Fusion protein containing IgA protease truncations and uses thereof

Abstract

The present application relates to an IgA protease truncate, a fusion protein comprising the IgA protease truncate (eg, a fusion protein comprising the IgA protease truncate and Fc), and use thereof in treating IgA deposition diseases (eg, IgA nephropathy).

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically, to an IgA protease truncate, a fusion protein comprising an IgA protease truncate, a pharmaceutical composition comprising the IgA protease truncate or the fusion protein, a nucleic acid encoding the IgA protease truncate or the fusion protein, a method for preparing the IgA protease truncate or the fusion protein, and use of the IgA protease truncate or the fusion protein in preparing a drug for treating IgA deposition-related diseases. Background Art

[0002] IgA nephropathy is one of the most common primary glomerular diseases in the world, which brings a heavy burden to patients and society. There is currently no specific treatment for IgA nephropathy. In clinical practice, supportive treatment based on RAS blockers is often used to slow down the deterioration of renal function. For patients who are ineffective with supportive treatment, combined hormone immunosuppressant treatment is given. However, the long-term use of hormone immunosuppressants can cause serious side effects to patients.

[0003] There is an urgent need to develop effective therapeutic drugs with few side effects.

[0004] Brief description of the invention

[0005] In one aspect, the present application provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide is an IgA protease truncation, and the second polypeptide cannot be cleaved by IgA protease or an IgA protease truncation.

[0006] In certain embodiments, the IgA protease truncate comprises a non-natural truncated fragment obtained or derived from the wild-type IgA protease of Streptococcus pneumoniae TIGR4 strain, or has at least 70% sequence identity with the non-natural truncated fragment. In certain embodiments, the non-natural truncated fragment has an amino acid mutation, deletion, insertion or modification based on the wild-type IgA protease of Streptococcus pneumoniae TIGR4 strain, so that the IgA protease truncate loses or reduces the self-cleavage function. In certain embodiments, the amino acid mutation, deletion, insertion or modification occurs in the natural self-cleavage site of the wild-type IgA protease of Streptococcus pneumoniae TIGR4 strain, within 5 amino acid residues upstream and / or within 5 amino acid residues downstream of the natural self-cleavage site. In certain embodiments, the natural self-cleavage site is between the 43rd and 745th positions of the amino acid sequence shown in SEQ ID NO: 1. In certain embodiments, the natural self-cleavage site is between the 43rd and 745th positions of the amino acid sequence shown in SEQ ID NO: 1. between positions 71 and 72, between positions 210 and 211, between positions 228 and 229, between positions 239 and 240, between positions 245 and 246, between positions 382 and 383, between positions 418 and 419, between positions 439 and 440, between positions 490 and 491, between positions 506 and 507, between positions 511 and 512, between positions 513 and 514, between positions 515 and 516, between positions 517 and 518, between positions 520 and 529, between positions 521 and 529, between positions 521 and 529, between positions 523 and 529, between positions 530 and 531 between positions 09 and 510, between positions 514 and 515, between positions 533 and 534, between positions 536 and 537, between positions 563 and 564, between positions 594 and 595, between positions 613 and 614, between positions 616 and 617, between positions 639 and 640, between positions 645 and 646, or between positions 678 and 679.

[0007] In certain embodiments, the non-natural truncated fragment is an N-terminal truncated fragment or a C-terminal truncated fragment of a wild-type IgA protease obtained or derived from Streptococcus pneumoniae TIGR4 strain. In certain embodiments, the C-terminal truncated fragment comprises a polypeptide fragment of at least 703 consecutive amino acids from position 43 of the wild-type IgA protease obtained or derived from Streptococcus pneumoniae TIGR4 strain, or has at least 70% sequence identity with the polypeptide fragment.

[0008] In certain embodiments, the deposit number of the Streptococcus pneumoniae TIGR4 strain is ATCC BAA-334. In certain embodiments, the amino acid sequence of the wild-type IgA protease of the Streptococcus pneumoniae TIGR4 strain is shown in SEQ ID NO:1.

[0009] In some embodiments, the first polypeptide comprises a polypeptide fragment of at least 1300 (e.g., at least 1310, at least 1320, at least 1330, or at least 1340) consecutive amino acids starting from position 665 of the amino acid sequence as shown in SEQ ID NO: 1. In some embodiments, the first polypeptide comprises a polypeptide fragment of amino acids from position 665 to position 2004 of the amino acid sequence as shown in SEQ ID NO: 1 or a polypeptide fragment having at least 70% sequence identity therewith. In some embodiments, the fusion protein has conservative substitutions of amino acids at one or more sites based on the amino acid sequence of the polypeptide fragment. In some embodiments, the first polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 2. In some embodiments, the second polypeptide is located at the N-terminus or C-terminus of the first polypeptide.

[0010] In certain embodiments, the fusion protein has an enzyme activity that specifically cuts human IgA. In certain embodiments, the fusion protein has an enzyme activity that specifically cuts human IgA heavy chain. In certain embodiments, the fusion protein has an enzyme activity that specifically cuts human IgA heavy chain hinge region. In certain embodiments, the fusion protein has an enzyme activity that specifically cuts human IgA1. In certain embodiments, the fusion protein has an enzyme activity that specifically cuts human IgA1 heavy chain. In certain embodiments, the fusion protein has an enzyme activity that specifically cuts human IgA1 heavy chain hinge region.

[0011] In some embodiments, the first polypeptide and the second polypeptide are connected by a linker. In some embodiments, the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker. In some embodiments, the linker comprises a peptide linker. In some embodiments, the peptide linker comprises a linker containing glycine and serine. In some embodiments, the linker containing glycine and serine comprises one, two, three, four or more repeats of an amino acid sequence as shown in SEQ ID NO: 15 (GGGS), SEQ ID NO: 16 (GGGGS), SEQ ID NO: 17 (GGGGGGS) or SEQ ID NO: 19 (GSS). In some embodiments, the linker comprises an amino acid sequence as shown in SEQ ID NO: 18 (GGGGSGGGGSGGGGS), as shown in SEQ ID NO: 20 (GSSGSSG) or as shown in SEQ ID NO: 21 (RSGSSGSSG).

[0012] In certain embodiments, the first polypeptide and the second polypeptide are directly linked.

[0013] In certain embodiments, the second polypeptide comprises an amino acid sequence for extending the half-life of the first polypeptide in a subject. In certain embodiments, the second polypeptide is selected from: an Fc domain and an albumin. In certain embodiments, the Fc domain comprises a hinge region. In certain embodiments, the Fc domain is derived from a human IgG Fc domain. In certain embodiments, the Fc domain is derived from a human IgG4 Fc domain. In certain embodiments, the Fc domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity with SEQ ID NO: 14. In certain embodiments, the Fc domain comprises an amino acid sequence as shown in SEQ ID NO: 14. In certain embodiments, the Fc domain comprises one or more mutations that extend the half-life of the fusion protein. In certain embodiments, the Fc domain is connected to the C-terminus or N-terminus of the first polypeptide. In certain embodiments, the fusion protein comprises a first polypeptide and a second polypeptide, wherein the amino acid sequence of the first polypeptide is as shown in SEQ ID NO: 2, and the amino acid sequence of the second polypeptide is as shown in SEQ ID NO: 14. In some embodiments, the fusion protein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are directly connected by a linker as shown in SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 8. In some embodiments, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 24. In some embodiments, the fusion protein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are connected by a linker as shown in SEQ ID NO: 20, the amino acid sequence of the first polypeptide is shown in SEQ ID NO: 2, and the amino acid sequence of the second polypeptide is shown in SEQ ID NO: 14. In some embodiments, the fusion protein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are connected by a linker as shown in SEQ ID NO: 21, the amino acid sequence of the first polypeptide is shown in SEQ ID NO: 2, and the amino acid sequence of the second polypeptide is shown in SEQ ID NO: 14. 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.

[0014] In some embodiments, the fusion protein further comprises a tag. In some embodiments, the tag is selected from the group consisting of a fluorescent tag, a luminescent tag, a purification tag, and a chromogenic tag. In some embodiments, the tag is selected from the group consisting of a c-Myc tag, an HA tag, a VSV-G tag, a FLAG tag, a V5 tag, and a HIS tag. In some embodiments, the tag is a HIS tag comprising 6, 7, 8, 9, 10 or more histidines. In some embodiments, the second polypeptide is located at the C-terminus of the first polypeptide, and the tag is located at the C-terminus of the second polypeptide.

[0015] In certain embodiments, the half-life of the fusion protein 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.

[0016] In another aspect, the present application provides an isolated nucleic acid comprising a nucleotide sequence encoding the fusion protein described in the present application. In certain embodiments, the nucleic acid described in the present application comprises a nucleotide sequence as shown in SEQ ID NO: 12 or a nucleotide sequence having at least 70% sequence identity thereto. In certain embodiments, the nucleic acid described in the present application comprises a nucleotide sequence as shown in SEQ ID NO: 25 or a nucleotide sequence having at least 70% sequence identity thereto.

[0017] In another aspect, the present application provides a separated IgA protease truncate, which comprises a non-natural truncated fragment obtained from or derived from the wild-type IgA protease of Streptococcus pneumoniae TIGR4 strain, or has at least 70% sequence identity with the non-natural truncated fragment. In certain embodiments, the non-natural truncated fragment has an amino acid mutation, deletion, insertion or modification on the basis of the wild-type IgA protease of the Streptococcus pneumoniae TIGR4 strain, so that the IgA protease truncate loses or reduces the self-cleavage function. In certain embodiments, the amino acid mutation, deletion, insertion or modification occurs in the natural self-cleavage site of the wild-type IgA protease of the Streptococcus pneumoniae TIGR4 strain, within 5 amino acid residues upstream of the natural self-cleavage site and / or within 5 amino acid residues downstream.

[0018] In certain embodiments, the non-natural truncated fragment is an N-terminal truncated fragment or a C-terminal truncated fragment of a wild-type IgA protease obtained or derived from Streptococcus pneumoniae TIGR4 strain. In certain embodiments, the deposit number of the Streptococcus pneumoniae TIGR4 strain is ATCC BAA1334. In certain embodiments, the C-terminal truncated fragment comprises a polypeptide fragment of at least 703 consecutive amino acids from position 43 of the wild-type IgA protease obtained or derived from Streptococcus pneumoniae TIGR4 strain, or has at least 70% sequence identity with the polypeptide fragment. In certain embodiments, the amino acid sequence of the wild-type IgA protease of Streptococcus pneumoniae TIGR4 strain is shown in SEQ ID NO: 1.

