U.S. patent application number 13/116488 was filed with the patent office on 2011-12-01 for multivalent synthetic nanocarrier vaccines.
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 | 20110293701 13/116488 |
Document ID | / |
Family ID | 45004392 |
Filed Date | 2011-12-01 |
United States Patent
Application |
20110293701 |
Kind Code |
A1 |
Bratzler; Robert L. ; et
al. |
December 1, 2011 |
MULTIVALENT SYNTHETIC NANOCARRIER VACCINES
Abstract
The invention relates, at least in part, to compositions
comprising populations of synthetic nanocarriers that comprise
different sets of antigens 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.: |
13/116488 |
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; 424/202.1; 424/203.1; 424/274.1; 424/489; 424/501;
424/85.4 |
Current CPC
Class: |
A61K 47/6921 20170801;
A61P 43/00 20180101; A61P 31/12 20180101; A61K 2039/543 20130101;
A61P 37/02 20180101; A61P 17/04 20180101; A61P 31/14 20180101; A61P
31/22 20180101; A61K 39/0005 20130101; A61P 11/08 20180101; A61K
47/02 20130101; A61K 47/6931 20170801; A61P 35/00 20180101; A61P
1/16 20180101; A61P 25/28 20180101; A61P 37/04 20180101; A61K
47/6929 20170801; A61K 2039/6093 20130101; A61P 29/00 20180101;
A61P 31/16 20180101; A61P 37/00 20180101; A61K 33/06 20130101; A61P
31/00 20180101; Y02A 50/30 20180101; A61K 47/22 20130101; A61K
2039/542 20130101; A61P 11/00 20180101; A61P 31/04 20180101; A61K
45/06 20130101; A61K 47/646 20170801; A61K 2039/55555 20130101;
A61K 2039/70 20130101; A61K 47/60 20170801; A61K 2039/55522
20130101; A61P 3/00 20180101; A61P 5/00 20180101; A61K 2039/55511
20130101; A61P 11/06 20180101; A61P 37/08 20180101; A61K 47/24
20130101; A61P 39/02 20180101; A61K 2039/54 20130101; A61K 39/385
20130101; A61P 25/34 20180101; A61P 17/00 20180101; A61P 25/36
20180101; A61K 31/7115 20130101; A61P 31/20 20180101; A61P 31/10
20180101; A61K 39/35 20130101; A61P 25/30 20180101; A61K 9/5153
20130101; A61K 47/593 20170801; A61K 31/4745 20130101; A61K 39/0013
20130101; A61K 39/39 20130101; A61K 47/68 20170801; A61K 2039/555
20130101; A61K 47/58 20170801; A61K 2039/55561 20130101; A61K
2039/541 20130101; 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 ;
424/202.1; 424/203.1; 424/274.1; 424/184.1; 424/489; 424/501;
424/85.4 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 39/116 20060101 A61K039/116; A61K 39/00 20060101
A61K039/00; A61K 38/21 20060101 A61K038/21; A61P 31/00 20060101
A61P031/00; A61P 35/00 20060101 A61P035/00; A61P 25/30 20060101
A61P025/30; A61K 39/295 20060101 A61K039/295; A61K 9/14 20060101
A61K009/14 |
Claims
1. A composition comprising: a dosage form comprising: a first
population of synthetic nanocarriers that comprise a first set of
surface antigens; a second population of synthetic nanocarriers
that comprise a second set of surface antigens; and a
pharmaceutically acceptable excipient; wherein the first set of
surface antigens and the second set of surface antigens are
structurally different.
2-3. (canceled)
4. The composition of claim 1, wherein the first set of surface
antigens comprise antigens obtained or derived from a first
infectious genus and the second set of surface antigens comprise
antigens obtained or derived from a second infectious genus.
5-9. (canceled)
10. The composition of claim 1, wherein the first set of surface
antigens and/or second set of surface antigens comprise antigens
that are obtained or derived from a virus of the Adenoviridae,
Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae,
Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papillomaviridae,
Rhabdoviridae, Togaviridae or Paroviridae family.
11-12. (canceled)
13. The composition of claim 1, wherein the first set of surface
antigens and/or second set of surface antigens comprise antigens
that are obtained or derived from a bacteria of the Bordetella,
Borrelia, Brucella, Campylobacter, Chlamydia and Chlamydophila,
Clostridium, Corynebacterium, Enterococcus, Escherichia,
Francisella, Haemophilus, Helicobacter, Legionella, Leptospira,
Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas,
Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus,
Treponema Vibrio or Yersinia genus.
14-15. (canceled)
16. The composition of claim 1, wherein the first set of surface
antigens and/or second set of surface antigens comprise antigens
that are obtained or derived from a fungus of the Candida,
Aspergillus, Cryptococcus, Histoplasma, Pneumocystis or
Stachybotrys genus.
17-18. (canceled)
19. The composition of claim 1, wherein the first set of surface
antigens and second set of surface antigens comprise antigens
obtained or derived from an abused or addictive substance.
20-41. (canceled)
42. The composition of claim 1, further comprising one or more
adjuvants.
43. The composition of claim 42, wherein the first population of
synthetic nanocarriers and/or the second population of synthetic
nanocarriers further comprise an adjuvant coupled to the synthetic
nanocarriers.
44. The composition of claim 42, wherein the first population of
synthetic nanocarriers and/or the second population of synthetic
nanocarriers further comprise an adjuvant coupled to the synthetic
nanocarriers and the composition comprises one or more admixed
adjuvants.
45. The composition of claim 42, wherein each of the one or more
adjuvants comprises a mineral salt, alum, alum combined with
monphosphoryl lipid (MPL) A of Enterobacteria, MPL.RTM. (AS04),
AS15, a saponin, QS-21,Quil-A, ISCOMs, ISCOMATRIX.TM., MF59.TM.,
Montanide.RTM. ISA 51, Montanide.RTM. ISA 720, AS02, a liposome or
liposomal formulation, AS01, AS15, synthesized or specifically
prepared microparticles and microcarriers, bacteria-derived outer
membrane vesicles of N. gonorrheae or Chlamydia trachomatis,
chitosan particles, a depot-forming agent, Pluronic.RTM. block
co-polymers, specifically modified or prepared peptides, muramyl
dipeptide, an aminoalkyl glucosaminide 4-phosphate, RC529, a
bacterial toxoid, a toxin fragment, an agonist of Toll-Like
Receptors 2, 3, 4, 5, 7, 8 or 9, an adenine derivative,
immunostimulatory DNA, immunostimulatory RNA, an imidazoquinoline
amine, an imidazopyridine amine, a 6,7-fused
cycloalkylimidazopyridine amine, a 1,2-bridged imidazoquinoline
amine, imiquimod, resiquimod, an agonist for DC surface molecule
CD40, a type I interferon, poly I:C, a bacterial
lipopolysacccharide (LPS), VSV-G, HMGB-1, flagellin or portions or
derivatives thereof, an immunostimulatory DNA molecule comprising
CpG, proinflammatory stimuli released from necrotic cells, urate
crystals, an activated component of the complement cascade, an
activated component of immune complexes, a complement receptor
agonist, a cytokine, or a cytokine receptor agonist.
46. (canceled)
47. The composition of claim 43, wherein the adjuvant coupled to
the first population of synthetic nanocarriers and/or the adjuvant
coupled to the second population of synthetic nanocarriers
comprises a TLR-2, -3, -4, -7, -8 or -9 agonist.
48. The composition of claim 47, wherein the adjuvant coupled to
the first population of synthetic nanocarriers and/or the adjuvant
coupled to the second population of synthetic nanocarriers
comprises an immunostimulatory nucleic acid, imidazoquinoline,
oxoadenine, MPL, imiquimod or resiquimod.
49. The composition of claim 44, wherein the admixed adjuvant is an
immunostimulatory nucleic acid comprising CpG, AS01, AS02, AS04,
AS15, QS-21, a saponin, alum or MPL.
50. The composition of claim 1, wherein the first and second
populations of synthetic nanocarriers are present in an amount
effective to generate an immune response to the first set of
surface antigens and the second set of surface antigens in a
subject.
51-57. (canceled)
58. The composition of claim 1, wherein the first and/or second
population of synthetic nanocarriers further comprise a universal T
cell antigen coupled thereto.
59-62. (canceled)
63. A composition comprising: a dosage form comprising: a first
population of synthetic nanocarriers that comprise a first set of
surface antigens; a second population of synthetic nanocarriers
that comprise a second set of surface antigens; and a
pharmaceutically acceptable excipient; wherein the first set of
surface antigens and the second set of surface antigens are
immunologically different.
64-65. (canceled)
66. The composition of claim 63, wherein the first set of surface
antigens comprise antigens obtained or derived from a first
infectious genus and the second set of surface antigens comprise
antigens obtained or derived from a second infectious genus.
67-71. (canceled)
72. The composition of claim 63, wherein the first set of surface
antigens and/or second set of surface antigens comprise antigens
that are obtained or derived from a virus of the Adenoviridae,
Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae,
Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papillomaviridae,
Rhabdoviridae, Togaviridae or Paroviridae family.
73-74. (canceled)
75. The composition of claim 63, wherein the first set of surface
antigens and/or second set of surface antigens comprise antigens
that are obtained or derived from a bacteria of the Bordetella,
Borrelia, Brucella, Campylobacter, Chlamydia and Chlamydophila,
Clostridium, Corynebacterium, Enterococcus, Escherichia,
Francisella, Haemophilus, Helicobacter, Legionella, Leptospira,
Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas,
Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus,
Treponema Vibrio or Yersinia genus.
76-77. (canceled)
78. The composition of claim 63, wherein the first set of surface
antigens and/or second set of surface antigens comprise antigens
that are obtained or derived from a fungus of the Candida,
Aspergillus, Cryptococcus, Histoplasma, Pneumocystis or
Stachybotrys genus.
79-80. (canceled)
81. The composition of claim 63, further comprising one or more
adjuvants.
82. The composition of claim 81, wherein the first population of
synthetic nanocarriers and/or the second population of synthetic
nanocarriers further comprise an adjuvant coupled to the synthetic
nanocarriers.
83. The composition of claim 81, wherein the first population of
synthetic nanocarriers and/or the second population of synthetic
nanocarriers further comprise an adjuvant coupled to the synthetic
nanocarriers and the composition comprises one or more admixed
adjuvants.
84. The composition of claim 81, wherein each of the one or more
adjuvants comprises a mineral salt, alum, alum combined with
monphosphoryl lipid (MPL) A of Enterobacteria, MPL.RTM. (AS04),
AS15, a saponin, QS-21,Quil-A, ISCOMs, ISCOMATRIX.TM., MF59.TM.,
Montanide.RTM. ISA 51, Montanide.RTM. ISA 720, AS02, a liposome or
liposomal formulation, AS01, synthesized or specifically prepared
microparticles and microcarriers, bacteria-derived outer membrane
vesicles of N. gonorrheae or Chlamydia trachomatis, chitosan
particles, a depot-forming agent, Pluronic.RTM. block co-polymers,
specifically modified or prepared peptides, muramyl dipeptide, an
aminoalkyl glucosaminide 4-phosphate, RC529, a bacterial toxoid, a
toxin fragment, an agonist of Toll-Like Receptors 2, 3, 4, 5, 7, 8
or 9, an adenine derivative, immunostimulatory DNA,
immunostimulatory RNA, an imidazoquinoline amine, an
imidazopyridine amine, a 6,7-fused cycloalkylimidazopyridine amine,
a 1,2-bridged imidazoquinoline amine, imiquimod, resiquimod, an
agonist for DC surface molecule CD40, a type I interferon, poly
I:C, a bacterial lipopolysacccharide (LPS), VSV-G, HMGB-1,
flagellin or portions or derivatives thereof, an immunostimulatory
DNA molecule comprising CpG, proinflammatory stimuli released from
necrotic cells, urate crystals, an activated component of the
complement cascade, an activated component of immune complexes, a
complement receptor agonist, a cytokine, or a cytokine receptor
agonist.
85. (canceled)
86. The composition of claim 82, wherein the adjuvant coupled to
the first population of synthetic nanocarriers and/or the adjuvant
coupled to the second population of synthetic nanocarriers
comprises a TLR-2, -3, -4, -7, -8 or -9 agonist.
87. The composition of claim 86, wherein the adjuvant coupled to
the first population of synthetic nanocarriers and/or the adjuvant
coupled to the second population of synthetic nanocarriers
comprises an immunostimulatory nucleic acid, imidazoquinoline,
oxoadenine, MPL, imiquimod or resiquimod.
88. The composition of claim 83, wherein the admixed adjuvant is an
immunostimulatory nucleic acid comprising CpG, AS01, AS02, AS04,
AS15, QS-21, a saponin, alum or MPL.
89. The composition of claim 63, wherein the first and second
populations of synthetic nanocarriers are present in an amount
effective to generate an immune response to the first set of
surface antigens and the second set of surface antigens in a
subject.
90-96. (canceled)
97. The composition of claim 63, wherein the first and/or second
population of synthetic nanocarriers further comprise a universal T
cell antigen coupled thereto.
98-102. (canceled)
103. The composition of claim 1, wherein synthetic nanocarriers of
each of the populations of synthetic nanocarriers comprise
lipid-based nanoparticles, polymeric nanoparticles, metallic
nanoparticles, surfactant-based emulsions, dendrimers, buckyballs,
nanowires, virus-like particles, peptide or protein-based
particles, lipid-polymer nanoparticles, spheroidal nanoparticles,
cuboidal nanoparticles, pyramidal nanoparticles, oblong
nanoparticles, cylindrical nanoparticles, or toroidal
nanoparticles.
104. The composition of claim 103, wherein each of the populations
of synthetic nanocarriers comprise one or more polymers.
105-109. (canceled)
110. A composition comprising: a dosage form comprising: a first
synthetic nanocarrier means for presenting a first set of surface
antigens; a second synthetic nanocarrier means for presenting a
second set of surface antigens; and a pharmaceutically acceptable
excipient; wherein the first set of surface antigens and the second
set of surface antigens are structurally different.
111. (canceled)
112. A composition comprising: a dosage form comprising: a first
synthetic nanocarrier means for presenting a first set of surface
antigens; a second synthetic nanocarrier means for presenting a
second set of surface antigens; and a pharmaceutically acceptable
excipient; wherein the first set of surface antigens and the second
set of surface antigens are immunologically different.
113. (canceled)
114. A method comprising: administering the composition of claim 1
to a subject.
115. The method of claim 114, wherein the subject has or is at risk
of having an infection or infectious disease.
116. The method of claim 114, wherein the subject has or is at risk
of having cancer.
117. The method of claim 114, wherein the subject has or is at risk
of having an addiction.
118. (canceled)
119. A method comprising: preparing a first population of synthetic
nanocarriers that comprise a first set of surface antigens;
preparing a second population of synthetic nanocarriers that
comprise a second set of surface antigens; and combining the first
and second populations of synthetic nanocarriers into a dosage
form; wherein the first set of surface antigens and the second set
of surface antigens are structurally different.
120. A method comprising: preparing a first population of synthetic
nanocarriers that comprise a first set of surface antigens;
preparing a second population of synthetic nanocarriers that
comprise a second set of surface antigens; and combining the first
and second populations of synthetic nanocarriers into a dosage
form; wherein the first set of surface antigens and the second set
of surface antigens are immunologically different.
121. The method of claim 119, further comprising administering the
dosage form to a subject.
122-124. (canceled)
125. A process for producing a dosage form of a composition, the
process comprising the method steps as defined in claim 119.
126-132. (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.
BACKGROUND OF THE INVENTION
[0002] Multivalent vaccines are a useful way of generating an
immune response to certain foreign substances that otherwise would
not be desirably robust. For instance, vaccinating against multiple
strains of a virus may provide more robust cross-protection against
multiple strains of that virus as compared to vaccinating using a
monovalent vaccine.
[0003] However, current multivalent vaccines and methods of making
them need improvement. For instance, current approaches of
conjugating antigens to protein carriers are complex and provide
for low yields. Additionally, new techniques often need to be
developed to conjugate new antigens to carrier proteins because
conventional techniques can be unsuccessful due to the relative
fragility of conventional carrier proteins.
[0004] What is needed are compositions and methods that provide for
improved multivalent vaccines.
SUMMARY OF THE INVENTION
[0005] In one aspect, a composition comprising a dosage form
comprising a first population of synthetic nanocarriers that
comprise a first set of surface antigens; a second population of
synthetic nanocarriers that comprise a second set of surface
antigens; and a pharmaceutically acceptable excipient, wherein the
first set of surface antigens and the second set of surface
antigens are structurally different is provided.
[0006] In another aspect, a composition comprising a dosage form
comprising a first population of synthetic nanocarriers that
comprise a first set of surface antigens; a second population of
synthetic nanocarriers that comprise a second set of surface
antigens; and a pharmaceutically acceptable excipient, wherein the
first set of surface antigens and the second set of surface
antigens are immunologically different is also provided.
[0007] In yet another aspect, a composition comprising a dosage
form comprising a first synthetic nanocarrier means for presenting
a first set of surface antigens; a second synthetic nanocarrier
means for presenting a second set of surface antigens; and a
pharmaceutically acceptable excipient, wherein the first set of
surface antigens and the second set of surface antigens are
structurally different is also provided.
[0008] In still another aspect, a composition comprising a dosage
form comprising a first synthetic nanocarrier means for presenting
a first set of surface antigens; a second synthetic nanocarrier
means for presenting a second set of surface antigens; and a
pharmaceutically acceptable excipient, wherein the first set of
surface antigens and the second set of surface antigens are
immunologically different is provided.
[0009] In one embodiment of any of the compositions provided, the
first set of surface antigens comprises 1, 2, 3, 4, 5, 6, 7, 8, 9,
10 or more types of antigens. In another embodiment of any of the
compositions provided, the second set of surface antigens comprises
2, 3, 4, 5, 6, 7, 8, 9, 10 or more types of antigens.
[0010] In yet another embodiment of any of the compositions
provided, the first set of surface antigens comprise antigens
obtained or derived from a first infectious genus and the second
set of surface antigens comprise antigens obtained or derived from
a second infectious genus. In one embodiment, the first infectious
genus and the second infectious genus are the same. In still
another embodiment of any of the compositions provided, the first
set of surface antigens comprise antigens obtained or derived from
a first infectious species and the second set of surface antigens
comprise antigens obtained or derived from of a second infectious
species. In one embodiment, the first infectious species and the
second infectious species are the same. In a further embodiment,
the first set of surface antigens comprise antigens obtained or
derived from a first infectious strain and the second set of
surface antigens comprise antigens obtained or derived from a
second infectious strain. In one embodiment, the first infectious
strain and second infectious strain are the same.
[0011] In another embodiment of any of the compositions provided,
the first set of surface antigens and/or second set of surface
antigens comprise antigens that are obtained or derived from a
virus of the Adenoviridae, Picornaviridae, Herpesviridae,
Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae,
Paramyxoviridae, Papillomaviridae, Rhabdoviridae, Togaviridae or
Paroviridae family. In one embodiment, the first set of surface
antigens and/or second set of surface antigens comprise antigens
that are obtained or derived from adenovirus, coxsackievirus,
hepatitis A virus, poliovirus, Rhinovirus, Herpes simplex virus,
Varicella-zoster virus, Epstein-barr virus, Human cytomegalovirus,
Human herpesvirus, Hepatitis B virus, Hepatitis C virus, yellow
fever virus, dengue virus, West Nile virus, HIV, Influenza virus,
Measles virus, Mumps virus, Parainfluenza virus, Respiratory
syncytial virus, Human metapneumovirus, Human papillomavirus,
Rabies virus, Rubella virus, Human bocarivus or Parvovirus B19. In
another embodiment, the first set of surface antigens and/or second
set of surface antigens comprise antigens that are obtained or
derived from VI, VII, E1A, E3-19K, 52K, VP1, surface antigen, 3A
protein, capsid protein, nucleocapsid, surface projection,
transmembrane proteins, UL6, UL18, UL35, UL38, UL19, early antigen,
capsid antigen, Pp65, gB, p52, latent nuclear antigen-1, NS3,
envelope protein, envelope protein E2 domain, gp120, p24,
lipopeptides Gag (17-35), Gag (253-284), Nef (66-97), Nef
(116-145), Pol (325-355), neuraminidase, nucleocapsid protein,
matrix protein, phosphoprotein, fusion protein, hemagglutinin,
hemagglutinin-neuraminidase, glycoprotein, E6, E7, envelope
lipoprotein or non-structural protein (NS).
[0012] In still another embodiment of any of the compositions
provided, the first set of surface antigens and/or second set of
surface antigens comprise antigens that are obtained or derived
from a bacteria of the Bordetella, Borrelia, Brucella,
Campylobacter, Chlamydia and Chlamydophila, Clostridium,
Corynebacterium, Enterococcus, Escherichia, Francisella,
Haemophilus, Helicobacter, Legionella, Leptospira, Listeria,
Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia,
Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema
Vibrio or Yersinia genus. In one embodiment, the first set of
surface antigens and/or second set of surface antigens comprise
antigens that are obtained or derived from Bordetella pertussis,
Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella
melitensis, Brucella suis, Campylobacter jejuni, Chlamydia
pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci,
Clostridium botulinum, Clostridium difficile, Clostridium
perfringens, Clostridium tetani, Corynebacterium diphtheriae,
Enterococcus faecalis, Enterococcus faecium, Escherichia coli,
Francisella tularensis, Haemophilus influenzae, Helicobacter
pylori, Legionella pneumophila, Leptospira interrogans, Listeria
monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis,
Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria
gonorrhoeae, Neisseria meningitides, Pseudomonas aeruginosa,
Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium,
Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis,
Staphylococcus saprophyticus, Streptococcus agalactiae,
Streptococcus pneumoniae, Streptococcus pyogenes, Treponema
pallidum, Vibrio cholerae or Yersinia pestis. In another
embodiment, the first set of surface antigens and/or second set of
surface antigens comprise antigens that are obtained or derived
from pertussis toxin (PT), filamentous hemagglutinin (FHA),
pertactin (PRN), fimbriae (FIM 2/3), VlsE; DbpA, OspA, Hia, PrpA,
MltA, L7/L12, D15, 0187, VirJ, Mdh, AfuA, L7/L12, out membrane
protein, LPS, antigen type A, antigen type B, antigen type C,
antigen type D, antigen type E, FliC, FliD, Cwp84, alpha-toxin,
theta-toxin, fructose 1,6-biphosphate-aldolase (FBA),
glyceraldehydes-3-phosphate dehydrogenase (GPD),
pyruvate:ferredoxin oxidoreductase (PFOR), elongation factor-G
(EF-G), hypothetical protein (HP), T toxin, Toxoid antigen,
capsular polysaccharide, Protein D, Mip, nucleoprotein (NP), RD1,
PE35, PPE68, EsxA, EsxB, RD9, EsxV, Hsp70, lipopolysaccharide,
surface antigen, Sp1, Sp2, Sp3, Glycerophosphodiester
Phosphodiesterase, outer membrane protein, chaperone-usher protein,
capsular protein (F1) or V protein.
[0013] In yet another embodiment of any of the compositions
provided, the first set of surface antigens and/or second set of
surface antigens comprise antigens that are obtained or derived
from a fungus of the Candida, Aspergillus, Cryptococcus,
Histoplasma, Pneumocystis or Stachybotrys genus. In one embodiment,
the first set of surface antigens and/or second set of surface
antigens comprise antigens that are obtained or derived from C.
albicans, Aspergillus fumigatus, Aspergillus flavus, Cryptococcus
neoformans, Cryptococcus laurentii, Cryptococcus albidus,
Cryptococcus gattii, Histoplasma capsulatum, Pneumocystis jirovecii
or Stachybotrys chartarum. In another embodiment, the first set of
surface antigens and/or second set of surface antigens comprise
antigens that are obtained or derived from surface antigen,
capsular glycoprotein, Yps3P, Hsp60, Major surface protein, MsgC1,
MsgC3, MsgC8, MsgC9 or SchS34.
