WO2019140318A1

WO2019140318A1 - Nanoparticle systems

Info

Publication number: WO2019140318A1
Application number: PCT/US2019/013370
Authority: WO
Inventors: Howard B. Sosin; Michael J. Caplan; Robert K. Prud'homme; Mark Kastantin; April DOWER; Spencer DAHL
Original assignee: N-Fold Llc
Priority date: 2018-01-11
Filing date: 2019-01-11
Publication date: 2019-07-18

Abstract

The present disclosure provides nanoparticle compositions in which individual nanoparticles comprise 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.

Description

NANOPARTICLE SYSTEMS

BACKGROUND

[0001] Polymer nanoparticle systems have become an important drug delivery modality.

In nanoparticle formulations of drugs, 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. However, improved approaches are both desirable and needed.

SUMMARY

[0002] 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. Among other things, 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.).

[0003] Alternatively or additionally, according to various embodiments, methods provide certain advantages and/or solve one or more problems associated with prior nanoparticle technologies. For example, in some embodiments, 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. In some embodiments, 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). In some embodiments, 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.

[0004] Among other things, the present disclosure provides nanoparticle compositions in a dry ( e.g ., lyophilized) state. In some embodiments, 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.

[0005] The present disclosure demonstrates that certain provided nanoparticle compositions, specifically including certain provided dry 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. For example, in some embodiments, 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) are substantially maintained during the period of time.

[0006] In some embodiments, 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.

[0007] In some embodiments, 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. In some embodiments, concentration comprises evaporation. In some embodiments, a concentration step removes at least some water and at least some solvent. In some embodiments, a concentration step removes substantially all of at least one of water and solvent.

[0008] In some embodiments, 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. In some embodiments, the block material has a substantially uniform distribution of the hydrophilic payload with respect to the polymer.

[0009] In some embodiments, the present disclosure also provides methods r including grinding the lyophilized cake and resuspending the ground cake in at least one alcohol. In some embodiments, grinding occurs in the presence of liquid nitrogen. In some embodiments, the at least one alcohol in which the ground cake is resuspended is or comprises propanol.

[0010] In some embodiments, a block material comprising polymer and hydrophilic payload has a substantially uniform distribution of a hydrophilic payload with respect to the polymer.

[0011] In some embodiments, 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.

[0012] In some embodiments, 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.

[0013] In some embodiments, 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.

[0014] In some embodiments, 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. In some embodiments, 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.

[0015] In some embodiments, 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.

[0016] In some embodiments, 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.

[0017] In accordance with several embodiments, any of a variety of polymers may be desirable for use in provided methods and compositions. In some embodiments, a polymer may be hydrophobic. In some embodiments, a polymer may be amphiphilic. As described herein, in various embodiments, provided methods and compositions are amenable to the use of polymers of various sizes and properties. For example, in some embodiments, a polymer has a molecular weight within a range of 5,000-5,000,000 Daltons.

[0018] As described herein, and in accordance with various embodiments, provided methods and compositions are amenable to the inclusion of any of a wide variety of payloads.

For example, in some embodiments, a hydrophilic payload is selected from the group consisting of a protein, a nucleic acid, an antigen, and combinations thereof. In some embodiments, an antigen is or comprises an allergic antigen. In some embodiments, an antigen is or comprises an anaphylactic antigen. In some embodiments, an antigen is or comprises an infectious antigen. In some embodiments, an antigen is or comprises an autoantigen. In some embodiments, an antigen is or comprises a disease-associated antigen.

[0019] In accordance with various embodiments, any of a variety of amounts (e.g., weight ratios or absolute amounts) of hydrophilic payload to polymer may be used. For example, in some embodiments, 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. In some embodiments, 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.

[0020] As described herein, 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. In some embodiments, 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.

[0021] In some embodiments, a solvent system comprises an aqueous solution. In some embodiments, an aqueous solution comprises water and DMSO. In some embodiments, a polymer is present in water, while a payload is present in DMSO, prior to the combining step, or vice versa.

[0022] In some embodiments, a non-solvent system comprises water and alcohol (e.g., propanol). [0023] 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. By way of additional example, in some embodiments, a lyophilized cake or block material may be comminuted using a mortar and pestle.

[0024] In some embodiments, 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.

[0025] In accordance with several embodiments, provided nanoparticles have a mean size within a range of approximately 100-500 nm. In some embodiments, provided nanoparticles may be separated into different populations. In some embodiments 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

[0026] As described herein and in accordance with several embodiments, certain provided methods include the processing of microparticles into nanoparticles, for example, via microfluidic processes. In some embodiments, a flowable microparticle suspension is passed through a microfluidizer two or more times, so that the nanoparticles have substantially uniform size. In some embodiments, a microfluidizer applies shear gradient to a flowable microparticle suspension. In some embodiments, a shear gradient is within a range of 10-6 to 10-7 s-l. While it is specifically contemplated that a variety of processing conditions may be useful in certain embodiments, in some embodiments, 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.

[0027] In some embodiments, 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.

[0028] In some embodiments, provided methods may comprise lyophilizing a stabilizing agent solution and a nanoparticle suspension prior to applying a preparation of coating agents to the nanoparticles. In some embodiments, a preparation of coating agents comprises at least one microbial extract.

[0029] In several embodiments, the present disclosure provides methods including a step of centrifugation. In some embodiments, centrifugation is performed on a nanoparticle suspension. In some embodiments, centrifugation is performed at a speed between 300 and 600 xg·

[0030] In several embodiments, the present disclosure further provides methods including a step of subjecting supernatant from a centrifuged solution to a step of tangential flow filtration. In some embodiments, the supernatant is mixed with a solution comprising a coating agent after centrifugation. In some embodiments, centrifugation is performed on a nanoparticle suspension. In some embodiments, centrifugation is performed at a speed between 300 and 600 xg·

[0031] In some embodiments, the present disclosure also provides compositions comprising 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.

[0032] In accordance with several embodiments, 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.

[0033] In some embodiments, a plurality of nanoparticles is comprised of at least two populations of nanoparticles, each with a different mean size. In some embodiments, at least one population of nanoparticles has a mean size within a range of approximately 300-500 nm. In some embodiments, at least one population of nanoparticles has a mean size within a range of approximately 100-300 nm. [0034] In some embodiments, 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. In some embodiments, 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. In some embodiments, a subject is suffering from at least one of allergy, infection, and cancer.

[0035] In some embodiments where a provided composition is administered to a subject, any appropriate route of administration may be used. By way of specific example, in some embodiments, provided nanoparticles (e.g., nanoparticle compositions) may be administered intravenously, intradermally, transdermally, orally, subcutaneously, and/or transmucosally. In some embodiments, transmucosal administration may be or comprise buccal, nasal, bronchial, vaginal, rectal, and/or sublingual administration.

[0036] As used in this application, the terms“about” and“approximately” are used as equivalents. Any citations to publications, patents, or patent applications herein are incorporated by reference in their entirety. Any numerals used in this application with or without

about/approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art.

[0037] Other features, objects, and advantages of the present disclosure are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments of the present disclosure, is given by way of illustration only, not limitation. Various changes and modifications within the scope of the present disclosure will become apparent to those skilled in the art from the detailed description. DEFINITIONS

[0038] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

[0039] Administration: As used herein, the term“administration” refers to the administration of a composition to a subject. Administration may be by any appropriate route. For example, in some embodiments, 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,

subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal and vitreal.

[0040] Allergen: The term“allergen”, as used herein, refers to those antigens that induce an allergic reaction. In some embodiments, an allergen is or comprises a polypeptide. In some embodiments, an allergen is or comprises a small molecule. In some embodiments, an allergen is selected from the group consisting of food allergens, drug allergens, environmental allergens, insect venoms, animal allergens, and latex.

[0041] Allergic reaction : The phrase“allergic reaction,” as used herein, has its art- understood meaning and refers to an IgE-mediated immune response to an antigen. When 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. Typically, 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. For the purposes of the present disclosure, an asthmatic reaction is considered to be a form of allergic reaction. In some embodiments, allergic reactions are mild; typical symptoms of a mild reaction include, for example, hives (especially over the neck and face) itching, nasal congestion, rashes, watery eyes, red eyes, and combinations thereof. In some embodiments, 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. In some embodiments, allergic reactions are anaphylactic reactions. In some embodiments, allergic reactions are defined as a disorder characterized by an adverse local or general response from exposure to one or more allergens. In some embodiments, allergic reactions may be graded by a“toxicity grading” system, that will be known to those of skill in the art. For example, in some embodiments, a grading system (such as NCI-CTCAD v 4.03), will be used to grade allergic reactions, such as a system described in Table 1 and/or Table 2

Table 1.

Table 2.

[0042] Allergy The term“allergy”, as used herein, refers to a condition characterized by an IgE-mediated immune response to particular antigens. In some embodiments, the antigens are ones that do not elicit an IgE-mediated immune response in many or most individuals. In some embodiments, 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. Thus, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. , one from the same source or one for which shared allergies are common), that individual may be considered susceptible to allergy to (and/or to an allergic or anaphylactic reaction to) the relevant allergen. Similarly, if members of an individual’s family react to a particular allergen, the individual may be considered to be susceptible to allergy to (and/or to an allergic and/or anaphylactic reaction to) that allergen.

[0043] Amino acid: As used herein, the term“amino acid,” in its broadest sense, 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. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally- occurring amino acid. In some embodiments, 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. In some embodiments, 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. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, and/or substitution as compared with the general structure. In some embodiments, 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. In some embodiments, 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. As will be clear from context, in some embodiments, 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.

[0044] Alloantigen: The term“alloantigen”, as used herein, refers to an antigen associated with allorecognition and/or graft rejection (e.g., an antigen against which a rejection immune response is directed). In general, 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. In general, 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. In some embodiments, 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.

[0045] Allorecognition. The term“allorecognition”, as used herein, 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. Typically, allorecognition involves T cell recognition of the alloantigen. In many embodiments, T cells recognize an alloantigen peptide, for example, encoded by a polymorphic gene whose sequence differs between the donor and recipient individuals.

[0046] Anaphylactic antigen: The phrase“anaphylactic antigen”, as used herein, 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. For example, for the purposes of the present disclosure, pollens and animal danders or excretions (e.g, saliva, urine) are not considered to be anaphylactic antigens. On the other hand, 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).

[0047] Anaphylactic reaction: The phrase“anaphylactic reaction,” (e.g.,“anaphylaxis”) as used herein, 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

cardiovascular system. As noted above, symptoms of severe allergic reactions such as anaphylactic 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. The most severe anaphylactic reactions can result in loss of consciousness and/or death. In some embodiments, 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. Clinically, anaphylaxis may present with breathing difficulty, dizziness, hypotension, cyanosis and/or loss of consciousness and may lead to death. In some embodiments, 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. Table 3. Staging System of Severity of Anaphylaxis

[0048] In some embodiments, 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:

1. Acute onset of an illness (minutes to hours) with involvement of:

• Skin/mucosal tissue (e.g., generalized hives, itch or flush, swollen

lips/tongue/uvula) AND

• Airway compromise (e.g., dyspnea, stridor, wheeze/ bronchospasm, hypoxia, reduced PEF) AND/OR

• Reduced BP or associated symptoms (e.g., hypotonia, syncope, incontinence)

2. Two or more of the following that occur rapidly after exposure to the allergen (minutes to hours):

• Skin/mucosal tissue (e.g., generalized hives, itch/flush, swollen lips/tongue/uvula)

• Airway compromise (e.g., dyspnea, stridor wheeze/bronchospasm, hypoxia, reduced PEF)

• Reduced BP or associated symptoms (e.g., hypotonia, syncope, incontinence)

• Persistent GI symptoms (e.g., nausea, vomiting, crampy abdominal pain)

3. Reduced BP after exposure to the allergen (minutes to hours):

• Infants and Children: low systolic BP (age-specific) or > 30% drop in systolic

BP*

Adults: systolic BP < 90 mm Hg or > 30% drop from their baseline

[0049] In some embodiments, 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. In some embodiments, isolated skin or mucosal lesions following the ingestion of a food constitute a“food-induced allergic reaction”.

[0050] Animal: As used herein, the term“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.

[0051] Antige The term“antigen”, as used herein, 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). In some embodiments, 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). In general, 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. In some embodiments, an antigen is or comprises a polypeptide. Those of ordinary skill in the art will appreciate that, in general, 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). In some embodiments, antigens utilized in accordance with the present disclosure are provided in a crude form. In some embodiments, an antigen is a recombinant antigen.

[0052] 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. In some embodiments, 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). Alternatively or additionally, in some embodiments, 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). To give but one example, in some embodiments, 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. Those of ordinary skill will appreciate, for example, that 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).

[0053] Approximately: As used herein, the term“approximately” and“about” is intended to encompass normal statistical variation as would be understood by those of ordinary skill in the art. In certain embodiments, 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).

[0054] Associated with: 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. For example, a particular entity (e.g, polypeptide) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and/or form correlates with incidence of and/or susceptibility of the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, 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.

[0055] 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

[0056] Biocompatible: The term“biocompatible”, as used herein, 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.

[0057] Biodegradable. As used herein, the term“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. In certain embodiments, components generated by breakdown of a biodegradable material are biocompatible and therefore do not induce significant inflammation and/or other adverse effects in vivo. In some embodiments, biodegradable polymer materials break down into their component monomers. In some embodiments, breakdown of biodegradable materials (including, for example, biodegradable polymer materials) involves hydrolysis of ester bonds. Alternatively or additionally, in some embodiments, breakdown of biodegradable materials (including, for example, biodegradable polymer materials) involves cleavage of urethane linkages. Exemplary 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. Many naturally occurring 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. Those of ordinary skill in the art will appreciate or be able to determine when such polymers are 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).

[0058] Biologically active: As used herein, the phrase“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.). For instance, a substance that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active. It will be appreciated by those skilled in the art that often only a portion or fragment of 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.

[0059] Carrier: As used herein, refers to a diluent, adjuvant, excipient, or vehicle with which a composition is administered. In some exemplary embodiments, 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. In some embodiments, carriers are or include one or more solid components. For example, in some embodiments, a carrier may be or comprise a bead, film, rod, or similarly structured component.

[0060] Cellular lysate: As used herein, the term“cellular lysate” or“cell lysate” refers to a fluid containing contents of one or more disrupted cells (i.e., cells whose membrane has been disrupted). In some embodiments, a cellular lysate includes both hydrophilic and hydrophobic cellular components. In some embodiments, 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. In some embodiments, a cellular lysate is a lysate of one or more abnormal cells, such as cancer cells. In some embodiments, 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. In some embodiments, one or more isolation or purification steps are performed on the primary lysate. However, the term “lysate” refers to a preparation that includes multiple cellular components and not to pure preparations of any individual component.

[0061] Combination therapy: As used herein, the term“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.

[0062] Corresponding to: As used herein, 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. Those of ordinary skill will appreciate that, for purposes of simplicity, 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

commercially-available algorithms specifically designed for polymer sequence comparisons.

[0063] Derivative: As used herein, the term“derivative” refers to a structural analogue substance that is produced or formed from another substance of similar structure in one or more steps. In some embodiments, a derivative refers to a second chemical substance related structurally to a first chemical substance and theoretically derivable from the first chemical substance. Examples of 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. Examples of cellulose organic esters include, but are not limited to cellulose acetate, cellulose triacetate, cellulose propionate, cellulose acetate propionate and cellulose acetate butyrate. Examples of cellulose inorganic esters include, but are not limited to, cellulose nitrate and cellulose sulfate. Examples of cellulose alkyl ethers include, but are not limited to, methylcellulose, ethylcellulose and ethyl methyl cellulose. Examples of cellulose hydroxyalkyl ethers include, but are not limited to, hydroxy ethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose and ethyl hydroxyethyl cellulose. Examples of cellulose carboxyalkyl ethers include, but are not limited to carboxymethyl cellulose.

[0064] Dosage form: As used herein, the term“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).

[0065] Dosing regimen: As used herein, the term“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. In some embodiments, a given therapeutic agent has a

recommended dosing regimen, which may involve one or more doses. In some embodiments, 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).

[0066] Encapsulated: The term“encapsulated” is used herein to refer to substances that are completely surrounded by another material.

[0067] Expression·. As used herein,“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.

[0068] 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.

[0069] Graft rejection: The term“graft rejection” as used herein, refers to rejection of tissue transplanted from a donor individual to a recipient individual. In some embodiments, graft rejection refers to an allograft rejection, wherein the donor individual and recipient individual are of the same species. Typically, allograft rejection occurs when the donor tissue carries an alloantigen against which the recipient immune system mounts a rejection response. In some embodiments, graft rejection refers to a xenograft rejection, wherein the donor and recipient are of different species. Typically, xenograft rejection occurs when the donor species tissue carries a xenoantigen against which the recipient species immune system mounts a rejection response.

[0070] Homology: As used herein, the term“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. In some embodiments, 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. In some embodiments, 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). For example, as is well known by those of ordinary skill in the art, 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:

As will be understood by those skilled in the art, a variety of algorithms are available that permit comparison of sequences in order to determine their degree of homology, including by permitting gaps of designated length in one sequence relative to another when considering which residues“correspond” to one another in different sequences. Calculation of the percent homology between two nucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes ( e.g ., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-corresponding sequences can be disregarded for comparison purposes). In certain embodiments, 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. [0071] Humarr. In some embodiments, a human is an embryo, a fetus, an infant, a child, a teenager, an adult, or a senior citizen.

[0072] Hydrophilic: As used herein, the term“hydrophilic” and/or“polar” refers to a tendency to mix with, or dissolve easily in, water.

[0073] 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.

[0074] Identity: As used herein, the term“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. In some embodiments, 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. As will be understood by those skilled in the art, a variety of algorithms are available that permit comparison of sequences in order to determine their degree of homology, including by permitting gaps of designated length in one sequence relative to another when considering which residues“correspond” to one another in different sequences. Calculation of the percent identity between two nucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g, gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non

corresponding sequences can be disregarded for comparison purposes). In certain embodiments, 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.

[0075] Infection: As used herein, the term“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.

[0076] Isolated: As used herein, the term“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. In some embodiments, 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. As used herein, a substance is “pure” if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, 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.

[0077] Nanoparticle: As used herein, the term“nanoparticle” refers to a particle having at least one dimension (e.g., diameter) of less than 1000 nanometers. In some embodiments, a nanoparticle may have at least two dimensions of less than 1000 nanometers (nm). In some embodiments, a nanoparticle has at least two dimensions of less than 300 nm. In some embodiments, a nanoparticle has at least two dimensions of less than 100 nm. In some embodiments, one or more measuring techniques may be used to calculate mean size (e.g.

hydrodynamic diameter) of a nanoparticle or population of nanoparticles. For example, in some embodiments, for nanoparticles with sizes less than 600 nm size may be determined by dynamic light scattering with size being reported as z-average diameter calculated by a deconvolution program. In some embodiments, for particles with average sizes greater than 600 nm 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. In some embodiments, a nanoparticle will have no dimension of more than 1000 nanometers.

[0078] Nanoparticle composition: As used herein, the term“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.

[0079] 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.

In some embodiments, a nucleic acid is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clear from context, in some embodiments,“nucleic acid” refers to individual nucleic acid residues ( e.g ., nucleotides and/or nucleosides); in some embodiments,“nucleic acid” refers to an

oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, a “nucleic acid” is or comprises RNA; in some embodiments, a“nucleic acid” is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, 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. Alternatively or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and/or 5’-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments, 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). In some embodiments, 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). In some embodiments, 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. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, a nucleic acid includes one or more introns. In some embodiments, 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. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,

25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long.

[0080] 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. In some embodiments, 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. In some embodiments, 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.

[0081] Patient: As used herein, the term“patient” or“subject” 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. In many embodiments, a patient is a human being. In some embodiments, a patient is a human presenting to a medical provider for diagnosis or treatment of a disease, disorder or condition. In some embodiments, a patient displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a patient does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a patient is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition.

[0082] Payload: As used herein, the term“payload” refers to an entity for delivery as described herein. In some embodiments, a payload may be or comprise a biologically active agent (e.g., a therapeutically active agent). In some embodiments, a payload may be or comprise one or more carbohydrates, lipids, metals, nucleic acids, polypeptides, small molecules and/or combinations thereof. In some embodiments, a payload may be or comprise a complex agent (e.g., protein, carbohydrate, lipid and/or nucleic acid mixtures, crude samples, cellular extracts, etc).

[0083] Pharmaceutically acceptable: The term“pharmaceutically acceptable” as used herein, refers to agents 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.

[0084] Polypeptide: The term“polypeptide”, as used herein, generally has its art- recognized meaning of a polymer of at least three amino acids. In some embodiments, 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. For each such class, 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. In such embodiments, the term“polypeptide” refers to any member of the class that shows significant sequence homology or identity with a relevant reference polypeptide. In many embodiments, such member also shares significant activity with the reference polypeptide. For example, in some embodiments, 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%,

94%, 95%, 96%, 97%, 98%, 99% or more and/or includes at least one region (i.e., a conserved region, often including a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such 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.

[0085] Protein: As used herein, the term“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. In some embodiments, 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. In some embodiments, proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.

[0086] 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.

[0087] Small molecule: As used herein, the term“small molecule” means a low molecular weight organic compound that may serve as an enzyme substrate or regulator of biological processes. In general, a“small molecule” is a molecule that is less than about 5 kilodaltons (kD) in size. In some embodiments, provided nanoparticles further include one or more small molecules. In some embodiments, the small molecule is less than about 4 kD, 3 kD, about 2 kD, or about 1 kD. In some embodiments, 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. In some embodiments, 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. In some embodiments, one or more small molecules are encapsulated within the nanoparticle. In some embodiments, small molecules are non-polymeric. In some embodiments, in accordance with the present disclosure, small molecules are not proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, polysaccharides, glycoproteins, proteoglycans, etc.

In some embodiments, 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.

[0088] Stable: The term“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. In some embodiments, 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. In some embodiments, a stable provided composition is one for which a biologically relevant activity is maintained for a period of time. In some embodiments, 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. For example, if a population of nanoparticles is subjected to prolonged storage, temperature changes, and/or pH changes, and a majority of the nanoparticles in the composition maintain a diameter within a stated range, the nanoparticle composition is stable. In some embodiments, a stable composition is stable at ambient conditions. In some embodiments, a stable composition is stable under biologic conditions (i.e., 37° C in phosphate buffered saline).

[0089] Subject: As used herein, the term“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. In many embodiments, 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.

[0090] Sublingual: As used herein, the term“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. In some embodiments, sublingual administration may be or comprise buccal mucosal administration.

[0091] Substantially: As used herein, 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.

[0092] Suffering from: An individual who is“suffering from” a disease, disorder, or condition has been diagnosed with and/or exhibits or has exhibited one or more symptoms or characteristics of the disease, disorder, or condition.

[0093] 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.).

[0094] 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: As used herein, the phrase“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.

[0096] Therapeutically effective amount: As used herein, the term“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). In some embodiments, 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. Those of ordinary skill in the art will appreciate that the term“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

administered to patients in need of such treatment. It is specifically understood that particular subjects may, in fact, be“refractory” to a“therapeutically effective amount.” To give but one example, a refractory subject may have a low bioavailability such that clinical efficacy is not obtainable. In some embodiments, 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.).

Those of ordinary skill in the art will appreciate that, in some embodiments, 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.

[0097] Therapeutic regimen : A“therapeutic regimen”, as that term is used herein, refers to a dosing regimen whose administration across a relevant population is correlated with a desired or beneficial therapeutic outcome. [0098] Treatment: As used herein, the term“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.

[0099] Uniform. The term“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. For example, in some embodiments, 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. In some embodiments, a uniform nanoparticle

composition contains nanoparticles with at least one dimension (e.g., diameter) within the range of about 100 nm to about 300 nm. In some embodiments, a uniform nanoparticle composition contains nanoparticles with a mean particle size that is under about 500 nm. In some

embodiments, a uniform nanoparticle composition contains nanoparticles with a mean particle size that is within a range of about 100 nm to about 500 nm. In some embodiments, 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. In some embodiments, 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. BRIEF DESCRIPTION OF THE DRAWING

[0100] The Drawing, which is comprised of at least the following Figures, is for illustration purposes only, not for limitation.

[0101] 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.

[0102] 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.

[0103] Figure 3 is a schematic showing an exemplary process to make a flowable microparticle suspension.

[0104] Figure 4 is a schematic showing an exemplary process to manufacture

nanoparticles.

[0105] Figure 5 is a schematic showing an exemplary process to manufacture

nanoparticles containing payloads and coating agents.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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. [0110] Figure 8C shows exemplary parameters described in Figure 8B, superimposed on

Lyol steps 1-5 of Figure 8A.

[0111] Figure 8D shows exemplary parameters described in Figure 8B, superimposed on

Lyo2 steps 5-9 of Figure 8 A.

[0112] Figure 8E shows exemplary parameters described in Figure 8B, superimposed on

Lyo3 steps 9-18 of Figure 8A.

[0113] 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.

[0114] Figure 8G shows exemplary data from additional centrifugation steps and calculation of safety factors of recovered nanoparticles.

[0115] 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.

[0116] Figures 10A-10F show NF-kB inducible secreted embryonic alkaline

phosphatase (SEAP) reporter gene expression in TLR4-negative HEK293 cells. Figures 10A (which corresponds to averages of screenings 1, 2, and 3 of Article 1 of 10D) and 10B (which corresponds to averages of screenings 1, 2, and 3 of Article 2 of 10E) 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.

[0117] Figures 11A-11R show NF-kB inducible secreted embryonic alkaline

phosphatase (SEAP) reporter gene expression in TLR4 positive HEK293 cells. Figure 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.

[0118] Figures 12A-12L show NF-kB inducible secreted embryonic alkaline

phosphatase (SEAP) reporter gene expression in TLR4 positive HEK293 cells. Figure 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). 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).

[0119] Figures 13A-13S show NF-kB inducible secreted embryonic alkaline

phosphatase (SEAP) reporter gene expression in TLR4 HEK293/Null2 negative control cells. Figure 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. [0120] 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). 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).

[0121] Figure 15 is a table showing an exemplary schedule of events in an exemplary clinical trial as described herein.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0122] The following description is for illustration and exemplification of the present disclosure only and is not intended to limit the present disclosure to the specific embodiments described herein. ETnless defined otherwise, technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.

[0123] In accordance with one or more embodiments, the present disclosure provides preparation of certain particles that may offer enhanced synthesis process, and/or consistent product quality as compared with other preparations. In some embodiments, 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). In some embodiments, 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. Manufacturing Nanoparticle Compositions

[0124] The present disclosure is based, in part, on a surprising insight that desirable nanoparticle compositions can be prepared by the manufacturing processes described herein. Among other things, the present disclosure identifies one or more problems ( e.g ., one or more sources of problems) in prior nanoparticle manufacturing technologies. Furthermore, in some embodiments, 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). Alternatively or additionally, in some embodiments, 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).

Initial polymer /payload solution

[0125] 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.

[0126] 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.

[0127] In many embodiments, methodologies provided by the present disclosure utilize an initial combination, e.g., solution, that includes both polymer and payload. As described herein, a variety of strategies can be utilized to provide or prepare an initial polymer/payload combination . For example, in some embodiments, 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. In some embodiments, 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). In some embodiments, a polymer solution is made by dissolving polymer (e.g., PLGA) into organic liquid (e.g.,

DMSO). In some embodiments, a payload solution is made by dissolving protein and DNA into water to produce an aqueous solution. In some embodiments, 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. In some embodiments, a

polymer/payload solution is made by combining a payload (aqueous) and a polymer (organic) solution. In some embodiments, a polymer/payload solution is made by adding a payload (aqueous) solution into a polymer (organic) solution.

[0128] Typically, 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. 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.

[0129] In some embodiments, 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.

[0130] In some embodiments, polymer and payload are present at a weight ratio within a range of 1 : 1 to 10²⁰: 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 10²⁰: 1 in an initial polymer/payload solution. In some embodiments, 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 10²⁰: 1 by weight (e.g., 50: 1 to 10²⁰: 1 by weight).

[0131] Among other things, 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. In some embodiments, technologies provided by the present disclosure achieve such uniform combination with or without application of disruptive energy or force ( e.g ., sonication).

[0132] In some embodiments, 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. In some such embodiments, additional steps as further described herein, may be employed to achieve a desirable distribution of payload with respect to polymer.

[0133] In some embodiments, a solvent system used to prepare an initial

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). In some embodiments, 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.

