Dipeptidyl peptidase and leucine aminopeptidase polypeptide variants

Abstract

To provide dipeptidyl peptidase IV (DPPIV) that retains activity and is resistant to at least partial degradation.SOLUTION: Disclosed is a polypeptide comprising a terminal truncated dipeptidyl peptidase IV (DPPIV) polypeptide. The polypeptide is useful in medical applications such as in the treatment of gluten-related disorders including celiac disease (CeD) and non-celiac gluten sensitivity (NCGS) as well as other diseases that may benefit from a gluten-free diet (GFD).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to polypeptides having peptidase activity that are truncated variants of dipeptidyl peptidase IV (DPPIV) and leucine aminopeptidase (LAP) polypeptides, nucleic acids such as vectors encoding them, and host cells comprising the nucleic acids described herein and, optionally, expressing the polypeptides described herein. The polypeptides and nucleic acids described herein are useful in medical applications, for example, in the treatment of gluten-related disorders, such as celiac disease (CeD) and non-celiac gluten sensitivity (NCGS), and other disorders that may benefit from a gluten-free diet (GFD). [Background technology]

[0002] Wheat, barley, and rye have been important staple foods in the human diet throughout history, but the interaction between their component gluten, a mixture of digestion-resistant immunogenic peptides, and the human body has led to an increasingly diverse range of clinical, serological, and morphological symptoms, as well as the manifestation of autoimmune reactions. Gluten consists of polymeric glutenin and monomeric alcohol-soluble gliadins, which have high immunogenic or toxic potential. Due to their high content of proline and glutamine and low content of lysine and methionine, gliadins remain largely stable against degradation by luminal proteases and intestinal brush border membrane enzymes.

[0003] Gliadin and glutenin account for 80% to 85% of gluten proteins. Gliadins are divided into α / β-, γ-, and ω-gliadins, which are monomeric proteins connected via intrachain disulfide bonds (α / β- and γ-gliadins) or not (ω-gliadins). The N-terminal domain of α-gliadins contains the proline- and glutamine-rich heptapeptide PQPQPFP and pentapeptide PQQPY, and the most characterized immunogenic fragments.

[0004] Gluten-related disorders refer to three main types of human disorders: autoimmune celiac disease, wheat allergy and non-celiac gluten sensitivity.

[0005] Celiac disease (CeD) is a chronic autoimmune disorder in which patients develop a variety of intestinal and extraintestinal symptoms, including specific neurological symptoms triggered by gluten immunogenic peptides (GIPs), which are generated by incomplete proteolysis during gluten digestion. These complex GIPs are rich in proline and glutamine and are insufficiently digested by endogenous gastric, pancreatic, and intestinal brush border proteases. GIPs function as T cell epitopes in genetically predisposed individuals (human leukocyte antigens HLA-DQ2 and / or DQ8) and induce CD4+ T cell-mediated immune responses. This leads to the generation of cytotoxic T cells and local and systemic inflammatory responses, which explain the destruction of the epithelial lining of the small intestine and extraintestinal clinical symptoms and complications.

[0006] Patients may experience diarrhea, vomiting, bloating, constipation, abdominal pain, weight loss, chronic fatigue, and various other symptoms. Secondary complications include, but are not limited to, increased risk of cancer, osteoporosis, and osteopenia, miscarriage and infertility in women, and childhood growth retardation syndrome. Approximately 40% of the population carries the HLA-DQ2 and / or HLA-DQ8 haplotype genotypes required for the development of CeD. Overall prevalence ranges from 4.5% to 0.75%.

[0007] The diagnosis of CeD is typically based on a combination of the patient's clinical history and symptom profile, serological testing, and findings from biopsies of the upper small intestine. In patients with typical symptoms, measurement of serum IgA antibodies against tissue transglutaminase (anti-tTG) is an excellent screening method with high sensitivity and specificity and is considered a primary screening test. Blood from suspected individuals can also be tested for the presence of anti-endomysial (EMA)-IgA, anti-gliadin (AGA), or deamidated gliadin-specific antibodies (DGP).

[0008] A further gluten-related complication is non-celiac gluten sensitivity (NCGS). NCGS is a syndrome characterized by intestinal and extraintestinal symptoms associated with the ingestion of gluten-containing foods in subjects who do not suffer from CeD or wheat allergy (WA). The prevalence of NCGS has not yet been clearly defined, but the prevalence has been estimated to be as high as 6% of the general population, depending on the population studied.

[0009] Clinically, in NCGS, symptoms range from intestinal disturbances (abdominal pain, diarrhea, nausea, body mass loss, bloating, and flatulence) to skin (erythema, eczema), general systemic symptoms (e.g., "foggy mind," headache, fatigue, bone and joint pain), anemia, behavior (attention disorders, depression, and hyperactivity), and chronic ulcerative stomatitis. In contrast to CeD, no specific genetic predisposition to NCGS has been identified to date, and no serological biomarkers are available for NCGS because measurement of celiac-associated antibodies is not sensitive or specific for NCGS.

[0010] In contrast to CeD, in which the adaptive immune system is activated, responses from the innate immune system appear to be upregulated in NCGS. Although the gastrointestinal tract and intestinal permeability of NCGS patients are normal, and histological lesions in their duodenal mucosa are minimal, increased infiltration of eosinophils and basophils into the duodenal lamina propria and activation of circulating basophils have been observed in NCGS patients. Studies have investigated the relationship between the consumption of gluten-containing foods and the emergence of neurological and psychiatric disorders / symptoms, such as ataxia, peripheral neuropathy, schizophrenia, autism, depression, anxiety, and hallucinations (so-called gluten psychosis), suggesting that gluten-related peptides may enter the systemic circulation and cause extraintestinal symptoms.

[0011] Therapeutic strategies are under development that aim to target various steps in disease pathogenesis or neutralize immunogenic peptides before they reach the small intestinal mucosa. Currently, the only treatment option for CeD and NCGS is a strict GFD. The goal of this treatment is strict avoidance of gluten ingestion and GIP production (the external immune trigger of gluten-related disorders). Clinical treatment goals are resolution and / or avoidance of symptoms, prevention of GIP-induced autoimmune reactions, and amelioration or avoidance of morphological and functional changes in the upper gastrointestinal (GI) tract (e.g., villous atrophy). Even very small amounts of gluten, which may be present in gluten-free products, can trigger immune responses and clinical symptoms due to non-adherence to a complete GFD. Despite best efforts to adhere to a GFD, a significant subgroup of patients remains symptomatic due to dietary errors and cross-contamination resulting in inadvertent gluten ingestion.

[0012] CeD patients become clinically, serologically, and morphologically asymptomatic after complete and strict gluten avoidance for a period of 3-4 months, indicating that persistent and complete enzymatic degradation of GIP qualifies as a treatment for CeD. Summary of the Invention

[0013] The combination of Trichophyton rubrum dipeptidyl peptidase IV (ruDPPIV) and Trichophyton rubrum leucine aminopeptidase II (ruLAPII) results in the complete degradation of GIP.

[0014] Described herein is the production of short variants of ruLAPII (ruLAPII short) and ruDPPIV (ruDPPIV short) in a P. pastoris expression platform. These variants of ruLAPII and ruDPPIV were purified and tested for enzymatic activity (U / mg purified protein) along with their full-length variants. The purified truncated variants were analyzed by mass spectrometry of the intact molecules.

[0015] It was found that the short variants of ruDPPIV exhibited similar enzymatic activity to the full-length variant. A slightly lower activity was measured for ruLAPII compared to the full-length variant: ruDPPIV_short: 12.7U / mg ·ruDPPIV total length: 12.4U / mg ruLAPII_short: 1.1U / mg ·ruLAPII total length: 1.5U / mg

[0016] Mass spectrometry studies to assess possible degradation revealed the following: for ruDPPIV-Short, the full-length product could be found, but also degradation products, in contrast to the full-length molecule ruDPPIV, for which only degradation products could be identified; and for ruLAPII-Short, no degradation products were observed, in contrast to the full-length molecule ruLAPII, for which only degradation products could be identified.

[0017] Based on the results presented, the short variants of ruDPPIV and ruLAPII represent an improvement, since the full-length molecules could be identified by mass spectrometry (only degradants were identified in the full-length molecules), and the truncated variants still showed similar enzymatic activity.

[0018] Thus, the present invention is based, at least in part, on the surprising finding that truncated ruDPPIV and ruLAPII polypeptides can be produced that retain activity and are at least partially resistant to degradation.

[0019] overview The present invention provides polypeptides having peptidase activity that are truncated variants of dipeptidyl peptidase IV (DPPIV), particularly ruDPPIV, or leucine aminopeptidase (LAP), particularly ruLAPII. ruDPPIV and ruLAPII are peptidases derived from the dermatophyte Trichophyton rubrum.

[0020] In one aspect, the present invention provides a polypeptide comprising a truncated dipeptidyl peptidase IV (DPPIV) polypeptide. In one embodiment, the truncation is an N-terminal and / or C-terminal truncation. In one embodiment, the truncated DPPIV polypeptide is a fragment of a wild-type DPPIV polypeptide.

[0021] 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 deleted at the C-terminus compared to the wild-type DPPIV polypeptide. In various embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids are deleted at the C-terminus compared to the wild-type DPPIV polypeptide. In these and other embodiments, at least 1 amino acid is deleted at the N-terminus compared to the wild-type DPPIV polypeptide. In these and other embodiments, 1 or 2 amino acids are deleted at the N-terminus compared to the wild-type DPPIV polypeptide. In one embodiment, at least 1 amino acid is deleted at the N-terminus compared to the wild-type DPPIV polypeptide and at least 14 amino acids are deleted at the C-terminus compared to the wild-type DPPIV polypeptide. In one embodiment, one amino acid is deleted at the N-terminus compared to the wild-type DPPIV polypeptide and 14 amino acids are deleted at the C-terminus compared to the wild-type DPPIV polypeptide.

[0022] In one embodiment, the truncated DPPIV polypeptide has (i) one or two amino acids deleted at the N-terminus and / or (ii) up to 15 amino acids deleted at the C-terminus compared to the wild-type DPPIV polypeptide. In one embodiment, 12 to 14 amino acids deleted at the C-terminus compared to the wild-type DPPIV polypeptide. In one embodiment, 13 or 14 amino acids deleted at the C-terminus compared to the wild-type DPPIV polypeptide.

[0023] In one embodiment, the wild-type DPPIV polypeptide has the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof.

[0024] In various embodiments, the polypeptides comprising the truncated DPPIV polypeptides described herein comprise a truncated DPPIV polypeptide consisting of the amino acid sequence represented by residues 1-745, 1-746, 1-747, 1-748, 1-749, 1-750, 1-751, 1-752, 1-753, 1-754, 1-755, 1-756, 1-757, 1-758, or 1-759 of SEQ ID NO: 1 or a variant thereof. In various embodiments, the polypeptides comprising the truncated DPPIV polypeptides described herein comprise a truncated DPPIV polypeptide consisting of the amino acid sequence represented by residues 2-745, 2-746, 2-747, 2-748, 2-749, 2-750, 2-751, 2-752, 2-753, 2-754, 2-755, 2-756, 2-757, 2-758, or 2-759 of SEQ ID NO: 1 or a variant thereof.

[0025] In a further aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) a polypeptide comprising a truncated DPPIV polypeptide consisting of the amino acid sequence represented by residues 1 to 748 of SEQ ID NO: 1; (ii) a polypeptide comprising a truncated DPPIV polypeptide consisting of the amino acid sequence represented by residues 1 to 747 of SEQ ID NO: 1; (iii) a polypeptide comprising a truncated DPPIV polypeptide consisting of the amino acid sequence represented by residues 1 to 746 of SEQ ID NO: 1; (iv) a polypeptide comprising a truncated DPPIV polypeptide consisting of the amino acid sequence represented by residues 1 to 745 of SEQ ID NO: 1; (v) a polypeptide comprising a truncated DPPIV polypeptide consisting of the amino acid sequence represented by residues 2 to 748 of SEQ ID NO: 1; (vi) a polypeptide comprising a truncated DPPIV polypeptide consisting of the amino acid sequence represented by residues 2 to 747 of SEQ ID NO: 1; (vii) a polypeptide comprising a truncated DPPIV polypeptide consisting of the amino acid sequence represented by residues 2 to 746 of SEQ ID NO: 1; and (viii) A polypeptide comprising a truncated DPPIV polypeptide consisting of the amino acid sequence represented by residues 2 to 745 of SEQ ID NO: 1. The present invention provides a polypeptide selected from the group consisting of:

[0026] In one embodiment, a polypeptide comprising a truncated DPPIV polypeptide described herein is derived from Trichophyton rubrum.

[0027] In one embodiment, a polypeptide comprising a truncated DPPIV polypeptide described herein cleaves the dipeptide motif NH2-X-Pro from the N-terminus of the polypeptide.

[0028] In one embodiment, a polypeptide comprising a truncated DPPIV polypeptide described herein is produced in Pichia pastoris.

[0029] In different embodiments, a polypeptide comprising a truncated DPPIV polypeptide described herein does not comprise the sequence of the wild-type DPPIV polypeptide from which it is derived, for example the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof, and preferably does not comprise a non-truncated sequence of the wild-type DPPIV polypeptide from which it is derived, for example the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof, in particular does not comprise a sequence of the wild-type DPPIV polypeptide from which it is derived that is truncated to a lesser extent compared to the truncated DPPIV polypeptide, for example the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof. For example, in the case of a polypeptide comprising a truncated DPPIV polypeptide in which 14 amino acids are deleted at the C-terminus compared to the wild-type DPPIV polypeptide, the polypeptide does not comprise the amino acid sequence of a non-truncated, i.e. wild-type, DPPIV polypeptide, and preferably does not comprise the amino acid sequence of a wild-type DPPIV polypeptide in which, for example, 13 or fewer amino acids are deleted at the C-terminus compared to the wild-type DPPIV polypeptide. Similarly, in the case of a polypeptide comprising a truncated DPPIV polypeptide, for example, including a truncated DPPIV polypeptide consisting of the amino acid sequence represented by residues 1 to 746 of a wild-type DPPIV polypeptide, the polypeptide does not comprise the amino acid sequence of a non-truncated, i.e., wild-type, DPPIV polypeptide, and preferably does not comprise the amino acid sequence represented by residues 1 to 747 or more of the wild-type DPPIV polypeptide. In other words, sequences that may be present at the N-terminus and / or C-terminus of a truncated DPPIV polypeptide in a polypeptide comprising a truncated DPPIV polypeptide described herein preferably do not correspond to amino acids that are truncated or deleted in the truncated DPPIV polypeptide compared to the wild-type DPPIV polypeptide. Thus, a polypeptide comprising a truncated DPPIV polypeptide described herein does not comprise the full-length sequence of the wild-type DPPIV polypeptide from which it is derived, or a continuous amino acid sequence of the full-length sequence of a wild-type DPPIV polypeptide that is longer than the amino acid sequence of the truncated DPPIV polypeptide.

[0030] In one embodiment, when a certain number of amino acids are deleted at the C-terminus compared to the wild-type DPPIV polypeptide, a polypeptide comprising a truncated DPPIV polypeptide described herein does not comprise the amino acid sequence represented by the amino acids deleted at the C-terminus compared to the wild-type DPPIV polypeptide. In various embodiments, a polypeptide comprising a truncated DPPIV polypeptide described herein does not comprise the amino acid sequence represented by residues 747-760, 748-760, 749-760, 750-760, 751-760, 752-760, 753-760, 754-760, or 755-760 of SEQ ID NO: 1 or a variant thereof.

[0031] Polypeptides, including the truncated DPPIV polypeptides described herein, retain the activity of the wild-type DPPIV polypeptide from which they are derived, and polypeptides, including the truncated DPPIV polypeptides described herein, retain at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the activity of the wild-type DPPIV polypeptide from which they are derived.

[0032] In a further aspect, the present invention provides a polypeptide consisting of a truncated DPPIV polypeptide as described herein, optionally fused to a signal peptide, for example a signal peptide useful for secretory expression in Pichia pastoris, wherein the signal peptide is preferably fused to the N-terminus of the truncated DPPIV polypeptide.

[0033] In one embodiment, the polypeptide comprising and / or the truncated DPPIV polypeptide consists of the amino acid sequence shown in SEQ ID NO:3.

