IgA protease truncates, fusion proteins containing IgA protease truncates and uses thereof

Abstract

This application relates to IgA protease truncates, fusion proteins comprising IgA protease truncates (eg, fusion proteins comprising IgA protease truncates and Fc) and their use in the treatment of IgA deposition diseases (eg, IgA nephropathy).

Description

[Technical field]

[0001] The present application relates to the field of biopharmaceuticals, in particular, the present application relates to IgA protease truncates, fusion proteins comprising the IgA protease truncates, pharmaceutical compositions comprising said IgA protease truncates or said fusion proteins, nucleic acids encoding said IgA protease truncates or said fusion proteins, methods for preparing said IgA protease truncates or said fusion proteins, and the use of IgA protease truncates or said fusion proteins in the preparation of medicaments for treating IgA deposition-associated diseases. [Background technology]

[0002] IgA nephropathy is currently one of the most common primary glomerular diseases in the world, and it places a great burden on patients and society. At present, there is no specific treatment for IgA nephropathy. In clinical practice, RAS inhibitor-based supportive therapy is often used, which slows the decline of renal function. For patients who do not respond to supportive therapy, a combination of steroids and immunosuppressants is used.

[0003] However, the use of steroids and immunosuppressants has low long-term efficacy and causes severe side effects in patients.

[0004] For this reason, there is an urgent need to develop effective therapeutic drugs with few side effects. Summary of the Invention

[0005] In one aspect, the present application provides an isolated IgA protease truncation comprising a non-naturally occurring truncated fragment of a wild-type IgA protease obtained or derived from Clostridium ramosum, or having at least 70% sequence identity to said non-naturally occurring truncated fragment. In some embodiments, said non-naturally occurring truncated fragment is based on said Clostridium ramosum wild-type IgA protease, and has an amino acid substitution, deletion, insertion, or modification that eliminates or reduces the autoenzymatic cleavage function of said IgA protease truncation. In some embodiments, said amino acid substitution, deletion, insertion, or modification occurs at the native autoenzymatic cleavage site of said Clostridium ramosum wild-type IgA protease, within 5 sites upstream of said native autoenzymatic cleavage site, and / or within 5 sites downstream of said native autoenzymatic cleavage site. In some embodiments, the Clostridium ramosum is Clostridium ramosum AK183 strain. In some embodiments, the amino acid sequence of the wild-type IgA protease of Clostridium ramosum is as set forth in SEQ ID NO: 1. In some embodiments, the natural autoenzyme cleavage site is between positions 730 and 840 (e.g., between positions 792 and 797) of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the natural autoenzyme cleavage site is at positions 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, or 800 of the amino acid sequence set forth in SEQ ID NO: 1.

[0006] In some embodiments, the non-naturally occurring truncated fragment is an N-terminally truncated fragment or a C-terminally truncated fragment of a wild-type IgA protease obtained or derived from Clostridium ramosum, in some embodiments, the N-terminally truncated fragment comprises a polypeptide fragment of at least 760 contiguous amino acids from position 31 N-terminal of a wild-type IgA protease obtained or derived from Clostridium ramosum, or has at least 70% sequence identity thereto. In some embodiments, the IgA protease truncates provided herein comprise at least 760 (e.g., at least 761, at least 762, at least 763, at least 764, at least 765, at least 766, at least 767, at least 768, at least 769, at least 770, at least 771, at least 772, at least 773, at least 774, at least 775, at least 776, at least 777, at least 778, at least 779, at least 780, at least 781, at least 782, at least 783, at least 784, at least 785, at least 786 at least 787, at least 788, at least 789, at least 790, at least 791, at least 792, at least 793, at least 794, at least 795, at least 796, at least 797, at least 798, at least 799, at least 800, at least 801, at least 802, at least 803, at least 804, at least 805, at least 806, at least 807, at least 808, at least 809, at least 810, at least 900, at least 950, at least 1000, at least 1100, at least 1150 or at least 1200 consecutive amino acids.In some embodiments, the IgA protease truncate provided by the present application comprises a polypeptide fragment selected from the group consisting of amino acids 31 to 790 of the amino acid sequence shown in SEQ ID NO:1, amino acids 31 to 792 of the amino acid sequence shown in SEQ ID NO:1, amino acids 31 to 798 of the amino acid sequence shown in SEQ ID NO:1, amino acids 31 to 807 of the amino acid sequence shown in SEQ ID NO:1, amino acids 31 to 816 of the amino acid sequence shown in SEQ ID NO:1, amino acids 31 to 833 of the amino acid sequence shown in SEQ ID NO:1, and polypeptide fragments having at least 70% sequence identity thereto.

[0007] In some embodiments, the non-naturally occurring truncated fragment comprises a polypeptide fragment of at least 456 contiguous amino acids from position 335 towards the N-terminus of a wild-type IgA protease obtained or derived from Clostridium ramosum, or has at least 90% or at least 95% sequence identity thereto. In some embodiments, the IgA protease truncates provided herein comprise at least 456 (e.g., at least 457, at least 458, at least 459, at least 460, at least 461, at least 462, at least 463, at least 464, at least 465, at least 466, at least 467, at least 468, at least 469, at least 470, at least 471, at least 472, at least 473, at least 474, at least 475, at least 476, at least 477, at least 478, at least 479, at least 48 In some embodiments, the polypeptide comprises a polypeptide fragment of at least 0, at least 481, at least 482, at least 483, at least 484, at least 485, at least 486, at least 487, at least 488, at least 489, at least 490, at least 491, at least 492, at least 493, at least 494, at least 495, at least 496, at least 497, at least 498, at least 499, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850 or at least 900 consecutive amino acids.In some embodiments, the IgA protease truncate provided by the present application comprises a polypeptide fragment selected from the group consisting of amino acids 335 to 790 of the amino acid sequence shown in SEQ ID NO:1, amino acids 335 to 791 of the amino acid sequence shown in SEQ ID NO:1, amino acids 335 to 792 of the amino acid sequence shown in SEQ ID NO:1, amino acids 285 to 790 of the amino acid sequence shown in SEQ ID NO:1, amino acids 285 to 791 of the amino acid sequence shown in SEQ ID NO:1, amino acids 285 to 792 of the amino acid sequence shown in SEQ ID NO:1, amino acids 330 to 790 of the amino acid sequence shown in SEQ ID NO:1, amino acids 330 to 791 of the amino acid sequence shown in SEQ ID NO:1, amino acids 330 to 792 of the amino acid sequence shown in SEQ ID NO:1, amino acids 285 to 816 of the amino acid sequence shown in SEQ ID NO:1, and polypeptide fragments having at least 90% or at least 95% sequence identity thereto.

[0008] In some embodiments, the IgA protease truncates provided herein have conservative amino acid substitutions at one or more sites based on the amino acid sequence of the polypeptide fragment. In some embodiments, the polypeptide fragment has amino acid mutations at one or more of positions 844, 862, 931, 933, 978, 1002, and 1004 of SEQ ID NO:1. In some embodiments, the polypeptide fragment has a mutation to glycine at one or more of positions 844, 862, 931, 933, 978, 1002, and 1004 of SEQ ID NO:1. In some embodiments, the polypeptide fragment has an amino acid mutation at a position corresponding to 844, an amino acid mutation at a position corresponding to 862, an amino acid mutation at positions corresponding to 931 and 933, an amino acid mutation at a position corresponding to 978, or an amino acid mutation at positions corresponding to 1002 and 1004 of SEQ ID NO:1. In some embodiments, the amino acid sequence of the polypeptide fragment is set forth in SEQ ID NO:53 (also referred to as "PA-GA Mut"), SEQ ID NO:54 (also referred to as "PI-GI Mut"), SEQ ID NO:55 (also referred to as "PAP-GAG Mut"), SEQ ID NO:56 (also referred to as "PAT-GAT Mut") or SEQ ID NO:57 (also referred to as "PIP-GIG Mut").

[0009] In some embodiments, the IgA protease truncates provided herein have the enzymatic activity of specifically cleaving human IgA. In some embodiments, the IgA protease truncates provided herein have the enzymatic activity of specifically cleaving human IgA heavy chain. In some embodiments, the IgA protease truncates provided herein have the enzymatic activity of specifically cleaving the junction of the human IgA heavy chain CH1 and the hinge region. In some embodiments, the IgA protease truncates provided herein have the enzymatic activity of specifically cleaving human IgA1.

[0010] In another aspect, the present application provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a full-length wild-type IgA protease obtained or derived from Clostridium ramosum, a polypeptide obtained by removing the signal peptide of a wild-type IgA protease obtained or derived from Clostridium ramosum, or an IgA protease truncate as described herein, and the second polypeptide comprises an amino acid sequence for extending the half-life of the first polypeptide in a subject. In some embodiments, the first polypeptide comprises a sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 42. In some embodiments, the second polypeptide is located at the N-terminus or C-terminus of the first polypeptide.

[0011] In some embodiments, the first polypeptide and the second polypeptide are linked by a linker. In some embodiments, the first polypeptide and the second polypeptide are directly linked. In some embodiments, the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker. In some embodiments, the linker comprises a peptide linker. In some embodiments, the peptide linker comprises a linker containing glycine and serine. In some embodiments, the linker containing glycine and serine comprises one, two, three, four, or more repeats of SEQ ID NO:21 (GGGS), SEQ ID NO:22 (GGGGS), SEQ ID NO:86 (GGGGGS), or SEQ ID NO:87 (GGGGGGGS). In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 23 (GGCGGCGGTGGATCC), SEQ ID NO: 58 (EEKKKEKEKEEQEERETK), or SEQ ID NO: 59 (HHHHHHHHHH).

[0012] In some embodiments, the second polypeptide is selected from an Fc domain and albumin. In some embodiments, the Fc domain comprises a hinge region. In some embodiments, the Fc domain is derived from a human IgG Fc domain. In some embodiments, the Fc domain is derived from a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, or a human IgG4 Fc domain. In some embodiments, the Fc domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:32, or SEQ ID NO:77. In some embodiments, the Fc domain comprises an amino acid sequence as set forth in SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:32, or SEQ ID NO:77. In some embodiments, the Fc domain comprises an amino acid mutation at a position corresponding to position 7 of SEQ ID NO:25. In some embodiments, the Fc domain comprises an amino acid mutation at a position corresponding to position 7 of SEQ ID NO:25 (e.g., an alanine) to a valine, glycine, serine, or leucine. In some embodiments, the Fc domain comprises one or more mutations that extend the half-life of the fusion protein. In some embodiments, the Fc domain is linked to the C-terminus or N-terminus of the first polypeptide. In some embodiments, the albumin comprises one or more domains of human serum albumin. In some embodiments, the albumin comprises the D3 domain of human serum albumin.

[0013] In some embodiments, the fusion protein described herein further comprises a tag. In some embodiments, the tag is selected from the group consisting of a fluorescent tag, a luminescent tag, a purification tag, and a chromogenic tag. In some embodiments, the tag is selected from the group consisting of a c-Myc tag, an HA tag, a VSV-G tag, a FLAG tag, a V5 tag, and a HIS tag. In some embodiments, the tag is a HIS tag comprising 6, 7, 8, 9, or 10 histidines. In some embodiments, the second polypeptide is located at the C-terminus of the first polypeptide and the tag is located at the C-terminus of the second polypeptide.

[0014] In some embodiments, the half-life of the fusion proteins described herein in the blood circulation of a subject is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days.

[0015] In another aspect, the present application provides an isolated nucleic acid comprising a nucleotide sequence encoding an IgA protease truncate described herein or comprising a nucleotide sequence encoding a fusion protein described herein. In some embodiments, the nucleic acid described herein comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, and nucleotide sequences having at least 70% sequence identity thereto.

[0016] In another aspect, the present application provides a vector comprising a nucleic acid described herein.

[0017] In another aspect, the present application provides a cell comprising a nucleic acid described herein or a vector described herein. In some embodiments, the cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the prokaryotic cell is an E. coli cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell or a Chinese Hamster Ovary (CHO) cell. In some embodiments, the mammalian cell is a Human Embryonic Kidney 293 (HEK293 cell).

[0018] In another aspect, the present application provides a pharmaceutical composition comprising an IgA protease truncate described herein, a fusion protein described herein, a nucleic acid described herein, a vector described herein, or a cell described herein, and a pharma- ceutically acceptable carrier.

[0019] In another aspect, the present application provides a method for producing a fusion protein, comprising culturing a cell described herein.

[0020] In another aspect, the present application provides a method for treating or preventing an IgA deposition associated disease, comprising administering to a subject in need of such treatment or prevention an IgA protease truncate described herein, a fusion protein described herein, or a pharmaceutical composition described herein.

[0021] In another aspect, the present application provides the use of an IgA protease truncate described herein, a fusion protein described herein, or a pharmaceutical composition described herein in the preparation of a medicament for treating or preventing an IgA deposition associated disease.

[0022] In another aspect, the present application provides an IgA protease truncate described herein, a fusion protein described herein, or a pharmaceutical composition described herein for treating or preventing an IgA deposition associated disease.

[0023] In another aspect, the present application provides a method of treating or preventing an IgA deposition associated disease, comprising administering to a subject in need of such treatment or prevention an IgA protease or a truncate thereof, a fusion protein comprising said IgA protease or a truncate thereof, or a pharmaceutical composition comprising said IgA protease or a truncate thereof or said fusion protein, wherein the amino acid sequence of said IgA protease is selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, or a combination thereof.

