IgA protease truncates, fusion proteins comprising iga protease truncates, and uses thereof

Abstract

The present application relates to an IgA protease truncate, a fusion protein comprising an IgA protease truncate (e.g., a fusion protein comprising an IgA protease truncate and an Fc), and uses thereof in the treatment of IgA deposition diseases (e.g., IgA nephropathy).

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to an IgA protease truncated form, a fusion protein comprising the IgA protease truncated form, a pharmaceutical composition comprising the IgA protease truncated form or the fusion protein, a nucleic acid encoding the IgA protease truncated form or the fusion protein, a method for preparing the IgA protease truncated form or the fusion protein, and the use of the IgA protease truncated form or the fusion protein in the preparation of a medicament for treating IgA deposition-related diseases. Background Technology

[0002] IgA nephropathy is one of the most common primary glomerular diseases worldwide, imposing a heavy burden on patients and society. Currently, there is no specific treatment for IgA nephropathy. Clinically, supportive care based on RAS blockers is commonly used to slow the deterioration of renal function. For patients who do not respond to supportive care, combined therapy with hormone immunosuppressants is administered. However, the long-term efficacy of hormone immunosuppressants is poor, and they cause serious side effects for patients.

[0003] There is an urgent need to develop effective treatments with low side effects.

[0004] Invention Summary

[0005] In one aspect, this application provides an isolated truncated IgA protease comprising a non-naturally truncated fragment obtained from or derived from the wild-type IgA protease of *Clostridium ramosum*, or having at least 70% sequence identity with said non-naturally truncated fragment. In some embodiments, the non-naturally truncated fragment has amino acid substitutions, deletions, insertions, or modifications based on the wild-type IgA protease of *Clostridium ramosum*, causing the truncated IgA protease to lose or reduce its auto-cleavage function. In some embodiments, the amino acid substitutions, deletions, insertions, or modifications occur at the natural auto-cleavage site of the wild-type IgA protease of *Clostridium ramosum*, within five sites upstream of the natural auto-cleavage site, and / or within five sites downstream of the natural auto-cleavage site. In some embodiments, the *Clostridium ramosum* strain is *Clostridium ramosum* AK183. In some embodiments, the amino acid sequence of the wild-type IgA protease of *Clostridium ramosum* is as shown in SEQ ID NO: 1. In some embodiments, the natural auto-cleavage site is located between positions 730 and 840 (e.g., between positions 792 and 797) of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the natural auto-cleavage site is located 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.

[0006] In some embodiments, the non-natural truncated fragment is an N-terminal or C-terminal truncated fragment of a wild-type IgA protease obtained from or derived from *Clostridium ramosum*. In some embodiments, the N-terminal truncated fragment comprises a polypeptide fragment of at least 760 consecutive amino acids starting from position 31 of the N-terminus of a wild-type IgA protease obtained from or derived from *Clostridium ramosum*, or has at least 70% sequence identity with said polypeptide fragment. In some embodiments, the truncated IgA protease provided in this application comprises, as shown in SEQ ID NO. NO: 1 contains at least 760 amino acids starting from position 31 (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). A polypeptide fragment containing at least 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 900, 950, 1000, 1100, 1150, or 1200 consecutive amino acids. In some embodiments, the IgA protease truncation provided in this 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 a polypeptide fragment having at least 70% sequence identity with it.

[0007] In some embodiments, the non-natural truncated fragment comprises a polypeptide fragment of at least 456 consecutive amino acids starting from position 335 of the N-terminus 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 said polypeptide fragment. In some embodiments, the IgA protease truncated variant provided in this application comprises, as shown in SEQ ID NO. NO:1 At least 456 amino acids starting from position 335 (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 480, at least 481) of the amino acid sequence shown. A polypeptide fragment consisting of 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 truncated derivative provided in this 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 330 to 792 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 335 to 791 ...5 to 792 of The amino acids 285 to 816 of the amino acid sequence shown in NO:1, and the polypeptide fragment having at least 90% or at least 95% sequence identity with it.

[0008] In some embodiments, the IgA protease truncated variant provided in this application has conserved 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 an amino acid mutation at one or more positions corresponding to positions 844, 862, 931, 933, 978, 1002, and 1004 of SEQ ID NO: 1. In some embodiments, the polypeptide fragment is mutated to glycine at one or more positions corresponding to 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 position 844, position 862, positions 931 and 933, position 978, or positions 1002 and 1004 of SEQ ID NO: 1. In some embodiments, the amino acid sequence of the polypeptide fragment is as shown in 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”).

[0009] In some embodiments, the IgA protease truncated variant provided in this application has enzymatic activity that specifically cleaves human IgA. In some embodiments, the IgA protease truncated variant provided in this application has enzymatic activity that specifically cleaves the human IgA heavy chain. In some embodiments, the IgA protease truncated variant provided in this application has enzymatic activity that specifically cleaves the junction of the CH1 and hinge regions of the human IgA heavy chain. In some embodiments, the IgA protease truncated variant provided in this application has enzymatic activity that specifically cleaves human IgA1.

[0010] On the other hand, this application provides 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 formed by removing the signal peptide from the wild-type IgA protease obtained from or derived from Clostridium ramosum, or a truncated form of the IgA protease as described in this application, 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 the sequence shown 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: cleavable linkers, non-cleavable linkers, peptide linkers, flexible linkers, rigid linkers, helical linkers, and non-helical linkers. In some embodiments, the linker includes a peptide linker. In some embodiments, the peptide linker includes a linker containing glycine and serine. In some embodiments, the linker containing glycine and serine includes one, two, three, four, or more repeats as shown in 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 an amino acid sequence as shown 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 includes 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 contains 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. In some embodiments, the Fc domain has an amino acid mutation at position 7 corresponding to SEQ ID NO: 25. In some embodiments, the Fc domain is mutated at amino acid position 7 (e.g., alanine) corresponding to SEQ ID NO: 25 to valine, glycine, serine, or leucine. In some embodiments, the Fc domain contains 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 in this application further includes a tag. In some embodiments, the tag is selected from the group consisting of fluorescent tags, luminescent tags, purification tags, and chromogenic tags. In some embodiments, the tag is selected from the group consisting of c-Myc tags, HA tags, VSV-G tags, FLAG tags, V5 tags, and HIS tags. In some embodiments, the tag is an HIS tag containing 6, 7, 8, 9, or 10 histidine residues. 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 protein described in this application 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] On the other hand, this application provides an isolated nucleic acid comprising a nucleotide sequence encoding a truncated IgA protease as described in this application or a nucleotide sequence encoding a fusion protein as described in this application. In some embodiments, the nucleic acid described in this application 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 a nucleotide sequence having at least 70% sequence identity with it.

[0016] On the other hand, this application provides a carrier containing the nucleic acid described in this application.

[0017] In another aspect, this application provides a cell comprising the nucleic acid described in this application or the vector described in this application. In some embodiments, the cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the prokaryotic cell is an *Escherichia 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 cell 293 (HEK293 cell).

[0018] In another aspect, this application provides a pharmaceutical composition comprising the IgA protease truncated form described in this application, the fusion protein described in this application, the nucleic acid described in this application, the vector described in this application, or the cell described in this application, as well as a pharmaceutically acceptable vector.

[0019] On the other hand, this application provides a method for producing a fusion protein, which includes the step of culturing the cells described in this application.

[0020] On the other hand, this application provides a method for treating or preventing IgA deposition-related diseases, comprising administering to a subject requiring treatment or prevention an IgA protease truncated form as described in this application, a fusion protein as described in this application, or a pharmaceutical composition as described in this application.

[0021] On the other hand, this application provides the use of the truncated IgA protease as described in this application, the fusion protein as described in this application, or the pharmaceutical composition as described in this application in the preparation of a medicament for treating or preventing IgA deposition-related diseases.

[0022] On the other hand, this application provides, as described in this application, truncated IgA protease proteins, fusion proteins, or pharmaceutical compositions for the treatment or prevention of IgA deposition-related diseases.

[0023] On the other hand, this application provides a method for treating or preventing IgA deposition-related diseases, comprising administering to a subject requiring treatment or prevention an IgA protease or a truncated form thereof, a fusion protein comprising the IgA protease or a truncated form thereof, or a pharmaceutical composition comprising the IgA protease or a truncated form thereof or the fusion protein, wherein the amino acid sequence of the 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, this application provides the use of an IgA protease or a truncated form thereof, a fusion protein comprising the IgA protease or a truncated form thereof, or a pharmaceutical composition comprising the IgA protease or a truncated form thereof or the fusion protein in the preparation of a medicament for treating or preventing IgA deposition-related diseases, wherein the amino acid sequence of the 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 combinations thereof.

[0025] In another aspect, this application provides an IgA protease or a truncated form thereof for treating or preventing IgA deposition-related diseases, a fusion protein comprising the IgA protease or a truncated form thereof, or a pharmaceutical composition comprising the IgA protease or a truncated form thereof or the fusion protein, wherein the amino acid sequence of the 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 combinations thereof.

[0026] In some embodiments, the IgA deposition-related diseases include IgA nephropathy, herpetic dermatitis, Henrönlein purpura (also known as IgA vasculitis), Kawasaki disease, purpuric nephritis, IgA vasculitis-related kidney damage, IgA rheumatoid factor-positive rheumatoid arthritis, IgA anti-GBM disease, or IgA ANCA-associated vasculitis. In some embodiments, the IgA deposition-related diseases are IgA nephropathy, IgA vasculitis, or Kawasaki disease.

[0027] Attached Figure Description

[0028] Figure 1 The results of in vitro enzymatic activity experiments on IgA1 by four IgA protease truncated variants, AK183(31-737), AK183(31-768), AK183(31-798) and AK183(31-833), are shown.

[0029] Figure 2 The results of in vitro enzymatic activity experiments on IgA1 by five IgA protease truncated variants, AK183(31-773), AK183(31-778), AK183(31-782), AK183(31-787), and AK183(31-792), are shown.

[0030] Figure 3 The results of in vitro enzymatic activity assays on IgA1 were shown for four truncated IgA protease variants, AK183(31-788), AK183(31-789), AK183(31-790), and AK183(31-791).

[0031] Figure 4The flowchart for constructing the PET30a-AK183(31-790)-Fc plasmid is shown.

[0032] Figure 5 The graph shows the expression results of the AK183(31-790)-Fc fusion protein.

[0033] Figure 6 Figure a shows the expression results of the AK183(31-792)-Fc fusion protein. Figure 6 b shows the results of in vitro enzymatic activity assays against IgA1 by the AK183(31-792)-Fc fusion protein.

[0034] Figure 7 The results of in vitro enzymatic activity assays of four fusion proteins, AK183(31-798)-Fc, AK183(31-807)-Fc, AK183(31-816)-Fc and AK183(31-833)-Fc, on IgA1 are shown.

[0035] Figure 8 The results of in vivo enzymatic activity assays against IgA1 by the AK183(31-807)-Fc fusion protein are shown.

[0036] Figure 9 The graph shows the expression results of the AK183(31-792)-Fc fusion protein in HEK293 cells.

[0037] Figure 10 The results of in vitro enzymatic activity experiments on IgA1 by seven IgA protease truncated variants, AK183(285-792), AK183(330-792), AK183(380-792), AK183(430-792), AK183(480-792), AK183(530-792), and AK183(580-792), are shown.

[0038] Figure 11 The results of in vitro enzymatic activity experiments on IgA1 by nine IgA protease truncated variants, 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), are shown.

[0039] Figure 12The results of in vitro enzymatic activity experiments on IgA1 by four IgA protease truncated variants, AK183(336-792), AK183(337-792), AK183(338-792), and AK183(339-792), are shown.

[0040] Figure 13 The results of in vitro enzymatic activity assays against IgA1 were shown, re-validating the effects of 10 IgA protease truncated variants, 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).

[0041] Figure 14 The graph shows the expression results of the two fusion proteins, AK183(285-816)-Fc and Fc-AK183(285-816).

[0042] Figure 15 The results of in vitro enzymatic activity assays of the two fusion proteins AK183(285-816)-Fc and Fc-AK183(285-816) against IgA1 are shown.

