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EP3218001A2 - Nanodispositifs modulaires pour vaccins intelligents adaptables - Google Patents

Nanodispositifs modulaires pour vaccins intelligents adaptables

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Publication number
EP3218001A2
EP3218001A2 EP15802242.6A EP15802242A EP3218001A2 EP 3218001 A2 EP3218001 A2 EP 3218001A2 EP 15802242 A EP15802242 A EP 15802242A EP 3218001 A2 EP3218001 A2 EP 3218001A2
Authority
EP
European Patent Office
Prior art keywords
composition
cells
antigen
elements
nanoparticles
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP15802242.6A
Other languages
German (de)
English (en)
Inventor
Ira S. Mellman
Tarek M. Fahmy
William Mark Saltzman
Michael J. Caplan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yale University
Original Assignee
Yale University
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 claimed from US14/537,541 external-priority patent/US10265407B2/en
Application filed by Yale University filed Critical Yale University
Publication of EP3218001A2 publication Critical patent/EP3218001A2/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6933—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained by reactions only involving carbon to carbon, e.g. poly(meth)acrylate, polystyrene, polyvinylpyrrolidone or polyvinylalcohol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A61K47/6937—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol the polymer being PLGA, PLA or polyglycolic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6939—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
    • C12N2760/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present disclosure generally relates to the field of modular scale vaccine compositions and methods of making and using these compositions.
  • the first variable is the form of the antigen itself, which can be whole inactivated or attenuated organisms, purified proteins and peptides, or DNA encoded antigens.
  • a second necessary component of a vaccine involves providing an adjuvant or other means for potentiating or stimulating both the innate and adaptive arms of the immune system to the antigen subunit (Pashine, et al., Nat. Med., 11(4 Suppl):S63-8 (2005), Bramwell and Perrie, Drug Discovery Today, 10(22):1527-34 (2005)).
  • Immune potentiators may include bacterial products, toxins or other molecules that augment specific immunity. Potentiators have various benefits, but also attendant risks such as triggering deleterious inflammatory responses. To affect optimal stimulation to a given antigen, a formulation is needed that delivers the correct amount of antigen in a repetitive or sustained fashion, to the appropriate immune cells and to the appropriate
  • adjuvant should target the vaccine antigen and facilitate delivery of both antigen and immune potentiating molecules selectively to target cells of the immune system. This is highly reminiscent of the strategy taken by viruses that inactivate specific components of the immune system during infection.
  • Traditional methods for increasing the effectiveness of vaccines have focused on co-administration of adjuvants or use of a delivery system. While the adjuvant role is critical, there are obvious risks, costs and limitations associated with this traditional approach. For example, currently available adjuvants, represented predominately by colloidal alum (aluminum sulfate or aluminum hydroxide) or montanide polymers, have a limited capacity to adsorb many antigens and have greatly limited
  • mucosal immunity is essential for protective responses to cellular and viral pathogens that are transmitted through mucosal surfaces (e.g. human immunodeficiency virus, HIV; herpes simplex virus, HSV; enteric pathogens).
  • pathogens e.g. human immunodeficiency virus, HIV; herpes simplex virus, HSV; enteric pathogens.
  • nanoparticulate vaccine compositions which provide for flexible addition and subtraction of elements.
  • Modular nanoparticle vaccine compositions and methods of making and using them have been developed.
  • the modular design of these nanoparticle vaccine compositions which involves flexible addition and subtraction of antigen, adjuvant and/or immune potentiators, molecular recognition factors, and transport mediation elements, as well as intracellular uptake mediators, allows foraki control over many of the variables that are important for optimizing an effective vaccine delivery system.
  • a key feature of these nanodevices is their ability to be selectively targeted to those cells of the immune system that are most closely associated with producing the desired immunological response for a given vaccine. This is
  • nanoparticle surface is then modified by the direct or indirect coupling of targeting molecules, such as antibodies, that guide the entire nanodevice to specific cell types (such as dendritic cells) associated with stimulating or suppressing immune responses.
  • targeting molecules such as antibodies
  • the targeted particles are constructed to bind to the intended cell type, to be internalized by endocytosis, and then to dissociate, thereby releasing the encapsulated antigen and immune activators
  • the modular nature of the nanodevice enables rapid production and the ability to modify the nanoparticle surface with any of a variety of targeting molecules, enabling targeting to different cell types, such as various dendritic cell subsets, epithelial cells, or macrophages.
  • the adjuvant composition can also be easily altered to enable the systematic assessment of optimal targeting and composition for any desired application.
  • the nanodevices can be easily characterized biochemically using conventional ELIS A and flow cytometry assays, and by in vitro or in vivo assays for antigen presentation and immune stimulation.
  • Modular nanoparticle vaccine compositions include an antigen incorporated or encapsulated in a polymeric nanoparticle.
  • Antigens may be viral, bacterial, parasitic, allergen, toxoid, tumor-specific or tumor-associated antigens, which can be one or more proteins, carbohydrates, lipids, nucleic acids, or combinations thereof.
  • the nanoparticle further includes adaptor elements which modularly couple functional elements to the particle.
  • the adaptor elements are fatty acids, hydrophobic or amphipathic peptides, or hydrophobic polymers.
  • Adaptor elements can be conjugated to affinity tags, which allow for modular assembly and disassembly of functional elements which are conjugated to complementary affinity tags to the nanoparticle.
  • Functional elements impart useful functions to the nanoparticle compositions. Functional elements may include, for example, dendritic cell targeting molecules, epithelial cell targeting molecules, pH-sensitive or non-pH-sensitive molecules which protect the vaccine composition from hydrolysis and degradation in low pH
  • Nanoparticle vaccine compositions may further include adjuvants, contrast agents and other markers and pharmaceutically acceptable excipients.
  • the ability to target exogenous antigens to internalizing surface molecules on antigen-presenting cells facilitates the uptake of antigens and their presentation to lymphocytes and thus overcomes a major rate-limiting step in vaccination.
  • the ability to target vaccine compositions to epithelial cells in the digestive tract greatly facilitates the ability of a vaccine to induce mucosal and systemic immunity when administered orally.
  • Molecules which protect the vaccine composition from hydrolysis and degradation in low pH environments also enhance the efficacy of vaccines administered orally.
  • Endosome-disrupting agents function to cause limited disruption of endosome-lysosome membranes during antigen uptake by antigen-presenting cells.
  • the modular nanoparticulate vaccine compositions offer several advantages over other vaccines: 1) targeting of different cells, thereby enabling optimal selection of different tissue and priming for antigen presentation; 2) delivery of a wide variety of antigens of clinical importance; and 3) rapid assembly of different combinations of protective, recognition and antigen modules to affect a broad-spectrum potent vaccine response.
  • Figure 1 is a graph demonstrating inhibition of CD3 -stimulated T- cell proliferation (number of cells) when T-cells are exposed to doxorubicin- loaded particles modified with an antibody (- ⁇ -) that recognizes T-cells at the indicated concentration (mg/ml).
  • Controls are doxorubicin-loaded nanoparticles without antibody (- ⁇ -) and blank nanoparticles (-A-).
  • Figure 2 A is a graph showing that spleen cells obtained from mice three days after subcutaneous immunization with ovalbumin-encapsulated, LPS -modified, nanoparticles (- ⁇ -) proliferated (number of cells x 10 6 ) in response to immobilized ovalbumin, thus demonstrating memory to the antigen. Controls are immobilized antigen (-o-) and blank nanoparticles
  • Figure 2B is a graph showing that spleen cells obtained from mice following oral immunization with ovalbumin-encapsulated, LPS-modified, nanoparticles (- ⁇ -) proliferated (absorbance) in response to immobilized ovalbumin, demonstrating the efficacy of the particles in inducing immunity through oral administration. Controls are immobilized (-o-) antigen and blank nanoparticles (- ⁇ -).
  • Figure 3 is a graph showing release of IL-2 (ng/ml) by CD8 (OT-1) (- ⁇ -) and CD4 (OT-II) (- ⁇ -) positive T-cells as a function of the
  • Endoporter concentration of Endoporter ( ⁇ /ml) incubated with mouse bone marrow- derived dendritic cells. This graph demonstrates that inclusion of increasing concentrations of Endoporter enhanced cross presentation of antigen to MHC class I-restricted CD8 T-cells, while presentation to MHC class II-restricted CD4 T-cells was not diminished.
  • Figure 5 is a diagram showing the mechanism of endosomal disruption following uptake of cargo by cells using endoporter as the exemplary endosomal targeting agent.
  • Figure 6 is a bar graph showing endosomal disruption as percentage (%) of bone marrow derived dendritic cells positive for 26.D16-PE when incubated for 24 hours with PLGA or liposomal nanoparticles encapsulating a positively charged macromolecule dendrimer (G5) and the antigen ovalbumin.
  • Figure 7 is a dot plot showing endosomal disruption as percentage (%) of bone marrow derived macrophages positive for 26.D16-PE when incubated for 24 hours with PLGA-PEG or liposomal nanoparticles encapsulating a positively charged macromolecule dendrimer and the antigen ovalbumin.
  • Figure 8 is a bar graph showing percentage (%) of dendritic cells with disrupted endosomes when the cells are incubated with PLGA- dendrimer (G4) or PLGA-cyclodextrin nanoparticles without (1) or with (2) carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP).
  • G4 PLGA- dendrimer
  • FCCP carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone
  • Figure 9 is a bar graph showing percentage (%) of green fluorescent protein (GFP)-positive bone marrow derived dendritic cells transfected with monophosphoryl lipid A (MPLA) or liposomal nanoparticles co- encapsulating GFP, the dendrimer G5 and/or CpG DNA.
  • GFP green fluorescent protein
  • MPLA monophosphoryl lipid A
  • LED stands for "Lipid Encapsulated Dendrimer”.
  • the different groups being: -/G5 LED is a Lipid Encapsulating Dendrimer of Generation 5 with no surface
  • (-/G5+CpG) LED is the same with co-encapsulated CpG associated with the encapsulated dendrimer
  • MPLA/G5 LED is the same with MPLA surface modified lipid and encapsulating G5 Dendrimer
  • MPLA/(G5+CpG) LED is MPLA surface modified lipid encapsulating CpG associated with the G5 Dendrimer; MPLA/G5 LED & -/(G5+CpG) LED are two particles with and without MPLA and unmodified particle encapsulating CpG; Lipofectamine is a gold standard for gene transfection.
