U.S. patent application number 13/116556 was filed with the patent office on 2011-12-01 for synthetic nanocarrier combination 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 | 20110293723 13/116556 |
Document ID | / |
Family ID | 45004392 |
Filed Date | 2011-12-01 |
United States Patent
Application |
20110293723 |
Kind Code |
A1 |
Bratzler; Robert L. ; et
al. |
December 1, 2011 |
SYNTHETIC NANOCARRIER COMBINATION VACCINES
Abstract
Disclosed are dosage forms and related methods, that include a
first population of synthetic nanocarriers that have one or more
first antigens coupled to them, one or more second antigens that
are not coupled to the synthetic nanocarriers, and a
pharmaceutically acceptable excipient.
Inventors: |
Bratzler; Robert L.;
(Concord, MA) ; Lipford; Grayson B.; (Watertown,
MA) ; Johnston; Lloyd; (Belmont, MA) ; Zepp;
Charles; (Hardwick, MA) |
Assignee: |
Selecta Biosciences, Inc.
Watertown
MA
|
Family ID: |
45004392 |
Appl. No.: |
13/116556 |
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: |
424/489 ;
424/184.1; 424/193.1; 424/204.1; 424/209.1; 424/211.1; 424/212.1;
424/215.1; 424/216.1; 424/217.1; 424/218.1; 424/219.1; 424/224.1;
424/226.1; 424/227.1; 424/228.1; 424/230.1; 424/232.1; 424/233.1;
424/240.1; 424/243.1; 424/244.1; 424/245.1; 424/247.1; 424/248.1;
424/249.1; 424/253.1; 424/256.1; 424/257.1; 424/258.1; 424/260.1;
424/261.1; 424/262.1; 424/263.1; 424/274.1; 424/85.4; 977/754;
977/762; 977/773; 977/915 |
Current CPC
Class: |
A61K 39/0013 20130101;
A61P 31/22 20180101; A61K 9/5153 20130101; A61K 39/385 20130101;
A61K 47/6921 20170801; A61K 47/6931 20170801; A61P 37/02 20180101;
A61P 37/04 20180101; A61P 1/16 20180101; A61P 39/02 20180101; A61K
47/68 20170801; A61P 25/28 20180101; A61P 31/20 20180101; A61K
2039/70 20130101; A61P 29/00 20180101; A61K 39/39 20130101; A61K
2039/542 20130101; A61P 25/34 20180101; A61K 2039/541 20130101;
A61K 2039/555 20130101; A61K 47/6929 20170801; A61P 11/08 20180101;
A61K 47/60 20170801; A61K 47/22 20130101; A61P 11/00 20180101; A61P
31/12 20180101; A61P 37/08 20180101; A61K 47/24 20130101; A61P 3/00
20180101; A61P 31/14 20180101; A61K 39/35 20130101; A61K 45/06
20130101; A61P 25/30 20180101; A61K 2039/543 20130101; A61P 31/04
20180101; A61K 2039/54 20130101; A61P 35/00 20180101; A61K
2039/55561 20130101; A61P 43/00 20180101; A61K 47/58 20170801; A61K
2039/55555 20130101; A61K 2039/6093 20130101; A61P 5/00 20180101;
A61P 31/16 20180101; A61K 31/4745 20130101; A61K 47/02 20130101;
A61K 2039/55522 20130101; A61P 17/04 20180101; A61P 25/36 20180101;
A61P 31/10 20180101; Y02A 50/30 20180101; A61K 31/7115 20130101;
A61P 17/00 20180101; A61K 39/0005 20130101; A61K 47/646 20170801;
A61K 2039/55511 20130101; A61P 11/06 20180101; A61P 31/00 20180101;
A61P 37/00 20180101; A61K 47/593 20170801; A61K 33/06 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: |
424/489 ;
424/184.1; 424/193.1; 424/204.1; 424/240.1; 424/209.1; 424/226.1;
424/227.1; 424/228.1; 424/230.1; 424/218.1; 424/212.1; 424/219.1;
424/244.1; 424/217.1; 424/224.1; 424/215.1; 424/232.1; 424/233.1;
424/216.1; 424/211.1; 424/253.1; 424/263.1; 424/247.1; 424/245.1;
424/257.1; 424/248.1; 424/249.1; 424/260.1; 424/258.1; 424/243.1;
424/256.1; 424/262.1; 424/261.1; 424/274.1; 424/85.4; 977/915;
977/773; 977/762; 977/754 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61K 39/385 20060101 A61K039/385; A61K 39/12 20060101
A61K039/12; A61K 39/10 20060101 A61K039/10; A61K 39/145 20060101
A61K039/145; A61K 39/29 20060101 A61K039/29; A61K 39/25 20060101
A61K039/25; A61K 39/165 20060101 A61K039/165; A61K 39/20 20060101
A61K039/20; A61K 39/09 20060101 A61K039/09; A61K 39/13 20060101
A61K039/13; A61K 39/205 20060101 A61K039/205; A61K 39/15 20060101
A61K039/15; A61K 39/285 20060101 A61K039/285; A61K 39/235 20060101
A61K039/235; A61K 39/125 20060101 A61K039/125; A61K 39/155 20060101
A61K039/155; A61K 39/106 20060101 A61K039/106; A61K 39/118 20060101
A61K039/118; A61K 39/08 20060101 A61K039/08; A61K 39/05 20060101
A61K039/05; A61K 39/108 20060101 A61K039/108; A61K 39/04 20060101
A61K039/04; A61K 39/095 20060101 A61K039/095; A61K 39/104 20060101
A61K039/104; A61K 39/112 20060101 A61K039/112; A61K 39/085 20060101
A61K039/085; A61P 35/00 20060101 A61P035/00; A61P 31/00 20060101
A61P031/00; A61P 31/04 20060101 A61P031/04; A61P 31/10 20060101
A61P031/10; A61P 31/12 20060101 A61P031/12; A61P 31/14 20060101
A61P031/14; A61P 31/16 20060101 A61P031/16; A61P 31/20 20060101
A61P031/20; A61P 31/22 20060101 A61P031/22; A61K 38/21 20060101
A61K038/21; A61K 39/00 20060101 A61K039/00 |
Claims
1. A dosage form comprising: (1) a first population of synthetic
nanocarriers that have one or more first antigens coupled to them,
(2) one or more second antigens that are not coupled to the
synthetic nanocarriers, and (3) a pharmaceutically acceptable
excipient.
2. The dosage form of claim 1, further comprising one or more
adjuvants that are coupled to the synthetic nanocarriers of the
first population of synthetic nanocarriers.
3. The dosage form of claim 2, wherein the one or more coupled
adjuvants comprise Pluronic.RTM. block co-polymers, specifically
modified or prepared peptides, muramyl dipeptide, aminoalkyl
glucosaminide 4-phosphates, RC529, bacterial toxoids, toxin
fragments, agonists of Toll-Like Receptors 2, 3, 4, 5, 7, 8, 9
and/or combinations thereof; adenine derivatives; immunostimulatory
DNA; immunostimulatory RNA; imidazoquinoline amines,
imidazopyridine amines, 6,7-fused cycloalkylimidazopyridine amines,
1,2-bridged imidazoquinoline amines; imiquimod; resiquimod; type I
interferons; poly I:C; bacterial lipopolysacccharide (LPS); VSV-G;
HMGB-1; flagellin or portions or derivatives thereof; or
immunostimulatory DNA molecules comprising CpGs.
4. The dosage form of claim 2, wherein the one or more coupled
adjuvants comprise an agonist of Toll-Like Receptor 2, 3, 4, 7, 8
or 9.
5. The dosage form of claim 2, wherein the one or more coupled
adjuvants comprise an imidazoquinoline or oxoadenine.
6. The dosage form of claim 5, wherein the imidazoquinoline
comprises resiquimod or imiquimod.
7-11. (canceled)
12. The dosage form of claim 1, wherein the one or more first
antigens comprise a B cell antigen or a T cell antigen.
13-18. (canceled)
19. The dosage form of claim 1, wherein the dosage form comprises a
vaccine that comprises the second antigen that is not coupled to
the synthetic nanocarriers.
20. The dosage form of claim 19, wherein the vaccine comprises a
hapten-carrier conjugate, a virus-like particle, a synthetic
nanocarrier vaccine, a subunit protein vaccine, or an attenuated
virus.
21. The dosage form of claim 19, wherein the vaccine is against
Anthrax; Diphtheria, Tetanus and/or Pertussis; Haemophilus
influenzae type B; Hepatitis B; Hepatitis A; Hepatitis C; Herpes
zoster (shingles); Human Papillomavirus (HPV); Influenza; Japanese
Encephalitis; Tick-borne Encephalitis; Measles, Mumps and/or
Rubella; Meningococcal disease; Pneumococcal disease; Polio;
Rabies; Rotavirus; Typhoid; Varicella; Vaccinia (Smallpox); or
Yellow Fever.
22. (canceled)
23. The dosage form of claim 1, wherein the one or more first
antigens and/or one or more second antigens are obtained or derived
from a virus of the Adenoviridae, Picornaviridae, Herpesviridae,
Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae,
Paramyxoviridae, Papillomaviridae, Rhabdoviridae, Togaviridae or
Paroviridae family.
24-25. (canceled)
26. The dosage form of claim 1, wherein the one or more first
antigens and/or one or more second antigens 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.
27-28. (canceled)
29. The dosage form of claim 1, wherein the one or more first
antigens and/or one or more second antigens are obtained or derived
from a fungus of the Candida, Aspergillus, Cryptococcus,
Histoplasma, Pneumocystis or Stachybotrys genus.
30-31. (canceled)
32. The dosage form of claim 1, wherein the one or more first
antigens and/or one or more second antigens are obtained or derived
from one or more proteins of human papilloma virus.
33-34. (canceled)
35. The dosage form of claim 1, wherein the one or more first
antigens and/or one or more second antigens are obtained or derived
from one or more proteins of hepatitis B virus.
36-37. (canceled)
38. The dosage form of claim 35, wherein when the one or more first
antigens are obtained or derived from hepatitis B virus, the one or
more second antigens are obtained or derived from one or more
proteins of human papilloma virus.
39. The dosage form of claim 35, wherein when the one or more
second antigens are obtained or derived from hepatitis B virus, the
one or more first antigens are obtained or derived from one or more
proteins of human papilloma virus.
40. (canceled)
41. The dosage form of claim 1, wherein the one or more first
antigens and/or one or more second antigens are obtained or derived
from one or more proteins of influenza virus.
42-47. (canceled)
48. The dosage form of claim 1, wherein the first 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.
49. The dosage form of claim 48, wherein the first synthetic
nanocarriers comprise one or more polymers.
50-54. (canceled)
55. A method comprising: administering the dosage form of claim 1
to a subject.
56. The method of claim 55, wherein the subject has or is at risk
of having an infection or infectious disease.
57. The method of claim 55, wherein the subject has or is at risk
of having cancer.
58-64. (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] To either minimize the number of childhood vaccinations
and/or to provide broader immune protection against different
strains of a given pathogen, there often is a desire to combine
multiple antigens in a single dosage form, the resulting vaccine
being termed a multivalent vaccine. The number of antigens that can
be combined in a single dosage form may be limited by the amount of
each antigen required to elicit the desired immune response and the
aqueous solubility of the antigen.
