Pharmaceutical compositions comprising exopeptidases and uses thereof

Abstract

Described herein are compositions which include exopeptidases and which are designed to reduce one or more symptoms of peptide-related disorders, in particular disorders related to immunogenic peptides. Also described herein is a method for treating a subject using compositions described herein.

Description

[0001] PHARMACEUTICAL COMPOSITIONS COMPRISING EXOPEPTIDASES AND USES THEREOF

[0002] Described herein are compositions which include exopeptidases and which are designed to reduce one or more symptoms of peptide-related disorders, in particular disorders related to immunogenic peptides. Also described herein is a method for treating a subject using compositions described herein.

[0003] Among the ~20 proteinogenic amino acids residues, proline is unique owing to its side chain R group being bonded with both the amino group and the a-carbon, creating a cyclic structure (Cunningham, D. F. & O'Connor, B., Biochim. Biophys. Acta BBA - Protein Struct. Mol. Enzymol. 1343, 160-186 (1997)). Proline therefore introduces a conformational change that confers particular biological properties to proline-rich or proline-containing peptides (PCPs). Of note, proline plays an important role in the proteolytic regulation of bioactive peptides and hormones (Cunningham, D. F. & O'Connor, B., Biochim. Biophys. Acta BBA - Protein Struct. Mol. Enzymol. 1343, 160-186 (1997); Mentlein, R., FEBS Lett. 234, 251-256 (1988)).

[0004] Interestingly, certain PCPs have been shown to exhibit significant biological activity as triggers of proinflammatory and immune modulating pathways (Misiura, M. & Miltyk, W., BioFactors 45, 857-866 (2019)). Glyprolines, for instance, play a role in chronic inflammation where they display a chemotactic effect on neutrophils in chronic obstructive pulmonary disease (Weathington, N. M. et al., Nat. Med. 12, 317-323 (2006)), cystic fibrosis (Sharma, N. S. et al., Am. J. Physiol. Lung Cell. Mol. Physiol. 315, L653-L661 (2018)), ischemic brain stroke (Hill, J. W. & Nemoto, E. M., J. Neuroinflammation 12, 214 (2015)), chorioamnionitis (Shibata, A. et al., Clin. Chim. Acta int. J. Clin. Chem. 402, 124-128 (2009)), and inflammatory bowel diseases (IBD) (Pim J Koelink et al., Gut 63, 578 (2014)). While the pathophysiology of IBD is multifaceted and poorly understood (Sugihara, K., Morhardt, T. L. & Kamada, N., Front. Immunol. 9, (2019)), neutrophil recruitment and chronic inflammation is thought to originate, in part, from endogenous PCPs derived from the self-digestion of the extracellular collagen matrix by an overexpression of prolyl endopeptidase and matrix metalloproteinases (Naito, Y. & Yoshikawa, T., Mol. Aspects Med. 26, 379-390 (2005); Pim J Koelink et al., Gut 63, 578 (2014); Gaggar, A. et al., J. Immunol. 180, 5662-5669 (2008)). For instance, the collagen- derived tripeptide N-acetyl Pro-Gly-Pro (PGP) was shown to recruit neutrophils via CXCR2 (Weathington, N. M. et al., Nat. Med. 12, 317-323 (2006)), creating a proinflammatory cycle in the intestine of patients with IBD (Pim J Koelink et al., Gut 63, 578 (2014)). With the ensuing damage and compromised integrity of the intestinal mucosal layer and epithelium, it is unclear if secretory proteases in the lumen and exogenous PCPs from dietary protein further contribute to this cycle. Interestingly, dietary supplementation of proline-rich collagen peptides (of various lengths) exacerbated ulcerative colitis in mice by increasing proinflammatory cytokines, promoting macrophage recruitment, and disrupting gut microbiota (Li, X. et al., Front. Nutr. 9, 957391 (2022)). A compounded effect could be created with damage-induced loss of brush border enterocytes, and the subsequent reduction or loss of peptide cleaving function.

[0005] Though the aetiology of dietary nutrients in IBD is lacking, there has been a growing emphasis in recent years on the role dietary proteins play in the symptoms and pathophysiology of functional gastrointestinal disorders (Boettcher, E. & Crowe, S. E., Am. J. Gastroenterol. 108, 728-736 (2013)). Celiac disease, for instance, is the only autoimmune disorder with known dietary triggers and presents an interesting case in which dietary bioactive PCPs, referred to in this field as gluten immunogenic peptides (GIPs), play a key disease-driving role. 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 not just proline but also glutamine residues, which together are credited for their proteolytic resistance to gastric, pancreatic, and intestinal digestive enzymes (Sharma, N. et al., Front. Nutr. 7, 6 (2020); Stamnaes, J. & Sollid, L. M., Semin. Immunol. 27, 343-352 (2015); Shan, L. et al., Implications for Celiac Sprue. J. Proteome Res. 4, 1732-1741 (2005)). The ensuing build-up of partially digested GIPs and the acquired immune response to these peptides in genetically susceptible individuals is the hallmark of celiac disease (CD). Other gluten-related disorders include wheat allery and non-celiac gluten sensitivity.

[0006] Digestion of protein into peptides and peptides into amino acids is dependent on the synergistic action of various enzyme classes such as proteases, peptidases, and proteinases. One of the defining characteristics of these enzymes is their substrate specificity between the amino (N-) and carboxyl (C-) termini, with endopeptidase (EPs) hydrolysing nonterminal amino acids, and exopeptidases (EXPs) cleaving amino acids at either terminus (Velden, V. D. & Hulsmann, A. R., Clin. Exp. Allergy 29, 445-456 (1999)). The conformational changes introduced by proline influence how PCPs interact with enzymes. Notably, proline confers proteolytic resistance through stop sequences (e.g. Xaa-Pro, Pro-Xaa, Xaa-Pro-Pro), and due to proline-induced supramolecular peptide configuration, proline may affect amino acids not directly attached to it (Cunningham, D. F. & O'Connor, B., Biochim. Biophys. Acta BBA - Protein Struct. Mol. Enzymol. 1343, 160-186 (1997)). Consequently, and due to their assumed proteolytic resistance to human digestive enzymes, significant efforts have been put into the investigation and application of exogenous proteases and peptidases (e.g., of fungal, bacterial, or plant origin) for the digestion of gluten protein and GIPs (Baharin, A., Ting, T.-Y. & Goh, H.- H., Plants 11, 1330 (2022)).

[0007] Interestingly, these efforts have almost exclusively focused on endoprotease and EPs supplementation in the stomach. Research approaches centred on EPs likely stem from their mode of action, as cleaving action on non-terminal amino acid motifs does not require a complex interplay of proteolytic enzymes. Unfortunately, treatment approaches using EPs present fundamental flaws, as their ability to completely neutralize peptides is limited. On the one hand, some exogenously administered EPs have been shown to increase the inflammatory potential of gluten as they generated more immunogenic peptides than pepsin alone (Siegel, M. et al., Chem. Biol. 13, 649-658 (2006)). On the other hand, and despite claims of cleaving through immunogenic epitopes, incomplete digestion of peptides may result in the spontaneous production of novel PCPs (Herrera, M. G. & Dodero, V. I., Biophys. Rev. 13, 1147- 1154 (2021)). Indeed, since pepsin digests of gliadins have been recently demonstrated to generate peptides that self-assemble into supramolecular structures with immunogenic potential (Herrera, M. G. et al., Mol. Nutr. Food Res. 65, 2100200 (2021)), it stands to reason that EP-based treatment approaches resulting in multiple PCP fragments may also lead to an increase in novel bioactive peptides.

[0008] Consequently, a novel treatment modality is needed in order to complement, or replace, the brush border membrane's ability to thoroughly hydrolyse PCPs and significantly reduce peptide self-assembly and / or immunogenicity. An enzymatic treatment that supports the hydrolysis of dietary peptides into amino acids is further warranted by the therapeutic use of amino acid supplementation as an alternative or ancillary treatment strategy for IBD patients due to their role in maintaining immunocompetence and protecting against disease (Sugihara, K., Morhardt, T. L. & Kamada, N., Immunol. 9, (2019); Liu, Y., Wang, X. & Hu, C.-A. A., Nutrients 9, 920 (2017)). Glutamine supplementation for instance, a crucial amino acid for enterocytes, can improve disease symptoms, intestinal structure, and barrier function (Sido, B., Seel, C., Hochlehnert, A., Breitkreutz, R. & Droge, W., Dig. Dis. Sci. 51, 2170-2179 (2006); Fasina, Y. O., Bowers, J. B., Hess, J. B. & McKee, S. R„ Poult. Sci. 89, 1042-1048 (2010)).

[0009] In view of the above, it has been the object of the present disclosure to provide a composition which is suitable for peptidase replacement therapy, and which is capable of degrading clinically relevant and disease-causing peptides, in particular proline-containing peptides, into harmless peptides.

