U.S. patent application number 12/697917 was filed with the patent office on 2011-02-10 for inhibitors of dna immunostimulatory sequence activity.
Invention is credited to Eyal Raz, Mark Roman.
Application Number | 20110034541 12/697917 |
Document ID | / |
Family ID | 21956478 |
Filed Date | 2011-02-10 |
United States Patent
Application |
20110034541 |
Kind Code |
A1 |
Raz; Eyal ; et al. |
February 10, 2011 |
Inhibitors of DNA Immunostimulatory Sequence Activity
Abstract
The invention consists of oligonucleotides which inhibit the
immunostimulatory activity of ISS-ODN (immunostimulatory sequence
oligodeoxynucleotides) as well as methods for their identification
and use. The oligonucleotides of the invention are useful in
controlling therapeutically intended ISS-ODN adjuvant activity as
well as undesired ISS-ODN activity exerted by recombinant
expression vectors, such as those used for gene therapy and gene
immunization. The oligonucleotides of the invention also have
anti-inflammatory activity useful in reducing inflammation in
response to infection of a host with ISS-ODN containing microbes,
in controlling autoimmune disease and in boosting host Th2 type
immune responses to an antigen. The invention also encompasses
pharmaecutically useful conjugates of the oligonucleotides of the
invention (including conjugate partners such as antigens and
antibodies).
Inventors: |
Raz; Eyal; (Del Mar, CA)
; Roman; Mark; (San Diego, CA) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
1900 UNIVERSITY AVENUE, SUITE 200
EAST PALO ALTO
CA
94303
US
|
Family ID: |
21956478 |
Appl. No.: |
12/697917 |
Filed: |
February 1, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09770943 |
Jan 25, 2001 |
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12697917 |
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09092314 |
Jun 5, 1998 |
6225292 |
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09770943 |
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60048793 |
Jun 6, 1997 |
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Current U.S.
Class: |
514/44R ;
435/455; 435/6.14; 506/10; 530/402; 536/25.5 |
Current CPC
Class: |
A61K 38/00 20130101;
C12N 2310/334 20130101; C12N 2310/33 20130101; A61P 37/06 20180101;
A61K 2039/55561 20130101; C12N 2310/3513 20130101; A61K 2039/57
20130101; Y02A 50/423 20180101; A61P 43/00 20180101; A61P 37/02
20180101; C12N 2310/111 20130101; C12N 2310/315 20130101; C12N
2310/18 20130101; A61K 39/39 20130101; C12N 2310/17 20130101; C07H
21/00 20130101; C07K 14/4703 20130101; Y02A 50/41 20180101; C12N
15/117 20130101; C12Q 1/6883 20130101 |
Class at
Publication: |
514/44.R ;
536/25.5; 530/402; 435/455; 506/10; 435/6 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C07H 21/00 20060101 C07H021/00; C12N 15/85 20060101
C12N015/85; C40B 30/06 20060101 C40B030/06; C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A pharmaceutically useful compound for inhibiting
immunostimulation by immunostimulatory sequence
oligodeoxynucleotides (ISS-ODN), wherein the ISS-ODN contain a
hexamer region consisting of at least one CpG nucleotide motif
flanked by two 5' purines and two 3' pyrimidines (ISS-ODN),
comprising: an oligonucleotide containing a hexamer region having
the nucleotide sequence
5'-Purine-Purine-[Y]-[Z]-Pyrmidine-Pyrimidine-3' or
5'-Purine-Purine-[Y]-[Z]-Pyrmidine-poly(Pyrimidine)-3'; where Y is
any naturally occurring or synthetic nucleotide except cytosine and
Z is any naturally occurring or synthetic nucleotide; however, when
Y is not guanosine or inosine, Z is guanosine or inosine.
2. The compound according to claim 1 where Y is guanosine or
inosine.
3. The compound according to claim 1 where Y is inosine and Z is
inosine or guanosine.
4. The compound according to claim 1 where Y is guanosine and Z is
guanosine or an unmethylated cytosine.
5. A pharmaceutically useful compound for inhibiting
immunostimulation by immunostimulatory sequence
oligodeoxynucleotides comprising an oligonucleotide containing a
hexamer region having the nucleotide sequence AAGGTT.
6. A pharmaceutically useful compound for inhibiting
immunostimulation by immunostimulatory sequence
oligodeoxynucleotides comprising an oligonucleotide containing a
hexamer region having a nucleotide sequence consisting of
AAGCTT.
7. A pharmaceutically useful compound for inhibiting
immunostimulation by immunostimulatory sequence
oligodeoxynucleotides comprising an oligonucleotide containing a
hexamer region having a nucleotide sequence consisting of
AGGGCT
8. A pharmaceutically useful compound for inhibiting
immunostimulation by immunostimulatory sequence
oligodeoxynucleotides comprising an oligonucleotide containing a
hexamer region having a nucleotide sequence consisting of
GAGGTT.
9. A pharmaceutically useful compound for inhibiting
immunostimulation by immunostimulatory sequence
oligodeoxynucleotides comprising an oligonucleotide containing a
hexamer region having a nucleotide sequence selected from the group
of sequences consisting of TABLE-US-00001 AAGCTT, AGGCTC, GAGCTT,
GGGCTT, AAGCTC, AGGCTC, GAGCTC, GGGCTC, AAGCCC, AGGCCC, GAGCCC,
GGGCCC, AGGCCT, GAGCCT, GGGGCT, TTGCAA, AATGTT, GGGGTT and
AAGCCC.
10. The compound according to any of claims 1 through 9 wherein the
hexamer region is flanked by nucleotides in a sequence identical to
the sequence of nucleotides which flank the hexamer region of any
known ISS-ODN.
11. The compound according to any of claims 1 through 9 wherein the
oligonucleotide compound is conjugated to a peptide.
12. A kit for use in gene therapy or gene immunization consisting
of any of the immunoinhibitory compounds of claims 1 through 11 in
a sterile vial and a recombinant expression vector in a sterile
vial.
13. The kit according to claim 12 wherein the immunoinhibitory
compound and the recombinant expression vector are contained in the
same sterile vial.
14. A method for inhibiting the immunostimulatory activity of
ISS-ODN in contact with a population of vertebrate cells which
includes lymphocytes or monocytes comprising contacting the
population of vertebrate cells with an immunoinhibitory amount of
an oligonucleotide containing a hexamer region having the
nucleotide sequence
5'-Purine-Purine-[Y]-[Z]-Pyrmidine-Pyrimidine-3' or
5'-Purine-Purine-[Y]-[Z]-Pyrimidine-poly(Pyrimidine)-3', where Y is
any naturally occurring or synthetic nucleotide except cytosine and
Z is any naturally occurring or synthetic nucleotide; however, when
Y is not guanosine or inosine, Z is guanosine or inosine; wherein a
reduction in Th1 type immune responses measured in the population
of vertebrate cells indicates that the desired inhibtion of ISS-ODN
immunostimulatory activity has been achieved.
15. The method according to claim 14 wherein the ISS-ODN are
believed to be present in a recombinant expression vector.
16. The method according to claim 15 wherein both the recombinant
expression vector and the immunoinhibitory oligonucleotide are
administered to a vertebrate host.
17. The method according to claim 14 wherein the ISS-ODN are
believed to be present in a microbe.
18. The method according to claim 17 wherein the microbe has
infected a vertebrate host and the microbe is contacted with the
immunoinhibitory oligonucleotide by adminstering the
oligonucleotide in an immunoinhibitory amount to the host.
19. The method according to claim 18 wherein the vertebrate host
has an autoimmune disease believed to be clinically related to
infection of the host by the microbe.
20. A method for prolonging gene expression in a recombinant
expression vector believed to contain at least one ISS-ODN
comprising contacting the recombinant expression vector with an
immunoinhibitory amount of an oligonucleotide containing a hexamer
region having the nucleotide sequence
5'-Purine-Purine-[Y]-[Z]-Pyrmidine-Pyrimidine-3' or
5'-Purine-Purine-[Y]-[Z]-Pyrmidine-poly(Pyrimidine)-3', where Y is
any naturally occurring or synthetic nucleotide except cytosine and
Z is any naturally occurring or synthetic nucleotide; however, when
Y is not guanosine or inosine, Z is guanosine or inosine; wherein
gene expression for a longer period of time than is obtained from
the same recombinant expression vector in the absence of contact
with the immunoinhibitory oligonucleotide indicates that the
desired prolongation of gene expression has been achieved.
21. The method according to claim 20 wherein both the recombinant
expression vector and the immunoinhibitory oligonucleotide are
administered to a vertebrate host.
22. A method for reducing inflammation in a host in response to a
microbial infection of the host comprising adminstering an
immunoinhibitory amount of an oligonucleotide containing a hexamer
region having the nucleotide sequence
5'-Purine-Purine-[Y]-[Z]-Pyrmidine-Pyrimidine-3' or
5'-Purine-Purine-[Y]-[Z]-Pyrmidine-poly(Pyrimidine)-3' to the host,
where Y is any naturally occurring or synthetic nucleotide except
cytosine and Z is any naturally occurring or synthetic nucleotide;
however, when Y is not guanosine or inosine, Z is guanosine or
inosine; wherein a reduction in Th1 type immune responses against
the infectious microbe measured in the host or a reduction in other
clinical signs of inflammation in the host indicates that the
desired reduction in host inflammation has been achieved.