[0043] Figure 16 The results of the enzymatic activity assays of Fc-AK183(285-816) fusion protein, AK183(285-816)-Fc fusion protein, and AK183(285-816) IgA protease truncated variants against IgA1 are shown.

[0044] Figure 17 The results of in vivo enzymatic activity assays against IgA1 by the Fc-AK183(285-816) fusion protein are shown.

[0045] Figure 18 The results of the enzymatic activity assays of AK183(285-816)-Fc fusion protein and Fc-AK183(31-1203) fusion protein against IgA1 are shown.

[0046] Figure 19 The results of the enzymatic activity assays of AK183(31-816)-IgG1 Fc fusion protein, AK183(31-816)-IgG4 Fc fusion protein, and AK183(31-816)-albumin fusion protein against IgA1 are shown.

[0047] Figure 20The results of enzymatic cleavage activity assays of AK183(285-816)-Fc fusion proteins with different linkers (SEQ ID NO: 59, SEQ ID NO: 58, SEQ ID NO: 22, SEQ ID NO: 78, SEQ ID NO: 79 or SEQ ID NO: 80) against IgA1 are shown.

[0048] Figure 21 The results of enzyme cleavage activity assays on IgA1 are shown for mutants of five truncated IgA proteases, as shown in SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 or SEQ ID NO: 57.

[0049] Figure 22 The results of IgA1 cleavage activity assays were shown for four mutants formed by four different mutations in the Fc region of the AK183(31-816)-Fc fusion protein.

[0050] Figure 23a and Figure 23b The results of enzyme digestion activities of 16 AK183 homologs on IgA1 are shown. Invention Details

[0052] Although this application discloses various aspects and embodiments below, it will be apparent to those skilled in the art that various equivalent changes and modifications can be made thereto without departing from the spirit and scope of the subject matter. The various aspects and embodiments disclosed in this application are for illustrative purposes only and are not intended to limit the scope of this application; the actual scope of protection of this application is determined by the claims. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. All references, patents, and patent applications cited in this application are incorporated herein by reference in their entirety.

[0053] definition

[0054] The terms "clostridium multibranchs" or "clostridium ramosum" used in this application refer to Clostridium ramosum, also known as Ramibacterium ramosum, which is a human intestinal commensal bacterium that produces IgA protease.

[0055] As used in this application, the term "protease" refers to an enzyme capable of breaking down proteins and peptides. Proteases break down proteins by hydrolyzing peptide bonds that link amino acids together in the peptide or polypeptide chain that forms a protein. Various methods are known in the art for testing the proteolytic activity of a protease. For example, the proteolytic activity of a protease can be determined by analyzing a comparative assay of the ability of various proteases to hydrolyze suitable substrates. Exemplary substrates used for proteolytic activity analysis include, for example, dimethylcasein, bovine collagen, bovine elastin, etc. Colorimetric assays using these substrates are also known in the art (see, for example, WO99 / 34011 and US 6,376,450).

[0056] As used in this application, the term "IgA protease" refers to an enzyme capable of specifically cleaving or breaking down IgA immunoglobulin molecules (e.g., IgA1 or IgA2) in a subject (e.g., a human). For example, an IgA protease obtained from or derived from Clostridium ramosum is capable of specifically cleaving the peptide bond between proline (Pro) at position 221 and valine (Val) at position 222 of IgA1 and IgA2, thereby breaking down IgA1 and IgA2.

[0057] When referring to polypeptides or proteins, the term "wild-type" as used in this application refers to a naturally occurring polypeptide or protein that does not contain artificial substitutions, insertions, deletions, or modifications at one or more amino acid positions. When referring to nucleic acids, nucleotides, or polynucleotides, the term "wild-type" as used in this application refers to a naturally occurring nucleic acid, nucleotide, or polynucleotide that does not contain artificial substitutions, insertions, deletions, or modifications at one or more nucleotide positions. However, polynucleotides encoding wild-type polypeptides are not limited to naturally occurring polynucleotides, but also include any polynucleotide encoding wild-type polypeptides.

[0058] As used in this application, the term "AK183" refers to strain AK183 of Clostridium ramosum. The amino acid sequence of the wild-type IgA protease produced by Clostridium ramosum strain AK183 is shown in SEQ ID NO: 1 (wherein, amino acids 1-30 are signal peptides).

[0059]

[0060]

[0061] As used in this application, the term "signal peptide" refers to an amino acid residue sequence that can participate in the secretion or directed transport of a protein in its mature or precursor form. Signal peptides are typically located at the N-terminus of a precursor or mature protein sequence. Signal peptides can be endogenous or exogenous. Mature proteins generally do not contain signal peptides. Typically, after protein transport, the signal peptide is cleaved from the protein by a signal peptidase. For example, the amino acid sequence shown in SEQ ID NO: 1, after removing the N-terminal signal peptide, results in the amino acid sequence shown in SEQ ID NO: 42.

[0062]

[0063] As used in this application, the term "subject" includes both human and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals. "Subject" can also be livestock, such as cattle, pigs, sheep, poultry, and horses; or rodents, such as rats and mice; or primates, such as apes, monkeys, chimpanzees, gorillas, orangutans, and baboons; or domesticated animals, such as dogs and cats. "Subject" can be male or female, and can be elderly, adult, adolescent, child, or infant. Human "subjects" can be Caucasian, African, Asian, Semitic, or of other races or a mixture of said racial backgrounds.

[0064] The terms "protein," "polypeptide," and "peptide" used in this application are interchangeable and refer to polymers of amino acids. The proteins, polypeptides, or peptides described in this application may contain natural amino acids, non-natural amino acids, or amino acid analogs or mimics. The proteins, polypeptides, or peptides described in this application can be obtained by any method known in the art, such as, but not limited to, natural isolation, recombinant expression, and chemical synthesis.

[0065] As used in this application, the term "amino acid" refers to an organic compound containing amino (-NH2) and carboxyl (-COOH) functional groups, as well as the unique side chain of each amino acid. The names of amino acids are also represented in this application using standard single-letter or three-letter codes, summarized as follows:

[0066] name Three-letter code Single-letter codes alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C glutamic acid Glu E glutamine Gln Q glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F proline Pro P Serine Ser S threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

[0067] In this application, when "conservative substitution" is used to refer to an amino acid sequence, it means replacing one amino acid residue with another amino acid residue with a side chain having 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 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, and Phe). It is known in the art that conservative substitutions generally do not cause significant changes in protein conformation and structure, thus preserving the protein's biological activity.

[0068] The term "homologous" as used in this application means that, when optimally aligned, a nucleic acid sequence (or its complementary strand) or amino acid sequence 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.

[0069] When "percentage (%) sequence identity" is used for amino acid sequences (or nucleic acid sequences), it 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, where necessary, the introduction of intervals 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 by the total number of amino acid residues (or bases) in either the candidate or reference sequence (whichever is shorter). Conservative substitutions of amino acid residues may or may not be considered identical residues. Sequences can be aligned to determine the percentage sequence identity of amino acid (or nucleic acid) sequences using tools publicly available in the field, such as BLASTN, BLASTp (National Center for Biotechnology Information (NCBI) website, 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 Institute for Bioinformatics website, see Higgins DGet 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. Those skilled in the art can use the default parameters of the tool or adjust the parameters appropriately as needed for comparison, for example by selecting a suitable algorithm.

[0070] "Separated" substances have been artificially altered from their natural state. If a "separated" composition or substance appears in nature, it has been altered or deviated from its original state, or both. For example, a naturally occurring polynucleotide or polypeptide in a living animal is not "separated," but if these polynucleotides or polypeptides are sufficiently separated from the substances that coexist in their natural state and exist in a substantially pure state, they can be considered "separated." "Separated nucleic acid sequence" refers to the sequence of a separated nucleic acid molecule. In some embodiments, "isolated IgA protease truncated isolates" refer to IgA protease truncated isolates with a purity of 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%, wherein the purity is determined by electrophoretic methods (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatographic methods (e.g., ion exchange chromatography or reversed-phase HPLC).

[0071] The term "vector" as used in this application refers to a delivery vehicle in which a genetic element can be operatively inserted and expressed, for example, to produce a protein, RNA, or DNA encoded by the genetic element, or to replicate the genetic element. Vectors can be used to transform, transduce, or transfect host cells, enabling the expression of the genetic element they carry within the host cells. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages such as λ phage or M13 phage, and animal viruses. Vectors may contain various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. Vectors may also include components that facilitate their entry into cells, including but not limited to viral particles, liposomes, or protein coats. Vectors can be expression vectors or cloning vectors. The vector (e.g., expression vector) provided in this application contains the nucleic acid sequence encoding the truncated IgA protease or fusion protein described in this application, at least one promoter operatively linked to the nucleic acid sequence (e.g., SV40, CMV, EF-1α), and at least one select marker.

[0072] The term "treatment" or "therapeutic method" used in this application for a disease, condition, or symptom includes preventing or alleviating a disease, condition, or symptom; slowing the onset or progression of a disease, condition, or symptom; reducing the risk of developing a disease, condition, or symptom; preventing or delaying the development of symptoms associated with a disease, condition, or symptom; reducing or terminating symptoms associated with a disease, condition, or symptom; causing complete or partial reversal of a disease, condition, or symptom; curing a disease, condition, or symptom; or a combination thereof.

[0073] The term “pharmaceutically acceptable” means that the specified carrier, medium, diluent, excipient and / or salt is generally chemically and / or physically compatible with the other components of the formulation and physiologically compatible with its receptors.

[0074] The term "IgA deposition-related disease" refers to diseases associated with the accumulation of IgA immunoglobulin in the tissues or organs of a subject, whether in aggregated or non-aggregated forms. Examples include, but are not limited to, IgA nephropathy, herpetic dermatitis, Henoch-Schönlein purpura (also known as IgA vasculitis), Kawasaki disease, purpuric nephritis, IgA vasculitis-related kidney damage, IgA rheumatoid factor-positive rheumatoid arthritis, IgA-type anti-GBM disease, or IgA-type ANCA-associated vasculitis.

[0075] The term "IgA nephropathy" refers to a kidney disease characterized by the deposition of IgA in the kidneys.

[0076] IgA protease truncated

[0077] In one aspect, this application provides an isolated truncated form of IgA protease comprising a non-naturally truncated fragment of a wild-type IgA protease obtained from or derived from Clostridium ramosum, or having at least 70% sequence identity with said non-naturally truncated fragment (e.g., 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%, or at least 99% sequence identity). In some embodiments, the IgA protease truncated body having at least 70% sequence identity with the non-natural truncated fragment (e.g., 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) still retains the function or activity of the IgA protease (e.g., proteolytic activity, enzyme activity that specifically cleaves IgA, etc.).

[0078] As used in this application, the terms "truncated form" or "truncated fragment" refer to a peptide formed by removing one or more amino acids from one or both ends of a wild-type polypeptide. Therefore, "truncated form" or "truncated fragment" in this application does not include the full length of its corresponding wild-type polypeptide, but may have one or more amino acid substitutions, deletions, insertions, or modifications compared to the truncated form of the wild-type polypeptide. For example, "truncated form of IgA protease" or "truncated fragment of IgA protease" may include a peptide formed by removing one or more amino acids from one or both ends of a wild-type IgA protease, or it may include a peptide with one or more amino acid substitutions, deletions, insertions, or modifications compared to the truncated form of the wild-type IgA protease.

[0079] In some embodiments, the truncated IgA protease described in this application has one or more amino acid substitutions, deletions, insertions, or modifications compared to its corresponding wild-type IgA protease. For example, in some embodiments, the truncated IgA protease described in this application comprises a non-naturally truncated fragment of a wild-type IgA protease obtained from or derived from Clostridium ramosum, wherein the non-naturally truncated fragment has amino acid substitutions, deletions, insertions, or modifications based on the wild-type IgA protease of Clostridium ramosum, causing the truncated IgA protease to lose or reduce its self-cleavage function.