[0003] At some point, the total liquid volume of the dosage form
becomes too large to comfortably and/or safely administer the
vaccine by an intramuscular and/or subcutaneous route. This
limitation is especially noticeable in the case of multivalent
conjugate vaccines such as Prevnar.TM., wherein each different
oligosaccharide antigen is conjugated to a protein carrier (e.g.,
with 7 or 13 oligosaccharide antigens conjugated to CRM197, a
non-toxic mutant of diphtheria toxin); or tetravalent Meningococcal
vaccines wherein the antigens are also conjugated to CRM197 or
other detoxified forms of diphtheria toxin.
[0004] Another limitation of existing vaccine formulations is their
limited coverage or their physical incompatibility with one another
which may preclude simple blending of two existing vaccines to
create a new, combination vaccine. For example, vaccines can
consist of virus like particles comprising one or more antigens
which self assemble or are linked to self assembling proteins.
Examples include Cervarix.TM. and Gardasil.TM., which are vaccines
against human papilloma virus (HPV). Both of these vaccines target
antigens derived from L1 protein of a limited number of HPV
strains. These vaccines do not provide protection against all
strains of HPV. To expand the strain coverage of these vaccines, it
is desirable to be able to admix additional viral antigens which
are compatible with the existing vaccine formulations, which
provide broader coverage and thereby create a new, expanded
multivalent vaccine. It certain circumstances it may not be
possible to simply blend in additional conventionally produced
antigens to an existing vaccine because of undesirable interactions
between the additional conventionally produced antigens and the
existing vaccine (which may lead to precipitation, aggregation,
etc.).
[0005] Therefore, what is needed are compositions and methods that
could address the problems noted above that are associated with
producing vaccines.
SUMMARY OF THE INVENTION
[0006] In one aspect, a dosage form comprising (1) a first
population of synthetic nanocarriers that have one or more first
antigens coupled to them, (2) one or more second antigens that are
not coupled to the synthetic nanocarriers, and (3) a
pharmaceutically acceptable excipient is provided.
[0007] In one embodiment, any of the dosage forms provided further
comprises one or more adjuvants that are coupled to the synthetic
nanocarriers of the first population of synthetic nanocarriers. In
another embodiment, the one or more coupled adjuvants comprise any
of the adjuvants as provided herein. In one embodiment, the one or
more adjuvants comprise Pluronic.RTM. block co-polymers,
specifically modified or prepared peptides, muramyl dipeptide,
aminoalkyl glucosaminide 4-phosphates, RC529, bacterial toxoids,
toxin fragments, agonists of Toll-Like Receptors 2, 3, 4, 5, 7, 8,
9 and/or combinations thereof; adenine derivatives;
immunostimulatory DNA; immunostimulatory RNA; imidazoquinoline
amines, imidazopyridine amines, 6,7-fused cycloalkylimidazopyridine
amines, 1,2-bridged imidazoquinoline amines; imiquimod; resiquimod;
type I interferons; poly I:C; bacterial lipopolysacccharide (LPS);
VSV-G; HMGB-1; flagellin or portions or derivatives thereof; or
immunostimulatory DNA molecules comprising CpGs. In another
embodiment, the one or more coupled adjuvants comprise an agonist
of Toll-Like Receptor 2, 3, 4, 7, 8 or 9. In yet another
embodiment, the one or more coupled adjuvants comprise an
imidazoquinoline or oxoadenine. In still another embodiment, the
imidazoquinoline comprises resiquimod or imiquimod.
[0008] In another embodiment, any of the dosage forms provided
further comprises one or more adjuvants that are not coupled to the
synthetic nanocarriers of the first population of synthetic
nanocarriers. In one embodiment, the one or more not coupled
adjuvants comprise stimulators or agonists of pattern recognition
receptors, mineral salts, alum, alum combined with monphosphoryl
lipid A of Enterobacteria (MPL), MPL.RTM. (AS04), AS15, saponins,
QS-21, Quil-A, ISCOMs, ISCOMATRIX.TM., MF59.TM., Montanide.RTM. ISA
51, Montanide.RTM. ISA 720, AS02, liposomes and liposomal
formulations, AS01, synthesized or specifically prepared
microparticles and microcarriers, bacteria-derived outer membrane
vesicles of N. gonorrheae or Chlamydia trachomatis, chitosan
particles, depot-forming agents, Pluronic.RTM. block co-polymers,
specifically modified or prepared peptides, muramyl dipeptide,
aminoalkyl glucosaminide 4-phosphates, RC529, bacterial toxoids,
toxin fragments, agonists of Toll-Like Receptors 2, 3, 4, 5, 7, 8,
9 and/or combinations thereof; adenine derivatives;
immunostimulatory DNA; immunostimulatory RNA; imidazoquinoline
amines, imidazopyridine amines, 6,7-fused cycloalkylimidazopyridine
amines, 1,2-bridged imidazoquinoline amines; imiquimod; resiquimod;
agonist for DC surface molecule CD40; type I interferons; poly I:C;
bacterial lipopolysacccharide (LPS); VSV-G; HMGB-1; flagellin or
portions or derivatives thereof; immunostimulatory DNA molecules
comprising CpGs; proinflammatory stimuli released from necrotic
cells; urate crystals; activated components of the complement
cascade; activated components of immune complexes; complement
receptor agonists; cytokines; or cytokine receptor agonists. In
another embodiment, the one or more not coupled adjuvants comprise
alum, AS01, AS02, AS04, AS15, MPL, QS-21, a saponin, or an
immunostimulatory nucleic acid comprising CpG.
[0009] In yet another embodiment of any of the dosage forms
provided, the one or more first antigens are identical to the one
or more second antigens.
[0010] In a further embodiment, any of the dosage forms further
comprises a second population of synthetic nanocarriers that have
one or more third antigens coupled to them; wherein the first and
third antigens are not identical.
[0011] In yet a further embodiment, the one or more first antigens
of any of the dosage forms comprise a B cell antigen or a T cell
antigen. In one embodiment, the T cell antigen is a universal T
cell antigen or T-helper cell antigen. In another embodiment, the
one or more first antigens comprise a B cell antigen or a T cell
antigen and a a universal T cell antigen or 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 an
embodiment of any of the dosage forms, the a universal T cell
antigen or T helper cell antigen is coupled by encapsulation. In
another embodiment, the one or more second antigens of any of the
dosage forms comprise a B cell antigen or a T cell antigen.
[0012] In one embodiment, any of the dosage forms provided
comprises a vaccine that comprises the second antigen that is not
coupled to the synthetic nanocarriers. In another embodiment, the
vaccine comprises a hapten-carrier conjugate, a virus-like
particle, a synthetic nanocarrier vaccine, a subunit protein
vaccine, or an attenuated virus. In still another embodiment, the
vaccine is any vaccine provided herein. In yet another embodiment,
the vaccine is against any infectious agent provided herein. In
still another embodiment, the vaccine is against Anthrax;
Diphtheria, Tetanus and/or Pertussis; Haemophilus influenzae type
B; Hepatitis B; Hepatitis A; Hepatitis C; Herpes zoster (shingles);
Human Papillomavirus (HPV); Influenza; Japanese Encephalitis;
Tick-borne Encephalitis; Measles, Mumps and/or Rubella;
Meningococcal disease; Pneumococcal disease; Polio; Rabies;
Rotavirus; Typhoid; Varicella; Vaccinia (Smallpox); or Yellow
Fever. In a further embodiment, the vaccine comprises BIOTHRAX,
DAPTACEL, INFANRIX, TRIPEDIA, TRIHIBIT, KINRIX, PEDIARIX, PENTACEL,
PEDVAXHIB, ACTHIB, HIBERIX, COMVAX, HAVRIX, VAQTA, ENGERIX-B,
RECOMBIVAX HB, TWINRIX, ZOSTAVAX, GARDASIL, CERVARIX, FLUARIX,
FLUVIRIN, FLUZONE, FLULAVAL, AFLURIA, AGRIFLU, FLUMIST, JE-VAX,
IXIARO, M-M-R II, PROQUAD, MENOMUNE, MENACTRA, MENVEO, PNEUMOVAX
23, PREVNAR, PCV13, IPOL, IMOVAX RABIES, RABAVERT, ROTATEQ,
ROTARIX, DECAVAC, BOOSTRIX, ADACEL, TYPHIM VI, VIVOTIF BERNA,
VARIVAX, ACAM2000 or YF-VAX.
[0013] In another embodiment, the one or more first antigens and/or
one or more second antigens are obtained or derived from any of the
infectious agents provided herein. In one embodiment, the
infectious agent is a virus of the Adenoviridae, Picornaviridae,
Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae,
Orthomyxoviridae, Paramyxoviridae, Papillomaviridae, Rhabdoviridae,
Togaviridae or Paroviridae family. In still another embodiment, the
one or more first antigens and/or one or more second antigens 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
yet another embodiment, the one or more first antigens and/or one
or more second antigens 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 a further
embodiment, the one or more first antigens and/or one or more
second antigens 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 one or more first antigens and/or one or more
second antigens are obtained or derived from a fungus of the
Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis or
Stachybotrys genus. In still another embodiment, the one or more
first antigens and/or one or more second antigens 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.
[0014] In yet another embodiment, the one or more first antigens
and/or one or more second antigens comprise or are obtained or
derived from any of the antigens provided herein. In one
embodiment, the antigen comprises 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). In another embodiment,
the one or more first antigens and/or one or more second antigens
comprise or 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. In yet
another embodiment, the one or more first antigens and/or one or
more second antigens comprise or are obtained or derived from
surface antigen, capsular glycoprotein, Yps3P, Hsp60, Major surface
protein, MsgC1, MsgC3, MsgC8, MsgC9 or SchS34.
[0015] In one embodiment of any of the dosage forms provided, the
one or more first antigens and/or one or more second antigens
comprise or are obtained or derived from one or more proteins of
human papilloma virus. In another embodiment of any of the dosage
forms provided, the one or more first antigens comprise or are
obtained or derived from L1 protein of human papilloma virus, and
the one or more second antigens are obtained or derived from L2
protein of human papilloma virus. In yet another embodiment of any
of the dosage forms provided, the one or more first antigens
comprise or are obtained or derived from L2 protein of human
papilloma virus, and the one or more second antigens are obtained
or derived from L1 protein of human papilloma virus. In still
another embodiment of any of the dosage forms provided, the one or
more first antigens and/or one or more second antigens comprise or
are obtained or derived from one or more proteins of hepatitis B
virus. In another embodiment of any of the dosage forms provided,
the one or more first antigens and/or one or more second antigens
comprise or are obtained or derived from hepatitis B surface
antigen (HBsAg). In one embodiment, the HBsAg is from strain ayw
produced in Saccharomyces cerevisiae. In another embodiment, when
the one or more first antigens are obtained or derived from
hepatitis B virus, the one or more second antigens comprise or are
obtained or derived from one or more proteins of human papilloma
virus. In a further embodiment, when the one or more second
antigens are obtained or derived from hepatitis B virus, the one or
more first antigens comprise or are obtained or derived from one or
more proteins of human papilloma virus. In one embodiment, the one
or more proteins of human papilloma virus is the L1 and/or L2
protein of human papilloma virus. In another embodiment of any of
the dosage forms provided, the one or more first antigens and/or
one or more second antigens comprise or are obtained or derived
from one or more proteins of influenza virus. In one embodiment,
the influenza virus is influenza A virus, H5N1 avian influenza
virus, or H1N1 influenza A virus. In another embodiment of any of
the dosage forms provided, the one or more first antigens are
obtained or derived from M2 protein of influenza A virus, and the
one or more second antigens are obtained or derived from
hemagglutinin of H5N1 avian influenza virus. In a further
embodiment of any of the dosage forms provided, the one or more
first antigens are obtained or derived from hemagglutinin of H5N1
avian influenza virus, and the one or more second antigens are
obtained or derived from M2 protein of influenza A virus. In yet
another embodiment of any of the dosage forms provided, the one or
more first antigens are obtained or derived from M2 protein of
influenza A virus, and the one or more second antigens are obtained
or derived from beta-propiolactone-inactivated influenza A virus
H1N1. In still another embodiment of any of the dosage forms
provided, the one or more first antigens are obtained or derived
from beta-propiolactone-inactivated influenza A virus H1N1, and the
one or more second antigens are obtained or derived from M2 protein
of influenza A virus.
