Nanoparticle manufacturing

Abstract

The present disclosure provides nanoparticle compositions in which individual nanoparticles comprise payloads, 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

Docket No.: 2006517-0315 NANOPARTICLE MANUFACTURING 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 a payload (e.g., a therapeutic or diagnostic agent). Diverse approaches have been applied in order to produce polymer nanoparticles containing a payload. However, improved approaches are both desirable and needed. SUMMARY

[0002] The present disclosure provides nanoparticle compositions in which individual nanoparticles comprise polymers and (one or more) payloads, and optionally may comprise a coating, etc.; the present disclosure also provides various technologies for making, using, and / or characterizing such nanoparticle compositions (and / or components and / or intermediates thereof).

[0003] 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.) and / or one or more fragile payloads (e.g., that maybe particularly susceptible to degradation and / or inactivation, etc. when exposed to particular conditions; in some embodiments, nucleic acids, particularly RNA and most particularly longer RNAs such as mRNAs, polypeptides, and / or saccharides may be considered to be “fragile” payloads).

[0004] Alternatively or additionally, according to various embodiments, provided technologies display certain advantages and / or solve one or more problems associated with conventional nanoparticle technologies, specifically including certain nanoparticle manufacturing technologies.

[0005] 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) that involve few steps and / or particularly gentle reagents and Page 1 of 340 12613923v1Docket No.: 2006517-0315 conditions. In some embodiments, provided manufacturing technologies involve simple procedures requiring fewer steps than conventional nanoparticle production methods. In some embodiments, provided manufacturing technologies do not involve harsh manufacturing conditions (e.g., high temperature, pressure and / or shear force). Among other things, therefore, the present disclosure provides technologies that may be particularly suitable and / or may offer particular advantages, for use with fragile (e.g., susceptible to damage from energy input and / or exposure to during manufacturing) and / or complex payloads (e.g., wherein individual nanoparticles may contain one or more payloads and / or may be coated with one or more coating agents, and further wherein one or more such payloads and / or coating agents may itself be or comprise a complex [e.g., multi-component, and / or crude] composition). In some embodiments, technologies described herein may be particularly useful for the manufacturing of nanoparticles (e.g., comprised of polymers and including payloads and / or coating agents as described herein) at a greater scale than conventional methodologies.

[0006] 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).

[0007] In some embodiments, the present disclosure provides insights regarding material behaviors at fluid interfaces and provide strategies for harnessing such insights in production technologies. For example, in certain embodiments, the present disclosure provides and / or utilizes mild mixing at a fluid interface of a heterogeneous, layered two-fluid system. The present disclosure teaches that mixing which is (a) mild, as described herein; and / or (b) occurs at the interface can be particularly useful and effective and can overcome various challenges otherwise encountered in nanoparticle production. Moreover, the present disclosure demonstrates that such processes can achieve surprising results, including for example, remarkable uniformity in nanoparticle production (e.g., in average size and / or in polydispersity (see, for example, Example 10, which exemplifies a heterogeneous, layered two-fluid process referred to herein as a “Tequila Sunrise” process).

[0008] In some embodiments, provided technologies achieve nanoparticle compositions in which payloads are not significantly surface exposed (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 Page 2 of 340 12613923v1Docket No.: 2006517-0315 encapsulated payloads are substantially wholly encapsulated. In some embodiments, payloads are considered to be “encapsulated” when they are not detectable as “free” (e.g., when nanoparticles have not been disrupted); in some such embodiments, at least 80% or at least 85% or at least 90% of payload is encapsulated. For example, in some embodiments, assessment of total payload utilized in nanoparticle manufacturing, and of “free” payload detectable when nanoparticles have not been disrupted reveal that no more than about 10%, 15%, or 20% of the total payload is detected as “free” payload.

[0009] In various embodiments, provided technologies embody, permit, and / or achieve one or more surprising features such as, for example, efficiency and / or simplicity of nanoparticle production, conditions amenable to fragile payloads, production of complex nanoparticle compositions (e.g., wherein individual nanoparticles may contain one or more payloads and / or may be coated with one or more coating agents, and further wherein one or more such payloads and / or coating agents may itself be or comprise a complex [e.g., multi-component, and / or crude] composition), production of nanoparticle compositions characterized by desirable attributes such as, for example, one or more of desirable particle average size and / or size distribution, particular zeta potential, particular immunomodulatory effects, particular release characteristics, etc.

[0010] In some embodiments, provided nanoparticle compositions can achieve immune modulation. For example, among other things, the present disclosure documents stimulation of Th1-type immune reactions with nanoparticle compositions containing lipids (e.g., an E. coli lipid extract and / or lipopolysaccharide) on their surfaces. Without wishing to be bound by any particular theory, we propose that such nanoparticle compositions may be viewed by a recipient’s immune system as analogous to bacterial agents (e.g., to bacterial cells). The present disclosure proposes and demonstrates that this ability to direct a Th1-type immune response to an administered nanoparticle composition presents an opportunity to shift or otherwise bias a recipient’s immune response to one or more antigens included in the nanoparticle composition toward such a Th1-type response; such an effect is particularly useful in the treatment of allergy to an encapsulated allergen. The present disclosure documents effectiveness of this approach with encapsulated peanut allergens, which are well known to trigger particularly potent (e.g., anaphylactic) immune responses in certain allergic individuals. Those skilled in the art, reading the present disclosure, therefore, will appreciate that success encapsulating peanut allergens (including in the form of a crude peanut extract), and furthermore modulating a subject’s Page 3 of 340 12613923v1Docket No.: 2006517-0315 immune response to them, provides significant evidence that comparable results can be achieved with other allergens (including specifically other food allergens, but also allergens generally.

[0011] In some embodiments, provided nanoparticle compositions may be used as and / or incorporated into pharmaceutical composition(s) (e.g., into dosage forms); in some embodiments, provided nanoparticle compositions are amenable to formulation for delivery via any of a variety of routes such as, for example, mucosal, oral, parenteral, topical and / or transdermal etc.

[0012] In particular embodiments, provided nanoparticles are amenable to oral administration and / or are administered orally e.g., into the mouth. In some embodiments, provided nanoparticles are amenable to mucosal delivery and / or are administered buccally (e.g., via the oral mucosa). In some embodiments, provided nanoparticles are administered sublingually.

[0013] In some embodiments, provided nanoparticle compositions are suitable for formulation into any of a variety of liquid, solid, or gel compositions including, for example, dispersions, emulsions, solutions, etc., tablets, capsules, gums, lozenges, suppositories, etc.; and / or incorporation into any of a variety of devices such as, for example, patches, syringes, etc.

[0014] In some embodiments a device (e.g., a patch, roller, etc.) may be or comprise a plurality of needles (e.g., microneedles).

[0015] In some embodiments a formulation comprising nanoparticles as described herein may be stable to storage. In some embodiments, stability to storage refers to stability of the nanoparticle structure of the formulation – e.g., as may be reflected, for example, in one or more characteristics such as average particle size, polydispersity etc. Alternatively or additionally, in some embodiments, stability to storage refers to stability of individual nanoparticles, e.g., their ability to encapsulate payload, such as an allergen.

[0016] In some embodiments, a formulation comprising nanoparticles may be stored at room temperature. In some embodiments, such a formulation may be stored under cooling, e.g., at a temperature below about 4oC. In some embodiments, such a formulation may be stored at a temperature at or below about 0oC, -20oC, -80oC, -196oC, etc. (i.e., under freezing conditions). In some embodiments, a formulation comprising nanoparticles may be stored in a conventional freezer; in some embodiments, such freezer may undergo periodic defrost cycles.

[0017] In some embodiments, the present disclosure provides nanoparticle compositions including at least one antigen substantially co-localized with at least one adjuvant agent; in some Page 4 of 340 12613923v1Docket No.: 2006517-0315 embodiments, provided nanoparticle compositions are characterized in that administration to a subject in need thereof achieves a desired immunological effect in the subject with respect to the antigen. In some embodiments, provided nanoparticle compositions may be designed and constructed to deliver both an antigen and an adjuvant to a population of a subject’s cells (e.g., immune cells, [e.g., antigen presenting cells (“APCs”)]).

[0018] In various embodiments, the present disclosure provides technologies for manufacturing a population of nanoparticles.

[0019] In some embodiments, provided manufacturing technologies comprise steps of (i) providing a first preparation, which comprises a payload (e.g,. a hydrophilic payload) in a first aqueous solvent system and a second preparation, which comprises a polymer (e.g., a hydrophobic polymer) in a second solvent system, wherein the second solvent system is non- aqueous, and the polymer is not fully (and in many embodiments is not significantly) soluble in the first aqueous solvent system; (ii) combining the first and second preparations to form a mixture that comprises the payload and the polymer in a combined solvent system; and (iii) adding a non-solvent system to the mixture, so that a population of nanoparticles comprising the payload and the polymer is formed in a nanoparticle suspension (e.g., wherein the non-solvent system is a non-solvent of the polymer and the payload) (e.g., wherein the non-solvent system precipitates the payload and the polymer, so that each of the nanoparticles comprises the payload and the polymer).

[0020] In some embodiments, the present disclosure provides technologies in which a nanoparticle preparation is manufactured by (i) providing a first liquid preparation, which comprises a payload (e.g., a fragile payload) in a first aqueous solvent system and a second liquid preparation, which comprises a polymer (e.g., a hydrophobic polymer) in a second solvent system; (ii) combining the first and second preparations to form a mixture that comprises the payload and the polymer in a combined solvent system, and (iii) adding a liquid non-solvent system to the mixture, so that a population of nanoparticles comprising the payload and the polymer is formed (e.g., wherein the method does not involve energy input) (e.g., wherein the non-solvent system does not significantly degrade the payload, or decrease one or more biological or pharmaceutical activities of the payload) (e.g., wherein one or more biological or pharmaceutical activities of fragile payload are substantially same before and after the step of adding). Page 5 of 340 12613923v1Docket No.: 2006517-0315

[0021] Alternatively or additionally, in some embodiments, the present disclosure provides manufacturing technologies that include mild mixing at a fluid interface (e.g., in a heterogeneous, layered two-fluid system).

[0022] In some embodiments, the present disclosure provides nanoparticle manufacturing technologies in which (i) payload materials and polymer materials are combined in the presence of a solvent / antisolvent system; typically at least the payload material(s) are sufficiently hydrophilic to be provided in water or other aqueous system (the present disclosure provides an insight that use of an organic antisolvent can reduce payload loss during the encapsulation process); and (ii) combined materials are mixed in an intentionally heterogeneous, layered two- fluid process, that typically involves mild mixing (quite different from conventional teachings of desirability or even necessity of intense mixing to homogenize a solvent / antisolvent mixture) at the fluid interface. The present disclosure demonstrates that this approach achieves surprising and remarkable consistency in nanoparticle size (e.g., average size) and / or polydispersity. Furthermore, the approach is scalable and, thanks to its gentle conditions, is particularly useful for the incorporation of fragile payloads (e.g., nucleic acid, polypeptide and / or carbohydrate (e.g., polysaccharide) payloads).

[0023] In certain embodiments, provided nanoparticle manufacturing technologies may include one or more homogenization steps.

[0024] In certain embodiments, provided nanoparticle manufacturing technologies may utilize one or more stabilizers. For example, in some embodiments, deoxycholate may be utilized (e.g., being included at least in a homogenization step).

[0025] In some embodiments, provided nanoparticle manufacturing technologies may include one or more concentration and / or purification steps. In some embodiments, provided technologies utilize one or more tangential flow filtration (TFF) steps. Among other things, the present disclosure provides an insight that, particularly when TFF is utilized, if a stabilizing agent is desired, deoxycholate is a particularly useful stabilizing agent (and / or that other standard stabilizing agents, such as polyvinyl alcohol, PVA, may be less useful or not useful and, in fact, may damage a TFF membrane.

[0026] In some embodiments, provided nanoparticle manufacturing technologies achieve a ratio of payload to polymer in the nanoparticles that is between about 0.1 to about 0.9 of the ratio of payload to polymer in the original mixture from which nanoparticles are precipitated. Page 6 of 340 12613923v1Docket No.: 2006517-0315

[0027] As noted, in some embodiments, provided technologies include one or more steps that remove solvent (e.g., the combined solvent / antisolvent system).

[0028] In some embodiments, provided manufacturing technologies utilize a stabilizing agent. In some embodiments, a stabilizing may be or comprise PVA. In some embodiments, however, particularly when one or more TFF steps is utilized, PVA is not used. In some embodiments, particularly in embodiments that utilize one or more TFF steps, deoxycholate is utilized as a stabilizing agent.

[0029] In some embodiments, provided manufacturing technologies include one or more steps of purifying nanoparticles (e.g., by one or more of filtration, (e.g., tangential flow filtration), sonication, dilution).

[0030] In some embodiments, provided technologies include one or more steps of drying nanoparticles.

[0031] In some embodiments, a nanoparticle preparation in accordance with the present disclosure (e.g., manufactured as described herein) has a mean size within a range of approximately 100-500 nm. In some embodiments, mean size is within a range of about 225 nm to about 450 nm. In many embodiments, mean size is determined by dynamic light scattering.

[0032] In some embodiments, a provided nanoparticle preparation has a mean diameter within a range of about 50 nm to about 150 nm.

[0033] In some embodiments, provided nanoparticle compositions include a payload selected from the group consisting of polypeptides, nucleic acids, carbohydrates (e.g., polysaccharides), and combinations thereof. In many embodiments, a payload is or comprises a polypeptide. In many embodiments, a payload is or comprises a nucleic acid; in some such embodiments, a payload is or comprises a long nucleic acid (e.g., a gene therapy vector, an mRNA, etc); in some embodiments, a payload is or comprises a partly or wholly single stranded nucleic acid).

[0034] In some embodiments, a payload is or comprises a RNA. In some embodiments, and RNA payload is an mRNA. In some embodiments, an RNA payload has a length within a range of about 15 to about 3,000,000 residues. In some embodiments, an RNA payload has a length within a range of about 500 to 50000 residues. In some embodiments, an RNA payload has a length within a range of about 1000 to about 10000 residues. In some embodiments, an RNA payload is an mRNA encoding a polypeptide having a length within a range of about 50 to about 5000 amino acids; in some embodiments, such encoded polypeptide has a length within a range Page 7 of 340 12613923v1Docket No.: 2006517-0315 of about 100 to about 3000 amino acids; in some embodiments, such encoded polypeptide polypeptide has a length within a range of about 200 to about 1500 amino acids.

[0035] In some embodiments, a provided nanoparticle preparation is manufactured using one or more relatively complex components (e.g., a payload that is a relatively crude extract or combination of components).

[0036] In some embodiments, a payload is or comprises one or more antigens. In some embodiments, an antigen is an allergic antigen, an infectious antigen, and / or a disease-associated (e.g., a cancer-associated) antigen.

[0037] In some embodiments, provided manufacturing technologies utilize a solvent system that comprises water and DMSO. In some such embodiments, a volume ratio of water and DMSO is within a range of 1:99 to 20:80, or 1:99 to 10:90.

[0038] In some embodiments, provided technologies utilize an anti-solvent system (which may in some embodiments be referred to as a non-solvent system). In some embodiments, an anti- solvent system is or comprises an alcohol. In some embodiments, an anti-solvent system is or comprises propanol, ethanol, methanol, or combination thereof. In some embodiments, an anti- solvent is or comprises IPA.

[0039] In some embodiments, provided nanoparticles include (e.g., are manufactured) from a polymer that is or comprises Poly (lactic-co-glycolic acid) (PLGA or PLG) .

[0040] In some embodiments, provided nanoparticles utilize (e.g., are manufactured from) a polymer preparation (e.g., a PLG preparation) where the polymer has a molecular weight within a range of 5,000 – 5,000,000 Daltons.

[0041] In some embodiments, a provided nanoparticle composition includes one or more payloads on (e.g., in some embodiments attached to; in some embodiments otherwise associated) nanoparticle surface(s).

[0042] In some embodiments, the present disclosure provides vaccine compositions comprising a nanoparticle population comprising one or more payloads, or precursor(s) thereof, that activate antigen-specific T cells, for example wherein such one or more payloads is / are displayed (or, if a nucleic acid, may encode an agent that is displayed) by an MHC class I complex or an MHC class II complex.

[0043] In some embodiments, a provided vaccine composition comprises an immune adjuvant. In some embodiments, an immune adjuvant is provided from one or more bacterial sources. In Page 8 of 340 12613923v1Docket No.: 2006517-0315 some embodiments, an immune adjuvant comprises a cellular lysate (e.g., microbial lysate) or a cellular lysate fraction. In some embodiments, an immune adjuvant is a mucosal immune adjuvant.

[0044] In some embodiments, a provided vaccine comprises a pore forming toxin.

[0045] In some embodiments, the present disclosure provides a vaccine composition comprising first and / or second nanoparticle populations, wherein the first nanoparticle population comprises a first payload, or precursor(s) thereof, that activates first antigen-specific T cells; and the second nanoparticle population comprises a second payload, or precursor(s) thereof, that activates second antigen-specific T cells. In some embodiments, the first payload is displayed by (or encodes an agent that is displayed by) an MHC class I complex. Alternatively or additionally, in some embodiments, the second payload is displayed by (or encodes an agent that is displayed by) an MHC class II complex. In some such embodiments, the first and second nanoparticle populations are included in a same composition.

[0046] In some embodiments, the present disclosure provides a method comprising steps of administering to a subject in need thereof a nanoparticle composition comprising a nanoparticle population having one or more payloads, or precursor(s) thereof, that activate antigen-specific T cells, wherein the nanoparticle composition is administered orally, sublingually, or buccally.

[0047] In some embodiments, the present disclosure provides a method comprising steps of administering to a subject in need thereof a nanoparticle composition comprising a first nanoparticle population having a first payload, or precursor(s) thereof, that activate a first antigen-specific T cell, and a second nanoparticle population having a second payload, or precursor(s) thereof, that activate a second antigen-specific T cell, wherein the nanoparticle composition is administered orally, sublingually or buccally.

