U.S. patent application number 17/066134 was filed with the patent office on 2021-04-08 for spherical nucleic acid-based constructs as immunostimulatory agents for prophylactic and therapeutic use.
This patent application is currently assigned to Exicure, Inc.. The applicant listed for this patent is Exicure, Inc.. Invention is credited to Sergei Gryaznov, Warefta Hasan, Aaron Love, Christopher C. Mader, Subbarao Nallagatla, Aleksandar Filip Radovic-Moreno.
Application Number | 20210102211 17/066134 |
Document ID | / |
Family ID | 1000005287522 |
Filed Date | 2021-04-08 |
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United States Patent
Application |
20210102211 |
Kind Code |
A1 |
Radovic-Moreno; Aleksandar Filip ;
et al. |
April 8, 2021 |
SPHERICAL NUCLEIC ACID-BASED CONSTRUCTS AS IMMUNOSTIMULATORY AGENTS
FOR PROPHYLACTIC AND THERAPEUTIC USE
Abstract
Aspects of the invention relate to spherical nucleic acid-based
constructs and related methods and compositions thereof. The
compositions of the invention are useful for activating agonists of
nucleic acid interacting complexes, such as TLRs, stimulating an
immune response, and treating diseases such as infectious disease,
cancer, allergies, allergic diseases, and autoimmune disease
Inventors: |
Radovic-Moreno; Aleksandar
Filip; (Evanston, IL) ; Mader; Christopher C.;
(Mendham, NJ) ; Nallagatla; Subbarao; (Chicago,
IL) ; Hasan; Warefta; (Houston, TX) ; Love;
Aaron; (Chicago, IL) ; Gryaznov; Sergei; (San
Mateo, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Exicure, Inc. |
Chicago |
IL |
US |
|
|
Assignee: |
Exicure, Inc.
Chicago
IL
|
Family ID: |
1000005287522 |
Appl. No.: |
17/066134 |
Filed: |
October 8, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15980428 |
May 15, 2018 |
10837018 |
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17066134 |
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14907430 |
Jan 25, 2016 |
10894963 |
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PCT/US2014/048291 |
Jul 25, 2014 |
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15980428 |
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61858558 |
Jul 25, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2310/17 20130101;
C12N 2320/30 20130101; C12N 15/117 20130101; A61K 2039/55561
20130101; B82Y 5/00 20130101; A61K 39/39 20130101; C12N 2320/32
20130101 |
International
Class: |
C12N 15/117 20060101
C12N015/117; A61K 39/39 20060101 A61K039/39 |
Claims
1. A nanoscale construct comprising: a corona comprised of an
exterior shell composed of nucleic acid molecules arranged in a
geometrical position and forming a spherical shape around a
nanoparticle core, wherein the nucleic acid molecules are CpG
oligonucleotides, wherein the nanoscale construct is about 1 nm to
about 40 nm in diameter, and wherein the nanoparticle core at the
center of the corona is not metallic.
2. The nanoscale construct of claim 1, wherein the surface density
of the nucleic acid molecules is at least 0.3 pmol/cm.sup.2.
3. The nanoscale construct of claim 1, wherein the nucleic acid
molecules comprise a spacer.
4. The nanoscale construct of claim 3, wherein the spacer consists
of oligoethylene.
5. The nanoscale construct of claim 1, wherein the nanoparticle
core at the center of the corona is hollow.
6. The nanoscale construct of claim 1, wherein the nanoscale
construct is degradable.
7. The nanoscale construct of claim 1, wherein the CpG
oligonucleotide has a modified backbone.
8. The nanoscale construct of claim 1, further comprising an
antigen.
9. A method for delivering a therapeutic agent to a cell comprising
delivering the nanoscale construct of claim 1 to the cell.
10. A method of treating a subject, comprising administering to the
subject a nanoscale construct in an effective amount to stimulate
an immune response, wherein the nanoscale construct comprises a
corona having an exterior shell composed of nucleic acid molecules
arranged in a geometrical position and forming a spherical shape
around a nanoparticle core, wherein the nucleic acid molecules are
CpG oligonucleotides, and wherein the nanoscale construct is about
1 nm to about 40 nm in [[mean]] diameter, and wherein the
nanoparticle core at the center of the corona is not metallic.
11. The method of claim 10, wherein the subject has an infectious
disease.
12. The method of claim 10, wherein the subject has cancer.
13. A method for activating a toll-like receptor (TLR), the method
comprising delivering to a cell a nanoscale construct, wherein the
nanoscale construct comprises a corona having an exterior shell
composed of nucleic acid molecules arranged in a geometrical
position and forming a spherical shape around a nanoparticle core,
wherein the nucleic acid molecules are CpG oligonucleotides,
wherein the nanoscale construct is about 1 nm to about 40 nm in
diameter, and wherein the nanoparticle core at the center of the
corona is not metallic.
14. The method of claim 13, wherein the cell is in vivo.
15. The method of claim 14, wherein the nanoscale construct is
administered to a subject having cancer.
16. The nanoscale construct of claim 4, wherein the oligoethylene
is hexaethylene glycol.
17. The nanoscale construct of claim 4, wherein the spacer consists
of two or three hexaethylene glycols.
18. The nanoscale construct of claim 3, wherein the spacer does not
comprise an oligonucleotide. (New) The nanoscale construct of claim
1, wherein the CpG oligonucleotide has a phosphorothioate (PS)
backbone.
20. A nucleic acid molecule comprising a CpG oligonucleotide having
the nucleotide sequence of SEQ ID NO: 30, wherein the nucleic acid
molecule comprises a spacer consisting of oligoethylene.
21. The nucleic acid molecule of claim 20, wherein the CpG
oligonucleotide has a phosphorothioate (PS) backbone.
22. The nucleic acid molecule of claim 20, wherein the
oligoethylene is hexaethylene glycol.
23. The nucleic acid molecule of claim 20, wherein the spacer
consists of two or three hexaethylene glycols.
24. A plurality of nanoscale constructs, each nanoscale construct
comprising: a corona comprised of an exterior shell composed of
nucleic acid molecules arranged in a geometrical position and
forming a spherical shape around a nanoparticle core, wherein the
nucleic acid molecules are CpG oligonucleotides, wherein the
nanoscale constructs have a mean diameter of about 1 nm to about 40
nm, and wherein the nanoparticle core at the center of the corona
is not metallic.
Description
RELATED APPLICATIONS
[0001] This Application is a continuation of U.S. application Ser.
No. 14/907,430, filed Jan. 25, 2016, entitled "SPHERICAL NUCLEIC
ACID-BASED CONSTRUCTS AS IMMUNOSTIMULATORY AGENTS FOR PROPHYLACTIC
AND THERAPEUTIC USE", which is a national stage filing under 35
U.S.C. 371 of International Patent Application Serial No.
PCT/US2014/048291, filed Jul. 25, 2014, entitled "SPHERICAL NUCLEIC
ACID-BASED CONSTRUCTS AS IMMUNOSTIMULATORY AGENTS FOR PROPHYLACTIC
AND THERAPEUTIC USE", which is a Non-Prov of Prov (35 USC 119(e))
of U.S. application Ser. No. 61/858,558, filed Jul. 25, 2013,
entitled "SPHERICAL NUCLEIC ACID-BASED CONSTRUCTS AS
IMMUNOSTIMULATORY AGENTS FOR PROPHYLACTIC AND THERAPEUTIC USE". The
entire contents of these applications are incorporated herein by
reference in their entirety.
FIELD OF INVENTION
[0002] The invention relates to nanoscale constructs of agonists of
nucleic acid-interacting complexes, such as agonists of TLR, as
well as methods and compositions thereof.
BACKGROUND OF INVENTION
[0003] The immune system is a highly evolved, exquisitely precise
endogenous mechanism for clearing foreign, harmful, and unnecessary
material including pathogens and senescent or malignant host cells.
It is known that modulating the immune system for therapeutic or
prophylactic purposes is possible by introducing compounds that
modulate the activity of specific immune cells. A primary example
is vaccines, which have shown the ability to induce protection
against pathogens as well as cancerous cells. The first modern
vaccine formulations included live/attenuated or inactivated
pathogens, but these were deemed too toxic in many instances or did
not provide protective immunity. Purified protein derivatives and
other antigenic subunit vaccine strategies have been pursued, but
these typically lead to mildly protective or inefficient responses.
It is now appreciated that effective immunity, in most instances,
is known to require use of immunostimulatory compounds, which,
among other things, provide the necessary signals to induce more
robust, specific, and long-lived responses, including cell-mediated
immunity and immunologic memory. The nature of these responses can
be modulated by the type of immunostimulatory compound(s)
introduced. Indeed, it has been postulated that immunostimulatory
compounds administered together in the presence of appropriate
antigenic stimuli can be used to elicit a wide variety of immune
responses, with the potential to treat or prevent various ailments,
including infectious diseases and cancer. These can also
potentially be used to vaccinate immunocompromised populations,
such as children and the elderly..sup.1
[0004] Existing vaccines fail to induce effective immune responses
in a variety of diseases with critical worldwide impact, including
AIDS, malaria, chlamydia, various malignancies and allergies or
allergic diseases, such as asthma. Among the immunostimulatory
compounds being developed, agonists of Toll-like receptors (TLR)
have demonstrated outstanding potential. Agonists of TLR4, such as
monophosphoryl lipid A (MPL) have reached late stages of clinical
trials and approval in various countries in some instances..sup.2
Despite these promising results, there is still a clear and
significant need for compounds which can safely and effective
induce responses that can clear intracellular pathogens and
cancers, such as inducers of cell-mediated immunity. Agonists of
TLR 3, TLR 7/8 and TLR 9 have excellent potential due to their
potent ability to induce Thl cell-mediated immune responses. A
synthetic TLR 7/8 agonist, imiquimod, has been approved to treat
various skin diseases, including superficial carcinomas and genital
warts, and is being developed for a variety of other indications.
Similarly, agonists of TLR 9 are in various stages of clinical
development, for treatment of various diseases with large unmet
medical needs. However, concerns due to lack of efficacy,
off-target phosphorothioate effects, and toxicity have slowed
effective clinical translation of TLR 7/8 and 9 agonists.
SUMMARY OF INVENTION
[0005] Described herein are novel methods and compositions for
enhancing immune responses and activating nucleic acid interacting
complexes such as TLRs using a nanoscale construct. Aspects of the
invention relate to nanoscale constructs having a corona of an
agonist of nucleic acid-interacting complexes wherein the surface
density of the agonist of nucleic acid-interacting complexes is at
least 0.3 pmol/cm.sup.2.
[0006] In other aspects the invention is a nanoscale construct
having a corona of an agonist of nucleic acid-interacting
complexes, and an antigen incorporated into the corona. In some
embodiments the surface density of the antigen is at least 0.3
pmol/cm.sup.2. In other embodiments the antigen includes at least
two different types of antigen.
[0007] In yet other aspects the invention is a nanoscale construct
having a corona with at least two agonists of nucleic
acid-interacting complexes incorporated, wherein the agonists are
selected from the group consisting of TLR 3, 7/8, and/or 9
agonists.
[0008] In some embodiments the agonist of nucleic acid-interacting
complexes contains a spacer.
[0009] In other embodiments the agonist of nucleic acid-interacting
complexes is RNA or DNA. The agonists of nucleic acid-interacting
complexes may be, for instance, a double stranded RNA, such as
poly(I:C). Alternatively the agonist of nucleic acid-interacting
complexes may be a single stranded RNA such as an RNA containing
UUG-motifs. In some embodiments the agonist of nucleic
acid-interacting complexes is an unmethylated deoxyribonucleic
acid, such as a CpG oligonucleotide.
[0010] The nanoscale construct, in some embodiments, contains a
nanoparticle core at the center of the corona which is optionally
metallic. The metallic core may be selected from the group
consisting of gold, silver, platinum, aluminum, palladium, copper,
cobalt, indium, nickel and mixtures thereof. In some embodiments
the nanoparticle core comprises gold. In other embodiments the
nanoscale construct is degradable.
[0011] In certain embodiments, the diameter of the nanoscale
construct is from 1 nm to about 250 nm in mean diameter, about 1
ran to about 240 nm in mean diameter, about 1 nm to about 230 nm in
mean diameter, about 1 nm to about 220 nm in mean diameter, about 1
nm to about 210 nm in mean diameter, about 1 nm to about 200 nm in
mean diameter, about 1 nm to about 190 nm in mean diameter, about 1
nm to about 180 nm in mean diameter, about 1 nm to about 170 nm in
mean diameter, about 1 nm to about 160 nm in mean diameter, about 1
nm to about 150 nm in mean diameter, about 1 nm to about 140 nm in
mean diameter, about 1 nm to about 130 nm in mean diameter, about 1
nm to about 120 nm in mean diameter, about 1 nm to about 110 nm in
mean diameter, about 1 nm to about 100 nm in mean diameter, about 1
nm to about 90 nm in mean diameter, about 1 nm to about 80 nm in
mean diameter, about 1 nm to about 70 nm in mean diameter, about 1
nm to about 60 nm in mean diameter, about 1 nm to about 50 nm in
mean diameter, about 1 nm to about 40 nm in mean diameter, about 1
nm to about 30 nm in mean diameter, or about 1 nm to about 20 nm in
mean diameter, or about 1 nm to about 10 nm in mean diameter.
[0012] In other aspects the invention is a nanoscale construct of a
corona of an agonist of nucleic acid-interacting complexes, wherein
the agonist is nucleic acid having at least one phosphodiester
internucleotide linkage. In some embodiments the agonist is a CpG
oligonucleotide. In other embodiments each internucleotide linkage
of the nucleic acid is a phosphodiester linkage.
[0013] In embodiments of the invention the corona is a spherical
corona.
[0014] A vaccine composed of a nanoscale construct described herein
and a carrier is provided according to other aspects of the
invention.
[0015] A method for delivering a therapeutic agent to a cell by
delivering the nanoscale construct of the invention to the cell is
provided in other aspects.
[0016] A method for regulating expression of a target molecule is
provided in other aspects of the invention. The method involves
delivering the nanoscale construct of the invention to the cell. In
some embodiments the target molecule is a TLR selected from the
group consisting of TLR3, 7, 8, and 9.
