WO2023143563A1 - IgA蛋白酶截短体、包含IgA蛋白酶截短体的融合蛋白及其用途 - Google Patents

IgA蛋白酶截短体、包含IgA蛋白酶截短体的融合蛋白及其用途 Download PDF

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WO2023143563A1
WO2023143563A1 PCT/CN2023/073698 CN2023073698W WO2023143563A1 WO 2023143563 A1 WO2023143563 A1 WO 2023143563A1 CN 2023073698 W CN2023073698 W CN 2023073698W WO 2023143563 A1 WO2023143563 A1 WO 2023143563A1
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seq
amino acid
fusion protein
iga
iga protease
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PCT/CN2023/073698
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English (en)
French (fr)
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吕继成
张宏
舒楚天
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北京大学第一医院
上海君祉医药科技有限公司
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Publication of WO2023143563A1 publication Critical patent/WO2023143563A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea

Definitions

  • the present application relates to the field of biomedicine, specifically, the present application relates to an IgA protease truncation, a fusion protein comprising the IgA protease truncation, a pharmaceutical composition comprising the IgA protease truncation or the fusion protein, an encoding
  • the nucleic acid of the IgA protease truncation or the fusion protein, the preparation method of the IgA protease truncation or the fusion protein, and the preparation of the IgA protease truncation or the fusion protein are related to the treatment of IgA deposition Use in medicine for disease.
  • IgA nephropathy is one of the most common primary glomerular diseases in the world, which brings a heavy burden to patients and society.
  • Clinically, supportive treatment based on RAS blockers is often used to slow down the deterioration of renal function.
  • combined hormone immunosuppressant therapy was given.
  • the long-term curative effect of hormone immunosuppressants is not good and brings serious side effects to patients.
  • the application provides an isolated IgA protease truncation, which comprises a non-native truncated fragment obtained or derived from a wild-type IgA protease of Clostridium ramosum, or combined with said non-natural Natural truncated fragments have at least 70% sequence identity.
  • the non-natural truncated fragment has amino acid substitutions, deletions, insertions or modifications based on the wild-type IgA protease of Clostridium ramosum, such that the IgA protease is truncated The body loses or reduces the self-cleavage function.
  • the amino acid substitution, deletion, insertion or modification occurs at the natural self-cleavage site, the natural self-cleavage site of the wild-type IgA protease of Clostridium ramosum Within 5 sites upstream and/or within 5 sites downstream of .
  • the Clostridium ramosum is Clostridium ramosum AK183 strain.
  • the amino acid sequence of the wild-type IgA protease of Clostridium ramosum As shown in SEQ ID NO:1.
  • the natural self-cleavage site is between the 730th and the 840th (for example, between the 792nd and the 797th) of the amino acid sequence shown in SEQ ID NO: 1 .
  • the natural self-cleavage site is at position 790, position 791, position 792, position 793, position 794, position 795 of the amino acid sequence shown in SEQ ID NO:1 bit, 796th, 797th, 798th, 799th, or 800th.
  • the non-natural truncation is an N-terminal truncation or a C-terminal truncation obtained or derived from a wild-type IgA protease from Clostridium ramosum.
  • the N-terminal truncated fragment comprises a polypeptide fragment of at least 760 consecutive amino acids obtained or derived from the 31st position of the N-terminal of the wild-type IgA protease of Clostridium ramosum, Or have at least 70% sequence identity with said polypeptide fragment.
  • the IgA protease truncated body provided by the application comprises at least 760 (for example, at least 761, at least 762, at least 763 at least 764, at least 765, at least 766, at least 767, at least 768, at least 769, at least 770, at least 771, at least 772, at least 773, at least 774, at least 775, At least 776, at least 777, at least 778, at least 779, at least 780, at least 781, at least 782, at least 783, at least 784, at least 785, at least 786, at least 787, at least 788 at least 789, at least 790, at least 791, at least 792, at least 793, at least 794, at least 795, at least 796, at least 797, at least 798, at least 799, at least 800, At least 801, at least 802, at least 803, at least 804, at least 805, at least 806, at least
  • the IgA protease truncated body provided by the application comprises a polypeptide fragment selected from the group consisting of the 31st to 790th amino acids of the amino acid sequence shown in SEQ ID NO: 1, such as SEQ ID NO The 31st to the 792nd amino acid of the amino acid sequence shown in: 1, the 31st to the 798th amino acid of the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 1 The 31st to the 807th amino acid, the 31st to the 816th amino acid of the amino acid sequence shown in SEQ ID NO:1, the 31st to the 833rd amino acid of the amino acid sequence shown in SEQ ID NO:1 and polypeptide fragments having at least 70% sequence identity thereto.
  • the non-natural truncated fragment comprises a A polypeptide fragment of at least 456 contiguous amino acids starting from position 335 of the N-terminal of wild-type IgA protease of Clostridium ramosum, or having at least 90% or at least 95% sequence identity with said polypeptide fragment.
  • the IgA protease truncated body provided by the application comprises at least 456 (for example, at least 457, at least 458, at least 459 at least 460, at least 461, at least 462, at least 463, at least 464, at least 465, at least 466, at least 467, at least 468, at least 469, at least 470, at least 471, At least 472, at least 473, at least 474, at least 475, at least 476, at least 477, at least 478, at least 479, at least 480, at least 481, at least 482, at least 483, at least 484 at least 485, at least 486, at least 487, at least 488, at least 489, at least 490, at least 491, at least 492, at least 493, at least 494, at least 495, at least 496, at least 497, at least 498, at least 499, at least 500, at least 550, at least 600, at least 650, at least 700,
  • the IgA protease truncated body provided by the application comprises a polypeptide fragment selected from the group consisting of the 335th to 790th amino acids of the amino acid sequence shown in SEQ ID NO: 1, such as SEQ ID NO
  • Amino acids 285 to 790 amino acids 285 to 791 of the amino acid sequence shown in SEQ ID NO: 1, amino acids 285 to 792 of the amino acid sequence shown in SEQ ID NO: 1 , the 330th to the 790th amino acid of the amino acid sequence shown in SEQ ID NO:1, the 330th to the 791st amino acid of the amino acid sequence shown in SEQ ID NO:1, as shown in SEQ ID NO:1
  • the 330th to the 792nd amino acid of the amino acid sequence shown, the 285th
  • the IgA protease truncate provided in the present application has conservative amino acid substitutions at one or more positions on the basis of the amino acid sequence of the polypeptide fragment.
  • the polypeptide fragment corresponds to one of the 844th, 862nd, 931st, 933rd, 978th, 1002nd, and 1004th positions of SEQ ID NO:1 Amino acid mutations at one or more positions.
  • the polypeptide fragment corresponds to one of the 844th, 862nd, 931st, 933rd, 978th, 1002nd, and 1004th positions of SEQ ID NO:1 or multiple positions mutated to glycine.
  • the polypeptide fragment has an amino acid mutation at position 844 corresponding to SEQ ID NO: 1, an amino acid mutation at position 862, an amino acid mutation at position 931 and 933, and an amino acid mutation at position 978 Mutations, or amino acid mutations at positions 1002 and 1004.
  • the amino acid sequence of the polypeptide fragment is such as SEQ ID NO:53 (also known as "PA-GA Mut"), SEQ ID NO:54 (also known as “PI-GI Mut”), SEQ ID NO:54 (also known as “PI-GI Mut”), SEQ ID NO:55 (also known as "PAP-GAG Mut”), SEQ ID NO:56 (also known as “PAT-GAT Mut”) or SEQ ID NO:57 (also known as "PIP-GIG Mut”) Show.
  • the IgA protease truncate provided herein has the enzymatic activity of specifically cleaving human IgA. In certain embodiments, the IgA protease truncate provided herein has the enzymatic activity of specifically cleaving the heavy chain of human IgA. In certain embodiments, the IgA protease truncate provided herein has the enzymatic activity of specifically cleaving the junction of human IgA heavy chain CH1 and the hinge region. In certain embodiments, the IgA protease truncate provided herein has the enzymatic activity of specifically cleaving human IgA1.
  • the application provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a wild-type polypeptide obtained or derived from Clostridium ramosum The full length of IgA protease, the polypeptide obtained or derived from the wild-type IgA protease of Clostridium ramosum after removing the signal peptide or the IgA protease truncation as described in the present application, the second polypeptide Comprising an amino acid sequence for prolonging the half-life of the first polypeptide in a subject.
  • the first polypeptide comprises the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 42.
  • the second polypeptide is N-terminal or C-terminal to the first polypeptide.
  • the first polypeptide and the second polypeptide are linked by a linker.
  • 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.
  • the linker comprises a peptide linker.
  • the peptide linker comprises a linker comprising glycine and serine.
  • the linker containing glycine and serine comprises such as SEQ ID NO: 21 (GGGS), SEQ ID NO: 22 (GGGGS), SEQ ID NO: 86 (GGGGGS) or SEQ ID NO: 87 One, two, three, four or more repeats as indicated by (GGGGGGGS).
  • the linker comprises an amino acid sequence as shown in SEQ ID NO: 23 (GGCGGCGGTGGATCC), as shown in SEQ ID NO: 58 (EEKKKEKEKEEQEERETK) or as shown in SEQ ID NO: 59 (HHHHHHHHHH) .
  • the second polypeptide is selected from the group consisting of an Fc domain and albumin.
  • the Fc domain comprises a hinge region.
  • the Fc domain is derived from a human IgG Fc domain.
  • 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.
  • the Fc domain comprises at least 70%, at least 75%, at least 80%, Amino acid sequences having at least 85%, at least 90%, or at least 95% sequence identity.
  • the Fc domain comprises an amino acid sequence as set forth in SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:32 or SEQ ID NO:77. In certain embodiments, the Fc domain has an amino acid mutation at position 7 corresponding to SEQ ID NO:25. In certain embodiments, the Fc domain is mutated to valine, glycine, serine, or leucine at the amino acid corresponding to position 7 of SEQ ID NO: 25 (e.g., alanine). In certain embodiments, the Fc domain comprises one or more mutations that extend the half-life of the fusion protein. In certain embodiments, the Fc domain is linked to the C-terminus or the N-terminus of the first polypeptide. In certain embodiments, the albumin comprises one or more domains of human serum albumin. In certain embodiments, the albumin comprises the D3 domain of human serum albumin.
  • the fusion proteins described herein further comprise a tag.
  • the tag is selected from the group consisting of fluorescent tags, luminescent tags, purification tags, and chromogenic tags.
  • the tag is selected from the group consisting of c-Myc tag, HA tag, VSV-G tag, FLAG tag, V5 tag and HIS tag.
  • the tag is a HIS tag comprising 6, 7, 8, 9 or 10 histidines.
  • the second polypeptide is at the C-terminus of the first polypeptide and the tag is at the C-terminus of the second polypeptide.
  • the half-life of the fusion protein described in the present application in the blood circulation of the subject is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days.
  • the present application provides an isolated nucleic acid comprising a nucleotide sequence encoding the IgA protease truncation described in the present application or comprising a nucleotide sequence encoding the fusion protein described in the present application.
  • the nucleic acid described in the application comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 and nucleotide sequences having at least 70% sequence identity thereto.
  • the present application provides a vector comprising the nucleic acid described in the present application.
  • the present application provides a cell comprising the nucleic acid described herein or the vector described herein.
  • the cell is a prokaryotic cell or a eukaryotic cell.
  • the prokaryotic cells are E. coli cells.
  • the eukaryotic cells are mammalian cells.
  • the mammalian cells are human cells or Chinese Hamster Ovary (CHO) cells.
  • the mammalian cell is a human embryonic kidney cell 293 (HEK293 cell).
  • the application provides a pharmaceutical composition, which comprises the IgA protease truncation body described in the application, the fusion protein described in the application, the nucleic acid described in the application, the nucleic acid described in the application.
  • a pharmaceutical composition which comprises the IgA protease truncation body described in the application, the fusion protein described in the application, the nucleic acid described in the application, the nucleic acid described in the application.
  • the carrier or the cell described in this application and a pharmaceutically acceptable carrier.
  • the present application provides a method of producing a fusion protein comprising the step of culturing the cells described herein.
  • the present application provides a method for treating or preventing IgA deposition-related diseases, which comprises administering the IgA protease truncation as described in the present application, the The fusion protein or the pharmaceutical composition as described in this application.
  • the application provides the IgA protease truncated body as described in the application, the fusion protein as described in the application or the pharmaceutical composition as described in the application in the preparation for the treatment or prevention of IgA deposition related diseases use in medicines.
  • the present application provides the IgA protease truncation as described in the present application, the fusion protein as described in the present application or the pharmaceutical composition as described in the present application for treating or preventing diseases related to IgA deposition.
  • the present application provides a method for treating or preventing IgA deposition-related diseases, which comprises administering IgA protease or a truncated body thereof, a method comprising the IgA protease or a truncated body thereof, to a subject in need of treatment or prevention.
  • a fusion protein of the body or a pharmaceutical composition comprising the IgA protease or its truncated body or the fusion protein, wherein the amino acid sequence of the IgA protease is selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62 , SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76 or a combination thereof.
  • the application provides IgA protease or its truncated body, a fusion protein comprising the IgA protease or its truncated body or a pharmaceutical composition comprising the IgA protease or its truncated body or the fusion protein
  • the amino acid sequence of the IgA protease is selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:63, ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO :72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76 or a combination thereof.
  • the present application provides an IgA protease or a truncated body thereof, a fusion protein comprising the IgA protease or a truncated body thereof, or a fusion protein comprising the IgA protease or a truncated body thereof for use in the treatment or prevention of diseases related to IgA deposition.
  • amino acid sequence of the IgA protease is selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO :73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76 or a combination thereof.
  • the IgA deposition-related diseases include IgA nephropathy, dermatitis herpetiformis, Henoch-Schoenlein purpura (also known as IgA vasculitis), Kawasaki disease, purpura nephritis, IgA vasculitis and renal damage , IgA rheumatoid factor-positive rheumatoid arthritis, IgA anti-GBM disease, or IgA ANCA-associated vasculitis.
  • the disease associated with IgA deposition is IgA nephropathy, IgA vasculitis, or Kawasaki disease.
  • Figure 1 shows the results of the in vitro enzyme cleavage activity of four IgA protease truncates, AK183(31-737), AK183(31-768), AK183(31-798) and AK183(31-833), on IgA1.
  • Figure 2 shows the effect of five IgA protease truncations, AK183(31-773), AK183(31-778), AK183(31-782), AK183(31-787) and AK183(31-792), on IgA1 in vitro The results of enzyme cleavage activity experiments.
  • Figure 3a-c shows the results of in vitro enzyme cleavage activity of four IgA protease truncations, AK183(31-788), AK183(31-789), AK183(31-790) and AK183(31-791), on IgA1 .
  • Figure 4 shows the flow chart of the construction of PET30a-AK183(31-790)-Fc plasmid.
  • Figure 5 shows the expression results of AK183(31-790)-Fc fusion protein.
  • Figure 6a shows the expression results of the AK183(31-792)-Fc fusion protein
  • Figure 6b shows the results of the in vitro enzyme cleavage activity of the AK183(31-792)-Fc fusion protein on IgA1.
  • Figure 7 shows the four fusion proteins of AK183(31-798)-Fc, AK183(31-807)-Fc, AK183(31-816)-Fc and AK183(31-833)-Fc in vitro digestion of IgA1 Activity test results.
  • Figure 8 shows the experimental results of the in vivo enzyme cutting activity of AK183(31-807)-Fc fusion protein on IgA1.
  • Fig. 9 shows the expression results of AK183(31-792)-Fc fusion protein in HEK293 cells.
  • Figure 10 shows AK183(285-792), AK183(330-792), AK183(380-792), AK183(430-792), AK183(480-792), AK183(530-792) and AK183(580- 792)
  • IgA IgA protease truncated bodies
  • Figure 11 shows AK183(335-792), AK183(340-792), AK183(345-792), AK183(350-792), AK183(355-792), AK183(360-792), AK183(365- 792), AK183(370-792) and AK183(375-792), the in vitro enzyme cleavage activity of nine IgA protease truncations to IgA1.
  • Figure 12 shows the results of the in vitro enzyme-cleavage activity of four IgA protease truncates, AK183(336-792), AK183(337-792), AK183(338-792) and AK183(339-792), on IgA1.
  • Figure 13 shows the revalidation of AK183(285-792), AK183(330-792), AK183(335-792), AK183(336-792), AK183(337-792), AK183(338-792), AK183( 339-792), AK183(340-792), AK183(345-792) and AK183(350-792) IgA protease truncations of 10 kinds of IgA protease truncation in vitro activity experiment results on IgA1.
  • Figure 14 shows the expression results of two fusion proteins, AK183(285-816)-Fc and Fc-AK183(285-816).
  • Figure 15 shows that two fusion proteins, AK183(285-816)-Fc and Fc-AK183(285-816), have a negative effect on IgA1
  • AK183(285-816)-Fc and Fc-AK183(285-816) have a negative effect on IgA1
  • Figure 16 shows the experimental results of the enzymatic activity of Fc-AK183(285-816) fusion protein, AK183(285-816)-Fc fusion protein and AK183(285-816) IgA protease truncation on IgA1.
  • Figure 17 shows the experimental results of the in vivo enzyme cutting activity of the Fc-AK183(285-816) fusion protein on IgA1.
  • Figure 18 shows the experimental results of the enzyme cutting activity of AK183(285-816)-Fc fusion protein and Fc-AK183(31-1203) fusion protein on IgA1.
  • Figure 19 shows the results of enzyme cleavage activity experiments of AK183(31-816)-IgG1 Fc fusion protein, AK183(31-816)-IgG4 Fc fusion protein and AK183(31-816)-albumin fusion protein on IgA1.
  • Figure 20 shows AK183 (285- 816)-Fc fusion protein enzyme cutting activity experiment results on IgA1.
  • Figure 21 has shown the mutant of five kinds of IgA protease truncated body as shown in SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 or SEQ ID NO:57 to IgA1 The results of enzyme cleavage activity experiments.
  • Fig. 22 shows the results of enzyme-cleavage activity experiments on IgA1 of four mutants formed by performing four different mutations on the Fc region of the AK183(31-816)-Fc fusion protein.
  • Figure 23a and Figure 23b show the experimental results of 16 kinds of AK183 homologous enzymes' enzyme cutting activity on IgA1.
  • Clostridium or “Clostridium” as used in this application both refer to the bacterium Clostridium ramosum, also known as Ramibacterium ramosum, which is a commensal bacterium in the human gut that produces IgA protease.
  • proteolytic activity refers to an enzyme having the ability to break down proteins and peptides.
  • proteases can break down proteins by hydrolyzing the peptide bonds that link amino acids together in the peptide or polypeptide chains that form them.
  • Various methods are known in the art to test the proteolytic activity of a certain protease.
  • the proteolytic activity of a protease can be determined by comparative assays that analyze the ability of various proteases to hydrolyze a suitable substrate.
  • Exemplary substrates for proteolytic activity assays include, eg, dimethyl casein, bovine collagen, bovine elastin, and the like. Colorimetric assays using these substrates are also known in the art (see, eg, WO99/34011 and US 6,376,450).
  • IgA protease refers to an enzyme capable of specifically cleaving or decomposing an IgA immunoglobulin molecule (eg, IgA1 or IgA2) in a subject (eg, human).
  • IgA protease obtained or derived from Clostridium ramosum can specifically cleave the peptide bond between the 221st proline (Pro) and the 222nd valine (Val) of IgA1 and IgA2 , thereby decomposing IgA1 and IgA2.
  • wild-type refers to a naturally occurring polypeptide or protein that does not include artificial substitutions, insertions, deletions or modifications at one or more amino acid positions;
  • wild-type when referring to nucleic acids, nucleotides or polynucleotides refers to nucleotides that do not include artificial substitutions, insertions, deletions or modifications at one or more nucleotide positions.
  • naturally occurring nucleic acid, nucleotide or polynucleotide naturally occurring nucleic acid, nucleotide or polynucleotide.
  • polynucleotides encoding wild-type polypeptides are not limited to naturally occurring polynucleotides, but also include any polynucleotides encoding wild-type polypeptides.
  • AK183 refers to the AK183 strain of Clostridium ramosum.
  • the amino acid sequence of wild-type IgA protease produced by Clostridium ramosum AK183 strain is shown in SEQ ID NO: 1 (wherein, amino acids 1-30 are signal peptides).
  • signal peptide refers to a sequence of amino acid residues that can participate in the secretion or directed transport of the mature or precursor form of a protein.
  • Signal peptides are usually located N-terminal to the precursor or mature protein sequence.
  • Signal peptides can be endogenous or exogenous.
  • Signal peptides are generally absent from mature proteins.
  • the signal peptide is cleaved from the protein by a signal peptidase.
  • the amino acid sequence formed after removing the N-terminal signal peptide from the amino acid sequence shown in SEQ ID NO:1 is shown in SEQ ID NO:42.
  • subject as used in this application includes humans and non-human animals.
  • Non-human animals include all vertebrates, such as mammals and non-mammals.
  • a "subject” may also be a livestock animal, such as cattle, pigs, sheep, poultry, and horses; or a rodent, such as a rat, mouse; or a primate, such as an ape, monkey, chimpanzee ( chimpanzee), gorilla, orangutan, baboon; or domestic animals such as dogs and cats.
  • a “subject” can be male or female, and can be elderly, adult, adolescent, child or infant.
  • a human “subject” can be Caucasian, African, Asian, Semitic, or other race or mixture of said race backgrounds.
  • protein As used herein, the terms “protein”, “polypeptide” and “peptide” are used interchangeably to refer to a polymer of amino acids.
  • the proteins, polypeptides or peptides described in this application may contain natural amino acids, or non-natural amino acids, or amino acid analogs and mimics.
  • the protein, polypeptide or peptide described in the present application can be obtained by any method known in the art, such as but not limited to, natural isolation, recombinant expression, chemical synthesis and the like.
  • amino acid refers to an organic compound containing amino ( -NH2 ) and carboxyl (-COOH) functional groups and side chains unique to each amino acid.
  • Amino acid names are also indicated in this application by standard one-letter or three-letter codes, summarized below:
  • conservative substitution refers to replacing one amino acid residue with another amino acid residue with a side chain having similar physicochemical properties.
  • amino acid residues with hydrophobic side chains such as Met, Ala, Val, Leu, and Ile
  • amino acid residues with neutral hydrophilic side chains such as Cys, Ser, Thr, Asn, and Gln
  • amino acid residues with acidic side chains e.g. Asp, Glu
  • amino acid residues with basic side chains e.g. His, Lys and Arg
  • amino acid residues with aromatic side chains eg Trp, Tyr and Phe
  • homologous refers to a nucleic acid sequence (or its complementary strand) or an amino acid sequence that shares 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.
  • percent (%) sequence identity is used for amino acid sequences (or nucleic acid sequences), it means that after sequence alignment and, if necessary, spacing is introduced to maximize the number of identical amino acids (or nucleic acids), the , the percentage of amino acid (or nucleic acid) residues identical to the reference sequence to the amino acid (or nucleic acid) residues of the candidate sequence. In other words, it can be calculated by dividing the number of amino acid residues (or bases) identical to the reference sequence it is compared with the total number of amino acid residues (or bases) in the candidate sequence or the reference sequence (whichever is shorter). The percent (%) sequence identity of amino acid sequences (or nucleic acid sequences) is calculated.
  • Conservative substitutions of such amino acid residues may or may not be considered identical residues. It can be obtained by tools disclosed in the art, such as BLASTN, BLASTp (National Center for Biotechnology Information website (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 (European Bioinformatics Institute website, see Higgins D.G.
  • isolated substance has been artificially altered from its natural state. If an “isolated” composition or substance occurs in nature, it has been altered or removed from its original state, or both.
  • polynucleotides or polypeptides naturally occurring in a living animal are not “isolated”, but if these polynucleotides or polypeptides are sufficiently separated from the materials with which they occur in nature and are present in a substantially pure state, then can be considered “separated”.
  • isolated nucleic acid sequence refers to the sequence of an isolated nucleic acid molecule.
  • isolated IgA protease truncation refers to 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% IgA protease truncated body, the purity of which was determined by electrophoresis (eg, SDS-PAGE, isoelectric focusing, capillary electrophoresis), or chromatographically (eg, ion exchange chromatography or reversed-phase HPLC).
  • electrophoresis eg, SDS-PAGE, isoelectric focusing, capillary electrophoresis
  • chromatographically eg, ion exchange chromatography or reversed-phase HPLC.
  • vector in this application refers to a vehicle into which a genetic element can be operatively inserted and expressed, such as to produce the protein, RNA or DNA encoded by the genetic element, or to replicate the genetic element.
  • Vectors can be used to transform, transduce or transfect host cells, so that the genetic elements carried by them can be expressed in the host cells.
  • Vectors include, for example: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived human Worker chromosome (PAC), bacteriophage such as lambda phage or M13 phage, and animal viruses, etc.
  • a vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication. A vector may also include components to facilitate its entry into cells, including, but not limited to, viral particles, liposomes, or protein coats. A vector may be an expression vector or a cloning vector.
  • the vector provided by the application (such as an expression vector) contains the nucleic acid sequence encoding the IgA protease truncation or fusion protein described in the application, at least one promoter (such as SV40, CMV, EF-1 ⁇ ), and at least one selectable marker.
  • treatment or “therapy” of a disease, disorder or condition includes preventing or alleviating a disease, disorder or condition, reducing the rate at which a disease, disorder or condition develops or risk of a disease, disorder or condition, preventing or delaying the development of symptoms associated with a disease, disorder or condition, reducing or terminating symptoms associated with a disease, disorder or condition, producing a disease, disorder or condition Complete or partial reversal, cure of a disease, disorder or condition, or a combination of the above.
  • pharmaceutically acceptable means that the specified carrier, vehicle, diluent, excipient and/or salt is generally chemically and/or physically compatible with the other ingredients making up the formulation and physiologically compatible with its receptors compatible.
  • IgA deposition-associated disease refers to a disease associated with the accumulation of IgA immunoglobulin in aggregated or non-aggregated form in a tissue or organ of a subject.
  • IgA nephropathy dermatitis herpetiformis
  • Henoch-Schoenlein purpura also known as IgA vasculitis
  • Kawasaki disease purpura nephritis
  • IgA vasculitis renal damage IgA rheumatoid factor positive Rheumatoid arthritis
  • IgA anti-GBM disease or IgA ANCA-associated vasculitis.
  • IgA nephropathy refers to a kidney disease characterized by IgA deposition in the kidney.
  • the application provides an isolated IgA protease truncation, which comprises a non-native truncated fragment obtained or derived from a wild-type IgA protease of Clostridium ramosum, or combined with said non-natural
  • the native truncated fragment has at least 70% sequence identity (e.g., has 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).
  • the non-native truncated fragment has at least 70% sequence identity (e.g., has 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) IgA protease truncations still maintain the function or activity of IgA protease (eg, proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.).
