Pharmaceutical compositions comprising aminopeptidase and dipeptidylpeptidase polypeptides and uses thereof

Abstract

The present disclosure relates to compositions comprising polypeptides with peptidase activity that are aminopeptidase (AP) and dipeptidylpeptidase (DPP) polypeptides which are useful for degradation of disease-promoting peptides such as gluten-immunogenic peptides. The formulation of the compositions protects the peptidases from acidic conditions in the stomach when administered orally. The compositions described herein are useful in medical applications such as in the treatment of gluten-related disorders including celiac disease (CeD) and non-celiac gluten sensitivity (NCGS) as well as other diseases that may profit from a gluten-free diet (GFD).

Description

[0001] PHARMACEUTICAL COMPOSRIONS COMPRISING AMINOPEPTIDASE AND DIPEPTIDYLPEPT1DASE POLYPEPTIDES AND USES THEREOF

[0002] The present disclosure relates to compositions comprising polypeptides with peptidase activity that are aminopeptidase (AP) and dipeptidylpeptidase (DPP) polypeptides which are useful for degradation of disease-promoting peptides such as gluten-immunogenic peptides. The formulation of the compositions protects the peptidases from acidic conditions in the stomach when administered orally. The compositions described herein are useful in medical applications such as in the treatment of gluten-related disorders including celiac disease (CeD) and non-celiac gluten sensitivity (NCGS) as well as other diseases that may profit from a gluten-free diet (GFD).

[0003] Gluten refers to the main storage proteins found in wheat, rye, and barley, known largely for their viscoelastic properties playing a crucial part in dough formation (Bailey, A. J. et al., Philos. Trans. R. Soc. Lond. B. Biol. Sci. 357, 133-142 (2002)). Gluten protein contains an unusually high content of proline and glutamine polypeptide residues which are credited for the proteolytic resistance of these proteins to gastric, pancreatic, and intestinal secretions containing digestive proteases (Sharma, N. et al., Front. Nutr. 7, 6 (2020); Stamnaes, J. & Sollid, L M., Semin. Immunol. 27, 343-352 (2015); Shan, L et al., J. Proteome Res. 4, 1732-1741 (2005)). The ensuing build-up of partially digested polypeptides, referred to as gluten immunogenic peptides (GIPs), and the acquired immune response to these peptides in genetically susceptible individuals is the hallmark of celiac disease (CD). The immune response to GIPs in CD is driven by CD4+T cells that bind to human leukocyte antigen (HLA)-DQ. allotypes (Goel, G. et al, Sci. Adv. 5, eaaw7756 (2019); Jabri, B, & Sollid, L M., J. Immunol. Baltim. Md 1950 198, 3005-3014 (2017)). Removing gluten from one's diet, and therefore GIPs, is seen as the only "effective" treatment for CD, though research shows that adhering to a strict gluten-free diet (GFD) is extremely challenging, and up to 80% of GFD-adhering patients experience inadvertent gluten contamination in their diet.

[0004] Approximately 1% of the global population suffers from CD and mucosal healing on a GFD occurs in less than half of adults (Singh, P. et al., Clin. Gastroenterol. Hepatol. 16, 823-836.e2 (2018); Silvester, J. A. et al., Aliment. Pharmacol. Ther. 52, 1469-1479 (2020)). Though the triggers of CD are well understood, it is important to note that there is a greater number of people consuming gluten-free food and adhering to a GFD than people diagnosed with CD. This population avoiding gluten suffers from a clinical entity referred to as "noncoeliac gluten sensitivity" (NCGS), for which the exact triggers are unknown but thought to revolve around innate immune system activation due to gluten or gluten-like immunogenic peptides, and / or FODMAPs (fermentable oligosaccharides, disaccharides, monosaccharides, and polyols) (Gibson, P. R., Skodje, G. I. & Lundin, K. E. A., J. Gastroenterol. Hepatol. 32 Suppl 1, 86-89 (2017)). The clinical presentations of NCGS are varied and include IBS-like symptoms, behavioural changes, and systemic manifestations of inflammation (Biesiekierski, J. R. & Iven, J., United Eur. Gastroenterol. J. 3, 160-165 (2015); Catassi, C. et al., Nutrients 5, 3839-3853 (2013)). Interestingly, gluten-like proteins (and associated peptides) are seen as one of the most relevant triggers in people suffering from IBS, with patients labelled as either NCGS or more broadly "noncoeliac wheat or wheat protein sensitive" (Roberto De Giorgio, Umberto Volta, & Peter R Gibson, Gut 65, 169 (2016)). Furthermore, a high prevalence of NCGS (c. 28%) is reported amongst IBS patients (Catassi, C. et al., Nutrients 5, 3839-3853 (2013)). Overall, there is increasing interest in the role dietary protein, and specifically gluten protein, plays in the symptoms and pathophysiology of functional gastrointestinal disorders (Boettcher, E. & Crowe, S. E., Am. J. Gastroenterol. 108, 728-736 (2013)).

[0005] Since human proteases are thought to be ineffective at degrading proline and glutamine-rich gluten peptides, the consensus approach for enzymatic treatment strategies has been to supplement gluten protein digestion in the stomach with administration of exogenous endoproteases and / or endopeptidases. Despite over 10 years of research and clinical trials on endoproteases, this form of enzyme supplementation has yielded no effective treatments for gluten-related health disorders (chiefly NCGS and CD) (Nemteanu, R. et al., Int. J. Mol. Sci. 23, (2022); Machado, M. V., Int. J. Mol. Sci. 24, (2023)). Limitations of these enzymes can be demonstrated through Latiglutenase by ImmunogenX, the most studied endoprotease to date. Latiglutenase is composed of a two-enzyme combination with alleged complementary action. The first enzyme, EP-B2, is an endoprotease targeting gluten-protein in the stomach. Subsequently, the second enzyme (SC-PEP) is an endopeptidase targeting the ensuing gluten peptides in the small intestine. SC-PEP suffers from severe chain-length restrictions and is therefore unable to cleave peptides unless these have been shortened by EP-B2. Furthermore, it was shown that EP-B2 increases the inflammatory characteristics of gluten compared to placebo as it generated more immunogenic peptides than pepsin alone (Siegel, M. et al., Chem. Biol. 13, 649-658 (2006)). Concerns were also raised on the ability of SC-PEP to adequately detoxify the peptides generated by EP-B2, particularly due to the vulnerability of SC-PEP to acidic pH in the stomach (Siegel, M. et al., Chem. Biol.- 13, 649-658 (2006)). Moreover, there is growing evidence that peptide fragments generated through endopeptidase digestion can self-assemble into immunogenic structures (Herrera, M. G. & Dodero, V. I., Biophys. Rev. 13, 1147-1154 (2021); Herrera, M. G. et al., Mol. Nutr. Food Res. 65, 2100200 (2021)).

[0006] Phase 2 clinical trials with Latiglutenase have failed to show convincing evidence of efficacy, with discrepancies reported on the assessed endpoints. In their latest Phase 2b trial, ImmunogenX tried to address these limitations in part by measuring gluten-immunogenic peptides in the urine of patients using immunoassays. However, questions were raised on dosage (at least 1.2 g of Latiglutenase needed for 2 g of gluten per day) and reliability of the qualitative analytical methods used to measure gluten peptides (ELISA kits) (Machado, M. V., Int. J. Mol. Sci. 24, (2023); Monachesi, C. et al., Clin. Transl. Gastroenterol. 12, (2021)).

[0007] The present disclosure is based on the insight that enzymatic approaches relying solely on gluten digestion in the stomach are necessarily ineffective. Gastric emptying of meals is a complex process that starts immediately after food intake. Whilst the digestion of large solids (particle size above 2-4 mm) involves the process of gastric grinding providing time for peptic hydrolysis of gluten, gastric emptying of small particle and liquids is largely dependent on volume and energy density of intragastric contents and starts almost immediately after food ingestion (Sanaka, M., Yamamoto, T. & Kuyama, Y., Dig. Dis. Sci. 55, 2431-2440 (2010)). Furthermore, solid food in the stomach is segmented into poorly mixing layers, whereas liquids taken together with solid meals are subject to rapid gastric emptying (Koziolek, M. et al., Underst. Chall. -Rule--5 Compd. 101, 75-88 (2016); Schulze, K., Neurogastroenterol. Motil. 18, 172-183 (2006)). It is therefore highly unlikely that the entirety of ingested gluten protein can be readily digested inside the stomach. These digestive processes, with fast emptying of dissolved material, further highlight the limited efficacy of stomach-only acting water-soluble enzyme treatments, as these are unlikely to encounter their target substrate due to both poor stomach mixing and vulnerability to "premature" gastric emptying. Once in the small intestine, endoproteases experience either greatly reduced activity or complete denaturation, thereby missing their window of opportunity for gluten peptide digestion. it would be desirable to provide a stable composition of enzymes that is effective at degrading proline and glutamine-rich gluten peptides following administration to a patient.

[0008] It is demonstrated herein that two complementary polypeptides with exopeptidase activity, an aminopeptidase (AP) and a dipeptidylpeptidase (DPP), which are sensitive to the highly acidic environment of the stomach, can be protected from gastric inactivation. Such protection may be achieved, for example, by buffering gastric pH or using enteric-coated formulations with targeted release in the small intestine. This enzyme combination is also shown to have long-lasting activity and efficacy at intestinal pH. This, together with the fact that the combination of polypeptides with exopeptidase activity can degrade proteins to single amino acids and dipeptidases, allows the expectation that oral administration of the polypeptides with exopeptidase activity so that they are protected from inactivation by the acidic environment in the stomach, e.g. administration in an enteric-coated formulation, can provide sufficient proteolytic activity for complete degradation of gluten peptides in the small intestine.

[0009] Summary

[0010] The present invention provides pharmaceutical compositions comprising polypeptides with aminopeptidase (AP) and dipeptidylpeptidase (DPP) activity. In one embodiment, the polypeptide with AP activity is leucine aminopeptidase 2 from Trichophyton rubrum (ruLAP2). In one embodiment, the polypeptide with DPP activity is dipeptidylpeptidase IV from Trichophyton rubrum (ruDPPIV).

[0011] The AP and DPP enzymes described herein are proteases which are labile to the harsh conditions in stomach, but are active at the conditions of the intestine. Inactivation and furthermore degradation of the enzymes is observed during their passage through the stomach. Application of oral drug delivery systems protecting the enzymes from gastric inactivation allows the delivery of the enzymes to the intestine without substantial loss of activity.

[0012] In some embodiments, this is realized with delayed release formulations. In some embodiments, the release at acidic conditions of the stomach is inhibited by pH sensitive polymers which are only soluble at high pH values.

[0013] In some embodiments, this is realized with immediate release formulations. In some embodiments, the acidic conditions of the stomach are buffered.

[0014] In some embodiments, mucoadhesivity is added to the formulation to increase the time spent at the site of action, i.e., intestine. Charged polymers such as alginate and chitosan are useful in this respect. In some embodiments, particulate formulations are used combining the enzymes and the mucoadhesive polymers in a single particle. In these embodiments, particles are attached to the mucus and then release the enzymes so that the time spent at the site of action is increased.

[0015] In one aspect, the present disclosure provides a pharmaceutical composition comprising a polypeptide having aminopeptidase (AP) activity and a polypeptide having dipeptidylpeptidase (DPP) activity for oral administration, wherein the pharmaceutical composition protects the polypeptides against gastric inactivation.

[0016] In some embodiments, the pharmaceutical composition, when orally administered to a mammal, releases the polypeptide having AP activity and the polypeptide having DPP activity in the small intestine, in particular the upper small intestine.