  • Figure 10 is a bar graph showing change in MFI targeted/non targeted antigen presenting cells (APCs) when BDCA3+ and DC SIGN+ DC subsets are targeted with nanoparticles surface modified with anti-BDCA3 and anti-DC-SIGN antibodies, respectively.
  • APCs MFI targeted/non targeted antigen presenting cells
  • Figures 11A-11D are bar graphs showing mean cytokine expression levels (IL-15, IFN- ⁇ and TNF-a in pg/ml, and IL-6 and IL-8 in pg/dl, ⁇ SEM) per 30,000 APCs for BDCA3+ or DC-SIGN+ DC subsets obtained from three different healthy donors.
  • Figure 12 is a bar graph showing mean percentage (%, ⁇ SEM) of CD8+ cells producing IFN- ⁇ when stimulated by Mo-DCs or BDCA3+ MDCs loaded with either blank NP or NP-CEF.
  • a solution to the vaccine problem requires a systematic approach that addresses each of the design challenges discussed above.
  • Viruses and pathogens that elicit or subvert immune responses are, in essence, small particles endowed with the ability to interact with or avoid cells of the immune system in a variety of ways.
  • the vaccines described herein are based on an approach in which nanoscale modules are assembled into units that are optimized for stimulating immune responses to a specific pathogen.
  • the principles of nanoassembly is used to design safe vaccine vectors that are highly optimized to protect against disease and provide new treatment options for disorders such as asthma, allergy, and cancer.
  • Affinity tags are defined herein as molecular species which form highly specific, non-covalent, physiochemical interactions with defined binding partners. Affinity tags which form highly specific, non-covalent, physiochemical interactions with one another are defined herein as
  • Adaptor elements are defined herein as molecular entities which associate with polymeric nanoparticles and provide substrates that facilitate the modular assembly and disassembly of functional elements onto the nanoparticle. Adaptor elements can be conjugated to affinity tags. Affinity tags allow for flexible assembly and disassembly of functional elements which are conjugated to affinity tags that form highly specific, noncovalent, physiochemical interactions with affinity tags conjugated to adaptor elements. Adaptor elements can also be covalently coupled to functional elements in the absence of affinity tags.
  • Functional elements are defined herein as molecular entities which associate with nanoparticles and impart a particular function to the nanoparticle. Functional elements can associate with nanoparticles through adaptor elements, or through direct association with the nanoparticle surface. Functional elements can be conjugated to affinity tags which form highly specific, noncovalent, physiochemical interactions with complementary affinity tags conjugated to adaptor elements. Thus, functional elements can be coupled to adaptor elements noncovalently through affinity tags.
  • functional elements can be covalently coupled to adaptor elements in the absence of affinity tags.
  • Functional elements can also be covalently or noncovalently associated with the surface of nanoparticles without the use of adaptor elements.
  • an "antigen” is defined herein as a molecule which contains one or more epitopes that will stimulate a host's immune system to make a cellular antigen-specific immune response, and/or a humoral antibody response.
  • Antigens can be peptides, proteins, polysaccharides, saccharides, lipids, nucleic acids, and combinations thereof.
  • the antigen can be derived from a virus, bacterium, parasite, plant, protozoan, fungus, tissue or transformed cell such as a cancer or leukemic cell and can be a whole cell or immunogenic component thereof, e.g., cell wall components.
  • An antigen may be an oligonucleotide or polynucleotide which expresses an antigen.
  • Antigens can be natural or synthetic antigens, for example, haptens, polyepitopes, flanking epitopes, and other recombinant or synthetically derived antigens
  • tumor-specific antigen is defined herein as an antigen that is unique to tumor cells and does not occur in or on other cells in the body.
  • tumor-associated antigen is defined herein as an antigen that is not unique to a tumor cell and is also expressed in or on a normal cell under conditions that fail to induce an immune response to the antigen.
  • an “adjuvant” is defined herein as a substance increasing the immune response to other antigens when administered with other antigens.
  • Adjuvants are also referred to herein as “immune potentiators” and “immune modulators”.
  • Antigen-presenting cells are defined herein as highly specialized cells that can process antigens and display their peptide fragments on the cell surface together with molecules required for lymphocyte activation.
  • the major antigen-presenting cells for T cells are dendritic cells, macrophages and B cells.
  • the major antigen-presenting cells for B cells are follicular dendritic cells.
  • Cross-presentation is defined herein as the ability of antigen- presenting cells to take up, process and present extracellular antigens with MHC class I molecules to CD8 T cells (cytotoxic T cells). This process induces cellular immunity against most tumors and against viruses that do not infect antigen-presenting cells. Cross-presentation is also required for induction of cytotoxic immunity by vaccination with protein antigens, for example in tumor vaccination.
  • Endosome-disrupting agent is defined herein as any agent which causes disruption of endosomal membranes during endocytosis. Endosome-disrupting agents facilitate the transit of extracellular antigens into the cytoplasm of antigen-presenting cells, where they can be imported into the endoplasmic reticulum and processed for cross-presentation on MHC class I molecules at the cell surface.
  • Dendritic cell targeting molecules are defined herein as molecules that target and facilitate endocytosis of nanoparticles by dendritic cells. Dendritic cell targeting molecules may be directly coupled to nanoparticles, or may be coupled to nanoparticles through adaptor elements. In a preferred embodiment the dendritic cell targeting molecules are functionally coupled to adaptor elements.
  • Epithelial cell targeting molecules are defined herein as molecules that target the nanoparticles to epithelium and mediate transcytosis to underlying antigen-presenting cells. Epithelial cell targeting molecules may be directly coupled to nanoparticles, or may be coupled to nanoparticles through adaptor elements. In a preferred embodiment the epithelial cell targeting molecules are functionally coupled to adaptor elements.
  • a molecule "specifically binds" to a target refers to a binding reaction which is determinative of the presence of the molecule in the presence of a heterogeneous population of other biologies.
  • a specified molecule binds preferentially to a particular target and does not bind in a significant amount to other biologies present in the sample.
  • Specific binding of an antibody to a target under such conditions requires the antibody be selected for its specificity to the target.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein.
  • antibody or “immunoglobulin” are used to include intact antibodies and binding fragments thereof. Typically, fragments compete with the intact antibody from which they were derived for specific binding to an antigen fragment including separate heavy chains, light chains Fab, Fab' F(ab')2, Fabc, and Fv. Fragments are produced by recombinant DNA techniques, or by enzymatic or chemical separation of intact immunoglobulins.
  • antibody also includes one or more immunoglobulin chains that are chemically conjugated to, or expressed as, fusion proteins with other proteins.
  • antibody also includes bispecific antibody. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites.
  • Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al, J. Immunol. 148, 1547-1553 (1992).
  • epitopes As used herein, the terms “epitope” or “antigenic determinant” refer to a site on an antigen to which B and/or T cells respond.
  • B-cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids, in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance.
  • T-cells recognize continuous epitopes of about nine amino acids for CD8 cells or about 13-15 amino acids for CD4 cells.
  • T cells that recognize the epitope can be identified by in vitro assays that measure antigen- dependent proliferation, as determined by H-thymidine incorporation by primed T cells in response to an epitope (Burke et al., J. Inf. Dis. 170, 1110- 19 (1994)), by antigen-dependent killing (cytotoxic T lymphocyte assay, Tigges et al., J. Immunol. 156, 3901-3910) or by cytokine secretion.
  • the terms "immunologic", “immunological” or “immune” response is the development of a humoral (antibody mediated) and/or a cellular (mediated by antigen-specific T cells or their secretion products) response directed against an antigen.
  • a humoral (antibody mediated) and/or a cellular (mediated by antigen-specific T cells or their secretion products) response directed against an antigen can be an active response induced by administration of immunogen or a passive response induced by administration of antibody or primed T-cells.
  • a cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II MHC molecules to activate antigen- specific CD4 + T helper cells and/or CD8 + cytotoxic T cells.
  • the response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia cells, eosinophils or other components of innate immunity.
  • the presence of a cell-mediated immunological response can be determined by proliferation assays (CD4 + T cells) or CTL (cytotoxic T lymphocyte) assays.
  • proliferation assays CD4 + T cells
  • CTL cytotoxic T lymphocyte
  • a "costimulatory polypeptide” or a “costimulatory molecule” is a polypeptide that, upon interaction with a cell-surface molecule on T cells, enhances T cell responses, enhances proliferation of T cells, enhances production and/or secretion of cytokines by T cells, stimulates differentiation and effector functions of T cells or promotes survival of T cells relative to T cells not contacted with a costimulatory peptide.
  • the terms “individual”, “host”, “subject”, and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, murines, simians, humans, mammalian farm animals, mammalian sport animals, and mammalian pets.
  • Modular nanodevice vaccine systems are constructed from nanoparticles. The modular design of these nanoparticle vaccine
  • compositions which involves flexible addition and subtraction of antigen, adjuvant, immune potentiators, molecular recognition, and/or transport mediation elements, as well as intracellular uptake mediators, allows for extremely control over many of the variables that are important for optimizing an effective vaccine delivery system.
  • nanoparticles generally refers to particles in the range of between 500 nm to less than 0.5 nm, preferably having a diameter that is between 50 and 500 nm.
  • the polymer that forms the core of the modular vaccine nanoparticle may be any biodegradable or non-biodegradable synthetic or natural polymer.
  • the polymer is a biodegradable polymer.
  • biodegradable polymers include synthetic polymers that degrade by hydrolysis such as poly(hydroxy acids), such as polymers and copolymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butic acid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates), and poly(lactide-co- caprolactone).
  • poly(hydroxy acids) such as polymers and copolymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butic acid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates), and poly(lactide-co- caprolactone).
  • Preferred natural polymers include alginate and other
  • polysaccharides collagen, albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.
  • non-biodegradable polymers can be used, especially hydrophobic polymers.
  • preferred non-biodegradable polymers include ethylene vinyl acetate, poly(meth) acrylic acid, copolymers of maleic anhydride with other unsaturated polymerizable monomers, poly(butadiene maleic anhydride), polyamides, copolymers and mixtures thereof, and dextran, cellulose and derivatives thereof.