  • IgA protease truncations still maintain the function or activity of IgA protease (eg, proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.).
  • truncate or “truncated fragment” as used in this application refers to a peptide formed after one or more amino acids are removed from one or both ends of a wild-type polypeptide. Therefore, the "truncated body” or “truncated fragment” in the present application does not include the full length of its corresponding wild-type polypeptide, but may have one or more amino acid substitutions, deletions, insertions or modifications, etc.
  • IgA protease truncates or "IgA protease truncated fragments” may include peptides formed by removing one or more amino acids from one or both ends of wild-type IgA protease, and may also include peptides formed with wild-type IgA protease Peptides with one or more amino acid substitutions, deletions, insertions or modifications compared to truncated forms.
  • the IgA protease truncation described herein has one or more amino acid substitutions, deletions, insertions or modifications compared to its corresponding wild-type IgA protease.
  • the IgA protease truncations described herein comprise a non-native truncated fragment obtained or derived from a wild-type IgA protease from Clostridium ramosum, wherein the non-natural The truncated fragment has amino acid substitutions, deletions, insertions or modifications on the basis of the wild-type IgA protease of Clostridium ramosum, so that the truncated IgA protease loses or reduces the self-cleavage function.
  • the terms “obtained from” and “derived from” include not only proteins produced or producible by the organisms mentioned, but also proteins encoded by DNA sequences isolated from such organisms and contained in such Proteins produced in the host organism of the DNA sequences also include proteins encoded by DNA sequences of synthetic and/or cDNA origin and having the identifying characteristics of the proteins mentioned.
  • a wild-type IgA protease obtained or derived from Clostridium mycobacterium includes both the IgA protease naturally produced by Clostridium mycobacterium, as well as other host cells transformed with a nucleic acid encoding an IgA protease by using genetic engineering techniques (such as , Escherichia coli) produced IgA protease.
  • non-natural truncated fragment refers to a truncated fragment with different amino acids from the truncated fragment formed by the wild-type IgA protease of Clostridium ramosum in natural environment Fragments of sequence (eg, different amino acid lengths, different amino acid types, etc.).
  • the amino acid substitution, deletion, insertion or modification occurs at a native autocleavage site of the wild-type IgA protease of Clostridium ramosum. In some embodiments, the amino acid substitution, deletion, insertion or modification occurs within 5 sites upstream of the natural self-cleavage site of the wild-type IgA protease of Clostridium ramosum (e.g. , 1 site, 2 sites, 3 sites, 4 sites or 5 sites upstream of the natural self-cleavage site). In certain embodiments, the amino acid substitution, deletion, insertion or modification occurs within 5 sites downstream of the natural self-cleavage site of the wild-type IgA protease of Clostridium ramosum (e.g.
  • the amino acid substitution, deletion, insertion or modification occurs at 5 sites upstream of the natural self-cleavage site of the wild-type IgA protease of Clostridium ramosum (for example, Within 1 site, 2 sites, 3 sites, 4 sites or 5 sites upstream of said natural self-cleavage site and within 5 sites downstream (for example, within said 1 site, 2 sites, 3 sites, 4 sites, or 5 sites downstream of the native restriction site).
  • the non-natural truncation is an N-terminal truncation or a C-terminal truncation obtained or derived from a wild-type IgA protease from Clostridium ramosum.
  • N-terminal truncation refers to a truncated fragment comprising the amino acid sequence at the amino terminus of the wild-type IgA protease of Clostridium ramosum.
  • the starting position of the "amino terminus” may be any position close to the amino terminus of the amino acid sequence of the wild-type IgA protease of Clostridium mycobacterium, for example, it may be the first position counted from the amino terminus, or it may be the position counted from the amino terminus other locations.
  • the N-terminal starting position of its N-terminal truncated fragment can be the 1st to 500th position from the amino-terminus of its amino acid sequence anywhere in between.
  • C-terminal truncation refers to a truncated fragment comprising the amino acid sequence of the carboxy-terminal of the wild-type IgA protease of Clostridium ramosum.
  • the termination position of the "carboxy terminus” may be any position close to the carboxyl terminus of the amino acid sequence of the wild-type IgA protease of Clostridium mycobacterium, for example, it may be the first position counted from the carboxy terminus, or it may be other positions counted from the carboxy terminus Location.
  • the carboxy-terminal termination position of its C-terminal truncated fragment can be between the 501st and 1000th positions from the amino-terminus of its amino acid sequence any location.
  • Clostridium ramosum is one of many species in the genus Clostridium, including various strains, such as AK183, VPI-0496A, and NCTC 10474 strains. In some embodiments, the Clostridium ramosum is Clostridium ramosum AK183 strain.
  • the N-terminal truncated fragment comprises a polypeptide fragment of at least 760 consecutive amino acids obtained or derived from the 31st position of the N-terminal of the wild-type IgA protease of Clostridium ramosum, or having at least 70% sequence identity (e.g., having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, At least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 % sequence identity).
  • 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 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% sequence identity to the polypeptide fragment) %, 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%, N-terminal truncated fragments with at least 98%, at least 99% sequence identity) still maintain the function or activity of IgA protease (eg, proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.).
  • IgA protease e.g, proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.
  • the non-native truncated fragment of the IgA protease described herein comprises at least 456 positions from position 335 of the N-terminal of the wild-type IgA protease obtained or derived from Clostridium ramosum A polypeptide fragment of contiguous amino acids, or having at least 90% or at least 95% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%) to said polypeptide fragment , at least 97%, at least 98%, at least 99% sequence identity).
  • At least 90% or at least 95% sequence identity e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%) to the polypeptide fragment , at least 97%, at least 98%, at least 99% sequence identity
  • the non-naturally truncated fragments still maintain the function or activity of IgA protease (eg, proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.).
  • amino acid sequence of the wild-type IgA protease of Clostridium ramosum is shown in SEQ ID NO:1.
  • the amino acid position of the IgA protease mentioned in this application is the amino acid position corresponding to the wild-type AK183 IgA protease (its amino acid sequence is shown in SEQ ID NO: 1).
  • the 790th position of the AK183 IgA protease mentioned in this application corresponds to the 790th position of SEQ ID NO:1.
  • the nomenclature of the AK183 IgA protease truncated body mentioned in this application is AK183 (corresponding to the start site of SEQ ID NO: 1-corresponding to the termination site of SEQ ID NO: 1).
  • AK183(31-790) refers to the IgA protease truncation formed from the 31st to 790th amino acids of SEQ ID NO:1.
  • the natural self-cleavage site of the IgA protease described in the application is between the 730th and 840th positions of the amino acid sequence shown in SEQ ID NO:1. In some embodiments, the natural self-cleavage site of the IgA protease described in the present application is between the 710th to the 830th, the 720th to the 820th of the amino acid sequence shown in SEQ ID NO:1 Between, 730th to 810th, 740th to 800th, 750th to 790th, 791st to 780th, or 792nd to 797th .
  • the natural self-cleavage site is at position 790, position 791, position 792, position 793, position 794, position 795 of the amino acid sequence shown in SEQ ID NO:1 bit, 796th, 797th, 798th, 799th, or 800th.
  • the IgA protease truncated body provided by the present application comprises a polypeptide fragment of at least 760 consecutive amino acids starting from position 31 of the amino acid sequence shown in SEQ ID NO:1.
  • the IgA protease truncated body provided by the application comprises at least 761, at least 762, at least 763, at least 764 at least 765, at least 766, at least 767, at least 768, at least 769, at least 770, at least 771, at least 772, at least 773, at least 774, at least 775, at least 776, At least 777, at least 778, at least 779, at least 780, at least 781, at least 782, at least 783, at least 784, at least 785, at least 786, at least 787, at least 788, at least 789 at least 790, at least 791, at least 792, at least 793, at least 794, at least 795
  • the IgA protease truncated body provided by the application comprises a polypeptide fragment of 760 consecutive amino acids starting from position 31 of the amino acid sequence shown in SEQ ID NO:1. In certain embodiments, the IgA protease truncated body provided by the application comprises a polypeptide fragment of 761 consecutive amino acids starting from position 31 of the amino acid sequence shown in SEQ ID NO:1. In some embodiments, the IgA protease truncated body provided by the application comprises a polypeptide fragment of 762 consecutive amino acids starting from position 31 of the amino acid sequence shown in SEQ ID NO:1.
  • the IgA protease truncated body provided by the application comprises a polypeptide fragment of 768 consecutive amino acids starting from position 31 of the amino acid sequence shown in SEQ ID NO:1. In certain embodiments, the IgA protease truncated body provided by the application comprises a polypeptide fragment of 777 consecutive amino acids starting from position 31 of the amino acid sequence shown in SEQ ID NO:1. In certain embodiments, the IgA protease truncated body provided by the application comprises a polypeptide fragment of 786 consecutive amino acids starting from position 31 of the amino acid sequence shown in SEQ ID NO:1. In certain embodiments, the IgA protease truncated body provided by the application comprises a polypeptide fragment of 803 consecutive amino acids starting from position 31 of the amino acid sequence shown in SEQ ID NO:1.
  • the IgA protease truncated body provided by the application comprises a polypeptide fragment selected from the group consisting of the 31st to 790th amino acids of the amino acid sequence shown in SEQ ID NO: 1, such as SEQ ID NO The 31st to the 792nd amino acid of the amino acid sequence shown in: 1, the 31st to the 798th amino acid of the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 1 Amino acids 31 to 807, amino acids 31 to 816 of the amino acid sequence shown in SEQ ID NO: 1, amino acids 31 to 833 of the amino acid sequence shown in SEQ ID NO: 1 and at least 70% sequence identity thereto (e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, At least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 9
  • 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% sequence identity to the polypeptide fragment
  • IgA protease truncations still retain the function or activity of IgA protease (e.g., proteolytic activity, specificity Cleavage of IgA enzyme activity, etc.).
  • the IgA protease truncated body provided by the application comprises a polypeptide fragment of at least 456 consecutive amino acids starting from position 335 of the amino acid sequence shown in SEQ ID NO:1.
  • the IgA protease truncated body provided by the application comprises at least 457, at least 458, at least 459, at least 460 at least 461, at least 462, at least 463, at least 464, at least 465, at least 466, at least 467, at least 468, at least 469, at least 470, at least 471, at least 472, At least 473, at least 474, at least 475, at least 476, at least 477, at least 478, at least 479, at least 480, at least 481, at least 482, at least 483, at least 484, at least 485 at least 486, at least 487, at least 488, at least 489, at least 490, at least 491, at least 492, at least 493, at
  • the IgA protease truncated body provided by the application comprises a polypeptide fragment selected from the group consisting of the 335th to 790th amino acids of the amino acid sequence shown in SEQ ID NO: 1, such as SEQ ID NO
  • Amino acids 285 to 790 amino acids 285 to 791 of the amino acid sequence shown in SEQ ID NO: 1, amino acids 285 to 792 of the amino acid sequence shown in SEQ ID NO: 1 , the 330th to the 790th amino acid of the amino acid sequence shown in SEQ ID NO:1, the 330th to the 791st amino acid of the amino acid sequence shown in SEQ ID NO:1, as shown in SEQ ID NO:1
  • the polypeptide fragment has at least 90% or at least 95% sequence identity (e.g., at least 91%, at least 92%, at least 93%, IgA protease truncations of at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity) still retain IgA protease function or activity (e.g., proteolytic activity, specificity enzyme activity that cleaves IgA, etc.).
  • sequence identity e.g., at least 91%, at least 92%, at least 93%, IgA protease truncations of at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity
  • IgA protease function or activity e.g., proteolytic activity, specificity enzyme activity that cleaves IgA, etc.
  • the present application provides AK183 (31-790) truncation, the amino acid sequence of which is shown in SEQ ID NO:14.
  • the present application provides AK183(31-791) truncation, the amino acid sequence of which is shown in SEQ ID NO:15.
  • the present application provides AK183 (31-792) truncation, the amino acid sequence of which is shown in SEQ ID NO:16.
  • the present application provides AK183(31-798) truncation, the amino acid sequence of which is shown in SEQ ID NO:17.
  • the present application provides AK183(31-807) truncation, the amino acid sequence of which is shown in SEQ ID NO:18.
  • the present application provides AK183(31-816) truncation, the amino acid sequence of which is shown in SEQ ID NO:19.
  • the present application provides AK183 (31-833) truncation, the amino acid sequence of which is shown in SEQ ID NO:20.
  • the application provides AK183 (285-790) truncation body, its amino acid sequence is as follows Shown in SEQ ID NO:43.
  • the present application provides AK183 (285-791) truncation, the amino acid sequence of which is shown in SEQ ID NO:44.
  • the present application provides AK183 (285-792) truncation, the amino acid sequence of which is shown in SEQ ID NO:45.
  • the present application provides AK183 (285-816) truncation, the amino acid sequence of which is shown in SEQ ID NO:46.
  • the present application provides AK183 (330-790) truncation, the amino acid sequence of which is shown in SEQ ID NO:47. In certain embodiments, the present application provides AK183 (330-791) truncation, the amino acid sequence of which is shown in SEQ ID NO:48. In certain embodiments, the present application provides AK183 (330-792) truncation, the amino acid sequence of which is shown in SEQ ID NO:49. In certain embodiments, the present application provides AK183 (335-790) truncation, the amino acid sequence of which is shown in SEQ ID NO:50.
  • the present application provides AK183 (335-791) truncation, the amino acid sequence of which is shown in SEQ ID NO:51. In some embodiments, the present application provides AK183 (335-792) truncation, the amino acid sequence of which is shown in SEQ ID NO:52.
  • sequences of SEQ ID NO: 43-52 are shown below.
  • the IgA protease truncation body provided by the application is based on the amino acid sequence of the above-mentioned polypeptide fragment, at one or more positions (for example, at 1, 2, 3, 4, 5 one or more positions) with conservative amino acid substitutions.
  • the conservative substitution of amino acid residues refers to the substitution between amino acids with similar properties, such as the substitution between polar amino acids (such as the substitution between glutamine and asparagine), the substitution between hydrophobic amino acids (such as leuco amino acid, isoleucine, methionine, and valine), and between amino acids with the same charge (such as between arginine, lysine, and histidine, or between glutamine acid and aspartic acid) etc.
  • the IgA protease truncated body described in the application is related to SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO: 49.
  • SEQ ID NO:50, SEQ ID NO:51 or SEQ ID NO:52 in 1, 2, 3, 4, 5, 6, 7, 8 1, 9, 10, 11, 15, 20 or more positions have conservative substitutions of amino acids.
  • the polypeptide fragment corresponds to one of the 844th, 862nd, 931st, 933rd, 978th, 1002nd, and 1004th positions of SEQ ID NO:1 Amino acid mutations at one or more positions.
  • the polypeptide fragment has an amino acid mutation at position 844 corresponding to SEQ ID NO:1.
  • the polypeptide fragment has an amino acid mutation at position 862 corresponding to SEQ ID NO:1.
  • the polypeptide fragment has amino acid mutations corresponding to positions 931 and 933 of SEQ ID NO:1.
  • the polypeptide fragment has an amino acid mutation corresponding to position 978 of SEQ ID NO:1.
  • the polypeptide fragment has amino acid mutations corresponding to positions 1002 and 1004 of SEQ ID NO:1.
  • the polypeptide fragment corresponds to one of the 844th, 862nd, 931st, 933rd, 978th, 1002nd, and 1004th positions of SEQ ID NO:1 or multiple positions mutated to glycine.
  • the polypeptide fragment is at position 844 corresponding to SEQ ID NO:1 Proline (P) at one or more of positions 1, 862, 931, 933, 978, 1002, 1004 is mutated to glycine (G).
  • the proline corresponding to position 844 of SEQ ID NO:1 is mutated to glycine in the polypeptide fragment.
  • the proline corresponding to position 862 of SEQ ID NO:1 is mutated to glycine in the polypeptide fragment.
  • the polypeptide fragments have mutations at prolines corresponding to positions 931 and 933 of SEQ ID NO:1 to glycines.
  • the proline corresponding to position 978 of SEQ ID NO:1 is mutated to glycine in the polypeptide fragment.
  • the polypeptide fragments are mutated to glycine at the prolines corresponding to positions 1002 and 1004 of SEQ ID NO:1.
  • the amino acid sequence of the polypeptide fragment is such as SEQ ID NO:53 (also known as "PA-GA Mut"), SEQ ID NO:54 (also known as “PI-GI Mut”), SEQ ID NO:54 (also known as “PI-GI Mut”), SEQ ID NO:55 (also known as "PAP-GAG Mut”), SEQ ID NO:56 (also known as “PAT-GAT Mut”), or SEQ ID NO:57 (also known as "PIP-GIG Mut”) Show.
  • the IgA protease truncation body provided by this application may also contain unnatural amino acids.
  • Unnatural amino acids include, for example, ⁇ -fluoroalanine, 1-methylhistidine, ⁇ -methyleneglutamic acid, ⁇ -methylleucine, 4,5-dehydrolysine, Hydroxyproline, 3-fluorophenylalanine, 3-aminotyramine acid, 4-methyltryptophan, etc.
  • the IgA protease truncations provided herein can also be modified using methods known in the art. For example, but not limited to, PEGylation, glycosylation, amino-terminal modification, fatty acylation, carboxy-terminal modification, phosphorylation, methylation, and the like. Those skilled in the art can understand that after the IgA protease truncation body provided in the present application is modified by methods known in the art, it still retains functions substantially similar to IgA protease or IgA protease truncation body.
  • the IgA protease truncate provided herein has the enzymatic activity of specifically cleaving human IgA. In certain embodiments, the IgA protease truncate provided herein has the enzymatic activity of specifically cleaving the heavy chain of human IgA. In certain embodiments, the IgA protease truncate provided herein has the enzymatic activity of specifically cleaving the junction of human IgA heavy chain CH1 and the hinge region. In certain embodiments, the IgA protease truncate provided herein has the enzymatic activity of specifically cleaving human IgA1.
  • the IgA protease truncated body provided by the present application has conservative amino acid substitutions at one or more positions based on the amino acid sequence of the above-mentioned polypeptide fragment, but still has the ability to cleave human IgA (for example, IgA1) of enzyme activity.
  • the IgA protease truncated body provided herein has at least 70% sequence identity (for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83% sequence identity with the above-mentioned polypeptide fragments).
  • the application provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a wild-type polypeptide obtained or derived from Clostridium ramosum
  • the first polypeptide comprises a wild-type polypeptide obtained or derived from Clostridium ramosum
  • the second polypeptide comprises An amino acid sequence for prolonging the half-life of the first polypeptide in a subject.
  • the first polypeptide comprises the sequence shown in SEQ ID NO:1 or SEQ ID NO:42.
  • the second polypeptide is N-terminal to the first polypeptide.
  • the second polypeptide is C-terminal to the first polypeptide.
  • first polypeptide and the second polypeptide are linked by a linker. In certain embodiments, the first polypeptide and the second polypeptide are linked directly (ie, not via a linker).
  • linker or “linker” as used in this application refers to a linker having 1, 2, 3, 4 or 5 amino acid residues, or between 5 and 15, 20, 30, 50 or more amino acid residues in length. Artificial amino acid sequences between bases, connected by peptide bonds, and used to link one or more polypeptides. Linkers may or may not have secondary structure. Linker sequences are known in the art, see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al., Structure 2:1121-1123 (1994 ).
  • 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.
  • the linker comprises a peptide linker.
  • linkers useful in this application may be rich in glycine and serine residues.
  • linkers with single or repeated sequences comprising threonine/serine and glycine such as GGGS (SEQ ID NO:21) or GGGGS (SEQ ID NO:22), GGGGGS (SEQ ID NO:86) or GGGGGGGS ( SEQ ID NO:87) or tandem repeats thereof (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeats).
  • the linker used in the present application comprises GGCGGCGGTGGATCC (SEQ ID NO: 23).
  • the linker may be a long peptide chain comprising one or more sequential or tandem repeats of the amino acid sequence shown as GGCGGCGGTGGATCC (SEQ ID NO: 23).
  • the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sequential or tandem repeats of SEQ ID NO: 23.
  • the linker comprises or consists of an amino acid sequence selected from the group consisting of at least 80%, at least Amino acid sequences having 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.
  • the linker used in the present application includes the amino acid sequence shown in SEQ ID NO: 58 (EEKKKEKEKEEQEERETK).
  • the linker may be a long peptide chain comprising one or more sequential or tandem repeats of the amino acid sequence shown in SEQ ID NO:58.
  • the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sequential or tandem repeats of SEQ ID NO:58.
  • the linker comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% of SEQ ID NO:58 , at least 96%, at least 97%, at least 98%, at least 99% sequence identity of amino acid sequences List.
  • the linker used in the present application comprises the amino acid sequence shown in SEQ ID NO: 59 (HHHHHHHHHH). In certain embodiments, the linker comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% of SEQ ID NO:59 , amino acid sequences having at least 96%, at least 97%, at least 98%, at least 99% sequence identity.
  • the second polypeptide is selected from an Fc domain and albumin.
  • the Fc domain comprises a hinge region.
  • the Fc domain comprises a lower hinge.
  • the Fc domain comprises a core hinge region and a lower hinge.
  • the Fc domain comprises an upper hinge region, a core hinge region and a lower hinge.
  • the Fc domain does not comprise a hinge region.
  • the Fc domain is derived from a human IgG Fc domain.
  • 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.
  • the Fc domain includes the amino acid sequence shown in SEQ ID NO:24. In certain embodiments, the Fc domain consists of the amino acid sequence shown in SEQ ID NO:24. In certain embodiments, the amino acid sequence of the Fc domain has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the amino acid sequence shown in SEQ ID NO:24. % sequence identity.
  • the nucleic acid sequence encoding the Fc domain includes the nucleotide sequence shown in SEQ ID NO:39. In certain embodiments, the nucleic acid sequence encoding the Fc domain consists of the nucleotide sequence shown in SEQ ID NO:39. In some embodiments, the nucleic acid sequence encoding the Fc domain shares at least 70%, at least 75%, at least 80%, at least 70%, at least 80%, and at least 70% of the nucleotide sequence as shown in SEQ ID NO:39. At least 85%, at least 90%, or at least 95% sequence identity.
  • the Fc domain includes the amino acid sequence shown in SEQ ID NO:25. In certain embodiments, the Fc domain consists of the amino acid sequence shown in SEQ ID NO:25. In certain embodiments, the amino acid sequence of the Fc domain is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the amino acid sequence shown in SEQ ID NO:25. % sequence identity.
  • the nucleic acid sequence encoding the Fc domain includes the nucleotide sequence shown in SEQ ID NO:40. In certain embodiments, the nucleic acid sequence encoding the Fc domain consists of the nucleotide sequence shown in SEQ ID NO:40. In certain embodiments, the nucleic acid sequence encoding the Fc domain has at least 70%, at least 75%, at least 80%, at least 85%, at least 90% of the nucleotide sequence shown in SEQ ID NO:40 Or at least 95% sequence identity.
  • the Fc domain includes the amino acid sequence shown in SEQ ID NO:32. In certain embodiments, the Fc domain consists of the amino acid sequence shown in SEQ ID NO:32. In certain embodiments, the amino acid sequence of the Fc domain has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the amino acid sequence shown in SEQ ID NO:32. % sequence identity.
  • the Fc domain includes the amino acid sequence shown in SEQ ID NO:77. In certain embodiments, the Fc domain consists of the amino acid sequence shown in SEQ ID NO:77. In certain embodiments, the amino acid sequence of the Fc domain is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the amino acid sequence shown in SEQ ID NO:77. % sequence identity.
  • the Fc domain has one or more amino acid mutations. In certain embodiments, the Fc domain has an amino acid mutation at position 7 corresponding to SEQ ID NO:25. In certain embodiments, the Fc domain is mutated to valine at the amino acid corresponding to position 7 of SEQ ID NO: 25 (e.g., alanine). In certain embodiments, the Fc domain is mutated to glycine at the amino acid corresponding to position 7 of SEQ ID NO: 25 (e.g., alanine). In certain embodiments, the Fc domain is mutated to a serine at the amino acid corresponding to position 7 of SEQ ID NO: 25 (e.g., alanine). In certain embodiments, the Fc domain is mutated to leucine at the amino acid corresponding to position 7 of SEQ ID NO: 25 (e.g., alanine).
  • the Fc domain comprises one or more mutations that extend the half-life of the fusion protein. In certain embodiments, the Fc domain is linked to the C-terminus of the first polypeptide. In certain embodiments, the Fc domain is linked to the N-terminus of the first polypeptide.
  • the second polypeptide is albumin.
  • the amino acid sequence of the albumin is shown in SEQ ID NO:60.
  • the albumin comprises one or more domains of human serum albumin.
  • the albumin comprises the D3 domain of human serum albumin.
  • the fusion protein provided herein further comprises a tag.
  • the tag is selected from the group consisting of fluorescent tags, luminescent tags, purification tags, and chromogenic tags.
  • the tag is selected from the group consisting of c-Myc tag, HA tag, VSV-G tag, FLAG tag, V5 tag and HIS tag.
  • the tag is a HIS tag.
  • the tag is a HIS tag comprising 6, 7, 8, 9 or 10 histidines.
  • the second polypeptide is at the C-terminus of the first polypeptide and the tag is at the C-terminus of the second polypeptide.
  • the fusion protein provided by the application includes such as SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 , the amino acid sequence shown in SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84 or SEQ ID NO:85.
  • the fusion protein provided by the application consists of an amino acid sequence selected from the group consisting of: SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO :30, SEQ ID NO:31, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84 or SEQ ID NO:85, or at least 70%, at least 75%, At least 80%, at least 85%, at least 90%, at least 95% sequence identity.
  • SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84 or SEQ ID NO:85 have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity
  • the non-specific fusion protein still maintains the function or activity of IgA protease (eg, proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.).
  • the fusion protein provided by the application includes such as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 Amino acid sequence shown.
  • the fusion protein provided by the application consists of an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO : 10, SEQ ID NO: 12, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity thereto.
  • the half-life of the fusion protein provided by the application in the blood circulation of the subject is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days. 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.
  • the present application provides an isolated nucleic acid comprising a nucleotide sequence encoding the IgA protease truncation described in the present application or comprising a nucleotide sequence encoding the fusion protein described in the present application.
  • nucleic acid refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single- or double-stranded form. Unless otherwise stated, a particular nucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as expressly indicated the sequence of.