[0024] In another aspect, the present application provides the use of an IgA protease or a truncate thereof, a fusion protein comprising said IgA protease or a truncate thereof, or a pharmaceutical composition comprising said IgA protease or a truncate thereof or said fusion protein in the preparation of a medicament for treating or preventing an IgA deposition associated disease, wherein the amino acid sequence of said IgA protease is selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76 or a combination thereof.

[0025] In another aspect, the present application provides an IgA protease or a truncate thereof, a fusion protein comprising said IgA protease or a truncate thereof, or a pharmaceutical composition comprising said IgA protease or a truncate thereof or said fusion protein for treating or preventing an IgA deposition associated disease, wherein the amino acid sequence of said IgA protease is selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76 or a combination thereof.

[0026] In some embodiments, the IgA deposition associated disease includes IgA nephropathy, dermatitis herpetiformis, Henoch-Scholein purpura (also known as IgA vasculitis), Kawasaki disease, purpura nephritis, kidney damage due to IgA vasculitis, IgA rheumatoid factor positive rheumatoid arthritis, IgA anti-GBM disease, or IgA ANCA associated vasculitis. In some embodiments, the IgA deposition associated disease is IgA nephropathy, IgA vasculitis, or Kawasaki disease. [Brief description of the drawings]

[0027] [Figure 1] FIG. 1 shows the results of an in vitro enzyme cleavage activity experiment against IgA1 of four IgA protease truncates, AK183(31-737), AK183(31-768), AK183(31-798), and AK183(31-833). [Diagram 2] FIG. 2 shows the results of an in vitro enzyme cleavage activity experiment against IgA1 of five IgA protease truncates, AK183(31-773), AK183(31-778), AK183(31-782), AK183(31-787), and AK183(31-792). [Diagram 3] 3a to 3c show the results of in vitro enzyme cleavage activity experiments against IgA1 of four IgA protease truncates, AK183(31-788), AK183(31-789), AK183(31-790), and AK183(31-791). [Figure 4] FIG. 4 shows a flow diagram of the construction of the PET30a-AK183(31-790)-Fc plasmid. [Diagram 5] FIG. 5 shows the expression results of the AK183(31-790)-Fc fusion protein. [Figure 6] Figure 6a shows the expression results of the AK183(31-792)-Fc fusion protein. Figure 6b shows the results of an in vitro enzyme cleavage activity experiment of the AK183(31-792)-Fc fusion protein against IgA1. [Figure 7] FIG. 7 shows the results of an in vitro enzyme cleavage activity experiment against IgA1 of four types of fusion proteins, AK183(31-798)-Fc, AK183(31-807)-Fc, AK183(31-816)-Fc, and AK183(31-833)-Fc. [Figure 8] FIG. 8 shows the results of an experiment on the in vivo enzymatic cleavage activity of the AK183(31-807)-Fc fusion protein against IgA1. [Figure 9] FIG. 9 shows the results of expression of the AK183(31-792)-Fc fusion protein in HEK293 cells. [Figure 10] FIG. 10 shows the results of an in vitro enzyme cleavage activity experiment against IgA1 of seven IgA protease truncates, AK183(285-792), AK183(330-792), AK183(380-792), AK183(430-792), AK183(480-792), AK183(530-792) and AK183(580-792). [Figure 11] FIG. 11 shows the results of an in vitro enzyme cleavage activity experiment against IgA1 of nine IgA protease truncates, AK183(335-792), AK183(340-792), AK183(345-792), AK183(350-792), AK183(355-792), AK183(360-792), AK183(365-792), AK183(370-792), and AK183(375-792). [Figure 12] FIG. 12 shows the results of an in vitro enzyme cleavage activity experiment against IgA1 of four IgA protease truncates, AK183(336-792), AK183(337-792), AK183(338-792) and AK183(339-792). [Figure 13]Figure 13 shows the results of a re-examination experiment of the in vitro enzymatic cleavage activity of 10 types of IgA protease truncates, AK183(285-792), AK183(330-792), AK183(335-792), AK183(336-792), AK183(337-792), AK183(338-792), AK183(339-792), AK183(340-792), AK183(345-792) and AK183(350-792), against IgA1. [Figure 14] FIG. 14 shows the expression results of two types of fusion proteins, AK183(285-816)-Fc and Fc-AK183(285-816). [Figure 15] FIG. 15 shows the results of an in vitro enzyme cleavage activity experiment against IgA1 of two types of fusion proteins, AK183(285-816)-Fc and Fc-AK183(285-816). [Figure 16] FIG. 16 shows the results of an experiment on the enzymatic cleavage activity of the Fc-AK183(285-816) fusion protein, the AK183(285-816)-Fc fusion protein, and the AK183(285-816) IgA protease truncate against IgA1. [Figure 17] FIG. 17 shows the results of an experiment on the in vivo enzymatic cleavage activity of the Fc-AK183(285-816) fusion protein against IgA1. [Figure 18] FIG. 18 shows the results of an experiment on the enzymatic cleavage activity of AK183(285-816)-Fc fusion protein and Fc-AK183(31-1203) fusion protein against IgA1. [Figure 19] FIG. 19 shows the results of an experiment on the enzymatic cleavage activity of the AK183(31-816)-IgG1 Fc fusion protein, the AK183(31-816)-IgG4 Fc fusion protein, and the AK183(31-816)-albumin fusion protein against IgA1. [Figure 20]FIG. 20 shows the results of an enzyme cleavage activity experiment against IgA1 of AK183(285-816)-Fc fusion proteins having different linkers (sequence number 59, sequence number 58, sequence number 22, sequence number 78, sequence number 79, or sequence number 80). [Figure 21] FIG. 21 shows the results of an experiment on the enzymatic cleavage activity of five IgA protease truncation mutants shown in SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, or SEQ ID NO:57 against IgA1. [Figure 22] FIG. 22 shows the results of an enzyme cleavage activity experiment against IgA1 of four mutants each of which has four different mutations in the Fc region of the AK183(31-816)-Fc fusion protein. [Figure 23a] FIG. 23a shows the results of an experiment on the enzyme cleavage activity of 16 types of AK183 homologous enzymes against IgA1. [Figure 23b] FIG. 23b shows the results of an experiment on the enzyme cleavage activity of 16 types of AK183 homologous enzymes against IgA1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] In the present application, various aspects and embodiments are disclosed below, but it is obvious to those skilled in the art that various equivalent changes and modifications can be made without departing from the spirit and scope of the subject matter of the present application. The various aspects and embodiments disclosed by the present application are merely examples for explanation and do not limit the present application. The actual scope of protection of the present application is based on the claims. Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs. All references, patents, and patent applications cited in this application are incorporated herein by reference in their entirety.

[0029] definition As used herein, the terms "Clostridium ramosum" or "Clostridium" refer to the bacterium Clostridium ramosum (also called Ramibacterium ramosum), a commensal bacterium present in the human intestine that is capable of producing IgA protease.

[0030] The term "protease" as used herein refers to an enzyme capable of degrading proteins and peptides. Proteases can degrade proteins by hydrolyzing the peptide bonds that connect the amino acids in the peptide or polypeptide chain that forms the protein. In the prior art, there are many methods known for measuring the proteolytic activity of a particular type of protease. For example, the proteolytic activity of a protease can be determined by comparatively measuring and analyzing the hydrolytic ability of various proteases on a suitable substrate. Examples of substrates for proteolytic activity analysis include, for example, dimethylcasein, bovine collagen, bovine elastin, etc. Colorimetric methods using these substrates are also known in the prior art (see, for example, WO99 / 34011 and US6,376,450).

[0031] As used herein, the term "IgA protease" refers to an enzyme capable of specifically cleaving or degrading a subject's (e.g., human) IgA immunoglobulin molecule (e.g., IgA1 or IgA2). For example, IgA protease obtained or derived from Clostridium ramosum can specifically cleave the peptide bond between proline (Pro) at position 221 and valine (Val) at position 222 of IgA1 and IgA2 to degrade IgA1 and IgA2.

[0032] As used herein, the term "wild type" when referring to a polypeptide or protein refers to a naturally occurring polypeptide or protein that does not contain an artificial substitution, insertion, deletion, or modification at one or more amino acid positions. As used herein, the term "wild type" when referring to a nucleic acid, nucleotide, or polynucleotide refers to a naturally occurring nucleic acid, nucleotide, or polynucleotide that does not contain an artificial substitution, insertion, deletion, or modification at one or more nucleotide positions. However, a polynucleotide encoding a wild type polypeptide is not limited to naturally occurring polynucleotides, but includes any polynucleotide that encodes a wild type polypeptide.

[0033] The term "AK183" used in this application refers to the AK183 strain of Clostridium ramosum. The amino acid sequence of the wild-type IgA protease produced by the Clostridium ramosum AK183 strain is as shown in SEQ ID NO: 1 (among which, amino acids 1 to 30 are a signal peptide).

[0034] [ka] [ka]

[0035] The term "signal peptide" as used herein refers to a sequence of amino acid residues that may be involved in the secretion or directional transport of a protein in its mature or precursor form. A signal peptide is usually located at the N-terminus of a precursor or mature protein sequence. A signal peptide may be endogenous or exogenous. A mature protein generally does not have a signal peptide. After protein transport, a signal peptide is usually cleaved from the protein by a signal peptidase. For example, the amino acid sequence of SEQ ID NO: 1 excluding the N-terminal signal peptide is as shown in SEQ ID NO: 42.

[0036]

[0037] The term "subject" as used herein includes humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals. A "subject" may be a livestock animal, e.g., cows, pigs, sheep, poultry, and horses, or a rodent, e.g., a rat, a mouse, or a primate, e.g., an ape, monkey, chimpanzee, gorilla, orangutan, or baboon, or a domestic animal, e.g., a dog or a cat. A "subject" may be male or female, elderly, adult, adolescent, child, or infant. A human "subject" may be of Caucasian, African, Asian, Semitic, or other race, or a mixture of said racial backgrounds.

[0038] As used herein, the terms "protein," "polypeptide," and "peptide" are used interchangeably to refer to a polymer of amino acids. The proteins, polypeptides, or peptides described herein may contain naturally occurring amino acids, non-naturally occurring amino acids, or amino acid analogs or mimetics. The proteins, polypeptides, or peptides described herein may be obtained by any method known in the art, including, but not limited to, natural isolation, recombinant expression, or chemical synthesis.

[0039] As used herein, the term "amino acid" refers to an organic compound containing amino (-NH2) and carboxyl (-COOH) functional groups and a side chain characteristic of each amino acid. In this application, the names of amino acids may be abbreviated to their standard one-letter or three-letter abbreviations, as summarized below.

[0040] [Table 1]

[0041] In this application, "conservative substitution" when used in reference to an amino acid sequence refers to the replacement of one amino acid residue with another amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can be made between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between amino acid residues with neutral and hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between amino acid residues with acidic side chains (e.g., Asp, Glu), between amino acid residues with basic side chains (e.g., His, Lys, and Arg), or between amino acid residues with aromatic side chains (e.g., Trp, Tyr, Phe). As is known in the art, conservative substitutions usually do not cause significant changes in the conformation of a protein, and therefore the biological activity of the protein is maintained.

[0042] As used herein, the term "homologous" refers to a nucleic acid sequence (or its complementary strand) or amino acid sequence that has at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with another sequence when optimally aligned.

[0043] "Percentage (%) sequence identity" when used with respect to an amino acid sequence (or nucleic acid sequence) refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in a reference sequence, after sequence alignment and introducing spacing as necessary to maximize the number of identical amino acids (or nucleic acids). In other words, the percentage (%) sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of identical amino acid residues (or bases) in the reference sequence to be compared by the total number of amino acid residues (or bases) in the candidate sequence or the reference sequence (the shorter one is taken as the reference). Conservative substitutions of the amino acid residues may or may not be considered as identical residues. The sequences can be aligned and the percentage sequence identity of amino acid (or nucleic acid) sequences can be determined using tools disclosed in the art, such as BLASTN, BLASTp (National Center for Biotechnology Information website (NCBI), see also Altschul SF et al., J. Mol. Biol., 215: 403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25: 3389-3402 (1997)), ClustalW2 (European Bioinformatics Institute website, see also Higgins DG et al., Methods in Enzymology, 266: 383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23 (21): 2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. A person skilled in the art can use the default parameters of said tools or can suitably adjust the parameters as required for the alignment, for example by selecting an appropriate algorithm.

[0044] An "isolated" material is altered from its natural state by the human eye. If an "isolated" composition or material occurs in nature, it has already been altered, deviated from its original state, or both. For example, a polynucleotide or polypeptide that naturally occurs in the body of an animal is not "isolated," but if such a polynucleotide or polypeptide is sufficiently separated from the coexisting materials in nature to exist in a substantially pure state, it is considered to be "isolated." An "isolated nucleic acid sequence" refers to a sequence of an isolated nucleic acid molecule. In some embodiments, an "isolated IgA protease truncate" refers to an IgA protease truncate that is at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure. Purity is determined by electrophoresis (eg, SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatography (eg, ion exchange chromatography or reverse phase HPLC).

[0045] The term "vector" as used herein refers to a delivery tool into which a genetic element is operably inserted to express the genetic element (e.g., to produce a protein, RNA, or DNA encoded by the genetic element, or to replicate the genetic element). A vector is used to transform, transduce, or transfect a host cell, and to express the genetic element it carries within the host cell. For example, vectors include plasmids, phagemids, cosmids, artificial chromosomes (e.g., yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs)), bacteriophages (e.g., lambda phage or M13 phage), and animal viruses. A vector may contain various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. A vector may also contain an origin of replication. A vector may also contain components that aid in entry into a cell, including, but not limited to, viral particles, liposomes, or protein coats. The vector may be an expression vector or a cloning vector. The vectors provided herein (e.g., expression vectors) contain a nucleic acid sequence encoding an IgA protease truncated or fusion protein described herein, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker.