[0017] A method for activating a TLR by delivering the nanoscale
construct as described herein to the cell is provided in other
aspects of the invention.
[0018] According to other aspects the invention is a method of
treating a subject, involving administering to the subject the
nanoscale construct as described herein in an effective amount to
stimulate an immune response. In some embodiments the subject has
an infectious disease, a cancer, an autoimmune disease, allergy, or
an allergic disease such as asthma.
[0019] In yet other embodiments, the invention is a method of
inducing an immune response in a subject, by administering to the
subject a nanoscale construct of a corona of an agonist of nucleic
acid-interacting complexes, wherein the agonist is nucleic acid
having at least one phosphodiester internucleotide linkage in an
effective amount to stimulate an immune response.
[0020] In certain embodiments, the method involves delivering a
therapeutic or detection modality to a cell.
[0021] Further aspects of the invention relate to a kit comprising:
a nanoscale construct optionally including a nanoparticle core; and
having an agonist of nucleic acid-interacting complexes and
instructions for assembly of an agonist of nucleic acid-interacting
complexes-corona. In certain embodiments, the kit further comprises
instructions for use.
[0022] Each of the limitations of the invention can encompass
various embodiments of the invention. It is, therefore, anticipated
that each of the limitations of the invention involving any one
element or combinations of elements can be included in each aspect
of the invention. This invention is not limited in its application
to the details of construction and the arrangement of components
set forth in the following description or illustrated in the
drawings. The invention is capable of other embodiments and of
being practiced or of being carried out in various ways.
BRIEF DESCRIPTION OF DRAWINGS
[0023] The accompanying drawings are not intended to be drawn to
scale. In the drawings, each identical or nearly identical
component that is illustrated in various figures is represented by
a like numeral. For purposes of clarity, not every component may be
labeled in every drawing. In the drawings:
[0024] FIGS. 1A-1B show a schematic non-limiting example of a
nanoscale construct of the invention. FIG. 1A. A general structure
of an adjuvant nanoscale construct having a core and one or more
agonists of nucleic acid interacting complexes, such as TLR
agonists bound thereto is shown. FIG. 1B. A general structure of an
adjuvant nanoscale construct having a core and one or more TLR
agonists and one or more antigens bound thereto is shown.
[0025] FIG. 2 is a set of graphs depicting markedly enhanced
potency in macrophages of the nanoscale constructs of the invention
over agonists of nucleic acid-interacting complexes (CpG
oligonucleotides) in solution.
[0026] FIG. 3 is a set of graphs depicting markedly enhanced
potency in macrophages of the nanoscale constructs of the invention
over agonists of nucleic acid-interacting complexes (CpG
oligonucleotides) in solution following overnight incubation.
[0027] FIG. 4 is a set of graphs depicting an enhanced level of
cytokine secretion with the nanoscale constructs of the invention
over agonists of nucleic acid-interacting complexes (CpG
oligonucleotides) in solution. The effect on cytokine induction was
examined for both oligonucleotides having phosphodiester and
phosphorothioate internucleotide linkages in both the nanoscale
construct and the TRL agonist groups.
[0028] FIG. 5 is a line and bar graph depicting TLR9 activation in
response to stimulation with a nanoscale construct of the invention
having a phosphodiester CpG oligonucleotide in comparison with
phosphodiester and phosphorothioate CpG oligonucleotides in
solution.
[0029] FIG. 6 is a set of graphs depicting multiple fold increase
in potency of a nanoscale construct of the invention over several
different CpG oligonucleotide sequences. CpG 1826 is SEQ ID NO: 1
and the rp1V below it is SEQ ID NO: 2. CpG 1668 is SEQ ID NO: 3 and
the rp1V below it is SEQ ID NO: 4.
[0030] FIGS. 7A-7B are a set of graphs depicting the effects of
modulating nanoscale construct core size suggests on the
enhancement of agonist activity. FIG. 8 shows a graph depicting
more rapid and sustained activation than CpG oligonucleotide.
[0031] FIG. 9 is a set of graphs depicting the ability of
phosphorothioate modifications to modulate agonist activity in a
sequence-dependent manner. For CpG 1668: PO is SEQ ID NO: 5, C*G is
SEQ ID NO: 6, 5PS2/C*G is SEQ ID NO: 7, and PS is SEQ ID NO: 8. For
CpG 1826: PO is SEQ ID NO: 9, C*G is SEQ ID NO: 10, 5PS2/C*G is SEQ
ID NO: 11, and PS is SEQ ID NO: 12.
[0032] FIG. 10 is a set of graphs depicting the ability of
oligonucleotide loading density to affect agonist activity.
[0033] FIG. 11 shows a graph depicting a time course of activation
of CpG PO/PO nanoscale constructs. The tested constructs are not
activated until >4 hr of incubation.
[0034] FIG. 12 shows a graph demonstrating that 5'Chol CpG PO
nanoscale constructs show activation in low nM range, and 5'C18
abrogated the activity.
[0035] FIG. 13 is a set of graphs demonstrating that pre-plated
macrophages are more primed for subsequent activation.
[0036] FIG. 14 is a set of graphs demonstrating low levels of
IFN-gamma secretion by macrophages.
[0037] FIG. 15 shows a representation of an immunotherapeutic SNA
(AST-008). FIG. 15 shows that the SNAs can co-present a therapeutic
vaccine antigen and adjuvant on a single nanoparticle, and may
simultaneously target multiple immunostimulatory receptors (e.g.
TLR 3, 4, 7/8, 9).
[0038] FIG. 16 is a schematic demonstrating how AST-008 can enter
endosomes via triggered endocytosis, where it then can be used for
versatile immune system stimulation. Within the endosome, AST-008
stimulates immune system signaling via the TLR 9 receptor, a
molecular target for SNA therapy, leading to both innate and
adaptive immune responses. AST-008 may also target TLR 3, 4, 7/8,
resulting in innate and adaptive immune responses.
[0039] FIGS. 17A-17B are a set of graphs showing that AST-008
induces higher pro-inflammatory responses than corresponding CpG
oligodeoxynucleotides (oligo) in vitro. FIG.
[0040] 17A shows the expression levels of TNF, IL-12, and IL-6
induced by CTL oligo, CTL SNA, CpG 1826, and AST-008. FIG. 17B
presents the NF-KB activation stemming from the indicated
agents.
[0041] FIG. 18 demonstrates that AST-008 targets draining lymph
nodes after administration of a single subcutaneous dose. AST-008
was silver-stained to enhance light scattering of the gold core,
and then counterstained with eosin. 4X bright field magnification
was used.
[0042] FIG. 19 is a graph illustrating the in vivo activity of
AST-008. Mice were given a 50 .mu.L bolus tail vein (intravenous)
injection of 5.1 nmol solution (AST-008-po, AST-008-ps, CpG
1826-po, CpG 1826-ps, GpC-po SNA, GpC-ps SNA, GpC-po, or GpC-ps)
and then analyzed for IL-12 expression 1, 3, and 6 hours after
injection (24 mice per group, 3 per each time point). IL-12 levels
are expressed as the fold over PBS. AST-008 architecture enhances
the induction of IL-12 by approximately 20-fold over free
oligodeoxynucleotides, and the effect was sustained for over six
hours after the initial administration.
[0043] FIGS. 20A-20C consist of a pair of graphs and a chart that
demonstrate that AST-008 induces both a balanced Th1/Th2 response
(FIG. 20A) and a higher IgG2a antibody (FIG. 20B) response than
alum or CpG oligonucleotides. The results are tabulated in FIG.
20C. **p<0.01.
[0044] FIGS. 21A-21B show that AST-008 induces cellular responses
more effectively than alum or CpG oligonucleotides. FIG. 21A
schematically represents the protocol: splenocytes were grown for
28 days, challenged on Day 0 and Day 21, and then restimulated with
SIINFEKL and probed for INF-.gamma. with ELISPOT on Day 28. FIG.
21B is a graph depicting the results. ****p<0.0001.
[0045] FIGS. 22A-22B demonstrate that AST-008 induces a profound
tumor-clearing immune response in an in vivo lymphoma model. FIG.
22A illustrates the protocol: the right flanks of C57BL/6 mice were
injected with 1.times.10.sup.6E.G7-OVA lymphoma (11 per group). The
mice were then challenged three times with 100 .mu.g OVA s.c., 1.8
.mu.g OVA.sub.257-264 s.c., and 0.92 nmol oligo in AST-008, and
sacrificed at 2000 mm.sup.3. FIG. 22B is a graph of the results.
*p<0.05 using Two-way ANOVA.
[0046] FIGS. 23A-23B show that AST-008 exhibits superior anti-tumor
activity and longer survival than CpG oligodeoxynucleotides. The
graphs show the tumor volume (FIG. 23A) and percent survival (FIG.
23B) after C57BL/6 mice were injected with 1.times.10.sup.6
E.G7-OVA lymphoma in their right flanks (11 per group) and then
were challenged three times with PBS, PBS and OVA, CpG 1826 and
OVA, or AST-008 and OVA. *p<0.05.
DETAILED DESCRIPTION
[0047] This invention is not limited in its application to the
details of construction and the arrangement of components set forth
in the following description or illustrated in the drawings. The
invention is capable of other embodiments and of being practiced or
of being carried out in various ways. Also, the phraseology and
terminology used herein is for the purpose of description and
should not be regarded as limiting. The use of "including,"
"comprising," or "having," "containing," "involving," and
variations thereof herein, is meant to encompass the items listed
thereafter and equivalents thereof as well as additional items.
[0048] The present invention, in some aspects, overcomes several
major hurdles encountered by conventional TLR 3, TLR 7/8, and TLR 9
agonists by achieving faster activation, creating a multivalent
structure, changing cellular distribution, and facilitating simple
and scalable synthesis of various adjuvant and antigen-containing
structures, among others. The constructs of the invention result in
more effective vaccines for prophylactic or therapeutic uses in
treating a wide variety of diseases/infections including, for
example, AIDS, malaria, chlamydia, campylobacter, cytomegalovirus,
dengue, Epstein-Barr mononucleosis, foot and mouth disease, rabies,
Helicobacter pylori gastric ulcers, hepatitis A, B, C, herpes
simplex, influenza, leishmaniasis, cholera, diphtheria, Haemophilus
influenza, meningococcal meningitis, plague, pneumococcal
pneumonia, tetanus, typhoid fever, respiratory synctial virus,
rhinovirus, schistosomiasis, shigella, streptococcus group A and B,
tuberculosis, vibrio cholera, salmonella, aspergillus, blastomyces,
histoplasma, candida, cryptococcus, pneumocystis, and urinary tract
infections; various food allergies such as peanut, fruit, garlic,
oats, meat, milk, fish, shellfish, soy, tree nut, wheat, gluten,
egg, sulphites; various drug allergies such as to tetracycline,
Dilantin, carbamazepine, penicillins, cephalosporins, sulfonamides,
NSAIDs, intravenous contrast dye, local anesthetics; autoimmune
diseases such as multiple sclerosis, lupus, inflammatory bowel
disease, Crohn's disease, ulcerative colitis, asthma, and COPD; and
cancers such as melanoma, breast cancer, prostate cancer, bladder
cancer, NSCLC, glioblastoma multiforme, among others. A set of
exemplary nanoscale constructs of the invention is shown in the
schematic of
[0049] FIGS. 1A-1B. The platform described herein is useful for
loading one or multiple agonists of nucleic acid-interacting
complexes (A) or one or multiple agonists of nucleic
acid-interacting complexes and antigen (B). Optimization of: (1)
nucleic acid interacting complex, such as a TLR that is targeted
(TLR 3, 7/8, and/or 9), (2) density of agonist of nucleic
acid-interacting complexes, (3) antigen density, (4) multiple
antigen presentation, (5) core composition, size, and charge, and
(6) core linker chemistry "L", and (7) agonist chemical structure
is expected to yield novel paradigms in vaccine development. In
particular, agonists of nucleic acid-interacting complexes include
double stranded RNA (such as poly(I:C), TLR 3), single stranded RNA
(such as strands containing UUG-motifs, TLR 7/8), and unmethylated
deoxyribonucleic acid and derivatives (such as strands containing
CpG motifs).
[0050] Aspects of the invention relate to nanoscale constructs. A
nanoscale construct refers to a nanometer sized construct having
one or more nucleic acids held in a geometrical position. The
nanoscale construct typically is referred to as a corona of a set
of nucleic acids. A corona, as used herein, refers to an exterior
shell composed of nucleic acid molecules. The corona may have a
nanoparticle core composed of nucleic acids or other materials,
such as metals. Alternatively, the corona may simply be a set of
nucleic acids arranged in a geometric shape with a hollow core,
i.e. a 3-dimensionally shaped layer of nucleic acids. Typically,
but not always, the corona has a spherical shape.
[0051] In the instance, when the corona includes a nanoparticle
core the nucleic acids may be linked directly to the core. Some or
all of the nucleic acids may be linked to other nucleic acids
either directly or indirectly through a covalent or non-covalent
linkage. The linkage of one nucleic acid to another nucleic acid
may be in addition to or alternatively to the linkage of that
nucleic acid to a core. One or more of the nucleic acids may also
be linked to other molecules such as an antigen.
[0052] When the corona does not include a nanoparticle core, the
nucleic acids may be linked to one another either directly or
indirectly through a covalent or non-covalent linkage. In some
embodiments the corona that does not include a nanoparticle core
may be formed by layering the nucleic acids on a lattice or other
dissolvable structure and then dissolving the lattice or other
structure to produce an empty center.