[0048] In another aspect, the present disclosure provides a method comprising steps of (i) administering to a subject in need thereof a first nanoparticle composition comprising a first nanoparticle population having a first payload, or precursor(s) thereof, that activate a first antigen-specific T cell; and administering to the subject a second nanoparticle composition comprising a second nanoparticle population having a second payload, or precursor(s) thereof, that activate a second antigen-specific T cell, wherein the first and / or second nanoparticle compositions are administered orally, sublingually or buccally. Page 9 of 340 12613923v1Docket No.: 2006517-0315

[0049] In some embodiments, the present disclosure provides a nanoparticle preparation prepared by the methods provided herein, and the nanoparticle preparation comprises a plurality of nanoparticles, each of which comprises a payload (e.g., a hydrophilic payload) in a polymer. DEFINITIONS

[0050] 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.

[0051] Administration: As used herein, the term “administration” refers to 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.

[0052] Aggregation: The term “aggregation”, as used herein, refers to the formation of higher molecular weight entities, rather than the desired, defined species of nanoparticles. Aggregation may pose a problem during nanoparticle formation and / or manufacturing, e.g., as described herein. Aggregation may involve entities “sticking” or “clumping” together. In some embodiments, aggregation is prevented and / or reduced during the nanoparticle formation and / or manufacturing, e.g., as described herein. In some embodiments, aggregates (e.g., entities formed by the process of aggregation) are excluded from nanoparticle compositions comprising desired, defined species of nanoparticles. Aggregates may comprise nanoparticles and / or components of nanoparticles as described herein. Aggregates may comprise biological material such as, for example, protein, DNA, and / or RNA. Aggregates may be defined by size (e.g., as characterized by molecular weight and / or diameter). In some embodiments, aggregates are greater than or equal to 1000, 1250, 1500, 1750, 2000, 2500, or 3000 nm in diameter. In some embodiments, aggregates are excluded via methods such as size exclusion chromatography.

[0053] 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 carbohydrate (e.g., polysaccharide). In some Page 10 of 340 12613923v1Docket No.: 2006517-0315 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.

[0054] 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 surfaces 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. Allergic Reaction Grading System. Grade 1 - Mild Grade 2 - Moderate Grade 3 – Severe Grade 4 – Life- Grade 5 -Page 11 of 340 12613923v1Docket No.: 2006517-0315 drug fever <38 degrees responds promptly to medication and / or brief consequences; urgent C (<100.4 degrees F); symptomatic treatment interruption of infusion); intervention indicated intervention not (e.g., antihistamines, recurrence of symptomsGrade 1 - Mild Grade 2 - Moderate Grade 3 – Severe Grade 4 - Life Grade 5 - threatening Death

[0055] 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 Page 12 of 340 12613923v1Docket No.: 2006517-0315 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.

[0056] 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.

[0057] 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 Page 13 of 340 12613923v1Docket No.: 2006517-0315 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.

[0058] 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.

[0059] 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).

[0060] 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 Page 14 of 340 12613923v1Docket No.: 2006517-0315 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 Stage Characterized By 1 Mild (skin & subcutaneous tissues GI Flushin urticaria eriorbital or facial an ioedemafollowing 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 Page 15 of 340 12613923v1Docket No.: 2006517-0315 • 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

[0062] 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”.

[0063] 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.

[0064] Antigen: 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 Page 16 of 340 12613923v1Docket No.: 2006517-0315 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.

[0065] 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, Th1 / Th17 cytokines and TLR2 / TLR4 expressing dendritic cells predominate the microenvironment within distinct oral mucosal sites. Allergy 66: 532, 2011).

[0066] 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 Page 17 of 340 12613923v1Docket No.: 2006517-0315 reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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 Page 18 of 340 12613923v1Docket No.: 2006517-0315 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)(PLG or 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 carbohydrates (e.g., 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).

[0071] 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.

[0072] 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, Page 19 of 340 12613923v1Docket No.: 2006517-0315 in some embodiments, a carrier may be or comprise a bead, film, rod, or similarly structured component.

[0073] 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.

[0074] 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.

[0075] 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 190thresidue in the first polymer but rather corresponds to the residue found at the 190thposition 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.

[0076] 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 Page 20 of 340 12613923v1Docket No.: 2006517-0315 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, hydroxyethyl 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.

[0077] 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 (i.e., with a therapeutic dosing regimen).

[0078] 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 (i.e., is a therapeutic dosing regimen).

[0079] Encapsulated: The term “encapsulated” is used herein to refer to substances that are completely surrounded by another material. Page 21 of 340 12613923v1Docket No.: 2006517-0315

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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: Alanine Ala A nonpolar neutral 1.812613923v1Docket No.: 2006517-0315 Glutamic acid Glu E polar negative -3.5 Glutamine Gln Q polar neutral -3.5 AmbAsparagine or aspartic acid Asx BAs 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 Page 23 of 340 12613923v1Docket No.: 2006517-0315 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.

[0084] Human: In some embodiments, a human is an embryo, a fetus, an infant, a child, a teenager, an adult, or a senior citizen.

[0085] Hydrophilic: As used herein, the term “hydrophilic” and / or “polar” refers to a tendency to mix with, or dissolve easily in, water.

[0086] 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.

[0087] 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. Page 24 of 340 12613923v1Docket No.: 2006517-0315 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.

[0088] 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.

[0089] 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, etc.); in such embodiments, percent isolation or purity of the substance is calculated without including such carriers or excipients. Page 25 of 340 12613923v1Docket No.: 2006517-0315

[0090] 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.

[0091] 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 may be characterized by a particular distribution of particle sizes.

[0092] 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 Page 26 of 340 12613923v1Docket No.: 2006517-0315 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, O(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, 200, 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.

[0093] 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. Page 27 of 340 12613923v1Docket No.: 2006517-0315

[0094] 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.

[0095] 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., may comprise a plurality of, and / or combination(s) of one or more different materials – e.g., carbohydrates, lipids, nucleic acids, proteins, small molecules, etc.; e.g., may be or comprise a mixture, a crude sample, a cellular extract, etc., and / or a combination or mixture of any with one or more other agents or substances).

[0096] 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.

[0097] 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 Page 28 of 340 12613923v1Docket No.: 2006517-0315 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 stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids.

[0098] Protein: As used herein, the term “protein” refers to a polypeptide (i.e., a string of at least three 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.

[0099] Precipitation: As used herein, the term “precipitation” refers to the formation of a solid in a solution.

[0100] 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. Page 29 of 340 12613923v1Docket No.: 2006517-0315

[0101] Sample: As used herein, the term “sample” typically refers to an aliquot of material obtained or derived from a source of interest, as described herein. In some embodiments, a source of interest is a biological or environmental source. In some embodiments, a source of interest may be or comprise a cell or an organism, such as a microbe, a plant, or an animal (e.g., a human). In some embodiments, a source of interest is or comprises biological tissue or fluid. In some embodiments, a biological tissue or fluid may be or comprise amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humor, vomit, and / or combinations or component(s) thereof. In some embodiments, a biological fluid may be or comprise an intracellular fluid, an extracellular fluid, an intravascular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, and / or a transcellular fluid. In some embodiments, a biological fluid may be or comprise a plant exudate. In some embodiments, a biological tissue or sample may be obtained, for example, by aspirate, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g., brocheoalvealar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage). In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and / or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques such as amplification or reverse transcription of nucleic acid, isolation and / or purification of certain components, etc. In some embodiments, a sample may be a “crude” sample in that it has been subjected to relatively little processing and / or is complex in that it includes components of relatively varied chemical classes.

[0102] Small molecule: As used herein, the term “small molecule” has its art-understood meaning of being an organic compound that is typically is less than about 5 kilodaltons (kD) in size. In some embodiments, a small molecule is less than about 4 kD, 3 kD, about 2 kD, or about Page 30 of 340 12613923v1Docket No.: 2006517-0315 1 kD. In some embodiments, a 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 has a molecular weight that 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, small molecules are non-polymeric (e.g., are not polymers such as, for example, not nucleic acids, polypeptides, polysaccharides, etc).

[0103] 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., 37º C in phosphate buffered saline).

[0104] 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 Page 31 of 340 12613923v1Docket No.: 2006517-0315 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.).

[0109] 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.

[0110] 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, Page 32 of 340 12613923v1Docket No.: 2006517-0315 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.

[0111] 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.

[0112] 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 Page 33 of 340 12613923v1Docket No.: 2006517-0315 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

[0113] The Drawing, which is comprised of at least the following Figures, is for illustration purposes only, not for limitation.

[0114] FIG.1A is a schematic showing the structure of an exemplary nanoparticle.

[0115] FIG.1B is a schematic showing an exemplary process to produce nanoparticles containing a polymer and payload(s).

[0116] FIG.1C is an exemplary image of the fluid bilayer produced by some exemplary methods.

[0117] FIG.1D is a graph of the size distribution of nanoparticles.

[0118] FIG.1E is an exemplary image of nanoparticles.

[0119] FIG.1F is a graph of TLR expression.

[0120] FIGs.2A- 2C demonstrates activation of basophils from peanut allergic individuals.2A, Representative dose-response curve of basophil activation obtained with one of the study subject samples. Basophil activation is shown as percentage of CD63+ basophils in response to increasing concentration of the peanut extract protein. The blue dotted line is unmasked peanut extract (PNE) compared with encapsulated peanut PNs (PN+PNE) (green) and empty NPs (Empty NP) (dotted green). The concentration of the empty NPs used for the stimulation was calculated based on the ratio of PNE to the nanoparticle’s material in the PN+PNE formulation. “0” on the X-axis corresponds to the negative (no stimulant) control condition.2B, The percentage of the CD63+ basophils when stimulated with formyl-methionyl-leucylphenylalanine (fMLP, gray Bar) or anti-human IgE (red Bar) as positive controls for basophil activation.2c, EC50 values (Y-axis, logarithmic scale) obtained with the 7 study samples stimulated with PNE (blue bars) or with PN+PNE (green bars).

[0121] FIGs.3A-3N show comparison of T cell activation (proliferation and cytokine production) induced by bone marrow derived dendritic cells (BMDC), pulsed with OVA PLG NPs and unencapsulated OVA in vitro. Bone marrow derived FLT3L dendritic cells (BMDCs) were pulsed with OVA PLG NPs including incorporated adjuvants (NP+OVA+adjuvant), Page 34 of 340 12613923v1Docket No.: 2006517-0315 unencapsulated OVA alone (OVA), and no antigen control (Ctr) at various doses of antigen for 1 hour. The adjuvant includes both E. coli lipid extract that coats the NPs and sheared DNA embedded within the NPs. CD4 (OT2) and CD8 (OT1) T cells derived from spleens of OT 2 and OT1 transgenic mice and were labelled with CFSE. The BMDCs and T cells were cocultured for 3 days, and the proliferative response of the CD4 (OT2) and CD8 (OT1) T cells were analyzed 3 days later. 3b-3d, Bar graphs show CD4T cell proliferation (%CFSE CD4 T cells) in response to BMDCs, pulsed with (100, 10, 1 µg / mL OVA or no OVA).3a, 3D Summary of CD4 T cell proliferation shown in 3b-3d.3e-3g, CD8 T cell proliferation (%CFSE CD8 T cells) shown in bar graphs as described above.3h.3D summary of CD8 T cell proliferation shown in e-g. Data are representative of three independent experiments.3i-3l, CD4 T cell cytokines production (IFNγ, IL-10, IL17, IL4). 3m-3n, CD8 T cell cytokines production (IFNγ, and granzyme B (Gzb)).3h, 3D summary of CD4 and CD8 T cell cytokine production. BMDC were pulsed with NP+OVA+adjuvant, NP+adjuvant, unencapsulated OVA along with adjuvants (OVA&adjuvant), and unencapsulated OVA alone (OVA). Data are representative of three independent experiments. P-values were calculated by an unpaired t test.

[0122] FIGs.4a-4b shows the proliferative response of CD4 (OT2) and CD8 (OT1) T cells to OVA nanoparticles in vivo.4a, Isolated CD8 (OT1) and CD4 (OT2) T cells were adoptively transferred into WT mice.18 hours later the mice were immunized (oral gavage) with Enano+OVA+adjuvant or Enano+adjuvant. The proliferation of T cells from mesenteric lymph nodes was measured at day 3 in 4 independent experiments.4a, 3D summary of the 4 experiments shown in 4b. P-values were calculated by an unpaired t test.

[0123] FIGs.5a-5b show T cell responses to OVA NP are OVA specific. BMDCs pulsed with either NP+OVA+adjuvant or NP+adjuvant were tested for their ability to induce CD4 (OT2) and CD8 (OT1) T cell proliferation. The NP+adjuvant pulsed BMDCs were pulsed with an equivalent amount of adjuvant as used in the NP+OVA+adjuvant group.5b. The proliferation of CD4 (OT2) and CD8 (OT1) T cells was analyzed 3 days later (bar graphs).5a, 3D summary of data shown in 5b. Data are representative of three independent experiments. P-values were calculated by an unpaired t test.

[0124] FIG.6 is a schematic showing an exemplary process to produce a nanoparticle. Page 35 of 340 12613923v1Docket No.: 2006517-0315

[0125] FIG.7 is a representative scanning electron microscope (SEM) image of an exemplary protein loaded nanoparticle sample (e.g., NP-PN1). Distance between white bars in the bottom right of the figure correspond to 2 µm.

[0126] FIG.8 shows comparison of total protein content in µg / mL of exemplary protein loaded nanoparticle batches 1-11 between Bicinchoninic Acid (BCA) assay measurements made in Laboratory 1 (left bar for each batch) and Laboratory 2 right bar for each batch). Batch 1 contained exemplary protein loaded nanoparticles at a concentration of 0.125 mg / mL, Batch 2 contained exemplary protein loaded nanoparticles at a concentration of 0.5 mg / mL, and batches 3-11 contained exemplary protein loaded nanoparticles at a concentration of 2.0 mg / mL.

[0127] FIG.9 comparison of free protein content in µg / mL of exemplary protein loaded nanoparticle batches 1-11 between BCA assay measurements made in Laboratory 1 (left bar for each batch) and Laboratory 2 right bar for each batch). Batch 1 contained exemplary protein loaded nanoparticles at a concentration of 0.125 mg / mL, Batch 2 contained exemplary protein loaded nanoparticles at a concentration of 0.5 mg / mL, and batches 3-11 contained exemplary protein loaded nanoparticles at a concentration of 2.0 mg / mL.

[0128] FIG.10 shows total protein content of exemplary protein loaded nanoparticle batches 12- 20 obtained from a BCA assay. Region delineated with dashed lines represents a target total protein concentration between 1800 and 3500 µg / mL.

[0129] FIG.11 shows free protein content of exemplary protein loaded nanoparticle batches 12- 20 obtained from a BCA assay.

[0130] FIG.12 shows safety factor calculated for exemplary protein loaded nanoparticle batches 12-20 from total protein content and free protein content obtained from a BCA assay. A target safety factor of 10 or greater, as delineated with a dashed line, is desired.

[0131] FIG.13 shows Z-average diameter of exemplary protein loaded nanoparticles from batches 12-20. Z-average diameter was measured with a dynamic light scattering (DLS) instrument. Region delineated with dashed lines represents a target z-average diameter between 225 and 450 nm.

[0132] FIG.14 shows polydispersity index (PDI) of exemplary protein loaded nanoparticles from batches 12-20. PDI was calculated based on repeated diameter measurements obtained with a DLS instrument. Region delineated with dashed lines represents a target PDI between 0.1 and 0.4. Page 36 of 340 12613923v1Docket No.: 2006517-0315

[0133] FIG.15 shows Z-average diameter of exemplary protein loaded nanoparticles from batches 1-11. Batch 1 contained exemplary protein loaded nanoparticles at a concentration of 0.125 mg / mL, Batch 2 contained exemplary protein loaded nanoparticles at a concentration of 0.5 mg / mL, and batches 3-11 contained exemplary protein loaded nanoparticles at a concentration of 2.0 mg / mL. Z-average diameter was measured with a DLS instrument.

[0134] FIG.16 shows poly(lactic-co-glycolic) acid (PLGA) concentration in exemplary protein loaded nanoparticles from batches 1-11. Batch 1 contained exemplary protein loaded nanoparticles at a concentration of 0.125 mg / mL, Batch 2 contained exemplary protein loaded nanoparticles at a concentration of 0.5 mg / mL, and batches 3-11 contained exemplary protein loaded nanoparticles at a concentration of 2.0 mg / mL. PLGA concentration was measured via mass spectrometry.

[0135] FIG.17 shows percent measured free protein content relative to measured total protein content of exemplary protein loaded nanoparticles from batches 1-11. Batch 1 contained exemplary protein loaded nanoparticles at a concentration of 0.125 mg / mL, Batch 2 contained exemplary protein loaded nanoparticles at a concentration of 0.5 mg / mL, and batches 3-11 contained exemplary protein loaded nanoparticles at a concentration of 2.0 mg / mL. Shaded region (e.g., 0-20%) shows a range of desired values. Free protein content and total protein content were measured with a BCA assay.

[0136] FIG.18 shows percent measured total protein content relative to a target total protein content of exemplary protein loaded nanoparticles from batches 1-11. Batch 1 contained exemplary protein loaded nanoparticles at a concentration of 0.125 mg / mL, Batch 2 contained exemplary protein loaded nanoparticles at a concentration of 0.5 mg / mL, and batches 3-11 contained exemplary protein loaded nanoparticles at a concentration of 2.0 mg / mL. Shaded region (e.g., 70-130%) shows a range of desired values. Total protein content was measured with a BCA assay.

[0137] FIG.19 shows in vitro protein release from exemplary protein loaded nanoparticles from batches 1-10 at three timepoints at 0 hours, 8 hours, and 24 hours after beginning of experiment. Batch 1 contained exemplary protein loaded nanoparticles at a concentration of 0.125 mg / mL, Batch 2 contained exemplary protein loaded nanoparticles at a concentration of 0.5 mg / mL, and batches 3-10 contained exemplary protein loaded nanoparticles at a concentration of 2.0 mg / mL. Page 37 of 340 12613923v1Docket No.: 2006517-0315 Each timepoint measurement is represented with a black circle, and dashed line connects each timepoint with a subsequent timepoint. In vitro protein release was measured with a BCA assay.

