U.S. patent application number 13/116453 was filed with the patent office on 2011-12-01 for nanocarrier compositions with uncoupled adjuvant.
This patent application is currently assigned to Selecta Biosciences, Inc.. Invention is credited to Robert L. Bratzler, Lloyd Johnston, Grayson B. Lipford, Charles Zepp.
Application Number | 20110293700 13/116453 |
Document ID | / |
Family ID | 45004392 |
Filed Date | 2011-12-01 |
United States Patent
Application |
20110293700 |
Kind Code |
A1 |
Bratzler; Robert L. ; et
al. |
December 1, 2011 |
NANOCARRIER COMPOSITIONS WITH UNCOUPLED ADJUVANT
Abstract
Disclosed are synthetic nanocarrier compositions with separate
adjuvant compositions as well as related methods.
Inventors: |
Bratzler; Robert L.;
(Concord, MA) ; Johnston; Lloyd; (Belmont, MA)
; Lipford; Grayson B.; (Watertown, MA) ; Zepp;
Charles; (Hardwick, MA) |
Assignee: |
Selecta Biosciences, Inc.
Watertown
MA
|
Family ID: |
45004392 |
Appl. No.: |
13/116453 |
Filed: |
May 26, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61348713 |
May 26, 2010 |
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61348717 |
May 26, 2010 |
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61348728 |
May 26, 2010 |
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61358635 |
Jun 25, 2010 |
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Current U.S.
Class: |
424/450 ;
424/184.1; 424/193.1; 424/278.1; 424/280.1; 424/489; 424/85.2;
424/85.4 |
Current CPC
Class: |
A61K 39/0005 20130101;
A61P 37/04 20180101; A61P 31/16 20180101; A61K 47/6921 20170801;
A61K 39/385 20130101; A61K 47/22 20130101; A61K 47/58 20170801;
A61K 47/646 20170801; A61K 39/39 20130101; A61K 2039/54 20130101;
A61K 2039/542 20130101; A61K 2039/555 20130101; A61P 31/04
20180101; A61P 37/00 20180101; A61K 39/35 20130101; Y02A 50/30
20180101; A61P 35/00 20180101; A61K 2039/55561 20130101; A61K
2039/70 20130101; A61P 31/12 20180101; A61K 39/0013 20130101; A61K
9/5153 20130101; A61P 37/02 20180101; A61K 2039/55522 20130101;
A61P 31/22 20180101; A61K 2039/541 20130101; A61P 5/00 20180101;
A61K 31/7115 20130101; A61K 47/593 20170801; A61K 47/68 20170801;
A61K 2039/55555 20130101; A61P 17/04 20180101; A61P 25/36 20180101;
A61K 33/06 20130101; A61K 2039/543 20130101; A61P 17/00 20180101;
A61P 29/00 20180101; A61K 31/4745 20130101; A61K 47/02 20130101;
A61K 47/60 20170801; A61P 1/16 20180101; A61P 31/14 20180101; A61P
43/00 20180101; A61P 37/08 20180101; A61K 47/6931 20170801; A61P
11/00 20180101; A61P 11/06 20180101; A61P 3/00 20180101; A61P 25/28
20180101; A61P 25/30 20180101; A61K 47/24 20130101; A61K 47/6929
20170801; A61P 11/08 20180101; A61P 25/34 20180101; A61K 2039/55511
20130101; A61P 31/10 20180101; A61K 2039/6093 20130101; A61P 39/02
20180101; A61K 45/06 20130101; A61P 31/00 20180101; A61P 31/20
20180101; A61K 31/4745 20130101; A61K 2300/00 20130101; A61K
31/7115 20130101; A61K 2300/00 20130101; A61K 33/06 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
424/450 ;
424/278.1; 424/184.1; 424/280.1; 424/85.2; 424/85.4; 424/193.1;
424/489 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 47/30 20060101 A61K047/30; A61K 47/42 20060101
A61K047/42; A61K 39/385 20060101 A61K039/385; A61P 29/00 20060101
A61P029/00; A61K 9/14 20060101 A61K009/14; A61P 35/00 20060101
A61P035/00; A61P 31/00 20060101 A61P031/00; A61P 17/00 20060101
A61P017/00; A61P 11/06 20060101 A61P011/06; A61P 11/00 20060101
A61P011/00; A61K 39/00 20060101 A61K039/00; A61P 37/00 20060101
A61P037/00 |
Claims
1. A composition comprising: a dosage form that comprises (1) a
population of synthetic nanocarriers, (2) a first adjuvant that is
not coupled to any synthetic nanocarriers, and (3) a
pharmaceutically acceptable excipient.
2. The composition of claim 1, wherein the composition comprises a
systemic dose of the first adjuvant.
3. The composition of claim 1, wherein the composition further
comprises a second adjuvant.
4. The composition of claim 3, wherein the first adjuvant and
second adjuvant are different.
5. The composition of claim 3, wherein the composition comprises a
systemic dose of the first adjuvant and/or second adjuvant.
6. The composition of claim 2, wherein the systemic dose results in
the systemic release of TNF-.alpha., IL-6 and/or IL-12.
7. The composition of claim 2, wherein the systemic dose results in
the systemic release of IFN-.gamma., IL-12 and/or IL-18.
8. The composition of claim 3, wherein the second adjuvant is
coupled to the synthetic nanocarriers.
9-10. (canceled)
11. The composition of claim 1, further comprising one or more
antigens.
12-14. (canceled)
15. The composition of claim 11, wherein the one or more antigens
comprise a B cell antigen and/or a T cell antigen.
16-18. (canceled)
19. The composition of claim 1, wherein the first adjuvant and/or
second adjuvant comprises a mineral salt, gel-type adjuvant, a
microbial adjuvant, an oil-emulsion or emulsifier-based adjuvant, a
particulate adjuvant, a synthetic adjuvant, a phosphate adjuvant, a
polymer, a liposome, a microcarrier, an immunostimulatory nucleic
acid, alum, a saponin, an interleukin, an interferon, a cytokine, a
toll-like receptor (TLR) agonist, an imidazoquinoline, a cytokine
receptor agonist, a CD40 agonist, an Fc receptor agonist, a
complement receptor agonist, QS21, vitamin E, squalene, tocopherol,
Quil A, ISCOMs, ISCOMATRIX, Ribi Detox, CRL-1005, L-121,
tetrachlorodecaoxide, alum, MF59, AS02, AS15, cholera toxin,
monophosphoryl lipid A, incomplete Freund's adjuvant, complete
Freund's adjuvant, muramyl dipeptide or montanide.
20-31. (canceled)
32. The composition of claim 1, wherein the synthetic nanocarriers
comprise lipid nanoparticles, polymeric nanoparticles, metallic
nanoparticles, surfactant-based emulsions, dendrimers, buckyballs,
nanowires, virus-like particles, peptide or protein particles,
nanoparticles that comprise a combination of nanomaterials,
spheroidal nanoparticles, cuboidal nanoparticles, pyramidal
nanoparticles, oblong nanoparticles, cylindrical nanoparticles or
toroidal nanoparticles.
33. The composition of claim 32, wherein the second adjuvant is
coupled to the synthetic nanocarriers and comprises resiquimod.
34. The composition of claim 33, wherein the one or more antigens
comprise nicotine and a T-helper cell antigen, each of which are
coupled to the synthetic nanocarriers.
35-37. (canceled)
38. The composition of claim 32, wherein the synthetic nanocarriers
comprise one or more polymers.
39-43. (canceled)
44. A method comprising: administering the composition of claim 1
to a subject.
45. (canceled)
46. A method comprising: administering the composition of claim 1
and a second adjuvant to a subject, wherein the second adjuvant is
administered at a time different from the administration of the
composition.
47-52. (canceled)
53. The method of claim 44, wherein the method further comprises
administering one or more antigens.
54. The method of claim 53, wherein the composition further
comprises one or more antigens.
55-58. (canceled)
59. The method of claim 53, wherein the one or more antigens
comprise a B cell antigen and/or a T cell antigen.
60-65. (canceled)
66. The method of claim 44, wherein the synthetic nanocarriers
comprise one or more polymers.
67-71. (canceled)
72. The method of claim 44, wherein the subject is in need of an
inflammatory response.
73. The method of claim 44, wherein the subject is in need of a Th1
immune response.
74. The method of claim 44, wherein the subject is in need of a
humoral immune response.
75. The method of claim 44, wherein the subject is in need of a
specific local cytotoxic T lymphocyte response.
76. The method of claim 44, wherein the subject has or is at risk
of having cancer.
77. The method of claim 44, wherein the subject has or is at risk
of having an infection or infectious disease.
78. The method of claim 44, wherein the subject has or is at risk
of having an atopic condition, asthma, COPD or a chronic
infection.
79-88. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119 of U.S. provisional applications 61/348,713, filed May
26, 2010, 61/348,717, filed May 26, 2010, 61/348,728, filed May 26,
2010, and 61/358,635, filed Jun. 25, 2010, the entire contents of
each of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to synthetic nanocarrier and separate
adjuvant compositions, and related methods, such as for treating
diseases in which generating an immune response is desirable.
BACKGROUND OF THE INVENTION
[0003] Adjuvants are generally important components for the
majority of currently used vaccination regimens. The optimal
approach for augmenting the immune response with adjuvant, however,
in a number of cases, is not yet known. Therefore, improved
compositions and therapeutic methods are needed to provide improved
therapies for diseases in which generating an immune response
and/or augmenting it is desirable.
SUMMARY OF THE INVENTION
[0004] In one aspect, a composition comprising a dosage form that
comprises (1) a population of synthetic nanocarriers, (2) a first
adjuvant that is not coupled to any synthetic nanocarriers, and (3)
a pharmaceutically acceptable excipient is provided. In another
aspect, a method comprising administering the composition to a
subject is provided.
[0005] In one embodiment, the compositions provided herein,
including those of the methods provided, comprise a systemic dose
of the first adjuvant. In another embodiment, said compositions
further comprise a second adjuvant. In still another embodiment,
the first adjuvant and second adjuvant are different. In yet
another embodiment, the first adjuvant and the second adjuvant are
the same. In still another embodiment, the compositions provided
herein, including those of the methods provided, comprise a
systemic dose of the first adjuvant and/or second adjuvant. In one
embodiment, the systemic dose results in the systemic release of
TNF-.alpha., IL-6 and/or IL-12. In another embodiment, the systemic
dose results in the systemic release of IFN-.gamma., IL-12 and/or
IL-18.
[0006] In one embodiment, the second adjuvant of the compositions
provided, including those of the methods provided, is coupled to
the synthetic nanocarriers. In another embodiment, the second
adjuvant is not coupled to any synthetic nanocarriers. In yet
another embodiment, the second adjuvant is coupled to another
population of synthetic nanocarriers.
[0007] In another embodiment, the compositions provided herein,
including those of the methods provided, comprise one or more
antigens. In a further embodiment, the one or more antigens are
coupled to the synthetic nanocarriers. In yet a further embodiment,
the one or more antigens are coupled to another population of
synthetic nanocarriers. In another embodiment, the one or more
antigens are not coupled to any synthetic nanocarriers.
[0008] In one embodiment, the one or more antigens of the
compositions provided, including those of the methods provided,
comprise a B cell antigen and/or a T cell antigen. In another
embodiment, the T cell antigen is a universal T cell antigen or T
helper cell antigen. In a further embodiment, the one or more
antigens comprise a B cell antigen and/or a T cell antigen and a
universal T cell antigen or T helper cell antigen. In one
embodiment, the B cell antigen is nicotine. In yet another
embodiment, the compositions provided, including those of the
methods provided, do not comprise an antigen.
[0009] In one embodiment, of the compositions provided, including
those of the methods provided, the first adjuvant and/or second
adjuvant comprises a mineral salt, gel-type adjuvant, a microbial
adjuvant, an oil-emulsion or emulsifier-based adjuvant, a
particulate adjuvant, a synthetic adjuvant, a phosphate adjuvant, a
polymer, a liposome, a microcarrier, an immunostimulatory nucleic
acid, alum, a saponin, an interleukin, an interferon, a cytokine, a
toll-like receptor (TLR) agonist, an imidazoquinoline, a cytokine
receptor agonist, a CD40 agonist, an Fc receptor agonist, a
complement receptor agonist, QS21, vitamin E, squalene, tocopherol,
Quil A, ISCOMs, ISCOMATRIX, Ribi Detox, CRL-1005, L-121,
tetrachlorodecaoxide, alum, MF59, AS02, AS15, cholera toxin,
monophosphoryl lipid A, incomplete Freund's adjuvant, complete
Freund's adjuvant, muramyl dipeptide or montanide. In one
embodiment, the immunostimulatory nucleic acid comprises a
CpG-containing nucleic acid. In another embodiment, the
imidazoquinoline comprises resiquimod or imiquimod. In still
another embodiment, the first and/or second adjuvant comprises
alum. In one embodiment, when the first adjuvant comprises a
CpG-containing nucleic acid, the second adjuvant comprises an
imidazoquinoline or alum. In another embodiment, the
imidazoquinoline is resiquimod. In still another embodiment, when
the first adjuvant comprises an imidazoquinoline, the second
adjuvant comprises a CpG-containing nucleic acid or alum. In a
further embodiment, the imidazoquinoline is resiquimod. In one
embodiment, when the first adjuvant comprises alum, the second
adjuvant comprises an imidazoquinoline or a CpG-containing nucleic
acid. In another embodiment, the imidazoquinoline is resiquimod. In
a further embodiment, the TLR agonist comprises a TLR-1, TLR-2,
TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8, TLR-9, TLR-10, TLR-11
agonist or a combination thereof. In another embodiment, the first
adjuvant and/or second adjuvant does not comprise a TLR agonist. In
yet another embodiment, the first adjuvant and/or second adjuvant
does not comprise a TLR-3, TLR-7, TLR-8 or TLR-9 agonist. In one
embodiment, the second adjuvant is coupled to the synthetic
nanocarriers and comprises resiquimod.
[0010] In one embodiment, the synthetic nanocarriers of the
compositions provided herein, including those of the methods
provided, comprise lipid nanoparticles, polymeric nanoparticles,
metallic nanoparticles, surfactant-based emulsions, dendrimers,
buckyballs, nanowires, virus-like particles, peptide or protein
particles, nanoparticles that comprise a combination of
nanomaterials, spheroidal nanoparticles, cuboidal nanoparticles,
pyramidal nanoparticles, oblong nanoparticles, cylindrical
nanoparticles or toroidal nanoparticles.
[0011] In one embodiment, the synthetic nanocarriers comprise one
or more polymers. In another embodiment, the one or more polymers
comprise a polyester. In yet another embodiment, the one or more
polymers comprise or further comprise a polyester coupled to a
hydrophilic polymer. In a further embodiment, the polyester
comprises a poly(lactic acid), poly(glycolic acid),
poly(lactic-co-glycolic acid), or polycaprolactone. In still a
further embodiment, the hydrophilic polymer comprises a polyether.
In another embodiment, the polyether comprises polyethylene
glycol.
[0012] In another embodiment, the one or more antigens of the
compositions provided herein, including those of the methods
provided, comprise nicotine and a universal T cell antigen or
T-helper cell antigen, each of which are coupled to the synthetic
nanocarriers.
[0013] In a further embodiment, the universal T cell antigen or T
helper cell antigen of the compositions provided herein, including
those of the methods provided, is coupled by encapsulation. In
still another embodiment, the T-helper cell antigen comprises a
peptide obtained or derived from ovalbumin. In a further
embodiment, the peptide obtained or derived from ovalbumin
comprises the sequence as set forth in SEQ ID NO: 1.
[0014] In another aspect, a method comprising administering any of
the compositions provided herein to a subject is provided. In one
embodiment, the subject is a human.
[0015] In a further aspect, a method comprising administering any
of the compositions provided and a second adjuvant to a subject,
wherein the second adjuvant is administered at a time different
from the administration of the composition, is provided. In one
embodiment, the subject is a human. In another embodiment, the
composition and second adjuvant are coadministered. In yet another
embodiment, the composition and second adjuvant are not
coadministered. In still another embodiment, the second adjuvant is
administered prior to the composition.
[0016] In one embodiment, any of the methods provided further
comprises administering one or more antigens. In another
embodiment, any of the the compositions provided, including those
of the methods provided, further comprises one or more antigens. In
one embodiment, the one or more antigens are coadministered.
[0017] In one embodiment, the subject of any of the methods
provided or to which any of the compositions provided is
administered is in need of an inflammatory response. In another
embodiment, the subject is in need of a Th1 immune response. In yet
another embodiment, the subject is in need of a humoral immune
response. In still another embodiment, the subject is in need of a
specific local cytotoxic T lymphocyte response. In a further
embodiment, the subject has or is at risk of having cancer. In
still a further embodiment, the subject has or is at risk of having
an infection or infectious disease. In yet a further embodiment,
the subject has or is at risk of having an atopic condition,
asthma, COPD or a chronic infection.
[0018] In one embodiment, any of the compositions can be for use in
therapy or prophylaxis. In another embodiment, any of the
compositions can be for use in any of the methods provided. In yet
another embodiment, any of the compositions can be for use in a
method of inducing an inflammatory response in a subject. In still
another embodiment, any of the compositions can be for use in a
method of inducing a Th1 immune response in a subject. In yet
another embodiment, any of the compositions can be for use in a
method of inducing a humoral immune response in a subject. In a
further embodiment, any of the compositions can be for use in a
method of inducing a specific local cytotoxic T lymphocyte response
in a subject. In still a further embodiment, any of the
compositions can be for use in a method of treating or preventing
cancer. In yet a further embodiment, any of the compositions can be
for use in a method of treating or preventing infection or
infectious disease. In another embodiment, any of the compositions
can be for use in a method of treating or preventing an atopic
condition, asthma, COPD or a chronic infection.
[0019] In another aspect, a use of any of the compositions provided
for the manufacture of a medicament for use in any of the methods
provided is provided herein.
BRIEF DESCRIPTION OF FIGURES
[0020] FIG. 1 shows the systemic cytokine production in mice after
nanocarrier (NC) inoculation. FIG. 1A, FIG. 1B and FIG.
1C--TNF-.alpha., IL-6, and IL-12 production in experimental groups
shown, respectively. Sera from groups of three mice were pooled and
analyzed by ELISA.
[0021] FIG. 2 demonstrates the systemic IFN-.gamma. production in
mice after NC inoculation. Sera from groups of three mice were
pooled and analyzed by ELISA.
[0022] FIG. 3 demonstrates that utilization of entrapped R848
within NCs generates an immune response, which is superior to one
induced by NC without R848.
[0023] FIG. 4 shows anti-nicotine antibody titers in mice immunized
with NC containing surface nicotine and T-helper ovalbumin-derived
peptide OP-II (NC-Nic) without adjuvant or with the same NC-Nic
admixed with 20 .mu.g of free R848 (5 animals/group; s.c., 100
.mu.g of NC per injection, 3 times with 4-wk intervals). Titers for
days 26 and 40 after the first immunization are shown (ELISA
against polylysine-nicotine) (group 1: immunized with NC[Nic,O
(i.e., no adjuvant), OP-II] (2.2% of OP-II); group 2: immunized
with NC[Nic,O,OP-II] admixed with 20 .mu.g of free R848).
[0024] FIG. 5 shows anti-nicotine antibody titers in mice immunized
with NC containing surface nicotine and T-helper ovalbumin-derived
peptide OP-II (NC-Nic) with R848 adjuvant or with the same NC-Nic
admixed with 80 .mu.g of free alum or 25 .mu.g of free CpG-1826 (5
animals/group; s.c., 100 .mu.g of NC per injection, 3 times with
4-wk intervals). Titers for days 26 and 40 after the first
immunization are shown (ELISA against polylysine-nicotine) (all
groups: immunized with NC[Nic,R848,OP-II]; group 2: NC admixed with
80 .mu.g of free alum; group 3: admixed with 25 .mu.g of free
CpG-1826).
[0025] FIG. 6 shows anti-nicotine antibody titers in mice immunized
with NC containing surface nicotine, R848 and T-helper
ovalbumin-derived peptide OP-II NC[Nic,R848,OP-II] or with the same
NC-Nic admixed with 80 .mu.g of free alum (5 animals/group; s.c.,
100 .mu.g of NC per injection, 3 times with 4-wk intervals). Titers
for days 40 and 70 after the first immunization are shown (ELISA
against polylysine-nicotine) (group 1: immunized with
NC[Nic,R848,OP-II] (3.1% of R848, 1.5% of OP-II); group 2:
immunized with NC[Nic,R848,OP-II] admixed with 80 .mu.g of free
alum).
[0026] FIG. 7 shows specific local CTL response in mice immunized
with NC containing ovalbumin or free ovalbumin. Mice were immunized
once (s.c., 100 .mu.g of NC, containing 2.8% of OVA, or with 2.5
.mu.g of OVA; both immunogens admixed with 10 .mu.g of free
1826-CpG).
[0027] FIG. 8 shows anti-nicotine antibody titers in mice immunized
with NC containing surface nicotine and T-helper ovalbumin-derived
peptide OP-II (NC-Nic) (no adjuvant within NC) admixed with 20
.mu.g of free CpG (PS) or 20 .mu.g of free CpG (PO) (5
animals/group; s.c., 100 .mu.g of NC per injection, 3 times with
2-wk intervals). Control mice received PBS alone. Titers for days
26 and 40 are shown (ELISA against polylysine-nicotine) (group 1:
immunized with NC-Nic (no adjuvant)+free CpG (PS); group 2:
immunized with NC-Nic (no adjuvant)+free CpG (PO); group 3:
immunized with PBS only).
