WO2019140318A1 - Systèmes de nanoparticules - Google Patents

Systèmes de nanoparticules Download PDF

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Publication number
WO2019140318A1
WO2019140318A1 PCT/US2019/013370 US2019013370W WO2019140318A1 WO 2019140318 A1 WO2019140318 A1 WO 2019140318A1 US 2019013370 W US2019013370 W US 2019013370W WO 2019140318 A1 WO2019140318 A1 WO 2019140318A1
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Prior art keywords
nanoparticles
polymer
payload
antigen
range
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English (en)
Inventor
Howard B. Sosin
Michael J. Caplan
Robert K. Prud'homme
Mark Kastantin
April DOWER
Spencer DAHL
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N-Fold LLC
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N-Fold LLC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5015—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5138—Organic macromolecular compounds; Dendrimers obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)

Definitions

  • biodegradable polymers are commonly used as a matrix to carry the drugs.
  • Diverse approaches have been applied in order to produce polymer
  • nanoparticles containing one or more therapeutic agents are both desirable and needed.
  • the present disclosure provides nanoparticle compositions in which individual nanoparticles comprise polymers, payloads, and coating agents, as well as methods of making and using such nanoparticle compositions, and various compositions and/or technologies relating to such nanoparticle compositions, their production, and/or their use.
  • the present disclosure identifies the source of at least one problem in certain polymer nanoparticle technologies, particularly when utilized to prepare compositions for delivering and/or otherwise including one or more complex payloads (e.g ., protein, carbohydrate, lipid and/or nucleic acid mixtures, crude samples, cellular extracts, etc.).
  • complex payloads e.g ., protein, carbohydrate, lipid and/or nucleic acid mixtures, crude samples, cellular extracts, etc.
  • methods provide certain advantages and/or solve one or more problems associated with prior nanoparticle technologies.
  • the present disclosure provides technologies for manufacturing nanoparticles (e.g., comprised of polymers and including payloads and/or coating agents as described herein) with minimal waste.
  • provided manufacturing technologies utilize and/or benefit from attributes of non-solvent systems (e.g, non-solvent systems of polymers, payloads, and/or coating agents).
  • provided technologies for manufacturing nanoparticles allow for production of nanoparticles encapsulating one or more payloads, wherein one or more payloads are not exposed to the surface of a nanoparticle (e.g., not exposed to the environment surrounding the nanoparticle). In some embodiments, provided technologies for manufacturing nanoparticles allow for production of nanoparticles encapsulating one or more payloads, such that encapsulated payloads are substantially wholly encapsulated.
  • the present disclosure provides nanoparticle compositions in a dry ( e.g ., lyophilized) state.
  • provided dry compositions are amenable suspension (e.g., re-suspension); in particular embodiments, provided dry compositions are amenable to suspension without significant deterioration of one or more relevant properties (e.g, load, release rate) of nanoparticles in the composition as compared with that observed prior to the nanoparticles being dried.
  • compositions are amenable to storage, in some embodiments, under atmospheric conditions, for a period of time (e.g, in some embodiments that may extend for at least 6 months, at least 9 months, at least 12 months, at least 2 years, at least 3 years or more); in some embodiments, such compositions are stable throughout the period of storage.
  • one or more stability characteristics e.g, size, redispersability, protein loading, antigenicity and/or other bioactivity of payload, and substantially intact encapsulation of payload of a polymer nanoparticle
  • stability characteristics e.g, size, redispersability, protein loading, antigenicity and/or other bioactivity of payload, and substantially intact encapsulation of payload of a polymer nanoparticle
  • the present disclosure provides methods including steps of combining a hydrophilic payload and a polymer that is not soluble in the same solvent as a hydrophilic payload, together in a solvent system characterized in that a mixture of a hydrophilic payload, a polymer and a solvent system is generated, and lyophilizing the mixture to form a lyophilized cake.
  • the present disclosure also provides methods including prior to generation of the lyophilized cake, subjecting the solvent system to a concentration step to remove at least one of water and solvent prior to lyophilization.
  • concentration comprises evaporation.
  • a concentration step removes at least some water and at least some solvent.
  • a concentration step removes substantially all of at least one of water and solvent.
  • the present disclosure also provides methods including steps of exposing a lyophilized cake to a temperature sufficient to melt the polymer to form a melted cake, cooling the melted cake to form a block material, wherein the block material has a porosity of less than 5%; and wherein the temperature is not so high that it damages one or more biological or pharmaceutical activities of the payload.
  • the block material has a substantially uniform distribution of the hydrophilic payload with respect to the polymer.
  • the present disclosure also provides methods r including grinding the lyophilized cake and resuspending the ground cake in at least one alcohol.
  • grinding occurs in the presence of liquid nitrogen.
  • the at least one alcohol in which the ground cake is resuspended is or comprises propanol.
  • a block material comprising polymer and hydrophilic payload has a substantially uniform distribution of a hydrophilic payload with respect to the polymer.
  • the present disclosure also provides methods of making a flowable microparticle suspension including the steps of comminuting a lyophilized cake or a block material comprising a hydrophilic payload and a polymer to form microparticles, and introducing the microparticles in a non-solvent system comprising a carrier, characterized in that neither the hydrophilic payload nor the polymer is miscible in the carrier used, to form a flowable microparticle suspension.
  • the present disclosure also provides methods of making nanoparticles including the steps of comminuting a lyophilized cake or a block material comprising a hydrophilic payload and a polymer to form microparticles, introducing the microparticles in a non-solvent system comprising a carrier, characterized in that neither the hydrophilic payload nor the polymer is miscible in the carrier used, to form a flowable microparticle suspension, and microfluidizing the flowable microparticle suspension at an elevated temperature to form nanoparticles in a nanoparticle suspension, wherein the flowable microparticle suspension is introduced to the microfluidizer under a shear gradient.
  • the present disclosure also provides methods include the steps of combining a hydrophilic payload and a polymer together in a solvent system to form a mixture, lyophilizing the mixture to form a lyophilized cake, comminuting the lyophilized cake to form microparticles, introducing the microparticles to a non-solvent system comprising a carrier, characterized in that neither the hydrophilic payload or the polymer are miscible in the carrier used, to form a flowable microparticle suspension, microfluidizing the flowable microparticle suspension at an elevated temperature to form nanoparticles in a nanoparticle suspension, wherein the flowable microparticle suspension is introduced to the microfluidizer applying shear gradient to the flowable microparticle suspension, adding a stabilizing agent solution to the nanoparticle suspension, and applying a preparation of coating agents to the nanoparticles.
  • the present disclosure also provides compositions including a plurality of nanoparticles, each of which is comprised of a polymer and a hydrophilic payload substantially uniformly disposed within the polymer, wherein the nanoparticles do not comprise a lumen, and wherein substantially no hydrophilic payload is exposed on the surface of the nanoparticles.
  • a plurality of nanoparticles has a size within a range of 500 nm or less. In some embodiments, a plurality of nanoparticles has a size within a range of 450 nm or less.
  • compositions including a plurality of nanoparticles, each of which is comprised of a polymer and a hydrophilic payload wherein the hydrophilic payload is disposed within the polymer such that nanoparticles of a size larger than approximately 500 nm have substantially less of the hydrophilic payload than nanoparticles of a size smaller than approximately 500 nm, and wherein the nanoparticles do not comprise a lumen, and wherein substantially no hydrophilic payload is exposed on the surface of the nanoparticles.
  • the present disclosure also provides compositions including a plurality of nanoparticles, each of which is comprised of a polymer and a hydrophilic payload wherein the hydrophilic payload is disposed within the polymer such that nanoparticles of a size larger than approximately 500 nm have substantially less of the hydrophilic payload than nanoparticles of a size smaller than approximately 500 nm, and wherein the nanoparticles do not comprise a lumen, and wherein substantially no hydrophilic payload is exposed on the surface of the nanoparticles and the composition comprises less than approximately 20% free protein.
  • any of a variety of polymers may be desirable for use in provided methods and compositions.
  • a polymer may be hydrophobic.
  • a polymer may be amphiphilic.
  • provided methods and compositions are amenable to the use of polymers of various sizes and properties.
  • a polymer has a molecular weight within a range of 5,000-5,000,000 Daltons.
  • a hydrophilic payload is selected from the group consisting of a protein, a nucleic acid, an antigen, and combinations thereof.
  • an antigen is or comprises an allergic antigen.
  • an antigen is or comprises an anaphylactic antigen.
  • an antigen is or comprises an infectious antigen.
  • an antigen is or comprises an autoantigen.
  • an antigen is or comprises a disease-associated antigen.
  • any of a variety of amounts (e.g., weight ratios or absolute amounts) of hydrophilic payload to polymer may be used.
  • a weight ratio of the hydrophilic payload to polymer is within a range of 1 :99 to 20:80, or 1 :99 to 10:90.
  • a weight ratio of the hydrophilic payload and the polymer is within a range of about 0.001 : 1 to 0.1 : 1, or 0.01 : 1 to 0.1 : 1.
  • various embodiments are amendable to the inclusion of any of a variety of solvents and non-solvents at various points in the production of provided compositions.
  • the inclusion of a non-solvent at specific step(s) in a process may allow for a higher degree of uniformity of distribution of a payload in a polymer than was possible using previously known methods.
  • a solvent system comprises an aqueous solution.
  • an aqueous solution comprises water and DMSO.
  • a polymer is present in water, while a payload is present in DMSO, prior to the combining step, or vice versa.
  • a non-solvent system comprises water and alcohol (e.g., propanol).
  • Various embodiments include the comminution of a lyophilized cake or block material into microparticles. While any application-appropriate manner and/or condition may be used, in some embodiments, a lyophilized cake or block material is comminuted at a temperature within a range of about -210 to -l96°C, about -175 to 0 °C, about -150 to 0 °C, about -125 to 0 °C, about -100 to 0 °C, about -75 to 0 °C, about -50 to 0 °C, about -30 to 0 °C, 0 to 20 °C, about 0 to 15 °C, about 0 to 5 °C, or about 5 to 15 °C.
  • a lyophilized cake or block material may be comminuted
  • provided microparticles have a size within a range of about 10 pm to 800 pm, about 50 pm to 800 pm, about 100 pm to 800 pm, or about 100 pm to 500 pm.
  • provided nanoparticles have a mean size within a range of approximately 100-500 nm.
  • provided nanoparticles may be separated into different populations.
  • a given population of nanoparticles has a mean size within a range of approximately 100-300 nm, and a different population of the nanoparticles has a mean size within a range of approximately 300-500 nm
  • certain provided methods include the processing of microparticles into nanoparticles, for example, via microfluidic processes.
  • a flowable microparticle suspension is passed through a microfluidizer two or more times, so that the nanoparticles have substantially uniform size.
  • a microfluidizer applies shear gradient to a flowable microparticle suspension.
  • a shear gradient is within a range of 10-6 to 10-7 s-l.
  • a flowable microparticle suspension is homogenized at a temperature within a range of about 80 °C to 110 °C, about 85 °C to 110 °C, about 90 °C to 110 °C, about 80 °C to 105 °C, about 80 °C to 100 °C, or about 90 °C to 100 °C.
  • provided methods may comprise adding a stabilizing agent solution to the nanoparticle suspension. In some embodiments, provided methods further comprise applying one or more coating agents to the nanoparticles (e.g., a preparation of one or more coating agents). In some embodiments, a coating agent may be or comprise at least one microbial extract.
  • provided methods may comprise lyophilizing a stabilizing agent solution and a nanoparticle suspension prior to applying a preparation of coating agents to the nanoparticles.
  • a preparation of coating agents comprises at least one microbial extract.
  • the present disclosure provides methods including a step of centrifugation.
  • centrifugation is performed on a nanoparticle suspension.
  • centrifugation is performed at a speed between 300 and 600 xg ⁇
  • the present disclosure further provides methods including a step of subjecting supernatant from a centrifuged solution to a step of tangential flow filtration.
  • the supernatant is mixed with a solution comprising a coating agent after centrifugation.
  • centrifugation is performed on a nanoparticle suspension. In some embodiments, centrifugation is performed at a speed between 300 and 600 xg ⁇
  • compositions comprising a plurality of nanoparticles, each of which is comprised of a polymer and a hydrophilic payload substantially uniformly disposed within the polymer,
  • nanoparticles do not comprise a lumen, and wherein substantially no hydrophilic payload is exposed on the surface of the nanoparticles.
  • a plurality of nanoparticles has a mean size within a range of 500 nm or less. In some embodiments, a plurality of nanoparticles has a mean size within a range of 100-500 nm, 300-500 nm, 100-300 nm, or 100-250 nm.
  • a plurality of nanoparticles is comprised of at least two populations of nanoparticles, each with a different mean size.
  • at least one population of nanoparticles has a mean size within a range of approximately 300-500 nm.
  • at least one population of nanoparticles has a mean size within a range of approximately 100-300 nm.
  • the present disclosure also provides methods including the step of administering to a subject in need thereof a nanoparticle composition comprising a plurality of nanoparticles, each of which is comprised of a polymer, a hydrophilic payload substantially uniformly disposed within the polymer, and at least one coating agent, wherein the nanoparticles do not comprise a lumen, and wherein substantially no hydrophilic payload is exposed on the surface of the nanoparticles.
  • the present disclosure also provides methods including the step of administering to a subject in need thereof a nanoparticle composition comprising a plurality of nanoparticles, each of which is comprised of a polymer, a hydrophilic payload disposed within the polymer, and at least one coating agent, wherein the nanoparticles do not comprise a lumen, and wherein substantially no hydrophilic payload is exposed on the surface of the nanoparticles.
  • a subject is suffering from at least one of allergy, infection, and cancer.
  • provided composition is administered to a subject
  • any appropriate route of administration may be used.
  • provided nanoparticles e.g., nanoparticle compositions
  • transmucosal administration may be or comprise buccal, nasal, bronchial, vaginal, rectal, and/or sublingual administration.
  • Administration refers to the administration of a composition to a subject. Administration may be by any appropriate route.
  • administration may be bronchial (including by bronchial instillation), buccal, (e.g., between teeth and cheek, includes lower and upper teeth), enteral, interdermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal,
  • allergen refers to those antigens that induce an allergic reaction.
  • an allergen is or comprises a polypeptide.
  • an allergen is or comprises a small molecule.
  • an allergen is selected from the group consisting of food allergens, drug allergens, environmental allergens, insect venoms, animal allergens, and latex.
  • Allergic reaction has its art- understood meaning and refers to an IgE-mediated immune response to an antigen.
  • an antigen induces IgE antibodies, they will bind to IgE receptors on the surface of basophils and mast cells. Subsequent exposures to the antigen trigger cross-linking of such surface-bound anti allergen IgEs, which trigger release of histamine from stores within the cells. This histamine release triggers the allergic reaction.
  • an allergic reaction involves one or more of the cutaneous (e.g, urticaria, angioedema, pruritus), respiratory (e.g, wheezing, coughing, laryngeal edema, rhinorrhea, watery/itching eyes), gastrointestinal (e.g, vomiting, abdominal pain, diarrhea), and/or cardiovascular (e.g, if a systemic reaction occurs) systems.
  • cutaneous e.g, urticaria, angioedema, pruritus
  • respiratory e.g, wheezing, coughing, laryngeal edema, rhinorrhea, watery/itching eyes
  • gastrointestinal e.g, vomiting, abdominal pain, diarrhea
  • cardiovascular e.g, if a systemic reaction occurs
  • an asthmatic reaction is considered to be a form of allergic reaction.
  • allergic reactions are mild; typical symptoms of a mild reaction include, for example, hives (especially over the neck and face) itching, nasal congestion, rashes, water
  • allergic reactions are severe and/or life threatening; in some embodiments, symptoms of severe allergic reactions (e.g ., anaphylactic reactions) are selected from the group consisting of abdominal pain, abdominal breathing sounds (typically high-pitched), anxiety, chest discomfort or tightness, cough, diarrhea, difficulty breathing, difficulty swallowing, dizziness or light-headedness, flushing or redness of the face, nausea or vomiting, palpitations, swelling of the face, eyes or tongue, unconsciousness, wheezing, and combinations thereof.
  • allergic reactions are anaphylactic reactions.
  • allergic reactions are defined as a disorder characterized by an adverse local or general response from exposure to one or more allergens.
  • allergic reactions may be graded by a“toxicity grading” system, that will be known to those of skill in the art.
  • a grading system such as NCI-CTCAD v 4.03
  • NCI-CTCAD v 4.03 will be used to grade allergic reactions, such as a system described in Table 1 and/or Table 2
  • allergy refers to a condition characterized by an IgE-mediated immune response to particular antigens.
  • the antigens are ones that do not elicit an IgE-mediated immune response in many or most individuals.
  • the term“allergy” is used to refer to those situations where an individual has a more dramatic IgE-mediated immune response when exposed to a particular antigen than is typically observed by members of the individual’s species when comparably exposed to the same antigen.
  • an individual who is suffering from or susceptible to“allergy” is one who experiences or is at risk of experiencing an allergic reaction when exposed to one or more allergens.
  • symptoms of allergy include, for example, presence of IgE antibodies, reactive with the allergen(s) to which the individual is allergic, optionally above a particular threshold, in blood or serum of the individual.
  • symptoms of allergy include development of a wheal/flare larger than a control wheal/flare when a preparation of the antigen is injected subcutaneously under the individual’s skin.
  • an individual can be considered susceptible to allergy without having suffered an allergic reaction to the particular allergen in question. For example, if the individual has suffered an allergic reaction, and particularly if the individual has suffered an anaphylactic reaction, to a related allergen (e.g.
  • that individual may be considered susceptible to allergy to (and/or to an allergic or anaphylactic reaction to) the relevant allergen.
  • the individual may be considered to be susceptible to allergy to (and/or to an allergic and/or anaphylactic reaction to) that allergen.
  • amino acid refers to any compound and/or substance that can be incorporated into a polypeptide chain, e.g. , through formation of one or more peptide bonds.
  • an amino acid has the general structure H2N-C(H)(R)-COOH.
  • an amino acid is a naturally- occurring amino acid.
  • an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L- amino acid.
  • “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides.“Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source.
  • an amino acid, including a carboxy- and/or amino-terminal amino acid in a polypeptide can contain a structural modification as compared with the general structure above.
  • an amino acid may be modified by methylation, amidation, acetylation, and/or substitution as compared with the general structure.
  • such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid.
  • such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid.
  • the term“amino acid” is used to refer to a free amino acid; in some embodiments it is used to refer to an amino acid residue of a polypeptide.
  • Alloantigen refers to an antigen associated with allorecognition and/or graft rejection (e.g., an antigen against which a rejection immune response is directed).
  • alloantigens are agents that are present in or on tissue from one individual (e.g., a donor individual) of a particular species, but not in or on tissue from another individual (e.g., a recipient individual, for example who is genetically different from the donor individual) of the species, so that transfer of tissue from the donor individual to the recipient individual risks and/or results in a rejection immune response.
  • an antigen may be or include any chemical entity such as, for example, a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, etc.
  • an alloantigen is or comprises a polypeptide.
  • a variety of polypeptides are known in the art whose amino acid sequences can vary between and among individuals of the same species such that they might act as alloantigens.
  • Allorecognition typically refers to an immune response mounted by the immune system of an individual (i.e., a recipient) who receives a tissue graft from another individual (i.e., a donor, who for example is genetically distinct from the recipient individual) of the same species, which immune response involves recognition of an alloantigen on the grafted tissue.
  • allorecognition involves T cell recognition of the alloantigen.
  • T cells recognize an alloantigen peptide, for example, encoded by a polymorphic gene whose sequence differs between the donor and recipient individuals.
  • Anaphylactic antigen refers to an antigen (e.g., an allergen) that is recognized to present a risk of anaphylactic reaction in allergic individuals when encountered in its natural state, under normal conditions.
  • an antigen e.g., an allergen
  • pollens and animal danders or excretions e.g, saliva, urine
  • certain food antigens, insect antigens, drugs, and rubber (e.g, latex) antigens latex are generally considered to be anaphylactic antigens.
  • Exemplary anaphylactic antigens include those to which reactions are so severe as to create a risk of death (e.g, nuts, seeds, and fish).
  • Anaphylactic reaction refers to a severe, whole body allergic reaction to an allergen, characterized by pathological responses in multiple target organs, e.g., airway, skin digestive tract, and
  • symptoms of severe allergic reactions typically develop quickly, often within minutes of exposure to the allergen, and can include, for example, abdominal pain, abdominal breathing sounds (typically high-pitched), anxiety, chest discomfort or tightness, cough, diarrhea, difficulty breathing, difficulty swallowing, dizziness or light-headedness, flushing or redness of the face, nausea or vomiting, palpitations, swelling of the face, eyes or tongue, unconsciousness, wheezing, and combinations thereof.
  • Particular signs of anaphylaxis may include, for example, abnormal heart rhythm (arrhythmia), fluid in the lungs (pulmonary edema), hives, low blood pressure, mental confusion, rapid pulse, skin that is blue from lack of oxygen or pale (e.g., from shock), swelling (angioedema) in the throat that may be severe enough to block the airway, swelling of the eyes and/or face, weakness, wheezing.
  • arrhythmia abnormal heart rhythm
  • fluid in the lungs pulmonary edema
  • hives low blood pressure
  • mental confusion e.g., from shock
  • swelling angioedema
  • the most severe anaphylactic reactions can result in loss of consciousness and/or death.
  • anaphylactic reactions may be defined as a disorder characterized by an acute inflammatory reaction resulting from the release of histamine and histamine-like substances from mast cells, causing a hypersensitivity immune response.
  • anaphylaxis may present with breathing difficulty, dizziness, hypotension, cyanosis and/or loss of consciousness and may lead to death.
  • a grading system (such as NCI-CTCAD v 4.03), will be used to grade anaphylactic reactions, such as a system described in Table 3.
  • anaphylactic reactions may be diagnosed according to the following criteria, wherein an anaphylactic reaction is likely to have occurred or be occurring when any one of the three following sets of criteria are fulfilled:
  • Skin/mucosal tissue e.g., generalized hives, itch or flush, swollen
  • Airway compromise e.g., dyspnea, stridor, wheeze/ bronchospasm, hypoxia, reduced PEF
  • Skin/mucosal tissue e.g., generalized hives, itch/flush, swollen lips/tongue/uvula
  • Airway compromise e.g., dyspnea, stridor wheeze/bronchospasm, hypoxia, reduced PEF
  • Persistent GI symptoms e.g., nausea, vomiting, crampy abdominal pain
  • low systolic BP for children is defined as ⁇ 70 mmHg from 1 month to 1 year; less than (70 mmHg + [2 x age]) from 1-10 years; and ⁇ 90 mmHg from age 11-17 years.
  • isolated skin or mucosal lesions following the ingestion of a food constitute a“food-induced allergic reaction”.
  • animal refers to any member of the animal kingdom. In some embodiments,“animal” refers to humans, at any stage of development. In some embodiments,“animal” refers to non-human animals, at any stage of development. In some embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or a clone.
  • a mammal e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig.
  • Antige refers to an agent that elicits an immune response; and/or (ii) an agent that binds to a T cell receptor (e.g, when presented by an MHC molecule) or to an antibody (e.g, produced by a B cell).
  • an antigen elicits a humoral response (e.g, including production of antigen-specific antibodies); in some embodiments, an antigen elicits cellular response (e.g, involving T-cells whose receptors specifically interact with the antigen).
  • an antigen may be or include any chemical entity such as, for example, a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, etc.
  • an antigen is or comprises a polypeptide.
  • an antigen may be provided in isolated or pure form, or alternatively may be provided in crude form (e.g, together with other materials, for example in an extract such as a cellular extract or other relatively crude preparation of an antigen-containing source).
  • antigens utilized in accordance with the present disclosure are provided in a crude form.
  • an antigen is a recombinant antigen.
  • Antigen presenting cell The phrase“antigen presenting cell” or“APC,” as used herein, has its art understood meaning referring to cells which process and/or present antigen(s) to T-cells.
  • Exemplary antigen presenting cells include dendritic cells, macrophages and certain activated epithelial cells.
  • an antigen presenting cell is a cell that processes and/or presents antigen(s) to a particular T-cell population (e.g., to T-cells of a particular type and/or T-cells that may be present in and/or localized to a particular site).
  • an antigen presenting cell may be a member of a particular cell population (e.g., a particular type of cell and/or a member of a cell population that is present in and/or localized to a particular site).
  • an antigen presenting cell may present antigen(s) to a T-cell population that is present in and/or localized to a particular site and/or may itself be present in and/or localized to a particular site.
  • TLR2/TLR4-expressing dendritic cells have been described as particularly prevalent in the microenvironment within certain oral mucosal sites (see, for example Allam, et al, Tolerogenic T cells, Thl/Thl7 cytokines and TLR2/TLR4 expressing dendritic cells predominate the microenvironment within distinct oral mucosal sites. Allergy 66: 532, 2011).
  • the term“approximately” and“about” is intended to encompass normal statistical variation as would be understood by those of ordinary skill in the art.
  • the term“approximately” or“about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
  • Two events or entities are“associated” with one another, as that term is used herein, if the presence, level and/or form of one are correlated with that of the other.
  • a particular entity e.g, polypeptide
  • two or more entities are“associated” with one another if they interact, directly or indirectly, so that they are and remain in physical proximity with one another.
  • Autoantigen As used herein, the term“autoantigen” is used to refer to antigens produced by an individual that are recognized by the immune system of that individual. In some embodiments, an autoantigen is one whose recognition by the individual’s immune system is associated with an autoimmune disease, disorder or condition. In general, an autoantigen may be or include any chemical entity such as, for example, a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, etc. In some embodiments, an autoantigen is or comprises a polypeptide. Those of skill in the art are familiar with a variety of agents, including
  • polypeptides that can act as autoantigens, and particular that are recognized in immune reactions associated with autoimmunity diseases, disorders and/or conditions
  • Biocompatible refers to materials that do not cause significant harm to living tissue when placed in contact with such tissue, e.g ., in vivo. In certain embodiments, materials are“biocompatible” if they are not toxic to cells. In certain embodiments, materials are“biocompatible” if their addition to cells in vitro results in less than or equal to 20% cell death, and/or their administration in vivo does not induce significant inflammation or other such adverse effects.
  • Biodegradable refers to materials that, are broken down in biological systems. The degradation may occur inside cells, e.g., where cellular machinery, such as by enzymatic degradation, by hydrolysis, and/or by combinations thereof is active, or it may occur elsewhere in vivo by means of, e.g. hydrolysis or enzymatic action. In either case, the resultant degradation components do not cause significant toxic effects on the cells.
  • components generated by breakdown of a biodegradable material are biocompatible and therefore do not induce significant inflammation and/or other adverse effects in vivo.
  • biodegradable polymer materials break down into their component monomers.
  • breakdown of biodegradable materials involves hydrolysis of ester bonds.
  • breakdown of biodegradable materials involves cleavage of urethane linkages.
  • biodegradable polymers include, for example, polymers of hydroxy acids such as lactic acid and glycolic acid, including but not limited to poly(hydroxyl acids), poly(lactic acid)(PLA), poly(glycolic acid)(PGA), poly(lactic-co-glycolic acid)(PLGA), and copolymers with PEG, polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butyric acid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates, poly(lactide-co- caprolactone), blends and copolymers thereof.
  • polymers are also biodegradable, including, for example, proteins such as albumin, collagen, gelatin and prolamines, for example, zein, and polysaccharides such as alginate, cellulose derivatives and polyhydroxyalkanoates, for example, polyhydroxybutyrate blends and copolymers thereof.
  • proteins such as albumin, collagen, gelatin and prolamines, for example, zein
  • polysaccharides such as alginate, cellulose derivatives and polyhydroxyalkanoates, for example, polyhydroxybutyrate blends and copolymers thereof.
  • biocompatible and/or biodegradable derivatives thereof e.g ., related to a parent polymer by substantially identical structure that differs only in substitution or addition of particular chemical groups as is known in the art).
  • biologically active refers to a substance that has activity in a biological system (e.g, in a cell (e.g, isolated, in culture, in a tissue, in an organism), in a cell culture, in a tissue, in an organism, etc.).
  • a substance that, when administered to an organism, has a biological effect on that organism is considered to be biologically active.
  • a biologically active substance is required (e.g, is necessary and sufficient) for the activity to be present; in such circumstances, that portion or fragment is considered to be a“biologically active” portion or fragment.
  • Carrier refers to a diluent, adjuvant, excipient, or vehicle with which a composition is administered.
  • carriers can include sterile liquids, such as, for example, water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • carriers are or include one or more solid components.
  • a carrier may be or comprise a bead, film, rod, or similarly structured component.
  • Cellular lysate refers to a fluid containing contents of one or more disrupted cells (i.e., cells whose membrane has been disrupted).
  • a cellular lysate includes both hydrophilic and hydrophobic cellular components.
  • a cellular lysate is a lysate of one or more cells selected from the group consisting of plant cells, microbial (e.g, bacterial or fungal) cells, animal cells (e.g, mammalian cells), human cells, and combinations thereof.
  • a cellular lysate is a lysate of one or more abnormal cells, such as cancer cells.
  • a cellular lysate is a crude lysate in that little or no purification is performed after disruption of the cells, which generates a“primary” lysate.
  • one or more isolation or purification steps are performed on the primary lysate.
  • lysate refers to a preparation that includes multiple cellular components and not to pure preparations of any individual component.
  • Combination therapy refers to those situations in which a subject is simultaneously exposed to two or more therapeutic agents. In some embodiments, such agents are administered simultaneously; in some embodiments, such agents are administered sequentially; in some embodiments, such agents are administered in overlapping regimens.
  • the term“corresponding to” is often used to designate the position/identity of a residue in a polymer, such as an amino acid residue in a polypeptide or a nucleotide residue in a nucleic acid.
  • residues in such a polymer are often designated using a canonical numbering system based on a reference related polymer, so that a residue in a first polymer “corresponding to” a residue at position 190 in the reference polymer, for example, need not actually be the l O* 11 residue in the first polymer but rather corresponds to the residue found at the l O* 11 position in the reference polymer; those of ordinary skill in the art readily appreciate how to identify“corresponding” amino acids, including through use of one or more
  • Derivative refers to a structural analogue substance that is produced or formed from another substance of similar structure in one or more steps.
  • a derivative refers to a second chemical substance related structurally to a first chemical substance and theoretically derivable from the first chemical substance.
  • cellulose derivatives include, but are not limited to, cellulose esters (such as organic and inorganic esters), cellulose ethers (such as alkyl, hydroxyalkyl and carboxyalkyl ethers), sodium carboxymethyl cellulose and cellulose acetate.