23. A method for modulating the immunostimulatory activity of an
ISS-ODN in contact with a population of vertebrate cells which
includes lymphocytes or monocytes comprising contacting the
population of vertebrate cells with an immunoinhibitory amount of
an oligonucleotide containing a hexamer region having the
nucleotide sequence
5'-Purine-Purine-[Y]-[Z]-Pyrmidine-Pyrimidine-3' or
5'-Purine-Purine-[Y]-[Z]-Pyrmidine-poly(Pyrimidine)-3', where Y is
any naturally occurring or synthetic nucleotide except cytosine and
Z is any naturally occurring or synthetic nucleotide; however, when
Y is not guanosine or inosine, Z is guanosine or inosine; wherein a
reduction in Th1 type immune responses measured in the population
of vertebrate cells indicates that the desired inhibtion of ISS-ODN
immunostimulatory activity has been achieved.
24. The method according to claim 23 wherein both the ISS-ODN and
the immunoinhibitory oligonucleotide are administered to a
vertebrate host.
25. A method for boosting a Th2 type immune response to an antigen
comprising contacting a population of antigen stimulated vertebrate
cells including lymphocytes with an immunostimulatory amount of an
oligonucleotide containing a hexamer region having the nucleotide
sequence 5'-Purine-Purine-[Y]-[Z]-Pyrmidine-Pyrimidine-3' or
5'-Purine-Purine-[Y]-[Z]-Pyrmidine-poly(Pyrimidine)-3', where Y is
any naturally occurring or synthetic nucleotide except cytosine and
Z is any naturally occurring or synthetic nucleotide; however, when
Y is not guanosine or inosine, Z is guanosine or inosine; wherein
wherein a reduction in Th1 type immune responses or increase in
antigen stimulated IgG1 production measured in the population of
vertebrate cells indicates that the desired boost in Th2 type
immune responses to the antigen has been achieved.
26. A method for identifying IIS-ON which inhibit the
immunostimulatory activity of ISS-ODN comprising: (a) contacting a
population of antigen stimulated immune cells with an ISS-ODN to
induce lymphocyte proliferation in; IFN.beta., IFN-.alpha.,
IFN-.gamma., IL-12 and IL-18 cytokine secretion from; IgG1 antibody
production by; or IgE suppression in, the population of antigen
stimulated immune cells; (b) measuring any change in the number of
lymphocytes or levels of secreted cytokines and/or levels of IgE or
IgG1 antibodies in the population of antigen stimulated cells after
contact with the ISS-ODN; (c) contacting the population of antigen
stimulated cells with a candidate IIS-ON inhibitory
oligonucleotide; and, (d) measuring any change in the number of
lymphocytes or levels of secreted IFN.beta., IFN-.alpha.,
IFN-.gamma., IL-12 and IL-18 cytokines and/or levels of IgE or IgG1
antibodies in the population of antigen stimulated cells after
contact with the oligonucleotide, wherein a decline in any of the
measured values for lymphocyte proliferation, cytokine secretion or
IgG1 antibody production, as well as an increase in IgE antibody
production, as compared to the measurements taken in step (b)
indicates that the oligonucleotide inhibits the immunostimulatory
activity of the ISS-ODN of step (a).
27. The method according to claim 26 wherein the candidate
inhibitory oligonucleotide contains a hexamer region having the
nucleotide sequence
5'-Purine-Purine-[Y]-[Z]-Pyrmidine-Pyrimidine-3' or
5'-Purine-Purine-[Y]-[Z]-Pyrmidine-poly(Pyrimidine)-3', where Y is
any naturally occurring or synthetic nucleotide except cytosine and
Z is any naturally occurring or synthetic nucleotide; however, when
Y is not guanosine, adenosine or inosine, Z is guanosine or
inosine.
28. A pharmaceutically useful compound comprising an
oligonucleotide identified according to the method of claim 26 as
one which inhibits the immunostimulatory activity of ISS-ODN.
29. A method for detecting ISS-ODN immunostimulatory activity in a
host comprising: (a) obtaining a sample of immune cells from the
host, which cells are believed to been exposed to an antigen or
autoantigen; (b) measuring the levels of lymphocyte proliferation
in; IFN.beta., IFN-.alpha., IFN-.gamma., IL-12 and IL-18 cytokine
secretion from; IgG1 antibody production by; or IgE suppression in,
the sample of host immune cells; (c) contacting the sample of host
immune cells with an immunoinhibitory oligonucleotide (IIS-ON);
and, (d) measuring any change in the number of lymphocytes or
levels of secreted IFN.beta., IFN-.alpha., IFN-.gamma., IL-12 and
IL-18 cytokines and/or levels of IgE, IgG2 or IgG1 antibodies in
the sample of host immune cells after contact with the IIS-ON,
wherein a decline in any of the measured values for lymphocyte
proliferation, cytokine secretion or IgG2 antibody production, as
well as an increase in IgG1 or IgE antibody production, as compared
to the measurements taken in step (b), indicates that an ISS-ODN
subject to inhibition by the IIS-ON is present in the sample of
host immune cells.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/048,794 filed on Jun. 6, 1997, which is
incorporated herein by reference.
BACKGROUND FOR THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to immunostimulatory sequences in DNA.
The invention further relates to recombinant expression vectors for
use in gene therapy.
[0004] 2. History of the Related Art
[0005] Recombinant expression vectors are the tools which
researchers and clinicians use to achieve the goals of gene therapy
and gene immunization. In gene therapy, viral and non-viral vectors
are used to deliver an expressible gene into a host to replace a
missing or defective gene, or to otherwise supply the host with a
therapeutically beneficial polypeptide. In gene immunization,
mostly non-viral vectors are used to induce an immune response by
the host to an expressed antigen.
[0006] One of the obstacles to successful clinical practice of both
gene therapy and gene immunization has been the often transient
nature of the gene expression achieved in vivo. Transient gene
expression is less problematic in gene immunization, where immune
responses sufficient for certain immunization schemes may be
stimulated by even short-term exposure to expressed antigen. In
addition, several options are available to boost the host immune
response to antigen, including use of the vector itself as an
adjuvant for the desired immune response by exposing the host to
non-coding, immunostimulatory nucleotide sequences (ISS-ODN)
present in the vector (Sato, et al., Science, 273:352-354
(1996)).
[0007] However, in a gene therapy protocol, premature loss of gene
expression deprives the host of the potential benefits of the
therapy (Friedmann, Scientific American, "Making Gene Therapy Work"
(June 1997)). Repetitive dosing to extend exposure of the host to a
therapeutic polypeptide can require that different vectors be used
to deliver each dose so the host immune response to vector antigens
is minimized (Tripathy, et al., Nature Med., 2:545-550 (1996)).
[0008] One potential source of vector immunogenicity are ISS-ODN in
the genome of the microbial species used to construct recombinant
expression vectors. To explain, the CpG motifs which characterize
ISS-ODN are present in bacteria and viruses (including
retroviruses) at a much greater frequency than is seen in
vertebrate genomes. One consequence of ISS-ODN activity is the
ISS-ODN induced production of cytokines such as interferon-.alpha.
(INF-.alpha.), INF-.gamma. and interleukin-12 (IL-12). This ISS-ODN
induced inflammation is believed to be defensive against microbial
infection in vertebrates and is also believed to be produced in
response to ISS-ODN introduced into a host as oligonucleotides or
as part of a recombinant expression vector.
SUMMARY OF THE INVENTION
[0009] The invention provides compounds consisting of
oligodeoxynucleotides, ribonucleotides or analogs thereof which
specifically inhibit the immunostimulatory activity of ISS-ODN.
ISS-ODN induced secretion of INF-.alpha. in particular can suppress
recombinant gene expression and directly impedes mRNA and protein
synthesis in transfected cells. Thus, inhibition of ISS-ODN
activity substantially avoids ISS-ODN induced loss of gene
expression, thereby prolonging the availability of the expressed
polypeptide to a host undergoing gene therapy or gene immunization
with an ISS-ODN containing recombinant expression vector. Further,
the need for repetitive dosing to prolong availability of expressed
proteins and for extensive reengineering of recombinant expression
vectors to eliminate ISS-ODN sequences is avoided through use of
the compounds of the invention.
[0010] The compounds of the invention are also useful in modulating
the immunostimulatory activity of ISS-ODN administered as adjuvants
to boost host immune responses to antigen in, for example,
immunotherapy. In this respect, the compounds of the invention
permit exquisite control over the effect of ISS-ODN based adjuvants
in a host.
[0011] Further, the compounds of the invention reduce host
inflammation generated in response to an infection by an ISS-ODN
containing bacteria or virus. Advantageously, the compounds of the
invention can be administered to inhibit ISS-ODN activity exerted
by a microbe even if the identity of the particular ISS-ODN present
in the microbe is unknown. Thus, the compounds of the invention can
be considered to be broad spectrum anti-inflammatory agents.
