Fusion protein containing IgA protease truncate and its use

Abstract

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

Description

Technical Field

[0001] The present application relates to the field of biopharmaceuticals. Specifically, the present application relates to an IgA protease truncate, a fusion protein containing the IgA protease truncate, a pharmaceutical composition containing 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 the use of the IgA protease truncate or the fusion protein in the preparation of a medicament for treating IgA deposition-related diseases.

Background Art

[0002] IgA nephropathy is one of the most common primary glomerular diseases in the world today, imposing a heavy burden on patients and society. Currently, there is still no specific treatment for IgA nephropathy. Clinically, supportive therapy based on RAS inhibitors is often used, which delays the decline of renal function. For patients who do not respond well to supportive therapy, a combination therapy of steroids and immunosuppressive drugs is administered. However, the long-term use of steroids and immunosuppressive drugs brings serious side effects to patients.

[0003] Therefore, the development of effective therapeutic drugs with few side effects is urgently needed.

Summary of the Invention

[0004] In one aspect, the present application provides a fusion protein comprising a first polypeptide that is an IgA protease truncate and a second polypeptide that cannot be cleaved by IgA protease or an IgA protease truncate.

[0005] In some embodiments, the IgA protease truncate comprises a non-native truncated fragment of wild-type IgA protease obtained from or derived from the Streptococcus pneumoniae TIGR4 strain, or has at least 70% sequence identity with the non-native truncated fragment. In some embodiments, the non-native truncated fragment is subjected to amino acid mutations, deletions, insertions, or modifications based on the wild-type IgA protease of the Streptococcus pneumoniae TIGR4 strain, thereby losing or reducing the autoenzymatic cleavage function of the IgA protease truncate. In some embodiments, the amino acid mutations, deletions, insertions, or modifications occur at the native autoenzymatic cleavage site of the wild-type IgA protease of the Streptococcus pneumoniae TIGR4 strain, within 5 amino acid residues upstream and / or within 5 amino acid residues downstream of the native autoenzymatic cleavage site. In some embodiments, the native autoenzymatic cleavage site is between positions 43 and 745 of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the native autoenzymatic 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 509 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 of the amino acid sequence shown in SEQ ID NO: 1.

[0006] In some 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 from or derived from the Streptococcus pneumoniae TIGR4 strain. In some embodiments, the C-terminal truncated fragment comprises a polypeptide fragment of at least 703 consecutive amino acids starting from position 43 of a wild-type IgA protease obtained from or derived from the Streptococcus pneumoniae TIGR4 strain, or has at least 70% sequence identity with the polypeptide fragment.

[0007] In some embodiments, the deposit number of the Streptococcus pneumoniae TIGR4 strain is ATCC BAA-334. In some embodiments, the amino acid sequence of the wild-type IgA protease of the Streptococcus pneumoniae TIGR4 strain is as shown in SEQ ID NO: 1.

[0008] 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 shown in SEQ ID NO: 1. In some embodiments, the first polypeptide comprises a polypeptide fragment of amino acids at positions 665 to 2004 of the amino acid sequence 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 the amino acid sequence 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.

[0009] In some embodiments, the fusion protein has an enzymatic activity that specifically cleaves human IgA. In some embodiments, the fusion protein has an enzymatic activity that specifically cleaves the heavy chain of human IgA. In some embodiments, the fusion protein has an enzymatic activity that specifically cleaves the hinge region of the heavy chain of human IgA. In some embodiments, the fusion protein has an enzymatic activity that specifically cleaves human IgA1. In some embodiments, the fusion protein has an enzymatic activity that specifically cleaves the heavy chain of human IgA1. In some embodiments, the fusion protein has an enzymatic activity that specifically cleaves the hinge region of the heavy chain of human IgA1.

[0010] In some embodiments, the first polypeptide and the second polypeptide are linked 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 the amino acid sequence shown in SEQ ID NO: 15 (GGGS), SEQ ID NO: 16 (GGGGS), SEQ ID NO: 17 (GGGGGGGS), or SEQ ID NO: 19 (GSS). In some embodiments, the linker comprises the amino acid sequence shown in SEQ ID NO: 18 (GGGGSGGGGSGGGGS), SEQ ID NO: 20 (GSSGSSG), or SEQ ID NO: 21 (RSGSSGSSG).

[0011] In some embodiments, the first polypeptide and the second polypeptide are directly linked.

[0012] In some embodiments, the second polypeptide comprises an amino acid sequence for extending the half-life of the first polypeptide in the body of a subject. In some embodiments, the second polypeptide is selected from an Fc domain and albumin. In some embodiments, the Fc domain comprises a hinge region. In some embodiments, the Fc domain is derived from a human IgG Fc domain. In some embodiments, the Fc domain is derived from a human IgG4 Fc domain. In some embodiments, the Fc domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 14. In some embodiments, the Fc domain comprises the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the Fc domain comprises one or more mutations that extend the half-life of the fusion protein. In some embodiments, the Fc domain is linked to the C-terminus or N-terminus of the first polypeptide. In some embodiments, the fusion protein comprises a first polypeptide and a second polypeptide, 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, and the first polypeptide and the second polypeptide are directly linked by a linker shown in SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 8. In some embodiments, the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 24. In some embodiments, the fusion protein comprises a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide are linked by a linker shown in SEQ ID NO: 20, 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, the first polypeptide and the second polypeptide are linked by the linker shown in SEQ ID NO: 21, 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 albumin comprises one or more domains of human serum albumin. In some embodiments, the albumin comprises the D3 domain of human serum albumin.

[0013] In some embodiments, the fusion protein 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.

[0014] In some embodiments, the half-life of the fusion protein in the bloodstream in the body 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, at least 14 days.

[0015] 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 some embodiments, the nucleic acid described in the present application comprises the nucleotide sequence shown in SEQ ID NO: 12, or a nucleotide sequence having at least 70% sequence identity thereto. In some embodiments, the nucleic acid described in the present application comprises the nucleotide sequence shown in SEQ ID NO: 25, or a nucleotide sequence having at least 70% sequence identity thereto.

[0016] In another aspect, the present application provides an isolated IgA protease truncate comprising a non-native truncated fragment of wild-type IgA protease obtained from or derived from the Streptococcus pneumoniae TIGR4 strain, or having at least 70% sequence identity with the non-native truncated fragment. In some embodiments, the non-native truncated fragment is subjected to amino acid mutation, deletion, insertion or modification based on the wild-type IgA protease of the Streptococcus pneumoniae TIGR4 strain, thereby causing the loss or reduction of the autoenzymatic cleavage function of the IgA protease truncate. In some embodiments, the amino acid mutation, deletion, insertion or modification occurs at the native autoenzymatic cleavage site of the wild-type IgA protease of the Streptococcus pneumoniae TIGR4 strain, within 5 amino acid residues upstream and / or within 5 amino acid residues downstream of the native autoenzymatic cleavage site.

[0017] In some embodiments, the non-natural truncated fragment is an N-terminal truncated fragment or a C-terminal truncated fragment of wild-type IgA protease obtained from or derived from Streptococcus pneumoniae strain TIGR4. In some embodiments, the deposit number of the Streptococcus pneumoniae strain TIGR4 is ATCC BAA-334. In some embodiments, the C-terminal truncated fragment comprises a polypeptide fragment of at least 703 consecutive amino acids starting from position 43 of wild-type IgA protease obtained from or derived from Streptococcus pneumoniae strain TIGR4, or has at least 70% sequence identity with the polypeptide fragment. In some embodiments, the amino acid sequence of the wild-type IgA protease of the Streptococcus pneumoniae strain TIGR4 is as shown in SEQ ID NO: 1.

[0018] In some embodiments, the natural autoenzyme cleavage site is between positions 43 and 745 of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the natural autoenzyme 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 509 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 of the amino acid sequence shown in SEQ ID NO: 1.

[0019] 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 shown in SEQ ID NO: 1. In some embodiments, the IgA protease truncate comprises a polypeptide fragment of amino acids at positions 665 to 2004 of the amino acid sequence shown in SEQ ID NO: 1, or a polypeptide fragment having at least 70% sequence identity thereto. In some embodiments, based on the amino acid sequence of the polypeptide fragment, it has conservative substitutions of amino acids at one or more sites. In some embodiments, the IgA protease truncate comprises the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the IgA protease truncate consists of the amino acid sequence shown in SEQ ID NO: 2.

[0020] In some embodiments, the IgA protease truncate provided by the present application has an enzymatic activity of specifically cleaving human IgA. In some embodiments, the IgA protease truncate provided by the present application has an enzymatic activity of specifically cleaving the human IgA heavy chain. In some embodiments, the IgA protease truncate provided by the present application has an enzymatic activity of specifically cleaving the junction between the CH1 and hinge regions of the human IgA heavy chain. In some embodiments, the IgA protease truncate provided by the present application has an enzymatic activity of specifically cleaving human IgA1. In some embodiments, the IgA protease truncate provided by the present application has an enzymatic activity of specifically cleaving the human IgA1 heavy chain. In some embodiments, the IgA protease truncate provided by the present application has an enzymatic activity of specifically cleaving the junction between the CH1 and hinge regions of the human IgA1 heavy chain.

[0021] 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.

[0022] In another aspect, the present application provides a vector comprising the nucleic acid described in the present application.

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

[0024] In another aspect, the present application provides a pharmaceutical composition comprising 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.

[0025] In another aspect, the present application provides a method for producing a fusion protein, comprising the step of culturing the cell described in the present application.

[0026] In another aspect, the present application provides a method for treating or preventing an IgA deposition-related disease, comprising administering to a subject in need of treatment or prevention 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.

[0027] In another aspect, the present application provides the 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 medicament for treating or preventing an IgA deposition-related disease.