[0028] FIG. 9 shows anti-ovalbumin (OVA) antibody titers in mice
immunized with NC containing surface OVA (NC-OVA) (no adjuvant
within NC) admixed with 20 .mu.g of free R848 or CpG (PS) (5
animals/group; s.c., 100 .mu.g of NC per injection, 3 times with
2-wk intervals). Control mice were immunized with 2.5 .mu.g of
soluble OVA admixed with 20 .mu.g of CpG (PS). Titers for days 26
and 44 are shown (ELISA against OVA protein) (group 1: immunized
with NC-OVA (no adjuvant)+free R848; group 2: immunized with NC-OVA
(no adjuvant)+free CpG (PS); group 3: immunized with soluble
OVA+CpG (PS)).
[0029] FIG. 10 shows anti-nicotine antibody titers in mice injected
with CpG (20 .mu.g per injection, 2 times with 2-wk intervals)
followed by immunization at day 35 with NC containing surface
nicotine and T-helper ovalbumin-derived peptide OP-II (NC-Nic)
either with or without NC-contained R848 (5 animals/group; s.c.,
100 .mu.g of NC per injection, 2 times with 2-wk intervals). Titers
for days 12, 26, and 40 after immunization with NC are shown (ELISA
against polylysine-nicotine) (group 1: immunized with CpG followed
by NC-Nic (R848+OP-II); group 2: immunized with CpG followed by
NC-Nic (OP-II only)).
DETAILED DESCRIPTION OF THE INVENTION
[0030] Before describing the present invention in detail, it is to
be understood that this invention is not limited to particularly
exemplified materials or process parameters as such may, of course,
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments of the
invention only, and is not intended to be limiting of the use of
alternative terminology to describe the present invention.
[0031] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety for all purposes.
[0032] As used in this specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the content clearly dictates otherwise. For example, reference to
"a polymer" includes a mixture of two or more such molecules,
reference to "a solvent" includes a mixture of two or more such
solvents, reference to "an adhesive" includes mixtures of two or
more such materials, and the like.
Introduction
[0033] The inventors have unexpectedly and surprisingly discovered
that the problems and limitations noted above can be overcome by
practicing the invention disclosed herein. The inventors have
unexpectedly and surprisingly discovered that the administration of
a population of synthetic nanocarriers and an adjuvant that is not
coupled to any of the synthetic nanocarriers provides stronger and
more rapid immune responses. In particular, the inventors have
unexpectedly discovered that it is possible to provide compositions
and methods that relate to a composition comprising a dosage form
that comprises (1) a population of synthetic nanocarriers, (2) a
first adjuvant that is not coupled to any synthetic nanocarriers,
and (3) a pharmaceutically acceptable excipient.
[0034] In embodiments, the administration of adjuvant separate from
synthetic nanocarriers leads to a rapid and strong systemic
induction of pro-inflammatory cytokines, such as TNF-.alpha., IL-6
and/or IL-12. The dose of the adjuvant or adjuvants in the
compositions in some embodiments, therefore, is a systemic dose. In
embodiments, the systemic dose results in the release of
TNF-.alpha., IL-6 or IL-12. In other embodiments, the systemic dose
results in the systemic release of TNF-.alpha., IL-6 and IL-12. As
such cytokines are pro-inflammatory, the administration of
compositions provided herein can be beneficial to subjects where an
inflammatory response is desired. In some embodiments, therefore,
the compositions provided are administered to such subjects. In
embodiments, such subjects have or are at risk of having cancer. In
other embodiments, such subjects have or are at risk of having an
infection or an infectious disease. Methods for the administration
of the compositions to such subjects are also provided.
[0035] In other embodiments, the administration of adjuvant
separate from synthetic nanocarriers leads to a rapid and strong
systemic induction of cytokines that are important for a Th1 immune
response, such as IFN-.gamma., IL-12 and/or IL-18. Therefore, the
dose of the adjuvant or adjuvants in the compositions in some
embodiments is a systemic dose that results in the systemic release
of IFN-.gamma., IL-12 and/or IL-18. As such cytokines are important
for a Th1 immune response, the administration of compositions
provided herein can be beneficial to subjects where a Th1 immune
response is desired. In some embodiments, the compositions provided
are administered to such subjects. In embodiments, such subjects
have or are at risk of having an atopic condition, asthma, chronic
obstructive pulmonary disease (COPD) or a chronic infection.
Methods for the administration of the compositions to such subjects
are also provided.
[0036] The inventors have also unexpectedly discovered that it is
possible to administer a second adjuvant with the aforementioned
compositions to provide a strong humoral response. The
aforementioned compositions, therefore, can further comprise a
second adjuvant. In some embodiments, the second adjuvant is
coupled to the synthetic nanocarriers. In other embodiments, the
second adjuvant is not coupled to any synthetic nanocarriers. In
still other embodiments, the second adjuvant is coupled to another
population of synthetic nanocarriers. In some embodiments, however,
the second adjuvant is administered to a subject at a time
different from when the composition that comprises a population of
synthetic nanocarriers and a first adjuvant that is not coupled to
any synthetic nanocarriers is administered. In some embodiments,
the second adjuvant is administered at a different time but is
coadministered. In other embodiments, the second adjuvant is not
coadministered. In still other embodiments, the second adjuvant is
administered prior to or after the administration of the
composition that comprises a population of synthetic nanocarriers
and a first adjuvant that is not coupled to any synthetic
nanocarriers. In some embodiments, the second adjuvant is also not
coupled to any synthetic nanocarriers. In other embodiments, the
second adjuvant is coupled to another population of synthetic
nanocarriers. The compositions provided herein, therefore, can be
beneficial to subjects where a humoral immune response is desired.
In some embodiments, the compositions provided are administered to
such subjects. In embodiments, such subjects have or are at risk of
having cancer, an infection or infectious disease. Methods for the
administration of the compositions to such subjects are also
provided.
[0037] In further embodiments, it is demonstrated that the
administration of one or more antigens with the compositions
provided above provides a strong specific local cytotoxic T
lymphocyte (CTL) response. In embodiments, the antigen(s) are
coadministered with the compositions provided. In some embodiments,
the antigen(s) are coupled to the synthetic nanocarriers. In other
embodiments, the antigen(s) are not coupled to the synthetic
nanocarriers but to another population of synthetic nanocarriers.
The antigen(s) can comprise a B cell or T cell antigen. In some
embodiments, the T cell antigen is a T helper cell antigen. In
other embodiments, the antigen(s) comprise a B cell or T cell
antigen as well as a T helper cell antigen. Therefore, the
compositions provided can be beneficial to subjects where a
specific local CTL response is desired. In some embodiments, the
compositions provided are administered to such subjects. Methods
for the administration of the compositions to such subjects are
also provided.
[0038] The present invention will now be described in more
detail.
Definitions
[0039] "Adjuvant" means an agent that does not constitute a
specific antigen, but boosts the strength and longevity of immune
response to a concomitantly administered antigen. Such adjuvants
may include, but are not limited to stimulators of pattern
recognition receptors, such as Toll-like receptors, RIG-1 and
NOD-like receptors (NLR), mineral salts, such as alum, alum
combined with monphosphoryl lipid (MPL) A of Enterobacteria, such
as Escherihia coli, Salmonella minnesota, Salmonella typhimurium,
or Shigella flexneri or specifically with MPL.RTM. (ASO4), MPL A of
above-mentioned bacteria separately, saponins, such as
QS-21,Quil-A, ISCOMs, ISCOMATRIX.TM., emulsions such as MF59.TM.,
Montanide.RTM. ISA 51 and ISA 720, AS02 (QS21+squalene+MPL.RTM.),
AS15, liposomes and liposomal formulations such as AS01,
synthesized or specifically prepared microparticles and
microcarriers such as bacteria-derived outer membrane vesicles
(OMV) of N. gonorrheae, Chlamydia trachomatis and others, or
chitosan particles, depot-forming agents, such as Pluronic.RTM.
block co-polymers, specifically modified or prepared peptides, such
as muramyl dipeptide, aminoalkyl glucosaminide 4-phosphates, such
as RC529, or proteins, such as bacterial toxoids or toxin
fragments.
[0040] In embodiments, adjuvants comprise agonists for pattern
recognition receptors (PRR), including, but not limited to
Toll-Like Receptors (TLRs), specifically TLRs 2, 3, 4, 5, 7, 8, 9
and/or combinations thereof. In other embodiments, adjuvants
comprise agonists for Toll-Like Receptors 3, agonists for Toll-Like
Receptors 7 and 8, or agonists for Toll-Like Receptor 9; preferably
the recited adjuvants comprise imidazoquinolines; such as R848;
adenine derivatives, such as those disclosed in U.S. Pat. No.
6,329,381 (Sumitomo Pharmaceutical Company), US Published Patent
Application 2010/0075995 to Biggadike et al., or WO 2010/018132 to
Campos et al.; immunostimulatory DNA; or immunostimulatory RNA. In
specific embodiments, synthetic nanocarriers incorporate as
adjuvants compounds that are agonists for toll-like receptors
(TLRs) 7 & 8 ("TLR 7/8 agonists"). Of utility are the TLR 7/8
agonist compounds disclosed in U.S. Pat. No. 6,696,076 to Tomai et
al., including but not limited to imidazoquinoline amines,
imidazopyridine amines, 6,7-fused cycloalkylimidazopyridine amines,
and 1,2-bridged imidazoquinoline amines. Preferred adjuvants
comprise imiquimod and resiquimod (also known as R848). In specific
embodiments, an adjuvant may be an agonist for the DC surface
molecule CD40. In certain embodiments, to stimulate immunity rather
than tolerance, a synthetic nanocarrier incorporates an adjuvant
that promotes DC maturation (needed for priming of naive T cells)
and the production of cytokines, such as type I interferons, which
promote antibody immune responses. In embodiments, adjuvants also
may comprise immunostimulatory RNA molecules, such as but not
limited to dsRNA, poly I:C or poly I:poly C12U (available as
Ampligen .RTM., both poly I:C and poly I:polyC12U being known as
TLR3 stimulants), and/or those disclosed in F. Heil et al.,
"Species-Specific Recognition of Single-Stranded RNA via Toll-like
Receptor 7 and 8" Science 303(5663), 1526-1529 (2004); J. Vollmer
et al., "Immune modulation by chemically modified ribonucleosides
and oligoribonucleotides" WO 2008033432 A2; A. Forsbach et al.,
"Immunostimulatory oligoribonucleotides containing specific
sequence motif(s) and targeting the Toll-like receptor 8 pathway"
WO 2007062107 A2; E. Uhlmann et al., "Modified oligoribonucleotide
analogs with enhanced immunostimulatory activity" U.S. Pat. Appl.
Publ. US 2006241076; G. Lipford et al., "Immunostimulatory viral
RNA oligonucleotides and use for treating cancer and infections" WO
2005097993 A2; G. Lipford et al., "Immunostimulatory G,U-containing
oligoribonucleotides, compositions, and screening methods" WO
2003086280 A2. In some embodiments, an adjuvant may be a TLR-4
agonist, such as bacterial lipopolysacccharide (LPS), VSV-G, and/or
HMGB-1. In some embodiments, adjuvants may comprise TLR-5 agonists,
such as flagellin, or portions or derivatives thereof, including
but not limited to those disclosed in U.S. Pat. Nos. 6,130,082,
6,585,980, and 7,192,725. In specific embodiments, synthetic
nanocarriers incorporate a ligand for Toll-like receptor (TLR)-9,
such as immunostimulatory DNA molecules comprising CpGs, which
induce type I interferon secretion, and stimulate T and B cell
activation leading to increased antibody production and cytotoxic T
cell responses (Krieg et al., CpG motifs in bacterial DNA trigger
direct B cell activation. Nature. 1995. 374:546-549; Chu et al. CpG
oligodeoxynucleotides act as adjuvants that switch on T helper 1
(Th1) immunity. J. Exp. Med. 1997. 186:1623-1631; Lipford et al.
CpG-containing synthetic oligonucleotides promote B and cytotoxic T
cell responses to protein antigen: a new class of vaccine
adjuvants. Eur. J. Immunol. 1997. 27:2340-2344; Roman et al.
Immunostimulatory DNA sequences function as T helper-1-promoting
adjuvants. Nat. Med. 1997. 3:849-854; Davis et al. CpG DNA is a
potent enhancer of specific immunity in mice immunized with
recombinant hepatitis B surface antigen. J. Immunol. 1998.
160:870-876; Lipford et al., Bacterial DNA as immune cell
activator. Trends Microbiol. 1998. 6:496-500; U.S. Pat. No.
6,207,646 to Krieg et al.; U.S. Pat. No. 7,223,398 to Tuck et al.;
U.S. Pat. No. 7,250,403 to Van Nest et al.; or U.S. Pat. No.
7,566,703 to Krieg et al.
[0041] In some embodiments, adjuvants may be proinflammatory
stimuli released from necrotic cells (e.g., urate crystals). In
some embodiments, adjuvants may be activated components of the
complement cascade (e.g., CD21, CD35, etc.). In some embodiments,
adjuvants may be activated components of immune complexes. The
adjuvants also include complement receptor agonists, such as a
molecule that binds to CD21 or CD35. In some embodiments, the
complement receptor agonist induces endogenous complement
opsonization of the synthetic nanocarrier. In some embodiments,
adjuvants are cytokines, which are small proteins or biological
factors (in the range of 5 kD-20 kD) that are released by cells and
have specific effects on cell-cell interaction, communication and
behavior of other cells. In some embodiments, the cytokine receptor
agonist is a small molecule, antibody, fusion protein, or
aptamer.
[0042] In embodiments, at least a portion of the dose of adjuvant
is not coupled to any synthetic nanocarriers, preferably, all of
the dose of adjuvant is not coupled to any synthetic nanocarriers.
In embodiments, the dose of adjuvant comprises two or more types of
adjuvants, and at least a portion of at least one of the types of
adjuvant is not coupled to any synthetic nanocarriers. For
instance, and without limitation, adjuvants that act on different
receptors, such as different TLR receptors, may be combined. As an
example, in an embodiment a TLR 7/8 agonist may be combined with a
TLR 9 agonist. In another embodiment, a TLR 7/8 agonist may be
combined with a TLR 4 agonist. In yet another embodiment, a TLR 9
agonist may be combined with a TLR 3 agonist.
[0043] "Administering" or "administration" means providing a
substance (e.g., a drug) to a subject in a manner that is
pharmacologically useful.
[0044] An "allergy" also referred to herein as an "allergic
condition", is any condition where there is an undesired immune
response to an allergen (i.e., allergic reaction). Allergies or
allergic conditions include, but are not limited to, allergic
asthma, hay fever, hives, eczema, plant allergies, bee sting
allergies, pet allergies, latex allergies, mold allergies, cosmetic
allergies, food allergies, allergic rhinitis or coryza, topic
allergic reactions, anaphylaxis, atopic dermatitis,
hypersensitivity reactions and other allergic conditions. The
allergic reaction may be the result of an immune reaction to any
allergen.
[0045] "Amount effective" is any amount of a composition provided
herein that produces one or more desired immune responses. This
amount can be for in vitro or in vivo purposes. For in vivo
purposes, the amount can be one that a clinician would believe may
have a clinical benefit for a subject in need of an inflammatory, a
Th1, a humoral or specific local CTL immune response. Such subjects
include those that have or are at risk of having cancer, an
infection or infectious disease, an atopic condition, asthma,
chronic obstructive pulmonary disease (COPD) or a chronic
infection.
[0046] Amounts effective include those that involve the systemic
release of one or more cytokines. In embodiments, the amounts
effective include those that involve the production of a systemic
cytokine release profile. In some embodiments, the one or more
cytokines or cytokine release profile comprises the systemic
release of TNF-.alpha., IL-6 and/or IL-12. In other embodiments,
the one or more cytokines or cytokine release profile comprises the
systemic release of IFN-.gamma., IL-12 and/or IL-18. This can be
monitored by routine methods. An amount that is effective to
produce one or more desired immune responses can also be an amount
of a composition provided herein that produces a desired
therapeutic endpoint or a desired therapeutic result.
[0047] Amounts effective will depend, of course, on the particular
subject being treated; the severity of a condition, disease or
disorder; the individual patient parameters including age, physical
condition, size and weight; the duration of the treatment; the
nature of concurrent therapy (if any); the specific route of
administration and like factors within the knowledge and expertise
of the health practitioner. These factors are well known to those
of ordinary skill in the art and can be addressed with no more than
routine experimentation. It is generally preferred that a maximum
dose be used, that is, the highest safe dose according to sound
medical judgment. It will be understood by those of ordinary skill
in the art, however, that a patient may insist upon a lower dose or
tolerable dose for medical reasons, psychological reasons or for
virtually any other reasons.
[0048] In general, doses of the compositions of the invention can
range from about 10 .mu.g/kg to about 100,000 .mu.g/kg. In some
embodiments, the doses can range from about 0.1 mg/kg to about 100
mg/kg. In still other embodiments, the doses can range from about
0.1 mg/kg to about 25 mg/kg, about 25 mg/kg to about 50 mg/kg,
about 50 mg/kg to about 75 mg/kg or about 75 mg/kg to about 100
mg/kg. Alternatively, the dose can be administered based on the
number of synthetic nanocarriers. For example, useful doses include
greater than 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9 or 10.sup.10
synthetic nanocarriers per dose. Other examples of useful doses
include from about 1.times.10.sup.6 to about 1.times.10.sup.10,
about 1.times.10.sup.7 to about 1.times.10.sup.9 or about
1.times.10.sup.8 to about 1.times.10.sup.9 synthetic nanocarriers
per dose. In some embodiments, the doses of the compositions
provided are systemic doses.
[0049] "Antigen" means a B cell antigen or T cell antigen. In
embodiments, antigens are coupled to the synthetic nanocarriers. In
other embodiments, antigens are not coupled to the synthetic
nanocarriers. In embodiments, antigens are coadministered with the
synthetic nanocarriers. In other embodiments, antigens are not
coadministered with the synthetic nanocarriers. "Type(s) of
antigens" means molecules that share the same, or substantially the
same, antigenic characteristics. In embodiments, antigens of the
compositions provided are associated with the disease or condition
that is being treated. For examples, the antigen can be an allergen
(for the treatment of an allergy or allergic condition), a
cancer-associated antigen (for the treatment of cancer or a tumor),
an infectious agent antigen (for the treatment of an infection, an
infectious disease or a chronic infectious disease), etc.
[0050] "At least a portion of the dose" means at least some part of
the dose, ranging up to including all of the dose.
[0051] An "at risk" subject is one in which a health practitioner
believes has a chance of having as disease or condition as provided
herein.
[0052] "B cell antigen" means any antigen that is recognized by a B
cell, and triggers an immune response in a B cell (e.g., an antigen
that is specifically recognized by a B cell receptor on a B cell).
In some embodiments, an antigen that is a T cell antigen is also a
B cell antigen. In other embodiments, the T cell antigen is not
also a B cell antigen. B cell antigens include, but are not limited
to proteins, peptides, small molecules, and carbohydrates. In some
embodiments, the B cell antigen comprises a non-protein antigen
(i.e., not a protein or peptide antigen). In some embodiments, the
B cell antigen comprises a carbohydrate associated with an
infectious agent. In some embodiments, the B cell antigen comprises
a glycoprotein or glycopeptide associated with an infectious agent.
The infectious agent can be a bacterium, virus, fungus, protozoan,
parasite or prion. In some embodiments, the B cell antigen
comprises a poorly immunogenic antigen. In some embodiments, the B
cell antigen comprises an abused substance or a portion thereof. In
some embodiments, the B cell antigen comprises an addictive
substance or a portion thereof. Addictive substances include, but
are not limited to, nicotine, a narcotic, a cough suppressant, a
tranquilizer, and a sedative. In some embodiments, the B cell
antigen comprises a toxin, such as a toxin from a chemical weapon
or natural source, or a pollutant. The B cell antigen may also
comprise a hazardous environmental agent. In other embodiments, the
B cell antigen comprises an alloantigen, an allergen, a contact
sensitizer, a degenerative disease antigen, a hapten, an infectious
disease antigen, a cancer antigen, an atopic disease antigen, an
autoimmune disease antigen, an addictive substance, a xenoantigen,
or a metabolic disease enzyme or enzymatic product thereof.
[0053] "Coadministered" means administering two or more substances
to a subject in a manner that is correlated in time, preferably
sufficiently correlated in time so as to provide a modulation in an
immune response. In embodiments, coadministration may occur through
administration of two or more substances in the same dosage form.
In other embodiments, coadministration may encompass administration
of two or more substances in different dosage forms, but within a
specified period of time, preferably within 1 month, more
preferably within 1 week, still more preferably within 1 day, and
even more preferably within 1 hour.
[0054] "Couple" or "Coupled" or "Couples" (and the like) means to
chemically associate one entity (for example a moiety) with
another. In some embodiments, the coupling is covalent, meaning
that the coupling occurs in the context of the presence of a
covalent bond between the two entities. In non-covalent
embodiments, the non-covalent coupling is mediated by non-covalent
interactions including but not limited to charge interactions,
affinity interactions, metal coordination, physical adsorption,
host-guest interactions, hydrophobic interactions, TT stacking
interactions, hydrogen bonding interactions, van der Waals
interactions, magnetic interactions, electrostatic interactions,
dipole-dipole interactions, and/or combinations thereof. In
embodiments, encapsulation is a form of coupling. In embodiments,
at least a portion of a dose of adjuvant(s) is not coupled to any
synthetic nanocarriers, preferably all of a dose of adjuvant(s) is
not coupled to any synthetic nanocarriers.
