ZEIN nanoparticles for use in the treatment of allergies and as immunoadjuvants

Abstract

The present disclosure relates to zein nanoparcles and their use as immunoadjuvants. The zein nanoparcles described herein exhibit desirable properes and can be used in immunotherapy and in the treatment of allergies, such as food allergies.

Description

[0001] ZEIN NANOPARTICLES FOR USE IN THE TREATMENT OF ALLERGIES AND AS IMMUNOADJUVANTS FIELD

[0002] The present disclosure relates to zein nanoparticles and their use in the treatment of allergies and as immunoadjuvants. The zein nanoparticles described herein exhibit desirable properties and can be used in immunotherapy and in the treatment of allergies, such as food allergies.

[0003] BACKGROUND

[0004] Food allergies, particularly peanut allergy, represent a significant health burden, affecting millions of people globally. Traditional approaches to managing allergies involve strict avoidance, emergency treatment with epinephrine, and, more recently, oral immunotherapy (OIT). OIT aims to desensitize the immune system to allergens by exposing individuals to gradually increasing doses of the allergen. However, current OIT methods have limitations, including the risk of severe allergic reactions, inconsistent efficacy, and the need for long-term maintenance dosing to sustain desensitization. Nanotechnology offers significant advantages in allergen-specific immunotherapy (AIT) by improving targeted delivery, and controlled release of allergens. Nanoparticles can serve as carriers for allergens, such as peanut allergen, ensuring gradual and controlled release, reducing exposure to sudden high doses that might trigger anaphylaxis. Furthermore, encapsulation technologies protect allergens from premature degradation, improve their bioavailability, and reduce adverse effects.

[0005] A key limitation of current nanoparticle-based immunotherapies is their inability to boost the immune system to support the allergic response. In particular, one of the critical challenges in food allergy immunotherapy is the lack of a long-lasting tolerance effect while minimizing adverse reactions.

[0006] The present invention represents a novel nanoparticle-based oral immunotherapy that addresses these limitations. In particular, the present invention utilizes a nanoparticle-based delivery systemthat synergistically activates TLR2 and TLR4. This synergistic activation leads to a robust induction of IL-10, a cytokine critical for promoting regulatory T cell (Treg) responses, thereby enhancing immune tolerance.

[0007] The synergistic TLR activation facilitated by the nanoparticles of the invention enhances the shift from an allergic Th2-dominant response to a more regulatory and tolerogenic immune environment. This effect reduces the need for continuous exposure to the allergen, potentially offering long-term immune tolerance even after discontinuation of therapy.

[0008] SUMMARY

[0009] In a first aspect, the present disclosure is directed to nanoparticles which comprise zein and an allergen in a weight ratio allergemzein of at most 1:20, meaning no more than 1 part of allergen per 20 parts of zein, preferably from 1:20 to 1:200. Preferably, the present disclosure relates to a nanoparticle comprising zein and an allergen, wherein the allergen: zein weight ratio is of at most 1: 50 , meaning no more than 1 part of allergen per 50 parts of zein, preferably from 1:50 to 1:150. In some embodiments, the allergen is comprised in an allergen extract and the protein in the allergen extract: zein weight ratio is of at most 1:50, preferably from 1:50 to 1:150. It further discloses in a related aspect a nanoparticle comprising zein and an allergen extract, wherein the protein in the allergen extract: zein weight ratio is of at most 1:50, preferably from 1:50 to 1:150. In a second aspect, the present disclosure is directed to a composition comprising at most one nanoparticle of the first aspect, and a carrier acceptable in food, pharmacy or cosmetics. In other aspects, the present disclosure is directed to the use of the nanoparticle of the first aspect or the composition according to the second aspect in immunotherapy. In particular, for inducing immune tolerance to the allergen.

[0010] In a third aspect, the present disclosure relates to a nanoparticle or a composition comprising thereof, wherein said nanoparticles comprises zein, for use in immunotherapy or for inducing immune tolerance to an allergen, wherein the zein in the nanoparticle form induces a TLR2 immune response. In some embodiments, the nanoparticle is according to the first aspect.In other embodiments, the nanoparticle comprises zein and a basic amino acid, preferably in the ratios described herein, and does not comprise an allergen. In related embodiments, the nanoparticle substantially comprises or consists of zein and a basic amino acid, preferably in the ratios described herein. In preferred embodiments of the later, the nanoparticle is for administration in combination with an allergen.

[0011] Advantageous Effect

[0012] The nanoparticles according to the invention represent a novel allergy immunotherapy addressing the need for a safe, effective, and long-lasting immunotolerance effect. The inventors have found for peanut extract encapsulated zein nanoparticles as described herein a synergistic activation of TLR2 and TLR4. By leveraging this synergistic activation, this nanoparticle-based platform enhances immune tolerance and reduces the risk of adverse reactions to allergen immunization treatments, making it a promising solution for allergies, such as food allergies and in particular for peanut allergy. The synergistic activation ofTLRs enhances oral tolerance, reduces systemic allergic inflammation, and promotes long-term desensitization to allergens, in particular to peanut allergens.

[0013] BRIEF DESCRIPTION OF THE FIGURES

[0014] Figure 1. Experimental protocol for the protective study. CD1 mice were sensitized i ntragastrica I ly (i.g.) with peanut extract and cholera toxin for four weeks. The animals received treatments (each containing 1 mg of peanut protein) on days 26, 31, and 36. Peanut-hypersensitive mice were treated i.g. with either free peanut extract or peanut-loaded nanoparticles.

[0015] Figure 2. Survival rate of mice after the challenge. Mice were treated with either peanut-loaded nanoparticles or free peanut extract. A group of sensitized but untreated mice was also included as a control.

[0016] Figure 3. Clinical trial experimental design.Figure 4. AEs frequency and AEs duration within the 5 cohorts developed (Cohort A: 0.15mg / day; Cohort B: 1.5mg / day; Cohort C: 5mg / day; Cohort D: lOmg / day; Cohort E: 20mg / day) Figure 5. Toll-like receptor (TLR) activation in TLR-transfected human embryonic kidney (HEK-293) reporter cells, engineered to sense individual TLR ligands. Cells were incubated with control medium, nanoparticles comprising peanut extract, or free peanut extract.

[0017] Figure 6. Cytokine release profile of monocytes isolated from peripheral blood mononuclear cells (PBMCs) after incubation with: PBS (control); TLR2 agonist (Pam3CSK4); TLR4 agonist (LPS); Two different clinical batches of nanoparticles comprising peanut extract.

[0018] Figure 7: TLR activation assays:

[0019] • (A) TLR4 activation by:

[0020] o TLR4 agonist LPS K12 (positive control)

[0021] o Free defatted peanut extract

[0022] o Defatted peanut extract loaded into nanoparticles with peanut extract proteimzein ratios of 1:35 and 1:100 (samples NP2 and NP4)

[0023] o Nanoparticles without peanut extract (placebo nanoparticles)

[0024] • (B) TLR2 activation by:

[0025] o TLR2 agonist PAM2 (positive control)

[0026] o Free defatted peanut extract

[0027] o Defatted peanut extract loaded into nanoparticles with peanut extract proteimzein ratios of 1:35 and 1:100 (samples NP2 and NP4)

[0028] o Nanoparticles without peanut extract

[0029] (C) TLR4 activation by:

[0030] o TLR4 agonist LPS K12 (positive control)o Free complete peanut extract

[0031] o Complete peanut extract loaded into nanoparticles with peanut extract proteimzein ratios of 1:35 and 1:100 (samples NP1 and NP3)

[0032] o Nanoparticles without peanut extract (placebo nanoparticles)

[0033] • (D) TLR2 activation by:

[0034] o TLR2 agonist PAM2 (positive control)

[0035] o Free complete peanut extract

[0036] o Complete peanut extract loaded into nanoparticles with peanut extract proteimzein ratios of 1:35 and 1:100 (samples NP1 and NP3)

[0037] o Nanoparticles without peanut extract (placebo nanoparticles)

[0038] Figure 8. TLR activation assays involving different formulations of peanut extract nanoparticles:

[0039] • (A) TLR4 activation by:

[0040] o TLR4 agonist LPS K12 (positive control)

[0041] o Defatted peanut extract nanoparticles with D-Lysine:zein ratios of 1:2, 1:6, and 1:25 (samples NP5, NP4 and NP6)

[0042] • (B) TLR2 activation by:

[0043] o TLR2 agonist PAM2 (positive control)

[0044] o Defatted peanut extract nanoparticles with D-Lysine:zein ratios of 1:2, 1:6, and 1:25 (samples NP5, NP4 and NP6)

[0045] • (C) TLR4 activation by:

[0046] o TLR4 agonist LPS K12 (positive control)

[0047] o Defatted peanut extract nanoparticles with L-Arginine:zein ratios of 1:2 and 1:6 (samples NP7 and NP8)(D) TLR2 activation by:

[0048] o TLR2 agonist PAM2 (positive control)

[0049] o Defatted peanut extract nanoparticles with L-Arginine:zein ratios of 1:2 and 1:6 (samples NP7 and NP8)

[0050] Figure 9. TLR4 activation of: TLR4 agonist LPS K12 (positive control), defatted peanut extract loaded in nanoparticles with a ratio protein in the extract: zein of 1:35 (NP2); defatted peanut extract loaded in nanoparticles with a ratio protein in the extract: zein of 1:100 (NP4); ovalbumin loaded in nanoparticles with a ratio allergen: zein of 1:10 (NP9); and ovalbumin loaded in nanoparticles with a ratio allergen: zein of 1:100 (NP10); B)TLR2 activation of:TLR2 agonist PAM2 (positive control), defatted peanut extract loaded in nanoparticles with a ratio protein in the extract:zein of 1:35 (NP2); defatted peanut extract loaded in nanoparticles with a ratio protein in the extract:zein of 1:100 (NP4); ovalbumin loaded in nanoparticles with a ratio allergen: zein of 1:10 (NP9); and ovalbumin loaded in nanoparticles with a ratio allergen: zein of 1:100 (NP10).