[0017] In some embodiments, the polypeptide having AP activity and the polypeptide having DPP activity in the pharmaceutical composition remain essentially intact during their transit through the stomach and into the duodenum.

[0018] In some embodiments, the polypeptide having AP activity and the polypeptide having DPP activity in the pharmaceutical composition exhibit a loss of enzyme activity of no more than 50%, 40%, 30%, 20%, or 10% during their transit through the stomach and into the duodenum. In some embodiments, the pharmaceutical composition is a solid formulation.

[0019] In some embodiments, the pharmaceutical composition is selected from the group consisting of granules, powders, tablets, capsules and pellets. In some embodiments, the pharmaceutical composition comprises a carrier material on which the polypeptide having AP activity and the polypeptide having DPP activity is immobilized.

[0020] In some embodiments, the polypeptide having AP activity and the polypeptide having DPP activity is immobilized on the carrier material by applying the polypeptide having AP activity and the polypeptide having DPP activity onto the carrier material and drying.

[0021] In some embodiments, the polypeptide having AP activity and the polypeptide having DPP activity is applied onto the carrier material by spraying.

[0022] In some embodiments, drying is performed at a temperature of between 20°C and 60°C for 0.5 to 3 hours.

[0023] In some embodiments, drying is performed at a temperature of between 30°C and 50°C for about 1 hour.

[0024] In some embodiments, the carrier material comprises a material selected from the group consisting of sucrose, microcrystalline cellulose, calcium carbonate, mannitol, maltodextrine and silicon dioxide.

[0025] In some embodiments, the carrier material comprises a material selected from the group consisting of sucrose and mannitol.

[0026] In some embodiments, the polypeptide having AP activity and the polypeptide having DPP activity are encapsulated within or coated by an enteric material.

[0027] In some embodiments, the enteric material comprises a pH-sensitive polymer.

[0028] In some embodiments, the enteric material comprises a material selected from the group consisting of cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, shellac, acrylic acid polymers or copolymers, methacrylic acid polymers or copolymers, and methacrylic acid / methylmethacrylate copolymers.

[0029] In some embodiments, the enteric material comprises a methacrylic acid polymer or copolymer.

[0030] In some embodiments, the enteric material comprises an EUDRAGUARD® resin.

[0031] In some embodiments, the enteric material comprises EUDRAGUARD® Control.

[0032] In some embodiments, the enteric material further comprises a mucoadhesive polymer. in some embodiments, the mucoadhesive polymer comprises a polymer selected from the group consisting of chitosans, alginates, pectins, hyaluronans, lectins, and derivatives thereof. In some embodiments, the mucoadhesive polymer comprises chitosan, or a derivative thereof.

[0033] In some embodiments, the enteric material comprises a methacrylic acid polymer or copolymer and chitosan,

[0034] In some embodiments, the pharmaceutical composition comprises a core comprising the polypeptide having AP activity and the polypeptide having DPP activity, coated with an enteric coating.

[0035] In some embodiments, the core further comprises a binder.

[0036] In some embodiments, the binder is selected from the group consisting of starches, sugars, lactose, sugar alcohols, xylitol, sorbitol, maltitol, cellulose, microcrystalline cellulose, modified celluloses, hydroxypropylcellulose, carboxymethylcellulose sodium, alginic acid, polyvinyl pyrrolidone, and mixtures thereof.

[0037] In some embodiments, the core further comprises a stabilizer.

[0038] In some embodiments, the stabilizer is selected from the group consisting of proline, trehalose, dextran, maltose, sucrose, mannitol, polyols, silica gel, aminoguanidine, pyridoxamine, anhydrous metal salts, magnesium oxide, calcium oxide, aluminum oxide, and mixtures thereof.

[0039] In some embodiments, the core further comprises a disintegrant.

[0040] In some embodiments, the disintegrant is selected from the group consisting of dibasic calcium phosphate, dibasic calcium phosphate dihydrate, tribasic calcium phosphate, alginic acid, hydroxypropylcellulose, carboxymethylcellulose calcium, carboxymethylcellulose sodium, cross-linked carboxymethylcellulose sodium, swellable ion exchange resins, alginates, formaldehyde-casein, cellulose, croscarmellose sodium, crospovidone, microcrystalline cellulose, sodium carboxymethyl starch, sodium starch glycolate, starches, corn starch, rice starch, and mixtures thereof.

[0041] In some embodiments, the polypeptide having AP activity cleaves single amino acids off the N-terminal end of polypeptides, except when these are connected to proline in an NH2-X-Pro sequence. In some embodiments, the polypeptide having AP activity is derived from a fungus.

[0042] In some embodiments, the polypeptide having AP activity is derived from Trichophyton rub rum.

[0043] In some embodiments, the polypeptide having AP activity is a polypeptide having leucine- aminopeptidase activity.

[0044] In some embodiments, the polypeptide having AP activity comprises the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof.

[0045] In some embodiments, the polypeptide having AP activity has been expressed in a fungus.

[0046] In some embodiments, the polypeptide having AP activity has been expressed in Pichia pastoris.

[0047] In some embodiments, the polypeptide having DPP activity cleaves the dipeptide motive NH2- X-Pro off the N-terminal end of polypeptides.

[0048] In some embodiments, the polypeptide having DPP activity is derived from a fungus.

[0049] In some embodiments, the polypeptide having DPP activity is derived from Trichophyton rubrum.

[0050] In some embodiments, the polypeptide having DPP activity is a polypeptide having dipeptidylpeptidase IV (DPPIV) activity.

[0051] In some embodiments, the polypeptide having DPP activity comprises the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof.

[0052] In some embodiments, the polypeptide having DPP activity has been expressed in a fungus.

[0053] In some embodiments, the polypeptide having DPP activity has been expressed in Pichia pastoris.

[0054] In some embodiments, the weight ratio of the polypeptide having AP activity and the polypeptide having DPP activity is between 20:1 and 1:5.

[0055] In some embodiments, the pharmaceutical composition completely degrades the following 33-mer peptide:

[0056] LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF

[0057] In a further aspect, the present disclosure provides the pharmaceutical composition described herein for pharmaceutical use. In a further aspect, the present disclosure provides the pharmaceutical composition described herein for use in treating a peptide-related disorder.

[0058] In a further aspect, the present disclosure provides the pharmaceutical composition described herein for use in treating a gluten-related disorder.

[0059] In some embodiments, the gluten-related disorder is selected from the group consisting of celiac disease, wheat allergy and non-celiac gluten sensitivity.

[0060] In a further aspect, the present disclosure provides a method of treating peptide-related disorder comprising orally administering the pharmaceutical composition described herein to a mammal in need thereof.

[0061] In a further aspect, the present disclosure provides a method of treating a gluten-related disorder comprising orally administering the pharmaceutical composition described herein to a mammal in need thereof.

[0062] In some embodiments, the gluten-related disorder is selected from the group consisting of celiac disease, wheat allergy and non-celiac gluten sensitivity.

[0063] In some embodiments of any method described herein, the mammal is a human.

[0064] In some embodiments of any method described herein, the pharmaceutical composition is administered in combination with a substance which increases Gl tract pH.

[0065] In some embodiments, the substance which increases Gl tract pH is selected from the group consisting of proton pump inhibitors and antacids.

[0066] In some embodiments of any method described herein, the pharmaceutical composition is administered to a mammal prior to, simultaneously with or following dietary intake. In some embodiments of any method described herein, the pharmaceutical composition is administered to a mammal prior to dietary intake.

[0067] In some embodiments of any method described herein, the pharmaceutical composition is administered to a mammal up to 3 hours prior to dietary intake.

[0068] In some embodiments of any method described herein, the pharmaceutical composition is administered to a mammal 1 to 3 hours prior to dietary intake.

[0069] In some embodiments, the diet is supposed to be gluten-free.

[0070] In some embodiments, the dietary intake comprises inadvertent gluten intake.

[0071] Brief description of the drawings

[0072] Figure 1. LC profile for gradient elution of free amino acids and dipeptides resulting from 33- mer degradation by tandem ruLAP-2 and ruDPP-4 activity. Schematic presentation of mobile phase composition over the entire period of the LC method (above) and detailed time and corresponding composition of mobile phases A and B (below).

[0073] Figure 2. Two-stage release of microencapsulated enzymes. Enzyme release from microparticle encapsulation is expressed as dissolution percentage. Both ruLAP-2 and ruDPP- IV demonstrated negligible release at acidic pH (0-60 min, 0.1 M HCI), and sustained release when pH was increased to pH 6.8.

[0074] Figure 3. ruDPP-IV enzyme activity following simulated gastrointestinal passage using two- stage dissolution assay. ruDPP-IV activity of microparticle formulations (NP-5 Lyo, NP-5 Spray Dry) were compared with enzymes dissolved directly in pH 6.8 (square line, no stomach passage), and enzymes dissolved first in 0.1 M HCI (triangle line, stomach passage).

[0075] Figure 4. ruLAP-2 enzyme activity following simulated gastrointestinal passage using two- stage dissolution assay. ruLAP-2 activity of microparticle formulations (NP-5 Lyo, NP-5 Spray Dry) were compared with enzymes dissolved directly in pH 6.8 (square line, no stomach passage), and enzymes dissolved first in 0.1 M HCI (triangle line, stomach passage).

[0076] Figure 5. Predicted 33-mer degradation by ruLAP-2 and ruDPP-IV exopeptidase combination. The gluten derived immunogenic 33-mer peptide is expected to be completely cleaved down to single amino acids and dipeptides by the combined action of ruLAP-2 (black) and ruDPP-IV (white). Proposed cleaving sides are shown by arrows. The length of the remaining peptide after each cleavage is indicated below.

[0077] Figure 6. Representative chromatogram of single amino acids and X-Pro dipeptides detected after complete 33-mer degradation by exopeptidases ruLAP-2 and ruDPP-IV Figure 7. Enzyme stability was assessed over time through a series of pre-incubation periods before addition of 33-mer peptide to the solution. Enzymes were dissolved in TRIS buffer at pH 7 at a 1:1 ratio (8.6 mg / L ruLAP-2; 8.6 mg / L ruDPP-IV). The 33-mer peptide (100 mg / L) was either added directly to the enzyme (Oh, black circle ) or after pre-incubation of the enzymes for Ih (black square), 3h (white circle), or 5h (white square) at 37°C. Digestion efficacy measured by detecting the single amino acids that result from ruLAP-2-mediated cleavage of the 33-mer. Identified cleaved amino acids are indicated in bold in the 33-mer structure below. Data represented as means ± SEM of three replicates.

[0078] Figure 8. Determination of complete 33-mer degradation by sucrose formulation after different periods of preincubation. 33-mer was digested in TRIS buffer at pH 7 with an enzyme ratio of ruLAP2:ruDPP4 of 8.6 mg / l to 8.6 mg / l (1:1). The 33-mer peptide (100 mg / L) was either added directly to the enzyme (Oh) or added after previous incubation of the enzymes for Ih, 3h, or 5h at 37°C. Complete degradation of the 33-mer peptide was monitored by quantification of the C-terminal single amino acid phenylalanine (F) that is only cleaved in the last step of enzyme-mediated 33-mer degradation by tandem action of ruLAP-2 and ruDPP-4. Degradation efficiency at each timepoint was compared to enzyme without preincubation (Oh). Shown are means ± SEM of three replicates.

[0079] Detailed description

[0080] Although the present disclosure is further described in more detail below, it is to be understood that this disclosure is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0081] In the following, the elements of the present disclosure will be described in more detail. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present disclosure to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0082] The practice of the present disclosure will employ, unless otherwise indicated, conventional chemistry, biochemistry, pharmaceutical, cell biology, immunology, and recombinant DNA techniques which are explained in the literature in the field.