[0028] In another aspect, the present application provides 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 for treating or preventing an IgA deposition-related disease.

[0029] In another aspect, the present application provides a method for treating or preventing an IgA deposition-related disease, comprising administering to a subject in need of treatment or prevention 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, 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.

[0030] In another aspect, the present application provides the use of 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 the preparation of a medicament for treating or preventing an IgA deposition-related disease, 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.

[0031] In another aspect, 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 an IgA deposition-related disease, 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.

[0032] In some embodiments, the IgA deposition-related diseases include IgA nephropathy, dermatitis herpetiformis, Henoch-Schönlein purpura (also known as IgA vasculitis), Kawasaki disease, purpuric nephritis, kidney injury due to IgA vasculitis, rheumatoid arthritis with IgA rheumatoid factor positivity, IgA anti-GBM disease, or IgA ANCA-associated vasculitis. In some embodiments, the IgA deposition-related disease is IgA nephropathy, IgA vasculitis, or Kawasaki disease.

Brief Description of the Drawings

[0033]

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[0034] In the present application, various aspects and embodiments are disclosed below. It is obvious to those skilled in the art that various equivalent changes and modifications can be implemented without departing from the spirit and scope of the subject matter of the present application. The various aspects and embodiments disclosed by the present application are merely examples for explanation and do not limit the present application. The actual protection scope of the present application is based on the scope of the claims. Unless otherwise indicated, all technical terms and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the technical field to which the present application belongs. All references, patents, and patent applications cited in the present application are hereby incorporated by reference in their entirety into this specification.

[0035] Definition As used herein, the term "pneumococcus" refers to all Streptococcus pneumoniae bacteria. This is an opportunistic pathogen belonging to Gram-positive cocci and can produce IgA protease.

[0036] As used herein, the term "protease" refers to an enzyme having the ability to degrade proteins and peptides. Proteases can degrade proteins by hydrolyzing the peptide bonds that connect amino acids in the peptide or polypeptide chains that form proteins. In the prior art, numerous methods for measuring the protein hydrolysis activity of specific species of proteases are known. For example, the protein hydrolysis activity of proteases can be determined by comparatively measuring and analyzing the hydrolysis ability of various proteases against appropriate substrates. Examples of substrates for protein hydrolysis activity analysis include, for example, dimethyl casein, bovine collagen, bovine elastin, etc. Colorimetric quantification methods using these substrates are also known in the prior art (see, for example, WO99 / 34011 and US6376450).

[0037] As used herein, the term "IgA protease" refers to an enzyme that can specifically cleave or degrade IgA immunoglobulin molecules (such as IgA1 or IgA2) of a subject (such as a human). For example, the IgA protease obtained from or derived from the Streptococcus pneumoniae TIGR4 strain can specifically cleave the peptide bond between proline (Pro) at position 227 and threonine (Thr) at position 228 of IgA1 to degrade IgA1.

[0038] When referring to a polypeptide or protein, the term "wild-type" as used in the present application refers to a naturally occurring polypeptide or protein that does not contain 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" as used in the present application refers to a naturally occurring nucleic acid, nucleotide, or polynucleotide that does not contain artificial mutations, insertions, deletions, or modifications at one or more nucleotide positions. However, the polynucleotide encoding the wild-type polypeptide is not limited to a naturally occurring polynucleotide, and any polynucleotide encoding the wild-type polypeptide (for example, an artificially synthesized polynucleotide) is also included.

[0039] The term "TIGR4" as used in the present application refers to the TIGR4 strain of Streptococcus pneumoniae. In some embodiments, the amino acid sequence of the wild-type IgA protease produced by the Streptococcus pneumoniae TIGR4 strain is as shown in SEQ ID NO: 1, of which the amino acids at positions 1 to 42 (underlined) are the signal peptide.

[0040] MEKYFGEKQERFSFRKLSVGLVSATISSLFFMSVLASSSVDA

[0041] As used herein, the term "signal peptide" refers to an amino acid residue sequence that may be involved in the secretion or directed transport of a protein in its mature or precursor form. A signal peptide is usually located at the N-terminus of the sequence of a precursor or mature protein. The signal peptide may be endogenous or exogenous. Generally, a mature protein does not have a signal peptide. Usually, after protein transport, the signal peptide is cleaved from the protein by signal peptidase. For example, the amino acid sequence obtained by removing the N-terminal signal peptide from the amino acid sequence shown in SEQ ID NO: 1 is as shown in SEQ ID NO: 22.

[0042]

[0043] As used herein, the term "subject" includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals). The "subject" may be a domestic animal (e.g., cows, pigs, sheep, poultry, and horses), or a rodent (e.g., rats, mice), or a primate (e.g., ape, monkey, chimpanzee, gorilla, orangutan, baboon), or a household pet (e.g., dogs and cats). The "subject" may be male or female, and may be elderly, adult, young, pediatric, or infant. The human "subject" may be Caucasian, African, Asian, Semitic or other ethnic group, or a mixture of the foregoing ethnic backgrounds.

[0044] As used herein, the terms "protein", "polypeptide", and "peptide" are used interchangeably and refer to polymers of amino acids. The proteins, polypeptides, or peptides described herein may contain natural amino acids, or non-natural amino acids, or amino acid analogs or mimetics. The proteins, polypeptides, or peptides described herein can be obtained by any method known in the art. For example, natural isolation, recombinant expression, chemical synthesis, etc., but are not limited thereto.

[0045] As used herein, the term "amino acid" refers to an organic compound containing a functional group of an amino group (-NH2) and a carboxyl group (-COOH), and a side chain unique to each amino acid. In this application, the names of amino acids may also be represented by the standard one-letter or three-letter abbreviations summarized below.

[0046] [Table 1]

[0047] As used herein, "conservative substitution" when used with respect to an amino acid sequence refers to the replacement of one amino acid residue with another amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can be made between amino acid residues having hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between amino acid residues having neutral / hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between amino acid residues having acidic side chains (e.g., Asp, Glu), between amino acid residues having basic side chains (e.g., His, Lys, and Arg), or between amino acid residues having aromatic side chains (e.g., Trp, Tyr, Phe). As is known in the art, in conservative substitutions, usually no significant change in the three-dimensional structure of the protein occurs, so the biological activity of the protein is retained.

[0048] As used herein, the term "homologous" refers to a nucleic acid sequence (or its complementary strand) or an amino acid sequence having 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.

[0049] "Percent (%) sequence identity" when used with respect to an amino acid sequence (or nucleic acid sequence) refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are the same as the reference sequence in the candidate sequence after performing a sequence alignment and introducing gaps as necessary to maximize the number of identical amino acids (or nucleic acids). In other words, the percent (%) sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of amino acid residues (or bases) that are the same as the reference sequence being 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 the same residue. Tools disclosed in the art, such as BLASTN, BLASTp (website of the National Center for Biotechnology Information, NCBI. See also Altschul S.F. et al., J. Mol. Biol., 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (website of the European Bioinformatics Institute. See also Higgins D.G. et al., Methods in Enzymology, 266:383-402 (1996); Larkin M.A. et al., Bioinformatics (Oxford, England), 23(21):2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software can be used to perform sequence alignments and determine the percent sequence identity of amino acid (or nucleic acid) sequences. Those skilled in the art can use the default parameters of the said tools or appropriately adjust the parameters as necessary for the alignment, for example, by selecting an appropriate algorithm.

[0050] An "isolated" substance has been artificially modified from its natural state. If an "isolated" composition or substance occurs in nature, it has already been modified, deviated from its original state, or both. For example, a polynucleotide or polypeptide that naturally exists in the body of an animal is not "isolated", but if such a polynucleotide or polypeptide is sufficiently separated from the substances coexisting in the natural state and exists in a substantially pure state, it is considered "isolated". An "isolated nucleic acid sequence" refers to the sequence of an isolated nucleic acid molecule. In some embodiments, an "isolated IgA protease truncate" refers to an IgA protease truncate having a purity of at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. The purity is determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing electrophoresis, capillary electrophoresis) or chromatography (e.g., ion exchange chromatography or reverse phase HPLC).

[0051] As used herein, the term "vector" refers to a vehicle into which a gene is operably inserted and which can be used to express the gene (e.g., to produce a protein, RNA, or DNA encoded by the gene, or to replicate the gene). Vectors are used for the transformation, transduction, or transfection of host cells and to express the genes they carry within the host cells. For example, vectors include plasmids, phagemids, cosmids, artificial chromosomes (e.g., yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs)), bacteriophages (e.g., λ phage or M13 phage), and animal viruses. Vectors may contain various factors for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection factors, and reporter genes. Vectors may further contain an origin of replication. Vectors may further contain components that assist in entry into cells, including, but not limited to, viral particles, liposomes, or protein coats. Vectors may be expression vectors or cloning vectors. The vectors provided herein (e.g., expression vectors) contain a nucleic acid sequence encoding an IgA protease truncate or fusion protein described herein, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker.

[0052] "Treatment" or "therapy" of a particular disease, condition, or disorder, as used herein, includes preventing or alleviating the particular disease, condition, or disorder, reducing the rate at which the particular disease, condition, or disorder occurs or progresses, decreasing the risk that the particular disease, condition, or disorder will occur, preventing or delaying the progression of symptoms associated with the particular disease, condition, or disorder, reducing or stopping the symptoms associated with the particular disease, condition, or disorder, effecting a complete or partial improvement of the particular disease, condition, or disorder, curing the particular disease, condition, or disorder, or combinations thereof.

[0053] The term "pharmaceutically acceptable" means that a specified vector, vehicle, diluent, excipient, and / or salt is usually chemically and / or physically compatible with the other components that make up the formulation and is physiologically compatible with its receptor.