[0046] "Treatment" or "therapy" of a particular disease, symptom, or condition, as used herein, includes preventing or alleviating the particular disease, symptom, or condition, reducing the rate at which a particular disease, symptom, or condition occurs or progresses, reducing the risk of developing a particular disease, symptom, or condition, preventing or slowing the progression of symptoms associated with a particular disease, symptom, or condition, reducing or halting symptoms associated with a particular disease, symptom, or condition, causing a complete or partial improvement of a particular disease, symptom, or condition, curing a particular disease, symptom, or condition, or combinations thereof.

[0047] The term "pharmaceutical acceptable" means that a specified vector, vehicle, diluent, excipient, and / or salt is compatible, usually chemically and / or physically, with the other ingredients that make up the formulation, and physiologically compatible with its recipient.

[0048] The term "IgA deposition-associated disease" refers to a disease associated with the accumulation of IgA immunoglobulins in aggregated or nonaggregated form in tissues or organs of a subject, including, but not limited to, IgA nephropathy, dermatitis herpetiformis, Henoch-Scholein purpura (also known as IgA vasculitis), Kawasaki disease, purpura nephritis, renal damage due to IgA vasculitis, IgA rheumatoid factor-positive rheumatoid arthritis, IgA anti-GBM disease, or IgA ANCA-associated vasculitis.

[0049] The term "IgA nephropathy" refers to a kidney disease characterized by the deposition of IgA in the kidney.

[0050] IgA Protease Truncates In one aspect, the present application provides an isolated IgA protease truncation comprising or having at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% sequence identity) to a non-naturally occurring truncated fragment of a wild-type IgA protease obtained or derived from Clostridium ramosum. In some embodiments, the IgA protease truncates having at least 70% sequence identity to the non-naturally occurring truncated fragments (e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% sequence identity) retain a function or activity of the IgA protease (e.g., proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.).

[0051] The term "truncate" or "truncate fragment" as used herein refers to a peptide obtained by removing one or more amino acids from one or both termini of a wild-type polypeptide. Thus, a "truncate" or "truncate fragment" as used herein does not include the full length of the corresponding wild-type polypeptide, but may include one or more amino acid substitutions, deletions, insertions, or modifications, etc., compared to the truncated form of the wild-type polypeptide. For example, an "IgA protease truncate" or an "IgA protease truncated fragment" may include a peptide obtained by removing one or more amino acids from one or both termini of a wild-type IgA protease, or may include a peptide in which one or more amino acids have been substituted, deleted, inserted, or modified compared to the truncated form of the wild-type IgA protease.

[0052] In some embodiments, the IgA protease truncates described herein have one or more amino acid substitutions, deletions, insertions, or modifications compared to the corresponding wild-type IgA protease. For example, in some embodiments, the IgA protease truncates described herein include a non-naturally occurring truncated fragment of a wild-type IgA protease obtained or derived from Clostridium ramosum, the non-naturally occurring truncated fragment being based on the wild-type IgA protease of Clostridium ramosum with an amino acid substitution, deletion, insertion, or modification that eliminates or reduces the autoenzymatic cleavage function of the IgA protease truncate.

[0053] As used herein, the terms "obtained from" and "derived from" include not only proteins produced or capable of being produced by the organisms referenced, but also proteins encoded by DNA sequences isolated from such organisms and produced in host organisms carrying such DNA sequences, as well as proteins encoded by synthetic and / or cDNA-derived DNA sequences and having characteristics characteristic of the proteins referenced. For example, wild-type IgA protease obtained or derived from Clostridium ramosum includes the IgA protease naturally produced by Clostridium ramosum, as well as the IgA protease produced by other host cells (e.g., E. coli) transformed with a nucleic acid encoding the IgA protease using genetic engineering techniques.

[0054] As used herein, the term "non-natural truncated fragment" refers to a fragment having an amino acid sequence (e.g., a different amino acid length or a different type of amino acid) different from the truncated fragment produced by autoenzymatic cleavage of wild-type IgA protease of Clostridium ramosum in a natural environment.

[0055] In some embodiments, the amino acid substitution, deletion, insertion, or modification occurs at the native autoenzyme cleavage site of the wild-type IgA protease of Clostridium ramosum. In some embodiments, the amino acid substitution, deletion, insertion, or modification occurs within 5 sites upstream of the native autoenzyme cleavage site of the wild-type IgA protease of Clostridium ramosum (e.g., 1 site, 2 sites, 3 sites, 4 sites, or 5 sites upstream of the native autoenzyme cleavage site). In some embodiments, the amino acid substitution, deletion, insertion, or modification occurs within 5 sites downstream of the native autoenzyme cleavage site of the wild-type IgA protease of Clostridium ramosum (e.g., 1 site, 2 sites, 3 sites, 4 sites, or 5 sites downstream of the native autoenzyme cleavage site). In some embodiments, the amino acid substitution, deletion, insertion, or modification occurs within 5 sites upstream (e.g., 1, 2, 3, 4, or 5 sites upstream of the native autoenzyme cleavage site) and within 5 sites downstream (e.g., 1, 2, 3, 4, or 5 sites downstream of the native autoenzyme cleavage site) of the wild-type Clostridium ramosum IgA protease.

[0056] In some embodiments, the non-native truncated fragment is an N-terminally truncated fragment or a C-terminally truncated fragment of a wild-type IgA protease obtained or derived from Clostridium ramosum.

[0057] The term "N-terminal truncated fragment" as used herein refers to a truncated fragment of an amino acid sequence that includes the amino terminus of wild-type IgA protease of Clostridium ramosum. The start position of the "amino terminus" may be any position near the amino terminus of the amino acid sequence of wild-type IgA protease of Clostridium ramosum. For example, it may be position 1 counting from the amino terminus, or it may be any other position counting from the amino terminus. For example, if the full-length amino acid sequence of wild-type IgA protease consists of 1000 amino acids, the start position of the amino terminus of the N-terminal truncated fragment may be any position between positions 1 and 500 from the amino terminus of the amino acid sequence.

[0058] The term "C-terminal truncated fragment" as used herein refers to a truncated fragment of an amino acid sequence that includes the carboxyl terminus of wild-type IgA protease of Clostridium ramosum. The end position of the "carboxyl terminus" may be any position near the carboxyl terminus of the amino acid sequence of wild-type IgA protease of Clostridium ramosum. For example, it may be the first position counting from the carboxyl terminus, or any other position counting from the carboxyl terminus. For example, if the full-length amino acid sequence of wild-type IgA protease consists of 1000 amino acids, the end position of the carboxyl terminus of the C-terminal truncated fragment may be any position between 501 and 1000 from the amino terminus of the amino acid sequence.

[0059] Clostridium ramosum is one of many species in the genus Clostridium, including many strains such as AK183, VPI-0496A, and NCTC 10474. In some embodiments, the Clostridium ramosum is Clostridium ramosum AK183.

[0060] In some embodiments, the N-terminal truncated fragment comprises a polypeptide fragment of at least 760 contiguous amino acids from position 31 towards the N-terminus of a wild-type IgA protease obtained or derived from Clostridium ramosum, or has at least 70% sequence identity thereto (e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% sequence identity thereto). In some embodiments, N-terminally truncated fragments having at least 70% sequence identity to the polypeptide fragment (e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% sequence identity) retain a function or activity of an IgA protease (e.g., proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.).

[0061] In some embodiments, the non-naturally occurring truncated fragment of the IgA protease described herein comprises a polypeptide fragment of at least 456 contiguous amino acids from position 335 towards the N-terminus of a wild-type IgA protease obtained or derived from Clostridium ramosum, or has at least 90% or at least 95% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity) thereto. In some embodiments, non-naturally occurring truncated fragments having at least 90% or at least 95% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to the polypeptide fragment retain a function or activity of an IgA protease (e.g., proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.).

[0062] In some embodiments, the amino acid sequence of the Clostridium ramosum wild-type IgA protease is as set forth in SEQ ID NO:1.

[0063] Unless otherwise stated, the amino acid site of the IgA protease referred to herein corresponds to the amino acid site of the wild-type AK183 IgA protease (whose amino acid sequence is as set forth in SEQ ID NO: 1). For example, position 790 of the AK183 IgA protease referred to herein corresponds to the 790th site of SEQ ID NO: 1. Unless otherwise stated, the naming rule of the AK183 IgA protease truncates referred to herein is "AK183 (start point corresponding to SEQ ID NO: 1-end point corresponding to SEQ ID NO: 1)". For example, AK183 (31-790) is an IgA protease truncate formed by amino acids 31 to 790 of SEQ ID NO: 1.

[0064] In some embodiments, the natural autoenzyme cleavage site of the IgA protease described in the application is between positions 730 and 840 of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the natural autoenzyme cleavage site of the IgA protease described in the present application is between positions 710 and 830, between positions 720 and 820, between positions 730 and 810, between positions 740 and 800, between positions 750 and 790, between positions 791 and 780, or between positions 792 and 797 of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the natural autoenzyme cleavage site is at positions 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, or 800 of the amino acid sequence shown in SEQ ID NO: 1.

[0065] In some embodiments, the IgA protease truncates provided herein comprise a polypeptide fragment of at least 760 contiguous amino acids from position 31 of the amino acid sequence set forth in SEQ ID NO:1. For example, in some embodiments, the IgA protease truncates provided herein comprise at least 761, at least 762, at least 763, at least 764, at least 765, at least 766, at least 767, at least 768, at least 769, at least 770, at least 771, at least 772, at least 773, at least 774, at least 775, at least 776, at least 777, at least 778, at least 779, at least 780, at least 781, at least 782, at least 783, at least 784, at least 785, at least 786, at least 787, at least 788, at least 789, at least 790, at least 791, at least 792, at least 793, at least at least 794, at least 795, at least 796, at least 797, at least 798, at least 799, at least 800, at least 801, at least 802, at least 803, at least 804, at least 805, at least 806, at least 807, at least 808, at least 809, at least 810, at least 850, at least 860, at least 870, at least 880, at least 890, at least 900, at least 910, at least 920, at least 930, at least 940, at least 950, at least 960, at least 970, at least 980, at least 990, at least 1000, at least 1050, at least 1100, at least 1150, at least 1200 consecutive amino acids.

[0066] In some embodiments, the IgA protease truncate provided herein comprises a polypeptide fragment of 760 contiguous amino acids from position 31 of the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the IgA protease truncate provided herein comprises a polypeptide fragment of 761 contiguous amino acids from position 31 of the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the IgA protease truncate provided herein comprises a polypeptide fragment of 762 contiguous amino acids from position 31 of the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the IgA protease truncate provided herein comprises a polypeptide fragment of 768 contiguous amino acids from position 31 of the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the IgA protease truncate provided herein comprises a polypeptide fragment of 777 contiguous amino acids from position 31 of the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the IgA protease truncate provided herein comprises a polypeptide fragment of 786 contiguous amino acids from position 31 of the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the IgA protease truncates provided herein comprise a polypeptide fragment of 803 contiguous amino acids from position 31 of the amino acid sequence set forth in SEQ ID NO:1.

[0067] In some embodiments, the IgA protease truncates provided herein are those having amino acids at positions 31 to 790 of the amino acid sequence shown in SEQ ID NO:1, amino acids at positions 31 to 792 of the amino acid sequence shown in SEQ ID NO:1, amino acids at positions 31 to 798 of the amino acid sequence shown in SEQ ID NO:1, amino acids at positions 31 to 807 of the amino acid sequence shown in SEQ ID NO:1, amino acids at positions 31 to 816 of the amino acid sequence shown in SEQ ID NO:1, amino acids at positions 31 to 833 of the amino acid sequence shown in SEQ ID NO:1, or amino acids having at least 70% sequence identity thereto (e.g., at least The polypeptide fragments include polypeptide fragments selected from the group consisting of polypeptide fragments having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% sequence identity thereto. In some embodiments, the IgA protease truncates having at least 70% sequence identity to the polypeptide fragments (e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% sequence identity) retain a function or activity of the IgA protease (e.g., proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.).

[0068] In some embodiments, the IgA protease truncates provided herein comprise a polypeptide fragment of at least 456 contiguous amino acids from position 335 of the amino acid sequence set forth in SEQ ID NO: 1. For example, in some embodiments, the IgA protease truncates provided herein comprise a polypeptide fragment of at least 457, at least 458, at least 459, at least 460, at least 461, at least 462, at least 463, at least 464, at least 465, at least 466, at least 467, at least 468, at least 469, at least 470, at least 471, at least 472, at least 473, at least 474, at least 475, at least 476, at least 477, at least 478, at least 479, at least 480, at least 481, at least 482, at least 483, at least 484, at least 485, at least 486, at least 487, at least 488, at least 489, at least 490, at least 500, at least 501, at least 502, at least 503, at least 504, at least 505, at least 506, at least 507, at least 508, at least 509, at least 510, at least 511, at least 512, at least 513, at least 514, at least 515, at least 516, at least 517, at least 518, at least 519, at least 520, at least 521, at least 522, at least 523, at least 524, at least 525, at least 526, at least 52 at least 481, at least 482, at least 483, at least 484, at least 485, at least 486, at least 487, at least 488, at least 489, at least 490, at least 491, at least 492, at least 493, at least 494, at least 495, at least 496, at least 497, at least 498, at least 499, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850 or at least 900 consecutive amino acids.