[0019] In some embodiments, the natural self-cleavage site is between positions 43 and 745 of the amino acid sequence as shown in SEQ ID NO: 1. In some embodiments, the natural self-cleavage site is between positions 71 and 72, between positions 210 and 211, between positions 228 and 229, between positions 239 and 240, between positions 245 and 246, between positions 382 and 383, between positions 418 and 419, between positions 439 and 440, between positions 490 and 491, between positions 506 and 507, between positions 511 and 512, between positions 513 and 514, between positions 515 and 516, between positions 517 and 518, between positions 519 and 520, between positions 521 and 522, between positions 523 and 524, between positions 525 and 526, between positions 527 and 528, between positions 529 and 529, between positions 530 and 531, between positions 532 and 533, between positions 534 and 535, between positions 536 and 537, between positions 538 and 539, between positions 540 and 541 between positions 09 and 510, between positions 514 and 515, between positions 533 and 534, between positions 536 and 537, between positions 563 and 564, between positions 594 and 595, between positions 613 and 614, between positions 616 and 617, between positions 639 and 640, between positions 645 and 646, or between positions 678 and 679.

[0020] In some embodiments, the IgA protease truncate comprises a polypeptide fragment of at least 1300 (e.g., at least 1310, at least 1320, at least 1330, or at least 1340) consecutive amino acids starting from position 665 of the amino acid sequence as shown in SEQ ID NO: 1. In some embodiments, the IgA protease truncate comprises a polypeptide fragment of amino acids from position 665 to position 2004 of the amino acid sequence as shown in SEQ ID NO: 1 or a polypeptide fragment having at least 70% sequence identity therewith. In some embodiments, based on the amino acid sequence of the polypeptide fragment, there is a conservative substitution of amino acids at one or more positions. In some embodiments, the IgA protease truncate comprises the amino acid sequence as shown in SEQ ID NO: 2. In some embodiments, the IgA protease truncate consists of the amino acid sequence as shown in SEQ ID NO: 2.

[0021] In certain embodiments, the IgA protease truncate provided herein has an enzyme activity that specifically cuts human IgA. In certain embodiments, the IgA protease truncate provided herein has an enzyme activity that specifically cuts human IgA heavy chain. In certain embodiments, the IgA protease truncate provided herein has an enzyme activity that specifically cuts the intersection of human IgA heavy chain CH1 and hinge region. In certain embodiments, the IgA protease truncate provided herein has an enzyme activity that specifically cuts human IgA1. In certain embodiments, the IgA protease truncate provided herein has an enzyme activity that specifically cuts into IgA1 heavy chain. In certain embodiments, the IgA protease truncate provided herein has an enzyme activity that specifically cuts the intersection of human IgA1 heavy chain CH1 and hinge region.

[0022] In another aspect, the present application provides an isolated nucleic acid comprising a nucleotide sequence encoding the IgA protease truncate described in the present application.

[0023] In another aspect, the present application provides a vector comprising the nucleic acid described in the present application.

[0024] In another aspect, the application provides a kind of cell, it comprises nucleic acid or vector described in the 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).

[0025] In another aspect, the present application provides a pharmaceutical composition comprising the fusion protein described herein, the nucleic acid described herein, the vector described herein, or the cell described herein, and a pharmaceutically acceptable carrier.

[0026] In another aspect, the present application provides a method for producing a fusion protein, which comprises the step of culturing the cells described in the present application.

[0027] In another aspect, the present application provides a method for treating or preventing IgA deposition-related diseases, comprising administering the IgA protease truncate as described herein, the fusion protein as described herein, or the pharmaceutical composition as described herein to a subject in need of treatment or prevention.

[0028] In another aspect, the present application provides use of the IgA protease truncate as described herein, the fusion protein as described herein, or the pharmaceutical composition as described herein in the preparation of a medicament for treating or preventing IgA deposition-related diseases.

[0029] In another aspect, the present application provides an IgA protease truncate as described herein, a fusion protein as described herein, or a pharmaceutical composition as described herein for treating or preventing IgA deposition-related diseases.

[0030] On the other hand, the present application provides a method for treating or preventing IgA deposition-related diseases, which comprises administering IgA protease or a truncation thereof, a fusion protein comprising the IgA protease or a truncation thereof, or a pharmaceutical composition comprising the IgA protease or a truncation thereof or the fusion protein to a subject in need of treatment or prevention, wherein the amino acid sequence of the IgA protease is selected from the following group: SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or a combination thereof.

[0031] On the other hand, the present application provides the use of IgA protease or its truncation, a fusion protein comprising the IgA protease or its truncation, or a pharmaceutical composition comprising the IgA protease or its truncation 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 following group: SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or a combination thereof.

[0032] On the other hand, the present application provides an IgA protease or a truncation thereof, a fusion protein comprising the IgA protease or a truncation thereof, or a pharmaceutical composition comprising the IgA protease or a truncation thereof or the fusion protein for treating or preventing IgA deposition-related diseases, wherein the amino acid sequence of the IgA protease is selected from the following group: SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or a combination thereof.

[0033] In certain embodiments, the IgA deposition-related diseases include IgA nephropathy, dermatitis herpetiformis, Henoch-Schönlein purpura (also known as IgA vasculitis), Kawasaki disease, purpuric nephritis, IgA vasculitis renal damage, IgA rheumatoid factor-positive rheumatoid arthritis, IgA anti-GBM disease or IgA ANCA-associated vasculitis. In certain embodiments, the IgA deposition-related diseases are IgA nephropathy, IgA vasculitis or Kawasaki disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The results of the in vitro enzymatic cleavage activity experiments of different TIGR4 IgA truncations, TIGR4(745-2004), TIGR4(845-2004) and TIGR4(945-2004), on IgA1 are shown.

[0035] Figure 2 a shows the results of the in vitro enzyme cleavage activity test of fusion protein 1 on IgA1. Figure 2 b shows the expression of fusion protein 1 in prokaryotic cells BL21 (DE3).

[0036] Figure 3 a shows the results of the in vitro enzyme cleavage activity test of fusion protein 2 on IgA1. Figure 3 b shows the expression of fusion protein 2 in prokaryotic cells BL21 (DE3).

[0037] Figure 4 a shows the results of the in vitro enzyme cleavage activity test of fusion protein 3 on IgA1. Figure 4 b shows the expression of fusion protein 3 in prokaryotic cells BL21 (DE3).

[0038] Figure 5 The results of the in vitro enzymatic cleavage activity experiment on IgA1 of fusion protein 3 purified using nickel and S200 respectively are shown.

[0039] Figure 6The graph shows the aggregation state of the fusion protein 3 purified by nickel at different time points at 4°C, the aggregation state of different flow-through peaks of the fusion protein 3 purified by S200, and the liquid chromatogram of the purified product.

[0040] Figure 7 a shows the stability of the first flow-through peak F1 of fusion protein 3 purified by S200 at 37°C for different time periods; Figure 7 b shows the stability of fusion protein 3 purified by S200 at 37°C for different time periods.

[0041] Figure 8 a shows the aggregation of fusion protein 4 purified using nickel and S200, respectively; Figure 8 b shows the stability of fusion protein 4 obtained by nickel purification at 37°C for different time periods.

[0042] Fig. 9 The in vivo activity of fusion protein 4 in humanized IgA1 (α1KI-Tg) C57BL / 6 mice was shown.

[0043] Fig.10 a shows the results of the in vitro enzyme cleavage activity test of fusion protein 5 on IgA1; Fig.10 b shows the stability of fusion protein 5 obtained by nickel purification at 37°C for different time periods.

[0044] Fig.11 The in vivo activity of fusion protein 5 in humanized IgA1 (α1KI-Tg) C57BL / 6 mice was shown.

[0045] Fig.12 Shows the results of the enzymatic activity experiment of five TIGR4 homologous enzymes on IgA1. DETAILED DESCRIPTION OF THE INVENTION

[0047] Although the present application will disclose multiple aspects and embodiments below, it is obvious that various equivalent changes and modifications can be made to it by those skilled in the art without violating the spirit and scope of the subject matter of the present application. The multiple aspects and embodiments disclosed in the present application are only for illustration, and are not intended to limit the present application. The actual scope of protection of the present application is subject to the claims. Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the field to which the present application belongs. All references, patents, and patent applications cited in this application are incorporated herein by reference in their entirety.

[0048] definition

[0049] The term "Streptococcus pneumoniae" as used in this application refers to Streptococcus pneumoniae, which is an opportunistic pathogen, a Gram-positive coccus, and can produce IgA protease.

[0050] The term "protease" used in this application refers to an enzyme with the ability to decompose proteins and peptides. Protease can hydrolyze the peptide bonds that connect amino acids together in the peptide or polypeptide chain that forms the protein, thereby decomposing the protein. The proteolytic activity of a certain protease is tested in a variety of methods known in the prior art. For example, the proteolytic activity of the protease can be determined by comparing the ability of various protease hydrolysis suitable substrates. Exemplary substrates for proteolytic activity analysis include, for example, dimethyl casein, bovine collagen, bovine elastin, etc. The colorimetric assay using these substrates is also known in the prior art (see, for example, WO99 / 34011 and US 6,376,450).

[0051] The term "IgA protease" used in this application refers to an enzyme that can specifically cut or decompose the IgA immunoglobulin molecules (e.g., IgA1 or IgA2) of a subject (e.g., a human). For example, the IgA protease obtained from or derived from the TIGR4 strain of Streptococcus pneumoniae can specifically cut the peptide bond between the 227th proline (Pro) and the 228th threonine (Thr) of IgA1, thereby decomposing IgA1.

[0052] When referring to a polypeptide or protein, the term "wild type" used in this application refers to a naturally occurring polypeptide or protein that does not include artificial mutations, insertions, deletions or modifications at one or more amino acid positions; when referring to a nucleic acid, nucleotide or polynucleotide, the term "wild type" used in this application refers to a naturally occurring nucleic acid, nucleotide or polynucleotide that does not include artificial mutations, 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 polynucleotides encoding wild-type polypeptides (e.g., artificially synthesized polynucleotides).