[0012] In one aspect, the ISS-ODN inhibitory compounds of the
invention are synthesized oligonucleotides (I-ON) which are
comprised of the following general primary structures: [0013]
5'-Purine-Purine-[Y]-[Z]-Pyrmidine-Pyrimidine-3' or [0014]
5'-Purine-Purine-[Y]-[Z]-Pyrmidine-pPyrimidine-3' where Y is any
naturally occurring or synthetic nucleotide except cytosine and is
preferably guanosine or inosine (for RNA I-ON). In general, Z is
any naturally occurring or synthetic nucleotide or repeat of the
same nucleotide. Preferably, when Y is inosine, Z is inosine or one
or more guanosine(s). Where Y is guanosine, Z is preferably
guanosine or one or more unmethylated cytosine(s). However, when Y
is not guanosine or inosine, Z is guanosine or inosine. Most
preferably, the 5' purines are the same nucleotide, as are the 3'
pyrimidines. For example, where ** is YZ, the 5' purines and 3'
pyrimidines may be AA**TT, AG**TT, GA**TT, GG**TT, AA**TC, AG**TC,
and so forth. Any sequences present which flank the hexamer core
sequence are constructed to substantially match the flanking
sequences present in any known ISS-ODN.
[0015] Inhibitory I-ON of the invention are prepared in a
pharmaceutically acceptable composition for use in a host. I-ON may
be mixed into the composition singly, in multiple copies or in a
cocktail of different I-ON. Alternatively, the inhibitory I-ON of
the invention may be incorporated into a recombinant expression
vector. The inhibitory I-ON can also be provided in the form of a
kit, comprising inhibitory I-ON and recombinant expression vector
constructs which contain, or are susceptible to insertion of, a
gene of interest.
[0016] A particular advantage of the I-ON of the invention is that
they can be used to target ISS-ODN in any ISS-ODN containing
recombinant expression vector or microbe, whether or not the
nucleotide composition of the vector or microbe is known. Indeed,
it is not necessary that the existence, identity or location of
ISS-ODN in the vector or microbe be known. If ISS-ODN are not
present in the vector or microbe, the I-ON of the invention will
simply have no effect. However, if ISS-ODN are present in the
vector or microbe, it can be expected that their immunostimulatory
activity will be inhibited in a dose-dependent manner by the I-ON
even if the specific structure or location of the ISS-ODN in the
vector or microbe is not known.
[0017] Thus, in another aspect, the invention provides a simple and
effective alternative to the arduous task of eliminating ISS-ODN
activity from recombinant expression vectors by identifying all
ISS-ODN present in the vector and removing them.
[0018] Further in this regard, the invention provides methods for
screening recombinant expression vectors for the presence of
ISS-ODN and for identifying additional inhibitory I-ON. In the
former respect, the presence of ISS-ODN in a recombinant expression
vector is confirmed by incubating the vector in a population of
lymphocytes with an I-ON of known inhibitory activity and comparing
the difference, if any, in the level of ISS-induced cytokine
production by the lymphocytes before and after I-ON incubation.
[0019] In the latter respect, additional inhibitory I-ON having the
characteristics disclosed herein are identified by their ability to
inhibit the immunostimulatory activity of a known ISS-containing
polynucleotide or recombinant expression vector.
[0020] In yet another aspect, the invention further provides a
useful anti-inflammatory agent for use in inhibiting the
immunostimulatory activity of any ISS-ODN present in an infectious
bacterium or virus.
[0021] In addition, the invention provides useful means for
modulating the immunostimulatory activity of ISS-ODN supplied to a
host for immunostimulation (e.g., as an adjuvant).
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 is a graph which represents in vivo inhibition of
ISS-ODN immunostimulatory activity by inhibitory I-ON of the
invention (I-ON DY1019 and DY1041 (having hexamer regions
consisting of, respectively, AAGGTT and AAGCTT)). Lymphocyte
proliferation stimulated in a murine model by the ISS-ODN (DY1038,
having a hexamer region consisting of AACGTT) was compared in the
presence or absence of the I-ON. A decline in measured
counts-per-minute (CPM; vertical axis) represents inhibition of
ISS-ODN immunostimulatory activity in the Figure. Dosages for each
I-ON tested are shown along the horizontal axis. DY1039 (an ISS-ODN
with the cytosine), DY1040, DY1042 and DY1043 (all with CC
dinucleotides in place of the CG dinucleotide of DY1038) served as
controls. To confirm the location of potential competition with
DY1038, all of the oligonucleotides were identical to DY1038 except
for the hexamer regions identified and DY1043 (an irrelevant
sequence control).
[0023] FIG. 2 is a graph which confirms in vivo dose dependent
inhibition of ISS-ODN immunostimulatory activity by the DY1019 and
DY1041 I-ON of the invention. Lymphocyte proliferation stimulated
in a murine model by a different ISS-ODN than the one tested in the
experiment of FIG. 1 (DY 1018) was compared in the presence or
absence of the I-ON. A decline in measured counts-per-minute (CPM;
vertical axis) represents inhibition of ISS-ODN immunostimulatory
activity in the Figure. Dosages for each I-ON tested are shown
along the horizontal axis. Inhibitory activity of I-ON DY1019 and
DY-1041 increased with dosage, with the increase in activity of
DY1019 being proportional to the increase in dosage. To confirm the
location of potential competition with DY1018, DY1019 and DY1041
are identical to DY1018 except for the hexamer regions
identified.
[0024] FIG. 3 is a graph which represents in vivo dose dependent
inhibition of ISS-ODN immunostimulatory activity by several
inhibitory I-ON of the invention. Lymphocyte proliferation
stimulated in a murine model by DY1038 was compared in the presence
or absence of the I-ON. A decline in measured counts-per-minute
(CPM; vertical axis) represents inhibition of ISS-ODN
immunostimulatory activity in the Figure. Dosages for each I-ON
tested are shown along the horizontal axis. In descending order,
the most inhibitory activity was displayed by I-ON DY1019, DY-1041,
DY1048, DY1050 and DY1060 (the latter have hexamer regions
consisting of, respectively, AGGGTT, GAGGTC and TTGCAA). DY1039 (an
ISS-ODN with the cytosine methylated), DY1040 and DY1043 (the
latter with CC dinucleotides in place of the CG dinucleotide of
DY1038) served as controls. To confirm the location of potential
competition with DY1038, all of the oligonucleotides were identical
to DY1038 except for the hexamer regions identified and DY1043 (an
irrelevant sequence control).
[0025] FIG. 4 is a graph which represents in viva dose dependent
inhibition of ISS-ODN immunostimulatory activity by inhibitory I-ON
of the invention. INF-.gamma. production stimulated by DY1018
ISS-ODN in a murine model was compared in the presence or absence
of the I-ON. A decline in measured INF-.gamma. (vertical axis)
represents inhibition of ISS-ODN immunostimulatory activity in the
Figure. Dosages for each I-ON tested are shown along the horizontal
axis. Some inhibitory activity was observed for all but one I-ON,
with the most activity being displayed by I-ON DY1019 and DY-1041,
as well as DY1042 (having a hexamer region consisting of TTCCTT).
The insert separates out the data for inhibition of INF-.gamma.
production by DY1019. To confirm the location of potential
competition with DY1018, all of the oligonucleotides were identical
to DY1018 except for the hexamer regions identified and DY1043 (an
irrelevant sequence control).
[0026] FIG. 5 is a graph which represents the adjuvant properties
of IIS-ODN, whereby a Th2-type cellular immune response in antigen
(.beta.-galactosidase) immunized mice is induced by
co-administration of the antigen and IIS-ODN DY1019 (identified in
the Figure as .beta.-gal/M-ODN). Th2 responses are represented by
IgE levels measured post-boosting. The values obtained are compared
to IgE levels measured in mice immunized with antigen and the
ISS-ODN composition .beta.-gal/ISS-ODN (5'-AATTCAACGTTCGC-3),
pKISS-3 (a plasmid having three copies of the AACGTT ISS-ODN
hexamer in the backbone) and pKISS-0 (a plasmid having no copies of
the AACGTT ISS-ODN hexamer in the backbone), as well as mice which
received only saline. Potent IgE responses (Th2-type responses)
above 1000 CPM were obtained only in the mice which received saline
(approximately 1200 CPM at 1 week post-boosting) and
.beta.-gal/M-ODN (approximately 1750 CPM at 1 week
post-boosting).
DETAILED DESCRIPTION OF THE INVENTION
A. Activity and Structure of IIS-ON
[0027] 1. IIS-ON Activity and Screening Assay
[0028] The IIS-ON of the invention reduce the immunostimulatory
effect of ISS-ODN. Structurally, ISS-ODN are non-coding
oligonucleotides 6 mer or greater in length which may include at
least one unmethylated CG motif. The relative position of each CG
sequence in ISS-ODN with immunostimulatory activity in certain
mammalian species (e.g., rodents) is 5'-CG-3' (i.e., the C is in
the 5' position with respect to the G in the 3' position). Many
known ISS-ODN flank the CG motif with at least two purine
nucleotides (e.g., GA or AA) and at least two pyrimidine
nucleotides (e.g., TC or TT) to enhance the B lymphocyte
stimulatory activity of the immunostimulatory polynucleotide (see,
e.g., Krieg, et al., Nature, 374:546-549, 1995).