[0080] The terms “obtained from” and “derived from” as used in this application include not only proteins produced or that can be produced by the mentioned organisms, but also proteins encoded by DNA sequences isolated from such organisms and produced in host organisms containing such DNA sequences, as well as proteins encoded by synthetic and / or cDNA-derived DNA sequences and having the identifying characteristics of the mentioned proteins. For example, wild-type IgA proteases obtained from or derived from Clostridium multiflorum include both IgA proteases naturally produced by Clostridium multiflorum and IgA proteases produced by other host cells (e.g., Escherichia coli) transformed with nucleic acids encoding IgA proteases using genetic engineering techniques.

[0081] As used in this application, the term "non-natural truncated fragment" refers to a fragment that has a different amino acid sequence (e.g., different amino acid length, different amino acid type, etc.) from the truncated fragment formed by the natural self-digestion of wild-type IgA protease from Clostridium ramosum.

[0082] In some embodiments, the amino acid substitution, deletion, insertion, or modification occurs at the natural autocleavage site of the wild-type IgA protease of *Clostridium ramosum*. In some embodiments, the amino acid substitution, deletion, insertion, or modification occurs within five sites upstream of the natural autocleavage site of the wild-type IgA protease of *Clostridium ramosum* (e.g., one, two, three, four, or five sites upstream of the natural autocleavage site). In some embodiments, the amino acid substitution, deletion, insertion, or modification occurs within five sites downstream of the natural autocleavage site of the wild-type IgA protease of *Clostridium ramosum* (e.g., one, two, three, four, or five sites downstream of the natural autocleavage site). In some embodiments, the amino acid substitution, deletion, insertion, or modification occurs within five sites upstream (e.g., one, two, three, four, or five sites upstream of the natural autocleavage site) and within five sites downstream (e.g., one, two, three, four, or five sites downstream of the natural autocleavage site) of the wild-type IgA protease of Clostridium ramosum.

[0083] In some embodiments, the non-natural truncated fragment is an N-terminal or C-terminal truncated fragment of a wild-type IgA protease obtained from or derived from Clostridium ramosum.

[0084] As used in this application, the term "N-terminal truncated fragment" refers to a truncated fragment comprising the amino acid sequence at the N-terminus of the wild-type IgA protease from Clostridium ramosum. The starting position of the "N-terminus" can be any position close to the N-terminus of the wild-type IgA protease's amino acid sequence; for example, it could be the first position counting from the N-terminus, or any other position counting from the N-terminus. For instance, if the full-length amino acid sequence of the wild-type IgA protease consists of 1000 amino acids, then the starting position of the N-terminus of its N-terminal truncated fragment can be any position between the first and 500th positions counting from the N-terminus.

[0085] As used in this application, the term "C-terminal truncated fragment" refers to a truncated fragment comprising the amino acid sequence at the carboxyl terminus of the wild-type IgA protease from Clostridium ramosum. The "carboxyl terminus" can be located anywhere near the carboxyl terminus of the wild-type IgA protease's amino acid sequence; for example, it could be the first position counting from the carboxyl terminus, or any other position counting from the carboxyl terminus. For instance, if the full-length amino acid sequence of the wild-type IgA protease consists of 1000 amino acids, then the carboxyl terminus of its C-terminal truncated fragment can be located anywhere between positions 501 and 1000 from the amino terminus.

[0086] Clostridium ramosum is one of the many species in the genus Clostridium, including various strains such as AK183, VPI-0496A, and NCTC 10474. In some embodiments, the Clostridium ramosum is Clostridium ramosum AK183.

[0087] In some embodiments, the N-terminal truncated fragment comprises a polypeptide fragment of at least 760 consecutive amino acids starting from position 31 of the N-terminus of a wild-type IgA protease obtained from or derived from Clostridium ramosum, or has at least 70% sequence identity with said polypeptide fragment (e.g., 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%, or at least 99% sequence identity). In some embodiments, the N-terminal truncated fragment having at least 70% sequence identity with the polypeptide fragment (e.g., 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) still retains the function or activity of the IgA protease (e.g., proteolytic activity, enzyme activity that specifically cleaves IgA, etc.).

[0088] In some embodiments, the non-natural truncated fragment of the IgA protease described in this application comprises a polypeptide fragment of at least 456 consecutive amino acids starting from position 335 of the N-terminus of the wild-type IgA protease obtained from or derived from *Clostridium ramosum*, or has at least 90% or at least 95% sequence identity with said polypeptide fragment (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 non-natural truncated fragment having at least 90% or at least 95% sequence identity with said polypeptide fragment (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) still retains the function or activity of the IgA protease (e.g., proteolytic activity, IgA-specific cleavage enzyme activity, etc.).

[0089] In some embodiments, the amino acid sequence of the wild-type IgA protease of the Clostridium ramosum is shown in SEQ ID NO: 1.

[0090] Unless otherwise specified, the amino acid sites of the IgA protease mentioned in this application correspond to the amino acid sites of the wild-type AK183IgA protease (whose amino acid sequence is shown in SEQ ID NO: 1). For example, position 790 of the AK183IgA protease mentioned in this application corresponds to position 790 of SEQ ID NO: 1. Unless otherwise specified, the naming rule for the truncated form of the AK183 IgA protease mentioned in this application is AK183 (corresponding to the start site of SEQ ID NO: 1 - corresponding to the end site of SEQ ID NO: 1). For example, AK183(31-790) refers to the truncated form of the IgA protease formed from amino acids 31 to 790 of SEQ ID NO: 1.

[0091] In some embodiments, the natural autocleavage site of the IgA protease described in this application is located between positions 730 and 840 of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the natural autocleavage site of the IgA protease described in this application is located between positions 710 and 830, 720 and 820, 730 and 810, 740 and 800, 750 and 790, 791 and 780, or 792 and 797 of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the natural autocleavage site is located 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.

[0092] In some embodiments, the IgA protease truncated derivative provided in this application comprises a polypeptide fragment of at least 760 consecutive amino acids starting from position 31 of the amino acid sequence shown in SEQ ID NO: 1. For example, in some embodiments, the IgA protease truncated derivative provided in this application comprises a polypeptide fragment of at least 760 consecutive amino acids starting from position 31 of the amino acid sequence shown in SEQ ID NO: 1. The amino acid sequence shown in NO:1 contains at least 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, and 795 amino acids starting from position 31. A polypeptide fragment of 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, or at least 1200 consecutive amino acids.

[0093] In some embodiments, the IgA protease truncated polypeptide provided in this application comprises a polypeptide fragment of 760 consecutive amino acids starting from position 31 of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the IgA protease truncated polypeptide provided in this application comprises a polypeptide fragment of 761 consecutive amino acids starting from position 31 of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the IgA protease truncated polypeptide provided in this application comprises a polypeptide fragment of 762 consecutive amino acids starting from position 31 of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the IgA protease truncated polypeptide provided in this application comprises a polypeptide fragment of 768 consecutive amino acids starting from position 31 of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the IgA protease truncated polypeptide provided in this application comprises a polypeptide fragment of 777 consecutive amino acids starting from position 31 of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the IgA protease truncated polypeptide provided in this application comprises a polypeptide fragment of 786 consecutive amino acids starting from position 31 of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the IgA protease truncated variant provided in this application comprises a polypeptide fragment of 803 consecutive amino acids starting from position 31 of the amino acid sequence shown in SEQ ID NO: 1.

[0094] In some embodiments, the IgA protease truncated derivative provided in this 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 816 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 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 ...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 816 of the amino acid sequence shown in SEQ ID NO: 1, amino acids 31 The amino acid sequence shown in NO:1 contains amino acids 31 to 833 and polypeptide fragments having at least 70% sequence identity with it (e.g., 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%, or at least 99% sequence identity). In some embodiments, the IgA protease truncated form having at least 70% sequence identity with the polypeptide fragment (e.g., 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%, or at least 99% sequence identity) still retains the function or activity of the IgA protease (e.g., proteolytic activity, enzyme activity that specifically cleaves IgA, etc.).

[0095] In some embodiments, the IgA protease truncated derivative provided in this application comprises a polypeptide fragment of at least 456 consecutive amino acids starting from position 335 of the amino acid sequence shown in SEQ ID NO: 1. For example, in some embodiments, the IgA protease truncated derivative provided in this application comprises 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... A polypeptide fragment of at least 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 550, 600, 650, 700, 750, 800, 850, or 900 consecutive amino acids.

[0096] In some embodiments, the IgA protease truncated derivative provided in this 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 330 to 792 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 335 to 791 ...5 to 792 of The amino acid sequence shown in NO:1, amino acids 285 to 816, and a polypeptide fragment 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). In some embodiments, the IgA protease truncated variant 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) retains the function or activity of the IgA protease (e.g., proteolytic activity, enzyme activity that specifically cleaves IgA, etc.).

[0097] In some embodiments, this application provides a truncated form of AK183(31-790), the amino acid sequence of which is shown in SEQ ID NO: 14.

[0098] ASKPDIKVGDYVKMGVYNNASILWRCVSIDNNGPLMLADKIVDTLAYDAKTNDNSNSKSHSRSYKRDDYGSNYWKDSNMRSWLNSTAAEGKVDWLCGNPPKDGYVSGVGAYNEKAGFLNAFSKSEIAAMKTVTQRSLVSHPEYNKGIVDGDANSDLLYYTDISEAVANYDSSYFETTTEKVFLLDVKQANAVWKNLKGYYVAYNNDGMAWPYWLRTPVTDCNHDMRYISSSGQVGRYAPWYSDLGVRPAFYLDSEYFVTTSGSGSQSSPYIGSAPNKQEDDYTISEPAEDANPDWNVSTEQSIQLTLGPWYSNDGKYSNPTIPVYTIQKTRSDTENMVVVVCGEGYTKSQQGKFINDVKRLWQDAMKYEPYRSYADRFNVYALCTASESTFDNGGSTFFDVIVDKYNSPVISNNLHGSQWKNHIFERCIGPEFIEKIHDAHIKKKCDPNTIPSGSEYEPYYYVHDYIAQFAMVVNTKSDFGGAYNNREYGFHYFISPSDSYRASKTFAHEFGHGLLGLGDEYSNGYLLDDKELKSLNLSSVEDPEKIKWRQLLGFRNTYTCRNAYGSKMLVSSYECIMRDTNYQFCEVCRLQGFKRMSQLVKDVDLYVATPEVKEYTGAYSKPSDFTDLETSSYYNYTYNRNDRLLSGNSKSRFNTNMNGKKIELRTVIQNISDKNARQLKFKMWIKHSDGSVATDSSGNPLQTVQTFDIPVWNDKANFWPLGALDHIKSDFNSGLKSCSLIYQIPSDAQLKSGDTVAFQ (SEQ ID NO: 14)

[0099] In certain embodiments, the present application provides an AK183(31-791) truncation, the amino acid sequence of which is shown in SEQ ID NO: 15.

[0100] ASKPDIKVGDYVKMGVYNNASILWRCVSIDNNGPLMLADKIVDTLAYDAKTNDNSNSKSHSRSYKRDDYGSNYWKDSNMRSWLNSTAAEGKVDWLCGNPPKDGYVSGVGAYNEKAGFLNAFSKSEIAAMKTVTQRSLVSHPEYNKGIVDGDANSDLLYYTDISEAVANYDSSYFETTTEKVFLLDVKQANAVWKNLKGYYVAYNNDGMAWPYWLRTPVTDCNHDMRYISSSGQVGRYAPWYSDLGVRPAFYLDSEYFVTTSGSGSQSSPYIGSAPNKQEDDYTISEPAEDANPDWNVSTEQSIQLTLGPWYSNDGKYSNPTIPVYTIQKTRSDTENMVVVVCGEGYTKSQQGKFINDVKRLWQDAMKYEPYRSYADRFNVYALCTASESTFDNGGSTFFDVIVDKYNSPVISNNLHGSQWKNHIFERCIGPEFIEKIHDAHIKKKCDPNTIPSGSEYEPYYYVHDYIAQFAMVVNTKSDFGGAYNNREYGFHYFISPSDSYRASKTFAHEFGHGLLGLGDEYSNGYLLDDKELKSLNLSSVEDPEKIKWRQLLGFRNTYTCRNAYGSKMLVSSYECIMRDTNYQFCEVCRLQGFKRMSQLVKDVDLYVATPEVKEYTGAYSKPSDFTDLETSSYYNYTYNRNDRLLSGNSKSRFNTNMNGKKIELRTVIQNISDKNARQLKFKMWIKHSDGSVATDSSGNPLQTVQTFDIPVWNDKANFWPLGALDHIKSDFNSGLKSCSLIYQIPSDAQLKSGDTVAFQV (SEQ ID NO: 15)

[0101] In certain embodiments, the present application provides an AK183(31-792) truncation, the amino acid sequence of which is shown in SEQ ID NO: 16.