[0053] The term "TIGR4" used in this application refers to the TIGR4 strain of Streptococcus pneumoniae. In certain embodiments, the amino acid sequence of the wild-type IgA protease produced by the TIGR4 strain of Streptococcus pneumoniae is shown in SEQ ID NO: 1, wherein amino acids 1 to 42 (underlined) are signal peptides.

[0054]

[0055]

[0056] The term "signal peptide" as used in this application refers to an amino acid residue sequence that can participate in the secretion or directional transport of the mature or precursor form of a protein. The signal peptide is usually located at the N-terminus of the precursor or mature protein sequence. The signal peptide can be endogenous or exogenous. Signal peptides are generally not present in mature proteins. Usually, after protein transport, the signal peptide is cut off from the protein by a signal peptidase. For example, the amino acid sequence shown in SEQ ID NO: 1 minus the signal peptide at the N-terminus is formed as shown in SEQ ID NO: 22.

[0057]

[0058]

[0059] The term "subject" as used in this application includes humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals. "Subjects" can also be livestock animals, such as cattle, pigs, sheep, poultry and horses; or rodents, such as rats, mice; or primates, such as apes, monkeys, chimpanzees, gorillas, orangutans, baboons; or domestic animals, such as dogs and cats. "Subjects" can be male or female, and can be elderly, adults, teenagers, children or infants. Human "subjects" can be Caucasians, Africans, Asians, Semites, or other races or mixtures of said racial backgrounds.

[0060] The terms "protein", "polypeptide" and "peptide" used in this application are used interchangeably and refer to polymers of amino acids. The protein, polypeptide or peptide described in this application may contain natural amino acids, or may contain non-natural amino acids, or analogs or mimetics of amino acids. The protein, polypeptide or peptide described in this application may be obtained by any method known in the art, such as, but not limited to, natural isolation, recombinant expression, chemical synthesis, etc.

[0061] The term "amino acid" as used herein refers to an organic compound containing amino (-NH2) and carboxyl (-COOH) functional groups and side chains unique to each amino acid. The amino acid names are also represented in this application by standard single-letter or three-letter codes, which are summarized as follows:

[0062] name Three letter code Single letter code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamate 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

[0063] In the present application, when "conservative substitution" is used for an amino acid sequence, it refers to replacing an amino acid residue with another amino acid residue having a side chain of 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 usually do not cause significant changes in the conformational structure of the protein, and therefore the biological activity of the protein can be retained.

[0064] The term "homologous" as used herein refers to a nucleic acid sequence (or its complementary strand) or an amino acid sequence that has at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with another sequence when optimally aligned.

[0065] When "percent (%) sequence identity" is used for an amino acid sequence (or nucleic acid sequence), it means that after the sequence is aligned and, if necessary, intervals are introduced to maximize the number of identical amino acids (or nucleic acids), in the candidate sequence, the percentage of amino acid (or nucleic acid) residues identical to those in the reference sequence as a percentage of the amino acid (or nucleic acid) residues in the candidate sequence. In other words, the percentage (%) sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of amino acid residues (or bases) identical to the reference sequence to be compared by the total number of amino acid residues (or bases) in the candidate sequence or the reference sequence (whichever is shorter). Conservative substitutions of the amino acid residues may or may not be considered identical residues. The sequences can be aligned to determine the percentage sequence identity of the amino acid (or nucleic acid) sequences using tools disclosed in the art, such as BLASTN, BLASTp (National Center for Biotechnology Information (NCBI), see also Altschul SF et al., J. Mol. Biol., 215: 403-410 (1990); Stephen F et al., Nucleic Acids Res., 25: 3389-3402 (1997)), ClustalW2 (European Bioinformatics Institute website, see Higgins DG et al., Methods in Enzymology, 266: 383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23 (21): 2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can use the default parameters of the tool or appropriately adjust the parameters according to the needs of the comparison, for example, by selecting a suitable algorithm.

[0066] An "isolated" substance has been artificially altered from its natural state. If an "isolated" composition or substance occurs in nature, it has been altered or removed from its original state, or both. For example, a polynucleotide or polypeptide naturally present in a living animal is not "isolated", but it may be considered "isolated" if the substance with which it coexists in nature is sufficiently separated and present in a substantially pure state. An "isolated nucleic acid sequence" refers to the sequence of an isolated nucleic acid molecule. In some embodiments, "isolated IgA protease truncation" refers to an IgA protease truncation that is at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% pure, wherein the purity is determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis), or chromatography (e.g., ion exchange chromatography or reverse phase HPLC).

[0067] The term "vector" in this application refers to a vehicle into which a genetic element can be operatively inserted and the genetic element can be expressed, such as producing a protein, RNA or DNA encoded by the genetic element, or replicating the genetic element. The vector can be used to transform, transduce or transfect a host cell so that the genetic element it carries is expressed in the host cell. For example, vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), phages such as lambda phage or M13 phage, and animal viruses, etc. The vector can contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector can also contain a replication initiation site. The vector can also include components that assist it in entering the cell, including but not limited to, viral particles, liposomes or protein shells. The vector can be an expression vector or a cloning vector. The vector (e.g., expression vector) provided by the present application contains the nucleic acid sequence encoding the IgA protease truncation or fusion protein described in the present application, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker.

[0068] As used in this application, "treatment" or "therapy" for a disease, disorder or condition includes preventing or alleviating a disease, disorder or condition, reducing the rate of occurrence or development of a disease, disorder or condition, reducing the risk of developing a disease, disorder or condition, preventing or delaying the development of symptoms associated with a disease, disorder or condition, reducing or stopping symptoms associated with a disease, disorder or condition, producing complete or partial reversal of a disease, disorder or condition, curing a disease, disorder or condition, or a combination of the above.

[0069] The term "pharmaceutically acceptable" means that the specified carrier, vehicle, diluent, excipient and / or salt is generally chemically and / or physically compatible with the other ingredients making up the formulation and physiologically compatible with the recipient thereof.

[0070] The term "IgA deposition-related disease" refers to a disease associated with the accumulation of IgA immunoglobulins in an aggregated or non-aggregated form in a tissue or organ of a subject. For example, it includes, but is not limited to, IgA nephropathy, dermatitis herpetiformis, Henoch-Schönlein purpura (also known as IgA vasculitis), Kawasaki disease, purpuric nephritis, IgA vasculitis renal damage, IgA rheumatoid factor-positive rheumatoid arthritis, IgA anti-GBM disease or IgA ANCA-associated vasculitis.

[0071] The term "IgA nephropathy" refers to a kidney disease characterized by the deposition of IgA in the kidneys.

[0072] IgA protease truncations

[0073] In one aspect, the present application provides an isolated IgA protease truncation, which comprises a non-native truncated fragment of a wild-type IgA protease obtained or derived from Streptococcus pneumoniae TIGR4 strain, or has at least 70% sequence identity with the non-native 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). In certain embodiments, an IgA protease truncation having at least 70% sequence identity to 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, enzymatic activity that specifically cleaves IgA, etc.).

[0074] The term "truncate" or "truncated fragment" used in the present application refers to a peptide formed by removing one or more amino acids from one or both ends of a wild-type polypeptide. Therefore, the "truncate" or "truncated fragment" in the present application does not include the full length of the corresponding wild-type polypeptide, but may have one or more amino acid mutations, deletions, insertions or modifications compared to the truncated form of the wild-type polypeptide. For example, "IgA protease truncate" or "IgA protease truncated fragment" may include a peptide formed by removing one or more amino acids from one or both ends of the wild-type IgA protease, and may also include a peptide that has one or more amino acid mutations, deletions, insertions or modifications compared to the truncated form of the wild-type IgA protease.

[0075] In certain embodiments, the IgA protease truncate described in the present application has one or more amino acid mutations, deletions, insertions or modifications compared to its corresponding wild-type IgA protease. For example, in certain embodiments, the IgA protease truncate described in the present application comprises a non-natural truncated fragment of the wild-type IgA protease obtained from or derived from the TIGR4 strain of Streptococcus pneumoniae, wherein the non-natural truncated fragment has an amino acid mutation, deletion, insertion or modification based on the wild-type IgA protease of the TIGR4 strain of Streptococcus pneumoniae, so that the IgA protease truncate loses or reduces the self-enzyme cleavage function.

[0076] The terms "obtained from" and "derived from" as used in this application include not only proteins produced or producible by the organisms mentioned, but also proteins encoded by DNA sequences isolated from such organisms and produced in host organisms containing such DNA sequences, and proteins encoded by synthetic and / or cDNA-derived DNA sequences and having the identifying characteristics of the proteins mentioned. For example, wild-type IgA proteases obtained from or derived from Streptococcus pneumoniae TIGR4 strain include both IgA proteases naturally produced by Streptococcus pneumoniae TIGR4 strain and IgA proteases produced by other host cells (e.g., E. coli) transformed with nucleic acids encoding IgA proteases by using genetic engineering techniques.

[0077] The term "non-natural truncated fragment" used in this application refers to a fragment having an amino acid sequence different from that of the truncated fragment formed after self-cleavage of the wild-type IgA protease of Streptococcus pneumoniae TIGR4 strain in a natural environment (e.g., different amino acid length, different amino acid type, etc.).

[0078] In certain embodiments, the amino acid mutation, deletion, insertion or modification occurs at the natural autolytic site of the wild-type IgA protease of the Streptococcus pneumoniae TIGR4 strain.

[0079] The term "natural autocleavage site" refers to the ability of IgA protease to recognize certain specific peptide bonds of itself, thereby performing autocatalytic cleavage to release the C-terminal mature IgA protease.