[0029] Functionally, ISS-ODN enhance the cellular and humoral
immune responses in a host, particularly lymphocyte proliferation
and the release of cytokines (including IFN) by host monocytes and
natural killer (NK) cells. Bacterial DNA contains unmethylated CpG
dinucleotides at a frequency of about one per every 16 bases. These
dinucleotides are also present in certain viral species, but are
notably underrepresented in vertebrate species.
[0030] It is believed that the ability of mycobacteria as well as
other bacterial and viral species to stimulate lymphocyte
proliferation, IL-12 production, tumor necrosis factor (TNF)
production, natural killer (NK) cell activity and IFN-.gamma.
secretion is owed to the presence of ISS-ODN in bacterial and viral
DNA (see, e.g., Krieg, Trends in Microbiology, 4:73-76 (1996)). In
contrast, CpG suppression and methylation in vertebrates may be an
evolutionary response to the threat of bacterial and viral
infection. Interestingly, a CpG containing oligonucleotide
comparable to bacterial ISS-ODN has also recently been implicated
in the onset and exacerbation of autoimmune disease through an
IL-12 dependent pathway (Segal, et al., J. Immunol., 158:5087
(1997)).
[0031] Immuostimulation by synthetic ISS-ODN in vivo occurs by
contacting host lymphocytes with, for example, ISS-ODN
oligonucleotides, ISS-ODN oligonucleotide-conjugates and
ISS-containing recombinant expression vectors (data regarding the
activity of ISS-ODN conjugates and ISS-ODN vectors are set forth in
co-pending, commonly assigned U.S. patent applications Ser. Nos.
60/028,118 and 08/593,554; data from which is incorporated herein
by reference solely to demonstrate ISS-ODN immunostimulatory
activity in vivo). Thus, while native microbial ISS-ODN stimulate
the host immune system to respond to infection, synthetic analogs
of these ISS-ODN may be useful therapeutically to modulate the host
immune response not only to microbial antigens, but also to tumor
antigens, allergens and other substances (id.).
[0032] Although the invention is not limited by any theory
regarding the mechanism of action of the IIS-ON, it is believed
that they compete with ISS-ODN for binding to the cellular membrane
of host lymphocytes. The region of ISS-ODN which confers their
immunostimulatory activity is believed to be the 6 mer or greater
length of nucleotides which include an unmethylated dinucleotide
(e.g., CpG). Therefore, it is believed that the presence of a
region of about 6 mer or greater length having at least one
competing dinucleotide (defined as [Y]-[Z] and [Y]-poly[Z] in the
formulae set forth below) therein confers ISS-inhibitory activity
on the IIS-ON of the invention.
[0033] Thus, the inhibitory compounds of the invention are
synthesized oligonucleotides (IIS-ON) which inhibit the
immunostimulatory activity of ISS-ODN in vertebrates and vertebrate
immune cells. To identify IIS-ON from a pool of synthesized
candidate IIS-ONs, the following steps provide a simple and
efficient means of rapidly screening the candidate pool: [0034] a.
A population of cultured, antigen stimulated lymphocytes and/or
monocytes is contacted with an ISS-ODN to induce lymphocyte
proliferation, IFN.beta., IFN-.alpha., IFN-.gamma., IL-12 and IL-18
cytokine secretion and/or IgG2 antibody production. [0035] b. Any
change in the number of lymphocytes, levels of secreted IFN.beta.,
IFN-.alpha., IFN-.gamma., IL-12 and IL-18 cytokines, IgG1 or IgG2
antibody levels or IgE antibody levels in the cell culture after
contact with the ISS-ODN is measured. [0036] c. The cells are
contacted with the candidate IIS-ON. [0037] d. Any change in the
number of lymphocytes, levels of secreted cytokines, IgG2 antibody
levels or IgE antibody levels in the population of cells after
contact with the oligonucleotide is measured.
[0038] A decline in any of these values (except IgG1 and IgE
antibodies) as compared to the measurements taken in step (2)
indicates that the candidate oligonucleotide is an IIS-ON of the
invention; i.e., it inhibits the immunostimulatory activity of
ISS-ODN. Alternatively, a rise in measured levels of IgG1 or IgE
antibodies is an indirect indicator of a rise in a Th2-type
lymphocyte response, indicating that the Th1 stimulatory activity
of ISS-ODN has declined in the presence of the IIS-ODN. Assay
techniques suitable for use in performing the steps above are
illustrated in the Examples below. In view of the teaching of this
disclosure, other assay techniques for measuring changes in ISS-ODN
induced lymphocyte proliferation or cytokine secretion will be
apparent to those of ordinary skill in the art.
[0039] The screening method can also be used to detect ISS-ODN in a
sample of immune cells taken from the host. This aspect of the
invention is useful in confirming the presence of ISS-ODN
containing antigens (e.g., microbial antigens) and autoantigens in
the host. To this end, the steps of the above-described screening
method are modified to include the steps of: [0040] a. Obtaining a
sample of immune cells from the host, which cells are believed to
been exposed to an antigen or autoantigen. [0041] b. Measuring the
levels of lymphocyte proliferation in; IFN.beta., IFN-.gamma.,
IL-12 and IL-18 cytokine secretion from; IgG1 and IgG2 antibody
production by; or IgE antibody production by, the sample of host
immune cells. [0042] c. Contacting the sample of host immune cells
with an IIS-ON. [0043] d. Measuring any change in the number of
lymphocytes or levels of secreted IFN.beta., IFN-.alpha.,
IFN-.gamma., IL-12 and IL-18 cytokines and/or levels of IgE or IgG1
antibodies in the sample of host immune cells after contact with
the IIS-ON, wherein a decline in any of the measured values for
lymphocyte proliferation, cytokine secretion or IgG2 antibody
production, as well as an increase in IgG1 and IgE antibody
production, as compared to the measurements taken in step (b)
indicates that an ISS-ODN subject to inhibition by the IIS-ON is
present in the sample of host immune cells.
[0044] 2. Exemplary IIS-ON Structure
[0045] Particular IIS-ON which inhibit the activity of CpG
motif-containing ISS-ODN include those oligonucleotides which are
comprised of the following general primary structure: [0046]
5'-Purine-Purine-[Y]-[Z]-Pyrmidine-Pyrimidine-3' or [0047]
5'-Purine-Purine-[Y]-[Z]-Pyrimidine-polyPyrimidine-3' where Y is
any naturally occurring or synthetic nucleotide except cytosine and
is preferably guanosine, adenosine or inosine (for RNA IIS-ON),
most preferably guanosine. In general, Z is any naturally occurring
or synthetic nucleotide or repeat of the same nucleotide.
Preferably, where Y is inosine, Z is inosine or one or more
guanosine(s). Where Y is guanosine, Z is preferably guanosine or
one or more unmethylated cytosine(s). Where Y is adenosine, Z is
preferably guanosine. However, when Y is not guanosine, adenosine
or inosine, Z is guanosine, adenosine or inosine. Most preferably,
the 5' purines are the same nucleotide, as are the 3' pyrimidines.
For example, where ** is YZ, the 5' purines and 3' pyrimidines may
be AA**TT, AG**TT, GA**TT, GG**TT, AA**TC, AG**TC, and so
forth.
[0048] The core hexamer structure of the foregoing IIS-ON may be
flanked upstream and/or downstream by any number or composition of
nucleotides or nucleosides. However, IIS-ON will preferably be
either 6 mer in length, or between 6 and 45 mer in length, to
enhance uptake of the IIS-ON and to minimize non-specific
interactions between the IIS-ON and the target recombinant
expression vector or host cells. Preferably, any IIS-ON flanking
sequences present are constructed to match the flanking sequences
present in any known ISS-ODN (such as the flanking sequence DY1038
(TTGACTGTG******AGAGATGA), where ****** is the immunostimulatory
hexamer sequence. Those of ordinary skill in the art will be
familiar with, or can readily identify, reported nucleotide
sequences of known ISS-ODN. For ease of reference in this regard,
the following sources are especially helpful: [0049] Yamamoto, et
al., Microbiol. Immunol., 36:983 (1992) [0050] Ballas, et al, J.
Immunol., 157:1840 (1996) [0051] Klinman, et al., J. Immunol.,
158:3635 (1997) [0052] Sato, et al., Science, 273:352 (1996)
[0053] Each of these articles are incorporated herein by reference
for the purpose of illustrating the level of knowledge in the art
concerning the nucleotide composition of ISS-ODN
[0054] Particular inhibitory IIS-ON of the invention include those
having the following hexamer sequences: [0055] 1. IIS-ODN having
"GG" dinucleotides: AAGGTT, AGGGTT, GGGGTT, GGGGTC, AAGGTC, AAGGCC,
AGGGTT, AGGGTC, GAGGTT, GAGGTC, GAGGCC, GGGGCT and so forth. [0056]
2. IIS-ODN having "GC" dinucleotides: AAGCTT, AGGCTC, AGGCCC,
GAGCTT, GAGCTC, GAGCCC, GGGCTT, GGGCTC, GGGCCC, AAGCCC, AAGCCT,
AGGCCT, GAGCCT and so forth. [0057] 3. Inosine and/or adenosine
subsitutions for nucleotides in the foregoing hexamer sequences
made according to the formulae set forth above.
