U.S. patent application number 11/128127 was filed with the patent office on 2005-11-03 for immunomodulatory oligonucleotides.
This patent application is currently assigned to University of Iowa Research Foundation. Invention is credited to Klinman, Dennis, Krieg, Arthur M., Steinberg, Alfred D..
Application Number | 20050244380 11/128127 |
Document ID | / |
Family ID | 23524008 |
Filed Date | 2005-11-03 |
United States Patent
Application |
20050244380 |
Kind Code |
A1 |
Krieg, Arthur M. ; et
al. |
November 3, 2005 |
Immunomodulatory oligonucleotides
Abstract
Oligonucleotides containing unthylated CpG dinucleotides and
therapeutic utilities based on their ability to stimulate an immune
response in a subject are disclosed. Also disclosed are therapies
for treating diseases associated with immune system activation that
are initiated by unthylated CpG dinucleotides in a subject
comprising administering to the subject oligonucleotides that do
not contain unmethylated CpG sequences (i.e. methylated CpG
sequences or no CpG sequence) to outcompete unmethylated CpG
nucleic acids for binding. Further disclosed are methylated CpG
containing dinucleotides for use antisense therapies or as in vivo
hybridization probes, and immunoinhibitory oligonucleotides for use
as antiviral therapeutics.
Inventors: |
Krieg, Arthur M.;
(Wellesley, MA) ; Klinman, Dennis; (Potomac,
MD) ; Steinberg, Alfred D.; (Potomac, MD) |
Correspondence
Address: |
WOLF GREENFIELD & SACKS, PC
FEDERAL RESERVE PLAZA
600 ATLANTIC AVENUE
BOSTON
MA
02210-2211
US
|
Assignee: |
University of Iowa Research
Foundation
Iowa City
IA
Coley Pharmaceutical Group, Inc.
Wellesley
MA
United States of America, as represented by the Secretary,
Department of Health and Human Services
Bethesda
MD
|
Family ID: |
23524008 |
Appl. No.: |
11/128127 |
Filed: |
May 11, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11128127 |
May 11, 2005 |
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10690495 |
Oct 21, 2003 |
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10690495 |
Oct 21, 2003 |
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09415142 |
Oct 9, 1999 |
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09415142 |
Oct 9, 1999 |
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08386063 |
Feb 7, 1995 |
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6194388 |
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08386063 |
Feb 7, 1995 |
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08276358 |
Jul 15, 1994 |
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Current U.S.
Class: |
424/93.2 ;
424/450; 514/44A; 536/23.1 |
Current CPC
Class: |
A61K 39/39 20130101;
C07H 21/00 20130101; A61K 31/4706 20130101; C12Q 1/68 20130101;
A61K 2039/55561 20130101 |
Class at
Publication: |
424/093.2 ;
514/044; 536/023.1; 424/450 |
International
Class: |
C07H 021/02; A61K
048/00; A61K 009/127 |
Goverment Interests
[0001] The work resulting in this invention was supported in part
by National Institute of Health Grant No. R29-ARP42556-01. The U.S.
Government may therefore be entitled to certain rights in the
invention.
Claims
1-36. (canceled)
37. A composition comprising: an immunostimulatory oligonucleotide
of 8 to 40 nucleotides in length, comprising:
5'X.sub.1X.sub.2CGX.sub.3X.sub.4.su- p.3', wherein C and G are
unmethylated and X.sub.1, X.sub.2, X.sub.3, and X.sub.4 are
nucleotides, wherein the immunostimulatory oligonucleotide includes
a phosphorothioate backbone modification and an antigen in a
pharmaceutically acceptable carrier.
38. The composition of claim 37, wherein the immunostimulatory
oligonucleotide does not include a GCG trinucleotide at a 5' and/or
3' terminal.
39. The composition of claim 37, wherein the immunostimulatory
oligonucleotide does not contain a
5'X.sub.1X.sub.2CGX.sub.3X.sub.4.sup.3- ' palindrome.
40. The composition of claim 38, wherein the immunostimulatory
oligonucleotide does not contain a
5'X.sub.1X.sub.2CGX.sub.3X.sub.4.sup.3- ' palindrome.
41. A method of inducing an antigen-specific immune response in a
subject comprising: administering a vaccine to a subject wherein
the vaccine includes an antigen in combination with an
immunostimulatory oligonucleotide of claim 37, in an amount
effective to induce the antigen-specific immune response.
42. A method of inducing an antigen-specific immune response in a
subject comprising: administering a vaccine to a subject wherein
the vaccine includes an antigen in combination with an
immunostimulatory oligonucleotide of claim 38, in an amount
effective to induce the antigen-specific immune response.
43. A method of inducing an antigen-specific immune response in a
subject comprising: administering a vaccine to a subject wherein
the vaccine includes an antigen in combination with an
immunostimulatory oligonucleotide of claim 39, in an amount
effective to induce the antigen-specific immune response.
44. A method of inducing an antigen-specific immune response in a
subject comprising: administering a vaccine to a subject wherein
the vaccine includes an antigen in combination with an
immunostimulatory oligonucleotide of claim 40, in an amount
effective to induce the antigen-specific immune response.
Description
BACKGROUND OF THE INVENTION
[0002] DNA binds to cell membrane and is internalized
[0003] In the 1970's, several investigators reported the binding of
high molecular weight DNA to cell membranes (Lemer, R. A., W.
Meinke, and D. A. Goldstein. 1971. "Membrane-associated DNA in the
cytoplasm of diploid human lymphocyates, Proc. Natl. Acad Sci. USA
68:1212; Agrawal, S. K, R. W. Wagner, P. K. McAllister, and B.
Rosenberg, 1975. "Cell-surface-associated nucleic acid in
tumorigenic cells made visible with platinum-pyrimdine complexes by
electron microscopy". Proc. Natl. Acad. Sci. USA 72:928). In 1985
Bennett et al. presented the first evidence that DNA binding to
lymphocytes is similar to a ligand receptor interaction: binding is
saturable, competitive, and leads to DNA endocytosis and
degradation (Bennett, R. M., G. T. Gabor, and M. M. Merritt 1985.
"DNA binding to human leukocytes. Evidence for a receptor-mediated
association internalization, and degradation of DNA". J. Clin.
Invest. 76:2182). Like DNA, oligodeoxyribonucleotides (ODNs) are
able to enter cells in a saturable, sequence independent and
temperature and energy dependent fashion (reviewed in Jaroszewski,
J. W., and J. S. Cohen 1991. "Cellular uptake of antisense
oligodeoxynucleotides". Advanced Drug Delivery Reviews 6:235;
Akhtar, S., Y. Shoji; and R. L. Juliano. 1992. "Pharmaceutical
aspects of the biological stability and membrane transport
characteristics of antisense oligonucleotides". In: Gene
Regulation: Biology of Antisense RNA and DNA. R. P. Erickson, and
J. G. Izant, eds. Raven Press, Ltd. New York, pp. 133; and Zhao,
Q., T. Waldschmidt, E. Fisher, C. J. Herrera, and A. M. Krieg.,
1994. "Stage specific oligonucleotide uptake in murine bone marrow
B cell precursors". Blood, 84:3660). No receptor for DNA or ODN
uptake has yet been cloned, and it is not yet clear whether ODN
binding and cell uptake occurs through the same or a different
mechanism from that of high molecular weight DNA.
[0004] Lymphocyte ODN uptake has been shown to be regulated by cell
activation. Spleen cells stimulated with the B cell mitogen LPS had
dramatically enhanced ODN uptake in the B cell population, while
spleen cells treated with the T cell mitogen Con A showed enhanced
ODN uptake by T but not B cells (Krieg, A. M., F. Gmelig-Meyling,
M. F. Gourley, W. J. Kisch, L. A. Chrisey, and A. D. Steinberg.
1991. "Uptake of oligodeoxyribonucleotides by lymphoid cells is
heterogeneous and inducible". Antisense Research and Development
1:161).
[0005] Immune effects of nucleic acid
[0006] Several polynucleotides have been extensively evaluated as
biological response modifiers. Perhaps the best example is poly
(I,C) which is a potent inducer of IFN production as well as a
macrophage activator and inducer of NK activity (Talmadge, J. E.,
J. Adams, H. Phillips, M. Collins, B. Lenz, M. Schneider, E.
Schlick, R. Ruffmann, R. H. Wiltrout, and M. A. Chirigos. 1985.
"Inmunomodulatory effects in mice of polyinosinic-polycytidylic
acid complexed with poly-L:-lysine and carboxymethylcellulose".
Cancer Res. 45:1058; Viltrout, R. H., R. R. Salup, T. A. Twilley,
and J. E. Talmadge. 1985. "Immunomodulation of natural killer
activity by polyribonucleotides". J. Biol. Resp. Mod. 4:512; Krown,
S. E. 1986. "Interferons and interferon inducers in cancer
treatment". Sem. Oncol. 13:207; and Ewel, C. H., S. J. Urba, W. C.
Kopp, J. W. Smith II, R. G. Steis, J. L. Rossio, D. L. Longo, M. J.
Jones, W. G. Alvord, C. M. Pinsky, J. M. Beveridge, K. L. McNitt,
and S. P. Creekrnore. 1992. "Polyinosinic-polycytidylic acid
complexed with poly-L-lysine and carboxymethylcellulose in
combination with interleukin 2 in patients with cancer: clinical
and immunological effects". Canc. Res. 52:3005). It appears that
this murine NK activation may be due solely to induction of IFN
.beta. secretion (Ishikawa, R., and C. A. Biron. 1993. "IFN
induction and associated changes in splenic leukocyte
distribution". J. Immunol. 150:3713). This activation was specific
for the ribose sugar since deoxyribose was ineffective. Its potent
in vitro antitumor activity led to several clinical trials using
poly (I,C) complexed with poly-L-lysine and carboxymethylcellulose
(to reduce degradation by RNAse) (Talmadge, J. E., et al., 1985.
cited supra; Wiltrout, R. H., et al., 1985. cited supra); Krown, S.