[0083] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated feature, element, member, integer or step or group of features, elements, members, integers or steps but not the exclusion of any other feature, element, member, integer or step or group of features, elements, members, integers or steps. The term "consisting essentially of" limits the scope of a claim or disclosure to the specified features, elements, members, integers, or steps and those that do not materially affect the basic and novel ch a racterist ic(s) of the claim or disclosure. The term "consisting of" limits the scope of a claim or disclosure to the specified features, elements, members, integers, or steps. The term "comprising” encompasses the term "consisting essentially of" which, in turn, encompasses the term "consisting of". Thus, at each occurrence in the present application, the term "comprising" may be replaced with the term "consisting essentially of" or "consisting of". Likewise, at each occurrence in the present application, the term "consisting essentially of" may be replaced with the term "consisting of".

[0084] The terms "a", "an" and "the" and similar references used in the context of describing the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context.

[0085] AH methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by the context.

[0086] The use of any and all examples, or exemplary language (including "such as", "for example" and "e.g."), provided herein is intended merely to better illustrate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0087] The term "optional" or "optionally" as used herein means that the subsequently described event, circumstance or condition may or may not occur, and that the description includes instances where said event, circumstance, or condition occurs and instances in which it does not occur.

[0088] Where used herein, "and / or" is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "X and / or Y" is to be taken as specific disclosure of each of (i) X, (ii) Y, and (iii) X and Y, just as if each is set out individually herein.

[0089] In the context of the present disclosure, the term "about" denotes an interval of accuracy that the person of ordinary skill will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value by ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, and for example ±0.01%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±10%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±5%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±4%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±3%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±2%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±1%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.9%, In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.8%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.7%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.6%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.5%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.4%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.3%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.2%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.1%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.05%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.01%. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect.

[0090] Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0091] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0092] In the following, definitions and embodiments will be provided which apply to all aspects of the present disclosure. Terms which are defined in the following have the meanings as defined unless otherwise indicated. Any undefined terms have their art recognized meanings.

[0093] Terms such as "reduce" or "inhibit" as used herein means the ability to cause an overall decrease, for example, of about 5% or greater, about 10% or greater, about 15% or greater, about 20% or greater, about 25% or greater, about 30% or greater, about 40% or greater, about 50% or greater, or about 75% or greater, in the level. The term "inhibit" or similar phrases includes a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero.

[0094] Terms such as "enhance" or "promote" as used herein means the ability to cause an overall increase, or enhancement, for example, by at least about 5% or greater, about 10% or greater, about 15% or greater, about 20% or greater, about 25% or greater, about 30% or greater, about 40% or greater, about 50% or greater, about 75% or greater, or about 100% or greater in the level.

[0095] Amino acids, peptides, polypeptides, peptidases

[0096] As used herein, the terms "polypeptide having peptidase activity", "peptidase", "protease", "proteolytic enzyme" and "peptide hydrolase" are synonyms and may be used interchangeably. Peptidases include all enzymes that catalyse the cleavage of the peptide bonds (CO-NH) of peptides, digesting these peptides into smaller peptides or free amino acids. A peptidase described herein may be a naturally occurring or non-naturally occurring polypeptide having the corresponding activity.

[0097] As used herein, the term "exopeptidase" relates to enzymes which catalyse the hydrolytic cleavage of peptide bonds at the ends (amino (N)- or carboxy (C)-terminus) of peptides. The products are free amino acids, dipeptides or tripeptides. Exopeptidases which act at the N- terminus are called aminopeptidases (APs) (cleavage of a single amino acid), dipeptidyl peptidases (DPPs) (release of a dipeptide), or tripeptidyl peptidases (TPPs) (release of a tripeptide), depending on the fragment cleaved. Exopeptidases also include carboxypeptidases, however, preferred according to the disclosure are exopeptidases which act at the N-terminus.

[0098] In some embodiments, an enzymatic activity cleaving amino acids from the N-terminal end of peptides is contributed by a polypeptide having aminopeptidase activity, or simply aminopeptidase, e.g., leucine aminopeptidase.

[0099] Leucine aminopeptidase (LAP) is an aminopeptidase that preferentially catalyzes the hydrolysis of leucine residues at the N-terminus of peptides. Other N-terminal residues such as proline can also be cleaved, however.

[0100] In some embodiments, an enzymatic activity cleaving dipeptides from the N-terminal end of peptides having a proline residue in the second amino acid position is contributed by a a polypeptide having d i peptidyl peptidase activity, or simply d ipeptidyl peptidase.

[0101] Dipeptidyl peptidase IV or dipeptidyl peptidase 4 (DPPIV or DPP4) is a dipeptidyl peptidase that cleaves N-terminal dipeptides from peptides, preferentially when the second amino acid is proline, provided the third amino acid is neither proline nor hydroxyproline.

[0102] Preferred embodiments of aminopeptidases and dipeptidyl peptidases are leucine aminopeptidase 2 of Trichophyton rubrum (ruLAP2) and dipeptidyl peptidase IV of Trichophyton rubrum (ruDPPIV), respectively. ruLAP2, a leucine aminopeptidase, cleaves single amino acids off the N-terminal end of polypeptides, except when these are connected to proline in an NH2-X-Pro sequence. ruDPPIV selectively cleaves the dipeptide motif NH2-X-Pro off the N-terminal end of polypeptides.

[0103] Simultaneous application of ruLAP2 and ruDPPIV can be used to degrade proline-rich, digestion-resistant, GIPs. None of both, ruLAP2 and ruDPPIV is capable on its own to completely degrade the a-gliadin 33-mer (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQ.PQ.PF), a gluten-derived highly immunogenic peptide, which represents a well described immunoreactive GIP and functions as accepted model peptide. The a-gliadin 33-mer contains two of the most potent celiac disease-related T-cell epitopes in multiple copies and is described as being highly degradation resistant. While ruLAP2 can remove the first 3 amino acids from the N-terminus, the degradation cannot proceed beyond that point due to the QP- motif that follows next. ruDPPIV on the other hand does not affect the 33-mer since the enzyme specifically cleaves X-Pro dipeptide motifs but not the amino acids present at the N- terminus of the 33-mer. When present in combination, however, ruLAP2 and ruDPPIV can completely degrade the gliadin 33-mer.

[0104] A ruLAP2 polypeptide may have an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 99 %, 98 %, 97 %, 96 %, 95 %, 90 %, 85 %, or 80 % identity to the amino acid sequence of SEQ ID NO: 1.

[0105] A ruDPPIV polypeptide may have an amino acid sequence comprisingthe amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 99 %, 98 %, 97 %, 96 %, 95 %, 90 %, 85 %, or 80 % identity to the amino acid sequence of SEQ ID NO: 2. ruLAP2 (SEQ ID NO: 1)

[0106] HPVVGQEPFGWPFKPMVTQDDLQNKIKLKDIMAGVEKLQSFSDAHPEKNRVFGGNGHKDTVEWIYNEI KATGYYDVKKQEQVHLWSHAEAALNANGKDLKASAMSYSPPASKIMAELWAKNNGCNATDYPANTQ GKIVLVERGVCSFGEKSAQAGDAKAAGAIVYNNVPGSLAGTLGGLDKRHVPTAGLSQEDGKNLATLVAS GKIDVTMNVISLFENRTTWNVIAETKGGDHNNVIMLGAHSDSVDAGPGINDNGSGSIGIMTVAKALTNF KLNNAVRFAWWTAEEFGLLGSTFYVNSLDDRELHKVKLYLNFDMIGSPNFANQIYDGDGSAYNMTGPA GSAEIEYLFEKFFDDQGIPHQPTAFTGRSDYSAFIKRNVPAGGLFTGAEVVKTPEQVKLFGGEAGVAYDKN YHRKGDTVANINKGAIFLNTRAIAYAIAEYARSLKGFPTRPKTGKRDVNPQYSKMPGGGCGHHTVFM ruDPPIV (SEQ ID NO: 2)

[0107] IVPPREPRSPTGGGNKLLTYKECVPRATISPRSTSLAWINSEEDGRYISQSDDGALILQNIVTNTNKTLVAAD KVPKGYYDYWFKPDLSAVLWATNYTKQYRHSYFANYFILDIKKGSLTPLAQDQAGDIQYAQWSPMNNSI AYVRGNDLYIWNNGKTKRITENGGPDIFNGVPDWVYEEEIFGDRFALWFSPDGEYLAYLRFNETGVPTYT IPYYKNKQKIAPAYPRELEIRYPKVSAKNPTVQFHLLNIASSQETTIPVTAFPENDLVIGEVAWLSSGHDSVA YRAFNRVQDREKIVSVKVESKESKVIRERDGTDGWIDNLLSMSYIGNVNGKEYYVDISDASGWAHIYLYPV DGGKEIALTKGEWEWAILKVDTKKKLIYFTSTKYHSTTRHVYSVSYDTKVMTPLVNDKEAAYYTASFSAKG GYYILSYQGPNVPYQELYSTKDSKKPLKTITSNDALLEKLKEYKLPKVSFFEIKLPSGETLNVKQRLPPNFNPH KKYPVLFTPYGGPGAQEVSQAWNSLDFKSYITSDPELEYVTWTVDNRGTGYKGRKFRSAVAKRLGFLEAQ DQVFAAKEVLKNRWADKDHIGIWGWSYGGFLTAKTLETDSGVFTFGISTAPVSDFRLYDSMYTERYMKT VELNADGYSETAVHKVDGFKNLKGHYLIQHGTGDDNVHFQNAAVLSNTLMNGGVTADKLTTQWFTDS

[0108] DHGIRYDMDSTYQYKQLSKMVYDQKQRRPESPPMHQWSKRVLAALFGERAEE

[0109] In some embodiments, a peptidase comprises a truncated peptidase polypeptide.

[0110] In some embodiments, a leucine aminopeptidase comprises a truncated LAP polypeptide. In some embodiments, a leucine aminopeptidase comprises a truncated LAP2 polypeptide. In some embodiments, the truncation is an N-terminal and / or C-terminal truncation. In some embodiments, the truncated LAP polypeptide is a fragment of a wildtype LAP polypeptide. In some embodiments, the truncated LAP polypeptide is a fragment of a wildtype LAP2 polypeptide.

[0111] In various embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 amino acids are missing at the C-terminus compared to the wildtype LAP polypeptide. In various embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acids are missing at the C-terminus compared to the wildtype LAP polypeptide. In these and other embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 amino acids are missing at the N-terminus compared to the wildtype LAP polypeptide. In these and other embodiments, 1, 2, 3, 4, 5, 6, or 7 amino acids are missing at the N-terminus compared to the wildtype LAP polypeptide.

[0112] In some embodiments, in the truncated LAP polypeptide (i) up to 7 amino acids are missing at the N-terminus, and / or (ii) up to 23 amino acids are missing at the C-terminus compared to wildtype LAP polypeptide. In some embodiments, in the truncated LAP polypeptide (i) up to 6 amino acids are missing at the N-terminus, and / or (ii) up to 22 amino acids are missing at the C-terminus compared to wildtype LAP polypeptide.

[0113] In some embodiments, the wildtype LAP polypeptide has the amino acid sequence represented by SEQ, ID NO: 1 or a variant thereof.