[0034] In a further aspect, the present invention provides a polypeptide comprising a truncated leucine aminopeptidase (LAP) polypeptide. In one embodiment, the truncation is an N-terminal and / or C-terminal truncation. In one embodiment, the truncated LAP polypeptide is a fragment of a wild-type LAP polypeptide.

[0035] 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 deleted at the C-terminus compared to the wild-type 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 deleted at the C-terminus compared to the wild-type 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 deleted at the N-terminus compared to the wild-type LAP polypeptide. In these and other embodiments, 1, 2, 3, 4, 5, 6, or 7 amino acids are deleted at the N-terminus compared to the wild-type LAP polypeptide. In one embodiment, at least 6 amino acids are deleted at the N-terminus compared to the wild-type LAP polypeptide and at least 22 amino acids are deleted at the C-terminus compared to the wild-type LAP polypeptide. In one embodiment, 6 amino acids are deleted at the N-terminus compared to the wild-type LAP polypeptide and 22 amino acids are deleted at the C-terminus compared to the wild-type LAP polypeptide.

[0036] In one embodiment, the truncated LAP polypeptide has (i) up to 7 amino acids deleted at the N-terminus and / or (ii) up to 23 amino acids deleted at the C-terminus compared to the wild-type LAP polypeptide. In one embodiment, 4 to 6 amino acids deleted at the N-terminus compared to the wild-type LAP polypeptide. In one embodiment, 6 amino acids deleted at the N-terminus compared to the wild-type LAP polypeptide. In one embodiment, between 20 and 22 amino acids deleted at the C-terminus compared to the wild-type LAP polypeptide. In one embodiment, 8, 16, 21, or 22 amino acids deleted at the C-terminus compared to the wild-type LAP polypeptide. In one embodiment, 22 amino acids deleted at the C-terminus compared to the wild-type LAP polypeptide.

[0037] In one embodiment, the wild-type LAP polypeptide has the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof.

[0038] In various embodiments, polypeptides comprising a truncated LAP polypeptide described herein include a truncated LAP polypeptide consisting of the amino acid sequence represented by residues 1-456, 1-457, 1-458, 1-459, 1-460, 1-461, 1-462, 1-463, 1-464, 1-465, 1-466, 1-467, 1-468, 1-469, 1-470, 1-471, 1-472, 1-473, 1-474, 1-475, 1-476, 1-477, or 1-478 of SEQ ID NO:2 or a variant thereof. In various embodiments, polypeptides comprising a truncated LAP polypeptide described herein include a truncated LAP polypeptide consisting of an amino acid sequence represented by residues 2-456, 2-457, 2-458, 2-459, 2-460, 2-461, 2-462, 2-463, 2-464, 2-465, 2-466, 2-467, 2-468, 2-469, 2-470, 2-471, 2-472, 2-473, 2-474, 2-475, 2-476, 2-477, or 2-478 of SEQ ID NO: 2 or a variant thereof. In various embodiments, polypeptides comprising a truncated LAP polypeptide described herein include a truncated LAP polypeptide consisting of an amino acid sequence represented by residues 3-456, 3-457, 3-458, 3-459, 3-460, 3-461, 3-462, 3-463, 3-464, 3-465, 3-466, 3-467, 3-468, 3-469, 3-470, 3-471, 3-472, 3-473, 3-474, 3-475, 3-476, 3-477, or 3-478 of SEQ ID NO: 2 or a variant thereof. In various embodiments, polypeptides comprising a truncated LAP polypeptide described herein include a truncated LAP polypeptide consisting of an amino acid sequence represented by residues 4-456, 4-457, 4-458, 4-459, 4-460, 4-461, 4-462, 4-463, 4-464, 4-465, 4-466, 4-467, 4-468, 4-469, 4-470, 4-471, 4-472, 4-473, 4-474, 4-475, 4-476, 4-477, or 4-478 of SEQ ID NO: 2 or a variant thereof.In various embodiments, polypeptides comprising a truncated LAP polypeptide described herein include a truncated LAP polypeptide consisting of an amino acid sequence represented by residues 5-456, 5-457, 5-458, 5-459, 5-460, 5-461, 5-462, 5-463, 5-464, 5-465, 5-466, 5-467, 5-468, 5-469, 5-470, 5-471, 5-472, 5-473, 5-474, 5-475, 5-476, 5-477, or 5-478 of SEQ ID NO: 2 or a variant thereof. In various embodiments, polypeptides comprising a truncated LAP polypeptide described herein include a truncated LAP polypeptide consisting of an amino acid sequence represented by residues 6-456, 6-457, 6-458, 6-459, 6-460, 6-461, 6-462, 6-463, 6-464, 6-465, 6-466, 6-467, 6-468, 6-469, 6-470, 6-471, 6-472, 6-473, 6-474, 6-475, 6-476, 6-477, or 6-478 of SEQ ID NO: 2 or a variant thereof. In various embodiments, polypeptides comprising a truncated LAP polypeptide described herein include a truncated LAP polypeptide consisting of an amino acid sequence represented by residues 7-456, 7-457, 7-458, 7-459, 7-460, 7-461, 7-462, 7-463, 7-464, 7-465, 7-466, 7-467, 7-468, 7-469, 7-470, 7-471, 7-472, 7-473, 7-474, 7-475, 7-476, 7-477, or 7-478 of SEQ ID NO: 2 or a variant thereof. In various embodiments, polypeptides comprising a truncated LAP polypeptide described herein include a truncated LAP polypeptide consisting of an amino acid sequence represented by residues 8-456, 8-457, 8-458, 8-459, 8-460, 8-461, 8-462, 8-463, 8-464, 8-465, 8-466, 8-467, 8-468, 8-469, 8-470, 8-471, 8-472, 8-473, 8-474, 8-475, 8-476, 8-477, or 8-478 of SEQ ID NO: 2 or a variant thereof.

[0039] In a further aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) a polypeptide comprising a truncated LAP polypeptide consisting of the amino acid sequence represented by residues 7 to 471 of SEQ ID NO: 2; (ii) a polypeptide comprising a truncated LAP polypeptide consisting of the amino acid sequence represented by residues 7 to 463 of SEQ ID NO: 2; (iii) a polypeptide comprising a truncated LAP polypeptide consisting of the amino acid sequence represented by residues 7 to 458 of SEQ ID NO: 2; (iv) a polypeptide comprising a truncated LAP polypeptide consisting of the amino acid sequence represented by residues 7 to 457 of SEQ ID NO: 2, and (v) a polypeptide comprising a truncated LAP polypeptide consisting of the amino acid sequence represented by residues 7 to 456 of SEQ ID NO: 2 The present invention provides a polypeptide selected from the group consisting of:

[0040] In one embodiment, a polypeptide comprising a truncated LAP polypeptide described herein is derived from Trichophyton rubrum.

[0041] In one embodiment, a polypeptide comprising a truncated LAP polypeptide described herein has a single amino acid truncated from the N-terminus of the polypeptide, except where the single amino acid is connected to a proline by the sequence NH2-X-Pro.

[0042] In one embodiment, a polypeptide comprising a truncated LAP polypeptide described herein is produced in Pichia pastoris.

[0043] In different embodiments, a polypeptide comprising a truncated LAP polypeptide described herein does not comprise the sequence of the wild-type LAP polypeptide from which it is derived, e.g., the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof, and preferably does not comprise a non-truncated sequence of the wild-type LAP polypeptide from which it is derived, e.g., the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof, in particular does not comprise a sequence of the wild-type LAP polypeptide from which it is derived that is less truncated than the truncated LAP polypeptide, e.g., the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof. For example, in the case of a polypeptide comprising a truncated LAP polypeptide having 22 amino acids deleted at the C-terminus compared to the wild-type LAP polypeptide, the polypeptide does not comprise the amino acid sequence of a non-truncated, i.e., wild-type, LAP polypeptide, and preferably does not comprise the amino acid sequence of a wild-type LAP polypeptide having, e.g., 21 or fewer amino acids deleted at the C-terminus compared to the wild-type LAP polypeptide. Similarly, in the case of a polypeptide comprising a truncated LAP polypeptide, for example, a truncated LAP polypeptide consisting of the amino acid sequence represented by residues 1-457 of a wild-type LAP polypeptide, the polypeptide does not comprise the amino acid sequence of a non-truncated, i.e., wild-type, LAP polypeptide, and preferably does not comprise the amino acid sequence represented by residues 1-458 or more of a wild-type LAP polypeptide. Similarly, in the case of a polypeptide comprising a truncated LAP polypeptide, for example, a truncated LAP polypeptide consisting of the amino acid sequence represented by residues 7-457 of a wild-type LAP polypeptide, the polypeptide does not comprise the amino acid sequence of a non-truncated, i.e., wild-type, LAP polypeptide, and preferably does not comprise the amino acid sequence represented by residues 6-457, 7-458, or 6-458 or more of a wild-type LAP polypeptide.In other words, sequences that may be present at the N-terminus and / or C-terminus of the truncated LAP polypeptide in a polypeptide comprising a truncated LAP polypeptide described herein preferably do not correspond to amino acids that are truncated or deleted in the truncated LAP polypeptide compared to the wild-type LAP polypeptide. Thus, a polypeptide comprising a truncated LAP polypeptide described herein does not contain the full-length sequence of the wild-type LAP polypeptide from which it is derived, or a longer contiguous amino acid sequence of the full-length sequence of the wild-type LAP polypeptide than the amino acid sequence of the truncated LAP polypeptide.

[0044] In one embodiment, where a particular number of amino acids are deleted at the N-terminus and / or C-terminus compared to a wild-type LAP polypeptide, a polypeptide comprising a truncated LAP polypeptide described herein does not comprise the amino acid sequence represented by the amino acids deleted at the N-terminus and / or C-terminus compared to a wild-type LAP polypeptide. In various embodiments, polypeptides comprising the truncated LAP polypeptides described herein do not comprise the amino acid sequence represented by residues 1-6 of SEQ ID NO:2 or a variant thereof, and / or do not comprise the amino acid sequence represented by residues 458-479, 459-479, 460-479, 461-479, 462-479, 463-479, 464-479, 465-479, 466-479, 467-479, 468-479, 469-479, 470-479, 471-479, 472-479, 473-479, or 474-479 of SEQ ID NO:2 or a variant thereof.

[0045] Polypeptides, including truncated LAP polypeptides described herein, retain the activity of the wild-type LAP polypeptide from which they are derived, and polypeptides, including truncated LAP polypeptides described herein, retain at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the activity of the wild-type LAP polypeptide from which they are derived.

[0046] In a further aspect, the present invention provides a polypeptide consisting of a truncated LAP polypeptide described herein, optionally fused to a signal peptide, for example a signal peptide useful for secretory expression in Pichia pastoris, wherein the signal peptide is preferably fused to the N-terminus of the truncated LAP polypeptide.

[0047] In one embodiment, the polypeptide comprising and / or the truncated LAP polypeptide consists of the amino acid sequence set forth in SEQ ID NO:4.

[0048] In a further aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) a polypeptide comprising a truncated DPPIV polypeptide as described herein; (ii) a polypeptide comprising a truncated LAP polypeptide described herein, or (iii) A combination of (i) and (ii) A composition comprising:

[0049] In a further aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) a polypeptide comprising a truncated DPPIV polypeptide described herein, and (ii) a polypeptide comprising a truncated LAP polypeptide described herein A composition comprising:

[0050] In one embodiment, in the compositions described herein, the truncated DPPIV polypeptides described herein constitute at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the DPPIV polypeptides in the composition. In one embodiment, in the compositions described herein, the truncated LAP polypeptides described herein constitute at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the LAP polypeptides in the composition. For example, in the compositions described herein, the truncated DPPIV polypeptides described herein constitute at least 70% of the DPPIV polypeptides in the composition, and the truncated LAP polypeptides described herein constitute at least 70% of the LAP polypeptides in the composition. For example, in the compositions described herein, the truncated DPPIV polypeptides described herein constitute at least 80% of the DPPIV polypeptides in the composition, and the truncated LAP polypeptides described herein constitute at least 80% of the LAP polypeptides in the composition. For example, in the compositions described herein, the truncated DPPIV polypeptides described herein constitute at least 90% of the DPPIV polypeptides in the composition, and the truncated LAP polypeptides described herein constitute at least 90% of the LAP polypeptides in the composition. For example, in the compositions described herein, the truncated DPPIV polypeptides described herein constitute at least 95% of the DPPIV polypeptides in the composition, and the truncated LAP polypeptides described herein constitute at least 95% of the DPPIV polypeptides in the composition. For example, in the compositions described herein, the truncated DPPIV polypeptides described herein comprise at least 98% of the DPPIV polypeptides in the composition, and the truncated LAP polypeptides described herein comprise at least 98% of the LAP polypeptides in the composition.

[0051] In one embodiment, the weight ratio of a polypeptide comprising a truncated DPPIV polypeptide described herein to a polypeptide comprising a truncated LAP polypeptide described herein is between 1:20 and 1:5, preferably between 1:15 and 1:7.5, more preferably about 1:9.5.

[0052] In one embodiment, the compositions described herein include, for example, the following alpha-gliadin 33-mer peptide: LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF Completely disassemble.

[0053] In one embodiment, the compositions described herein are pharmaceutical compositions.

[0054] In a further aspect, the present invention provides a composition as described herein for pharmaceutical use.

[0055] In a further aspect, the present invention provides a composition as described herein for use in treating a gluten-related disorder.

[0056] In one embodiment, the compositions described herein are oral compositions, particularly liquid oral compositions.

[0057] In a further aspect, the present invention provides a nucleic acid encoding a polypeptide comprising a truncated DPPIV polypeptide as described herein. In a further aspect, the present invention provides a cell transfected with said nucleic acid.

[0058] In a further aspect, the present invention provides a nucleic acid encoding a polypeptide comprising a truncated LAP polypeptide described herein, hi a further aspect, the present invention provides a cell transfected with said nucleic acid.

[0059] In a further aspect, the present invention provides the polypeptides and compositions described herein for pharmaceutical use. In one embodiment, the pharmaceutical use comprises the therapeutic or prophylactic treatment of a gluten-related disorder in a subject.

[0060] In a further aspect, the present invention provides a method of treating or preventing a gluten-related disorder in a subject, comprising administering to the subject a polypeptide or composition described herein.