[0058] IIS-ON hexamers with especially strong expected inhibitory
activity are those with GG and GC competing dinucleotides,
particularly AAGGTT (DY1019 in the Figures), AAGCTT (DY1041 in the
Figures), AGGGCT, and GAGGTT (including their 3'
Pyrimidine-pPyrimidine analogs).
[0059] IIS-ON may be single-stranded or double-stranded DNA, single
or double-stranded RNA and/or oligonucleosides. The nucleotide
bases of the IIS-ON which flank the competing dinucleotides may be
the known naturally occurring bases or synthetic non-natural bases
(e.g., TCAG or, in RNA, UACGI). Oligonucleosides may be
incorporated into the internal region and/or termini of the IIS-ON
using conventional techniques for use as attachment points for
other compounds (e.g., peptides). The base(s), sugar moiety,
phosphate groups and termini of the IIS-ON may also be modified in
any manner known to those of ordinary skill in the art to construct
an IIS-ON having properties desired in addition to the inhibitory
activity of the IIS-ON. For example, sugar moieties may be attached
to nucleotide bases of IIS-ON in any steric configuration. In
addition, backbone phosphate group modifications (e.g.,
methylphosphonate, phosphorothioate, phosphoroamidate and
phosphorodithioate internucleotide linkages) can confer
anti-microbial activity on the IIS-ON, making them particuarly
useful in therapeutic applications.
[0060] The techniques for making these phosphate group
modifications to oligonucleotides are known in the art and do not
require detailed explanation. For review of one such useful
technique, the an intermediate phosphate triester for the target
oligonucleotide product is prepared and oxidized to the naturally
occurring phosphate triester with aqueous iodine or with other
agents, such as anhydrous amines. The resulting oligonucleotide
phosphoramidates can be treated with sulfer to yield
phophorothioates. The same general technique (excepting the sulfer
treatment step) can be applied to yield methylphosphoamidites from
methylphosphonates. For more details concerning phosphate group
modification techniques, those of ordinary skill in the art may
wish to consult U.S. Pat. Nos. 4,425,732; 4,458,066; 5,218,103 and
5,453,496, as well as Tetrahedron Lett. at 21:4149 (1995), 7:5575
(1986), 25:1437 (1984) and Journal Am. Chem Soc., 93:6657 (1987),
the disclosures of which are incorporated herein for the sole
purpose of illustrating the standard level of knowledge in the art
concerning preparation of these compounds.
[0061] A particularly useful phosphate group modification is the
conversion to the phosphorothioate or phosphorodithioate forms of
the IIS-ON oligonucleotides. In addition to their potentially
anti-microbial properties, phosphorothioates and
phosphorodithioates are more resistant to degradation in vivo than
their unmodified oligonucleotide counterparts, making the IIS-ON of
the invention more available to the host.
[0062] IIS-ON can be synthesized using techniques and nucleic acid
synthesis equipment which are well-known in the art. For reference
in this regard, see, e.g., Ausubel, et al., Current Protocols in
Molecular Biology, Chs. 2 and 4 (Wiley Interscience, 1989);
Maniatis, et al., Molecular Cloning: A Laboratory Manual (Cold
Spring Harbor Lab., New York, 1982); U.S. Pat. No. 4,458,066 and
U.S. Pat. No. 4,650,675. These references are incorporated herein
by reference for the sole purpose of demonstrating knowledge in the
art concerning production of synthetic oligonucleotides.
[0063] Alternatively, IIS-ON can be obtained by mutation of
isolated microbial ISS-ODN to substitute a competing dinucleotide
for the naturally occurring CpG motif. Screening procedures which
rely on nucleic acid hybridization make it possible to isolate any
polynucleotide sequence from any organism, provided the appropriate
probe or antibody is available. Oligonucleotide probes, which
correspond to a part of the sequence encoding the protein in
question, can be synthesized chemically. This requires that short,
oligo-peptide stretches of amino acid sequence must be known. The
DNA sequence encoding the protein can also be deduced from the
genetic code, however, the degeneracy of the code must be taken
into account.
[0064] For example, a cDNA library believed to contain an
ISS-containing polynucleotide of interest can be screened by
injecting various mRNA derived from cDNAs into oocytes, allowing
sufficient time for expression of the cDNA gene products to occur,
and testing for the presence of the desired cDNA expression
product, for example, by using antibody specific for a peptide
encoded by the polynucleotide of interest or by using probes for
the repeat motifs and a tissue expression pattern characteristic of
a peptide encoded by the polynucelotide of interest. Alternatively,
a cDNA library can be screened indirectly for expression of
peptides of interest having at least one epitope using antibodies
specific for the peptides. Such antibodies can be either
polyclonally or monoclonally derived and used to detect expression
product indicative of the presence of cDNA of interest.
[0065] Once the ISS-containing polynucleotide has been obtained, it
can be shortened to the desired length by, for example, enzymatic
digestion using conventional techniques. The CpG motif in the
ISS-ODN oligonucleotide product is then mutated to substitute a
competing dinucleotide for the CpG motif. Techniques for making
substitution mutations at particular sites in DNA having a known
sequence are well known, for example M13 primer mutagenesis through
PCR. Because the IIS-ON is non-coding, there is no concern about
maintaining an open reading frame in making the substitution
mutation. However, for in vivo use, the polynucleotide starting
material, ISS-ODN oligonucleotide intermediate or IIS-ON mutation
product should be rendered substantially pure (i.e., as free of
naturally occurring contaminants and LPS as is possible using
available techniques known to and chosen by one of ordinary skill
in the art).
[0066] The IIS-ON of the invention may be used alone or may be
incorporated in cis or in trans into a recombinant expression
vector (plasmid, cosmid, virus or retrovirus) which may in turn
code for any therapeutically beneficial protein deliverable by a
recombinant expression vector. For the sake of convenience, the
IIS-ON are preferably administered without incorporation into an
expression vector. However, if incorporation into an expression
vector is desired, such incorporation may be accomplished using
conventional techniques which do not require detailed explanation
to one of ordinary skill in the art. For review, however, those of
ordinary skill may wish to consult Ausubel, Current Protocols in
Molecular Biology, supra.
[0067] Briefly, construction of recombinant expression vectors
employs standard ligation techniques. For analysis to confirm
correct sequences in vectors constructed, the ligation mixtures may
be used to transform a host cell and successful transformants
selected by antibiotic resistance where appropriate. Vectors from
the transformants are prepared, analyzed by restriction and/or
sequenced by, for example, the method of Messing, et al., (Nucleic
Acids Res., 9:309, 1981), the method of Maxam, et al., (Methods in
Enzymology, 65:499, 1980), or other suitable methods which will be
known to those skilled in the art. Size separation of cleaved
fragments is performed using conventional gel electrophoresis as
described, for example, by Maniatis, et al., (Molecular Cloning,
pp. 133-134, 1982).
[0068] Host cells may be transformed with the expression vectors of
this invention and cultured in conventional nutrient media modified
as is appropriate for inducing promoters, selecting transformants
or amplifying genes. The culture conditions, such as temperature,
pH and the like, are those previously used with the host cell
selected for expression, and will be apparent to the ordinarily
skilled artisan.
[0069] If a recombinant expression vector is utilized as a carrier
for the IIS-ON of the invention, plasmids and cosmids are
particularly preferred for their lack of pathogenicity. However,
plasmids and cosmids are subject to degradation in vivo more
quickly than viruses and therefore may not deliver an adequate
dosage of IIS-ON to substantially inhibit ISS-ODN immunostimulatory
activity exerted by a systemically administered gene therapy
vector. Of the viral vector alternatives, adeno-associated viruses
would possess the advantage of low pathogenicity. The relatively
low capacity of adeno-associated viruses for insertion of foreign
genes would pose no problem in this context due to the relatively
small size in which IIS-ON of the invention can be synthesized.
[0070] Other viral vectors that can be utilized in the invention
include adenovirus, adeno-associated virus, herpes virus, vaccinia
or an RNA virus such as a retrovirus. Retroviral vectors are
preferably derivatives of a murine, avian or human HIV retrovirus.
Examples of retroviral vectors in which a single foreign gene can
be inserted include, but are not limited to: Moloney murine
leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),
murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A
number of additional retroviral vectors can incorporate multiple
genes. All of these vectors can transfer or incorporate a gene for
a selectable marker so that transduced cells can be identified and
generated.
[0071] Since recombinant retroviruses are defective, they require
assistance in order to produce infectious vector particles. This
assistance can be provided, for example, by using helper cell lines
that contain plasmids encoding all of the structural genes of the
retrovirus under the control of regulatory sequences within the
LTR. These plasmids are missing a nucleotide sequence that enables
the packaging mechanism to recognize an RNA transcript for
encapsidation. Helper cell lines that have deletions of the
packaging signal include, but are not limited to, .PSI.2, PA317 and
PA12, for example. These cell lines produce empty virions, since no
genome is packaged. If a retroviral vector is introduced into such
helper cells in which the packaging signal is intact, but the
structural genes are replaced by other genes of interest, the
vector can be packaged and vector virion can be produced.
