WO2013019669A2 - Synthetic nanocarriers that generate humoral and cytotoxic t lymphocyte (ctl) immune responses - Google Patents

Synthetic nanocarriers that generate humoral and cytotoxic t lymphocyte (ctl) immune responses Download PDF

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Publication number
WO2013019669A2
WO2013019669A2 PCT/US2012/048670 US2012048670W WO2013019669A2 WO 2013019669 A2 WO2013019669 A2 WO 2013019669A2 US 2012048670 W US2012048670 W US 2012048670W WO 2013019669 A2 WO2013019669 A2 WO 2013019669A2
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WO
WIPO (PCT)
Prior art keywords
synthetic nanocarriers
infection
population
protein
epitope
Prior art date
Application number
PCT/US2012/048670
Other languages
French (fr)
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WO2013019669A3 (en
Inventor
David H. Altreuter
Conlin O'NEIL
Petr Ilyinskii
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Selecta Biosciences, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to EA201490381A priority Critical patent/EA201490381A1/en
Priority to MX2014001142A priority patent/MX2014001142A/en
Priority to JP2014523996A priority patent/JP2014521687A/en
Priority to BR112014002139A priority patent/BR112014002139A2/en
Application filed by Selecta Biosciences, Inc. filed Critical Selecta Biosciences, Inc.
Priority to CN201280037441.8A priority patent/CN103702687A/en
Priority to EP12819411.5A priority patent/EP2736537A4/en
Priority to KR1020147005410A priority patent/KR20140050698A/en
Priority to CA2843274A priority patent/CA2843274A1/en
Priority to AU2012290306A priority patent/AU2012290306B2/en
Publication of WO2013019669A2 publication Critical patent/WO2013019669A2/en
Publication of WO2013019669A3 publication Critical patent/WO2013019669A3/en
Priority to IL230269A priority patent/IL230269B/en
Priority to AU2017261562A priority patent/AU2017261562A1/en
Priority to AU2019236653A priority patent/AU2019236653A1/en
Priority to IL273674A priority patent/IL273674A/en