[0053] As used herein, the nanoscale construct is a construct
having an average diameter on the order of nanometers (i.e.,
between about 1 nm and about 1 micrometer. For example, in some
instances, the diameter of the nanoparticle is from about 1 nm to
about 250 nm in mean diameter, about 1 nm to about 240 nm in mean
diameter, about 1 nm to about 230 nm in mean diameter, about 1 nm
to about 220 nm in mean diameter, about 1 nm to about 210 nm in
mean diameter, about 1 nm to about 200 nm in mean diameter, about 1
nm to about 190 nm in mean diameter, about 1 nm to about 180 nm in
mean diameter, about 1 nm to about 170 ran in mean diameter, about
1 nm to about 160 nm in mean diameter, about 1 nm to about 150 nm
in mean diameter, about 1 nm to about 140 nm in mean diameter,
about 1 nm to about 130 nm in mean diameter, about 1 nm to about
120 nm in mean diameter, about 1 nm to about 110 nm in mean
diameter, about 1 nm to about 100 nm in mean diameter, about 1 nm
to about 90 nm in mean diameter, about 1 nm to about 80 nm in mean
diameter, about 1 nm to about 70 nm in mean diameter, about 1 nm to
about 60 nm in mean diameter, about 1 nm to about 50 nm in mean
diameter, about 1 nm to about 40 nm in mean diameter, about 1 nm to
about 30 nm in mean diameter, about 1 nm to about 20 nm in mean
diameter, about 1 nm to about 10 nm in mean diameter, about 5 nm to
about 150 nm in mean diameter, about 5 to about 50 nm in mean
diameter, about 10 to about 30 nm in mean diameter, about 10 to 150
nm in mean diameter, about 10 to about 100 nm in mean diameter,
about 10 to about 50 nm in mean diameter, about 30 to about 100 nm
in mean diameter, or about 40 to about 80 nm in mean diameter.
[0054] In some instances the corona includes a nanoparticle core
that is attached to one or more agonists of nucleic
acid-interacting complexes and/or antigens. As used herein, a
nanoparticle core refers to the nanoparticle component of a
nanoparticle construct, without any attached modalities. In some
instances, the nanoparticle core is metallic. It should be
appreciated that the nanoparticle core can comprise any metal.
Several non-limiting examples of metals include gold, silver,
platinum, aluminum, palladium, copper, cobalt, indium, nickel and
mixtures thereof. In some embodiments, the nanoparticle core
comprises gold. For example, the nanoparticle core can be a lattice
structure including degradable gold. Nanoparticles can also
comprise semiconductor and magnetic materials.
[0055] Non-limiting examples of nanoparticles compatible with
aspects of the invention are described in and incorporated by
reference from: US Patent No. 7,238,472, US Patent Publication No.
2003/0147966, US Patent Publication No. 2008/0306016, US Patent
Publication No. 2009/0209629, US Patent Publication No.
2010/0136682, US Patent Publication No. 2010/0184844, US Patent
Publication No. 2010/0294952, US Patent Publication No.
2010/0129808, US Patent Publication No. 2010/0233270, US Patent
Publication No. 2011/0111974, PCT Publication No. WO 2002/096262,
PCT Publication No. WO 2003/08539, PCT Publication No. WO
2006/138145, PCT Publication No. WO 2008/127789, PCT Publication
No. WO 2008/098248, PCT Publication No. WO 2011/079290, PCT
Publication No. WO 2011/053940, PCT Publication No. WO 2011/017690
and PCT Publication No. WO 2011/017456. Nanoparticles associated
with the invention can be synthesized according to any means known
in the art or can be obtained commercially. For example, several
non-limiting examples of commercial suppliers of nanoparticles
include: Ted Pella, Inc., Redding, Calif., Nanoprobes, Inc.,
Yaphank, N.Y., Vacuum Metallurgical Co,. Ltd., Chiba, Japan and
Vector Laboratories, Inc., Burlington, Calif.
Agonists of Nucleic Acid-Interacting Complexes
[0056] A nucleic acid-interacting complex as used herein refers to
a molecule or complex of molecules that interact with a nucleic
acid molecule and are stimulated to produce an immune response in
response to that interaction. The molecule or complex of molecules
may be a receptor, for instance. In some embodiments a nucleic
acid-interacting complex is a pattern recognition receptor (PRR)
complex. PRRs are a primitive part of the immune system composed of
proteins expressed by cells of the innate immune system to identify
pathogen-associated molecular patterns (PAMPs), which are
associated with microbial pathogens or cellular stress, as well as
damage-associated molecular patterns (DAMPs), which are associated
with cell components released during cell damage. PRRs include but
are not limited to membrane-bound PRRs, such as receptor kinases,
toll-like receptors (TLR), and C-type lectin Receptors (CLR)
(mannose receptors and asialoglycoprotein receptors); Cytoplasmic
PRRs such as RIG-I-like receptors (RLR), RNA Helicases, Plant PRRs,
and NonRD kinases; and secreted PRRs.
[0057] Nucleic acid-interacting complexes include but are not
limited to TLRs, RIG-I, transcription factors, cellular translation
machinery, cellular transcription machinery, nucleic-acid acting
enzymes, and nucleic acid associating autoantigens. Nucleic acid
molecules that are agonists of a nucleic acid-interacting complex
include but are not limited to TLR agonists, and agonists of RIG-I,
transcription factors, cellular translation machinery, cellular
transcription machinery, nucleic-acid acting enzymes, and nucleic
acid associating autoantigens.
[0058] In some embodiments an agonist of a nucleic acid-interacting
complex is a TLR agonist. A TLR agonist, as used herein is a
nucleic acid molecule that interacts with and stimulates the
activity of a TLR.
[0059] Toll-like receptors (TLRs) are a family of highly conserved
polypeptides that play a critical role in innate immunity in
mammals. At least ten family members, designated TLR1-TLR10, have
been identified. The cytoplasmic domains of the various TLRs are
characterized by a Toll-interleukin 1 (IL-1) receptor (TIR) domain.
Medzhitov R et al. (1998) Mol Cell 2:253-8. Recognition of
microbial invasion by TLRs triggers activation of a signaling
cascade that is evolutionarily conserved in Drosophila and mammals.
The TIR domain-containing adaptor protein MyD88 has been reported
to associate with TLRs and to recruit IL-1 receptor-associated
kinase (IRAK) and tumor necrosis factor (TNF) receptor-associated
factor 6 (TRAF6) to the TLRs. The MyD88-dependent signaling pathway
is believed to lead to activation of NF-.kappa.B transcription
factors and c-Jun NH2 terminal kinase (Jnk) mitogen-activated
protein kinases (MAPKs), critical steps in immune activation and
production of inflammatory cytokines. For a review, see Aderem A et
al. (2000) Nature 406:782-87.
[0060] TLRs are believed to be differentially expressed in various
tissues and on various types of immune cells. For example, human
TLR7 has been reported to be expressed in placenta, lung, spleen,
lymph nodes, tonsil and on plasmacytoid precursor dendritic cells
(pDCs). Chuang T-H et al. (2000) Eur Cytokine Netw 11:372-8);
Kadowaki Net al. (2001) J Exp Med 194:863-9. Human TLR8 has been
reported to be expressed in lung, peripheral blood leukocytes
(PBL), placenta, spleen, lymph nodes, and on monocytes. Kadowaki N
et al. (2001) J Exp Med 194:863-9; Chuang T-H et al. (2000) Eur
Cytokine Netw 11:372-8. Human TLR9 is reportedly expressed in
spleen, lymph nodes, bone marrow, PBL, and on pDCs, and B cells.
Kadowaki N et al. (2001) J Exp Med 194:863-9; Bauer S et al. (2001)
Proc Natl Acad Sci USA 98:9237-42; Chuang T-H et al. (2000) Eur
Cytokine Netw 11:372-8.
[0061] Nucleotide and amino acid sequences of human and murine TLR7
are known. See, for example, GenBank Accession Nos. AF240467,
AF245702, NM_016562, AF334942, NM_133211; and AAF60188, AAF78035,
NP_057646, AAL73191, and AAL73192, the contents of all of which are
incorporated herein by reference. Human TLR7 is reported to be 1049
amino acids long. Murine TLR7 is reported to be 1050 amino acids
long. TLR7 polypeptides include an extracellular domain having a
leucine-rich repeat region, a transmembrane domain, and an
intracellular domain that includes a TIR domain.
[0062] Nucleotide and amino acid sequences of human and murine TLR8
are known. See, for example, GenBank Accession Nos. AF246971,
AF245703, NM_016610, XM_045706, AY035890, NM_133212; and AAF64061,
AAF78036, NP_057694, XP_045706, AAK62677, and NP_573475, the
contents of all of which is incorporated herein by reference. Human
TLR8 is reported to exist in at least two isoforms, one 1041 amino
acids long and the other 1059 amino acids long. Murine TLR8 is 1032
amino acids long. TLR8 polypeptides include an extracellular domain
having a leucine-rich repeat region, a transmembrane domain, and an
intracellular domain that includes a TIR domain.
[0063] Nucleotide and amino acid sequences of human and murine TLR9
are known. See, for example, GenBank Accession Nos. NM_017442,
AF259262, AB045180, AF245704, AB045181, AF348140, AF314224,
NM_031178; and NP_059138, AAF72189, BAB19259, AAF78037, BAB19260,
AAK29625, AAK28488, and NP_112455, the contents of all of which are
incorporated herein by reference. Human TLR9 is reported to exist
in at least two isoforms, one 1032 amino acids long and the other
1055 amino acids. Murine TLR9 is 1032 amino acids long. TLR9
polypeptides include an extracellular domain having a leucine-rich
repeat region, a transmembrane domain, and an intracellular domain
that includes a TIR domain.
[0064] As used herein, the term "TLR signaling" refers to any
aspect of intracellular signaling associated with signaling through
a TLR. As used herein, the term "TLR-mediated immune response"
refers to the immune response that is associated with TLR
signaling.
[0065] A TLR7-mediated immune response is a response associated
with TLR7 signaling. TLR7-mediated immune response is generally
characterized by the induction of IFN-.alpha. and IFN-inducible
cytokines such as IP-10 and I-TAC. The levels of cytokines IL-1
.alpha./.beta., IL-6, IL-8, MIP-1 .alpha./.beta. and MIP-3
.alpha./.beta. induced in a TLR7-mediated immune response are less
than those induced in a TLR8-mediated immune response.
[0066] A TLR8-mediated immune response is a response associated
with TLR8 signaling. This response is further characterized by the
induction of pro-inflammatory cytokines such as IFN-.gamma.,
IL-12p40/70, TNF-.alpha., IL-1 .alpha./.beta., IL-6, IL-8, MIP-1
.alpha./.beta. and MIP-3 .alpha./.beta..
[0067] A TLR9-mediated immune response is a response associated
with TLR9 signaling. This response is further characterized at
least by the production/secretion of IFN-.gamma. and IL-12, albeit
at levels lower than are achieved via a TLR8-mediated immune
response.
[0068] As used herein, a "TLR7/8 agonist" collectively refers to
any nucleic acid that is capable of increasing TLR7 and/or TLR8
signaling (i.e., an agonist of TLR7 and/or TLR8). Some TLR7/8
ligands induce TLR7 signaling alone (e.g., TLR7 specific agonists),
some induce TLR8 signaling alone (e.g., TLR8 specific agonists),
and others induce both TLR7 and TLR8 signaling.
[0069] The level of TLR7 or TLR8 signaling may be enhanced over a
pre-existing level of signaling or it may be induced over a
background level of signaling. TLR7 ligands include, without
limitation, guanosine analogues such as C8-substituted guanosines,
mixtures of ribonucleosides consisting essentially of G and U,
guanosine ribonucleotides and RNA or RNA-like molecules
(PCT/US03/10406), and adenosine-based compounds (e.g.,
6-amino-9-benzyl-2-(3-hydroxy-propoxy)-9H-purin-8-ol, and similar
compounds made by Sumitomo (e.g., CL-029)).
[0070] As used herein, the term "guanosine analogues" refers to a
guanosine-like nucleotide (excluding guanosine) having a chemical
modification involving the guanine base, guanosine nucleoside
sugar, or both the guanine base and the guanosine nucleoside sugar.
Guanosine analogues specifically include, without limitation,
7-deaza-guanosine.
[0071] Guanosine analogues further include C8-substituted
guanosines such as 7-thia-8-oxoguanosine (immunosine),
8-mercaptoguanosine, 8-bromoguanosine, 8-methylguanosine,
8-oxo-7,8-dihydroguanosine, C8-arylamino-2'-deoxyguanosine,
C8-propynyl-guanosine, C8- and N7-substituted guanine
ribonucleosides such as 7-allyl-8-oxoguanosine (loxoribine) and
7-methyl-8-oxoguanosine, 8-aminoguanosine,
8-hydroxy-2'-deoxyguanosine, 8-hydroxyguanosine, and 7-deaza
8-substituted guanosine.
[0072] TLR8 ligands include mixtures of ribonucleosides consisting
essentially of G and U, guanosine ribonucleotides and RNA or
RNA-like molecules (PCT/US03/10406). Additional TLR8 ligands are
also disclosed in Gorden et al. J. Immunol. 2005, 174:1259-1268).
As used herein, the term "TLR9 agonist" refers to any agent that is
capable of increasing
[0073] TLR9 signaling (i.e., an agonist of TLR9). TLR9 agonists
specifically include, without limitation, immunostimulatory nucleic
acids, and in particular CpG immunostimulatory nucleic acids.
[0074] As used herein, the term "immunostimulatory CpG nucleic
acids" or "immunostimulatory CpG oligonucleotides" refers to any
CpG-containing nucleic acid that is capable of activating an immune
cell. At least the C of the CpG dinucleotide is typically, but not
necessarily, unmethylated. Immunostimulatory CpG nucleic acids are
described in a number of issued patents and published patent
applications, including U.S. Pat. Nos. 6,194,388; 6,207,646;
6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199.
[0075] In some embodiments the agonists of nucleic acid-interacting
complexes is an immunostimulatory oligonucleotide. An
"immunostimulatory oligonucleotide" as used herein is any nucleic
acid (DNA or RNA) containing an immunostimulatory motif or backbone
that is capable of inducing an immune response. An induction of an
immune response refers to any increase in number or activity of an
immune cell, or an increase in expression or absolute levels of an
immune factor, such as a cytokine. Immune cells include, but are
not limited to, NK cells, CD4+T lymphocytes, CD8+T lymphocytes, B
cells, dendritic cells, macrophage and other antigen-presenting
cells. Cytokines include, but are not limited to, interleukins,
TNF-.alpha., IFN-.alpha.,.beta. and .gamma., Flt-ligand, and
co-stimulatory molecules. Immunostimulatory motifs include, but are
not limited to CpG motifs and T-rich motifs.