[0051] DETAILED DESCRIPTION

[0052] Definitions

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "zein" refers to any globular protein belonging to the group of prolamines; said protein is generally synthesized during the development of the endosperm (nutritive tissue formed in the embryo sac of seed plants and usually forms a food deposit for the embryo of the seeds of various angiosperm plants). Zein can be obtained from any suitable source, although it is preferably obtained from corn. Various methods and techniques for extracting zein from corn endosperm are known; commercial zein is generally extracted from corn gluten meal (US 2009 / 0258050). As used herein, the term "zein" includes both native zein and modified zein. The term "modified zein" includes any zein having an amino acid sequence which is normally not naturally-occurring, but which behaves similarly to natural zeins and which are soluble in alcohol. Amino acid substitutions, especially those which do not substantially modifythe hydrophobicity, may be introduced. By way of illustration, amino acid substitutions can be performed within the repeated sections, or a single amino acid can be substituted, and substitutions can also be performed in the segments connecting the domains of repeated sequences. Insertions and substitutions can also be introduced in the carboxyl terminus and the amino terminus of the zein molecule. Additionally, deletions can be performed in the amino acid sequence provided that the resulting protein is functionally equivalent to zein, i.e., that it maintains its properties. In the present disclosure, the source or the grade of zein is not limited to a single zein and, in fact, any zein can be used to put the present disclosure into practice. By way of example, commercial zein supplied by Flo enterprise (F4400C-Pharmaceutical grade (USP39 / NF34) can be used. This is a natural type of zein which is eco-friendly, biobased, vegan, and food-safe. Preferably, in the present disclosure non modified zein is used.

[0054] As used herein, the term "allergen" can refer to any naturally occurring protein allergen that has been reported to induce allergic, i.e. IgE mediated, reactions upon their repeated exposure to an individual. The term "an allergen" can refer to one or more allergenic proteins. In some nonlimiting examples, the allergens according to the disclosure can be comprised in allergenic pollen extracts, allergenic insect extracts, allergenic fungi extracts, allergenic food or food product extracts. In some embodiments, the allergen is an isolated or purified allergenic protein.The term "immunostimulating agent" refers to a substance capable of specifically or non-specifically enhancing an immune response, for example, proteins or peptides working as natural adjuvants stimulating immune system response to the allergen or antigen, bacterial lipopolysaccharides, components of the cell wall of Gram-positive bacteria, DNA CpG sequences, plant extracts, mainly saponin plant extracts, etc. Immunostimulanting agents can be used in the nanoparticles of the disclosure. Surprisingly, the inventors found that the nanoparticles of the invention have immunostimulating properties. In preferred embodiments, the nanoparticles of the invention are used as an oral allergy vaccine in a dosage regimen in the absence of immunoadjuvants in the same or separate compositions.

[0055] As used herein, the term "allergen extract" refers to the product comprising the allergen obtained further to incubation of an allergen source material with an extraction buffer, such as with phosphate-buffer saline (PBS). The extraction process may optionally be followed by purification.Extraction and purification methods are well known in the art. The allergen extract can be a lyophilized allergen extract or an allergen extract solution or suspension. An allergen extract is susceptible to induce an allergenic reaction and is thus sometimes referred herein as allergenic extract. The amount of protein in the allergen extract (also referred as "allergen extract protein") can be determined by established methods like for example Bradford.

[0056] In preferred embodiments, the "allergen extract" is "peanut extract". The term "peanut extract" preferably refers to defatted and / or complete peanut extract. As used herein, "basic amino acid" refers to an organic molecule containing an amino group (-NHz) and a carboxyl group (-COOH) and positive charge, said basic amino acid is preferably a basic alpha-amino acid such as lysine, arginine and histidine.

[0057] The term "nanoparticle" can be used both in the singular and plural forms, as the characteristics described apply equally to individual particles or multiple particles, and the usage of either form is interchangeable without affecting the meaning.

[0058] As used herein, hydroalcoholic solution labelled as X% w / v alcohol means that there are X grams of alcohol dissolved in every 100 mL of the total solution. This percentage expresses the weight of alcohol (e.g. ethanol) (solute) relative to the final volume of the mixture, which includes both ethanol and water.

[0059] Zein nanoparticles

[0060] The nanoparticles of the disclosure are nanoparticles which comprise zein and an allergen in a weight ratio allergemzein of at most 1:20, preferably from 1:20 to 1:200. Preferably, the nanoparticles of the disclosure are nanoparticles which comprise zein and an allergen in a weight ratio allergemzein of at most 1:50, preferably from 1:50 to 1:150. In some embodiments, the allergen is comprised in an allergen extract and the protein in the allergen extract: zein weight ratio is of at most 1:50, preferably from 1:50 to 1:150. In a related aspect, a nanoparticle comprises zein and an allergen extract, wherein the protein in the allergen extract: zein weight ratio is of at most 1:50, preferably from 1:50 to 1:150.Preferably, the nanoparticle comprises zein and an allergen in a weight ratio allergemzein of from 1:60 to 1:140, preferably from 1:70 to 1:130, more preferably from 1:80 to 1:120, further preferably from 1:90 to 1:110, still more preferably from 1:95 to 1:105, even more preferably about 1:100. In a particular embodiment, the nanoparticle comprises zein and an allergen in a weight ratio allergemzein of from 1:70 to 1:100.

[0061] In some embodiments, the nanoparticle comprises zein and an allergen extract in a protein in the allergen extract:zein weight ratio of from 1:60 to 1:140, preferably from 1:70 to 1:130, more preferably from 1:80 to 1:120, further preferably from 1:90 to 1:110, still more preferably from 1:95 to 1:105, even more preferably about 1:100. In a particular embodiment, the nanoparticle comprises zein and an allergen extract in a protein in the allergen extract weight ratio of from 1:70 to 1:100.

[0062] According to the present disclosure, any allergen can be used in preparing allergen-loaded nanoparticles of the disclosure; nevertheless, in a particular embodiment, said allergen is comprised in a food product or food product extract (and is generally referred as "food allergen"). Additional details on the molecular properties of food allergens are described for instance at Breiteneder H, Mills EN. J Allergy Clin Immunol. 2005, 115(l):14-23. Most food allergens are soluble in water and or saline solutions, thus belonging to the classes known as albumins (water soluble) and globulins (salt soluble) (Bargman et al., 1992). However, many food proteins fall into these two solubility categories (RIVM report 640400001 / 2002, Food, novel foods, and allergenicity, H. van Loveren). In some embodiments, the food allergens are albumins or globulins. In an embodiment, the food allergen is selected from a group consisting of peanut, tree nuts, milk, egg, wheat, soy, fish and shellfish allergens and mixtures thereof. In some embodiments, the allergen has low to very high-water solubility. Examples of allergens with low water solubility are peanut allergens (e.g. Ara h 1-8); examples of moderate water solubility allergens are tree nuts / legumes allergens (e.g., 7S / 11S globulins); examples of allergens with high solubility allergens are pollen or fruits allergens (e.g., Bet v 1 family) and examples of very high water solubility are milk whey proteins (e.g., (3-lactoglobulin, a-lactalbumin) and egg allergens (e.g., ovalbumin, ovomucoid, lysozyme). In some embodiments, the allergen is not a water insolubleallergen such as cereal allergens (e.g., gliadins and glutelins) or milk casein allergens (e.g., otsl-, ots2-, P-, K-casein).

[0063] In a particular embodiment, the food allergen extract can be a tree nut extract selected from walnut, almond, hazelnut, cashew, pistachio, Brazil nuts or peanut. A peanut extract is particularly preferred. In a preferred embodiment, the allergen extract is peanut extract. In particularly preferred embodiments, the peanut extract is not dialyzed peanut extract. In some preferred embodiments, the allergen extract is not dialyzed peanut extract at a weight ratio dialyzed peanut extract zein of 1:10.

[0064] In some embodiments, the allergen is one or more of the peanut allergens, preferably all, and is comprised in peanut extract. According to the Allergen Nomenclature subcommittee of the World Health Organisation / International Union of Immunological Societies (WHO / IUIS), there are 20 allergens which have been described in peanut (Arachis hypogaea), see Table 1 below. These are referred as Ara hl to Ara h20, the major allergens being: Ara hl, Ara h2,

[0065] Table 1. List of allergens for Arachis hypogaea (peanut) according to the WHO / IUIS allergen nomenclature database

[0066]

[0067]

[0068]

[0069] The peanut extract according to the present disclosure can be obtained by commonly known aqueous extraction procedures.

[0070] In some embodiments, the peanut extract according to the present disclosure can be defatted or complete. In the defatted peanut extract the fat is removed from the peanut extract. This process can concentrate the protein. The defatting process can be done using standard defatting method commonly known in the art. For example, defatting of peanuts can be done using a solvent extraction process or mechanical pressing to remove the fat while keeping the protein intact. An example of defatting process used in the present disclosure is reported in the experimental part. In an embodiment, the nanoparticles comprise zein and peanut extract as allergen extract in a weight ratio allergemzein of from 1:70 to 1:100. In a particular embodiment, the nanoparticles comprise zein and defatted peanut extract as allergen in a weight ratio allergemzein of from 1:70 to 1:100. In a particular embodiment, the nanoparticles comprise zein and complete peanut extract as allergen extract in a weight ratio allergemzein of from 1:70 to 1:100.