[0010] The present disclosure is based on the insight that only an EXP-based treatment would be able to systematically hydrolyse peptides into trimers, dimers, or monomers. Treatments using EXPs, however, have their own challenges as each EXP possesses its own substrate specificity and proteolytic stop sequence. A complementary combination is therefore required to thoroughly trim peptides into harmless digestion products. It has been shown that fungal dipeptidyl peptidases (DPPs) act synergistically with mono aminopeptidases (APs) in order to cleave large peptides into amino acids and dipeptides (Monod, M. & Beauvais, A. Chapter 749 - Dipeptidyl-peptidases IV and V of Aspergillus, in Handbook of Proteolytic Enzymes (Third Edition) (eds. Rawlings, N. D. & Salvesen, G.) 3392-3394 (Academic Press, 2013). doi:10.1016 / B978-0-12-382219-2.00749-3; Byun, T., Kofod, L. & Blinkovsky, A., J. Agric. Food Chem. 49, 2061-2063 (2001)). In this scenario, APs cleave single amino acids from the N- terminal of peptides until they encounter a dipeptide motif which acts as a stop sequence, which in turn would be complementarily cleaved by DPPs. Since DPPs activity is limited to specific N-terminal dipeptide motifs, it requires synergistic APs for access to these dipeptides (Monod, M. & Beauvais, A. Chapter 749 - Dipeptidyl-peptidases IV and V of Aspergillus, in Handbook of Proteolytic Enzymes (Third Edition) (eds. Rawlings, N. D. & Salvesen, G.) 3392- 3394 (Academic Press, 2013). doi:10.1016 / B978-0-12-382219-2.00749-3; Byun, T., Kofod, L. & Blinkovsky, A., J. Agric. Food Chem. 49, 2061-2063 (2001)). Consequently, a combination of APs and DPPs is able to completely digest certain immunogenic PCP or GIP chains into harmless but useful amino acids and dipeptides, as seen with a combination of leucine aminopeptidase 2 ( LAP2) and dipeptidyl peptidase IV (DPPIV) of Trichophyton rubrum and the digestion of a,2-gliadin 33mer peptide in the human small intestine (Romer et al., in prep}.

[0011] Unfortunately, DPPs are also susceptible to proline-based proteolytic stop sequences. It is demonstrated herein that N-terminal Phe-Pro-Pro tripeptide motifs act as a stop sequence for mammalian DPPII despite claims of DPPI I being able to cleave Xaa-Pro-Pro (Duve, H., Johnsen, A. H., Scott, A. G. & Thorpe, A., Regul. Pept. 57, 237-245 (1995)). As such, two-form combinations of APs and DPPs are insufficient for complete digestion of immunogenic PCPs, as Xaa-Pro-Pro stop sequences are found in proteins and peptides such as gliadins and may be generated in situ through self-assembly mechanisms. Our data highlights the complexity of exopeptidase substrate specificity, and the need for a novel exopeptidase combination based on a better understanding of proline-based proteolytic stop sequences and the enzymes required to overcome them.

[0012] The present disclosure provides a novel treatment modality comprising three exopeptidase activities: a generalist mono aminopeptidase (AP) activity, a Xaa-Pro dipeptidyl peptidase (DPP) activity with a broad dipeptidyl substrate spectrum, and a Xaa-Pro-Pro tripeptidyl peptidase (TPP) activity that efficiently cleaves any remaining motifs that would otherwise represent stop codons for the AP / DPP tandem.

[0013] The exopeptidase combination provided herein is able to complement or replace exopeptidases of luminal or brush border enterocytes origin in the intestine, and based on its comprehensive cleaving of PCPs into harmless digestive by-products, offers an ancillary treatment approach for the mitigation of peptide-derived chronic inflammation, and supplementation of crucial amino acids, such as proline and glutamine, to patients suffering from functional gastrointestinal disorders.

[0014] Summary

[0015] Certain proline-rich or proline-containing peptides (PCPs) have been shown to trigger proinflammatory and immunomodulatory signaling pathways. In celiac disease, for example, dietary bioactive PCPs, referred to in this field as gluten immunogenic peptides (GIPs), play an important disease-promoting role. It is desirable to eliminate these peptides by appropriate treatment before they can exert their disease-promoting or disease-inducing role. An exopeptidase-based treatment would in principle be able to systematically hydrolyze peptides into trimers, dimers, or monomers. For example, fungal dipeptidyl peptidases (DPPs) have been shown to act synergistically with monoaminopeptidases (APs) to completely cleave certain immunogenic PCP or GIP chains into harmless ones.

[0016] The present disclosure is based on the insight that a combination of APs and DPPs may not be sufficient for complete digestion of certain disease-promoting peptides because further degradation of these peptides may be blocked by Xaa-Pro-Pro stop sequences. A combination of exopeptidase activities is provided herein that enables extensive degradation of disease- promoting peptides. The compositions described herein are useful for exopeptidase supplementation or replacement therapy in medical applications such as in the treatment of peptide-related disorders such as peptide-related immune disorders and functional gastrointestinal disorders associated with immunogenic or bioactive peptides. Such disorders may involve inflammation caused by dietary peptides and include gluten-related disorders such as celiac disease (CeD) and non-celiac gluten sensitivity (NCGS), as well as other diseases that may profit from the removal of immunogenic peptides, in particular PCPs, such as inflammatory bowel diseases (IBD).

[0017] In one aspect, the present disclosure provides a pharmaceutical composition comprising the following enzymatic activities: an enzymatic activity cleaving amino acids from the N-terminal end of peptides, an enzymatic activity cleaving dipeptides from the N-terminal end of peptides having a proline residue in the second amino acid position, and an enzymatic activity cleaving tripeptides from the N-terminal end of peptides having proline residues in the second and third amino acid positions.

[0018] In some embodiments, the enzymatic activity cleaving amino acids from the N-terminal end of peptides comprises an enzymatic activity cleaving at least leucine and glutamine from the N-terminal end of a peptide unless a proline residue is present in the second amino acid position. in some embodiments, the enzymatic activity cleaving amino acids from the N-terminal end of peptides comprises an enzymatic activity cleaving at least 5, at least 10, at least 15, at least 16, at least 17, or at least 18 different amino acids from the N-terminal end of a peptide unless a proline residue is present in the second amino acid position.

[0019] In some embodiments, the enzymatic activity cleaving amino acids from the N-terminal end of peptides comprises an enzymatic activity cleaving at least Leu, lie, Vai, Ala, Gly, Ser, Thr, Cys, Met, Asn, Gin, Asp, Glu, Lys, Arg, His, Phe, and Pro from the N-terminal end of a peptide unless a proline residue is present in the second amino acid position.

[0020] In some embodiments, the enzymatic activity cleaving amino acids from the N-terminal end of peptides is contributed by an aminopeptidase present in the composition.

[0021] In some embodiments, the aminopeptidase is a leucine-aminopeptidase.

[0022] In some embodiments, the aminopeptidase comprises an amino acid sequence derived from an aminopeptidase of a fungus.

[0023] In some embodiments, the aminopeptidase comprises an amino acid sequence derived from an aminopeptidase of Trichophyton rubrum.

[0024] In some embodiments, the aminopeptidase comprises an amino acid sequence derived from leucine-aminopeptidase 2 of Trichophyton rubrum.

[0025] In some embodiments, the aminopeptidase comprises the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof.

[0026] In some embodiments, the enzymatic activity cleaving dipeptides from the N-terminal end of peptides having a proline residue in the second amino acid position comprises an enzymatic activity cleaving at least glutamine-proline, phenylalanine-proline, leucine-proline, and tyrosine-proline from the N-terminal end of a peptide unless proline residues are present in the second and third amino acid positions.

[0027] In some embodiments, the enzymatic activity cleaving dipeptides from the N-terminal end of peptides having a proline residue in the second amino acid position comprises an enzymatic activity cleaving at least 4, at least 5, at least 10, at least 15, at least 16, at least 17, or at least 18 different dipeptides having a proline residue in the second amino acid position from the N- terminal end of a peptide unless proline residues are present in the second and third amino acid positions. In some embodiments, the 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 dipeptidyl peptidase present in the composition.

[0028] In some embodiments, the dipeptidyl peptidase comprises an amino acid sequence derived from a dipeptidyl peptidase of a fungus.

[0029] In some embodiments, the dipeptidyl peptidase comprises an amino acid sequence derived from a dipeptidyl peptidase of Trichophyton rubrum.

[0030] In some embodiments, the dipeptidyl peptidase comprises an amino acid sequence derived from dipeptidyl peptidase IV of Trichophyton rubrum.

[0031] In some embodiments, the dipeptidyl peptidase comprises the amino acid sequence represented by SEQ. ID NO: 2 or a variant thereof.

[0032] In some embodiments, the enzymatic activities are maintained at the pH of the small intestine, in particular the upper small intestine.

[0033] In some embodiments, the enzymatic activities are maintained at the conditions of the small intestine, in particular the upper small intestine.

[0034] In some embodiments, the enzymatic activity cleaving amino acids from the N-terminal end of peptides and the enzymatic activity cleaving dipeptides from the N-terminal end of peptides having a proline residue in the second amino acid position in combination do not have an enzymatic activity cleaving amino acids or dipeptides from the N-terminal end of a peptide having proline residues in the second and third amino acid positions.

[0035] In a further aspect, the present disclosure provides a pharmaceutical composition comprising an aminopeptidase and a dipeptidyl peptidase, the pharmaceutical composition further comprising an enzyme having an enzymatic activity cleaving tripeptides from the N-terminal end of peptides having proline residues in the second and third amino acid positions.

[0036] In some embodiments, the aminopeptidase comprises an enzymatic activity cleaving amino acids from the N-terminal end of peptides.

[0037] In some embodiments, the aminopeptidase comprises an enzymatic activity cleaving at least leucine and glutamine from the N-terminal end of a peptide unless a proline residue is present in the second amino acid position. In some embodiments, the aminopeptidase comprises an enzymatic activity cleaving at least 5, at least 10, at least 15, at least 16, at least 17, or at least 18 different amino acids from the N-terminal end of a peptide unless a proline residue is present in the second amino acid position.