  • cellulose organic esters include, but are not limited to cellulose acetate, cellulose triacetate, cellulose propionate, cellulose acetate propionate and cellulose acetate butyrate.
  • cellulose inorganic esters include, but are not limited to, cellulose nitrate and cellulose sulfate.
  • cellulose alkyl ethers include, but are not limited to, methylcellulose, ethylcellulose and ethyl methyl cellulose.
  • cellulose hydroxyalkyl ethers include, but are not limited to, hydroxy ethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose and ethyl hydroxyethyl cellulose.
  • cellulose carboxyalkyl ethers include, but are not limited to carboxymethyl cellulose.
  • Dosage form refers to a physically discrete unit of a therapeutic agent for administration to a subject. Each unit contains a predetermined quantity of active agent. In some embodiments, such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (z.e., with a therapeutic dosing regimen).
  • Dosing regimen refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time.
  • a given therapeutic agent has a
  • a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (z.e., is a therapeutic dosing regimen).
  • Encapsulated The term“encapsulated” is used herein to refer to substances that are completely surrounded by another material.
  • expression of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and/or 3’ end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.
  • Functional As used herein, the term“functional” is used to refer to a form or fragment of an entity that exhibits a particular property and/or activity.
  • Graft rejection refers to rejection of tissue transplanted from a donor individual to a recipient individual.
  • graft rejection refers to an allograft rejection, wherein the donor individual and recipient individual are of the same species.
  • allograft rejection occurs when the donor tissue carries an alloantigen against which the recipient immune system mounts a rejection response.
  • graft rejection refers to a xenograft rejection, wherein the donor and recipient are of different species.
  • xenograft rejection occurs when the donor species tissue carries a xenoantigen against which the recipient species immune system mounts a rejection response.
  • homology refers to the overall relatedness between polymeric molecules, e.g. , between nucleic acid molecules (e.g, DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • polymeric molecules are considered to be“homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical.
  • polymeric molecules are considered to be“homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g, containing residues with related chemical properties at corresponding positions).
  • certain amino acids are typically classified as similar to one another as“hydrophobic” or“hydrophilic” amino acids, and/or as having“polar” or“non-polar” side chains. Substitution of one amino acid for another of the same type may often be considered a“homologous” substitution.
  • Typical amino acid categorizations are summarized below:
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence.
  • the nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position; when a position in the first sequence is occupied by a similar nucleotide as the corresponding position in the second sequence, then the molecules are similar at that position.
  • the percent homology between the two sequences is a function of the number of identical and similar positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • Representative algorithms and computer programs useful in determining the percent homology between two nucleotide sequences include, for example, the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent homology between two nucleotide sequences can, alternatively, be determined for example using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.
  • a human is an embryo, a fetus, an infant, a child, a teenager, an adult, or a senior citizen.
  • Hydrophilic As used herein, the term“hydrophilic” and/or“polar” refers to a tendency to mix with, or dissolve easily in, water.
  • Hydrophobic As used herein, the term“hydrophobic” and/or“non-polar”, refers to a tendency to repel, not combine with, or an inability to dissolve easily in, water.
  • Identity refers to the overall relatedness between polymeric molecules, e.g ., between nucleic acid molecules (e.g, DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • polymeric molecules are considered to be“substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical.
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence.
  • the nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • Representative algorithms and computer programs useful in determining the percent identity between two nucleotide sequences include, for example, the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two nucleotide sequences can, alternatively, be determined for example using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.
  • Infection refers to the invasion of a host organism’s body by a disease-causing organism that multiplies in the host. Symptoms of an infection may result from action of toxins produced by the disease-causing organism and/or be reaction of host tissues to the organisms and/or to toxins they produce.
  • Isolated refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated.
  • isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
  • a substance is “pure” if it is substantially free of other components.
  • a substance may still be considered“isolated” or even “pure”, after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g ., buffer, solvent, water, etcf in such embodiments, percent isolation or purity of the substance is calculated without including such carriers or excipients.
  • Nanoparticle refers to a particle having at least one dimension (e.g., diameter) of less than 1000 nanometers.
  • a nanoparticle may have at least two dimensions of less than 1000 nanometers (nm).
  • a nanoparticle has at least two dimensions of less than 300 nm.
  • a nanoparticle has at least two dimensions of less than 100 nm.
  • one or more measuring techniques may be used to calculate mean size (e.g.
  • hydrodynamic diameter of a nanoparticle or population of nanoparticles may be determined by dynamic light scattering with size being reported as z-average diameter calculated by a deconvolution program.
  • the average size may be determined from electron microscopy measurements of the particles where more than 200 particles are counted and the z-average diameter is reported.
  • a nanoparticle will have no dimension of more than 1000 nanometers.
  • Nanoparticle composition refers to a composition that contains at least one nanoparticle and at least one additional agent or ingredient. In some embodiments, a nanoparticle composition contains a substantially uniform collection of nanoparticles as described herein.
  • nucleic acid As used herein, the term“nucleic acid,” in its broadest sense, refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain.
  • a nucleic acid is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage.
  • “nucleic acid” refers to individual nucleic acid residues ( e.g ., nucleotides and/or nucleosides); in some embodiments,“nucleic acid” refers to an
  • a “nucleic acid” is or comprises RNA; in some embodiments, a“nucleic acid” is or comprises DNA.
  • a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues.
  • a nucleic acid is, comprises, or consists of one or more nucleic acid analogs.
  • a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone.
  • a nucleic acid is, comprises, or consists of one or more“peptide nucleic acids”, which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present disclosure.
  • a nucleic acid has one or more phosphorothioate and/or 5’-N-phosphoramidite linkages rather than phosphodiester bonds.
  • a nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine).
  • adenosine thymidine
  • guanosine guanosine
  • cytidine uridine
  • deoxyadenosine deoxythymidine
  • deoxyguanosine deoxycytidine
  • a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g, 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof
  • a nucleic acid comprises one or more modified sugars (e.g., 2’-fluororibose, ribose, 2’-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids.
  • a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein.
  • a nucleic acid includes one or more introns.
  • nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis.
  • a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,
  • Non-solvent As used herein the term“non-solvent” is used in reference to a particular substance and refers to a liquid system (which may be a single liquid or mixture of liquids) in which the substance is relatively insoluble.
  • a liquid system is considered to be a“non-solvent” with respect to a particular substance if the substance does not dissolve in the liquid at room temperature and under atmospheric conditions and/or without investment of mechanical, electrical, or other energy, for example, to a weight/volume percent above about 1, 0.5, or 0.1.
  • a liquid system is considered to be a“non solvent” with respect to a particular substance if the substance aggregates in, coagulates in, or precipitates from the liquid, and/or cannot readily be maintained in solution in the liquid.
  • a patient refers to a human or any non-human animal (e.g ., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate) to whom therapy is administered.
  • a patient is a human being.
  • a patient is a human presenting to a medical provider for diagnosis or treatment of a disease, disorder or condition.
  • a patient displays one or more symptoms or characteristics of a disease, disorder or condition.
  • a patient does not display any symptom or characteristic of a disease, disorder, or condition.
  • a patient is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition.
  • Payload refers to an entity for delivery as described herein.
  • a payload may be or comprise a biologically active agent (e.g., a therapeutically active agent).
  • a payload may be or comprise one or more carbohydrates, lipids, metals, nucleic acids, polypeptides, small molecules and/or combinations thereof.
  • a payload may be or comprise a complex agent (e.g., protein, carbohydrate, lipid and/or nucleic acid mixtures, crude samples, cellular extracts, etc).
  • compositions that, within the scope of sound medical judgment, are suitable for use in contact with tissues of human beings and/or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • Polypeptide generally has its art- recognized meaning of a polymer of at least three amino acids.
  • the term is used to refer to specific functional classes of polypeptides, such as, for example, autoantigen polypeptides, nicotinic acetylcholine receptor polypeptides, alloantigen polypeptides, etc.
  • the present specification provides several examples of amino acid sequences of known exemplary polypeptides within the class; in some embodiments, such known polypeptides are reference polypeptides for the class.
  • the term“polypeptide” refers to any member of the class that shows significant sequence homology or identity with a relevant reference polypeptide.
  • such member also shares significant activity with the reference polypeptide.
  • a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%,
  • a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stre/ch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids.
  • Protein refers to a polypeptide (i.e., a string of at least two amino acids linked to one another by peptide bonds). Proteins may include moieties other than amino acids (e.g ., may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means.
  • Polypeptides may contain L-amino acids, D- amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc.
  • proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof.
  • the term“peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids.
  • proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.
  • Refractory As used herein, the term“refractory” refers to any subject that does not respond with an expected clinical efficacy following the administration of provided compositions as normally observed by practicing medical personnel.
  • Small molecule means a low molecular weight organic compound that may serve as an enzyme substrate or regulator of biological processes.
  • a“small molecule” is a molecule that is less than about 5 kilodaltons (kD) in size.
  • provided nanoparticles further include one or more small molecules.
  • the small molecule is less than about 4 kD, 3 kD, about 2 kD, or about 1 kD.
  • the small molecule is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D.
  • a small molecule is less than about 2000 g/mol, less than about 1500 g/mol, less than about 1000 g/mol, less than about 800 g/mol, or less than about 500 g/mol.
  • one or more small molecules are encapsulated within the nanoparticle.
  • small molecules are non-polymeric.
  • small molecules are not proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, polysaccharides, glycoproteins, proteoglycans, etc.
  • a small molecule is a therapeutic. In some embodiments, a small molecule is an immune adjuvant. In some embodiments, a small molecule is a drug.
  • Stable when applied to compositions herein, means that the compositions maintain one or more aspects of their physical structure (e.g ., size range and/or distribution of particles) over a period of time.
  • a stable nanoparticle composition is one for which the average particle size, the maximum particle size, the range of particle sizes, and/or the distribution of particle sizes (i.e., the percentage of particles above a designated size and/or outside a designated range of sizes) is maintained for a period of time under specified conditions.
  • a stable provided composition is one for which a biologically relevant activity is maintained for a period of time.
  • the period of time is at least about one hour; in some embodiments the period of time is about 5 hours, about 10 hours, about one (1) day, about one (1) week, about two (2) weeks, about one (1) month, about two (2) months, about three (3) months, about four (4) months, about five (5) months, about six (6) months, about eight (8) months, about ten (10) months, about twelve (12) months, about twenty-four (24) months, about thirty-six (36) months, or longer. In some embodiments, the period of time is within the range of about one (1) day to about twenty-four (24) months, about two (2) weeks to about twelve (12) months, about two (2) months to about five (5) months, etc.
  • a stable composition is stable at ambient conditions.
  • a stable composition is stable under biologic conditions (i.e., 37° C in phosphate buffered saline).
  • Subject refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate).
  • a human includes pre and post-natal forms.
  • a subject is a human being.
  • a subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease.
  • a subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.
  • Sublingual refers to the route of administration where a substance is placed in the oral cavity (e.g., sublingual (e.g. buccal mucosal space)) to be absorbed through the oral mucosa.
  • sublingual administration may be or comprise buccal mucosal administration.
  • the term“substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
  • One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result.
  • the term“substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
  • Susceptible to An individual who is“susceptible to” a disease, disorder, or condition is at risk for developing the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition does not display any symptoms of the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition has not been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition is an individual who has been exposed to conditions associated with development of the disease, disorder, or condition. In some embodiments, a risk of developing a disease, disorder, and/or condition is a population-based risk (e.g, family members of individuals suffering from allergy, etc.).
  • a population-based risk e.g, family members of individuals suffering from allergy, etc.
  • Symptoms are reduced. According to the present disclosure,“symptoms are reduced” when one or more symptoms of a particular disease, disorder or condition is reduced in magnitude (e.g., intensity, severity, etc.) and/or frequency. For purposes of clarity, a delay in the onset of a particular symptom is considered one form of reducing the frequency of that symptom [0095]
  • Therapeutic agent refers to any agent that has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect, when administered to a subject. In some embodiments, an agent is considered to be a therapeutic agent if its administration to a relevant population is statistically correlated with a desired or beneficial therapeutic outcome in the population, whether or not a particular subject to whom the agent is administered experiences the desired or beneficial therapeutic outcome.
  • therapeutically effective amount means an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and/or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and/or condition (e.g. , allergy).
  • a therapeutically effective amount is one that reduces the incidence and/or severity of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition.
  • a therapeutically effective amount does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when
  • reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g, a tissue affected by the disease, disorder or condition) or fluids (e.g, blood, saliva, serum, sweat, tears, urine, etc.).
  • a therapeutically effective agent may be formulated and/or administered in a single dose. In some embodiments, a therapeutically effective agent may be formulated and/or administered in a plurality of doses, for example, as part of a dosing regimen.
  • Therapeutic regimen refers to a dosing regimen whose administration across a relevant population is correlated with a desired or beneficial therapeutic outcome.
  • treatment also“treat” or“treating” refers to any administration of a substance that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces frequency, incidence or severity of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition.
  • Such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition.
  • Uniform when used herein in reference to a nanoparticle composition, refers to a nanoparticle composition in which individual nanoparticles have at least one dimension (e.g., dimension of nanoparticle’s cross-section, e.g., diameter) within a specified range.
  • a uniform nanoparticle composition is one in which the difference between the minimum dimension of the smallest nanoparticle and maximum dimension of the biggest nanoparticle.
  • a uniform nanoparticle is one in which the difference between the minimum dimension of the smallest nanoparticle and maximum dimension of the biggest nanoparticle.
  • composition contains nanoparticles with at least one dimension (e.g., diameter) within the range of about 100 nm to about 300 nm.
  • a uniform nanoparticle composition contains nanoparticles with a mean particle size that is under about 500 nm.
  • a uniform nanoparticle composition contains nanoparticles with a mean particle size that is within a range of about 100 nm to about 500 nm.
  • a uniform nanoparticle composition is one in which a majority of the particles within the composition have at least one dimension below a specified size or within a specified range. In some embodiments, the majority is more than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more of the particles in the composition.
  • a mean dimension or mean cross-section of nanoparticles is measured by dynamic light scattering (DLS), for example based on the scattering intensity distribution measured by photon correlation spectroscopy.
  • DLS dynamic light scattering
  • Figure l is a schematic showing an exemplary process to produce shelf-stable nanoparticles containing a payload and coating agent.
  • a coating agent is represented as OEE, however, coating agents can take any of several forms and may be coated on provided compositions in any of a variety of amounts and degrees, as described herein.
  • Figure 2 is a schematic showing an exemplary process to produce a lyophilized cake or block by having a hydrophilic payload and a polymer that is not soluble in the same solvent as the hydrophilic payload.
  • Figure 3 is a schematic showing an exemplary process to make a flowable microparticle suspension.
  • Figure 4 is a schematic showing an exemplary process to manufacture
  • Figure 5 is a schematic showing an exemplary process to manufacture
  • nanoparticles containing payloads and coating agents are nanoparticles containing payloads and coating agents.
  • Figure 6 is a schematic showing an exemplary nanoparticle containing polymer and hydrophilic payload dispersed throughout the particle, and absence of hydrophilic payload associated with the surface.
  • Figure 7 is a schematic showing an exemplary nanoparticle containing polymer and hydrophilic payload dispersed throughout the particle, and absence of hydrophilic payload associated with the surface, and a coating agent on the particle.
  • Figure 8A is a schematic showing an exemplary manufacturing process to produce shelf-stable nanoparticles comprising a payload and coating agent.
  • a payload is represented as DNA and protein and a coating agent as OEE.
  • Figure 8B shows exemplary estimates of process details for each of Lyo 1, Lyo 2, and Lyo 3 of Figure 8 A for encapsulation of 1 g of peanut protein.
  • Figure 8C shows exemplary parameters described in Figure 8B, superimposed on
  • Figure 8D shows exemplary parameters described in Figure 8B, superimposed on
  • Figure 8E shows exemplary parameters described in Figure 8B, superimposed on
  • Figure 8F shows exemplary estimates of process details for each of Lyo 1, Lyo 2, and Lyo 3 of Figure 8 A for encapsulation of 1 g of peanut protein.
  • Figure 8G shows exemplary data from additional centrifugation steps and calculation of safety factors of recovered nanoparticles.
  • Figures 9A-9E show quantification of NF-kB inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene expression in TLR4 positive HEK293 cells.
  • Figure 9A is a dose response curve for increasing concentrations of coated nanoparticles, which corresponds to averages of screenings 1, 2, and 3 of Article 1 in Figure 9D.
  • Figure 9B is a dose response curve for increasing concentrations of heat killed E. coli (HKEB), which corresponds to averages of screenings 1, 2, and 3 of Article 2 in Figure 9E.
  • Figure 9C is a dose response curve for increasing concentrations of a control ligand, LPS-EK, which corresponds to averages of screenings 1, 2, and 3 for LPS-EK in Figure 9F.
  • FIGS 10A-10F show NF-kB inducible secreted embryonic alkaline
  • FIGS 10A and 10B are dose response curves for Article 1 (coated nanoparticles) and Article 2 (HKEB), respectively.
  • Figure 10C (which corresponds to averages of screenings 1, 2, and 3 of TNFoc control ligand of 10F) shows a dose response curve for TNFoc control substrate.
  • FIGS 11A-11R show NF-kB inducible secreted embryonic alkaline
  • FIG. 11 A shows dose response curves for increasing concentrations of pre-centrifugation articles: coated nanoparticles pre-centrifugation (Article A), coated nanoparticles with 11 :1 trehalose:OEE formulation pre-centrifugation (Article D), nanoparticles with no OEE added pre-centrifugation (Article G), and LPS-EK, which each correspond to averages of screenings 1 (Figure 11B), 2 ( Figure 11C), and 3 ( Figure 11D).
  • Figure 11E shows fold induction (ratio of average induced value to average non-induced value) of each of Articles A, D, G, and LPS-EK at different concentrations of coated nanoparticles.
  • Figure 11F shows dose response curves for increasing concentrations of supernatant post-centrifugation of coated nanoparticles (Article B), supernatant post-centrifugation of coated nanoparticles with 11 : 1 trehalose: OEE formulation (Article E), supernatant post-centrifugation of un-coated nanoparticles (Article H), and LPS-EK, which each correspond to averages of screenings 1 (Figure 11G), 2 ( Figure 11H), and 3 ( Figure 1 II).
  • Figure 11 J shows fold induction (ratio of average induced value to average non-induced value) of each of Articles B, E, H and LPS-EK at different concentrations of coated nanoparticles.
  • Figure 11K shows dose response curves for increasing concentrations of pellet from centrifugation of coated nanoparticles (Article C), pellet from centrifugation of coated nanoparticles with 11 : 1 trehalose:OEE formulation (Article F), pellet from centrifugation of un-coated nanoparticles (Article I), and LPS-EK, which each correspond to averages of screenings 1 (Figure 11L), 2 ( Figure 11M), and 3 ( Figure 11N).
  • Figure 110 shows fold induction (ratio of average induced value to average non-induced value) of each of Articles C, F, I, and LPS-EK at different concentrations of coated nanoparticles.
  • Figure 11P shows a dose response curve of Article F tested at eight concentrations.
  • Figure 11Q shows a dose response curve of LPS-EK control at three different concentrations (corresponding to averages of screenings 1, 2, and 3 of Figure 11R).
  • Figure 11R shows results of screenings 1, 2, 3, and fold induction.
  • Figures 12A-12L show NF-kB inducible secreted embryonic alkaline
  • FIG. 12A shows dose response curves for increasing concentrations of Article A (pre-centrifugation; raw data and fold induction shown in Figure 12B), Article B (supernatant; raw data and fold induction shown in Figure 12C), and Article C (pellet; raw data and fold induction shown in Figure 12D).
  • Article A pre-centrifugation; raw data and fold induction shown in Figure 12B
  • Article B supernatant; raw data and fold induction shown in Figure 12C
  • Article C pellet; raw data and fold induction shown in Figure 12D.
  • Figure 12E shows dose response curves for increasing concentrations of nanoparticles with OEE, 11 : 1 trehalose:OEE formulation pre-centrifugation of Article D (pre centrifugation; raw data and fold induction shown in Figure 12F), Article E (supernatant; raw data and fold induction shown in Figure 12G), and Article F (pellet; raw data and fold induction shown in Figure 12H).
  • Figure 121 shows dose response curves for increasing concentrations of nanoparticles with no OEE added, of Article G (pre-centrifugation; raw data and fold induction shown in Figure 12J), Article H (supernatant; raw data and fold induction shown in Figure 12K), and Article I (pellet; raw data and fold induction shown in Figure 12L).
  • Figures 13A-13S show NF-kB inducible secreted embryonic alkaline
  • FIG. 13 A shows dose response curves for increasing concentrations of pre-centrifugation articles: coated nanoparticles pre-centrifugation (Article A), coated nanoparticles with 11 : 1 trehalose: OEE formulation pre-centrifugation (Article D), nanoparticles with no OEE added pre- centrifugation (Article G), and TNFoc, which each correspond to averages of screenings 1 (Figure 13B), 2, ( Figure 13C), and 3 ( Figure 13D).
  • Figure 13E shows fold induction (ratio of average induced value to average non-induced value) of each of Articles A, D, G, and TNFoc at different concentrations of coated nanoparticles.
  • Figure 13F shows dose response curves for increasing concentrations of supernatant post-centrifugation of coated nanoparticles (Article B), supernatant post-centrifugation of coated nanoparticles with 11 : 1 trehalose: OEE formulation (Article E), supernatant post-centrifugation of un-coated nanoparticles (Article H), and TNFoc, which each correspond to averages of screenings 1 (Figure 13G), 2 ( Figure 13H), and 3 ( Figure 131).
  • Figure 13 J shows fold induction (ratio of average induced value to average non-induced value) of each of Articles B, E, H, and TNFoc at different concentrations of coated nanoparticles.
  • Figure 13K shows dose response curves for increasing concentrations of pellet from centrifugation of coated nanoparticles (Article C), pellet from centrifugation of coated nanoparticles with 11 : 1 trehalose:OEE formulation (Article F), pellet from centrifugation of un-coated nanoparticles (Article I), and TNFoc, which each correspond to averages of screenings 1 (Figure 13L), 2 ( Figure 13M), and 3 ( Figure 13N).
  • Figure 130 shows fold induction (ratio of average induced value to average non-induced value) of each of Articles C, F, I, and LPS-EK at different concentrations of coated nanoparticles.
  • Figure 13P shows a dose response curve of TNFoc: control HEK293/Null2 tested at eight concentrations.
  • Figure 13Q shows a dose response curve of TNFoc: control HEK293/Null2 at three different concentrations (corresponding to averages of screenings 1, 2, and 3 of Figure 13R).
  • Figures 13R and 13S show results of screenings 1, 2, 3, and fold induction.
  • Figures 14A-14L show NF-kB inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene expression in TLR4 HEK293/Null2 negative control cells.
  • Figure 14A shows dose response curves for increasing concentrations of Article A (pre- centrifugation; raw data and TNFoc fold change shown in Figure 14B), Article B (supernatant; raw data and TNFoc fold-change shown in Figure 14C), and Article C (pellet; raw data and TNF fold-change shown in Figure 14D).
  • SEAP embryonic alkaline phosphatase
  • Figure 14E shows dose response curves for increasing concentrations of nanoparticles with OEE, 11 :1 trehalose:OEE formulation pre-centrifugation of Article D (pre-centrifugation; raw data shown in Figure 14F), Article E (supernatant; raw data shown in Figure 14G), and Article F (pellet; raw data shown in Figure 14H).
  • Figure 141 shows dose response curves for increasing concentrations of nanoparticles with no OEE added, of Article G (pre-centrifugation; raw data shown in Figure 14J), Article H (supernatant; raw data shown in Figure 14K), and Article I (pellet; raw data shown in Figure 14L).
  • Figure 15 is a table showing an exemplary schedule of events in an exemplary clinical trial as described herein.
  • the present disclosure provides preparation of certain particles that may offer enhanced synthesis process, and/or consistent product quality as compared with other preparations.
  • disclosed preparations may offer different or unique properties that, for example, may address previously unmet requirements associated with production yield (e.g ., amount of waste).
  • provided preparations are characterized by more stable formation (e.g., can be stored longer), and/or other attributes relative to a standard preparation (e.g, using emulsions), as described herein.
  • the present disclosure is based, in part, on a surprising insight that desirable nanoparticle compositions can be prepared by the manufacturing processes described herein.
  • the present disclosure identifies one or more problems (e.g ., one or more sources of problems) in prior nanoparticle manufacturing technologies.
  • the present disclosure provides insights that permit preparation of nanoparticle compositions that comprise payloads (e.g. complex payloads) and/or coating agents (e.g., complex coating agents).
  • the present disclosure provides insights that permit preparation of nanoparticle compositions that incorporate two or more materials having different physicochemical properties (e.g., hydrophobic polymer and hydrophilic payloads).
  • Teachings provided by the present disclosure are particularly applicable to preparations of polymer nanoparticles. As discussed herein, those skilled in the art are aware of a variety of polymers that can be utilized in the preparation of nanoparticles, and of solvent systems that can be utilized to prepare appropriate solutions of such polymers.
  • Teachings provided by the present disclosure are particularly applicable to preparations of polymer nanoparticles that comprise a payload. As discussed herein, those skilled in the art are aware of a variety of payloads that can be included in the preparation of nanoparticles, and of solvent systems that can be utilized to prepare appropriate solutions of such payloads and/or polymers.
  • an initial combination e.g., solution
  • an initial polymer/payload solution is made from mixing a polymer solution and a payload solution as shown in, e.g., FIGS. 1 and 8A, steps 1-2.
  • an initial polymer/payload solution is made by dissolving dry polymer and dry payload in a solvent system (see, e.g. FIG. 1, steps 1-3).
  • a polymer solution is made by dissolving polymer (e.g., PLGA) into organic liquid (e.g.,
  • a payload solution is made by dissolving protein and DNA into water to produce an aqueous solution.
  • preparation of an aqueous solution also involves pH adjustment (e.g., using NaOH), and/or application of disruptive energy and/or force such as, e.g., sonication, and/or homogenization.
  • a payload solution is made by dissolving protein and DNA into water to produce an aqueous solution.
  • preparation of an aqueous solution also involves pH adjustment (e.g., using NaOH), and/or application of disruptive energy and/or force such as, e.g., sonication, and/or homogenization.
  • a pH adjustment e.g., using NaOH
  • disruptive energy and/or force such as, e.g., sonication, and/or homogenization.
  • a payload solution is made by dissolving protein and DNA into water to produce an aqueous solution.
  • preparation of an aqueous solution also involves pH adjustment (e.g.
  • polymer/payload solution is made by combining a payload (aqueous) and a polymer (organic) solution.
  • a polymer/payload solution is made by adding a payload (aqueous) solution into a polymer (organic) solution.
  • At least one polymer is present in a polymer/payload combination (e.g., solution) as described herein, at a concentration within a range of about 0.01 to 20 weight %, 0.1 to 20 weight %, 1.0 to 20 weight %, 0.01 to 15 weight %, 0.1 to 15 weight %, 1.0 to 15 weight%, 0.91 to 10 weight %, 0.1 to 10 weight%, 1.0 to 10 weight %, 0.01 to 1 weight %, 0.1 to 1 weight %, 1.0 to 5 weight %, 5 to 10 weight %, 5 to 15 weight %, or 5 to 20 weight % in an appropriate solvent system.
  • a polymer/payload combination e.g., solution
  • Payloads will commonly be present in such a solution at a concentration within a range of about 0.01 to 20 weight %, 0.1 to 20 weight %, 1.0 to 20 weight %, 0.01 to 15 weight %, 0.1 to 15 weight %, 1.0 to 15 weight%, 0.91 to 10 weight %, 0.1 to 10 weight%, 1.0 to 10 weight %, 0.01 to 1 weight %, 0.1 to 1 weight %, 1.0 to 5 weight %, 5 to 10 weight %, 5 to 15 weight %, or 5 to 20 weight % in an appropriate solvent system.
  • polymer and payload, and/or relative amounts thereof are selected so that, when processed, a payload is encapsulated within polymer matrix, distributed throughout and/or coated by polymer.
  • polymer and payload are present at a weight ratio within a range of 1 : 1 to 10 20 : 1 (e.g., 1 :99 to 20:80; 1 :99 to 10:90) in an initial polymer/payload solution. In some embodiments, polymer and payload are present at a weight ratio within a range of 50: 1 to 10 20 : 1 in an initial polymer/payload solution.
  • polymer and payload are present in an initial polymer/payload solution in relative amounts such that, when the solution is processed as described herein, they are present in a dry material as described herein and/or in a processed material as described herein, at a weight ratio of polymer to payload within a range of 1 : 1 to 10 20 : 1 by weight (e.g., 50: 1 to 10 20 : 1 by weight).
  • the present disclosure provides, in some embodiments, technologies that achieve sufficiently uniform combinations of polymer and payload in an initial polymer/payload solution that drying of the solution produces a material that has a substantially homogenous distribution of payload with respect to polymer.
  • technologies provided by the present disclosure achieve such uniform combination with or without application of disruptive energy or force (e.g ., sonication).
  • the present disclosure provides technologies that achieve a material comprising a combination of polymer and payload(s) that does not have a substantially homogenous distribution of payload with respect to polymer (e.g., before and/or after one or more post-combining steps) in an initial polymer/payload solution.
  • additional steps as further described herein may be employed to achieve a desirable distribution of payload with respect to polymer.
  • polymer/payload solution as described herein utilizes only a single solvent (e.g., when both polymer and payload are sufficiently soluble in the single solvent).
  • an initial polymer/payload solution that utilizes only a single solvent may include one or more additional components, for example, that may improve or facilitate solubilization of one or both of the polymer and the payload in the single solvent.
  • polymer/payload solution utilizes two or more solvents.
  • a solvent system comprising two or more solvents may be particularly useful when polymer and payload do not readily dissolve together in a single solvent.
  • a solvent system comprising two or more solvents may be useful when either a polymer is substantially hydrophobic (i.e., relatively insoluble in water or other aqueous media) and a payload is substantially hydrophilic, or vice versa.
  • Many embodiments exemplified or otherwise described herein utilize a substantially hydrophobic polymer and one or more substantially hydrophilic payloads.
  • an initial polymer/payload solution may include one or more other components in addition to polymer and payload.
  • an initial polymer/payload solution may include one or more emulsifiers, preservatives, solubilizers, surfactants, viscosity modifiers, salt, buffers (e.g., volatile buffers [e.g., ammonium bicarbonate]) etc.