[0014] In still another embodiment of any of the compositions
provided, the first set of surface antigens and/or second set of
surface antigens comprise antigens that are obtained or derived
from any of the infectious agents, viruses, bacteria, proteins,
peptides, polypeptides, small molecules, polysaccharides or
oligosaccharides provided herein.
[0015] In a further embodiment of any of the compositions provided,
the first set of surface antigens and second set of surface
antigens comprise antigens obtained or derived from an abused or
addictive substance. In one embodiment, the abused or addictive
substance is cocaine or nicotine.
[0016] In yet a further embodiment of any of the compositions
provided, the first set of surface antigens and the second set of
surface antigens comprise the same surface antigens, and wherein at
least one antigen of the first set of surface antigens is presented
in a different orientation than as presented in the second set of
surface antigens.
[0017] In still a further embodiment of any of the compositions
provided, the first set of surface antigens and the second set of
surface antigens comprise the same surface antigens, and wherein at
least one antigen of the first set of surface antigens is presented
in a different conformation than as presented in the second set of
surface antigens.
[0018] In another embodiment of any of the compositions provided,
the molecular structure of the first set of surface antigens and
the second set of surface antigens are different.
[0019] In yet another embodiment of any of the compositions
provided, the first set of surface antigens and/or the second set
of surface antigens comprise surface antigens with a molecular
weight of less than 10,000 Da.
[0020] In still another embodiment of any of the compositions
provided, the first set of surface antigens and/or the second set
of surface antigens comprise surface antigens that comprise
peptides, proteins, oligosaccharides, polysaccharides and/or small
molecules.
[0021] In a further embodiment of any of the compositions provided,
at least one surface antigen of the first set of surface antigens
and/or at least one surface antigen of the second set of surface
antigens has a molecular weight of less than 10,000 Da.
[0022] In a yet a further embodiment of any of the compositions
provided, the first set of surface antigens comprises surface
antigens comprising peptides, and the second set of surface
antigens comprises surface antigens with a molecular weight of less
than 10,000 Da.
[0023] In still a further embodiment of any of the compositions
provided, the first set of surface antigens comprises surface
antigens comprising peptides, and the second set of surface
antigens comprises surface antigens comprising peptides, proteins,
oligosaccharides, polysaccharides and/or small molecules. In one
embodiment, at least one surface antigen of the second set of
surface antigens has a molecular weight of less than 10,000 Da.
[0024] In another embodiment of any of the compositions provided,
the first set of surface antigens comprises surface antigens
comprising proteins, and the second set of surface antigens
comprises surface antigens with a molecular weight of less than
10,000 Da.
[0025] In yet another embodiment of any of the compositions
provided, the first set of surface antigens comprises surface
antigens comprising proteins, and the second set of surface
antigens comprises surface antigens comprising peptides, proteins,
oligosaccharides, polysaccharides and/or small molecules. In one
embodiment, at least one surface antigen of the second set of
surface antigens has a molecular weight of less than 10,000 Da.
[0026] In still another embodiment of any of the compositions
provided, the first set of surface antigens comprises surface
antigens comprising oligosaccharides, and the second set of surface
antigens comprises surface antigens with a molecular weight of less
than 10,000 Da.
[0027] In a further embodiment of any of the compositions provided,
the first set of surface antigens comprises surface antigens
comprising oligosaccharides, and the second set of surface antigens
comprises surface antigens comprising peptides, proteins,
oligosaccharides, polysaccharides and/or small molecules. In one
embodiment, at least one surface antigen of the second set of
surface antigens has a molecular weight of less than 10,000 Da.
[0028] In yet a further embodiment of any of the compositions
provided, the first set of surface antigens comprises surface
antigens comprising polysaccharides, and the second set of surface
antigens comprises surface antigens with a molecular weight of less
than 10,000 Da.
[0029] In still a further embodiment of any of the compositions
provided, the first set of surface antigens comprises surface
antigens comprising polysaccharides, and the second set of surface
antigens comprises surface antigens comprising peptides, proteins,
oligosaccharides, polysaccharides and/or small molecules. In one
embodiment, at least one surface antigen of the second set of
surface antigens has a molecular weight of less than 10,000 Da.
[0030] In another embodiment of any of the compositions provided,
the first set of surface antigens comprises surface antigens
comprising small molecules, and the second set of surface antigens
comprises surface antigens with a molecular weight of less than
10,000 Da.
[0031] In still another embodiment of any of the compositions
provided, the first set of surface antigens comprises surface
antigens comprising small molecules, and the second set of surface
antigens comprises surface antigens comprising peptides, proteins,
oligosaccharides, polysaccharides and/or small molecules. In one
embodiment, at least one surface antigen of the second set of
surface antigens has a molecular weight of less than 10,000 Da.
[0032] In one embodiment of any of the compositions provided, the
compositions further comprise one or more adjuvants. In one
embodiment, the first population of synthetic nanocarriers and/or
the second population of synthetic nanocarriers further comprise an
adjuvant coupled to the synthetic nanocarriers. In another
embodiment, the first population of synthetic nanocarriers and/or
the second population of synthetic nanocarriers further comprise an
adjuvant coupled to the synthetic nanocarriers and the composition
comprises one or more admixed adjuvants.
[0033] In one embodiment, each of the one or more adjuvants of any
of the compositions provided comprises a mineral salt, alum, alum
combined with monphosphoryl lipid (MPL) A of Enterobacteria,
MPL.RTM. (AS04), AS15, a saponin, QS-21,Quil-A, ISCOMs,
ISCOMATRIX.TM., MF59.TM., Montanide.RTM. ISA 51, Montanide.RTM. ISA
720, AS02, a liposome or liposomal formulation, AS01, AS15,
synthesized or specifically prepared microparticles and
microcarriers, bacteria-derived outer membrane vesicles of N.
gonorrheae or Chlamydia trachomatis, chitosan particles, a
depot-forming agent, Pluronic.RTM. block co-polymers, specifically
modified or prepared peptides, muramyl dipeptide, an aminoalkyl
glucosaminide 4-phosphate, RC529, a bacterial toxoid, a toxin
fragment, an agonist of Toll-Like Receptors 2, 3, 4, 5, 7, 8 or 9,
an adenine derivative, immunostimulatory DNA, immunostimulatory
RNA, an imidazoquinoline amine, an imidazopyridine amine, a
6,7-fused cycloalkylimidazopyridine amine, a 1,2-bridged
imidazoquinoline amine, imiquimod, resiquimod, an agonist for DC
surface molecule CD40, a type I interferon, poly I:C, a bacterial
lipopolysaccharide (LPS), VSV-G, HMGB-1, flagellin or portions or
derivatives thereof, an immunostimulatory DNA molecule comprising
CpG, proinflammatory stimuli released from necrotic cells, urate
crystals, an activated component of the complement cascade, an
activated component of immune complexes, a complement receptor
agonist, a cytokine, or a cytokine receptor agonist. In one
embodiment, the adjuvants are different. In another embodiment, the
adjuvant coupled to the first population of synthetic nanocarriers
and/or the adjuvant coupled to the second population of synthetic
nanocarriers comprises a TLR-2, -3, -4, -7, -8 or -9 agonist. In
yet another embodiment, the adjuvant coupled to the first
population of synthetic nanocarriers and/or the adjuvant coupled to
the second population of synthetic nanocarriers comprises an
immunostimulatory nucleic acid, imidazoquinoline, oxoadenine, MPL,
imiquimod or resiquimod. In one embodiment, the admixed adjuvant is
an immunostimulatory nucleic acid comprising CpG, AS01, AS02, AS04,
AS15, QS-21, a saponin, alum or MPL.
[0034] In one embodiment of any of the compositions provided, the
first and second populations of synthetic nanocarriers are present
in an amount effective to generate an immune response to the first
set of surface antigens and the second set of surface antigens in a
subject. In one embodiment, the immune response is the generation
of antibody titers specific for the first set of surface antigens
and the second set of surface antigens.
[0035] In another embodiment of any of the compositions provided,
the compositions comprise one or more additional populations of
synthetic nanocarriers, wherein each additional population of
synthetic nanocarriers comprises a set of surface antigens
structurally different from the other sets of surface antigens in
the composition. In one embodiment, at least one of the one or more
additional populations of synthetic nanocarriers further comprise
an adjuvant coupled thereto. In another embodiment, the adjuvant
coupled to the at least one of the one or more additional
populations of synthetic nanocarriers is different from the other
adjuvants in the composition.
[0036] In yet another embodiment of any of the compositions
provided, the first set of surface antigens comprises a first set
of monovalent or oligovalent surface antigens; and the second set
of surface antigens comprises a second set of monovalent or
oligovalent surface antigens.
[0037] In still another embodiment of any of the compositions
provided, each set of surface antigens is a monovalent or
oligovalent set of surface antigens.
[0038] In a further embodiment of any of the compositions provided,
the populations of synthetic nanocarriers are present in an amount
effective to generate an immune response to each set of surface
antigens. In one embodiment, the immune response is the generation
of antibody titers specific for each set of surface antigens.
[0039] In one embodiment of any of the compositions provided, the
first and/or second population of synthetic nanocarriers further
comprise a universal T cell antigen coupled thereto. In another
embodiment, the universal T cell antigen comprises a T helper cell
antigen. In yet another embodiment, the T-helper cell antigen
comprises a peptide obtained or derived from ovalbumin. In still
another embodiment, the peptide obtained or derived from ovalbumin
comprises the sequence as set forth in SEQ ID NO: 1. In a further
embodiment, the universal T cell antigen is coupled by
encapsulation.
[0040] In one embodiment of any of the compositions provided, the
pharmaceutically acceptable excipient comprises a preservative, a
buffer, saline, phosphate buffered saline, a colorant, or a
stabilizer.
[0041] In another embodiment of any of the compositions provided,
synthetic nanocarriers of each of the populations of synthetic
nanocarriers comprise lipid-based nanoparticles, polymeric
nanoparticles, metallic nanoparticles, surfactant-based emulsions,
dendrimers, buckyballs, nanowires, virus-like particles, peptide or
protein-based particles, lipid-polymer nanoparticles, spheroidal
nanoparticles, cuboidal nanoparticles, pyramidal nanoparticles,
oblong nanoparticles, cylindrical nanoparticles, or toroidal
nanoparticles. In one embodiment, each of the populations of
synthetic nanocarriers comprise one or more polymers. In another
embodiment, the one or more polymers comprise a polyester. In still
another embodiment, the one or more polymers comprise or further
comprise a polyester coupled to a hydrophilic polymer. In yet
another embodiment, the polyester comprises a poly(lactic acid),
poly(glycolic acid), poly(lactic-co-glycolic acid), or
polycaprolactone. In a further embodiment, the hydrophilic polymer
comprises a polyether. In still a further embodiment, the polyether
comprises polyethylene glycol.
[0042] In another aspect, a method comprising administering any of
the compositions provided to a subject is provided. In one
embodiment, the subject has or is at risk of having an infection or
infectious disease. In another embodiment, the subject has or is at
risk of having cancer. In yet another embodiment, the subject has
or is at risk of having an addiction. In a further embodiment, the
composition is administered by oral, subcutaneous, pulmonary,
intranasal, intradermal or intramuscular administration.
[0043] In yet another aspect, a method comprising preparing a first
population of synthetic nanocarriers that comprise a first set of
surface antigens; preparing a second population of synthetic
nanocarriers that comprise a second set of surface antigens; and
combining the first and second populations of synthetic
nanocarriers into a dosage form, wherein the first set of surface
antigens and the second set of surface antigens are structurally
different is provided.
[0044] In a further aspect, a method comprising preparing a first
population of synthetic nanocarriers that comprise a first set of
surface antigens; preparing a second population of synthetic
nanocarriers that comprise a second set of surface antigens; and
combining the first and second populations of synthetic
nanocarriers into a dosage form, wherein the first set of surface
antigens and the second set of surface antigens are immunologically
different is provided. In one embodiment, the method further
comprises administering the dosage form to a subject. In another
embodiment, the method further comprises determining whether or not
an immune response to each set of surface antigens is generated. In
one embodiment, the immune response is the generation of antibody
titers specific for each set of surface antigens. In a further
embodiment, the method further comprises determining the amount
effective to generate the immune response to each set of surface
antigens.
[0045] In still a further aspect, a process for producing a dosage
form of a composition, the process comprising the method steps as
defined in any of the methods provided herein is provided.
[0046] In one embodiment, any of the compositions provided is for
use in therapy or prophylaxis. In another embodiment, any of the
compositions provided is for use in any of the methods provided
herein. In yet another embodiment, any of the compositions provided
is for use in a method of treating or preventing infection or
infectious disease. In still another embodiment, any of the
compositions provided is for use in a method of treating or
preventing cancer. In a further embodiment, any of the compositions
provided is for use in a method of treating or preventing an
addiction.
[0047] In another embodiment, any of the methods comprise
administration of any of the compositions by oral, subcutaneous,
pulmonary, intranasal, intradermal or intramuscular
administration.
[0048] In yet another aspect, the use of any of the compositions
provided for the manufacture of a medicament for use in any of the
methods provided is provided.
BRIEF DESCRIPTION OF FIGURES
[0049] FIG. 1 shows anti-nicotine (dark gray bars) and
anti-ovalbumin (light gray bars) antibody titers in unimmunized
mice and mice injected with NC-Nic and NC-OVA (5 animals/group;
s.c., 100 .mu.g of each NC per injection, 2 times at 3-wk
intervals).
[0050] FIG. 2 shows anti-nicotine, anti-ovalbumin, and anti-L2
peptide antibody titers in unimmunized mice and mice injected with
NC-Nic-OVA and NC-L2 (5 animals/group; s.c., 100 .mu.g of each NC
per injection, 2 times at 3-wk intervals).
[0051] FIG. 3 shows anti-nicotine, anti-ovalbumin, anti-M2e
peptide, and anti-L2 peptide antibody titers in unimmunized mice
and mice injected with NC-Nic-OVA and NC-M2e-L2 (5 animals/group;
s.c., 100 .mu.g of each NC per injection, 2 times at 3-wk
intervals).
[0052] FIG. 4 shows anti-M2e peptide and anti-L2 peptide antibody
titers in unimmunized mice and mice injected with NC-M2e and NC-L2
(5 animals/group; s.c., 100 .mu.g of each NC per injection, 2 times
at 3-wk intervals).
[0053] FIG. 5 shows anti-HA5 protein and anti-ovalbumin protein
antibody titers in unimmunized mice and mice injected with NC-HA5
and NC-OVA (5 animals/group; s.c., 100 .mu.g of each NC per
injection, 2 times at 3-wk intervals).
[0054] FIG. 6 shows anti-HA, anti-ovalbumin, anti-M2e peptide, and
anti-L2 peptide antibody titers in unimmunized mice and mice
injected with NC-HA5, NC-OVA, and NC-M2e-L2 (5 animals/group; s.c.,
100 .mu.g of each NC per injection, 2 times at 3-wk intervals).
[0055] FIG. 7 shows antibody titers in mice immunized with a
combination of NC-M2e, NC-L2 peptide and NC-nicotine-ovalbumin.
[0056] FIG. 8 shows antibody titers in mice immunized with a
combination of NC-3'-nicotine and NC-1'-nicotine.
DETAILED DESCRIPTION OF THE INVENTION
[0057] 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.
[0058] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety for all purposes.
[0059] 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
[0060] The inventors have unexpectedly and surprisingly discovered
that the problems and limitations noted above can be overcome by
practicing the invention disclosed herein. In particular, the
inventors have unexpectedly discovered that it is possible to
provide inventive compositions, and related methods, that address
the problems and limitations in the art by providing a composition
comprising a dosage form comprising: a first population of
synthetic nanocarriers that comprise a first set of surface
antigens; a second population of synthetic nanocarriers that
comprise a second set of surface antigens; and a pharmaceutically
acceptable excipient; wherein the first set of surface antigens and
the second set of surface antigens are structurally different.
[0061] In another aspect, the invention provides a composition
comprising: a dosage form comprising: a first population of
synthetic nanocarriers that comprise a first set of surface
antigens; a second population of synthetic nanocarriers that
comprise a second set of surface antigens; and a pharmaceutically
acceptable excipient; wherein the first set of surface antigens and
the second set of surface antigens are immunologically
different.
[0062] In an aspect, the invention provides a composition
comprising: a dosage form comprising: a first synthetic nanocarrier
means for presenting a first set of surface antigens; a second
synthetic nanocarrier means for presenting a second set of surface
antigens; and a pharmaceutically acceptable excipient; wherein the
first set of surface antigens and the second set of surface
antigens are structurally different.
[0063] In an aspect, the invention provides a composition
comprising: a dosage form comprising: a first synthetic nanocarrier
means for presenting a first set of surface antigens; a second
synthetic nanocarrier means for presenting a second set of surface
antigens; and a pharmaceutically acceptable excipient; wherein the
first set of surface antigens and the second set of surface
antigens are immunologically different.
[0064] In another aspect, the invention provides a method
comprising: preparing a first population of synthetic nanocarriers
that comprise a first set of surface antigens; preparing a second
population of synthetic nanocarriers that comprise a second set of
surface antigens; and combining the first and second populations of
synthetic nanocarriers into a dosage form; wherein the first set of
surface antigens and the second set of surface antigens are
structurally different.
[0065] In yet another aspect, the invention provides a method
comprising: preparing a first population of synthetic nanocarriers
that comprise a first set of surface antigens; preparing a second
population of synthetic nanocarriers that comprise a second set of
surface antigens; and combining the first and second populations of
synthetic nanocarriers into a dosage form; wherein the first set of
surface antigens and the second set of surface antigens are
immunologically different.
[0066] It has been discovered that it is possible to generate a
first and second population of synthetic nanocarriers that comprise
a first and second set of surface antigens, respectively, which can
be combined together with a pharmaceutically acceptable excipient
to create a dosage form. This dosage form can, in certain
embodiments, be useful as a multivalent vaccine. The inventors have
further noted certain advantages in the creation of the inventive
dosage forms, particularly with respect to conventional multivalent
vaccines. These include, but are not limited to, minimizing vaccine
volumes which is a problem in conventional multivalent vaccines,
and minimizing protein-protein interactions present in conventional
protein carrier-hapten multivalent vaccines that can lead to
non-specific binding and precipitation.
[0067] A further advantage of the present invention is that
combining different populations of synthetic nanocarriers that
comprise sets of surface antigens allows different methods to be
used to couple different sets of surface antigens to different
populations of synthetic nanocarriers. This can be a significant
advantage for embodiments wherein incompatible coupling methods are
required to couple sets of surface antigens to populations of
synthetic nanocarriers. As an example, vaccines for Streptococcus
pneumonia (U.S. Pat. No. 6,132,723 to Alberta Research Council and
WO 2008/143709 to Wyeth) contain multiple antigens. Since the
attachment chemistry conditions are not the same for all of the
polysaccharide antigens (WO 2008/143709) coupling methods that
would attach all of the surface antigens to a single population of
synthetic nanocariers in a single coupling environment would be
undesirable. Practicing embodiments of the present invention
wherein different populations of synthetic nanocarriers are first
coupled to certain sets of surface antigens and then combined could
ameliorate the problem noted in the art.
[0068] Another example of multivalent vaccines that could benefit
from this embodiment of the invention comprise vaccines against N.
meningitides which is polysaccharide-based and multivalent. Such
embodiments may be aimed either at N. meningitidis groups A and C
(bivalent) or groups A, C, W135 and Y (tetravalent).
[0069] The examples illustrate certain embodiments according to the
invention, wherein peptides, polysaccharides, small molecules,
etc., are conjugated to a first population of synthetic
nanocarriers and/or a second population of synthetic nanocarriers.
These populations are then combined to form a composition according
to the invention.
[0070] The present invention will now be described in more
detail.
Definition
[0071] "Abused substance" is any substance taken by a subject
(e.g., a human) for purposes other than those for which it is
indicated or in a manner or in quantities other than directed by a
physician. The abused substance, in some embodiments, is an
addictive substance. In some embodiments, the abused substance for
inclusion in a nanocarrier is the complete molecule, analog or a
portion thereof. "Addictive substance" is a substance that causes
obsession, compulsion, or physical dependence or psychological
dependence. In some embodiments, the addictive substance for
inclusion in a nanocarrier is the complete molecule, analog or a
portion thereof.
[0072] "Adjuvant" means an agent that does not constitute a
specific antigen, but boosts the strength and longevity of immune
response to an administered antigen (e.g., 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 Escherichia 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.) , liposomes and liposomal formulations
such as AS01, AS15, 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.
[0073] 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.
[0074] 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, synthetic nanocarriers incorporate a ligand for
toll-like receptor (TLR)-9, such as CpGs, which induce type I
interferon production, 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
(Thi) 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.).
[0075] 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 and
anti-viral immunity. In embodiments, adjuvants also may comprise
immunostimulatory RNA molecules, such as but not limited to dsRNA,
ssRNA, 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 oligcoribonucleotide
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.
[0076] In some embodiments, an adjuvant may be a TLR-4 agonist,
such as bacterial lipopolysacharide (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.
[0077] 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.
[0078] In embodiments, at least a portion of the dose of adjuvant
may be coupled to synthetic nanocarriers, preferably, all of the
dose of adjuvant is coupled to synthetic nanocarriers. In other
embodiments, at least a portion of the dose of the adjuvant is not
coupled to the synthetic nanocarriers. In embodiments, the dose of
adjuvant comprises two or more types of adjuvants. For instance,
and without limitation, adjuvants that act on 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.
[0079] "Administering" or "administration" means providing a
substance to a subject in a manner that is pharmacologically
useful.
[0080] "Amount effective" is any amount of a composition 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 health practitioner would believe may have a
clinical benefit for a subject in need of an antibody response
specific to one or more antigens. "Antibody response" means any
immune response that results in the production or stimulation of B
cells and/or the production of antibodies. In embodiments,
therefore, an amount effective is one that a health practitioner
would believe may generate an antibody response against the surface
antigen(s) of the inventive compositions provided herein. Effective
amounts 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.
Therefore, in other embodiments, the amount effective in one that a
clinician would believe would provide a therapeutic benefit
(including a prophylactic benefit) to a subject provided herein.
Such subjects include those that have or are at risk of having
cancer, an infection or infectious disease.
[0081] The antigen(s) of any of the inventive compositions provided
herein can in embodiments be in an amount effective. In some
embodiments, the amount effective is one that a health practitioner
would believe may generate antibody titers against the sets of
surface antigens of the compositions provided herein. "Antibody
titer" means the production of a measurable level of antibodies.
Preferably, the antibody response or generation of the antibody
titer is in a human. In some embodiments, the antibodies are
antibodies are of a certain isotype, such as IgG or a subclass
thereof. Methods for measuring antibody titers are known in the art
and include Enzyme-linked Immunosorbent Assay (ELISA). Methods for
measuring antibody response are also described in some detail in
the Examples. Preferably, the antibody response or antibody titer
is specific to a set of surface antigens. In some embodiments where
the synthetic nanocarriers also comprise a universal antigen in
addition to a set of surface antigens against which a specific
immune response, such as an antibody response or antibody titer,
the immune response is specific to the set of surface antigens but
not to the universal antigen.
[0082] 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.
[0083] "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.
[0084] "At least a portion of the dose" means at least some part of
the dose, ranging up to including all of the dose.
[0085] An "at risk" subject is one in which a health practitioner
believes has a chance of having the disease or condition provided
herein including, but not limited to, an infection, infectious
disease, cancer or an addiction.
[0086] "B cell antigen" means any antigen that is or recognized by
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, carbohydrates,
oligosaccharides and polysaccharides. 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, oligosaccharide or polysaccharide
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, or parasite. In some embodiments, the B
cell antigen comprises a poorly immunogenic antigen. In some
embodiments, the B cell antigen comprises an abused or addictive
substance or a portion or analog 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 sources. The B cell antigen may also
comprise a hazardous environmental agent. In some embodiments, the
B cell antigen comprises a self antigen. 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.
[0087] "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.
[0088] "Derived" means adapted or modified from the original
source. For example, as a non-limiting example, a peptide antigen
derived from an infectious strain may have several non-natural
amino acid residues substituted for the natural amino acid residues
found in the original antigen found in the infectious strain. The
adaptations or modifications may be for a variety of reasons,
including but not limited to increased specificity, easier antigen
processing, or improved safety.
