Gluten-degrading protein and uses thereof

Abstract

The present invention relates to a protein comprising the amino acid sequence SEQ ID NO: 1, compositions comprising the protein, and uses of said compositions in the treatment of diseases caused by the presence of peptide antigens in gluten, in the treatment of gluten-containing foods, or in the preparation of gluten-free food products.

Description

[0001] DESCRIPTION

[0002] Gluten-degrading protein and its uses

[0003] The present invention falls within the field of biomedicine and food biotechnology. Specifically, the present invention relates to a protein comprising the amino acid sequence SEQ ID NO: 1, as well as uses thereof, such as the treatment of diseases caused by the presence of peptide antigens present in gluten or in the pretreatment of foods containing gluten.

[0004] STATE OF THE ART

[0005] Gluten proteins (gliadins and prolamines and glutenin analogs) are the main environmental factors that trigger celiac disease and other associated disorders. These proteins contain peptide sequences rich in proline and glutamine, which makes them more resistant to digestive enzymes than other dietary proteins, and they can persist in the intestinal lumen. In susceptible individuals, these peptides are responsible for an abnormal reaction involving both innate and adaptive immunity, which together causes chronic inflammation of the intestinal mucosa, an increase in intraepithelial lymphocytes, crypt hyperplasia, and progressive deterioration of the intestinal villi, even their total disappearance.

[0006] Gluten-related disorders, such as celiac disease, are becoming a major public health problem worldwide. Although celiac disease was previously thought to occur only or predominantly in northern and western Europe, it is now recognized to be present worldwide. A systematic review of the global prevalence of celiac disease found a seroprevalence rate of 1.4%, with the prevalence varying by continent, ranging from 1.3% (South America, 11 studies) to 1.8% (Asia, 20 studies). Singh P. et al. Global Prevalence of Celiac Disease: Systematic Review and Meta-analysis. Clin Gastroenterol Hepatol. 2018 Jun;16(6):823-836. Other associated disorders are non-celiac gluten sensitivity (NCGS) and irritable bowel syndrome (IBS), which together affect more than 13% of the world's population.Some strategies aimed at improving the health of individuals with gluten-related disorders include the use of microorganisms as probiotics. ES2343499B1 describes the use of a strain of the species Bifidobacterium longum to improve the health of patients with related diseases. Another known approach or strategy is the use of microorganisms capable of hydrolyzing immunotoxic peptides in bread doughs (DiCagno et al. 2004. Sourdough bread made from wheat and nontoxic flours and selected lactobacilli is tolerated in celiac sprue patients. Appl Environ Microbiol 70:1088-96).

[0007] On the other hand, the use of recombinant proteins for the treatment and / or prevention of disorders associated with gluten intake has also been described in the state of the art. In patent documents such as ES2883347T3 or WO201519221 1A1 , belonging to the same patent family, compositions and methods are described for the attenuation or prevention of intestinal inflammation caused by the presence of peptide food antigens, based on the use of an enzyme called Neprosin, capable of carrying out the digestion of gliadins.

[0008] In Piper et al. 2004 (Effect of prolyl endopeptidase on digestive-resistant gliadin peptides in vivo. J Pharmacol Exp Ther.311:213-9) or Tack et al. 2013 (Consumption of gluten with gluten-degrading enzyme by celiac patients: a pilot-study. World J Gastroenterol. 19(35):5837-47; Gass et al. 2007), the hydrolysis of immunotoxic gluten peptides during the digestive process is disclosed, preventing their arrival to the small intestine by means of the oral administration of other purified enzymes of vegetal or bacterial origin capable of degrading gluten peptides (Piper et al. 2004. Effect of prolyl endopeptidase on digestive-resistant gliadin peptides in vivo. J Pharmacol Exp Ther.31 1 :213-9; Tack et al. 2013. Consumption of gluten with gluten-degrading enzyme by celiac patients: a pilot-study. World J Gastroenterol. 19(35):5837-47; Gass et al. 2007).

[0009] However, despite the severity of the symptoms and the high prevalence of celiac disease, the development of effective therapies is limited, and completely avoiding gluten consumption is a complicated task because traces of gluten are found in more than 80% of the foods we consume.

[0010] Therefore, it is necessary to have alternative treatments, preferably improved ones compared to those described in the state of the art, as well as alternative strategies aimed at processing and preparing gluten-free or low-gluten food products.

[0011] DESCRIPTION OF THE INVENTION

[0012] As explained in the background, an enzyme known in the state of the art as Neprosin (also referred to herein as neprosin), originating from Nepenthes ventrata, is known, capable of carrying out the digestion of gliadins (which as will be described later in this document, is a group of proteins present in gluten), whose amino acid sequence is as follows:

[0013] MQAKFFTFVILSSVFYFNYPLAEARSIQARLANKPKGTIKTIKGDDGEVVDCVDIYKQPA FDHPLLKNHTLQMQPSSYASKVGEYNKLEQPWHKNGECPKGSIPIRQVITGLPVVK KQFPNLKFAPPSANTNHQYAVIAYFYGNASLQGANATINIWEPNLKNPNGDFSLTQIWI SAGSGSSLNTIEAGWQVYPGRTGDSQPRFFIYWTADGYTSTGCYDLTCPGFVQTNN YYAIGMALQPSVYGGQQYELNESIQRDPATGNWWLYLWGTVVGYWPASIYNSITNG ADTVEWGGEIYDSSGTGGFHTTTQMGSGHFPTEGYGKASYVRDLQCVDTYGNVISP TANSFQGIAPAPNCYNYQFQQGSSELYLFYGGPGCQ (SEQ ID NO: 2)

[0014] The inventors of the present invention have designed and obtained a recombinant protein, referred to herein as celiacase or protein of the invention, whose sequence, SEQ ID NO: 1, presents the C334V mutation with respect to the Neprosin protein sequence. This mutation was designed by the inventors when determining the crystallographic structure of Neprosin and verifying that the cisterna in position 334 could be responsible for the yield losses that were observed recurrently when producing, purifying and activating the enzyme. The inventors have demonstrated in a series of tests that Celiacase is more effective than Neprosin in carrying out a cleavage reaction of the QPQL sequence (SEQ ID NO: 3) that is part of peptides present in gluten such as the 33-mer (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF; SEQ ID NO: 4), as shown in Fig. 3.

[0015] The genetic modification carried out on the enzyme thus increased its production yield and also its activity and capacity to degrade gluten, specifically gliadins and peptides derived from them that cannot be degraded in normal digestion, being the main components associated with celiac disease, having different applications of interest as will be described later in this report.

[0016] Furthermore, they have also experimentally demonstrated that among all the mutants generated from Neprosin, Celiacase is the protein that showed the best activity, stability and performance (Fig. 3).

[0017] Protein of the invention

[0018] One aspect of the invention relates to a protein, which comprises, preferably consists of, the amino acid sequence SEQ ID NO: 1, hereinafter the “protein of the invention”, also referred to as Celiacase.

[0019] SEQ ID NO: 1

[0020] MQAKFFTFVILSSVFYFNYPLAEARSIQARLANKPKGTIKTIKGDDGEVVDCVDIYKQPA FDHPLLKNHTLQMQPSSYASKVGEYNKLEQPWHKNGECPKGSIPIRQVITGLPVVK KQFPNLKFAPPSANTNHQYAVIAYFYGNASLQGANATINIWEPNLKNPNGDFSLTQIWI SAGSGSSLNTIEAGWQVYPGRTGDSQPRFFIYWTADGYTSTGCYDLTCPGFVQTNN YYAIGMALQPSVYGGQQYELNESIQRDPATGNWWLYLWGTVVGYWPASIYNSITNG ADTVEWGGEIYDSSGTGGFHTTTQMGSGHFPTEGYGKASYVRDLQVVDTYGNVISP TANSFQGIAPAPNCYNYQFQQGSSELYLFYGGPGCQ

[0021] In the context of the present invention, a protein is understood to be a molecule formed by joining amino acids together via peptide bonds. In a preferred embodiment, the protein of the invention has a length of 380 to 500 amino acids (including the termini).