  • buffers e.g., volatile buffers [e.g., ammonium bicarbonate]
  • An initial polymer/payload solution as described herein may be prepared, for example, by combining separate solutions of polymer and payload, by combining a solution of polymer with solid payload (or vice versa), or by solubilizing a dry material containing both polymer and payload.
  • solid (e.g., dry) polymer and/or payload are added to solvent system (e.g., to a premeasured amount of one or more solvents).
  • dry material is added slowly or in steps; in some embodiments, added dry material is permitted to solubilize substantially completely before a further addition of dry material is made.
  • stirring is performed (e.g., during dissolution of a solid material in a solvent system and/or during combination of two or more solvents or solutions). Stirring rate and/or time can be controlled.
  • stirring is performed with a mixer.
  • a mixer may be or comprise a stir bar or other device that, for example, utilizes an axial or radial flow impeller (e.g., a bar, paddle, or blade that may, for example, be magnetic), and/or any other impeller or propeller) to achieve mixing.
  • a mixer may be or comprise a magnetic stirrer, a turbine, or any electrical or mechanical impeller or propeller.
  • mixing is done in a high intensity mixing device, e.g. a
  • Microfluidizer for example, to ensure homogenization of polymer and payload.
  • substantially uniform distribution of payload into polymer matrix is an important step to ensure substantially uniform distribution of payload into a final nanoparticle formulation.
  • mixing is performed for one or more time periods (which may be consecutive and/or may have gaps between them).
  • a time period maybe approximately 5, 10, 15, 20, 25, 30, 40, 45, 50, 55, or 60 minutes, or longer.
  • a time period may be approximately 1, 2, 3, 4, 5, 10, 12, 15, 20, or 24 hours, or longer.
  • mixing is performed at a temperature within a range of about 15 °C to 30 °C (e.g., 15 -25°C, l5-20°C, or 20-30°C). In some embodiments, mixing is performed without application of heat from an external source. In some embodiments, mixing is performed without application of cooling from an external source. In some embodiments, mixing is performed under conditions in which temperature is controlled (e.g., external heat and/or cooling may be applied).
  • sedimentation e.g., centrifugation
  • a solution may be centrifuged to remove aggregated, undissolved and/or partially dissolved solid material.
  • an initial polymer/payload solution is characterized by certain material properties. In some embodiments, an initial polymer/payload solution is not turbid (e.g., is substantially transparent).
  • the present disclosure identifies a source of a problem that may be encountered with certain technologies that involve combining organic and aqueous solutions to achieve a homogenous combination.
  • the present disclosure provides methodologies (e.g., steps) that can mitigate one or more such identified sources of problem(s).
  • the present disclosure provides technologies for preparing substantially homogenous combinations of organic and aqueous materials as described herein, as well as the substantially homogenous compositions generated thereby. In some such
  • resultant compositions are substantially homogenous even if combinations of one or more precursors/components of, or one or more precursors/components used in the making thereof is/are not homogenous.
  • removal of water and/or other solvent(s) may decrease inconsistencies and/or increase homogeneity of an organic/aqueous (polymer/payload) combination as described herein before, during, and after subsequent lyophilization steps.
  • the present disclosure provides methodologies that include step(s) of removing water and/or other solvent(s) (e.g., by concentration) and/or increasing temperature, for example as part of generating a lyophilized cake.
  • the present disclosure provides substantially homogenous lyophilized cakes, for example as may be achieved by such methodologies.
  • a polymer/payload (e.g., an initial polymer/payload) combination e.g., solution
  • concentration of e.g., removal of a certain percentage of water and/or other solvent(s) or non-solvent(s) (e.g., using, e.g., evaporation, e.g., rotary evaporation) from a polymer/payload combination (e.g., an initial polymer/payload combination) may ultimately increase encapsulation of payload in polymer, in subsequent steps (e.g., microfluidization as in steps 6 and 7 of Figure 8 A).
  • concentration of a polymer/payload solution may be performed (e.g., via evaporation (e.g. rotary evaporation as illustrated in Figure 8A, between steps 3 and 4)) before an initial
  • polymer/payload solution is further manipulated such as, e.g., transformed into a different (e.g. solid, semi-solid) phase (see, e.g., steps 4-5 of Figures 1 and 8 A).
  • polymer/payload combination e.g., solution
  • polymer/payload combination e.g., solution
  • present disclosure encompasses the recognition that such improved homogeneity may facilitate, and may even be required for reasonable performance of additional production steps.
  • polymer/payload combination e.g., solution
  • a non-homogenous combination e.g., solution
  • steps e.g., lyophilization
  • steps may be added (e.g., solution concentration such as, e.g., by water and/or other solvent(s) evaporation step(s)) such that homogeneity (e.g., extent of mixing) is improved.
  • a polymer/payload combination (e.g., solution) is concentrated.
  • concentration may be applied to any polymer/payload combination (e.g., solution) (i.e., at any stage in a manufacturing process).
  • a polymer/payload combination (e.g., solution) is concentrated using evaporation methods.
  • an initial polymer/payload combination (e.g., solution) (see, e.g., Steps 1 and 2 of Figure 8 A) is concentrated using evaporation methods.
  • a utilized evaporation method is or comprises rotary evaporation.
  • concentration of a polymer/payload combination is concentrated for a time and under conditions sufficient to remove a certain percentage of water and/or other solvent(s) or non-solvent(s).
  • percentage of water and/or other solvent(s) or non-solvent(s) e.g., individually or in aggregate
  • percentage of water and/or other solvent(s) or non-solvent(s) removed from a combination is approximately 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • percentage of water and/or other solvent(s) removed is approximately 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more.
  • time of concentration e.g., length of time of water and/or other solvent(s) evaporation process(es), such as by rotary evaporation
  • concentration is performed for a time period of at least or about one or more of: 1 hour, 2 hours, 3 hours, 4, hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or more.
  • a polymer/payload combination as described herein may comprise a ratio of polymer/payload may be expressed as about 0.01 to 10 weight %, 0.1 to 10 weight %, 1 to 10 weight %, 0.01 to 1 weight %, 0.1 to 1 weight %, or 1 to 5 weight % relative to one another.
  • a polymer: payload ratio in a polymer/payload combination e.g., solution
  • concentration may be expressed as a ratio, for example, as within a range of about 90: 10 and 100:0.
  • concentration may be applied to a polymer/payload solution having a polymer: payload ratio within a range of about 90:10 to about 97:3. In some embodiments, concentration may achieve a polymer: payload ratio within a range of about 98:2 to 99: 1.
  • a polymer: payload ratio is greater than about 40:60, greater than about 50:50, greater than about 60:40, greater than about 70:30, greater than about 80:20, greater than about 90: 10, or greater than about 95:5 or more, prior to concentration. In some embodiments, a polymer: payload ratio is greater than about 50:50, greater than about 60:40, greater than about 70:30, greater than about 80:20, greater than about 90: 10, greater than about 95:5, greater than about 99: 1, greater than about 99.5:0.5, or more after concentration.
  • a polymer: payload ratio in a polymer/payload combination is greater than about 90: 10 and less than 100:0 prior to concentration (e.g., evaporation, e.g., rotary evaporation) and greater than about 95:5 after concentration (e.g., evaporation, e.g., rotary evaporation).
  • a polymer: payload ratio in a polymer/payload combination is greater than about 50:50 and less than 100:0 prior to concentration (e.g., evaporation, e.g., rotary evaporation) and greater than about 50:50 after concentration (e.g., evaporation, e.g., rotary evaporation).
  • a polymer: payload ratio is about 96:4 prior to concentration (e.g., evaporation, e.g., rotary evaporation) and about 99: 1 after concentration (e.g., evaporation, e.g., rotary evaporation).
  • concentration is achieved, at least in part, by water evaporation. In some such embodiments, approximately 50% to approximately 75% of water is removed by concentration (e.g., after evaporation, e.g., after rotary evaporation), relative to water in the polymer/payload combination (e.g., solution) prior to concentration.
  • concentration is achieved by solvent or non-solvent (i.e., not water) evaporation.
  • solvent or non-solvent i.e., not water
  • approximately 50% to approximately 75% of solvent is removed by concentration (e.g., after evaporation, e.g., after rotary evaporation), relative to solvent in polymer/payload combination (e.g., solution) prior to concentration.
  • conditions under which concentration e.g., water and/or other solvent or non-solvent evaporation, such as by rotary evaporation
  • concentration e.g., water and/or other solvent or non-solvent evaporation, such as by rotary evaporation
  • concentration techniques e.g., pressure, temperature, time
  • concentration may be performed at variable temperatures.
  • concentration e.g., evaporation, e.g., rotary evaporation
  • concentration may be performed at temperatures between 20 0 C and 120 0 C.
  • concentration may involve rotation at a particular speed or speeds.
  • a plurality of distinct speeds e.g., variable speed
  • speeds may vary between approximately 40 rpm and 100 rpm.
  • speeds may vary between approximately 50 rpm and 90 rpm.
  • speeds may vary between approximately 60 rpm and 80 rpm.
  • concentration may be performed at a particular pressure or pressures (e.g., approximately 50 mbar - 250 mbar). In some such embodiments pressure may vary between approximately 50 mbar and 175 mbar. In some embodiments, pressure may vary between approximately 50 mbar and 150 mbar. In some embodiments, pressure may vary between approximately 75 mbar and 150 mbar.
  • concentration e.g., by water evaporation such as by rotary evaporation
  • concentration results in increased encapsulation of protein in the polymer.
  • manufacturing of nanoparticles involves one or more concentration steps before or after one or more lyophilization steps.
  • concentration is performed prior to and/or following one or more lyophilization steps.
  • concentration is not performed prior to and/or following one or more lyophilization steps.
  • concentration may replace, in whole or part, one or more lyophilization steps.
  • concentration e.g., by water evaporation such as by rotary evaporation
  • concentration may be used to remove water and solvent from an initial polymer/payload combination (e.g., solution) before placing the combination (e.g., solution) into a microfluidization system.
  • a lyophilization step may be performed, follow by grinding a lyophilization product and resuspending in a liquid (e.g., to make a combination, e.g., solution) before placing into a microfluidizer.
  • concentration e.g., by water evaporation such as by rotary evaporation
  • concentration may follow microfluidization in order to remove one or more components (e.g., solvent (e.g., propanol)) used during production of nanoparticles in the microfluidizer.
  • solvent e.g., propanol
  • a dry material that includes both polymer and payload (and optionally any additional components that may have been included in an initial polymer/payload solution) is prepared from an initial polymer/payload combination (e.g., solution) as described herein.
  • an initial polymer/payload combination e.g., solution
  • drying e.g., freeze-drying or other drying strategy
  • drying is performed at a temperature within a range of -
  • drying is performed under conditions in which temperature is controlled (e.g., external heat and/or cooling may be applied). In some embodiments, drying is performed with application of cooling or heat from an external source. In some embodiments, drying is performed without application of cooling or heat from an external source.
  • drying is performed at a pressure within a range of 10 8 to
  • drying is performed under conditions in which pressure is controlled (e.g., using an external pressure controller).
  • drying is performed under inert gas (e.g., N 2 , Ar).
  • drying is performed under atmospheric conditions.
  • a material is considered to be dry when a solvent content is lower than 1 weight % (e.g., approximately 0 weight %). In some embodiments, a material is considered to be dry when a glass temperature of the material is above room temperature (e.g., above 25°C) , and preferably above 30°C. In some embodiments, where high boiling solvents such as DMSO are used, it may not be required to remove all residual solvent.
  • one or more steps or measures may be taken to reduce or substantially eliminate aggregation of payload within a polymer matrix. For example, in some embodiments, selecting a polymer with an adequately high glass transition temperature may be sufficient to ensure that the payload does not diffuse and aggregate within the polymer matrix.
  • a dry or glassy material e.g., polymer/payload cake, for example, a lyophilized polymer/payload cake
  • a payload is substantially homogenous with respect to polymer.
  • a dry material is sufficiently brittle so that the dry material can be milled.
  • a polymer/payload cake may optionally be processed as described herein, to generate a processed material.
  • processing e.g., heating
  • processing of a polymer/payload cake may remove one or more voids in the polymer/payload cake, and/or assist polymer being in proximity of payload.
  • processing of a polymer/payload cake may reduce porosity.
  • processing of a polymer/payload cake may decrease the volume of the
  • optional processing of polymer/payload cake comprises heating.
  • a polymer/payload cake is heated to a temperature that exceeds the glass temperature of the polymer in the polymer/payload cake.
  • a polymer/payload cake is heated to a temperature within a range of about 70 °C to 150 °C.
  • a polymer/payload cake is heated to a temperature within a range of about 80 °C to 150 °C.
  • a polymer/payload cake is heated to a temperature within a range of about 90 °C to 150 °C.
  • a polymer/payload cake is heated to a temperature within a range of about 95 °C to 150 °C. In some embodiments, a polymer/payload cake is heated to a temperature within a range of about 100 °C to 150 °C. In some embodiments, a polymer/payload cake is heated for period of time within a range of approximately 0.1 to 10 mins. In some embodiments, a polymer/payload cake is heated to a temperature within 5°C (e.g., within 4°C, 3°C, 2°C, l°C) of the glass transition temperature of the polymer.
  • heating is performed at a pressure within a range of 10 8 to
  • polymer in a polymer/payload cake is melted during processing (e.g., heating).
  • morphology and/or configuration of polymer in a polymer/payload cake may be changed during processing (e.g, heating).
  • heated polymer may wrap payload.
  • a polymer/payload cake may become homogeneous (e.g, payload is evenly distributed in polymer) during processing (e.g, heating).
  • optional processing of polymer/payload cake comprises cooling (e.g, after heating).
  • a polymer/payload cake is cooled to room temperature.
  • a polymer/payload cake is cooled to a temperature within a range of about -15 to 40 °C or lower.
  • a polymer/payload cake is cooled to a temperature within a range of about -15 to 35 °C.
  • a polymer/payload cake is cooled to a temperature within a range of about -15 to 30 °C.
  • a polymer/payload cake is cooled to a temperature within a range of about -15 to 25 °C.
  • a polymer/payload cake is cooled to a temperature within a range of about -15 to 20 °C. In some embodiments, a polymer/payload cake is cooled to a temperature within a range of about -15 to 15 °C. In some embodiments, a polymer/payload cake is cooled to a temperature within a range of about -15 to 10 °C.
  • cooling is performed at a pressure within a range of 10 8 to
  • a processed polymer/payload cake as described herein is characterized in that a payload is distributed in a substantially homogenous manner with respect to polymer.
  • provided technologies include a step of generating microparticles from a dried and/or lyophilized polymer/payload cake, or a processed
  • a dried and/or lyophilized polymer/payload cake, or a processed polymer/payload cake is comminuted.
  • a dried and/or lyophilized polymer/payload cake, or a processed polymer/payload cake is pulverized using mortar and pestle, grinding machines (e.g ., ball mill, rod mill, autogenous mill, semi-autogenous mill, pebble mill, high pressure grinding rolls, buhrstone mill, vertical shaft impactor mill, tower mill) or combinations thereof.
  • a dried and/or lyophilized polymer/payload cake, or a processed polymer/payload cake may be cooled prior to and/or during comminuting to reduce condensation.
  • a solvent e.g., alcohol (e.g, propanol)
  • granulation may be performed under a dry condition that may reduce condensation.
  • a dried and/or lyophilized polymer/payload cake, or a processed polymer/payload cake is cooled to a temperature within a range of -40 to 0 °C prior to grinding.
  • dry ice and/or liquid nitrogen may be provided to cool a dried and/or lyophilized polymer/payload cake, or a processed polymer/payload cake.
  • temperatures may be adjusted to accommodate materials, such as, e.g., cooling materials (liquid nitrogen).
  • a material temperature during freezing with liquid nitrogen may be lower than -40 °C, to a temperature somewhere above a range of approximately -210 °C to approximately -196 °C.
  • grinding is performed within the same range as temperature preceding a grinding step (e.g., freezing with liquid nitrogen).
  • grinding is performed at a different temperature than a preceding freezing step.
  • a grinding process is performed at a temperature within a range of about 210 to about -l96°C, about -175 to 0 °C, about -150 to 0 °C, about -125 to 0 °C, about -100 to 0 °C, about -75 to 0 °C, about -50 to 0 °C, about -30 to 0 °C, about 0 to 20°C, about 0 to l5°C, about 0 to 5°C, or about 5 to l5°C.
  • granulated microparticles e.g., comminuted
  • polymer/payload material have a dimension/size (e.g, average or mean diameter) within a range of about 1 pm to 1000 pm.
  • granulated microparticles have a size (e.g, average or mean diameter) within a range of about 1 pm to 800 pm.
  • granulated microparticles have a size (e.g, average or mean diameter) within a range of about 1 pm to 500 pm.
  • granulated microparticles e.g, comminuted
  • polymer/payload material have a size (e.g, average or mean diameter) within a range of about 1 pm to 100 pm.
  • granulated microparticles e.g, comminuted
  • polymer/payload material have a size (e.g, average or mean diameter) within a range of about 1 pm to 50 pm.
  • granulated microparticles as described herein are characterized as having a substantially uniform distribution of payload with respect to polymer.
  • microparticles as described herein are characterized as not having a substantially uniform distribution of payload with respect to polymer.
  • one or more populations of granulated microparticles is present.
  • a particular population of microparticles comprises a disproportionate amount of payload with respect to polymer versus another population of microparticles.
  • provided technologies include methods for creating a flowable microparticle suspension from granulated microparticles (e.g ., comprised of polymer and payload).
  • a liquid system is added to granulated microparticles ( e.g ., comprising polymer and payload) to form a flowable microparticle suspension.
  • a liquid system for a flowable microparticle suspension is substantially in accordance with several embodiments as described herein.
  • a liquid system for a flowable microparticle suspension may be a non-solvent for the microparticle matrix (e.g., polymer) and/or a payload.
  • the solvent system may be a non-solvent for the polymer matrix, and either a solvent or partial solvent for a payload miscible with water.
  • a liquid system for a flowable microparticle suspension is selected from the group consisting of propanol, ethanol, methanol, and
  • granulated microparticles are present in a suspension with a concentration within a range of about 1 to 20 mg/mL (e.g., 2 to 20 mg/mL, 5 to 20 mg/mL, 10 to 20 mg/mL, 1 to 15 mg/mL, 1 to 10 mg/mL, 1 to 5 mg/mL, or 5 to 15 mg/mL).
  • a solvent system for a flowable microparticle suspension is or comprises propanol.
  • Propanol has a relatively high molecular weight (e.g., among alcohols which are miscible with water).
  • propanol may have a high enough boiling point such that it can accommodate heat during a homogenization process (e.g, described herein) without boiling.
  • a solvent system for a flowable microparticle suspension further comprises one or more agents to assist in dissolution of protein and/or DNA and/or to improve homogeneity of suspension components.
  • a solvent system further comprises one or more detergents and/or surfactants.
  • solvent systems e.g, non-solvent system of microparticles, e.g, propanol
  • a non-solvent system can minimize dissolution of polymer and/or payload during homogenization of a flowable microparticle suspension.
  • a flowable microparticle suspension is created by adding ground microparticles as described herein to propanol.
  • an initial propanol concentration is higher than a final propanol concentration.
  • an initial n-propanol concentration in an initial flowable microparticle suspension is approximately 5-10 mg/mL.
  • a concentration of n-propanol is reduced when a flowable microparticle/propanol suspension is added to a homogenizer already having hot propanol inside. In some such embodiments, concentration of propanol may be reduced to approximately 2-6 mg/mL.
  • a starting concentration of n- propanol is approximately 7 mg/mL, diluted down to a final concentration of about 3.25 mg/mL after addition to hot propanol in a homogenizer.
  • a final concentration of n-propanol may be within a range of approximately 0.25 to 20 mg/mL.
  • a final concentration of n-propanol may be within a range of approximately 0.3 - 15 mg/mL.
  • a final concentration of n-propanol may be within a range of approximately 0.5-10 mg/mL.
  • provided technologies include methods of making nanoparticles from a flowable microparticle suspension (e.g, homogenization).
  • homogenization is performed by blender, bead mills, sonicator, rotor-stator mechanical homogenizer, microfluidizer, or combinations thereof.
  • a flowable microparticle suspension may be homogenized by a microfluidizer.
  • a microfluidizer converts a high fluid pressure into a shear force applied to a suspension.
  • a pump of a microfluidizer drives a suspension at constant pressure through a chamber.
  • the suspension may be accelerated to a high velocity, as it goes through a fixed-geometry microchannel, creating high shear force.
  • a shear force applied to a suspension may produce homogenized particles.
  • a microfluidizer applies a shear gradient within a range of about 10 '5 to 10 '8 , 10 '6 to 10 '8 , 10 '5 to 10 '7 or 10 '6 to lO ' V 1 to a flowable microparticle suspension.
  • a flowable microparticle (e.g., comprising polymer and payload) suspension is homogenized into nanoparticles at a temperature above the glass transition temperature of a polymer. In some embodiments, a flowable microparticle suspension is homogenized at a temperature within a range of about 80 °C to 110 °C. In some
  • a flowable microparticle suspension is homogenized at a temperature within a range of about 85 °C to 110 °C. In some embodiments, a flowable microparticle suspension is homogenized at a temperature within a range of about 90 °C to 110 °C. In some embodiments, a flowable microparticle suspension is homogenized at a temperature within a range of about 80 °C to 105 °C. In some embodiments, a flowable microparticle suspension is homogenized at a temperature within a range of about 80 °C to 100 °C. In some embodiments, a flowable microparticle suspension is homogenized at a temperature within a range of about 90 °C to 100 °C. In some embodiments, a flowable microparticle suspension is homogenized at a temperature of about 95 °C.
  • a flowable microparticle suspension may be passed through a homogenizer any number of times, as is appropriate for a particular application. For example, in some embodiments, a flowable microparticle suspension passes through a homogenizer once. In some embodiments, a flowable microparticle suspension passes through a homogenizer twice. In some embodiments, a flowable microparticle suspension passes through a homogenizer three times. In some embodiments, a flowable microparticle suspension passes through a homogenizer four, five, six, seven, eight, nine or ten times. In some
  • a flowable microparticle suspension passes through a homogenizer between ten and twenty times. In some embodiments, a flowable microparticle suspension passes through a homogenizer between ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty or more times.
  • a solution/suspension of homogenized nanoparticles is stabilized using one or more additives (e.g., one or more liquid or powder additives to, e.g., stabilize a combination comprising nanoparticles).
  • one or more additives e.g., one or more liquid or powder additives to, e.g., stabilize a combination comprising nanoparticles.
  • a solution/suspension of homogenized nanoparticles is stabilized to prevent homogenized nanoparticles from agglomeration.
  • dilution may stabilize homogenized nanoparticles. Without wishing to be held to a particular theory, it is contemplated that dilution (e.g., through use of a diluting solvent or non-solvent system) would dilute a combination comprising homogenized nanoparticles, thereby reducing agglomeration.
  • a diluting solvent system may be or comprise a non solvent of polymer.
  • a diluting solvent system is miscible with at least one of water and DMSO.
  • a diluting solvent system is the same as or comprises the original solvent used for nanoparticle formulation.
  • a diluting solvent system is at a temperature within a range of 0 °C to 40 °C, 0 °C to 30 °C, 0 °C to 35 °C, 0 °C to 30 °C, 0 °C to 25 °C, 5 °C to 40 °C, 10 °C to 40 °C, 15 °C to 40 °C, 20 °C to 40 °C, 10 °C to 30 °C, 20 °C to 30 °C, or 15 °C to 25 °C, when it is added to a solution/suspension of homogenized nanoparticles.
  • a stabilizing agent may be or comprise a surfactant based on sugar units, or polyethylene glycol units, or ionic units, or combinations thereof.
  • the hydrophobic units of the surfactant will be alkane or alkyene units.
  • the surfactants may be biologically sourced or synthetic. An example of a biologically based surfactant would be tocopherol units derivatized with polyethylene oxide units. In some embodiments amphiphilic copolymers may be used.
  • Exemplary surfactants would include ionic surfactants (e.g., sodium dodecyl sulfate, cetrimonium bromide, etc.), sugar based surfactants such as TWEEN® or SPAN® , and combinations thereof.
  • a stabilizing agent may be or comprise an amphiphilic copolymer (i.e., a copolymer of a hydrophilic block coupled with a hydrophobic block).
  • an amphiphilic copolymer i.e., a copolymer of a hydrophilic block coupled with a hydrophobic block.
  • nanoparticles formed by the process of the present disclosure can be formed with graft, block or random amphiphilic copolymers. These copolymers can have a molecular weight between 1,000 g/mole and 50,000 g/mole or more, or between about 3,000 g/mole to about 25,000 g/mole, or at least 2,000 g/mole.
  • Suitable hydrophobic blocks in an amphiphilic copolymer include but are not limited to the following: acrylates including methyl acrylate, ethyl acrylate, propyl acrylate, n-butyl acrylate (BA), isobutyl acrylate, 2-ethyl acrylate, and t-butyl acrylate;
  • methacrylates including ethyl methacrylate, n-butyl methacrylate, and isobutyl methacrylate; acrylonitriles; methacrylonitrile; vinyls including vinyl acetate, vinylversatate, vinylpropionate, vinylformamide, vinylacetamide, vinylpyridines, and vinylimidazole; aminoalkyls including aminoalkylacrylates, aminoalkylmethacrylates, and aminoalkyl(meth)acrylamides; styrenes; cellulose acetate phthalate, cellulose acetate succinate, hydroxypropylmethylcellulose phthalate, poly(D,L lactide), poly (D,L-lactide-co-glycolide), poly(glycolide), poly(hydroxybutyrate), poly(alkylcarbonate) and poly(orthoesters), polyesters, poly(hydroxyvaleric acid),
  • polydioxanone poly(ethylene terephthalate), poly(malic acid), poly(tartronic acid),
  • polyanhydrides polyphosphazenes, poly(amino acids) and their copolymers (see generally,
  • hydrophobic peptide-based polymers and copolymers based on poly(L-amino acids) (Lavasanifar, A., et al, Advanced Drug Delivery Reviews (2002) 54: 169-190), poly(ethylene-vinyl acetate) (“EVA”) copolymers, silicone rubber, polyethylene, polypropylene, polydienes (polybutadiene, polyisoprene and hydrogenated forms of these polymers), maleic anhydride copolymers of vinyl methylether and other vinyl ethers, polyamides (nylon 6,6), polyurethane, poly(ester urethanes), poly(ether urethanes), polyester- urea).
  • Particularly preferred polymeric blocks include poly(ethylenevinyl acetate), poly
  • (D,L-lactic acid) oligomers and polymers poly (L-lactic acid) oligomers and polymers, poly (glycolic acid), copolymers of lactic acid and glycolic acid, poly (caprolactone), poly
  • particularly preferred polymeric blocks include polystyrene, polyacrylates, and butadienes.
  • suitable hydrophilic blocks in an amphiphilic copolymer include but are not limited to the following: carboxylic acids including acrylic acid, methacrylic acid, itaconic acid, and maleic acid; polyoxyethylenes or poly ethylene oxide; polyacrylamides and copolymers thereof with dimethylaminoethylmethacrylate, diallyldimethylammonium chloride, vinylbenzylthrimethylammonium chloride, acrylic acid, methacrylic acid, 2-acrylamido-2- methylpropane sulfonic acid and styrene sulfonate, polyvinyl pyrrolidone, starches and starch derivatives, dextran and dextran derivatives; polypeptides, such as polylysines, polyarginines, polyglutamic acids; poly hyaluronic acids, alginic acids, polylactides, polyethyleneimines, polyionenes, polyacrylic acids, and polyiminocarboxylates,
  • blocks of a particular copolymer may be either diblock or triblock repeats.
  • block copolymers include blocks of polystyrene, polyethylene, polybutyl acrylate, polybutyl methacrylate, polylactic acid, polycaprolactone, polyacrylic acid, polyoxyethylene and polyacrylamide.
  • suitable hydrophilic polymers compatible with some embodiments can be found in Handbook of Water-Soluble Gums and Resins. R. Davidson, McGraw-Hill (1980).
  • the length of a grafted moiety can vary.
  • the grafted segments are alkyl chains of 12 to 32 carbons or equivalent to 6 to 16 ethylene units in length.
  • the grafting of the polymer backbone can be useful to enhance solvation or nanoparticle stabilization properties.
  • a grafted butyl group on the hydrophobic backbone of a diblock copolymer of a polyethylene and polyethylene glycol may increase the solubility of the polyethylene block.
  • suitable chemical moieties grafted to the block unit of the copolymer comprise alkyl chains containing species such as amides, imides, phenyl, carboxy, aldehyde or alcohol groups.
  • a commercially available stabilizer is the Hypermer family marketed by Uniqema Co.
  • an amphiphilic stabilizer could also be of the gelatin family such as the gelatins derived from animal or fish collagen.
  • a stabilizing agent may be used to reduce agglomeration of homogenized nanoparticles.
  • surfaces of homogenized nanoparticles may be modified by a stabilizing agent.
  • a stabilizing agent may be or comprise a poloxamer, or small ionic surfactant.
  • a stabilizing agent is selected from the group consisting of polyvinyl alcohol (PVA1), ionic surfactants (e.g., sodium dodecyl sulfate, cetrimonium bromide, etc.), and combinations thereof.
  • PVA1 polyvinyl alcohol
  • ionic surfactants e.g., sodium dodecyl sulfate, cetrimonium bromide, etc.
  • a stabilizing agent solution is added to a solution/suspension of homogenized nanoparticles.
  • a solvent system of a stabilizing agent solution may comprise water. In some embodiments, a solvent system of a stabilizing agent solution is same as a diluting solvent system.
  • a stabilizing agent e.g., PVA1
  • a stabilizing agent is present in a solution at mass ratio within a range of about 10: 1 to 1 : 10, relative to the mass of one or more components of a given nanoparticle composition (e.g., polymer, protein, DNA, etc.) in the solution.
  • a stabilizing agent e.g., PVA1
  • a stabilizing agent is present in a solution at mass ratio within a range of about 10: 1 to 1 : 10, relative to the mass of nanoparticles in the solution.
  • a stabilizing solution is at a temperature within a range of
  • 0°C to 40°C 0°C to 30°C, 0°C to 35°C, 0°C to 30°C, 0°C to 25°C, 5°C to 40°C, l0°C to 40°C, l5°C to 40°C, 20°C to 40°C, l0°C to 30°C, 20°C to 30°C, or l5°C to 25°C, when it is added to a nanoparticle suspension (e.g., a substantially homogenized nanoparticle suspension).