[0089] In embodiments, 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. In
other embodiments, the material is substantially modified.
Substantially modified material means a material that is modified
such that the modification 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.
[0090] "Dosage form" means a pharmacologically and/or
immunologically active material, such as a vaccine, in a medium,
carrier, vehicle, or device suitable for administration to a
subject.
[0091] "Encapsulate" or "encapsulated" 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
adsorption, 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.
[0092] "Immunologically different" refers to a difference between
certain surface antigens that can be noted if a sera generated by
immunization generates a distinct antibody response spectrum for
each of the surface antigens. Surface antigen specific antibodies
will only recognize a specific set of surface antigens, and will
bind in distinguishable binding patterns to other sets of surface
antigens. For example, if immunized with a set of surface antigens
A, the antiserum generated will bind to the set of surface antigens
A, but not to the set of surface antigens B. If two or more surface
antigens are combined on a single synthetic nanocarrier, a panning
assay can be designed which will distinguish the binding patterns
of the sera relative to the two sets of surface antigens. In
embodiments, a first set of surface antigens and a second set of
surface antigens are immunologically different. In other
embodiments, a first set of surface antigens, a second set of
surface antigens, and a third set of surface antigens all are
immunologically different.
[0093] 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. The surface antigens if the
inventive compositions provided herein can be obtained or derived
from any infectious agent, such as those that can cause the
infections or infectious diseases provided herein.
[0094] "Infectious genus" means a genus that comprises organisms
capable of infecting a subject. In embodiments, surface antigens
may be obtained or derived from a first infectious genus, or
obtained or derived from a second infectious genus. In embodiments,
the first infectious genus and the second infectious genus are the
same. In other embodiments, the first infectious genus and the
second infectious genus are different.
[0095] "Infectious species" means a species that comprises
organisms capable of infecting a subject. In embodiments, surface
antigens may be obtained or derived from a first infectious
species, or obtained or derived from a second infectious species.
In embodiments, the first infectious species and the second
infectious species are of the same genus. In other embodiments, the
first infectious species and the second infectious species are also
the same. In some embodiments, the first infectious species and the
second infectious species are different but are of the same genus.
In other embodiments, the different infectious species are of
different genera.
[0096] "Infectious strain" means a strain that comprises organisms
capable of infecting a subject. In embodiments, surface antigens
may be obtained or derived from a first infectious strain, or
obtained or derived from a second infectious strain. In
embodiments, the first infectious strain and the second infectious
strain are of the same species. In other embodiments, the first
infectious strain and the second infectious strain are also the
same. In still other embodiments, they are of the same species but
are of a different strain. In some embodiments, the first
infectious strain and the second infectious strain are of different
species but of the same genus.
[0097] "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.
[0098] "Isolated peptide, polypeptide or protein" means the
peptide, polypeptide or protein is separated from its native
environment and present in sufficient quantity to permit its
identification or use. This means, for example, the peptide,
polypeptide or protein may be (i) selectively produced by
expression cloning or (ii) purified as by chromatography or
electrophoresis. Isolated peptides, polypeptides or proteins 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 peptide, polypeptide or protein may comprise only
a small percentage by weight of the preparation. The peptide,
polypeptide 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 peptides, polypeptides or
proteins. Any of the peptides, polypeptides or proteins provided
herein may be isolated. In some embodiments, the antigens in the
compositions provided herein are peptides, polypeptides or
proteins.
[0099] "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 spheroidal 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 a
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 equal to or greater than 110 nm,
more preferably equal to or greater than 120 nm, more preferably
equal to or greater than 130 nm, and more preferably still equal to
or 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
100 nm, more preferably equal to or greater than 120 nm, 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).
[0100] "Molecular weight less than 10,000" means a molecular weight
calculated based on the molecular structure of a molecule of less
than 10,000.
[0101] "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. In embodiments, the
obtained material has been taken from the original source and has
not been adapted or modified. For example, in embodiments, antigens
obtained from a source may comprise the original amino acid residue
sequence found in that source. In other embodiments, for example,
antigens obtained from a source may comprise the original molecular
structure found in that source.
[0102] "Oligosaccharide(s)" means a saccharide polymer containing a
small number (typically two to twenty) of saccharide units linked
by glycosidic bonds. At a high number of saccharide units,
oligosaccharides may comprise polysaccharides.
[0103] "Peptide(s)" means compounds comprising amino acid residues
joined together primarily by peptide bonds between the carboxyl and
amino groups of adjacent amino acid residues, and possessing 100 or
less amino acid residues. Certain of the peptide bonds in peptide
may be replaced by other bond types, for various purposes, such as
stabilization or coupling.
[0104] "Pharmaceutically acceptable excipient (or carrier)" means a
pharmacologically inactive material used together with the recited
synthetic nanocarriers to formulate the inventive compositions.
Pharmacologically inactive materials can be added to an inventive
dosage form to further facilitate administration of the
composition. Pharmaceutically acceptable 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. Examples,
without limitation, of pharmaceutically acceptable excipients
include calcium carbonate, calcium phosphate, various diluents,
various sugars and types of starch, cellulose derivatives, gelatin,
vegetable oils, polyethylene glycols, preservatives, various
pharmaceutical carriers, sterile saline, lyophilization
stabilizers, and the like. 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.
[0105] "Polysaccharide(s)" means a saccharide polymer made of many
saccharide units linked by glycosidic bonds. At a low number of
saccharide units, polysaccharides may comprise
oligosaccharides.
[0106] "Population" means a defined group of synthetic nanocarriers
that share one or more common physical or chemical characteristics.
Common physical or chemical characteristics may comprise having a
common set of surface antigens, common coupled adjuvant(s), common
materials making up the bulk nanocarrier, a common shape, a common
particle size, and the like. Multiple populations of synthetic
nanocarriers may be identified, for example a first population, a
second population, a third population, a fourth population, and the
like. In an embodiment, three or more populations of synthetic
nanocarriers may be present, preferably wherein each population of
synthetic nanocarriers comprises a set of surface antigens; and
wherein each set of surface antigens is structurally or
immunologically different from one another.
[0107] "Protein(s)" means compounds, typically having a molecular
weight greater than 1000 daltons, comprising amino acid residues
joined together primarily by peptide bonds between the carboxyl and
amino groups of adjacent amino acid residues. Proteins may also
comprise additional bonding structures such as secondary
structures, tertiary structures, and the like. Certain of the
peptide bonds in proteins may be replaced by other bond types, for
various purposes, such as stabilization or coupling.
[0108] "Set of monovalent surface antigens" means a set of surface
antigens in which the surface antigens are not different,
preferably not different either structurally and/or
immunologically. In embodiments, the set of monovalent surface
antigens is composed of multiple copies of one type of surface
antigen that is not different structurally or immunologically
(i.e., multiple copies of the same antigen). The multiple copies of
this same antigen can be in some embodiments strung together, such
as that illustrated in US Publication 2003/0223938. A set of
monovalent surface antigens that is composed of multiple copies of
one type of surface antigen that is not different structurally or
immunologically is not a set of oligovalent (or multivalent)
surface antigens.
[0109] "Set of oligovalent (or multivalent) surface antigens" means
a set of surface antigens in which there are a limited number, that
is greater than one, of different types of surface antigens,
preferably wherein the difference comprises structural difference
and/or immunological difference. In preferred embodiments, the
limited number of surface antigens in the set comprise 2 to 15
types of surface antigens, preferably 2 to 10 types of surface
antigens, more preferably 2 to 8 types of surface antigens, more
preferably 2 to 7 types of surface antigens, more preferably 2 to 6
types of surface antigens, more preferably 2 to 5 types of surface
antigens, more preferably 2 to 4 types of surface antigens, more
preferably 2 to 3 types of surface antigens, and even more
preferably 2 types of surface antigens. In other embodiments, the
set of oligovalent (or multivalent surface antigens) comprise at
least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19 or 20 or more types of surface antigens.
[0110] "Set of surface antigens" means a group of surface antigens
that are identified, preferably identified through measurement
and/or prediction, based on their properties, preferably their
structural and/or their immunological properties. A set of surface
antigens may be identified, in part or in whole, based on
prediction using the chemical synthetic methods used to synthesize,
along with the chemical methods used to couple, the set of surface
antigens and/or the population of synthetic nanocarriers of which
the set of surface antigens are comprised. Multiple sets of surface
antigens can be identified; e.g. a first set, a second set, a third
set, and so on.
[0111] "Structurally different" or "structural difference" means
presenting different molecular structures for interaction with a B
cell receptor. In embodiments, this difference can be expressed by
comparing the prevalence and types of presented antigens in a set
of surface antigens to the prevalence and types of presented
antigens in a different set of surface antigens. If the prevalence
and/or types of presented antigens are different between the sets,
then the sets of surface antigens can be said to be structurally
different. In embodiments, the difference in the prevalence and/or
types of presented antigens can be ascertained by comparison of the
chemical synthetic strategies and formulation strategies used to
generate the surface antigens and/or couple the surface antigens to
a surface of the synthetic nanocarriers. For example, in an
embodiments, if a set of surface antigens was generated using a
particular chemical compound or compounds, and another set of
surface antigens was generated using a different chemical compound
or compounds, then the two sets of surface antigens could be
ascertained to be different. In a different embodiment, if surface
antigens were generated using three chemical compounds to form a
set of three surface antigens, and other surface antigens were
generated using two chemical compounds to form a set of two surface
antigens, then the two sets of surface antigens could be
ascertained to be different. In yet another embodiment, if
non-differing chemical synthetic strategies and formulation
strategies (including using non-different amounts of
materials--within experimental error--in the strategies) are used
to generate two sets of surface antigens and (as appropriate)
couple the two sets of surface antigens to surfaces of synthetic
nanocarriers, and the two sets of surface antigens possessed the
same conformation and orientation, then the two sets of surface
antigens likely would not be structurally different. In
embodiments, the structural difference between a first set of
surface antigens and a second set of surface antigens comprises
non-differing sets of molecules that are presented in orientations
that differ between the first and second sets of surface antigens.
In embodiments, the structural difference between a first set of
surface antigens and a second set of surface antigens comprises
non-differing sets of molecules that are presented in conformations
that differ between the first and second sets of surface antigens.
In embodiments, the structural difference between a first set of
surface antigens and a second set of surface antigens comprises
sets of molecules whose molecular structure is different between
the first and second sets of surface antigens.
[0112] "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.
[0113] "Surface antigen(s)" means an antigen found on or around a
surface of a synthetic nanocarrier. In preferable embodiments,
surface antigens comprise B cell antigens. In embodiments, surface
antigens are coupled to a surface of the synthetic
nanocarriers.
[0114] "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, the inventive synthetic
nanocarriers do not comprise chitosan.
[0115] 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. 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.
[0116] 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 exclude virus-like
particles. In embodiments, when synthetic nanocarriers comprise
virus-like particles, the virus-like particles comprise non-natural
adjuvant (meaning that the VLPs comprise an adjuvant other than
naturally occurring RNA generated during the production of the
VLPs). 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.
[0117] "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 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.
[0118] 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.
[0119] 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).
[0120] 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.
[0121] "Types of surface antigens," "surface antigen types," etc.
means a defined group of surface antigens that share one or more
common chemical and/or immunological characteristics.
[0122] "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). In embodiments, a vaccine may
comprise dosage forms according to the invention.
Nanocarrier Populations and Sets of Surface Antigens
[0123] In embodiments, populations of synthetic nanocarriers share
common physical or chemical characteristics. In embodiments, such
common physical or chemical characteristics may comprise a common
set of surface antigens, common coupled adjuvant(s), common
materials making up the bulk nanocarrier, a common shape, a common
particle size, common surface charge, and the like. Types of
adjuvants, materials, shapes and particle sizes are discussed
throughout the present application.
[0124] In embodiments, a population may share a set of common
surface antigens. These common surface antigens may be grouped
together based on common physical or chemical characteristics, such
as, but not limited to, structural or immunological properties. In
embodiments, the common characteristics may comprise common
orientation or conformation, or sets of molecules sharing a common
molecular structure, or all of the foregoing. In embodiments,
common surface antigens may comprise those having a molecular
weight less than 10,000. In other embodiments, common surface
antigens may comprise being peptides, proteins, oligosaccharides,
polysaccharides, or small molecules. In still other embodiments,
common surface antigens may comprise those having a molecular
weight less than 10,000 and that comprise being peptides, proteins,
oligosaccharides, polysaccharides, or small molecules. In other
embodiments, the common surface antigens may be grouped together
based on the infectious organisms that they were obtained or
derived from; and would be categorized as sharing a common genus,
species, and/or strain. In embodiments wherein the surface antigens
have a molecular weight less than 10,000, common surface antigens
may be grouped together based on classes of molecules such as
chemical warfare agents, environmental toxins, addictive or abused
substances, and physiologically endogenous molecules including but
not limited to hormones, lipids and neurotransmitters. In
embodiments, sets of common surface antigens may be defined by the
strength of their ability to induce an antibody response in vivo.
For example one set of surface antigens may have the ability to
ability to induce high affinity antibody production in vivo, while
another set of common surface antigens may induce low affinity
antibody production in vivo.
[0125] In embodiments, a set of surface antigens (e.g., a first
and/or second set of surface antigens) can comprise antigens
obtained or derived from an infectious agent. In some embodiments,
the infectious agent is a bacterium, fungus, virus, protozoan, or
parasite. In other embodiments, the virus is a pox virus, smallpox
virus, ebola virus, marburg virus, measles virus (in embodiments,
the antigen can be obtained or derived from hemagglutinin protein,
hemagglutinin noose epitope, hemagglutining amino acids 106-114
and/or 519-550, etc.), dengue fever virus, influenza virus,
influenza A virus (in embodiments, the antigen can be obtained or
derived from HA protein, M2e protein, etc.), influenza H5N1 virus,
influenza H1N1 virus, infectious salmon anemia virus, parainfluenza
virus, respiratory syncytial virus, rubeola virus, human
immunodeficiency virus, human papillomavirus, varicella-zoster
virus, herpes simplex virus, cytomegalovirus, Epstein-Barr virus,
JC virus, rhabdovirus, rotavirus, rhinovirus, adenovirus,
papillomavirus (in embodiments, the antigen can be obtained or
derived from L1 or L2 protein), parvovirus, picornavirus,
poliovirus, virus that causes mumps, virus that causes rabies,
reovirus, rubella virus, togavirus, orthomyxovirus, retrovirus,
hepadnavirus, coxsackievirus, equine encephalitis virus, tick-borne
encephalitis, Japanese encephalitis virus, yellow fever virus, Rift
Valley fever virus, hepatitis A virus, hepatitis B virus, hepatitis
C virus, hepatitis D virus, or hepatitis E virus.
[0126] In embodiments, a set of surface antigens (e.g., a first
and/or second set of surface antigens) comprises or is obtained or
derived from a virus of a family of viruses shown below in Table 1.
In another embodiment, a set of surface antigens (e.g., a first
and/or second set of surface antigens) comprises or is obtained or
derived from a virus of a species provided in Table 1. In still
another embodiment, a set of surface antigens (e.g., a first and/or
second set of surface antigens) comprises or is obtained or derived
from an antigen provided in Table 1.
TABLE-US-00001 TABLE 1 Viral Infectious Agents Family Exemplary
Species Exemplary Antigens Adenoviridae adenovirus VI, VII, E1A,
E3-19K, 52K Picornaviridae coxsackievirus, , VP1 hepatitis A virus
Surface antigen poliovirus, 3A protein, capsid protein Rhinovirus
(e.g., nucleocapsid, surface projection, type 16) and transmembrane
proteins Herpesviridae Herpes simplex Capsid proteins (e.g., UL6,
(type 1 and type 2) UL18, UL35, UL38, and UL19) Varicella-zoster
Early antigen virus Epstein-barr virus, Early antigen, capsid
antigen Human Pp65, gB, p52 cytomegalovirus Human herpesvirus,
Latent nuclear antigen-1 (e.g., type 8) Hepadnaviridae Hepatitis B
virus surface antigen Flaviviridae Hepatitis C virus, NS3, Envelop
protein (e.g., E2 yellow fever virus, domain) dengue virus, West
Nile virus Retroviridae HIV gp120, p24, and lipopeptides Gag
(17-35), Gag (253-284), Nef (66-97), Nef (116-145), and Pol
(325-355); see Roberts et al., J. Immunol. Methods, 365(1-2):
27-37, 2011 Orthomyxoviridae Influenza virus neuraminidase, surface
antigen, Measles virus, Nucleocapsid protein, matrix Mumps virus
protein, phosphoprotein, fusion Parainfluenza virus protein,
hemagglutinin, hemagglutinin-neuraminidase, glycoprotein,
Paramyxoviridae Respiratory syncytial virus Human metapneumovirus
Papillomaviridae Human E6, E7, capsid antigen papillomavirus (e.g.,
type 16 and 18) Rhabdoviridae Rabies virus Envelope lipoprotein
Togaviridae Rubella virus Capsid protein Paroviridae Human
bocarivus, Capsid protein, non-structural Parvovirus B19 protein
(NS)
[0127] In other embodiments, a set of surface antigens (e.g., a
first and/or second set of surface antigens) can comprise antigens
obtained or derived from bacterial organisms such as Borrelia
species, Bacillus anthracis, Borrelia burgdorferi, Bordetella
pertussis, Bordetella parapertussis, Camphylobacter jejuni,
Chlamydia species, Chlamydial psittaci, Chlamydial trachomatis,
Clostridium species, Clostridium tetani, Clostridium botulinum,
Clostridium perfringens, Corynebacterium diphtheriae, Coxiella
species, an Enterococcus species, Erlichia species, Escherichia
coli, Francisella tularensis, Haemophilus species, Haemophilus
influenzae, Haemophilus parainfluenzae, Lactobacillus species, a
Legionella species, Legionella pneumophila, Leptospirosis
interrogans, Listeria species, Listeria monocytogenes,
Mycobacterium species, Mycobacterium tuberculosis, Mycobacterium
leprae, Mycoplasma species, Mycoplasma pneumoniae, Neisseria
species, Neisseria meningitidis, Neisseria gonorrhoeae,
Pneumococcus species (e.g., type 6A, 6B, 3, 4, 14, 19F, etc.),
Pseudomonas species, Pseudomonas aeruginosa, Salmonella species,
Salmonella typhi, Salmonella enterica, Rickettsia species,
Rickettsia ricketsii, Rickettsia typhi, Shigella species,
Staphylococcus species, Staphylococcus aureus, Streptococcus
species, Streptococccus pnuemoniae, Streptococcus pyrogenes,
Streptococcus mutans, Treponema species, Treponema pallidum, a
Vibrio species, Vibrio cholerae, Yersinia pestis, and the like.
[0128] In embodiments, a set of surface antigens (e.g., a first
and/or second set of surface antigens) comprises or is obtained or
derived from a bacteria of a genera of bacteria shown below in
Table 2. In another embodiment, a set of surface antigens (e.g., a
first and/or second set of surface antigens) comprises or is
obtained or derived from a bacterial species provided in Table 2.
In still another embodiment, a set of surface antigens (e.g., a
first and/or second set of surface antigens) comprises or is
obtained or derived from an antigen provided in Table 2.
TABLE-US-00002 TABLE 2 Bacterial Infectious Agents Pathogenic
Bacterial Genera Exemplary Species Exemplary Antigens Bordetella
Bordetella pertussis pertussis toxin (PT), filamentous
hemagglutinin (FHA), pertactin (PRN), and fimbriae (FIM 2/3)
Borrelia Borrelia burgdorferi VlsE; DbpA and OspA Brucella Brucella
abortus Hia, PrpA, MltA, L7/L12, D15, 0187, VirJ, Mdh, AfuA
Brucella canis L7/L12 Brucella melitensis Out membrane proteins
such as Omp28 Brucella suis Campylobacter Campylobacter jejuni;
LPS, an 100-kD antigen Chlamydia and Chlamydia pneumoniae See
Richard et al., J. Infectious Chlamydophila Chlamydia trachomatis
Diseases. 181: S521 (2000) Chlamydophila psittaci Clostridium
Clostridium botulinum antigen types A, B, C, D, and E Clostridium
difficile FliC, FliD, and Cwp84 Clostridium perfringens
alpha-toxin, theta-toxin, fructose 1,6- biphosphate-aldolase (FBA),
glyceraldehydes-3-phosphate dehydrogenase (GPD), pyruvate:
ferredoxin oxidoreductase (PFOR), elongation factor-G (EF-G), and a
hypothetical protein (HP) Clostridium tetani T toxin
Corynebacterium Corynebacterium diphtheriae Toxoid antigen
Enterococcus Enterococcus faecalis capsular polysaccharides
Enterococcus faecium Escherichia Escherichia coli See Moriel et
al., PNAS 107(20): 9072- 9077 (2010) Francisella Francisella
tularensis See Havlasova et al., Proteomics 2(7): 857-867, 2002
Haemophilus Haemophilus influenzae capsular polysaccharides,
Protein D, Helicobacter Helicobacter pylori See Bumann et al.,
Proteomics 4(10): 2843-2843, 2004 Legionella Legionella pneumophila
Mip Leptospira Leptospira interrogans See Brown et al., Infect Immu
59(5): 1772-1777, 1991 Listeria Listeria monocytogenes
nucleoprotein (NP) Mycobacterium* Mycobacterium leprae
Mycobacterium tuberculosis RD1, PE35, PPE68, EsxA, EsxB, RD9, and
EsxV Mycobacterium ulcerans Mycoplasma Mycoplasma pneumoniae Hsp70
Neisseria Neisseria gonorrhoeae Neisseria meningitidis See Litt et
al., J. Infectious Disease 190(8): 1488-1497, 2004 Pseudomonas
Pseudomonas aeruginosa Lipopolysaccharides Rickettsia Rickettsia
rickettsii Surface antigen Salmonella Salmonella typhi Salmonella
typhimurium Shigella Shigella sonnei Staphylococcus Staphylococcus
aureus See Vytvtska et al., Proteomics 2(5): 580-590, 2002; Etz et
al., PNAS 99(10): 6573-6578; 2002 Staphylococcus epidermidis
Staphylococcus saprophyticus Streptococcus Streptococcus agalactiae
Streptococcus pneumoniae Sp1, Sp2, Sp3 Streptococcus pyogenes Lei
et al., J. Infectious Disease 189(1): 79-89, 2004 Treponema
Treponema pallidum Glycerophosphodiester Phosphodiesterase Vibrio
Vibrio cholerae Outer membrane proteins such as OmpK Yersinia
Yersinia pestis Chaperone-usher protein, capsular protein (F1), and
V protein
[0129] In still other embodiments, a set of surface antigens (e.g.,
a first and/or second set of surface antigens) can comprise
antigens obtained or derived from antigens of fungal, protozoan,
and/or parasitic organisms such as Aspergillus species, Candida
species, Candida albicans, Candida tropicalis, Cryptococcus
species, Cryptococcus neoformans, Entamoeba histolytica,
Histoplasma capsulatum, Leishmania species, Nocardia asteroides,
Plasmodium falciparum, Toxoplasma gondii, Trichomonas vaginalis,
Toxoplasma species, Trypanosoma brucei, Schistosoma mansoni, and
the like.
[0130] In still other embodiments, a set of surface antigens (e.g.,
a first and/or second set of surface antigens) can comprise
antigens obtained or derived from a toxin, such as O-Alkyl
(<C10, incl. cycloalkyl) alkyl (Me, Et, n-Pr or
i-Pr)-phosphonofluoridates (e.g. Sarin: O-Isopropyl
methylphosphonofluoridate, Soman: O-Pinacolyl
methylphosphonofluoridate), O-Alkyl (<C10, incl. cycloalkyl)
N,N-dialkyl (Me, Et, n-Pr or i-Pr) phosphoramidocyanidates (e.g.