[0055] "Derived" means taken from a source and subjected to
substantial modification. For instance, a peptide or nucleic acid
with a sequence with only 50% identity to a natural peptide or
nucleic acid, preferably a natural consensus peptide or nucleic
acid, would be said to be derived from the natural peptide or
nucleic acid. Substantial modification is modification that
significantly affects the chemical or immunological properties of
the material in question. Derived peptides and nucleic acids can
also include those with a sequence with greater than 50% identity
to a natural peptide or nucleic acid sequence if said derived
peptides and nucleic acids have altered chemical or immunological
properties as compared to the natural peptide or nucleic acid.
These chemical or immunological properties comprise hydrophilicity,
stability, affinity, and ability to couple with a carrier such as a
synthetic nanocarrier.
[0056] "Dosage form" means a pharmacologically and/or
immunologically active material in a medium, carrier, vehicle, or
device suitable for administration to a subject.
[0057] "Encapsulate" means to enclose within a synthetic
nanocarrier, preferably enclose completely within a synthetic
nanocarrier. Most or all of a substance that is encapsulated is not
exposed to the local environment external to the synthetic
nanocarrier. Encapsulation is distinct from absorption, which
places most or all of a substance on a surface of a synthetic
nanocarrier, and leaves the substance exposed to the local
environment external to the synthetic nanocarrier.
[0058] "Humoral response" means any immune response that results in
the production or stimulation of B cells and/or the production of
antibodies. Preferably, the humoral immune response is specific to
an antigen comprised within an inventive composition or
administered during the practice of an inventive method. Methods
for assessing whether a humoral response is induced are known to
those of ordinary skill in the art. Examples of such methods are
provided below in the Examples.
[0059] An "infection" or "infectious disease" is any condition or
disease caused by a microorganism, pathogen or other agent, such as
a bacterium, fungus, prion or virus. Examples of infectious disease
include, but are not limited to, viral infectious diseases, such as
AIDS, Chickenpox (Varicella), Common cold, Cytomegalovirus
Infection, Colorado tick fever, Dengue fever, Ebola hemorrhagic
fever, Hand, foot and mouth disease, Hepatitis, Herpes simplex,
Herpes zoster, HPV, Influenza (Flu), Lassa fever, Measles, Marburg
hemorrhagic fever, Infectious mononucleosis, Mumps, Norovirus,
Poliomyelitis, Progressive multifocal leukencephalopathy, Rabies,
Rubella, SARS, Smallpox (Variola), Viral encephalitis, Viral
gastroenteritis, Viral meningitis, Viral pneumonia, West Nile
disease and Yellow fever; bacterial infectious diseases, such as
Anthrax, Bacterial Meningitis, Botulism, Brucellosis,
Campylobacteriosis, Cat Scratch Disease, Cholera, Diphtheria,
Epidemic Typhus, Gonorrhea, Impetigo, Legionellosis, Leprosy
(Hansen's Disease), Leptospirosis, Listeriosis, Lyme disease,
Melioidosis, Rheumatic Fever, MRSA infection, Nocardiosis,
Pertussis (Whooping Cough), Plague, Pneumococcal pneumonia,
Psittacosis, Q fever, Rocky Mountain Spotted Fever (RMSF),
Salmonellosis, Scarlet Fever, Shigellosis, Syphilis, Tetanus,
Trachoma, Tuberculosis, Tularemia, Typhoid Fever, Typhus and
Urinary Tract Infections; parasitic infectious diseases, such as
African trypanosomiasis, Amebiasis, Ascariasis, Babesiosis, Chagas
Disease, Clonorchiasis, Cryptosporidiosis, Cysticercosis,
Diphyllobothriasis, Dracunculiasis, Echinococcosis, Enterobiasis,
Fascioliasis, Fasciolopsiasis, Filariasis, Free-living amebic
infection, Giardiasis, Gnathostomiasis, Hymenolepiasis,
Isosporiasis, Kala-azar, Leishmaniasis, Malaria, Metagonimiasis,
Myiasis, Onchocerciasis, Pediculosis, Pinworm Infection, Scabies,
Schistosomiasis, Taeniasis, Toxocariasis, Toxoplasmosis,
Trichinellosis, Trichinosis, Trichuriasis, Trichomoniasis and
Trypanosomiasis; fungal infectious disease, such as Aspergillosis,
Blastomycosis, Candidiasis, Coccidioidomycosis, Cryptococcosis,
Histoplasmosis, Tinea pedis (Athlete's Foot) and Tinea cruris;
prion infectious diseases, such as Alpers' disease, Fatal Familial
Insomnia, Gerstmann-Straussler-Scheinker syndrome, Kuru and Variant
Creutzfeldt-Jakob disease.
[0060] "Inflammatory response" means any immune response involved
in the body's innate immune defense system that operates in
response to, for example, exposure to an infectious agent, cell
injury, etc. In embodiments, the inflammatory response includes the
systemic release of cytokines, such as TNF-.alpha., IL-6 and/or
IL-12. Methods for assessing whether an inflammatory response is
induced, such as an assessment of the production of proinflammatory
cytokines, are known to those of ordinary skill in the art.
Examples of such methods are provided below in the Examples.
[0061] "Isolated nucleic acid" means a nucleic acid that is
separated from its native environment and present in sufficient
quantity to permit its identification or use. An isolated nucleic
acid may be one that is (i) amplified in vitro by, for example,
polymerase chain reaction (PCR); (ii) recombinantly produced by
cloning; (iii) purified, as by cleavage and gel separation; or (iv)
synthesized by, for example, chemical synthesis. An isolated
nucleic acid is one which is readily manipulable by recombinant DNA
techniques well known in the art. Thus, a nucleotide sequence
contained in a vector in which 5' and 3' restriction sites are
known or for which polymerase chain reaction (PCR) primer sequences
have been disclosed is considered isolated but a nucleic acid
sequence existing in its native state in its natural host is not.
An isolated nucleic acid may be substantially purified, but need
not be. For example, a nucleic acid that is isolated within a
cloning or expression vector is not pure in that it may comprise
only a tiny percentage of the material in the cell in which it
resides. Such a nucleic acid is isolated, however, as the term is
used herein because it is readily manipulable by standard
techniques known to those of ordinary skill in the art. Any of the
nucleic acids provided herein may be isolated. In some embodiments,
the antigens in the compositions provided herein are present in the
form of an isolated nucleic acid, such as an isolated nucleic acid
that encodes an antigenic peptide, polypeptide or protein.
[0062] "Isolated peptide, polypeptide or protein" means the
polypeptide (or peptide or protein) is separated from its native
environment and present in sufficient quantity to permit its
identification or use. This means, for example, the polypeptide (or
peptide or protein) may be (i) selectively produced by expression
cloning or (ii) purified as by chromatography or electrophoresis.
Isolated peptides, proteins or polypeptides may be, but need not
be, substantially pure. Because an isolated peptide, polypeptide or
protein may be admixed with a pharmaceutically acceptable carrier
in a pharmaceutical preparation, the polypeptide (or peptide or
protein) may comprise only a small percentage by weight of the
preparation. The polypeptide (or peptide or protein) is nonetheless
isolated in that it has been separated from the substances with
which it may be associated in living systems, i.e., isolated from
other proteins (or peptides or polypeptides). Any of the peptides,
polypeptides or proteins provided herein may be isolated. In some
embodiments, the antigens in the compositions provided herein are
in the form of isolated peptides, polypeptides or proteins.
[0063] "Maximum dimension of a synthetic nanocarrier" means the
largest dimension of a nanocarrier measured along any axis of the
synthetic nanocarrier. "Minimum dimension of a synthetic
nanocarrier" means the smallest dimension of a synthetic
nanocarrier measured along any axis of the synthetic nanocarrier.
For example, for a spheriodal synthetic nanocarrier, the maximum
and minimum dimension of a synthetic nanocarrier would be
substantially identical, and would be the size of its diameter.
Similarly, for a cuboidal synthetic nanocarrier, the minimum
dimension of a synthetic nanocarrier would be the smallest of its
height, width or length, while the maximum dimension of a synthetic
nanocarrier would be the largest of its height, width or length. In
an embodiment, a minimum dimension of at least 75%, preferably at
least 80%, more preferably at least 90%, of the synthetic
nanocarriers in a sample, based on the total number of synthetic
nanocarriers in the sample, is greater than 100 nm. In an
embodiment, a maximum dimension of at least 75%, preferably at
least 80%, more preferably at least 90%, of the synthetic
nanocarriers in a sample, based on the total number of synthetic
nanocarriers in the sample, is equal to or less than 5 .mu.m.
Preferably, a minimum dimension of at least 75%, preferably at
least 80%, more preferably at least 90%, of the synthetic
nanocarriers in a sample, based on the total number of synthetic
nanocarriers in the sample, is greater than 110 nm, more preferably
greater than 120 nm, more preferably greater than 130 nm, and more
preferably still greater than 150 nm. Aspects ratios of the maximum
and minimum dimensions of inventive synthetic nanocarriers may vary
depending on the embodiment. For instance, aspect ratios of the
maximum to minimum dimensions of the synthetic nanocarriers may
vary from 1:1 to 1,000,000:1, preferably from 1:1 to 100,000:1,
more preferably from 1:1 to 1000:1, still preferably from 1:1 to
100:1, and yet more preferably from 1:1 to 10:1. Preferably, a
maximum dimension of at least 75%, preferably at least 80%, more
preferably at least 90%, of the synthetic nanocarriers in a sample,
based on the total number of synthetic nanocarriers in the sample
is equal to or less than 3 .mu.m, more preferably equal to or less
than 2 .mu.m, more preferably equal to or less than 1 .mu.m, more
preferably equal to or less than 800 nm, more preferably equal to
or less than 600 nm, and more preferably still equal to or less
than 500 nm. In preferred embodiments, a maximum dimension of at
least 75%, preferably at least 80%, more preferably at least 90%,
of the synthetic nanocarriers in a sample, based on the total
number of synthetic nanocarriers in the sample, is equal to or
greater than 100nm, more preferably equal to or greater than 120nm,
more preferably equal to or greater than 130 nm, more preferably
equal to or greater than 140 nm, and more preferably still equal to
or greater than 150 nm. Measurement of synthetic nanocarrier sizes
is obtained by suspending the synthetic nanocarriers in a liquid
(usually aqueous) media and using dynamic light scattering (e.g.
using a Brookhaven ZetaPALS instrument).
[0064] "Obtained" means taken from a source without substantial
modification. Substantial modification is modification that
significantly affects the chemical or immunological properties of
the material in question. For example, as a non-limiting example, a
peptide or nucleic acid with a sequence with greater than 90%,
preferably greater than 95%, preferably greater than 97%,
preferably greater than 98%, preferably greater than 99%,
preferably 100%, identity to a natural peptide or nucleotide
sequence, preferably a natural consensus peptide or nucleotide
sequence, and chemical and/or immunological properties that are not
significantly different from the natural peptide or nucleic acid
would be said to be obtained from the natural peptide or nucleotide
sequence. These chemical or immunological properties comprise
hydrophilicity, stability, affinity, and ability to couple with a
carrier such as a synthetic nanocarrier.
[0065] "Pharmaceutically acceptable carrier or excipient" means a
pharmacologically inactive material used together with the recited
synthetic nanocarriers to formulate the inventive compositions.
Pharmaceutically acceptable carriers or excipients comprise a
variety of materials known in the art, including but not limited
to, saccharides (such as glucose, lactose and the like),
preservatives such as antimicrobial agents, reconstitution aids,
colorants, saline (such as phosphate buffered saline) and buffers.
In some embodiments, pharmaceutically acceptable carriers or
excipients comprise calcium carbonate, calcium phosphate, various
diluents, various sugars and types of starch, cellulose
derivatives, gelatin, vegetable oils and polyethylene glycols.
[0066] "Specific local cytotoxic T lymphocyte (CTL) response" means
any stimulation, induction or proliferation of cytotoxic T cells,
preferably cytotoxic T cells that are specific to an antigen. In
embodiments, the antigen is associated with any of the diseases or
conditions provided herein. In some embodiments, the antigen is
comprised within an inventive composition or is administered in an
inventive method provided herein. Methods for assessing CTL
response are known to those of skill in the art. An examples of
such a method is provided in the Examples.
[0067] "Subject" means animals, including warm blooded mammals such
as humans and primates; avians; domestic household or farm animals
such as cats, dogs, sheep, goats, cattle, horses and pigs;
laboratory animals such as mice, rats and guinea pigs; fish;
reptiles; zoo and wild animals; and the like.
[0068] "Synthetic nanocarrier(s)" means a discrete object that is
not found in nature, and that possesses at least one dimension that
is less than or equal to 5 microns in size. Albumin nanoparticles
are generally included as synthetic nanocarriers, however in
certain embodiments the synthetic nanocarriers do not comprise
albumin nanoparticles. In embodiments, inventive synthetic
nanocarriers do not comprise chitosan.
[0069] A synthetic nanocarrier can be, but is not limited to, one
or a plurality of lipid-based nanoparticles(e.g. liposomes) (also
referred to herein as lipid nanoparticles, i.e., nanoparticles
where the majority of the material that makes up their structure
are lipids), polymeric nanoparticles, metallic nanoparticles,
surfactant-based emulsions, dendrimers, buckyballs, nanowires,
virus-like particles(i.e., particles that are primarily made up of
viral structural proteins but that are not infectious or have low
infectivity), peptide or protein-based particles (also referred to
herein as protein particles, i.e., particles where the majority of
the material that makes up their structure are peptides or
proteins) (such as albumin nanoparticles) and/or nanoparticles that
are developed using a combination of nanomaterials such as
lipid-polymer nanoparticles. Synthetic nanocarriers may be a
variety of different shapes, including but not limited to
spheroidal, cuboidal, pyramidal, oblong, cylindrical, toroidal, and
the like. Synthetic nanocarriers according to the invention
comprise one or more surfaces, including but not limited to
internal surfaces (surfaces generally facing an interior portion of
the synthetic nanocarrier) and external surfaces (surfaces
generally facing an external environment of the synthetic
nanocarrier). Exemplary synthetic nanocarriers that can be adapted
for use in the practice of the present invention comprise: (1) the
biodegradable nanoparticles disclosed in U.S. Pat. No. 5,543,158 to
Gref et al., (2) the polymeric nanoparticles of Published US Patent
Application 20060002852 to Saltzman et al., (3) the
lithographically constructed nanoparticles of Published US Patent
Application 20090028910 to DeSimone et al., (4) the disclosure of
WO 2009/051837 to von Andrian et al., (5) the nanoparticles
disclosed in Published US Patent Application 2008/0145441 to
Penades et al., (6) the protein nanoparticles disclosed in
Published US Patent Application 20090226525 to de los Rios et al.,
(7) the virus-like particles disclosed in published US Patent
Application 20060222652 to Sebbel et al., (8) the nucleic acid
coupled virus-like particles disclosed in published US Patent
Application 20060251677 to Bachmann et al., (9) the virus-like
particles disclosed in WO2010047839A1 or WO2009106999A2, or (10)
the nanoprecipitated nanoparticles disclosed in P. Paolicelli et
al., "Surface-modified PLGA-based Nanoparticles that can
Efficiently Associate and Deliver Virus-like Particles"
Nanomedicine. 5(6):843-853 (2010). In embodiments, synthetic
nanocarriers may possess an aspect ratio greater than 1:1, 1:1.2,
1:1.5, 1:2, 1:3, 1:5, 1:7, or greater than 1:10.
[0070] Synthetic nanocarriers according to the invention that have
a minimum dimension of equal to or less than about 100 nm,
preferably equal to or less than 100 nm, do not comprise a surface
with hydroxyl groups that activate complement or alternatively
comprise a surface that consists essentially of moieties that are
not hydroxyl groups that activate complement. In a preferred
embodiment, synthetic nanocarriers according to the invention that
have a minimum dimension of equal to or less than about 100 nm,
preferably equal to or less than 100 nm, do not comprise a surface
that substantially activates complement or alternatively comprise a
surface that consists essentially of moieties that do not
substantially activate complement. In a more preferred embodiment,
synthetic nanocarriers according to the invention that have a
minimum dimension of equal to or less than about 100 nm, preferably
equal to or less than 100 nm, do not comprise a surface that
activates complement or alternatively comprise a surface that
consists essentially of moieties that do not activate complement.
In embodiments, synthetic nanocarriers may possess an aspect ratio
greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or greater than
1:10.
[0071] "Systemic dose" means a dose of an adjuvant that provides a
particular systemic cytokine release, preferably a particular
systemic cytokine release profile. In some embodiments, the
particular systemic cytokine release, preferably a particular
systemic cytokine release profile, is in a human. In embodiments,
the compositions and methods provided herein (where at least a
portion of a dose of adjuvant is not coupled to any nanocarriers)
result in a particular systemic cytokine release profile in a
subject. The term "separately" is also used to mean adjuvant that
is not coupled to any synthetic nanocarriers. Additionally,
"systemic cytokine release profile" means a pattern of systemic
cytokine release, wherein the pattern comprises cytokine levels
measured for several different systemic cytokines. In some
embodiments, the particular systemic cytokine release profile
comprises the systemic release of TNF-.alpha., IL-6 and/or IL-12.
In other embodiments, the particular systemic cytokine release
profile comprises the systemic release of IFN-.gamma., IL12 and/or
IL-18.
[0072] "T cell antigen" means any antigen that is recognized by and
triggers an immune response in a T cell (e.g., an antigen that is
specifically recognized by a T cell receptor on a T cell or an NKT
cell via presentation of the antigen or portion thereof bound to a
Class I or Class II major histocompatability complex molecule
(MHC), or bound to a CD1 complex.) In some embodiments, an antigen
that is a T cell antigen is also a B cell antigen. In other
embodiments, the T cell antigen is not also a B cell antigen. T
cell antigens generally are proteins, polypeptides or peptides. T
cell antigens may be an antigen that stimulates a CD8+ T cell
response, a CD4+ T cell response, or both. The nanocarriers,
therefore, in some embodiments can effectively stimulate both types
of responses.
[0073] In some embodiments the T cell antigen is a `universal` T
cell antigen, or T cell memory antigen, (i.e., one to which a
subject has a pre-existing memory and that can be used to boost T
cell help to an unrelated antigen, for example an unrelated B cell
antigen). Universal T cell antigens include tetanus toxoid, as well
as one or more peptides derived from tetanus toxoid, Epstein-Barr
virus, or influenza virus. Universal T cell antigens also include a
components of influenza virus, such as hemagglutinin,
neuraminidase, or nuclear protein, or one or more peptides derived
therefrom. In some embodiments, the universal T cell antigen is not
one that is presented in a complex with a MHC molecule. In some
embodiments, the universal T cell antigen is not complexed with a
MHC molecule for presentation to a T helper cell. Accordingly, in
some embodiments, the universal T cell antigen is not a T helper
cell antigen. However, in other embodiments, the universal T cell
antigen is a T helper cell antigen.
[0074] In embodiments, a T-helper cell antigen may comprise one or
more peptides obtained or derived from tetanus toxoid, Epstein-Barr
virus, influenza virus, respiratory syncytial virus, measles virus,
mumps virus, rubella virus, cytomegalovirus, adenovirus, diphtheria
toxoid, or a PADRE peptide (known from the work of Sette et al.
U.S. Pat. No. 7,202,351). In other embodiments, a T-helper cell
antigen may comprise ovalbumin or a peptide obtained or derived
therefrom. Preferably, the ovalbumin comprises the amino acid
sequence as set forth in Accession No. AAB59956, NP.sub.--990483.1,
AAA48998, or CAA2371. In other embodiments, the peptide obtained or
derived from ovalbumin comprises the following amino acid sequence:
H-Ile-Ser-Gln-Ala-Val-His-Ala-Ala-His-Ala-Glu-Ile-Asn-Glu-Ala-Gly-Arg-OH
(SEQ ID NO: 1). In other embodiments, a T-helper cell antigen may
comprise one or more lipids, or glycolipids, including but not
limited to: .alpha.-galactosylceramide (.alpha.-GalCer),
.alpha.-linked glycosphingolipids (from Sphingomonas spp.),
galactosyl diacylglycerols (from Borrelia burgdorferi),
lypophosphoglycan (from Leishmania donovani), and
phosphatidylinositol tetramannoside (PIM4) (from Mycobacterium
leprae). For additional lipids and/or glycolipids useful as
T-helper cell antigen, see V. Cerundolo et al., "Harnessing
invariant NKT cells in vaccination strategies." Nature Rev Immun,
9:28-38 (2009).
[0075] In embodiments, CD4+ T-cell antigens may be derivatives of a
CD4+ T-cell antigen that is obtained from a source, such as a
natural source. In such embodiments, CD4+ T-cell antigen sequences,
such as those peptides that bind to MHC II, may have at least 70%,
80%, 90%, or 95% identity to the antigen obtained from the source.
In embodiments, the T cell antigen, preferably a universal T cell
antigen or T-helper cell antigen, may be coupled to, or uncoupled
from, a synthetic nanocarrier. In some embodiments, the universal T
cell antigen or T-helper cell antigen is encapsulated in the
synthetic nanocarriers of the inventive compositions.
[0076] "Th1 immune response" means any immune response that results
in the production of Th1 cells and Th1-associated cytokines,
IFN-.gamma., IL-12 and/or IL-18, or that counteracts the
differentiation of Th2 cells and the action of Th2 cytokines.
Methods for assessing whether a Th1 immune response is induced are
known to those of ordinary skill in the art. Examples of such
methods are provided below in the Examples.
[0077] "Time different from administration" or "a time different
from a time when the composition is administered" means a time more
than about 30 seconds either before or after administration,
preferably more than about 1 minute either before or after
administration, more preferably more than 5 minutes either before
or after administration, still more preferably more than 1 day
either before or after administration, still more preferably more
than 2 days either before or after administration, still more
preferably more than 1 week either before or after administration,
still more preferably more than 2 weeks either before or after
administration, still more preferably more than 3 weeks either
before or after administration, still more preferably more than 1
month either before or after administration, and still more
preferably more than 2 months either before or after
administration.