  • a nanoparticle suspension e.g., a substantially homogenized nanoparticle suspension
  • an aqueous PVA1 solution is added to a homogenized nanoparticle suspension to reduce aggregation of nanoparticles. In some embodiments, no PVA1 is added.
  • aqueous PVA1 solution and nanoparticle suspension are mixed for about 10 to 45 mins (e.g., approximately 10, 20, 30, or 40 minutes).
  • one or more solutes or solvents is added to a combination
  • nanoparticles are stabilized when one or more such solutes or solvents is/are present.
  • nanoparticles are stabilized by addition of polyvinyl alcohol (PVA1) in water to the solution of nanoparticles in hot propanol.
  • PVA1 in water is added to the nanoparticle containing solution, and the solution is cooled to room temperature before proceeding to any further steps
  • a stabilized nanoparticle suspension e.g., comprising polymer/payload nanoparticles with a stabilizing agent
  • a microfluidizer is further homogenized (e.g., by a microfluidizer).
  • a stabilized nanoparticle suspension passes through a homogenizer between one and twenty, or more, times.
  • a stabilized nanoparticle suspension passes through a homogenizer one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more times.
  • a homogenizer does not supply heat while homogenizing the stabilized nanoparticle suspension.
  • a homogenizer is cooled (e.g., actively cooled) while homogenizing the stabilized nanoparticle suspension.
  • polymeric nanoparticles comprising protein and DNA are loaded with protein/DNA (protein/DNA is encapsulated in polymer), as compared to polymeric nanoparticles without any protein and/or DNA (polymer without encapsulated protein/DNA).
  • a composition is a combination comprising polymeric nanoparticles loaded with protein and DNA.
  • a provided composition is or comprises a combination as described herein comprising loaded nanoparticles, PVA1, water and propanol, with a certain ratio of water to propanol present after microfluidic processing.
  • percent of water is in a range between about 50% to about 99% of the combination.
  • a ratio of propanol to water e.g., volume: volume, e.g., volume: volume determined before combining, e.g., propanol, water, and/or other components
  • nanoparticles is in a range of approximately 1 :99 to 99: 1.
  • a ratio of water to propanol is 10:90 to 30:70.
  • a ratio of water to propanol is 50:50.
  • a ratio of water to propanol is approximately 75:25.
  • a ratio of water to propanol is approximately 85: 15.
  • a ratio of water to propanol is approximately 80:20.
  • the present disclosure provides the insight that manufacturing protocols as described herein may produce one or more populations of nanoparticles.
  • the term“population” refers to a group of nanoparticles sharing a particular characteristic (e.g., size, payload, payload concentration, coating agent, amount of coating agent, etc).
  • a population of nanoparticles may have a mean size of between
  • nm approximately 100-500 nm (e.g., mean average size of, e.g., 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm).
  • different populations of nanoparticles are represented by different sizes (e.g., mean size, e.g., mean range of
  • a population of nanoparticles is represented by a particular mean size (e.g., 300 nm), but is itself comprised of more than one population of nanoparticles (e.g., a population with a mean size of 200 nm and another population with a mean size of 400 nm).
  • payload encapsulation results in one or more populations of nanoparticles, e.g., one or more sets of sizes, e.g., one or more of nanoparticles with higher encapsulation percentages than other sets of nanoparticles.
  • certain larger nanoparticles e.g., greater than 400-500 nm mean size
  • are less concentrated in protein payload relative to smaller nanoparticles e.g., smaller than 500 nm mean size.
  • nanoparticles between 100-500 nm in at least one dimension comprise
  • nanoparticles between 100-500 nm in at least one dimension comprise approximately 30-90 mg protein/mg PLGA. In some such embodiments, nanoparticles between 100-500 nm in at least one dimension comprise approximately 50-75 mg protein/mg PLGA. [0228] In some embodiments, nanoparticles larger than approximately 300 nm (and smaller than approximately 500 nm) comprise approximately 5-20 pg protein/mg PLGA.
  • At least two populations of nanoparticles are producing during a single manufacturing process.
  • At least two populations of nanoparticles are produced in separate manufacturing processes.
  • purification procedures are altered to selectively eliminate and/or selectively enrich for a particular population of nanoparticles.
  • encapsulation (relative to 100% of starting protein amount at, e.g., step 1 of Figure 8A) is between approximately 10-95%. In some embodiments, encapsulation of protein is
  • encapsulation of protein is approximately 10-20%. In some embodiments, encapsulation of protein is approximately 20- 30%. In some embodiments, encapsulation of protein is approximately 30-40%. In some embodiments, encapsulation of protein is approximately 40-50%. In some embodiments, encapsulation of protein is approximately 50-90%. In some embodiments, encapsulation of protein is approximately 60-90%. In some embodiments, encapsulation of protein is
  • compositions as described herein may include one or more additional components not specifically named in the description above.
  • additional components may comprise one or more dissolution aids, emulsifiers, preservatives, solubilizers, surfactants, viscosity modifiers, salts, sugars, buffers, etc. It will be understood by those of skill in the art that any additional components may desirably be modified to maintain a particular composition or portion thereof.
  • an additional component may naturally occur in a crystalline form that is not particularly compatible with a nanoparticle formulation.
  • one of skill in the art will recognize and know how to modify such a component (e.g., by obtaining a more granulated form, or by using processing methods, such as, e.g., lyophilization of an aqueous solution containing the component), to make the component more amenable to a particular nanoparticle formulation as described herein.
  • processing methods such as, e.g., lyophilization of an aqueous solution containing the component
  • a dissolution aid may be added to a nanoparticle suspension (e.g., comprising nanoparticles coated with a stabilizing agent).
  • a nanoparticle suspension e.g., comprising nanoparticles coated with a stabilizing agent.
  • a dissolution aid is selected from the group consisting of sugars (e.g., trehalose, mannitol, lactose, glucose), hydrophilic polymers (e.g., polyethylene glycol,
  • polyvinylpyrrolidone polyvinylpyrrolidone vinyl acetate copolymer
  • a dissolution aid may be pre-processed in order to facilitate incorporation into a suspension and/or production of a product for use in a
  • trehalose granules are crystalline and larger than nanoparticles of suspensions disclosed herein. In some such embodiments, it is
  • micronized trehalose is used anywhere that trehalose or an equivalent is used, in accordance with the present disclosure. In some such embodiments, if micronized trehalose is not available, trehalose is mixed with water, lyophilized and ground to produce a micronized equivalent of trehalose.
  • a weight ratio of a dissolution aid to polymer is within a range of about 20:0.5 to 0.5:20, 15:5 to 5: 15, 11 : 1 to 1 : 11, 7: 1 to 1 :7, 5: 1 to 1 :5, 5: 1 to 1 : 1, or 3 : 1 to 1 : 1.
  • Post-yrocessins nanoparticles are within a range of about 20:0.5 to 0.5:20, 15:5 to 5: 15, 11 : 1 to 1 : 11, 7: 1 to 1 :7, 5: 1 to 1 :5, 5: 1 to 1 : 1, or 3 : 1 to 1 : 1.
  • provided methods further include a post-processing step applicable to provided nanoparticles.
  • certain post-processing parameters and/or procedures may be altered in order to accommodate conditions such as, e.g., materials used, and/or scale of processing (e.g., in larger scale processing different parameters may be desirable).
  • the present disclosure provides the insight that post-processing may reduce a burst rate (e.g., payload amount released in first 15 minutes, when nanoparticles are exposed to a physiological condition).
  • a burst rate e.g., payload amount released in first 15 minutes, when nanoparticles are exposed to a physiological condition.
  • certain processing steps may result in improved encapsulation and/or yield/retention of nanoparticles, as well as improved safety factor of solutions comprising loaded nanoparticles.
  • a post-processing step may remove a payload that is weakly associated with a nanoparticle and/or a payload associated with and/or exposed to an outer surface of a nanoparticle (e.g. free payload).
  • the present disclosure also provides the insight that effective post-processing of nanoparticles may minimize waste.
  • post-processing steps may change (e.g., increase or decrease) recovery of solids during a nanoparticle manufacturing procedure.
  • post-processing may comprise one or more centrifugation steps.
  • two or more centrifugation steps may be performed (e.g., in a serial manner).
  • two or more centrifugation steps are performed with one or more optional steps in between centrifugations.
  • each centrifugation step may be at the same or different speed, same or different temperature and/or for the same or different amount of time.
  • one or more low speed centrifugation step(s) is/are performed using a nanoparticle solution.
  • low speed centrifugation may aid in collecting (e.g., into a pellet) large particles which are considered undesirable for a final nanoparticle composition (e.g., large polymer particles with low protein encapsulation).
  • a low speed centrifugation step is within a range of approximately 750 xg, 700 xg, 600 xg, 500 xg, 400 xg, 300 xg, 200 xg, 100 xg, or less.
  • a low speed centrifugation step is performed within a temperature ranges of approximately 4 °C to approximately 37 °C. In some embodiments, a low speed centrifugation step is approximately 15 mins to approximately 20 hours or more in duration. In some embodiments, a low speed centrifugation step is
  • one or more additional centrifugation steps may be performed using supernatant from an initial low-speed centrifugation step. It is contemplated that subsequent centrifugation steps, following an initial low-speed centrifugation step, will further pellet any residual large particles and facilitate collection and removal. In some embodiments, one or more subsequent low speed centrifugation steps is/are performed for at speeds of approximately 100-750 xg, for approximately 15 minutes to 20 hours at approximately 4-37 °C.
  • centrifugation is insufficient to fully separate and/or collect desired nanoparticle populations (e.g., nanoparticles in a range of 100-500 nm mean size, e.g., 100-300 nm, etc.), thus, additional purification steps (e.g., tangential flow filtration) may be performed to collect smaller (e.g., 100-500 nm mean size, e.g., 100-300 nm, etc.) nanoparticles.
  • additional purification steps e.g., tangential flow filtration
  • one or more additional processing, purification, and/or separation methods e.g., lyophilization, filtration, centrifugation, tangential flow filtration, protease digestion, ion exchange and use of other resins
  • one or more purification and/or separation methods may be performed prior to an initial or subsequent (relative to initial) low speed centrifugation step.
  • an intermediate speed centrifugation step is performed alone, or in addition to (i.e., before or after) another centrifugation step in the same or different (e.g., low speed or high speed) range, on a nanoparticle solution to pellet the desired
  • an intermediate speed centrifugation step is performed after a low speed centrifugation step.
  • one or more purification and/or separation methods e.g., filtration, centrifugation, tangential flow filtration, protease digestion, ion exchange and use of other resins are performed prior to or following an intermediate speed centrifugation step.
  • an intermediate speed centrifugation step is performed at speeds of approximately 750 xg - approximately 7500 x g.
  • an intermediate speed centrifugation step is performed at speeds of approximately 1000 xg, 1500 xg, 2000 xg, 2500 xg, 3000 xg, 3500 xg, 4000 xg, 4500 xg, 5000 xg, 5500 xg, 60000 xg, 6500 xg, 7000 xg, or 7500 x g.
  • an intermediate speed centrifugation step is performed at temperature ranges of approximately 4-37 °C.
  • one or more additional centrifuge steps are performed on a given supernatant to further pellet nanoparticles present in the solution.
  • an intermediate speed centrifuge step is performed for approximately 15 minutes to approximately 20 hours. In some embodiments, an intermediate speed centrifuge step is performed for approximately 15 mins - 30 mins, 15 mins - 1 hour, 30 mins - 2 hours, 1 hour - 3 hours, 3 hours -5 hours, 5 hours - 8 hours, 5 hours - 10 hours, 10 hours - 15 hours, or 15 hours - 20 hours, or more, in duration.
  • a high speed centrifugation step is performed alone, or in addition to (i.e., before or after) another centrifugation step in the same or different (e.g., low speed or intermediate speed) range, on a nanoparticle solution to pellet the desired nanoparticles from the solution.
  • a high speed centrifugation step is performed after a low and/or intermediate speed centrifugation step.
  • one or more purification and/or separation methods e.g., filtration, centrifugation, tangential flow filtration, protease digestion, ion exchange and use of other resins are performed prior to or following a high speed centrifugation step.
  • a high speed centrifugation step is performed at speeds of approximately 8000 xg - 25,000 x g or greater. In some embodiments, a high speed centrifugation step is performed at speeds of approximately 8000 xg, 9000 xg, 10000 xg, 11000 xg, 12000 xg, 13000 xg, 14000 xg, 15000 xg, 16000 xg, 17000 xg, 18000 xg, 19000 xg, 20000 xg, 21000 xg, 22000 xg, 23000 xg, 24000 xg, or 25,000 x g or greater.
  • a high speed centrifugation step is performed at temperature ranges of approximately 4-37 °C. In some other embodiments, after an initial high speed centrifuge step one or more additional centrifuge steps are performed on a given supernatant to further pellet residual nanoparticles from a solution. In some embodiments, a high speed centrifuge step is performed for 15 minutes to approximately 20 hours.
  • a high speed centrifuge step is performed for 15 mins - 30 mins, 15 mins - 1 hour, 30 mins - 1 hour, 30 mins - 2 hours, 1 hour - 3 hours, 3 hours -5 hours, 5 hours - 8 hours, 5 hours - 10 hours, 10 hours - 15 hours, or 15 hours - 20 hours, or more, in duration
  • post-processing steps may increase yields of particular populations of nanoparticles and/or improve purity of nanoparticle populations/solutions. For example, in some embodiments, post-processing steps may improve yield of nanoparticles that are approximately 100-500 nm in at least one dimension. In some embodiments, post-processing steps may increase yield of nanoparticles that are approximately 100-200 nm in at least a single dimension. In some embodiments, post-processing steps may increase yield of nanoparticles that are approximately 100-300 nm in at least a single dimension. In some embodiments, post processing steps may increase yield of nanoparticles that are approximately 100-400 nm in at least a single dimension.
  • post-processing may be or comprise warming provided nanoparticles to a temperature above room temperature (e.g., within a range of about 30 to 50°C).
  • a payload weakly associated with a nanoparticle may be released at a temperature near the glass transition temperature of a provided payload.
  • a payload weakly associated with a nanoparticle may be released at a temperature within a range of about 30 to 50°C.
  • post-processing may be or comprise one or more of lyophilization, electrodialysis, collection of nanoparticles by separation of one or more components of a provided composition (e.g., filtration, e.g., ultrafiltration, tangential flow filtration; e.g., centrifugation (including, e.g.
  • continuous flow centrifugation which may be or comprise flow in an aqueous buffer and extraction while spinning, potentially with nanoparticles under centrifugal force for extended periods of time, such as several hours; e.g., use of column and/or resin purification, e.g., ion exchange resin), and/or removal of free or weakly associated payload by protease digestion,.
  • post-processing does not comprise one or more of electrodialysis, collection of nanoparticles by filtration, tangential flow filtration, removal of free or weakly associated payload by protease digestion, centrifugation (including, e.g.
  • nanoparticle compositions may be further separated using filtration.
  • nanoparticles may be filtered.
  • filtration may occur through a column comprising a medium (e.g., a resin).
  • filtration may occur through a membrane.
  • nanoparticles when nanoparticles are filtered, a consistent mass of nanoparticles per filtration medium surface area is maintained. For example, in some embodiments, a particular concentration of nanoparticles per square centimeter of membrane is maintained during filtration.
  • filtration parameters may be altered to accommodate features such as, e.g., type or material of membrane, scale of procedure (e.g., maintaining a particular ratio of nanoparticles to membrane such that a membrane does not get overloaded and, e.g., clogged or, e.g., allow through unfiltered materials, etc.).
  • filtration may be or comprise tangential flow filtration.
  • tangential flow filtration may be performed by contacting a surface (e.g., filter, membrane) with a composition.
  • a surface area of a membrane is relative to a volume of initial input solution to be filtered.
  • a surface area of a membrane is between a range of approximately 0.01 - 0.1 m 2 /L.
  • a surface has a surface area of approximately 75 1000 cm 2 .
  • tangential flow filtration may be performed using a filter with a surface area of approximately 1000-5000 cm 2 .
  • a surface area of a filter may be between 100-750 cm 2 .
  • a surface used in tangential flow filtration has a molecular weight cut-off (MWCO) of about approximately 100 kilodaltons to approximately 1000 kilodaltons.
  • a MWCO is approximately 200 kilodaltons to approximately 600 kilodaltons.
  • a MWCO is approximately 300 kilodaltons to approximately 500 kilodaltons.
  • surface area and/or MWCO may be altered according to desired output (e.g., higher recovery of a particular composition or portion thereof, recovery of a particular size range of materials of a composition or portion thereof, etc.) of filtering a provided composition.
  • desired output e.g., higher recovery of a particular composition or portion thereof, recovery of a particular size range of materials of a composition or portion thereof, etc.
  • desired output e.g., higher recovery of a particular composition or portion thereof, recovery of a particular size range of materials of a composition or portion thereof, etc.
  • a membrane when tangential flow filtration is used, a membrane is washed with between 1 and 30 volume washes. In some embodiments, a membrane is washed with 1-5, 5-10, 10-15, 15-20, 20-25, 25-30 or more volume washes. In some embodiments, a membrane is washed with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more, volume washes. Without being bound by any particular theory, it is contemplated that increased numbers of volume washes will also increase safety factor of a suspension subjected to tangential flow filtration. For example, in some
  • a safety factor may reach 90. In some embodiments, at least 15 washes are used to reach a safety factor of 20 using a 500 kD membrane, or 31 washes to reach a safety factor of 20 using a 750 kD membrane.
  • wash volumes and times will be altered due to factors such as membrane type and contents of material (e.g., suspension) to be filtered.
  • post-processing may be or comprise addition of one or more agents or additional components and/or one or more additional steps as described herein.
  • a sugar e.g., trehalose
  • trehalose may be added to a composition comprising nanoparticles.
  • trehalose may be added to a combination comprising nanoparticles, PVA1, water and propanol.
  • sugar e.g., trehalose
  • a ratio of a sugar (e.g., trehalose) to PLGA is 1 : 1 to 2: 1 mg/ mg PLGA. In some embodiments, a ratio of a sugar (e.g., trehalose) to PLGA is 5: 1 - 15: 1. In some embodiments, a ratio of a sugar (e.g., trehalose) to PLGA is 7: 1 - 11 : 1. In some such embodiments, following addition of one or more components to a composition comprising nanoparticles (e.g., trehalose), one or more additional steps (e.g., lyophilization) may be performed.
  • one or more additional steps e.g., lyophilization
  • lyophilization of a nanoparticle solution will produce a dry cake comprising nanoparticles, PVA1, and trehalose.
  • a dry cake comprising nanoparticles is resuspended in a buffer.
  • a buffer comprises ammonium bicarbonate.
  • a 10 mM ammonium bicarbonate buffer is used to resuspend a lyophilized cake comprising nanoparticles, PVA1, and trehalose.
  • buffers may be altered in composition and concentration in accordance with a given process and in consideration of factors such as components of given compositions.
  • post-processing of nanoparticles includes one or more lyophilization steps.
  • lyophilizations are not serially performed, rather, are separated by additional post-processing steps.
  • post-processing may comprise an ion exchange step (e.g., through filtration), chromatography (e.g., an ion exchange chromatography)), which may be performed on a nanoparticle suspension.
  • ion exchange and/or chromatography may separate a payload that is weakly associated with a nanoparticle.
  • chromatography separates ions and polar molecules based on their affinity, for example, to the ion exchanger.
  • water-soluble and charged molecules bind to moieties which are oppositely charged by forming ionic bonds to the insoluble stationary phase (e.g., ion exchange resin).
  • a resin, for example, in a column may be used for post- processing of nanoparticles.
  • post-processing comprising a column e.g. an ion exchange column
  • an amount of resin in a column may vary relative to an amount of polymer, payload, or even resin used in a different step of a particular embodiment.
  • post-processing may include incubating nanoparticles with an amount of resin (e.g., ion exchange resin) for a period of time.
  • resin e.g., ion exchange resin
  • a period of time may be, e.g. 15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, or more)
  • nanoparticles may be incubated with a stationary phase longer to achieve a higher degree of separation of weakly associated payloads.
  • an ion exchange resin with a higher affinity to nanoparticles e.g., higher retention time
  • the present disclosure provides the insight that one or more post-processing steps may be combined, for example to optimize yield of particular populations of nanoparticles.
  • additional separation steps may be needed to collect desired populations of nanoparticles.
  • separation by centrifugation at any speed may not sufficiently collect desired nanoparticles from a suspension; thus a combination of separation and collection methods (e.g., centrifugation followed by filtration, e.g., tangential flow filtration) may be used to optimize yield of all and/or desired populations (e.g., particular size and/or protein content) nanoparticles.
  • a first separation (e.g., low speed centrifugation) step may be performed in order to remove nanoparticles and/or aggregates within a particular size range (e.g., 300-500 nm; 500-1000 nm, or greater than 1000 nm), followed by a second separation (e.g., filtration, e.g., tangential flow filtration) to collect nanoparticles in a desirable size range such as, e.g., 100- 200 nm, 100-300 nm, 100-400 m, 200-400 nm, or 200-300 nm.
  • a particular size range e.g., 300-500 nm; 500-1000 nm, or greater than 1000 nm
  • a second separation e.g., filtration, e.g., tangential flow filtration
  • the amount of free payload to encapsulated payload may be any of a variety of ratios.
  • a ratio of free payload to encapsulated payload (before post processing) may be approximately 10: 1, 9: 1, 8: 1 :7: 1, 6: 1, 5: 1, 4: 1, 3 : 1, 2: 1, 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 : 10, or any range of such ratios.
  • the ratio of free payload to encapsulated payload is greater than 10: 1.
  • the ratio of free payload to encapsulated payload is less than 1 : 10.
  • a certain percentage of nanoparticles comprising encapsulated payload may be lost (e.g. destroyed, retained in a column) during a process of contacting nanoparticles to a resin for, e.g. removal of weakly associated or unassociated payload.
  • a percentage of nanoparticles lost is at least about 5% to at most about 25%.
  • a percentage of nanoparticles lost is at least about 25% to at most about 50%.
  • a percentage of nanoparticles lost is at least about 50% to at most about 75%.
  • post-processing may be or comprise treating provided nanoparticles ( e.g ., comprising a polymer and a payload) with at least one protease (e.g ., papain) to remove (e.g., partially or totally digest) some or substantially all of a payload associated with an outer surface of a nanoparticle.
  • a protease may be in a suspension or solution.
  • a protease may be associated with a carrier (e.g., a bead).
  • chromatography is performed to separate the digested payload by the protease.
  • provided nanoparticles are treated with at least one protease prior to a chromatography step. In some embodiments, provided nanoparticles are treated with at least one protease during or substantially simultaneously with a chromatography step. In some embodiments, provided nanoparticles are treated with at least one protease and not subjected to a chromatography step.
  • a protease may be selected from the group consisting of papain, proteinase K, trypsin, chymotrypsin, any other protease derived from plant, animal or bacterial sources that could be deemed pharmaceutically compatible, and combinations thereof.
  • a protease may be used at a weight ratio of nanoparticles to protease within a range of about 1000: 1 to 1 :1 (e.g., 100: 1, 10: 1, 5: 1, etc.).
  • an amount e.g., concentration in a suspension or solution, ratio of molecules of protease to nanoparticles
  • an amount of protease may be chosen to ensure that it is sufficient to digest payload associated with a surface of a nanoparticle within 30 minutes or one hour.
  • nanoparticles are treated with a protease for between 5 and
  • provided nanoparticles are treated with a protease for a time period sufficient to at least partially degrade any payload that is exposed on the surface of the nanoparticle, while not compromising the integrity of the polymer such that additional payload is released.
  • nanoparticles are treated with a protease at a temperature within a range of between 0 and 37 °C.
  • post-processing is performed under conditions in which temperature is controlled (e.g., external heat and/or cooling may be applied). In some embodiments, post-processing is performed under ambient conditions.
  • any one or combination of post-processing steps e.g., those discussed above may be used to isolate nanoparticle species with one or more desirable characteristics (e.g., maximum desirable level of protein encapsulation).
  • a nanoparticle species with desirable (e.g., elevated, including maximized) protein encapsulation has approximately 10 - 90 mg protein/mg PLGA. In some such embodiments, a nanoparticle with desirable protein encapsulation is in a size range of approximately 100 - 500 nm. In some embodiments, an amount of protein encapsulation is in a range of approximately 40 - 80 mg/mg of polymer. In some embodiments, a desirable size range is approximately 100-300 nm.
  • an amount of free (e.g., unencapsulated) protein in a given composition comprising nanoparticles is low enough that there is little to no risk of inducing an allergic reaction when administered to a subject with an allergy to the protein.
  • amount of protein encapsulation corresponds to a safety factor.
  • a safety factor indicates that a quantity of free protein is not great enough to result in risk of anaphylaxis, when administered to a subject with an allergy to the protein.
  • an increased safety factor corresponds to a higher encapsulation rate and/or higher percentage of removal of any remaining free protein from a provided nanoparticle composition prior to administration.
  • a desirable protein encapsulation range corresponds to a particular safety factor (e.g., as measured by an equation, e.g., Equation 1 as described in Example 7B).
  • free protein may be reduced by one or more separation steps as provided herein and/or one or more wash steps. It will be understood by those of skill in the art that separation and/or wash steps may be altered to both optimize free protein reduction and nanoparticle retention.
  • provided methods further include a step of coating nanoparticles.
  • a dry coating agent is directly added to a nanoparticle suspension.
  • a coating agent solution is added to a nanoparticle suspension.
  • those skilled in the art are aware of a variety of coating agents that can be utilized in the preparation of nanoparticles, and of solvent systems that can be utilized to prepare appropriate solutions of such coating agents.
  • a combination of nanoparticles and coating agents is stirred and/or sonicated to form coated nanoparticles.
  • a combination of nanoparticles and coating agents may be sonicated for time within a range of about 0.1 to 10 seconds per mL of the combination.
  • coating is performed at a temperature within a range of about 0 to 25 °C. In some embodiments, coating is performed without application of heat from an external source. In some embodiments, coating is performed without application of cooling from an external source. In some embodiments, coating is performed under conditions in which temperature is controlled (e.g., external heat and/or cooling may be applied).
  • a solution comprising coated nanoparticles is lyophilized to form a solid dispersion (e.g., a powder).
  • a coated nanoparticle suspension is subjected to freeze-drying, lyophilization, or other drying strategy so that such solid nanoparticle dispersion is obtained.
  • a solid dispersion of coated nanoparticles may be milled, sifted, or sieved, so that the solid dispersion may have a desired particle size distribution.
  • Nanoparticle compositions useful in accordance with the present disclosure include those in which the nanoparticles are comprised of at least one polymer and at least one payload.
  • payloads are homogeneously or substantially homogenously distributed in a polymer matrix.
  • nanoparticles are comprised of at least one polymer that is a homopolymer, a diblock polymer, a triblock polymer, a multiblock copolymer, a linear polymer, a dendritic polymer, a branched polymer, a random block, etc ., or combinations thereof. In some embodiments, nanoparticles are comprised of a blend and/or mixture of polymers.
  • nanoparticles are comprised of one or more biocompatible polymers and/or one or more biodegradable polymers. In some embodiments, nanoparticles are comprised of one or more synthetic polymers, or derivatives thereof. In some embodiments, nanoparticles are comprised of one or more natural polymers, or derivatives thereof. In some embodiments, nanoparticles are comprised of combinations of synthetic and natural polymers, or derivatives thereof.
  • nanoparticles are comprised of one or more polymers selected from the group consisting of poly(hydroxy acids) such as poly(lactic acid), poly(glycolic acid), poly(lactic acid-co-glycolic acid), poly(lactic-co-glycolic acid), and derivatives of poly(lactic-co-glycolic acid), PEGylated poly(lactic-co-glycolic acid), poly(lactide),
  • poly(hydroxy acids) such as poly(lactic acid), poly(glycolic acid), poly(lactic acid-co-glycolic acid), poly(lactic-co-glycolic acid), and derivatives of poly(lactic-co-glycolic acid), PEGylated poly(lactic-co-glycolic acid), poly(lactide),
  • polyphosphazenes polyamino acids, polyethers, polyacetals, polylactides,
  • polyhydroxyalkanoates polyglycolides, polyketals, polyesteramides, poly(dioxanones), polyhydroxybutyrates, polyhydroxyvalyrates, polycarbonates, polyorthocarbonates, poly(vinyl pyrrolidone), polycyanoacrylates, polyalkylene oxalates, polyalkylene succinates, poly(malic acid), poly(methyl vinyl ether), poly(ethylene imine), poly(acrylic acid), poly(maleic anhydride), poly(ethylene imine), derivatives of poly(ethylene imine), PEGylated poly(ethylene imine), poly(acrylic acid), derivatives of poly(acrylic acid), PEGylated poly(acrylic acid),
  • polyalkylene oxides such as poly(ethylene oxide), polyalkylene terepthalates such as
  • nanoparticles are comprised of one or more natural polymers.
  • natural polymers include, but are not limited to, proteins (such as albumin, collagen, gelatin), prolamines (for example, zein), polysaccharides (such as alginate), cellulose derivatives (such as hydroxypropyl cellulose, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate), polyhydroxyalkanoates (for example, polyhydroxybutyrate), and/or combinations thereof.
  • a natural polymer may comprise or consist of chitosan.
  • nanoparticles are comprised of one or more polymers such as poly(lactide-co-glycolide) copolymerized with polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the present disclosure encompasses the recognition that viscosity of polymer may be an important factor for producing nanoparticles.
  • viscosity of a polymer solution is a function of the molecular weight of the polymer and operating temperature.
  • a polymer with a high molecular weight requires high operation temperature to have low enough viscosity to be processed.
  • provided nanoparticles and/or nanoparticle compositions include at least one payload (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more).
  • a payload may be or comprise an agent or entity that elicits a particular biological response when delivered to an appropriate subject.
  • a payload may be or comprise an agent or entity that modulates a particular biological response to another, different, agent or entity.
  • a payload may be or comprise an agent or entity with respect to which a particular biological response is desired.
  • a biological response elicited by or desired with respect to a particular payload may be or comprise an immune response.
  • a payload that modifies a biological response is or comprises an immune adjuvant.
  • presence of an immune adjuvant may modify (e.g., amplify, bias, or alter) an immune response to another entity (e.g., to an antigen).
  • One feature of certain embodiments of the present disclosure is that it permits delivery of an antigen to a subject in a context that minimizes exposure of the antigen to immune system component(s) that might induce or mediate an undesirable reaction or response to the antigen while achieving its exposure to immune system component(s) that might induce or mediate a beneficial response.