[0016] In one embodiment of any of the dosage forms provided, the
pharmaceutically acceptable excipient comprises a preservative, a
buffer, saline, phosphate buffered saline, a colorant, or a
stabilizer.
[0017] In another embodiment of any of the dosage forms provided,
the first 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, cubic
nanoparticles, pyramidal nanoparticles, oblong nanoparticles,
cylindrical nanoparticles, or toroidal nanoparticles. In one
embodiment, the first synthetic nanocarriers comprise one or more
polymers. In another embodiment, the one or more polymers comprise
a polyester. In yet another embodiment, the one or more polymers
comprise or further comprise a polyester coupled to a hydrophilic
polymer. In still 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 yet a further
embodiment, the polyether comprises polyethylene glycol.
[0018] In another aspect, a method comprising administering any of
the dosage forms 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.
[0019] In one embodiment of any of the methods provided, the dosage
form is administered by oral, subcutaneous, pulmonary, intranasal,
intradermal or intramuscular administration. In yet another aspect,
any of the dosage forms is provided for use in therapy or
prophylaxis. In still another aspect, any of the dosage forms for
use in any of the methods provided is provided. In yet another
aspect, any of the dosage forms for use in a method of treating or
preventing cancer is provided. In a further aspect, any of the
dosage forms for use in a method of treating or preventing
infection or infectious disease is provided. In one embodiment of
any of the dosage forms, the method comprises administration of the
dosage form by oral, subcutaneous, pulmonary, intranasal,
intradermal or intramuscular administration. In still another
aspect, use of any of the dosage forms for the manufacture of a
medicament for use in any of the methods is provided.
BRIEF DESCRIPTION OF FIGURES
[0020] FIG. 1 shows antibody titers in mice immunized with a
combination of NC-M2e and free hemagglutinin from H5N1 avian
influenza strain (Vietnam).
[0021] FIG. 2 shows antibody titers in mice immunized with a
combination of NC-M2e and free hemagglutinin from H5N1 avian
influenza strain (Vietnam) admixed with 80 .mu.g of alum.
[0022] FIG. 3 shows antibody titers in mice immunized with a
combination of NC-M2e and beta-propiolactone-inactivated influenza
A virus H1N1 (H1N1 New Caledonia/20/99/IVR 116) admixed with 80
.mu.g of alum.
[0023] FIG. 4 shows antibody titers in mice immunized with a
combination of NC-L2-peptide and HBsAg strain ayw produced in the
yeast Saccharomyces cerevisiae admixed with 80 .mu.g of alum.
DETAILED DESCRIPTION OF THE INVENTION
[0024] 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.
[0025] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety for all purposes.
[0026] 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
[0027] 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 compositions, and related methods, that comprise a dosage
form comprising a first population of synthetic nanocarriers that
have one or more first antigens coupled to them, one or more second
antigens that are not coupled to the synthetic nanocarriers, and a
pharmaceutically acceptable excipient.
[0028] In embodiments, the populations of synthetic nanocarriers
may be combined with the one or more second antigens (which may be
incorporated in a wide variety of ways) to form dosage forms
according to the present invention. The one or more second antigens
may be provided in solution form, suspension form, powder form,
etc., and may be provided as a vaccine formulation. For instance,
in an embodiment, the one or more second antigens may be provided
in the form of a hapten-carrier protein or live attenuated virus
vaccine formulation, and the population of synthetic nanocarriers
admixed with the hapten-carrier protein or live attenuated virus
vaccine formulations form a multivalent vaccine dosage form (or
increase the valency of the hapten-carrier protein or live
attenuated virus vaccine formulations). In another embodiment, the
population of synthetic nanocarriers may be combined with proteins
taken from an infectious organism to form a multivalent vaccine
dosage form according to the invention. In another embodiment, the
population of synthetic nanocarriers may be added to another
population of synthetic nanocarriers that comprise the one or more
second antigens to form a multivalent synthetic nanocarrier vaccine
dosage form. In other embodiments, the population of synthetic
nanocarriers may be combined with protein antigens in the form of
virus like particles to form a multivalent vaccine dosage form
according to the invention. In other embodiments, additional
antigens beyond the one or more first and/or second antigens can be
incorporated into the dosage form (through admixing, and other
techniques disclosed herein or known conventionally).
[0029] In an embodiment, synthetic nanocarriers comprising one or
more first antigens and optionally a a universal T cell antigen or
T helper antigen and/or an adjuvant, can be added to one or more
second antigens (e.g., an existing vaccine) to create a combination
vaccine with expanded breadth of antigen coverage.
[0030] For example, the vaccines Gardasil.RTM. and Cervarix.RTM.
for protection against HPV comprise protein antigen epitopes from
the major structural protein L1 protein derived from 4 and 2 sets
of HPV strains, correspondingly. Vaccines with L1 peptide antigens
from as many as 9 different HPV strains are known. Such a vaccine
with multiple peptide antigens would potentially protect the
individual against most, but not all, HPV strains. If a population
of synthetic nanocarriers comprising one or more peptide epitopes
from another HPV structural protein, L2, is added to an existing L1
protein-based vaccine, broader protection from an HPV challenge
would be obtained with the potential of creating a "universal HPV
vaccine." This population of water-dispersed L2 peptide synthetic
nanocarriers can simply be admixed to the existing aqueous vaccine
formulation much as one would add an excipient, or diluent, to the
formulation. This simple method for expanding the breadth of
coverage avoids having to engineer the L2 peptide into a
recombinant protein antigen form as is conventionally done. This is
illustrated in Examples 1 and 2 below, which together illustrate
the formation of a combination HPV vaccine containing conventional
Gardasil.RTM. augmented by synthetic nanocarriers comprising a
peptide derived from L2 protein.
[0031] The inventive synthetic nanocarrier combination vaccine
approach can be generalized to include other infectious disease
prophylactic or therapeutic vaccines with less than 100% protection
against the various strains of the infectious agent. It can also be
used for prophylactic and/or therapeutic vaccines directed against
non-infectious disease targets, such as cancer or small molecule
agents. In embodiments, the inventive compositions provide for
combinations of synthetic nanocarriers with existing "conventional"
vaccines that can be formulated easily without the limitations of
protein antigen solubility at higher concentrations. This can
reduce multivalent vaccine volumes, and enhance ease of
formulation.
[0032] Examples 3-6 and 8-11 show different embodiments of the
present invention. Examples 3 and 4 illustrate a combination
vaccine of conventional hepatitis B vaccines augmented by synthetic
nanocarriers which comprise surface adsorbed heparin as a first
antigen. Examples 5 and 6 illustrate an oral combination vaccine of
a conventional anti-rotaviral vaccine augmented by synthetic
nanoparticles that comprise peptides derived from the L2 protein of
HPV. Examples 8 and 9 illustrate a combination vaccine of free
hemagglutinin from H5N1 avian influenza strain (Vietnam) augmented
by synthetic nanocarriers that comprise M2e, OP-II T-helper peptide
and R848 without or with admixed adjuvant, respectively. Example 10
illustrates a combination of inactivated influenza A virus H1N1
vaccine and augmented with synthetic nanocarriers that comprise
M2e, OP-II T-helper peptide and R848 adjuvant with admixed alum.
Example 11 illustrates a combination of recombinant hepatitis B
surface antigen augmented with synthetic nanocarriers that comprise
L2 peptide, OP-II T-helper peptide and R848 with admixed alum. The
compositions exemplified in the Examples are also provided herein
as are methods of their administration to a subject.
[0033] The invention will now be described in more detail
below.
Definitions
[0034] "Adjuvant" means an agent that does not constitute a
specific antigen, but boosts the strength and longevity of immune
response to a co-administered antigen, preferably an antigen
present in a dosage form together with the antigen, and more
preferably a concomitantly administered antigen. Such adjuvants may
include, but are not limited to stimulators of pattern recognition
receptors, such as Toll-like receptors, RIG-1 and NOD-like
receptors (NLR), mineral salts, such as alum, alum combined with
monphosphoryl lipid (MPL) A of Enterobacteria, such as Escherihia
coli, Salmonella minnesota, Salmonella typhimurium, or Shigella
flexneri or specifically with MPL.RTM. (AS04), MPL A of
above-mentioned bacteria separately, saponins, such as QS-21,
Quil-A, ISCOMs, ISCOMATRIX.TM., emulsions such as MF59.TM.,
Montanide.RTM. ISA 51 and ISA 720, AS02 (QS21+squalene+MPL.RTM.),
AS15, liposomes and liposomal formulations such as AS01,
synthesized or specifically prepared microparticles and
microcarriers such as bacteria-derived outer membrane vesicles
(OMV) of N. gonorrheae, Chlamydia trachomatis and others, or
chitosan particles, depot-forming agents, such as Pluronic.RTM.
block co-polymers, specifically modified or prepared peptides, such
as muramyl dipeptide, aminoalkyl glucosaminide 4-phosphates, such
as RC529, or proteins, such as bacterial toxoids or toxin
fragments.
[0035] 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
(resiquimod); adenine derivatives, such as those disclosed in U.S.
Pat. No. 6,329,381 (Sumitomo Pharmaceutical Company), US Published
Patent Application 2010/0075995 to Biggadike et al., or WO
2010/018132 to Campos et al.; immunostimulatory DNA; or
immunostimulatory RNA. In specific embodiments, synthetic
nanocarriers incorporate as adjuvants compounds that are agonists
for toll-like receptors (TLRs) 7 & 8 ("TLR 7/8 agonists"). Of
utility are the TLR 7/8 agonist compounds disclosed in U.S. Pat.
No. 6,696,076 to Tomai et al., including but not limited to
imidazoquinoline amines, imidazopyridine amines, 6,7-fused
cycloalkylimidazopyridine amines, and 1,2-bridged imidazoquinoline
amines. Preferred adjuvants comprise imiquimod and resiquimod. In
specific embodiments, an adjuvant may be an agonist for the DC
surface molecule CD40. In certain embodiments, to stimulate
immunity rather than tolerance, a synthetic nanocarrier
incorporates an adjuvant that promotes DC maturation (needed for
priming of naive T cells) and the production of cytokines, such as
type I interferons, which promote antibody immune responses. In
embodiments, adjuvants also may comprise immunostimulatory RNA
molecules, such as but not limited to dsRNA or poly I:poly C12U
(available as Ampligen.RTM., both poly I:C and poly I:polyC12U
being known as TLR3 stimulants), and/or those disclosed in F. Heil
et al., "Species-Specific Recognition of Single-Stranded RNA via
Toll-like Receptor 7 and 8" Science 303(5663), 1526-1529 (2004); J.