[0022] The protein of the invention can be obtained by techniques widely known in the art. Examples of protein production techniques include, but are not limited to, chemical or biological synthesis, genetic recombination, or expression of polynucleotides encoding the protein of the invention.

[0023] Furthermore, once obtained, the protein of the invention can be subjected to a lyophilization process. Lyophilization is a technique or process known in the state of the art, carried out in a vacuum and at a low temperature to prevent protein denaturation. Thus, in a preferred embodiment, the protein of the invention is lyophilized.

[0024] The protein of the invention may also comprise a his-tag sequence at the C-terminus to facilitate its purification (to which three spacer amino acids, such as AIA, may be added, giving rise to the sequence AIAHHHHHH (SEQ ID NO: 5). Thus, in a preferred embodiment, alone or in combination with other preferred embodiments, the protein of the invention further comprises the sequence SEQ ID NO: 5.

[0025] Furthermore, the protein of the invention can be linked to a signal peptide that directs the protein to the secretory pathway in a cell. Thus, in a preferred embodiment, alone or in combination with other preferred embodiments, the protein of the invention further comprises a signal peptide or signal sequence. Techniques for adding signal peptides to the protein of interest are known in the art.

[0026] Effective signal peptide sequences for bacterial host cells are the signal peptide obtained from maltogenic amylase of Bacillus stearothermophilus, subtilisin of B. licheniformis, beta-lactamase of B. licheniformis, alpha-amylase of Bacillus stearothermophilus, neutral proteases of B. stearothermophilus (nprT, nprS, nprM), prsA of Bacillus subtilis, etc. Effective signal peptide sequences for filamentous fungal host cells are the signal peptide obtained from Aspergillus niger neutral amylase, A. niger glucoamylase A, Aspergillus shirousami glucoamylase A, Aspergillus kawachü glucoamylase A, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, H. insolens endoglucanase V, Humicola lanuginosa lipase and Rhizomucor miehei aspartic proteinase.

[0027] Techniques for producing fusion polypeptides, for example for adding signal peptides to the protein of interest, are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under the control of the same promoter(s) and terminator. Fusion polypeptides can also be constructed using protein technology in which the fusion polypeptides are created post-translationally.

[0028] Polynucleotide, gene constructs and cell of the invention As previously indicated, the protein of the invention can be obtained by techniques widely known in the state of the art, such as the expression in a cell of the polynucleotide encoding the protein of the invention, and its subsequent isolation. Thus, in another aspect, the invention relates to a polynucleotide, hereinafter the "polynucleotide of the invention", which encodes the protein of the invention.

[0029] The term "polynucleotide," as used herein, refers to a polymeric form of nucleotides of any length, composed of ribonucleotides and / or deoxyribonucleotides. The term includes both single-stranded and double-stranded polynucleotides, as well as modified (nucleated, protected, and the like) polynucleotides. The polynucleotide of the invention may be DNA, RNA, or cDNA.

[0030] The polynucleotide of the invention can be obtained by a person skilled in the art using techniques widely known in the art. The sequence of the polynucleotide of the invention can be included in a gene construct.

[0031] Thus, in another aspect, the invention relates to a gene construct, hereinafter the “gene construct of the invention”, which comprises the polynucleotide of the invention.

[0032] Preferably, the construct comprises the polynucleotide of the invention operably linked to sequences regulating the expression of the polynucleotide of the invention. In principle, any promoter may be used in the gene constructs of the present invention, provided that said promoter is compatible with the cells in which the polynucleotide is to be expressed, the cells preferably being human, more preferably Exp¡293F cells.

[0033] A promoter, or promoter region, is a nucleotide sequence that controls the transcription of a given gene (nucleotide sequences). In the present invention, it refers to a nucleotide sequence that controls the transcription of the polynucleotide of the invention. Promoter sequences can be unidirectional or bidirectional. A unidirectional promoter is one that controls the transcription of one or more genes that are located in tandem with the first. "In tandem" refers to the 3' end of the first gene being followed, either consecutively or separated by a certain nucleotide sequence, by the 5' end of the second gene. A bidirectional promoter refers to the promoter region that controls transcription in two opposite directions.That is, a bidirectional promoter directs the transcription of two genes located divergently, i.e., in opposite directions, with the 5' end of both nucleotide sequences being closer to each other than the 3' end. The terms "promoter" and "promoter region" are used interchangeably in the present invention.

[0034] Other appropriate sequences that may be included in the gene construct are sequences that control and regulate said transcription and, where appropriate, the translation of the product of interest, such as, for example, transcription initiation and termination signals, polyadenylation signal, origin of replication, or ribosome binding sequences (RBS), coding sequences for transcriptional regulators, among others.

[0035] Additionally, the gene construct of the invention may contain markers or tags that allow the isolation of the protein of the invention once it is synthesized in the cell.

[0036] On the other hand, the polynucleotide of the invention or gene construct comprising it may also comprise a coding sequence for a signal peptide. Thus, in a preferred embodiment, the polynucleotide or gene construct of the invention also comprises a coding sequence for a signal peptide, preferably wherein said sequence is linked to the polynucleotide of the invention. Signal peptides that may be linked to the protein of the invention have been described above in this document.

[0037] On the other hand, the polynucleotide or gene construct of the invention may be part of a vector. Thus, in another aspect, the invention relates to a vector, hereinafter "vector of the invention," comprising the polynucleotide or gene construct of the invention.

[0038] Examples of appropriate expression vectors can be selected according to the conditions and needs of each specific case. The choice of vector will depend on the host cell and the type of intended use. Such a vector can be obtained by conventional methods known to those skilled in the art, such as, but not limited to, chemical transformation, electroporation, microinjection, among others, in the case of a eukaryotic host cell. Examples of suitable vectors in the context of the present invention include, but are not limited to, the pET-28a(+) vector or the pCMV vector.

[0039] The vector of the invention can be used, for example, to transform, transfect, or infect cells susceptible to being transformed, transfected, or infected by said vector. In the context of the present invention, the cells are preferably human and are transfected with the vector of the invention.

[0040] Therefore, another aspect of the invention relates to a cell, preferably a human cell, even more preferably Exp¡293F human cells, hereinafter the “cell of the invention”, comprising the protein, the polynucleotide, the gene construct or the vector of the invention, for which said cell may have been transformed, transfected, transduced or infected with a construct or a vector provided by this invention. Transformed, transfected, transduced or infected cells can be obtained by conventional methods known to those skilled in the art. In a preferred embodiment, said host cell is transfected with an appropriate vector.

[0041] The term "expression" includes any step involved in the production of the protein of the invention including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0042] On the other hand, as understood by one skilled in the art, the protein of the invention can be purified by a variety of procedures known in the art that include, but are not limited to, chromatography (for example, ion exchange, affinity, hydrophobic, chromatofocusing and size exclusion), electrophoretic procedures (for example, preparative isoelectric focusing), differential solubility (for example, ammonium sulfate precipitation), SDS-PAGE, or extraction, in order to obtain substantially pure celiacase, which can be included, for example, in a composition.

[0043] Composition of the invention and derived formulations

[0044] Likewise, the protein, polynucleotide, gene construct, vector, or cell of the invention may be part of a composition. Thus, another aspect of the invention relates to a composition, hereinafter the "composition of the invention," comprising the protein, polynucleotide, gene construct, vector, or cell of the invention.

[0045] In a preferred embodiment, the composition of the invention is a pharmaceutical composition.