[0102] ASKPDIKVGDYVKMGVYNNASILWRCVSIDNNGPLMLADKIVDTLAYDAKTNDNSNSKSHSRSYKRDDYGSNYWKDSNMRSWLNSTAAEGKVDWLCGNPPKDGYVSGVGAYNEKAGFLNAFSKSEIAAMKTVTQRSLVSHPEYNKGIVDGDANSDLLYYTDISEAVANYDSSYFETTTEKVFLLDVKQANAVWKNLKGYYVAYNNDGMAWPYWLRTPVTDCNHDMRYISSSGQVGRYAPWYSDLGVRPAFYLDSEYFVTTSGSGSQSSPYIGSAPNKQEDDYTISEPAEDANPDWNVSTEQSIQLTLGPWYSNDGKYSNPTIPVYTIQKTRSDTENMVVVVCGEGYTKSQQGKFINDVKRLWQDAMKYEPYRSYADRFNVYALCTASESTFDNGGSTFFDVIVDKYNSPVISNNLHGSQWKNHIFERCIGPEFIEKIHDAHIKKKCDPNTIPSGSEYEPYYYVHDYIAQFAMVVNTKSDFGGAYNNREYGFHYFISPSDSYRASKTFAHEFGHGLLGLGDEYSNGYLLDDKELKSLNLSSVEDPEKIKWRQLLGFRNTYTCRNAYGSKMLVSSYECIMRDTNYQFCEVCRLQGFKRMSQLVKDVDLYVATPEVKEYTGAYSKPSDFTDLETSSYYNYTYNRNDRLLSGNSKSRFNTNMNGKKIELRTVIQNISDKNARQLKFKMWIKHSDGSVATDSSGNPLQTVQTFDIPVWNDKANFWPLGALDHIKSDFNSGLKSCSLIYQIPSDAQLKSGDTVAFQVL (SEQ ID NO: 16)

[0103] In certain embodiments, the present application provides an AK183(31-798) truncation, the amino acid sequence of which is shown in SEQ ID NO: 17.

[0104] ASKPDIKVGDYVKMGVYNNASILWRCVSIDNNGPLMLADKIVDTLAYDAKTNDNSNSKSHSRSYKRDDYGSNYWKDSNMRSWLNSTAAEGKVDWLCGNPPKDGYVSGVGAYNEKAGFLNAFSKSEIAAMKTVTQRSLVSHPEYNKGIVDGDANSDLLYYTDISEAVANYDSSYFETTTEKVFLLDVKQANAVWKNLKGYYVAYNNDGMAWPYWLRTPVTDCNHDMRYISSSGQVGRYAPWYSDLGVRPAFYLDSEYFVTTSGSGSQSSPYIGSAPNKQEDDYTISEPAEDANPDWNVSTEQSIQLTLGPWYSNDGKYSNPTIPVYTIQKTRSDTENMVVVVCGEGYTKSQQGKFINDVKRLWQDAMKYEPYRSYADRFNVYALCTASESTFDNGGSTFFDVIVDKYNSPVISNNLHGSQWKNHIFERCIGPEFIEKIHDAHIKKKCDPNTIPSGSEYEPYYYVHDYIAQFAMVVNTKSDFGGAYNNREYGFHYFISPSDSYRASKTFAHEFGHGLLGLGDEYSNGYLLDDKELKSLNLSSVEDPEKIKWRQLLGFRNTYTCRNAYGSKMLVSSYECIMRDTNYQFCEVCRLQGFKRMSQLVKDVDLYVATPEVKEYTGAYSKPSDFTDLETSSYYNYTYNRNDRLLSGNSKSRFNTNMNGKKIELRTVIQNISDKNARQLKFKMWIKHSDGSVATDSSGNPLQTVQTFDIPVWNDKANFWPLGALDHIKSDFNSGLKSCSLIYQIPSDAQLKSGDTVAFQVLDENGNV (SEQ ID NO: 17)

[0105] In certain embodiments, the present application provides an AK183(31-807) truncation, the amino acid sequence of which is shown in SEQ ID NO: 18.

[0106] ASKPDIKVGDYVKMGVYNNASILWRCVSIDNNGPLMLADKIVDTLAYDAKTNDNSNSKSHSRSYKRDDYGSNYWKDSNMRSWLNSTAAEGKVDWLCGNPPKDGYVSGVGAYNEKAGFLNAFSKSEIAAMKTVTQRSLVSHPEYNKGIVDGDANSDLLYYTDISEAVANYDSSYFETTTEKVFLLDVKQANAVWKNLKGYYVAYNNDGMAWPYWLRTPVTDCNHDMRYISSSGQVGRYAPWYSDLGVRPAFYLDSEYFVTTSGSGSQSSPYIGSAPNKQEDDYTISEPAEDANPDWNVSTEQSIQLTLGPWYSNDGKYSNPTIPVYTIQKTRSDTENMVVVVCGEGYTKSQQGKFINDVKRLWQDAMKYEPYRSYADRFNVYALCTASESTFDNGGSTFFDVIVDKYNSPVISNNLHGSQWKNHIFERCIGPEFIEKIHDAHIKKKCDPNTIPSGSEYEPYYYVHDYIAQFAMVVNTKSDFGGAYNNREYGFHYFISPSDSYRASKTFAHEFGHGLLGLGDEYSNGYLLDDKELKSLNLSSVEDPEKIKWRQLLGFRNTYTCRNAYGSKMLVSSYECIMRDTNYQFCEVCRLQGFKRMSQLVKDVDLYVATPEVKEYTGAYSKPSDFTDLETSSYYNYTYNRNDRLLSGNSKSRFNTNMNGKKIELRTVIQNISDKNARQLKFKMWIKHSDGSVATDSSGNPLQTVQTFDIPVWNDKANFWPLGALDHIKSDFNSGLKSCSLIYQIPSDAQLKSGDTVAFQVLDENGNVLADDNTETQ (SEQ ID NO: 18)

[0107] In certain embodiments, the present application provides an AK183(31-816) truncation, the amino acid sequence of which is shown in SEQ ID NO: 19.

[0108] ASKPDIKVGDYVKMGVYNNASILWRCVSIDNNGPLMLADKIVDTLAYDAKTNDNSNSKSHSRSYKRDDYGSNYWKDSNMRSWLNSTAAEGKVDWLCGNPPKDGYVSGVGAYNEKAGFLNAFSKSEIAAMKTVTQRSLVSHPEYNKGIVDGDANSDLLYYTDISEAVANYDSSYFETTTEKVFLLDVKQANAVWKNLKGYYVAYNNDGMAWPYWLRTPVTDCNHDMRYISSSGQVGRYAPWYSDLGVRPAFYLDSEYFVTTSGSGSQSSPYIGSAPNKQEDDYTISEPAEDANPDWNVSTEQSIQLTLGPWYSNDGKYSNPTIPVYTIQKTRSDTENMVVVVCGEGYTKSQQGKFINDVKRLWQDAMKYEPYRSYADRFNVYALCTASESTFDNGGSTFFDVIVDKYNSPVISNNLHGSQWKNHIFERCIGPEFIEKIHDAHIKKKCDPNTIPSGSEYEPYYYVHDYIAQFAMVVNTKSDFGGAYNNREYGFHYFISPSDSYRASKTFAHEFGHGLLGLGDEYSNGYLLDDKELKSLNLSSVEDPEKIKWRQLLGFRNTYTCRNAYGSKMLVSSYECIMRDTNYQFCEVCRLQGFKRMSQLVKDVDLYVATPEVKEYTGAYSKPSDFTDLETSSYYNYTYNRNDRLLSGNSKSRFNTNMNGKKIELRTVIQNISDKNARQLKFKMWIKHSDGSVATDSSGNPLQTVQTFDIPVWNDKANFWPLGALDHIKSDFNSGLKSCSLIYQIPSDAQLKSGDTVAFQVLDENGNVLADDNTETQRYTTVSIQY (SEQ ID NO: 19)

[0109] In certain embodiments, the present application provides an AK183(31-833) truncation, the amino acid sequence of which is shown in SEQ ID NO: 20.

[0110] ASKPDIKVGDYVKMGVYNNASILWRCVSIDNNGPLMLADKIVDTLAYDAKTNDNSNSKSHSRSYKRDDYGSNYWKDSNMRSWLNSTAAEGKVDWLCGNPPKDGYVSGVGAYNEKAGFLNAFSKSEIAAMKTVTQRSLVSHPEYNKGIVDGDANSDLLYYTDISEAVANYDSSYFETTTEKVFLLDVKQANAVWKNLKGYYVAYNNDGMAWPYWLRTPVTDCNHDMRYISSSGQVGRYAPWYSDLGVRPAFYLDSEYFVTTSGSGSQSSPYIGSAPNKQEDDYTISEPAEDANPDWNVSTEQSIQLTLGPWYSNDGKYSNPTIPVYTIQKTRSDTENMVVVVCGEGYTKSQQGKFINDVKRLWQDAMKYEPYRSYADRFNVYALCTASESTFDNGGSTFFDVIVDKYNSPVISNNLHGSQWKNHIFERCIGPEFIEKIHDAHIKKKCDPNTIPSGSEYEPYYYVHDYIAQFAMVVNTKSDFGGAYNNREYGFHYFISPSDSYRASKTFAHEFGHGLLGLGDEYSNGYLLDDKELKSLNLSSVEDPEKIKWRQLLGFRNTYTCRNAYGSKMLVSSYECIMRDTNYQFCEVCRLQGFKRMSQLVKDVDLYVATPEVKEYTGAYSKPSDFTDLETSSYYNYTYNRNDRLLSGNSKSRFNTNMNGKKIELRTVIQNISDKNARQLKFKMWIKHSDGSVATDSSGNPLQTVQTFDIPVWNDKANFWPLGALDHIKSDFNSGLKSCSLIYQIPSDAQLKSGDTVAFQVLDENGNVLADDNTETQRYTTVSIQYKFEDGSEIPNTAGGTFT(SEQ ID NO:20)

[0111] In some embodiments, this application provides a truncated form of AK183 (285-790), the amino acid sequence of which is shown in SEQ ID NO: 43. In some embodiments, this application provides a truncated form of AK183 (285-791), the amino acid sequence of which is shown in SEQ ID NO: 44. In some embodiments, this application provides a truncated form of AK183 (285-792), the amino acid sequence of which is shown in SEQ ID NO: 45. In some embodiments, this application provides a truncated form of AK183 (285-816), the amino acid sequence of which is shown in SEQ ID NO: 46. In some embodiments, this application provides a truncated form of AK183 (330-790), the amino acid sequence of which is shown in SEQ ID NO: 47. In some embodiments, this application provides a truncated form of AK183 (330-791), the amino acid sequence of which is shown in SEQ ID NO: 48. In some embodiments, this application provides a truncated form of AK183 (330-792), the amino acid sequence of which is shown in SEQ ID NO: 49. In some embodiments, this application provides a truncated form of AK183 (335-790), the amino acid sequence of which is shown in SEQ ID NO: 50. In some embodiments, this application provides a truncated form of AK183 (335-791), the amino acid sequence of which is shown in SEQ ID NO: 51. In some embodiments, this application provides a truncated form of AK183 (335-792), the amino acid sequence of which is shown in SEQ ID NO: 52.

[0112] The sequences of SEQ ID NO: 43-52 are shown below.