[0038] In some embodiments, the aminopeptidase comprises an enzymatic activity cleaving at least Leu, IIe, Val, Ala, Gly, Ser, Thr, Cys, Met, Asn, Gln, Asp, Glu, Lys, Arg, His, Phe, and Pro from the N-terminal end of a peptide unless a proline residue is present in the second amino acid position.

[0039] In some embodiments, the aminopeptidase is a leucine-aminopeptidase.

[0040] In some embodiments, the aminopeptidase comprises an amino acid sequence derived from an aminopeptidase of a fungus.

[0041] In some embodiments, the aminopeptidase comprises an amino acid sequence derived from an aminopeptidase of Trichophyton rubrum.

[0042] In some embodiments, the aminopeptidase comprises an amino acid sequence derived from leucine-aminopeptidase 2 of Trichophyton rubrum.

[0043] In some embodiments, the aminopeptidase comprises the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof.

[0044] In some embodiments, the dipeptidyl peptidase comprises an enzymatic activity cleaving dipeptides from the N-terminal end of peptides having a proline residue in the second amino acid position.

[0045] In some embodiments, the dipeptidyl peptidase comprises an enzymatic activity cleaving at least glutamine-proline, phenylalanine-proline, leucine-proline, and tyrosine-proline from the N-terminal end of a peptide unless proline residues are present in the second and third amino acid positions.

[0046] In some embodiments, the dipeptidyl peptidase comprises an enzymatic activity cleaving at least 4, at least 5, at least 10, at least 15, at least 16, at least 17, or at least 18 different dipeptides having a proline residue in the second amino acid position from the N-terminal end of a peptide unless proline residues are present in the second and third amino acid positions. In some embodiments, the dipeptidyl peptidase comprises an amino acid sequence derived from a dipeptidyl peptidase of a fungus. In some embodiments, the dipeptidyl peptidase comprises an amino acid sequence derived from a dipeptidyl peptidase of Trichophyton rubrum.

[0047] In some embodiments, the dipeptidyl peptidase comprises an amino acid sequence derived from dipeptidyl peptidase IV of Trichophyton rubrum.

[0048] In some embodiments, the dipeptidyl peptidase comprises the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof.

[0049] In some embodiments, the enzymatic activities of the enzymes are maintained at the pH of the small intestine, in particular the upper small intestine.

[0050] In some embodiments, the enzymatic activities of the enzymes are maintained at the conditions of the small intestine, in particular the upper small intestine.

[0051] In some embodiments, the aminopeptidase and the dipeptidyl peptidase in combination do not have an enzymatic activity cleaving amino acids or dipeptides from the N-terminal end of a peptide having proline residues in the second and third amino acid positions.

[0052] In a further aspect, the present disclosure provides a pharmaceutical composition comprising an aminopeptidase which comprises an amino acid sequence derived from leucineaminopeptidase 2 of Trichophyton rubrum and a dipeptidyl peptidase which comprises an amino acid sequence derived from dipeptidyl peptidase IV of Trichophyton rubrum, the pharmaceutical composition further comprising an enzymatic activity cleaving tripeptides from the N-terminal end of peptides having proline residues in the second and third amino acid positions.

[0053] In some embodiments, the aminopeptidase comprises an enzymatic activity cleaving amino acids from the N-terminal end of peptides.

[0054] In some embodiments, the aminopeptidase comprises an enzymatic activity cleaving at least leucine and glutamine from the N-terminal end of a peptide unless a proline residue is present in the second amino acid position.

[0055] In some embodiments, the aminopeptidase comprises an enzymatic activity cleaving at least 5, at least 10, at least 15, at least 16, at least 17, or at least 18 different amino acids from the N-terminal end of a peptide unless a proline residue is present in the second amino acid position. In some embodiments, the aminopeptidase comprises an enzymatic activity cleaving at least Leu, He, Vai, Ala, Gly, Ser, Thr, Cys, Met, Asn, Gin, Asp, Glu, Lys, Arg, His, Phe, and Pro from the N-terminal end of a peptide unless a proline residue is present in the second amino acid position.

[0056] In some embodiments, the aminopeptidase comprises the amino acid sequence represented by SEQ. ID NO: 1 or a variant thereof.

[0057] In some embodiments, the dipeptidyl peptidase comprises an enzymatic activity cleaving dipeptides from the N-terminal end of peptides having a proline residue in the second amino acid position.

[0058] In some embodiments, the dipeptidyl peptidase comprises an enzymatic activity cleaving at least glutamine-proline, phenylalanine-proline, leucine-proline, and tyrosine-proiine from the N-terminal end of a peptide unless proline residues are present in the second and third amino acid positions.

[0059] In some embodiments, the dipeptidyl peptidase comprises an enzymatic activity cleaving at least 4, at least 5, at least 10, at least 15, at least 16, at least 17, or at least 18 different dipeptides having a proline residue in the second amino acid position from the N-terminal end of a peptide unless proline residues are present in the second and third amino acid positions. In some embodiments, the dipeptidyl peptidase comprises the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof.

[0060] In some embodiments, the enzymatic activities of the enzymes are maintained at the pH of the small intestine, in particular the upper small intestine.

[0061] In some embodiments, the enzymatic activities of the enzymes are maintained at the conditions of the small intestine, in particular the upper small intestine.

[0062] In some embodiments, the aminopeptidase and the dipeptidyl peptidase in combination do not have an enzymatic activity cleaving amino acids or dipeptides from the N-terminal end of a peptide having proline residues in the second and third amino acid positions.

[0063] In a further aspect, the present disclosure provides a pharmaceutical composition comprising exoproteolytic activities which in combination cleave amino acids, dipeptides, and tripeptides from the N-terminal end of peptides (i) not having a proline residue in the second amino acid position, (ii) having a proline residue in the second amino acid position but not having a proline residue in the third amino acid position, and (iii) having proline residues in the second and third amino acid positions.

[0064] In some embodiments, the pharmaceutical composition comprises the following enzymatic activities: an enzymatic activity cleaving amino acids from the N-terminal end of peptides, an enzymatic activity cleaving dipeptides from the N-terminal end of peptides having a proline residue in the second amino acid position, and an enzymatic activity cleaving tripeptides from the N-terminal end of peptides having proline residues in the second and third amino acid positions.

[0065] In some embodiments, the enzymatic activity cleaving amino acids from the N-terminal end of peptides comprises an enzymatic activity cleaving at least leucine and glutamine from the N-terminal end of a peptide unless a proline residue is present in the second amino add position.

[0066] In some embodiments, the enzymatic activity cleaving amino acids from the N-terminal end of peptides comprises an enzymatic activity cleaving at least 5, at least 10, at least 15, at least 16, at least 17, or at least 18 different amino acids from the N-terminal end of a peptide unless a proline residue is present in the second amino acid position.

[0067] In some embodiments, the enzymatic activity cleaving amino acids from the N-terminal end of peptides comprises an enzymatic activity cleaving at least Leu, He, Vai, Ala, Gly, Ser, Thr, Cys, Met, Asn, Gin, Asp, Glu, Lys, Arg, His, Phe, and Pro from the N-terminal end of a peptide unless a proline residue is present in the second amino acid position.

[0068] In some embodiments, the enzymatic activity cleaving amino acids from the N-terminal end of peptides is contributed by an aminopeptidase present in the composition.

[0069] In some embodiments, the aminopeptidase is a leucine-aminopeptidase.

[0070] In some embodiments, the aminopeptidase comprises an amino acid sequence derived from an aminopeptidase of a fungus.

[0071] In some embodiments, the aminopeptidase comprises an amino acid sequence derived from an aminopeptidase of Trichophyton rubrum.

[0072] In some embodiments, the aminopeptidase comprises an amino acid sequence derived from leucine-aminopeptidase 2 of Trichophyton rubrum. In some embodiments, the aminopeptidase comprises the amino acid sequence represented by SEQ. ID NO: 1 or a variant thereof.

[0073] In some embodiments, the enzymatic activity cleaving dipeptides from the N-terminal end of peptides having a proline residue in the second amino acid position comprises an enzymatic activity cleaving at least glutamine-proline, phenylalanine-proline, leucine-proline, and tyrosine-proline from the N-terminal end of a peptide unless proline residues are present in the second and third amino acid positions.

[0074] In some embodiments, the enzymatic activity cleaving dipeptides from the N-terminal end of peptides having a proline residue in the second amino acid position comprises an enzymatic activity cleaving at least 4, at least 5, at least 10, at least 15, at least 16, at least 17, or at least 18 different dipeptides having a proline residue in the second amino acid position from the N- terminal end of a peptide unless proline residues are present in the second and third amino acid positions.

[0075] In some embodiments, the 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 dipeptidyl peptidase present in the composition.

[0076] In some embodiments, the dipeptidyl peptidase comprises an amino acid sequence derived from a dipeptidyl peptidase of a fungus.

[0077] In some embodiments, the dipeptidyl peptidase comprises an amino acid sequence derived from a dipeptidyl peptidase of Trichophyton rubrum.

[0078] In some embodiments, the dipeptidyl peptidase comprises an amino acid sequence derived from dipeptidyl peptidase IV of Trichophyton rubrum.

[0079] In some embodiments, the dipeptidyl peptidase comprises the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof.

[0080] In some embodiments, the enzymatic activities are maintained at the pH of the small intestine, in particular the upper small intestine.

[0081] In some embodiments, the enzymatic activities are maintained at the conditions of the small intestine, in particular the upper small intestine.

