine, 100 units/ml penicillin, and 100 .mu.g/ml streptomycin
(all from GIBCO/BRL) (complete medium). All compounds were
purchased endotoxin-tested. Freshly prepared IPCs (final
concentration 5.times.10.sup.5 cells per ml) were cultured for two
days in complete medium alone or complete medium supplemented with
6 .mu.g/ml phosphorothioate CpG ODN 2006
(5'-tcgtcgttttgtcgttttgtcgt- t-3'; SEQ ID NO:147), 100 ng/ml LPS
(from Salmonella typhimurium, Sigma catalog no. L2262), 800
units/ml GM-CSF (1.25.times.10.sup.4 units/mg, Genzyme), or CpG
oligonucleotide in combination with GM-CSF. Expression of CD80 and
MHC class II on IPCs was examined by flow cytometry (see Example
1).
[0187] Results. Representative results of five independent
experiments are depicted in FIG. 2. A single addition of CpG ODN
2006 (SEQ ID NO:147, 2 .mu.g/ml) to freshly prepared IPCs was
superior to GM-CSF (800 units/ml) in promoting cell survival (74.3
percent.+-.5.2 percent vs. 57.1 percent.+-.2.3 percent). The
combination of GM-CSF and CpG ODN 2006 (SEQ ID NO:147) further
increased the number of viable cells (81.0 percent.+-.6.7 percent).
Freshly isolated IPCs placed into culture for two days without IL-3
or GM-CSF remained unactivated, as indicated by the lack of
staining for CD80, even when LPS was added to the media. Addition
of GM-CSF induced CD80. Addition of CpG ODN 2006 (SEQ ID NO:147, 6
.mu.g/ml) activated IPCs to an even greater degree than GM-CSF (800
units/ml). Further activation of IPCs occurred when GM-CSF and CpG
oligonucleotide were present together. This demonstrates that CpG
substitutes for IL-3 and GM-CSF in supporting the survival of IPCs.
LPS did not contribute either to survival or activation of IPCs
(FIG. 2).
Example 3
CpG Oligonucleotide, but not Poly IC, Activates IPCs In Vitro
[0188] IL-3 provides excellent survival of IPCs but does not
activate IPCs. When IL-3 was combined with CpG oligonucleotide,
expression of CD80 increased by 5 to 20-fold (FIG. 3). Poly IC,
another polynucleotide with well known immunostimulatory functions
on myeloid cells (dendritic cells, macrophages), did not stimulate
IPCs.
[0189] Freshly prepared IPCs (see Example 1, final concentration
3.times.10.sup.5 cells per ml) were cultured for three days in
complete medium (see Example 2) supplemented with 10 ng/ml IL-3.
Cultures of IPCs were then continued for a further 24 hours (a)
without any additional supplements, (b) following the addition of 6
.mu.g/ml CpG ODN 2006 (SEQ ID NO:147), and (c) following the
addition of 10 .mu.g/ml poly IC. Forward scattering (FSC), side
scattering (SSC), and expression of CD80 and MHC class II on IPCs
were examined by flow cytometry (see Example 1).
[0190] Results. Representative results of three independent
experiments are shown in FIG. 3. IPCs cultured in complete media
supplemented with IL-3 and CpG ODN 2006 (SEQ ID NO:147, 6 .mu.g/ml)
were larger and more granular than IPCs cultured in complete media
containing IL-3 alone or in complete media supplemented with IL-3
and poly IC. In addition, IPCs cultured in complete media
supplemented with IL-3 and CpG ODN 2006 (SEQ ID NO:147, 6 .mu.g/ml)
were more highly activated than IPCs cultured in complete media
containing IL-3 alone or in complete media supplemented with IL-3
and poly IC. This demonstrates that CpG oligonucleotide activates
IPCs in vitro.
Example 4
CpG Oligonucleotide Induces IFN-.alpha. Production by IPCs
[0191] Induction of type I interferons by CpG in whole PBMC has
been previously demonstrated. Sun S et al. J Exp Med 188:2335-42
(1998). Here is shown for the first time that IFN-.alpha. is
induced in IPCs by CpG oligonucleotide within 48 hours using an
ELISA specific for 9 out of 10 subspecies of IFN-.alpha.
tested.
[0192] Freshly prepared IPCs (see Example 1, final concentration
3.times.10.sup.5 cells per ml) were cultured for two days in
complete medium (see Example 2) supplemented with 10 ng/ml IL-3 and
800 units/ml GM-CSF (1.25.times.10.sup.4 units/mg, Genzyme). Half
of the cultures were supplemented with 6 .mu.g/ml CpG ODN 2006 (SEQ
ID NO:147). IFN-.alpha. was measured in the supernatant using a
combination of separate ELISAs specific for IFN-.alpha. (human
IFN-.alpha. multispecies ELISA, PBL Biomedical Laboratories, New
Brunswick, N.J.) and for IFN-.beta. (PBL Biomedical Laboratories,
New Brunswick, N.J.) performed according to the supplier's
instructions. The multispecies IFN-.alpha. ELISA has a range from
100 to 5000 pg/ml, detects all of the human IFN-.alpha. subtypes
except IFN-.alpha.F, and does not detect IFN-.beta. or IFN-.gamma..
The IFN-.beta. ELISA has a range of 250-10,000 pg/ml.
[0193] Results. Representative results of three independent
experiments appear in FIG. 4. IPCs cultured for 48 hours in
complete media supplemented with 10 ng/ml IL-3, 800 units/ml GM-CSF
and 6 .mu.g/ml CpG ODN 2006 (SEQ ID NO:147) were strongly induced
to secrete IFN-.alpha. compared to similar cultures without added
CpG oligonucleotide. This result demonstrates CpG oligonucleotide
induces human IPCs to secrete multiple subspecies of IFN-.alpha..
This result also indicates CpG oligonucleotide would permit the in
vitro production of natural interferons using a permanent cell line
derived from IPCs.
Example 5
Identification of CpG ODN with IFN-.alpha. and IFN-.beta. Inducing
Activity
[0194] The 24 mer CpG ODN 2006 (SEQ ID NO:147) which contains three
consecutive "human" CpG motifs (5' GTCGTT 3') is one of the most
potent CpG sequences to activate human B cells. Hartmann G et al. J
Immunol 164:944-53 (2000); Hartmann G et al. J Immunol 164:1617-24
(2000). In contrast to other microbial stimuli such as LPS and poly
(I:C), ODN 2006 strongly promotes survival and activation of pDC
precursors. However, compared to its strong ability to activate NK
cells, the ability of ODN 2006 to induce type I IFN in pDC is
relatively poor.
[0195] In order to test the hypothesis that other CpG ODN may
activate NK cells by inducing type I IFN in pDC, a panel of CpG ODN
with known NK cell activity were tested for their capability to
stimulate IFN-.alpha. production in PBMC. The panel of CpG ODN
included the following, where lower case letters signify
phosphorothioate linkages, upper case letters signify
phosphodiester linkages, and m signifies 7-deaza-guanosine:
11 tcgtcgttttgtcgttttgtcgtt ODN 2006 (SEQ ID NO:147)
ggGGTCAACGTTGAgggggG ODN 1585 (SEQ ID NO:1) gmGGTCAACGTTGAgggmggG
ODN 2197 (SEQ ID NO:148) ggGGAGTTCGTTGAgggggG ODN 2198 (SEQ ID
NO:149)
[0196] All ODN were dissolved in TE buffer (10 mM Tris-HCl, 1 mM
EDTA, pH 8) at a concentration of 20 mg/ml. Aliquots diluted in PBS
(0.4 mg/ml) were stored at -20.degree. C. and thawed prior to use.
Pyrogen-free reagents were used for all dilutions. ODN were tested
for endotoxin using the LAL assay (BioWhittaker, Walkersville, Md.;
lower detection limit 0.1 EU/ml).
[0197] Freshly isolated PBMC were incubated with CpG ODN (3
.mu.g/ml) for 48 hours. IFN-.alpha. was measured in the supernatant
by an ELISA which detects 10 of 13 isoforms of IFN-.alpha.. Among
all sequences initially examined, CpG ODN 1585 (SEQ ID NO:1) showed
the highest activity to induce IFN-.alpha. in PBMC. ODN 1585 is a
chimeric ODN (mixed phosphorothioate-phosphodiester backbone) with
poly G on both ends and a central CpG-dinucleotide within a 10 mer
palindrome. Hartmann G et al. J Pharmacol Exp Ther 285:920 (1998).