[0134] In some embodiments, a solvent system used to prepare an initial

polymer/payload solution utilizes two or more solvents. For example, 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. In some particular embodiments, 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.

[0135] In some embodiments, an initial polymer/payload solution may include one or more other components in addition to polymer and payload. To give but a few examples, in some embodiments, 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. Those skilled in the art will be aware of a variety of such agents that may be useful in the practice of certain embodiments as disclosed herein. [0136] 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.

[0137] In some embodiments, solid (e.g., dry) polymer and/or payload are added to solvent system (e.g., to a premeasured amount of one or more solvents). In some embodiments, 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.

[0138] In some embodiments, during preparation of an initial polymer/payload solution, 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.

[0139] In some embodiments, stirring is performed with a mixer. In some embodiments, 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. In some embodiments, a mixer may be or comprise a magnetic stirrer, a turbine, or any electrical or mechanical impeller or propeller.

[0140] In some embodiments mixing is done in a high intensity mixing device, e.g. a

Microfluidizer, for example, to ensure homogenization of polymer and payload. In some embodiments, substantially uniform distribution of payload into polymer matrix is an important step to ensure substantially uniform distribution of payload into a final nanoparticle formulation.

[0141] In some embodiments, mixing is performed for one or more time periods (which may be consecutive and/or may have gaps between them). In some embodiments, a time period maybe approximately 5, 10, 15, 20, 25, 30, 40, 45, 50, 55, or 60 minutes, or longer. In some embodiments, a time period may be approximately 1, 2, 3, 4, 5, 10, 12, 15, 20, or 24 hours, or longer.

[0142] In some embodiments, 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).

[0143] In some embodiments, sedimentation (e.g., centrifugation) is performed during preparation of an initial polymer/payload solution. For example, after dissolution of a solid material (e.g., payload or polymer) in a solvent system (e.g., prior to mixing payload and polymer), a solution may be centrifuged to remove aggregated, undissolved and/or partially dissolved solid material.

[0144] In some embodiments, 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).

[0145] 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. In some embodiments, the present disclosure provides methodologies (e.g., steps) that can mitigate one or more such identified sources of problem(s). Among other things, in some embodiments, 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

embodiments, 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.

[0146] In some embodiments, it is contemplated that 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. In some embodiments, combining organic and aqueous

materials/components, such as described in, e.g., Figure 8A, Lyo 1, may result in difficulty obtaining a homogenous lyophilized cake and/or a lyophilized cake that can be easily

homogenized. Accordingly, it is contemplated that in some such embodiments, removal of water and/or other solvent(s) and/or increased temperature during creation of a lyophilized cake (see, e.g., Lyo 1 of Figure 8 A) will be helpful in providing more homogeneity and/or easier solubilization. In some embodiments, 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. In some embodiments, the present disclosure provides substantially homogenous lyophilized cakes, for example as may be achieved by such methodologies.

[0147] In some embodiments, a polymer/payload (e.g., an initial polymer/payload) combination (e.g., solution) is concentrated. In some embodiments, 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).

[0148] In some embodiments, concentration of an initial polymer/payload combination

(e.g., solution) is performed by removal of water and/or other solvent(s) or non-solvent(s).

[0149] Without wishing to be held to a particular theory, it is contemplated that excess water and/or other solvent(s) may interfere with obtaining a homogenous dried combination and/or obtaining a dried mixture that can be resuspended to create a homogenate (e.g., a uniform homogenate) from manipulation of a polymer/payload solution. Accordingly, in some embodiments, concentration of a polymer/payload solution (removal of a portion of water and/or other solvent(s)) or non-solvent(s) 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).

[0150] In some embodiments, polymer/payload combination (e.g., solution) homogeneity may desirably be improved; the present disclosure encompasses the recognition that such improved homogeneity may facilitate, and may even be required for reasonable performance of additional production steps. For example, without being bound to any particular theory, it is contemplated that a particular concentration of water and/or other solvent(s) in a

polymer/payload combination (e.g., solution) may result in a non-homogenous combination (e.g., solution) during subsequent steps (e.g., lyophilization) such as, e.g., bubbling and/or unequal freezing, etc. Therefore, in some embodiments, the present disclosure provides technologies in which 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.

[0151] In some embodiments, a polymer/payload combination (e.g., solution) is concentrated. In some such embodiments, concentration may be applied to any polymer/payload combination (e.g., solution) (i.e., at any stage in a manufacturing process). In some

embodiments, a polymer/payload combination (e.g., solution) is concentrated using evaporation methods. In some embodiments, an initial polymer/payload combination (e.g., solution) (see, e.g., Steps 1 and 2 of Figure 8 A) is concentrated using evaporation methods. In some embodiments, a utilized evaporation method is or comprises rotary evaporation.

[0152] In some embodiments, concentration of a polymer/payload combination (e.g., solution) 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). In some embodiments, percentage of water and/or other solvent(s) or non-solvent(s) (e.g., individually or in aggregate) removed from a combination (e.g., solution, e.g., relative to amount present prior to concentration) is approximately 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, percentage of water and/or other solvent(s) removed (e.g., either alone or in aggregate) is approximately 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more.

[0153] In some embodiments, time of concentration (e.g., length of time of water and/or other solvent(s) evaporation process(es), such as by rotary evaporation) is within a range of about one hour to about 24 hours or more. In some embodiments, 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.

[0154] As described herein, a polymer/payload combination (e.g., solution) 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. In some embodiments, a polymer: payload ratio in a polymer/payload combination (e.g., solution) such as, e.g., prior to or after concentration may be expressed as a ratio, for example, as within a range of about 90: 10 and 100:0. In some embodiments, 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.

[0155] In some embodiments, 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. In some embodiments, 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). In some embodiments, 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). In some embodiments, 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). In some embodiments, 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.

[0156] In some embodiments, concentration is achieved by solvent or non-solvent (i.e., not water) evaporation. In some such embodiments, 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.

[0157] In some embodiments, conditions under which concentration (e.g., water and/or other solvent or non-solvent evaporation, such as by rotary evaporation) is performed may be altered according to particular solvents or non-solvents and/or components in a combination. It will be understood to those of skill in the art that certain parameters used in concentration techniques (e.g., pressure, temperature, time), will be altered to desirably achieve concentration of provided combinations..

[0158] In some embodiments, concentration may be performed at variable temperatures.

For example, in some embodiments, concentration (e.g., evaporation, e.g., rotary evaporation) may be performed at temperatures between 20 ⁰ C and 120 ⁰ C.

[0159] In some embodiments, concentration (e.g., evaporation, e.g., rotary evaporation) may involve rotation at a particular speed or speeds. In some such embodiments, a plurality of distinct speeds (e.g., variable speed) may be employed. In some embodiments, where rotation is used, speeds may vary between approximately 40 rpm and 100 rpm. In some embodiments, where rotation is used, speeds may vary between approximately 50 rpm and 90 rpm. In some embodiments, where rotation is used, speeds may vary between approximately 60 rpm and 80 rpm.

[0160] In some embodiments, concentration (e.g., evaporation, e.g., rotary evaporation) 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.

[0161] In certain embodiments, concentration (e.g., by water evaporation such as by rotary evaporation) of a polymer/payload combination (e.g., solution) results in increased encapsulation of protein in the polymer.

[0162] In some embodiments, for example, as described herein, manufacturing of nanoparticles involves one or more concentration steps before or after one or more lyophilization steps. In some embodiments, concentration is performed prior to and/or following one or more lyophilization steps. In some embodiments, concentration is not performed prior to and/or following one or more lyophilization steps.

[0163] In certain alternative embodiments, it is contemplated that concentration (e.g., by water evaporation such as by rotary evaporation) may replace, in whole or part, one or more lyophilization steps. For example, in some embodiments, concentration (e.g., by water evaporation such as by rotary evaporation)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. In some embodiments, after concentration (e.g., by water evaporation such as by rotary evaporation), 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.

[0164] In some embodiments, when microfluidization is used, concentration (e.g., by water evaporation such as by rotary evaporation) 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.

Polymer /payload cake

[0165] In some embodiments, 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. For example, in some embodiments, an initial polymer/payload combination (e.g., solution) is subjected to drying (e.g., freeze-drying or other drying strategy) so that such a dry material, referred to herein as a“polymer/payload cake”, is obtained (see, e.g., Figure 1, steps 3- 4; Figure 8A, steps 4-5).

[0166] In some embodiments, drying is performed at a temperature within a range of -

100 to 25°C (e.g., between -100 to 20°C, -100 to l5°C, -100 to l0°C, -100 to 5°C, -100 to 0°C, - 75 to 0 °C, -65 to 0 °C, -50 to 25°C, -25 to 25°C, or 0 to 25°C). In some embodiments, 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.

[0167] In some embodiments, drying is performed at a pressure within a range of 10 ⁸ to

0 bar (e.g., 10 ⁸ to 10 ¹ bar, 10 ⁸ to 10 ⁴ bar, or 10 ⁴ bar to 0 bar). In some embodiments, drying is performed under conditions in which pressure is controlled (e.g., using an external pressure controller). [0168] In some embodiments, drying is performed under inert gas (e.g., N₂, Ar). In some embodiments, drying is performed under atmospheric conditions.

[0169] In some embodiments, 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.

[0170] In some embodiments, 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.

[0171] In some embodiments, a dry or glassy material (e.g., polymer/payload cake, for example, a lyophilized polymer/payload cake) is characterized in that a payload is substantially homogenous with respect to polymer. In some embodiments, a dry material is sufficiently brittle so that the dry material can be milled.

Optional processing of polymer /payload cake

[0172] In some embodiments, a polymer/payload cake may optionally be processed as described herein, to generate a processed material. Among other things, the present disclosure provides the insight that processing (e.g., heating) 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. In some embodiments, processing of a polymer/payload cake may reduce porosity. In some embodiments, processing of a polymer/payload cake may decrease the volume of the

polymer/payload cake.

[0173] In some embodiments, optional processing of polymer/payload cake comprises heating. In some embodiments, a polymer/payload cake is heated to a temperature that exceeds the glass temperature of the polymer in the polymer/payload cake. In some embodiments, a polymer/payload cake is heated to a temperature within a range of about 70 °C to 150 °C. In some embodiments, a polymer/payload cake is heated to a temperature within a range of about 80 °C to 150 °C. In some embodiments, a polymer/payload cake is heated to a temperature within a range of about 90 °C to 150 °C. In some embodiments, 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.

[0174] In some embodiments, heating is performed at a pressure within a range of 10 ⁸ to

10 ⁴ bar.

[0175] In some embodiments, polymer in a polymer/payload cake is melted during processing (e.g., heating). In some embodiments, morphology and/or configuration of polymer in a polymer/payload cake may be changed during processing (e.g, heating). In some embodiments, heated polymer may wrap payload. In some embodiments, a polymer/payload cake may become homogeneous (e.g, payload is evenly distributed in polymer) during processing (e.g, heating).

[0176] In some embodiments, optional processing of polymer/payload cake comprises cooling (e.g, after heating). In some embodiments, a polymer/payload cake is cooled to room temperature. In some embodiments, a polymer/payload cake is cooled to a temperature within a range of about -15 to 40 °C or lower. In some embodiments, a polymer/payload cake is cooled to a temperature within a range of about -15 to 35 °C. In some embodiments, a polymer/payload cake is cooled to a temperature within a range of about -15 to 30 °C. In some embodiments, a polymer/payload cake is cooled to a temperature within a range of about -15 to 25 °C. In some embodiments, 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.

[0177] In some embodiments, cooling is performed at a pressure within a range of 10 ⁸ to

10 bar. [0178] In some embodiments, 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.

Granulated microparticles

[0179] In some embodiments, provided technologies include a step of generating microparticles from a dried and/or lyophilized polymer/payload cake, or a processed

polymer/payload cake.

[0180] In some embodiments, a dried and/or lyophilized polymer/payload cake, or a processed polymer/payload cake is comminuted. In some embodiments, 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.

[0181] Without wishing to be held to a particular theory, condensation during granulation

(e.g., generation of microparticles) may cause adhesion between granulates. In some

embodiments, 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. In some embodiments, a solvent (e.g., alcohol (e.g, propanol)) may be added during granulation to reduce condensation. In some embodiments, granulation may be performed under a dry condition that may reduce condensation.

[0182] In some embodiments, 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. In some embodiments, 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. One of skill in the art will recognize that temperatures may be adjusted to accommodate materials, such as, e.g., cooling materials (liquid nitrogen). For example, in some embodiments, where liquid nitrogen is used to freeze and/or process a material (e.g., polymer/payload cake) 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. [0183] In some embodiments, grinding is performed within the same range as temperature preceding a grinding step (e.g., freezing with liquid nitrogen). In some

embodiments, grinding is performed at a different temperature than a preceding freezing step. In some embodiments, 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.

[0184] In some embodiments, 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. In some embodiments, granulated microparticles have a size (e.g, average or mean diameter) within a range of about 1 pm to 800 pm. In some embodiments, granulated microparticles have a size (e.g, average or mean diameter) within a range of about 1 pm to 500 pm. In some embodiments, 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. In some embodiments, 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.

[0185] In some embodiments, granulated microparticles as described herein are characterized as having a substantially uniform distribution of payload with respect to polymer.

[0186] In some embodiments, microparticles as described herein are characterized as not having a substantially uniform distribution of payload with respect to polymer. In some such embodiments, one or more populations of granulated microparticles is present. For example, in some embodiments, a particular population of microparticles comprises a disproportionate amount of payload with respect to polymer versus another population of microparticles.

Flowable microparticle suspension

[0187] In some embodiments, provided technologies include methods for creating a flowable microparticle suspension from granulated microparticles ( e.g ., comprised of polymer and payload). [0188] In some embodiments, a liquid system is added to granulated microparticles ( e.g ., comprising polymer and payload) to form a flowable microparticle suspension. In some embodiments, a liquid system for a flowable microparticle suspension is substantially in accordance with several embodiments as described herein. In some embodiments, a liquid system for a flowable microparticle suspension may be a non-solvent for the microparticle matrix (e.g., polymer) and/or a payload. In some embodiments, 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. In some embodiments, a liquid system for a flowable microparticle suspension is selected from the group consisting of propanol, ethanol, methanol, and

combinations thereof.

[0189] In some embodiments, 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).

[0190] For example, in some embodiments, 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). In some embodiments, propanol may have a high enough boiling point such that it can accommodate heat during a homogenization process (e.g, described herein) without boiling.

[0191] In some embodiments, 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. In some embodiments, a solvent system further comprises one or more detergents and/or surfactants.

[0192] Among other things, the present disclosure demonstrates the surprising discovery that provided solvent systems (e.g, non-solvent system of microparticles, e.g, propanol) can achieve advantages in homogenization of a flowable microparticle suspension. For example, in some embodiments, a non-solvent system can minimize dissolution of polymer and/or payload during homogenization of a flowable microparticle suspension.

[0193] In some embodiments, a flowable microparticle suspension is created by adding ground microparticles as described herein to propanol. In some embodiments, an initial propanol concentration is higher than a final propanol concentration. For example, in some embodiments, an initial n-propanol concentration in an initial flowable microparticle suspension is approximately 5-10 mg/mL. In some embodiments, 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. For example, in some embodiments, 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. In some embodiments, a final concentration of n-propanol may be within a range of approximately 0.25 to 20 mg/mL. In some embodiments, a final concentration of n-propanol may be within a range of approximately 0.3 - 15 mg/mL. In some embodiments, a final concentration of n-propanol may be within a range of approximately 0.5-10 mg/mL.

Homogenized nanoyarticles

[0194] In some embodiments, provided technologies include methods of making nanoparticles from a flowable microparticle suspension (e.g, homogenization).

[0195] In some embodiments, homogenization is performed by blender, bead mills, sonicator, rotor-stator mechanical homogenizer, microfluidizer, or combinations thereof.

[0196] In some embodiments, a flowable microparticle suspension may be homogenized by a microfluidizer. Typically, a microfluidizer converts a high fluid pressure into a shear force applied to a suspension. For example, 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. Without wishing to be bound by any particular theory, a shear force applied to a suspension may produce homogenized particles.

[0197] In some embodiments, 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^'V¹ to a flowable microparticle suspension.

[0198] In some embodiments, 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

embodiments, 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.

[0199] In accordance with various embodiments, 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

embodiments, 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.

Stabilized nanoparticles

[0200] In some embodiments, 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).

[0201] In some embodiments, a solution/suspension of homogenized nanoparticles is stabilized to prevent homogenized nanoparticles from agglomeration. [0202] In some embodiments, it is contemplated that 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.

[0203] In some embodiments, a diluting solvent system may be or comprise a non solvent of polymer. In some embodiments, a diluting solvent system is miscible with at least one of water and DMSO. In some embodiments, a diluting solvent system is the same as or comprises the original solvent used for nanoparticle formulation. In some embodiments, 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.

[0204] In some embodiments, 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.

Amphiphilic Stabilizers

[0205] In some embodiments, a stabilizing agent may be or comprise an amphiphilic copolymer (i.e., a copolymer of a hydrophilic block coupled with a hydrophobic block). In some embodiments, 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. [0206] Examples of 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,

Illum, L., Davids, S. S. (eds.) Polymers in Controlled Drug Delivery. Wright, Bristol, 1987; Arshady, J Controlled Release (1991) 17:1-22; Pitt, Int. J. Phar. (1990) 59: 173-196; Holland, et al, J. Controlled Release (1986) 4: 155-180); 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

(valerolactone), polyanhydrides, copolymers of poly (caprolactone) or poly (lactic acid) In some embodiments, for non-biologically related applications particularly preferred polymeric blocks include polystyrene, polyacrylates, and butadienes.

[0207] Examples of 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, gelatin, and unsaturated ethylenic mono or dicarboxylic acids.

[0208] In some embodiments, blocks of a particular copolymer may be either diblock or triblock repeats. In some embodiments, block copolymers include blocks of polystyrene, polyethylene, polybutyl acrylate, polybutyl methacrylate, polylactic acid, polycaprolactone, polyacrylic acid, polyoxyethylene and polyacrylamide. A listing of suitable hydrophilic polymers compatible with some embodiments can be found in Handbook of Water-Soluble Gums and Resins. R. Davidson, McGraw-Hill (1980).

[0209] In some embodiments including one or more graft copolymers, the length of a grafted moiety can vary. In some embodiments, the grafted segments are alkyl chains of 12 to 32 carbons or equivalent to 6 to 16 ethylene units in length. In some embodiments, the grafting of the polymer backbone can be useful to enhance solvation or nanoparticle stabilization properties. In some embodiments, 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. In some embodiments, 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. One example of a commercially available stabilizer is the Hypermer family marketed by Uniqema Co. In some embodiments, an amphiphilic stabilizer could also be of the gelatin family such as the gelatins derived from animal or fish collagen.

[0210] In some embodiments, a stabilizing agent may be used to reduce agglomeration of homogenized nanoparticles. Without wishing to be bound by any particular theory, surfaces of homogenized nanoparticles may be modified by a stabilizing agent.

[0211] In some embodiments, a stabilizing agent may be or comprise a poloxamer, or small ionic surfactant. In some embodiments, 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. [0212] In some embodiments, a stabilizing agent is directly added to a

solution/suspension of homogenized nanoparticles. In some embodiments, a stabilizing agent solution is added to a solution/suspension of homogenized nanoparticles.

[0213] In some embodiments, 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.

[0214] In some embodiments, a stabilizing agent (e.g., PVA1) 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. In some embodiments, a stabilizing agent (e.g., PVA1) 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.

[0215] In some embodiments, 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).

[0216] In some embodiments, an aqueous PVA1 solution is added to a homogenized nanoparticle suspension to reduce aggregation of nanoparticles. In some embodiments, no PVA1 is added.

[0217] In some embodiments, aqueous PVA1 solution and nanoparticle suspension are mixed for about 10 to 45 mins (e.g., approximately 10, 20, 30, or 40 minutes).

[0218] In some embodiments, one or more solutes or solvents is added to a combination

(e.g., solution, e.g., suspension) comprising of homogenized nanoparticles; in some such embodiments, nanoparticles are stabilized when one or more such solutes or solvents is/are present.

[0219] In some embodiments, nanoparticles are stabilized by addition of polyvinyl alcohol (PVA1) in water to the solution of nanoparticles in hot propanol. In some such embodiments, PVA1 in water is added to the nanoparticle containing solution, and the solution is cooled to room temperature before proceeding to any further steps [0220] In some embodiments, a stabilized nanoparticle suspension (e.g., comprising polymer/payload nanoparticles with a stabilizing agent) is further homogenized (e.g., by a microfluidizer). In some embodiments, a stabilized nanoparticle suspension (e.g, comprising polymer/payload nanoparticles coated with a stabilizing agent) passes through a homogenizer between one and twenty, or more, times. In some embodiments, a stabilized nanoparticle suspension (e.g, comprising polymer/payload nanoparticles coated with a stabilizing agent) 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. In some embodiments, a homogenizer does not supply heat while homogenizing the stabilized nanoparticle suspension. In some embodiments, a homogenizer is cooled (e.g., actively cooled) while homogenizing the stabilized nanoparticle suspension.

Loaded nanoyarticles

[0221] The present disclosure describes certain“loaded” nanoparticles. For example, in some embodiments, 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).

[0222] In some embodiments, after stabilization of nanoparticles following microfluidic processing, a composition is a combination comprising polymeric nanoparticles loaded with protein and DNA.

[0223] In some embodiments, 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. In some embodiments, in a combination comprising loaded nanoparticles, propanol, and water, percent of water is in a range between about 50% to about 99% of the combination. In some embodiments, 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) in a combination comprising loaded

nanoparticles is in a range of approximately 1 :99 to 99: 1. In some embodiments, a ratio of water to propanol is 10:90 to 30:70. In some embodiments, a ratio of water to propanol is 50:50. In some embodiments, a ratio of water to propanol is approximately 75:25. In some embodiments, a ratio of water to propanol is approximately 85: 15. In some embodiments, a ratio of water to propanol is approximately 80:20.

[0224] The present disclosure provides the insight that manufacturing protocols as described herein may produce one or more populations of nanoparticles. As used herein, 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). For example, in some embodiments, a population of nanoparticles may have a mean size of between

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). In some embodiments, different populations of nanoparticles are represented by different sizes (e.g., mean size, e.g., mean range of

approximately 100-200 nm in at least one dimension, 100-300 nm in at least one dimension, 100- 400 nm in at least one dimension, 100-500 nm in at least one dimension, etc.) and/or different protein concentrations (e.g., 20-90 mg protein/mg PLGA, 5-20 mg protein/mg PLGA, etc.).

[0225] In some embodiments, 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).

[0226] In some embodiments, 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. For example, in some embodiments, 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).

[0227] In some embodiments, a population of nanoparticles with the highest

encapsulation rate (relative to other populations of nanoparticles produced during a

manufacturing process) is a population between approximately 100-500 nm. In some such embodiments, nanoparticles between 100-500 nm in at least one dimension comprise

approximately 20-90 mg protein/mg PLGA. In some such embodiments, 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.

[0229] In some embodiments, at least two populations of nanoparticles (e.g., a population with a higher protein/PLGA concentration and a population with a lower protein/PLGA concentration either objectively as described herein, or relative to one another) are producing during a single manufacturing process.

[0230] In some embodiments, at least two populations of nanoparticles are produced in separate manufacturing processes.

[0231] In some embodiments, purification procedures (e.g., see Figure 8A, steps 10-12) are altered to selectively eliminate and/or selectively enrich for a particular population of nanoparticles.

[0232] The present disclosure provides the insight that certain steps may be taken in order to improve encapsulation of payload in loaded nanoparticles. In some embodiments, 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

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

approximately 70-90%.

Additional components

[0233] In some embodiments, compositions as described herein may include one or more additional components not specifically named in the description above. Those skilled in the art, reading the present disclosure, will be aware of a variety of additional components that can be included in such compositions. To give but some examples, in some embodiments, 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. For example, in some embodiments, an additional component may naturally occur in a crystalline form that is not particularly compatible with a nanoparticle formulation. In some such embodiments, 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.

[0234] In some embodiments, a dissolution aid may be added to a nanoparticle suspension (e.g., comprising nanoparticles coated with a stabilizing agent). In some

embodiments, 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) and combinations thereof.

[0235] In some embodiments, 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

pharmaceutical composition. The present disclosure provides the insight that, for example, in production and subsequent administration of a pharmaceutical composition, an important feature in any clinical trial is for an active ingredient to have a similar texture and/or appearance as inactive ingredients, such that, e.g., a placebo will not be readily distinguishable from an active compound. For example, in some embodiments, trehalose granules are crystalline and larger than nanoparticles of suspensions disclosed herein. In some such embodiments, it is

contemplated that a favorable approach is to have a dissolution agent that is more similarly sized and textured to nanoparticles as produced according to the methods described herein. Therefore, in some embodiments, 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.

[0236] In some embodiments, 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

[0237] In some embodiments, provided methods further include a post-processing step applicable to provided nanoparticles. As will be apparent to one of ordinary skill in the art, 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). Among other things, 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). The present disclosure also recognizes that 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. Without wishing to be bound by any particular theory, 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. In some embodiments, post-processing steps may change (e.g., increase or decrease) recovery of solids during a nanoparticle manufacturing procedure.

[0238] In some embodiments, post-processing may comprise one or more centrifugation steps. In some embodiments, two or more centrifugation steps may be performed (e.g., in a serial manner). In some embodiments, two or more centrifugation steps are performed with one or more optional steps in between centrifugations. In some embodiments, when two or more centrifugation steps are performed, 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.

[0239] In some embodiments, one or more low speed (e.g., 100-750 xg) centrifugation step(s) is/are performed using a nanoparticle solution. Without wishing to be bound to any particular theory, it is contemplated that 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). In some embodiments, 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. In some embodiments, 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

approximately 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.

[0240] In some embodiments, after an initial low speed centrifugation step using a solution comprising nanoparticles, 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. In some embodiments, it is contemplated that 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.

[0241] In some embodiments, after an initial low speed centrifugation step, 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) may be performed. In some embodiments, one or more purification and/or separation methods may be performed prior to an initial or subsequent (relative to initial) low speed centrifugation step.

[0242] In some embodiments, 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

nanoparticles from a solution. In some embodiments, an intermediate speed centrifugation step is performed after a low speed centrifugation step. In other embodiments, 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. In some embodiments, an intermediate speed centrifugation step is performed at speeds of approximately 750 xg - approximately 7500 x g. In some embodiments, 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. In some embodiments, an intermediate speed centrifugation step is performed at temperature ranges of approximately 4-37 °C. In some other embodiments, after an initial intermediate speed centrifuge step one or more additional centrifuge steps are performed on a given supernatant to further pellet nanoparticles present in the solution. In some embodiments, 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.

[0243] In some embodiments, 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. In some embodiments, a high speed centrifugation step is performed after a low and/or intermediate speed centrifugation step. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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

[0244] In some embodiments, 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.

[0245] In some embodiments, 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). Without wishing to be held by a particular theory, a payload weakly associated with a nanoparticle may be released at a temperature near the glass transition temperature of a provided payload. For example, in some embodiments, a payload weakly associated with a nanoparticle may be released at a temperature within a range of about 30 to 50°C. In some embodiments, 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,. In some embodiments, 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. continuous flow centrifugation which may be or comprise flow in an aqueous buffer and extraction while spinning, with nanoparticles under centrifugal force for many hours), and/or use of an ion exchange resin. [0246] For example, in some embodiments, components of nanoparticle compositions may be further separated using filtration. In some such embodiments, nanoparticles may be filtered. In some embodiments, filtration may occur through a column comprising a medium (e.g., a resin). In some embodiments, filtration may occur through a membrane.

[0247] In some embodiments, 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.

[0248] As will be appreciated and understood by one of skill in the art, 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.).

[0249] In some embodiments, filtration may be or comprise tangential flow filtration. In some such embodiments, tangential flow filtration may be performed by contacting a surface (e.g., filter, membrane) with a composition. In some embodiments, a surface area of a membrane is relative to a volume of initial input solution to be filtered. In some such embodiments, a surface area of a membrane is between a range of approximately 0.01 - 0.1 m²/L. In some such embodiments, a surface has a surface area of approximately 75 1000 cm². In some embodiments, tangential flow filtration may be performed using a filter with a surface area of approximately 1000-5000 cm². In some embodiments, a surface area of a filter may be between 100-750 cm². In some embodiments, a surface used in tangential flow filtration has a molecular weight cut-off (MWCO) of about approximately 100 kilodaltons to approximately 1000 kilodaltons. In some embodiments, a MWCO is approximately 200 kilodaltons to approximately 600 kilodaltons. In some embodiments, a MWCO is approximately 300 kilodaltons to approximately 500 kilodaltons. In some embodiments, 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. One of skill in the art will bring with them an understanding of surface area and MWCO sizes appropriate to filter a provided composition.