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119] In some embodiments, the IgA protease truncated variant provided in this application has conserved substitutions of amino acids at one or more sites (e.g., 1, 2, 3, 4, 5 or more sites) based on the amino acid sequence of the aforementioned polypeptide fragment. Conserved substitutions of amino acid residues refer to substitutions between amino acids with similar properties, such as substitutions between polar amino acids (e.g., between glutamine and asparagine), substitutions between hydrophobic amino acids (e.g., between leucine, isoleucine, methionine and valine), and substitutions 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 truncated variants described in this application have conserved amino acid substitutions at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 15, 20 or more sites compared to the amino acid sequences shown 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.

[0120] In some embodiments, the polypeptide fragment has an amino acid mutation at one or more of the following positions corresponding to SEQ ID NO: 1: 844, 862, 931, 933, 978, 1002, and 1004. In some embodiments, the polypeptide fragment has an amino acid mutation at position 844 of SEQ ID NO: 1. In some embodiments, the polypeptide fragment has an amino acid mutation at position 862 of SEQ ID NO: 1. In some embodiments, the polypeptide fragment has amino acid mutations at positions 931 and 933 of SEQ ID NO: 1. In some embodiments, the polypeptide fragment has an amino acid mutation at position 978 of SEQ ID NO: 1. In some embodiments, the polypeptide fragment has amino acid mutations at positions 1002 and 1004 of SEQ ID NO: 1.

[0121] In some embodiments, the polypeptide fragment is mutated to glycine at one or more positions corresponding to positions 844, 862, 931, 933, 978, 1002, and 1004 of SEQ ID NO: 1. In some embodiments, the polypeptide fragment is mutated to glycine (G) at one or more positions corresponding to positions 844, 862, 931, 933, 978, 1002, and 1004 of SEQ ID NO: 1. In some embodiments, the polypeptide fragment is mutated to glycine at position 844 of SEQ ID NO: 1. In some embodiments, the polypeptide fragment is mutated to glycine at position 862 of SEQ ID NO: 1. In some embodiments, the polypeptide fragment is mutated to glycine at positions 931 and 933 of SEQ ID NO: 1. In some embodiments, the polypeptide fragment has a proline mutated to glycine at position 978 of SEQ ID NO: 1. In some embodiments, the polypeptide fragment has a proline mutated to glycine at positions 1002 and 1004 of SEQ ID NO: 1.

[0122] In some embodiments, the amino acid sequence of the polypeptide fragment is as shown in SEQ ID NO: 53 (also known as “PA-GAMut”), 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”).

[0123] The sequences of SEQ ID NO: 53-57 are shown below.

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130] Without affecting activity, the IgA protease truncated form provided in this application may also contain non-natural amino acids. Non-natural amino acids include, for example, β-fluoroalanine, 1-methylhistidine, γ-methyleneglutamate, α-methylleucine, 4,5-dehydrolysine, hydroxyproline, 3-fluorophenylalanine, 3-aminotyrosine, 4-methyltryptophan, etc.

[0131] The IgA protease truncated form provided in this application can also be modified using methods known in the art. For example, but not limited to, PEGylation, glycosylation, amino-terminal modification, fatty acid acylation, carboxyl-terminal modification, phosphorylation, methylation, etc. Those skilled in the art will understand that the IgA protease truncated form provided in this application, after modification using methods known in the art, still retains functions substantially similar to the IgA protease or the IgA protease truncated form.

[0132] In some embodiments, the IgA protease truncated variant provided in this application has enzymatic activity that specifically cleaves human IgA. In some embodiments, the IgA protease truncated variant provided in this application has enzymatic activity that specifically cleaves the human IgA heavy chain. In some embodiments, the IgA protease truncated variant provided in this application has enzymatic activity that specifically cleaves the junction of the CH1 and hinge regions of the human IgA heavy chain. In some embodiments, the IgA protease truncated variant provided in this application has enzymatic activity that specifically cleaves human IgA1.

[0133] In some embodiments, the IgA protease truncated variant provided in this application has conserved substitutions of amino acids at one or more sites based on the amino acid sequence of the aforementioned polypeptide fragment, but still possesses enzymatic activity for cleaving human IgA (e.g., IgA1). In some embodiments, the IgA protease truncated variant provided in this application has at least 70% sequence identity with the aforementioned 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), and still possesses enzymatic activity for cleaving human IgA (e.g., IgA1).

[0134] Fusion protein

[0135] On the other hand, this application provides 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 formed by removing the signal peptide from the wild-type IgA protease obtained from or derived from *Clostridium ramosum*, or a truncated form of the IgA protease described in this application, 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 the sequence shown 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.

[0136] 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 by a linker). As used herein, the term "linker" or "connector" refers to an artificial amino acid sequence having 1, 2, 3, 4, or 5 amino acid residues, or a length between 5 and 15, 20, 30, 50, or more amino acid residues, linked by peptide bonds, and used to link one or more polypeptides. Linkers may or may not have secondary structures. Linker sequences are known in the art, for example, see Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); Poljak et al., Structure 2: 1121-1123 (1994).

[0137] 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, useful linkers in this application may be rich in glycine and serine residues. Examples include linkers having a single or repeating sequence comprising threonine / serine and glycine, such as GGGS (SEQ ID NO: 21) or GGGGS (SEQ ID NO: 22), GGGGGS (SEQ ID NO: 86) or GGGGGGGGS (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, the linker used in this application includes GGCGGCGGTGGATCC (SEQ ID NO: 23). Optionally, the linker may be a long peptide chain comprising one or more sequential or tandem repeats of the amino acid sequence shown in GGCGGCGGTGGATCC (SEQ ID NO: 23). In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sequential 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 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%, or at least 99% sequence identity with any of SEQ ID No: 21, 22, or 23.

[0138] In some embodiments, the linker used in this application comprises the amino acid sequence shown in SEQ ID NO: 58 (EEKKKEKEKEEQEERETK). Optionally, the linker may be a long peptide chain comprising one or more sequential or tandem repeats of the amino acid sequence shown in SEQ ID NO: 58. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sequential 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%, or at least 99% sequence identity with SEQ ID NO: 58.

[0139] In some embodiments, the linker used in this application comprises an amino acid sequence as shown 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 with SEQ ID NO: 59.

[0140] In some embodiments, the second polypeptide is selected from an Fc domain and albumin. In some embodiments, the Fc domain includes a hinge region. In some embodiments, the Fc domain includes a lower hinge region. In some embodiments, the Fc domain includes a core hinge region and a lower hinge region. In some embodiments, the Fc domain includes an upper hinge region, a core hinge region, and a lower hinge region. In some embodiments, the Fc domain does not include 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.

[0141] In some embodiments, the Fc domain comprises the amino acid sequence shown in SEQ ID NO: 24. In some embodiments, the Fc domain is composed of the amino acid sequence shown 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 with the amino acid sequence shown in SEQ ID NO: 24.

[0142] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 24)

[0143] In some embodiments, the nucleic acid sequence encoding the Fc domain comprises the nucleotide sequence shown in SEQ ID NO: 39. In some embodiments, the nucleic acid sequence encoding the Fc domain consists of the nucleotide sequence shown in SEQ ID NO: 39. In some embodiments, the nucleic acid sequence encoding 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 with the nucleotide sequence shown in SEQ ID NO: 39.

[0144] (SEQ ID NO: 39)

[0145] In some embodiments, the Fc domain comprises the amino acid sequence shown in SEQ ID NO: 25. In some embodiments, the Fc domain is composed of the amino acid sequence shown 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 with the amino acid sequence shown in SEQ ID NO: 25.

[0146] TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 25)

[0147] In some embodiments, the nucleic acid sequence encoding the Fc domain comprises the nucleotide sequence shown in SEQ ID NO: 40. In some embodiments, the nucleic acid sequence encoding the Fc domain consists of the nucleotide sequence shown in SEQ ID NO: 40. In some embodiments, the nucleic acid sequence encoding 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 with the nucleotide sequence shown in SEQ ID NO: 40.

[0148] (SEQ IDNO:40).

[0149] In some embodiments, the Fc domain comprises the amino acid sequence shown in SEQ ID NO: 32. In some embodiments, the Fc domain is composed of the amino acid sequence shown 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 with the amino acid sequence shown in SEQ ID NO: 32.

[0150] ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQID NO: 32).

[0151] In some embodiments, the Fc domain comprises the amino acid sequence shown in SEQ ID NO: 77. In some embodiments, the Fc domain is composed of the amino acid sequence shown 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 with the amino acid sequence shown in SEQ ID NO: 77.

[0152] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 77)

[0153] In some embodiments, the Fc domain has one or more amino acid mutations. In some embodiments, the Fc domain has an amino acid mutation at position 7 corresponding to SEQ ID NO: 25. In some embodiments, the Fc domain mutates to valine at position 7 (e.g., alanine) of SEQ ID NO: 25. In some embodiments, the Fc domain mutates to glycine at position 7 (e.g., alanine) of SEQ ID NO: 25. In some embodiments, the Fc domain mutates to serine at position 7 (e.g., alanine) of SEQ ID NO: 25. In some embodiments, the Fc domain mutates to leucine at position 7 (e.g., alanine) of SEQ ID NO: 25.

[0154] In some embodiments, the Fc domain contains 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 peptide. In some embodiments, the Fc domain is linked to the N-terminus of the first peptide.

[0155] In some embodiments, the second polypeptide is albumin. In some embodiments, the amino acid sequence of the albumin is as shown 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.

[0156] (SEQ ID NO:60)

[0157] In some embodiments, the fusion protein provided in this application further includes a tag. In some embodiments, the tag is selected from the group consisting of fluorescent tags, luminescent tags, purification tags, and chromogenic tags. In some embodiments, the tag is selected from the group consisting of c-Myc tags, HA tags, VSV-G tags, FLAG tags, V5 tags, and HIS tags. In some embodiments, the tag is an HIS tag. In some embodiments, the tag is an HIS tag containing 6, 7, 8, 9, or 10 histidine residues. 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.

[0158] In some embodiments, the fusion protein provided in this application comprises the amino acid sequences shown 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 in this application consists of amino acid sequences 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, or having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with them. In some embodiments, the fusion protein 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: 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 still retains the function or activity of the IgA protease (e.g., proteolytic activity, enzyme activity that specifically cleaves IgA, etc.).

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168] In some embodiments, the fusion protein provided in this application comprises the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12. In some embodiments, the fusion protein provided in this application consists of amino acid sequences 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, and SEQ ID NO: 12, or having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with them.

[0169]

[0170]

[0171]

[0172]

[0173]

[0174] In some embodiments, the half-life of the fusion protein provided in this application 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.

[0175] Nucleic acid

[0176] On the other hand, this application provides an isolated nucleic acid comprising a nucleotide sequence encoding the truncated IgA protease of this application or a nucleotide sequence encoding the fusion protein of this application.

[0177] As used herein, the term “nucleic acid” or “nucleotide” refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form and polymers thereof. Unless otherwise stated, a particular nucleotide sequence also implicitly encompasses variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences, as well as explicitly stated sequences. Specifically, degenerate codon substitutions can be achieved by producing sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases 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)).

[0178] Using conventional procedures, the DNA encoding the truncated IgA protease variant or the DNA encoding the fusion protein described in this application can be easily isolated and sequenced (e.g., by using oligonucleotide probes capable of specifically binding to the gene encoding the truncated IgA protease variant or the fusion protein). The encoding DNA can also be obtained by synthetic methods.

[0179] In some embodiments, the nucleic acid provided in this application comprises nucleic acid sequences as shown in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38. In some embodiments, the nucleic acid provided in this application consists of nucleotide sequences 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, and SEQ ID NO: 38, or having 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 with them.

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190] In some embodiments, the nucleic acid provided in this application comprises nucleic acid sequences as shown in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13. In some embodiments, the nucleic acid provided in this application consists of nucleotide sequences 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, and SEQ ID NO: 13, or having 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 with them.

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204] carriers and cells

[0205] On the other hand, this application provides a vector comprising nucleic acid encoding the truncated IgA protease of the present application or nucleic acid encoding the fusion protein of the present application.