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Definitions

  • This invention relates to methods for generating humoral and cytotoxic T lymphocyte
  • the humoral and CTL immune responses are generated with synthetic nanocarrier compositions that comprise a protein that comprises at least one humoral epitope and at least one MHC Class I-restricted epitope.
  • vaccines have promoted a single arm of the immune system, for example, the generation of a humoral immune response consisting of antibodies to an antigen or, alternatively, activation of a CTL response to an antigen.
  • conventional vaccines generally do not target the sites of action of cells of interest, such as APCs, in an optimal manner. Methods and compositions for effectively activating both of these arms of the immune system optimally to effectively generate immune responses and/or reduce off- target effects and toxicity are needed.
  • a method comprising identifying a subject in need of a humoral and CTL immune response to a first protein, and administering to the subject a composition comprising a population of synthetic nanocarriers coupled to the first protein, wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin-cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm is provided.
  • the mean of a DLS distribution is determined by any of the examples of such a method provided herein. Such examples are described in more detail below.
  • the composition is administered in an amount effective to generate a humoral and CTL immune response to the first protein.
  • a method comprising administering to the subject a composition comprising a population of synthetic nanocarriers coupled to a first protein; wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin-cholesterol adjuvant, wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm, and wherein the composition is administered according to a vaccination regimen is provided.
  • a method comprising administering to the subject a composition comprising a population of synthetic nanocarriers coupled to a first protein; wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin-cholesterol adjuvant, wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm, and wherein the composition is administered according to a protocol that was previously shown to result in a humoral and CTL immune response specific to the first protein in one or more test subjects is provided.
  • the methods provided herein further comprise identifying a subject in need of a humoral and cytotoxic T lymphocyte (CTL) immune response to the first protein.
  • CTL cytotoxic T lymphocyte
  • the composition is administered according to a vaccination regimen.
  • the composition is administered according to a protocol that was previously shown to result in a humoral and CTL immune response specific to the first protein in one or more test subjects
  • the composition further comprises one or more adjuvants.
  • the method further comprises administering one or more adjuvants.
  • the one or more adjuvants comprise stimulators or agonists of pattern recognition receptors, mineral salts, alum, alum combined with monphosphoryl lipid A of Enterobacteria (MPL), MPL® (AS04), AS15, saponins, QS-21,Quil-A, ISCOMs,
  • ISCOMATRIXTM MF59TM, Montanide® ISA 51, Montanide® 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® 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; immuno stimulatory DNA; immuno stimulatory 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; immuno stimulatory DNA molecules comprising CpGs; proinflammatory stimuli released from necrotic cells; urate crystals; activated components of the complement cascade;
  • the one or more adjuvants comprise an agonist of Toll-Like Receptor 2, 3, 4, 7, 8 or 9.
  • the one or more adjuvants comprise an imidazoquinoline or oxoadenine.
  • the imidazoquinoline comprises resiquimod or imiquimod.
  • the one or more adjuvants are coupled to the synthetic nanocarriers of the population of synthetic nanocarriers.
  • composition further comprises or the method further comprises administering another population of synthetic nanocarriers, and the one or more adjuvants are coupled to the synthetic nanocarriers of the other population of synthetic nanocarriers.
  • the one or more adjuvants are not coupled to a synthetic nanocarrier.
  • the composition further comprises one or more additional antigens or the method further comprises administering one or more additional antigens.
  • the one or more additional antigens comprise at least one humoral epitope and/or at least one MHC Class I-restricted epitope.
  • the one or more additional antigens comprise at least one humoral epitope and at least one MHC Class I- restricted epitope that are not the same epitope.
  • the one or more additional antigens comprise a second protein.
  • the one or more additional antigens comprise a humoral epitope and/or a MHC Class I-restricted epitope.
  • the one or more additional antigens comprise a humoral epitope and a MHC Class I-restricted epitope. In yet another embodiment, the one or more additional antigens comprise at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope.
  • the one or more additional antigens are coupled to the synthetic nanocarriers.
  • composition further comprises or the method further comprises administering another population of synthetic nanocarriers, and the one or more additional antigens are coupled to the synthetic nanocarriers of the other population of synthetic nanocarriers.
  • the one or more additional antigens are not coupled to a synthetic nanocarrier.
  • the synthetic nanocarriers and/or other synthetic nanocarriers comprise a polymeric nanoparticle, a metallic nanoparticle, a dendrimer, a buckyball, a nanowire, a virus-like particle or a peptide or protein particle.
  • the synthetic nanocarriers and/or other synthetic nanocarriers comprise one or more polymers.
  • the one or more polymers comprise a polyester, polyamino acid, polycarbonate, polyacetal, polyketal, polysaccharide, polyethyloxazoline or
  • the one or more polymers comprise a polyester.
  • the polyester comprises a poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid) or polycaprolactone.
  • the polyester is coupled to a polyether.
  • the polyether comprises polyethylene glycol.
  • the first protein and/or one or more additional antigens are antigens associated with cancer, an infection or infectious disease or a non- autoimmune or degenerative disease.
  • the first protein and/or one or more additional antigens are antigens associated with human immunodeficiency virus (HIV), malaria, leischmaniasis, a human filovirus infection, a togavirus infection, a alphavirus infection, an arenavirus infection, a bunyavirus infection, a flavivirus infection, a human papillomavirus infection, a human influenza A virus infection, a hepatitis B infection or a hepatitis C infection.
  • HIV human immunodeficiency virus
  • the subject has or is at risk of having cancer, an infection or infectious disease or a non- autoimmune or degenerative disease.
  • the subject has or is at risk of having HIV, malaria, leischmaniasis, a human filovirus infection, a togavirus infection, a alphavirus infection, an arenavirus infection, a bunyavirus infection, a flavivirus infection, a human papillomavirus infection, a human influenza A virus infection, a hepatitis B infection or a hepatitis C infection.
  • the humoral and CTL immune responses that are generated are clinically effective.
  • the immune responses are effective to treat or prevent cancer, an infection or infectious disease or a non- autoimmune or
  • the immune responses are effective to treat or prevent HIV, malaria, leischmaniasis, a human filovirus infection, a togavirus infection, a alphavirus infection, an arenavirus infection, a bunyavirus infection, a flavivirus infection, a human papillomavirus infection, a human influenza A virus infection, a hepatitis B infection or a hepatitis C infection in the subject.
  • the composition further comprises a pharmaceutically acceptable excipient.
  • the composition is sterile.
  • composition is reconstituted from a lyophilized form.
  • dosage form comprising any of the compositions provided is provided.
  • a vaccine comprising any of the compositions and dosage forms provided is provided.
  • composition is administered by intravenous, oral, subcutaneous, pulmonary, intranasal, intradermal, transmucosal, intramucosal or
  • a method comprising administering any of the compositions provided herein to a subject in need thereof.
  • the subject is a human.
  • the subject has or is at risk of having cancer.
  • the subject has or is at risk of having an infection or infectious disease.
  • the subject has or is at risk of having a non- autoimmune or
  • the subject has or is at risk of having HIV.
  • the subject has or is at risk of having malaria, leischmaniasis, a human filovirus infection, a togavirus infection, a alphavirus infection, an arenavirus infection, a bunyavirus infection, a flavivirus infection, a human papillomavirus infection, a human influenza A virus infection, a hepatitis B infection or a hepatitis C infection.
  • any of the compositions provided can be administered to a subject, such as a human, according to a vaccination regimen.
  • any of the compositions provided can be administered to a subject according to a protocol that was previously shown to result in a humoral and CTL immune response specific to the first protein in one or more test subjects is provided
  • any of the methods provided can further comprise assessing the humoral and CTL immune response in the subject.
  • the methods for assessing the humoral and CTL immune response can be any of the methods provided herein.
  • the composition comprises synthetic nanocarriers coupled to a first protein that comprises at least one humoral epitope and at least one MHC Class I- restricted epitope that are not the same epitope.
  • the population of synthetic nanocarriers coupled to the first protein does not comprise a saponin-cholesterol adjuvant and/or the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm.
  • composition comprising a population of synthetic nanocarriers coupled to a first protein, wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin-cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm, for use in therapy or prophylaxis is provided.
  • composition comprising a population of synthetic nanocarriers coupled to a first protein, wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin-cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm, for use in any of the methods provided herein is provided.
  • composition comprising a population of synthetic nanocarriers coupled to a first protein, wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin-cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm, for use in vaccination is provided.
  • composition comprising a population of synthetic compounds
  • nanocarriers coupled to a first protein wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin- cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm, for use in generating a humoral and CTL immune response to the first protein in a subject is provided.
  • these immune responses are clinically effective.
  • these immune responses are each effective in achieving immunity against a disease.
  • a composition comprising a population of synthetic nanocarriers coupled to a first protein, wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin-cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm, for use in a method of therapy or prophylaxis of cancer, an infection or infectious disease, a non- autoimmune or degenerative disease, HIV, malaria, leischmaniasis, a human filovirus infection, a togavirus infection, a alphavirus infection, an arenavirus infection, a bunyavirus infection, a flavivirus infection, a human papillomavirus infection, a human influenza A virus infection, a hepatitis B infection or a
  • a composition comprising a population of synthetic nanocarriers coupled to a first protein, wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin- cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm, for use in a method of therapy or prophylaxis comprising administration by intravenous, oral, subcutaneous, pulmonary, intranasal, intradermal, transmucosal, intramucosal or intramuscular administration is provided.
  • composition comprising a population of synthetic nanocarriers coupled to a first protein, wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin- cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm, for the manufacture of a medicament, for example a vaccine, for use in any of the methods provided herein is provided.
  • compositions for use as defined for any of the compositions or methods provided herein wherein the composition is any of the compositions provided herein is provided.
  • Fig. 1 shows the antibody titers generated at days 12, 26, 43 and 57 following a prime and one boost (on day 28) vaccination regimen.
  • Fig. 2 shows the antibody titers generated at days 26 and 34 following a prime and one boost (on day 14) vaccination regimen.
  • Fig. 3 shows the induction of local memory CTL response by synthetic nanocarriers coupled to ovalbumin (OVA).
  • Fig. 4 shows the induction of central CTL response by synthetic nanocarriers coupled to ovalbumin.
  • Fig. 5 shows the induction of central CTL induction by synthetic nanocarrier compositions with ovalbumin and adjuvant.
  • Fig. 6 shows the antibody titers generated at days 25 and 42 following a prime and two boosts (on days 14 and 28) vaccination regimen.
  • Fig. 7 shows the development of antibody titers after a single injection by NC-R848 +
  • NC-OVA NC-OVA. Individual titers and averages for each experimental group are shown.
  • Fig. 8 shows the specific cytotoxicity in vivo after a single immunization with NC- R848 + NC-OVA. Averages for each group with standard deviation are shown.
  • Fig. 9 shows the anti-ovalbumin antibody titers upon the immunization with nanocarriers carrying CpG and OVA vs. free CpG (5x) and OVA (5x).
  • Fig. 10 shows the induction of OVA- specific CTL response in draining lymph nodes and spleens by the immunization with nanocarriers carrying CpG and OVA vs. free CpG (5x) and OVA (5x).
  • Fig. 11 shows the expansion of systemically induced OVA- specific CTLs in vitro upon the immunization with nanocarriers carrying CpG and OVA vs. free CpG (5x) and OVA (5x).
  • Left Y axis (dark striped bars) - fraction of SIINFEKL (SEQ ID NO: l)-specific CD8+ cells after expansion; right Y axis (light striped bars) - expansion potential presented as proportion of post- and pre-expansion SIINFEKL (SEQ ID NO: l)-specific CTLs.
  • nanocarrier includes a mixture of two or more such synthetic nanocarriers or a plurality of such synthetic nanocarriers
  • reference to "a DNA molecule” includes a mixture of two or more such DNA molecules or a plurality of such DNA molecules
  • reference to "an adjuvant” includes mixture of two or more such adjuvant molecules or a plurality of such adjuvant molecules, and the like.
  • the term “comprise” or variations thereof such as “comprises” or “comprising” are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, elements, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers.
  • any recited integer e.g. a feature, element, characteristic, property, method/process step or limitation
  • group of integers e.g. features, elements, characteristics, properties, method/process steps or limitations
  • compositions and related compositions, that are believed to have not been previously shown to generate strong and effective humoral and CTL immune responses.
  • Such compositions can effectively target immune cells of interest to generate more effective immune responses.
  • nanocarriers is a maximum dimension of from 20 nm to 250 nm.
  • a method comprising administering such a composition.
  • the method comprises identifying a subject in need of a humoral and CTL immune response to a first protein, and administering to the subject a composition comprising such synthetic nanocarriers.
  • the composition is in an amount effective to generate a humoral and CTL immune response to the first protein.
  • the method comprises administering such a composition to a subject according to a protocol that was previously shown to result in a humoral and CTL immune response specific to the first protein in one or more test subjects.
  • the immune responses that are generated are clinically effective.
  • the subject to which the compositions are administered may have or be at risk of having cancer, an infection or infectious disease or a non-autoimmune or degenerative disease.
  • the subject may have or be at risk of having HIV, malaria, leischmaniasis, a human filovirus infection, a togavirus infection, a alphavirus infection, an arenavirus infection, a bunyavirus infection, a flavivirus infection, a human papillomavirus infection, a human influenza A virus infection, a hepatitis B infection or a hepatitis C infection.
  • compositions are administered to a subject, such as a human, according to a vaccination regimen.
  • adjuvant means an agent that does not constitute a specific antigen, but boosts the strength and longevity of an immune response to a concomitantly administered antigen.
  • 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® (AS04), MPL A of above-mentioned bacteria separately, saponins, such as QS-21,Quil-A, ISCOMs, ISCOMATRIXTM, emulsions such as MF59TM, Montanide® ISA 51 and ISA 720, AS02 (QS21+squalene+ MPL®) , 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® 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.
  • Saponin-cholesterol adjuvants are saponin adjuvants that are stabilized by admixture with cholesterol. Such adjuvants include, for example, ISCOMs and ISCOMATRIX adjuvants.
  • the synthetic nanocarriers provided herein are not or do not comprise such adjuvants.
  • compositions provided herein do not comprise such adjuvants.
  • 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.
  • adjuvants comprise agonists for Toll- Like Receptors 3, agonists for Toll-Like Receptors 7 and 8, or agonists for Toll-Like
  • Receptor 9 preferably the recited adjuvants comprise imidazoquinolines; such as R848; adenine derivatives, such as those disclosed in US patent 6,329,381 (Sumitomo
  • synthetic nanocarriers incorporate as adjuvants compounds that are agonists for toll-like receptors (TLRs) 7 & 8 ("TLR 7/8 agonists").
  • TLR 7/8 agonists are agonists for toll-like receptors (TLRs) 7 & 8
  • TLR 7/8 agonists are the TLR 7/8 agonist compounds disclosed in US Patent 6,696,076 to Tomai et al., including but not limited to imidazoquinoline amines, imidazopyridine amines, 6,7-fused
  • Preferred adjuvants comprise imiquimod and resiquimod (also known as R848).
  • an adjuvant may be an agonist for the DC surface molecule CD40.
  • a synthetic nanocarrier to stimulate immunity rather than tolerance, a synthetic nanocarrier
  • adjuvants 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.
  • adjuvants also may comprise immuno stimulatory RNA molecules, such as but not limited to dsRNA, poly I:C or poly Lpoly C12U (available as Ampligen ®, 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.,
  • an adjuvant may be a TLR-4 agonist, such as bacterial lipopolysacccharide (LPS), VSV-G, and/or HMGB-1.
  • adjuvants may comprise TLR-5 agonists, such as flagellin, or portions or derivatives thereof, including but not limited to those disclosed in US Patents 6,130,082, 6,585,980, and 7,192,725.
  • 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
  • adjuvants may be proinflammatory stimuli released from necrotic cells (e.g., urate crystals).
  • adjuvants may be activated components of the complement cascade (e.g., CD21, CD35, etc.).
  • 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.
  • the complement receptor agonist induces endogenous complement
  • 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.
  • the cytokine receptor agonist is a small molecule, antibody, fusion protein, or aptamer.
  • 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.
  • 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.
  • administering means providing a material, such as a drug, to a subject in a manner that is pharmacologically useful.
  • Amount effective is any amount of a composition provided herein that produces one or more desired responses, such as one or more desired immune responses. This amount can be for in vitro or in vivo purposes. For in vivo purposes, the amount can be one that a clinician would believe may have a clinical benefit for a subject in need of a humoral immune response and a CTL immune response to a single protein. An effective amount that a clinician would believe may have a clinical benefit for such a subject is also referred to herein as a "clinically effective amount". In embodiments, both the humoral immune response and the CTL immune response that is elicited by a composition provided herein results in a clinical effect from each of these arms of the immune system.
  • clinically effective amounts are effective amounts that can be helpful in the treatment of a subject with a disease or condition in which a humoral immune response and a CTL immune response to a single protein would provide a benefit.
  • Such subjects include, in some embodiments, those that have or are at risk of having cancer, an infection or infectious disease or a non- autoimmune or degenerative disease.
  • such subjects include those that have or are at risk of having HIV, malaria, leischmaniasis, a human filovirus infection, a togavirus infection, a alphavirus infection, an arenavirus infection, a bunyavirus infection, a flavivirus infection, a human papillomavirus infection, a human influenza A virus infection, a hepatitis B infection or a hepatitis C infection.
  • Amounts effective include those that involve the production of a humoral immune response against a humoral epitope and a CTL immune response against a MHC Class I- restricted epitope administered in one of the compositions provided herein.
  • a subject's humoral and CTL immune response can be monitored by routine methods.
  • An amount that is effective to produce the desired immune responses as provided herein can also be an amount of a composition provided herein that produces a desired therapeutic endpoint or a desired therapeutic result.
  • the amount that is effective is one that provides effective immunity against a disease or agent that causes a disease as provided herein.
  • the immunity persists in the subject for at least 6, 12, 18, 24, 36, 48, 60 or more months.
  • the immunity results or persists due to the administration of a composition provided herein according to a vaccination regimen.
  • 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,
  • doses of the compositions of the invention can range from about 10 g/kg to about 100,000 ⁇ g/kg. In some embodiments, the doses can range from about 0.1 mg/kg to about 100 mg/kg. In still other embodiments, the doses can range from about 0.1 mg/kg to about 25 mg/kg, about 25 mg/kg to about 50 mg/kg, about 50 mg/kg to about 75 mg/kg or about 75 mg/kg to about 100 mg/kg. Alternatively, the dose can be administered based on the number of synthetic nanocarriers. For example, useful doses include greater than 10 6 ,
  • Antigen means any antigen that can generate one or more immune responses.
  • the antigen may be one that generates a humoral and/or CTL immune response.
  • Such antigens include, but are not limited to proteins, peptides, small molecules, oligosaccharides, and carbohydrates.
  • such an antigen comprises a non-protein antigen (i.e., not a protein or peptide antigen).
  • an antigen that generates a humoral immune response comprises a carbohydrate associated with an infectious agent.
  • an antigen that generates a humoral immune response comprises a
  • glycoprotein or glycopeptide associated with an infectious agent can be a bacterium, virus, fungus, protozoan, or parasite.
  • Antigens may be B cell or T cell antigens.
  • compositions for use in the inventive methods provided herein are coupled to an antigen that is a protein that comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that is not the same epitope.
  • Such compositions can, in some embodiments, comprise one or more additional antigens that may also be so limited but not necessarily so.
  • the one or more additional antigens for use in the methods and compositions provided herein can be any antigen, which include antigens that comprise humoral epitopes and/or MHC Class I-restricted epitopes.
  • the one or more additional antigens may also include MHC Class II-restricted epitopes.
  • the one or more additional antigens may be any antigen that generates a humoral immune response.
  • the one or more additional antigens may be any of the T cell antigens described herein, including a CD-I restricted antigen.
  • the one or more additional antigens may be a protein, peptide, small molecule, oligosaccharide and carbohydrate.
  • antigens including a protein that comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, are coupled to the synthetic nanocarriers.
  • the antigens are not coupled to the synthetic nanocarriers.
  • the antigens are encapsulated in the synthetic nanocarriers.
  • Type(s) of antigens means molecules that share the same, or substantially the same, antigenic characteristics.
  • Antigens associated with a disease, disorder or condition are antigens that can generate an undesired immune response against, as a result of, or in conjunction with the disease, disorder or condition; the cause of the disease, disorder or condition (or a symptom or effect thereof); and/or can generate an undesired immune response that is a symptom, result or effect of the disease, disorder or condition.
  • antigens are expressed in or on diseased cells, such as cancer or tumor cells, but not in or on normal or healthy cells (or non-diseased cells).
  • antigens can also comprise an antigen that is expressed in or on diseased cells and on normal or healthy cells (or non-diseased cells) but is expressed in or on diseased cells at a greater level than on normal or healthy cells (or non-diseased cells).
  • an antigen associated with a disease or condition provided herein will not lead to a substantial or detrimental immune response against normal or healthy cells or will lead to a beneficial immune response against the disease or condition that outweighs any immune response against normal or healthy cells (or non-diseased cells).
  • the antigens associated with a disease or condition provided herein are proteins that are coupled to synthetic nanocarriers that comprise humoral and/or MHC Class I-restricted epitopes.
  • such proteins comprise a humoral and a MHC Class I-restricted epitope. In still other embodiments, such proteins comprise at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope. Examples of antigens, including the foregoing proteins, are provided elsewhere herein.
  • At least a portion of the dose means at least some part of the dose, ranging up to including all of the dose.
  • An "at risk" subject is one in which a health practitioner believes has a chance of having a disease or condition as provided herein.
  • B cell antigen means any antigen that is recognized by or triggers an immune response in a B cell (e.g., an antigen that is specifically recognized by a B cell or a receptor thereon).
  • an antigen that is a T cell antigen is also a B cell antigen.
  • the T cell antigen is not also a B cell antigen.
  • B cell antigens include, but are not limited to proteins, peptides, small molecules, oligosaccharides and
  • Couple or “Coupled” or “Couples” (and the like) means to chemically associate one entity (for example a moiety) with another.
  • the coupling is covalent, meaning that the coupling occurs in the context of the presence of a covalent bond between the two entities.
  • the non-covalent coupling is mediated by non-covalent interactions including but not limited to charge interactions, affinity
  • encapsulation is a form of coupling.
  • Cytotoxic T lymphocyte (CTL) immune response means any stimulation, induction or proliferation of cytotoxic T cells, preferably cytotoxic T cells that are specific to an epitope, such as a MHC Class I-restricted epitope.
  • the epitope is or is of an antigen that is associated with any of the diseases or conditions provided herein.
  • Dosage form means a pharmacologically and/or immunologically active material in a medium, carrier, vehicle, or device suitable for administration to a subject.
  • Encapsulate means to enclose at least a portion of a substance within a synthetic nanocarrier. In some embodiments, a substance is enclosed completely within a synthetic nanocarrier. In other embodiments, most or all of a substance that is encapsulated is not exposed to the local environment external to the synthetic nanocarrier. In other
  • no more than 50%, 40%, 30%, 20%, 10% or 5% (weight/weight) is exposed to the local environment.
  • 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.
  • Epitope also known as an antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by, for example, antibodies, B cells, or T cells.
  • a “humoral epitope” is one that is recognized by antibodies or B cells
  • a “MHC Class I-restricted epitope” is one that is presented to immune cells by MHC class I molecules found on nucleated cells.
  • MHC Class II-restricted epitopes are epitopes that are presented to immune cells by MHC class II molecules found on antigen presenting cells (APCs), for example, on professional antigen-presenting immune cells, such as on macrophages, B cells, and dendritic cells, or on non-hematopoietic cells, such as hepatocytes.
  • APCs antigen presenting cells
  • professional antigen-presenting immune cells such as on macrophages, B cells, and dendritic cells
  • non-hematopoietic cells such as hepatocytes.
  • epitopes are known to those of skill in the art, and exemplary epitopes suitable according to some aspects of this invention include, but are not limited to those listed in the Immune Epitope Database (www.immuneepitope.org, Vita R, Zarebski L, Greenbaum JA, Emami H, Hoof I, Salimi N, Damle R, Sette A, Peters B.
  • Epitopes can also be identified with publicly available algorithms, for example, the algorithms described in Wang P, Sidney J, Kim Y, Sette A, Lund O, Nielsen M, Peters B. 2010. peptide binding predictions for HLA DR, DP and DQ molecules. BMC Bioinformatics 2010, 11:568; Wang P, Sidney J, Dow C, Mothe B, Sette A, Peters B. 2008. A systematic assessment of MHC class II peptide binding predictions and evaluation of a consensus approach. PLoS Comput Biol. 4(4):el000048; Nielsen M, Lund O. 2009. NN-align. An artificial neural network-based alignment algorithm for MHC class II peptide binding prediction. BMC Bioinformatics.
  • Geneating means causing an action, such as an immune response (e.g., a humoral immune response or a CTL immune response) against an epitope to occur, either directly oneself or indirectly, such as, but not limited to, an unrelated third party that takes an action through reliance on one's words or deeds.
  • an immune response e.g., a humoral immune response or a CTL immune response
  • Human immune response means any immune response that results in the production or stimulation of B cells and/or the production of antibodies.
  • the humoral immune response is specific to an epitope comprised within an inventive
  • antibody response means the production of a measurable level of antibodies.
  • the antibody response or generation of the antibody titer is in a human.
  • the antibodies are antibodies of a certain isotype, such as IgG or a subclass thereof.
  • Methods for measuring antibody titers are known in the art and include Enzyme-linked Immunosorbent Assay (ELISA). Methods for measuring antibody titers are also described in some detail in the EXAMPLES.
  • the antibody response or antibody titer is specific to an epitope as provided herein.
  • the antibody response can be quantitated, for example, as the number of antibodies, concentration of antibodies or titer. The values can be absolute or they can be relative.
  • Assays for quantifying an antibody response include antibody capture assays, enzyme-linked immunosorbent assays (ELISAs), inhibition liquid phase absorption assays (ILPAAs), rocket Immunoelectrophoresis (RIE) assays and line
  • Immunoelectrophoresis (LIE) assays When an antibody response is compared to another antibody response the same type of quantitative value (e.g., titer) and method of measurement (e.g., ELISA) is preferably used to make the comparison.
  • titer e.g., titer
  • ELISA e.g., ELISA
  • An ELISA method for measuring an antibody titer may consist of the following steps (i) preparing an ELISA-plate coating material such that the antibody target of interest is coupled to a substrate polymer or other suitable material (ii) preparing the coating material in an aqueous solution (such as PBS) and delivering the coating material solution to the wells of a multiwell plate for overnight deposition of the coating onto the multiwell plate (iii) thoroughly washing the multiwell plate with wash buffer (such as 0.05% Tween-20 in PBS) to remove excess coating material (iv) blocking the plate for nonspecific binding by applying a diluent solution (such as 10% fetal bovine serum in PBS), (v) washing the blocking/diluent solution from the plate with wash buffer (vi) diluting the serum sample(s) containing antibodies and appropriate standards (positive controls) with diluent as required to obtain a concentration that suitably saturates the ELISA reponse (vii) serially diluting the plasma samples on the following steps (
  • Identifying is any action or set of actions that allows a clinician to recognize a subject as one who may benefit from the methods and compositions provided herein.
  • the identified subject is one who is in need of a humoral immune response and CTL immune response to a single protein.
  • Such subjects include any subject that has or is at risk of having any of the disease or conditions provided herein.
  • the action or set of actions may be either directly oneself or indirectly, such as, but not limited to, an unrelated third party that takes an action through reliance on one's words or deeds.
  • infectious disease is any condition or disease caused by a microorganism, pathogen or other agent, such as a bacterium, fungus, prion or virus.
  • “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.
  • a minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample is equal to or greater than 100 nm.
  • 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 ⁇ .
  • 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.
  • 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 10,000: 1, more preferably from 1: 1 to 1000: 1, still more preferably from 1: 1 to 100: 1, and yet more preferably from 1: 1 to 10: 1.
  • 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 ⁇ , more preferably equal to or less than 2 ⁇ , more preferably equal to or less than 1 ⁇ , 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.
  • a minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample is equal to or greater than lOOnm, 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 dimensions e.g., diameter
  • DLS dynamic light scattering
  • a suspension of synthetic nanocarriers can be diluted from an aqueous buffer into purified water to achieve a final synthetic nanocarrier suspension concentration of approximately 0.01 to 0.1 mg/mL.
  • the diluted suspension may be prepared directly inside, or transferred to, a suitable cuvette for DLS analysis.
  • the cuvette may then be placed in the DLS, allowed to equilibrate to the controlled temperature, and then scanned for sufficient time to acquire a stable and reproducible distribution based on appropriate inputs for viscosity of the medium and refractive indicies of the sample.
  • the effective diameter, or mean of the distribution is then reported.
  • "Dimension" or "size” or “diameter” of synthetic nanocarriers means the mean of a particle size distribution obtained using dynamic light scattering.
  • MHC refers to major histocompatibility complex, a large genomic region or gene family found in most vertebrates that encodes MHC molecules that display fragments or epitopes of processed proteins on the cell surface.
  • the presentation of MHC:peptide on cell surfaces allows for surveillance by immune cells, usually a T cell.
  • immune cells usually a T cell.
  • Class I MHC molecules are found on nucleated cells and present peptides to cytotoxic T cells.
  • Class II MHC molecules are found on certain immune cells, chiefly macrophages, B cells and dendritic cells, collectively known as APCs.
  • APCs dendritic cells
  • the best-known genes in the MHC region are the subset that encodes antigen-presenting proteins on the cell surface. In humans, these genes are referred to as human leukocyte antigen (HLA) genes.
  • HLA human leukocyte antigen
  • “Pharmaceutically acceptable excipient” means a pharmacologically inactive material used together with the recited synthetic nanocarriers to formulate the compositions.
  • compositions 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.
  • saccharides such as glucose, lactose, and the like
  • preservatives such as antimicrobial agents
  • reconstitution aids such as phosphate buffered saline
  • colorants such as phosphate buffered saline
  • saline such as phosphate buffered saline
  • Protein(s) means compounds, typically having a molecular weight greater than 1000 daltons, comprising amino acid residues joined together primarily by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. Proteins may also comprise additional bonding structures such as secondary structures, tertiary structures, and the like. Certain of the peptide bonds in proteins may be replaced by other bond types, for various purposes, such as stabilization or coupling.
  • synthetic nanocarriers preferably, there are multiple copies of the protein that are coupled to each synthetic nanocarrier.
  • Protocol refers to any dosing regimen of one or more substances to a subject.
  • a dosing regimen may include the amount, frequency and/or mode of administration.
  • such a protocol may be used to administer one or more compositions of the invention to one or more test subjects. Immune responses in these test subjects can then be assessed to determine whether or not the protocol was effective in generating desired immune response(s). Any other therapeutic and/or prophylactic effects may also be assessed instead of or in addition to the aforementioned immune responses. Whether or not a protocol had a desired effect can be determined using any of the methods provided herein or otherwise known in the art.
  • a population of cells may be obtained from a subject to which a composition provided herein has been administered according to a specific protocol in order to determine whether or not specific immune cells, cytokines, antibodies, etc. were generated, activated, etc.
  • Useful methods for detecting the presence and/or number of immune cells include, but are not limited to, flow cytometric methods (e.g., FACS) and
  • kits typically include staining reagents for multiple antigens that allow for FACS-based detection, separation and/or quantitation of a desired cell population from a heterogeneous population of cells.
  • 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.
  • 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
  • the synthetic nanocarriers do not comprise albumin nanoparticles. In embodiments, synthetic nanocarriers do not comprise chitosan. In certain other
  • the synthetic nanocarriers do not comprise chitosan. In other embodiments, inventive synthetic nanocarriers are not lipid-based nanoparticles. In further embodiments, inventive synthetic nanocarriers do not comprise a phospholipid.
  • a synthetic nanocarrier can be, but is not limited to, one or a plurality of lipid-based nanoparticles (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, viruslike 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.
  • lipid-based nanoparticles also referred to herein as lipid nanoparticles, i.
  • Synthetic nanocarriers may be a variety of different shapes, including but not limited to spheroidal, cuboidal, pyramidal, oblong, cylindrical, toroidal, and the like. Synthetic nanocarriers according to the invention comprise one or more surfaces.
  • Exemplary synthetic nanocarriers that can be adapted for use in the practice of the present invention comprise: (1) the biodegradable nanoparticles disclosed in US Patent 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
  • 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.
  • 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.
  • 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.
  • 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.
  • synthetic nanocarriers exclude 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).
  • 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.
  • 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).
  • an antigen that is a T cell antigen is also a B cell antigen.
  • 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 combination of T cell antigen that is 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+
  • embodiments can effectively stimulate both types of responses.
  • the T cell antigen is a T helper cell antigen (i.e. one that can generate an enhanced response to a B cell antigen, preferably an unrelated B cell antigen, through stimulation of T cell help).
  • 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. US Patent 7,202,351).
  • a T helper cell antigen may comprise one or more lipids, or glycolipids, including but not limited to: cc-galactosylceramide (cc-GalCer), cc- linked glycosphingolipids (from Sphingomonas spp.), galactosyl diacylglycerols (from Borrelia burgdorferi), lypophosphoglycan (from Leishmania donovani), and
  • CD4+ T-cell antigens may be derivatives of a CD4+ T-cell antigen that is obtained from a source, such as a natural source.
  • 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.
  • the T cell antigen preferably a T helper cell antigen
  • the T cell antigen may be coupled to, or uncoupled from, a synthetic nanocarrier.
  • the T cell antigen is encapsulated in the synthetic nanocarriers of the compositions.
  • 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).
  • a vaccine may comprise dosage forms according to the invention.
  • Vaccine regimen or “vaccination regimen” is a schedule of one or more
  • vaccinations that includes the number and timing of doses of a vaccine.
  • vaccination regimens are intended to achieve immunity against the development of a disease or condition.
  • the vaccine regimen is one that achieves immunity via both the humoral and CTL arms of the immune system.
  • compositions for effective humoral and CTL immune response generation are methods and related compositions for effective humoral and CTL immune response generation. It has been found that synthetic nanocarriers to which a protein that comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, is coupled, wherein the synthetic nanocarriers do not comprise a saponin-cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm can be used to generate effective and strong humoral and CTL immune responses.
  • the compositions provided can be used for a variety of desired clinical endpoints such as for vaccination.
  • synthetic nanocarriers are spheres or spheroids. In some embodiments, synthetic nanocarriers are flat or plate-shaped. In some embodiments, synthetic nanocarriers are cubes or cubic. In some embodiments, synthetic nanocarriers are ovals or ellipses. In some embodiments, synthetic nanocarriers are cylinders, cones, or pyramids.
  • a population of synthetic nanocarriers that is relatively uniform in terms of size, shape, and/or composition so that each synthetic nanocarner 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.
  • 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).
  • 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.
  • synthetic nanocarriers may comprise metal particles, quantum dots, ceramic particles, etc.
  • a non-polymeric synthetic nanocarrier is an aggregate of non-polymeric components, such as an aggregate of metal atoms (e.g., gold atoms).
  • synthetic nanocarriers may optionally comprise one or more lipids.
  • a synthetic nanocarrier may comprise a liposome.
  • a synthetic nanocarrier may comprise a lipid bilayer.
  • a synthetic nanocarrier may comprise a lipid monolayer.
  • a synthetic nanocarrier may comprise a micelle.
  • a synthetic nanocarrier may comprise a core comprising a polymeric matrix surrounded by a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.).
  • 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.).
  • synthetic nanocarriers can comprise one or more polymers.
  • such a polymer can be surrounded by a coating layer (e.g., liposome, lipid monolayer, micelle, etc.).
  • various elements (i.e., components) of the synthetic nanocarriers can be coupled with the polymer.
  • a component can be covalently associated with a polymeric matrix. In some embodiments, covalent association is mediated by a linker. In some embodiments, a component can be noncovalently associated with a polymeric matrix. For example, in some embodiments, a component can be encapsulated within, surrounded by, and/or dispersed throughout a polymeric matrix. Alternatively or additionally, a component can be associated with a polymeric matrix by hydrophobic interactions, charge interactions, van der Waals forces, etc.
  • a polymeric matrix comprises one or more polymers.
  • the synthetic nanocarriers provided herein may be polymeric nanocarriers.
  • 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.
  • polymers in accordance with the present invention are organic polymers.
  • the synthetic nanocarriers comprise one or more polymers that comprise a polyester, polycarbonate, polyamide, or polyether, or unit thereof.
  • the polymer comprises poly(ethylene glycol) (PEG), poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), or a polycaprolactone, or unit thereof.
  • PEG poly(ethylene glycol)
  • the polymer is biodegradable. Therefore, in these embodiments, it is preferred that if the polymer comprises a polyether, such as poly(ethylene glycol) or unit thereof, the polymer comprises a block-co-polymer of a polyether and a biodegradable polymer such that the polymer is biodegradable.
  • the polymer does not solely comprise a polyether or unit thereof, such as poly(ethylene glycol) or unit thereof.
  • the one or more polymers may be comprised within a polymeric synthetic nanocarrier or may be comprised in a number of other different types of synthetic
  • polymers suitable for use in the present invention also include, but are not limited to polyethylenes, polycarbonates (e.g. poly(l,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.
  • polymers in accordance with the present invention include polymers which have been approved for use in humans by the U.S. Food and Drug
  • polyesters e.g., polylactic acid, poly(lactic-co-glycolic acid), polycaprolactone, polyvalerolactone, poly(l,3-dioxan-2one)
  • polyanhydrides e.g., poly(sebacic anhydride)
  • polyethers e.g., polyethylene glycol
  • polyurethanes polymethacrylates; polyacrylates; and
  • polymers can be hydrophilic.
  • 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).
  • a synthetic nanocarrier comprising a hydrophilic polymeric matrix generates a hydrophilic environment within the synthetic nanocarrier.
  • polymers can be hydrophobic.
  • a synthetic nanocarrier comprising a hydrophobic polymeric matrix generates a hydrophobic
  • hydrophobicity of the polymer may have an impact on the nature of materials that are incorporated (e.g. coupled) within the synthetic nanocarrier.
  • polymers may be modified with one or more moieties and/or functional groups.
  • moieties or functional groups can be used in accordance with the present invention.
  • polymers may be modified with polyethylene glycol (PEG), with a carbohydrate, and/or with acyclic polyacetals derived from
  • polymers may be modified with a lipid or fatty acid group.
  • a fatty acid group may be one or more of butyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, arachidic, behenic, or lignoceric acid.
  • 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.
  • 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.”
  • 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.
  • polyesters include, for example,
  • 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.
  • 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.
  • polymers may be one or more acrylic polymers.
  • 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.
  • polymers can be cationic polymers.
  • 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.,
  • the synthetic nanocarriers may not comprise (or may exclude) cationic polymers.
  • 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).
  • polyesters include poly(L-lactide-co-L-lysine) (Barrera et al., 1993, J. Am. Chem.
  • 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
  • polymers can be used in accordance with the present invention without undergoing a cross-linking step.
  • synthetic nanocarriers may comprise block copolymers, graft copolymers, blends, mixtures, and/or adducts of any of the foregoing and other polymers.
  • synthetic nanocarriers may optionally comprise one or more amphiphilic entities.
  • an amphiphilic entity can promote the production of synthetic nanocarriers with increased stability, improved uniformity, or increased viscosity.
  • amphiphilic entities can be associated with the interior surface of a lipid membrane (e.g., lipid bilayer, lipid monolayer, etc.).
  • lipid membrane e.g., lipid bilayer, lipid monolayer, etc.
  • amphiphilic entities known in the art are suitable for use in making synthetic nanocarriers in accordance with the present invention.
  • 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®85) glycocholate; sorbitan monolaurate (Span®20); polysorbate 20 (Tween®20); polysorbate 60 (Tween®60);
  • polysorbate 65 (Tween®65); polysorbate 80 (Tween®80); polysorbate 85 (Tween®85); polyoxyethylene monostearate; surfactin; a poloxomer; a sorbitan fatty acid ester such as sorbitan trioleate; lecithin; lysolecithin; phosphatidylserine;
  • phosphatidylinositol phosphatidylinositol
  • sphingomyelin phosphatidylethanolamine (cephalin); cardiolipin
  • phosphatidic acid cerebrosides
  • dicetylphosphate dipalmitoylphosphatidylglycerol
  • 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.
  • synthetic nanocarriers may optionally comprise one or more carbohydrates.
  • Carbohydrates may be natural or synthetic.
  • a carbohydrate may be a derivatized natural carbohydrate.
  • 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.
  • a carbohydrate is a polysaccharide, including but not limited to pullulan, cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextran, glycogen,
  • the synthetic nanocarriers do not comprise (or specifically exclude) carbohydrates, such as a polysaccharide.
  • the carbohydrate may comprise a carbohydrate derivative such as a sugar alcohol, including but not limited to mannitol, sorbitol, xylitol, erythritol, maltitol, and lactitol.
  • a carbohydrate derivative such as a sugar alcohol, including but not limited to mannitol, sorbitol, xylitol, erythritol, maltitol, and lactitol.
  • compositions for use in the methods according to the invention comprise 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.
  • synthetic nanocarriers are suspended in sterile saline solution for injection together with a preservative.
  • the components when preparing synthetic nanocarriers as carriers for use in vaccines, methods for coupling the components to the synthetic nanocarriers may be useful. If the component is a small molecule it may be of advantage to attach the component 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 component to the synthetic nanocarrier through the use of these surface groups rather than attaching the component to a polymer and then using this polymer conjugate in the construction of synthetic nanocarriers. In certain embodiments, the coupling can be a covalent linker.
  • components 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 the component containing an alkyne group or by the 1,3- dipolar cycloaddition reaction of alkynes on the surface of the nanocarrier with components containing an azido group.
  • 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.
  • 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.
  • 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.
  • An amide linker is formed via an amide bond between an amine on one component 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.
  • 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 component containing activated thiol group.