Vollmer et al., "Immune modulation by chemically modified
ribonucleosides and oligoribonucleotides" WO 2008033432 A2; A.
Forsbach et al., "Immunostimulatory oligoribonucleotides containing
specific sequence motif(s) and targeting the Toll-like receptor 8
pathway" WO 2007062107 A2; E. Uhlmann et al., "Modified
oligoribonucleotide analogs with enhanced immunostimulatory
activity" U.S. Pat. Appl. Publ. US 2006241076; G. Lipford et al.,
"Immunostimulatory viral RNA oligonucleotides and use for treating
cancer and infections" WO 2005097993 A2; G. Lipford et al.,
"Immunostimulatory G,U-containing oligoribonucleotides,
compositions, and screening methods" WO 2003086280 A2. In some
embodiments, an adjuvant may be a TLR-4 agonist, such as bacterial
lipopolysacccharide (LPS), VSV-G, and/or HMGB-1. In some
embodiments, adjuvants may comprise TLR-5 agonists, such as
flagellin, or portions or derivatives thereof, including but not
limited to those disclosed in U.S. Pat. Nos. 6,130,082, 6,585,980,
and 7,192,725. In specific embodiments, synthetic nanocarriers
incorporate a ligand for Toll-like receptor (TLR)-9, such as
immunostimulatory DNA molecules comprising CpGs, which induce type
I interferon secretion, and stimulate T and B cell activation
leading to increased antibody production and cytotoxic T cell
responses (Krieg et al., CpG motifs in bacterial DNA trigger direct
B cell activation. Nature. 1995. 374:546-549; Chu et al. CpG
oligodeoxynucleotides act as adjuvants that switch on T helper 1
(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.
[0036] 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.
[0037] 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.
[0038] "Administering" or "administration" means providing a dosage
form to a subject in a manner that is pharmacologically useful.
[0039] "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. In embodiments, therefore, an
amount effective is one that a health practitioner would believe
may generate an antibody response against the 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.
[0040] 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. The antigen(s) of any of the inventive
compositions provided herein can in embodiments be in an amount
effective.
[0041] "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, dosage forms according to the
invention comprise one or more antigens, for example, one or more
first antigens, one or more second antigens, one or more third
antigens, one or more fourth antigens, and one or more additional
antigens. 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.
[0042] "At least a portion of the dose" means at least some part of
the dose, ranging up to including all of the dose.
[0043] An "at risk" subject is one in which a health practitioner
believes has a chance of having a disease or condition provided
herein including, but not limited to, an infection, infectious
disease or cancer.
[0044] "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, and carbohydrates. In some
embodiments, the B cell antigen comprises a non-protein antigen
(i.e., not a protein or peptide antigen). In some embodiments, the
B cell antigen comprises a carbohydrate associated with an
infectious agent. In some embodiments, the B cell antigen comprises
a glycoprotein or glycopeptide associated with an infectious agent.
The infectious agent can be a bacterium, virus, fungus, protozoan,
parasite or prion. In some embodiments, the B cell antigen
comprises a poorly immunogenic antigen. In some embodiments, the B
cell antigen comprises an abused substance or a portion thereof. In
some embodiments, the B cell antigen comprises an addictive
substance or a portion thereof. Addictive substances include, but
are not limited to, nicotine, a narcotic, a cough suppressant, a
tranquilizer, and a sedative. In some embodiments, the B cell
antigen comprises a toxin, such as a toxin from a chemical weapon
or natural sources, or a pollutant. 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.
[0045] "Couple" or "Coupled" or "Couples" (and the like) means to
chemically associate one entity (for example a moiety) with
another. In some embodiments, the coupling is covalent, meaning
that the coupling occurs in the context of the presence of a
covalent bond between the two entities. In non-covalent
embodiments, the non-covalent coupling is mediated by non-covalent
interactions including but not limited to charge interactions,
affinity interactions, metal coordination, physical adsorption,
host-guest interactions, hydrophobic interactions, TT stacking
interactions, hydrogen bonding interactions, van der Waals
interactions, magnetic interactions, electrostatic interactions,
dipole-dipole interactions, and/or combinations thereof. In
embodiments, encapsulation is a form of coupling. In embodiments,
populations of synthetic nanocarriers have one or more antigens
and/or adjuvants coupled to them, meaning that a plurality,
preferably a majority, of the synthetic nanocarriers within the
population have coupled to them one or more antigens and/or
adjuvants that are similar to one another. In other embodiments,
inventive dosage forms may comprise antigens and/or adjuvants that
are not coupled to synthetic nanocarriers within a population of
synthetic nanocarriers.
[0046] "Derived" means taken from a source and subjected to
substantial modification. For instance, a peptide or nucleic acid
with a sequence with only 50% identity to a natural peptide or
nucleic acid, preferably a natural consensus peptide or nucleic
acid, would be said to be derived from the natural peptide or
nucleic acid. Substantial modification is modification that
significantly affects the chemical or immunological properties of
the material in question. Derived peptides and nucleic acids can
also include those with a sequence with greater than 50% identity
to a natural peptide or nucleic acid sequence if said derived
peptides and nucleic acids have altered chemical or immunological
properties as compared to the natural peptide or nucleic acid.
These chemical or immunological properties comprise hydrophilicity,
stability, affinity, and ability to couple with a carrier such as a
synthetic nanocarrier.
[0047] "Dosage form" means a pharmacologically and/or
immunologically active material in a medium, carrier, vehicle, or
device suitable for administration to a subject.
[0048] "Encapsulate" means to enclose within a synthetic
nanocarrier, preferably enclose completely within a synthetic
nanocarrier. Most or all of a substance that is encapsulated is not
exposed to the local environment external to the synthetic
nanocarrier. Encapsulation is distinct from absorption, which
places most or all of a substance on a surface of a synthetic
nanocarrier, and leaves the substance exposed to the local
environment external to the synthetic nanocarrier.
[0049] "Identical" means that a substance shares one or more common
chemical and/or immunological characteristics with another
substance. For instance, one or more antigens are identical to one
or more other antigens when both sets of antigens share one or more
common chemical and/or immunological characteristics. Substances,
such as antigens, are not identical when they fail to meet the
criteria for being identical. Certain biologically active
macromolecules may be described as having a percent identity with
respect to one another, which is a measure of the matching of their
sequences, as is conventionally known in the art. Such biologically
active macromolecules are identical within the scope of this
invention when they share greater than 20% identity, preferably
greater than 30% identity, preferably greater than 40% identity,
preferably greater than 50% identity, preferably greater than 60%
identity, preferably greater than 70% identity, preferably greater
than 80% identity, or preferably greater than 90% identity, with
one another.
[0050] 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.
[0051] "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.
[0052] "Isolated peptide, polypeptide or protein" means the
polypeptide (or peptide or protein) is separated from its native
environment and present in sufficient quantity to permit its
identification or use. This means, for example, the polypeptide (or
peptide or protein) may be (i) selectively produced by expression
cloning or (ii) purified as by chromatography or electrophoresis.
Isolated peptides, proteins or polypeptides may be, but need not
be, substantially pure. Because an isolated peptide, polypeptide or
protein may be admixed with a pharmaceutically acceptable carrier
in a pharmaceutical preparation, the polypeptide (or peptide or
protein) may comprise only a small percentage by weight of the
preparation. The polypeptide (or peptide or protein) is nonetheless
isolated in that it has been separated from the substances with
which it may be associated in living systems, i.e., isolated from
other proteins (or peptides or polypeptides). Any of the peptides,
polypeptides or proteins provided herein may be isolated. In some
embodiments, the antigens in the compositions provided herein are
peptides, polypeptides or proteins.
[0053] "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 an
embodiment, a maximum dimension of at least 75%, preferably at
least 80%, more preferably at least 90%, of the synthetic
nanocarriers in a sample, based on the total number of synthetic
nanocarriers in the sample, is equal to or less than 5 .mu.m.
Preferably, a minimum dimension of at least 75%, preferably at
least 80%, more preferably at least 90%, of the synthetic
nanocarriers in a sample, based on the total number of synthetic
nanocarriers in the sample, is greater than 110 nm, more preferably
greater than 120 nm, more preferably greater than 130 nm, and more
preferably still greater than 150 nm. Aspects ratios of the maximum
and minimum dimensions of inventive synthetic nanocarriers may vary
depending on the embodiment. For instance, aspect ratios of the
maximum to minimum dimensions of the synthetic nanocarriers may
vary from 1:1 to 1,000,000:1, preferably from 1:1 to 100,000:1,
more preferably from 1:1 to 1000:1, still preferably from 1:1 to
100:1, and yet more preferably from 1:1 to 10:1. Preferably, a
maximum dimension of at least 75%, preferably at least 80%, more
preferably at least 90%, of the synthetic nanocarriers in a sample,
based on the total number of synthetic nanocarriers in the sample
is equal to or less than 3 .mu.m, more preferably equal to or less
than 2 .mu.m, more preferably equal to or less than 1 .mu.m, more
preferably equal to or less than 800 nm, more preferably equal to
or less than 600 nm, and more preferably still equal to or less
than 500 nm. In preferred embodiments, a maximum dimension of at
least 75%, preferably at least 80%, more preferably at least 90%,
of the synthetic nanocarriers in a sample, based on the total
number of synthetic nanocarriers in the sample, is equal to or
greater than 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).
[0054] "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.
[0055] "Pharmaceutically acceptable carrier(s) or excipient(s)"
means materials that are contained within the dosage form, but do
not contribute substantially to the primary pharmacological
activity of the dosage form. In embodiments, the materials are
pharmacologically inactive. In embodiments, pharmaceutically
acceptable excipients comprise preservatives, buffers, saline, or
phosphate buffered saline, colorants, or stabilizers.
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.
[0056] "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 coupled antigen(s), 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.
[0057] "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.
[0058] "Synthetic nanocarrier(s)" means a discrete object that is
not found in nature, and that possesses at least one dimension that
is less than or equal to 5 microns in size. Albumin nanoparticles
are generally included as synthetic nanocarriers, however in
certain embodiments the synthetic nanocarriers do not comprise
albumin nanoparticles. In embodiments, inventive synthetic
nanocarriers do not comprise chitosan.