[0078] "Vaccine" means a composition of matter that improves the
immune response to a particular pathogen or disease. A vaccine
typically contains factors that stimulate a subject's immune system
to recognize a specific antigen as foreign and eliminate it from
the subject's body. A vaccine also establishes an immunologic
`memory` so the antigen will be quickly recognized and responded to
if a person is re-challenged. Vaccines can be prophylactic (for
example to prevent future infection by any pathogen), or
therapeutic (for example a vaccine against a tumor specific antigen
for the treatment of cancer or against an antigen derived from an
infectious agent for the treatment of an infection or infectious
disease). In embodiments, a vaccine may comprise dosage forms
according to the invention. Preferably, in some embodiments, these
vaccines comprise an adjuvant not coupled to any synthetic
nanocarriers.
[0079] In specific embodiments, the inventive compositions
incorporate adjuvants that comprise agonists for toll-like
receptors (TLRs) 7 & 8 ("TLR 7/8 agonists"). Of utility are the
TLR 7/8 agonist compounds disclosed in U.S. Pat. No. 6,696,076 to
Tomai et al., including but not limited to imidazoquinoline amines,
imidazopyridine amines, 6,7-fused cycloalkylimidazopyridine amines,
and 1,2-bridged imidazoquinoline amines. Preferred adjuvants
comprise imiquimod and R848.
[0080] In specific embodiments, the inventive compositons
incorporate adjuvants that comprise a ligand for Toll-like receptor
(TLR)-9, such as immunostimulatory DNA molecules comprising CpGs,
which induce type I interferon secretion, and stimulate T and B
cell activation leading to increased antibody production and
cytotoxic T cell responses (Krieg et al., CpG motifs in bacterial
DNA trigger direct B cell activation. Nature. 1995. 374:546-549;
Chu et al. CpG oligodeoxynucleotides act as adjuvants that switch
on T helper 1 (Th1) immunity. J. Exp. Med. 1997. 186:1623-1631;
Lipford et al. CpG-containing synthetic oligonucleotides promote B
and cytotoxic T cell responses to protein antigen: a new class of
vaccine adjuvants. Eur. J. Immunol. 1997. 27:2340-2344; Roman et
al. Immunostimulatory DNA sequences function as T
helper-1-promoting adjuvants. Nat. Med. 1997. 3:849-854; Davis et
al. CpG DNA is a potent enhancer of specific immunity in mice
immunized with recombinant hepatitis B surface antigen. J. Immunol.
1998. 160:870-876; Lipford et al., Bacterial DNA as immune cell
activator. Trends Microbiol. 1998. 6:496-500. In embodiments, CpGs
may comprise modifications intended to enhance stability, such as
phosphorothioate linkages, or other modifications, such as modified
bases. See, for example, U.S. Pat. Nos. 5,663,153, 6,194,388,
7,262,286, or 7,276,489. In certain embodiments, to stimulate
immunity rather than tolerance, a composition provided herein
incorporates an adjuvant that promotes DC maturation (needed for
priming of naive T cells) and the production of cytokines, such as
type I interferons, which promote antibody responses and anti-viral
immunity. In some embodiments, the adjuvant comprises a TLR-4
agonist, such as bacterial lipopolysacharide (LPS), VSV-G, and/or
HMGB-1. In some embodiments, adjuvants comprise cytokines, which
are small proteins or biological factors (in the range of 5 kD-20
kD) that are released by cells and have specific effects on
cell-cell interaction, communication and behavior of other cells.
In some embodiments, adjuvants comprise proinflammatory stimuli
released from necrotic cells (e.g., urate crystals). In some
embodiments, adjuvants comprise activated components of the
complement cascade (e.g., CD21, CD35, etc.). In some embodiments,
adjuvants comprise activated components of immune complexes. The
adjuvants also include those that comprise complement receptor
agonists, such as a molecule that binds to CD21 or CD35. In some
embodiments, the complement receptor agonist induces endogenous
complement opsonization of the nanocarrier. Adjuvants also include
those that comprise cytokine receptor agonists, such as a
cytokine.
[0081] In some embodiments, the cytokine receptor agonist is a
small molecule, antibody, fusion protein, or aptamer. In
embodiments, adjuvants also may comprise immunostimulatory RNA
molecules, such as but not limited to dsRNA or poly I:C (a TLR3
stimulant), and/or those disclosed in F. Heil et al.,
"Species-Specific Recognition of Single-Stranded RNA via Toll-like
Receptor 7 and 8" Science 303(5663), 1526-1529 (2004); J. Vollmer
et al., "Immune modulation by chemically modified ribonucleosides
and oligoribonucleotides" WO 2008033432 A2; A. Forsbach et al.,
"Immunostimulatory oligoribonucleotides containing specific
sequence motif(s) and targeting the Toll-like receptor 8 pathway"
WO 2007062107 A2; E. Uhlmann et al., "Modified oligoribonucleotide
analogs with enhanced immunostimulatory activity" U.S. Pat. Appl.
Publ. US 2006241076; G. Lipford et al., "Immunostimulatory viral
RNA oligonucleotides and use for treating cancer and infections" WO
2005097993 A2; G. Lipford et al., "Immunostimulatory G,U-containing
oligoribonucleotides, compositions, and screening methods" WO
2003086280 A2.
[0082] In some embodiments, the adjuvants comprise gel-type
adjuvants (e.g., aluminum hydroxide, aluminum phosphate, calcium
phosphate, etc.), microbial adjuvants (e.g., immunomodulatory DNA
sequences that include CpG motifs; immunostimulatory RNA molecules;
endotoxins such as monophosphoryl lipid A; exotoxins such as
cholera toxin, E. coli heat labile toxin, and pertussis toxin;
muramyl dipeptide, etc.); oil-emulsion and emulsifier-based
adjuvants (e.g., Freund's Adjuvant, MF59 [Novartis], SAF, etc.);
particulate adjuvants (e.g., liposomes, biodegradable microspheres,
saponins, etc.); synthetic adjuvants (e.g., nonionic block
copolymers, muramyl peptide analogues, polyphosphazene, synthetic
polynucleotides, etc.), and/or combinations thereof.
Synthetic Nanocarrier Compositions
[0083] A wide variety of synthetic nanocarriers can be used
according to the invention. In some embodiments, synthetic
nanocarriers are spheres or spheroids. In some embodiments,
synthetic nanocarriers are flat or plate-shaped. In some
embodiments, synthetic nanocarriers are cubes, cuboidal or cubic.
In some embodiments, synthetic nanocarriers are ovals or ellipses.
In some embodiments, synthetic nanocarriers are cylinders, cones,
or pyramids.
[0084] In some embodiments, it is desirable to use a population of
synthetic nanocarriers that is relatively uniform in terms of size,
shape, and/or composition so that each synthetic nanocarrier has
similar properties. For example, at least 80%, at least 90%, or at
least 95% of the synthetic nanocarriers, based on the total number
of synthetic nanocarriers, may have a minimum dimension or maximum
dimension that falls within 5%, 10%, or 20% of the average diameter
or average dimension of the synthetic nanocarriers. In some
embodiments, a population of synthetic nanocarriers may be
heterogeneous with respect to size, shape, and/or composition.
[0085] Synthetic nanocarriers can be solid or hollow and can
comprise one or more layers. In some embodiments, each layer has a
unique composition and unique properties relative to the other
layer(s). To give but one example, synthetic nanocarriers may have
a core/shell structure, wherein the core is one layer (e.g. a
polymeric core) and the shell is a second layer (e.g. a lipid
bilayer or monolayer). Synthetic nanocarriers may comprise a
plurality of different layers.
[0086] In some embodiments, synthetic nanocarriers may optionally
comprise one or more lipids. In some embodiments, a synthetic
nanocarrier may comprise a liposome. In some embodiments, a
synthetic nanocarrier may comprise a lipid bilayer. In some
embodiments, a synthetic nanocarrier may comprise a lipid
monolayer. In some embodiments, a synthetic nanocarrier may
comprise a micelle. In some embodiments, a synthetic nanocarrier
may comprise a core comprising a polymeric matrix surrounded by a
lipid layer (e.g., lipid bilayer, lipid monolayer, etc.). In some
embodiments, a synthetic nanocarrier may comprise a non-polymeric
core (e.g., metal particle, quantum dot, ceramic particle, bone
particle, viral particle, proteins, nucleic acids, carbohydrates,
etc.) surrounded by a lipid layer (e.g., lipid bilayer, lipid
monolayer, etc.).
[0087] In some embodiments, synthetic nanocarriers can comprise one
or more polymers or polymeric matrices. In some embodiments, such a
polymer or polymeric matrix can be surrounded by a coating layer
(e.g., liposome, lipid monolayer, micelle, etc.). In some
embodiments, various elements of the synthetic nanocarriers can be
coupled with the polymer or polymeric matrix.
[0088] In some embodiments, an element, such as a targeting moiety,
oligonucleotide, antigen, adjuvant, etc. can be covalently
associated with a polymeric matrix. In some embodiments, covalent
association is mediated by a linker. In some embodiments, an
element can be noncovalently associated with a polymeric matrix.
For example, in some embodiments, an element can be encapsulated
within, surrounded by, and/or dispersed throughout a polymeric
matrix. Alternatively or additionally, an element can be associated
with a polymeric matrix by hydrophobic interactions, charge
interactions, van der Waals forces, etc.
[0089] A wide variety of polymers and methods for forming polymeric
matrices therefrom are known conventionally. In general, a
polymeric matrix comprises one or more polymers. Polymers may be
natural or unnatural (synthetic) polymers. Polymers may be
homopolymers or copolymers comprising two or more monomers. In
terms of sequence, copolymers may be random, block, or comprise a
combination of random and block sequences. Typically, polymers in
accordance with the present invention are organic polymers.
[0090] Examples of polymers suitable for use in the present
invention include, but are not limited to polyethylenes,
polycarbonates (e.g. poly(1,3-dioxan-2one)), polyanhydrides (e.g.
poly(sebacic anhydride)), polypropylfumerates, polyamides (e.g.
polycaprolactam), polyacetals, polyethers, polyesters (e.g.,
polylactide, polyglycolide, polylactide-co-glycolide,
polycaprolactone, polyhydroxyacid (e.g.,
poly(.beta.-hydroxyalkanoate)), poly(orthoesters),
polycyanoacrylates, polyvinyl alcohols, polyurethanes,
polyphosphazenes, polyacrylates, polymethacrylates, polyureas,
polystyrenes, polyamines, polylysine, polylysine-PEG copolymers,
and poly(ethyleneimine), poly(ethylene imine)-PEG copolymers.
[0091] In some embodiments, polymers in accordance with the present
invention include polymers which have been approved for use in
humans by the U.S. Food and Drug Administration (FDA) under 21
C.F.R. .sctn.177.2600, including but not limited to polyesters
(e.g., polylactic acid, poly(lactic-co-glycolic acid),
polycaprolactone, polyvalerolactone, poly(1,3-dioxan-2one));
polyanhydrides (e.g., poly(sebacic anhydride)); polyethers (e.g.,
polyethylene glycol); polyurethanes; polymethacrylates;
polyacrylates; and polycyanoacrylates.
[0092] In some embodiments, polymers can be hydrophilic. For
example, polymers may comprise anionic groups (e.g., phosphate
group, sulphate group, carboxylate group); cationic groups (e.g.,
quaternary amine group); or polar groups (e.g., hydroxyl group,
thiol group, amine group). In some embodiments, a synthetic
nanocarrier comprising a hydrophilic polymeric matrix generates a
hydrophilic environment within the synthetic nanocarrier. In some
embodiments, polymers can be hydrophobic. In some embodiments, a
synthetic nanocarrier comprising a hydrophobic polymeric matrix
generates a hydrophobic environment within the synthetic
nanocarrier. Selection of the hydrophilicity or hydrophobicity of
the polymer may have an impact on the nature of materials that are
incorporated (e.g. coupled) within the synthetic nanocarrier.
[0093] In some embodiments, polymers may be modified with one or
more moieties and/or functional groups. A variety of moieties or
functional groups can be used in accordance with the present
invention. In some embodiments, polymers may be modified with
polyethylene glycol (PEG), with a carbohydrate, and/or with acyclic
polyacetals derived from polysaccharides (Papisov, 2001, ACS
Symposium Series, 786:301). Certain embodiments may be made using
the general teachings of U.S. Pat. No. 5,543,158 to Gref et al., or
WO publication WO2009/051837 by Von Andrian et al.
[0094] In some embodiments, polymers may be modified with a lipid
or fatty acid group. In some embodiments, a fatty acid group may be
one or more of butyric, caproic, caprylic, capric, lauric,
myristic, palmitic, stearic, arachidic, behenic, or lignoceric
acid. In some embodiments, a fatty acid group may be one or more of
palmitoleic, oleic, vaccenic, linoleic, alpha-linoleic,
gamma-linoleic, arachidonic, gadoleic, arachidonic,
eicosapentaenoic, docosahexaenoic, or erucic acid.
[0095] In some embodiments, polymers may be polyesters, including
copolymers comprising lactic acid and glycolic acid units, such as
poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide),
collectively referred to herein as "PLGA"; and homopolymers
comprising glycolic acid units, referred to herein as "PGA," and
lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid,
poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and
poly-D,L-lactide, collectively referred to herein as "PLA." In some
embodiments, exemplary polyesters include, for example,
polyhydroxyacids; PEG copolymers and copolymers of lactide and
glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers, PLGA-PEG
copolymers, and derivatives thereof. In some embodiments,
polyesters include, for example, poly(caprolactone),
poly(caprolactone)-PEG copolymers, poly(L-lactide-co-L-lysine),
poly(serine ester), poly(4-hydroxy-L-proline ester),
poly[.alpha.-(4-aminobutyl)-L-glycolic acid], and derivatives
thereof.
[0096] In some embodiments, a polymer may be PLGA. PLGA is a
biocompatible and biodegradable co-polymer of lactic acid and
glycolic acid, and various forms of PLGA are characterized by the
ratio of lactic acid:glycolic acid. Lactic acid can be L-lactic
acid, D-lactic acid, or D,L-lactic acid. The degradation rate of
PLGA can be adjusted by altering the lactic acid:glycolic acid
ratio. In some embodiments, PLGA to be used in accordance with the
present invention is characterized by a lactic acid:glycolic acid
ratio of approximately 85:15, approximately 75:25, approximately
60:40, approximately 50:50, approximately 40:60, approximately
25:75, or approximately 15:85.
[0097] In some embodiments, polymers may be one or more acrylic
polymers. In certain embodiments, acrylic polymers include, for
example, acrylic acid and methacrylic acid copolymers, methyl
methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl
methacrylate, aminoalkyl methacrylate copolymer, poly(acrylic
acid), poly(methacrylic acid), methacrylic acid alkylamide
copolymer, poly(methyl methacrylate), poly(methacrylic acid
anhydride), methyl methacrylate, polymethacrylate, poly(methyl
methacrylate) copolymer, polyacrylamide, aminoalkyl methacrylate
copolymer, glycidyl methacrylate copolymers, polycyanoacrylates,
and combinations comprising one or more of the foregoing polymers.
The acrylic polymer may comprise fully-polymerized copolymers of
acrylic and methacrylic acid esters with a low content of
quaternary ammonium groups.
[0098] In some embodiments, polymers can be cationic polymers. In
general, cationic polymers are able to condense and/or protect
negatively charged strands of nucleic acids (e.g. DNA, or
derivatives thereof). Amine-containing polymers such as
poly(lysine) (Zauner et al., 1998, Adv. Drug Del. Rev., 30:97; and
Kabanov et al., 1995, Bioconjugate Chem., 6:7), poly(ethylene
imine) (PEI; Boussif et al., 1995, Proc. Natl. Acad. Sci., USA,
1995, 92:7297), and poly(amidoamine) dendrimers (Kukowska-Latallo
et al., 1996, Proc. Natl. Acad. Sci., USA, 93:4897; Tang et al.,
1996, Bioconjugate Chem., 7:703; and Haensler et al., 1993,
Bioconjugate Chem., 4:372) are positively-charged at physiological
pH, form ion pairs with nucleic acids, and mediate transfection in
a variety of cell lines. In embodiments, the inventive synthetic
nanocarriers may not comprise (or may exclude) cationic
polymers.
[0099] In some embodiments, polymers can be degradable polyesters
bearing cationic side chains (Putnam et al., 1999, Macromolecules,
32:3658; Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Kwon
et al., 1989, Macromolecules, 22:3250; Lim et al., 1999, J. Am.
Chem. Soc., 121:5633; and Zhou et al., 1990, Macromolecules,
23:3399). Examples of these polyesters include
poly(L-lactide-co-L-lysine) (Barrera et al., 1993, J. Am. Chem.
Soc., 115:11010), poly(serine ester) (Zhou et al., 1990,
Macromolecules, 23:3399), poly(4-hydroxy-L-proline ester) (Putnam
et al., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am.
Chem. Soc., 121:5633), and poly(4-hydroxy-L-proline ester) (Putnam
et al., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am.
Chem. Soc., 121:5633).
[0100] The properties of these and other polymers and methods for
preparing them are well known in the art (see, for example, U.S.
Pat. Nos. 6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404;
6,095,148; 5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600;
5,399,665; 5,019,379; 5,010,167; 4,806,621; 4,638,045; and
4,946,929; Wang et al., 2001, J. Am. Chem. Soc., 123:9480; Lim et
al., 2001, J. Am. Chem. Soc., 123:2460; Langer, 2000, Acc. Chem.
Res., 33:94; Langer, 1999, J. Control. Release, 62:7; and Uhrich et
al., 1999, Chem. Rev., 99:3181). More generally, a variety of
methods for synthesizing certain suitable polymers are described in
Concise Encyclopedia of Polymer Science and Polymeric Amines and
Ammonium Salts, Ed. by Goethals, Pergamon Press, 1980; Principles
of Polymerization by Odian, John Wiley & Sons, Fourth Edition,
2004; Contemporary Polymer Chemistry by Allcock et al.,
Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and in
U.S. Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.
[0101] In some embodiments, polymers can be linear or branched
polymers. In some embodiments, polymers can be dendrimers. In some
embodiments, polymers can be substantially cross-linked to one
another. In some embodiments, polymers can be substantially free of
cross-links. In some embodiments, polymers can be used in
accordance with the present invention without undergoing a
cross-linking step. It is further to be understood that inventive
synthetic nanocarriers may comprise block copolymers, graft
copolymers, blends, mixtures, and/or adducts of any of the
foregoing and other polymers. Those skilled in the art will
recognize that the polymers listed herein represent an exemplary,
not comprehensive, list of polymers that can be of use in
accordance with the present invention.
[0102] In some embodiments, the synthetic nanocarriers comprise one
or more polymers. The polymeric synthetic nanocarriers, therefore,
can also include those described in WO publication WO2009/051837 by
Von Andrian et al., including, but not limited to those, with one
or more hydrophilic components. Preferably, the one or more
polymers comprise a polyester, such as a poly(lactic acid),
poly(glycolic acid), poly(lactic-co-glycolic acid), or
polycaprolactone. More preferably, the one or more polymers
comprise or further comprise a polyester coupled to a hydrophilic
polymer, such as a polyether. In embodiments, the polyether
comprises polyethylene glycol. Still more preferably, the one or
more polymers comprise a polyester and a polyester coupled to a
hydrophilic polymer, such as a polyether. In other embodiments, the
one or more polymers are coupled to one or more antigens and/or one
or more adjuvants. In embodiments, at least some of the polymers
are coupled to the antigen(s) and/or at least some of the polymers
are coupled to the adjuvant(s). Preferably, when there are more
than one type of polymer, one of the types of polymer is coupled to
the antigen(s). In embodiments, one of the other types of polymer
is coupled to the adjuvant(s). For example, in embodiments, when
the nanocarriers comprise a polyester and a polyester coupled to a
hydrophilic polymer, such as a polyether, the polyester is coupled
to the adjuvant, while the polyester coupled to the hydrophilic
polymer, such as a polyether, is coupled to the antigen(s). In
embodiments, where the nanocarriers comprise a T helper cell
antigen, the T helper cell antigen can be encapsulated in the
nanocarrier.
[0103] In some embodiments, synthetic nanocarriers may not comprise
a polymeric component. In some embodiments, synthetic nanocarriers
may comprise metal particles, quantum dots, ceramic particles, etc.
In some embodiments, a non-polymeric synthetic nanocarrier is an
aggregate of non-polymeric components, such as an aggregate of
metal atoms (e.g., gold atoms).