ODN 1585 stimulated IFN-.alpha. in the nanogram range (1.3.+-.0.4
ng/ml; n=7) as compared to ODN 2006 which did not induce
significant amounts of IFN-.alpha. in PBMC (0.021.+-.0.015 ng/ml;
n=8) (FIG. 5). The control ODN 2197 (7-deaza-guanosine
substitutions in poly G ends, unable to form G tetrads) and ODN
2198 (CG and poly G ends but no palindrome) were essentially
inactive (FIG. 5). Based on the sequence of ODN 1585, a new panel
of CpG ODN were then designed. ODN 2216 (ggGGGACGATCGTCgggggG; SEQ
ID NO:7), which contains poly G ends and three CG dinucleotides
within a palindrome, is one example among several sequences with
pronounced IFN-.alpha.-inducing activity in PBMC (23.7.+-.5.2
ng/ml; n=7).
[0198] CpG ODN stimulated IFN-.alpha. production in a
concentration-dependent manner (FIG. 6). The activities of ODN 2216
and ODN 1585 were tested for concentrations up to 12 .mu.g/ml,
confirming that the higher potency of ODN 2216 was not a
concentration-dependent effect. As little as 0.4 .mu.g/ml ODN 2216
induced considerable amounts of IFN-.alpha. (0.7 ng/ml) in PBMC,
whereas ODN 2006 and the GC control to ODN 2216
(ggGGGAGCATGCTCgggggG; ODN 2243; SEQ ID NO:150) had no effect even
at higher concentrations. Maximum activity was reached at 3
.mu.g/ml. Production of IFN-.alpha. could already be detected after
6 hours of incubation (0.2 ng/ml) and reached a plateau after 48
hours.
[0199] The natural interferon-producing cell in PBMC upon virus
infection is identical to the pDC precursor with a frequency of
less than 0.5%. PBMC were enriched 10- to 70-fold for pDC
precursors by depletion of T-cells, NK-cells and monocytes (2-18%
CD123.sup.++ pDC; 3-10% CD11c.sup.+ myeloid DC (mDC); 50-90%
B-cells; n=4). To increase the viability of pDC, IL-3 was added to
all samples. This procedure resulted in a 30- to 60-fold increase
in IFN-.alpha. production (up to 428.3.+-.56.8 ng/ml with ODN 2216;
n=4; FIG. 7, upper panel, left side). The most active CpG ODN in
PBMC were also the most active in the samples enriched for pDC. ODN
2006, ODN 2197, or IL-3 alone induced only little IFN-.alpha.
(means: 0.8, 0.4 and 0.6 ng/ml respectively, n=4). Poly (I:C) (7
.mu.g/ml), which mimics double-stranded RNA and which is known to
induce IFN-.alpha. in macrophages, was an even weaker stimulus of
IFN-.alpha. in cells enriched for pDC (0.3 ng/ml, not in figure).
The same CpG ODN which induced high amounts of IFN-.alpha. also
stimulated IFN-.beta. production (up to 2.8.+-.0.8 ng/ml, n=4; FIG.
7, lower panel, left side). Considering that IFN-.beta. represents
a single isoform and that IFN-.alpha. consists of at least 13
isoforms, remarkable amounts of IFN-.beta. are produced.
[0200] To determine whether cellular uptake of CpG ODN was critical
for the induction of IFN-.alpha. and IFN-.beta. by CpG ODN, the
effects of the cationic lipid lipofectin were examined (FIG. 7,
upper and lower panel, right side). Positively charged cationic
lipids form complexes with negatively charged ODN, which increase
cellular uptake of ODN. Lipofectin enhanced the production of
IFN-.alpha. and IFN-.beta. induced by CpG ODN (up to 786 ng/ml
IFN-.alpha., n=3; and up to 9 ng/ml IFN-.beta., n=3). The increase
was seen for all CpG ODN examined but was most prominent for ODN
1585 (20-fold).
Example 6
CpG ODN-Induced IFN-.alpha. is Exclusively Produced by Plasmacytoid
Dendritic Precursor Cells
[0201] To examine which cell type within PBMC produces IFN-.alpha.
in response to CpG ODN, a protocol was developed which allowed the
detection of intracellular IFN-.alpha. on a single cell basis by
flow cytometry. PBMC were incubated with ODN 2216 (SEQ ID NO:7) or
ODN 2006 (SEQ ID NO:147) at 3 .mu.g/ml. After five hours, cells
were harvested and intracellular staining of IFN-.alpha. was
performed.
[0202] For the analysis of intracellular IFN-.alpha., no brefeldin
A was added during the incubation period to block protein
secretion. PBMC were harvested (approximately 600,000 cells/tube),
incubated with anti-CD123-biotin (Pharmingen), washed in PBS (400
g, 5 minutes, 4.degree. C.) and stained with Streptavidin-APC
(Pharmingen), FITC-conjugated anti-lineage cocktail (consisting of
anti-CD3, -CD14, -CD16, -CD19, -CD20, and -CD56; Becton Dickinson),
and anti-HLA DR-PerCP (Becton Dickinson). Then cells were washed in
PBS, resuspended in 100 .mu.l of fixation buffer A (Fix and Perm
Kit, Caltag Laboratories, Burlingame, Calif.) and incubated at room
temperature for 15 min. Cells were washed in 2 ml PBS again and
then resuspended in 100 .mu.l permeabilization buffer B (Fix and
Perm Kit). 4 .mu.g/ml mouse anti-human IFN-.alpha. mAb (MMHA-11,
PBL Biomedical Laboratories) was added. PE-labeled mouse IgG1
(MOPC-21, Pharmingen) was used as control antibody. After 15 min
incubation at room temperature in the dark, cells were washed in 2
ml PBS. For detection of intracellular IFN-.alpha. cells were again
resuspended in 100 .mu.l permeabilization buffer B (Fix and Perm
Kit) and stained with PE-labeled rat anti-mouse Ig .kappa. light
chain (R8-140, Pharmingen) as secondary antibody. After washing in
PBS, cells were analyzed by four-color flow cytometry on a Becton
Dickinson FACS Calibur equipped with two lasers (excitation at 488
nm and 635 nm). Spectral overlap was corrected by appropriate
compensation and gates were set using isotype control antibodies.
Analysis was performed on viable cells within a morphologic gate
(FSC, SSC, >97% of viable cells as confirmed by propidium iodide
staining). Data were analysed using CELLQUEST (Becton Dickinson) or
FLOWJO software (version 2.5.1, Tree Star, Inc., Stanford,
Calif.).
[0203] Results. As shown in FIG. 8A, lineage.sup.+ and lineage
(lin.sup.+ and lin.sup.-) cells were defined by lineage marker
expression and forward scatter characteristics. After stimulation
with ODN 2216 intracellular IFN-.alpha. was not detectable in
lin.sup.+ cells which contain monocytes and macrophages as
potential IFN-.alpha. producing cells (FIG. 8C). Within the
lin.sup.- cells, which contain mainly pDC and mDC, a distinct
population with intermediate HLA DR (MHC class II) expression
stained positive for IFN-.alpha. (FIG. 8D).
[0204] Within the lin.sup.- population, mDC and pDC can be
distinguished by their HLA DR/CD123 phenotype (FIG. 8B). The mDC
are CD123.sup.+/- and HLA DR.sup.++ (gate II); pDC are CD123.sup.++
and HLA DR.sup.+ (gate III). The CD123.sup.++/HLA DR- population
are basophils. FIG. 9A shows intracellular staining for IFN-.alpha.
in lin.sup.-/HLA DR.sup.+ cells. IFN-.alpha. was exclusively
produced by pDC in response to ODN 2216 but not in response to ODN
2006. Among pDC 46% stained positive for IFN-.alpha., corresponding
to a frequency of 0.25% cells within PBMC which produced
IFN-.alpha. at this particular time point of staining. In three
other experiments frequencies of IFN-.alpha. producing cells in
response to ODN 2216 were 0.08%, 0.05% and 0.22% of PBMC (16%, 8%
and 63% of pDC).