[0082] In some embodiments, the enzymatic activity cleaving amino acids from the N-terminal end of peptides and the enzymatic activity cleaving dipeptides from the N-terminal end of peptides having a proline residue in the second amino acid position in combination do not have an enzymatic activity cleaving amino acids or dipeptides from the N-terminal end of a peptide having proline residues in the second and third amino acid positions.

[0083] In some embodiments of the composition of any aspect described herein, the enzymatic activities in combination cleave the peptide LGQQQ.PFPPQQPY into amino acids, dipeptides, and tripeptides.

[0084] In some embodiments of the composition of any aspect described herein, the pharmaceutical composition is for oral administration.

[0085] In a further aspect, the present disclosure provides a pharmaceutical composition comprising exoproteolytic activities which in combination cleave peptides comprising Xaa-Pro and Xaa- Pro-Pro motifs into amino acids, dipeptides, and tripeptides. In a further aspect, the present disclosure provides a pharmaceutical composition comprising exoproteolytic activities which in combination cleave the peptide LGQQQPFPPQQPY into amino acids, dipeptides, and tripeptides. In a further aspect, the present disclosure provides a pharmaceutical composition comprising an enzymatic activity cleaving amino acids from the N-terminal end of peptides not having a proline residue in the second amino acid position, wherein the pharmaceutical composition further comprises an enzymatic activity cleaving amino acids, dipeptides, or tripeptides from the N-terminal end of a peptide having a proline residue in the second amino acid position, or having proline residues in the second and third amino acid positions. In a further aspect, the present disclosure provides a pharmaceutical composition comprising an enzymatic activity cleaving amino acids from the N-terminal end of peptides not having a proline residue in the second amino acid position, an enzymatic activity cleaving dipeptides from the N-terminal end of peptides having a proline residue in the second amino acid position but not having a proline residue in the third amino acid position, and an enzymatic activity cleaving tripeptides from the N-terminal end of peptides having proline residues in the second and third amino acid positions. In some embodiments, the composition of any of these aspects comprises the enzymatic activities and / or enzymes described above for the other aspects.

[0086] In a further aspect, the present disclosure provides the pharmaceutical composition of any aspect described herein for pharmaceutical use.

[0087] In a further aspect, the present disclosure provides the pharmaceutical composition of any aspect described herein for use in treating a peptide-related condition or disorder.

[0088] In some embodiments, the peptide-related condition or disorder is an immunogenic peptide- related condition or disorder.

[0089] In a further aspect, the present disclosure provides the pharmaceutical composition of any aspect described herein for use in treating a gluten-related condition or disorder.

[0090] In some embodiments, the gluten-related condition or disorder is selected from the group consisting of celiac disease, wheat allergy and non-celiac gluten sensitivity.

[0091] In a further aspect, the present disclosure provides a method of treating a peptide-related condition or disorder comprising administering the pharmaceutical composition of any aspect described herein to a mammal in need thereof.

[0092] In some embodiments, the peptide-related condition or disorder is an immunogenic peptide- related disorder.

[0093] In a further aspect, the present disclosure provides a method of treating a gluten-related condition or disorder comprising administering the pharmaceutical composition of any aspect described herein to a mammal in need thereof.

[0094] In some embodiments, the gluten-related condition or disorder is selected from the group consisting of celiac disease, wheat allergy and non-celiac gluten sensitivity.

[0095] In some embodiments of any method described herein the mammal is a human. In some embodiments of any method described herein the pharmaceutical composition is administered orally.

[0096] Brief description of the drawings

[0097] Figure 1: LAP2, DPPIV, and DPPII (0005) are unable to cleave the N-terminal Phe-Pro-Pro tripeptide motif of a 7-mer peptide (FPPQQPY) found in gluten. Assay parameters correspond to Example 1 and Example 2, section 2. Fluorescence Peptide Cleavage Assay Development.

[0098] Figure 2: The tripeptidyl peptidase 0010.1 can hydrolyze the N-terminal Phe-Pro-Pro tripeptide motif of a 7-mer peptide (FPPQQPY) found in gluten. Assay parameters correspond to Example 2, section 2. Fluorescence Peptide Cleavage Assay Development.

[0099] Figure 3: Fluorescence measurement with peptide quantification kit from Thermo Fisher Scientific on degradation of 33-mer (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF) and 13-mer ( LG QQQP FPPQQPY) in PIPES buffer. Assay parameters correspond to Example 2, section 2. Fluorescence Peptide Cleavage Assay Development, except use of 33-mer peptide at a concentration of 16.7g / μl and concentrations of ruLAP-2 and ruDPP-4, which were set to 0.91g / μl and 0.47ng / μl.

[0100] Figure 4: Fluorescence measurement with peptide quantification kit from Thermo Fisher Scientific on degradation of 13-mer peptide (LGQQQPFPPQQPY) by ruLAP-2 and ruDPP-4 with the prospective XPP protease 0009. Assay parameters correspond to Example 2, section 2. Fluorescence Peptide Cleavage Assay Development, except concentration of 0009 which was set to 0.85ng / μl .

[0101] Figure 5: Fluorescence measurement with peptide quantification kit from Thermo Fisher Scientific on degradation of 13-mer peptide (LGQQQPFPPQQPY) by ruLAP-2 and ruDPP-4 with the prospective XPP protease 0033. Assay parameters correspond to Example 2, section 2. Fluorescence Peptide Cleavage Assay Development.

[0102] Figure 6: Fluorescence measurement with peptide quantification kit from Thermo Fisher Scientific on degradation of 13-mer peptide (LGQQQPFPPQQPY) by ruLAP-2 and ruDPP-4 with the prospective XPP protease 0010.1. Assay parameters correspond to Example 2, section 2.

[0103] Fluorescence Peptide Cleavage Assay Development.

[0104] Figure 7: Fluorescence measurement with peptide quantification kit from Thermo Fisher Scientific on degradation of 13-mer peptide (LGQQQPFPPQQPY) by ruLAP-2 and ruDPP-4 with the prospective XPP protease 0019. Assay parameters correspond to Example 2, section 2. Fluorescence Peptide Cleavage Assay Development.

[0105] Figure 8: Fluorescence measurement with peptide quantification kit from Thermo Fisher Scientific on degradation of 13-mer peptide (LGQQQPFPPQQPY) by ruLAP-2 and ruDPP-4 with the prospective XPP protease 0005. Assay parameters correspond to Example 2, section 2. Fluorescence Peptide Cleavage Assay Development.

[0106] Figure 9: Fluorescence measurement with peptide quantification kit from Thermo Fisher Scientific on degradation of 7-mer peptide (FPPQQPY) by ruLAP-2 and ruDPP-4 with prospective XPP proteases. Assay parameters correspond to Example 2, section 2. Fluorescence Peptide Cleavage Assay Development.

[0107] Figure 10: Fluorescence measurement with peptide quantification kit from Thermo Fisher Scientific on degradation of 7-mer peptide (FPPQQPY) by ruLAP-2 and ruDPP-4 with the prospective XPP protease 0009. Assay parameters correspond to Example 2, section 2. Fluorescence Peptide Cleavage Assay Development.

[0108] Figure 11: Fluorescence measurement with peptide quantification kit from Thermo Fisher Scientific on degradation of 7-mer peptide (FPPQQPY) by ruLAP-2 and ruDPP-4 with the prospective XPP protease 0033. Assay parameters correspond to Example 2, section 2. Fluorescence Peptide Cleavage Assay Development.

[0109] Figure 12: Fluorescence measurement with peptide quantification kit from Thermo Fisher Scientific on degradation of 7-mer peptide (FPPQQPY) by ruLAP-2 and ruDPP-4 with the prospective XPP protease 0010.1. Assay parameters correspond to Example 2, section 2.

[0110] Fluorescence Peptide Cleavage Assay Development.

[0111] Figure 13: Fluorescence measurement with peptide quantification kit from Thermo Fisher Scientific on degradation of 7-mer peptide (FPPQQPY) by ruLAP-2 and ruDPP-4 with the prospective XPP protease 0019. Assay parameters correspond to Example 2, section 2. Fluorescence Peptide Cleavage Assay Development.

[0112] Figure 14: Fluorescence measurement with peptide quantification kit from Thermo Fisher Scientific on degradation of 7-mer peptide (FPPQQPY) by ruLAP-2 and ruDPP-4 with the prospective XPP protease 0005. Assay parameters correspond to Example 2, section 2. Fluorescence Peptide Cleavage Assay Development.

[0113] Detailed description

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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 characteristic(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".

[0118] 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.

[0119] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by the context.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] Amino acids, peptides, polypeptides, peptidases

[0130] As used herein, the terms "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.

[0131] 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.

[0132] In some embodiments, an enzymatic activity cleaving amino acids from the N-terminal end of peptides is contributed by an aminopeptidase, e.g., leucine aminopeptidase.

[0133] 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.

[0134] 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 dipeptidyl peptidase.

[0135] 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.

[0136] In some embodiments, an enzymatic activity cleaving tripeptides from the N-terminal end of peptides having proline residues in the second and third amino acid positions is contributed by a tripeptidyl peptidase.

[0137] In some embodiments, a tripeptidyl peptidase for use herein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 6 or a variant thereof. In some embodiments, a tripeptidylpeptidase for use herein comprises the amino acid sequence according to SEQ ID NO: 5 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 referenced amino acid sequence.

[0138] 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. 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 (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF), 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.

[0139] 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.