[0104] In some embodiments, synthetic nanocarriers may optionally
comprise one or more amphiphilic entities. In some embodiments, an
amphiphilic entity can promote the production of synthetic
nanocarriers with increased stability, improved uniformity, or
increased viscosity. In some embodiments, amphiphilic entities can
be associated with the interior surface of a lipid membrane (e.g.,
lipid bilayer, lipid monolayer, etc.). Many amphiphilic entities
known in the art are suitable for use in making synthetic
nanocarriers in accordance with the present invention. Such
amphiphilic entities include, but are not limited to,
phosphoglycerides; phosphatidylcholines; dipalmitoyl
phosphatidylcholine (DPPC); dioleylphosphatidyl ethanolamine
(DOPE); dioleyloxypropyltriethylammonium (DOTMA);
dioleoylphosphatidylcholine; cholesterol; cholesterol ester;
diacylglycerol; diacylglycerolsuccinate; diphosphatidyl glycerol
(DPPG); hexanedecanol; fatty alcohols such as polyethylene glycol
(PEG); polyoxyethylene-9-lauryl ether; a surface active fatty acid,
such as palmitic acid or oleic acid; fatty acids; fatty acid
monoglycerides; fatty acid diglycerides; fatty acid amides;
sorbitan trioleate (Span.RTM.85) glycocholate; sorbitan monolaurate
(Span.RTM.20); polysorbate 20 (Tween.RTM.20); polysorbate 60
(Tween.RTM.60); polysorbate 65 (Tween.RTM.65); polysorbate 80
(Tween.RTM.80); polysorbate 85 (Tween.RTM.85); polyoxyethylene
monostearate; surfactin; a poloxomer; a sorbitan fatty acid ester
such as sorbitan trioleate; lecithin; lysolecithin;
phosphatidylserine; phosphatidylinositol;sphingomyelin;
phosphatidylethanolamine(cephalin); cardiolipin; phosphatidic acid;
cerebrosides; dicetylphosphate; dipalmitoylphosphatidylglycerol;
stearylamine; dodecylamine; hexadecyl-amine; acetyl palmitate;
glycerol ricinoleate; hexadecyl sterate; isopropyl myristate;
tyloxapol; poly(ethylene glycol)5000-phosphatidylethanolamine;
poly(ethylene glycol)400-monostearate; phospholipids; synthetic
and/or natural detergents having high surfactant properties;
deoxycholates; cyclodextrins; chaotropic salts; ion pairing agents;
and combinations thereof. An amphiphilic entity component may be a
mixture of different amphiphilic entities. Those skilled in the art
will recognize that this is an exemplary, not comprehensive, list
of substances with surfactant activity. Any amphiphilic entity may
be used in the production of synthetic nanocarriers to be used in
accordance with the present invention.
[0105] In some embodiments, synthetic nanocarriers may optionally
comprise one or more carbohydrates. Carbohydrates may be natural or
synthetic. A carbohydrate may be a derivatized natural
carbohydrate. In certain embodiments, a carbohydrate comprises
monosaccharide or disaccharide, including but not limited to
glucose, fructose, galactose, ribose, lactose, sucrose, maltose,
trehalose, cellbiose, mannose, xylose, arabinose, glucoronic acid,
galactoronic acid, mannuronic acid, glucosamine, galatosamine, and
neuramic acid. In certain embodiments, a carbohydrate is a
polysaccharide, including but not limited to pullulan, cellulose,
microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC),
hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextran,
glycogen, starch, hydroxyethylstarch, carageenan, glycon, amylose,
chitosan, N,O-carboxylmethylchitosan, algin and alginic acid,
starch, chitin, heparin, inulin, konjac, glucommannan, pustulan,
heparin, hyaluronic acid, curdlan, and xanthan. In embodiments, the
inventive synthetic nanocarriers do not comprise (or specifically
exclude) carbohydrates, such as a polysaccharide. In certain
embodiments, the carbohydrate may comprise a carbohydrate
derivative such as a sugar alcohol, including but not limited to
mannitol, sorbitol, xylitol, erythritol, maltitol, and
lactitol.
[0106] Compositions according to the invention comprise inventive
synthetic nanocarriers in combination with pharmaceutically
acceptable excipients, such as preservatives, buffers, saline, or
phosphate buffered saline. The compositions may be made using
conventional pharmaceutical manufacturing and compounding
techniques to arrive at useful dosage forms. In an embodiment,
inventive synthetic nanocarriers are suspended in sterile saline
solution for injection together with a preservative.
[0107] In embodiments, when preparing synthetic nanocarriers as
carriers for agents (e.g., antigen or adjuvant) for use in vaccines
methods for coupling the agents to the synthetic nanocarriers may
be useful. If the agent is a small molecule it may be of advantage
to attach the agent to a polymer prior to the assembly of the
synthetic nanocarriers. In embodiments, it may also be an advantage
to prepare the synthetic nanocarriers with surface groups that are
used to couple the agent to the synthetic nanocarrier through the
use of these surface groups rather than attaching the agent to a
polymer and then using this polymer conjugate in the construction
of synthetic nanocarriers. A variety of reactions can be used for
the purpose of attaching agents to synthetic nanocarriers.
[0108] In certain embodiments, the coupling can be a covalent
linker. In embodiments, peptides according to the invention can be
covalently coupled to the external surface via a 1,2,3-triazole
linker formed by the 1,3-dipolar cycloaddition reaction of azido
groups on the surface of the nanocarrier with antigen or adjuvant
containing an alkyne group or by the 1,3-dipolar cycloaddition
reaction of alkynes on the surface of the nanocarrier with antigens
or adjuvants containing an azido group. Such cycloaddition
reactions are preferably performed in the presence of a Cu(I)
catalyst along with a suitable Cu(I)-ligand and a reducing agent to
reduce Cu(II) compound to catalytic active Cu(I) compound. This
Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) can also be
referred as the click reaction.
[0109] Additionally, the covalent coupling may comprise a covalent
linker that comprises an amide linker, a disulfide linker, a
thioether linker, a hydrazone linker, a hydrazide linker, an imine
or oxime linker, an urea or thiourea linker, an amidine linker, an
amine linker, and a sulfonamide linker.
[0110] An amide linker is formed via an amide bond between an amine
on one component such as the antigen or adjuvant with the
carboxylic acid group of a second component such as the
nanocarrier. The amide bond in the linker can be made using any of
the conventional amide bond forming reactions with suitably
protected amino acids or antigens or adjuvants and activated
carboxylic acid such N-hydroxysuccinimide-activated ester.
[0111] A disulfide linker is made via the formation of a disulfide
(S--S) bond between two sulfur atoms of the form, for instance, of
R.sub.1--S--S--R.sub.2. A disulfide bond can be formed by thiol
exchange of an antigen or adjuvant containing thiol/mercaptan
group(--SH) with another activated thiol group on a polymer or
nanocarrier or a nanocarrier containing thiol/mercaptan groups with
a antigen or adjuvant containing activated thiol group.
[0112] A triazole linker, specifically a 1,2,3-triazole of the
form
##STR00001##
wherein R.sub.1 and R.sub.2 may be any chemical entities, is made
by the 1,3-dipolar cycloaddition reaction of an azide attached to a
first component such as the nanocarrier with a terminal alkyne
attached to a second component such as the peptide. The 1,3-dipolar
cycloaddition reaction is performed with or without a catalyst,
preferably with Cu(I)-catalyst, which links the two components
through a 1,2,3-triazole function. This chemistry is described in
detail by Sharpless et al., Angew. Chem. Int. Ed. 41(14), 2596,
(2002) and Meldal, et al, Chem. Rev., 2008, 108(8), 2952-3015 and
is often referred to as a "click" reaction or CuAAC.
[0113] In embodiments, a polymer containing an azide or alkyne
group, terminal to the polymer chain is prepared. This polymer is
then used to prepare a synthetic nanocarrier in such a manner that
a plurality of the alkyne or azide groups are positioned on the
surface of that nanocarrier. Alternatively, the synthetic
nanocarrier can be prepared by another route, and subsequently
functionalized with alkyne or azide groups. The antigen or adjuvant
is prepared with the presence of either an alkyne (if the polymer
contains an azide) or an azide (if the polymer contains an alkyne)
group. The antigen or adjuvant is then allowed to react with the
nanocarrier via the 1,3-dipolar cycloaddition reaction with or
without a catalyst which covalently couples the antigen or adjuvant
to the particle through the 1,4-disubstituted 1,2,3-triazole
linker.
[0114] A thioether linker is made by the formation of a
sulfur-carbon (thioether) bond in the form, for instance, of
R.sub.1--S--R.sub.2. Thioether can be made by either alkylation of
a thiol/mercaptan (--SH) group on one component such as the antigen
or adjuvant with an alkylating group such as halide or epoxide on a
second component such as the nanocarrier. Thioether linkers can
also be formed by Michael addition of a thiol/mercaptan group on
one component such as a antigen or adjuvant to an
electron-deficient alkene group on a second component such as a
polymer containing a maleimide group or vinyl sulfone group as the
Michael acceptor. In another way, thioether linkers can be prepared
by the radical thiol-ene reaction of a thiol/mercaptan group on one
component such as a antigen or adjuvant with an alkene group on a
second component such as a polymer or nanocarrier.
[0115] A hydrazone linker is made by the reaction of a hydrazide
group on one component such as the antigen or adjuvant with an
aldehyde/ketone group on the second component such as the
nanocarrier.
[0116] A hydrazide linker is formed by the reaction of a hydrazine
group on one component such as the antigen or adjuvant with a
carboxylic acid group on the second component such as the
nanocarrier. Such reaction is generally performed using chemistry
similar to the formation of amide bond where the carboxylic acid is
activated with an activating reagent.
[0117] An imine or oxime linker is formed by the reaction of an
amine or N-alkoxyamine (or aminooxy) group on one component such as
the antigen or adjuvant with an aldehyde or ketone group on the
second component such as the nanocarrier.
[0118] An urea or thiourea linker is prepared by the reaction of an
amine group on one component such as the antigen or adjuvant with
an isocyanate or thioisocyanate group on the second component such
as the nanocarrier.
[0119] An amidine linker is prepared by the reaction of an amine
group on one component such as the antigen or adjuvant with an
imidoester group on the second component such as the
nanocarrier.
[0120] An amine linker is made by the alkylation reaction of an
amine group on one component such as the antigen or adjuvant with
an alkylating group such as halide, epoxide, or sulfonate ester
group on the second component such as the nanocarrier.
Alternatively, an amine linker can also be made by reductive
amination of an amine group on one component such as the antigen or
adjuvant with an aldehyde or ketone group on the second component
such as the nanocarrier with a suitable reducing reagent such as
sodium cyanoborohydride or sodium triacetoxyborohydride.
[0121] A sulfonamide linker is made by the reaction of an amine
group on one component such as the antigen or adjuvant with a
sulfonyl halide (such as sulfonyl chloride) group on the second
component such as the nanocarrier.
[0122] A sulfone linker is made by Michael addition of a
nucleophile to a vinyl sulfone. Either the vinyl sulfone or the
nucleophile may be on the surface of the nanoparticle or attached
to the antigen or adjuvant.
[0123] The antigen or adjuvant can also be conjugated to the
nanocarrier via non-covalent conjugation methods. For examples, a
negative charged antigen or adjuvant can be conjugated to a
positive charged nanocarrier through electrostatic adsorption. An
antigen or adjuvant containing a metal ligand can also be
conjugated to a nanocarrier containing a metal complex via a
metal-ligand complex.
[0124] In embodiments, the antigen or adjuvant can be attached to a
polymer, for example polylactic acid-block-polyethylene glycol,
prior to the assembly of the synthetic nanocarrier or the synthetic
nanocarrier can be formed with reactive or activatible groups on
its surface. In the latter case, the antigen or adjuvant is
prepared with a group which is compatible with the attachment
chemistry that is presented by the synthetic nanocarriers' surface.
In other embodiments, agents, such as a peptide antigen, can be
attached to VLPs or liposomes using a suitable linker. A linker is
a compound or reagent that capable of coupling two molecules
together. In an embodiment, the linker can be a homobifuntional or
heterobifunctional reagent as described in Hermanson 2008. For
example, an VLP or liposome synthetic nanocarrier containing a
carboxylic group on the surface can be treated with a
homobifunctional linker, adipic dihydrazide (ADH), in the presence
of EDC to form the corresponding synthetic nanocarrier with the ADH
linker. The resulting ADH linked synthetic nanocarrier is then
conjugated with an agent containing an acid group via the other end
of the ADH linker on the NC to produce the corresponding VLP or
liposome peptide conjugate.
[0125] For detailed descriptions of available conjugation methods,
see Hermanson G T "Bioconjugate Techniques", 2nd Edition Published
by Academic Press, Inc., 2008. In addition to covalent attachment
the antigen or adjuvant can be coupled by adsorbtion to a
pre-formed synthetic nanocarrier or it can be coupled by
encapsulation during the formation of the synthetic
nanocarrier.
Methods of Making and Using the Compositions and Related
Methods
[0126] Synthetic nanocarriers may be prepared using a wide variety
of methods known in the art. For example, synthetic nanocarriers
can be formed by methods as nanoprecipitation, flow focusing using
fluidic channels, spray drying, single and double emulsion solvent
evaporation, solvent extraction, phase separation, milling,
microemulsion procedures, microfabrication, nanofabrication,
sacrificial layers, simple and complex coacervation, and other
methods well known to those of ordinary skill in the art.
Alternatively or additionally, aqueous and organic solvent
syntheses for monodisperse semiconductor, conductive, magnetic,
organic, and other nanomaterials have been described (Pellegrino et
al., 2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci.,
30:545; and Trindade et al., 2001, Chem. Mat., 13:3843). Additional
methods have been described in the literature (see, e.g., Doubrow,
Ed., "Microcapsules and Nanoparticles in Medicine and Pharmacy,"
CRC Press, Boca Raton, 1992; Mathiowitz et al., 1987, J. Control.
Release, 5:13; Mathiowitz et al., 1987, Reactive Polymers, 6:275;
and Mathiowitz et al., 1988, J. Appl. Polymer Sci., 35:755, U.S.
Pat. Nos. 5,578,325 and 6,007,845; P. Paolicelli et al.,
"Surface-modified PLGA-based Nanoparticles that can Efficiently
Associate and Deliver Virus-like Particles" Nanomedicine.
5(6):843-853 (2010)).
[0127] Various materials may be encapsulated into synthetic
nanocarriers as desirable using a variety of methods including but
not limited to C. Astete et al., "Synthesis and characterization of
PLGA nanoparticles" J. Biomater. Sci. Polymer Edn, Vol. 17, No. 3,
pp. 247-289 (2006); K. Avgoustakis "Pegylated Poly(Lactide) and
Poly(Lactide-Co-Glycolide) Nanoparticles: Preparation, Properties
and Possible Applications in Drug Delivery" Current Drug Delivery
1:321-333 (2004); C. Reis et al., "Nanoencapsulation I. Methods for
preparation of drug-loaded polymeric nanoparticles" Nanomedicine
2:8-21 (2006); P. Paolicelli et al., "Surface-modified PLGA-based
Nanoparticles that can Efficiently Associate and Deliver Virus-like
Particles" Nanomedicine. 5(6):843-853 (2010). Other methods
suitable for encapsulating materials, such as oligonucleotides,
into synthetic nanocarriers may be used, including without
limitation methods disclosed in U.S. Pat. No. 6,632,671 to Unger
Oct. 14, 2003.
[0128] In certain embodiments, synthetic nanocarriers are prepared
by a nanoprecipitation process or spray drying. Conditions used in
preparing synthetic nanocarriers may be altered to yield particles
of a desired size or property (e.g., hydrophobicity,
hydrophilicity, external morphology, "stickiness," shape, etc.).
The method of preparing the synthetic nanocarriers and the
conditions (e.g., solvent, temperature, concentration, air flow
rate, etc.) used may depend on the materials to be coupled to the
synthetic nanocarriers and/or the composition of the polymer
matrix.
[0129] If particles prepared by any of the above methods have a
size range outside of the desired range, particles can be sized,
for example, using a sieve.
[0130] Elements of the inventive synthetic nanocarriers (such as
targeting moieties, polymeric matrices, antigens, adjuvants, and
the like), may be coupled to the synthetic nanocarrier, e.g., by
one or more covalent bonds, or may be coupled by means of one or
more linkers. Additional methods of functionalizing synthetic
nanocarriers may be adapted from Published U.S. Patent Application
2006/0002852 to Saltzman et al., Published U.S. Patent Application
2009/0028910 to DeSimone et al., or Published International Patent
Application WO/2008/127532 A1 to Murthy et al.
[0131] Alternatively or additionally, synthetic nanocarriers can be
coupled to an element, such as targeting moieties, adjuvants,
various antigens, etc., directly or indirectly via non-covalent
interactions. In non-covalent embodiments, the non-covalent
coupling is mediated by non-covalent interactions including but not
limited to charge interactions, affinity interactions, metal
coordination, physical adsorption, host-guest interactions,
hydrophobic interactions, TT stacking interactions, hydrogen
bonding interactions, van der Waals interactions, magnetic
interactions, electrostatic interactions, dipole-dipole
interactions, and/or combinations thereof. Such couplings may be
arranged to be on an external surface or an internal surface of an
inventive synthetic nanocarrier. In embodiments, encapsulation
and/or absorption is a form of coupling.
[0132] In embodiments, the inventive synthetic nanocarriers can be
combined with other adjuvants by admixing in the same vehicle or
delivery system. Such adjuvants may include, but are not limited to
mineral salts, such as alum, alum combined with monphosphoryl lipid
(MPL) A of Enterobacteria, such as Escherihia coli, Salmonella
minnesota, Salmonella typhimurium, or Shigella flexneri or
specifically with MPL.RTM. (AS04), MPL A of above-mentioned
bacteria separately, saponins, such as QS-21,Quil-A, ISCOMs,
ISCOMATRIX.TM., emulsions such as MF59.TM., Montanide.RTM. ISA 51
and ISA 720, AS02 (QS21+squalene+MPL.RTM.), AS15, liposomes and
liposomal formulations such as AS01, synthesized or specifically
prepared microparticles and microcarriers such as bacteria-derived
outer membrane vesicles (OMV) of N. gonorrheae, Chlamydia
trachomatis and others, or chitosan particles, depot-forming
agents, such as Pluronic.RTM. block co-polymers, specifically
modified or prepared peptides, such as muramyl dipeptide,
aminoalkyl glucosaminide 4-phosphates, such as RC529, or proteins,
such as bacterial toxoids or toxin fragments. Additional useful
adjuvants may be found in WO 2002/032450; U.S. Pat. No. 7,357,936
"Adjuvant Systems and Vaccines"; U.S. Pat. No. 7,147,862 "Vaccine
composition containing adjuvants"; U.S. Pat. No. 6,544,518
"Vaccines"; U.S. Pat. No. 5,750,110 "Vaccine composition containing
adjuvants." The doses of such other adjuvants can be determined
using conventional dose ranging studies. In embodiments, adjuvant
that is not coupled to the recited synthetic nanocarriers may be
the same or different from adjuvant that is coupled to the
synthetic nanocarriers, if any. In other embodiments, the doses of
such adjuvants may also be the same or different.
[0133] In embodiments, any adjuvant coupled to the inventive
synthetic nanocarriers can be different, similar or identical to
those not coupled to any nanocarriers. The adjuvants (coupled and
not coupled) can be administered separately at a different
time-point and/or at a different body location and/or by a
different immunization route. Additionally, the separate adjuvant
and population of nanocarriers can be administered separately at a
different time-point and/or at a different body location and/or by
a different immunization route.
[0134] Populations of synthetic nanocarriers may be combined to
form pharmaceutical dosage forms according to the present invention
using traditional pharmaceutical mixing methods. These include
liquid-liquid mixing in which two or more suspensions, each
containing one or more subset of nanocarriers, are directly
combined or are brought together via one or more vessels containing
diluent. As synthetic nanocarriers may also be produced or stored
in a powder form, dry powder-powder mixing could be performed as
could the re-suspension of two or more powders in a common media.
Depending on the properties of the nanocarriers and their
interaction potentials, there may be advantages conferred to one or
another route of mixing.
[0135] Typical inventive compositions that comprise synthetic
nanocarriers may comprise inorganic or organic buffers (e.g.,
sodium or potassium salts of phosphate, carbonate, acetate, or
citrate) and pH adjustment agents (e.g., hydrochloric acid, sodium
or potassium hydroxide, salts of citrate or acetate, amino acids
and their salts) antioxidants (e.g., ascorbic acid,
alpha-tocopherol), surfactants (e.g., polysorbate 20, polysorbate
80, polyoxyethylene 9-10 nonyl phenol, sodium desoxycholate),
solution and/or cryo/lyo stabilizers (e.g., sucrose, lactose,
mannitol, trehalose), osmotic adjustment agents (e.g., salts or
sugars), antibacterial agents (e.g., benzoic acid, phenol,
gentamicin), antifoaming agents (e.g., polydimethylsilozone),
preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymeric
stabilizers and viscosity-adjustment agents (e.g.,
polyvinylpyrrolidone, poloxamer 488, carboxymethylcellulose) and
co-solvents (e.g., glycerol, polyethylene glycol, ethanol).
[0136] Compositions according to the invention comprise inventive
synthetic nanocarriers in combination with pharmaceutically
acceptable excipients. The compositions may be made using
conventional pharmaceutical manufacturing and compounding
techniques to arrive at useful dosage forms. Techniques suitable
for use in practicing the present invention may be found in
Handbook of Industrial Mixing: Science and Practice, Edited by
Edward L. Paul, Victor A. Atiemo-Obeng, and Suzanne M. Kresta, 2004
John Wiley & Sons, Inc.; and Pharmaceutics: The Science of
Dosage Form Design, 2nd Ed. Edited by M. E. Auten, 2001, Churchill
Livingstone. In an embodiment, inventive synthetic nanocarriers are
suspended in sterile saline solution for injection together with a
preservative.
[0137] It is to be understood that the compositions of the
invention can be made in any suitable manner, and the invention is
in no way limited to compositions that can be produced using the
methods described herein. Selection of an appropriate method may
require attention to the properties of the particular moieties
being associated.
[0138] In some embodiments, inventive synthetic nanocarriers are
manufactured under sterile conditions or are terminally sterilized.
This can ensure that resulting composition are sterile and
non-infectious, thus improving safety when compared to non-sterile
compositions. This provides a valuable safety measure, especially
when subjects receiving synthetic nanocarriers have immune defects,
are suffering from infection, and/or are susceptible to infection.
In some embodiments, inventive synthetic nanocarriers may be
lyophilized and stored in suspension or as lyophilized powder
depending on the formulation strategy for extended periods without
losing activity.