[0069] In some embodiments, the IgA protease truncate provided by the present application is selected from the group consisting of amino acids 335 to 790 of the amino acid sequence shown in SEQ ID NO: 1, amino acids 335 to 791 of the amino acid sequence shown in SEQ ID NO: 1, amino acids 335 to 792 of the amino acid sequence shown in SEQ ID NO: 1, amino acids 285 to 790 of the amino acid sequence shown in SEQ ID NO: 1, amino acids 285 to 791 of the amino acid sequence shown in SEQ ID NO: 1, amino acids 285 to 792 of the amino acid sequence shown in SEQ ID NO: 1, amino acids 330 to 790 of the amino acid sequence shown in SEQ ID NO: 1, and amino acids 335 to 791 of the amino acid sequence shown in SEQ ID NO: 1. The IgA protease truncates include polypeptide fragments selected from the group consisting of amino acids 330 to 791 of the amino acid sequence shown in SEQ ID NO: 1, amino acids 330 to 792 of the amino acid sequence shown in SEQ ID NO: 1, amino acids 285 to 816 of the amino acid sequence shown in SEQ ID NO: 1, and polypeptide fragments having at least 90% or at least 95% sequence identity thereto (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity). In some embodiments, the IgA protease truncates having at least 90% or at least 95% sequence identity thereto (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity) retain the function or activity of IgA protease (e.g., proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.).

[0070] In some embodiments, the present application provides an AK183(31-790) truncate having the amino acid sequence set forth in SEQ ID NO:14. (SEQ ID NO:14)

[0071] In some embodiments, the present application provides an AK183(31-791) truncate having the amino acid sequence set forth in SEQ ID NO:15. (SEQ ID NO:15)

[0072] In some embodiments, the present application provides an AK183(31-792) truncate having the amino acid sequence set forth in SEQ ID NO:16. (SEQ ID NO:16)

[0073] In some embodiments, the present application provides an AK183(31-798) truncate having the amino acid sequence set forth in SEQ ID NO:17. (SEQ ID NO:17)

[0074] In some embodiments, the present application provides an AK183(31-807) truncate having the amino acid sequence set forth in SEQ ID NO:18. (SEQ ID NO:18)

[0075] In some embodiments, the present application provides an AK183(31-816) truncate having the amino acid sequence set forth in SEQ ID NO:19. (SEQ ID NO:19)

[0076] In some embodiments, the present application provides an AK183(31-833) truncate having the amino acid sequence set forth in SEQ ID NO:20. (SEQ ID NO:20)

[0077] In some embodiments, the present application provides an AK183(285-790) truncate having an amino acid sequence as set forth in SEQ ID NO: 43. In some embodiments, the present application provides an AK183(285-791) truncate having an amino acid sequence as set forth in SEQ ID NO: 44. In some embodiments, the present application provides an AK183(285-792) truncate having an amino acid sequence as set forth in SEQ ID NO: 45. In some embodiments, the present application provides an AK183(285-816) truncate having an amino acid sequence as set forth in SEQ ID NO: 46. In some embodiments, the present application provides an AK183(330-790) truncate having an amino acid sequence as set forth in SEQ ID NO: 47. In some embodiments, the present application provides an AK183(330-791) truncate having an amino acid sequence as set forth in SEQ ID NO: 48. In some embodiments, the present application provides an AK183(330-792) truncate having an amino acid sequence as set forth in SEQ ID NO: 49. In some embodiments, the present application provides an AK183(335-790) truncate having an amino acid sequence as set forth in SEQ ID NO: 50. In some embodiments, the present application provides an AK183(335-791) truncate having an amino acid sequence as set forth in SEQ ID NO: 51. In some embodiments, the present application provides an AK183(335-792) truncate having an amino acid sequence as set forth in SEQ ID NO: 52.

[0078] The sequences of SEQ ID NOs: 43 to 52 are as follows. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0079] In some embodiments, the IgA protease truncates provided herein have conservative substitutions of amino acids at one or more sites (e.g., one, two, three, four, five or more sites) based on the amino acid sequence of the aforementioned polypeptide fragment. Conservative substitutions of amino acid residues refer to substitutions between amino acids with similar properties, such as between polar amino acids (e.g., between glutamine and asparagine), between hydrophobic amino acids (e.g., between leucine, isoleucine, methionine, and valine), and between amino acids with the same charge (e.g., between arginine, lysine, and histidine, or between glutamic acid and aspartic acid). In some embodiments, the IgA protease truncates described herein have conservative amino acid substitutions at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 15, 20 or more positions compared to the amino acid sequence set forth in SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:52.

[0080] In some embodiments, the polypeptide fragment has an amino acid mutation at a position corresponding to one or more of positions 844, 862, 931, 933, 978, 1002, and 1004 of SEQ ID NO:1. In some embodiments, the polypeptide fragment has an amino acid mutation at a position corresponding to position 844 of SEQ ID NO:1. In some embodiments, the polypeptide fragment has an amino acid mutation at a position corresponding to position 862 of SEQ ID NO:1. In some embodiments, the polypeptide fragment has an amino acid mutation at positions corresponding to positions 931 and 933 of SEQ ID NO:1. In some embodiments, the polypeptide fragment has an amino acid mutation at a position corresponding to position 978 of SEQ ID NO:1. In some embodiments, the polypeptide fragment has an amino acid mutation at positions corresponding to positions 1002 and 1004 of SEQ ID NO:1.

[0081] In some embodiments, the polypeptide fragment is mutated to glycine at positions corresponding to one or more of positions 844, 862, 931, 933, 978, 1002, and 1004 of SEQ ID NO:1. In some embodiments, the polypeptide fragment is mutated from proline (P) to glycine (G) at positions corresponding to one or more of positions 844, 862, 931, 933, 978, 1002, and 1004 of SEQ ID NO:1. In some embodiments, the polypeptide fragment is mutated from proline to glycine at position corresponding to position 844 of SEQ ID NO:1. In some embodiments, the polypeptide fragment is mutated from proline to glycine at position corresponding to position 862 of SEQ ID NO:1. In some embodiments, the polypeptide fragment is mutated from proline to glycine at positions corresponding to positions 931 and 933 of SEQ ID NO:1. In some embodiments, the polypeptide fragment is mutated from proline to glycine at position corresponding to position 978 of SEQ ID NO:1. In some embodiments, the polypeptide fragment has proline mutated to glycine at positions corresponding to positions 1002 and 1004 of SEQ ID NO:1.

[0082] In some embodiments, the amino acid sequence of the polypeptide fragment is set forth in SEQ ID NO:53 (also referred to as "PA-GA Mut"), SEQ ID NO:54 (also referred to as "PI-GI Mut"), SEQ ID NO:55 (also referred to as "PAP-GAG Mut"), SEQ ID NO:56 (also referred to as "PAT-GAT Mut") or SEQ ID NO:57 (also referred to as "PIP-GIG Mut").

[0083] The sequences of SEQ ID NOs: 53 to 57 are as follows. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0084] Provided that activity is not affected, the IgA protease truncates provided herein may further include unnatural amino acids, including, for example, β-fluoroalanine, 1-methylhistidine, γ-methylene glutamic acid, α-methylleucine, 4,5-dehydrolysine, hydroxyproline, 3-fluorophenylalanine, 3-aminotyrosine, 4-methyltryptophan, and the like.

[0085] The IgA protease truncates provided herein may be modified using methods known in the art, including, but not limited to, PEGylation, glycosylation, amino-terminal modification, fatty acylation, carboxyl-terminal modification, phosphorylation, methylation, etc. As will be appreciated by those skilled in the art, the IgA protease truncates provided herein retain substantially similar functionality to the IgA protease or IgA protease truncates after modification using methods known in the art.

[0086] In some embodiments, the IgA protease truncates provided herein have the enzymatic activity of specifically cleaving human IgA. In some embodiments, the IgA protease truncates provided herein have the enzymatic activity of specifically cleaving human IgA heavy chain. In some embodiments, the IgA protease truncates provided herein have the enzymatic activity of specifically cleaving the junction of the human IgA heavy chain CH1 and the hinge region. In some embodiments, the IgA protease truncates provided herein have the enzymatic activity of specifically cleaving human IgA1.

[0087] In some embodiments, the IgA protease truncates provided herein have conservative amino acid substitutions at one or more sites based on the amino acid sequence of the aforementioned polypeptide fragments, but retain the enzymatic activity to cleave human IgA (e.g., IgA1). In some embodiments, the IgA protease truncates provided herein have at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% sequence identity) to the aforementioned polypeptide fragments, and retain the enzymatic activity to cleave human IgA (e.g., IgA1).

[0088] Fusion proteins In another aspect, the present application provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a full-length wild-type IgA protease obtained or derived from Clostridium ramosum, a polypeptide obtained by removing the signal peptide of a wild-type IgA protease obtained or derived from Clostridium ramosum, or an IgA protease truncate as described herein, and the second polypeptide comprises an amino acid sequence for extending the half-life of the first polypeptide in a subject. In some embodiments, the first polypeptide comprises a sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 42. In some embodiments, the second polypeptide is located at the N-terminus of the first polypeptide. In some embodiments, the second polypeptide is located at the C-terminus of the first polypeptide.

[0089] In some embodiments, the first polypeptide and the second polypeptide are linked by a linker. In some embodiments, the first polypeptide and the second polypeptide are directly linked (i.e., not linked via a linker). As used herein, the term "linker" refers to an artificial amino acid sequence having 1, 2, 3, 4, or 5 amino acid residues, or between 5 and 15, 20, 30, 50 or more amino acid residues in length, linked by peptide bonds, and used to link one or more polypeptides. Linkers may or may not have secondary structure. Linker sequences are known in the art. See, for example, Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al., Structure 2:1121-1123 (1994).

[0090] In some embodiments, the linker is selected from the group consisting of cleavable linkers, non-cleavable linkers, peptide linkers, flexible linkers, rigid linkers, helical linkers, and non-helical linkers. Any suitable linker known in the art can be used. In some embodiments, the linker comprises a peptide linker. For example, a linker useful in the present application can be rich in glycine and serine residues. Examples include linkers having single or repeating sequences containing threonine / serine and glycine, such as GGGS (SEQ ID NO:21) or GGGGS (SEQ ID NO:22), GGGGGS (SEQ ID NO:86) or GGGGGGGS (SEQ ID NO:87), or tandem repeats thereof (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeats). In some embodiments, a linker used in the present application comprises GGCGGCGGTGGATCC (SEQ ID NO:23). Alternatively, the linker may be a long peptide chain comprising one or more sequences or tandem repeats of the amino acid sequence set forth in GGCGGCGGTGGATCC (SEQ ID NO: 23). In some embodiments, the linker comprises one, two, three, four, five, six, seven, eight, nine, ten or more sequences or tandem repeats of SEQ ID NO: 23. In some embodiments, the linker comprises or consists of an amino acid sequence selected from the group consisting of amino acid sequences having 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%, at least 99% sequence identity to any of SEQ ID NOs: 21, 22, 23.

[0091] In some embodiments, a linker for use herein comprises the amino acid sequence set forth in SEQ ID NO:58 (EEKKKEKEKEEQEERETK). Optionally, the linker may be a longer peptide chain comprising one or more sequences or tandem repeats of the amino acid sequence set forth in SEQ ID NO:58. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sequences or tandem repeats of SEQ ID NO:58. In some embodiments, the linker comprises an amino acid sequence having 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%, at least 99% sequence identity to SEQ ID NO:58.

[0092] In some embodiments, a linker for use herein comprises the amino acid sequence set forth in SEQ ID NO: 59 (HHHHHHHHHH). In some embodiments, the linker comprises an amino acid sequence having 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% sequence identity to SEQ ID NO: 59.

[0093] In some embodiments, the second polypeptide is selected from an Fc domain and albumin. In some embodiments, the Fc domain comprises a hinge region. In some embodiments, the Fc domain comprises a lower hinge region. In some embodiments, the Fc domain comprises a core hinge region and a lower hinge region. In some embodiments, the Fc domain comprises an upper hinge region, a core hinge region, and a lower hinge region. In some embodiments, the Fc domain does not comprise a hinge region. In some embodiments, the Fc domain is derived from a human IgG Fc domain. In some embodiments, the Fc domain is derived from a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, or a human IgG4 Fc domain.

[0094] In some embodiments, the Fc domain comprises the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, the Fc domain consists of the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, the amino acid sequence of the Fc domain has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:24. EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 24)

[0095] In some embodiments, the encoding nucleic acid sequence for the Fc domain comprises the nucleotide sequence set forth in SEQ ID NO: 39. In some embodiments, the encoding nucleic acid sequence for the Fc domain consists of the nucleotide sequence set forth in SEQ ID NO: 39. In some embodiments, the encoding nucleic acid sequence for the Fc domain has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO:39. (SEQ ID NO:39)

[0096] In some embodiments, the Fc domain comprises the amino acid sequence set forth in SEQ ID NO: 25. In some embodiments, the Fc domain consists of the amino acid sequence set forth in SEQ ID NO: 25. In some embodiments, the amino acid sequence of the Fc domain has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:25. TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 25)

[0097] In some embodiments, the encoding nucleic acid sequence for the Fc domain comprises the nucleotide sequence set forth in SEQ ID NO: 40. In some embodiments, the encoding nucleic acid sequence for the Fc domain consists of the nucleotide sequence set forth in SEQ ID NO: 40. In some embodiments, the encoding nucleic acid sequence for the Fc domain has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO:40. (Sequence number 40).