[0138] FIG.20 shows measured concentration on E. Coli DNA in exemplary protein loaded nanoparticles from batches 1-11. Batch 1 contained exemplary protein loaded nanoparticles at a concentration of 0.125 mg / mL, Batch 2 contained exemplary protein loaded nanoparticles at a concentration of 0.5 mg / mL, and batches 3-11 contained exemplary protein loaded nanoparticles at a concentration of 2.0 mg / mL. For each batch, a left bar represents total E. Coli DNA, and a right bar represents free E. Coli DNA. E. Coli DNA concentration was measured via polymerase chain reaction (PCR). DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0139] 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. Unless 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.

[0140] In accordance with one or more embodiments, the present disclosure provides technologies for preparation of certain particle compositions, particularly including particles comprised of a polymer (e.g., PLG). Provided technologies achieve production of desirable particle compositions, including, for example, compositions characterized by one or more of particular particle size (e.g., average size, size range, size distribution, etc.), zeta potential, payload, ratio of polymer:payload, concentration of unit of payload per unit of polymer, safety factor, outer surface decoration (e.g., type of decoration, density of decoration, complexity of decoration, etc.), and / or immunological properties, etc. Moreover, provided technologies are particularly useful for preparation of nanoparticle compositions in which nanoparticles comprise one or more payloads and / or coating agents.

[0141] In some embodiments, certain aspects of provided technologies make them particularly useful and / or effective for production of nanoparticle compositions comprising fragile payload(s). Alternatively or additionally, in some embodiments, certain aspects of provided technologies make them particularly useful and / or effective for production of nanoparticle Page 38 of 340 12613923v1Docket No.: 2006517-0315 compositions comprising complex payload(s) and / or complex coating agent(s). Polymer nanoparticle systems, and particularly PLG nanoparticle systems, have proven to be highly useful in the delivery of a variety of payload agents, including various therapeutic payloads. See, for example review article by Danheir et al., J Controlled Release 161, available online February 4, 2012. Danaheir et al. list among the properties for which PLG nanoparticles are celebrated “(i) biodegradability and biocompatibility, (ii) FDA and European Medicine Agency approval in drug delivery systems for parenteral administration, (iii) well described formulations and methods of production adapted to various types of drugs e.g., hydrophilic or hydrophobic small molecules or macromolecules, (iv) protection of drug from degradation, (v) possibility of sustained release, (vi) possibility to modify surface properties to provide stealthiness and / or better interaction with biological materials and (vii) possibility to target nanoparticles to specific organs or cells”. See Abstract of Danheir et al., J Controlled Release 161, available online February 4, 2012.

[0142] In some embodiments, the present disclosure provides technologies that may offer an enhanced synthesis process, and / or consistent product quality as compared with other nanoparticle preparations (e.g., prepared by other technologies and / or including other component(s) and / or not sharing one or more characteristics as described herein). 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), and / or fragile / complex material. In some embodiments, provided preparations are characterized by more stable formations (e.g., can be stored longer), and / or other attributes relative to a standard preparation (e.g., using emulsions), as described herein. Components of Nanoparticle Compositions

[0143] In many embodiments, nanoparticle compositions to which the present disclosure relates are formed from: a) A polymer component; b) A payload component; c) An optional coating component; and d) One or more optional additional components. Page 39 of 340 12613923v1Docket No.: 2006517-0315

[0144] In some embodiments, a payload component is incorporated into, or otherwise combined with, a polymer component so that the payload component is protected from one or more aspects of an external environment. For example, in some embodiments, a payload component is protected from degrading or otherwise damaging aspect(s) of an external environment (e.g., enzymes, temperature, immune system components, etc.)

[0145] In some embodiments, a payload is incorporated into, or otherwise combined with, a polymer component, and / or is otherwise incorporated into nanoparticles of the preparation (e.g., which may be coated), so that, when such nanoparticles are administered, e.g., orally, to a subject, such payload component is protected from exposure, for example to such subject’s immune system (e.g., so that such subject does not experience a systemic allergic reaction to such payload component).

[0146] In some embodiments of nanoparticle composition(s) to which the present disclosure relates, one or more payload components is / are homogeneously or substantially homogenously distributed in a polymer matrix.

[0147] In some embodiments, provided nanoparticle compositions are useful, for example, in desensitizing a subject to a payload component. Polymer component

[0148] In some embodiments, a polymer component of nanoparticles to which the present disclosure relates is or comprises 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.

[0149] 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.

[0150] 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), Page 40 of 340 12613923v1Docket No.: 2006517-0315 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, poly(propylene 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 poly(ethylene 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 Page 41 of 340 12613923v1Docket No.: 2006517-0315 acids"), poly(butyric acid), poly(valeric acid), and poly(lactide-co-caprolactone) and / or derivatives thereof.

[0151] 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), carbohydrates (e.g., polysaccharides)s (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.

[0152] 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.

[0153] In many embodiments, a polymer component comprises or consists of poly(lactic-co- glycolic acid)(“PLGA” or “PLG”).

[0154] In some embodiments, the present disclosure encompasses the recognition that viscosity of polymer preparation may impact its usefulness in producing nanoparticles as described herein. As those skilled in the art are aware, 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 as described herein. Payload components

[0155] In some embodiments, provided nanoparticles and / or nanoparticle compositions include and / or deliver at least one payload (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In some embodiments, a payload may be or comprise a polypeptide agent. In some embodiments, a composition may deliver such payload by including a nucleic acid that encodes a polypeptide, where in the nucleic acid is expressed upon / after administration. In some embodiments, a payload may be or comprise a particular form of an agent or entity – e.g., a glycosylated or phosphorylated form or a truncated form or a complexed form or an otherwise modified form. In Page 42 of 340 12613923v1Docket No.: 2006517-0315 some embodiments, a composition may deliver such payload if it comprises the precursor (e.g., unmodified, uncomplexed etc) form, and conversion to the particular form occurs upon / after administration of the composition.

[0156] 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, comprise, and / or deliver (e.g., encode) an agent or entity with respect to which a particular biological response is desired.

[0157] 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 antigen to which an immune response in generated. 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).

[0158] One feature of certain embodiments of the present disclosure is that it permits delivery of an antigen to a subject in a context that both (a) minimizes exposure of the antigen to immune system component(s) that might induce or mediate an undesirable reaction or response to the antigen while (b) 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, basophil cells, IgE and / 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., Th1 and / or Treg cells) that might mediate an allergy- suppressing (e.g., Th1 or Treg) response.

[0159] In some embodiments, a payload comprises one or more carbohydrates, lipids, metals, nucleic acids, polypeptides, small molecules and / or combinations thereof.

[0160] Those skilled in the art will appreciate that a variety of technologies may be utilized to provide payload(s) useful in accordance with the present disclosure. For example, in some embodiments, 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 Page 43 of 340 12613923v1Docket No.: 2006517-0315 system. In some such embodiments, DNA may be in the form of vector DNA such as plasmid DNA. Alternatively or additionally, in some embodiments, a payload that is or comprises a polypeptide may be prepared by isolation from another source (e.g., a natural source).

[0161] In some embodiments, a payload may be provided in combination with another substance. In some embodiments, a payload may be provided as a complex mixture (e.g., including different classes of compounds – e.g., both polypeptides and nucleic acids, etc.) (e.g., protein, carbohydrate, lipid and / or nucleic acid mixtures, which in some embodiments may represent or include one or more crude samples, cellular extracts, etc.). In some embodiments, nucleic acids (and in some embodiments, particularly RNA, which in some embodiments may specifically be or comprise longer RNAs such as mRNAs), polypeptides, and / or saccharides (which may, in some embodiments, be incorporated into other entities, such as glyopeptides) may be considered to be “fragile” payloads.

[0162] Indeed, one feature of certain embodiments of the present disclosure is that it permits utilization of relatively complex payloads, specifically including payloads that are or comprise relatively crude preparations (e.g., only modestly processed samples). In some embodiments, a payload may be or comprise a crude preparation and / or other complex material (e.g., an extract, etc.).

[0163] In some embodiments, provided nanoparticles comprise microbial and / or cellular components (e.g., that are or comprise a microbial or other cellular extract). Without wishing to be bound by a particular theory, some embodiments of the present disclosure including one or more microbial cellular component(s) 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.

[0164] The present disclosure also provides an 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. Page 44 of 340 12613923v1Docket No.: 2006517-0315

[0165] In some embodiments, provided nanoparticles comprise microbial or other cellular extracts – e.g., hydrophilic or hydrophobic extracts of cells (e.g., microbial) 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.

[0166] In many embodiments, cellular (e.g., microbial) extracts are prepared from cell (e.g., microbial cell) preparations such as cell cultures. Those skilled in the art will appreciate that, in some embodiments, cell preparations (e.g., cell cultures) may be 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, cell (e.g., 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.

[0167] In some embodiments, the present disclosure provides and / or utilizes (e.g., as payload components) hydrophilic cell (e.g., microbial cell) extracts, for example extracts prepared by contacting a 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. Page 45 of 340 12613923v1Docket No.: 2006517-0315

[0168] In some embodiments, provided nanoparticle compositions include a payload selected from the group consisting of polypeptides, nucleic acids, carbohydrates (e.g., polysaccharides), and combinations thereof. In many embodiments, a payload is or comprises a polypeptide. In many embodiments, a payload is or comprises a nucleic acid; in some such embodiments, a payload is or comprises a long nucleic acid (e.g., a gene therapy vector, an mRNA, etc); in some embodiments, a payload is or comprises a partly or wholly single stranded nucleic acid).

[0169] In some embodiments, a payload is or comprises a RNA. In some embodiments, and RNA payload is an mRNA. In some embodiments, an RNA payload has a length within a range of about 15 to about 3,000,000 residues. In some embodiments, an RNA payload has a length within a range of about 500 to 50000 residues. In some embodiments, an RNA payload has a length within a range of about 1000 to about 10000 residues. In some embodiments, an RNA payload is an mRNA encoding a polypeptide having a length within a range of about 50 to about 5000 amino acids; in some embodiments, such encoded polypeptide has a length within a range of about 100 to about 3000 amino acids; in some embodiments, such encoded polypeptide has a length within a range of about 200 to about 1500 amino acids.

[0170] In some embodiments, a provided nanoparticle preparation is manufactured using one or more relatively complex components (e.g., a payload that is a relatively crude extract or combination of components).

[0171] In some embodiments, a payload is or comprises one or more antigens. In some embodiments, an antigen is an allergic antigen, an infectious antigen, and / or a disease-associated (e.g., a cancer-associated) antigen.

[0172] In some embodiments, a provided nanoparticle composition includes one or more payloads on (e.g., attached) nanoparticle surface(s). Fragile payloads

[0173] In some embodiments, a payload may be fragile (e.g., susceptible to damage from energy input (e.g., increased temperature, pressure, applied shear force, etc.)) or other operation of an element of an environment to which the payload is or might be exposed (e.g., but for its incorporation within a nanoparticle composition as described herein). In some embodiments, a fragile payload may be decomposed or (partly or fully) inactivated when exposed to energy input or certain environmental conditions (e.g., high temperature, high or low pH, high pressure, high Page 46 of 340 12613923v1Docket No.: 2006517-0315 shear force, high ionic strength, etc.). In some embodiments, one or more biological or pharmaceutical activities of a fragile payload may be decreased when exposed to energy input or such environmental condition(s) (e.g., high temperature, high or low pH, high pressure, high shear force, high ionic strength, etc.).

[0174] In some embodiments, a fragile payload may be or comprise a polypeptide, a nucleic acid, or a combination thereof. In some embodiments, a fragile payload may be or comprise a DNA, a RNA, or a combination thereof. In some embodiments, a fragile payload comprises anti-micro RNA, antisense RNA (asRNA), circular RNA (circRNA), enhancer RNA (eRNA), long non-coding RNA (lncRNA), messenger RNA (mRNA), micro RNA (miRNA), Piwi- interacting RNA (piRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNAs), small rDNA-derived RNA (srRNA), transfer RNA (tRNA), tRNA-derived small RNA (tsRNA), or a combination thereof.

[0175] In some embodiments, a fragile payload comprises a mRNA encoding a polypeptide, e.g., a polypeptide that elicits (or is intended to elicit) a particular immune response (i.e., that is an immunologically relevant polypeptide).

[0176] In some embodiments, a fragile payload is or comprises a gene therapy vector. In some embodiments a gene therapy vector is a viral vector. In some embodiments, a viral vector is an adenoviral vector, an adenoviral associated viral (AAV) vector; or a lentiviral vector. In some embodiments, a gene therapy vector encodes a therapeutic agent. In some embodiments, a gene therapy vector encodes an immunologically relevant polypeptide. In some embodiments, a gene therapy vector targets antigen presenting cells (APCs). In some embodiments an immunologically relevant polypeptide encoded by a gene therapy vector is presented on an APC.

[0177] In some embodiments, an immunologically relevant polypeptide is one with respect to which a particular immune response (e.g., a protective immune response such a Th1-type immune response, a tolerized immune response, etc.) is elicited or desired.

[0178] To give but a few examples, in some embodiments, an immunologically relevant polypeptide may be or comprise at least one (and, in many embodiments, a plurality) of epitopes associated with or characteristic of a pathogen or disease state. In some embodiments, such epitope(s) may be or comprise tumor-associated and / or tumor-specific epitope(s). Alternatively or additionally, in some embodiments, such epitope(s) may be or comprise epitope(s) of an infectious agent (e.g., a microbe or virus). In some embodiments, immunologically relevant Page 47 of 340 12613923v1Docket No.: 2006517-0315 polypeptide may be or comprise one or more infectious agent antigens, cancer antigens, alloantigens, or other antigens as described hereinbelow.

[0179] In some embodiments, a fragile payload comprises an RNA (e.g., an mRNA or other RNA that encodes a polypeptide) having 200 to 100,000 residues, 200 to 50,000 residues, 200 to 10,000 residues, 500 to 100,000 residues, 500 to 50,000 residues, or 500 to 10,000 residues in length. In some embodiments, a fragile payload comprises an RNA (e.g., an mRNA or other RNA that encodes a polypeptide) having 200 to 100,000 residues, 200 to 50,000 residues, 200 to 10,000 residues, 500 to 100,000 residues, 500 to 50,000 residues, or 500 to 10,000 residues length.

[0180] In some embodiments, a payload is or comprises (or otherwise delivers, e.g., by expression of a payload construct) an siRNA. In some embodiments, an siRNA targets a disease-associated gene. To give but a few examples, an siRNA may target genes selected from the group consisting of Eg5 / KSP, PCSK9, Serpina1, TTR, VEGF, XBP-1, and combinations thereof.

[0181] In some embodiments, a fragile payload comprises a RNA pre-complexed with chaperone proteins.

[0182] In some embodiments, a nanoparticle composition comprising an RNA payload further comprises one or more RNAse inhibitors.

[0183] In some embodiments, a fragile payload is or comprises one or more carbohydrates or carbohydrate structures (e.g., a glycosylated polypeptide), or a construct that delivers such carbohydrate or an agent (e.g., a polypeptide) including such carbohydrate structure (e.g., a glycosylated polypeptide, which may be delivered, for example, by administering a nucleic acid encoding the polypeptide to a system that will glycosylate it). Antigens

[0184] In some embodiments, a payload is or comprises an antigen, or a construct that delivers (e.g., encodes) an antigen. In some embodiments, an antigen may be or comprise a polypeptide (e.g., a peptide, a protein, a glycoprotein, etc.), a carbohydrate (e.g., polysaccharide), a lipid (e.g., glycolipid) a nucleic acid, or combinations thereof.

[0185] 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 Page 48 of 340 12613923v1Docket No.: 2006517-0315 (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).

[0186] Those skilled in the art, reading the present disclosure, will appreciate that, in some embodiments, antigen payload component(s), like other payload components may be utilized in a pure form and in other embodiments may be or comprise one or more crude (e.g., 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.

[0187] In some embodiments, suitable antigens are known in the art and are available from commercial, government, scientific or other sources. In some embodiments, antigens are provided as or obtained from whole, inactivated or attenuated organisms.

[0188] One of skill in the art will recognize that, in certain embodiments, multiple antigens may be delivered by nanoparticles simultaneously and / or sequentially in accordance with the present disclosure. Indeed, one feature of certain embodiments described herein is that they surprisingly achieve incorporation of multiple antigens (and / or other components) in nanoparticle compositions; in some such embodiments, some or all such antigens are provided together, e.g., as part of an extract, e.g., a relatively crude extract, from an appropriate source (e.g., an allergen or other antigen source).

[0189] In some embodiments, provided nanoparticle compositions incorporate multiple antigens (e.g., multiple antigen polypeptides) from a single source. In some embodiments, provided nanoparticle compositions incorporate multiple epitopes of a single antigen, and / or different epitopes of different antigens, in some embodiments as part of one or more natural antigens (e.g., one or more antigen proteins) and in some embodiments as part of one or more engineered antigen(s) (e.g., a polypeptide that may be engineered to includes epitopes from one than one antigen and / or to link together two or more epitopes from a single antigen but in a different arrangement than that which they have in the antigen.

[0190] Alternatively or additionally, in some embodiments, provided nanoparticle compositions incorporate one or more antigens together with one or more other agents (e.g., cytokines or adjuvants or other immune modulators or biologically active agents). For example, including as Page 49 of 340 12613923v1Docket No.: 2006517-0315 exemplified herein, in some embodiments, provided nanoparticle compositions include (e.g., enfold and / or are coated with) one or more immunomodulatory agents such as, for example, one or more adjuvants, one or more interleukins, one or more TLR receptor agonists, etc.

[0191] 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.).

[0192] 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.

[0193] In some embodiments, a payload component is or comprises an antigen 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. a. Allergens

[0194] In some embodiments, an antigen is or comprises an allergen. In some embodiments, an allergen may be or comprise an environmental allergen. For example, in some embodiments, an environmental antigen may be or comprise one or more pollen allergens (grass-, tree-, and weed- pollen allergens), insect allergens (inhalant, saliva and venom allergens), animal hair and / or dander allergens.

[0195] In some embodiments, an allergen for use in accordance with the present disclosure may, for example, be an allergen found in certain foods, venom, drugs or rubber that elicits an allergic response; in some embodiments, an allergen is one that elicits an anaphylactic allergic response.

[0196] Those skilled in the art will be aware of various allergens that may induce anaphylaxis, including, for example, various allergens found in food (e.g., egg, meat, milk, peanut, tree nuts, wheat), insect venom (e.g., bees, reptiles), drugs, and latex.