  • an antigen may be or comprise an allergic antigen and provided systems may minimize its exposure during delivery to mast cells, IgE or other immune system components that might mediate an anaphylactic response (and might be present, for example, in blood), while permitting its exposure to immune components (e.g., Thl and/or Treg cells) that might mediate an allergy-suppressing (e.g., Thl or ThO) response.
  • IgE immune system components
  • Thl and/or Treg cells that might mediate an allergy-suppressing (e.g., Thl or ThO) response.
  • a payload comprises one or more carbohydrates, lipids, metals, nucleic acids, polypeptides, small molecules and/or combinations thereof.
  • a payload as described and/or utilized herein may be prepared or manufactured by any appropriate technology.
  • a payload that is or comprises a polypeptide may be produced recombinantly (e.g., by expressing DNA encoding all or part of the polypeptide antigen in an appropriate expression system; in some such embodiments, the DNA may be in the form of vector DNA such as plasmid DNA).
  • a payload may be provided in combination with another substance.
  • a payload may also be provided as a complex mixture (e.g., including different classes of compounds - e.g., both polypeptides and nucleic acids, etc.).
  • a payload may be or comprise a crude preparation and/or other complex material (e.g., an extract, etc.).
  • provided nanoparticles comprise microbial hydrophobic and/or hydrophilic cellular components (e.g., from a crude microbial extract).
  • microbial hydrophobic and/or hydrophilic cellular components e.g., from a crude microbial extract.
  • some embodiments of the present disclosure including one or more of a microbial hydrophilic cellular component and/or a microbial hydrophobic cellular component may permit development and/or production of useful immunomodulatory
  • nanoparticle compositions at least in part because they utilize various evolved attributes of microbial cells relating to their ability to modulate or evade human or animal immune reactions.
  • the present disclosure also captures the insight that combining such evolved attributes with various features of certain nanoparticle systems such as, for example, ability to sequester antigens and/or cellular hydrophilic components from immune system elements, tunable degradation rates and/or locations, and/or modular association with targeting, immune adjuvant, or other surface entities, permits development and/or production of particularly useful immunomodulatory compositions.
  • provided nanoparticles comprise microbial extracts - e.g., hydrophilic or hydrophobic extracts of microbial cells for use in or with nanoparticle
  • such microbial extracts may contain a collection of microbial components that share a chemical feature, so that they associate with other included components and not with excluded components during production of the extract.
  • extracts may contain at least some cellular components at relative levels comparable to those at which they are present in the cells.
  • microbial extracts are prepared from microbial cell preparations.
  • Microbial cell preparations are prepared by culturing microbial cells for a period of time and under conditions sufficient to achieve cell growth to a desirable level (e.g ., optical density, concentration, colony size, total protein, total DNA, and colony forming units).
  • microbial cell preparations contain intact cells, and optionally are substantially free of lysed cells.
  • microbial cell preparations contain lysed cells, and optionally are substantially free of intact cells.
  • the present disclosure provides hydrophilic microbial extracts, for example extracts prepared by contacting a microbial cell preparation with a hydrophilic solvent so that hydrophilic cellular components partition into solution in the hydrophilic solvent.
  • a hydrophilic solvent can then be separated from non-solubilized components which may, for example, be precipitated, solubilized in a hydrophobic solvent (optionally not miscible with the hydrophilic solvent), or otherwise separable from the hydrophilic solvent.
  • hydrophilic cellular components that partition into a hydrophilic solvent include, for example, components that are miscible and/or soluble in such solvent.
  • a payload is or comprises an antigen.
  • an antigen may be or comprise a polypeptide (e.g., a peptide, a protein, a glycoprotein, etc.), a polysaccharide, a lipid (e.g., glycolipid) a nucleic acid, or combinations thereof.
  • a polypeptide e.g., a peptide, a protein, a glycoprotein, etc.
  • a polysaccharide e.g., a polysaccharide
  • a lipid e.g., glycolipid
  • an antigen may be obtained from (or otherwise found in) a source such as, for example, a microbe (e.g., a bacterium, fungus, protozoan, etc.), a virus, an organism (e.g., a plant, fish, mammal, reptile, etc.), or a cell or tissue thereof.
  • a microbe e.g., a bacterium, fungus, protozoan, etc.
  • a virus e.g., a plant, fish, mammal, reptile, etc.
  • an antigen may be obtained from (or otherwise found in) a cell in culture (e.g., a cancer cell, a cell of a graft to be transplanted, etc.).
  • an antigen may be or comprise whole cells and/or one or more intact cellular structures (e.g., cell walls, organelles, and/or portions thereof).
  • provided nanoparticles and/or nanoparticle compositions may include one or more crude (i.e., unpurified or substantially unpurified) antigenic extracts.
  • crude extract can be a useful and inexpensive alternative to using individual antigens in provided nanoparticle compositions.
  • suitable antigens are known in the art and are available from commercial government and scientific sources. In some embodiments, antigens are provided or obtained from whole inactivated or attenuated organisms.
  • antigens may be delivered by nanoparticles simultaneously and/or sequentially in accordance with methods of the present disclosure.
  • different antigens for one antigenic protein may be delivered.
  • Different antigens from different antigenic proteins may also be delivered.
  • multiple antigenic polypeptides and proteins may be delivered in accordance with the present disclosure.
  • single or multiple antigenic polypeptides and single or multiple cytokines may be delivered to individuals by nanoparticles in accordance with the present disclosure.
  • allergenic antigens of the present disclosure and immunomodulatory molecules such as interleukins may be delivered by nanoparticles using methods in accordance with the present disclosure.
  • a particular provided composition may contain a combination of antigens.
  • a particular provided composition may contain a combination of antigens (e.g., at least two antigens) associated with a particular disease, disorder or condition (e.g., with a particular cancer, a particular infectious disease, a particular graft v host or host v graft syndrome, etc.).
  • compositions comprising an antigen may comprise the antigen in any of a variety of forms.
  • exemplary forms include, without limitation, RNA, DNA, protein, and combinations thereof.
  • an antigen may be provided as a portion of a cell, tissue or extract thereof.
  • an antigen is selected from the group consisting of an allergen, an infectious antigen, a disease-associated antigen (e.g ., a cancer antigen), an autoantigen, or combinations thereof.
  • an antigen is or comprises an allergen.
  • provided nanoparticles and/or nanoparticle compositions may include one or more environmental antigens.
  • Exemplary environmental antigens include, but are not limited to, those derived from naturally occurring allergens such as pollen allergens (tree-, weed-, and grass pollen allergens), insect allergens (inhalant, saliva and venom allergens), animal hair and/or dander allergens.
  • an antigen may be an allergen, for example as may be found in certain foods, venom, drugs or rubber that are capable of eliciting allergic responses, and in particular anaphylactic allergic responses in an individual.
  • allergens that may induce anaphylaxis include several protein allergens found in food (peanut, milk, egg, wheat), insect venom (e.g, bees, reptiles), drugs, and latex.
  • an environmental antigen may be one or more venoms. Stings from organisms that inject venoms, such as insect stings are known to cause anaphylaxis in individuals with allergies to the venom.
  • insect venom includes venom from Hymenoptera such as bees, hornets, wasps, yellow jackets, velvet ants, and fire ants.
  • venom from honey bees of the genus Apis can cause anaphylaxis in stung victims who are allergic (Weber et al. Allergy 42:464-470).
  • honey bees contains numerous compounds which have been extensively studied and characterized (see for a reference, Banks and Shipolini. Chemistry and Pharmacology of Honey bee Venom. Chapter 7 of Venoms of the Hymenoptera. Ed. T. Piek. Academic Press. London. 1986).
  • the two main components of bee venom are phospholipase A2 and melittin and may be used in some embodiments for treating and preventing allergies to bee venom.
  • protein allergens found in food include proteins found in nuts (e.g, peanut, walnut, almond, pecan, cashew, hazelnut, pistachio, pine nut, brazil nut), fish (e.g., cod, salmon, tuna), seafood (e.g, shrimp, crab, lobster, clams), fruit (e.g, plums, peaches, nectarines; Ann Allergy Asthma Immunol 7(6):504-8 (1996); cherries, Allergy 5l(l0):756-7 (1996)), seeds ( e.g sesame, poppy, mustard, sunflower), and legume (e.g., soy, lupine, peanut, lentil, pea) and dairy products (e.g, egg, milk).
  • nuts e.g, peanut, walnut, almond, pecan, cashe
  • protein antigens found in pollen-related food allergies may be used (e.g., birch pollen related to apple allergies).
  • Important pollen allergens from trees, grasses and herbs originate from the taxonomic orders of Fagales, Oleales, Pinales and platanaceae including e.g.
  • birch (Betula), alder (Alnus), hazel (Corylus), hornbeam (Carpinus) and olive (Olea), cedar (Cryptomeriaand Juniperus) , Plane tree (Platanus), the order of Poales including e.g., grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris, Secale, and Sorghum, the orders of Asterales and Urticales including e.g., herbs of the genera Ambrosia, Artemisia, and Parietaria.
  • an antigen may be one or more allergens from house dust mites of the genus Dermatophagoides and Euroglyphus, storage mite e.g., Lepidoglyphys, Glycyphagus and Tyrophagus, cockroaches, midges and fleas e.g., Blatella, Periplaneta, Chironomus and Ctenocepphalides, 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 Hymenoptera including bees (superfamily Apidae), wasps (superfamily Vespided), 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.
  • an antigen may comprise more complex allergens and/or crude allergenic extracts. Therefore, collections of more than one antigen may be used so that immune responses to multiple antigens may be modulated with a single embodiment.
  • provided nanoparticles and/or nanoparticle compositions may include one or more allergens listed in Table 4.
  • Exemplary crude extracts include, but are not limited to, to extracts derived from the Allergen Source listed in Table 4. Table 4.
  • Exemplary Antigens include, but are not limited to, to extracts derived from the Allergen Source listed in Table 4.
  • the present disclosure encompasses the recognition that a particular subject may benefit from being exposed to a combination of antigens, such as multiple allergens.
  • a nanoparticle composition comprising multiple antigens relevant to a specific subject, and/or to a population of subjects.
  • a particular provided composition will contain a combination of allergens to address some or all of a particular subject’s allergies and/or a combination of allergens to address some or all allergies commonly present within a population.
  • a nanoparticle composition may be designed and manufactured to address both allergies.
  • nanoparticle compositions including antigens from a plurality of allergens (i) to which members of a particular community are commonly exposed ( e.g ., by virtue of geographic location); (ii) to which subjects are exposed by a common route (e.g., inhalation, injection, contact, etc.); (iii) to which incidence of allergy within a relevant population (e.g, a geographic population, an age population, an ethnic population, etc.) is above a designated threshold; (iv) to which subjects allergic to one allergen also tend to have allergy to, for example, subjects allergic to tree nuts tend to also be allergic to pecans, walnuts, and pistachios, subjects with allergy to crustaceans (e.g, lobster, crab, shrimp, or crayfish) or mollusks (e.g, clams, mussels, oysters, or scallops) tend to have allergy to various types, not just a single crustacean or mollusk.
  • crustaceans e.g, lobster, crab, shrimp
  • antigens may be provided from infectious organisms, such as viruses, parasites and bacteria.
  • the antigens may be purified or partially purified polypeptides derived from viral or bacterial sources. Exemplary criteria for identifying and selecting effective antigenic peptides (e.g. , minimal peptide sequences capable of eliciting an immune response) may be found in the art. For example, protestopoulos, et al.
  • provided nanoparticles and/or nanoparticle compositions may include one or more viral antigens.
  • a virus consists of either two or three parts:
  • a viral antigen may be provided from any component of a virus.
  • a viral antigen may 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, Coronavtridae (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, and Dengue virus 4), Hepadnaviridae, Herpesviridae (e.g., Human herpesvirus 1, 3, 4, 5, and 6, and Cytomegalovirus), Hypoviridae, Iridoviridae , Leviviridae, Lipothrixviridae, Microviridae , Orthomyxoviridae (e.g, Influenza virus A and B and C), Papovaviridae, Paramyxovirida
  • HIV immunodeficiency virus 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 D 1 NS 1, Dengue D 1 NS2, and Dengue D1NS3.
  • a viral antigen may comprise or consist of fragments of one or more viruses, such as fragments from an influenza virus, for example.
  • viral fragments are provided from one or more of 1) viral genetic material 2) a portion of a viral protein coat, and/or 3) a portion of a viral lipid envelope. In some embodiments, viral fragments may be provided from two or more of 1) viral genetic material 2) a portion of a viral protein coat, and/or 3) a portion of a viral lipid envelope.
  • Exemplary viral antigens include, but are not limited to, those found in the following viral strains such as an adenovirus, borrelia, chagas, coxsackieviruses,
  • cytomegalovirus dengue, Epstein-Barr (EBV), encephalitis (e.g, equine encephalitis and Japanese encephalitis), hantavirus, hepatitis A (HAV), hepatitis B (HBV), hepatitis C (HCV), delta hepatitis D (HDV), hepatitis E (HEV), hepatitis G virus (HGV), herpes simplex virus (HSV)(/.e., HSV1 and HSV2), human immunodeficiency virus (HIV), human T-lymphotrophic virus (HTLV), influenza, lymphocytic choriomeningitis (LCMV), malaria, measles,
  • HAV hepatitis A
  • HBV hepatitis B
  • HCV hepatitis C
  • HDV delta hepatitis D
  • HEV hepatitis E
  • HGV herpes simplex virus
  • HSV herpes simple
  • mycoplasma papillomavirus (e.g, human papillomavirus, HPV), parainfluenza, parvovirus, rhinovirus, Rift Valley fever, rotavirus, rubella, SARS, toxoplasma, treponema, varicella-zoster (VZV), west nile virus (WNV), yellow fever, and combinations thereof.
  • papillomavirus e.g, human papillomavirus, HPV
  • parainfluenza parvovirus
  • rhinovirus e.g., Rift Valley fever, rotavirus, rubella, SARS, toxoplasma, treponema, varicella-zoster (VZV), west nile virus (WNV), yellow fever, and combinations thereof.
  • VZV varicella-zoster
  • WNV west nile virus
  • provided nanoparticles and/or nanoparticle compositions may include one or more bacterial antigens.
  • Bacterial antigens may originate from any bacteria including, but not limited to Actinomyces, Aeromonas, Anabaena, Arthrobacter , Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Citrobacter, Clostridium, Corynebacterium, Cytophaga,
  • HAB Hemophilus influenza type B
  • Hyphomicrobium Klebsiella, Lactococcus, Legionella, Leptospirosis, Listeria, Meningococcus A, B and C, Methanobacterium
  • Micrococcus Morganella, Mycoplasma, Myobacterium, Myxococcus, Neisseria, Nitrobacter, Oscillatoria , Peptococcus, Phodospirillum , Plesiomonas , Prochloron , Proteus , Providencia, Pseudomonas , Rickettsia , Salmonella , Serratia, Shigella , Spirillum , Spirochaeta,
  • provided nanoparticles and/or nanoparticle compositions may include one or more parasite antigens.
  • Parasite antigens can be obtained from parasites such as, but not limited to, an antigen derived from Candida albicans , Candida tropicalis , Chlamydia trachomatis , Chlamydial psittaci , Cryptococcus neoformans , Entamoeba histolytica ,
  • Histoplasma capsulatum Histoplasma capsulatum , Mycoplasma pneumoniae , Nocardia asteroides,
  • Plasmodiumfalciparum Rickettsia ricketsii , Rickettsia typhi , Schistosoma mansoni, Toxoplasma gondii , Trichomonas vaginalis and Trypanosoma brucei.
  • 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.
  • provided nanoparticles and/or nanoparticle compositions may include cancer antigens.
  • cancer antigens may be provided from tumor cells.
  • the cancer antigens may be purified or partially purified polypeptides derived from tumors.
  • antigens can be a cancer antigen, including a cancer-associated or cancer-specific antigen, such as, but not limited to, alpha- actinin-4, Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-l, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferaseAS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-l, 2, and 3, neo-PAP, myosin class I, OS-9, pmlRARa fusion protein, PTPRK, K-ras, N-ras, Triosephosphate isomeras, Bage-l, Gage 3, 4, 5, 6, 7, GnTV, Herv-K-mel, Lü-l, MageAl, 2,3,4,6,10
  • cancer antigens are provided in crude form such as a cellular lysate or cellular fraction.
  • exemplary cellular lysates and/or cellular lysate fractions include, but are not limited to, cancer cells from acute lymphoblastic leukemia (ALL);
  • adrenocortical carcinoma AIDS-related cancers including AIDS-related lymphoma; anal cancer; appendix cancer; astrocytomas; basal cell carcinoma; bile duct cancer; bladder cancer; bone cancer (e.g ., osteosarcoma and malignant fibrous histiocytoma); brainstem glioma; brain cancer; brain tumors; breast cancer; bronchial adenomas/carcinoids; Burkitt lymphoma; carcinoid tumors (e.g., childhood and gastrointestinal tumors); carcinoma (including carcinoma of unknown primary (CLIP) whose origin or developmental lineage is unknown but that possess specific molecular, cellular, and histological characteristics of epithelial cells); central nervous system lymphoma; cerebellar astrocytoma; malignant glioma; cervical cancer; childhood cancers;
  • CIP carcinoma of unknown primary
  • chronic lymphocytic leukemia chronic myelogenous leukemia; chronic myeloproliferative disorders; colon Cancer; cutaneous T-cell lymphoma; desmoplastic small round cell tumor;
  • endometrial cancer ependymoma; esophageal cancer; Ewing's sarcoma in the Ewing family of tumors; extracranial germ cell tumor; extragonadal germ cell tumor; ovarian germ cell tumor; extrahepatic bile duct cancer; eye cancer; intraocular melanoma; retinoblastoma; gallbladder cancer; gastric cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal tumor (GIST); gestational trophoblastic tumor; gastric carcinoid; hairy cell leukemia; head and neck cancer; heart cancer; hepatocellular (liver) cancer; Hodgkin lymphoma; hypopharyngeal cancer;
  • hypothalamic and visual pathway glioma glioma; intraocular Melanoma; Islet Cell Carcinoma
  • kaposi sarcoma soft tissue sarcoma; uterine sarcoma; kidney cancer (renal cell carcinoma); laryngeal cancer; leukemias (including acute lymphoblastic or acute lymphocytic leukemia, acute myeloid or acute myelogenous leukemia, chronic lymphocytic or chronic lymphocytic leukemia, chronic myelogenous or chronic myeloid leukemia); Lip and Oral Cavity Cancer; liposarcoma; liver cancer; lung cancer (including non-small cell and small cell); lymphomas (e.g, AIDS-related, Burkitt, cutaneous T-Cell, Hodgkin, non-Hodgkin, Primary Central Nervous System); macroglobulinemia; medulloblastoma; melanoma; Merkel Cell Carcinoma; mesothelioma (e.g, adult malignant mesothelioma, childhood mesothelioma);
  • leukemias including acute lympho
  • Myelodysplastic/Myeloproliferative Diseases Myelogenous Leukemia; Myeloid Leukemia;
  • neuroblastoma oral cancer; oropharyngeal cancer; ovarian cancer; ovarian epithelial cancer (Surface epithelial-stromal tumor); ovarian germ cell tumor; ovarian low malignant potential tumor; pancreatic cancer; paranasal sinus and nasal cavity cancer; parathyroid cancer; penile cancer; pharyngeal cancer; pheochromocytoma; pineal astrocytoma; pineal germinoma;
  • pineoblastoma and supratentorial primitive neuroectodermal tumors pineoblastoma and supratentorial primitive neuroectodermal tumors; pituitary adenoma;
  • pleuropulmonary blastoma prostate cancer; rectal cancer; renal pelvis and ureter and transitional cell cancer; rhabdomyosarcoma; Sezary syndrome; skin cancer (including melanoma and nonmelanoma); skin carcinoma; small intestine cancer; squamous cell carcinoma; stomach cancer; testicular cancer; throat cancer; thymoma and thymic carcinoma; thyroid cancer; urethral cancer; endometrial uterine cancer; vaginal cancer; vulvar cancer; and/or combinations thereof.
  • provided nanoparticles include one or more alloantigens.
  • an alloantigen refers to an antigen associated with allorecognition and/or graft rejection (e.g ., an antigen against which a rejection immune response is directed).
  • Alloantigens are generally polypeptides expressed by an individual that are genetically different from another individual of the same species.
  • the term“alloantigen polypeptide” refers to a polypeptide whose amino acid sequence includes at least one characteristic sequence of an alloantigen. A wide variety of alloantigen sequences are known in the art.
  • an alloantigen for use in accordance with the present disclosure is a major histocompatibility complex (MHC) polypeptide.
  • MHC major histocompatibility complex
  • an alloantigen for use in accordance with the present disclosure is a Class I MHC polypeptide.
  • an alloantigen for use in accordance with the present disclosure is a Class II MHC polypeptide.
  • an alloantigen for use in accordance with the present disclosure contains part of or all of an extracellular domain of an MHC polypeptide.
  • an alloantigen for use in accordance with the present disclosure is a minor histocompatibility complex polypeptide.
  • an alloantigen for use in accordance with the present disclosure is a co-stimulatory entity (e.g. , CD28, CD80, and CD86, among others).
  • an alloantigen for use in accordance with the present disclosure is a non-MHC protein produced by or present in graft tissue and not produced by or present in a host.
  • alloantigens described herein are exemplary. Any polypeptide that is associated with an allorecognition and/or graft rejection can be classified as an alloantigen.
  • alloantigen polypeptides may have a complete sequence, or alternatively may be polypeptides that represent functional fragments (i.e., fragments retaining at least one activity and/or one characteristic sequence or portion) of such complete polypeptides.
  • functional fragments i.e., fragments retaining at least one activity and/or one characteristic sequence or portion
  • protein sequences generally tolerate some substitution without destroying activity.
  • an exemplary list of antigens and/or antigenic extracts (such as one or more allergens and/or allergenic extracts) that may be used in some embodiments include, but are not limited to, Acarus siro (mite) fatty acid-binding protein (Aca s 13); Actinidia chinensis (kiwi) cysteine protease (Act c 1); Aedes aegyptii (mosquito) antigen (Aed a 2); Aedes aegyptii (mosquito) antigen (Aed a 2); Aedes aegyptii (mosquito) apyrase (Aed a 1); Aedes aegyptii (mosquito) apyrase (Aed a 1); Alnus glutinosa (alder) antigen (Aln g 1); Altern
  • Aspergillus Fumigatus (fungus) antigen (Asp f 9); Aspergillus Fumigatus (fungus) aspartis protease (Asp f 10); Aspergillus Fumigatus (fungus) heat shock protein P70 (Asp f 12);
  • Dermatophagoides pteronyssinus (mite) antigen Dermatophagoides pteronyssinus (mite) antigen (Der p 2); Dermatophagoides pteronyssinus (mite) antigen (Der p 5); Dermatophagoides pteronyssinus (mite) antigen (Der p 7);
  • Euroglyphus maynei mite
  • Felis domesticus cat
  • cat-l antigen Fel d 1
  • Fraxinus excelsior (ash) antigen (Fra e 1); Gadus callarias (cod) allergen M (Gad c 1); Gallus domesticus (chicken) conalbumin; A22 (Gal d 3); Gallus domesticus (chicken) lysozyme (Gal d 4); Gallus domesticus (chicken) ovalbumin (Gal d 2); Gallus domesticus (chicken) ovomucoid (Gal d 1); Gallus domesticus (chicken) serum albumin (Gal d 5); Glycine max (soybean) antigen (Gly m 2); Glycine max (soybean) HPS (Gly m 1.0101); Glycine max
  • Hevea brasiliensis (rubber) antigen Hev b 3); Hevea brasiliensis (rubber) antigen (Hev b 5); Hevea brasiliensis (rubber) component of microhelix protein complex (Hev b 4); Hevea brasiliensis (rubber) C-terminal fragment antigen (Hev b 6.03); Hevea brasiliensis (rubber) elongation factor (Hev b 1); Hevea brasiliensis (rubber) enolase (Hev b 9); Hevea brasiliensis (rubber) hevein (Hev b 6.02); Hevea brasiliensis (rubber) hevein precursor (Hev b 6.01); Hevea brasiliensis (rubber) Mn-superoxide dismut (Hev b 10); Hevea brasiliensis (rubber) patatin homologue (Hev b 7); Hevea brasiliensis (rubber) profil
  • Lepidoglyphus destructor (storage mite) antigen (Lep d 2.0101); Lepidoglyphus destructor (storage mite) antigen (Lep d 2.0102); Ligustrum vulgare (privet) antigen (Lig v 1); Lolium perenne (rye grass) antigen (Lol p lb); Lolium perenne (rye grass) group I antigen (Lol p 1); Lolium perenne (rye grass) group II antigen (Lol p 2); Lolium perenne (rye grass) group III antigen (Lol p 3); Lolium perenne (rye grass) group IX antigen (Lol p 5); Lolium perenne (rye grass) trypsin (Lol p 11); Malassezia furfur (fungus) antigen (Mal f 1); Malassezia furfur (fungus) antigen (Mal f 4); Malassezia furfur (fungus) antigen (Mal
  • Myrmecia pilosula (Australian jumper ant) antigen (Myr p 1); Myrmecia pilosula (Australian jumper ant) antigen (Myr p 2); Olea europea (olive) antigen (Ole e 1); Olea europea (olive) antigen (Ole e 3); Olea europea (olive) antigen (Ole e 4); Olea europea (olive) antigen (Ole e 6); Olea europea (olive) profilin (Ole e 2); Olea europea (olive) superoxide dismutase (Ole e 5); Oryza sativa (rice) antigen (Ory s 1); Penaeus aztecus (shrimp) tropomyosin (Pen a 1); Penaeus indicus (shrimp) tropomyosin (Pen i 1); Penicillium brevicompactum (fungus) al
  • Penicillium citrinum (fungus) heat shock protein P70 (Pen c 1); Penicillium citrinum (fungus) peroxisomal membrane protein (Pen c 3); Penicillium notatum (fungus) alkaline serine proteinase (Pen n 13); Penicillium notatum (fungus) N-acetyl glucosaminidase (Pen n 1);
  • Polistes annularies (wasp) phospholipase Al (Pol a 1); Polistes dominulus (Mediterranean paper wasp) antigen (Pol d 1); Polistes dominulus (Mediterranean paper wasp) antigen (Pol d 5); Polistes dominulus (Mediterranean paper wasp) serine protease (Pol d 4); Polistes exclamans (wasp) antigen 5 (Pol e 5); Polistes exclamans (wasp)
  • provided nanoparticles and/or nanoparticle compositions may include one or more other agents (e.g., agents which do not elicit a humoral immune response in a subject).
  • compositions comprising one or more other agents may comprise one or more other agents in any of a variety of forms.
  • Exemplary forms include, without limitation, RNA, DNA, protein, and combinations thereof.
  • one or more other agents may be provided as a portion of a cell, tissue or extract thereof.
  • one or more other agents may comprise
  • immunomodulatory polypeptides include cytokines which are small proteins or biological factors (in the range of 5-20 kD) that are released by cells and have specific effects on cell-cell interaction, communication and behavior of other cells. Cytokines are proteins that are secreted to T-cells to induce a Thl or Th2 response. In some embodiments, cytokine(s) may be selected to reduce production of a Th2 response to antigens associated with anaphylaxis. Cytokines that, when expressed during antigen delivery into cells, induce a Thl response in T cells include IL-12, IL-2, 1-18, IL-l or fragments thereof, IFN, and/or IFNy.
  • one or more other agents may comprise immunological inducing agents.
  • Inducing agents may prompt the expression of Thl stimulating cytokines by T- cells and include factors such as, CD40, CD40 ligand, oligonucleotides containing CpG motifs, TNF, and microbial extracts such as preparations of Staphylococcus aureus, heat killed Listeria, and modified cholera toxin, etc.
  • one or more other agents may include preparations
  • microorganisms such as Listeria monocytogenes or others (e.g., Bacille Calmette- Guerin[BCG], Corynebacterium species, Mycobacterium species, Rhodococcus species, Eubacte ria species, Bortadella species, and Nocardia species), and preparations of nucleic acids that include unmethylated CpG motifs.
  • BCG Bacille Calmette- Guerin[BCG], Corynebacterium species, Mycobacterium species, Rhodococcus species, Eubacte ria species, Bortadella species, and Nocardia species
  • BCG Bacille Calmette- Guerin[BCG]
  • Corynebacterium species e.g., Corynebacterium species, Mycobacterium species, Rhodococcus species, Eubacte ria species, Bortadella species, and Nocardia species
  • nucleic acids that include unmethylated CpG motifs.
  • one or more other agents include, for example, Aviridine (N,N-dioctadecyl-N'N'-bis(2-hydroxy ethyl) propanediamine) and CRL 1005.
  • one or more other agents induce IL-12 production, including microbial extracts such as fixed Staphylococcus aureus, Streptococcal preparations, Mycobacterium tuberculosis, lipopolysaccharide (LPS), monophosphoryl lipid A (MPLA) from gram negative bacterial lipopolysaccharides (Richards et al. Infect Immun 1998 June; 66(6):2859-65), listeria monocytogenes, toxoplasma gondii, leishmania major.
  • microbial extracts such as fixed Staphylococcus aureus, Streptococcal preparations, Mycobacterium tuberculosis, lipopolysaccharide (LPS), monophosphoryl lipid A (MPLA) from
  • one or more other agents may be or comprise one or more immune adjuvants.
  • immune adjuvants may be provided from one or more bacterial sources, including, by way of non-limiting example, certain bacterial cellular lysates, cellular lysate fractions, or specific components thereof.
  • bacterial cellular lysate fractions comprise entities known as pathogen-associated molecular patterns (“PAMPs”).
  • PAMPs pathogen-associated molecular patterns
  • one or more of a hydrophobic bacterial cellular lysate fraction and/or hydrophilic bacterial cellular lysate fraction include one or more PAMPs as a hydrophilic cellular component and/or hydrophobic cellular component.
  • an immune adjuvant is a mucosal immune adjuvant (i.e., an immune adjuvant capable of eliciting or enhancing an immune response to a mucosally administered antigen).
  • mucosal antigens include, but are not limited to, TLR4 ligands (e.g., LPS, MPL), cytokines (e.g., IL-la), c48/80, R848, Pam3CSK4, CpG(ODN 1826), lethal factor (LF), and cholera toxin.
  • PAMPs are entities associated with bacterial cells that are recognized by cells of the innate immune system.
