WO2021254479A1 - 一种免疫球蛋白降解酶IdeE的突变体 - Google Patents

一种免疫球蛋白降解酶IdeE的突变体 Download PDF

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WO2021254479A1
WO2021254479A1 PCT/CN2021/100844 CN2021100844W WO2021254479A1 WO 2021254479 A1 WO2021254479 A1 WO 2021254479A1 CN 2021100844 W CN2021100844 W CN 2021100844W WO 2021254479 A1 WO2021254479 A1 WO 2021254479A1
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mutant
protein
idee
degrading enzyme
last
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PCT/CN2021/100844
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English (en)
French (fr)
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刘彦君
王征
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上海宝济药业有限公司
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Priority to US18/001,876 priority Critical patent/US20230364207A1/en
Priority to CN202180013436.2A priority patent/CN115443288A/zh
Priority to EP21825273.2A priority patent/EP4169934A1/en
Priority to JP2022577764A priority patent/JP2023532219A/ja
Publication of WO2021254479A1 publication Critical patent/WO2021254479A1/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)
    • A61K38/4873—Cysteine endopeptidases (3.4.22), e.g. stem bromelain, papain, ficin, cathepsin H
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • 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
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/22—Cysteine endopeptidases (3.4.22)
    • C12Y304/2201—Streptopain (3.4.22.10)

Definitions

  • the invention relates to the field of biotechnology, in particular to a mutant of an immunoglobulin degrading enzyme.
  • Streptococcus pyogenes is one of the common pathogens in humans and animals. It is widely present in nature and in the oropharyngeal cavity, respiratory tract and intestinal tract of humans or animals. Streptococcal infection can cause related diseases, such as purulent dermatitis and pharyngitis, and more serious diseases such as sepsis, necrotizing fasciitis and toxic shock syndrome.
  • Immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) from Streptococcus pyogenes is a common type A Streptococcus (Group A Streptococcus pyogenes, GAS) cysteine protease with hydrolysis Peptidase activity within the peptide chain of IgG (Agniswamy J, Lei B, Musser JM etc., J Biol Chem, 2004, 279: 52789-52796. Lei B, DeLeo FR, Reid SD etc., Infect Immun, 2002, 70: 6880- 6890.
  • IdeS Streptococcus pyogenes
  • Immunoglobulin G is the main antibody component of serum, accounting for about 75% of serum immunoglobulin. It mainly plays a protective role in the body's immunity and can effectively prevent infectious diseases. In addition to its protective effect, IgG is also related to disease. In some autoimmune diseases, IgG antibodies react with the body's own molecules. In organ transplantation, IgG can cause acute transplant rejection. IdeS specifically degrades IgG, so that IgG loses its due function and achieves immunosuppression.
  • IdeS currently used in clinical practice has the problems of poor activity and high pre-existing antibodies in the human body. IdeS is a virulence factor of human pathogens. Clinical studies have found that normal people have nearly 100% of anti-IdeS antibodies detected under normal physiological conditions, which leads to inefficient use of IdeS and safety issues.
  • the IdeE protease which has a sequence homology of about 70% with IdeS, is derived from Streptococcus equi ssp.equi, a pathogenic bacteria of horses (Jonas Bengt Guss. FEMS Microbiol Lett, 2006, 262:230-235).
  • the two enzymes, IdeE and IdeS cut IgG at exactly the same position. The cutting is highly reproducible and specific, and has a very similar substrate range. It is better than IdeE from equine pathogenic bacteria. It is speculated that its pre-existing antibodies in the human body may be far lower than IdeS, and it is more suitable for the development of immunosuppressive agents for the treatment and prevention of diseases mediated by IgG antibodies.
  • wild-type IdeE like IdeS, also has the problem of low activity. Therefore, it is necessary to improve the activity of IdeE through molecular design and mutation screening, and reduce the clinical dosage, thereby reducing the risk of high-dose bacterial-derived protein. This is exactly what the present invention aims to achieve.
  • the first aspect of the present invention relates to a mutant of the immunoglobulin degrading enzyme IdeE, which is characterized in that the immunoglobulin degrading enzyme IdeE comprises the amino acid sequence shown in SEQ ID NO: 2 in the sequence table or is composed of the amino acid sequence shown in SEQ ID NO: 2 Sequence composition; the mutation is selected from the following group:
  • the immunoglobulin degrading enzyme IdeE is truncated, and the first 1, the first 2, the first 3, the first 4, the first 5, the first 6, the first 7 and the first 8 of the N-terminal are deleted.
  • immunoglobulin degrading enzyme IdeE is truncated, and the last 1, last 2, last 3, last 4, last 5, last 6, last 7, and last 8 of the C-terminal are deleted.
  • the mutant has higher activity and/or thermal stability than the immunoglobulin degrading enzyme IdeE.
  • the second aspect of the invention relates to a protein comprising the mutant of the invention.
  • the protein has a secretion signal sequence and/or methionine attached to the N-terminus of the mutant; and/or the protein has a histidine tag attached to the C-terminus of the mutant.
  • the third to fifth aspects of the present invention relate to a nucleotide encoding the mutant or protein of the present invention, an expression vector containing the nucleotide, and an expression vector containing the expression vector or expressing the mutant or protein of the present invention Host cell.
  • the sixth aspect of the present invention relates to a composition or kit, which comprises: the mutant or protein of the present invention; and optionally a substance selected from the group consisting of: a pharmaceutically acceptable carrier or excipient, an antibody, or Containing Fc protein, viral vector drugs.
  • Figure 8 shows SDS-PAGE gel electrophoresis diagrams of the cleavage products produced by the E97D_del18 mutant and IdeS cleaving human IgG1 at different concentrations.
  • Figure 9 shows SDS-PAGE gel electrophoresis diagrams of the cleavage products produced by E97D_del18 mutants and IdeZ cleaving human IgG1 at different concentrations.
  • Figure 10 is an SDS-PAGE gel electrophoresis diagram of the cleavage product produced by the E97D_del18 mutant cleaving human IVIg in mouse serum and plasma.
  • Figure 11 is an SDS-PAGE gel electrophoresis diagram of the cleavage product produced by the E97D_del18 mutant in mouse and human serum.
  • Figures 12A-12D are SDS-PAGE gel electrophoresis diagrams of cleavage products produced by cleavage of IgG with different concentrations of E97D_del18 in the serum of beagle dogs, rats, mice, rabbits, monkeys and pigs.
  • Figure 13 is an SDS-PAGE gel electrophoresis diagram of the cleavage product produced by E97D_del18 cleaving human IVIg at different times in mice.
  • the first aspect of the present invention provides a functional polypeptide that has the activity of an immunoglobulin degrading enzyme and contains a mutant based on the amino acid sequence shown in SEQ ID NO: 2, the mutant Selected from:
  • the mutant of the present invention has the function of the immunoglobulin degrading enzyme IdeE, and preferably also has improved IgG cleavage activity and thermal stability.