[0250] In some embodiments, 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

embodiments, if a suspension is filtered through a 500 kD membrane with 18 volume washes, 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. One of skill in the art will understand that wash volumes and times will be altered due to factors such as membrane type and contents of material (e.g., suspension) to be filtered.

[0251] In some embodiments 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. For example, in some embodiments, a sugar (e.g., trehalose) may be added to a composition comprising nanoparticles. For example, in some embodiments, trehalose may be added to a combination comprising nanoparticles, PVA1, water and propanol. In some embodiments, sugar (e.g., trehalose) is added in a ratio of approximately 0.5:20 mg /mg PLGA to approximately 20: 0.5 mg / mg PLGA. In some embodiments, 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.

[0252] In some embodiments, lyophilization of a nanoparticle solution will produce a dry cake comprising nanoparticles, PVA1, and trehalose. In some embodiments, a dry cake comprising nanoparticles is resuspended in a buffer. In some embodiments, a buffer comprises ammonium bicarbonate. In some embodiments, a 10 mM ammonium bicarbonate buffer is used to resuspend a lyophilized cake comprising nanoparticles, PVA1, and trehalose. One of skill in the art will recognize that 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.

[0253] In some embodiments, post-processing of nanoparticles includes one or more lyophilization steps. In some embodiments, lyophilizations are not serially performed, rather, are separated by additional post-processing steps.

[0254] In some embodiments, 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. In some embodiments, ion exchange and/or chromatography may separate a payload that is weakly associated with a nanoparticle.

Typically, chromatography separates ions and polar molecules based on their affinity, for example, to the ion exchanger. For example, 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).

[0255] In some embodiments a resin, for example, in a column may be used for post- processing of nanoparticles. In some embodiments, post-processing comprising a column (e.g. an ion exchange column) may occur in more than one step of certain provided methods and/or with more than one column. In some embodiments, 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.

[0256] In some embodiments, post-processing may include incubating nanoparticles with an amount of resin (e.g., ion exchange resin) for a period of time.

[0257] In some embodiments, a period of time may be, e.g. 15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, or more)

[0258] In some embodiments, it may be desirable to modify certain key parameters of an ion exchange chromatography to produce desired nanoparticles. For example, a relative amount of ion exchange resin to nanoparticles may be increased, which may be helpful to separate a payload that is weakly associated with a nanoparticle. In some embodiments, nanoparticles may be incubated with a stationary phase longer to achieve a higher degree of separation of weakly associated payloads. In some embodiments, an ion exchange resin with a higher affinity to nanoparticles (e.g., higher retention time) may be helpful to separate payloads that are weakly associated with nanoparticles.

[0259] 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. For example, the present disclosure recognizes that, in some embodiments, additional separation steps may be needed to collect desired populations of nanoparticles. For example, in some embodiments, 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. In some embodiments, 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.

[0260] In some embodiments, before nanoparticles are subject to post-processing (e.g., contact with a resin), the amount of free payload to encapsulated payload may be any of a variety of ratios. In some embodiments, 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. In some embodiments, the ratio of free payload to encapsulated payload is greater than 10: 1. In some embodiments, the ratio of free payload to encapsulated payload is less than 1 : 10.

[0261] In some embodiments, 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. In some embodiments, a percentage of nanoparticles lost is at least about 5% to at most about 25%. In some embodiments, a percentage of nanoparticles lost is at least about 25% to at most about 50%. In some embodiments, a percentage of nanoparticles lost is at least about 50% to at most about 75%. In some embodiments, 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. In some embodiments, a protease may be in a suspension or solution. In some embodiments, a protease may be associated with a carrier (e.g., a bead). In some embodiments, chromatography is performed to separate the digested payload by the protease. In some embodiments, 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.

[0262] In some embodiments, 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.

[0263] In some embodiments, 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.).

[0264] In some embodiments, an amount (e.g., concentration in a suspension or solution, ratio of molecules of protease to nanoparticles) 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.

[0265] In some embodiments, nanoparticles are treated with a protease for between 5 and

60 mins (e.g., approximately 10, 20, 30, 40, or 50 minutes). In some embodiments, 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.

[0266] In some embodiments, nanoparticles are treated with a protease at a temperature within a range of between 0 and 37 °C. In some embodiments, 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. [0267] In some embodiments, 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). In some embodiments, 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.

[0268] In some embodiments, 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. In some embodiments, amount of protein encapsulation corresponds to a safety factor. In some such embodiments, 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. In some embodiments, 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. In some embodiments, 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). In some embodiments, 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.

Coating Nanoyarticles

[0269] In some embodiments, provided methods further include a step of coating nanoparticles. In some embodiments, a dry coating agent is directly added to a nanoparticle suspension. In some embodiments, a coating agent solution is added to a nanoparticle suspension. As discussed further herein, 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. In some embodiments, a combination of nanoparticles and coating agents is stirred and/or sonicated to form coated nanoparticles. In some embodiments, 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.

[0270] In some embodiments, 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).

[0271] In some embodiments, a solution comprising coated nanoparticles is lyophilized to form a solid dispersion (e.g., a powder). In some embodiments, a coated nanoparticle suspension is subjected to freeze-drying, lyophilization, or other drying strategy so that such solid nanoparticle dispersion is obtained.

[0272] In some embodiments, a solid dispersion of coated nanoparticles may be milled, sifted, or sieved, so that the solid dispersion may have a desired particle size distribution.

Components of Nanoparticle Compositions

Polymers

[0273] 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. In some embodiments, payloads are homogeneously or substantially homogenously distributed in a polymer matrix.

[0274] In some embodiments, 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.

[0275] In some embodiments, 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.

[0276] In some embodiments, 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(glycolide), poly(lactide-co-glycolide), poly(anhydrides), PEGylated poly(anhydrides), poly (ortho esters), derivatives of poly(ortho esters), PEGylated poly(ortho esters),

poly(caprolactones), derivatives of poly(caprolactone), PEGylated poly(caprolactones), polyamines ( e.g ., spermine, spermidine, polylysine, and derivatives thereof), PEGylated polylysine, polyamides, polycarbonates, polypropylene fumarates), polyamides,

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),

poly(urethane), PEGylated poly(urethane), derivatives of poly(urethane), poly(lactide), poly(glycolide), poly(hydroxy acids), polyesters, poly(acrylates), polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as polyethylene glycol),

polyalkylene oxides such as poly(ethylene oxide), polyalkylene terepthalates such as

poly(ethylene terephthalate), polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides such as poly(vinyl chloride), polyvinylpyrrolidone, polysiloxanes, poly(vinyl alcohols), poly(vinyl acetate), polystyrene, polyurethanes and co-polymers thereof, derivativized celluloses such as alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, and cellulose sulfate sodium salt (jointly referred to herein as "synthetic celluloses"), polymers of acrylic acid, methacrylic acid or copolymers or derivatives thereof including esters, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly (isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate) (jointly referred to herein as "polyacrylic acids"), poly(butyric acid), poly(valeric acid), and poly(lactide-co-caprolactone) and/or derivatives thereof.

[0277] In some embodiments, nanoparticles are comprised of one or more natural polymers. Exemplary 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. In some embodiments, a natural polymer may comprise or consist of chitosan.

[0278] In some embodiments, nanoparticles are comprised of one or more polymers such as poly(lactide-co-glycolide) copolymerized with polyethylene glycol (PEG). Without wishing to be held to a particular theory, it is proposed that arrangement of a nanoparticle so that PEG is exposed on the external surface, may increase stability of the nanoparticle in blood, perhaps at least in part due to the hydrophilicity of PEG.

[0279] In some embodiments, the present disclosure encompasses the recognition that viscosity of polymer may be an important factor for producing nanoparticles. Without wishing to be bound by any particular theory, viscosity of a polymer solution is a function of the molecular weight of the polymer and operating temperature. In some embodiments, a polymer with a high molecular weight requires high operation temperature to have low enough viscosity to be processed.

Payloads

[0280] In some embodiments, 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). [0281] In some embodiments, a payload may be or comprise an agent or entity that elicits a particular biological response when delivered to an appropriate subject. Alternatively or additionally, in some embodiments, a payload may be or comprise an agent or entity that modulates a particular biological response to another, different, agent or entity. In some embodiments, a payload may be or comprise an agent or entity with respect to which a particular biological response is desired.

[0282] In some embodiments, a biological response elicited by or desired with respect to a particular payload may be or comprise an immune response. In some embodiments, a payload that modifies a biological response is or comprises an immune adjuvant. In some embodiments, presence of an immune adjuvant may modify (e.g., amplify, bias, or alter) an immune response to another entity (e.g., to an antigen).

[0283] 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. For instance, in some embodiments, 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.

[0284] In some embodiments, a payload comprises one or more carbohydrates, lipids, metals, nucleic acids, polypeptides, small molecules and/or combinations thereof.

[0285] Those skilled in the art will appreciate that a payload as described and/or utilized herein may be prepared or manufactured by any appropriate technology. For example, 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). [0286] In some embodiments, a payload may be provided in combination with another substance. In some embodiments, 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.).

[0287] Alternatively or additionally, one feature of certain embodiments of the present disclosure is that it permits utilization of relatively crude payload preparations. In some embodiments, a payload may be or comprise a crude preparation and/or other complex material (e.g., an extract, etc.).

[0288] In some embodiments, provided nanoparticles comprise microbial hydrophobic and/or hydrophilic cellular components (e.g., from a crude microbial extract). Without wishing to be held by a particular theory, 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.

[0289] 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.

[0290] In some embodiments, provided nanoparticles comprise microbial extracts - e.g., hydrophilic or hydrophobic extracts of microbial cells for use in or with nanoparticle

compositions. In some embodiments, 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. In some embodiments, 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. Moreover, 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.

[0291] In general, 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). In some embodiments, microbial cell preparations contain intact cells, and optionally are substantially free of lysed cells. In some embodiments, microbial cell preparations contain lysed cells, and optionally are substantially free of intact cells.

[0292] In some embodiments, 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. In some embodiments, hydrophilic cellular components that partition into a hydrophilic solvent include, for example, components that are miscible and/or soluble in such solvent.

Antigens

[0293] In some embodiments, a payload is or comprises an antigen. In some

embodiments, 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.

[0294] In some embodiments, 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. In some embodiments, 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.). In some embodiments, 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).

[0295] In some embodiments, provided nanoparticles and/or nanoparticle compositions may include one or more crude (i.e., unpurified or substantially unpurified) antigenic extracts. In some embodiments, crude extract can be a useful and inexpensive alternative to using individual antigens in provided nanoparticle compositions.

[0296] In some embodiments, 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.

[0297] One of skill in the art will recognize that multiple antigens may be delivered by nanoparticles simultaneously and/or sequentially in accordance with methods of the present disclosure. Without limitation, different antigens for one antigenic protein may be delivered. Different antigens from different antigenic proteins may also be delivered. Further, multiple antigenic polypeptides and proteins may be delivered in accordance with the present disclosure.

It is also recognized that single or multiple antigenic polypeptides and single or multiple cytokines may be delivered to individuals by nanoparticles in accordance with the present disclosure. For example, but without limitation, 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.

[0298] In some embodiments, a particular provided composition may contain a combination of antigens. For example, in some embodiments, 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.).

[0299] Those of skill in the art will recognize a wide variety of potential applications utilizing combinations of antigens; each of these is contemplated as within the scope of the present disclosure.

[0300] According to various embodiments, provided 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. In some embodiments, an antigen may be provided as a portion of a cell, tissue or extract thereof.

[0301] In some embodiments, 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.

Allergens

[0302] In some embodiments, an antigen is or comprises an allergen. In some embodiments, 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.

[0303] In some embodiments, 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. Exemplary 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. In some embodiments, 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. In general, insect venom includes venom from Hymenoptera such as bees, hornets, wasps, yellow jackets, velvet ants, and fire ants. For example, venom from honey bees of the genus Apis can cause anaphylaxis in stung victims who are allergic (Weber et al. Allergy 42:464-470). The venom from 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. Non-limiting examples of 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).

[0304] In some embodiments, 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.

[0305] In some embodiments, 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.

[0306] In some embodiments, it may be desirable to work in systems in which a single compound (e.g, a single protein) is responsible for an observed allergy. In some embodiments, 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.

[0307] In some embodiments, 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

[0308] The present disclosure encompasses the recognition that a particular subject may benefit from being exposed to a combination of antigens, such as multiple allergens. In some embodiments, it may be desirable to provide a nanoparticle composition comprising multiple antigens relevant to a specific subject, and/or to a population of subjects. For example, in some embodiments, 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. For example, if a particular subject is allergic to peanuts and to dust mites, a nanoparticle composition may be designed and manufactured to address both allergies. Alternatively or additionally, in some embodiments it may be desirable to prepare 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.

Infectious Antigens

[0309] In some embodiments, antigens may be provided from infectious organisms, such as viruses, parasites and bacteria. In some embodiments, 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, Apostolopoulos, et al.

(Curr. Opin. Mol. Ther ., 2:29-36 (2000)), discusses a strategy for identifying minimal antigenic peptide sequences based on an understanding of the three dimensional structure of an antigen- presenting molecule and its interaction with both an antigenic peptide and T-cell receptor.

Shastri, (Curr. Opin. Immunol ., 8:271-7 (1996)), discloses how to distinguish rare peptides that serve to activate T cells from the thousands peptides normally bound to MHC molecules.

[0310] In some embodiments, provided nanoparticles and/or nanoparticle compositions may include one or more viral antigens. Generally, a virus consists of either two or three parts:

1) genetic material, which may be DNA or RNA, depending on the virus, 2) a protein coat that surrounds and protects the genetic material, and, in some viruses, 3) a lipid envelope that surrounds the protein coat. In some embodiments, a viral antigen may be provided from any component of a virus. In some embodiments, 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, Paramyxoviridae (e.g., measles, mumps, and human respiratory syncytial virus), Parvoviridae, Picornaviridae (e.g., poliovirus, rhinovirus, hepatovirus, and aphthovirus), Poxviridae (e.g, vaccinia and smallpox virus), Reoviridae (e.g., rotavirus), Retroviridae (e.g, lentivirus, such as human

immunodeficiency virus (HIV) 1 and HIV 2), Rhabdoviridae (for example, rabies virus, measles virus, respiratory syncytial virus, etc.), Togaviridae (for example, rubella virus, dengue virus, etc), and 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. In some embodiments, a viral antigen may comprise or consist of fragments of one or more viruses, such as fragments from an influenza virus, for example. In some embodiments, 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.

[0311] 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,

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.

[0312] In some embodiments, 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,

Deinococcus, Enterobacter, Escherichia, Francisella, Haemophilus, Halobacterium,

Heliobacter, Hemophilus influenza type B (HIB), 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,

Sporolactohacillu , Staphylococcus , Streptococcus , Streptomyces, Sulfolohus , Thermoplasma , Thiohacillus , Treponema , Vibrio , Yersinia , and combinations thereof

[0313] In some embodiments, 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 , Mycoplasma pneumoniae , Nocardia asteroides,

Plasmodiumfalciparum , Rickettsia ricketsii , Rickettsia typhi , Schistosoma mansoni, Toxoplasma gondii , Trichomonas vaginalis and Trypanosoma brucei. These include Sporozoan antigens, Plasmodian antigens, such as all or part of a Circumsporozoite protein, a Sporozoite surface protein, a liver stage antigen, an apical membrane associated protein, or a Merozoite surface protein.

Cancer Antigens

[0314] In some embodiments, provided nanoparticles and/or nanoparticle compositions may include cancer antigens. In some embodiments, cancer antigens may be provided from tumor cells. In some embodiments, the cancer antigens may be purified or partially purified polypeptides derived from tumors. In some embodiments, 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, Lage-l, MageAl, 2,3,4,6,10,12, Mage-C2, NA-88, NY-Eso- l/Lage-2, SP17, SSX-2, and TRP2-Int2, MelanA (MART -I), gplOO (Pmell7), tyrosinase, TRP- 1, TRP-2, MAGE-l, MAGE-3, BAGE, GAGE-l, GAGE-2, pl5(58), CEA, RAGE, NY-ESO (LAGE), SCP-l, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4- RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, pl85erbB2, pl80erbB-3, c-met, nm-25 23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, pCatenin, CDK4, Mum-l, pl6, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 79lTgp72, a-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA15-3 (CA 27.29VBCAA), CA 195, CA 242, CA-50, CAM43, CD68\KPl, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-l75, M344, MA-50, MG7-30 Ag, MOV18, NB\70K, NY-C0-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin C-associated protein), TAAL6, TAG72, TLP, and TPS.

[0315] In some embodiments, 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;

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; intraocular Melanoma; Islet Cell Carcinoma

(Endocrine Pancreas); 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);

metastatic squamous neck cancer; mouth cancer; Multiple Endocrine Neoplasia Syndrome; Multiple Myeloma; Mycosis Fungoides; Myelodysplastic Syndromes;

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; and/or combinations thereof.

Alloantigens

[0316] In some embodiments, provided nanoparticles include one or more alloantigens.

As described herein, 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.

[0317] In some embodiments, an alloantigen for use in accordance with the present disclosure is a major histocompatibility complex (MHC) polypeptide. In some embodiments, an alloantigen for use in accordance with the present disclosure is a Class I MHC polypeptide. In some embodiments, an alloantigen for use in accordance with the present disclosure is a Class II MHC polypeptide. In some embodiments, an alloantigen for use in accordance with the present disclosure contains part of or all of an extracellular domain of an MHC polypeptide. In some embodiments, an alloantigen for use in accordance with the present disclosure is a minor histocompatibility complex polypeptide. In some embodiments, an alloantigen for use in accordance with the present disclosure is a co-stimulatory entity ( e.g. , CD28, CD80, and CD86, among others). In some embodiments, 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. One of ordinary skill in the art will recognize that alloantigens described herein are exemplary. Any polypeptide that is associated with an allorecognition and/or graft rejection can be classified as an alloantigen.

[0318] It will be appreciated that 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. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and further usually including at least one region of much higher identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions, usually encompassing at least 3-4 and often up to 20 or more amino acids, with another alloantigen polypeptide of the same class, is encompassed within the relevant term“alloantigen polypeptide” as used herein.

Exemplary Antigens

[0319] In an effort to better exemplify some embodiments, 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); Alternaria alternata (fungus) acid ribosomal protein Pl (Alt a 12); Alternaria alternata (fungus) aldehyde dehydrogenase (Alt a 10); Alternaria alternata (fungus) antigen (Alt a 1); Alternaria alternata (fungus) antigen (Alt a 2); Alternaria alternata (fungus) enloase (Alt a l l); Alternaria alternata (fungus) heat shock protein (Alt a 3); Alternaria alternata (fungus) ribosomal protein (Alt a 6); Alternaria alternata (fungus) YCP4 protein (Alt a 7); Ambrosia artemisiifolia (short ragweed) antigen E (Amb a 1); Ambrosia artemisiifolia (short ragweed) antigen K (Amb a 2); Ambrosia artemisiifolia (short ragweed) Ra3 antigen (Amb a 3); Ambrosia artemisiifolia (short ragweed) Ra5 antigen (Amb a 5); Ambrosia artemisiifolia (short ragweed) Ra6 antigen (Amb a 6); Ambrosia artemisiifolia (short ragweed) Ra7 antigen (Amb a 7); Ambrosia trifida (giant ragweed) Ra5G antigen (Amb t 5); Anisakis simplex (nematode) antigen (Ani s 1); Anisakis simplex (nematode) paramyosin (Ani s 2); Apis mellifera (honey bee) antigen (Api m 6); Apis mellifera (honey bee) hyaluronidase (Api m 2); Apis mellifera (honey bee) melittin (Api m 4); Apis mellifera (honey bee) phospholipase A2 (Api m l); Apium graveolens (celery) antigen (Api g 5); Apium graveolens (celery) Bet v 1 homologue (Api g 1); Apium graveolens (celery) profilin (Api g 4); Arachis hypogaea (peanut) (conglutin Ar a h 2); Arachis hypogaea (peanut) (profilin Ar a h 5); Arachis hypogaea (peanut) conglutin homologue (Ar a h 6); Arachis hypogaea (peanut) conglutin homologue (Ar a h 7); Arachis hypogaea (peanut) glycinin (Ar a h 3); Arachis hypogaea (peanut) glycinin (Ar a h 4); Arachis hypogaea (peanut) vicilin (Ar a h 1); Artemisia vulgaris (mugwort) antigen (Art v 1); Artemisia vulgaris (mugwort) antigen (Art v 2); Ascaris suum (worm) antigen (Asc s 1); Aspergillus flavus (fungus) alkaline serine proteinase (Asp fl 13); Aspergillus Fumigatus (fungus) alkaline serine proteinase (Asp f 13); Aspergillus Fumigatus (fungus) antigen (Asp f 1); Aspergillus Fumigatus (fungus) antigen (Asp f 15); Aspergillus Fumigatus (fungus) antigen (Asp f 16); Aspergillus Fumigatus (fungus) antigen (Asp f 17); Aspergillus Fumigatus (fungus) antigen (Asp f 2); Aspergillus Fumigatus (fungus) antigen (Asp f 4); Aspergillus Fumigatus (fungus) antigen (Asp f 7);

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);

Aspergillus Fumigatus (fungus) metalloprotease (Asp f 5); Aspergillus Fumigatus (fungus) Mn superoxide dismutase (Asp f 6); Aspergillus Fumigatus (fungus) peptidyl-prolyl isomerase (Asp f 11); Aspergillus Fumigatus (fungus) peroxisomal protein (Asp f 3); Aspergillus Fumigatus (fungus) ribosomal protein P2 (Asp f 8); Aspergillus Fumigatus (fungus) vacuolar serine (Asp f 18); Aspergillus niger (fungus) antigen (Asp n 18); Aspergillus niger (fungus) beta-xylosidase (Asp n 14); Aspergillus niger (fungus) vacuolar serine proteinase; Aspergillus oryzae (fungus) alkaline serine proteinase (Asp o 13); Aspergillus oryzae (fungus) TAKA-amylase A (Asp o 2); Bertholletia excelsa (Brazil nut) 2S albumin (Ber e 1); Betula verrucosa (birch) antigen (Bet v 1); Betula verrucosa (birch) antigen (Bet v 3); Betula verrucosa (birch) antigen (Bet v 4); Betula verrucosa (birch) cyclophilin (Bet v 7); Betula verrucosa (birch) isoflavone reductase homologue (Bet v 5); Betula verrucosa (birch) profilin (Bet v 2); Blattella germanica (German cockroach) aspartic protease (Bla g 2); Blattella germanica (German cockroach) Bd90k (Bla g 1); Blattella germanica (German cockroach) calycin (Bla g 4); Blattella germanica (German cockroach) glutathione transferase (Bla g 5); Blattella germanica (German cockroach) troponin C (Bla g 6); Blomia tropicalis (mite) antigen (Blo t 5); Blomia tropicalis (mite) Btl la antigen (Blo 1 12); Blomia tropicalis (mite) Bt6 fatty acid-binding protein (Blo t); Bombus

pennsylvanicus (bumble bee) phospholipase (Bom p 1); Bombus pennsylvanicus (bumble bee) protease (Bom p 4); Bos domesticus (cow) Ag3, lipocalin (Bos d 2); Bos domesticus (cow) alpha- lactalbumin (Bos d 4); Bos domesticus (cow) beta-lactalbumin (Bos d 5); Bos domesticus (cow) casein (Bos d 8); Bos domesticus (cow) immunoglobulin (Bos d 7); Bos domesticus (cow) serum albumin (Bos d 6); Brassica juncea (oriental mustard) 2S albumin (Bra j 1); Brassica rapa (turnip) prohevein-like protein (Bar r 2); Candida albicans (fungus) antigen (Cand a 1); Candida boidinii (fungus) antigen (Cand b 2); Canis familiaris (dog) albumin (Can f ?); Canis familiaris (dog) antigen (Can f 1); Canis familiaris (dog) antigen (Can f 2); Carpinus betulus (hornbeam) antigen (Car b 1); Castanea sativa (chestnut) Bet v 1 homologue (Cas s 1); Castanea sativa (chestnut) chitinase (Cas s 5); Chironomus thummi thummi (midge) component I (Chi 12.0101); Chironomus thummi thummi (midge) component IA (Chi 12.0102); Chironomus thummi thummi (midge) component II-beta (Chi t 3); Chironomus thummi thummi (midge) component III (Chi t 1.01); Chironomus thummi thummi (midge) component IIIA (Chi 14); Chironomus thummi thummi (midge) component IV (Chi 1 1.02); Chironomus thummi thummi (midge) component IX (Chi t 6.02); Chironomus thummi thummi (midge) component VI (Chi t 5); Chironomus thummi thummi (midge) component VIIA (Chi t 6.01); Chironomus thummi thummi (midge) component VIIB (Chi t 7); Chironomus thummi thummi (midge) component VIII (Chi t 8); Chironomus thummi thummi (midge) component X (Chi t 9); Chironomus thummi thummi (midge) hemoglobin (Chi 1 1-9); Cladosporium herbarum (fungus) acid ribosomal protein Pl (Cla h 12); Cladosporium herbarum (fungus) aldehyde dehydrogenase (Cla h 3); Cladosporium herbarum (fungus) antigen (Cla h 1); Cladosporium herbarum (fungus) antigen (Cla h 2); Cladosporium herbarum (fungus) enolase (Cla h 6); Cladosporium herbarum (fungus) ribosomal protein); Cladosporium herbarum (fungus) YCP4 protein (Cla h 5); Coprinus comatus (shaggy cap) antigen (Cop c l); Coprinus comatus (shaggy cap) antigen (Cop c 2); Coprinus comatus (shaggy cap) antigen (Cop c 3); Coprinus comatus (shaggy cap) antigen (Cop c 5); Coprinus comatus (shaggy cap) antigen (Cop c 7); Corylus avellana (hazel) antigen (Cor a 1); Corylus avellana (hazelnut) Bet v 1 homologue (Cor a 1.0401); Cryptomeria japonica (sugi) antigen (Cry j 1); Cryptomeria japonica (sugi) antigen (Cry j 2); Ctenocephalides felis felis (cat flea) antigen (Cte f

I); Cynodon dactylon (Bermuda grass) antigen (Cyn d 1); Cynodon dactylon (Bermuda grass) antigen (Cyn d 7); Cynodon dactylon (Bermuda grass) profilin (Cyn d 12); Dactylis glomerata (orchard grass) AgDgl antigen (Dac g 1); Dactylis glomerata (orchard grass) antigen (Dac g 2); Dactylis glomerata (orchard grass) antigen (Dac g 3); Dactylis glomerata (orchard grass) antigen (Dac g 5); Dermatophagoides farinae (mite) antigen (Der f 1); Dermatophagoides farinae (mite) antigen (Der f 2); Dermatophagoides farinae (mite) antigen (Der f 3); Dermatophagoides farinae (mite) Mag 3, apolipophorin (Der f 14); Dermatophagoides farinae (mite) paramyosin (Der f

I I); Dermatophagoides farinae (mite) tropomyosin (Der f 10); Dermatophagoides microceras (mite) antigen (Der m l); Dermatophagoides pteronyssinus (mite) amylase (Der p 4);

Dermatophagoides pteronyssinus (mite) antigen (Der p 2); Dermatophagoides pteronyssinus (mite) antigen (Der p 5); Dermatophagoides pteronyssinus (mite) antigen (Der p 7);

Dermatophagoides pteronyssinus (mite) antigen Pl (Der p 1); Dermatophagoides pteronyssinus (mite) apolipophorin like p (Der p 14); Dermatophagoides pteronyssinus (mite) chymotrypsin (Der p 6); Dermatophagoides pteronyssinus (mite) collagenolytic serine prot. (Der p 9);

Dermatophagoides pteronyssinus (mite) glutathione transferase (Der p 8); Dermatophagoides pteronyssinus (mite) tropomyosin (Der p 10); Dermatophagoides pteronyssinus (mite) trypsin (Der p 3); Dolichovespula arenaria (yellow hornet) antigen 5 (Dol a 5); Dolichovespula maculata (white face hornet) antigen 5 (Dol m 5); Dolichovespula maculata (white face hornet) phospholipase (Dol m l); Dolichovespula maculate (white face hornet) hyaluronidase (Dol m 2); Equus caballus (horse) lipocalin (Equ c l); Equus caballus (horse) lipocalin (Equ c 2);