[0061] In one aspect, the invention relates to a polypeptide or composition described herein for use in a method described herein. [Brief explanation of the drawings]

[0062] [Figure 1] Overview of the P. pastoris AOX1 screening. Glucose release is achieved by enzymatic digestion of polysaccharides (EnPump200 system, EnPresso GmbH). The promoter is induced by limiting methanol concentration. [Figure 2] ruLAPII_Supply strategy for short. [Figure 3] Supply strategy for ruDPPIV_short. [Figure 4]Coomassie staining of culture supernatant isolated from a clone with the LP1(pFJP6015, ruDPPIV_short) strain background. 7.5 μL of culture supernatant was loaded onto a 12% Bis-Tris SDS gel, 26 wells, in MES buffer under reducing conditions. A) SDS PAGE / Coomassie staining. The black arrow indicates the position of the ruDPPIV product, and the blue arrow indicates the position of the higher molecular weight variant of ruDPPIV. B) Loading scheme. Black: clone selected for subsequent high-cell-density fermentation; green: reference strain. [Figure 5] Coomassie staining of culture supernatant isolated from a clone with the LP1(pFJP6014, ruLAPII_short) strain background. 7.5 μL of culture supernatant was loaded onto a 12% Bis-Tris SDS gel, 26 wells, in MES buffer under reducing conditions. A) SDS PAGE / Coomassie staining. The black arrow indicates the position of the ruLAPII product; B) Loading scheme. Black: clone selected for subsequent high-cell-density fermentation; green: reference strain. [Figure 6] Biomass production of clones with the LP1(pFJP6015,ruDPPIV_short) strain background: LP1(pFJP6015.8), LP1(pFJP6015.6), LP1(pFJP6015.18), LP1(pFJP6015.12), LP1(pFJP6015.15), LP1(pCKP6003.1). A) Optical density OD600; B) Dry cell weight DCW. [Figure 7] Enzyme activity of clones with the LP1(pFJP6015, ruDPPIV_short) strain background: LP1(pFJP6015.8), LP1(pFJP6015.6), LP1(pFJP6015.18), LP1(pFJP6015.12), LP1(pFJP6015.15), and LP1(pCKP6003.1). Activity measurements (AMC assay) were performed according to the SOP provided by AMYRA. [Figure 8]Biomass production of clones with the LP1(pFJP6014,ruLAPII_short) strain background: LP1(pFJP6014.13), LP1(pFJP6014.20), LP1(pFJP6014.6), LP1(pFJP6014.12), LP1(pFJP6014.15), LP1(pCKP6011.4). A) Optical density OD; B) Dry cell weight DCW. [Figure 9] Enzyme activity of clones with the LP1(pFJP6014,ruLAPII_short) strain background: LP1(pFJP6014.13), LP1(pFJP6014.20), LP1(pFJP6014.6), LP1(pFJP6014.12), LP1(pFJP6014.15), LP1(pCKP6011.4). Activity measurements (AMC assay) were performed according to the SOP provided by AMYRA. [Figure 10] Coomassie staining of purified material isolated from a clone with the LP1(pFJP6015, ruDPPIV_short) strain background. 7.5 μL of culture supernatant was loaded onto a 12% Bis-Tris SDS gel, 26 wells, in MES buffer under reducing conditions. A) SDS PAGE / Coomassie staining. The black arrow indicates the position of the ruDPPIV product, and the blue arrow indicates the position of a higher molecular weight variant of ruDPPIV. B) Loading scheme. [Figure 11] Coomassie staining of purified material isolated from a clone with the LP1(pFJP6014.13, ruLAPII_short) strain background. 7.5 μL of culture supernatant was loaded onto a 12% Bis-Tris SDS gel, 26 wells, in MES buffer under reducing conditions. A) SDS PAGE / Coomassie staining. The black arrow indicates the position of the ruLAPII product, and the blue arrow indicates the position of the molecular weight variant of ruLAPII. B) Loading scheme. [Figure 12]Mass spectrometry (Maldi-TOF) of ruDPPIV. 40 μL of the PNGase-digested elution sample was analyzed by Maldi-TOF (see Example 1, section 7.10). A) ruDPPIV_short; expected mass: 84,802; B) ruDPPIV; expected mass: 86,486. [Figure 13] Mass spectrometry of ruLAPII. 40 μL of PNGase-digested eluted sample was analyzed by Maldi-TOF (see Example 1, section 7.10). A) ruLAPII_short; expected mass: 48,696; B) ruLAPII; expected mass: 51,714. Numbers in red indicate ESI data. [Figure 14] Partial degradation of the 33-mer by ruLAPII (A) / incomplete degradation of the 33-mer by ruDPPIV (B). [Figure 15] Synergistic mechanism of action of ruLAPII and ruDPPIV DETAILED DESCRIPTION OF THE INVENTION

[0063] Although the present disclosure is described in detail below, it should be understood that the present disclosure is not limited to the specific methodology, protocols, and reagents described herein, which may vary. It should also 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 is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0064] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)," H.G. W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0065] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques as described in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0066] The elements of the present disclosure are described below. While these elements are listed with specific embodiments, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The various described examples and embodiments should not be construed as limiting the disclosure to only the explicitly described embodiments. This description should be understood to disclose and encompass embodiments combining the explicitly described embodiments with any number of the disclosed elements. Furthermore, any permutation and combination of all described elements should be considered to be disclosed by this description unless the context indicates otherwise.

[0067] The term "about," in the context of numerical values ​​or ranges described herein, means approximately or near, and in one embodiment, means ±20%, ±10%, ±5%, or ±3% of the recited or claimed numerical value or range.

[0068] As used in the context of describing this disclosure (particularly in the context of the claims), the terms "a," "an," "the," and similar references should be construed to encompass both the singular and the plural unless otherwise indicated herein or clearly contradicted by context. The 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 separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any examples or exemplary language (e.g., "such as") provided herein is intended merely to better describe the disclosure and does not limit the scope of the claims. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0069] Unless otherwise specified, the term "comprising" is used in the context of this document to indicate that in addition to the members of the list introduced by "comprising," further members may optionally be present. However, for certain embodiments of the present disclosure, the term "comprising" is intended to encompass the possibility that further members are not present, i.e., for the purposes of this embodiment, "comprising" should be understood to have the meaning of "consisting of" or "consisting essentially of."

[0070] Throughout the text of this specification, several documents are cited. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such disclosure.

[0071] definition The following provides definitions that apply to all aspects of this disclosure. The following terms have the following meanings unless otherwise indicated: Any undefined terms have their art-recognized meanings.

[0072] Terms such as "reduce," "reduce," "inhibit," or "impair," as used herein, relate to an overall reduction in levels, or the ability to cause an overall reduction, preferably by at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or even more. These terms include complete or essentially complete inhibition, i.e., a reduction to zero or essentially zero.

[0073] Terms such as "increase," "enhance," or "exceed" preferably relate to an increase or enhancement of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or even more.

[0074] As used herein, the terms "peptidase," "protease," "proteolytic enzyme," and "peptide hydrolase" are synonymous and may be used interchangeably. Peptidases include all enzymes that catalyze the cleavage of peptide bonds (CO-NH) in proteins or peptides, digesting these proteins or peptides into peptides or free amino acids. Exopeptidases act near the ends of polypeptide chains, at the amino (N) or carboxy (C) termini. Those acting at the free N-terminus liberate a single amino acid residue and are called aminopeptidases.

[0075] Dipeptidyl peptidase IV or dipeptidyl peptidase 4 (abbreviated as DPPIV or DPP4) is a serine exopeptidase that cleaves X-proline or X-alanine dipeptides from the N-terminus of polypeptides.

[0076] Leucine aminopeptidase (LAP) is an enzyme that preferentially catalyzes the hydrolysis of leucine residues at the N-terminus of peptides and proteins, but is also capable of cleaving other N-terminal residues.

[0077] Trichophyton rubrum dipeptidyl peptidase IV (ruDPPIV) and Trichophyton rubrum leucine aminopeptidase II (ruLAPII) are preferred embodiments of the DPPIV and LAP polypeptides, respectively. The leucine aminopeptidase ruLAPII cleaves single amino acids from the N-terminus of polypeptides, except when the single amino acid is connected to proline by the NH2-X-Pro sequence. The dipeptidyl peptidase ruDPPIV selectively cleaves the dipeptide motif NH2-X-Pro from the N-terminus of polypeptides. Therefore, simultaneous application of both enzymes can be used to degrade proline-rich, digestion-resistant GIP.

[0078] Neither ruDPPIV nor ruLAPII can completely degrade the α-gliadin 33-mer (a highly immunogenic peptide derived from gluten), which represents the well-described immunoreactive GIP and serves as a recognized model peptide. The α-gliadin 33-mer contains multiple copies of two of the most potent celiac disease-associated T cell epitopes and is described as highly resistant to degradation. While ruLAPII can remove the first three amino acids from the N-terminus, degradation cannot proceed beyond that point due to the subsequent QP motif (see Figure 14A). On the other hand, ruDPPIV specifically cleaves the X-Pro dipeptide motif but does not cleave the amino acids present at the N-terminus of the 33-mer, leaving the 33-mer unaffected (see Figure 14B). However, when present in combination, ruDPPIV and ruLAPII can completely degrade the gliadin 33-mer (Figure 15). For complete degradation, ruDPPIV and ruLAPII must act synergistically.

[0079] The ruDPPIV 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.

[0080] The ruLAPII polypeptide may have an amino acid sequence comprising the 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.

[0081] The polypeptides described herein include truncated dipeptidyl peptidase IV (DPPIV) polypeptides, such as truncated ruDPPIV, or truncated leucine aminopeptidase (LAP) polypeptides, such as truncated ruLAPII. The truncated DPPIV polypeptides, such as truncated ruDPPIV, or truncated leucine aminopeptidase (LAP) polypeptides, such as truncated ruLAPII, may be part of a chimeric or fusion protein.

[0082] A "chimeric protein" or "fusion protein" comprises a truncated DPPIV polypeptide, e.g., truncated ruDPPIV, or a truncated leucine aminopeptidase (LAP) polypeptide, e.g., truncated ruLAPII, linked at the N-terminus and / or C-terminus to one or more other amino acid sequences.

[0083] The term "one or more other amino acid sequences" refers to, in the case of a truncated DPPIV polypeptide, e.g., truncated ruDPPIV, an amino acid sequence corresponding to a protein that is not substantially homologous to the DPPIV polypeptide, e.g., ruDPPIV. Within the fusion protein, the truncated DPPIV polypeptide, e.g., truncated ruDPPIV, and one or more other amino acid sequences are fused to each other. One or more other amino acid sequences may be fused to the N-terminus and / or C-terminus of the truncated DPPIV polypeptide, e.g., truncated ruDPPIV. However, fusing one or more other amino acid sequences to a truncated DPPIV polypeptide, e.g., truncated ruDPPIV, does not result in the addition of an amino acid sequence present in the complete, i.e., non-truncated, DPPIV polypeptide, e.g., ruDPPIV, to the truncated DPPIV polypeptide, e.g., truncated ruDPPIV.

[0084] The term "one or more other amino acid sequences," in the case of a truncated leucine aminopeptidase (LAP) polypeptide, e.g., truncated ruLAPII, refers to amino acid sequences corresponding to proteins that are not substantially homologous to the leucine aminopeptidase (LAP) polypeptide, e.g., ruLAPII. Within a fusion protein, the truncated leucine aminopeptidase (LAP) polypeptide, e.g., truncated ruLAPII, and one or more other amino acid sequences are fused to each other. The one or more other amino acid sequences may be fused to the N-terminus and / or C-terminus of the truncated leucine aminopeptidase (LAP) polypeptide, e.g., truncated ruLAPII. However, the fusion of one or more other amino acid sequences to a truncated leucine aminopeptidase (LAP) polypeptide, such as truncated ruLAPII, does not result in the addition of an amino acid sequence present in the intact, i.e., non-truncated, leucine aminopeptidase (LAP) polypeptide, such as ruLAPII, to the truncated leucine aminopeptidase (LAP) polypeptide, such as truncated ruLAPII.

[0085] In one embodiment, the one or more other amino acid sequences comprises a signal sequence, eg, a heterologous signal sequence, fused to the truncated polypeptide, eg, to the N-terminus of the truncated polypeptide.

[0086] According to the present disclosure, the term "peptide" includes oligopeptides and polypeptides and refers to a substance comprising about 2 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 together via peptide bonds. The terms "protein" or "polypeptide" refer to large peptides, particularly peptides having more than about 150 amino acids, although the terms "peptide," "protein," and "polypeptide" are generally used synonymously herein.

[0087] In relation to an amino acid sequence (peptide or protein), a "fragment" refers to a portion of the amino acid sequence, i.e., a sequence representing an amino acid sequence truncated at the N-terminus and / or C-terminus. A C-terminally truncated fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. An N-terminally truncated fragment (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end of the open reading frame (as long as the truncated open reading frame contains the initiation codon responsible for initiating translation). A fragment of an amino acid sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues from the amino acid sequence. A fragment of an amino acid sequence preferably comprises at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from the amino acid sequence.

[0088] As used herein, "variant" refers to an amino acid sequence that differs from a parent amino acid sequence by at least one amino acid modification. The parent amino acid sequence may be a naturally occurring or wild-type (WT) amino acid sequence, or may be a modified version of the wild-type amino acid sequence. Preferably, the amino acid sequence of a variant has at least one amino acid modification compared to the parent amino acid sequence, for example, 1 to about 20 amino acid modifications compared to the parent, preferably 1 to about 10 or 1 to about 5 amino acid modifications.

[0089] "Wild-type" or "WT" or "native" herein refers to an amino acid sequence found in nature, including allelic variations. A wild-type amino acid sequence, peptide, or protein has an amino acid sequence that has not been intentionally modified.

[0090] For the purposes of this disclosure, a "variant" of an amino acid sequence (peptide, protein, or polypeptide) includes 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, particularly those that occur naturally. The term "variant" particularly includes fragments of an amino acid sequence.

[0091] Amino acid insertion variants include the insertion of a single or two or more amino acids in a specific amino acid sequence. In amino acid sequence variants with insertions, one or more amino acid residues are inserted at specific sites in the amino acid sequence, although random insertion is also possible with appropriate screening of the resulting products. Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, for example, 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, for example, the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion may occur at any position in the protein. Amino acid deletion variants containing deletions at the N-terminus and / or C-terminus of the protein are also called N- and / or C-terminal truncation variants. Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Modifications at positions in the amino acid sequence that are not conserved between homologous proteins or peptides and / or that substitute amino acids with other amino acids with similar properties are preferred. Preferably, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve the substitution of one member of a family of amino acids that are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (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 collectively classified as aromatic amino acids. In one embodiment, conservative amino acid substitutions include substitutions within the following groups: Glycine, Alanine, Valine, isoleucine, leucine, Aspartic acid, glutamic acid, Asparagine, glutamine, Serine, threonine, Lysine, arginine, and Phenylalanine, tyrosine.

[0092] Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of said given amino acid sequence is 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 degree of similarity or identity is preferably given over an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, 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 amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably given for at least about 20, at least about 40, at least about 60, at least about 80, 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, consecutive amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using best sequence alignment, for example, using Align with standard settings, preferably EMBOSS:needle, matrix: Blosum62, gap open 10.0, gap extend 0.5.

[0093] "Sequence similarity" indicates the percentage of amino acids that are identical or 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.

[0094] The terms "% identical", "% identity" or similar terms are intended to refer in particular to the percentage of nucleotides or amino acids that are identical in optimal alignment between the sequences being compared. The percentage is purely statistical; the differences between the two sequences may, but are not necessarily, randomly distributed over the entire length of the sequences being compared. Comparison of two sequences is usually performed by comparing the sequences after optimal alignment over a segment or "comparison window" to identify local regions of corresponding sequences. Optimal alignment for comparison may be performed manually, or using the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, or the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, or the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 88, 2444, or using computer programs employing said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, available at the National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq).In some embodiments, the algorithm parameters used for the BLASTN algorithm at the NCBI website include: (i) setting the Expect Threshold to 10; (ii) setting the word size to 28; (iii) setting the maximum matches in the query range to 0; (iv) setting the match / mismatch score to 1, -2; (v) setting the gap cost to linear; and (vi) using a filter for low complexity regions. In some embodiments, the algorithm parameters used for the BLASTP algorithm at the NCBI website include: (i) setting the Expect Threshold to 10; (ii) setting the word size to 3; (iii) setting the maximum matches in the query range to 0; (iv) setting the matrix to BLOSUM62; (v) setting the gap costs to 11 for presence and 1 for extension; and (vi) using a conditional compositional score matrix adjustment.

[0095] The percentage identity is obtained by determining the number of corresponding identical positions in the compared sequences, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100.

[0096] In some embodiments, the degree of similarity or identity is provided over a region that 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 nucleic acid sequence consists of 200 nucleotides, the degree of identity is provided over at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments, consecutive nucleotides. In some embodiments, the degree of similarity or identity is provided over the entire length of the reference sequence.

[0097] According to the present disclosure, homologous amino acid sequences exhibit at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably at least 95%, at least 98%, or at least 99% identity of the amino acid residues.

[0098] According to the present invention, the term "truncated" in reference to a particular peptide or polypeptide refers to a peptide or polypeptide in which one or more amino acids have been deleted at the N-terminus and / or C-terminus of the parent peptide or polypeptide, e.g., the wild-type peptide or polypeptide. According to the present invention, a truncated form or variant of an amino acid sequence is a fragment of said amino acid sequence.

[0099] The amino acid sequence variants described herein can be readily prepared by one of skill in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to prepare peptides or proteins with substitutions, additions, insertions, or deletions is described in detail, for example, in Sambrook et al. (1989). Furthermore, the peptides and amino acid variants described herein can be readily prepared using known peptide synthesis techniques, for example, by solid-phase synthesis and similar methods.

[0100] In one embodiment, a fragment or variant of an amino acid sequence (peptide or protein) is preferably a "functional fragment" or "functional variant." The term "functional fragment" or "functional variant" of an amino acid sequence relates to any fragment or variant that exhibits 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 a peptidase, such as a DPPIV or LAP sequence, one particular function is one or more peptidase activities exhibited by the amino acid sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant," as used herein, particularly refers to a variant molecule or sequence that comprises an amino acid sequence in which one or more amino acids have been altered compared to the amino acid sequence of the parent molecule or sequence, and that is still capable of performing one or more of the functions of the parent molecule or sequence, such as peptidase activity. In one embodiment, the alterations 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, for example the peptidase activity of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence, however in other embodiments the peptidase activity of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0101] An amino acid sequence (peptide, protein, or polypeptide) "derived from" a specified amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the initial amino acid sequence. Preferably, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to the particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of the particular sequence or a fragment thereof. For example, it will be understood by those skilled in the art that sequences suitable for use herein can be altered to differ in sequence from the naturally occurring or native sequence from which they are derived while maintaining the desired activity of the native sequence.