[0197] In some embodiments, an allergen may be found in 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 some embodiments, an allergen may be from venom allergens including such originating from stinging or biting insects such as those from the taxonomic order Page 50 of 340 12613923v1Docket No.: 2006517-0315 of Hymenoptera including and ants (superfamily Formicoidae; e.g, fire ants, velvet ants), bees (superfamily Apidae; e.g., honey bees), and wasps (superfamily Vespidea; e.g, hornets, wasps, yellow jackets). For example, venom from honeybees of the genus Apis can cause anaphylaxis in stung victims who are allergic (Weber et al. Allergy 42:464-470). The venom from honeybees 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.

[0198] Non-limiting examples of allergens found in food include proteins found in dairy products (e.g., egg, milk), fish (e.g., cod, salmon, tuna), fruit (e.g., nectarines, peaches, plums; Ann Allergy Asthma Immunol 7(6):504-8 (1996); cherries, Allergy 51(10):756-7 (1996)), legume (e.g., lentil, lupine, pea, peanut, soy), nuts (e.g., almond, Brazil nut, cashew, hazelnut, macadamia, peanut, pecan, pine nut, pistachio, walnut), seafood (e.g., clams, crab, lobster, shrimp), seeds (e.g., mustard, poppy, sesame, sunflower), and Alpha-gal (galactose-a-1,3- galactose) found in certain meats (e.g., beef, lamb, port, rabbit, venison, etc.) can also trigger allergic reactions in certain individuals.

[0199] In some embodiments an allergen may be in meat. In some embodiments an allergen may be in meat from mammals. In some embodiments an allergen may be a protein found in meat. In some embodiments an allergen may be a sugar group found in meat In some embodiments, an allergen may be a sugar group on a protein found in meat. In some embodiments, a sugar group that is an allergen comprises a hexose. In some embodiments, a sugar group that is an allergen comprises galactose. In some embodiments, a sugar group that is an allergen is a disaccharide. In some embodiments, a sugar group that is an allergen is galactose-alpha-1,3-galactose.

[0200] In some embodiments, an allergen is found in pollen-related food allergies (e.g., birch pollen related to apple allergies) may be utilized in the practice of the present invention. In some embodiments, an allergen is a pollen allergens from grasses, herbs, and trees such as pollens of the taxonomic orders of Asterales (e.g., Ambrosia and Artemisia), Cupressales (e.g., cedar (Cryptomeria and Juniperus), Fagales (e.g., alder (Alnus), birch (Betula), hazel (Corylus), and hornbeam (Carpinus)), Lamiales (e.g., olive (Olea)), Pinales, Poales (e.g., grasses (Cynodon, Page 51 of 340 12613923v1Docket No.: 2006517-0315 Dactylis, Holcus Lolium, Phalaris, Phleum, Poa, Secale, and Sorghum), Proteales (e.g., Platanaceae, Plane tree (Platanus)), and Urticales (e.g., Parietaria).

[0201] In some embodiments, an allergen may be from cockroaches, fleas (e.g., Blatella, Periplaneta, Chironomus and Ctenocepphalides), house dust mites (e.g., Dermatophagoides and Euroglyphus), midges, and storage mite (e.g., Lepidoglyphys, Glycyphagus, and Tyrophagus).

[0202] In some embodiments, an allergen may be from mammals such as cat, dog and horse. In some embodiments, an allergen may be from birds.

[0203] Still other allergens that may be used include inhalation allergens from fungi such as from the genera Alternaria and Cladosporium.

[0204] 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.

[0205] 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 Allergic Antigens ALLERGEN SYSTEMATIC AND ORIGINAL MW SEQ ACCESSION PMID R E NAME KDA N ( ITATI N)Page 52 of 340 12613923v1Docket No.: 2006517-0315 ALLERGEN SYSTEMATIC AND ORIGINAL MW SEQ ACCESSION PMID SOURCE NAMES KDA NO. (CITATION)Page 53 of 340 12613923v1Docket No.: 2006517-0315 ALLERGEN SYSTEMATIC AND ORIGINAL MW SEQ ACCESSION PMID SOURCE NAMES KDA NO. (CITATION)12613923v1Docket No.: 2006517-0315 ALLERGEN SYSTEMATIC AND ORIGINAL MW SEQ ACCESSION PMID SOURCE NAMES KDA NO. (CITATION)age o 12613923v1Docket No.: 2006517-0315 ALLERGEN SYSTEMATIC AND ORIGINAL MW SEQ ACCESSION PMID SOURCE NAMES KDA NO. (CITATION)Page 56 of 340 12613923v1Docket No.: 2006517-0315 ALLERGEN SYSTEMATIC AND ORIGINAL MW SEQ ACCESSION PMID SOURCE NAMES KDA NO. (CITATION)Page 57 of 340 12613923v1Docket No.: 2006517-0315 ALLERGEN SYSTEMATIC AND ORIGINAL MW SEQ ACCESSION PMID SOURCE NAMES KDA NO. (CITATION)12613923v1Docket No.: 2006517-0315 ALLERGEN SYSTEMATIC AND ORIGINAL MW SEQ ACCESSION PMID SOURCE NAMES KDA NO. (CITATION)Page 59 of 340 12613923v1Docket No.: 2006517-0315 ALLERGEN SYSTEMATIC AND ORIGINAL MW SEQ ACCESSION PMID SOURCE NAMES KDA NO. (CITATION)Page 60 of 340 12613923v1Docket No.: 2006517-0315 ALLERGEN SYSTEMATIC AND ORIGINAL MW SEQ ACCESSION PMID SOURCE NAMES KDA NO. (CITATION)age o 12613923v1Docket No.: 2006517-0315 ALLERGEN SYSTEMATIC AND ORIGINAL MW SEQ ACCESSION PMID SOURCE NAMES KDA NO. (CITATION)age o 12613923v1Docket No.: 2006517-0315 ALLERGEN SYSTEMATIC AND ORIGINAL MW SEQ ACCESSION PMID SOURCE NAMES KDA NO. (CITATION)Page 63 of 340 12613923v1Docket No.: 2006517-0315 ALLERGEN SYSTEMATIC AND ORIGINAL MW SEQ ACCESSION PMID SOURCE NAMES KDA NO. (CITATION)

[0206] 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 Page 64 of 340 12613923v1Docket No.: 2006517-0315 with allergy to crustaceans (e.g., crab, crayfish, lobster, or shrimp) or mollusks (e.g., clams, mussels, oysters, or scallops) tend to have allergy to various types, not just a single crustacean or mollusk.

[0207] 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 P1 (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 11); 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 1); 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 Page 65 of 340 12613923v1Docket No.: 2006517-0315 (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) Bt11a antigen (Blo t 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 (midge) component I (Chi t 2.0101); Chironomus thummi thummi Page 66 of 340 12613923v1Docket No.: 2006517-0315 (midge) component IA (Chi t 2.0102); 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 t 4); Chironomus thummi thummi (midge) component IV (Chi t 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 t 1-9); Cladosporium herbarum (fungus) acid. ribosomal protein P1 (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 1); 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 1); 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) AgDg1 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 11); Dermatophagoides farinae (mite) tropomyosin (Der f 10); Dermatophagoides microceras (mite) antigen (Der m 1); 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 P1 (Der p 1); Dermatophagoides pteronyssinus (mite) apolipophorin like p (Der p 14); Dermatophagoides pteronyssinus (mite) chymotrypsin Page 67 of 340 12613923v1Docket No.: 2006517-0315 (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 1); Dolichovespula maculate (white face hornet) hyaluronidase (Dol m 2); Equus caballus (horse) lipocalin (Equ c 1); Equus caballus (horse) lipocalin (Equ c 2); Euroglyphus maynei (mite) apolipophorin (Eur m 14); Felis domesticus (cat) cat-1 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 1); Helianthus annuus (sunflower) antigen (Hel a 1); Helianthus annuus (sunflower) profilin (Hel a 2); Hevea brasiliensis (rubber) 1,3-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 l 1); Homo sapiens (human autoallergen) antigen (Hom s 1); Homo sapiens (human autoallergen) antigen (Hom s 2); Homo sapiens (human autoallergen) antigen (Hom s 3); Homo sapiens (human autoallergen) antigen (Hom s 4); Homo sapiens (human autoallergen) antigen (Hom s 5); Hordeum vulgare (barley) BMAI-1 (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); Page 68 of 340 12613923v1Docket No.: 2006517-0315 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 Ib); 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 1); Mus musculus (mouse) MUP antigen (Mus m 1); 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 Page 69 of 340 12613923v1Docket No.: 2006517-0315 (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 A1 (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 A1 (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); Psilocybe cubensis (fungus) antigen (Psi c 1); 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 t 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 pacificus (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 t 1); Trichophyton tonsurans (fungus) serine protease (Tri t 4); 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 A1 Page 70 of 340 12613923v1Docket No.: 2006517-0315 (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 A1 (Ves v 1); Zea mays (maize, corn) lipid transfer protein (Zea m 14); and / or combinations thereof. b. Infectious Antigens

[0208] In some embodiments, provided nanoparticles and / or nanoparticle compositions may include a payload that comprises or delivers (e.g., encodes, releases, or otherwise becomes or provides) one or more epitopes of one or more antigens may be provided from an infectious agent or organisms, such as a virus, parasite and / or bacterium.

[0209] In some embodiments, an infectious antigen may be or comprise an epitope of one or more protein antigens derived from viral or bacterial sources.

[0210] Exemplary criteria for identifying and selecting effective antigenic sequences (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 of peptides normally bound to MHC molecules.

[0211] 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 provided from the viral envelope. In some embodiments, a viral antigen may be provided from a glycoprotein of the viral envelope.

[0212] Representative viruses that may be the source of viral antigens relevant to the present disclosure may be from a viral family such as, for example: Adenoviridae, Arenaviridae (e.g., Lymphocytic choriomeningitis mammarenavirus), Arteriviridae, Astroviridae, Baculoviridae, , Barnaviridae, Betaflexiviridae (e.g., Capillovirus and Carlavirus), Birnaviridae, Bromoviridae, Page 71 of 340 12613923v1Docket No.: 2006517-0315 Bunyaviridae, Caliciviridae, Caulimoviridae (e.g., Badnavirus and Caulimovirus), Circoviridae, Closteroviridae (e.g., Closterovirus), Comoviridae, Coronaviridae (e.g., Coronavirus, such as severe acute respiratory syndrome (SARS) virus, Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), Middle Eastern Respiratory Syndrome (MERS) coronavirus), Corticoviridae, Cystoviridae, , Filoviridae (e.g., Marburg virus and Ebola virus (e.g., Zaire, Reston, Ivory Coast, or Sudan strain)), Flaviviridae (e.g., Dengue virus 1, Dengue virus 2, Dengue virus 3, and Dengue virus 4, Hepatitis C virus, hepatitis G virus (HGV), West Nile virus (WNV), yellow fever), Hantaviridae, Hepadnaviridae (e.g., hepatitis B (HBV)), Hepeviridae (e.g., Hepatitis E virus (HEV)), Herpesviridae (e.g., Human herpesvirus 1, 2, 3, 4, 5, 6A, 6B, 7 and 8), Hypoviridae, Iridoviridae, Kolmioviridae (e.g., Deltavirus (e.g., hepatitis D virus)), Leviviridae, Lipothrixviridae, Matonaviridae (e.g., rubella virus), Microviridae, Orthomyxoviridae (e.g., Influenza virus A and B and C), Papillomaviridae (e.g., Papillomavirus, human papillomavirus (HPV)), Polyomaviridae, Paramyxoviridae (e.g., measles, mumps, hepatitis A virus (HAV), parainfluenza viruses), Parvoviridae (e.g., parvovirus), Phenuiviridae (e.g., Phlebovirus, Rift Valley fever), Picornaviridae (e.g., aphthovirus, coxsackievirus, hepatovirus, poliovirus, rhinovirus), Pneumoviridae (e.g., human respiratory syncytial virus, human metapneumovirus), Poxviridae (e.g., vaccinia virus and smallpox virus), Reoviridae (e.g., rotavirus), Retroviridae (e.g., lentivirus, such as human immunodeficiency virus (HIV) 1 and HIV 2, human T-lymphotrophic virus (HTLV)), Rhabdoviridae (e.g., rabies virus, Indiana vesiculovirus (VSV)), Solemoviridae (e.g., Enamovirus) Togaviridae (e.g., eastern equine encephalitis virus, Japanese encephalitis virus), Tombusviridae (e.g., Dianthovirus), and Totiviridae.

[0213] In some embodiments, a viral antigen may be or comprise epitopes of one or more viruses. In some embodiments, viral epitope may be comprised of 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, a payload for use in accordance with the present disclosure may comprise, consist of, or otherwise deliver 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.

[0214] In some embodiments, provided nanoparticles and / or nanoparticle compositions may include one or more bacterial antigens. Bacterial antigens may originate from a pathogenic bacterium. Exemplary such pathogenic bacteria may include, but not be limited to, those of a Page 72 of 340 12613923v1Docket No.: 2006517-0315 genus such as 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 (e.g., Haemophilus influenzae type B(HIB)), Hyphomicrobium, Klebsiella, Lactococcus, Legionella, Leptospirosis, Listeria, Methanobacterium, Micrococcus, Morganella, Mycoplasma, Myobacterium (e.g., Mycoplasma pneumoniae), Myxococcus, Neisseria (Neisseria meningitidis), Nitrobacter, Norcardia (e.g., Nocardia asteroids), Oscillatoria, Peptococcus, Phodospirillum, Plesiomonas, Prochloron, Proteus, Providencia, Pseudomonas, Rickettsia (e.g., Rickettsia ricketsii, Rickettsia typhi), Salmonella, Serratia, Shigella, Spirillum, Spirochaeta, Sporolactobacillus, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, Treponema, Vibrio, Yersinia, and combinations thereof.

[0215] In some embodiments, provided nanoparticles and / or nanoparticle compositions may include one or more parasite antigens. In some embodiments, a parasite is a fungus. In some embodiments, a parasite is a protozoan. In some embodiments, a parasite is a helminth. 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, Plasmodium falciparum, Schistosoma mansoni, Toxoplasma gondii, Trichomonas vaginalis, Trypanosoma brucei and Trypanosoma cruzi. 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. c. Cancer Antigens

[0216] In some embodiments, provided nanoparticles and / or nanoparticle compositions may include a payload that comprises or delivers (e.g., encodes, releases, or otherwise becomes or provides) one or more epitopes of one or more cancer antigens. In some embodiments, cancer antigens may be provided from tumor cells.

[0217] In some embodiments, a payload may be or comprise one or more polypeptides that is or comprises one or more tumor-associated or tumor-specific epitopes, or a construct that encodes Page 73 of 340 12613923v1Docket No.: 2006517-0315 such polypeptide or another entity that delivers or becomes it. In some embodiments, a tumor- associated or tumor-specific epitope is a neoepitope.

[0218] In some embodiments, a payload may be or comprise a polypeptide (e.g., that is recombinantly expressed or that is purified or partially purified from a tumor source. In some embodiments, a payload is or comprises a nucleic acid that encodes such polypeptide (e.g., a DNA or RNA construct, e.g., an mRNA).

[0219] In some embodiments, provided nanoparticles and / or nanoparticle compositions may include one or more crude (i.e., unpurified or substantially unpurified) cancer antigenic extracts. In some embodiments, cancer antigens are provided in a crude form such as a cellular lysate or cellular fraction.

[0220] In some embodiments, an exemplary cancer antigen may be, for example, 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-All, hsp70-2, KIAAO205, Mart2, Mum-1, 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-1, MageAl, 2,3,4,6,10,12, Mage-C2, NA-88, NY-Eso- 1 / 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, p15(58), CEA, RAGE, NY-ESO (LAGE), SCP-1, 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, p180erbB-3, c-met, nm-2523Hl, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, pCatenin, CDK4, Mum-1, pl6, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, a-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, C0-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-30 Ag, MOV18, NB\70K, NY-C0-1, RCASl, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin C-associated protein), TAAL6, TAG72, TLP, and TPS.

[0221] Representative cancer cells that may be the source of cancer antigens relevant to the present disclosure may be from, for example, 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 Page 74 of 340 12613923v1Docket No.: 2006517-0315 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; 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; endometrial uterine cancer; ependymoma; esophageal cancer; Ewing's sarcoma in the Ewing family of tumors; extracranial germ cell tumor; extragonadal germ cell tumor; extrahepatic bile duct cancer; eye cancer; intraocular melanoma; gallbladder cancer; gastric carcinoid; gastric cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal tumor (GIST); gestational trophoblastic tumor; 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; kidney cancer (renal cell carcinoma); laryngeal cancer; leiomyosarcoma; 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; malignant glioma; 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; 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; pleuropulmonary blastoma; Page 75 of 340 12613923v1Docket No.: 2006517-0315 prostate cancer; rectal cancer; renal pelvis and ureter and transitional cell cancer; retinoblastoma; rhabdomyosarcoma; Sézary syndrome; skin cancer (including melanoma and nonmelanoma); skin carcinoma; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma; stomach cancer; testicular cancer; throat cancer; thymoma and thymic carcinoma; thyroid cancer; urethral cancer; uterine sarcoma; vaginal cancer; vulvar cancer; and / or combinations thereof. d. Alloantigens

[0222] In some embodiments, provided nanoparticles and / or nanoparticle compositions may include a payload that comprises or delivers (e.g., encodes, releases, or otherwise becomes or provides) 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.

[0223] 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. Page 76 of 340 12613923v1Docket No.: 2006517-0315

[0224] 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. Coating agents

[0225] 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). In some embodiments, a coating agent may have adjuvant properties (e.g., may be or act as an immune adjuvant). In some embodiments, a coating agent may differ depending upon payload and / or target and / or desired immune response (e.g., Th1 vs Th2). For example, in some embodiments, if a nanoparticle composition is delivering a payload in order to desensitize a subject to an autoantigen, a coating agent may act to stimulate or assist in producing a desired response. In some embodiments, if a nanoparticle composition is delivering a payload to treat, e.g., cancer or infectious disease, a different type of coating agent may be desirable.

[0226] 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), Page 77 of 340 12613923v1Docket No.: 2006517-0315 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., Th1 and / or Treg cells) that might mediate an allergy- suppressing (e.g., Th1 or Treg) response.