[0114] In some embodiments, a dipeptidyl peptidase comprises a truncated DPP polypeptide. In some embodiments, a dipeptidyl peptidase comprises a truncated DPPIV polypeptide. In some embodiments, the truncation is an N-terminal and / or C-terminal truncation. In some embodiments, the truncated DPP polypeptide is a fragment of a wildtype DPP polypeptide. In some embodiments, the truncated DPP polypeptide is a fragment of a wildtype DPPIV polypeptide. In various embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 amino acids are missing at the C-terminus compared to the wildtype DPP polypeptide. In various embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids are missing at the C-terminus compared to the wildtype DPP polypeptide. In these and other embodiments, at least 1 amino acid is missing at the N-terminus compared to the wildtype DPP polypeptide. In these and other embodiments, 1, or 2 amino acids are missing at the N- terminus compared to the wildtype DPP polypeptide. In some embodiments, in the truncated DPP polypeptide (i) one or two amino acids are missing at the N-terminus and / or (ii) up to 15 amino acids are missing at the C-terminus compared to the wildtype DPP polypeptide. In some embodiments, in the truncated DPP polypeptide (i) one or two amino acids are missing at the N-terminus and / or (ii) up to 14 amino acids are missing at the C-terminus compared to the wildtype DPP polypeptide.

[0115] In some embodiments, the wildtype DPP polypeptide has the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof.

[0116] In some embodiments, an aminopeptidase for use herein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 or a variant thereof. In some embodiments, a variant comprises an amino acid sequence having at least 99 %, 98 %, 97 %, 96 %, 95 %, 90 %, 85 %, or 80 % identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, such variant comprises an amino acid sequence which is a truncation of the amino acid sequence of SEQ ID NO: 1, e.g., the amino acid sequence of SEQ ID NO: 1 wherein 12 amino acids are missing at the N-terminus and 22 amino adds are missing at the C- terminus, or an amino acid sequence having at least 99 %, 98 %, 97 %, 96 %, 95 %, 90 %, 85 %, or 80 % identity to the amino acid sequence which is a truncation of the amino acid sequence of SEQ ID NO: 1.

[0117] In some embodiments, a dipeptidyl peptidase for use herein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 or a variant thereof. In some embodiments, a variant comprises an amino acid sequence having at least 99 %, 98 %, 97 %, 96 %, 95 %, 90 %, 85 %, or 80 % identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, such variant comprises an amino acid sequence which is a truncation of the amino acid sequence of SEQ ID NO: 2, e.g., the amino acid sequence of SEQ ID NO: 2 wherein 1 amino acid is missing at the N-terminus and 16 amino acids are missing at the C-terminus, or an amino acid sequence having at least 99 %, 98 %, 97 %, 96 %, 95 %, 90 %, 85 %, or 80 % identity to the amino acid sequence which is a truncation of the amino acid sequence of SEQ ID NO: 2.

[0118] A peptidase polypeptide may be part of a chimeric or fusion protein comprising the peptidase polypeptide linked at the N-terminus and / or C-terminus to one or more other amino acid sequences.

[0119] The term "one or more other amino acid sequences" in the case of a peptidase polypeptide refers to (an) amino acid sequence(s) corresponding to a protein that is not substantially homologous to the peptidase polypeptide. Within the fusion protein, the peptidase polypeptide and the one or more other amino acid sequences are fused with one another. The one or more other amino acid sequences can be fused to the N-terminus and / or C- terminus of the peptidase polypeptide.

[0120] In some embodiments, the one or more other amino acid sequences comprise a signal sequence, e.g. a heterologous signal sequence, that is fused to a peptidase polypeptide, e.g., to the N-terminus of the peptidase polypeptide.

[0121] In its broadest sense, as used herein, the term "amino acid" refers to a compound and / or substance that can be, is, or has been incorporated into a peptide, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid. In some embodiments, an amino acid is an L-amino acid. In some embodiments, an amino acid is a D-amino acid. In general, the term "amino acid" as used herein refers to a standard amino acid.

[0122] "Standard amino acid", "natural amino acid" or "proteinogenic amino acid" refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides and polypeptides. "Nonstandard amino acid" refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source.

[0123] As will be clear from context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid. In some embodiments it may be used to refer to an amino acid residue of a peptide or polypeptide.

[0124] The following table lists the 20 natural amino acids and their abbreviations:

[0125] According to the disclosure, the term "peptide" refers to substances which comprise about two or more, about 3 or more, about 4 or rnnm ahniit A or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100 or about 150, consecutive amino acids linked to one another via peptide bonds. The term "polypeptide" refers to large peptides, in particular peptides having more than about 150 amino acids. "Peptides" and "polypeptides" are both protein molecules. Thus, the terms "peptide", "protein" and "polypeptide" are used herein usually as synonyms and the term "peptide" when used herein is to include polypeptides if not contradicted by the context.

[0126] The term "disease-promoting peptide" refers to any peptide that can trigger, promote, or exacerbate a disease, disorder or condition. In particular, the term includes peptides that are present in food or that are degradation products of food. In some embodiments, the term "disease-promoting peptide" includes immunogenic peptides, i.e., peptides that have an immunogenic effect. Such peptides, in some embodiments, have an inflammation-inducing or promoting effect and may be the cause of or a factor in chronic inflammation. Examples of disease-promoting peptides include gluten-immunogenic peptides.

[0127] As used herein, the term "immunogenic peptide" includes any peptide that can elicit, facilitate, or produce an immune response.

[0128] "Fragment", with reference to an amino acid sequence (peptide or polypeptide), relates to a part of an amino acid sequence, i.e., a sequence which represents the amino acid sequence shortened at the N-terminus and / or C-terminus. A fragment shortened at the C-terminus (N- terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 3'-end of the open reading frame. A fragment shortened at the N-terminus (C- terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 5'-end of the open reading frame, as long as the truncated open reading frame comprises a start codon that serves to initiate translation. A fragment of an amino acid sequence comprises, e.g., at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 % of the amino acid residues from an amino acid sequence. A fragment of an amino acid sequence comprises, e.g., at least 6, in particular at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from an amino acid sequence. A fragment of an amino acid sequence comprises, e.g., a sequence of up to 8, in particular up to 10, up to 12, up to 15, up to 20, up to 30 or up to 50, consecutive amino acids of the amino acid sequence. "Variant," as used herein and with reference to an amino acid sequence (peptide or polypeptide), means an amino acid sequence that differs from a parent amino acid sequence by virtue of at least one amino acid (e.g., a different amino acid, or a modification of the same amino acid). The parent amino acid sequence may be a naturally occurring or wild type (WT) amino acid sequence, or may be a modified version of a wild type amino acid sequence. In some embodiments, the variant amino acid sequence has at least one amino acid difference as compared to the parent amino acid sequence, e.g., from 1 to about 20 amino acid differences, such as from 1 to about 10 or from 1 to about 5 amino acid differences compared to the parent.

[0129] By "wild type" or "WT" or "native" as used herein and with reference to an amino acid sequence (peptide or polypeptide) is meant an amino acid sequence that is found in nature, including allelic variations. A wild type amino acid sequence, peptide or polypeptide has an amino acid sequence that has not been intentionally modified.

[0130] For the purposes of the present disclosure, "variants" of an amino acid sequence (peptide or polypeptide) may comprise amino acid insertion variants, amino acid addition variants, amino acid deletion variants and / or amino acid substitution variants. The term "variant" includes all mutants, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, in particular those which are naturally occurring. The term "variant" includes, in particular, fragments of an amino acid sequence.

[0131] Amino acid insertion variants comprise insertions of single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted into a particular site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible. Amino acid addition variants comprise amino- and / or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions may be in any position of the protein. Amino acid deletion variants that comprise the deletion at the N-terminal and / or C-terminal end of the protein are also called N-terminal and / or C- terminal truncation variants. Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Preference is given to the modifications being in positions in the amino acid sequence which are not conserved between homologous peptides or polypeptides and / or to replacing amino acids with other ones having similar properties. In some embodiments, amino acid changes in peptide and polypeptide variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.

[0132] In some embodiments, the degree of similarity, such as identity between a given amino acid sequence and an amino acid sequence which is a variant of said given amino acid sequence will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The alignment for determining sequence similarity, such as sequence identity can be done with art known tools, such as using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS: needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0133] "Sequence similarity" indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.

[0134] The terms "% identical" and "% identity" or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or "window of comparison", in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of algorithms, e.g., the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, the local homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website (e.g., at blast. ncbi.nlm. nih.gov / Blast. cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC =align2seq). In some embodiments, the algorithm parameters used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match / Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used. In some embodiments, the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment.

[0135] Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.

[0136] In some embodiments, the degree of similarity or identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence. For example, if the reference sequence consists of 200 amino acids, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments continuous amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence. According to the disclosure, the term "truncated" with respect to a certain peptide or polypeptide refers to the peptide or polypeptide with one or more amino acids deleted at the N- and / or C-terminus of the parent peptide or polypeptide such as wildtype peptide or polypeptide. According to the disclosure, a truncated form or variant of an amino acid sequence is a fragment of said amino acid sequence.

[0137] The peptides and polypeptides described herein may readily be prepared by the skilled person, for example, by recombinant DNA manipulation or with the aid of known peptide synthesis techniques such as, for example, by solid phase synthesis and similar methods.

[0138] In some embodiments, a fragment or variant of an amino acid sequence (peptide or polypeptide) is a "functional fragment" or "functional variant". The term "functional fragment" or "functional variant" of an amino acid sequence relates to any fragment or variant exhibiting one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., it is functionally equivalent. With respect to sequences of peptidase polypeptides, one particular function is one or more proteolytic activities displayed by the amino add sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant", as used herein, in particular refers to a variant molecule or sequence that comprises an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and that is still capable of fulfilling one or more of the functions of the parent molecule or sequence, e.g., proteolytic activity. In some embodiments, the modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced but still significantly present, e.g., function of the functional fragment or functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the parent molecule or sequence. However, in other embodiments, function of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0139] An amino acid sequence (peptide or polypeptide) "derived from" a designated amino acid sequence (peptide or polypeptide) refers to the origin of the first amino acid sequence. In some embodiments, the amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to that particular sequence or a fragment thereof. Amino acid sequences derived from a particular amino acid sequence may be variants of that particular sequence or a fragment thereof. For example, it will be understood by one of ordinary skill in the art that the sequences suitable for use herein may be altered such that they vary in sequence from the naturally occurring or native sequences from which they were derived, while retaining the desirable activity of the native sequences.

[0140] As used herein, an "instructional material" or "instructions" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the disclosure. The instructional material of a kit comprising a composition described herein may, for example, be affixed to a container which contains the composition or be shipped together with a container which contains the composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the composition be used cooperatively by the recipient.

[0141] In some embodiments, "isolated" means removed (e.g., purified) from the natural state or from an artificial composition, such as a composition from a production process. For example, a nucleic acid, peptide or polypeptide naturally present in a living animal is not "isolated", but the same nucleic acid, peptide or polypeptide partially or completely separated from the coexisting materials of its natural state is "isolated". An isolated nucleic acid, peptide or polypeptide can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0142] The term "recombinant" in the context of the present disclosure means "made through genetic engineering". Preferably, a "recombinant object" such as a recombinant nucleic acid in the context of the present disclosure is not occurring naturally.

[0143] The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.

[0144] The term "genetic modification" or simply "modification" includes the transfection of cells with nucleic acid. The term "transfection" relates to the introduction of nucleic acids into a cell. For purposes of the present disclosure, the term "transfection" also includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by such cell. According to the present disclosure, a cell for transfection of a nucleic acid described herein can be present in vitro. According to the disclosure, transfection can be transient or stable. For some applications of transfection, it is sufficient if the transfected genetic material is only transiently expressed. RNA can be transfected into cells to transiently express its coded protein. Since the nucleic acid introduced in the transfection process is usually not integrated into the nuclear genome, the foreign nucleic acid will be diluted through mitosis or degraded. Cells allowing episomal amplification of nucleic acids greatly reduce the rate of dilution. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, a stable transfection must occur. Such stable transfection can be achieved by using virus-based systems or transposon-based systems for transfection.