  • a triazole linker specifically a 1,2,3-triazole of the form , wherein RI 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.
  • 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.
  • 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.
  • the synthetic nanocarrier can be prepared by another route, and subsequently functionalized with alkyne or azide groups.
  • the component is prepared with the presence of either an alkyne (if the polymer contains an azide) or an azide (if the polymer contains an alkyne) group.
  • the component is then allowed to react with the nanocarrier via the 1,3-dipolar cycloaddition reaction with or without a catalyst which covalently couples the component to the particle through the 1,4-disubstituted 1,2,3-triazole linker.
  • 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 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 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.
  • thioether linkers can be prepared by the radical thiol-ene reaction of a thiol/mercaptan group on one component with an alkene group on a second component such as a polymer or nanocarrier.
  • a hydrazone linker is made by the reaction of a hydrazide group on one component with an aldehyde/ketone group on the second component such as the nanocarrier.
  • a hydrazide linker is formed by the reaction of a hydrazine group on one component 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.
  • An imine or oxime linker is formed by the reaction of an amine or N-alkoxyamine (or aminooxy) group on one component with an aldehyde or ketone group on the second component such as the nanocarrier.
  • An urea or thiourea linker is prepared by the reaction of an amine group on one component with an isocyanate or thioisocyanate group on the second component such as the nanocarrier.
  • An amidine linker is prepared by the reaction of an amine group on one component with an imidoester group on the second component such as the nanocarrier.
  • An amine linker is made by the alkylation reaction of an amine group on one component with an alkylating group such as halide, epoxide, or sulfonate ester group on the second component such as the nanocarrier.
  • an amine linker can also be made by reductive amination of an amine group on one component 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.
  • a sulfonamide linker is made by the reaction of an amine group on one component with a sulfonyl halide (such as sulfonyl chloride) group on the second component such as the nanocarrier.
  • a sulfonyl halide such as sulfonyl chloride
  • 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 nanocarrier or attached to a component.
  • the component can also be conjugated to the nanocarrier via non-covalent conjugation methods.
  • a negative charged component can be conjugated to a positive charged nanocarrier through electrostatic adsorption.
  • a component containing a metal ligand can also be conjugated to a nanocarrier containing a metal complex via a metal- ligand complex.
  • the component 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.
  • the component may be prepared with a group which is compatible with the attachment chemistry that is presented by the synthetic nanocarriers' surface.
  • a component 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.
  • the linker can be a homobifuntional or heterobifunctional reagent as described in Hermanson 2008.
  • 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.
  • ADH adipic dihydrazide
  • the resulting ADH linked synthetic nanocarrier is then conjugated with a component containing an acid group via the other end of the ADH linker on NC to produce the corresponding VLP or liposome peptide conjugate.
  • a component containing an acid group via the other end of the ADH linker on NC to produce the corresponding VLP or liposome peptide conjugate.
  • the component 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.
  • a component such as an antigen or adjuvant
  • Isolated refers to the element being separated from its native environment and present in sufficient quantities to permit its identification or use. This means, for example, the element may be (i) selectively produced by expression cloning or (ii) purified as by chromatography or electrophoresis. Isolated elements may be, but need not be, substantially pure. Because an isolated element may be admixed with a pharmaceutically acceptable excipient in a pharmaceutical preparation, the element may comprise only a small percentage by weight of the preparation. The element 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 lipids or proteins. Any of the elements provided herein may be isolated. Any of the antigens provided herein can be included in the compositions in isolated form.
  • Synthetic nanocarriers may be prepared using a wide variety of methods known in the art.
  • synthetic nanocarriers can be formed by methods as nanoprecipitation, flow focusing using fluidic channels, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, milling, microemulsion procedures, microfabrication, nanofabrication, sacrificial layers, simple and complex coacervation, and other methods well known to those of ordinary skill in the art.
  • 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.
  • 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.
  • 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.
  • 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.
  • Elements of the synthetic nanocarriers 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 Al to Murthy et al.
  • synthetic nanocarriers can be coupled to elements directly or indirectly via non-covalent interactions.
  • 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 synthetic nanocarrier.
  • encapsulation and/or absorption is a form of coupling.
  • the synthetic nanocarriers can be combined with adjuvants by admixing in the same vehicle or delivery system.
  • 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® (AS04), MPL A of above-mentioned bacteria separately, saponins, such as QS-21,Quil-A, ISCOMs, ISCOMATRIXTM, emulsions such as MF59TM, Montanide® ISA 51 and ISA 720, AS02 (QS21+squalene+ MPL®) , liposomes and liposomal formulations such as AS01, synthesized or specifically prepared microparticles and microcarriers such as bacteria-derived outer membrane vesicles (OMV) of N.
  • Pluronic® 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.
  • the synthetic nanocarriers can be combined with an antigen different, similar or identical to those coupled to a nanocarrier (with or without adjuvant, utilizing or not utilizing another delivery vehicle) administered separately at a different time- point and/or at a different body location and/or by a different immunization route or with another antigen and/or adjuvant-carrying synthetic nanocarrier administered separately at a different time-point and/or at a different body location and/or by a different immunization route.
  • Synthetic nanocarriers may be combined to form pharmaceutical dosage forms according to the present invention using traditional pharmaceutical mixing methods. These include liquid-liquid mixing in which two or more suspensions, each containing one or more subsets of nanocarriers, are directly combined or are brought together via one or more vessels containing diluent. As synthetic nanocarriers may also be produced or stored in a powder form, dry powder-powder mixing could be performed as could the re- suspension of two or more powders in a common media. Depending on the properties of the nanocamers and their interaction potentials, there may be advantages conferred to one or another route of mixing.
  • 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., thoxy
  • carboxymethylcellulose carboxymethylcellulose
  • co-solvents e.g., glycerol, polyethylene glycol, ethanol
  • compositions according to the invention comprise 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.
  • synthetic nanocarriers are suspended in sterile saline solution for injection together with a preservative.
  • compositions of synthetic nanocarriers can be made in any suitable manner, and the invention is in no way limited to the use of compositions that can be produced using the methods described herein. Selection of an appropriate method may require attention to the properties of the particular elements being associated.
  • 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.
  • synthetic nanocarriers may be lyophilized and stored in suspension or as lyophilized powder depending on the formulation strategy for extended periods without losing activity.
  • compositions of the invention can be administered by a variety of routes, including or not limited to subcutaneous, intranasal, oral, intravenous, intraperitoneal, intramuscular, transmucosal, transmucosal, sublingual, rectal, ophthalmic, pulmonary, intradermal, transdermal, transcutaneous or intradermal or by a combination of these routes.
  • Routes of administration also include administration by inhalation or pulmonary aerosol. Techniques for preparing aerosol delivery systems are well known to those of skill in the art (see, for example, Sciarra and Cutie, "Aerosols," in Remington's Pharmaceutical Sciences, 18th edition, 1990, pp. 1694-1712; incorporated by reference).
  • Doses of dosage forms contain varying amounts of populations of synthetic nanocarriers and varying amounts of the proteins and/or adjuvants and/or additional antigens, according to the invention.
  • the amount of synthetic nanocarriers and/or proteins and/or adjuvants and/or additional antigens present in the dosage forms can be varied according to the nature of the elements present, the therapeutic benefit to be accomplished, and other such parameters.
  • dose ranging studies can be conducted to establish optimal therapeutic amount of the population of synthetic nanocarriers and the amount of proteins and/or adjuvants and/or additional antigens to be present in the dosage form.
  • the synthetic nanocarriers and the proteins and/or adjuvants and/or additional antigens are present in the dosage form in an amount effective to generate an immune response to the proteins and/or additional antigens upon administration to a subject. It may be possible to determine amounts effective to generate an immune response using
  • Dosage forms may be administered at a variety of frequencies.
  • administration of the dosage form is sufficient to generate a pharmacologically relevant response.
  • 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.
  • 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, non- autoimmune diseases, HIV, malaria, hepatitis B or any of the other disorders and/or conditions provided herein.
  • infectious disease examples 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, Diphth
  • Fasciolopsiasis Fasciolopsiasis, Filariasis, Free-living amebic infection, Giardiasis, Gnathostomiasis,
  • Hymenolepiasis Isosporiasis, Kala-azar, Leishmaniasis, Malaria, Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Pinworm Infection, Scabies, Schistosomiasis, Taeniasis, Toxocariasis, Toxoplasmosis, Trichinellosis, Trichinosis, Trichuriasis, Trichomoniasis and Trypanosomiasis; fungal infectious disease, such as Aspergillosis, Blastomycosis,
  • cancers include, but are not limited to breast cancer; biliary tract cancer; bladder cancer; brain cancer including glioblastomas and meduUoblastomas; 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 osteo
  • metabolic diseases include, but are not limited to, disorders of carbohydrate metabolism, amino acid metabolism, organic acid metabolism, fatty acid oxidation and mitochondrial metabolism, prophyrin metabolism, purine or pyrimidine metabolism, steroid metabolism, lysosomal mitochondrial function, peroxisomal function, lysosomal storage, urea cycle disorders (e.g., N-acetyl glutamate synthetase deficiency, carbamylphosphate synthase deficiency, ornithine carbamyl transferase deficiency, crgino succinic aciduria, citrullinaemia, arginase deficiency), amino acid disorders (e.g., Non- ketotic hyperglycinaemia, tyrosinaemia (Type I), Maple syrup urine disease), organic acidemias (e.g, isovaleric acidemia, methylmalonic acidemia, propionic acidemia, glutaric aciduria type I, glutaric acidemia type I & II),
  • mucopolysaccharoidosis II mucopolysaccharoidosis VI.
  • degenerative diseases include, but are not limited to,
  • mesenchyme/mesoderm degenerative disease e.g., mesenchyme/mesoderm degenerative disease, muscle degenerative disease, endothelial degenerative disease, neurodegenerative disease, degenerative joint disease (e.g., CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD, CAD
  • osteoarthritis a major type of degenerative heart disease (e.g., coronary heart disease, congenital heart disease, rheumatic heart disease, angina pectoris), neurodegenerative disease (e.g., Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, Lewy body disease, Parkinson's disease, spinal muscular atrophy), neuromuscular disorders (e.g., muscular dystrophy, duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, myotonic muscular dystrophy, congenital myopathy, familial cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, or coronary artery disease).
  • degenerative heart disease e.g., coronary heart disease, congenital heart disease, rheumatic heart disease, angina pectoris
  • neurodegenerative disease e.g., Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich's at
  • the proteins for coupling to the synthetic nanocarriers and/or the additional antigens provided herein can be antigens associated with any of the diseases or conditions provided herein. These include antigens associated with cancer, infections or infectious disease or degenerative or non-autoimmune disease. Antigens associated with HIV, malaria, leischmaniasis, a human filovirus infection, a togavirus infection, a alphavirus infection, an arenavirus infection, a bunyavirus infection, a flavivirus infection, a human papillomavirus infection, a human influenza A virus infection, a hepatitis B infection or a hepatitis C infection are also included.
  • cancer antigens examples include HER 2 (pi 85), CD20, CD33, GD3 ganglioside, GD2 ganglioside, carcinoembryonic antigen (CEA), CD22, milk mucin core protein, TAG- 72, Lewis A antigen, ovarian associated antigens such as OV-TL3 and MOvl8, high Mr melanoma antigens recognized by antibody 9.2.27, HMFG-2, SM-3, B72.3, PR5C5, PR4D2, and the like.
  • CEA carcinoembryonic antigen
  • CD22 milk mucin core protein
  • TAG- 72 Lewis A antigen
  • Lewis A antigen ovarian associated antigens
  • OV-TL3 and MOvl8 high Mr melanoma antigens recognized by antibody 9.2.27, HMFG-2, SM-3, B72.3, PR5C5, PR4D2, and the like.
  • MAGE MART-l/Melan-A, gplOO, Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), FAP, cyclophilin b, Colorectal associated antigen (CRC)— C017-1A/GA733, Carcinoembryonic Antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, amll, prostatic acid phosphatase (PAP), Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, pro state- specific membrane antigen (PSMA), T-cell receptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-I or MAGE-II families) (e.g., MAGE-A1, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A5, MAGE-A6,
  • antigens associated with infection or infectious disease are associated with any of the infectious agents provided herein.
  • the infectious agent is a virus of the Adenoviridae, Picornaviridae, Herpesviridae,
  • the infectious agent is 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,
  • the infectious agent is 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.
  • the infectious agent is 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,
  • the infectious agent is a fungus of the Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis or Stachybotrys genus. In still another embodiment, the infectious agent is C.
  • the antigen associated with infection or infectious disease is one that 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, gpl20, 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).
  • NS structural protein
  • the antigen comprises 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),
  • PT pertussis toxin
  • HP hypothetical protein
  • T toxin T toxin
  • Toxoid antigen capsular polysaccharide
  • Protein D capsular polysaccharide
  • Mip capsular polysaccharide
  • NP nucleoprotein
  • RD1 PE35
  • PPE68 EsxA
  • EsxB EsxB
  • RD9 EsxV
  • Hsp70 hypothetical protein
  • lipopolysaccharide surface antigen, Spl, Sp2, Sp3, Glycerophosphodiester
  • the antigen is one that comprises capsular glycoprotein, Yps3P, Hsp60, Major surface protein, MsgCl, MsgC3, MsgC8, MsgC9 or SchS34.
  • Ovalbumin protein was purchased from Worthington Biochemical Corporation (730
  • PLGA-R848 of approximately 5,200 Da made from PLGA of 3: 1 lactide to glycolide ratio and having 12.7% w/w conjugated R848 content was synthesized.
  • Polyvinyl alcohol (Mw 11,000 - 31,000, 87-89% hydrolyzed) was purchased from J.T. Baker (Part Number U232-08).
  • Solution 1 Ovalbumin protein at 20 mg/mL in lOmM phosphate buffer.
  • Solution 2 PLGA-R848 at 50 mg/mL, PLA-PEG-Nicotine at 25 mg/mL, PLA at 25 mg/ml in dichloromethane.
  • the solution was prepared by separately dissolving each polymer as a 100 mg/mL in dichloromethane, then mixing the solutions by adding 2 parts PLGA- R848 solution to 1 part each PLA-PEG-Nicotine solution and PLA solution.
  • Solution 3 Polyvinyl alcohol @ 50 mg/mL in 100 mM in lOOmM phosphate buffer, pH 8.
  • Solution 4 70 mM phosphate buffer, pH 8.
  • a primary (Wl/O) emulsion was first created using Solution 1 & Solution 2.
  • Solution 1 (0.2 mL) and Solution 2 (1.0 mL) were combined in a small glass pressure tube and sonicated at 50% amplitude for 40 seconds using a Branson Digital Sonifier 250.
  • a secondary (W1/0/W2) emulsion was then formed by adding Solution 3 (2.0 mL) to the primary emulsion, and then sonicating at 30% amplitude for 40 seconds using the Branson Digital Sonifier 250.
  • the secondary emulsion was 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 13,823g for one hour, removing the supernatant, and re-suspending the pellet in phosphate buffered saline. This washing procedure was repeated and then the pellet was re-suspended in phosphate buffered saline to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. The suspension was stored frozen at -20°C until use.
  • Ovalbumin protein was purchased from Worthington Biochemical Corporation (730 Vassar Avenue, Lakewood, NJ 08701. Product Code 3048.) Ovalbumin peptide 323-339 amide acetate salt, was purchased from Bachem Americas Inc. (3132 Kashiwa Street, Torrance CA 90505. Product code 4065609.) PLGA-R848 of approximately 5,200 Da made from PLGA of 3: 1 lactide to glycolide ratio and having 12.7% w/w conjugated R848 content was synthesized. PLA-PEG-Nicotine with a nicotine-terminated PEG block of
  • Solution 1A Ovalbumin protein at 40 mg/mL in lOmM phosphate buffer.
  • Solution IB Ovalbumin peptide amide 323 - 339 @ 40 mg/mL in dilute hydrochloric acid aqueous solution.
  • Solution 2 PLGA-R848 at 50 mg/mL, PLA-PEG-Nicotine at 25 mg/mL, PLA at 25 mg/ml in dichloromethane.
  • the solution was prepared by separately dissolving each polymer at 100 mg/mL in dichloromethane, then mixing the solutions by adding 2 parts PLGA-R848 solution to 1 part each PLA-PEG-Nicotine solution and PLA solution.
  • Solution 3 Polyvinyl alcohol @ 50 mg/mL in 100 mM in lOOmM phosphate buffer, pH 8.
  • Solution 4 70 mM phosphate buffer, pH 8.
  • the first primary (Wl/O) emulsion was created using Solution 1A & Solution 2.
  • Solution 1A (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 second primary (Wl/O) emulsion was created using Solution IB & Solution 2.
  • Solution IB (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.
  • 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 13,823g for one hour, removing the supernatant, and re-suspending the pellet in phosphate buffered saline. This washing procedure was repeated and then the pellet was re- suspended in phosphate buffered saline to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. The suspension was stored frozen at -20°C until use. Table 2: Nanocarrier Characterization
  • Solution 1 Ovalbumin protein at 20 mg/mL in lOmM phosphate buffer.
  • Solution 2 PLGA-R848 at 50 mg/mL, PLA-PEG-OMe at 25 mg/mL, PLA at 25 mg/ml in dichloromethane.
  • the solution was prepared by separately dissolving each polymer as a 100 mg/mL in dichloromethane, then mixing the solutions by adding 2 parts PLGA- R848 solution to 1 part each PLA-PEG-OMe solution and PLA solution.
  • Solution 3 Polyvinyl alcohol @ 50 mg/mL in 100 mM in lOOmM phosphate buffer, pH 8.
  • Solution 4 70 mM phosphate buffer, pH 8.
  • a primary (Wl/O) emulsion was first created using Solution 1 & Solution 2.
  • Solution 1 (0.2 mL) and Solution 2 (1.0 mL) were combined in a small glass pressure tube and sonicated at 50% amplitude for 40 seconds using a Branson Digital Sonifier 250.
  • a secondary (W1/0/W2) emulsion was then formed by adding Solution 3 (2.0 mL) to the primary emulsion, and then sonicating at 30% amplitude for 40 seconds using the Branson Digital Sonifier 250.
  • the secondary emulsion was 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 13,823g for one hour, removing the supernatant, and re-suspending the pellet in phosphate buffered saline. This washing procedure was repeated and then the pellet was re-suspended in phosphate buffered saline to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. The suspension was stored frozen at -20°C until use.
  • Ovalbumin protein was purchased from Worthington Biochemical Corporation (730 Vassar Avenue, Lakewood, NJ 08701. Product Code 3048.) Ovalbumin peptide 323-339 amide acetate salt, was purchased from Bachem Americas Inc. (3132 Kashiwa Street,
  • Product code 4065609. PLGA-R848 of approximately 5,200 Da made from PLGA of 3: 1 lactide to glycolide ratio and having 12.7% w/w conjugated R848 content was synthesized.
  • PLA with an inherent viscosity of 0.21 dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211.
  • Polyvinyl alcohol (Mw 11,000 - 31,000, 87-89% hydrolyzed) was purchased from J.T. Baker (Part Number U232-08).
  • Solution 1A Ovalbumin protein at 40 mg/mL in lOmM phosphate buffer.
  • Solution IB Ovalbumin peptide amide 323 - 339 @ 40 mg/mL in dilute hydrochloric acid aqueous solution.
  • Solution 2 PLGA-R848 at 50 mg/mL, PLA-PEG-OMe at 25 mg/mL, PLA at 25 mg/ml in dichloromethane.
  • the solution was prepared by separately dissolving each polymer as a 100 mg/mL in dichloromethane, then mixing the solutions by adding 2 parts PLGA- R848 solution to 1 part each PLA-PEG-OMe solution and PLA solution.
  • Solution 3 Polyvinyl alcohol @ 50 mg/mL in 100 mM in lOOmM phosphate buffer, pH 8.
  • Solution 4 70 mM phosphate buffer, pH 8.
  • the first primary (Wl/O) emulsion was created using Solution 1A & Solution 2.
  • Solution 1A (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 second primary (Wl/O) emulsion was created using Solution IB & Solution 2.
  • Solution IB (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.
  • the two primary emulsions were combined, in part, by transferring 0.6 mL of each into a third glass pressure tube to create a 1 : 1 mixture of the two primary emulsions in a total volume of approximately 1.2 mL.
  • a secondary (W1/0/W2) emulsion was then formed by adding Solution 3 (2.0 mL) to the primary emulsion, and then sonicating at 30% amplitude for 40 seconds using the Branson Digital Sonifier 250.
  • the secondary emulsion was 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 13,823g for one hour, removing the supernatant, and re-suspending the pellet in phosphate buffered saline. This washing procedure was repeated and then the pellet was re-suspended in phosphate buffered saline to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. The suspension was stored frozen at -20°C until use.
  • SIINFEKL (SEQ ID NO: l) (ovalbumin peptide [257 - 264]), was purchased from Bachem Americas Inc. (3132 Kashiwa Street, Torrance CA 90505. Product code H-4866.) Ovalbumin peptide 323-339 amide acetate salt, was purchased from Bachem Americas Inc. (3132 Kashiwa Street, Torrance CA 90505. Product code 4065609.) PLGA-R848 of approximately 4,500 Da made from PLGA of 3: 1 lactide to glycolide ratio and having 15% w/w conjugated R848 content was synthesized.
  • PLA-PEG-Nicotine with a nicotine- terminated PEG block of approximately 5,000 Da and DL-PLA block of approximately 17,000 Da was synthesized.
  • PLA with an inherent viscosity of 0.21 dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211. Product Code 100 DL 2A.)
  • Solution 1A SIINFEKL (SEQ ID NO: l) @ 200 mg/mL DMSO.
  • Solution IB Ovalbumin peptide amide 323 - 339 @ 20 mg/mL in dilute hydrochloric acid aqueous solution.
  • Solution 2 PLGA-R848 at 50 mg/mL, PLA-PEG-Nicotine at 25 mg/mL, PLA at 25 mg/ml in dichloromethane.
  • the solution was prepared by separately dissolving each polymer as a 100 mg/mL in dichloromethane, then mixing the solutions by adding 2 parts PLGA- R848 solution to 1 part each PLA-PEG-Nicotine solution and PLA solution.
  • Solution 3 Polyvinyl alcohol @ 50 mg/mL in 100 mM in lOOmM phosphate buffer, pH 8.
  • Solution 4 70 mM phosphate buffer, pH 8.
  • the first primary (S/O) emulsion was created using Solution 1A & Solution 2.
  • Solution 1A (0.025 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 serial primary (Wl/(S/0)) emulsion was created using Solution IB and the first primary emulsion.
  • Solution IB (0.25 mL) was added to the small glass pressure tube containing the first primary emulsion and then sonicated at 50% amplitude for 40 seconds using a Branson Digital Sonifier 250.
  • a secondary ((S+Wl)/0/W2) emulsion was then formed by adding Solution 3 (2.0 mL) to the serial primary emulsion, and then sonicating at 30% amplitude for 40 seconds using the Branson Digital Sonifier 250.
  • the secondary emulsion was added to an open 50 mL beaker containing 70 mM phosphate buffer solution (30 mL) and stirred at room temperature for 2 hours to allow the dichloromethane to evaporate and the nanocarriers to form in suspension.
  • a portion of the suspended nanocarriers was washed by transferring the nanocarrier suspension to a centrifuge tube, spinning at 13,823g for one hour, removing the supernatant, and re-suspending the pellet in phosphate buffered saline. This washing procedure was repeated and then the pellet was re-suspended in phosphate buffered saline to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. The suspension was stored frozen at -20°C until use.
  • Example 6 Synthetic Nanocarrier Compositions Generate High Antibody Titers and Strong Antigen- Specific CTL Activity
  • mice were bled at dates indicated and antibodies to ovalbumin measured in standard ELISA using serial dilutions of test sera.
  • Biotinylated goat anti-mouse Ig was used as a detection antibody (BD Biosciences, San Diego, CA).
  • EC50 was determined based on titration curves.
  • CTL activity was measured as follows. 4-5 days after the final injection (subcutaneous, s.c, or intranasal, i.n.) with the nanocarrier preparations or protein controls draining lymph nodes (LNs) were removed, treated with collagenase, homogenized, washed and incubated with 10-100 units/ml of IL-2 for 4-5 days.
  • LNs draining lymph nodes
  • Syngeneic EL-4 cells pulsed with SIINFEKL (SEQ ID NO: l) peptide or EG.7-OVA cells (stably transfected with ovalbumin) served as targets with intact EL-4 cells providing for background control.
  • Cytoxicity at various effectontarget ratios was measured over 24 hours (37°C) using CytoTox-ONETM Homogenuous Membrane Integrity Assay (Promega, Madison, WI) according to manufacturer's recommendations.
  • Ovalbumin protein was purchased from Worthington Biochemical Corporation (730 Vassar Avenue, Lakewood, NJ 08701). Product Code LS003054.
  • PLA with an inherent viscosity of 0.21 dL/g was purchased from SurModics
  • Phosphate-buffered saline IX (PBS IX). From Mediatech Inc. (9345 Discovery Boulevard. Manassas, VA 20109.) Product Code 21- 040-CV.
  • Solution 1 Ovalbumin protein @ 20 mg/mL was prepared in PBS IX at room temperature.
  • Solution 2 PLA was prepared by dissolving PLA at 100 mg per 1 mL of
  • PLA-PEG-OMe was prepared by dissolving PLA-PEG-OMe at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
  • PLGA-R848 was prepared by dissolving PLGA-R848 at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
  • Solution 5 Polyvinyl alcohol @ 50 mg/mL in 100 mM in lOOmM phosphate buffer, pH 8.
  • Solution 6 70 mM phosphate buffer, pH 8.
  • a primary (Wl/O) emulsion was first created by mixing Solutions 1 through 4.
  • Solution 1 (0.2 mL), Solution 2 (0.50 mL), Solution 3 (0.25mL) and Solution 4 (0.25mL) were combined in a small glass pressure tube and sonicated at 50% amplitude for 40 seconds using a Branson Digital Sonifier 250.
  • a secondary (W1/0/W2) emulsion was then formed by adding Solution 5 (2.0 mL) to the primary emulsion, vortexing to create a crude dispersion, and then sonicating at 30% amplitude for 60 seconds using the Branson Digital Sonifier 250.
  • the secondary emulsion was added to an open 50 mL beaker containing Solution 6 (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 PBS IX to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. The suspension was stored frozen at -20C until use.
  • Ovalbumin protein was purchased from Worthington Biochemical Corporation (730 Vassar Avenue, Lakewood, NJ 08701). Product Code LS003054.
  • PLA with an inherent viscosity of 0.21 dL/g was purchased from SurModics
  • Phosphate-buffered saline IX (PBS IX). From Mediatech Inc. (9345 Discovery Boulevard. Manassas, VA 20109.) Product Code 21- 040-CV.
  • Solution 1 Ovalbumin protein @ 5 mg/mL was prepared in PBS IX at room temperature.
  • Solution 2 PLA was prepared by dissolving PLA at 100 mg per 1 mL of
  • PLA-PEG-OMe was prepared by dissolving PLA-PEG-OMe at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
  • PLGA-R848 was prepared by dissolving PLGA-R848 at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
  • Solution 5 Polyvinyl alcohol @ 50 mg/mL in 100 mM in lOOmM phosphate buffer, pH 8.
  • Solution 6 70 mM phosphate buffer, pH 8.
  • a primary (Wl/O) emulsion was first created by mixing Solutions 1 through 4.
  • Solution 1 (0.2 mL), Solution 2 (0.50 mL), Solution 3 (0.25mL) and Solution 4 (0.25mL) were combined in a small glass pressure tube and sonicated at 50% amplitude for 40 seconds using a Branson Digital Sonifier 250.
  • a secondary (W1/0/W2) emulsion was then formed by adding Solution 5 (2.0 mL) to the primary emulsion, vortexing to create a crude dispersion, and then sonicating at 30% amplitude for 60 seconds using the Branson Digital Sonifier 250.
  • the secondary emulsion was added to an open 50 mL beaker containing Solution 6 (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 PBS IX to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. The suspension was stored frozen at -20C until use.
  • Table 10 Nanocarrier Characterization
  • Ovalbumin protein was purchased from Worthington Biochemical Corporation (730 Vassar Avenue, Lakewood, NJ 08701). Product Code LS003054.
  • EMPROVE® Polyvinyl Alcohol 5-88, USP (85-89% hydrolyzed, viscosity of 4.3-5.7 mPa.s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027. Part Number 1.41354). Phosphate-buffered saline IX (PBS IX). From Mediatech Inc. (9345 Discovery Boulevard. Manassas, VA 20109.) Product Code 21 -040-CV.
  • Solution 1 Ovalbumin protein @ 20 mg/mL was prepared in PBS IX at room temperature.
  • Solution 2 PLA was prepared by dissolving PLA at 100 mg per 1 mL of
  • Solution 3 PLA-PEG-OMe was prepared by dissolving PLA-PEG-OMe at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
  • Solution 4 PLGA-R848 was prepared by dissolving PLGA-R848 at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
  • Solution 5 Polyvinyl alcohol @ 50 mg/mL in 100 mM in lOOmM phosphate buffer, pH 8.
  • Solution 6 70 mM phosphate buffer, pH 8.
  • a primary (Wl/O) emulsion was first created by mixing Solutions 1 through 4.
  • Solution 1 (0.2 mL), Solution 2 (0.50 mL), Solution 3 (0.25mL) and Solution 4 (0.25mL) were combined in a small glass pressure tube and sonicated at 50% amplitude for 40 seconds using a Branson Digital Sonifier 250.
  • a secondary (W1/0/W2) emulsion was then formed by adding Solution 5 (2.0 mL) to the primary emulsion, vortexing to create a crude dispersion, and then sonicating at 30% amplitude for 60 seconds using the Branson Digital Sonifier 250.
  • the secondary emulsion was added to an open 50 mL beaker containing Solution 6 (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 PBS IX to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. The suspension was stored frozen at -20C until use.
  • Example 10 Synthetic Nanocarrier Compositions Generate High Antibody Titers and Strong Antigen- Specific CTL Activity
  • Synthetic nanocarriers delivering R848 and OVA was as successful as a positive comparator control consisting of high dose of PS-CpG plus a 6x higher dose of free OVA at generating a central (spleen) OVA-specific CTL response and also in creating as strong (or stronger) OVA-specific humoral response.
  • 4-5 days after s.c. injection with nanocarrier preparations or controls draining lymph nodes (LNs) were removed, treated with collagenase, homogenized, washed and incubated with 10-100 units/ml of IL-2 for 4-5 days. Then resulting cell populations were counted and used as effector cells in cytotoxicity assay.
  • Nanocarrier-encapsulated Antigen After a Single Injection of NC-OVA + NC-R848 Mix
  • PLGA-R848 (S-205), Poly-D/L-lactide-co-glycolide, 4-amino-2-(ethoxymethyl)-a,a- dimethyl-lH-imidazo[4,5-c]quinoline-l-ethanol amide of approximately 7,800 Da made from PLGA of 3: 1 lactide to glycolide ratio and having 8.5% w/w conjugated resiquimod content was custom manufactured at Princeton Global Synthesis (300 George Patterson Drive #206, Bristol, PA 19007.). PLA with an inherent viscosity of 0.19 dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211. Product Code 100 DL 2A).
  • PLA-PEG-OMe block co-polymer with a methyl ether terminated PEG block of approximately 5,000 Da and PLA block of approximately 28,000 Da was purchased from SurModics Pharmaceuticals (Product Code 100 DL mPEG 5000 5CE).
  • EMPROVE® Polyvinyl Alcohol 4-88, USP (85-89% hydrolyzed, viscosity of 3.4-4.6 mPa.s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027).
  • Phosphate- buffered saline IX (PBS IX). From Mediatech Inc. (9345 Discovery Boulevard. Manassas, VA 20109.) Product Code 21 -040-CV. Method - Lot #9
  • Solution 1 PLGA-R848 was prepared by weighing out PLGA-R848, PLA, and PLA- PEG-OMe powders in 2: 1: 1 weight ratio and then dissolving the mixed polymers in dichloromethane to achieve a total polymer concentration of 100 mg per 1 mL.
  • Solution 2 Polyvinyl alcohol @ 35 mg/mL in lOOmM phosphate buffer, pH 8.
  • An O/W emulsion was created by mixing Solutions 1 and 2, and then creating a coarse emulsion prior to a fine emulsion.
  • Solution 1 (2 mL) was coarsely emulsified with Solution 2 (8 mL), using 10 passes through an 18G emulsification needle.
  • a fine emulsion was made by loading the coarse emulsion into a primed and ice-water-chilled high pressure homogenizer (Microfluidics LV1) and performing three passes at 5000 psi.
  • the fine O/W emulsion was added to an open 100 mL beaker containing IX PBS (60 mL) and stirred at room temperature for more than 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 75,600 rcf for 35 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 PBS IX to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis.
  • the nanocarrier formation process was repeated another three times at lx or 2x the same scale.
  • the four suspensions were combined and then filtered through 0.22 micron PES syringe filter and then stored frozen at -20C until use.
  • PLGA-R848 (S-205), Poly-D/L-lactide-co-glycolide, 4-amino-2-(ethoxymethyl)-a,a- dimethyl-lH-imidazo[4,5-c]quinoline-l-ethanol amide of approximately 7,800 Da made from PLGA of 3: 1 lactide to glycolide ratio and having 8.5% w/w conjugated resiquimod content was custom manufactured at Princeton Global Synthesis (300 George Patterson Drive #206, Bristol, PA 19007.). PLA with an inherent viscosity of 0.19 dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211. Product Code 100 DL 2A).
  • PLA-PEG-OMe block co-polymer with a methyl ether terminated PEG block of approximately 5,000 Da and PLA block of approximately 28,000 Da was purchased from SurModics Pharmaceuticals (Product Code 100 DL mPEG 5000 5CE).
  • EMPROVE® Polyvinyl Alcohol 4-88, USP (85-89% hydrolyzed, viscosity of 3.4-4.6 mPa.s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027).
  • Phosphate- buffered saline IX (PBS IX). From Mediatech Inc. (9345 Discovery Boulevard. Manassas, VA 20109.) Product Code 21 -040-CV.
  • PLGA-R848 was prepared by dissolving PLGA-R848 at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
  • Solution 2 PLA was prepared by dissolving PLA at 100 mg per 1 mL of
  • PLA-PEG-OMe was prepared by dissolving PLA-PEG-OMe at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
  • Solution 4 Polyvinyl alcohol @ 50 mg/mL in lOOmM phosphate buffer, pH 8.
  • a primary (O/W) emulsion was created by mixing Solutions 1 through 4 and creating a coarse emulsion prior to a fine emulsion.
  • Solution 1 (1 mL), Solution 2 (0.5 mL), and Solution 3 (0.5mL) were combined first and then coarsely emulsified with Solution 4 (8 mL), by stirring together at 350 rpm in a 50 mL beaker for two minutes and by repeat pipetting.
  • a fine emulsion was made by loading the coarse emulsion into a primed high pressure homogenizer (Microfluidics LV1) and performing three passes at 5000 psi.
  • a primed high pressure homogenizer Microfluidics LV1
  • the fine O/W emulsion was added to an open 50 mL beaker containing IX PBS (30 mL) and stirred at room temperature for more than 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 75,600 rcf for 35 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 PBS IX to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. Nanocarrier suspension was then filtered through a 0.22 micron PES syringe filter and then stored frozen at -20C until use. Materials - Lot #11
  • Ovalbumin protein was purchased from Worthington Biochemical Corporation (730 Vassar Avenue, Lakewood, NJ 08701). Product Code LS003054. PLGA with 75% lactide and 25% glycolide content and an inherent viscosity of 0.24 dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211. Product Code 7525 DLG 2.5A). PLA with an inherent viscosity of 0.2 dL/g was purchased from
  • Solution 1 Ovalbumin protein @ 50 mg/mL was prepared in PBS IX at room temperature.
  • Solution 2 PLGA, PLA, and PLA-PEG-OMe were weighed out in 2: 1: 1 weight ratio and dissolved in dichloromethane in the chemical fume hood to achieve a final total polymer concentration of 100 mg per 1 mL.
  • Solution 3 Polyvinyl alcohol @ 50 mg/mL in lOOmM phosphate buffer, pH 8.
  • Solution 4 70 mM phosphate buffer, pH 8.
  • a primary (Wl/O) emulsion was first created by mixing Solutions 1 and 2.
  • Solution 1 (66 mL) and Solution 2 (264 mL) were combined and formed into a coarse emulsion using an overhead mixer in a 1 L beaker.
  • the coarse emulsion was transferred to a custom-made temperature-controlled homogenization vessel and homogenized using a high- shear rotor stator.
  • a coarse secondary (W1/0/W2) emulsion was then formed by transferring 1 ⁇ 2 of the primary emulsion into a beaker containing 330 mL of Solution 3 and mixing with an overhead mixer.
  • the coarse secondary emulsion was then returned to a custom
  • mice C57BL/6 female mice (2-3/group) were immunized once by a mix of 100 ⁇ g NC- OVA and 100 ⁇ g of NC-R848 containing 4.3 ⁇ g R848 and 6.2 ⁇ g of OVA (per each mouse).
  • OVA antibody
  • mice were bled and their antibody (IgG) titer against OVA determined by ELISA.
  • mice were injected (i.v.) by syngeneic splenocytes pulsed by a peptide representing a dominant CTL epitope of OVA (SIINFEKL (SEQ ID NO: l)) and differentially labeled by CSFE.
  • NC-OVA + NC-R848 leads to rapid induction of cellular and humoral immune responses with the former being detected as early as four days after injection and then persisting for at least ten days with a peak at day 7, and the latter being detected at seven days after injection and reaching a significant level at 10 days after inoculation.
  • Example 12 Synthetic Nanocarriers Delivering CpG and OVA Materials - Lot #12
  • PO-1826 DNA oligonucleotide with phosphodiester backbone having nucleotide sequence 5'-TCC ATG ACG TTC CTG ACG TT-3' (SEQ ID NO:2) with a sodium counter- ion was purchased from Oligo Factory (120 Jeffrey Avenue, Holliston, MA 01746.)
  • PLGA having 54% lactide and 46% glycolide content and an inherent viscosity of 0.24 dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211. Product Code 5050 DLG 2.5 A).
  • PLGA-PEG-OMe block co-polymer with a methyl ether terminated PEG block of approximately 2,000 Da and 75% lactide/25% glycolide PLGA block of approximately 88,000 Da was purchased from SurModics Pharmaceuticals (Product Code 7525 DLG PEG 2000 7E-P).
  • EMPROVE® Polyvinyl Alcohol 4-88, USP (85- 89% hydrolyzed, viscosity of 3.4-4.6 mPa.s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027).
  • Phosphate-buffered saline IX PBS IX
  • From Mediatech Inc. (9345 Discovery Blvd. Manassas, VA 20109.) Product Code 21-040- CV.
  • Solution 1 PO-1826 was prepared by dissolving at 40 mg per 1 mL of an aqueous solution containing 250 mg Na cholate per 1 mL endotoxin-free water.
  • Solution 2 PLGA was prepared by dissolving PLA at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
  • Solution 3 PLGA-PEG-OMe was prepared by dissolving PLGA-PEG-OMe at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
  • Solution 4 Polyvinyl alcohol @ 50 mg/mL in lOOmM phosphate buffer, pH 8.
  • Solution 5 70 mM phosphate buffer, pH 8.
  • a primary (W/O) emulsion was created by mixing Solutions 1 through 3 and creating a coarse emulsion prior to a fine emulsion.
  • Solution 1 (0.2 mL), Solution 2 (0.5 mL), and Solution 3 (0.5mL) were combined in a small glass pressure tube, coarsely emulsified by repeat pipetting, and sonicated at 50% amplitude for 40 seconds over an ice bath using a Branson Digital Sonifier 250.
  • a secondary (W1/0/W2) emulsion was then formed by adding Solution 4 (3.0 mL) to the primary emulsion, vortexing to create a crude dispersion, and then sonicating at 30% amplitude for 60 seconds over an ice bath using the Branson Digital Sonifier 250.
  • the fine W1/0/W2 emulsion was added to an open 50 mL beaker containing 70 mM phosphate buffer (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 90 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 PBS IX to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. Nanocarrier suspension was then filtered through 0.22 micron PES syringe filters, stored refrigerated until the concentration was determined, and then concentration adjusted and stored frozen at -20C until use. Materials - Lot #13
  • Ovalbumin protein was purchased from Worthington Biochemical Corporation (730 Vassar Avenue, Lakewood, NJ 08701). Product Code LS003054. PLGA with 75% lactide and 25% glycolide content and an inherent viscosity of 0.2 dL/g was purchased from
  • Phosphate-buffered saline IX (PBS IX). From Mediatech Inc. (9345 Discovery Boulevard. Manassas, VA 20109.) Product Code 21-040-CV.
  • Solution 1 Ovalbumin protein @ 50 mg/mL was prepared in PBS IX at room temperature.
  • Solution 2 PLGA was prepared by dissolving PLGA at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
  • PLA-PEG-OMe was prepared by dissolving PLA-PEG-OMe at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
  • Solution 4 Polyvinyl alcohol @ 50 mg/mL in lOOmM phosphate buffer, pH 8.
  • Solution 5 70 mM phosphate buffer, pH 8.
  • a primary (Wl/O) emulsion was first created by mixing Solutions 1 through 3.
  • Solution 1 (0.2 mL), Solution 2 (0.75 mL), and Solution 3 (0.25mL) were combined in a small glass pressure tube and sonicated at 50% amplitude for 40 seconds over an ice bath using a Branson Digital Sonifier 250.
  • a secondary (W1/0/W2) emulsion was then formed by adding Solution 4 (3.0 mL) to the primary emulsion, vortexing to create a crude dispersion, and then sonicating at 30% amplitude for 60 seconds over an ice bath using the Branson Digital Sonifier 250.
  • the secondary emulsion was added to an open 50 mL beaker containing Solution 5 (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 130 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 PBS IX to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. The suspension was stored frozen at -20C until use.
  • Ovalbumin protein was purchased from Worthington Biochemical Corporation (730 Vassar Avenue, Lakewood, NJ 08701. Product Code LS003054).
  • PLGA with 76% lactide and 24% glycolide content and an inherent viscosity of 0.69 dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211. Product Code 7525 DLG 7A).
  • PLA with an inherent viscosity of 0.22 dL/g was purchased from SurModics Pharmaceuticals (Product Code 100 DL 2A).
  • PLA-PEG-OMe block co-polymer with a methyl ether terminated PEG block of approximately 5,000 Da and PLA block of
  • Solution 1 Ovalbumin protein @ 50 mg/mL was prepared in PBS IX at room temperature.
  • Solution 2 PLGA was prepared by dissolving PLGA at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
  • Solution 3 PLA was prepared by dissolving PLGA at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
  • PLA-PEG-OMe was prepared by dissolving PLA-PEG-OMe at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
  • Solution 5 Polyvinyl alcohol @ 50 mg/mL in lOOmM phosphate buffer, pH 8.
  • Solution 6 70 mM phosphate buffer, pH 8.
  • a primary (Wl/O) emulsion was first created by mixing Solutions 1 through 4.
  • Solution 1 (0.2 mL), Solution 2 (0.5 mL), Solution 3 (0.25mL) and Solution 4 (0.25mL) were combined in a small glass pressure tube and sonicated at 50% amplitude for 40 seconds over an ice bath using a Branson Digital Sonifier 250.
  • a secondary (W1/0/W2) emulsion was then formed by adding Solution 5 (3.0 mL) to the primary emulsion, vortexing to create a crude dispersion, and then sonicating at 30% amplitude for 60 seconds over an ice bath using the Branson Digital Sonifier 250.
  • the secondary emulsion was added to an open 50 mL beaker containing Solution 6 (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 25,600 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 PBS IX to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. The suspension was stored frozen at -20C until use. Materials - Lot #15
  • PO-1826 DNA oligonucleotide with phosphodiester backbone having nucleotide sequence 5'-TCC ATG ACG TTC CTG ACG TT-3' (SEQ ID NO:2) with a sodium counter- ion was purchased from Oligo Factory (120 Jeffrey Avenue, Holliston, MA 01746.)
  • PLGA having 54% lactide and 46% glycolide content and an inherent viscosity of 0.24 dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL
  • Solution 1 PO-1826 was prepared by dissolving at 40 mg per 1 mL of an aqueous solution containing 150 mg KC1 per 1 mL of endotoxin-free water.
  • Solution 2 Na Cholate was prepared by dissolving dry powder at 200 mg per 1 mL IX PBS at room temperature.
  • Solution 3 PLGA was prepared by dissolving PLGA at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
  • PLGA-PEG-OMe was prepared by dissolving PLGA-PEG-OMe at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
  • Solution 5 Polyvinyl alcohol @ 50 mg/mL in lOOmM phosphate buffer, pH 8.
  • Solution 6 70 mM phosphate buffer, pH 8.
  • a primary (W/O) emulsion was created by mixing Solutions 1 through 4 and creating a coarse emulsion prior to a fine emulsion.
  • Solution 1 (0.25 mL), Solution 2 (0.25 mL), Solution 3 (0.5 mL), and Solution 4 (0.5mL) were combined in a small glass pressure tube, coarsely emulsified by repeat pipetting, and sonicated at 50% amplitude for 40 seconds over an ice bath using a Branson Digital Sonifier 250.
  • a secondary (W1/0/W2) emulsion was then formed by adding Solution 5 (3.0 mL) to the primary emulsion, vortexing to create a coarse dispersion, and then sonicating at 30% amplitude for 60 seconds over an ice bath using the Branson Digital Sonifier 250.
  • the fine W1/0/W2 emulsion was added to an open 50 mL beaker containing 70 mM phosphate buffer (30 mL) and stirred at room temperature for 2 hours to allow the dichloromethane to evaporate and the nanocarriers to form in suspension.
  • Nanocarrier suspension was then stored refrigerated until the concentration was determined, and then concentration adjusted and stored frozen at -20C until use.
  • PO-1826 DNA oligonucleotide with phosphodiester backbone having nucleotide sequence 5'-TCC ATG ACG TTC CTG ACG TT-3' (SEQ ID NO:2) with a sodium counter- ion was purchased from Oligo Factory (120 Jeffrey Avenue, Holliston, MA 01746.)
  • PLGA having 54% lactide and 46% glycolide content and an inherent viscosity of 0.24 dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211. Product Code 5050 DLG 2.5A).
  • PBS IX Phosphate-buffered saline IX
  • Solution 1 PO-1826 was prepared by dissolving at 40 mg per 1 mL of an aqueous solution containing 150 mg KC1 per 1 mL of endotoxin-free water.
  • Solution 2 Polyvinyl alcohol @ 100 mg/mL in lOOmM phosphate buffer, pH 8.
  • Solution 3 PLGA was prepared by dissolving PLGA at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
  • PLGA-PEG-OMe was prepared by dissolving PLGA-PEG-OMe at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
  • Solution 5 Polyvinyl alcohol @ 100 mg/mL in lOOmM phosphate buffer, pH 8.
  • Solution 6 70 mM phosphate buffer, pH 8.
  • a primary (W/O) emulsion was created by mixing Solutions 1 through 4 and creating a coarse emulsion prior to a fine emulsion.
  • Solution 1 (0.25 mL), Solution 2 (0.25 mL), Solution 3 (0.5 mL), and Solution 4 (0.5mL) were combined in a small glass pressure tube, coarsely emulsified by repeat pipetting, and sonicated at 50% amplitude for 40 seconds over an ice bath using a Branson Digital Sonifier 250.
  • a secondary (W1/0/W2) emulsion was then formed by adding Solution 5 (3.0 mL) to the primary emulsion, vortexing to create a coarse dispersion, and then sonicating at 30% amplitude for 60 seconds over an ice bath using the Branson Digital Sonifier 250.
  • the fine W1/0/W2 emulsion was added to an open 50 mL beaker containing 70 mM phosphate buffer (30 mL) and stirred at room temperature for 2 hours to allow the dichloromethane to evaporate and the nanocarriers to form in suspension.
  • Nanocarrier suspension was then stored refrigerated until the concentration was determined, and then concentration adjusted and stored frozen at -20C until use.
  • Synthetic nanocarriers delivering CpG and OVA were superior in rapid antibody induction capacity than free high-dose CpG and OVA (both free CpG and free OVA used in 5x higher dose) and as successful or superior in induction of local and systemic antigen- specific CTLs as the same high dose of free CpG and free OVA.
  • Groups of three animals (C57BL/6 mice, females) were immunized (prime-boost, days 0 and 10; hind limb, s.c.) by three combinations of nanocarrier-incorporated CpG (ODN 1826) and OVA in parallel with immunization with 5-fold excess of free CpG and OVA.
  • Three different formulations of NC-CpG were used with the same formulation of NC-OVA (see Table 13 for details).
  • At 4 days after the second immunization animals were sacrificed and their serum, draining (popliteal) lymph nodes (LNs) and spleens were taken.
  • MHC Class-I-restricted immunodominant OVA-derived peptide SIINFEKL SEQ ID NO: l. Then differential cell populations were analyzed by FACS with those cells exhibiting CD8 expression (CD8 + ), no CD 19 expression (CD 19 " ) and bound to MHC Class I-complexed SIINFEKL (SEQ ID NO: l) (SIINFEKL + (SEQ ID NO: l)) considered representing a major species of OVA-specific CTLs.
  • At least one of NC-CpG formulations used has equal or higher capacity to produce short-term CTLs locally and systemically when coupled with NC- OVA than 5-fold higher amounts of free CpG and OVA.
  • different NC-CpG formulations have been especially beneficial for induction either of local or of systemic CTL response against nanocarrier-incorporated ovalbumin antigen.
  • purified splenocytes from immunized animals were also expanded in vitro (100 u/mL IL-2) by being stimulated with mitomycin-treated EG.7-OVA cells
  • NC-CpG single-cells stably transfected with ovalbumin
  • OVA-specific CD8 + cells At 11 days of in vitro incubation expanded cultures were labeled as described above and analyzed by FACS. All NC-CpG formulations tested resulted in induction of Ag-specific CTLs with a higher expansion potential than those induced by 5x doses of free CpG and OVA.
  • one CpG formulation (NC-CpG) had especially strong potential for CTL expansion and another (NC-CpG) has exceeded systemic CTL induction levels by free high-dose CpG and OVA both when analyzed ex vivo and upon in vitro expansion.