[0076] A non-limiting set of immunostimulatory oligonucleotides
includes:
TABLE-US-00001 dsRNA (TLR 3): poly(A:C) and poly(I:C) ssRNA
(TLR7/8): (SEQ ID NO: 13) CCGUCUGUUGUGUGACUC (SEQ ID NO: 14)
GCCACCGAGCCGAAGGCACC (SEQ ID NO: 15) UAUAUAUAUAUAUAUAUAUA (SEQ ID
NO: 16) UUAUUAUUAUUAUUAUUAUU (SEQ ID NO: 17) UUUUAUUUUAUUUUAUUUUA
(SEQ ID NO: 18) UGUGUGUGUGUGUGUGUGUG (SEQ ID NO: 19)
UUGUUGUUGUUGUUGUUGUU (SEQ ID NO: 20) UUUGUUUGUUUGUUUGUUUG (SEQ ID
NO: 21) UUAUUUAUUUAUUUAUUUAUUUAU (SEQ ID NO: 22)
UUGUUUGUUUGUUUGUUUGUUUGU (SEQ ID NO: 23) GCCCGUCUGUUGUGUGACUC (SEQ
ID NO: 24) GUCCUUCAAGUCCUUCAA DNA (TLR9): (SEQ ID NO: 25)
GGTGCATCGATGCAGGGGGG (SEQ ID NO: 26) TCCATGGACGTTCCTGAGCGTT (SEQ ID
NO: 27) TCGTCGTTCGAACGACGTTGAT (SEQ ID NO: 28)
TCGTCGACGATCCGCGCGCGCG (SEQ ID NO: 29) GGGGTCAACGTTGAGGGGGG (SEQ ID
NO: 30) TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID NO: 31)
TCGTCGTTGTCGTTTTGTCGTT (SEQ ID NO: 32) GGGGGACGATCGTCGGGGGG (SEQ ID
NO: 33) GGGGACGACGTCGTGGGGGGG (SEQ ID NO: 34)
TCGTCGTTTTCGGCGCGCGCCG (SEQ ID NO: 35)
TCGTCGTCGTTCGAACGACGTTGAT
[0077] The immunostimulatory oligonucleotides may be linked to the
core or to one another or to other molecules such an antigens. For
instance, the oligonucleotides may be conjugated to a linker via
the 5' end or the 3' end. E.g. [Sequence, 5'-3']-Linker or
Linker-[Sequence, 5'-3'].
[0078] The terms "oligonucleotide" and "nucleic acid" are used
interchangeably to 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), thymidine (T)
or uracil (U)) or a substituted purine (e.g., adenine (A) or
guanine (G)). Thus, the term embraces both DNA and RNA
oligonucleotides. The terms shall also include polynucleosides
(i.e., a polynucleotide 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 nucleic acid synthesis). A
polynucleotide of the nanoscale construct and optionally attached
to a nanoparticle core can be single stranded or double stranded. A
double stranded polynucleotide is also referred to herein as a
duplex. Double-stranded oligonucleotides of the invention can
comprise two separate complementary nucleic acid strands.
[0079] As used herein, "duplex" includes a double-stranded nucleic
acid molecule(s) in which complementary sequences are hydrogen
bonded to each other. The complementary sequences can include a
sense strand and an antisense strand. The antisense nucleotide
sequence can be identical or sufficiently identical to the target
gene to mediate effective target gene inhibition (e.g., at least
about 98% identical, 96% identical, 94%, 90% identical, 85%
identical, or 80% identical) to the target gene sequence.
[0080] A double-stranded polynucleotide can be double-stranded over
its entire length, meaning it has no overhanging single-stranded
sequences and is thus blunt-ended. In other embodiments, the two
strands of the double-stranded polynucleotide can have different
lengths producing one or more single-stranded overhangs. A
double-stranded polynucleotide of the invention can contain
mismatches and/or loops or bulges. In some embodiments, it is
double-stranded over at least about 70%, 80%, 90%, 95%, 96%, 97%,
98% or 99% of the length of the oligonucleotide. In some
embodiments, the double-stranded polynucleotide of the invention
contains at least or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, or 15 mismatches.
[0081] Polynucleotides associated with the invention can be
modified such as at the sugar moiety, the phosphodiester linkage,
and/or the base. As used herein, "sugar moieties" includes natural,
unmodified sugars, including pentose, ribose and deoxyribose,
modified sugars and sugar analogs. Modifications of sugar moieties
can include replacement of a hydroxyl group with a halogen, a
heteroatom, or an aliphatic group, and can include
functionalization of the hydroxyl group as, for example, an ether,
amine or thiol.
[0082] Modification of sugar moieties can include 2'-O-methyl
nucleotides, which are referred to as "methylated." In some
instances, polynucleotides associated with the invention may only
contain modified or unmodified sugar moieties, while in other
instances, polynucleotides contain some sugar moieties that are
modified and some that are not.
[0083] In some instances, modified nucleomonomers include sugar- or
backbone-modified ribonucleotides. Modified ribonucleotides can
contain a non-naturally occurring base such as uridines or
cytidines modified at the 5'-position, e.g., 5'-(2-amino)propyl
uridine and 5'-bromo uridine; adenosines and guanosines modified at
the 8-position, e.g., 8-bromo guanosine; deaza nucleotides, e.g.,
7-deaza-adenosine; and N-alkylated nucleotides, e.g., N6-methyl
adenosine. Also, sugar-modified ribonucleotides can have the 2'-OH
group replaced by an H, alkoxy (or OR), R or alkyl, halogen, SH,
SR, amino (such as NH.sub.2, NHR, NR.sub.2,), or CN group, wherein
R is lower alkyl, alkenyl, or alkynyl. In some embodiments,
modified ribonucleotides can have the phosphodiester group
connecting to adjacent ribonucleotides replaced by a modified
group, such as a phosphorothioate group.
[0084] In some aspects, 2'-O-methyl modifications can be beneficial
for reducing undesirable cellular stress responses, such as the
interferon response to double-stranded nucleic acids. Modified
sugars can include D-ribose, 2'-O-alkyl (including 2'-O-methyl and
2'-O-ethyl), i.e., 2'-alkoxy, 2'-amino, 2'-S-alkyl, 2'-halo
(including 2'-fluoro), 2'-methoxyethoxy, 2'-allyloxy
(--OCH.sub.2CH.dbd.CH.sub.2), 2'-propargyl, 2'-propyl, ethynyl,
ethenyl, propenyl, and cyano and the like. The sugar moiety can
also be a hexose.
[0085] The term "alkyl" includes saturated aliphatic groups,
including straight-chain alkyl groups (e.g., methyl, ethyl, propyl,
butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.),
branched-chain alkyl groups (isopropyl, tert-butyl, isobutyl,
etc.), cycloalkyl (alicyclic) groups (cyclopropyl, cyclopentyl,
cyclohexyl, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl
groups, and cycloalkyl substituted alkyl groups. In some
embodiments, a straight chain or branched chain alkyl has 6 or
fewer carbon atoms in its backbone (e.g., C.sub.1-C.sub.6 for
straight chain, C.sub.3-C.sub.6 for branched chain), and more
preferably 4 or fewer. Likewise, preferred cycloalkyls have from
3-8 carbon atoms in their ring structure, and more preferably have
5 or 6 carbons in the ring structure. The term C.sub.1-C.sub.6
includes alkyl groups containing 1 to 6 carbon atoms.
[0086] Unless otherwise specified, the term alkyl includes both
"unsubstituted alkyls" and "substituted alkyls," the latter of
which refers to alkyl moieties having independently selected
substituents replacing a hydrogen on one or more carbons of the
hydrocarbon backbone. Such substituents can include, for example,
alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
cyano, amino (including alkyl amino, dialkylamino, arylamino,
diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moiety. Cycloalkyls can be further
substituted, e.g., with the substituents described above. An
"alkylaryl" or an "arylalkyl" moiety is an alkyl substituted with
an aryl (e.g., phenylmethyl (benzyl)). The term "alkyl" also
includes the side chains of natural and unnatural amino acids. The
term "n-alkyl" means a straight chain (i.e., unbranched)
unsubstituted alkyl group.
[0087] The term "alkenyl" includes unsaturated aliphatic groups
analogous in length and possible substitution to the alkyls
described above, but that contain at least one double bond. For
example, the term "alkenyl" includes straight-chain alkenyl groups
(e.g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl,
octenyl, nonenyl, decenyl, etc.), branched-chain alkenyl groups,
cycloalkenyl (alicyclic) groups (cyclopropenyl, cyclopentenyl,
cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl
substituted cycloalkenyl groups, and cycloalkyl or cycloalkenyl
substituted alkenyl groups. In some embodiments, a straight chain
or branched chain alkenyl group has 6 or fewer carbon atoms in its
backbone (e.g., C.sub.2-C.sub.6 for straight chain, C.sub.3-C.sub.6
for branched chain). Likewise, cycloalkenyl groups may have from
3-8 carbon atoms in their ring structure, and more preferably have
5 or 6 carbons in the ring structure. The term C2-C6 includes
alkenyl groups containing 2 to 6 carbon atoms.
[0088] Unless otherwise specified, the term alkenyl includes both
"unsubstituted alkenyls" and "substituted alkenyls," the latter of
which refers to alkenyl moieties having independently selected
substituents replacing a hydrogen on one or more carbons of the
hydrocarbon backbone. Such substituents can include, for example,
alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
cyano, amino (including alkyl amino, dialkylamino, arylamino,
diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moiety.
[0089] The term "hydrophobic modifications` refers to modification
of bases such that overall hydrophobicity is increased and the base
is still capable of forming close to regular Watson-Crick
interactions. Non-limiting examples of base modifications include
5-position uridine and cytidine modifications like phenyl,
4-pyridyl, 2-pyridyl, indolyl, and isobutyl, phenyl
(C.sub.6H.sub.5OH); tryptophanyl
(C.sub.8H.sub.6N)CH.sub.2CH(NH.sub.2)CO), Isobutyl, butyl,
aminobenzyl; phenyl; naphthyl,
[0090] The term "heteroatom" includes atoms of any element other
than carbon or hydrogen. In some embodiments, preferred heteroatoms
are nitrogen, oxygen, sulfur and phosphorus. The term "hydroxy" or
"hydroxyl" includes groups with an--OH or --O.sup.-- (with an
appropriate counterion). The term "halogen" includes fluorine,
bromine, chlorine, iodine, etc. The term "perhalogenated" generally
refers to a moiety wherein all hydrogens are replaced by halogen
atoms.
[0091] The term "substituted" includes independently selected
substituents which can be placed on the moiety and which allow the
molecule to perform its intended function. Examples of substituents
include alkyl, alkenyl, alkynyl, aryl, (CR'R'').sub.0-3NR'R'',
(CR'R'').sub.0-3CN, NO2, halogen, (CR'R'').sub.0-3C(halogen).sub.3,
(CR'R'').sub.0-3CH(halogen)2, (CR'R'').sub.0-3CH2(halogen),
(CR'R'').sub.0-3CONR'R'', (CR'R'').sub.0-3S(O).sub.1-2NR'R'',
(CR'R'').sub.0-3CHO, (CR'R'').sub.0-3O(CR'R'').sub.0-3H,
(CR'R'').sub.0-3S(O).sub.0-2R', (CR'R'').sub.0-3O(CR'R'').sub.0-3H,
(CR'R'').sub.0-3COR', (CR'R'').sub.0-3CO.sub.2R', or
(CR'R'').sub.0-3' groups; wherein each R' and R'' are each
independently hydrogen, a C.sub.1-C.sub.5 alkyl, C.sub.2-C.sub.5
alkenyl, C.sub.2-C.sub.5 alkynyl, or aryl group, or R' and R''
taken together are a benzylidene group or a
--(CH.sub.2).sub.2O(CH2).sub.2-- group.
[0092] The term "amine" or "amino" includes compounds or moieties
in which a nitrogen atom is covalently bonded to at least one
carbon or heteroatom. The term "alkyl amino" includes groups and
compounds wherein the nitrogen is bound to at least one additional
alkyl group. The term "dialkyl amino" includes groups wherein the
nitrogen atom is bound to at least two additional alkyl groups.
[0093] The term "ether" includes compounds or moieties which
contain an oxygen bonded to two different carbon atoms or
heteroatoms. For example, the term includes "alkoxyalkyl," which
refers to an alkyl, alkenyl, or alkynyl group covalently bonded to
an oxygen atom which is covalently bonded to another alkyl
group.
[0094] The term "base" includes the known purine and pyrimidine
heterocyclic bases, deazapurines, and analogs (including
heterocyclic substituted analogs, e.g., aminoethyoxy phenoxazine),
derivatives (e.g., 1-alkyl-, 1-alkenyl-, heteroaromatic- and
1-alkynyl derivatives) and tautomers thereof. Examples of purines
include adenine, guanine, inosine, diaminopurine, and xanthine and
analogs (e.g., 8-oxo-N.sup.6-methyladenine or 7-diazaxanthine) and
derivatives thereof. Pyrimidines include, for example, thymine,
uracil, and cytosine, and their analogs (e.g., 5-methylcytosine,
5-methyluracil, 5-(1-propynyl)uracil, 5-(1-propynyl)cytosine and
4,4-ethanocytosine). ethanocytosine). Other examples of suitable
bases include non-purinyl and non-pyrimidinyl bases such as
2-aminopyridine and triazines.
[0095] In some aspects, the nucleomonomers of a polynucleotide of
the invention are RNA nucleotides, including modified RNA
nucleotides. The term "nucleoside" includes bases which are
covalently attached to a sugar moiety, preferably ribose or
deoxyribose. Examples of preferred nucleosides include
ribonucleosides and deoxyribonucleosides. Nucleosides also include
bases linked to amino acids or amino acid analogs which may
comprise free carboxyl groups, free amino groups, or protecting
groups. Suitable protecting groups are well known in the art (see
P. G. M. Wuts and T. W. Greene, "Protective Groups in Organic
Synthesis", 2.sup.nd Ed., Wiley-Interscience, New York, 1999).
[0096] The term "nucleotide" includes nucleosides which further
comprise a phosphate group or a phosphate analog.