[0145] Nucleic acids

[0146] The term "polynucleotide" or "nucleic acid", as used herein, is intended to include DNA and RNA such as genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. A nucleic acid may be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (I VT RNA) or synthetic RNA.

[0147] In some embodiments, the nucleic acid described herein may have modified and / or non- naturally occurring nucleosides.

[0148] Nucleic acids may be comprised in a vector. The term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and includes any vectors known to the skilled person including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retroviral, adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC). Said vectors include expression as well as cloning vectors. Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may lack functional sequences needed for expression of the desired DNA fragments.

[0149] "Plasmid" refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. A "viral vector" is a vector wherein additional DNA segments can be ligated into a viral genome.

[0150] Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

[0151] "Expression vectors" are capable of directing the expression of genes to which they are operatively linked. In general, expression vectors used in recombinant DNA techniques are often present in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the disclosure is intended to include other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0152] The production of a functional protein is intimately related to the cellular machinery of the organism producing the protein. E.coli has typically been the "factory" of choice for the expression of many proteins because its genome has been fully mapped and the organism is easy to handle; grows rapidly; requires an inexpensive, easy-to-prepare medium for growth; and secretes protein into the medium which facilitates recovery of the protein. However, E.coli is a prokaryote and lacks intracellular organelles, such as the endoplasmic reticulum and the golgi apparatus that are present in eukaryotes, which contain enzymes which modify the proteins being produced. Many eukaryotic proteins can be produced in E.coli but these may be produced in a nonfunctional, unfinished form, since glycosylation or post-translational modifications do not occur.

[0153] Therefore, eukaryotic yeast, mammalian and plant expression systems are frequently used for protein production. For example, the methanoltrophic yeast P. pastoris has become a powerful host for the heterologous expression of proteins and has been established as an alternative eukaryotic host for the expression of human proteins with high-throughput technologies.

[0154] As another example, plants are being used as expression hosts for large-scale heterologous expression of proteins and offer potential advantages of cost-effectiveness, scalability and safety over traditional expression systems. There are currently a variety of plant heterologous expression systems including transient expression, plant cell-suspension cultures, recombinant plant viruses and chloroplast transgenic systems. While proteins expressed in plants have some variations from mammalian proteins (e.g., glycosylation), there is currently no evidence that these differences result in adverse reactions in human patients.

[0155] Another suitable heterologous expression system uses insect cells, often in combination with baculovirus expression vectors. Baculovirus vectors are available for expressing proteins in cultured insect cells.

[0156] One particularly preferred expression system for production of polypeptides decribed herein is the Pichia pastoris expression system. P. pastoris has been developed to be an outstanding host for the production of foreign proteins since its alcohol oxidase promoter was isolated and cloned. Compared to other eukaryotic expression systems, Pichia offers many advantages, because it does not have the endotoxin problem associated with bacteria nor the viral contamination problem of proteins produced in animal cell cultures. Furthermore, P. pastoris can utilize methanol as a carbon source in the absence of glucose. The P. pastoris expression system uses the methanol-induced alcohol oxidase (A0X1) promoter, which controls the gene that codes for the expression of alcohol oxidase, the enzyme that catalyzes the first step in the metabolism of methanol. This promoter has been characterized and incorporated into a series of P. pastoris expression vectors. Since the proteins produced in P. pastoris are typically folded correctly and secreted into the medium, the fermentation of genetically engineered P. pastoris provides an excellent alternative to E.coli expression systems. Furthermore, P. pastoris has the ability to spontaneously glycosylate expressed proteins, which also is an advantage over E.coli. A number of proteins have been produced using this system, including tetanus toxin fragment, Bordatella pertussis pertactin, human serum albumin and lysozyme. A nucleic acid encoding a peptide or polypeptide described herein may be expressed in a cell transfected with the nucleic acid to provide the peptide or polypeptide. In some embodiments, expression is into the extracellular space, i.e., the encoded polypeptide is secreted.

[0157] "Encoding" refers to the inherent property of specific sequences of nucleotides in a nucleic acid to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0158] In the context of the present disclosure, the term "transcription" relates to a process, wherein the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA may be translated into peptide or protein.

[0159] According to the present disclosure, the term "transcription" comprises "in vitro transcription", wherein the term "in vitro transcription" relates to a process wherein RNA, in particular mRNA, is in vitro synthesized in a cell-free system, preferably using appropriate cell extracts. Preferably, cloning vectors are applied for the generation of transcripts. These cloning vectors are generally designated as transcription vectors and are according to the present disclosure encompassed by the term "vector". The promoter for controlling transcription can be any promoter for any RNA polymerase. Particular examples of RNA polymerases are the T7, T3, and SP6 RNA polymerases. Preferably, the in vitro transcription according to the disclosure is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA.

[0160] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence. With respect to RNA, the term "expression" or "translation" relates to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of a sequence of amino acids to make a peptide or protein.

[0161] As used herein "endogenous" refers to any material from or produced inside an organism, cell, tissue or system.

[0162] As used herein, the term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0163] As used herein, the terms "linked," "fused", or "fusion" are used interchangeably. These terms refer to the joining together of two or more elements or components or domains.

[0164] Pharmaceutical compositions

[0165] The term "pharmaceutical composition" relates to a formulation comprising a therapeutically effective agent, preferably together with pharmaceutically acceptable carriers, diluents and / or excipients. A pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease or disorder by administration of said pharmaceutical composition to a subject. A pharmaceutical composition is also known in the art as a pharmaceutical formulation.

[0166] The total amount of peptidase polypeptides (by weight) in the pharmaceutical compositions described herein can be 20-98%, 20-95%, 20-90%, 20-80%, 20-70%, 20-60%, 20-50%, 20-40%, or 20-30%.

[0167] A pharmaceutical compositions described herein is an oral dosage form.

[0168] In some embodiments, the pharmaceutical compositions described herein exhibit a loss of enzyme activity of no more than about 25%, no more than about 20%, no more than about 15%, no more than about 10%, or no more than about 5%, during their transit through the stomach and into the duodenum.

[0169] Non-limiting examples of suitable oral dosage forms include granules, powders, tablets, capsules, sachets, pellets and liquid or semi-liquid formulations such as suspensions and gels. Since the peptidase enzymes need to be protected against gastric inactivation prior to release in the duodenum, it is desirable that the oral dosage forms be provided as a delayed release formulation. Delayed release formulations may minimize or prevent release of the enzymes in the stomach and delay their release until the dosage form has emptied from the stomach into the small intestine. Alternatively, the formulations can include capsules filled with the enzymes, whereby the capsule protects the contents against gastric inactivation, yet releases the enzymes in the small intestine.

[0170] As used herein, the term "delayed release formulation" indicates any formulation technique where release of the active substance from the dosage form is modified so that release occurs at a later time than that from a conventional immediate release product. One example of a delayed release formulation is an enteric coated formulation which includes enteric-coated tablets, enteric-coated capsules, enteric-coated granules, and enteric-coated spheres (commonly referred to as multiparticulate dosage forms). Enteric coatings on the dosage form are intended to control the region of the gastrointestinal tract where dissolution of the active substance from the enteric coated dosage form occurs. Enteric coatings can be prepared to substantially prevent dissolution of the dosage form contents in the stomach. These coatings function by incorporating materials in the enteric coating which allow the enteric coating to remain substantially intact in the acidic environment of the stomach. This substantially intact enteric coating minimizes or prevents the dissolution of active substance in stomach contents. Enteric coatings are formulated to release the contents of the dosage form when the pH of the gastrointestinal fluid increases. This increase in pH typically occurs when the dosage form passes out of the stomach into the small intestine. That is, the coating remains intact in the relatively more acidic stomach pH (pH<2) and disintegrates, dissolves, or is otherwise removed in the relatively less acidic pH of the intestine (pH values from about 2 to about 5 for the upper regions of the small intestine and pH values from about 7 to about 8.5 in the lower regions of the intestines). Formulations can be prepared using enteric coatings intended to release active substance at pH values of about 5.5 to about 6.5 or at higher pH values that typically occur in the lower regions of the intestines. In those delayed release formulations intended to dissolve at pH 5.5 to about 6.5 or higher, active substance release occurs substantially only upon reaching the duodenum (the upper portion of the small intestine) so that substantially no active substance is released in the stomach, thus minimizing or eliminating adverse effects.

[0171] Enzymes formulated as delayed release formulations may contain an enteric coating which disintegrates, dissolves, or erodes at neutral or slightly acidic or slightly alkaline pH, and thereby allows dissolution of the enzymes upon leaving the stomach, that is, upon stomach emptying into the small intestine. The release of the enzymes in the intestine prevents or reduces loss of enzymatic activity associated with the low pH of the stomach.

[0172] Other types of delayed release formulations are available, and the above examples are not limiting. Furthermore, methods of preparing delayed release formulations are known to one skilled in the art.

[0173] When the compositions of the present disclosure are formulated as tablets, the enzymes can be "tabletted" (i.e., formed into tablets) using methods known in the art, and subsequently coated with an enteric coating, again using methods known in the art.

[0174] When the compositions of the present disclosure are formulated as capsules, the contents of the capsule can be formulated using methods known in the art. For example, the enzyme composition can be provided in the form of particles or tablets suited to incorporation in a capsule.

[0175] The term "particles" as used herein includes fine powders (having particle diameters in the range of about 1 pm) up to pellets having a diameter of about 5 mm.

[0176] The enzyme composition can also be formed into particles in which it is embedded in or coated with an enteric polymer.

[0177] The term "enteric polymer" means a polymer that protects the enzymes from gastric contents, for example a polymer that is stable at acidic pH, but can break down rapidly at higher pH (pH- sensitive polymer) or a polymer whose rate of hydration or erosion is slow enough to ensure that contact of gastric contents with the enzymes is relatively minor while it is in the stomach, as opposed to the remainder of the gastro-intestinal tract. Non-limiting examples of enteric polymers include those known in the art, such as modified or unmodified natural polymers such as cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, and shellac; or synthetic polymers such as acrylic polymers or copolymers methacrylic acid polymers and copolymers, methylmethacrylate copolymers, and methacrylic acid / methylmethacrylate copolymers.

[0178] The enteric polymer coating can be a synthetic polymer, optionally including an inorganic material, such as an alkalinizing agent. The resulting coated particles provide delayed release beads comprising a core which comprises the enzymes and an enteric coating encapsulating the core. The coated enzyme particles can then be formulated into tablets or capsules.

[0179] The enteric polymer and the at least one inorganic material impart enteric properties to the coating of the present disclosure. That is, when used as a medication, the coating will act as a barrier protecting the medication from the acidic environment of the stomach and substantially prevent the release of the medication before it reaches the small intestine (i.e., the release of enzyme in the stomach is less than about 10-20% of the total amount of enzyme in the composition).

[0180] The inorganic material can include, for example, an alkali n izi ng agent. Non-limiting examples of alkalinizing agents include silicon dioxide, sodium salts, calcium salts, magnesium salts, aluminum salts, aluminum hydroxides, calcium hydroxides magnesium hydroxides, talc, and combinations thereof. In some embodiments, the alkalinizing agent is talc.

[0181] In some embodiments, the pharmaceutical compositions or oral dosage forms of the present disclosure comprise enzyme particles coated with an enteric coating comprising an enteric polymer and an inorganic material such as talc.

[0182] In some embodiments of the present disclosure, the coating further comprises a plasticizer. Examples of suitable plasticizers include, but are not limited to triacetin, tributyl citrate, tri- ethyl citrate, acetyl tri-n-butyl citrate, diethyl phthalate, dibutyl sebacate, polyethylene glycol, polypropylene glycol, castor oil, acetylated mono-glyceride, acetylated di-glyceride, and mixtures thereof.