Euroglyphus maynei (mite) apolipophorin (Eur m 14); 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

(soybean) HPS (Gly m 1.0102); Glycine max (soybean) profilin (Gly m 3); Haliotis Midae (abalone) antigen (Hal m l); Helianthus annuus (sunflower) antigen (Hel a 1); Helianthus annuus (sunflower) profilin (Hel a 2); Hevea brasiliensis (rubber) l,3-glucanase (Hev b 2);

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) profilin (Hev b 8); Holcus lanatus (velvet grass) antigen (Hol 1 1); Homo sapiens (human autoallergen) antigen (Horn s 1); Homo sapiens (human autoallergen) antigen (Horn s 2); Homo sapiens (human autoallergen) antigen (Horn s 3); Homo sapiens (human autoallergen) antigen (Horn s 4); Homo sapiens (human autoallergen) antigen (Horn s 5); Hordeum vulgare (barley) BMAI-l (Hor v 1); Juglans regia (English walnut) 2S albumin (Jug r 1); Juglans regia (English walnut) vicilin (Jug r 2); Juniperus ashei (mountain cedar) antigen (Jun a 1); Juniperus ashei (mountain cedar) antigen (Jun a 3); Juniperus oxycedrus (prickly juniper) calmodulin-like antigen (Jun o 2); Juniperus sabinoides (mountain cedar) antigen (Jun s 1); Juniperus virginiana (eastern red cedar) antigen (Jun v 1);

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 f 5); Malassezia furfur (fungus) cyclophilin homologue (Mal f 6); Malassezia furfur (fungus) MF1 peroxisomal membrane protein (Mal f 2); Malassezia furfur (fungus) MF2 peroxisomal membrane protein (Mal f 3); Malus domestica (apple) Bet v 1 homologue (Mal d 1); Malus domestica (apple) lipid transfer protein (Mal d 3); Mercurialis annua (annual mercury) profilin (Mer a 1); Metapenaeus ensis (shrimp) tropomyosin (Met e l); Mus musculus (mouse) MUP antigen (Mus m l);

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) alkaline serine proteinase (Pen b 13); Penicillium citrinum (fungus) alkaline serine proteinase (Pen c 13);

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);

Penicillium notatum (fungus) vacuolar serine proteinase (Pen n 18); Penicillium oxalicum (fungus) vacuolar serine proteinase (Pen o 18); Periplaneta americana (American cockroach) Cr-PI (Per a 3); Periplaneta americana (American cockroach) Cr-PII (Per a 1); Periplaneta americana (American cockroach) tropomyosin (Per a 7); Persea americana (avocado) endochitinase (Pers a 1); Phalaris aquatica (canary grass) antigen (Pha a 1); Phleum pratense (timothy grass) antigen (Phl p 1); Phleum pratense (timothy grass) antigen (Phl p 2); Phleum pratense (timothy grass) antigen (Phl p 4); Phleum pratense (timothy grass) antigen (Phl p 6); Phleum pratense (timothy grass) antigen Ag 25 (Phl p 5); Phleum pratense (timothy grass) polygalacturonase (Phl p 13); Phleum pratense (timothy grass) profilin (Phl p 12); Poa pratensis (Kentucky blue grass) antigen (Poa p 5); Poa pratensis (Kentucky blue grass) group I antigen (Poa p 1); Polistes annularies (wasp) antigen 5 (Pol a 5); Polistes annularies (wasp)

hyaluronidase (Pol a 2); 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)

phospholipase Al (Pol e 1); Polistes fuscatus (wasp) antigen 5 (Pol f 5); Polistes metricus (wasp) antigen 5 (Pol m 5); Prunus armeniaca (apricot) Bet v 1 homologue (Pru ar 1); Prunus armeniaca (apricot) lipid transfer protein (Pru ar 3); Prunus avium (sweet cherry) Bet v 1 homologue (Pru av 1); Prunus avium (sweet cherry) profilin (Pru av 4); Prunus avium (sweet cherry) thaumatin homologue (Pru av 2); Prunus persica (peach) lipid transfer protein (Pru p 3); Psilocyhe cubensis (fungus) antigen (Psi c l); Psilocybe cubensis (fungus) cyclophilin (Psi c 2); Pyrus communis (pear) Bet v 1 homologue (Pyr c 1); Pyrus communis (pear) isoflavone reductase homologue (Pyr c 5); Pyrus communis (pear) profilin (Pyr c 4); Quercus alba (white oak) antigen (Que a 1); Rattus norvegius (rat) antigen (Rat n 1); Ricinus communis (castor bean) 2S albumin (Ric c 1); Salmo salar (Atlantic salmon) parvalbumin (Sal s 1); Sinapis alba (yellow mustard) 2S albumin (Sin a 1); Solanum tuberosum (potato) patatin (Sol 1 1); Solenopsis geminata (tropical fire ant) antigen (Sol g 2); Solenopsis geminata (tropical fire ant) antigen (Sol g 4); Solenopsis invicta (fire ant) antigen (Sol i 2); Solenopsis invicta (fire ant) antigen (Sol i 3); Solenopsis invicta (fire ant) antigen (Sol i 4); Solenopsis saevissima (Brazilian fire ant) antigen (Sol s 2); Sorghum halepense (Johnson grass) antigen (Sor h 1); Syringa vulgaris (lilac) antigen (Syr v 1); Todarodes pacifwus (squid) tropomyosin (Tod p 1); Trichophyton rubrum (fungus) antigen (Tri r 2); Trichophyton rubrum (fungus) serine protease (Tri r 4); Trichophyton tonsurans (fungus) antigen (Tri 1 1); Trichophyton tonsurans (fungus) serine protease (Tri 14); Vespa crabo (European hornet) antigen 5 (Vesp c 5.0101); Vespa crabo (European hornet) antigen 5 (Vesp c 5.0102); Vespa crabo (European hornet) phospholipase (Vesp c 1); Vespa mandarina (giant Asian hornet) antigen (Vesp m 1.01); Vespa mandarina (giant Asian hornet) antigen (Vesp m 1.02); Vespa mandarina (giant Asian hornet) antigen (Vesp m 5); Vespula flavopilosa (yellowjacket) antigen 5 (Ves f 5); Vespula germanica (yellowjacket) antigen 5 (Ves g 5); Vespula maculifrons (yellowjacket) antigen 5 (Ves m 5); Vespula maculifrons

(yellowjacket) hyaluronidase (Ves m 2); Vespula maculifrons (yellowjacket) phospholipase Al (Ves m 1); Vespula pennsylvanica (yellowjacket) (antigen 5Ves p 5); Vespula squamosa (yellowjacket) antigen 5 (Ves s 5); Vespula vidua (wasp) antigen (Ves vi 5); Vespula vulgaris (yellowjacket) antigen 5 (Ves v 5); Vespula vulgaris (yellowjacket) hyaluronidase (Ves v 2); Vespula vulgaris (yellowjacket) phospholipase Al (Ves v 1); Zea mays (maize, corn) lipid transfer protein (Zea m 14); and/or combinations thereof.

Other agents [0320] In some embodiments, 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).

[0321] According to various embodiments, provided 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. In some embodiments, one or more other agents may be provided as a portion of a cell, tissue or extract thereof.

[0322] In some embodiments, one or more other agents may comprise

immunomodulatory polypeptides or immunostimulatory factors to modulate an individual's immune response. In some embodiments, 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.

[0323] In some embodiments, 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.

[0324] In some embodiments, one or more other agents may include preparations

(including heat-killed samples, extracts, partially purified isolates, or any other preparation of a microorganism or macroorganism component sufficient to display immune adjuvant activity) of 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. In some embodiments, one or more other agents (e.g., immune adjuvant) include, for example, Aviridine (N,N-dioctadecyl-N'N'-bis(2-hydroxy ethyl) propanediamine) and CRL 1005. In some embodiments, one or more other agents (e.g., immune adjuvant) 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.

[0325] In some embodiments, one or more other agents may be or comprise one or more immune adjuvants. In some embodiments, 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. In some embodiments, bacterial cellular lysate fractions comprise entities known as pathogen-associated molecular patterns (“PAMPs”). In some embodiments, 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. In some embodiments, a hydrophilic bacterial cellular lysate fraction and/or hydrophilic cellular component may be encapsulated within or substantially encapsulated within provided nanoparticles. In some embodiments, 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). Exemplary 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. It will be recognized by those of skill in the art that particular mucosal immune adjuvants may induce different immune responses. The skilled artisan will understand and be aware of technologies that may be used to select particular immune adjuvant(s) for use in a particular product or products and such variation is specifically contemplated as within the scope of the present disclosure.

[0326] In some embodiments, PAMPs are entities associated with bacterial cells that are recognized by cells of the innate immune system. In some embodiments, PAMPs are recognized by Toll-like receptors (TLRs) and other pattern recognition receptors (PRRs) in both plants and animals. In some embodiments, PAMPs are recognized by C-type lectin receptors (CLRs). In some embodiments, a CLR is a type I or type II CLR. In some embodiments, 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.

Table 5. Exemplary TLRs and TLR Ligands

Coating agents

[0327] In some embodiments, nanoparticle compositions may be partially or wholly coated with a coating agent. In some embodiments, 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.). Alternatively or additionally, in some embodiments, 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).

[0328] 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. For instance, in some embodiments including one or more coating agent(s), 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.

[0329] In some embodiments, 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. For example, in some embodiments, a coating agent comprises a hydrophobic cellular component.

[0330] In some embodiments, a hydrophobic cellular component preparation may be provided from a microbial cellular lysate. In some embodiments, a hydrophilic bacterial cellular lysate fraction and/or hydrophilic cellular component may be encapsulated within or

substantially coat provided nanoparticles.

[0331] Alternatively or additionally, one feature of certain embodiments of the present disclosure is that it permits utilization of relatively crude coating agents and/or coating agent preparations. In some embodiments, a coating agent may be or comprise a crude preparation and/or other complex material (e.g., an extract, etc.). [0332] In some embodiments, coating agents may comprise microbial hydrophobic and/or hydrophilic cellular components (e.g., from a crude microbial extract, for example, an E. coli extract). Without wishing to be held by a particular theory, 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.

[0333] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. Moreover, 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.

[0334] In general, 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). In some embodiments, microbial cell preparations contain intact cells, and optionally are substantially free of lysed cells. In some embodiments, microbial cell preparations contain lysed cells, and optionally are substantially free of intact cells.

[0335] In some embodiments, one or more coating agents (e.g., extracts, preparations and/or agents) is associated covalently with a nanoparticle surface. In some embodiments, one or more coating agents (e.g., extracts, preparations and/or agents) is associated non-covalently with a nanoparticle surface. In some embodiments, non-covalent association involves incorporation of one or more components into the nanoparticle membrane. In some

embodiments, non-covalent association involves specific binding with the nanoparticle membrane or an element incorporated therein. In some specific embodiments, one or more particular components of a coating agent (e.g., an extract, preparation and/or agent) may be coupled with a ligand that specifically binds with a target in the nanoparticle membrane. In some embodiments, 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.

[0336] In some embodiments, 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). For example, in some embodiments, organic E. coli extract (OEE) powder is mixed with water (see, e.g., Figure 8A, steps 13 and 14). In some such embodiments, a combination of water and OEE powder is sonicated, producing OEE micelles in water.

[0337] In some embodiments, OEE micelles in water are coated onto nanoparticles of the present disclosure using a spray-drying method. In some embodiments, after spray drying of nanoparticles is completed a certain percentage of solid material (e.g., coated nanoparticles), is recovered. In some such embodiments, approximately 50 to 95% of solids are recovered. In some such embodiments, approximately 60-85% of solids are recovered. In some such embodiments, approximately 65-80% of solids are recovered.

[0338] In some embodiments, OEE micelles in water are combined with a nanoparticle mixture, sonicated, and lyophilized (see, e.g., Figure 8A, steps 12-17). In some such

embodiments, combining OEE micelles with a provided nanoparticle mixture, and lyophilizing results in an association of a coating (OEE) with a nanoparticle surface. [0339] In some embodiments, 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. In some embodiments, 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. In some such embodiments, it is contemplated that a coating applied using spray drying may be more concentrated than a coated applied using lyophilization procedures. For example, in some embodiments, 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). In some embodiments, 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). Without being bound by any particular theory, it is contemplated that in some such embodiments, 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. In some embodiments, higher amount(s) of LPS relative to wile-type E.coli may be desirable. In some embodiments, lower amounts of LPS than found on wild-type E. coli may be beneficial and/or desirable.

[0340] Additional methods and parameters suitable for the preparation of crude and/or microbial extract-based coating agents may be found in PCT. Application No. PCT/US14/32838, filed April 3, 2014.

Nanoparticle compositions

[0341] In certain embodiments, provided nanoparticle compositions comprise

nanoparticles (e.g., comprised of polymer) combined with one or more payloads, one or more coating agents, and/or one or more other agents so that certain combined elements are encapsulated within a polymer matrix. In certain embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0342] In certain particular embodiments, 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. In some embodiments, provided nanoparticle compositions comprise a population of nanoparticles. In some embodiments, a population of nanoparticles comprises nanoparticles of a uniform size. In some embodiments, a population of nanoparticles comprises nanoparticles of different sizes; in some embodiments showing a particular size distribution. In many embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, provided nanoparticle compositions comprise

nanoparticles having sizes (e.g., average, or mean size) similar to the size of bacterial cells. For example, in some embodiments, 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.

[0343] In some embodiments, 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. In some embodiments, 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.

[0344] In some embodiments, 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.

[0345] In some embodiments, 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. In some embodiments, a weight ratio of a payload to a polymer in a nanoparticle composition may be represented in, e.g. mg (payload) / mg (polymer). For example, in some embodiments, a payload to polymer ratio is no less than 30 mg/mg and no greater than 250 mg/mg. In some embodiments, a ratio of payload to polymer is between 30 mg/mg and 150 mg/mg. In some embodiments, a ratio of payload to polymer is between 50 mg/mg and 100 mg/mg.

[0346] In some embodiments, provided compositions may also contain a certain amount

(e.g., relative to initial protein starting material input) of free (e.g., unencapsulated) protein. For example, in some embodiments, an amount of unencapsulated protein is 5-30% of an originally input amount of protein. In some embodiments, a certain amount of free protein is allowed to remain in a given composition (e.g., approximately 20% or less).

[0347] In some embodiments, free protein is removed from a preparation comprising nanoparticles using one or more separation methods as described herein. In some embodiments, 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. In some such embodiments, 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. Without wishing to be bound by any particular theory, it is contemplated that a certain amount of free protein in a given composition as described herein may be desirable. For example, in some embodiments, 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. [0348] Without being bound by any particular theory, it is contemplated that, in some embodiments, methods of the present disclosure produce one or more populations of

nanoparticles. In some embodiments, 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

approximately 300-500 nm or greater. In some embodiments, 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. In some embodiments, 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).

[0349] In some embodiments, 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.

[0350] In some embodiments, 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. [0351] In some embodiments, a nanoparticle composition is biodegradable. In some embodiments, a polymer of a nanoparticle composition is decomposed (e.g., nanoparticles release payloads), when they are exposed to a physiological environment.

[0352] In some embodiments, a nanoparticle composition is capable of interacting with biological systems and/or of inducing one or more desired biological responses. For example, in some embodiments, 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.).

[0353] In some embodiments, provided nanoparticle compositions may include a plurality of sets of nanoparticles that share one or more structural and/or functional

characteristics. For example, in some embodiments, 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. Alternatively or additionally, in some

embodiments, 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.).

[0354] In some embodiments, a provided nanoparticle composition may be characterized by a safety factor (e.g., when measured as described in Example 7B, for instance). In some embodiments, a safety factor may be between 5-100 or more. In some embodiments, a safety factor is between approximately 5 and 20. In some embodiments, a safety factor is between approximately 25 and 100. In some embodiments, a safety factor is between a range of approximately 30-90. In some embodiments, a safety factor is between a range of approximately 40-80. In some embodiments, a lower safety factor may be desirable. In some embodiments, a higher safety factor may be desirable. In some such embodiments, 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. Pharmaceutical Compositions

[0355] In some embodiments, the present disclosure provides pharmaceutical compositions comprising one or more provided nanoparticle compositions together with one or more pharmaceutically acceptable excipients.

[0356] In some embodiments, provided pharmaceutical compositions may be prepared by any appropriate method, for example as known or hereafter developed in the art of

pharmacology. In general, 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.

[0357] In some embodiments, compositions may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, 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.

[0358] In many embodiments, provided pharmaceutical compositions are specifically formulated for mucosal delivery ( e.g ., oral, nasal, rectal or sublingual delivery).

[0359] In some embodiments, 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. Alternatively or additionally, 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.

[0360] In some embodiments, 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.

[0361] In some embodiments, liquid dosage forms ( e.g ., for oral and/or parenteral administration) include, but are not limited to, emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs. In addition to provided nanoparticle compositions, 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,

dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents. In certain embodiments for parenteral administration, compositions are mixed with solubilizing agents such a CREMOPHOR^®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.

[0362] In some embodiments, injectable preparations, for example, sterile aqueous or oleaginous suspensions, may be formulated according to known methods using suitable dispersing agents, wetting agents, and/or suspending agents. In some embodiments, provided injectable preparations 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. Among the 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. For this purpose 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.

[0363] 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.

[0364] In some embodiments, one or more strategies may be utilized prolong and/or delay the effect of a provided nanoparticle composition after delivery.

[0365] In some embodiments, provided pharmaceutical compositions may be formulated as suppositories, for example for rectal or vaginal delivery. In some embodiments, 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.

[0366] In some embodiments, solid dosage forms (e.g., for oral administration) include one or more portions of a provided nanoparticle composition that may be or comprise capsules, tablets, pills, powders, and/or granules. In such solid dosage forms, 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, quaternary ammonium compounds), wetting agents (e.g, acetyl alcohol and glycerol monostearate), absorbents (e.g, kaolin and bentonite clay), and lubricants (e.g, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate), and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may comprise buffering agents.

[0367] In some embodiments, 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. 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.

[0368] 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;

polymethacrylic acid/acrylic acid copolymer; hydroxyethyl ethyl cellulose phthalate;

hydroxypropyl methyl cellulose acetate succinate; cellulose acetate tetrahydrophtalate; acrylic resin; shellac, and combinations thereof.

[0369] In some embodiments, 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. Examples of 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.

[0370] In some embodiments, the present disclosure provides 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.

[0371] In some embodiments, 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. In some embodiments, an adjuvant is provided in a separate formulation. Separate adjuvant may be administered prior to,

simultaneously with, or subsequent to provided nanoparticle composition administration.

[0372] In some embodiments, 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.

In some embodiments, 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.

[0373] In some embodiments, it may be advantageous to release a payload, for example, an antigen, at various locations along a subject’s gastrointestinal (GI) tract. In some

embodiments, it may be advantageous to release a payload, for example, an antigen, in a subject’s mouth as well as one or more locations along the subject’s GI tract. Accordingly, in some embodiments, a plurality of provided compositions (e.g., two or more) may be

administered to a single subject to facilitate release of a payload at multiple locations. In some embodiments, 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.

[0374] In some embodiments, 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. In some such embodiments, 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

[0375] In some embodiments, provided compositions may be characterized in order to determine, for example, protein content per nanoparticle. As will be appreciated by one of skill in the art, there are several methods by which compositions as described herein may be characterized. For example, in some embodiments, 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. In some embodiments, characterization includes measurements of nanoparticles within provided compositions including, e.g. quantification of amount of payload ^g) per amount of polymer (mg). In some embodiments, as described herein, 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. In some embodiments, a weight ratio of a payload to a polymer in a nanoparticle composition may be represented in, e.g. mg (payload) / mg (polymer). For example, in some embodiments, a payload to polymer ratio is no less than 30 mg/mg and no greater than 250 mg/mg. In some embodiments, a ratio of payload to polymer is between 30 mg/mg and 150 mg/mg. In some embodiments, a ratio of payload to polymer is between 50 mg/mg and 100 mg/mg.

[0376] In some embodiments, 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). In some embodiments, encapsulation efficiency is no lower than 40%. In some embodiments, encapsulation efficiency is substantially 100%. In some embodiments, encapsulation efficiency is between 50% and 100%; 60% and 100%; 70% and 100%; 75% and l00%;80% and 100%;

90% and 100%; and 95% and 100%. In some embodiments, encapsulation is between 75% and 95%; 80% and 90%; 85% and 95%.

[0377] In some embodiments, 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).

[0378] In some embodiments, characterization may include, e.g. taking an aliquot from a composition during and/or at various points throughout the production process. In some embodiments, an aliquot of a nanoparticle composition, as described herein, is removed, e.g. after step 8/before step 9 in Figure 1. By way of non-limiting example, when an aliquot of nanoparticle suspension solution 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. For example, 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.). Without wishing to be bound by any theory, it is contemplated that in some embodiments, such a low speed spin prior to hydrolysis accomplishes separation of nanoparticles from free protein without damaging any already formed nanoparticles. Once a protein assay has been performed, the resulting number (s) represent quantification of total protein per volume of suspension. Remaining suspension (i.e. suspension that has not been analyzed via protein measurement assay) can then be spun down using an ultracentrifuge (e.g. spinning at or about 350, 000 ref), and resulting supernatant analyzed for free protein, resulting in another measurement of total protein per volume of solution (a method involving a high-speed spin as described herein may be referred to as “Method 1”). Results from the initial aliquot and the ultracentrifuged sample are then compared to determine encapsulation percentage. In some embodiments, 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.

[0379] In some embodiments, rather than spinning in an ultracentrifuge [e.g. after initial aliquot removal, spinning, hydrolysis and protein analysis], 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”). One of skill in the art, depending on context, will be able to determine when low and/or higher speed centrifugation steps will desirably to be performed. [0380] In some embodiments, 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.

[0381] In some embodiments, amounts of excipients in provided compositions may be quantified. In some embodiments, excipients means components that are part of or used in making compositions as described herein that do not comprise nanoparticle payload. In some embodiments, excipients include, e.g. PLGA, PVA1, trehalose, residual water, etc.

[0382] In some embodiments, characterization of nanoparticles includes evaluation using dynamic light scattering (“DLS”). For example, in some embodiments, dynamic light scattering may be used to evaluate one or more aliquots of solution from one or more stages of

manufacturing processes as described herein (e.g., Figure 8A). In some embodiments, 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.

Characterization of payload

[0383] In some embodiments, a payload of nanoparticle compositions is evaluated in one or more ways at one or more times. For example, in some embodiments, protein encapsulated by a provided nanoparticle composition is evaluated both before and after incorporation into nanoparticles (and compared to protein not encapsulated by nanoparticles). In some such embodiments, evaluation is performed to ensure that processing into nanoparticles has not materially altered payload components. In some embodiments, evaluation methods comprise standard procedures for evaluation of proteins and/or nucleic acids (e.g., gel analysis, western blots, etc.). In some embodiments, provided nanoparticle compositions examined for presence of expected protein and/or nucleic acid components. It will be understood by those of skill in the art as to which methods are most appropriate to evaluate protein (e.g., western blot) and/or DNA (e.g., gel analysis, sequencing, etc.). In some embodiments, evaluation is performed by isolating payload from loaded nanoparticles and using standard methods to separate components. To give but one example, in evaluation of peanut protein, 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. In some embodiments, 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

manufacturing process.

Release Testing

[0384] In some embodiments, 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. In some embodiments, 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.

[0385] In some embodiments, 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). In some embodiments, 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). In some embodiments, 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. In some embodiments, 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. [0386] In some embodiments, 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. For example, in some embodiments, 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. , by labeling it, sequestering it, or destroying it; passed on to a subsequent step in manufacture; reprocessed (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. For example, in some embodiments, based on a result of a determination or whether one or more subject entities is present, or upon comparison to a reference standard, a batch from which a preparation is taken can be processed, e.g, as just described.

[0387] In some embodiments, methods disclosed herein may include making a decision:

(a) as to whether 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. In some embodiments, 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). [0388] In some embodiments, methods (i.e., evaluation, identification, and production 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. Pharmacopoeia (USP)) or insurance company) that 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.

[0389] In some embodiments, methods (i.e., evaluation, identification, and production methods) include taking action (e.g., physical action) in response to methods disclosed herein.

For example, in some embodiments, 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.

[0390] In some embodiments, processing may include formulating, packaging (e.g., in a vial or other container), labeling, or shipping at least a portion of the nanoparticle preparation. In some embodiments, 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). In some embodiments, processing can include directing and/or contracting another party to process as described herein. Routes of Administration

[0391] In some embodiments, provided nanoparticle compositions may be formulated for any appropriate route of delivery. In some embodiments, 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,

intraperitoneal (IP), intrathecal, intratracheal instillation (by), intravenous (IV), intraventricular, mucosal, nasal spray, and/or aerosol, oral (PO), as an oral spray, rectal (PR), subcutaneous (SQ), sublingual; topical and/or transdermal ( e.g ., by lotions, creams, liniments, ointments, powders, gels, drops, etc.), transdermal, vaginal, vitreal, and/or through a portal vein catheter; and/or combinations thereof. In some embodiments, the present disclosure provides methods of administration of provided nanoparticle compositions via mucosal administration. In some embodiments, the present disclosure provides methods of administration of provided

nanoparticle compositions via oral administration. In some embodiments, the present disclosure provides methods of administration of provided nanoparticle compositions via sublingual administration.

[0392] In some embodiments, provided nanoparticles and/or nanoparticle compositions may be formulated for oral administration. In some embodiments, oral administration may be or comprise enteral administration. In some embodiments, oral administration is buccal, sublabial, and/or sublingual administration. In some embodiments, 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.

Methods of Treatment

[0393] 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. [0394] In some embodiments, 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. In some embodiments, 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. In some such embodiments, 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. In some such embodiments, 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. In some such embodiments, 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. Without being bound by any particular theory, it is contemplated that such administration will alter immune responses in subjects such that responses that mediate anaphylactic reactions will occur less frequently or will not occur, and responses that mediate tolerance to one or more proteins will function more frequently or at every exposure to one or more components of a payload in a provided nanoparticle composition.

[0395] In some embodiments, the present disclosure provides methods of treating various diseases, disorders and/or conditions. In some embodiments, 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.

Treating Allergy

[0396] The present disclosure provides, among other things, methods and compositions for the treatment and/or prevention of allergy. In some embodiments, provided nanoparticle compositions are useful as vaccines to prevent and/or delay the onset of an allergic reaction. In some embodiments, provided nanoparticle compositions are useful as vaccines to lessen the severity and/or duration of a future allergic reaction. In some embodiments, provided nanoparticle compositions are useful as therapeutics to alleviate and/or arrest an allergic reaction in progress. In some embodiments, 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.

[0397] In some embodiments, 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).

[0398] In some embodiments, 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. Examples of 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).

[0399] In some embodiments, provided nanoparticle compositions may be used for treatment and/or prevention of allergies associated with insect allergens. Examples of common insect allergens include, but are not limited to, proteins from insects such as fleas, ticks, ants, cockroaches, and bees.

[0400] In some embodiments, 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).

[0401] In some embodiments, 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.

[0402] In some embodiments, provided nanoparticle compositions may function to suppress and/or decrease a subject’s T_H2-type responses and/or enhance and/or increase a subject’s T_Hl-type responses. In some embodiments, provided nanoparticle compositions may function to enhance and/or increase a subject’s T_H2-type responses and/or suppress and/or decrease a subject’s T_Hl-type responses. In some embodiments, a subject’s T_H2-type responses are enhanced through targeting of a cell surface receptor for CpG oligonucleotides (e.g.,

DEC205).

[0403] In some embodiments, provided nanoparticle compositions effectively treat and/or prevent all of a subject’s allergies falling into a particular class of allergy. In some embodiments, exemplary“classes” of allergies include, but are not limited to, anaphylactic allergies and non-anaphylactic allergies. In some embodiments, 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. Thus, in some embodiments, provided nanoparticle compositions may be useful for treating all of a subject’s food allergies. In some embodiments, exemplary“classes” of allergies include, but are not limited to, particular individual foods which contain multiple allergens. For example, there are at least eleven known peanut allergen proteins. Thus, in some embodiments, a“class” of allergies is“peanut” allergy, and provided nanoparticle compositions may be useful for treating all of a subject’s allergies associated with all seven different peanut allergen proteins.