Tabun: O-Ethyl N,N-dimethylphosphoramidocyanidate), O-Alkyl (H or
<C10, incl. cycloalkyl) S-2-dialkyl (Me, Et, n-Pr or
i-Pr)-aminoethyl alkyl (Me, Et, n-Pr or i-Pr) phosphonothiolates
and corresponding alkylated or protonated salts (e.g. VX: O-Ethyl
S-2-diisopropylaminoethyl methylphosphonothiolate), Sulfur
mustards: 2-Chloroethylchloromethylsulfide, Mustard gas:
Bis(2-chloroethyl)sulfide, Bis(2-chloroethylthio)methane,
Sesquimustard: 1,2-Bis(2-chloroethylthio)ethane,
1,3-Bis(2-chloroethylthio)-n-propane,
1,4-Bis(2-chloroethylthio)-n-butane,
1,5-Bis(2-chloroethylthio)-n-pentane,
Bis(2-chloroethylthiomethyl)ether, O-Mustard:
Bis(2-chloroethylthioethyl)ether, Lewisites: Lewisite 1:
2-Chlorovinyldichloroarsine, Lewisite 2:
Bis(2-chlorovinyl)chloroarsine, Lewisite 3:
Tris(2-chlorovinyl)arsine, Nitrogen mustards: HN1:
Bis(2-chloroethyl)ethylamine, HN2: Bis(2-chloroethyl)methylamine,
HN3: Tris(2-chloroethyl)amine, Saxitoxin, Ricin, Amiton:
O,O-Diethyl S-(2-(diethylamino)ethyl)phosphorothiolate and
corresponding alkylated or protonated salts, PFIB:
1,1,3,3,3-Pentafluoro-2-(trifluoromethyl)-1-propene,
3-Quinuclidinyl benzilate (BZ), Phosgene: Carbonyl dichloride,
Cyanogen chloride, Hydrogen cyanide and Chloropicrin:
Trichloronitromethane.
[0131] In other embodiments, a set of surface antigens (e.g., a
first and/or second set of surface antigens) comprises or is
obtained or derived from a fungus of a genera of fungi shown below
in Table 3. In another embodiment, a set of surface antigens (e.g.,
a first and/or second set of surface antigens) comprises or is
obtained or derived from a fungal species provided in Table 3. In
still another embodiment, a set of surface antigens (e.g., a first
and/or second set of surface antigens) comprises or is obtained or
derived from an antigen provided in Table 3.
TABLE-US-00003 TABLE 3 Fungal Infectious Agents Genera Exemplary
Species Exemplary Antigens Candida C. albicans Surface antigens,
see also Thomas et al., Proteomics 6(22): 6033-6041, 2006
Aspergillus Aspergillus fumigatus Stevens et al., Medical and
Aspergillus flavus. Mycology 49 (Suppl. 1): S170-S176, 2011
Cryptococcus Cryptococcus Capsular glycoproteins, neoformans,
Cryptococcus laurentii and Cryptococcus albidus, Cryptococcus
gattii Histoplasma Histoplasma Yps3P, Hsp60 capsulatum Pneumocystis
Pneumocystis Major surface proteins jirovecii (Msg) such as MsgC1,
MsgC3, MsgC8, and MsgC9 Stachybotrys Stachybotrys SchS34,
chartarum
[0132] In still further embodiments, a set of surface antigens
(e.g., a first and/or second set of surface antigens) can comprise
antigens obtained or derived from an abused or addictive substance.
In some embodiments, the abused or addictive substance is a drug,
such as an illegal drug, an over-the-counter drug or a prescription
drug. In other embodiments, the abused or addictive substance has
mood-altering effects, and, therefore, includes inhalants and
solvents. In other embodiments, the abused or addictive substance
is one that has no mood-altering effects or intoxication
properties, and, therefore, includes anabolic steroids. Abused or
addictive substances include, but are not limited to, cannabinoids
(e.g., hashish, marijuana), depressants (e.g., barbituates,
benodiazepines, flunitrazepam (Rohypnol), GHB, methaqualone
(quaaludes)), dissociative anesthetics (e.g., ketamine, PCP),
hallucinogens (e.g, LSD, mescaline, psilocybin), opioids and
morphine derivatives (e.g., codeine, fentanyl, heroin, morphine,
opium), stimulants (amphetamine, cocaine, Ecstacy (MDMA),
methamphetamine, methylphenidate (Ritalin), nicotine), anabolic
steriods, and inhalants. In embodiments, the antigen comprises a
cocaine analog, such as norcocaine. In other embodiments, the
antigen comprises cotinine.
[0133] In embodiments of the present invention, different
populations of synthetic nanocarriers that each comprise a set of
surface antigens may be combined. The difference between the
populations is based on the differences between the sets of surface
antigens.
[0134] In certain embodiments, these differences can comprise
differences in physical or chemical characteristics, such as, but
not limited to, structural or immunological properties. In
embodiments, the differences may comprise differences in surface
antigen orientation or conformation, or differences in molecular
structure between sets of surface antigens. In still other
embodiments, the difference in surface antigens may be based on the
infectious organisms that they were obtained or derived from; and
would be categorized as being from a different genus, species,
and/or strain. In embodiments wherein the surface antigens have a
molecular weight less than 10,000, surface antigens may be
different based on chemical classes such as chemical warfare
agents, addictive or abused substances, and endogenous molecules
including but not limited to hormones, lipids and
neurotransmitters.
[0135] In embodiments, the differences may comprise differences in
surface antigen orientation or conformation. For instance,
different points of attachment of a surface antigen to a synthetic
nanocarrier would give rise to different presentations of that
surface antigen. These different presentations may produce
antibodies that recognize different epitopes of the surface
antigen. Surface antigens may be presented with different
conformations, and may be synthesized or modified to achieve those
conformations. For example, peptide or protein truncations may be
performed in which results in modified conformational changes in
the peptide or protein antigen of interest. Alternatively amino
acids or chemical linkers may be added in order to add length or
stabilize a specific orientation that alters peptide or protein
antigen exposure. Similarly antigens such those as having a
molecular weight less than 10,000, or oligosaccharides, or
polysaccharides may be altered by addition of a chemical linker, or
chemical modification.
[0136] In other embodiments, differences between populations of
synthetic nanocarriers may be based on differences between sets of
surface antigens based on different molecular structures and/or
prevalence of the antigens. In some embodiments, the difference may
comprise a difference in the prevalence of one or more of the types
of surface antigens between the sets.
[0137] In embodiments wherein a population comprises a monovalent
set of surface antigens, the molecular structure, preferably the
antigen type, of its set of surface antigens may be different from
the molecular structure of a set of monovalent surface antigens of
another population or other populations. In certain embodiments,
wherein a population comprises a monovalent set of surface
antigens, the prevalence of surface antigens of which its set of
surface antigens is comprised may be different from the prevalence
of surface antigens of which a set of monovalent surface antigens
of another population or other populations is comprised.
[0138] In embodiments wherein a population of synthetic
nanocarriers comprises a set of oligovalent (or multivalent)
surface antigens, various antigen types at different prevalences
may be combined within the set to form combinations of such surface
antigen types. Accordingly, in embodiments wherein at least one
population of synthetic nanocarriers comprises a set of oligovalent
(or multivalent) surface antigens, the molecular structure of the
set of oligovalent (or multivalent) surface antigens (which can be
expressed as a function of both the molecular structure of each
type of antigen, along with their prevalence within the set) can be
different from the molecular structure of the set of surface
antigens of another population or other populations. In
embodiments, this may be because another population comprises a
monovalent set of surface antigens (wherein the sets of surface
antigens would be different by definition) or because another
population comprises a set of oligovalent (or multivalent) surface
antigens wherein the molecular structures of the two sets of
surface antigens (expressed as a molecular structure of each type
of antigen and/or a prevalence of each antigen type within the set)
are different.
[0139] For example the sets of surface antigens can be comprised of
a set of enantiomers such as (R) and (S) nicotine. The enantiomers
can be present in equal amounts on the same or different
nanocarriers and can be present in unequal amounts on the same or
different populations of synthetic nanocarriers. In another
embodiment, a set of surface antigens may comprise two structurally
different but related molecules such as cotinine and nicotine,
either optically pure or racemic. The cotinine and nicotine can be
present in equal amounts on the same or different populations of
synthetic nanocarriers and can be present in unequal amounts on the
same or different populations of synthetic nanocarriers. In
addition, sets of surface antigens can be comprised of antigens
from a single organism comprised of several serotypes such as the
capsular antigenic polysaccharides from Streptococcus Pneumoniae
serotypes 4, 6B, 9V,14, 18C, 19F, and 23F. The various antigens can
be present in equal amounts on the same or different populations of
synthetic nanocarriers and can be present in unequal amounts on the
same or different populations of synthetic nanocarriers. In
embodiments, sets of surface antigens can comprise a family of
different antigens from a single organism such as the capsid
proteins L1 and L2 of the human papillomavirus. The sets of surface
antigens can be present in equal amounts on the same or different
populations of synthetic nanocarriers and can be present in unequal
amounts on the same or different populations of synthetic
nanocarriers. In embodiments, sets of surface antigens can comprise
several small molecules of diverse structures such as the war gases
VX, sarin and soman. The different compounds can be present in
equal amounts on the same or different populations of synthetic
nanocarriers and can be present in unequal amounts on the same or
different populations of synthetic nanocarriers. For instance, in
an embodiment, one set of surface antigens may comprise 50% of VX
and 50% sarin, while another set of surface antigens may comprise
80% of VX and 20% sarin, where the percent of surface antigens may
be a weight percent or mole percent, and based on the total weight
or total number of moles of surface antigens.
[0140] In embodiments, differences in sets of surface antigens may
comprise providing a population of synthetic nanocarriers that
comprises a set of surface antigens from a type or types such as
having a molecular weight less than 10,000 and/or being peptides,
proteins, oligosaccharides, polysaccharides, or small molecules;
and then providing another population of synthetic nanoparticles
comprising a different set of surface antigens from a type or types
such as having a molecular weight less than 10,000 and/or or being
peptides, proteins, oligosaccharides, polysaccharides, or small
molecules.
[0141] In embodiments, when a first set of surface antigens
comprises surface antigens having a molecular weight less than
10,000, a second set of surface antigens comprises peptides,
proteins, oligosaccharides, polysaccharides or small molecules
(provided the sets are structurally or immunologically different).
In embodiments, when a first set of surface antigens comprises
surface antigens comprising peptides, a second set of surface
antigens comprises surface antigens comprising those having a
molecular weight less than 10,000 and/or comprising proteins,
oligosaccharides, polysaccharides or small molecules. In
embodiments, when a first set of surface antigens comprises surface
antigens comprising proteins, a second set of surface antigens
comprises surface antigens comprising those having a molecular
weight less than 10,000 and/or comprising peptides,
oligosaccharides, polysaccharides or small molecules. In
embodiments, when a first set of surface antigens comprises surface
antigens comprising oligosaccharides, a second set of surface
antigens comprises surface antigens comprising those having a
molecular weight less than 10,000 and/or comprising peptides,
proteins, polysaccharides or small molecules. In embodiments, when
a first set of surface antigens comprises surface antigens
comprising polysaccharides, a second set of surface antigens
comprises surface antigens comprising those having a molecular
weight less than 10,000 and/or comprising peptides, proteins,
oligosaccharides or small molecules. In embodiments, when a first
set of surface antigens comprises surface antigens comprising small
molecules, a second set of surface antigens comprises surface
antigens comprising those having a molecular weight less than
10,000 and/or comprising peptides, proteins, oligosaccharides or
polysaccharides (provided the sets are structurally or
immunologically different).
[0142] In embodiments, when a second set of surface antigens
comprises surface antigens having a molecular weight less than
10,000, a first set of surface antigens comprises peptides,
proteins, oligosaccharides, polysaccharides or small molecules
(provided the sets are structurally or immunologically different).
In embodiments, when a second set of surface antigens comprises
surface antigens comprising peptides, a first set of surface
antigens comprises surface antigens comprising those having a
molecular weight less than 10,000 and/or comprising proteins,
oligosaccharides, polysaccharides or small molecules. In
embodiments, when a second set of surface antigens comprises
surface antigens comprising proteins, a first set of surface
antigens comprises surface antigens comprising those having a
molecular weight less than 10,000 and/or comprising peptides,
oligosaccharides, polysaccharides or small molecules. In
embodiments, when a second set of surface antigens comprises
surface antigens comprising oligosaccharides, a first set of
surface antigens comprises surface antigens comprising those having
a molecular weight less than 10,000 and/or comprising peptides,
proteins, polysaccharides or small molecules. In embodiments, when
a second set of surface antigens comprises surface antigens
comprising polysaccharides, a first set of surface antigens
comprises surface antigens comprising those having a molecular
weight less than 10,000 and/or comprising peptides, proteins,
oligosaccharides or small molecules. In embodiments, when a second
set of surface antigens comprises surface antigens comprising small
molecules, a first set of surface antigens comprises surface
antigens comprising those having a molecular weight less than
10,000 and/or comprising peptides, proteins, oligosaccharides or
polysaccharides (provided the sets are structurally or
immunologically different).
[0143] Other differences between populations of synthetic
nanocarriers may be based on differences between sets of surface
antigens based on the source of the antigens. For instance, in an
embodiment, such differences may be based on differences of the
infectious genera, species and/or strains from which the surface
antigens were obtained or derived. For example, one population of
synthetic nanocarriers may comprise a set of surface antigens
obtained or derived from a bacterial source, such as E. Coli,
mycobacterium tuberculosis, clostridium tetani or bacillus
anthracis while another population may comprise a set of surface
antigens obtained or derived from a viral source, such as influenza
virus, hepatitis B virus, hepatitis C virus, and human herpesvirus.
In embodiments, one population of synthetic nanocarriers may
comprise a set of surface antigens obtained or derived from a
bacterial source, such as those noted above, while another
population may comprise a set of surface antigens obtained or
derived from fungi such as candida albicans, or pneumocystis
jiroveci. In other embodiments, one population of synthetic
nanocarriers may comprise a set of surface antigens obtained or
derived from a viral source, while another population may comprise
a set of surface antigens obtained or derived from parasites such
as plasmodium falciparum. Other combinations and sub-combinations
along the lines of the illustrations above are contemplated to be
within the scope of the present invention.
[0144] In other embodiments, the various sets of surfaces antigens
may be obtained or derived from infectious genera, species or
strains that are not different. In embodiments, those differences
may arise from selection of different antigens within the
infectious genus, species or strain. For example, one set of
surface antigens may be obtained or derived from one viral coat
protein, while another set of surface antigens may be obtained or
derived from different epitopes from the same or another viral coat
protein from the same virus. In an embodiment, different sets of
surface antigens may be obtained or derived from one viral protein,
for example, the cytomegalovirus (CMV) capsid protein, or other CMV
proteins, which may comprise several distinct epitopes. Likewise,
different sets of surface antigens may be obtained or derived from
different epitopes of diphtheria or tetanus toxin.
[0145] In other embodiments, differences between populations of
synthetic nanocarriers may be based on differences between sets of
surface antigens based on different chemical classes of the
antigens. For instance, in the case of molecules having a molecular
weight less than 10,000 such differences may be based on
differences of the chemical scaffold, or the overall molecular
structure, or the activity exhibited by such molecules. For
instance, sets of different surface antigens may be obtained or
derived from sets of surface antigens of differing structure but
with similar activities like opiods such as morphine and heroin. In
other embodiments, different sets of surface antigens may be
comprised of molecules with similar structures but with differing
activities exemplified by enantiomers such as (R) and (S) Ritalin,
or (R) and (S) nicotine. In embodiments, difference between sets of
surface antigens may comprise compounds and their metabolites such
as terfenadine and fexofenadine or astemazole and norastemazole.
Differences between sets of surface antigens may also be based on
the un-relatedness of the structure of compounds such as nicotine
and methamphetamine.
[0146] In other embodiments, differences between populations of
synthetic nanocarriers may be based on differences between sets of
surface antigens based on immunological differences between the
sets of surface antigens. In embodiments, sets of surface antigens
may be defined by their ability to induce an immune response in
vivo. For example, one set of surface antigens may have the ability
to induce high levels of high affinity antibody production to an
antigen of interest in vivo, while a second set of surface antigens
may not induce high levels of high affinity antibody production in
vivo to that antigen. As another example a first set of surface
antigens may have the ability to generate antibody titers specific
to the antigens of the first set of surface antigens, while a
second set of surface antigens may have the ability generate
antibody titers specific to the antigens of the second set of
surface antigens. In embodiments, the second set of surface
antigens generate antibody titers specific to the antigens of the
second set of surface antigens but not to the antigens of the first
set.
Inventive Compositions and Related Methods
[0147] 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, and
also 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)).
[0148] In embodiments, the present invention comprises synthetic
nanocarrier means for presenting sets of surface antigens,
preferably sets of monovalent or oligovalent surface antigens. A
particular inventive embodiment comprises a first synthetic
nanocarrier means for presenting a first set of surface antigens,
preferably a first set of monovalent or oligovalent surface
antigens; and a second synthetic nanocarrier means for presenting a
second set of surface antigens; preferably a second set of
monovalent or oligovalent surface antigens. Such synthetic
nanocarrier means for presenting surface antigens are disclosed
throughout the present disclosure, and encompass the embodiments
disclosed herein.
[0149] 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.
[0150] 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.
[0151] 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, differential centrifugation or
settling.
[0152] Elements of the inventive synthetic nanocarriers such as
moieties of which an immunofeature surface is comprised, 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 US Patent Application 2006/0002852 to
Saltzman et al., Published US Patent Application 2009/0028910 to
DeSimone et al., or Published International Patent Application
WO/2008/127532 A1 to Murthy et al.
[0153] Alternatively or additionally, synthetic nanocarriers can be
coupled to moieties of which an immunofeature surface is comprised,
targeting moieties, adjuvants, antigens and/or other elements
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.
[0154] 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 or cuboidal. In some
embodiments, synthetic nanocarriers are ovals or ellipses. In some
embodiments, synthetic nanocarriers are cylinders, cones, or
pyramids.
[0155] 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.
[0156] 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.
[0157] 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.).
[0158] In some embodiments, synthetic nanocarriers can comprise one
or more polymers. In some embodiments, such a polymer 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.
[0159] In some embodiments, an immunofeature surface, targeting
moiety, antigens, adjuvants, and/or oligonucleotide can be
covalently associated with a polymeric matrix. In some embodiments,
covalent association is mediated by a linker. In some embodiments,
an immunofeature surface, targeting moiety, antigens, adjuvants,
and/or oligonucleotide can be noncovalently associated with a
polymeric matrix. For example, in some embodiments, an
immunofeature surface, targeting moiety, antigens, adjuvants,
and/or oligonucleotide can be encapsulated within, surrounded by,
and/or dispersed throughout a polymeric matrix. Alternatively or
additionally, an immunofeature surface, targeting moiety, antigens,
adjuvants, and/or nucleotide can be associated with a polymeric
matrix by hydrophobic interactions, charge interactions, van der
Waals forces, etc.
[0160] 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.
[0161] 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, polyhydroxyacids (e.g.
poly(.beta.-hydroxyalkanoate))), poly(orthoesters),
polycyanoacrylates, polyvinyl alcohols, polyurethanes,
polyphosphazenes, polyacrylates, polymethacrylates, polyureas,
polystyrenes, polyamines, polylysine, polylysine-PEG copolymers,
poly(ethyleneimine), poly(ethylene imine)-PEG copolymers, and
polyphosphazines.
[0162] 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.
[0163] 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.
[0164] 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.
[0165] 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.
[0166] 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.
[0167] 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.
[0168] 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.
[0169] 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.
[0170] 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).
[0171] 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.
[0172] 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.
[0173] 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 universal T cell
antigen, such as a T helper cell antigen, the universal T cell
antigen can be encapsulated in the nanocarrier.
[0174] In some embodiments, synthetic nanocarriers do 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).
[0175] 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.
[0176] 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, hydroxyethylstarch, carageenan, glycon, amylose,
chitosan, N,O-carboxylmethylchitosan, algin and alginic acid,
starch, chitin, 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.
[0177] 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.
[0178] In embodiments, when preparing synthetic nanocarriers as
carriers for antigens and/or adjuvants for use in vaccines, methods
for coupling the antigens and/or adjuvants to the synthetic
nanocarriers may be useful. If the antigen and/or adjuvant is a
small molecule it may be of advantage to attach the antigen and/or
adjuvant 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 antigen and/or adjuvant to the synthetic
nanocarrier through the use of these surface groups rather than
attaching the antigen and/or adjuvant to a polymer and then using
this polymer conjugate in the construction of synthetic
nanocarriers.
[0179] Surface antigens can be coupled to the synthetic
nanocarriers by a variety of methods. In embodiments, the surface
antigen is coupled to an external surface of the synthetic
nanocarrier covalently or non-covalently.
[0180] In certain embodiments, the coupling can be via a covalent
linker. In embodiments, surface antigens and/or adjuvants 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
surface antigens and/or adjuvants containing an alkyne group or by
the 1,3-dipolar cycloaddition reaction of alkynes on the surface of
the nanocarrier with surface antigens and/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.
[0181] 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.
[0182] An amide linker is formed via an amide bond between an amine
on one component such as the peptide 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 peptides
and activated carboxylic acid such N-hydroxysuccinimide-activated
ester.
[0183] 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 a surface antigens and/or adjuvants containing
thiol/mercaptan group (--SH) with another activated thiol group on
a polymer or nanocarrier or a nanocarrier containing
thiol/mercaptan groups with an antigen and/or adjuvants containing
activated thiol group.
[0184] 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 antigen and/or adjuvant.
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 "click" reaction or
CuAAC.
[0185] 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 and/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 and/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
and/or adjuvant to the particle through the 1,4-disubstituted
1,2,3-triazole linker.
[0186] 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
and/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 an antigen and/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 an antigen and/or adjuvant with an alkene group
on a second component such as a polymer or nanocarrier.
[0187] A hydrazone linker is made by the reaction of a hydrazide
group on one component such as the antigen and/or adjuvant with an
aldehyde/ketone group on the second component such as the
nanocarrier.
[0188] A hydrazide linker is formed by the reaction of a hydrazine
group on one component such as the antigen and/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.
[0189] 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 and/or adjuvant with an aldehyde or ketone group on the
second component such as the nanocarrier.
[0190] An urea or thiourea linker is prepared by the reaction of an
amine group on one component such as the antigen and/or adjuvant
with an isocyanate or thioisocyanate group on the second component
such as the nanocarrier.
[0191] An amidine linker is prepared by the reaction of an amine
group on one component such as the antigen and/or adjuvant with an
imidoester group on the second component such as the
nanocarrier.
[0192] An amine linker is made by the alkylation reaction of an
amine group on one component such as the antigen and/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
and/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.
[0193] A sulfonamide linker is made by the reaction of an amine
group on one component such as the antigen and/or adjuvant with a
sulfonyl halide (such as sulfonyl chloride or sulfonyl fluoride)
group on the second component such as the nanocarrier.
[0194] 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.
[0195] 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. A
antigen or adjuvant containing a metal ligand can also be
conjugated to a nanocarrier containing a metal complex via a
metal-ligand complex.
[0196] 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 may be
prepared with a group which is compatible with the attachment
chemistry that is presented by the synthetic nanocarriers' surface.
In other embodiments, 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 a peptide antigen and/or adjuvant containing an
acid group via the other end of the ADH linker on NC to produce the
corresponding VLP or liposome peptide conjugate.
[0197] 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 and/or adjuvant can be coupled by adsorbtion to a
pre-formed synthetic nanocarrier or it/they can be coupled by
encapsulation during the formation of the synthetic
nanocarrier.
[0198] In embodiments, surface antigens can be non-covalently
coupled to synthetic nanocarriers using various 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. When coupling
charged surface antigens, the synthetic nanocarriers can be
produced in the presence of surfactants which become adsorbed to
surfaces of the synthetic nanocarrier and in doing so they impart a
charge to the synthetic nanocarrier. Charged surface antigens can
then be non-covalently attached to the charged synthetic
nanocarrier by a charge-charge interaction (see for example O'Hagen
WO2000006123A1).
[0199] In embodiments, the inventive synthetic nanocarriers can be
combined with one or more adjuvants by admixing in the same vehicle
or delivery system. Such adjuvants may include, but are not limited
to the adjuvant provided herein, such as 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.), liposomes and liposomal formulations
such as AS01, AS15, 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. The doses of such other adjuvants can be
determined using conventional dose ranging studies.