[0102] As used herein, "instructional material" or "instructions" includes publications, records, diagrams, or any other medium of expression that can be used to communicate the utility of the compositions and methods of the invention. The instructional material of the kits of the invention may, for example, be attached to a container containing the composition of the invention or may be shipped together with a container containing 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.

[0103] The polypeptides and nucleic acids described herein may be isolated and / or recombinant molecules.

[0104] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as a host cell.

[0105] The term "recombinant" in the context of the present invention means "produced by genetic engineering." Preferably, a "recombinant entity," such as a recombinant nucleic acid, in the context of the present invention is not naturally occurring.

[0106] 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 natural source and has not been intentionally modified by humans in the laboratory is naturally occurring.

[0107] The term "genetic modification" or simply "modification" includes transfection of cells with nucleic acids. The term "transfection" refers to the introduction of nucleic acids into cells. For purposes of the present invention, the term "transfection" also includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by the cell. According to the present invention, cells for transfection of nucleic acids described herein can be in vitro. According to the present invention, transfection can be transient or stable. For some applications of transfection, it is sufficient that the transfected genetic material is only transiently expressed. RNA can be transfected into cells to transiently express its encoded protein. Because nucleic acids introduced during the transfection process are not usually integrated into the nuclear genome, the foreign nucleic acid is diluted or degraded through mitosis. Cells that allow episomal amplification of nucleic acids significantly reduce the dilution rate. If it is desired that the transfected nucleic acid actually remain in the genome of the cell and its daughter cells, stable transfection must occur. Such stable transfection can be achieved by using viral-based or transposon-based systems for transfection.

[0108] nucleic acid The terms "polynucleotide" or "nucleic acid," as used herein, are intended to include DNA and RNA, e.g., genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. Nucleic acids may be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA.

[0109] In one embodiment, the nucleic acids described herein may have modified and / or non-naturally occurring nucleosides.

[0110] The nucleic acid may be contained in a vector. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked, and includes any vector known to those skilled in the art, such as a plasmid vector, a cosmid vector, a phage vector, such as lambda phage, a viral vector, such as a retroviral, adenoviral, or baculoviral vector, or an artificial chromosome vector, such as a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), or a P1 artificial chromosome (PAC). Such vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and generally contain a desired coding sequence and appropriate DNA sequences required for expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are generally used to manipulate and amplify specific desired DNA fragments and may lack functional sequences required for expression of the desired DNA fragment.

[0111] A "plasmid" refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. A "viral vector" is a vector into which additional DNA segments can be ligated into the viral genome.

[0112] 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.

[0113] "Expression vectors" are capable of directing the expression of genes to which they are operatively linked. In general, expression vectors of use in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of expression vectors that serve equivalent functions, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses).

[0114] The production of functional proteins is closely linked to the cellular machinery of the organism that produces them. Escherichia coli (E. coli) is typically the "factory" of choice for the expression of many proteins because its genome has been completely mapped, the organism is easy to handle, grows rapidly, requires inexpensive and easy-to-prepare media for growth, and secretes proteins into the media for easy protein recovery. However, E. coli is a prokaryotic organism and lacks the intracellular organelles present in eukaryotes, such as the endoplasmic reticulum and Golgi apparatus, which contain enzymes that modify the proteins being produced. Many eukaryotic proteins can be produced in E. coli, but because glycosylation or post-translational modifications do not occur, they may be produced in a non-functional, untranslated form.

[0115] Therefore, eukaryotic yeast, mammalian, and plant expression systems are frequently used for protein production. For example, the methylotrophic yeast P. pastoris has become a powerful host for heterologous protein expression and has been established as an alternative eukaryotic host for the expression of human proteins using high-throughput techniques.

[0116] As another example, plants have been used as expression hosts for large-scale heterologous expression of proteins, offering potential advantages of cost-effectiveness, scalability, and safety over traditional expression systems. Currently, there are a variety of plant heterologous expression systems, such as transient expression, plant cell suspension cultures, recombinant plant viruses, and chloroplast transgenic systems. Proteins expressed in plants have some variations from mammalian proteins (e.g., glycosylation), but there is currently no evidence that these differences cause adverse reactions in human patients.

[0117] 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. One particularly preferred expression system for producing the polypeptides described herein is the Pichia pastoris expression system. P. pastoris has been developed as an excellent 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, as it is free from the endotoxin issues associated with bacteria and the viral contamination of proteins produced in animal cell culture. Furthermore, P. pastoris can utilize methanol as a carbon source in the absence of glucose. The P. pastoris expression system uses the methanol-inducible alcohol oxidase (AOX1) promoter, which controls the expression of the gene encoding 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. Because proteins produced in P. pastoris are typically correctly folded and secreted into the medium, fermentation of genetically engineered P. pastoris offers a superior alternative to E. coli expression systems. Furthermore, P. pastoris has the ability to spontaneously glycosylate expressed proteins, another advantage over E. coli. Several proteins have been produced using this system, including tetanus toxin fragments, Bordetella pertussis pertactin, human serum albumin, and lysozyme.

[0118] In the present disclosure, the term "RNA" refers to a nucleic acid molecule containing ribonucleotide residues. In a preferred embodiment, the RNA contains all or most of the ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA, e.g., partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may refer to the addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of the RNA. It is also contemplated herein that the nucleotides in the RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the purposes of the present disclosure, these modified RNAs are considered analogs of naturally occurring RNA.

[0119] In certain embodiments of the present disclosure, the RNA is messenger RNA (mRNA), which refers to an RNA transcript that encodes a peptide or protein. As established in the art, mRNA generally contains a 5' untranslated region (5'-UTR), a peptide-coding region, and a 3' untranslated region (3'-UTR). In some embodiments, the RNA is produced by in vitro transcription or chemical synthesis. In one embodiment, the mRNA is produced by in vitro transcription using a DNA template, where DNA refers to a nucleic acid containing deoxyribonucleotides.

[0120] In some embodiments, an RNA according to the present disclosure comprises a 5'-cap. The term "5'-cap" refers to the structure found at the 5' end of an mRNA molecule and generally consists of a guanosine nucleotide attached to the mRNA via a 5'-to-5' triphosphate linkage. In one embodiment, the guanosine is methylated at position 7. Providing an RNA with a 5'-cap or 5'-cap analog may be achieved by in vitro transcription, in which the 5'-cap is co-transcriptionally expressed onto the RNA strand, or may be attached to the RNA post-transcriptionally using a capping enzyme.

[0121] In some embodiments, an RNA according to the present disclosure comprises a 5'-UTR and / or a 3'-UTR. The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or a corresponding region in an RNA molecule, such as an mRNA molecule. An untranslated region (UTR) can be located 5' (upstream) of an open reading frame (5'-UTR) and / or 3' (downstream) of an open reading frame (3'-UTR). If present, the 5'-UTR is located at the 5' end upstream of the start codon of a protein-coding region. If present, the 5'-UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 5'-cap. If present, the 3'-UTR is located at the 3' end downstream of the stop codon of a protein-coding region, although the term "3'-UTR" preferably does not include a poly(A) sequence. Thus, the 3'-UTR is upstream of the poly(A) sequence (if present), e.g., directly adjacent to the poly(A) sequence.

[0122] As used herein, the term "poly(A) sequence" or "poly(A tail)" refers to a sequence of adenylic acid residues typically located at the 3' end of an RNA molecule. Poly(A) sequences are known to those of skill in the art and may follow the 3'-UTR in the RNAs described herein.

[0123] In one embodiment of all aspects of the invention, the nucleic acids described herein are expressed in a cell transfected with the nucleic acid to provide the encoded polypeptide, hi one embodiment, expression is into the extracellular space, i.e., the encoded polypeptide is secreted.

[0124] "Encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have 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 the mRNA corresponding to the gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of the gene or cDNA.

[0125] In the context of the present disclosure, the term "transcription" relates to the process by which the genetic code in a DNA sequence is transcribed into RNA, which can then be translated into peptides or proteins.

[0126] According to the present invention, the term "transcription" includes "in vitro transcription," which refers to a process in which RNA, particularly mRNA, is synthesized in vitro in a cell-free system, preferably using a suitable cell extract. Preferably, a cloning vector is used to generate the transcription product. These cloning vectors are commonly called transcription vectors and are encompassed by the term "vector" according to the present invention. The promoter for controlling transcription can be any promoter for any RNA polymerase. Specific examples of RNA polymerases are T7, T3, and SP6 RNA polymerases. Preferably, in vitro transcription according to the present invention is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription may be obtained by cloning a nucleic acid, particularly cDNA, and introducing it into a suitable vector for in vitro transcription. cDNA may also be obtained by reverse transcription of RNA.

[0127] The term "expression," as used herein, is defined as the transcription and / or translation of a particular nucleotide sequence.

[0128] With respect to RNA, the terms "expression" or "translation" refer to the process in a cell's ribosomes by which an mRNA chain directs the assembly of a sequence of amino acids to make a peptide or protein.

[0129] As used herein, "endogenous" refers to any substance that originates from or is produced within an organism, cell, tissue, or system.

[0130] As used herein, the term "exogenous" refers to any substance introduced into or produced outside of an organism, cell, tissue, or system.

[0131] As used herein, the terms "linked," "fused," or "fusion" are used interchangeably and refer to the joining of two or more elements or components or domains.

[0132] Pharmaceutical Composition In one embodiment of all aspects of the invention, the components described herein, such as polypeptides, may be administered in a pharmaceutical composition, which may include a pharmaceutically acceptable carrier, and optionally, stabilizers, etc. In one embodiment, the pharmaceutical composition is for therapeutic or prophylactic treatment, for example, for use in treating or preventing a gluten-related disorder.

[0133] The term "pharmaceutical composition" relates to a formulation comprising a therapeutically active agent, preferably together with a pharmaceutically acceptable carrier, diluent and / or excipient. Said pharmaceutical composition is useful for treating, preventing or lessening the severity of a disease or disorder upon administration of said pharmaceutical composition to a subject. Pharmaceutical compositions are also known in the art as pharmaceutical formulations.

[0134] Pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and in a "pharmaceutically acceptable preparation."

[0135] The term "pharmaceutically acceptable" refers to the non-toxicity of a material that does not interact with the action of the active ingredients of the pharmaceutical composition.

[0136] The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount that achieves the desired response or desired effect, either alone or together with further doses. In the case of the treatment of a specific disease, the desired response preferably relates to the inhibition of the course of the disease. This includes slowing the progression of the disease, particularly halting or reversing the progression of the disease. The desired response in the treatment of a disease may also be delaying the onset or preventing the onset of the disease or condition. The effective amount of the compositions described herein depends on the condition to be treated, the severity of the disease, individual parameters of the patient, such as age, physiological state, size, and weight, duration of treatment, type of concomitant therapy (if any), specific route of administration, and similar factors. Thus, the dosage of the compositions described herein may depend on various such parameters. If the patient's response is insufficient with the initial dose, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) may be used.

[0137] The pharmaceutical compositions of the present disclosure may contain salts, buffering agents, preservatives, and optionally other therapeutic agents. In one embodiment, the pharmaceutical compositions of the present disclosure include one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0138] Suitable preservatives for use in pharmaceutical compositions of the present disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0139] The term "excipient," as used herein, refers to a substance that may be present in a pharmaceutical composition of the present disclosure but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.

[0140] The term "diluent" refers to an agent that dilutes and / or thins. Furthermore, the term "diluent" includes any one or more of a fluid, liquid, or solid suspension and / or mixing medium. Examples of suitable diluents include ethanol, glycerol, and water.

[0141] The term "carrier" refers to a component, which may be natural, synthetic, organic, or inorganic, in which the active ingredient is combined to facilitate, enhance, or enable administration of the pharmaceutical composition. As used herein, a carrier may be one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes, and biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers, among others. In one embodiment, the pharmaceutical composition of the present disclosure comprises isotonic saline.

[0142] Pharmaceutically acceptable carriers, excipients, or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).

[0143] Pharmaceutical carriers, excipients, or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0144] In one embodiment, the pharmaceutical compositions described herein may be administered orally, intravenously, intraarterially, subcutaneously, intradermally, or intramuscularly. In certain embodiments, the pharmaceutical compositions are formulated for local or systemic administration. Systemic administration may include enteral administration with absorption via the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to administration in any manner other than via the gastrointestinal tract, for example, administration by intravenous injection. In a preferred embodiment, the pharmaceutical compositions are formulated for oral administration.

[0145] The term "co-administering," as used herein, refers to the process by which different compounds or compositions are administered to the same patient. The different compounds or compositions may be administered simultaneously, essentially simultaneously, or sequentially.

[0146] treatment The present invention provides methods and medicaments for treating pathological conditions in a subject, such as gluten-related disorders (including celiac disease and non-celiac gluten sensitivity), gastrointestinal malabsorption, sprue, allergic reactions, and enzyme deficiencies. For example, an allergic reaction can be a reaction to gluten. The present invention particularly provides methods and medicaments for treating celiac disease (CeD) and non-celiac gluten sensitivity. The methods described herein can include administering an effective amount of a polypeptide or composition described herein.

[0147] The cocktail of ruLAPII and ruDPPIV degrades gluten immunogenic peptides (GIPs) in situ within minutes as they are produced upon digestion of gluten into non-immunogenic single amino acids and dipeptides, thereby preventing the immune response and subsequent symptoms of gluten-related disorders. Because the truncated polypeptide variants described herein retain biological activity, they have the same or similar utilities as the parent polypeptides from which they are derived.

[0148] Therapeutic compounds or compositions of the invention may be administered prophylactically (i.e., to prevent a disease or disorder) or therapeutically (i.e., to treat a disease or disorder) to a subject suffering from a disease or disorder or at risk of (or susceptible to) developing a disease or disorder. Such subjects can be identified using standard clinical methods. In the context of the present invention, prophylactic administration occurs prior to the manifestation of overt clinical symptoms of the disease, thereby preventing the disease or disorder or slowing its progression. In the context of the medical field, the term "preventing" encompasses any activity that reduces the mortality or morbidity burden due to a disease. Prevention can occur at primary, secondary, and tertiary levels. Primary prevention avoids the onset of the disease, while secondary and tertiary levels include activities aimed at preventing the progression of the disease and the appearance of symptoms, as well as reducing the adverse effects of an already established disease by restoring function and reducing disease-related complications.

[0149] In some embodiments, administration of an agent or composition of the invention may be by single administration or may be boosted by multiple administrations.

[0150] The term "disease" refers to an abnormal condition affecting an individual's body. Disease is often interpreted as a medical condition associated with specific symptoms and signs. A disease may be caused by factors originally derived from external causes, such as an infection, or it may be caused by an internal dysfunction, such as an autoimmune disease. In humans, "disease" is often used more broadly to refer to any condition that causes pain, impairment, distress, social problems, or death to the afflicted individual or causes similar problems to those who come into contact with the individual. In this broader sense, it may include injuries, disabilities, disorders, syndromes, clinical entities, infections, isolated symptoms, deviant behavior, and atypical variations in structure and function, while in other contexts and for other purposes, these may be considered distinct categories. Because suffering from and living with many illnesses can alter one's outlook on life and personality, illnesses typically affect individuals not only physically but also emotionally.

[0151] The term "gluten-related disorders" refers to conditions and diseases caused by the ingestion of gluten-containing foods, such as wheat-based foods. This term includes celiac disease (CeD), wheat allergy (WA) and non-celiac gluten sensitivity (NCGS), as well as other diseases that may benefit from a gluten-free diet (GFD).

[0152] Celiac disease (CeD) is a chronic immune-mediated enteropathy triggered by gluten immunogenic peptides (GIPs) in genetically predisposed individuals. GIPs are produced during the digestion of gluten and play a central role in disease pathology; they consist of digestion-resistant, proline-rich peptides that function as T cell epitopes in HLA-DQ2- and HLA-DQ8-positive patients, whereby CD4+ T cells engage with HLA molecules and initiate the typical autoimmune pathology, which explains the clinical symptoms and long-term complications. Current diagnosis is based on the presence of clinical symptoms of enteropathy, villous atrophy, crypt hyperplasia, and intraepithelial lymphocytosis, as well as the presence of circulating CeD-specific antibodies against tissue transglutaminase (tTG), deamidated gliadin peptide (DGP), and endomysial lymphocyte antigen (EMA) in the small intestine.