  • PAMPs are recognized by Toll-like receptors (TLRs) and other pattern recognition receptors (PRRs) in both plants and animals.
  • PAMPs are recognized by C-type lectin receptors (CLRs).
  • CLRs C-type lectin receptors
  • a CLR is a type I or type II CLR.
  • PAMPs are or comprise entities associated with the outer surface of a bacterial cell, including, but not limited to, membrane-associated proteins and/or peptides, receptors embedded in bacterial membranes, etc.
  • Exemplary PAMPs include, but are not limited to, bacterial lipopolysaccharide (LPS), bacterial flagellin, lipoteichoic acid from gram positive bacteria, peptidoglycan, double-stranded RNAs (dsRNAs), unmethylated CpG motifs, any of the TLR ligands presented in Table 5, characteristic portions thereof, and/or combinations thereof.
  • LPS bacterial lipopolysaccharide
  • dsRNAs double-stranded RNAs
  • CpG motifs unmethylated CpG motifs
  • nanoparticle compositions may be partially or wholly coated with a coating agent.
  • a coating agent may be or comprise one or more entities that target nanoparticles to a particular site (e.g., to a specific cell, tissue, cell surface marker, etc.).
  • a coating agent may be or comprise a payload (e.g., nanoparticles may be partially or wholly coated with a payload entity - e.g., with an antigen and/or an immune adjuvant as described herein).
  • One feature of certain embodiments of the present disclosure is that it permits delivery of an antigen to a subject in a context that minimizes exposure of the antigen to immune system component(s) that might induce or mediate an undesirable reaction or response to the antigen while achieving its exposure to immune system component(s) that might induce or mediate a beneficial response.
  • an antigen may be or comprise an allergic antigen and provided systems may minimize its exposure during delivery to mast cells, IgE or other immune system components that might mediate an anaphylactic response (and might be present, for example, in blood), while permitting its exposure to immune components (e.g., Thl and/or Treg cells) that might mediate an allergy-suppressing (e.g., Thl or ThO) response.
  • IgE immune system components
  • Thl and/or Treg cells that might mediate an allergy-suppressing (e.g., Thl or ThO) response.
  • a coating agent comprises a hydrophobic component selected from the group consisting of peptide, protein, small molecule (e.g., synthetic folate- PEG-lipid conjugates), polymer, and combinations thereof.
  • a coating agent comprises a hydrophobic cellular component.
  • a hydrophobic cellular component preparation may be provided from a microbial cellular lysate.
  • a hydrophilic bacterial cellular lysate fraction and/or hydrophilic cellular component may be encapsulated within or
  • a coating agent may be or comprise a crude preparation and/or other complex material (e.g., an extract, etc.).
  • coating agents may comprise microbial hydrophobic and/or hydrophilic cellular components (e.g., from a crude microbial extract, for example, an E. coli extract).
  • some embodiments of the present disclosure including a coating agent comprising one or more of a microbial hydrophilic cellular components and/or a microbial hydrophobic cellular components may permit development and/or production of useful immunomodulatory nanoparticle compositions at least in part because they utilize various evolved attributes of microbial cells relating to their ability to modulate or evade human or animal immune reactions.
  • the present disclosure also captures the insight that combining such evolved attributes with various features of certain nanoparticle systems such as, for example, ability to sequester antigens and/or cellular hydrophilic components from immune system elements, tunable degradation rates and/or locations, and/or modular association with targeting, immune adjuvant, or other surface entities, permits development and/or production of particularly useful immunomodulatory compositions.
  • coating agents may comprise microbial extracts - e.g., hydrophilic or hydrophobic extracts of microbial cells (e.g., E. coli ) for use in or with provided nanoparticle compositions.
  • microbial extracts may contain a collection of microbial components that share a chemical feature, so that they associate with other included components and not with excluded components during production of the extract.
  • extracts may contain at least some cellular components at relative levels comparable to those at which they are present in the cells.
  • Those skilled in the art will be aware of a variety of techniques available to determine presence and/or level of particular components, and to compare such determined level(s) with those observed in intact cells.
  • those of ordinary skill in the art will readily appreciate reasonable and expected experimental variation and therefore will be able to determine whether components are present in absolute or relative levels or concentrations in an extract that are reasonably comparable to those at which they are present in cells.
  • microbial extracts are prepared from microbial cell preparations.
  • Microbial cell preparations are prepared by culturing microbial cells for a period of time and under conditions sufficient to achieve cell growth to a desirable level (e.g, optical density, concentration, colony size, total protein, total DNA, and colony forming units).
  • microbial cell preparations contain intact cells, and optionally are substantially free of lysed cells.
  • microbial cell preparations contain lysed cells, and optionally are substantially free of intact cells.
  • one or more coating agents is associated covalently with a nanoparticle surface.
  • one or more coating agents e.g., extracts, preparations and/or agents
  • non-covalent association involves incorporation of one or more components into the nanoparticle membrane.
  • non-covalent association involves specific binding with the nanoparticle membrane or an element incorporated therein.
  • a coating agent e.g., an extract, preparation and/or agent
  • a ligand-target combination utilized in such an embodiment may be, for example, biotin-avidin, antibody-antigen, toll-like receptor 4 (TLR4) and lipopolysaccharide (LPS), GST-glutathione, mannose binding protein-mannose, Protein A-IgG, and/or S-tag, or components thereof.
  • one or more coating agents is prepared using a process that involves mixture of a dry coating agent with water, followed by application of disruptive energy force (e.g., sonication).
  • a dry coating agent e.g., water
  • disruptive energy force e.g., sonication
  • OEE organic E. coli extract
  • a combination of water and OEE powder is sonicated, producing OEE micelles in water.
  • OEE micelles in water are coated onto nanoparticles of the present disclosure using a spray-drying method.
  • a certain percentage of solid material e.g., coated nanoparticles
  • approximately 50 to 95% of solids are recovered.
  • approximately 60-85% of solids are recovered.
  • approximately 65-80% of solids are recovered.
  • OEE micelles in water are combined with a nanoparticle mixture, sonicated, and lyophilized (see, e.g., Figure 8A, steps 12-17). In some such
  • combining OEE micelles with a provided nanoparticle mixture, and lyophilizing results in an association of a coating (OEE) with a nanoparticle surface.
  • concentration of coating agents is quantified and/or compared to one or more natural organisms. For example, in some embodiments, quantity of TLR4 ligand (LPS) present per nanoparticle as compared to LPS present in a given, wild-type E. coli cell may be calculated.
  • nanoparticles may have a lesser (e.g., 10%, 25%, 50%, 75%), substantially equivalent, or greater (e.g., 110%, 125,%, 150%, 200%, 250%, 300% or more) amount of LPS than a given wild-type E. coli.
  • a coating applied using spray drying may be more concentrated than a coated applied using lyophilization procedures.
  • nanoparticles coated with OEE using spray drying may have an LPS-equivalent of approximately 5-7 E. coli (e.g., approximately 6.5-7 E. coli).
  • nanoparticles coated with OEE using a lyophilization procedure may have an LPS-equivalent of approximately 1-5 E. coli cells (e.g., approximately 3-3.5 E. coli).
  • LPS-equivalent of approximately 1-5 E. coli cells e.g., approximately 3-3.5 E. coli.
  • higher amount(s) of LPS relative to what is present on wild-type E. coli is/are favorable and will assist in function of a given nanoparticle composition.
  • higher amount(s) of LPS relative to wile-type E.coli may be desirable.
  • lower amounts of LPS than found on wild-type E. coli may be beneficial and/or desirable.
  • provided nanoparticle compositions comprise
  • nanoparticles e.g., comprised of polymer
  • provided nanoparticle compositions comprise nanoparticles combined with one or more payloads, one or more coating agents, and/or one or more other agents so that certain combined elements are distributed (e.g., substantially homogenously) within a polymer matrix.
  • provided nanoparticle compositions comprise nanoparticles combined with one or more payloads, one or more coating agents, and/or one or more other agents so that certain combined elements are associated with the external surface of nanoparticles.
  • provided nanoparticle compositions comprise nanoparticles combined with one or more payloads, one or more coating agents, and/or one or more other agents so that certain combined elements are present both in and on nanoparticles.
  • provided nanoparticle compositions comprise nanoparticles combined with one or more payloads, one or more coating agents, and/or one or more other agents so that certain combined elements are mixed with, but not specifically associated with any site on or in, nanoparticles.
  • the present disclosure provides nanoparticle compositions in which a coating agent is localized on the external surface of nanoparticle; in some such embodiments, a coating agent is preferentially localized on the nanoparticle external surface; in some such embodiments, a coating agent is substantially exclusively localized on the external surface.
  • provided nanoparticle compositions comprise a population of nanoparticles.
  • a population of nanoparticles comprises nanoparticles of a uniform size.
  • a population of nanoparticles comprises nanoparticles of different sizes; in some embodiments showing a particular size distribution.
  • provided nanoparticle compositions comprise nanoparticles having sizes (e.g ., average, or mean size) within a range defined by a lower limit and an upper limit.
  • the lower limit is 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 150 nm, 200 nm, or more.
  • the upper limit is 1000 nm, 950 nm, 900 nm, 850 nm, 800 nm, 750 nm, 700 nm, 650 nm, 600 nm, 550 nm, 500 nm, 450 nm, 400 nm, 350 nm, 300 nm, 250 nm or less.
  • provided nanoparticle compositions comprise
  • nanoparticles having sizes having sizes (e.g., average, or mean size) similar to the size of bacterial cells.
  • provided nanoparticle compositions comprise nanoparticles having sizes (e.g, average, or mean size) within a range of 100 nm to 2000 nm, 100 nm to 1000 nm, 100 nm to 500 nm, 100 nm to 400 nm, 100 nm to 300 nm, or 100 nm to 200 nm.
  • provided nanoparticle compositions are substantially free of nanoparticles larger than about 2000 nm, about 1000 nm, about 900 nm, about 800 nm, about 700 nm, about 600 nm, about 500 nm, about 400 nm, or about 300 nm.
  • provided nanoparticle compositions comprise no more than about 50%, about 25%, about 10%, about 5%, or about 1% of nanoparticles larger than about 2000 nm, about 1000 nm, about 900 nm, about 800 nm, about 700 nm, about 600 nm, about 500 nm, about 400 nm, or about 300 nm.
  • a weight ratio of a payload to a polymer in a nanoparticle composition is within a range of about 0.001 : 1 to 1 : 1; 0.001 to 0.1 :1, or 0.01 : 1 to 0.1 : 1.
  • a weight ratio of a coating to a polymer in a nanoparticle composition is within a range of about 0.001 : 1 to 1 : 1; 0.001 to 0.1 :1, or 0.01 : 1 to 0.1 :l.
  • a weight ratio of a payload to a polymer in a nanoparticle composition may be represented in, e.g. mg (payload) / mg (polymer).
  • a payload to polymer ratio is no less than 30 mg/mg and no greater than 250 mg/mg.
  • a ratio of payload to polymer is between 30 mg/mg and 150 mg/mg.
  • a ratio of payload to polymer is between 50 mg/mg and 100 mg/mg.
  • provided compositions may also contain a certain amount
  • free protein e.g., unencapsulated protein
  • an amount of unencapsulated protein is 5-30% of an originally input amount of protein.
  • a certain amount of free protein is allowed to remain in a given composition (e.g., approximately 20% or less).
  • free protein is removed from a preparation comprising nanoparticles using one or more separation methods as described herein.
  • free protein is reduced to approximately no greater than 1-5% of total protein relative to that originally put into an initial polymer/payload combination. In some embodiments, free protein is reduced to approximately no greater than 2.5-5%, 5-10%, 10-15%, 15-20%, or 20-25% of total protein relative to that originally put into an initial polymer/payload combination.
  • an amount of free protein in a provided composition is not sufficient to trigger an allergic reaction when administered to a subject allergic to the protein. In some embodiments, an amount of free protein is not sufficient to increase risk of anaphylaxis when administered to a subject allergic to the protein.
  • a certain amount of free protein in a given composition as described herein may be desirable.
  • a certain amount of free protein may act synergistically with administered nanoparticles such that a desirable immune response is activated in an individual to whom the nanoparticles are administered.
  • methods of the present disclosure produce one or more populations of
  • nanoparticles different populations of nanoparticles may be represented by different size ranges (e.g., approximately 100-200 nm and 300-500 nm or more). In some such embodiments, two or more populations of nanoparticles are produced during a single manufacturing cycle. In some such embodiments, nanoparticles in a range of approximately 100-200 nm contain a higher payload: polymer ratio than nanoparticles in a range of
  • nanoparticles between 100-400 nm have higher ratios of payload: polymer than nanoparticles larger than 400 nm (i.e., higher encapsulation percentage). In some embodiments, nanoparticles with the higher payload:
  • polymer ratio are between 100-200 nm.
  • nanoparticles greater than approximately 400 nm have a lower payload: polymer ratios than nanoparticles smaller than 400 nm (i.e., larger than 400 nm have a lower encapsulation percentage than smaller than 400 nm).
  • a nanoparticle composition comprises at least one polymer having a concentration within a range of about 10 to 90 %, 20 to 80%, 25 to 70%, or 25 to 65% by weight. In some embodiments, a nanoparticle composition comprises a plurality of polymers with a total concentration of polymer within a range of about 10 to 90 %, 20 to 80%, 25 to 70%, or 25 to 65% by weight. In some embodiments, a nanoparticle composition comprises one or more payloads having a concentration within a range of, by way of non-limiting example, about 0.1 to 10 %, 0.1 to 5, 0.5 to 10%, 0.5 to 5%, or 1 to 3 % by weight. In some embodiments, a nanoparticle composition comprises a coating agent having a concentration within a range of about 0.1 to 5 %, 0.1 to 3, 0.5 to 5, 0.5 to 3, or 1 to 3 % by weight.
  • a nanoparticle composition is characterized with respect to size of nanoparticles, uniformity of a payload within a nanoparticle, payload content (e.g., DNA and/or protein), release rate of payload and/or surface exposure of payloads (e.g., how much of the payload(s) are exposed at/accessible from the surface of the nanoparticle). Surface exposure of payloads may be assessed using a proteolysis assay (e.g., surface exposed payloads are susceptible to protease added to the media, whereas materials encapsulated within particle are protected) or by an antibody binding assay.
  • a nanoparticle composition is biodegradable.
  • a polymer of a nanoparticle composition is decomposed (e.g., nanoparticles release payloads), when they are exposed to a physiological environment.
  • a nanoparticle composition is capable of interacting with biological systems and/or of inducing one or more desired biological responses.
  • a nanoparticle composition may be i) susceptible to uptake by macrophages and/or antigen presenting cells, ii) able to activate Toll Like Receptors, or iii) able to induce relevant responses in vitro or in vivo assays of immunological parameters (e.g., cytokine release, proliferation, etc.).
  • provided nanoparticle compositions may include a plurality of sets of nanoparticles that share one or more structural and/or functional
  • provided nanoparticle compositions may comprise a plurality of sets of nanoparticles, each of which includes a coating agent that localizes members of the set to a particular target site.
  • a coating agent that localizes members of the set to a particular target site.
  • provided nanoparticle compositions may comprise a plurality of sets each of which is designed to have and/or is characterized by a different half-life (e.g., in a relevant tissue or organ of interest) and/or different components (e.g. in the lumen or associated with external surface, different populations of antigens, etc.).
  • a provided nanoparticle composition may be characterized by a safety factor (e.g., when measured as described in Example 7B, for instance).
  • a safety factor may be between 5-100 or more.
  • a safety factor is between approximately 5 and 20.
  • a safety factor is between approximately 25 and 100.
  • a safety factor is between a range of approximately 30-90.
  • a safety factor is between a range of approximately 40-80.
  • a lower safety factor may be desirable.
  • a higher safety factor may be desirable.
  • a particular safety factor indicates that a quantity of free protein is not great enough to result in risk of anaphylaxis, when administered to a subject with an allergy to the protein.
  • the present disclosure provides pharmaceutical compositions comprising one or more provided nanoparticle compositions together with one or more pharmaceutically acceptable excipients.
  • provided pharmaceutical compositions may be prepared by any appropriate method, for example as known or hereafter developed in the art of
  • Such preparatory methods include the step of bringing a provided nanoparticle composition into association with one or more pharmaceutically acceptable excipients, and then, if necessary and/or desirable, shaping and/or packaging the product into an appropriate form for administration, for example as or in a single- or multi-dose unit.
  • compositions may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a“unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the provided nanoparticle composition.
  • the amount of the provided nanoparticle composition is generally equal to the dosage of the provided nanoparticle which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • provided pharmaceutical compositions are specifically formulated for mucosal delivery (e.g ., oral, nasal, rectal or sublingual delivery).
  • appropriate excipients for use in provided pharmaceutical compositions may, for example, include one or more pharmaceutically acceptable solvents, dispersion media, granulating media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents and/or emulsifiers, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, disintegrating agents, binding agents, preservatives, buffering agents and the like, as suited to the particular dosage form desired.
  • pharmaceutically acceptable excipients such as cocoa butter and/or suppository waxes, coloring agents, sweetening, flavoring, and/or perfuming agents can be utilized.
  • Remington s The Science and Practice of Pharmacy, 2 I st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2005; incorporated herein by reference) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof.
  • an appropriate excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved by United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or other International Pharmacopoeia.
  • USP United States Pharmacopoeia
  • EP European Pharmacopoeia
  • British Pharmacopoeia the British Pharmacopoeia
  • liquid dosage forms include, but are not limited to, emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs.
  • liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -butylene glycol,
  • compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents.
  • solubilizing agents such as a CREMOPHOR ® , alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
  • injectable preparations for example, sterile aqueous or oleaginous suspensions
  • suitable dispersing agents, wetting agents, and/or suspending agents may be stored in a pre-filled syringe.
  • Sterile liquid preparations may be, for example, solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, as a solution in l,3-butanediol.
  • acceptable vehicles and solvents that may be employed, for example, are water, Ringer’s solution, U.S.P., and isotonic sodium chloride solution.
  • Sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • Fatty acids such as oleic acid can be used in the preparation of liquid formulations.
  • Liquid formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • one or more strategies may be utilized prolong and/or delay the effect of a provided nanoparticle composition after delivery.
  • provided pharmaceutical compositions may be formulated as suppositories, for example for rectal or vaginal delivery.
  • suppository formulations can be prepared by mixing utilizing suitable non-irritating excipients such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the body ( e.g ., in the rectum or vaginal cavity) and release the provided nanoparticle composition.
  • solid dosage forms include one or more portions of a provided nanoparticle composition that may be or comprise capsules, tablets, pills, powders, and/or granules.
  • the provided nanoparticle composition may be mixed with at least one inert, pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and/or fillers or extenders (e.g, starches, lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g, glycerol), disintegrating agents (e.g, agar, calcium carbonate, potato starch, tapioca starch, alginic acid, certain silicates, and sodium carbonate), solution retarding agents (e.g, paraffin), absorption accelerators (e.g, paraffin), absorption accelerators (e.g, paraffin), absorption accelerators
  • solid compositions of a similar type may be employed as fillers in soft and/or hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, impregnated filter paper, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art.
  • Exemplary enteric coatings include, but are not limited to, one or more of the following: cellulose acetate phthalate; methyl acrylate-methacrylic acid copolymers; cellulose acetate succinate; hydroxy propyl methyl cellulose phthalate; hydroxy propyl methyl cellulose acetate succinate (hypromellose acetate succinate); HP55; polyvinyl acetate phthalate (PVA1P); methyl methacrylate-methacrylic acid copolymers; methacrylic acid copolymers, cellulose acetate (and its succinate and phthalate version); styrol maleic acid co-polymers;
  • solid dosage forms may optionally comprise opacifying agents and can be of a composition that they release the provided nanoparticle composition(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.
  • embedding compositions which can be used include polymeric substances and waxes.
  • Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • compositions for topical and/or transdermal delivery e.g. , as a cream, liniment, ointment, oil, foam, spray, lotion, liquid, powder, thickening lotion, or gel.
  • Particular exemplary such formulations may be prepared, for example, as products such as skin softeners, nutritional lotion type emulsions, cleansing lotions, cleansing creams, skin milks, emollient lotions, massage creams, emollient creams, make-up bases, lipsticks, facial packs or facial gels, cleaner formulations such as shampoos, rinses, body cleansers, hair-tonics, or soaps, or dermatological compositions such as lotions, ointments, gels, creams, liniments, patches, deodorants, or sprays.
  • products such as skin softeners, nutritional lotion type emulsions, cleansing lotions, cleansing creams, skin milks, emollient lotions, massage creams, emollient creams, make-up bases, lipsticks, facial packs or facial gels, cleaner formulations such as shampoos, rinses, body cleansers, hair-tonics, or soaps, or dermatological compositions such as lotions, ointments, gels, creams,
  • an adjuvant is provided in the same formulation with provided nanoparticle composition(s) so that adjuvant and provided nanoparticle composition are delivered substantially simultaneously to the individual.
  • an adjuvant is provided in a separate formulation. Separate adjuvant may be administered prior to,
  • nanoparticle composition administration simultaneously with, or subsequent to provided nanoparticle composition administration.
  • provided compositions are stable for extended periods of time, such as 1 week, 2 weeks, 1 month, 2 months, 6 months, 1 year, 2 years, 3 years, or more.
  • provided compositions are easily transportable and may even be sent via traditional courier or other package delivery service. Accordingly, some embodiments may be useful in situations of disease outbreak, such as epidemics, or attacks with biological agents (e.g ., anthrax, smallpox, viral hemorrhagic fevers, plague, and others) at least in part due to their ability to be stored for long periods of time and transported quickly, easily, and safely. Such attributes may allow for rapid distribution of provided compositions to those in need.
  • biological agents e.g ., anthrax, smallpox, viral hemorrhagic fevers, plague, and others
  • a payload for example, an antigen
  • GI gastrointestinal
  • a payload for example, an antigen
  • a plurality of provided compositions may be
  • each of the plurality of compositions has a different release profile, such as provided by various enteric coatings, for example. In some embodiments, each of the plurality of compositions has a similar release profile. In some embodiments, the plurality of
  • compositions comprises one or more antigens. In some embodiments, each of the plurality of administered compositions comprises a different antigen. In some embodiments, each of the plurality of compositions comprises the same antigen.
  • a provided pharmaceutical composition is characterized in that the composition does not comprise an amount of free protein that is expected to and/or does increase risk of allergic reaction (e.g., anaphylaxis) when administered to a subject allergic to the protein.
  • a provided pharmaceutical composition is characterized by a particular safety factor as described herein, including, e.g., in Example 7B (e.g., 5-20, e.g., 20- 100, e.g., 20-80, etc.). Characterization of compositions and components thereof
  • compositions may be characterized in order to determine, for example, protein content per nanoparticle.
  • characterization may include, e.g., quantifying payload encapsulation efficiency, assessing content of payload (e.g., determining if payload contains expected protein and/or DNA in expected amounts and/or forms), evaluating a surface coating (e.g., evaluating OEE on surface of nanoparticles), etc.
  • characterization includes measurements of nanoparticles within provided compositions including, e.g.
  • a weight ratio of a payload to a polymer in a nanoparticle composition is within a range of about 0.001 : 1 to 1 : 1; 0.001 to 0.1 :l, or 0.01 : 1 to 0.1 : 1.
  • a weight ratio of a payload to a polymer in a nanoparticle composition may be represented in, e.g. mg (payload) / mg (polymer).
  • a payload to polymer ratio is no less than 30 mg/mg and no greater than 250 mg/mg.
  • a ratio of payload to polymer is between 30 mg/mg and 150 mg/mg.
  • a ratio of payload to polymer is between 50 mg/mg and 100 mg/mg.
  • characterization includes an evaluation of encapsulation efficiency (e.g. amount of payload provided during production of nanoparticles versus amount of payload encapsulated by polymer measured during or after nanoparticles are forming or formed).
  • encapsulation efficiency is no lower than 40%.
  • encapsulation efficiency is substantially 100%.
  • encapsulation efficiency is between 50% and 100%; 60% and 100%; 70% and 100%; 75% and l00%;80% and 100%;
  • encapsulation is between 75% and 95%; 80% and 90%; 85% and 95%.
  • characterization includes analysis of certain properties or features of compositions as provided herein. Such characterization for, e.g. nanoparticles or pharmaceutical compositions will be known to one of skill in the art. For example, in some embodiments, characterization includes visualization by microscopy (e.g., fluorescent microscopy, scanning electron microscopy, etc.). In some embodiments, microscopic evaluation is performed after each of multiple steps (e.g., to evaluate status of composition and any nanoparticles therein, e.g., see Fig. 8A, after nanoparticle formation in Lyo 2 and/or after centrifugation and/or tangential flow filtration in Lyo 3).
  • microscopy e.g., fluorescent microscopy, scanning electron microscopy, etc.
  • microscopic evaluation is performed after each of multiple steps (e.g., to evaluate status of composition and any nanoparticles therein, e.g., see Fig. 8A, after nanoparticle formation in Lyo 2 and/or after centrifugation and/or tang
  • characterization may include, e.g. taking an aliquot from a composition during and/or at various points throughout the production process.
  • an aliquot of a nanoparticle composition as described herein, is removed, e.g. after step 8/before step 9 in Figure 1.
  • the aliquot can be analyzed to determine, e.g. free protein and/or payload encapsulation efficiency.
  • an aliquot of nanoparticle suspension may be analyzed in a method that comprises steps of removing an aliquot of nanoparticle suspension, centrifuging at low speed (e.g. 1500- 2500 ref), hydrolyzing said suspension with NaOH, and then analyzing using an assay that measures protein content (e.g. BCA, Bradford, etc.).
  • a low speed spin prior to hydrolysis accomplishes separation of nanoparticles from free protein without damaging any already formed nanoparticles.
  • the resulting number (s) represent quantification of total protein per volume of suspension. Remaining suspension (i.e.
  • a sample may be filtered through a 100 nm centrifuge filter, prior to ultracentrifugation. In some embodiments, a sample is not filtered through a centrifuge filter, prior to centrifugation.
  • an additional low speed spin (e.g. spin at or about 1500-2500 ref) may be performed (a method involving a second, low-speed spin as described herein may be referred to as“Method 2”).
  • Method l is a preferred method for characterizing quantity of free protein and/or encapsulation efficiency of payload in compositions as described herein. Without wishing to be bound by any theory, it is contemplated that a second, low speed spin may not recover all nanoparticles and/or protein in a given nanoparticle suspension or aliquot thereof.
  • excipients in provided compositions may be quantified.
  • excipients means components that are part of or used in making compositions as described herein that do not comprise nanoparticle payload.
  • excipients include, e.g. PLGA, PVA1, trehalose, residual water, etc.
  • characterization of nanoparticles includes evaluation using dynamic light scattering (“DLS”).
  • DLS dynamic light scattering
  • dynamic light scattering may be used to evaluate one or more aliquots of solution from one or more stages of
  • dynamic light scattering may provide information that can be used to alter manufacturing protocols. For example, if dynamic light scattering shows nanoparticles of particular sizes that are not found in later samples, additional or different steps may be inserted into manufacturing processes.
  • a payload of nanoparticle compositions is evaluated in one or more ways at one or more times.
  • protein encapsulated by a provided nanoparticle composition is evaluated both before and after incorporation into nanoparticles (and compared to protein not encapsulated by nanoparticles).
  • evaluation is performed to ensure that processing into nanoparticles has not materially altered payload components.
  • evaluation methods comprise standard procedures for evaluation of proteins and/or nucleic acids (e.g., gel analysis, western blots, etc.).
  • provided nanoparticle compositions examined for presence of expected protein and/or nucleic acid components.
  • evaluation is performed by isolating payload from loaded nanoparticles and using standard methods to separate components.
  • nanoparticle payloads are exposed, isolated, and then proteins separated using gel-separation. Proteins separated on gels may then be transferred onto membranes and probed for particular components, for example, in the case of peanut extract, Ara hl, Ara h2, and Ara h3 components.
  • payload contents are detectable both before and after incorporation into nanoparticles, and can be used to confirm whether payload starting material is or is not materially changed by the nanoparticle
  • methods disclosed herein can be used to confirm the identity and/or quality of a given composition and/or its components protein, e.g ., nanoparticles and/or nanoparticle payload.
  • methods can include assessing preparations (e.g, samples, lots, and/or batches) of a given composition, e.g, to confirm whether a composition comprises all necessary components, and, optionally, qualifying a compoistion as acceptable for use in administration if qualifying criteria (e.g, predefined qualifying criteria) are met; thereby evaluating, identifying, and/or producing (e.g, manufacturing) a nanoparticle composition.
  • methods as disclosed herein can have a variety of applications and can include, e.g, quality control at different stages of manufacture, analysis of a nanoparticle preparation prior to and/or after completion of manufacture (e.g, prior to or after distribution to a fill/finish environment or facility), and/or prior to and/or after release into commerce (e.g, before distribution to a pharmacy, a caregiver, a patient, or other end-user).
  • a nanoparticle preparation may be a drug substance (i.e., an active pharmaceutical ingredient or“API”) or a drug product (i.e., an API formulated for use in a subject such as a human patient).
  • a given nanoparticle preparation may be from a stage of manufacture or use that is prior to release to end-users; prior to packaging into individual dosage forms, such as single portions of powder or tablets; prior to determination that a batch can be commercially released, prior to production of a Certificate of Testing, Material Safety Data Sheet (MSDS) or Certificate of Analysis (CofA) of a preparation.
  • a nanoparticle preparation may be from an intermediate step in production, e.g, after formation of a nanoparticle comprising one or more payloads, but prior to further modification and/or purification of a drug substance.
  • evaluations of methods described in the present disclosure can be useful for guiding, controlling or implementing one or more of a number of activities or steps in a process of making, distributing, and monitoring and providing for a safe and efficacious use of a nanoparticle preparation. Accordingly, in some embodiments, e.g ., responsive to an evaluation, e.g. , depending on whether a criterion is met, a decision or step is taken. In some embodiments, methods can further include one or both of a decision to take a step and/or carrying out the step itself.
  • a step can include one in which a preparation (or another preparation for which the preparation is representative, or an intermediate of a preparation) is: classified; selected; accepted or discarded; released or processed into a drug product; rendered unusable for commercial release, e.g.
  • a preparation may undergo a repetition of a previous process step or subjected to a corrective process
  • formulated e.g, into drug substance or drug product; combined with another component, e.g, an excipient, buffer or diluent; disposed into a container; divided into smaller aliquots, e.g, unit doses, or multi-dose containers; combined with another nanoparticle preparation (e.g, nanoparticles with the same or different payloads); packaged; shipped; moved to a different location; combined with another element to form a kit; combined, e.g, placed into a package with a delivery device, diluent, or package insert; released into commerce; sold or offered for sale; delivered to an end-user; or administered to a subject.