  • degenerate codon substitutions can be achieved by generating sequences in which one or more selected (or all) codons are substituted at the third position by mixed bases and/or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985) and Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994)).
  • DNA encoding the IgA protease truncations described herein or the DNA encoding the fusion proteins described herein can be easily isolated and sequenced using conventional procedures (e.g., by using Oligonucleotide probes for gene-specific binding of body or fusion proteins). coding DNA can also be obtained synthetically.
  • the nucleic acid provided by the application comprises as shown in SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 The nucleic acid sequence shown.
  • the nucleic acid provided by the application consists of a nucleotide sequence selected from the group consisting of: SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO: 37, SEQ ID NO: 38, or 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.
  • the nucleic acid provided by the application comprises as shown in SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13 The nucleic acid sequence shown.
  • the nucleic acid provided by the application consists of a nucleotide sequence selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, or 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.
  • the present application provides a vector comprising a nucleic acid encoding the IgA protease truncation described in the present application or comprising a nucleic acid encoding the fusion protein described in the present application.
  • the isolated polynucleotide encoding the IgA protease truncation or fusion protein can be inserted into a vector for further cloning (amplification of DNA) or for expression.
  • vectors typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1 ⁇ ), and a transcription termination sequence.
  • the nucleic acid provided herein encodes an IgA protease truncation or fusion protein, at least one promoter (e.g., SV40, CMV, EF-1 ⁇ ) operably linked to the nucleic acid sequence, and at least one selection tag .
  • promoter e.g., SV40, CMV, EF-1 ⁇
  • vectors include, but are not limited to: retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40), lambda 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,
  • pCDM8 pCDNA1.1/amp, pcDNA3.1, pRc/RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.
  • the vector comprising the nucleic acid sequence encoding the IgA protease truncation or fusion protein can be introduced into host cells for cloning or gene expression.
  • the host cells suitable for cloning or expressing the DNA in the vectors described in this application are the above-mentioned prokaryotic, yeast or higher eukaryotic cells.
  • Prokaryotic cells suitable for use in the present application include eubacteria, such as Gram-negative bacteria or Gram-positive bacteria, for example, Enterobacteriaceae (Enterobacteriaceae), for example, Escherichia (Escherichia) (for example, Escherichia coli ( E.coli)), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (for example, Salmonella typhimurium ( Salmonella typhimurium), Serratia (for example, Serratia marcescans), Shigella, Bacilli (for example, subtilis Bacillus (B.subtilis) and lichen buds B. licheniformis), Pseudomonas (e.g., P. aeruginosa), and Streptomyces.
  • the cell is E. coli cell.
  • eukaryotic cells such as eukaryotic microorganisms such as filamentous fungi or yeast
  • eukaryotic microorganisms such as filamentous fungi or yeast
  • Saccharomyces cerevisiae, or baker's yeast, is the most commonly used lower eukaryotic host microorganism.
  • Kluyveromyces hosts for example, Kluyveromyces lactis (K .lactis), K.fragilis (ATCC 12,424), K.bulgaricus (ATCC 16,045), K.wickeramii (ATCC 24,178) , K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans and K.
  • the eukaryotic cells are mammalian cells.
  • the mammalian cells are human cells or Chinese Hamster Ovary (CHO) cells.
  • the mammalian cell is a human embryonic kidney cell 293 (HEK293 cell).
  • the application provides a pharmaceutical composition, which comprises the IgA protease truncation body described in the application, the fusion protein described in the application, the nucleic acid described in the application, the nucleic acid described in the application.
  • a pharmaceutical composition which comprises the IgA protease truncation body described in the application, the fusion protein described in the application, the nucleic acid described in the application, the nucleic acid described in the application.
  • the carrier or the cell described in this application and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers used in the pharmaceutical compositions disclosed in this application may include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial substances, etc. osmotic 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 of the above.
  • 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, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butyl methyl Anisole, Butylated Hydroxytoluene and/or Propyl Gallate.
  • antioxidants such as methionine
  • a composition comprising an IgA protease truncation or fusion protein disclosed herein reduces the protein oxidation.
  • the present application further provides various methods for preventing the oxidation of the IgA protease truncation or fusion protein, prolonging its shelf life and/or improving its activity, for example, by combining the IgA protease truncation or fusion protein provided in the present application with a One or more antioxidants (eg, methionine) mixed to achieve.
  • the pharmaceutically acceptable carrier may include, for example, an aqueous medium such as sodium chloride injection, Ringer's solution injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's solution.
  • an aqueous medium such as sodium chloride injection, Ringer's solution injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's solution.
  • non-aqueous media such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antibacterial substances at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, Buffers such as: phosphate or citrate buffer, antioxidants such as: sodium bisulfate, local anesthetics such as: procaine hydrochloride, suspending and dispersing agents such as: sodium carboxymethylcellulose, hydroxypropyl methylcellulose or polyvinylpyrrolidone, emulsifiers such as polysorbate 80 (Tween-80), chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol bis(2-aminoethylene ether) tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid.
  • isotonic agents such as sodium chloride or dex
  • Antimicrobial agents as carriers can be added to pharmaceutical compositions in multi-dose containers and include phenols or cresols, mercury agents, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thiophene Mercury sprinkles, chlorobenzalkonium and chlorobenzethonium.
  • Suitable excipients may include, for example, water, saline, dextrose, glycerol or ethanol.
  • Suitable nontoxic auxiliary substances may include, for example, wetting agents, emulsifiers, pH buffering agents, stabilizers, solubilizers, or sodium acetate, sorbitan laurate, triethanolamine oleate, or cyclodextrin and the like.
  • the pharmaceutical composition may be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation or powder.
  • Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • the pharmaceutical composition is formulated as an injectable composition.
  • injectable pharmaceutical compositions can be prepared in any conventional form, for example, liquid solvents, suspensions, emulsifications or solid forms suitable for liquid solvents, suspensions, or emulsifications.
  • Preparations for injection may include ready-to-use sterile and/or pyrogen-free solutions, sterile dry solubles that are combined with solvents just before use, such as lyophilized powders, including subcutaneous tablets, sterile suspensions ready for injection, Sterile dry insoluble products, and sterile and/or pyrogen-free emulsions, to be combined with a vehicle just before use.
  • the solvent can be aqueous or non-aqueous.
  • the unit dose injection formulation is packaged in an ampoule, a tube or a syringe with a needle.
  • all preparations for parenteral administration should be sterile and pyrogen-free.
  • sterile lyophilized powders can be prepared by dissolving the IgA protease truncations or fusion proteins disclosed in this application in an appropriate solvent.
  • the solvent may contain an additional pharmacological component that increases the stability of the powder or reconstituted solution prepared from the powder, or improves the powder or reconstituted solution. Suitable excipients include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, dextrose, sucrose, or other suitable substances.
  • the solvent may contain a buffer, such as citrate buffer, sodium or potassium phosphate buffer or other buffers known to those skilled in the art. In one embodiment, the pH of the buffer is neutral.
  • the resulting solvent is aliquoted into vials and lyophilized.
  • Each vial can contain a single dose or multiple doses of the IgA protease truncation or fusion protein or a combination thereof.
  • the amount loaded into each vial can be slightly higher than that required for each dose or multiple doses (eg, 10% excess) to ensure accurate sampling and accurate dosing.
  • the lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature.
  • the lyophilized powder is redissolved with water for injection to obtain a preparation for injection.
  • the lyophilized powder can be added to sterile pyrogen-free water or other suitable liquid carriers for reconstitution. The precise amount is determined by the chosen therapy and can be determined empirically.
  • the present application provides a method for treating or preventing IgA deposition-related diseases, which comprises administering the IgA protease truncation described in the present application, the fusion described in the present application to a subject in need of treatment or prevention protein or the pharmaceutical composition described in this application.
  • the present application provides a method for treating or preventing IgA deposition-related diseases, which comprises administering IgA protease or a truncated body thereof, a method comprising the IgA protease or a truncated body thereof, to a subject in need of treatment or prevention.
  • a fusion protein of the body or a pharmaceutical composition comprising the IgA protease or its truncated body or the fusion protein, wherein the amino acid sequence of the IgA protease is selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62 , SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, or a combination thereof.
  • the amino acid sequence of the IgA protease is such as SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO: 66. SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, The amino acid sequence formed after removing the signal peptide sequence from the amino acid sequence shown in SEQ ID NO:75 or SEQ ID NO:76.
  • the IgA protease truncation is identical to, for example, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO: 66.
  • polypeptides set forth in SEQ ID NO:75 or SEQ ID NO:76 have at least 70% sequence identity (e.g., have 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).
  • the IgA protease truncation is identical to, for example, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO: 66.
  • polypeptides set forth in SEQ ID NO:75 or SEQ ID NO:76 have at least 70% sequence identity (e.g., have at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84% , at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity), and still maintain the function or activity of IgA protease (eg, proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.).
  • IgA protease e.g, proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.
  • the application provides the IgA protease truncated body described in the application, the fusion protein described in the application or the pharmaceutical composition described in the application in the preparation of medicines for treating or preventing IgA deposition-related diseases the use of.
  • the application provides IgA protease or its truncated body, a fusion protein comprising the IgA protease or its truncated body or a pharmaceutical composition comprising the IgA protease or its truncated body or the fusion protein
  • the amino acid sequence of the IgA protease is selected from the group consisting of: SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO :72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76 or a combination thereof.
  • the amino acid sequence of the IgA protease is such as SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO: 66. SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, The amino acid sequence formed after removing the signal peptide sequence from the amino acid sequence shown in SEQ ID NO:75 or SEQ ID NO:76.
  • the IgA protease truncation is combined with such as SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID
  • the polypeptides shown in NO:74, SEQ ID NO:75 or SEQ ID NO:76 have at least 70% sequence identity (e.g., have 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
  • the IgA protease truncation is identical to, for example, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO: 66.
  • polypeptides set forth in SEQ ID NO:75 or SEQ ID NO:76 have at least 70% sequence identity (e.g., have at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84% , at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity), and still maintain the function or activity of IgA protease (eg, proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.).
  • IgA protease e.g, proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.
  • the present application provides the IgA protease truncation, fusion protein or pharmaceutical composition as described in the present application for treating or preventing IgA deposition-related diseases.
  • the present application provides an IgA protease or a truncated body thereof, a fusion protein comprising the IgA protease or a truncated body thereof, or a fusion protein comprising the IgA protease or a truncated body thereof for use in the treatment or prevention of diseases related to IgA deposition.
  • amino acid sequence of the IgA protease is selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO :73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76 or a combination thereof.
  • the amino acid sequence of the IgA protease is such as SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO: 66. SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, The amino acid sequence formed after removing the signal peptide sequence from the amino acid sequence shown in SEQ ID NO:75 or SEQ ID NO:76.
  • the IgA protease truncated body and such as SEQ ID NO:61, SEQ ID NO: ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO :70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75 or SEQ ID NO:76 have 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%
  • the IgA protease truncation is identical to, for example, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO: 66.
  • polypeptides set forth in SEQ ID NO:75 or SEQ ID NO:76 have at least 70% sequence identity (e.g., have at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84% , at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity), and still maintain the function or activity of IgA protease (eg, proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.).
  • IgA protease e.g, proteolytic activity, enzymatic activity that specifically cleaves IgA, etc.
  • the diseases associated with IgA deposition described herein include IgA nephropathy, dermatitis herpetiformis, Henoch-Schoenlein purpura (also known as IgA vasculitis), Kawasaki disease, purpuric nephritis, IgA vascular Inflammatory kidney damage, IgA rheumatoid factor-positive rheumatoid arthritis, IgA anti-GBM disease, or IgA ANCA-associated vasculitis.
  • the IgA deposition-associated disease described herein is IgA nephropathy.
  • the IgA deposition-associated disease described herein is IgA1 nephropathy.
  • the IgA deposition-associated disease described herein is IgA vasculitis.
  • the IgA deposition-associated disease described herein is Kawasaki disease.
  • the inventors will come from the signal peptide of the N-terminal of the wild-type IgA protease (its amino acid sequence is shown in SEQ ID NO: 1) from Clostridium ramosum (Clostridium ramosum) AK183 strain (that is, the 1st to 30th of SEQ ID NO: 1 amino acid) and the C-terminal transmembrane region plus the intracellular region (i.e., amino acids 1205 to 1234 of SEQ ID NO: 1) were removed, and then the Fc sequence of human IgG1 (HR-CH2-CH3, the amino acid sequence of which was as follows Shown in SEQ ID NO:24) added to the amino acid sequence of the IgA protease that has removed the signal peptide, transmembrane region and intracellular region (that is, the IgA protease composed of the 31st to 1204 amino acids of SEQ ID NO:1 The N-terminus of the truncated body) was used to construct the PET30a-Fc
  • the inventors used the PET30a-Fc-AK183 plasmid as a template to carry out termination mutations and constructed a series of Fc-AK183 truncations to study the shortest active site at the C-terminus of AK183 IgA protease. Based on previous research results, the inventors believe that there is a self-cleavage site between the 730th and 840th amino acids of the AK183 IgA protease. Therefore, the inventors carried out the first round of termination mutations.
  • the termination mutation sites are the four amino acid positions 738, 769, 799 and 834 of AK183 IgA protease. The results are shown in FIG. 1 .
  • the conclusion of the first round of termination mutation is that the shortest active site of the C-terminal of AK183 IgA protease is located between the 768th and 798th amino acids;
  • IgA protease truncated fragments obtained after mutation have no in vitro enzyme cleavage activity, and the amino acid at position 793 terminates the mutation
  • the truncated fragment of AK183(31-792) IgA protease obtained later is still active.
  • the conclusion of the second round of termination mutation is that the shortest active site of the C-terminal of AK183 IgA protease is located between the 787th and 792nd amino acids; The 789th, 790th, 791st or 792nd amino acid positions of the protease, the results are shown in Figure 3, the AK183 (31-788 ), AK183(31-789) IgA protease truncated fragments have no in vitro enzyme cleavage activity, and the AK183(31-790) and AK183(31-791) IgA protease truncated fragments obtained after the 791st or 792nd amino acid termination mutation Short fragments are still active (of which the 791st Possibly due to the protease conformation problem, the activity is not complete, and only shows a slight enzymatic cleavage). Therefore, the third round of termination mutation concluded that the shortest active fragment at the C-terminus of AK183 IgA protease
  • the inventors performed three rounds of truncating mutations to investigate the N-terminal shortest active site of AK183 IgA protease.
  • the inventors performed the first round of truncation mutations, removing a domain of unknown (DUF) at the N-terminus of AK183 on the basis of AK183 (31-792), and fixing the amino acid position at the C-terminus At the 792nd place.
  • the N-terminal DUF corresponding to amino acids 31 to 284 of SEQ ID NO:1 is removed to obtain the AK183(285-792) IgA protease truncated fragment.
  • IgA protease Truncated fragments Take a similar method to obtain AK183 (330-792), AK183 (380-792), AK183 (430-792), AK183 (480-792), AK183 (530-792), AK183 (580-792) IgA protease Truncated fragments.
  • the experimental results of the in vitro enzyme-cleavage activity of the obtained IgA protease truncated fragments on IgA1 are shown in FIG. 10 .
  • AK183(285-792), AK183(330-792) IgA protease truncated fragments still have in vitro enzyme cleavage activity, while AK183(380-792), AK183(430-792), AK183(480- 792), AK183(530-792), AK183(580-792) IgA protease truncated fragments have no in vitro enzyme cleavage activity. Therefore, the conclusion of the first round of truncation mutations is that the N-terminal shortest active site of AK183 IgA protease is located between the 330th and 380th amino acids.
  • the results of the in vitro enzyme digestion activity test of the obtained IgA protease truncated fragments on IgA1 are shown in FIG. 11 .
  • AK183(335-792) IgA protease truncated fragments still have in vitro enzyme cleavage activity, while AK183(340-792), AK183(345-792), AK183(350-792), AK183(355- 792), AK183(360-792), AK183(365-792), AK183(370-792), AK183(375-792) IgA protease truncated fragments have no in vitro enzyme cleavage activity. Therefore, the conclusion of the second round of truncation mutations is that the N-terminal shortest active site of AK183 IgA protease is located between the 335th and 340th amino acids.
  • the inventors carried out the third round of truncation mutations, and constructed truncations one by one between the 335th and 340th amino acids, and obtained AK183 (336-792), AK183 (337-792), AK183 ( 338-792), AK183 (339-792) IgA protease truncated fragments.
  • the results of the in vitro enzyme-cleavage activity test of the obtained IgA protease truncated fragments on IgA1 are shown in FIG. 12 . As shown in FIG.
  • IgA protease truncated fragments have no in vitro enzyme cleavage activity. Therefore, the conclusion of the third round of truncating mutations is: The N-terminal shortest active site of AK183 IgA protease is located at the 335th amino acid.
  • AK183(285-792), AK183(330-792), AK183(335-792) IgA protease truncated fragments still have in vitro enzyme cleavage activity, while AK183(336-792), AK183(337- 792), AK183(338-792), AK183(339-792), AK183(340-792), AK183(345-792), AK183(350-792) IgA protease truncated fragments have no in vitro enzyme cleavage activity, and the aforementioned
  • the conclusions of the three rounds of truncation mutations were consistent, that is, the shortest active site of the N-terminal of AK183 IgA protease was located at the 335th amino acid.
  • AK183 IgA protease the shortest active fragment of AK183 IgA protease is AK183(335-790).
  • Embodiment 2 Preparation comprises the fusion protein of AK183 IgA protease truncated body or AK183 IgA protease full length
  • the inventors used the PET30a-AK183(31-790)-Fc plasmid as a template, and added the 791st and 792nd amino acids behind the AK183(31-790) truncation by PCR to construct the PET30a-AK183(31 -792)-Fc plasmid.
  • the applicant also commissioned Beijing Liuhe Huada Gene Technology Co., Ltd. to construct PET30a-AK183(31-798)-Fc, PET30a-AK183(31-807)-Fc, PET30a-AK183(31-816)-Fc, PET30a-AK183 (31-833)-Fc four alternative subclones, the Fc (CH2-CH3) of the alternative subclones has removed the hinge region, and its amino acid sequence is shown in SEQ ID NO: 6 (SEQ ID NO: 6 and SEQ ID Compared with NO:2, the first 9 amino acids of SEQ ID NO:2 (EPKSCDKTH) are missing, and 10 His are added between the IgA protease truncated body and Fc (located behind the linker GGGGS and in front of Fc).
  • Four alternative subclones were used as alternatives for later protease yield and purity screening.
  • the inventors In order to investigate whether the connection method between the AK183 IgA protease truncated fragment and the Fc region will affect its enzyme-cleaving activity on IgA, the inventors also constructed PET30a-AK183(285-816)-Fc, PET30a-Fc-AK183(285-816 ) These two alternative subclones, wherein the amino acid sequence of Fc is shown in SEQ ID NO:25.
  • the inventors In order to compare the IgA cleavage activity of the fusion protein formed by the truncated fragment of AK183 IgA protease and Fc, and the fusion protein formed by the full length of AK183 IgA protease and Fc, the inventors also constructed an alternative subclone PET30a-Fc-AK183(31- 1203), wherein the amino acid sequence of Fc is as shown in SEQ ID NO:24.
  • the inventors In order to investigate the influence of IgG1 Fc, IgG4 Fc and albumin on the IgA cleavage activity of the fusion protein comprising the AK183 IgA protease truncation, the inventors also constructed PET30a-AK183(31-816)-IgG4 Fc, PET30a-AK183( 31-816)-the two alternative subclones of albumin, wherein the amino acid sequence of IgG4 Fc is shown in SEQ ID NO:77, and the amino acid sequence of albumin is shown in SEQ ID NO:60.
  • the inventors also constructed six alternative subclones PET30a-AK183(285-816)-linker-Fc, which Among the fusion protein sequences expressed by the six alternative subclones, except for the different linkers, the amino acid sequences of AK183 (285-816) and Fc are the same, wherein the amino acid sequence of AK183 (285-816) is shown in SEQ ID NO:46 As shown, the amino acid sequence of Fc is shown in SEQ ID NO: 25, and the amino acid sequences of the linker are HHHHHHHHHH (SEQ ID NO: 59, also known as "10xHis”), EEKKKEKEKEEQEERETK (SEQ ID NO: 58, also known as "IgD linker"), GGGGS (SEQ ID NO: 22, also known as "1xlinker”), GGGGSGGGGS (SEQ ID
  • the amino acid sequence of the AK183 (31-792)-Fc fusion protein expressed by the PET30a-AK183 (31-792)-Fc plasmid is shown in SEQ ID NO: 2, and its encoding nucleic acid sequence is shown in SEQ ID NO: 3;
  • the amino acid sequence of the AK183 (31-798)-Fc fusion protein expressed by the PET30a-AK183 (31-798)-Fc plasmid is shown in SEQ ID NO: 6, and its encoding nucleic acid sequence is shown in SEQ ID NO: 7;
  • the amino acid sequence of the AK183 (31-807)-Fc fusion protein expressed by the PET30a-AK183 (31-807)-Fc plasmid is shown in SEQ ID NO: 8, and its encoding nucleic acid sequence is shown in SEQ ID NO: 9;
  • the amino acid sequence of the AK183 (31-816)-Fc fusion protein expressed by the PET30a-AK183 (31-816)-Fc plasmid is shown in SEQ ID NO: 10, and its encoding nucleic acid sequence is shown in SEQ ID NO: 11;
  • the amino acid sequence of the AK183 (31-833)-Fc fusion protein expressed by the PET30a-AK183 (31-833)-Fc plasmid is shown in SEQ ID NO: 12, and its encoding nucleic acid sequence is shown in SEQ ID NO: 13;
  • the amino acid sequence of the AK183(285-816)-Fc fusion protein expressed by the PET30a-AK183(285-816)-Fc plasmid is shown in SEQ ID NO:81;
  • the amino acid sequence of the Fc-AK183 (285-816) fusion protein expressed by the PET30a-Fc-AK183 (285-816) plasmid is shown in SEQ ID NO: 82;
  • the amino acid sequence of the Fc-AK183 (31-1203) fusion protein expressed by the PET30a-Fc-AK183 (31-1203) plasmid is shown in SEQ ID NO: 83;
  • the amino acid sequence of the AK183(31-816)-IgG4 Fc fusion protein expressed by the PET30a-AK183(31-816)-IgG4 Fc plasmid is shown in SEQ ID NO:84;
  • the amino acid sequence of the AK183(31-816)-albumin fusion protein expressed by the PET30a-AK183(31-816)-albumin particle is shown in SEQ ID NO:85.
  • the obtained fusion protein containing AK183 IgA protease truncated body was mixed with the substrate IgA1 purified from the plasma of IgA nephropathy patients in vitro, reacted at 37°C for 2-12h, and then performed Western blot to verify its enzyme activity to the substrate IgA1 cutting activity.
  • the obtained fusion protein containing the AK183 IgA protease truncated body was injected into the humanized IgA1 alpha chain knock-in ( ⁇ 1KI-Tg) C57BL/6 mice through the tail vein, and collected before injection, 5min, 2h after injection, respectively. 4h, 24h blood samples, and then Western blot verification.
  • PET30a-AK183(31-798)-Fc PET30a-AK183(31-807)-Fc
  • PET30a-AK183(31-816)-Fc PET30a-AK183(31-833)-Fc Both express the fusion protein, and both have in vitro enzyme cleavage activity against IgA1 (as shown in FIG. 7 ).
  • PET30a-AK183(285-816)-Fc and PET30a-Fc-AK183(285-816) both expressed fusion proteins (as shown in Figure 14), and both had in vitro enzyme cleavage activity (as shown in Figure 15 Show).
  • the inventors also verified the AK183(31-807)-Fc fusion protein expressed by the subclone PET30a-AK183(31-807)-Fc, the Fc-AK183(285- 816)
  • the in vivo activity of the fusion protein is shown in Figure 8 (AK183(31-807)-Fc, under reducing conditions) and Figure 17 (Fc-AK183(285-816), under non-reducing conditions).
  • Figure 8 As shown in Figure 8, after humanized IgA1 mice ( ⁇ 1KI-Tg) C57BL/6 received a single-needle tail vein injection of AK183(31-807)-Fc fusion protein, the complete IgA1 heavy chain (H) in the blood was all Disappeared and persisted for at least 24 hours.
  • the inventor also compared the enzymatic cleavage activity of Fc-AK183 (285-816) fusion protein, AK183 (285-816)-Fc fusion protein, and AK183 (285-816) IgA protease truncated body to IgA1, and the results were as follows: Figure 16 shows. As shown in Figure 16, these three proteins all have enzymatic cleavage activity on IgA1.
  • the inventors also compared the enzymatic activity of AK183(285-816)-Fc fusion protein and Fc-AK183(31-1203) fusion protein to IgA1, and the results are shown in FIG. 18 .
  • both the AK183(285-816)-Fc fusion protein and the Fc-AK183(31-1203) fusion protein have enzymatic cleavage activity on IgA1.
  • the inventors also compared AK183(31-816)-IgG1 Fc fusion protein, AK183(31-816)-IgG4 Fc fusion
  • the enzymatic cleavage activity of fusion protein and AK183(31-816)-albumin fusion protein on IgA1 are shown in Figure 19.
  • all three fusion proteins have enzymatic cleavage activity on IgA1.
  • the inventors also compared the enzymatic activity of AK183(285-816)-Fc fusion proteins with different linkers (10xHis, IgD linker, 1xlinker, 2xlinker, 3xlinker or 4xlinker) on IgA1, and the results are shown in Figure 20. As shown in Figure 20, these six fusion proteins all have enzymatic cleavage activity on IgA1.
  • the codon optimization for the eukaryotic expression system was carried out on the Fc sequence.
  • the amino acid sequence of the IL2-AK183 (31-792)-Fc fusion protein expressed by pcDNA3.1/hygro (+)-IL2-AK183 (31-792)-Fc is shown in SEQ ID NO: 4, and its coding nucleic acid sequence is shown in Shown in SEQ ID NO:5.
  • the expression results of AK183(31-792)-Fc fusion protein in HEK293 cells are shown in FIG. 9 .
  • the results showed that the AK183(31-792)-Fc fusion protein had expected full-length expression, and the fusion protein expressed in the eukaryotic system existed in dimer form.
  • Embodiment 3 Preparation and activity test of AK183 IgA protease mutant
  • the inventor screened several amino acid sequences with a certain homology to the wild-type IgA enzyme of AK183 from the metagenomic database, and synthesized 16 AK183 homologous enzymes. Their amino acid sequences are respectively shown in SEQ ID NO:61 ⁇ SEQ ID NO:76. According to the in vitro activity test method described in Example 2.3, the inventors respectively tested the enzymatic activity of these AK183 homologous enzymes on IgA1. The results are shown in Figures 23a and 23b.