[0200] In embodiments, the inventive synthetic nanocarriers can be
combined with other antigens different, similar or identical to
those coupled to a nanocarrier (with or without adjuvant, utilizing
or not utilizing another delivery vehicle) administered separately
at a different time-point and/or at a different body location
and/or by a different immunization route or with another antigen
and/or adjuvant-carrying synthetic nanocarrier administered
separately at a different time-point and/or at a different body
location and/or by a different immunization route.
[0201] Populations of synthetic nanocarriers may be combined to
form 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.
[0202] 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, polyoxyethylene9-10 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).
[0203] Compositions according to the invention comprise inventive
synthetic nanocarriers in combination with pharmaceutically
acceptable excipients or carriers. 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.
[0204] Doses of dosage forms contain varying amounts of populations
of synthetic nanocarriers according to the invention. The amount of
synthetic nanocarriers present in the inventive dosage forms can be
varied according to the nature of the sets of surface antigens, the
therapeutic benefit to be accomplished, and other such parameters.
In embodiments, dose ranging studies can be conducted to establish
optimal therapeutic amount of synthetic nanocarriers to be present
in the dosage form. In embodiments, first and second populations
are present in an amount effective to generate an immune response
to the first set of surface antigens and the second set of surface
antigens upon administration to a subject. It may be possible to
determine amounts of the first, second, and/or subsequent
populations 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.
[0205] 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. In embodiments, methods of manufacture comprise
preparing a first population of synthetic nanocarriers that
comprise a first set of surface antigens; preparing a second
population of synthetic nanocarriers that comprise a second set of
surface antigens; and combining the first and second populations of
synthetic nanocarriers into a pharmaceutical dosage form; wherein
the first set of surface antigens and the second set of surface
antigens are structurally or immunologically different.
[0206] 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.
[0207] The inventive compositions may be administered by a variety
of routes of administration, including but not limited to
parenteral (such as subcutaneous, intramuscular, intravenous, or
intradermal); oral; transnasal, intranasal, transmucosal,
sublingual, rectal, ophthalmic, transdermal, transcutaneous or by a
combination of these routes.
[0208] The compositions and methods described herein can be used to
induce, enhance, modulate, stimulate, suppress, 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.
[0209] The subjects provided herein can have or be at risk of
having an addiction to an abused or addictive substance.
[0210] 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.
[0211] 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.
EXAMPLES
[0212] The invention will be more readily understood by reference
to the following examples, which are included merely for purposes
of illustration of certain aspects and embodiments of the present
invention and not as limitations.
[0213] Those skilled in the art will appreciate that various
adaptations and modifications of the just-described embodiments can
be configured without departing from the scope and spirit of the
invention. Other suitable techniques and methods known in the art
can be applied in numerous specific modalities by one skilled in
the art and in light of the description of the present invention
described herein.
[0214] Therefore, it is to be understood that the invention can be
practiced other than as specifically described herein. The above
description is intended to be illustrative, and not restrictive.
Many other embodiments will be apparent to those of skill in the
art upon reviewing the above description. The scope of the
invention should, therefore, be determined with reference to the
appended claims, along with the full scope of equivalents to which
such claims are entitled.
Example 1
Formulation for First Population of Nanocarriers (Prophetic)
[0215] Synthetic nanocarriers comprising PLGA-R848 conjugate
(adjuvant), PLA-PEG-N3 conjugate (linker to peptide antigen) and
ova peptide (T-cell antigen) are prepared via a double emulsion
method wherein the ova peptide is encapsulated in the synthetic
nanocarriers. To a suspension of the synthetic nanocarriers (10
mg/mL in PBS (pH 7.4 buffer), 5 mL, containing about 12.5 mg (MW:
20, 000; 0.000625 mmol) of PLA-PEG-N3) is added HPV L1-peptide
comprising an acetylene linker (33 mg) under gentle stirring. A
solution of sodium ascorbate (100 mM in H2O, 0.3 mL) is added,
followed by CuSO4 solution (10 mM in water, 0.6 mL). The resulting
light yellow suspension is stirred at 20 C for 15 h and additional
CuSO4 solution (0.3 mL) and sodium ascorbate solution (0.15 mL) are
added. The suspension is stirred for 5 h at 20 C and diluted with
PBS buffer (pH 7.4) to 10 mL and is centrifuged to remove the
supernatant. The residual nanocarrier pellets are washed twice with
PBS buffer. The washed NCs are then re-suspended in 5 mL of PBS
buffer and stored frozen. The conjugation of L1 peptide on the
surface of the synthetic nanocarriers is confirmed by HPLC analysis
of the digested synthetic nanocarriers and by bioassay.
Example 2
Formulation for Second Population of Nanocarriers (Prophetic)
[0216] Using the general procedures outlines in Example 1 above,
synthetic nanocarriers comprising PLA-R848, PLA-PEG-N3 and
encapsulated ova peptide are prepared and conjugated with an HPV L2
peptide to provide L2 peptide conjugated synthetic
nanocarriers.
Example 3
Formulation Combining First and Second Populations of Nanocarriers
(Prophetic)
[0217] The synthetic nanocarrier preparations from Examples 1 and 2
above are thawed and diluted in PBS to a final concentration of 5
mg of nanocarriers per milliliter. Equal aliquots of each (0.5 mL)
are combined to provide a population of nanocarriers that contain
both the HPV L1 and L2 peptides.
Example 4
Preparations of Nanocarriers
Preparation of NC-Nic-OVA
[0218] PLGA-R848, poly-D/L-lactide-co-glycolide,
4-amino-2-(ethoxymethyl)-.alpha.,.alpha.-dimethyl-1H-imidazo[4,5-c]quinol-
ine-1-ethanol amide of approximately 7,000 Da made from PLGA of 3:1
lactide to glycolide ratio and having approximately 8.5% w/w
conjugated resiquimod content was custom manufactured at Princeton
Global Synthesis (300 George Patterson Drive #206, Bristol, Pa.
19007.) PLA-PEG-Nicotine (S-642), poly-D/L
lactide-block-poly(ethylene
glycol)-(.+-.)-trans-3'-hydroxymethylnicotine ether with PEG block
of approximately 5,000 Da and PLA block of approximately 21,000 Da
was custom manufactured at Princeton Global Synthesis (300 George
Patterson Drive #206, Bristol, Pa. 19007.) PLA-PEG-Maleimide, block
co-polymer consisting of a poly-D/L-lactide (PLA) block of
approximately 22000 Da and a polyethylene glycol (PEG) block of
approximately 2900 Da that is terminated by a maleimide functional
group was synthesized from commercial starting materials by
generating the PLA block by ring-opening polymerization of
dl-lactide with HO-PEG-Maleimide with dl-lactide. Polyvinyl alcohol
PhEur, USP (85-89% hydrolyzed, viscosity of 3.4-4.6 mPas) was
purchased from EMD Chemicals Inc. (480 South Democrat Road
Gibbstown, N.J. 08027. Part Number 4-88).
[0219] Solutions were prepared as follows:
[0220] Solution 1: 0.13N HCl in purified water.
[0221] Solution 2: PLGA-R848 @ 50 mg/mL, PLA-PEG-Nicotine @ 25
mg/mL, and PLA-PEG-Maleimide @ 25 mg/mL in dichloromethane was
prepared by dissolving each polymer separately in dichloromethane
at 100 mg/mL then combining 2 parts PLGA-R848 solution to 1 part
each PLA-PEG-Nicotine solution and PLA-PEG-Maleimide solution.
[0222] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM
phosphate buffer, pH 8.
[0223] Solution 4: 70 mM phosphate buffer, pH 8.
[0224] A primary (W1/O) emulsion was first created using Solution 1
and 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
(2.0 mL) to the primary emulsion, vortexing to create a course
dispersion, and then sonicating at 30% amplitude for 40 seconds
using the Branson Digital Sonifier 250.
[0225] 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 nanocarrier
suspension was stored frozen at -20 C until further use.
TABLE-US-00004 TABLE 4 NC-Nic-OVA Characterization Effective TLR
Agonist, T-cell agonist, Nanocarrier Diameter (nm) % w/w % w/w
NC-Nic-OVA 215 R848, 4.2 None
[0226] (1) NC with PEG-Nicotine and PEG-MAL on the surface,
prepared as above; 6.5 mg/mL suspension in PBS buffer. [0227] (2)
OVA protein (Ovalbumin from egg white): Worthington, Lot# POK12101,
MW: 46000. [0228] (3) Traut's reagent (2-iminothiolane.HCl): MP
Biomedical, Lot#8830KA, MW: 137.6 [0229] (4) pH 8 buffer (sodium
phosphate, 20 mM with 0.5 mM EDTA). [0230] (5) pH 7 1.times. PBS
buffer. OVA protein (10 mg) was dissolved in 1 mL pH 8 buffer. A
freshly made solution of Traut's reagent in pH 8 buffer (0.25 mL, 2
mg/mL) was added to the OVA protein solution. The resulting
solution was stirred under argon in the dark for 1.5 h. The
solution was diafiltered with MWCO 3K diafilter tube and washed
with pH 8 buffer twice. The resulting modified OVA with thiol group
was dissolved in 1 mL pH 8 buffer under argon. The NC suspension (3
mL, 6.5 mg/mL) was centrifuged to remove the supernatant. The
modified OVA solution was then mixed with the NC pellets. The
resulting suspension was stirred at rt under argon in the dark for
12 h. The NC suspension was then diluted to 10 mL with pH 7 PBS and
centrifuged. The resulting NC was pellet washed with 2.times.10 mL
pH 7 PBS. The NC-Nic-OVA conjugates were then resuspended in pH 7
PBS (ca. 6 mg/mL, 3 mL) stored at 4.degree. C.
Preparation of NC-OVA
[0231] PLGA-R848, poly-D/L-lactide-co-glycolide,
4-amino-2-(ethoxymethyl)-.alpha.,.alpha.-dimethyl-1H-imidazo[4,5-c]quinol-
ine-1-ethanol amide of approximately 7,000 Da made from PLGA of 3:1
lactide to glycolide ratio and having approximately 8.5% w/w
conjugated resiquimod content was custom manufactured at Princeton
Global Synthesis (300 George Patterson Drive #206, Bristol, Pa.
19007.) PLA-PEG-Maleimide, block co-polymer consisting of a
poly-D/L-lactide (PLA) block of approximately 22000 Da and a
polyethylene glycol (PEG) block of approximately 2900 Da that is
terminated by a maleimide functional group was synthesized from
commercial starting materials by generating the PLA block by
ring-opening polymerization of dl-lactide with HO-PEG-Maleimide.
Polyvinyl alcohol PhEur, USP (85-89% hydrolyzed, viscosity of
3.4-4.6 mPas) was purchased from EMD Chemicals Inc. (480 South
Democrat Road Gibbstown, N.J. 08027. Part Number 4-88).
[0232] Solutions were prepared as follows:
[0233] Solution 1: 0.13N HCl in purified water.
[0234] Solution 2: PLGA-R848 @ 50 mg/mL and PLA-PEG-Maleimide @ 50
mg/mL in dichloromethane was prepared by dissolving each polymer
separately in dichloromethane at 100 mg/mL then combining 1 part
PLGA-R848 solution to 1 part PLA-PEG-Maleimide solution.
[0235] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM
phosphate buffer, pH 8.
[0236] Solution 4: 70 mM phosphate buffer, pH 8.
[0237] A primary (W1/O) emulsion was first created using Solution 1
and 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
(2.0 mL) to the primary emulsion, vortexing to create a course
dispersion, and then sonicating at 30% amplitude for 40 seconds
using the Branson Digital Sonifier 250.
[0238] 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
nanocarrier suspension was stored frozen at -20.degree. C. until
further use.
TABLE-US-00005 TABLE 5 NC-OVA Characterization Effective TLR
Agonist, T-cell agonist, Nanocarrier Diameter (nm) % w/w % w/w
NC-OVA 208 R848, 4.3 None
[0239] (1) NC with PEG-MAL on the surface, prepared as above; 6
mg/mL suspension in PBS buffer. [0240] (2) OVA protein (Ovalbumin
from egg white): Worthington, Lot#POK12101, MW: 46000. [0241] (3)
Traut's reagent (2-iminothiolane.HCl): MP Biomedical, Lot# 8830KA,
MW: 137.6. [0242] (4) pH 8 buffer (sodium phosphate, 20 mM with 0.5
mM EDTA). [0243] (5) pH 7 1.times. PBS buffer.
[0244] OVA protein (20 mg) was dissolved in 1 mL pH 8 buffer. A
freshly made solution of Traut's reagent in pH 8 buffer (0.5 mL, 2
mg/mL) was added to the OVA protein solution. The resulting
solution was stirred under argon in the dark for 1.5 h. The
solution was diafiltered with MWCO 3K diafilter tube and washed
with pH 8 buffer twice. The resulting modified OVA with thiol group
was dissolved in 1 mL pH 8 buffer under argon. The NC suspension (4
mL, 6 mg/mL) was centrifuged to remove the supernatant. The
modified OVA solution was then mixed with the NC pellets. The
resulting suspension was stirred at rt under argon in the dark for
12 h. The NC suspension was then diluted to 10 mL with pH 7 PBS and
centrifuged. The resulting NC was pellet washed with 2.times.10 mL
pH 7 PBS. The NC-OVA conjugates were then resuspended in pH 7 PBS
(ca. 6 mg/mL, 4 mL) stored at 4.degree. C.
Preparation of NC-HA5
[0245] Ovalbumin peptide 323-339 amide acetate salt, was purchased
from Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif.
90505. Product code 4065609.) PLGA-R848,
poly-D/L-lactide-co-glycolide,
4-amino-2-(ethoxymethyl)-.alpha.,.alpha.-dimethyl-1H-imidazo[4,5-c]quinol-
ine-1-ethanol amide of approximately 7,000 Da made from PLGA of 3:1
lactide to glycolide ratio and having approximately 8.5% w/w
conjugated resiquimod content was custom manufactured at Princeton
Global Synthesis (300 George Patterson Drive #206, Bristol, Pa.
19007.) PLA-PEG-Maleimide, block co-polymer consisting of a
poly-D/L-lactide (PLA) block of approximately 22000 Da and a
polyethylene glycol (PEG) block of approximately 2900 Da that is
terminated by a maleimide functional group, was synthesized from
commercial starting materials by generating the PLA block by
ring-opening polymerization of dl-lactide with HO-PEG-Maleimide.
Polyvinyl alcohol PhEur, USP (85-89% hydrolyzed, viscosity of
3.4-4.6 mPas) was purchased from EMD Chemicals Inc. (480 South
Democrat Road Gibbstown, N.J. 08027. Part Number 4-88).
[0246] Solutions were prepared as follows:
[0247] Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL was
prepared in 0.13N HCl at room temperature.
[0248] Solution 2: PLGA-R848 @ 50 mg/mL and PLA-PEG-Maleimide @ 50
mg/mL in dichloromethane was prepared by dissolving each polymer
separately in dichloromethane at 100 mg/mL then combining 1 part
PLGA-R848 solution to 1 part PLA-PEG-Maleimide solution.
[0249] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM
phosphate buffer, pH 8.
[0250] Solution 4: 70 mM phosphate buffer, pH 8.
[0251] A primary (W1/O) emulsion was first created using Solution 1
and 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
(2.0 mL) to the primary emulsion, vortexing to create a course
dispersion, and then sonicating at 30% amplitude for 40 seconds
using the Branson Digital Sonifier 250.
[0252] 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
nanocarrier suspension was stored frozen at -20.degree. C. until
further use.
TABLE-US-00006 TABLE 6 NC-HA5 Characterization Effective TLR
Agonist, T-cell agonist, Nanocarrier Diameter (nm) % w/w % w/w
NC-HA5 216 R848, 3.6 Ova peptide 323-339, 2.0
[0253] (1) NC with PEG-MAL on the surface, prepared as above; 6.7
mg/mL suspension in PBS buffer. [0254] (2) HAS protein: Recombinant
Hemagglutinin, A/Vietnam/1203/2004, MW: 72000, supplied as a
solution in pH 7 PBS-tween buffer (0.55 mg/mL). [0255] (3) Traut's
reagent (2-iminothiolane.HCl): MP Biomedical, Lot# 8830KA, MW:
137.6. [0256] (4) pH 8 buffer (sodium phosphate, 20 mM with 0.5 mM
EDTA). [0257] (5) pH 7 1.times. PBS buffer.
[0258] HA5 protein (0.21 g in 0.38 mL pH 7.1 PBS-tween buffer) was
diluted to 0.5 mL with pH 8 buffer. A freshly made solution of
Traut's reagent in pH 8 buffer (0.02 mL, 2 mg/mL) was added to the
HA5 protein solution. The resulting solution was stirred under
argon in the dark for 1.5 h. The solution was diafiltered with MWCO
3K diafilter tube and washed with pH 8 buffer twice. The resulting
modified HAS protein with thiol group was dissolved in 0.5 mL pH 8
buffer under argon. The NC suspension (3 mL, 6.7 mg/mL) was
centrifuged to remove the supernatant. The modified HAS solution
was then mixed with the NC pellets. The resulting suspension was
stirred at rt under argon in the dark for 12 h. The NC suspension
was then diluted to 10 mL with pH 7 PBS and centrifuged. The
resulting NC was pellet washed with 2.times.10 mL pH 7 PBS. The
NC-HA5 conjugates were then resuspended in pH 7 PBS (ca. 6 mg/mL, 3
mL) stored at 4.degree. C.
Preparation of NC-L2, NC-M2e or NC-M2e-L2
[0259] Ovalbumin peptide 323-339 amide acetate salt, was purchased
from Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif.
90505. Product code 4065609.) PLGA-R848,
poly-D/L-lactide-co-glycolide,
4-amino-2-(ethoxymethyl)-.alpha.,.alpha.-dimethyl-1H-imidazo[4,5-c]quinol-
ine-1-ethanol amide of approximately 7,000 Da made from PLGA of 3:1
lactide to glycolide ratio and having approximately 8.5% w/w
conjugated resiquimod content was custom manufactured at Princeton
Global Synthesis (300 George Patterson Drive #206, Bristol, Pa.
19007.) PLA-PEG-C6-N.sub.3, block co-polymer consisting of a
poly-D/L-lactide (PLA) block of approximately 23000 Da and a
polyethylene glycol (PEG) block of approximately 2000 Da that is
terminated by an amide-conjugated C.sub.6H.sub.12 linker to an
azide, was synthesized by conjugating HO-PEG-COOH to an
amino-C.sub.6H.sub.12-azide and then generating the PLA block by
ring-opening polymerization of the resulting HO-PEG-C6-N3 with
dl-lactide. Polyvinyl alcohol PhEur, USP (85-89% hydrolyzed,
viscosity of 3.4-4.6 mPas) was purchased from EMD Chemicals Inc.
(480 South Democrat Road Gibbstown, N.J. 08027. Part Number
4-88).
[0260] Solutions were prepared as follows:
[0261] Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL was
prepared in 0.13N HCl at room temperature.
[0262] Solution 2: PLGA-R848 @ 50 mg/mL and PLA-PEG-C6-N.sub.3 @ 50
mg/mL in dichloromethane was prepared by dissolving each separately
at 100 mg/mL in dichloromethane then combining in equal parts by
volume.
[0263] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM
phosphate buffer, pH 8.
[0264] Solution 4: 70 mM phosphate buffer, pH 8.
[0265] A primary (W1/O) emulsion was first created using Solution 1
and 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
(2.0 mL) to the primary emulsion, vortexing to create a course
dispersion, and then sonicating at 30% amplitude for 40 seconds
using the Branson Digital Sonifier 250.
[0266] 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. Two identical
batches were created and then combined to form a single homogenous
suspension at which was stored frozen at -20.degree. C. until
further use.
TABLE-US-00007 TABLE 7 NC-L2, NC-M2e or NC-M2e-L2 Characterization
Effective TLR Agonist, Antigen, Nanocarrier Diameter (nm) % w/w %
w/w NC-L2, NC- 209 R848, 4.2 Ova 323-339 peptide, 2.4 M2e or NC-
M2e-L2
[0267] (1) Nanocarriers with surface PEG-C6-N3 containing PLGA-R848
and Ova-peptide, prepared as above, 7 mg/mL suspension in PBS.
[0268] (2) HPV16 L2 peptide modified with an alkyne linker attached
to C-terminal Lys amino group; Bachem Americas, Inc, Lot B06055, MW
2595, TFA salt; Sequence:
H-Ala-Thr-Gln-Leu-Tyr-Lys-Thr-Cys-Lys-Gln-Ala-Gly-Thr-Cys-Pro-P-
ro-Asp-Ile-Ile-Pro-Lys-Val-Lys(5-hexynoyl)-NH2(with Cys-Cys
disulfide bond). [0269] (3) Catalysts: CuSO4 , 100 mM in DI water;
THPTA ligand, 200 mM in DI water; sodium ascorbate, 200 mM in DI
water freshly prepared. [0270] (4) pH 7.4 PBS buffer.
[0271] The NC suspension (7 mg/mL, 4 mL) was concentrated to ca. 1
mL in volume by centrifugation. A solution of L2 peptide (20 mg) in
2 mL PBS buffer was added. A pre-mixed solution of 0.2 mL of CuSO4
(100 mM) and 0.2 mL of THPTA ligand (200 mM) was added, followed by
0.4 mL of sodium ascorbate (200 mM). The resulting light yellow
suspension was stirred in dark at ambient room temperature for 18
h. The suspension was then diluted with PBS buffer to 10 mL and
centrifuged to remove the supernatant. The NC-L2 conjugates were
further pellet washed twice with 10 mL PBS buffer and resuspended
in pH 7.4 buffer at final concentration of ca. 6 mg/mL (ca. 4 mL)
and stored at 4.degree. C. [0272] (1) Nanocarriers with surface
PEG-C6-N3 containing PLGA-R848 and Ova-peptide, prepared as above,
7 mg/mL suspension in PBS. [0273] (2) M2e peptide modified with an
alkyne linker attached to C-terminal Gly; CS Bio Co, Catalog No.
CS4956, Lot: H308, MW 2650, TFA salt; Sequence:
H-Met-Ser-Leu-Leu-Thr-Glu-Val-Glu-Thr-Pro-Thy-Arg-Asn-Glu-Trp-Glu-Cys-Arg-
-Cys-Ser-Asp-Gly-Gly-NHCH2CCH. [0274] (3) Catalysts: CuSO4, 100 mM
in DI water; THPTA ligand, 200 mM in DI water; sodium ascorbate,
200 mM in DI water freshly prepared. [0275] (4) pH 7.4 PBS
buffer.
[0276] The NC suspension (7 mg/mL, 4 mL) was concentrated to ca. 1
mL in volume by centrifugation. A solution of M2e peptide (20 mg)
in 2 mL PBS buffer was added. A pre-mixed solution of 0.2 mL of
CuSO4 (100 mM) and 0.2 mL of THPTA ligand (200 mM) was added,
followed by 0.4 mL of sodium ascorbate (200 mM). The resulting
light yellow suspension was stirred in dark at ambient room
temperature for 18 h. The suspension was then diluted with PBS
buffer to 10 mL and centrifuged to remove the supernatant. The
NC-M2e conjugates were further pellet washed twice with 10 mL PBS
buffer and resuspended in pH 7.4 buffer at final concentration of
ca. 6 mg/mL (ca. 4 mL) and stored at 4.degree. C. [0277] (1)
Nanocarriers with surface PEG-C6-N3 containing PLGA-R848 and
Ova-peptide, prepared as above, 7 mg/mL suspension in PBS. [0278]
(2) HPV16 L2 peptide modified with an alkyne linker attached to
C-terminal Lys amino group; Bachem Americas, Inc, Lot B06055, MW
2595, TFA salt; Sequence:
H-Ala-Thr-Gln-Leu-Tyr-Lys-Thr-Cys-Lys-Gln-Ala-Gly-Thr-Cys-Pro-Pro-Asp-Ile-
-Ile-Pro-Lys-Val-Lys(5-hexynoyl)-NH2(with Cys-Cys disulfide bond).