[0205] In contrast to the results with pDC, in mDC no IFN-.alpha.
synthesis was detected after stimulation with ODN 2006 or ODN
2216.
[0206] Thus, pDC were the only cells within PBMC which produced
IFN-.alpha. in response to CpG ODN. Of note, intracellular
IFN-.alpha. staining was performed without brefeldin A. Thus the
amount of IFN-.alpha. detected represented the actual IFN-.alpha.
production at the time point of harvest and not the cumulative
amount of IFN-.alpha. over several hours. When brefeldin A was
added during incubation to block protein secretion (standard
protocol for intracellular cytokine staining), no
IFN-.alpha.-producing cells could be detected.
Example 7
Both IFN-.alpha.-Inducing and non-IFN-.alpha.-Inducing CpG ODN
Stimulate Early TNF Production in Plasmacytoid Dendritic Cells
[0207] It has been reported that pDC produce TNF-.alpha. in
response to IL-3 and thus promote their own maturation in an
autocrine fashion. Hartmann G et al. Antisense Nucleic Acid Drug
Dev 6:291-9 (1996). The intracellular accumulation of TNF-.alpha.
in pDC therefore was examined in response to different CpG ODN
(FIG. 9B). PBMC were incubated for five hours with with ODN 2216
(SEQ ID NO:7) or ODN 2006 (SEQ ID NO:147) at 3 .mu.g/ml in the
absence of IL-3. Brefeldin A (1 .mu.g/ml, Sigma) was added during
the five hour stimulation period to block cytokine secretion. PBMC
were harvested (approximately 600,000 cells/tube), incubated with
anti-CD123-biotin, washed in PBS (400 g, 5 minutes, 4.degree. C.)
and stained with Streptavidin-APC (Pharmingen), FITC-conjugated
anti-lineage cocktail and anti-HLA DR-PerCP (Becton Dickinson).
Then cells were washed in PBS, resuspended in 100 .mu.l of fixation
buffer A (Fix and Perm Kit, Caltag Laboratories, Burlingame,
Calif.) and incubated at room temperature for 15 min. Cells were
washed in 2 ml PBS again and then resuspended in 100 .mu.l
permeabilization buffer B (Fix and Perm Kit). 5 .mu.g/ml PE-labeled
mouse anti-human TNF-.alpha. mAb (MAb11, Pharmingen) was added as
primary antibody. PE-labeled mouse IgG1 (MOPC-21, Pharmingen) was
used as control antibody. After 15 min incubation at room
temperature in the dark, cells were washed in 2 ml PBS and then
analyzed by four-color flow cytometry as described above.
[0208] Results. In contrast to IFN-.alpha., the percentage of
TNF-.alpha. producing pDC in response to ODN 2006 and ODN 2216 was
similar (59% versus 56%). Two other experiments showed comparable
results (26 vs 22% and 8 vs 6% TNF-.alpha..sup.+ pDC with ODN 2006
and ODN 2216, respectively). The production of TNF-.alpha. per cell
(MFI, mean fluorescence intensity) was consistently higher with ODN
2216 than with ODN 2006 (FIG. 9B). No TNF-.alpha. was detected in
mDC. Lineage.sup.+ cells did not produce significant amounts of
TNF-.alpha. in response to either ODN 2006 or ODN 2216.
Example 8
Upregulation of Costimulatory Molecules on pDC in Response to
IFN-.alpha.-Inducing CpG ODN
[0209] In previous studies ODN 2006 was reported to stimulate the
expression of costimulatory molecules on CD4.sup.+ peripheral blood
DC. Zhong R K et al. J Immunol 163:1354 (1999). In order to examine
the capacity of different CpG ODN to upregulate CD80 and CD86 on
pDC, PBMC were depleted of monocytes, T cells and NK cells, and the
remaining cells were stimulated with different CpG ODN in the
presence of IL-3. After 48 hours, expression of CD80 and CD86 was
examined on pDC (CD123.sup.++/HLA DR.sup.+) by three-color flow
cytometry. B-cells (CD123.sup.-/HLA DR.sup.+) and mDC
(CD123.sup.+/-/HLA DR.sup.++) were excluded from the analysis. As
shown in FIG. 10, expression of CD86 on pDC was increased by ODN
2006 (SEQ ID NO:147) as well as by ODN 1585 (SEQ ID NO:1) and ODN
2216 (SEQ ID NO:7). The effect of ODN 1585 was abolished by
substitution of the poly G tails with 7-deaza-guanosine (ODN 2197;
SEQ ID NO:148). The non-palindrome CpG ODN 2198 (SEQ ID NO:149) was
inactive. Upregulation of CD80 and HLA DR was similar to CD86.
Increased expression of CD80 and CD86 in response to the weakly
IFN-.alpha.-inducing ODN 2006 was detectable after 6 hours. In
contrast, ODN 1585 and ODN 2216 showed a delayed response, starting
later than 12 hours. For both strongly IFN-.alpha.-inducing CpG ODN
(ODN 1585, ODN 2216) and weakly IFN-.alpha.-inducing CpG ODN (ODN
2006), a plateau was reached after 48 hours. At later time points,
identification of pDC among PBMC by flow cytometry was hampered by
downregulation of CD123 during cell culture.
Example 9
Stimulation of IFN-.alpha. and IFN-.beta. Production by CpG ODN is
a Direct Effect on Plasmacytoid Dendritic Cells and is Partially
Blocked by Anti-CD4 Magnetic Beads
[0210] In order to examine whether CpG ODN induce IFN-.alpha.
production directly, purified pDC, rather than pDC-enriched PBMC
were studied. PBMC were depleted of monocytes, T-cells, NK cells
and B cells. CD123.sup.++ and HLA DR.sup.+ pDC were sorted by FACS
from the remaining cell population to yield purified (97%) pDC
(FIG. 11A). Purified pDC (160,000 cells/ml) were incubated with or
without ODN 2216 (SEQ ID NO:7) in the presence of IL-3. After 48
hours IFN-.alpha. and IFN-.beta. were measured in the supernatant
by ELISA. As shown in FIG. 11B, ODN 2216 stimulated the production
of high levels of IFN-.alpha. (146 ng/ml; 1 pg per single pDC) and
IFN-.beta. (1 ng/ml), as compared to IL-3 alone (<10 pg/ml).
[0211] Within PBMC as well as within pDC-enriched PBMC, 4.2.+-.0.8
pg IFN-.alpha. (0.8 to 1.4 U; n=4) was produced per single pDC.
IFN-.alpha. production of pDC was much lower when pDC were enriched
by using magnetically labeled anti-CD4 mAb. This was not due to a
loss of IFN-.alpha. producing cells in the CD4 fraction, since the
CD4 fraction did not produce IFN-.alpha.. Adding the CD4.sup.-
fraction back to CD4.sup.+ DC did not restore the IFN-.alpha.
response, thereby excluding a secondary effect of CpG ODN via
accessory cells in the CD4 fraction. Thus, crosslinking of CD4 on
the surface of pDC appeared to be responsible for the reduced
activity.
Example 10
IFN-.alpha.-Inducing CpG ODN Provide Superior Indirect Activation
of NK Cells Compared to non-IFN-.alpha.-Inducing CpG ODN
[0212] To examine if CpG ODN which induce high amounts of
IFN-.alpha. also show higher activation of NK cells, PBMC were
incubated with different CpG ODN. NK cell activation was measured
in terms of CD69 expression (FACS) and by in vitro NK-cell lytic
activity. For determination of NK-cell lytic activity, PBMC were
incubated with different ODN at various concentrations. After 18
hours cells were harvested and used as effector cells in a standard
4 hour .sup.51Cr-release assay against K562 target cells as
previously described. Hartmann G et al. Gene Therapy 6:893 (1999).
Positive controls included recombinant IL-2 (100 IU/ml) and
negative controls included media alone. Results are expressed as %
specific lysis: specific lysis (%)=((experimental
counts-spontaneous release counts)/(maximal release
counts-spontaneous release counts)).times.100%.