E., 1986. cited supra); and Ewel, C. H., et al., 1992. cited
supra). Unfortunately, toxic side effects have thus far prevented
poly (I,C) from becoming a useful therapeutic agent
[0007] Guanine ribonucleotides substituted at the C8 position with
either a bromine or a thiol group are B cell mitogens and may
replace "B cell differentiation factors" (Feldbush, T. L., and Z.
K. Ballas. 1985. "Lymphokine-like activity of 8-mercaptoguanosine:
induction of T and B cell differentiation". J. Immunol. 134:3204;
and Goodman, M. G. 1986. "Mechanism of synergy between T cell
signals and C8-substituted guanine nucleosides in humoral immunity:
B lymphotropic cytokines induce responsiveness to
8-mercaptoguanosine". J. Immunol. 136:3335). 8-mercaptoguanosine
and 8-bromoguanosine also can substitute for the cytokine
requirement for the generation of MHC restricted CTI (Feldbush, T.
L., 1985. cited supra), augment murine NK activity (Koo, G. C., M.
E. Jewell, C. L. Manyak, N. H. Sigal, and L. S. Wicker. 1988.
"Activation of murine natural killer cells and macrophages by
8-bromoguanosine". J. Immunol. 140:3249), and synergize with IL-2
in inducing murine LAK generation (Thompson, R. A., and Z. K.
Ballas. 1990. "Lymphokine-activated killer (LAK) cells.
V.8-Mercaptoguanosine as an IL-2-sparing agent in LAK generation".
J. Immunol. 145:3524). The NK and LAK augmenting activities of
these C8-substituted guanosines appear to be due to their induction
of IFN (Thompson, R. A., et al. 1990. cited supra). Recently, a 5'
triphosphorylated thymidine produced by a mycobacterium was found
to be mitogenic for a subset of human .gamma..delta. T cells
(Constant, P., F. Davodeau, M.-A. Peyrat, Y. Poquet, G. Puzo, M.
Bonneville, and J.-J. Fournie. 1994. "Stimulation of human
.gamma..delta. T cells by nonpeptidic mycobacteral ligands" Science
264:267). This report indicated the possibility that the immune
system may have evolved ways to preferentially respond to microbial
nucleic acids.
[0008] Several observations suggest that certain DNA structures may
also have the potential to activate lymphocytes. For example, Bell
et al. reported that nucleosomal protein-DNA complexs (but not
naked DNA) in spleen cell supernatants caused B cell-proliferation
and immunoglobulin secretion Bell, D. A., B. Morrison, and P.
VandenBygaart 1990. "Immunogenic DNA-related factors". J. Clin.
Invest. 85:1487). In other cases, naked DNA has been reported to
have immune effects. For example, Messina et al. have recently
reported that 260 to 800 bp fragments of poly (dG)o(dC) and poly
(dGodC) were mitogenic for B cells (Messina, J. P., O. S. Gilkeson,
and D. S. Pisetsky. 1993. "The influence of DNA structure on the in
vitro stimulation of murine lymphocytes by natural and synthetic
polynucleotide antigens". Cell Immunol.147:148 ). Tokunaga, et al.
have reported that dGo dC induces .gamma.-IFN and NK activity
(Tokunaga, S. Yamamoto, and KY Narnba 1988. "A synthetic
single-stranded DNA, poly(dG,dC), induces interferon-.alpha./.beta.
and -.gamma., augments natural killer activity, and suppresses
tumor growth" Jpn. J. Cancer Res. 79:682). Aside from such
artificial homopolymer sequences, Pisetsky et al. reported that
pure mammalian DNA has no detectable immune effects, but that DNA
from certain bacteria induces B cell activation and immunoglobulin
secretion (Messina, J. P., G. S. Gilkeson, and D. S. Pisetsky.
1991. "Stimulation of in vitro murine lymphocyte proliferation by
bacterial DNA". J. Immunol. 147:1759). Assuming that these data did
not result from some unusual contaminant, these studies suggested
that a particular structure or other characteristic of bacterial
DNA renders it capable of triggering B cell activation.
Investigations of mycobacterial DNA sequences have demonstrated
that ODN which contain certain palindrome sequences canl activate
NK cells (Yamamoto, S., T. Yamamoto, T. Kataoka, E. Kuramoto, O.
Yano, and T. Tokunaga. 1992. "Unique palindromic sequences in
synthetic oligonucleotides are required to induce INF and augment
INF-mediated natural killer activity". J. Immunol. 148:4072;
Kuramoto, E., O. Yano, Y. Kimura, M. Baba, T. Makino, S. Yamamoto,
T. Yamamoto, T. Kataoka, and T. Tokunaga 1992. "Oligonucleotide
sequences required for natural killer cell activation". Jpn. J.
Cancer Res. 83:1128).
[0009] Several phosphorothioate modified ODN have been reported to
induce in vitro or invivo Becell stimulation (Tanaka, T., C. C.
Chu, and W. E. Paul. 1992. "An antisense oligonucleotide
complementary to a sequence in I.gamma.2b increases .gamma.2b
germline transcripts, stimulates B cell DNA synthesis, and inhibits
immunoglobulin secretion". J Ep. Med 175:597; Branda, R F., A. L.
Moore, L. Mathews, J. J. McCormack, and G. Zon. 1993. "Immune
stilmulation by an antisense oligomer complementary to the rev gene
of HIV-1". Biochem. Pharmacol. 45:2037; McIntyre, K. W., K
Lomabard-Gillooly, J. R. Perez, C. Kunsch, U. M. Sarmiento, J. D.
Larigan, K-T. Landreth, and R. Narayanan 1993. "A sense
phosphorothioate-oligonucleotide directed to the initiation; codon
of trainscription factor NF-.kappa. .beta. T65 causes
sequence-specific immune stimulation". Antisene Res. Develop.
3:309; and Pisetsky, D. S., and C. F. Reich 1993. "Stimulation of
murine lymphocyte proliferation by a phosphorothioate
oligonucleotide with antisense activity for herpes simplex virus".
Life Sciences 54:101). These reports do not suggest a common
structural motif or sequence element in these ODN that might
explain their effects.
[0010] The CREB/ATF family of transcription factors and their role
in replication
[0011] The cAMP response element binding protein (CREB) and
activating transcription factor (ATF) or CREB/ATF fanily of
transcription factors is a ubiquitously expressed class of
transcription factors of which 11 members have so far been cloned
(reviewed in de Groot, R. P., and P. Sassone-Corsi: "Hormonal
control of gene expression: Multiplicity and versatility of cyclic
adenosine 3',5'-monophosphate-responsive nuclear regulators". Mol.
Endocrin. 7:145, 1993; Lee, K. A. W., and N. Masson:
"Transcriptional regulation by CREB and its relatives". Biochim
Biophys. Acta 1174:221, 1993.). They all belong to the basic
region/leucine zipper (bZip) class of proteins. All cells appear to
express one or more CREB/ATF proteins, but the members expressed
and the regulation of mRNA splicing appear to be tissue-specific.
Differential splicing of activation domains can determine whether a
particular CRUBIATF protein will be atanscriptional inhibitor or
activator. Many CREB/ATF proteins activate viral transcription, but
some splicing variants which lack the activation domain are
inhibitory. CREB/ATF proteins can bind DNA as homo- or
hetero-dimers through the cAMP response element, the CRE, the
consensus form of which is the unmethylated sequence TGACGTC
(binding is abolished if the CpG is methylated) (Uguchi-Ariga, S.
M. M., and W. Schaffaer: "CpG methylation of the cAMP-responsive
enhancer/promoter sequence TGACGTCA abolishes specific factor
binding as well as transcriptional activation". Genes &
Develop. 3:612, 1989.).
[0012] The transcriptional activity of the CRE is increased during
B cell activation (Xie, H. T. C. Chiles, and T. L. Rothstein:
"Induction of CREB activity via the surface Ig receptor of B
cells". J. Immunol. 151:880, 1993.). CREB/ATF proteins appear to
regulate the expression of multiple genes through the CRE including
immunologically important genes such as fos, jun B, Rb1, IL-6, IL-1
(Tsukada, J., K. Saito, W. R. Waterman, A. C. Webb, and P. E.
Auron: "Transcription factors NF-IL6 and CREB recognize a common
essential site in the human prointerleukin 1.beta. gene". Mol.
Cell. Biol. 14-7295, 1994; Gray, G. D., O. M. Hernandez, D. Hebel,
M. Root, J. M. Pow-Sang, and E. Wickstrom: "Antisense DNA
inhibition of tumor growth induced by c-Ha-ras oncogene in nude
mice". Cancer Res. 53:577, 1993), IFN-.beta. (Du, W., and T.
Maniatis: "An AT/CREB binding site protein is required for virus
induction of the human interferon B gene". Proc. Natl. Acad. Sci.
USA 89:2150, 1992), TGF-.beta.1 (Asiedu, C. K., L. Scott, R. K.
Assoian, M. Ehrlich: "Binding of AP-1/CREB proteins and of MDBP to
contiguous sites downstream of the human TGF-B1 gene". Biochim.
Biophys. Acta 1219:55, 1994.), TGF-.beta.2, class II MHC (Cox, P.
M., and C. R. Goding: "An ATF/CREB binding motif is required for
aberrant constitutive expression of the MHC class II DRa promoter
and activation by SV40 T-antigen". Nucl. Acids Res. 20:4881,
1992.), E-selectin, GM-CSF, CD-8.alpha.; the germline Ig.alpha.
constant region gene, the TCR V.beta. gene, and the proliferating
cell nuclear antigen Huang, D., P. M. Shipman-Appasamy, D. J.
Orten, S. H. Hinrichs, and M. B. Prystowsky: "Promoter activity of
the proliferating-cell nuclear antigen gene is associated with
inducible CRE-binding proteins in interleukin 2-stimulated T
lymphocytes". Mol. Cell. Biol. 14:4233, 1994.). In addition to
activation through the cAMP pathway, CREB can also mediate
transcriptional responses to changes in intracellular Ca.sup.++
concentration (Sheng, M., G. McFadden, and M. E. Greenberg:
"Membrane depolarization and calcium induce c-fos transcription via
phosphorylation of transcription factor CREB". Neuron 4:571,
1990).