[0080] In certain embodiments, the amino acid mutation, deletion, insertion or modification occurs within 5 amino acid residues upstream of the natural self-cleavage site of the wild-type IgA protease of the Streptococcus pneumoniae TIGR4 strain (e.g., 1 amino acid residue, 2 amino acid residues, 3 amino acid residues, 4 amino acid residues or 5 amino acid residues upstream of the natural self-cleavage site). In certain embodiments, the amino acid mutation, deletion, insertion or modification occurs within 5 amino acid residues downstream of the natural self-cleavage site of the wild-type IgA protease of the Streptococcus pneumoniae TIGR4 strain (e.g., 1 amino acid residue, 2 amino acid residues, 3 amino acid residues, 4 amino acid residues or 5 amino acid residues downstream of the natural self-cleavage site). In certain embodiments, the amino acid mutation or deletion occurs within 5 amino acid residues upstream (for example, 1 amino acid residue, 2 amino acid residues, 3 amino acid residues, 4 amino acid residues or 5 amino acid residues upstream of the natural self-cleavage site) and within 5 amino acid residues downstream (for example, 1 amino acid residue, 2 amino acid residues, 3 amino acid residues, 4 amino acid residues or 5 amino acid residues downstream of the natural self-cleavage site) of the wild-type IgA protease of the Streptococcus pneumoniae TIGR4 strain.

[0081] In certain embodiments, the amino acid mutation, deletion, insertion or modification occurs at one or more amino acid positions (corresponding to SEQ ID NO: 1) of the wild-type IgA protease of Streptococcus pneumoniae TIGR4 strain: 71, 97, 175, 177, 210, 222, 228, 239, 245, 254, 262, 292, 303, 315, 338, 382, ​​384, 401, 418, 425, 435, 439, 442, 456, 462, 470, 481, 483, 484, 490, 501, 512, 524, 536, 537, 541, 558, 562, 563, 571, 572, 573, 584, 585, 591, 603, 614, 615, 626, 638, 640, 657, 661, 673, 685 459th, 469th, 474th, 476th, 487th, 490th, 499th, 506th, 509th, 514th, 517th, 533rd, 536th, 549th, 563rd, 576th, 594th, 597th, 613th, 616th, 639th, 644th, 645th, 652nd and 661st.

[0082] In certain embodiments, the non-natural truncated fragment is an N-terminal truncated fragment or a C-terminal truncated fragment of a wild-type IgA protease obtained or derived from the TIGR4 strain of Streptococcus pneumoniae.

[0083] The term "N-terminal truncated fragment" used in the present application refers to a truncated fragment of the amino acid sequence of the amino terminus of the wild-type IgA protease of Streptococcus pneumoniae TIGR4 strain. The starting position of the "amino terminus" can be any position of the amino terminus of the amino acid sequence of the wild-type IgA protease of Streptococcus pneumoniae TIGR4 strain near the amino terminus, for example, it can be the 1st position counted from the amino terminus, or it can be other positions counted from the amino terminus. For another example, if the amino acid sequence of the full length of the wild-type IgA protease is composed of 1000 amino acids, then the amino terminus starting position of its N-terminal truncated fragment can be any position between the 1st and the 500th position of its amino acid sequence from the amino terminus.

[0084] In certain embodiments, the N-terminal starting site of the non-natural truncated fragment of IgA protease described in the present application is within 5 amino acid residues upstream of the natural self-cleavage site of the wild-type IgA protease of Streptococcus pneumoniae TIGR4 strain (for example, 1 amino acid residue upstream of the natural self-cleavage site, 2 amino acid residues, 3 amino acid residues, 4 amino acid residues or 5 amino acid residues). In certain embodiments, the N-terminal starting site of the non-natural truncated fragment of IgA protease described in the present application is within 5 amino acid residues downstream of the natural self-cleavage site of the wild-type IgA protease of Streptococcus pneumoniae TIGR4 strain (for example, 1 amino acid residue downstream of the natural self-cleavage site, 2 amino acid residues, 3 amino acid residues, 4 amino acid residues or 5 amino acid residues).

[0085] In certain embodiments, the N-terminal start site of the non-natural truncated fragment of IgA protease described in the present application is a natural self-cleavage site of the wild-type IgA protease of Streptococcus pneumoniae TIGR4 strain (e.g., corresponding to position 71, 97, 175, 177, 210, 222, 228, 239, 245, 254, 262, 292, 303, 315, 338, 382, ​​384, 401, 418, 425, 435, 439, 442, 456, 462, 470, 481, 483, 484, 490, 501, 512, 524, 536, 537, 540, 551, 552, 563, 571, 572, 573, 584, 585, 586, 587, 591, 603, 614, 615, 626, 627, 638, 640, 651, 661, 673, 687, 691, 715, 720, 739, 741, 752, 760, 771, 780, 791, 803 In some embodiments, the N-terminal start site of the non-natural truncated fragment of IgA protease described in the present application is one amino acid residue downstream of the natural self-cleavage site of the wild-type IgA protease of Streptococcus pneumoniae TIGR4 strain (for example, corresponding to SEQ ID NO: 1). IDNO: 1st 72nd, 98th, 176th, 178th, 211th, 223rd, 229th, 240th, 246th, 255th, 263rd, 293rd, 304th, 316th, 339th, 383rd, 385th, 402nd, 419th, 426th, 436th, 440th, 443rd, 457th, 460, 470, 475, 477, 488, 491, 500, 507, 510, 515, 518, 534, 537, 550, 564, 577, 595, 598, 614, 617, 640, 645, 646, 653 or 662).

[0086] The term "C-terminal truncated fragment" used in the present application refers to a truncated fragment of the amino acid sequence of the carboxyl terminus of the wild-type IgA protease of Streptococcus pneumoniae TIGR4 strain. The termination position of "carboxyl terminus" can be any position of the carboxyl terminus of the amino acid sequence of the wild-type IgA protease of Streptococcus pneumoniae TIGR4 strain near the carboxyl terminus, for example, can be the 1st position counted from the carboxyl terminus, or can be other positions counted from the carboxyl terminus. For another example, if the amino acid sequence of the wild-type IgA protease full length is composed of 1000 amino acids, the carboxyl terminus termination position of its C-terminal truncated fragment can be any position between the 501st and the 1000th position of its amino acid sequence from the amino terminus.

[0087] In certain embodiments, the deposit number of the Streptococcus pneumoniae TIGR4 strain is ATCC BAA-334.

[0088] In certain embodiments, the C-terminal truncated fragment comprises at least 703 (e.g., at least 710, at least 750, at least 800, at least 850, at least 900, at least 950, at least 1000, at least 1050, at least 1100, at least 1150, at least 1200, at least 1250, at least 1300, at least 1350, at least 1400, at least 1450, at least 1500, at least 1550, at least 1600, at least 1650, at least 1700, at least 1750, at least 1800, at least 1850, at least 1900) of the wild-type IgA protease obtained or derived from position 43 of the Streptococcus pneumoniae TIGR4 strain. , at least 1910, at least 1920, at least 1930, at least 1940, at least 1950, at least 1960, at least 1961, at least 1962) consecutive amino acids in a polypeptide fragment, or having at least 70% sequence identity (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) thereto. In certain embodiments, an N-terminal truncated fragment having at least 70% sequence identity to 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 IgA protease (e.g., proteolytic activity, enzyme activity that specifically cleaves IgA, etc.). In certain embodiments, the amino acid sequence of the wild-type IgA protease of Streptococcus pneumoniae TIGR4 strain is shown in SEQ ID NO: 1.

[0089] Unless otherwise specified, the amino acid sites of TIGR4 IgA protease mentioned in this application are the amino acid sites corresponding to the wild-type TIGR4 IgA protease (whose amino acid sequence is shown in SEQ ID NO: 1). For example, the 665th position of TIGR4 IgA protease mentioned in this application corresponds to the 665th position of SEQ ID NO: 1. Unless otherwise specified, the naming convention of the truncation of TIGR4 IgA protease mentioned in this application is TIGR4 (corresponding to the start site of SEQ ID NO: 1-corresponding to the end site of SEQ ID NO: 1). For example, TIGR4 (665-2004) refers to the truncation of TIGR4 IgA protease formed by amino acids from the 665th to the 2004th position of SEQ ID NO: 1.

[0090] In certain embodiments, the natural self-cleavage site of the IgA protease described in the present application is between positions 43 and 745 of the amino acid sequence as shown in SEQ ID NO: 1. In certain embodiments, the natural self-cleavage site of the IgA protease described in the present application is between positions 43 and 665 of the amino acid sequence as shown in SEQ ID NO: 1. In certain embodiments, the natural self-cleavage site is between positions 71 and 72, between positions 210 and 211, between positions 228 and 229, between positions 239 and 240, between positions 245 and 246, between positions 382 and 383, between positions 418 and 419, between positions 439 and 440, between positions 490 and 491, between positions 506 and 507, between positions 511 and 512 of the amino acid sequence as shown in SEQ ID NO: 1. between positions 09 and 510, between positions 514 and 515, between positions 533 and 534, between positions 536 and 537, between positions 563 and 564, between positions 594 and 595, between positions 613 and 614, between positions 616 and 617, between positions 639 and 640, between positions 645 and 646, or between positions 678 and 679.

[0091] In certain embodiments, the IgA protease truncate comprises a polypeptide fragment of at least 1300 consecutive amino acids starting from position 665 of the amino acid sequence as shown in SEQ ID NO: 1. For example, in certain embodiments, the IgA protease truncate provided herein comprises a polypeptide fragment of at least 1310, at least 1320, at least 1321, at least 1322, at least 1323, at least 1324, at least 1325, at least 1326, at least 1327, at least 1328, at least 1329, at least 1330, at least 1331, at least 1332, at least 1333, at least 1334, at least 1335, at least 1336, at least 1337, at least 1338, at least 1339, at least 1340 consecutive amino acids starting from position 665 of the amino acid sequence as shown in SEQ ID NO: 1. In certain embodiments, the IgA protease truncate provided herein comprises a polypeptide fragment of 1340 consecutive amino acids starting from position 665 of the amino acid sequence as shown in SEQ ID NO:1.

[0092] In certain embodiments, the IgA protease truncation comprises a polypeptide fragment of amino acids 665 to 2004 of the amino acid sequence as shown in SEQ ID NO: 1, or a polypeptide fragment having at least 70% (e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) sequence identity thereto. In certain embodiments, an IgA protease truncation having at least 70% sequence identity to 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, enzymatic activity that specifically cleaves IgA, etc.).

[0093] In certain embodiments, the IgA protease truncation described in the present application is a TIGR4 (665-2004) truncation, and its amino acid sequence is shown in SEQ ID NO:2.