[0213] Results. The IFN-.alpha.-inducing ODN 2216 and ODN 1585
increased the percentage of CD69-positive (early marker of
activation) NK cells within 24 hours (38.+-.12% with ODN 2216; n=5)
as compared to the control without stimulus (8.+-.2%; n=5). ODN
2006 showed a lower response (19%.+-.6%). In agreement with
increased CD69 expression, NK cell-mediated lysis of K562 cells was
markedly enhanced when PBMC were incubated with CpG ODN. Even at
the low concentration of 0.6 .mu.g/ml, ODN 2216 (SEQ ID NO:7) was
still as effective as IL-2 (100 IU/ml) to stimulate NK cell lytic
activity (FIG. 12). ODN 1585 (SEQ ID NO:1) and ODN 2006 (SEQ ID
NO:147) were less effective. Even at higher concentrations (6
.mu.g/ml) the GC control to ODN 1585 (5' ggGGTCAAGCTTGAgggggG 3';
ODN 2118; SEQ ID NO:151) was completely inactive compared to medium
alone (FIG. 12). When purified NK cells were incubated with CpG
ODN, CD69 expression and lysis of K562 cells was not increased,
demonstrating an indirect effect of CpG ODN on NK cells.
Example 11
CpG Oligonucleotide Induces Production of High Amounts of IL-8 by
IPCs
[0214] IL-8 is a chemokine which attracts other immune cells. IPCs
grown in IL-3 produce no IL-8, while CpG oligonucleotide stimulates
the production by IPCs of large amounts of IL-8 (mean 23 ng/ml,
FIG. 13).
[0215] Freshly prepared IPCs (see Example 1, final concentration
2.times.10.sup.5-5.times.10.sup.5 cells per ml) were cultured for
two days in complete medium supplemented with 10 ng/ml IL-3. One
set of parallel cultures was supplemented with 10 .mu.g/ml poly IC,
and another set of parallel cultures supplemented with 6 .mu.g/ml
CpG ODN 2006 (SEQ ID NO:147). Supernatants were analyzed for IL-8
using an ELISA specific for human IL-8 (R&D Systems,
Minneapolis, Minn.) according to the supplier's instructions.
[0216] Results. Representative data from three different donors are
shown in FIG. 13. IL-8 secretion by IPCs was strongly induced by
the addition of CpG oligonucleotide to complete media containing
IL-3. In contrast, addition of poly IC to the media had no effect.
This result demonstrates that CpG oligonucleotide induces IPCs to
produce high amounts of IL-8.
Example 12
Type I IFN Induces Activation and Proliferation of .gamma..delta. T
Cells
[0217] The .gamma..delta. T cells (V.gamma.9/V.delta.2) are
antigen-specific T cells in a preactivated stage which respond to
common non-peptidic phosphoantigens. Exposure of .gamma..delta. T
cells to these antigens stimulates IFN-.gamma. production in the
absence of APC. To examine .gamma..delta. T cell activation, PBMC
(2.times.10.sup.6/ml) from healthy donors were stimulated for three
days with 6 .mu.g/ml CpG ODN (2006, 1585, or 2216) or medium alone
in the presence or absence of 15 .mu.M isopentenyl pyrophosphate
(IPP, specific phosphoantigen for V.gamma.9/V.delta.2 cells).
Brefeldin A was added for the last 4 hours. After surface staining
for V.gamma.9 TCR and CD3, cells were fixed, permeabilized and
stained with mAb against IFN-.gamma.. In three-color flow cytometry
.gamma..delta. T cells were gated using their FSC/SSC profile, CD3
and TCR V.gamma.9 expression and analyzed for IFN-.gamma.
expression. To compare the results from different donors, data were
calculated first as x-fold increase compared to medium or IPP
controls and then multiplied with the mean of medium and IPP,
respectively. Between 14 and 20 donors were analyzed for each CpG
ODN. Data are presented as mean+SEM; * (p<0,01) and **
(p<0,001) indicate p values calculated by Student's t-test for
paired samples comparing medium control to CpG ODN and IPP alone to
IPP+CpG ODN.
[0218] To examine .gamma..delta. T cell proliferation, PBMC from
healthy donors were stimulated with IPP (30 .mu.M) in the presence
or absence of different CpG ODN (2006, 1585, or 2216, each at 6
.mu.g/ml). The expansion of .gamma..delta. TCR positive cells was
assessed by flow cytometry with an anti V.gamma.9 antibody and is
shown as % TCR V.gamma.9 positive cells within viable PBMC. Between
9 and 16 donors were analyzed for each ODN. Data are presented as
x-fold increase compared to IPP alone (mean+SEM); * indicates
p<0,05 (IPP versus IPP+CpG ODN.
[0219] Results. Within PBMCs both .gamma..delta. T cells and NK
cells but not .alpha..beta. T cells responded to CpG ODN with
increased CD69 expression, IFN-.gamma. and TNF-.alpha. production,
perforin content and lytic activity. CpG ODN in combination with
IPP synergistically induced the production of IFN-.gamma. (FIG. 14)
and perforin in .gamma..delta. T cells. The synergistic effect was
more pronounced for ODN 2216 and 1585, i.e., ODN that are strong
inducers of type I IFN, than for ODN 2006. In purified
.gamma..delta. T cells or NK cells, CpG ODN showed no activity or
even reduced IPP-stimulated activity.
[0220] Furthermore CpG ODN synergistically enhanced the
proliferative response of .gamma..delta. T cells to IPP (FIG. 15).
FIG. 15A shows the kinetics of .gamma..delta. T cell expansion from
one representative experiment. FIG. 15B shows the expansion of
.gamma..delta. T cells 10 days after stimulation with IPP alone or
in combination with different CpG ODN.
[0221] The addition of recombinant IFN-.alpha./.beta. or IL-12
mimicked the stimulatory effects of CpG ODN within PBMC. Functional
IL12p70 could not be detected in the supernatants of PBMCs
stimulated with CpG ODN. The potential of CpG ODN to activate
.gamma..delta. T-cells and NK cells correlated well with their
ability to induce IFN-.alpha./.beta.. The blockade of
IFN-.alpha./.beta. function by a combination of neutralizing
antibody to IFN-.alpha./.beta. protein and the corresponding
receptor inhibited CpG ODN-induced activation of .gamma..delta. T
cells and NK cells. A neutralizing antibody to IL-12 or the
addition of IL-18 binding protein reduced baseline IFN-.gamma. but
not CpG ODN-stimulated IFN-.gamma.. Neutralizing TNF-.alpha., IL-10
or IL-15 showed no effect. In conclusion the results demonstrated
that (i) IFN-.alpha./.beta. is a potent activator of .gamma..delta.
T cells; (ii) CpG ODN activates .gamma..delta. T cells and NK cells
via induction of IFN-.alpha./.beta.; (iii) CpG ODN which are strong
inducers of type I IFN are more potent than ODN which are not
strong inducers of type I IFN to activate .gamma..delta. T cells
and NK cells; (iv) CpG ODN costimulate antigen-specific T cell
responses in .gamma..delta. T cells; and (v) CpG ODN-induced
nonspecific activation of .gamma..delta. T cells and NK cells
provides early IFN-.gamma. which promotes Th1 responses.
Example 13
Type I IFN-Inducing ISNA Inhibit IL-12 Production
[0222] IFN-.beta. has been described to downregulate IL-12
production. The effect of type I IFN-inducing and non-type I
IFN-inducing ISNA on IL-12 production was therefore studied as
follows. PBMC (2.times.10.sup.6/ml) from healthy donors were
stimulated with 25 .mu.g/ml of a stimulating anti-CD40 antibody in
the presence of IL-4 (100 U/ml), GM-CSF (10 U/ml) and IFN-.gamma.
(10 ng/ml). Either medium, 6 .mu.g/ml ODN 2006 (SEQ ID NO:147), 6
.mu.g/ml ODN 1585 (SEQ ID NO:1), or a combination of 5000 U/ml
recombinant IFN-.alpha. and 500 U/ml IFN-.beta. were added. After
48 hours IL12p70 was measured in the supernatant by ELISA. Data are
shown as x-fold of IL12p70 production by anti-CD40 alone (mean=143
pg/ml) and represent the mean (+ SEM) of three different
donors.
[0223] Results. FIG. 16 shows that ODN 1585 in combination with
anti-CD40 inhibited IL12p70 production compared to anti-CD40
control, to an extent similar to the inhibition by addition of
recombinant IFN-.alpha. and recombinant IFN-.beta.. In contrast,
ODN 2006 in combination with anti-CD40 boosted IL12p70 production
beyond anti-CD40 positive control. These results show that, in
PBMC, ISNA that induce type I IFN can suppress production of
IL-12p70, and conversely ISNA that do not induce type I IFN can
boost production of IL-12p70.