Abstract

Disclosed are methods for generating humoral and cytotoxic T lymphocyte (CTL) immune responses in a subject and related compositions.

Description

SYNTHETIC NANO CARRIERS THAT GENERATE HUMORAL AND
CYTOTOXIC T LYMPHOCYTE (CTL) IMMUNE RESPONSES
RELATED APPLICATIONS
This application claims the benefit under 35 U.S.C. § 119 of United States provisional application 61/513,496, 61/513,526 and 61/513,527, each filed July 29, 2011, the entire contents of each of which are incorporated herein by reference.
FIELD OF THE INVENTION
This invention relates to methods for generating humoral and cytotoxic T lymphocyte
(CTL) immune responses in a subject and related compositions. Generally, the humoral and CTL immune responses are generated with synthetic nanocarrier compositions that comprise a protein that comprises at least one humoral epitope and at least one MHC Class I-restricted epitope.
BACKGROUND OF THE INVENTION
Classically, vaccines have promoted a single arm of the immune system, for example, the generation of a humoral immune response consisting of antibodies to an antigen or, alternatively, activation of a CTL response to an antigen. Additionally, conventional vaccines generally do not target the sites of action of cells of interest, such as APCs, in an optimal manner. Methods and compositions for effectively activating both of these arms of the immune system optimally to effectively generate immune responses and/or reduce off- target effects and toxicity are needed. SUMMARY OF THE INVENTION
Provided herein are methods, and related compositions, for generating humoral and CTL immune responses in a subject. In one aspect, a method comprising identifying a subject in need of a humoral and CTL immune response to a first protein, and administering to the subject a composition comprising a population of synthetic nanocarriers coupled to the first protein, wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin-cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm is provided. In another embodiment, the mean of a DLS distribution is determined by any of the examples of such a method provided herein. Such examples are described in more detail below. In one embodiment, the composition is administered in an amount effective to generate a humoral and CTL immune response to the first protein.
In another aspect, a method comprising administering to the subject a composition comprising a population of synthetic nanocarriers coupled to a first protein; wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin-cholesterol adjuvant, wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm, and wherein the composition is administered according to a vaccination regimen is provided.
In another aspect, a method comprising administering to the subject a composition comprising a population of synthetic nanocarriers coupled to a first protein; wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin-cholesterol adjuvant, wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm, and wherein the composition is administered according to a protocol that was previously shown to result in a humoral and CTL immune response specific to the first protein in one or more test subjects is provided.
In one embodiment, the methods provided herein further comprise identifying a subject in need of a humoral and cytotoxic T lymphocyte (CTL) immune response to the first protein. In another embodiment, the composition is administered according to a vaccination regimen. In yet another embodiment, the composition is administered according to a protocol that was previously shown to result in a humoral and CTL immune response specific to the first protein in one or more test subjects
In another embodiment, the composition further comprises one or more adjuvants. In another embodiment, the method further comprises administering one or more adjuvants. In a further embodiment, the one or more adjuvants comprise stimulators or agonists of pattern recognition receptors, mineral salts, alum, alum combined with monphosphoryl lipid A of Enterobacteria (MPL), MPL® (AS04), AS15, saponins, QS-21,Quil-A, ISCOMs,
ISCOMATRIX™, MF59™, Montanide® ISA 51, Montanide® 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® 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; immuno stimulatory DNA; immuno stimulatory 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; immuno stimulatory 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 yet another embodiment, the one or more adjuvants comprise an agonist of Toll-Like Receptor 2, 3, 4, 7, 8 or 9. In still another embodiment, the one or more adjuvants comprise an imidazoquinoline or oxoadenine. In one embodiment, the imidazoquinoline comprises resiquimod or imiquimod.
In another embodiment, the one or more adjuvants are coupled to the synthetic nanocarriers of the population of synthetic nanocarriers.
In still another embodiment, the composition further comprises or the method further comprises administering another population of synthetic nanocarriers, and the one or more adjuvants are coupled to the synthetic nanocarriers of the other population of synthetic nanocarriers.
In a further embodiment, the one or more adjuvants are not coupled to a synthetic nanocarrier.
In another embodiment, the composition further comprises one or more additional antigens or the method further comprises administering one or more additional antigens. In an embodiment, the one or more additional antigens comprise at least one humoral epitope and/or at least one MHC Class I-restricted epitope. In another embodiment, the one or more additional antigens comprise at least one humoral epitope and at least one MHC Class I- restricted epitope that are not the same epitope. In one embodiment, the one or more additional antigens comprise a second protein. In another embodiment, the one or more additional antigens comprise a humoral epitope and/or a MHC Class I-restricted epitope. In still another embodiment, the one or more additional antigens comprise a humoral epitope and a MHC Class I-restricted epitope. In yet another embodiment, the one or more additional antigens comprise at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope.
In one embodiment, the one or more additional antigens are coupled to the synthetic nanocarriers.
In another embodiment, the composition further comprises or the method further comprises administering another population of synthetic nanocarriers, and the one or more additional antigens are coupled to the synthetic nanocarriers of the other population of synthetic nanocarriers.
In still another embodiment, the one or more additional antigens are not coupled to a synthetic nanocarrier.
In one embodiment, the synthetic nanocarriers and/or other synthetic nanocarriers comprise a polymeric nanoparticle, a metallic nanoparticle, a dendrimer, a buckyball, a nanowire, a virus-like particle or a peptide or protein particle. In another embodiment, the synthetic nanocarriers and/or other synthetic nanocarriers comprise one or more polymers. In yet another embodiment, the one or more polymers comprise a polyester, polyamino acid, polycarbonate, polyacetal, polyketal, polysaccharide, polyethyloxazoline or
polyethyleneimine. In still another embodiment, the one or more polymers comprise a polyester. In one embodiment, the polyester comprises a poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid) or polycaprolactone. In another embodiment, the polyester is coupled to a polyether. In yet another embodiment, the polyether comprises polyethylene glycol.
In one embodiment, the first protein and/or one or more additional antigens are antigens associated with cancer, an infection or infectious disease or a non- autoimmune or degenerative disease. In another embodiment, the first protein and/or one or more additional antigens are antigens associated with human immunodeficiency virus (HIV), malaria, leischmaniasis, a human filovirus infection, a togavirus infection, a alphavirus infection, an arenavirus infection, a bunyavirus infection, a flavivirus infection, a human papillomavirus infection, a human influenza A virus infection, a hepatitis B infection or a hepatitis C infection.
In another embodiment, the subject has or is at risk of having cancer, an infection or infectious disease or a non- autoimmune or degenerative disease. In yet another embodiment, the subject has or is at risk of having HIV, malaria, leischmaniasis, a human filovirus infection, a togavirus infection, a alphavirus infection, an arenavirus infection, a bunyavirus infection, a flavivirus infection, a human papillomavirus infection, a human influenza A virus infection, a hepatitis B infection or a hepatitis C infection.
In still another embodiment, the humoral and CTL immune responses that are generated are clinically effective. In one embodiment, the immune responses are effective to treat or prevent cancer, an infection or infectious disease or a non- autoimmune or
degenerative disease in the subject. In another embodiment, the immune responses are effective to treat or prevent HIV, malaria, leischmaniasis, a human filovirus infection, a togavirus infection, a alphavirus infection, an arenavirus infection, a bunyavirus infection, a flavivirus infection, a human papillomavirus infection, a human influenza A virus infection, a hepatitis B infection or a hepatitis C infection in the subject.
In one embodiment, the composition further comprises a pharmaceutically acceptable excipient.
In another embodiment, the composition is sterile.
In another embodiment, the composition is reconstituted from a lyophilized form. In another aspect, a dosage form comprising any of the compositions provided is provided.
In yet another aspect, a vaccine comprising any of the compositions and dosage forms provided is provided.
In yet a further embodiment, the composition is administered by intravenous, oral, subcutaneous, pulmonary, intranasal, intradermal, transmucosal, intramucosal or
intramuscular administration.
In still another aspect, a method comprising administering any of the compositions provided herein to a subject in need thereof is provided. In one embodiment, the subject is a human. In another embodiment, the subject has or is at risk of having cancer. In still another embodiment, the subject has or is at risk of having an infection or infectious disease. In yet another embodiment, the subject has or is at risk of having a non- autoimmune or
degenerative disease. In yet a further embodiment, the subject has or is at risk of having HIV. In another embodiment, the subject has or is at risk of having malaria, leischmaniasis, a human filovirus infection, a togavirus infection, a alphavirus infection, an arenavirus infection, a bunyavirus infection, a flavivirus infection, a human papillomavirus infection, a human influenza A virus infection, a hepatitis B infection or a hepatitis C infection. In one embodiment of any of the methods provided herein, any of the compositions provided can be administered to a subject, such as a human, according to a vaccination regimen.
In another embodiment of any of the method provided herein, any of the compositions provided can be administered to a subject according to a protocol that was previously shown to result in a humoral and CTL immune response specific to the first protein in one or more test subjects is provided
In another aspect, any of the methods provided can further comprise assessing the humoral and CTL immune response in the subject. The methods for assessing the humoral and CTL immune response can be any of the methods provided herein.
In yet another aspect, a method comprising preparing any of the compositions provided herein and assessing the generation of a humoral and CTL immune response is provided. In one embodiment, the composition comprises synthetic nanocarriers coupled to a first protein that comprises at least one humoral epitope and at least one MHC Class I- restricted epitope that are not the same epitope. In another embodiment, the population of synthetic nanocarriers coupled to the first protein does not comprise a saponin-cholesterol adjuvant and/or the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm.
In one aspect, a composition comprising a population of synthetic nanocarriers coupled to a first protein, wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin-cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm, for use in therapy or prophylaxis is provided.
In another aspect, a composition comprising a population of synthetic nanocarriers coupled to a first protein, wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin-cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm, for use in any of the methods provided herein is provided. In yet another aspect, a composition comprising a population of synthetic nanocarriers coupled to a first protein, wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin-cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm, for use in vaccination is provided.
In still another aspect, a composition comprising a population of synthetic
nanocarriers coupled to a first protein, wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin- cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm, for use in generating a humoral and CTL immune response to the first protein in a subject is provided. In one embodiment, these immune responses are clinically effective. In another embodiment, these immune responses are each effective in achieving immunity against a disease.
In a further aspect, a composition comprising a population of synthetic nanocarriers coupled to a first protein, wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin-cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm, for use in a method of therapy or prophylaxis of cancer, an infection or infectious disease, a non- autoimmune or degenerative disease, HIV, malaria, leischmaniasis, a human filovirus infection, a togavirus infection, a alphavirus infection, an arenavirus infection, a bunyavirus infection, a flavivirus infection, a human papillomavirus infection, a human influenza A virus infection, a hepatitis B infection or a hepatitis C infection I is provided.
In still a further aspect, a composition comprising a population of synthetic nanocarriers coupled to a first protein, wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin- cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm, for use in a method of therapy or prophylaxis comprising administration by intravenous, oral, subcutaneous, pulmonary, intranasal, intradermal, transmucosal, intramucosal or intramuscular administration is provided.
In yet a further aspect, a composition comprising a population of synthetic nanocarriers coupled to a first protein, wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin- cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm, for the manufacture of a medicament, for example a vaccine, for use in any of the methods provided herein is provided.
In another aspect, a composition for use as defined for any of the compositions or methods provided herein wherein the composition is any of the compositions provided herein is provided.
BRIEF DESCRIPTION OF FIGURES
Fig. 1 shows the antibody titers generated at days 12, 26, 43 and 57 following a prime and one boost (on day 28) vaccination regimen.
Fig. 2 shows the antibody titers generated at days 26 and 34 following a prime and one boost (on day 14) vaccination regimen.
Fig. 3 shows the induction of local memory CTL response by synthetic nanocarriers coupled to ovalbumin (OVA).
Fig. 4 shows the induction of central CTL response by synthetic nanocarriers coupled to ovalbumin.
Fig. 5 shows the induction of central CTL induction by synthetic nanocarrier compositions with ovalbumin and adjuvant.
Fig. 6 shows the antibody titers generated at days 25 and 42 following a prime and two boosts (on days 14 and 28) vaccination regimen.
Fig. 7 shows the development of antibody titers after a single injection by NC-R848 +
NC-OVA. Individual titers and averages for each experimental group are shown.
Nanocarrier=NC=NP. Fig. 8 shows the specific cytotoxicity in vivo after a single immunization with NC- R848 + NC-OVA. Averages for each group with standard deviation are shown.
Fig. 9 shows the anti-ovalbumin antibody titers upon the immunization with nanocarriers carrying CpG and OVA vs. free CpG (5x) and OVA (5x).
Fig. 10 shows the induction of OVA- specific CTL response in draining lymph nodes and spleens by the immunization with nanocarriers carrying CpG and OVA vs. free CpG (5x) and OVA (5x).
Fig. 11 shows the expansion of systemically induced OVA- specific CTLs in vitro upon the immunization with nanocarriers carrying CpG and OVA vs. free CpG (5x) and OVA (5x). Left Y axis (dark striped bars) - fraction of SIINFEKL (SEQ ID NO: l)-specific CD8+ cells after expansion; right Y axis (light striped bars) - expansion potential presented as proportion of post- and pre-expansion SIINFEKL (SEQ ID NO: l)-specific CTLs.
DETAILED DESCRIPTION OF THE INVENTION
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.
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety for all purposes.
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 or a mixture of differing molecular weights of a single polymer species, reference to "a synthetic
nanocarrier" includes a mixture of two or more such synthetic nanocarriers or a plurality of such synthetic nanocarriers, reference to "a DNA molecule" includes a mixture of two or more such DNA molecules or a plurality of such DNA molecules, reference to "an adjuvant" includes mixture of two or more such adjuvant molecules or a plurality of such adjuvant molecules, and the like.
As used herein, the term "comprise" or variations thereof such as "comprises" or "comprising" are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, elements, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein, the term
"comprising" is inclusive and does not exclude additional, unrecited integers or
method/process steps.
In embodiments of any of the compositions and methods provided herein,
"comprising" may be replaced with "consisting essentially of or "consisting of. The phrase "consisting essentially of is used herein to require the specified integer(s) or steps as well as those which do not materially affect the character or function of the claimed invention. As used herein, the term "consisting" is used to indicate the presence of the recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, elements, characteristics, properties, method/process steps or limitations) alone.
A. INTRODUCTION
Treatment of challenging diseases such as of HIV, malaria, hepatitis B, and cancer with therapeutic or prophylactic vaccines may be enhanced by, or in some circumstances require, combined humoral and CTL immune responses. While vaccine approaches have been proposed for creating a combined CTL and humoral immune response, alternative approaches could provide valuable improvements in clinical efficacy, safety, and/or manufacturability. Provided herein are methods for using synthetic nanocarrier
compositions, and related compositions, that are believed to have not been previously shown to generate strong and effective humoral and CTL immune responses. Such compositions can effectively target immune cells of interest to generate more effective immune responses.
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, it has been unexpectedly and surprisingly discovered that effective humoral and CTL immune responses can be generated with synthetic nanocarriers to which a protein, that comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, is coupled, wherein the population of synthetic nanocarriers does not comprise a saponin-cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic
nanocarriers is a maximum dimension of from 20 nm to 250 nm. In one aspect, therefore, a method comprising administering such a composition is provided. In one embodiment, the method comprises identifying a subject in need of a humoral and CTL immune response to a first protein, and administering to the subject a composition comprising such synthetic nanocarriers. In some embodiments, the composition is in an amount effective to generate a humoral and CTL immune response to the first protein. In another embodiment, the method comprises administering such a composition to a subject according to a protocol that was previously shown to result in a humoral and CTL immune response specific to the first protein in one or more test subjects.
In embodiments, the immune responses that are generated are clinically effective. In some embodiments, the subject to which the compositions are administered may have or be at risk of having cancer, an infection or infectious disease or a non-autoimmune or degenerative disease. In other embodiments, the subject may have or be at risk of having HIV, malaria, leischmaniasis, a human filovirus infection, a togavirus infection, a alphavirus infection, an arenavirus infection, a bunyavirus infection, a flavivirus infection, a human papillomavirus infection, a human influenza A virus infection, a hepatitis B infection or a hepatitis C infection.
In other embodiments, the compositions are administered to a subject, such as a human, according to a vaccination regimen.
The invention will now be described in more detail below. B. DEFINITIONS
"Adjuvant" means an agent that does not constitute a specific antigen, but boosts the strength and longevity of an immune response to a concomitantly administered antigen. Such adjuvants may include, but are not limited to stimulators of pattern recognition receptors, such as Toll-like receptors, RIG-1 and NOD-like receptors (NLR), mineral salts, such as alum, alum combined with monphosphoryl lipid (MPL) A of Enterobacteria, such as
Escherihia coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri or specifically with MPL® (AS04), MPL A of above-mentioned bacteria separately, saponins, such as QS-21,Quil-A, ISCOMs, ISCOMATRIX™, emulsions such as MF59™, Montanide® ISA 51 and ISA 720, AS02 (QS21+squalene+ MPL®) , 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® 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. "Saponin-cholesterol adjuvants" are saponin adjuvants that are stabilized by admixture with cholesterol. Such adjuvants include, for example, ISCOMs and ISCOMATRIX adjuvants. Preferably, in some embodiments, the synthetic nanocarriers provided herein are not or do not comprise such adjuvants. In other
embodiments, the compositions provided herein do not comprise such adjuvants.
In embodiments, adjuvants comprise agonists for pattern recognition receptors (PRR), including, but not limited to Toll-Like Receptors (TLRs), specifically TLRs 2, 3, 4, 5, 7, 8, 9 and/or combinations thereof. In other embodiments, adjuvants comprise agonists for Toll- Like Receptors 3, agonists for Toll-Like Receptors 7 and 8, or agonists for Toll-Like
Receptor 9; preferably the recited adjuvants comprise imidazoquinolines; such as R848; adenine derivatives, such as those disclosed in US patent 6,329,381 (Sumitomo
Pharmaceutical Company), US Published Patent Application 2010/0075995 to Biggadike et al., or WO 2010/018132 to Campos et al.; immuno stimulatory DNA; or immuno stimulatory 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 US Patent 6,696,076 to Tomai et al., including but not limited to imidazoquinoline amines, imidazopyridine amines, 6,7-fused
cycloalkylimidazopyridine amines, and 1,2-bridged imidazoquinoline amines. Preferred adjuvants comprise imiquimod and resiquimod (also known as R848). In specific
embodiments, an adjuvant may be an agonist for the DC surface molecule CD40. In certain embodiments, to stimulate immunity rather than tolerance, a synthetic nanocarrier
incorporates an adjuvant that promotes DC maturation (needed for priming of naive T cells) and the production of cytokines, such as type I interferons, which promote antibody immune responses. In embodiments, adjuvants also may comprise immuno stimulatory RNA molecules, such as but not limited to dsRNA, poly I:C or poly Lpoly C12U (available as Ampligen ®, 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., "Immuno stimulatory 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., "Immuno stimulatory 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 US Patents 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 (Thl) 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; US Patent 6,207,646 to Krieg et al.; US Patent 7,223,398 to Tuck et al.; US Patent 7,250,403 to Van Nest et al.; or US Patent 7,566,703 to Krieg et al.
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.
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.
"Administering" or "administration" means providing a material, such as a drug, to a subject in a manner that is pharmacologically useful.
"Amount effective" is any amount of a composition provided herein that produces one or more desired responses, such as one or more desired immune responses. This amount can be for in vitro or in vivo purposes. For in vivo purposes, the amount can be one that a clinician would believe may have a clinical benefit for a subject in need of a humoral immune response and a CTL immune response to a single protein. An effective amount that a clinician would believe may have a clinical benefit for such a subject is also referred to herein as a "clinically effective amount". In embodiments, both the humoral immune response and the CTL immune response that is elicited by a composition provided herein results in a clinical effect from each of these arms of the immune system. In other embodiments, clinically effective amounts are effective amounts that can be helpful in the treatment of a subject with a disease or condition in which a humoral immune response and a CTL immune response to a single protein would provide a benefit. Such subjects include, in some embodiments, those that have or are at risk of having cancer, an infection or infectious disease or a non- autoimmune or degenerative disease. In other embodiments, such subjects include those that have or are at risk of having HIV, malaria, leischmaniasis, a human filovirus infection, a togavirus infection, a alphavirus infection, an arenavirus infection, a bunyavirus infection, a flavivirus infection, a human papillomavirus infection, a human influenza A virus infection, a hepatitis B infection or a hepatitis C infection.
Amounts effective include those that involve the production of a humoral immune response against a humoral epitope and a CTL immune response against a MHC Class I- restricted epitope administered in one of the compositions provided herein. A subject's humoral and CTL immune response can be monitored by routine methods. An amount that is effective to produce the desired immune responses as provided herein can also be an amount of a composition provided herein that produces a desired therapeutic endpoint or a desired therapeutic result. In one embodiment, the amount that is effective is one that provides effective immunity against a disease or agent that causes a disease as provided herein. In another embodiment, the immunity persists in the subject for at least 6, 12, 18, 24, 36, 48, 60 or more months. In still another embodiment, the immunity results or persists due to the administration of a composition provided herein according to a vaccination regimen.
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 reason.
In general, doses of the compositions of the invention can range from about 10 g/kg to about 100,000 μg/kg. In some embodiments, the doses can range from about 0.1 mg/kg to about 100 mg/kg. In still other embodiments, the doses can range from about 0.1 mg/kg to about 25 mg/kg, about 25 mg/kg to about 50 mg/kg, about 50 mg/kg to about 75 mg/kg or about 75 mg/kg to about 100 mg/kg. Alternatively, the dose can be administered based on the number of synthetic nanocarriers. For example, useful doses include greater than 106,
10 7, 108, 109 or 1010 synthetic nanocarriers per dose. Other examples of useful doses include from about lxlO6 to about lxlO10, about lxlO7 to about lxlO9 or about lxlO8 to about lxlO9 synthetic nanocarriers per dose.
"Antigen" means any antigen that can generate one or more immune responses. The antigen may be one that generates a humoral and/or CTL immune response. Such antigens include, but are not limited to proteins, peptides, small molecules, oligosaccharides, and carbohydrates. In some embodiments, such an antigen comprises a non-protein antigen (i.e., not a protein or peptide antigen). In some embodiments, an antigen that generates a humoral immune response comprises a carbohydrate associated with an infectious agent. In some embodiments, an antigen that generates a humoral immune response comprises a
glycoprotein or glycopeptide associated with an infectious agent. The infectious agent can be a bacterium, virus, fungus, protozoan, or parasite. Antigens may be B cell or T cell antigens.
The synthetic nanocarrier compositions for use in the inventive methods provided herein are coupled to an antigen that is a protein that comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that is not the same epitope. Such compositions can, in some embodiments, comprise one or more additional antigens that may also be so limited but not necessarily so. The one or more additional antigens for use in the methods and compositions provided herein can be any antigen, which include antigens that comprise humoral epitopes and/or MHC Class I-restricted epitopes. The one or more additional antigens may also include MHC Class II-restricted epitopes. In other
embodiments, the one or more additional antigens may be any antigen that generates a humoral immune response. In still other embodiments, the one or more additional antigens may be any of the T cell antigens described herein, including a CD-I restricted antigen. In yet other embodiments, the one or more additional antigens may be a protein, peptide, small molecule, oligosaccharide and carbohydrate.
In embodiments, antigens, including a protein that comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, are coupled to the synthetic nanocarriers. In other embodiments, the antigens are not coupled to the synthetic nanocarriers. In yet other embodiments, the antigens are encapsulated in the synthetic nanocarriers. "Type(s) of antigens" means molecules that share the same, or substantially the same, antigenic characteristics.
"Antigens associated" with a disease, disorder or condition provided herein are antigens that can generate an undesired immune response against, as a result of, or in conjunction with the disease, disorder or condition; the cause of the disease, disorder or condition (or a symptom or effect thereof); and/or can generate an undesired immune response that is a symptom, result or effect of the disease, disorder or condition. In some embodiments, such as with cancer, such antigens are expressed in or on diseased cells, such as cancer or tumor cells, but not in or on normal or healthy cells (or non-diseased cells). Such antigens can also comprise an antigen that is expressed in or on diseased cells and on normal or healthy cells (or non-diseased cells) but is expressed in or on diseased cells at a greater level than on normal or healthy cells (or non-diseased cells). Preferably, the use of an antigen associated with a disease or condition provided herein will not lead to a substantial or detrimental immune response against normal or healthy cells or will lead to a beneficial immune response against the disease or condition that outweighs any immune response against normal or healthy cells (or non-diseased cells). The antigens associated with a disease or condition provided herein, in some embodiments, are proteins that are coupled to synthetic nanocarriers that comprise humoral and/or MHC Class I-restricted epitopes. In other embodiments, such proteins comprise a humoral and a MHC Class I-restricted epitope. In still other embodiments, such proteins comprise at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope. Examples of antigens, including the foregoing proteins, are provided elsewhere herein.
"At least a portion of the dose" means at least some part of the dose, ranging up to including all of the dose.
An "at risk" subject is one in which a health practitioner believes has a chance of having a disease or condition as provided herein.
"B cell antigen" means any antigen that is recognized by or triggers an immune response in a B cell (e.g., an antigen that is specifically recognized by a B cell or a receptor thereon). 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, oligosaccharides and
carbohydrates.
"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.
"Cytotoxic T lymphocyte (CTL) immune response" means any stimulation, induction or proliferation of cytotoxic T cells, preferably cytotoxic T cells that are specific to an epitope, such as a MHC Class I-restricted epitope. In embodiments, the epitope is or is of an antigen that is associated with any of the diseases or conditions provided herein. Methods for assessing CTL immune responses are known to those of skill in the art. Examples of such a method are provided in the EXAMPLES.
"Dosage form" means a pharmacologically and/or immunologically active material in a medium, carrier, vehicle, or device suitable for administration to a subject.
"Encapsulate" means to enclose at least a portion of a substance within a synthetic nanocarrier. In some embodiments, a substance is enclosed completely within a synthetic nanocarrier. In other embodiments, most or all of a substance that is encapsulated is not exposed to the local environment external to the synthetic nanocarrier. In other
embodiments, no more than 50%, 40%, 30%, 20%, 10% or 5% (weight/weight) is exposed to the local environment. 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.
"Epitope", also known as an antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by, for example, antibodies, B cells, or T cells. As used herein, a "humoral epitope" is one that is recognized by antibodies or B cells, while a "MHC Class I-restricted epitope" is one that is presented to immune cells by MHC class I molecules found on nucleated cells. "MHC Class II-restricted epitopes" are epitopes that are presented to immune cells by MHC class II molecules found on antigen presenting cells (APCs), for example, on professional antigen-presenting immune cells, such as on macrophages, B cells, and dendritic cells, or on non-hematopoietic cells, such as hepatocytes.
A number of epitopes are known to those of skill in the art, and exemplary epitopes suitable according to some aspects of this invention include, but are not limited to those listed in the Immune Epitope Database (www.immuneepitope.org, Vita R, Zarebski L, Greenbaum JA, Emami H, Hoof I, Salimi N, Damle R, Sette A, Peters B. The immune epitope database 2.0. Nucleic Acids Res. 2010 Jan;38(Database issue):D854-62; the entire contents of which as well as all database entries of IEDB version 2.4, August 2011, and particularly all epitopes disclosed therein, are incorporated herein by reference). Epitopes can also be identified with publicly available algorithms, for example, the algorithms described in Wang P, Sidney J, Kim Y, Sette A, Lund O, Nielsen M, Peters B. 2010. peptide binding predictions for HLA DR, DP and DQ molecules. BMC Bioinformatics 2010, 11:568; Wang P, Sidney J, Dow C, Mothe B, Sette A, Peters B. 2008. A systematic assessment of MHC class II peptide binding predictions and evaluation of a consensus approach. PLoS Comput Biol. 4(4):el000048; Nielsen M, Lund O. 2009. NN-align. An artificial neural network-based alignment algorithm for MHC class II peptide binding prediction. BMC Bioinformatics. 10:296; Nielsen M, Lundegaard C, Lund O. 2007. Prediction of MHC class II binding affinity using SMM-align, a novel stabilization matrix alignment method. BMC Bioinformatics. 8:238; Bui HH, Sidney J, Peters B, Sathiamurthy M, Sinichi A, Purton KA, Mothe BR, Chisari FV, Watkins DI, Sette A. 2005. Immunogenetics. 57:304-314; Sturniolo T, Bono E, Ding J, Raddrizzani L, Tuereci O, Sahin U, Braxenthaler M, Gallazzi F, Protti MP, Sinigaglia F, Hammer J. 1999. Generation of tissue-specific and promiscuous HLA ligand databases using DNA microarrays and virtual HLA class II matrices. Nat Biotechnol. 17(6):555-561; Nielsen M, Lundegaard C, Worning P, Lauemoller SL, Lamberth K, Buus S, Brunak S, Lund O. 2003. Reliable prediction of T-cell epitopes using neural networks with novel sequence representations. Protein Sci 12: 1007-1017; Bui HH, Sidney J, Peters B, Sathiamurthy M, Sinichi A, Purton KA, Mothe BR, Chisari FV, Watkins DI, Sette A. 2005. Automated generation and evaluation of specific MHC binding predictive tools: ARB matrix applications.
Immunogenetics 57:304-314; Peters B, Sette A. 2005. Generating quantitative models describing the sequence specificity of biological processes with the stabilized matrix method. BMC Bioinformatics 6: 132; Chou PY, Fasman GD. 1978. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol 47:45-148; Emini EA, Hughes JV, Perlow DS, Boger J. 1985. Induction of hepatitis A virus- neutralizing antibody by a virus-specific synthetic peptide. J Virol 55:836-839; Karplus PA, Schulz GE. 1985. Prediction of chain flexibility in proteins. Naturwissenschaften 72:212-213; Kolaskar AS, Tongaonkar PC. 1990. A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett276: 172-174; Parker JM, Guo D, Hodges RS. 1986. New hydrophilicity scale derived from high-performance liquid chromatography peptide retention data: correlation of predicted surface residues with antigenicity and X-ray- derived accessible sites. Biochemistry 25:5425-5432; Larsen JE, Lund O, Nielsen M. 2006. Improved method for predicting linear B-cell epitopes. Immunome Res 2:2; Ponomarenko JV, Bourne PE. 2007. Antibody-protein interactions: benchmark datasets and prediction tools evaluation. BMC Struct Biol 7:64; Haste Andersen P, Nielsen M, Lund O. 2006. Prediction of residues in discontinuous B-cell epitopes using protein 3D structures. Protein Sci 15:2558- 2567; Ponomarenko JV, Bui H, Li W, Fusseder N, Bourne PE, Sette A, Peters B. 2008. ElliPro: a new structure-based tool for the prediction of antibody epitopes. BMC
Bioinformatics 9:514; Nielsen M, Lundegaard C, Blicher T, Peters B, Sette A, Justesen S, Buus S, and Lund O. 2008. PLoS Comput Biol.4(7)el000107. Quantitative predictions of peptide binding to any HLA-DR molecule of known sequence: NetMHCIIpan; the entire contents of each of which are incorporated herein by reference for disclosure of methods and algorithms for the identification of epitopes.
"Generating" means causing an action, such as an immune response (e.g., a humoral immune response or a CTL immune response) against an epitope to occur, either directly oneself or indirectly, such as, but not limited to, an unrelated third party that takes an action through reliance on one's words or deeds.
"Humoral immune response" means any immune response that results in the production or stimulation of B cells and/or the production of antibodies. Preferably, the humoral immune response is specific to an epitope comprised within an inventive
composition or administered during the practice of an inventive method. Methods for assessing whether a humoral response is induced are known to those of ordinary skill in the art. The production of antibodies is referred to herein as an "antibody response". "Antibody titer" means the production of a measurable level of antibodies. Preferably, the antibody response or generation of the antibody titer is in a human. In some embodiments, the antibodies are antibodies of a certain isotype, such as IgG or a subclass thereof. Methods for measuring antibody titers are known in the art and include Enzyme-linked Immunosorbent Assay (ELISA). Methods for measuring antibody titers are also described in some detail in the EXAMPLES. Preferably, the antibody response or antibody titer is specific to an epitope as provided herein. In embodiments, the antibody response can be quantitated, for example, as the number of antibodies, concentration of antibodies or titer. The values can be absolute or they can be relative. Assays for quantifying an antibody response include antibody capture assays, enzyme-linked immunosorbent assays (ELISAs), inhibition liquid phase absorption assays (ILPAAs), rocket Immunoelectrophoresis (RIE) assays and line
Immunoelectrophoresis (LIE) assays. When an antibody response is compared to another antibody response the same type of quantitative value (e.g., titer) and method of measurement (e.g., ELISA) is preferably used to make the comparison.
An ELISA method for measuring an antibody titer, for example, may consist of the following steps (i) preparing an ELISA-plate coating material such that the antibody target of interest is coupled to a substrate polymer or other suitable material (ii) preparing the coating material in an aqueous solution (such as PBS) and delivering the coating material solution to the wells of a multiwell plate for overnight deposition of the coating onto the multiwell plate (iii) thoroughly washing the multiwell plate with wash buffer (such as 0.05% Tween-20 in PBS) to remove excess coating material (iv) blocking the plate for nonspecific binding by applying a diluent solution (such as 10% fetal bovine serum in PBS), (v) washing the blocking/diluent solution from the plate with wash buffer (vi) diluting the serum sample(s) containing antibodies and appropriate standards (positive controls) with diluent as required to obtain a concentration that suitably saturates the ELISA reponse (vii) serially diluting the plasma samples on the multiwell plate such to cover a range of concentrations suitable for generating an ELISA response curve (viii) incubating the plate to provide for antibody-target binding (ix) washing the plate with wash buffer to remove antibodies not bound to antigen (x) adding an appropriate concentration of a secondary detection antibody in same diluent such as a biotin-coupled detection antibody capable of binding the primary antibody (xi) incubating the plate with the applied detection antibody, followed by washing with wash buffer (xii) adding an enzyme such as streptavidin-HRP (horse radish peroxidase) that will bind to biotin found on biotinylated antibodes and incubating (xiii) washing the multiwell plate (xiv) adding substrate(s) (such as TMB solution) to the plate (xv) applying a stop solution (such as 2N sulfuric acid) when color development is complete (xvi) reading optical density of the plate wells at a specific wavelength for the substrate (450 nm with subtraction of readings at 570 nm) (xvi) applying a suitable multiparameter curve fit to the data and defining half-maximal effective concentration (EC50) as the concentration on the curve at which half the maximum OD value for the plate standards is achieved.
"Identifying" is any action or set of actions that allows a clinician to recognize a subject as one who may benefit from the methods and compositions provided herein.
Preferably, the identified subject is one who is in need of a humoral immune response and CTL immune response to a single protein. Such subjects include any subject that has or is at risk of having any of the disease or conditions provided herein. The action or set of actions may be either directly oneself or indirectly, such as, but not limited to, an unrelated third party that takes an action through reliance on one's words or deeds.
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.
"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 equal to or 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 μιη. 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 10,000: 1, more preferably from 1: 1 to 1000: 1, still more 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 μιη, more preferably equal to or less than 2 μιη, more preferably equal to or less than 1 μιη, 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 minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is equal to or greater than lOOnm, 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 dimensions (e.g., diameter) is obtained by suspending the synthetic nanocarriers in a liquid (usually aqueous) media and using dynamic light scattering (DLS) (e.g. using a Brookhaven ZetaPALS instrument). For example, a suspension of synthetic nanocarriers can be diluted from an aqueous buffer into purified water to achieve a final synthetic nanocarrier suspension concentration of approximately 0.01 to 0.1 mg/mL. The diluted suspension may be prepared directly inside, or transferred to, a suitable cuvette for DLS analysis. The cuvette may then be placed in the DLS, allowed to equilibrate to the controlled temperature, and then scanned for sufficient time to acquire a stable and reproducible distribution based on appropriate inputs for viscosity of the medium and refractive indicies of the sample. The effective diameter, or mean of the distribution, is then reported. "Dimension" or "size" or "diameter" of synthetic nanocarriers means the mean of a particle size distribution obtained using dynamic light scattering.
"MHC" refers to major histocompatibility complex, a large genomic region or gene family found in most vertebrates that encodes MHC molecules that display fragments or epitopes of processed proteins on the cell surface. The presentation of MHC:peptide on cell surfaces allows for surveillance by immune cells, usually a T cell. There are two general classes of MHC molecules: Class I and Class II. Generally, Class I MHC molecules are found on nucleated cells and present peptides to cytotoxic T cells. Class II MHC molecules are found on certain immune cells, chiefly macrophages, B cells and dendritic cells, collectively known as APCs. The best-known genes in the MHC region are the subset that encodes antigen-presenting proteins on the cell surface. In humans, these genes are referred to as human leukocyte antigen (HLA) genes.
"Pharmaceutically acceptable excipient" means a pharmacologically inactive material used together with the recited synthetic nanocarriers to formulate the compositions.
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.
"Protein(s)" means compounds, typically having a molecular weight greater than 1000 daltons, comprising amino acid residues joined together primarily by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. Proteins may also comprise additional bonding structures such as secondary structures, tertiary structures, and the like. Certain of the peptide bonds in proteins may be replaced by other bond types, for various purposes, such as stabilization or coupling. When coupled to synthetic nanocarriers, preferably, there are multiple copies of the protein that are coupled to each synthetic nanocarrier.
"Protocol " refers to any dosing regimen of one or more substances to a subject. A dosing regimen may include the amount, frequency and/or mode of administration. In some embodiments, such a protocol may be used to administer one or more compositions of the invention to one or more test subjects. Immune responses in these test subjects can then be assessed to determine whether or not the protocol was effective in generating desired immune response(s). Any other therapeutic and/or prophylactic effects may also be assessed instead of or in addition to the aforementioned immune responses. Whether or not a protocol had a desired effect can be determined using any of the methods provided herein or otherwise known in the art. For example, a population of cells may be obtained from a subject to which a composition provided herein has been administered according to a specific protocol in order to determine whether or not specific immune cells, cytokines, antibodies, etc. were generated, activated, etc. Useful methods for detecting the presence and/or number of immune cells include, but are not limited to, flow cytometric methods (e.g., FACS) and
immunohistochemistry methods. Antibodies and other binding agents for specific staining of immune cell markers, are commercially available. Such kits typically include staining reagents for multiple antigens that allow for FACS-based detection, separation and/or quantitation of a desired cell population from a heterogeneous population of cells.
"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.
"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, synthetic nanocarriers do not comprise chitosan. In certain other
embodiments, the synthetic nanocarriers do not comprise chitosan. In other embodiments, inventive synthetic nanocarriers are not lipid-based nanoparticles. In further embodiments, inventive synthetic nanocarriers do not comprise a phospholipid.
A synthetic nanocarrier can be, but is not limited to, one or a plurality of lipid-based nanoparticles (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, viruslike particles (i.e., particles that are primarily made up of viral structural proteins but that are not infectious or have low infectivity), peptide or protein-based particles (also referred to herein as protein particles, i.e., particles where the majority of the material that makes up their structure are peptides or proteins) (such as albumin nanoparticles) and/or nanoparticles that are developed using a combination of nanomaterials such as lipid-polymer nanoparticles. Synthetic nanocarriers may be a variety of different shapes, including but not limited to spheroidal, cuboidal, pyramidal, oblong, cylindrical, toroidal, and the like. Synthetic nanocarriers according to the invention comprise one or more surfaces. Exemplary synthetic nanocarriers that can be adapted for use in the practice of the present invention comprise: (1) the biodegradable nanoparticles disclosed in US Patent 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 WO2009106999 A2, (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) or (11) apoptotic cells, apoptotic bodies or the synthetic or semisynthetic mimics disclosed in U.S. Publication 2002/0086049. 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.
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.
"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.
In some embodiments the T cell antigen is a T helper cell antigen (i.e. one that can generate an enhanced response to a B cell antigen, preferably an unrelated B cell antigen, through stimulation of T cell help). 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. US Patent 7,202,351). In other embodiments, a T helper cell antigen may comprise one or more lipids, or glycolipids, including but not limited to: cc-galactosylceramide (cc-GalCer), cc- 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 a T helper cell antigen, see V. Cerundolo et al., "Harnessing invariant NKT cells in vaccination strategies." Nature Rev Immun, 9:28-38 (2009). 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 T helper cell antigen, may be coupled to, or uncoupled from, a synthetic nanocarrier. In some embodiments, the T cell antigen is encapsulated in the synthetic nanocarriers of the compositions.
"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.
"Vaccine regimen" or "vaccination regimen" is a schedule of one or more
vaccinations that includes the number and timing of doses of a vaccine. Generally, vaccination regimens are intended to achieve immunity against the development of a disease or condition. Preferably, the vaccine regimen is one that achieves immunity via both the humoral and CTL arms of the immune system.
C. COMPOSITIONS FOR USE IN THE INVENTIVE METHODS
Provided herein are methods and related compositions for effective humoral and CTL immune response generation. It has been found that synthetic nanocarriers to which a protein that comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, is coupled, wherein the synthetic nanocarriers do not comprise a saponin-cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm can be used to generate effective and strong humoral and CTL immune responses. The compositions provided can be used for a variety of desired clinical endpoints such as for vaccination. 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 cubic. In some embodiments, synthetic nanocarriers are ovals or ellipses. In some embodiments, synthetic nanocarriers are cylinders, cones, or pyramids.
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 nanocarner 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.
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.
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).
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.). 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 (i.e., components) of the synthetic nanocarriers can be coupled with the polymer.
In some embodiments, a component can be covalently associated with a polymeric matrix. In some embodiments, covalent association is mediated by a linker. In some embodiments, a component can be noncovalently associated with a polymeric matrix. For example, in some embodiments, a component can be encapsulated within, surrounded by, and/or dispersed throughout a polymeric matrix. Alternatively or additionally, a component can be associated with a polymeric matrix by hydrophobic interactions, charge interactions, van der Waals forces, etc.
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.
The synthetic nanocarriers provided herein may be polymeric nanocarriers. 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.
In some embodiments, the synthetic nanocarriers comprise one or more polymers that comprise a polyester, polycarbonate, polyamide, or polyether, or unit thereof. In other embodiments, the polymer comprises poly(ethylene glycol) (PEG), poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), or a polycaprolactone, or unit thereof. In some embodiments, it is preferred that the polymer is biodegradable. Therefore, in these embodiments, it is preferred that if the polymer comprises a polyether, such as poly(ethylene glycol) or unit thereof, the polymer comprises a block-co-polymer of a polyether and a biodegradable polymer such that the polymer is biodegradable. In other embodiments, the polymer does not solely comprise a polyether or unit thereof, such as poly(ethylene glycol) or unit thereof. The one or more polymers may be comprised within a polymeric synthetic nanocarrier or may be comprised in a number of other different types of synthetic
nanocarriers.
Examples of polymers suitable for use in the present invention also include, but are not limited to polyethylenes, polycarbonates (e.g. poly(l,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(P-hydroxyalkanoate))), poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polyureas, polystyrenes, and polyamines, polylysine, polylysine-PEG copolymers, and poly(ethyleneimine), poly(ethylene imine)-PEG copolymers.
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. § 177.2600, including but not limited to polyesters (e.g., polylactic acid, poly(lactic-co-glycolic acid), polycaprolactone, polyvalerolactone, poly(l,3-dioxan-2one)); polyanhydrides (e.g., poly(sebacic anhydride)); polyethers (e.g., polyethylene glycol); polyurethanes; polymethacrylates; polyacrylates; and
polycyanoacrylates.
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.
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 US Patent No. 5543158 to Gref et al., or WO publication WO2009/051837 by Von Andrian et al.
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.
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[a-(4-aminobutyl)-L-glycolic acid], and derivatives thereof.
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.
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. 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), polyethylene 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 synthetic nanocarriers may not comprise (or may exclude) cationic polymers.
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).
The properties of these and other polymers and methods for preparing them are well known in the art (see, for example, U.S. Patents 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 AUcock et al., Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and in U.S. Patents 6,506,577, 6,632,922, 6,686,446, and 6,818,732.
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 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.
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®85) glycocholate; sorbitan monolaurate (Span®20); polysorbate 20 (Tween®20); polysorbate 60 (Tween®60);
polysorbate 65 (Tween®65); polysorbate 80 (Tween®80); polysorbate 85 (Tween®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; chao tropic 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.
In some embodiments, synthetic nanocarriers may optionally comprise one or more carbohydrates. Carbohydrates may be natural or synthetic. A carbohydrate may be a derivatized natural carbohydrate. In certain embodiments, a carbohydrate comprises monosaccharide or disaccharide, including but not limited to glucose, fructose, galactose, ribose, lactose, sucrose, maltose, trehalose, cellbiose, mannose, xylose, arabinose, glucoronic acid, galactoronic acid, mannuronic acid, glucosamine, galatosamine, and neuramic acid. In certain embodiments, a carbohydrate is a polysaccharide, including but not limited to pullulan, cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextran, glycogen,
hydroxyethylstarch, carageenan, glycon, amylose, chitosan, Ν,Ο-carboxylmethylchitosan, algin and alginic acid, starch, chitin, inulin, konjac, glucommannan, pustulan, heparin, hyaluronic acid, curdlan, and xanthan. In embodiments, the 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.
Compositions for use in the methods according to the invention comprise synthetic nanocarriers in combination with pharmaceutically acceptable excipients, such as
preservatives, buffers, saline, or phosphate buffered saline. The compositions may be made using conventional pharmaceutical manufacturing and compounding techniques to arrive at useful dosage forms. In an embodiment, synthetic nanocarriers are suspended in sterile saline solution for injection together with a preservative.
In embodiments, when preparing synthetic nanocarriers as carriers for use in vaccines, methods for coupling the components to the synthetic nanocarriers may be useful. If the component is a small molecule it may be of advantage to attach the component 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 component to the synthetic nanocarrier through the use of these surface groups rather than attaching the component to a polymer and then using this polymer conjugate in the construction of synthetic nanocarriers. In certain embodiments, the coupling can be a covalent linker. In embodiments, components 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 the component containing an alkyne group or by the 1,3- dipolar cycloaddition reaction of alkynes on the surface of the nanocarrier with components 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.
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.
An amide linker is formed via an amide bond between an amine on one component 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.
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 component containing activated thiol group.
A triazole linker, specifically a 1,2,3-triazole of the form
Figure imgf000037_0001
, wherein RI 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. 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.
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 component is prepared with the presence of either an alkyne (if the polymer contains an azide) or an azide (if the polymer contains an alkyne) group. The component is then allowed to react with the nanocarrier via the 1,3-dipolar cycloaddition reaction with or without a catalyst which covalently couples the component to the particle through the 1,4-disubstituted 1,2,3-triazole linker.
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 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 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 with an alkene group on a second component such as a polymer or nanocarrier.
A hydrazone linker is made by the reaction of a hydrazide group on one component with an aldehyde/ketone group on the second component such as the nanocarrier.
A hydrazide linker is formed by the reaction of a hydrazine group on one component 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.
An imine or oxime linker is formed by the reaction of an amine or N-alkoxyamine (or aminooxy) group on one component with an aldehyde or ketone group on the second component such as the nanocarrier.
An urea or thiourea linker is prepared by the reaction of an amine group on one component with an isocyanate or thioisocyanate group on the second component such as the nanocarrier. An amidine linker is prepared by the reaction of an amine group on one component with an imidoester group on the second component such as the nanocarrier.
An amine linker is made by the alkylation reaction of an amine group on one component 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 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.
A sulfonamide linker is made by the reaction of an amine group on one component with a sulfonyl halide (such as sulfonyl chloride) group on the second component such as the nanocarrier.
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 nanocarrier or attached to a component.
The component can also be conjugated to the nanocarrier via non-covalent conjugation methods. For examples, a negative charged component can be conjugated to a positive charged nanocarrier through electrostatic adsorption. A component containing a metal ligand can also be conjugated to a nanocarrier containing a metal complex via a metal- ligand complex.
In embodiments, the component 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 component may be prepared with a group which is compatible with the attachment chemistry that is presented by the synthetic nanocarriers' surface. In other embodiments, a component 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 component containing an acid group via the other end of the ADH linker on NC to produce the corresponding VLP or liposome peptide conjugate. 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 component 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.
In some embodiments, a component, such as an antigen or adjuvant, may be isolated. Isolated refers to the element being separated from its native environment and present in sufficient quantities to permit its identification or use. This means, for example, the element may be (i) selectively produced by expression cloning or (ii) purified as by chromatography or electrophoresis. Isolated elements may be, but need not be, substantially pure. Because an isolated element may be admixed with a pharmaceutically acceptable excipient in a pharmaceutical preparation, the element may comprise only a small percentage by weight of the preparation. The element 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 lipids or proteins. Any of the elements provided herein may be isolated. Any of the antigens provided herein can be included in the compositions in isolated form.