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Abstract

本申请涉及一种IgA蛋白酶截短体、包含IgA蛋白酶截短体的融合蛋白(例如，包含IgA蛋白酶截短体和Fc的融合蛋白)及其在治疗IgA沉积疾病(例如，IgA肾病)中的用途。

Description

IgA蛋白酶截短体、包含IgA蛋白酶截短体的融合蛋白及其用途 技术领域
本申请涉及生物医药领域，具体地，本申请涉及一种IgA蛋白酶截短体、包含IgA蛋白酶截短体的融合蛋白、包含所述IgA蛋白酶截短体或所述融合蛋白的药物组合物、编码所述IgA蛋白酶截短体或所述融合蛋白的核酸、所述IgA蛋白酶截短体或所述融合蛋白的制备方法、以及IgA蛋白酶截短体或所述融合蛋白在制备用于治疗IgA沉积相关疾病的药物中的用途。
背景技术
IgA肾病是目前世界上最常见的原发性肾小球疾病之一，给患者和社会带来沉重的负担。目前针对IgA肾病尚缺乏特异性的治疗。临床上多用RAS阻断剂为基础的支持治疗，以减缓肾功能恶化。对于支持治疗无效的患者予以联合激素免疫抑制剂治疗。但激素免疫抑制剂的使用长期疗效不佳且给患者带来严重的副作用。
亟需开发有效并且具有低副作用的治疗药物。
发明简述
在一个方面，本申请提供了一种分离的IgA蛋白酶截短体，其包含获得自或衍生自多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的非天然截短片段，或者与所述非天然截短片段具有至少70％的序列同一性。在某些实施方式中，所述非天然截短片段在所述多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的基础上有氨基酸取代、缺失、插入或修饰，使得所述IgA蛋白酶截短体丧失或降低自酶切功能。在某些实施方式中，所述氨基酸取代、缺失、插入或修饰发生在所述多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的天然自酶切位点、所述天然自酶切位点的上游5个位点以内和/或下游5个位点以内。在某些实施方式中，所述多枝梭菌(Clostridium ramosum)为Clostridium ramosum AK183株。在某些实施方式中，所述多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的氨基酸序列 如SEQ ID NO:1所示。在某些实施方式中，所述天然自酶切位点在如SEQ ID NO:1所示的氨基酸序列的第730位至第840位之间(例如，第792位至第797位之间)。在某些实施方式中，所述天然自酶切位点在如SEQ ID NO:1所示的氨基酸序列的第790位、第791位、第792位、第793位、第794位、第795位、第796位、第797位、第798位、第799位或第800位。
在某些实施方式中，所述非天然截短片段为获得自或衍生自多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的N末端截短片段或C末端截短片段。在某些实施方式中，所述N末端截短片段包含获得自或衍生自多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的N末端第31位起的至少760个连续氨基酸的多肽片段，或者与所述多肽片段具有至少70％的序列同一性。在某些实施方式中，本申请提供的IgA蛋白酶截短体包含如SEQ ID NO:1所示的氨基酸序列的第31位起的至少760个(例如，至少761个、至少762个、至少763个、至少764个、至少765个、至少766个、至少767个、至少768个、至少769个、至少770个、至少771个、至少772个、至少773个、至少774个、至少775个、至少776个、至少777个、至少778个、至少779个、至少780个、至少781个、至少782个、至少783个、至少784个、至少785个、至少786个、至少787个、至少788个、至少789个、至少790个、至少791个、至少792个、至少793个、至少794个、至少795个、至少796个、至少797个、至少798个、至少799个、至少800个、至少801个、至少802个、至少803个、至少804个、至少805个、至少806个、至少807个、至少808个、至少809个、至少810个、至少900个、至少950个、至少1000个、至少1100个、至少1150个或者至少1200个)连续氨基酸的多肽片段。在某些实施方式中，本申请提供的IgA蛋白酶截短体包含选自下组的多肽片段：如SEQ ID NO:1所示的氨基酸序列的第31位至第790位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第31位至第792位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第31位至第798位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第31位至第807位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第31位至第816位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第31位至第833位氨基酸以及与其具有至少70％的序列同一性的多肽片段。
在某些实施方式中，所述非天然截短片段包含获得自或衍生自多枝梭菌 (Clostridium ramosum)的野生型IgA蛋白酶的N末端第335位起的至少456个连续氨基酸的多肽片段，或者与所述多肽片段具有至少90％或至少95％的序列同一性。在某些实施方式中，本申请提供的IgA蛋白酶截短体包含如SEQ ID NO:1所示的氨基酸序列的第335位起的至少456个(例如，至少457个、至少458个、至少459个、至少460个、至少461个、至少462个、至少463个、至少464个、至少465个、至少466个、至少467个、至少468个、至少469个、至少470个、至少471个、至少472个、至少473个、至少474个、至少475个、至少476个、至少477个、至少478个、至少479个、至少480个、至少481个、至少482个、至少483个、至少484个、至少485个、至少486个、至少487个、至少488个、至少489个、至少490个、至少491个、至少492个、至少493个、至少494个、至少495个、至少496个、至少497个、至少498个、至少499个、至少500个、至少550个、至少600个、至少650个、至少700个、至少750个、至少800个、至少850个或者至少900个)连续氨基酸的多肽片段。在某些实施方式中，本申请提供的IgA蛋白酶截短体包含选自下组的多肽片段：如SEQ ID NO:1所示的氨基酸序列的第335位至第790位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第335位至第791位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第335位至第792位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第285位至第790位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第285位至第791位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第285位至第792位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第330位至第790位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第330位至第791位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第330位至第792位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第285位至第816位氨基酸以及与其具有至少90％或至少95％的序列同一性的多肽片段。
在某些实施方式中，本申请提供的IgA蛋白酶截短体在所述多肽片段的氨基酸序列基础上，在一个或多个位点具有氨基酸的保守替换。在某些实施方式中，所述多肽片段在对应于SEQ ID NO:1的第844位、第862位、第931位、第933位、第978位、第1002位、第1004位中的一个或多个位置有氨基酸突变。在某些实施方式中，所述多肽片段在对应于SEQ ID NO:1的第844位、第862位、第931位、第933位、第978位、第1002位、第1004位中的一个或多个位置突变为甘氨酸。 在某些实施方式中，所述多肽片段在对应于SEQ ID NO:1的第844位有氨基酸突变、第862位有氨基酸突变、第931位和第933位有氨基酸突变、第978位有氨基酸突变、或者第1002位和第1004位有氨基酸突变。在某些实施方式中，所述多肽片段的氨基酸序列如SEQ ID NO:53(也称为“PA-GA Mut”)、SEQ ID NO:54(也称为“PI-GI Mut”)、SEQ ID NO:55(也称为“PAP-GAG Mut”)、SEQ ID NO:56(也称为“PAT-GAT Mut”)或者SEQ ID NO:57(也称为“PIP-GIG Mut”)所示。
在某些实施方式中，本申请提供的IgA蛋白酶截短体具有特异性切割人IgA的酶活性。在某些实施方式中，本申请提供的IgA蛋白酶截短体具有特异性切割人IgA重链的酶活性。在某些实施方式中，本申请提供的IgA蛋白酶截短体具有特异性切割人IgA重链CH1与铰链区交汇处的酶活性。在某些实施方式中，本申请提供的IgA蛋白酶截短体具有特异性切割人IgA1的酶活性。
在另一方面，本申请提供了一种融合蛋白，其包含第一多肽和第二多肽，其中所述第一多肽包含获得自或衍生自多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的全长、获得自或衍生自多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶去掉信号肽之后形成的多肽或者如本申请所述的IgA蛋白酶截短体，所述第二多肽包含用于延长所述第一多肽在受试者体内半衰期的氨基酸序列。在某些实施方式中，所述第一多肽包含如SEQ ID NO:1或者SEQ ID NO:42所示的序列。在某些实施方式中，所述第二多肽位于所述第一多肽的N末端或者C末端。
在某些实施方式中，所述第一多肽和所述第二多肽之间通过连接子连接。在某些实施方式中，所述第一多肽和所述第二多肽之间直接连接。在某些实施方式中，所述连接子选自下组：可切割连接子、不可切割连接子、肽连接子、柔性连接子、刚性连接子、螺旋连接子和非螺旋连接子。在某些实施方式中，所述连接子包括肽连接子。在某些实施方式中，所述肽连接子包括含有甘氨酸和丝氨酸的连接子。在某些实施方式中，所述含有甘氨酸和丝氨酸的连接子包括如SEQ ID NO:21(GGGS)、SEQ ID NO:22(GGGGS)、SEQ ID NO:86(GGGGGS)或SEQ ID NO:87(GGGGGGGS)所示的一个、两个、三个、四个或更多个重复。在某些实施方式中，所述连接子包括如SEQ ID NO:23(GGCGGCGGTGGATCC)所示、如SEQ ID NO:58(EEKKKEKEKEEQEERETK)所示或者如SEQ ID NO:59(HHHHHHHHHH)所示的氨基酸序列。
在某些实施方式中，所述第二多肽选自：Fc结构域和白蛋白。在某些实施方式中，所述Fc结构域包含铰链区。在某些实施方式中，所述Fc结构域来源于人IgG Fc结构域。在某些实施方式中，所述Fc结构域来源于人IgG1 Fc结构域、人IgG2Fc结构域、人IgG3 Fc结构域或人IgG4 Fc结构域。在某些实施方式中，所述Fc结构域包含与SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:32或SEQ ID NO:77具有至少70％、至少75％、至少80％、至少85％、至少90％或至少95％的序列同一性的氨基酸序列。在某些实施方式中，所述Fc结构域包含如SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:32或SEQ ID NO:77所示的氨基酸序列。在某些实施方式中，所述Fc结构域在对应于SEQ ID NO:25的第7位有氨基酸突变。在某些实施方式中，所述Fc结构域在对应于SEQ ID NO:25的第7位的氨基酸(例如，丙氨酸)突变为缬氨酸、甘氨酸、丝氨酸或者亮氨酸。在某些实施方式中，所述Fc结构域包含一个或多个延长所述融合蛋白的半衰期的突变。在某些实施方式中，所述Fc结构域与所述第一多肽的C末端或者N末端连接。在某些实施方式中，所述白蛋白包含人血清白蛋白的一个或多个结构域。在某些实施方式中，所述白蛋白包含人血清白蛋白的D3结构域。
在某些实施方式中，本申请所述的融合蛋白进一步包含标签。在某些实施方式中，所述标签选自下组：荧光标签、发光标签、纯化标签和生色标签。在某些实施方式中，所述标签选自下组：c-Myc标签、HA标签、VSV-G标签、FLAG标签、V5标签和HIS标签。在某些实施方式中，所述标签是包含6个、7个、8个、9个或10个组氨酸的HIS标签。在某些实施方式中，所述第二多肽位于所述第一多肽的C末端，所述标签位于所述第二多肽的C末端。
在某些实施方式中，本申请所述的融合蛋白在受试者体内的血循环中的半衰期为至少1天、至少2天、至少3天、至少4天、至少5天、至少6天、至少7天、至少8天、至少9天、至少10天、至少11天、至少12天、至少13天、至少14天。
在另一方面，本申请提供了一种分离的核酸，其包含编码本申请所述的IgA蛋白酶截短体的核苷酸序列或包含编码本申请所述的融合蛋白的核苷酸序列。在某些实施方式中，本申请所述的核酸包含选自下组的核苷酸序列：SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ  ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38以及与其具有至少70％的序列同一性的核苷酸序列。
在另一方面，本申请提供了一种载体，其包含本申请所述的核酸。
在另一方面，本申请提供了一种细胞，其包含本申请所述的核酸或如本申请所述的载体。在某些实施方式中，所述细胞是原核生物细胞或真核生物细胞。在某些实施方式中，所述原核生物细胞是大肠杆菌细胞。在某些实施方式中，所述真核生物细胞是哺乳动物细胞。在某些实施方式中，所述哺乳动物细胞是人细胞或中国仓鼠卵巢(CHO)细胞。在某些实施方式中，所述哺乳动物细胞是人胚胎肾细胞293(HEK293细胞)。
在另一方面，本申请提供了一种药物组合物，其包含本申请所述的IgA蛋白酶截短体、包含本申请所述的融合蛋白、包含本申请所述的核酸、包含本申请所述的载体或者包含本申请所述的细胞，以及药学上可接受的载体。
在另一方面，本申请提供了一种产生融合蛋白的方法，其包括培养本申请所述的细胞的步骤。
在另一方面，本申请提供了一种治疗或预防IgA沉积相关疾病的方法，其包括向需要治疗或预防的受试者施用如本申请所述的IgA蛋白酶截短体、如本申请所述的融合蛋白或者如本申请所述的药物组合物。
在另一方面，本申请提供了如本申请所述的IgA蛋白酶截短体、如本申请所述的融合蛋白或者如本申请所述的药物组合物在制备用于治疗或预防IgA沉积相关疾病的药物中的用途。
在另一方面，本申请提供了用于治疗或预防IgA沉积相关疾病的如本申请所述的IgA蛋白酶截短体、如本申请所述的融合蛋白或者如本申请所述的药物组合物。
在另一方面，本申请提供了一种治疗或预防IgA沉积相关疾病的方法，其包括向需要治疗或预防的受试者施用IgA蛋白酶或其截短体、包含所述IgA蛋白酶或其截短体的融合蛋白或者包含所述IgA蛋白酶或其截短体或者所述融合蛋白的药物组合物，其中，所述IgA蛋白酶的氨基酸序列选自下组：SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66、 SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76或其组合。
在另一方面，本申请提供了IgA蛋白酶或其截短体、包含所述IgA蛋白酶或其截短体的融合蛋白或者包含所述IgA蛋白酶或其截短体或者所述融合蛋白的药物组合物在制备用于治疗或预防IgA沉积相关疾病的药物中的用途，其中，所述IgA蛋白酶的氨基酸序列选自下组：SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76或其组合。
在另一方面，本申请提供了用于治疗或预防IgA沉积相关疾病的IgA蛋白酶或其截短体、包含所述IgA蛋白酶或其截短体的融合蛋白或者包含所述IgA蛋白酶或其截短体或者所述融合蛋白的药物组合物，其中，所述IgA蛋白酶的氨基酸序列选自下组：SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76或其组合。
在某些实施方式中，所述IgA沉积相关疾病包括IgA肾病、疱疹样皮炎、亨诺赫-舍恩莱因紫癜(又称IgA血管炎)、川崎病、紫癜性肾炎、IgA血管炎肾损害、IgA类风湿因子阳性的类风湿性关节炎、IgA型抗GBM病或IgA型ANCA相关血管炎。在某些实施方式中，所述IgA沉积相关疾病为IgA肾病、IgA血管炎或川崎病。
附图描述
图1显示了AK183(31-737)、AK183(31-768)、AK183(31-798)和AK183(31-833)这四种IgA蛋白酶截短体对IgA1的体外酶切活性实验结果。
图2显示了AK183(31-773)、AK183(31-778)、AK183(31-782)、AK183(31-787)和AK183(31-792)这五种IgA蛋白酶截短体对IgA1的体外酶切活性实验结果。
图3a-c显示了AK183(31-788)、AK183(31-789)、AK183(31-790)和AK183(31-791)这四种IgA蛋白酶截短体对IgA1的体外酶切活性实验结果。
图4显示了PET30a-AK183(31-790)-Fc质粒的构建流程图。
图5显示了AK183(31-790)-Fc融合蛋白的表达结果图。
图6a显示了AK183(31-792)-Fc融合蛋白的表达结果图，图6b显示了AK183(31-792)-Fc融合蛋白对IgA1的体外酶切活性实验结果。
图7显示了AK183(31-798)-Fc、AK183(31-807)-Fc、AK183(31-816)-Fc和AK183(31-833)-Fc这四种融合蛋白对IgA1的体外酶切活性实验结果。
图8显示了AK183(31-807)-Fc融合蛋白对IgA1的体内酶切活性实验结果。
图9显示了AK183(31-792)-Fc融合蛋白在HEK293细胞中的表达结果图。
图10显示了AK183(285-792)、AK183(330-792)、AK183(380-792)、AK183(430-792)、AK183(480-792)、AK183(530-792)和AK183(580-792)这7种IgA蛋白酶截短体对IgA1的体外酶切活性实验结果。
图11显示了AK183(335-792)、AK183(340-792)、AK183(345-792)、AK183(350-792)、AK183(355-792)、AK183(360-792)、AK183(365-792)、AK183(370-792)和AK183(375-792)这9种IgA蛋白酶截短体对IgA1的体外酶切活性实验结果。
图12显示了AK183(336-792)、AK183(337-792)、AK183(338-792)和AK183(339-792)这四种IgA蛋白酶截短体对IgA1的体外酶切活性实验结果。
图13显示了再次验证AK183(285-792)、AK183(330-792)、AK183(335-792)、AK183(336-792)、AK183(337-792)、AK183(338-792)、AK183(339-792)、AK183(340-792)、AK183(345-792)和AK183(350-792)这10种IgA蛋白酶截短体对IgA1的体外酶切活性实验结果。
图14显示了AK183(285-816)-Fc和Fc-AK183(285-816)这两种融合蛋白的表达结果图。
图15显示了AK183(285-816)-Fc和Fc-AK183(285-816)这两种融合蛋白对IgA1 的体外酶切活性实验结果。
图16显示了Fc-AK183(285-816)融合蛋白、AK183(285-816)-Fc融合蛋白和AK183(285-816)IgA蛋白酶截短体对IgA1的酶切活性实验结果。
图17显示了Fc-AK183(285-816)融合蛋白对IgA1的体内酶切活性实验结果。
图18显示了AK183(285-816)-Fc融合蛋白和Fc-AK183(31-1203)融合蛋白对IgA1的酶切活性实验结果。
图19显示了AK183(31-816)-IgG1 Fc融合蛋白、AK183(31-816)-IgG4 Fc融合蛋白和AK183(31-816)-白蛋白融合蛋白对IgA1的酶切活性实验结果。
图20显示了具有不同连接子(SEQ ID NO:59、SEQ ID NO:58、SEQ ID NO:22、SEQ ID NO:78、SEQ ID NO:79或SEQ ID NO:80)的AK183(285-816)-Fc融合蛋白对IgA1的酶切活性实验结果。
图21显示了如SEQ ID NO:53、SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:56或SEQ ID NO:57所示的五种IgA蛋白酶截短体的突变体对IgA1的酶切活性实验结果。
图22显示了对AK183(31-816)-Fc融合蛋白的Fc区分别进行4种不同突变形成的四种突变体对IgA1的酶切活性实验结果。
图23a和图23b显示了16种AK183同源酶对IgA1的酶切活性实验结果。
发明详述
尽管本申请将在以下公开多个方面和实施方式，但是在不违背本申请主题精神和范围的前提下，本领域技术人员显然可以对其进行各种等同改变和修改。本申请公开的多个方面和实施方式仅用于举例说明，其并非旨在限制本申请，本申请的实际保护范围以权利要求为准。除非另外指出，本文中使用的所有技术和科学术语均具有与本申请所属领域中的普通技术人员通常所理解的相同的含义。本申请中引用的所有参考文献、专利、专利申请均通过整体引用并入本文。
定义
本申请中使用的术语“多枝梭菌”或“梭状芽孢杆菌”均指的是Clostridium ramosum菌，也称为Ramibacterium ramosum，它是一种人体肠道共生菌，可以产生IgA蛋白酶。
本申请中使用的术语“蛋白酶”指的是具有分解蛋白质和肽的能力的酶。蛋白酶可以通过在形成蛋白质的肽或多肽链中将氨基酸连接在一起的肽键进行水解，从而分解蛋白质。现有技术中已知多种方法测试某种蛋白酶的蛋白水解活性。例如，可以通过分析各种蛋白酶水解合适底物的能力的比较测定来确定蛋白酶的蛋白水解活性。用于蛋白水解活性分析的示例性底物包括，例如，二甲基酪蛋白、牛胶原、牛弹性蛋白等等。使用这些底物的比色测定也是现有技术中已知的(参见，例如WO99/34011和US 6,376,450)。
本申请中使用的术语“IgA蛋白酶”是指能够特异性切割或分解受试者(例如，人)的IgA免疫球蛋白分子(例如，IgA1或IgA2)的酶。例如，获得自或衍生自多枝梭菌(Clostridium ramosum)的IgA蛋白酶能够特异性切割IgA1和IgA2的第221位脯氨酸(Pro)和第222位缬氨酸(Val)之间的肽键，从而分解IgA1和IgA2。
当提及的是多肽或蛋白质时，本申请中使用的术语“野生型”指的是在一个或多个氨基酸位置处不包括人为的取代、插入、缺失或修饰的天然存在的多肽或蛋白质；当提及的是核酸、核苷酸或多核苷酸时，本申请中使用的术语“野生型”指的是在一个或多个核苷酸位置处不包括人为的替换、插入、缺失或修饰的天然存在的核酸、核苷酸或多核苷酸。但是编码野生型多肽的多核苷酸不仅限于天然存在的多核苷酸，也包括编码野生型多肽的任何多核苷酸。
本申请中使用的术语“AK183”是指多枝梭菌(Clostridium ramosum)的AK183株。Clostridium ramosum AK183株产生的野生型IgA蛋白酶的氨基酸序列如SEQ ID NO:1所示(其中，第1-30位氨基酸为信号肽)。