[0224] Analysis of mRNA levels by quantitative real-time PCR
revealed the induction of small but equal copy numbers of IL-12p40
and IL-12p35 mRNA by ISNA that do not induce type I IFN. In
contrast, ISNA that induce type I IFN induced a higher number of
copies of IL-12 p35 mRNA, but IL-12p40 mRNA could not be detected.
ISNA that do not induce type I IFN enhanced (170%), and ISNA that
induce type I IFN blocked (25%) IL-12p70 synthesis. Inhibition of
IL-12p70 could be mimicked by recombinant IFN-.beta.. A combination
of neutralizing antibodies to IFN-.alpha./.beta. protein and
receptor reversed the type I IFN-inducing ISNA-mediated inhibition
of IL-12p70. These results demonstrate that CpG ODN which are
strong inducers of type I IFN suppress CD40-dependent IL-12p70
production by an IFN-.alpha./.alpha.-mediated negative feedback
mechanism on IL-12p40 mRNA production. Thus the interaction of T
cells and antigen presenting cells via CD40L leads to a cytokine
milieu dominated by IL-12 (ISNA that do not induce type I IFN) or
IFN-.alpha./.beta. (ISNA that induce type I IFN). Although both
promote Th1 responses, ISNA that do not induce type I IFN may be
superior for priming naive T cells, and ISNA that induce type I IFN
may have higher activity to support preactivated and memory T
cells.
Example 14
Effect of CpG ODN on Primary and Recall Peptide-Specific Human CTL
Responses
[0225] CD8+ T cells (1.times.10.sup.6) from HLA A2 positive healthy
donors were stimulated in 24 well plates in the presence or absence
of CpG ODN 2006 (SEQ ID NO:147), 1585 (SEQ ID NO:1), or 2216 (SEQ
ID NO:7) at 6 .mu.g/ml with either a HLA A2-restricted peptide
derived from the influenza matrix protein (GILGFVFTL) or a peptide
derived from the melan A/mart-1 protein (ELAGIGILTV). Autologous
PBMC (3.times.10.sup.6) were used as APCs. After 14 days cells were
harvested, washed, and restimulated with influenza matrix or
melan-A peptides for 6 hours. Brefeldin A was added for the last 4
hours. Cells were stained for CD8 and CD3, subsequently fixed,
permeabilized and stained with mAb against IFN-.gamma.. Also after
14 days the percentage of tetramer-positive CD8.sup.+ T cells
(HLA-A2/melan-A-peptide and HLA-A2/influenza matrix-peptide) was
determined by flow cytometry. Tetramers are fluorochrome-labeled
MHC-peptide tetramers which are designed to bind specifically to a
peptide-specific T cell receptor, making it possible to identify
peptide-specific T cells using flow cytometry. Altman J D et al.
Science 274:94-96 (1996); U.S. Pat. No. 5,635,363.
[0226] Results. In three-color flow cytometry CD8.sup.+ T cells
(CTL) were analyzed for IFN-.gamma. expression. Results are
presented in FIG. 17A and 17C as % IFN-.gamma. positive cells of
all CD8.sup.+ T cells. Peptide specificity was tested by
stimulating with an irrelevant HLA A2 peptide derived from HIV pol
and was <0.2% for all samples. Data from 7 donors are presented
as mean+SEM. These results show that ODN 2006 increased both
primary and recall CTL responses to melan A/mart-1 peptide and to
influenza peptide, respectively, in contrast to ODN 1585 and ODN
2216, which induced less recall and had no effect or even inhibited
the development of primary CTLs.
[0227] Results from the quantification of antigen-specific CTLs
using MHC-tetramer staining are shown for influenza peptide and
melan A/mart-1 peptide in FIG. 17B and 17D, respectively. Data from
7 donors are presented as mean+SEM; * indicate p values<0,05
calculated by Student's t-test for paired samples (medium compared
to stimulation with CpG ODN).
Example 15
IFN-.alpha. Secretion in "High Responders"
[0228] PBMC from 12 different donors were incubated with varying
concentrations of ODN selected from a panel ODN including: ODN 2336
(SEQ ID NO:37), ODN 2334 (SEQ ID NO:36), ODN 2295 (SEQ ID NO:20),
ODN 2255 (SEQ ID NO:16), ODN 2247 (SEQ ID NO:11), ODN 2216 (SEQ ID
NO:7), and ODN 2006 (SEQ ID NO:147). Results from this study
indicated that 6 of the donors could be classified as "high
responders" in that the cells of these blood donors secreted more
than 500 pg/ml (up to 7000 pg/ml) IFN-.alpha. after incubation with
the selected ODN. A discrimination between "high" and "low"
responders could be made because the cells of the 6 remaining
donors only secreted amounts of IFN-.alpha. between 10 and 500
pg/ml. One reason for these differing results might come about from
using buffy coats that are at least 24 h old. pDC, as the main cell
type secreting IFN-.alpha., survive only about 3 days in cell
culture so that PBMC from buffy coats at least 24 hours old may
contain very low numbers of this cell type.
[0229] IFN-.alpha. was measured in the above experiments by using
an ELISA kit that recognizes all subtypes of IFN-.alpha.. Most
other ELISA kits, in contrast, only measure IFN-.alpha.2B.
Therefore, the amounts of IFN-.alpha.2B were compared against all
IFN-.alpha. subtypes in several experiments to obtain information
about possible differences in induction of different IFN-.alpha.
subtypes. In addition, the amounts of IFN-.alpha. were compared
against IFN-.gamma.. Based on the results of this study, there was
a correlation between the induction of IFN-.alpha.2B and all
IFN-.alpha. subtypes. In contrast, however, ther was no clear
correlation between IFN-.alpha. and IFN-.gamma..
Example 16
Induction of IFN-.alpha. Secretion by Select CpG ODN
[0230] Human PBMCs from a single donor were enriched for DC by
going through the first step of the Miltenyi DC isolation kit which
depletes monocytes, NK cells, and T cells, leaving mostly B cells,
RBCs, and DCs. These were then incubated for two days in the
presence of IL-3 (10 ng/ml) and various ODN at 6 .mu.g/ml: ODN 1585
(SEQ ID NO:1), ODN 2022 (SEQ ID NO:2), ODN 2118 (SEQ ID NO:151),
ODN 2184 (SEQ ID NO:3), ODN 2185 (SEQ ID NO:4), ODN 2192 (SEQ ID
NO:5), ODN 2197 (SEQ ID NO:148), ODN 2198 (SEQ ID NO:149), ODN 2204
(SEQ ID NO:6), ODN 2216 (SEQ ID NO:7), or ODN 2217 (SEQ ID NO:8).
In parallel samples, IFN-.gamma. was added at 1000 U/ml.
Supernatants were collected and analyzed in an ELISA specific for
IFN-.alpha..
[0231] Results. ODN induced IFN-.alpha. to varying degrees, with
some augmentation by the addition of IFN-.gamma.. Some of the ODN
induced IFN-.alpha. to an exceptional degree (>50,000 pg/ml)
even in the absence of added IFN-.gamma..