  • Other suitable biodegradable and non-biodegradable polymers include, but are not limited to, polyanhydrides, polyamides, polycarbonates, polyalkylenes, polyalkylene oxides such as polyethylene glycol,
  • polyalkylene terepthalates such as poly(ethylene terephthalate), polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyethylene, polypropylene, poly(vinyl acetate), poly vinyl chloride, polystyrene, polyvinyl halides, polyvinylpyrrolidone, polymers of acrylic and methacrylic esters, polysiloxanes, polyurethanes and copolymers thereof, modified celluloses, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate, cellulose sulfate sodium salt, and polyacrylates such as
  • the nanoparticle is formed of polymers fabricated from polylactides (PLA) and copolymers of lactide and glycolide (PLGA). These have established commercial use in humans and have a long safety record (Jiang, et al., Adv. Drug Deliv. Rev. , 57(3):391-410); Aguado and Lambert, Immunobiology, 184(2-3): 113-25 (1992); Bramwell, et al., A/v. Drug Deliv. Rev., 57(9): 1247-65 (2005)).
  • PLA polylactides
  • PLGA lactide and glycolide
  • the polymer may be a bioadhesive polymer that is hydrophilic or hydrophobic.
  • Hydrophilic polymers include CARBOPOLTM (a high molecular weight, crosslinked, acrylic acid-based polymers manufactured by NOVEONTM), polycarbophil, cellulose esters, and dextran.
  • Rate controlling polymers may be included in the polymer matrix or in the coating on the formulation.
  • rate controlling polymers that may be used are hydroxypropylmethylcellulose (HPMC) with viscosities of either 5, 50, 100 or 4000 cps or blends of the different viscosities, ethylcellulose, methylmethacrylates, such as EUDRAGIT® RSI 00,
  • EUDRAGIT® RL100 EUDRAGIT® NE 30D (supplied by Rohm
  • Gastrosoluble polymers such as EUDRAGIT® El 00 or enteric polymers such as EUDRAGIT® L100-55D, LI 00 and SI 00 may be blended with rate controlling polymers to achieve pH dependent release kinetics.
  • Other hydrophilic polymers such as alginate, polyethylene oxide,
  • carboxymethylcellulose, and hydroxyethylcellulose may be used as rate controlling polymers.
  • Antigens can be peptides, proteins, polysaccharides, saccharides, lipids, glycolipids, nucleic acids, or combinations thereof.
  • the antigen can be derived from any source, including, but not limited to, a virus, bacterium, parasite, plant, protozoan, fungus, tissue or transformed cell such as a cancer or leukemic cell and can be a whole cell or immunogenic component thereof, e.g., cell wall components or molecular components thereof.
  • Suitable antigens are known in the art and are available from commercial government and scientific sources.
  • the antigens are whole inactivated or attenuated organisms. These organisms may be infectious organisms, such as viruses, parasites and bacteria. These organisms may also be tumor cells.
  • the antigens may be purified or partially purified polypeptides derived from tumors or viral or bacterial sources.
  • Criteria for identifying and selecting effective antigenic peptides can be found in the art. For example, Pontopoulos, et al. (Curr. Opin. Mol. Ther., 2:29-36 (2000)), discusses the strategy for identifying minimal antigenic peptide sequences based on an understanding of the three- dimensional structure of an antigen-presenting molecule and its interaction with both an antigenic peptide and T-cell receptor. Shastri, (Curr. Opin. Immunol, 8:271-7 (1996)), disclose how to distinguish rare peptides that serve to activate T cells from the thousands peptides normally bound to MHC molecules.
  • the antigens can be recombinant polypeptides produced by expressing DNA encoding the polypeptide antigen in a heterologous expression system.
  • the antigens can be DNA encoding all or part of an antigenic protein.
  • the DNA may be in the form of vector DNA such as plasmid DNA.
  • Antigens may be provided as single antigens or may be provided in combination. Antigens may also be provided as complex mixtures of polypeptides or nucleic acids.
  • a viral antigen can be isolated from any virus including, but not limited to, a virus from any of the following viral families: Arenaviridae, Arterivirus, Astroviridae, Baculoviridae, Badnavirus, Barnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Capillovirus, Carlavirus, Caulimovirus, Circoviridae, Closterovirus, Comoviridae, Coronaviridae (e.g., Coronavirus, such as severe acute respiratory syndrome (SARS) virus), Corticoviridae, Cystoviridae, Deltavirus, Dianthovirus, Enamovirus, Filoviridae (e.g., Marburg virus and Ebola virus (e.g., Zaire, Reston, Ivory Coast, or Sudan strain)), Flaviviridae, (e.g., Hepatitis C virus, Dengue virus 1, Dengue virus 2, Dengue virus 3, Dengue virus
  • Iridoviridae Iridoviridae, Leviviridae, Lipothrixviridae, Microviridae, Orthomyxoviridae (e.g., Influenzavirus A and B and C), Papovaviridae, Paramyxoviridae (e.g., measles, mumps, and human respiratory syncytial virus), Parvoviridae, Picornaviridae (e.g., poliovirus, rhinovirus, hepatovirus, and aphthovirus), Poxviridae (e.g.
  • Retroviridae e.g., lentivirus, such as human immunodeficiency virus (HIV) 1 and HIV 2
  • Rhabdoviridae for example, rabies virus, measles virus, respiratory syncytial virus, etc.
  • Togaviridae for example, rubella virus, dengue virus, etc.
  • Totiviridae Suitable viral antigens also include all or part of Dengue protein M, Dengue protein E, Dengue D1NS1, Dengue D1NS2, and Dengue D1NS3.
  • Viral antigens may be derived from a particular strain such as a papilloma virus, a herpes virus, i.e. herpes simplex 1 and 2; a hepatitis virus, for example, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), the delta hepatitis D virus (HDV), hepatitis E virus (HEV) and hepatitis G virus (HGV), the tick-borne encephalitis viruses; parainfluenza, varicella-zoster, cytomeglavirus, Epstein-Barr, rotavirus, rhinovirus, adenovirus, coxsackieviruses, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, and lymphocytic choriomeningitis.
  • a hepatitis virus for example, hepatitis A virus (HAV), hepatit
  • Bacterial antigens can originate from any bacteria including, but not limited to, Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio,
  • Bordetella Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Hemophilus influenza type B (HIB), Hyphomicrobium, Legionella,
  • Leptspirosis Listeria, Meningococcus A, B and C, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria,
  • Phodospirillum Rickettsia, Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, and Treponema, Vibrio, and Yersinia.
  • Parasite antigens can be obtained from parasites such as, but not limited to, an antigen derived from Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis and Schistosoma mansoni.
  • parasites such as, but not limited to, an antigen derived from Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickett
  • Sporozoan antigens include Sporozoan antigens, Plasmodian antigens, such as all or part of a Circumsporozoite protein, a Sporozoite surface protein, a liver stage antigen, an apical membrane associated protein, or a Merozoite surface protein.
  • the antigen can be an allergen or environmental antigen, such as, but not limited to, an antigen derived from naturally occurring allergens such as pollen allergens (tree-, herb, weed-, and grass pollen allergens), insect allergens (inhalant, saliva and venom allergens), animal hair and dandruff allergens, and food allergens.
  • pollen allergens tree-, herb, weed-, and grass pollen allergens
  • insect allergens inhalant, saliva and venom allergens
  • animal hair and dandruff allergens and food allergens.
  • Important pollen allergens from trees, grasses and herbs originate from the taxonomic orders of Fagales, Oleales, Pinoles and platanaceae including, i.e., birch (Bet ld), alder (Alnus), hazel
  • Lepidoglyphys, Glycyphagus and Tyrophagus those from cockroaches, midges and fleas e.g., Blatella, Periplaneta, Chironomus and
  • Ctenocepphalides those from mammals such as cat, dog and horse, birds, venom allergens including such originating from stinging or biting insects such as those from the taxonomic order of Hymenopter a including bees (superfamily Apidae), wasps (superfamily Vespidea), and ants (superfamily Formicoidae).
  • venom allergens including such originating from stinging or biting insects such as those from the taxonomic order of Hymenopter a including bees (superfamily Apidae), wasps (superfamily Vespidea), and ants (superfamily Formicoidae).
  • Still other allergen antigens that may be used include inhalation allergens from fungi such as from the genera Alternaria and Cladosporium.
  • the antigen can be a tumor antigen, including a tumor-associated or tumor-specific antigen, such as, but not limited to SOX2, alpha-actinin-4, Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR- fucosyltransferaseAS fusion protein, HLA-A2, HLA-A11, hsp70-2,
  • a tumor-associated or tumor-specific antigen such as, but not limited to SOX2, alpha-actinin-4, Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR- fucosyltransferaseAS
  • Adaptor elements associate with the nanoparticle and provide substrates that facilitate the modular assembly and disassembly of functional elements to the nanoparticle.
  • Adaptor elements may associate with nanoparticles through a variety of interactions including, but not limited to, hydrophobic interactions, electrostatic interactions and covalent coupling.
  • the adaptor elements associate with the polymeric nanoparticles noncovalently through hydrophobic interactions.
  • Examples of adaptor elements which may associate with nanoparticles via hydrophobic interactions include, but are not limited to, fatty acids, hydrophobic or amphipathic peptides or proteins, and polymers. These classes of adaptor elements may also be used in any combination or ratio.
  • nanoparticles facilitates a prolonged presentation of functional elements which can last for several weeks.
  • Adaptor elements can also be attached to polymeric nanoparticles through covalent interactions through various functional groups.
  • Functionality refers to conjugation of a molecule to the surface of the particle via a functional chemical group (carboxylic acids, aldehydes, amines, sulfhydryls and hydroxyls) present on the surface of the particle and present on the molecule to be attached.
  • a functional chemical group carboxylic acids, aldehydes, amines, sulfhydryls and hydroxyls
  • Functionality may be introduced into the particles in two ways.
  • the first is during the preparation of the nanoparticles, for example during the emulsion preparation of nanoparticles by incorporation of stabilizers with functional chemical groups.
  • Suitable stabilizers include hydrophobic or amphipathic molecules that associate with the outer surface of the nanoparticles.
  • a second is post-particle preparation, by direct crosslinking particles and ligands with homo- or heterobifunctional crosslinkers.
  • This second procedure may use a suitable chemistry and a class of crosslinkers (CDI, ED AC, glutaraldehydes, etc. as discussed in more detail below) or any other crosslinker that couples ligands to the particle surface via chemical modification of the particle surface after preparation.
  • This second class also includes a process whereby amphiphilic molecules such as fatty acids, lipids or functional stabilizers may be passively adsorbed and adhered to the particle surface, thereby introducing functional end groups for tethering to ligands.