[0098] In some embodiments, the Fc domain comprises the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the Fc domain consists of the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the amino acid sequence of the Fc domain has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:32. ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (sequence number 32).

[0099] In some embodiments, the Fc domain comprises the amino acid sequence set forth in SEQ ID NO: 77. In some embodiments, the Fc domain consists of the amino acid sequence set forth in SEQ ID NO: 77. In some embodiments, the amino acid sequence of the Fc domain has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:77. ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 77)

[0100] In some embodiments, the Fc domain comprises one or more amino acid mutations. In some embodiments, the Fc domain comprises an amino acid mutation at a position corresponding to position 7 of SEQ ID NO:25. In some embodiments, the Fc domain has the amino acid (e.g., alanine) at the position corresponding to position 7 of SEQ ID NO:25 mutated to valine. In some embodiments, the Fc domain has the amino acid (e.g., alanine) at the position corresponding to position 7 of SEQ ID NO:25 mutated to glycine. In some embodiments, the Fc domain has the amino acid (e.g., alanine) at the position corresponding to position 7 of SEQ ID NO:25 mutated to serine. In some embodiments, the Fc domain has the amino acid (e.g., alanine) at the position corresponding to position 7 of SEQ ID NO:25 mutated to leucine.

[0101] In some embodiments, the Fc domain comprises one or more mutations that extend the half-life of the fusion protein. In some embodiments, the Fc domain is linked to the C-terminus of the first polypeptide. In some embodiments, the Fc domain is linked to the N-terminus of the first polypeptide.

[0102] In some embodiments, the second polypeptide is albumin. In some embodiments, the amino acid sequence of the albumin is as set forth in SEQ ID NO: 60. In some embodiments, the albumin comprises one or more domains of human serum albumin. In some embodiments, the albumin comprises the D3 domain of human serum albumin. (SEQ ID NO:60)

[0103] In some embodiments, the fusion protein provided herein further comprises a tag. In some embodiments, the tag is selected from the group consisting of a fluorescent tag, a luminescent tag, a purification tag, and a chromogenic tag. In some embodiments, the tag is selected from the group consisting of a c-Myc tag, an HA tag, a VSV-G tag, a FLAG tag, a V5 tag, and a HIS tag. In some embodiments, the tag is a HIS tag. In some embodiments, the tag is a HIS tag comprising 6, 7, 8, 9, or 10 histidines. In some embodiments, the second polypeptide is located at the C-terminus of the first polypeptide, and the tag is located at the C-terminus of the second polypeptide.

[0104] In some embodiments, the fusion protein provided herein comprises an amino acid sequence as set forth in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, or SEQ ID NO:85. In some embodiments, the fusion protein provided herein consists of, or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to, an amino acid sequence selected from the group consisting of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, or SEQ ID NO:85. In some embodiments, a fusion protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, or SEQ ID NO:85 retains a function or activity of an IgA protease (e.g., proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.).

[0105] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10] [Table 4-11]

[0106] In some embodiments, the fusion protein provided herein comprises an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12. In some embodiments, the fusion protein provided herein consists of, or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to, an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12.

[0107] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6]

[0108] In some embodiments, the half-life of the fusion protein provided herein in the blood circulation of a subject is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days.

[0109] nucleic acid In another aspect, the present application provides an isolated nucleic acid comprising a nucleotide sequence encoding an IgA protease truncate described herein, or comprising a nucleotide sequence encoding a fusion protein described herein.

[0110] As used herein, the term "nucleic acid" or "nucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single- or double-stranded form, and polymers thereof. Unless otherwise indicated, a particular nucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and / or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0111] DNA encoding the IgA protease truncates described herein, or the fusion proteins described herein, can be readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes capable of specifically binding to the genes encoding the IgA protease truncates or fusion proteins). Encoding DNA can also be obtained by synthetic methods.

[0112] In some embodiments, the nucleic acid provided herein comprises a nucleic acid sequence set forth in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38. In some embodiments, the nucleic acid provided herein consists of, or has 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%, at least 99% sequence identity to, a nucleotide sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38.

[0113] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 6-8] [Table 6-9] [Table 6-10]

[0114] In some embodiments, the nucleic acids provided herein comprise the nucleic acid sequences set forth in SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13. In some embodiments, the nucleic acids provided herein consist of, or have 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%, at least 99% sequence identity to, a nucleotide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13.

[0115] [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Table 7-8] [Table 7-9] [Table 7-10] [Table 7-11] [Table 7-12] [Table 7-13]

[0116] Vectors and cells In another aspect, the present application provides a vector comprising a nucleic acid encoding an IgA protease truncate described herein or a nucleic acid encoding a fusion protein described herein.

[0117] Using recombinant techniques known in the art, the isolated polynucleotides encoding the IgA protease truncated or fusion proteins may be inserted into a vector for further cloning (DNA amplification) or for expression. A variety of vectors can be selected. The vector components usually include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0118] In some embodiments, the nucleic acid provided herein encodes an IgA protease truncated or fusion protein, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection tag. Illustrative vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, papova viruses (e.g., SV40), lambda and M13 phages, and plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, p These include, but are not limited to, cDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, and the like.

[0119] The vectors containing the nucleic acid sequences encoding the IgA protease truncates or fusion proteins may be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or expressing the DNA in the vectors described herein are prokaryotic, yeast, or higher eukaryotic cells as described above. Prokaryotic cells suitable for use herein include eubacteria, such as gram-negative or gram-positive bacteria. For example, Escherichia (e.g., E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescens), and the like. marcescans), Enterobacteriaceae such as Shigella, Bacilli (e.g., B. subtilis and B. licheniformis), Pseudomonas (e.g., P. aeruginosa), and Streptomyces. In some embodiments, the cell is an E. coli cell.

[0120] In addition to prokaryotic cells, eukaryotic cells, e.g., eukaryotic microbes such as filamentous fungi or yeast, can be used as suitable cloning or expression hosts for vectors encoding IgA protease truncates or fusion proteins. Saccharomyces cerevisiae, or baker's yeast, is the most commonly used lower eukaryotic host microorganism. However, many other genera, species and strains are more commonly used and suitable for use herein. For example, Schizosaccharomyces cerevisiae, pombe, and hosts of Kluyveromyces species (e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. Kluyveromyces waltii (ATCC 56,500), Kluyveromyces drosophilarum (ATCC 36,906), Kluyveromyces thermotolerans, Kluyveromyces marxianus, Yarrowia (EP 402,226), Pichia pastoris pastoris (EP 183,070), Candida, Trichoderma reesia (EP 244,234), Neurospora crassa, Schwanniomyces (e.g. Schwanniomyces occidentalis), and filamentous fungi (e.g. Neurospora, Penicillium), Tolypocladium, and Aspergillus (e.g. Aspergillus nidulans and Aspergillus niger).In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell or a Chinese Hamster Ovary (CHO) cell. In some embodiments, the mammalian cell is a Human Embryonic Kidney 293 (HEK293 cell).

[0121] Pharmaceutical Compositions In another aspect, the present application provides a pharmaceutical composition comprising an IgA protease truncate described herein, a fusion protein described herein, a nucleic acid described herein, a vector described herein, or a cell described herein, and a pharma- ceutically acceptable carrier.

[0122] Pharmaceutically acceptable carriers for the pharmaceutical compositions disclosed herein can include, for example, pharma- ceutically acceptable liquid, gel or solid carriers, aqueous solvents, non-aqueous solvents, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0123] Suitable ingredients may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers, or stabilizers (e.g., sugars and cyclodextrins). Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylmethylanisole, butylhydroxytoluene, and / or propyl gallate. As disclosed herein, the inclusion of one or more antioxidants, such as methionine, in compositions comprising the IgA protease truncates or fusion proteins disclosed herein can reduce oxidation of the IgA protease truncates or fusion proteins. The present application further provides methods for preventing oxidation, extending shelf life, and / or improving activity of the IgA protease truncates or fusion proteins. For example, this can be achieved by combining the IgA protease truncates or fusion proteins provided herein with one or more antioxidants (eg, methionine).

[0124] Additionally, pharma- ceutically acceptable carriers may include, for example, aqueous vehicles (e.g., Sodium Chloride Injection, Ringer's Solution Injection, Isotonic Dextrose Injection, Sterile Water Injection, or Lactated Ringer's Dextrose Injection), non-aqueous vehicles (e.g., fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antibacterial agents in bacterial- or fungal-inhibiting concentrations), isotonic agents (e.g., sodium chloride or glucose), buffers (e.g., phosphate or citrate buffers), antioxidants (e.g., sodium bisulfate), local anesthetics (e.g., procaine hydrochloride), suspending and dispersing agents (e.g., sodium carboxymethylcellulose, hypromellose, or polyvinylpyrrolidone), emulsifying agents (e.g., Polysorbate 80 (Tween 80)), chelating agents (e.g., EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol bis(2-aminoethyl ether)tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid). Antimicrobial agents as carriers can be added to the pharmaceutical compositions in multi-dose containers and include phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients can include, for example, water, salts, glucose, glycerin, or ethanol. Suitable non-toxic auxiliary substances can include, for example, wetting agents, emulsifiers, pH buffers, stabilizers, solubilizers, or substances such as sodium acetate, sorbitan laurate, triethanolamine oleate, or cyclodextrins.

[0125] The pharmaceutical compositions may be in the form of liquid solutions, suspensions, emulsions, pills, capsules, tablets, sustained release formulations or powders. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0126] In some embodiments, the pharmaceutical composition is formulated as an injectable composition. The injectable pharmaceutical composition may be prepared in any conventional form, such as a liquid vehicle, suspension, emulsion, or a solid form suitable for forming a liquid vehicle, suspension, or emulsion. The injectable preparation may include a sterile and / or pyrogen-free solution that can be used immediately, a sterile dry solubilized product (such as a lyophilized powder) that is combined with a solvent when used. This includes a tablet for subcutaneous injection, a sterile suspension that can be used immediately for injection, a sterile dry insoluble product that is combined with a vehicle when used, and a sterile and / or pyrogen-free emulsion. The solvent may be aqueous or non-aqueous.

[0127] In some embodiments, the unit dose of the injectable formulation is packaged in an ampoule, a vial, or a syringe with a needle. As is known in the art, all formulations administered by injection must be sterile and pyrogen-free.

[0128] In some embodiments, a sterile lyophilized powder may be prepared by dissolving the IgA protease truncated or fusion protein disclosed herein in a suitable solvent. The solvent may contain other pharmacological ingredients that can improve the stability of the powder or the reconstituted solution prepared from the powder, or that can improve the powder or the reconstituted solution. Suitable excipients include, but are not limited to, water, glucose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable substances. The solvent may contain a buffer, such as a citrate buffer, a sodium or potassium phosphate buffer, or other buffer known to those skilled in the art, and in one embodiment, the buffer has a neutral pH. The dissolution is followed by sterilization by filtration under standard conditions known in the art, followed by lyophilization to obtain the ideal formulation. In one embodiment, the resulting solvent is dispensed into mini-vials and lyophilized. Each mini-vial may contain a single or multiple doses of the IgA protease truncated or fusion protein or composition thereof. Each minivial may be filled with a slightly larger amount (e.g., 10% more) than is needed for a single dose or multiple doses to ensure accuracy of sampling and dosing. The lyophilized powder may be stored under appropriate conditions, such as at a temperature ranging from about 4° C. to room temperature.

[0129] The lyophilized powder is reconstituted with water for injection to provide a formulation for injectable administration. In one embodiment, the lyophilized powder may be reconstituted by adding sterile, pyrogen-free water or other suitable liquid carrier. The exact amount depends on the selected therapeutic agent and may be determined empirically.

[0130] Methods for Treating or Preventing Disease In another aspect, the present application provides a method for treating or preventing an IgA deposition associated disease, comprising administering to a subject in need of such treatment or prevention an IgA protease truncate described herein, a fusion protein described herein, or a pharmaceutical composition described herein.

[0131] In another aspect, the present application provides a method of treating or preventing an IgA deposition associated disease, comprising administering to a subject in need of such treatment or prevention an IgA protease or a truncate thereof, a fusion protein comprising said IgA protease or a truncate thereof, or a pharmaceutical composition comprising said IgA protease or a truncate thereof or said fusion protein, wherein the amino acid sequence of said IgA protease is selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, or a combination thereof. In some embodiments, the amino acid sequence of the IgA protease is the amino acid sequence of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or SEQ ID NO:76, with the signal peptide removed. In some embodiments, the IgA protease truncates have at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% sequence identity) to the polypeptide set forth in SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or SEQ ID NO:76.In some embodiments, the IgA protease truncates have at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% sequence identity) to the polypeptide set forth in SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or SEQ ID NO:76) and retain a function or activity of an IgA protease (e.g., proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.).

[0132] [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6] [Table 8-7] [Table 8-8] [Table 8-9] [Table 8-10] [Table 8-11] [Table 8-12] [Table 8-13] [Table 8-14] [Table 8-15] [Table 8-16]

[0133] In another aspect, the present application provides the use of an IgA protease truncate described herein, a fusion protein described herein, or a pharmaceutical composition described herein in the preparation of a medicament for treating or preventing an IgA deposition associated disease.

[0134] In another aspect, the present application provides the use of an IgA protease or a truncate thereof, a fusion protein comprising said IgA protease or a truncate thereof, or a pharmaceutical composition comprising said IgA protease or a truncate thereof or said fusion protein in the preparation of a medicament for treating or preventing an IgA deposition associated disease, wherein the amino acid sequence of said IgA protease is selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76 or a combination thereof. In some embodiments, the amino acid sequence of the IgA protease is the amino acid sequence of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or SEQ ID NO:76, with the signal peptide removed. In some embodiments, the IgA protease truncates have at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% sequence identity) to the polypeptide set forth in SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or SEQ ID NO:76.In some embodiments, the IgA protease truncates have at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% sequence identity) to the polypeptide set forth in SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or SEQ ID NO:76) and retain a function or activity of an IgA protease (e.g., proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.).