[0279] (3) M2e peptide modified with an alkyne linker attached to
C-terminal Gly; CS Bio Co, Catalog No. CS4956, Lot: H308, MW 2650,
TFA salt; Sequence:
H-Met-Ser-Leu-Leu-Thr-Glu-Val-Glu-Thr-Pro-Thy-Arg-Asn-Glu-Trp-G-
lu-Cys-Arg-Cys-Ser-Asp-Gly-Gly-NHCH2CCH. [0280] (4) Catalysts:
CuSO4, 100 mM in DI water; THPTA ligand, 200 mM in DI water; sodium
ascorbate, 200 mM in DI water freshly prepared. [0281] (5) pH 7.4
PBS buffer.
[0282] The NC suspension (7 mg/mL, 2 mL) was concentrated to ca.
0.5 mL in volume by centrifugation. A mixture of L2 peptide (5 mg)
and M2e peptide (5 mg) in 1 mL PBS buffer was added. A pre-mixed
solution of 0.2 mL of CuSO4 (100 mM) and 0.2 mL of THPTA ligand
(200 mM) was added, followed by 0.4 mL of sodium ascorbate (200
mM). The resulting light yellow suspension was stirred in dark at
ambient room temperature for 18 h. The suspension was then diluted
with PBS buffer to 10 mL and centrifuged to remove the supernatant.
The NC-M2e-L2 conjugates were further pellet washed twice with 10
mL PBS buffer and resuspended in pH 7.4 buffer at final
concentration of ca. 6 mg/mL (ca. 2 mL) and stored at 4.degree.
C.
Example 5
Immunization with Two Monovalent Antigen Nanocarriers Leads to
Immune Response to Both Antigens
[0283] Anti-nicotine (dark gray bars) and anti-ovalbumin (light
gray bars) antibody titers in unimmunized mice and mice injected
with NC-Nic and NC-OVA (as prepared in Example 4) (5 animals/group;
s.c., 100 .mu.g of each NC per injection, 2 times at 3-wk
intervals) were measured. Titers for day 33 after immunization with
NC are shown in FIG. 1 (ELISA against polylysine-nicotine or
ovalbumin protein) (group 1: unimmunized; group 2: immunized with
NC-Nic and NC-OVA).
[0284] Mice were injected with 100 .mu.g of NC-Nic (nanocarrier
exhibiting nicotine on the outer surface and containing OP-II
helper peptide and R848 adjuvant in the NC) and 100 .mu.g of NC-OVA
(nanocarrier exhibiting ovalbumin on the outer surface and
containing OP-II helper peptide and R848 adjuvant in the NC)
(subcutaneously, hind limbs) at 3-week intervals (days 0 and 21).
Serum anti-nicotine and anti-ovalbumin antibody titers were
measured at day 33 after immunization with NC. Anti-nicotine and
anti-ovalbumin antibody titers (EC.sub.50) as measured by ELISA
against polylysine-nicotine or ovalbumin protein are shown (FIG.
1). Titers for control unimmunized mice are also shown. The results
demonstrate that mice immunized with a combination of two
monovalent antigen nanocarriers (NC-Nic and NC-OVA) generate
antibodies to both antigens.
Example 6
Immunization with Monovalent and Bivalent Antigen Nanocarriers Lead
to Immune Response to All Three Antigens
[0285] Anti-nicotine, anti-ovalbumin, and anti-L2 peptide antibody
titers in unimmunized mice and mice injected with NC-Nic-OVA and
NC-L2 (as prepared in Example 4) (5 animals/group; s.c., 100 .mu.g
of each NC per injection, 2 times at 3-wk intervals) were measured.
Titers for day 33 after immunization with NC are shown in FIG. 2
(ELISA against polylysine-nicotine, ovalbumin protein, or
PLA-PEG-L2 peptide) (group 1: unimmunized; group 2: immunized with
NC-Nic-OVA and NC-L2).
[0286] Mice were injected with 100 .mu.g of NC-Nic-OVA (nanocarrier
exhibiting nicotine and ovalbumin on the outer surface and
containing OP-II helper peptide and R848 adjuvant in the NC) and
100 .mu.g of NC-L2 (nanocarrier exhibiting HPV L2 peptide (aa17-36)
on the outer surface and containing OP-II helper peptide and R848
adjuvant in the NC) (subcutaneously, hind limbs) at 3-week
intervals (days 0 and 21). Serum anti-nicotine, anti-ovalbumin, and
anti-L2 peptide antibody titers were measured at day 33 after
immunization with NC. Anti-nicotine, anti-ovalbumin, and anti-L2
peptide antibody titers (EC.sub.50) as measured by ELISA against
polylysine-nicotine, ovalbumin protein, and L2 peptide are shown
(FIG. 2). Titers for control unimmunized mice are also shown. The
results demonstrate that mice immunized with a combination of one
monovalent and one bivalent antigen nanocarrier (NC-Nic-OVA and
NC-L2) generate antibodies to all three antigens.
Example 7
Immunization with Two Bivalent Antigen Nanocarriers Leads to Immune
Response to all Four Antigens
[0287] Anti-nicotine, anti-ovalbumin, anti-M2e peptide, and anti-L2
peptide antibody titers in unimmunized mice and mice injected with
NC-Nic-OVA and NC-M2e-L2 (as prepared in Example 4) (5
animals/group; s.c., 100 .mu.g of each NC per injection, 2 times at
3-wk intervals) were measured. Titers for day 33 after immunization
with NC are shown in FIG. 3 (ELISA against polylysine-nicotine,
ovalbumin protein, PLA-PEG-M2e peptide, or PLA-PEG-L2 peptide)
(group 1: unimmunized; group 2: immunized with NC-Nic-OVA and
NC-M2e-L2).
[0288] Mice were injected with 100 .mu.g of NC-Nic-OVA (nanocarrier
exhibiting nicotine and ovalbumin on the outer surface and
containing OP-II helper peptide and R848 adjuvant in the NC) and
100 .mu.g of NC-M2e-L2 (nanocarrier exhibiting influenza M2e
peptide (aa2-27) and HPV L2 peptide (aa17-36) on the outer surface
and containing OP-II helper peptide and R848 adjuvant in the NC)
(subcutaneously, hind limbs) with 3-week intervals (days 0 and 21).
Serum anti-nicotine, anti-ovalbumin, anti-M2e peptide, and anti-L2
peptide antibody titers were measured at day 33 after immunization
with NC. Anti-nicotine, anti-ovalbumin, anti-M2e peptide, and
anti-L2 peptide antibody titers (EC.sub.50) as measured by ELISA
against polylysine-nicotine, ovalbumin protein, M2e peptide, and L2
peptide are shown (FIG. 3). Titers for control unimmunized mice are
also shown. The results demonstrate that mice immunized with a
combination of two bivalent antigen nanocarriers (NC-Nic-OVA and
NC-M2e-L2) generate antibodies to all four antigens.
Example 8
Immunization with Two Monovalent Peptide Antigen Nanocarriers Leads
to Immune Response to Both Peptide Antigens
[0289] Anti-M2e peptide and anti-L2 peptide antibody titers in
unimmunized mice and mice injected with NC-M2e and NC-L2 (as
prepared in Example 4) (5 animals/group; s.c., 100 .mu.g of each NC
per injection, 2 times at 3-wk intervals) were measured. Titers for
day 33 after immunization with NC are shown in FIG. 4 (ELISA
against PLA-PEG-M2e peptide or PLA-PEG-L2 peptide) (group 1:
unimmunized; group 2: immunized with NC-M2e and NC-L2).
[0290] Mice were injected with 100 .mu.g of NC-M2e (nanocarrier
exhibiting influenza M2e peptide (aa2-27) on the outer surface and
containing OP-II helper peptide and R848 adjuvant in the NC) and
100 .mu.g of NC-L2 (nanocarrier exhibiting HPV L2 peptide (aa17-36)
on the outer surface and containing OP-II helper peptide and R848
adjuvant in the NC) (subcutaneously, hind limbs) at 3-week
intervals (days 0 and 21). Serum anti-M2e peptide and anti-L2
peptide antibody titers were measured at day 33 after immunization
with NC. Anti-M2e peptide and anti-L2 peptide antibody titers
(EC.sub.50) as measured by ELISA against M2e peptide and L2 peptide
are shown (FIG. 4). Titers for control unimmunized mice are also
shown. These results demonstrate that mice immunized with a
combination of two monovalent peptide antigen nanocarriers (NC-M2e
and NC-L2) generate antibodies to both peptide antigens.
Example 9
Immunization with Two Monovalent Protein Antigen Nanocarriers Leads
to Immune Response to Both Protein Antigens
[0291] Anti-HA5 protein and anti-ovalbumin protein antibody titers
in unimmunized mice and mice injected with NC-HA5 and NC-OVA (as
prepared in Example 4) (5 animals/group; s.c., 100 .mu.g of each NC
per injection, 2 times with 3-wk intervals) were measured. Titers
for day 33 after immunization with NC are shown in FIG. 5 (ELISA
against H5N1 HA protein or ovalbumin protein) (group 1:
unimmunized; group 2: immunized with NC-HA5 and NC-OVA).
[0292] Mice were injected with 100 .mu.g of NC-HA5 protein
(nanocarrier exhibiting influenza H5N1 HA protein on the outer
surface and containing OP-II helper peptide and R848 adjuvant in
the NC) and 100 .mu.g of NC-OVA (nanocarrier exhibiting ovalbumin
on the outer surface and containing OP-II helper peptide and R848
adjuvant in the NC) (subcutaneously, hind limbs) at 3-week
intervals (days 0 and 21). Serum anti-HA5 and anti-ovalbumin
antibody titers were measured at day 33 after immunization with NC.
Anti-HA5 and anti-ovalbumin antibody titers (EC.sub.50) as measured
by ELISA against H5N1 HA protein and ovalbumin protein are shown
(FIG. 5). Titers for control unimmunized mice are also shown. These
results demonstrate that mice immunized with a combination of two
monovalent protein antigen nanocarriers (NC-HA5 and NC-OVA)
generate antibodies to both protein antigens.
Example 10
Immunization with Two Monovalent and One Bivalent Antigen
Nanocarriers Leads to Immune Response to all Four Antigens
[0293] Anti-HA, anti-ovalbumin, anti-M2e peptide, and anti-L2
peptide antibody titers in unimmunized mice and mice injected with
NC-HA5, NC-OVA, and NC-M2e-L2 (as prepared in Example 4) (5
animals/group; s.c., 100 .mu.g of each NC per injection, 2 times at
3-wk intervals) were measured. Titers for day 33 after immunization
with NC are shown in FIG. 6 (ELISA against HA protein, ovalbumin
protein, PLA-PEG-M2e peptide, or PLA-PEG-L2 peptide) (group 1:
unimmunized; group 2: immunized with NC-HA5, NC-OVA, and
NC-M2e-L2).
[0294] Mice were injected with 100 .mu.g of NC-HA5 protein
(nanocarrier exhibiting influenza H5N1 HA protein on the outer
surface and containing OP-II helper peptide and R848 adjuvant in
the NC), 100 .mu.g of NC-OVA (nanocarrier exhibiting ovalbumin on
the outer surface and containing OP-II helper peptide and R848
adjuvant in the NC), and 100 .mu.g of NC-M2e-L2 (nanocarrier
exhibiting influenza M2e peptide (aa2-27) and HPV L2 peptide
(aa17-36) on the outer surface and containing OP-II helper peptide
and R848 adjuvant in the NC) (subcutaneously, hind limbs) at 3-week
intervals (days 0 and 21). Serum anti-HA, anti-ovalbumin, anti-M2e
peptide, and anti-L2 peptide antibody titers were measured at day
33 after immunization with NC. Anti-HA, anti-ovalbumin, anti-M2e
peptide, and anti-L2 peptide antibody titers (EC.sub.50) as
measured by ELISA against HA protein, ovalbumin protein, M2e
peptide, and L2 peptide are shown (FIG. 6). Titers for control
unimmunized mice are also shown. These results demonstrate that
mice immunized with a combination of two monovalent and one
bivalent antigen nanocarrier (NC-HA5, NC-OVA, and NC-M2e-L2)
generate antibodies to all four antigens.
Example 11
Immunization with Two Monovalent and One Bivalent Antigen
Nanocarriers Leads to Immune Response to all Four Antigens
[0295] Antibody titers in mice immunized with a combination of
NC-M2e, NC-L2 peptide and NC-nicotine-ovalbumin (as prepared in
Example 4) were measured. NC-M2e and NC-L2 peptide contained OP-II
T-helper peptide (2.0% and 2.4%, correspondingly) and R848 adjuvant
(3.6% and 4.3%, correspondingly); NC-nicotine-ovalbumin contained
R848 adjuvant (4.2%). Each bar of FIG. 7 represents the titer
against antigen. Five animals per group were immunized s.c. with
120 .mu.g of each NC per injection, 2 times at 3-wk intervals.
Titers for day 33 after the first immunization are shown (ELISA
done against PLA-PEG-M2e, PLA-PEG-L2, ovalbumin and
polylysine-nicotine, correspondingly).
[0296] These results demonstrate that immunization with a
combination of two NCs each carrying a different peptide antigen
together with a NC carrying another two antigens results in
generation of antibodies to all four NC-carried antigens. When
identical amounts of three NC, the first containing surface M2e
peptide from influenza A virus (ectodomain of M2 matrix protein,
amino acids 2-27), the second containing surface L2 peptide from
HPV virus (amino acids 17-36 from L2 capsid protein of HPV-16), and
the third carrying surface nicotine and ovalbumin protein were used
for animal immunization, a strong humoral response was induced in
all animals against all four NC-coupled antigens (FIG. 7). No
reactivity was detected in the sera of preimmune mice.
Example 12
Immunization with Two Monovalent Nanocarriers with Antigen in
Different Steric Orientations Leads to Immune Response both
Orientations
Preparation of NC-3'-Nicotine
[0297] Ovalbumin peptide 323-339 amide acetate salt, was purchased
from Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif.
90505. Product code 4065609.) PLGA-R848,
poly-D/L-lactide-co-glycolide,
4-amino-2-(ethoxymethyl)-.alpha.,.alpha.-dimethyl-1H-imidazo[4,5-c]quinol-
ine-1-ethanol amide of approximately 7,000 Da made from PLGA of 3:1
lactide to glycolide ratio and having approximately 8.5% w/w
conjugated resiquimod content was custom manufactured at Princeton
Global Synthesis (300 George Patterson Drive #206, Bristol, Pa.
19007.) PLA-PEG-Nicotine (S-642), poly-D/L
lactide-block-poly(ethylene
glycol)-(.+-.)-trans-3'-hydroxymethylnicotine ether with PEG block
of approximately 5,000 Da and PLA block of approximately 21,000 Da
was custom manufactured at Princeton Global Synthesis (300 George
Patterson Drive #206, Bristol, Pa. 19007.) Polyvinyl alcohol PhEur,
USP (85-89% hydrolyzed, viscosity of 3.4-4.6 mPas) was purchased
from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, N.J.
08027. Part Number 4-88).
[0298] Solutions were prepared as follows:
[0299] Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL was
prepared in 0.13N HCl at room temperature.
[0300] Solution 2: PLGA-R848 @ 50 mg/mL, PLA-PEG-Nicotine @ 25
mg/mL, and PLA @ 25 mg/mL in dichloromethane were prepared by
dissolving each polymer separately in dichloromethane at 100 mg/mL
then combining 2 parts PLGA-R848 solution to 1 part each
PLA-PEG-Nicotine solution and PLA solution.
[0301] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM
phosphate buffer, pH 8.
[0302] Solution 4: 70 mM phosphate buffer, pH 8.
[0303] A primary (W1/O) emulsion was first created using Solution 1
and 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
(2.0 mL) to the primary emulsion, vortexing to create a course
dispersion, and then sonicating at 30% amplitude for 40 seconds
using the Branson Digital Sonifier 250.
[0304] 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
nanocarrier suspension was stored frozen at -20.degree. C. until
further use.
TABLE-US-00008 TABLE 8 NC-3'-Nicotine Characterization Effective
TLR Agonist, T-cell agonist, Nanocarrier Diameter (nm) % w/w % w/w
NC-3'- 193 R848, 4.2 Ova 323-339 peptide, 2.1 Nicotine
Preparation of NC-1'-Nicotine
[0305] Ovalbumin peptide 323-339 amide acetate salt, was purchased
from Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif.
90505. Product code 4065609.) PLGA-R848,
poly-D/L-lactide-co-glycolide,
4-amino-2-(ethoxymethyl)-.alpha.,.alpha.-dimethyl-1H-imidazo[4,5-c]quinol-
ine-1-ethanol amide of approximately 7,000 Da made from PLGA of 3:1
lactide to glycolide ratio and having approximately 8.5% w/w
conjugated resiquimod content was custom manufactured at Princeton
Global Synthesis (300 George Patterson Drive #206, Bristol, Pa.
19007.) PLA-PEG-1'-Nic, a block co-polymer consisting of a
poly-D/L-lactide (PLA) block of approximately 23000 Da and a
polyethylene glycol (PEG) block of approximately 2000 Da that is
conjugated to nicotine via a 4-carbon linkage to the 1' amino group
on nicotine was synthesized. In brief, nicotine with a
butyl-alcohol linker at the 1' position was made into HO-PEG-1'-Nic
by polymerization with ethylene oxide, and the PLA extension was
then generated by ring-opening polymerization of the HO-PEG-1'-Nic
with dl-lactide. PLA with an inherent viscosity of 0.22 dL/g was
purchased from SurModics Pharmaceuticals (756 Tom Martin Drive,
Birmingham, Ala. 35211. Product Code 100 DL 2A.) Polyvinyl alcohol
PhEur, USP (85-89% hydrolyzed, viscosity of 3.4-4.6 mPas) was
purchased from EMD Chemicals Inc. (480 South Democrat Road
Gibbstown, N.J. 08027. Part Number 4-88).
[0306] Solutions were prepared as follows:
[0307] Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL was
prepared in 0.13N HCl at room temperature.
[0308] Solution 2: PLGA-R848 @ 50 mg/mL, PLA-PEG-1'-Nic @ 25 mg/mL,
and PLA @ 25 mg/mL in dichloromethane was prepared by dissolving
each polymer separately in dichloromethane at 100 mg/mL then
combining 2 parts PLGA-R848 solution to 1 part each PLA-PEG-1'-Nic
solution and PLA solution.
[0309] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM
phosphate buffer, pH 8.
[0310] Solution 4: 70 mM phosphate buffer, pH 8.
[0311] A primary (W1/O) emulsion was first created using Solution 1
and 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
(2.0 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.
[0312] 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
nanocarrier suspension was stored frozen at -20.degree. C. until
further use.
TABLE-US-00009 TABLE 9 NC-1'-Nicotine Characterization Effective
TLR Agonist, T-cell agonist, Nanocarrier Diameter (nm) % w/w % w/w
NC-1'- 238 R848, 3.9 Ova 323-339 peptide, 2.8 Nicotine
Immunization and Results
[0313] Antibody titers in mice immunized with a combination of
NC-3'-nicotine and NC-1'-nicotine were measured. NC-3'-nicotine and
NC-1'-nicotine contained OP-II T-helper peptide (2.1%) and R848
adjuvant (4.2%). Each bar of FIG. 8 represents the titer against
antigen. Five animals per group were immunized s.c. with 120 .mu.g
of each NC per injection, 2 times at 3-wk intervals. Titers for day
33 after the first immunization are shown (ELISA done against
polylysine-nicotine, respectively).
[0314] These results show that immunization with a combination of
two NCs each carrying the same antigen but in different steric
orientations results in the generation of antibodies against both
of these different orientations of the same antigen. When identical
amounts of two NCs, the first containing surface nicotine attached
to NC in the 3'-position and the second attached to NC in the
1'-position were used for animal immunization, a strong humoral
response was induced in all animals against both orientations of
nicotine (FIG. 8). No reactivity was detected in the sera of
preimmune mice.
Example 13
Preparations of Polymers and Nanocarriers
Preparation of PLGA-R848
[0315] PLGA-R848 was prepared by reaction of PLGA polymer
containing an acid end group with R848 in the presence of coupling
agent such as HBTU as follows. A mixture of PLGA (Lakeshores
Polymers, MW .about.5000, 7525DLG1A, acid number 0.7 mmol/g, 10 g,
7.0 mmol) and HBTU (5.3 g, 14 mmol) in anhydrous EtOAc (160 mL) was
stirred at room temperature under argon for 50 minutes. Compound
R848 (2.2 g, 7 mmol) was added, followed by diisopropylethylamine
(DIPEA) (5 mL, 28 mmol). The mixture was stirred at room
temperature for 6 h and then at 50-55.degree. C. overnight (about
16 h). After cooling, the mixture was diluted with EtOAc (200 mL)
and washed with saturated NH4Cl solution (2.times.40 mL), water (40
mL) and brine solution (40 mL). The solution was dried over Na2SO4
(20 g) and concentrated to a gel-like residue. Isopropyl alcohol
(IPA) (300 mL) was then added and the polymer conjugate
precipitated out of solution. The polymer was then washed with IPA
(4.times.50 mL) to remove residual reagents and dried under vacuum
at 35-40.degree. C. for 3 days as a white powder (10.26 g, MW by
GPC is 5200, R848 loading is 12% by HPLC).
[0316] In a similar manner, PLA-R848 was prepared by the reaction
of PLA-CO2H (polylactide with acid ending group) with R848.
Preparation of PLA-PEG-CO2H
[0317] A mixture of HO-PEG-CO2H (MW: 2000, 1.0 g, 0.5 mmol),
dl-lactide (10.8 g, 75 mmol) and Na2SO4 (15 g) in a 100 mL round
bottom flask was dried under vacuum at 60.degree. C. for 2 days.
Anhydrous toluene (30 ML) was added, and the mixture was heated to
reflux under argon. Sn(Oct)2 (0.162 mL, 0.5 mmol) was added. The
mixture was refluxed under argon overnight and cooled to ambient
room temperature. The mixture was diluted with CH2Cl2 (200 mL) and
filtered through a pad of Celite. The filtrate was concentrated to
a dense sticky residue. 10% MeOH in diethyl ether (200 mL) was
added to precipitate out the polymer with vigorous stirring. The
polymer was further washed with 10% MeOH in ether (100 mL) and
dried under vacuum at 30.degree. C. to give the PLA-PEG-CO2H
copolymer as an off-white foamy solid (10.0 g, H NMR in CDCl3
showed the polymer has MW of 21000).
Preparation of PLA-PEG-NH2
[0318] A mixture of HO-PEG-NH2.HCl (MW: 3500, 1.0 g, 0.28 mmol),
dl-lactide (6.1 g, 42 mmol) and Na2SO4 (10 g) in a 100 mL round
bottom flask was dried under vacuum at 60.degree. C. for 1 day.
Anhydrous toluene (30 ML) was added and the mixture was heated to
90.degree. C. under argon. Sn(Oct)2 (0.1 mL, 0.28 mmol) was added.
The mixture was refluxed under argon overnight and cooled to
ambient room temperature. The mixture was diluted with ethyl
acetate (200 mL) and filtered through a pad of Celite. The filtrate
was concentrated to a dense sticky residue. 10% MeOH in t-butyl
methyl ether (MTBE) (200 mL) was added to precipitate out the
polymer with vigorous stirring. The polymer was further washed with
5% MeOH in MTBE (50 mL) and MTBE (50 mL) and dried under vacuum at
30.degree. C. to give the PLA-PEG-NH2.HCl copolymer as an off-white
foamy solid (5.0 g, H NMR in CDCl3 showed the polymer has MW of
18000).