  • One useful protocol involves the "activation" of hydroxyl groups on polymer chains with the agent, carbonyldiimidazole (CDI) in aprotic solvents such as DMSO, acetone, or THF.
  • CDI forms an imidazolyl carbamate complex with the hydroxyl group which may be displaced by binding the free amino group of a molecule such as a protein.
  • the reaction is an N- nucleophilic substitution and results in a stable N-alkylcarbamate linkage of the molecule to the polymer.
  • the "coupling" of the molecule to the "activated" polymer matrix is maximal in the pH range of 9-10 and normally requires at least 24 hrs.
  • the resulting molecule-polymer complex is stable and resists hydrolysis for extended periods of time.
  • Another coupling method involves the use of l-ethyl-3-(3- dimethylaminopropyl) carbodiimide (ED AC) or "water-soluble CDI" in conjunction with N-hydroxylsulfosuccinimide (sulfo NHS) to couple the exposed carboxylic groups of polymers to the free amino groups of molecules in a totally aqueous environment at the physiological pH of 7.0.
  • ED AC and sulfo-NHS form an activated ester with the carboxylic acid groups of the polymer which react with the amine end of a molecule to form a peptide bond.
  • the resulting peptide bond is resistant to hydrolysis.
  • the use of sulfo-NHS in the reaction increases the efficiency of the ED AC coupling by a factor of ten-fold and provides for exceptionally gentle conditions that ensure the viability of the molecule-polymer complex.
  • a useful coupling procedure for attaching molecules with free hydroxyl and carboxyl groups to polymers involves the use of the cross- linking agent, divinylsulfone. This method would be useful for attaching sugars or other hydroxylic compounds with bioadhesive properties to hydroxylic matrices.
  • the activation involves the reaction of divinylsulfone to the hydroxyl groups of the polymer, forming the vinylsulfonyl ethyl ether of the polymer.
  • the vinyl groups will couple to alcohols, phenols and even amines.
  • Activation and coupling take place at pH 11.
  • the linkage is stable in the pH range from 1-8 and is suitable for transit through the intestine.
  • Any suitable coupling method known to those skilled in the art for the coupling of molecules and polymers with double bonds including the use of UV crosslinking, may be used for attachment of molecules to the polymer.
  • adaptor elements can be conjugated to affinity tags.
  • Affinity tags are any molecular species which form highly specific, noncovalent, physiochemical interactions with defined binding partners. Affinity tags which form highly specific, noncovalent, physiochemical interactions with one another are defined herein as "complementary”.
  • Suitable affinity tag pairs are well known in the art and include
  • epitope/antibody biotin/avidin, biotin/streptavidin, biotin/neutravidin, glutathione-S-transferase/glutathione, maltose binding protein/amylase and maltose binding protein/maltose.
  • suitable epitopes which may be used for epitope/antibody binding pairs include, but are not limited to, HA, FLAG, c-Myc, glutatione-S-transferase, His 6 , GFP, DIG, biotin and avidin.
  • Antibodies both monoclonal and polyclonal and antigen-binding fragments thereof which bind to these epitopes are well known in the art.
  • Affinity tags that are conjugated to adaptor elements allow for highly flexible, modular assembly and disassembly of functional elements which are conjugated to affinity tags which form highly specific, noncovalent, physiochemical interactions with complementary affinity tags which are conjugated to adaptor elements.
  • Adaptor elements may be conjugated with a single species of affinity tag or with any combination of affinity tag species in any ratio. The ability to vary the number of species of affinity tags and their ratios conjugated to adaptor elements allows foraki control over the number of functional elements which may be attached to the
  • adaptor elements are coupled directly to functional elements in the absence of affinity tags, such as through direct covalent interactions.
  • Adaptor elements can be covalently coupled to at least one species of functional element.
  • Adaptor elements can be covalently coupled to a single species of functional element or with any combination of species of functional elements in any ratio.
  • adaptor elements are conjugated to at least one affinity tag that provides for assembly and disassembly of modular functional elements which are conjugated to complementary affinity tags.
  • adaptor elements are fatty acids that are conjugated with at least one affinity tag.
  • the adaptor elements are fatty acids conjugated with avidin or streptavidin. Such avidin/streptavidin-conjugated fatty acids allow for the attachment of a wide variety of biotin-conjugated functional elements.
  • the adaptor elements are provided on, or in the surface of, nanoparticles at a high density. This high density of adaptor elements allows for coupling of the nanoparticle to a variety of species of functional elements while still allowing for the functional elements to be present in high enough numbers to be efficacious.
  • the adaptor elements may include fatty acids.
  • Fatty acids may be of any acyl chain length and may be saturated or unsaturated. In a particularly preferred embodiment the fatty acid is palmitic acid.
  • Other suitable fatty acids include, but are not limited to, saturated fatty acids such as butyric, caproic, caprylic, capric, lauric, myristic, stearic, arachidic and behenic acid.
  • Still other suitable fatty acids include, but are not limited to, unsaturated fatty acids such as oleic, linoleic, alpha-linolenic, arachidonic, eicosapentaenoic, docosahexaenoic and erucic acid.
  • the adaptor elements may include hydrophobic or amphipathic peptides. Preferred peptides should be sufficiently hydrophobic to preferentially associate with the polymeric nanoparticle over the aqueous environment. Amphipathic polypeptides useful as adaptor elements may be mostly hydrophobic on one end and mostly hydrophilic on the other end. Such amphipathic peptides may associate with polymeric nanoparticles through the hydrophobic end of the peptide and be conjugated on the hydrophilic end to a functional group.
  • Adaptor elements may include hydrophobic polymers.
  • hydrophobic polymers include, but are not limited to, polyanhydrides, poly(ortho)esters, and polyesters such as polycaprolactone.
  • D. Functional elements include, but are not limited to, polyanhydrides, poly(ortho)esters, and polyesters such as polycaprolactone.
  • Functional elements which associate with the nanoparticles provide a number of different functions to the composition. These functions include protection of the nanoparticle vaccine from hostile environments during transit in the gastrointestinal tract, transport through epithelial barriers, targeting antigen presenting cells (APCs) with high avidity, and transport of mediators that facilitate uptake and presentation of antigen by antigen- presenting cells through disruption of intracellular antigen-sequestering compartments.
  • Functional elements may include dendritic cell recognition elements, epithelial cell recognition elements, pH-sensitive molecules which protect the composition from hydrolysis and degradation in low-pH environments, non-pH-sensitive molecules which protect the composition from hydrolysis and degradation in low-pH environments, and/or endosome- disrupting agents.
  • Nanoparticles may be associated with a single species of functional element or may be associated with any combination of disclosed functional elements in any ratio.
  • functional elements are directly associated with nanoparticles in the absence of adaptor elements.
  • Functional elements may be directly associated with nanoparticles through covalent or noncovalent interactions, including, but not limited to, hydrophobic interactions and electrostatic interactions. Covalent attachment of functional elements can be achieved by introducing functionality to the polymeric nanoparticles using methods described above with respect to adaptor elements.
  • functional elements are associated with nanoparticles through adaptor elements which directly associate with the nanoparticles.
  • Functional elements may be directly, covalently coupled to adaptor elements or may couple to adaptor elements through complementary affinity tags conjugated to the adaptor and functional elements.
  • Multiple different species of functional elements may be associated with nanoparticles in any desired ratio, for instance, by conjugating each species of functional element to a separate species of affinity tag. These functional elements may then associate with nanoparticles coated with adaptor elements conjugated to an appropriate ratio of complementary affinity tags.
  • Multiple species of functional elements may also be associated with nanoparticles by covalently coupling each species of functional element at a desired ratio to adaptor elements.
  • functional elements are conjugated to biotin.
  • Biotin conjugation allows the functional elements to interact with adaptor elements conjugated with avidin, neutravidin or streptavidin.
  • Targeting molecules for professional antigen presenting cells i. Targeting molecules for professional antigen presenting cells
  • DC antigen-presenting cells
  • One biological feature of DCs is their ability to sense conditions under which antigen is encountered, initiating a process of "DC maturation".
  • DCs respond to antigen exposure in different ways depending on the nature of the pathogen (virus, bacteria, protozoan) encountered. This information is transmitted to T cells by altered patterns of cytokine release at the time of antigen presentation in lymph nodes, altering the type of T cell response elicited.
  • targeting DCs provides the opportunity not only to quantitatively enhance the delivery of antigen and antigen responses in general, but to qualitatively control the nature of the immune response depending on the desired vaccination outcome.
  • Dendritic cells express a number of cell surface receptors that can mediate the endocytosis of bound antigen. Targeting exogenous antigens to internalizing surface molecules on systemically-distributed antigen presenting cells facilitates uptake of antigens and thus overcomes a major rate-limiting step in immunization and thus in vaccination.
  • Dendritic cell targeting molecules include monoclonal or polyclonal antibodies or fragments thereof that recognize and bind to epitopes displayed on the surface of dendritic cells. Dendritic cell targeting molecules also include ligands which bind to a cell surface receptor on dendritic cells.
  • One such receptor, the lectin DEC-205 has been used in vitro and in mice to boost both humoral (antibody-based) and cellular (CD8 T cell) responses by 2-4 orders of magnitude (Hawiger, et al., J. Exp. Med., 194(6):769-79 (2001); Bonifaz, et al, J. Exp.
  • endocytic receptors including a mannose-specific lectin (mannose receptor) and IgG Fc receptors, have also been targeted in this way with similar enhancement of antigen presentation efficiency.
  • suitable molecules which may be targeted include, but are not limited to, DC-SIGN, BDCA3 (CD141), 33D1, SIGLEC-H, DCIR, CD1 lc, heat shock protein receptors and scavenger receptors.
  • TLRs toll-like receptors
  • PAMPs pathogen-associated molecular patterns
  • PAMPs target the TLR on the surface of the dendritic cell and signals internally, thereby potentially increasing DC antigen uptake, maturation and T-cell stimulatory capacity.
  • PAMPs conjugated to the particle surface or co-encapsulated include unmethylated CpG DNA
  • bacterial bacterial
  • double-stranded RNA viral
  • lipopolysacharride bacterial
  • peptidoglycan bacterial
  • lipoarabinomannin bacterial
  • zymosan zymosan
  • mycoplasmal lipoproteins such as MALP-2 (bacterial), flagellin (bacterial) poly(inosinic-cytidylic) acid (bacterial), lipoteichoic acid (bacterial) or imidazoquinolines (synthetic).