[0140] 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)

[0141] HPWGQEPFGWPFKPMVTQDDLQNKIKLKDIMAGVEKLQSFSDAHPEKNRVFGGNGHKDTVEWIYNEI KATGYYDVKKQEQVHLWSHAEAALNANGKDLKASAMSYSPPASKIMAELWAKNNGCNATDYPANTQ GKIVLVERGVCSFGEKSAQAGDAKAAGAIVYNNVPGSLAGTLGGLDKRHVPTAGLSQEDGKNLATLVAS GKIDVTMNVISLFENRTTWNVIAETKGGDHNNVIMLGAHSDSVDAGPGINDNGSGSIGIMTVAKALTNF KLNNAVRFAWWTAEEFGLLGSTFYVNSLDDRELHKVKLYLNFDMIGSPNFANQIYDGDGSAYNMTGPA GSAEIEYLFEKFFDDQGIPHQPTAFTGRSDYSAFIKRNVPAGGLFTGAEWKTPEQVKLFGGEAGVAYDKN YHRKGDTVANINKGAIFLNTRAIAYAIAEYARSLKGFPTRPKTGKRDVNPQYSKMPGGGCGHHTVFM ruDPPIV (SEQ ID NO: 2) IVPPREPRSPTGGGNKLLTYKECVPRATISPRSTSLAWINSEEDGRYISQSDDGALILQNIVTNTNKTLVAAD KVPKGYYDYWFKPDLSAVLWATNYTKQ.YRHSYFANYFILDIKKGSLTPLAQDQAGDIQYAQWSPMNNSI AYVRGNDLYIWNNGKTKRITENGGPDIFNGVPDWVYEEEIFGDRFALWFSPDGEYLAYLRFNETGVPTYT IPYYKNKQKIAPAYPRELEIRYPKVSAKNPTVQFHLLNIASSQETTIPVTAFPENDLVIGEVAWLSSGHDSVA YRAFNRVQDREKIVSVKVESKESKVIRERDGTDGWIDNLLSMSYIGNVNGKEYYVDISDASGWAHIYLYPV DGGKEIALTKGEWEWAILKVDTKKKLIYFTSTKYHSTTRHVYSVSYDTKVMTPLVNDKEAAYYTASFSAKG GYYILSYQGPNVPYQELYSTKDSKKPLKTITSNDALLEKLKEYKLPKVSFFEIKLPSGETLNVKQRLPPNFNPH KKYPVLFTPYGGPGAQEVSQAWNSLDFKSYITSDPELEYVTWTVDNRGTGYKGRKFRSAVAKRLGFLEAQ. DQVFAAKEVLKNRWADKDHIGIWGWSYGGFLTAKTLETDSGVFTFGISTAPVSDFRLYDSMYTERYMKT VELNADGYSETAVHKVDGFKNLKGHYLIQHGTGDDNVHFQNAAVLSNTLMNGGVTADKLTTQWFTDS DHGIRYDMDSTYQ.YKQLSKMVYDQKQRRPESPPMHQWSKRVLAALFGERAEE

[0142] In some embodiments, a peptidase comprises a truncated peptidase polypeptide.

[0143] 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.

[0144] 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. 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. in some embodiments, the wildtype LAP polypeptide has the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof.

[0145] 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.

[0146] In some embodiments, the wildtype DPP polypeptide has the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof.

[0147] 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 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: 1.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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. 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.

[0152] "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.

[0153] "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.

[0154] 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.

[0155] The following table lists the 20 natural amino acids and their abbreviations:

[0156] According to the disclosure, the term "peptide" refers to substances which comprise about two or more, about 3 or more, about 4 or more, about 6 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.

[0157] 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. In some embodiments, disease-promoting peptide are proline-rich or proline-containing peptides (PCPs). Examples of disease-promoting peptides include gluten-immunogenic peptides.

[0158] As used herein, the term "immunogenic peptide" includes any peptide that can elicit, facilitate, or produce an immune response.

[0159] "Fragment", with reference to an amino add 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.

[0160] "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 add 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.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] "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.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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 acid 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.

[0172] 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.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] Nucleic acids

[0179] 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 (IVT RNA) or synthetic RNA.

[0180] In some embodiments, the nucleic acid described herein may have modified and / or non- naturally occurring nucleosides.

[0181] 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.

[0182] "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.

[0183] 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.

[0184] "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.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 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 (AOX1) 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.

[0190] "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. 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.

[0191] 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.

[0192] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence.

[0193] 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.

[0194] As used herein "endogenous" refers to any material from or produced inside an organism, cell, tissue or system.

[0195] As used herein, the term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0196] 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.

[0197] Pharmaceutical compositions

[0198] 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. 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%.

[0199] In some embodiments, a pharmaceutical composition described herein is an oral dosage form. 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.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] 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.

[0207] The enzyme composition can also be formed into particles in which it is embedded in or coated with an enteric polymer. 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.

[0208] 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.

[0209] 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).

[0210] The inorganic material can include, for example, an alkalinizing 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.

[0211] 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.

[0212] 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, triethyl citrate, acetyl t ri-n-butyl citrate, diethyl phthalate, dibutyl sebacate, polyethylene glycol, polypropylene glycol, castor oil, acetylated mono-glyceride, acetylated di-glyceride, and mixtures thereof.

[0213] 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 al kalinizing 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.

[0214] 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.

[0215] 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.

[0216] In some embodiments, a composition described herein comprises particles formed from precipitating a mixture comprising the peptidase enzymes and a pH sensitive polymer.

[0217] 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.

[0218] As solvent for the pH sensitive polymer methanol, ethanol, t-butanol, acetone, tetrahydrofuran, isopropyl alcohol may be used.

[0219] 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.

[0220] Particles can be designed to be used in variety of different pharmaceutical compositions and formulations such as for tablets, capsules or suspensions. 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 microenvironment, buffering the macro-environment involves buffering the entire stomach by buffering agents. In some embodiments, such formulations utilize the concept of microenvironment 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).

[0221] Pharmaceutical compositions described herein may contain a proton pump inhibitor.

[0222] 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.

[0223] Pharmaceutical compositions described herein may contain a H2 blocker.

[0224] 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.

[0225] As used herein, the term "antacid" refers to a substance having the ability to neutralize and / or to buffer an add. 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.

[0226] Pharmaceutical compositions described herein may contain a mucoadhesive polymer.

[0227] 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.

[0228] 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.

[0229] 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.

[0230] 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.

[0231] 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, formaldehydecasein, cellulose, croscarmellose sodium, crospovidone (e.g., cross-linked polyvinyl pyrrolidone), microcrystalline cellulose, sodium carboxymethyl starch, sodium starch glycolate, starches (corn starch, rice starch), and mixtures thereof. 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.

[0232] Non-limiting examples of suitable glidants include colloidal silicon dioxide, talc, and mixtures thereof.

[0233] Non-limiting examples of suitable diluents include microcrystalline cellulose, starch, calcium phosphate, lactose, sucrose, mannitol, sorbitol, and combinations thereof.

[0234] 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.

[0235] The pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and in "a pharmaceutically acceptable preparation".

[0236] 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.

[0237] 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. In some embodiments, a pharmaceutical composition described herein is administered orally. In some embodiments, a pharmaceutical composition described herein is formulated for oral administration.

[0238] Treatments

[0239] 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).

[0240] 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.

[0241] In some embodiments, administration of a composition described herein may be performed by single administration or boosted by multiple administrations. 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 referto 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.

[0242] 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 (I BD).

[0243] 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).

[0244] Celiac disease (CeD) is a chronic immune-mediated enteropathy precipitated by glutenimmunogenic peptides (G I Ps) in genetically predisposed individuals. GIPs are generated in the digest of gluten and play a central role in the disease pathology; they consist of digestionresistant 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 autoimmune 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.

[0245] 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.

[0246] 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.

[0247] 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.

[0248] 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.

[0249] 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.

[0250] 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".

[0251] The term "patient" means an individual or subject for treatment, in particular a diseased individual or subject.

[0252] 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.

[0253] 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. Examples

[0254] Experimental Approach

[0255] The gliadin p31-43 is a potential trigger of the innate immune response in non-celiac gluten sensitivity (Lundin, K. E. A., Brottveit, M. & Skodje, G. Chapter 9 - Noncoeliac gluten sensitivity, in Coeliac Disease and Gluten-Related Disorders (eds. Schieptti, A. & Sanders, D.) 177-195 (Academic Press, 2022). doi:10.1016 / B978-0-12-821571-5.00004-0), and celiac disease (Gomez Castro, M. F. et al., Front. Immunol. 10, (2019)). Variants of this peptide, with a core sequence including the proteolytic resistant Phe-Pro-Pro motif (XPP), are the most prevalent peptides found in the urine of gluten consuming individuals regardless of their clinical status (Palanski, B. A. et al., Nat. Commun. 13, 888 (2022)). The p31-43 peptide was shown to induce several proinflammatory pathways in humans (Barone, M. V., Troncone, R. & Auricchio, S., Int. J. Mol. Sci. 15, 20518-20537 (2014)) and mice (Araya, R. E. et al. Mechanisms of innate immune activation by gluten peptide p31-43 in mice. Am. J. Physiol. -Gastrointest. Liver Physiol. 311, G40-G49 (2016)), and to be prone to spontaneous self-assembly into bioactive supramolecular structures (Gomez Castro, M. F. et a!., Front. Immunol. 10, (2019)). Furthermore, p31-43 and its variants are naturally produced from the gastropancreatic digestion of gluten and resistant to hydrolysis by brush border membrane enzymes (Gianfrani, C. et al., Mol. Nutr. Food Res. 59, 1844-1854 (2015); lacomino, G. et al., J. Agric. Food Chem. 61, 1088-1096 (2013)), such as APs and DPPs as discussed above.