[0206] Using recombination techniques known in the art, isolated polynucleotides encoding the truncated IgA protease or fusion protein can be inserted into a vector for further cloning (DNA amplification) or for expression. A variety of vectors are available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, a replication origin, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0207] In some embodiments, the nucleic acid provided in this application encodes a truncated or fusion protein of the IgA protease, at least one promoter (e.g., SV40, CMV, EF-1α) operatively linked to the nucleic acid sequence, and at least one select tag. Examples of vectors include, but are not limited to: retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (e.g., SV40), λ phages and M13 phages, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, and pGEMEX. , pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p1 5TV-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, etc.

[0208] Vectors containing nucleic acid sequences encoding the truncated form or fusion protein of the IgA protease can be introduced into host cells for cloning or gene expression. Host cells suitable for cloning or expressing the DNA in the vectors described in this application are the aforementioned prokaryotic, yeast, or higher eukaryotic cells. Prokaryotic cells suitable for the purposes of this application include eubacteria, such as Gram-negative or Gram-positive bacteria, for example, Enterobacteriaceae, such as Escherichia (e.g., E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), and Serratia (e.g., Serratia marcescens). The bacteria include *Bacillus subtilis* (e.g., *Bacillus subtilis* and *Bacillus licheniformis*), *Pseudomonas* (e.g., *Pseudomonas aeruginosa*), and *Streptomyces*. In some embodiments, the cells are *Escherichia coli* cells.

[0209] Besides prokaryotic cells, eukaryotic cells, such as eukaryotic microorganisms like filamentous fungi or yeast, can also serve as suitable cloning or expression hosts for vectors encoding truncated IgA proteases 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 commonly used and applicable in this application, such as *Schizosaccharomyces pombe*; hosts of the genus *Kluyveromyces*, such as *Kluyveromyces lactis*, *Kluyveromyces fragilis* (ATCC 12, 424), *Kluyveromyces bulgaricus* (ATCC 16, 045), *Kluyveromyces wickeramii* (ATCC 24, 178), *Kluyveromyces waltii* (ATCC 56, 500), *Kluyveromyces drosophilarum* (ATCC 36, 906), *Kluyveromyces thermomotolerans*, and *Kluyveromyces marxianus*; *Yarrowia* (EP402, 226); and *Pichia pastoris* (EP402, 226). 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 cell 293 (HEK293 cell).

[0210] Pharmaceutical Composition

[0211] In another aspect, this application provides a pharmaceutical composition comprising the IgA protease truncated form described in this application, the fusion protein described in this application, the nucleic acid described in this application, the vector described in this application, or the cell described in this application, as well as a pharmaceutically acceptable vector.

[0212] Pharmaceutically acceptable carriers used in the pharmaceutical compositions disclosed in this application may include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous solvents, non-aqueous solvents, antimicrobial substances, isotonic substances, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients or non-toxic excipients, other components known in the art, or combinations thereof.

[0213] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, mercaptoacetic acid, mercaptosorbitol, butylated methyl anisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed in this application, including one or more antioxidants such as methionine in a composition comprising the IgA protease truncated or fusion protein disclosed in this application can reduce the oxidation of the IgA protease truncated or fusion protein. This application further provides various methods for preventing the oxidation of the IgA protease truncated or fusion protein, extending its shelf life, and / or improving its activity, for example, by mixing the IgA protease truncated or fusion protein provided in this application with one or more antioxidants (e.g., methionine).

[0214] Furthermore, pharmaceutically acceptable carriers may include, for example, aqueous media such as sodium chloride injection, Ringer's solution injection, isotonic glucose injection, sterile water injection, or glucose and lactated Ringer's solution injection; non-aqueous media such as non-volatile plant-derived oils, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial substances at bacterial or fungal inhibitory concentrations; isotonic agents such as sodium chloride or glucose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; emulsifiers such as polysorbate 80 (Tween-80); chelating agents such as 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 serving as delivery carriers can be incorporated into pharmaceutical compositions in multi-dose containers, including phenols or cresols, mercury preparations, benzyl alcohol, chlorobutanol, methyl and propylparabens, thiamethoxam, chlorobenzamide, and chlorophenoxyacetamide. Suitable excipients may include, for example, water, salts, glucose, glycerol, or ethanol. Suitable non-toxic excipients may include, for example, wetting agents, emulsifiers, pH buffers, stabilizers, solubilizers, or substances such as sodium acetate, dehydrosorbitan laurate, triethanolamine oleate, or cyclodextrin.

[0215] The pharmaceutical composition may be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained-release formulation, or powder. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0216] In some embodiments, the pharmaceutical composition is formulated as an injectable composition. Injectable pharmaceutical compositions can be prepared in any conventional form, such as liquid solvents, suspensions, emulsifiers, or solid forms suitable for producing liquid solvents, suspensions, or emulsifiers. Injectable formulations may include pre-existing sterile and / or pyrogen-free solutions, sterile dried soluble products bound to a solvent before use, such as lyophilized powders, including subcutaneous tablets, sterile suspensions ready for injection, sterile dried insoluble products bound to a medium before use, and sterile and / or pyrogen-free emulsions. The solvent may be aqueous or non-aqueous.

[0217] In some embodiments, a unit dose of the injectable formulation is packaged in an ampoule, a tube, or a syringe with a needle. It is known in the art that all injectable formulations should be sterile and pyrogen-free.

[0218] In some embodiments, a sterile lyophilized powder can be prepared by dissolving the truncated or fusion protein of the IgA protease disclosed in this application in a suitable solvent. The solvent may contain other pharmacological components that improve the stability of the powder or the recombinant solution derived from the powder, or enhance the powder or the recombinant solution. Suitable excipients include, but are not limited to, water, glucose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable substances. The solvent may contain a buffer solution, such as citrate buffer, sodium or potassium phosphate buffer, or other buffer solutions known to those skilled in the art; in one embodiment, the buffer solution is neutral at pH. The dissolution is subsequently sterilized by filtration under standard conditions known in the art, and then lyophilized to obtain the desired formulation. In one embodiment, the resulting solvent is aliquoted into tubes and lyophilized. Each tube may contain a single or multiple dose of the truncated or fusion protein of the IgA protease or a combination thereof. The amount packed into each vial can be slightly higher than required for each dose or multiple doses (e.g., 10% overdose) to ensure accurate sampling and administration. The lyophilized powder can be stored under appropriate conditions, such as in the range of about 4°C to room temperature.

[0219] The lyophilized powder is reconstituted with water for injection to obtain a formulation for injection. In one embodiment, the lyophilized powder can be reconstituted in sterile, pyrogen-free water or other suitable liquid carrier. The precise amount depends on the chosen therapy and can be determined empirically.

[0220] Methods of treating or preventing diseases

[0221] On the other hand, this application provides a method for treating or preventing IgA deposition-related diseases, comprising administering to a subject requiring treatment or prevention the IgA protease truncated form, the fusion protein, or the pharmaceutical composition described in this application.

[0222] On the other hand, this application provides a method for treating or preventing IgA deposition-related diseases, comprising administering to a subject requiring treatment or prevention an IgA protease or a truncated form thereof, a fusion protein comprising the IgA protease or a truncated form thereof, or a pharmaceutical composition comprising the IgA protease or a truncated form thereof or the fusion protein, wherein the amino acid sequence of the 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 formed by removing the signal peptide sequence from the amino acid sequences shown 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 truncated IgA protease is associated with, but is not identical to, 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: 66. The polypeptide shown in NO:76 has 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).In some embodiments, the IgA protease truncated variant is associated with, but is not identical to, 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: 66. The polypeptide shown in NO:76 has 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%), and still retains the function or activity of the IgA protease (e.g., proteolytic activity, enzyme activity that specifically cleaves IgA, etc.).

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239] On the other hand, this application provides the use of the truncated IgA protease of this application, the fusion protein of this application, or the pharmaceutical composition of this application in the preparation of a medicament for treating or preventing IgA deposition-related diseases.

[0240] In another aspect, this application provides the use of an IgA protease or a truncated form thereof, a fusion protein comprising the IgA protease or a truncated form thereof, or a pharmaceutical composition comprising the IgA protease or a truncated form thereof or the fusion protein in the preparation of a medicament for treating or preventing IgA deposition-related diseases, wherein the amino acid sequence of the 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 combinations thereof. In some embodiments, the amino acid sequence of the IgA protease is the amino acid sequence formed by removing the signal peptide sequence from the amino acid sequences shown 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 truncated IgA protease is associated with, but is not identical to, 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: 66. The polypeptide shown in NO:76 has 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).In some embodiments, the truncated IgA protease is associated with, but is not identical to, 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: 66. The polypeptide shown in NO:76 has 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%), and still retains the function or activity of the IgA protease (e.g., proteolytic activity, enzyme activity that specifically cleaves IgA, etc.).

[0241] On the other hand, this application provides IgA protease truncated forms, fusion proteins, or pharmaceutical compositions as described in this application for the treatment or prevention of IgA deposition-related diseases.

[0242] In another aspect, this application provides an IgA protease or a truncated form thereof for treating or preventing IgA deposition-related diseases, a fusion protein comprising the IgA protease or a truncated form thereof, or a pharmaceutical composition comprising the IgA protease or a truncated form thereof or the fusion protein, wherein the amino acid sequence of the 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 combinations thereof. In some embodiments, the amino acid sequence of the IgA protease is the amino acid sequence formed by removing the signal peptide sequence from the amino acid sequences shown 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 truncated variant is associated with, but is not identical to, 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: 66. The polypeptide shown in NO:76 has 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).In some embodiments, the truncated IgA protease is associated with, but is not identical to, 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: 66. The polypeptide shown in NO:76 has 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%), and still retains the function or activity of the IgA protease (e.g., proteolytic activity, enzyme activity that specifically cleaves IgA, etc.).

[0243] In some embodiments, the IgA deposition-related diseases described in this application include IgA nephropathy, herpetic dermatitis, Henrönlein purpura (also known as IgA vasculitis), Kawasaki disease, purpuric nephritis, IgA vasculitis-related kidney damage, IgA rheumatoid factor-positive rheumatoid arthritis, IgA-type anti-GBM disease, or IgA-type ANCA-related vasculitis. In some embodiments, the IgA deposition-related disease described in this application is IgA nephropathy. In some embodiments, the IgA deposition-related disease described in this application is IgA1 nephropathy. In some embodiments, the IgA deposition-related disease described in this application is IgA vasculitis. In some embodiments, the IgA deposition-related disease described in this application is Kawasaki disease. Detailed Implementation

[0244] All biological materials involved in the embodiments, such as Escherichia coli strains, various cloning and expression plasmids, culture media, tool enzymes, buffers, and various culture methods, protein extraction and purification methods, and other molecular biology operations, are familiar to those skilled in the art. For reference, see "Molecular Cloning" (Laboratory Handbook, Cold Spring Harbor, 1989) edited by Sambrook et al. and "A Concise Guide to Molecular Biology" (US / F. Osber et al., translated by Yan Ziying et al., Beijing: Science Press, 1998).

[0245] Example 1: Study on the shortest active site of AK183 IgA protease

[0246] The inventors removed the signal peptide (i.e., amino acids 1 to 30 of SEQ ID NO: 1) and the transmembrane and intracellular regions (i.e., amino acids 1205 to 1234 of SEQ ID NO: 1) at the N-terminus of the wild-type IgA protease (its amino acid sequence is shown in SEQ ID NO: 1) from Clostridium ramosum AK183 strain. Then, they added the Fc sequence of human IgG1 (HR-CH2-CH3, its amino acid sequence is shown in SEQ ID NO: 24) to the N-terminus of the amino acid sequence of the IgA protease (i.e., the truncated IgA protease composed of amino acids 31 to 1204 of SEQ ID NO: 1) after removing the signal peptide, transmembrane and intracellular regions, thus constructing the PET30a-Fc-AK183 plasmid.