[0072] By inserting one or more sequences of interest into the
viral vector, along with another gene which encodes the ligand for
a receptor on a specific target cell, for example, the vector can
be rendered target specific. Retroviral vectors can be made target
specific by inserting, for example, a polynucleotide encoding a
sugar, a glycolipid, or a protein. Preferred targeting is
accomplished by using an antibody to target the retroviral vector.
Those of skill in the art will know of, or can readily ascertain
without undue experimentation, specific polynucleotide sequences
which can be inserted into the retroviral genome to allow target
specific delivery of the retroviral vector containing the
polynucleotides of interest.
[0073] Alternatively, a colloidal dispersion system may be used for
targeted delivery. Colloidal dispersion systems include
macromolecule complexes, nanocapsules, microspheres, beads, and
lipid-based systems including oil-in-water emulsions, micelles,
mixed micelles, and liposomes. The preferred colloidal system of
this invention is a liposome.
[0074] Liposomes are artificial membrane vesicles which are useful
as delivery vehicles in vitro and in vivo. It has been shown that
large unilamellar vesicles (LUV), which range in size from 0.2-4.0
.mu.m can encapsulate a substantial percentage of an aqueous buffer
containing large macromolecules. RNA, DNA and intact virions can be
encapsulated within the aqueous interior and be delivered to cells
in a biologically active form (Fraley, et al., Trends Biochem.
Sci., 6:77, 1981). In addition to mammalian cells, liposomes have
been used for delivery of polynucleotides in plant, yeast and
bacterial cells. In order for a liposome to be an efficient gene
transfer vehicle, the following characteristics should be present:
(1) encapsulation of the genes encoding the antisense
polynucleotides at high efficiency while not compromising their
biological activity; (2) preferential and substantial binding to a
target cell in comparison to non-target cells; (3) delivery of the
aqueous contents of the vesicle to the target cell cytoplasm at
high efficiency; and (4) accurate and effective expression of
genetic information (Mannino, et al., Biotechniques, 6:682,
1938).
[0075] The composition of the liposome is usually a combination of
phospholipids, particularly high-phase-transition-temperature
phospholipids, usually in combination with steroids, especially
cholesterol. Other phospholipids or other lipids may also be used.
The physical characteristics of liposomes depend on pH, ionic
strength, and the presence of divalent cations.
[0076] Examples of lipids useful in liposome production include
phosphatidyl compounds, such as phosphatidylglycerol,
phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,
sphingo-lipids, cerebrosides, and gangliosides. Particularly useful
are diacylphosphatidylglycerols, where the lipid moiety contains
from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and
is saturated. Illustrative phospholipids include egg
phosphatidylcholine, dipalmitoylphosphatidylcholine and
distearoylphosphatidylcholine.
[0077] The targeting of liposomes can be classified based on
anatomical and mechanistic factors. Anatomical classification is
based on the level of selectivity, for example, organ-specific,
cell-specific, and organelle-specific. Mechanistic targeting can be
distinguished based upon whether it is passive or active. Passive
targeting utilizes the natural tendency of Liposomes to distribute
to cells of the reticulo-endothelial system (RES) in organs which
contain sinusoidal capillaries. Active targeting, on the other
hand, involves alteration of the liposome by coupling the liposome
to a specific ligand such as a monoclonal antibody, sugar,
glycolipid, or protein, or by changing the composition or size of
the liposome in order to achieve targeting to organs and cell types
other than the naturally occurring sites of localization.
[0078] The surface of the targeted delivery system may be modified
in a variety of ways. In the case of a liposomal targeted delivery
system, lipid groups can be incorporated into the lipid bilayer of
the liposome in order to maintain the targeting ligand in stable
association with the liposomal bilayer. Various well known linking
groups can be used for joining the lipid chains to the, targeting
ligand (see, e.g., Yanagawa, et al., Nuc. Acids Symp. Ser., 19:189
(1988); Grabarek, et al., Anal. Biochem., 185:131 (1990); Staros,
et al., Anal. Biochem., 156:220 (1986) and Boujrad, et al., Proc.
Natl. Acad. Sci. USA, 90:5728 (1993), the disclosures of which are
incorporated herein by reference solely to illustrate the standard
level of knowledge in the art concerning conjugation of
oligonucleotides to lipids).
[0079] Targeted delivery of IIS-ON can also be achieved by
conjugation of the IIS-ON to a the surface of viral and non-viral
recombinant expression vectors, to an antigen or other ligand, to a
monoclonal antibody or to any molecule which has the desired
binding specificity. A particular IIS-ODN conjugate of interest is
one in which an autoantigen or autoantibody is the IIS-ODN
conjugate partner. IIS-ODN autoantigen conjugates are useful in
boosting host Th2 type immune responses to the autoantigen
(suppressing the Th1 responses induced by the autoantigen itself;
see, e.g., Conboy, et al., J. Exp. Med., 185:439-451 (1997)), while
IIS-ODN autoantibody conjugates are useful in inducing passive
immunity in a host suffering from an autoimmune condition. Specific
methods for delivery of such conjugates, as well as IIS-ON in
general, are described in greater detail infra.
[0080] Those of ordinary skill in the art will be familiar with, or
can readily determine, sources for autoantigens and autoantibodies
useful as IIS-ON conjugates. Examples of such conjugate materials
include myelin basic protein (see, e.g., sequence and sourcing
information provided in Segal, et al., J. Immunol., 158:5087
(1997); Matsuo, et al., Am. J. Pathol., 150:1253 (1997); and
Schluesener, FEMS Immunol. Med. Microbiol., 17:179 (1997));
Sjorgen's syndrome autoantigen (see, e.g., Hanjei, et al., Science,
276:604 (1997)); hemochromatosis autoantigen (see, e.g., Ruddy, et
al., Genome Res., 7:441 (1997)), La/SSB protein (see, e.g., Castro,
et al., Cell Calcium, 20:493 (1996)); HsEg5 lupus autoantigen (see,
e.g., Whitehead, et al., Arthritis Rheum., 39:1635 (1996)); Ki
nuclear lupus autoantigen (see, e.g., Paesen and Nuttal, Biochem.
Biophys. Acta, 1309:9 (1996)); and antibodies thereto (see, e.g.,
Menon, et al., J. Autoimmun., 10:43 (1997) and Rahman, et al.,
Semin. Arthritis Rheum., 26:515 (1996) [human antiphospholipid
(anti-DNA) monoclonal antibodies]; and, Kramers, et al., J.
Autoimmun., 9:723 (1997) [monoclonal anti-nucleosome lupus
autoantibodies]). Each of the cited references is incorporated
herein solely to illustrate the level of knowledge and skill in the
art concerning the identity, activity and structure of autoantigens
and autoantibodies.
[0081] Examples of other useful conjugate partners include any
immunogenic antigen (including allergens, live and attenuated viral
particles and tumor antigens), targeting peptides (such as receptor
ligands, antibodies and antibody fragments, hormones and enzymes),
non-peptidic antigens (coupled via a peptide linkage, such as
lipids, polysaccharides, glycoproteins, gangliosides and the like)
and cytokines (including interleukins, interferons, erythorpoietin,
tumor necrosis factor and colony stimulating factors). Such
conjugate partners can be prepared according to conventional
techniques (e.g., peptide synthesis) and many are commercially
available.
[0082] Those of ordinary skill in the art will also be familiar
with, or can readily determine, methods useful in preparing
oligonucleotide-peptide conjugates. Conjugation can be accomplished
at either termini of the IIS-ON or at a suitably modified base in
an internal position (e.g., a cytosine or uracil). For reference,
methods for conjugating oligonucleotides to proteins and to
oligosaccharide moieties of Ig are known (see, e.g., O'Shannessy,
et al., J. Applied Biochem., 7:347 (1985), the is disclosure of
which is incorporated herein by reference solely to illustrate the
standard level of knowledge in the art concerning oligonucleotide
conjugation). Another useful reference is Kessler: "Nonradioactive
Labeling Methods for Nucleic Acids", in Kricka (ed.), Nonisotopic
DNA Probe Techniques (Acad. Press, 1992)).
[0083] Briefly, examples of known, suitable conjugation methods
include: conjugation through 3' attachment via solid support
chemistry (see, e.g., Haralambidis, et al., Nuc. Acids Res., 18:493
(1990) and Haralambidis, et al., Nuc. Acids Res., 18:501 (1990)
[solid support synthesis of peptide partner]; Zuckermann, et al.,
Nuc. Acids Res., 15:5305 (1987), Corey, et al., Science, 238:1401
(1987) and Nelson, et al., Nuc. Acids Res., 17:1781 (1989) [solid
support synthesis of oligonucleotide partner]). Amino-amino group
linkages may be performed as described in Benoit, et al.,
Neuromethods, 6:43 (1987), while thiol-carboxyl group linkages may
be performed as described in Sinah, et al., Oligonucleotide
Analogues: A Practical Approach (IRL Press, 1991). In these latter
methods, the oligonucleotide partner is synthesized on a solid
support and a linking group comprising a protected amine, thiol or
carboxyl group opposite a phosphoramidite is covalently attached to
the 5'-hydroxyl (see, e.g., U.S. Pat. Nos. 4,849,513; 5,015,733;
5,118,800 and 5,118,802).