[0094]

[0095] In certain embodiments, the IgA protease truncation described in the present application is a TIGR4 (745-2004) truncation, and its amino acid sequence is shown in SEQ ID NO:3.

[0096]

[0097] In certain embodiments, the IgA protease truncation described in the present application is a TIGR4 (845-2004) truncation, and its amino acid sequence is shown in SEQ ID NO:4.

[0098]

[0099]

[0100] In certain embodiments, the IgA protease truncate has conservative substitutions of amino acids at one or more amino acid residues (e.g., at 1, 2, 3, 4, 5 or more amino acid residues) based on the amino acid sequence of the above-mentioned polypeptide fragment. Conservative substitutions of amino acid residues refer to substitutions between amino acids with similar properties, such as substitutions between polar amino acids (e.g., substitutions between glutamine and asparagine), substitutions between hydrophobic amino acids (e.g., substitutions between leucine, isoleucine, methionine and valine), and substitutions between amino acids with the same charge (e.g., substitutions between arginine, lysine and histidine, or substitutions between glutamic acid and aspartic acid), etc. In certain embodiments, the IgA protease truncate described in the present application has conservative substitutions of amino acids at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 15, 20 or more amino acid residues compared to the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0101] Under the premise of not affecting the activity, the IgA protease truncate described in the present application may also contain non-natural amino acids. Non-natural amino acids include, for example, β-fluoroalanine, 1-methylhistidine, γ-methyleneglutamic acid, α-methylleucine, 4,5-dehydrolysine, hydroxyproline, 3-fluorophenylalanine, 3-aminotyrosine, 4-methyltryptophan, etc.

[0102] The IgA protease truncations described herein can also be modified using methods known in the art, such as, but not limited to, PEGylation, glycosylation, amino-terminal modification, fatty acylation, carboxyl-terminal modification, phosphorylation, methylation, etc. It will be appreciated by those skilled in the art that the IgA protease truncations provided herein still retain substantially similar functions to the IgA protease or IgA protease truncations after being modified using methods known in the art.

[0103] In certain embodiments, the IgA protease truncates described in the present application have an enzyme activity that specifically cuts human IgA. In certain embodiments, the IgA protease truncates described in the present application have an enzyme activity that specifically cuts human IgA heavy chain. In certain embodiments, the IgA protease truncates described in the present application have an enzyme activity that specifically cuts the hinge region of human IgA heavy chain. In certain embodiments, the IgA protease truncates described in the present application have an enzyme activity that specifically cuts human IgA1. In certain embodiments, the IgA protease truncates described in the present application have an enzyme activity that specifically cuts human IgA1 heavy chain. In certain embodiments, the IgA protease truncates described in the present application have an enzyme activity that specifically cuts the hinge region of human IgA1 heavy chain.

[0104] In certain embodiments, the IgA protease truncate described in the present application has conservative substitution of amino acids at one or more amino acid residues based on the amino acid sequence of the above-mentioned polypeptide fragment, but still has the enzymatic activity of cleaving human IgA (e.g., IgA1). In certain embodiments, the IgA protease truncate described in the present application has at least 70% sequence identity with the above-mentioned 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), and still has the enzymatic activity of cleaving human IgA (e.g., IgA1).

[0105] Fusion Protein

[0106] In another aspect, the present application provides a fusion protein, which comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the full length of the wild-type IgA protease obtained or derived from the TIGR4 strain of Streptococcus pneumoniae, the polypeptide obtained or derived from the wild-type IgA protease of the TIGR4 strain of Streptococcus pneumoniae without the signal peptide, or the IgA protease truncate described in the present application, and the second polypeptide cannot be cut by the IgA protease or the IgA protease truncate. Not limited by any theory, it is believed that the second polypeptide cannot be cut by the IgA protease or the IgA protease truncate, because this can not only make the fusion protein maintain its integrity and stability, but also maintain the activity of the first polypeptide in cutting IgA.

[0107] In some embodiments, the first polypeptide and the second polypeptide of the fusion protein described in the present application are connected by a linker. In some embodiments, the first polypeptide and the second polypeptide are directly connected (i.e., not connected by a linker). The term "linker" or "linker" used in the present application 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, connected by peptide bonds, and used to connect one or more polypeptides. The linker may or may not have a secondary structure. 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).

[0108] In certain embodiments, the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker. Any suitable linker known in the art can be used. In certain embodiments, the linker comprises a peptide linker. For example, useful linkers in the present application may be rich in glycine and serine residues. Examples include linkers having a single or repeated sequence comprising threonine / serine and glycine, such as GGGS (SEQ ID NO: 15), GGGGS (SEQ ID NO: 16), GGGGGGS (SEQ ID NO: 17) or GSS (SEQ ID NO: 19), or tandem repeats thereof (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeats). In certain embodiments, the linker used in the present application includes GGGGSGGGGSGGGGS (SEQ ID NO: 18). In certain embodiments, the linker used in the present application includes GSSGSSG (SEQ ID NO: 20). In certain embodiments, the linker used in the present application comprises RSGSSGSSG (SEQ ID NO: 21). In certain embodiments, the linker used in the present application comprises RSGGGGS (SEQ ID NO: 31). In certain embodiments, the linker used in the present application comprises or consists of an amino acid sequence selected from the group consisting of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any one of SEQ ID NOs: 18, 20, 21, 31.

[0109] In certain embodiments, the second polypeptide comprises an amino acid sequence for extending the half-life of the first polypeptide in the subject. In certain embodiments, the second polypeptide is selected from an Fc domain and albumin. In certain embodiments, the Fc domain comprises a hinge region. In certain embodiments, the Fc domain comprises a lower hinge region. In certain embodiments, the Fc domain comprises a core hinge region and a lower hinge region. In certain embodiments, the Fc domain comprises an upper hinge region, a core hinge region and a lower hinge region. In certain embodiments, the Fc domain does not comprise a hinge region. In certain embodiments, the Fc domain is derived from an IgG Fc domain. In certain 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.

[0110] In certain embodiments, the Fc domain is derived from a human IgG1 Fc domain. In certain embodiments, the Fc domain comprises the amino acid sequence as shown in SEQ ID NO: 13. In certain embodiments, the Fc domain consists of the amino acid sequence as shown in SEQ ID NO: 13. In certain embodiments, the amino acid sequence of the Fc domain has at least 70%, 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 with the amino acid sequence as shown in SEQ ID NO: 13.

[0111]

[0112] In certain embodiments, the Fc domain is derived from a human IgG4 Fc domain. In certain embodiments, the Fc domain comprises the amino acid sequence shown in SEQ ID NO: 14. In certain embodiments, the Fc domain consists of the amino acid sequence shown in SEQ ID NO: 14. In certain embodiments, the amino acid sequence of the Fc domain has at least 70%, 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 with the amino acid sequence shown in SEQ ID NO: 14.

[0113]

[0114] The inventors of the present application unexpectedly found that although the fusion protein formed by the TIGR4 IgA protease truncate and the IgG4 Fc domain or the IgG1 Fc domain has the enzymatic activity of cleaving IgA, the stability of the fusion protein formed by the TIGR4 IgA protease truncate and the IgG4 Fc domain is better than the stability of the fusion protein formed by the TIGR4 IgA protease truncate and the IgG1 Fc domain. In certain embodiments, the half-life of the fusion protein formed by the TIGR4 IgA protease truncate and the IgG4 Fc domain is at least 10 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours or at least 96 hours longer than the half-life of the fusion protein formed by the TIGR4 IgA protease truncate and the IgG1 Fc domain.

[0115] In some embodiments, the Fc domain comprises one or more mutations that extend the half-life of the fusion protein. In some embodiments, the Fc domain is connected to the C-terminus of the first polypeptide. In some embodiments, the Fc domain is connected to the N-terminus of the first polypeptide.

[0116] In some embodiments, the second polypeptide is albumin. In some embodiments, the amino acid sequence of the albumin is shown in SEQ ID NO: 23. 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.

[0117]

[0118]

[0119] In certain embodiments, the fusion protein provided by the present application further comprises a tag. In certain embodiments, the tag is selected from the group consisting of a fluorescent tag, a luminescent tag, a purification tag, and a chromogenic tag. In certain embodiments, the tag is selected from the group consisting of a c-Myc tag, an HA tag, a VSV-G tag, a FLAG tag, a V5 tag, and a HIS tag. In certain embodiments, the tag is a HIS tag. In certain embodiments, the tag is a HIS tag comprising 6, 7, 8, 9, 10 or more histidines. In certain 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.

[0120] In certain embodiments, the present application provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, and the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 13 or SEQ ID NO: 14.

[0121] In certain embodiments, the present application provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 2, and the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 13. In certain embodiments, the present application provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide consists of an amino acid sequence as shown in SEQ ID NO: 2, and the second polypeptide consists of an amino acid sequence as shown in SEQ ID NO: 13. In certain embodiments, the present application provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide consists of an amino acid sequence as shown in SEQ ID NO: 2, the second polypeptide consists of an amino acid sequence as shown in SEQ ID NO: 13, and the first polypeptide is located at the N-terminus of the second polypeptide. In certain embodiments, the present application provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide consists of an amino acid sequence as shown in SEQ ID NO: 2, the second polypeptide consists of an amino acid sequence as shown in SEQ ID NO: 13, and the first polypeptide is located at the C-terminus of the second polypeptide.

[0122] In certain embodiments, the present application provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 2, and the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 14. In certain embodiments, the present application provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide consists of an amino acid sequence as shown in SEQ ID NO: 2, and the second polypeptide consists of an amino acid sequence as shown in SEQ ID NO: 14. In certain embodiments, the present application provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide consists of an amino acid sequence as shown in SEQ ID NO: 2, the second polypeptide consists of an amino acid sequence as shown in SEQ ID NO: 14, and the first polypeptide is located at the N-terminus of the second polypeptide. In certain embodiments, the present application provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide consists of an amino acid sequence as shown in SEQ ID NO: 2, the second polypeptide consists of an amino acid sequence as shown in SEQ ID NO: 14, and the first polypeptide is located at the C-terminus of the second polypeptide.