[0013] The role of protein-protein interactions in transcriptional
activation by CREB/ATF proteins appears to be extremely important.
Activation of CREB through the cyclic AMP pathway requires protein
kinase A (PKA), which phosphorylates CREB.sup.341 on ser.sup.133
and allows it to bind to a recently cloned protein, CBP (Kwok, R.
P. S., J. R. Lundblad, J. C. Chrivia, J. P. Richards,. H. P.
Bachinger, R. G. Brennan, S. G. E. Roberts, M. R. Green, and R. H.
Goodman: "Nuclear protein CBP is a coactivator for the
transcription factor CREB". Nature 370:223, 1994; Arias, J., A. S.
Alberts, P. Brindle, F. X. Claret, T. Smea, M. Karin, J. Feramisco,
and M. Montminy: "Activation of cAMP and mitogen responsive genes
relies on a common nuclear factor". Nature 370:226, 1994.). CBP in
turn interacts with the basal transcription factor TFIIB causing
increased transcription. CREB also has been reported to interact
with dTAFII 110, a TATA binding protein-associated factor whose
binding may regulate transcription (Ferreri, K., G. Gill, and M.
Montminy: "The cAMP-regulated transcription factor CREB interacts
with a component of the TFIID complex". Proc. Natl. Acad. Sci. USA
91:1210, 1994.). In addition to these interactions, CREB/ATF
proteins can specifically bind multiple other nuclear factors
(Hoeffler, J. P., J. W. Lustbader, and C.-Y. Chen: "Identification
of multiple nuclear factors that interact with cyclic adenosine
3',5'-monophosphate response element-binding protein and activating
transcription factor-2 by protein-protein interactions". Mol.
Endocrinol. 5:256,1991) but the biologic significance of most of
these interactions is unknown. CREB is normally thought to bind DNA
either as a homodimer or as a heterodimer with several other
proteins. Surprisingly, CREB monomers constitutively activate
transcription (Krajewski, W., and K. A. W. Lee: "A monomeric
derivative of the cellular transcription factor CREB functions as a
constitutive activator". Mol. Cell. Biol. 14:7204, 1994.).
[0014] Aside from their critical role in regulating cellular
transcription, it has recently been shown that CREB/ATF proteins
are subverted by some infectious viruses and retroviruses, which
require them for viral replication. For example, the
cytomegalovirus immediate early promoter, one of the strongest
known mammalian promoters, contains eleven copies of the CRE which
are essential for promoter function (Chang, Y.-N., S. Crawford, J.
Stall, D.R. Rawlins, K.-T. Jeang, and G.S. Hayward: "The palmdromic
series I repeats in the simian cytomegalovirus major
immediate-early promoter behave as both strong basal enhancers and
cyclic AMP response elements", J. Virol. 64:264, 1990). At least
some of the transcriptional activating effects of the adenovirus
E1A protein, which induces many promoters, are due to its binding
to the DNA binding domain of the CREB/ATF protein, ATF-2, which
mediates E1A inducible transcription activation (Liu, F., and M. R.
Green: "Promoter targeting by adenovirus E1a through interaction
with different cellular DNA-binding domains". Nature 368:520,
1994). It has also been suggested that E1A binds to the
CREB-binding protein, CBP (Arany, Z., W. R. Sellers, D. M.
Livingston, and R. Eckner: "E1A-associated p300 and CREB-associated
CBP belong to a conserved family of coactivators". Cell 77:799,
1994). Human T lymphotropic virus-I (HTLV-1), the retrovirus which
causes human T cell leukemia and tropical spastic paresis, also
requires CREB/ATF proteins for replication. In this case, the
retrovirus produces a protein, Tax, which binds to CREB/ATF
proteins and redirects them from their normal cellular binding
sites to different DNA sequences (flanked by G- and C-rich
sequences) present within the HTLV transcriptional enhancer
(Paca-Uccaralertkum, S., L.-J. Zhao, N. Adya, J. V. Cross, B. R.
Cullen, I. N. Boros, and C.-Z. Giam: "In vitro selection of DNA
elements highly responsive to the human T-cell lymphotropic virus
type I transcriptional activator, Tax". Mol. Cell. Biol. 14:456,
1994; Adya, N., L.-J. Zhao, W. Huang, I. Boros, and C.-Z. Giam:
"Expansion of CREB's DNA recognition specificity by Tax results
from interaction with Ala-Ala-Arg at positions 282-284 near the
conserved DNA-binding domain of CREB". Proc. Natl. Acad Sci. USA
91:5642, 1994).
SUMMARY OF THE INVENTION
[0015] The instant invention is based on the finding that certain
oligonucleotides containing unmethylated cytosine-guanine (CpG)
dinucleotides activate lymphocytes as evidenced by in vitro and in
vivo data. Based on this finding, the invention features, in one
aspect, novel immnunostimulatory oligonucleotide compositions.
[0016] In a preferred embodiment, an immunostimulatory
oligonucleotide is synthetic, between 2 to 100 base pairs in size
and contains a consensus mitogenic CpG motif represented by the
formula:
1 5' X.sub.1X.sub.2CGX.sub.3X.sub.4 3'
[0017] wherein C and G are unmethylated, X.sub.1, X.sub.2, X.sub.3
and X.sub.4 are nucleotides and a GCG trinucleotide sequence is not
present at or near the 5' and 3' termini.
[0018] For facilitating uptake into cells, CpG containing
immunostimulatory oligonucleotides are preferably in the range of 8
to 40 base pairs in size. Prolonged immunostimulation can be
obtained using stabilized oligonucleotides, particularly
phosphorothioate stabilized oligonucleotides. Enhanced
immunostimulatory activity has been observed where X.sub.1X.sub.2
is the dinucleotide GpA and/or X.sub.3X.sub.4 is the dinucleotide
is most preferably TpC or also TpT. Further enhanced
immunostimulatory activity has been observed where the consensus
motif X.sub.1X.sub.2CGX.sub.3X.sub.4 is preceded on the 5' end by a
T.
[0019] In a second aspect, the invention features useful methods,
which are based on the immunostimulatory activity of the
oligonucleotides. For example, lymphocytes can either be obtained
from a subject and stimulated ex vivo upon contact with an
appropriate oligonucleotide; or a non-methylated CpG containing
oligonucleotide can be administered to a subject to facilitate in
vivo activation of a subject's lymphocytes. Activated lymphocytes,
stimulated by the methods described herein (e.g. either ex vivo or
in vivo), can boost a subject's immune response. The
immunostimulatory oligonucleotides can therefore be used to treat,
prevent or ameliorate an immune system deficiency (e.g., a tumor or
cancer or a viral, fungal, bacterial or parasitic infection in a
subject. In addition, immunostimulatory oligonucleotides can also
be administered as a vaccine adjuvant, to stimulate a subject's
response to a vaccine. Further, the ability of immunostimulatory
cells to induce leukemic cells to enter the cell cycle, suggests a
utility for treating leukemia by increasing the sensitivity of
chronic leukemia cells and then administering conventional ablative
chemotherapy.
[0020] In a third aspect, the invention features neutral
oligonucleotides (i.e. oligonucleotide that do not contain an
unmethylated CpG or which contain a methylated CpG dinucleotide).
In a preferred embodiment, a neutralizing oligonucleotide is
complementary to an immunostimulatory sequence, but contains a
methylated instead of an unmethylated CpG dinucleotide sequence and
therefore can compete for binding with unmethylated CpG containing
oligonucleotides. In a preferred embodiment, the methylation occurs
at one or more of the four carbons and two nitrogens comprising the
cytosine six member ring or at one or more of thefive carbons and
four nitrogens comprising the guanine nine member double ring. 5'
methyl cytosine is a preferred methylated CpG.
[0021] In a fourth aspect, the invention features useful methods
using the neutral oligonucleotides. For example, in vivo
administration of neutral oligonucleotides should prove useful for
treating diseases such as systemic lupus erythematosus, sepsis and
autoimmune diseases, which are caused or exacerbated by the
presence of unmethylated CpG dimers in a subject. In addition,
methylation CpG containing antisense oligonucleotides or
oligonucleotide probes would not initiate an immune reaction when
administered to a subject in vivo and therefore would be safer than
corresponding unmethylated oligonucleotides.
[0022] In a fifth aspect, the invention features immunoinhibitory
oligonucleotides, which are capable of interfering with the
activity of viral or cellular transcription factors. In a preferred
embodiment, immunoinhibitory oligonucleotides are between 2 to 100
base pairs in size and contain a consensus immunoinhibitory CpG
motif represented by the formula:
2 5'GCGXnGCG3'
[0023] wherein X=a nucleotide and n=in the range of 0-50. In a
preferred embodiment, X is a pyrimidine.
[0024] For facilitating uptake into cells, immunoinhibitory
oligonucleotides are preferably in the range of 8 to 40 base pairs
in size. Prolonged inmunostimulation can be obtained using
stabilized oligonucleotides, particularly phosphorothioate
stabilized oligonucleotides.
[0025] In a sixth and final aspect, the invention features various
uses for immunoinhibitory oligonucleotides. Immunoinhibitory
oligonucleotides have antiviral activity, independent of any
antisense effect due to complementarity between the oligonucleotide
and the viral sequence being targeted.
[0026] Other features and advantages of the invention will become
more apparent from the following detailed description and
claims.
DETAILED DESCRIPTION OF THE INVENTION
[0027] Definitions
[0028] As used herein, the following terms and phrases shall have
the meanings set forth below:
[0029] An "oligonucleotide" or "oligo" shall mean multiple
nucleotides (i.e. molecules comprising a sugar (e.g. ribose or
deoxyribose) linked to a phosphate group and to an exchangeable
organic base, which is either a substituted pyrimidine (e.g.
cytosine (C), thymine (T) or uracil (U)) or a substituted purine
(e.g. adenine (A) or guanine (G)). The term "oligonucleotide" as
used herein refers to both oligoribonucleotides (ORNs) and
oligodeoxyribonucleotides (ODNs). The term oligonucleotide shall
also include oligonucleosides (i.e. an oligonucleotide minus the
phosphate) and any other organic base containing polymer.