Example 17
Donor and Sequence Dependence of IFN-.alpha. Response to Various
ODN
[0232] PBMC taken from four different donors were incubated for two
day with a variety of ODN at 0.1 .mu.g/ml. The panel of ODN
included the following:
12 ggGGTCAACGTTGAgggggG ODN 1585 SEQ ID NO:1 ggggtcgtcgttttgggggg
ODN 2184 SEQ ID NO:3 tcgtcgttttgtcgttttgggggg ODN 2185 SEQ ID NO:4
ggggtcgacgtcgagggggg ODN 2192 SEQ ID NO:5 gmGGTCAACGTTGAgggmggG ODN
2197 SEQ ID NO:148 ggGGAGTTCGTTGAgggggG ODN 2198 SEQ ID NO:149
ggggtcatcgatgagggggg ODN 2204 SEQ ID NO:6 ggGGGACGATCGTCgggggG ODN
2216 SEQ ID NO:7 gggggtcgtacgacgggggg ODN 2217 SEQ ID NO:8
ggggacgtcgacgtgggggg ODN 2229 SEQ ID NO:152 ggggtcgttcgaacgagggggg
ODN 2237 SEQ ID NO:153 ggggacgttcgaacgtgggggg ODN 2238 SEQ ID
NO:154 ggGGGAGCATGCTGgggggG ODN 2243 SEQ ID NO:155
ggGGGACGATATCGTCgggggG ODN 2245 SEQ ID NO:9 ggGGGACGACGTCGTCgggggG
ODN 2246 SEQ ID NO:10 ggGGGACGAGCTCGTCgggggG ODN 2247 SEQ ID NO:11
ggGGGACGTACGTCgggggG ODN 2248 SEQ ID NO:12 ggGGGACGATCGTTGggggG ODN
2252 SEQ ID NO:13 ggGGAACGATCGTCgggggG ODN 2253 SEQ ID NO:14
ggGGGGACGATCGTCgggggG ODN 2254 SEQ ID NO:15 ggGGGACGATCGTCGgggggG
ODN 2255 SEQ ID NO:16 ggGGGTCATCGATGAgggggG ODN 2260 SEQ ID NO:17
ggGGGTCAACGTTGAgggggG ODN 2261 SEQ ID NO:156 ggGGTCGTCGACGAgggggG
ODN 2293 SEQ ID NO:18 ggGGTCGTTCGAACGAgggggG ODN 2294 SEQ ID NO:19
ggGGACGTTCGAACGTgggggG ODN 2295 SEQ ID NO:20 ggGGAACGACGTCGTTgggggG
ODN 2297 SEQ ID NO:21 ggGGAACGTACGTCgggggG ODN 2298 SEQ ID NO:22
ggGGAACGTACGTACGTTgggggG ODN 2299 SEQ ID NO:23 ggGGTCACCGGTGAgggggG
ODN 2300 SEQ ID NO:24 ggGGTCGACGTACGTCGAgggggG ODN 2301 SEQ ID
NO:25 ggGGACCGGTACCGGTgggggG ODN 2302 SEQ ID NO:26
ggGTCGACGTCGAgggggG ODN 2303 SEQ ID NO:27 ggGGTCGACGTCGagggg ODN
2304 SEQ ID NO:28 ggGGAACGTTAACGTTgggggG ODN 2305 SEQ ID NO:29
ggGGACGTCGACGTggggG ODN 2306 SEQ ID NO:30 ggGGGTCGTTCGTTgggggG ODN
2311 SEQ ID NO:31 ggGGGATGATTGTTgggggG ODN 2312 SEQ ID NO:157
ggGGGAZGATZGTTgggggG ODN 2313 SEQ ID NO:158 ggGGGAGCTAGCTTgggggG
ODN 2314 SEQ ID NO:159 ggGACGATCGTCGgggggG ODN 2328 SEQ ID NO:32
ggGTCGTCGACGAggggggG ODN 2329 SEQ ID NO:33 ggTCGTCGACGAGgggggG ODN
2330 SEQ ID NO:34 ggGTCGTCGTCGTGgggggG ODN 2331 SEQ ID NO:160
ggGGACGATCGTCGgggggG ODN 2332 SEQ ID NO:35 ggGGACGTCGTCGTgggggG ODN
2333 SEQ ID NO:161 ggGGTCGACGTCGACGTCGAGgggggG ODN 2334 SEQ ID
NO:36 ggGGAACCGCGGTTgggggG ODN 2335 SEQ ID NO:162 (Z in ODN 2313
represents 5-methyl cytosine)
[0233] Supernatants were collected and assayed for IFN-.alpha. by
ELISA. In a parallel set of experiments PBMC taken from the same
four different donors were incubated for two day with the dame
panel of ODN at 1 .mu.g/ml.
[0234] Results. The results for PBMC derived from the four donors
and incubated with ODN at 0.1 .mu.g/ml and with ODN at 1 .mu.g/ml
again showed dose and donor variation, with several ODN inducing
IFN-.alpha. to levels of at least 5000 pg/ml and some inducing
IFN-.alpha. to levels well in excess of 5000 pg/ml.
[0235] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
169 1 20 DNA Artificial Sequence Synthetic Oligonucleotide 1
ggggtcaacg ttgagggggg 20 2 24 DNA Artificial Sequence Synthetic
Oligonucleotide 2 tcgtcgtttt gtcgttttgt cgtt 24 3 20 DNA Artificial
Sequence Synthetic Oligonucleotide 3 ggggtcgtcg ttttgggggg 20 4 24
DNA Artificial Sequence Synthetic Oligonucleotide 4 tcgtcgtttt
gtcgttttgg gggg 24 5 20 DNA Artificial Sequence Synthetic
Oligonucleotide 5 ggggtcgacg tcgagggggg 20 6 20 DNA Artificial
Sequence Synthetic Oligonucleotide 6 ggggtcatcg atgagggggg 20 7 20
DNA Artificial Sequence Synthetic Oligonucleotide 7 gggggacgat
cgtcgggggg 20 8 20 DNA Artificial Sequence Synthetic
Oligonucleotide 8 gggggtcgta cgacgggggg 20 9 22 DNA Artificial
Sequence Synthetic Oligonucleotide 9 gggggacgat atcgtcgggg gg 22 10
22 DNA Artificial Sequence Synthetic Oligonucleotide 10 gggggacgac
gtcgtcgggg gg 22 11 22 DNA Artificial Sequence Synthetic
Oligonucleotide 11 gggggacgag ctcgtcgggg gg 22 12 20 DNA Artificial
Sequence Synthetic Oligonucleotide 12 gggggacgta cgtcgggggg 20 13
20 DNA Artificial Sequence Synthetic Oligonucleotide 13 gggggacgat
cgttgggggg 20 14 20 DNA Artificial Sequence Synthetic
Oligonucleotide 14 ggggaacgat cgtcgggggg 20 15 21 DNA Artificial
Sequence Synthetic Oligonucleotide 15 ggggggacga tcgtcggggg g 21 16
21 DNA Artificial Sequence Synthetic Oligonucleotide 16 gggggacgat
cgtcgggggg g 21 17 21 DNA Artificial Sequence Synthetic
Oligonucleotide 17 ggggtcatc gatgaggggg g 21 18 20 DNA Artificial
Sequence Synthetic Oligonucleotide 18 ggggtcgtcg acgagggggg 20 19
22 DNA Artificial Sequence Synthetic Oligonucleotide 19 ggggtcgttc
gaacgagggg gg 22 20 22 DNA Artificial Sequence Synthetic
Oligonucleotide 20 ggggacgttc gaacgtgggg gg 22 21 22 DNA Artificial
Sequence Synthetic Oligonucleotide 21 ggggaacgac gtcgttgggg gg 22
22 20 DNA Artificial Sequence Synthetic Olignucleotide 22
ggggaacgta cgtcgggggg 20 23 24 DNA Artificial Sequence Synthetic
Oligonucleotide 23 ggggaacgta cgtacgttgg gggg 24 24 20 DNA
Artificial Sequence Synthetic Oligonucleotide 24 ggggtcaccg
gtgagggggg 20 25 24 DNA Artificial Sequence Synthetic
Oligonucleotide 25 ggggtcgacg tacgtcgagg gggg 24 26 22 DNA
Artificial Sequence Synthetic Oligonucleotide 26 ggggaccggt
accggtgggg gg 22 27 19 DNA Artificial Sequence Synthetic
Oligonucleotide 27 gggtcgacgt cgagggggg 19 28 18 DNA Artificial
Sequence Synthetic Oligonucleotide 28 ggggtcgacg tcgagggg 18 29 22
DNA Artificial Sequence Synthetic Oligonucleotide 29 ggggaacgtt
aacgttgggg gg 22 30 19 DNA Artificial Sequence Synthetic
Oligonucleotide 30 ggggacgtcg acgtggggg 19 31 20 DNA Artificial
Sequence Synthetic Oligonucleotide 31 gggggtcgtt cgttgggggg 20 32
19 DNA Artificial Sequence Synthetic Oligonucleotide 32 gggacgatcg
tcggggggg 19 33 20 DNA Artificial Sequence Synthetic