[0046] In the present invention, "pharmaceutical composition" refers to any preparation or pharmaceutical form, the composition formula of which, expressed in units of the international system, consists of a substance or mixture of substances, with constant weight, volume and percentages, prepared in legally established pharmaceutical laboratories, packaged or labeled to be distributed and marketed as effective for the diagnosis, treatment, mitigation and prophylaxis of a disease, physical anomaly or symptom, or the restoration, correction or modification of the balance of organic functions in humans and animals. The preparation of the pharmaceutical composition can be carried out by any of the methods described in the state of the art.

[0047] In another preferred embodiment, the composition of the invention further comprises a carrier, which, in the case of a pharmaceutical composition, will be a pharmaceutically acceptable carrier.

[0048] The term "vehicle" refers to a diluent or excipient with which the active ingredient is administered. Such vehicles may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as vehicles, particularly for injectable solutions. Preferably, pharmaceutical vehicles are approved by a state or federal regulatory agency or are listed in the United States Pharmacopoeia or another generally recognized pharmacopoeia for use in animals, and more particularly in humans.The vehicles and auxiliary substances required to manufacture the desired pharmaceutical form of administration of the pharmaceutical composition of the invention will depend, among other factors, on the pharmaceutical form of administration chosen. These pharmaceutical forms of administration of the pharmaceutical composition will be manufactured according to conventional methods known to those skilled in the art.

[0049] In a preferred embodiment, the composition of the invention comprises the protein, polynucleotide, gene construct, vector or cell of the invention in a therapeutically effective amount.

[0050] As used in this description, the term "therapeutically effective amount" refers to the quantity of the active agent or ingredient, or of the pharmaceutical composition of the invention, that produces the desired effect and, in general, will be determined, among other causes, by the characteristics of said compound or pharmaceutical composition and the therapeutic effect to be achieved. The dosage to obtain a therapeutically effective amount depends on a variety of factors, such as, for example, the age, weight, sex, or tolerance of the mammal. The "adjuvants" and "pharmaceutically acceptable carriers" that can be used in said compositions are those known in the state of the art.

[0051] In a preferred embodiment, the concentration of the protein of the invention in the composition of the invention is from 0.000001 to 500 mg / mL (including the extreme values). In a more preferred embodiment, the concentration of the protein of the invention in the composition of the invention is from 0.00001 mg / mL to 200 mg / mL (including the extreme values) or from 0.0001 mg / mL to 100 mg / mL (including the extreme values).

[0052] Additionally, the composition of the invention may comprise an adjuvant. By "adjuvant" is meant any substance that enhances the effectiveness of the pharmaceutical composition of the invention. Examples of adjuvants include, but are not limited to, adjuvants formed from aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, or aluminum sulfate; oil-in-water or water-in-oil emulsion formulations such as Complete Freund's Adjuvant (CFA) as well as Incomplete Freund's Adjuvant (IFA); mineral gels; block copolymers, Avridine™ , SEAM62; adjuvants formed from bacterial cell wall components such as adjuvants including liposaccharides (e.g.,lipid A or monophosphoryl lipid A (MLA), trehalose dimycolate (TDM), and cell wall skeleton components (CWS)), heat shock proteins or their derivatives, adjuvants derived from ADPribosylatinized bacterial toxins, including diphtheria toxin (DT), pertussis toxin (PT), cholera toxin (CT), the heat-labile E. coli toxins (LT 1 and LT2), Pseudomonas endotoxin A and exotoxin, B. cereus exoenzyme B, B. sphaerieus toxin, C. botulinum toxins C2 and C3, C. limosum exoenzyme as well as the toxins of C. perfringens, C. spiriforma and C. diffieile, S.aureus, EDIM and mutant toxin mutants such as CRM-197, non-toxic mutant diphtheria toxin; saponins such as ISCOMs (immunostimulatory complexes), chemokines, chemokines and cytokines such as interleukins (IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-12, etc.), interferons (such as interferon gamma), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), defensins 102, RANTES, MIPI-.alpha, and MEP-2, muramyl peptides; adjuvants derived from the CpG family of molecules, CpG dinucleotides and synthetic oligonucleotides comprising CpG motifs, C. lysosum exoenzymeLimosum and synthetic adjuvants such as PCPP, cholera toxin, Salmonella toxin, alum and the like, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, MTP-PE and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 12% squalene / Tween 80 emulsion. Other examples of adjuvants include DDA (dimethyldioctadecylammonium bromide), Freund's complete and incomplete adjuvants or QuilA.

[0053] In addition, the composition of the invention may comprise other additional active or inactive components. Additional components include salts to modulate ionic strength, preservatives including, but not limited to, antimicrobial agents, antioxidants, chelating agents, or the like, or nutrients, including glucose, dextrose, vitamins, and minerals. Other components, such as inert carriers or excipients, include, but are not limited to, binders; excipients; dispersing agents, such as alginic acid; lubricants, such as magnesium stearate; glidants such as colloidal silicon dioxide; sweetening agents; or flavoring agents, such as peppermint or methyl salicylate.

[0054] In a preferred embodiment, alone or in combination with other preferred embodiments, the composition of the invention further comprises binders.

[0055] Binders are used in solid dosage forms, e.g., to hold the ingredients in a tablet together, to ensure that tablets and granules can be formed with the required mechanical strength, and to provide bulk to low-active tablets. Binders in solid dosage forms such as tablets include: lactose, sucrose, maize (corn) starch, modified starches, microcrystalline cellulose, modified cellulose (e.g., hydroxypropyl methylcellulose (HPMC) and hydroxyethylcellulose), other water-soluble cellulose ethers, polyvinylpyrrolidone (PVP), also known as povidone, polyethylene glycol, sorbitol, maltitol, xylitol, and dibasic calcium phosphate; or mixtures of these substances.

[0056] The term "pharmaceutically acceptable" means that the vehicle or excipient must allow the activity of the compounds of the pharmaceutical composition, particularly the protein of the invention, that is, it must be compatible with said components, so that it does not cause harm to the organisms to which it is administered.

[0057] Such compositions and / or their formulations can be administered orally to an animal, including a mammal and, therefore, a human. Thus, in a preferred embodiment, alone or in combination with other preferred embodiments, the composition of the invention is formulated for oral administration.

[0058] Furthermore, the composition of the invention can be formulated in solid, semi-solid, or liquid preparations. In a preferred embodiment, the composition of the invention is formulated in solid or liquid form.

[0059] In a more preferred embodiment, the solid formulation is selected from the list consisting of tablets, pills, chewable tablets, capsules (including hard-shell capsules), sachets, powders, gels, granules, coated particles, dispersible strips and films.

[0060] In another more preferred embodiment, the liquid formulation is selected from the list consisting of oral solutions, suspensions, emulsions, drops and syrup.

[0061] Furthermore, the composition of the invention may, in certain embodiments, be buffered.

[0062] As previously explained, the protein of the invention requires an acidic pH, similar to that of the stomach, to enter its active form.

[0063] Thus, in some embodiments of the present invention, the composition comprises a suitable buffer, such that the pH of the composition is less than 6, preferably such that the pH of the composition is 3 to 5.

[0064] Examples of suitable buffers include, but are not limited to, 100mM glycine pH 2.5, 100mM glycine pH 3, 20mM HCl pH 2.5, 20mM HCl pH 3. Thus, in a more preferred embodiment, the buffer is selected from the list consisting of 100mM glycine pH 2.5 buffer, 100mM glycine pH 3 buffer, 20mM HCl pH 2.5 buffer, and 20mM HCl pH 3 buffer.