Oligonucleotides can be obtained from existing nucleic acid sources
(e.g. genomic or cDNA), but are preferably synthetic (e.g. produced
by oligonucleotide synthesis).
[0030] A "stabilized oligonucleotide" shall mean an oligonucleotide
that is relatively resistant to in vivo degradation (e.g. via an
exo- or endo-nuclease). Preferred stabilized oligonucleotides of
the instant invention have a modified phosphate backbone.
Especially preferred oligonucleotides have a phosphorothioate
modified phosphate backbone (i.e. at least one of the phosphate
oxygens is replaced by sulfur). Other stabiized oligonucleotides
include: nonionic DNA analogs, such as alkyl- and aryl-phosphonates
(in which the charged phosphonate oxygen is replaced by an alkyl or
aryl group), phosphodiester and alkylphosphotriesters, in which the
charged oxygen moiety is alkylated. Oligonucleotides which contain
a diol, such as tetraethyleneglycol or hexaethyleneglycol, at
either or both termini have also been shown to be substantially
resistant to nuclease degradation.
[0031] An "immunostimulatory oligonucleotide", "immunostimulatory
CpG containing oligonucleotide", or "CpG ODN" refer to an
oligonucleotide, which contains a cytosine, guanine dinucleotide
sequence and stimulates (e.g. has a mitogenic effect) on vertebrate
lymphocyte. Preferred immunostimulatory oligonucleotides are
between 2 to 100 base pairs in size and contain a consensus
mitogenic CpG motif represented by the formula:
3 5' X.sub.1X.sub.2CGX.sub.3X.sub.4 3'
[0032] wherein C and G are unmethylated, X.sub.1, X.sub.2, X.sub.3
and X.sub.4 are nucleotides and a GCG trinucleotide sequence is not
present at or near the 5' and 3' termini.
[0033] Preferably the immunostimulatory oligonucleotides range
between 8 to 40 base pairs in size. In addition, the
immunostimulatory oligonucleotides are preferably stabilized
oligonucteotides, particularly preferred are phosphorothioate
stabilized oligonucleotides. In one preferred embodiment, X.sub.16l
X.sub.2 is the dinucleotide GpA. In another preferred embodiment,
X.sub.3X.sub.4 is preferably the dinucleotide TpC or also TpT. In a
particularly preferred embodiment, the consensus motif
X.sub.1X.sub.2CGX.sub.3X.sub.4 preceded on the 5' end by a T.
Particularly preferred consensus sequences are TGACGTT or
TGACGTC.
[0034] A "neutral oligonucleotide" refers to an oligonucleotide
that does not contain an umethylated CpG or an oligonucleotide
which contains a methylated CpG dinucleotide. In a preferred
embodiment, a neutraliing oligonucleotide is complementary to an
immunostimulatory sequence, but contains a methylated instead of an
unmethylated CpG dinucleotide sequence and therefore can compete
for binding with unmethylated CpG containing oligonucleotides. In a
preferred embodiment, the methylation occurs at one or more of the
four carbons and two nitrogens comprising the cytosine six member
ring or at one or more of the five carbons and four nitrogens
comprising the guanine nine member double ring. 5' methyl cytosine
is a preferred methylated CpG.
[0035] An "immunoinhibitory oligonucleotide" or "immunoinhibitory
CpG containing oligonucleotide" is an oligonucleotide that.
Preferable immunoinhibitory oligonucleotides are between 2 to 100
base pairs in size and can be represented by the formula:
4 5'GCGXnGCG3'
[0036] wherein X=a nucleotide and n=in the range of 0-50. In a
preferred embodiment, X is a pyrimidine.
[0037] For facilitating uptake into cells, immunoinhibitory
oligonucleotides are preferably in the range of 8 to 40 base pairs
in size. Prolonged immunostimulation can be obtained using
stabilized oligonucleotides, particularly phosphorothioate
stabilized
[0038] "Palindromic sequence" shall mean an inverted repeat (i.e. a
sequence such as ABCDEE'D'C'B'A' in which A and A' are bases
capable of forming the usual Watson-Crick base pairs. In vivo, such
sequences may form double stranded structures.
[0039] An "oligonucleotide delivery complex" shall mean an
oligonucleotide 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 and
natural killer (NK) cell) surfaces and/or increased cellular uptake
by target cells). Examples of oligonucleotide delivery complexes
include oligonucleotides 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
must be sufficiently stable in vivo to prevent significant
uncoupling prior to internalization by the target cell. However,
the complex should be cleavable under appropriate conditions within
the cell so that the oligonucleotide is released in a functional
form.
[0040] An "immune system deficiency" shall mean a disease or
disorder in which the subject's immune system is not functioning in
normal capacity or in which it would be useful to boost a subject's
immune response for example to eliminate a tumor or cancer (e.g.
tumors of the brain, lung (e.g. small cell and non-small cell),
ovary, breast, prostate, colon, as well as other carcinomas and
sarcomas) or a viral (e,.g. HIV, herpes), fungal (e.g. Candida
sp.), bacterial or parasitic (e.g. Leishmania, Toxoplasma)
infection in a subject.
[0041] A "disease associated with immune system activation" shall
mean a disease or condition caused or exacerbated by activation of
the subject's immune system. Examples include systemic lupus
erythematosus, sepsis and autoimmune diseases such as rheumatoid
arthritis and multiple sclerosis.
[0042] A "subject" shall mean a human or vertebrate animals
including a dog, cat, horse, cow, pig, sheep, goat, chicken,
monkey, rat, mouse, etc.
[0043] Certain Unmethylated CpG Containing Oligos Have B Cell
Stimulatory Activity As Shown in vitro and in vivo
[0044] In the course of investigating the lymphocyte stimulatory
effects of two antisense oligonucleotides specific for endogenous
retroviral sequences, using protocols described in the attached
Examples 1 and 2, it was surprisingly found that two out of
twenty-four "controls" (including various scrambled, sense, and
mismatch controls for a panel of "antisense" ODN) also mediated B
cell activation and IgM secretion, while the other "controls" had
no effect
[0045] Two observations suggested that the mechanism of this B cell
activation by the "control" ODN may not involve antisense effects
1) comparison of vertebrate DNA sequences listed in GenBank showed
no greater homology than that seen with non-stimulatory ODN and 2)
the two controls showed no hybridization to Northern blots with 10
.mu.g of spleen poly A+ RNA. Resynthesis of these ODN on a
different synthesizer or extensive purification by polyacrylamide
gel electrophoresis or high pressure liquid chromatography gave
identical stimulation, eliminating the possibility of an impurity.
Similar stimulation was seen using B cells from C3H/HeJ mice,
eliminating the possibility that lipopolysaccharide (LPS)
contamination could account for the results.
[0046] The fact that two "control" ODN caused B cell activation
similar to that of the two "antisense" ODN raised the possibility
that all four ODN were stimulating B cells through some
non-antisense mechanism involving a sequence motif that was absent
in all of the other nonstimulatory control ODN. In comparing these
sequences, it was discovered that all of the four stimulatory ODN
contained ODN dinucleotides that were in a different sequence
context from the nonstimulatory control.
[0047] To determine whether the CpG motif present in the
stimiulatory ODN was responsible for the observed stimulation, over
300 ODN ranging in length from 5 to 42 bases that contained
methylated, usnmethylated, or no CpG dinucleotides in various
sequence contexts were synthesized. These ODNs, including the two
original "controls" (ODN 1 and 2) and two originally synthesized as
"antisense" (ODN 3D and 3M; Krieg, A. M. J Immunol. 143:2448
(1989)), were then examined for in vitro effects on spleen cells
(representative sequences are listed in Table 1). Several ODN that
contained CpG dinucleotides induced B cell activation and IgM
secretion; the magnitude of this stimulation typically could be
increased by adding more CpG dinucleotides (Table 1; compare ODN 2
to 2a or 3D to 3Da and 3Db). Stimaulation did not appear to result
from an antisense mechanism or impurity. ODN caused no detectable
activation of .gamma..delta. or other T cell populations.
[0048] Mitogenic ODN sequences uniformly became nonstimulatory if
the CpG dinucleotide was mutated (Table 1; compare ODN 1 to 1a; 3D
to 3Dc; 3M to 3Ma; and 4 to 4a) or if the cytosine of the CpG
dinucleotide was replaced by 5-methylcytdsine (Table 1; ODN 1b, 2b,
2c, 3Dd, and 3Mb). In contrast, methylation of other cytosines did
not reduce ODN activity (ODN 1c, 2d, 3De and 3Mc). These data
confirmed that a CpG motif is the essential element present in ODN
that activate B cells.
[0049] In the course of these studies, it became clear that the
bases flanking the CpG dinucleotide played an important role in
determining the B cell activation induced by an ODN. The optimal
stimulatory motif was determined to consist of a CpG flanked by two
5' purines (preferably a OpA dinucleotide) and two 3' pyrimidines
(preferably a TpT or TpC dinucleotide). Mutations of ODN to bring
the CpG motif closer to this ideal improved stimulation (e.g.
compare ODN 2 to 2e; 3M to 3Md) while mutations that disturbed the
motif reduced stimulation (e.g. compare ODN 3D to 3Df;,4 to4b, 4c
and 4d). On the other hand, mutations outside the CpG motif did not
reduce stimulation (e.g. compare ODN 1 to 1d; 3D to 3Dg; 3M to
3Me).