[0123] In certain embodiments, the fusion protein provided herein includes the amino acid sequence shown in SEQ ID NO: 5. In certain embodiments, the fusion protein provided herein consists of the amino acid sequence shown in SEQ ID NO: 5, or has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity thereto. In certain embodiments, a fusion protein having at least 70%, 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 to the amino acid sequence shown in SEQ ID NO:5 still retains the function or activity of IgA protease (e.g., proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.).

[0124]

[0125]

[0126] In certain embodiments, the fusion protein provided herein includes the amino acid sequence shown in SEQ ID NO: 6. In certain embodiments, the fusion protein provided herein consists of the amino acid sequence shown in SEQ ID NO: 6, or has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity thereto. In certain embodiments, a fusion protein having at least 70%, 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 to the amino acid sequence shown in SEQ ID NO:6 still retains the function or activity of IgA protease (e.g., proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.).

[0127]

[0128]

[0129] In certain embodiments, the fusion protein provided herein includes the amino acid sequence shown in SEQ ID NO: 7. In certain embodiments, the fusion protein provided herein consists of the amino acid sequence shown in SEQ ID NO: 7, or has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity thereto. In certain embodiments, a fusion protein having at least 70%, 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 to the amino acid sequence shown in SEQ ID NO:7 still retains the function or activity of IgA protease (e.g., proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.).

[0130]

[0131]

[0132] In certain embodiments, the fusion protein provided herein includes the amino acid sequence shown in SEQ ID NO: 8. In certain embodiments, the fusion protein provided herein consists of the amino acid sequence shown in SEQ ID NO: 8, or has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity thereto. In certain embodiments, a fusion protein having at least 70%, 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 to the amino acid sequence shown in SEQ ID NO:8 still retains the function or activity of IgA protease (e.g., proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.).

[0133]

[0134]

[0135] In certain embodiments, the fusion protein provided herein includes the amino acid sequence shown in SEQ ID NO: 24. In certain embodiments, the fusion protein provided herein consists of the amino acid sequence shown in SEQ ID NO: 24, or has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity thereto. In certain embodiments, a fusion protein having at least 70%, 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 to the amino acid sequence shown in SEQ ID NO: 24 still retains the function or activity of IgA protease (e.g., proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.).

[0136]

[0137]

[0138] In certain embodiments, the half-life of the fusion protein provided herein in the blood circulation of a subject is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days.

[0139] Nucleic Acids

[0140] In another aspect, the present application provides an isolated nucleic acid comprising a nucleotide sequence encoding the IgA protease truncate described in the present application or a nucleotide sequence encoding the fusion protein described in the present application.

[0141] The term "nucleic acid" or "nucleotide" as used herein refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise indicated, a particular nucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed 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)).

[0142] DNA encoding the IgA protease truncations or fusion proteins described herein can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to the gene encoding the fusion protein). Encoding DNA can also be obtained synthetically.

[0143] In certain embodiments, the nucleic acids provided herein include a nucleic acid sequence as shown in SEQ ID NO: 9. In certain embodiments, the nucleic acids provided herein consist of a nucleotide sequence as shown in SEQ ID NO: 9, or have at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity thereto.

[0144]

[0145]

[0146]

[0147]

[0148]

[0149] In certain embodiments, the nucleic acids provided herein include a nucleic acid sequence as shown in SEQ ID NO: 10. In certain embodiments, the nucleic acids provided herein consist of a nucleotide sequence as shown in SEQ ID NO: 10, or have at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity thereto.

[0150]

[0151]

[0152]

[0153]

[0154] In certain embodiments, the nucleic acids provided herein include a nucleic acid sequence as shown in SEQ ID NO: 11. In certain embodiments, the nucleic acids provided herein consist of a nucleotide sequence as shown in SEQ ID NO: 11, or have at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity thereto.

[0155]

[0156]

[0157]

[0158] In certain embodiments, the nucleic acids provided herein include a nucleic acid sequence as shown in SEQ ID NO: 12. In certain embodiments, the nucleic acids provided herein consist of a nucleotide sequence as shown in SEQ ID NO: 12, or have at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity thereto.

[0159]

[0160]

[0161]

[0162] In certain embodiments, the nucleic acids provided herein include a nucleic acid sequence as shown in SEQ ID NO: 25. In certain embodiments, the nucleic acids provided herein consist of a nucleotide sequence as shown in SEQ ID NO: 25, or have at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity thereto.

[0163]

[0164]

[0165]

[0166] Vectors and cells

[0167] In another aspect, the present application provides a vector comprising a nucleic acid encoding the IgA protease truncate described in the present application or comprising a nucleic acid encoding the fusion protein described in the present application.

[0168] Using recombinant techniques known in the art, the isolated polynucleotide encoding the IgA protease truncate or fusion protein can be inserted into a vector for further cloning (amplification of the DNA) or for expression. A variety of vectors are available. Vector components generally 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.

[0169] In certain embodiments, the nucleic acid provided herein encodes an IgA protease truncate or fusion protein, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection tag. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (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, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUS E. pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0170] The vector containing the nucleic acid sequence encoding the IgA protease truncate or fusion protein can be introduced into the host cell for cloning or gene expression. The host cell suitable for cloning or expressing the DNA in the vector described in the present application is the above-mentioned prokaryotic, yeast or higher eukaryotic cell. Prokaryotic cells suitable for the purposes of the present application include true bacteria, such as Gram-negative bacteria or Gram-positive bacteria, for example, Enterobacteriaceae, for example, Escherichia (Escherichia) (for example, Escherichia coli (E.coli)), Enterobacter (Enterobacter), Erwinia (Erwinia), Klebsiella (Klebsiella), Proteus (Proteus), Salmonella (Salmonella) (for example, Salmonella typhimurium (Salmonella typhimurium)), Serratia (Serratia) (for example, Serratia marcescens (Serratia marcescans), Shigella, Bacilli (e.g., B. subtilis and B. licheniformis), Pseudomonas (e.g., Pseudomonas aeruginosa), and Streptomyces. In certain embodiments, the cell is an Escherichia coli cell.

[0171] In addition to prokaryotes, eukaryotic cells, for example eukaryotic microorganisms such as filamentous fungi or yeast can also be used as suitable cloning or expression hosts for vectors encoding 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 suitable for use in this application, for example, Schizosaccharomyces pombe; Kluyveromyces hosts, for example, K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans and K. marxianus; Yarrowia lipolytica (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, for example, Schwanniomyces occidentalis; and filamentous fungi, for example, Neurospora, Penicillium, Tolypocladium, and Aspergillus (for example, A. nidulans and A. 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).

[0172] Pharmaceutical composition

[0173] On the other hand, the present application provides a pharmaceutical composition comprising the IgA protease truncate described in the present application, the fusion protein described in the present application, the nucleic acid described in the present application, the vector described in the present application, or the cell described in the present application, and a pharmaceutically acceptable carrier.

[0174] The pharmaceutically acceptable carrier used in the pharmaceutical composition disclosed in the present application may include, for example, a pharmaceutically acceptable liquid, gel or solid carrier, an aqueous solvent, a non-aqueous solvent, an antimicrobial substance, an isotonic substance, a buffer, an antioxidant, an anesthetic, a suspending agent / dispersing agent, a chelating agent, a diluent, an adjuvant, an auxiliary material or a non-toxic auxiliary substance, other components well known in the art, or a combination of the above.

[0175] Applicable components may include, for example, antioxidants, fillers, adhesives, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers or stabilizers such as sugars and cyclodextrins. Applicable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylmethylanisole, butylated hydroxytoluene and / or propyl gallate. As disclosed in the present application, one or more antioxidants such as methionine are included in the composition comprising the IgA protease truncate or fusion protein disclosed in the present application, which can reduce the oxidation of the IgA protease truncate or fusion protein. The present application further provides a variety of methods for preventing the oxidation of the fusion protein, extending its shelf life and / or improving its activity, for example, by mixing the IgA protease truncate or fusion protein provided in the present application with one or more antioxidants (e.g., methionine) to achieve.

[0176] Further, pharmaceutically acceptable carriers may include, for example, aqueous media such as sodium chloride injection, Ringer's solution injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, non-aqueous media such as fixed oils of plant origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antibacterial substances at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, 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 carboxymethylcellulose, hydroxypropyl methylcellulose or polyvinyl pyrrolidone, 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 as carriers may be added to the pharmaceutical compositions in multiple dose containers and include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, saline, glucose, glycerol or ethanol. Suitable nontoxic auxiliary substances may include, for example, wetting agents, emulsifiers, pH buffers, stabilizers, solubilizers, or substances such as sodium acetate, sorbitan laurate, triethanolamine oleate or cyclodextrins.

[0177] The pharmaceutical composition can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation or powder. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, polyvinyl pyrrolidone, saccharin sodium, cellulose, magnesium carbonate, etc.

[0178] In certain embodiments, the pharmaceutical composition is formulated into an injectable composition. Injectable pharmaceutical compositions can be prepared in any conventional form, for example, a liquid solvent, a suspending agent, an emulsifier, or a solid form suitable for producing a liquid solvent, a suspending agent, or an emulsifier. Injectable formulations can include sterile and / or pyrogen-free solutions, sterile dried solubles that are combined with solvents before use, such as lyophilized powders, including subcutaneous tablets, sterile suspensions for injection, sterile dried insoluble products that are combined with media before use, and sterile and / or pyrogen-free emulsions. The solvent can be an aqueous phase or a non-aqueous phase.

[0179] In certain embodiments, the unit dose of the injection preparation is packaged in an ampoule, a tube or a syringe with a needle. As is known in the art, all preparations for injection should be sterile and pyrogen-free.

[0180] In certain embodiments, a sterile lyophilized powder can be prepared by dissolving the IgA protease truncate or fusion protein disclosed in the present application in a suitable solvent. The solvent may contain a component that can improve the stability of the powder or a reconstituted solution obtained from the powder, or improve other pharmacological components of the powder or the reconstituted 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, such as a citrate buffer, a sodium phosphate or potassium phosphate buffer or other buffer known to those skilled in the art, and in one embodiment, the pH of the buffer is neutral. 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 dispensed into vials and lyophilized. Each vial can accommodate a single dose or multiple doses of the IgA protease truncate or fusion protein or a combination thereof. Each vial may be filled with slightly more than required for each dose or multiple doses (eg, 10% overdose) to ensure accurate sampling and dosing. The lyophilized powder may be stored under appropriate conditions, such as at about 4°C to room temperature.