[0065] Other aspects of the invention relate to a pharmaceutical product, a veterinary product, a medical food, a food product, a food ingredient or additive, a food supplement, a probiotic food composition, a dietary supplement, a symbiotic composition, or a biotherapeutic composition, comprising an effective amount of the composition of the invention, preferably further comprising appropriate amounts of acceptable excipients.

[0066] In the context of the present invention, the aforementioned products are tolerated by a celiac subject, comprising an effective amount of the composition of the invention. The term "tolerated by a celiac subject," as used herein, refers to said products being suitable for celiac subjects, being gluten-free or containing reduced or trace levels thereof, allowing them to be tolerated by so many subjects suffering from this disorder.

[0067] A "probiotic food composition" is any combination of ingredients, a composition that constitutes a gluten-free solid or liquid food product, which, in addition to its nutritional characteristics, includes specific characteristics that help improve health and reduce the risk of contracting the disease.

[0068] A “dietary supplement” is an additional nutritional supplement intended to complement the diet.

[0069] A “biotherapeutic composition” is understood to be a product with a beneficial effect on health or a therapeutic effect.

[0070] "Symbiotic composition" refers to a functional food containing a mixture of prebiotic and probiotic food products. The term "pharmaceutical product" is understood in its broadest sense in this description, without being limited to a medication, and includes any composition comprising an active ingredient, in this case, the protein and / or derived aspects such as the cell of the invention, together with pharmaceutically acceptable excipients / vehicles.

[0071] The term "food product" is used herein in its broadest meaning, including any type of product, in any form of presentation, that can be ingested by an animal, but excluding pharmaceutical and veterinary products. Examples of food products include, but are not limited to, dairy products (yogurt, cheese, fermented milk, powdered milk, fermented milk-based product, ice cream, fermented cereal-based product, milk-based powder), bread, bars, spreads, biscuits and cereals, a beverage, various types of oil, or a dressing. Examples of other food products are meat products (e.g., sausages or spreadable meat products), spreadable chocolate products, fillings and icings, chocolate, pastries, baked goods (buns, puff pastries), sauces and soups, fruit juices, and coffee whiteners.

[0072] In a preferred embodiment, the food product is selected from the list consisting of yogurt, cheese, fermented milk, powdered milk, fermented milk-based product, ice cream, a fermented cereal-based product, milk-based powder, bread, bars, spreads, biscuits, cereals, beverages, oil, sausages, spreadable meat products, spreadable chocolate products, fillings and icings, chocolate, baked goods, including scones or puff pastries, sauces, and soups. More preferably, the food product is bread or chocolate.

[0073] "Food ingredient" refers to any substance used in the manufacture or preparation of a food product and still present in the finished product, albeit in a modified form. In one particular embodiment, the food ingredient is flour.

[0074] An "additive" refers to substances intentionally added to a food during its manufacturing or preparation process to change, enhance, or maintain its color, texture, flavor, shelf life, etc. In a preferred embodiment, the additive is a preservative. The terms "medical food" or "special medical purpose food" refer to a specially formulated food intended for the dietary management of a condition that has distinctive nutritional needs that cannot be met through a normal diet alone. Medical foods are distinct from the broader category of dietary supplements and traditional foods that make a health claim.

[0075] The term “dietary supplement” can be defined as a preparation or product intended to supplement the diet, prepared from compounds commonly used in food products, which provides nutrients or beneficial ingredients that are not normally ingested in the normal diet or that may not be consumed in sufficient quantities.

[0076] The term “effective amount” refers to any amount of the composition of the invention that, when administered to a subject, is sufficient to produce the desired effect.

[0077] Therapeutic uses of the invention

[0078] As previously explained herein, the protein of the invention, as well as the cell or composition of the invention, preferably the pharmaceutical composition of the invention, have different activities with potential therapeutic use in subjects.

[0079] Thus, another aspect of the invention relates to the protein, cell or composition of the invention for use as a medicine.

[0080] In another aspect, the invention relates to the use of the protein, cell or composition of the invention, in the preparation of a medicament or pharmaceutical composition.

[0081] The term "medicament" as used herein refers to any composition / substance used for the prevention, diagnosis, alleviation, treatment, or cure of diseases in a subject or that can be administered to the subject in order to restore, correct, or modify its physiological functions by exerting a pharmacological, immunological, or metabolic action. In another aspect, the invention relates to the protein, cell, or composition of the invention for use in the prevention, attenuation, and / or treatment of a disease in a subject due to the presence of peptide antigens, wherein the peptide antigen is present in gluten.

[0082] In another aspect, the invention relates to the use of the protein, cell or composition of the invention in the preparation of a medicament or pharmaceutical composition for the prevention, attenuation and / or treatment of a disease in a subject due to the presence of peptide antigens, wherein the peptide antigen is present in gluten.

[0083] In another aspect, the invention relates to a method for the prevention, attenuation and / or treatment of a disease in a subject due to the presence of peptide antigens, wherein the peptide antigen is present in gluten, which comprises the administration to said subject of the protein, cell or composition of the invention.

[0084] In the present invention, “treatment” is understood to mean the set of means used to treat or cure a disease.

[0085] The term “prevention” can be defined as the set of means or measures aimed at avoiding the onset of a disease, disorder or disturbance, or delaying its progress.

[0086] The term “attenuation” can be defined as alleviating the disease or disorder, that is, causing the regression of abnormal biological reactions and / or symptoms.

[0087] In the present invention, "subject" refers to any animal, preferably a mammal, more preferably a primate, particularly a human, of any race, sex, or age. Thus, in a preferred embodiment, the subject is a mammal, preferably a human. In another preferred embodiment, alone or in combination with other preferred embodiments, the subject suffers from celiac disease and / or disorders associated with gluten intake.

[0088] As used herein, the term “gluten” generally refers to proteins present in wheat or related grain species, including barley and rye, that have a potentially harmful effect on certain individuals. Gluten proteins include gliadins, such as α-gliadins, p-gliadins, y-gliadins, and β-gliadins, which are monomeric proteins, and glutenins, which are highly heterogeneous mixtures of aggregates of high molecular weight and low molecular weight subunits linked by disulfide bonds. Many wheat gluten proteins have been characterized. See, e.g., Woychik et al., Amino Acid Composition of Proteins in Wheat Gluten, J. Agrie. Food Chem., 9(4), 307–310 (1961). The term gluten, as used herein, also includes oligopeptides that can be obtained from normal human digestion of gluten proteins from gluten-containing foods and elicit the abnormal immune response.Some of these oligopeptides are resistant to normal digestive enzymes. Gluten, including the aforementioned proteins and oligopeptides, is thought to act as an antigen for T lymphocytes in susceptible patients (e.g., patients with celiac disease or gluten intolerance). The term gluten also refers to denatured gluten, such as would be found in baked goods.

[0089] The term "peptide antigen" in the present invention refers to a molecule formed by a chain of amino acids that has the ability to trigger an immune response in an organism. Specifically, antigens present a region, portion, or fragment known as an epitope or antigenic determinant, which is recognized by the immune system, triggering an immune response. Antigenic peptides are presented on the surface of antigen-presenting cells, such as dendritic cells, along with molecules of the major histocompatibility complex (MHC). This presentation allows lymphocytes to specifically recognize and bind to the presented peptide antigen, leading to the activation of the immune response.

[0090] In a preferred embodiment, alone or in combination with other preferred embodiments, the peptide antigen is a food antigen.

[0091] In another preferred embodiment, alone or in combination with other preferred embodiments, the disease is an intestinal disease.

[0092] In another preferred embodiment, alone or in combination with other preferred embodiments, the peptide antigen is rich in proline and / or glutamine residues. In another preferred embodiment, alone or in combination with other preferred embodiments, the peptide antigen is present in a gliadin or fragment thereof, preferably wherein the gliadin is an α-gliadin.