本申请中使用的术语“信号肽”是指可以参与蛋白质的成熟或前体形式的分泌或定向转运的氨基酸残基序列。信号肽通常位于前体或成熟蛋白序列的N末端。信号肽可以是内源的或外源的。成熟蛋白中一般不存在信号肽。通常情况下，在蛋白转运后，信号肽通过信号肽酶从所述蛋白中被切割掉。例如，SEQ ID NO:1所示的氨基酸序列去除掉N末端的信号肽之后形成的氨基酸序列如SEQ ID NO:42所示。

本申请中使用的术语“受试者”包括人类和非人类的动物。非人类的动物包括所有的脊椎动物，例如哺乳动物和非哺乳动物。“受试者”也可以是家畜动物，例如牛、猪、羊、家禽和马；或啮齿类动物，例如大鼠、小鼠；或灵长类动物，例如猿(ape)、猴子、黑猩猩(chimpanzee)、大猩猩(gorilla)、猩猩(orangutan)、狒狒(baboon)；或家养动物，例如狗和猫。“受试者”可以是雄性或者雌性，可以是老年、成年、青少年、儿童或者婴儿。人类“受试者”可以是高加索人、非洲人、亚洲人、闪族人，或其他种族或所述种族背景的混合。
本申请中使用的术语“蛋白”、“多肽”以及“肽”可以互换使用，是指氨基酸的聚合物。本申请所述的蛋白、多肽或肽可以含有天然的氨基酸，也可以含有非天然的氨基酸，或氨基酸的类似物、模拟物。本申请所述的蛋白、多肽或肽可以通过本领域公知的任何方法获得，例如但不限于，通过天然分离、重组表达、化学合成等。
本申请所用的术语“氨基酸”是指含有氨基(-NH2)和羧基(-COOH)官能团以及每个氨基酸特有的侧链的有机化合物。氨基酸名称在本申请中也以标准的单字母或三字母代码表示，总结如下：
在本申请中当“保守替换”用于氨基酸序列时，是指将一个氨基酸残基用另一个具有相似理化性质的侧链的氨基酸残基替代。例如，可以在具有疏水侧链的氨基酸残基之间(例如Met、Ala、Val、Leu和Ile)、具有中性亲水侧链的氨基酸残基之间(例如Cys、Ser、Thr、Asn和Gln)、具有酸性侧链的氨基酸残基之间(例如Asp、Glu)、具有碱性侧链的氨基酸残基之间(例如His、Lys和Arg)或具有芳香侧链的氨基酸残基之间(例如Trp、Tyr和Phe)进行保守替换。本领域已知，保守替换通常不会引起蛋白构象结构的显著变化，因此能够保留蛋白质的生物活性。
本申请所述的术语“同源的”指当最佳比对时核酸序列(或其互补链)或氨基酸序列与另一条序列具有至少60％(例如，至少65％、70％、75％、80％、85％、88％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)的序列同一性。
当“百分比(％)序列同一性”用于氨基酸序列(或核酸序列)时，是指在进行序列比对，并且必要时引入间隔使相同氨基酸(或核酸)数目达到最多后，在候选序列中，与参比序列相同的氨基酸(或核酸)残基占所述候选序列的氨基酸(或核酸)残基的百分比。换言之，可以通过用与其比较的参比序列相同的氨基酸残基(或碱基)数除以候选序列或参比序列(以较短者为准)中的氨基酸残基(或碱基)总数来计算氨基酸序列(或核酸序列)的百分比(％)序列同一性。所述氨基酸残基的保守替换可以认为或可以不认为是相同残基。可以通过本领域公开的工具，例如BLASTN、BLASTp(美国国家生物技术信息中心网站(NCBI)，也可参见Altschul S.F.et al.，J.Mol.Biol.，215：403–410(1990)；Stephen F.et al.，Nucleic Acids Res.，25：3389–3402(1997))、ClustalW2(欧洲生物信息研究所网站，可参见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))和ALIGN或Megalign(DNASTAR)软件，对序列进行比对以确定氨基酸(或核酸)序列的百分比序列同一性。本领域技术人员可以使用所述工具的默认参数或根据比对的需要适当调整参数，例如通过挑选合适的算法。
“分离的”物质已经经人工由自然状态改变。如果自然界中出现某种“分离的”组合物或物质，那么其已经被改变或脱离其原始状态，或二者均有发生。例如，某一活体动物体内天然存在的多核苷酸或多肽不是“分离的”，但如果这些多核苷酸或多肽与之在天然状态下共存的物质足够分离并以基本上纯的状态存在，则可以认为是“分离的”。“分离的核酸序列”是指分离的核酸分子的序列。在一些实施方式中，“分离的IgA蛋白酶截短体”是指纯度为至少60％、70％、75％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的IgA蛋白酶截短体，其中纯度由电泳方法(例如，SDS-PAGE、等电聚焦、毛细管电泳)，或色谱法(例如，离子交换色谱或反相HPLC)确定。
本申请中的术语“载体”是指可将遗传元件操作性地插入其中并使该遗传元件获得表达的一种运载工具，例如生产由该遗传元件编码的蛋白质、RNA或DNA，或者复制所述遗传元件。载体可用于转化、转导或转染宿主细胞，使其携带的遗传元件在宿主细胞内得以表达。举例来说，载体包括：质粒、噬菌粒、柯斯质粒(cosmid)、人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1衍生的人 工染色体(PAC)、噬菌体如λ噬菌体或M13噬菌体，以及动物病毒等。载体可含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体还可包括协助其进入细胞的成分，包括但不限于，病毒颗粒、脂质体或蛋白外壳。载体可以是表达载体或克隆载体。本申请提供的载体(例如表达载体)含有本申请所述的编码IgA蛋白酶截短体或融合蛋白的核酸序列、至少一个可操作地连接至所述核酸序列的启动子(例如，SV40、CMV、EF-1α)，以及至少一个选择标记。
本申请中使用的对某种疾病、病症或状况的“治疗”或“疗法”包括预防或减轻某种疾病、病症或状况，降低某种疾病、病症或状况发生或发展的速度，降低发展出某种疾病、病症或状况的风险，预防或延迟与某种疾病、病症或状况相关的症状发展，减少或终止与某种疾病、病症或状况相关的症状，产生某种疾病、病症或状况的完全或部分的逆转，治愈某种疾病、病症或状况，或以上的组合。
术语“药学上可接受的”表示指定的载体、媒介、稀释剂、赋形剂和/或盐通常在化学和/或物理上与组成该制剂的其他成分相容，并且在生理上与其受体相容。
术语“IgA沉积相关疾病”是指与IgA免疫球蛋白以聚集或非聚集形式在受试者的组织或器官中积累相关的疾病。例如，包括但不限于，IgA肾病、疱疹样皮炎、亨诺赫-舍恩莱因紫癜(又称IgA血管炎)、川崎病、紫癜性肾炎、IgA血管炎肾损害、IgA类风湿因子阳性的类风湿性关节炎、IgA型抗GBM病或IgA型ANCA相关血管炎。
术语“IgA肾病”是指肾脏内以IgA沉积为特征的肾脏疾病。
IgA蛋白酶截短体
在一个方面，本申请提供了一种分离的IgA蛋白酶截短体，其包含获得自或衍生自多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的非天然截短片段，或者与所述非天然截短片段具有至少70％的序列同一性(例如，具有至少75％、至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性)。在 某些实施方式中，与所述非天然截短片段具有至少70％的序列同一性(例如，具有至少75％、至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性)的IgA蛋白酶截短体仍然保持IgA蛋白酶的功能或活性(例如，蛋白水解活性、特异性切割IgA的酶活性等)。
本申请中使用的术语“截短体”或“截短片段”是指从野生型多肽的一端或两端去除一个或多个氨基酸之后形成的肽。因此，本申请中的“截短体”或“截短片段”并不包括其对应的野生型多肽的全长，但是可以与野生型多肽的截短形式相比有一个或多个氨基酸取代、缺失、插入或修饰等。例如，“IgA蛋白酶截短体”或“IgA蛋白酶截短片段”可以包括从野生型的IgA蛋白酶的一端或者两端去除一个或多个氨基酸之后形成的肽，也可以包括与野生型的IgA蛋白酶的截短形式相比有一个或多个氨基酸取代、缺失、插入或修饰的肽。
在某些实施方式中，本申请所述的IgA蛋白酶截短体与其对应的野生型IgA蛋白酶相比有一个或多个氨基酸取代、缺失、插入或修饰。例如，在某些实施方式中，本申请所述的IgA蛋白酶截短体包含获得自或衍生自多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的非天然截短片段，其中所述非天然截短片段在所述多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的基础上有氨基酸取代、缺失、插入或修饰，使得所述IgA蛋白酶截短体丧失或降低自酶切功能。
本申请中使用的术语“获得自”和“衍生自”不仅包括由所提及的生物体生产或可由其生产的蛋白质，而且还包括由从此类生物体分离的DNA序列编码并且在含有此类DNA序列的宿主生物体中生产的蛋白质，也包括由合成的和/或cDNA来源的DNA序列编码并且具有所提及的蛋白质的鉴定特征的蛋白质。例如，获得自或衍生自多枝梭菌的野生型IgA蛋白酶既包括由多枝梭菌天然生产的IgA蛋白酶，也包括通过使用遗传工程技术由用编码IgA蛋白酶的核酸转化的其他宿主细胞(例如，大肠杆菌)生产的IgA蛋白酶。
本申请中使用的术语“非天然截短片段”是指与多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶在自然环境下自酶切之后形成的截短片段具有不同的氨基酸 序列(例如，不同的氨基酸长度、不同的氨基酸类型等等)的片段。
在某些实施方式中，所述氨基酸取代、缺失、插入或修饰发生在所述多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的天然自酶切位点。在某些实施方式中，所述氨基酸取代、缺失、插入或修饰发生在所述多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的天然自酶切位点的上游5个位点以内(例如，在所述天然自酶切位点的上游1个位点、2个位点、3个位点、4个位点或5个位点)。在某些实施方式中，所述氨基酸取代、缺失、插入或修饰发生在所述多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的天然自酶切位点的下游5个位点以内(例如，在所述天然自酶切位点的下游1个位点、2个位点、3个位点、4个位点或5个位点)。在某些实施方式中，所述氨基酸取代、缺失、插入或修饰发生在所述多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的天然自酶切位点的上游5个位点(例如，在所述天然自酶切位点的上游1个位点、2个位点、3个位点、4个位点或5个位点)以内和下游5个位点以内(例如，在所述天然自酶切位点的下游1个位点、2个位点、3个位点、4个位点或5个位点)。
在某些实施方式中，所述非天然截短片段为获得自或衍生自多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的N末端截短片段或C末端截短片段。
本申请中使用的术语“N末端截短片段”是指包括多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的氨基端的氨基酸序列的截短片段。“氨基端”的起始位置可以是靠近多枝梭菌的野生型IgA蛋白酶的氨基酸序列的氨基端的任何位置，例如，可以是从氨基端数起的第1位，也可以是从氨基端数起的其他位置。再例如，如果野生型IgA蛋白酶全长的氨基酸序列由1000个氨基酸组成，那么它的N末端截短片段的氨基端起始位置可以是它的氨基酸序列从氨基端起第1位至第500位之间的任何位置。
本申请中使用的术语“C末端截短片段”是指包括多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的羧基端的氨基酸序列的截短片段。“羧基端”的终止位置可以是靠近多枝梭菌的野生型IgA蛋白酶的氨基酸序列的羧基端的任何位置，例如，可以是从羧基端数起的第1位，也可以是从羧基端数起的其他位置。再 例如，如果野生型IgA蛋白酶全长的氨基酸序列由1000个氨基酸组成，那么它的C末端截短片段的羧基端终止位置可以是它的氨基酸序列从氨基端起第501位至第1000位之间的任何位置。
多枝梭菌(Clostridium ramosum)为Clostridium属中的众多菌种(species)之一，包括多种菌株(strain)，例如AK183、VPI-0496A、NCTC 10474菌株等。在某些实施方式中，所述多枝梭菌(Clostridium ramosum)为Clostridium ramosum AK183株。
在某些实施方式中，所述N末端截短片段包含获得自或衍生自多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的N末端第31位起的至少760个连续氨基酸的多肽片段，或者与所述多肽片段具有至少70％的序列同一性(例如，具有至少75％、至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性)。在某些实施方式中，与所述多肽片段具有至少70％的序列同一性(例如，具有至少75％、至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性)的N末端截短片段仍然保持IgA蛋白酶的功能或活性(例如，蛋白水解活性、特异性切割IgA的酶活性等)。
在某些实施方式中，本申请所述的IgA蛋白酶的非天然截短片段包含获得自或衍生自多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的N末端第335位起的至少456个连续氨基酸的多肽片段，或者与所述多肽片段具有至少90％或至少95％的序列同一性(例如，至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性)。在某些实施方式中，与所述多肽片段具有至少90％或至少95％的序列同一性(例如，至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性)的非天然截短片段仍然保持IgA蛋白酶的功能或活性(例如，蛋白水解活性、特异性切割IgA的酶活性等)。
在某些实施方式中，所述多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的氨基酸序列如SEQ ID NO:1所示。
除特殊说明外，本申请所提到的IgA蛋白酶的氨基酸位点即为对应于野生型AK183 IgA蛋白酶(其氨基酸序列如SEQ ID NO:1所示)的氨基酸位点。例如，本申请所提到的AK183 IgA蛋白酶的第790位对应于SEQ ID NO:1的第790个位点。除特殊说明外，本申请所提到的AK183 IgA蛋白酶截短体的命名规则为AK183(对应于SEQ ID NO:1的起始位点-对应于SEQ ID NO:1的终止位点)。例如，AK183(31-790)指的是SEQ ID NO:1的第31位至第790位氨基酸形成的IgA蛋白酶截短体。
在某些实施方式中，申请所述的IgA蛋白酶的天然自酶切位点在如SEQ ID NO:1所示的氨基酸序列的第730位至第840位之间。在某些实施方式中，本申请所述的IgA蛋白酶的天然自酶切位点在如SEQ ID NO:1所示的氨基酸序列的第710位至830位之间、第720位至第820位之间、第730位至810位之间、第740位至800位之间、第750位至第790位之间、第791位至第780位之间或者第792位至第797位之间。在某些实施方式中，所述天然自酶切位点在如SEQ ID NO:1所示的氨基酸序列的第790位、第791位、第792位、第793位、第794位、第795位、第796位、第797位、第798位、第799位或第800位。
在某些实施方式中，本申请提供的IgA蛋白酶截短体包含如SEQ ID NO:1所示的氨基酸序列的第31位起的至少760个连续氨基酸的多肽片段。例如，在某些实施方式中，本申请提供的IgA蛋白酶截短体包含如SEQ ID NO:1所示的氨基酸序列的第31位起的至少761个、至少762个、至少763个、至少764个、至少765个、至少766个、至少767个、至少768个、至少769个、至少770个、至少771个、至少772个、至少773个、至少774个、至少775个、至少776个、至少777个、至少778个、至少779个、至少780个、至少781个、至少782个、至少783个、至少784个、至少785个、至少786个、至少787个、至少788个、至少789个、至少790个、至少791个、至少792个、至少793个、至少794个、至少795个、至少796个、至少797个、至少798个、至少799个、至少800个、至少801个、至少802个、至少803个、至少804个、至少805个、至少806个、至少807个、至少808个、至少809个、至少810个、至少850个、至少860个、至少870 个、至少880个、至少890个、至少900个、至少910个、至少920个、至少930个、至少940个、至少950个、至少960个、至少970个、至少980个、至少990个、至少1000个、至少1050个、至少1100个、至少1150个、至少1200个连续氨基酸的多肽片段。
在某些实施方式中，本申请提供的IgA蛋白酶截短体包含如SEQ ID NO:1所示的氨基酸序列的第31位起的760个连续氨基酸的多肽片段。在某些实施方式中，本申请提供的IgA蛋白酶截短体包含如SEQ ID NO:1所示的氨基酸序列的第31位起的761个连续氨基酸的多肽片段。在某些实施方式中，本申请提供的IgA蛋白酶截短体包含如SEQ ID NO:1所示的氨基酸序列的第31位起的762个连续氨基酸的多肽片段。在某些实施方式中，本申请提供的IgA蛋白酶截短体包含如SEQ ID NO:1所示的氨基酸序列的第31位起的768个连续氨基酸的多肽片段。在某些实施方式中，本申请提供的IgA蛋白酶截短体包含如SEQ ID NO:1所示的氨基酸序列的第31位起的777个连续氨基酸的多肽片段。在某些实施方式中，本申请提供的IgA蛋白酶截短体包含如SEQ ID NO:1所示的氨基酸序列的第31位起的786个连续氨基酸的多肽片段。在某些实施方式中，本申请提供的IgA蛋白酶截短体包含如SEQ ID NO:1所示的氨基酸序列的第31位起的803个连续氨基酸的多肽片段。
在某些实施方式中，本申请提供的IgA蛋白酶截短体包含选自下组的多肽片段：如SEQ ID NO:1所示的氨基酸序列的第31位至第790位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第31位至第792位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第31位至第798位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第31位至第807位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第31位至第816位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第31位至第833位氨基酸以及与其具有至少70％的序列同一性(例如，具有至少75％、至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性)的多肽片段。在某些实施方式中，与所述多肽片段具有至少70％的序列同一性(例如，具有至少75％、至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少 94％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性)的IgA蛋白酶截短体仍然保持IgA蛋白酶的功能或活性(例如，蛋白水解活性、特异性切割IgA的酶活性等)。
在某些实施方式中，本申请提供的IgA蛋白酶截短体包含如SEQ ID NO:1所示的氨基酸序列的第335位起的至少456个连续氨基酸的多肽片段。例如，在某些实施方式中，本申请提供的IgA蛋白酶截短体包含如SEQ ID NO:1所示的氨基酸序列的第335位起的至少457个、至少458个、至少459个、至少460个、至少461个、至少462个、至少463个、至少464个、至少465个、至少466个、至少467个、至少468个、至少469个、至少470个、至少471个、至少472个、至少473个、至少474个、至少475个、至少476个、至少477个、至少478个、至少479个、至少480个、至少481个、至少482个、至少483个、至少484个、至少485个、至少486个、至少487个、至少488个、至少489个、至少490个、至少491个、至少492个、至少493个、至少494个、至少495个、至少496个、至少497个、至少498个、至少499个、至少500个、至少550个、至少600个、至少650个、至少700个、至少750个、至少800个、至少850个或者至少900个连续氨基酸的多肽片段。
在某些实施方式中，本申请提供的IgA蛋白酶截短体包含选自下组的多肽片段：如SEQ ID NO:1所示的氨基酸序列的第335位至第790位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第335位至第791位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第335位至第792位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第285位至第790位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第285位至第791位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第285位至第792位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第330位至第790位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第330位至第791位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第330位至第792位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第285位至第816位氨基酸以及与其具有至少90％或至少95％的序列同一性(例如，至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性)的多肽片段。在某些实施方式中，与所述多肽片段具有至少90％或至少95％的序列同一性(例如，至少91％、至少92％、至少93％、 至少94％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性)的IgA蛋白酶截短体仍然保持IgA蛋白酶的功能或活性(例如，蛋白水解活性、特异性切割IgA的酶活性等)。
在某些实施方式中，本申请提供了AK183(31-790)截短体，其氨基酸序列如SEQ ID NO:14所示。
在某些实施方式中，本申请提供了AK183(31-791)截短体，其氨基酸序列如SEQ ID NO:15所示。