[0054] The term "IgA deposition-related disease" refers to a disease associated with the accumulation of IgA immunoglobulins in the form of aggregated or non-aggregated forms in the tissue or organ of interest. For example, it includes, but is not limited to, IgA nephropathy, dermatitis herpetiformis, Henoch-Schönlein purpura (also called IgA vasculitis), Kawasaki disease, purpuric nephritis, kidney injury due to IgA vasculitis, rheumatoid arthritis positive for IgA rheumatoid factor, IgA anti-GBM disease, or IgA ANCA-associated vasculitis.

[0055] The term "IgA nephropathy" refers to a kidney disease characterized by IgA deposition in the kidney.

[0056] IgA protease truncate In one aspect, the present application provides an isolated IgA protease truncate comprising a non-natural truncated fragment of wild-type IgA protease obtained from or derived from Streptococcus pneumoniae strain TIGR4, or having at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity) with the non-natural truncated fragment. In some embodiments, the IgA protease truncate having at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity) with the non-natural truncated fragment retains the function or activity of IgA protease (e.g., proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.).

[0057] As used herein, the term "truncate" or "truncated fragment" refers to a peptide formed by removing one or more amino acids from one or both ends of a wild-type polypeptide. Thus, the "truncate" or "truncated fragment" herein does not include the full length of its 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, an "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 a wild-type IgA protease, or may include a peptide in which one or more amino acids are mutated, deleted, inserted, or modified compared to the truncated form of the wild-type IgA protease.

[0058] In some embodiments, the IgA protease truncates described herein have one or more amino acid mutations, deletions, insertions, or modifications compared to their corresponding wild-type IgA proteases. For example, in some embodiments, the IgA protease truncates described herein include non-natural truncated fragments of a wild-type IgA protease obtained from or derived from the Streptococcus pneumoniae TIGR4 strain, wherein the non-natural truncated fragments have amino acid mutations, deletions, insertions, or modifications based on the wild-type IgA protease of the Streptococcus pneumoniae TIGR4 strain, thereby causing the autoenzymatic cleavage function of the IgA protease truncates to be lost or reduced.

[0059] As used herein, the terms "obtained from" and "derived from" include not only proteins produced or producible by the organisms recited, but also proteins encoded by DNA sequences isolated from such organisms and produced in host organisms having such DNA sequences, as well as proteins encoded by synthetic and / or cDNA-derived DNA sequences and having characteristics specific to the recited proteins. For example, the wild-type IgA protease obtained from or derived from Streptococcus pneumoniae strain TIGR4 includes the IgA protease naturally produced by Streptococcus pneumoniae strain TIGR4, as well as IgA protease produced by other host cells (e.g., Escherichia coli) transformed with a nucleic acid encoding IgA protease using genetic engineering techniques.

[0060] As used herein, the term "non-natural truncated fragment" refers to a fragment having an amino acid sequence different (e.g., different in amino acid length or amino acid type) from the truncated fragment formed by the self-enzyme cleavage of the wild-type IgA protease of Streptococcus pneumoniae strain TIGR4 in a natural environment.

[0061] In some embodiments, the amino acid mutations, deletions, insertions, or modifications occur at the natural self-enzyme cleavage site of the wild-type IgA protease of Streptococcus pneumoniae strain TIGR4.

[0062] The term "natural self-enzyme cleavage site" refers to the ability of IgA protease to recognize a specific peptide bond of itself, thereby performing self-catalytic cleavage and releasing the mature IgA protease at the C-terminus.

[0063] In some embodiments, the amino acid mutation, deletion, insertion, or modification occurs within 5 amino acid residues upstream of the natural autocleavage site of the wild-type IgA protease of the Streptococcus pneumoniae TIGR4 strain (e.g., 1 amino acid residue upstream, 2 amino acid residues upstream, 3 amino acid residues upstream, 4 amino acid residues upstream, or 5 amino acid residues upstream of the natural autocleavage site). In some embodiments, the amino acid mutation, deletion, insertion, or modification occurs within 5 amino acid residues downstream of the natural autocleavage site of the wild-type IgA protease of the Streptococcus pneumoniae TIGR4 strain (e.g., 1 amino acid residue downstream, 2 amino acid residues downstream, 3 amino acid residues downstream, 4 amino acid residues downstream, or 5 amino acid residues downstream of the natural autocleavage site). In some embodiments, the amino acid mutation or deletion occurs within 5 amino acid residues upstream (e.g., 1 amino acid residue upstream, 2 amino acid residues upstream, 3 amino acid residues upstream, 4 amino acid residues upstream, or 5 amino acid residues upstream of the natural autocleavage site) and within 5 amino acid residues downstream (e.g., 1 amino acid residue downstream, 2 amino acid residues downstream, 3 amino acid residues downstream, 4 amino acid residues downstream, or 5 amino acid residues downstream of the natural autocleavage site) of the natural autocleavage site of the wild-type IgA protease of the Streptococcus pneumoniae TIGR4 strain.

[0064] In some embodiments, the amino acid mutation, deletion, insertion, or modification occurs at one or more amino acid positions (corresponding to SEQ ID NO: 1) selected from the group consisting of positions 71, 97, 175, 177, 210, 222, 228, 239, 245, 254, 262, 292, 303, 315, 338, 382, 384, 401, 418, 425, 435, 439, 442, 456, 459, 469, 474, 476, 487, 490, 499, 506, 509, 514, 517, 533, 536, 549, 563, 576, 594, 597, 613, 616, 639, 644, 645, 652, and 661 of the wild-type IgA protease of the Streptococcus pneumoniae TIGR4 strain.

[0065] In some 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 from or derived from the Streptococcus pneumoniae TIGR4 strain.

[0066] As used herein, the term "N-terminal truncated fragment" refers to a truncated fragment that includes the amino acid sequence at the amino terminus of the wild-type IgA protease of the Streptococcus pneumoniae TIGR4 strain. The starting position of the "amino terminus" may be any position near the amino terminus of the amino acid sequence of the wild-type IgA protease of the Streptococcus pneumoniae TIGR4 strain. For example, it may be position 1 counted from the amino terminus, or it may be other positions counted from the amino terminus. Also, for example, if the full-length amino acid sequence of the wild-type IgA protease consists of 1000 amino acids, the amino terminus starting position of its N-terminal truncated fragment may be any position between positions 1 and 500 from the amino terminus of the amino acid sequence.

[0067] In some embodiments, the N-terminal starting point of the non-native truncated fragment of the IgA protease described in the present application is within 5 amino acid residues upstream of the natural autoenzyme cleavage site of the wild-type IgA protease of Streptococcus pneumoniae strain TIGR4 (e.g., 1 amino acid residue upstream, 2 amino acid residues upstream, 3 amino acid residues upstream, 4 amino acid residues upstream, or 5 amino acid residues upstream of the amino acid residue of the natural autoenzyme cleavage site). In some embodiments, the N-terminal starting point of the non-native truncated fragment of the IgA protease described in the present application is within 5 amino acid residues downstream of the natural autoenzyme cleavage site of the wild-type IgA protease of Streptococcus pneumoniae strain TIGR4 (e.g., 1 amino acid residue downstream, 2 amino acid residues downstream, 3 amino acid residues downstream, 4 amino acid residues downstream, or 5 amino acid residues downstream of the amino acid residue of the natural autoenzyme cleavage site).

[0068] In some embodiments, the N-terminal starting point of the non-natural truncated fragment of the IgA protease described herein corresponds to the native autoenzymatic cleavage site of the wild-type IgA protease of Streptococcus pneumoniae strain TIGR4 (e.g., positions 71, 97, 175, 177, 210, 222, 228, 239, 245, 254, 262, 292, 303, 315, 338, 382, 384, 401, 418, 425, 435, 439, 442, 456, 459, 469, 474, 476, 487, 490, 499, 506, 509, 514, 517, 533, 536, 549, 563, 576, 594, 597, 613, 616, 639, 644, 645, 652, or 661 of SEQ ID NO: 1). In some embodiments, the N-terminal starting point of the non-natural truncated fragment of the IgA protease described herein is one amino acid residue downstream of the native autoenzymatic cleavage site of the wild-type IgA protease of Streptococcus pneumoniae strain TIGR4 (e.g., positions 72, 98, 176, 178, 211, 223, 229, 240, 246, 255, 263, 293, 304, 316, 339, 383, 385, 402, 419, 426, 436, 440, 443, 457, 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 of SEQ ID NO: 1).

[0069] As used herein, the term "C-terminal truncated fragment" refers to a truncated fragment that includes the amino acid sequence at the carboxyl terminus of the wild-type IgA protease of Streptococcus pneumoniae strain TIGR4. The ending position of the "carboxyl terminus" may be any position near the carboxyl terminus of the amino acid sequence of the wild-type IgA protease of Streptococcus pneumoniae strain TIGR4. For example, it may be the 1st position counted from the carboxyl terminus, or it may be other positions counted from the carboxyl terminus. Also, for example, if the full-length amino acid sequence of the wild-type IgA protease consists of 1000 amino acids, the carboxyl-terminal ending position of its C-terminal truncated fragment may be any position between the 501st and 1000th positions from the amino terminus of the amino acid sequence.

[0070] In some embodiments, the deposit number of the Streptococcus pneumoniae strain TIGR4 is ATCC BAA-334.

[0071] In some embodiments, the C-terminal truncated fragment comprises a polypeptide fragment of 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, 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 obtained from or derived from the wild-type IgA protease of Streptococcus pneumoniae strain TIGR4, or has at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity) with the polypeptide fragment.In some embodiments, an N-terminal truncated fragment having at least 70% sequence identity with the polypeptide fragment (e.g., having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity) retains the function or activity of IgA protease (e.g., proteolytic activity, enzyme activity that specifically cleaves IgA, etc.). In some embodiments, the amino acid sequence of the wild-type IgA protease of the Streptococcus pneumoniae TIGR4 strain is as shown in SEQ ID NO: 1.