[0093] The primary structure of gliadins has a high percentage of proline and glutamine residues, which confers a high resistance to degradation by gastrointestinal enzymes. As a result, peptides resistant to the action of gastrointestinal enzymes accumulate in the intestinal lumen (Shan et al. 2005. Identification and analysis of multivalent proteolytically resistant peptides from gluten: implications for celiac sprue. J Proteome Res. 4, 1732–1741). Among these, there is a 33 amino acid peptide (33-mer) (LQLQPFPQPQLPYPQPLPYPQPQLPYPQPQPF; SEQ ID NO: 4) that has been described as the most immunogenic peptide of gluten, because it has six overlapping toxic epitopes (Qiao et al. 2004. Antigen presentation to celiac lesion- derived T cells of a 33-mer gliadin peptide naturally formed by gastrointestinal digestion. J Immunol 173:1757-1762).In susceptible individuals, these peptides are responsible for an abnormal reaction that eventually leads to chronic inflammation of the intestinal mucosa, crypt hyperplasia, and progressive deterioration of the intestinal villi, even their total disappearance.

[0094] In another preferred embodiment, alone or in combination with other preferred embodiments, the peptide antigen, the peptide antigen comprises the amino acid sequence SEQ ID NO: 3. More preferably, the antigen comprises the amino acid sequence SEQ ID NO: 4, which in turn comprises the amino acid sequence SEQ ID NO: 3.

[0095] In another preferred embodiment, alone or in combination with other preferred embodiments, the peptide antigen is 33-mer of α-gliadin or a fragment thereof.

[0096] Since the protein of the invention is capable of cutting the amino acid sequence QPQL (SEQ ID NO: 3) that is part of the peptides that generate the celiac disease response in celiac patients, the protein of the invention, the cell or the composition of the invention, preferably wherein the composition of the invention is the pharmaceutical composition of the invention, have application in the prevention, attenuation and / or treatment of celiac disease and / or disorders associated with gluten intake. Thus, in a preferred embodiment, the disease is celiac disease and / or disorders associated with gluten intake.

[0097] Celiac disease can be defined as a chronic autoimmune disorder induced by, or primarily triggered by, gluten ingestion in genetically predisposed individuals. Typical symptoms are associated with intestinal disorders due to villous atrophy and crypt hyperplasia, which cause a drastic reduction in the surface area of ​​the intestinal wall (malabsorption, diarrhea, anemia, fatigue, weight loss, abdominal bloating, etc.).

[0098] The term “celiac disease and / or disorders associated with gluten intake” includes, but is not limited to, any condition or disorder triggered by gluten proteins or peptides, including, but not limited to, gluten intolerance, wheat allergy, gluten sensitivity, gluten-sensitive enteropathy, idiopathic gluten sensitivity, and dermatitis herpetiformis.

[0099] In many cases, it may also have associated extraintestinal symptoms (osteoporosis, depression, infertility, etc.).

[0100] In addition, gluten intake and celiac disease are closely associated with other disorders such as, but not limited to, type I diabetes mellitus, Down syndrome, multiple sclerosis, dermatitis herpetiformis, myopathy, arthritis, including rheumatoid arthritis, autism, schizophrenia, lymphomas, attention deficit hyperactivity disorder (ADHD), fibromyalgia, Crohn's disease, nutrient malabsorption, and irritable bowel syndrome (IBS).

[0101] In a preferred embodiment, alone or in combination with other preferred embodiments, the protein, composition, or cell of the invention is administered to the subject prior to ingestion of a potentially antigenic food or protein containing gluten. In another preferred embodiment, the protein, composition, or cell of the invention is administered to the subject with ingestion of a potentially antigenic food or protein containing gluten. In another preferred embodiment, the protein, composition, or cell of the invention is administered to the subject after ingestion of a potentially antigenic food or protein containing gluten. In another preferred embodiment, the protein, composition, or cell of the invention is administered to the subject regardless of consumption of a potentially antigenic food or protein.

[0102] Industrial uses

[0103] In addition to the previously mentioned therapeutic uses, the protein of the invention, as well as the composition or cell of the invention, have other applications of interest, derived from the capacity of the protein of the invention to carry out cuts in peptide sequences that comprise the sequence QPQL (SEQ ID NO: 3), present for example in gluten peptides such as gliadins.

[0104] The protein, composition, or cell of the invention is capable of degrading peptides present in gluten. Thus, another aspect of the invention relates to the in vitro use of the protein, composition, or cell of the invention in the degradation of a peptide or fragment thereof present in gluten.

[0105] In a preferred embodiment, the peptide is a gliadin, preferably wherein the gliadin is α-gliadin.

[0106] In another preferred embodiment, alone or in combination with other preferred embodiments, the peptide or fragment thereof comprises the amino acid sequence SEQ ID NO: 3.

[0107] In another preferred embodiment, alone or in combination with other preferred embodiments, the peptide or fragment thereof comprises the sequence SEQ ID NO: 4, which in turn comprises the amino acid sequence SEQ ID NO: 3.

[0108] In another preferred embodiment, alone or in combination with other preferred embodiments, the peptide is 33-mer of α-gliadin.

[0109] Another aspect of the invention relates to the use of the protein, composition, or cell of the invention in the treatment of a food, food product, ingredient, or food additive containing gluten, wherein said treatment comprises degrading gluten from the food, food product, ingredient, or food additive, hereinafter referred to as "the use in the pretreatment of foods of the invention." In a preferred embodiment of the use in the pretreatment of foods of the invention, the gluten in turn comprises gliadins, preferably α-gliadins.

[0110] In this aspect of the invention, the term "treatment", referring to food, food product, ingredient or food additive, is understood as the processing of these prior to consumption by a subject, in order to modify the content or composition of its components, in the present invention gluten, reducing or eliminating it partially or totally.

[0111] Methods or techniques to evaluate whether gluten degradation has occurred include, but are not limited to, the use of ELISA kits for specific antigens derived from gluten ingestion such as G12 or R5, spectrophotometric, chromatographic and mass spectrometry techniques, among others.

[0112] In another aspect, the invention relates to the use of the protein, composition or cell of the invention in the preparation of a gluten-free food, food product, ingredient or food additive, hereinafter "the use in the preparation of gluten-free foods of the invention."

[0113] The term "gluten-free" refers to the food, food product, ingredient or food additive that has gluten levels less than 20 ppm. In a preferred embodiment of this aspect of the invention, the food, food product, ingredient or food additive has gluten levels less than 20 ppm. Methods or techniques for determining gluten levels in a food or food product, ingredient or food additive include, but are not limited to, the use of ELISA kits for specific antigens derived from gluten intake such as G12 or R5, spectrophotometric, chromatographic and mass spectrometry techniques, among others.

[0114] In principle, any food, food product, ingredient or food additive can be prepared as gluten-free by using the protein, composition or cell of the invention.

[0115] The terms food product, ingredient or additive have been previously explained in this document, being applicable, as well as their preferred embodiments, to the present aspect of the invention. technological

[0116] Since the protein of the invention is capable of degrading peptides present in gluten, such as gliadin peptides, it can be used as a processing aid in the preparation of foods with reduced gluten content. A processing aid is understood to be any substance that is not consumed as food in itself, but is intentionally used in the processing of raw materials, foods, or their ingredients to fulfill a specific technological purpose during treatment or processing, and which may give rise to the unintentional, but technically unavoidable, presence in the final product of residues of the processing aid itself or its derivatives, provided that they do not present any risk to health and that they do not have any technological effect on the final product.

[0117] Thus, another aspect of the present invention relates to the use of the protein, the cell of the invention or the composition of the invention as a technological adjuvant, preferably, in the preparation of foods.

[0118] Thus, its use as a processing aid promotes the degradation of gliadin and immunotoxic peptides in the food or product, which are responsible for triggering celiac disease and disorders associated with gluten ingestion.