[0181] The lyophilized powder is reconstituted with water for injection to obtain a preparation for injection. In one embodiment, the lyophilized powder can be added to sterile pyrogen-free water or other applicable liquid carriers for reconstitution. The exact amount is determined by the selected therapy and can be determined based on empirical values.

[0182] Methods of treating or preventing disease

[0183] On the other hand, the present application provides a method for treating or preventing IgA deposition-related diseases, which comprises administering the IgA protease truncate, the fusion protein or the pharmaceutical composition described herein to a subject in need of treatment or prevention.

[0184] On the other hand, the present application provides a method for treating or preventing IgA deposition-related diseases, which comprises administering IgA protease or its truncation, a fusion protein comprising the IgA protease or its truncation, or a pharmaceutical composition comprising the IgA protease or its truncation or the fusion protein to a subject in need of treatment or prevention, wherein the amino acid sequence of the IgA protease is selected from the following group: SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or a combination thereof. In certain embodiments, the IgA protease truncation has at least 70% sequence identity to the polypeptide set forth in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30 (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 certain embodiments, the IgA protease truncation has at least 70% sequence identity to the polypeptide set forth in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 or SEQ ID NO:30 (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 retains the function or activity of the IgA protease (e.g., proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.).

[0185] The amino acid sequences of SEQ ID NOs: 26 to 30 are shown in the following table.

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194] On the other hand, the present application provides use of the IgA protease truncate described in the present application, the fusion protein described in the present application, or the pharmaceutical composition described in the present application in the preparation of a drug for treating or preventing IgA deposition-related diseases.

[0195] In another aspect, the present application provides the use of IgA protease or its truncation, a fusion protein comprising the IgA protease or its truncation, or a pharmaceutical composition comprising the IgA protease or its truncation 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: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or a combination thereof. In certain 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 sequence shown in SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30. In certain embodiments, the IgA protease truncation has at least 70% sequence identity to the polypeptide set forth in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30 (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 certain embodiments, the IgA protease truncation has at least 70% sequence identity to the polypeptide set forth in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 or SEQ ID NO:30 (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 retains the function or activity of the IgA protease (e.g., proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.).

[0196] In another aspect, the present application provides an IgA protease truncate as described herein, a fusion protein as described herein, or a pharmaceutical composition as described herein for treating or preventing IgA deposition-related diseases.

[0197] In another aspect, the present application provides an IgA protease or a truncate thereof, a fusion protein comprising the IgA protease or a truncate thereof, or a pharmaceutical composition comprising the IgA protease or a truncate thereof or the fusion protein 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: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or a combination thereof. In certain embodiments, the amino acid sequence of the IgA protease is an amino acid sequence formed by removing a signal peptide sequence from the amino acid sequence shown in SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30. In certain embodiments, the IgA protease truncation has at least 70% sequence identity to the polypeptide set forth in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30 (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 certain embodiments, the IgA protease truncation has at least 70% sequence identity to the polypeptide set forth in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 or SEQ ID NO:30 (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 retains the function or activity of the IgA protease (e.g., proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.).

[0198] In certain embodiments, the IgA deposition-related diseases described in the present application include IgA nephropathy, dermatitis herpetiformis, Henoch-Schönlein purpura (also known as IgA vasculitis), Kawasaki disease, purpuric nephritis, IgA vasculitis renal damage, rheumatoid arthritis with positive IgA rheumatoid factor, IgA anti-GBM disease or IgA ANCA-associated vasculitis. In certain embodiments, the IgA deposition-related diseases described in the present application are IgA nephropathy. In certain embodiments, the IgA deposition-related diseases described in the present application are IgA1 nephropathy. In certain embodiments, the IgA deposition-related diseases described in the present application are IgA vasculitis. In certain embodiments, the IgA deposition-related diseases described in the present application are Kawasaki disease. DETAILED DESCRIPTION

[0199] The biological materials involved in all 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 operation methods, are all familiar to technicians in this field. Reference can be made to "Molecular Cloning" compiled by Sambrook et al. (Laboratory Manual, Cold Spring Harbor, 1989) and "The Concise Molecular Biology Experiment Guide" (US / F. Osber et al., translated by Yan Ziying et al., Beijing, Science Press, 1998).

[0200] Example 1: Study on the active site of TIGR4 IgA protease

[0201] The inventors removed the signal peptide at the N-terminus (i.e., amino acids 1 to 42 of SEQ ID NO: 1) of the wild-type IgA protease from the TIGR4 strain of Streptococcus pneumoniae (whose amino acid sequence is shown in SEQ ID NO: 1) and constructed the PET30a-TIGR4 plasmid. Then, the inventors used the PET30a-TIGR4 plasmid as a template to construct a series of TIGR4 IgA protease truncations and expressed them in prokaryotic cells (Escherichia coli) to study the active site of TIGR4 IgA protease. Figure 1As shown, starting from the N-terminal of SEQ ID NO: 1, the IgA enzymatic activity of the C-terminal truncation TIGR4 (745-2004) (whose amino acid sequence is shown in SEQ ID NO: 3) formed when truncated to the 745th amino acid begins to weaken; the C-terminal truncation TIGR4 (845-2004) (whose amino acid sequence is shown in SEQ ID NO: 4) formed when truncated to the 845th amino acid and the C-terminal truncation TIGR4 (945-2004) formed when truncated to the 945th amino acid have no in vitro enzymatic activity. Therefore, it can be concluded that the active site of TIGR4 IgA protease is located between the 43rd and 745th amino acids.

[0202] Example 2: Preparation of fusion protein containing TIGR4 IgA protease truncate

[0203] 2.1 Plasmid construction

[0204] The inventors removed the signal peptide (i.e., amino acids 1 to 42 of SEQ ID NO: 1) from the N-terminus of the wild-type IgA protease from the TIGR4 strain of Streptococcus pneumoniae (whose amino acid sequence is shown in SEQ ID NO: 1), and then added the Fc sequence of human IgG1 (HR-CH2-CH3, whose amino acid sequence is shown in SEQ ID NO: 13) to the N-terminus of the amino acid sequence of the IgA protease from which the signal peptide was removed (i.e., the IgA protease truncate consisting of amino acids 43 to 2004 of SEQ ID NO: 1), thereby constructing the PET30a-IgG1 Fc-TIGR4 (43-2004) plasmid. According to a similar strategy, the inventors also constructed the PET30a-IgG1 Fc-TIGR4 (665-2004) plasmid.

[0205] The inventors removed the amino acids 1 to 664 (i.e., amino acids 1 to 664 of SEQ ID NO: 1) from the N-terminus of the wild-type IgA protease from the TIGR4 strain of Streptococcus pneumoniae (whose amino acid sequence is shown in SEQ ID NO: 1), and then added the Fc sequence of human IgG1 (whose amino acid sequence is shown in SEQ ID NO: 13) to the C-terminus of the TIGR4 (665-2004) IgA protease truncate to construct the pET30a-TIGR4 (665-2004)-IgG1 Fc plasmid. According to a similar strategy, the inventors also constructed two pET30a-TIGR4 (665-2004)-IgG4 Fc plasmids (the difference is that different linkers are used).

[0206] 2.2 Fusion protein preparation method

[0207] The five plasmid expression vectors constructed in Example 2.1 were transfected into competent cells of Escherichia coli (BL21-DE3) respectively, and selected for resistance by LB agar culture plates containing 50 μg / ml kanamycin, and then single clone colonies were picked and shaken in LB culture medium containing corresponding antibiotics until the exponential growth phase (OD600: 0.6-0.8). After the exponential growth phase, 0.1-0.5mM isopropyl-β-D-thiogalactoside (IPTG) was added for induction, and the expression was induced at low temperature of 16°C for 24h (or induced at 37°C for 3h). The pET30a-TIGR4(665-2004)-IgG1 Fc plasmid was also transfected into HEK293 cells of the eukaryotic expression system. After the expression was completed, the Escherichia coli cell bodies were treated according to the conventional method, ultrasonically fragmented, and then centrifuged at high speed and the supernatant was retained, and then affinity chromatography and molecular sieve purification were used to obtain the recombinant fusion protein.

[0208] The amino acid sequence of the fusion protein expressed by the PET30a-IgG1 Fc-TIGR4 (43-2004) plasmid (hereinafter referred to as "fusion protein 1") is shown in SEQ ID NO: 5, and the encoding nucleic acid sequence thereof is shown in SEQ ID NO: 9; wherein, fusion protein 1 comprises the IgG1 Fc domain shown in SEQ ID NO: 13 and the truncation of TIGR4 (43-2004);

[0209] The amino acid sequence of the fusion protein expressed by the PET30a-IgG1 Fc-TIGR4(665-2004) plasmid (hereinafter referred to as "fusion protein 2") is shown in SEQ ID NO: 6, and the encoding nucleic acid sequence thereof is shown in SEQ ID NO: 10; wherein, fusion protein 2 comprises the IgG1 Fc domain shown in SEQ ID NO: 13 and the TIGR4(665-2004) truncation shown in SEQ ID NO: 2;

[0210] The amino acid sequence of the fusion protein expressed by the pET30a-TIGR4(665-2004)-IgG1 Fc plasmid (hereinafter referred to as "fusion protein 3") is shown in SEQ ID NO: 7, and the encoding nucleic acid sequence thereof is shown in SEQ ID NO: 11; wherein, fusion protein 3 comprises the TIGR4(665-2004) truncation shown in SEQ ID NO: 2 and the IgG1 Fc domain shown in SEQ ID NO: 13;

[0211] The amino acid sequence of a fusion protein expressed by a pET30a-TIGR4(665-2004)-IgG4 Fc plasmid (hereinafter referred to as "fusion protein 4") is shown in SEQ ID NO: 8, and the encoding nucleic acid sequence thereof is shown in SEQ ID NO: 12; wherein, fusion protein 4 comprises a TIGR4(665-2004) truncate as shown in SEQ ID NO: 2 and an IgG4 Fc domain as shown in SEQ ID NO: 14;

[0212] The amino acid sequence of another fusion protein expressed by pET30a-TIGR4(665-2004)-IgG4 Fc plasmid (hereinafter referred to as "fusion protein 5") is shown in SEQ ID NO: 24, and its encoding nucleic acid sequence is shown in SEQ ID NO: 25; wherein, fusion protein 5 comprises the TIGR4(665-2004) truncation shown in SEQ ID NO: 2 and the IgG4Fc domain shown in SEQ ID NO: 14.