Oligonucleotide 33 gggtcgtcga cgaggggggg 20 34 19 DNA Artificial
Sequence Synthetic Oligonucleotide 34 ggtcgtcgac gaggggggg 19 35 20
DNA Artificial Sequence Synthetic Oligonucleotide 35 ggggacgatc
gtcggggggg 20 36 27 DNA Artificial Sequence Synthetic
Oligonucleotide 36 ggggtcgacg tcgacgtcga ggggggg 27 37 21 DNA
Artificial Sequence Synthetic Oligonucleotide 37 ggggacgacg
tcgtgggggg g 21 38 8 DNA Artificial Sequence Synthetic
Oligonucleotide 38 aacgttct 8 39 24 DNA Artificial Sequence
Synthetic Oligonucleotide 39 accatggacg aactgtttcc cctc 24 40 24
DNA Artificial Sequence Synthetic Oligonucleotide 40 accatggacg
acctgtttcc cctc 24 41 24 DNA Artificial Sequence Synthetic
Oligonucleotide 41 accatggacg agctgtttcc cctc 24 42 24 DNA
Artificial Sequence Synthetic Oligonucleotide 42 accatggacg
atctgtttcc cctc 24 43 24 DNA Artificial Sequence Synthetic
Oligonucleotide 43 accatggacg gtctgtttcc cctc 24 44 24 DNA
Artificial Sequence Synthetic Oligonucleotide 44 accatggacg
tactgtttcc cctc 24 45 24 DNA Artificial Sequence Synthetic
Oligonucleotide 45 accatggacg ttctgtttcc cctc 24 46 18 DNA
Artificial Sequence Synthetic Oligonucleotide 46 agctatgacg
ttccaagg 18 47 20 DNA Artificial Sequence Synthetic Oligonucleotide
47 ataggaggtc caacgttctc 20 48 20 DNA Artificial Sequence Synthetic
Oligonucleotide 48 atcgactctc gaacgttctc 20 49 20 DNA Artificial
Sequence Synthetic Oligonucleotide 49 atcgactctc gagcgttctc 20 50
17 DNA Artificial Sequence Synthetic Oligonucleotide 50 atgacgttcc
tgacgtt 17 51 20 DNA Artificial Sequence Synthetic Oligonucleotide
51 atggaaggtc caacgttctc 20 52 20 DNA Artificial Sequence Synthetic
Oligonucleotide 52 atggaaggtc cagcgttctc 20 53 20 DNA Artificial
Sequence Synthetic Oligonucleotide 53 atggactctc cagcgttctc 20 54
20 DNA Artificial Sequence Synthetic Oligonucleotide 54 atggaggctc
catcgttctc 20 55 7 DNA Artificial Sequence Synthetic
Oligonucleotide 55 caacgtt 7 56 15 DNA Artificial Sequence
Synthetic Oligonucleotide 56 cacgttgagg ggcat 15 57 8 DNA
Artificial Sequence Synthetic Oligonucleotide 57 ccaacgtt 8 58 20
DNA Artificial Sequence Synthetic Oligonucleotide 58 gagaacgatg
gaccttccat 20 59 20 DNA Artificial Sequence Synthetic
Oligonucleotide 59 gagaacgctc cagcactgat 20 60 20 DNA Artificial
Sequence Synthetic Oligonucleotide 60 gagaacgctc gaccttccat 20 61
20 DNA Artificial Sequence Synthetic Oligonucleotide 61 gagaacgctc
gaccttcgat 20 62 20 DNA Artificial Sequence Synthetic
Oligonucleotide 62 gagaacgctg gaccttccat 20 63 15 DNA Artificial
Sequence Synthetic Oligonucleotide 63 gcatgacgtt gagct 15 64 21 DNA
Artificial Sequence Synthetic Oligonucleotide 64 gcgtgcgttg
tcgttgtcgt t 21 65 15 DNA Artificial Sequence Synthetic
Oligonucleotide 65 gctagacgtt agcgt 15 66 15 DNA Artificial
Sequence Synthetic Oligonucleotide 66 gctagacgtt agtgt 15 67 15 DNA
Artificial Sequence Synthetic Oligonucleotide 67 gctagatgtt agcgt
15 68 19 DNA Artificial Sequence Synthetic Oligonucleotide 68
ggggtcaacg ttgacgggg 19 69 19 DNA Artificial Sequence Synthetic
Oligonucleotide 69 ggggtcagtc gtgacgggg 19 70 6 DNA Artificial
Sequence Synthetic Oligonucleotide 70 gtcgyt 6 71 8 DNA Artificial
Sequence Synthetic Oligonucleotide 71 tcaacgtc 8 72 8 DNA
Artificial Sequence Synthetic Oligonucleotide 72 tcaacgtt 8 73 8
DNA Artificial Sequence Synthetic Oligonucleotide 73 tcagcgct 8 74
12 DNA Artificial Sequence Synthetic Oligonucleotide 74 tcagcgtgcg
cc 12 75 8 DNA Artificial Sequence Synthetic Oligonucleotide 75
tcatcgat 8 76 20 DNA Artificial Sequence Synthetic Oligonucleotide
76 tccacgacgt tttcgacgtt 20 77 20 DNA Artificial Sequence Synthetic
Oligonucleotide 77 tccataacgt tcctgatgct 20 78 20 DNA Artificial
Sequence Synthetic Oligonucleotide 78 tccatagcgt tcctagcgtt 20 79
20 DNA Artificial Sequence Synthetic Oligonucleotide 79 tccatcacgt
gcctgatgct 20 80 20 DNA Artificial Sequence Synthetic
Oligonucleotide 80 tccatgacgg tcctgatgct 20 81 20 DNA Artificial
Sequence Synthetic Oligonucleotide 81 tccatgacgt ccctgatgct 20 82
20 DNA Artificial Sequence Synthetic Oligonucleotide 82 tccatgacgt
gcctgatgct 20 83 20 DNA Artificial Sequence Synthetic
Oligonucleotide 83 tccatgacgt tcctgacgtt 20 84 20 DNA Artificial
Sequence Synthetic Oligonucleotide 84 tccatgacgt tcctgatgct 20 85
20 DNA Artificial Sequence Synthetic Oligonucleotide 85 tccatgccgg
tcctgatgct 20 86 20 DNA Artificial Sequence Synthetic
Oligonucleotide 86 tccatgcgtg cgtgcgtttt 20 87 20 DNA Artificial
Sequence Synthetic Oligonucleotide 87 tccatgcgtt gcgttgcgtt 20 88
20 DNA Artificial Sequence Synthetic Oligonucleotide 88 tccatggcgg
tcctgatgct 20 89 20 DNA Artificial Sequence Synthetic
Oligonucleotide 89 tccatgtcga tcctgatgct 20 90 20 DNA Artificial
Sequence Synthetic Oligonucleotide 90 tccatgtcgc tcctgatgct 20 91
20 DNA Artificial Sequence Synthetic Oligonucleotide 91 tccatgtcgg
tcctgacgca 20 92 20 DNA Artificial Sequence Synthetic
Oligonucleotide 92 tccatgtcgg tcctgatgct 20 93 20 DNA Artificial
Sequence Synthetic Oligonucleotide 93 tccatgtcgg tcctgctgat 20 94
20 DNA Artificial Sequence Synthetic Oligonucleotide 94 tccatgtcgt
ccctgatgct 20 95 20 DNA Artificial Sequence Synthetic
Oligonucleotide 95 tccatgtcgt tcctgtcgtt 20 96 20 DNA Artificial
Sequence Synthetic Oligonucleotide 96 tccatgtcgt ttttgtcgtt 20 97
19 DNA Artificial Sequence Synthetic Oligonucleotide 97 tcctgacgtt
cctgacgtt 19 98 19 DNA Artificial Sequence Synthetic
Oligonucleotide 98 tcctgtcgtt cctgtcgtt 19 99 20 DNA Artificial
Sequence Synthetic Oligonucleotide 99 tcctgtcgtt ccttgtcgtt 20 100
20 DNA Artificial Sequence Synthetic Oligonucleotide 100 tcctgtcgtt
ttttgtcgtt 20 101 20 DNA Artificial Sequence Synthetic
Oligonucleotide 101 tccttgtcgt tcctgtcgtt 20 102 21 DNA Artificial
Sequence Synthetic oligonucleotide 102 tcgtcgctgt ctccccttct t 21
103 21 DNA Artificial Sequence Synthetic Oligonucleotide 103