  • based on a result of a determination or whether one or more subject entities is
  • methods disclosed herein may include making a decision:
  • a nanoparticle preparation may be formulated into drug substance or drug product; (b) as to whether a nanoparticle preparation may be reprocessed (e.g, a preparation may undergo a repetition of a previous process step, e.g., at any point in the manufacture process, e.g., another homogenization pass during microfluidization and nanoparticle formation); and/or (c) that a nanoparticle preparation may not be suitable for formulation into drug substance or drug product.
  • methods can include: formulating as referred to in step (a), reprocessing as referred to in step (b), or rendering a preparation unusable for commercial release, e.g, by labeling it or destroying it, as referred to in step (c).
  • methods can further include, e.g., one or more of: providing or obtaining a nanoparticle preparation (e.g., such as a nanoparticle drug substance or a precursor thereof); memorializing confirmation or identification of the nanoparticle preparation as comprising expected and sufficient payload (e.g., protein and DNA) using a recordable medium (e.g., on paper or in a computer readable medium, e.g., in a Certificate of Testing, Certificate of Analysis, Material Safety Data Sheet (MSDS), batch record, or Certificate of Analysis (CofA)); informing a party or entity (e.g., a contractual or manufacturing partner, a care giver or other end-user, a regulatory entity, e.g., the FDA or other U.S., European, Japanese, Chinese or other governmental agency, or another entity, e.g., a compendial entity (e.g., U.S.
  • a party or entity e.g., a contractual or manufacturing partner, a care giver or other end-
  • a nanoparticle preparation contains the expected payload in the expected quantity; selecting the nanoparticle preparation for further processing (e.g., processing (e.g., formulating) the nanoparticle preparation as a drug product (e.g., a pharmaceutical product) if the nanoparticle preparation is identified as containing the expected identiy and quantity of payload; reprocessing or disposing of the nanoparticle preparation if the nanoparticle preparation is not identified as containing the expected identity and/or quantity of payload and/or if the preparation contains something unexpected as detected through quality control analysis and release assays.
  • processing e.g., formulating
  • the nanoparticle preparation is identified as containing the expected identiy and quantity of payload
  • reprocessing or disposing of the nanoparticle preparation if the nanoparticle preparation is not identified as containing the expected identity and/or quantity of payload and/or if the preparation contains something unexpected as detected through quality control analysis and release assays.
  • methods include taking action (e.g., physical action) in response to methods disclosed herein.
  • a given nanoparticle preparation is classified, selected, accepted or discarded, released or withheld, processed into a drug product, shipped, moved to a different location, formulated, labeled, packaged, released into commerce, or sold or offered for sale, depending on whether the preselected relationship is met.
  • processing may include formulating, packaging (e.g., in a vial or other container), labeling, or shipping at least a portion of the nanoparticle preparation.
  • processing may include formulating, packaging (e.g., in a vial or other container), and labeling at least a portion of the nanoparticle as a particular drug product (e.g., ENP-501).
  • processing can include directing and/or contracting another party to process as described herein. Routes of Administration
  • provided nanoparticle compositions may be formulated for any appropriate route of delivery.
  • provided nanoparticles and/or nanoparticle compositions may be formulated for any route of delivery, including, but not limited to, bronchial instillation, and/or inhalation; buccal, enteral, interdermal, intra-arterial (IA), intradermal, intragastric (IG), intramedullary, intramuscular (IM), intranasal,
  • IP intraperitoneal
  • IV intravenous
  • IV intraventricular
  • mucosal mucosal
  • nasal spray and/or aerosol
  • oral PO
  • PR rectal
  • SQ subcutaneous
  • topical and/or transdermal e.g ., by lotions, creams, liniments, ointments, powders, gels, drops, etc.
  • transdermal e.g ., vaginal, vitreal, and/or through a portal vein catheter; and/or combinations thereof.
  • the present disclosure provides methods of administration of provided nanoparticle compositions via mucosal administration.
  • the present disclosure provides methods of administration of provided
  • nanoparticle compositions via oral administration.
  • present disclosure provides methods of administration of provided nanoparticle compositions via sublingual administration.
  • provided nanoparticles and/or nanoparticle compositions may be formulated for oral administration.
  • oral administration may be or comprise enteral administration.
  • oral administration is buccal, sublabial, and/or sublingual administration.
  • dosage forms for oral administration include tablets (e.g., to swallow, chew or dissolve in water or sublingually), capsules (e.g., chewable capsules e.g., with a coating that dissolves in the stomach or bowel to release the medication there), time-release or sustained-release tablets and capsules, powders, granules, teas, drops, liquid medications, and syrups.
  • the present disclosure provides, among other things, methods of administering to a subject in need thereof a nanoparticle composition including a plurality of nanoparticles, each of which is comprised of a biodegradable or biocompatible polymer, and at least one of a preparation of a payload and/or at least one preparation of a coating agent associated with the external surface of the nanoparticle.
  • a nanoparticle composition including a plurality of nanoparticles, each of which is comprised of a biodegradable or biocompatible polymer, and at least one of a preparation of a payload and/or at least one preparation of a coating agent associated with the external surface of the nanoparticle.
  • provided nanoparticle compositions are administered to a subject in need thereof so that, when administered, the payload (i.e., comprising protein to which subject is allergic) is hidden from immune system components for at least a period of time.
  • encapsulated contents of provided nanoparticle compositions are released into the system of a subject to whom a composition has been administered over a period of time.
  • payload of compositions (comprising protein to which a subject is allergic) are released over a period of time such that the subject does not have an anaphylactic reaction when exposed to encapsulated contents of the nanoparticle.
  • it is contemplated that such administration and exposure, repeated and with payload amount increased over a period of time, will result in a desensitization to one or more components of a payload.
  • treatment of a subject (e.g., for a period of time) with a sensitization and/or allergy (e.g., history of anaphylactic reaction to) a payload in a provided composition will result in decreased incidence and/or risk of reaction when exposed to one or more components of a payload of a provided nanoparticle composition.
  • a sensitization and/or allergy e.g., history of anaphylactic reaction to
  • a payload in a provided composition will result in decreased incidence and/or risk of reaction when exposed to one or more components of a payload of a provided nanoparticle composition.
  • the present disclosure provides methods of treating various diseases, disorders and/or conditions.
  • provided compositions may be administered to a subject for treatment and/or prevention of allergy, infection, cancer, and combinations thereof.
  • Exemplary suitable compositions include those described herein.
  • provided nanoparticle compositions are useful as vaccines to prevent and/or delay the onset of an allergic reaction.
  • provided nanoparticle compositions are useful as vaccines to lessen the severity and/or duration of a future allergic reaction.
  • provided nanoparticle compositions are useful as therapeutics to alleviate and/or arrest an allergic reaction in progress.
  • the subject in need thereof is suffering from an allergic condition as herein described, including, but not limited to allergic rhinitis, asthma, atopic eczema, anaphylaxis, insect venom, drug allergies, food allergies, and/or combinations thereof.
  • provided nanoparticle compositions may be used for treatment and/or prevention of allergies associated with anaphylactic allergens, e.g ., food allergens, insect allergens, and rubber allergens (e.g, from latex).
  • anaphylactic allergens e.g ., food allergens, insect allergens, and rubber allergens (e.g, from latex).
  • provided nanoparticle compositions may be used for treatment and/or prevention of allergies associated with food.
  • Food allergies are mediated through the interaction of IgE to specific proteins contained within the food.
  • common food allergens include proteins from nuts (e.g, from peanut, walnut, almond, pecan, cashew, hazelnut, pistachio, pine nut, brazil nut), dairy products (e.g, from egg, milk), seeds (e.g, from sesame, poppy, mustard), soybean, wheat, and fish (e.g, shrimp, crab, lobster, clams, mussels, oysters, scallops, crayfish).
  • provided nanoparticle compositions may be used for treatment and/or prevention of allergies associated with insect allergens.
  • insect allergens include, but are not limited to, proteins from insects such as fleas, ticks, ants, cockroaches, and bees.
  • allergens elicit a reaction when ingested, inhaled, and/or injected. Allergens can also elicit a reaction based solely on contact with the skin.
  • Latex is a well-known example. Latex products are manufactured from a milky fluid derived from the rubber tree (Hevea brasiliensis) and other processing chemicals. A number of the proteins in latex can cause a range of allergic reactions. Many products contain latex, such as medical supplies and personal protective equipment. Two types of reactions can occur in persons sensitive to latex: local allergic dermatitis and immediate systemic hypersensitivity (or anaphylaxis).
  • provided nanoparticle compositions may be used for treatment and/or prevention of allergies associated with local allergic dermatitis.
  • Local allergic dermatitis may develop within a short time after exposure to latex and generally includes symptoms of urticaria or hives.
  • the reaction is thought to be allergic and triggered by direct contact, not inhalation (Sussman et al, 1991, JAMA, 265:2844; incorporated herein by reference).
  • Symptoms of immediate systemic hypersensitivity vary from skin and respiratory problems (e.g ., urticaria, hives, rhinoconjunctivitis, swelling of lips, eyelids, and throat, wheezing, and coughing) to anaphylaxis which may progress to hypotension and shock.
  • Such a reaction may be triggered by inhalation or skin exposure to the allergen.
  • provided nanoparticle compositions may function to suppress and/or decrease a subject’s T H 2-type responses and/or enhance and/or increase a subject’s T H l-type responses.
  • provided nanoparticle compositions may function to enhance and/or increase a subject’s T H 2-type responses and/or suppress and/or decrease a subject’s T H l-type responses.
  • a subject’s T H 2-type responses are enhanced through targeting of a cell surface receptor for CpG oligonucleotides (e.g.,
  • provided nanoparticle compositions effectively treat and/or prevent all of a subject’s allergies falling into a particular class of allergy.
  • exemplary“classes” of allergies include, but are not limited to, anaphylactic allergies and non-anaphylactic allergies.
  • exemplary“classes” of allergies include, but are not limited to food allergies, insect allergies, pet dander allergies, pollen allergies, grass allergies, rubber allergies, and so forth.
  • provided nanoparticle compositions may be useful for treating all of a subject’s food allergies.
  • exemplary“classes” of allergies include, but are not limited to, particular individual foods which contain multiple allergens.
  • a“class” of allergies is“peanut” allergy
  • provided nanoparticle compositions may be useful for treating all of a subject’s allergies associated with all seven different peanut allergen proteins.
  • provided nanoparticle compositions may be useful for treating and/or preventing a single allergy, even though no allergy-specific antigen is included.
  • provided nanoparticle compositions may be useful for treating and/or preventing multiple different allergies.
  • provided nanoparticles may be useful for treating and/or preventing multiple different allergies.
  • compositions may be useful for treating and/or preventing substantially all of a subject’s allergies.
  • subjects suffering from and/or susceptible to allergy are frequently allergic to more than one allergen, e.g, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or more different allergens.
  • an provided nanoparticle composition may be used for treating and/or preventing at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or more different allergies in a single patient.
  • an provided nanoparticle composition is administered to a subject suffering from and/or susceptible to multiple different allergies, e.g ., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or more different allergies, such that the subject’s symptoms are reduced and/or improved.
  • an provided nanoparticle composition is administered to a subject suffering from and/or susceptible to multiple different allergies, e.g. , at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or more different allergies, such that onset of the subject’s symptoms is delayed.
  • a provided composition maybe used as an oral vaccine to treat allergy.
  • One of the major benefits of oral vaccines is the ability to generate both mucosal and systemic immunity. While oral vaccines have been developed previously, but they have been almost entirely directed to prevention of infectious disease, and have met with widely varying levels of success. For example, oral vaccines have been developed for anthrax, cholera, gastroenteritis, infant diarrhea, malaria, measles, and tuberculosis, among others (see Aziz et al., Oral Vaccines: New Needs, New Possibilities, 2007, BioEssays 29.6: 591-604; see also Silin et al., Oral Vaccination: Where are we?, Exp.
  • T-cells in the gut mucosa are either ab or gd types. Both CD4 and CD8 cells are found in the gut mucosa, which also carries B cells, monocytes/macrophages, dendrocytes and other immune cells. In fact, the gut is known to house -90% of the total number of
  • any therapy that is exposed to the gut environment has the potential to engender a wide variety of responses and be affected by any of several immune or other cells.
  • APC antigen presenting cell
  • M-cells and Peyer’s patches are popular targets of oral therapies
  • additional targets include, but are not limited to, enterocytes, mesenteric lymph nodes, and intestinal epithelial cells.
  • Each APC may be targeted by various embodiments.
  • Oral immunization is known to generate significant quantities of secretory IgA (slgA), which is known to play a major role in mucosal defense against pathogens.
  • slgA secretory IgA
  • the value of slgA is questionable when one considers non-mucosal pathogens or conditions.
  • Various embodiments recognize this and do not trigger large amounts of slgA release, instead substantially generating a Th2 response.
  • oral tolerance is a phenomenon where oral antigen exposure can lead to immune tolerance and a suppression of the systemic immune response to subsequent challenges.
  • the development of oral tolerance is not an automatic feature of oral antigen exposure, but rather depends on several factors including, but not limited to, age of subject, MHC restriction, delivery site, nature, size and dose of antigen, degree of antigenic uptake, and processing and frequency of administration of antigen.
  • Oral tolerance is thought to be mediated by several immunological mechanisms including: induction of regulatory T-cells (suppressors) that downregulate specific cytokines including IL-4, IL-10, and TGF-b, functional of clonal deletion of effector cells, and antibody-mediated suppression (see Silin et al.).
  • compositions are able to present antigen to APCs without inducing oral tolerance. Without wishing to be held to a particular theory, it is possible certain embodiments are able to present larger quantities of antigen to the immune system than traditionally known methods of oral immunization. It is suspected that oral tolerance may manifest, at least in part, due to very small amounts of antigen being presented to APCs (see Silin et ah, Overcoming immune tolerance during oral vaccination against actinobacillus pleuropneumoniae, 2002, J Vet. Med. 49: 169-175). In some embodiments, provided
  • compositions present antigens to APCs in such a manner as to promote immune tolerance.
  • provided nanoparticle compositions are useful as vaccines to prevent and/or delay the onset of an infectious disease.
  • provided nanoparticle compositions are useful as vaccines to lessen the severity and/or duration of a future infectious disease.
  • provided nanoparticle compositions are useful as therapeutics to alleviate and/or arrest an infectious disease in progress.
  • the subject in need thereof is suffering from an infection caused by, but not limited to viruses, prions, bacteria, viroids, macroparasites, fungi, and/or combinations thereof.
  • the subject is suffering from a primary infection.
  • the subject is suffering from a secondary infection. In some embodiments, the subject is suffering from an active symptomatic infection. In some embodiments, the subject is suffering from an active asymptomatic infection (i.e ., infection is active, but does not produce noticeable symptoms; e.g., silent or subclinical infection). In some embodiments, the subject is suffering from a latent infection (i.e., inactive or dormant infection).
  • Exemplary infections that may be treated by some embodiments include, but are not limited to actinomycosis, African sleeping sickness, AIDS, anthrax, hemorrhagic fevers, bacterial pneumonia, candidiasis, cellulitis, Chagas disease, chickenpox, cholera, C.
  • provided nanoparticle compositions are useful as vaccines to prevent and/or delay the onset of a cancer.
  • provided nanoparticle compositions are useful as therapeutics to alleviate and/or arrest an cancer in progress.
  • the subject in need thereof is suffering from a cancer including, but not limited to acute lymphoblastic leukemia (ALL); adrenocortical carcinoma; AIDS-related cancers including AIDS-related lymphoma; anal cancer; appendix cancer; astrocytomas; basal cell carcinoma; bile duct cancer; bladder cancer; bone cancer (e.g., osteosarcoma and malignant fibrous histiocytoma); brainstem glioma; brain cancer; brain tumors; breast cancer; bronchial adenomas/carcinoids; Burkitt lymphoma; carcinoid tumors (e.g., childhood and gastrointestinal tumors); carcinoma (including carcinoma of unknown primary (CUP) whose origin or developmental lineage is unknown but that possess specific molecular, cellular, and histological characteristics of epithelial cells); central nervous system lymphoma; cerebellar astrocytoma; malignant glioma; cervical cancer; childhood cancers; chronic lympho
  • ALL acute
  • extracranial germ cell tumor extracranial germ cell tumor; extragonadal germ cell tumor; ovarian germ cell tumor;
  • extrahepatic bile duct cancer eye cancer; intraocular melanoma; retinoblastoma; gallbladder cancer; gastric cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal tumor (GIST); gestational trophoblastic tumor; gastric carcinoid; hairy cell leukemia; head and neck cancer; heart cancer; hepatocellular (liver) cancer; Hodgkin lymphoma; hypopharyngeal cancer;
  • hypothalamic and visual pathway glioma glioma; intraocular Melanoma; Islet Cell Carcinoma
  • kaposi sarcoma soft tissue sarcoma; uterine sarcoma; kidney cancer (renal cell carcinoma); laryngeal cancer; leukemias (including acute lymphoblastic or acute lymphocytic leukemia, acute myeloid or acute myelogenous leukemia, chronic lymphocytic or chronic lymphocytic leukemia, chronic myelogenous or chronic myeloid leukemia); Lip and Oral Cavity Cancer; liposarcoma; liver cancer; lung cancer (including non-small cell and small cell); lymphomas (e.g., AIDS-related, Burkitt, cutaneous T-Cell, Hodgkin, non-Hodgkin, Primary Central Nervous System); macroglobulinemia; medulloblastoma; melanoma; Merkel Cell Carcinoma; mesothelioma (e.g., adult malignant mesothelioma, childhood mesothelioma
  • Myelodysplastic/Myeloproliferative Diseases Myelogenous Leukemia; Myeloid Leukemia; (e.g. Adult Acute; nasal cavity and paranasal sinus cancer; nasopharyngeal carcinoma; neuroblastoma; oral cancer; oropharyngeal cancer; ovarian cancer; ovarian epithelial cancer (Surface epithelial- stromal tumor); ovarian germ cell tumor; ovarian low malignant potential tumor; pancreatic cancer; paranasal sinus and nasal cavity cancer; parathyroid cancer; penile cancer; pharyngeal cancer; pheochromocytoma; pineal astrocytoma; pineal germinoma; pineoblastoma and supratentorial primitive neuroectodermal tumors; pituitary adenoma; pleuropulmonary blastoma; prostate cancer; rectal cancer; renal pelvis and ureter and transitional cell cancer;
  • rhabdomyosarcoma Sezary syndrome; skin cancer (including melanoma and nonmelanoma); skin carcinoma; small intestine cancer; squamous cell carcinoma; stomach cancer; testicular cancer; throat cancer; thymoma and thymic carcinoma; thyroid cancer; urethral cancer;
  • endometrial uterine cancer vaginal cancer
  • vulvar cancer a malignant uterine cancer
  • provided nanoparticle and/or pharmaceutical compositions are administered according to a dosing regimen sufficient to achieve a desired immunological reaction.
  • a dosing regimen is sufficient to achieve a desired immunological reaction if its administration to a relevant patient population shows a statistically significant correlation with achievement of the desired immunological reaction.
  • the desired immunological reaction is a reduction in the degree and/or prevalence of symptoms of a disease, disorder or condition (e.g., allergy, infection and/or cancer) of at least about 20%, about 25%; about 30%; about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 70%, about 71%, about 72%
  • composition is administered according to a dosing regimen sufficient to achieve a reduction in the degree and/or prevalence of symptoms of a disease, disorder or condition (e.g., allergy, infectious disease, cancer) of a specified percentage of a population of patients to which the composition is administered.
  • a disease, disorder or condition e.g., allergy, infectious disease, cancer
  • the specified percentage of population of patients to which the composition was administered is at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%
  • administration of at least one provided nanoparticle and/or pharmaceutical composition according to a dosing regimen is sufficient to achieve a reduction in the degree and/or prevalence of a disease, disorder or conditions (e.g., allergy, infectious disease, cancer) of at least about 20% in at least about 50% of the population of patients to which the composition was administered.
  • administration of at least one provided nanoparticle and/or pharmaceutical composition according to a dosing regimen is sufficient to achieve a reduction in the degree and/or prevalence of a disease, disorder or conditions (e.g., allergy, infectious disease, cancer) of at least about 30% in at least about 50% of the population of patients to which the composition was administered.
  • At least one provided nanoparticle and/or pharmaceutical composition is administered according to a dosing regimen sufficient to achieve a delay in the onset of symptoms of a disease, disorder or conditions (e.g., allergy, infectious disease, cancer). In some embodiments, at least one provided nanoparticle and/or pharmaceutical composition is administered according to a dosing regimen sufficient to prevent the onset of one or more symptoms of a disease, disorder or conditions (e.g., allergy, infectious disease, cancer).
  • a provided dosing regimen comprises or consists of a single dose. In some embodiments, a provided dosing regimen comprises or consists of multiple doses, separated from one another by intervals of time that may or may not vary. In some embodiments, a provided dosing regimen comprises or consists of dosing once every 20 years, once every 10 years, once every 5 years, once every 4 years, once every 3 years, once every 2 years, once per year, twice per year, 3 times per year, 4 times per year, 5 times per year, 6 times per year, 7 times per year, 8 times per year, 9 times per year, 10 times per year, 11 times per year, once per month, twice per month, three times per month, once per week, twice per week, three times per week, 4 times per week, 5 times per week, 6 times per week, daily, twice daily, 3 times daily, 4 times daily, 5 times daily, 6 times daily, 7 times daily, 8 times daily, 9 times daily, 10 times daily, 11 times daily, 12 times daily, or hourly
  • a provided dosing regimen comprises or consists of an initial dose with one or more booster doses.
  • one or more booster doses are administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 1 month, 2 months, 6 months, 1 year, 2 years, 5 years, 10 years, or longer than 10 years after the initial dose.
  • an initial dose comprises a series of doses administered over a period of time.
  • an initial dose comprises a series of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more doses administered at regular intervals, e.g., intervals that are close in time to one another, such as 5 minute intervals,
  • an initial dose and booster doses contain the same amount of provided nanoparticles and/or nanoparticle composition. In some embodiments, an initial dose and booster doses contain different amounts of provided nanoparticles and/or nanoparticle composition.
  • provided nanoparticles and/or nanoparticle compositions are administered at dosage levels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg (e.g., of payload, nanoparticles, or nanoparticle composition), from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day.
  • provided nanoparticles and/or nanoparticle compositions are formulated into a unit dose.
  • a unit dosage is about 10 mg, about 25 mg, about 50 mg, about 100 mg, about 250 mg, about 500 mg, about 1 g, about 5 g, about 10 g, about 25 g, about 50 g, about 100 g, or more than about 100 g.
  • the amount of provided nanoparticles and/or nanoparticle composition present in a particular unit dose depends on the subject to which the composition is to be administered. To give but a few examples, in some embodiments, a unit dose appropriate for a mouse is smaller than a unit dose that is appropriate for a rat, which is smaller than a unit dose that is appropriate for a dog, is smaller than a unit dose that is appropriate for a human.
  • a provided dosing regimen comprises or consists of administration of multiple doses over the course of the subject’s entire lifespan.
  • a provided dosing regimen comprises administration of multiple doses over the course of several years (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 years).
  • a provided dosing regimen comprises or consists of multiple doses over the course of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.
  • a subject’s baseline allergic response is determined by one or more of a variety of methods, including, but not limited to, (1) performing a prick skin test (PST) of one or more of the subject’s 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 allergens, and measuring the wheal and flare response to the PST; (2) measuring blood serum IgE levels; (3) noting the subject’s own description of her typical symptoms (e.g, nature, severity, and/or duration of symptoms) upon exposure to one or more of her 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 allergens; (4) exposing the subject to a certain dose of one or more of her 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 allergens (e.g.
  • a subject’s allergic response is monitored using any combination of methods, e.g, methods (1) - (6) described above, throughout the course of the treatment regimen and/or after the treatment regimen is completed, e.g, at regular intervals.
  • allergic response is monitored daily, weekly, bi-weekly, monthly, 6 times per year, 4 times per year, 3 times per year, 2 times per year, once per year, every 2 years, every 5 years, and/or every 10 years, etc.
  • a subject is challenged with a single allergen and/or multiple allergens, e.g, a subset of the subject’s allergens (e.g, allergens to which the subject is known to be allergic) and/or all of the subject’s allergens (e.g, allergens to which the subject is known to be allergic).
  • allergy challenge is performed after 1 week, 2 weeks, 1 month, 2 months, 6 months, and 1 year after initiation of treatment.
  • provided nanoparticles and/or compositions may be administered via any medically acceptable route.
  • a provided composition may be administered via intravenous administration; intradermal administration; transdermal administration; oral administration; subcutaneous administration; transmucosal administration; and/or combinations thereof.
  • exemplary routes of transmucosal administration include, but are not limited to buccal administration; nasal administration; bronchial administration; vaginal administration; rectal administration;
  • sublingual administration and/or combinations thereof.
  • provided therapy e.g., provided nanoparticles and compositions
  • a subject may have previously received or be currently receiving at least one other therapy.
  • the at least one other therapy is administered to a subject who has previously received or is currently receiving nanoparticle therapy as described herein.
  • a provided nanoparticle composition is utilized in a pharmaceutical formulation that is separate from and distinct from the pharmaceutical formulation containing another therapeutic agent.
  • a provided nanoparticle composition is admixed with the composition comprising another therapeutic agent.
  • a provided nanoparticle composition is produced individually, and the provided nanoparticle composition is simply mixed with another composition comprising another therapeutic agent.
  • provided nanoparticle compositions can be administered concurrently with, prior to, or subsequent to, one or more other therapeutic agents (e.g ., desired known allergy therapeutics).
  • a provided nanoparticle composition useful for treating allergy may be administered concurrently with a known allergy therapeutic that is also useful for treating allergy), or they may achieve different effects (for example, a provided nanoparticle composition that is useful for treating allergy may be administered concurrently with a therapeutic agent that is useful for alleviating adverse side effects, for instance, inflammation, nausea, etc.).
  • provided nanoparticle compositions in accordance with the present disclosure are administered with a second therapeutic agent that is approved by the U.S. Food and Drug Administration (FDA).
  • FDA U.S. Food and Drug Administration
  • the terms“in combination with” and“in conjunction with” mean that the provided nanoparticle compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics. In general, each substance will be administered at a dose and/or on a time schedule determined for that agent. Allergy Therapies
  • provided nanoparticles and/or compositions for the treatment of allergy may, in some embodiments, be administered in combination with, for example, one or more antihistamines ⁇ i.e., histamine antagonist), corticosteroids including glucocorticoids; epinephrine (adrenaline); theophylline (dimethylxanthine); cromolyn sodium; anti-leukotrienes; anti-cholinergics; decongestants; mast cell stabilizers; immunotherapy
  • antihistamines ⁇ i.e., histamine antagonist
  • corticosteroids including glucocorticoids; epinephrine (adrenaline); theophylline (dimethylxanthine); cromolyn sodium; anti-leukotrienes; anti-cholinergics; decongestants; mast cell stabilizers; immunotherapy
  • monoclonal anti-IgE antibodies e.g, omalizumab
  • combinations thereof e.g, omalizumab
  • antihistamines include, but are not limited to Azelastine;
  • Diphenhydramine (Benadryl); Doxylamine; Ebastine; Embramine; Fexofenadine; Levocetirizine; Loratadine; Olopatadine (Patanol); Phenindamine (Nolahist and Thephorin); Pheniramine (Avil); Phenyltoloxamine; Promethazine; Pyrilamine; Rupatadine; Tripelennamine; Triprolidine; and/or combinations thereof.
  • Exemplary corticosteroids and glucocorticoids include, but are not limited to
  • Beclometasone dipropionate and Beclomethasone (Clenil, Qvar, Beconase AQ, Alanase, Vancenase); Budesonide (Rhinocort, Rhinosol, Pulmicort, Budicort, Symbicort, Noex);
  • Ciclesonide (Alvesco, Omnaris, Omniair); Flunisolide (Aerobid); Fluticasone (Veramyst);
  • Exemplary forms of cromolyn sodium include, but are not limited to, Rynacrom;
  • Nasalcrom Prevalin; Intal; Optocrom; Optrex; Gastrocrom; Intercron; and/or combinations thereof.
  • Exemplary anti-leukotrienes and leukotriene inhibitors include, but are not limited to Montelukast (Singulair, Montelo-lO, and Monteflo); Zafirlukast (Accolate, Accoleit, Vanticon); Pranlukast; Zileuton (Zyflo, Zyflo CR); and/or combinations thereof.
  • Exemplary anti-cholinergics include, but are not limited to, Ipratropium bromide
  • Benztropine (Cogentin); Oxitropium (Oxivent); Tiotropium (Spiriva); Glycopyrrolate (Robinul); Oxybutinin (Ditropan, Driptane, Lyrinel XL); Tolterodine (Detrol, Detrusitol); Chlorphenamine (Chlor-Trimeton); Diphenhydramine (Benadryl, Sominex, Advil PM, etc.) Dimenhydrinate (Dramamine); Bupropion (Zyban, Wellbutrin); Hexamethonium; Tubocurarine;
  • Dextromethorphan Mecamylamine
  • Doxacurium Doxacurium
  • Exemplary decongestants include, but are not limited to, Ephedrine; Levo- methamphetamine; Naphazoline; Oxymetazoline; Phenylephrine; Phenylpropanolamine;
  • Exemplary mast cell stabilizers include, but are not limited to, Cromoglicic acid;
  • Ketotifen and Ketotifen fumarate (Zaditor, Zaditen, Alaway, Zyrtec Itchy-Eye Drops, Claritin Eye); Methyl xanthines; and/or combinations thereof.
  • exemplary known allergy therapeutics that can be administered in combination with provided nanoparticle compositions in accordance with the present disclosure include, but are not limited to, any of the therapeutics described in US Patent Numbers 5,558,869, 5,973,121, 6,835,824, 6,486,311, and/or 7,485,708, and/or in US Patent Publication Numbers 2003/0035810, 2003/0202980, 2004/0208894, 2004/0234548,
  • provided nanoparticles and/or compositions for the treatment of infectious disease may, in some embodiments, be administered in combination with, for example, one or more sulfaniliamides; folic acid analogs; beta-lactams such as penicillins, cephalosporins, and carbapenems; aminoglycosides such as streptomycin, kanamycin, neomycin, and gentamycin; tetracyclines such as chlortetracycline, oxytetracycline, and doxycycline; macrolides; lincosamides; streptogramins; fluoroquinolones, rifampin, mupirocin, cycloserine, aminocyclitols, glycopeptides, oxazolidinones, and derivatives/analogs and/or combinations thereof.