[0097] As used herein, the term "linkage" includes a naturally
occurring, unmodified phosphodiester moiety
(--O--(PO.sup.2--)--O--) that covalently couples adjacent
nucleomonomers. As used herein, the term "substitute linkage"
includes any analog or derivative of the native phosphodiester
group that covalently couples adjacent nucleomonomers. Substitute
linkages include phosphodiester analogs, e.g., phosphorothioate,
phosphorodithioate, and P-ethyoxyphosphodiester,
P-ethoxyphosphodiester, P-alkyloxyphosphotriester,
methylphosphonate, and nonphosphorus containing linkages, e.g.,
acetals and amides. Such substitute linkages are known in the art
(e.g., Bjergarde et al. 1991. Nucleic Acids Res. 19:5843; Caruthers
et al. 1991. Nucleosides Nucleotides. 10:47). In certain
embodiments, non-hydrolizable linkages are preferred, such as
phosphorothioate linkages.
[0098] In some aspects, polynucleotides of the invention comprise
3' and 5' termini (except for circular oligonucleotides). The 3'
and 5' termini of a polynucleotide can be substantially protected
from nucleases, for example, by modifying the 3' or 5' linkages
(e.g., U.S. Pat. No. 5,849,902 and WO 98/13526). Oligonucleotides
can be made resistant by the inclusion of a "blocking group." The
term "blocking group" as used herein refers to substituents (e.g.,
other than OH groups) that can be attached to oligonucleotides or
nucleomonomers, either as protecting groups or coupling groups for
synthesis (e.g., FITC, propyl (CH.sub.2--CH.sub.2--CH.sub.3),
glycol (--O--CH.sub.2--CH.sub.2--O--) phosphate (PO.sub.3.sup.2--),
hydrogen phosphonate, or phosphoramidite). "Blocking groups" also
include "end blocking groups" or "exonuclease blocking groups"
which protect the 5' and 3' termini of the oligonucleotide,
including modified nucleotides and non-nucleotide exonuclease
resistant structures.
[0099] Exemplary end-blocking groups include cap structures (e.g.,
a 7-methylguanosine cap), inverted nucleomonomers, e.g., with 3'-3'
or 5'-5' end inversions (see, e.g., Ortiagao et al. 1992. Antisense
Res. Dev. 2:129), methylphosphonate, phosphoramidite,
non-nucleotide groups (e.g., non-nucleotide linkers, amino linkers,
conjugates) and the like. The 3' terminal nucleomonomer can
comprise a modified sugar moiety. The 3' terminal nucleomonomer
comprises a 3'-0 that can optionally be substituted by a blocking
group that prevents 3'-exonuclease degradation of the
oligonucleotide. For example, the 3'-hydroxyl can be esterified to
a nucleotide through a 3'.fwdarw.3' internucleotide linkage. For
example, the alkyloxy radical can be methoxy, ethoxy, or
isopropoxy, and preferably, ethoxy. Optionally, the
3'.fwdarw.31'linked nucleotide at the 3' terminus can be linked by
a substitute linkage. To reduce nuclease degradation, the 5' most
3'.fwdarw.5' linkage can be a modified linkage, e.g., a
phosphorothioate or a P-alkyloxyphosphotriester linkage.
Preferably, the two 5' most 3'.fwdarw.5' linkages are modified
linkages. Optionally, the 5' terminal hydroxy moiety can be
esterified with a phosphorus containing moiety, e.g., phosphate,
phosphorothioate, or P-ethoxyphosphate.
[0100] In some aspects, polynucleotides can comprise both DNA and
RNA.
[0101] In some aspects, at least a portion of the contiguous
polynucleotides are linked by a substitute linkage, e.g., a
phosphorothioate linkage. The presence of substitute linkages can
improve pharmacokinetics due to their higher affinity for serum
proteins.
[0102] CpG sequences, while relatively rare in human DNA are
commonly found in the DNA of infectious organisms such as bacteria.
The human immune system has apparently evolved to recognize CpG
sequences as an early warning sign of infection and to initiate an
immediate and powerful immune response against invading pathogens
without causing adverse reactions frequently seen with other immune
stimulatory agents. Thus CpG containing nucleic acids, relying on
this innate immune defense mechanism can utilize a unique and
natural pathway for immune therapy. The effects of CpG nucleic
acids on immune modulation have been described extensively in U.S.
Pat. No. 6,194,388, and published patent applications, such as PCT
US95/01570), PCT/US97/19791, PCT/US98/03678; PCT/US98/10408;
PCT/US98/04703; PCT/US99/07335; and PCT/US99/09863.
[0103] A "CpG oligonucleotide" is a nucleic acid which includes at
least one unmethylated CpG dinucleotide. In some embodiments, the
nucleic acid includes three or more unmethylated CpG dinucleotides.
A nucleic acid containing at least one "unmethylated CpG
dinucleotide" is a nucleic acid molecule which contains an
unmethylated cytosine in a cytosine-guanine dinucleotide sequence
(i.e. "CpG DNA" or DNA containing a 5' cytosine followed by 3'
guanosine and linked by a phosphate bond) and activates the immune
system.
[0104] The immunostimulatory oligonucleotides of the nanoscale
construct are preferably in the range of 6 to 100 bases in length.
However, nucleic acids of any size greater than 6 nucleotides (even
many kb long) are capable of inducing an immune response according
to the invention if sufficient immunostimulatory motifs are
present. Preferably the immunostimulatory nucleic acid is in the
range of between 8 and 100 and in some embodiments between 8 and 50
or 8 and 30 nucleotides in size.
[0105] In some embodiments the immunostimulatory oligonucleotides
have a modified backbone such as a phosphorothioate (PS) backbone.
In other embodiments the immunostimulatory oligonucleotides have a
phosphodiester (PO) backbone. In yet other embodiments
immunostimulatory oligonucleotides have a mixed PO and PS
backbone.
Attachment of Modalities to Nanoparticle Cores
[0106] Modalities associated with the invention, including agonists
of nucleic acid-interacting complexes and antigens, can be attached
to nanoparticle cores by any means known in the art. Methods for
attaching oligonucleotides to nanoparticles are described in detail
in and incorporated by reference from US Patent Publication No.
2010/0129808.
[0107] A nanoparticle can be functionalized in order to attach a
polynucleotide. Alternatively or additionally, the polynucleotide
can be functionalized. One mechanism for functionalization is the
alkanethiol method, whereby oligonucleotides are functionalized
with alkanethiols at their 3' or 5' termini prior to attachment to
gold nanoparticles or nanoparticles comprising other metals,
semiconductors or magnetic materials. Such methods are described,
for example Whitesides, Proceedings of the Robert A. Welch
Foundation 39th Conference On Chemical Research Nanophase
Chemistry, Houston, Tex., pages 109-121 (1995), and Mucic et al.
Chem. Commun. 555-557 (1996). Oligonucleotides can also be attached
to nanoparticles using other functional groups such as
phosophorothioate groups, as described in and incorporated by
reference from U.S. Pat. No. 5,472,881, or substituted
alkylsiloxanes, as described in and incorporated by reference from
Burwell, Chemical Technology, 4, 370-377 (1974) and Matteucci and
Caruthers, J. Am. Chem. Soc., 103, 3185-3191 (1981). In some
instances, polynucleotides are attached to nanoparticles by
terminating the polynucleotide with a 5' or 3' thionucleoside. In
other instances, an aging process is used to attach polynucleotides
to nanoparticles as described in and incorporated by reference from
U.S. Pat. Nos. 6,361,944, 6,506, 569, 6,767,702 and 6,750,016 and
PCT Publication Nos. WO 1998/004740, WO 2001/000876, WO 2001/051665
and WO 2001/073123.
[0108] In some instances, the nucleic acid and/or antigen are
covalently attached to the nanoparticle core, such as through a
gold-thiol linkage. A spacer sequence can be included between the
attachment site and the uptake control moiety and/or the binding
moiety. In some embodiments, a spacer sequence comprises or
consists of an oligonucleotide, a peptide, a polymer or an
oligoethylene.
[0109] Nanoscale constructs can be designed with multiple
chemistries. For example, a DTPA (dithiol phosphoramidite) linkage
can be used. The DTPA resists intracellular release of flares by
thiols and can serve to increase signal to noise ratio.
[0110] The conjugates produced by the methods described herein are
considerably more stable than those produced by other methods. This
increased stability is due to the increased density of the
oligonucleotides on the surfaces of a nanoparticle core or forming
the surface of the corona. By performing the salt additions in the
presence of a surfactant, for example approximately 0.01% sodium
dodecylsulfate (SDS), Tween, or polyethylene glycol (PEG), the salt
aging process can be performed in about an hour.
[0111] The surface density may depend on the size and type of
nanoparticles and on the length, sequence and concentration of the
oligonucleotides. A surface density adequate to make the
nanoparticles stable and the conditions necessary to obtain it for
a desired combination of nanoparticles and oligonucleotides can be
determined empirically. Generally, a surface density of at least 10
picomoles/cm will be adequate to provide stable
nanoparticle-oligonucleotide conjugates. Preferably, the surface
density is at least 15 picomoles/cm. Since the ability of the
oligonucleotides of the conjugates to hybridize with targets may be
diminished if the surface density is too great, the surface density
optionally is no greater than about 35-40 picomoles/cm.sup.2.
Methods are also provided wherein the oligonucleotide is bound to
the nanoparticle at a surface density of at least 10 pmol/cm.sup.2,
at least 15 pmol/cm.sup.2, at least 20 pmol/cm.sup.2, at least 25
pmol/cm.sup.2, at least 30 pmol/cm.sup.2, at least 35
pmol/cm.sup.2, at least 40 pmol/cm.sup.2, at least 45 pmol/cm, at
least 50 pmol/cm.sup.2, or 50 pmol/cm.sup.2 or more.
Therapeutics
[0112] Aspects of the invention relate to delivery of nanoscale
constructs to a subject for therapeutic and/or diagnostic use. The
particles may be administered alone or in any appropriate
pharmaceutical carrier, such as a liquid, for example saline, or a
powder, for administration in vivo. They can also be co-delivered
with larger carrier particles or within administration devices. The
particles may be formulated. The formulations of the invention can
be administered in pharmaceutically acceptable solutions, which may
routinely contain pharmaceutically acceptable concentrations of
salt, buffering agents, preservatives, compatible carriers,
adjuvants, and optionally other therapeutic ingredients. In some
embodiments, nanoscale constructs associated with the invention are
mixed with a substance such as a lotion (for example, aquaphor) and
are administered to the skin of a subject, whereby the nanoscale
constructs are delivered through the skin of the subject. It should
be appreciated that any method of delivery of nanoparticles known
in the art may be compatible with aspects of the invention.
[0113] For use in therapy, an effective amount of the particles can
be administered to a subject by any mode that delivers the
particles to the desired cell. Administering pharmaceutical
compositions may be accomplished by any means known to the skilled
artisan. Routes of administration include but are not limited to
oral, parenteral, intramuscular, intravenous, subcutaneous,
mucosal, intranasal, sublingual, intratracheal, inhalation, ocular,
vaginal, dermal, rectal, and by direct injection.
[0114] Thus, the invention in one aspect involves the finding that
agonists of nucleic acid-interacting complexes are highly effective
in mediating immune stimulatory effects. These agonists of nucleic
acid-interacting complexes are useful therapeutically and
prophylactically for stimulating the immune system to treat cancer,
infectious diseases, allergy, asthma, autoimmune disease, and other
disorders and to help protect against opportunistic infections
following cancer chemotherapy. The strong yet balanced, cellular
and humoral immune responses that result from, for example, TLR
agonist stimulation reflect the body's own natural defense system
against invading pathogens and cancerous cells.
[0115] Thus the agonists of nucleic acid-interacting complexes
useful in some aspects of the invention as a vaccine for the
treatment of a subject at risk of developing or a subject having
allergy or asthma, an infection with an infectious organism or a
cancer in which a specific cancer antigen has been identified. The
agonists of nucleic acid-interacting complexes can also be given
without the antigen or allergen for protection against infection,
allergy or cancer, and in this case repeated doses may allow longer
term protection. A subject at risk as used herein is a subject who
has any risk of exposure to an infection causing pathogen or a
cancer or an allergen or a risk of developing cancer. For instance,
a subject at risk may be a subject who is planning to travel to an
area where a particular type of infectious agent is found or it may
be a subject who through lifestyle or medical procedures is exposed
to bodily fluids which may contain infectious organisms or directly
to the organism or even any subject living in an area where an
infectious organism or an allergen has been identified. Subjects at
risk of developing infection also include general populations to
which a medical agency recommends vaccination with a particular
infectious organism antigen. If the antigen is an allergen and the
subject develops allergic responses to that particular antigen and
the subject may be exposed to the antigen, i.e., during pollen
season, then that subject is at risk of exposure to the
antigen.
[0116] A subject having an infection is a subject that has been
exposed to an infectious pathogen and has acute or chronic
detectable levels of the pathogen in the body. The CpG
immunostimulatory oligonucleotides can be used with or without an
antigen to mount an antigen specific systemic or mucosal immune
response that is capable of reducing the level of or eradicating
the infectious pathogen. An infectious disease, as used herein, is
a disease arising from the presence of a foreign microorganism in
the body. It is particularly important to develop effective vaccine
strategies and treatments to protect the body's mucosal surfaces,
which are the primary site of pathogenic entry.
[0117] A subject having an allergy is a subject that has or is at
risk of developing an allergic reaction in response to an allergen.
An allergy refers to acquired hypersensitivity to a substance
(allergen). Allergic conditions include but are not limited to
eczema, allergic rhinitis or coryza, hay fever, conjunctivitis,
bronchial asthma, urticaria (hives) and food allergies, and other
atopic conditions.
[0118] A subject having a cancer is a subject that has detectable
cancerous cells. The cancer may be a malignant or non-malignant
cancer. Cancers or tumors include but are not limited to biliary
tract cancer; brain cancer; breast cancer; cervical cancer;
choriocarcinoma; colon cancer; endometrial cancer; esophageal
cancer; gastric cancer; intraepithelial neoplasms; lymphomas; liver
cancer; lung cancer (e.g. small cell and non-small cell); melanoma;
neuroblastomas; oral cancer; ovarian cancer; pancreas cancer;
prostate cancer; rectal cancer; sarcomas; skin cancer; testicular
cancer; thyroid cancer; and renal cancer, as well as other
carcinomas and sarcomas. In one embodiment the cancer is hairy cell
leukemia, chronic myelogenous leukemia, cutaneous T-cell leukemia,
multiple myeloma, follicular lymphoma, malignant melanoma, squamous
cell carcinoma, renal cell carcinoma, prostate carcinoma, bladder
cell carcinoma, or colon carcinoma.