[0059] A synthetic nanocarrier can be, but is not limited to, one
or a plurality of lipid-based nanoparticles (e.g. liposomes) (also
referred to herein as lipid nanoparticles, i.e., nanoparticles
where the majority of the material that makes up their structure
are lipids), polymeric nanoparticles, metallic nanoparticles,
surfactant-based emulsions, dendrimers, buckyballs, nanowires,
virus-like particles (i.e., particles that are primarily made up of
viral structural proteins but that are not infectious or have low
infectivity), peptide or protein-based particles (also referred to
herein as protein particles, i.e., particles where the majority of
the material that makes up their structure are peptides or
proteins) (such as albumin nanoparticles) and/or nanoparticles that
are developed using a combination of nanomaterials such as
lipid-polymer nanoparticles. Synthetic nanocarriers may be a
variety of different shapes, including but not limited to
spheroidal, cuboidal, pyramidal, oblong, cylindrical, toroidal, and
the like. Synthetic nanocarriers according to the invention
comprise one or more surfaces, including but not limited to
internal surfaces (surfaces generally facing an interior portion of
the synthetic nanocarrier) and external surfaces (surfaces
generally facing an external environment of the synthetic
nanocarrier). Exemplary synthetic nanocarriers that can be adapted
for use in the practice of the present invention comprise: (1) the
biodegradable nanoparticles disclosed in U.S. Pat. No. 5,543,158 to
Gref et al., (2) the polymeric nanoparticles of Published US Patent
Application 20060002852 to Saltzman et al., (3) the
lithographically constructed nanoparticles of Published US Patent
Application 20090028910 to DeSimone et al., (4) the disclosure of
WO 2009/051837 to von Andrian et al., (5) the nanoparticles
disclosed in Published US Patent Application 2008/0145441 to
Penades et al., (6) the protein nanoparticles disclosed in
Published US Patent Application 20090226525 to de los Rios et al.,
(7) the virus-like particles disclosed in published US Patent
Application 20060222652 to Sebbel et al., (8) the nucleic acid
coupled virus-like particles disclosed in published US Patent
Application 20060251677 to Bachmann et al., (9) the virus-like
particles disclosed in WO2010047839A1 or WO2009106999A2, or (10)
the nanoprecipitated nanoparticles disclosed in P. Paolicelli et
al., "Surface-modified PLGA-based Nanoparticles that can
Efficiently Associate and Deliver Virus-like Particles"
Nanomedicine. 5(6):843-853 (2010). In embodiments, synthetic
nanocarriers may possess an aspect ratio greater than 1:1, 1:1.2,
1:1.5, 1:2, 1:3, 1:5, 1:7, or greater than 1:10.
[0060] 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.
[0061] "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.
[0062] 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.
[0063] 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).
[0064] 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.
[0065] "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. In other
embodiments, the inventive dosage form may comprise a vaccine
comprising the second antigen that is not coupled to the synthetic
nanocarriers. Vaccines according to the invention may comprise a
hapten-carrier conjugate, a virus-like particle, a synthetic
nanocarrier vaccine, a subunit protein vaccine, or an attenuated
virus. In some embodiments, the vaccine comprises any of the
vaccines, including the commercially available vaccines, described
herein.
Inventive Compositions
[0066] 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.
[0067] 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.
[0068] 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.
[0069] 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.).
[0070] 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.
[0071] In some embodiments, an immunofeature surface, targeting
moiety, antigen, adjuvant 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, antigen, adjuvant and/or
oligonucleotide can be noncovalently associated with a polymeric
matrix. For example, in some embodiments, an immunofeature surface,
targeting moiety, antigen, adjuvant and/or oligonucleotide can be
adsorbed upon, encapsulated within, surrounded by, and/or dispersed
throughout a polymeric matrix. Alternatively or additionally, an
immunofeature surface, targeting moiety, antigen, adjuvant and/or
nucleotide can be associated with a polymeric matrix by hydrophobic
interactions, charge interactions, van der Waals forces, etc.
[0072] 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.
[0073] Examples of polymers suitable for use in the present
invention include, but are not limited to polyethylenes,
polycarbonates (e.g. poly(1,3-dioxan-2one)), polyanhydrides (e.g.
poly(sebacic anhydride)), polypropylfumerates, polyamides (e.g.
polycaprolactam), polyacetals, polyethers, polyesters (e.g.,
polylactide, polyglycolide, polylactide-co-glycolide,
polycaprolactone, polyhydroxyacid (e.g.
poly(.beta.-hydroxyalkanoate)), poly(orthoesters),
polycyanoacrylates, polyvinyl alcohols, polyurethanes,
polyphosphazenes, polyacrylates, polymethacrylates, polyureas,
polystyrenes, polyamines, polylysine, polylysine-PEG copolymers,
and poly(ethyleneimine), poly(ethylene imine)-PEG copolymers.
[0074] 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 (e.g., poly(1,3-dioxan-2one)); polyvalerolactone;
polyanhydrides (e.g., poly(sebacic anhydride)); polyethers (e.g.,
polyethylene glycol); polyurethanes; polymethacrylates;
polyacrylates; and polycyanoacrylates.
[0075] 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.
[0076] 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.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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.
[0081] 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.
[0082] 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).
[0083] 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.
[0084] 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.
[0085] In some embodiments, the synthetic nanocarriers comprise one
or more polymers. The polymeric synthetic nanocarriers, therefore,
can also include those described in WO publication WO2009/051837 by
Von Andrian et al., including, but not limited to those, with one
or more hydrophilic components. Preferably, the one or more
polymers comprise a polyester, such as a poly(lactic acid),
poly(glycolic acid), poly(lactic-co-glycolic acid), or
polycaprolactone. More preferably, the one or more polymers
comprise or further comprise a polyester coupled to a hydrophilic
polymer, such as a polyether. In embodiments, the polyether
comprises polyethylene glycol. Still more preferably, the one or
more polymers comprise a polyester and a polyester coupled to a
hydrophilic polymer, such as a polyether. In other embodiments, the
one or more polymers are coupled to one or more antigens and/or one
or more adjuvants. In embodiments, at least some of the polymers
are coupled to the antigen(s) and/or at least some of the polymers
are coupled to the adjuvant(s). Preferably, when there are more
than one type of polymer, one of the types of polymer is coupled to
the antigen(s). In embodiments, one of the other types of polymer
is coupled to the adjuvant(s). For example, in embodiments, when
the nanocarriers comprise a polyester and a polyester coupled to a
hydrophilic polymer, such as a polyether, the polyester is coupled
to the adjuvant, while the polyester coupled to the hydrophilic
polymer, such as a polyether, is coupled to the antigen(s). In
embodiments, where the nanocarriers comprise a T helper cell
antigen, the T helper cell antigen can be encapsulated in the
nanocarrier.
[0086] 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).
[0087] 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.
[0088] In some embodiments, synthetic nanocarriers may optionally
comprise one or more carbohydrates. Carbohydrates may be natural or
synthetic. A carbohydrate may be a derivatized natural
carbohydrate. In certain embodiments, a carbohydrate comprises
monosaccharide or disaccharide, including but not limited to
glucose, fructose, galactose, ribose, lactose, sucrose, maltose,
trehalose, cellbiose, mannose, xylose, arabinose, glucoronic acid,
galactoronic acid, mannuronic acid, glucosamine, galatosamine, and
neuramic acid. In certain embodiments, a carbohydrate is a
polysaccharide, including but not limited to pullulan, cellulose,
microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC),
hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextran,
glycogen, starch, hydroxyethylstarch, carageenan, glycon, amylose,
chitosan, N,O-carboxylmethylchitosan, algin and alginic acid,
starch, chitin, 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.
[0089] 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. Typical inventive
compositions 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). In an
embodiment, inventive synthetic nanocarriers are suspended in
sterile saline solution for injection together with a
preservative.
[0090] In embodiments, when preparing synthetic nanocarriers as
carriers for antigens or adjuvants for use in vaccines, methods for
coupling the antigens or adjuvants to the synthetic nanocarriers
may be useful. If the antigens or adjuvant is a small molecule it
may be of advantage to attach the antigens 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
antigens or adjuvant to the synthetic nanocarrier through the use
of these surface groups rather than attaching the antigens or
adjuvant to a polymer and then using this polymer conjugate in the
construction of synthetic nanocarriers.
[0091] In certain embodiments, the coupling can be a covalent
linker. In embodiments, peptides according to the invention can be
covalently coupled to the external surface via a 1,2,3-triazole
linker formed by the 1,3-dipolar cycloaddition reaction of azido
groups on the surface of the nanocarrier with antigen or adjuvant
containing an alkyne group or by the 1,3-dipolar cycloaddition
reaction of alkynes on the surface of the nanocarrier with antigens
or adjuvants containing an azido group. Such cycloaddition
reactions are preferably performed in the presence of a Cu(I)
catalyst along with a suitable Cu(I)-ligand and a reducing agent to
reduce Cu(II) compound to catalytic active Cu(I) compound. This
Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) can also be
referred as the click reaction.
[0092] 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.
[0093] An amide linker is formed via an amide bond between an amine
on one component such as the antigen or adjuvant with the
carboxylic acid group of a second component such as the
nanocarrier. The amide bond in the linker can be made using any of
the conventional amide bond forming reactions with suitably
protected amino acids or antigens or adjuvants and activated
carboxylic acid such N-hydroxysuccinimide-activated ester.
[0094] A disulfide linker is made via the formation of a disulfide
(S--S) bond between two sulfur atoms of the form, for instance, of
R1-S--S--R2. A disulfide bond can be formed by thiol exchange of an
antigen or adjuvant containing thiol/mercaptan group (--SH) with
another activated thiol group on a polymer or nanocarrier or a
nanocarrier containing thiol/mercaptan groups with a antigen or
adjuvant containing activated thiol group.
[0095] A triazole linker, specifically a 1,2,3-triazole of the
form
##STR00001##
wherein R1 and R2 may be any chemical entities, is made by the
1,3-dipolar cycloaddition reaction of an azide attached to a first
component such as the nanocarrier with a terminal alkyne attached
to a second component such as the peptide. The 1,3-dipolar
cycloaddition reaction is performed with or without a catalyst,
preferably with Cu(I)-catalyst, which links the two components
through a 1,2,3-triazole function. This chemistry is described in
detail by Sharpless et al., Angew. Chem. Int. Ed. 41(14), 2596,
(2002) and Meldal, et al, Chem. Rev., 2008, 108(8), 2952-3015 and
is often referred to as a "click" reaction or CuAAC.
[0096] In embodiments, a polymer containing an azide or alkyne
group, terminal to the polymer chain is prepared. This polymer is
then used to prepare a synthetic nanocarrier in such a manner that
a plurality of the alkyne or azide groups are positioned on the
surface of that nanocarrier. Alternatively, the synthetic
nanocarrier can be prepared by another route, and subsequently
functionalized with alkyne or azide groups. The antigen or
adjuvantis prepared with the presence of either an alkyne (if the
polymer contains an azide) or an azide (if the polymer contains an
alkyne) group. The antigen or adjuvant is then allowed to react
with the nanocarrier via the 1,3-dipolar cycloaddition reaction
with or without a catalyst which covalently couples the antigen or
adjuvant to the particle through the 1,4-disubstituted
1,2,3-triazole linker.
[0097] A thioether linker is made by the formation of a
sulfur-carbon (thioether) bond in the form, for instance, of
R1-S--R2. Thioether can be made by either alkylation of a
thiol/mercaptan (--SH) group on one component such as the antigen
or adjuvant with an alkylating group such as halide or epoxide on a
second component such as the nanocarrier. Thioether linkers can
also be formed by Michael addition of a thiol/mercaptan group on
one component such as a antigen or adjuvant to an
electron-deficient alkene group on a second component such as a
polymer containing a maleimide group or vinyl sulfone group as the
Michael acceptor. In another way, thioether linkers can be prepared
by the radical thiol-ene reaction of a thiol/mercaptan group on one
component such as a antigen or adjuvant with an alkene group on a
second component such as a polymer or nanocarrier.
[0098] A hydrazone linker is made by the reaction of a hydrazide
group on one component such as the antigen or adjuvant with an
aldehyde/ketone chemistrygroup on the second component such as the
nanocarrier.