[0227] In some embodiments, a coating agent comprises a hydrophobic component. For example, in some embodiments, a coating agent comprises a hydrophobic cellular component. In some embodiments, a hydrophobic component is or comprises a lipid component. In some embodiments, a hydrophobic component is or comprises LPS.

[0228] In some embodiments, a hydrophobic cellular component preparation may be provided from a cellular lysate (e.g., microbial lysate) or other extract.

[0229] 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.).

[0230] 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 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 an insight that combining such evolved attributes with various features of certain nanoparticle systems such as, for example, ability to sequester antigens and / or cellular 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.

[0231] 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 Page 78 of 340 12613923v1Docket No.: 2006517-0315 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.

[0232] 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.

[0233] 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.

[0234] 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) Page 79 of 340 12613923v1Docket No.: 2006517-0315 powder is mixed with water. In some such embodiments, a combination of water and OEE powder is sonicated, producing OEE micelles in water.

[0235] 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.

[0236] In some embodiments, OEE micelles in water are combined with a nanoparticle mixture, sonicated, and lyophilized. 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.

[0237] 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 wild-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.

[0238] In some embodiments, nanoparticles of the present disclosure are coated with a “shell” (e.g., that is or comprises a lipid component such as an LPS component and, in some Page 80 of 340 12613923v1Docket No.: 2006517-0315 embodiments, may be an organic cellular extract such as an organic microbial extract such as an organic E. coli extract) that is 5-7 nm thickness. In some such embodiments, a 5-7 nm thick shell approximates an amount of LPS on a single E. coli.

[0239] In some embodiments, nanoparticles of the present disclosure have a mass equivalent of OEE to that of LPS on E. coli. In some embodiments, nanoparticles of the present disclosure have a mass equivalent of OEE greater than that of LPS on E. coli. In some embodiments, nanoparticles of the present disclosure have a mass equivalent of OEE less than that of LPS on E. coli. For example, in some embodiments, mass of OEE on a population of particles approximates mass of OEE associated with an E. coli. By way of non-limiting example, in some embodiments, a mass / mass equivalent of 38 mg of OEE is used to approximate an amount of LPS on a single E. coli.

[0240] 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. Other agents

[0241] 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, and / or agents that may promote or sustain a particular immune response, e.g., to an included antigen).

[0242] 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. For example, a nanoparticle comprising an RNA may further comprise RNAse inhibitors.

[0243] 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 have specific effects on cell-cell interaction, communication and behavior of other cells. Cytokines are proteins that are secreted to T-cells to Page 81 of 340 12613923v1Docket No.: 2006517-0315 induce a Th1 or Th2 response. In some embodiments, cytokine(s) may be selected to reduce production of a Th2 response to antigens associated with anaphylaxis. In some embodiments, cytokine(s) may be selected to reduce production of a Th1 response to antigens. Cytokines that, when presented during antigen delivery into cells, induce a Th1 response in T cells include IL- 12, IL-2, I-18, IL-1 or fragments thereof, IFN, and / or IFNγ.

[0244] In some embodiments, one or more other agents may comprise immunological inducing agents. Inducing agents may prompt the expression of Th1 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.

[0245] 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., Bacillus Calmette- Guérin[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-hydroxyethyl) 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.

[0246] 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 cellular lysate (e.g., microbial lysate (e.g., bacterial lysate)), cellular lysate fractions, or specific components thereof. In some embodiments, cellular lysate fractions comprise entities known as pathogen-associated molecular patterns (“PAMPs”). In some embodiments, one or more of a hydrophobic bacterial lysate fraction and / or hydrophilic bacterial lysate fraction include one or more PAMPs as a Page 82 of 340 12613923v1Docket No.: 2006517-0315 hydrophilic cellular component and / or hydrophobic cellular component. In some embodiments, a hydrophilic bacterial 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-1α), c48 / 80, R848, Pam3CSK4, CpG(ODN1826), 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.

[0247] 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 recognized by RIG-I-like receptors (RLRs). In some embodiments, PAMPs are recognized by NOD-like receptors (NLRs). 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, sheared E. coli genomic DNA, any of the TLR ligands presented in Table 5, characteristic portions thereof, and / or combinations thereof. Table 5. Exemplary TLRs and TLR Ligands TLR TLR Ligand(s)age o 12613923v1Docket No.: 2006517-0315 TLR TLR Ligand(s) HSP70 Z

[0248] In some embodiments, one or more other agents may comprise a pore forming toxin (PFT). In some embodiments, a PFT may be or comprise a bacterial cytotoxic protein for virulence. A PFT may disrupt host cell membranes. For example, in some embodiments, a nanoparticle preparation comprising a payload displayed by an MHC class I complex may comprise one or more other agents comprising a PFT. In some embodiments, an exemplary PFT may be, for example, α-pore-forming toxin, β-barrel pore-forming toxin, large β-barrel pore- forming toxin, binary toxin, small pore-forming toxin, etc.

[0249] In some embodiments, one or more other agents may be incorporated within nanoparticles. In some embodiments, one or more other agents may be coated on nanoparticles. Those skilled in the art will appreciate desirability of incorporating particular other agents within or on nanoparticles, or both. Without wishing to be bound by any particular theory, where it is Page 84 of 340 12613923v1Docket No.: 2006517-0315 desirable to provide nanoparticle preparations that mimic one or more features of cells, e.g., of microbial cells, it may be desirable to incorporate other agents accordingly (for example, to include nucleic acids, and particularly nucleic acids containing unmethylated CpG motifs, within nanoparticles and / or lipids and / or other cell surface components on surface(s) thereof). Nanoparticle compositions

[0250] 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. In certain embodiments, certain combined elements are encapsulated within a polymer matrix.

[0251] 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. For example, in some embodiments, one or more payloads are distributed substantially homogenously within a polymer matrix.

[0252] 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.

[0253] 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.

[0254] 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.

[0255] In certain particular embodiments, the present disclosure provides nanoparticle compositions in which a coating agent is localized on the external surface of the 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 Page 85 of 340 12613923v1Docket No.: 2006517-0315 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.

[0256] 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. Without wishing to be held to a particular theory, it is contemplated that smaller nanoparticles contain more payloads than larger particles.

[0257] 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.

[0258] 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 0.1:1.

[0259] In some embodiments, a weight ratio of a payload to a polymer in a nanoparticle composition may be represented in, e.g., µg (payload) / mg (polymer). For example, in some embodiments, a payload to polymer ratio is no less than 20 µg / mg and no greater than 250 µg / mg. In some embodiments, a ratio of payload to polymer is between 20 µg / mg and 200 µg / mg. In some embodiments, a ratio of payload to polymer is between 20 µg / mg and 150 Page 86 of 340 12613923v1Docket No.: 2006517-0315 µg / mg. In some embodiments, a ratio of payload to polymer is between 20 µg / mg and 100 µg / mg. In some embodiments, a ratio of payload to polymer is between 30 µg / mg and 150 µg / mg. In some embodiments, a ratio of payload to polymer is between 30 µg / mg and 100 µg / mg. In some embodiments, a ratio of payload to polymer is between 50 µg / mg and 100 µg / mg.

[0260] In some embodiments, a weight ratio of a payload on a surface of nanoparticles to a payload in nanoparticles (e.g., encapsulated, mixed, associated within nanoparticles) 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. Without wishing to be held to a particular theory, it is contemplated that nanoparticles with a low ratio of a payload on a surface to a payload in nanoparticles are beneficial, when a payload need to be protected from endogenous RNases (e.g., a nanoparticle preparation is given by sublingual or oral administration). In some embodiments, a high ratio of a payload on a surface of to a payload in nanoparticles are beneficial when a fast release of a payload is required (e.g., for uptake by skeletal muscle cells).

[0261] 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).

[0262] 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 Page 87 of 340 12613923v1Docket No.: 2006517-0315 synergistically with administered nanoparticles such that a desirable immune response is activated in an individual to whom the nanoparticles are administered.

[0263] The present disclosure provides an 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.).

[0264] 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). In some embodiments, payloads are considered to be “encapsulated” when they are not detectable as “free” (e.g., when nanoparticles have not been disrupted); in some such embodiments, at least 80% or at least 85% or at least 90% of payload is encapsulated. For example, in some embodiments, assessment of total payload utilized in nanoparticle manufacturing, and of “free” payload detectable when nanoparticles have not been disrupted reveal that no more than about 10%, 15%, or 20% of the total payload is detected as “free” payload.

[0265] 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 Page 88 of 340 12613923v1Docket No.: 2006517-0315 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.

[0266] In some embodiments, a nanoparticle composition is characterized with respect to the size of nanoparticles, uniformity of a payload within a nanoparticle, payload content, 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.

[0267] 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.

[0268] 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.).

[0269] In some embodiments, a provided preparation may include a plurality of nanoparticle populations, each of which shares one or more structural and / or functional characteristics. For example, in some embodiments, each nanoparticle population has substantially uniform size distribution, common payload(s), and / or the same amount of the common payload(s). In some embodiments, each of nanoparticle populations comprises one or more payloads. In some embodiments, nanoparticle populations comprise different payloads or different payload combinations from each other.

[0270] In some embodiments, each of nanoparticle populations may be manufactured separately. In some embodiments, nanoparticle populations may be manufactured together initially, and then divided for post-processing, coating, adding, drying, and / or freezing.

[0271] In some embodiments, each of nanoparticle populations may include 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 Page 89 of 340 12613923v1Docket No.: 2006517-0315 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.).

[0272] In some embodiments, provided nanoparticle compositions can achieve immune modulation. Those skilled in the art, reading the present disclosure, will appreciate that, in some embodiments, it may be desirable to prepare and / or utilize nanoparticle compositions that modulate an immune response to an antigen payload toward or away from a particular type of response. For example, in some embodiments, it may be desirable to stimulate or sustain a Th1 response (e.g., when a payload is an allergen), a Th2 response (e.g., when the payload is an antigen derived from an extracellular parasite or extracellular bacteria, a Treg response (e.g., when the payload is an antigen derived from an autoantigen or alloantigen, such as antigens involved in autoimmune disease or graft-versus-host disease), etc.

[0273] In some embodiments, nanoparticle compositions with allergen payload modulate a recipient’s immune response away from a Th2 response and / or toward a Th1 and / or Treg response to such allergen. In some embodiments, nanoparticle compositions with infectious antigens modulate a recipient’s immune response toward immunity to the infectious agent from which the antigen(s) is / are derived. In some embodiments, nanoparticle compositions with cancer antigens modulate a recipient’s immune response toward a T-cell response effective against cancer cells displaying or releasing such cancer antigen(s). In some embodiments, nanoparticle compositions containing alloantigens modulate a recipient’s immune response toward desensitization to such alloantigen(s).

[0274] Among other things, the present disclosure documents stimulation of Th1-type immune reactions with nanoparticle compositions containing lipids (e.g., an E. coli lipid extract and / or lipopolysaccharide) on their surfaces. Without wishing to be bound by any particular theory, we propose that such nanoparticle compositions may be viewed by a recipient’s immune system as analogous to bacterial agents (e.g., to bacterial cells). The present disclosure proposes and demonstrates that this ability to direct a Th1-type immune response to an administered nanoparticle composition presents an opportunity to shift or otherwise bias a recipient’s immune response to one or more antigens included in the nanoparticle composition toward such a Th1- type response; such an effect is particularly useful in the treatment of allergy in an encapsulated allergen. Page 90 of 340 12613923v1Docket No.: 2006517-0315

[0275] In some embodiments, a particular subject may benefit from being exposed to a combination of antigens. For example, in some embodiments, a combination of antigens may promote an immune response to one agent (e.g., infectious agent, tumor, etc.). In some embodiments, a combination of antigens may promote an immune response to two or more agents (e.g., infectious agent, tumor, etc.). In some embodiments, a nanoparticle preparation may comprise a first nanoparticle population comprising a first payload, or precursor(s) thereof, that activate first antigen-specific T cells, and a second payload, or precursor(s) thereof, that activate second antigen-specific T cells. In some embodiments, the first payload is displayed by an MHC class I complex. In some embodiments, the second payload is displayed by an MHC class II complex. In some embodiments, a first nanoparticle population and a second nanoparticle population are included in a same composition (e.g., capsules, tablets, pills, powders, and / or granules). In some embodiments, a first nanoparticle population and a second nanoparticle population are included in different compositions. In some embodiments, a multi- NP system comprises an immune adjuvant. In some embodiments, provided nanoparticle preparation comprises one or more immune adjuvants of each of one or more antigen.

[0276] In some embodiments, when a nanoparticle preparation is desired to comprise multiple combinations of payloads (e.g., the first combination of the first antigen and the first adjuvant, the second combination of the second antigen and the second adjuvant) and the multiple combinations are chosen to be separated from each other, the nanoparticle preparation may include two or more nanoparticle populations for each combination, and two or more nanoparticle populations may be processed differently.

[0277] In some embodiments, manufacturing process of each nanoparticle population can be adapted to incorporate a particular payload effectively and / or accurately, maintaining biological activities of the payload. For example, in some embodiments, when a nanoparticle preparation is desired to have two or more payloads, and the payloads are chosen to be separated from each other or the payloads require different manufacturing processes, the nanoparticle preparation may include two or more nanoparticle populations for each payloads (e.g., the first nanoparticle population for the first payload, and the second nanoparticle population for the second payload), and two or more nanoparticle populations may be processed separately and / or differently.

[0278] In some embodiments, when a nanoparticle preparation is desired to include a payload having multiple concentrations and / or amounts, a nanoparticle preparation may include two or Page 91 of 340 12613923v1Docket No.: 2006517-0315 more nanoparticle populations for each concentration / amount, and two or more nanoparticle populations may be processed separately to facilitate multiple concentrations and / or amounts.

[0279] In some embodiments, a provided nanoparticle composition may be characterized by a safety factor (e.g., when measured as described in Example 3, 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 target safety factor is greater than about 10. 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.

[0280] The present disclosure provides an 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). The mean size of nanoparticles may be measured using a Z-average. 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.). In some preferred embodiments, a population of nanoparticles are about 225nm to about 450nm.

[0281] In some embodiments, a population of nanoparticles is represented by a particular mean size (e.g., 150 nm), but is itself comprised of more than one population of nanoparticles.

[0282] 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. In some embodiments, nanoparticles comprise approximately 20-90 µg, 30-90 µg, or 50-75 µg payload / mg polymer. Page 92 of 340 12613923v1Docket No.: 2006517-0315

[0283] In some embodiments, a nanoparticle composition has a total protein concentration of 100-10,000 ug / mL. In some embodiments, a nanoparticle composition has a total protein concentration of 500-5,000 ug / mL. In some preferred embodiments, a nanoparticle composition has a total protein concentration of 1,800-3,500 ug / mL.

[0284] The present disclosure provides an 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) 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%.

[0285] In some embodiments, purification procedures are altered to selectively eliminate and / or selectively enrich for a particular population of nanoparticles.

[0286] In some embodiments, a provided nanoparticle preparation may have a zeta potential range of about – 50 mV to about 0 mV, about – 40 mV to about 0 mV, about – 30 mV to about 0 mV, about – 20 mV to about 0 mV, or about – 15 mV to about 0 mV.

[0287] In some embodiments, a provided nanoparticle preparation may be suitable to be stored (e.g., stable) at temperature at or above -80 °C, -70 °C, -60 °C, -50 °C, -40 °C, -30 °C, -20 °C, -4 °C, 0 °C, or room temperature.

[0288] In some embodiments, the present disclosure provides a nanoparticle preparation prepared by the methods provided herein, and the nanoparticle preparation comprises a plurality of nanoparticles, each of which comprises a payload (e.g., a hydrophilic payload) in a polymer.

[0289] In some embodiments, a separation step comprising centrifugation results in a composition comprising nanoparticles with improved PDI (e.g., a lower PDI as compared to a population of nanoparticles produced via control or unoptimized methods) as compared to a separation step not comprising centrifugation. For example, in some embodiments, a composition comprising nanoparticles of the present disclosure has a PDI of less than about 0.5, 0.4, 0.3, 0.2, 0.1. In some preferred embodiments, a PDI is less than about 0.3, 0.2, 0.1. In some preferred embodiment, a PDI is from about 0.1 to about 0.4. Page 93 of 340 12613923v1Docket No.: 2006517-0315 Pharmaceutical Compositions

[0290] In some embodiments, the present disclosure provides pharmaceutical compositions comprising one or more provided nanoparticle compositions together with one or more pharmaceutically acceptable excipients.

[0291] 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.

[0292] 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.

[0293] In many embodiments, provided pharmaceutical compositions are specifically formulated for mucosal delivery (e.g., oral, nasal, rectal or sublingual delivery).

[0294] 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, 21stEdition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2005; incorporated herein by reference) discloses various Page 94 of 340 12613923v1Docket No.: 2006517-0315 excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof.

[0295] 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.

[0296] 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.

[0297] 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 1,3-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 Page 95 of 340 12613923v1Docket No.: 2006517-0315 mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of liquid formulations.

[0298] 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.

[0299] In some embodiments, one or more strategies may be utilized prolong and / or delay the effect of a provided nanoparticle composition after delivery.

[0300] 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.

[0301] 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, pills and tablets, the dosage form may comprise buffering agents.

[0302] 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 capsules, Page 96 of 340 12613923v1Docket No.: 2006517-0315 pills, and tablets, 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.

[0303] 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 (PVAlP); 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.

[0304] 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.

[0305] 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.

[0306] 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. Page 97 of 340 12613923v1Docket No.: 2006517-0315

[0307] 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.

[0308] 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.

[0309] 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.).

[0310] In some embodiments, a pharmaceutical composition may include one or more stabilizing agents, such as one or more cryoprotectants. In some embodiments, a stabilizing agent may be or comprise a sugar such as sucrose and / or trehalose. The present disclosure teaches that such agent(s) may be particularly useful for emulsion formulations and / or liquid formulations, and particularly for liquid emulsion formulations. Page 98 of 340 12613923v1Docket No.: 2006517-0315

[0311] Alternatively or additionally, in some embodiments, a pharmaceutical composition may include an agent such as TCA and / or PVA, one or both of which may be particularly useful for emulsion formulations and / or liquid formulations, and particularly for liquid emulsion formulations. Characterization of compositions and components thereof

[0312] In some embodiments, provided compositions may be characterized in order to determine, for example, protein content per nanoparticle. Those skilled in the art will be aware of a variety of technologies available to characterize nanoparticle compositions provided in accordance with the present disclosure.