[0084] Linkage of the oligonucleotide partner to a peptide may also
be made via incorporation of a linker arm (e.g., amine or carboxyl
group) to a modified cytosine or uracil base (see, e.g., Ruth, 4th
Annual Congress for Recombinant DNA Research at 123). Affinity
linkages (e.g., biotin-streptavidin) may also be used (see, e.g.,
Roget, et al., Nuc. Acids Res., 17:7643 (1989)).
[0085] Methods for linking oligonucleotides to lipids are also
known and include synthesis of oligo-phospholipid conjugates (see,
e.g., Yanagawa, et al., Nuc. Acids Symp. Ser., 19:189 (1988)),
synthesis of oligo-fatty acids conjugates (see, e.g., Grabarek, et
al., Anal. Biochem., 185:131 (1990)) and oligo-sterol conjugates
(see, e.g., Boujrad, et al., Proc. Natl. Acad. Sci USA, 90:5728
(1993)).
[0086] Each of the foregoing references is incorporated herein by
reference for the sole purpose of illustrating the level of
knowledge and skill in the art with respect to oligonucleotide
conjugation methods.
[0087] If to be delivered without use of a vector or other delivery
system, IIS-ON will be prepared in a pharmaceutically acceptable
composition. Pharmaceutically acceptable carriers preferred for use
with the IIS-ON of the invention may include sterile aqueous of
non-aqueous solutions, suspensions, and emulsions. Examples of
non-aqueous solvents are propylene glycol, polyethylene glycol,
vegetable oils such as olive oil, and injectable organic esters
such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's or fixed oils. Intravenous vehicles include fluid
and nutrient replenishers, electrolyte replenishers (such as those
based on Ringer's dextrose), and the like. Preservatives and other
additives may also be present such as, for example, antimicrobials,
antioxidants, chelating agents, and inert gases and the like. A
composition of IIS-ON may also be lyophilized using means well
known in the art, for subsequent reconstitution and use according
to the invention.
B. Methods for Administering and Using IIS-ON of the Invention
[0088] The IIS-ON of the invention are useful in inhibiting the
immunostimulatory activity of ISS, wherever present. Thus, IIS-ON
are useful as, for example, anti-inflammatory agents for reducing
host immune responses to ISS-ODN in bacteria and viruses. IIS-ON
are also useful as agents for suppressing the immunostimulatory
activity of any ISS-ODN, known or unknown, present in recombinant
expression vectors, especially those used for gene therapy and
immunization. In addition, IIS-ODN are useful in inhibiting host
autoimmune responses stimulated by microbial ISS-ODN and in
boosting Th2 type responses to antigen.
[0089] In this context, "inhibition" refers to a reduction in the
host immune response as compared to the level of ISS-ODN stimulated
host immune response prior to IIS-ODN administration. Because
ISS-ODN stimulate secretion of certain cytokines (e.g., IL-12,
IL-18 and IFNs) and tend to shift the host cellular immune response
to the Th1 repertoire, measurements of cytokine levels,
cytokine-stimulated lymphocyte proliferation, IgG2 antibody levels
(the production of which is indicative of a Th1 lymphocyte
response), IgE levels (the suppression of which is indicative of a
Th1 lymphocyte response) and IgG1 antibody levels (the production
of which is indicative of a Th2 lymphocyte response) are all
suitable values for use in detecting IIS-ODN inhibitory activity.
Specific examples and details of methods for determining such
values are described further infra.
[0090] With respect to shifts in the Th1/Th2 repertoire and
consequent changes in cytokine levels, it is helpful to recall that
CD4+ lymphocytes generally fall into one of two distinct subsets;
i.e., the Th1 and Th2 cells. Th1 cells principally secrete IL-2,
IFN.gamma. and TNF.beta. (the latter two of which mediate
macrophage activation and delayed type hypersensitivity) while Th2
cells principally secrete IL-4 (which stimulates production of IgE
antibodies), IL-5, IL-6 and IL-10. These CD4+ subsets exert a
negative influence on one another; i.e., secretion of Th1
lymphokines inhibits secretion of Th2 lymphokines and vice versa.
In addition, it is believed that exposure of Th2 cells to cytotoxic
T lymphocytes (CTLs) also suppresses TH2 cell activity.
[0091] Factors believed to favor Th1 activation resemble those
induced by viral infection and include intracellular pathogens,
exposure to IFN-.beta., IFN-.alpha., IFN.gamma., IL-12 and IL-18,
as well as the presence of APCs and exposure to low doses of
antigen. Th1 type immune responses also predominate in autoimmune
disease. Factors believed to favor Th2 activation include exposure
to IL-4 and IL-10, APC activity on the part of B lymphocytes and
high doses of antigen. Active Th1 (IFN.gamma.) cells enhance
cellular immunity and are therefore of particular value in
responding to intracellular infections, while active Th2 cells
enhance antibody production and are therefore of value in
responding to extracellular infections (albeit at the risk of
anaphylactic events associated with IL-4 stimulated induction of
IgE antibody production). Thus, the ability to shift host immune
responses from the Th1 to the Th2 repertoire and vice versa has
substantial clinical significance for enhancing and controlling
host immunity against infection and allergy. Further, control over
Th1/Th2 mediated cytokine release enables one to control host
immune responses to self-antigens (having clinical significance for
treatment of autoimmune disease) and to recombinant expression
vector antigens (having clinical significance for control of gene
expression for gene therapy and gene immunization).
[0092] For use in modulating the immunogenicity of a recombinant
expression vector, the IIS-ON of the invention will be administered
according to any means and route by which the target recombinant
expression vector is administered to a host, including in vivo and
ex vivo routes. Uptake of IIS-ON by host cells occurs at least as
robustly as does uptake of therapy and immunization vectors, if not
more so due to the small size of IIS-ON as compared to the total
dimensions of plasmid, viral and retroviral nucleic acids.
[0093] A particular goal of IIS-ON administration in this context
is the inhibition of ISS-ODN stimulated, Th1 mediated cytokine
production. Thus, a measurable reduction of such cytokine levels in
a treated host constitutes IIS-ON therapeutic activity in this
embodiment of the invention. IIS-ON therapeutic activity is also
demonstrated in this context by prolongation of gene expression as
compared to expression levels obtained in the absence of IIS-ON.
Those of ordinary skill in the gene therapy and immunization arts
will be very familiar with, or can readily ascertain, clinically
acceptable means and routes for administration of therapy and
immunization vectors and, by extension, IIS-ON.
[0094] For use as anti-inflammatory agents, IIS-ON and IIS-ON
conjugates will be administered according to any means and route by
which known anti-inflammatories and antibiotics are administered. A
particular goal of IIS-ODN administration in this context is the
inhibition of ISS-ODN stimulated, Th1 mediated cytokine production.
Thus, a measurable reduction of such cytokine levels in a treated
host constitutes IIS-ODN therapeutic activity in this embodiment of
the invention. Those of ordinary skill in the art of treating
infectious disease will be very familiar with, or can readily
ascertain, clinically acceptable means and routes for
administration of anti-inflammatories and antibiotics and, by
extension, IIS-ON and their conjugates.
[0095] For use as autoimmune modulators, IIS-ON and IIS-ON
autoantigen or autoantibody conjugates will be administered
according to any means and route by which known therapies for
autoimmune disease are practiced. A particular goal of IIS-ODN
administration in this context is the inhibition of ISS-ODN
stimulated, Th1 mediated IL-12 production. Thus, a measurable
reduction of IL-12 levels in an autoimmune host constitutes IIS-ODN
therapeutic activity in this embodiment of the invention. Those of
ordinary skill in the art of treating autoimmune disease will be
very familiar with, or can readily ascertain, clinically acceptable
means and routes for administration of IIS-ON and their
conjugates.
[0096] For use as modulators of ISS-ODN administered as
immunostimulants, the IIS-ON and IIS-ON conjugates of the invention
will be administered according to any means and route by which the
target ISS-ODN is administered to a host, including in vivo and ex
vivo routes. For example, where ISS-ODN are administered as
adjuvants in an immunization protocol (see, co-pending and commonly
assigned U.S. Patent Applications Ser. Nos. 60/028,118 and
08/593,554), it may be desirable to be able to subsequently reduce
or eliminate the ISS-ODN immunostimulatory activity to modify the
course of therapy. In this context, therefore, IIS-ON serve as
ISS-ODN "off" switches, whereby IIS-ON and IIS-ON conjugate
activity is demonstrated by a measured reduction in ISS-ODN
stimulated cytokine production, ISS-ODN stimulated lymphocyte
production, or a shift away from the Th1 lymphocyte repertoire.
[0097] For use as adjuvants for Th2 immune responses to
extracellular antigen, the IIS-ON of the invention will be
administered according to any means and route by which
antigen-based vaccines may be administered to a host. Shifts away
from the Th1 lymphocyte repertoire are a measure of efficacy for
use of IIS-ON and IIS-ON conjugates as Th2 lymphocyte stimulatory
adjuvants in the presence of antigen.