[0119] A subject shall mean a human or vertebrate animal including
but not limited to a dog, cat, horse, cow, pig, sheep, goat,
turkey, chicken, primate, e.g., monkey, and fish (aquaculture
species), e.g. salmon. Thus, the invention can also be used to
treat cancer and tumors, infections, and allergy/asthma in non
human subjects.
[0120] As used herein, the term treat, treated, or treating when
used with respect to an disorder such as an infectious disease,
cancer, allergy, or asthma refers to a prophylactic treatment which
increases the resistance of a subject to development of the disease
(e.g., to infection with a pathogen) or, in other words, decreases
the likelihood that the subject will develop the disease (e.g.,
become infected with the pathogen) as well as a treatment after the
subject has developed the disease in order to fight the disease
(e.g., reduce or eliminate the infection) or prevent the disease
from becoming worse.
[0121] An antigen as used herein is a molecule capable of provoking
an immune response. Antigens include but are not limited to cells,
cell extracts, proteins, polypeptides, peptides, polysaccharides,
polysaccharide conjugates, peptide and non-peptide mimics of
polysaccharides and other molecules, small molecules, lipids,
glycolipids, carbohydrates, viruses and viral extracts and
muticellular organisms such as parasites and allergens. The term
antigen broadly includes any type of molecule which is recognized
by a host immune system as being foreign. Antigens include but are
not limited to cancer antigens, microbial antigens, and
allergens.
[0122] As used herein, the terms "cancer antigen" and "tumor
antigen" are used interchangeably to refer to antigens which are
differentially expressed by cancer cells and can thereby be
exploited in order to target cancer cells. Cancer antigens are
antigens which can potentially stimulate apparently tumor-specific
immune responses. Some of these antigens are encoded, although not
necessarily expressed, by normal cells. These antigens can be
characterized as those which are normally silent (i.e., not
expressed) in normal cells, those that are expressed only at
certain stages of differentiation and those that are temporally
expressed such as embryonic and fetal antigens. Other cancer
antigens are encoded by mutant cellular genes, such as oncogenes
(e.g., activated ras oncogene), suppressor genes (e.g., mutant
p53), fusion proteins resulting from internal deletions or
chromosomal translocations. Still other cancer antigens can be
encoded by viral genes such as those carried on RNA and DNA tumor
viruses. A cancer antigen is a compound, such as a peptide or
protein, associated with a tumor or cancer cell surface and which
is capable of provoking an immune response when expressed on the
surface of an antigen presenting cell in the context of an MHC
molecule. Cancer antigens can be prepared from cancer cells either
by preparing crude extracts of cancer cells, for example, as
described in Cohen, et al., 1994, Cancer Research, 54:1055, by
partially purifying the antigens, by recombinant technology, or by
de novo synthesis of known antigens.
[0123] A microbial antigen as used herein is an antigen of a
microorganism and includes but is not limited to virus, bacteria,
parasites, and fungi. Such antigens include the intact
microorganism as well as natural isolates and fragments or
derivatives thereof and also synthetic compounds which are
identical to or similar to natural microorganism antigens and
induce an immune response specific for that microorganism. A
compound is similar to a natural microorganism antigen if it
induces an immune response (humoral and/or cellular) to a natural
microorganism antigen. Such antigens are used routinely in the art
and are well known to those of ordinary skill in the art.
[0124] Examples of viruses that have been found in humans include
but are not limited to: Retroviridae (e.g. human immunodeficiency
viruses, such as HIV-1 (also referred to as HDTV-III, LAVE or
HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP;
Picornaviridae (e.g. polio viruses, hepatitis A virus;
enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses);
Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae
(e.g. equine encephalitis viruses, rubella viruses); Flaviridae
(e.g. dengue viruses, encephalitis viruses, yellow fever viruses);
Coronoviridae (e.g. coronaviruses); Rhabdoviradae (e.g. vesicular
stomatitis viruses, rabies viruses); Filoviridae (e.g. ebola
viruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus,
measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g.
influenza viruses); Bungaviridae (e.g. Hantaan viruses, bunga
viruses, phleboviruses and Nairo viruses); Arena viridae
(hemorrhagic fever viruses); Reoviridae (e.g. reoviruses,
orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae
(Hepatitis B virus); Parvovirida (parvoviruses); Papovaviridae
(papilloma viruses, polyoma viruses); Adenoviridae (most
adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2,
varicella zoster virus, cytomegalovirus (CMV), herpes virus;
Poxviridae (variola viruses, vaccinia viruses, pox viruses); and
Iridoviridae (e.g. African swine fever virus); and unclassified
viruses (e.g. the agent of delta hepatitis (thought to be a
defective satellite of hepatitis B virus), the agents of non-A,
non-B hepatitis (class 1=internally transmitted; class
2=parenterally transmitted (i.e. Hepatitis C); Norwalk and related
viruses, and astroviruses).
[0125] Both gram negative and gram positive bacteria serve as
antigens in vertebrate animals. Such gram positive bacteria
include, but are not limited to, Pasteurella species, Staphylococci
species, and Streptococcus species. Gram negative bacteria include,
but are not limited to, Escherichia coli, Pseudomonas species, and
Salmonella species. Specific examples of infectious bacteria
include but are not limited to, Helicobacter pyloris, Borelia
burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g. M.
tuberculosis, M. avium, M. intracellulare, M. kansaii, M.
gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria
meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group
A Streptococcus), Streptococcus agalactiae (Group B Streptococcus),
Streptococcus (viridans group), Streptococcus faecalis,
Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus
pneumoniae, pathogenic Campylobacter sp., Enterococcus sp.,
Haemophilus influenzae, Bacillus antracis, corynebacterium
diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae,
Clostridium perfringers, Clostridium tetani, Enterobacter
aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides
sp., Fusobacterium nucleatum, Streptobacillus moniliformis,
Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia,
and Actinomyces israelli.
[0126] Examples of fungi include Cryptococcus neoformans,
Histoplasma capsulatum, Coccidioides immitis, Blastomyces
dermatitidis, Chlamydia trachomatis, Candida albicans.
[0127] Other infectious organisms (i.e., protists) include
Plasmodium spp. such as Plasmodium falciparum, Plasmodium malariae,
Plasmodium ovale, and Plasmodium vivax and Toxoplasma gondii.
Blood-borne and/or tissues parasites include Plasmodium spp.,
Babesia microti, Babesia divergens, Leishmania tropica, Leishmania
spp., Leishmania braziliensis, Leishmania donovani, Trypanosoma
gambiense and Trypanosoma rhodesiense (African sleeping sickness),
Trypanosoma cruzi (Chagas' disease), and Toxoplasma gondii.
[0128] Other medically relevant microorganisms have been described
extensively in the literature, e.g., see C. G. A Thomas, Medical
Microbiology, Bailliere Tindall, Great Britain 1983, the entire
contents of which is hereby incorporated by reference.
[0129] An allergen refers to a substance (antigen) that can induce
an allergic or asthmatic response in a susceptible subject. The
list of allergens is enormous and can include pollens, insect
venoms, animal dander dust, fungal spores and drugs (e.g.
penicillin). Examples of natural, animal and plant allergens
include but are not limited to proteins specific to the following
genuses: Canine (Canis familiaris); Dermatophagoides (e.g.
Dermatophagoides farinae); Felis (Felis domesticus); Ambrosia
(Ambrosia artemiisfolia; Lolium (e.g. Lolium perenne or Lolium
multiflorum); Cryptomeria (Cryptomeria japonica); Alternaria
(Alternaria alternata); Alder; Alnus (Alnus gultinoasa); Betula
(Betula verrucosa); Quercus (Quercus alba); Olea (Olea europa);
Artemisia (Artemisia vulgaris); Plantago (e.g. Plantago
lanceolata); Parietaria (e.g. Parietaria officinalis or Parietaria
judaica); Blattella (e.g. Blattella germanica); Apis (e.g. Apis
multiflorum); Cupressus (e.g. Cupressus sempervirens, Cupressus
arizonica and Cupressus macrocarpa); Juniperus (e.g. Juniperus
sabinoides, Juniperus virginiana, Juniperus communis and Juniperus
ashei); Thuya (e.g. Thuya orientalis); Chamaecyparis (e.g.
Chamaecyparis obtusa); Periplaneta (e.g. Periplaneta americana);
Agropyron (e.g. Agropyron repens); Secale (e.g. Secale cereale);
Triticum (e.g. Triticum aestivum); Dactylis (e.g. Dactylis
glomerata); Festuca (e.g. Festuca elatior); Poa (e.g. Poa pratensis
or Poa compressa); Avena (e.g. Avena sativa); Holcus (e.g. Holcus
lanatus); Anthoxanthum (e.g. Anthoxanthum odoratum); Arrhenatherum
(e.g. Arrhenatherum elatius); Agrostis (e.g. Agrostis alba); Phleum
(e.g. Phleum pratense); Phalaris (e.g. Phalaris arundinacea);
Paspalum (e.g. Paspalum notatum); Sorghum (e.g. Sorghum
halepensis); and Bromus (e.g. Bromus inermis).
[0130] The nanoscale constructs of the invention may also be coated
with or administered in conjunction with an anti-microbial agent.
An anti-microbial agent, as used herein, refers to a
naturally-occurring or synthetic compound which is capable of
killing or inhibiting infectious microorganisms. The type of
anti-microbial agent useful according to the invention will depend
upon the type of microorganism with which the subject is infected
or at risk of becoming infected. Anti-microbial agents include but
are not limited to anti-bacterial agents, anti-viral agents,
anti-fungal agents and anti-parasitic agents. Phrases such as
"anti-infective agent", "anti-bacterial agent", "anti-viral agent",
"anti-fungal agent", "anti-parasitic agent" and "parasiticide" have
well-established meanings to those of ordinary skill in the art and
are defined in standard medical texts. Briefly, anti-bacterial
agents kill or inhibit bacteria, and include antibiotics as well as
other synthetic or natural compounds having similar functions.
Antibiotics are low molecular weight molecules which are produced
as secondary metabolites by cells, such as microorganisms. In
general, antibiotics interfere with one or more bacterial functions
or structures which are specific for the microorganism and which
are not present in host cells. Anti-viral agents can be isolated
from natural sources or synthesized and are useful for killing or
inhibiting viruses. Anti-fungal agents are used to treat
superficial fungal infections as well as opportunistic and primary
systemic fungal infections. Anti-parasite agents kill or inhibit
parasites.
[0131] Antibacterial agents kill or inhibit the growth or function
of bacteria. A large class of antibacterial agents is antibiotics.
Antibiotics, which are effective for killing or inhibiting a wide
range of bacteria, are referred to as broad spectrum antibiotics.
Other types of antibiotics are predominantly effective against the
bacteria of the class gram-positive or gram-negative. These types
of antibiotics are referred to as narrow spectrum antibiotics.
Other antibiotics which are effective against a single organism or
disease and not against other types of bacteria, are referred to as
limited spectrum antibiotics. Antibacterial agents are sometimes
classified based on their primary mode of action. In general,
antibacterial agents are cell wall synthesis inhibitors, cell
membrane inhibitors, protein synthesis inhibitors, nucleic acid
synthesis or functional inhibitors, and competitive inhibitors.
[0132] Antiviral agents are compounds which prevent infection of
cells by viruses or replication of the virus within the cell. There
are many fewer antiviral drugs than antibacterial drugs because the
process of viral replication is so closely related to DNA
replication within the host cell, that non-specific antiviral
agents would often be toxic to the host. There are several stages
within the process of viral infection which can be blocked or
inhibited by antiviral agents. These stages include, attachment of
the virus to the host cell (immunoglobulin or binding peptides),
uncoating of the virus (e.g. amantadine), synthesis or translation
of viral mRNA (e.g. interferon), replication of viral RNA or DNA
(e.g. nucleotide analogues), maturation of new virus proteins (e.g.
protease inhibitors), and budding and release of the virus.
[0133] The constructs of the invention may also be administered in
conjunction with a therapeutic or diagnostic antibody. In one
embodiment, the antibody may be selected from the group consisting
of Ributaxin, Herceptin, Quadramet, Panorex, IDEC-Y2B8, BEC2, C225,
Oncolym, SMART M195, ATRAGEN, Ovarex, Bexxar, LDP-03, ior t6,
MDX-210, MDX-11, MDX-22, OV103, 3622W94, anti-VEGF, Zenapax,
MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE-1, CEACIDE, Pretarget,
NovoMAb-G2, TNT, Gliomab-H, GNI-250, EMD-72000, LymphoCide, CMA
676, Monopharm-C, 4B5, ior egf.r3, ior c5, BABS, anti-FLK-2,
MDX-260, ANA Ab, SMART 1D10 Ab, SMART ABL 364 Ab, rituxan,
bevacizumab, and ImmuRAIT-CEA.
[0134] The agonists of nucleic acid-interacting complexes are also
useful for treating and preventing autoimmune disease. Autoimmune
disease is a class of diseases in which an subject's own antibodies
react with host tissue or in which immune effector T cells are
autoreactive to endogenous self peptides and cause destruction of
tissue. Thus an immune response is mounted against a subject's own
antigens, referred to as self antigens. Autoimmune diseases include
but are not limited to rheumatoid arthritis, Crohn's disease,
multiple sclerosis, systemic lupus erythematosus (SLE), autoimmune
encephalomyelitis, myasthenia gravis (MG), Hashimoto's thyroiditis,
Goodpasture's syndrome, pemphigus (e.g., pemphigus vulgaris),
Grave's disease, autoimmune hemolytic anemia, autoimmune
thrombocytopenic purpura, scleroderma with anti-collagen
antibodies, mixed connective tissue disease, polymyositis,
pernicious anemia, idiopathic Addison's disease,
autoimmune-associated infertility, glomerulonephritis (e.g.,
crescentic glomerulonephritis, proliferative glomerulonephritis),
bullous pemphigoid, Sjogren's syndrome, insulin resistance, and
autoimmune diabetes mellitus.