[0404] In some embodiments, provided nanoparticle compositions may be useful for treating and/or preventing a single allergy, even though no allergy-specific antigen is included.

In some embodiments, provided nanoparticle compositions may be useful for treating and/or preventing multiple different allergies. In some embodiments, provided nanoparticle

compositions may be useful for treating and/or preventing substantially all of a subject’s allergies. For example, 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. Thus, in some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0405] In some embodiments, 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. Opin. Drug Deliv., 2007, 4(4):323-340, both of which are hereby incorporated by reference in their entirety). Part of the reason for such unpredictable results is the complex nature of the gut mucosa. Briefly, the base of the mucosa in the gut is lined by gut- or mucosa-associated lymphoid tissue, with underlying lamina propria that is rich in intraepithelial lymphocytes (sometimes referred to as diffuse lymphoid tissue).

The majority of 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

immunocompetent cells in the human body, with circulating lymphocytes only comprising -2% of the total lymphocytes (see Silin et al.). Furthermore, the gut is known to accommodate -80% of all immunoglobin or Ig-producing cells and releases 2 to 3 times more secretory IgA that the total output of circulating IgG (see Silin et al.). Accordingly, 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.

[0406] In order to have an effective oral vaccine to treat allergy, effective presentation of one or more antigens to an antigen presenting cell (APC) is required. While 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. However, 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.

[0407] Major known barriers to providing effective oral vaccines include proteolytic degradation of antigens in the gut, tuning of proper release profile in the intestine, and problems delivering enough antigen in a reasonable sized dose. Additionally, the development of oral tolerance to an antigen is thought to be a major point of concern in developing oral vaccines in general. 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.).

[0408] In some embodiments, provided 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.

Without wishing to be held to a particular theory, it may be advantageous to promote immune tolerance in some clinical circumstances, such as in cases of anaphylaxis, autoimmune disease, or certain infectious diseases including, but not limited to, dengue fever and RSV.

Treating Infectious Disease

[0409] The present disclosure provides, among other things, methods and compositions for the treatment and/or prevention of an infectious disease. In some embodiments, provided nanoparticle compositions are useful as vaccines to prevent and/or delay the onset of an infectious disease. In some embodiments, provided nanoparticle compositions are useful as vaccines to lessen the severity and/or duration of a future infectious disease. In some embodiments, provided nanoparticle compositions are useful as therapeutics to alleviate and/or arrest an infectious disease in progress. In some embodiments, 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. In some embodiments, the subject is suffering from a primary infection. In some embodiments, 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).

[0410] 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. difficile infection, Creutzfeldt-Jakob disease, dengue fever, diphtheria, ebola, enterococcus infection, food poisoning, gangrene, gonorrhea, streptococcal infections, hepatitis A-E, herpes, hookworm, mononucleosis, leishmaniosis, leprosy, Lyme disease, malaria, measles, meningitis, mumps, conjunctivitis, pertussis, rabies, respiratory syncytial virus, rhinovirus, rubella, SARS, scabies, sepsis, shingles, syphilis, tetanus, trichinellosis, tuberculosis, tularemia, viral pneumonia, west nile fever, and yellow fever.

Treating Cancer

[0411] The present disclosure provides, among other things, methods and compositions for the treatment and/or prevention of cancer. In some embodiments, provided nanoparticle compositions are useful as vaccines to prevent and/or delay the onset of a cancer. In some embodiments, provided nanoparticle compositions are useful as therapeutics to alleviate and/or arrest an cancer in progress. In some embodiments, 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 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;

(Endocrine Pancreas); 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);

metastatic squamous neck cancer; mouth cancer; Multiple Endocrine Neoplasia Syndrome;

Multiple Myeloma; Mycosis Fungoides; Myelodysplastic Syndromes;

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; and/or combinations thereof.

Dosins

[0412] In some embodiments, provided nanoparticle and/or pharmaceutical compositions are administered according to a dosing regimen sufficient to achieve a desired immunological reaction. For example, in some embodiments, 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.

[0413] In some embodiments, 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 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more.

[0414] In some embodiments, a provided nanoparticle and/or pharmaceutical

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. In some embodiments, 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%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more.

[0415] To give but a few illustrative examples, in some embodiments, 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. In some embodiments, 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.

[0416] In some embodiments, 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).

[0417] In some embodiments, 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.

[0418] In some embodiments, a provided dosing regimen comprises or consists of an initial dose with one or more booster doses. In some embodiments, 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. In some embodiments, an initial dose comprises a series of doses administered over a period of time. For example, in some embodiments, 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,

10 minute intervals, 15 minute intervals, 20 minute intervals, 25 minute intervals, 30 minute intervals, 45 minute intervals, hourly intervals, every 2 hours, etc. [0419] In some embodiments, 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. In certain embodiments, 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. In some embodiments, provided nanoparticles and/or nanoparticle compositions are formulated into a unit dose. In some embodiments, 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. In some embodiments, 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.

[0420] In some embodiments, a provided dosing regimen comprises or consists of administration of multiple doses over the course of the subject’s entire lifespan. In some embodiments, 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). In some embodiments, 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.

[0421] In some embodiments, when provided nanoparticles and/or compositions are used in the treatment of allergy, prior to the first dose, 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. , if only a small or nonexistent risk of anaphylaxis); (5) measuring expression (e.g, levels, spatial distribution, temporal distribution, etc.), of one or more molecular markers, including, but not limited to, T- cell markers CD4+ and/or CD8+; (6) performing a basophil histamine release assay; and/or combinations thereof. In some embodiments, 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. In some embodiments, 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.

[0422] In some embodiments, 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). In some embodiments, allergy challenge is performed after 1 week, 2 weeks, 1 month, 2 months, 6 months, and 1 year after initiation of treatment.

[0423] In some embodiments, provided nanoparticles and/or compositions may be administered via any medically acceptable route. For example, in some embodiments, a provided composition may be administered via intravenous administration; intradermal administration; transdermal administration; oral administration; subcutaneous administration; transmucosal administration; and/or combinations thereof. In some embodiments, 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.

Combination Therapy

[0424] In some embodiments, provided therapy (e.g., provided nanoparticles and compositions) can be administered in combination with at least one other therapy, so that the subject receives at least some benefit from both. In some embodiments, a subject may have previously received or be currently receiving at least one other therapy. In some embodiments, the at least one other therapy is administered to a subject who has previously received or is currently receiving nanoparticle therapy as described herein. For example, useful in the treatment of one or more diseases, disorders, or conditions treated by the relevant provided pharmaceutical composition, so the subject is simultaneously exposed to both. In some embodiments, a provided nanoparticle composition is utilized in a pharmaceutical formulation that is separate from and distinct from the pharmaceutical formulation containing another therapeutic agent. In some embodiments, a provided nanoparticle composition is admixed with the composition comprising another therapeutic agent. In other words, in some embodiments, a provided nanoparticle composition is produced individually, and the provided nanoparticle composition is simply mixed with another composition comprising another therapeutic agent.

[0425] The particular combination of therapies (substances and/or procedures) to employ in a combination regimen will take into account compatibility of the desired substances and/or procedures and the desired therapeutic effect to be achieved. In some embodiments, 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).

[0426] It will be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, 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.). In some embodiments, 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).

[0427] As used herein, 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

[0428] For example, in some embodiments, 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

(progressively larger doses of a specific allergen); monoclonal anti-IgE antibodies (e.g, omalizumab); and/or combinations thereof.

[0429] Exemplary antihistamines include, but are not limited to Azelastine;

Brompheniramine; Buclizine; Bromodiphenhydramine; Carbinoxamine; Cetirizine; Cyclizine; Chlorpheniramine; Chlorodiphenhydramine; Clemastine; Cyproheptadine; Desloratadine;

Dexbrompheniramine; Deschlorpheniramine; Dexchlorpheniramine; Dimetindene;

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.

[0430] 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);

Fluticasone (Flonase); Mometasone and Mometasone furoate (Nasonex); Triamcinolone

(Nasacort AQ); Prednisone; Methylprednisolone (Depo-Medrol); Triamcinolone (Kenalog); and/or combinations thereof.

[0431] Exemplary forms of cromolyn sodium include, but are not limited to, Rynacrom;

Nasalcrom; Prevalin; Intal; Optocrom; Optrex; Gastrocrom; Intercron; and/or combinations thereof.

[0432] Exemplary anti-leukotrienes and leukotriene inhibitors (or modifiers) 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. [0433] Exemplary anti-cholinergics include, but are not limited to, Ipratropium bromide

(Atrovent, Apovent, Ipraxa , Aervoent); Combivent (Ipratropium bromide and Albuterol);

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; and/or combinations thereof.

[0434] Exemplary decongestants include, but are not limited to, Ephedrine; Levo- methamphetamine; Naphazoline; Oxymetazoline; Phenylephrine; Phenylpropanolamine;

Propylhexedrine; Synephrine; Tetrahydrozoline; and/or combinations thereof.

[0435] 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.

[0436] In some embodiments, 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,

2007/0213507, 2010/0166802, and/or 2011/0027298, all of which are incorporated herein by reference.

Infectious Disease Therapies

[0437] For example, in some embodiments, 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.

[0438] Exemplary antiviral agents include, but are not limited to Abacavir, Aciclovir,

Acyclovir, Adefovir, Amantadine, Amprenavir, Ampligen, Arbidol, Atazanavir, Atripla, Boceprevirertet, Cidofovir, Combivir, Darunavir, Delavirdine, Didanosine, Docosanol,

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,

Lopinavir, Loviride, Maraviroc, Moroxydine, Methisazone, Nelfmavir, Nevirapine, Nexavir, Nucleoside analogues, Oseltamivir, Peginterferon alfa-2a, Penciclovir, Peramivir, Pleconaril, Podophyllotoxin, Raltegravir, Reverse transcriptase inhibitors, Ribavirin, Rimantadine,

Ritonavir, Pyramidine, Saquinavir, Stavudine, Tea tree oil, Telaprevir, Tenofovir, Tenofovir disoproxil, Tipranavir, Trifluridine, Trizivir, Tromantadine, Truvada, Valaciclovir,

Valganciclovir, Vicriviroc, Vidarabine, Viramidine, Zalcitabine, Zanamivir, Zidovudine, and derivatives/analogs and/or combinations thereof.

[0439] Exemplary 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 echinocandins such as anidulafungin, caspofungin, and micafungin and derivatives/analogs and/or combinations thereof.

[0440] As an additional example, in some embodiments, 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. In some embodiments, provided pharmaceutical compositions may be administered in combination with a vaccine. Cancer Therapies

[0441] As an additional example, in some embodiments, 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.

[0442] Exemplary 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.

[0443] Exemplary 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.

[0444] 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

[0445] The present disclosure provides kits comprising provided nanoparticles, nanoparticle compositions, and/or pharmaceutical compositions. In some embodiments, a kit may comprise (i) at least one provided nanoparticle composition; and (ii) at least one

pharmaceutically acceptable excipient; and, optionally, (iii) instructions for use.

[0446] In some embodiments, 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) doses of provided nanoparticles and/or nanoparticle compositions. In some embodiments, 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). In some embodiments, multiple populations of provided nanoparticles are packaged separately from one another in provided kits. In some embodiments, provided kits may include provided compositions and one or more other therapeutic agents intended for administration with the provided compositions.

[0447] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0448] In some embodiments, 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).

EXEMPLIFICATION

Example 1: Preparation of polymer nanoparticles comprising protein and DNA and coated with OEE

[0449] 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] 1. Preparation of solid block material comprising PLGA, protein, and DNA (see, e.g., Figures 1 (steps 1-4), and Figure 2)

Steps:

1) A solution comprising polymer (e.g. PLGA, which makes a“PLGA solution”) was prepared by dissolving PLGA in organic solvent (e.g. DMSO), for example, at 3.2 mg/mL. 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.

2) 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. In this Example, 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

3) 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.

4) The resulting solution was freeze-dried in a lyophilizer to generate a frozen, solid block material (“block material”), wherein the block material is comprised of protein, DNA, and polymer (here, crude peanut extract, sheared E. coli DNA, and PLGA).

4 A) Optionally, 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.

[0451] 2. Preparation of nanoparticles comprising PLGA, protein, DNA (see, e.g.,

Figure 1 (steps 4 - 9) and Figures 3-6)

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.

6) 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. In some embodiments, for example, 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. In some embodiments, for example, 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.

7) An aqueous polyvinyl alcohol (PVA1) solution (0.8125 mg/mL) was added to the

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.

8) 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) includes nanoparticles, PVA1, and trehalose.

8A) Without wishing to be bound by any particular theory, it is contemplated that an additional step to remove any free protein will be helpful in formation of a nanoparticle suspension, wherein substantially no hydrophilic payload is exposed on a surface of a nanoparticle. The dried cake may then be dissolved in, e.g. 10 mM ammonium bicarbonate, followed by removal of free protein by, e.g. centrifugation. As described herein, 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. Accordingly, as an optional step, centrifugation may be performed after lyophilization in step 8. Alternatively, in some embodiments, 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

centrifugation

9) The dry mixture (e.g. dried cake) was dissolved in water, creating a solution comprised of nanoparticles, PVA1, and trehalose.

[0452] 3. Preparation of nanoparticles coated with an agent(e.g. OEE) (Figure 1, steps

10-17)

Steps:

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.

10) An aqueous solution of Organic E. Coli. Extract (“OEE”) (as prepared in accordance with Example 2, below) was prepared by adding water to OEE powder (in this Example, to a concentration of 2 g/L OEE).

11) The mixture was sonicated to make OEE micelles in water (“OEE Solution”).

12) 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).

13) The solution containing OEE micelles, water, and trehalose was spray dried.

14) The dry OEE and trehalose (OEE/trehalose spray dried solution, or“OEE/Trehalose SDD”) from step 13 was added to the solution of nanoparticles from step 9.

15) The aqueous solution containing PLGA/protein/DNA nanoparticles coated with OEE and PVA1, free OEE, and trehalose was lyophilized, resulting in a solid dispersion of nanoparticles with OEE coating.

16) Optionally, 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. 17) Optionally, a flowable powder may be obtained from step 16

Example 2: Preparation of OEE

[0453] 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 master and working cell bank of the production ( E.coli ) strain may be established prior to clinical manufacture.

[0454] Briefly, 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. 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.

[0455] Exemplary specifications for the OEE are presented in Table 6. It is expected that substantially no proteins or nucleic acids will be extracted into the OEE.

Table 6. Specification for Organic E. Coli Extract

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

Example 3: Preparation of Arachis hyyosaea ( Peanut ) Allersen Extracts

[0456] The present Example describes preparation of Arachis hypogaea (Peanut)

Allergen Extract {i.e., crude peanut extract) for use in accordance with the present disclosure.

[0457] 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.

[0458] 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.

[0459] A representative method of Arachis hypogaea (Peanut) Allergen Extract (i.e., crude peanut 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.

[0460] 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

[0461] 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%

Tween 20)), for one hour at room temperature. All subsequent incubations were performed at room temperature. Membranes were then incubated with 1 :20 human sera (pooled from seven highly peanut-allergic subjects) in blocking buffer. All sera (from each of seven patients) had been previously confirmed to show reactivity to different epitopes on Ara hl, h2, and h3 in Luminex based peptide epitope assay. Membranes were then incubated with 1 : 10000 goat Anti human IgE (affinity purified) - HRP conjugate, in blocking buffer, incubated with

chemiluminescent (ECL) substrate, and processed for visual detection of bands.

[0462] Crude peanut extract was analyzed both before and after formation of

nanoparticles, demonstrating that each of Ara hl, Ara h2, and Ara h3 are detectable both before and after incorporation into nanoparticles, confirming that the starting material crude peanut extract is not materially changed by the nanoparticle manufacturing process (data not shown). This experiment also confirms that hydrolyzation of nanoparticles (releasing contents) results in release of Ara hl, h2, and h3 which appears substantially similar to the material put into the nanoparticles, confirming that peanut protein may be successfully encapsulated into

nanoparticles.

[0463] 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). [0464] In the following Example, 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.

[0465] 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.

Phase I Study Design and Investigational Product Details

[0466] “Investigational Product”: ENP-501 (peanut allergy vaccine) orally

disintegrating product (e.g., powder or tablet). In this Example, the form of the Investigational Product is an orally-disintegrating formulation such as a powder for buccal mucosal

administration, which is comprised of 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. 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. 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.

Test Products and Mode of Administration

[0467] 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.

[0468] 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

Packaging and Labeling

[0469] All Investigational Product and orally-disintegrating placebo will be supplied to a pharmacy (“Investigational Site pharmacy”) in high-density polyethylene (HDPE) bottles with child resistant closures.

[0470] On dose escalation visit days, a pharmacist will dispense the maximum required number and strengths of doses required for the planned escalation of the respective visit into individual portions. 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.

[0471] Once dose escalation for a given visit has been completed and the appropriate dose level determined, the Investigational Site pharmacist will dispense a sufficient number of Investigational Product or placebo to complete dosing at home until the next visit. The

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.

[0472] 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.

[0473] The Investigational Site pharmacist will not be blinded to the Investigational

Product, and thus labeling of bottles supplied to the Investigational Site pharmacy will not be blinded. Both participants and Investigational Site staff will be blinded to the Investigational Product or placebo in part II of the trial, therefore labeling of bottles supplied to the

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.

Storage and Handling

[0474] At the Investigational Site, all 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).

[0475] At the time of dispensing, the Investigational Site pharmacist will remove the

Investigational Product or placebo bottle from refrigeration, allow the bottle to warm to room temperature for 30 minutes, dispense the required amount of Investigational Product or placebo into containers as described above, and put any remaining Investigational Product or placebo (in the original packaging) back into refrigeration. [0476] 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.

Study Design, Methodology, and Details

[0477] The study period will be 18 months.

[0478] The study is a phase I study that will be conducted in two sequential parts, Part I

(Phase I A) and Part II (Phase IB).

[0479] The number of planned participants for part I (non-peanut allergic participants) is

4.

[0480] The number of planned participants for part II (peanut allergic participants) is 12.

[0481] 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).

Parti

[0482] 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

Investigational Product for follow-up visits. Part II

[0483] 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).

Participants will return to the CRU 4 weeks after their last dose of Investigational Product for a follow-up visit.

Enrollment and Randomization

[0484] 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.

[0485] 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.

Blinding

[0486] The Investigational Site pharmacist will be unblinded to Investigational Product, whereas, the remainder of the Investigational Site staff and the participant will be blinded.

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.

Requirements for Unblinding

[0487] Prior to the DBPCFC, a subject can only be unblinded for safety reasons. If a clinically significant event occurs prior to the DBPCFC, immediate medical attention will be provided irrespective of the treatment assignment. In the event of a life-threatening event that requires unblinding, notify the medical monitor before unblinding occurs, if time allows. For all other unscheduled events that require unblinding, the Investigator will contact the medical monitor before unblinding. Any participant that experiences a serious suspected adverse reaction will be unblinded prior to filing an expedited report to determine treatment assignment and thereby determine whether an expedited report is required (only if on active treatment).

Guidelines for Study Enrollment Pause and/or Study Discontinuation

[0488] 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.

Diagnosis and Main Criteria for Inclusion/Exclusion

[0489] Participants in Parts I and II must meet certain inclusion criteria and must not meet any single one of the exclusion criteria. 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:

Inclusion Criteria

Part I (Non-Peanut Allergic Participants)

[0490] The main criteria for inclusion (of which participants must meet all of in order to be enrolled in Part I of the study) 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.

Part II (Peanut Allergic Participants)

[0491] The main criteria for inclusion (of which participants must meet all of in order to be enrolled in Part II of the study) 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

(ImmunoCAP®) within 1 year of enrollment (including at Screening); (iv) ability to perform spirometry maneuvers in accordance with the ATS guidelines (2005); (v) 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; (vi) signed and dated written informed consent obtained from the participant and/or parent guardian and (vii) signed and dated assent obtained from participants under 18 years of age, in accordance with local IRB regulations.

[0492] For both Parts I and II, 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. For this study, a woman who has been surgically sterilized or who has been in a state of amenorrhea for more than two years will be deemed not to be of childbearing potential; Exclusion Criteria

[0493] Participants meeting any of the following criteria will be excluded from the trial

(these exclusion criteria apply to both Part I and Part II of the trial):

1. History of severe anaphylactic event requiring mechanical ventilation or use of intravenous (IV) vasopressor drugs (i.e., patient underwent cardio-respiratory arrest);

2. FEV1 value < 80% predicted at Screening;

3. Poor control or persistent activation of atopic dermatitis;

4. Any hospitalization in the past year for asthma, or >1 course of oral steroids for asthma or any emergency room visit in the past 6 months for asthma;

5. Eosinophilic gastrointestinal disease;

6. ETse of oral or IV corticosteroids within 30 days of Screening;

7. Inability to discontinue antihistamines for skin testing;

8. ETse of omalizumab or other non-traditional forms of allergen immunotherapy (e.g., oral or sublingual) or immunomodulatory therapy (not including corticosteroids) or biologic therapy within one year of Screening;

9. Use of any other food or specific allergen immunotherapy within one year of Screening;

10. Use of immunosuppressive drugs within 30 days of Screening;

11. Use of B-blockers (oral), angiotensin-converting enzyme (ACE) inhibitors, angiotensin- receptor blockers (ARBs), or calcium channel blockers;

12. Evidence of clinically significant immunosuppressive, neurologic, cardiac, pulmonary, hepatic, rheumatologic, autoimmune, or renal disease by history, physical examination, and/or laboratory studies including urinalysis;

13. Pregnancy or breast-feeding (if female);

14. Behavioral, cognitive, or psychiatric disease that in the opinion of the Investigator affects the ability of the participant to understand and cooperate with the study protocol;

15. Known allergy to inactive ingredients of Investigational Product or placebo;

16. Participation in another investigational vaccine or drug trial within 30 days of Screening. Dosing

Part I: Non-Peanut Allergic Participants

[0494] 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.

Part II: Peanut Allergic Participants·.

[0495] 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.

[0496] For the first dose escalation visit (Day 1), up to 3 dose levels will be attempted

(0.25, 0.5, and 1 pg peanut protein), with 30 minutes between each dose and two hours of monitoring after the last dose. Participants randomized to the placebo arm will receive the same number of portions as planned for the ENP-501 dose escalation; however, the portions will contain no peanut protein.

[0497] The following modifications to the Day 1 dose escalation will be required in the event of the listed symptoms. These are participant-specific and only apply to the participant for which the symptoms were observed.

Optional, Exemplary Modifications for subjects on a case-by-case basis after dose escalation

Possible Modifications to Dosing [0498] Extend duration between dose levels: If mild localized skin hives (< 5 hives), mild lip swelling or abdominal discomfort is observed after administration of a dose, the next dose level in the escalation will be administered 1 hour after the dose level that resulted in the symptoms. Mild oral or pharyngeal pruritus will not require a delay between dose levels (the duration to the next dose level will remain 30 minutes).

[0499] Stop the escalation for the day: If a mild or moderate symptom listed below is observed after administration of a dose, the dose escalation for that day will be stopped. o Mild symptoms: generalized skin flushing or pruritus, rhinorrhea, sneezing, nasal congestion, occasional cough, mild abdominal pain, one episode of vomiting; or persistence or recurrence of localized hives/swelling (< 5 hives) as described above o Moderate symptoms: systemic skin hives (> 5) or swelling, throat tightness without hoarseness, persistent cough, wheezing without dyspnea, persistent abdominal pain, gastrointestinal (GI) cramping, more than 1 episode of vomiting

[0500] Stop dosing entirely: If a severe symptom listed below is observed after administration of a dose, the participant will not be administered any further Investigational Product or placebo(administration will be discontinued). o Severe symptoms: laryngeal edema, throat tightness with hoarseness, wheezing with dyspnea, severe abdominal pain, significant or persistent GI cramping, repetitive vomiting, change in mental status, hypotension

[0501] Participants who can escalate to the highest planned Day 1 dose (i.e., 1 pg peanut protein or the corresponding number of matched placebo tablets) will take that dose qd for the next 13 days, for a total of two weeks at the 1 pg peanut protein dose level.

[0502] Participants who stop the Day 1 escalation prior to reaching the highest planned dose, but are not required to discontinue Investigational Product or placebo, will take the highest tolerated dose for the next 13 days.

[0503] Participants that are required to discontinue Investigational Product or placebo entirely due to severe symptoms during the Day 1 dose escalation will not take any further Investigational Product and will return in 4 weeks (± 3 days) for the follow-up visit. [0504] For the remaining dose escalations (i.e., biweekly visits after Day 1), up to 3 dose levels will be attempted, with 30 minutes between each dose and two hours of monitoring after the last dose. The same dose escalation modifications described for Day 1 will apply for these dose escalations. The planned dose escalation levels for the biweekly dose escalations are 2, 4,

8, 16, 32, 64, 128, 256, 500, 1,000, and 2,000 pg peanut protein (or corresponding number of matched placebo tablets). If three doses are taken in the CRU, the highest dose will be taken at home (e.g., administration of 1, 2, and 4 pg and participants take 4 pg at home; then 8, 16, 32 pg at the next visit and participants take 32 pg dose home, etc.)

[0505] If a participant is required to stop the dose escalation on Day 1 or any of the biweekly dose escalation visits, but is not required to stop Investigational Product or placebo entirely, 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).

[0506] Participants will complete Investigational Product or placebo treatment at

52 weeks and will return to the CRU 24 hours (+7 days) for a DBPCFC. The participant will return in 4 weeks (± 3 days) after their last dose of Investigational Product or placebo for a follow-up visit.

[0507] 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 to dose for the remainder of the study at the maximum tolerated dose.

[0508] 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.

[0509] Participants will complete Investigational Product or placebo treatment in

52 weeks and return to the CRU 24 hours (+7 days) for a peanut DBCPCFC and 4 weeks (± 3 days) after their last dose of Investigational Product or placebo for a follow-up visit. [0510] The total treatment duration will depend on the subject’s tolerance of the drug and whether delays in the planned dose escalation are required. A description of the planned dose escalation and criteria for modification, including the dose level to be taken between the dose escalation visits is described herein.

[0511] 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.

Table 7. Study Day(s) and Dose(s)

[0512] For participants where the dose escalation is interrupted, but not discontinued,

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.

Mode of Administration

Phase I Non-Peanut Allergic Participants

[0514] ENP-501 will be taken in a CRU or comparable monitored clinical site on Day 1,

Day 2, and at each weekly dose escalation visit. All remaining doses will be self-administered at home. For doses taken at home, 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.

Phase 2 : Peanut Allergic Participants

[0515] 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.

[0516] ENP-501 or placebo will be administered sublingually (e.g. buccal mucosal).

Participants will be instructed to hold the portions (e.g., powder or tablets) sublingually (e.g. in the buccal mucosal space) for two minutes, and then swallow. For doses that require more than one portion (i.e., when using tablets instead of powders), 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. Methodology and Timeline

Part I: Non-Peanut Allergic Participants

[0517] Four non-peanut allergic participants will receive sublingual administrations of

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.

Part II: Peanut Allergic Participants

[0518] Part II will occur as long as no safety concerns are raised during Part I. In Part II,

12 peanut-allergic participants will be randomized 9:3 to receive sublingual administrations of 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 highest dose for the remaining 26 weeks of treatment. Each dose escalation will occur in the CRET. Participants will complete Investigational Product or placebo dosing at 52 weeks and will return to the CRET 24 hours later for a DBPCFC. Participants will return to the CRET 4 weeks after their last dose of Investigational Product or placebo for follow-up visits.

Schedule of Events in Parts I and II Screening (Parts I and II)

[0519] 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.