[0256] Interestingly, we showed in a recent molecular dynamic study that peptides larger than tripeptides are particularly susceptible to self-assembly, i.e. to producing unspecified and potentially harmful surface paratopes. Consequently, we set out to identify exopeptidases that are complementary to APs and DPPs as they (A) are capable of efficiently cleaving Xaa- Pro-Pro motifs, (B) have a broad substrate promiscuity, i.e. are capable of cleaving all possible PCP digestion by-products, and (C) optionally perform well in the conditions of the designated application setting (small intestine).

[0257] The core XPP motif found in all variants of p31-43 presents a uniquely challenging stop sequencing in PCPs for exopeptidases and was used as a template substrate to identify peptidase candidates that are capable of efficiently cleaving Xaa-Pro-Pro motifs. We generated a seed profile of viable sequences from available literature and database annotations to experimentally validate activity of sequences and to correlate mechanistic, topological, and structural enzyme features with catalytic performance.

[0258] Example 1: LAP2, DPPIV, and DPPII (0005) are unable to cleave the N-terminal Phe-Pro-Pro tripeptide motif of a 7-mer peptide (FPPQQPY) found in gluten.

[0259] A degradation product of the gluten-derived peptide a-gliadin 31-43 (p31-43), with an N- terminal Phe-Pro-Pro motif (XPP), was used to assess the cleaving efficacy of the XPP stop sequence by DPP2. The 7-mer peptide, and a combination of LAP2 and DPPIV were used as negative control. Cleaving efficacy was screened with the Thermo Scientific Pierce Quantitative Fluorometric Peptide Assay. No significant differences were observed between the peptide negative control (7-mer), the peptide with DPPIV and LAP2, and the peptide with DPPII (0005), DPPIV, and LAP2; see Figure 1. These data demonstrate that no cleaving events are taking place on the 7-mer peptide, and the N-terminal Phe-Pro-Pro motif therefore constitutes a stop sequence for all enzymes tested.

[0260] Example 2: The tripeptidyl peptidase 0010.1 can hydrolyze the N-terminal Phe-Pro-Pro tripeptide motif of a 7-mer peptide (FPPQQPY) found in gluten.

[0261] A degradation product of the gluten-derived peptide a-gliadin 31-43 (p31-43), with an N- terminal Phe-Pro-Pro motif (XPP), was used to assess the cleaving efficacy of the XPP stop sequence by the TPP exopeptidase 0010.1. The 7-mer peptide, and a combination of LAP2, DPPII and DPPIV were used as negative control. Cleaving efficacy was screened with the Thermo Scientific Pierce Quantitative Fluorometric Peptide Assay. No significant differences were observed between the peptide negative control (7-mer), the peptide with DPPIV and LAP2, and the peptide with DPPII (0005), DPPIV, and LAP2; see Figure 1 and Figure 2. Conversely, a significant increase in fluorescence was observed when the 7-mer peptide was incubated with ruLAP2, ruDPPIV, and the TPP 0010.1 (Figure 2). These data demonstrate that the N-terminal XPP stop sequence of the 7-mer peptide can be hydrolysed by a tripeptidyl peptidase exopeptidase.

[0262] Methods

[0263] 1. Prospective XPP proteases

[0264] A library of prospective XPP proteases was prepared consisting of enzymes that were procured through commercial supply and enzymes that were recombinantly produced in E. coli and purified (Table 1). The general process from gene synthesis to purification and screening is outlined below (Table 2). The selection of gene sequences, plasmids, strains or expression media was based on literature (if available) or standard expression protocols for similar proteins in E. coli.

[0265] Synthesis of protease sequences (after codon-optimization) at GenScript and cloning into vectors: pET26b(+) for expression with disulfide bonds, pGEX-5X-l for expression with GST fusion protein or pQE-60 for expression under weaker promotor.

[0266] - Transformation in standard NEB E. coli BL21(DE3) expression host (pET26b(+)), in NEB E. coli BL21 (pGEX-5X-l) or in NEBExpress® Iq Competent E. coli (High Efficiency) (pQE- 60 for non-leaky expression) via heat-shock protocol (42°C).

[0267] Expression based on literature (if available) in TB media (var. 010.1, 0019) or in LB media (var. 0033), at 26°C overnight (20h), induced at OD600 = 0.7-1.0 with 0.2mM - ImM IPTG, addition of ImM metal cofactors if required (ref. Table 1).

[0268] Cell lysis (in 50mM Tris-HCI, 300mM NaCl, pH=7.5) via sonication or in lOmM PBS at pH=7.4 (pGEX-5X-l), followed by Purification via HIS TALON gravity flow columns (Cytiva) or via GST gravity flow columns (Cytiva), respectively.

[0269] - Buffer Exchange via PD-10 disposable desalting columns (Cytiva) into storage buffer (50mM Tris-HCI, 150mM NaCl, pH=7.5) and concentrating purified protein with VivaSpin 30kDa or 50kDa (depending on protein size) centrifugal concentrators at 4000rcf and 4°C (+ TEV cleavage for eGFP fusion protein expression with second HIS purification to remove cleaved of eGFP and TEV protease, both HIS-tagged).

[0270] Concentration measurement via Thermo Fisher NanoDrop One via A280 and SDS- PAGE evaluation of expression / purification success and purity. Screening of FPP cleavage activity with 13-mer / 7-mer peptides in fluorescence peptide cleavage assay (based on Thermo Fisher kit). All prospective XPP proteases were additionally prescreened for basic protease activity as a QC measure of functional expression (data not shown).

[0271] 2. Fluorescence Peptide Cleavage Assay Development

[0272] A fluorescence-based assay was developed to enable the detection of successful peptide cleavage events. A commercial kit was applied that enables quantification of peptides based on N-terminal labelling of the primary amine: https: / / www.thermofisher.com / order / catalog / product / 23290.

[0273] Cleavage events coincide with the increase in the number of primary amines, which are then labeled, and the corresponding increase in fluorescence signal is detected.

[0274] Assay protocols were adapted accordingto guidelines and limitations described in the supplier manual: https: / / www.thermofisher.com / document-connect / document- connect.html?url=https: / / assets.thermofisher.com / TFS- Assets%2FLSG%2Fmanuals%2F23290_quantpeptide_fluor_UG.pdf.

[0275] Notably, the assay requires that the reaction does not contain free amino acids and that TRIS buffer cannot be applied, as this would interfere with the fluorescence signal. PIPES buffer was used as the best performing system after extensive testing in this project.

[0276] 10 pl of sample are combined with 70|_il of "Fluorometric Peptide Assay Buffer" (supplied by the kit) and with 20 pl of "Fluorometric Peptide Assay Reagent" (supplied by the kit). After incubation for 5 min at RT, signals can be measured at 390 nm excitation and 475 nm emission wavelengths.

[0277] This procedure was applied after setting up a general peptide degradation assay, that exemplarily showed degradation of p31-43 (13-mer; LGQQQPFPPQQPY) cleavage and 33-mer (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF) cleavage by the ruLAP-2 and ruDPP-4 tandem (Figure 3). It was shown that the tandem of ruLAP-2 and ruDPP-4 is capable of cleaving 33-mer and the 13-mer, whereas the reaction with the 13-mer (containing FPP motif) seems to stop between 15-30min of reaction time.

[0278] This data confirms functionality and applicability of the fluorometric peptide cleavage assay and its application for detecting peptide cleavage events within the context of this project.

[0279] The general method for fluorescence peptide cleavage assays can be described by the following parameters:

[0280] • 37°C reaction temperature

[0281] • 200 μl total reaction volume

[0282] • 16.7 ng / μl 13-mer or 28.7 g / μl 7-mer

[0283] • 35 mM PIPES buffer pH=7.00 at 37°C

[0284] • 0.86 g / μl ruLAP-2 and 0.86 g / μl ruDPP-4

[0285] • 0.86 g / μl prospective XPP proteases

[0286] • excitation 390 nm and emission 475 nm (N-terminal primary amines labelled with fluorophore)

[0287] Any changes applied within specific assays in comparison to above-listed assay parameters are described within the respective figure legends.

[0288] Table 1: Overview of expressed enzymes, their expression strains, plasmids, induction conditions, literature references, accessions, used promotors, purification methods and cofactors if needed.

[0289] Variants Type Origin Accession Plasmid Promotor Strain InductionPurificationCofactor Literature

[0290] Table 2: Overview of prospective XPP proteases, their source organisms, accessions and native amino acid sequences.

[0291]

[0292]

[0293] 3. Prospective XPP Proteases 13-mer Cleavage

[0294] For benchmarking of prospective XPP proteases, the peptide degradation assays were performed with a mixture of ruLAP-2, ruDPP-4 and 13-mer substrate. Prospective XPP proteases were individually added as a third enzyme to evaluate their efficacy in cleaving the FPP motif, to determine potentially cross-reactivity as well as ranking effective enzyme combination. As ruLAP-2 and ruDPP-4 enzymes are unable to cleave the FPP motif, in the experiment, a higher fluorescence signal than ruLAP-2 and ruDPP-4 baseline infers FPP cleaving capability when adding additional prospective XPP proteases to the mixture, see Figures 4 - 8.