[0153] Non-celiac gluten sensitivity (NCGS) is a gluten-related disorder associated with gluten ingestion. In contrast to CeD, no specific genetic predisposition to NCGS has been identified, and serological biomarkers are not available for NCGS because measurements of celiac-associated antibodies are not sensitive or specific for NCGS. NCGS patients have normal gastrointestinal tracts and normal intestinal permeability, and their duodenal mucosa exhibits minimal histological lesions. However, increased infiltration of eosinophils and basophils into the duodenal lamina propria and activation of circulating basophils have been observed in NCGS patients, which may contribute to the symptoms associated with the syndrome.

[0154] Because gluten-derived GIP is a trigger for gluten-related disorders, a strict, lifelong gluten-free diet is the mainstay of treatment, but it is very difficult to achieve in daily life.

[0155] In the present context, the terms "treatment", "treating" or "therapeutic intervention" relate to the management and care of a subject for the purpose of combating a condition, such as a disease or disorder. This term is intended to include the whole range of treatments for a given condition from which a subject is suffering, such as the administration of therapeutically effective compounds to alleviate the symptoms or complications, delay the progression of the disease, disorder or condition, relieve or relieve the symptoms and complications, and / or cure or eliminate the disease, disorder or condition, and to prevent the condition, where 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 active compounds to prevent the onset of symptoms or complications.

[0156] The term "therapeutic treatment" relates to any treatment that improves the health and / or prolongs (increases) the lifespan of an individual. The treatment may eliminate the disease in an individual, prevent or delay the onset of the disease in an individual, inhibit or delay the onset of the disease in an individual, reduce the frequency or severity of symptoms in an individual, and / or reduce recurrence in an individual who currently has or previously had the disease.

[0157] The term "prophylactic treatment" or "preventative treatment" relates to any treatment intended to prevent the occurrence of a disease in an individual. The terms "prophylactic treatment" or "preventative treatment" are used interchangeably herein.

[0158] The terms "individual" and "subject" are used interchangeably herein. They refer to a human or another mammal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate) that may be afflicted with or susceptible to a disease or disorder, but may or may not have the disease or disorder. In many embodiments, the individual is a human. Unless otherwise specified, the terms "individual" and "subject" do not denote a particular age and, therefore, encompass adults, elderly people, children, and newborns. In embodiments of the present disclosure, an "individual" or "subject" is a "patient."

[0159] The term "patient" means an individual or subject for treatment, particularly an individual or subject suffering from a disease.

[0160] Citation of documents and works referred to herein is not intended as an admission that any of the foregoing is pertinent prior art. All statements regarding the contents of these documents are based on information available to applicant and do not constitute an admission of the accuracy of the contents of these documents.

[0161] The following description is presented to enable any person skilled in the art to make and use various embodiments. Descriptions of specific devices, techniques, and applications are provided by way of example only. Various modifications to the examples described herein will be readily apparent to those skilled 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. Accordingly, the various embodiments are not intended to be limited to the examples described and shown herein, but are to be accorded the scope consistent with the appended claims. [Example]

[0162] The following example describes a screening study conducted to test the production of short variants of ruLAPII (ruLAPII_short) and ruDPPIV (ruDPPIV_short) in Lonza's P. pastoris expression platform. These variants of ruLAPII and ruDPPIV were purified and tested for enzymatic activity (U / mg purified protein) along with their full-length variants. The purified truncated variants were analyzed by mass spectrometry of the intact molecules.

[0163] ruDPPIV_Short Twenty-three PAOX1 clones in the strain background LP1 were analyzed for secretion of ruDPPIV_short into the culture medium using SDS PAGE / Coomassie staining and for enzymatic activity (AMC assay, SOP provided by Amyra). Several promising clones were identified with enzymatic activities up to 2,032 U / L [reference strain LP1(pCKP6003.1) = 4,325 U / L] and lower product titers than the reference strain. Five clones were tested in high-cell-density fermentations (Ambr250) together with the reference strain. Enzymatic activity was up to 9,056 U / L [reference strain LP1(pCKP6003.1) = 21,586 U / L], a lower product titer than the reference strain. The products ruDPPIV [expressed in strain LP1(pCKP6003.1)] and ruDPPIV_short [expressed in strain LP1(pFJP6015.6)] were purified from the culture supernatant and their activities were measured. Similar specific productivities were measured. ruDPPIV_short: 12.7U / mg ·ruDPPIV total length: 12.4U / mg

[0164] Purified ruDPPIV and ruDPPIV_short were deglycosylated and analyzed by mass spectrometry for possible degradation. ruDPPIV_short: Full-length product and degradation products were observed Full-length ruDPPIV: Only degradation products were observed

[0165] ruLAPII_Short Twenty-three PAOX1 clones in the strain background LP1 were analyzed for secretion of ruLAPII_short into the culture medium using SDS PAGE / Coomassie staining and for enzymatic activity (AMC assay, SOP provided by Amyra). Several promising clones were identified with enzymatic activities up to 26 U / L [reference strain LP1(pCKP6011.4) = 174 U / L] and lower product titers than the reference strain. Five clones were tested in high-cell-density fermentations (Ambr250) together with the reference strain. Enzymatic activity was up to 371 U / L [reference strain LP1(pCKP6011.4) = 1,212 U / L]. The products ruLAPII [expressed in strain LP1(pCKP6011.4)] and ruLAPII_short [expressed in strain LP1(pFJP6014.13)] were purified from the culture supernatant, and activity was measured. The specific activity of the short variant was slightly lower than that of wild-type ruLAPII. ruLAPII_short: 1.1U / mg ·ruLAPII total length: 1.5U / mg

[0166] Purified ruLAPII and ruLAPII_short were deglycosylated and analyzed by mass spectrometry for possible degradation. ruLAPII_short: Only full-length products were observed, and no degradation products were observed. ruLAPII full length: only degradation products were observed

[0167] Example 1: Materials and Methods 1. Strains and Plasmids 1.1 Escherichia coli strains DH10B (Invitrogen, Cat. No. 18297-00) was used for all cloning steps.

[0168] 1.2 Pichia pastoris strains All P. pastoris expression studies were performed using strain LP1. Genotype description can be shared upon request.

[0169] 1.3 Plasmids The PAOX1 plasmid pFJP6014 was constructed by ligation of a linear approximately 3085 bp SbfI / SfiI fragment of the pXSP603 vector with a linear 1480 bp SbfI / SfiI digested fragment (containing the short ruLAPII variant) from ATUM400860.

[0170] The PAOX1 plasmid pFJP6015 was constructed by ligation of a linear approximately 3085 bp SbfI / SfiI fragment of the pXSP603 vector with a linear 2314 bp SbfI / SfiI digested fragment from ATUM400861 (containing the short ruDPPIV variant).

[0171] Chemically competent DH10B cells were then transformed with the ligation mix. After restriction digestion analysis, the target gene in the newly generated plasmid was confirmed by sequencing, and strain LP1 was transformed with plasmids pFJP6014 and pFJP6015 for subsequent multicopy screening.

[0172] [Table 1]

[0173] 2. Genes DNA sequence of the ruLAPII_short product derived from the original ruLAPII sequence (i.e., plasmid pCKP6011) except for the missing codons at the 5' and 3' ends (see sections 3.1 and 3.2).

[0174] The DNA sequence of the ruDPPIV_short product derived from the original ruDPPIV sequence (i.e., plasmid pCKP6003) except for the missing codons at the 5′ and 3′ ends (see sections 3.3 and 3.4).

[0175] 3. Protein Sequence 3.1 ruLAPII Italics indicate amino acids missing in the corresponding short variants. TIFF2025143246000002.tif55166 Theoretical Mw: 51843.34 Theoretical pI: 6.73

[0176] 3.2 ruLAPII short variant TIFF2025143246000003.tif55166 Theoretical Mw: 48695.79 Theoretical pI: 6.62

[0177] 3.3 ruDPPIV Italics indicate amino acids missing in the corresponding short variants. TIFF2025143246000004.tif86166 Theoretical Mw: 86486.34 Theoretical pI: 8.05

[0178] 3.4 ruDPPIV short variant TIFF2025143246000005.tif33166TIFF2025143246000006.tif55166 Theoretical Mw: 84931.48 Theoretical pI: 7.77

[0179] 4. Primary screening 4.1 Multicopy screening After linearization of the plasmids with DraI, strain LP1 was transformed with plasmids pFJP6014 (ruLAPII_short) and pFJP6015 (ruDPPIV_short) and plated onto agar plates containing various concentrations of Zeocin™ (Invitrogen, catalog no. ant-zn-1) (500 and 1000 μg / mL). After 48 h of incubation at 30°C, 23 clones per host / plasmid integration were picked and streaked onto master plates (containing 100 μg / mL Zeocin™) for subsequent expression screening in 24-well plates.

[0180] 4.2 Expression in 24-well plates Expression experiments were performed in 24-well plates (GE Healthcare Life Sciences, catalog no. 7701-5102) containing YPC (yeast extract, peptone, 100 mM sodium citrate, pH 6.0) medium supplemented with concentrated polysaccharide (50 g / L, EnPump200, EnPresso, Germany) and 14 U / L concentrated enzyme mixture (EnPump200, EnPresso, Germany). Main cultures (2 mL) were inoculated to a starting OD of 2 with cultures grown overnight at 30 °C in YPG (yeast peptone glycerol). Cultures were then incubated at 25 °C with 260 rpm shaking and induced with repeated methanol shots (1%) every 12 h for 72 h.

[0181] 5. High cell density fermentation The medium is 3g kg -1 It is based on a medium modified from Gasser (Gasser et al. 2013; Future Microbiol., 8(2): 191-208) and Prielhofer (Prielhofer et al. 2013, Microbial Cell Factories 12:5), containing peptone (Biokar, Cat. No. A1601). Reagents and solutions are stored at room temperature, except for PTM1, which is stored at 4°C in the dark, biotin, which is stored at -4°C, and the biotin solution, which is stored at 4°C. Reagents were supplied by Merck (Darmstadt, Germany) unless otherwise stated.

[0182] 5.1 Preculture Cultures in shake flasks were performed to generate inoculum for the main fermentation. For each strain, 100 mL of preculture medium was placed in a 500 mL baffled shake flask and inoculated with 1 mL of a -80°C glycerol stock culture. The shake flasks were incubated for 21 hours (±2 hours) at 170 rpm (φ25 mm) and 30°C on an orbital shaker (Kuhner ISF-1-W). The axenic status of the cultures was confirmed by microscopic examination (Nikon Eclipse) immediately before transferring the inoculum to the bioreactor. The main fermentation was initiated from these precultures by inoculating the fermentor to an initial optical density of 1.

[0183] 5.2 Main fermentation After preparing the periphery, medium, and solutions, the bioreactor was filled with 90 mL of batch medium, autoclaved at 121 °C for 30 min, and then attached to the Ambr250 station. The feed systems for carbon substrate and base addition were cleaned in place by rinsing with 70% ethanol, 2 M sodium hydroxide, and then demineralized water for 30 min each. Acid (phosphate) and base (ammonia) were used for pH control in the PAOX1 strain. Setpoints for agitation and aeration with air and oxygen were determined by a cascade controller that maintained dissolved oxygen levels between 25% and 40%.

[0184] 5.3 Fermentation Setup and Strategy The duration of the batch phase (length of the batch phase) is predetermined by an initial optical density of about 1 and the glycerol concentration.

[0185] ruLAPII_Short Fermentation was carried out according to Lonza SOP143961-2 and report 2014.157 (S. Bieli and N. Krumov, 2014).

[0186] After the batch phase, the main fermentation is divided into a biomass production phase followed by a target protein production phase (see Figure 2). 5 h 12 mL L -1 BV h -1A constant glycerol feed (45%) of 10000 kJ / ml is carried out during the fed-batch phase 1. This phase is followed by a short starvation period of about 30 min during which no feed is introduced into the fermenter, aiming at complete glycerol depletion on the one hand and adaptation of the cell metabolism to the upcoming methanol feed on the other hand. The production phase begins with a temperature shift from 30 °C to 26 °C and an addition of 5 mL L -1 BV h -1 The process begins with the start of a constant methanol feed.

[0187] ruDPPIV_Short Fermentation was carried out according to Lonza SOP143601-2 and report 2014.157 (S. Bieli and N. Krumov, 2014).

[0188] After the batch phase (pH 5.2), the main fermentation is divided into a biomass production phase followed by a target protein production phase (see Figure 3). The fermentation time is 6 h and the yield is 12 mL L. -1 BV h -1 A constant glycerol feed (45%) of 10000 kJ / ml is administered during fed-batch phase 1. This phase is followed by a short starvation period of approximately 30 min during which no feed is introduced into the fermenter, aiming at complete glycerol depletion on the one hand and adaptation of the cell metabolism to the upcoming methanol feed on the other hand. The production phase begins with a temperature shift from 30 °C to 24.2 °C and an addition of 6.5 mL L -1 BV h -1 The reaction mixture was fed at a constant rate starting with the initiation of exponential methanol feeding until a feed rate of 0.1% was achieved, which was continued until the end of the fermentation.

[0189] Fermentations using the Ambr250 system were carried out using custom programming scripts that allowed for automatic initiation of various feeds (upon detection of DO spikes at the end of the batch phase) and programmed duration of the various feed phases. Fermentation parameters were controlled and recorded by the IRIS process control system.

[0190] 6. Downstream purification Purification was performed using a simplified batch mode based on the presentation “GLH-003: DPP4 and LAP2 step elution feasibility” (presentation; A. Zurbriggen et al., 2016) and the block flow diagram of the demonstration run.

[0191] ruLAPII_Short Purification was adapted to batch mode according to "LAP2_BFD_Demonstration Runs_01" (A. Zurbriggen, 2016). After fermentation and centrifugation of the culture broth at 4000 rpm and 4°C for 45 min, 100 μL of Halt protease inhibitor-single-use cocktail (100x) (Thermo Scientific, catalog no. 78430) was added to 12 mL of EoF culture supernatant.

[0192] resin preparation One mL of SP Sepharose® XL (GE Healthcare, Catalog No. GE17-5073-01, binding capacity 5-10 mg / mL) was added to a 15 mL Falcon tube and mixed. The Falcon tube was then centrifuged at 1000 rpm for 5 minutes. The resin was washed with 10 mL of MQ water and centrifuged at 1000 rpm for 5 minutes. The supernatant was then removed by pipetting, and 10 mL of 50 mM Na acetate, pH 5.0 was added for equilibration. After centrifugation at 1000 rpm for 5 minutes, the supernatant was removed from the resin by pipetting, and the resin was washed again with 10 mL of 50 mM Na acetate, pH 5.0.

[0193] Sample incubation After centrifugation at 1000 rpm for 5 min, the resin supernatant was removed by pipetting, and the resin was incubated with 12 mL of EoF culture supernatant on a rocking shaker for 30 min at room temperature. After incubation, the Falcon tube was centrifuged at 1000 rpm for 5 min, and the supernatant was removed by pipetting.

[0194] Cleaning The resin was washed with 10 mL of 20 mM Na phosphate, pH 6.0, centrifuged at 1000 rpm for 5 minutes, and the supernatant was removed by pipetting. After washing, the Falcon tube was centrifuged at 1000 rpm for 5 minutes, and the supernatant was removed by pipetting.

[0195] Elution 2 mL of 20 mM Na phosphate, pH 6.0, 130 mM NaCl was added to the resin and incubated on a rocking shaker at room temperature for 10 minutes. The Falcon tube was then centrifuged at 1000 rpm for 5 minutes, and the supernatant was isolated and stored at -20°C for later analysis.

[0196] ruDPPIV_Short Purification was adapted to batch mode according to "DPP4_BFD_Demonstration Runs_01" (A. Zurbriggen, 2016). After fermentation and centrifugation of the culture broth at 4000 rpm and 4°C for 45 min, 100 μL of Halt protease inhibitor-single-use cocktail (100x) (Thermo Scientific, catalog no. 78430) was added to 12 mL of EoF culture supernatant.