在某些实施方式中，本申请提供了AK183(31-792)截短体，其氨基酸序列如SEQ ID NO:16所示。
在某些实施方式中，本申请提供了AK183(31-798)截短体，其氨基酸序列如SEQ ID NO:17所示。

在某些实施方式中，本申请提供了AK183(31-807)截短体，其氨基酸序列如SEQ ID NO:18所示。
在某些实施方式中，本申请提供了AK183(31-816)截短体，其氨基酸序列如SEQ ID NO:19所示。

在某些实施方式中，本申请提供了AK183(31-833)截短体，其氨基酸序列如SEQ ID NO:20所示。
在某些实施方式中，本申请提供了AK183(285-790)截短体，其氨基酸序列如 SEQ ID NO:43所示。在某些实施方式中，本申请提供了AK183(285-791)截短体，其氨基酸序列如SEQ ID NO:44所示。在某些实施方式中，本申请提供了AK183(285-792)截短体，其氨基酸序列如SEQ ID NO:45所示。在某些实施方式中，本申请提供了AK183(285-816)截短体，其氨基酸序列如SEQ ID NO:46所示。在某些实施方式中，本申请提供了AK183(330-790)截短体，其氨基酸序列如SEQ ID NO:47所示。在某些实施方式中，本申请提供了AK183(330-791)截短体，其氨基酸序列如SEQ ID NO:48所示。在某些实施方式中，本申请提供了AK183(330-792)截短体，其氨基酸序列如SEQ ID NO:49所示。在某些实施方式中，本申请提供了AK183(335-790)截短体，其氨基酸序列如SEQ ID NO:50所示。在某些实施方式中，本申请提供了AK183(335-791)截短体，其氨基酸序列如SEQ ID NO:51所示。在某些实施方式中，本申请提供了AK183(335-792)截短体，其氨基酸序列如SEQ ID NO:52所示。
SEQ ID NO:43～52的序列如下所示。





在某些实施方式中，本申请提供的IgA蛋白酶截短体在上述多肽片段的氨基酸序列基础上，在一个或多个位点(例如，在1个、2个、3个、4个、5个或更多个位点)具有氨基酸的保守替换。氨基酸残基的保守替换是指性质相似的氨基酸之间的替换，例如极性氨基酸之间的替换(如谷氨酰胺和天冬酰胺之间的替换)，疏水性氨基酸之间的替换(如亮氨酸、异亮氨酸、蛋氨酸和缬氨酸之间的替换)，以及带相同电荷的氨基酸之间的替换(如精氨酸、赖氨酸和组氨酸之间的替换，或者谷氨酸和天冬氨酸之间的替换)等。在某些实施方式中，本申请所述的IgA蛋白酶截短体与SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51或者SEQ ID NO:52所示的氨基酸序列相比，在1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、15个、20个或更多个位点具有氨基酸的保守替换。
在某些实施方式中，所述多肽片段在对应于SEQ ID NO:1的第844位、第862位、第931位、第933位、第978位、第1002位、第1004位中的一个或多个位置有氨基酸突变。在某些实施方式中，所述多肽片段在对应于SEQ ID NO:1的第844位有氨基酸突变。在某些实施方式中，所述多肽片段在对应于SEQ ID NO:1的第862位有氨基酸突变。在某些实施方式中，所述多肽片段在对应于SEQ ID NO:1的第931位和第933位有氨基酸突变。在某些实施方式中，所述多肽片段在对应于SEQ ID NO:1的第978位有氨基酸突变。在某些实施方式中，所述多肽片段在对应于SEQ ID NO:1第1002位和第1004位有氨基酸突变。
在某些实施方式中，所述多肽片段在对应于SEQ ID NO:1的第844位、第862位、第931位、第933位、第978位、第1002位、第1004位中的一个或多个位置突变为甘氨酸。在某些实施方式中，所述多肽片段在对应于SEQ ID NO:1的第844 位、第862位、第931位、第933位、第978位、第1002位、第1004位中的一个或多个位置的脯氨酸(P)突变为甘氨酸(G)。在某些实施方式中，所述多肽片段在对应于SEQ ID NO:1的第844位的脯氨酸突变为甘氨酸。在某些实施方式中，所述多肽片段在对应于SEQ ID NO:1的第862位的脯氨酸突变为甘氨酸。在某些实施方式中，所述多肽片段在对应于SEQ ID NO:1的第931位和第933位的脯氨酸突变为甘氨酸。在某些实施方式中，所述多肽片段在对应于SEQ ID NO:1的第978位的脯氨酸突变为甘氨酸。在某些实施方式中，所述多肽片段在对应于SEQ ID NO:1第1002位和第1004位的脯氨酸突变为甘氨酸。
在某些实施方式中，所述多肽片段的氨基酸序列如SEQ ID NO:53(也称为“PA-GA Mut”)、SEQ ID NO:54(也称为“PI-GI Mut”)、SEQ ID NO:55(也称为“PAP-GAG Mut”)、SEQ ID NO:56(也称为“PAT-GAT Mut”)或者SEQ ID NO:57(也称为“PIP-GIG Mut”)所示。
SEQ ID NO:53～57的序列如下所示。





在不影响活性的前提下，本申请提供的IgA蛋白酶截短体还可以含有非天然的氨基酸。非天然的氨基酸包括例如，β-氟代丙氨酸、1-甲基组氨酸、γ-亚甲基谷氨酸、α-甲基亮氨酸、4,5-脱氢赖氨酸、羟基脯氨酸、3-氟代苯基丙氨酸、3-氨基酪氨 酸、4-甲基色氨酸等。
本申请提供的IgA蛋白酶截短体也可以使用本领域公知的方法进行修饰。例如，但不限于，PEG化、糖基化、氨基端修饰、脂肪酰化、羧基端修饰、磷酸化、甲基化等。本领域技术人员可以理解，本申请提供的IgA蛋白酶截短体使用本领域公知的方法进行修饰之后，仍然保留了与IgA蛋白酶或IgA蛋白酶截短体基本上相似的功能。
在某些实施方式中，本申请提供的IgA蛋白酶截短体具有特异性切割人IgA的酶活性。在某些实施方式中，本申请提供的IgA蛋白酶截短体具有特异性切割人IgA重链的酶活性。在某些实施方式中，本申请提供的IgA蛋白酶截短体具有特异性切割人IgA重链CH1与铰链区交汇处的酶活性。在某些实施方式中，本申请提供的IgA蛋白酶截短体具有特异性切割人IgA1的酶活性。
在某些实施方式中，本申请提供的IgA蛋白酶截短体在上述多肽片段的氨基酸序列基础上，在一个或多个位点具有氨基酸的保守替换，但仍然具有切割人IgA(例如，IgA1)的酶活性。在某些实施方式中，本申请提供的IgA蛋白酶截短体与上述多肽片段具有至少70％的序列同一性(例如，具有至少75％、至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性)，并且仍然具有切割人IgA(例如，IgA1)的酶活性。
融合蛋白
在另一方面，本申请提供了一种融合蛋白，其包含第一多肽和第二多肽，其中所述第一多肽包含获得自或衍生自多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的全长、获得自或衍生自多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶去掉信号肽之后形成的多肽或者本申请所述的IgA蛋白酶截短体，所述第二多肽包含用于延长所述第一多肽在受试者体内半衰期的氨基酸序列。在某些实施方式中，所述第一多肽包含如SEQ ID NO:1或者SEQ ID NO:42所示的序列。在某些实施方式中，所述第二多肽位于所述第一多肽的N末端。在某些实施方式中，所述第二多肽位于所述第一多肽的C末端。
在某些实施方式中，所述第一多肽和所述第二多肽之间通过连接子连接。在某些实施方式中，所述第一多肽和所述第二多肽之间直接连接(即，不通过连接子连接)。本申请中使用的术语“连接子”或“接头”是指具有1、2、3、4或5个氨基酸残基，或长度介于5和15、20、30、50或更多个氨基酸残基之间的人工氨基酸序列，通过肽键连接，并用于连接一个或多个多肽。连接子可能有也可能没有二级结构。连接子序列在本领域是已知的，例如，参见Holliger et al.,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)；Poljak et al.,Structure 2:1121-1123(1994)。
在某些实施方式中，所述连接子选自下组：可切割连接子、不可切割连接子、肽连接子、柔性连接子、刚性连接子、螺旋连接子和非螺旋连接子。可以使用本领域已知的任何合适的连接子。在某些实施方式中，所述连接子包含肽连接子。例如，本申请中的有用连接子可能富含甘氨酸和丝氨酸残基。示例包括具有包含苏氨酸/丝氨酸和甘氨酸的单个或重复序列的连接子，例如GGGS(SEQ ID NO:21)或GGGGS(SEQ ID NO:22)、GGGGGS(SEQ ID NO:86)或GGGGGGGS(SEQ ID NO:87)或其串联重复(例如，2、3、4、5、6、7、8、9、10或更多重复)。在某些实施方式中，本申请中使用的连接子包括GGCGGCGGTGGATCC(SEQ ID NO:23)。可选地，所述连接子可能是包含如GGCGGCGGTGGATCC(SEQ ID NO:23)所示的氨基酸序列的一个或多个顺序或串联重复的长肽链。在某些实施方式中，所述连接子包括SEQ ID NO:23的1、2、3、4、5、6、7、8、9、10或更多顺序或串联重复。在某些实施方式中，所述连接子包括选自下组的氨基酸序列或由选自下组的氨基酸序列组成：与SEQ ID No:21、22、23中的任何一个具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性的氨基酸序列。
在某些实施方式中，本申请中使用的连接子包括如SEQ ID NO:58(EEKKKEKEKEEQEERETK)所示的氨基酸序列。可选地，所述连接子可能是包含如SEQ ID NO:58所示的氨基酸序列的一个或多个顺序或串联重复的长肽链。在某些实施方式中，所述连接子包括SEQ ID NO:58的1、2、3、4、5、6、7、8、9、10或更多顺序或串联重复。在某些实施方式中，所述连接子包括与SEQ ID NO:58具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性的氨基酸序 列。
在某些实施方式中，本申请中使用的连接子包括如SEQ ID NO:59(HHHHHHHHHH)所示的氨基酸序列。在某些实施方式中，所述连接子包括与SEQ ID NO:59具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性的氨基酸序列。
在某些实施方式中，所述第二多肽选自Fc结构域和白蛋白。在某些实施方式中，所述Fc结构域包含铰链区。在某些实施方式中，所述Fc结构域包含下游铰链区(lower hinge)。在某些实施方式中，所述Fc结构域包含核心铰链区(core hinge region)和下游铰链区(lower hinge)。在某些实施方式中，所述Fc结构域包含上游铰链区(upper hinge region)、核心铰链区(core hinge region)和下游铰链区(lower hinge)。在某些实施方式中，所述Fc结构域不包含铰链区。在某些实施方式中，所述Fc结构域来源于人IgG Fc结构域。在某些实施方式中，所述Fc结构域来源于人IgG1 Fc结构域、人IgG2 Fc结构域、人IgG3 Fc结构域或人IgG4 Fc结构域。
在某些实施方式中，所述Fc结构域包括如SEQ ID NO:24所示的氨基酸序列。在某些实施方式中，所述Fc结构域由如SEQ ID NO:24所示的氨基酸序列组成。在某些实施方式送，所述Fc结构域的氨基酸序列与如SEQ ID NO:24所示的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％或至少95％的序列同一性。
在某些实施方式中，所述Fc结构域的编码核酸序列包括如SEQ ID NO:39所示的核苷酸序列。在某些实施方式中，所述Fc结构域的编码核酸序列由如SEQ ID NO:39所示的核苷酸序列组成。在某些实施方式中，所述Fc结构域的编码核酸序列与如SEQ ID NO:39所示的核苷酸序列具有至少70％、至少75％、至少80％、至 少85％、至少90％或至少95％的序列同一性。
在某些实施方式中，所述Fc结构域包括如SEQ ID NO:25所示的氨基酸序列。在某些实施方式中，所述Fc结构域由如SEQ ID NO:25所示的氨基酸序列组成。在某些实施方式送，所述Fc结构域的氨基酸序列与如SEQ ID NO:25所示的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％或至少95％的序列同一性。
在某些实施方式中，所述Fc结构域的编码核酸序列包括如SEQ ID NO:40所示的核苷酸序列。在某些实施方式中，所述Fc结构域的编码核酸序列由如SEQ ID NO:40所示的核苷酸序列组成。在某些实施方式中，所述Fc结构域的编码核酸序列与如SEQ ID NO:40所示的核苷酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％或至少95％的序列同一性。
在某些实施方式中，所述Fc结构域包括如SEQ ID NO:32所示的氨基酸序列。在某些实施方式中，所述Fc结构域由如SEQ ID NO:32所示的氨基酸序列组成。在某些实施方式送，所述Fc结构域的氨基酸序列与如SEQ ID NO:32所示的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％或至少95％的序列同一性。
在某些实施方式中，所述Fc结构域包括如SEQ ID NO:77所示的氨基酸序列。在某些实施方式中，所述Fc结构域由如SEQ ID NO:77所示的氨基酸序列组成。在某些实施方式送，所述Fc结构域的氨基酸序列与如SEQ ID NO:77所示的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％或至少95％的序列同一性。

在某些实施方式中，所述Fc结构域具有一个或多个氨基酸突变。在某些实施方式中，所述Fc结构域在对应于SEQ ID NO:25的第7位有氨基酸突变。在某些实施方式中，所述Fc结构域在对应于SEQ ID NO:25的第7位的氨基酸(例如，丙氨酸)突变为缬氨酸。在某些实施方式中，所述Fc结构域在对应于SEQ ID NO:25的第7位的氨基酸(例如，丙氨酸)突变为甘氨酸。在某些实施方式中，所述Fc结构域在对应于SEQ ID NO:25的第7位的氨基酸(例如，丙氨酸)突变为丝氨酸。在某些实施方式中，所述Fc结构域在对应于SEQ ID NO:25的第7位的氨基酸(例如，丙氨酸)突变为亮氨酸。
在某些实施方式中，所述Fc结构域包含一个或多个延长所述融合蛋白的半衰期的突变。在某些实施方式中，所述Fc结构域与所述第一多肽的C末端连接。在某些实施方式中，所述Fc结构域与所述第一多肽的N末端连接。
在某些实施方式中，所述第二多肽为白蛋白。在某些实施方式中，所述白蛋白的氨基酸序列如SEQ ID NO:60所示。在某些实施方式中，所述白蛋白包含人血清白蛋白的一个或多个结构域。在某些实施方式中，所述白蛋白包含人血清白蛋白的D3结构域。
在某些实施方式中，本申请提供的融合蛋白进一步包含标签。在某些实施方式中，所述标签选自下组：荧光标签、发光标签、纯化标签和生色标签。在某些实施方式中，所述标签选自下组：c-Myc标签、HA标签、VSV-G标签、FLAG标签、V5标签和HIS标签。在某些实施方式中，所述标签为HIS标签。在某些实施方式中，所述标签是包含6个、7个、8个、9个或10个组氨酸的HIS标签。在某些实施方式中，所述第二多肽位于所述第一多肽的C末端，所述标签位于所述第二多肽的C末端。
在某些实施方式中，本申请提供的融合蛋白包括如SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:81、SEQ ID NO:82、SEQ ID NO:83、SEQ ID NO:84或SEQ ID NO:85所示的氨基酸序列。在某些实施方式中，本申请提供的融合蛋白由选自下组的氨基酸序列组成：SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:81、SEQ ID NO:82、SEQ ID NO:83、SEQ ID NO:84或SEQ ID NO:85，或者与其具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％的序列同一性。在某些实施方式中，与SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:81、SEQ ID NO:82、SEQ ID NO:83、SEQ ID NO:84或SEQ ID NO:85具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％的序列同一性的融合蛋白仍然保持IgA蛋白酶的功能或活性(例如，蛋白水解活性、特异性切割IgA的酶活性等)。








在某些实施方式中，本申请提供的融合蛋白包括如SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12所示的氨基酸序列。在某些实施方式中，本申请提供的融合蛋白由选自下组的氨基酸序列组成：SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12，或者与其具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％的序列同一性。




在某些实施方式中，本申请提供的融合蛋白在受试者体内的血循环中的半衰期为至少1天、至少2天、至少3天、至少4天、至少5天、至少6天、至少7天、至少8天、至少9天、至少10天、至少11天、至少12天、至少13天、至少14天。
核酸
在另一方面，本申请提供了一种分离的核酸，其包含编码本申请所述的IgA蛋白酶截短体的核苷酸序列或包含编码本申请所述的融合蛋白的核苷酸序列。
本申请所用的术语“核酸”或“核苷酸”是指单链或双链形式的脱氧核糖核酸(DNA)或核糖核酸(RNA)及其聚合物。除非另有说明，否则特定的核苷酸序列还隐含地涵盖其保守修饰的变体(例如简并的密码子取代)、等位基因、直向同源物、SNP和互补序列以及明确指出的序列。具体而言，简并的密码子取代可通过产生这样的序列来实现：其中一个或多个选定的(或全部)密码子的第三位置被混合碱基和/或脱氧肌苷残基取代(参见Batzer et al.,Nucleic Acid Res.19:5081(1991)；Ohtsuka et al.,J.Biol.Chem.260:2605-2608(1985)以及Rossolini et al.,Mol.Cell.Probes 8:91-98(1994))。
使用传统的步骤，可以容易地对编码本申请所述的IgA蛋白酶截短体的DNA或者编码本申请所述的融合蛋白的DNA进行分离和测序(例如通过使用能够与编码所述IgA蛋白酶截短体或融合蛋白的基因特异性结合的寡核苷酸探针)。编码 DNA也可以通过合成方法获得。
在某些实施方式中，本申请提供的核酸包括如SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38所示的核酸序列。在某些实施方式中，本申请提供的核酸由选自下组的核苷酸序列组成：SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38，或者与其具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性。









在某些实施方式中，本申请提供的核酸包括如SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13所示的核酸序列。在某些实施方式中，本申请提供的核酸由选自下组的核苷酸序列组成：SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13，或者与其具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性。