[0313] For example, in some embodiments, characterization may include, e.g., one or more of assessing (e.g., identifying and / or quantifying) presence of polymer component, payload component(s), coating agent(s) and / or other agent(s), determining intactness of such polymer component, payload component(s), coating agent(s) and / or other agent(s), assessing degree of encapsulation of one or more payload component(s), determining extent of coating, assessing relative amount(s) (e.g., weight percent) of different components – e.g., payload to polymer, coating to polymer, coating to payload, etc., assessing one or more features of particle size and / or particle size distribution, determining microbial load, quantifying payload encapsulation efficiency, assessing content of payload (e.g., determining if payload contains expected amounts and / or forms), evaluating a surface coating, etc.

[0314] 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 0.1:1, 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., µg (payload) / mg (polymer). For example, in some embodiments, a payload to polymer ratio is no less than 30 µg / mg and no greater than 250 µg / mg. In some embodiments, a ratio of payload to polymer is between 30 µg / mg and 150 µg / mg. In some embodiments, a ratio of payload to polymer is between 50 µg / mg and 100 µg / mg.

[0315] In some embodiments, as described herein, a weight of payload in an individual dose of the nanoparticles is within a range of about 1-10µg, 5-50µg, 10-100µg, 50-500µg, 100-1000µg, 500-5000µg, 1000-10,000µg. Page 99 of 340 12613923v1Docket No.: 2006517-0315

[0316] 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 100%; 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%.

[0317] 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).

[0318] 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. By way of non-limiting example, when an aliquot of nanoparticle suspension solution is removed, the aliquot can be analyzed to determine, e.g., free payload 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 rcf), hydrolyzing said suspension with NaOH, and then analyzing using an assay that measures payload content (e.g., BCA, Bradford, etc. when a payload is or comprises protein). 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 payload without damaging any already formed nanoparticles. Once an assay has been performed, the resulting number(s) represent quantification of total payload per volume of suspension. Remaining suspension (i.e., suspension that has not been analyzed via payload measurement assay) can then be spun down using an ultracentrifuge (e.g., spinning at or about 350,000 rcf), and resulting supernatant analyzed for free payload, resulting in another measurement of total Page 100 of 340 12613923v1Docket No.: 2006517-0315 payload 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.

[0319] 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 rcf) 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.

[0320] In some embodiments, method 1 is a preferred method for characterizing quantity of free payload 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.

[0321] In some embodiments, presence, amount, form and / or integrity of one or more non- payload components is determined (e.g., quantified). For example, in some embodiments, polymer component (e.g., PLG) is detected, quantified and / or assessed for integrity. Alternatively or additionally, presence, amount, form and / or integrity of one or more other components (e.g., solvent or medium, such as water; cryoprotectant such as trehalose, stabilizer such as PVA, etc., is quantified.

[0322] In certain particular embodiments, nanoparticle compositions may be or include nanoparticles that are substantially free of a payload component; in some such examples, presence, amount, form and / or integrity of one or more non-payload components can be determined.

[0323] Dynamic Light Scattering (“DLS”) is a method that can be used to characterize nanoparticles of the present disclosure and can allow for measures such as polydispersity index (“PDI”), which is a measure of size distribution of a given population of, for example, nanoparticles. In some embodiments, a separation step comprising centrifugation results in a composition comprising nanoparticles with improved PDI (e.g., a lower PDI as compared to a population of nanoparticles produced via control or unoptimized methods) as compared to a Page 101 of 340 12613923v1Docket No.: 2006517-0315 separation step not comprising centrifugation. For example, in some embodiments, a composition comprising nanoparticles of the present disclosure has a PDI of less than about 0.5, 0.4, 0.3, 0.2, 0.1. In some preferred embodiments, a PDI is less than about 0.3, 0.2, 0.1.

[0324] In some embodiments, characterization of nanoparticles includes evaluation using dynamic light scattering (“DLS”) and / or polydispersity index (“PDI”). 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. In some embodiments, dynamic light scattering may provide information that can be used to alter manufacturing protocols. For example, in some embodiments, 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. In some embodiments, PDI may be used to evaluate the breadth of distribution of nanoparticle sizes in a given sample. In some embodiments, if PDI shows increased numbers (e.g., greater than 0.5, 0.6, 0.7, 0.8), DLS data may not be able to accurately quantify the size distribution of a sample of nanoparticles.

[0325] In some embodiments, DLS data are expressed as z-averages. In some embodiments, when two populations are compared, a lower z-average in one population indicates a more uniform / homogenous set of nanoparticles as compared to the other population.

[0326] PDI measurements are unitless and, in some embodiments, when comparing two nanoparticle populations, a lower PDI in one population indicates a more uniform / homogenous sample as compared to the other.

[0327] In some embodiments, a z-average is lower than 500 nm. In some embodiments, a z- average is lower than 450, 400, 350, 300, 250 nm.

[0328] In some embodiments, a PDI measurement is lower than 0.7, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, and lower. Characterization of payload

[0329] In some embodiments, a payload of nanoparticle compositions is evaluated in one or more ways at one or more times.

[0330] For example, in some embodiments, payload 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, Page 102 of 340 12613923v1Docket No.: 2006517-0315 for example to ensure that processing into nanoparticles has not materially altered payload components.

[0331] Alternatively or additionally, in some embodiments, payload component(s) may be characterized while nanoparticles are still intact, for example to assess extent to which payload may be present on nanoparticle surface(s) and / or may have been released from nanoparticles.

[0332] Those skilled in the art will be aware of a variety of technologies available for evaluating payloads of different types (e.g., proteins, nucleic acids, carbohydrates, etc.), and will be able to select appropriate such technologies depending on, for example, type and / or identity of payload component(s) to be assessed.

[0333] In some embodiments, it may be desirable to assess overall amount of one or more payload component(s). In some embodiments, it may be desirable to assess “intactness” of one or more payload components. In some embodiments, it may be desirable to assess specific identity of a payload component(s); in some embodiment(s) detection of payload type (e.g., protein, nucleic acid) may be sufficient. Again, those skilled in the art will be aware of appropriate analysis technologies (e.g., ELISA to assess identity of a payload component, particularly of a protein payload component, vs BCA for protein content generally). Release Testing

[0334] 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 to subjects (e.g., human subjects) if qualifying criteria (e.g., predefined qualifying criteria) are met; thereby evaluating, identifying, and / or producing (e.g., manufacturing) a nanoparticle composition.

[0335] 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 (e.g., of a therapeutic drug substance or drug product), 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 Page 103 of 340 12613923v1Docket No.: 2006517-0315 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 someembodiments, a given nanoparticle 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.

[0336] In some embodiments, evaluations 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 / or 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 Page 104 of 340 12613923v1Docket No.: 2006517-0315 comparison to a reference standard, a batch from which a preparation is taken can be processed, e.g., as just described.

[0337] 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).

[0338] In some embodiments, methods (e.g.,., evaluation, identification, and / or 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.

[0339] In some embodiments, methods (e.g.,., evaluation, identification, and / or 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, Page 105 of 340 12613923v1Docket No.: 2006517-0315 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.

[0340] 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., NP-PN1). In some embodiments, processing can include directing and / or contracting another party to process as described herein. Protein Quantification

[0341] In many embodiments, provided nanoparticular compositions comprise a protein component (e.g., a protein payload). In some embodiments, it may be desirable to assess one or more features (e.g., extent of encapsulation, intactness and / or activity while encapsulated, degree and / or timing of release, intactness and / or activity when released, etc) of such protein.

[0342] Those skilled in the art will be aware of various technologies for assessing one or more aspects of a protein component, such as for example its level, form, degree of intactness, activity, extent of encapsulation, timing and / or extent of release, etc. Moreover, those skilled in the art reading the present disclosure will appreciate that it may be desirable to do such assessments at one or more times during manufacture, storage and / or use of provided nanoparticle compositions.

[0343] In some embodiments, protein is assessed using a BCA assay. For example, in some embodiments, a BCA assay may be used to quantify protein in a particular sample (e.g., in a nanoparticle preparation supernatant, and / or after disruption of nanoparticles in a preparation).The present disclosure appreciates that, in some embodiments, performance of one or more assays, specifically including BCA assays, can be impacted by one or more features of nanoparticle production. For example, the present disclosure identifies the source of a problem that can be encountered when PVA is utilized during manufacturing of nanoparticle compositions. PVA solubility is reduced in cold solutions. Without wishing to be bound by any particular theory, the present disclosure proposes that PVA, at least when present in amounts above a particular threshold, may have a tendency to gel under certain circumstances, for Page 106 of 340 12613923v1Docket No.: 2006517-0315 example when made cold and / or when exposed to basic conditions (and / or specifically to NaOH). The present disclosure appreciates that such insolubility and / or gelling may interfere with TFF and / or with other aspect(s) of nanoparticle composition producing, processing and / or assessment. The present disclosure specifically teaches that, in some embodiments, it may be desirable or even necessary to reduce PVA in, or remove PVA from, a nanoparticle preparation prior to performance of a BCA assay thereon.

[0344] In some embodiments, PVA is present during protein measurement.

[0345] In some embodiments, PVA is at least partially removed prior to measuring protein quantity. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of PVA is removed prior to protein measurement.

[0346] In some embodiments, PVA is removed using TFF.

[0347] In some embodiments, if a TFF step is performed to remove PVA, the material is dried (e.g., lyophilized) prior to performing the TFF step.

[0348] In some embodiments, PVA needs to be removed from a sample prior to dissolving the sample in cold buffer. In some such embodiments, if an amount of PVA is greater than 1%, a sample will not be likely to dissolve in cold buffer, leaving undissolved solid material.

[0349] In some embodiments, one or more lyophilization steps that may otherwise be performed, is / are not performed if the amount of PVA present in a particular sample or intermediate composition is above a particular threshold. For example, in some embodiments, if PVA is not removed, one or more lyophilization steps that may otherwise be performed is skipped. In some such embodiments, if one or more lyophilization steps is skipped, it occurs prior to performing filtration. In some embodiments, not completing one or more lyophilization steps occurs before performing a TFF step.

[0350] In some embodiments, a certain amount or concentration of PVA (e.g., 0.5, 1, 2, 3, 4, 5%) may negatively impact accuracy of a BCA assay.

[0351] In some embodiments, concentrations of reagents (e.g., NaOH) may be varied (e.g., 0.5M, 1M, 1.5M, 2M, 2.5M) when performing a BCA assay or any steps (e.g., preparation of sample) related thereto.

[0352] In some embodiments, a standard curve may need to be adjusted, for example to accommodate conditions employed in the production of a particular batch of composition (e.g., of nanoparticles). For example, in some embodiments, one or more standard curves may be Page 107 of 340 12613923v1Docket No.: 2006517-0315 generated using, e.g., NaOH, Ammonium Bicarbonate buffer, or a mixture of NaOH and Ammonium Bicarbonate buffer. In some such embodiments, standard curve adjustments will be determined by measuring total and free protein concentrations (TPC and FPC) in different solvent mixtures. For example, in some embodiments, a standard curve for a BCA assay may be generated using 2 / 3 Ammonium Bicarbonate buffer and 1 / 3 NaOH. In some embodiments, these standard curves may reflect conditions used to process samples for BCA assay analyses (e.g., samples comprising a certain ratio of NaOH and / or ammonium bicarbonate buffer). The present disclosure also provides the insight that BCA assay accuracy may be impacted (e.g., improved or reduced) due to one or more components of a solvent. Preparation and testing of solvent mixtures for standard curves. Accordingly, in some embodiments, it is important that standard curves with components of solvent mixtures (e.g., NaOH and ammonium bicarbonate) are prepared.

[0353] In some embodiments, more NaOH than is used in standard protocols may be used in samples as described herein. For example, the present disclosure provides the insight that, in some embodiments, higher concentrations of NaOH or ammonium bicarbonate may be required to process samples for BCA analysis. In some such embodiments, standard curves will be prepared and tested to reflect changes in processing steps (e.g., changes in solvent mixtures used in a given assay and / or preparation). Assessing Payload Exposure

[0354] In some embodiments, technologies provided by the present disclosure permit or otherwise include one or more assessments of payload exposure – i.e., the degree to which a subject receiving a nanoparticle composition becomes exposed to its payload while nanoparticles remain intact.

[0355] In some embodiments, a payload included in a nanoparticle composition and / or otherwise delivered through administration of such nanoparticle composition is or has the potential to be harmful to the recipient. For example, in some embodiments, a payload included in and / or otherwise delivered through administration of such nanoparticle composition is an allergen to which the subject is or may be allergic; in some such embodiments, the allergen is an anaphylactic allergen (e.g., a food allergen such as a peanut and / or milk allergen, a venom, etc). Page 108 of 340 12613923v1Docket No.: 2006517-0315 Assessment of degree of payload encapsulation, and / or of subject exposure to payload upon administration may be particularly useful or important in such contexts.

[0356] In some embodiments, the present disclosure provides and / or utilizes one or more assessments of allergen encapsulation. For example, in some embodiments, a composition comprising nanoparticles encapsulating a payload is assayed to determine quantity of payload; in some embodiments, encapsulated payload is determined, for example, by quantifying “free” (outside of nanoparticles) payload and “total” payload (amount detectable after nanoparticles have been disrupted); the difference is encapsulated payload. Encapsulated payload as a percentage of total payload may be referred to as the “Encapsulation Ratio”.

[0357] In some embodiments, a nanoparticle preparation in accordance with the present disclosure is substantially free of detectable unencapsulated payload (in a relevant assay – e.g., a detection assay such as an ELISA, or an activity assay such as an assessment of immune impact or toxicity).

[0358] In some embodiments, a nanoparticle preparation is characterized in that it includes less detectable “free” payload than does a comparable preparation of payload (e.g., containing an equivalent amount of payload to that used to generate the nanoparticle composition) that is not so encapsulated. In some such embodiments, amount of detectable “free” payload is assessed through binding detection of the payload. Alternatively or additionally, in some embodiments, amount of detectable “free” payload is determined by assessing impact of such payload on a relevant system. To give but one specific example, in some embodiments, where the payload is or comprises an antigen, amount of detectable “free” payload can be or comprise detection of an immune response in an appropriate system – e.g., an allergic response to an allergen in a system (e.g., cell, tissue, organism) reactive to such allergen.

[0359] In some embodiments, reactivity of a composition is determined using an assay in which cells (e.g., immune cells, e.g., basophils) are exposed to either encapsulated or free payload and reactivity of the cells is measured. In some embodiments, reactivity of a composition is determined using whole blood basophil activation test (BAT). In some embodiments, reactivity to encapsulated payload is reduced as compared to reactivity to an equivalent quantity of free payload (e.g., weight / weight equivalents). That is, in some embodiments, a subject may consume a greater quantity of encapsulated payload without reaction as compared to the same or lesser amount of free payload (unencapsulated). In some embodiments, reactivity to Page 109 of 340 12613923v1Docket No.: 2006517-0315 encapsulated payload is reduced by a certain amount relative to reactivity to an equivalent (ug) quantity of free payload. In some embodiments, the reduction of reactivity of encapsulated payload is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 85, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000-fold or greater, as compared to payload that is unencapsulated. Manufacturing Nanoparticle Compositions

[0360] 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.

[0361] Furthermore, in some embodiments, the present disclosure provides insights that permit preparation of nanoparticle compositions that comprise payloads and / or coating agents (e.g., complex payloads and / or 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). Still further alternatively or additionally, in some embodiments, the present disclosure provides technologies that permit preparation of nanoparticle compositions incorporating one or more fragile payloads. Yet further alternatively or additionally, in some embodiments, the present disclosure provides scalable technologies, amenable to commercial scale production of nanoparticle preparations as described here.

[0362] As noted, the present disclosure provides an insight that nanoparticles comprising fragile (e.g., susceptible to damage from energy input during manufacturing) and / or complex payloads can be prepared by manufacturing processes described herein. Among other things, the present disclosure identifies a problem with conventional nanoparticle manufacturing technologies in that they typically involve one or more steps that utilize harsh manufacturing conditions (e.g., high temperature, pressure, shear force, etc.). The present disclosure appreciates that such conditions can degrade fragile payloads, and / or can decrease one or more biological or pharmaceutical activities of fragile and / or complex payloads. The present disclosure notes that, Page 110 of 340 12613923v1Docket No.: 2006517-0315 as one skilled in the art will be aware, many such existing nanoparticle manufacturing technologies could only incorporate fragile payloads if such payloads could be introduced after nanoparticle manufacture were complete; the present disclosure appreciates that such strategies would be unlikely to achieve encapsulation of such fragile payloads and therefore would not achieve certain advantages provided by preparations as provided by the present disclosure.

[0363] Teachings provided by the present disclosure are particularly applicable to preparations of polymer nanoparticles comprising payload(s). 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 and / or payloads.

[0364] In many embodiments, methodologies provided by the present disclosure utilize an initial polymer / payload preparation, 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 preparation. For example, in some embodiments, an initial polymer / payload preparation is made from mixing a polymer solution and a payload solution. In some embodiments, an initial polymer / payload preparation is made by dissolving dry polymer and dry payload in a solvent system. In some embodiments, a polymer solution is made by dissolving polymer (e.g., that is or comprises PLG) into organic liquid (e.g., that is or comprises dimethyl sulfoxide (DMSO), acetone, acetonitrile, tetrahydrofuran, or a combination thereof). In some embodiments, a payload solution is made by dissolving payload into water to produce an aqueous solution. In some embodiments, preparation of an aqueous solution also involves pH adjustment (e.g., using buffers, NaOH, etc.), and / or application of disruptive energy and / or force such as, e.g., sonication, and / or homogenization. In some embodiments, an initial polymer / payload preparation is prepared by combining a payload (aqueous) and a polymer (organic) solution. In some embodiments, an initial polymer / payload preparation is prepared by adding a payload (aqueous) solution into a polymer (organic) solution. In some embodiments, an initial polymer / payload preparation is prepared by adding a polymer (organic) solution into a payload (aqueous) solution. In some embodiments, an initial polymer / payload preparation is prepared by solubilizing a dry material containing both polymer and payload. In some embodiments, dry material is added slowly or in steps; in some embodiments, added dry material Page 111 of 340 12613923v1Docket No.: 2006517-0315 is permitted to solubilize substantially completely before a further addition of dry material is made.