[0520] Participants that consent to participate in the study will undergo the eligibility assessments listed below. The procedures are the same for Part 1 and Part 2, except where noted. All procedures must be completed within 14 days of the first dose of Investigational Product or placebo (Day 1): 1. Record demographic data (date of birth/age at Screening, gender, and race);

2. Comprehensive medical history;

3. Record medications taken within 30 days of Screening visit. Participants must discontinue use of antihistamines for an appropriate length of time (5 half-lives of the antihistamine) prior to the SPT;

4. Comprehensive physical examination, including height and weight;

5. Vital signs (blood pressure, pulse rate, respiration rate, and temperature);

6. Spirometry (FEV1, FVC, and PEF);

7. Safety labs, including CBC with differential, serum chemistry, and urinalysis (see Appendix 3 for list of tests);

8. Urine pregnancy test [beta-chorionic gonadotropin (b-hCG)] for WCBP only;

9. Blood collection for total IgE and peanut-specific IgE and IgG₄ levels;

10. SPT to peanut - Part I Only;

11. SPT to peanut, milk, egg, cashew, walnut, sesame, cottonwood, rocky mountain juniper, Kentucky and timothy grasses, sagebrush, and/or ragweed (western) - Part II Only;

12. Assessment of inclusion/exclusion criteria.

[0521] Participants who meet eligibility criteria based on the completion of the above

Screening assessments will be instructed as follows:

1. Do not take any of the prohibited medications listed in Section 5.8 of the protocol for the duration of the study (through final follow-up visit);

2. For males and WCBP: Abstain from sex or use an adequate method of contraception for the duration of the study and through 30 days after the last dose of Investigational Product or placebo;

3. Return to the CRU within 14 days for baseline procedures (Day 1).

Baseline Pre-Dose on Study Day 1 (Parts I and II)

[0522] Participants who meet eligibility criteria after completing all Screening evaluations will return to the CRU on Day 1. The following baseline procedures will be performed prior to enrollment to confirm that a participant continues to meet eligibility criteria. The procedures are the same for Parts I and II.

1. Update of medical history;

2. Review and record medications taken since Screening visit;

3. Targeted physical examination;

4. Weight;

5. Vital signs (blood pressure, pulse rate, respiration rate, and temperature);

6. PEF;

7. Urine pregnancy test (b-hCG) for WCBP only;

8. Review of inclusion/exclusion criteria.

Participants that continue to meet all eligibility requirements will be enrolled in the study.

Treatment Period arf I (Non-Peanut Allergic Participants) Study Day 1

[0523] Participants that continue to meet eligibility requirements after baseline assessments and that are enrolled in Part 1 of the trial will be given a single buccal mucosal dose of ENP-501 on Day 1. This visit is to be completed within 14 days of Screening.

[0524] The following procedures will be performed on Day 1 after administration of

Investigational Product or placebo:

1. Monitor for treatment emergent AEs beginning immediately following the administration of Investigational Product or placebo. Participants will remain in the CRU under observation for 2 hours post- dose;

2. Vital signs (blood pressure, pulse rate, respiration rate, and temperature) at 15 minutes, 30 minutes, 1 hour and 2 hours post-dose;

3. Participants will be instructed on the identification and treatment of systemic reactions, including indications for self-injectable epinephrine in the event of a severe allergic reaction between study visits;

4. Participants will be provided with a diary and instructed to record any AEs or concomitant medications taken between visits, and to bring the completed diary to each visit for review; 5. Participants will be given a 24-hour emergency telephone number and instructed to call the investigational site immediately should an AE occur between visits;

6. Participants will be instructed to return to the CRU the following day (24 hours +3 days post dose).

Study Day 2

[0525] Participants will return to the CRU on Day 2, and the following procedures will be performed prior to Investigational Product or placebo administration:

1. Monitoring of AEs and review of concurrent illnesses (treatment-emergent or worsening illness must be recorded as an AE), including a review of diary;

2. Review and update of concomitant medications, including a review of diary;

3. Targeted physical examination;

4. Vital signs (blood pressure, pulse rate, respiration rate, and temperature);

5. PEF.

[0526] Pending no safety issues, participants will be administered their second dose of

ENP-501.

[0527] The following procedures will be performed after Investigational Product administration on Day 2:

1. Monitor for AEs. Participants will remain in the CRU under observation for 2 hours post-dose;

3. Pending no safety issues, the participants will be provided with sufficient

Investigational Product or placebo to complete the first week of dosing (Day 3 to Day 7). The participants will be instructed on how to administer the Investigational Product or placebo, and will be instructed to administer the Investigational Product or placebo qd in the buccal mucosal space at approximately the same time each day. The participants will be instructed to return the Investigational Product or placebo container and any unused Investigational Product or placebo at the next CRU visit;

4. Participants will be provided back their diary for recording the details of

Investigational Product or placebo administration, AEs or concomitant medications taken between visits, and will be instructed to bring the completed diary to the next study visit;

5. Participants will be instructed to return to the CRU on Day 8.

Weekly Dose Escalation Visits (Study Days 8 and 15 )

[0528] Participants will return to the CRU for weekly dose escalation visits on Days 8 and 15. The following procedures will be performed prior to Investigational Product or placebo administration at each weekly dose escalation, except where noted:

2. Review and update of concomitant medications, including a review of diary;

3. Collect Investigational Product or placebo container. Unused Investigational Product or placebo will be counted for compliance and recorded;

4. Review of the Investigational Product or placebo administration diary;

5. Targeted physical examination;

6. Weight;

7. Vital signs (blood pressure, pulse rate, respiration rate, and temperature);

8. PEF;

9. Safety labs, including CBC with differential, serum chemistry, and urinalysis (see Appendix 3 for list of tests) - Study Day 8 Only.

[0529] Pending no safety issues, Investigational Product or placebo will be administered.

[0530] The following procedures will be performed after administration of

Investigational Product or placebo: 1. Monitor for AEs. Participants will remain in the CRET under observation for 2 hours post-dose;

3. Pending no safety issues, the participants will be provided with sufficient Investigational Product or placebo to complete the next week of dosing at home (Day 9 to Day 14 and Day 16 to Day 21, respectively). The participants will be reminded on how to administer the Investigational Product or placebo in the buccal mucosal space, and will be instructed to take the Investigational Product or placebo qd at approximately the same time each day. The participants will be instructed to return the Investigational Product or placebo container and any unused Investigational Product or placebo at the next study visit;

4. Participants will be provided back their diary for recording the details of Investigational Product or placebo administration, AEs or concomitant medications taken between visits, and will be instructed to bring the completed diary to the next study visit;

5. Participants will be instructed to return to the CRET in 7 days.

A phone interview will be conducted the day after each weekly dose escalation visit to assess for any AEs.

Treatment Period Part II (Peanut Allergic Participants)

Study Day 1

[0531] Participants that continue to meet eligibility requirements after the baseline assessments and that are enrolled in Part II of the trial will be randomized to treatment and administered Investigational Product placebo. This visit is to be completed within 14 days of Screening. [0532] Up to 3 dose levels will be attempted (0.25, 0.5, and 1 pg peanut protein) on Day

1, with 30 minutes between each dose. Participants randomized to the placebo arm will receive the same number of tablets as planned for the Investigational Product dose escalation; however, the tablets will contain no peanut protein. Protocol-required dose escalation modifications may be made in the event of symptoms.

[0533] Monitoring for treatment emergent AEs will begin immediately following administration of the first dose of Investigational Product or placebo and will continue throughout the study. Participants will remain in the CRU under observation for 2 hours post- dose.

[0534] The following procedures will be performed on Day 1 after administration of the last dose of Investigational Product or placebo for the day:

1. Vital signs (blood pressure, pulse rate, respiration rate, and temperature) at 15 minutes, 30 minutes, 1 hour and 2 hours post-dose;

2. Participants will be instructed on the identification and treatment of systemic reactions, including indications for self-injectable epinephrine in the event of a severe allergic reaction between study visits;

3. Participants will be provided with a diary and instructed to record any AEs or concomitant medications taken between visits, and to bring the completed diary to each visit for review;

4. Participants will be given a 24-hour emergency telephone number and instructed to call the investigational site immediately should an AE occur between visits;

5. Participants will be instructed to return to the CRU on the following day (Day 2). Study Day 2

[0535] Participants will return to the CRU on Day 2, and the following procedures will be performed prior to Investigational Product or placebo administration:

2. Review and update of concomitant medications, including a review of diary;

3. Targeted physical examination; 4. Vital signs (blood pressure, pulse rate, respiration rate, and temperature);

5. PEF.

[0536] Pending no safety issues, the participants will be administered Investigational

Product or placebo at the daily dose level determined on Day 1.

[0537] The following procedures will be performed after Investigational Product or placebo administration on Day 2:

1. Monitor for AEs. Participants will remain in the CRET under observation for 2 hours post-dose;

3. Pending no safety issues, the participants will be provided with sufficient Investigational Product or placebo to complete the first two weeks of dosing (Day 3 to Day 14). The participants will be instructed on how to administer the Investigational Product or placebo in the buccal mucosal space, and will be instructed to take the Investigational Product or placebo qd at approximately the same time each day. The participants will be instructed to return the Investigational Product container and any unused Investigational Product or placebo at the next CRU visit;

5. Participants will be instructed to return to the CRU on Day 15.

Biweekly Dose Escalation Visits (Study Days 15, 29, 43, and 57)

[0538] Participants will return to the CRU for weekly dose escalation visits on Days 15,

29, 43 and 57. Participants with dose escalation delays will have additional weekly dose escalation (e.g. visits at Days 71, 85, and 99, as needed) up through 26 weeks. [0539] The following procedures will be performed prior to Investigational Product or placebo administration at each biweekly dose escalation visit, except where noted:

2. Review and update of concomitant medications, including a review of diary;

3. Collect Investigational Product container. ETnused Investigational Product or placebo will be counted for compliance and recorded;

4. Review of the Investigational Product or placebo administration diary;

5. Targeted physical examination;

6. Weight;

7. Vital signs (blood pressure, pulse rate, respiration rate, and temperature);

8. PEF;

9. Safety labs, including CBC with differential, serum chemistry, and urinalysis (see Appendix 3 for list of tests) - Study Days 15 and 29 Only.

10. ETrine pregnancy test [beta-chorionic gonadotropin (b-hCG)] for WCBP only - Study Days 29 and 57 Only;

[0540] Pending no safety issues, Investigational Product or placebo will be administered.

Tip to 3 dose levels will be attempted, with 30 minutes between each dose.

[0541] The following procedures will be performed after administration of the last dose of Investigational Product or placebo for the visit:

2. Vital signs (blood pressure, pulse rate, respiration rate, and temperature) at 15 minutes, 30 minutes, 1 hour and 2 hours post-dose; 3. Pending no safety issues, the participants will be provided with sufficient Investigational Product or placebo to complete the next two weeks of dosing at home (e.g., Day 16 to Day 28, Day 30 to Day 42, Day 44 to Day 56, and Day 58 to Day 70). The participants will be reminded on how to administer the Investigational Product or placebo in the buccal mucosal space, and will be instructed to take the Investigational Product or placebo qd at approximately the same time each day. The participants will be instructed to return the Investigational Product or placebo container and any unused Investigational Product or placebo at the next study visit;

5. Participants will be instructed to return to the CRU in 14 days.

A phone interview will be conducted the day after each biweekly dose escalation visit to assess for any AEs.

Maintenance Visits (Study Days 85 and 168)

[0542] Participants will return to the CRET for visits on Days 85 and 168. Participants still in dose escalation at Day 85 will have the escalation procedures above conducted in addition to a blood collection.

[0543] The following procedures will be performed prior to Investigational Product or placebo administration:

2. Review and update of concomitant medications, including a review of diary;

3. Collect Investigational Product or placebo container. ETnused Investigational Product or placebo will be counted for compliance and recorded;

4. Review of the Investigational Product or placebo administration diary;

5. Targeted physical examination; 6. Weight;

7. Vital signs (blood pressure, pulse rate, respiration rate, and temperature);

8. PEF;

9. Safety labs, including CBC with differential, serum chemistry, and urinalysis (see Appendix 3 for list of tests)

10. Urine pregnancy test [beta-chorionic gonadotropin (b-hCG)] for WCBP only;

11. Blood collection for total IgE and peanut-specific IgE and IgG₄ levels;

[0544] Pending no safety issues, Investigational Product or placebo will be administered.

Participants still in dose escalation at Day 85 will have up to 3 dose levels will be attempted, with 30 minutes between each dose.

[0545] The following procedures will be performed after administration of the last dose of Investigational Product or placebo for the visit:

3. Pending no safety issues, the maintenance participants will be provided with sufficient Investigational Product or placebo to complete dosing at home until the next visit (e.g., Day 86 to Day 167 and Day 169 to Day 365). The participants will be instructed to take the Investigational Product or placebo via buccal mucosal administration qd at approximately the same time each day. The final dose will be administered the day before the DBPCFC.

Participants will be provided back their diary for recording the details of Investigational Product or placebo administration, AEs or concomitant medications taken between visits, and will be instructed to bring the completed diary to the next study visit; Oral Food Challenge (Study Day 366 + 7 Days)

[0546] Optionally, in place of or in addition to a final blood test, participants will return to the CRU for a double-blind placebo controlled oral food challenge at Day 365 (i.e. ~52 weeks). Prior to the DBPCFC, the following study procedures will be conducted:

1. Monitoring of AEs and review of concurrent illnesses (treatment-emergent or worsening illness must be recorded as an AE), including a review of diary. If the participant has had an upper respiratory infection (URI) within 7 days of either part of the DBPCFC, the challenge will be rescheduled within 7 days following resolution of the URI;

2. Review and update of concomitant medications, including a review of diary. Participants must discontinue use of antihistamines for an appropriate length of time (5 half-lives of the antihistamine) prior to the DBPCFC and SPT;

4. Review of the Investigational Product or placebo administration diary;

5. Targeted physical examination;

6. Weight;

7. Vital signs (blood pressure, pulse rate, respiration rate, and temperature);

8. PEF to assess for an exacerbation of asthma as defined by active wheezing or a PEF <80% of predicated;

9. Safety labs, including CBC with differential, serum chemistry, and urinalysis (see Appendix 3 for list of tests);

10. Urine pregnancy test [beta-chorionic gonadotropin (b-hCG)] for WCBP only;

11. Blood collection for total IgE and antigen-specific IgE and IgG₄ levels;

12. SPT to peanut, milk, egg, walnut, cashew, sesame, cottonwood, rocky mountain juniper, Kentucky and timothy grasses, sagebrush, and/or ragweed (western).

[0547] The DBPCFC may be conducted over a 1-2 day period and will consist of giving a total of 4444 mg of peanut protein in gradually increasing doses at 20-30 minute intervals. If conducted in a single day, at least 2 hours must separate the first half of the challenge from the second half. The DBPCFC will take place in a CRU or comparable monitored clinical site with experience in treating severe allergic reactions. Specifically, a crash cart will be available in the facility and there will be medical personnel present to treat anaphylaxis.

[0548] The investigator may use clinical judgment to increase the intervals between doses, if there is a concern that a reaction may be developing. The doses are distributed in the following increments, for a total of 4444 mg of peanut protein (1, 3, 10, 30, 100, 300, 1000, and 3000 mg). The Investigational Site registered dietitian will prepare the challenge material using the site standard operating procedure (SOP). Records of the challenge preparation will be maintained in the Investigational Site dietary kitchen.

Frequent assessments are made for symptoms affecting the skin, gastrointestinal tract, cardiovascular system, and/or respiratory tract. The OFC will be stopped when the investigator determines that symptoms indicate a positive reaction and the dosing will be terminated or if the maximum cumulative dose is reached.

First Post Dose Visit (Part I, and Part II for participants with early discontinuation)

[0549] Participants in Part I and participants in Part II that discontinue dosing prior to week 52 will return to the CRU 24 hours (+3 day visit window) after their last dose of

Investigational Product or placebo. The following procedures will be performed.

2. Review and update of concomitant medications, including a review of diary;

4. Review of the Investigational Product or placebo administration diary;

5. Targeted physical examination;

6. Weight;

7. Vital signs (blood pressure, pulse rate, respiration rate, and temperature);

8. PEF;

10. ETrine pregnancy test (b-hCG) for WCBP only;

11. Blood collection for total IgE and peanut-specific IgE and IgG₄ levels;

12. SPT to peanut - Part I only;

13. SPT to peanut, milk, egg, cashew, walnut, sesame, cottonwood, rocky mountain juniper, Kentucky and timothy grasses, sagebrush, and/or ragweed (western) - Part II only. Following the above procedures, the participants will be provided back their diary for recording any AEs or concomitant medications taken between visits, and reminded to bring the completed diary to the next study visit. The participants will be instructed to return to the CRU in four weeks (± 3 days).

Second Post Dose Visit/Final Study Visit (Part I and Part II)

[0550] Participants will return to the CRU 4 weeks ± 3 days after their last dose of

Investigational Product or placebo (i.e., Day 49 ± 3 days for Part I, Day 394 ± 3 days for Part II, or sooner if Investigational Product or placebo is prematurely discontinued). If a participant is withdrawn from the study early, all evaluations described below for the final study visit will be performed if feasible.

[0551] The following procedures will be performed, and will be the same for part I and part II, except where noted.

2. Review and update of concomitant medications, including a review of diary;

3. Comprehensive physical examination;

4. Weight;

5. Vital signs (blood pressure, pulse rate, respiration rate, and temperature);

6. PEF;

8. Urine pregnancy test (b-hCG) for WCBP only;

9. Blood collection for total IgE and peanut-specific IgE and IgG₄ levels;

10. SPT to peanut - Part I Only;

11. SPT to peanut, milk, egg, walnut, cashew, sesame cottonwood, rocky mountain juniper, Kentucky and timothy grasses, sagebrush, and/or ragweed (western) - Part II Only; [0552] Any participant with a suspected Investigational Product or placebo-related AE at the final study visit will be followed until resolution or stabilization of the event.

Measuring Treatment Compliance

Part I (Non-Peanut Allergic Participants)

[0553] For Part I of this trial, participants will take qd buccal mucosal doses of ENP-501 for 3 weeks. The Investigational Product or placebo will be taken in a CRU or comparable monitored clinical site on Day 1, Day 2, and at each weekly dose escalation visit (Day 8 and Day 15). Treatment compliance at these visits will be ascertained by inspection of the oral cavity (mouth check) following Investigational Product or placebo administration.

[0554] All remaining Investigational Product or placebo doses will be self-administered at home. The participants will be provided with a diary to record the details of Investigational Product or placebo administration, and they will be instructed to return the completed diary, along with the Investigational Product or placebo bottle at the next visit. The Investigational Site staff will collect the Investigational Product or placebo bottle at each visit, and will count and record any unused portions while the participant is at the clinic. Further, they will review the Investigational Product or placebo administration diary to confirm consistency with the planned Investigational Product or placebo dosing regimen. Participants will be counseled and re- educated on appropriate dosing for compliance <80% or >110%.

Part II (Peanut Allergic Participants)

[0555] For Part II of this trial, participants will take qd buccal mucosal doses of

Investigational Product (ENP-501 or placebo) for up to 52 weeks. The Investigational Product or placebo will be taken in a CRU or comparable monitored clinical site on Day 1, Day 2, and at each biweekly dose escalation visit (up to 26 weeks). Treatment compliance at these visits will be ascertained by inspection of the oral cavity (mouth check) following Investigational Product or placebo administration.

[0556] All remaining Investigational Product doses will be self-administered at home.

The participants will be provided with a diary to record the details of Investigational Product or placebo administration, and they will be instructed to return the completed diary, along with the Investigational Product bottle at the next visit. The Investigational Site staff will collect the Investigational Product bottle at each visit, and will count and record any unused portions while the participant is at the clinic. Further, they will review the Investigational Product or placebo administration diary to confirm consistency with the planned Investigational Product or placebo dosing regimen. Participants will be counseled and re-educated on appropriate dosing for compliance <80% or >110%.

Concomitant Medications

[0557] All medications (or treatments) other than Investigational Product or placebo taken or received by the participant at any time during the study from the first dose of

Investigational Product or placebo through the final study visit assessment will be considered concomitant medications. Use of all concomitant medications, including any change in therapy, will be recorded and updated.

[0558] Participants may continue their usual medications, including those taken for asthma, allergic rhinitis, and atopic dermatitis, during the study, except for the prohibited medications listed below. Participants must be able to temporarily discontinue antihistamines (5 half-lives of the antihistamine) prior to skin testing. Regular topical steroids use is permitted at the time of skin testing.

[0559] The medications listed below are prohibited during this trial. If they are used, regardless of whether they are deemed necessary for the treatment of an intercurrent medical condition, the participant will be discontinued from the trial.

1. IV vasopressor drugs;

2. b-blockers (oral), ACE inhibitors, ARBs, or calcium channel blockers;

3. Oral or IV corticosteroids;

4. Any other allergen immunotherapy, including non-traditional forms (e.g., oral or sublingual);

5. Any other biologic therapy;

6. Omalizumab or any other immunomodulatory therapy (not including topical or nasal corticosteroids);

7. Any other investigational vaccine or drug. Criteria for Evaluation

Part I (Non-Peanut Allergic Cohort)

[0560] Primary Endpoint: Safety and tolerability of buccal mucosal administrations of

ENP-501 when administered qd for 3 weeks in non-peanut allergic participants who are 18 to 50 years of age.

[0561] Secondary Endpoint: Total IgE, peanut-specific IgE, and peanut-specific IgG4 levels after 3 weeks of treatment and at 4 weeks post-treatment relative to Screening.

Part II (Peanut Allergic Cohort)

[0562] Primary Endpoint: Safety and tolerability of buccal mucosal administrations of

ENP-501 compared to placebo when administered daily for up to 8 weeks in peanut allergic participants who are 12 to 50 years of age.

[0563] Secondary Endpoints: (i) safety and tolerability of buccal mucosal

administrations (qd) of ENP-501 compared to placebo when administered daily for up to 52 weeks in peanut allergic participants; (ii) changes in total IgE, peanut-specific IgE, and peanut- specific IgG₄ levels after up to 52 weeks of treatment; and (iii) changes in wheal diameter on SPT to peanut after up to 52 weeks of treatment.

[0564] Exploratory Endpoints: (i) evaluation of successfully consumed dose of peanut protein on the DBPCFC after up to 52 weeks; (ii) evaluation of changes in peanut-specific IgE to other common allergens (e.g. milk, egg, walnut, cashew sesame, cottonwood, rocky mountain juniper, Kentucky and timothy grasses, sagebrush and/or ragweed (western)) as a result of up to 52 weeks of treatment; and/or (iii) evaluation of changes in wheal diameter on the endpoint titration SPT to other common allergens (e.g., milk, egg, walnut, cashew, sesame, cottonwood, rocky mountain juniper, Kentucky and timothy grasses, sagebrush, and/or ragweed (western)) as a result of up to 52 weeks of treatment with ENP-501.

Statistical Methods Part I (Non-Peanut Allergic Participants)

[0565] Analysis will be primarily descriptive and results will be summarized as appropriate (e.g., mean, median, range, and standard deviation for continuous data and frequency for categorical data). All participants who receive at least one dose of Investigational Product or placebo will be included in the safety analysis population.

Part II (Peanut Allergic Participants)

[0566] The study is not powered to detect differences between active and placebo for either safety or immunological changes. Safety analysis will be primarily descriptive and results will be summarized as appropriate (e.g., mean, median, range, and standard deviation for continuous data and frequency for categorical data). All participants who receive at least one dose of Investigational Product or placebo will be included in the safety analysis population. A blinded interim safety review will take place after all participants have been followed for 8 weeks or withdrawn from the study.

[0567] For immunological and SCD outcomes, analysis will also be primarily descriptive with results summarized as appropriate (e.g., mean, median, range, and standard deviation for continuous data and frequency for categorical data) and by treatment group (e.g., active or placebo). Change from baseline/screening for immune parameters will also be described.

[0568] The following Example describes a representative Phase 2 Trial and is designed to investigate ENP-501 for treatment of subjects suffering from or susceptible to peanut allergy. A Phase 2 trial will be conducted only if a Phase 1 trial is successfully completed (e.g. no safety issues present during Phase 1 trial). This Phase 2 trial will be a double blind, placebo-controlled food challenge (DBPCFC) and will use portions with the same ingredients (active and inactive ingredients) as in the ENP-501 tablets and placebo tables described in Example 4.

Participants and Sample Size

[0569] The target sample size for this Phase 2 study will be between 45 and 51 participants if conducting a 1 :2 randomization. The lower sample size of 45 is sufficient to test a roughly 45% success rate difference between the arms. Expansion to 51, however, provides protection against dropout (e.g., 10% dropout would still leave 45-46 completing the Week 52 OFC) and if all participants complete is sufficient to test an approximately 40% difference thus guarding against a slightly smaller treatment effect than expected. Treatment will occur in a blinded fashion for 52 weeks with assessment of efficacy based on change from a baseline OFC to the Week 52 OFC. This Phase 2 study may also include a crossover component (e.g., participants receiving placebo will cross over to receiving ENP-501) or extension of dosing for both active and placebo through an open-label long term follow-up study.

[0570] We expect the success rate in the placebo arm of this study will range from about lO-to about 20%. We expect that the active (ENP-501 receiving participants) arm will show an increase of about 35- about 45%, with a range of about 25% to about 75% success in the active arm. Table 8 below shows sample sizes for a balanced (1 : 1) and unbalanced (1 :2) randomization. The primary endpoint for this trial will be powered to achieve success or failure. Additional endpoints may include those as described for Part II, primary and secondary. Achievement may be considered upon, e.g. a lO-fold increase in successful consumption of a dose at a

predetermined time point (e.g. 44 or 52 weeks from commencement of the study), as compared to baseline consumption or, e.g. an increase of 45% on each active arm as compared to that of placebo. Success may also be determined if, e.g. an eliciting dose greater than 1,000 mg of peanut occurs independently of even a lO-fold increase as described above.

[0571] An entry DBPCFC would establish the successfully consumed dose (or eliciting dose). A treatment success would demonstrate a 10 fold increase in SCD after 52 weeks of treatment. Consideration could be given to a minimum SCD (e.g. 10 fold plus a SCD of 144 mg), to avoid minor changes in SCD being considered successes.

[0572] Table 8. Required sample sizes to achieve at least 85% or 90% power with various underlying success rates in each arm, using a two-sided unconditional exact test at the a=0.05 -level

Number of Centers

[0573] As described in Example 4, the Phase 1 trial will take place at two study sites.

For the Phase 2 study, a larger number of centers (e.g. more than 2) will be used. Such expansion has advantages including, e.g. i) increased accrual; ii) allowing to assess logistics prior to a Phase 3 trial; and/or iii) generalizability of the results to a wider population to allow for better Phase 3 estimates and design. Based on previous studies, accrual in a peanut-allergic patient population appears to be about 1 participant/month/site. With a 51 participant trial, expansion to 5 sites would assume accrual completion in roughly a year. A two-center trial would project >2 years of enrollment.

Other Considerations

[0574] Depending upon the enrollment, this Phase 2 trial may also incorporate a crossover component (e.g. participants that begin the study on Placebo will then move to the treatment arm) and/or a separate long term follow-up protocol to allow for, e.g., assessment of continued dosing and building of a safety database.

[0575] In some embodiments, an Investigational Product will be a powder formulation comprised of nanoparticles and one or more excipients as described in Example 5A. In some such embodiments, trials will be performed using the formulation as described in accordance with this disclosure including Example 5A, with the following specifications: The trial period will be 14 months (60 weeks of treatment), will have 50 peanut-allergic participants and is a randomized, double-blind, placebo-controlled, dose escalation study to evaluate safety, tolerability, and pharmacodynamics of the Investigational Product compared to placebo in peanut allergic participants who are 12 to 50 years of age. 50 peanut allergic participants will be randomized 1 : 1 to receive buccal mucosal administrations of Investigational Product or placebo daily for up to 60 weeks (25 active; 25 placebo with 80% power to detect 41% difference). The dose will be escalated on Day 1 and every two weeks thereafter. After 8 weeks, participants will return to the clinic on Day 57 for the final escalation visit and maintain the highest tolerated dose for the remaining 44 weeks. Each dose escalation will occur in the CRU. Participants will return to the CRU 24 hours and 52 weeks after their last dose of investigational product for follow-up visits. The Investigational Product and placebo will be in the form of a bulk drug powder for buccal mucosal administration. Ingredients for Investigational Product and placebo will be as described in Example 4.

[0576] Diagnosis and Inclusion/Exclusion Criteria will be as follows:

Inclusion Criteria

[0577] Participants 12 to 50 years of age with a convincing clinical history of peanut allergy. The main criteria for inclusion are: A convincing clinical history of peanut allergy at age 2 or older, 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; At least two of the three following requirements: i) Positive SPT (wheal diameter of > 5 mm) to peanut,

ii) An elevated serum peanut-specific IgE level > 8 kUA/L (ImmunoCAP®) within 1 year of enrollment (including at Screening),

iii) An Ara-h2 IgE level of > 2 kUA/L within 1 year of enrollment (including at

Screening).