D. METHODS OF USING AND MAKING SYNTHETIC NANOCARRIER
COMPOSITIONS
Synthetic nanocarriers may be prepared using a wide variety of methods known in the art. For example, synthetic nanocarriers can be formed by methods as nanoprecipitation, flow focusing using fluidic channels, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, milling, microemulsion procedures, microfabrication, nanofabrication, sacrificial layers, simple and complex coacervation, and other methods well known to those of ordinary skill in the art. Alternatively or additionally, aqueous and organic solvent syntheses for monodisperse semiconductor, conductive, magnetic, organic, and other nanomaterials have been described (Pellegrino et al., 2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci., 30:545; and Trindade et al., 2001, Chem. Mat., 13:3843). Additional methods have been described in the literature (see, e.g., Doubrow, Ed., "Microcapsules and Nanoparticles in Medicine and Pharmacy," CRC Press, Boca Raton, 1992; Mathiowitz et al., 1987, J. Control. Release, 5: 13; Mathiowitz et al., 1987, Reactive Polymers, 6:275; and Mathiowitz et al., 1988, J. Appl. Polymer Sci., 35:755; US Patents 5578325 and 6007845; P. Paolicelli et al., "Surface-modified PLGA-based Nanoparticles that can Efficiently Associate and Deliver Virus-like Particles" Nanomedicine. 5(6):843-853 (2010)).
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 into synthetic nanocarriers may be used, including without limitation methods disclosed in United States Patent 6,632,671 to Unger October 14, 2003.
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.
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.
Elements of the synthetic nanocarriers 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 Al to Murthy et al.
Alternatively or additionally, synthetic nanocarriers can be coupled to 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 synthetic nanocarrier. In embodiments, encapsulation and/or absorption is a form of coupling.
In embodiments, the synthetic nanocarriers can be combined with adjuvants by admixing in the same vehicle or delivery system. Such adjuvants may include, but are not limited to mineral salts, such as alum, alum combined with monphosphoryl lipid (MPL) A of Enterobacteria, such as Escherihia coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri or specifically with MPL® (AS04), MPL A of above-mentioned bacteria separately, saponins, such as QS-21,Quil-A, ISCOMs, ISCOMATRIX™, emulsions such as MF59™, Montanide® ISA 51 and ISA 720, AS02 (QS21+squalene+ MPL®) , 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® 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, the synthetic nanocarriers can be combined with an antigen different, similar or identical to those coupled to a nanocarrier (with or without adjuvant, utilizing or not utilizing another delivery vehicle) administered separately at a different time- point and/or at a different body location and/or by a different immunization route or with another antigen and/or adjuvant-carrying synthetic nanocarrier administered separately at a different time-point and/or at a different body location and/or by a different immunization route.
Populations of synthetic nanocarriers may be combined to form pharmaceutical dosage forms according to the present invention using traditional pharmaceutical mixing methods. These include liquid-liquid mixing in which two or more suspensions, each containing one or more subsets of nanocarriers, are directly combined or are brought together via one or more vessels containing diluent. As synthetic nanocarriers may also be produced or stored in a powder form, dry powder-powder mixing could be performed as could the re- suspension of two or more powders in a common media. Depending on the properties of the nanocamers and their interaction potentials, there may be advantages conferred to one or another route of mixing.
Typical 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).
Compositions according to the invention comprise 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, synthetic nanocarriers are suspended in sterile saline solution for injection together with a preservative.
It is to be understood that the compositions of synthetic nanocarriers can be made in any suitable manner, and the invention is in no way limited to the use of compositions that can be produced using the methods described herein. Selection of an appropriate method may require attention to the properties of the particular elements being associated.
In some embodiments, 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, synthetic nanocarriers may be lyophilized and stored in suspension or as lyophilized powder depending on the formulation strategy for extended periods without losing activity.
The compositions of the invention can be administered by a variety of routes, including or not limited to subcutaneous, intranasal, oral, intravenous, intraperitoneal, intramuscular, transmucosal, transmucosal, sublingual, rectal, ophthalmic, pulmonary, intradermal, transdermal, transcutaneous or intradermal or by a combination of these routes. Routes of administration also include administration by inhalation or pulmonary aerosol. Techniques for preparing aerosol delivery systems are well known to those of skill in the art (see, for example, Sciarra and Cutie, "Aerosols," in Remington's Pharmaceutical Sciences, 18th edition, 1990, pp. 1694-1712; incorporated by reference).
Doses of dosage forms contain varying amounts of populations of synthetic nanocarriers and varying amounts of the proteins and/or adjuvants and/or additional antigens, according to the invention. The amount of synthetic nanocarriers and/or proteins and/or adjuvants and/or additional antigens present in the dosage forms can be varied according to the nature of the elements present, 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 proteins and/or adjuvants and/or additional antigens to be present in the dosage form. In
embodiments, the synthetic nanocarriers and the proteins and/or adjuvants and/or additional antigens are present in the dosage form in an amount effective to generate an immune response to the proteins and/or additional antigens upon administration to a subject. It may be possible to determine amounts effective to generate an immune response using
conventional dose ranging studies and techniques in subjects. 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.
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, non- autoimmune diseases, HIV, malaria, hepatitis B or any of the other disorders and/or conditions provided herein.
Examples of infectious disease include, but are not limited to, viral infectious diseases, such as AIDS, Chickenpox (Varicella), Common cold, Cytomegalovirus Infection, Colorado tick fever, Dengue fever, Ebola hemorrhagic fever, Hand, foot and mouth disease, Hepatitis, Herpes simplex, Herpes zoster, HPV, Influenza (Flu), Lassa fever, Measles, Marburg hemorrhagic fever, Infectious mononucleosis, Mumps, Norovirus, Poliomyelitis, Progressive multifocal leukencephalopathy, Rabies, Rubella, SARS, Smallpox (Variola), Viral encephalitis, Viral gastroenteritis, Viral meningitis, Viral pneumonia, West Nile disease and Yellow fever; bacterial infectious diseases, such as Anthrax, Bacterial Meningitis, Botulism, Brucellosis, Campylobacteriosis, Cat Scratch Disease, Cholera, Diphtheria, Epidemic Typhus, Gonorrhea, Impetigo, Legionellosis, Leprosy (Hansen's Disease), Leptospirosis, Listeriosis, Lyme disease, Melioidosis, Rheumatic Fever, MRSA infection, Nocardiosis, Pertussis (Whooping Cough), Plague, Pneumococcal pneumonia, Psittacosis, Q fever, Rocky Mountain Spotted Fever (RMSF), Salmonellosis, Scarlet Fever, Shigellosis, Syphilis, Tetanus, Trachoma, Tuberculosis, Tularemia, Typhoid Fever, Typhus and Urinary Tract Infections; parasitic infectious diseases, such as African trypanosomiasis, Amebiasis, Ascariasis, Babesiosis, Chagas Disease, Clonorchiasis, Cryptosporidiosis, Cysticercosis, Diphyllobothriasis, Dracunculiasis, Echinococcosis, Enterobiasis, Fascioliasis,
Fasciolopsiasis, Filariasis, Free-living amebic infection, Giardiasis, Gnathostomiasis,
Hymenolepiasis, Isosporiasis, Kala-azar, Leishmaniasis, Malaria, Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Pinworm Infection, Scabies, Schistosomiasis, Taeniasis, Toxocariasis, Toxoplasmosis, Trichinellosis, Trichinosis, Trichuriasis, Trichomoniasis and Trypanosomiasis; fungal infectious disease, such as Aspergillosis, Blastomycosis,
Candidiasis, Coccidioidomycosis, Cryptococcosis, Histoplasmosis, Tinea pedis (Athlete's Foot) and Tinea cruris; prion infectious diseases, such as Alpers' disease, Fatal Familial Insomnia, Gerstmann-Straussler-Scheinker syndrome, Kuru and Variant Creutzfeldt-Jakob disease.
Examples of cancers include, but are not limited to breast cancer; biliary tract cancer; bladder cancer; brain cancer including glioblastomas and meduUoblastomas; 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.
Examples of metabolic diseases include, but are not limited to, disorders of carbohydrate metabolism, amino acid metabolism, organic acid metabolism, fatty acid oxidation and mitochondrial metabolism, prophyrin metabolism, purine or pyrimidine metabolism, steroid metabolism, lysosomal mitochondrial function, peroxisomal function, lysosomal storage, urea cycle disorders (e.g., N-acetyl glutamate synthetase deficiency, carbamylphosphate synthase deficiency, ornithine carbamyl transferase deficiency, crgino succinic aciduria, citrullinaemia, arginase deficiency), amino acid disorders (e.g., Non- ketotic hyperglycinaemia, tyrosinaemia (Type I), Maple syrup urine disease), organic acidemias (e.g, isovaleric acidemia, methylmalonic acidemia, propionic acidemia, glutaric aciduria type I, glutaric acidemia type I & II), mitochondrial disorders (e.g., carboxylase defects, mitochondrial myopathies, lactic acidosis (pyruvate dehydrogenase complex defects), congenital lactic acidosis, mitochondrial respiratory chain defects, cystinosis, Gaucher' s disease, Fabry's disease, Pompe's disease, mucopolysaccharoidosis I,
mucopolysaccharoidosis II, mucopolysaccharoidosis VI).
Examples of degenerative diseases include, but are not limited to,
mesenchyme/mesoderm degenerative disease, muscle degenerative disease, endothelial degenerative disease, neurodegenerative disease, degenerative joint disease (e.g.,
osteoarthritis), major types of degenerative heart disease (e.g., coronary heart disease, congenital heart disease, rheumatic heart disease, angina pectoris), neurodegenerative disease (e.g., Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, Lewy body disease, Parkinson's disease, spinal muscular atrophy), neuromuscular disorders (e.g., muscular dystrophy, duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, myotonic muscular dystrophy, congenital myopathy, familial cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, or coronary artery disease).
The proteins for coupling to the synthetic nanocarriers and/or the additional antigens provided herein can be antigens associated with any of the diseases or conditions provided herein. These include antigens associated with cancer, infections or infectious disease or degenerative or non-autoimmune disease. Antigens associated with HIV, malaria, leischmaniasis, a human filovirus infection, a togavirus infection, a alphavirus infection, an arenavirus infection, a bunyavirus infection, a flavivirus infection, a human papillomavirus infection, a human influenza A virus infection, a hepatitis B infection or a hepatitis C infection are also included.
Examples of cancer antigens include HER 2 (pi 85), CD20, CD33, GD3 ganglioside, GD2 ganglioside, carcinoembryonic antigen (CEA), CD22, milk mucin core protein, TAG- 72, Lewis A antigen, ovarian associated antigens such as OV-TL3 and MOvl8, high Mr melanoma antigens recognized by antibody 9.2.27, HMFG-2, SM-3, B72.3, PR5C5, PR4D2, and the like. Further examples include MAGE, MART-l/Melan-A, gplOO, Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), FAP, cyclophilin b, Colorectal associated antigen (CRC)— C017-1A/GA733, Carcinoembryonic Antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, amll, prostatic acid phosphatase (PAP), Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, pro state- specific membrane antigen (PSMA), T-cell receptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-I or MAGE-II families) (e.g., MAGE-A1, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-Al l, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-family of tumor antigens (e.g., GAGE-1, GAGE-2, GAGE- 3, GAGE-4, GAGE- 5, GAGE-6, GAGE-7, GAGE- 8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1, a-fetoprotein, E- cadherin, a-catenin, β-catenin and γ-catenin, pl20ctn, gplOOPmell 17, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, pl5, gp75, GM2 and GD2 gangliosides, viral products such as human papilloma virus proteins, Smad family of tumor antigens, lmp-1, PIA, EBV-encoded nuclear antigen (EBNA)-l, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, CD20 and c-erbB-2.
In another embodiment, antigens associated with infection or infectious disease are associated with 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 infectious agent is 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 infectious agent is 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 infectious agent is 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 infectious agent is a fungus of the Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis or Stachybotrys genus. In still another embodiment, the infectious agent is C. albicans, Aspergillus fumigatus, Aspergillus flavus, Cryptococcus neoformans, Cryptococcus laurentii, Cryptococcus albidus, Cryptococcus gattii, Histoplasma capsulatum, Pneumocystis jirovecii or Stachybotrys chartarum.
In yet another embodiment, the antigen associated with infection or infectious disease is one that 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, gpl20, 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 antigen comprises 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, Spl, Sp2, Sp3, Glycerophosphodiester
Phosphodiesterase, outer membrane protein, chaperone-usher protein, capsular protein (Fl) or V protein. In yet another embodiment, the antigen is one that comprises capsular glycoprotein, Yps3P, Hsp60, Major surface protein, MsgCl, MsgC3, MsgC8, MsgC9 or SchS34. EXAMPLES
Example 1: Synthetic Nanocarrier Formulation Lot #1 Materials
Ovalbumin protein, was purchased from Worthington Biochemical Corporation (730
Vassar Avenue, Lakewood, NJ 08701. Product Code 3048.) PLGA-R848 of approximately 5,200 Da made from PLGA of 3: 1 lactide to glycolide ratio and having 12.7% w/w conjugated R848 content was synthesized. PLA-PEG-Nicotine with a nicotine-terminated PEG block of approximately 5,000 Da and DL-PLA block of approximately 19,000 Da was synthesized. PLA with an inherent viscosity of 0.21 dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211. Product Code 100 DL 2A.) Polyvinyl alcohol (Mw = 11,000 - 31,000, 87-89% hydrolyzed) was purchased from J.T. Baker (Part Number U232-08).
Method
Solutions were prepared as follows:
Solution 1: Ovalbumin protein at 20 mg/mL in lOmM phosphate buffer.
Solution 2: PLGA-R848 at 50 mg/mL, PLA-PEG-Nicotine at 25 mg/mL, PLA at 25 mg/ml in dichloromethane. The solution was prepared by separately dissolving each polymer as a 100 mg/mL in dichloromethane, then mixing the solutions by adding 2 parts PLGA- R848 solution to 1 part each PLA-PEG-Nicotine solution and PLA solution.
Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in lOOmM phosphate buffer, pH 8.
Solution 4: 70 mM phosphate buffer, pH 8.
A primary (Wl/O) emulsion was first created using Solution 1 & Solution 2. Solution 1 (0.2 mL) and Solution 2 (1.0 mL) were combined in a small glass pressure tube and sonicated at 50% amplitude for 40 seconds using a Branson Digital Sonifier 250. A secondary (W1/0/W2) emulsion was then formed by adding Solution 3 (2.0 mL) to the primary emulsion, and then sonicating at 30% amplitude for 40 seconds using the Branson Digital Sonifier 250. The secondary emulsion was 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 13,823g for one hour, removing the supernatant, and re-suspending the pellet in phosphate buffered saline. This washing procedure was repeated and then the pellet was re-suspended in phosphate buffered saline to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. The suspension was stored frozen at -20°C until use.
Table 1 : Nanocarrier Characterization Nanocarrier Effective R848 (% Ovalbumin (%
ID Diameter (nm) w/w) w/w)
1 214 4.0 1.1
Example 2: Synthetic Nanocarrier Formulation Lot #2 Materials
Ovalbumin protein, was purchased from Worthington Biochemical Corporation (730 Vassar Avenue, Lakewood, NJ 08701. Product Code 3048.) Ovalbumin peptide 323-339 amide acetate salt, was purchased from Bachem Americas Inc. (3132 Kashiwa Street, Torrance CA 90505. Product code 4065609.) PLGA-R848 of approximately 5,200 Da made from PLGA of 3: 1 lactide to glycolide ratio and having 12.7% w/w conjugated R848 content was synthesized. PLA-PEG-Nicotine with a nicotine-terminated PEG block of
approximately 5,000 Da and DL-PLA block of approximately 19,000 Da was synthesized. PLA with an inherent viscosity of 0.21 dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211. Product Code 100 DL 2A.) Polyvinyl alcohol (Mw = 11,000 - 31,000, 87-89% hydrolyzed) was purchased from J.T. Baker (Part Number U232-08).
Method
Solutions were prepared as follows:
Solution 1A: Ovalbumin protein at 40 mg/mL in lOmM phosphate buffer.
Solution IB: Ovalbumin peptide amide 323 - 339 @ 40 mg/mL in dilute hydrochloric acid aqueous solution.
Solution 2: PLGA-R848 at 50 mg/mL, PLA-PEG-Nicotine at 25 mg/mL, PLA at 25 mg/ml in dichloromethane. The solution was prepared by separately dissolving each polymer at 100 mg/mL in dichloromethane, then mixing the solutions by adding 2 parts PLGA-R848 solution to 1 part each PLA-PEG-Nicotine solution and PLA solution.
Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in lOOmM phosphate buffer, pH 8.
Solution 4: 70 mM phosphate buffer, pH 8.
The first primary (Wl/O) emulsion was created using Solution 1A & Solution 2.
Solution 1A (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 second primary (Wl/O) emulsion was created using Solution IB & Solution 2. Solution IB (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. Approximately ½ of the second primary emulsion was removed from its pressure tube and discarded and then ½ of the first primary emulsion (0.500 mL), was added to the tube to create a 1: 1 mixture of the two primary emulsions in a total volume of approximately 1 mL. A secondary (W1/0/W2) emulsion was then formed by adding Solution 3 (2.0 mL) to the primary emulsion, and then sonicating at 30% amplitude for 40 seconds using the Branson Digital Sonifier 250. The secondary emulsion was 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 13,823g for one hour, removing the supernatant, and re-suspending the pellet in phosphate buffered saline. This washing procedure was repeated and then the pellet was re- suspended in phosphate buffered saline to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. The suspension was stored frozen at -20°C until use. Table 2: Nanocarrier Characterization
Figure imgf000052_0001
Example 3: Synthetic Nanocarrier Formulation Lot #3 Materials
Ovalbumin protein, was purchased from Worthington Biochemical Corporation (730 Vassar Avenue, Lakewood, NJ 08701. Product Code 3048.) PLGA-R848 of approximately 5,200 Da made from PLGA of 3: 1 lactide to glycolide ratio and having 12.7% w/w conjugated R848 content was synthesized. PLA-PEG-OMe block co-polymer with a methyl ether terminated PEG block of 2,000 Da and DL-PLA block of approximately 19,000 Da was synthesized. PLA with an inherent viscosity of 0.21 dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211. Product Code 100 DL 2A.) Polyvinyl alcohol (Mw = 11,000 - 31,000, 87-89% hydrolyzed) was purchased from J.T. Baker (Part Number U232-08).
Method
Solutions were prepared as follows:
Solution 1: Ovalbumin protein at 20 mg/mL in lOmM phosphate buffer.
Solution 2: PLGA-R848 at 50 mg/mL, PLA-PEG-OMe at 25 mg/mL, PLA at 25 mg/ml in dichloromethane. The solution was prepared by separately dissolving each polymer as a 100 mg/mL in dichloromethane, then mixing the solutions by adding 2 parts PLGA- R848 solution to 1 part each PLA-PEG-OMe solution and PLA solution.
Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in lOOmM phosphate buffer, pH 8.
Solution 4: 70 mM phosphate buffer, pH 8.
A primary (Wl/O) emulsion was first created using Solution 1 & Solution 2. Solution 1 (0.2 mL) and Solution 2 (1.0 mL) were combined in a small glass pressure tube and sonicated at 50% amplitude for 40 seconds using a Branson Digital Sonifier 250. A secondary (W1/0/W2) emulsion was then formed by adding Solution 3 (2.0 mL) to the primary emulsion, and then sonicating at 30% amplitude for 40 seconds using the Branson Digital Sonifier 250. The secondary emulsion was 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 13,823g for one hour, removing the supernatant, and re-suspending the pellet in phosphate buffered saline. This washing procedure was repeated and then the pellet was re-suspended in phosphate buffered saline to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. The suspension was stored frozen at -20°C until use.
Table 3: Nanocarrier Characterization Nanocarrier ID Effective R848 (% w/w) Ovalbumin (% w/w)
Diameter (nm)
3 217 4.3 0.8
Example 4: Synthetic Nanocarrier Formulation Lot #4
Ovalbumin protein, was purchased from Worthington Biochemical Corporation (730 Vassar Avenue, Lakewood, NJ 08701. Product Code 3048.) Ovalbumin peptide 323-339 amide acetate salt, was purchased from Bachem Americas Inc. (3132 Kashiwa Street,
Torrance CA 90505. Product code 4065609.) PLGA-R848 of approximately 5,200 Da made from PLGA of 3: 1 lactide to glycolide ratio and having 12.7% w/w conjugated R848 content was synthesized. PLA-PEG-OMe block co-polymer with a methyl ether terminated PEG block of 2,000 Da and DL-PLA block of approximately 19,000 Da was synthesized. PLA with an inherent viscosity of 0.21 dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211. Product Code 100 DL 2A.) Polyvinyl alcohol (Mw = 11,000 - 31,000, 87-89% hydrolyzed) was purchased from J.T. Baker (Part Number U232-08). Method
Solutions were prepared as follows:
Solution 1A: Ovalbumin protein at 40 mg/mL in lOmM phosphate buffer.
Solution IB: Ovalbumin peptide amide 323 - 339 @ 40 mg/mL in dilute hydrochloric acid aqueous solution.
Solution 2: PLGA-R848 at 50 mg/mL, PLA-PEG-OMe at 25 mg/mL, PLA at 25 mg/ml in dichloromethane. The solution was prepared by separately dissolving each polymer as a 100 mg/mL in dichloromethane, then mixing the solutions by adding 2 parts PLGA- R848 solution to 1 part each PLA-PEG-OMe solution and PLA solution.
Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in lOOmM phosphate buffer, pH 8.
Solution 4: 70 mM phosphate buffer, pH 8.
The first primary (Wl/O) emulsion was created using Solution 1A & Solution 2. Solution 1A (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 second primary (Wl/O) emulsion was created using Solution IB & Solution 2. Solution IB (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. The two primary emulsions were combined, in part, by transferring 0.6 mL of each into a third glass pressure tube to create a 1 : 1 mixture of the two primary emulsions in a total volume of approximately 1.2 mL. A secondary (W1/0/W2) emulsion was then formed by adding Solution 3 (2.0 mL) to the primary emulsion, and then sonicating at 30% amplitude for 40 seconds using the Branson Digital Sonifier 250. The secondary emulsion was 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 13,823g for one hour, removing the supernatant, and re-suspending the pellet in phosphate buffered saline. This washing procedure was repeated and then the pellet was re-suspended in phosphate buffered saline to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. The suspension was stored frozen at -20°C until use.
Table 4: Nanocarrier Characterization
Figure imgf000055_0001
Example 5: Synthetic Nanocarrier Formulation Lot #5 Materials
SIINFEKL (SEQ ID NO: l) (ovalbumin peptide [257 - 264]), was purchased from Bachem Americas Inc. (3132 Kashiwa Street, Torrance CA 90505. Product code H-4866.) Ovalbumin peptide 323-339 amide acetate salt, was purchased from Bachem Americas Inc. (3132 Kashiwa Street, Torrance CA 90505. Product code 4065609.) PLGA-R848 of approximately 4,500 Da made from PLGA of 3: 1 lactide to glycolide ratio and having 15% w/w conjugated R848 content was synthesized. PLA-PEG-Nicotine with a nicotine- terminated PEG block of approximately 5,000 Da and DL-PLA block of approximately 17,000 Da was synthesized. PLA with an inherent viscosity of 0.21 dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211. Product Code 100 DL 2A.) Polyvinyl alcohol (Mw = 11,000 - 31,000, 87-89% hydrolyzed) was purchased from J.T. Baker (Part Number U232-08).
Method
Solutions were prepared as follows:
Solution 1A: SIINFEKL (SEQ ID NO: l) @ 200 mg/mL DMSO.
Solution IB: Ovalbumin peptide amide 323 - 339 @ 20 mg/mL in dilute hydrochloric acid aqueous solution.
Solution 2: PLGA-R848 at 50 mg/mL, PLA-PEG-Nicotine at 25 mg/mL, PLA at 25 mg/ml in dichloromethane. The solution was prepared by separately dissolving each polymer as a 100 mg/mL in dichloromethane, then mixing the solutions by adding 2 parts PLGA- R848 solution to 1 part each PLA-PEG-Nicotine solution and PLA solution.
Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in lOOmM phosphate buffer, pH 8.
Solution 4: 70 mM phosphate buffer, pH 8.
The first primary (S/O) emulsion was created using Solution 1A & Solution 2.
Solution 1A (0.025 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 serial primary (Wl/(S/0)) emulsion was created using Solution IB and the first primary emulsion. Solution IB (0.25 mL) was added to the small glass pressure tube containing the first primary emulsion and then sonicated at 50% amplitude for 40 seconds using a Branson Digital Sonifier 250. A secondary ((S+Wl)/0/W2) emulsion was then formed by adding Solution 3 (2.0 mL) to the serial primary emulsion, and then sonicating at 30% amplitude for 40 seconds using the Branson Digital Sonifier 250. The secondary emulsion was added to an open 50 mL beaker containing 70 mM phosphate buffer solution (30 mL) and stirred at room temperature for 2 hours to allow the dichloromethane to evaporate and the nanocarriers to form in suspension. A portion of the suspended nanocarriers was washed by transferring the nanocarrier suspension to a centrifuge tube, spinning at 13,823g for one hour, removing the supernatant, and re-suspending the pellet in phosphate buffered saline. This washing procedure was repeated and then the pellet was re-suspended in phosphate buffered saline to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. The suspension was stored frozen at -20°C until use.
Table 5: Nanocarrier Characterization
Figure imgf000057_0002
Example 6: Synthetic Nanocarrier Compositions Generate High Antibody Titers and Strong Antigen- Specific CTL Activity
3rd and 4th in vivo (C57BL/6 mice) immunization studies were performed. The above polymeric nanocarrier formulation (#3) delivering a TLR agonist (R848) and entrapped ovalbumin protein (OVA) was introduced and created high antibody titers (e.g., anti-OVA IgG titers of ~le6) and strong antigen- specific CTL activity from local lymph and spleen cells.
Immunized mice were bled at dates indicated and antibodies to ovalbumin measured in standard ELISA using serial dilutions of test sera. Biotinylated goat anti-mouse Ig was used as a detection antibody (BD Biosciences, San Diego, CA). EC50 was determined based on titration curves. CTL activity was measured as follows. 4-5 days after the final injection (subcutaneous, s.c, or intranasal, i.n.) with the nanocarrier preparations or protein controls draining lymph nodes (LNs) were removed, treated with collagenase, homogenized, washed and incubated with 10-100 units/ml of IL-2 for 4-5 days. Then resulting cell populations were counted and used as effector cells in cytotoxicity assays. Syngeneic EL-4 cells pulsed with SIINFEKL (SEQ ID NO: l) peptide or EG.7-OVA cells (stably transfected with ovalbumin) served as targets with intact EL-4 cells providing for background control.
Cytoxicity at various effectontarget ratios was measured over 24 hours (37°C) using CytoTox-ONE™ Homogenuous Membrane Integrity Assay (Promega, Madison, WI) according to manufacturer's recommendations.
Table 6. Formulation of Nanocarrier
Figure imgf000057_0001
TLR Agonist PLGA-R848 (50%)
Matrix polymer 1 PLA-PEG (25%)
Matrix Polymer 2 100 DL 2A (25%)
Table 7. Experiment 1 Layout
Figure imgf000058_0001
Table 8. Experiment 2 layout
Figure imgf000058_0002
Example 7: Synthetic Nanocarrier Formulation Lot #6 Materials
Ovalbumin protein, was purchased from Worthington Biochemical Corporation (730 Vassar Avenue, Lakewood, NJ 08701). Product Code LS003054. PLGA-R848, Poly-D/L- lactide-co-glycolide, 4-amino-2-(ethoxymethyl)-a,a-dimethyl- lH-imidazo[4,5-c]quinoline- 1- ethanol amide of approximately 7,800 Da made from PLGA of 3: 1 lactide to glycolide ratio and having 8.5% w/w conjugated resiquimod content was custom manufactured at Princeton Global Synthesis (300 George Patterson Drive #206, Bristol, PA 19007.) Lot number PGS 16-52. PLA with an inherent viscosity of 0.21 dL/g was purchased from SurModics
Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211. Product Code 100 DL 2A.) PLA-PEG-OMe block co-polymer with a methyl ether terminated PEG block of approximately 5,000 Da and PLA block of approximately 21,000 Da by 1H-NMR (Mn of 21kDa) was synthesized. EMPROVE® Polyvinyl Alcohol 5-88, USP (85-89% hydrolyzed, viscosity of 4.3-5.7 mPa.s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027. Part Number 1.41354). Phosphate-buffered saline IX (PBS IX). From Mediatech Inc. (9345 Discovery Blvd. Manassas, VA 20109.) Product Code 21- 040-CV.
Method
Solutions were prepared as follows:
Solution 1: Ovalbumin protein @ 20 mg/mL was prepared in PBS IX at room temperature.
Solution 2: PLA was prepared by dissolving PLA at 100 mg per 1 mL of
dichloromethane in the chemical fume hood.
Solution 3: PLA-PEG-OMe was prepared by dissolving PLA-PEG-OMe at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 4: PLGA-R848 was prepared by dissolving PLGA-R848 at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 5: Polyvinyl alcohol @ 50 mg/mL in 100 mM in lOOmM phosphate buffer, pH 8.
Solution 6: 70 mM phosphate buffer, pH 8.
This lot was prepared in duplicate, then combined after washing. A primary (Wl/O) emulsion was first created by mixing Solutions 1 through 4. Solution 1 (0.2 mL), Solution 2 (0.50 mL), Solution 3 (0.25mL) and Solution 4 (0.25mL) were combined in a small glass pressure tube and sonicated at 50% amplitude for 40 seconds using a Branson Digital Sonifier 250. A secondary (W1/0/W2) emulsion was then formed by adding Solution 5 (2.0 mL) to the primary emulsion, vortexing to create a crude dispersion, and then sonicating at 30% amplitude for 60 seconds using the Branson Digital Sonifier 250. The secondary emulsion was added to an open 50 mL beaker containing Solution 6 (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 PBS IX to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. The suspension was stored frozen at -20C until use.
Table 9: Nanocarrier Characterization
Figure imgf000060_0001
Example 8: Synthetic Nanocarrier Formulation Lot #7 Materials
Ovalbumin protein, was purchased from Worthington Biochemical Corporation (730 Vassar Avenue, Lakewood, NJ 08701). Product Code LS003054. PLGA-R848, Poly-D/L- lactide-co-glycolide, 4-amino-2-(ethoxymethyl)-a,a-dimethyl- lH-imidazo[4,5-c]quinoline- 1- ethanol amide of approximately 7,800 Da made from PLGA of 3: 1 lactide to glycolide ratio and having 8.5% w/w conjugated resiquimod content was custom manufactured at Princeton Global Synthesis (300 George Patterson Drive #206, Bristol, PA 19007.) Lot number PGS 16-52. PLA with an inherent viscosity of 0.21 dL/g was purchased from SurModics
Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211. Product Code 100 DL 2A.) PLA-PEG-OMe block co-polymer with a methyl ether terminated PEG block of approximately 5,000 Da and PLA block of approximately 21,000 Da by 1H-NMR (Mn of 21kDa) was synthesized. EMPROVE® Polyvinyl Alcohol 5-88, USP (85-89% hydrolyzed, viscosity of 4.3-5.7 mPa.s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027. Part Number 1.41354). Phosphate-buffered saline IX (PBS IX). From Mediatech Inc. (9345 Discovery Blvd. Manassas, VA 20109.) Product Code 21- 040-CV.
Method
Solutions were prepared as follows:
Solution 1: Ovalbumin protein @ 5 mg/mL was prepared in PBS IX at room temperature.
Solution 2: PLA was prepared by dissolving PLA at 100 mg per 1 mL of
dichloromethane in the chemical fume hood.
Solution 3: PLA-PEG-OMe was prepared by dissolving PLA-PEG-OMe at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 4: PLGA-R848 was prepared by dissolving PLGA-R848 at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 5: Polyvinyl alcohol @ 50 mg/mL in 100 mM in lOOmM phosphate buffer, pH 8.
Solution 6: 70 mM phosphate buffer, pH 8.
This lot was prepared in duplicate, then combined after washing. A primary (Wl/O) emulsion was first created by mixing Solutions 1 through 4. Solution 1 (0.2 mL), Solution 2 (0.50 mL), Solution 3 (0.25mL) and Solution 4 (0.25mL) were combined in a small glass pressure tube and sonicated at 50% amplitude for 40 seconds using a Branson Digital Sonifier 250. A secondary (W1/0/W2) emulsion was then formed by adding Solution 5 (2.0 mL) to the primary emulsion, vortexing to create a crude dispersion, and then sonicating at 30% amplitude for 60 seconds using the Branson Digital Sonifier 250. The secondary emulsion was added to an open 50 mL beaker containing Solution 6 (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 PBS IX to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. The suspension was stored frozen at -20C until use. Table 10: Nanocarrier Characterization
Figure imgf000062_0001
Example 9: Synthetic Nanocarrier Formulation Lot #8
Materials
Ovalbumin protein, was purchased from Worthington Biochemical Corporation (730 Vassar Avenue, Lakewood, NJ 08701). Product Code LS003054. PLGA-R848, Poly-D/L- lactide-co-glycolide, 4-amino-2-(ethoxymethyl)-a,a-dimethyl- lH-imidazo[4,5-c]quinoline- 1- ethanol amide of approximately 7,800 Da made from PLGA of 3: 1 lactide to glycolide ratio and having 8.5% w/w conjugated resiquimod content was custom manufactured at Princeton Global Synthesis (300 George Patterson Drive #206, Bristol, PA 19007.) PLA with an inherent viscosity of 0.21 dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211. Product Code 100 DL 2A.) PLA-PEG-OMe block co-polymer with a methyl ether terminated PEG block of approximately 5,000 Da and PLA block of approximately 21,000 Da by 1H-NMR (Mn of 21kDa) was synthesized.
EMPROVE® Polyvinyl Alcohol 5-88, USP (85-89% hydrolyzed, viscosity of 4.3-5.7 mPa.s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027. Part Number 1.41354). Phosphate-buffered saline IX (PBS IX). From Mediatech Inc. (9345 Discovery Blvd. Manassas, VA 20109.) Product Code 21 -040-CV.
Method
Solutions were prepared as follows:
Solution 1: Ovalbumin protein @ 20 mg/mL was prepared in PBS IX at room temperature.
Solution 2: PLA was prepared by dissolving PLA at 100 mg per 1 mL of
dichloromethane in the chemical fume hood.
Solution 3: PLA-PEG-OMe was prepared by dissolving PLA-PEG-OMe at 100 mg per 1 mL of dichloromethane in the chemical fume hood. Solution 4: PLGA-R848 was prepared by dissolving PLGA-R848 at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 5: Polyvinyl alcohol @ 50 mg/mL in 100 mM in lOOmM phosphate buffer, pH 8.
Solution 6: 70 mM phosphate buffer, pH 8.
This lot was prepared in duplicate, then combined after washing. A primary (Wl/O) emulsion was first created by mixing Solutions 1 through 4. Solution 1 (0.2 mL), Solution 2 (0.50 mL), Solution 3 (0.25mL) and Solution 4 (0.25mL) were combined in a small glass pressure tube and sonicated at 50% amplitude for 40 seconds using a Branson Digital Sonifier 250. A secondary (W1/0/W2) emulsion was then formed by adding Solution 5 (2.0 mL) to the primary emulsion, vortexing to create a crude dispersion, and then sonicating at 30% amplitude for 60 seconds using the Branson Digital Sonifier 250. The secondary emulsion was added to an open 50 mL beaker containing Solution 6 (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 PBS IX to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. The suspension was stored frozen at -20C until use.
Table 11 : Nanocarrier Characterization
Figure imgf000063_0001
Example 10: Synthetic Nanocarrier Compositions Generate High Antibody Titers and Strong Antigen- Specific CTL Activity
Synthetic nanocarriers delivering R848 and OVA was as successful as a positive comparator control consisting of high dose of PS-CpG plus a 6x higher dose of free OVA at generating a central (spleen) OVA-specific CTL response and also in creating as strong (or stronger) OVA-specific humoral response. 4-5 days after s.c. injection with nanocarrier preparations or controls draining lymph nodes (LNs) were removed, treated with collagenase, homogenized, washed and incubated with 10-100 units/ml of IL-2 for 4-5 days. Then resulting cell populations were counted and used as effector cells in cytotoxicity assay. Syngeneic EL-4 cells pulsed with SIINFEKL (SEQ ID NO: l) peptide or EG.7-OVA cells (stably transfected with ovalbumin) served as targets with intact EL-4 cells providing for background control. Cytoxicity at various effectontarget ratios was measured over 24 hours (37°C) using CytoTox-ONE™
Homogenuous Membrane Integrity Assay (Promega, Madison, WI) according to
manufacturer's recommendations.
Table 12
Figure imgf000064_0001
Example 11 : Measure Development of Humoral and Cellular Immune Responses to
Nanocarrier-encapsulated Antigen After a Single Injection of NC-OVA + NC-R848 Mix
Materials - Lot #9
PLGA-R848 (S-205), Poly-D/L-lactide-co-glycolide, 4-amino-2-(ethoxymethyl)-a,a- dimethyl-lH-imidazo[4,5-c]quinoline-l-ethanol amide of approximately 7,800 Da made from PLGA of 3: 1 lactide to glycolide ratio and having 8.5% w/w conjugated resiquimod content was custom manufactured at Princeton Global Synthesis (300 George Patterson Drive #206, Bristol, PA 19007.). PLA with an inherent viscosity of 0.19 dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211. Product Code 100 DL 2A). PLA-PEG-OMe block co-polymer with a methyl ether terminated PEG block of approximately 5,000 Da and PLA block of approximately 28,000 Da was purchased from SurModics Pharmaceuticals (Product Code 100 DL mPEG 5000 5CE). EMPROVE® Polyvinyl Alcohol 4-88, USP (85-89% hydrolyzed, viscosity of 3.4-4.6 mPa.s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027). Phosphate- buffered saline IX (PBS IX). From Mediatech Inc. (9345 Discovery Blvd. Manassas, VA 20109.) Product Code 21 -040-CV. Method - Lot #9
Solutions were prepared as follows:
Solution 1: PLGA-R848 was prepared by weighing out PLGA-R848, PLA, and PLA- PEG-OMe powders in 2: 1: 1 weight ratio and then dissolving the mixed polymers in dichloromethane to achieve a total polymer concentration of 100 mg per 1 mL.
Solution 2: Polyvinyl alcohol @ 35 mg/mL in lOOmM phosphate buffer, pH 8.
An O/W emulsion was created by mixing Solutions 1 and 2, and then creating a coarse emulsion prior to a fine emulsion. Solution 1 (2 mL) was coarsely emulsified with Solution 2 (8 mL), using 10 passes through an 18G emulsification needle. A fine emulsion was made by loading the coarse emulsion into a primed and ice-water-chilled high pressure homogenizer (Microfluidics LV1) and performing three passes at 5000 psi. The fine O/W emulsion was added to an open 100 mL beaker containing IX PBS (60 mL) and stirred at room temperature for more than 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 75,600 rcf for 35 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 PBS IX to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. The nanocarrier formation process was repeated another three times at lx or 2x the same scale. The four suspensions were combined and then filtered through 0.22 micron PES syringe filter and then stored frozen at -20C until use.
Materials - Lot #10
PLGA-R848 (S-205), Poly-D/L-lactide-co-glycolide, 4-amino-2-(ethoxymethyl)-a,a- dimethyl-lH-imidazo[4,5-c]quinoline-l-ethanol amide of approximately 7,800 Da made from PLGA of 3: 1 lactide to glycolide ratio and having 8.5% w/w conjugated resiquimod content was custom manufactured at Princeton Global Synthesis (300 George Patterson Drive #206, Bristol, PA 19007.). PLA with an inherent viscosity of 0.19 dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211. Product Code 100 DL 2A). PLA-PEG-OMe block co-polymer with a methyl ether terminated PEG block of approximately 5,000 Da and PLA block of approximately 28,000 Da was purchased from SurModics Pharmaceuticals (Product Code 100 DL mPEG 5000 5CE). EMPROVE® Polyvinyl Alcohol 4-88, USP (85-89% hydrolyzed, viscosity of 3.4-4.6 mPa.s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027). Phosphate- buffered saline IX (PBS IX). From Mediatech Inc. (9345 Discovery Blvd. Manassas, VA 20109.) Product Code 21 -040-CV.
Method - Lot #10
Solutions were prepared as follows:
Solution 1: PLGA-R848 was prepared by dissolving PLGA-R848 at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 2: PLA was prepared by dissolving PLA at 100 mg per 1 mL of
dichloromethane in the chemical fume hood.
Solution 3: PLA-PEG-OMe was prepared by dissolving PLA-PEG-OMe at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 4: Polyvinyl alcohol @ 50 mg/mL in lOOmM phosphate buffer, pH 8.
A primary (O/W) emulsion was created by mixing Solutions 1 through 4 and creating a coarse emulsion prior to a fine emulsion. Solution 1 (1 mL), Solution 2 (0.5 mL), and Solution 3 (0.5mL) were combined first and then coarsely emulsified with Solution 4 (8 mL), by stirring together at 350 rpm in a 50 mL beaker for two minutes and by repeat pipetting. A fine emulsion, was made by loading the coarse emulsion into a primed high pressure homogenizer (Microfluidics LV1) and performing three passes at 5000 psi. The fine O/W emulsion was added to an open 50 mL beaker containing IX PBS (30 mL) and stirred at room temperature for more than 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 75,600 rcf for 35 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 PBS IX to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. Nanocarrier suspension was then filtered through a 0.22 micron PES syringe filter and then stored frozen at -20C until use. Materials - Lot #11
Ovalbumin protein, was purchased from Worthington Biochemical Corporation (730 Vassar Avenue, Lakewood, NJ 08701). Product Code LS003054. PLGA with 75% lactide and 25% glycolide content and an inherent viscosity of 0.24 dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211. Product Code 7525 DLG 2.5A). PLA with an inherent viscosity of 0.2 dL/g was purchased from
SurModics Pharmaceuticals (Product Code 100 DL 2A). PLA-PEG-OMe block co-polymer with a methyl ether terminated PEG block of approximately 5,000 Da and PLA block of approximately 21,000 Da was synthesized. EMPROVE® Polyvinyl Alcohol 4-88, USP (85- 89% hydrolyzed, viscosity of 3.4-4.6 mPa.s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027). Phosphate-buffered saline IX (PBS IX). From Mediatech Inc. (9345 Discovery Blvd. Manassas, VA 20109.) Product Code 21-040- CV.
Method - Lot #11
Solutions were prepared as follows:
Solution 1: Ovalbumin protein @ 50 mg/mL was prepared in PBS IX at room temperature.
Solution 2: PLGA, PLA, and PLA-PEG-OMe were weighed out in 2: 1: 1 weight ratio and dissolved in dichloromethane in the chemical fume hood to achieve a final total polymer concentration of 100 mg per 1 mL.
Solution 3: Polyvinyl alcohol @ 50 mg/mL in lOOmM phosphate buffer, pH 8.
Solution 4: 70 mM phosphate buffer, pH 8.
A primary (Wl/O) emulsion was first created by mixing Solutions 1 and 2. Solution 1 (66 mL) and Solution 2 (264 mL) were combined and formed into a coarse emulsion using an overhead mixer in a 1 L beaker. The coarse emulsion was transferred to a custom-made temperature-controlled homogenization vessel and homogenized using a high- shear rotor stator. A coarse secondary (W1/0/W2) emulsion was then formed by transferring ½ of the primary emulsion into a beaker containing 330 mL of Solution 3 and mixing with an overhead mixer. The coarse secondary emulsion was then returned to a custom
homogenization vessel and homogenized to a fine emulsion using high shear. The secondary emulsion was then added to a purged 6L vessel containing Solution 4 (3.2 L) and shaken overnight at room temperature to evaporate and the nanocarriers to form in suspension. Portions (30 mL each) of the suspended nanocarriers were washed by transferring the nanocarrier suspension to a centrifuge tube, spinning at 75,600 rcf for 35 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 PBS IX to achieve the target nanocarrier concentration. The suspension was filtered and stored frozen at -20C until use.
C57BL/6 female mice (2-3/group) were immunized once by a mix of 100 μg NC- OVA and 100 μg of NC-R848 containing 4.3 μg R848 and 6.2 μg of OVA (per each mouse). At days 4, 7, 10 and 14 after nanocarrier inoculation mice were bled and their antibody (IgG) titer against OVA determined by ELISA. Additionally, at the same time-points mice were injected (i.v.) by syngeneic splenocytes pulsed by a peptide representing a dominant CTL epitope of OVA (SIINFEKL (SEQ ID NO: l)) and differentially labeled by CSFE. The next day, splenocytes from immunized mice were taken and analyzed by FACS and specific cytotoxicity in each animal determined compared to basic cytotoxicity level in PBS-injected (naive) animals (%=100x[l-RRnaive/RRimm]).
A single immunization with NC-OVA + NC-R848 leads to rapid induction of cellular and humoral immune responses with the former being detected as early as four days after injection and then persisting for at least ten days with a peak at day 7, and the latter being detected at seven days after injection and reaching a significant level at 10 days after inoculation.
Example 12: Synthetic Nanocarriers Delivering CpG and OVA Materials - Lot #12
PO-1826 DNA oligonucleotide with phosphodiester backbone having nucleotide sequence 5'-TCC ATG ACG TTC CTG ACG TT-3' (SEQ ID NO:2) with a sodium counter- ion was purchased from Oligo Factory (120 Jeffrey Avenue, Holliston, MA 01746.) PLGA having 54% lactide and 46% glycolide content and an inherent viscosity of 0.24 dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211. Product Code 5050 DLG 2.5 A). PLGA-PEG-OMe block co-polymer with a methyl ether terminated PEG block of approximately 2,000 Da and 75% lactide/25% glycolide PLGA block of approximately 88,000 Da was purchased from SurModics Pharmaceuticals (Product Code 7525 DLG PEG 2000 7E-P). EMPROVE® Polyvinyl Alcohol 4-88, USP (85- 89% hydrolyzed, viscosity of 3.4-4.6 mPa.s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027). Phosphate-buffered saline IX (PBS IX). From Mediatech Inc. (9345 Discovery Blvd. Manassas, VA 20109.) Product Code 21-040- CV.
Method - Lot #12
Solutions were prepared as follows:
Solution 1: PO-1826 was prepared by dissolving at 40 mg per 1 mL of an aqueous solution containing 250 mg Na cholate per 1 mL endotoxin-free water.
Solution 2: PLGA was prepared by dissolving PLA at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 3: PLGA-PEG-OMe was prepared by dissolving PLGA-PEG-OMe at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 4: Polyvinyl alcohol @ 50 mg/mL in lOOmM phosphate buffer, pH 8.
Solution 5: 70 mM phosphate buffer, pH 8.
A primary (W/O) emulsion was created by mixing Solutions 1 through 3 and creating a coarse emulsion prior to a fine emulsion. Solution 1 (0.2 mL), Solution 2 (0.5 mL), and Solution 3 (0.5mL) were combined in a small glass pressure tube, coarsely emulsified by repeat pipetting, and sonicated at 50% amplitude for 40 seconds over an ice bath using a Branson Digital Sonifier 250. A secondary (W1/0/W2) emulsion was then formed by adding Solution 4 (3.0 mL) to the primary emulsion, vortexing to create a crude dispersion, and then sonicating at 30% amplitude for 60 seconds over an ice bath using the Branson Digital Sonifier 250. The fine W1/0/W2 emulsion was added to an open 50 mL beaker containing 70 mM phosphate buffer (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 90 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 PBS IX to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. Nanocarrier suspension was then filtered through 0.22 micron PES syringe filters, stored refrigerated until the concentration was determined, and then concentration adjusted and stored frozen at -20C until use. Materials - Lot #13
Ovalbumin protein, was purchased from Worthington Biochemical Corporation (730 Vassar Avenue, Lakewood, NJ 08701). Product Code LS003054. PLGA with 75% lactide and 25% glycolide content and an inherent viscosity of 0.2 dL/g was purchased from
SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211. Product Code 7525 DLG 2A). PLA-PEG-OMe block co-polymer with a methyl ether terminated PEG block of approximately 5,000 Da and PLA block of approximately 21,000 Da by 1H-NMR (Mn of 21kDa) was synthesized. EMPROVE® Polyvinyl Alcohol 4-88, USP (85-89% hydrolyzed, viscosity of 3.4-4.6 mPa.s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027). Phosphate-buffered saline IX (PBS IX). From Mediatech Inc. (9345 Discovery Blvd. Manassas, VA 20109.) Product Code 21-040-CV.
Method - Lot #13
Solutions were prepared as follows:
Solution 1: Ovalbumin protein @ 50 mg/mL was prepared in PBS IX at room temperature.
Solution 2: PLGA was prepared by dissolving PLGA at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 3: PLA-PEG-OMe was prepared by dissolving PLA-PEG-OMe at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 4: Polyvinyl alcohol @ 50 mg/mL in lOOmM phosphate buffer, pH 8.
Solution 5: 70 mM phosphate buffer, pH 8.
A primary (Wl/O) emulsion was first created by mixing Solutions 1 through 3.
Solution 1 (0.2 mL), Solution 2 (0.75 mL), and Solution 3 (0.25mL) were combined in a small glass pressure tube and sonicated at 50% amplitude for 40 seconds over an ice bath using a Branson Digital Sonifier 250. A secondary (W1/0/W2) emulsion was then formed by adding Solution 4 (3.0 mL) to the primary emulsion, vortexing to create a crude dispersion, and then sonicating at 30% amplitude for 60 seconds over an ice bath using the Branson Digital Sonifier 250. The secondary emulsion was added to an open 50 mL beaker containing Solution 5 (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 130 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 PBS IX to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. The suspension was stored frozen at -20C until use.
Materials - Lot #14
Ovalbumin protein, was purchased from Worthington Biochemical Corporation (730 Vassar Avenue, Lakewood, NJ 08701. Product Code LS003054). PLGA with 76% lactide and 24% glycolide content and an inherent viscosity of 0.69 dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211. Product Code 7525 DLG 7A). PLA with an inherent viscosity of 0.22 dL/g was purchased from SurModics Pharmaceuticals (Product Code 100 DL 2A). PLA-PEG-OMe block co-polymer with a methyl ether terminated PEG block of approximately 5,000 Da and PLA block of
approximately 21,000 Da was synthesized. EMPROVE® Polyvinyl Alcohol 4-88, USP (85- 89% hydrolyzed, viscosity of 3.4-4.6 mPa.s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027). Phosphate-buffered saline IX (PBS IX) was purchased from Mediatech Inc. (9345 Discovery Blvd. Manassas, VA 20109.) Product Code 21-040-CV. Method - Lot #14
Solutions were prepared as follows:
Solution 1: Ovalbumin protein @ 50 mg/mL was prepared in PBS IX at room temperature.
Solution 2: PLGA was prepared by dissolving PLGA at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 3: PLA was prepared by dissolving PLGA at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 4: PLA-PEG-OMe was prepared by dissolving PLA-PEG-OMe at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 5: Polyvinyl alcohol @ 50 mg/mL in lOOmM phosphate buffer, pH 8.
Solution 6: 70 mM phosphate buffer, pH 8.
A primary (Wl/O) emulsion was first created by mixing Solutions 1 through 4.
Solution 1 (0.2 mL), Solution 2 (0.5 mL), Solution 3 (0.25mL) and Solution 4 (0.25mL) were combined in a small glass pressure tube and sonicated at 50% amplitude for 40 seconds over an ice bath using a Branson Digital Sonifier 250. A secondary (W1/0/W2) emulsion was then formed by adding Solution 5 (3.0 mL) to the primary emulsion, vortexing to create a crude dispersion, and then sonicating at 30% amplitude for 60 seconds over an ice bath using the Branson Digital Sonifier 250. The secondary emulsion was added to an open 50 mL beaker containing Solution 6 (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 25,600 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 PBS IX to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. The suspension was stored frozen at -20C until use. Materials - Lot #15
PO-1826 DNA oligonucleotide with phosphodiester backbone having nucleotide sequence 5'-TCC ATG ACG TTC CTG ACG TT-3' (SEQ ID NO:2) with a sodium counter- ion was purchased from Oligo Factory (120 Jeffrey Avenue, Holliston, MA 01746.) PLGA having 54% lactide and 46% glycolide content and an inherent viscosity of 0.24 dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL
35211. Product Code 5050 DLG 2.5A). PLA-PEG-OMe block co-polymer with a methyl ether terminated PEG block of approximately 5,000 Da and PLA block of approximately 21,000 Da was synthesized. EMPROVE® Polyvinyl Alcohol 4-88, USP (85-89%
hydrolyzed, viscosity of 3.4-4.6 mPa.s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027). Na cholate was purchased from Sigma Aldrich LLC. (3050 Spruce St. St. Louis, MO 6310. Product Code C6445-100G.) Phosphate-buffered saline IX (PBS IX) was purchased from Mediatech Inc. (9345 Discovery Blvd. Manassas, VA 20109. Product Code 21-040-CV.) Method - Lot #15
Solutions were prepared as follows:
Solution 1: PO-1826 was prepared by dissolving at 40 mg per 1 mL of an aqueous solution containing 150 mg KC1 per 1 mL of endotoxin-free water. Solution 2: Na Cholate was prepared by dissolving dry powder at 200 mg per 1 mL IX PBS at room temperature.
Solution 3: PLGA was prepared by dissolving PLGA at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 4: PLGA-PEG-OMe was prepared by dissolving PLGA-PEG-OMe at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 5: Polyvinyl alcohol @ 50 mg/mL in lOOmM phosphate buffer, pH 8.
Solution 6: 70 mM phosphate buffer, pH 8.
A primary (W/O) emulsion was created by mixing Solutions 1 through 4 and creating a coarse emulsion prior to a fine emulsion. Solution 1 (0.25 mL), Solution 2 (0.25 mL), Solution 3 (0.5 mL), and Solution 4 (0.5mL) were combined in a small glass pressure tube, coarsely emulsified by repeat pipetting, and sonicated at 50% amplitude for 40 seconds over an ice bath using a Branson Digital Sonifier 250. A secondary (W1/0/W2) emulsion was then formed by adding Solution 5 (3.0 mL) to the primary emulsion, vortexing to create a coarse dispersion, and then sonicating at 30% amplitude for 60 seconds over an ice bath using the Branson Digital Sonifier 250. The fine W1/0/W2 emulsion was added to an open 50 mL beaker containing 70 mM phosphate buffer (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 90 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 PBS IX to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. Nanocarrier suspension was then stored refrigerated until the concentration was determined, and then concentration adjusted and stored frozen at -20C until use.
Materials - Lot #16
PO-1826 DNA oligonucleotide with phosphodiester backbone having nucleotide sequence 5'-TCC ATG ACG TTC CTG ACG TT-3' (SEQ ID NO:2) with a sodium counter- ion was purchased from Oligo Factory (120 Jeffrey Avenue, Holliston, MA 01746.) PLGA having 54% lactide and 46% glycolide content and an inherent viscosity of 0.24 dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211. Product Code 5050 DLG 2.5A). PLA-PEG-OMe block co-polymer with a methyl ether terminated PEG block of approximately 5,000 Da and PLA block of approximately 21,000 Da was synthesized. EMPROVE® Polyvinyl Alcohol 4-88, USP (85-89%
hydrolyzed, viscosity of 3.4-4.6 mPa.s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027). Phosphate-buffered saline IX (PBS IX) was purchased from Mediatech Inc. (9345 Discovery Blvd. Manassas, VA 20109. Product Code 21-040-CV.)
Method - Lot #16
Solutions were prepared as follows:
Solution 1: PO-1826 was prepared by dissolving at 40 mg per 1 mL of an aqueous solution containing 150 mg KC1 per 1 mL of endotoxin-free water.
Solution 2: Polyvinyl alcohol @ 100 mg/mL in lOOmM phosphate buffer, pH 8.
Solution 3: PLGA was prepared by dissolving PLGA at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 4: PLGA-PEG-OMe was prepared by dissolving PLGA-PEG-OMe at 100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 5: Polyvinyl alcohol @ 100 mg/mL in lOOmM phosphate buffer, pH 8.
Solution 6: 70 mM phosphate buffer, pH 8.
A primary (W/O) emulsion was created by mixing Solutions 1 through 4 and creating a coarse emulsion prior to a fine emulsion. Solution 1 (0.25 mL), Solution 2 (0.25 mL), Solution 3 (0.5 mL), and Solution 4 (0.5mL) were combined in a small glass pressure tube, coarsely emulsified by repeat pipetting, and sonicated at 50% amplitude for 40 seconds over an ice bath using a Branson Digital Sonifier 250. A secondary (W1/0/W2) emulsion was then formed by adding Solution 5 (3.0 mL) to the primary emulsion, vortexing to create a coarse dispersion, and then sonicating at 30% amplitude for 60 seconds over an ice bath using the Branson Digital Sonifier 250. The fine W1/0/W2 emulsion was added to an open 50 mL beaker containing 70 mM phosphate buffer (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 90 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 PBS IX to achieve a nanocarrier suspension having a nominal concentration of 10 mg/mL on a polymer basis. Nanocarrier suspension was then stored refrigerated until the concentration was determined, and then concentration adjusted and stored frozen at -20C until use.
Synthetic nanocarriers delivering CpG and OVA were superior in rapid antibody induction capacity than free high-dose CpG and OVA (both free CpG and free OVA used in 5x higher dose) and as successful or superior in induction of local and systemic antigen- specific CTLs as the same high dose of free CpG and free OVA.
Groups of three animals (C57BL/6 mice, females) were immunized (prime-boost, days 0 and 10; hind limb, s.c.) by three combinations of nanocarrier-incorporated CpG (ODN 1826) and OVA in parallel with immunization with 5-fold excess of free CpG and OVA. Three different formulations of NC-CpG were used with the same formulation of NC-OVA (see Table 13 for details). At 4 days after the second immunization animals were sacrificed and their serum, draining (popliteal) lymph nodes (LNs) and spleens were taken. Sera from immunized mice were used to determine antibody titer against ovalbumin, which was measured in standard ELISA with serial dilutions of test sera. Biotinylated goat anti-mouse Ig was used as a detection antibody (BD Biosciences, San Diego, CA). EC50 was determined based on titration curves. All NC-CpG formulations combined with NC-OVA induced more than 30-fold higher early antibody response against OVA than 5-fold higher doses of free CpG and OVA.
In parallel, the induction of OVA-specific CTLs was assessed ex vivo (without in vitro expansion) in draining LNs (locally) or spleens (systemically) via FACS analysis. Briefly, both tissues were treated with collagenase, homogenized, washed, cells counted using Trypan exclusion (Countess, Invitrogen, CA, USA) and labeled with antibodies or MHC class I-restricted pentamers coupled with fluorescent dyes capable of recognizing surface CD 8 (T cell marker), CD 19 (B cell marker) and T cell receptor (TCR) specific to
MHC Class-I-restricted immunodominant OVA-derived peptide SIINFEKL (SEQ ID NO: l). Then differential cell populations were analyzed by FACS with those cells exhibiting CD8 expression (CD8+), no CD 19 expression (CD 19") and bound to MHC Class I-complexed SIINFEKL (SEQ ID NO: l) (SIINFEKL+ (SEQ ID NO: l)) considered representing a major species of OVA-specific CTLs. At least one of NC-CpG formulations used has equal or higher capacity to produce short-term CTLs locally and systemically when coupled with NC- OVA than 5-fold higher amounts of free CpG and OVA. Of note, different NC-CpG formulations have been especially beneficial for induction either of local or of systemic CTL response against nanocarrier-incorporated ovalbumin antigen.
Furthermore, purified splenocytes from immunized animals were also expanded in vitro (100 u/mL IL-2) by being stimulated with mitomycin-treated EG.7-OVA cells
(syngeneic cells stably transfected with ovalbumin), which should result in preferential expansion of OVA-specific CD8+ cells. At 11 days of in vitro incubation expanded cultures were labeled as described above and analyzed by FACS. All NC-CpG formulations tested resulted in induction of Ag-specific CTLs with a higher expansion potential than those induced by 5x doses of free CpG and OVA. Of note, one CpG formulation (NC-CpG) had especially strong potential for CTL expansion and another (NC-CpG) has exceeded systemic CTL induction levels by free high-dose CpG and OVA both when analyzed ex vivo and upon in vitro expansion.
Table 13. Experimental layout for testing of humoral and cellular immune response induced by nanocarrier-incorporated CpG and OVA vs. free CpG and OVA.
Gr. # Immunized w. NC Lot CpG ^g) OVA (μ§) Regimen
1 NC-OVA + NC-CpG #12/#13 4.0 10 0/10 d
(1)
2 NC-OVA + NC-CpG 4.0 10 Same
#14/#15
(2)
3 NC-OVA + NC-CpG 4.0 10 Same
#14/#16
(3)
4 OVA + CpG (free) N/A 20 50 Same