[0119] Another aspect of the present invention relates to the protein, the cell of the invention or the composition of the invention for use as a processing aid.

[0120] Method of obtaining the invention

[0121] Another aspect of the invention relates to a method for obtaining the protein of the invention, which comprises subjecting a protein comprising the amino acid sequence SEO ID NO: 2 to a mutagenesis step, wherein said mutagenesis step comprises the use of two specific primers whose nucleotide sequences comprise, preferably consist of, GTGACCTGCAGGTCGTTGATACCTATG (SEQ ID NO: 13) and

[0122] GCACATAGCTCGCCTTGCCGT (SEQ ID NO: 14). Techniques for carrying out protein mutagenesis are known to those skilled in the art, including, but not limited to, PCR or the use of Stratagene's QuickChange site-directed mutagenesis kit, which are preferred ways of carrying out the mutagenesis step in the context of the present invention.

[0123] The terms used to define the present inventive aspect have been explained in previous inventive aspects, and both they and their preferred embodiments are applicable to the present inventive aspect.

[0124] DESCRIPTION OF THE FIGURES

[0125] Fig. 1. Analysis of the expression and purification of Neprosin / Neprosin variants.

[0126] Fig. 2. Analysis of Neprosin / Neprosin and Celiacase activation at pH 3 and 37 e C.

[0127] Fig. 3. Activity of Celiacase, Neprosin / Neprosina and the mutant vahantes at 37 e C and pH 3 against the fluorogenic peptide MCA-QPQL-Dpa-AR-NH2.

[0128] Fig. 4. Neprosin / Neprosin and Celiacase activity at 37 e C and pH 3 against the fluorogenic peptide MCA-QPQL-Dpa-AR-NH2, after having been at 4 for a week e C, - 80 e C and freeze-dried.

[0129] EXAMPLES

[0130] The invention will then be illustrated by tests carried out by the inventors, which demonstrate the effectiveness of the invention.

[0131] To design Celiacase, we first generated various mutants and studied both their activity and their performance during enzyme activation. The synthetic neprosin gene we used comes from Nepenthes ventrata and is 91% identical to the Nephentes alata ortholog (uniprot ID: A0A1 L7NZU4). The coding sequence for the neprosin zymogen without the original signal peptide (SEQ. ID 12) was transferred into the pCMV-sp6-splgk vector, which included a signal peptide that would remain at the N-terminus of Celiacase for protein export.

[0132] SEQ ID 12: CGTAGCATTCAGGCGCGTCTGGCGAATAAACCGAAAGGCACCATCAAAACCATCA AGGGCGACGACGGCGAAGTTGTGGATTGCGTTGACATCTACAAGCAGCCGGCGT TCGATCACCCGCTGCTGAAAAACCACACCCTGCAGATGCAACCGAGCAGCTATGC

[0133] GAGCAAGGTTGGCGAGTACAACAAACTGGAACAACCGTGGCACAAGAACGGCGA GTGCCCGAAAGGCAGCATCCCGATTCGTCGTCAGGTGATCACCGGTCTGCCGGT GGTTAAGAAACAATTCCCGAACCTGAAGTTTGCTCCGCCGAGCGCGAACACCAAC

[0134] CACCAGTATGCGGTTATTGCGTACTTCTATGGTAACGCGAGCCTGCAAGGCGCGA ACGCGACCATCAACATTTGGGAGCCGAACCTGAAAAACCCGAACGGTGACTTTAG CCTGACCCAGATCTGGATTAGCGCGGGTAGCGGCAGCAGCCTGAACACCATTGA

[0135] AGCGGGTTGGCAGGTGTATCCGGGTCGTACCGGTGATAGCCAACCGCGTTTCTT TATTTACTGGACCGCGGACGGTTATACCAGCACCGGCTGCTACGATCTGACCTGC CCGGGTTTCGTTCAGACCAACAACTACTATGCGATCGGCATGGCGCTGCAACCGA

[0136] GCGTGTACGGTGGCCAGCAATATGAGCTGAACGAAAGCATTCAGCGTGACCCGG CGACCGGTAACTGGTGGCTGTATCTGTGGGGTACCGTGGTTGGCTATTGGCCGG CGAGCATCTACAACAGCATTACCAACGGTGCGGACACCGTTGAGTGGGGTGGCG

[0137] AAATCTACGATAGCAGCGGTACCGGTGGCTTCCACACCACCACCCAAATGGGTAG CGGCCACTTTCCGACCGAAGGTTACGGCAAGGCGAGCTATGTGCGTGACCTGCA GTGCGTTGATACCTATGGTAACGTGATCAGCCCGACCGCGAACAGCTTTCAAGGC

[0138] ATTGCGCCGGCGCCGAACTGCTACAACTATCAATTTCAGCAGGGCAGCAGCGAA CTGTACCTGTTTTACGGTGGTCCGGGTTGCCAA

[0139] In this way, the pS6-proNEP construct is generated which adds a his-tag at the C- terminus (AIAHHHHHH; SEQ ID NO: 5 corresponding to the his-tag sequence) for its purification, resulting in SEQ ID NO: 6 sequence

[0140] SEQ ID NO:6

[0141] MQAKFFTFVILSSVFYFNYPLAEARSIQARLANKPKGTTIKGDDGEVVDCVDIYKQPA FDHPLLKNHTLQMQPSSYASKVGEYNKLEQPWHKNGECPKGSIPIRRQVITGLPVVK KQFPNLKFAPPSANTNHQYAVIAYFYGNASLQGANATINIWEPNLKNPNGDFSLTQIWI

[0142] SAGSGSSLNTIEAGWQVYPGRTGDSQPRFFIYWTADGYTSTGCYDLTCPGFVQTNN YYAIGMALQPSVYGGQQYELNESIQRDPATGNWWLYLWGTVVGYWPASIYNSITNG ADTVEWGGEIYDSSGTGGFHTTTQMGSGHFPTEGYGKASYVRDLQCVDTYGNWISP

[0143] TANSFQGIAPAPNCYNYQFQQGSSELYLFYGGPGCQAIAHHHHHH

[0144] To generate the different mutants, on the neprosin sequence (SEQ ID NO: 2), or from the construct that additionally includes the his-tag sequence at the C-terminal (SEQ ID NO: 5), we made the following mutations: R25K, S26T, I27L, Q28N, R30E, A32S, K36R, T41 S, V49M, L65K, H69P, T70S, S76N, E89K, P91 S, N95S, K100E, G101 V, T1 11 S, L113V, K117R, N161T, N166D, G167K, S182T, S183N, T215S, T217S, T230V, Q241 K, V244T, G247A, Q248E, G271 S, T272V, G279S, S284T, N287E, T304S, F306Y, T320R, K325R, C334V, V335T, T337E, Y338R, N340Q, V341 T, S343T, T345P, A346T, N347E, S348K, N357Q, N360K, Q362E, Q365E, S367K, and S368K.

[0145] The XGR3 mutant was synthesized containing all the mutations mentioned above. In addition, additional point mutations were made in both neprosin and XGR3 through PCR-based mutagenesis or by using the Stratagene QuickChange site-directed mutagenesis kit. The final mutants we generated are: XGR3, XGR3+N152T, XGR3+N145Q. In addition, we generated the simple mutant C334V (celiac case; SEQ ID NO: 1) and the mutants C334V+N145Q, C334V+N152T and C334V+N145Q+N152T.