[0135] In another aspect, the present application provides an IgA protease truncate, a fusion protein, or a pharmaceutical composition described herein for treating or preventing an IgA deposition associated disease.

[0136] In another aspect, the present application provides an IgA protease or a truncate thereof, a fusion protein comprising said IgA protease or a truncate thereof, or a pharmaceutical composition comprising said IgA protease or a truncate thereof or said fusion protein for treating or preventing an IgA deposition associated disease, wherein the amino acid sequence of said IgA protease is selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76 or a combination thereof. In some embodiments, the amino acid sequence of the IgA protease is the amino acid sequence of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or SEQ ID NO:76, with the signal peptide removed. In some embodiments, the IgA protease truncates have at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% sequence identity) to the polypeptide set forth in SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or SEQ ID NO:76.In some embodiments, the IgA protease truncates have at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% sequence identity) to the polypeptide set forth in SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or SEQ ID NO:76) and retain a function or activity of an IgA protease (e.g., proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.).

[0137] In some embodiments, the IgA deposition-associated disease described herein includes IgA nephropathy, dermatitis herpetiformis, Henoch-Scholein purpura (also known as IgA vasculitis), Kawasaki disease, purpura nephritis, kidney damage due to IgA vasculitis, IgA rheumatoid factor positive rheumatoid arthritis, IgA anti-GBM disease, or IgA ANCA-associated vasculitis. In some embodiments, the IgA deposition-associated disease described herein is IgA nephropathy. In some embodiments, the IgA deposition-associated disease described herein is IgA1 nephropathy. In some embodiments, the IgA deposition-associated disease described herein is IgA vasculitis. In some embodiments, the IgA deposition-associated disease described herein is Kawasaki disease. EXAMPLES

[0138] In all examples, the biological materials mentioned (such as E. coli strains, various cloning and expression plasmids, culture media, enzymes as manipulation tools, buffer solutions), as well as various culture methods, protein extraction and purification methods, and other molecular biological manipulation methods are all well known to those skilled in the art. See Molecular Cloning, edited by Sambrook et al. (Laboratory Manual, Cold Spring Harbor, 1989) and Selected Molecular Biology Experimental Protocols (Mi F. Ausubel et al., translated by Yan Ziying et al., Beijing, Science Press, 1998).

[0139] [Example 1] Study of the shortest active site of AK183 IgA protease The inventors removed the N-terminal signal peptide (i.e., amino acids 1 to 30 of SEQ ID NO: 1) and the C-terminal transmembrane domain + intracellular domain (i.e., amino acids 1205 to 1234 of SEQ ID NO: 1) of wild-type IgA protease derived from Clostridium ramosum AK183 strain (the amino acid sequence of which is shown in SEQ ID NO: 1), and then added a human IgG1 Fc sequence (HR-CH2-CH3, the amino acid sequence of which is shown in SEQ ID NO: 24) to the N-terminus of the amino acid sequence of IgA protease from which the signal peptide, transmembrane domain, and intracellular domain had been removed (i.e., an IgA protease truncate consisting of amino acids 31 to 1204 of SEQ ID NO: 1), thereby constructing the PET30a-Fc-AK183 plasmid.

[0140] Next, the inventors introduced nonsense mutations using the PET30a-Fc-AK183 plasmid as a template to construct a series of Fc-AK183 truncations to investigate the shortest C-terminal active site of AK183 IgA protease. Based on the results of preliminary studies, the inventors considered that a self-cleavage site exists between amino acids 730 and 840 of AK183 IgA protease. Therefore, the inventors introduced a first round of nonsense mutations, and the nonsense mutation sites were set at four amino acid sites at positions 738, 769, 799, and 834 of AK183 IgA protease. The results are shown in Figure 1. The results show that the AK183(31-737) and AK183(31-768) IgA protease truncated fragments obtained after nonsense mutations at amino acids 738 and 769 have no in vitro enzyme cleavage activity, while the AK183(31-798) and AK183(31-833) IgA protease truncated fragments obtained after nonsense mutations at amino acids 799 and 834 have activity. Therefore, the conclusion of the first round of nonsense mutations is that the shortest C-terminal active site of AK183 IgA protease is between amino acids 768 and 798. Subsequently, the second round of nonsense mutations was introduced, and the mutation sites were set at five amino acid sites at positions 774, 779, 783, 788, or 793 of AK183 IgA protease. The results are shown in Figure 2. The AK183(31-773), AK183(31-778), AK183(31-782), and AK183(31-787) IgA protease truncated fragments obtained after nonsense mutations at amino acids 774, 779, 783, or 788 had no in vitro enzyme cleavage activity, while the AK183(31-792) IgA protease truncated fragment obtained after nonsense mutation at amino acid 793 retained activity. Therefore, the conclusion of the second round of nonsense mutations is that the C-terminal shortest active site of AK183 IgA protease is between amino acids 787 and 792.Next, the inventors introduced a third round of nonsense mutations, with the mutation sites being four amino acid sites at positions 789, 790, 791, or 792 of the AK183 IgA protease. The results are shown in FIG. 3, where the AK183 (31-788) and AK183 (31-789) IgA protease truncated fragments obtained after nonsense mutations at positions 789 and 790 had no in vitro enzyme cleavage activity, and the AK183 (31-790) and AK183 (31-791) IgA protease truncated fragments obtained after nonsense mutations at positions 791 and 792 retained activity (among these, position 791 may have exhibited incomplete activity and only a slight enzyme cleavage effect due to the conformation of the protease). Therefore, the conclusion of the third round of nonsense mutations is that the shortest active C-terminal fragment of AK183 IgA protease is AK183(31-790).

[0141] The present inventors similarly performed three rounds of truncation mutation to examine the shortest N-terminal active site of AK183 IgA protease. First, the inventors introduced a first round of truncation mutation to remove the domain of unknown function (DUF) at the N-terminus of AK183 based on AK183 (31-792), and fixed the amino acid site at the C-terminus at position 792. For example, by removing the DUF consisting of amino acids corresponding to positions 31 to 284 of SEQ ID NO: 1 at the N-terminus based on AK183 (31-792), an AK183 (285-792) IgA protease truncated fragment is obtained. Using a similar method, IgA protease truncated fragments of AK183(330-792), AK183(380-792), AK183(430-792), AK183(480-792), AK183(530-792), and AK183(580-792) were obtained. The results of the in vitro enzyme cleavage activity experiment of the obtained IgA protease truncated fragments against IgA1 are shown in Figure 10. As shown in Figure 10, AK183(285-792) and AK183(330-792) IgA protease truncated fragments retain in vitro enzyme cleavage activity, while AK183(380-792), AK183(430-792), AK183(480-792), AK183(530-792), and AK183(580-792) IgA protease truncated fragments have no in vitro enzyme cleavage activity. Therefore, the conclusion of the first round of truncation mutation is that the shortest N-terminal active site of AK183 IgA protease is between amino acids 330 and 380. A second round of truncation mutations was then introduced, constructing one truncation at every fifth amino acid between amino acids 330 and 380, to obtain AK183(335-792), AK183(340-792), AK183(345-792), AK183(350-792), AK183(355-792), AK183(360-792), AK183(365-792), AK183(370-792), and AK183(375-792) IgA protease truncated fragments, respectively.The results of the in vitro enzyme cleavage activity experiment on IgA1 of the obtained IgA protease truncated fragments are shown in Figure 11. As shown in Figure 11, the AK183 (335-792) IgA protease truncated fragment retains the in vitro enzyme cleavage activity, while the AK183 (340-792), AK183 (345-792), AK183 (350-792), AK183 (355-792), AK183 (360-792), AK183 (365-792), AK183 (370-792), and AK183 (375-792) IgA protease truncated fragments have no in vitro enzyme cleavage activity. Therefore, the conclusion of the second round of truncation mutation is that the shortest N-terminal active site of the AK183 IgA protease is between amino acids 335 and 340. Next, the inventors introduced a third round of truncation mutations, and constructed a truncation at each amino acid between the 335th and 340th amino acids, thereby obtaining AK183(336-792), AK183(337-792), AK183(338-792), and AK183(339-792) IgA protease truncated fragments, respectively. The results of the in vitro enzyme cleavage activity experiment for IgA1 of the obtained IgA protease truncated fragments are shown in FIG. 12. As shown in FIG. 12, none of the AK183(336-792), AK183(337-792), AK183(338-792), and AK183(339-792) IgA protease truncated fragments have in vitro enzyme cleavage activity. Therefore, the conclusion of the third round of truncation mutations is that the shortest N-terminal active site of AK183 IgA protease is at amino acid position 335. Finally, the inventors re-examined the results of these three rounds and once again simultaneously expressed AK183(285-792), AK183(330-792), AK183(335-792), AK183(336-792), AK183(337-792), AK183(338-792), AK183(339-792), AK183(340-792), AK183(345-792), and AK183(350-792) IgA protease truncated fragments.The results of the in vitro enzyme cleavage activity experiment on IgA1 of the obtained IgA protease truncated fragments are shown in Figure 13. As shown in Figure 13, the AK183 (285-792), AK183 (330-792), and AK183 (335-792) IgA protease truncated fragments retain in vitro enzyme cleavage activity, while the AK183 (336-792), AK183 (337-792), AK183 (338-792), AK183 (339-792), AK183 (340-792), AK183 (345-792), and AK183 (350-792) IgA protease truncated fragments have no in vitro enzyme cleavage activity. This is consistent with the conclusion of the three rounds of truncation mutations described above, namely, that the shortest N-terminal active site of AK183 IgA protease is located at amino acid 335.

[0142] Taken together, the shortest active fragment of AK183 IgA protease is AK183(335-790).

[0143] [Example 2] Preparation of fusion protein containing AK183 IgA protease truncate or full-length AK183 IgA protease 2.1 Plasmid construction After identifying the C-terminal shortest active fragment AK183 (31-790) of AK183 IgA protease, the inventors placed the Fc domain at the C-terminus of the 790th amino acid of AK183 IgA protease, added GGGGS between them to link them, and added a 6XHis tag to the C-terminus of Fc for use in protein purification to construct the PET30a-AK183 (31-790)-Fc plasmid. The construction flow is shown in Figure 4. Next, the inventors used the PET30a-AK183 (31-790)-Fc plasmid as a template and added the 791st and 792nd amino acids to the rear of the AK183 (31-790) truncation by PCR to construct the PET30a-AK183 (31-792)-Fc plasmid.

[0144] At the same time, the applicant entrusted Beijing Liuhehua Dagene Technology Co., Ltd. to construct four candidate subclones, PET30a-AK183(31-798)-Fc, PET30a-AK183(31-807)-Fc, PET30a-AK183(31-816)-Fc, and PET30a-AK183(31-833)-Fc. The Fc(CH2-CH3) of the candidate subclones has a hinge region removed, and its amino acid sequence is as shown in SEQ ID NO:6 (compared to SEQ ID NO:2, SEQ ID NO:6 lacks the first nine amino acids EPKSCDKTH of SEQ ID NO:2), and 10 His (after the linker GGGGS and before Fc) are added between the IgA protease truncation and Fc. The four candidate subclones were used as candidates for subsequent protease yield and purity screening.

[0145] To examine whether the linking method between the AK183IgA protease truncated fragment and the Fc region affects its enzymatic cleavage activity against IgA, the inventors further constructed two candidate subclones, PET30a-AK183(285-816)-Fc and PET30a-Fc-AK183(285-816). The amino acid sequence of Fc is shown in SEQ ID NO:25.

[0146] In order to compare the enzymatic cleavage activity of the fusion protein formed by the AK183 IgA protease truncated fragment and Fc with that of the fusion protein formed by the full-length AK183 IgA protease and Fc against IgA, the inventors further constructed a candidate subclone PET30a-Fc-AK183 (31-1203). The amino acid sequence of Fc is shown in SEQ ID NO:24.

[0147] To examine the effects of IgG1 Fc, IgG4 Fc, and albumin on the IgA enzyme cleavage activity of the fusion protein containing the AK183 IgA protease truncated, the inventors further constructed two candidate subclones, PET30a-AK183(31-816)-IgG4 Fc and PET30a-AK183(31-816)-albumin. The amino acid sequence of IgG4 Fc is shown in SEQ ID NO:77, and the amino acid sequence of albumin is shown in SEQ ID NO:60.

[0148] To examine the effect of different linkers on the IgA enzyme cleavage activity of fusion proteins containing AK183 IgA protease truncated, the inventors further constructed six candidate subclones PET30a-AK183(285-816)-linker-Fc. In the fusion protein sequences expressed by these six candidate subclones, except for the different linkers, the amino acid sequences of AK183(285-816) and Fc are all identical, and the amino acid sequence of AK183(285-816) is shown in SEQ ID NO:46, and the amino acid sequence of Fc is shown in SEQ ID NO:25. The amino acid sequences of the linkers are HHHHHHHHH (SEQ ID NO:59, also referred to as "10xHis"), EEKKKEKEKEEQEERETK (SEQ ID NO:58, also referred to as "IgD linker"), GGGGS (SEQ ID NO:22, also referred to as "1xlinker"), GGGGSGGGGS (SEQ ID NO:78, also referred to as "2xlinker"), GGGSGGGGSGGGGGS (SEQ ID NO:79, also referred to as "3xlinker"), and GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:80, also referred to as "4xlinker").