[0050] Of those tested, ODNs shorter than 8 bases were
non-stimulatory (e.g. ODN 4e). Among the forty-eight 8 base ODN
tested, the most stimulatory sequence identified was TCAACGTT (ODN
4) which contains the self complementary-"palindrome" AACGTT. In
further optimizing this motif, it was found that ODN containing Gs
at both ends showed increased stimulation, particularly if the the
ODN were rendered nuclease resistant by phosphorothioate
modification of the terminal internucleotide linkages. ODN 1585 (5'
GGGGTCAACGTACAGGGGG3' (SEQ ID NO:1)), in which the first two and
last five internucleotide linkages are phosphorothioate modified
caused an average 25.4 fold increase in mouse spleen cell
proliferation compared to an average 3.2 fold increase in
proliferation induced by ODN 1638, which has the same sequence as
ODN 1585 except that the 10 Gs at the two ends are replaced by 10
As. The effect of the G-rich ends is cis; addition of an ODN with
poly G ends but no CpG motif to cells along with 1638 gave no
increased proliferation.
[0051] Other octamer ODN containing a 6 base palindrome with a TpC
dinucleotide at the 5' end were also active if they were close to
the optimal motif (e.g. ODN 4b,4c). Other dinucleotides at the 5'
end gave reduced stimulation (eg ODN 4f; all sixteen possible
dinucleotides were tested). The presence of a 3' dinucleotide was
insufficient to compensate for the lack of a 5' dinucleotide (eg.
ODN 4g). Disruption of the palindrome eliminated stimulation in
octamer ODN (eg., ODN 4h), but palindromes were not required in
longer ODN.
5TABLE 1 Oligonucleotide Stimulation of B Cells Stimulation Index'
ODN Sequence (5' to 3').dagger. .sup.3H Uridine IgM Production 1
(SEQ ID NO:2) GCTAGACGTTAGCGT 6.1 .+-. 0.8 17.9 .+-. 3.6 1a(SEQ ID
NO:3) ......T........ 1.2 .+-. 0.2 1.7 .+-. 0.5 1b(SEQ ID NO:4)
......Z........ 1.2 .+-. 0.1 1.8 .+-. 0.0 1c(SEQ ID NO:5)
............Z.. 10.3 .+-. 4.4 9.5 .+-. 1.8 1d(SEQ ID NO:6)
..AT......GAGC. 13.0 .+-. 2.3 18.3 .+-. 7.5 2 (SEQ ID NO:7)
ATGGAAGGTCCAGCGTTCTC 2.9 .+-. 0.2 13.6 .+-. 2.0 2a(SEQ ID NO:8)
..C..CTC..G......... 7.7 .+-. 0.8 24.2 .+-. 3.2 2b(SEQ ID NO:9)
..Z..CTC.ZG..Z...... 1.6 .+-. 0.5 2.8 .+-. 2.2 2c(SEQ ID NO:10)
..Z..CTC..G......... 3.1 .+-. 0.6 7.3 .+-. 1.4 2d(SEQ ID NO:11)
..C..CTC..G......Z.. 7.4 .+-. 1.4 27.7 .+-. 5.4 2e(SEQ ID NO:12)
............A....... 5.6 .+-. 2.0 ND 3D (SEQ ID NO:13)
GAGAACGCTGGACCTTCCAT 4.9 .+-. 0.5 19.9 .+-. 3.6 3Da(SEQ ID NO:14)
.........C.......... 6.6 .+-. 1.5 33.9 .+-. 6.8 3Db(SEQ ID NO:15)
.........C.......G.. 10.1 .+-. 2.8 25.4 .+-. 0.8 3Dc(SEQ ID NO:16)
...C.A.............. 1.0 .+-. 0.1 1.2 .+-. 0.5 3Dd(SEQ ID NO:17)
.....Z.............. 1.2 .+-. 0.2 1.0 .+-. 0.4 3De(SEQ ID NO:18)
.............Z...... 4.4 .+-. 1.2 18.8 .+-. 4.4 3Df(SEQ ID NO:19)
.......A............ 1.6 .+-. 0.1 7.7 .+-. 0.4 3Dg(SEQ ID NO:20)
.........CC.G.ACTG.. 6.1 .+-. 1.5 18.6 .+-. 1.5 3M (SEQ ID NO:21)
TCCATGTCGGTCCTGATGCT 4.1 .+-. 0.2 23.2 .+-. 4.9 3Ma(SEQ ID NO:22)
......CT............ 0.9 .+-. 0.1 1.8 .+-. 0.5 3Mb(SEQ ID NO:23)
.......Z............ 1.3 .+-. 0.3 1.5 .+-. 0.6 3Mc(SEQ ID NO:24)
...........Z........ 5.4 .+-. 1.5 8.5 .+-. 2.6 3Md(SEQ ID NO:25)
......A..T.......... 17.2 .+-. 9.4 ND 3Me(SEQ ID NO:26)
...............C..A. 3.6 .+-. 0.2 14.2 .+-. 5.2 4 TCAACGTT 6.1 .+-.
1.4 19.2 .+-. 5.2 4a ....GC.. 1.1 .+-. 0.2 1.5 .+-. 1.1 4b ...GCGC.
4.5 .+-. 0.2 9.6 .+-. 3.4 4c ...TCGA. 2.7 .+-. 1.0 ND 4d ..TT..AA
1.3 .+-. 0.2 ND 4e -....... 1.3 .+-. 0.2 1.1 .+-. 0.5 4f C.......
3.9 .+-. 1.4 ND 4g --......CT 1.4 .+-. 0.3 ND 4h .......C 1.2 .+-.
0.2 ND LPS 7.8 .+-. 2.5 4.8 .+-. 1.0 'Stimulation indexes are the
means and std. dev. derived from at least 3 separate experiments,
and are compared to wells cultured with no added ODN. ND = not
done. CpG dinucleotides are underlined. Dots indicate identity;
dashes indicate deletions. Z indicates 5 methyl cytosine.)
[0052] The kinetics of lymphocyte activation were investigated
using mouse spleen cells. When the cells were pulsed at the same
time as ODN addition and harvested just four hours later, there was
already a two-fold increase in .sup.3H uridine incorporation.
Stimulation peaked at 12048 hours and then decreased. After 24
hours, no intact ODN were detected, perhaps accounting for the
subsequent fall in stimulation when purified B cells with or
without anti-IgM (at a submitogenic dose) were cultured with CpG
ODN, proliferation was found to synergistically increase about
10-fold by the two mitogens in combination after 48 hours. The
magnitude of stimulation was concentration dependent and
consistently exceeded that of LPS under optimal conditions for
both. Oligonucleotides containing a nuclease resistant
phosphorothioate backbone were approximately two hundred times more
potent than unmodified oligonucleotides.
[0053] Cell cycle analysis was used to determine the proportion of
B cells activated by CpG-ODN. CpG-ODN induced cycling in more than
95% of B cells (Table 2). Splenic B lymphocytes sorted by flow
cytometry into CD23- (marginal zone) and CD23+ (follicular)
subpopulations were equally responsive to ODN-induced stimulation,
as were both resting and activated populations of B cells isolated
by fractionation over Percoll gradients. These studies demonstrated
that CpG-ODN induce essentially all B cells to enter the cell
cycle.
6TABLE 2 Cell Cycle Analysis with CpG ODN Percent of cells in
Treatment G0 G1 SA + G2 + M Media 97.6 2.4 0.02 ODN 1a 95.2 4.8
0.04 ODN 1d 2.7 74.4 22.9 ODN 3Db 3.5 76.4 20.1 LPS (30 .mu.g/ml)
17.3 70.5 12.2
[0054] The mitogenic effects of CpG ODN on human cells, were tested
on peripheral blood mononuclear cells (PBMCs) obtained from two
patients with chronic lymphocytic leukemia (CLL), as described in
Example 1. Control ODN containing no CpG-dinucleotide sequence
showed no effect on the basal proliferation of 442 cpm and 874 cpm
(proliferation measured by .sup.3H thymidine incorporation) of the
human cells. However, a phosphorothioate modified CpG ODN 3Md (SEQ
ID NO: 25) induced increased proliferation of 7210 and 86795 cpm
respectively in the two patients at a concentration of just 1
.mu.M. Since these cells had been frozen, they may have been less
responsive to the oligos than fresh cells in vivo. In addition,
cells from CLL patients typically are non-proliferating, which is
why traditional chemotherapy is not effective.
[0055] Certain B cell lines such as WEHI-231 are induced to undergo
growth arrest and/or apoptosis in response to crosslinking of their
antigen receptor by anti-IgM (Jakway, J. P. et al., "Growth
regulation of the B lymphoma cell line WEHI-231 by
anti-immunoglobulin, lipopolysaccharide and other bacterial
products" J. Immunol. 137: 2225 (1986); Tsubata, T., J. Wu and T.
Honjo: Bcell apoptosis induced by antigen receptor crosslinking is
blocked by a T-cell signal through CD40." Nature 364: 645 (1993)).
WEHI-231 cells are rescued from this growth arrest by certain
stimuli such as LPS and by the CD40 ligand. ODN containing the CpG
motif were also found to protect WEHI-231 from anti-IgM induced
growth arrest, indicating that accessory cell populations are not
required for the effect.
[0056] To better understand the immune effects of unmethylated CpG
ODN, the levels of cytokines and prostaglandins in vitro and in
vivo were measured. Unlike LPS, CpG ODN were not found to induce
purified macrophages to produce prostaglandin PGE2. In fact, no
apparent direct effect of CpG ODN was detected on either
macrophages or T cells. In vivo or in whole spleen cells, no
significant increase in the following interleukins: IL-2, IL-3,
IL-4, or IL-10 was detected within the first six hours. However,
the level of IL-6 increased strikingly within 2 hours in the serum
of mice injected with CpG ODN. Increased expression of IL-12 and
interferon gamma (IFN-.gamma.) by spleen cells was also detected
within the first two hours.
[0057] To determine whether CpG ODN can cause in vivo immune
stimulation, DBA/2 mice were injected once intraperitoneally with
PBS or phosphorothioate CpG or non-CpG ODN at a dose of 33 mg/kg
(approximately 500 .mu.g/mouse). Pharmacokinetic studies in mice
indicate that this dose of phosphorothioate gives levels of
approximately 10 .mu.g/g in spleen tissue (within the effective
concentration range determined from the in vitro studies described
herein) for longer than twenty-four-hours (Agrawal, S. et al.