[0071] In some embodiments, the allergen (e.g., peanut) extract:zein ratio refers to the ratio of protein in the extract to zein in weight. Accordingly, in some embodiments, the nanoparticles comprisezein and peanut extract in a protein in the extract:zein weight ratio of from 1:70 to 1:100. In a particular embodiment, the nanoparticles comprise zein and defatted peanut extract in a protein in the extract:zein weight ratio of from 1:70 to 1:100. In a particular embodiment, the nanoparticles comprise zein and complete peanut extract in a protein in the extract:zein weight ratio of from 1:70 to 1:100. In some embodiments the allergen is at least about 50%, 55%, 60%, 65%, 70% or 75%, preferably at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% of the protein in the extract, more preferably at least about 85%. It has been described that in peanut extract about 85% or more of the protein in the extract are allergenic proteins (Yu J, Eghbali M. 2025, a Review, under "Characteristics of Allergenic Proteins in Peanut", Nutrients 17(19):3078; and Hebling CM et al. 2012, Proteome Res. ll(ll):5384-95).

[0072] The total protein content quantification in the extract can be made using different methods commercially available. Preferably, the amount of protein in the extract is calculated using the Bradford method. The quantification of total protein content in peanut extract can be performed using various commercially available methods. Preferably, the protein amount in the peanut extract is determined using the Bradford method. This is a widely recognized protein quantification assay that uses Coomassie protein assay reagent to bind proteins, causing a color shift that can be measured spectrophotometrically. It is commonly used for protein quantification in various biological samples, including peanut extracts. Specific allergens in an extract (e.g., Ara h 1-20) can be quantified by well-known methods such as ELISA or LC-MS quantitation.

[0073] Preferably, the nanoparticles according to the present disclosure comprise a basic amino acid. In an embodiment, the basic amino is selected from the group consisting of arginine, lysine, histidine and mixtures thereof.

[0074] Preferably, the basic amino acid is selected from arginine and / or lysine.

[0075] In an embodiment, the nanoparticles comprise the basic amino acid in a basic amino acid:zein weight ratio of from 1:4 to 1:8. In a particular embodiment, the nanoparticles comprise the basic amino acid in a basic amino acid:zein weight ratio of from 1:5 to 1:7. For example of about 1:6.In a preferred embodiment, the nanoparticles comprise zein and an allergen or allergen extract in a weight ratio allergen and / or protein in the extract:zein of from 1:70 to 1:100, the nanoparticles further comprise lysine as basic amino acid.

[0076] In a preferred embodiment, the nanoparticles comprise zein and an allergen or allergen extract in a weight ratio allergen and / or protein in the extract:zein of from 1:70 to 1:100, the nanoparticles further comprise lysine as basic amino acid in a lysine:zein weight ratio of from 1:5 to 1:7, such as 1:6.

[0077] In another preferred embodiment, the nanoparticles comprise zein and an allergen or allergen extract in a weight ratio allergen and / or protein in the extract:zein of from 1:70 to 1:100, the nanoparticles further comprise arginine as basic amino acid.

[0078] In a preferred embodiment, the nanoparticles comprise zein and an allergen or allergen extract in a weight ratio allergen and / or protein in the extract:zein of from 1:70 to 1:100, the nanoparticles further comprise arginine as basic amino acid in a arginine:zein weight ratio of from 1:5 to 1:7, such as 1:6.

[0079] In preferred embodiments, of any of the above the allergen extract is peanut extract.

[0080] In a preferred embodiment, the nanoparticles comprise zein and peanut extract in a weight ratio peanut extract:zein of from 1:70 to 1:100, the nanoparticles further comprise lysine as basic amino acid.

[0081] In a preferred embodiment, the nanoparticles comprise zein and peanut extract in a weight ratio peanut extract:zein of from 1:70 to 1:100, the nanoparticles further comprise lysine as basic amino acid in a lysine:zein weight ratio of from 1:5 to 1:7.

[0082] In a preferred embodiment, the nanoparticles comprise zein and peanut extract in a weight ratio peanut extract:zein of from 1:70 to 1:100, the nanoparticles further comprise lysine as basic amino acid in a lysine:zein weight ratio of 1:6.In another preferred embodiment, the nanoparticles comprise zein and peanut extract in a weight ratio peanut extract:zein of from 1:70 to 1:100, the nanoparticles further comprise argine as basic amino acid.

[0083] In a preferred embodiment, the nanoparticles comprise zein and peanut extract in a weight ratio peanut extract:zein of from 1:70 to 1:100, the nanoparticles further comprise arginine as basic amino acid in a arginine:zein weight ratio of from 1:5 to 1:7.

[0084] In a preferred embodiment, the nanoparticles comprise zein and peanut extract as allergen extract in a weight ratio allergen or peanut extract:zein of from 1:70 to 1:100, the nanoparticles further comprise arginine as basic amino acid in a arginine:zein weight ratio of 1:6.

[0085] The inventor surprisingly found that a basic amino acid to zein ratio within the above ranges is particularly advantageous in terms of synergistic activation of TLRs which enhances oral tolerance, reduces systemic allergic inflammation, and promotes long-term desensitization to allergens, in particular to peanut allergens.

[0086] In the present disclosure, the zein is preferably non modified zein.

[0087] The nanoparticles according to the disclosure can comprise a saccharide or a sugar alcohol, which is an organic compound derived from a monosaccharide or disaccharide, where the carbonyl group (C=O) of the sugar is reduced to a hydroxyl (-OH) group.

[0088] In an embodiment, the nanoparticles can comprise a saccharide or a sugar alcohol selected from the group consisting of lactose, trehalose, mannitol, sucrose, maltodextrin, glucose, sorbitol, maltose and mixtures thereof. In a particular embodiment, the sugar alcohol is mannitol.

[0089] Preferably, the nanoparticles of the disclosure comprise the saccharide or the sugar alcohol in a zein to saccharide / sugar alcohol weight ratio of from 1:0.1 to 1:25, preferably from 1:1 to 1:10, more preferably from 1:1 to 1:4, still preferably about 1:2 or 1:3. In a particular embodiment, the nanoparticles comprise mannitol in a zein to mannitol weight ratio of froml:l to 1:4, preferably about 1:2 or 1:3.The nanoparticles of the present disclosure can be considered nanocarriers in which the zein form a solid continuous network (matrix) capable of encapsulating a compound, such as the allergen. The nanoparticles can encapsulate additional compounds, such as biologically active compounds, such as immunomodulating agent. Said compound(s) can be distributed in the zein matrix. Preferably, said compound(s) is / are homogeneously distributed throughout the zein matrix. The compound(s) is / are then released in a controlled manner.

[0090] The nanoparticles of the present disclosure can have a spherical or spheroidal shape. Preferably, the nanoparticles of the disclosure have an average size of 100 nm to 600 nm, 120 nm to 400 nm, more preferably of 120 nm to 300 nm, even more preferably of 140 nm to 300 nm, for example of 140 nm to 250 nm. The term spheroidal herewith refers to a shape that approximates a sphere but with slight elongation, flattening, or irregularity.

[0091] In some embodiments, the nanoparticles of the present disclosure have a negative zeta-potential. Preferably, the nanoparticles of the disclosure have an average Zeta potential of -200mV to -lOmV, -150mV to -20 mV, more preferably of -lOOmV to -30 mV and even more preferably of -70 mV to -35 mV.

[0092] In some embodiments, the nanoparticles of the present disclosure have a particle size distribution with a polydispersity index (PDI) less than 0.6, 0.5, 0.4, preferably less than or equal to 0.3, preferably determined by photon correlation spectroscopy (PCS) technique.

[0093] As used herein, "average size" refers to the average diameter of the population of nanoparticles in an aqueous medium. The average size of these systems can be measured by standard processes known by the person skilled in the art, for example, in the present disclosure, the average particle size can be measured using dynamic light scattering (DLS). In preferred embodiments, the particle size, polydispersity index (PDI) and zeta-potential were determined by photon correlation spectroscopy and electrophoretic laser Doppler anemometry, respectively, using a Zetasizer analyzer system (Brookhaven Instruments Corporation, Holtsville, NY, USA). The diameter and PDI of the nanoparticles were determined after dispersion in ultrapure water at a concentration of lmg / mL and measured at 25 °C by dynamic light scattering angle of 90°.Process

[0094] The nanoparticles of the disclosure may be obtained by any suitable means known in the art. Preferably, however, the nanoparticles are obtained via a process for producing zein loaded with an allergen comprising the dissolution of the protein (zein) in a hydroalcoholic medium, preferably together with a particular amount of basic amino acid followed by the addition, under magnetic stirring, of a separately prepared alcoholic solution of said allergen (e.g., peanut extract). After incubating the mixture for a few minutes, a particular volume of water is added to give rise to the formation of the nanoparticle's suspension.

[0095] In the following, the process for preparing loaded nanoparticles comprising an allergen is described. However, the process can be used to encapsulate different compounds (e.g. biologically active compounds). Depending on the nature of the compound, slight routinary modification of the process are possible. These are however within the expertise of the skilled person.

[0096] In an embodiment, the disclosure relates to a process for producing nanoparticles comprising zein and a compound encapsulated in it. Preferably, the compound is an allergen, which may be a mixture of allergens.