[0149] 2.2 Method for preparing fusion proteins The expression vector was transfected into E. coli (BL21-DE3) competent cells, and after resistance selection on an LB agar medium petri dish containing 50ug / ml kanamycin, a monoclonal colony was selected and cultured with shaking in an LB medium containing the appropriate antibiotic until it reached the exponential growth phase (OD600: 0.6-0.8). After the exponential growth phase was reached, 0.1-0.5mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added for induction, and low-temperature induction expression was performed at 16°C for 24 hours. After expression was completed, the E. coli cell bodies were treated in the usual manner, ultrasonicated, centrifuged at high speed, and the supernatant was retained, and then purified by affinity chromatography and molecular sieve to obtain the recombinant fusion protein.

[0150] The amino acid sequence of the AK183(31-792)-Fc fusion protein expressed by the PET30a-AK183(31-792)-Fc plasmid is as shown in SEQ ID NO:2, and the encoding nucleic acid sequence thereof is as shown in SEQ ID NO:3.

[0151] The amino acid sequence of the AK183(31-798)-Fc fusion protein expressed by the PET30a-AK183(31-798)-Fc plasmid is as shown in SEQ ID NO:6, and the encoding nucleic acid sequence thereof is as shown in SEQ ID NO:7.

[0152] The amino acid sequence of the AK183(31-807)-Fc fusion protein expressed by the PET30a-AK183(31-807)-Fc plasmid is as shown in SEQ ID NO:8, and the encoding nucleic acid sequence thereof is as shown in SEQ ID NO:9.

[0153] The amino acid sequence of the AK183(31-816)-Fc fusion protein expressed by the PET30a-AK183(31-816)-Fc plasmid is as shown in SEQ ID NO: 10, and the encoding nucleic acid sequence thereof is as shown in SEQ ID NO: 11.

[0154] The amino acid sequence of the AK183(31-833)-Fc fusion protein expressed by the PET30a-AK183(31-833)-Fc plasmid is as shown in SEQ ID NO: 12, and the encoding nucleic acid sequence thereof is as shown in SEQ ID NO: 13.

[0155] The amino acid sequence of the AK183(285-816)-Fc fusion protein expressed by the PET30a-AK183(285-816)-Fc plasmid is shown in SEQ ID NO:81.

[0156] The amino acid sequence of the Fc-AK183(285-816) fusion protein expressed by the PET30a-Fc-AK183(285-816) plasmid is as shown in SEQ ID NO:82.

[0157] The amino acid sequence of the Fc-AK183(31-1203) fusion protein expressed by the PET30a-Fc-AK183(31-1203) plasmid is as shown in SEQ ID NO:83.

[0158] The amino acid sequence of the AK183(31-816)-IgG4 Fc fusion protein expressed by the PET30a-AK183(31-816)-IgG4 Fc plasmid is shown in SEQ ID NO:84.

[0159] The amino acid sequence of the AK183(31-816)-albumin fusion protein expressed by the PET30a-AK183(31-816)-albumin plasmid is shown in SEQ ID NO:85.

[0160] 2.3 In vitro activity measurement method The fusion protein containing the obtained AK183 IgA protease truncate was mixed in vitro with substrate IgA1 purified from the plasma of a patient with IgA nephropathy and reacted at 37°C for 2 to 12 hours, after which Western blotting was performed to verify the enzymatic cleavage activity against the substrate IgA1.

[0161] 2.4 In vivo activity measurement method The obtained fusion protein containing the AK183 IgA protease truncated was injected into the tail vein of humanized IgA1 alpha chain knock-in (α1KI-Tg) C57BL / 6 mice, and blood samples were taken before injection and 5 min, 2 h, 4 h, and 24 h after injection and examined by Western blot.

[0162] 2.5 Results The experiment shows that PET30a-AK183(31-790)-Fc plasmid successfully expresses AK183(31-790)-Fc fusion protein (as shown in Figure 5), and AK183(31-792)-Fc fusion protein has expected full-length protein expression (as shown in Figure 6a) and in vitro enzymatic cleavage activity against IgA1 (as shown in Figure 6b).

[0163] The four candidate subclones, PET30a-AK183(31-798)-Fc, PET30a-AK183(31-807)-Fc, PET30a-AK183(31-816)-Fc, and PET30a-AK183(31-833)-Fc, all expressed the fusion protein and all had in vitro enzymatic cleavage activity against IgA1 (see Figure 7).

[0164] In addition, both of the subclones PET30a-AK183(285-816)-Fc and PET30a-Fc-AK183(285-816) expressed the fusion protein (as shown in Figure 14), and both had in vitro enzymatic cleavage activity (as shown in Figure 15).

[0165] The inventors further verified the in vivo activity of the AK183(31-807)-Fc fusion protein expressed by the subclone PET30a-AK183(31-807)-Fc and the Fc-AK183(285-816) fusion protein expressed by the subclone PET30a-Fc-AK183(285-816). The results are shown in Figure 8 (AK183(31-807)-Fc, under reducing conditions) and Figure 17 (Fc-AK183(285-816), under non-reducing conditions). As shown in Figure 8, after the AK183(31-807)-Fc fusion protein was injected into the tail vein of humanized IgA1 mice (α1KI-Tg) C57BL / 6 with a single needle, all intact IgA1 heavy chains (H) in the blood disappeared and persisted for at least 24 hours. As shown in Figure 17, after single-needle tail vein injection of Fc-AK183(285-816) fusion protein into humanized IgA1 mice (α1KI-Tg) C57BL / 6, all intact IgA1 heavy chains (H) disappeared from the blood and persisted for at least 2 weeks.

[0166] The inventors further compared the enzymatic cleavage activity of the Fc-AK183(285-816) fusion protein, the AK183(285-816)-Fc fusion protein, and the AK183(285-816)IgA protease truncated protein against IgA1. The results are shown in Figure 16. As shown in Figure 16, all of these three proteins have enzymatic cleavage activity against IgA1.

[0167] The inventors further compared the enzymatic cleavage activity of the AK183(285-816)-Fc fusion protein and the Fc-AK183(31-1203) fusion protein against IgA1. The results are shown in Figure 18. As shown in Figure 18, both the AK183(285-816)-Fc fusion protein and the Fc-AK183(31-1203) fusion protein have enzymatic cleavage activity against IgA1.

[0168] The inventors further compared the enzymatic cleavage activity of AK183(31-816)-IgG1 Fc fusion protein, AK183(31-816)-IgG4 Fc fusion protein, and AK183(31-816)-albumin fusion protein against IgA1. The results are shown in Figure 19. As shown in Figure 19, all of these three types of fusion proteins have enzymatic cleavage activity against IgA1.

[0169] The inventors further compared the enzymatic cleavage activity of AK183(285-816)-Fc fusion proteins with different linkers (10xHis, IgD linker, 1x linker, 2x linker, 3x linker, or 4x linker) against IgA1. The results are shown in Figure 20. As shown in Figure 20, all of these six types of fusion proteins have enzymatic cleavage activity against IgA1.

[0170] 2.6 Eukaryotic Expression Systems All the above experiments were carried out in E. coli (BL21-DE3) competent cells (i.e., prokaryotic expression system). Next, the inventors cloned the AK183(31-792)-Fc fusion cDNA sequence into the pcDNA3.1 / hygro(+) expression vector, and added ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCG (SEQ ID NO: 41) to the N-terminus of the fusion protein to encode a signal peptide sequence expressing human IL-2, and constructed the pcDNA3.1 / hygro(+)-IL2-AK183(31-792)-Fc plasmid, which was used to transfect eukaryotic expression system HEK293 cells. In the plasmid, the Fc sequence was codon-optimized for eukaryotic expression systems. The amino acid sequence of the IL2-AK183(31-792)-Fc fusion protein expressed by pcDNA3.1 / hygro(+)-IL2-AK183(31-792)-Fc is shown in SEQ ID NO:4, and the encoding nucleic acid sequence thereof is shown in SEQ ID NO:5.

[0171] The expression results of the AK183(31-792)-Fc fusion protein in HEK293 cells are shown in Figure 9. The results show that the AK183(31-792)-Fc fusion protein has the expected full-length expression, and that the fusion protein expressed in a eukaryotic system has a dimeric form.

[0172] [Example 3] Preparation of AK183 IgA protease mutant and activity measurement The inventors performed site-directed mutagenesis of prolines (P) at positions 844, 862, 931 and 933, 978, 1002 and 1004 (all of which correspond to SEQ ID NO: 1) of AK183(31-1203) IgA protease truncated base to glycine (G), to obtain five mutants of AK183(31-1173) IgA protease truncated base. The amino acid sequences of the mutants are shown in SEQ ID NO: 53 (also called "PA-GA Mut"), SEQ ID NO: 54 (also called "PI-GI Mut"), SEQ ID NO: 55 (also called "PAP-GAG Mut"), SEQ ID NO: 56 (also called "PAT-GAT Mut") and SEQ ID NO: 57 (also called "PIP-GIG Mut").

[0173] The inventors measured the enzymatic cleavage activity of each of these five mutants against IgA1, and the results are shown in Figure 21. As shown in Figure 21, all of these five mutants have enzymatic cleavage activity against IgA1.

[0174] Furthermore, the inventors further performed site-specific mutagenesis on alanine (A) at position 7 (relative to SEQ ID NO: 25) of the Fc region based on the amino acid sequence of the AK183(31-816)-Fc fusion protein prepared in Example 2 (i.e., SEQ ID NO: 10), mutating it to valine (V), glycine (G), serine (S) and leucine (L), respectively, to obtain four mutants of the AK183(31-816)-Fc fusion protein, which they designated as AV Mut, AG Mut, AS Mut and AL Mut, respectively.

[0175] The inventors measured the enzymatic cleavage activity of these four mutants against IgA1, and the results are shown in Figure 22. As shown in Figure 22, all of these four mutants have enzymatic cleavage activity against IgA1.

[0176] [Example 4] Search for other IgA proteases The inventors screened several amino acid sequences having a certain degree of homology with the wild-type IgA enzyme of AK183 from a metagenomic database, and synthesized 16 types of AK183 homologous enzymes. The amino acid sequences are as shown in SEQ ID NO: 61 to SEQ ID NO: 76, respectively. The inventors measured the enzyme cleavage activity of these AK183 homologous enzymes against IgA1 according to the in vitro activity measurement method described in Example 2.3. The results are shown in Figures 23a and 23b. In Figure 23a, "1+IgA1" represents an in vitro mixture of the polypeptide shown in SEQ ID NO: 61 and the substrate IgA1, "2+IgA1" represents an in vitro mixture of the polypeptide shown in SEQ ID NO: 62 and the substrate IgA1, and so on. In Figure 23b, "16+IgA1" represents an in vitro mixture of the polypeptide shown in SEQ ID NO: 76 and the substrate IgA1.

[0177] As can be seen from FIG. 23a and FIG. 23b, all of the polypeptides shown in SEQ ID NOs: 61 to 76 have enzymatic cleavage activity against IgA1.

[0178] Although the present application has been particularly expressive and illustrative of the invention with reference to specific embodiments, it will be understood by those skilled in the art that the above content may be modified in form and detail without departing from the spirit and scope of protection disclosed herein.

Claims

1. An isolated IgA protease truncate comprising a non-natural truncated fragment of a wild-type IgA protease obtained from or derived from Clostridium ramosum, or having at least 70% sequence identity with the said non-natural truncated fragment.

2. The isolated IgA protease truncate according to claim 1, wherein the non-natural truncate fragment is based on the wild-type IgA protease of Clostridium ramosum and subjected to amino acid substitution, deletion, insertion, or modification, thereby resulting in the loss or reduction of the autoenzymatic cleavage function of the IgA protease truncate.

3. The isolated IgA protease truncate according to claim 2, wherein the substitution, deletion, insertion, or modification of the amino acids occurs at the natural autoenzymatic cleavage site of the wild-type IgA protease of Clostridium ramosum, within five sites upstream of the natural autoenzymatic cleavage site, and / or within five sites downstream of the natural autoenzymatic cleavage site.

4. The isolated IgA protease truncate according to claim 1, wherein the non-natural truncate fragment is the N-terminal or C-terminal truncate fragment of a wild-type IgA protease obtained from or derived from Clostridium ramosum.

5. The isolated IgA protease truncate according to claim 1, wherein the Clostridium ramosum is strain Clostridium ramosum AK183.

6. The isolated IgA protease truncate according to claim 4, wherein the N-terminal truncate fragment comprises a polypeptide fragment of at least 760 consecutive amino acids from the N-terminal 31st position of a wild-type IgA protease obtained from or derived from Clostridium ramosum, or has at least 70% sequence identity with the polypeptide fragment.

7. The isolated IgA protease truncate according to claim 1, wherein the non-natural truncate fragment comprises a polypeptide fragment of at least 456 consecutive amino acids from the N-terminal 335 position of a wild-type IgA protease obtained from or derived from Clostridium ramosum, or has at least 90% or at least 95% sequence identity with the polypeptide fragment.

8. The isolated IgA protease truncate according to claim 1, wherein the amino acid sequence of the wild-type IgA protease of Clostridium ramosum is as shown in SEQ ID NO:

1.

9. The isolated IgA protease truncate according to claim 3, wherein the aforementioned natural autoenzyme cleavage site is between positions 730 and 840 (for example, between positions 792 and 797) of the amino acid sequence shown in SEQ ID NO:

1.