Claims

What is claimed is:
CLAIMS 1. A method comprising:
identifying a subject in need of a humoral and cytotoxic T lymphocyte (CTL) immune response to a first protein, and
administering to the subject a composition comprising a population of synthetic nanocarners coupled to the first protein;
wherein the first protein comprises at least one humoral epitope and at least one MHC
Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarners does not comprise a saponin-cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarners is a maximum dimension of from 20 nm to 250 nm.
2. A method comprising:
administering to the subject a composition comprising a population of synthetic nanocarners coupled to a first protein;
wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarners does not comprise a saponin-cholesterol adjuvant, wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarners is a maximum dimension of from 20 nm to 250 nm, and wherein the
composition is administered according to a vaccination regimen.
3. A method comprising:
administering to the subject a composition comprising a population of synthetic nanocarners coupled to a first protein;
wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarners does not comprise a saponin-cholesterol adjuvant, wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarners is a maximum dimension of from 20 nm to 250 nm, and wherein the composition is administered according to a protocol that was previously shown to result in a humoral and CTL immune response specific to the first protein in one or more test subjects.
4. The method of claim 2 or 3, wherein the method further comprises identifying a subject in need of a humoral and cytotoxic T lymphocyte (CTL) immune response to the first protein.
5. The method of claim 1 or 3, wherein the composition is administered according to a vaccination regimen.
6. The method of claim 1 or 2, wherein the composition is administered according to a protocol that was previously shown to result in a humoral and CTL immune response specific to the first protein in one or more test subjects.
7. The method of any of claims 1-6, wherein the composition is administered in an amount effective to generate a humoral and CTL immune response to the first protein.
8. The method of any of claims 1-7, wherein the composition further comprises one or more adjuvants.
9. The method of any of claims 1-7, wherein the method further comprises administering one or more adjuvants.
10. The method of claims 8 or 9, wherein the one or more adjuvants comprise stimulators or agonists of pattern recognition receptors, mineral salts, alum, alum combined with monphosphoryl lipid A of Enterobacteria (MPL), MPL® (AS04), AS 15, saponins, QS- 21,Quil-A, ISCOMs, ISCOMATRIX™, MF59™, Montanide® ISA 51, Montanide® 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® 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; immuno stimulatory DNA; immuno stimulatory 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; immuno stimulatory 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.
11. The method of claim 10, wherein the one or more adjuvants comprise an agonist of Toll-Like Receptor 2, 3, 4, 7, 8 or 9.
12. The method of claim 10, wherein the one or more adjuvants comprise an
imidazoquinoline or oxoadenine.
13. The method of claim 12, wherein the imidazoquinoline comprises resiquimod or imiquimod.
14. The method of any of claims 8-13, wherein the one or more adjuvants are coupled to the synthetic nanocarriers of the population of synthetic nanocarriers.
15. The method of any of claims 8-13, wherein the composition further comprises or the method further comprises administering another population of synthetic nanocarriers, and wherein the one or more adjuvants are coupled to the synthetic nanocarriers of the other population of synthetic nanocarriers.
16. The method of any of claims 8-13, wherein the one or more adjuvants are not coupled to a synthetic nanocarrier.
17. The method of any of claims 1-16, wherein the composition further comprises one or more additional antigens.
18. The method of any of claims 1-16, wherein the method further comprises
administering one or more additional antigens.
19. The method of claim 17 or 18, wherein the one or more additional antigens comprise a second protein.
20. The method of any of claims 17-19, wherein the one or more additional antigens comprise a humoral epitope and/or a MHC Class I-restricted epitope.
21. The method of claim 20, wherein the one or more additional antigens comprise a humoral epitope and a MHC Class I-restricted epitope.
22. The method of claim 20 or 21, wherein the one or more additional antigens comprise at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope.
23. The method of any of claims 17-22, wherein the one or more additional antigens are coupled to the synthetic nanocarriers.
24. The method of any of claims 17-22, wherein the composition further comprises or the method further comprises administering another population of synthetic nanocarriers, and wherein the one or more additional antigens are coupled to the synthetic nanocarriers of the other population of synthetic nanocarriers.
25. The method of any of claims 17-22, wherein the one or more additional antigens are not coupled to a synthetic nanocarrier.
26. The method of any of claims 1-25, wherein the synthetic nanocarriers and/or other synthetic nanocarriers comprise a polymeric nanoparticle, a metallic nanoparticle, a dendrimer, a buckyball, a nanowire, a virus-like particle or a peptide or protein particle.
27. The method of any of claims 1-26, wherein the synthetic nanocarriers and/or other synthetic nanocarriers comprise one or more polymers.
28. The method of claim 27, wherein the one or more polymers comprise a polyester, polyamino acid, polycarbonate, polyacetal, polyketal, polysaccharide, polyethyloxazoline or polyethyleneimine.
29. The method of claim 27 or 28, wherein the one or more polymers comprise a polyester.
30. The method of claim 29, wherein the polyester comprises a poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid) or polycaprolactone.
31. The method of claim 29 or 30, wherein the polyester is coupled to a polyether.
32. The method of claim 31, where in the polyether comprises polyethylene glycol.
33. The method of any of claims 1-32, wherein the first protein and/or one or more additional antigens are antigens associated with cancer, an infection or infectious disease, a non-autoimmune or degenerative disease, HIV, malaria, leischmania, human filovirus, togavirus, alphavirus, arenavirus, bunyavirus, flavivirus, human papillomavirus, human influenza A virus, hepatitis B or hepatitis C.
34. The method of any of claims 1-33, wherein the subject has or is at risk of having cancer, an infection or infectious disease or a non- autoimmune or degenerative disease.
35. The method of claim 34, wherein the subject has or is at risk of having HIV, malaria, leischmaniasis, a human filovirus infection, a togavirus infection, a alphavirus infection, an arenavirus infection, a bunyavirus infection, a flavivirus infection, a human papillomavirus infection, a human influenza A virus infection, a hepatitis B infection or a hepatitis C infection.
36. The method of any of claims 1-35, wherein the humoral and CTL immune responses that are generated are clinically effective.
37. The method of claim 36, wherein the humoral and CTL immune responses are effective to treat or prevent cancer, an infection or infectious disease or a non-autoimmune or degenerative disease in the subject.
38. The method of claim 37, wherein the humoral and CTL immune responses are effective to treat or prevent HIV, malaria, leischmaniasis, a human filovirus infection, a togavirus infection, a alphavirus infection, an arenavirus infection, a bunyavirus infection, a flavivirus infection, a human papillomavirus infection, a human influenza A virus infection, a hepatitis B infection or a hepatitis C infection.
39. The method of any of claims 1-38, wherein the composition further comprises a pharmaceutically acceptable excipient.
40. The method of any of claims 1-39, wherein the composition is sterile.
41. The method of any of claims 1-40, wherein the composition is reconstituted from lyophilized form.
42. The method of any of claims 1-41, wherein the composition is a dosage form.
43. The method of any of claims 1-42, wherein the composition is a vaccine.
44. The method of any of claims 1-43, wherein the composition is administered by intravenous, oral, subcutaneous, pulmonary, intranasal, intradermal, transmucosal, intramucosal or intramuscular administration.
45. A composition comprising a population of synthetic nanocarriers coupled to a first protein, wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin-cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm, for use in therapy or prophylaxis.
46. A composition comprising a population of synthetic nanocarriers coupled to a first protein, wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin-cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm, for use in a method as defined in any one of claims 1-44.
47. A composition comprising a population of synthetic nanocarriers coupled to a first protein, wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin-cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm, for use in vaccination.
48. A composition comprising a population of synthetic nanocarriers coupled to a first protein, wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin-cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm, for use in generating a humoral and CTL immune response to the first protein in a subject.
49. A composition comprising a population of synthetic nanocarriers coupled to a first protein, wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin-cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm, for use in a method of therapy or prophylaxis of cancer, an infection or infectious disease, a non- autoimmune or degenerative disease, HIV, malaria, leischmaniasis, a human filovirus infection, a togavirus infection, a alphavirus infection, an arenavirus infection, a bunyavirus infection, a flavivirus infection, a human papillomavirus infection, a human influenza A virus infection, a hepatitis B infection or a hepatitis C infection.
50. A composition comprising a population of synthetic nanocarriers coupled to a first protein, wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin-cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm, for use in a method of therapy or prophylaxis comprising administration by intravenous, oral, subcutaneous, pulmonary, intranasal, intradermal, intramucosal, transmucosal or
intramuscular administration.
51. Use of a composition comprising a population of synthetic nanocarriers coupled to a first protein, wherein the first protein comprises at least one humoral epitope and at least one MHC Class I-restricted epitope that are not the same epitope, wherein the population of synthetic nanocarriers does not comprise a saponin-cholesterol adjuvant, and wherein the mean of a particle size distribution obtained using dynamic light scattering of the population of synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm, for the manufacture of a medicament, for example a vaccine, for use in a method as defined in any one of claims 1-44.
52. A composition for use as defined in any one of claims 45-50 or use of claim 51 wherein the composition is as defined in any one of claims 1-44.
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Cited By (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013151666A2 (en) 2012-04-02 2013-10-10 modeRNA Therapeutics Modified polynucleotides for the production of biologics and proteins associated with human disease
WO2013151736A2 (en) 2012-04-02 2013-10-10 modeRNA Therapeutics In vivo production of proteins
US8664194B2 (en) 2011-12-16 2014-03-04 Moderna Therapeutics, Inc. Method for producing a protein of interest in a primate
US8710200B2 (en) 2011-03-31 2014-04-29 Moderna Therapeutics, Inc. Engineered nucleic acids encoding a modified erythropoietin and their expression
US8822663B2 (en) 2010-08-06 2014-09-02 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
WO2015034925A1 (en) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Circular polynucleotides
WO2015034928A1 (en) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Chimeric polynucleotides
US8980864B2 (en) 2013-03-15 2015-03-17 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
WO2015075557A2 (en) 2013-11-22 2015-05-28 Mina Alpha Limited C/ebp alpha compositions and methods of use
US9107886B2 (en) 2012-04-02 2015-08-18 Moderna Therapeutics, Inc. Modified polynucleotides encoding basic helix-loop-helix family member E41
WO2016014846A1 (en) 2014-07-23 2016-01-28 Moderna Therapeutics, Inc. Modified polynucleotides for the production of intrabodies
US9283287B2 (en) 2012-04-02 2016-03-15 Moderna Therapeutics, Inc. Modified polynucleotides for the production of nuclear proteins
US9334328B2 (en) 2010-10-01 2016-05-10 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9428535B2 (en) 2011-10-03 2016-08-30 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9464124B2 (en) 2011-09-12 2016-10-11 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
CN106065047A (en) * 2016-07-14 2016-11-02 山东省肿瘤防治研究院 A kind of Liver targeting cationic polymer and preparation method and application
US9572897B2 (en) 2012-04-02 2017-02-21 Modernatx, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US9597380B2 (en) 2012-11-26 2017-03-21 Modernatx, Inc. Terminally modified RNA
WO2017112943A1 (en) 2015-12-23 2017-06-29 Modernatx, Inc. Methods of using ox40 ligand encoding polynucleotides
WO2017120612A1 (en) 2016-01-10 2017-07-13 Modernatx, Inc. Therapeutic mrnas encoding anti ctla-4 antibodies
WO2018104538A1 (en) 2016-12-08 2018-06-14 Curevac Ag Rna for treatment or prophylaxis of a liver disease
WO2018104540A1 (en) 2016-12-08 2018-06-14 Curevac Ag Rnas for wound healing
WO2019048632A1 (en) 2017-09-08 2019-03-14 Mina Therapeutics Limited Stabilized hnf4a sarna compositions and methods of use
WO2019048645A1 (en) 2017-09-08 2019-03-14 Mina Therapeutics Limited Stabilized cebpa sarna compositions and methods of use
US10323076B2 (en) 2013-10-03 2019-06-18 Modernatx, Inc. Polynucleotides encoding low density lipoprotein receptor
WO2019197845A1 (en) 2018-04-12 2019-10-17 Mina Therapeutics Limited Sirt1-sarna compositions and methods of use
WO2020208361A1 (en) 2019-04-12 2020-10-15 Mina Therapeutics Limited Sirt1-sarna compositions and methods of use
US10815291B2 (en) 2013-09-30 2020-10-27 Modernatx, Inc. Polynucleotides encoding immune modulating polypeptides
EP4011451A1 (en) 2015-10-22 2022-06-15 ModernaTX, Inc. Metapneumovirus mrna vaccines
WO2022122872A1 (en) 2020-12-09 2022-06-16 Ucl Business Ltd Therapeutics for the treatment of neurodegenerative disorders
WO2022200810A1 (en) 2021-03-26 2022-09-29 Mina Therapeutics Limited Tmem173 sarna compositions and methods of use
WO2023006999A2 (en) 2021-07-30 2023-02-02 CureVac SE Mrnas for treatment or prophylaxis of liver diseases
WO2023099884A1 (en) 2021-12-01 2023-06-08 Mina Therapeutics Limited Pax6 sarna compositions and methods of use
WO2023104964A1 (en) 2021-12-09 2023-06-15 Ucl Business Ltd Therapeutics for the treatment of neurodegenerative disorders
WO2023144193A1 (en) 2022-01-25 2023-08-03 CureVac SE Mrnas for treatment of hereditary tyrosinemia type i
WO2023161350A1 (en) 2022-02-24 2023-08-31 Io Biotech Aps Nucleotide delivery of cancer therapy
WO2023170435A1 (en) 2022-03-07 2023-09-14 Mina Therapeutics Limited Il10 sarna compositions and methods of use

Families Citing this family (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BRPI1011836A2 (en) * 2009-04-21 2017-05-16 Selecta Biosciences Inc immunotherapeutic agents that provide a th1-induced response
AU2010254551B2 (en) 2009-05-27 2016-10-20 Selecta Biosciences, Inc. Immunomodulatory agent-polymeric compounds
BR112012003977A2 (en) * 2009-08-26 2017-06-06 Selecta Biosciences Inc t-cell aid-inducing compositions
CA2798323A1 (en) 2010-05-26 2011-12-01 Selecta Biosciences, Inc. Dose selection of adjuvanted synthetic nanocarriers
US9994443B2 (en) 2010-11-05 2018-06-12 Selecta Biosciences, Inc. Modified nicotinic compounds and related methods
CN103429232B (en) 2010-11-12 2016-03-16 盖茨咨询和项目管理公司 The immunomodulating particle modified
CN103517707A (en) 2011-04-29 2014-01-15 西莱克塔生物科技公司 Controlled release of immunosuppressants from synthetic nanocarriers
US20130028857A1 (en) 2011-07-29 2013-01-31 Selecta Biosciences, Inc. Synthetic nanocarriers comprising polymers comprising multiple immunomodulatory agents
CA3211102A1 (en) 2012-06-21 2013-12-27 Northwestern University Peptide conjugated particles
JP6725413B2 (en) 2013-03-13 2020-07-15 クール ファーマシューティカルズ ディベロップメント カンパニー インコーポレイテッド Immunomodulating particles for the treatment of inflammation
US10314917B2 (en) * 2013-03-15 2019-06-11 The Brigham And Women's Hospital, Inc. Targeted polymeric inflammation-resolving nanoparticles
KR20220025909A (en) * 2013-05-03 2022-03-03 셀렉타 바이오사이언시즈, 인크. Delivery of immunosuppressants having a specified pharmacodynamic effective-life and antigen for the inducation of immune tolerance
WO2014179771A1 (en) * 2013-05-03 2014-11-06 Selecta Biosciences, Inc. Dosing combinations for reducing undesired humoral immune responses
AU2014292926B2 (en) 2013-07-25 2020-03-05 Exicure Operating Company Spherical nucleic acid-based constructs as immunostimulatory agents for prophylactic and therapeutic use
EP3033102B2 (en) 2013-08-13 2023-12-20 Northwestern University Peptide conjugated particles
CA2932122C (en) 2013-12-03 2022-04-19 Northwestern University Liposomal particles, methods of making same and uses thereof
TR201908550T4 (en) 2014-06-04 2019-07-22 Exicure Inc Multivalent delivery of immune modulators by liposomal spherical nucleic acids for prophylactic or therapeutic applications.
US20150359865A1 (en) * 2014-06-17 2015-12-17 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers for t-cell-mediated autoimmune disease
US10336968B2 (en) 2014-07-09 2019-07-02 Conopco, Inc. Laundry liquid composition comprising a polyester/butyl glycol/water active blend
EP2966160A1 (en) 2014-07-09 2016-01-13 Clariant International Ltd. Storage-stable compositions comprising soil release polymers
WO2016010788A1 (en) 2014-07-15 2016-01-21 The United States Of America, As Represented By The Secretary, Dept. Of Health And Human Services Polyketal particles including a cpg oligodeoxynucleotide for the treatment of lung cancer
WO2016025668A1 (en) * 2014-08-13 2016-02-18 The Regents Of The University Of California Biodegradable trehalose glycopolymers
BR112017001470A2 (en) 2014-09-07 2018-02-20 Selecta Biosciences Inc methods and compositions for attenuating the immune responses of the gene therapy antiviral transfer vector
KR101892689B1 (en) * 2014-10-14 2018-08-28 삼성전기주식회사 Chip electronic component and board having the same mounted thereon
CN107106493A (en) 2014-11-21 2017-08-29 西北大学 The sequence-specific cellular uptake of spherical nucleic acid nano particle conjugate
US20170025028A1 (en) * 2015-07-23 2017-01-26 Rhythmalytics LLC Actigraphy based biological rhythm modification methods and systems that result in a greater efficacy of applied medical treatment to a patient
WO2017106630A1 (en) 2015-12-18 2017-06-22 The General Hospital Corporation Polyacetal polymers, conjugates, particles and uses thereof
WO2017205338A1 (en) * 2016-05-23 2017-11-30 University Of Miami Compositions for selective humoral responses and methods of use thereof
US11364304B2 (en) 2016-08-25 2022-06-21 Northwestern University Crosslinked micellar spherical nucleic acids
CN110612122A (en) 2017-03-11 2019-12-24 西莱克塔生物科技公司 Methods and compositions related to combination therapy with anti-inflammatory agents and synthetic nanocarriers comprising immunosuppressants
WO2018201090A1 (en) 2017-04-28 2018-11-01 Exicure, Inc. Synthesis of spherical nucleic acids using lipophilic moieties
SG11201912480SA (en) 2017-06-23 2020-01-30 Pathovax Llc Chimeric virus-like particles and uses thereof as antigen-specific redirectors of immune responses
CA3069909A1 (en) 2017-07-13 2019-02-14 Northwestern University General and direct method for preparing oligonucleotide-functionalized metal-organic framework nanoparticles
US10457681B2 (en) 2017-08-16 2019-10-29 Bristol_Myers Squibb Company Toll-like receptor 7 (TLR7) agonists having a tricyclic moiety, conjugates thereof, and methods and uses therefor
US10472361B2 (en) 2017-08-16 2019-11-12 Bristol-Myers Squibb Company Toll-like receptor 7 (TLR7) agonists having a benzotriazole moiety, conjugates thereof, and methods and uses therefor
US10494370B2 (en) 2017-08-16 2019-12-03 Bristol-Myers Squibb Company Toll-like receptor 7 (TLR7) agonists having a pyridine or pyrazine moiety, conjugates thereof, and methods and uses therefor
US10508115B2 (en) 2017-08-16 2019-12-17 Bristol-Myers Squibb Company Toll-like receptor 7 (TLR7) agonists having heteroatom-linked aromatic moieties, conjugates thereof, and methods and uses therefor
US10487084B2 (en) 2017-08-16 2019-11-26 Bristol-Myers Squibb Company Toll-like receptor 7 (TLR7) agonists having a heterobiaryl moiety, conjugates thereof, and methods and uses therefor
BR112020005565A2 (en) 2017-09-21 2020-10-06 Emergex Vaccines Holding Limited class i mhc-associated peptides for prevention and treatment of zika virus
US11554120B2 (en) 2018-08-03 2023-01-17 Bristol-Myers Squibb Company 1H-pyrazolo[4,3-d]pyrimidine compounds as toll-like receptor 7 (TLR7) agonists and methods and uses therefor
EP3902527A4 (en) 2018-12-27 2022-09-28 VerImmune Inc. Conjugated virus-like particles and uses thereof as anti-tumor immune redirectors
CN110938200B (en) * 2019-12-05 2021-04-20 大连理工大学 Preparation method of amine polyester containing dimethyl pyridine on side chain
MX2019015508A (en) 2019-12-18 2021-06-21 Univ Guadalajara Nanoparticles for treating cancer.
CN111122429B (en) * 2019-12-31 2022-04-22 华南理工大学 Rapid detection method for nanocellulose conversion degree
US20220193225A1 (en) * 2020-08-31 2022-06-23 Bruce Lyday Compositions and methods for sars-2 vaccine with virus replicative particles and recombinant glycoproteins
CN114177282B (en) * 2020-09-14 2024-03-15 苏州大学 Use of fluorinated polyethylenimine for preparing vaccine or preparation for preventing/treating diseases caused by virus/bacteria
IL302190A (en) 2020-10-19 2023-06-01 Verimmune Inc Virus-inspired compositions and methods of redirecting preexisting immune responses using the same for treatment of cancer