[0247] Then, using the PET30a-Fc-AK183 plasmid as a template, the inventors performed termination mutations to construct a series of Fc-AK183 truncated variants to study the shortest C-terminal active site of the AK183 IgA protease. Based on previous research results, the inventors believed that there was an autocleavage site between amino acids 730 and 840 of the AK183 IgA protease. Therefore, the inventors performed the first round of termination mutations, targeting amino acid positions 738, 769, 799, and 834 of the AK183 IgA protease. The results are as follows. Figure 1 As shown in the figure. The results showed that the AK183(31-737) and AK183(31-768) IgA protease truncated fragments obtained after amino acid termination mutations at positions 738 and 769 had no in vitro enzymatic activity, while the AK183(31-798) and AK183(31-833) IgA protease truncated fragments obtained after amino acid termination mutations at positions 799 or 834 had activity. Therefore, the conclusion of the first round of termination mutations was that the shortest active site at the C-terminus of the AK183 IgA protease is located between amino acids 768 and 798. A second round of termination mutations was then performed, targeting amino acid positions 774, 779, 783, 788, or 793 of the AK183 IgA protease. The results are shown in the figure. Figure 2As shown, the AK183(31-773), AK183(31-778), AK183(31-782), and AK183(31-787) IgA protease truncated fragments obtained by amino acid termination mutations at positions 774, 779, 783, or 788 showed no in vitro enzymatic activity, while the AK183(31-792) IgA protease truncated fragment obtained by amino acid termination mutation at position 793 still exhibited activity. Therefore, the conclusion of the second round of termination mutations is that the shortest active site at the C-terminus of the AK183 IgA protease is located between amino acids 787 and 792. Then, the inventors conducted a third round of termination mutations, targeting amino acid positions 789, 790, 791, or 792 of the AK183 IgA protease, with the following results: Figure 3 As shown, the AK183(31-788) and AK183(31-789) IgA protease truncated fragments obtained after amino acid termination mutations at positions 789 and 790 showed no in vitro cleavage activity, while the AK183(31-790) and AK183(31-791) IgA protease truncated fragments obtained after amino acid termination mutations at positions 791 or 792 still showed activity (among which position 791 may have incomplete activity due to protease conformation issues, exhibiting only slight cleavage). Therefore, the conclusion of the third round of termination mutations is that the shortest active C-terminal fragment of the AK183 IgA protease is AK183(31-790).

[0248] Similarly, the inventors performed three rounds of truncation mutations to study the shortest N-terminal active site of the AK183 IgA protease. First, the inventors performed a first round of truncation mutations, removing a domain of unknown function (DUF) from the N-terminus of AK183 (31-792), fixing the C-terminal amino acid position at 792. For example, removing the DUF corresponding to amino acids 31 to 284 of SEQ ID NO: 1 from the N-terminus of AK183 (31-792) yielded a truncated fragment of the AK183 (285-792) IgA protease. Using a similar method, truncated fragments of AK183(330-792), AK183(380-792), AK183(430-792), AK183(480-792), AK183(530-792), and AK183(580-792) IgA proteases were obtained. The in vitro enzymatic activity of the obtained truncated IgA protease fragments against IgA1 was shown in the following results. Figure 10 As shown. Figure 10As shown, truncated fragments of AK183(285-792) and AK183(330-792) IgA proteases still retain in vitro cleavage activity, while truncated fragments of AK183(380-792), AK183(430-792), AK183(480-792), AK183(530-792), and AK183(580-792) IgA proteases do not exhibit in vitro cleavage activity. Therefore, the conclusion of the first round of truncation mutations is that the shortest active site at the N-terminus of the AK183 IgA protease is located between amino acids 330 and 380. A second round of truncation mutations was then performed, constructing truncated fragments every 5 amino acids between amino acid positions 330 and 380, resulting in 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. The in vitro enzymatic activity of the obtained IgA protease truncated fragments against IgA1 was shown in the following figures. Figure 11 As shown. Figure 11 As shown, the truncated fragments of AK183(335-792)IgA protease still possess in vitro cleavage activity, while the truncated fragments of 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 do not. Therefore, the conclusion of the second round of truncated mutations is that the shortest active site at the N-terminus of the AK183 IgA protease is located between amino acids 335 and 340. Then, the inventors performed a third round of truncation mutations, constructing truncated versions amino acid-by-amino acid between amino acids 335 and 340, obtaining AK183(336-792), AK183(337-792), AK183(338-792), and AK183(339-792) IgA protease truncated fragments, respectively. The in vitro enzymatic activity experiments of the obtained IgA protease truncated fragments against IgA1 are shown below. Figure 12 As shown. Figure 12As shown, the truncated fragments of AK183 (336-792), AK183 (337-792), AK183 (338-792), and AK183 (339-792) IgA proteases all lacked in vitro cleavage activity. Therefore, the conclusion of the third round of truncated mutations is that the shortest active site at the N-terminus of the AK183 IgA protease is located at amino acid 335. Finally, the inventors re-verified the results of the first three rounds by simultaneously expressing truncated fragments of 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. The in vitro enzymatic activity of the obtained truncated IgA protease fragments against IgA1 was as follows... Figure 13 As shown. Figure 13 As shown, the truncated fragments of AK183(285-792), AK183(330-792), and AK183(335-792) IgA proteases still have in vitro enzymatic cleavage activity, while the truncated fragments of AK183(336-792), AK183(337-792), AK183(338-792), AK183(339-792), AK183(340-792), AK183(345-792), and AK183(350-792) IgA proteases do not have in vitro enzymatic cleavage activity. This is consistent with the conclusion of the aforementioned three rounds of truncated mutations, namely, that the shortest active site at the N-terminus of the AK183 IgA protease is located at amino acid 335.

[0249] In summary, the shortest active fragment of the AK183 IgA protease is AK183 (335-790).

[0250] Example 2: Preparation of fusion proteins containing either a truncated form of AK183 IgA protease or the full length of AK183 IgA protease

[0251] 2.1 Plasmid Construction

[0252] After determining the shortest active C-terminal fragment AK183(31-790) of the AK183 IgA protease, the inventors placed the Fc domain at the C-terminus of amino acid 790 of the AK183 IgA protease, added a GGGGS linker, and added a 6XHis tag to the C-terminus of the Fc domain for protein purification, thus constructing the PET30a-AK183(31-790)-Fc plasmid. The construction procedure is as follows: Figure 4As shown. Then, using the PET30a-AK183(31-790)-Fc plasmid as a template, the inventors added the 791st and 792nd amino acids after the truncated AK183(31-790) by PCR to construct the PET30a-AK183(31-792)-Fc plasmid.

[0253] The applicant also commissioned Beijing Liuhe BGI Genomics 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 hinge region of the Fc (CH2-CH3) subclones in these candidate subclones has been removed, and its amino acid sequence is shown in SEQ ID NO: 6 (SEQ ID NO: 6 lacks the first 9 amino acids EPKSCDKTH of SEQ ID NO: 2 compared to SEQ ID NO: 2). Furthermore, 10 His molecules (located after the linker GGGGS and before Fc) have been added between the truncated IgA protease and Fc. These four candidate subclones are intended as alternative schemes for subsequent protease yield and purity screening.

[0254] To investigate whether the connection mode between the AK183 IgA protease truncated fragment and the Fc region would affect its IgA cleavage activity, the inventors also constructed two candidate subclones, PET30a-AK183(285-816)-Fc and PET30a-Fc-AK183(285-816), in which the amino acid sequence of Fc is shown in SEQ ID NO: 25.

[0255] To compare the enzymatic cleavage activity of the fusion protein formed by the truncated fragment of AK183 IgA protease and Fc, and the fusion protein formed by the full-length AK183 IgA protease and Fc, the inventors also constructed a candidate subclone PET30a-Fc-AK183(31-1203), wherein the amino acid sequence of Fc is shown in SEQ ID NO: 24.

[0256] To investigate the effects of IgG1 Fc, IgG4 Fc, and albumin on the IgA cleavage activity of the fusion protein containing the truncated form of the AK183 IgA protease, the inventors also 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.

[0257] To investigate the effect of different linkers on the IgA cleavage activity of the fusion protein containing the AK183 IgA protease truncated variant, the inventors constructed six candidate subclones PET30a-AK183(285-816)-linker-Fc. The fusion protein sequences expressed by these six candidate subclones were identical to those of AK183(285-816) and Fc, except for the linkers. 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 were HHHHHHHHHH (SEQ ID NO: 59, also known as "10xHis"), EEKKKEKEKEEQEERETK (SEQ ID NO: 58, also known as "IgD linker"), GGGGS (SEQ ID NO: 22, also known as "1xlinker"), GGGGSGGGGS (SEQ ID NO: 58, also known as "IgD linker"), and GGGGSGGGGS (SEQ ID NO: 59, also known as "1xlinker"). NO: 78, also known as "2xlinker"), GGGGSGGGGSGGGGS (SEQ ID NO: 79, also known as "3xlinker") and GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 80, also known as "4xlinker").

[0258] 2.2 Fusion Protein Preparation Method

[0259] The expression vector was transfected into competent *E. coli* (BL21-DE3) cells. After resistance selection using LB agar plates containing 50 μg / ml kanamycin, single colonies were picked and cultured in LB medium containing the appropriate antibiotic until the exponential growth phase (OD600: 0.6-0.8). After reaching the exponential growth phase, 0.1-0.5 mM isopropyl-β-D-thiogalactoside (IPTG) was added for induction, and expression was induced at 16°C for 24 h. After expression, *E. coli* cells were processed using standard methods, sonicated, centrifuged at high speed, and the supernatant was retained. The recombinant fusion protein was then purified using affinity chromatography and molecular sieve filtration.

[0260] The amino acid sequence of the AK183(31-792)-Fc fusion protein expressed by the PET30a-AK183(31-792)-Fc plasmid is shown in SEQ ID NO: 2, and its encoded nucleic acid sequence is shown in SEQ ID NO: 3;

[0261] The amino acid sequence of the AK183(31-798)-Fc fusion protein expressed by the PET30a-AK183(31-798)-Fc plasmid is shown in SEQ ID NO: 6, and its encoding nucleic acid sequence is shown in SEQ ID NO: 7;

[0262] The amino acid sequence of the AK183(31-807)-Fc fusion protein expressed by the PET30a-AK183(31-807)-Fc plasmid is shown in SEQ ID NO: 8, and its encoded nucleic acid sequence is shown in SEQ ID NO: 9;

[0263] The amino acid sequence of the AK183(31-816)-Fc fusion protein expressed by the PET30a-AK183(31-816)-Fc plasmid is shown in SEQ ID NO: 10, and its encoding nucleic acid sequence is shown in SEQ ID NO: 11;

[0264] The amino acid sequence of the AK183(31-833)-Fc fusion protein expressed by the PET30a-AK183(31-833)-Fc plasmid is shown in SEQ ID NO: 12, and its encoding nucleic acid sequence is shown in SEQ ID NO: 13;

[0265] 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;

[0266] The amino acid sequence of the Fc-AK183(285-816) fusion protein expressed by the PET30a-Fc-AK183(285-816) plasmid is shown in SEQ ID NO: 82;

[0267] The amino acid sequence of the Fc-AK183(31-1203) fusion protein expressed by the PET30a-Fc-AK183(31-1203) plasmid is shown in SEQ ID NO: 83;

[0268] 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;

[0269] The amino acid sequence of the AK183(31-816)-albumin fusion protein expressed by PET30a-AK183(31-816)-albumin particles is shown in SEQ ID NO: 85.

[0270] 2.3 In vitro activity testing methods

[0271] The obtained fusion protein containing the truncated form of AK183 IgA protease was mixed in vitro with substrate IgA1 purified from the plasma of IgA nephropathy patients and reacted at 37°C for 2-12 h. Then, Western blot was performed to verify its enzymatic activity against substrate IgA1.

[0272] 2.4 In vivo activity assay methods

[0273] The obtained fusion protein containing the truncated AK183 IgA protease was injected into humanized IgA1alpha chain knock-in (α1KI-Tg) C57BL / 6 mice via tail vein injection. Blood samples were collected before injection and at 5 min, 2 h, 4 h, and 24 h after injection, and then Western blot was performed for verification.