[0097] The process of the present disclosure can comprise the following steps:

[0098] • a) preparing a hydroalcoholic solution (i) comprising zein and, optionally, a basic amino acid;

[0099] • b) preparing an hydroalcoholic solution comprising the allergen (ii);

[0100] • c) mixing said hydroalcoholic solution (i) containing zein and optionally a basic amino acid with said hydroalcoholic solution (ii) comprising the allergen; and

[0101] • d) optionally, adding water to the mixture resulting from step c).

[0102] Step a) and b) can be performed in any order or simultaneously as these correspond to the preparation of two separate hydroalcoholic solutions. Step c) and d) are sequential, i.e. one after the other and both after steps a) and b).The hydroalcoholic solution (i), used in step (a), contains zein and, optionally, a basic amino acid, and comprises water and alcohol, preferably ethanol. In one embodiment, this solution contains between 50% and 90% (w / v) alcohol, preferably between 60% and 80% (w / v), and more preferably around 70% (w / v). The solution is prepared by mixing its components in appropriate amounts. The hydroalcoholic solution is prepared before the zein is added.

[0103] The concentration of zein in hydroalcoholic solution (i) depends on the desired final ratio. Preferably, it ranges between 0.1% and 10% (w / v), more preferably between 0.2% and 2.5% (w / v), and most preferably between 0.5% and 2% (w / v). The amount of basic amino acid in this solution (i) is not particularly limited and may vary based on the amount of zein. In particular, the weight ratio of basic amino acid to zein can be between 1:4 and 1:8, preferably between 1:5 and 1:7, and most preferably around 1:6.

[0104] The hydroalcoholic solution (ii), used in step (b), contains the compound to be encapsulated (e.g., an allergen) and can be prepared by mixing water and alcohol (preferably ethanol) in specific w / v amounts. Therefore, this solution contains both water and ethanol. In a preferred embodiment, hydroalcoholic solution (ii) contains between 25% and 90% (w / v) alcohol, preferably between 30% and 70%, and most preferably around 60%. Alternatively, the allergen can be dissolved in water and then the solution can be diluted with alcohol (e.g., ethanol) to obtain a specific w / v%. This is done before the mixing step c). This step can be tweaked depending on the nature of the allergen. In a preferred embodiment, peanut extract is used and it is first dissolved into water and then diluted with alcohol.

[0105] The hydroalcoholic solution (ii) may optionally contain further a second basic amino acid. The amount of the second basic amino acid in this solution (i) is not particularly limited and may vary based on the amount of zein. In particular, the weight ratio of the second basic amino acid to zein can be between 1:4 and 1:8, preferably between 1:5 and 1:7, and most preferably around 1:6. The hydroalcoholic solutions (i) and (ii) can be different or the same. In a preferred embodiment solutions (i) and (ii) are the same.

[0106] In step (c) of the process, hydroalcoholic solution (i) (containing zein and, optionally, a basic amino acid) is mixed with hydroalcoholic solution (ii) (containing the compound to be encapsulated and,optionally, a second basic amino acid). This results in a mixture comprising zein, one or more basic amino acids, and the allergen. The allergen-to-zein weight ratio in this mixture can vary, but it is preferably of from at most 1:50, preferably between 1:50 to 1:150, more preferably between 1:60 and 1:140, more preferably between 1:70 to 1:130, more preferably between 1:80 and 1:120, even more preferably between 1:90 and 1:110, and most preferably between 1:95 and 1:105, with an optimal ratio of 1:70 to 1:100, preferably of about 1:100.

[0107] In some embodiments the allergen is comprised in an allergen extract, such as peanut extract, and the protein in the extract: zein weight ratio in this mixture can vary, but it is preferably of from at most 1:50, preferably between 1:50 to 1:150, more preferably between 1:60 and 1:140, more preferably between 1:70 to 1:130, more preferably between 1:80 and 1:120, even more preferably between 1:90 and 1:110, and most preferably between 1:95 and 1:105, with an optimal ratio of 1:70 to 1:100, preferably of about 1:100.

[0108] In step (d) of the process, water is added dropwise to the mixture obtained in step (c), triggering a solvation process that forms the nanoparticles described in this disclosure. The amount of water added can vary, but in a preferred embodiment, it is sufficient to achieve a final alcohol concentration between 55% and 75% (w / v), preferably between 45% and 65% (w / v), and most preferably around 35% (w / v). The skilled person would understand that this step d) of adding water can be done manually in a lab seting or using an apparatus or a device that allows to obtain a continuous and reproducible formation of nanoparticles formation in an industrial seting. In one embodiment, the present disclosure relates to nanoparticles obtainable by the process described above. The nanoparticles obtainable by the process of the disclosure fall within the definition of the nanoparticles of the disclosure. Additional preparation steps can be included depending on the final use of the nanoparticles, and on the compound incapsulated.

[0109] For example, the nanoparticles can be dried for long-term storage, optionally in the presence of a protective agent. Therefore, the process can further comprise a drying step for drying the suspension comprising the nanoparticles of the disclosure therefore obtaining a powder. This is particularly useful to enhance stability and for the application in solid foods, such as flour, bread,pastry products, cereals, milk powder, etc., as well as in cosmetic and / or pharmaceutical products.

[0110] The drying step is not particularly limited, conventional methods for drying suspensions containing nanoparticles can be used. A preferred method for drying the nanoparticles suspension is by aspiration or spraying (spray drying) or by means of or freeze-drying (lyophilization). Standard cryop rotective agents can be used, preferably at a concentration comprised between 0.1 and 10% by weight with respect to the total composition weight, to facilitate drying.

[0111] In one particular embodiment, the disclosure refers to a process comprising the steps a) to d) as defined above and additionally a step e) of adding a water solution comprising a saccharide or a sugar alcohol, preferably mannitol; and a step f) of drying, preferably spray drying the solution obtained in step e).

[0112] In one embodiment, the disclosure refers to a process for producing a nanoparticle comprising zein and an allergen which comprises:

[0113] a) preparing a hydroalcoholic solution (i) comprising zein and, preferably, a basic amino acid; b) preparing an hydroalcoholic solution (ii) comprising an allergen and, optionally, a second basic amino acid;

[0114] c) mixing said hydroalcoholic solution (i) comprising zein and preferably a basic amino acid with said solution (ii) comprising an allergen , and, optionally, a second basic amino acid, and; d) optionally, adding water to the mixture resulting from step c);

[0115] e) adding a saccharide or a sugar alcohol, preferably in a water solution, the sugar alcohol preferably being mannitol;

[0116] f) optionally, drying, preferably spray drying or freeze drying the solution obtained in step e). In another embodiment, the disclosure refers to a process for producing a nanoparticle comprising zein and an allergen which comprises: a) preparing a hydroalcoholic solution (i) comprising zein and, preferably, a basic amino acid;b) preparing an aqueous solution comprising an allergen (e.g., peanut extract) and, optionally, a second basic amino acid, and diluting it with an alcohol to obtain a hydroalcoholic solution (ii) comprising an allergen, and, optionally, a second basic amino acid;

[0117] c) mixing said hydroalcoholic solution (i) comprising zein and preferably a basic amino acid with said hydroalcoholic solution (ii) comprising an allergen, and, optionally, a second basic amino acid, and;

[0118] d) optionally, adding water to the mixture resulting from step c);

[0119] e) adding a saccharide or a sugar alcohol, preferably in a water solution, the sugar alcohol preferably being mannitol;

[0120] f) drying, preferably spray drying or freeze drying the solution obtained in step e).

[0121] Composition

[0122] The nanoparticles of the disclosure can be incorporated into various compositions or products. Non-limiting examples include dietary supplements, food, feed, nutraceuticals, cosmetics, cosmeceuticals, and pharmaceutical products. Accordingly, in one aspect, the disclosure relates to a composition comprising the nanoparticles of the disclosure and at most a suitable carrier or vehicle acceptable in food, pharmacy or cosmetics. The following includes examples of compositions in which the nanoparticles of the disclosure can be used.

[0123] Pharmaceutical compositions: These compositions include nanoparticles formulated with a pharmaceutically acceptable carrier, ensuring compatibility and safety per industry standards. Suitable carriers include buffered solutions, emulsions, and excipients for various routes of administration, including oral and mucosal delivery.

[0124] Food and Feed compositions: These include solid or liquid substances intended for human or animal nutrition, whether as regular food, dietetic products, or functional ingredients. The term "feed" encompasses all materials and products suitable for animal consumption.Nutraceutical compositions: These compositions include natural bioactive ingredients that provide health benefits or contribute to disease prevention. They may be formulated as dietary supplements in non-food matrices such as capsules or powders.

[0125] Cosmetic compositions: These include formulations for personal care, hygiene, and appearance enhancement. Cosmeceuticals, in particular, contain bioactive ingredients with skincare benefits beyond conventional cosmetics.

[0126] An embodiment of the present disclosure refers to a composition comprising the nanoparticles according to the disclosure, and a carrier acceptable in food, pharmacy or cosmetics.

[0127] An embodiment of the present disclosure refers to a composition as disclosed herewith, wherein said composition is a pharmaceutical composition and the carrier is a pharmaceutically acceptable carrier.

[0128] An embodiment of the present disclosure refers to a composition comprising the nanoparticles of the disclosure, and the composition is administered in a dosage form suitable for oral or subcutaneous administration, preferably wherein said composition is in an oral form such as powders, tablets, capsules, granules, suspensions or emulsions.

[0129] An embodiment of the present disclosure refers to a composition comprising the nanoparticles as disclosed herewith, wherein the composition comprises a saccharide or a sugar alcohol, preferably wherein the sugar alcohol is mannitol. Preferably, the zein to sugar alcohol weight ratio is of froml:l to 1:4, preferably about 1:2 or 1:3.