[0099] A hydrazide linker is formed by the reaction of a hydrazine
group on one component such as the antigen or adjuvant with a
carboxylic acid group on the second component such as the
nanocarrier. Such reaction is generally performed using chemistry
similar to the formation of amide bond where the carboxylic acid is
activated with an activating reagent.
[0100] An imine or oxime linker is formed by the reaction of an
amine or N-alkoxyamine (or aminooxy) group on one component such as
the antigen or adjuvant with an aldehyde or ketone group on the
second component such as the nanocarrier.
[0101] An urea or thiourea linker is prepared by the reaction of an
amine group on one component such as the antigen or adjuvant with
an isocyanate or thioisocyanate group on the second component such
as the nanocarrier.
[0102] An amidine linker is prepared by the reaction of an amine
group on one component such as the antigen or adjuvant with an
imidoester group on the second component such as the
nanocarrier.
[0103] An amine linker is made by the alkylation reaction of an
amine group on one component such as the antigen or adjuvant with
an alkylating group such as halide, epoxide, or sulfonate ester
group on the second component such as the nanocarrier.
Alternatively, an amine linker can also be made by reductive
amination of an amine group on one component such as the antigen or
adjuvant with an aldehyde or ketone group on the second component
such as the nanocarrier with a suitable reducing reagent such as
sodium cyanoborohydride or sodium triacetoxyborohydride.
[0104] A sulfonamide linker is made by the reaction of an amine
group on one component such as the antigen or adjuvant with a
sulfonyl halide (such as sulfonyl chloride) group on the second
component such as the nanocarrier.
[0105] 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.
[0106] The antigen or adjuvant can also be conjugated to the
nanocarrier via non-covalent conjugation methods. For examples, a
negative charged antigen or adjuvant can be conjugated to a
positive charged nanocarrier through electrostatic adsorption. An
antigen or adjuvant containing a metal ligand can also be
conjugated to a nanocarrier containing a metal complex via a
metal-ligand complex.
[0107] In embodiments, an 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 nanocarrier's 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 containing an acid group via the
other end of the ADH linker on NC to produce the corresponding VLP
or liposome peptide conjugate.
[0108] For detailed descriptions of available conjugation methods,
see Hermanson G T "Bioconjugate Techniques", 2nd Edition Published
by Academic Press, Inc., 2008. In addition to covalent attachment
the antigen or adjuvant can be coupled by adsorption to a
pre-formed synthetic nanocarrier or it can be coupled by
encapsulation during the formation of the synthetic
nanocarrier.
Methods of Making and Using the Inventive Dosage Forms and Related
Methods
[0109] 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
fluidic channels, spray drying, single and double emulsion solvent
evaporation, solvent extraction, phase separation, milling,
microemulsion procedures, microfabrication, nanofabrication,
sacrificial layers, simple and complex coacervation, and other
methods well known to those of ordinary skill in the art.
Alternatively or additionally, aqueous and organic solvent
syntheses for monodisperse semiconductor, conductive, magnetic,
organic, and other nanomaterials have been described (Pellegrino et
al., 2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci.,
30:545; and Trindade et al., 2001, Chem. Mat., 13:3843). Additional
methods have been described in the literature (see, e.g., Doubrow,
Ed., "Microcapsules and Nanoparticles in Medicine and Pharmacy,"
CRC Press, Boca Raton, 1992; Mathiowitz et al., 1987, J. Control.
Release, 5:13; Mathiowitz et al., 1987, Reactive Polymers, 6:275;
and Mathiowitz et al., 1988, J. Appl. Polymer Sci., 35:755, U.S.
Pat. Nos. 5,578,325 and 6,007,845; P. Paolicelli et al.,
"Surface-modified PLGA-based Nanoparticles that can Efficiently
Associate and Deliver Virus-like Particles" Nanomedicine.
5(6):843-853 (2010)).
[0110] 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).
[0111] 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.
[0112] 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.
[0113] 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 overall 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.
[0114] Alternatively or additionally, synthetic nanocarriers can be
coupled to immunofeature surfaces, targeting moieties, adjuvants,
various 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.
[0115] A wide variety of the one or more second antigens (or
additional antigens that are not coupled to the population of
synthetic nanocarriers) may be incorporated into the dosage form,
and may be incorporated in a wide variety of ways. Types of one or
more second antigens (or additional antigens that are not coupled
to the population of synthetic nanocarriers) suitable for use with
the present invention have been discussed elsewhere herein.
[0116] There are a wide variety of ways of incorporating the one or
more first or more second antigens (or additional antigens that are
not coupled to the population of synthetic nanocarriers) into the
inventive dosage form. In an embodiment, the one or more second
antigens may be admixed into the dosage form together with the
population of synthetic nanocarriers. For instance, in an
embodiment, a vaccine that comprises the one or more second
antigens may be admixed with the population of synthetic
nanocarriers to form the inventive dosage forms. In embodiments,
the inventive synthetic nanocarriers can be incorporated into the
inventive dosage forms together with one or more first antigen that
are different, similar or the same as the one or more second
antigens in a wide variety of ways, including but not limited to:
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.
[0117] In embodiments, the populations of synthetic nanocarriers
may be combined with the one or more second antigens (which may be
incorporated in a wide variety of ways) to form dosage forms
according to the present invention. The one or more second antigens
may be provided in solution form, suspension form, powder form,
etc., and may be provided as a vaccine formulation. For instance,
in an embodiment, the one or more second antigen may be provided in
the form of a hapten-carrier protein, oligosaccharide,
oligosaccharide complex, oligosaccharide-carrier protein fusion,
live attenuated, or recombinant virus vaccine formulation, and the
population of synthetic nanocarriers admixed with the
hapten-carrier protein, oligosaccharide, oligosaccharide complex,
oligosaccharide-carrier protein fusion, live attenuated, or
recombinant virus vaccine formulation, to form a multivalent
vaccine dosage form (or increase the valency of the hapten-carrier
protein or live attenuated virus vaccine formulations). In
embodiments, the one or more second antigens can be comprised in a
vaccine against Anthrax; Diphtheria, Tetanus and/or Pertussis;
Haemophilus influenzae type B; Hepatitis B; Hepatitis A; Hepatitis
C; Herpes zoster (shingles); Human Papillomavirus (HPV); Influenza;
Japanese Encephalitis; Tick-borne Encephalitis; Measles, Mumps
and/or Rubella; Meningococcal disease; Pneumococcal disease; Polio;
Rabies; Rotavirus; Typhoid; Varicella; Vaccinia (Smallpox); or
Yellow Fever. In other embodiments, the one or more second antigens
are comprised in a commercially available vaccine, including but
not limited to, BIOTHRAX, DAPTACEL, INFANRIX, TRIPEDIA, TRIHIBIT,
KINRIX, PEDIARIX, PENTACEL, PEDVAXHIB, ACTHIB, HIBERIX, COMVAX,
HAVRIX, VAQTA, ENGERIX-B, RECOMBIVAX HB, TWINRIX, ZOSTAVAX,
GARDASIL, CERVARIX, FLUARIX, FLUVIRIN, FLUZONE, FLULAVAL, AFLURIA,
AGRIFLU, FLUMIST, JE-VAX, IXIARO, M-M-R II, PROQUAD, MENOMUNE,
MENACTRA, MENVEO, PNEUMOVAX 23, PREVNAR, PCV13, IPOL, IMOVAX
RABIES, RABAVERT, ROTATEQ, ROTARIX, DECAVAC, BOOSTRIX, ADACEL,
TYPHIM VI, VIVOTIF BERNA, VARIVAX, ACAM2000 or YF-VAX.
[0118] In another embodiment, the population of synthetic
nanocarriers may be combined with proteins taken from an infectious
organism, such as human influenza A virus HA protein, either in
proteinaceous form or in virus-like particles, to form a
multivalent vaccine dosage form according to the invention. In
another embodiment, the population of synthetic nanocarriers may be
added to another population of synthetic nanocarriers that comprise
the one or more second antigens to form a multivalent synthetic
nanocarrier vaccine dosage form. In other embodiments, additional
antigens beyond the one or more first and/or second antigens can be
incorporated into the dosage form (through admixing, and other
techniques disclosed herein or known conventionally). In
embodiments, the inventive compositions provided herein comprise at
least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15 or more different
antigens.
[0119] In embodiments, the one or more first antigens and/or one or
more second antigens comprise or are obtained or derived from a
virus of a family of viruses shown below in Table 1. In another
embodiment, the one or more first antigens and/or one or more
second antigens comprise or are obtained or derived from a virus of
a species provided in Table 1. In still another embodiment, the one
or more first antigens and/or one or more second antigens comprise
or are 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, Paramyxoviridae Measles virus, Nucleocapsid protein,
matrix Mumps virus protein, phosphoprotein, Parainfluenza virus
fusion protein, hemagglutinin, Respiratory
hemagglutinin-neuraminidase, syncytial virus glycoprotein, 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)
[0120] In embodiments, the one or more first antigens and/or one or
more second antigens comprise or are obtained or derived from a
bacteria of a genera of bacteria shown below in Table 2. In another
embodiment, the one or more first antigens and/or one or more
second antigens comprise or are obtained or derived from a
bacterial species provided in Table 2. In still another embodiment,
the one or more first antigens and/or one or more second antigens
comprise or are 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, M1tA, 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 F1iC, F1iD, 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 Sp 1, 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
[0121] In other embodiments, the one or more first antigens and/or
one or more second antigens comprise or are obtained or derived
from a fungus of a genera of fungi shown below in Table 3. In
another embodiment, the one or more first antigens and/or one or
more second antigens comprise or are obtained or derived from a
fungal species provided in Table 3. In still another embodiment,
the one or more first antigens and/or one or more second antigens
comprise or are 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 Mycology
49 and Aspergillus (Suppl. 1):5170-5176, 2011 flavus. Cryptococcus
Cryptococcus Capsular glycoproteins, neoformans, Cryptococcus
laurentii and Cryptococcus albidus, Cryptococcus gattii Histoplasma
Histoplasma Yps3P, Hsp60 capsulatum Pneumocystis Pneumocystis Major
surface proteins (Msg) such as jirovecii MsgC1, MsgC3, MsgC8, and
MsgC9 Stachybotrys Stachybotrys SchS34, chartarum
[0122] Combination of the population of synthetic nanocarriers and
the one or more second antigens may be accomplished using
traditional pharmaceutical mixing methods. These include
liquid-liquid mixing in which two or more suspensions, containing a
population of synthetic nanocarrier or the one or more second
antigens, 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 if the one or more second antigens are available
in powder, as could the re-suspension of two or more powders in a
common media. Depending on the properties and the interaction
potential of the synthetic nanocarriers and the one or more second
antigens, there may be advantages conferred to one or another route
of mixing. 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.
[0123] Doses of dosage forms contain varying amounts of populations
of synthetic nanocarriers and varying amounts of one or more second
antigens, according to the invention. The amount of synthetic
nanocarriers and/or one or more second antigens present in the
inventive dosage forms can be varied according to the nature of the
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 the population
of synthetic nanocarriers and the amount of one or more second
antigens to be present in the dosage form. In embodiments, the
population of synthetic nanocarriers and the one or more second
antigens are present in the dosage form in an amount effective to
generate an immune response to the one or more first antigens and
the one or more second antigens upon administration to a subject.