[0197] resin preparation One mL of SP Sepharose® XL (GE Healthcare, catalog number GE17-5073-01), binding capacity 5-10 mg / mL, was added to a 15 mL Falcon tube and mixed. The Falcon tube was then centrifuged at 1000 rpm for 5 minutes. The resin was washed with 10 mL of MQ water, centrifuged at 1000 rpm for 5 minutes, the supernatant was removed by pipetting, and 10 mL of 25 mM Tris, pH 7.2, was added for equilibration. After centrifugation at 1000 rpm for 5 minutes, the resin supernatant was removed by pipetting, and the resin was washed again with 10 mL of 25 mM Tris, pH 7.2.

[0198] Sample incubation After centrifugation at 1000 rpm for 5 min, the resin supernatant was removed by pipetting, and the resin was incubated with 12 mL of EoF culture supernatant on a rocking shaker for 30 min at room temperature. After incubation, the Falcon tube was centrifuged at 1000 rpm for 5 min, and the supernatant was removed by pipetting.

[0199] Cleaning The resin was washed with 10 mL of 25 mM Tris, pH 7.2, 20 mM NaCl, centrifuged at 1000 rpm for 5 minutes, and the supernatant was removed by pipetting. After washing, the Falcon tube was centrifuged at 1000 rpm for 5 minutes and the supernatant was removed by pipetting.

[0200] Elution 2 mL of 25 mM Tris, pH 7.2, 130 mM NaCl was added to the resin and incubated on a rocking shaker at room temperature for 10 minutes. The Falcon tube was then centrifuged at 1000 rpm for 5 minutes, and the supernatant was isolated and stored at -20°C for later analysis.

[0201] 7. Analysis method 7.1 Sample processing Cells (1 mL samples) were removed by centrifugation at 6000 g for 5 minutes at 4° C. and the supernatant was collected. Samples were either subjected to analysis directly or stored at −80° C. for later testing.

[0202] 7.2 Wet cell weight To measure the wet cell weight, 1 mL of the fermentation suspension was transferred to a pre-weighed tube. The sample was centrifuged at 15,000 g for 15 minutes and the supernatant was isolated. The tube was then weighed and the wet cell weight (g L -1 ) (biomass per current fermentation volume) was calculated.

[0203] 7.3 Dry cell weight For determination of dry cell weight, 1 mL of sample was centrifuged at 17,000 g (4°C) for 15 min. The supernatant was discarded and the resulting pellet was dried at 100°C for 48 h. The resulting dry cell weight (g L -1 ) (biomass per current fermentation volume) was calculated.

[0204] 7.4 Optical density Biomass concentration was determined by measuring the optical density at 600 nm within the linear range of the photospectrometer (between OD600 = 0.05 and OD = 0.5). Briefly, 10 μL of culture was added to a 96-well plate (VWR, catalog number 732-2746) and diluted (dilution factor 1:20) in PBS (190 μL) using a Starlet liquid handler (Hamilton, Bonaduz, Switzerland). Subsequently, the optical density at 600 nm was measured using a Tecan Infinite reader. 200 μL of YPC medium was used as a blank.

[0205] 7.5 Product detection by SDS PAGE / Coomassie staining SDS-PAGE was performed under reducing conditions. Precast Criterion 12% Bis-Tris SDS gels (Bio-Rad, catalog no. 567-1124) were used with MES buffer (Bio-Rad, catalog no. 161-0796). Samples were mixed with NuPAGE 4x LDS loading buffer (Invitrogen, catalog no. NP0007) and incubated at 95°C for 5 minutes. 7.5 μL of sample (10 μL of culture supernatant plus 5 μL of 4x LDS loading buffer) was loaded per lane. Mark12 (Invitrogen, catalog no. LC5677) was loaded as a molecular weight standard. 1 μg of recombinant ruLAPII (Biomeva LAP-2 100.6 mg / ml) was loaded as a reference. Electrophoresis was performed at 200 V for approximately 80 minutes. The separated proteins were visualized by staining with GelCode Blue staining reagent (Thermo Scientific, Cat. No. 24592) for 1-2 hours and destained overnight with water.

[0206] 7.6 AMC activity assay Activity tests were performed in 96-well plates (GreinerOne, catalog no. 655900). As substrates, 10 mM Gly-Pro-AMC (Bachem, catalog no. I1225) for ruDPPIV or 10 mM Leu-AMC hydrochloride (Bachem, catalog no. I1245) for ruLAPII were used. As reaction buffer, 50 mM Tris-HCl buffer (pH 7.5, 1 mM CoCl2, 1% BSA) was used. As a standard, 1 mM AMC (solubilized in EtOH, Bachem, catalog no. Q-1025) was used at final concentrations of 0 nM to 10,000 nM diluted in reaction buffer. Samples (cell-free medium) were diluted between 1 / 400 and 1 / 2500, and fluorescence was within the AMC standard curve. Briefly, 10 μL of sample was added to 90 μL of reaction buffer along with 10 μL of substrate (final concentration: 1 mM). Measurements were performed using the following settings: Temperature: 25℃ Excitation: 370nm Radiation: 460nm Reaction period: 30 minutes Measurement interval: 30 seconds

[0207] 7.7 BCA Assay (Protein Concentration) Protein determination was performed according to the manufacturer (Pierce BCA Protein Assay Kit, Cat. No. 23227) adapted for 96-well microtiter plates.

[0208] 7.8 PNGase Digestion (Deglycosylation) Deglycosylation was performed according to the manufacturer's instructions (NEB Rapid PNGase F, catalog number P0710S). Briefly, 15 μL of eluted sample was added to 5 μL of Rapid PNGase F buffer (5×) and incubated at 80°C for 2 minutes. After cooling, 1 μL of Rapid PNGase F was added and incubated at 50°C for 10 minutes.

[0209] 7.9 Sample desalting Briefly, desalting was performed on an Amicon Ultra -0.5 mL, 30K according to the manufacturer's instructions (Merck, catalog number UFC503096). Briefly, five PNGase F-digested samples were pooled (100 μL total) and filled to 500 μL with 25 mM Tris, pH 7.2. The tubes were then centrifuged at 14,000 g for 10 minutes at 4°C. The supernatant was isolated. 20 μL of 25 mM Tris, pH 7.2 was then added to the filter, which was then centrifuged at 14,000 g for 10 minutes at 4°C. Both samples were pooled for further analysis.

[0210] 7.10 Mass spectrometry Analyses were performed at B-Fabric (Functional Genomics Center Zurich). Briefly, prior to ESI-MS analysis, samples were desalted using C4 Zip Tips (Millipore, USA) and analyzed in MeOH:2-PrOH:0.2% FA (30:20:50). The solution was injected at a flow rate of 1 μL / min through a fused silica capillary (ID 75 μm) and sprayed through PicoTips (ID 30 μm), the latter obtained from New Objective (Woburn, MA). Nano ESI-MS analysis of the samples was performed on a Synapt G2_Si mass spectrometer, and data were recorded using MassLynx 4.2 software (both Waters, UK). Mass spectra were acquired in positive ion mode by scanning the m / z range from 100 to 5000 da with a 1-second scan duration and a 0.1-second interscan delay. The spray voltage was set to 3 kV, the cone voltage was set to 50 V, and the source temperature was set to 80° C. All four samples were also analyzed by MALDI-MS without further sample preparation, i.e., simply applied onto a steel target.

[0211] 7.11 Glycerol Stock Cultures The -80°C stock cultures used for experiments in the secondary screen were prepared according to the following protocol: Inoculation of the main culture: Transfer 20 mL of YPD medium into a 100 mL shake flask. Add 20 μL of Zeocin™ (InvivoGen, Cat. No. ant-zn-1) (100 mg / mL); final concentration: 100 μg / mL Inoculate using a loop of colonies from a plate Incubate at 30°C, 200 rpm for approximately 20 hours. Preparation of main culture for storage at -80°C: Measure the optical density (OD600) (the expected OD600 is 40~50) Add 850 μL of culture to a Nunc 1.8 mL cryotube containing 150 μL glycerol (100%) Mix by inversion Store at -80°C

[0212] Example 2: Primary Screening After transformation of P. pastoris strains with linearized plasmids pFJP6015 (ruDPPIV_short) and pFJP6014 (ruLAPII_short), colonies were picked and inoculated into 24-well plates containing 2 mL of YPC medium. Only clearly separated colonies were picked. In total, 46 clones (23 clones per product) were incubated at 25°C, 260 rpm, grown at pH 6.0, and induced by repeated shots of methanol (1%) every 12 hours for 72 hours. As references, precursor strains LP1 (pCKP6003.1, ruDPPIV) and LP1 (pCKP6011.4, ruLAPII) were included.

[0213] Culture supernatants of the clones were then analyzed by SDS PAGE / Coomassie staining and enzyme activity (AMC assay).

[0214] 1. ruDPPIV_short variant Culture supernatants of all LP1(pCKP6015) clones grown at pH 6.0 were loaded onto a 12% SDS-PAGE gel under reducing conditions. As a reference, culture supernatant of the reference strain LP1(pCKP6003.1, ruDPPIV) was loaded (Figure 4, lane 26).

[0215] As shown in Figure 4A, the culture supernatants of several clones showed bands migrating at the same height as the reference strain LP1(pCKP6003.1) (Figure 4, lane 26). A strong ruDPPIV_short product could be found in the culture supernatants of clones LP1(pCKP6015.8) (Figure 4, lane 6), LP1(pCKP6015.6) (Figure 4, lane 13), LP1(pCKP6015.12) (Figure 4, lane 14), LP1(pCKP6015.15) (Figure 4, lane 19), and LP1(pCKP6015.18) (Figure 4, lane 25). For the clone with the most intense product band, aggregate formation was observed, similar to that seen in the reference strain LP1(pCKP6003.1) (Fig. 4, compare lanes 6, 13, 14, 19, and 25 with lane 26), indicating that the short variants are capable of forming aggregates (most likely ruDPPIV dimers) similar to full-length ruDPPIV. The most intense product amount was found in clone LP1(pFJP6015.8), which was visually determined to be approximately half the amount of product found in the reference strain LP1(pCKP6003.1).

[0216] The culture supernatants of all clones were analyzed for enzymatic activity using the AMC assay (see Example 1, section 7.6). As expected, the culture supernatants of the clones showing the highest product titers also showed the highest enzymatic activity. The highest activity was found in the culture supernatant of clone LP1(pFJP6015.8) (2032 U / L), which was approximately half the activity measured in the reference strain LP1(pCKP6003.1) (4325 U / L). The reference clone LP1(pCKP6003.1) showed activity reproducible to that in the initial screening (3977 U / L, ResRep2014.111). In summary, the data suggested that the short variants of ruDPPIV were still enzymatically active.

[0217] 2. ruLAPII_short variant Culture supernatants of all LP1(pCKP6014) clones grown at pH 6.0 were loaded onto a 12% SDS-PAGE gel under reducing conditions. As a reference, culture supernatants of the reference strain LP1(pCKP6011.4, ruLAPII) (Figure 5, lane 26) and purified ruLAPII (Biomeva LAP-2 100.6 mg / mL) were loaded.

[0218] In contrast to the ruDPPIV-producing clones, very little or no product was observed in the culture supernatant of the ruLAPII-producing clones (Figure 5A). This may be explained by the strong glycosylation of ruLAPII, which makes quantification difficult (i.e., there is no clear band, but rather a faint band). Despite the difficulty in identifying high-producing strains, several clones showed promising results: LP1(pFJP6014.6) (Figure 5, lane 13), LP1(pFJP6014.12) (Figure 5, lane 14), LP1(pFJP6014.13) (Figure 5, lane 15), LP1(pFJP6014.20) (Figure 5, lane 18), and LP1(pFJP6014.15) (Figure 5, lane 19). Overall, the amount of product secreted by the ruLAPII_short clones was significantly lower compared to the reference strain LP1 (pCKP6011.4).

[0219] The culture supernatants of all clones were analyzed for enzymatic activity using the AMC assay (see Example 1, section 7.6). As expected, the culture supernatants of the clones showing the highest product titers also showed the highest enzymatic activity. The highest activity was found in the culture supernatant of clone LP1(pFJP6014.13) (26.1 U / L), which was approximately 15% of the activity measured in the reference strain LP1(pCKP6011.4) (173.8 U / L). The reference clone showed similar activity as in the initial screening (158.4 U / L, ResRep2014.111). In summary, the data suggested that the short variants were still enzymatically active.

[0220] Example 3: Secondary Screening Expression driven by the AOX1 promoter is induced by a limited methanol feed. Approximately 24–28 h of the batch phase (indicated by a pO spike) is followed by a glycerol fed-batch phase to increase biomass. After 12 h of glycerol feeding, the methanol feed is initiated depending on the fermentation protocol for either ruDPPIV or ruLAPII. Five clones of short variants of either ruDPPIV or ruLAPII were included, as well as a reference strain for direct comparison.

[0221] [Table 2]

[0222] 1. ruDPPIV_short 1.1 Biomass production The cell density and dry cell weight of all fermentation runs were compared with each other. The duration of the batch phase for all fermentations was adjusted to 45 g L for all strains. -1 The batch phase was completed between 18 and 20 hours, as previously determined by the initial glycerol concentration and starting OD of 1 (indicated by a pO spike). Subsequently, a 12-hour glycerol feed was initiated, followed by a methanol feed. The total fermentation duration was 78 hours.

[0223] No significant differences in biomass were observed at the end of fermentation (less than 10% deviation from the median WCW and less than 1% deviation from the median DCW), indicating that the strains were not affected by the short variants of ruDPPIV. Small differences in optical density were observed, but this was most likely an analytical error due to the high cell density of the cultures (Figure 6).

[0224] 1.2 Product Generation Several samples were taken midway through the fermentation run to investigate the ruDPPIV_short product growth and to identify the most promising strains. Cells were centrifuged, and the culture supernatant was isolated. Representative samples were then analyzed by SDS-PAGE / Coomassie staining.

[0225] As expected from the primary screening data, the product titer of ruDPPIV_short was significantly reduced compared to the full-length ruDPPIV (Figure 7, compare the green line with the other lines). When the culture supernatants of the highest-producing ruDPPIV_short clones [without clone LP1(pFJP6015.15)] were compared with each other, no significant differences in activity were observed; i.e., enzyme activity ranged between 7,276.6 U / L and 9,055.6 U / L. When compared with the wild-type strain LP1(pCKP6003.1), the activity of the highest-producing ruDPPIV_short clone was approximately 50% of that of the reference strain LP1(pCKP6003.1) (21,586 U / L) (Figure 7). The activity of the reference clone LP1 (pCKP6003.1) was consistent with previous data (25,000 U / L, 10 L Infors, report 2014.157).

[0226] 2. ruLAPII_Short 2.1 Biomass production The cell density and dry cell weight of all fermentation runs were compared with each other. The duration of the batch phase for all fermentations was adjusted to 45 g L for all strains. -1 The batch phase was completed between 18 and 20 hours, as previously determined by the initial glycerol concentration and starting OD of 1 (indicated by a pO spike). Subsequently, a 12-hour glycerol feed was initiated, followed by a methanol feed. The total fermentation duration was 78 hours.

[0227] No significant differences in biomass were observed at the end of fermentation (less than 10% deviation from the median WCW and less than 1% deviation from the median DCW), indicating that clones producing short variants grow similarly to clones producing full-length ruLAPII. Small differences in optical density were observed, but were most likely analytical errors due to the high cell density of the culture medium (Figure 8).

[0228] 2.2 Product Generation Several samples were taken midway through the fermentation run to investigate the ruLAPII_short product growth and to identify the most promising strains. Cells were centrifuged, and the culture supernatant was isolated. Representative samples were then analyzed by SDS-PAGE / Coomassie staining and AMC assay.

[0229] Similar to ruDPPIV_short, and as expected from the primary screening data, the product titer of ruLAPII_short was significantly reduced compared to full-length ruLAPII (Figure 9, compare the green line with the other lines). The strain ranking was identical to that observed in the primary screening. When the culture supernatants of the two most productive ruLAPII_short clones were compared with each other, no significant differences in activity were observed; i.e., enzyme activity ranged between 320 U / L and 371.2 U / L. When compared with the wild-type strain LP1(pCKP6011.4), the activity of the most productive ruLAPII_short clone was approximately 30% of that of the reference strain LP1(pCKP6011.4) (1,212.3 U / L) (Figure 9). The activity of the reference clone LP1 (pCKP6011.4) was consistent with previous data (1,500 U / L, 10 L Infors, report 2014.157).