[0130] A particular embodiment of the present disclosure refers to the following composition with a peanut extract protei zein weight ratio of 1:100:

[0131]

[0132] Medical uses

[0133] The disclosure also relates to a nanoparticle, a nanoparticle obtainable by the disclosed process, or a pharmaceutical composition comprising the nanoparticles for medical use.

[0134] The inventors have surprisingly found that the nanoparticles of the disclosure can modulate the immune response, making them suitable for immunotherapy and vaccine applications through various administration routes.

[0135] More specifically, these nanoparticles have demonstrated to have a safe, effective, and long-lasting immunoadjuvant effect. Thus, the nanoparticles of the disclosure can be used in immunotherapy and vaccination.

[0136] An embodiment of the disclosure relates to the nanoparticle as described herewith for use in immunotherapy. A particular embodiment relates to the nanoparticle as described herewith orthe composition for use in inducing immune tolerance to an allergen (e.g. a food allergen, such as peanut allergen in peanut extract).

[0137] An embodiment of the disclosure relates to a nanoparticle comprising or consisting of zein for use in immunotherapy or for inducing immune tolerance to an allergen, wherein the zein in the nanoparticle induces a TLR2 immune response. In some embodiments thereto, the nanoparticle substantially comprises or consists of zein and a basic amino acid. A particular embodiment of the disclosure relates to a nanoparticle comprising or consisting of zein for use in immunotherapy or for inducing immune tolerance to an allergen, wherein the zein in nanoparticle form acts as an immune adjuvant. Preferably the nanoparticles of these embodiments comprise zein and an allergen, more preferably zein, a basic amino acid and an allergen. In other embodiments, the nanoparticle substantially comprises or consists of zein and a basic amino acid. In preferred embodiments of the later, the nanoparticle is for administration in combination with an allergen. A particular embodiment of the disclosure is directed to the nanoparticle described herewith, preferably wherein the allergen extract is peanut extract, for use in inducing immune tolerance, wherein said nanoparticle induces a TLR2 and TLR4 immune response. Induction of a TLR2 and / or TLR4 mediated immune response can be determined by in vitro methods as described in the experimental part. In some embodiments, there is an activation of TLR2 and / or TLR4 when there is a release of cytokines and / or chemokines associated with these TLRs, such as IL6, TNFoc, IL8 and IL10 for TLR2 or TGF , IL6, IL8, IL12 and I LIO for TLR4. In some embodiments, there is an activation when the level of expression of one or more of these cytokines is in the same range (e.g. ±10%) as that of known positive controls, such as TLR2 agonist:Pam3CSK4 or TLR4 agonist: LPS. TLR2 agonists promote an increase in IL-10, which is responsible for T-regulatory responses that inhibit IgE-mediated Th2 responses in allergic reactions. They play a crucial role in intestinal epithelial homeostasis and integrity. Treatment with TLR2 agonists has shown the ability to suppress intestinal mucosal inflammation and protect the integrity of tight junctions in the intestine. These effects are particularly beneficial in food allergy pathologies, where intestinal epithelial cells are often damaged. On the other hand, TLR4 agonists also promote IL-10 secretion and trigger T-regulatory responses. Furthermore, the simultaneous activation of multiple TLRs provides long-lasting protection through IL-10-mediated T-regulatory responses. Based on theinformation discussed above, one could consider these zein nanoparticles of the invention as potent activators of IL-10. The IL-10 rich environment supported by TLR2 or TLR4 activation has been described to promote class switching to lgG4 in humans, the canonical blocking antibody in successful allergen immunotherapy, while reducing IgE production. TLR4 agonists used as vaccine adjuvants may increase IgG subclasses and reduce immediate hypersensitivity on challenge. An embodiment of the disclosure is directed to the use of the nanoparticles of the disclosure or composition thereof for use in the treatment of allergies and allergy-related diseases.

[0138] An embodiment of the disclosure is directed to the use of the nanoparticles of the disclosure or composition thereof for use in the treatment of immunodeficiencies and associated pathologies. Nanoparticles can be administered through various routes depending on the intended application. Oral administration includes solid dosage forms such as tablets, capsules, powders, and granules, as well as liquid forms like solutions, suspensions, and emulsions. Parenteral administration involves injection-based delivery, including intravenous (IV) for direct bloodstream access, intramuscular (IM) for depot release in muscle tissue, subcutaneous (SC) for administration under the skin, intradermal (ID) for localized delivery into the dermis, and intraperitoneal (IP) for administration into the peritoneal cavity. Mucosal administration targets various mucosal surfaces, including intranasal delivery via sprays, drops, or inhalable powders; sublingual or buccal routes for absorption through the oral mucosa; ocular administration through eye drops or ophthalmic formulations; pulmonary or inhalation delivery via nebulizers, dry powder inhalers (DPIs), or metered-dose inhalers (MDIs); rectal administration through suppositories or enemas; and vaginal administration via gels, suppositories, or vaginal rings. Transdermal administration includes topical application in the form of creams, gels, patches, lotions, or emulsions, as well as transdermal patches for controlled drug release through the skin. An embodiment of the disclosure refers to nanoparticles comprising zein and peanut extract as allergen extract in a weight ratio peanut extract:zein or protein in the peanut extract: zein of from 1:70 to 1:100, the nanoparticles preferably comprise a basic amino acid, and are for use in the treatment of allergies and allergy-related diseases.A particular embodiment of the disclosure refers to nanoparticles or a composition comprising the same, the nanoparticles comprising zein and peanut extract as allergen extract in a weight ratio peanut extract:zein or protein in the peanut extract: zein of from 1:70 to 1:100 or from 1:90 to 1:105, more preferably about 1:100, where the nanoparticles preferably comprise a basic amino acid, and are for use in the treatment of allergies and allergy-related diseases, the nanoparticles or the composition thereof are administered at a daily dosage of from 0.25 mg to 300 mg of peanut extract protein, preferably of from 5 mg to 100 mg, even more preferably of from 10 mg to 50 mg, still more preferably of 20 mg.

[0139] In preferred embodiments, the nanoparticles are peanut extract:zein nanoparticles at a protein in the peanut extract:zein ratio of about 1:100, having a basic amino acid at a 1:6 ratio, preferably D-lysine, and are administered at a daily dosage of 20 mg. In preferred embodiments, said nanoparticles are the NP4 nanoparticles described herein.

[0140] An embodiment of the disclosure refers to nanoparticles comprising zein and peanut extract as allergen extract in a weight ratio peanut extract:zein or protein in the peanut extract: zein of from 1:70 to 1:100, the nanoparticles preferably comprise a basic amino acid and are for use in the treatment of immunodeficiencies and associated pathologies.

[0141] An embodiment of the present disclosure refers to nanoparticles (or compositions comprising the same), wherein the nanoparticles comprise zein and an allergen in a weight ratio allerge zein of at most 1:50, preferably from 1:50 to 1:150, the nanoparticles further comprise a basic amino acid and are for use as immunoadjuvants. In some embodiments, the allergen is found in an allergen extract in a protein in the allergen extract:zein weight ratio of at most 1:50, preferably from 1:50 to 1:150.

[0142] An embodiment of the present disclosure refers to nanoparticles comprising zein and an allergen in a weight ratio allergemzein of at most 1:50, preferably from 1:50 to 1:150, or composition comprising the same for use as immunoadjuvants. In some embodiments, the allergen is found in an allergen extract in a protein in the allergen extract:zein weight ratio of at most 1:50, preferably from 1:50 to 1:150.An embodiment of the present disclosure refers to nanoparticles comprising zein and an allergen in a weight ratio allergemzein of at most 1:50, preferably from 1:50 to 1:150, or composition comprising the same for inducing a TLR2 and TLR4 immune response. In some embodiments, the allergen is found in an allergen extract in a protein in the allergen extract:zein weight ratio of at most 1:50, preferably from 1:50 to 1:150.

[0143] An embodiment of the present disclosure refers to nanoparticles comprising zein and an allergen in a weight ratio allergemzein of 1:70 to 1:100, or composition comprising the same for use as immunoadjuvants. In some embodiments, the allergen is found in an allergen extract in a protein in the allergen extract:zein weight ratio of at 1:70 to 1:100.

[0144] An embodiment of the present disclosure refers to nanoparticles comprising zein and an allergen, preferably peanut extract, in a weight ratio allergemzein or protein in the peanut extract:zein of 1:70 to 1:100, or composition comprising the same, for inducing a TLR2 and TLR4 immune response. Preferably the nanoparticles comprise a basic amino acid.

[0145] Methods of treatment

[0146] The disclosure additionally refers to a method of using the nanoparticles described herewith as immunoadjuvant, the method comprising:

[0147] • administering to a subject an effective amount of zein nanoparticles or composition thereof, wherein the nanoparticles comprise zein and an allergen, preferably zein and peanut extract, in a weight ratio allergemzein or protein in the extract:zein of at most 1:50, preferably from 1:50 to 1:150 and are configured to stimulate TLR2 and TLR4 receptors, thereby enhancing the immune response.

[0148] Preferably, the zein nanoparticles are administered at a daily dosage of from 0.25 mg to 300 mg of peanut extract protein, preferably of from 5 mg to 100 mg, even more preferably of from 10 mg to50 mg.

[0149] Preferably, the nanoparticles or the composition thereof is for use in a method for treating allergies and allergy-related diseases. Alternatively, the nanoparticles or the composition thereof are for use in a method for treating immunodeficiencies and associated pathologies.It is to be understood that the methods described herein are applicable to all compositions and nanoparticles disclosed in this application, whether explicitly combined or not. The methods may be practiced with any of the described compositions individually or in any combination, as would be recognized by a person skilled in the art.lt will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

[0150] All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The use of the word "a" or "an" may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one". The use of the term "another" may also refer to one or more. The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to referto alternatives only orthe alternatives are mutually exclusive.