[0183] The dosage forms of the present disclosure can be capsules containing the composition of the present disclosure (e.g., controlled-release particles of the enzyme composition, coated with an enteric polymer and an alkalinizing agent). The capsules themselves can be comprised of any conventional biodegradable material known in the art, for example, gelatin, polysaccharides such as pullulan, or modified cellulosic materials such as hydroxypropylmethylcellulose.

[0184] In some embodiments, the formulations and compositions described herein are particulate formulations. A preferred size range of the particles is less than 100 pm, preferably between 0.01 and 10 pm, more preferably between 0.05 and 1 pm. In some embodiments, the polydispersity index of the particles is below 2.0. The polydispersity index (PDI) generally is a measure of the distribution of mass in a given sample. It indicates the distribution of individual masses in a batch of nanoparticles. In some embodiments, the polydispersity index is below 1.0, preferably below 0.4.

[0185] In some embodiments, a composition described herein comprises particles formed from precipitating a mixture comprising the peptidase enzymes and a pH sensitive polymer.

[0186] Particles described herein can be produced, for example, by a method comprising the following steps: a) providing a fluid containing pH sensitive polymer and a solvent (e.g., an alcohol); and a fluid (e.g., aqueous fluid) containing enzyme and optionally mucoadhesive polymer; b) precipitating particles containing pH sensitive polymer, enzyme and optionally mucoadhesive polymer by colliding fluid streams.

[0187] As solvent for the pH sensitive polymer methanol, ethanol, t-butanol, acetone, tetrahydrofuran, isopropyl alcohol may be used.

[0188] Particles may be isolated by a method comprising evaporation, preferably under vacuum, concentration using cross flow filtration, granulation using a suitable excipient, spray drying and / or lyophilization.

[0189] Particles can be designed to be used in variety of different pharmaceutical compositions and formulations such as for tablets, capsules or suspensions.

[0190] In some embodiments, the oral dosage forms described herein may be provided as an immediate release formulation. These formulations may contain agents which buffer the acidic environment of the stomach and increase the stability of the enzymes in the stomach. For example, compositions comprising buffers, e.g., sodium bicarbonate, may be used to prevent the degradation of the enzymes in the stomach. The buffer used may be water soluble or water insoluble. Water insoluble buffers include aluminum hydroxide, dihydroxy aluminum sodium carbonate, calcium carbonate, aluminum phosphate, aluminum carbonate, magnesium hydroxide dihydroxy aluminum amino acetate, magnesium oxide, magnesium trisilicate, aluminum phosphate, magnesium carbonate and their combinations. Water soluble buffers include tripotassium phosphate, meglumine, sodium carbonate, sodium bicarbonate, sodium citrate, calcium gluconate, disodium hydrogen phosphate, sodium bicarbonate dipotassium hydrogen phosphate, sodium tartarate, sodium acetate, calcium glycerophosphate, tromethamine and their combinations. Two approaches can be used, either buffering the micro- or the macro-environment. In contrast to buffering the micro- environment, buffering the macro-environment involves buffering the entire stomach by buffering agents. In some embodiments, such formulations utilize the concept of micro- environment pH buffering and alkali is used to neutralize the acid in the stomach so as to protect the enzymes from acid degradation. Non-limiting examples of suitable immediate release oral dosage forms include buffered tablets, buffered capsules or buffered microspheres (e.g., comprising particles having a particle size less than 200pm).

[0191] Pharmaceutical compositions described herein may contain a proton pump inhibitor.

[0192] The term "proton pump inhibitor", as used herein, refers to compounds that can reduce the release of acid in the stomach and intestines. Inhibitors of H+-K+ATPase (proton pump) can bind irreversibly or reversibly to the enzyme. Agents referred to as Proton Pump Inhibitors (PPIs) typically include irreversible inhibitors. The most commonly known irreversible proton pump inhibitors include: Omeprazole (brand names: Losec®, Prilosec®, Lansoprazole (brand names: Prevacid®, Zoton®), Esomeprazole (brand names: Nexium®), Pantoprazole (brand names: Protonix®, Somac®) and Rabeprazole (brand names: Aciphex®, Pariet®). Reversible inhibitors are also referred to as Acid Pump Antagonists (APAs). APAs differ from the classical PPIs listed above in the way they inhibit H+-K+ATPase. Examples of suitable APAs include, but are not limited to, CS-526 (Sankyo), AZD0865 (Astra Zeneca), Soraprazan (Altana AG); other APAs are described by Sachs et al. U.S. Pat. No. 6,132,768, the disclosure of which is hereby incorporated by reference in its entirety. Accordingly, within the meaning of the present disclosure, the term "proton pump inhibitor" includes all the compounds that can suppress H+-K+ATPase activity acting either as irreversible or reversible inhibitors.

[0193] Pharmaceutical compositions described herein may contain a H2 blocker.

[0194] The term "H2 blocker", as used herein, refers to an agent that blocks the histamine H2 receptor thereby reducing or eliminating the production of hydrochloric acid in the stomach. Pharmaceutical compositions described herein may contain an antacid.

[0195] As used herein, the term "antacid" refers to a substance having the ability to neutralize and / or to buffer an acid. Antacids suitable for use with the present disclosure include, but are not limited to, aluminum carbonate, aluminum hydroxy carbonate, aluminum hydroxide, aluminum phosphate, aluminum citrate, dihydroxyaluminum sodium carbonate, aluminum magnesium glycinate, dihydroxyaluminum aminoacetic acid, dihydroxyaluminum aminoacetate, bismuth aluminate, bismuth carbonate, bismuth subcarbonate, bismuth subgallate, bismuth subnitrate, calcium carbonate, calcium hydroxide, calcium phosphate, calcium citrate, calcium citrate malate, activated sulfate, magnesium aluminate, hydrated magnesium aluminate, magnesium aluminosilicates, magnesium carbonate, magnesium glycinate, magnesium hydroxide, magnesium oxide, magnesium trisilicate, potassium carbonate, potassium phosphate, potassium citrate, sodium carbonate, sodium bicarbonate, sodium phosphate, sodium citrate, and combinations thereof. Preferred antacids include, but are not limited to, hydrated magnesium aluminate, magnesium hydroxide, aluminum phosphate, calcium phosphate, magnesium carbonate, magnesium oxide, magnesium trisilicate, aluminum hydroxide, dihydroxy aluminum amino acetate, sodium bicarbonate, calcium carbonate, and combinations thereof. More preferred antacids include, but are not limited to, calcium phosphate, magnesium carbonate, magnesium oxide, magnesium trisilicate, aluminum hydroxide, dihydroxy aluminum amino acetate, sodium bicarbonate, calcium carbonate, and combinations thereof. Even more preferred antacids include, but are not limited to, aluminum hydroxide, dihydroxy aluminum amino acetate, sodium bicarbonate, calcium carbonate, and combinations thereof. Yet even more preferred antacids include, but are not limited to, calcium carbonate and magnesium hydroxide.

[0196] Pharmaceutical compositions described herein may contain a mucoadhesive polymer.

[0197] The term "mucoadhesive polymer" refers to polymers with affinity to mucus. Such polymers include, but are not limited to, alginates, chitosans, pectins, hyaluronans, lectins or derivatives thereof.

[0198] In some embodiments, the pharmaceutical compositions described herein comprise one or more pharmaceutically acceptable excipients. The term "excipients" includes other pharmaceutically acceptable ingredients added to the active component(s) of a composition in order to improve processing, stability, palatability, etc. Non-limiting examples of suitable excipients include pharmaceutically acceptable binders, stabilizers, disintegrants, lubricants, glidants, diluents, and mixtures thereof etc. It will be appreciated by those skilled in the art of pharmaceutical formulations that a particular excipient may carry out multiple functions in the composition. So, for example a binder may also function as a diluent, etc.

[0199] Non-limiting examples of suitable binders include starches, sugars (e.g. lactose), sugar alcohols (e.g. xylitol, sorbitol, maltitol), cellulose (e.g. microcrystalline cellulose), modified celluloses (e.g., hydroxypropylcellulose, carboxymethylcellulose sodium), alginic acid, polyvinyl pyrrolidone (povidone), and mixtures thereof.

[0200] The pharmaceutical compositions can comprise one or more stabilizers which enhance or improve the stability of the pharmaceutical compositions. Non-limiting examples of suitable stabilizers include proline, trehalose, dextran, maltose, sucrose, mannitol, polyols, silica gel, aminoguanidine, pyridoxamine, anhydrous metal salts, such as sodium hydrogen carbonate magnesium oxide, calcium oxide, aluminium oxide and mixtures thereof.

[0201] Non-limiting examples of suitable disintegrants include dibasic calcium phosphate, dibasic calcium phosphate dihydrate, tribasic calcium phosphate, alginic acid, hydroxypropylcellulose, carboxymethylcellulose calcium, carboxymethylcellulose sodium, cross-linked carboxymethylcellulose sodium, swellable ion exchange resins, alginates, formaldehyde- casein, cellulose, croscarmellose sodium, crospovidone (e.g., cross-linked polyvinyl pyrrolidone), microcrystalline cellulose, sodium carboxymethyl starch, sodium starch glycolate, starches (com starch, rice starch), and mixtures thereof.

[0202] Non-limiting examples of suitable lubricants include calcium stearate, magnesium stearate, sodium stearyl fumarate, stearic acid, zinc stearate, talc, waxes, Sterotex®, Stearowet®, and mixtures thereof.

[0203] Non-limiting examples of suitable glidants include colloidal silicon dioxide, talc, and mixtures thereof.

[0204] Non-limiting examples of suitable diluents include microcrystalline cellulose, starch, calcium phosphate, lactose, sucrose, mannitol, sorbitol, and combinations thereof.

[0205] In some embodiments, a pharmaceutical composition described herein comprises a pharmaceutically acceptable carrier and may optionally comprise stabilizers etc. In some embodiments, a pharmaceutical composition described herein is for therapeutic or prophylactic treatment, e.g., for use in treating or preventing gluten-related disorders. The pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and in "a pharmaceutically acceptable preparation".

[0206] The term "pharmaceutically acceptable" refers to the non-toxicity of a material which does not interact with the action of the active component of the pharmaceutical composition.

[0207] The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In the case of the treatment of a particular disease, the desired reaction preferably relates to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in particular, interrupting or reversing the progress of the disease. The desired reaction in a treatment of a disease may also be delay of the onset or a prevention of the onset of said disease or said condition. An effective amount of the compositions described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered of the compositions described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

[0208] A pharmaceutical composition described herein is administered orally. A pharmaceutical composition described herein is formulated for oral administration.

[0209] Treatments

[0210] The present disclosure provides methods and compositions for treating a pathological state in a subject such as peptide-related disorders, e.g., gluten-related disorders (including celiac disease and non-celiac gluten sensitivity), digestive tract malabsorption, sprue, an allergic reaction and an enzyme deficiency. For example, the allergic reaction can be a reaction to gluten. The present disclosure, in particular, provides methods and agents for treating celiac disease (CeD) and non-celiac gluten sensitivity. Methods described herein may comprise administering an effective amount of a composition described herein. The methods and compositions described herein may also be useful in treating inflammatory bowel disease (IBD).

[0211] The therapeutic compositions described herein may be administered prophylactically (i.e., to prevent a disease or disorder) or therapeutically (i.e., to treat a disease or disorder) to subjects suffering from, or at risk of (or susceptible to) developing a disease or disorder. Such subjects may be identified using standard clinical methods. In the context of the present disclosure, prophylactic administration occurs prior to the manifestation of overt clinical symptoms of disease, such that a disease or disorder is prevented or alternatively delayed in its progression. In the context of the field of medicine, the term "prevent" encompasses any activity, which reduces the burden of mortality or morbidity from disease. Prevention can occur at primary, secondary and tertiary prevention levels. While primary prevention avoids the development of a disease, secondary and tertiary levels of prevention encompass activities aimed at preventing the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications.