[0072] Unless otherwise specified, the amino acid sites of the TIGR4 IgA protease referred to in this application correspond to the amino acid sites of the wild-type TIGR4 IgA protease (whose amino acid sequence is as shown in SEQ ID NO: 1). For example, the 665th site of the TIGR4 IgA protease referred to in this application corresponds to the 665th site of SEQ ID NO: 1. Unless otherwise specified, the naming rule of the TIGR4 IgA protease truncation referred to in this application is "TIGR4 (starting point corresponding to SEQ ID NO: 1 - ending point corresponding to SEQ ID NO: 1)". For example, TIGR4(665-2004) is a TIGR4 IgA protease truncation formed by the amino acids at positions 665 to 2004 of SEQ ID NO: 1.

[0073] In some embodiments, the native autoenzymatic cleavage site of the IgA protease described in the present application is between positions 43 and 745 of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the native autoenzymatic cleavage site of the IgA protease described in the present application is between positions 43 and 665 of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the native autoenzymatic 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 509 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 of the amino acid sequence shown in SEQ ID NO: 1.

[0074] In some embodiments, the IgA protease truncate comprises a polypeptide fragment of at least 1300 contiguous amino acids starting from position 665 of the amino acid sequence shown in SEQ ID NO: 1. For example, in some embodiments, the IgA protease truncate provided by the present application comprises 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 contiguous amino acids of the polypeptide fragment starting from position 665 of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the IgA protease truncate provided by the present application comprises a polypeptide fragment of 1340 contiguous amino acids starting from position 665 of the amino acid sequence shown in SEQ ID NO: 1.

[0075] In some embodiments, the IgA protease truncate comprises a polypeptide fragment of amino acids at positions 665 to 2004 of the amino acid sequence 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 some embodiments, the IgA protease truncate 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) with the polypeptide fragment retains the function or activity of the IgA protease (e.g., proteolytic activity, enzyme activity that specifically cleaves IgA, etc.).

[0076] In some embodiments, the IgA protease truncate described in the present application is a TIGR4(665-2004) truncate having the amino acid sequence shown in SEQ ID NO: 2.

[0077]

[0078] In some embodiments, the IgA protease truncate described herein is the TIGR4(745-2004) truncate having the amino acid sequence shown in SEQ ID NO: 3.

[0079]

[0080] In some embodiments, the IgA protease truncate described herein is the TIGR4(845-2004) truncate having the amino acid sequence shown in SEQ ID NO: 4.

[0081]

[0082] In some embodiments, the IgA protease truncate has conservative substitutions of amino acids at the position of one or more amino acid residues (e.g., the position of one, two, three, four, five or more amino acid residues) based on the amino acid sequence of the aforementioned polypeptide fragment. Conservative substitution of an amino acid residue refers to substitution between amino acids with similar properties, for example, substitution between polar amino acids (such as substitution between glutamine and asparagine), substitution between hydrophobic amino acids (such as substitution among leucine, isoleucine, methionine, and valine), and substitution between amino acids with the same charge (such as substitution among arginine, lysine, and histidine, or substitution between glutamic acid and aspartic acid). In some embodiments, the IgA protease truncate described in the present application has conservative substitutions of amino acids at the position of one, two, three, four, five, six, seven, eight, nine, ten, eleven, fifteen, twenty or more amino acid residues as compared with the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0083] On the premise of not affecting the activity, the IgA protease truncate described in the present application may further contain non-natural amino acids. Non-natural amino acids include, for example, β-fluoroalanine, 1-methylhistidine, γ-methyleneglutamic acid, α-methyleucine, 4,5-dehydrolysine, hydroxyproline, 3-fluorophenylalanine, 3-aminotyrosine, 4-methyltryptophan, and the like.

[0084] The IgA protease truncate described in the present application may be modified by methods known in the art. For example, but not limited to, PEGylation, glycosylation, amino-terminal modification, fatty acylation, carboxyl-terminal modification, phosphorylation, methylation and the like. As can be understood by those skilled in the art, after the IgA protease truncate provided by the present application is modified by methods known in the art, it still retains a function substantially similar to that of IgA protease or IgA protease truncate.

[0085] In some embodiments, the IgA protease truncates described herein have enzymatic activity that specifically cleaves human IgA. In some embodiments, the IgA protease truncates described herein have enzymatic activity that specifically cleaves the human IgA heavy chain. In some embodiments, the IgA protease truncates described herein have enzymatic activity that specifically cleaves the human IgA heavy chain hinge region. In some embodiments, the IgA protease truncates described herein have enzymatic activity that specifically cleaves human IgA1. In some embodiments, the IgA protease truncates described herein have enzymatic activity that specifically cleaves the human IgA1 heavy chain. In some embodiments, the IgA protease truncates described herein have enzymatic activity that specifically cleaves the human IgA1 heavy chain hinge region.

[0086] In some embodiments, the IgA protease truncates described herein have conservative substitutions of amino acids at the positions of one or more amino acid residues based on the amino acid sequence of the aforementioned polypeptide fragment, but retain the enzymatic activity of cleaving human IgA (e.g., IgA1). In some embodiments, the IgA protease truncates described herein have at least 70% sequence identity with the aforementioned polypeptide fragment (e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity), and retain the enzymatic activity of cleaving human IgA (e.g., IgA1).

[0087] Fusion protein In another aspect, the present application provides a fusion protein comprising a first polypeptide and a second polypeptide. Here, the first polypeptide comprises the full length of wild-type IgA protease obtained from or derived from Streptococcus pneumoniae strain TIGR4, a polypeptide obtained by removing the signal peptide from wild-type IgA protease obtained from or derived from Streptococcus pneumoniae strain TIGR4, or an IgA protease truncate described in the present application, and the second polypeptide is not cleavable by IgA protease or an IgA protease truncate. Without being bound by any theory, it would be beneficial for the second polypeptide to be non-cleavable by IgA protease or an IgA protease truncate. This is because in that case, the fusion protein can retain its integrity and stability, and the first polypeptide can also retain its activity to cleave IgA.

[0088] In some embodiments, the first polypeptide and the second polypeptide of the fusion protein described in the present application are linked by a linker. In some embodiments, the first polypeptide and the second polypeptide are directly linked (i.e., linked without passing through a linker). As used herein, the term "linker" or "linker" refers to an artificial amino acid sequence having 1, 2, 3, 4, or 5 amino acid residues, or having a length between 5 and 15, 20, 30, 50, or more amino acid residues, linked by peptide bonds and used to link one or more polypeptides. The linker may or may not have a secondary structure. Linker sequences are known in the art. See, for example, Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), Poljak et al., Structure 2:1121-1123 (1994).

[0089] 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. Any suitable linker known in the art can be used. In some embodiments, the linker includes a peptide linker. For example, useful linkers in the present application may be rich in glycine and serine residues. Examples thereof include linkers having a single or repetitive sequence containing threonine / serine and glycine. For example, GGGS (SEQ ID NO: 15) or 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 some embodiments, the linker used in the present application includes GGGGSGGGGSGGGGS (SEQ ID NO: 18). In some embodiments, the linker used in the present application includes GSSGSSG (SEQ ID NO: 20). In some embodiments, the linker used in the present application includes RSGSSGSSG (SEQ ID NO: 21). In some embodiments, the linker used in the present application includes RSGGGGS (SEQ ID NO: 31). In some embodiments, the linker used in the present application comprises 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 of SEQ ID NOs: 18, 20, 21, 31, or consists of an amino acid sequence selected from the group.

[0090] In some embodiments, the second polypeptide comprises an amino acid sequence for extending the half-life of the first polypeptide in the body of a subject. In some embodiments, the second polypeptide is selected from an Fc domain and albumin. In some embodiments, the Fc domain comprises a hinge region. In some embodiments, the Fc domain comprises a lower hinge region. In some embodiments, the Fc domain comprises a core hinge region and a lower hinge region. In some embodiments, the Fc domain comprises an upper hinge region, a core hinge region, and a lower hinge region. In some embodiments, the Fc domain does not comprise a hinge region. In some embodiments, the Fc domain is derived from a human IgG Fc domain. In some embodiments, the Fc domain is derived from a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, or a human IgG4 Fc domain.

[0091] In some embodiments, the Fc domain is derived from a human IgG1 Fc domain. In some embodiments, the Fc domain comprises the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the Fc domain consists of the amino acid sequence shown in SEQ ID NO: 13. In some 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: 13.

[0092] TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 13)

[0093] In some embodiments, the Fc domain is derived from a human IgG4 Fc domain. In some embodiments, the Fc domain comprises the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the Fc domain consists of the amino acid sequence shown in SEQ ID NO: 14. In some 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.

[0094] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 14)

[0095] According to the accidental discovery made by the inventors of the present application, both fusion proteins formed from the TIGR4 IgA protease truncate and the IgG4 Fc domain or the IgG1 Fc domain have the enzyme activity to cleave IgA, but the stability of the fusion protein formed from the TIGR4 IgA protease truncate and the IgG4 Fc domain is superior to that of the fusion protein formed from the TIGR4 IgA protease truncate and the IgG1 Fc domain. In some embodiments, the half-life of the fusion protein formed from 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 from the TIGR4 IgA protease truncate and the IgG1 Fc domain.

[0096] In some embodiments, the Fc domain contains one or more mutations that extend the half-life of the fusion protein. In some embodiments, the Fc domain is linked to the C-terminus of the first polypeptide. In some embodiments, the Fc domain is linked to the N-terminus of the first polypeptide.

[0097] In some embodiments, the second polypeptide is albumin. In some embodiments, the amino acid sequence of the albumin is as shown in SEQ ID NO: 23. In some embodiments, the albumin contains one or more domains of human serum albumin. In some embodiments, the albumin contains the D3 domain of human serum albumin.