[0135] A "self-antigen" as used herein refers to an antigen of a
normal host tissue. Normal host tissue does not include cancer
cells. Thus an immune response mounted against a self-antigen, in
the context of an autoimmune disease, is an undesirable immune
response and contributes to destruction and damage of normal
tissue, whereas an immune response mounted against a cancer antigen
is a desirable immune response and contributes to the destruction
of the tumor or cancer. Thus, in some aspects of the invention
aimed at treating autoimmune disorders it is not recommended that
the CpG immunostimulatory nucleic acids be administered with self
antigens, particularly those that are the targets of the autoimmune
disorder.
[0136] In other instances, the CpG immunostimulatory nucleic acids
may be delivered with low doses of self-antigens. A number of
animal studies have demonstrated that mucosal administration of low
doses of antigen can result in a state of immune hyporesponsiveness
or "tolerance." The active mechanism appears to be a
cytokine-mediated immune deviation away from a Thl towards a
predominantly Th2 and Th3 (i.e., TGF-.beta. dominated) response.
The active suppression with low dose antigen delivery can also
suppress an unrelated immune response (bystander suppression) which
is of considerable interest in the therapy of autoimmune diseases,
for example, rheumatoid arthritis and SLE. Bystander suppression
involves the secretion of Th1-counter-regulatory, suppressor
cytokines in the local environment where proinflammatory and Th1
cytokines are released in either an antigen-specific or
antigen-nonspecific manner. "Tolerance" as used herein is used to
refer to this phenomenon. Indeed, oral tolerance has been effective
in the treatment of a number of autoimmune diseases in animals
including: experimental autoimmune encephalomyelitis (EAE),
experimental autoimmune myasthenia gravis, collagen-induced
arthritis (CIA), and insulin-dependent diabetes mellitus. In these
models, the prevention and suppression of autoimmune disease is
associated with a shift in antigen-specific humoral and cellular
responses from a Th1 to Th2/Th3 response.
[0137] In another aspect, the present invention is directed to a
kit including one or more of the compositions previously discussed.
A "kit," as used herein, typically defines a package or an assembly
including one or more of the compositions of the invention, and/or
other compositions associated with the invention, for example, as
previously described. Each of the compositions of the kit, if
present, may be provided in liquid form (e.g., in solution), or in
solid form (e.g., a dried powder). In certain cases, some of the
compositions may be constitutable or otherwise processable (e.g.,
to an active form), for example, by the addition of a suitable
solvent or other species, which may or may not be provided with the
kit. Examples of other compositions that may be associated with the
invention include, but are not limited to, solvents, surfactants,
diluents, salts, buffers, emulsifiers, chelating agents, fillers,
antioxidants, binding agents, bulking agents, preservatives, drying
agents, antimicrobials, needles, syringes, packaging materials,
tubes, bottles, flasks, beakers, dishes, frits, filters, rings,
clamps, wraps, patches, containers, tapes, adhesives, and the like,
for example, for using, administering, modifying, assembling,
storing, packaging, preparing, mixing, diluting, and/or preserving
the compositions components for a particular use, for example, to a
sample and/or a subject.
[0138] In some embodiments, a kit associated with the invention
includes one or more nanoparticle cores, such as a nanoparticle
core that comprises gold. A kit can also include one or more
agonists of nucleic acid-interacting complexes. A kit can also
include one or more antigens.
[0139] A kit of the invention may, in some cases, include
instructions in any form that are provided in connection with the
compositions of the invention in such a manner that one of ordinary
skill in the art would recognize that the instructions are to be
associated with the compositions of the invention. For instance,
the instructions may include instructions for the use,
modification, mixing, diluting, preserving, administering,
assembly, storage, packaging, and/or preparation of the
compositions and/or other compositions associated with the kit. In
some cases, the instructions may also include instructions for the
use of the compositions, for example, for a particular use, e.g.,
to a sample. The instructions may be provided in any form
recognizable by one of ordinary skill in the art as a suitable
vehicle for containing such instructions, for example, written or
published, verbal, audible (e.g., telephonic), digital, optical,
visual (e.g., videotape, DVD, etc.) or electronic communications
(including Internet or web-based communications), provided in any
manner.
[0140] In some embodiments, the present invention is directed to
methods of promoting one or more embodiments of the invention as
discussed herein. As used herein, "promoting" includes all methods
of doing business including, but not limited to, methods of
selling, advertising, assigning, licensing, contracting,
instructing, educating, researching, importing, exporting,
negotiating, financing, loaning, trading, vending, reselling,
distributing, repairing, replacing, insuring, suing, patenting, or
the like that are associated with the systems, devices,
apparatuses, articles, methods, compositions, kits, etc. of the
invention as discussed herein. Methods of promotion can be
performed by any party including, but not limited to, personal
parties, businesses (public or private), partnerships,
corporations, trusts, contractual or sub-contractual agencies,
educational institutions such as colleges and universities,
research institutions, hospitals or other clinical institutions,
governmental agencies, etc. Promotional activities may include
communications of any form (e.g., written, oral, and/or electronic
communications, such as, but not limited to, e-mail, telephonic,
Internet, Web-based, etc.) that are clearly associated with the
invention.
[0141] In one set of embodiments, the method of promotion may
involve one or more instructions. As used herein, "instructions"
can define a component of instructional utility (e.g., directions,
guides, warnings, labels, notes, FAQs or "frequently asked
questions," etc.), and typically involve written instructions on or
associated with the invention and/or with the packaging of the
invention. Instructions can also include instructional
communications in any form (e.g., oral, electronic, audible,
digital, optical, visual, etc.), provided in any manner such that a
user will clearly recognize that the instructions are to be
associated with the invention, e.g., as discussed herein.
[0142] The present invention is further illustrated by the
following Examples, which in no way should be construed as further
limiting. The entire contents of all of the references (including
literature references, issued patents, published patent
applications, and co pending patent applications) cited throughout
this application are hereby expressly incorporated by
reference.
EXAMPLES
Example 1
[0143] Materials and Methods
[0144] Synthesis of Immunostimulatory SNAs
[0145] Synthesis of immunostimulatory SNAs (isSNA) is achieved as
described elsewhere.sup.7-12 with the following essential
modifications. In brief, 20 mL 13 nm gold colloid is mixed with 10%
Tween 20 and TLR agonist sequence of sulfhydryl-modified nucleic
acid (TLR 3, 7/8, 9) at appropriate concentration, such as 5 uM and
allowed to react overnight. Addition of antigen can be achieved
similar to methods as described elsewhere..sup.10 Purification of
SNAs can be achieved by repeated ultracentrifugation at
75,000.times.g for 30 min.
[0146] Cell Lines
[0147] RAW 264.7 cell lines were obtained from ATCC. RAW-Blue
macrophages were obtained from InVivoGen. Ramos-Blue and THP1-XBlue
cells were obtained from InVivoGen. All were cultured according to
the distributor recommendations.
[0148] Results and Discussion
[0149] It was found that the nanoscale constructs of the invention
markedly enhance potency in macrophages over unformulated agonists
of nucleic acid-interacting complexes (CpG oligonucleotides) in
solution (FIG. 2). RAW-Blue macrophages were plated at 65 k cells
per well and allowed to adhere overnight. On the day of
experiments, cells were treated with AST-008-ps or CpG 1826-ps at
the indicated concentration of oligo for 30 min (top panel), 4 h
(middle panel), or overnight (bottom panel). For 30 min and 4 h
time points, the entire supernatant was aspirated, the cells were
washed, and complete growth medium without test agents were
administered. At the overnight time point, the activation state of
the cells was determined using the QuantiBlue assay kit. The
results show that particularly at short time points, AST-008-ps
demonstrates a significantly lower EC50 than CpG 1826-ps of 188 nM
as compared to 17800 nM (9-1000-fold reduction one standard
deviation from the mean). At 4 h, AST-008-ps demonstrates a lower
EC50 (32 nM vs 57 nm) and greater activation state than CpG
1826-ps. This difference became narrower following overnight
incubation, at which point the EC50s were not statistically
different from each other. This suggests that under conditions
where residence time of the agent with the target cell is limited,
particularly to 30 minutes or below, the AST-008-ps formulation may
result in more rapid and robust immune activation.
[0150] It was also demonstrated that the nanoscale constructs of
the invention markedly enhanced potency in macrophages over
unformulated agonists of nucleic acid-interacting complexes (CpG
oligonucleotides) in solution following overnight incubation (FIG.
3). RAW-Blue macrophages were plated at 65k cells per well and
allowed to adhere overnight. On the day of experiments, AST-007-po,
AST-007-ps, AST-008-po, AST-008-ps, CpG 1826-po, CpG 1826-ps were
incubated with the cells overnight. The degree of activation was
then determined using the QuantiBlue assay kit. The results show
that for compounds containing only phosphodiester (-po) linkages
(top panel), AST-007-po and AST-008-po are .about.50-150 fold more
potent as determined by their EC50 values than CpG 1826-po (191 nM
and 194 nM as compared to 16032 nM, respectively). With
phosphorothioate-modified compounds (-ps, bottom panel), AST-007-ps
and AST-008-ps are approximately equivalent to CpG 1826-ps, with an
EC50 of 29 nM, 27 nM, and 20 nM, respectively.
[0151] Levels of cytokine secretion following exposure to the
nanoscale constructs of the invention versus unformulated agonists
of nucleic acid-interacting complexes (CpG oligonucleotides) in
solution was examined. The effect on cytokine induction was
examined for both oligonucleotides having phosphodiester and
phosphorothioate internucleotide linkages in both the nanoparticle
and the TRL agonist groups (FIG. 4). RAW-Blue macrophages were
plated at 65k cells per well and allowed to adhere overnight. On
the day of experiments, the indicated compounds were incubated with
the cells overnight. The degree of cytokine secretion was
determined by collecting the supernatant and measuring the
concentration of the indicated cytokines by ELISA (TNF-alpha- top
panel, IL-12- bottom right panel, IL-6- bottom left panel). The
results show significantly higher cytokine secretion at lower doses
are possible using AST-008-ps and AST-008-po than CpG 1826-ps, CpG
1826-po, and the indicated controls. For example, to achieve
greater than 2000 pg/mL TNF-alpha, less than 100 nM AST-008-ps was
needed and less than 1000 nM AST-007-po was needed, but greater
than 1000 nM CpG 1826-ps was required.
[0152] Next, TLR9 activation in response to stimulation with a
nanoscale of the invention having a phosphodiester CpG
oligonucleotide in comparison with phosphodiester and
phosphorothioate CpG oligonucleotides in solution was examined
(FIG. 5). Ramos-Blue or THP1-XBlue cells were seeded and activated
according to the manufacturer's recommended protocol using the
indicated compounds and controls. Remarkably, AST-007-po,
AST-008-po, and AST-009-po demonstrate comparable activation at
similar doses than CpG 7909-ps, a known and optimized TLR 9
agonist. In addition, activation appeared to be dependent on TLR 9,
as the TLR 9 agonist insensitive THP1-XBlue cells did not
demonstrate any activation.
[0153] It was determined that a nanoscale construct of the
invention had a multiple fold increase in potency of over several
different CpG oligo sequences (FIG. 6). Ramos-Blue cells were
seeded and activated according to the manufacturer's recommended
protocol using the indicated compounds and controls. Oligo 1826
(top panel) and 1668 (bottom panel) were tested. Notably, SNA
compounds demonstrate significantly lower EC50 values than free
oligos, independent of chemistry, as compared to controls. This
suggests that for these sequences, the SNA formulation of oligos is
several fold more potent.
[0154] The effects of modulating nanoparticle core size was
examined (FIGS. 7A-7B). Raw Blue cells were plated and treated with
the indicated agonists with different gold core sizes, ranging from
3.5 nm to 13 nm using the indicated oligos. The results show that
smaller gold core sizes appear to demonstrate the potential to
enhance agonist activity in vitro.
[0155] The nanoscale constructs of the invention were observed to
have a more rapid and sustained activation than CpG oligo (FIG. 8).
Cells were plated as described and activation was measured using
QuantiBlue. The results show that at 6 nM oligo, the PS SNAs
demonstrate significantly more activation than free 1668 PS
oligos.
[0156] The ability of phosphorothioate modifications to modulate
agonist activity in a sequence-dependent manner was examined (FIG.
9). Raw Blue cells were plated and treated with the indicated
agonists. Oligo 1826 (top panel) and 1668 (bottom panel) were
tested. The results show that internal phosphorothioate
modifications (C*G) and two 5' phosphorothioate linkages (5'PS2)
have an effect on the activity of immunostimulatory SNAs that
appears to be sequence dependent.
[0157] The ability of oligonucleotide loading density to affect
agonist activity was assessed (FIG. 10). V2 indicates that the
construct was completely gold coated prior to oligo addition to the
gold core. Phosphodiester oligonucleotides (top panel) and
phosphorothioate oligonucleotides (bottom panel) were tested. The
data shows that the density of oligonucleotide on the surface of
the gold will modulate the activity of the immunostimulatory
construct.
[0158] A time course of activation of CpG PO/PO nanoscale
constructs was studied (FIG. 11). The tested constructs are not
activated until >4 hr of incubation. Raw Blue cells were plated
and treated with the indicated agonists. The results show that PO
and PO SNAs do not robustly activate adherent RAW Blue cells until
greater than 4 hours of incubation.
[0159] 5'Chol CpG PO nanoscale constructs showed activation in low
nM range, while 5'C18 abrogated the activity (FIG. 12). A 5'
cholesterol modification (5'Chol) may increase the potency of the
agonist, particularly at low concentrations in a
oligo-dose-independent manner. Modification of the 5' end with a
C18 molecule (5'C18) appears to eliminate the activity
altogether.
[0160] Pre-plated macrophages are more primed for subsequent
activation (FIG. 13). RAW Blue macrophages that were plated
overnight prior to addition of the agonist compounds (top)
generally demonstrate greater activation than when the cells are
plated at the same time as the agonist compound is added
(bottom).
[0161] It was demonstrated that low levels of IFN-gamma secretion
by macrophages (FIG. 14). RAW-Blue macrophages were plated at 65 k
cells per well and allowed to adhere overnight. On the day of
experiments, the indicated compounds were incubated with the cells
overnight. The degree of cytokine secretion (either 24 hours--left
panel or 48 hours--right panel after treatment) was determined by
collecting the supernatant and measuring the concentration of the
indicated cytokines by ELISA. The results show that IFN-gamma is
not produced to an appreciable extent by RAW Blue macrophages
stimulated with these compounds.