(1991) Proc. Natl. Acad. Sci. USA 91:7595). Spleen cells from mice
were examined twenty-four hours after ODN injection for expression
of B cells surface activation markers Ly-6A/E, Bla-1, and class II
MHCC using three color flow cytometry and for their spontaneous
proliferation using .sup.3H thymidine. Expression of all three
activation markers was significantly increased in B cells from mice
injected with CpG ODN, but not from mice injected with PBS or
non-CpG ODN. Spontaneous .sup.3H thymidine incorporation was
increased by 2-6 fold in spleen cells from mice injected with the
stimulatory ODN compared to PBS or non-CpG ODN-injected mice. After
4 days, serum IgM levels in mice injected with CpG ODN in vivo were
increased by approximately 3-fold compared to controls. Consistent
with the inability of these agents to activate T cells, there was
minimal change in T cell expression of the IL-2R or CD-44.
[0058] Degradation of phophodiester ODN in serum is predominantly
mediated by 3' exonucleases, while intracellular ODN degradation is
more complex, involving 5' and 3' exonucleases and endonucleases.
Using a panel of ODN bearing the 3D sequence with varying numbers
of phosphorothioate modified linkages at the 5' and 3' ends, it was
empirically determined that two 5' and five 3' modified linkages
are required to provide optimal stimulation with this CpG ODN.
[0059] Unmethylated CpG Containing Oligos Have NK Cell Stimulatory
Activity
[0060] As described in further detail in Example 4, experiments
were conducted to determine whether CpG containing oligonucleotides
stimulated the activity of natural killer (NK) cells in addition to
B cells. As shown in Table 3, a marked induction of NK activity
among spleen cells cultured with CpG ODN 1 and 3Dd was observed. In
contrast, there was relatively no induction in effectors that had
been treated with non-CpG control ODN.
7TABLE 3 Induction Of NK Activity By CpG Oligodeoxynucleotides
(ODN) % YAC-1 Specific Lysis* % 2C11 Specific Lysis Effector:Target
Effector:Target ODN 50:1 100:1 50:1 100:1 None -1.1 -1.4 15.3 16.6
1 16.1 24.5 38.7 47.2 3Dd 17.1 27.0 37.0 40.0 non-CpG ODN -1.6 -1.7
14.8 15.4
[0061] Neutralizing Activity of Methylated CpG Containing
Oligos
[0062] B cell mitogenicity of ODN in which cytosines in CpG motifs
or elsewhere were replaced by 5-methylcytosine were tested as
described in Example 1. As shown in Table 1 above, ODN containing
methylated CpG motifs were non-mitogenic (Table 1; ODN 1c, 2f, 3De,
and 3Mc). However, methylation of cytosines other than in a CpG
dinucleotide retained their stimulatory properties (Table 1, ODN
1d, 2d, 3Df, and 3Md).
[0063] Immunoinhibitory Activity of Oligos Containing a GCG
Trinucleotide Sequence at or near both termini
[0064] In some cases, ODN containing CpG dinucleotides that are not
in the stimulatory motif described above were found to block the
stimulatory effect of other mitogenic CpG ODN. Specifically the
addition of an a typical CpG motif consisting of a GCG near or at
the 540 and/or 3' end of CpG ODN actually inhibited stimulation of
proliferation by other CpG motifs. Methylation or substitution of
the cytosine in a GCG motif reverses this effect. By itself, a GCG
motif in an ODN has a modest mitogenic effect, though far lower
than that seen with the preferred CpG motif.
[0065] Proposed Mechanisms of Action of Immunostimulatory
Neutralizing and Immunoinhibitory Oligonucleotides
[0066] Unlike antigens that trigger B cells through their surface
Ig receptor, CpG-ODN did not induce any detectable Ca.sup.2+ flux,
changes in protein tyrosine phosphorylation, or IP 3 generation.
Flow cytometry with FITC-conjugated ODN with or without a CpG motif
was performed as described in Zhao, Q et al., (Antisense Research
and Development 3:53-66 (1993)), and showed equivalent membrane
binding, cellular uptake, efflux, and intracellular localization.
This suggests that there may not be cell membrane proteins specific
for CpG ODN. Rather than acting through the cell membrane, that
data suggests that unmethylated CpG containing oligonucleotides
require cell uptake for activity: ODN covalently linked to a solid
Teflon support were nonstimulatory, as were biotinylated ODN
immobilized on either avidin beads or avidin coated petri dishes.
CpG ODN conjugated to either FITC or biotin retained full mitogenic
properties, indicating no steric hindrance.
[0067] The optimal CpG motif (TGACGTT/C is identical to the CRE
(cyclic AMP response element). Like the mitogenic effects of CpG
ODN, binding of CREB to the CRE is abolished if the central CpG is
methylated. Electrophoretic mobility shift assays were used to
determine whether CpG ODN, which are-single stranded, could compete
with the binding of B cell CREB/ATF proteins to their normal
binding site, the doublestranded CRE. Competition assays
demonstrated that single stranded ODN containing CO motifs could
completely compete the binding of CREB to its binding site, while
ODN without CpG motifs could not. The data support the conclusion
that CpG ODN exert their mitgenic effects through interacting with
one or more B cell CREB/ATF proteins in some way. Conversely, the
presence of GCG sequences or other atypical CPG motifs near the 5'
and/or 3' ends of ODN likely interact with CREB/ATF proteins in a
way that does not cause activation, and may even prevent it.
[0068] The stimulatory CpG motif is common in microbial genomic
DNA, but quite rare in vertebrate DNA. In addition, bacterial DNA
has been reported to induce B cell proliferation and immunoglobulin
(Ig) production, while mammalian DNA does not (Messina, J. P. et
al., J. Immunol. 147:1759 (1991)). Experiments further described in
Example 3, in which methylation of bacterial DNA with CpG methylase
was found to abolish mitogenicity, demonstrates that the difference
in CpG status is the cause of B cell stimulation by bacterial DNA.
This data supports the following conclusion: that unmethylated CpG
dinucleotides present within bacterial DNA are responsible for the
stimulatory effects of bacterial DNA.
[0069] Teleologically, it appears likely that lymphocyte activation
by the CpG motif represents an immune defense mechanism that can
thereby distinguish bacterial from host DNA. Host DNA would induce
little or no lymphocyte activation due to it CpG suppression and
methylation. Bacterial DNA would cause selective lymphocyte
activation in infected tissues. Since the CpG pathway synergizes
with B cell activation through the antigen receptor, B cells
bearing antigen receptor specific for bacterial antigens would
receive one activation signal through cell membrane Ig and a second
signal from bacterial DNA, and would therefore tend to be
preferentially activated. The interrelationship of this pathway
with other pathways of B cell activation provide a physiologic
mechanism employing a polyclonal antigen to induce antigen-specific
responses.
[0070] Method for Making Immunostimulatory Oligos
[0071] For use in the instant invention, oligonucleotides can be
synthesized de novo using any of a number of procedures well known
in the art. For example, the .beta.-cyanoethyl phosphoramidite
method (S. L. Beaucage and M. H. Caruthers, (1981) Tet. Let.
22:1859); nucleoside H-phosphonate method (Garegg et al., (1986)
Tet. Let. 27: 40514054; Froehler et al., (1986) Nucl. Acid Res. 14:
5399-5407; Garegg et al., (1986) Tet. Let. 27: 4055-4058, Gaffney
et al., (1988) Tet. Let. 29:2619-2622). These chemistries can be
performed by a variety of automated oligonucleotide synthesizers
available in the market. Alternatively, oligonucleotides can be
prepared from existing nucleic acid sequences (e.g. genomic or
cDNA) using known techniques, such as those employing restriction
enzymes, exonucleases or endonucleases.
[0072] For use in vivo, oligonucleotides are preferably relatively
resistant to degradation (e.g. via endo- and exo-nucleases).
Oligonucleotide stabilization can be accomplished via phosphate
backbone modifications. A preferred stabilized oligonucleotide has
a phosphorothioate modified backbone. The pharmacokinetics of
phosphorothioate ODN show that they have a systemic half-life of
forty-eight hours in rodents and suggest that they may be useful
for in vivo applications (Agrawal, S. et al. (1991) Proc. Natl.
Acad. Sci. USA 88:7595). Phosphorothioates may be synthesized using
automated techniques employing either phosphoramidate or H
phosphonate chemistries. Aryl- and alky-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. (1990) Chem. Rev. 90:544;
Goodchild, J. (1990) Bioconjugate Chem. 1:165).
[0073] For administration invivo, oligonucleotides may be
associated with a molecule that results in higher affinity binding
to target cell (e.g. B-cell and natural killer (NK) cell) surfaces
and/or increased cellular uptake by target cells to form an
"oligonucleotide delivery complex". Oligonucleotides can be
ionically, or covalently associated with appropriate molecules
using techniques which are well known in the art. A variety of
coupling or crosslinking agents can be used e.g. protein A,
carbodiimide, and N-succinimidyl-3-(2-pyridyldithio) propionate
(SPDP). Oligonucleotides can alternatively be encapsulated in
liposomes or virosomes using well-known techniques.
[0074] 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.
[0075] Therapeutic Uses of Immunostimulatory Oligos
[0076] Based on their immunostimulatory properties oligonucleotides
containing at least one unmethylated CpG dinucleotide can be
administered to a subject in vivo to treat an "immune system
deficiency". Alternatively, oligonucleotides containing at least
one unmethylated CpG dinucleotide can be contacted with lymphocytes
(e.g. B cells or NK cells) obtained from a subject having
deficiency ex vivo and activated lymphocytes can then be
reimplanted in the subject.
[0077] Immunostimulatory oligonucleotides can also be administered
to a subject in conjunction with a vaccine, as an adjuvant, to
boost a subjects immune system to effect better response from the
vaccine. Preferably the unmethylated CpG dinucleotide is
administered slightly before or at the same time as the
vaccine.