It may be possible to determine amounts of the first, second,
and/or subsequent antigens effective to generate an immune response
using conventional dose ranging studies and techniques in
subjects.
[0124] In embodiments, the inventive dosage forms can be formulated
by admixing uncoupled adjuvants in the same vehicle or delivery
system as the population of synthetic nanocarriers and the one or
more second antigens. Such adjuvants may include, but are not
limited to mineral salts, such as alum, alum combined with
monphosphoryl lipid (MPL) A of Enterobacteria, such as Escherihia
coli, Salmonella minnesota, Salmonella typhimurium, or Shigella
flexneri or specifically with MPL.RTM. (AS04), MPL A of
above-mentioned bacteria separately, saponins, such as QS-21,
Quil-A, ISCOMs, ISCOMATRIX.TM., emulsions such as MF59.TM.,
Montanide.RTM. ISA 51 and ISA 720, AS02 (QS21+squalene+MPL.RTM.),
AS15, liposomes and liposomal formulations such as AS01,
synthesized or specifically prepared microparticles and
microcarriers such as bacteria-derived outer membrane vesicles
(OMV) of N. gonorrheae, Chlamydia trachomatis and others, or
chitosan particles, depot-forming agents, such as Pluronic.RTM.
block co-polymers, specifically modified or prepared peptides, such
as muramyl dipeptide, aminoalkyl glucosaminide 4-phosphates, such
as RC529, or proteins, such as bacterial toxoids or toxin
fragments. The doses of such other adjuvants can be determined
using conventional dose ranging studies. In embodiments, adjuvant
that is not coupled to the recited population synthetic
nanocarriers may be the same or different from adjuvant that is
coupled to the synthetic nanocarriers.
[0125] 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).
[0126] Compositions according to the invention comprise inventive
synthetic nanocarriers in combination with pharmaceutically
acceptable excipients. The compositions may be made using
conventional pharmaceutical manufacturing and compounding
techniques to arrive at useful dosage forms. Techniques suitable
for use in practicing the present invention may be found in
Handbook of Industrial Mixing: Science and Practice, Edited by
Edward L. Paul, Victor A. Atiemo-Obeng, and Suzanne M. Kresta, 2004
John Wiley & Sons, Inc.; and Pharmaceutics: The Science of
Dosage Form Design, 2nd Ed. Edited by M. E. Auten, 2001, Churchill
Livingstone. In an embodiment, inventive synthetic nanocarriers are
suspended in sterile saline solution for injection together with a
preservative.
[0127] 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.
[0128] 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.
[0129] The inventive compositions may be administered by a variety
of routes of administration, including but not limited to
subcutaneous, intramuscular, intradermal, oral, intranasal,
transmucosal, sublingual, rectal, ophthalmic, transdermal,
transcutaneous or by a combination of these routes.
[0130] Doses of dosage forms contain varying amounts of synthetic
nanocarriers or populations thereof and varying amounts of antigens
and/or adjuvants, according to the invention. The amount of
synthetic nanocarriers and/or antigens and/or adjuvants present in
the inventive dosage forms can be varied according to the nature of
the 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 the synthetic
nanocarriers or population thereof and the amount of antigens
and/or adjuvant to be present in the dosage form. In embodiments,
the synthetic nanocarriers and the antigens and/or adjuvants are
present in the dosage form in an amount effective to generate an
immune response to the antigens upon administration to a subject.
It may be possible to determine amounts of the antigens and/or
adjuvants 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.
[0131] The compositions and methods described herein can be used to
induce, enhance, suppress, modulate, direct, or redirect an immune
response. The compositions and methods described herein can be used
in the diagnosis, prophylaxis and/or treatment of conditions such
as cancers, infectious diseases, metabolic diseases, degenerative
diseases, autoimmune diseases, inflammatory diseases, immunological
diseases, or other disorders and/or conditions. The compositions
and methods described herein can also be used for the prophylaxis
or treatment of an addiction, such as an addiction to nicotine or a
narcotic. The compositions and methods described herein can also be
used for the prophylaxis and/or treatment of a condition resulting
from the exposure to a toxin, hazardous substance, environmental
toxin, or other harmful agent.
[0132] 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.
[0133] 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, Kalaazar, 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
Example 1
Synthetic Nanocarriers with Covalently Coupled Adjuvant
[0134] Nanocarriers comprising PLGA-R848, PLA-PEG-N3, and ova
peptide were prepared via double emulsion method wherein the ova
peptide was encapsulated in the nanocarriers.
[0135] The polyvinyl alcohol (Mw=11 KD-31 KD, 87-89% partially
hydrolyzed) was purchased from JT Baker. Ovalbumin peptide 323-339
was obtained from Bachem Americas Inc. (3132 Kashiwa Street,
Torrance Calif. 90505. Part # 4065609). PLGA-R848, and PLA-PEG-N3
conjugates were synthesized and purified.
[0136] The above materials were used to prepare the following
solutions:
[0137] 1. PLGA-R848 conjugate in methylene chloride @ 100 mg/mL
[0138] 2. PLA-PEG-N3 in methylene chloride @ 100 mg/mL
[0139] 3. Ovalbumin peptide 323-339 in 0.13N HCl @ 70 mg/mL
[0140] 4. Polyvinyl alcohol in 100 mM pH 8 phosphate buffer @50
mg/mL
[0141] Solution #1 (0.75 mL) and solution #2 (0.25 mL) were
combined and solution #3 (0.1 mL) or 0.13N HCl (0.1 mL) 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 # 4 (2.0 mL) and sonication at 30%
amplitude for 40 seconds using the Branson Digital Sonifier 250
formed the second emulsion. This was added to a stiffing 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.
[0142] To wash the nanocarriers, a portion of the nanoparticle
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.
[0143] 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) was added L2 derived peptide
H-Ala-Thr-Gln-Leu-Tyr-Lys-Thr-Cys-Lys-Gln-Ala-Gly-Thr-Cys-Pro-Pro-Asp-Ile-
-Ile-Pro-Lys-Val-X (SEQ ID NO:2); wherein X is a linker group
comprising an acetylene linker (33 mg) with gentle stiffing. A
solution of sodium ascorbate (100 mM in H2O, 0.3 mL) was added,
followed by CuSO4 solution (10 mM in water, 0.6 mL). The resulting
light yellow suspension was stirred at 20 C for 15 h and additional
CuSO4 solution (0.3 mL) and sodium ascorbate solution (0.15 mL)
were added. The suspension was stirred for 5 h at 20 C and diluted
with PBS buffer (pH 7.4) to 10 mL and centrifuged to remove the
supernatant. The residual nanocarrier pellets were washed twice
with PBS buffer. The washed nanocarriers were then re-suspended in
5 mL of PBS buffer and stored frozen. The conjugation of L2 peptide
on the surface of the synthetic nanocarriers was confirmed by HPLC
analysis of the digested nanocarriers and by bioassay.
Example 2
Composition with Synthetic Nanocarriers and Uncoupled Antigen
(Prophetic)
[0144] A 4 mL portion of the synthetic nanocarrier suspension from
Example 1 containing 8 mg of L2 substituted nanocarriers is
centrifuged to settle the particles. The supernatant is discarded
and a 0.5-mL suspension of Gardasil.RTM., Human Papillomavirus
Quadrivalent (Types 6, 11, 16, and 18) Vaccine containing purified
virus-like particles (VLPs) of the major capsid (L1) protein of HPV
Types 6, 11, 16, and 18 is added. The combination vaccine is
agitated to re-suspend the nanocarriers and the resulting
suspension is stored at -20.degree. C. prior to use.
Example 3
Synthetic Nanocarriers with Non-Covalently Coupled Adjuvant
[Prophetic]
[0145] DNA containing cationic disulfide PRINT nanocarriers are
produced by the method described in the patent application of
DeSimone, WO2008118861, example 16 with the exception that the
ssDNA-fluorescein of example 16 is replaced by the
phosphorothioated DNA CpG 7909. After isolation, the cationic
nanocarriers are suspended in 1.0 mL of PBS solution containing 10
mg/mL of heparin. After stirring at room temperature for 2 hours,
the nanocarriers are isolated by centrifugation and are washed
twice with PBS by centrifugation and decantation. The nanocarriers
containing CpG 7909 with surface adsorbed heparin are re-suspended
in 1.0 mL of PBS and are stored at -20.degree. C. prior to use.
Example 4
Composition with Synthetic Nanocarriers and Uncoupled Antigen
(Prophetic)
[0146] A 1 mL portion of the synthetic nanocarrier suspension from
Example 3 containing 10 mg of heparin substituted nanocarriers is
centrifuged to settle the particles. The supernatant is discarded
and a 1-mL suspension of Recombivax HB.RTM. or Engerix-B.RTM.,
human hepatitis B Virus (HBV) vaccines containing purified
proteinaceous particles consisting of the major surface antigen
(HBsAg) protein of HBV is added. The combination vaccine is
agitated to re-suspend the nanocarriers and the resulting
suspension is stored at -20.degree. C. prior to use. A similar
process is used to combine the heparin-substituted nanocarriers of
Example 3 with a 1 mL suspension of bivalent vaccine against human
hepatitis A and B viruses (Twinrix.RTM.), consisting of purified
HBsAg and inactivated human hepatitis A virus.
Example 5
Synthetic Nanocarriers with Covalently Coupled Adjuvant
[Prophetic]
Example 5A
Preparation of R848 Covalently Attached to a Thiol
##STR00002##
[0148] 3,3'-dithio bis-propionic acid (cat #109010) is purchased
from Aldrich Chemical Company. R848 is synthesized at Selecta
Biosciences. A solution of 3,3'-dithio bis-propionic acid (2.10 gm,
1.0.times.10.sup.-2 moles) and HBTU (15.2 g, 4.times.10.sup.-2
moles) in EtOAc (450 mL) is stirred at room temperature under argon
for 45 min. Compound R848 (6.28 g, 2.times.10.sup.-2 moles)) is
added, followed by DIPEA (20.9 mL, 1.2.times.10.sup.-1 moles). The
mixture is stirred at room temperature for 6 h and then at
50-55.degree. C. for 15 h. After cooling, the mixture is washed
with 1% citric acid solution (2.times.40 mL), water (40 mL) and
brine solution (40 mL). The solution is dried over Na.sub.2SO.sub.4
(10 g) and, after filtration, the ethyl acetate is removed under
vacuum. The product is recrystallized from 2-methoxyethanol to
provide 6.5 gm (78%) of a white solid product.
[0149] The disulfide from above (5.0 gm) is dissolved in chloroform
(200 mL) and the solution is treated with dithiothreitol (1.0 gm).
After stiffing at room temperature for 2 hours, the chloroform
solution is washed with water (100 mL) and is then dried over
sodium sulfate. After filtration to remove the drying agent, the
chloroform is removed under vacuum and the solid remaining is
purified by chromatography on silica using 10% methanol in
methylene chloride as eluent. The fractions containing the
thiol-R848 conjugate are pooled and evaporated to give 3.5 gm (70%)
of the thiol-R848 conjugate as a white solid.