[0139] The inventive compositions may be administered by a variety
of routes of administration, including but not limited to
subcutaneous, intramuscular, intradermal, oral, intranasal,
transmucosal, sublingual, rectal, ophthalmic, transdermal,
transcutaneous or by a combination of these routes.
[0140] Doses of dosage forms contain varying amounts of populations
of synthetic nanocarriers and/or varying amounts of adjuvants
and/or antigens, according to the invention. The amount of
synthetic nanocarriers and/or adjuvants and/or antigens present in
the inventive dosage forms can be varied according to the nature of
the adjuvants and/or antigens, the therapeutic benefit to be
accomplished, and other such parameters. In some embodiments, the
doses of the dosage forms are systemic doses. In embodiments, dose
ranging studies can be conducted to establish optimal therapeutic
amount of the population of synthetic nanocarriers and/or the
amount of adjuvants and/or antigens to be present in the dosage
form. In embodiments, the synthetic nanocarriers and/or the
adjuvants and/or antigens are present in the dosage form in an
amount effective to generate an immune response upon administration
to a subject. It may be possible to determine amounts of the
adjuvants and/or antigens effective to generate an immune response
using conventional dose ranging studies and techniques in subjects.
Inventive dosage forms may be administered at a variety of
frequencies. In a preferred embodiment, at least one administration
of the dosage form is sufficient to generate a pharmacologically
relevant response. In more preferred embodiment, at least two
administrations, at least three administrations, or at least four
administrations, of the dosage form are utilized to ensure a
pharmacologically relevant response.
[0141] The compositions and methods described herein can be used to
induce, enhance, stimulate, modulate, direct or redirect an immune
response. The compositions and methods described herein can be used
in the diagnosis, prophylaxis and/or treatment of conditions such
as cancers, infectious diseases, metabolic diseases, degenerative
diseases, autoimmune diseases, inflammatory diseases, immunological
diseases, or other disorders and/or conditions. The compositions
and methods described herein can also be used for the prophylaxis
or treatment of an addiction, such as an addiction to nicotine or a
narcotic. The compositions and methods described herein can also be
used for the prophylaxis and/or treatment of a condition resulting
from the exposure to a toxin, hazardous substance, environmental
toxin, or other harmful agent.
[0142] In embodiments, the compositions provided can be used to
induce a rapid and strong systemic induction of pro-inflammatory
cytokines, such as TNF-.alpha., IL-6 and/or IL-12. The compositions
provided, therefore, can be administered to subjects in need of an
inflammatory response, preferably a systemic inflammatory response.
In other embodiments, the compositions provided can be used for the
rapid and strong systemic induction of cytokines that are important
for a TH1 immune response, such as IFN-.gamma., IL-12 and/or IL-18.
The compositions provided, therefore, can be administered to
subjects in need of a TH1 response, preferably a systemic TH1
response. In still other embodiments, the compositions provided can
be used to induce a strong humoral response. The compositions
provided, therefore, can be administered to subjects in need of a
humoral response. In still further embodiments, the compositions
provided can be used to induce a strong specific local CTL
response. The compositions provided, therefore, can be administered
to subjects in need of a specific local CTL response. Such a
response can be specific to any of the antigens provided herein,
preferably to one or more antigens in an inventive composition or
that is administered according to an inventive method provided
herein.
[0143] The subjects provided herein can have or be at risk of
having cancer. Cancers include, but are not limited to, breast
cancer; biliary tract cancer; bladder cancer; brain cancer
including glioblastomas and medulloblastomas; cervical cancer;
choriocarcinoma; colon cancer; endometrial cancer; esophageal
cancer; gastric cancer; hematological neoplasms including acute
lymphocytic and myelogenous leukemia, e.g., B Cell CLL; T-cell
acute lymphoblastic leukemia/lymphoma; hairy cell leukemia; chronic
myelogenous leukemia, multiple myeloma; AIDS-associated leukemias
and adult T-cell leukemia/lymphoma; intraepithelial neoplasms
including Bowen's disease and Paget's disease; liver cancer; lung
cancer; lymphomas including Hodgkin's disease and lymphocytic
lymphomas; neuroblastomas; oral cancer including squamous cell
carcinoma; ovarian cancer including those arising from epithelial
cells, stromal cells, germ cells and mesenchymal cells; pancreatic
cancer; prostate cancer; rectal cancer; sarcomas including
leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and
osteosarcoma; skin cancer including melanoma, Merkel cell
carcinoma, Kaposi's sarcoma, basal cell carcinoma, and squamous
cell cancer; testicular cancer including germinal tumors such as
seminoma, non-seminoma (teratomas, choriocarcinomas), stromal
tumors, and germ cell tumors; thyroid cancer including thyroid
adenocarcinoma and medullar carcinoma; and renal cancer including
adenocarcinoma and Wilms tumor.
[0144] The subjects provided herein can have or be at risk of
having an infection or infectious disease. Infections or infectious
diseases include, but are not limited to, viral infectious
diseases, such as AIDS, Chickenpox (Varicella), Common cold,
Cytomegalovirus Infection, Colorado tick fever, Dengue fever, Ebola
hemorrhagic fever, Hand, foot and mouth disease, Hepatitis, Herpes
simplex, Herpes zoster, HPV, Influenza (Flu), Lassa fever, Measles,
Marburg hemorrhagic fever, Infectious mononucleosis, Mumps,
Norovirus, Poliomyelitis, Progressive multifocal
leukencephalopathy, Rabies, Rubella, SARS, Smallpox (Variola),
Viral encephalitis, Viral gastroenteritis, Viral meningitis, Viral
pneumonia, West Nile disease and Yellow fever; bacterial infectious
diseases, such as Anthrax, Bacterial Meningitis, Botulism,
Brucellosis, Campylobacteriosis, Cat Scratch Disease, Cholera,
Diphtheria, Epidemic Typhus, Gonorrhea, Impetigo, Legionellosis,
Leprosy (Hansen's Disease), Leptospirosis, Listeriosis, Lyme
disease, Melioidosis, Rheumatic Fever, MRSA infection, Nocardiosis,
Pertussis (Whooping Cough), Plague, Pneumococcal pneumonia,
Psittacosis, Q fever, Rocky Mountain Spotted Fever (RMSF),
Salmonellosis, Scarlet Fever, Shigellosis, Syphilis, Tetanus,
Trachoma, Tuberculosis, Tularemia, Typhoid Fever, Typhus and
Urinary Tract Infections; parasitic infectious diseases, such as
African trypanosomiasis, Amebiasis, Ascariasis, Babesiosis, Chagas
Disease, Clonorchiasis, Cryptosporidiosis, Cysticercosis,
Diphyllobothriasis, Dracunculiasis, Echinococcosis, Enterobiasis,
Fascioliasis, Fasciolopsiasis, Filariasis, Free-living amebic
infection, Giardiasis, Gnathostomiasis, Hymenolepiasis,
Isosporiasis, Kala-azar, Leishmaniasis, Malaria, Metagonimiasis,
Myiasis, Onchocerciasis, Pediculosis, Pinworm Infection, Scabies,
Schistosomiasis, Taeniasis, Toxocariasis, Toxoplasmosis,
Trichinellosis, Trichinosis, Trichuriasis, Trichomoniasis and
Trypanosomiasis; fungal infectious disease, such as Aspergillosis,
Blastomycosis, Candidiasis, Coccidioidomycosis, Cryptococcosis,
Histoplasmosis, Tinea pedis (Athlete's Foot) and Tinea cruris;
prion infectious diseases, such as Alpers' disease, Fatal Familial
Insomnia, Gerstmann-Straussler-Scheinker syndrome, Kuru and Variant
Creutzfeldt-Jakob disease.
[0145] Subject provided here also include those that have or are at
risk of having an atopic condition, such as but not limited to
allergy, allergic asthma, or atopic dermatitis; asthma; chronic
obstructive pulmonary disease (COPD, e.g. emphysema or chronic
bronchitis); and chronic infections due to chronic infectious
agents such as chronic leishmaniasis, candidiasis or
schistosomiasis and infections caused by plasmodia, Toxoplasma
gondii, mycobacteria, HIV, HBV, HCV EBV or CMV, or any one of the
above, or any subset of the above. Other indications treatable
using the inventive compositions include but are not limited to
indications in which a subject's TH1 response is suboptimal and/or
ineffective. Use of the present invention can enhance a subject's
TH1 immune response with an adjuvant that can stimulate a TH1
immune response. The subjects, therefore, also include those that
have or are at risk of having cancer, subjects with compromised or
suboptimal immunity, such as infants, the elderly, cancer patients,
individuals receiving immunosuppressive drugs or irradiation,
hemodialysis patients and those with genetic or idiopathic immune
dysfunction.
EXAMPLES
Example 1
Administration of Nanocarrier and Admixed R848 Adjuvant Results in
Strong Systemic Production of Inflammatory Cytokines
Materials for NC-R848-1 Nanocarrier Formulations
[0146] Ovalbumin peptide 323-339 amide acetate salt, was purchased
from Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif.
90505. Part #4065609.) PLGA-R848 conjugate of 75/25
lactide/glycolide monomer composition and of approximately 4100 Da
molecular weight having 5.2% w/w R848 content was synthesized.
PLA-PEG-Nicotine with a nicotine-terminated PEG block of
approximately 3,500 Da and DL-PLA block of approximately 15,000 Da
was synthesized Polyvinyl alcohol (Mw=11,000-31,000, 87-89%
hydrolyzed) was purchased from J.T. Baker (Part Number
U232-08).
Methods for NC-R848-1 Nanocarrier Production
[0147] Solutions were prepared as follows:
[0148] Solution 1: Ovalbumin peptide 323-339 @ 70 mg/mL was
prepared in 0.13N hydrochloric acid at room temperature.
[0149] Solution 2: PLGA-R848 @ 75 mg/mL and PLA-PEG-Nicotine @ 25
mg/mL in dichloromethane was prepared by dissolving PLGA-R848 at
100 mg/mL in dichloromethane and PLA-PEG-Nicotine at 100 mg/mL in
dichloromethane, then combining 3 parts of the PLGA-R848 solution
to 1 part of the PLA-PEG-Nicotine solution.
[0150] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM
phosphate buffer, pH 8.
[0151] Solution 4: 70 mM phosphate buffer, pH 8.
[0152] A primary (W1/O) emulsion was first created using Solution 1
& Solution 2. Solution 1 (0.1 mL) and Solution 2 (1.0 mL) were
combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. A
secondary (W1/O/W2) emulsion was then formed by adding Solution 3
(2.0 mL) to the primary emulsion and sonicating at 30% amplitude
for 40 seconds using the Branson Digital Sonifier 250. The
secondary emulsion was added to an open 50 mL beaker containing 70
mM phosphate buffer solution (30 mL) and stirred at room
temperature for 2 hours to allow the dichloromethane to evaporate
and the nanocarriers to form in suspension. A portion of the
suspended nanocarriers was washed by transferring the nanocarrier
suspension to a centrifuge tube, spinning at 13800 rcf for 60
minutes, removing the supernatant, and re-suspending the pellet in
phosphate buffered saline. This washing procedure was repeated, and
then the pellet was re-suspended in phosphate buffered saline to
achieve a nanocarrier suspension having a nominal concentration of
10 mg/mL on a polymer basis. The suspension was stored frozen at
-20.degree. C. until use.
TABLE-US-00001 TABLE 1 Characterization of the Nanocarriers
Produced According to the Above Nanocarrier Effective TLR Agonist,
T-cell helper peptide, ID Diameter (nm) % w/w % w/w NC-R848-1 220
R848, 1.3 Ova 323-339, 2.0
Materials for NC-R848-2 Formulations
[0153] Ovalbumin peptide 323-339 amide acetate salt was purchased
from Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif.
90505. Part #4065609.) PLGA-R848 conjugate of 75/25
lactide/glycolide monomer composition and of approximately 4100 Da
molecular weight having 5.2% w/w R848 content was synthesized.
PLA-PEG-Nicotine with a nicotine-terminated PEG block of
approximately 5,000 Da and DL-PLA block of approximately 17,000 Da
was synthesized. Polyvinyl alcohol (Mw=11,000-31,000, 87-89%
hydrolyzed) was purchased from J.T. Baker (Part Number
U232-08).
Methods for NC-R848-2 Nanocarrier Production
[0154] Solutions were prepared as follows:
[0155] Solution 1: Ovalbumin peptide 323-339 @ 70 mg/mL was
prepared in 0.13N hydrochloric acid at room temperature.
[0156] Solution 2: PLGA-R848 @ 75 mg/mL and PLA-PEG-Nicotine @ 25
mg/mL in dichloromethane was prepared by dissolving PLGA-R848 at
100 mg/mL in dichloromethane and PLA-PEG-Nicotine at 100 mg/mL in
dichloromethane, then combining 3 parts of the PLGA-R848 solution
to 1 part of the PLA-PEG-Nicotine solution.
[0157] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100mM
phosphate buffer, pH 8.
[0158] Solution 4: 70 mM phosphate buffer, pH 8.
[0159] A primary W1/O) emulsion was first created using Solution 1
& Solution 2. Solution 1 (0.1 mL) and Solution 2 (1.0 mL) were
combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. A
secondary (W1/O/W2) emulsion was then formed by adding Solution 3
(2.0 mL) to the primary emulsion and sonicating at 30% amplitude
for 40 seconds using the Branson Digital Sonifier 250. The
secondary emulsion was added to an open 50 mL beaker containing 70
mM phosphate buffer solution (30 mL) and stirred at room
temperature for 2 hours to allow the dichloromethane to evaporate
and the nanocarriers to form in suspension. A portion of the
suspended nanocarriers was washed by transferring the nanocarrier
suspension to a centrifuge tube, spinning at 13800 rcf for 60
minutes, removing the supernatant, and re-suspending the pellet in
phosphate buffered saline. This washing procedure was repeated, and
then the pellet was re-suspended in phosphate buffered saline to
achieve a nanocarrier suspension having a nominal concentration of
10 mg/mL on a polymer basis. The suspension was stored frozen at
-20.degree. C. until use.
TABLE-US-00002 TABLE 2 Characterization of the Nanocarriers
Produced According to the Above Effective TLR Agonist, T-cell
helper peptide, Nanocarrier ID Diameter (nm) % w/w % w/w NC-R848-2
229 R848, 3.3 Ova 323-339, 1.6
Materials for NC Only Formulations
[0160] Ovalbumin peptide 323-339 amide acetate salt, was purchased
from Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif.
90505. Part #4065609.) PLGA with 73% lactide and 27% glycolide
content and an inherent viscosity of 0.12 dL/g was purchased from
SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala.
35211. Product Code 7525 DLG 1A.) PLA-PEG-Nicotine with a
nicotine-terminated PEG block of approximately 3,500 Da and DL-PLA
block of approximately 15,000 Da was synthesized. Polyvinyl alcohol
(Mw=11,000-31,000, 87-89% hydrolyzed) was purchased from J.T. Baker
(Part Number U232-08).
Methods for NC Only Production
[0161] Solutions were prepared as follows:
[0162] Solution 1: Ovalbumin peptide 323-339 @ 70 mg/mL was
prepared in 0.13N hydrochloric acid at room temperature.
[0163] Solution 2: PLGA @ 75 mg/mL and PLA-PEG-Nicotine @ 25 mg/mL
in dichloromethane was prepared by dissolving PLGA at 100 mg/mL in
dichloromethane and PLA-PEG-Nicotine at 100 mg/mL in
dichloromethane, then combining 3 parts of the PLGA solution to 1
part of the PLA-PEG-Nicotine solution.
[0164] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100mM
phosphate buffer, pH 8.
[0165] Solution 4: 70 mM phosphate buffer, pH 8.
[0166] A primary W1/O) emulsion was first created using Solution 1
& Solution 2. Solution 1 (0.1 mL) and Solution 2 (1.0 mL) were
combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. A
secondary (W1/O/W2) emulsion was then formed by adding Solution 3
(2.0 mL) to the primary emulsion and sonicating at 30% amplitude
for 40 seconds using the Branson Digital Sonifier 250. The
secondary emulsion was added to an open 50 mL beaker containing 70
mM phosphate buffer solution (30 mL) and stirred at room
temperature for 2 hours to allow the dichloromethane to evaporate
and the nanocarriers to form in suspension. A portion of the
suspended nanocarriers was washed by transferring the nanocarrier
suspension to a centrifuge tube, spinning at 13800 rcf for 60
minutes, removing the supernatant, and re-suspending the pellet in
phosphate buffered saline. This washing procedure was repeated, and
then the pellet was re-suspended in phosphate buffered saline to
achieve a nanocarrier suspension having a nominal concentration of
10 mg/mL on a polymer basis. The suspension was stored frozen at
-20.degree. C. until use.
TABLE-US-00003 TABLE 3 Characterization of the Nanocarriers
Produced According to the Above Effective TLR Agonist, T-cell
helper peptide, Nanocarrier Diameter (nm) % w/w % w/w NC only 176
None 1.1
Results
[0167] Groups of mice were injected subcutaneously into hind limbs
with 100 .mu.g of nanocarriers (NC) coupled, non-coupled or admixed
with small molecule nucleoside analogue and known TLR7/8 agonist
and adjuvant, R848. R848 amounts in nanocarrier were 2-3% resulting
in 2-3 .mu.g of coupled R848 per injection; amount of free R848
used was 20 .mu.g per injection. Mouse serum was taken by terminal
bleed and systemic cytokine production in serum was measured at
different time-points by ELISA (BD Biosciences). As seen in FIGS.
1A-1C, strong systemic production of major pro-inflammatory
cytokines TNF-.alpha., IL-6 and IL-12 was observed when admixed
R848 (NC+R848) was used, while no expression of TNF-.alpha., IL-6
and IL-12 was detected when two separate preparations of NC coupled
with R848 (NC-R848-1 and NC-R848-2) were used. The difference in
peak cytokine expression levels was >100-fold for TNF-.alpha.
and IL-6, and >50-fold for IL-12. NC not coupled to R848
(labeled as NC only) did not induce any systemic cytokines when
used without admixed R848.
Example 2
Coupling of Nanocarrier to R848 Adjuvant does not Inhibit Systemic
Production of Immune Cytokine IFN-.gamma.
Materials for NC-R848 Nanocarrier Formulations
[0168] Ovalbumin peptide 323-339 amide acetate salt was purchased
from Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif.
90505. Part #4065609.) PLGA-R848 conjugate of 75/25
lactide/glycolide monomer composition and of approximately 4100 Da
molecular weight having 5.2% w/w R848 content was synthesized.
PLA-PEG-Nicotine with a nicotine-terminated PEG block of
approximately 3,500 Da and DL-PLA block of approximately 15,000 Da
was synthesized. Polyvinyl alcohol (Mw=11,000-31,000, 87-89%
hydrolyzed) purchased from J.T. Baker (Part Number U232-08).
Methods for NC-R848 Nanocarrier Production
[0169] Solutions were prepared as follows:
[0170] Solution 1: Ovalbumin peptide 323-339 @ 70 mg/mL was
prepared in 0.13N hydrochloric acid at room temperature.
[0171] Solution 2: PLGA-R848 @ 75 mg/mL and PLA-PEG-Nicotine @ 25
mg/mL in dichloromethane was prepared by dissolving PLGA-R848 at
100 mg/mL in dichloromethane and PLA-PEG-Nicotine at 100 mg/mL in
dichloromethane, then combining 3 parts of the PLGA-R848 solution
to 1 part of the PLA-PEG-Nicotine solution.
[0172] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100mM
phosphate buffer, pH 8.
[0173] Solution 4: 70 mM phosphate buffer, pH 8.
[0174] A primary W1/O) emulsion was first created using Solution 1
& Solution 2. Solution 1 (0.1 mL) and Solution 2 (1.0 mL) were
combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. A
secondary (W1/O/W2) emulsion was then formed by adding Solution 3
(2.0 mL) to the primary emulsion and sonicating at 30% amplitude
for 40 seconds using the Branson Digital Sonifier 250. The
secondary emulsion was added to an open 50 mL beaker containing 70
mM phosphate buffer solution (30 mL) and stirred at room
temperature for 2 hours to allow for the dichloromethane to
evaporate and for the nanocarriers to form in suspension. A portion
of the suspended nanocarriers was washed by transferring the
nanocarrier suspension to a centrifuge tube, spinning at 13800 rcf
for 60 minutes, removing the supernatant, and re-suspending the
pellet in phosphate buffered saline. This washing procedure was
repeated, and then the pellet was re-suspended in phosphate
buffered saline to achieve a nanocarrier suspension having a
nominal concentration of 10 mg/mL on a polymer basis. The
suspension was stored frozen at -20.degree. C. until use.
TABLE-US-00004 TABLE 4 Characterization of the Nanocarriers
Produced According to the Above Effective TLR Agonist, T-cell
helper peptide, Nanocarrier ID Diameter (nm) % w/w % w/w NC-R848
213 R848, 2.6 Ova 323-339, 0.9
Materials for NC Only Nanocarrier Formulations
[0175] Ovalbumin peptide 323-339 amide acetate salt was purchased
from Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif.
90505. Part #4065609.) PLGA with 73% lactide and 27% glycolide
content and an inherent viscosity of 0.12 dL/g was purchased from
SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala.
35211. Product Code 7525 DLG 1A.) PLA-PEG-Nicotine with a
nicotine-terminated PEG block of approximately 3,500 Da and DL-PLA
block of approximately 15,000 Da was synthesized. Polyvinyl alcohol
(Mw=11,000-31,000, 87-89% hydrolyzed) was purchased from J.T. Baker
(Part Number U232-08).
Methods for NC Only Nanocarrier Production
[0176] Solutions were prepared as follows:
[0177] Solution 1: Ovalbumin peptide 323-339 @ 70 mg/mL was
prepared in 0.13N hydrochloric acid at room temperature.