[0078] Preceding chemotherapy with an immunostimulatory
oligonucleotide should prove useful for increasing the
responsiveness of the malignant cells to subsequent chemotherapy.
CpG ODN also increased natural killer cell activity in both human
and murine cells. Induction of NK activity may likewise be
beneficial in cancer immunotherapy.
[0079] Therapeutic Uses for Neutral Oligonucleotides
[0080] Oligonucleotides that are complementary to certain target
sequences can be synthesized and administered to a subject in vivo.
For example, antisense oligonucleotides hybridize to complementary
mRNA, thereby preventing expression of a specific target gene. The
sequence-specific effects of antisense oligonucleotides have made
them useful research tools for the investigation of protein
function. Phase I/II human trials of systemic antisense therapy are
now underway for acute myelogenous leukemia and HIV.
[0081] In addition, oligonucleotide probes (i.e. oligonucleotides
with a detectable label) can be administered to a subject to detect
the presence of a complementary sequence based on detection of
bound label. In vivo administration and detection of
oligonucleotide probes may be useful for diagnosing certain
diseases that are caused or exacerbated by certain DNA sequences
(e.g. systemic lupus erythematosus, sepsis and autoimmune
diseases).
[0082] Antisense oligonucleotides or oligonucleotide probes in
which any or all CpG dinucleotide is methylated, would not produce
an immune reaction when administered to a subject in vivo and
therefore would be safer than the corresponding non-methylated CpG
containing oligonucleotide.
[0083] For use in therapy, an effective amount of an appropriate
oligonucleotide alone or formulated as an oligonucleotide delivery
complex can be administered to a subject by any mode allowing the
oligonucleotide to be taken up by the appropriate target cells
(e.g. B-cells and NK cells). Preferred routes of administration
include oral and transdermal (e.g. via a patch). Examples of other
routes of administration include injection (subcutaneous,
intravenous, parenteral, intraperitoneal, intrathecal, etc.). The
injection can be in a bolus or a continuous infusion.
[0084] An oligonucleotide alone or as an oligonucleotide delivery
complex can be administered in conjunction with a pharmaceutically
acceptable carrier. As used herein, the phrase "pharmaceutically
acceptable carrier" is intended to include substances that can be
coadministered with an oligonucleotide or an -oligonucleotide
delivery complex and allows the oligonucleotide to perform its
intended function. Examples of such carriers include solutions,
solvents, dispersion media, delay agents, emulsions and the like.
The use of such media for pharmaceutically active substances are
well known in the art. Any other conventional carrier suitable for
use with the oligonucleotides falls within the scope of the instant
invention.
[0085] The language "effective amount" of an oligonucleotide refers
to that amount necessary or sufficient to realize a desired
biologic effect. For example, an effective amount of an
oligonucleotide containing at least one methylated CpG for treating
an immune system deficiency could be that amount necessary to
eliminate a tumor, cancer, or bacterial, viral or fungal infection.
An effective amount for use as a vaccine adjuvant could be that
amount useful for boosting a subject's immune response to a
vaccine. An "effective amount" of an oligonucleotide lacking a
non-methylated CpG for use in treating a disease associated with
immune system activation, could be that amount necessary to
outcompete non-methylated CpG containing nucleotide sequences. The
effective amount for any particular application can vary depending
on such factors as the disease or condition being treated, the
particular oligonucleotide being administered, 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 oligonucleotide without necessitating undue
experimentation.
[0086] The studies reported above indicate that unmethylated CpG
containing oligonucleotides are directly mitogenic for lymphocytes
(e.g. B cells and NK cells). Together with the presence of these
sequences in bacterial DNA, these results suggest that the
underrepresentation of CpG dinucleotides in animal genomes, and the
extensive methylation of cytosines present in such dinucleotides,
may be explained by the existence of an immune defense mechanism
that can distinguish bacterial from host DNA. Host DNA would
commonly be present in many anatomic regions and areas of
inflammation due to apoptosis (cell death), but generally induces
little or no lymphocyte activation. However, the presence of
bacterial DNA containing unmethylated CpG motifs can cause
lymphocyte activation precisely in infected anatomic regions, where
it is beneficial. This novel activation pathway provides a rapid
alternative to T cell dependent antigen specific B-cell activation.
However, it is likely that B cell activation would not be totally
nonspecific. B cells bearing antigen receptors specific for
bacterial products could receive one activation signal through cell
membrane Ig, and a second from bacterial DNA, thereby more
vigorously triggering antigen specific immune responses.
[0087] As with other immune defense mechanisms, the response to
bacterial DNA could have -undesirable consequences in some
settings. For example, autoimmune responses to self antigens would
also tend to be preferentially triggered by bacterial infections,
since autoantigens could also provide a second activation signal to
autoreactive B cells triggered by bacterial DNA. Indeed the
induction of autoimmunity by bacterial infections is a common
clinical observance. For example, the autoimmune disease systemic
lupus erythematosus, which is: i) characterized by the production
of anti-DNA antibodies; ii) induced by drugs which inhibit DNA
methyltransferase (Cornacchia, E. J. et al., J. Clin. Invest. 92:38
(1993)); and iii) associated with reduced DNA methylation
(Richardson, B., L. et al., Arth. Rheum 35:647 (1992)), is likely
triggered at least in part by activation of DNA-specific B cells
through stimulatory signals provided by CpG motifs, as well as by
binding of bacterial DNA to antigen receptors.
[0088] Further, sepsis, which is characterized by high morbidity
and mortality due to massive and nonspecific activation of the
immune system may be initiated by bacterial DNA and other products
released from dying bacteria that reach concentrations sufficient
to directly activate many lymphocytes.
[0089] Lupus, sepsis and other "diseases associated with immune
system activation" may be treated, prevented or ameliorated by
administering to a subject oligonucleotides lacking an unmethylated
CpG dinucleotide (e.g. oligonucleotides that do not include a CpG
motif or oligonucleotides in which the CpG motif is methylated) to
block the binding of unmethylated CpG containing nucleic acid
sequences. Oligonucleotides lacking an unmethylated CpG motif can
be administered alone or in conjunction with compositions that
block an immune cells response to other mitogenic bacterial
products (e.g. LPS).
[0090] The following serves to illustrate mechanistically how
oligonucleotides containing an unmethylated CpG dinucleotide can
treat, prevent or ameliorate the disease lupus. Lupus is commonly
thought to be triggered by bacterial or viral infections. Such
infections have been reported to stimulate the production of
nonpathogenic antibodies to single stranded DNA. These antibodies
likely recognize primarily bacterial sequences including
unmethylated CpGs. As disease develops in lupus, the anti-DNA
antibodies shift to pathogenic antibodies that are specific for
double-stranded DNA. These antibodies would have increased binding
for methylated CpG-sequences and their production would result from
a breakdown of tolerance in lupus. Alternatively, lupus may result
when a patient's DNA becomes hypomethylated, thus allowing anti-DNA
antibodies specific for unmethylated CpGs to bind to self DNA and
trigger more widespread autoimmunity through the process referred
to as "epitope spreading".
[0091] In either case, it may be possible to restore tolerance in
lupus patients by coupling antigenic oligonucleotides to a protein
carrier such as gamma globulin (IgG). Calf-thymus DNA complexed to
gamma globulin has been reported to reduce anti-DNA antibody
formation.
[0092] Therapeutic Uses of Oligos Containing GCG Trinucleotide
Sequences at or Near Both Termini
[0093] Based on their interaction with CREB/ATF, oligonucleotides
containing GCG trinucleotide sequences at or near both termini have
antiviral activity, independent of any antisense effect due to
complementarity between the oligonucleotide and the viral sequence
being targeted. Based on this activity, an effective amount of
inhibitory oligonucleotides can be administered to a subject to
treat or prevent a viral infection.
EXAMPLES
Example 1
[0094] Effects of ODNs on B Cell Total RNA Synthesis and Cell
Cycle
[0095] B cells were purified from spleens obtained from 6-12 wk old
specific pathogen free DBA/2 or BXSB mice (bred in the University
of Iowa animal care facility; no substantial strain differences
were noted) that were depleted of T cells with anti-Thy-1.2 and
complement and centrifugation over lympholyte M (Cedarlane
Laboratories, Hornby, Ontario, Canada) ("B cells"). B cells
contained fewer than 1% CD4.sup.+ or CD8.sup.+ cells.
8.times.10.sup.4 B cells were dispensed in triplicate into 96 well
microtiter plates in 100 .mu.l RPMI containing 10% FBS (heat
inactivated to 65.degree. C. for 30 min), 50 .mu.M
2-mercaptoethanol, 100 U/ml penicillin, 100 ug/ml streptomycin, and
2 mM L-glutamate. 20 .mu.M-ODN were added at the start of culture
for 20 h at 37.degree. C., cells pulsed with 1 .mu.Ci of .sup.3H
uridine, and harvested and counted 4 hr later. Ig secreting B cells
were enumerated using the ELISA spot assay after culture of whole
spleen cells with ODN at 20 .mu.M for 48 hr. Data, reported in
Table 1, represent the stimulation index compared to cells cultured
without ODN. Cells cultured without ODN gave 687 cpm, while cells
cultured with 20 .mu.g/ml LPS (determined by titration to be the
optimal concentration) gave 99,699 cpm in this experiment. .sup.3H
thymidine incorporation assays showed similar results, but with
some nonspecific inhibition by thymidine released from degraded ODN
(Matson. S and A. M. Krieg (1992) Nonspecific suppression of
.sup.3H-thymidine incorporation by control oligonucleotides.
Antisense Research and Development 2:325).
[0096] For cell cycle-analysis, 2.times.10.sup.6 B cells were
cultured for 48 hr. in 2 ml tissue culture medium alone, or with 30
.mu.g/ml LPS or with the indicated phosphorothioate modified ODN at
1 .mu.M. Cell cycle analysis was performed as described in
(Darzynkiewicz, Z. et al., Proc. Natl. Acad. Sci. USA 78:2881
(1981)).