Preparation of PLA-PEG-PEGS-N3
[0319] PLA-PEG-N3 polymer was prepared by ring opening
polymerization of HO-PEG-azide with dl-lactide in the presence of a
catalyst such as Sn(Oct)2 as follows. HO-PEG-CO2H (MW 3500, 1.33 g,
0.38 mmol) was treated with NH2-PEG3-N3 (MW 218.2, 0.1 g, 0.458
mmol) in the presence of DCC (MW 206, 0.117 g, 0.57 mmol) and NHS
(MW 115, 0.066 g, 0.57 mmol) in dry DCM (10 mL) overnight. After
filtration to remove insoluble byproduct (DCC-urea), the solution
was concentrated and then diluted with ether to precipitate out the
polymer, HO-PEG-PEG3-N3 (1.17 g). After drying, HO-PEG-PEG3-N3 (MW
3700, 1.17 g, 0.32 mmol) was mixed with dl-lactide (recrystallized
from EtOAc, MW 144, 6.83 g, 47.4 mmol) and Na2SO4 (10 g) in a 100
mL flask. The solid mixture was dried under vacuum at 45.degree. C.
overnight and dry toluene (30 mL) was added. The resulting
suspension was heated to 110.degree. C. under argon and Sn(Oct)2
(MW 405, 0.1 mL, 0.32 mmol) was added. The mixture was heated at
reflux for 18 h and cooled to rt. The mixture was diluted with DCM
(50 mL) and filtered. After concentration to an oily residue, MTBE
(200 mL) was added to precipitate out the polymer which was washed
once with 100 mL of 10% MeOH in MTBE and 50 mL of MTBE. After
drying, PLA-PEG-PEG3-N3 was obtained as a white foam (7.2 g,
average MW: 23,700 by H NMR).
Preparation of PLA-PEG-C6-N3
[0320] HO-PEG-CO2H (MW 3500, 1.00 g, 0.29 mmol) was treated with
6-azido-1-hexylamine (H2N-C6-N3) (MW 142, 0.081 g, 0.57 mmol) in
the presence of DCC (MW 206, 0.118 g, 0.57 mmol) and NHS (MW 115,
0.066 g, 0.57 mmol) in dry DCM (10 mL) overnight. After filtration
to remove insoluble byproduct (DCC-urea), the solution was
concentrated and then diluted with MTBE to precipitate out the
polymer which was then washed twice with MTBE and dried under
vacuum at 30.degree. C. overnight to give HO-PEG-C6-N3 polymer (1.1
g). HO-PEG-C6-N3 polymer (1.1 g, 0.29 mmol) and dl-lactide (6.5 g,
45 mmol) were mixed in dry toluene (60 mL). The mixture was heated
to reflux while 30 mL of toluene was removed by azeotrope
distillation. The resulting solution was cooled to 100.degree. C.
and Sn(Oct)2 (0.095 mL, 0.29 mmol) was added. The solution was
heated at reflux under argon overnight and cooled to rt. The
solution was then added to 150 mL of 2-propanol to precipitate out
the polymer which was washed with 2-propanol (100 mL) and dried
under vacuum at 30.degree. C. for 2 days to give PLA-PEG-C6-N3
copolymer as an off-white solid (6.8 g, MW by GPC is 27000 with DPI
of 1.5).
Preparation of PLA-PEG(5K)-CONH2NH2
[0321] A mixture of HO-PEG(5 k)-CO2H (JenKem Technology, USA) (MW:
5000, 1.0 g, 0.2 mmol), tert-butyl carbazate (Boc-hydrazide) (MW:
132, 0.053 g, 0.4 mmol), DCC (MW 206, 0.083 g, 0.4 mmol) and
N-hydroxysuccinimide (NHS) (MW 115, 0.05 g, 0.4 mmol) in dry DCM
(15 mL) was stirred at rt for 25 h. The insoluble DCC-urea was
removed by filtration and the filtrate was concentrated. The
residual was added to 50 mL of MTBE to precipitate out the polymer
which was washed twice with 40 mL of MTBE and dried under vacuum
for 2 days to give HO-PEG(5 k)-CONHNHtBoc as a white powder (1.07
g). HO-PEG(5 k)-CONHNHtBoc polymer (1.07 g, 0.20 mmol) and
dl-lactide (4.32 g, 30 mmol) were mixed in dry toluene (70 mL). The
mixture was heated to reflux while 50 mL of toluene was removed by
azeotrope distillation. The resulting solution was cooled to
100.degree. C. and Sn(Oct)2 (0.065 mL, 0.20 mmol) was added. The
solution was heated at reflux under argon for 22 h and cooled to
rt. The solution was then added to 150 mL of 2-propanol to
precipitate out the polymer which was washed with 2-propanol (60
mL) and dried under vacuum at 30.degree. C. for 2 days to give
PLA-PEG(5 k)-CONHNHtBoc copolymer as a white solid chunk. The
polymer was dissolved in 50 mL of dry DCM and cooled with ice
water. Trifluoroacetic acid (TFA) (15 mL) was added and the
resulting solution was stirred at rt overnight. The yellowish
solution was concentrated to dryness. The residual was added to 200
mL of 2-propanol to precipitate out the polymer which was washed
with 100 mL of 2-propanol. The polymer was dried at 30.degree. C.
under vacuum to give the desired polymer as PLA-PEG(5 k)-CONHNH2
(3.4 g, MW by NMR: 24000).
Preparation of PLA-PEG-MAL
[0322] HO-PEG(3K)-maleimide (HO-PEG-MAL) (Laysan Bio, Inc) (MW:
3000, 0.6 g, 0.2 mmol) was mixed with dl-lactide (recrystallized
from EtOAc, MW 144, 4.32 g, 30 mmol) and Na2SO4 (4 g) in a 100 mL
flask. The solid mixture was dried under vacuum at 60.degree. C.
overnight and dry toluene (20 mL) was added. The resulting
suspension was heated to 110.degree. C. under argon and Sn(Oct)2
(MW 405, 0.065 mL, 0.2 mmol) was added. The mixture was heated at
reflux for 20 h and cooled to rt. The mixture was diluted with DCM
(50 mL) and filtered. After concentration to an oily residue, 10%
MeOH in ethyl ether (80 mL) was added to precipitate out the
polymer which was washed once with 80 mL of 10% MeOH in ether and
60 mL of ether. After drying at 30.degree. C. under vacuum
overnight, PLA-PEG(3K)-MAL was obtained as a white foam (3.26 g,
average MW: 24,000 by H NMR).
Preparation of PLA-PEG-SH (Prophetic)
[0323] PLA-PEG-SH copolymer is prepared according to the literature
(Nisha C. Kalarickal, et al; Macromolecules 2007, 40:1874-1880).
Briefly, the following steps are performed.
[0324] Step-1. Preparation of tBuS-PEG: Anhydrous THF (22 mL),
potassium naphthalene (0.2 M solution in THF, 12 mL), and tBu-SH
(0.54 mL, 4.8 mmol) are charged into a sealed 100 mL round-bottom
flask. The components are stirred for at least 15 min to ensure the
formation of thiolates, at which point liquid ethylene oxide (EO)
(11.5 mL, 0.230 mol) is added using a two-headed needle. The
polymerization reaction is carried out for 48 h, and the product is
recovered by precipitation in cold diethyl ether. MW of the polymer
by GPC is about 2100.
[0325] Step-2. Preparation of (PEG-S)2: tBu-S-PEG from Step-1 (1.0
g) is dissolved in DMSO (19 mL) followed by addition of TFA (106
mL, 15/85 v/v) to a final polymer concentration of 8 mg/mL. The
reaction is stirred for 20 min, after which TFA is removed by
rotary evaporation. The residual is then precipitated twice in cold
diethyl ether to recover the crude PEG disulfide. The crude
(PEG-S)2 is further purified by fractional precipitation. Thus, the
polymer (1.0 g) is dissolved in dichloromethane (100 mL), and then
cold diethyl ether is added stepwise with stirring until the
appearance of a precipitate. The solution is further stirred for 30
min, and the precipitated mass is isolated by filtration and dried
in vacuo. The recovery yield of PEG disulfide, (PEG-S)2, at the end
of two to three fractional precipitations is in the range
55-60%.
[0326] Step-3. Preparation of (PLA-b-PEG-S)2 by ring-opening
polymerization of dl-lactide: (PEG-S)2 (0.4 g, 0.10 mmol) and
dl-lactide (4.32 g, 30 mmol) are mixed in dry toluene (70 mL). The
mixture is heated to reflux while 50 mL of toluene is removed by
azeotrope distillation. The resulting solution was cooled to
100.degree. C. and Sn(Oct)2 (0.065 mL, 0.20 mmol) was added. The
solution is heated at reflux under argon for 18-20 h and cooled to
rt. The solution is then added to 150 mL of 2-propanol to
precipitate out the polymer which is washed with 2-propanol (60 mL)
and ether (60 mL) and dried under vacuum at 30.degree. C. for 2
days to give (PLA-PEG-S)2 (ca. 4.0 g, MW: 46000).
[0327] Step-4. Preparation of PLA-PEG-SH by reduction of
(PLA-PEG-S)2: The (PLA-PEG-S)2 from Step-3 (3.2 g, 0.07 mmol) is
dissolved in deoxygenated THF (25 mL), and Bu3P (1.7 mL, 7.0 mmol,
100 equiv with respect to disulfide units) is added. The reaction
mixture is stirred under argon at room temperature overnight. The
reduced thiolated polymer is recovered by precipitation in cold
diethyl ether followed by filtration under argon atmosphere and
further dried under vacuum to give PLA-PEG-SH as an off white
chunky solid (ca. 3.0, MW: 23000).
Preparation of Nanocarriers with Surface PEG-X Containing
Encapsulated Ova Peptide
[0328] Nanocarriers comprising PLGA-R848, PLA-PEG-X (where
X=carboxylic acid (CO2H), amine (NH2), C6-azide (C6-N3) or
PEG3-azide (PEG3-N3), hydrazide (CONHNH2), maleimide (MAL), thiol
(SH) and nitrilotriacetic acid group (NTA)) containing ova peptide
were prepared via a double emulsion method wherein the ova peptide
was encapsulated in the nanocarriers. Polyvinyl alcohol (Mw=11 KD
-31 KD, 87-89% partially hydrolyzed) was purchased from J T Baker.
Ovalbumin peptide 323-339, (sequence:
H-Ile-Ser-Gln-Ala-Val-His-Ala-Ala-His-Ala-Glu-Ile-Asn-Glu-Ala-Gly-Arg-NH2-
, acetate salt, Lot#B06395) was obtained from Bachem Americas Inc.
(3132 Kashiwa Street, Torrance Calif. 90505), PLA with acid end
group (100DL2A) was obtained from SurModics Pharmaceuticals (756
Tom Martin Drive, Birmingham, Ala. 35211); PLGA-R848, and PLA-PEG-X
conjugates were prepared as described above in this same
example.
[0329] The above materials were used to prepare the following
solutions: [0330] 1. PLGA-R848 conjugate in methylene chloride @
100 mg/mL, [0331] 2. PLA-PEG-X in methylene chloride @ 100 mg/mL,
[0332] 3. PLA (100DL2A) in methylene chloride @ 100 mg/mL, [0333]
4. Ovalbumin peptide 323-339 in 0.13N HCl @ 70 mg/mL, and [0334] 5.
Polyvinyl alcohol in 100 mM pH 8 phosphate buffer @50 mg/mL.
[0335] Solution #1 (0.50 mL), solution #2 (0.25 mL) and solution #3
(0.25 mL) were combined and solution #4 in 0.13N HCl (0.1mL) was
added in a small vessel and the mixture was sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. To
this emulsion was added solution # 5 (2.0 mL) and sonication at 30%
amplitude for 40 seconds using the Branson Digital Sonifier 250 was
performed on the second emulsion. This was then added to a stirring
beaker containing a 70 mM pH 8 phosphate buffer solution (30 mL),
and this mixture was stirred at room temperature for 2 hours to
form the nanocarriers.
[0336] To wash the nanocarriers, a portion of the nanocarrier
dispersion (26.5 mL) was transferred to a 50 mL centrifuge tube and
spun at 9500 rpm (13,800 g) for one hour at 4.degree. C. The
supernatant was removed, and the pellet was re-suspended in 26.5 mL
of phosphate buffered saline. The centrifuge procedure was repeated
and the pellet was re-suspended in 8.3 g of phosphate buffered
saline for a final nanocarrier dispersion of about 10 mg/mL
containing encapsulated ova peptide.
Preparation of Nanocarriers with surface PEG-X Without Encapsulated
Ova Peptide
[0337] In a similar manner to the procedure described immediately
above, nanocarriers without ova peptide were prepared where
solution #4 was eliminated in the preparation.
Example 14
Nanocarriers with Obtained Versus Derived Antigen (Prophetic)
[0338] Nanocarriers with PTH: Nanocarriers with surface PEG-CONHNH2
hydrazide groups are prepared as described above in Example 13. PTH
(parathyroid hormone) protein is acylated via the lysine amino
group with 4-formyl-benzoic acid in the presence of EDC. HCl and
NHS are used to generate PTH containing benzaldehyde groups. After
purification via dialfiltration with a MWCO 1K filter, the modified
PTH is conjugated with the NCs containing the hydrazide on the
surface in PBS buffer (pH 8-9). After purification by pellet
washing with PBS buffer, the resulting NC-modified PTH conjugate is
suspended in pH 7.4 buffer.
[0339] Nanocarriers with Modified PTH: Nanocarriers with surface
PEG-CO2H group are prepared as described above in Example 13. The
NCs are then activated with excess EDC/NHS in pH 6 PBS buffer at
4.degree. C. for 1-2 h. The activated NCs are then pellet washed
with pH 6.0 buffer to remove un-reacted EDC/NHS. Modified PTH
dissolved in the same PBS buffer is then added to the resulting NC
suspension. The conjugation is allowed to proceed at 4.degree. C.
overnight. After pellet washing with PBS buffer, the resulting
NC-Modified PTH conjugate is suspended in pH 7.4 PBS buffer.
[0340] Equal portions of the two nanocarriers can then be combined
to form a NC suspension for further testing.
Example 15
Monovalent and Bivalent Nanocarriers with Antigens from the Same
Genus of Infectious Agent (Prophetic)
[0341] Nanocarriers with surface PEG-CO2H groups are prepared as
described above in Example 13. The NCs are then activated with
excess EDC/NHS in pH 6 PBS buffer at 4.degree. C. for 1-2 h. The
activated NCs are then pellet washed with pH 6.0 buffer to remove
un-reacted EDC/NHS and suspended in pH 6.0 buffer. Human Influenza
A virus HA protein trimer and HA M2e protein dissolved in pH 6.0
buffer is then added to the resulting NC suspension. The
conjugation is allowed to proceed at 4.degree. C. overnight. After
pellet washing with PBS buffer, the resulting NC-HA protein
trimer/M2e protein conjugate is suspended in pH 7.4 PBS buffer.
[0342] In the same fashion, a NC-HA monomer protein conjugate is
prepared using monomeric Human Influenza A virus HA protein.
[0343] The nanocarriers can then be combined to form a NC
suspension for further testing.
Example 16
Monovalent and Bivalent Nanocarriers with Antigens from a Different
Genus of Infectious Agent (Prophetic)
[0344] Nanocarriers with surface PEG-CO2H groups are prepared as
described above in Example 13. The NCs are then activated with
excess EDC/NHS in pH 6 PBS buffer at 4.degree. C. for 1-2 h. The
activated NCs are then pellet washed with pH 6.0 buffer to remove
un-reacted EDC/NHS and suspended in pH 6.0 buffer. Human Influenza
A virus HA protein trimer and HA M2e protein dissolved in pH 6.0
buffer is then added to the resulting NC suspension. The
conjugation is allowed to proceed at 4.degree. C. overnight. After
pellet washing with PBS buffer, the resulting NC-HA protein
trimer/M2e protein conjugate is suspended in pH 7.4 PBS buffer.
[0345] In the same fashion, a NC-infectious salmon anemia virus
conjugate is prepared using inactivated infectious salmon anemia
virus.
[0346] The nanocarriers can then be combined to form a NC
suspension for further testing.
Example 17
Monovalent Nanocarriers with Antigens from the Same Species of
Infectious Agent (Prophetic)
[0347] Nanocarriers with surface PEG-CO2H groups are prepared as
described above in Example 13. The NCs are then activated with
excess EDC/NHS in pH 6 PBS buffer at 4.degree. C. for 1-2 h. The
activated NCs are then pellet washed with pH 6.0 buffer to remove
un-reacted EDC/NHS and suspended in pH 6.0 buffer. Measles
hemaglutinin antigen (a recombinant fragment containing the measles
hemagglutinin immunodominant regions, amino acids 106-114 and
519-550) is dissolved in pH 6.0 buffer and then added to the
resulting NC suspension. The conjugation is allowed to proceed at
4.degree. C. overnight. After pellet washing with PBS buffer, the
resulting NC-measles hemaglutinin conjugate is suspended in pH 7.4
PBS buffer.
[0348] In the same fashion, NC-measles fusion antigen conjugate is
prepared using a fragment of measles fusion protein (a recombinant
fragment corresponding to amino acids 399-525 of measles large
fusion protein).
[0349] The nanocarriers can then be combined to form a NC
suspension for further testing.
Example 18
Monovalent Nanocarriers with Antigens from Different Species of
Infectious Agent (Prophetic)
[0350] Nanocarriers with surface PEG-CO2H groups are prepared as
described above in Example 13. The NCs are then activated with
excess EDC/NHS in pH 6 PBS buffer at 4.degree. C. for 1-2 h. The
activated NCs are then pellet washed with pH 6.0 buffer to remove
un-reacted EDC/NHS and suspended in pH 6.0 buffer. Human Influenza
A virus HA protein trimer dissolved in pH 6.0 buffer is then added
to the resulting NC suspension. The conjugation is allowed to
proceed at 4.degree. C. overnight. After pellet washing with PBS
buffer, the resulting NC-HA protein trimer conjugate is suspended
in pH 7.4 PBS buffer for further testing.
[0351] Streptococcus pneumonia polysaccharide (PnPs) 6B is selected
as a representative PnPs serotype. Purified native (i.e., no post
purification size reduction) PnPs-6B is dissolved in 2 M NaCl. A
solution of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate
(CDAP) in CH3CN (100 mg /mL) is added (ratio of CDAP/PnPs: 1.0
mg/mg). The pH of the resulting solution is adjusted to 9 with 0.2
M of aqueous Et3N or dilutes of NaOH solution. After 3-4 min, the
resulting activated PnPs-6B solution is added to NCs with surface
PEG-CONHNH2 (PEG-hydrazide) groups prepared as described above in
Example 13 in pH 9 buffer. The resulting NCs and PnPs-6B suspension
is shaken for 1 h and quenched with 2 M glycine solution. After
pellet washing with PBS buffer, the resulting NC-PnPs-6B conjugate
is suspended in pH 7.4 PBS buffer.
[0352] The nanocarriers can then be combined to form a NC
suspension for further testing.
Example 19
Monovalent Nanocarriers with Antigens from the Same Strain of
Infectious Agent (Prophetic)
[0353] Nanocarriers with PEG-X on the surface are prepared as
follows. Monodisperse PRINT nanocarriers (PRINT NCs) comprising
PLGA-R848, PLA-PEG-X (where X=carboxylic acid (CO2H), amine (NH2),
C6-azide (C6-N3) or PEG3-azide (PEG3-N3), hydrazide (CONHNH2),
maleimide (MAL) and thiol (SH)) containing ova peptide are prepared
by the Particle Replication in Non-wetting Templates (PRINT) method
as described in the literature ((1) "Direct Fabrication and
Harvesting of Monodisperse, Shape Specific Nano-Biomaterials";
Rolland, J. P.; Maynor, B. W.; Euliss, L. E.; Exner, A. E.;
Denison, G. M.; DeSimone, J. M J. Am. Chem. Soc. 2005, 127, 10096;
(2) "The Complex Role of Multivalency in Nanoparticles Targeting
the Transferrin Receptor for Cancer Therapies" Jin Wang, Shaomin
Tian, Robby A. Petros, Mary E. Napier and Joseph M. DeSimone; J.
Am. Chem. Soc., 2010, 132 (32), pp 11306-11313). PRINT-NCs with
surface PEG-CO2H groups are activated with excess EDC/NHS in pH 6
PBS buffer at 4.degree. C. for 1-2 h. The activated NCs are then
pellet washed with pH 6.0 buffer to remove un-reacted EDC/NHS and
suspended in pH 6.0 buffer. Pneumococcal surface protein A (PspA)
dissolved in pH 6.0 buffer is then added to the resulting NC
suspension. The conjugation is allowed to proceed at 4.degree. C.
overnight. After pellet washing with PBS buffer, the resulting
NC-PsPA conjugate is suspended in pH 7.4 PBS buffer.
[0354] Purified native PnPs-6B is dissolved in 2 M NaCl. A solution
of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) in
CH3CN (100 mg /mL) is added (ratio of CDAP/PnPs: 1.0 mg/mg). The pH
of the resulting solution is adjusted to 9 with 0.2 M of aqueous
Et3N or dilutes of NaOH solution. After 3-4 min, the resulting
activated PnPs-6B solution is added to PRINT NCs with surface
PEG-CONHNH2 (PEG-hydrazide) groups prepared as described above in
pH 9 buffer. The resulting NCs and PnPs-6B suspension is shaken for
1 h and quenched with 2 M glycine solution. After pellet washing
with PBS buffer, the resulting NC-PnPs-6B conjugates are suspended
in pH 7.4 PBS buffer.
[0355] The nanocarriers can then be combined to form a NC
suspension for further testing.
Example 20
Monovalent Nanocarriers with Antigens from Different Strains of
Infectious Agent (Prophetic)
[0356] Purified native PnPs-6B is dissolved in 2 M NaCl. A solution
of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) in
CH3CN (100 mg /mL) is added (ratio of CDAP/PnPs: 1.0 mg/mg). The pH
of the resulting solution is adjusted to 9 with 0.2 M of aqueous
Et3N or dilutes of NaOH solution. After 3-4 min, the resulting
activated PnPs-6B solution is added to NCs with surface PEG-CONHNH2
(PEG-hydrazide) groups prepared as described above in Example 13 in
pH 9 buffer. The resulting NCs and PnPs-6B suspension is shaken for
1 h and quenched with 2 M glycine solution. After pellet washing
with PBS buffer, the resulting NC-PnPs-6B conjugates are suspended
in pH 7.4 PBS buffer.
[0357] Purified native PnPs14 from is dissolved in 2 M NaCl. A
solution of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate
(CDAP) in CH3CN (100 mg /mL) is added (ratio of CDAP/PnPs: 1.0
mg/mg). The pH of the resulting solution is adjusted to 9 with 0.2
M of aqueous Et3N or dilutes of NaOH solution. After 3-4 min, the
resulting activated PnPs14 solution is added to PRINT NCs with
surface PEG-CONHNH2 (PEG-hydrazide) groups prepared as described
above in pH 9 buffer. The resulting PRINT NCs and PnPs14 suspension
is shaken for 1 h and quenched with 2 M glycine solution. After
pellet washing with PBS buffer, the resulting PRINT NC-PnPs14
conjugates are suspended in pH 7.4 PBS buffer.
[0358] Gold NCs with surface PEG-X (where X=carboxylic acid (CO2H),
amine (NH2), azide (N3), hydrazide (CONHNH2) and aldehyde (CHO))
are prepared as follows.
[0359] Step-1. Formation of Gold NCs (AuNCs): A aq. solution of 500
mL of 1 mM HAuCl4 is heated to reflux for 10 min with vigorous
stirring in a 1 L round-bottom flask equipped with a condenser. A
solution of 50 mL of 40 mM of trisodium citrate is then rapidly
added to the stirring solution. The resulting deep wine red
solution is kept at reflux for 25-30 min and the heat is withdrawn
and the solution is cooled to room temperature. The solution is
then filtered through a 0.8 .mu.m membrane filter to give the AuNCs
solution. The AuNCs are characterized using visible spectroscopy
and transmission electron microscopy. The AuNCs are ca. 20 nm
diameter capped by citrate with peak absorption at 520 nm.
[0360] Step-2. AuNCs functionalized with PEG-X using HS-PEG-X:
AuNCs are functionalized with HS-PEG-X (MW range: 1500-5000) (where
X=carboxylic acid (CO2H), amine (NH2), azide (N3), hydrazide
(CONHNH2) and aldehyde (CHO)) as follows. A solution of 150 .mu.l
of HS-PEG-X (10 .mu.M in 10 mM pH 9.0 carbonate buffer) is added to
1 mL of 20 nm diameter citrate-capped gold nanocarriers (1.16 nM)
to produce a molar ratio of thiol to gold of 2500:1. The mixture is
stirred at room temperature under argon for 1 hour to allow
complete exchange of thiol with citrate on the gold nanocarriers.