  • the term "having higher activity than immunoglobulin degrading enzyme IdeE" in the present invention means that the mutant has a better ability to degrade immunoglobulin than the wild-type immunoglobulin degrading enzyme IdeE.
  • higher thermal stability than IdeE means that the mutant has a better ability to degrade immunoglobulins than wild-type immunoglobulins under the same conditions after being maintained at a certain temperature for a period of time. Enzyme IdeE.
  • mutants of the present invention are preferably produced through genetic engineering recombination.
  • the mutant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least the sequence shown in SEQ ID NO: 2 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.
  • amino acid at position 8, 10, 24, 59, 97 or 280 is replaced, for example, the amino acid sequence of the obtained mutant is shown in any one of SEQ ID NO: 3-17, SEQ ID NO: 35 ;
  • the first 18 amino acids are preferably deleted.
  • the amino acid sequence of the obtained mutant is shown in any one of SEQ ID NOs: 25-29.
  • amino acid substitution is selected from the following group:
  • threonine at position 8 of SEQ ID NO: 2 is replaced with cysteine, phenylalanine, tryptophan, tyrosine, aspartic acid, glutamic acid, alanine, Any one of glycine, histidine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, valine, arginine and lysine;
  • alanine at position 10 of SEQ ID NO: 2 is replaced with cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, and lysine Any one of acid, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan and tyrosine;
  • threonine at position 24 of SEQ ID NO: 2 is replaced with alanine, cysteine, aspartic acid, asparagine, glutamic acid, phenylalanine, glycine, histamine Any one of acid, isoleucine, lysine, leucine, methionine, proline, glutamine, arginine, serine, valine, tryptophan and tyrosine;
  • alanine at position 59 of SEQ ID NO: 2 is replaced with cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, and lysine Any one of acid, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan and tyrosine;
  • the glutamic acid at position 97 of SEQ ID NO: 2 is replaced with alanine, cysteine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, and lysine Any one of acid, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan and tyrosine;
  • arginine at position 280 of SEQ ID NO: 2 is replaced with alanine, aspartic acid, glutamic acid, cysteine, serine, phenylalanine, histidine, and isoleuc Any one of acid, lysine, leucine, methionine, asparagine, proline, glutamine, threonine, valine, tryptophan, and tyrosine.
  • amino acid substitution is selected from the following group:
  • SEQ ID NO: 9 based on the 5 amino acids of SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 16.
  • the sequence obtained by substitution further deletes the first 18 amino acids at the N-terminus, and the amino acid sequence of the obtained mutant is shown in SEQ ID NO: 25-29 in the sequence listing.
  • the 5 or 10 amino acids at the C-terminus are further deleted based on the 5 amino acid substitutions of SEQ ID NO: 26-29, and the amino acid sequence of the resulting mutant is as shown in SEQ ID NO in the sequence table. : Shown from 30 to 34.
  • a combined mutation is further performed, and the amino acid sequence of the obtained mutant is shown in SEQ ID NO: 35 in the sequence table.
  • SEQ ID NO: 29 based on the 5 amino acids of SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 16.
  • the sequence obtained by substitution further deletes the first 18 amino acids at the N-terminus, and the amino acid sequence of the obtained mutant is shown in SEQ ID NO: 25-29 in the sequence listing.
  • the mutant described in the present invention can be further mutated, and the sequence of the variant obtained after further mutating has at least 60%, at least 65%, at least 70%, at least 75% with the sequence of SEQ ID NO: 2. %, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% Identity, while having the function of immunoglobulin degrading enzyme IdeE.
  • the full sequence of IdeE used in the present invention is publicly available as GenBank accession number ABF57910.1, and its sequence is provided herein as SEQ ID NO:1.
  • the sequence includes the N-terminal methionine, followed by the 33 amino acid secretion signal sequence, followed by the IdeE coding sequence.
  • the N-terminal methionine and signal sequence are usually removed to form a mature IdeE protein, the sequence of which is provided as SEQ ID NO: 2 herein.
  • all references to the numbering of amino acid positions in the immunoglobulin degrading enzyme sequences disclosed herein are based on the numbering of the corresponding positions in SEQ ID NO: 2 starting from the N-terminus.
  • the present invention also provides a protein comprising the mutant as described above.
  • the protein contains a signal peptide at the N-terminus of the mutant; preferably, the protein has a secretion signal sequence connected to the N-terminus of the mutant and is located at the N-terminus of the secretion sequence.
  • a methionine is attached to the end and/or a histidine tag is attached to the C-terminus of the mutant; more preferably, the protein from the N-terminus to the C-terminus contains or consists of the following: methionine, The secretion signal sequence and the mutant.
  • the second aspect of the present invention provides a nucleotide encoding the protein or mutant as described above.
  • the present invention also provides an expression vector containing the nucleotide.
  • the present invention also provides a host cell containing the above-mentioned expression vector, or a host cell expressing the above-mentioned protein or mutant.
  • the host cell can be a cell used for expressing proteins or polypeptides conventionally in the art, and is selected from E. coli cells, yeast cells and the like.
  • a composition comprising an immunoglobulin degrading enzyme or a mutant thereof or a protein comprising an immunoglobulin degrading enzyme or a mutant thereof, and optionally a pharmaceutically acceptable carrier or excipient.
  • the immunoglobulin degrading enzyme is selected from IdeE, IdeS and IdeZ.
  • the mutant of the immunoglobulin degrading enzyme is the mutant as described above, and the protein is a protein comprising the mutant as described above.
  • the composition of the present invention further comprises: an antibody or an Fc-containing protein.
  • the target of the antibody is selected from the group consisting of cell surface proteins, cytokines, hormones, enzymes, intracellular messengers, intercellular messengers, and immune checkpoints.
  • the composition of the present invention further comprises: a viral vector drug, preferably, the viral vector drug is selected from the group consisting of oncolytic viruses, gene therapy viruses and viral vector vaccines.
  • the composition of the present invention further comprises: a drug capable of lowering blood IgG levels, preferably, the drug lowering blood IgG levels is selected from the group consisting of FcRn antibodies, Fc fragments with high affinity to FcRn Variants.