[0151] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. As used herein, the phrase "consisting essentially of" limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. As used herein, the phrase "consisting of excludes any element, step, or ingredient not specified in the claim except for, e.g., impurities ordinarily associated with the element or limitation.

[0152] The term "or combinations thereof" as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" is intendedto include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0153] As used herein, words of approximation such as, without limitation, "about", "around", "approximately" refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as "about" or "around" may vary from the stated value by ±1, 2, 3, 4, 5, 6, 7,8, 9 or 10%.

[0154] Embodiments (Eb)

[0155] Embodiment 1: A nanoparticle comprising zein and an allergen,

[0156] (i) wherein the allergen: zein weight ratio is of at most 1:50, preferably from 1:50 to 1:150, more preferably, with the proviso that when the allergen is comprised in an extract, the extract is not dialyzed peanut extract at a weight ratio dialyzed peanut extract: zein of 1:10; and / or (ii) wherein the allergen is comprised in an allergen extract and the protein in the allergen extract:zein weight ratio is of at most 1:50, preferably from 1:50 to 1:150, and optionally wherein at most about 80%, preferably at most about 85%, 90%, 95%, 96%, 97%, 98% or 99% of the protein in the extract is the allergen.Embodiment 2: The nanoparticle according to Eb 1, wherein the allergen is a food allergen, preferably the food allergen is selected from the group consisting of peanut, tree nuts, milk, egg, wheat, soy, fish and shellfish allergens and mixtures thereof.

[0157] Embodiment 3: The nanoparticle according to Ebs 1 or 2, wherein the allergemzein weight ratio is of from 1:60 to 1:140, preferably from 1:70 to 1:130, more preferably from 1:80 to 1:120, further preferably from 1:90 to 1:110, still more preferably from 1:95 to 1:105, even more preferably about 1:100.

[0158] Embodiment 4: The nanoparticle according to any of Ebs 1 to 3, wherein the food allergen is a peanut allergen.

[0159] Embodiment 5: The nanoparticle according to any of Ebs 1 to Eb 4, wherein the allergen is comprised in peanut extract, such as complete or defatted peanut extract, and the protein in the peanut extract :zein weight ratio is of from about 1:70 to about 1:100.

[0160] Embodiment 6: The nanoparticle according to any of Ebs 1 to 5, wherein the allergen is comprised in peanut extract and the nanoparticle is administered at a daily dosage of from 0.25 mg to 300 mg of peanut extract protein, preferably of from 5 mg to 100 mg, even more preferably of from 10 mg to 50 mg, still more preferably of 20 mg.

[0161] Embodiment 7: The nanoparticle according to any of Ebs 1 to 6, wherein the nanoparticle further comprises a basic amino acid, preferably selected from the group consisting of: arginine, lysine, histidine and mixtures thereof.

[0162] Embodiment 8: The nanoparticle according to Ebs 7, wherein the basic amino acid is lysine. Embodiment 9: The nanoparticle according to Ebs 7 or 8, wherein the basic amino acid:zein weight ratio is of from 1:4 to 1:8, preferably of 1:5 to 1:7, more preferably about 1:6.

[0163] Embodiment 10: The nanoparticle according to any of Ebs 1 to 9, wherein the nanoparticle has an average particle size of the nanoparticle of from 120 nm to 350 nm.

[0164] Embodiment 11: A composition comprising one or more nanoparticles according to any of Ebs 1 to 10, and a carrier acceptable in food, pharmacy or cosmetics.Embodiment 12: The composition according to Eb 11, wherein said composition is a pharmaceutical composition and the carrier is a pharmaceutically acceptable carrier.

[0165] Embodiment 13: The composition according to Eb 11 or 12, wherein said composition is in a dosage form suitable for oral or subcutaneous administration, preferably wherein said composition is in an oral form such as powders, tablets, capsules, granules, suspensions or emulsions.

[0166] Embodiment 14: The composition according to any of Ebs 9 to 13, wherein the composition comprises a sugar alcohol, preferably wherein the sugar alcohol is mannitol.

[0167] Embodiment 15: The composition according to Eb 14, wherein the zein to sugar alcohol weight ratio is of froml:l to 1:4, preferably about 1:2 or 1:3.

[0168] Embodiment 16: The composition according to Ebs 11 to 15, wherein the composition comprises:

[0169]

[0170] Embodiment 17: The composition according to any of Eb 11 to 16, wherein the allergen is comprised in peanut extract and the composition is administered at a daily dosage of from 0.25 mg to 300 mg of peanut extract protein, preferably of from 5 mg to 100 mg, even more preferably of from 10 mg to 50 mg, still more preferably of 20 mg.

[0171] Embodiment 18: A process for producing a nanoparticle according to Ebs 1 to 10 which comprises:

[0172] a) preparing a hydroalcoholic solution (i) comprising zein and, optionally, a basic amino acid;

[0173] b) preparing an aqueous solution comprising an allergen and diluting it with an alcohol to obtain a hydroalcoholic solution (ii) comprising the allergen;

[0174] c) mixing said hydroalcoholic solution (i) comprising zein and optionally a basic amino acid with said hydroalcoholic solution (ii) comprising the allergen; and

[0175] d) optionally, adding water to the mixture resulting from step c).

[0176] Embodiment 19: The nanoparticle according to any of Ebs 1 to 10 or the composition of any of Ebs 11 to 17 for use as a medicament.

[0177] Embodiment 20: The nanoparticle according to any of Ebs 1 to 10 or the composition of any of Ebs 11 to 17 for use in immunotherapy.

[0178] Embodiment 21: The nanoparticle according to any of Ebs 1 to 10 or the composition of any of Ebs 11 to 17 for use in inducing immune tolerance to the allergen.

[0179] Embodiment 22: A nanoparticle or a composition comprising thereof, wherein said nanoparticles comprises or consists of zein, for use in immunotherapy or for inducing immune tolerance to an allergen, wherein the zein in the nanoparticle form induces a TLR2 immune response.

[0180] Embodiment 23: A nanoparticle or a composition comprising thereof, wherein said nanoparticle comprises or consists of zein, for use in immunotherapy or for inducing immune tolerance to an allergen, wherein the zein in nanoparticle form acts as an immune adjuvant.Embodiment 24: The nanoparticle or composition comprising thereof for use according to Eb 22 or 23, wherein said nanoparticle further comprises an allergen.

[0181] Embodiment 25: The nanoparticle or composition comprising thereof for use according to Eb 24, wherein said nanoparticle induces a TLR2 and TLR4 immune response.

[0182] Embodiment 26: The nanoparticle or composition comprising thereof for use according to Eb 22 or Eb 23, wherein said nanoparticle is as defined in any of Ebs 1 to 10 and / or the composition is as defined in any of Ebs 11 to 17.

[0183] Embodiment 27: The nanoparticle or composition comprising thereof for use according to Eb 22 or Eb 23, wherein said nanoparticle is as defined in any of Ebs 1 to 10 and / or the composition is as defined in any of Ebs 11 to 17, wherein sain nanoparticle or composition is administered at a daily dosage of from 0.25 mg to 300 mg of peanut extract protein, preferably of from 5 mg to 100 mg, even more preferably of from 10 mg to 50 mg, more preferably of 20 mg.

[0184] Alternative embodiments

[0185] Embodiment Al: A nanoparticle comprising zein, an allergen, and lysine wherein the wherein the basic amino acid:zein weight ratio is of from 1:4 to 1:8, preferably of 1:5 to 1:7, more preferably about 1:6.

[0186] Embodiment A2: The nanoparticle according to Eb Al, wherein the allergen is a food allergen, preferably the food allergen is selected from the group consisting of peanut, tree nuts, milk, egg, wheat, soy, fish and shellfish allergens and mixtures thereof.

[0187] Embodiment A3: The nanoparticle according to Eb Al or 2, wherein the food allergen is peanut allergen, preferably is comprised in a peanut extract, such as complete or defatted peanut extract.

[0188] Embodiment A4: The nanoparticle according to any of Ebs Al to 3, wherein the nanoparticle comprises peanut extract and the nanoparticle is administered at a daily dosage of from 0.25 mg to 300 mg of peanut extract protein, preferably of from 5 mg to 100 mg, even more preferably of from 10 mg to 50 mg.

[0189] Embodiment A5: The nanoparticle according to any of EbsA 1 to 4 for use as a medicament.Embodiment A6: The nanoparticle according to any of EbsA 1 to 4 for use in immunotherapy. Embodiment A7: The nanoparticle according to any of Ebs 1 to 4 for use in inducing immune tolerance to the allergen.

[0190] It is to be understood that the nanoparticles of embodiments as described above are applicable to all compositions and methods disclosed in this application, whether explicitly combined or not, as would be recognized by a person skilled in the art.

[0191] EXPERIMENTAL PART

[0192] Example 1 - Nanoparticles production and characterization

[0193] Zein (commercially available from Flo enterprise) was dissolved in an hydroalcoholic solution (i) comprising 3.05 mLof purified water and 7.58 mLof ethanol (99% purity) (hydroalcoholic solution at 56.3 w / v% of alcohol) at room temperature (RT) under magnetic stirring (1 hour of magnetic stirring). An amino acid as specified in Table 1 was also dissolved in the hydroalcoholic solution. The resulting mixture was stirred for 1-2 minutes until a clear solution was obtained (step a)).