The AuNCs with PEG-X on the surface is then purified by centrifuge
at 12,000g for 30 minutes. The supernatant is decanted and the
pellet containing AuNC-PEG-X is re-suspended in appropriate PBS
buffer for further bioconjugation with biomolecules. Purified
native PnPs-19F from is dissolved in 2 M NaCl. A solution of
1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) in CH3CN
(100 mg /mL) is added (ratio of CDAP/PnPs: 1.0 mg/mg). The pH of
the resulting solution is adjusted to 9 with 0.2 M of aqueous Et3N
or dilutes of NaOH solution. After 3-4 min, the resulting activated
PnPs-19F solution is added to AuNCs with surface PEG-CONHNH2
(PEG-hydrazide) groups prepared as described above in pH 9 buffer.
The resulting AuNCs and PnPs-19F suspension is shaken for 1 h and
quenched with 2 M glycine solution. After pellet washing with PBS
buffer, the resulting AuNC-PnPs-19F conjugates are suspended in pH
7.4 PBS buffer.
[0361] The nanocarriers can then be combined to form a NC
suspension for further testing.
Example 21
Monovalent Nanocarriers with the Same Antigen but Different
Orientation (Prophetic)
[0362] PRINT NCs with surface nicotine analog attached via the
3'-position are prepared from PLA-PEG-3-HO-MeNic copolymer derived
from trans-3'-hydroxymethylnicotine (3-HO-MeNic), PLGA-R848 and ova
peptide as described above. The resulting PRINT NCs containing
surface 3'-substituted nicotine analog are suspended in pH 7.4
buffer.
[0363] In a similar manner, PRINT NCs with surface nicotine analog
attached via the 1'-position are prepared from PLA-PEG-1-butyl-Nic
copolymer derived from 1'-butyl nicotine (1-butyl-Nic), PLGA-R848
and ova peptide as described above. The resulting PRINT NCs
containing surface 1'-substituted nicotine analog are suspended in
pH 7.4 buffer.
[0364] The nanocarriers can then be combined to form a NC
suspension for further testing.
Example 22
Monovalent Nanocarriers with the Same Antigen but Different
Conformation (Prophetic)
[0365] Nanocarriers with surface PEG-CO2H groups are prepared as
described above in Example 13. The NCs are then activated with
excess EDC/NHS in pH 6 PBS buffer at 4.degree. C. for 1-2 h. The
activated NCs are then pellet washed with pH 6.0 buffer to remove
un-reacted EDC/NHS and suspended in pH 6.0 buffer. Measles virus
hemagglutinin noose epitope (HNE, H379-410, disulfide intact)
dissolved in pH 6.0 buffer is then added to the resulting NC
suspension. The conjugation is allowed to proceed at 4.degree. C.
overnight. After pellet washing with PBS buffer, the resulting
NC-HNE conjugates are suspended in pH 7.4 PBS buffer.
[0366] The highly conserved hemagglutinin noose epitope (HNE,
H379-410) of the measles virus contains three cysteine residues,
two of which (Cys386 and Cys394) form a disulfide bridge. The HNE
peptide containing the disulfide bridge is reduced using
dithiothreitol (DTT) in PBS buffer to give reduced HNE. NCs with
surface PEG-CO2H groups are prepared as described above in Example
13. The NCs are then activated with excess EDC/NHS in pH 6 PBS
buffer at 4.degree. C. for 1-2 h. The activated NCs are then pellet
washed with pH 6.0 buffer to remove un-reacted EDC/NHS and
suspended in pH 6.0 buffer. The reduced HNE dissolved in pH 6.0
buffer is then added to the resulting NC suspension under argon in
the presence of DTT. The conjugation is allowed to proceed at
4.degree. C. overnight under argon. After pellet washing with PBS
buffer, the resulting NC-reduced HNE conjugates are suspended in pH
7.4 PBS buffer.
[0367] The nanocarriers can then be combined to form a NC
suspension for further testing.
Example 23
Monovalent and Bivalent Nanocarriers with the Small Molecule
Antigens of Different Structure (Prophetic)
[0368] AuNCs with surface PEG-CO2H groups are prepared as described
above. The AuNCs are then activated with excess EDC/NHS in pH 6 PBS
buffer at 4.degree. C. for 1-2 h. The activated NCs are then pellet
washed with pH 6.0 buffer to remove un-reacted EDC/NHS and
suspended in pH 6.0 buffer. Trans-3'-aminomethylnicotine prepared
from commercially available 4-cotininecarboxylic acid (US Patent
Application: US2007/0129551 A1) in pH 6.0 buffer is added to the
activated AuNCs. The conjugation is allowed to proceed at 4.degree.
C. overnight. After pellet washing with PBS buffer, the resulting
AuNC-nicotine conjugates are suspended in pH 7.4 PBS buffer.
[0369] VLPs with surface functional groups such as azide or alkyne
for CuAAC click chemistry are prepared as described in the
literature ("Surface Functionalization of Virus-Like Particles by
Direct Conjugation Using Azide--Alkyne Click Chemistry", Kedar G.
Patel and James R. Swartz; Bioconjugate Chem., 2011, 22 (3), pp
376-387). Cocaine analog containing alkyne or azide linker and
methamphetamine analog containing alkyne or azide linker are
prepared according to literature procedures as surface B-cell
antigen epitopes. An equal molar mixture of cocaine analog and
methamphetamine analog with azide linker is treated with VLPs
containing surface alkyne group under standard CuAAC condition to
give VLP-cocaine-methamphetamine conjugates.
[0370] The nanocarriers can then be combined to form a NC
suspension for further testing.
Example 24
Bivalent Nanocarriers with Oligosaccharide Antigens of Different
Structure (Prophetic)
[0371] Purified PnPs-6B is size reduced with dilute acid or under
sonication to give oligomeric PnPs-6B which is dissolved in 2 M
NaCl. Similarly, purified PnPs-3 is size reduced with dilute acid
or under sonication to give oligomeric PnPs-3 which is dissolved in
2 M NaCl. An equal molar mixed solution of oligomeric PnPs-6B and
PnPs-3 is prepared from these solutions. A solution of
1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) in CH3CN
(100 mg /mL) is added (ratio of CDAP/PnPs: 1.5 mg/mg) to the mixed
PnPs solution. The pH of the resulting solution is adjusted to 9
with 0.2 M of aqueous Et3N or dilutes of NaOH solution. After 3-4
min, the resulting activated oligomeric PnPs-6B/PnPs-3 solution is
added to AuNCs with surface PEG-CONHNH2 (PEG-hydrazide) groups
prepared as described above in pH 9 buffer. The resulting AuNCs and
activated PnPs-6B/PnPs-3 suspension is shaken for 1 h and quenched
with 2 M glycine solution. After pellet washing with PBS buffer,
the resulting AuNC-PnPs-6B/3 conjugates are suspended in pH 7.4 PBS
buffer.
[0372] VLPs containing carboxylic acid (CO2H) groups on the surface
are activated with excess EDC/NHS in pH 6 PBS buffer at 4.degree.
C. for 1-2 h. The activated VLPs are then pellet washed with pH 6.0
buffer to remove un-reacted EDC/NHS and suspended in pH 6.0 buffer.
Purified PnPs-4 is size reduced with dilute acid or under
sonication to give oligomeric PnPs-4 which is dissolved in 2 M
NaCl. Similarly, purified PnPs-19F is size reduced with dilute acid
or under sonication to give oligomeric PnPs-19F which is dissolved
in 2 M NaCl. An equal molar mixed solution of oligomeric PnPs-4 and
PnPs-19F is prepared from these solutions. A solution of
1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) in CH3CN
(100 mg/mL) is added (ratio of CDAP/PnPs: 1.5 mg/mg) to the mixed
PnPs solution. The pH of the resulting solution is adjusted to 9
with 0.2 M of aqueous Et3N or dilutes of NaOH solution. After 3-4
min, a solution of adipic acid dihydrazide (ADH) linker in pH 9
buffer is added to the activated mixed PnPs-4/19F solution. The
resulting solution is mixed for 1 h and quenched with 2 M glycine
solution and purified by dialysis. The purified oligomeric
PnPs-4/19F with ADH linker in pH 6.0 buffer is then added to the
activated VLPs in pH 6.0 buffer and the resulting suspension is
mixed at 4.degree. C. overnight and purified by dialysis or pellet
wash to give VLP-PnPs-4/19F conjugates for further testing.
[0373] The nanocarriers can then be combined to form a NC
suspension for further testing.
Example 25
Bivalent Nanocarriers with Polysaccharide Antigens of Different
Structure (Prophetic)
[0374] Purified native PnPs-6B is dissolved in 2 M NaCl. A solution
of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) in
CH3CN (100 mg /mL) is added (ratio of CDAP/PnPs: 1.0 mg/mg). The pH
of the resulting solution is adjusted to 9 with 0.2 M of aqueous
Et3N or dilutes of NaOH solution. After 3-4 min, a solution of
adipic acid dihydrazide (ADH) linker in pH 9 buffer is added to the
activated PnPs-6B solution. The resulting solution is mixed for 1 h
and purified by dialysis. The purified PnPs-6B with ADH linker is
dissolved in pH 6.0 buffer for NC conjugation.
[0375] Purified N. meningitidis meningococcal polysaccharide
serogroup A (NmA) is dissolved in 1 M NaCl. A solution of
1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) in CH3CN
(100 mg /mL) is added (ratio of CDAP/NmA: 1.5 mg/mg). The pH of the
resulting solution is adjusted to 9 with 0.2 M of aqueous Et3N or
dilutes of NaOH solution. After 3-4 min, a solution of adipic acid
dihydrazide (ADH) linker in pH 9 buffer is added to the activated
NmA solution. The resulting solution is mixed for 1-2 h and
purified by dialysis. The purified NmA with ADH linker is dissolved
in pH 6.0 buffer for NC conjugation.
[0376] NCs with surface PEG-CO2H groups are prepared as described
above in Example 13. The NCs are then activated with excess EDC/NHS
in pH 6 PBS buffer at 4.degree. C. for 1-2 h. The activated NCs are
then pellet washed with pH 6.0 buffer to remove un-reacted EDC/NHS
and suspended in pH 6.0 buffer. An equal molar mixed solution of
PnPs-6B with ADH linker and NmA with ADH linker in pH 6.0 buffer is
added to the activated NC solution, and the resulting suspension is
mixed at 4.degree. C. overnight. After pellet washing with PBS
buffer, the resulting NC-PnPs6B/NmA conjugates are suspended in pH
7.4 PBS buffer.
[0377] Purified native PnPs-19F is dissolved in 2 M NaCl. A
solution of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate
(CDAP) in CH3CN (100 mg /mL) is added (ratio of CDAP/PnPs: 1.0
mg/mg). The pH of the resulting solution is adjusted to 9 with 0.2
M of aqueous Et3N or dilutes of NaOH solution. After 3-4 min, a
solution of adipic acid dihydrazide (ADH) linker in pH 9 buffer is
added to the activated PnPs-19F solution. The resulting solution is
mixed for 1 h and purified by dialysis. The purified PnPs-19F with
ADH linker is dissolved in pH 6.0 buffer for NC conjugation.
[0378] Purified N. meningitidis meningococcal polysaccharide
serogroup C (NmC) is dissolved in 1 M NaCl. A solution of
1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) in CH3CN
(100 mg /mL) is added (ratio of CDAP/NmC: 1.5 mg/mg). The pH of the
resulting solution is adjusted to 9 with 0.2 M of aqueous Et3N or
dilutes of NaOH solution. After 3-4 min, a solution of adipic acid
dihydrazide (ADH) linker in pH 9 buffer is added to the activated
NmC solution. The resulting solution is mixed for 1-2 h and
purified by dialysis. The purified NmC with ADH linker is dissolved
in pH 6.0 buffer for NC conjugation.
[0379] NCs with surface PEG-CO2H groups are prepared as described
above in Example 13. The NCs are then activated with excess EDC/NHS
in pH 6 PBS buffer at 4.degree. C. for 1-2 h. The activated NCs are
then pellet washed with pH 6.0 buffer to remove un-reacted EDC/NHS
and suspended in pH 6.0 buffer. An equal molar mixed solution of
PnPs-19F with ADH linker and NmC with ADH linker in pH 6.0 buffer
is added to the activated NC solution, and the resulting suspension
is mixed at 4.degree. C. overnight. After pellet washing with PBS
buffer, the resulting NC-PnPs-19F/NmC conjugates are suspended in
pH 7.4 PBS buffer.
[0380] The nanocarriers can then be combined to form a NC
suspension for further testing.
Example 26
Bivalent and Monovalent Nanocarriers with Small Molecule Antigens
in Different Orientations (Prophetic)
[0381] Nanocarriers with surface PEG-CONHNH2 (PEG-hydrazide) are
prepared as described above in Example 13 and suspended in pH 6.0
buffer at 4.degree. C. A cocaine analog GNC
(6-(2R,3S)-3-(benzoyloxy)-8-methyl-8-azabicyclo [3.2.11
octane-2-carbonyloxy-hexanoic acid) is prepared according to a
reported procedure ("Cocaine Analog Coupled to Disrupted
Adenovirus: A Vaccine Strategy to Evoke High-titer Immunity Against
Addictive Drugs" Martin J Hicks, et al, Mol Ther 2011, 19:
612-619). This compound is activated with EDC/NHS in DMF, and the
activated GNC-NHS ester is isolated and purified for NC
conjugation. Another cocaine analog, AI1 is prepared according to a
reported procedure ("Positional linker effects in haptens for
cocaine immunopharmacotherapy", Akira Ino, Tobin J. Dickerson, and
Kim D. Janda; Bioorganic & Medicinal Chemistry Letters 17
(2007) 4280-4283) and activated with EDC/NHS as above. An equal
molar portion of each activated cocaine analog in excess to NC
surface PEG-hydrazide is mixed with the NCs in pH 6.0 buffer. The
resulting suspension is mixed at 4.degree. C. overnight. After
pellet washing with PBS buffer, the resulting NC-GNC/AI1 cocaine
conjugates are suspended in pH 7.4 PBS buffer.
[0382] Norcocaine is treated with succinic anhydride to give
cocaine containing a succinic acid linker, SNC, and then activated
with EDC/NHS according to a reported procedure (Fox B S, Kantak K
M, Edwards M A et al. Efficacy of a therapeutic cocaine vaccine in
rodent models. Nat. Med. 2(10), 1129-1132 (1996). NCs with surface
PEG-CONHNH2 (PEG-hydrazide) are prepared as described above in
Example 13 and suspended in pH 6.0 buffer at 4.degree. C. Excess
amounts of activated cocaine analog, SNC, is added to the NCs. The
resulting suspension is mixed at 4.degree. C. overnight. After
pellet washing with PBS buffer, the resulting NC-SNC cocaine
conjugates are suspended in pH 7.4 PBS buffer.
[0383] The nanocarriers can then be combined to form a NC
suspension for further testing.
Example 27
Bivalent and Monovalent Nanocarriers with Peptide Antigens with
Different Attachments (Prophetic)
[0384] Nanocarriers with surface PEG-azide (PEG-N3) are prepared
according to Example 13 and suspended in de-gassed pH 7 buffer with
argon. Ovalbumin (325-336) peptide with a C-terminal propargyl
amide group (a C-alkyne group) is prepared by standard solid phase
peptide synthesis, and the resulting purified Ova
(325-336)-C-alkyne peptide is dissolved in pH 7 buffer under argon.
Ovalbumin (325-336) peptide with the N-terminal amine acylated with
5-hexynoic acid (an N-terminal alkyne group) is prepared by
standard solid phase peptide synthesis, and the resulting purified
Ova (325-336)-N-alkyne peptide is dissolved in pH 7 buffer under
argon. NCs with surface PEG-N3is mixed with an equal molar amount
of each ova --C-alkyne and N-alkyne peptide in pH 7 buffer under
argon, and the resulting suspension is subjected to the CuAAC click
reaction according to a reported protocol ("Analysis and
optimization of copper-catalyzed azide-alkyne cycloaddition for
bioconjugation", Hong V, Presolski S I, Ma C, Finn M G.; Angew Chem
Int Ed Engl. 2009;48(52):9879-83). The resulting NC-Ova
peptide-C-linked/Ova peptide-N-linked conjugates are purified by
pellet wash with pH 7 buffer and suspended in pH 7 buffer.
[0385] Recombinant virus-like particles (VLP) are prepared
according to a standard procedure. In particular, VLPs from rabbit
hemorrhagic disease virus is prepared and conjugated with Ova
(323-339) peptide via a heterobifunctional linker such as
Sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate
(Sulfo-SMCC) as described by Matthew Peacey, et al. ((1) Peacey M,
Wilson S, Baird M A, Ward V K. "Versatile RHDV virus-like
particles: incorporation of antigens by genetic modification and
chemical conjugation" Biotechnol Bioeng; 2007; 98:968-77; (2)
Peacey M, Wilson S, Perret R, Ronchese F, Ward V K, Young V, Young
S, Baird, M A. "Virus-like particles from rabbit hemorrhagic
disease virus can induce ananti-tumor response" Vaccine; 2008;
26:5334-5337). The resulting VLP-ova peptide conjugates are
purified and suspended in pH 7 buffer.
[0386] The nanocarriers can then be combined to form a NC
suspension for further testing.
Example 28
Monovalent Nanocarriers with Protein Antigens Coupled at Different
Attachment Points on the Protein (Activated Versus Protein Tag)
(Prophetic)
[0387] Nanocarriers with surface PEG-CO2H groups are prepared as
described in Example 13. The NCs are then activated with excess
EDC/NHS in pH 6 PBS buffer at 4.degree. C. for 1-2 h. The activated
NCs are then pellet washed with pH 6.0 buffer to remove un-reacted
EDC/NHS and suspended in pH 6.0 buffer. Measles hemaglutinin
protein (MHP) dissolved in pH 6.0 buffer is then added to the
resulting NC suspension. The conjugation is allowed to proceed at
4.degree. C. overnight. After pellet washing with PBS buffer, the
resulting NC-MHP conjugate is suspended in pH 7.4 PBS buffer.
[0388] Nanocarriers with surface PEG-NTA group for Ni-His tag
complexation are prepared as described in Example 13. The NCs are
then treated with a solution of NiCl2 in a binding buffer (50 mM
phosphate buffer system, 300 mM NaCl, 10 mM imidazole, pH 8.0) to
form the NCs with surface NTA-Ni complex. After pellet washing with
PBS buffer, the resulting NCs are suspended in the binding buffer
under argon. A solution of His6-tagged recombinant measles
hemaglutinin protein in the binding buffer is added to the NC
suspension, and the suspension is incubated at 4.degree. C.
overnight under argon. The resulting NC-NTA-His6-MHP conjugates are
pellet washed with pH 7 buffer and suspended in PBS buffer.
[0389] The nanocarriers can then be combined to form a NC
suspension for further testing.
Example 29
Monovalent Nanocarriers with Oligosaccharide Antigens Coupled at
Different Attachment Points (Activated Hydroxyl Group Versus
Linker) (Prophetic)
[0390] Nanocarriers with surface PEG-CONHNH2 (PEG-hydrazide) groups
are prepared as described in Example 13 and suspended in pH 9
buffer under argon. Purified PnPs-6B is size reduced with dilute
acid or under sonication to give oligomeric PnPs-6B which is
dissolved in 2 M NaCl. A solution of
1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) in CH3CN
(100 mg /mL) is added (ratio of CDAP/PnPs: 1.5 mg/mg) to the
PnPs-6B solution. The pH of the resulting solution is adjusted to 9
with 0.2 M of aqueous Et3N or dilutes of NaOH solution. After 3-4
min, the resulting activated oligomeric PnPs-6B solution is added
to the NCs with surface PEG-CONHNH2 (PEG-hydrazide) groups. The
resulting NCs and activated PnPs-6B suspension is shaken for 1 h
and quenched with 2 M glycine solution. After pellet washing with
PBS buffer, the resulting NC-PnPs-6B conjugates are suspended in pH
7.4 PBS buffer.
[0391] Nanocarriers with surface PEG-CO2H groups are prepared as
described in Example 13. The NCs are then activated with excess
EDC/NHS in pH 6 PBS buffer at 4.degree. C. for 1-2 h. The activated
NCs are then pellet washed with pH 6.0 buffer to remove un-reacted
EDC/NHS and suspended in pH 6.0 buffer. Oligo PnPs-6B with a
3-aminopropyl linker is prepared according to a reported method
("Synthetic 6B Di-, Tri-, and Tetrasaccharide-Protein Conjugates
Contain Pneumococcal Type 6A and 6B Common and 6B-Specific Epitopes
That Elicit Protective Antibodies in Mice", Jansen W T M, et al.
Infect Immun. 2001; 69(2): 787-793). The oligomeric
PnPs-6B-3-propylamine in pH 6 buffer is added to the activated NCs.
The resulting suspension is mixed at 4.degree. C. overnight under
argon. After pellet washing with PBS buffer, the NC-PnPs-6B
conjugates are suspended in pH 7.4 PBS buffer.
[0392] The nanocarriers can then be combined to form a NC
suspension for further testing.
Example 30
Monovalent Nanocarriers with Polysaccharide Antigens Coupled at
Different Attachment Points on the Polysaccharide (Prophetic)
[0393] NmA is attached via CDAP activated hydroxyl groups to NCs
with multiple attachment points. Purified N. meningitidis
meningococcal polysaccharide serogroup A (NmA) is dissolved in 1 M
NaCl. A solution of 1-cyano-4-dimethylaminopyridinium
tetrafluoroborate (CDAP) in CH3CN (100 mg/mL) is added (ratio of
CDAP/NmA: 1.5 mg/mg). The pH of the resulting solution is adjusted
to 9 with 0.2 M of aqueous Et3N or dilutes of NaOH solution. After
3-4 min, a solution of adipic acid dihydrazide (ADH) linker in pH 9
buffer is added to the activated NmA solution. The resulting
solution is mixed for 1-2 h and purified by dialysis. The purified
NmA with ADH linker is dissolved in pH 6.0 buffer for NC
conjugation.
[0394] NCs with surface PEG-CO2H groups are prepared as described
in Example 13. The NCs are then activated with excess EDC/NHS in pH
6 PBS buffer at 4.degree. C. for 1-2 h. The activated NCs are then
pellet washed with pH 6.0 buffer to remove un-reacted EDC/NHS and
suspended in pH 6.0 buffer. A solution of NmA with ADH linkers in
pH 6.0 buffer is added to the activated NC solution, and the
resulting suspension is mixed at 4.degree. C. overnight. After
pellet washing with PBS buffer, the resulting NC-NmA conjugates are
suspended in pH 7.4 PBS bufferS.
[0395] NmA is attached to NCs via a terminal amino group. NCs with
surface PEG-CO2H groups are prepared as described in Example 13.
The NCs are then activated with excess EDC/NHS in pH 6 PBS buffer
at 4.degree. C. for 1-2 h. The activated NCs are then pellet washed
with pH 6.0 buffer to remove un-reacted EDC/NHS and suspended in pH
6.0 buffer. Purified NmA is subjected to reductive amination with
NH4Cl and sodium cyanoborohydride (NaCNBH3) in pH 7 buffer to give
the amino-NmA according to a reported procedure ("Development and
phase 1 clinical testing of a conjugate vaccine against
meningococcus A and C", Costantino P, Viti S, Podda A, Velmonte M
A, Nencioni L, Rappuoli R. Vaccine. 1992; 10(10):691-8). The
amino-NmA is then added to the activated NCs suspension, and and
the resulting suspension is mixed at 4.degree. C. overnight. After
pellet washing with PBS buffer, the resulting NC-NmA conjugates are
suspended in pH 7.4 PBS buffer.
[0396] The nanocarriers can then be combined to form a NC
suspension for further testing.
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
* * * * *