  • the composition as described above, wherein the target of the antibody can be a cell surface protein, including but not limited to: AFP, ⁇ v integrin, ⁇ 4 ⁇ 7 integrin, BCMA, CD2, CD3, CD19, CD20, CD22, CD25, CD30, CD32, CD33, CD36, CD40, CD46, CD52, CD56, CD64, CD70, CD74, CD79, CD80, CD86, CD105, CD121, CD123, CD133, CD138, CD174, CD205, CD227, CD326, CD340, CEA, c-Met, Cripto, CA1X, Claudin18.2, ED-B, EGFR, EpCAM, EphA2, EphB2, FAP, FOLR1, GD2, Globo H, GPC3, GPNMB, HER-1, HER -2, HER-3, MAGE-A3, Mesothelin, MUC16, GPNMB, PSMA, TMEFF2, TAG-72, 5T4, ROR-1
  • the target of the antibody may be a cytokine, including but not limited to: interleukins IL-1 to IL-13, tumor necrosis factor ⁇ and ⁇ , interferon ⁇ , ⁇ and ⁇ , tumor growth factor ⁇ (TGF- ⁇ ), Colony stimulating factor (CSF) or granulocyte monocyte colony stimulating factor (GM-CSF).
  • cytokine including but not limited to: interleukins IL-1 to IL-13, tumor necrosis factor ⁇ and ⁇ , interferon ⁇ , ⁇ and ⁇ , tumor growth factor ⁇ (TGF- ⁇ ), Colony stimulating factor (CSF) or granulocyte monocyte colony stimulating factor (GM-CSF).
  • the target of the antibody can be hormones, enzymes, intracellular and intercellular messengers, such as adenosine cyclase, guanosine cyclase or phospholipase C.
  • the target of the antibody may be an immune checkpoint, and the immune checkpoint includes: CTLA-4, PD-1, PD-L1, TIM-3, LAG3, Siglec15, 4-1BB, GITR, OX40, CD40L, CD28, TIGIT, VISTA.
  • the composition as described above wherein the composition further comprises a targeted drug or a chemotherapeutic drug or an immune checkpoint blocker, and the targeted drug is selected from epigenetic drugs, targeted PI3K/Akt/ mTOR signaling pathway inhibitors and tyrosine kinase inhibitors, the chemotherapeutic drugs are selected from immunosuppressants, proteasome inhibitors, cytotoxic drugs and cell cycle non-specific drugs, and the immune checkpoint blockers are selected from anti- CTLA-4 antibody, anti-PD-1 antibody, anti-TIM-3 antibody, anti-LAG3 antibody, anti-Siglec15 antibody, anti-4-1BB antibody, anti-GITR antibody, anti-OX40 antibody, anti-CD40L antibody, anti-CD28 antibody, anti-TIGIT antibody Anti-VISTA antibody; the epigenetic drugs such as histone deacetylase inhibitors, the inhibitors that target the PI3K/Akt/mTOR signaling pathway such as Tricibine, and the tyrosine kinase inhibitor
  • the composition as described above, wherein the polypeptide drug capable of reducing blood IgG levels can block the binding of blood IgG and FcRn protein.
  • the affinity of the polypeptide to human FcRn protein is higher than that of blood IgG and human FcRn protein; the IgG is selected from IgG1, IgG2, IgG3, and IgG4.
  • the polypeptide comprises a variant of an antibody Fc fragment, the variant comprising a mutation capable of increasing the affinity of Fc and FcRn, the mutation is preferably YTE, YTEKF, LS, NHS, and the antibody Fc fragment such as Efgartigimod.
  • the variants can be monomers, dimers, and multimers.
  • the YTE, YTEKF, LS, NHS and other mutations that can be used in the present invention, and the positions of the mutations are as described by Dall'Acqua et al. (WF, DA et al. (2002). Journal of immunology (Baltimore, Md.: 1950)) 169(9): 5171-5180.), Lee et al. (Lee, CH, etc. (2019). Nat Commun 10(1): 5031.).
  • the mutation target is selected from human IgG, and the IgG is selected from IgG1, IgG2, IgG3, and IgG4.
  • Fc fragment variants that can be used in the present invention include, but are not limited to, the mutations described by Dall'Acqua et al. (WF, DA et al. (2002). Journal of immunology (Baltimore, Md.: 1950)) 169 (9 ): 5171-5180.), the mutation described by Shan et al. (Shan, L. et al. (2016).PLoS One 11(8): e0160345.), the mutation described by Lee et al. (Lee, CH et al. (2019). Nat Commun) 10(1):5031.), the mutation described by Mackness et al. (Mackness, BC et al. (2019).
  • the polypeptide comprises an antibody Fc fragment variant, the variant comprising a mutation capable of increasing the affinity of Fc and Fc ⁇ R, and the variant is preferably S239D/I322E, S239D/I322E/A330L, K326W/E333S, R214K mutations;
  • the variant is preferably a fucose-free modification.
  • the variants can be monomers, dimers, and multimers.
  • Other Fc fragment variants that can be used in the present invention include, but are not limited to, the mutations described by Wang et al. (Wang Xinhua., Mathieu Mary., Brezski Randall J. (2016). Protein Cell, 9(1), 63-73.doi:10.1007/s13238-017-0473-8).
  • the variants that can increase the affinity of Fc and FcRn also include mutations that can increase the affinity of Fc and Fc ⁇ R.
  • the variants can be monomers, dimers, and multimers.
  • the drug combination as described above, wherein the polypeptide is selected from anti-FcRn antibodies, such as Nipocalimab, Rozanolixizumab, RVT-1401, HBM9161, ALXN1830, SYNT001, Nirsevimab.
  • anti-FcRn antibodies such as Nipocalimab, Rozanolixizumab, RVT-1401, HBM9161, ALXN1830, SYNT001, Nirsevimab.
  • the pharmaceutical combination as described above wherein the polypeptide is selected from small peptide fragments that can specifically bind to FcRn, and the small peptide fragment has a length of 10-70 amino acids; the small peptide fragment is, for example, ABY-039 .
  • the polypeptide is selected from Fc multimers capable of specifically binding FcRn, such as GL-2045, M230, PRIM, HexaGard TM , CSL777, Hexavalent molecules by UCB.
  • Fc multimers capable of specifically binding FcRn, such as GL-2045, M230, PRIM, HexaGard TM , CSL777, Hexavalent molecules by UCB.
  • the polypeptide includes, but is not limited to, the polypeptide fragments described by Sockolosky et al. (Sockolosky Jonathan T, Szoka Francis C. Adv. Drug Deliv. Rev., 2015, 91: 109-24).
  • the composition as described above wherein, in the viral vector drug, the virus used in the viral vector drug is selected from the group consisting of ssDNA virus, dsDNA virus, ssRNA virus or dsRNA virus; and/or,
  • the virus used in the viral vector medicine is selected from a wild-type virus strain or a naturally attenuated strain, a genetically engineered selective attenuated strain, a gene-loaded virus strain, and a gene transcription-targeted virus strain.
  • the wild-type virus strain or naturally attenuated strain is selected from Newcastle disease virus, reovirus, mumps virus, West Nile virus, adenovirus, vaccinia virus and the like.