Citations (52)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4638045A (en) 1985-02-19 1987-01-20 Massachusetts Institute Of Technology Non-peptide polyamino acid bioerodible polymers
US4806621A (en) 1986-01-21 1989-02-21 Massachusetts Institute Of Technology Biocompatible, bioerodible, hydrophobic, implantable polyimino carbonate article
US4946929A (en) 1983-03-22 1990-08-07 Massachusetts Institute Of Technology Bioerodible articles useful as implants and prostheses having predictable degradation rates
US5010167A (en) 1989-03-31 1991-04-23 Massachusetts Institute Of Technology Poly(amide-and imide-co-anhydride) for biological application
US5019379A (en) 1987-07-31 1991-05-28 Massachusetts Institute Of Technology Unsaturated polyanhydrides
US5399665A (en) 1992-11-05 1995-03-21 Massachusetts Institute Of Technology Biodegradable polymers for cell transplantation
US5512600A (en) 1993-01-15 1996-04-30 Massachusetts Institute Of Technology Preparation of bonded fiber structures for cell implantation
US5514378A (en) 1993-02-01 1996-05-07 Massachusetts Institute Of Technology Biocompatible polymer membranes and methods of preparation of three dimensional membrane structures
US5543158A (en) 1993-07-23 1996-08-06 Massachusetts Institute Of Technology Biodegradable injectable nanoparticles
US5578325A (en) 1993-07-23 1996-11-26 Massachusetts Institute Of Technology Nanoparticles and microparticles of non-linear hydrophilic-hydrophobic multiblock copolymers
US5716404A (en) 1994-12-16 1998-02-10 Massachusetts Institute Of Technology Breast tissue engineering
US5736372A (en) 1986-11-20 1998-04-07 Massachusetts Institute Of Technology Biodegradable synthetic polymeric fibrous matrix containing chondrocyte for in vivo production of a cartilaginous structure
US5770417A (en) 1986-11-20 1998-06-23 Massachusetts Institute Of Technology Children's Medical Center Corporation Three-dimensional fibrous scaffold containing attached cells for producing vascularized tissue in vivo
US5804178A (en) 1986-11-20 1998-09-08 Massachusetts Institute Of Technology Implantation of cell-matrix structure adjacent mesentery, omentum or peritoneum tissue
US5837752A (en) 1997-07-17 1998-11-17 Massachusetts Institute Of Technology Semi-interpenetrating polymer networks
US5902599A (en) 1996-02-20 1999-05-11 Massachusetts Institute Of Technology Biodegradable polymer networks for use in orthopedic and dental applications
US6007845A (en) 1994-07-22 1999-12-28 Massachusetts Institute Of Technology Nanoparticles and microparticles of non-linear hydrophilic-hydrophobic multiblock copolymers
US6095148A (en) 1995-11-03 2000-08-01 Children's Medical Center Corporation Neuronal stimulation using electrically conducting polymers
US6123727A (en) 1995-05-01 2000-09-26 Massachusetts Institute Of Technology Tissue engineered tendons and ligaments
US6130082A (en) 1988-05-05 2000-10-10 American Cyanamid Company Recombinant flagellin vaccines
US6207646B1 (en) 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6329381B1 (en) 1997-11-28 2001-12-11 Sumitomo Pharmaceuticals Company, Limited Heterocyclic compounds
US20020086049A1 (en) 2000-09-18 2002-07-04 Bolton Anthony E. Apoptosis-mimicking synthetic entities and use thereof in medical treatment
US6506577B1 (en) 1998-03-19 2003-01-14 The Regents Of The University Of California Synthesis and crosslinking of catechol containing copolypeptides
US6585980B1 (en) 1997-04-11 2003-07-01 The University Of Toronto Flagellin gene, FlaC of Campylobacter
US6632922B1 (en) 1998-03-19 2003-10-14 The Regents Of The University Of California Methods and compositions for controlled polypeptide synthesis
US6632671B2 (en) 2000-02-28 2003-10-14 Genesegues, Inc. Nanoparticle encapsulation system and method
WO2003086280A2 (en) 2002-04-04 2003-10-23 Coley Pharmaceutical Gmbh Immunostimulatory g,u-containing oligoribonucleotides
US6686446B2 (en) 1998-03-19 2004-02-03 The Regents Of The University Of California Methods and compositions for controlled polypeptide synthesis
US6696076B2 (en) 1996-10-25 2004-02-24 3M Innovative Properties Company Immune response modifier compounds for treatment of TH2 mediated and related diseases
US6818732B2 (en) 2001-08-30 2004-11-16 The Regents Of The University Of California Transition metal initiators for controlled poly (beta-peptide) synthesis from beta-lactam monomers
WO2005097993A2 (en) 2004-02-19 2005-10-20 Coley Pharmaceutical Group, Inc. Immunostimulatory viral rna oligonucleotides
US20060002852A1 (en) 2004-07-01 2006-01-05 Yale University Targeted and high density drug loaded polymeric materials
US20060222652A1 (en) 2000-05-05 2006-10-05 Cytos Biotechnology Ag Molecular antigen array
US20060241076A1 (en) 2005-04-26 2006-10-26 Coley Pharmaceutical Gmbh Modified oligoribonucleotide analogs with enhanced immunostimulatory activity
US20060251677A1 (en) 2003-03-26 2006-11-09 Cytos Biotechnology Ag Packaging of immunostimulatory oligonucleotides into virus-like particles: method of preparation and use
US7192725B2 (en) 2000-05-19 2007-03-20 University Of Toronto Flagellin gene, flaC of Campylobacter
US7202351B1 (en) 1993-09-14 2007-04-10 Pharmexa Inc. Alteration of immune response using pan DR-binding peptides
US7223398B1 (en) 1999-11-15 2007-05-29 Dynavax Technologies Corporation Immunomodulatory compositions containing an immunostimulatory sequence linked to antigen and methods of use thereof
WO2007062107A2 (en) 2005-11-25 2007-05-31 Coley Pharmaceutical Gmbh Immunostimulatory oligoribonucleotides
US7250403B2 (en) 2000-03-10 2007-07-31 Dynavax Technologies Corporation Biodegradable immunomodulatory formulations and methods for use thereof
WO2008033432A2 (en) 2006-09-12 2008-03-20 Coley Pharmaceutical Group, Inc. Immune modulation by chemically modified ribonucleosides and oligoribonucleotides
US20080145441A1 (en) 2000-10-16 2008-06-19 Midatech Limited Nanoparticles
WO2008127532A1 (en) 2007-04-12 2008-10-23 Emory University Novel strategies for delivery of active agents using micelles and particles
US20090028910A1 (en) 2003-12-19 2009-01-29 University Of North Carolina At Chapel Hill Methods for Fabrication Isolated Micro-and Nano-Structures Using Soft or Imprint Lithography
WO2009051837A2 (en) 2007-10-12 2009-04-23 Massachusetts Institute Of Technology Vaccine nanotechnology
US7566703B2 (en) 2004-10-20 2009-07-28 Coley Pharmaceutical Group, Inc. Semi-soft C-class immunostimulatory oligonucleotides
WO2009106999A2 (en) 2008-02-28 2009-09-03 Deutsches Krebsforschungszentrum, Stiftung Des Öffentlichen Rechts Hollow nanoparticles and uses thereof
US20090226525A1 (en) 2007-04-09 2009-09-10 Chimeros Inc. Self-assembling nanoparticle drug delivery system
WO2010018132A1 (en) 2008-08-11 2010-02-18 Smithkline Beecham Corporation Compounds
US20100075995A1 (en) 2008-08-11 2010-03-25 Smithkline Beecham Corporation Compounds
WO2010047839A1 (en) 2008-10-25 2010-04-29 Aura Biosciences Modified plant virus particles and uses therefor

Family Cites Families (280)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR7461M (en) 1968-06-19 1970-01-05
GB1355961A (en) 1970-02-27 1974-06-12 Wellcome Found Preparation of immunosuppressive antilymphocytic serum
CH594444A5 (en) 1972-12-04 1978-01-13 Gerd Birrenbach
DK143689C (en) 1975-03-20 1982-03-15 J Kreuter PROCEDURE FOR THE PREPARATION OF AN ADVERTISED VACCINE
US4756907A (en) 1978-10-17 1988-07-12 Stolle Research & Development Corp. Active/passive immunization of the internal female reproductive organs
US6309669B1 (en) 1984-03-16 2001-10-30 The United States Of America As Represented By The Secretary Of The Army Therapeutic treatment and prevention of infections with a bioactive materials encapsulated within a biodegradable-biocompatible polymeric matrix
US4631211A (en) 1985-03-25 1986-12-23 Scripps Clinic & Research Foundation Means for sequential solid phase organic synthesis and methods using the same
JPS63122620A (en) 1986-11-12 1988-05-26 Sanraku Inc Polylactic acid microsphere and production thereof
FR2608988B1 (en) 1986-12-31 1991-01-11 Centre Nat Rech Scient PROCESS FOR THE PREPARATION OF COLLOIDAL DISPERSIBLE SYSTEMS OF A SUBSTANCE, IN THE FORM OF NANOPARTICLES
US5912017A (en) 1987-05-01 1999-06-15 Massachusetts Institute Of Technology Multiwall polymeric microspheres
US5229490A (en) 1987-05-06 1993-07-20 The Rockefeller University Multiple antigen peptide system
US4929624A (en) 1989-03-23 1990-05-29 Minnesota Mining And Manufacturing Company Olefinic 1H-imidazo(4,5-c)quinolin-4-amines
US5114703A (en) 1989-05-30 1992-05-19 Alliance Pharmaceutical Corp. Percutaneous lymphography using particulate fluorocarbon emulsions
US5733572A (en) 1989-12-22 1998-03-31 Imarx Pharmaceutical Corp. Gas and gaseous precursor filled microspheres as topical and subcutaneous delivery vehicles
GB9016885D0 (en) 1990-08-01 1990-09-12 Scras Sustained release pharmaceutical compositions
US6699474B1 (en) 1990-08-20 2004-03-02 Erich Hugo Cerny Vaccine and immunserum against drugs of abuse
US5175296A (en) 1991-03-01 1992-12-29 Minnesota Mining And Manufacturing Company Imidazo[4,5-c]quinolin-4-amines and processes for their preparation
US5389640A (en) 1991-03-01 1995-02-14 Minnesota Mining And Manufacturing Company 1-substituted, 2-substituted 1H-imidazo[4,5-c]quinolin-4-amines
NZ242220A (en) 1991-04-02 1994-04-27 Biotech Australia Pty Ltd Complex for oral delivery of a substance to the circulatory or lymphatic drainage system comprising microparticle coupled to at least one carrier, the substance being encapsulated by the microparticle
US5811447A (en) 1993-01-28 1998-09-22 Neorx Corporation Therapeutic inhibitor of vascular smooth muscle cells
IL105325A (en) 1992-04-16 1996-11-14 Minnesota Mining & Mfg Immunogen/vaccine adjuvant composition
US6235313B1 (en) 1992-04-24 2001-05-22 Brown University Research Foundation Bioadhesive microspheres and their use as drug delivery and imaging systems
SG49909A1 (en) 1992-06-25 1998-06-15 Smithkline Beecham Biolog Vaccine composition containing adjuvants
WO1994002068A1 (en) 1992-07-21 1994-02-03 The General Hospital Corporation System of drug delivery to the lymphatic tissues
GB9216082D0 (en) 1992-07-28 1992-09-09 Univ Nottingham Lymphatic delivery composition
US6608201B2 (en) 1992-08-28 2003-08-19 3M Innovative Properties Company Process for preparing 1-substituted, 2-substituted 1H-imidazo[4,5-c]quinolin-4-amines
FR2695563B1 (en) 1992-09-11 1994-12-02 Pasteur Institut Microparticles carrying antigens and their use for the induction of humoral or cellular responses.
WO1994007469A1 (en) 1992-09-25 1994-04-14 Dynagen, Inc. An immunobooster for delayed release of immunogen
US5928647A (en) 1993-01-11 1999-07-27 Dana-Farber Cancer Institute Inducing cytotoxic T lymphocyte responses
JPH08507070A (en) 1993-02-22 1996-07-30 アルザ・コーポレーション Compositions for oral administration of active substances
ATE156706T1 (en) 1993-03-17 1997-08-15 Silica Gel Gmbh SUPERPARAMAGNETIC PARTICLES, METHOD FOR THE PRODUCTION AND USE OF THE SAME
WO1995003035A1 (en) 1993-07-23 1995-02-02 Massachusetts Institute Of Technology Polymerized liposomes with enhanced stability for oral delivery
US5798340A (en) 1993-09-17 1998-08-25 Gilead Sciences, Inc. Nucleotide analogs
US5500161A (en) 1993-09-21 1996-03-19 Massachusetts Institute Of Technology And Virus Research Institute Method for making hydrophobic polymeric microparticles
AU1947395A (en) 1994-02-28 1995-09-11 Medinova Medical Consulting Gmbh Drug targeting system, method for preparing same and its use
WO1995026204A1 (en) 1994-03-25 1995-10-05 Isis Pharmaceuticals, Inc. Immune stimulation by phosphorothioate oligonucleotide analogs
GB9412273D0 (en) 1994-06-18 1994-08-10 Univ Nottingham Administration means
EP0772619B2 (en) 1994-07-15 2010-12-08 The University of Iowa Research Foundation Immunomodulatory oligonucleotides
US6239116B1 (en) 1994-07-15 2001-05-29 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
JPH10511957A (en) 1995-01-05 1998-11-17 ザ ボード オブ リージェンツ オブ ザ ユニヴァーシティ オブ ミシガン Surface-modified nanoparticles and methods for their production and use
US5876727A (en) 1995-03-31 1999-03-02 Immulogic Pharmaceutical Corporation Hapten-carrier conjugates for use in drug-abuse therapy and methods for preparation of same
US5866132A (en) 1995-06-07 1999-02-02 Alberta Research Council Immunogenic oligosaccharide compositions
WO1997004747A1 (en) 1995-07-27 1997-02-13 Dunn James M Drug delivery systems for macromolecular drugs
AU710347B2 (en) 1995-08-31 1999-09-16 Alkermes Controlled Therapeutics, Inc. Composition for sustained release of an agent
US6946133B1 (en) 1996-03-20 2005-09-20 The United States Of America As Represented By The Department Of Health And Human Services Prostate specific antigen oligo-epitope peptide
WO1997041440A1 (en) 1996-04-26 1997-11-06 Rijksuniversiteit Te Leiden Methods for selecting and producing t cell peptide epitopes and vaccines incorporating said selected epitopes
US5874064A (en) 1996-05-24 1999-02-23 Massachusetts Institute Of Technology Aerodynamically light particles for pulmonary drug delivery
US5922695A (en) 1996-07-26 1999-07-13 Gilead Sciences, Inc. Antiviral phosphonomethyoxy nucleotide analogs having increased oral bioavarilability
EP1006798A4 (en) 1996-09-05 2003-03-05 Massachusetts Inst Technology Compositions and methods for treatment of neurological disorders and neurodegenerative diseases
US6042820A (en) 1996-12-20 2000-03-28 Connaught Laboratories Limited Biodegradable copolymer containing α-hydroxy acid and α-amino acid units
WO2000006123A1 (en) 1998-07-29 2000-02-10 Chiron Corporation Microparticles with adsorbent surfaces, methods of making same, and uses thereof
CA2281838A1 (en) 1997-02-28 1998-09-03 University Of Iowa Research Foundation Use of nucleic acids containing unmethylated cpg dinucleotide in the treatment of lps-associated disorders
ES2333069T3 (en) 1997-03-10 2010-02-16 Ottawa Hospital Research Institute USE OF NUCLEIC ACIDS CONTAINING NON-METHYLED CPG DINUCLEOTIDE IN COMBINATION WITH ALUMINUM AS AN ASSISTANT.
US6060082A (en) 1997-04-18 2000-05-09 Massachusetts Institute Of Technology Polymerized liposomes targeted to M cells and useful for oral or mucosal drug delivery
CA2301575C (en) 1997-05-20 2003-12-23 Ottawa Civic Hospital Loeb Research Institute Vectors and methods for immunization or therapeutic protocols
US6989435B2 (en) 1997-09-11 2006-01-24 Cambridge University Technical Services Ltd. Compounds and methods to inhibit or augment an inflammatory response
DE69840600D1 (en) 1997-09-16 2009-04-09 Univ Oregon Health & Science RECOMBINANT MHC MOLECULES WHICH ARE USEFUL FOR THE MANIPULATION OF ANTIGEN-SPECIFIC T CELLS
DE19745950A1 (en) 1997-10-17 1999-04-22 Dds Drug Delivery Service Ges Drug carrier particle for site specific drug delivery, especially to CNS
US6197229B1 (en) 1997-12-12 2001-03-06 Massachusetts Institute Of Technology Method for high supercoiled DNA content microspheres
US6254890B1 (en) 1997-12-12 2001-07-03 Massachusetts Institute Of Technology Sub-100nm biodegradable polymer spheres capable of transporting and releasing nucleic acids
FR2775435B1 (en) 1998-02-27 2000-05-26 Bioalliance Pharma NANOPARTICLES COMPRISING AT LEAST ONE POLYMER AND AT LEAST ONE COMPOUND CAPABLE OF COMPLEXING ONE OR MORE ACTIVE INGREDIENTS
US6232287B1 (en) 1998-03-13 2001-05-15 The Burnham Institute Molecules that home to various selected organs or tissues
CA2323929C (en) 1998-04-03 2004-03-09 University Of Iowa Research Foundation Methods and products for stimulating the immune system using immunotherapeutic oligonucleotides and cytokines
CA2328602A1 (en) 1998-05-06 1999-11-11 University Of Iowa Research Foundation Methods for the prevention and treatment of parasitic infections and related diseases using cpg oligonucleotides
SE9801923D0 (en) 1998-05-29 1998-05-29 Independent Pharmaceutical Ab Nicotine vaccine
US6693086B1 (en) 1998-06-25 2004-02-17 National Jewish Medical And Research Center Systemic immune activation method using nucleic acid-lipid complexes
US6242589B1 (en) 1998-07-14 2001-06-05 Isis Pharmaceuticals, Inc. Phosphorothioate oligonucleotides having modified internucleoside linkages
DE19839214C1 (en) 1998-08-28 2000-05-25 Aventis Res & Tech Gmbh & Co Process for the production of spherical microparticles with a smooth surface which consist wholly or partly of at least one water-insoluble linear polysaccharide, and microparticles obtainable by this process and their use
US6306640B1 (en) 1998-10-05 2001-10-23 Genzyme Corporation Melanoma antigenic peptides
SK4272001A3 (en) 1998-10-05 2003-02-04 Pharmexa As Methods for therapeutic vaccination
US7357936B1 (en) 1998-10-16 2008-04-15 Smithkline Beecham Biologicals, Sa Adjuvant systems and vaccines
WO2000025752A1 (en) 1998-11-02 2000-05-11 Church, Marla, J. Multiparticulate modified release composition
US7521068B2 (en) 1998-11-12 2009-04-21 Elan Pharma International Ltd. Dry powder aerosols of nanoparticulate drugs
WO2000032626A1 (en) 1998-11-25 2000-06-08 Regents Of The University Of Minnesota Methods of using epitope peptides of human pathogens
WO2000032227A2 (en) 1998-11-30 2000-06-08 Cytos Biotechnology Ag Ordered molecular presentation of antigens, method of preparation and use
US6232082B1 (en) 1998-12-01 2001-05-15 Nabi Hapten-carrier conjugates for treating and preventing nicotine addiction
SK287112B6 (en) 1999-01-08 2009-12-07 3M Innovative Properties Company Use of immune response modifiying compound for cervical dysplasias treatment
US7238711B1 (en) 1999-03-17 2007-07-03 Cambridge University Technical Services Ltd. Compounds and methods to inhibit or augment an inflammatory response
US6444192B1 (en) 1999-02-05 2002-09-03 The Regents Of The University Of California Diagnostic imaging of lymph structures
US6558951B1 (en) 1999-02-11 2003-05-06 3M Innovative Properties Company Maturation of dendritic cells with immune response modifying compounds
JP4812942B2 (en) 1999-02-26 2011-11-09 ノバルティス ヴァクシンズ アンド ダイアグノスティクス エスアールエル Enhancement of Neisseria antigen bactericidal activity using oligonucleotides containing CG motif
IL145982A0 (en) 1999-04-19 2002-07-25 Smithkline Beecham Biolog Vaccines
PL360915A1 (en) 1999-05-06 2004-09-20 Genetics Institute, Llc Use of soluble costimulatory molecules to enhance immune responses
US6800296B1 (en) 1999-05-19 2004-10-05 Massachusetts Institute Of Technology Modification of surfaces using biological recognition events
US6815170B1 (en) 1999-06-30 2004-11-09 John Wayne Cancer Institute Methods for lymph node identification
AU6635900A (en) 1999-08-13 2001-03-13 Point Biomedical Corporation Microparticles useful as ultrasonic contrast agents and for lymphatic system
EP1700603A3 (en) 1999-09-25 2007-06-13 Coley Pharmaceutical GmbH Immunostimulatory nucleic acids
CA2391534A1 (en) 1999-11-15 2001-05-25 Drug Innovation & Design, Inc. Selective cellular targeting: multifunctional delivery vehicles
US7462354B2 (en) 1999-12-28 2008-12-09 Pharmexa Inc. Method and system for optimizing minigenes and peptides encoded thereby
AU774593C (en) 2000-01-13 2005-06-23 Nanosphere, Inc. Nanoparticles having oligonucleotides attached thereto and uses therefor
AT409085B (en) * 2000-01-28 2002-05-27 Cistem Biotechnologies Gmbh PHARMACEUTICAL COMPOSITION FOR IMMUNULATING AND PRODUCING VACCINES
US20050032733A1 (en) 2001-05-18 2005-02-10 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (SiNA)
US20050020525A1 (en) 2002-02-20 2005-01-27 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US8202979B2 (en) 2002-02-20 2012-06-19 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid
US7157437B2 (en) 2000-03-10 2007-01-02 Dynavax Technologies Corporation Methods of ameliorating symptoms of herpes infection using immunomodulatory polynucleotide sequences
US7129222B2 (en) 2000-03-10 2006-10-31 Dynavax Technologies Corporation Immunomodulatory formulations and methods for use thereof
SE0000933D0 (en) 2000-03-21 2000-03-21 Independent Pharmaceutica Ab Method of producing 6-substituted (S) -nicotine derivatives and intermediate compounds
US6610713B2 (en) 2000-05-23 2003-08-26 North Shore - Long Island Jewish Research Institute Inhibition of inflammatory cytokine production by cholinergic agonists and vagus nerve stimulation
US20020099013A1 (en) * 2000-11-14 2002-07-25 Thomas Piccariello Active agent delivery systems and methods for protecting and administering active agents
AU9475001A (en) 2000-09-26 2002-04-08 Hybridon Inc Modulation of immunostimulatory activity of immunostimulatory oligonucleotide analogs by positional chemical changes
AU2001297913A1 (en) 2000-10-13 2002-12-23 Ligocyte Pharmaceuticals, Inc. Polyvalent nanoparticles
CA2425358C (en) 2000-10-18 2012-08-21 Glaxosmithkline Biologicals S.A. A vaccine composition comprising qs21, mpl, a cpg oligonucleotide and a cancer antigen
AU2002245205B2 (en) 2000-10-19 2007-07-19 Ecole Polytechnique Federale De Lausanne Block copolymers for multifunctional self-assembled systems
US7592008B2 (en) 2000-11-20 2009-09-22 The Board Of Trustees Of The University Of Illinois, A Body Corporate And Politic Of The State Of Illinois Membrane scaffold proteins
WO2006091720A2 (en) 2000-12-08 2006-08-31 3M Innovative Properties Company Compositions and methods for targeted delivery of immune response modifiers
NZ526322A (en) 2000-12-27 2005-06-24 Dynavax Tech Corp Immunomodulatory polynucleotides and methods of using the same
US7320793B2 (en) 2001-01-19 2008-01-22 Cytos Biotechnology Ag Molecular antigen array
US7097837B2 (en) 2001-02-19 2006-08-29 Pharmexa A/S Synthetic vaccine agents
US20030175950A1 (en) 2001-05-29 2003-09-18 Mcswiggen James A. RNA interference mediated inhibition of HIV gene expression using short interfering RNA
US7314624B2 (en) 2001-06-05 2008-01-01 The Regents Of The University Of Michigan Nanoemulsion vaccines
NZ530315A (en) 2001-07-10 2007-01-26 Corixa Corp Compositions and methods for delivery of proteins and adjuvants encapsulated in microspheres
JP4607452B2 (en) 2001-08-07 2011-01-05 ダイナバックス テクノロジーズ コーポレイション Immunomodulating composition, formulation and method of use thereof
US7276489B2 (en) 2002-10-24 2007-10-02 Idera Pharmaceuticals, Inc. Modulation of immunostimulatory properties of oligonucleotide-based compounds by optimal presentation of 5′ ends
CN1321695C (en) 2001-11-07 2007-06-20 赛托斯生物技术公司 Antigen arrays presenting il-5, il-13 or eotaxin for treatment of allergic eosinophilic diseases
NZ532515A (en) 2001-11-07 2005-10-28 Cytos Biotechnology Ag Compositions comprising a RANKL peptide attached to a core or virus-like particle for treatment of bone disease and immunization
US8088388B2 (en) 2002-02-14 2012-01-03 United Biomedical, Inc. Stabilized synthetic immunogen delivery system
ES2607431T3 (en) 2002-02-20 2017-03-31 Glaxosmithkline Biologicals Sa Microparticles with molecules containing adsorbed polypeptides
US20030232013A1 (en) 2002-02-22 2003-12-18 Gary Sieckman Therapeutic and diagnostic targeting of cancers cells with tumor homing peptides
US20040038303A1 (en) 2002-04-08 2004-02-26 Unger Gretchen M. Biologic modulations with nanoparticles
US20080233181A1 (en) 2002-04-12 2008-09-25 Nagy Jon O Nanoparticle adjuvants for sub-unit vaccines
US7285289B2 (en) 2002-04-12 2007-10-23 Nagy Jon O Nanoparticle vaccines
US7524630B2 (en) 2002-04-22 2009-04-28 University Of Florida Research Foundation, Inc. Functionalized nanoparticles and methods of use
AU2003276131A1 (en) 2002-06-18 2003-12-31 Epigenesis Pharmaceuticals, Inc. A dry powder oligonucleotide formulation, preparation and its uses
WO2004005325A2 (en) 2002-07-10 2004-01-15 The Ohio State University Research Foundation Antigen-polymer compositions
CA2489410C (en) 2002-07-17 2015-01-13 Cytos Biotechnology Ag Molecular antigen arrays
KR20120041739A (en) 2002-07-18 2012-05-02 사이토스 바이오테크놀로지 아게 Hapten-carrier conjugates and uses thereof
DE60336902D1 (en) 2002-07-19 2011-06-09 Cytos Biotechnology Ag VACCINE COMPOSITIONS CONTAINING AMYLOID BETA 1-6 ANTIGENARRAYS
MXPA05001647A (en) 2002-08-15 2005-04-19 3M Innovative Properties Co Immunostimulatory compositions and methods of stimulating an immune response.
US7488792B2 (en) 2002-08-28 2009-02-10 Burnham Institute For Medical Research Collagen-binding molecules that selectively home to tumor vasculature and methods of using same
US20040152105A1 (en) 2002-09-06 2004-08-05 Cytos Biotechnology Ag. Immune modulatory compounds and methods
WO2004053056A2 (en) 2002-09-24 2004-06-24 University Of Kentucky Research Foundation Nanoparticle-based vaccine delivery system containing adjuvant
US7008411B1 (en) 2002-09-30 2006-03-07 Advanced Cardiovascular Systems, Inc. Method and apparatus for treating vulnerable plaque
NO20024755D0 (en) 2002-10-03 2002-10-03 Amersham Health As Method
WO2004053104A2 (en) 2002-12-11 2004-06-24 Coley Pharmaceutical Group, Inc. 5’ cpg nucleic acids and methods of use
SE0203687D0 (en) 2002-12-13 2002-12-13 Ian Harwigsson Med Adagit Fa Pharmaceutical Porous Particles
SI1575977T1 (en) 2002-12-23 2010-01-29 Dynavax Tech Corp Immunostimulatory sequence oligonucleotides and methods of using the same
AU2003300184B8 (en) 2002-12-30 2009-12-03 3M Innovative Properties Company Immunostimulatory combinations
WO2004063963A2 (en) 2003-01-08 2004-07-29 Xencor, Inc. Novel proteins with altered immunogenicity
US20040156846A1 (en) 2003-02-06 2004-08-12 Triton Biosystems, Inc. Therapy via targeted delivery of nanoscale particles using L6 antibodies
EP1592302A4 (en) 2003-02-13 2007-04-25 3M Innovative Properties Co Methods and compositions related to irm compounds and toll-like receptor 8
ATE371437T1 (en) * 2003-02-17 2007-09-15 Peter Burkhard PEPTIDIC NANOPARTICLES AS DRUG DELIVERY AND ANTIGEN DISPLAY SYSTEMS
US20040191215A1 (en) 2003-03-25 2004-09-30 Michael Froix Compositions for induction of a therapeutic response
JP2006521367A (en) 2003-03-26 2006-09-21 株式会社Ｌｔｔバイオファーマ Nanoparticles for intravenous injection for targeting and sustained release
WO2004091500A2 (en) 2003-04-10 2004-10-28 3M Innovative Properties Company Delivery of immune response modifier compounds
US7731967B2 (en) 2003-04-30 2010-06-08 Novartis Vaccines And Diagnostics, Inc. Compositions for inducing immune responses
US7727969B2 (en) 2003-06-06 2010-06-01 Massachusetts Institute Of Technology Controlled release nanoparticle having bound oligonucleotide for targeted delivery
US7149574B2 (en) 2003-06-09 2006-12-12 Palo Alto Investors Treatment of conditions through electrical modulation of the autonomic nervous system
US20060182793A1 (en) 2003-07-22 2006-08-17 Cytos Biotechnology Ag Cpg-packaged liposomes
US20050042298A1 (en) 2003-08-20 2005-02-24 Pardridge William M. Immunonanoparticles
EP1670509A4 (en) 2003-09-03 2007-10-31 Dendritherapeutics Inc Multiplex vaccines
US7943179B2 (en) 2003-09-23 2011-05-17 Massachusetts Institute Of Technology pH triggerable polymeric particles
US7771726B2 (en) 2003-10-08 2010-08-10 New York University Use of synthetic glycolipids as universal adjuvants for vaccines against cancer and infectious diseases
DE10347710B4 (en) 2003-10-14 2006-03-30 Johannes-Gutenberg-Universität Mainz Recombinant vaccines and their use
US20080160089A1 (en) 2003-10-14 2008-07-03 Medivas, Llc Vaccine delivery compositions and methods of use
JP2007514519A (en) 2003-10-20 2007-06-07 ウィリアム・マーシュ・ライス・ユニバーシティ Method for producing microcapsules comprising polymer and charged nanoparticles
US8188254B2 (en) 2003-10-30 2012-05-29 Coley Pharmaceutical Gmbh C-class oligonucleotide analogs with enhanced immunostimulatory potency
WO2005051351A2 (en) 2003-11-21 2005-06-09 Alza Corporation Gene delivery mediated by liposome-dna complex with cleavable peg surface modification
WO2005055949A2 (en) 2003-12-09 2005-06-23 The Children's Hospital Of Philadelphia Sustained release preparations composed of biocompatible complex microparticles
WO2005065418A2 (en) 2003-12-31 2005-07-21 Board Of Regents, The University Of Texas System Compositions and methods of use of targeting peptides for diagnosis and therapy
US20070087986A1 (en) 2004-01-26 2007-04-19 Brett Premack Compositions and methods for enhancing immunity by chemoattractant adjuvants
EP1735010A4 (en) 2004-04-09 2008-08-27 3M Innovative Properties Co Methods, compositions, and preparations for delivery of immune response modifiers
ES2246695B1 (en) 2004-04-29 2007-05-01 Instituto Cientifico Y Tecnologico De Navarra, S.A. STIMULATING COMPOSITION OF THE IMMUNE RESPONSE THAT INCLUDES NANOPARTICLES BASED ON A COPYLIMER OF METHYL VINYL ETER AND MALEIC ANHYDRIDE.
WO2005108425A1 (en) 2004-05-10 2005-11-17 Cytos Biotechnology Ag Il-23 p19 antigen array and uses thereof
JP2008509654A (en) 2004-06-01 2008-04-03 イノジェネティックス・ナムローゼ・フェンノートシャップ Peptides for inducing CTL and / or HTL responses against hepatitis C virus
CA2569590C (en) 2004-06-11 2014-04-01 Riken Drug having regulatory cell ligand contained in liposome
WO2006014579A2 (en) 2004-07-08 2006-02-09 The Regents Of California Enhancing class i antigen presentation with synthetic sequences
US8017151B2 (en) 2004-09-07 2011-09-13 Board Of Regents Of The University Of Nebraska By And Behalf Of The University Of Nebraska Medical Center Amphiphilic polymer-protein conjugates and methods of use thereof
US20080213308A1 (en) 2004-09-14 2008-09-04 Nicholas Valiante Imidazoquinoline Compounds
CN1692943A (en) 2004-09-17 2005-11-09 四川大学 Preparation and application of CpG DNA molecule anti-infection and immunity prepn
JP5117191B2 (en) 2004-10-01 2013-01-09 ミダテック リミテッド Nanoparticles containing antigens and adjuvants, and immunogenic structures
EP1812864A2 (en) 2004-10-07 2007-08-01 Emory University Multifunctional nanoparticles conjugates and their use
CN101119749A (en) 2004-10-25 2008-02-06 赛托斯生物技术公司 Gastric inhibitory polypeptide (gip) antigen arrays and uses thereof
US20090123471A1 (en) 2004-10-29 2009-05-14 Cytos Biotechnology Ag T-Cadherin antigen arrays and uses thereof
WO2007001448A2 (en) 2004-11-04 2007-01-04 Massachusetts Institute Of Technology Coated controlled release polymer particles as efficient oral delivery vehicles for biopharmaceuticals
CA2586765A1 (en) 2004-11-05 2006-12-28 The General Hospital Corporation Purposeful movement of human migratory cells away from an agent source
ES2434029T3 (en) 2004-11-15 2013-12-13 Novartis Vaccines And Diagnostics, Inc. Immunogenic compositions containing anthrax antigen, biodegradable polymer microparticles, and immunological adjuvant containing polynucleotide
US20060111271A1 (en) 2004-11-24 2006-05-25 Cerny Erich H Active and passive immunization against pharmacologically active hapten molecules using a synthetic carrier compound composed of similar elements
US20070292386A9 (en) 2004-12-02 2007-12-20 Campbell Robert L Vaccine formulations for intradermal delivery comprising adjuvants and antigenic agents
EP1830875A2 (en) 2004-12-13 2007-09-12 Cytos Biotechnology AG Il-15 antigen arrays and uses thereof
US7361752B2 (en) 2004-12-14 2008-04-22 Alnylam Pharmaceuticals, Inc. RNAi modulation of MLL-AF4 and uses thereof
US20060257359A1 (en) 2005-02-28 2006-11-16 Cedric Francois Modifying macrophage phenotype for treatment of disease
CN101189339A (en) 2005-03-22 2008-05-28 美得思达健康有限公司 Delivery systems and methods for diagnosing and treating cardiovascular diseases
US7709001B2 (en) 2005-04-08 2010-05-04 Wyeth Llc Multivalent pneumococcal polysaccharide-protein conjugate composition
US20080305161A1 (en) 2005-04-13 2008-12-11 Pfizer Inc Injectable depot formulations and methods for providing sustained release of nanoparticle compositions
BRPI0610367A2 (en) 2005-05-04 2010-06-15 Noxxon Pharma Ag use of a nucleic acid, method for binding an intracellular receptor, composition, pharmaceutical composition, hmga binding nucleic acid, method for selecting an hmga antagonist or agonist, hmga detection kit, hmga antagonist and agonist and complex
CA2608086A1 (en) 2005-05-10 2006-11-16 Emory University Strategies for delivery of active agents using micelles and particles
US9290617B2 (en) 2005-07-06 2016-03-22 Molly S. Shoichet Method of biomolecule immobilization on polymers using click-type chemistry
WO2007019678A1 (en) 2005-08-12 2007-02-22 University Health Network Methods and devices for lymphatic targeting
JP2009507049A (en) 2005-09-09 2009-02-19 北京徳科瑞医薬科技有限公司 Nanomicelle formulation of vinca alkaloid anticancer drug encapsulated in polyethylene glycol derivative of phospholipid
JP5806444B2 (en) 2005-12-02 2015-11-10 ノバルティス アーゲー Nanoparticles for use in immunogenic compositions
EP1973608A1 (en) 2005-12-14 2008-10-01 Cytos Biotechnology AG Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity
WO2007070682A2 (en) 2005-12-15 2007-06-21 Massachusetts Institute Of Technology System for screening particles
US7842312B2 (en) 2005-12-29 2010-11-30 Cordis Corporation Polymeric compositions comprising therapeutic agents in crystalline phases, and methods of forming the same
EP1986685A4 (en) 2006-01-31 2009-02-25 Medivas Llc Vaccine delivery compositions and methods of use
US8021689B2 (en) 2006-02-21 2011-09-20 Ecole Polytechnique Federale de Lausanne (“EPFL”) Nanoparticles for immunotherapy
WO2007100699A2 (en) 2006-02-24 2007-09-07 Novartis Ag Microparticles containing biodegradable polymer and cationic polysaccharide for use in immunogenic compositions
EP2010530A2 (en) 2006-03-23 2009-01-07 Novartis AG Methods for the preparation of imidazole-containing compounds
CA2648099C (en) 2006-03-31 2012-05-29 The Brigham And Women's Hospital, Inc System for targeted delivery of therapeutic agents
US20100247653A1 (en) 2006-04-11 2010-09-30 Hans Lautenschlager Nanoparticles containing nicotine and/or cotinine, dispersions, and use thereof
CA2652280C (en) 2006-05-15 2014-01-28 Massachusetts Institute Of Technology Polymers for functional particles
US20110052697A1 (en) 2006-05-17 2011-03-03 Gwangju Institute Of Science & Technology Aptamer-Directed Drug Delivery
DK2032592T3 (en) 2006-06-12 2013-09-02 Cytos Biotechnology Ag PROCEDURES FOR PACKING OLIGONUCLEOTIDES FOR VIRUS LIKE PARTICLES OF RNA BACTERY PHAGES
US20080014281A1 (en) 2006-06-16 2008-01-17 Florida Atlantic University Chitin Micro-Particles As An Adjuvant
US20100028381A1 (en) 2006-06-19 2010-02-04 3M Innovative Properties Company Formulation for delivery of immune response modifiers
WO2007150030A2 (en) 2006-06-23 2007-12-27 Massachusetts Institute Of Technology Microfluidic synthesis of organic nanoparticles
WO2008019142A2 (en) 2006-08-04 2008-02-14 Massachusetts Institute Of Technology Oligonucleotide systems for targeted intracellular delivery
WO2008019366A2 (en) 2006-08-07 2008-02-14 Ludwig Institute For Cancer Research Methods and compositions for increased priming of t-cells through cross-presentation of exogenous antigens
AR062334A1 (en) 2006-08-11 2008-10-29 Panacea Biotec Ltd PARTICLES TO SUPPLY ACTIVE INGREDIENTS, PROCESS TO MAKE THEM AND COMPOSITIONS OF THE SAME
CA2665090A1 (en) 2006-09-22 2008-03-27 Dana-Farber Cancer Institute, Inc. Methods for treating mica-related disorders
EP2077821B1 (en) 2006-10-12 2019-08-14 The University Of Queensland Compositions and methods for modulating immune responses
WO2008147456A2 (en) 2006-11-20 2008-12-04 Massachusetts Institute Of Technology Drug delivery systems using fc fragments
AU2007333225B2 (en) 2006-12-08 2014-06-12 Massachusetts Institute Of Technology Delivery of nanoparticles and/or agents to cells
WO2008071774A1 (en) 2006-12-14 2008-06-19 Cytos Biotechnology Ag Purification process for coat protein of rna bacteriophages
US20080149123A1 (en) 2006-12-22 2008-06-26 Mckay William D Particulate material dispensing hairbrush with combination bristles
AU2007345952C1 (en) 2007-01-31 2017-06-08 Chongxi Yu Positively charged water-soluble prodrugs of 1H-imidazo[4, 5-c]quinolin-4-amines and related compounds with very high skin penetration rates
ES2552471T3 (en) 2007-02-07 2015-11-30 The Regents Of The University Of California Conjugates of synthetic TLR agonists and their uses
EP2134830A2 (en) 2007-02-09 2009-12-23 Massachusetts Institute of Technology Oscillating cell culture bioreactor
US8889117B2 (en) 2007-02-15 2014-11-18 Yale University Modular nanoparticles for adaptable vaccines
JP5650406B2 (en) 2007-03-07 2015-01-07 ユーティーアイ リミテッド パートナーシップ Compositions and methods for prevention and treatment of autoimmune conditions
WO2008118861A2 (en) 2007-03-23 2008-10-02 The University Of North Carolina At Chapel Hill Discrete size and shape specific organic nanoparticles designed to elicit an immune response
US11246831B2 (en) 2007-03-30 2022-02-15 Particle Sciences, Inc. Particle formulations and uses thereof
WO2008124634A1 (en) 2007-04-04 2008-10-16 Massachusetts Institute Of Technology Polymer-encapsulated reverse micelles
WO2008124639A2 (en) 2007-04-04 2008-10-16 Massachusetts Institute Of Technology Poly (amino acid) targeting moieties
EP1982729A1 (en) 2007-04-20 2008-10-22 Cytos Biotechnology AG Vaccination Regimen for B-Cell Vaccines
EP2757109B1 (en) 2007-05-31 2019-07-10 Academisch Ziekenhuis Leiden h.o.d.n. LUMC Hpv epitopes targeted by t cells infiltrating cervical malignancies for use in vaccines
US8778847B2 (en) 2007-06-13 2014-07-15 The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Immunogenic peptides of influenza virus
MX2010001779A (en) 2007-08-15 2010-07-06 Circassia Ltd Peptides for desensibilization against allergens.
US8394914B2 (en) 2007-08-24 2013-03-12 Board Of Trustees Of Michigan State University Functional polyglycolide nanoparticles derived from unimolecular micelles
ES2664753T3 (en) 2007-12-07 2018-04-23 Glaxosmithkline Biologicals Sa Immune response induction compositions
JP6088123B2 (en) 2008-02-01 2017-03-01 アルファ−オー・ペプチドズ・アーゲーＡｌｐｈａ−Ｏ Ｐｅｐｔｉｄｅｓ Ａｇ Self-assembling peptide nanoparticles useful as vaccines
KR101617790B1 (en) 2008-02-26 2016-05-03 아파르나 바이오사이언시스 Engineered Tunable Nanoparticles for Delivery of Therapeutics, Diagnostics, and Experimental Compounds and Related Compositions for Therapeutic Use
EP2250189A4 (en) * 2008-02-26 2012-07-04 Univ California Glycopeptides and methods of making and using them
JP2011525477A (en) 2008-03-04 2011-09-22 リクイディア・テクノロジーズ・インコーポレーテッド Immunomodulator particles and method of treatment
US20090297621A1 (en) 2008-06-03 2009-12-03 Abbott Cardiovascular Systems Inc. Microparticles For The Treatment Of Disease
EP2309990B2 (en) 2008-06-16 2017-03-15 Pfizer Inc. Drug loaded polymeric nanoparticles and methods of making and using same
US8613951B2 (en) 2008-06-16 2013-12-24 Bind Therapeutics, Inc. Therapeutic polymeric nanoparticles with mTor inhibitors and methods of making and using same
AU2009266940A1 (en) 2008-07-01 2010-01-07 Emory University Synergistic induction of humoral and cellular immunity by combinatorial activation of toll-like receptors
WO2010017330A1 (en) 2008-08-06 2010-02-11 Novartis Ag Microparticles for use in immunogenic compositions
WO2010018130A1 (en) 2008-08-11 2010-02-18 Smithkline Beecham Corporation Purine derivatives for use in the treatment of allergic, inflammatory and infectious diseases
US20110135671A1 (en) 2008-08-11 2011-06-09 Glaxosmithkline Llc Purine derivatives for use in the treatment of allergic, inflammatory and infectious diseases
US20120034156A1 (en) 2010-08-03 2012-02-09 Searete Llc, A Limited Liability Corporation Of The State Of Delaware Artificial cells
EP2331124A1 (en) 2008-08-15 2011-06-15 Circassia Limited T-cell antigen peptide from allergen for stimulation of il-10 production
US8323696B2 (en) 2008-08-29 2012-12-04 Ecole Polytechnique Federale De Lausanne Nanoparticles for immunotherapy
US8889635B2 (en) 2008-09-30 2014-11-18 The Regents Of The University Of Michigan Dendrimer conjugates
WO2010037402A1 (en) 2008-10-02 2010-04-08 Dako Denmark A/S Molecular vaccines for infectious disease
US8343497B2 (en) 2008-10-12 2013-01-01 The Brigham And Women's Hospital, Inc. Targeting of antigen presenting cells with immunonanotherapeutics
US8591905B2 (en) 2008-10-12 2013-11-26 The Brigham And Women's Hospital, Inc. Nicotine immunonanotherapeutics
US8343498B2 (en) * 2008-10-12 2013-01-01 Massachusetts Institute Of Technology Adjuvant incorporation in immunonanotherapeutics
US8277812B2 (en) 2008-10-12 2012-10-02 Massachusetts Institute Of Technology Immunonanotherapeutics that provide IgG humoral response without T-cell antigen
BRPI1016133A2 (en) 2009-04-01 2016-04-19 Univ Miami vaccine, method for dispensing an antigen to a mammal, composition, methods for dispensing siren within professional antigen presenting cells, for inhibiting proliferation of tumor cells, and for dispensing a nucleic acid to a cell.
BRPI1011836A2 (en) 2009-04-21 2017-05-16 Selecta Biosciences Inc immunotherapeutic agents that provide a th1-induced response
GB0907989D0 (en) * 2009-05-08 2009-06-24 Hybrid Systems Ltd Multivalent adjuvant display
AU2010254551B2 (en) 2009-05-27 2016-10-20 Selecta Biosciences, Inc. Immunomodulatory agent-polymeric compounds
EP2451485B1 (en) 2009-07-07 2016-03-02 The Research Foundation Of State University Of New York Lipidic compositions for induction of immune tolerance
US8653155B2 (en) * 2009-08-13 2014-02-18 Boston Scientific Scimed, Inc. Polymers having lipophilic hydrocarbon and biodegradable polymeric segments
BR112012003977A2 (en) 2009-08-26 2017-06-06 Selecta Biosciences Inc t-cell aid-inducing compositions
ES2780156T3 (en) 2009-12-15 2020-08-24 Pfizer Therapeutic compositions of polymeric nanoparticles with high glass transition temperature or high molecular weight copolymers
US20110171248A1 (en) 2010-01-08 2011-07-14 Selecta Biosciences, Inc. Synthetic virus-like particles conjugated to human papillomavirus capsid peptides for use as vaccines
US20110229556A1 (en) 2010-03-19 2011-09-22 Massachusetts Institute Of Technology Lipid-coated polymer particles for immune stimulation
US20110272836A1 (en) 2010-04-12 2011-11-10 Selecta Biosciences, Inc. Eccentric vessels
US20110262491A1 (en) 2010-04-12 2011-10-27 Selecta Biosciences, Inc. Emulsions and methods of making nanocarriers
CA2798323A1 (en) 2010-05-26 2011-12-01 Selecta Biosciences, Inc. Dose selection of adjuvanted synthetic nanocarriers
US20120064110A1 (en) 2010-08-20 2012-03-15 Selecta Biosciences, Inc. Synthetic nanocarrier vaccines comprising peptides obtained or derived from human influenza a virus hemagglutinin
CA2809029A1 (en) 2010-08-23 2012-03-01 Selecta Biosciences, Inc. Targeted multi-epitope dosage forms for induction of an immune response to antigens
US9994443B2 (en) 2010-11-05 2018-06-12 Selecta Biosciences, Inc. Modified nicotinic compounds and related methods
WO2012092552A1 (en) 2010-12-30 2012-07-05 Selecta Biosciences, Inc. Synthetic nanocarriers with reactive groups that release biologically active agents
JP6320912B2 (en) 2011-03-25 2018-05-09 セレクタ バイオサイエンシーズ インコーポレーテッドＳｅｌｅｃｔａ Ｂｉｏｓｃｉｅｎｃｅｓ，Ｉｎｃ． Osmotically mediated release synthetic nanocarrier
CN103517707A (en) 2011-04-29 2014-01-15 西莱克塔生物科技公司 Controlled release of immunosuppressants from synthetic nanocarriers
US20130028857A1 (en) 2011-07-29 2013-01-31 Selecta Biosciences, Inc. Synthetic nanocarriers comprising polymers comprising multiple immunomodulatory agents
US20130058902A1 (en) 2011-09-06 2013-03-07 Selecta Biosciences, Inc. Dendritic cell subsets for generating induced tolerogenic dendritic cells and related compositions and methods
KR20220025909A (en) 2013-05-03 2022-03-03 셀렉타 바이오사이언시즈, 인크. Delivery of immunosuppressants having a specified pharmacodynamic effective-life and antigen for the inducation of immune tolerance
KR102631173B1 (en) 2013-06-04 2024-01-31 셀렉타 바이오사이언시즈, 인크. Repeated administration of non-immunosupressive antigen specific immunotherapeutics
US20160220501A1 (en) 2015-02-03 2016-08-04 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers to reduce immune responses to therapeutic proteins
US20150359865A1 (en) 2014-06-17 2015-12-17 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers for t-cell-mediated autoimmune disease
WO2015200728A1 (en) 2014-06-25 2015-12-30 Selecta Biosciences, Inc. Methods and compositions for treatment with synthetic nanocarriers and immune checkpoint inhibitors
BR112017001470A2 (en) 2014-09-07 2018-02-20 Selecta Biosciences Inc methods and compositions for attenuating the immune responses of the gene therapy antiviral transfer vector
FI3834823T3 (en) 2014-11-05 2024-04-02 Cartesian Therapeutics Inc Methods and compositions related to the use of low hlb surfactants in the production of synthetic nanocarriers comprising a rapalog
CN109152819A (en) 2016-03-11 2019-01-04 西莱克塔生物科技公司 The preparation and dosage of Pegylation uricase
US20180071394A1 (en) 2016-08-25 2018-03-15 Selecta Biosciences, Inc. Polyester polymer matrices for the delivery of allergens
WO2018064215A1 (en) 2016-09-27 2018-04-05 Selecta Biosciences, Inc. Recombinant immunotoxins for use in the treatment of cancer