[0295] Data suggests that variants 0009, 0010.1, 0019, 0033 (combined with ruLAP-2 and ruDPP-4) produce significantly higher signals in comparison to the ruLAP-2 and ruDPP-4 baseline, and thus, those seem capable of FPP cleavage. Variant 0005 does not show clear results and has been evaluated on the 7-mer for more precise results (see section 4. Prospective XPP Proteases 7-mer (FPPQQPY) Cleavage).

[0296] Furthermore, no signal increase can be detected in any enzyme controls that does not contain the 13-mer, and thus, no proteolytic cleavage between the third enzymes and ruLAP-2 / ruDPP-4 occurs.

[0297] 4. Prospective XPP Proteases 7-mer (FPPQQPY) Cleavage

[0298] Furthermore, the assay was applied to a minimalist XPP peptide system, the 7-mer peptide (FPPQQPY), the sequence of which is contained in the 13-mer, and which is assumed to be byproduct of incomplete digestion of the 13-mer with the ruLAP-2 / ruDPP-4 tandem.

[0299] If activity is detected on the 7-mer peptide under assay conditions, FPP cleaving capability from prospective XPP proteases can be inferred (Figure 9).

[0300] It was demonstrated that variant 0010.1 (combined with ruLAP-2 and ruDPP-4) has the highest signal compared to with ruLAP-2 / ruDPP-4 baseline. Variants 0009, 0019 and 0033 produce higher signals as well, and thus, variants 0010.1, 0009, 0019 and 0033 are capable of FPP cleavage. Conversely, no signal increase can be detected for the rul_AP-2 / ruDPP-4 tandem, as well as variant 0005, and thus, ruLAP-2 / ruDPP-4 and 0005 are not capable of cleaving the FPP motif. The individual performance of each variant in comparison to the ruLAP-2 / ruDPP-4 tandem is displayed in Figures 10 to 14.

Claims

CLAIMS1. A pharmaceutical composition comprising the following enzymatic activities: an enzymatic activity cleaving amino acids from the N-terminal end of peptides, an enzymatic activity cleaving dipeptides from the N-terminal end of peptides having a proline residue in the second amino acid position, and an enzymatic activity cleaving tripeptides from the N- terminal end of peptides having proline residues in the second and third amino acid positions.

2. The pharmaceutical composition of claim 1, wherein the enzymatic activity cleaving amino acids from the N-terminal end of peptides comprises an enzymatic activity cleaving at least leucine and glutamine from the N-terminal end of a peptide unless a proline residue is present in the second amino acid position.

3. The pharmaceutical composition of claim 1 or 2, wherein the enzymatic activity cleaving amino acids from the N-terminal end of peptides comprises an enzymatic activity cleaving at least 5, at least 10, at least 15, at least 16, at least 17, or at least 18 different amino acids from the N-terminal end of a peptide unless a proline residue is present in the second amino acid position.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the enzymatic activity cleaving amino acids from the N-terminal end of peptides is contributed by an aminopeptidase present in the composition.

5. The pharmaceutical composition of claim 3, wherein the aminopeptidase is a leucineaminopeptidase.

6. The pharmaceutical composition of claim 4 or 5, wherein the aminopeptidase comprises an amino acid sequence derived from an aminopeptidase of a fungus.

7. The pharmaceutical composition of any one of claims 4 to 6, wherein the aminopeptidase comprises an amino acid sequence derived from an aminopeptidase of Trichophyton rubrum.

8. The pharmaceutical composition of any one of claims 4 to 7, wherein the aminopeptidase comprises an amino acid sequence derived from leucine-aminopeptidase 2 of Trichophyton rubrum.

9. The pharmaceutical composition of any one of claims 4 to 8, wherein the aminopeptidase comprises the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof.

10. The pharmaceutical composition of any one of claims 1 to 9, wherein the enzymatic activity cleaving dipeptides from the N-terminal end of peptides having a proline residue in the second amino acid position comprises an enzymatic activity cleaving at least glutamineproline, phenylalanine-proline, leucine-proline, and tyrosine-proline from the N-terminal end of a peptide unless proline residues are present in the second and third amino acid positions.

11. The pharmaceutical composition of any one of claims 1 to 10, wherein the enzymatic activity cleaving dipeptides from the N-terminal end of peptides having a proline residue in the second amino acid position comprises an enzymatic activity cleaving at least 4, at least 5, at least 10, at least 15, at least 16, at least 17, or at least 18 different dipeptides having a proline residue in the second amino acid position from the N-terminal end of a peptide unless proline residues are present in the second and third amino acid positions.

12. The pharmaceutical composition of any one of claims 1 to 11, wherein the 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 dipeptidyl peptidase present in the composition.

13. The pharmaceutical composition of claim 12, wherein the dipeptidyl peptidase comprises an amino acid sequence derived from a dipeptidyl peptidase of a fungus.

14. The pharmaceutical composition of claim 12 or 13, wherein the dipeptidyl peptidase comprises an amino acid sequence derived from a dipeptidyl peptidase of Trichophyton rubrum.

15. The pharmaceutical composition of any one of claims 12 to 14, wherein the dipeptidyl peptidase comprises an amino acid sequence derived from dipeptidyl peptidase IV of Trichophyton rubrum.

16. The pharmaceutical composition of any one of claims 12 to 15, wherein the dipeptidyl peptidase comprises the amino acid sequence represented by SEQ, ID NO: 2 or a variant thereof.

17. The pharmaceutical composition of any one of claims 1 to 16, wherein the enzymatic activities are maintained at the pH of the small intestine, in particular the upper small intestine.

18. The pharmaceutical composition of any one of claims 1 to 17, wherein the enzymatic activities are maintained at the conditions of the small intestine, in particular the upper small intestine.

19. The pharmaceutical composition of any one of claims 1 to 18, wherein the enzymatic activity cleaving amino acids from the N-terminal end of peptides and the enzymatic activity cleaving dipeptides from the N-terminal end of peptides having a proline residue in the second amino acid position in combination do not have an enzymatic activity cleaving amino acids or dipeptides from the N-terminal end of a peptide having proline residues in the second and third amino acid positions.

20. & pharmaceutical composition comprising an aminopeptidase and a di peptidyl peptidase, the pharmaceutical composition further comprising an enzyme having an enzymatic activity cleaving tripeptides from the N-terminal end of peptides having proline residues in the second and third amino acid positions.

21. The pharmaceutical composition of claim 20, wherein the aminopeptidase comprises an enzymatic activity cleaving amino acids from the N-terminal end of peptides.

22. The pharmaceutical composition of claim 20 or 21, wherein the aminopeptidase comprises an enzymatic activity cleaving at least leucine and glutamine from the N-terminal end of a peptide unless a proline residue is present in the second amino acid position.

23. The pharmaceutical composition of any one of claims 20 to 22, wherein the aminopeptidase comprises an enzymatic activity cleaving at least 5, at least 10, at least 15, at least 16, at least 17, or at least 18 different amino acids from the N-terminal end of a peptide unless a proline residue is present in the second amino acid position.

24. The pharmaceutical composition of any one of claims 20 to 23, wherein the aminopeptidase is a leucine-aminopeptidase.

25. The pharmaceutical composition of any one of claims 20 to 24, wherein the aminopeptidase comprises an amino acid sequence derived from an aminopeptidase of a fungus.

26. The pharmaceutical composition of any one of claims 20 to 25, wherein the aminopeptidase comprises an amino acid sequence derived from an aminopeptidase of Trichophyton rubrum.

27. The pharmaceutical composition of any one of claims 20 to 26, wherein the aminopeptidase comprises an amino acid sequence derived from leucine-aminopeptidase 2 of Trichophyton rubrum.

28. The pharmaceutical composition of any one of claims 20 to 27, wherein the aminopeptidase comprises the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof.

29. The pharmaceutical composition of any one of claims 20 to 28, wherein the dipeptidyl peptidase comprises an enzymatic activity cleaving dipeptides from the N-terminal end of peptides having a proline residue in the second amino acid position.

30. The pharmaceutical composition of any one of claims 20 to 29, wherein the dipeptidyl peptidase comprises an enzymatic activity cleaving at least glutamine-proline, phenylalanineproline, leucine-proline, and tyrosine-proline from the N-terminal end of a peptide unless proline residues are present in the second and third amino acid positions.

31. The pharmaceutical composition of any one of claims 20 to 30, wherein the dipeptidyl peptidase comprises an enzymatic activity cleaving at least 4, at least 5, at least 10, at least 15, at least 16, at least 17, or at least 18 different dipeptides having a proline residue in the second amino acid position from the N-terminal end of a peptide unless proline residues are present in the second and third amino acid positions.

32. The pharmaceutical composition of any one of claims 20 to 31, wherein the dipeptidyl peptidase comprises an amino acid sequence derived from a dipeptidyl peptidase of a fungus.

33. The pharmaceutical composition of any one of claims 20 to 32, wherein the dipeptidyl peptidase comprises an amino acid sequence derived from a dipeptidyl peptidase of Trichophyton rubrum.

34. The pharmaceutical composition of any one of claims 20 to 33, wherein the dipeptidyl peptidase comprises an amino acid sequence derived from dipeptidyl peptidase IV of Trichophyton rubrum.

35. The pharmaceutical composition of any one of claims 20 to 34, wherein the dipeptidyl peptidase comprises the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof.

36. The pharmaceutical composition of any one of claims 20 to 35, wherein the enzymatic activities of the enzymes are maintained at the pH of the small intestine, in particular the upper small intestine.