  • the genetically engineered selective attenuated strain achieves tumor selectivity of virus replication by manually deleting key genes, such as thymidine kinase (Thymidine kinase, TK) knockout genetically modified human herpes simplex virus I (HSV-1) ),
  • the genetically engineered selectively attenuated strains are, for example, ONYX-015, G207.
  • ONYX-015 deleted 827bp in the E1b region, and carried out a point mutation in the gene for E1B55K protein, so that the expression gene was terminated prematurely, and the E1B55K protein could not be expressed.
  • G207 deleted the ⁇ 34.5 gene, which is the determinant of HSV-1 neurotoxicity.
  • the gene-loaded virus strain is loaded with a foreign gene
  • the foreign gene is, for example, granulocyte macrophage colony stimulating factor (GM-CSF)
  • GM-CSF granulocyte macrophage colony stimulating factor
  • the gene-loaded virus strain is, for example, JX-594 or JX-594. T-VEC.
  • the gene transcription-targeted virus strain that is, a tissue or tumor-specific promoter is inserted in front of the virus essential genes to control the replication of oncolytic virus in tumor cells
  • the gene transcription-targeted virus strain is, for example, G92A .
  • the dsDNA virus is selected from the group consisting of herpes simplex virus, adenovirus, and poxvirus; more preferably, the herpes simplex virus is preferably type I herpes simplex virus HSV-1, for example, R3616, T-VEC, HF10, G207, NV1020, OrienX010, the poxvirus is selected from Pexa-Vec (vaccinia viruse), JX-594 (vaccinia viruse), GL-ONC1, Myxoma; the adenovirus is selected from Enadenotucirev, DNX -2401, C-REV, NG-348, ProsAtak, CG0070, ADV-TK, EDS01, KH901, H101, H103, VCN-01, Telomelysin (OBP-301).
  • the herpes simplex virus is preferably type I herpes simplex virus HSV-1, for example, R3616, T-VEC, HF10, G207,
  • the ssRNA virus is selected from Piconavirus, alphavirus, Retroviruses, Paramyxoviruses, Rhabdoviruses; preferably, the Piconavirus is selected from CAVATAK, PVS-RIPO, CVA21 (enterovirus), RIGVIR, and the alphavirus is selected from M1, Sindbis AR339 , Semliki Forest virus, the Retroviruses are selected from Toca511, the Paramyxoviruses are selected from MV-NIS, PV701 (Newcastle disease virus), and the Rhabdoviruses are selected from VSV-IFN ⁇ , MG1-MAGEA3, VSV-GP.
  • the Piconavirus is selected from CAVATAK, PVS-RIPO, CVA21 (enterovirus), RIGVIR
  • the alphavirus is selected from M1, Sindbis AR339 , Semliki Forest virus
  • the Retroviruses are selected from Toca511
  • the Paramyxoviruses are selected
  • the dsRNA virus is selected from Reoviruses; preferably, the Reoviruses are selected from Pelareorep, Reolysin, vaccinia virus, mumps virus, human immunodeficiency virus, HIV);
  • the RNA virus is selected from the group consisting of reovirus, coxsackievirus, polio virus, seneca valley virus, Measles virus (measles virus), Newcastle disease virus (newcastle disease virus), vesicular stomatitis virus (vesicular stomatitis virus), influenza virus.
  • the BiTE can bind to CD3 and other molecules that activate T cells, and at the same time can bind to antigen targets on the surface of cancer cells; the scFv targets immune checkpoints; the immune checkpoints include CTLA-4, PD-1, and TIM-3 , LAG3, Siglec15, 4-1BB, GITR, OX40, CD40L, CD28, TIGIT, VISTA.
  • the cytokines, chemokines such as GM-CSF, interleukin-2 (IL-2), interleukin-12 (IL-12), interferon (IFN), tumor necrosis factor (TNF), soluble CD80 , CCL3.
  • the composition as described above, wherein the gene therapy virus expresses a foreign gene the foreign gene encodes a protein required for a gene defect disease, and the protein is selected from acid ⁇ -glucosidase, copper transport ATPase2, ⁇ -galactosidase, arginine succinate synthase, ⁇ -glucocerebrosidase, ⁇ -hexosaminidase A, Cl protease inhibitor or Cl esterase inhibitor, glucose 6 phosphatase, insulin , Glucagon, Growth Hormone, Parathyroid Hormone, Growth Hormone Releasing Factor, Follicle Stimulating Hormone, Luteinizing Hormone, Human Chorionic Gonadotropin, Vascular Endothelial Growth Factor, Angiopoietin, Angiostatin, Granulocyte Colony stimulating factor, erythropoietin, connective tissue growth factor, basic fibroblast growth factor, acidic fibroblast growth factor
  • the inhibitory nucleic acid binds to a polynucleotide repeating a disease-related gene, a transcript of the gene or a polynucleotide repeat of the transcript of the gene.
  • the disease gene encodes a related protein, and the protein is selected from the group consisting of Huntingtin (HTT), androgen receptor on the X chromosome of spinal muscular atrophy, human Ataxin-1/-2/-3/-7, Cav2 .1P/Q voltage-dependent calcium channel (CACNA1A), TATA binding protein, Ataxin8 reverse chain (ATXN80S), serine/threonine protein phosphatase 2A55kDa subtype B subtype ⁇ subtype ( 1, 2, 3, 6, 7, 8, 1217), FMR1 (fragile X syndrome fragility 1), fragile X-related tremor/ataxia syndrome FMR1 (fragile X mental retardation 1), fragility XE mental retardation FMR1 (fragile X mental retardation 2) or AF4/FMR2 family member 2; troponin kinase (MT-PK) and Frataxin in myotonic dystrophy.
  • HTT Huntingtin
  • CACNA1A Cav2 .1P/
  • the disease gene is selected from, superoxide dismutase 1 (SOD1) gene mutants, genes related to the pathogenesis of Parkinson's disease and/or Alzheimer's disease, apolipoprotein B (APOB), PCSK9 , HIV infection related genes (HIVTat, TAR, HIVTAR, CCR5), influenza A virus genome/gene sequence in influenza virus infection, severe acute respiratory syndrome (SARS) coronavirus genome/gene sequence in SARS infection, respiratory tract synergy Respiratory syncytial virus genome/gene sequence in Ebola virus infection, Ebola virus genome/gene sequence in Ebola virus infection, Hepatitis B and C virus genome/gene in hepatitis B and C viruses Sequence, HSV infected herpes simplex virus (HSV) genome/gene sequence, Coxsackie virus B3 infected Coxsackie virus B3 genome/gene sequence, silencing the pathogenic alleles of genes in primary dystonia ( Allele-specific silencing) such
  • the present invention also provides a product containing the mutant or protein as described above and a therapeutic agent; the therapeutic agent is selected from viral carrier drugs, antibodies, and polypeptide drugs capable of lowering blood IgG levels.