  • nanoparticle vaccine systems are determined in part by their route of administration into the body. While injection (intradermal, intramuscular, intravenous) is an acceptable solution in many cases, having a vaccine product that is orally available will greatly extend its ease of use and applicability on a global scale.
  • injection intradermal, intramuscular, intravenous
  • epithelial cells constitute the principal barrier that separates an organism's interior from the outside world. Epithelial cells such as those that line the gastrointestinal tract form continuous monolayers that
  • mucosal- associated lymphoid tissue sample the environment for the presence of pathogens.
  • This sampling is carried out by an apical to basolateral transcytotic event and is mediated by M cells located in lymphoid follicle- associated epithelium (FAE) throughout the GI tract.
  • FAE lymphoid follicle- associated epithelium
  • absorptive enterocytes may transport microorganisms or other nanoparticulates to intraepithelial lymphocytes.
  • DCs may perform this function directly, with a population of DCs being intercalated between epithelial cells and extending processes into the gut lumen to sample the microorganisms present.
  • modular nanoparticle vaccines further include epithelial cell recognition elements.
  • Epithelial cell targeting molecules include monoclonal or polyclonal antibodies or bioactive fragments thereof that recognize and bind to epitopes displayed on the surface of epithelial cells.
  • Epithelial cell targeting molecules also include ligands which bind to a cell surface receptor on epithelial cells.
  • Ligands include, but are not limited to, molecules such as polypeptides, nucleotides and polysaccharides.
  • a variety of receptors on epithelial cells may be targeted by epithelial cell targeting molecules.
  • suitable receptors to be targeted include, but are not limited to, IgE Fc receptors, EpCAM, selected carbohydrate specificities, dipeptidyl peptidase, and E-cadherin.
  • Vaccine particles administered orally will encounter a corrosive environment in the gastrointestinal (GI) tract with areas of low and high pH, as well as resident degradative enzymes and solubilizing agents.
  • Biodegradable particulates have gained attention as oral vaccines because of their ability to protect antigens on route to immune sites across the intestinal epithelium (O'Hagan and Valiante, Nat. Rev. Drug Discov., 2(9):727-35 (2003); van der Lubben, et al, Adv. DrugDeliv. Rev., 52(2): 139-44 (2001); Wikingsson and Sjoholm, Vaccine, 20(27-28):3355-63 (2002); Moser, et al., Expert Rev.
  • modular nanoparticle vaccines further include pH-sensitive molecules which protect the composition from hydrolysis and degradation in low pH environments. Such pH-sensitive protecting molecules are preferred because subsequent to the particles transit through a low pH environment, upon reaching its destination in the higher pH intestinal site, particles should expose epithelial targeting molecules to allow for specific interactions with target epithelial cells, followed by transcytosis through the epithelium and subsequent interactions with subepithelial dendritic cells.
  • Preferred non-pH-sensitive molecules which protect the composition from hydrolysis and degradation in low pH environments are poly(ethylene) glycol, gelatin and albumins.
  • Other pH-sensitive molecules which may be used include poly(acrylic acid), poly(methyl methacrylic acid) and poly(N-alkyl acrylamides), or other enteric coatings discussed above.
  • pH-sensitive or pH-insensitive protective molecules may be directly coupled to nanoparticles, or may be coupled to nanoparticles through adaptor elements, such as those described above.
  • the epithelial cell targeting molecules are functionally coupled to adaptor elements.
  • nanoparticle vaccines to dendritic cells have the property of delivering antigens to late endosomal elements that serve as efficient sites for the formation of immunogenic peptides and their loading onto MHC class II molecules (which are needed for CD4 T cell and antibody responses) (Mellman, Adv. Exp. Med. Biol, 560:63-7; Mellman and Steinman, Cell, 106(3):255-8 (2001)).
  • Effective vaccination however, often requires the production of CD8 cytotoxic T cell responses which occurs only when antigen is present in the cytoplasm.
  • DCs are adept at this function by the process of "cross-presentation", whereby exogenous antigens escape endocytic vesicles and enter the cytoplasm where they are cleaved into peptides by the proteasome, imported into the endoplasmic reticulum, and loaded onto newly synthesized MHC class I molecules (which are required for stimulation of CD 8 T cells).
  • the modular nanoparticulate vaccine compositions include an agent which causes the disruption of endosomal membranes.
  • Endosomal membrane disrupting agents include, but are not limited to, small molecule drugs, peptides, polypeptides, including elastin, and synthetic agents that disrupt intracellular pH or vesicular membranes.
  • Osmotic delivery is an endocytosis-mediated system for delivering large polar molecules into the cytosol of cells.
  • the mechanism behind this process is also referred to as the "proton-sponge effect".
  • the process renders the endosome fragile, releasing the contents of the endosome into the cytosol.
  • Osmotic delivery and endosomal disruption work as follows:
  • the endosome-disrapting agent is a low pH-activated, amphipathic, pore-forming peptide.
  • the low pH-activated, amphipathic, pore-forming peptide is the commercially-available Endoporter (Endoporter; GeneTools, Philomath, OR) (Summerton, Ann. N. Y. Acad. Scl, 1058:1-14 (2005)).
  • Agents other than Endoporter can mediate endosome disruption.
  • Other agents include charged macromolecules such as poly(amido amine)
  • the endosome-disrupting agent may be encapsulated into the polymeric core of the nanoparticle. Additionally, or alternatively, the endosome-disrapting agent may be attached to the surface of the nanoparticle by association with attached adaptor elements.
  • the modular nanoparticulate vaccines can include adjuvants. These can be incorporated into, administered with, or administered separately from, the vaccine nanoparticles.
  • Adjuvants may be provided encapsulated or otherwise entrapped in the polymeric core of the nanoparticle vaccine, or may be associated with the surface of the nanoparticle either through direct association with the polymeric core, or through association with adaptor elements.
  • Adjuvant may be in the form of separate nanoparticles or in a suspension or solution administered with the vaccine nanoparticles.
  • the adjuvant is the synthetic glycolipid alpha- galactosylceramide (aGalCer).
  • AGalCer synthetic glycolipid alpha- galactosylceramide
  • Dendritic cells presenting antigens in the context of CD Id can lead to rapid innate and prolonged production of cytokines such as interferon and IL-4 by natural killer T cells (NKT cells).
  • CD Id is a major histocompatibility complex class I-like molecule that presents glycolipid antigens to a subset of NKT cells.
  • aGalCer is not toxic to humans and has been shown to act as an adjuvant, priming both antigen-specific CD4+ and CD8+ T cell responses.
  • aGalCer in conjunction with a malaria vaccine can lead to cytotoxic responses against infected cells, which is an ideal scenario for vaccines against infectious diseases.
  • other glycolipids that function as adjuvants to activate NKT cell- mediated immune responses can be used.
  • the adjuvant can be, but is not limited to, one or more of the following: oil emulsions (e.g., Freund's adjuvant); saponin formulations; virosomes and viral-like particles; bacterial and microbial derivatives including, but not limited to, carbohydrates such as
  • lipopolysachharide LPS
  • immunostimulatory oligonucleotides ADP- ribosylating toxins and detoxified derivatives
  • alum BCG
  • mineral- containing compositions e.g., mineral salts, such as aluminium salts and calcium salts, hydroxides, phosphates, sulfates, etc.
  • bioadhesives and/or mucoadhesives e.g., liposomes; polyoxyethylene ether and polyoxyethylene ester formulations; polyphosphazene; muramyl peptides; imidazoquinolone compounds; and surface active substances (e.g.
  • lysolecithin pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • Adjuvants may also include immunomodulators such as cytokines, interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., interferon-. gamma.), macrophage colony stimulating factor, and tumor necrosis factor; and co-stimulatory molecules, such as those of the B7 family.
  • immunomodulators such as cytokines, interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., interferon-. gamma.), macrophage colony stimulating factor, and tumor necrosis factor; and co-stimulatory molecules, such as those of the B7 family.
  • Such proteinaceous adjuvants may be provided as the full-length polypeptide or an active fragment thereof, or in the form of DNA, such as
  • modular nanoparticulate vaccine may further include agents useful for determining the location of administered particles.
  • Agents useful for this purpose include fluorescent tags, radionuclides and contrast agents.
  • Suitable imaging agents include, but are not limited to, fluorescent molecules such as those described by Molecular Probes (Handbook of fluorescent probes and research products), such as Rhodamine, fluorescein, Texas red, Acridine Orange, Alexa Fluor (various), Allophycocyanin, 7- aminoactinomycin D, BOBO-1, BODIPY (various), Calcien, Calcium Crimson, Calcium green, Calcium Orange, 6-carboxyrhodamine 6G, Cascade blue, Cascade yellow, DAPI, DiA, DiD, Dil, DiO, DiR, ELF 97, Eosin, ER Tracker Blue- White, EthD-1, Ethidium bromide, Fluo-3, Fluo4, FM1-43, FM4-64, Fura-2, Fura Red, Hoechst 33258, Hoechst 33342, 7- hydroxy-4-methylcoumarin, Indo-1, JC-1, JC-9, JOE dye, Lissamine rhodamine B, Lucifer Yellow
  • Rhodamine 110 Propidium iodide
  • Rhodamine 110 Rhodamine Red
  • R-Phycoerythrin Resorfm
  • Rhod-2 Rhodamine Green
  • Rhodamine 123 ROX dye
  • Sodium Green SYTO blue (various)
  • SYTO green Various
  • SYTO orange SYTO orange
  • radionuclides can be used as imaging agents. Suitable radionuclides include, but are not limited to radioactive species of Fe(III), Fe(II), Cu(II), Mg(II), Ca(II), and Zn(Il) Indium, Gallium and Technetium.
  • Other suitable contrast agents include metal ions generally used for chelation in paramagnetic Tl-type MIR contrast agents, and include di- and tri-valent cations such as copper, chromium, iron, gadolinium, manganese, erbium, europium, dysprosium and holmium.
  • Metal ions that can be chelated and used for radionuclide imaging include, but are not limited to metals such as gallium, germanium, cobalt, calcium, indium, iridium, rubidium, yttrium, ruthenium, yttrium, technetium, rhenium, platinum, thallium and samarium. Additionally metal ions known to be useful in neutron-capture radiation therapy include boron and other metals with large nuclear cross-sections. Also suitable are metal ions useful in ultrasound contrast, and X-ray contrast compositions.
  • contrast agents examples include gases or gas emitting compounds, which are radioopaque.