[0098] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL(SEQ ID NO: 23)

[0099] In some embodiments, the fusion protein provided by the present application further comprises a tag. In some embodiments, the tag is selected from the group consisting of a fluorescent tag, a luminescent tag, a purification tag, and a chromogenic tag. In some embodiments, the tag is selected from the group consisting of a c-Myc tag, an HA tag, a VSV-G tag, a FLAG tag, a V5 tag, and a HIS tag. In some embodiments, the tag is a HIS tag. In some embodiments, the tag is a HIS tag containing 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.

[0100] In some embodiments, the present application provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, and the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 14.

[0101] In some embodiments, the present application provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2, and the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the present application provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide consists of the amino acid sequence shown in SEQ ID NO: 2, and the second polypeptide consists of the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the present application provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide consists of the amino acid sequence shown in SEQ ID NO: 2, the second polypeptide consists of the amino acid sequence shown in SEQ ID NO: 13, and the first polypeptide is located at the N-terminus of the second polypeptide. In some embodiments, the present application provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide consists of the amino acid sequence shown in SEQ ID NO: 2, the second polypeptide consists of the amino acid sequence shown in SEQ ID NO: 13, and the first polypeptide is located at the C-terminus of the second polypeptide.

[0102] In some embodiments, the present application provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2, and the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the present application provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide consists of the amino acid sequence shown in SEQ ID NO: 2, and the second polypeptide consists of the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the present application provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide consists of the amino acid sequence shown in SEQ ID NO: 2, the second polypeptide consists of the amino acid sequence shown in SEQ ID NO: 14, and the first polypeptide is located at the N-terminus of the second polypeptide. In some embodiments, the present application provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide consists of the amino acid sequence shown in SEQ ID NO: 2, the second polypeptide consists of the amino acid sequence shown in SEQ ID NO: 14, and the first polypeptide is located at the C-terminus of the second polypeptide.

[0103] In some embodiments, the fusion protein provided by the present application comprises the amino acid sequence shown in SEQ ID NO: 5. In some embodiments, the fusion protein provided by the present application 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 therewith. In some 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%, at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 5 retains the function or activity of IgA protease (such as proteolytic activity, enzyme activity that specifically cleaves IgA, etc.).

[0104]

[0105] In some embodiments, the fusion protein provided by the present application comprises the amino acid sequence shown in SEQ ID NO: 6. In some embodiments, the fusion protein provided by the present application 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 therewith. In some 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%, at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 6 retains the function or activity of IgA protease (e.g., protein hydrolysis activity, enzyme activity that specifically cleaves IgA, etc.).

[0106]

[0107] In some embodiments, the fusion protein provided by the present application comprises the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the fusion protein provided by the present application 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 therewith. In some 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%, at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7 retains the function or activity of IgA protease (for example, protein hydrolysis activity, enzyme activity that specifically cleaves IgA, etc.).

[0108]

[0109] In some embodiments, the fusion protein provided by the present application comprises the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the fusion protein provided by the present application 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 therewith. In some 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%, at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8 retains the function or activity of IgA protease (e.g., proteolytic activity, enzyme activity that specifically cleaves IgA, etc.).

[0110]

[0111] In some embodiments, the fusion protein provided by the present application comprises the amino acid sequence shown in SEQ ID NO: 24. In some embodiments, the fusion protein provided by the present application 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 therewith. In some 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%, at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 24 retains the function or activity of IgA protease (such as protein hydrolysis activity, enzyme activity that specifically cleaves IgA, etc.).

[0112]

[0113] In some embodiments, the half-life of the fusion protein provided by the present application in the blood circulation in the body of the subject is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days.

[0114] Nucleic acid 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.

[0115] As used herein, the terms "nucleic acid" or "nucleotide" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form, and polymers thereof. Unless otherwise specified, a particular nucleotide sequence implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly recited sequences. 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 a mixed base and / or a deoxyinosine residue (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)).

[0116] The DNA encoding the IgA protease truncate or fusion protein described in this application can be easily isolated and sequenced using conventional methods (e.g., by using an oligonucleotide probe that can specifically bind to the gene encoding the fusion protein). The encoding DNA can also be obtained by synthetic methods.

[0117] In some embodiments, the nucleic acid provided by this application comprises the nucleic acid sequence shown in SEQ ID NO: 9. In some embodiments, the nucleic acid provided by this application consists of, 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 with the nucleotide sequence shown in SEQ ID NO: 9.

[0118]

[0119] In some embodiments, the nucleic acid provided by the present application comprises the nucleic acid sequence shown in SEQ ID NO: 10. In some embodiments, the nucleic acid provided by the present application consists of the nucleotide sequence shown in SEQ ID NO: 10, 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 therewith.

[0120]

[0121] In some embodiments, the nucleic acid provided by the present application comprises the nucleic acid sequence shown in SEQ ID NO: 11. In some embodiments, the nucleic acid provided by the present application consists of the nucleotide sequence shown in SEQ ID NO: 11, 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 therewith.

[0122]

[0123] In some embodiments, the nucleic acid provided by the present application comprises the nucleic acid sequence shown in SEQ ID NO: 12. In some embodiments, the nucleic acid provided by the present application consists of the nucleotide sequence shown in SEQ ID NO: 12, 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 therewith.

[0124]

[0125] In some embodiments, the nucleic acid provided by the present application comprises the nucleic acid sequence shown in SEQ ID NO: 25. In some embodiments, the nucleic acid provided by the present application consists of the nucleotide sequence shown in SEQ ID NO: 25, 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 therewith.

[0126] GGATCAAGCGGGTCATCTGGA GAATCGAAGTACGGTCCGCCGTGTCCGAGTTGCCCGGCTCCGGAGTTTCTGGGTGGCCCTAGCGTGTTCTTATTTCCGCCAAAGCCGAAGGACACCCTGATGATCAGCCGCACCCCGGAGGTAACTTGCGTCGTCGTGGATGTTTCCCAAGAAGATCCGGAGGTGCAGTTTAACTGGTATGTGGACGGCGTTGAAGTTCATAATGCGAAAACCAAGCCGCGTGAAGAGCAGTTCAACAGCACCTATCGTGTGGTCTCCGTGTTGACGGTTCTTCACCAGGATTGGTTGAATGGTAAAGAATATAAGTGCAAAGTTTCCAATAAAGGCCTGCCGTCGAGCATTGAAAAAACGATTAGCAAGGCAAAGGGCCAACCTCGCGAGCCGCAAGTGTACACTCTGCCGCCGAGCCAAGAGGAAATGACCAAAAACCAGGTTTCATTGACCTGCCTGGTTAAGGGTTTTTACCCGAGCGACATCGCCGTTGAGTGGGAATCTAACGGCCAGCCGGAGAACAACTACAAGACCACCCCACCGGTTCTGGACAGCGATGGTTCCTTCTTCCTGTACTCCCGTCTCACCGTGGACAAAAGCCGTTGGCAAGAGGGAAACGTGTTCTCTTGTAGCGTGATGCATGAAGCGCTGCACAATCATTATACGCAGAAATCTCTGAGCCTTTCTCTGGGCAAA(SEQ ID NO: 25)

[0127] Vectors and Cells In another aspect, the present application provides a vector comprising a nucleic acid encoding an IgA protease truncate described herein or encoding a fusion protein described herein.

[0128] Using recombinant techniques known in the art, the isolated polynucleotide encoding the IgA protease truncate or fusion protein may be inserted into a vector for further cloning (DNA amplification) or expression. The vector can be selected from various types. Vector components typically include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, enhancer elements, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0129] In some embodiments, the nucleic acid provided by the present application 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, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), λ phage and M13 phage, plasmid pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0130] A vector containing a nucleic acid sequence encoding the IgA protease truncate or fusion protein may be introduced into a host cell for cloning or gene expression. Host cells suitable for cloning or expression of DNA in the vectors described herein are the prokaryotic, yeast, or higher eukaryotic cells described above. Prokaryotic cells suitable for the uses of the present application include eubacteria such as Gram-negative or Gram-positive bacteria. For example, Escherichia (e.g., E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescens), Enterobacteriaceae such as Shigella, Bacilli (e.g., B. subtilis and B. licheniformis), Pseudomonas (e.g., P. aeruginosa), and Streptomyces. In some embodiments, the cell is a cell of E. coli.

[0131] In addition to prokaryotic cells, eukaryotic cells, such as eukaryotic microorganisms like filamentous fungi or yeasts, 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 also relatively commonly used and suitable for use in the present application. For example, Schizosaccharomyces pombe, hosts of the genus Kluyveromyces (e.g., Kluyveromyces lactis, Kluyveromyces fragilis (ATCC 12,424), Kluyveromyces bulgaricus (ATCC 16,045), Kluyveromyces wickeramii (ATCC 24,178), Kluyveromyces waltii (ATCC 56,500), Kluyveromyces drosophilarum (ATCC 36,906), Kluyveromyces thermotolerans, Kluyveromyces marxianus), the genus Yarrowia (EP 402,226), Pichia pastoris (EP 183,070), the genus Candida, Trichoderma reesia (EP 244,234), Neurospora crassa, the genus Schwanniomyces (e.g., Schwanniomyces occidentalis), and filamentous fungi (e.g., the genus Neurospora, the genus Penicillium), the genus Tolypocladium, and the genus Aspergillus (e.g., Aspergillus nidulans and Aspergillus niger), etc.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).

[0132] Pharmaceutical composition In another aspect, 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.