10. The isolated IgA protease truncate according to claim 9, wherein the natural autoenzymatic cleavage sites are positions 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, or 800 of the amino acid sequence shown in SEQ ID NO:

1.

11. At least 760 amino acids from position 31 of the amino acid sequence shown in Sequence ID No. 1 (for example, at least 761, at least 762, at least 763, at least 764, at least 765, at least 766, at least 767, at least 768, at least 769, at least 770, at least 771, at least 772, at least 773, at least 774, at least 775, at least 776, at least 777, at least 778, at least 779, at least 780, at least 781, at least 782, at least 783, at least 784, at least 785, at least 786, at least 787, at least 788, at least 789, at least The isolated IgA protease truncate according to claim 1, comprising a polypeptide fragment of a sequence of amino acids (at least 790, at least 791, at least 792, at least 793, at least 794, at least 795, at least 796, at least 797, at least 798, at least 799, at least 800, at least 801, at least 802, at least 803, at least 804, at least 805, at least 806, at least 807, at least 808, at least 809, at least 810, at least 900, at least 950, at least 1000, at least 1100, at least 1150, or at least 1200).

12. The isolated IgA protease truncate according to claim 1, comprising a polypeptide fragment selected from the group consisting of amino acids at positions 31 to 790 of the amino acid sequence shown in SEQ ID NO: 1, amino acids at positions 31 to 792 of the amino acid sequence shown in SEQ ID NO: 1, amino acids at positions 31 to 798 of the amino acid sequence shown in SEQ ID NO: 1, amino acids at positions 31 to 807 of the amino acid sequence shown in SEQ ID NO: 1, amino acids at positions 31 to 816 of the amino acid sequence shown in SEQ ID NO: 1, amino acids at positions 31 to 833 of the amino acid sequence shown in SEQ ID NO: 1, and polypeptide fragments having at least 70% sequence identity with them.

13. At least 456 amino acids from position 335 of the amino acid sequence shown in Sequence ID No. 1 (for example, at least 457, at least 458, at least 459, at least 460, at least 461, at least 462, at least 463, at least 464, at least 465, at least 466, at least 467, at least 468, at least 469, at least 470, at least 471, at least 472, at least 473, at least 474, at least 475, at least 476, at least 477, at least 478, at least 479, at least 480, at least 481, at least 482, at least 483, The isolated IgA protease truncate according to claim 1, comprising a polypeptide fragment of at least 484, at least 485, at least 486, at least 487, at least 488, at least 489, at least 490, at least 491, at least 492, at least 493, at least 494, at least 495, at least 496, at least 497, at least 498, at least 499, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, or at least 900 consecutive amino acids.

14. The isolated IgA protease truncate according to claim 13, comprising a polypeptide fragment selected from the group consisting of amino acids at positions 335 to 790 of the amino acid sequence shown in SEQ ID NO: 1, amino acids at positions 335 to 791 of the amino acid sequence shown in SEQ ID NO: 1, amino acids at positions 335 to 792 of the amino acid sequence shown in SEQ ID NO: 1, amino acids at positions 285 to 790 of the amino acid sequence shown in SEQ ID NO: 1, amino acids at positions 285 to 791 of the amino acid sequence shown in SEQ ID NO: 1, amino acids at positions 285 to 792 of the amino acid sequence shown in SEQ ID NO: 1, amino acids at positions 330 to 790 of the amino acid sequence shown in SEQ ID NO: 1, amino acids at positions 330 to 791 of the amino acid sequence shown in SEQ ID NO: 1, amino acids at positions 330 to 792 of the amino acid sequence shown in SEQ ID NO: 1, amino acids at positions 285 to 816 of the amino acid sequence shown in SEQ ID NO: 1, and polypeptide fragments having at least 90% or at least 95% sequence identity with them.

15. The isolated IgA protease truncate according to claim 1, having one or more conserved amino acid substitutions at a site based on the amino acid sequence of the polypeptide fragment.

16. The isolated IgA protease truncate according to claim 1, wherein the polypeptide fragment has amino acid mutations at one or more positions among positions 844, 862, 931, 933, 978, 1002, and 1004 of SEQ ID NO:

1.

17. The isolated IgA protease truncate according to claim 16, wherein the polypeptide fragment is mutated with glycine at one or more positions corresponding to positions 844, 862, 931, 933, 978, 1002, and 1004 of SEQ ID NO:

1.

18. The isolated IgA protease truncate according to claim 16, wherein the polypeptide fragment has an amino acid mutation at the position corresponding to position 844 of SEQ ID NO: 1, an amino acid mutation at the position corresponding to position 862, an amino acid mutation at the positions corresponding to positions 931 and 933, an amino acid mutation at the position corresponding to position 978, or an amino acid mutation at the positions corresponding to positions 1002 and 1004.

19. The isolated IgA protease truncate according to claim 16, wherein the amino acid sequence of the polypeptide fragment is represented by SEQ ID NO: 53 (also known as "PA-GA Mut"), SEQ ID NO: 54 (also known as "PI-GI Mut"), SEQ ID NO: 55 (also known as "PAP-GAG Mut"), SEQ ID NO: 56 (also known as "PAT-GAT Mut"), or SEQ ID NO: 57 (also known as "PIP-GIG Mut").

20. The isolated IgA protease truncate according to claim 1, having enzymatic activity that specifically cleaves human IgA.

21. The isolated IgA protease truncate according to claim 20, having enzymatic activity that specifically cleaves human IgA heavy chains.

22. The isolated IgA protease truncate according to claim 21, having enzymatic activity that specifically cleaves the intersection of the human IgA heavy chain CH1 and the hinge region.

23. The isolated IgA protease truncate according to claim 20, having enzymatic activity that specifically cleaves human IgA1.

24. The isolated IgA protease truncate according to claim 1, wherein the IgA protease truncate is modified by PEGylation, glycosylation, amino-terminal modification, lipoacylation, carboxyl-terminal modification, phosphorylation, or methylation.

25. The isolated IgA protease truncate according to claim 24, wherein the IgA protease truncate is modified by PEGylation.

26. The isolated IgA protease truncate according to claim 24, wherein the unnatural truncated fragment of the wild-type IgA protease is truncated at the C-terminus or N-terminus.

27. ​​The isolated IgA protease truncate according to claim 26, wherein the site of truncation at the C-terminus is between positions 720 and 820 of the wild-type IgA protease, or the unnatural truncated fragment of the wild-type IgA protease has a deletion of at least 31 amino acids at the N-terminus of the wild-type IgA protease.

28. A fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the full-length wild-type IgA protease obtained from or derived from Clostridium ramosum, a polypeptide obtained by removing the signal peptide of wild-type IgA protease obtained from or derived from Clostridium ramosum, or the IgA protease truncate according to any one of claims 1 to 23, and the second polypeptide comprises an amino acid sequence for extending the half-life of the first polypeptide in the body of a subject.

29. The fusion protein according to claim 28, wherein the first polypeptide comprises the sequence shown in SEQ ID NO: 1 or SEQ ID NO:

42.

30. The fusion protein according to claim 28, wherein the second polypeptide is located at the N-terminus or C-terminus of the first polypeptide.

31. The fusion protein according to claim 28, wherein the first polypeptide and the second polypeptide are linked directly or linked by a linker.

32. The fusion protein according to claim 31, wherein the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.

33. The fusion protein according to claim 32, wherein the linker comprises a peptide linker.

34. The fusion protein according to claim 33, wherein the peptide linker comprises a linker containing glycine and serine.

35. The fusion protein according to claim 34, wherein the linker containing glycine and serine comprises one, two, three, four or more repeats as shown in SEQ ID NO: 21 (GGGS), SEQ ID NO: 22 (GGGGGS), SEQ ID NO: 86 (GGGGGGGS), or SEQ ID NO: 87 (GGGGGGGGGS).

36. The fusion protein according to claim 33, wherein the linker comprises the amino acid sequence shown in SEQ ID NO: 23 (GGCGGCGGGTGGATCC), SEQ ID NO: 58 (EEKKKEKEKEKEEQREETK), or SEQ ID NO: 59 (HHHHHHHHHH).

37. The fusion protein according to claim 28, wherein the second polypeptide is selected from an Fc domain and albumin.

38. The fusion protein according to claim 37, wherein the Fc domain includes a hinge region.

39. The fusion protein according to claim 38, wherein the Fc domain is derived from a human IgG Fc domain.

40. The fusion protein according to claim 39, wherein the Fc domain is derived from a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, or a human IgG4 Fc domain.

41. The fusion protein according to claim 37, wherein the Fc domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 32, or SEQ ID NO:

77.

42. The fusion protein according to claim 41, wherein the Fc domain comprises the amino acid sequence shown in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 32, or SEQ ID NO:

77.

43. The fusion protein according to claim 42, wherein the Fc domain has an amino acid mutation at the position corresponding to position 7 of SEQ ID NO:

25.

44. The fusion protein according to claim 43, wherein the Fc domain is mutated in which the amino acid at the position corresponding to position 7 of SEQ ID NO: 25 (e.g., alanine) is changed to valine, glycine, serine, or leucine.

45. The fusion protein according to claim 37, wherein the Fc domain includes one or more mutations that extend the half-life of the fusion protein.

46. The fusion protein according to claim 37, wherein the Fc domain is ligated to the C-terminus or N-terminus of the first polypeptide.

47. The fusion protein according to claim 37, wherein the albumin comprises one or more domains of human serum albumin.

48. The fusion protein according to claim 47, wherein the albumin comprises the D3 domain of human serum albumin.

49. The fusion protein according to claim 28, further including a tag.

50. The fusion protein according to claim 49, wherein the tag is selected from the group consisting of fluorescent tags, luminescent tags, purification tags, and chromogenic tags.

51. The fusion protein according to claim 49, wherein the tag is selected from the group consisting of c-Myc tag, HA tag, VSV-G tag, FLAG tag, V5 tag, and HIS tag.

52. The fusion protein according to claim 51, wherein the tag is a HIS tag containing 6, 7, 8, 9, or 10 histidines.

53. The fusion protein according to claim 49, wherein the second polypeptide is located at the C-terminus of the first polypeptide, and the tag is located at the C-terminus of the second polypeptide.

54. The fusion protein according to claim 49, wherein the half-life of the fusion protein in the blood circulation within the subject's body is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, and at least 14 days.

55. An isolated nucleic acid comprising a nucleotide sequence encoding the IgA protease truncate described in claim 1, or a nucleotide sequence encoding the fusion protein described in claim 28.

56. The isolated nucleic acid according to claim 55, comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, and nucleotide sequences having at least 70% sequence identity therewith.

57. A vector comprising the nucleic acid described in claim 55.

58. A cell comprising the nucleic acid described in claim 55, or the vector described in claim 57.

59. The cell according to claim 58, wherein the cell is a prokaryotic cell or a eukaryotic cell.

60. The cell according to claim 59, wherein the prokaryotic cell is a cell of Escherichia coli, or the eukaryotic cell is a mammalian cell.

61. The cell according to claim 60, wherein the mammalian cell is a human cell or a Chinese hamster ovary (CHO) cell.

62. The cell according to claim 60, wherein the mammalian cell is human fetal kidney cell 293 (HEK293 cell).

63. (i) an IgA protease truncate according to any one of claims 1 to 27, a fusion protein according to any one of claims 28 to 54, a nucleic acid according to claim 55 or 56, a vector according to claim 57, or a cell according to any one of claims 58 to 62, and (ii) a pharmaceutically acceptable carrier.

64. A method for producing a fusion protein, comprising the step of culturing cells according to any one of claims 58 to 62.

65. A pharmaceutical composition according to claim 63 for use in a method for treating or preventing an IgA deposition-related disease, wherein the method comprises administering to a subject in need of treatment or prevention an IgA protease truncate according to any one of claims 1 to 27 or a fusion protein according to any one of claims 28 to 54 contained in the pharmaceutical composition.

66. The pharmaceutical composition according to claim 65, wherein the IgA deposition-related disease is selected from the group consisting of IgA nephropathy, herpetiform dermatitis, Henoch-Schönein purpura (also known as IgA vasculitis), Kawasaki disease, Henoch-Schönein purpura nephritis, renal injury due to IgA vasculitis, IgA rheumatoid factor-positive rheumatoid arthritis, IgA-type anti-GBM disease, and IgA-type ANCA-associated vasculitis.

67. The pharmaceutical composition according to claim 65, wherein the IgA deposition-related disease is IgA nephropathy, IgA vasculitis, or Kawasaki disease.

68. A pharmaceutical composition for use in a method of treating or preventing an IgA deposition-related disease, comprising an IgA protease or its truncate or a fusion protein comprising the IgA protease or its truncate, wherein the method comprises administering the IgA protease or its truncate or the fusion protein comprising the IgA protease or its truncate contained in the pharmaceutical composition to a subject in need of treatment or prevention, the amino acid sequence of the IgA protease being selected from the group consisting of SEQ ID NOs: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 or a combination thereof.

69. The pharmaceutical composition according to claim 68, wherein the IgA deposition-related disease is selected from the group consisting of IgA nephropathy, herpetiform dermatitis, Henoch-Schönein purpura (also known as IgA vasculitis), Kawasaki disease, Henoch-Schönein purpura nephritis, renal injury due to IgA vasculitis, IgA rheumatoid factor-positive rheumatoid arthritis, IgA-type anti-GBM disease, and IgA-type ANCA-associated vasculitis.

70. The pharmaceutical composition according to claim 68, wherein the IgA deposition-related disease is IgA nephropathy, IgA vasculitis, or Kawasaki disease.