  • compositions may be administered in combination with a physiologically or pharmaceutically acceptable carrier, excipient, or stabilizer.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.
  • pharmaceutically-acceptable carrier means one or more compatible solid or liquid fillers, dilutants or encapsulating substances which are suitable for administration to a human or other vertebrate animal.
  • carrier refers to an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • compositions may be formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • Optional pharmaceutically acceptable excipients include, but are not limited to, diluents, binders, lubricants, disintegrants, colorants, stabilizers, and surfactants.
  • Diluents also referred to as "fillers,” are typically necessary to increase the bulk of a solid dosage form so that a practical size is provided for compression of tablets or formation of beads and granules.
  • Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose,
  • microcrystalline cellulose kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinized starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powdered sugar.
  • Binders are used to impart cohesive qualities to a solid dosage formulation, and thus ensure that a tablet or bead or granule remains intact after the formation of the dosage forms.
  • Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia, tragacanth, sodium alginate, cellulose, including hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, and veegum, and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid/polymethacrylic acid and polyvinylpyrrolidone.
  • Lubricants are used to facilitate tablet manufacture.
  • suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, and mineral oil.
  • Disintegrants are used to facilitate dosage form disintegration or "breakup" after administration, and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginine, gums or cross linked polymers, such as cross- linked PVP (POLYPLASDONE® XL from GAF Chemical Corp).
  • starch sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginine, gums or cross linked polymers, such as cross- linked PVP (POLYPLASDONE® XL from GAF Chemical Corp).
  • Stabilizers are used to inhibit or retard decomposition reactions which include, by way of example, oxidative reactions.
  • Surfactants may be anionic, cationic, amphoteric or nonionic surface active agents.
  • Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions.
  • anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2- ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate.
  • Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine.
  • nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG- 1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401 , stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide.
  • amphoteric surfactants include sodium N-dodecyl-b-alanine, sodium N-lauryl-b-iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.
  • the particles may also contain minor amount of nontoxic auxiliary substances such as wetting or emulsifying agents, dyes, pH buffering agents, or preservatives.
  • nontoxic auxiliary substances such as wetting or emulsifying agents, dyes, pH buffering agents, or preservatives.
  • the particles may be complexed with other agents.
  • compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., acacia, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone (Povidone), hydroxypropyl methylcellulose, sucrose, starch, and ethylcellulose); fillers (e.g., corn starch, gelatin, lactose, acacia, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, calcium carbonate, sodium chloride, or alginic acid); lubricants (e.g.
  • binding agents e.g., acacia, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone (Povidone), hydroxypropyl methylcellulose, sucrose, starch, and ethylcellulose
  • fillers e.g., corn starch, gelatin, lactose, acacia, suc
  • disintegrators e.g. micro-crystalline cellulose, corn starch, sodium starch glycolate and alginic acid.
  • water-soluble, such formulated complex then may be formulated in an appropriate buffer, for example, phosphate buffered saline or other physiologically compatible solutions.
  • a non-ionic surfactant such as TWEENTM, or polyethylene glycol.
  • the compounds and their physiologically acceptable solvates may be formulated for administration.
  • Liquid formulations for oral administration prepared in water or other aqueous vehicles may contain various suspending agents such as
  • liquid formulations may also include solutions, emulsions, syrups and elixirs containing, together with the active compound(s), wetting agents, sweeteners, and coloring and flavoring agents.
  • Various liquid and powder formulations can be prepared by conventional methods for inhalation by the patient.
  • the particles may be further coated.
  • Suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name EUDRAGIT® (Rohm Pharma, Darmstadt, Germany), zein, shellac, and polysaccharides. Additionally, the coating material may contain conventional carriers such as plasticizers, pigments, colorants, glidants, stabilization agents, pore formers and surfactants.
  • A. Methods of making antigen-encapsulated nanoparticles Many different processes can be used to form the nanoparticles. If the process does not produce particles having a homogenous size range, then the particles can be separated using standard techniques such as sieving to produce a population of particles having the desired size range. i. Solvent Evaporation
  • a substance to be incorporated optionally is added to the solution, and the mixture is suspended in an aqueous solution that contains a surface active agent such as poly(vinyl alcohol).
  • a surface active agent such as poly(vinyl alcohol).
  • Substances which can be incorporated in the nanoparticles include, but are not limited to, antigens, adjuvants, imaging agents, endosome-disrupting agents and contrast agents. The resulting emulsion is stirred until most of the organic solvent evaporated, leaving solid nano- and microparticles.
  • antigen- loaded, spherical PLGA nanoparticles with a mean diameter of 100-200 nm and protein loadings of up to 40% are produced by a modified version of this technique.
  • 100 mg of PLGA is dissolved in 2 ml of methylene chloride in a short glass test tube (5.8 cm long, diameter 1.2 cm) overnight.
  • approximately 100-200 ul of the concentrated antigen solution is added and vortexed rapidly. This solution is added drop wise to 4 ml of an aqueous solution of 5% poly (vinyl alcohol) while vortexing.
  • the emulsion formed is further sonicated three times for intervals of 10 seconds each at 38% amplitude (Tekmar Soni Disrupter model TM300, 40% duty cycle, microtip # 4) to yield a homogeneous milky mixture.
  • the single emulsion is poured into 100 ml of PVA 0.3%.
  • the polymer/ PVA dispersion is stirred on a magnetic stir plate for 3 hours at room temperature to allow for adequate solvent evaporation. Once solidified, the nanospheres are isolated by centrifugation (12000 rpm, 4 °C, 10 minutes). The supernatant is discarded.
  • Nanospheres are washed three times with deionized water (10 ml) to remove excess of PVA before they are frozen at - 80°C and then lyophilized for 48 hours. All parameters of this method are easily scaled to produce different batch sizes of nanoparticles.
  • Microspheres can be formed from polymers such as polyesters and polyanhydrides using hot melt microencapsulation methods as described in Mathiowitz, et al., Reactive Polymers, 6:275 (1987). In this method, the use of polymers with molecular weights between 3-75,000 daltons is preferred.
  • the polymer first is melted and then mixed with the solid particles o f a substance to be incorporated that have been sieved to less than 50 microns. The mixture is suspended in a non-miscible solvent (like silicon oil), and, with continuous stirring, heated to above the melting point of the polymer, for example, 5° C. Once the emulsion is stabilized, it is cooled until the polymer particles solidify. The resulting microspheres are washed by decanting with petroleum ether to give a free-flowing powder.
  • a non-miscible solvent like silicon oil
  • Microspheres with sizes between one to 1000 microns are obtained with this method.
  • Microspheres can be formed from polymers using a phase inversion method wherein a polymer is dissolved in a "good" solvent, fine particles of a substance to be incorporated, such as a drug, are mixed or dissolved in the polymer solution, and the mixture is poured into a strong non-solvent for the polymer, to spontaneously produce, under favorable conditions, polymeric microspheres, wherein the polymer is either coated with the particles or the particles are dispersed in the polymer.
  • the method can be used to produce microparticles in a wide range of sizes, including, for example, about 100 nanometers to about 10 microns.
  • Exemplary polymers which can be used include polyvinylphenol and polylactic acid.
  • Substances which can be incorporated include, for example, imaging agents such as fluorescent dyes, or biologically active molecules such as proteins or nucleic acids.
  • imaging agents such as fluorescent dyes
  • biologically active molecules such as proteins or nucleic acids.
  • the polymer is dissolved in an organic solvent and then contacted with a non-solvent, which causes phase inversion of the dissolved polymer to form small spherical particles, with a narrow size distribution optionally incorporating an antigen or other substance.
  • Adaptor elements may be conjugated to affinity tags prior to, or after their association with polymeric nanoparticles.
  • the adaptor elements are fatty acids and the affinity tag is avidin/streptavidin.
  • palmitic acid is conjugated to avidin.
  • avidin is dissolved at a concentration of 5 mg/ml in 37° C pre warmed 2 ml solution of 2% deoxycholate in IX PBS. To this solution, a 10 fold molar excess of NHS-Palmitic acid is added and the solution is stirred and sonicated in 37° C water bath (Branson, 50kHz freq.).
  • the reaction is maintained at 37° C for 24 hours after which excess palmitic acid is removed by dialysis against a 0.15% deoxycholate-PBS buffer prewarmed to 37° C.
  • the avidin-palmitic acid conjugate is verified by reverse phase HPLC on a Prevail CI 8 column with a linear methanol gradient in IX PBS as the mobile phase and UV detection at 280 nm. This method is easily adapted to conjugate avidin to any fatty acid of choice.
  • Avidin may be coupled to peptides and polymers by similar techniques. The chemistry involved in the coupling reaction will depend on the nature of available functional groups on the fatty acid, peptide or polymer. Methods for conjugating avidin to fatty acids, peptides and polymers are well known in the art. Methods for conjugating other affinity tags such as biotin, epitope tags (HA, FLAG, c-myc) and antibodies to fatty acids, peptides and polymers are well known in the art.
  • adaptor elements such as those described above, including fatty acids, hydrophobic or aliphatic peptides, and polymers, are conjugated onto the surface of nanoparticles at the emulsion stage of nanoparticle preparation.
  • the nanoparticles include PLGA and the adaptor elements include avidin- conjugated palmitic acid.
  • dissolved PLGA solution is added to a 4 ml solution of 2 parts avidin-palmitic acid, 2 parts 5% PVA.
  • a 50:50 mixture of protein-palmitic acid conjugates and 5% PVA has been found to yield optimal surface coverage of avidin groups on nanosized particles.
  • Functional elements can routinely be assembled onto adaptor elements incorporated onto the nanoparticle surface by conjugating the functional elements to affinity tags which are complementary to the affinity tags conjugated to the adaptor elements.
  • affinity tag pairs for use in coupling adaptor elements to functional elements are biotin-avidin and biotin-streptavidin.
  • Affinity tag-conjugated functional elements are incubated with nanoparticles pre-coated with adaptor elements conjugated to complementary affinity tags under any appropriate buffer, salt and detergent conditions. For example, typical incubations may be performed at 4° C for 2-4 hours, 37° C for 20 minutes or room temperature for 1 hour. Incubations may be performed in phosphate buffered saline or other buffer compositions adjusted to a pH between 6.0 and 7.4. Incubation may occur with gentle shaking, rocking or rotation. Nanoparticles may then be washed with excess incubation buffer to remove unbound or non-specifically bound functional elements.