[0133] Pharmaceutically acceptable carriers for the pharmaceutical compositions disclosed in the present application may include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous solvents, non-aqueous solvents, antimicrobial substances, isotonic substances, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0134] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorants, thickeners, colorants, emulsifiers, or stabilizers (such as sugars and cyclodextrins). Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylmethyl anisole, butylhydroxytoluene, and / or propyl gallate. As disclosed in the present application, by including one or more antioxidants such as methionine in a composition containing the IgA protease truncate or fusion protein disclosed in the present application, oxidation of the IgA protease truncate or fusion protein can be reduced. The present application further provides a plurality of methods for preventing oxidation, extending the shelf life, and / or improving the activity of the fusion protein. For example, it is achieved by mixing the IgA protease truncate or fusion protein provided in the present application with one or more antioxidants (such as methionine).

[0135] Furthermore, pharmaceutically acceptable carriers may include, for example, aqueous media (such as sodium chloride injection, Ringer's injection, isotonic glucose injection, sterile water injection, or glucose plus lactated Ringer's injection), non-aqueous media (such as plant-derived non-volatile oils, cottonseed oil, corn oil, sesame oil, or peanut oil, antibacterial substances at bacteriostatic or fungistatic concentrations), tonicity agents (such as sodium chloride or glucose), buffer solutions (such as phosphate or citrate buffer solutions), antioxidants (such as sodium bisulfate), local anesthetics (such as procaine hydrochloride), suspending and dispersing agents (such as sodium carboxymethylcellulose, hypromellose or polyvinylpyrrolidone), emulsifiers (such as polysorbate 80 (Tween 80)), chelating agents (such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol bis(2-aminoethyl ether) tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid). Antibacterial agents as carriers can be added to pharmaceutical compositions in multi-dose containers, which include phenol or cresol, mercury agents, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients may include, for example, water, salts, glucose, glycerin or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting agents, emulsifiers, pH buffers, stabilizers, solubilizing agents, or substances such as sodium acetate, sorbitan laurate, triethanolamine oleate, or cyclodextrin.

[0136] The pharmaceutical composition may be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained-release formulation or powder. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate and the like.

[0137] In some embodiments, the pharmaceutical composition is formulated as an injectable composition. The injectable pharmaceutical composition may be prepared in any conventional form, for example, a liquid solvent, a suspension, an emulsion, or a solid form suitable for generating a liquid solvent, a suspension or an emulsion. Injectable formulations may include sterile and / or pyrogen-free solutions that can be used as such, sterile dry solubilized products (such as lyophilized powders) to be combined with a solvent at the time of use. Here, it includes tablets for subcutaneous injection, sterile suspensions that can be used for injection as such, sterile dry insoluble products to be combined with a medium at the time of use, and sterile and / or pyrogen-free emulsions. The solvent may be aqueous or non-aqueous.

[0138] In some embodiments, a unit dose of the injectable formulation is packaged in one ampoule, one vial, or one syringe with a needle. As is known in the art, all formulations administered by injection must be sterile and pyrogen-free.

[0139] In some 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 another pharmacological component capable of improving the stability of the powder or the reconstitution solution prepared from the powder, or capable of improving the powder or the reconstitution solution. Suitable excipients include, but are not limited to, water, glucose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable substances. The solvent can contain a buffer such as, for example, citrate buffer, sodium phosphate or potassium phosphate buffer, or other buffers known to those skilled in the art. In one embodiment, the pH of the buffer is neutral. Under standard conditions known in the art, filter sterilization is performed after the dissolution, and then lyophilization is carried out to obtain an ideal formulation. In one embodiment, the obtained solvent is aliquoted into vials and lyophilized. Each vial can contain a single dose or multiple doses of the IgA protease truncate or fusion protein or its composition. By making the loading amount per vial slightly more (e.g., 10% more) than the amount required for a single administration or multiple administrations, the accuracy of sample collection and dosing can be ensured. The lyophilized powder can be stored under suitable conditions, such as in the range of about 4°C to room temperature.

[0140] The lyophilized powder is reconstituted with water for injection to obtain a formulation for injection administration. In one embodiment, the lyophilized powder may be added to sterile, pyrogen-free water or other suitable liquid carrier and reconstituted. The exact amount is determined according to the selected treatment method and may be determined empirically.

[0141] Method for treating or preventing a disease In another aspect, the present application provides a method for treating or preventing IgA deposition-related diseases, which includes administering to a subject in need of treatment or prevention 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.

[0142] In another aspect, the present application provides a method for treating or preventing an IgA deposition-related disease, which comprises administering 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 to a subject in need of treatment or prevention, 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 some embodiments, the IgA protease truncate has at least 70% sequence identity with the polypeptide shown 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 some embodiments, the IgA protease truncate has at least 70% sequence identity with the polypeptide shown 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 retains the function or activity of the IgA protease (e.g., proteolytic activity, enzyme activity that specifically cleaves IgA, etc.).

[0143] The amino acid sequences of SEQ ID NOs: 26 to 30 are as shown in the following table.

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

[0144] In another aspect, the present application provides the use of the IgA protease truncate, the fusion protein, or the pharmaceutical composition described in the present application in the preparation of a medicament for treating or preventing IgA deposition-related diseases.

[0145] In another aspect, the present application provides the use of 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, in the preparation of a medicament for treating or preventing an IgA deposition-related disease, 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 some embodiments, the amino acid sequence of the IgA protease is an amino acid sequence obtained by removing the signal peptide sequence from the amino acid sequences shown in SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30. In some embodiments, the IgA protease truncation has at least 70% sequence identity with the polypeptide shown 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 some embodiments, the IgA protease truncate has at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity) with the polypeptide shown in SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30, and retains the function or activity of IgA protease (e.g., proteolytic activity, enzyme activity that specifically cleaves IgA, etc.).

[0146] In another aspect, the present application provides the IgA protease truncate described herein, the fusion protein described herein, or the pharmaceutical composition described herein for treating or preventing IgA deposition-related diseases.

[0147] In another aspect, the present application provides an IgA protease or a truncate thereof for treating or preventing IgA deposition-related diseases, 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, 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 some embodiments, the amino acid sequence of the IgA protease is an amino acid sequence obtained by removing the signal peptide sequence from the amino acid sequences shown in SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30. In some embodiments, the IgA protease truncate has at least 70% sequence identity with the polypeptide shown 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 some embodiments, the IgA protease truncate has at least 70% sequence identity with the polypeptide shown 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 retains the function or activity of the IgA protease (e.g., proteolytic activity, enzyme activity that specifically cleaves IgA, etc.).

[0148] In some embodiments, the IgA deposition-related diseases described in the present application include IgA nephropathy, dermatitis herpetiformis, Henoch-Schönlein purpura (also referred to as IgA vasculitis), Kawasaki disease, purpuric nephritis, kidney injury due to IgA vasculitis, rheumatoid arthritis with IgA rheumatoid factor positivity, IgA anti-GBM disease, or IgA ANCA-associated vasculitis. In some embodiments, the IgA deposition-related disease described in the present application is IgA nephropathy. In some embodiments, the IgA deposition-related disease described in the present application is IgA1 nephropathy. In some embodiments, the IgA deposition-related disease described in the present application is IgA vasculitis. In some embodiments, the IgA deposition-related disease described in the present application is Kawasaki disease.

Example

[0149] In all examples, the biological materials mentioned (such as E. coli strains, various cloning and expression plasmids, media, enzymes as operation tools, buffers), as well as various culture methods, protein extraction and purification methods, and other molecular biology operation methods are all well-known to those skilled in the art. Reference may be made to "Molecular Cloning" (Laboratory Manual, Cold Spring Harbor, 1989) edited by Sambrook et al. and "Selected Molecular Biology Experimental Protocols" (authored by F. Ausubel et al., translated by Yan Ziying et al., Beijing, Science Press, 1998).

[0150] [Example 1] Study on the Active Site of TIGR4 IgA Protease The inventors removed the N-terminal signal peptide of wild-type IgA protease derived from Streptococcus pneumoniae TIGR4 strain (the amino acid sequence thereof is as shown in SEQ ID NO: 1), i.e., amino acids 1 to 42 of SEQ ID NO: 1, and constructed the PET30a-TIGR4 plasmid. Next, using the PET30a-TIGR4 plasmid as a template, the inventors constructed a series of TIGR4 IgA protease truncates, expressed them in prokaryotic cells (Escherichia coli), and examined the active site of TIGR4 IgA protease. As shown in FIG. 1, the IgA enzymatic cleavage activity began to attenuate from the C-terminal truncate TIGR4(745-2004) (the amino acid sequence thereof is as shown in SEQ ID NO: 3) formed when truncated to amino acid 745 counted from the N-terminus of SEQ ID NO: 1, and the C-terminal truncate TIGR4(845-2004) (the amino acid sequence thereof is as shown in SEQ ID NO: 4) formed when truncated to amino acid 845 and the C-terminal truncate TIGR4(945-2004) formed when truncated to amino acid 945 all had no in vitro enzymatic cleavage activity. Therefore, it can be concluded that the active site of TIGR4 IgA protease is between amino acids 43 and 745.

[0151] [Example 2] Preparation of fusion protein containing TIGR4 IgA protease truncate 2.1 Plasmid construction The inventors removed the N-terminal signal peptide (i.e., amino acids 1 to 42 of SEQ ID NO: 1) of wild-type IgA protease derived from Streptococcus pneumoniae strain TIGR4 (the amino acid sequence thereof is as shown in SEQ ID NO: 1), and then added the Fc sequence of human IgG1 (HR-CH2-CH3, the amino acid sequence thereof is as shown in SEQ ID NO: 13) to the N-terminus of the amino acid sequence of IgA protease from which the signal peptide was removed (i.e., IgA protease truncate consisting of amino acids 43 to 2004 of SEQ ID NO: 1) to construct the PET30a-IgG1 Fc-TIGR4(43-2004) plasmid. By the same strategy, the inventors further constructed the PET30a-IgG1 Fc-TIGR4(665-2004) plasmid.