  • the present invention also provides a kit or kit of medicine, the kit comprising: 1) a therapeutically effective amount of a drug comprising the mutant as described above; and 2) a therapeutically effective amount of a therapeutic agent; the therapeutic agent It is selected from viral vector drugs, antibodies, and polypeptide drugs that can reduce blood IgG levels; the viral vector drugs are preferably oncolytic viruses and gene therapy viruses.
  • the kit may also include 3) targeted drugs or chemotherapeutic drugs or immune checkpoint blockers.
  • the targeted drugs are selected from epigenetic drugs, inhibitors targeting the PI3K/Akt/mTOR signaling pathway, and tyrosine kinase inhibitors, and the chemotherapeutic drugs are selected from immunosuppressants, proteasome inhibitors, and cytotoxic drugs.
  • the immune checkpoint blocker is selected from the group consisting of anti-CTLA-4 antibody, anti-PD-1 antibody, anti-TIM-3 antibody, anti-LAG3 antibody, anti-Siglec15 antibody, anti-4-1BB antibody, Anti-GITR antibody, anti-OX40 antibody, anti-CD40L antibody, anti-CD28 antibody, anti-TIGIT antibody, anti-VISTA antibody; the epigenetic drugs such as histone deacetylase inhibitors, the targeting PI3K/Akt/mTOR Signal pathway inhibitors such as Tricibine, the tyrosine kinase inhibitors such as sunitinib, the immunosuppressants such as cyclophosphamide, the proteasome inhibitors such as bortezomib, the immunosuppressants such as sa Lidamide, pomalidomide, the cytotoxic drugs such as gemcitabine, temozolomide, and the cell cycle non-specific drugs such as mitoxantrone.
  • the epigenetic drugs such as histone deace
  • the kit or kit includes a kit A and a kit B, the kit A includes a therapeutically effective amount of the mutant or protein described above, and the kit B includes a therapeutically effective amount of a therapeutic agent;
  • the therapeutic agent is selected from viral vector drugs, antibodies, and polypeptide drugs that can reduce blood IgG levels; the viral vector drugs are preferably oncolytic viruses and gene therapy viruses.
  • the kit medicine box may also include a medicine box C.
  • the kit C includes targeted drugs or chemotherapeutic drugs or immune checkpoint blockers.
  • the targeted drugs are selected from epigenetic drugs, inhibitors targeting the PI3K/Akt/mTOR signaling pathway, and tyrosine kinase inhibitors, and the chemotherapeutic drugs are selected from immunosuppressants, proteasome inhibitors, and cytotoxic drugs.
  • the immune checkpoint blocker is selected from the group consisting of anti-CTLA-4 antibody, anti-PD-1 antibody, anti-TIM-3 antibody, anti-LAG3 antibody, anti-Siglec15 antibody, anti-4-1BB antibody, Anti-GITR antibody, anti-OX40 antibody, anti-CD40L antibody, anti-CD28 antibody, anti-TIGIT antibody, anti-VISTA antibody; the epigenetic drugs such as histone deacetylase inhibitors, the targeting PI3K/Akt/mTOR Signal pathway inhibitors such as Tricibine, the tyrosine kinase inhibitors such as sunitinib, the immunosuppressants such as cyclophosphamide, the proteasome inhibitors such as bortezomib, the immunosuppressants such as sa Lidamide, pomalidomide, the cytotoxic drugs such as gemcitabine, temozolomide, and the cell cycle non-specific drugs such as mitoxantrone.
  • the epigenetic drugs such as histone deace
  • the kit may include instructions concerning the administration of a therapeutically effective amount of the mutant or protein as described above and a therapeutically effective amount of the therapeutic agent (e.g., dosage information, dosing interval information).
  • the therapeutic agent is selected from viral vector drugs, antibodies, and polypeptide drugs that can reduce blood IgG levels; the viral vector drugs are preferably oncolytic viruses and gene therapy viruses.
  • a mature and complete expression system can be used to manufacture viral vector drugs.
  • Some examples of methods include the use of mammalian cell expression systems to produce viral particles, such as the use of HEK293 cells to produce adenovirus-like virus vector drugs (Freedman Joshua D, Duffy Margaret R, Lei-Rossmann Janet, etc., An Oncolytic Virus Expressing a T-cell Engager Simultaneously Targets Cancer and Immunosuppressive Stromal Cells.[J].Cancer Res.,2018,78:6852-6865).
  • the pharmaceutical carrier may be liquid, and the pharmaceutical composition may be in the form of a solution.
  • Liquid carriers are used to prepare solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions.
  • the active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier, such as water, an organic solvent, a mixture of the two, or a pharmaceutically acceptable oil or fat.
  • the pharmaceutical composition for parenteral administration is sterile, substantially isotonic, pyrogen-free, and prepared in accordance with the GMP of the FDA or similar agencies.
  • Viral vector drugs can be administered as an injectable form of a solution or suspension of the substance, where the substance is in a physiologically acceptable diluent and drug carrier (which can be a sterile liquid, such as water, oil, saline, glycerin) Or ethanol).
  • auxiliary substances such as wetting or emulsifying agents, surfactants, and pH buffering substances may be present in the composition.
  • Other components of the pharmaceutical composition are those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil and mineral oil.
  • glycols such as propylene glycol or polyethylene glycol are the preferred liquid carriers, especially for injectable solutions.
  • Viral vector drugs can be administered in the form of depot injections or implant preparations, which can be formulated to allow sustained release of the active ingredient.
  • the composition is prepared as an injectable, that is, a liquid solution or suspension; it can also be prepared in a solid form suitable for dissolution or suspension in a liquid carrier before injection.
  • nucleotide or “polynucleotide” means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally-occurring nucleotides.
  • oligonucleotide analogs which include PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, etc.) .
  • PNA peptide nucleic acid
  • DNA analogs used in antisense technology phosphorothioates, phosphoramidates, etc.
  • a specific nucleic acid sequence also implicitly encompasses conservatively modified variants (including but not limited to degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences.
  • degenerate codon substitution can be achieved by generating a sequence in which one or more selected (or all) codons are substituted with mixed bases and/or deoxyinosine residues at position 3 (Batzer et al., Nucleic Acid Res.
  • polypeptide and protein are used interchangeably herein to mean a polymer of amino acid residues. That is, the description for polypeptides is equally applicable to describing peptides and describing proteins, and vice versa.
  • the term applies to naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids.
  • the term encompasses amino acid chains of any length, including full-length proteins (ie, antigens) in which the amino acid residues are linked via covalent peptide bonds.
  • the term "host cell” means a cell containing the nucleotides of the present invention, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, pairing, or other methods known in the art.