[0194] A peanut extract (defatted or complete as shown in the Tables below) was dissolved in 1.1 mL of a 60% hydroalcoholic solution (water and ethanol) (step b)) before being added to the solution prepared above. The resulting mixture was then stirred at RT for 5 minutes (step c). Complete or defatted peanut extract was incorporated at a protein in the extract-to-zein ratio of 1:35 or 1:100, depending on the desired formulation. The total protein content quantification in the peanut extract (PE) was made with the Bradford method. The quantity of PE in the nanoparticles was determined according to the indicated peanut extract protein: zein weight ratio.

[0195] Nanoparticles were obtained following the desolvation procedure. To this end, 12.5 mL of water were added dropwise to the previously prepared solution into a glass container under magnetic stirring. Once mixed, the nanoparticles were formed (step d).

[0196] Finally, 1.96 mL of a mannitol aqueous solution (204.6 mg / mL) was added to the suspension of nanoparticles obtained in step d), and the mixture was dried in a Buchi Mini Spray Drier B-290 apparatus (Buchi Labortechnik AG, Switzerland). For the purpose of spray drying the obtainedmixture, the following parameters were selected: inlet temperature of 90 °C, outlet temperature of 60 °C, spray-flow of 500 mL / h, and aspirator at 80 % of the maximum capacity.

[0197]

[0198] 1.1.2 Physicochemical characterization

[0199] The particle size (diameter), polydispersity index (PDI) and zeta-potential ( ) were determined by dynamic light scattering and photon correlation spectroscopy (PCS) and electrophoretic laser Doppler anemometry, respectively, using a Zetasizer analyser system (Brookhaven Instruments Corporation, New York, USA). The diameter and polydispersity index (PDI) of the nanoparticles were determined after dispersion in ultrapure water (lmg / mL) and measured at 255C by dynamic light scattering angle of 90°. The zeta potential was determined as follows: 200 pL of the samples were diluted in 2 mL of purified water. See the obtained results in Tables 2-4.

[0200] Defatted peanut extract:

[0201] The process for obtaining defatted peanut extract was carried out using salt-free, roasted, and shelled peanuts (commercially available). First, the peanuts were crushed and transferred to a beaker. Acetone was then added, and the mixture was stirred for 90 minutes at 300 rpm and a temperature of 0-8°C (preferably 4-8°C).The resulting suspension was filtered through a Buchnerfunnel with filter paper. The remaining peanut solids were returned to the beaker, and fresh acetone was added. The stirring was repeated for another 90 minutes at the same temperature and speed. The suspension was filtered again using a Buchner funnel with a second filter paper. The remaining solids were returned to the beaker, fresh acetone was added, and stirring was repeated for a final 90 minutes at 300 rpm. The suspension was filtered again through a Buchner funnel with a third filter paper. The obtained defatted peanut solids were air-dried for 48 hours to allow any residual acetone to evaporate. After drying, the defatted peanut solids were transferred to a bottle, and water was added. The mixture was stirred for 18 hours at 250 rpm and 4-8°C. The contents were then centrifuged at 4,000 rpm for 30 minutes at 0-8°C. The supernatant was filtered under vacuum through 1.2 pm glass microfiber filters, followed by 0.7 pm glass microfiber filters, 0.45 pm cellulose acetate filters, and finally 0.20 pm cellulose acetate filters.

[0202] Complete peanut extract:

[0203] The process for obtaining complete peanut extract was carried out using salt-free, roasted, and shelled peanuts (commercially available). First, the peanuts were crushed and transferred to a bottle. Water was then added, and the mixture was stirred for 18 hours at 250 rpm and a temperature of 0-8°C (preferably 4-8°C). The contents of the bottle are then centrifuged at 4,000 rpm for 30 minutes at 0-8°C. The supernatant was filtered first through a Buchner funnel with filter paper, followed by vacuum filtration using 1.2 pm glass microfiber filters, then 0.7 pm glass microfiber filters, 0.45 pm cellulose acetate filters, and finally 0.20 pm cellulose acetate filters.

[0204] Bradford method

[0205] The prepared peanut extract (defatted or complete) was analyzed to determine the protein (allergen) content. Protein determination was conducted using the Pierce™ Bradford Plus Protein Assay Kit (ThermoFisher Scientific, A55866) following the provider's instructions. In brief, a known concentration of Bovine Serum Albumin (BSA) standard was used to prepare the calibration curve, with a previously prepared lysis buffer serving as the diluent. Once the calibration curve was prepared, a known volume of peanut extract was also diluted with the same lysis buffer. Then, Coomassie protein assay reagent was added to each sample (both calibration curve and samplewells) and incubated for 10 minutes at room temperature, protected from light. Finally, the absorbance was measured at 595 nm using a spectrophotometer, thus determining the protein concentration.

[0206] 1.2 Results

[0207] 1.2.1Physicochemical characterization

[0208]

[0209]

[0210]

[0211] As shown in the tables above, all formulations exhibited similar physicochemical properties in terms of size, polydispersity, and zeta potential. No significant differences were observed when varying the protein in the extract:zein or amino acid:zein ratios. Furthermore, changing the aminoacid from L-lysine to D-arginine did not result in significant alterations in the physicochemical characteristics of the formulations.

[0212] Example 2: Efficacy study in sensitised mice

[0213] 2.1 Material and Methods:

[0214] Experiments were conducted in compliance with the regulations of the Ethics Committee of the University of Navarra, in accordance with European legislation on animal experiments (approved protocol 006 / 15).

[0215] CDI female mice, weighing approximately 20 grams, were sensitized via oral administration of a mixture containing peanut butter (CAPITAN MANI soft peanut butter; 4.35 mg with approximately 1 mg of protein) and 5 pg of cholera toxin in a total volume of 200 pL of saline solution. This sensitization was carried out on days 0, 7, 14, and 21. Additionally, tape stripping was applied to enhance sensitization. Mice were shaved, and the skin on their backs was barrier-disrupted. Percutaneous sensitization was then performed by topically applying 100 pg of peanut extract (PE) in 100 pL of saline solution to the damaged skin. To confirm successful sensitization, plasma IgE levels were quantified.

[0216] On days 26, 31, and 36, the animals received a single oral dose of 1 mg of peanut protein, either resuspended in purified water or incorporated into zein nanoparticles corresponding to sample NP4. Finally, on day 45, the animals were challenged with an intraperitoneal injection of 0.25 mg of peanut protein to induce an anaphylactic reaction in the sensitized mice (see Figure 1).

[0217] To assess the severity of anaphylactic shock, various parameters were observed, including mobility, bristly fur, and cyanosis, while the mortality rate was recorded.

[0218] 2.2 Results

[0219] Figure 2 illustrates the survival rate of mice after being challenged with an intraperitoneal injection of 0.25 mg of peanut protein. As shown in the figure, the PE: zein nanoparticles according to the disclosure (NP4) effectively protected peanut-sensitized mice from mortality.Specifically, the group treated with the PE: zein nanoparticles (NP4) exhibited a survival rate of 75%, whereas the untreated sensitized control group had a survival rate of only 30%.

[0220] Additionally, the free peanut extract group provided slight protection compared to the control group. At the same dose, the PE: zein nanoparticles (NP4) demonstrated a higher protective effect than the free peanut extract, further supporting our hypothesis that encapsulating peanut extract in nanoparticles enhances the immune response.

[0221] Example 3: double-blind placebo-controlled phase I clinical trial on peanut-allergic patients 3.1 Material and Methods

[0222] 52 patients from 12 years old with a history of immediate hypersensitive reaction to peanut protein are being recruited for this double-blind placebo-controlled phase I clinical trial. After demonstrating hypersensitivity through methods, such as specific IgE, positive skin prick test, and double-blind challenge test, patients are treated with increasing doses of peanut protein (6 cohorts) daily for 14 days to observe the safety of this oral peanut immunotherapy (see Figure 3).

[0223] 3.2 Results

[0224] 6 cohorts of patients reaching 30 mg dose of peanut protein have already finished the treatment with very good safety outcomes. No severe adverse effects have been observed. Treatment-related reactions were mild and mainly consisting of oral allergy syndrome in 29 of 52 patients. Most adverse events, including those of higher severity, occurred during the initial hospital-supervised treatment phase. Thereafter, adverse events in both the active and placebo groups were predominantly mild. The safety threshold appeared to be reached at cohort F (30 mg), where 2 epinephrine administrations were needed, showing an increased frequency of treatment-related adverse events, indicating a potential ceiling dose. Based on safety considerations, the maximum tolerated dose was determined to be 20 mg and was selected as the recommended oral dose of the PE: zein nanoparticles according to the disclosure (sample NP4). A phase II study is planned to be developed with the recommended dose from this phase I study.. A phase II study is planned to be developed with the recommended dose from this phase I study.As observed in the data (Figure 4), the PE:zein nanoparticles according to the disclosure (sample NP4) has been administered orally to 40 peanut-allergic patients, and its safety profile to date appears promising. Furthermore, the PE:zein nanoparticles of the disclosure offer several notable advantages, including a relatively brief induction period, the convenience of home selfadministration, and the oral route of administration.

[0225] In conclusion, administration of PE:zein nanoparticles of the disclosure according to the disclosure (NP4) as oral immunotherapy to 40 peanut-allergic patients has resulted in an acceptable safety profile with no serious adverse events.

[0226] Example 4: PE: zein nanoparticles induce in vitro TLR2 & TLR4 simultaneous activation

[0227] 4.1 Material and Methods

[0228] TLR-transfected human embryonic kidney reporter cells (HEK-293), engineered to detect individual TLR ligands were challenged with the PE: zein nanoparticles corresponding to sample NP4.