[0213] 2.3 In vitro activity test method

[0214] The obtained fusion proteins 1, 2, 3, 4 and 5 containing TIGR4 IgA protease truncations were mixed with substrate IgA purified from healthy human plasma in vitro, reacted overnight at 37°C, and then subjected to Western blot to verify their enzymatic cleavage activity on substrate IgA.

[0215] 2.4 In vivo activity test method

[0216] The obtained fusion proteins 4 and 5 containing TIGR4 IgA protease truncation were injected into humanized IgA1 alpha chain knock-in (α1KI-Tg) C57BL / 6 mice through the tail vein, and blood samples were collected before injection, 2 hours, 1 day, 2 days, 3 days, 5 days, and 7 days after injection, and then verified by Western blot.

[0217] 2.5 Results

[0218] In vitro activity experiments showed that fusion protein 1 and fusion protein 2 had enzymatic activity against IgA in vitro (respectively Figure 2 a and Figure 3 a), but fusion protein 1 and fusion protein 2 have a lot of non-full-length protease expression (as shown in Figure 2 b and Figure 3 b).

[0219] In vitro activity experiments showed that the pET30a-TIGR4(665-2004)-IgG1 Fc plasmid successfully expressed fusion protein 3 in prokaryotic cells (e.g. Figure 4 b), and also has in vitro enzymatic activity against IgA (as shown in Figure 4 a). The pET30a-TIGR4(665-2004)-IgG1 Fc plasmid was expressed in HEK293 eukaryotic cells, but no enzymatic activity against IgA was detected in vitro (data not shown). In addition, activity verification after purification using a nickel column and S200 showed that the fusion protein 3 purified using nickel still had enzymatic activity against IgA in vitro after being placed at 4°C overnight or for 24 hours; the F1, F2 and F3 flow-through peaks purified using S200 also had enzymatic activity against IgA in vitro (as shown in FIG. Figure 5 Among them, the proportion of fusion protein 3 forming dimers in F1 and F2 is the highest (as shown in Figure 6 The resulting flow-through peak was placed at 37°C. The results showed that F1 still had a clear band of fusion protein 3 after 6 hours. However, after being placed for more than 21 hours, fusion protein 3 was cleaved, leaving only the IgA protease truncated form TIGR4 (665-2004) with the Fc cut off and some residual amino acids on the IgG1 Fc (such as Figure 7 a, as shown in 7b).

[0220] In vitro activity experiments showed that the pET30a-TIGR4(665-2004)-IgG4 Fc plasmid also successfully expressed fusion protein 4, and a portion of F1, F2 and F3 formed dimers (such as Figure 8 In addition, the stability test showed that the nickel-purified sample still had a clear target band after being placed at 37°C for 40 hours or even 80 hours, indicating that the fusion protein 4 has good stability (as shown in Fig. Figure 8 b).

[0221] In vitro activity experiments also showed that another pET30a-TIGR4(665-2004)-IgG4 Fc plasmid also successfully expressed fusion protein 5 and also had in vitro enzymatic activity against IgA (e.g. Fig.10 In addition, the stability test showed that the nickel-purified sample still had a clear target band after being placed at 37°C for 9 days, indicating that the fusion protein 5 has good stability (as shown in FIG. Fig.10 b).

[0222] In vivo activity experiments also showed that after a single tail vein injection of fusion protein 4, IgA in the blood of humanized IgA1 (α1KI-Tg) C57BL / 6 mice was cleaved into Fc and Fab fragments by fusion protein 4, and the activity lasted for at least 5 days (e.g. Fig. 9 In vivo activity experiments also showed that after a single tail vein injection of fusion protein 5, IgA in the blood of humanized IgA1 (α1KI-Tg) C57BL / 6 mice was cleaved into Fc and Fab fragments by fusion protein 5, and the activity lasted for at least 5 days (as shown in Figure 2A). Fig.11 shown).

[0223] Example 3: Exploring other IgA proteases

[0224] The inventors screened several amino acid sequences with certain homology to the wild-type IgA protease of Streptococcus pneumoniae TIGR4 strain from the metagenomic database, and synthesized 5 TIGR4 homologous enzymes. Their amino acid sequences are shown in SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively. The inventors tested the enzymatic activity of these TIGR4 homologous enzymes on IgA1 according to the in vitro activity test method described in Example 2.3. The results are shown in FIG. Fig.12 shown. Fig.12 "Streptococcus pneumoniae-1" means that the polypeptide shown in SEQ ID NO: 26 is mixed with the substrate IgA1 in vitro; "Streptococcus pneumoniae-2" means that the polypeptide shown in SEQ ID NO: 27 is mixed with the substrate IgA1 in vitro; "Oral Streptococcus-1" means that the polypeptide shown in SEQ ID NO: 28 is mixed with the substrate IgA1 in vitro; "Oral Streptococcus-2" means that the polypeptide shown in SEQ ID NO: 29 is mixed with the substrate IgA1 in vitro; "Streptococcus mitis-2" means that the polypeptide shown in SEQ ID NO: 30 is mixed with the substrate IgA1 in vitro.

[0225] Depend on Fig.12 It can be seen that the polypeptides shown in SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30 all have enzymatic activity against IgA1.

[0226] Although the present application specifically presents and describes the invention by referring to specific embodiments, those skilled in the art should understand that the above content may be changed in various forms and details without departing from the subject matter and scope of protection disclosed in the present application.

Claims

1. A fusion protein comprising a first polypeptide and a second polypeptide, wherein: a) the amino acid sequence of the first polypeptide is shown in SEQ ID NO: 2; b) the second polypeptide is an IgG4 Fc domain; c) the second polypeptide is located at the C-terminus of the first polypeptide; and d) The first polypeptide and the second polypeptide are connected via a linker. The fusion protein according to claim 1 , which has an enzyme activity that specifically cleaves human IgA1. The fusion protein according to claim 1 , which has an enzyme activity that specifically cleaves human IgA1 heavy chain. The fusion protein according to claim 1 , which has an enzyme activity that specifically cleaves the hinge region of the human IgA1 heavy chain. The fusion protein of claim 1 , wherein the linker comprises a cleavable linker or a non-cleavable linker. The fusion protein of claim 1 , wherein the linker comprises a flexible linker or a rigid linker.

7. The fusion protein of claim 1, wherein the linker comprises a helical linker or a non-helical linker.

8. The fusion protein of claim 1, wherein the linker comprises a peptide linker.

9. The fusion protein of claim 8, wherein the peptide linker comprises a linker containing glycine and serine.

10. The fusion protein of claim 9, wherein the linker containing glycine and serine comprises one, two, three or four repeats of an amino acid sequence as shown in SEQ ID NO: 15 (GGGS), SEQ ID NO: 16 (GGGGS), SEQ ID NO: 17 (GGGGGGS) or SEQ ID NO: 19 (GSS).

11. The fusion protein of claim 1, wherein the linker comprises an amino acid sequence as shown in SEQ ID NO: 18 (GGGGSGGGGSGGGGS), as shown in SEQ ID NO: 20 (GSSGSSG), or as shown in SEQ ID NO: 21 (RSGSSGSSG).

12. The fusion protein of claim 1, wherein the IgG4 Fc domain comprises a hinge region. The fusion protein of claim 1 , wherein the Fc domain is derived from a human IgG4 Fc domain.

14. The fusion protein of claim 1, wherein the amino acid sequence of the Fc domain is shown in SEQ ID NO:

14.

15. The fusion protein of claim 14, wherein the first polypeptide and the second polypeptide are directly connected via a linker as shown in SEQ ID NO: 20 or SEQ ID NO:

21.

16. The fusion protein of claim 1, wherein the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 8 or as shown in SEQ ID NO:

24.

17. The fusion protein of any one of the preceding claims, further comprising a tag. The fusion protein of claim 17 , wherein the tag is selected from the group consisting of a fluorescent tag and a purification tag.

19. The fusion protein of claim 17, wherein the tag is selected from the group consisting of a c-Myc tag, a HA tag, a VSV-G tag, a FLAG tag, a V5 tag, and a HIS tag.

20. The fusion protein of claim 19, wherein the tag is a HIS tag comprising 6, 7, 8, 9 or 10 histidines. The fusion protein of claim 17 , 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.

22. The fusion protein of any one of claims 1-16, wherein the half-life of the fusion protein in the blood circulation in a subject is 1 to 5 days.

23. An isolated nucleic acid comprising a nucleotide sequence encoding the fusion protein of any one of claims 1-22.

24. The nucleic acid of claim 23, comprising the nucleotide sequence shown in SEQ ID NO: 12 or SEQ ID NO:

25.

25. A vector comprising the nucleic acid of claim 23 or 24.

26. A cell comprising the nucleic acid of claim 23 or 24 or the vector of claim 25.

27. The cell of claim 26, wherein the cell is a prokaryotic cell or a eukaryotic cell.

28. The cell of claim 27, wherein the prokaryotic cell is an E. coli cell.

29. The cell of claim 27, wherein the eukaryotic cell is a mammalian cell.

30. The cell of claim 29, wherein the mammalian cell is a human cell or a Chinese Hamster Ovary (CHO) cell.

31. The cell of claim 30, wherein the mammalian cell is a human embryonic kidney 293 cell (HEK293 cell).

32. A pharmaceutical composition comprising the fusion protein of any one of claims 1-22, the nucleic acid of claim 23 or 24, the vector of claim 25 or the cell of any one of claims 26-31, and a pharmaceutically acceptable carrier.

33. A method for producing a fusion protein, comprising the step of culturing the cell according to any one of claims 26-31.

34. Use of the fusion protein according to any one of claims 1 to 22 or the pharmaceutical composition according to claim 32 in the preparation of a medicament for treating or preventing IgA nephropathy.

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