37. The pharmaceutical composition of any one of claims 20 to 36, wherein the enzymatic activities of the enzymes are maintained at the conditions of the small intestine, in particular the upper small intestine.

38. The pharmaceutical composition of any one of claims 20 to 37, wherein the aminopeptidase and the dipeptidyl peptidase in combination do not have an enzymatic activity cleaving amino acids or dipeptides from the N-terminal end of a peptide having proline residues in the second and third amino acid positions.

39. A pharmaceutical composition comprising an aminopeptidase which comprises an amino acid sequence derived from leucine-aminopeptidase 2 of Trichophyton rubrum and a dipeptidyl peptidase which comprises an amino acid sequence derived from dipeptidyl peptidase IV of Trichophyton rubrum, the pharmaceutical composition further comprising an enzymatic activity cleaving tripeptides from the N-terminal end of peptides having proline residues in the second and third amino acid positions.

40. The pharmaceutical composition of claim 39, wherein the aminopeptidase comprises an enzymatic activity cleaving amino acids from the N-terminal end of peptides.

41. The pharmaceutical composition of claim 39 or 40, wherein the aminopeptidase comprises an enzymatic activity cleaving at least leucine and glutamine from the N-terminal end of a peptide unless a proline residue is present in the second amino acid position.

42. The pharmaceutical composition of any one of claims 39 to 41, wherein the aminopeptidase comprises an enzymatic activity cleaving at least 5, at least 10, at least 15, at least 16, at least 17, or at least 18 different amino acids from the N-terminal end of a peptide unless a proline residue is present in the second amino acid position.

43. The pharmaceutical composition of any one of claims 39 to 42, wherein the aminopeptidase comprises the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof.

44. The pharmaceutical composition of any one of claims 39 to 43, wherein the dipeptidyl peptidase comprises an enzymatic activity cleaving dipeptides from the N-terminal end of peptides having a proline residue in the second amino acid position.

45. The pharmaceutical composition of any one of claims 39 to 44, wherein the dipeptidyl peptidase comprises an enzymatic activity cleaving at least glutamine-proline, phenylalanineproline, leucine-proline, and tyrosine-proline from the N-terminal end of a peptide unless proline residues are present in the second and third amino acid positions.

46. The pharmaceutical composition of any one of claims 39 to 45, wherein the dipeptidyl peptidase comprises an enzymatic activity cleaving at least 4, at least 5, at least 10, at least 15, at least 16, at least 17, or at least 18 different dipeptides having a proline residue in the second amino acid position from the N-terminal end of a peptide unless proline residues are present in the second and third amino acid positions.

47. The pharmaceutical composition of any one of claims 39 to 46, wherein the dipeptidyl peptidase comprises the amino acid sequence represented by SEQ. ID NO: 2 or a variant thereof.

48. The pharmaceutical composition of any one of claims 39 to 47, wherein the enzymatic activities of the enzymes are maintained at the pH of the small intestine, in particular the upper small intestine.

49. The pharmaceutical composition of any one of claims 39 to 48, wherein the enzymatic activities of the enzymes are maintained at the conditions of the small intestine, in particular the upper small intestine.

50. The pharmaceutical composition of any one of claims 39 to 49, wherein the aminopeptidase and the dipeptidyl peptidase in combination do not have an enzymatic activity cleaving amino acids or dipeptides from the N-terminal end of a peptide having proline residues in the second and third amino acid positions.

51. A pharmaceutical composition comprising exoproteolytic activities which in combination cleave amino acids, dipeptides, and tripeptides from the N-terminal end of peptides (i) not having a proline residue in the second amino acid position, (ii) having a proline residue in the second amino acid position but not having a proline residue in the third amino acid position, and (iii) having proline residues in the second and third amino acid positions.

52. The pharmaceutical composition of claim 51 comprising the following enzymatic activities: an enzymatic activity cleaving amino acids from the N-terminal end of peptides, an enzymatic activity cleaving dipeptides from the N-terminal end of peptides having a proline residue in the second amino acid position, and an enzymatic activity cleaving tripeptidesfrom the N-terminal end of peptides having proline residues in the second and third amino acid positions.

53. The pharmaceutical composition of claim 52, wherein the enzymatic activity cleaving amino acids from the N-terminal end of peptides comprises an enzymatic activity cleaving at least leucine and glutamine from the N-terminal end of a peptide unless a proline residue is present in the second amino acid position.

54. The pharmaceutical composition of claim 52 or 53, wherein the enzymatic activity cleaving amino acids from the N-terminal end of peptides comprises an enzymatic activity cleaving at least 5, at least 10, at least 15, at least 16, at least 17, or at least 18 different amino acids from the N-terminal end of a peptide unless a proline residue is present in the second amino acid position.

55. The pharmaceutical composition of any one of claims 52 to 54, wherein the enzymatic activity cleaving amino acids from the N-terminal end of peptides is contributed by an aminopeptidase present in the composition.

56. The pharmaceutical composition of claim 55, wherein the aminopeptidase is a leucineaminopeptidase.

57. The pharmaceutical composition of claim 55 or 56, wherein the aminopeptidase comprises an amino acid sequence derived from an aminopeptidase of a fungus.

58. The pharmaceutical composition of any one of claims 55 to 57, wherein the aminopeptidase comprises an amino acid sequence derived from an aminopeptidase of Trichophyton rubrum.

59. The pharmaceutical composition of any one of claims 55 to 58, wherein the aminopeptidase comprises an amino acid sequence derived from leucine-aminopeptidase 2 of Trichophyton rubrum.

60. The pharmaceutical composition of any one of claims 55 to 59, wherein the aminopeptidase comprises the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof.

61. The pharmaceutical composition of any one of claims 52 to 60, wherein the enzymatic activity cleaving dipeptides from the N-terminal end of peptides having a proline residue in the second amino acid position comprises an enzymatic activity cleaving at least glutamineproline, phenylalanine-proline, leucine-proline, and tyrosine-proline from the N-terminal end of a peptide unless proline residues are present in the second and third amino acid positions.

62. The pharmaceutical composition of any one of claims 52 to 61, wherein the enzymatic activity cleaving dipeptides from the N-terminal end of peptides having a proline residue in the second amino acid position comprises an enzymatic activity cleaving at least 4, at least 5, at least 10, at least 15, at least 16, at least 17, or at least 18 different dipeptides having a proline residue in the second amino acid position from the N-terminal end of a peptide unless proline residues are present in the second and third amino acid positions.

63. The pharmaceutical composition of any one of claims 52 to 62, wherein the 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 di peptidyl peptidase present in the composition.

64. The pharmaceutical composition of claim 63, wherein the di peptidyl peptidase comprises an amino acid sequence derived from a di peptidyl peptidase of a fungus.

65. The pharmaceutical composition of claim 63 or 64, wherein the dipeptidyl peptidase comprises an amino acid sequence derived from a dipeptidyl peptidase of Trichophyton rubrum.

66. The pharmaceutical composition of any one of claims 63 to 65, wherein the dipeptidyl peptidase comprises an amino acid sequence derived from dipeptidyl peptidase IV of Trichophyton rubrum.

67. The pharmaceutical composition of any one of claims 63 to 66, wherein the dipeptidyl peptidase comprises the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof.

68. The pharmaceutical composition of any one of claims 51 to 67, wherein the enzymatic activities are maintained at the pH of the small intestine, in particular the upper small intestine.

69. The pharmaceutical composition of any one of claims 51 to 68, wherein the enzymatic activities are maintained at the conditions of the small intestine, in particular the upper small intestine.

70. The pharmaceutical composition of any one of claims 52 to 69, wherein the enzymatic activity cleaving amino acids from the N-terminal end of peptides and the enzymatic activity cleaving dipeptides from the N-terminal end of peptides having a proline residue in the second amino acid position in combination do not have an enzymatic activity cleaving amino acids or dipeptides from the N-terminal end of a peptide having proline residues in the second and third amino add positions.

71. The pharmaceutical composition of any one of claims 1 to 70, wherein the enzymatic activities in combination cleave the peptide LGQQQPFPPQQPY into amino acids, dipeptides, and tripeptides.

72. The pharmaceutical composition of any one of claims 1 to 71, which is for oral administration.

73. The pharmaceutical composition of any one of claims 1 to 72 for pharmaceutical use.

74. The pharmaceutical composition of any one of claims 1 to 73 for use in treating a peptide-related condition or disorder.

75. The pharmaceutical composition of claim 74, wherein the peptide-related condition or disorder is an immunogenic peptide-related condition or disorder.

76. The pharmaceutical composition of any one of claims 1 to 75 for use in treating a gluten-related condition or disorder.

77. The pharmaceutical composition of claim 76, wherein the gluten-related condition or disorder is selected from the group consisting of celiac disease, wheat allergy and non-celiac gluten sensitivity.

78. A method of treating a peptide-related condition or disorder comprising administering the pharmaceutical composition of any one of claims 1 to 72 to a mammal in need thereof.

79. The method of claim 78, wherein the peptide-related condition or disorder is an immunogenic peptide-related disorder.

80. A method of treating a gluten-related condition or disorder comprising administering the pharmaceutical composition of any one of claims 1 to 72 to a mammal in need thereof.

81. The method of claim 80, wherein the gluten-related condition or disorder is selected from the group consisting of celiac disease, wheat allergy and non-celiac gluten sensitivity.

82. The method of any one of claims 78 to 81, wherein the mammal is a human.

83. The method of any one of claims 78 to 82, wherein said pharmaceutical composition is administered orally.

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