[0097] To test the mitogenic effects of CpG ODN on human cells,
perpheral blood monocyte cells (PBMCs) were obtained from two
patients with chronic lymphocytic leukemia (CLL), a disease in
which the circulating cells are malignant B cells. Cells were
cultured for 48 hrs and pulsed for 4 hours with tritiated thymidine
as described above.
Example 2
[0098] Effects of ODN on Production of IgM from B cells
[0099] Single cell suspensions from the spleens of freshly killed
mice were treated with anti-Thy1, anti-CD4, and anti-CD8 and
complement by the method of Leibson et al., J. Exp. Med. 154:1681
(1981)). Resting B cells (<,02% T cell contamination) were
isolated from the 63-70% band of a discontinuous Percoll gradient
by the procedure of DeFranco et al, J. Exp. Med. 155:1523 (1982).
These were cultured as described above in 30 .mu.M ODN or 20
.mu.g/ml LPS for 48 hr. The number of B cells actively secreting
IgM was maximal at this time point, as determined by ELIspot assay
(Klinman, D. M. et al. J. Immunol. 144:506 (1990)). In that assay,
B cells were incubated for 6 hrs on anti-Ig coated microtiter
plates. The Ig they produced (>99% IgM) was detected using
phosphatase-labelled anti-Ig (Southern Biotechnology Associated,
Birmingham, Ala.). The antibodies produced by individual B cells
were visualized by addition of BCIP (Sigma Chemical Co., St. Louis
Mo.) which forms an insoluble blue precipitate in the presence of
phosphatase. The dilution of cells producing 20-40 spots/well was
used to determine the total number of antibody-secreting B
cells/sample. All assays were performed in triplicate. In some
experiments, culture supernatants were assayed for IgM by ELISA,
and showed similar increases in response to CpG-ODN.
[0100] table 1
Example 3
[0101] B cell Stimulation by Bacterial DNA
[0102] DBA/2 B cells were cultured with no DNA or 50 .mu.g/ml of a)
Micrococcus lysodeikticus; b) NZB/N mouse spleen; and c) NFS/N
mouse spleen genomic DNAs for 48 hours, then pulsed with .sup.3H
thymidine for 4 hours prior to cell harvest. Duplicate DNA samples
were digested with DNAse I for 30 minutes at 37 C. prior to
addition to cell cultures. E coli DNA also induced an 8.8 fold
increase in the number of IgM secreting B cells by 48 hours using
the ELISA-spot assay.
[0103] DBA/2 B cells were cultured with either no additive, 50
.mu.g/ml LPS or the ODN 1; 1a; 4; or 4a at 20 uM. Cells were
cultured and harvested at4, 8, 24 and48 hours. BXSB cells were
cultured as in Example 1 with 5, 10, 20, 40 or 80 .mu.M of ODN 1;
1a; 4; or 4a or LPS. In this experiment, wells with no ODN had 3833
cpm. Each experiment was performed at least three times with
similar results. Standard deviations of the triplicate wells were
<5%.
Example 4
[0104] Effects of ODN on natural killer (NK) activity
[0105] 10.times.10.sup.6 C57BL/6 spleen cells were cultured in two
ml RPMI (supplemented as described for Example 1) with or without
40 .mu.M CpG or non-CpG ODN for forty-eight hours. Cells were
washed, and then used as effector cells in a short term .sup.51Cr
release assay with YAC-1 and 2C11, two NK sensitive target cell
lines (Ballas, Z. K. et al. (1993) J. Immunol. 150:17). Effector
cells were added at various concentrations to 10.sup.4 51Cr-labeled
target cells in V-bottom microtiter plates in 0.2 ml, and incubated
in 5% CO.sub.2 for 4 hr. at 37.degree. C. Plates were then
centrifuged, and an aliquot of the supernatant counted for
radioactivity. Percent specific lysis was determined by calculating
the ratio of the .sup.51Cr released in the presence of effector
cells minus the .sup.51Cr released when the target cells are
cultured alone, over the total counts released after cell lysis in
2% acetic acid minus the .sup.51Cr cpm released when the cells are
cultured alone.
Example 5
[0106] In-vivo studies with CpG phosphorothioate ODN
[0107] Mice were weighed and injected IP with 0.25 ml of sterile
PBS or the indicated phophorothioate ODN dissolved in PBS. Twenty
four hours later, spleen cells were harvested, washed, and stained
for flow cytometry using phycoerythrin conjugated 6B2 to gate on B
cells in conjunction with biotin conjugated anti Ly-6A/E or
anti-Ia.sup.d (Pharmingen, San Diego, Calif.) or anti-Bla-1 (Hardy,
R. R. et al., J. Exp. Med. 159:1169 (1984). Two mice were studied
for each condition and analyzed individually.
Example 6
[0108] Titration of Phosphorothioate ODN for B Cell Stimulation
[0109] B cells were cultured with phosphorothioate ODN with the
sequence of control ODN 1a or the CpG ODN 1d and 3Db and then
either pulsed after 20 hr with .sup.3H uridine or after 44 hr with
.sup.3H thymidine before harvesting and determining cpm.
Example 7
[0110] Rescue of B Cells From Apoptosis
[0111] WEHI-231 cells (5.times.10.sup.4/well) were cultured for 1
hr. at 37 C. in the presence or absence of LPS or the control ODN
1a or the CpG ODN 1d and 3Db before addition of anti-IgM (1
.mu./ml). Cells were cultured for a further 20 hr. before a 4 hr.
pulse with 2 .mu.Ci/well .sup.3H thymidine. In this experiment,
cells with no ODN or anti-IgM gave 90.4.times.10.sup.3 by addition
of anti-IgM. The phosphodiester ODN shown in Table 1 gave similar
protection, though with some nonspecific suppression due to ODN
degradation. Each experiment was repeated at least 3 times with
similar results.
Example 8
[0112] In vivo induction of IL-6
[0113] DBA/2 female mice (2 mos. old) were injected IP with 500
.mu.g CpG or control phosphorothioate ODN. At various time points
after injection, the mice were bled. Two mice were studied for each
time point. IL-6 was measured by Elisa, and IL-6 concentration was
calculated by comparison to a standard curve generated using
recombinant IL-6. The sensitivity of the assay was 10 pg/ml. Levels
were undetectable after 8 hr.
Example 9
[0114] Binding of B cell CREB/ATF to a radiolabelled doublestranded
CRE probe CREB).
[0115] Whole cell extracts from CH12.LX B cells showed 2 retarded
bands when analyzed by EMSA with the CRE probe (free probe is off
the bottom of the figure). The CREB/ATF protein(s) binding to the
CRE were competed by the indicated amount of cold CRE, and by
single-stranded CpG ODN, but not by non-CpG ODN.
[0116] Equivalents
[0117] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents of the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
27 1 20 DNA Artificial Sequence Synthetic oligonucleotide 1
ggggtcaacg ttcagggggg 20 2 15 DNA Artificial Sequence Synthetic
oligonucleotide 2 gctagacgtt agcgt 15 3 15 DNA Artificial Sequence
Synthetic oligonucleotide 3 gctagatgtt agcgt 15 4 15 DNA Artificial
Sequence Synthetic oligonucleotide 4 gctagangtt agcgt 15 5 15 DNA
Artificial Sequence Synthetic oligonucleotide 5 gctagacgtt agngt 15
6 15 DNA Artificial Sequence Synthetic oligonucleotide 6 gcatgacgtt
gagct 15 7 20 DNA Artificial Sequence Synthetic oligonucleotide 7
atggaaggtc cagcgttctc 20 8 20 DNA Artificial Sequence Synthetic
oligonucleotide 8 atcgactctc gagcgttctc 20 9 20 DNA Artificial
Sequence Synthetic oligonucleotide 9 atngactctn gagngttctc 20 10 20
DNA Artificial Sequence Synthetic oligonucleotide 10 atngactctc
gagcgttctc 20 11 20 DNA Artificial Sequence Synthetic
oligonucleotide 11 atcgactctc gagcgttntc 20 12 20 DNA Artificial
Sequence Synthetic oligonucleotide 12 atggaaggtc caacgttctc 20 13
20 DNA Artificial Sequence Synthetic oligonucleotide 13 gagaacgctg
gaccttccat 20 14 20 DNA Artificial Sequence Synthetic
oligonucleotide 14 gagaacgctc gaccttccat 20 15 20 DNA Artificial
Sequence Synthetic oligonucleotide 15 gagaacgctc gaccttcgat 20 16
20 DNA Artificial Sequence Synthetic oligonucleotide 16 gagcaagctg
gaccttccat 20 17 20 DNA Artificial Sequence Synthetic
oligonucleotide 17 gagaangctg gaccttccat 20 18 20 DNA Artificial
Sequence Synthetic oligonucleotide 18 gagaacgctg gacnttccat 20 19
20 DNA Artificial Sequence Synthetic oligonucleotide 19 gagaacgatg
gaccttccat 20 20 20 DNA Artificial Sequence Synthetic
oligonucleotide 20 gagaacgctc cagcactgat 20 21 20 DNA Artificial
Sequence Synthetic oligonucleotide 21 tccatgtcgg tcctgatgct 20 22
20 DNA Artificial Sequence Synthetic oligonucleotide 22 tccatgctgg
tcctgatgct 20 23 20 DNA Artificial Sequence Synthetic
oligonucleotide 23 tccatgtngg tcctgatgct 20 24 20 DNA Artificial
Sequence Synthetic oligonucleotide 24 tccatgtcgg tnctgatgct 20 25
20 DNA Artificial Sequence Synthetic oligonucleotide 25 tccatgacgt
tcctgatgct 20 26 20 DNA Artificial Sequence Synthetic
oligonucleotide 26 tccatgtcgg tcctgctgat 20 27 19 DNA Artificial
Sequence Synthetic oligonucleotide 27 gggtcaagtc tgagggggg 19
* * * * *