Exclusion Criteria

[0578] Participants meeting any of the following criteria will be excluded from the trial

2. FEV1 value < 80% predicted at Screening;

3 Poor control or persistent activation of atopic dermatitis;

4 Any hospitalization in the past year for asthma, or >1 course of oral steroids for asthma or any emergency room visit in the past 6 months for asthma; 5. Eosinophilic gastrointestinal disease;

6. Use of oral or IV corticosteroids within 30 days of Screening;

7. Inability to discontinue antihistamines for skin testing;

8. Use of omalizumab or other non-traditional forms of allergen immunotherapy (e.g., oral or sublingual) or immunomodulatory therapy (not including corticosteroids) or biologic therapy within one year of Screening;

10. Use of immunosuppressive drugs within 30 days of Screening;

13. Pregnancy or breast-feeding (if female);

16. Participation in another investigational vaccine or drug trial within 30 days of Screening.

Criteria for Evaluation

[0579] Criteria for evaluation will include the following primary endpoints:

[0580] The primary endpoint is the response rate, defined as the proportion of subjects who are able to successfully consume a single dose of a pre-determined quantity of peanut protein with no dose-limiting symptoms, as defined by PRACTALL guidelines at exit DBPCFC.

[0581] Criteria for evaluation will include the following secondary endpoints:

1. Changes in total IgE, peanut-specific IgE, and peanut-specific IgG4 levels after up to 60 weeks of treatment;

2. Changes in wheal diameter on the Skin Prick Test (SPT) to peanut after up to 60 weeks of treatment; and 3. Safety and tolerability of buccal mucosal administrations of ENP-501 compared to placebo when administered daily for up to 12 weeks in peanut allergic participants who are 12 to 50 years of age.

[0582] A schedule of trial events can be seen in Figure 15.

LABORATORY BLOOD TESTS (“Safety Tests”)

[0583] Tests listed below will be performed at each visit where“safety labs” are specified. 5 ml of blood will be collected.

EXAMPLE 6 A: Optimized Method of Preparing polymer nanoparticles comyrisins protein and DNA and coated with OEE

[0584] This Example describes an exemplary 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 in Figure 8A in Figure 8. One of skill in the art will appreciate that certain conditions and specific values as described herein may be changed as desired.

[0585] 1. Preparation of solid block material comprising PLGA, protein, and DNA (see, e.g., column“ Lyo 1” of Figure 8A (steps 1-5))

Steps:

1) An organic polymer solution was prepared by dissolving polymer (here, PLGA) into organic solvent (here, DMSO), at 1.9 mg/mL using magnetic stirring to generate an organic PLGA solution. The temperature was maintained between approximately 25°C - 30°C, which prevents DMSO from freezing, lowers viscosity of the PLGA solution, and increases speed at which PLGA dissolves.

2) An aqueous payload solution was prepared at room temperature by dissolving payload comprising protein (e.g., peanut protein as, e.g., crude peanut extract) and DNA (e.g., sheared E. coli DNA) in water to achieve a 25: 1 ratio of protein to DNA (e.g., here, 6 mg/mL crude peanut extract and 0.06 mg/mL sheared E. coli DNA.. The payload solution was then diluted to 3 mg/mL of protein and 0.03 mg/mL of DNA, and adjusted to pH 9 using NaOH. Sonication and/or homogenization were used as required to obtain a substantially homogenous solution of protein, DNA and water.

3) The aqueous payload solution of step 2 was then added to the organic polymer solution of step 1, and combined to achieve a mixture of substantial homogeneity. The volumetric ratio of DMSO (organic/”payload”) solution: aqueous (water/”payload” solution) was 94:6. This ratio may be varied to suit other applications or desired embodiments, including as described in the present disclosure.

4) The mixture of step 3 (organic solution of step 1 and aqueous solution of step 2) was then subjected to rotary evaporation (represented as“rotovap” in Figure 8, between steps 3 and 4) at between 50-85 rpms, 160-250 mbar, and approximately 65-75 °C until approximately 75% of the water was evaporated (approximately 10-12.5 hours). The volumetric ratio of the solution after rotary evaporation was approximately 98.5: 1.5. DMSO (organic/”payload”) solution:

aqueous (water/”payload” solution).

5) The solution of step 4 was frozen at a temperature of -65 °C and lyophilized to generate a block material (“dry cake”), containing crude peanut extract, sheared E. coli DNA, and PLGA. 5 A) Optionally, the dry cake may be heated, for example, to approximately 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.

[0586] 2. Preparation of nanoparticles comprising PLGA, protein, DNA (see, e.g., column“Lyo 2” in Figure 8A (steps 6 - 9))

Steps:

6) The block material of step 5 resulting from Lyol (Steps 1-5) (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)) and ground using a mortar and pestle under dry conditions. Resulting micro-sized granules were suspended in n-propanol to produce, forming a flowable microparticle suspension, which was added to a microfluidic homogenizer. The starting concentration of granules in n-propanol was approximately 7 mg/mL, which was diluted to approximately 3.25 mg/mL after addition of the PLGA/protein/DNA suspension in n-propanol to the hot propanol already present in the microfluidic homogenizer.

7) Nanoparticles were produced by microfluidic homogenization of the granule/propanol solution of step 6 at temperatures between 85 and 90 °C . The solution in the homogenizer may be either recycled through the chamber. In some embodiments, for example, a recycled solution scenario may use a volume of about 200 mL which flows at a liquid flow rate of 200 mL/min, with the homogenizer run for 5 mins for an overall ratio of solution volume to pumped volume of 1 :5. In some embodiments, for example, 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. 8) An aqueous polyvinyl alcohol (PVA1) solution (0.8125 mg/mL) was added to the 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 and/or otherwise stabilizing formed nanoparticles. The volumetric ratio of nanoparticle suspension (loaded nanoparticles with PVA1 in water, i.e., aqueous solution): organic solution(propanol) was 4: 1. The resulting suspension was passed through the microfluidic homogenizer. Homogenization heating was turned off, but

homogenization was continued. 9) Trehalose dehydrate granules were added directly to the nanoparticle suspension of step 8 and the mixture was lyophilized to form a dried mixture (e.g., in this Example, a dried cake).

The dried cake includes nanoparticles, PVA1, and trehalose.

[0587] 3. Preparation of nanoparticles coated with an agent(e.g. OEE) (scheme Lyo 3 in

Figure 8 (steps 10-18))

Steps:

10) The dried cake obtained in step 9/Lyo2 was then re-suspended in 10 mM ammonium bicarbonate buffer, resulting in a solution comprising buffer, big particles (i.e., larger than 100- 400 nm), free protein (e.g. protein freely suspended and/or loosely associated with

nanoparticles), nanoparticles, PVA1, and trehalose Alternatively, if desirable, the dried cake may be suspended in propanol and diluted by a factor of 50 or more in an aqueous buffer such as water.

11) The re-suspended solution of step 10 was then subjected to a low speed centrifugation (e.g. approximately 250 to approximately 500 x g) to pellet non-homogenous large particles (e.g., clumped, unencapsulated protein). After centrifugation, the supernatant solution comprising nanoparticles, free protein, PVA1, and trehalose was decanted from the pellet produced during the low speed centrifugation. It is contemplated that, as necessary, large particles (i.e., particles greater than approximately 400 nm)) could also be initially removed using other or additional purification and/or separation methods (e.g., filtration (e.g., syringe filter), centrifugation, and/or others).

11 A) Without being bound by any particular theory, it is contemplated that the low speed centrifugation is insufficient to remove all remaining free protein (i.e., protein that is not loaded into nanoparticles) and/or clusters and/or clumped together material or nanoparticles

Accordingly, the supernatant solution of step 10 was then subjected to tangential flow filtration for further purification. By way of non-limiting example, the suspension was filtered through a TFF membrane with a MWCO of 500 kilodaltons (e.g., MiniKros/KrosFlo/etc.) at a flux rate of between 7 and 8 mL/min for 1 hour. After filtration the mean hydrodynamic diameter of the nanoparticles in the permeate was measured by dynamic light scattering and determined to have a range of approximately 200 to approximately 230 nm. Optionally, the supernatant suspension obtained after low speed centrifugation and/or tangential flow filtration may be followed by one or more additional centrifugation steps to yield nanoparticles of different size ranges. In some embodiments, the supernatant suspension is centrifuged at an intermediate speed (e.g., approximately 5000 x g to approximately 9000 x g) resulting in a pellet of nanoparticles, with each nanoparticle measuring approximately 300 - 400 nm in diameter. In some embodiments, the supernatant suspension is centrifuged at a higher speed (e.g., approximately 17,000 to approximately 20,000 x g) resulting a pellet containing nanoparticles that are each approximately 100-200 nm in diameter. In the embodiments described, the pellet of nanoparticles is then resuspended in aqueous buffer for further processing. In some additional embodiments, sequential centrifugation steps at variable speeds can be used to recover nanoparticles of a desired size.

12) After purification with Tangential Flow Filtration (TFF) methods, the resulting solution comprised nanoparticles, trace PVA1, and ammonium bicarbonate buffer. This solution was reserved to mix with concentrated OEE micelles in water, which are described in steps 13-15, below.

Steps 13-15 (preparing a coating agent, e.g. OEE Solution), may be performed either in parallel (concurrently) with steps 1-12, or sequentially, following, e.g. Steps 1-12.

13 and 14) An aqueous solution of Organic E. Coli. Extract (“OEE”) (as prepared in accordance with Example 2, above) was prepared by adding water to OEE powder (in this Example, to a concentration of 2 g/L OEE).

15) The mixture was sonicated to make a solution of concentrated OEE micelles in water (“OEE Solution”), which was then mixed with the loaded nanoparticle solution of step 12, sonicated, and then combined with trehalose at a concentration of such that the final amount of dehydrated trehalose was 2 times the mass of the purified nanoparticles (e.g. for 0.5 g/L OEE, the concentration of trehalose is 1 g/L). The addition of trehalose in this step constitutes the vast majority of trehalose in the final formulation because the prior TFF step reduces trehalose concentration to negligible levels.

16). The resulting solution comprising loaded nanoparticles, trace PVA1, trehalose, OEE micelles, and water was then lyophilized.

17) The resulting solid dispersion following lyophilization comprised loaded nanoparticles, which nanoparticles were coated with OEE. The solid dispersion was then ground and passed through a sieve. 18) Following grinding/sieving of the solid dispersion of step 17, an intermediate as a flowable powder was obtained. The powder contained OEE-coated, loaded nanoparticles.

20) Optionally, the flowable powder of step 18 may be further processed as desired for use in one or more pharmaceutical preparations.

EXAMPLE 6B: Scale Up Parameters and Protocols

[0S88J 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, at different scales. A representative nanoparticle manufacturing process is described below and shown in whole (e.g., Figure 8A) and in part, according to different portions of the process and different scales (e.g., Figures 8B-8D). One of skill in the art will appreciate that certain conditions and specific values as described herein may be changed as desired.

[0589] In order to scale up the manufacture of nanoparticle product, scaling production parameters were estimated for runs of scales of 1 g and 20 g of peanut protein using a manufacturing and/or purification protocol according to the present disclosure.

Phase 1 process : 1 g Peanut Protein

[0590] It is estimated that an overall manufacturing process to make loaded

nanoparticles containing an aggregate of 1 g of peanut protein (total of peanut protein loaded in all nanoparticles for Phase I) for a Phase I clinical trial, will take approximately three weeks (4-5 days of active labor and 11 days of lyophilization). To accomplish a manufacturing run on this scale, three lyophilizers will run in parallel (see Figure 8B for a summary of process details for each of three lyophilization steps). It will be understood to those of skill in the art that optimization of one or more scale-up steps may be performed as production scales are changed, and those skilled in the art will also appreciate insights that the present disclosure provides in planning larger scale manufacturing protocols.

[0591] As seen in the schematic of Figure 8 A, a general manufacturing procedure may include up to three lyophilization steps. According to the exemplary protocol illustrated in Figure 8A, a more detailed schematic of the three-week process is as follows: [0592] “Lyol” will require a few hours of solution prep, approximately 12 hours of rotary evaporation and five days of lyophilization (see Figure 8C for representative details and a schematic superimposed over Lyol/steps 1-5 of Figure 8A).

[0593] “Lyo2” will require homogenization to be performed over a course of two days

(to fill three lyophilizers), and three days for lyophilization. (see Figure 8D for representative details and a schematic superimposed over Lyol/steps 5 -9 of Figure 8A).

[0594] “Lyo3” will require approximately one day for purification and OEE addition and three days for lyophilization. (see Figure 8E for representative details and a schematic superimposed over Lyol/steps 9 -18 of Figure 8A).

[0595] It is contemplated that process improvements could result in shortened times, including, for example, shortening timelines used in Lyo3.

[0596] Lyo3 has a purification step that uses tangential flow filtration(TFF), which has different scale-up options that have been estimated and are considered for use in increasing manufacturing capacity.

[0597] For example, a single centrifuge will produce 15.5 liters of solution that may be filtered using TFF (which is an increase of 78-fold over the scale used to initially develop the procedures disclosed herein). In order to maintain a consistent mass of nanoparticles per membrane area, 0.9 m² of filter area will be required. The overall time for centrifugation will be 15 minutes of centrifugation time, plus additional time to decant eight buckets of centrifuged solution, which will then be filtered using TFF (over the course of three hours). In order to achieve a volume of 50 liters, three centrifugation and TFF batches will be used.

[0598] Optional parameters to consider for scale-up using TFF include: 4 MiniKros=

1.04 m2 (which would have a faster process but may be subject to slightly increases loss of nanoparticles on filter, and has more dead volume in tubing); 3 MiniKros= 0.78 m2 (which has a slower process, may be subject to slightly fewer losses of nanoparticles on filter, but has more dead volume in the tubing); and 1 KrosFlo= 1.25 m2 (which would allow a faster process, but more nanoparticles may be lost as a result). It is contemplated that use of centrifugation and TFF at higher concentrations would allow use of a single KrosFlo unit while maintaining the same mass per membrane area. One of skill in the art will appreciate that there are other options, known to those in the art, which may depend upon, e.g., how many centrifuges and/or lyophilizers are running in parallel.

Phase 2 process : 20 g Peanut Protein

[0599] In order to produce enough nanoparticles for a Phase II trial, as described in the present disclosure, 20 g of peanut protein will be loaded into nanoparticles using manufacturing protocol(s) as substantially disclosed herein, including, e.g., the exemplary protocol of Figure 8A/Example 6A. Figure 8F shows exemplary estimates of volumes, times, and options to reduce production time. Changes to protocols may be implemented, as necessary, during scale up and manufacturing.

EXAMPLE 7 A: Optimization of Centrifusation Steps During Nanoparticle Preparation

[0600] This Example describes use of centrifugation to improve protein recovery, potency, and safety of nanoparticle compositions as described herein (e.g., polymeric nanoparticles comprising protein, DNA, and/or an external coating). The following results are intended to be non-limiting and exemplify effect(s) of centrifugation (e.g., centrifugation speed and/or duration) on certain nanoparticle characteristics. It is understood by a person with ordinary skill in the art that certain variables during one or more centrifugation steps can be modified as desired for a given composition.

[0601] A nanoparticle solution prepared according to the procedure of Figure 8 A was subjected to centrifugation using the solution of step 10, Figure 8 A, and the solution was centrifuged at 500 RCF at 5-10 ⁰ C for 15 minutes. Without wishing to be held to a particular theory, it is contemplated that this centrifugation step collects (e.g., in a pellet) large particles (e.g., unencapsulated protein aggregates) from a centrifuged nanoparticle solution, allowing for removal of the particles from the solution. The resulting solution is then subjected to additional purification steps, such as tangential flow filtration to remove any residual contaminants such as free protein, as well as to collect desired populations of nanoparticles (e.g., nanoparticles of particular sizes and/or protein concentrations).

[0602] Supernatant was then carefully removed from resulting pellets (which comprise nanoparticles) reserving 1 mL surrounding the pellet to avoid disturbance, and the supernatant was then filtered using tangential flow filtration, as described in Example 6A. The pellet was found to contain large aggregates and protein“poor” nanoparticles (approximately 5-20 pg protein/mg PLGA e.g., free protein and/or aggregates of protein and nanoparticles and/or nanoparticles generally at or larger than 400 nm with payload: polymer ratios lower than nanoparticles smaller than 400 nm). The supernatant was found to contain protein“rich” nanoparticles, in that there was a high (45-70 pg protein / mg PLGA) encapsulation amount.

[0603] Optionally, after an initial centrifugation step, and if tangential flow filtration is not used subsequent to an initial sept, supernatant may be transferred to new centrifuge tube(s) and centrifuged at either 8,400 RCF (Spins A-C of Figure 8G) or 17,500 RCF (Spins D-F of Figure 8G) for a duration of 1, 2, or 3 hours at 5-10 ⁰ C. It is contemplated, however, that additional centrifugation steps beyond the initial centrifugation may condense remaining nanoparticles too tightly such that resuspension of nanoparticles is difficult and subjects the procedure to greater loss.

EXAMPLE 7B: Nanoparticle Dissolution Test

Measurement of Total and Free (Unencapsulated) Protein & Determination of Safety Factor

[0604] Safety factor of nanoparticles is a term used to quantify free protein as compared to encapsulated protein to determine how much protein is encapsulated versus not, with a greater amount of encapsulated protein being considered more safe. Determination of safety factor requires two preparations: a nanoparticle total protein preparation and a nanoparticle free protein preparation as described herein. Solutions and safety factor determination was conducted as follows:

Materials

[0605] The following tables contain the equipment and reagents used to analyze solutions for peanut protein content using the bicinchoninic acid(BCA) assay kit.

Table 5. Reagents utilized for the quantitation of peanut protein using BCA assay.

Table 6. Equipment utilized for the quantitation of peanut protein using BCA assay.

Table 7. Glassware and consumables utilized for the quantitation of peanut protein using BCA assay.

Table 8. Method variation summaries.

Low force centrifugation is inadequate in spinning down a fraction of the

Assay Procedure

[0606] The procedure used to analyze solutions as disclosed herein, for peanut is

described in the present example. This procedure also uses a Pierce BCA assay in accordance with manufacturer’s instructions and can be accessed at hypertext transfer protocol secure

as sets .therm ofi sher.com/TF S -

Assets/LSG/manuals/MANOOl l430_Pierce_BCA_Protein_Asy_UG.pdf), which also describes two manufacturer recommended protocols,“Standard” (sample incubation at 37°C) and

“Enhanced” (sample incubation 60°C). This Example uses the“Enhanced” protocol to analyze lyophilized peanut extract (LPE) .

[0607] Quantification of free and total protein entails thee parts, as follows:

1. Prepare materials for the standard curve;

2. Prepare nanoparticles for analysis (i.e. sample preparation, which differs for measurement of total protein versus free (unencapsulated) protein); and

3. Run the BCA assay on materials from A and B

Preparation of Standard Curve

1. Prepare 100% stock standard at 200 pg protein/mL using a 10 mL type A volumetric

pipette (Dissolve 2.8 mg of LPE in 10 mL 0.1N NaOH); note that lyophilized peanut extract has a 71.5% assay value so 2.8 mg LPE = 2.0 mg protein

2. Prepare standard solutions by diluting 100% standard as shown in Table 5.

Table 9. Description of standard solution preparation using type A volumetric pipettes.

* 5% Standard Dilution is a serial dilution using excess of the 10% standard. This can also be prepared by diluting 1 mL of 100% standard into 19 mL of diluent.

[0608] An examplary standard curve was prepared using LPE standards dissolved in

0.1N NaOH, demonstrating good linearity is observed between 2 to 200 pg/mL. Absorbance values can be found in Table 10. Samples for a standard curve are typically prepared using one replicate for each concentration, therefore any uncertainty in a given standard curve can be assessed using the standard error calculated for the slope and intercept of that curve.

Table 10. Absorbance values for standard curve at 562 nm (n = 1 replicates) with slope of 0.00322 ± 0.00003 (absorbance/pg/mL) and y-intercept of 0.0142 ± 0.003 (absorbance). Error is calculated using standard linear regression statistics.

Sample Preparation

[0609] Total protein content was determined as follows:

1. Weigh out approximately 4 mg of nanoparticles into an appropriately sized scintillation vial (e.g., 4 mL for 4 mg nanoparticles) in duplicate. Typically, nanoparticles are stabilized in PVA1 and trehalose, so the actual mass that is weighed out is corrected for the expected mass of the excipients.

a. A typical sample includes of -33% PLGA nanoparticles with a peanut loading of about 50 pg Peanut/mg PLGA. In the present Example, 12 mg of material is equivalent to 4 mg of nanoparticles.

2. Add 2 mL of 0.1N NaOH using a type A volumetric pipette and vortex for about 30 seconds to suspend the powder. The volume of 0.1N NaOH can be changed if expected potency of nanoparticles is not 50 pg Peanut/mg PLGA.

3. Allow samples to hydrolyze overnight (-16 hours) on rotating mixer or rocker table. Ensure all samples are visually fully hydrolyzed (samples should be clear with no evidence of undissolved solids). 4. Analyze solutions for peanut content using BCA assay. (Procedure can be found below)

[0610] Free protein content was determined as follows:

1. Weigh 12 mg of nanoparticles into a 4 mL scintillation vial in duplicate or triplicate. a. An exemplary sample includes -33% PLGA nanoparticles with a peanut loading of about 50 pg Peanut/mg PLGA. In this Example, 36 mg of material would be equivalent to 12 mg of nanoparticles.

b. Manufacturing aims to achieve low levels of un-encapsulated protein. As such, it is important to achieve a high enough nanoparticle concentration in ammonium bicarbonate such that low levels of un-encapsulated protein are detectable, and ideally quantifiable. It is contemplated that with more material available, it may desirable to increase nanoparticle concentration to better quantify low levels of free protein. An ideal final concentration of nanoparticle suspension would be 20 mg nanoparticles/mL.

2. Add 2 mL of 10 mM Ammonium bicarbonate and vortex until powder is evenly suspended. The volume of ammonium bicarbonate (and mass of nanoparticles) can be varied depending on the expected free protein (see lb, above).

3. Take a 1 mL aliquot and centrifuge at 380,000 RCF for 8 minutes at 25°C. This force is sufficiently high that the majority of nanoparticles spin down and only the solubilized peanut protein remains in the supernatant.

4. Take a portion of the supernatant using a 1.5 mL transfer pipette and filter through 0.1 pm PVDF centrifuge filter (Millipore, catalog number UFC40VV00) by spinning down sample at 10,000 RCF for 4 minutes. (Note: remove supernatant away from the pellet since the pellet is easily disturbed).

5. Sample the filtrate and analyze using BCA assay. (Procedure can be found below)

[0611] BCA assay was performed as follows:

1. Prepare assay working reagent (WR) by mixing 50 parts of regent A and 1 part of regent B (both regents provided in the BCA assay kit) into an Erlenmeyer flask. The amount of WR depends on the number of samples being analyzed. For example, 24 samples requires 50 mL of WR. This can be prepared by adding 50 mL of regent A and 1 mL of regent B into a 50 mL Erlenmeyer flask and allowing to stir for at least 1 minute on a stir plate (typically stirring at 300 RPM).

2. Add 100 pL of sample and 2 mL of reagent to 4 mL clear scintillation vials using a 100 pL Pipetman and a 2 mL variable volume Pipet-Plus. (Note: Samples are typically analyzed in the order in which WR is added. For instance, if the 100% standard sample is the first to be mixed with WR, the 100% standard sample would be the first sample analyzed using a spectrophotometer which minimizes error associated with continuation of the reaction between the BCA reagent and peanut protein at room temperature.)

3. Mix by vortexing samples for ~l0s using a Vortex Genie 2 at a setting of 8, and incubate for 30 minutes at 60°C in water bath. A water bath can be set up by adding water to a 100 mm x 190 mm crystallizing dish and heating to 60°C using a temperature controlled hot plate. The water level of the bath should be high enough such that all of the liquid in the 4 mL vials is submerged below the water line. Sample incubation time is critical and affects sensitivity range for the assay.

4. After 30 minutes, remove vials from the water bath and allow samples to equilibrate to RT. Samples are typically allowed to equilibrate for about 10 minutes.

5. Transfer the colored solutions into disposable 1.5 mL semi micro cuvettes (disposable or quartz) using disposable transfer pipettes and measure the absorbance spectra from 500 nm to 600 nm using UV-VIS spectrophotometer. A spectrum is collected for potential

troubleshooting. For example, baseline shifts can be indicative of scattering from undissolved solids.

6. Subtract the absorbance measurement of a blank solution (typically 0.1 N NaOH) from the 562 nm absorbance of all samples and standards. Use the slope and intercept of the standard curve to determine the protein concentration of each unknown sample.

Calculations and Interpretation

[0612] Prior to data interpretation, protein concentrations measured in part B must be converted to mass of protein per mass of starting material, taking into account any actual concentrations and dilutions. This correction enables conversion of the total protein concentration measured in the BCA assay to a potency value that reports mass of protein per mass of the solid intermediate. [0613] Calculation of Safety factor (SF) also requires normalizing both protein concentrations to a unit mass of solid intermediate. Safety factor is then calculated using Equation 1.

[0614] The following notes may be considered for this and future assays:

[0615] Potency (protein mass per mass of solid intermediate) is not the same as the mass of protein per mass of nanoparticles. To perform this measurement, a sample workup, analogous to those described in the present Example, must be performed to remove excipients from the nanoparticles including centrifuging the nanoparticles and washing the pellet to remove unbound excipients. The lyophilized pellet can then be weighed and hydrolyzed directly to release free protein from a known mass of nanoparticles.

[0616] Higher sensitivity and accuracy for quantitation of low peanut protein

concentrations can be achieved by forcing the standard curve through zero in addition to adding replicate preparation of standards at the lower range of protein concentrations.

[0617] The recommended masses in the sample preparation sections can be scaled up to higher concentrations or higher amounts for greater accuracy

[0618] The enhanced protocol is linear from 5 pg LPE/mL to 250 pg LPE/mL. This range can be changed by varying the incubation time at 60°C. Longer incubation times will result in higher sensitivity at the lower concentrations, but decreased sensitivity at the higher concentrations.

[0619] The assay is a kinetic assay that continually develops overtime even at room temperature. Although the rates significantly decrease at room temperature, there is a practical limitation to the number of samples that can be prepared and analyzed at a time. The time elapsed between your first and last sample is generally one hour or less, however, in some situations, more or less elapsed time may be desired.

[0620] The stability of the peanut protein in 0.1N is not well understood, and is an area that should be further developed. As such, solutions should be analyzed as soon as the PLGA appears to be fully hydrolyzed.

Nanoparticle Total Protein Preparation [0621] To determine safety factor, approximately 4 mg of loaded nanoparticles prepared in accordance with the protocol of Example 6A/Figure 8 A were weighed into a scintillation vial in replicates. To vials containing nanoparticles, 2 mL of 0.1 N NaOH were added using a type A volumetric pipette and vortexed for ~ 30 seconds to fully suspend nanoparticle powder. Volume of 0.1 N NaOH may be altered accordingly, including, e.g., in consideration of expected potency of nanoparticles being measured. Suspended nanoparticles in 0.1 N NaOH were allowed to hydrolyze overnight (approximately 16 hours) on a rotating mixer or rocker table prior to processing for protein quantification.

Nanoparticle Free Protein Preparation

[0622] Approximately 12 mg of nanoparticles were weighed into a scintillation vial in replicate. To vials containing nanoparticles, 2 mL of 10 mM ammonium bicarbonate were added and vortexed until nanoparticles were uniformly suspended. Volume of 10 mM ammonium bicarbonate may be altered accordingly, including, e.g., in consideration of expected amount of free protein. After resuspension, 1 mL of solution was centrifuged at 380,000 RCF for 8 minutes at 25 °C to pellet nanoparticles. An aliquot of supernatant was then removed with a 1.5 mL transfer pipette and filtered through a 0.1 um PVDF centrifuge filter by spinning the sample at 10,000 RCF for 4 minutes. The resulting filtrate was then used for protein quantification.

Protein Quantification

[0623] In the present Example, protein quantification was performed using a BCA assay kit. It is understood by a person of ordinary skill in the art that alternative methods may be used to quantify protein (e.g., Bradford assay). To 2 mL of BCA working reagent in a 4 mL clear scintillation vial, 100 pL of a sample (e.g., total protein from nanoparticles, free protein from nanoparticles) was added, vortexed for approximately 10 seconds and incubated for 30 minutes at 60 ⁰ C in a water bath, with a water level high enough such that all liquid inside the vial(s) was submerged below the water line. After 30 minutes, vials containing BCA reagent and sample were removed from the water bath and allowed to equilibrate to room temperature for approximately 10 minutes. The solutions were then transferred into disposable 1.5 mL semi microcuvettes (e.g., disposable or quartz) using disposable transfer pipettes and absorbance spectra were measured from 500 nm to 600 nm using a UV-VIS spectrophotometer.