[0212] In some embodiments, administration of a composition described herein may be performed by single administration or boosted by multiple administrations.

[0213] The term "disease" or "disorder" refers to an abnormal condition that affects the body of an individual. A disease is often construed as a medical condition associated with specific symptoms and signs. A disease may be caused by factors originally from an external source, such as infectious disease, or it may be caused by internal dysfunctions, such as autoimmune diseases. In humans, "disease" is often used more broadly to refer to any condition that causes pain, dysfunction, distress, social problems, or death to the individual afflicted, or similar problems for those in contact with the individual. In this broader sense, it sometimes includes injuries, disabilities, disorders, syndromes, clinical entities, infections, isolated symptoms, deviant behaviors, and atypical variations of structure and function, while in other contexts and for other purposes these may be considered distinguishable categories. Diseases usually affect individuals not only physically, but also emotionally, as contracting and living with many diseases can alter one's perspective on life, and one's personality. The term "peptide-related disorder" refers to any disease, disorder or condition that is triggered, promoted, or exacerbated by peptides, i.e., disease-promoting peptides. In some embodiments, a peptide-related disorder comprises an inflammatory component and may involve chronic inflammation. In some embodiments, the term "peptide-related disorder" or "peptide-related condition" includes gluten-related disorders such as celiac disease (CeD), or non-celiac gluten sensitivity (NCGS). In some embodiments, the term "peptide-related disorder" includes inflammatory bowel disease (IBD).

[0214] The term "gluten-related disorder" refers to any disease, disorder or condition caused by the ingestion of gluten-containing food, such as wheat-based food. The term includes celiac disease (CeD), wheat allergy (WA) and non-celiac gluten sensitivity (NCGS) as well as other diseases that may, profit from a gluten-free diet (GFD).

[0215] Celiac disease (CeD) is a chronic immune-mediated enteropathy precipitated by gluten- immunogenic peptides (GIPs) in genetically predisposed individuals. GIPs are generated in the digest of gluten and play a central role in the disease pathology; they consist of digestion- resistant proline-rich peptides, which act as T-cell epitopes in HLA-DQ2 and HLA-DQ8 positive patients, whereby CD4+ T-cells associate with HLA-molecules and initiate a typical auto- immune pathology, which explains the clinical symptomatology and long-term complications. Current diagnosis is based on the presence of clinical symptoms of enteropathy, villous atrophy, crypt hyperplasia and intraepithelial lymphocytosis, and the presence of circulating CeD-specific antibodies to tissue transglutaminase (tTG), deamidated gliadin peptides (DGP), and endomysium (EMA) in the small intestine.

[0216] Non-celiac gluten sensitivity is a gluten-related disorder related to the ingestion of gluten. In contrast to CeD no specific genetic predisposition factor for NCGS has been identified so far and serological biomarkers are not available for NCGS, since the determination of celiac- related antibodies is not sensitive or specific to NCGS. The NCGS patients' gastrointestinal tracts and their intestinal permeability are normal and the lesions in the histological picture of their duodenal mucosa are minor, but increased infiltration of eosinophils and basophils to the duodenal lamina propria and activation of circulating basophils have been observed in NCGS patients which may be responsible for syndrome-related symptomes. Since gluten-derived GIPs are the causative trigger of gluten-related disorders, a strict lifelong gluten-free diet has been the mainstay of treatment. However, a gluten-free diet is very difficult to achieve in day-to-day life.

[0217] In the present context, the term "treatment", "treating" or "therapeutic intervention" relates to the management and care of a subject for the purpose of combating a condition such as a disease or disorder. The term is intended to include the full spectrum of treatments for a given condition from which the subject is suffering, such as administration of the therapeutically effective compound to alleviate the symptoms or complications, to delay the progression of the disease, disorder or condition, to alleviate or relief the symptoms and complications, and / or to cure or eliminate the disease, disorder or condition as well as to prevent the condition, wherein prevention is to be understood as the management and care of an individual for the purpose of combating the disease, condition or disorder and includes the administration of the active compounds to prevent the onset of the symptoms or complications.

[0218] The term "therapeutic treatment" relates to any treatment which improves the health status and / or prolongs (increases) the lifespan of an individual. Said treatment may eliminate the disease in an individual, arrest or slow the development of a disease in an individual, inhibit or slow the development of a disease in an individual, decrease the frequency or severity of symptoms in an individual, and / or decrease the recurrence in an individual who currently has or who previously has had a disease.

[0219] The terms "prophylactic treatment" or "preventive treatment" relate to any treatment that is intended to prevent a disease from occurring in an individual. The terms "prophylactic treatment" or "preventive treatment" are used herein interchangeably.

[0220] The terms "individual" and "subject" are used herein interchangeably. They refer to a human or another mammal (e.g. mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate) that can be afflicted with or is susceptible to a disease or disorder but may or may not have the disease or disorder. In many embodiments, the individual is a human being. Unless otherwise stated, the terms "individual" and "subject" do not denote a particular age, and thus encompass adults, elderlies, children, and newborns. In embodiments of the present disclosure, the "individual" or "subject" is a "patient". The term "patient" means an individual or subject for treatment, in particular a diseased individual or subject.

[0221] Citation of documents and studies referenced herein is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the contents of these documents.

[0222] The following description is presented to enable a person of ordinary skill in the art to make and use the various embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. Various modifications to the examples described herein will be readily apparent to those of ordinary skill in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Thus, the various embodiments are not intended to be limited to the examples described herein and shown but are to be accorded the scope consistent with the claims.

[0223] Examples

[0224] Methods

[0225] Enteric coating for gastric protection and enteric release

[0226] Eudraguard control dissolved in ethanol (10 mg / mL) was mixed with an enzyme solution (5 mg / mL ruLAP-2 or ruDPP-IV) containing chitosan (0.25 mg / mL) at a ratio of 1:3. This resulted in a precipitation of enzymes encapsulated in Eudraguard / Chitosan particles. The suspension was washed with Tween80 solution using crossflow filtration (CFF) to remove the ethanol, acetic acid, and any free enzyme in solution. CFF was also used to concentrate the enzyme particles, and sucrose was then added as a cryoprotectant. The suspension was then subject to spray drying or lyophilized to obtain a powder formulation, and both enzyme powders were mixed at the required concentration.

[0227] Dried enzyme powders were characterized with two-stage dissolution to assess gastric resistance and enzyme release at small intestine pH values.

[0228] Two-stage dissolution assay

[0229] Dissolution tests of microparticle suspensions were performed through a two-stage approach mimicking the gastric and intestinal stages of digestions (Ph. Eur. 11.0). Enzyme powders are first dissolved in 0.1 M HCI (Stage 1, mimicking stomach pH) and incubated fortwo-hours. The pH of the solution is then raised to 6.8 using 50 mM Tris buffer (Stage 2, mimicking small intestine pH). For control groups, unencapsulated enzymes (no enteric protection) were either incubated through the two-stage assay (positive control) to demonstrate vulnerability to the gastric stage of digestion, or incubated directly into Stage 2 (negative control, no pH-mediated impact on efficacy). All samples were filtered through a 0.2 pm PES filter before analyses. Enzyme concentration was determined through HPLC-UV, and enzyme activity after release from particle through LC-UV-Vis.

[0230] LC-UV-Vis digestion kinetics Enzyme cleaving efficacy after simulated gastrointestinal passage was assessed using a fluorogenic assay. Briefly, the fluorophore 7-amido-4-methylcoumarin (AMC) was conjugated to either Leucine (H-Leu-AMC) as a substrate for ruLAP-2, or a Glycine-Proline dipeptide (H- Gly-Pro-AMC) as a substrate for ruDPP-IV. Kinetic analyses were performed on a UV-1600PC spectrophotometer (340 nm).

[0231] Filtered solutions obtained after Stage 2 of the two-stage dissolution assay were diluted with Buffer B (50 mM TRIS-HCI, 1 mM C0CI2, 1% w / v BSA pH 7.0) to reach a final concentration of 10 pg / mL of each enzyme. For blank measurement, 1 mL of 2 mM AMC substrate (either Leu or Gly-Pro) was added to 1 mL of Buffer B. For samples, 1 mL of 2 mM AMC substrate was added to 1 mL of sample diluted in Buffer B. Fluorescence measurements were collected from the start of the reaction, with datapoints every 60 seconds over a period of 20 minutes. AMC concentration was plotted over time, and the slope of the graph was used to determine enzyme activity after release.

[0232] Enzyme Stability

[0233] Enzyme stability was assessed in vitro by assessing digestion efficacy of the 33-mer peptide after various periods of enzyme pre-incubation. Exopeptidases ruLAP-2 and ruDPP-4 were dissolved with a concentration of 86 mg / L each in 50 mM Tris buffer at pH 7 and pre-incubated in a microtube shaking incubator (AccuTherm Inc., Monroe City MO U.S.A) at 37 °C, 300 rpm for 5, 3, 1 or 0 h. At the end of the pre-incubation period, solutions were diluted 10-fold using 110 mg / L 33-mer peptide in 50 mM Tris buffer at pH 7, achieving a final concentration of 8.6 mg / L for each exopeptidase and 100 mg / L of the 33-mer peptide.

[0234] 100 pL aliquots of the digestion solution were sampled after 0, 10, 30 and 60 min of the reaction starting, and immediately mixed with 400 pL 0.1 % TFA to stop the reaction then snap frozen LC-MS / MS analysis.

[0235] HPLC-MS / MS detection of 33-mer peptide degradation products

[0236] Tandem cleaving action of exopeptidases on their peptide substrate is expected to produce free single amino acids and dipeptides. Therefore, an HPLC-MS / MS-based detection method was developed to precisely identify and quantify these degradation products resulting from 33-mer digestion. The setup was composed of a Nexera LC40 high performance liquid chromatographical system coupled to a state-of-the-art SCIEX QTRAP 6500+ mass spectrometer. The single amino acids tyrosine and proline, in theory not produced by ruLAP- 2 and ruDPP-4 activity on the 33-mer (Figure 5), were used as negative control. To avoid preparation-based variability between samples, amino acids were analyzed without commonly used derivatization processes like AccQ-tag derivatization. All amino acids and dipeptides were manually tuned and optimized for declustering potential (DP), collision energy (CE), entry potential (EP) and cell exit potential (CXP, Table 1). Furthermore, MS Source and ionisation conditions were specifically optimized for single amino acids and dipeptides (Table 2).

[0237] Table 1. MS parameters for detection of single amino acids and dipeptides resulting from complete 33-mer digestion by exopeptidases ruLAP-2 and ruDPP-IV. Table 1 continued. MS parameters for detection of single amino acids and dipeptides resulting from complete 33-mer digestion by exopeptidases ruLAP-2 and ruDPP-IV.

[0238] Table 2. Optimized conditions of Q.-TRAP 6500+ MS source for ionization of amino acids and dipeptides, and evaporation of LC solvent.

[0239] Finally, optimal chromatographical separation of amino acids and dipeptides was achieved using PFP columns and a gradient elution profile (Table 3, Figure 1).

[0240] Table 3. LC conditions for chromatographic separation of amino acids and dipeptides resulting from complete 33-mer degradation by exopeptidases ru LAP-2 and ruDPP-IV.