  • Functional elements may also be conjugated directly to adaptor elements in the absence of affinity tags, either prior to, or after their association with polymeric nanoparticles.
  • Methods for conjugating functional elements such as peptides, polypeptides, polymers and antibodies to adaptor elements such as fatty acids, peptides and polymers are well known in the art.
  • fatty acids such as palmitic acid may be conjugated to the C-terminus of peptides, polypeptides and antibodies using a methodology similar to that described above for conjugation of palmitic acid to avidin.
  • the nanoparticle vaccine compositions disclosed herein are useful for activating T cells in subjects for prophylactic and therapeutic applications. Activation of T cells by nanoparticle vaccine compositions increases their proliferation, cytokine production, differentiation, effector functions and/or survival. Methods for measuring these are well known to those in the art.
  • the T cells activated by the nanoparticle vaccine compositions can be any cell which express the T cell receptor, including ⁇ / ⁇ and ⁇ / ⁇ T cell receptors.
  • T-cells include all cells which express CD3, including T-cell subsets which also express CD4 and CD8. Other markers of T cell subsets include
  • T-cells include both naive and memory cells and effector cells such as CTL. T-cells also include regulatory cells such as Thl, Tel, Th2, Tc2, Th3, Treg, and Trl cells. T- cells also include NKT-cells and similar unique classes of the T-cell lineage. In preferred embodiments the T cells that are activated are CD8 + T cells. As demonstrated in the examples below, the APCs disclosed herein
  • CD8 + T cells preferentially activate and expand CD8 + T cells when activated ex vivo.
  • compositions described herein are useful for treating a subject having or being predisposed to any disease or disorder to which the subject's immune system mounts an immune response.
  • the compositions are useful as prophylactic vaccines, which confer resistance in a subject to subsequent exposure to infectious agents.
  • the compositions are also useful as therapeutic vaccines, which can be used to initiate or enhance a subject's immune response to a pre-existing antigen, such as a tumor antigen in a subject with cancer, or a viral antigen in a subject infected with a virus.
  • the compositions are also useful as desensitizing vaccines, which function to "tolerize" an individual to an environmental antigen, such as an allergen.
  • the type of disease to be treated or prevented is a malignant tumor or a chronic infectious disease caused by a bacterium, virus, protozoan, helminth, or other microbial pathogen that enters intracellularly and is attacked, i.e., by the cytotoxic T lymphocytes.
  • the desired outcome of a prophylactic, therapeutic or de-sensitized immune response may vary according to the disease, according to principles well known in the art.
  • an immune response against an infectious agent may completely prevent colonization and replication of an infectious agent, affecting "sterile immunity" and the absence of any disease symptoms.
  • a vaccine against infectious agents may be considered effective if it reduces the number, severity or duration of symptoms; if it reduces the number of individuals in a population with symptoms; or reduces the transmission of an infectious agent.
  • immune responses against cancer, allergens or infectious agents may completely treat a disease, may alleviate symptoms, or may be one facet in an overall therapeutic
  • the stimulation of an immune response against a cancer may be coupled with surgical, chemotherapeutic, radiologic, hormonal and other immunologic approaches in order to affect treatment.
  • Subjects with or exposed to infectious agents can be treated therapeutically or prophylactically with nanoparticle vaccine compositions disclosed herein.
  • Infectious agents include bacteria, viruses and parasites.
  • the subject can be treated prophylactically, such as when there may be a risk of developing disease from an infectious agent.
  • An individual traveling to or living in an area of endemic infectious disease may be considered to be at risk and a candidate for prophylactic vaccination against the particular infectious agent.
  • Preventative treatment can be applied to any number of diseases where there is a known relationship between the particular disease and a particular risk factor, such as geographical location or work environment.
  • malignant tumors exhibit metastasis.
  • small clusters of cancerous cells dislodge from a tumor, invade the blood or lymphatic vessels, and are carried to other tissues, where they continue to proliferate. In this way a primary tumor at one site can give rise to a secondary tumor at another site.
  • the compositions and method described herein may be useful for treating subjects having malignant tumors.
  • Malignant tumors which may be treated are classified herein according to the embryonic origin of the tissue from which the tumor is derived.
  • Carcinomas are tumors arising from endodermal or ectodermal tissues such as skin or the epithelial lining of internal organs and glands.
  • a melanoma is a type of carcinoma of the skin for which this invention is particularly useful.
  • Sarcomas, which arise less frequently, are derived from mesodermal connective tissues such as bone, fat, and cartilage.
  • the leukemias and lymphomas are malignant tumors of hematopoietic cells of the bone marrow. Leukemias proliferate as single cells, whereas lymphomas tend to grow as tumor masses. Malignant tumors may show up at numerous organs or tissues of the body to establish a cancer.
  • the types of cancer that can be treated in with the provided compositions and methods include, but are not limited to, the following: bladder, brain, breast, cervical, colo-rectal, esophageal, kidney, liver, lung, nasopharangeal, pancreatic, prostate, skin, stomach, uterine, and the like.
  • Administration is not limited to the treatment of an existing tumor or infectious disease but can also be used to prevent or lower the risk of developing such diseases in an individual, i.e., for prophylactic use.
  • Potential candidates for prophylactic vaccination include individuals with a high risk of developing cancer, i.e., with a personal or familial history of certain types of cancer. iii. Subjects exposed to allergens
  • the vaccine compositions may be administered to subjects for the purpose of preventing and/or attenuating allergic reactions, such as allergic reactions which lead to anaphylaxis. Allergic reactions may be characterized by the T H 2 responses against an antigen leading to the presence of IgE antibodies. Stimulation of 3 ⁇ 41 immune responses and the production of IgG antibodies may alleviate allergic disease. Thus, the disclosed vaccine compositions may lead to the production of antibodies that prevent and/or attenuate allergic reactions in subjects exposed to allergens.
  • Nanoparticle vaccines disclosed herein can be used for treatment of disease conditions characterized by immunosuppression, including, but not limited to, AIDS or AIDS-related complex, idiopathic immunosuppression, drug induced immunosuppression, other virally or environmentally-induced conditions, and certain congenital immune deficiencies.
  • Nanoparticle vaccine compositions can also be employed to increase immune function that has been impaired by the use of radiotherapy of immunosuppressive drugs (e.g., certain chemotherapeutic agents), and therefore can be particularly useful when used in conjunction with such drugs or radiotherapy.
  • immunosuppressive drugs e.g., certain chemotherapeutic agents
  • Vaccines can be administered by a number of routes including, but not limited to: oral, inhalation (nasal or pulmonary), intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual, or rectal means.
  • Injections can be e.g., intravenous, intradermal, subcutaneous, intramuscular, or intraperitoneal. In some embodiments, the injections can be given at multiple locations.
  • nanoparticle vaccines disclosed herein are particularly suitable for enteral administration.
  • the ability to target vaccine compositions to epithelial cells in the digestive tract greatly facilitates the ability of a vaccine to induce mucosal and systemic immunity when administered orally.
  • Administration of the formulations may be accomplished by any acceptable method which allows an effective amount of the vaccine to reach its target.
  • the particular mode selected will depend upon factors such as the particular formulation, the severity of the state of the subject being treated, and the dosage required to induce an effective immune response.
  • an "effective amount" is that amount which is able to induce an immune response in the treated subject.
  • the actual effective amounts of vaccine can vary according to the specific antigen or
  • biotinylated antibodies and recombinant proteins that target different immune system cells were attached to the surface of the particles. These surface-modified particles interact specifically with cells and provide effective delivery of anti-proliferative drugs to intracellular compartments. For example, when modified with an antibody that recognizes T cells, Doxorubicin-loaded particles specifically reduced the proliferation of those cells ( Figure 1).
  • Nanoparticles were prepared by a water-oil- water emulsion method using 50:50 Poly(DL-lactide-co-glycolide) from Lactel ® with an inherent viscosity of 0.59dL/g.
  • PLGA was dissolved in methylene chloride.
  • LPS- coated particles were prepared with 20 mg/ml lipopolysaccharide (Sigma ® , from Escherichia coli) in the surfactant. Nanoparticles were stored after lyophilization at -20° C. Nanospheres were characterized using scanning electron microscopy. Protein encapsulation was quantified by dissolving the particles in DMSO for 24hr and performing a BCA Protein Assay (Pierce ® ).
  • Nanoparticulates encapsulating the model antigen, ovalbumin, were surface modified with lipopolysaccharide (LPS) and used to induce immunity in live animals against ovalbumin.
  • LPS lipopolysaccharide
  • LPS is a principal component of the cell wall of gram-negative bacteria and is a ligand for Toll like receptor 4, a major inducer of DC maturation and thus of T cell
  • LPS consists of a hydrophobic fatty acid chain conjugated to hydrophilic polysaccharide chains (Mayer, Methods in Microbiology, 18:157-207 (1985)). LPS is thus a similar composition to protein-fatty acid conjugates and serves as a model for incorporating protein- fatty acid conjugates onto PLGA nanoparticles for engineering high density protein display on the surface.
  • the nanoparticles' ability to elicit an immune response was significantly enhanced.
  • Addition of modules to enhance particle targeting, internalization, endosome escape, and extracellular protection will increase the degree to which these elements can further enhance their efficacy as vaccine vehicles.
  • Liposomal and polymeric particles prepared with OVA and poly(amido amine) dendrimer generation 5 (PAMAM dendrimer G5), were incubated with bone-marrow derived dendritic cells (BMDCs) or bone marrow derived macrophages (BMDMs) for 24 hours.
  • Endosomal disruption was measured indirectly through assessing the level of cross- presentation by the cells.
  • Cross-presentation is a process where antigen escapes the endosomal compartment and is presented by the MHC in the cytosol. This presentation can be measured with an antibody (25.D16-PE). The amount of 25.D16-PE-positive cells represents the degree of cross- presentation mediated by endosomal disruption.
  • the PLGA nanoparticles were prepared carrying carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) via a dendrimer (G4) or cyclodextrin.
  • the monophosphoryl lipid A (MPLA) nanoparticles were prepared carrying G5 and/or CpG.
  • the prepared nanoparticles were tested for their ability to disrupt endosomes.
  • Particles co-encapsulating the OVA peptide or GFP associated with the dendrimer were incubated at a concentration of 50 ⁇ g/ml with 1E5 BMDCs for 24 hours. Liposomal disruption was assessed both qualitatively via fluorescence microscopy of GFP in the cytosol or quantitatively by amount of OVA antigen presented via MHC Class I. Antigen presentation on Class I MHC takes place via cytosolic transport and OVA peptide/MHC Class I was detected specifically using the antibody 24D16-PE.