[0178] Solution 2: PLGA @ 75 mg/mL and PLA-PEG-Nicotine @ 25 mg/mL
in dichloromethane was prepared by dissolving PLGA at 100 mg/mL in
dichloromethane and PLA-PEG-Nicotine at 100 mg/mL in
dichloromethane, then combining 3 parts of the PLGA solution to 1
part of the PLA-PEG-Nicotine solution.
[0179] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM
phosphate buffer, pH 8.
[0180] Solution 4: 70 mM phosphate buffer, pH 8.
[0181] A primary W1/O) emulsion was first created using Solution 1
& Solution 2. Solution 1 (0.1 mL) and Solution 2 (1.0 mL) were
combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. A
secondary (W1/O/W2) emulsion was then formed by adding Solution 3
(2.0 mL) to the primary emulsion and sonicating at 30% amplitude
for 40 seconds using the Branson Digital Sonifier 250. The
secondary emulsion was added to an open 50 mL beaker containing 70
mM phosphate buffer solution (30 mL) and stirred at room
temperature for 2 hours to allow the dichloromethane to evaporate
and the nanocarriers to form in suspension. A portion of the
suspended nanocarriers was washed by transferring the nanocarrier
suspension to a centrifuge tube, spinning at 13800 rcf for 60
minutes, removing the supernatant, and re-suspending the pellet in
phosphate buffered saline. This washing procedure was repeated, and
then the pellet was re-suspended in phosphate buffered saline to
achieve a nanocarrier suspension having a nominal concentration of
10 mg/mL on a polymer basis. The suspension was stored frozen at
-20.degree. C. until use.
TABLE-US-00005 TABLE 5 Characterization of the Nanocarriers
Produced According to the Above Effective TLR Agonist, T-cell
helper peptide, Nanocarrier Diameter (nm) % w/w % w/w NC Only 176
None Ova 323-339, 1.1
Results
[0182] While early proinflammatory cytokines are mostly associated
with side effects during immunization, the production of other
cytokines, such as immune IFN-.gamma. is known to be involved in
the induction of effective immune response. Therefore, systemic
production of immune cytokine IFN-.gamma. after injection with NC
was measured 0-24 hours after inoculation. Briefly, groups of mice
were injected subcutaneously into hind limbs with 100 .mu.g of NCs
coupled or admixed with small molecule nucleoside analogue and
known TLR7/8 agonist and adjuvant, R848. R848 amounts in
nanocarrier were 2% resulting in 2 .mu.g of coupled R848 per
injection; amount of free R848 used was 20 .mu.g per injection.
Mouse serum was taken by terminal bleed and systemic cytokine
production in serum was measured at different time-points by ELISA
(BD Biosciences). IFN-.gamma., which is important for TH1 immune
response, was seen with both NC-R848 (containing 2 .mu.g of R848)
and NC with admixed R848 (20 .mu.g) (FIG. 2). Furthermore, higher
levels of IFN-.gamma. by NC with admixed R848 occurred earlier.
Example 3
Addition of Free Adjuvant Augments Immune Response
Materials for NC-Nic w/o R848 Nanocarrier Formulations
[0183] Ovalbumin peptide 323-339 amide TFA salt was purchased from
Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif. 90505.
Part #4064565.) PLA with an inherent viscosity of 0.19 dL/g was
purchased from Boehringer Ingelheim (Ingelheim Germany. Product
Code R202H). PLA-PEG-Nicotine with a nicotine-terminated PEG block
of approximately 3,500 Da and DL-PLA block of approximately 15,000
Da was synthesized. Polyvinyl alcohol (Mw=11,000-31,000, 87-89%
hydrolyzed) was purchased from J.T. Baker (Part Number
U232-08).
Methods for NC-Nic w/o R848 Nanocarrier Production
[0184] Solutions were prepared as follows:
[0185] Solution 1: Ovalbumin peptide 323-339 @ 69 mg/mL was
prepared in 0.13N hydrochloric acid at room temperature.
[0186] Solution 2: PLA @ 75 mg/mL and PLA-PEG-Nicotine @ 25 mg/mL
in dichloromethane was prepared by dissolving PLA @ 100 mg/mL in
dichloromethane and PLA-PEG-Nicotine at 100 mg/mL in
dichloromethane, then combining 3 parts of the PLA solution to 1
part of the PLA-PEG-Nicotine solution.
[0187] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in
deionized water.
[0188] Solution 4: 70 mM phosphate buffer, pH 8.
[0189] A primary W1/O) emulsion was first created using Solution 1
& Solution 2. Solution 1 (0.1 mL) and Solution 2 (1.0 mL) were
combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. A
secondary (W1/O/W2) emulsion was then formed by adding Solution 3
(2.0 mL) to the primary emulsion and sonicating at 35% amplitude
for 40 seconds using the Branson Digital Sonifier 250. The
secondary emulsion was added to a beaker containing 70 mM phosphate
buffer solution (30 mL) in an open 50 ml beaker and stirred at room
temperature for 2 hours to allow for the dichloromethane to
evaporate and for the nanocarriers to form in suspension. A portion
of the suspended nanocarriers were washed by transferring the
nanocarrier suspension to centrifuge tubes, spinning at 5300 rcf
for 60 minutes, removing the supernatant, and re-suspending the
pellet in phosphate buffered saline. This washing procedure was
repeated, and then the pellet was re-suspended in phosphate
buffered saline to achieve nanocarrier suspension having a nominal
concentration of 10 mg/mL on a polymer basis. The suspension was
stored frozen at -20.degree. C. until use.
TABLE-US-00006 TABLE 6 Characterization of the Nanocarriers
Produced According to the Above Effective TLR Agonist, T-cell
helper peptide, Nanocarrier Diameter (nm) % w/w % w/w NC-Nic w/o
248 None Ova 323-339, 2.2 R848
Materials for NC-Nic w/Entrapped R848 Nanocarrier Formulations
[0190] Ovalbumin peptide 323-339 amide TFA salt was purchased from
Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif. 90505.
Part #4064565.) R848 (Resiquimod) of approximately 98-99% purity
was synthesized and purified. PLA with an inherent viscosity of
0.19 dL/g was purchased from Boehringer Ingelheim (Ingelheim
Germany. Product Code R202H). PLA-PEG-Nicotine with a
nicotine-terminated PEG block of approximately 3,500 Da and DL-PLA
block of approximately 15,000 Da was synthesized. Polyvinyl alcohol
(Mw=11,000-31,000, 87-89% hydrolyzed) was purchased from J.T. Baker
(Part Number U232-08).
Methods for NC-Nic w/Entrapped R848 Nanocarrier Production
[0191] Solutions were prepared as follows:
[0192] Solution 1: Ovalbumin peptide 323-339 @ 69 mg/mL was
prepared in 0.13N hydrochloric acid at room temperature.
[0193] Solution 2: PLA @ 75 mg/mL, R848 @ 7.5 mg/mL, and
PLA-PEG-Nicotine @ 25 mg/mL in dichloromethane was prepared by
dissolving PLA @ 100 mg/mL in dicholoromethane and adding R848 at
10 mg/mL, also dissolving PLA-PEG-Nicotine at 100 mg/mL in
dichloromethane, then combining 3 parts of the PLA/R848 solution to
1 part of the PLA-PEG-Nicotine.
[0194] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in
deionized water.
[0195] Solution 4: 70 mM phosphate buffer, pH 8.
[0196] A primary W1/O) emulsion was first created using Solution 1
& Solution 2. Solution 1 (0.1 mL) and Solution 2 (1.0 mL) were
combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. A
secondary (W1/O/W2) emulsion was then formed by adding Solution 3
(2.0 mL) to the primary emulsion and sonicating at 35% amplitude
for 40 seconds using the Branson Digital Sonifier 250. The
secondary emulsion was added to a beaker containing 70 mM phosphate
buffer solution (30 mL) in an open 50 ml beaker and stirred at room
temperature for 2 hours to allow for the dichloromethane to
evaporate and for the nanocarriers to form in suspension. A portion
of the suspended nanocarriers were washed by transferring the
nanocarrier suspension to centrifuge tubes, spinning at 5300 rcf
for 60 minutes, removing the supernatant, and re-suspending the
pellet in phosphate buffered saline. This washing procedure was
repeated, and then the pellet was re-suspended in phosphate
buffered saline to achieve nanocarrier suspension having a nominal
concentration of 10 mg/mL on a polymer basis. The suspension was
stored frozen at -20.degree. C. until use.
TABLE-US-00007 TABLE 7 Characterization of the Nanocarriers
Produced According to the Above Effective TLR Agonist, T-cell
helper peptide, Nanocarrier ID Diameter (nm) % w/w % w/w NC-Nic w/
207 R848, 0.8 Ova 323-339, 1.6 Entrapped R848
Results
[0197] Mice were immunized with NC-Nic (nanocarrier exhibiting
nicotine on the outer surface) carrying entrapped (non-conjugated)
R848 with or without second adjuvant. Groups of five mice were
immunized three times (subcutaneously, hind limbs) at 2-week
intervals (days 0, 14 and 28) with 100 .mu.g of NC-Nic. Serum
anti-nicotine antibodies were then measured on days 26 and 40.
EC.sub.50 for anti-nicotine antibodies were measured by standard
ELISA against polylysine-nicotine (FIG. 3) (Group 1: NC-Nic w/o
entrapped R848; group 2: NC-Nic w. 1.5% of entrapped R848; group 3:
NC-Nic w. 1.5% of entrapped R848+80 .mu.g of alum; group 4: NC-Nic
w. 1.5% of entrapped R84+25 .mu.g of CpG-1826). This demonstrates
that utilization of entrapped R848 (Th1 adjuvant, TLR7/8 agonist)
within the nanocarriers (NC) generates an immune response, which is
superior to one induced by NC without R848 (group 2>group 1).
Moreover, addition of free Th2 adjuvant (alum) to NC-Nic with R848
further augments humoral immune response (group 3>group 2). At
the same time, addition of another free TH1 adjuvant (CpG, TLR9
agonist; group 4) also augments immune response, but is less potent
in this combination than alum (group 4>group 2 versus 4<group
3).
Example 4
Addition of Free Adjuvant Augments Immune Response to NC without
Adjuvant
Materials for NC-Nic Nanocarrier Formulations
[0198] Ovalbumin peptide 323-339 amide TFA salt, was purchased from
Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif. 90505.
Part #4064565.) PLA with an inherent viscosity of 0.19 dL/g was
purchased from Boehringer Ingelheim (Ingelheim Germany. Product
Code R202H). PLA-PEG-Nicotine with a nicotine-terminated PEG block
of approximately 3,500 Da and DL-PLA block of approximately 15,000
Da was synthesized. Polyvinyl alcohol (Mw=11,000-31,000, 87-89%
hydrolyzed) was purchased from J.T. Baker (Part Number
U232-08).
Methods for NC-Nic Nanocarrier Production
[0199] Solutions were prepared as follows:
[0200] Solution 1: Ovalbumin peptide 323-339 @ 69 mg/mL was
prepared in 0.13N hydrochloric acid at room temperature.
[0201] Solution 2: PLA @ 75 mg/mL and PLA-PEG-Nicotine @ 25 mg/mL
in dichloromethane was prepared by dissolving PLA @ 100 mg/mL in
dichloromethane and PLA-PEG-Nicotine at 100 mg/mL in
dichloromethane, then combining 3 parts of the PLA solution to 1
part of the PLA-PEG-Nicotine solution.
[0202] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in
deionized water.
[0203] Solution 4: 70 mM phosphate buffer, pH 8.
[0204] A primary W1/O) emulsion was first created using Solution 1
& Solution 2. Solution 1 (0.1 mL) and Solution 2 (1.0 mL) were
combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. A
secondary (W1/O/W2) emulsion was then formed by adding Solution 3
(2.0 mL) to the primary emulsion and sonicating at 35% amplitude
for 40 seconds using the Branson Digital Sonifier 250. The
secondary emulsion was added to a beaker containing 70 mM phosphate
buffer solution (30 mL) in an open 50m1 beaker and stirred at room
temperature for 2 hours to allow for the dichloromethane to
evaporate and for the nanocarriers to form in suspension. A portion
of the suspended nanocarriers were washed by transferring the
nanocarrier suspension to centrifuge tubes, spinning at 5300 rcf
for 60 minutes, removing the supernatant, and re-suspending the
pellet in phosphate buffered saline. This washing procedure was
repeated, and then the pellet was re-suspended in phosphate
buffered saline to achieve nanocarrier suspension having a nominal
concentration of 10 mg/mL on a polymer basis. The suspension was
stored frozen at -20.degree. C. until use.
TABLE-US-00008 TABLE 8 Characterization of the Nanocarriers
Produced According to the Above Effective TLR Agonist, T-cell
helper peptide, Nanocarrier ID Diameter (nm) % w/w % w/w NC-Nic 248
None Ova 323-339, 2.2
Materials for NC-Nic-R848 Nanocarrier Formulations
[0205] Ovalbumin peptide 323-339 amide TFA salt, was purchased from
Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif. 90505.
Part #4064565.) R848 (Resiquimod) of approximately 98-99% purity
was synthesized and purified. PLA with an inherent viscosity of
0.19 dL/g was purchased from Boehringer Ingelheim (Ingelheim
Germany. Product Code R202H). PLA-PEG-Nicotine with a
nicotine-terminated PEG block of approximately 3,500 Da and DL-PLA
block of approximately 15,000 Da was synthesized. Polyvinyl alcohol
(Mw=11,000-31,000, 87-89% hydrolyzed) was purchased from J.T. Baker
(Part Number U232-08).
Methods for NC-Nic-R848 Nanocarrier Production
[0206] Solutions were prepared as follows:
[0207] Solution 1: Ovalbumin peptide 323-339 @ 69 mg/mL was
prepared in 0.13N hydrochloric acid at room temperature.
[0208] Solution 2: PLA @ 75 mg/mL, R848 @ 7.5 mg/mL, and
PLA-PEG-Nicotine @ 25 mg/mL in dichloromethane was prepared by
dissolving PLA @ 100 mg/mL in dicholoromethane and adding R848 at
10 mg/mL, also dissolving PLA-PEG-Nicotine at 100 mg/mL in
dichloromethane, then combining 3 parts of the PLA/R848 solution to
1 part of the PLA-PEG-Nicotine.
[0209] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in
deionized water.
[0210] Solution 4: 70 mM phosphate buffer, pH 8
[0211] A primary W1/O) emulsion was first created using Solution 1
& Solution 2. Solution 1 (0.1 mL) and Solution 2 (1.0 mL) were
combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. A
secondary (W1/O/W2) emulsion was then formed by adding Solution 3
(2.0 mL) to the primary emulsion and sonicating at 35% amplitude
for 40 seconds using the Branson Digital Sonifier 250. The
secondary emulsion was added to a beaker containing 70 mM phosphate
buffer solution (30 mL) in an open 50 ml beaker and stirred at room
temperature for 2 hours to allow for the dichloromethane to
evaporate and for the nanocarriers to form in suspension.
[0212] A portion of the suspended nanocarriers were washed by
transferring the nanocarrier suspension to centrifuge tubes,
spinning at 5300 rcf for 60 minutes, removing the supernatant, and
re-suspending the pellet in phosphate buffered saline. This washing
procedure was repeated, and then the pellet was re-suspended in
phosphate buffered saline to achieve nanocarrier suspension having
a nominal concentration of 10 mg/mL on a polymer basis. The
suspension was stored frozen at -20.degree. C. until use.
TABLE-US-00009 TABLE 9 Characterization of the Nanocarriers
Produced According to the Above Effective TLR Agonist, T-cell
helper peptide, Nanocarrier ID Diameter (nm) % w/w % w/w
NC-Nic-R848 207 R848, 0.8 Ova 323-339, 1.6
Results
[0213] Mice were immunized with NC-Nic (nanocarrier exhibiting
nicotine on the outer surface) that did not have adjuvant in the NC
with or without admixed R848. Groups of five mice were immunized
three times (subcutaneously, hind limbs) at 2-week intervals (days
0, 14 and 28) with 100 .mu.g of NC-Nic. Serum anti-nicotine
antibodies were then measured on days 26 and 40. EC.sub.50 for
anti-nicotine antibodies were measured by standard ELISA against
polylysine-nicotine (FIG. 4) (group 1: NC-Nic w/o entrapped R848;
group 2: NC-Nic w/o entrapped R848+20 .mu.g of free R848). This
demonstrates that admixing of free R848 (Th1 adjuvant, TLR7/8
agonist) to antigen-carrying NCs generates immune response, which
is superior to one induced by NC without admixed R848 (group
2>group 1).
[0214] Similarly, the immune response to NC-carried antigen in mice
immunized with NC-Nic carrying encapsulated R848 adjuvant
(NC-Nic-R848) admixed with free TH1 adjuvant CpG-1826 (Enzo) or Th2
adjuvant alum (Pierce) was augmented. Groups of five mice were
immunized three times (s.c., hind limbs) at 2-week intervals (days
0, 14 and 28) with 100 .mu.g of NC-Nic-R848. Serum anti-nicotine
antibodies were then measured on days 26 and 40. EC.sub.50 for
anti-nicotine antibodies as measured in standard ELISA against
polylysine-nicotine (FIG. 5) (group 1: NC-Nic-R848; group 2:
NC-NicR848+80 .mu.g of free alum; group 3: NC-NicR848+25 .mu.g of
free CpG-1826). This demonstrates that admixing of a free adjuvant
to antigen/adjuvant-carrying NCs generates immune response, which
is superior to one induced by the same NC without admixed adjuvant
(group 2>group 1; group 3>group 1).
Example 5
Addition of Free Adjuvant Augments Immune Response to NC Containing
Encapsulated Adjuvant
Materials for Nanocarrier Formulations
[0215] Ovalbumin peptide 323-339 amide acetate salt, was purchased
from Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif.
90505. Part #4065609.) R848 (Resiquimod) of approximately 98-99%
purity was synthesized and purified. PLA-R848 conjugate having
molecular weight of approximately 1300 Da and R848 content of
approximately 9% by weight was synthesized at by a ring-opening
process. PLA-PEG-Nicotine with a nicotine-terminated PEG block of
approximately 3,500 Da and DL-PLA block of approximately 15,000 Da
was synthesized. Polyvinyl alcohol (Mw=11,000-31,000, 87-89%
hydrolyzed) was purchased from J.T. Baker (Part Number
U232-08).
Methods for Nanocarrier Production
[0216] Solutions were prepared as follows:
[0217] Solution 1: Ovalbumin peptide 323-339 @ 70 mg/mL was
prepared in 0.13N hydrochloric acid at room temperature.
[0218] Solution 2: PLA-R848 @ 75 mg/mL, PLA-PEG-Nicotine @ 25
mg/mL, and R848 @ 1.9 mg/mL in dichloromethane was prepared by
dissolving the polymers at 100 mg/mL, adding the R848 to the
PLA-PEG-Nicotine solution, and then combining 3 parts of the
PLA-R848 solution to 1 part of the PLA-PEG-Nicotine/R848
solution.
[0219] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in
deionized water.
[0220] Solution 4: 70 mM phosphate buffer, pH 8.
[0221] A primary W1/O) emulsion was first created using Solution 1
& Solution 2. Solution 1 (0.1 mL) and Solution 2 (1.0 mL) were
combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. A
secondary (W1/O/W2) emulsion was then formed by adding Solution 3
(2.0 mL) to the primary emulsion and sonicating at 35% amplitude
for 40 seconds using the Branson Digital Sonifier 250. The
secondary emulsion was added to a beaker containing 70 mM phosphate
buffer solution (30 mL) in an open 50 ml beaker and stirred at room
temperature for 2 hours to allow for the dichloromethane to
evaporate and for the nanocarriers to form in suspension. A portion
of the suspended nanocarriers were washed by transferring the
nanocarrier suspension to a centrifuge tube, spinning at 13,800 rcf
for 60 minutes, removing the supernatant, and re-suspending the
pellet in phosphate buffered saline. This washing procedure was
repeated, and then the pellet was re-suspended in phosphate
buffered saline to achieve nanocarrier suspension having a nominal
concentration of 10 mg/mL on a polymer basis. The suspension was
stored frozen at -20.degree. C. until use.
TABLE-US-00010 TABLE 10 Characterization of the Nanocarriers
Produced According to the Above Effective TLR Agonist, T-cell
helper peptide, Nanocarrier Diameter (nm) % w/w % w/w 231 R848, 3.3
Ova 323-339, 1.5
Results
[0222] Mice were immunized with the nanocarriers, NC-Nic
(nanocarrier exhibiting nicotine on the outer surface) which
carried R848 and OP-II helper peptide, with or without admixed
alum. Groups of five mice were immunized three times
(subcutaneously, hind limbs) at 2-week intervals (days 0, 14 and
28) with 100 .mu.g of NC[Nic,R848,OP-II]+80 .mu.g of admixed alum
(Pierce). Serum anti-nicotine antibodies were then measured on days
40 and 70. EC.sub.50 for anti-nicotine antibodies were measured by
standard ELISA against polylysine-nicotine (FIG. 6) (Group 1:
NC[Nic,R848,OP-II]; group 2: NC[Nic,R848,OP-II]+80 .mu.g of admixed
alum). This demonstrates that admixing of free alum (Th2 adjuvant)
to antigen-carrying adjuvant-containing NCs generates immune
response, which is superior to one induced by the same NC without
admixed alum (group 2>group 1).
Example 6
NC-encapsulated Antigen Generates a Stronger Cellular Immune
Response than Free Antigen (Free Adjuvant Admixed)
Materials for Nanocarrier Formulations
[0223] Ovalbumin protein, was purchased from Worthington
Biochemical Corporation (730 Vassar Avenue, Lakewood, N.J. 08701.