[0241] Example 1: Eudraguard / Chitosan Encapsulation and Two-Stage Dissolution Assay

[0242] Enzymes encapsulated in a Eudraguard Control / Chitosan formulation demonstrated negligible release from their microparticles in acidic solutions, and sustained release when pH was raised to 6.8 (Figure 2). Furthermore, when assessing enzyme activity after simulated gastrointestinal passage (two-stage dissolution assay), there was no appreciable loss of activity in encapsulated enzymes compared to unencapsulated enzymes (no enteric protection) dissolved directly in pH 6.8. Conversely, unencapsulated enzymes undergoing simulated gastrointestinal passage suffered irreversible loss of activity from the stomach stage (0.1 M HCI), as demonstrated by AMC fluorescence assay (Figure 3 and 4).

[0243] Example 2: Enzyme Stability

[0244] Degradation of the a2-gliadin 33-mer peptide by exopeptidases ruLAP-2 and ruDPP-IV is expected to act through a stepwise cleaving process beginning at the N-terminus (-NH2). It is therefore expected that each cleavage event will release a single amino acid or X-Pro dipeptide and a shortened peptide will remain (Figure 5).

[0245] Since LC-UV-Vis is unable to resolve this degradation process in detail, we used an LC-MS / MS based approach to assess the digestion efficacy over time. Optimization of LC conditions resulted in chromatograms with clear and detectable peaks for each expected free amino acid and dipeptide (Figure 6).

[0246] Using this method, ruLAP-2 and ruDPP-4 "longevity" was evaluated by pre-incubation for up to 5 hours before adding 33-mer peptide to the solution. Activity was compared to digestion kinetics without preincubation (0 h timepoint). At the amino acid level, no differences in degradation kinetics were observed between the different preincubation periods (Figure 7). Thus, the exopeptidases maintained their activity even after a 5 h preincubation period at 37 °C. This was confirmed with analysis of the C-terminal phenylalanine (F), with over 90% of enzyme efficacy maintained after a preincubation of 5 h (Figure 8).

Claims

CLAIMS1. A pharmaceutical composition comprising a polypeptide having aminopeptidase (AP) activity and a polypeptide having dipeptidylpeptidase (DPP) activity for oral administration, wherein the pharmaceutical composition protects the polypeptides against gastric inactivation.

2. The pharmaceutical composition of claim 1, which, when orally administered to a mammal, releases the polypeptide having AP activity and the polypeptide having DPP activity in the small intestine, in particular the upper small intestine.

3. The pharmaceutical composition of claim 1 or 2, wherein the polypeptide having AP activity and the polypeptide having DPP activity in the pharmaceutical composition remain essentially intact during their transit through the stomach and into the duodenum.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the polypeptide having AP activity and the polypeptide having DPP activity in the pharmaceutical composition exhibit a loss of enzyme activity of no more than 50% during their transit through the stomach and into the duodenum.

5. The pharmaceutical composition of any one of claims 1 to 4, which is a solid formulation.

6. The pharmaceutical composition of any one of claims 1 to 5, which is selected from the group consisting of granules, powders, tablets, capsules and pellets.

7. The pharmaceutical composition of any one of claims 1 to 6, which comprises a carrier material on which the polypeptide having AP activity and the polypeptide having DPP activity is immobilized.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the polypeptide having AP activity and the polypeptide having DPP activity is immobilized on the carrier material by applying the polypeptide having AP activity and the polypeptide having DPP activity onto the carrier material and drying.

9. The pharmaceutical composition of claim 8, wherein the polypeptide having AP activity and the polypeptide having DPP activity is applied onto the carrier material by spraying.

10. The pharmaceutical composition of claim 8 or 9, wherein drying is performed at a temperature of between 20°C and 60°C for 0.5 to 3 hours.

11. The pharmaceutical composition of any one of claims 8 to 10, wherein drying is performed at a temperature of between 30°C and 50°C for about 1 hour.

12. The pharmaceutical composition of any one of claims 7 to 11, wherein the carrier material comprises a material selected from the group consisting of sucrose, microcrystalline cellulose, calcium carbonate, mannitol, maltodextrine and silicon dioxide.

13. The pharmaceutical composition of any one of claims 7 to 12, wherein the carrier material comprises a material selected from the group consisting of sucrose and mannitol.

14. The pharmaceutical composition of any one of claims 1 to 13, wherein the polypeptide having AP activity and the polypeptide having DPP activity are encapsulated within or coated by an enteric material.

15. The pharmaceutical composition of claim 14, wherein the enteric material comprises a pH-sensitive polymer.

16. The pharmaceutical composition of claim 14 or 15, wherein the enteric material comprises a material selected from the group consisting of cellulose acetate phthalate,hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, shellac, acrylic acid polymers or copolymers, methacrylic acid polymers or copolymers, and methacrylic acid / methylmethacrylate copolymers.

17. The pharmaceutical composition of any one of claims 14 to 16, wherein the enteric material comprises a methacrylic acid polymer or copolymer.

18. The pharmaceutical composition of any one of claims 14 to 17, wherein the enteric material comprises an EUDRAGUARD® resin.

19. The pharmaceutical composition of any one of claims 14 to 18, wherein the enteric material comprises EUDRAGUARD® Control.

20. The pharmaceutical composition of any one of claims 14 to 19, wherein the enteric material further comprises a mucoadhesive polymer. •21. The pharmaceutical composition of claim 20, wherein the mucoadhesive polymer comprises a polymer selected from the group consisting of chitosans, alginates, pectins, hyaluronans, lectins, and derivatives thereof.

22. The pharmaceutical composition of claim 20 or 21, wherein the mucoadhesive polymer comprises chitosan, or a derivative thereof.

23. The pharmaceutical composition of any one of claims 14 to 22, wherein the enteric material comprises a methacrylic acid polymer or copolymer and chitosan.

24. The pharmaceutical composition of any one of claims 1 to 23, comprising a core comprising the polypeptide having AP activity and the polypeptide having DPP activity, coated with an enteric coating.

25. The pharmaceutical composition of claim 24, wherein the core further comprises a binder.

26. The pharmaceutical composition of claim 25, wherein the binder is selected from the group consisting of starches, sugars, lactose, sugar alcohols, xylitol, sorbitol, maltitol, cellulose, microcrystalline cellulose, modified celluloses, hydroxypropylcellulose, carboxymethylcellulose sodium, alginic acid, polyvinyl pyrrolidone, and mixtures thereof.

27. The pharmaceutical composition of any one of claims 24 to 26, wherein the core further comprises a stabilizer.

28. The pharmaceutical composition of claim 27, wherein the stabilizer is selected from the group consisting of proline, trehalose, dextran, maltose, sucrose, mannitol, polyols, silica gel, aminoguanidine, pyridoxamine, anhydrous metal salts, magnesium oxide, calcium oxide, aluminum oxide, and mixtures thereof.

29. The pharmaceutical composition of any one of claims 24 to 28, wherein the core further comprises a disintegrant.

30. The pharmaceutical composition of claim 29, wherein the disintegrant is selected from the group consisting of dibasic calcium phosphate, dibasic calcium phosphate dihydrate, tribasic calcium phosphate, alginic acid, hydroxypropylcellulose, carboxymethylcellulose calcium, carboxymethylcellulose sodium, cross-linked carboxymethylcellulose sodium, swellable ion exchange resins, alginates, formaldehyde-casein, cellulose, croscarmellose sodium, crospovidone, microcrystalline cellulose, sodium carboxymethyl starch, sodium starch glycolate, starches, corn starch, rice starch, and mixtures thereof.

31. The pharmaceutical composition of any one of claims 1 to 30, wherein the polypeptide having AP activity cleaves single amino acids off the N-terminal end of polypeptides, except when these are connected to proline in an NH2-X-Pro sequence.

32. The pharmaceutical composition of any one of claims 1 to 31, wherein the polypeptide having AP activity is derived from a fungus.

33. The pharmaceutical composition of any one of claims 1 to 32, wherein the polypeptide having AP activity is derived from Trichophyton rubrum.

34. The pharmaceutical composition of any one of claims 1 to 33, wherein the polypeptide having AP activity is a polypeptide having leucine-aminopeptidase activity.

35. The pharmaceutical composition of any one of claims 1 to 34, wherein the polypeptide having AP activity comprises the amino acid sequence represented by SEQ. ID NO: 1 or a variant thereof.

36. The pharmaceutical composition of any one of claims 1 to 35, wherein the polypeptide having AP activity has been expressed in a fungus.

37. The pharmaceutical composition of any one of claims 1 to 36, wherein the polypeptide having AP activity has been expressed in Pichia pastoris.

38. The pharmaceutical composition of any one of claims 1 to 37, wherein the polypeptide having DPP activity cleaves the dipeptide motive NH2-X-Pro off the N-terminal end of polypeptides.

39. The pharmaceutical composition of any one of claims 1 to 38, wherein the polypeptide having DPP activity is derived from a fungus.

40. The pharmaceutical composition of any one of claims 1 to 39, wherein the polypeptide having DPP activity is derived from Trichophyton rubrum.

41. The pharmaceutical composition of any one of claims 1 to 40, wherein the polypeptide having DPP activity is a polypeptide having dipeptidylpeptidase IV (DPPIV) activity.

42. The pharmaceutical composition of any one of claims 1 to 41, wherein the polypeptide having DPP activity comprises the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof.

43. The pharmaceutical composition of any one of claims 1 to 42, wherein the polypeptide having DPP activity has been expressed in a fungus.

44. The pharmaceutical composition of any one of claims 1 to 43, wherein the polypeptide having DPP activity has been expressed in Pichia pastoris.

45. The pharmaceutical composition of any one of claims 1 to 44, wherein the weight ratio of the polypeptide having AP activity and the polypeptide having DPP activity is between 20:1 and 1:5.

46. The pharmaceutical composition of any one of claims 1 to 45, which completely degrades the following 33-mer peptide:LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF47. The pharmaceutical composition of any one of claims 1 to 46 for pharmaceutical use.

48. The pharmaceutical composition of any one of claims 1 to 47 for use in treating a peptide-related disorder.

49. The pharmaceutical composition of any one of claims 1 to 48 for use in treating a gluten-related disorder.

50. The pharmaceutical composition of claim 49, wherein the gluten-related disorder is selected from the group consisting of celiac disease, wheat allergy and non-celiac gluten sensitivity.

51. A method of treating peptide-related disorder comprising orally administering the pharmaceutical composition of any one of claims 1 to 46 to a mammal in need thereof.

52. A method of treating a gluten-related disorder comprising orally administering the pharmaceutical composition of any one of claims 1 to 46 to a mammal in need thereof.

53. The method of claim 52, wherein the gluten-related disorder is selected from the group consisting of celiac disease, wheat allergy and non-celiac gluten sensitivity.

54. The method of any one of claims 51 to 53, wherein the mammal is a human.

55. The method of any one of claims 51 to 54, wherein said pharmaceutical composition is administered in combination with a substance which increases Gl tract pH.

56. The method of claim 55, wherein the substance which increases Gl tract pH is selected from the group consisting of proton pump inhibitors and antacids.

57. The method of any one of claims 51 to 56, wherein the pharmaceutical composition is administered to a mammal prior to, simultaneously with or following dietary intake.

58. The method of any one of claims 51 to 57, wherein the pharmaceutical composition is administered to a mammal prior to dietary intake.

59. The method of any one of claims 51 to 58, wherein the pharmaceutical composition is administered to a mammal up to 3 hours prior to dietary intake.

60. The method of any one of claims 51 to 59, wherein the pharmaceutical composition is administered to a mammal 1 to 3 hours prior to dietary intake.

61. The method of any one of claims 57 to 60, wherein the diet is supposed to be gluten- free.

62. The method of any one of claims 57 to 61, wherein the dietary intake comprises inadvertent gluten intake.

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