  • Figures 6-7 show amount of antibody bound to the surface of DCs after particle internalization and compared to PLGA nanoparticles encapsulating the same antigen.
  • Figure 8 shows endosomal disruption assessed by quantitating cytosolic fluorescence in the presence and absence of a trifluoromethoxy phenylhydrazone (FCCP) a protonophore and uncoupler of oxidative phosphorylation in the mitochondria.
  • FCCP is hosted in the dendrimer via a conjugated cyclodextrin molecule accommodating the hydrophobic FCCP molecule.
  • the dendrimer/Cyclodextrin ratio is 1 :5 and the figure shows that FCCP hosting can take place even without cyclodextrin attached since FCCP may be associated with the dendrimer cavity itself.
  • FCCP would further disrupt the endosomal/lysosomal compartment resulting in increased fluorescence compared to dendrimer alone or the drug alone as shown in Figure 7.
  • Figures 6 and 7 demonstrate that dendrimer (G5) encapsulation with antigen OVA increases cross-presentation in liposomal and polymeric nanoparticles (PLGA or PLGA-PEG nanoparticles) when co-incubated with mouse BMDCs or BMDMs.
  • dendrimer (G5) encapsulation with antigen OVA increases cross-presentation in liposomal and polymeric nanoparticles (PLGA or PLGA-PEG nanoparticles) when co-incubated with mouse BMDCs or BMDMs.
  • Figure 8 demonstrates that incorporation of FCCP significantly enhances the ability of PLGA-G4 or PLGA-G4-cyclodextrin nanoparticles to disrupt endosomes.
  • Figure 9 demonstrates that the highest percentage of transfection of bone marrow derived dendritic cells with GFP occurred when the cells were transfected with MPLA nanoparticles co-encapsulating GFP, the dendrimer G5 and CpG.
  • the graph also describes the importance of having both TLR ligands associated with the same particle in that MPLA/G5 LED & - /(G5+CpG) LED is not as efficient in transfection compared to
  • Particles were prepared as in Example 2. After lyophilization, biotin- elastin was prepared by biotinylation with NHS-LC-biotin (Pierce
  • NHS-LC-biotin was incubated with 10 mg of particles (room temperature, 1 hour) at a concentration of 20 mg/ml. Following incubation, particles were washed by centrifugation 3X with deionized water and freeze- dried for further use. Controlled release studies were performed at the indicated pH.
  • Polymers such as PMAA in conjunction with poly(ethylene glycol) (PEG) have been used in past applications for enhancing the oral delivery of chemotherapeutic drugs, and thus there is good precedence for the use of these polymers as 'shielding' components in oral delivery (Blanchette and Peppas, Ann. Biomed. Eng. , 33(2): 142-9 (2005); Blanchette and Peppas, J Biomed. Mater. Res. A, 72(4):381-8 (2005)).
  • NPs peptide-loaded nanoparticles
  • PLGA NPs containing avidin on the surface were prepared using methods described by Park (Park et al., J Control. Release, 156:109-115, 2011) and Fahmy (Fahmy et al., Biomaterials, 26:5727-5736, 2005).
  • the prepared NPs included blank NP (no peptide), coumarin-labeled blank NP (NP-coumarin), NP-influenza-matrix peptide (FMP) (incorporating HLA A2.1 FMP sequence GILGFVFTL), NP-CEF (incorporating CEF pool peptide, pool of 32 peptides from EBV, CMV, and influenza virus, Anaspec), and NP-SOX2 (22 15-mer SOX2 peptides; Table 1).
  • the amount of each peptide in the NPs was as follows: NP-FMP (9 ⁇ g/mg NP), NP-CEF (0.56 g/mg NP), and NP-SOX2 (4.1 ⁇ x jmg NP).
  • Table 1 A list of SOX2 overlapping peptide sequences loaded in nanoparticles.
  • TLR and/or antibody (Ab)-coated NPs were prepared by adding biotinylated LPS (InvivoGen), polyinosinic-polycytidylic acid [poly (I:C)] (InvivoGen), BDCA3 Ab (Miltenyi Biotec), or DC-SIGN Ab (Miltenyi Biotec) at the concentration of 5 ⁇ g Ab per milligram of NPs. The vials were gently rotated for 15 min. They were then centrifuged at 1200 rpm for 5 min to remove the supernatant and washed twice to remove any soluble ligand prior to use in experiments.
  • Figure 10 demonstrates specific targeting of BDCA3+ and DC- SIGN+ dendritic cells with targeted NPs.
  • the change in MFI of coumarin in targeted APCs over that in non-targeted APCs was greatest when BDCA3- targeted NPs were incubated with BDCA3+ myeloid dendritic cells (MDCs) and when DC-SIGN-targeted NPs were incubated with DC-SIGN+ DCs.
  • MDCs myeloid dendritic cells
  • DC-SIGN-targeted NPs were incubated with DC-SIGN+ DCs.
  • Example 7 Targeting the influenza antigen to dendritic cells potentiates the immune response
  • Mo-DCs Monocyte-derived DCs
  • PBMCs Monocyte-derived DCs
  • Dhodapkar Dhodapkar et al., Proc. Natl. Acad. Sci. USA, 102:2910:2915, 2005. Briefly, CD14+ monocytes were isolated from PBMCs by immunomagnetic bead selection using CD 14 beads following the manufacturer's protocol (Miltenyi Biotec).
  • CD14+ cells were suspended in 1% healthy donor plasma in RPMI 1640 (Cellgro), supplemented with IL-4 (25 ⁇ g/ml; R&D Systems) and GM-CSF (20 ng/ml sargramostim (Leukine); Genzyme) on days 0, 2, and 4 of culture. Immature Mo-DCs were harvested on days 5-6 and used for the experiments described below.
  • the CD 14- fraction of PBMCs was cultured in the presence of 5% pooled human serum (Labquip) in RPMI 1640.
  • BDCA3+ MDCs were isolated from the PBMCs using BDCA3 MACS beads (Miltenyi Biotec). NP uptake experiments
  • BDCA3+ MDCs and DC-SIGN+ Mo- DCs were studied by incubating the cells overnight with NP-FMP (peptide concentration 0.05 ⁇ g/ml) at 37°C.
  • BDCA3+ MDCs isolated from healthy donor buffy coats or DC-SIGN+ Mo-DC were loaded with NP-FMP at 37°C and supernatants were collected after 24 h.
  • Cytokines were quantified using VeriPlex Human Cytokine ELISA (PBL IFN source) and data were analyzed by Q-View 2.160 software (Quansys Biosciences).
  • BDCA3+ MDCs and DC-SIGN DCs incubated alone were used as negative controls.
  • FIG. 11 A-l ID demonstrate that targeting the influenza antigen (FMP) to BDCA3+ or DC-SIGN+ DC subsets potentiates the immune response.
  • the graph shows mean cytokine expression levels (IL-15, IFN- ⁇ and TNF-a in pg/ml, and IL-6 and IL-8 in pg/dl, ⁇ SEM) per 30,000 APCs for cells obtained from three different healthy donors. *p ⁇ 0.05.
  • Example 8 Delivery of combination of peptides by dendritic cells stimulates a specific and multivalent T cell response.
  • Nanoparticles loaded with CEF combination of 32 peptides were prepared as described in Example 6.
  • NP-CEF CEF pool peptide
  • CD8+ T cell response following stimulation by Mo-DCs or BDCA3+ MDCs loaded with either blank NP or NP-CEF was confirmed when the CD8+ T cells were re-stimulated with individual peptide components (Pepl-Pepl8; described in Table 2) of the CEF peptide pool. Blank NP and NP-CEF restimulated with CEF pool peptide were used as negative and positive controls, respectively.
  • Example 9 Delivery of combinations of tumor antigen peptides of SOX2 to dendritic cells leads to stimulation of antigen-specific CD4 well as CD8 T cells.
  • Nanoparticles loaded with tumor antigen peptides of SOX2 were prepared as described in Example 6. Antigen-specific T cell stimulation
  • T cells were restimulated with NP-SOX2 loaded DCs on days 7 and
  • DCs were matured overnight with LPS (50 ng/ml; Sigma-Aldrich) or poly(LC) (25 ⁇ g/ml; Sigma-Aldrich) or cytokine mixture [IL-6 (O.O ⁇ g/ml; R&D Systems), IL- ⁇ (0.01 ⁇ g/ml; R&D Systems), TNF-a (0.01 ⁇ g/ml; R&D Systems), and PGE2 (1 ⁇ g/ml, Sigma-Aldrich)] after loading with NPs.
  • LPS 50 ng/ml; Sigma-Aldrich
  • poly(LC) 25 ⁇ g/ml; Sigma-Aldrich
  • cytokine mixture [IL-6 (O.O ⁇ g/ml; R&D Systems), IL- ⁇ (0.01 ⁇ g/ml; R&D Systems), TNF-a (0.01 ⁇ g/ml; R&D Systems), and PGE2 (1 ⁇ g/ml, Sigma-Aldrich)]
  • NP-SOX2-loaded autologous DCs were used to stimulate T cells because SOX2 peptides are 15 aa long and require active processing for antigen presentation. DCs loaded with NP-SOX2 were able to stimulate both SOX2-specific CD4 and CD8+ T cells in culture. Taken together these data demonstrate that both BDCA3+ and Mo-DC-SIGN+ NP-loaded DCs are equally effective at generating antigen-specific human T cells in culture, including against complex peptide mixtures from viral and tumor antigens across multiple MHC molecules.

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Abstract

Des compositions de vaccins nanoparticulaires modulaires et des procédés de préparation et d'utilisation de celles-ci ont été mis au point. Lesdites compositions de vaccins nanoparticulaires modulaires comprennent un antigène encapsulé dans une particule polymère et des éléments adaptateurs qui relient de manière modulaire des éléments fonctionnels à la particule. La conception modulaire de ces compositions de vaccins, qui implique une addition et une élimination flexibles d'antigène, d'adjuvant, de potentialisateurs immunitaires, d'éléments de reconnaissance moléculaire et de médiation de transport, ainsi que de médiateurs d'absorption intracellulaire, permet une régulation exquise des variables qui sont importantes dans l'optimisation d'un système efficace d'administration de vaccin.
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