[0365] As discussed herein above, provided technologies are amenable to encapsulation of a variety of different payloads. To reiterate just a few examples here, in some embodiments, provided nanoparticle compositions include a payload selected from the group consisting of polypeptides, nucleic acids, carbohydrates (e.g., polysaccharides), and combinations thereof. In many embodiments, a payload is or comprises a polypeptide. In many embodiments, a payload is or comprises a nucleic acid; in some such embodiments, a payload is or comprises a long nucleic acid (e.g., a gene therapy vector, an mRNA, etc); in some embodiments, a payload is or comprises a partly or wholly single stranded nucleic acid).

[0366] In some embodiments, a payload is or comprises a RNA. In some embodiments, and RNA payload is an mRNA. In some embodiments, an RNA payload has a length within a range of about 15 to about 3,000,000 residues. In some embodiments, an RNA payload has a length within a range of about 500 to 50000 residues. In some embodiments, an RNA payload has a length within a range of about 1000 to about 10000 residues.

[0367] In some embodiments, a provided nanoparticle preparation is manufactured using one or more relatively complex components (e.g., a payload and / or a coating agent that is a relatively crude extract or combination of components).

[0368] In some embodiments, the present disclosure provides technologies in which a nanoparticle preparation is manufactured by (i) providing a first liquid preparation, which comprises a payload (e.g., a fragile payload and / or a complex payload, and / or a combination of payloads wherein one or more may be a fragile and / or complex payload) in a first aqueous solvent system and a second liquid preparation, which comprises a polymer (e.g., a hydrophobic polymer) in a second solvent system; (ii) combining the first and second preparations to form a mixture that comprises the payload and the polymer in a combined solvent system, and (iii) adding a liquid non-solvent system to the mixture, so that a population of nanoparticles comprising the payload and the polymer is formed (e.g., wherein the method does not involve energy input such as, for example, input of heat) (e.g., wherein the non-solvent system does not degrade the payload, or decrease one or more biological or pharmaceutical activities of the payload) (e.g., wherein one or more biological or pharmaceutical activities of payload are substantially same before and after the step of adding). Page 112 of 340 12613923v1Docket No.: 2006517-0315

[0369] Alternatively or additionally, in some embodiments, the present disclosure provides manufacturing technologies that include a heterogeneous, layered two-fluid process that achieves remarkable uniformity in nanoparticle production (e.g., in average size and / or in polydispersity); typically, the process utilizes mild mixing at the fluid interface. In some embodiments (see, for example, Example 10), this heterogeneous, layered two-fluid process is referred to as a “Tequila Sunrise” process. FIG.1B provides a flow diagram for an exemplary Tequila Sunrise process as utilized in Example 10 to produce PLG nanoparticles including crude peanut extract and fragmented E. coli DNA payloads.

[0370] In some such Tequila Sunrise embodiments, including as described in Example 10 and depicted in FIG.1C, an aqueous lipid preparation including payload(s) is combined with a hydrophobic (e.g., DMSO) liquid preparation including a hydrophobic polymer (e.g., PLG), and the combination is optionally concentrated (e.g., by rotovap) before Tequila Sunrise nanoprecipitation is performed.

[0371] In some embodiments, the present disclosure provides nanoparticle manufacturing technologies in which (i) payload materials and polymer materials are combined in the presence of a solvent / antisolvent system; typically at least the payload material(s) are sufficiently hydrophilic to be provided in water or other aqueous system (the present disclosure provides an insight that use of an organic antisolvent can reduce payload loss during the encapsulation process); and (ii) the combined materials are mixed in an intentionally heterogeneous, layered two-fluid process, that typically involves mild mixing (quite different from conventional teachings of desirability or even necessity of intense mixing to homogenize a solvent / antisolvent mixture) at the fluid interface. The present disclosure demonstrates that this approach achieves surprising and remarkable consistency in nanoparticle size (e.g., average size) and / or polydispersity. Furthermore, the approach is scalable and, thanks to its gentle conditions, is particularly useful for the incorporation of fragile payloads (e.g., nucleic acid, polypeptide and / or carbohydrate (e.g., polysaccharide) payloads).

[0372] In some embodiments, provided manufacturing technologies utilize a solvent system that comprises water and DMSO. In some such embodiments, a volume ratio of water and DMSO is within a range of 1:99 to 20:80, or 1:99 to 10:90.

[0373] In some embodiments, provided technologies utilize an anti-solvent system (which may in some embodiments be referred to as a non-solvent system). In some embodiments, an anti- Page 113 of 340 12613923v1Docket No.: 2006517-0315 solvent system is or comprises an alcohol. In some embodiments, an anti-solvent system is or comprises propanol, ethanol, methanol, or combination thereof. In some embodiments, an anti- solvent is or comprises IPA.

[0374] In certain embodiments, provided nanoparticle manufacturing technologies may include one or more homogenization steps.

[0375] In certain embodiments, provided nanoparticle manufacturing technologies may utilize one or more stabilizers. For example, in some embodiments, deoxycholate may be utilized (e.g., being included at least in a homogenization step).

[0376] In some embodiments, provided nanoparticle manufacturing technologies achieve a ratio of payload to polymer in the nanoparticles that is between about 0.1 to about 0.9 of the ratio of payload to polymer in the original mixture from which nanoparticles are precipitated.

[0377] Typically, at least one polymer is present in an initial polymer / payload preparation 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.

[0378] 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.

[0379] 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 preparation. 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 preparation. In some embodiments, polymer and payload are present in an initial polymer / payload preparation in relative amounts such that, when the solution is processed as described herein, they are present in a nanoparticles as described herein Page 114 of 340 12613923v1Docket No.: 2006517-0315 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).

[0380] 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 preparation (e.g., solution). 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).

[0381] 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 preparation. In some such embodiments, additional steps as further described herein, may be employed to achieve a desirable distribution of payload with respect to polymer.

[0382] In some embodiments, provided technologies include one or more steps that remove solvent (e.g., the combined solvent / antisolvent system).

[0383] In some embodiments, a solvent system used to prepare an initial polymer / payload preparation 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 preparation 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.

[0384] In some embodiments, a solvent system used to prepare an initial polymer / payload preparation 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 into 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.

[0385] In some embodiments, an initial polymer / payload preparation may include one or more other components in addition to polymer and payload. To give but a few examples, in some Page 115 of 340 12613923v1Docket No.: 2006517-0315 embodiments, an initial polymer / payload preparation 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.

[0386] In some embodiments, during preparation of an initial polymer / payload preparation, 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. For example, in some embodiments, stirring is conducted under conditions that do not exert a high shear force. In some embodiments, stirring is performed at room temperature. In some embodiment, stirring is performed below room temperature, but above a freezing temperature of any component present in a preparation being stirred.

[0387] 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.

[0388] 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.

[0389] In some embodiments, mixing is performed at a temperature within a range of about 15 °C to 30 °C (e.g., 15-25oC, 15-20oC, or 20-30oC). 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).

[0390] In some embodiments, sedimentation (e.g., centrifugation) is performed during preparation of an initial polymer / payload preparation. For example, after dissolution of a solid material (e.g., payload or polymer) in a solvent system (e.g., prior to mixing payload and Page 116 of 340 12613923v1Docket No.: 2006517-0315 polymer), a solution may be centrifuged to remove aggregated, undissolved and / or partially dissolved solid material.

[0391] In some embodiments, an initial polymer / payload preparation is characterized by certain material properties. In some embodiments, an initial polymer / payload preparation is not turbid (e.g., is substantially transparent).

[0392] 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. Optional Concentration

[0393] In some embodiments, a polymer / payload combination (e.g., an initial polymer / payload preparation) is concentrated. In some embodiments, concentration (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 preparation)) may ultimately increase encapsulation of payload in polymer, in subsequent steps. In some embodiments, concentration improves one or more properties of a nanoparticle in subsequent steps (e.g., shape, size, payload: polymer ratio, etc.).

[0394] Without wishing to be held to a particular theory, it is contemplated that excess water and / or other solvent(s) may interfere with obtaining homogenous nanoparticles. Accordingly, in some embodiments, concentration of an initial polymer / payload preparation (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)) before an initial polymer / payload preparation is further manipulated (e.g., addition of a non-solvent system) (see, e.g., FIG.6). Page 117 of 340 12613923v1Docket No.: 2006517-0315

[0395] In some embodiments, polymer / payload combination (e.g., solution) homogeneity may desirably be improved by concentration; 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., precipitation). 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.

[0396] In some embodiments, a polymer / payload combination (e.g., solution) is concentrated using evaporation methods. In some embodiments, an initial polymer / payload preparation (e.g., solution) is concentrated using evaporation methods. In some embodiments, a utilized evaporation method is or comprises rotary evaporation.

[0397] 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, a percentage of water and / or other solvent(s) removed from a polymer / payload 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%.

[0398] 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.

[0399] In some embodiments, polymer and payload are present at a weight ratio within a range of 40:60 to 99:1 in a polymer / payload combination after concentration. In some embodiments, polymer and payload are present at a weight ratio within a range of 40:60 to 95:1 in a polymer / payload combination after concentration. In some embodiments, polymer and payload are present at a weight ratio within a range of 50:50 to 99:1 in a polymer / payload combination Page 118 of 340 12613923v1Docket No.: 2006517-0315 after concentration. In some embodiments, polymer and payload are present at a weight ratio within a range of 50:50 to 95:1 in a polymer / payload combination after concentration. In some embodiments, polymer and payload are present at a weight ratio within a range of 60:40 to 99:1 in a polymer / payload combination after concentration. In some embodiments, polymer and payload are present at a weight ratio within a range of 60:40 to 99:1 in a polymer / payload combination after concentration. In some embodiments, polymer and payload are present at a weight ratio within a range of 60:40 to 95:1 in a polymer / payload combination after concentration. In some embodiments, polymer and payload are present at a weight ratio within a range of 70:30 to 99:1 in a polymer / payload combination after concentration. In some embodiments, polymer and payload are present at a weight ratio within a range of 70:30 to 95:1 in a polymer / payload combination after concentration. In some embodiments, polymer and payload are present at a weight ratio 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.

[0400] 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. 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 º C and 120 º C; in some embodiments, such temperature may be within a range of, for example, about 25 º C and about 90 º C, and / or within a range that does not exceed about 90 º C, about 85 º C, about 80 º C, or about 78 º C, and / or at a temperature of about 75 º C.

[0401] 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 Page 119 of 340 12613923v1Docket No.: 2006517-0315 rpm. In some embodiments, where rotation is used, a rotary evaporator may be set to achieve a speed of about 60 rpm.

[0402] 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.

[0403] In some embodiments, concentration (e.g., evaporation, e.g., rotary evaporation) may be performed at a particular pressure or pressures (e.g., approximately 1-5 torr), for example at about 1 torr or about 2 torr or about 3 torr.

[0404] 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, e.g., relative to otherwise comparable processes that do not include such concentration. Nanoparticle Precipitation

[0405] The present disclosure provides the insight that a non-solvent system can initiate and / or allow for the precipitation of polymer and payload from a polymer / payload combination (e.g., from a concentrated polymer / payload solution). In some embodiments, precipitation of polymer and payload in a polymer / payload combination creates a nanoparticle preparation (e.g., a nanoparticle suspension) comprising nanoparticles.

[0406] The present disclosure provides an insight that certain non-solvent system(s) may allow for the precipitation of nanoparticles in mild conditions (e.g., conditions do not require energy input, such as increasing temperature and / or pressure, applying shear force). In some embodiments, provided methods allow for the precipitation of fragile (e.g., susceptible to damage from energy input, such as increasing temperature, pressure, applying shear force, etc.) and / or complex payloads.

[0407] The present disclosure provides an insight that provided methods generate a uniform / homogenous set of nanoparticles (e.g., with respect to size of nanoparticles, uniformity of a payload within a nanoparticle, payload content, release rate of payload and / or surface Page 120 of 340 12613923v1Docket No.: 2006517-0315 exposure of payloads). In some embodiments, uniform / homogenous characteristics of provided nanoparticles allow for the removal of certain purification / sorting steps (e.g., centrifugation).

[0408] Without wishing to be bound by any particular theory, addition of a non-solvent system may allow for the simultaneous precipitation of polymer and payload. In some embodiments, it is contemplated simultaneous precipitation (e.g., co-precipitation) of polymer and payload generates nanoparticles comprising both polymer and payload. In some embodiments, it is contemplated polymer and payload nucleate and grow separately. In some embodiments, nucleated / grown polymer and payload aggregate into nanoparticles. In some embodiments, it is contemplated simultaneous precipitation (e.g., co-precipitation) of polymer and payload results in nanoparticles having uniform and / or homogeneous distribution of polymer and payload therein.

[0409] In some embodiments, payload is substantially insoluble in a non-solvent system. In some embodiments, polymer is substantially insoluble in a non-solvent system. In some embodiments, payload and polymer are substantially insoluble in a non-solvent system.

[0410] In some embodiments, a volume ratio of a solvent system for a polymer / payload preparation to a non-solvent system is within a range of 1:0.1 to 1:1000, 1:0.1 to 1:100, 1:0.1 to 1:10, 1:0.1 to 1:1000, 1:1 to 1:1000, 1:1 to 1:100, 1:1 to 1:10, or 1:5 to 1:10 in a nanoparticle suspension.

[0411] In some embodiments, a non-solvent system comprises one solvent. In some embodiments, a non-solvent system comprise two or more solvents. In some embodiments, a non-solvent system comprises a solvent selected from the group consisting of propanol, ethanol, methanol, and combinations thereof.

[0412] In some embodiments, a non-solvent system provides mild precipitation conditions (e.g., conditions do not require energy input, such as increasing temperature and / or pressure, applying shear force). In some embodiments, a non-solvent system allows for the precipitation of nanoparticles without requiring high temperature, high pressure, or high shear force. In some embodiments, a non-solvent system does not have high or low pH, and / or high ionic strength.

[0413] In some embodiments, nanoparticle precipitation is performed by combining a polymer / payload preparation and a non-solvent system. In some embodiments, a polymer / payload preparation is added (e.g., poured, injected, dropped) into a non-solvent system. Page 121 of 340 12613923v1Docket No.: 2006517-0315 In some embodiments, a non-solvent system is added (e.g., poured, injected, dropped) into a polymer / payload preparation.

[0414] In some embodiments, nanoparticle precipitation is performed by adding (e.g., injecting, inserting) a polymer / payload preparation into a non-solvent system. In some embodiments, adding (e.g., injecting, inserting) a polymer / payload preparation into a non-solvent system results in at least two separate layers: a polymer / payload preparation and a non-solvent system. In some embodiments, adding (e.g., injecting, inserting) a payload / polymer preparation into a non-solvent system comprises adding the payload / polymer preparation under the non-solvent system. For example, in some embodiments, a non-solvent system is present in a container (e.g., a beaker) and a payload / polymer preparation is added to the non-solvent system by dispensing the payload / polymer preparation at the interface between the non-solvent system and the container (e.g., the bottom of the container). In some embodiments, where a payload / polymer preparation is added to a non-solvent system, the non-solvent system comprises one or more or isopropyl alcohol (IPA), dimethyl-sulfoxide (DMSO).

[0415] Without wishing to be bound by any particular theory, adding (e.g., injecting, inserting) a polymer / payload preparation into a non-solvent system enables formation of a population of nanoparticles in a nanoparticle suspension. For example, in some embodiments, adding a polymer / payload preparation into a non-solvent system where the payload / polymer preparation and the non-solvent system are in separate layers, a population of nanoparticles is formed in a nanoparticle suspension at an interface of the payload / polymer preparation layer and the non- solvent system layer.

[0416] Without wishing to be bound by any particular theory, the present disclosure teaches that, in some embodiments, rate of addition (e.g., addition of a non-solvent system into a polymer / payload preparation, addition of a polymer / payload preparation into a non-solvent system) may affect characteristics of nanoparticles. For example, average size, size distribution, ratio of polymer and payload within nanoparticles, surface charge, and / or surface hydrophobicity may be affected by a rate of addition.

[0417] In some embodiments, conditions under which precipitation 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 Page 122 of 340 12613923v1Docket No.: 2006517-0315 combinations. In some embodiments, precipitation may be performed at variable temperatures. For example, in some embodiments, precipitation may be performed at temperatures between 20 ºC and 120 ºC. In some embodiments, precipitation may be performed for 5 mins to 120 mins, 5 mins to 100 mins, 5 mins to 80 mins, 5 mins to 60 mins, 5 mins to 40 mins, 5 mins to 20 mins, 20 mins to 120 mins, 40 mins to 120 mins, 60 mins to 120 mins, 80 mins to 120 mins, or 100 mins to 120 mins.

[0418] In certain embodiments, precipitation is performed according to a “Tequila Sunrise” process that involves non-homogenous, layered precipitation. Specifically, combined payload / polymer liquid preparation is carefully layered under liquid non-solvent system so that an interface is formed. A stirrer (e.g., stirring paddle) is placed at the interface and operated to perform gentle stirring so that the layering is eliminated (as can be detected, e.g., by presence of turbidity). Stirring speed can be adjusted – e.g., optionally being increased once turbidity has been established. The present disclosure teaches that, in some embodiments, adjustment of stirring speed and / or of component(s) of the utilized non-solved system may be adjusted, for example until a desired particle size range and / or distribution is achieved.

[0419] Note that a Tequila Sunrise process, as described herein, represents a departure from a conventional water-in-oil-in-water (W / O / W) emulsion precipitation process for nanoparticles and achieves very different results including, for example, improved payload loading (including specifically for protein payloads). In some embodiments, a provided Tequila Sunrise process achieves payload loading efficiency (e.g., for a protein payload) of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or more. Alternatively or additionally, in many embodiments, a Tequila Sunrise process achieves a Z-average diameter within a range of about 50 nm to about 450 nm, or about 100 nm to about 400 nm, or about 100 nM to about 300 nm, or about 100 nM to about 200 nM, or about 120 nM to about 180 nM; in some embodiments nanoparticles in a preparation have a size (and / or a preparation has an average size) within about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1. In some embodiments, a Tequila Sunrise process achieves a polydispersity of about 0.05 to about 0.3, or about 0.1 to about 0.3, or about 0.1 to about 0.2. In some embodiments, a Tequila Sunrise process produces a nanoparticle preparation characterized by a zeta potential below about -20, about -25, about -30, about -35, about -40, or less. Page 123 of 340 12613923v1Docket No.: 2006517-0315

[0420] In some embodiments, precipitation may be stopped in order to obtain a desired nanoparticle population. In some embodiments, a solvent system of a polymer / payload precipitation is added to a nanoparticle suspension to terminate precipitation. In some embodiments, a solvent system comprising a stabilizing agent is added to a nanoparticle suspension to terminate precipitation. In some embodiments, stirring is stopped to terminate precipitation.