Example 2
Immuno-Oncology and Immunotherapies
[0162] Immunotherapeutic SNAs (i.e., AST-008) provide a novel and
versatile technology platform. Their multi-valent immunomodulator
delivery optimizes responses, while profound tumor reduction in a
lymphoma model has been observed. Additionally, they trigger a
potent and balanced T cell response in vivo greater than that of
free oligonucleotide or alum. SNAs can co-present therapeutic
vaccine antigen and adjuvant on a single nanoparticle and have
enhanced activity and faster kinetics than free immunostimulatory
CpG oligodeoxynucleotides. Furthermore, SNAs have the potential to
simultaneously target multiple immunostimulatory receptors (e.g.
TLR 3, 4, 7/8, 9). They can be used, for example, in cancer
immunotherapy and vaccines (prophylactic or therapeutic).
[0163] A schematic of an immunotherapeutic SNA (AST-008) is shown
in FIG. 15. The SNAs can co-present a therapeutic vaccine antigen
and adjuvant on a single nanoparticle, and may simultaneous target
multiple immunostimulatory receptors (e.g. TLR 3, 4, 7/8, 9).
[0164] A schematic demonstrating how AST-008 can enter endosomes
via triggered endocytosis is shown in FIG. 16. AST-008 once in the
endosomes can be used for versatile immune system stimulation.
Within the endosome, AST-008 stimulates immune system signaling via
the TLR 9 receptor, a molecular target for SNA therapy, leading to
both innate and adaptive immune responses. AST-008 may also target
TLR 3, 4, 7/8, resulting in innate and adaptive immune
responses.
[0165] AST-008 induces higher pro-inflammatory responses than
corresponding CpG oligodeoxynucleotides (oligo) in vitro. An assay
demonstrating this finding was conducted. The data is shown as a
set of graphs in FIGS. 17A and 17B. FIG. 17A shows the expression
levels of TNF, IL-12, and IL-6 induced by CTL oligo, CTL SNA, CpG
1826, and AST-008. FIG. 17B presents the NF-.kappa.B activation
stemming from the indicated agents.
[0166] AST-008 also targets draining lymph nodes after
administration of a single subcutaneous dose. AST-008 was
silver-stained to enhance light scattering of the gold core, and
then counterstained with eosin. 4.times. bright field magnification
was used. The data is shown in FIG. 18.
[0167] The structures of the invention are useful for stimulating a
robust immune response in vivo. For instance, FIG. 19 is a graph
illustrating the in vivo activity of AST-008. Mice were given a 50
.mu.L bolus tail vein (intravenous) injection of 5.1 nmol solution
(AST-008-po, AST-008-ps, CpG 1826-po, CpG 1826-ps, GpC-po SNA,
GpC-ps SNA, GpC-po, or GpC-ps) and then analyzed for IL-12
expression 1, 3, and 6 hours after injection (24 mice per group, 3
per each time point). IL-12 levels are expressed as the fold over
PBS. AST-008 architecture enhances the induction of IL-12 by
approximately 20-fold over free oligodeoxynucleotides, and the
effect was sustained for over six hours after the initial
administration. FIGS. 20A-20C consist of a pair of graphs and a
chart that demonstrate that AST-008 induces both a balanced Th1/Th2
response (FIG. 20A) and a higher IgG2a antibody (FIG. 20B) response
than alum or CpG oligonucleotides. The results are tabulated in
FIG. 20C. **p<0.01. FIGS. 21A-21B show that AST-008 induces
cellular responses more effectively than alum or CpG
oligonucleotides. FIG. 21A schematically represents the protocol:
splenocytes were grown for 28 days, challenged on Day 0 and Day 21,
and then restimulated with SIINFEKL and probed for INF-.gamma. with
ELISPOT on Day 28. FIG. 21B is a graph depicting the results.
****p<0.0001.
[0168] The structures have been shown to produce a dramatic
anti-tumor response in vivo as well. FIGS. 22A-22B demonstrate that
AST-008 induces a profound tumor-clearing immune response in an in
vivo lymphoma model. FIG. 22A illustrates the protocol: the right
flanks of C57BL/6 mice were injected with 1.times.10.sup.6 E.G7-OVA
lymphoma (11 per group). The mice were then challenged three times
with 100 .mu.g OVA s.c., 1.8 .mu.g OVA.sub.257-264 s.c., and 0.92
nmol oligo in AST-008, and sacrificed at 2000 mm.sup.3. FIG. 13 is
a graph of the results. *p<0.05 using Two-way ANOVA. FIGS.
23A-23B show that AST-008 exhibits superior anti-tumor activity and
longer survival than CpG oligodeoxynucleotides. The graphs show the
tumor volume (FIG. 23A) and percent survival (FIG. 23B) after
C57BL/6 mice were injected with 1.times.10.sup.6 E.G7-OVA lymphoma
in their right flanks (11 per group) and then were challenged three
times with PBS, PBS and OVA, CpG 1826 and OVA, or AST-008 and OVA.
*p<0.05.
TABLE-US-00002 TABLE 1 Key to symbols SEQ ID Name Oligo Sequence
(5'-3') Formulation NO: AST- TCCATGACGTTCCTGATGCT/ Conjugated to 13
36 007-po, iSp18//iSp18//iSp18// nm gold core via CpG 3ThioMC3-D/
thio-gold bond 1668 PO SNA AST- TCCATGACGTTCCTGACGTT/ Conjugated to
13 37 008-po, iSp18//iSp18// nm gold core via CpG 3ThioMC3-D/
thio-gold bond 1826 PO SNA AST- TCGTCGTTTTGTCGTTTTGTCG Conjugated
to 13 38 009-po, TT/iSp18//iSp18// nm gold core via CpG 3ThioMC3-D/
thio-gold bond 7909 PO SNA AST- tccatgacgttcctgatgct/ Conjugated to
13 39 007-ps, iSp18//iSp18//iSp18// nm gold core via CpG
3ThioMC3-D/ thio-gold bond, 1668 PS backfilled with SNA
tetraethylene glycol AST- tccatgacgttcctgacgtt/ Conjugated to 13 40
008-ps, iSp18//iSp18//iSp18// nm gold core via CpG 3ThioMC3-D/
thio-gold bond, 1826 PS backfilled with SNA tetraethylene glycol
AST- tcgtcgttttgtcgttttgtcg Conjugated to 13 41 009-ps,
tt/iSp18//iSp18// nm gold core via CpG 3ThioMC3-D/ thio-gold bond,
7909 PS backfilled with SNA tetraethylene glycol CpG
TCCATGACGTTCCTGACGTT Free 42 1826-po CpG Tccatgacgttcctgacgtt Free
43 1826-ps CpG TCCATGACGTTCCTGATGCT Free 44 1668-po CpG
Tccatgacgttcctgatgct Free 45 1668-ps CpG TCGTCGTTTTGTCGTTTTGTCG
Free 46 7909-po TT CpG Tcgtcgttttgtcgttttgtcg Free 47 7909-ps tt
rplV-po GCTTTCTTGTTGGTGTAGGTC Free 48 rplV-ps Gctttcttgttggtgtaggtc
Free 49 Ctrl- Sequence containing Conjugated to 13 SNA-po, no CpG
motifs, all nm gold core via rplV phosphodiester thio-gold bond SNA
PO linkages Ctrl- Sequence containing Conjugated to 13 SNA-ps, no
CpG motifs, all nm gold core via rplV phosphorothioate thio-gold
bond SNA PS linkages Lowercase indicates phosphorothioate linkages
Capital indicates phosphodiester linkages Prefix "Ctrl" indicates
oligo used with no CpG motifs present /iSp18/ internal spacer-18
(hexaethylene glycol) /3ThioMC3-D/ terminal sulfhydryl group
REFERENCES
[0169] 1. Koff WC et al. Science 340:1232910-1 (2013)
[0170] 2. Cluff CW. Monophosphoryl Lipid A (MPL) as an Aduvant for
Anti-Cancer Vaccines: Clinical Results. Lipid A in Cancer Therapy,
Jeannin J Ed. Landes Bioscience (2000)
[0171] 3. Krieg AM. Proc Am Thorac Soc 4:289 (2007)
[0172] 4. Schmidt C. Nat Biotechnol 25:825 (2007)
[0173] 5. Ellis RD et al. PLOS One 10:e46094 (2012)
[0174] 6. Garcon N et al. Expert Rev. Vaccines 6:723 (2007)
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[0176] 8. Lytton-Jean AK et al. JACS 127:12754 (2005)
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[0179] 11. Seferos DS et al. Nano Lett 9:308 (2009)
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(2010)
EQUIVALENTS
[0181] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
[0182] All references, including patent documents, disclosed herein
are incorporated by reference in their entirety.
Sequence CWU 1
1
49120DNAArtificial SequenceSynthetic Polynucleotide 1tccatgacgt
tcctgacgtt 20221DNAArtificial SequenceSynthetic Polynucleotide
2gctttcttgt tggtgtaggt c 21320DNAArtificial SequenceSynthetic
Polynucleotide 3tccatgacgt tcctgatgct 20421DNAArtificial
SequenceSynthetic Polynucleotide 4gctttcttgt tggtgtaggt c
21520DNAArtificial SequenceSynthetic Polynucleotide 5tccatgacgt
tcctgatgct 20620DNAArtificial SequenceSynthetic Polynucleotide
6tccatgacgt tcctgatgct 20720DNAArtificial SequenceSynthetic
Polynucleotide 7tccatgacgt tcctgatgct 20820DNAArtificial
SequenceSynthetic Polynucleotide 8tccatgacgt tcctgatgct
20920DNAArtificial SequenceSynthetic Polynucleotide 9tccatgacgt
tcctgacgtt 201020DNAArtificial SequenceSynthetic Polynucleotide
10tccatgacgt tcctgacgtt 201120DNAArtificial SequenceSynthetic
Polynucleotide 11tccatgacgt tcctgacgtt 201220DNAArtificial
SequenceSynthetic Polynucleotide 12tccatgacgt tcctgacgtt
201318RNAArtificial SequenceSynthetic Polynucleotide 13ccgucuguug
ugugacuc 181420RNAArtificial SequenceSynthetic Polynucleotide
14gccaccgagc cgaaggcacc 201520RNAArtificial SequenceSynthetic
Polynucleotide 15uauauauaua uauauauaua 201620RNAArtificial
SequenceSynthetic Polynucleotide 16uuauuauuau uauuauuauu
201720RNAArtificial SequenceSynthetic Polynucleotide 17uuuuauuuua
uuuuauuuua 201820RNAArtificial SequenceSynthetic Polynucleotide
18ugugugugug ugugugugug 201920RNAArtificial SequenceSynthetic
Polynucleotide 19uuguuguugu uguuguuguu 202020RNAArtificial
SequenceSynthetic Polynucleotide 20uuuguuuguu uguuuguuug
202124RNAArtificial SequenceSynthetic Polynucleotide 21uuauuuauuu
auuuauuuau uuau 242224RNAArtificial SequenceSynthetic
Polynucleotide 22uuguuuguuu guuuguuugu uugu 242320RNAArtificial
SequenceSynthetic Polynucleotide 23gcccgucugu ugugugacuc
202418RNAArtificial SequenceSynthetic Polynucleotide 24guccuucaag
uccuucaa 182520DNAArtificial SequenceSynthetic Polynucleotide
25ggtgcatcga tgcagggggg 202622DNAArtificial SequenceSynthetic
Polynucleotide 26tccatggacg ttcctgagcg tt 222722DNAArtificial
SequenceSynthetic Polynucleotide 27tcgtcgttcg aacgacgttg at
222822DNAArtificial SequenceSynthetic Polynucleotide 28tcgtcgacga
tccgcgcgcg cg 222920DNAArtificial SequenceSynthetic Polynucleotide
29ggggtcaacg ttgagggggg 203024DNAArtificial SequenceSynthetic
Polynucleotide 30tcgtcgtttt gtcgttttgt cgtt 243122DNAArtificial
SequenceSynthetic Polynucleotide 31tcgtcgttgt cgttttgtcg tt
223220DNAArtificial SequenceSynthetic Polynucleotide 32gggggacgat
cgtcgggggg 203321DNAArtificial SequenceSynthetic Polynucleotide
33ggggacgacg tcgtgggggg g 213422DNAArtificial SequenceSynthetic
Polynucleotide 34tcgtcgtttt cggcgcgcgc cg 223525DNAArtificial
SequenceSynthetic Polynucleotide 35tcgtcgtcgt tcgaacgacg ttgat
253620DNAArtificial SequenceSynthetic Polynucleotide 36tccatgacgt
tcctgatgct 203720DNAArtificial SequenceSynthetic Polynucleotide
37tccatgacgt tcctgacgtt 203824DNAArtificial SequenceSynthetic
Polynucleotide 38tcgtcgtttt gtcgttttgt cgtt 243920DNAArtificial
SequenceSynthetic Polynucleotide 39tccatgacgt tcctgatgct
204020DNAArtificial SequenceSynthetic Polynucleotide 40tccatgacgt
tcctgacgtt 204124DNAArtificial SequenceSynthetic Polynucleotide
41tcgtcgtttt gtcgttttgt cgtt 244220DNAArtificial SequenceSynthetic
Polynucleotide 42tccatgacgt tcctgacgtt 204320DNAArtificial
SequenceSynthetic Polynucleotide 43tccatgacgt tcctgacgtt
204420DNAArtificial SequenceSynthetic Polynucleotide 44tccatgacgt
tcctgatgct 204520DNAArtificial SequenceSynthetic Polynucleotide
45tccatgacgt tcctgatgct 204624DNAArtificial SequenceSynthetic
Polynucleotide 46tcgtcgtttt gtcgttttgt cgtt 244724DNAArtificial
SequenceSynthetic Polynucleotide 47tcgtcgtttt gtcgttttgt cgtt
244821DNAArtificial SequenceSynthetic Polynucleotide 48gctttcttgt
tggtgtaggt c 214921DNAArtificial SequenceSynthetic Polynucleotide
49gctttcttgt tggtgtaggt c 21
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