[0152] The inventors removed the amino acids 1 to 664 at the N-terminus (i.e., amino acids 1 to 664 of SEQ ID NO: 1) of wild-type IgA protease derived from Streptococcus pneumoniae strain TIGR4 (the amino acid sequence thereof is as shown in SEQ ID NO: 1), and then added the Fc sequence of human IgG1 (the amino acid sequence thereof is as 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. By the same strategy, the inventors further constructed two types of pET30a-TIGR4(665-2004)-IgG4 Fc plasmids (distinguished by the different linkers used).

[0153] 2.2 Preparation method of fusion protein The five plasmid expression vectors constructed in Example 2.1 were each transfected into competent Escherichia coli (BL21-DE3) cells. After undergoing resistance selection on an LB agar plate containing 50 μg / ml kanamycin, monoclonal colonies were selected and cultured with shaking in LB medium containing the corresponding antibiotic until the exponential growth phase (OD600: 0.6 - 0.8). After reaching the exponential growth phase, 0.1 - 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added for induction, and low-temperature induction expression was carried out at 16°C for 24 hours (or induction at 37°C for 3 hours). Furthermore, the pET30a-TIGR4(665-2004)-IgG1 Fc plasmid was transfected into eukaryotic expression system HEK293 cells. After the expression was completed, the Escherichia coli cell bodies were treated by the usual method, sonicated, and then centrifuged at high speed, and the supernatant was retained. Subsequently, it was purified by affinity chromatography and molecular sieve to obtain the recombinant fusion protein.

[0154] The amino acid sequence of the fusion protein (hereinafter referred to as "fusion protein 1") expressed by the PET30a-IgG1 Fc-TIGR4(43-2004) plasmid is as shown in SEQ ID NO: 5, and its coding nucleic acid sequence is as shown in SEQ ID NO: 9. Fusion protein 1 contains the IgG1 Fc domain shown in SEQ ID NO: 13 and the TIGR4(43-2004) truncation, The amino acid sequence of the fusion protein (hereinafter referred to as "fusion protein 2") expressed by the PET30a-IgG1 Fc-TIGR4(665-2004) plasmid is as shown in SEQ ID NO: 6, and its coding nucleic acid sequence is as shown in SEQ ID NO: 10. Fusion protein 2 contains the IgG1 Fc domain shown in SEQ ID NO: 13 and the TIGR4(665-2004) truncation shown in SEQ ID NO: 2, The amino acid sequence of the fusion protein (hereinafter referred to as "fusion protein 3") expressed by the pET30a-TIGR4(665-2004)-IgG1 Fc plasmid is as shown in SEQ ID NO: 7, and its coding nucleic acid sequence is as shown in SEQ ID NO: 11. The fusion protein 3 contains the TIGR4(665-2004) truncation shown in SEQ ID NO: 2 and the IgG1 Fc domain shown in SEQ ID NO: 13. The amino acid sequence of the fusion protein (hereinafter referred to as "fusion protein 4") expressed by a pET30a-TIGR4(665-2004)-IgG4 Fc plasmid is as shown in SEQ ID NO: 8, and its coding nucleic acid sequence is as shown in SEQ ID NO: 12. The fusion protein 4 contains the TIGR4(665-2004) truncation shown in SEQ ID NO: 2 and the IgG4 Fc domain shown in SEQ ID NO: 14. The amino acid sequence of the fusion protein (hereinafter referred to as "fusion protein 5") expressed by another pET30a-TIGR4(665-2004)-IgG4 Fc plasmid is as shown in SEQ ID NO: 24, and its coding nucleic acid sequence is as shown in SEQ ID NO: 25. The fusion protein 5 contains the TIGR4(665-2004) truncation shown in SEQ ID NO: 2 and the IgG4 Fc domain shown in SEQ ID NO: 14.

[0155] 2.3 In vitro activity measurement method The obtained fusion proteins 1, 2, 3, 4 and 5 containing the TIGR4 IgA protease truncation were each mixed in vitro with the substrate IgA purified from the plasma of healthy individuals and reacted overnight at 37°C, and then Western blot was performed to verify the enzymatic cleavage activity against the substrate IgA.

[0156] 2.4 In vivo activity measurement method Fusion protein 4 and fusion protein 5, each containing the obtained TIGR4 IgA protease truncation, were respectively injected into the bodies of humanized IgA1 alpha-chain knock-in (α1KI-Tg) C57BL / 6 mice via the tail vein. Blood samples were collected before injection, 2 hours after injection, 1 day after injection, 2 days after injection, 3 days after injection, 5 days after injection, and 7 days after injection, and verified by Western blot.

[0157] 2.5 Results The in vitro activity experiments showed that although fusion protein 1 and fusion protein 2 had enzymatic cleavage activity against IgA in vitro (as shown in Figure 2a and Figure 3a respectively), fusion protein 1 and fusion protein 2 had a lot of non-full-length protease expression (as shown in Figure 2b and Figure 3b).

[0158] The in vitro activity experiment shows that the pET30a-TIGR4(665-2004)-IgG1 Fc plasmid successfully expresses the fusion protein 3 in prokaryotic cells (as shown in Figure 4b), and also has enzymatic cleavage activity against IgA in vitro (as shown in Figure 4a). Although the pET30a-TIGR4(665-2004)-IgG1 Fc plasmid was expressed in HEK293 eukaryotic cells, no enzymatic cleavage activity against IgA was detected in vitro (data not shown). In addition, in the activity verification after purification using a nickel column and S200, the fusion protein 3 purified using nickel still had enzymatic cleavage activity against IgA in vitro after being left at 4°C overnight or for 24 hours, and all of the flow-through peaks F1, F2, and F3 purified using S200 also had enzymatic cleavage activity against IgA in vitro (as shown in Figure 5). The proportion of the fusion protein 3 forming dimers was the highest in F1 and F2 (as shown in Figure 6). As a result of leaving the obtained flow-through peak at 37°C, F1 still had a clear band of the fusion protein 3 after 6 hours, but when left for more than 21 hours, the fusion protein 3 was cleaved, leaving only the IgA protease truncate TIGR4(665-2004) with Fc removed and some residual amino acids on IgG1 Fc (as shown in Figures 7a and 7b).

[0159] The in vitro activity experiment shows that the pET30a-TIGR4(665-2004)-IgG4 Fc plasmid similarly successfully expresses the fusion protein 4, and a part of each of F1, F2, and F3 formed a dimeric form (as shown in Figure 8a). In addition, in the stability test, the sample purified with nickel still had a clear target band even after being left at 37°C for 40 hours, and even after 80 hours, indicating that the fusion protein 4 has good stability (as shown in Figure 8b).

[0160] The in vitro activity experiment further shows that another pET30a-TIGR4(665-2004)-IgG4 Fc plasmid can also successfully express fusion protein 5 and has enzymatic cleavage activity against IgA in vitro (as shown in Figure 10a). In addition, in the stability test, the target band was still clear after the sample purified with nickel was left at 37 °C for 9 days, indicating that fusion protein 5 has good stability (as shown in Figure 10b).

[0161] The in vivo activity experiment further shows that after humanized IgA1(α1KI-Tg) C57BL / 6 mice were injected with fusion protein 4 via the tail vein with a single needle, IgA in the blood was enzymatically cleaved into the Fc region and the Fab region by fusion protein 4 and lasted for at least 5 days (as shown in Figure 9). The in vivo activity experiment also shows that after humanized IgA1(α1KI-Tg) C57BL / 6 mice were injected with fusion protein 5 via the tail vein with a single needle, IgA in the blood was enzymatically cleaved into the Fc region and the Fab region by fusion protein 5 and lasted for at least 5 days (as shown in Figure 11).

[0162] [Example 3] Search for other IgA proteases The inventors screened several amino acid sequences having a certain homology with the wild-type IgA protease of Streptococcus pneumoniae strain TIGR4 from a metagenome database and synthesized five types of TIGR4 homologous enzymes. Their amino acid sequences are as 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 measured the enzymatic cleavage activities of these TIGR4 homologous enzymes against IgA1 according to the in vitro activity measurement method described in Example 2.3. The results are as shown in FIG. 12. In FIG. 12, "Pneumococcus-1" shows the in vitro mixture of the polypeptide shown in SEQ ID NO: 26 and the substrate IgA1, "Pneumococcus-2" shows the in vitro mixture of the polypeptide shown in SEQ ID NO: 27 and the substrate IgA1, "Streptococcus oralis-1" shows the in vitro mixture of the polypeptide shown in SEQ ID NO: 28 and the substrate IgA1, "Streptococcus oralis-2" shows the in vitro mixture of the polypeptide shown in SEQ ID NO: 29 and the substrate IgA1, and "Streptococcus mitis-2" shows the in vitro mixture of the polypeptide shown in SEQ ID NO: 30 and the substrate IgA1.

[0163] As can be seen from FIG. 12, 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 cleavage activity against IgA1.

[0164] Although this application describes the invention with specific expressions and explanations while citing specific examples, as can be understood by those skilled in the art, the above content can be variously modified in form and details without departing from the spirit and scope of protection disclosed in this application.

Claims

1. A fusion protein comprising a first polypeptide which is an IgA protease truncate and a second polypeptide which is incapable of cleavage by IgA protease or an IgA protease truncate.

2. The fusion protein according to claim 1, wherein the IgA protease truncate comprises a non-natural truncated fragment of wild-type IgA protease obtained from or derived from the Streptococcus pneumoniae TIGR4 strain, or has at least 70% sequence identity with the non-natural truncated fragment.

3. The fusion protein according to claim 2, wherein the non-natural truncated fragment is based on the wild-type IgA protease of the Streptococcus pneumoniae TIGR4 strain, and undergoes amino acid mutation, deletion, insertion, or modification, thereby resulting in the loss or reduction of the autoenzymatic cleavage function of the IgA protease truncate.