  • the exogenous polynucleotide can be maintained as a non-integrating vector such as a plasmid or can be integrated into the host genome.
  • the host cell can be a prokaryotic cell or a eukaryotic cell.
  • transformation means a method of introducing a heterologous DNA sequence into a host cell or organism.
  • expression means the transcription and/or translation of an endogenous gene or transgene in a cell.
  • the positive progress effect of the present invention is that: the present invention provides an immunoglobulin degrading enzyme mutant whose activity and/or thermal stability is higher than that of wild-type IdeE, and its activity is also higher than that of IdeS and IdeZ (it is better than IdeS in cutting human IgG). And IdeZ is more effective, the activity is close to 2 times that of IdeS and 4 times that of IdeZ).
  • the polynucleotide sequence encoding the wild-type IdeE protein sequence (SEQ ID NO: 2) was synthesized, and the N-terminal signal peptide sequence and the C-terminal 6 ⁇ histidine tag were added. After the sequence was synthesized, it was inserted into the pET32a expression vector After the sequencing is correct, a recombinant plasmid for expressing wild-type IdeE is obtained. Based on the wild-type IdeE expression plasmid, design the degenerate primers required for the mutant library, amplify the original wild-type sequence, and insert the amplified sequence into the vector to obtain the mutant library recombinant plasmid.
  • the wild-type and mutant library recombinant plasmids were electrotransformed into E. coli BL21 Star (DE3) and inoculated on LB agarose plates containing 100ug/ml ampicillin. Cultivate overnight at 37°C until colonies grow. Pick a single colony, connect it to 200ul LB medium containing 100ug/ml ampicillin, and cultivate overnight at 37°C and 250rpm. The overnight culture was connected to 1ml of LB medium containing 100ug/ml ampicillin, cultured at 37°C for 4h, then 0.1mM IPTG was added, and the culture was continued overnight at 30°C. The overnight culture was centrifuged to collect the supernatant. SDS-PAGE was used to evaluate the concentration of mutant protein in the supernatant of mutant expression.
  • an activity assay method based on ELSIA was established.
  • the principle of the assay is to coat an ELISA plate with a human IgG1 specific antigen, and then incubate the supernatant sample containing a considerable concentration of mutant protein with human IgG1 in the well.
  • a human IgG1 detection antibody specific for the Fc portion of the antibody is used to measure the amount of intact or incompletely cleaved human IgG1 bound to the well.
  • a standard curve of IgG1 can be produced. According to the standard curve, the amount of intact or incompletely cleaved IgG1 can be calculated, and then the amount of completely cleaved IgG1 can be calculated. The ratio of fully cleaved IgG1 to the initial IgG1 was used to evaluate the activity of the mutant.
  • Example 1 In order to keep the mutant protein concentration in the supernatant harvested in Example 1 at a comparable level, SDS-PAGE detection was performed with the same loading amount, and Quantity One was used to analyze the optical density value of the target protein band in the electrophoresis pattern. In the case of the same sample amount, the higher the optical density value of the target protein band in the map, the higher the concentration. Using the IdeE supernatant as a control, the other mutant supernatants were concentrated or diluted so that the optical density values of the mutant protein bands were basically consistent with the optical density values of the IdeE control.
  • Standard curve preparation use 200ng/ml trastuzumab with reaction buffer (10mM PB, 10mM NaCl, pH6.5) at a ratio of 1:2 to gradually dilute to 3.125ng/ml, and 100ul trastuzumab of different concentrations
  • the monoclonal antibody is added to the wells of the ELISA plate for the preparation of the standard curve of the substrate (trastuzumab).
  • Cleavage reaction Dilute the supernatant after adjusting the protein concentration by 5 times with reaction buffer (10mM PB, 10mM NaCl, pH6.5), add 50ul 100ng/ml trastuzumab and 50ul diluted supernatant to enzyme label In the hole of the plate.
  • Table 1 shows the fold relationship of the activity of each mutant relative to the activity of the wild-type IdeE.
  • the 40 mutants screened in Example 1 all have higher activity than the wild-type IdeE, and 15 mutants have an activity that is twice or more than that of the wild-type IdeE.
  • a single colony was picked from each plate transformed by the above 5 single-point mutants in Example 1, and then inoculated into 3 ml of LB medium containing 100 ug/ml ampicillin, and cultured overnight at 37° C. and 250 rpm.
  • the overnight culture was connected to 50ml of LB medium containing 100ug/ml ampicillin, cultivated at 37°C until the OD600 reached 0.4-0.6, added 0.1mM IPTG, and continued to cultivate overnight at 30°C.
  • the overnight culture was centrifuged to collect the supernatant.
  • the supernatant was purified with IDA-Ni agarose magnetic beads, and the purified and eluted protein was changed into the PBS buffer system with an ultrafiltration centrifuge tube. SDS-PAGE was used to evaluate the purity of the purified mutant protein. Detect OD280 and calculate the purified mutant protein concentration based on the extinction coefficient.
  • the cleavage products produced by different concentrations of each mutant on human IgG1 were displayed on SDS-PAGE to further evaluate the activity of different mutants in cleaving human IgG1 relative to wild-type IdeE.
  • the reaction system is placed at 37°C for 30min. After mixing the sample with the same volume of 2 ⁇ SDS loading buffer, the cleavage product was detected by SDS-PAGE in a water bath at 75°C for 5 min.
  • the 7 single-point mutants are not worse than the wild-type IdeE at a concentration of 0.001 mg/ml in cleaving IgG1, indicating that the 7 single-point mutants have a cleavage activity of human IgG1 not lower than that of the wild-type IdeE. Times.
  • Example 5 Comparison of N-terminal truncation truncation mutants cleaved human IgG1 activity and thermal stability
  • Example 2 the mutant polynucleotide sequence in Table 3 was synthesized, the mutant expression recombinant plasmid was constructed, and E. coli BL21 Star (DE3) was transformed.
  • the purified mutant protein was prepared according to the method in Example 4.
  • the residual activity of the 5 truncated mutants after heat treatment at 50°C was significantly higher than that of the wild type, indicating that the thermal stability of the 5 truncated mutants was significantly improved compared to the wild type.
  • the mutant polynucleotide sequence in Table 4 was synthesized according to the method of Example 1, and the mutant expression recombinant plasmid was constructed to transform E. coli BL21 Star (DE3).
  • the purified mutant protein was prepared according to the method in Example 4.
  • the cleavage activity of the two truncated mutants was more than 2 times higher than that of the wild-type IdeE.
  • Example 7 Comparison of the activity and thermal stability of combined mutants cleaving human IgG1
  • the mutant polynucleotide sequence in Table 5 was synthesized according to the method of Example 1, and the mutant expression recombinant plasmid was constructed to transform E. coli BL21 Star (DE3).
  • the purified mutant protein was prepared according to the method in Example 4.