[0146] We then proceeded to evaluate the expression (Fig. 1), activity, and productivity of the different mutants. After analyzing the results, the C334V (Celiacase) mutant showed the best activity, stability, and performance, improving the capabilities of neprosin, whose sequence is as follows:

[0147] <h2 style=";text-align:left;direction:ltr">MQAKFFTFVILSSVFYFNYPLAEARSIQARLANKPKGTIKTIKGDDGEVVDCVDIYKQPA FDHPLLKNHTLQMQPSSYASKVGEYNKLEQPWHKNGECPKGSIPIRRQVITTGLPVVK KQFPNLKFAPPSANTNHQYAVIAYFYGNASLQGANATINIWEPNLKNPNGDFSLTQIWI SAGSGSSLNTIEAGWQVYPGRTGDSQPRFFIYWTADGYTSTGCYDLTCPGFVQTNN YYAIGMALQPSVYGGQQYELNESIQRDPATGNWWLYLWGTVVGYWPASIYNSITNG ADTVEWGGEIYDSSGTGGFHTTTQMGSGHFPTEGYGKASYVRDLQVVDTYGNVISP TANSFQGIAPAPNCYNYQFQQGSSELYLFYGGPGCQ (SEQ ID NO: 1 )<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0148] <h2 style=";text-align:left;direction:ltr"> In addition to the rest of the mutants the results are:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0149] <h2 style=";text-align:left;direction:ltr"> Secuencia de aminoácidos del mutante XGR3:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0150] <h2 style=";text-align:left;direction:ltr"> MQAKFFTFVILSSVFYFNYPLAEAKTLNAELSNKPRGTIKSIKGDDGEMVDCVDIYKQP AFDHPKLKNPSLQMQPNSYASKVGEYNKLKQSWHKSGECPEVSIPIRRQVISGVPVV RKQFPNLKFAPPSANTNHQYAVIAYFYGNASLQGANATINIWEPTLKNPDKDFSLTQI WISAGSGTNLNTIEAGWQVYPGRTGDSQPRFFIYWTADGYSSSGCYDLTCPGFVQV NNYYAIGMALKPSTYGAEQYELNESIQRDPATGNWWLYLWSVVVGYWPSSIYNTITE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0151] <h2 style=";text-align:left;direction:ltr"> GADTVEWGGEIYDSSGSGGYHTTTQMGSGHFPREGYGRASYVRDLQVTDERGQTIT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0152] PPTECFQGIAPQCYKYEFQEGKKELYLFYGGPGCQ (SEQ ID NO: 7)

[0153] Amino acid sequence of the XGR3-N145Q-N152T mutant:

[0154] MQAKFFTFVILSSVFYFNYPLAEAKTLNAELSNKPRGTIKSIKGDDGEMVDCVDIYKQP

[0155] AFDHPKLKNPSLQMQPNSYASKVGEYNKLKQSWHKSGECPEVSIPIRRQVISGVPVV

[0156] RKQFPNLKFAPPSANTNHQYAVIAYFYGQASLQGATATINIWEPTLKNPDKDFSLTQI

[0157] WISAGSGTNLNTIEAGWQVYPGRTGDSQPRFFIYWTADGYSSSGCYDLTCPGFVQV

[0158] NNYYAIGMALKPSTYGAEQYELNESIQRDPATGNWWLYLWSSVVVGYWPSSIYNTITE

[0159] GADTVEWGGEIYDSSGSGGYHTTTQMGSGHFPREGYGRASYVRDLQVTDERGQTIT

[0160] PPTEKFQGIAPQCYKYEFQEGKKELYLFYGGPGCQ (SEQ ID NO: 8)

[0161] Amino acid sequence of the C334V-N145Q mutant:

[0162] MQAKFFTFVILSSVFYFNYPLAEARSIQARLANKPKGTIKTIKGDDGEVVDCVDIYKQPA

[0163] FDHPLLKNHTLQMQPSSYASKVGEYNKLEQPWHKNGECPKCGSIPIRRQVITGLPVVK

[0164] <h2 style=";text-align:left;direction:ltr">KQFPNLKFAPPSANTNHQYAVIAYFYGQASLQGANATINIWEPNLKNPNGDFSLTQIWI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0165] <h2 style=";text-align:left;direction:ltr"> SAGSGSSLNTIEAGWQVYPGRTGDSQPRFFIYWTADGYTSTGCYDLTCPGFVQTNN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0166] <h2 style=";text-align:left;direction:ltr"> YYAIGMALQPSVYGGQQYELNESIQRDPATGNWWLYLWGTVVGYWPASIYNSITNG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0167] <h2 style=";text-align:left;direction:ltr"> ADTVEWGGEIYDSSGTGGFHTTTQMGSGHFPTEGYGKASYVRDLQVVDTYGNVISP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0168] <h2 style=";text-align:left;direction:ltr"> TANSFQGIAPAPNCYNYQFQQGSSELYLFYGGPGCQ (SEQ ID NO: 9)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0169] <h2 style=";text-align:left;direction:ltr"> Secuencia de aminoácidos del mutante C334V+N152T:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0170] <h2 style=";text-align:left;direction:ltr"> MQAKFFTFVILSSVFYFNYPLAEARSIQARLANKPKGTIKTIKGDDGEVVDCVDIYKQPA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0171] <h2 style=";text-align:left;direction:ltr"> FDHPLLKNHTLQMQPSSYASKVGEYNKLEQPWHKNGECPKGSIPIRRQVITGLPVVK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0172] <h2 style=";text-align:left;direction:ltr"> KQFPNLKFAPPSANTNHQYAVIAYFYGNASLQGATATINIWEPNLKNPNGDFSLTQIWI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0173] <h2 style=";text-align:left;direction:ltr"> SAGSGSSLNTIEAGWQVYPGRTGDSQPRFFIYWTADGYTSTGCYDLTCPGFVQTNN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0174] <h2 style=";text-align:left;direction:ltr"> YYAIGMALQPSVYGGQQYELNESIQRDPATGNWWLYLWGTVVGYWPASIYNSITNG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0175] <h2 style=";text-align:left;direction:ltr"> ADTVEWGGEIYDSSGTGGFHTTTQMGSGHFPTEGYGKASYVRDLQVVDTYGNVISP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0176] TANSFQGIAPAPNCYNYQFQQGSCELYLFYGGPGCQ (SEQ ID NO: 10)

[0177] Amino acid sequence of mutant C334V+N145Q+N152T: MQAKFFTFVILSSVFYFNYPLAEARSIQARLANKPKGTIKTIKGDDGEVVDCVDIYKQPA FDHPLLKNHTLQMQPSSYASKVGEYNKLEQPWHKNGECPKGSIPIRRQVITGLPVVK KQFPNLKFAPPSANTNHQYAVIAYFYGQASLQGATATINIWEPNLKNPNGDFSLTQIWI SAGSGSSLNTIEAGWQVYPGRTGDSQPRFFIYWTADGYTSTGCYDLTCPGFVQTNN YYAIGMALQPSVYGGQQYELNESIQRDPATGNWWLYLWGTVVGYWPASIYNSITNG ADTVEWGGEIYDSSGTGGFHTTTQMGSGHFTEGYGKASYVRDLQVVDTYGNVISP TANSFQGIAPAPNCYNYQFQQGSCELYLYLYLFYGGPGCQ (SEQ ID NO: 11 )

[0178] Both Celiacase, and the rest of mutants, can include the his-tag sequence at the C-terminus (for example, AIAHHHHHH; SEQ ID NO: 5 corresponding to the his-tag sequence) for their purification.