[0098] A particular advantage of the IIS-ON of the invention is
their capacity to exert an ISS-ODN inhibitory activity even at
relatively low dosages. Although the dosage used will vary
depending on the clinical goals to be achieved, a suitable dosage
range is one which provides up to about 1-200 .mu.g of IIS-ON/ml of
carrier in a single dosage. In view of the teaching provided by
this disclosure, those of ordinary skill in the clinical arts will
be familiar with, or can readily ascertain, suitable parameters for
administration of IIS-ON according to the invention.
[0099] In this respect, the inhibitory activity of IIS-ON is
essentially dose-dependent. Therefore, to increase IIS-ON potency
by a magnitude of two, each single dose is doubled in
concentration. For use in inhibiting ISS-ODN activity (including
activity of ISS-ODN in recombinant expression vectors), it is
useful to administer the IIS-ON and target ISS-ODN or vector in
equivalent dosages, then increase the dosage of IIS-ON as needed to
achieve the desired level of inhibition. For use as an
anti-inflammatory agent, it is useful to administer the IIS-ON in a
low dosage (e.g., about 1 .mu.g/ml to about 50 .mu.g/ml), then
increase the dosage as needed to achieve the desired therapeutic
goal. Alternatively, a target dosage of IIS-ON can be considered to
be about 1-10 .mu.M in a sample of host blood drawn within the
first 24-48 hours after administration of IIS-ON.
[0100] To maximize the effectiveness of IIS-ON to inhibit ISS-ODN
immunostimulatory activity, the IIS-ON are preferably
co-administered with the target ISS-ODN or recombinant expression
vector. In addition, IIS-ON may be pre-incubated with the target
recombinant expression vector prior to administration to the host
to reduce the latter's capacity to present ISS-ODN
immunostimulatory activity in the host during treatment in a
therapy or immunization regime. For use as an anti-inflammatory,
the IIS-ON may be co-administered with, or otherwise taken by a
host treated with, other anti-inflammatory pharmaceuticals.
[0101] To these ends, IIS-ON are conveniently supplied in single
dose vials and/or in kits together with suitable dosages of
ISS-ODN, recombinant expression vectors or anti-inflammatory
agents. In kits including recombinant expression vectors, the
IIS-ON and vectors can be pre-mixed in single dosage vials. Means
for administering each dosage to a host (e.g., syringes,
transdermal patches, iontophoresis devices and inhalers), if
required, are included in each kit.
[0102] Examples illustrating the immunoinhibitory activity of
IIS-ON are set forth below. The examples are for purposes of
reference only and should not be construed to limit the invention,
which is to be defined by the appended claims. All abbreviations
and terms used in the examples have their expected and ordinary
meaning unless otherwise specified.
Example I
Assay to Confirm IIS-ON Inhibitory Activity as Measured by a
Reduction in Lymphocyte Proliferation
[0103] Splenocytes from immunologically naive female Balb/c mice
(6-8 weeks of age) were harvested from each animal. Supernatants of
the harvested splenocytes were incubated with 1 .mu.g/ml of the
DY1018 ISS-ODN or 1 .mu.g/ml of the DY1038 ISS-ODN in normal saline
(all oligonucleotide sequences are set forth in the legend to the
FIGURES and in the Description of Drawings). The backbones of both
DY1018 and DY1038 were modified as phosphorothioates. In this
context, the ISS-ODN served as non-specific adjuvants for in vitro
stimulation of the immune system.
[0104] Within 4 hours of ISS-ODN contact, the supernatants were
incubated with various concentrations of IIS-ON or a control.
DY1039 (an ISS with the cytosine methylated), DY1040 and DY1043
(the latter with CC dinucleotides in place of the CG dinucleotide
of DY1018 and DY1038) served as controls. To confirm the location
of potential competition with DY1018 and DY1038, all of the
oligonucleotides were identical to DY1038 (FIGS. 1 and 3) or DY1018
(FIG. 2) except for the hexamer regions identified in the FIGURES
and DY1043 (an irrelevant sequence control).
[0105] Lymphocyte proliferation pre- and post-IIS-ODN
administration was measured (as a function of counts per minute)
using conventional assay techniques. Any observable changes in
lymphocyte proliferation among the supernatants were noted. Values
shown in FIGS. 1 through 3 are averages for each group of mice
tested.
[0106] The results of these assays are shown in FIGS. 1 through 3.
With respect to both DY1038 (FIGS. 1 and 3) and DY1018 (FIG. 2),
the strongest inhibition of ISS immunostimulatory activity by
inhibitory IIS-ON of the invention in these experiments was
demonstrated by IIS-ON DY1019 (having a hexamer region consisting
of AAGGTT). Other strongly inhibitory IIS-ON tested were DY1048
(hexamer region=GAGGTC), DY1050 (hexamer region=AGGGCT), DY1060
(hexamer region=TTGCAA) and DY1041 (hexamer region=AAGCTT) (FIG.
3). Inhibitory strength was dose-dependent in a generally
proportional relationship of dosage to reduction in lymphocyte
proliferation measured.
Example II
Assay to Confirm IIS-ON Inhibitory Activity as Measured by a
Reduction in INF-.gamma. Secretion
[0107] Groups of mice were immunized as described in Example I,
sacrificied and their splenocytes harvested. Supernatants of
harvested splenocytes was incubated with 1 .mu.g/ml of DY1018
ISS-ODN in saline as described in Example I. Within 4 hours, the
supernatants were incubated with various concentrations of IIS-ON
or a control. DY1039 (an ISS with the cytosine methylated), DY1040
and DY1043 (the latter with CC dinucleotides in place of the CG
dinucleotide of DY1018) served as controls (all oligonucleotide
sequences are set forth in the legend to the FIGURES and in the
Description of Drawings). To confirm the location of potential
competition with DY1018, all of the oligonucleotides were identical
to DY1018 except for the hexamer regions identified and DY1043 (an
irrelevant sequence control).
[0108] IFN-.gamma. levels were measured pre- and post-IIS-ODN
contact. Any observable changes in IFN-.gamma. secretion (pg/ml
supernatants) among the supernatants were noted. Values shown in
FIG. 4 are averages for each group of mice tested.
[0109] The results of these assays are shown in FIG. 4. Again, the
strongest inhibition of ISS immunostimulatory activity by
inhibitory IIS-ON of the invention in these experiments was
demonstrated by IIS-ON DY1019 (having a hexamer region consisting
of AAGGTT). DY1041 (hexamer region=AAGCTT) was also strongly
inhibitory, even at low dosage (1 .mu.g/ml saline). At a higher
dosage (10 .mu.g/ml), INF-.gamma. levels began to decline in
control mice as well.
Example III
IIS-ODN Boosting of Th2 Type Immune Responses to Antigen
[0110] Groups of four Balb/c mice were co-immunized with 10 .mu.g
.beta.-galactosidase antigen and 50 .mu.g (in 50 .mu.l normal
saline) of IIS-ODN DY1019 (identified in the Figure as
.beta.-gal/M-ODN), the ISS-ODN composition .beta.-gal/ISS-ODN
(5'-AATTCAACGTTCGC-3'), the .beta.-gal antigen and pKISS-3 (a
plasmid having three copies of the AACGTT ISS-ODN hexamer in the
backbone), the .beta.-gal antigen and pKISS-0 (a control plasmid
having no copies of the AACGTT ISS-ODN hexamer in the backbone), or
saline alone. Th2 responses in each group of mice were measured by
ELISA as a function of IgE levels obtained post-boosting. As shown
in FIG. 5, potent Th2-type responses (above 1000 CPM) were obtained
only in the mice which received saline (approximately 1200 CPM at 1
week post-boosting) and .beta.-gal/M-ODN (approximately 1750 CPM at
1 week post-boosting).
[0111] Further, high levels of IgG2a antibodies and low levels of
IgG1 antibodies (Th1 and Th2 type responses, respectively) were
induced in response to antigen in the ISS-ODN treated mice, while
the opposite responses were obtained in the IIS-ON treated mice,
thus showing a shift toward the Th2 repertoire in the latter
group.
[0112] The invention having been fully described, modifications of
the disclosed embodiments may become apparent to those of ordinary
skill in the art. All such modifications are considered to be
within the scope of the invention, which is defined by the appended
claims.
Sequence CWU 1
1
11122DNAArtificial SequenceOligonucleotide 1tgactgtgaa ggttagagat
ga 22222DNAArtificial SequenceOligonucleotide 2tgactgtgaa
cgttagagat ga 22322DNAArtificial SequenceOligonucleotide
3tgactgtgaa ccttagagat ga 22422DNAArtificial
SequenceOligonucleotide 4tgactgtgaa gcttagagat ga
22522DNAArtificial SequenceOligonucleotide 5tgactgtgtt ccttagagat
ga 22622DNAArtificial SequenceOligonucleotide 6tcactctctt
ccttactctt ct 22722DNAArtificial SequenceOligonucleotide
7tgactgtgga ggtcagagat ga 22822DNAArtificial
SequenceOligonucleotide 8tgactgtgag ggctagagat ga
22922DNAArtificial SequenceOligonucleotide 9tgactgtgtt gcaaagagat
ga 221022DNAArtificial SequenceOligonucleotide 10tgactgtgaa
tgttagagat ga 221114DNAArtificial SequenceOligonucleotide
11aattcaacgt tcgc 14
* * * * *