[0229] Free peanut extract and nanoparticle-encapsulated peanut extract were tested at different concentrations, and the activation of TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9 was evaluated. Known TLR agonists were used as positive controls (TLR2 agonist:Pam3CSK4 and TLR4 agonist: LPS).

[0230] Monocytes were isolated from PBMCs using CD14 Miltenyi beads and incubated for 24 hours with various formulations, along with Pam3CSK4 as the TLR2 ligand and LPS as the TLR4 ligand. The release of cytokines and chemokines into the supernatants was subsequently analyzed using the ProcartaPlex™ Plex 21 Storm, following the manufacturer's instructions.

[0231] 4.2 Results

[0232] The PE: zein nanoparticles (sample NP4) showed potent activation of TLR2 and TLR4-transfected cells, with both TLRs being activated synergistically in a highly potent manner. Moreover, reproducibility between different batches was observed in terms of TLR activation. Peanut extractalone showed no TLR activation, whereas empty nanoparticles activated TLR2 but failed to activate TLR4 (see Figures 5 and 6).

[0233] As illustrated in Figures 5 and 6, the composition of nanoparticles according to the disclosure enables synergistic activation when the peanut extract is encapsulated. A nanoparticle-based oral immunotherapy according to the disclosure is demonstrated to be a potent TLR activator, highlighting its potential as an immunotherapy with immunostimulatory properties, particularly through the robust release of IL-10. The nanoparticles according to the disclosure serves as a proof-of-concept platform that could be applied to all food allergies.

[0234] Example 5: Nanoparticles Characterization and TLR2 & TLR4 Activation

[0235] 5.1 Materials and Methods

[0236] TLR-transfected HEK-293 reporter cells, engineered to detect individual TLR ligands, were challenged with the nanoparticles according to the disclosure.

[0237] Free peanut extract and nanoparticle-encapsulated peanut extract with varying excipient ratios were tested at different concentrations, and the activation of TLR2 and TLR4 was evaluated. Known TLR4 and TLR2 agonists (LPS K12 and PAM2) were used as positive controls.

[0238] 5.2 Results

[0239] 5.2.1 Influence of the peanut extract protein-to-zein ratio on TLR2 and TLR4 activation

[0240] As demonstrated in Example 4, the composition of the nanoparticles enables synergistic activation when peanut extract is encapsulated. Given the crucial role of the composition, two different extracts— complete peanut extract and defatted peanut extract— at two different peanut extract protein-to-zein ratios (1:35 and 1:100) were evaluated.

[0241] According to the data, TLR4 activation was tested for samples NP1, NP2, NP3 and NP4. TLR4 activation was obtained for both the defatted and complete extracts (samples NP3 and NP4), see Figure 7A (defatted) and 7C (complete), and a similar trend was observed for TLR2 activation, seeFig. 7B (defatted) and 7D (complete). Particularly advantageous results were obtained for a ratio peanut extract proteimzein of 1:100.

[0242] Interestingly, although no significant differences were observed in the physicochemical characterization of the formulations (see Example 1), the peanut extract protein-to-zein ratio significantly influenced TLR activation. As shown in Figure 7, both defatted and complete peanut extracts slightly activated TLR4, whereas empty nanoparticles (without extract) did not activate TLR4 at all. This finding suggests that the 1:35 ratio, which contains a higher extract concentration, should theoretically induce the highest TLR4 activation. However, unexpectedly, the opposite was observed— indicating that the effect is significantly better for a ratio as claimed, in particular of 1:100.

[0243] Without being bind to theory, this finding seems to suggest that a specific composition, independent of the allergen type and physicochemical properties of the nanoparticles, contributes to optimal TLR4 activation.

[0244] 5.2.2 Influence of the ratio amino acid:zein on the activation of TLR4 and TLR2

[0245] The effect of lysine on TLR4 and TLR2 activation was evaluated at different lysine-to-zein ratios (samples NP4, 5, and 6). For TLR4 activation, a progressive increase was observed as the lysine:zein ratio increased, reaching a peak at a 1:6 ratio. Beyond this point, a significant decrease in TLR4 activation was noted, suggesting that the 1:6 ratio may further enhance the synergistic effect of the present disclosure (see Figure 8).

[0246] For TLR2 activation, the effect was even more pronounced. The 1:6 ratio demonstrated a better activation compared to the 1:2 and 1:25 ratios (see Figure 8).

[0247] For the formulations using arginine instead of lysine, a further improved effect was also found to be 1:6. In this case, TLR4 activation in response to the arginine:zein ratio remained low at 1:2 but increased significantly at 1:6, following a similar trend to that observed with the lysine:zein ratio (see Figure 8). As with lysine, the 1:6 ratio appears to further improve the effect of the invention. In contrast, the arginine:zein ratio seems to have less impact on TLR2 activation, although the 1:6 ratio still demonstrates a very good activation.Based on these results, we conclude that an amino acid-to-zein ratio of 1:6 is preferred.

[0248] It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the claims

[0249] 5.2.3 Influence of the allergen-to-zein ratio on TLR2 and TLR4 activation in peanut extract and ovalbumin nanoparticles

[0250] Two different allergens: defatted peanut extract (obtained as described above) and ovalbumin (CAS 9006-59-1, Sigma-Aldrich (USA)) were evaluated at different allergen to zein ratios to identify the optimal ratio of the formulation in terms of TLR2 &TLR4 activation. TLR2 & TLR4 Activation assays were conducted as described under Example 5. The tested peanut extract particles correspond to NP2 and NP4 as described in Example 1 above. Ovalbumin nanoparticles were prepared according to the process as described in Example 1 but using ovalbumin instead of peanut extract. Two types of ovalbumin-loaded nanoparticles were prepared: i) with a ratio allergen: zein of 1:10 (NP9) and ii) with a ratio allerge zein of 1:100 (NP10).

[0251] It was observed that TLR4 activation was highest at the 1:100 ratio for all tested extracts (Figure 9A). A similar trend was observed for TLR2 activation in the case of peanut extract; however, this pattern was not observed for ovalbumin (Fig. 9B).

Claims

44CLAIMS1. A nanoparticle comprising zein and an allergen, wherein the allergen: zein weight ratio is of at most 1:50, preferably from 1:50 to 1:150.

2. The nanoparticle according to claim 1, wherein the allergen is comprised in an allergen extract and wherein the protein in the allergen extract:zein weight ratio is of at most 1:50, preferably from 1:50 to 1:150.

3. The nanoparticle according to claim 2, wherein at least 80%, preferably at least 85%, 90%, 95%, 97%, 98% or 99% of the protein in the extract is the allergen.

4. The nanoparticle according to any of claims 1 to 3, wherein the allerge zein and / or the protein in the allergen extract:zein weight ratio is of from 1:60 to 1:140, preferably from 1:70 to 1:130, more preferably from 1:80 to 1:120, further preferably from 1:90 to 1:110, still more preferably from 1:95 to 1:105, even more preferably about 1:100.

5. The nanoparticle according to any one of claims 1 to 4, wherein the allergen is a food allergen, preferably the food allergen is selected from the group consisting of peanut, tree nuts, milk, egg, wheat, soy, fish and shellfish allergens and mixtures thereof.

6. The nanoparticle according to any one of claims 1 to 5, wherein the allergen is a peanut allergen or the allergen extract is peanut extract, such as complete or defatted peanut extract.

7. The nanoparticle according to claim 6„ wherein the allergen extract is peanut extract, such as complete or defatted peanut extract, and the protein in the peanut extract:zein weight ratio is of from 1:90 to 1:110, preferably from 1:95 to 1:105, more preferably about 1:100.

8. The nanoparticle according to any of claims 1 to 7, wherein the nanoparticle further comprises a basic amino acid, preferably selected from the group consisting of: arginine, lysine, histidine and mixtures thereof, more preferably wherein the basic amino acid is lysine.

9. The nanoparticle according to claim 8, wherein the basic aminoacid:zein weight ratio is of from 1:4 to 1:8, preferably of 1:5 to 1:7, more preferably about 1:6.

10. A process for producing a nanoparticle comprising zein and an allergen which comprises:45a) preparing a hydroalcoholic solution (i) comprising zein and, preferably, a basic amino acid; b) preparing an hydroalcoholic solution (ii) comprising an allergen and, optionally, a second basic amino acid;c) mixing said hydroalcoholic solution (i) comprising zein and preferably a basic amino acid with said hydroalcoholic solution (ii) comprising an allergen, and, optionally, a second basic amino acid.

11. A composition comprising one or more nanoparticles according to any of claims 1 to 9, and a carrier acceptable in food, pharmacy or cosmetics, optionally wherein said composition is a pharmaceutical composition and the carrier is a pharmaceutically acceptable carrier, and / or, optionally wherein said composition is in a dosage form suitable for oral or subcutaneous administration, preferably wherein said composition is in an oral form such as powders, tablets, capsules, granules, suspensions or emulsions.

12. The nanoparticle according to any of claims 1 to 9 or the composition of claim 11 for use as a medicament.

13. The nanoparticle according to any of claims 1 to 9 or the composition of claim 11 for use in immunotherapy.

14. The nanoparticle according to any of claims 1 to 9 or the composition of claim 11 for use in inducing immune tolerance to the allergen.

15. The nanoparticle or the composition for use according to claims 13 or 14, wherein the zein in the nanoparticle form induces a TLR2 immune response.

16. The nanoparticle or the composition for use according to any of claims 13 to 15, wherein the zein in nanoparticle form acts as an immune adjuvant.

17. The nanoparticle or the composition for use according to any of claims 13 to 16, wherein said nanoparticle induces a TLR2 and TLR4 immune response.

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