Normalization of sample readings was performed by subtracting the absorbance measurement of a blank solution (e.g., 0.1 N NaOH) from the 562 nm absorbance of all samples and standards. The slope and intercept of the standard curve was used to determine protein concentration in each measured sample.

Calculations and interpretation

[0624] Prior to data interpretation, measured protein concentrations measured were converted to mass of protein per mass of starting material. This was done by accounting for actual concentrations used when resuspending nanoparticles during preparation, in addition to taking into account the 1 :21 dilution factor from the BCA assay. This correction enables conversion of total protein concentration measured in a BCA assay to a potency value that reports mass of protein per mass of the solid intermediate. Calculation of Safety Factor (SF) also requires normalizing both protein concentrations to a unit mass of solid intermediate. After all transformations and normalizations were completed, SF was calculated using Equation 1. (See Figure 8G for results)

SF— 19

EXAMPLE 8: Quantification of Nanoyarticles on TLR stimulation

[0625] This Example describes an exemplary method of testing toll-like receptor (TLR) stimulation of certain polymer nanoparticles ( e.g polymer nanoparticles comprising a payload and/or a coating) in accordance with the present disclosure by assessing NF-kB activation in HEK293 cells expressing a given TLR.

Materials and Methods & Description

[0626] Toll-Like Receptor (TLR) stimulation is tested by assessing NF-kB activation in

HEK293 cells expressing a given TLR. The activities of the test articles are tested on human TLR4 as potential agonists. The test article was evaluated at eight concentrations, 15,000, 3,750, 937.5, 234.4, 58.6, 14.7, 3.7, and 0.9 ng/mL, and compared to control ligands (see list below). These steps are performed in triplicate. Control Ligand

[0627] hTLR4: E. coli K12 LPS at 1,000, 250, 62.5, 15.6, 3.9, 1.0, 0.24, and 0.06 ng/mL

TLR- Negative Control Cell Line

[0628] HEK293/Null2: Control for human TLR4

[0629] TNFa at 1,000, 250, 62.5, 15.6, 3.9, 1.0, 0.24, and 0.06 ng/mL

Negative Control

[0630] LPS - EK is used in various assays as a negative control. LPS-EK is

lipopolysaccharide (LPS) prepared from E. coli K12. LPS-EK also contains other bacterial components (e.g., lipopeptides) and is able to stimulate both TLR4 and TLR2.

Preparation of Test Articles

[0631] Article 1 was coated nanoparticles prepared according to methods described in the present disclosure. To prepare Article 1 for assay, the following procedure was followed: 1.5 mg/mL solution was prepared by weighing 3 mg of Article I (dry, powdered form) and resuspending in 2 mL of sterile PBS and vortexing; 150 pg/mL solution was prepared by mixing 100 pL of 1.5 mg/mL solution with 900 pL of sterile PBS and vortexing; serial dilutions were prepared by mixing 50 pL of the previous highest dilution with 150 pL sterile PBS.

[0632] Article 2 was heat-killed E.coli , prepared according to the manufacturer’s protocol (InVivogen®).

General Procedure

[0633] A secreted embryonic alkaline phosphatase (SEAP) reporter is under the control of a promoter inducible by the transcription factor NF-kB. This reporter gene allows the monitoring of signaling through the TLR based on the activation of NF-kB. In a 96-well plate (200 pL total volume) containing the appropriate cells (50,000-75,000 cells/well), 20 pL of the test article or the positive control ligand was added to the wells. The media added to the wells is designed for the detection of NF-kB induced SEAP expression. After a 16-24 hr incubation the optical density (OD)was read at 650 nm on a Molecular Devices SpectraMax 340PC absorbance detector.

Dose Response Screening [0634] The Test Articles (i.e., Article 1 and Article 2) were evaluated at eight concentrations and compared to a control ligand LPS-EK tested at concentrations of 1,000, 250,

62.5, 15.6, 3.9, 1.0, 0.24, and 0.06 ng/mL or. a TLR-negative cell line such as HEK293/TLR null, tested with a substrate that activates NF-kB (e.g., TNFoc) at concentrations of 1,000, 250, 62.5,

15.6, 3.9, 1.0, 0.24, and 0.06 ng/mL). All testing steps were performed in triplicate.

Table 11: Test Articles

Results

Dose response curves show that Article 1 had a significant stimulatory effect on human TLR4 at all concentrations tested except 0.9 ng/mL (see Figures 9A and 9D). Dose response curves also show that Article 2 had a significant stimulatory effect on human TLR4 at lxlO⁹, 2.5xl0⁸, 6.3xl0⁷, and l.6xl0⁷ cells/mL with a moderate stimulatory effect at 3.9xl0⁶, and 9.8xl0⁵ cells/mL (see Figures 9B and 9E). Dose response curves of LPS-EK control substrate appear to show a stimulatory effect on human TLR4 at all concentrations tested except for 0.06 ng/mL (see Figures 9C and 9F).

Results of Human TLR4- HEK293-/Null2 Negative Control Dose Response Screening

[0635] Dose response curves for Article 1 (coated nanoparticles) and Article 2

(HKEB)did not appear to show evidence of stimulatory effect, as evidenced by lack of detectable increase in optical density versus negative control (0 cells/mL) on HEK293 cells null for TLR4 (see Figure 10A, which corresponds to averages of screenings 1, 2, and 3 of Article 1 of 10D, and Figure 10B, which corresponds to averages of screenings 1, 2, and 3 of Article 2 of 10E . Figure 10C shows dose response for TNFoc control substrate, which appears to have higher Optical Density values (2.5, approximately 2.4, and 2.0 at concentrations of 1,000, 250, and 62.5 ng/mL TNFoc respectively) than at 15.6 and 3.9 ng/mL (each less than one), with almost no difference in Optical Density detected between 0 ng/mL TNFoc and 0.06, 0.2, and 1.0 ng/mL TNFoc (see Figure 10C, which corresponds to averages of screenings 1, 2, and 3 of TNFoc control ligand).

EXAMPLE 9: Quantification of various nanoparticle preparations on TLR stimulation

[0636] This Example describes an exemplary method of testing TLR stimulation of certain polymer nanoparticles ( e.g polymer nanoparticles comprising a payload and/or a coating) in accordance with the present disclosure by assessing NF-kB activation in HEK293 cells expressing a given TLR. Toll-Like Receptor (TLR) stimulation is tested by assessing NF- kB activation in HEK293 cells expressing a given TLR. The activities of the test articles are tested on human TLR4 as potential agonists. The test articles are evaluated at either eight or three concentrations, and compared to control ligands (see list below). These steps are performed in triplicate.

Control Ligand

[0637] hTLR4: E. coli K12 LPS; concentrations used in 8-point dose response: 100, 50,

20, 10, 5, 2, 1, 0.5 ng/mL; concentrations used in 3-point dose responses: 3, 1, and 0.5 ng/mL; 2, 1, 0.5 ng/mL; 20, 10, 5 ng/mL.

TLR4 Negative Control Cell Line

[0638] HEK293/Null2: TNFa; concentrations used in 8-point dose response: 100, 50, 20,

10, 5, 2, 1, 0.5 ng/mL; concentrations used in 3-point dose responses: 3, 1, and 0.5 ng/mL; 2, 1, 0.5 ng/mL; 20, 10, 5 ng/mL.

Table 12: Test Articles

Test articles started as dry powders.

Preparation of Test Articles Part 1: [0639] Test articles A, D and G were weighed out and resuspended in sterile PBS to generate 1 mg/mL stock solutions. Solutions of 30, 10 and 5 ng/mL were prepared for articles A, D and G

Part 2:

[0640] Test articles E and F were weighed out and resuspended in sterile PBS to generate

1 mg/mL stock solutions. Solutions of 1,000, 500, 200, 100, 50, 20, 10, 1 and 5 ng/mL were prepared for articles E and F.

Part 3:

[0641] After obtaining results from the dose responses of article E, the following concentrations for the remaining supernatant samples (Articles B and H) were selected for testing: 2, 1 and 0.5 ng/mL. After obtaining results from the dose responses of article F, the following concentrations for the remaining pellet samples (Articles C and I) were selected for testing: 20, 10 and 5 ng/mL. Test articles B, C, H and I were weighed out and resuspended in sterile PBS to generate 1 mg/mL stock solutions. Solutions of 20, 10 and 5 ng/mL were prepared for articles B and H. Solutions of 200, 100 and 50 ng/mL were prepared for articles C and I.

General Procedure

[0642] The secreted embryonic alkaline phosphatase (SEAP) reporter is under the control of a promoter inducible by the transcription factor NF-kB. This reporter gene allows the monitoring of signaling through the TLR based on the activation of NF-kB. In a 96-well plate (200 pL total volume) containing the appropriate cells (50,000-75,000 cells/well), 20 pL of the test article or the positive control ligand is added to the wells. The media added to the wells is designed for the detection of NF-kB induced SEAP expression. After a 16-24 hr incubation the optical density (OD) is read at 650 nm on a Molecular Devices SpectraMax 340PC absorbance detector.

Results of Human TLR4 Dose Response Screening

[0643] As a surrogate for TLR4-mediated activity, measurements of NF-kB inducible secreted embryonic alkaline phosphatase (SEAP) were performed on TLR4-positive HEK293 cells exposed to Articles A- 1 at pre-centrifugation, supernatant and pellet stages and compared to LPS-EK. Figure 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 111). Figure 11J 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 HR shows results of screenings 1,

2, 3, and fold induction.

[0644] As a surrogate for TLR4-mediated activity, measurements of NF-kB inducible secreted embryonic alkaline phosphatase (SEAP) were performed on TLR4-positive HEK293 cells exposed to Articles A- 1 at pre-centrifugation, supernatant and pellet stages. Figure 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). 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).

Results of Human TLR4 HEK293/Null2 Negative Control Dose Response Screening:

[0645] As a control for TLR4-mediated activity, measurements of NF-kB inducible secreted embryonic alkaline phosphatase (SEAP) were performed on TLR4 HEK293/Null2 negative cells exposed to Articles A- 1 at pre-centrifugation, supernatant and pellet stages.

Figures 13A-13S show NF-KB inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene expression in TLR4 HEK293/Null2 negative control cells. Figure 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 TNFD : control HEK293/Null2 tested at eight concentrations. Figure 13Q shows a dose response curve of TNFD : 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.

[0646] As a control for TLR4-mediated activity, measurements of NF-kB inducible secreted embryonic alkaline phosphatase (SEAP) were performed on TLR4 HEK293/Null2 negative cells exposed to Articles A- 1 at pre-centrifugation, supernatant and pellet stages.

Figure 14A shows dose response curves for increasing concentrations of Article A (pre- centrifugation; raw data and TNFa fold change shown in Figure 14B), Article B (supernatant; raw data and TNFa fold-change shown in Figure 14C) and Article C (pellet; raw data and TNF fold-change shown in Figure 14D). 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).

[0647] Overall Results of screening for TLR4 activity show that Article A (Pre-

Centrifugation) had a significant stimulatory effect on human TLR4 at 3 ng/mL, with moderate stimulatory effects at 1 and 0.5 ng/mL (see Figs. 11 A, 12A, 13A, and 14A). Article B

(Supernatant) showed significant stimulatory effects on human TLR4 at 2, 1, and 0.5 ng/mL(see Figs. 11F, 12A, 12C, 13F, 14A, and 14C). Article C (Pellet) showed significant stimulatory effect on human TLR4 at 20 ng/mL, with moderate stimulatory effects at 10 and 5 ng/mL(see Figs. 11K, 12A, 12D, 13K, 14A, and 14D). Article D: Pre-Centrifugation showed a significant stimulatory effect on human TLR4 at 3 ng/mL, with moderate stimulatory effects at 1 and 0.5 ng/mL(see Figs. 11 A, 12E, 12F, 13A, 14E, and 14F). Article E: Supernatant showed significant stimulatory effects on human TLR4 at 100, 50, 20, 10, 5, 2, 1, and 0.5 ng/mL(see Figs. 11F, 12E, 12G, 13F, 14E, and 14G). Article F: Pellet showed significant stimulatory effects on human TLR4 at 100, 50, 20 and 10 ng/mL, with moderate stimulatory effects at 5 and 2 ng/mL, and slight stimulatory effects at 1 and 0.5 ng/mL(see Figs. 11K, 11P, 12E, 12H, 13K, 14E, 14H). Article G: Pre-Centrifugation showed no stimulatory effect on human TLR4(see Figs. 11 A, 12G, 121, 12 A, 14E, and 141). Article H: Supernatant showed no stimulatory effect on human

TLR4(see Figs. 11F, 12G, 12J, 13F, 141, and 14K). Article I: Pellet showed no stimulatory effect on human TLR4(see Figs. 11K, 12G, 12K, 13K, 141 and 14L).

[0648] Overall results from Examples 8 and 9 demonstrate that 5 ng/ml of nanoparticles produced in accordance with the present disclosure produced the same TLR4-reporter signal as 2C10^L6 E. coli. The number of nanoparticles per gram is ~6C10^L13 (5 ng of nanoparticles there are ~3C10^L5 nanoparticles). Thus, TLR4 ligand activity in 3X10^L5 nanoparticles equals that in 2C10^L6 E. coli (1 nanoparticle = 6.7 E. coli with regard to its TLR4 ligand content). The data show that differences in OEE preparations alter TLR4 ligand content, such that preparation via lyophlization results in approximately 1 nanoparticle = 3.3 E. Coli. Further, 5 ng/mg

nanoparticles were tested and showed that nanoparticles contain 5.5 ng of LPS for every 1 ng of peanut protein (ratio of LPS to antigen protein on a weight/weight basis is 5.5: 1).

Example 10: Comparison of manufacturing protocols with and without rotary evaporation

[0649] In order to determine whether rotary evaporation of water from a

payload/polymer solution (e.g., Figure 8A, steps 1-3) would improve nanoparticle

characteristics, manufacturing was performed with and without a rotary evaporation step (see Figure 8A for identification of where rotary evaporation was added). Protein per mass of nanoparticles was determined to be higher in manufacturing that used rotary evaporation as compared to manufacturing without rotary evaporation.

[0650] A comparison of manufacturing of nanoparticles with and without rotary evaporation (“rotovap”)was performed as follows:

Samples: 1. BREC 2020-087 A - Lyo 1 without rotovap (see, e.g., Figure 8A but without rotovap step)

2. BREC 2020-087 B - Lyo 1 with rotovap; nanoparticles formed using the same process and purification as -087A (see, e.g., Figure 8A)

3. BREC 2020-087 C - Lyo 1 without rotovap; nanoparticles formed using a different homogenization process than -087 A and B

4. BREC 2020-087 D - Lyo 1 with rotovap; nanoparticles formed using the same process as -087C (see, e.g., Figure 8A)

5. Reference for analytical testing: BREC 0987-015

Methods:

[0651] Two lots of nanoparticles were formed by almost identical processes except that lot A did not receive treatment with rotovap and lot B did. Total protein content of purified nanoparticles was found to be higher in lot B (66 mg protein / mg nanos) than lot A (46 mg protein / mg nanos). When“free protein” was separated from encapsulated protein, 65 mg protein / mg nanoparticles remained in lot B versus 45 mg protein / mg nanoparticles in lot A.

[0652] In addition, Lyo 1 lots used to create lot A and lot B (described above) were processed into nanoparticles using a different variation on the homogenization process. Although the protein encapsulation is much lower than the A and B lots, it was again seen that the sample that was rotovapped had measurably higher protein encapsulation than the sample that was not. Lot D (rotovapped) had 23 mg protein / mg nanoparticles (22 mg protein / mg nanos after removal of free protein) whereas Lot C (not rotovapped) had 12.4 mg protein / mg nanos (12.0 mg protein / mg nanos after removal of free protein).

[0653] A consistent trend was observed in nanoparticles generated with and without rotovap, in that rotovap appeared to improve protein encapsulation, however, it should also be noted that another change in procedure (versus that described in Figure 8A) was made, by inserting a step of tangential flow filtration following low speed centrifugation, which appear to improve yield of nanoparticles with higher payload: polymer ratios than were being obtained prior to introduction of centrifugation and/or tangential flow filtration. [ANY FIGURES

HERE?]

Example 11: Characterization of Nanoparticles by Dynamic Light Scattering [0654] Dynamic light scattering was used to assess solutions from various steps of the process shown in Figure 8A. Dynamic light scattering was used to compare nanoparticles produced under varying conditions and from various steps in the process of 8A. Nanoparticles of approximately 114 nm were detected, but determined to be difficult to collect using only centrifugation. To determine an optimized approach to collecting nanoparticles, including nanoparticles of desirable size and/or protein concentration, centrifugation times were altered and varied 0.5 to 5.5 hours at 5300 ref. Supernatants were sampled throughout the range of time, and particle sizes measured using dynamic light scattering. The mean hydrodynamic diameter dropped, gradually from 180 nm to 140 nm, from 0.5 to 2 hours. At the end of 5.5 hours, the mean hydrodynamic diameter was about 125 nm. The originally detected 114 nm nanoparticles were not detected, thus a tangential flow filtration step was added, and particles within a range of 100-200 nm were captured.

Claims

CLAIMS What is claimed is:

1. A method comprising

combining a hydrophilic payload and a polymer that is not soluble in the same solvent as the hydrophilic payload together in a solvent system characterized in that a mixture of the hydrophilic payload, the polymer and the solvent system is generated; and lyophilizing the mixture to form a lyophilized cake.

2. The method of claim 1, wherein prior to generation of the lyophilized cake, the solvent system is subjected to a concentration step to remove at least one of water and solvent prior to lyophilization.

3. The method of claim 2, where concentration comprises evaporation.

4. The method of claim 2, wherein the concentration step removes at least some water and at least some solvent.

5. The method of claim 2, wherein the concentration step removes substantially all of at least one of water and solvent.

6. The method of claim 1, wherein the hydrophilic payload is selected from the group consisting of a protein, a nucleic acid, an antigen, and combinations thereof.

7. The method of any one of the preceding claims, wherein the weight ratio of the hydrophilic payload to polymer is within a range of 1 :99 to 20:80, or 1 :99 to 10:90.

8. The method of any one of the preceding claims, wherein the solvent system comprises an aqueous solution.

9. The method of claim 8, wherein the aqueous solution comprises water and DMSO.

10. The method of any one of the preceding claims, wherein the polymer is hydrophobic.

11. The method of any one of the preceding claims, wherein the polymer has a molecular weight within a range of 5,000-5,000,000 Daltons.

12. The method of any one of the above claims, further comprising

exposing the lyophilized cake to a temperature sufficient to melt the polymer to form a melted cake; and

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.

13. The method of any one of claims 1-12, further comprising

grinding the lyophilized cake and resuspending the ground cake in at least one alcohol.

14. The method of claim 13, wherein the grinding occurs in the presence of liquid nitrogen.

15. The method of claim 13 or 14, wherein the at least one alcohol is or comprises propanol.

16. The method of any one of claims 12-15, wherein the block material has a substantially uniform distribution of the hydrophilic payload with respect to the polymer.

17. A method of making a flowable microparticle suspension comprising:

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.

18. The method of claim 17, wherein the lyophilized cake or the 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 l5°C, about 0 to 5°C, or about 5 to l5°C.

19. The method of claim 17 or claim 18, wherein the lyophilized cake or the block material is comminuted by using a mortar and pestle.

20. The method of any one of claims 17-19, wherein the non-solvent system comprises water and alcohol.

21. The method of any one of claims 17-20, wherein the microparticles have a size within a range of about 10 pm to about 800 pm, about 50 pm to about 800 pm, about 100 pm to about 800 pm, or about 100 pm to about 500 pm.

22. A method of making nanoparticles comprising

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.

23. The method of claim 22, wherein the nanoparticles have a mean size within a range of approximately 100-500 nm.

24. The method of claim 23, wherein a portion of the nanoparticles has a mean size within a range of approximately 100-300 nm, and a different portion of the nanoparticles has a mean size within a range of approximately 300-500 nm.

25. The method of claim 22, wherein the flowable microparticle suspension is passed through a microfluidizer two or more times, so that the nanoparticles have substantially uniform size.

26. The method of claim 25, wherein the microfluidizer applies a shear gradient to the flowable microparticle suspension.

27. The method of claim 26, wherein the shear gradient is within a range of 10 ⁶ to 10 ⁷ s ¹.

28. The method of any one of claims 22-27, wherein the flowable microparticle suspension is homogenized at a temperature within a range of about 80°C to 1 lO°C, about 85°C to 1 lO°C, about 90°C to 1 lO°C, about 80°C to l05°C, about 80°C to l00°C, or about 90°C to l00°C.

29. The method of any one of claims 22-28, wherein the method further comprises adding a stabilizing agent solution to the nanoparticle suspension.

30. The method of any one of claims 22-29, further comprising applying a preparation of one or more coating agents to the nanoparticles.

31. The method of claim 30, wherein the preparation of coating agents comprises at least one microbial extract.

32. A method comprising

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.

33. The method of claim 32, wherein the nanoparticles have a mean size within a range of approximately 100-500 nm.

34. The method of claim 33, wherein a population of the 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.

35. The method of any one of claims 32-34, wherein the hydrophilic payload is selected from the group consisting of a protein, a nucleic acid, an antigen, and combinations thereof.

36. The method of any one of claims 32 -35, wherein the weight ratio of the hydrophilic payload to polymer is within a range of 1 :99 to 20:80, or 1 :99 to 10:90.

37. The method of any one of claims 32-36, wherein the solvent system comprises an aqueous solution.

38. The method of claim 37, wherein the aqueous solution comprises water and DMSO.

39. The method of claim 9 or claim 38, wherein a volume ratio of water and DMSO is within a range of 1 :99 to 20:80, or 1 :99 to 10:90.

40. The method of any one of claims 32-39, wherein the polymer is hydrophobic.

41. The method of any one of claims 32-40, wherein the polymer has a molecular weight within a range of 5,000 - 5,000,000 Daltons.

42. The method of any one of claims 32-41, wherein the lyophilized cake has a substantially uniform distribution of the hydrophilic payload with respect to the polymer.

43. The method of any one of claims 32-42, wherein the lyophilized cake is comminuted 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.

44. The method of any one of claims 32-43, wherein the lyophilized cake is comminuted by using a mortar and pestle.

45. The method of any one of claims 32-44, wherein the non-solvent system comprises water and alcohol.

46. The method of any one of claims 32-45, wherein the 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.

47. The method of any one of claims 32-46, wherein the flowable microparticle suspension is passed through the microfluidizer two or more times, so that the nanoparticles have substantially uniform size.

48. The method of any one of claims 32-47, wherein the microfluidizer applies shear gradient to the flowable microparticle suspension.

49. The method of any one of claims 32-48, wherein the shear gradient is within a range of 10 ⁶ to 10 ⁷ s ¹.

50. The method of any one of claims 32-49, wherein the flowable microparticle suspension is homogenized at a temperature within a range of about 80°C to 1 l0°C, about 85°C to 1 l0°C, about 90°C to 1 l0°C, about 80°C to l05°C, about 80°C to l00°C, or about 90°C to l00°C.

51. The method of any one of claims 32-50, wherein the method further comprises lyophilizing the stabilizing agent solution and the nanoparticle suspension prior to applying the preparation of the coating agents to the nanoparticles.

52. The method of any one of claims 32-51, wherein the preparation of coating agents comprises at least one microbial extract.

53. The method of any one of claims 32-52, further comprising centrifugation of the

nanoparticle suspension.

54. The method of claim 53, wherein the centrifuge speed is between 300 and 600 xg.

55. The method of claim 53, wherein the supernatant from the centrifuged solution is further subjected to a step of tangential flow filtration.

56. The method of claim 55, wherein the supernatant is mixed with a solution comprising the coating agent after centrifugation.

57. A composition comprising

a plurality of nanoparticles, each of which is comprised of a polymer and a hydrophilic payload substantially uniformly disposed within the polymer,

58. The composition of claim 57, wherein the plurality of nanoparticles has a mean size within a range of 450 nm or less.

59. The composition of claim 57, wherein the plurality of nanoparticles has a mean size within a range of 100-500 nm, 300-500 nm, 100-300 nm, or 100-250 nm.

60. The composition of claim 57, wherein the plurality of nanoparticles is comprised of at least two populations of nanoparticles, each with a different mean size.

61. The composition of claim 60, wherein at least one population of nanoparticles has a mean size within a range of approximately 300-500 nm.

62. The composition of claim 60, wherein at least one population of nanoparticles has a mean size within a range of approximately 100-300 nm.

63. The composition of any one of claims 57-62, further comprising centrifugation of the nanoparticle suspension.

64. The composition of claim 63, wherein the centrifugation speed is between 300 and 600 xg.

65. The composition of claim 63, wherein prior to centrifugation, the nanoparticle suspension comprises nanoparticles with a mean size within in a range of approximately 100-500 nm, and after centrifugation comprises nanoparticles with a mean size within in a range of approximately 100-300 nm.

66. The composition of claim 63, wherein a supernatant from the centrifuged suspension is further subjected to a step of tangential flow filtration.

67. The composition of claim 66, wherein the solution subjected to tangential flow filtration is mixed with a solution comprising the coating agent after centrifugation.

68. The composition of any one of claims 57-67, wherein the hydrophilic payload is selected from the group consisting of a protein, a nucleic acid, an antigen, and combinations thereof.

69. The composition of any one of claims 57-68, wherein 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.

70. The composition of claims 57-69, wherein the polymer is hydrophobic.

71. The composition of claims 57-70, the polymer has a molecular weight within a range of 5,000-5,000,000 Daltons.

72. The composition of any one of claims 57-71, further comprising at least one coating agent.

73. The composition of claim 72, wherein the at least one coating agents comprises at least one microbial extract.

74. A method comprising 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,

75. The method of claim 74, wherein the at least one coating agent comprises at least one microbial extract.

76. The method of claim 74 or claim 75, wherein the hydrophilic payload comprises an antigen.

77. The method of claim 76, wherein the antigen is or comprises an allergic antigen

78. The method of claim 76, wherein the antigen is or comprises an anaphylactic antigen.

79. The method of claim 76, wherein the antigen is or comprises an infectious antigen.

80. The method of claim 76, wherein the antigen is or comprises an autoantigen.

81. The method of claim 76, wherein the antigen is or comprises a disease-associated antigen.

82. The method of any one of claims 74-81, wherein the subject is suffering from at least one of allergy, infection, and cancer.

83. The method of any one of claims 74-82, wherein the nanoparticle composition is

administered intravenously, intradermally, transdermally, orally, subcutaneously, and/or transmucosally.

84. The method of claim 83, wherein the transmucosal administration is buccal, nasal, bronchial, vaginal, rectal, and/or sublingual administration.

85. A composition comprising

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

86. A composition comprising

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.

87. The composition of claim 85 or 86, wherein 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.

88. The composition of any one of claims 85-87, wherein the polymer is hydrophobic.

89. The composition of any one of claims 85-88, the polymer has a molecular weight within a range of 5,000-5,000,000 Daltons.

90. The composition of any one of claims 85-89, further comprising at least one coating agent.

91. The composition of claim 90, wherein the at least one coating agents comprises at least one microbial extract.

92. A method comprising administering to a subject in need thereof a nanoparticle composition comprising:

93. The method of claim 92, wherein the at least one coating agent comprises at least one microbial extract.

94. The method of claim 92 or claim 93, wherein the hydrophilic payload comprises an antigen.

95. The method of claim 94, wherein the antigen is or comprises an allergic antigen

96. The method of claim 94, wherein the antigen is or comprises an anaphylactic antigen.

97. The method of claim 94, wherein the antigen is or comprises an infectious antigen.

98. The method of claim 94, wherein the antigen is or comprises an autoantigen.

99. The method of claim 94 wherein the antigen is or comprises a disease-associated antigen.

100. The method of any one of claims 92-99, wherein the subject is suffering from at least one of allergy, infection, and cancer.

101. The method of any one of claims 92-100, wherein the nanoparticle composition is administered intravenously, intradermally, transdermally, orally, subcutaneously, and/or transmucosally.

102. The method of claim 101, wherein the transmucosal administration is buccal, nasal, bronchial, vaginal, rectal, and/or sublingual administration.