Patent Citations (53)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4946929A (en) 1983-03-22 1990-08-07 Massachusetts Institute Of Technology Bioerodible articles useful as implants and prostheses having predictable degradation rates
US4638045A (en) 1985-02-19 1987-01-20 Massachusetts Institute Of Technology Non-peptide polyamino acid bioerodible polymers
US4806621A (en) 1986-01-21 1989-02-21 Massachusetts Institute Of Technology Biocompatible, bioerodible, hydrophobic, implantable polyimino carbonate article
US5804178A (en) 1986-11-20 1998-09-08 Massachusetts Institute Of Technology Implantation of cell-matrix structure adjacent mesentery, omentum or peritoneum tissue
US5736372A (en) 1986-11-20 1998-04-07 Massachusetts Institute Of Technology Biodegradable synthetic polymeric fibrous matrix containing chondrocyte for in vivo production of a cartilaginous structure
US5770417A (en) 1986-11-20 1998-06-23 Massachusetts Institute Of Technology Children's Medical Center Corporation Three-dimensional fibrous scaffold containing attached cells for producing vascularized tissue in vivo
US5019379A (en) 1987-07-31 1991-05-28 Massachusetts Institute Of Technology Unsaturated polyanhydrides
US6130082A (en) 1988-05-05 2000-10-10 American Cyanamid Company Recombinant flagellin vaccines
US5010167A (en) 1989-03-31 1991-04-23 Massachusetts Institute Of Technology Poly(amide-and imide-co-anhydride) for biological application
US5399665A (en) 1992-11-05 1995-03-21 Massachusetts Institute Of Technology Biodegradable polymers for cell transplantation
US5512600A (en) 1993-01-15 1996-04-30 Massachusetts Institute Of Technology Preparation of bonded fiber structures for cell implantation
US5696175A (en) 1993-01-15 1997-12-09 Massachusetts Institute Of Technology Preparation of bonded fiber structures for cell implantation
US5514378A (en) 1993-02-01 1996-05-07 Massachusetts Institute Of Technology Biocompatible polymer membranes and methods of preparation of three dimensional membrane structures
US5543158A (en) 1993-07-23 1996-08-06 Massachusetts Institute Of Technology Biodegradable injectable nanoparticles
US5578325A (en) 1993-07-23 1996-11-26 Massachusetts Institute Of Technology Nanoparticles and microparticles of non-linear hydrophilic-hydrophobic multiblock copolymers
US7202351B1 (en) 1993-09-14 2007-04-10 Pharmexa Inc. Alteration of immune response using pan DR-binding peptides
US6207646B1 (en) 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6007845A (en) 1994-07-22 1999-12-28 Massachusetts Institute Of Technology Nanoparticles and microparticles of non-linear hydrophilic-hydrophobic multiblock copolymers
US5716404A (en) 1994-12-16 1998-02-10 Massachusetts Institute Of Technology Breast tissue engineering
US6123727A (en) 1995-05-01 2000-09-26 Massachusetts Institute Of Technology Tissue engineered tendons and ligaments
US6095148A (en) 1995-11-03 2000-08-01 Children's Medical Center Corporation Neuronal stimulation using electrically conducting polymers
US5902599A (en) 1996-02-20 1999-05-11 Massachusetts Institute Of Technology Biodegradable polymer networks for use in orthopedic and dental applications
US6696076B2 (en) 1996-10-25 2004-02-24 3M Innovative Properties Company Immune response modifier compounds for treatment of TH2 mediated and related diseases
US6585980B1 (en) 1997-04-11 2003-07-01 The University Of Toronto Flagellin gene, FlaC of Campylobacter
US5837752A (en) 1997-07-17 1998-11-17 Massachusetts Institute Of Technology Semi-interpenetrating polymer networks
US6329381B1 (en) 1997-11-28 2001-12-11 Sumitomo Pharmaceuticals Company, Limited Heterocyclic compounds
US6632922B1 (en) 1998-03-19 2003-10-14 The Regents Of The University Of California Methods and compositions for controlled polypeptide synthesis
US6686446B2 (en) 1998-03-19 2004-02-03 The Regents Of The University Of California Methods and compositions for controlled polypeptide synthesis
US6506577B1 (en) 1998-03-19 2003-01-14 The Regents Of The University Of California Synthesis and crosslinking of catechol containing copolypeptides
US7223398B1 (en) 1999-11-15 2007-05-29 Dynavax Technologies Corporation Immunomodulatory compositions containing an immunostimulatory sequence linked to antigen and methods of use thereof
US6632671B2 (en) 2000-02-28 2003-10-14 Genesegues, Inc. Nanoparticle encapsulation system and method
US7250403B2 (en) 2000-03-10 2007-07-31 Dynavax Technologies Corporation Biodegradable immunomodulatory formulations and methods for use thereof
US20060222652A1 (en) 2000-05-05 2006-10-05 Cytos Biotechnology Ag Molecular antigen array
US7192725B2 (en) 2000-05-19 2007-03-20 University Of Toronto Flagellin gene, flaC of Campylobacter
US20020086049A1 (en) 2000-09-18 2002-07-04 Bolton Anthony E. Apoptosis-mimicking synthetic entities and use thereof in medical treatment
US20080145441A1 (en) 2000-10-16 2008-06-19 Midatech Limited Nanoparticles
US6818732B2 (en) 2001-08-30 2004-11-16 The Regents Of The University Of California Transition metal initiators for controlled poly (beta-peptide) synthesis from beta-lactam monomers
WO2003086280A2 (en) 2002-04-04 2003-10-23 Coley Pharmaceutical Gmbh Immunostimulatory g,u-containing oligoribonucleotides
US20060251677A1 (en) 2003-03-26 2006-11-09 Cytos Biotechnology Ag Packaging of immunostimulatory oligonucleotides into virus-like particles: method of preparation and use
US20090028910A1 (en) 2003-12-19 2009-01-29 University Of North Carolina At Chapel Hill Methods for Fabrication Isolated Micro-and Nano-Structures Using Soft or Imprint Lithography
WO2005097993A2 (en) 2004-02-19 2005-10-20 Coley Pharmaceutical Group, Inc. Immunostimulatory viral rna oligonucleotides
US20060002852A1 (en) 2004-07-01 2006-01-05 Yale University Targeted and high density drug loaded polymeric materials
US7566703B2 (en) 2004-10-20 2009-07-28 Coley Pharmaceutical Group, Inc. Semi-soft C-class immunostimulatory oligonucleotides
US20060241076A1 (en) 2005-04-26 2006-10-26 Coley Pharmaceutical Gmbh Modified oligoribonucleotide analogs with enhanced immunostimulatory activity
WO2007062107A2 (en) 2005-11-25 2007-05-31 Coley Pharmaceutical Gmbh Immunostimulatory oligoribonucleotides
WO2008033432A2 (en) 2006-09-12 2008-03-20 Coley Pharmaceutical Group, Inc. Immune modulation by chemically modified ribonucleosides and oligoribonucleotides
US20090226525A1 (en) 2007-04-09 2009-09-10 Chimeros Inc. Self-assembling nanoparticle drug delivery system
WO2008127532A1 (en) 2007-04-12 2008-10-23 Emory University Novel strategies for delivery of active agents using micelles and particles
WO2009051837A2 (en) 2007-10-12 2009-04-23 Massachusetts Institute Of Technology Vaccine nanotechnology
WO2009106999A2 (en) 2008-02-28 2009-09-03 Deutsches Krebsforschungszentrum, Stiftung Des Öffentlichen Rechts Hollow nanoparticles and uses thereof
WO2010018132A1 (en) 2008-08-11 2010-02-18 Smithkline Beecham Corporation Compounds
US20100075995A1 (en) 2008-08-11 2010-03-25 Smithkline Beecham Corporation Compounds
WO2010047839A1 (en) 2008-10-25 2010-04-29 Aura Biosciences Modified plant virus particles and uses therefor

Non-Patent Citations (69)

* Cited by examiner, † Cited by third party
Title
"Concise Encyclopedia of Polymer Science and Polymeric Amines and Ammonium Salts", 1980, PERGAMON PRESS
"Handbook of Industrial Mixing: Science and Practice", 2004, JOHN WILEY & SONS, INC.
"Microcapsules and Nanoparticles in Medicine and Pharmacy", 1992, CRC PRESS
"Pharmaceutics: The Science of Dosage Form Design", 2001, CHURCHILL LIVINGSTONE
ALLCOCK ET AL.: "Contemporary Polymer Chemistry", 1981, PRENTICE-HALL
BARRERA ET AL., J. AM. CHEM. SOC., vol. 115, 1993, pages 11010
BOUSSIF ET AL., PROC. NATL. ACAD. SCI., USA, vol. 92, 1995, pages 7297
BUI HH; SIDNEY J; PETERS B; SATHIAMURTHY M; SINICHI A; PURTON KA; MOTHE BR; CHISARI FV; WATKINS DI; SETTE A, IMMUNOGENETICS, vol. 57, 2005, pages 304 - 314
BUI HH; SIDNEY J; PETERS B; SATHIAMURTHY M; SINICHI A; PURTON KA; MOTHE BR; CHISARI FV; WATKINS DI; SETTE A.: "Automated generation and evaluation of specific MHC binding predictive tools: ARB matrix applications", IMMUNOGENETICS, vol. 57, 2005, pages 304 - 314, XP019331658, DOI: doi:10.1007/s00251-005-0798-y
C. ASTETE ET AL.: "Synthesis and characterization of PLGA nanoparticles", J. BIOMATER. SCI. POLYMER EDN, vol. 17, no. 3, 2006, pages 247 - 289, XP009134610
C. REIS ET AL.: "Nanoencapsulation I. Methods for preparation of drug-loaded polymeric nanoparticles", NANOMEDICINE, vol. 2, 2006, pages 8 - 21
CHOU PY; FASMAN GD: "Prediction of the secondary structure of proteins from their amino acid sequence", ADV ENZYMOL RELAT AREAS MOL BIOL, vol. 47, 1978, pages 45 - 148
CHU ET AL.: "CpG oligodeoxynucleotides act as adjuvants that switch on T helper 1 (Thl) immunity", J. EXP. MED., vol. 186, 1997, pages 1623 - 1631, XP002910130, DOI: doi:10.1084/jem.186.10.1623
DAVIS ET AL.: "CpG DNA is a potent enhancer of specific immunity in mice immunized with recombinant hepatitis B surface antigen", J. IMMUNOL., vol. 160, 1998, pages 870 - 876
DEMING ET AL., NATURE, vol. 390, 1997, pages 386
EMINI EA; HUGHES JV; PERLOW DS; BOGER J: "Induction of hepatitis A virus-neutralizing antibody by a virus-specific synthetic peptide", J VIROL, vol. 55, 1985, pages 836 - 839
F. HEIL ET AL.: "Species-Specific Recognition of Single-Stranded RNA via Toll-like Receptor 7 and 8", SCIENCE, vol. 303, no. 5663, 2004, pages 1526 - 1529, XP002371479
HAENSLER ET AL., BIOCONJUGATE CHEM, vol. 4, 1993, pages 372
HASTE ANDERSEN P; NIELSEN M; LUND O: "Prediction of residues in discontinuous B-cell epitopes using protein 3D structures", PROTEIN SCI, vol. 15, 2006, pages 2558 - 2567, XP055113267, DOI: doi:10.1110/ps.062405906
HERMANSON G T: "Bioconjugate Techniques", 2008, ACADEMIC PRESS, INC.
K. AVGOUSTAKIS: "Pegylated Poly(Lactide) and Poly(Lactide-Co-Glycolide) Nanoparticles: Preparation, Properties and Possible Applications in Drug Delivery", CURRENT DRUG DELIVERY, vol. 1, 2004, pages 321 - 333, XP009134627
KABANOV ET AL., BIOCONJUGATE CHEM., vol. 6, 1995, pages 7
KARPLUS PA; SCHULZ GE: "Prediction of chain flexibility in proteins", NATURWISSENSCHAFTEN, vol. 72, 1985, pages 212 - 213
KOLASKAR AS; TONGAONKAR PC: "A semi-empirical method for prediction of antigenic determinants on protein antigens", FEBS LETT, vol. 276, 1990, pages 172 - 174, XP025603614, DOI: doi:10.1016/0014-5793(90)80535-Q
KRIEG ET AL.: "CpG motifs in bacterial DNA trigger direct B cell activation", NATURE, vol. 374, 1995, pages 546 - 549, XP002910391, DOI: doi:10.1038/374546a0
KUKOWSKA-LATALLO ET AL., PROC. NATL. ACAD. SCI., USA, vol. 93, 1996, pages 4897
KWON ET AL., MACROMOLECULES, vol. 22, 1989, pages 3250
LANGER, ACC. CHEM. RES., vol. 33, 2000, pages 94
LANGER, J. CONTROL. RELEASE, vol. 62, 1999, pages 7
LARSEN JE; LUND O; NIELSEN M: "Improved method for predicting linear B-cell epitopes", IMMUNOME RES, vol. 2, 2006, pages 2, XP021019965, DOI: doi:10.1186/1745-7580-2-2
LIM ET AL., J. AM. CHEM. SOC., vol. 121, 1999, pages 5633
LIM ET AL., J. AM. CHEM. SOC., vol. 121, pages 5633
LIM ET AL., J. AM. CHEM. SOC., vol. 123, 2001, pages 2460
LIPFORD ET AL.: "Bacterial DNA as immune cell activator", TRENDS MICROBIOL., vol. 6, 1998, pages 496 - 500, XP000952562, DOI: doi:10.1016/S0966-842X(98)01408-5
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., vol. 27, 1997, pages 2340 - 2344, XP002123073, DOI: doi:10.1002/eji.1830270931
MATHIOWITZ ET AL., J. APPL. POLYMER SCI., vol. 35, 1988, pages 755
MATHIOWITZ ET AL., J. CONTROL. RELEASE, vol. 5, 1987, pages 13
MATHIOWITZ ET AL., REACTIVE POLYMERS, vol. 6, 1987, pages 275
MELDAL ET AL., CHEM. REV., vol. 108, no. 8, 2008, pages 2952 - 3015
MURRAY ET AL., ANN. REV. MAT. SCI., vol. 30, 2000, pages 545
NIELSEN M; LUND O: "NN-align. An artificial neural network-based alignment algorithm for MHC class II peptide binding prediction", BMC BIOINFORMATICS, vol. 10, 2009, pages 296, XP021055730, DOI: doi:10.1186/1471-2105-10-296
NIELSEN M; LUNDEGAARD C; BLICHER T; PETERS B; SETTE A; JUSTESEN S; BUUS S; LUND 0, PLOS COMPUT BIOL, vol. 4, no. 7, 2008, pages e1000107
NIELSEN M; LUNDEGAARD C; LUND O: "Prediction of MHC class II binding affinity using SMM-align, a novel stabilization matrix alignment method", BMC BIOINFORMATICS, vol. 8, 2007, pages 238, XP021027565, DOI: doi:10.1186/1471-2105-8-238
NIELSEN M; LUNDEGAARD C; WORNING P; LAUEMOLLER SL; LAMBERTH K; BUUS S; BRUNAK S; LUND 0: "Reliable prediction of T-cell epitopes using neural networks with novel sequence representations", PROTEIN SCI, vol. 12, 2003, pages 1007 - 1017
ODIAN: "Principles of Polymerization", 2004, JOHN WILEY & SONS
P. PAOLICELLI ET AL.: "Surface-modified PLGA-based Nanoparticles that can Efficiently Associate and Deliver Virus-like Particles", NANOMEDICINE, vol. 5, no. 6, 2010, pages 843 - 853
PAPISOV, ACS SYMPOSIUM SERIES, vol. 786, 2001, pages 301
PARKER JM; GUO D; HODGES RS: "New hydrophilicity scale derived from high-performance liquid chromatography peptide retention data: correlation of predicted surface residues with antigenicity and X-ray-derived accessible sites", BIOCHEMISTRY, vol. 25, 1986, pages 5425 - 5432, XP001062495, DOI: doi:10.1021/bi00367a013
PELLEGRINO ET AL., SMALL, vol. 1, 2005, pages 48
PETERS B; SETTE A: "Generating quantitative models describing the sequence specificity of biological processes with the stabilized matrix method", BMC BIOINFORMATICS, vol. 6, 2005, pages 132, XP021000724, DOI: doi:10.1186/1471-2105-6-132
PONOMARENKO JV; BOURNE PE: "Antibody-protein interactions: benchmark datasets and prediction tools evaluation", BMC STRUCT BIOL, vol. 7, 2007, pages 64, XP021035783
PONOMARENKO JV; BUI H; LI W; FUSSEDER N; BOURNE PE; SETTE A; PETERS B: "ElliPro: a new structure-based tool for the prediction of antibody epitopes", BMC BIOINFORMATICS, vol. 9, 2008, pages 514
PUTNAM ET AL., MACROMOLECULES, vol. 32, 1999, pages 3658
ROMAN ET AL.: "Immunostimulatory DNA sequences function as T helper-1-promoting adjuvants", NAT. MED., vol. 3, 1997, pages 849 - 854, XP002188113, DOI: doi:10.1038/nm0897-849
SCIARRA; CUTIE: "Remington's Pharmaceutical Sciences", 1990, article "Aerosols", pages: 1694 - 1712
See also references of EP2736537A4
SHARPLESS ET AL., ANGEW. CHEM. INT. ED., vol. 41, no. 14, 2002, pages 2596
STANO ET AL.: "PPS nanoparticles as versatile delivery system to induce systemic and broad mucosal immunity after intranasal administration", VACCINE, vol. 29, no. 4, 2011, pages 804 - 812
STURNIOLO T; BONO E; DING J; RADDRIZZANI L; TUERECI O; SAHIN U; BRAXENTHALER M; GALLAZZI F; PROTTI MP; SINIGAGLIA F: "Generation of tissue-specific and promiscuous HLA ligand databases using DNA microarrays and virtual HLA class II matrices", NAT BIOTECHNOL., vol. 17, no. 6, 1999, pages 555 - 561, XP002168815
TANG ET AL., BIOCONJUGATE CHEM., vol. 7, 1996, pages 703
TRINDADE ET AL., CHEM. MAT., vol. 13, 2001, pages 3843
UHRICH ET AL., CHEM. REV., vol. 99, 1999, pages 3181
V. CERUNDOLO ET AL.: "Harnessing invariant NKT cells in vaccination strategies", NATURE REV IMMUN, vol. 9, 2009, pages 28 - 38
VITA R; ZAREBSKI L; GREENBAUM JA; EMAMI H; HOOF I; SALIMI N; DAMLE R; SETTE A; PETERS B: "The immune epitope database 2.0", NUCLEIC ACIDS RES., vol. 38, January 2010 (2010-01-01), pages D854 - 62, XP055204880, DOI: doi:10.1093/nar/gkp1004
WANG ET AL., J. AM. CHEM. SOC., vol. 123, 2001, pages 9480
WANG P; SIDNEY J; DOW C; MOTHE B; SETTE A; PETERS B: "A systematic assessment of MHC class II peptide binding predictions and evaluation of a consensus approach", PLOS COMPUT BIOL, vol. 4, no. 4, 2008, pages e1000048, XP002598739, DOI: doi:10.1371/journal.pcbi.1000048
WANG P; SIDNEY J; KIM Y; SETTE A; LUND O; NIELSEN M; PETERS B: "peptide binding predictions for HLA DR, DP and DQ molecules", BMC BIOINFORMATICS, vol. 11, 2010, pages 568, XP021085842, DOI: doi:10.1186/1471-2105-11-568
ZAUNER ET AL., ADV. DRUG DEL. REV., vol. 30, 1998, pages 97
ZHOU ET AL., MACROMOLECULES, vol. 23, 1990, pages 3399

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9447164B2 (en) 2010-08-06 2016-09-20 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US9937233B2 (en) 2010-08-06 2018-04-10 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
US9181319B2 (en) 2010-08-06 2015-11-10 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
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US9216205B2 (en) 2012-04-02 2015-12-22 Moderna Therapeutics, Inc. Modified polynucleotides encoding granulysin
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US9878056B2 (en) 2012-04-02 2018-01-30 Modernatx, Inc. Modified polynucleotides for the production of cosmetic proteins and peptides
US9675668B2 (en) 2012-04-02 2017-06-13 Moderna Therapeutics, Inc. Modified polynucleotides encoding hepatitis A virus cellular receptor 2
US9828416B2 (en) 2012-04-02 2017-11-28 Modernatx, Inc. Modified polynucleotides for the production of secreted proteins
US9827332B2 (en) 2012-04-02 2017-11-28 Modernatx, Inc. Modified polynucleotides for the production of proteins
US9782462B2 (en) 2012-04-02 2017-10-10 Modernatx, Inc. Modified polynucleotides for the production of proteins associated with human disease
US9814760B2 (en) 2012-04-02 2017-11-14 Modernatx, Inc. Modified polynucleotides for the production of biologics and proteins associated with human disease
US9597380B2 (en) 2012-11-26 2017-03-21 Modernatx, Inc. Terminally modified RNA
US8980864B2 (en) 2013-03-15 2015-03-17 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
WO2015034928A1 (en) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Chimeric polynucleotides
WO2015034925A1 (en) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Circular polynucleotides
US10815291B2 (en) 2013-09-30 2020-10-27 Modernatx, Inc. Polynucleotides encoding immune modulating polypeptides
US10323076B2 (en) 2013-10-03 2019-06-18 Modernatx, Inc. Polynucleotides encoding low density lipoprotein receptor
EP3985118A1 (en) 2013-11-22 2022-04-20 MiNA Therapeutics Limited C/ebp alpha short activating rna compositions and methods of use
WO2015075557A2 (en) 2013-11-22 2015-05-28 Mina Alpha Limited C/ebp alpha compositions and methods of use
EP3594348A1 (en) 2013-11-22 2020-01-15 Mina Therapeutics Limited C/ebp alpha short activating rna compositions and methods of use
WO2016014846A1 (en) 2014-07-23 2016-01-28 Moderna Therapeutics, Inc. Modified polynucleotides for the production of intrabodies
EP4349404A2 (en) 2015-10-22 2024-04-10 ModernaTX, Inc. Respiratory virus vaccines
EP4349405A2 (en) 2015-10-22 2024-04-10 ModernaTX, Inc. Respiratory virus vaccines
EP4011451A1 (en) 2015-10-22 2022-06-15 ModernaTX, Inc. Metapneumovirus mrna vaccines
EP4039699A1 (en) 2015-12-23 2022-08-10 ModernaTX, Inc. Methods of using ox40 ligand encoding polynucleotides
WO2017112943A1 (en) 2015-12-23 2017-06-29 Modernatx, Inc. Methods of using ox40 ligand encoding polynucleotides
WO2017120612A1 (en) 2016-01-10 2017-07-13 Modernatx, Inc. Therapeutic mrnas encoding anti ctla-4 antibodies
CN106065047A (en) * 2016-07-14 2016-11-02 山东省肿瘤防治研究院 A kind of Liver targeting cationic polymer and preparation method and application
WO2018104540A1 (en) 2016-12-08 2018-06-14 Curevac Ag Rnas for wound healing
WO2018104538A1 (en) 2016-12-08 2018-06-14 Curevac Ag Rna for treatment or prophylaxis of a liver disease
EP3808380A1 (en) 2016-12-08 2021-04-21 CureVac AG Rna for treatment or prophylaxis of a liver disease
WO2019048632A1 (en) 2017-09-08 2019-03-14 Mina Therapeutics Limited Stabilized hnf4a sarna compositions and methods of use
EP4183882A1 (en) 2017-09-08 2023-05-24 MiNA Therapeutics Limited Stabilized hnf4a sarna compositions and methods of use
WO2019048631A1 (en) 2017-09-08 2019-03-14 Mina Therapeutics Limited Hnf4a sarna compositions and methods of use
WO2019048645A1 (en) 2017-09-08 2019-03-14 Mina Therapeutics Limited Stabilized cebpa sarna compositions and methods of use
EP4233880A2 (en) 2017-09-08 2023-08-30 MiNA Therapeutics Limited Hnf4a sarna compositions and methods of use
EP4219715A2 (en) 2017-09-08 2023-08-02 MiNA Therapeutics Limited Stabilized cebpa sarna compositions and methods of use
EP4242307A2 (en) 2018-04-12 2023-09-13 MiNA Therapeutics Limited Sirt1-sarna compositions and methods of use
WO2019197845A1 (en) 2018-04-12 2019-10-17 Mina Therapeutics Limited Sirt1-sarna compositions and methods of use
WO2020208361A1 (en) 2019-04-12 2020-10-15 Mina Therapeutics Limited Sirt1-sarna compositions and methods of use
WO2022122872A1 (en) 2020-12-09 2022-06-16 Ucl Business Ltd Therapeutics for the treatment of neurodegenerative disorders
WO2022200810A1 (en) 2021-03-26 2022-09-29 Mina Therapeutics Limited Tmem173 sarna compositions and methods of use
WO2023006999A2 (en) 2021-07-30 2023-02-02 CureVac SE Mrnas for treatment or prophylaxis of liver diseases
WO2023099884A1 (en) 2021-12-01 2023-06-08 Mina Therapeutics Limited Pax6 sarna compositions and methods of use
WO2023104964A1 (en) 2021-12-09 2023-06-15 Ucl Business Ltd Therapeutics for the treatment of neurodegenerative disorders
WO2023144193A1 (en) 2022-01-25 2023-08-03 CureVac SE Mrnas for treatment of hereditary tyrosinemia type i
WO2023161350A1 (en) 2022-02-24 2023-08-31 Io Biotech Aps Nucleotide delivery of cancer therapy
WO2023170435A1 (en) 2022-03-07 2023-09-14 Mina Therapeutics Limited Il10 sarna compositions and methods of use

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