[0274] 2.5 Results

[0275] Experiments showed that the PET30a-AK183(31-790)-Fc plasmid successfully expressed the AK183(31-790)-Fc fusion protein (e.g., Figure 5 (As shown). Meanwhile, the AK183(31-792)-Fc fusion protein exhibits the expected full-length protein expression (as shown). Figure 6 As shown in a), it also has in vitro enzymatic activity against IgA1 (e.g., as shown in a). Figure 6 (as shown in b).

[0276] 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 fusion proteins and possessed in vitro enzymatic activity against IgA1 (e.g., Figure 7 (As shown).

[0277] In addition, subclones PET30a-AK183(285-816)-Fc and PET30a-Fc-AK183(285-816) both expressed fusion proteins (such as...). Figure 14 As shown), and all of them have in vitro enzymatic digestion activity (e.g. Figure 15 (As shown).

[0278] The inventors also 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), and the results are as follows. Figure 8 (AK183(31-807)-Fc, under reducing conditions) and Figure 17 (Fc-AK183(285-816), under non-reducing conditions) as shown. Figure 8As shown, in humanized IgA1 mice (α1KI-Tg) C57BL / 6, after receiving a single-needle tail vein injection of the AK183(31-807)-Fc fusion protein, the complete disappearance of the intact IgA1 heavy chain (H) in the blood persisted for at least 24 hours. Figure 17 As shown, in humanized IgA1 mice (α1KI-Tg) C57BL / 6, after receiving a single-needle tail vein injection of the Fc-AK183 (285-816) fusion protein, the complete IgA1 heavy chain (H) disappeared from the blood and persisted for at least 2 weeks.

[0279] The inventors also compared the enzymatic activities of Fc-AK183(285-816) fusion protein, AK183(285-816)-Fc fusion protein, and AK183(285-816) IgA protease truncated variant on IgA1, and the results are as follows: Figure 16 As shown. Figure 16 As shown, all three proteins exhibit enzymatic activity against IgA1.

[0280] The inventors also compared the enzymatic activity of AK183(285-816)-Fc fusion protein and Fc-AK183(31-1203) fusion protein against IgA1, and the results are as follows: Figure 18 As shown. Figure 18 As shown, both the AK183(285-816)-Fc fusion protein and the Fc-AK183(31-1203) fusion protein exhibit enzymatic activity against IgA1.

[0281] The inventors also compared the enzymatic activities of AK183(31-816)-IgG1 Fc fusion protein, AK183(31-816)-IgG4 Fc fusion protein, and AK183(31-816)-albumin fusion protein on IgA1, and the results are as follows: Figure 19 As shown. Figure 19 As shown, all three fusion proteins exhibit enzymatic activity against IgA1.

[0282] The inventors also compared the enzymatic activity of AK183(285-816)-Fc fusion proteins with different linkers (10xHis, IgD linker, 1xlinker, 2xlinker, 3xlinker, or 4xlinker) against IgA1, and the results are as follows: Figure 20 As shown. Figure 20 As shown, all six fusion proteins exhibit enzymatic activity against IgA1.

[0283] 2.6 Eukaryotic Expression System

[0284] All the above experiments were conducted in *E. coli* (BL21-DE3) competent cells (i.e., the prokaryotic expression system). Next, the inventors cloned the AK183(31-792)-Fc fusion cDNA sequence into the pcDNA3.1 / hygro(+) expression vector, and added the signal peptide sequence encoding human IL-2 (SEQ ID NO: 41) to the N-terminus of the fusion protein, constructing the pcDNA3.1 / hygro(+)-IL2-AK183(31-792)-Fc plasmid, which was used to transfect the eukaryotic expression system HEK293 cells. The Fc sequence underwent codon optimization specifically for the eukaryotic expression system. 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 its encoding nucleic acid sequence is shown in SEQ ID NO: 5.

[0285] The expression results of AK183(31-792)-Fc fusion protein in HEK293 cells are as follows: Figure 9 As shown in the figure. The results indicate that the AK183(31-792)-Fc fusion protein is expressed in its expected full length, and the fusion protein expressed in the eukaryotic system exists in a dimer form.

[0286] Example 3: Preparation and activity test of AK183 IgA protease mutant

[0287] Based on the truncated AK183(31-1203) IgA protease, the inventors performed site-directed mutagenesis on proline (P) at positions 844, 862, 931, 933, 978, 1002, and 1004 (all positions are relative to SEQ ID NO: 1), respectively, to mutate it to glycine (G), resulting in five mutants of the AK183(31-1173) IgA protease truncated AK183. Their amino acid sequences are shown in 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"), and SEQ ID NO: 57 (also known as "PIP-GIG Mut").

[0288] The inventors tested the IgA1 cleavage activity of these five mutants, and the results are as follows: Figure 21As shown. Figure 21 As shown, all five mutants exhibit enzymatic activity against IgA1.

[0289] In addition, based on the amino acid sequence of the AK183(31-816)-Fc fusion protein prepared in Example 2 (i.e., SEQ ID NO: 10), the inventors performed a site-directed mutation on the alanine (A) at position 7 of the Fc region (relative to SEQ ID NO: 25), which was mutated to valine (V), glycine (G), serine (S), and leucine (L), respectively, to obtain four mutants of the AK183(31-816)-Fc fusion protein, which were named AV Mut, AG Mut, AS Mut, and AL Mut, respectively.

[0290] The inventors tested the enzymatic activity of these four mutants against IgA1, and the results are as follows: Figure 22 As shown. Figure 22 As shown, all four mutants exhibit enzymatic activity against IgA1.

[0291] Example 4: Exploring other IgA proteases

[0292] The inventors screened several amino acid sequences with certain homology to the wild-type IgA enzyme of AK183 from a metagenomic database and synthesized 16 AK183 homologs. Their amino acid sequences are shown in SEQ ID NO: 61 to SEQ ID NO: 76. Following the in vitro activity assay method described in Example 2.3, the inventors tested the enzymatic activity of these AK183 homologs against IgA1. The results are as follows: Figure 23a and Figure 23b As shown. Figure 23a In this context, "1+IgA1" indicates that the peptide shown in SEQ ID NO: 61 is mixed with the substrate IgA1 in vitro, "2+IgA1" indicates that the peptide shown in SEQ ID NO: 62 is mixed with the substrate IgA1 in vitro, and so on. Figure 23b The “16+IgA1” indicates that the polypeptide shown in SEQ ID NO: 76 is mixed with the substrate IgA1 in vitro.

[0293] Depend on Figure 23a and Figure 23b It is known that the polypeptides shown in SEQ ID NO: 61-76 all have enzymatic activity against IgA1.

[0294] Although this application demonstrates and describes the invention in a specific way by referring to specific embodiments, those skilled in the art should understand that various changes in form and detail can be made to the above content without departing from the spirit and scope of protection disclosed in this application.

Claims

1. An isolated truncated form of IgA protease, the amino acid sequence of which is shown in SEQ ID NO: 16, 17, 18, 19, 20, 45, 46, 49 or 52.

2. A fusion protein comprising a first polypeptide and a second polypeptide, wherein: a) The first polypeptide is the IgA protease truncated form as described in claim 1; b) The second polypeptide is selected from: the Fc domain and albumin.

3. The fusion protein of claim 2, wherein the second polypeptide is located at the N-terminus or C-terminus of the first polypeptide.

4. A fusion protein comprising a first polypeptide, a linker, and a second polypeptide, wherein: a) The first polypeptide is the IgA protease truncated form as described in claim 1; b) The second polypeptide is selected from: the Fc domain and albumin; c) The first polypeptide and the second polypeptide are connected by the linker.

5. The fusion protein of claim 4, wherein 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.

6. The fusion protein of claim 5, wherein the linker is a peptide linker.

7. The fusion protein of claim 6, wherein the peptide linker is a linker containing glycine and serine.

8. The fusion protein of claim 7, wherein the linker containing glycine and serine is one, two, three, four or more repeats as shown in SEQ ID NO: 21 (GGGS), SEQ ID NO: 22 (GGGGS), SEQ ID NO: 86 (GGGGGS) or SEQ ID NO: 87 (GGGGGGGS).

9. The fusion protein of claim 6, wherein the linker is an amino acid sequence as shown in SEQ ID NO: 23 (GGCGGCGGTGGATCC), SEQ ID NO: 58 (EEKKKEKEKEEQEERETK), or SEQ ID NO: 59 (HHHHHHHHHH).

10. The fusion protein of claim 2 or 4, wherein the Fc domain comprises a hinge region.

11. The fusion protein of claim 10, wherein the Fc domain is derived from the human IgG Fc domain.

12. The fusion protein of claim 11, wherein the Fc domain is derived from the human IgG1 Fc domain, the human IgG2 Fc domain, the human IgG3 Fc domain, or the human IgG4 Fc domain.

13. The fusion protein of claim 2 or 4, wherein the amino acid sequence of the Fc domain is as shown in SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:32 or SEQ ID NO:

77.

14. The fusion protein of claim 13, wherein the Fc domain is mutated at position 7 of SEQ ID NO: 25 to valine, glycine, serine, or leucine.

15. The fusion protein of claim 2 or 4, wherein the Fc domain is linked to the C-terminus or N-terminus of the first polypeptide.

16. The fusion protein of claim 2, wherein the albumin comprises the D3 domain of human serum albumin.

17. The fusion protein of claim 16, wherein the amino acid sequence of the albumin is as shown in SEQ ID NO:

60.

18. The fusion protein of claim 2 or 4, further linked to a tag.

19. The fusion protein of claim 18, wherein the tag is selected from the group consisting of fluorescent tags, luminescent tags, purification tags, and chromogenic tags.

20. The fusion protein of claim 18, 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.

21. The fusion protein of claim 20, wherein the tag is an HIS tag comprising 6, 7, 8, 9, or 10 histidine residues.

22. The fusion protein of claim 18, 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.

23. A fusion protein having an amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 26, 27, 28, 29, 30, 31, 81, 82, 84 or 85.

24. The fusion protein of claim 2, 4 or 23, wherein the half-life of the fusion protein in the blood circulation of the 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.

25. An isolated nucleic acid, which is a nucleotide sequence encoding a truncated form of the IgA protease as described in claim 1 or a nucleotide sequence encoding a fusion protein as described in any one of claims 2 to 24.

26. The nucleic acid of claim 25, wherein the nucleotide sequence is selected from the nucleotide sequences shown 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, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 or SEQ ID NO:

38.

27. A vector comprising the nucleic acid as described in claim 25 or 26.

28. A cell comprising the nucleic acid as claimed in claim 25 or 26 or the vector as claimed in claim 27, wherein the cell is not an embryonic cell.

29. The cell of claim 28, wherein the cell is a prokaryotic cell or a eukaryotic cell.

30. The cell of claim 29, wherein the prokaryotic cell is an Escherichia coli cell.

31. The cell of claim 29, wherein the eukaryotic cell is a mammalian cell.

32. The cell of claim 31, wherein the mammalian cell is a human cell or a Chinese hamster ovary (CHO) cell.

33. The cell of claim 31, wherein the mammalian cell is human embryonic kidney cell 293 (HEK293 cell).

34. A pharmaceutical composition comprising an IgA protease truncated form as claimed in claim 1, a fusion protein as claimed in any one of claims 2 to 24, a nucleic acid as claimed in claim 25 or 26, a vector as claimed in claim 27, or a cell as claimed in any one of claims 28 to 33, and a pharmaceutically acceptable vector.

35. A method for producing a fusion protein, comprising the step of culturing cells as described in any one of claims 28 to 33.

36. Use of the truncated IgA protease of claim 1, the fusion protein of any one of claims 2 to 24, or the pharmaceutical composition of claim 34 in the preparation of a medicament for the treatment or prevention of IgA deposition-related diseases.

37. The use as described in claim 36, wherein the IgA deposition-related disease is IgA nephropathy, herpetic dermatitis, Henrönlein purpura, Kawasaki disease, purpuric nephritis, IgA vasculitis-related kidney damage, IgA rheumatoid factor-positive rheumatoid arthritis, IgA anti-GBM disease, or IgA ANCA-related vasculitis.

38. The use as described in claim 36, wherein the IgA deposition-related disease is IgA nephropathy, IgA vasculitis, or Kawasaki disease.

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