  • sulfaniliamides such as penicillins, cephalosporins, and carbapenems
  • antiviral agents include, but are not limited to Abacavir, Aciclovir,
  • Edoxudine Efavirenz, Emtricitabine, Enfuvirtide, Entecavir, Entry inhibitors, Famciclovir, Fomivirsen, Fosamprenavir, Foscarnet, Fosfonet, Imunovir, Idoxuridine, Imiquimod, Indinavir, Inosine, Interferon type III, Interferon type II, Interferon type I, Interferon, Lamivudine,
  • Valganciclovir Valganciclovir, Vicriviroc, Vidarabine, Viramidine, Zalcitabine, Zanamivir, Zidovudine, and derivatives/analogs and/or combinations thereof.
  • antifungal agents include, but are not limited to polyene agents such as amphotericin, candicidin, filipin, hamycin, natamycin, nystatin, and rimocidin; imidazole, triazole and thiazole agents such as bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, alboconazole, fluconazole, isavuconazole, posaconazole, ravuconazole, terconazole, voriconazole, and abafungin; allylamines such as amorolfm, butenafme, naftafme, and terbinafme; and echinocandin
  • provided nanoparticles and/or compositions for the treatment of infectious disease may be administered in combination with, for example, an antibiotic such as an antibacterial agent, an antiviral agent, and/or an antifungal agent.
  • provided pharmaceutical compositions may be administered in combination with a vaccine.
  • provided nanoparticles and/or compositions for the treatment of cancer may be administered in combination with, for example, alkylating agents, antimetabolite agents, and/or other anticancer medications.
  • alkylating agents include, but are not limited to polyfunctional alkylating agents such as cyclophosphamide (Cytoxan), mechlorethamine, melphan (Alkeran), chlorambucil (Leukeran), thiopeta (Thioplex), and busulfan (Myleran); procarbazine, dacarbazine, altretamine, cisplatin, and derivatives/analogs and/or combinations thereof.
  • polyfunctional alkylating agents such as cyclophosphamide (Cytoxan), mechlorethamine, melphan (Alkeran), chlorambucil (Leukeran), thiopeta (Thioplex), and busulfan (Myleran); procarbazine, dacarbazine, altretamine, cisplatin, and derivatives/analogs and/or combinations thereof.
  • antimetabolite agents include, but are not limited to methotrexate; purine antagonists such as mercaptopurine (6-MP), thioguanine (6-TG), fludarabine phosphate, cladribine, and pentostatin; pyrimidine antagonists such as fluorouracil, cytarabine, and azacitidine; plant alkaloids such as vinblastine (Velban), vincristine (Oncovin), etoposide (VP- 16), teniposide (Vimon), topotecan (Hycamtin), irinotecan (Camptosar), paclitaxel (Taxol), and docetaxel (Taxotere) and derivatives/analogs and/or combinations thereof.
  • purine antagonists such as mercaptopurine (6-MP), thioguanine (6-TG), fludarabine phosphate, cladribine, and pentostatin
  • pyrimidine antagonists such
  • Exemplary other anticancer agents include, but are not limited to amsacrine; hydroxyurea (Hydrea); asparaginase (El-spar); mitoxantrone (Novantrone); mitotane; retinoic acid, bone marrow growth factors, amifostine, and derivatives/analogs and/or combinations thereof.
  • kits comprising provided nanoparticles, nanoparticle compositions, and/or pharmaceutical compositions.
  • a kit may comprise (i) at least one provided nanoparticle composition; and (ii) at least one
  • kits include multiple ( e.g ., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
  • kits include multiple (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) populations of provided nanoparticles having different functional elements (e.g, microbial mimic entities).
  • multiple populations of provided nanoparticles are packaged separately from one another in provided kits.
  • provided kits may include provided compositions and one or more other therapeutic agents intended for administration with the provided compositions.
  • the present disclosure provides pharmaceutical packs or kits including provided nanoparticles and/or nanoparticle compositions to be used in treatment methods according to the present disclosure.
  • pharmaceutical packs or kits include preparations or pharmaceutical compositions containing provided nanoparticles and/or nanoparticle compositions in one or more containers filled with optionally one or more additional ingredients of pharmaceutical compositions in accordance with the present disclosure.
  • the pharmaceutical pack or kit includes an additional approved therapeutic agent for use in combination therapies, as described herein.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical products, which notice reflects approval by the agency of manufacture, use, or sale for human administration.
  • kits are provided that include provided nanoparticle compositions and instructions for use.
  • Pharmaceutical doses or instructions therefor may be provided in a kit for administration to an individual suffering from and/or susceptible to a disease, disorder or condition (e.g., allergy, infectious disease, cancer).
  • Example 1 Preparation of polymer nanoparticles comprising protein and DNA and coated with OEE
  • This Example describes an exemplary provided method for preparation of certain polymer nanoparticles (e.g polymer nanoparticles comprising a payload and/or a coating) in accordance with the present disclosure.
  • a representative nanoparticle manufacturing process is described below and shown in whole (e.g. Figure 1) and in part (e.g. Figures 2-5).
  • One of skill in the art will appreciate that certain conditions and specific values as described herein may be changed as desired.
  • [0450] Preparation of solid block material comprising PLGA, protein, and DNA (see, e.g., Figures 1 (steps 1-4), and Figure 2)
  • a solution comprising polymer e.g. PLGA, which makes a“PLGA solution”
  • PLGA polymer
  • organic solvent e.g. DMSO
  • the PLGA solution was prepared using magnetic stirring (without sonication). In this Example, the temperature was maintained between approximately 25 - 30°C, in order to prevent DMSO from freezing as well as to lower the viscosity of the solution and increase the speed at which PLGA dissolves.
  • a solution comprising a payload (“payload solution”) was prepared by dissolving payload comprising protein and DNA (e.g., crude peanut extract and sheared E. coli DNA) in water, wherein the protein concentration (e.g. crude peanut extract) in the solution was, by way of non limiting example, 6 mg/mL and the DNA concentration (e.g. sheared E. coli DNA) was, by way of non-limiting example, 0.06 mg/mL.
  • the payload solution was diluted to 3 mg/mL of protein and 0.03 mg/mL of DNA, and the pH of the payload solution was adjusted to pH 9 using NaOH. All the solutions were prepared at room temperature
  • the payload solution was then added to the polymer (e.g. PLGA) solution.
  • the volumetric ratio of PLGA solution: payload solution was 96:4, though this proportion may be varied to suit other applications or desired embodiments.
  • the mixture of PLGA and payload solutions was substantially homogenous.
  • block material a frozen, solid block material
  • the block material is comprised of protein, DNA, and polymer (here, crude peanut extract, sheared E. coli DNA, and PLGA).
  • the dry cake may be heated, for example, to 100 °C for approximately 1 minute and cooled to room temperature. The dry cake may then be cooled and frozen to form a block material.
  • Steps: 5) The block material of step 4 4 (comprising PLGA, crude peanut extract, and DNA) was maintained as frozen (at a temperature close to the boiling point of liquid nitrogen (about -190 °C)) while pulverized using a mortar and pestle. To minimize condensation, the grinding process was performed under dry conditions. Micro-sized granules resulted from grinding and were suspended in n-propanol, forming a flowable microparticle suspension. Here, the starting concentration of n-propanol was approximately 7 mg/mL, diluted down to a final concentration of about 3.25 mg/mL after addition to hot propanol in the homogenizer.
  • the suspension was homogenized in a microfluidic homogenizer at 95 °C, to produce nanoparticles.
  • the solution in the homogenizer may be either recycled through the chamber (rather than performing discrete passes) or run through the homogenizer in discrete passes.
  • a recycled solution scenario may use a volume of about 100 mL which flows at a liquid flow rate of 200 mL/min, with the homogenizer run for 30 mins for an overall ratio of solution volume to pumped volume of 1 :60.
  • a discrete pass scenario may use approximately 2-3 discrete passes through the chamber to produce an acceptable particle size . It is estimated that the shear rate in the system was between 10 6 to 10 7 s 1 .
  • nanoparticle suspension Without being held to a particular theory, it is contemplated that the addition of PVA1 is helpful in minimizing aggregation of the nanoparticles in suspension.
  • the volumetric ratio of nanoparticle suspension: PVA1 solution was 1 :4.
  • the resulting suspension (comprising nanoparticles, allergen protein extract, and DNA), propanol, water, and PVA1) was passed through the microfluidic homogenizer. Homogenization heating was turned off, but homogenization was continued.
  • Trehalose dehydrate granules were added directly to the nanoparticle suspension (comprising loaded nanoparticles, which comprise polymer, protein and DNA), which suspension may include residual PVA1, in water and propanol, here 80/20 water/propanol, and the mixture was lyophilized to form a dried mixture (e.g., in this Example, a dried cake).
  • the dried mixture e.g. cake
  • the dried mixture includes nanoparticles, PVA1, and trehalose.
  • the dried cake may then be dissolved in, e.g. 10 mM ammonium bicarbonate, followed by removal of free protein by, e.g. centrifugation.
  • free protein is protein that is not encapsulated within a nanoparticle and may be, e.g. freely suspended in a nanoparticle suspension and/or loosely associated with nanoparticles within a suspension.
  • centrifugation may be performed after lyophilization in step 8.
  • the lyophilized cake may be suspended in propanol (e.g., be diluted, for example, by a factor of 50 or more in an aqueous buffer such as water), followed by
  • Steps 10-14 (preparing a coating agent, e.g. OEE Solution), may be performed either in parallel (concurrently) with steps 1-9, or sequentially, following, e.g. Steps 1-9.
  • OEE Organic E. Coli. Extract
  • Trehalose dihydrate was added to the OEE Solution at a concentration of such that the final amount of dehydrated trehalose was 11 times the mass of the OEE (e.g. for 2 g/L OEE, the concentration of trehalose is 22 g/L).
  • the solid dispersion may then be further processed, for example, ground and sieved, or otherwise processed to make the composition suitable for storage or administration.
  • a flowable powder may be obtained from step 16
  • the present example describes preparation of a representative coating agent, (e.g. a representative hydrophobic coating), which, in this Example is hydrophobic (organic) E. coli extract (i.e., an organic extract of an E. coli cell culture,“OEE”), and was manufactured in accordance with standard procedures known in the art.
  • a representative coating agent e.g. a representative hydrophobic coating
  • hydrophobic (organic) E. coli extract i.e., an organic extract of an E. coli cell culture,“OEE”
  • a master and working cell bank of the production ( E.coli ) strain may be established prior to clinical manufacture.
  • an extract is prepared using the well-known phenol-chloroform- petroleum ether process, with the exception that hexane is used in lieu of petroleum ether, as petroleum ether is a pharmaceutically unacceptable solvent.
  • Dried bacterial cells are suspended in the phenol-chloroform-hexane (PCH) mixture for about 30 minutes.
  • PCH phenol-chloroform-hexane
  • the slurry is then centrifuged to remove the remaining cells.
  • the remaining cells are then treated with PCH twice more.
  • the combined organic extracts are evaporated to remove the volatile organic solvents. Water is added drop-wise to the phenol concentrate to precipitate the LPS and lipids.
  • the precipitated OEE is then washed with 95% phenol followed by acetone, suspended in water, lyophilized, and stored frozen prior to use in the nanoparticle manufacturing process. It is expected that the OEE will be comprised mainly of bacterial LPS and lipids.
  • CFU colony forming unit
  • KDO keto-deoxyoctulosonic acid
  • LAL limulus amebocyte lysate
  • LC/MS liquid chromatography/mass spectrometry
  • LPS lipopolysaccharide
  • NMT not more than
  • TBD to be determined
  • USP United Stated Pharmacopeia
  • Allergen Extract ⁇ i.e., crude peanut extract) for use in accordance with the present disclosure.
  • the present Example describes preparation of a Peanut Allergen Extract, containing Ara hl, Ara h2, and Ara h3 antigens, among others, for use in accordance with the present disclosure.
  • the present Example also describes preparation of recombinant modified peanut proteins (mAra hl, mAra h2, and mAra h3, wherein m stands for modified), for use in accordance with the present disclosure.
  • a representative method of Arachis hypogaea (Peanut) Allergen Extract is as follows: Commercially available roasted peanuts in shells (White Rose Brand, NJ) were de-shelled, homogenized in phosphate buffered saline (PBS, pH 7.4), and subjected to acetone extraction. The final concentration of peanut protein in PBS was determined by bicinchoninic acid (BCA) assay. Peanut allergen extract was then combined with aqueous E. coli extract (“AEE”) and prepared, e.g., substantially as described herein.
  • AEE aqueous E. coli extract
  • Recombinant peanut proteins were prepared as follows. The three recombinant modified peanut proteins (mAra hl, mAra h2, and mAra h3) were separately expressed in E. coli strain BLR(DE3), and the E.coli were subsequently killed using heat and phenol. The expressed proteins remained encapsulated within the dead E. coli., which resulted in three whole-cell suspensions referred to as EMP-l, EMP-2, and EMP-3 ⁇ i.e., encapsulated mAra h 1, mAra h 2, and mAra h 3, respectively). Each whole-cell suspension was then used to prepare AEE, which included the expressed recombinant peanut protein in the aqueous phase, for fabrication of a nanoparticle composition. Characterization of crude peanut extract
  • Crude peanut extract was evaluated both before and after incorporation into nanoparticles to ensure that processing into nanoparticles did not materially alter peanut extract used in compositions as described herein. Compositions were examined for presence of peanut components using western blot analysis. Crude peanut extract was isolated from nanoparticles, for comparison to crude peanut extract that had not been incorporated into nanoparticle, using standard methods for performing protein separation on agarose. Separated proteins were then transferred from the gels onto membranes and the membranes were probed for presence and quantity of Ara hl, Ara h2, and Ara h3. Briefly, membranes were dehydrated and incubated in a blocking buffer ( ⁇ l% normal goat serum in PBS-T (phosphate buffered saline with 0.05%
  • chemiluminescent (ECL) substrate and processed for visual detection of bands.
  • the present Example describes a representative procedure for a study (clinical trial) examining effect(s) of an exemplary nanoparticle composition, which composition comprises an exemplary payload and/or coating, and is administered to subjects with or without allergy (to, e.g., peanuts).
  • a study is performed using an exemplary nanoparticle composition in orally disintegrating tablet form, comprising an exemplary payload and a coating.
  • the exemplary nanoparticle composition may be made in accordance with, e.g. a procedure such as described in, e.g. Example 1.
  • ENP-501 (“Investigational Product”), which is further described herein, is a representative composition for use in clinical analysis.
  • This study may be conducted in one or more parts (e.g. two sequential parts) over a period of time (e.g. 18 months), wherein Part II occurs following, e.g., successful completion of Part I.
  • Successful completion of Part I may be determined, for example, by finding no cause for substantial safety concerns.
  • Part I will be an open-label, single-arm, dose escalation study to evaluate the safety and tolerability of ENP 501 in non- peanut allergic participants who are 18 to 50 years of age.
  • Part II will be a randomized, double-blind, placebo-controlled, dose escalation study to evaluate the safety, tolerability, and pharmacodynamics of ENP 501 compared to placebo in participants who are 12 to 50 years of age and have peanut allergy.
  • the Study (Parts I and II) will take place at two separate sites. A summary of the study design, including objectives and endpoints, as well as details regarding the Investigational Product is described herein.
  • disintegrating product e.g., powder or tablet
  • the form of the Investigational Product is an orally-disintegrating formulation such as a powder for buccal mucosal
  • ENP-501 a biologic that includes allergenic extract of common peanut ⁇ Arachis hypogaea
  • encapsulated along with sheared E. coli DNA within PLGA nanoparticles see, e.g. Figure 6) , which nanoparticles are coated with OEE (see, e.g., Figure 7).
  • Powders will be used in the initial trials, and other formulations, such as, e.g., tablets may be used if, for any reason, powders are found to be ineffective or otherwise problematic in any way.
  • the active ingredients in the Investigational Product include Organic Escherichia (E.) coli extract- (OEE-) coated poly(lactic-co-glycolic acid)- (PLGA-) encapsulated Arachis hypogaea peanut extract and sheared A. coli deoxyribonucleic acid (DNA) nanoparticles.
  • the OEE includes predominantly bacterial lipopolysaccharide (LPS).
  • the nanoparticles are manufactured as a spray-dried preparation with trehalose added as a bulking agent to prevent aggregation.
  • the inactive ingredients include D-mannitol, xylitol, microcrystalline cellulose, crospovidone, magnesium stearate, and dibasic calcium phosphate anhydrous.
  • the Investigational Product will be pharmacy compounded for this trial, including 0.25, 1, 8, 64, and 500 pg peanut protein/dose strengths.
  • the placebo to be used in this trial will be orally- disintegrating version of the Investigational Product (e.g., a tablet or powder) that matches the form of the Investigational Product, but that does not contain any active pharmaceutical ingredient.
  • the inactive ingredients will be the same as for the Investigational Product.
  • the dosage form of the investigational drug is an orally-disintegrating tablet or powder for buccal mucosal administration. Multiple strengths of the Investigational Product will be pharmacy compounded for this trial, including 0.25, 1, 8, 64, and 500 pg peanut protein/dose strengths.
  • Placebo to be used in this trial will be orally-disintegrating powder for buccal mucosal administration that matches the Investigational Product, but that does not contain any active pharmaceutical ingredient.
  • the inactive ingredients will be the same as for the
  • Investigational Product or placebo will be dispensed in HDPE bottles or individually packed tubes (e.g., 1 tube per individual dose) with child resistant closures, comparable to bottles used to supply the Investigational Product to the Investigational Site pharmacy. Actual numbers and strengths of Investigational Product or placebo taken at the visit will depend on how the participant tolerates the Investigational Product at the visit. If the participant takes fewer Investigational Product or placebo than dispensed, unused Investigational Product or placebo will be returned to the Investigational Site pharmacy and will not be reused.
  • Investigational Product or placebo will be dispensed in HDPE bottles or individually-packed tubes (e.g., 1 tube per individual dose), each with child resistant closures.
  • All Investigational Product or placebo bottles will be labeled according to the requirements of local law and legislation. Representative labels for the bottles supplied to the Investigational Site pharmacy will be provided by N-Fold for inclusion in the study files. The Investigational Site pharmacy will provide representative labels for the bottles supplied to the Investigational Site staff (dose escalation visits) and participants (at home administration) for inclusion in the study files.
  • Investigational Site staff for the dose escalation visits and to the participants for at home administration during Part II of the trial will be blinded.
  • Investigational Product and orally-disintegrating placebo will be stored in a locked, secure area to prevent unauthorized access with active and placebo products separated.
  • Investigational Product and placebo will be stored in their provided packaging (bottles) under refrigerated conditions (2 to 8°C; 36 to 46°F).
  • Bottles of dispensed Investigational Product or placebo will be held at room temperature until the protocol-specified in-clinic dosing is completed for the visit or until the bottle is given to a participant to take home. The participant will be instructed to store his/her bottle of Investigational Product or placebo in the refrigerator once home, and to avoid exposure to extreme heat or light during the transport of the Investigational Product or placebo between the CRU and home.
  • the study period will be 18 months.
  • the study is a phase I study that will be conducted in two sequential parts, Part I
  • Phase I A Part II (Phase IB).
  • the duration of treatment will be 3 weeks for Part I and 52 weeks for Part II.
  • the total duration of participation for participants enrolled in part I will be up to 9 weeks (Screening visit through final follow-up visit).
  • the total duration of participation for participants enrolled in part II of this study will be up to 58 weeks (Screening visit through final follow-up visit).
  • Part I is an open-label, single-arm, dose escalation study to evaluate the safety and tolerability of Investigational Product in non-peanut allergic participants who are 18 to 50 years of age. Non-peanut allergic participants will receive buccal mucosal administrations of
  • Investigational Product qd for 3 weeks.
  • the dose of Investigational Product will be escalated weekly.
  • the first two doses will be at the starting level and the first dose at each subsequent level will be taken in the Clinical Research Unit (CRU), and all remaining doses will be taken at home. Participants will return to the CRU 24 hours and 4 weeks after their last dose of
  • Part II is a randomized, double-blind, placebo-controlled, dose escalation study to evaluate the safety, tolerability, and pharmacodynamics of Investigational Product as compared to placebo in peanut allergic participants who are 12 to 50 years of age. Pending review by a Safety Review Committee (SRC) with no safety concerns in part I of the trial, peanut allergic participants will be randomized 3 : 1 for Part II to receive buccal mucosal administrations of ENP- 501 or placebo daily for up to 52 weeks. Dose will be escalated on Day 1 and every two weeks thereafter for a maximum of 26 weeks of escalation. After 26 weeks, participants will maintain their highest dose for the remaining 26 weeks. Each dose escalation will occur in the CRET. Participants will complete Investigational Product or placebo dosing at 52 weeks and will return to the CRU 24 hours for a Double-Blind Placebo-Controlled Food Challenge (DBPCFC).
  • DBPCFC Double-Blind Placebo-Controlled Food Challenge
  • Participants will be recruited over a 1 month period for Part I and over a 3 month period for Part II. All participants will be enrolled in the Advantage eClinical data system which will generate an email notification to the Investigational Site research coordinator and pharmacist. Randomization will also occur with enrollment for participants in Part II. The email notification for participants randomized in Part II will include a blinded treatment number. The Investigational Site pharmacist will match with the blinded treatment number with the unblinded treatment assignment list (Investigational Product and placebo) provided by the DCC to determine the treatment assignment for the randomized participant.
  • the Investigational Site pharmacist will match with the blinded treatment number with the unblinded treatment assignment list (Investigational Product and placebo) provided by the DCC to determine the treatment assignment for the randomized participant.
  • Participants will not be enrolled (and randomized for Part II) until they complete all Screening evaluations, all eligibility criteria are met based on Screening evaluations, and the eligibility criteria continue to be met following all baseline assessments on Day 1.
  • Investigational Product or placebo will be supplied to the Investigational Site pharmacist in an unblinded manner.
  • the Investigational Site pharmacist will prepare the appropriate Investigational Product or placebo dose and quantity in a blinded manner and dispense to the Investigational Site research coordinator to provide to the participant. All DBPCFCs will be performed in a double-blind manner.
  • Study enrollment will be paused or stopped and/or the study may be discontinued if any of the following occur: (i) any death related to Investigational Product or placebo; (ii) more than one severe anaphylactic reaction [cyanosis or peripheral capillary oxygen saturation (Sp02) ⁇ 90% at any stage, hypotension, confusion, collapse, loss of consciousness, or incontinence] related to Investigational Product or placebo occurs; and/or (iii) any case of confirmed EoE.
  • a Screening Visit which may also be referred to as Screening, means an initial visit to determine whether a participants meets inclusion or exclusion criteria.
  • Inclusion Criteria for each Part are substantially as listed herein:
  • the main criteria for inclusion are: (i) 18 to 50 years of age; (ii) regular consumption of meal sized portion (approximately 5 grams) of peanut at least twice per month during the preceding 6 months; (iii) Negative SPT (wheal diameter ⁇ 3 mm) to peanut at Screening; (iv) serum peanut- specific IgE level ⁇ 0.35 kUA/L (ImmunoCAP®) at Screening; (v) ability to perform spirometry maneuvers in accordance with the American Thoracic Society (ATS) guidelines (2005); (vi) males and all WCBP agree to abstain from sex or use an adequate method of contraception for the duration of the study and for 30 days after the last dose of Investigational Product or placebo; and (vii) signed and dated written informed consent obtained from the participant in accordance with local IRB regulations.
  • the main criteria for inclusion are: (i) 12 to 50 years of age; (ii) a convincing clinical history of peanut allergy, which includes the development of symptoms (e.g., urticaria, flushing, rhinorrhea and sneezing, throat tightness or hoarseness, wheezing, vomiting) within minutes to 2 hours of ingestion of peanut and verified by a physician; (iii) Positive SPT (wheal diameter of > 5 mm) to peanut AND an elevated serum peanut-specific IgE level > 5 kUA/L
  • adequate contraceptive methods include those with a low failure rate, i.e., less than 1% per year, when used consistently and correctly, such as complete abstinence from sexual intercourse with a potentially fertile partner, and some double barrier methods (condom with spermicide) in conjunction with use by the partner of an intrauterine device (IUD), diaphragm with spermicide, oral contraceptives, birth control patch or vaginal ring, oral, or injectable or implanted contraceptives.
  • IUD intrauterine device
  • IV vasopressor drugs i.e., patient underwent cardio-respiratory arrest
  • B-blockers oral
  • ACE angiotensin-converting enzyme
  • ARBs angiotensin- receptor blockers
  • calcium channel blockers calcium channel blockers
  • All participants enrolled in Part I will receive sublingual (e.g. buccal mucosal) administrations of ENP-501 qd for 3 weeks.
  • the dose will be escalated weekly, starting at 500 pg peanut protein for the first week and escalating to 1,000 and 2,000 pg peanut protein for the second and third weeks, respectively.
  • the doses will be given as one, two, and four 500 pg peanut protein strength tablets, respectively.
  • the first two doses at the starting dose level and the first dose at each subsequent dose level (e.g. Days 8 and 15) will be taken in the Clinical Research Unit (CRU), and all remaining doses will be taken at home, qd, at approximately the same time each day. Participants will return to the CRU 24 hours (+ 3 days) and 4 weeks ( ⁇ 3 days) after their last dose of Investigational Product or placebo for follow-up visits.
  • CRU Clinical Research Unit
  • Participants enrolled in Part II will be randomized at a ratio of 3 : 1 to receive daily sublingual administrations of either ENP-501 or placebo daily for up to 52 weeks. The dose will be escalated on Day 1 and every two weeks thereafter for a maximum of 26 weeks of escalation After 26 weeks, participants will maintain their top dose for the remaining 26 weeks of treatment. Each dose escalation will occur in the CRU. As this is a Phase 1 safety trial, no pre- therapy oral food challenge for baseline peanut allergy threshold will be assessed.
  • the planned dose escalation levels will be carried over to the subsequent dose escalation visits with no more than three dose level escalations in a single visit.
  • the objective is to achieve maintenance dosing at the highest dose level (2,000 pg peanut protein).
  • the planned dose escalation for a participant that experiences no symptom requiring an interruption in the escalation is outlined in the table below. These participants will complete Investigational Product or placebo treatment in 52 weeks (after up to 26 weeks of biweekly dose escalation visits followed by maintenance dosing). The peanut protein, number of portions, and portion strength per dose apply to Investigational Product. Participants randomized to the placebo arm will receive the same number of portions planned for the Investigational Product; however, the portions will contain no peanut protein.
  • the planned dose escalation for a subject that experiences no symptoms requiring an interruption in the escalation is outlined in the Table 7. These participants will complete product treatment in 52 weeks (after up to 26 weeks of weekly dose escalation visits followed by maintenance dosing). The peanut protein, number of portions, and portion strength per dose apply to Investigational Product. Participants randomized to the placebo arm will receive the same number of portions planned for the Investigational Product; however, the portions will contain no peanut protein.
  • ENP-501 or placebo will continue to be administered for up to 26 total weeks of biweekly dose escalation visits to allow the participants to achieve maintenance dosing at 2,000 pg. [0513] Dose escalation will not continue beyond 26 weeks, regardless of whether the highest dose was achieved. If the highest dose is not achieved at the final escalation visit, the participant will continue dosing at the maximum tolerated dose for up to 52 weeks of total treatment.
  • ENP-501 will be taken in a CRU or comparable monitored clinical site on Day 1,
  • ENP-501 will be taken qd at approximately the same time each day. ENP-501 will be administered sublingually (e.g. buccal mucosal) wherein participants will be instructed to hold the tablets sublingually (e.g. in the buccal mucosal space) for two minutes and then swallow.
  • sublingually e.g. buccal mucosal
  • participants will be instructed to hold the tablets sublingually (e.g. in the buccal mucosal space) for two minutes and then swallow.
  • ENP-501 or placebo will be taken in a CRET or comparable monitored clinical site on Day 1, Day 2, and at each biweekly dose escalation visit. All remaining doses will be self- administered at home. For the doses taken at home, ENP-501 or placebo will be taken qd at approximately the same time each day.
  • ENP-501 or placebo will be administered sublingually (e.g. buccal mucosal).
  • portions e.g., powder or tablets
  • portions will be placed sublingually two at a time, with two minutes between each pair (to allow time for the tablets to dissolve before the next pair is placed sublingually); this does not apply when powders are being used and administered sublingually, as, in such a case, the portion of powder will be increased. If a portion of powder exceeds a reasonable amount in the judgment of a physician, it can be administered in a split dose, separated by 2 minutes as in tablet administration. Participants will be instructed not to eat within 15 minutes before and 30 minutes after dosing.
  • ENP-501 qd polymer nanoparticle comprising a payload and/or coating [e.g., as prepared by the procedure described in Example 1]) for 3 weeks.
  • the dose will be escalated weekly, starting at 500 pg peanut protein for the first week and escalating to 1,000 and 2,000 pg peanut protein for the second and third weeks, respectively.
  • the first two doses at the starting dose level and the first dose at each subsequent dose level will be taken in the Clinical Research ETnit (CRET), and all remaining doses will be taken at home. Participants return to the CRET 24 hours and 4 weeks after their last dose of Investigational Product or placebo for follow-up visits.
  • CRET Clinical Research ETnit
  • Part II will occur as long as no safety concerns are raised during Part I. In Part II,
  • Each participant will be provided with oral and written information (ICF) describing the study and will have any questions answered.
  • the parent(s) will be provided with the ICF and assent for children if the participant is under the age of 18 (Part 2 only).
  • Written informed consent must be obtained prior to performing any screening evaluations.
  • Urine pregnancy test [beta-chorionic gonadotropin (b-hCG)] for WCBP only;

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Abstract

La présente invention concerne des compositions de nanoparticules dans lesquelles des nanoparticules individuelles comprennent des charges utiles et des agents de revêtement, ainsi que des procédés de préparation et d'utilisation de telles compositions de nanoparticules, et diverses compositions et/ou technologies associées à de telles compositions de nanoparticules, leur production et/ou leur utilisation.
PCT/US2019/013370 2018-01-11 2019-01-11 Systèmes de nanoparticules Ceased WO2019140318A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
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US11071776B2 (en) 2012-04-23 2021-07-27 N-Fold Llc Nanoparticles for treatment of allergy
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