[0230] Example 4: Specific Activity As shown in Examples 2 and 3, the volumetric activity (U / L) of the short variants of ruDPPIV or ruLAPII was significantly reduced. Because the product yield was also significantly reduced, it was unclear whether the reduced activity was due to lower specific activity or lower product yield in the culture supernatant. Therefore, to assess specific activity, it was decided to purify the proteins and evaluate their specific activity (i.e., U / mg). To this end, the purification strategy from "GLH-003: DPP4 and LAP2 step elution feasibility" (presentation; A. Zurbriggen et al., 2016) was adapted for use in batch mode.

[0231] 1. ruDPPIV_short Twelve mL of culture supernatant from LP1 (pFJP6015.6, ruDPPI_short, 9,056 U / L) and LP1 (pCKP6003.1, ruDPPIV, 21,586 U / L) was loaded onto 2 mL of SP Sepharose XL (GE Healthcare) and subsequently washed twice with 10 mL of 25 mM Tris, pH 7.2, 20 mM NaCl before elution in 2 mL of 25 mM Tris, pH 7.2, 130 mM NaCl. AMC assay (activity assay) and BCA assay (protein concentration) were performed on purified samples (load, flow-through, and elution). Three trials were performed: in the first purification trial, technical problems occurred, and therefore the data were unreliable. In the third trial, the aim was to generate more purified material for subsequent mass spectrometry analysis. Despite increasing sample volume and resin, binding capacity had already been reached, and no improvement in material delivery could be achieved. For convenience, data from the second purification run is presented.

[0232] After purification, the samples were loaded onto SDS-PAGE / Coomassie staining and a BCA assay was performed along with the AMC assay (Figure 10).

[0233] No significant difference in specific activity (12 U / mg) was found between ruDPPIV_short and ruDPPIV. For comparison, the ruDPPIV purified material from the tox material production (report ResRep2014.165) showed specific activities of 19.6 U / mg and 20 U / mg; the demonstration run showed activities of 18.3 U / mg and 17.9 U / mg. The difference in ruDPPIV between the data presented here and the material production report is most likely due to the different resin modes (batch mode vs. column mode).

[0234] 2. ruLAPII_Short Twelve mL of culture supernatant from LP1 (pFJP6014.13, ruLAPII_short, 371 U / L) and LP1 (pCKP6011.4, ruLAPII, 1,212 U / L) was loaded onto 2 mL of SP Sepharose XL (GE Healthcare), followed by two washes with 10 mL of 20 mM Na phosphate, pH 6.0, and then elution in 2 mL of 20 mM Na phosphate, pH 6.0, 130 mM NaCl. Purified samples (load, flow-through, and elution) were analyzed for AMC (activity assay) and BCA (protein concentration) assays. Three trials were performed: the first purification trial encountered technical problems, and therefore the data were unreliable. In the third trial, the goal was to generate more purified material for subsequent mass spectrometry analysis. As with the ruDPPIV_short purification, binding capacity had already been reached, and no improvement in material supply could be achieved. For convenience, data from the second purification trial are presented.

[0235] After purification, the samples were loaded onto SDS-PAGE / Coomassie staining and a BCA assay was performed along with the AMC assay (Figure 11).

[0236] A slightly lower specific activity was found in the purified sample of ruLAPII_short, approximately 26% lower than that of full-length ruLAPII (i.e., 1.1 U / mg vs. 1.5 U / mg). Comparing the samples on a Coomassie-stained SDS gel revealed fewer variants at the expected locations (Figure 11, compare lanes 3 & 6 or lanes 4 & 7, blue arrows). The specific activity of ruLAPII (1.5 U / mg) was slightly lower than that observed during tox material production (1.7 U / mg and reported in ResRep2014.165) and demo runs (1.9 U / mg and 2 U / mg, respectively). As with ruDPPIV purification, the difference may be explained by the use of slightly different purification strategies (i.e., batch mode vs. column mode).

[0237] Example 4: Product Degradation The goal of this study was to confirm that the short variants of ruDPPIV and ruLAPII do not undergo further degradation, as observed with the full-length molecules. To this end, mass spectrometry (ESI-MS and Maldi-TOF) was performed. Briefly, after purification, the eluted samples were further treated with PNGase F to cleave glycan structures (which interfere with mass spectrometry) and with Amicon desalting to remove free glycans.

[0238] 1. ruDPPIV_short First, ESI-MS and Maldi-TOF were performed using B-Fabric. For ESI-MS, the signal intensity was very low or no protein-like species were detected. Trace amounts of PNGaseF (34,874 Da and 34,887 Da) were detected.

[0239] For both variants, degradation products were observed in Maldi-TOF. In contrast to the full-length ruDPPIV, the full-length ruDPPIV_short molecule could be found, likely with an acetylation modification (+42) (Figure 12).

[0240] 2. ruLAPII_Short First, ESI-MS and Maldi-TOF were performed using B-Fabric. For ESI-MS, only the full-length molecule ruLAPII was detected. Trace amounts of PNGaseF (34,869 Da and 34,863 Da) were also detected.

[0241] Maldi-TOF detected only the full-length molecule for the ruLAPII short fragment. For the full-length ruLAPII molecule, only degradation products could be identified. For the full-length ruLAPII, the major cleavage events occurred after Val4 and Lys457 (48,884 da) and only after Val4 (51,284 da), consistent with previous data provided by Biomeva (Figure 13).

[0242] [Table 3] TIFF2025143246000009.tif29161

[0243] SEQUENCE LISTING <110> AMYRA Biotech AG <120> DIPEPTIDYLPEPTIDASE AND LEUCINE AMINOPEPTIDASE POLYPEPTIDE VARIANTS <130> PA25-181 <140> JP_2025-088107 <141> 2021-10-25 <150> PCT / EP2020 / 080170 <151> 2020-10-27 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 760 <212> PRT <213> Artificial Sequence <220> <223> Dipeptidylpeptidase <400> 1 Ile Val Pro Pro Arg Glu Pro Arg Ser Pro Thr Gly Gly Gly Asn Lys 1 5 10 15 Leu Leu Thr Tyr Lys Glu Cys Val Pro Arg Ala Thr Ile Ser Pro Arg 20 25 30 Ser Thr Ser Leu Ala Trp Ile Asn Ser Glu Glu Asp Gly Arg Tyr Ile 35 40 45 Ser Gln Ser Asp Asp Gly Ala Leu Ile Leu Gln Asn Ile Val Thr Asn 50 55 60 Thr Asn Lys Thr Leu Val Ala Ala Asp Lys Val Pro Lys Gly Tyr Tyr 65 70 75 80 Asp Tyr Trp Phe Lys Pro Asp Leu Ser Ala Val Leu Trp Ala Thr Asn 85 90 95 Tyr Thr Lys Gln Tyr Arg His Ser Tyr Phe Ala Asn Tyr Phe Ile Leu 100 105 110 Asp Ile Lys Lys Gly Ser Leu Thr Pro Leu Ala Gln Asp Gln Ala Gly 115 120 125 Asp Ile Gln Tyr Ala Gln Trp Ser Pro Met Asn Asn Ser Ile Ala Tyr 130 135 140 Val Arg Gly Asn Asp Leu Tyr Ile Trp Asn Asn Gly Lys Thr Lys Arg 145 150 155 160 Ile Thr Glu Asn Gly Gly Pro Asp Ile Phe Asn Gly Val Pro Asp Trp 165 170 175 Val Tyr Glu Glu Glu Ile Phe Gly Asp Arg Phe Ala Leu Trp Phe Ser 180 185 190 Pro Asp Gly Glu Tyr Leu Ala Tyr Leu Arg Phe Asn Glu Thr Gly Val 195 200 205 Pro Thr Tyr Thr Ile Pro Tyr Tyr Lys Asn Lys Gln Lys Ile Ala Pro 210 215 220 Ala Tyr Pro Arg Glu Leu Glu Ile Arg Tyr Pro Lys Val Ser Ala Lys 225 230 235 240 Asn Pro Thr Val Gln Phe His Leu Leu Asn Ile Ala Ser Ser Gln Glu 245 250 255 Thr Thr Ile Pro Val Thr Ala Phe Pro Glu Asn Asp Leu Val Ile Gly 260 265 270 Glu Val Ala Trp Leu Ser Ser Gly His Asp Ser Val Ala Tyr Arg Ala 275 280 285 Phe Asn Arg Val Gln Asp Arg Glu Lys Ile Val Ser Val Lys Val Glu 290 295 300 Ser Lys Glu Ser Lys Val Ile Arg Glu Arg Asp Gly Thr Asp Gly Trp 305 310 315 320 Ile Asp Asn Leu Leu Ser Met Ser Tyr Ile Gly Asn Val Asn Gly Lys 325 330 335 Glu Tyr Tyr Val Asp Ile Ser Asp Ala Ser Gly Trp Ala His Ile Tyr 340 345 350 Leu Tyr Pro Val Asp Gly Gly Lys Glu Ile Ala Leu Thr Lys Gly Glu 355 360 365 Trp Glu Val Val Ala Ile Leu Lys Val Asp Thr Lys Lys Lys Leu Ile 370 375 380 Tyr Phe Thr Ser Thr Lys Tyr His Ser Thr Thr Arg His Val Tyr Ser 385 390 395 400 Val Ser Tyr Asp Thr Lys Val Met Thr Pro Leu Val Asn Asp Lys Glu 405 410 415 Ala Ala Tyr Tyr Thr Ala Ser Phe Ser Ala Lys Gly Gly Tyr Tyr Ile 420 425 430 Leu Ser Tyr Gln Gly Pro Asn Val Pro Tyr Gln Glu Leu Tyr Ser Thr 435 440 445 Lys Asp Ser Lys Lys Pro Leu Lys Thr Ile Thr Ser Asn Asp Ala Leu 450 455 460 Leu Glu Lys Leu Lys Glu Tyr Lys Leu Pro Lys Val Ser Phe Phe Glu 465 470 475 480 Ile Lys Leu Pro Ser Gly Glu Thr Leu Asn Val Lys Gln Arg Leu Pro 485 490 495 Pro Asn Phe Asn Pro His Lys Lys Tyr Pro Val Leu Phe Thr Pro Tyr 500 505 510 Gly Gly Pro Gly Ala Gln Glu Val Ser Gln Ala Trp Asn Ser Leu Asp 515 520 525 Phe Lys Ser Tyr Ile Thr Ser Asp Pro Glu Leu Glu Tyr Val Thr Trp 530 535 540 Thr Val Asp Asn Arg Gly Thr Gly Tyr Lys Gly Arg Lys Phe Arg Ser 545 550 555 560 Ala Val Ala Lys Arg Leu Gly Phe Leu Glu Ala Gln Asp Gln Val Phe 565 570 575 Ala Ala Lys Glu Val Leu Lys Asn Arg Trp Ala Asp Lys Asp His Ile 580 585 590 Gly Ile Trp Gly Trp Ser Tyr Gly Gly Phe Leu Thr Ala Lys Thr Leu 595 600 605 Glu Thr Asp Ser Gly Val Phe Thr Phe Gly Ile Ser Thr Ala Pro Val 610 615 620 Ser Asp Phe Arg Leu Tyr Asp Ser Met Tyr Thr Glu Arg Tyr Met Lys 625 630 635 640 Thr Val Glu Leu Asn Ala Asp Gly Tyr Ser Glu Thr Ala Val His Lys 645 650 655 Val Asp Gly Phe Lys Asn Leu Lys Gly His Tyr Leu Ile Gln His Gly 660 665 670 Thr Gly Asp Asp Asn Val His Phe Gln Asn Ala Ala Val Leu Ser Asn 675 680 685 Thr Leu Met Asn Gly Gly Val Thr Ala Asp Lys Leu Thr Thr Gln Trp 690 695 700 Phe Thr Asp Ser Asp His Gly Ile Arg Tyr Asp Met Asp Ser Thr Tyr 705 710 715 720 Gln Tyr Lys Gln Leu Ser Lys Met Val Tyr Asp Gln Lys Gln Arg Arg 725 730 735 Pro Glu Ser Pro Pro Met His Gln Trp Ser Lys Arg Val Leu Ala Ala 740 745 750 Leu Phe Gly Glu Arg Ala Glu Glu 755 760 <210> 2 <211> 479 <212> PRT <213> Artificial Sequence <220> <223> Leucine aminopeptidase <400> 2 His Pro Val Val Gly Gln Glu Pro Phe Gly Trp Pro Phe Lys Pro Met 1 5 10 15 Val Thr Gln Asp Asp Leu Gln Asn Lys Ile Lys Leu Lys Asp Ile Met 20 25 30 Ala Gly Val Glu Lys Leu Gln Ser Phe Ser Asp Ala His Pro Glu Lys 35 40 45 Asn Arg Val Phe Gly Gly Asn Gly His Lys Asp Thr Val Glu Trp Ile 50 55 60 Tyr Asn Glu Ile Lys Ala Thr Gly Tyr Tyr Asp Val Lys Lys Gln Glu 65 70 75 80 Gln Val His Leu Trp Ser His Ala Glu Ala Ala Leu Asn Ala Asn Gly 85 90 95 Lys Asp Leu Lys Ala Ser Ala Met Ser Tyr Ser Pro Pro Ala Ser Lys 100 105 110 Ile Met Ala Glu Leu Val Val Ala Lys Asn Asn Gly Cys Asn Ala Thr 115 120 125 Asp Tyr Pro Ala Asn Thr Gln Gly Lys Ile Val Leu Val Glu Arg Gly 130 135 140 Val Cys Ser Phe Gly Glu Lys Ser Ala Gln Ala Gly Asp Ala Lys Ala 145 150 155 160 Ala Gly Ala Ile Val Tyr Asn Asn Val Pro Gly Ser Leu Ala Gly Thr 165 170 175 Leu Gly Gly Leu Asp Lys Arg His Val Pro Thr Ala Gly Leu Ser Gln 180 185 190 Glu Asp Gly Lys Asn Leu Ala Thr Leu Val Ala Ser Gly Lys Ile Asp 195 200 205 Val Thr Met Asn Val Ile Ser Leu Phe Glu Asn Arg Thr Thr Trp Asn 210 215 220 Val Ile Ala Glu Thr Lys Gly Gly Asp His Asn Asn Val Ile Met Leu 225 230 235 240 Gly Ala His Ser Asp Ser Val Asp Ala Gly Pro Gly Ile Asn Asp Asn 245 250 255 Gly Ser Gly Ser Ile Gly Ile Met Thr Val Ala Lys Ala Leu Thr Asn 260 265 270 Phe Lys Leu Asn Asn Ala Val Arg Phe Ala Trp Trp Thr Ala Glu Glu 275 280 285 Phe Gly Leu Leu Gly Ser Thr Phe Tyr Val Asn Ser Leu Asp Asp Arg 290 295 300 Glu Leu His Lys Val Lys Leu Tyr Leu Asn Phe Asp Met Ile Gly Ser 305 310 315 320 Pro Asn Phe Ala Asn Gln Ile Tyr Asp Gly Asp Gly Ser Ala Tyr Asn 325 330 335 Met Thr Gly Pro Ala Gly Ser Ala Glu Ile Glu Tyr Leu Phe Glu Lys 340 345 350 Phe Phe Asp Asp Gln Gly Ile Pro His Gln Pro Thr Ala Phe Thr Gly 355 360 365 Arg Ser Asp Tyr Ser Ala Phe Ile Lys Arg Asn Val Pro Ala Gly Gly 370 375 380 Leu Phe Thr Gly Ala Glu Val Val Lys Thr Pro Glu Gln Val Lys Leu 385 390 395 400 Phe Gly Gly Glu Ala Gly Val Ala Tyr Asp Lys Asn Tyr His Arg Lys 405 410 415 Gly Asp Thr Val Ala Asn Ile Asn Lys Gly Ala Ile Phe Leu Asn Thr 420 425 430 Arg Ala Ile Ala Tyr Ala Ile Ala Glu Tyr Ala Arg Ser Leu Lys Gly 435 440 445 Phe Pro Thr Arg Pro Lys Thr Gly Lys Arg Asp Val Asn Pro Gln Tyr 450 455 460 Ser Lys Met Pro Gly Gly Gly Cys Gly His His Thr Val Phe Met 465 470 475 <210> 3 <211> 745 <212> PRT <213> Artificial Sequence <220> <223> Dipeptidylpeptidase <400> 3 Val Pro Pro Arg Glu Pro Arg Ser Pro Thr Gly Gly Gly Asn Lys Leu 1 5 10 15 Leu Thr Tyr Lys Glu Cys Val Pro Arg Ala Thr Ile Ser Pro Arg Ser 20 25 30 Thr Ser Leu Ala Trp Ile Asn Ser Glu Glu Asp Gly Arg Tyr Ile Ser 35 40 45 Gln Ser Asp Asp Gly Ala Leu Ile Leu Gln Asn Ile Val Thr Asn Thr 50 55 60 Asn Lys Thr Leu Val Ala Ala Asp Lys Val Pro Lys Gly Tyr Tyr Asp 65 70 75 80 Tyr Trp Phe Lys Pro Asp Leu Ser Ala Val Leu Trp Ala Thr Asn Tyr 85 90 95 Thr Lys Gln Tyr Arg His Ser Tyr Phe Ala Asn Tyr Phe Ile Leu Asp 100 105 110 Ile Lys Lys Gly Ser Leu Thr Pro Leu Ala Gln Asp Gln Ala Gly Asp 115 120 125 Ile Gln Tyr Ala Gln Trp Ser Pro Met Asn Asn Ser Ile Ala Tyr Val 130 135 140 Arg Gly Asn Asp Leu Tyr Ile Trp Asn Asn Gly Lys Thr Lys Arg Ile 145 150 155 160 Thr Glu Asn Gly Gly Pro Asp Ile Phe Asn Gly Val Pro Asp Trp Val 165 170 175 Tyr Glu Glu Glu Ile Phe Gly Asp Arg Phe Ala Leu Trp Phe Ser Pro 180 185 190 Asp Gly Glu Tyr Leu Ala Tyr Leu Arg Phe Asn Glu Thr Gly Val Pro 195 200 205 Thr Tyr Thr Ile Pro Tyr Tyr Lys Asn Lys Gln Lys Ile Ala Pro Ala 210 215 220 Tyr Pro Arg Glu Leu Glu Ile Arg Tyr Pro Lys Val Ser Ala Lys Asn 225 230 235 240 Pro Thr Val Gln Phe His Leu Leu Asn Ile Ala Ser Ser Gln Glu Thr 245 250 255 Thr Ile Pro Val Thr Ala Phe Pro Glu Asn Asp Leu Val Ile Gly Glu 260 265 270 Val Ala Trp Leu Ser Ser Gly His Asp Ser Val Ala Tyr Arg Ala Phe 275 280 285 Asn Arg Val Gln Asp Arg Glu Lys Ile Val Ser Val Lys Val Glu Ser 290 295 300 Lys Glu Ser Lys Val Ile Arg Glu Arg Asp Gly Thr Asp Gly Trp Ile 305 310 315 320 Asp Asn Leu Leu Ser Met Ser Tyr Ile Gly Asn Val Asn Gly Lys Glu 325 330 335 Tyr Tyr Val Asp Ile Ser Asp Ala Ser Gly Trp Ala His Ile Tyr Leu 340 345 350 Tyr Pro Val Asp Gly Gly Lys Glu Ile Ala Leu Thr Lys Gly Glu Trp 355 360 365 Glu Val Val Ala Ile Leu Lys Val Asp Thr Lys Lys Lys Leu Ile Tyr 370 375 380 Phe Thr Ser Thr Lys Tyr His Ser Thr Thr Arg His Val Tyr Ser Val 385 390 395 400 Ser Tyr Asp Thr Lys Val Met Thr Pro Leu Val Asn Asp Lys Glu Ala 405 410 415 Ala Tyr Tyr Thr Ala Ser Phe Ser Ala Lys Gly Gly Tyr Tyr Ile Leu 420 425 430 Ser Tyr Gln Gly Pro Asn Val Pro Tyr Gln Glu Leu Tyr Ser Thr Lys 435 440 445 Asp Ser Lys Lys Pro Leu Lys Thr Ile Thr Ser Asn Asp Ala Leu Leu 450 455 460 Glu Lys Leu Lys Glu Tyr Lys Leu Pro Lys Val Ser Phe Phe Glu Ile 465 470 475 480 Lys Leu Pro Ser Gly Glu Thr Leu Asn Val Lys Gln Arg Leu Pro Pro 485 490 495 Asn Phe Asn Pro His Lys Lys Tyr Pro Val Leu Phe Thr Pro Tyr Gly 500 505 510 Gly Pro Gly Ala Gln Glu Val Ser Gln Ala Trp Asn Ser Leu Asp Phe 515 520 525 Lys Ser Tyr Ile Thr Ser Asp Pro Glu Leu Glu Tyr Val Thr Trp Thr 530 535 540 Val Asp Asn Arg Gly Thr Gly Tyr Lys Gly Arg Lys Phe Arg Ser Ala 545 550 555 560 Val Ala Lys Arg Leu Gly Phe Leu Glu Ala Gln Asp Gln Val Phe Ala 565 570 575 Ala Lys Glu Val Leu Lys Asn Arg Trp Ala Asp Lys Asp His Ile Gly 580 585 590 Ile Trp Gly Trp Ser Tyr Gly Gly Phe Leu Thr Ala Lys Thr Leu Glu 595 600 605 Thr Asp Ser Gly Val Phe Thr Phe Gly Ile Ser Thr Ala Pro Val Ser 610 615 620 Asp Phe Arg Leu Tyr Asp Ser Met Tyr Thr Glu Arg Tyr Met Lys Thr 625 630 635 640 Val Glu Leu Asn Ala Asp Gly Tyr Ser Glu Thr Ala Val His Lys Val 645 650 655 Asp Gly Phe Lys Asn Leu Lys Gly His Tyr Leu Ile Gln His Gly Thr 660 665 670 Gly Asp Asp Asn Val His Phe Gln Asn Ala Ala Val Leu Ser Asn Thr 675 680 685 Leu Met Asn Gly Gly Val Thr Ala Asp Lys Leu Thr Thr Gln Trp Phe 690 695 700 Thr Asp Ser Asp His Gly Ile Arg Tyr Asp Met Asp Ser Thr Tyr Gln 705 710 715 720 Tyr Lys Gln Leu Ser Lys Met Val Tyr Asp Gln Lys Gln Arg Arg Pro 725 730 735 Glu Ser Pro Pro Met His Gln Trp Ser 740 745 <210> 4 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> Leucine aminopeptidase <400> 4 Glu Pro Phe Gly Trp Pro Phe Lys Pro Met Val Thr Gln Asp Asp Leu 1 5 10 15 Gln Asn Lys Ile Lys Leu Lys Asp Ile Met Ala Gly Val Glu Lys Leu 20 25 30 Gln Ser Phe Ser Asp Ala His Pro Glu Lys Asn Arg Val Phe Gly Gly 35 40 45 Asn Gly His Lys Asp Thr Val Glu Trp Ile Tyr Asn Glu Ile Lys Ala 50 55 60 Thr Gly Tyr Tyr Asp Val Lys Lys Gln Glu Gln Val His Leu Trp Ser 65 70 75 80 His Ala Glu Ala Ala Leu Asn Ala Asn Gly Lys Asp Leu Lys Ala Ser 85 90 95 Ala Met Ser Tyr Ser Pro Pro Ala Ser Lys Ile Met Ala Glu Leu Val 100 105 110 Val Ala Lys Asn Asn Gly Cys Asn Ala Thr Asp Tyr Pro Ala Asn Thr 115 120 125 Gln Gly Lys Ile Val Leu Val Glu Arg Gly Val Cys Ser Phe Gly Glu 130 135 140 Lys Ser Ala Gln Ala Gly Asp Ala Lys Ala Ala Gly Ala Ile Val Tyr 145 150 155 160 Asn Asn Val Pro Gly Ser Leu Ala Gly Thr Leu Gly Gly Leu Asp Lys 165 170 175 Arg His Val Pro Thr Ala Gly Leu Ser Gln Glu Asp Gly Lys Asn Leu 180 185 190 Ala Thr Leu Val Ala Ser Gly Lys Ile Asp Val Thr Met Asn Val Ile 195 200 205 Ser Leu Phe Glu Asn Arg Thr Thr Trp Asn Val Ile Ala Glu Thr Lys 210 215 220 Gly Gly Asp His Asn Asn Val Ile Met Leu Gly Ala His Ser Asp Ser 225 230 235 240 Val Asp Ala Gly Pro Gly Ile Asn Asp Asn Gly Ser Gly Ser Ile Gly 245 250 255 Ile Met Thr Val Ala Lys Ala Leu Thr Asn Phe Lys Leu Asn Asn Ala 260 265 270 Val Arg Phe Ala Trp Trp Thr Ala Glu Glu Phe Gly Leu Leu Gly Ser 275 280 285 Thr Phe Tyr Val Asn Ser Leu Asp Asp Arg Glu Leu His Lys Val Lys 290 295 300 Leu Tyr Leu Asn Phe Asp Met Ile Gly Ser Pro Asn Phe Ala Asn Gln 305 310 315 320 Ile Tyr Asp Gly Asp Gly Ser Ala Tyr Asn Met Thr Gly Pro Ala Gly 325 330 335 Ser Ala Glu Ile Glu Tyr Leu Phe Glu Lys Phe Phe Asp Asp Gln Gly 340 345 350 Ile Pro His Gln Pro Thr Ala Phe Thr Gly Arg Ser Asp Tyr Ser Ala 355 360 365 Phe Ile Lys Arg Asn Val Pro Ala Gly Gly Leu Phe Thr Gly Ala Glu 370 375 380 Val Val Lys Thr Pro Glu Gln Val Lys Leu Phe Gly Gly Glu Ala Gly 385 390 395 400 Val Ala Tyr Asp Lys Asn Tyr His Arg Lys Gly Asp Thr Val Ala Asn 405 410 415 Ile Asn Lys Gly Ala Ile Phe Leu Asn Thr Arg Ala Ile Ala Tyr Ala 420 425 430 Ile Ala Glu Tyr Ala Arg Ser Leu Lys Gly Phe Pro Thr Arg Pro Lys 435 440 445 Thr Gly Lys 450 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 5 Pro Gln Pro Gln Pro Phe Pro 1 5 <210> 6 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 6 Pro Gln Gln Pro Tyr 1 5 <210> 7 <211> 33 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 7 Leu Gln Leu Gln Pro Phe Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro 1 5 10 15 Gln Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln Pro 20 25 30 Phe