[0421] In some embodiments, provided nanoparticle manufacturing technologies may include one or more concentration and / or purification steps. In some embodiments, provided technologies utilize one or more tangential flow filtration (TFF) steps. Among other things, the present disclosure provides an insight that, particularly when TFF is utilized, if a stabilizing agent is desired, deoxycholate is a particularly useful stabilizing agent (and / or that other standard stabilizing agents, such as polyvinyl alcohol, PVA, may be less useful or not useful and, in fact, may damage a TFF membrane.

[0422] In some embodiments, provided manufacturing technologies utilize a stabilizing agent. In some embodiments, a stabilizing may be or comprise PVA. In some embodiments, however, particularly when one or more TFF steps is utilized, PVA is not used. In some embodiments, particularly in embodiments that utilize one or more TFF steps, deoxycholate is utilized as a stabilizing agent.

[0423] In some embodiments, provided nanoparticles include (e.g., are manufactured) from a polymer that is or comprises Poly (lactic-co-glycolic acid) (PLGA or PLG) .

[0424] In some embodiments, provided nanoparticles utilize (e.g., are manufactured from) a polymer preparation (e.g., a PLG preparation) where the polymer has a molecular weight within a range of 5,000 – 5,000,000 Daltons.

[0425] In some embodiments, during precipitation of nanoparticles, stirring is performed. Stirring rate and / or time can be controlled. In some embodiments, stirring includes placing a blade (e.g., a ). A four-bladed stirring paddle with a diameter of 114 mm into the mixture. 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, mixing is performed at a temperature within a range of about 0 °C to 30 °C (e.g., 10 -25oC, 10-20oC, or 15-20oC). In some embodiments, stirring is performed by a magnetic Page 124 of 340 12613923v1Docket No.: 2006517-0315 stirrer and / or mechanical stirrer. In some embodiments, stirring is conducted under conditions that do not exert a high shear force. In some embodiments, stirring is performed at approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 revolutions per minute (rpm), or higher. In some embodiments, stirring is performed for one or more time periods (which may be consecutive and / or may have gaps between them). In some embodiments, each of the one or more time periods were stirring is performed involves stirring at different speeds. For example, in some embodiments, stirring includes one time period where stirring is performed at 80 rpm and a second time period were stirring is performed at 150 rpm. In some embodiments, stirring is performed until nanoprecipitation is complete.

[0426] In some embodiments, where a population of nanoparticles is formed a nanoparticle suspension as a result of adding (e.g., injecting, inserting) a payload / polymer preparation into a non-solvent system, stirring is performed before, concomitantly with, or after adding of the payload / polymer preparation into the non-solvent system. In some embodiments, stirring is performed using an overhead stirrer. For example, in some embodiments, stirring is performed using an overhead stirrer where the top edge of the fin is placed at the interface between the payload / polymer preparation layer and the non-solvent layer, the bottom edge of the fin is placed at the interface between the payload / polymer preparation layer and the non-solvent layer, or the midline of the fin is place at the interface between the payload / polymer preparation layer and the non-solvent layer.

[0427] Without being bound by any particular theory, the present disclosure contemplates that, some embodiments, after precipitation, nanoparticles may exist in more than one subtype. That is, for example, some nanoparticles may have a particular polymer:payload ratio while others may have a different polymer:payload ratio. For example, in some embodiments, after precipitation, a population of nanoparticles may be smaller than another population of nanoparticles. In some such embodiments, a population of nanoparticles with a small average size may contain more payload than a population of nanoparticles with a large average size. In some such embodiments, a population of nanoparticles with a small average size may have higher density than a population of nanoparticles with a large average size.

[0428] In some embodiments, nanoparticles may be dried prior to separation (e.g., using TFF). In some embodiments, nanoparticles may be separated (e.g., using TFF) directly after precipitation. Page 125 of 340 12613923v1Docket No.: 2006517-0315 Stabilized Nanoparticles

[0429] In some embodiments, a solution / suspension of 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. In some embodiments, a solution / suspension of nanoparticles is stabilized to prevent nanoparticles from agglomeration.

[0430] In some embodiments, a stabilizing agent may be used to reduce agglomeration of nanoparticles. Without wishing to be bound by any particular theory, surfaces of nanoparticles may be modified by a stabilizing agent.

[0431] 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 alkene units. The surfactants may be biologically sourced or synthetic. An example of a biologically based surfactant would be tocopherol units d...

Claims

CLAIMS What is claimed is:

1. A method of manufacturing a population of nanoparticles comprising steps of: providing a first preparation, which comprises a hydrophilic payload in a first aqueous solvent system and a second preparation, which comprises a hydrophobic polymer in a second solvent system, wherein: the second solvent system is non-aqueous, and the hydrophobic polymer is not soluble in the first aqueous solvent system; combining the first and second preparations to form a mixture that comprises the hydrophilic payload and the hydrophobic polymer in a combined solvent system; and adding a non-solvent system to the mixture, so that a population of nanoparticles comprising the hydrophilic payload and the hydrophobic polymer is formed in a nanoparticle suspension (e.g., wherein the non-solvent system is a non-solvent of the hydrophobic polymer and the hydrophilic payload) (e.g., wherein the non-solvent system precipitates the hydrophilic payload and the hydrophobic polymer, so that each of the nanoparticles comprises the hydrophilic payload and the hydrophobic polymer).

2. The method of claim 1, wherein a ratio of the hydrophilic payload and the hydrophobic polymer in the nanoparticles is between 0.1 to 0.9 of a ratio of the hydrophilic payload and the hydrophobic polymer in the mixture.

3. The method of any preceding claims, further comprising removing a portion of the combined solvent system from the mixture.

4. The method of any preceding claims, further comprising adding a stabilizing agent solution to the nanoparticle suspension.

5. The method of claim 4, wherein the stabilizing agent solution comprises PVA.Page 329 of 340 12613923v1Docket No.: 2006517-0315 6. The method of any preceding claims, further comprising purifying the population of nanoparticles (e.g., filtration, (e.g., tangential flow filtration), sonication, dilution).

7. The method of any preceding claims, further comprising drying the nanoparticle suspension.

8. The method of any preceding claims, wherein the population of nanoparticles has a mean size within a range of approximately 100-500 nm.

9. The method of any preceding claims, wherein the hydrophilic payload is selected from the group consisting of polypeptides, nucleic acids, and combinations thereof.

10. The method of claim 9, wherein the hydrophilic payload comprises a RNA.

11. The method of claim 10, wherein the hydrophilic payload comprises mRNA.

12. The method of claims 10-11, wherein the RNA comprises 200 to 1000000 residues.

13. The method of any preceding claims, wherein the hydrophilic payload is or comprises a crude preparation.

14. The method of any preceding claims, wherein the hydrophilic payload comprises antigen.

15. The method of claim 14, wherein the antigen is allergic antigen, infectious antigen, and / or disease-associated antigen.

16. The method of any preceding claims, wherein the combined solvent system comprises water and DMSO. Page 330 of 340 12613923v1Docket No.: 2006517-0315 17. The method of claim 16, wherein a volume ratio of water and DMSO is within a range of 1:99 to 20:80, or 1:99 to 10:

90.

18. The method of any preceding claims, wherein the non-solvent system comprises alcohol.

19. The method of claim 18, wherein the non-solvent system comprises propanol, ethanol, methanol, or combinations thereof.

20. The method of any preceding claims, wherein the hydrophobic polymer comprises PLG.

21. The method of any preceding claims, wherein the hydrophobic polymer has a molecular weight within a range of 5,000 – 5,000,000 Daltons.

22. A method comprising steps of: providing a first liquid preparation, which comprises a fragile payload in a first aqueous solvent system and a second liquid preparation, which comprises a hydrophobic polymer in a second solvent system; combining the first and second preparations to form a mixture that comprises the fragile payload and the hydrophobic polymer in a combined solvent system; and adding a liquid non-solvent system to the mixture, so that a population of nanoparticles comprising the hydrophilic payload and the polymer is formed (e.g., wherein the method does not involve energy input) (e.g., wherein the non-solvent system does not degrade the fragile payload, or decrease one or more biological or pharmaceutical activities of the fragile payload) (e.g., wherein one or more biological or pharmaceutical activities of fragile payload are substantially same before and after the step of adding).

23. The method of claim 22, wherein the fragile payload is selected from the group consisting of polypeptides, nucleic acids, and combinations thereof.

24. The method of claim 23, wherein the fragile payload comprises a RNA. Page 331 of 340 12613923v1Docket No.: 2006517-0315 25. The method of claim 24, wherein the fragile payload comprises mRNA.

26. The method of claims 24-25, wherein the RNA comprises 200 to 100000 residues.

27. The method of claim 23, wherein the fragile payload further comprises one or more structural proteins.

28. The method of claims 22-27, further comprising removing a portion of the combined solvent system from the mixture.

29. The method of claims 22-28, wherein a ratio of the fragile payload and the hydrophobic polymer in the nanoparticles is between 0.1 to 0.9 of a ratio of the fragile payload and the hydrophobic polymer in the mixture.

30. The method of claims 22-29, further comprising removing a portion of the combined solvent system from the mixture.

31. The method of claims 22-30, further comprising adding a stabilizing agent solution to a nanoparticle suspension comprising the population of nanoparticles.

32. The method of claim 31, wherein the stabilizing agent solution comprises PVA.

33. The method of claims 22-32, further comprising purifying the population of nanoparticles (e.g., filtration, (e.g., tangential flow filtration), sonication, dilution).

34. The method of claims 22-33, further comprising drying the nanoparticle suspension.

35. The method of claims 22-34, wherein the population of nanoparticles has a mean size within a range of approximately 100-500 nm. Page 332 of 340 12613923v1Docket No.: 2006517-0315 36. The method of claims 22-35, wherein the fragile payload comprises antigen.

37. The method of claim 36, wherein the antigen is allergic antigen, infectious antigen, and / or disease-associated antigen.

38. The method of claims 22-37, wherein the combined solvent system comprises water and DMSO.

39. The method of 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 claims 22-39, wherein the non-solvent system comprises alcohol.

41. The method of claim 40, wherein the non-solvent system comprises propanol, ethanol, methanol, or combinations thereof.

42. The method of claims 22-41, wherein the hydrophobic polymer has a molecular weight within a range of 5,000 – 5,000,000 Daltons.

43. The method of claims 22-42, wherein the hydrophobic polymer comprises PLG.

44. A vaccine composition comprising a nanoparticle population comprising: one or more payloads, or precursor(s) thereof, that activate antigen-specific T cells, wherein the one or more payloads is / are displayed by an MHC class I complex or an MHC class II complex.

45. The vaccine composition of claim 44, further comprising an immune adjuvant.

46. The vaccine composition of claim 44, wherein the immune adjuvant is provided from one or more bacterial sources. Page 333 of 340 12613923v1Docket No.: 2006517-0315 47. The vaccine composition of claims 45-46, wherein the immune adjuvant comprises cellular lysate (e.g., microbial lysate) or cellular lysate fractions.

48. The vaccine composition of claims 45-47, wherein the immune adjuvant is a mucosal immune adjuvant.

49. The vaccine composition of claims 44-48, wherein the nanoparticle population has a mean particle diameter of from 50 nm to 150 nm.

50. The vaccine composition of claims 44-49, wherein the one or more payloads are attached to a surface of nanoparticles of the nanoparticle population.

51. The vaccine composition of claims 44-50, wherein the hydrophobic polymer comprises PLG.

52. The vaccine composition of claims 44-51, further comprising a pore forming toxin.

53. A vaccine comprising first and second nanoparticle populations, wherein: the first nanoparticle population comprises a first payload, or precursor(s) thereof, that activate first antigen-specific T cells, wherein the first payload is displayed by an MHC class I complex; and the second nanoparticle population comprises a second payload, or precursor(s) thereof, that activate second antigen-specific T cells, wherein the second payload is displayed by an MHC class II complex.

54. The vaccine composition of claim 53, wherein the first and second nanoparticle populations are included in a same composition.

55. The vaccine composition of claims 53-55, wherein the vaccine further comprises an immune adjuvant. Page 334 of 340 12613923v1Docket No.: 2006517-0315 56. The vaccine composition of claim 55, wherein the immune adjuvant is provided from one or more bacterial sources.

57. The vaccine composition of claims 55-56, wherein the immune adjuvant comprises cellular lysate (e.g., microbial lysate) or cellular lysate fractions.

58. The vaccine composition of claims 55-57, wherein the immune adjuvant is a mucosal immune adjuvant.

59. The vaccine composition of claims 53-58, wherein the first and / or second nanoparticle populations have a mean particle diameter of from 50 nm to 100 nm.

60. The vaccine composition of claims 53-59, wherein the first and / or second payloads are attached to a surface of nanoparticles of the first and / or second nanoparticle populations.

61. The vaccine composition of claims 53-60, wherein the hydrophobic polymer comprises PLG.

62. The vaccine composition of claims 53-61, further comprising a pore forming toxin.

63. A method comprising steps of: administering to a subject in need thereof a nanoparticle composition comprising a nanoparticle population having one or more payloads, or precursor(s) thereof, that activate antigen-specific T cells, wherein the nanoparticle composition is administered orally, sublingually or buccally.

64. The method of claim 63, wherein the one or more payloads is / are displayed by an MHC class I complex.

65. The method of claim 63, wherein the one or more payloads is / are displayed by an MHC class II complex. Page 335 of 340 12613923v1Docket No.: 2006517-0315 66. The method of claim 63, wherein the one or more payloads is / are displayed by an MHC class I complex and an MHC class II complex.

67. The method of claims 63-66, wherein the nanoparticle composition further comprises an immune adjuvant.

68. The method of claim 67, wherein the immune adjuvant is provided from one or more bacterial sources.

69. The method of claim 68, wherein the immune adjuvant comprises cellular lysate (e.g., microbial lysate) or cellular lysate fractions.

70. The method of claims 67-69, wherein the immune adjuvant is a mucosal immune adjuvant.

71. The method of claims 63-70, wherein the nanoparticle population has a mean particle diameter of from 50 nm to 100 nm.

72. The method of claims 63-71, wherein the one or more payloads are attached to a surface of nanoparticles in the nanoparticle population.

73. The method of claims 63-72, wherein the hydrophobic polymer comprises PLG.

74. The method of claims 63-73, wherein the nanoparticle composition comprises a pore forming toxin.

75. A method comprising steps of: administering to a subject in need thereof a nanoparticle composition comprising a first nanoparticle population having a first payload, or precursor(s) thereof, that activate a first antigen-specific T cell, and a second nanoparticle population having a second Page 336 of 340 12613923v1Docket No.: 2006517-0315 payload, or precursor(s) thereof, that activate a second antigen-specific T cell, wherein the nanoparticle composition is administered orally, sublingually or buccally.

76. The method of claim 75, wherein the first payload is displayed by an MHC class I complex, and the second payload is displayed by an MHC class II complex.

77. The method of claims 75-76, wherein the nanoparticle composition further comprises an immune adjuvant.

78. The method of claim 77, wherein the immune adjuvant is provided from one or more bacterial sources.

79. The method of claims 77-78, wherein the immune adjuvant comprises cellular lysate (e.g., microbial lysate) or cellular lysate fractions.

80. The method of claims 77-79, wherein the immune adjuvant is a mucosal immune adjuvant.

81. The method of claims 75-80, wherein the first and / or second nanoparticle populations have a mean particle diameter of from 50 nm to 100 nm.

82. The method of claims 75-81, wherein the first and / or second payloads are attached to a surface of nanoparticles in the first and / or second nanoparticle populations.

83. The method of claims 75-82, wherein the hydrophobic polymer comprises PLG.

84. The method of claims 75-83, the nanoparticle composition comprises a pore forming toxin.

85. A method comprising steps of: Page 337 of 340 12613923v1Docket No.: 2006517-0315 administering to a subject in need thereof a first nanoparticle composition comprising a first nanoparticle population having a first payload, or precursor(s) thereof, that activate a first antigen-specific T cell; administering to the subject a second nanoparticle composition comprising a second nanoparticle population having a second payload, or precursor(s) thereof, that activate a second antigen-specific T cell, wherein the first and / or second nanoparticle compositions are administered orally, sublingually or buccally.

86. The method of claim 85, wherein the first payload is displayed by an MHC class I complex, and the second payload is displayed by an MHC class II complex.

87. The method of claims 85-86, wherein the first nanoparticle composition and / or the second nanoparticle composition further comprise an immune adjuvant.

88. The method of claims 85-87, wherein the immune adjuvant is provided from one or more bacterial sources.

89. The method of claim 88, wherein the immune adjuvant comprises cellular lysate (e.g., microbial lysate) or cellular lysate fractions.

90. The method of claims 87-88, wherein the immune adjuvant is a mucosal immune adjuvant.

91. The method of claims 85-90, wherein the first and / or second nanoparticle populations have a mean particle diameter of from 50 nm to 100 nm.

92. The method of claims 85-91, wherein the first and / or second payloads are attached to a surface of nanoparticles in the first and / or second nanoparticle populations.

93. The method of claims 85-92, wherein the hydrophobic polymer comprises PLG. Page 338 of 340 12613923v1Docket No.: 2006517-0315 94. The method of claims 85-93, the first and / or nanoparticle composition(s) comprises a pore forming toxin.

95. The method of any one of claims 1-43, wherein the mixture comprising the hydrophilic payload and the hydrophobic polymer in a combined solvent system is layered under the non- solvent system.

96. The method of claim 95, wherein a stirring paddle is placed at the interface of the solvent system and the non-solvent system and the stirring paddle is operated to perform gentle stirring.

97. A nanoparticle preparation prepared by the method of claims 1-43, the nanoparticle preparation comprising a plurality of nanoparticles, each of which comprises a hydrophilic payload in a polymer. Page 339 of 340 12613923v1

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