4. The fusion protein according to claim 3, wherein the mutation, deletion, insertion, or modification of the amino acid occurs at the natural autoenzymatic cleavage site of the wild-type IgA protease of the Streptococcus pneumoniae TIGR4 strain, within five amino acid residues upstream of the natural autoenzymatic cleavage site, and / or within five amino acid residues downstream of the natural autoenzymatic cleavage site.

5. The fusion protein according to claim 2, wherein the non-natural truncated fragment is the N-terminal or C-terminal truncated fragment of a wild-type IgA protease obtained from or derived from the Streptococcus pneumoniae TIGR4 strain.

6. The fusion protein according to claim 2, wherein the deposit number of the Streptococcus pneumoniae TIGR4 strain is ATCC BAA-334.

7. The fusion protein according to claim 5, wherein the C-terminal truncated fragment comprises a polypeptide fragment of at least 703 consecutive amino acids starting from position 43 of a wild-type IgA protease obtained from or derived from the TIGR4 strain of Streptococcus pneumoniae, or has at least 70% sequence identity with the polypeptide fragment.

8. The fusion protein according to claim 7, wherein the amino acid sequence of the wild-type IgA protease of the Streptococcus pneumoniae TIGR4 strain is as shown in SEQ ID NO:

1.

9. The fusion protein according to claim 8, wherein the aforementioned natural autoenzymatic cleavage site is located between positions 43 and 745 of the amino acid sequence shown in Sequence ID No.

1.

10. The aforementioned natural autoenzyme cleavage sites are located between positions 71 and 72, 210 and 211, 228 and 229, 239 and 240, 245 and 246, 382 and 383, 418 and 419, 439 and 440, 490 and 491, 506 and 507, and 509 of the amino acid sequence shown in Sequence ID No.

1. The fusion protein according to claim 8, which is located between position 510 and position 510, between position 514 and position 515, between position 533 and position 534, between position 536 and position 537, between position 563 and position 564, between position 594 and position 595, between position 613 and position 614, between position 616 and position 617, between position 639 and position 640, between position 645 and position 646, or between position 678 and position 679.

11. The fusion protein according to claim 1, wherein the first polypeptide comprises a polypeptide fragment of at least 1300 consecutive amino acids (for example, at least 1310, at least 1320, at least 1330, or at least 1340) starting from position 665 of the amino acid sequence shown in SEQ ID NO:

1.

12. The fusion protein according to claim 1, wherein the first polypeptide comprises a polypeptide fragment of the amino acids at positions 665 to 2004 of the amino acid sequence shown in SEQ ID NO: 1, or a polypeptide fragment having at least 70% sequence identity therewith.

13. The fusion protein according to claim 12, having one or more conserved amino acid substitutions at a site based on the amino acid sequence of the polypeptide fragment.

14. The fusion protein according to claim 1, having enzymatic activity that specifically cleaves human IgA.

15. The fusion protein according to claim 14, having enzymatic activity that specifically cleaves human IgA heavy chains.

16. The fusion protein according to claim 15, having enzymatic activity that specifically cleaves the human IgA heavy chain hinge region.

17. The fusion protein according to claim 14, having enzymatic activity that specifically cleaves human IgA1.

18. The fusion protein according to claim 1, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO:

2.

19. The fusion protein according to claim 1, wherein the second polypeptide is located at the N-terminus or C-terminus of the first polypeptide.

20. The fusion protein according to claim 1, wherein the first polypeptide and the second polypeptide are linked by a linker.

21. The fusion protein according to claim 1, wherein the first polypeptide and the second polypeptide are directly linked.

22. The fusion protein according to claim 20, wherein the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.

23. The fusion protein according to claim 22, wherein the linker comprises a peptide linker.

24. The fusion protein according to claim 23, wherein the peptide linker comprises a linker containing glycine and serine.

25. The fusion protein according to claim 24, wherein the linker containing glycine and serine comprises one, two, three, four, or more repeats of the amino acid sequence shown in SEQ ID NO: 15 (GGGS), SEQ ID NO: 16 (GGGGGS), SEQ ID NO: 17 (GGGGGGGS), or SEQ ID NO: 19 (GSS).

26. The fusion protein according to claim 25, wherein the linker comprises the amino acid sequence shown in SEQ ID NO: 18 (GGGGGSGGGGGGGGGS), SEQ ID NO: 20 (GSGSGSSG), or SEQ ID NO: 21 (RSGSGSGSSG).

27. The fusion protein according to claim 1, wherein the second polypeptide comprises an amino acid sequence for extending the half-life of the first polypeptide in the body of the subject.

28. The fusion protein according to claim 27, wherein the second polypeptide is selected from an Fc domain and albumin.

29. The fusion protein according to claim 28, wherein the Fc domain includes a hinge region.

30. The fusion protein according to claim 29, wherein the Fc domain is derived from a human IgG Fc domain.

31. The fusion protein according to claim 30, wherein the Fc domain is derived from a human IgG4 Fc domain.

32. The fusion protein according to claim 28, wherein the Fc domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO:

14.

33. The fusion protein according to claim 32, wherein the Fc domain comprises the amino acid sequence shown in SEQ ID NO:

14.

34. The fusion protein according to claim 28, wherein the Fc domain includes one or more mutations that extend the half-life of the fusion protein.

35. The fusion protein according to claim 28, wherein the Fc domain is ligated to the C-terminus or N-terminus of the first polypeptide.

36. The fusion protein according to claim 1, 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.

37. The fusion protein according to claim 36, wherein the first polypeptide and the second polypeptide are directly linked by a linker shown in SEQ ID NO: 20 or SEQ ID NO:

21.

38. The fusion protein according to claim 36, wherein the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 8 or SEQ ID NO:

24.

39. The fusion protein according to claim 28, wherein the albumin comprises one or more domains of human serum albumin.

40. The fusion protein according to claim 39, wherein the albumin comprises the D3 domain of human serum albumin.

41. The fusion protein according to claim 1, further including a tag.

42. The fusion protein according to claim 41, wherein the tag is selected from the group consisting of fluorescent tags, luminescent tags, purification tags, and color-developing tags.

43. The fusion protein according to claim 41, wherein the tag is selected from the group consisting of c-Myc tag, HA tag, VSV-G tag, FLAG tag, V5 tag, and HIS tag.

44. The fusion protein according to claim 43, wherein the tag is a HIS tag containing six, seven, eight, nine, ten or more histidines.

45. The fusion protein according to claim 41, 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.

46. The fusion protein according to claim 41, wherein the half-life in the blood circulation within the subject's body is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days.

47. An isolated nucleic acid comprising a nucleotide sequence encoding the fusion protein described in claim 1.

48. The nucleic acid according to claim 47, comprising the nucleotide sequence shown in SEQ ID NO: 12 or SEQ ID NO: 25, or a nucleotide sequence having at least 70% sequence identity therewith.

49. A vector comprising the nucleic acid described in claim 47.

50. A cell comprising the nucleic acid described in claim 47, or the vector described in claim 49.

51. The cell according to claim 50, which is a prokaryotic cell or a eukaryotic cell.

52. The cell according to claim 51, wherein the prokaryotic cell is a cell of Escherichia coli.

53. The cell according to claim 51, wherein the eukaryotic cell is a mammalian cell.

54. The cell according to claim 53, wherein the mammalian cell is a human cell or a Chinese hamster ovary (CHO) cell.

55. The cell according to claim 54, wherein the mammalian cell is human fetal kidney cell 293 (HEK293 cell).

56. A pharmaceutical composition comprising (i) a fusion protein according to claim 1, a nucleic acid according to claim 47, a vector according to claim 49, or a cell according to claim 50, and (ii) a pharmaceutically acceptable carrier.

57. A method for producing a fusion protein, comprising the step of culturing the cells described in claim 50.

58. A pharmaceutical composition according to claim 56 for use in a method for treating or preventing IgA deposition-related diseases, The method comprises administering the fusion protein described in claim 1, which is contained in the pharmaceutical composition, to a subject requiring treatment or prevention.

59. The pharmaceutical composition according to claim 58, wherein the IgA deposition-related disease is selected from the group consisting of IgA nephropathy, herpetiform dermatitis, Henoch-Schönein purpura (also known as IgA vasculitis), Kawasaki disease, Henoch-Schönein purpura nephritis, renal injury due to IgA vasculitis, IgA rheumatoid factor-positive rheumatoid arthritis, IgA-type anti-GBM disease, and IgA-type ANCA-associated vasculitis.

60. The pharmaceutical composition according to claim 59, wherein the IgA deposition-related disease is IgA nephropathy, IgA vasculitis, or Kawasaki disease.

61. A pharmaceutical composition for use in a method for treating or preventing an IgA deposition-related disease, comprising an IgA protease or its truncate or a fusion protein containing the IgA protease or its truncate, The method described above includes administering to a subject requiring treatment or prevention the IgA protease or its truncate contained in the pharmaceutical composition, or a fusion protein containing the IgA protease or its truncate. 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 this pharmaceutical composition.

62. The pharmaceutical composition according to claim 61, wherein the IgA deposition-related disease is selected from the group consisting of IgA nephropathy, herpetiform dermatitis, Henoch-Schönein purpura (also known as IgA vasculitis), Kawasaki disease, Henoch-Schönein purpura nephritis, renal injury due to IgA vasculitis, IgA rheumatoid factor-positive rheumatoid arthritis, IgA-type anti-GBM disease, and IgA-type ANCA-associated vasculitis.

63. The pharmaceutical composition according to claim 62, wherein the IgA deposition-related disease is IgA nephropathy, IgA vasculitis, or Kawasaki disease.

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