载体和细胞
在另一方面，本申请提供了一种载体，其包含编码本申请所述的IgA蛋白酶截短体的核酸或包含编码本申请所述的融合蛋白的核酸。
使用本领域公知的重组技术，可以将编码所述IgA蛋白酶截短体或融合蛋白的分离的多核苷酸插入载体，用于进一步的克隆(DNA的扩增)或用于表达。有多种载体可供选择。载体组分通常包括但不限于下列的一种或多种：信号序列、复制起始点、一种或多种标记基因、增强子元件、启动子(例如SV40、CMV、EF-1α)和转录终止序列。
在某些实施方式中，本申请提供的核酸编码IgA蛋白酶截短体或融合蛋白、与核酸序列可操作连接的至少一种启动子(例如SV40、CMV、EF-1α)和至少一种选择标签。载体的实例包括但不限于：逆转录病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(例如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳多空病毒(例如SV40)、λ噬菌体和M13噬菌体、质粒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等。
可以将包含编码所述IgA蛋白酶截短体或融合蛋白的核酸序列的载体引入宿主细胞，用于克隆或基因表达。适用于克隆或表达本申请中所述的载体中的DNA的宿主细胞为上述的原核、酵母或高等真核细胞。适用于本申请用途的原核细胞包括真细菌，如革兰氏阴性菌或革兰氏阳性菌，例如，肠杆菌科(Enterobacteriaceae)，例如，埃希氏菌属(Escherichia)(例如，大肠杆菌(E.coli))、肠杆菌属(Enterobacter)、欧文氏菌属(Erwinia)、克雷白氏杆菌属(Klebsiella)、变形杆菌属(Proteus)、沙门氏菌属(Salmonella)(例如，鼠伤寒沙门(氏)杆菌(Salmonella typhimurium))、沙雷氏菌属(Serratia)(例如，粘质沙雷氏菌(Serratia marcescans))、志贺氏菌属(Shigella)、杆菌属(Bacilli)(例如，枯草芽孢杆菌(B.subtilis)和地衣芽 孢杆菌(B.licheniformis))、假单胞菌属(Pseudomonas)(例如，绿脓杆菌(P.aeruginosa)、以及链霉菌属(Streptomyces)。在某些实施方式中，所述细胞是大肠杆菌细胞。
除了原核细胞以外，真核生物细胞，例如真核微生物如丝状真菌或酵母也可用作编码IgA蛋白酶截短体或融合蛋白的载体的合适的克隆或表达宿主。酿酒酵母(Saccharomyces cerevisiae)，或面包酵母是最常用的低等真核宿主微生物。但是，许多其他属、种和株都比较常用且在本申请中适用，例如，粟酒裂殖酵母(Schizosaccharomyces pombe)；克鲁维酵母属(Kluyveromyces)宿主，例如，乳酸克鲁维酵母(K.lactis)、脆壁克鲁维酵母(K.fragilis)(ATCC 12,424)、保加利亚克鲁维酵母(K.bulgaricus)(ATCC 16,045)、魏氏克鲁维酵母(K.wickeramii)(ATCC 24,178)、克鲁雄酵母(K.waltii)(ATCC 56,500)、果蝇克鲁维酵母(K.drosophilarum)(ATCC 36,906)、耐热克鲁维酵母(K.thermotolerans)和马克斯克鲁维酵母(K.marxianus)；解脂耶氏酵母(yarrowia)(EP 402,226)；巴斯德毕赤酵母(Pichia pastoris)(EP 183,070)；假丝酵母(Candida)；里氏木霉(Trichoderma reesia)(EP 244,234)；链孢霉(Neurospora crassa)；西方许旺酵母(Schwanniomyces)，例如，西方许旺酵母(Schwanniomyces occidentalis)；和丝状真菌(filamentous fungi)，例如，脉孢菌(Neurospora)、青霉菌(Penicillium)、弯颈霉(Tolypocladium)和曲霉菌(Aspergillus)(例如，钩巢曲霉(A.nidulans)和黑曲霉(A.niger))。在某些实施方式中，所述真核生物细胞是哺乳动物细胞。在某些实施方式中，所述哺乳动物细胞是人细胞或中国仓鼠卵巢(CHO)细胞。在某些实施方式中，所述哺乳动物细胞是人胚胎肾细胞293(HEK293细胞)。
药物组合物
在另一方面，本申请提供了一种药物组合物，其包含本申请所述的IgA蛋白酶截短体、包含本申请所述的融合蛋白、包含本申请所述的核酸、包含本申请所述的载体或者包含本申请所述的细胞，以及药学上可接受的载体。
用于本申请中公开的药物组合物的药学上可接受的载体可包括，例如，药学上可接受的液体、凝胶或固体载剂、水相溶媒、非水相溶媒、抗微生物物质、等渗物质、缓冲液、抗氧化剂、麻醉剂、悬浮剂/分散剂、螯合剂、稀释剂、佐剂、辅料或 无毒辅助物质，其他本领域公知的组分或以上的多种组合。
适用的组分可包括，例如，抗氧剂、填充剂、粘合剂、崩解剂、缓冲液、防腐剂、润滑剂、搅味剂、增稠剂、着色剂、乳化剂或稳定剂例如糖和环糊精。适用的抗氧剂可包括，例如，甲硫氨酸、抗坏血酸、EDTA、硫代硫酸钠、铂、过氧化氢酶、柠檬酸、半胱氨酸、巯基甘油、巯基乙酸、巯基山梨醇、丁基甲基茴香醚、丁基化羟基甲苯和/或没食子酸丙酯。如本申请所公开，在包含本申请公开的IgA蛋白酶截短体或融合蛋白的组合物中包括一种或多种抗氧剂如甲硫氨酸，可降低所述IgA蛋白酶截短体或融合蛋白的氧化。本申请进一步提供多种防止所述IgA蛋白酶截短体或融合蛋白氧化、延长其保质期和/或提高其活性的方法，例如，通过将本申请中提供的IgA蛋白酶截短体或融合蛋白与一种或多种抗氧剂(例如，甲硫氨酸)混合来实现。
进一步地说，药学上可接受的载体可包括，例如，水相介质如氯化钠注射液、林格氏液注射液、等渗葡萄糖注射液、无菌水注射液、或葡萄糖和乳酸林格注射液、非水介质例如：植物来源的不挥发性油、棉花子油、玉米油、芝麻油、或者花生油、细菌抑制或真菌抑制浓度下的抗菌物质、等渗剂如：氯化钠或葡萄糖、缓冲液如：磷酸盐或枸橼酸酸盐缓冲液，抗氧化剂如：硫酸氢钠，局部麻醉剂如：盐酸普鲁卡因，助悬剂和分散剂如：羧甲基纤维素钠、羟丙基甲基纤维素或聚乙烯吡咯烷酮，乳化剂如：聚山梨醇酯80(吐温-80)、螯合试剂如EDTA(乙二胺四乙酸)或EGTA(乙二醇双(2-氨基乙基醚)四乙酸)、乙醇、聚乙二醇、丙二醇、氢氧化钠、盐酸、柠檬酸或乳酸。作为运载体的抗菌剂可加入多剂量容器中的药物组合物中，其包括酚类或甲酚、汞制剂、苯甲醇、氯代丁醇、甲基和丙基对羟基苯甲酸酯、噻汞撒、氯苯甲烷铵和氯苯乙铵。适用的辅料可包括，例如，水、盐、葡萄糖、甘油或乙醇。适用的无毒辅助物质可包括，例如，润湿剂、乳化剂、pH缓冲剂、稳定剂、增溶剂，或者醋酸钠、去水山梨糖醇月桂酸酯、三乙醇胺油酸酯或者环糊精之类的物质。
所述药物组合物可以是液体溶液、悬浮液、乳剂、丸剂、胶囊、片剂、持续释放制剂或粉末。口服制剂可以包括标准运载体如药物级的甘露醇、乳糖、淀粉、硬脂酸镁、聚乙烯吡咯烷酮、糖精钠、纤维素、碳酸镁等。
在某些实施方式中，所述药物组合物被制剂成可注射的组合物。可注射的药物组合物可以任何常规的形式制备，例如，液体溶剂、悬浮剂、乳化剂或适用于产生液体溶剂、悬浮剂或乳化剂的固体形式。注射制剂可包括现用的无菌和/或无热原溶液、使用前现与溶剂结合的无菌干燥的可溶物，如冻干粉，包括皮下片、注射即用的无菌悬浮剂、使用前现与介质结合的无菌干燥不溶产品，和无菌和/或无热原的乳剂。溶剂可以为水相或非水相。
在某些实施方式中，单位剂量的注射制剂包装在一个安瓿、一支管或一支带有针的针筒中。本领域习知，所有注射给药的制剂应为无菌无热原。
在某些实施方式中，通过将本申请中公开的IgA蛋白酶截短体或融合蛋白溶解于某适当的溶剂中可制备无菌冻干的粉末。所述溶剂可含有一种可提高粉末或由粉末制得的重组溶液的稳定性，或改善粉末或重组溶液的其他药理组分。适用的辅料包括，但不限于，水、葡萄糖、三梨糖醇、果糖、玉米糖浆、木糖醇、甘油、葡萄糖、蔗糖或其他适用的物质。溶剂可含有缓冲液，如枸橼酸缓冲液、磷酸钠或磷酸钾缓冲液或其他本技术人员公知的缓冲液，在一种实施方式中，缓冲液的pH为中性。在本领域公知的标准条件下进行对所述溶解进行随后的过滤除菌，然后冻干制得理想的制剂。在一种实施方式中，将所得的溶剂分装至小管中冻干。每支小管可容纳单次剂量或多次剂量的所述IgA蛋白酶截短体或融合蛋白或其组合物。每支小管中的装入量可略微高于每次剂量所需或多次剂量所需(例如10％过量)，从而保证取样精确和给药精确。冻干粉可在适当的条件下储存，如在约4℃到室温范围。
用注射用水将冻干粉重溶得到用于注射给药的制剂。在一种实施方式中，可将冻干粉加至无菌无热原水或其他适用的液体载剂中重溶。精确的量由选择的疗法决定，可根据经验值决定。
治疗或预防疾病的方法
在另一方面，本申请提供了一种治疗或预防IgA沉积相关疾病的方法，其包括向需要治疗或预防的受试者施用本申请所述的IgA蛋白酶截短体、本申请所述的融合蛋白或者本申请所述的药物组合物。
在另一方面，本申请提供了一种治疗或预防IgA沉积相关疾病的方法，其包括向需要治疗或预防的受试者施用IgA蛋白酶或其截短体、包含所述IgA蛋白酶或其截短体的融合蛋白或者包含所述IgA蛋白酶或其截短体或者所述融合蛋白的药物组合物，其中，所述IgA蛋白酶的氨基酸序列选自下组：SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76或其组合。在某些实施方式中，所述IgA蛋白酶的氨基酸序列是如SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75或SEQ ID NO:76所示的氨基酸序列去掉信号肽序列之后形成的氨基酸序列。在某些实施方式中，所述IgA蛋白酶截短体与如SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75或SEQ ID NO:76所示的多肽具有至少70％的序列同一性(例如，具有至少75％、至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性)。在某些实施方式中，所述IgA蛋白酶截短体与如SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75或SEQ ID NO:76所示的多肽具有至少70％的序列同一性(例如，具有至少75％、至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性)，并且仍然保持IgA蛋白酶的功能或活性(例如，蛋白水解活性、特异性切割IgA的酶活性等)。















在另一方面，本申请提供了本申请所述的IgA蛋白酶截短体、本申请所述的融合蛋白或者本申请所述的药物组合物在制备用于治疗或预防IgA沉积相关疾病的药物中的用途。
在另一方面，本申请提供了IgA蛋白酶或其截短体、包含所述IgA蛋白酶或其截短体的融合蛋白或者包含所述IgA蛋白酶或其截短体或者所述融合蛋白的药物组合物在制备用于治疗或预防IgA沉积相关疾病的药物中的用途，其中，所述IgA蛋白酶的氨基酸序列选自下组：SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76或其组合。在某些实施方式中，所述IgA蛋白酶的氨基酸序列是如SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75或SEQ ID NO:76所示的氨基酸序列去掉信号肽序列之后形成的氨基酸序列。在某些实施方式中，所述IgA蛋白酶截短体与如SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、 SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75或SEQ ID NO:76所示的多肽具有至少70％的序列同一性(例如，具有至少75％、至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性)。在某些实施方式中，所述IgA蛋白酶截短体与如SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75或SEQ ID NO:76所示的多肽具有至少70％的序列同一性(例如，具有至少75％、至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性)，并且仍然保持IgA蛋白酶的功能或活性(例如，蛋白水解活性、特异性切割IgA的酶活性等)。
在另一方面，本申请提供了用于治疗或预防IgA沉积相关疾病的如本申请所述的IgA蛋白酶截短体、融合蛋白或者药物组合物。
在另一方面，本申请提供了用于治疗或预防IgA沉积相关疾病的IgA蛋白酶或其截短体、包含所述IgA蛋白酶或其截短体的融合蛋白或者包含所述IgA蛋白酶或其截短体或者所述融合蛋白的药物组合物，其中，所述IgA蛋白酶的氨基酸序列选自下组：SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76或其组合。在某些实施方式中，所述IgA蛋白酶的氨基酸序列是如SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75或SEQ ID NO:76所示的氨基酸序列去掉信号肽序列之后形成的氨基酸序列。在某些实施方式中，所述IgA蛋白酶截短体与如SEQ ID NO:61、SEQ  ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75或SEQ ID NO:76所示的多肽具有至少70％的序列同一性(例如，具有至少75％、至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性)。在某些实施方式中，所述IgA蛋白酶截短体与如SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75或SEQ ID NO:76所示的多肽具有至少70％的序列同一性(例如，具有至少75％、至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性)，并且仍然保持IgA蛋白酶的功能或活性(例如，蛋白水解活性、特异性切割IgA的酶活性等)。
在某些实施方式中，本申请所述的IgA沉积相关疾病包括IgA肾病、疱疹样皮炎、亨诺赫-舍恩莱因紫癜(又称IgA血管炎)、川崎病、紫癜性肾炎、IgA血管炎肾损害、IgA类风湿因子阳性的类风湿性关节炎、IgA型抗GBM病或IgA型ANCA相关血管炎。在某些实施方式中，本申请所述的IgA沉积相关疾病为IgA肾病。在某些实施方式中，本申请所述的IgA沉积相关疾病为IgA1肾病。在某些实施方式中，本申请所述的IgA沉积相关疾病为IgA血管炎。在某些实施方式中，本申请所述的IgA沉积相关疾病为川崎病。
具体实施方式
所有实施例中的涉及的生物学材料如大肠杆菌菌株、各种克隆与表达质粒、培养基、工具酶、缓冲液，和各种培养方法、蛋白提取和纯化方法、其它的分子生物学操作方法，均为该领域技术人员所熟悉，可以参考Sambrook等人编著的“分子克隆”(实验室手册，冷泉港，1989)及“精编分子生物学实验指南”(美/F.奥斯伯等著，颜子颖等译，北京，科学出版社，1998)。
实施例1：AK183 IgA蛋白酶的最短活性位点研究
发明人将来自多枝梭菌(Clostridium ramosum)AK183株的野生型IgA蛋白酶(其氨基酸序列如SEQ ID NO:1所示)N末端的信号肽(即，SEQ ID NO:1的第1至30位氨基酸)及C末端的跨膜区加胞内区(即，SEQ ID NO:1的第1205至1234位氨基酸)去掉，然后将人IgG1的Fc序列(HR-CH2-CH3，其氨基酸序列如SEQ ID NO:24所示)加在了去掉了信号肽、跨膜区和胞内区的IgA蛋白酶的氨基酸序列(即，由SEQ ID NO:1的第31位至1204位氨基酸组成的IgA蛋白酶截短体)的N末端，构建了PET30a-Fc-AK183质粒。
然后，发明人以PET30a-Fc-AK183质粒为模板，进行终止突变，构建了一系列Fc-AK183截短体，以研究AK183 IgA蛋白酶的C末端最短活性位点。基于前期研究结果，发明人认为在AK183 IgA蛋白酶的第730位至第840位氨基酸之间存在自切位点。因此，发明人进行了第一轮终止突变，终止突变位点为AK183 IgA蛋白酶的第738位、第769位、第799位、第834位这四个氨基酸位点，结果如图1所示。结果显示，第738位、第769位的氨基酸终止突变后得到的AK183(31-737)、AK183(31-768)IgA蛋白酶截短片段无体外酶切活性，而第799位或第834位的氨基酸终止突变后得到的AK183(31-798)、AK183(31-833)IgA蛋白酶截短片段有活性。因此，第一轮终止突变结论为：AK183 IgA蛋白酶的C末端最短活性位点位于第768位至第798位氨基酸之间；接着进行了第二轮终止突变，突变位点为AK183 IgA蛋白酶的第774位、第779位、第783位、第788位或者第793位这五个氨基酸位点，结果如图2所示，第774位、第779位、第783位或者第788位的氨基酸终止突变后得到的AK183(31-773)、AK183(31-778)、AK183(31-782)、AK183(31-787)IgA蛋白酶截短片段无体外酶切活性，而第793位的氨基酸终止突变后得到的AK183(31-792)IgA蛋白酶截短片段仍有活性。因此，第二轮终止突变结论为：AK183 IgA蛋白酶的C末端最短活性位点位于第787位至第792位氨基酸之间；然后，发明人进行了第三轮终止突变，突变位点为AK183 IgA蛋白酶的第789位、第790位、第791位或者第792位这四个氨基酸位点，结果如图3所示，第789位和第790位的氨基酸终止突变后得到的AK183(31-788)、AK183(31-789)IgA蛋白酶截短片段无体外酶切活性，而第791位或第792位的氨基酸终止突变后得到的AK183(31-790)、AK183(31-791)IgA蛋白酶截短片段仍有活性(其中第791 位可能由于蛋白酶构像问题，活性不完全，仅表现出轻微酶切作用)。因此，第三轮终止突变结论为：AK183 IgA蛋白酶的C末端最短活性片段为AK183(31-790)。
类似地，发明人进行了三轮截短突变，以研究AK183 IgA蛋白酶的N末端最短活性位点。首先，发明人进行了第一轮截短突变，在AK183(31-792)的基础上去掉AK183的N末端的一段功能未知的结构域(domain of unknown,DUF)，C末端的氨基酸位点固定在第792位。例如，在AK183(31-792)的基础上去掉N末端的对应于由SEQ ID NO:1的第31位至284位氨基酸组成的DUF，得到AK183(285-792)IgA蛋白酶截短片段。采取类似的方法，分别得到AK183(330-792)、AK183(380-792)、AK183(430-792)、AK183(480-792)、AK183(530-792)、AK183(580-792)IgA蛋白酶截短片段。得到的IgA蛋白酶截短片段对IgA1的体外酶切活性实验结果如图10所示。如图10所示，AK183(285-792)、AK183(330-792)IgA蛋白酶截短片段仍有体外酶切活性，而AK183(380-792)、AK183(430-792)、AK183(480-792)、AK183(530-792)、AK183(580-792)IgA蛋白酶截短片段没有体外酶切活性。因此，第一轮截短突变的结论为：AK183 IgA蛋白酶的N末端最短活性位点位于第330位至第380位氨基酸之间。接着进行了第二轮截短突变，在第330位至第380位氨基酸之间每隔5个氨基酸进行一个截短体构建，分别得到AK183(335-792)、AK183(340-792)、AK183(345-792)、AK183(350-792)、AK183(355-792)、AK183(360-792)、AK183(365-792)、AK183(370-792)、AK183(375-792)IgA蛋白酶截短片段。得到的IgA蛋白酶截短片段对IgA1的体外酶切活性实验结果如图11所示。如图11所示，AK183(335-792)IgA蛋白酶截短片段仍有体外酶切活性，而AK183(340-792)、AK183(345-792)、AK183(350-792)、AK183(355-792)、AK183(360-792)、AK183(365-792)、AK183(370-792)、AK183(375-792)IgA蛋白酶截短片段没有体外酶切活性。因此，第二轮截短突变的结论为：AK183 IgA蛋白酶的N末端最短活性位点位于第335位至第340位氨基酸之间。然后，发明人进行了第三轮截短突变，在第335位至第340位氨基酸之间逐个氨基酸进行截短体构建，分别得到AK183(336-792)、AK183(337-792)、AK183(338-792)、AK183(339-792)IgA蛋白酶截短片段。得到的IgA蛋白酶截短片段对IgA1的体外酶切活性实验结果如图12所示。如图12所示，AK183(336-792)、AK183(337-792)、AK183(338-792)、AK183(339-792)IgA蛋白酶截短片段均没有体外酶切活性。因此，第三轮截短突变的结论为： AK183 IgA蛋白酶的N末端最短活性位点位于第335位氨基酸。最后，发明人对前三轮的结果再次进行了验证，重新同时表达AK183(285-792)、AK183(330-792)、AK183(335-792)、AK183(336-792)、AK183(337-792)、AK183(338-792)、AK183(339-792)、AK183(340-792)、AK183(345-792)、AK183(350-792)IgA蛋白酶截短片段。得到的IgA蛋白酶截短片段对IgA1的体外酶切活性实验结果如图13所示。如图13所示，AK183(285-792)、AK183(330-792)、AK183(335-792)IgA蛋白酶截短片段仍有体外酶切活性，而AK183(336-792)、AK183(337-792)、AK183(338-792)、AK183(339-792)、AK183(340-792)、AK183(345-792)、AK183(350-792)IgA蛋白酶截短片段没有体外酶切活性，与前述三轮截短突变的结论一致，即，AK183 IgA蛋白酶的N末端最短活性位点位于第335位氨基酸。
综上，AK183 IgA蛋白酶的最短活性片段为AK183(335-790)。
实施例2：制备包含AK183 IgA蛋白酶截短体或AK183 IgA蛋白酶全长的融合蛋白
2.1质粒构建
在确定了AK183 IgA蛋白酶的C末端最短活性片段AK183(31-790)后，发明人将Fc结构域放在AK183 IgA蛋白酶的第790位氨基酸的C端，中间加GGGGS连接，Fc的C端加6XHis标签用于蛋白纯化，构建了PET30a-AK183(31-790)-Fc质粒，构建流程如图4所示。然后，发明人以PET30a-AK183(31-790)-Fc质粒为模板，通过PCR在AK183(31-790)截短体后面加了第791个及第792个氨基酸，构建了PET30a-AK183(31-792)-Fc质粒。
申请人同时委托北京六合华大基因科技有限公司构建了PET30a-AK183(31-798)-Fc、PET30a-AK183(31-807)-Fc、PET30a-AK183(31-816)-Fc、PET30a-AK183(31-833)-Fc四个备选亚克隆，备选亚克隆的Fc(CH2-CH3)除去了铰链区，其氨基酸序列如SEQ ID NO:6所示(SEQ ID NO:6与SEQ ID NO:2相比，缺少了SEQ ID NO:2的前9个氨基酸EPKSCDKTH)，且在IgA蛋白酶截短体与Fc中间添加了10个His(位于连接子GGGGS后面，Fc前面)。四个备选亚克隆用于后期蛋白酶产量及纯度筛选的备选方案。
为了考察AK183 IgA蛋白酶截短片段与Fc区的连接方式是否会影响其对IgA的酶切活性，发明人还构建了PET30a-AK183(285-816)-Fc、PET30a-Fc-AK183(285-816)这两个备选亚克隆，其中Fc的氨基酸序列如SEQ ID NO:25所示。
为了比较AK183 IgA蛋白酶截短片段与Fc形成的融合蛋白、AK183 IgA蛋白酶全长与Fc形成的融合蛋白对IgA的酶切活性，发明人还构建了备选亚克隆PET30a-Fc-AK183(31-1203)，其中Fc的氨基酸序列如SEQ ID NO:24所示。
为了考察IgG1 Fc、IgG4 Fc和白蛋白对包含AK183 IgA蛋白酶截短体的融合蛋白的IgA酶切活性的影响，发明人还构建了PET30a-AK183(31-816)-IgG4 Fc、PET30a-AK183(31-816)-白蛋白这两个备选亚克隆，其中IgG4 Fc的氨基酸序列如SEQ ID NO:77所示，白蛋白的氨基酸序列如SEQ ID NO:60所示。
为了考察不同的连接子对包含AK183 IgA蛋白酶截短体的融合蛋白的IgA酶切活性的影响，发明人还构建了六个备选亚克隆PET30a-AK183(285-816)-linker-Fc，这六个备选亚克隆表达的融合蛋白序列中，除了连接子不同之外，AK183(285-816)和Fc的氨基酸序列均相同，其中AK183(285-816)的氨基酸序列如SEQ ID NO:46所示，Fc的氨基酸序列如SEQ ID NO:25所示，连接子的氨基酸序列分别为HHHHHHHHHH(SEQ ID NO:59，也称为“10xHis”)、EEKKKEKEKEEQEERETK(SEQ ID NO:58，也称为“IgD linker”)、GGGGS(SEQ ID NO:22，也称为“1xlinker”)、GGGGSGGGGS(SEQ ID NO:78，也称为“2xlinker”)、GGGGSGGGGSGGGGS(SEQ ID NO:79，也称为“3xlinker”)和GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:80，也称为“4xlinker”)。
2.2融合蛋白制备方法
将表达载体转染入大肠杆菌(BL21-DE3)感受态细胞，经含50ug/ml的卡那霉素的LB琼脂培养皿抗性选择，然后挑取单克隆菌落至含相应抗生素的LB培养液中摇菌至指数生长期(OD600：0.6-0.8)，至指数生长期后，加入0.1-0.5mM的异丙基-β-D-硫代半乳糖苷(IPTG)诱导，16℃低温诱导表达24h。完成表达后按照常规方法处理大肠杆菌胞体超声碎裂后高速离心并保留上清，然后采用亲和层析及分子筛纯化得到所述重组融合蛋白。
PET30a-AK183(31-792)-Fc质粒表达的AK183(31-792)-Fc融合蛋白的氨基酸序列如SEQ ID NO:2所示，其编码核酸序列如SEQ ID NO:3所示；
PET30a-AK183(31-798)-Fc质粒表达的AK183(31-798)-Fc融合蛋白的氨基酸序列如SEQ ID NO:6所示，其编码核酸序列如SEQ ID NO:7所示；
PET30a-AK183(31-807)-Fc质粒表达的AK183(31-807)-Fc融合蛋白的氨基酸序列如SEQ ID NO:8所示，其编码核酸序列如SEQ ID NO:9所示；
PET30a-AK183(31-816)-Fc质粒表达的AK183(31-816)-Fc融合蛋白的氨基酸序列如SEQ ID NO:10所示，其编码核酸序列如SEQ ID NO:11所示；
PET30a-AK183(31-833)-Fc质粒表达的AK183(31-833)-Fc融合蛋白的氨基酸序列如SEQ ID NO:12所示，其编码核酸序列如SEQ ID NO:13所示；
PET30a-AK183(285-816)-Fc质粒表达的AK183(285-816)-Fc融合蛋白的氨基酸序列如SEQ ID NO:81所示；
PET30a-Fc-AK183(285-816)质粒表达的Fc-AK183(285-816)融合蛋白的氨基酸序列如SEQ ID NO:82所示；
PET30a-Fc-AK183(31-1203)质粒表达的Fc-AK183(31-1203)融合蛋白的氨基酸序列如SEQ ID NO:83所示；
PET30a-AK183(31-816)-IgG4 Fc质粒表达的AK183(31-816)-IgG4 Fc融合蛋白的氨基酸序列如SEQ ID NO:84所示；
PET30a-AK183(31-816)-白蛋白质粒表达的AK183(31-816)-白蛋白融合蛋白的氨基酸序列如SEQ ID NO:85所示。
2.3体外活性测试方法
将获得的包含AK183 IgA蛋白酶截短体的融合蛋白与从IgA肾病患者血浆中提纯的底物IgA1在体外混合，37℃下反应2-12h，然后进行Western blot，验证其对底物IgA1的酶切活性。
2.4体内活性测试方法
将获得的包含AK183 IgA蛋白酶截短体的融合蛋白通过尾静脉注射到人源化IgA1 alpha链敲入(α1KI-Tg)C57BL/6小鼠体内，分别在收集注射前，注射后5min，2h，4h，24h的血样，然后进行Western blot验证。
2.5结果
实验表明，PET30a-AK183(31-790)-Fc质粒成功表达AK183(31-790)-Fc融合蛋白(如图5所示)。同时，AK183(31-792)-Fc融合蛋白有预期的全长蛋白表达(如图6a所示)，也具有体外针对IgA1的酶切活性(如图6b所示)。
备选的四个亚克隆PET30a-AK183(31-798)-Fc、PET30a-AK183(31-807)-Fc、PET30a-AK183(31-816)-Fc、PET30a-AK183(31-833)-Fc均表达融合蛋白，并且均具有体外针对IgA1的酶切活性(如图7所示)。
另外，亚克隆PET30a-AK183(285-816)-Fc、PET30a-Fc-AK183(285-816)均表达了融合蛋白(如图14所示)，并且均具有体外酶切活性(如图15所示)。
发明人还验证了亚克隆PET30a-AK183(31-807)-Fc表达的AK183(31-807)-Fc融合蛋白、亚克隆PET30a-Fc-AK183(285-816)表达的Fc-AK183(285-816)融合蛋白的体内活性，结果如图8(AK183(31-807)-Fc，还原条件下)和图17(Fc-AK183(285-816)，非还原条件下)所示。如图8所示，人源化IgA1小鼠(α1KI-Tg)C57BL/6在接受单针尾静脉注射AK183(31-807)-Fc融合蛋白后，血液中完整的IgA1重链(H)全部消失，并且持续到至少24h。如图17所示，人源化IgA1小鼠(α1KI-Tg)C57BL/6在接受单针尾静脉注射Fc-AK183(285-816)融合蛋白后，血液中完整的IgA1重链(H)全部消失，并且持续到至少2周。
发明人还比较了Fc-AK183(285-816)融合蛋白、AK183(285-816)-Fc融合蛋白、AK183(285-816)IgA蛋白酶截短体等三者对IgA1的酶切活性，结果如图16所示。如图16所示，这三种蛋白对IgA1均具有酶切活性。
发明人还比较了AK183(285-816)-Fc融合蛋白、Fc-AK183(31-1203)融合蛋白对IgA1的酶切活性，结果如图18所示。如图18所示，AK183(285-816)-Fc融合蛋白、Fc-AK183(31-1203)融合蛋白对IgA1均具有酶切活性。
发明人还比较了AK183(31-816)-IgG1 Fc融合蛋白、AK183(31-816)-IgG4 Fc融 合蛋白和AK183(31-816)-白蛋白融合蛋白对IgA1的酶切活性，结果如图19所示。如图19所示，这三种融合蛋白对IgA1均具有酶切活性。
发明人还比较了具有不同连接子(10xHis、IgD linker、1xlinker、2xlinker、3xlinker或4xlinker)的AK183(285-816)-Fc融合蛋白对IgA1的酶切活性，结果如图20所示。如图20所示，这六种融合蛋白对IgA1均具有酶切活性。
2.6真核表达系统
以上实验均在大肠杆菌(BL21-DE3)感受态细胞(即，原核表达系统)中进行。接下来，发明人将AK183(31-792)-Fc融合cDNA序列克隆入pcDNA3.1/hygro(+)表达载体中，融合蛋白的N端加上ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCG(SEQ ID NO:41)编码表达人IL-2的信号肽序列，构建了pcDNA3.1/hygro(+)-IL2-AK183(31-792)-Fc质粒，用于转染真核表达系统HEK293细胞。其中对Fc序列进行了针对真核表达系统的密码子优化。pcDNA3.1/hygro(+)-IL2-AK183(31-792)-Fc表达的IL2-AK183(31-792)-Fc融合蛋白的氨基酸序列如SEQ ID NO:4所示，其编码核酸序列如SEQ ID NO:5所示。
AK183(31-792)-Fc融合蛋白在HEK293细胞中的表达结果如图9所示。结果表明，AK183(31-792)-Fc融合蛋白有预期的全长表达，且在真核系统中表达的融合蛋白存在二聚体(dimer)形式。
实施例3：AK183 IgA蛋白酶突变体的制备及活性测试
发明人在AK183(31-1203)IgA蛋白酶截短体的基础上，分别对第844位、第862位、第931位和第933位、第978位、第1002位和第1004位(上述位点均为相对于SEQ ID NO:1的位点)的脯氨酸(P)进行定点突变，突变为甘氨酸(G)，得到AK183(31-1173)IgA蛋白酶截短体的五种突变体，其氨基酸序列分别如SEQ ID NO:53(也称为“PA-GA Mut”)、SEQ ID NO:54(也称为“PI-GI Mut”)、SEQ ID NO:55(也称为“PAP-GAG Mut”)、SEQ ID NO:56(也称为“PAT-GAT Mut”)和SEQ ID NO:57(也称为“PIP-GIG Mut”)所示。
发明人分别测试了这五种突变体对IgA1的酶切活性，结果如图21所示。如图 21所示，这五种突变体对IgA1均具有酶切活性。
另外，发明人还在实施例2中制备的AK183(31-816)-Fc融合蛋白的氨基酸序列(即，SEQ ID NO:10)的基础上，对其Fc区第7位(该位点是相对于SEQ ID NO:25而言)的丙氨酸(A)进行定点突变，分别突变为缬氨酸(V)、甘氨酸(G)、丝氨酸(S)、亮氨酸(L)，得到AK183(31-816)-Fc融合蛋白的四种突变体，分别称为A-V Mut、A-G Mut、A-S Mut和A-L Mut。
发明人分别测试了这四种突变体对IgA1的酶切活性，结果如图22所示。如图22所示，这四种突变体对IgA1均具有酶切活性。
实施例4：探索其他IgA蛋白酶
发明人从宏基因组数据库里筛选了若干种与AK183的野生型IgA酶具有一定同源性的氨基酸序列，并合成了16种AK183同源酶。它们的氨基酸序列分别如SEQ ID NO:61～SEQ ID NO:76所示。发明人按照实施例2.3所述的体外活性测试方法，分别测试了这些AK183同源酶对IgA1的酶切活性。结果如图23a和图23b所示。图23a中的“1+IgA1”表示如SEQ ID NO:61所示的多肽与底物IgA1在体外混合，“2+IgA1”表示如SEQ ID NO:62所示的多肽与底物IgA1在体外混合，以此类推，图23b中的“16+IgA1”表示如SEQ ID NO:76所示的多肽与底物IgA1在体外混合。
由图23a和图23b可知，如SEQ ID NO:61～76所示的多肽均对IgA1具有酶切活性。
虽然本申请通过引用特定实施例的方式对发明进行特定的展现和描述，但是本领域技术人员都应该理解，在不脱离本申请披露的主旨和保护范围的情况下，上述内容还可以进行各种形式和细节上的变化。