Product Code 3048.) PLA with an inherent viscosity of 0.21 dL/g was
purchased from SurModics Pharmaceuticals (756 Tom Martin Drive,
Birmingham, Ala. 35211. Product Code 100 DL 2A.) PLA-PEG-OMe block
co-polymer with a methyl ether terminated PEG block of
approximately 2,000 Da and PLA block of approximately 19,000 Da was
synthesized. Polyvinyl alcohol (Mw=11,000-31,000, 87-89%
hydrolyzed) was purchased from J.T. Baker (Part Number
U232-08).
Methods for Nanocarrier Production
[0224] Solutions were prepared as follows:
[0225] Solution 1: Ovalbumin protein @ 20 mg/mL was prepared in
phosphate buffered saline at room temperature.
[0226] Solution 2: PLA @ 75 mg/mL and PLA-PEG-OMe @ 25 mg/mL in
dichloromethane was prepared by dissolving PLA at 100 mg/mL in
dichloromethane and PLA-PEG-OMe at 100 mg/mL in dichloromethane,
then combining 3 parts of the PLA solution to 1 part of the
PLA-PEG-OMe solution.
[0227] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM
phosphate buffer, pH 8.
[0228] Solution 4: 70 mM phosphate buffer, pH 8.
[0229] A primary W1/O) emulsion was first created using Solution 1
& Solution 2. Solution 1 (0.2 mL) and Solution 2 (1.0 mL) were
combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. A
secondary (W1/O/W2) emulsion was then formed by adding Solution 3
(3.0 mL) to the primary emulsion, vortexing to create a course
dispersion, and then sonicating at 30% amplitude for 60 seconds
using the Branson Digital Sonifier 250. The secondary emulsion was
added to an open 50 mL beaker containing 70 mM phosphate buffer
solution (30 mL) and stirred at room temperature for 2 hours to
allow the dichloromethane to evaporate and the nanocarriers to form
in suspension. A portion of the suspended nanocarriers was washed
by transferring the nanocarrier suspension to a centrifuge tube,
spinning at 21,000 rcf for 45 minutes, removing the supernatant,
and re-suspending the pellet in phosphate buffered saline. This
washing procedure was repeated, and then the pellet was
re-suspended in phosphate buffered saline to achieve a nanocarrier
suspension having a nominal concentration of 10 mg/mL on a polymer
basis. The suspension was stored frozen at -20.degree. C. until
use.
TABLE-US-00011 TABLE 11 Characterization of the Nanocarriers
Produced According to the Above Effective Nanocarrier Diameter (nm)
TLR Agonist, % w/w Antigen, % w/w 228 None OVA protein, 2.8
Results
[0230] Mice were immunized either with the nanocarriers, NC-OVA
(nanocarrier carrying encapsulated ovalbumin protein), or with free
ovalbumin (OVA) with a free adjuvant admixed. Groups of 3 mice were
immunized once (s.c., hind limbs) with 100 .mu.g of NC-OVA (2.8%
OVA) or with 2.5 .mu.g of free OVA admixed with 10 .mu.g of free
1826-CpG (TLR9 agonist). Draining popliteal lymph nodes were taken
at day 4 after immunization, meshed, incubated in vitro for 4 days
in complete RPMI medium supplemented with 10 units/ml of IL-2, and
specific CTL (cytotoxic T cell) activity was determined (as % of
lysis of ovalbumin-expressing cell line EG.7-OVA-% of lysis of its
parental cell line EL-4) at different effector/target (E:T) ratios
(FIG. 7). This demonstrates that utilization of NC-encapsulated
antigen results in the generation of a stronger local cellular
immune response than immunization with a free antigen.
Example 7
Addition of Free Adjuvant Augments Immune Response to NC without
Adjuvant
Materials for Nanocarrier Formulations
[0231] Ovalbumin peptide 323-339 amide acetate salt was purchased
from Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif.
90505. Product code #4065609.) PLA with an inherent viscosity of
0.19 dL/g was purchased from Boehringer Ingelheim (Ingelheim
Germany. Product Code R202H). PLA-PEG-Nicotine with a
nicotine-terminated PEG block of approximately 5,000 Da and DL-PLA
block of approximately 17,000 Da was synthesized. Polyvinyl alcohol
(Mw=11,000-31,000, 87-89% hydrolyzed) was purchased from J.T. Baker
(Part Number U232-08).
Methods for Nanocarrier Production
[0232] Solutions were prepared as follows:
[0233] Solution 1: Ovalbumin peptide 323-339 @ 17.5 mg/mL in dilute
hydrochloric acid aqueous solution. The solution was prepared by
dissolving ovalbumin peptide in 0.13N hydrochloric acid solution at
room temperature.
[0234] Solution 2: 0.19-IV PLA @ 75 mg/mL and PLA-PEG-nicotine @ 25
mg/ml in dichloromethane. The solution was prepared by separately
dissolving PLA @ 100 mg/mL in dichloromethane and PLA-PEG-nicotine
@ 100 mg/mL in dichloromethane, then mixing the solutions by adding
3 parts PLA solution for each part of PLA-PEG-nicotine
solution.
[0235] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8
phosphate buffer.
[0236] A primary W1/O) emulsion was first created using Solution 1
& Solution 2. Solution 1 (0.1 mL) and Solution 2 (1.0 mL) were
combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. A
secondary (W1/O/W2) emulsion was then formed by adding Solution 3
(2.0 mL) to the primary emulsion and then sonicating at 30%
amplitude for 40 seconds using the Branson Digital Sonifier 250.
The secondary emulsion was then added to an open 50 mL beaker
containing 70 mM pH 8 phosphate buffer solution (30 mL) and stirred
at room temperature for 2 hours to evaporate dichloromethane and to
form nanocarriers in aqueous suspension. A portion of the
nanocarriers was washed by transferring the suspension to a
centrifuge tube and spinning at 13,800 g for one hour, removing the
supernatant, and re-suspending the pellet in phosphate buffered
saline. The washing procedure was repeated, and the pellet was
re-suspended in phosphate buffered saline for a final nanocarrier
dispersion of about 10 mg/mL. The amounts of oligonucleotide and
peptide in the nanocarrier were determined by HPLC analysis. The
total dry-nanocarrier mass per mL of suspension was determined by a
gravimetric method and was adjusted to 5 mg/mL.
TABLE-US-00012 TABLE 12 Characterization of the Nanocarriers
Produced According to the Above Effective TLR Agonist, T-cell
helper peptide, Nanocarrier Diameter (nm) % w/w % w/w 211 None Ova
323-339, 0.7
Results
[0237] Mice were immunized with NC-Nic (nanocarrier exhibiting
nicotine on the outer surface and containing OP-II helper peptide,
no adjuvant in the NC) admixed with CpG in either the
phosphodiester (PO) or phosphorothioate (PS) form. The PO form is
degraded by nucleases and, therefore, is not stable once injected
into mice. The PS form is nuclease-resistant and, therefore, stable
once injected into mice. As a negative control, mice were immunized
with PBS only. Groups of five mice were immunized three times
(subcutaneously, hind limbs) at 2-week intervals (days 0, 14 and
28) with 100 .mu.g of NC-Nic+20 .mu.g of CpG (PS or PO) or PBS.
Serum anti-nicotine antibody titers were measured on days 26 and
40. Anti-nicotine antibody titers (EC.sub.50) were measured by
ELISA against polylysine-nicotine (FIG. 8) (group 1: NC-Nic (no
adjuvant)+free CpG (PS); group 2: NC-Nic (no adjuvant)+free CpG
(PO); group 3: PBS only). This demonstrates that admixing of free
CpG (PS) (Th1 adjuvant, TLR9 agonist) to antigen-carrying NCs
generates an immune response which is superior to those induced by
NC with admixed CpG (PO) or with PBS (group 1>group 2>group
3).
Example 8
Addition of Free Adjuvant Augments Immune Response to NC without
Adjuvant
Materials for Nanocarrier Formulations
[0238] Ovalbumin protein was purchased from Worthington Biochemical
Corporation (730 Vassar Avenue, Lakewood, N.J. 08701. Product Code
3048.) PLA with an inherent viscosity of 0.21 dL/g was purchased
from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham,
Ala. 35211. Product Code 100 DL 2A.) PLA-PEG-OMe block co-polymer
with a methyl ether terminated PEG block of approximately 2,000 Da
and PLA block of approximately 19,000 Da was synthesized. Polyvinyl
alcohol (Mw=11,000-31,000, 87-89% hydrolyzed) was purchased from
J.T. Baker (Part Number U232-08).
Methods for Nanocarrier Production
[0239] Solutions were prepared as follows:
[0240] Solution 1: Ovalbumin protein @ 20mg/mL was prepared in
phosphate buffered saline at room temperature.
[0241] Solution 2: PLA @ 75 mg/mL and PLA-PEG-OMe @ 25 mg/mL in
dichloromethane was prepared by dissolving PLA at 100 mg/mL in
dichloromethane and PLA-PEG-OMe at 100 mg/mL in dichloromethane,
then combining 3 parts of the PLA solution to 1 part of the
PLA-PEG-OMe solution.
[0242] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM
phosphate buffer, pH 8.
[0243] Solution 4: 70 mM phosphate buffer, pH 8.
[0244] A primary W1/O) emulsion was first created using Solution 1
& Solution 2. Solution 1 (0.2 mL) and Solution 2 (1.0 mL) were
combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. A
secondary (W1/O/W2) emulsion was then formed by adding Solution 3
(3.0 mL) to the primary emulsion, vortexing to create a course
dispersion, and then sonicating at 30% amplitude for 60 seconds
using the Branson Digital Sonifier 250. The secondary emulsion was
added to an open 50 mL beaker containing 70 mM phosphate buffer
solution (30 mL) and stirred at room temperature for 2 hours to
allow the dichloromethane to evaporate and the nanocarriers to form
in suspension. A portion of the suspended nanocarriers was washed
by transferring the nanocarrier suspension to a centrifuge tube,
spinning at 21,000 rcf for 45 minutes, removing the supernatant,
and re-suspending the pellet in phosphate buffered saline. This
washing procedure was repeated, and then the pellet was
re-suspended in phosphate buffered saline to achieve a nanocarrier
suspension having a nominal concentration of 10 mg/mL on a polymer
basis. The suspension was stored frozen at -20.degree. C. until
use.
TABLE-US-00013 TABLE 13 Characterization of the Nanocarriers
Produced According to the Above Effective Nanocarrier Diameter (nm)
TLR Agonist, % w/w Antigen, % w/w NC-OVA 228 None OVA protein,
2.8
Results
[0245] Mice were immunized with NC-OVA (nanocarrier exhibiting
ovalbumin (OVA) on the outer surface, no adjuvant in the NC)
admixed with either 20 .mu.g of R848 or CpG (PS;
nuclease-resistant). Control mice received 2.5 .mu.g of soluble
antigen (OVA) admixed with 20 .mu.g of CpG (PS). Groups of five
mice were immunized three times (subcutaneously, hind limbs) at
2-week intervals (days 0, 14 and 28) with 100 .mu.g of NC-OVA+20
.mu.g of R848 CpG (PS) or 2.5 .mu.g of soluble OVA+20 .mu.g of CpG
(PS). Serum anti-OVA antibody titers were measured on days 26 and
44. Anti-OVA antibody titers (EC.sub.50) were measured by ELISA
against OVA protein (FIG. 9) (group 1: NC-OVA (no adjuvant)+free
R848; group 2: NC-OVA (no adjuvant)+free CpG (PS); group 3: soluble
OVA+CpG (PS)). This demonstrates that admixing of free R848 (Th1
adjuvant, TLR7/8 agonist) or CpG (PS) (Th1 adjuvant, TLR9 agonist)
to antigen-carrying NCs generates an immune response, which is
superior to those induced by soluble antigen admixed with adjuvant
(CpG (PS)) (groups 1 and 2>group 3).
Example 9
Addition of Free Adjuvant Augments Immune Response to NC with
Adjuvant
Materials for Group 1 Nanocarrier Formulations
[0246] Ovalbumin peptide 323-339 amide acetate salt was purchased
from Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif.
90505. Part #4065609.) PLGA-R848 conjugate of 75/25
lactide/glycolide monomer composition and of approximately 4100 Da
molecular weight having 5.2% w/w R848 content was synthesized.
PLA-PEG-Nicotine with a nicotine-terminated PEG block of
approximately 3,500 Da and DL-PLA block of approximately 15,000 Da
was synthesized. Polyvinyl alcohol (Mw=11,000-31,000, 87-89%
hydrolyzed) was purchased from J.T. Baker (Part Number
U232-08).
Methods for Group 1 Nanocarrier Production
[0247] Solutions were prepared as follows:
[0248] Solution 1: Ovalbumin peptide 323-339 @ 70 mg/mL was
prepared in 0.13N hydrochloric acid at room temperature.
[0249] Solution 2: PLGA-R848 @ 75 mg/mL and PLA-PEG-Nicotine @ 25
mg/mL in dichloromethane was prepared by dissolving PLGA-R848 at
100 mg/mL in dichloromethane and PLA-PEG-Nicotine at 100 mg/mL in
dichloromethane, then combining 3 parts of the PLGA-R848 solution
to 1 part of the PLA-PEG-Nicotine solution.
[0250] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM
phosphate buffer, pH 8.
[0251] Solution 4: 70 mM phosphate buffer, pH 8.
[0252] A primary W1/O) emulsion was first created using Solution 1
& Solution 2. Solution 1 (0.1 mL) and Solution 2 (1.0 mL) were
combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. A
secondary (W1/O/W2) emulsion was then formed by adding Solution 3
(2.0 mL) to the primary emulsion and sonicating at 30% amplitude
for 40 seconds using the Branson Digital Sonifier 250. The
secondary emulsion was added to an open 50 mL beaker containing 70
mM phosphate buffer solution (30 mL) and stirred at room
temperature for 2 hours to allow for the dichloromethane to
evaporate and for the nanocarriers to form in suspension. A portion
of the suspended nanocarriers was washed by transferring the
nanocarrier suspension to a centrifuge tube, spinning at 13800 rcf
for 60 minutes, removing the supernatant, and re-suspending the
pellet in phosphate buffered saline. This washing procedure was
repeated, and then the pellet was re-suspended in phosphate
buffered saline to achieve a nanocarrier suspension having a
nominal concentration of 10 mg/mL on a polymer basis. The
suspension was stored frozen at -20.degree. C. until use.
TABLE-US-00014 TABLE 14 Characterization of the Nanocarriers
Produced According to the Above Effective TLR Agonist, T-cell
helper peptide, Nanocarrier Diameter (nm) % w/w % w/w Group 1 NC
213 R848, 2.6 Ova 323-339, 0.9
Materials for Group 2 Nanocarrier Formulations
[0253] Ovalbumin peptide 323-339 amide acetate salt was purchased
from Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif.
90505. Part #4065609.) PLGA with 73% lactide and 27% glycolide
content and an inherent viscosity of 0.12 dL/g was purchased from
SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala.
35211. Product Code 7525 DLG 1A.) PLA-PEG-Nicotine with a
nicotine-terminated PEG block of approximately 3,500 Da and DL-PLA
block of approximately 15,000 Da was synthesized. Polyvinyl alcohol
(Mw=11,000-31,000, 87-89% hydrolyzed) was purchased from J.T. Baker
(Part Number U232-08).
Methods for Group 2 Nanocarrier Production
[0254] Solutions were prepared as follows:
[0255] Solution 1: Ovalbumin peptide 323-339 @ 70 mg/mL was
prepared in 0.13N hydrochloric acid at room temperature.
[0256] Solution 2: PLGA @ 75 mg/mL and PLA-PEG-Nicotine @ 25 mg/mL
in dichloromethane was prepared by dissolving PLGA at 100 mg/mL in
dichloromethane and PLA-PEG-Nicotine at 100 mg/mL in
dichloromethane, then combining 3 parts of the PLGA solution to 1
part of the PLA-PEG-Nicotine solution.
[0257] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM
phosphate buffer, pH 8.
[0258] Solution 4: 70 mM phosphate buffer, pH 8.
[0259] A primary W1/O) emulsion was first created using Solution 1
& Solution 2. Solution 1 (0.1 mL) and Solution 2 (1.0 mL) were
combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. A
secondary (W1/O/W2) emulsion was then formed by adding Solution 3
(2.0 mL) to the primary emulsion and sonicating at 30% amplitude
for 40 seconds using the Branson Digital Sonifier 250. The
secondary emulsion was added to an open 50 mL beaker containing 70
mM phosphate buffer solution (30 mL) and stirred at room
temperature for 2 hours to allow the dichloromethane to evaporate
and the nanocarriers to form in suspension. A portion of the
suspended nanocarriers was washed by transferring the nanocarrier
suspension to a centrifuge tube, spinning at 13800 rcf for 60
minutes, removing the supernatant, and re-suspending the pellet in
phosphate buffered saline. This washing procedure was repeated, and
then the pellet was re-suspended in phosphate buffered saline to
achieve a nanocarrier suspension having a nominal concentration of
10 mg/mL on a polymer basis. The suspension was stored frozen at
-20.degree. C. until use.
TABLE-US-00015 TABLE 15 Characterization of the Nanocarriers
Produced According to the Above Effective TLR Agonist, T-cell
helper peptide, Nanocarrier Diameter (nm) % w/w % w/w Group 2 NC
176 None Ova 323-339, 1.1
[0260] Mice were injected with 20 .mu.g of CpG twice
(subcutaneously, hind limbs) at 2-week intervals (days 0 and 14).
At days 35 and 49, mice were immunized with 100 .mu.g of NC-Nic
(containing 2.6% R848 and 0.9% OP-II peptide) or 100 .mu.g of
NC-Nic (containing 1.1% OP-II peptide only). Serum anti-nicotine
antibody titers were measured at days 12, 26, and 40 after
immunization with NC. Anti-nicotine antibody titers (EC.sub.50)
were measured by ELISA against polylysine-nicotine (FIG. 10 (group
1: NC-Nic (R848+OP-II); group 2: NC-Nic (OP-II only)). This
demonstrates that mice immunized with a combination of CpG followed
at a later date by NC-Nic that contain R848 generate higher
antibody titers to nicotine than mice immunized with CpG followed
at a later date by NC-Nic that do not contain R848 (group
1>group 2).
Example 10
Addition of Two Free Adjuvants Augments Immune Response to NC-Nic
(Prophetic)
Materials for NC-Nic Nanocarrier Formulations
[0261] Ovalbumin peptide 323-339 amide acetate salt is purchased
from Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif.
90505. Part #4064565.) PLA with an inherent viscosity of 0.19 dL/g
is purchased from SurModics Pharmaceuticals (756 Tom Martin Drive,
Birmingham, Ala. 35211 (Product Code 100 DL 2A). PLA-PEG-Nicotine
with a nicotine-terminated PEG block of approximately 5,000 Da and
DL-PLA block of approximately 20,000 Da is synthesized. Polyvinyl
alcohol (Mw=11,000-31,000, 87-89% hydrolyzed) is purchased from
J.T. Baker (Part Number U232-08).
Methods for NC-Nic Nanocarrier Production
[0262] Solutions are prepared as follows:
[0263] Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL is prepared
in 0.13N hydrochloric acid at room temperature.
[0264] Solution 2: PLA @ 75 mg/mL and PLA-PEG-Nicotine @ 25 mg/mL
in dichloromethane is prepared by dissolving PLA @ 100 mg/mL in
dichloromethane and PLA-PEG-Nicotine at 100 mg/mL in
dichloromethane, then combining 3 parts of the PLA solution to 1
part of the PLA-PEG-Nicotine solution.
[0265] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in
deionized water.
[0266] Solution 4: 70 mM phosphate buffer, pH 8.
[0267] A primary W1/O) emulsion is first created using Solution 1
& Solution 2. Solution 1 (0.2 mL) and Solution 2 (1.0 mL) are
combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. A
secondary (W1/O/W2) emulsion is then formed by adding Solution 3
(2.0 mL) to the primary emulsion and sonicating at 30% amplitude
for 40 seconds using the Branson Digital Sonifier 250. The
secondary emulsion is added to a beaker containing 70 mM phosphate
buffer solution (30 mL) in an open 50 ml beaker and stirred at room
temperature for 2 hours to allow for the dichloromethane to
evaporate and for the nanocarriers to form in suspension. A portion
of the suspended nanocarriers are washed by transferring the
nanocarrier suspension to centrifuge tubes, spinning at 21,000 rcf
for 45 minutes, removing the supernatant, and re-suspending the
pellet in phosphate buffered saline. This washing procedure is
repeated, and then the pellet is re-suspended in phosphate buffered
saline to achieve nanocarrier suspension having a nominal
concentration of 10 mg/mL on a polymer basis. The suspension is
stored frozen at -20.degree. C. until use.
TABLE-US-00016 TABLE 16 Characterization of the Nanocarriers
Produced According to the Above Effective TLR Agonist, T-cell
helper peptide, Nanocarrier Diameter (nm) % w/w % w/w 200 None Ova
323-339, 1.5
Results
[0268] Mice are immunized with NC-Nic (nanocarrier exhibiting
nicotine on the outer surface) admixed with a first (R848) and
second adjuvant (alum). Groups of five mice are immunized three
times (subcutaneously, hind limbs) at 2-week intervals (days 0, 14
and 28) with 100 .mu.g of NC-Nic. Serum anti-nicotine antibodies
are then measured on days 26 and 40. EC.sub.50 for anti-nicotine
antibodies are measured by standard ELISA against
polylysine-nicotine.
Sequence CWU 1
1
1117PRTG. gallus 1Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile
Asn Glu Ala Gly1 5 10 15Arg
* * * * *