  • the purified mutants were diluted to 0.001 mg/mL respectively. Take 50ul of the diluted mutant or wild-type IdeE respectively and add 50ul of the reaction system containing 2mg/ml trastuzumab to start the cleavage reaction. The reaction system is placed at 37°C for 30 minutes. After mixing the sample with the same volume of 2 ⁇ SDS loading buffer, the cleavage product was detected by SDS-PAGE in a water bath at 75°C for 5 min.
  • the purified mutants were diluted to 0.1 mg/ml respectively, incubated at 50°C for 1 h, and then diluted to 0.001 mg/ml after incubation. Take 50ul of the diluted mutant or wild-type IdeE respectively and add 50ul of the reaction system containing 2mg/ml trastuzumab to start the cleavage reaction, and the reaction system is placed at 37°C for 30 minutes. After mixing the sample with the same volume of 2 ⁇ SDS loading buffer, the cleavage product was detected by SDS-PAGE in a water bath at 75°C for 5 min.
  • the E97D_del18 mutant purified in Example 7 was sequentially diluted to 20 ⁇ g/mL, 10 ⁇ g/mL, 5 ⁇ g/mL, 2.5 ⁇ g/mL and 1.25 ⁇ g/ml.
  • IdeS Item No.: A0-FRI-020, Genovis
  • IdeZ Product number: A0-FRZ-020, Genovis
  • IdeZ were diluted to 0.4U/ ⁇ l, 0.2U/ ⁇ l, 0.1U/ ⁇ l, 0.05U/ ⁇ l and 0.025U/ ⁇ l.
  • Figure 8 shows the electropherograms of the cleavage products produced by different concentrations of E97D_del18 mutants and IdeS cleavage of human IgG1. From the enzyme protein band on the electropherogram, it can be judged that the IdeS enzyme concentration in lane 1 is between the E97D_del18 mutant enzyme concentration in lanes 7 and 8, and the IdeS enzyme concentration in lane 3 is recursively between 9 and 10 The enzyme concentration of E97D_del18 mutant in lane, and the cleavage effect of IgG1 in lane 3 is between lanes 10 and 11. It can be inferred that the activity of E97D_del18 mutant in cleaving human IgG1 is close to twice that of IdeS.
  • Figure 9 shows the electropherograms of the cleavage products produced by different concentrations of E97D_del18 mutants and IdeZ cleavage of human IgG1. From the enzyme protein bands on the electrophoresis pattern, it can be judged that the IdeZ enzyme concentration in lane 1 is higher than the E97D_del18 mutant enzyme concentration in lane 7, and the IdeZ enzyme concentration in lane 3 is recursively higher than the E97D_del18 mutant enzyme concentration in lane 9, namely The enzyme concentration of the E97D_del18 mutant in lane 11 is 4 times higher than that of the E97D_del18 mutant in lane 11, and the digestion effect of IgG1 in lane 3 is close to that in lane 11. It can be inferred that the activity of E97D_del18 mutant in cutting human IgG1 is 4 times higher than that of IdeZ.
  • the in vitro cleavage activity of E97D_del18 mutant on human IgG1 was evaluated by detecting the amount of intact or single-cutting IVIg in the serum or plasma of mice treated with E97D_del18 mutant and human IVIg. According to Table 6, the different groups of mouse serum or plasma enzyme digestion system were prepared.
  • the effect of iodoacetic acid in the iodoacetic acid treatment group is to inhibit the activity of IgG degrading enzymes.
  • Figure 10 shows the electropherogram of the cleavage product produced by the E97D_del18 mutant cleaving human IVIg in mouse serum and plasma. The results show that E97D_del18 can effectively cleave human IVIg in mouse serum and plasma.
  • the system was placed at 37°C for 24h. Take 20 ⁇ l of the sample and mix it with the same volume of 2 ⁇ SDS reducing loading buffer, and then dilute it 20 times with 1 ⁇ SDS reducing loading buffer, and bathe it in a 75°C water bath for 5 minutes. SDS-PAGE detects the cleavage product.
  • Figure 11 shows the electropherogram of the cleavage product produced by the E97D_del18 mutant in mouse and human serum. The results show that E97D_del18 cleaves in human serum to produce a visible 25kD Fc fragment, but there is no such fragment in mouse serum, indicating that E97D_del18 can effectively and specifically cleave IgG1 in human serum, but has a cleavage activity on IgG1 in mouse serum. Very low or no cutting activity.
  • Example 10 E97D_del18 mutant cleaves immunoglobulins of different species
  • the in vitro cleavage activity of E97D_del18 mutant on serum immunoglobulins of different species of animals was evaluated by detecting the amount of intact or single-cut IgG in serum or plasma of E97D_del18 mutant and different species of animals. According to Table 8 and Table 9, the serum or antibody digestion system of different species was prepared.
  • the system was placed at 37°C for 1 hour, and the digested product was detected by SDS-PAGE.
  • Figures 12A-12D show the effect of E97D_del18 mutant in serum and antibodies of different species.
  • the results show that E97D_del18 can effectively cut canine IgG, rabbit IgG and mouse IgG2a, but cannot cut mouse IgG1;
  • E97D_del18 can effectively cut rabbit, dog and monkey serum IgG, of which rabbit serum IgG has the best cutting effect, while pig serum IgG has poor cutting effect , Rat and mouse serum IgG was almost untouched.
  • This assay is based on the competition between the E97D_del18 mutant and IdeS for binding to the anti-E97D_del18/IdeS antibody.
  • the pre-incubation of the test enzyme and human serum will enable the anti-E97D_del18/IdeS antibody to bind to the E97D_del18 mutant and IdeS.
  • mice Under aseptic conditions, two mice were injected intraperitoneally with human IVIg (intravenous human immunoglobulin) (two mice were parallel experiments, mouse No. 1 and No. 2), and the injection dose was 1 g/kg. 24 hours after the injection of human IVIg, IgG degrading enzyme (E97D_del18) was injected intravenously into mice at a dose of 5 mg/kg. Both mice were given blood and serum samples at 0h, 15min, 2h, 6h and 24h after the injection of E97D_del18. Take 20 ⁇ l of serum sample and mix it with the same volume of 2 ⁇ SDS non-reducing loading buffer, and then dilute it 20 times with 1 ⁇ SDS non-reducing loading buffer, bath in 75°C water for 5min, and detect by SDS-PAGE.
  • human IVIg intravenous human immunoglobulin
  • Figure 13 shows the electropherograms of the cleavage products produced by E97D_del18 cleaving human IVIg at different times in mice. The results show that E97D_del18 has a significant effect on cutting IVIg in mice, and it has basically reached complete digestion within 15 minutes.