Claims (68)

  1. 一种分离的IgA蛋白酶截短体，其包含获得自或衍生自多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的非天然截短片段，或者与所述非天然截短片段具有至少70％的序列同一性。
  2. 如权利要求1所述的IgA蛋白酶截短体，其中所述非天然截短片段在所述多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的基础上有氨基酸取代、缺失、插入或修饰，使得所述IgA蛋白酶截短体丧失或降低自酶切功能。
  3. 如权利要求2所述的IgA蛋白酶截短体，其中所述氨基酸取代、缺失、插入或修饰发生在所述多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的天然自酶切位点、所述天然自酶切位点的上游5个位点以内和/或下游5个位点以内。
  4. 如权利要求1-3中任一项所述的IgA蛋白酶截短体，其中所述非天然截短片段为获得自或衍生自多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的N末端截短片段或C末端截短片段。
  5. 如前述权利要求中任一项所述的IgA蛋白酶截短体，其中所述多枝梭菌(Clostridium ramosum)为Clostridium ramosum AK183株。
  6. 如权利要求4或5所述的IgA蛋白酶截短体，其中所述N末端截短片段包含获得自或衍生自多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的N末端第31位起的至少760个连续氨基酸的多肽片段，或者与所述多肽片段具有至少70％的序列同一性。
  7. 如前述权利要求中任一项所述的IgA蛋白酶截短体，其中所述非天然截短片段包含获得自或衍生自多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的N末端第335位起的至少456个连续氨基酸的多肽片段，或者与所述多肽片段具有至少90％或至少95％的序列同一性。
  8. 如前述权利要求中任一项所述的IgA蛋白酶截短体，其中所述多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的氨基酸序列如SEQ ID NO:1所示。
  9. 如权利要求3至8中任一项所述的IgA蛋白酶截短体，其中所述天然自酶切位点在如SEQ ID NO:1所示的氨基酸序列的第730位至第840位之间(例如，第792位至第797位之间)。
  10. 如权利要求9所述的IgA蛋白酶截短体，其中所述天然自酶切位点在如SEQ ID NO:1所示的氨基酸序列的第790位、第791位、第792位、第793位、第794位、第795位、第796位、第797位、第798位、第799位或第800位。
  11. 如前述权利要求中任一项所述的IgA蛋白酶截短体，其包含如SEQ ID NO:1所示的氨基酸序列的第31位起的至少760个(例如，至少761个、至少762个、至少763个、至少764个、至少765个、至少766个、至少767个、至少768个、至少769个、至少770个、至少771个、至少772个、至少773个、至少774个、至少775个、至少776个、至少777个、至少778个、至少779个、至少780个、至少781个、至少782个、至少783个、至少784个、至少785个、至少786个、至少787个、至少788个、至少789个、至少790个、至少791个、至少792个、至少793个、至少794个、至少795个、至少796个、至少797个、至少798个、至少799个、至少800个、至少801个、至少802个、至少803个、至少804个、至少805个、至少806个、至少807个、至少808个、至少809个、至少810个、至少900个、至少950个、至少1000个、至少1100个、至少1150个或者至少1200个)连续氨基酸的多肽片段。
  12. 如前述权利要求中任一项所述的IgA蛋白酶截短体，其包含选自下组的多肽片段：如SEQ ID NO:1所示的氨基酸序列的第31位至第790位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第31位至第792位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第31位至第798位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第31位至第807位氨基酸、如SEQ ID NO: 1所示的氨基酸序列的第31位至第816位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第31位至第833位氨基酸以及与其具有至少70％的序列同一性的多肽片段。
  13. 如前述权利要求中任一项所述的IgA蛋白酶截短体，其包含如SEQ ID NO:1所示的氨基酸序列的第335位起的至少456个(例如，至少457个、至少458个、至少459个、至少460个、至少461个、至少462个、至少463个、至少464个、至少465个、至少466个、至少467个、至少468个、至少469个、至少470个、至少471个、至少472个、至少473个、至少474个、至少475个、至少476个、至少477个、至少478个、至少479个、至少480个、至少481个、至少482个、至少483个、至少484个、至少485个、至少486个、至少487个、至少488个、至少489个、至少490个、至少491个、至少492个、至少493个、至少494个、至少495个、至少496个、至少497个、至少498个、至少499个、至少500个、至少550个、至少600个、至少650个、至少700个、至少750个、至少800个、至少850个或者至少900个)连续氨基酸的多肽片段。
  14. 如权利要求13所述的IgA蛋白酶截短体，其包含选自下组的多肽片段：如SEQ ID NO:1所示的氨基酸序列的第335位至第790位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第335位至第791位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第335位至第792位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第285位至第790位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第285位至第791位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第285位至第792位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第330位至第790位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第330位至第791位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第330位至第792位氨基酸、如SEQ ID NO:1所示的氨基酸序列的第285位至第816位氨基酸以及与其具有至少90％或至少95％的序列同一性的多肽片段。
  15. 如前述权利要求中任一项所述的IgA蛋白酶截短体，其在所述多肽片段的氨基酸序列基础上，在一个或多个位点具有氨基酸的保守替换。
  16. 如前述权利要求中任一项所述的IgA蛋白酶截短体，其中所述多肽片段在对应于SEQ ID NO:1的第844位、第862位、第931位、第933位、第978位、第1002位、第1004位中的一个或多个位置有氨基酸突变。
  17. 如权利要求16所述的IgA蛋白酶截短体，其中所述多肽片段在对应于SEQ ID NO:1的第844位、第862位、第931位、第933位、第978位、第1002位、第1004位中的一个或多个位置突变为甘氨酸。
  18. 如权利要求16或17所述的IgA蛋白酶截短体，其中所述多肽片段在对应于SEQ ID NO:1的第844位有氨基酸突变、第862位有氨基酸突变、第931位和第933位有氨基酸突变、第978位有氨基酸突变、或者第1002位和第1004位有氨基酸突变。
  19. 如权利要求16至18中任一项所述的IgA蛋白酶截短体，其中所述多肽片段的氨基酸序列如SEQ ID NO:53(也称为“PA-GA Mut”)、SEQ ID NO:54(也称为“PI-GI Mut”)、SEQ ID NO:55(也称为“PAP-GAG Mut”)、SEQ ID NO:56(也称为“PAT-GAT Mut”)或者SEQ ID NO:57(也称为“PIP-GIG Mut”)所示。
  20. 如前述权利要求中任一项所述的IgA蛋白酶截短体，其具有特异性切割人IgA的酶活性。
  21. 如权利要求20所述的IgA蛋白酶截短体，其具有特异性切割人IgA重链的酶活性。
  22. 如权利要求21所述的IgA蛋白酶截短体，其具有特异性切割人IgA重链CH1与铰链区交汇处的酶活性。
  23. 如权利要求20至22中任一项所述的IgA蛋白酶截短体，其具有特异性切割人IgA1的酶活性。
  24. 一种融合蛋白，其包含第一多肽和第二多肽，其中所述第一多肽包含获得自或衍生自多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶的全长、获得自或衍生自多枝梭菌(Clostridium ramosum)的野生型IgA蛋白酶去掉信号肽之后形成的多肽或者如前述权利要求中任一项所述的IgA蛋白酶截短体，所述第二多肽包含用于延长所述第一多肽在受试者体内半衰期的氨基酸序列。
  25. 根据权利要求24所述的融合蛋白，其中所述第一多肽包含如SEQ ID NO:1或者SEQ ID NO:42所示的序列。
  26. 如权利要求24所述的融合蛋白，其中所述第二多肽位于所述第一多肽的N末端或者C末端。
  27. 如权利要求24至26中任一项所述的融合蛋白，其中所述第一多肽和所述第二多肽之间通过连接子连接。
  28. 如权利要求24至26中任一项所述的融合蛋白，其中所述第一多肽和所述第二多肽之间直接连接。
  29. 如权利要求27所述的融合蛋白，其中所述连接子选自下组：可切割连接子、不可切割连接子、肽连接子、柔性连接子、刚性连接子、螺旋连接子和非螺旋连接子。
  30. 如权利要求29所述的融合蛋白，其中所述连接子包括肽连接子。
  31. 如权利要求30所述的融合蛋白，所述肽连接子包括含有甘氨酸和丝氨酸的连接子。
  32. 如权利要求31所述的融合蛋白，其中所述含有甘氨酸和丝氨酸的连接子包括如SEQ ID NO:21(GGGS)、SEQ ID NO:22(GGGGS)、SEQ ID NO:86(GGGGGS)或SEQ ID NO:87(GGGGGGGS)所示的一个、两个、三个、四个或更多个重复。
  33. 如权利要求30所述的融合蛋白，其中所述连接子包括如SEQ ID NO:23(GGCGGCGGTGGATCC)所示、如SEQ ID NO:58 (EEKKKEKEKEEQEERETK)所示或者如SEQ ID NO:59(HHHHHHHHHH)所示的氨基酸序列。
  34. 如权利要求24至33中任一项所述的融合蛋白，其中所述第二多肽选自：Fc结构域和白蛋白。
  35. 如权利要求34所述的融合蛋白，其中所述Fc结构域包含铰链区。
  36. 如权利要求35所述的融合蛋白，其中所述Fc结构域来源于人IgG Fc结构域。
  37. 如权利要求36所述的融合蛋白，其中所述Fc结构域来源于人IgG1Fc结构域、人IgG2Fc结构域、人IgG3Fc结构域或人IgG4Fc结构域。
  38. 如权利要求34至37中任一项所述的融合蛋白，其中所述Fc结构域包含与SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:32或SEQ ID NO:77具有至少70％、至少75％、至少80％、至少85％、至少90％或至少95％的序列同一性的氨基酸序列。
  39. 如权利要求38所述的融合蛋白，其中所述Fc结构域包含如SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:32或SEQ ID NO:77所示的氨基酸序列。
  40. 如权利要求39所述的融合蛋白，其中所述Fc结构域在对应于SEQ ID NO:25的第7位有氨基酸突变。
  41. 如权利要求40所述的融合蛋白，其中所述Fc结构域在对应于SEQ ID NO:25的第7位的氨基酸(例如，丙氨酸)突变为缬氨酸、甘氨酸、丝氨酸或者亮氨酸。
  42. 如权利要求34至41中任一项所述的融合蛋白，其中所述Fc结构域包含一个或多个延长所述融合蛋白的半衰期的突变。
  43. 如权利要求34至42中任一项所述的融合蛋白，其中所述Fc结构域与所述第一多肽的C末端或者N末端连接。
  44. 如权利要求34所述的融合蛋白，其中所述白蛋白包含人血清白蛋白的一个或多个结构域。
  45. 如权利要求44所述的融合蛋白，其中所述白蛋白包含人血清白蛋白的D3结构域。
  46. 如权利要求24至45中任一项所述的融合蛋白，其进一步包含标签。
  47. 如权利要求46所述的融合蛋白，其中所述标签选自下组：荧光标签、发光标签、纯化标签和生色标签。
  48. 如权利要求46或47所述的融合蛋白，其中所述标签选自下组：c-Myc标签、HA标签、VSV-G标签、FLAG标签、V5标签和HIS标签。
  49. 如权利要求48所述的融合蛋白，其中所述标签是包含6个、7个、8个、9个或10个组氨酸的HIS标签。
  50. 如权利要求46至49中任一项所述的融合蛋白，其中所述第二多肽位于所述第一多肽的C末端，所述标签位于所述第二多肽的C末端。
  51. 如权利要求46至50中任一项所述的融合蛋白，其中所述融合蛋白在受试者体内的血循环中的半衰期为至少1天、至少2天、至少3天、至少4天、至少5天、至少6天、至少7天、至少8天、至少9天、至少10天、至少11天、至少12天、至少13天、至少14天。
  52. 一种分离的核酸，其包含编码如权利要求1至23中任一项所述的IgA蛋白酶截短体的核苷酸序列或包含编码如权利要求24至51中任一项所述的融合蛋白的核苷酸序列。
  53. 如权利要求52所述的核酸，其包含选自下组的核苷酸序列：SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38以及与其具有至少70％的序列同一性的核苷酸序列。
  54. 一种载体，其包含如权利要求52或53所述的核酸。
  55. 一种细胞，其包含如权利要求52或53所述的核酸或如权利要求54所述的载体。
  56. 如权利要求55所述的细胞，其中所述细胞是原核生物细胞或真核生物细胞。
  57. 如权利要求56所述的细胞，其中所述原核生物细胞是大肠杆菌细胞。
  58. 如权利要求56所述的细胞，其中所述真核生物细胞是哺乳动物细胞。
  59. 如权利要求58所述的细胞，其中所述哺乳动物细胞是人细胞或中国仓鼠卵巢(CHO)细胞。
  60. 如权利要求58所述的细胞，其中所述哺乳动物细胞是人胚胎肾细胞293(HEK293细胞)。
  61. 一种药物组合物，其包含如权利要求1至23中任一项所述的IgA蛋白酶截短体、包含如权利要求24至51中任一项所述的融合蛋白、包含如权利要求52或53所述的核酸、包含如权利要求54所述的载体或者包含如权利要求55至60中任一项所述的细胞，以及药学上可接受的载体。
  62. 一种产生融合蛋白的方法，其包括培养如权利要求55至60中任一项所述的细胞的步骤。
  63. 一种治疗或预防IgA沉积相关疾病的方法，其包括向需要治疗或预防的受试者施用如权利要求1至23中任一项所述的IgA蛋白酶截短体、如权利要求24至51中任一项所述的融合蛋白或者如权利要求61所述的药物组合物。
  64. 一种治疗或预防IgA沉积相关疾病的方法，其包括向需要治疗或预防的受试者施用IgA蛋白酶或其截短体、包含所述IgA蛋白酶或其截短体的融合蛋白或者包含所述IgA蛋白酶或其截短体或者所述融合蛋白的药物组合物，其中，所述IgA蛋白酶的氨基酸序列选自下组：SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO: 70、SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76或其组合。
  65. 如权利要求1至23中任一项所述的IgA蛋白酶截短体、如权利要求24至51中任一项所述的融合蛋白或者如权利要求61所述的药物组合物在制备用于治疗或预防IgA沉积相关疾病的药物中的用途。
  66. IgA蛋白酶或其截短体、包含所述IgA蛋白酶或其截短体的融合蛋白或者包含所述IgA蛋白酶或其截短体或者所述融合蛋白的药物组合物在制备用于治疗或预防IgA沉积相关疾病的药物中的用途，其中，所述IgA蛋白酶的氨基酸序列选自下组：SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76或其组合。
  67. 如权利要求63或64所述的方法或者如权利要求65或66所述的用途，其中所述IgA沉积相关疾病包括IgA肾病、疱疹样皮炎、亨诺赫-舍恩莱因紫癜(又称IgA血管炎)、川崎病、紫癜性肾炎、IgA血管炎肾损害、IgA类风湿因子阳性的类风湿性关节炎、IgA型抗GBM病或IgA型ANCA相关血管炎。
  68. 如权利要求63或64所述的方法或者如权利要求65或66所述的用途，其中所述IgA沉积相关疾病为IgA肾病、IgA血管炎或川崎病。
PCT/CN2023/073698 2022-01-29 2023-01-29 IgA蛋白酶截短体、包含IgA蛋白酶截短体的融合蛋白及其用途 WO2023143563A1 (zh)

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CN1777673A (zh) * 2003-03-07 2006-05-24 新英格兰医学中心医院有限公司 IgA1沉积疾病的治疗
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