tcgtcgctgt ctgcccttct t 21 104 21 DNA Artificial Sequence Synthetic
Oligonucleotide 104 tcgtcgctgt tgtcgtttct t 21 105 14 DNA
Artificial Sequence Synthetic Oligonucleotide 105 tcgtcgtcgt cgtt
14 106 20 DNA Artificial Sequence Synthetic Oligonucleotide 106
tcgtcgttgt cgttgtcgtt 20 107 22 DNA Artificial Sequence Synthetic
Oligonucleotide 107 tcgtcgttgt cgttttgtcg tt 22 108 24 DNA
Artificial Sequence Synthetic Oligonucleotide 108 tcgtcgtttt
gtcgttttgt cgtt 24 109 17 DNA Artificial Sequence Synthetic
Oligonucleotide 109 tctcccagcg ggcgcat 17 110 18 DNA Artificial
Sequence Synthetic Oligonucleotide 110 tctcccagcg tgcgccat 18 111 8
DNA Artificial Sequence Synthetic Oligonucleotide 111 tcttcgaa 8
112 8 DNA Artificial Sequence Synthetic Oligonucleotide 112
tcttcgat 8 113 13 DNA Artificial Sequence Synthetic Oligonucleotide
113 tgtcgttgtc gtt 13 114 19 DNA Artificial Sequence Synthetic
Oligonucleotide 114 tgtcgttgtc gttgtcgtt 19 115 25 DNA Artificial
Sequence Synthetic Oligonucleotide 115 tgtcgttgtc gttgtcgttg tcgtt
25 116 21 DNA Artificial Sequence Synthetic Oligonucleotide 116
tgtcgtttgt cgtttgtcgt t 21 117 7 DNA Artificial Sequence Synthetic
Oligonucleotide 117 tgtcgyt 7 118 20 DNA Artificial Sequence
Synthetic Oligonucleotide 118 atggaaggtc caaggggctc 20 119 20 DNA
Artificial Sequence Synthetic Oligonucleotide 119 atggaaggtc
cagggggctc 20 120 20 DNA Artificial Sequence Synthetic
Oligonucleotide 120 atggaaggtc cggggttctc 20 121 20 DNA Artificial
Sequence Synthetic Oligonucleotide 121 atggactctc cggggttctc 20 122
20 DNA Artificial Sequence Synthetic Oligonucleotide 122 atggactctg
gagggggctc 20 123 20 DNA Artificial Sequence Synthetic
Oligonucleotide 123 atggactctg gaggggtctc 20 124 20 DNA Artificial
Sequence Synthetic Oligonucleotide 124 atggactctg gggggttctc 20 125
20 DNA Artificial Sequence Synthetic Oligonucleotide 125 atggaggctc
catggggctc 20 126 20 DNA Artificial Sequence Synthetic
Oligonucleotide 126 gagaaggggc cagcactgat 20 127 20 DNA Artificial
Sequence Synthetic Oligonucleotide 127 gagaaggggg gaccttccat 20 128
20 DNA Artificial Sequence Synthetic Oligonucleotide 128 gagaaggggg
gaccttggat 20 129 15 DNA Artificial Sequence Synthetic
Oligonucleotide 129 gcatgagggg gagct 15 130 14 DNA Artificial
Sequence Synthetic Oligonucleotide 130 gctagaggga gtgt 14 131 15
DNA Artificial Sequence Synthetic Oligonucleotide 131 gctagagggg
agggt 15 132 15 DNA Artificial Sequence Synthetic Oligonucleotide
132 gctagatgtt agggg 15 133 20 DNA Artificial Sequence Synthetic
Oligonucleotide 133 gggggacgat cgtcgggggg 20 134 20 DNA Artificial
Sequence Synthetic Oligonucleotide 134 gggggggggg gggggggggg 20 135
20 DNA Artificial Sequence Synthetic Oligonucleotide 135 ggggtcaacg
ttgagggggg 20 136 20 DNA Artificial Sequence Synthetic
Oligonucleotide 136 ggggtcgacg tcgagggggg 20 137 20 DNA Artificial
Sequence Synthetic Oligonucleotide 137 tccatcgggg gcctgatgct 20 138
20 DNA Artificial Sequence Synthetic Olignucleotide 138 tccatgaggg
gcctgatgct 20 139 20 DNA Artificial Sequence Synthetic
Oligonucleotide 139 tccatgcggg tggggatgct 20 140 20 DNA Artificial
Sequence Synthetic Oligonucleotide 140 tccatggggg tcctgatgct 20 141
20 DNA Artificial Sequence Synthetic Oligonucleotide 141 tccatggggt
ccctgatgct 20 142 20 DNA Artificial Sequence Synthetic
Oligonucleotide 142 tccatggggt gcctgatgct 20 143 20 DNA Artificial
Sequence Synthetic Oligonucleotide 143 tccatggggt tcctgatgct 20 144
20 DNA Artificial Sequence Synthetic Oligonucleotide 144 tccatgtggg
gcctgatgct 20 145 20 DNA Artificial Sequence Synthetic
Oligonucleotide 145 tccatgtggg gcctgctgat 20 146 20 DNA Artificial
Sequence Synthetic Oligonucleotide 146 tccatgtggg tggggatgct 20 147
24 DNA Artificial Sequence Synthetic Oligonucleotide 147 tcgtcgtttt
gtcgttttgt cgtt 24 148 21 DNA Artificial Sequence Synthetic
Oligonucleotide 148 gmggtcaacg ttgagggmgg g 21 149 20 DNA
Artificial Sequence Synthetic Oligonucleotide 149 ggggagttcg
ttgagggggg 20 150 20 DNA Artificial Sequence Synthetic
Oligonucleotide 150 gggggagcat gctcgggggg 20 151 20 DNA Artificial
Sequence Synthetic Oligonucleotide 151 ggggtcaagc ttgagggggg 20 152
20 DNA Artificial Sequence Synthetic Oligonucleotide 152 ggggacgtcg
acgtgggggg 20 153 22 DNA Artificial Sequence Synthetic
Oligonucleotide 153 ggggtcgttc gaacgagggg gg 22 154 22 DNA
Artificial Sequence Synthetic Oligonucleotide 154 ggggacgttc
gaacgtgggg gg 22 155 20 DNA Artificial Sequence Synthetic
Oligonucleotide 155 gggggagcat gctggggggg 20 156 21 DNA Artificial
Sequence Synthetic Oligonucleotide 156 gggggtcaac gttgaggggg g 21
157 20 DNA Artificial Sequence Synthetic Oligonucleotide 157
gggggatgat tgttgggggg 20 158 20 DNA Artificial Sequence Synthetic
Oligonucleotide 158 gggggangan tgttgggggg 20 159 20 DNA Artificial
Sequence Synthetic Oligonucleotide 159 gggggagcta gcttgggggg 20 160
20 DNA Artificial Sequence Synthetic Oligonucleotide 160 gggtcgtcgt
cgtggggggg 20 161 20 DNA Artificial Sequence Synthetic
Oligonucleotide 161 ggggacgtcg tcgtgggggg 20 162 20 DNA Artificial
Sequence Synthetic Oligonucleotide 162 ggggaaccgc ggttgggggg 20 163
45 DNA Artificial Sequence Synthetic Oligonucleotide 163 accgatgacg
tcgccggtga cggcaccacg acggccaccg tgctg 45 164 30 DNA Artificial
Sequence Synthetic Oligonucleotide 164 accgatgacg tcgccggtga
cggcaccacg 30 165 30 DNA Artificial Sequence Synthetic
Oligonucleotide 165 gggggggggg ggaacgttgg gggggggggg 30 166 9 PRT
Artificial Sequence Synthetic Peptide 166 Gly Ile Leu Gly Phe Val
Phe Thr Leu 1 5 167 10 PRT Artificial Sequence Synthetic Peptide
167 Glu Leu Ala Gly Ile Gly Ile Leu Thr Val 1 5 10 168 26 DNA
Artificial Sequence Synthetic Oligonucleotide 168 gggnnnnnnn
nnnnnnnnnn nnnggg 26 169 49 DNA Artificial Sequence Synthetic
Oligonucleotide 169 gggnnnnnnn nnnnnnnnnn nnngggnnnn nnnnnnnnnn
nnnnnnggg 49
* * * * *