[0179] Both neprosin and Celiacase are produced in their proform or zymogen state, and an acidic pH similar to that of the stomach (Fig. 2) is required for the enzyme to convert to its active form. To produce the enzyme, we used the human cell line Exp¡293 (Thermofisher Scientific), where the recombinant protein is excreted into the medium. These cells are grown at 37 °C. e C in the Multitron cell shaker Incubator (Infors HT) at 37 eC. Cells are transfected with plasmid DNA containing neprosin, celiacase, or any other variant. Once the protein is produced, the medium is clarified by centrifugation and supplemented with 20mM imidazole. The medium is then subjected to nickel affinity purification (Ni-NTA). Finally, the protein is eluted in 300mM imidazole. Purification is then performed by molecular exclusion using a Superdex75 10 / 300 GL column (GE Healthcare) and the ÁKTA Purifier liquid chromatography system (GE Healthcare). Neprosin, celiacase, and the rest of the variants were activated at pH 3.0. Sample purity was analyzed using both the gel filtration chromatographic profile and a 20% SDS-PAGE gel stained by Comassie. The production yield of both neprosin and Celiacase yields approximately 10 mg to 20 mg of protein per liter of culture, while the other variants have a lower yield.After producing both proteins, we compared how much enzyme is lost during activation at pH=3 and we were able to verify that less total amount of Celiacase was lost than neprosin during activation (Fig. 2).

[0180] We evaluated the activity of Celiacase and neprosin against the fluorogenic peptide MCA-QPQL-Dpa-AR-NH2 (Fig. 3). The QPQL sequence (SEQ ID NO: 3) is part of the target capable of cleaving Celiacase, which is part of the peptides that generate the celiac disease response in celiac patients, such as the 33-mer (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF; SEQ ID NO: 4). Specifically, the enzyme is capable of cleaving those peptide sequences between P and Q or Y, which human digestive enzymes are unable to cleave, as previously mentioned, and which are present in gliadin peptides of different lengths. Celiacase is more effective than neprosin in carrying out this cleavage reaction (Fig. 3).

[0181] To perform the activity assays with the fluorogenic peptide MCA-QPQL-Dpa-A-R-NH2 (GenScript), a concentration of 10pM of peptide per 10OnM of enzyme was used, either neprosin, celiacase or vahante. The fluorescence signal was measured during the digestion of the peptide by each enzyme until the curve reached its stationary phase. Once the measurement was completed, the slope of the linear zone of each activity was calculated. The activity assays with the fluorogenic peptide were performed at 37 e C for 30 minutes. All assays were performed in quadruplicate (Fig. 3).

[0182] Using the same peptide MCA-QPQL-Dpa-AR-NH2(GenScript), to evaluate stability and conservation, we evaluated the activity of the protein at 37 e C and pH 3.0, after having been at 4 for a week e C, frozen at -80 eC or lyophilized (Fig. 4). Both neprosin and Celiacase can be preserved using any of the three processes; in the activated form, Celiacase shows greater recovery of activity after freezing compared to neprosin. (Fig. 4)

Claims

CLAIMS 1. A protein comprising the amino acid sequence SEQ ID NO:

1.

2. Protein according to claim 1, wherein the protein has a length of 380 to 500 amino acids.

3. Protein according to claim 1, consisting of the amino acid sequence SEQ ID NO:

1.

4. A polynucleotide encoding a protein according to any one of claims 1 to 3.

5. A gene construct comprising a polynucleotide according to claim 4.

6. A vector comprising a polynucleotide according to claim 4, or a gene construct according to claim 5.

7. A cell comprising a protein according to any one of claims 1 to 3, a polynucleotide according to claim 4, a gene construct according to claim 5, or a vector according to claim 6.

8. Composition comprising a protein according to any one of claims 1 to 3, a polynucleotide according to claim 4, a gene construct according to claim 5, a vector according to claim 6, or a cell according to claim 7.

9. Composition according to the preceding claim, further comprising a vehicle.

10. Composition according to claim 8 or 9, wherein the composition is a pharmaceutical composition.

11. Composition according to any one of claims 8 to 10, wherein the composition is formulated for oral administration.

12. Composition according to any one of claims 8 to 11, wherein the composition is formulated in solid form or liquid form.

13. Composition according to claim 12, wherein the solid formulation is selected from the list consisting of tablets, pills, chewable tablets, capsules, sachets, powders, gels, granules, coated particles, dispersible strips and films.

14. Composition according to claim 13, wherein the liquid formulation is selected from the list consisting of oral solutions, suspensions, emulsions, drops and syrup.

15. Protein according to any one of claims 1 to 3, a cell according to claim 7, or a composition according to any one of claims 8 to 14 for use as a medicament.

16. Protein according to any one of claims 1 to 3, a cell according to claim 7, or a composition according to any one of claims 8 to 14, for use in the prevention, attenuation and / or treatment of a disease due to the presence of peptide antigens in a subject, wherein the peptide antigen is present in gluten.

17. Protein, cell or composition for use according to claim 16, wherein the peptide antigen is a food antigen.

18. Protein, cell or composition for use according to claim 16 or 17, wherein the peptide antigen is present in a gliadin or fragment thereof, preferably wherein the gliadin is an α-gliadin.

19. Protein, cell or composition for use according to any one of claims 16 to 18, wherein the peptide antigen comprises the amino acid sequence SEQ ID NO:

3.

20. Protein, cell or composition for use according to claim 19, wherein the antigen comprises the amino acid sequence SEQ ID NO: 4, which in turn comprises the amino acid sequence SEQ ID NO:

3.

21. Protein, cell or composition for use according to any one of claims 18 to 20, wherein the peptide antigen is 33-mer of α-gliadin or a fragment thereof.

22. Protein, cell or composition for use according to any one of claims 16 to 21, wherein the disease is celiac disease and / or disorders associated with gluten intake.

23. Protein, cell or composition for use according to any one of claims 16 to 22, wherein the disease is an intestinal disease.

24. A pharmaceutical product, a veterinary product, a medical food, a food product, a food ingredient or additive, a food supplement, a probiotic food composition, a dietary supplement, a symbiotic composition, or a biotherapeutic composition, comprising an effective amount of the composition according to any one of claims 8 to 14.

25. A pharmaceutical product, a veterinary product, a medical food, a food product, a food supplement, a food ingredient or additive, a probiotic food composition, a dietary supplement, a symbiotic composition, or a biotherapeutic composition according to claim 24, further comprising appropriate amounts of acceptable excipients.

26. Use of a protein according to any one of claims 1 to 3, a cell according to claim 7, or a composition according to any one of claims 8 to 14, as a processing aid.

27. In vitro use of a protein according to any one of claims 1 to 3, a cell according to claim 7, or a composition according to any one of claims 8 to 14, in the degradation of a peptide or fragment thereof present in gluten.

28. Use according to claim 27, wherein the peptide is a gliadin, preferably wherein the gliadin is α-gliadin.

29. Use according to claim 27 or 28, wherein the peptide or fragment thereof, comprises the amino acid sequence SEQ ID NO:

3.

30. Use according to claim 29, wherein the peptide or fragment thereof comprises the sequence SEQ ID NO: 4, which in turn comprises the amino acid sequence SEQ ID NO:

3.

31. Use according to any one of claims 28 to 30, wherein the peptide is 33-mer of α-gliadin.

32. Use of a protein according to any one of claims 1 to 3, a cell according to claim 7, or a composition according to any one of claims 8 to 14, in the treatment of a food, foodstuff, ingredient or food additive having gluten, wherein said treatment comprises degrading gluten from the food, foodstuff, ingredient or food additive.

33. Use according to claim 32, wherein the gluten in turn comprises gliadins, preferably α-gliadins.

34. Use of a protein according to any one of claims 1 to 3, a cell according to claim 7, or a composition according to any one of claims 8 to 14, in the preparation of a gluten-free food, food product, ingredient or food additive.

35. Method for obtaining a protein according to any one of claims 1 to 4, comprising subjecting a protein with an amino acid sequence SEQ ID NO: 2 to a mutagenesis step, wherein said mutagenesis comprises the use of two specific primers whose nucleotide sequence comprises, preferably consists of, the sequence SEQ ID NO: 13 and SEQ ID NO: 14.

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