Example 5B
Preparation of Nanocarriers
[0150] Gold synthetic nanocarriers are prepared as described in
example (a) of US patent application 2009 0104268 A1 to Midatech
Limited except that peptide BC11 is replaced with the thiol R848
conjugate from Example 5A above and the oligosaccharide antigens
are replaced with L2 derived peptide
H-Ala-Thr-Gln-Leu-Tyr-Lys-Thr-Cys-Lys-Gln-Ala-Gly-Thr-Cys-Pro-Pro-
-Asp-Ile-Ile-Pro-Lys-Val-X (SEQ ID NO:2); wherein X is a linker
group comprising a cysteine residue. After washing and
concentration as described in the Midatech application, the
particles weighing 1.0 mg are used as described in Example 6.
Example 6
Composition with Synthetic Nanocarriers and Uncoupled Antigen
(Prophetic)
[0151] A 1.0 mg portion of the gold nanocarriers from Example 5 are
added to a 1 mL of oral suspension of live recombinant
anti-rotaviral vaccine Rotarix.RTM. against gastroenteritis induced
by type G1 and non-G1 (G3, G4, and G9) rotavirus types. The
combination oral vaccine is agitated to re-suspend the nanocarriers
and the resulting suspension is stored at -20.degree. C. prior to
use as a combination oral vaccine.
Example 7
Synthetic Nanocarriers with T-helper Antigen and Adjuvant
[0152] 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-N3, 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 C6H12 linker to an azide, was
synthesized by conjugating HO-PEG-COOH to an amino-C6H12-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).
[0153] Solutions were prepared as follows:
[0154] Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL was
prepared in 0.13N HCl at room temperature.
[0155] Solution 2: PLGA-R848 @ 50 mg/mL and PLA-PEG-C6-N3 @ 50
mg/mL in dichloromethane was prepared by dissolving each separately
at 100 mg/mL in dichloromethane then combining in equal parts by
volume.
[0156] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM
phosphate buffer, pH 8
[0157] Solution 4: 70 mM phosphate buffer, pH 8
[0158] 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.
[0159] 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-00004 TABLE 4 Nanocarrier Characterization Effective TLR
Agonist, Nanocarrier Diameter (nm) % w/w Antigen, % w/w 209 R848,
4.2 Ova 323-339 peptide, 2.4
Example 8
Immunization with Synthetic Nanocarriers with Coupled Antigen and
Free Protein Without Admixed Adjuvant
Materials and Methods
[0160] (1) Nanocarriers with surface PEG-C6-N3 containing PLGA-R848
and Ova-peptide, prepared as above in Example 7, 7 mg/mL suspension
in PBS.
[0161] (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:
TABLE-US-00005 (SEQ ID NO: 3)
H-Met-Ser-Leu-Leu-Thr-Glu-Val-Glu-Thr-Pro-Thr-
Arg-Asn-Glu-Trp-Glu-Cys-Arg-Cys-Ser-Asp-Gly-Gly- NHCH2CCH
[0162] (3) Catalysts: CuSO4, 100 mM in DI water; THPTA ligand, 200
mM in DI water; sodium ascorbate, 200 mM in DI water freshly
prepared.
[0163] (4) pH 7.4 PBS buffer.
[0164] The NC suspension (7 mg/mL, 4 mL) was concentrated to ca. 1
mL in volume by centrifuge. 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.
Results
[0165] Antibody titers in mice immunized with a combination of
NC-M2e and free hemagglutinin from H5N1 avian influenza strain
(Vietnam) were measured. NC-M2e contained OP-II T-helper peptide
(2.4%) and R848 adjuvant (4.2%). Each bar represents the titer
against antigen. Five animals per group were immunized s.c. with
120 .mu.g of NC and 10 .mu.g of H5 hemaggutinin per injection, 2
times with 3-wk intervals. Titers for day 33 after the first
immunization are shown (ELISA against PLA-PEG-M2e and H5
hemagglutinin, respectively).
[0166] The results show that immunization with a combination of NC
carrying an antigen admixed to a free protein without an admixed
adjuvant results in generation of antibodies to both NC-carried
antigen and to a free protein. When a NC containing surface M2e
peptide from influenza A virus (ectodomain of M2 matrix protein,
amino acids 2-27) was admixed to free influenza A virus
hemagglutinin protein and used for animal immunization, a strong
humoral response was induced in all animals against both M2e
peptide and hemagglutinin (FIG. 1). No reactivity was detected in
the sera of preimmune mice.
Example 9
Immunization with Synthetic Nanocarriers with Coupled Antigen and
Free Protein with Admixed Adjuvant
Materials and Methods
[0167] (1) Nanocarriers with surface PEG-C6-N3 containing PLGA-R848
and Ova-peptide, prepared as above in Example 7, 7 mg/mL suspension
in PBS.
[0168] (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:
TABLE-US-00006 (SEQ ID NO: 3)
H-Met-Ser-Leu-Leu-Thr-Glu-Val-Glu-Thr-Pro-Thr-
Arg-Asn-Glu-Trp-Glu-Cys-Arg-Cys-Ser-Asp-Gly-Gly- NHCH2CCH.
[0169] (3) Catalysts: CuSO4, 100 mM in DI water; THPTA ligand, 200
mM in DI water; sodium ascorbate, 200 mM in DI water freshly
prepared.
[0170] (4) pH 7.4 PBS buffer.
[0171] The NC suspension (7 mg/mL, 4 mL) was concentrated to ca. 1
mL in volume by centrifuge. 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.
Results
[0172] Antibody titers in mice immunized with a combination of
NC-M2e and free hemagglutinin from H5N1 avian influenza strain
(Vietnam) admixed with 80 .mu.g of alum. NC-M2e contained OP-II
T-helper peptide (2.4%) and R848 adjuvant (4.2%). Each bar
represents the titer against antigen. Five animals per group were
immunized s.c. with 120 .mu.g of NC and 10 .mu.g of H5 hemaggutinin
per injection, 2 times with 3-wk intervals. Titers for day 33 after
the first immunization are shown (ELISA against PLA-PEG-M2e and H5
hemagglutinin, respectively).
[0173] The results show that immunization with a combination of a
NC carrying an antigen admixed to a second antigen (free protein)
with an admixed adjuvant results in generation of antibodies to
both NC-carried antigen and to the second antigen. When a NC
containing surface M2e peptide from influenza A virus (ectodomain
of M2 matrix protein, amino acids 2-27) was admixed to free
influenza A virus hemagglutinin protein and used for animal
immunization admixed to alum (Imject Alum, Pierce), a strong
humoral response was induced in all animals against both M2e
peptide and hemagglutinin (FIG. 2). No reactivity was detected in
the sera of preimmune mice.
Example 10
Immunization with Synthetic Nanocarriers with Coupled Antigen and
Virus Vaccine and Adjuvant
Materials and Methods
[0174] (1) Nanocarriers with surface PEG-C6-N3 containing PLGA-R848
and Ova-peptide, prepared as above in Example 7, 7 mg/mL suspension
in PBS.
[0175] (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:
TABLE-US-00007 (SEQ ID NO: 3)
H-Met-Ser-Leu-Leu-Thr-Glu-Val-Glu-Thr-Pro-Thr-
Arg-Asn-Glu-Trp-Glu-Cys-Arg-Cys-Ser-Asp-Gly-Gly- NHCH2CCH.
[0176] (3) Catalysts: CuSO4, 100 mM in DI water; THPTA ligand, 200
mM in DI water; sodium ascorbate, 200 mM in DI water freshly
prepared.
[0177] (4) pH 7.4 PBS buffer.
[0178] The NC suspension (7 mg/mL, 4 mL) was concentrated to ca. 1
mL in volume by centrifuge. 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.
Results
[0179] Antibody titers in mice immunized with a combination of
NC-M2e and beta-propiolactone-inactivated influenza A virus H1N1
(H1N1 New Caledonia/20/99/IVR 116) admixed with 80 .mu.g of alum
were measured. NC-M2e contained OP-II T-helper peptide (2.4%) and
R848 adjuvant (4.2%). Each bar represents the titer against
antigen. Five animals per group were immunized s.c. with 120 .mu.g
of NC and 1 .mu.g of inactivated, thimerosal-containing H1N1 New
Caledonia per injection, 2 times with 3-wk intervals. Titers for
day 33 after the first immunization are shown (ELISA against
PLA-PEG-M2e and H1N1 New Caledonia, respectively).
[0180] The results show that immunization with a combination of NC
carrying an antigen admixed with an inactivated virus vaccine and
an adjuvant results in generation of antibodies to both NC-carried
antigen and an inactivated virus. When a NC containing surface M2e
peptide from influenza A virus (ectodomain of M2 matrix protein,
amino acids 2-27) was admixed to inactivated influenza A virus H1N1
and used for animal immunization admixed to alum (Imject Alum,
Pierce), a strong humoral response was induced in all animals
against both M2e peptide and inactivated influenza A virus H1N1
(FIG. 3). No reactivity was detected in the sera of preimmune
mice.
Example 11
Immunization with Synthetic Nanocarriers with Coupled Antigen and
Recombinant Vaccine with Adjuvant
Materials and Methods
[0181] (1) Nanocarriers with surface PEG-C6-N3 containing PLGA-R848
and Ova-peptide, prepared as above in Example 7, 7 mg/mL suspension
in PBS.
[0182] (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:
TABLE-US-00008 (SEQ ID NO: 2)
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-hexynoy1)-NH2
(with Cys-Cys disulfide bond).
[0183] (3) Catalysts: CuSO4, 100 mM in DI water; THPTA ligand, 200
mM in DI water; sodium ascorbate, 200 mM in DI water freshly
prepared.
[0184] (4) pH 7.4 PBS buffer.
[0185] The NC suspension (7 mg/mL, 4 mL) was concentrated to ca. 1
mL in volume by centrifuge. 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.
Results
[0186] Antibody titers in mice immunized with a combination of
NC-L2-peptide and HBsAg strain ayw produced in the yeast
Saccharomyces cerevisiae admixed with 80 .mu.g of alum were
measured. NC-L2-peptide contained OP-II T-helper peptide (2.4%) and
R848 adjuvant (4.2%). Each bar represents the titer against antigen
indicated. Five animals per group were immunized s.c. with 120
.mu.g of NC and 0.6 .mu.g of recombinant HBsAg, per injection, 2
times with 3-wk intervals. Titers for day 33 after the first
immunization are shown (ELISA against PLA-PEG-L2 and HBsAg ayw,
respectively).
[0187] The results show that immunization with a combination of a
NC carrying an antigen admixed to a recombinant vaccine and an
adjuvant results in generation of antibodies to both NC-carried
antigen and to an inactivated virus. When a NC containing surface
L2 peptide from HPV-16 virus minor capsid L2 protein (amino acids
17-36) was admixed to recombinant hepatitis B surface antigen
(HBsAg) and used for animal immunization admixed to alum (Imject
Alum, Pierce), a strong humoral response was induced in all animals
against both L2 peptide and recombinant HBsAg (FIG. 4). No
reactivity was detected in the sera of preimmune mice.
Sequence CWU 1
1
3117PRTG. gallus 1Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile
Asn Glu Ala Gly1 5 10 15Arg222PRTArtificial SequenceHPV16 L2
peptide 2Ala Thr Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys
Pro Pro1 5 10 15Asp Ile Ile Pro Lys Val 20323PRTArtificial
SequenceH5N1 avian influenza M2e peptide 3Met Ser Leu Leu Thr Glu
Val Glu Thr Pro Thr Arg Asn Glu Trp Glu1 5 10 15Cys Arg Cys Ser Asp
Gly Gly 20
* * * * *