Claims

1. A polypeptide comprising a truncated dipeptidyl peptidase IV (DPPIV) polypeptide.

2. The polypeptide of claim 1, wherein the truncated DPPIV polypeptide has (i) one or two amino acids deleted at the N-terminus and / or (ii) up to 15 amino acids deleted at the C-terminus compared to the wild-type DPPIV polypeptide.

3. The DPPIV polypeptide according to claim 1 or 2, which has 12 to 14 amino acids deleted at the C-terminus compared to the wild-type DPPIV polypeptide.

4. The DPPIV polypeptide according to any one of claims 1 to 3, which has 13 or 14 amino acids deleted at the C-terminus compared to the wild-type DPPIV polypeptide.

5. The polypeptide according to any one of claims 1 to 4, wherein the wild-type DPPIV polypeptide has the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof.

6. (i) a polypeptide comprising a truncated DPPIV polypeptide consisting of the amino acid sequence represented by residues 1 to 748 of SEQ ID NO: 1; (ii) a polypeptide comprising a truncated DPPIV polypeptide consisting of the amino acid sequence represented by residues 1 to 747 of SEQ ID NO: 1; (iii) a polypeptide comprising a truncated DPPIV polypeptide consisting of the amino acid sequence represented by residues 1 to 746 of SEQ ID NO: 1; (iv) a polypeptide comprising a truncated DPPIV polypeptide consisting of the amino acid sequence represented by residues 1 to 745 of SEQ ID NO: 1; (v) a polypeptide comprising a truncated DPPIV polypeptide consisting of the amino acid sequence represented by residues 2 to 748 of SEQ ID NO: 1; (vi) a polypeptide comprising a truncated DPPIV polypeptide consisting of the amino acid sequence represented by residues 2 to 747 of SEQ ID NO: 1; (vii) a polypeptide comprising a truncated DPPIV polypeptide consisting of the amino acid sequence represented by residues 2 to 746 of SEQ ID NO: 1; and (viii) a polypeptide comprising a truncated DPPIV polypeptide consisting of the amino acid sequence represented by residues 2 to 745 of SEQ ID NO: 1 A polypeptide selected from the group consisting of:

7. The polypeptide according to any one of claims 1 to 6, which is derived from Trichophyton rubrum.

8. The polypeptide of any one of claims 1 to 7, which cleaves the dipeptide motif NH2-X-Pro from the N-terminus of the polypeptide.

9. The polypeptide according to any one of claims 1 to 8, which is produced in Pichia pastoris.

10. A polypeptide comprising a truncated leucine aminopeptidase (LAP) polypeptide.

11. 11. The polypeptide of claim 10, wherein the truncated LAP polypeptide has (i) up to 7 amino acids deleted at the N-terminus and / or (ii) up to 23 amino acids deleted at the C-terminus compared to the wild-type LAP polypeptide.

12. The polypeptide according to claim 10 or 11, which has 4 to 6 amino acids deleted at the N-terminus compared to the wild-type LAP polypeptide.

13. The polypeptide according to any one of claims 10 to 12, which has six amino acids deleted at the N-terminus compared to the wild-type LAP polypeptide.

14. The polypeptide according to any one of claims 10 to 13, which has between 20 and 22 amino acids deleted at the C-terminus compared to the wild-type LAP polypeptide.

15. The polypeptide of any one of claims 10 to 14, which has 8, 16, 21, or 22 amino acids deleted at the C-terminus compared to the wild-type LAP polypeptide.

16. The polypeptide according to any one of claims 10 to 15, which has 22 amino acids deleted at the C-terminus compared to the wild-type LAP polypeptide.

17. The polypeptide of any one of claims 10 to 16, wherein the wild-type LAP polypeptide has the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof.

18. (i) a polypeptide comprising a truncated LAP polypeptide consisting of the amino acid sequence represented by residues 7 to 471 of SEQ ID NO:2; (ii) a polypeptide comprising a truncated LAP polypeptide consisting of the amino acid sequence represented by residues 7 to 463 of SEQ ID NO:2; (iii) a polypeptide comprising a truncated LAP polypeptide consisting of the amino acid sequence represented by residues 7 to 458 of SEQ ID NO:2; (iv) a polypeptide comprising a truncated LAP polypeptide consisting of the amino acid sequence represented by residues 7 to 457 of SEQ ID NO:2; and (v) a polypeptide comprising a truncated LAP polypeptide consisting of the amino acid sequence represented by residues 7 to 456 of SEQ ID NO:

2. A polypeptide selected from the group consisting of:

19. The polypeptide according to any one of claims 10 to 18, which is derived from Trichophyton rubrum.

20. 20. The polypeptide of any one of claims 10 to 19, wherein a single amino acid is truncated from the N-terminus of the polypeptide, except when the single amino acid is connected to a proline by the sequence NH2-X-Pro.

21. The polypeptide according to any one of claims 10 to 20, produced in Pichia pastoris.

22. (i) a polypeptide according to any one of claims 1 to 9; (ii) a polypeptide according to any one of claims 10 to 21, or (iii) A combination of (i) and (ii) A composition comprising:

23. (i) a polypeptide according to any one of claims 1 to 9, and (ii) a polypeptide according to any one of claims 10 to 21 A composition comprising:

24. 24. The composition of claim 23, wherein the weight ratio of the polypeptide of any one of claims 1 to 9 to the polypeptide of any one of claims 10 to 21 is between 1:20 and 1:5, preferably between 1:15 and 1:7.5, more preferably about 1:9.

5.

25. The following 33-mer peptide: LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF 25. The composition according to claim 23 or 24, which completely decomposes

26. A composition according to any one of claims 23 to 25 for pharmaceutical use.

27. A composition according to any one of claims 23 to 26 for use in the treatment of celiac disease.

28. The composition according to any one of claims 23 to 27, which is an oral composition, in particular a liquid oral composition.

29. A nucleic acid encoding the polypeptide according to any one of claims 1 to 9.

30. A cell transfected with the nucleic acid of claim 29.

31. A nucleic acid encoding the polypeptide according to any one of claims 10 to 21.

32. A cell transfected with the nucleic acid of claim 31.

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