  • Example 1 the mutant polynucleotide sequence in Table 11 was synthesized, the mutant expression recombinant plasmid was constructed, and E. coli BL21 Star (DE3) was transformed.
  • the purified mutant protein was prepared according to the method in Example 4.
  • the purified mutants were diluted to 0.001 mg/mL respectively. Take 50ul of the diluted mutant or wild-type IdeE respectively and add 50ul of the reaction system containing 2mg/ml trastuzumab to start the cleavage reaction, and the reaction system is placed at 37°C for 30 minutes. After mixing the sample with the same volume of 2 ⁇ SDS loading buffer, the cleavage product was detected by SDS-PAGE in a water bath at 75°C for 5 min.

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Abstract

提供了一种免疫球蛋白降解酶IdeE的突变体。所述的免疫球蛋白降解酶IdeE包含如序列表中SEQ ID NO:2所示的氨基酸序列；至少对所述氨基酸序列的位置8、10、24、59、97和280中的一位或者多位进行替换后得到所述突变体；所述的突变体的功能至少包含所述免疫球蛋白降解酶IdeE的功能。提供的免疫球蛋白降解酶突变体的活性和热稳定性高于野生型IdeE。

Description

一种免疫球蛋白降解酶IdeE的突变体 技术领域
本发明涉及生物技术领域，具体涉及一种免疫球蛋白的降解酶的突变体。
背景技术
化脓链球菌是人畜常见的病原菌之一，广泛存在于自然界及人或动物的口咽腔、呼吸道及肠道中。链球菌感染会引发相关疾病，较轻的病症如化脓性皮炎、咽炎，比较严重的疾病如败血症、坏死性筋膜炎和中毒性休克综合症。来自酿脓链球菌的免疫球蛋白G降解酶(Immunoglobulin G-degrading enzyme of Streptococcus pyogenes,IdeS)，是一种常见的A型链球菌(Group A Streptococcus pyogenes，GAS)半胱氨酸蛋白酶，具有水解IgG的肽链内肽酶活性(Agniswamy J,Lei B,Musser J M等,J Biol Chem,2004,279:52789-52796.Lei B,DeLeo F R,Reid S D等,Infect Immun,2002,70:6880-6890.Von Pawel-Rammingen U,Johansson B P,Bjorck L..EMBO J,2002,21:1607-1615.)。作为一种致病菌的毒力因子，它可以识别抗体下铰链区CH1和CH2结构域并特异性降解IgG，获得同质的F(ab) 2和Fc片段，帮助GAS逃避抗体介导的吞噬及细胞毒作用，从而减弱宿主免疫系统对GAS的杀伤(Von Pawel-Rammingen U.J Innate Immunity,2012,4:132-140.Su,Y.-F.等,Molecular Immunology,2011,49:134-142.)。
免疫球蛋白G(Immunoglobulin G，IgG)，是血清主要的抗体成分，约占血清免疫球蛋白的75％，在机体免疫中主要起保护作用，能有效预防感染性疾病。除了具有保护性作用之外，IgG也和疾病相关。在一些自身免疫性疾病中，IgG抗体与人体自身分子发生反应，在器官移植中IgG会引起急性移植排斥反应。IdeS特异性降解IgG，从而使IgG失去其应有功能从而实现免疫抑制。
目前应用于临床的IdeS存在活性不佳，人体预存抗体高的问题。IdeS是人类病原菌的毒力因子，临床研究发现正常人在正常生理条件下接近100％检出抗IdeS抗体，导致IdeS的使用方式给药效率不高，同时存在安全性问题。
与IdeS序列同源性为70％左右的IdeE蛋白酶来源于马链球菌兽疫亚种Streptococcus equi ssp.equi，是一种马的致病菌(Jonas
Figure PCTCN2021100844-appb-000001
Bengt Guss.FEMS Microbiol Lett,2006,262:230-235)。IdeE与IdeS这两种酶在完全相同的位置切断IgG，切割具有高度可重复性和特异性，并且有着非常类似的底物范围。优于IdeE来自马致病菌，推测其在人体内预先存在的抗体可能远低于IdeS，更适合开发用于治疗和预防由IgG抗体介导疾病的免疫抑制剂。但野生型IdeE和IdeS一样，也存在活性低的问题。因此需要通过分子设计和突变筛选提高IdeE的活性，降低临床使用剂量，从而降低由高剂量细菌来源蛋白带来的风险。这正是本发明所要达到的目的。
发明内容
本发明的第一方面涉及免疫球蛋白降解酶IdeE的突变体，其特征在于，所述的免疫球蛋白降解酶IdeE包含如序列表中SEQ ID NO:2所示的氨基酸序列或由所述氨基酸序列组成；所述突变选自下组：
(1)对所述氨基酸序列的位置8、10、24、59、97和280中的一位或者多位进行替换后得到所述突变体；和/或，
(2)对所述免疫球蛋白降解酶IdeE进行截短，删除其N端的前1个、前2个、前3个、前4个、前5个、前6个、前7个、前8个、前9个，前10个、前11个、前12个、前13个、前14个、前15个、前16个、前17个、前18个或者前19个氨基酸序列；和/或，
(3)对所述免疫球蛋白降解酶IdeE进行截短，删除其C端的最后1个、最后2个、最后3个、最后4个、最后5个、最后6个、最后7个、最后8个、最后9个或者最后10个氨基酸序列；
其中所述突变体具有高于所述免疫球蛋白降解酶IdeE的活性和/或热稳定性。
本发明的第二方面涉及包含本发明的突变体的蛋白。所述蛋白在所述突变体的N末端连接有分泌信号序列和/或甲硫氨酸；和/或所述蛋白在所述突变体的C末端连接有组氨酸标签。
本发明的第三-第五方面涉及一种编码本发明的突变体或蛋白的核苷酸，包含所述核苷酸的表达载体，以及包含所述表达载体或者表达本发明的突变体或蛋白的宿主细胞。
本发明的第六方面涉及一种组合物或试剂盒，其包含：本发明的突变体或蛋白；和任选的选自下组的物质：药学上可接受的载体或赋形剂、抗体或者含有Fc的蛋白、病毒载体药物。
附图说明
图1为7个单点突变体及野生型IdeE切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图(酶:底物＝1:1000)。
图2为7个单点突变体及野生型IdeE切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图(酶:底物＝1:2000)。
图3为5个N端截短突变体切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图(酶:底物＝1:1000)。
图4为50℃条件下保温1h后5个N端截短突变体及野生型IdeE切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图(酶:底物＝1:1000)。
图5为2个C截短突变体切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图(酶:底物＝1:1000)。
图6为5个组合突变体切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图(酶:底物＝1:2000)。
图7为50℃条件下保温1h后5个组合突变体切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图(酶:底物＝1:2000)。
图8为不同浓度的E97D_del18突变体和IdeS切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图。
图9为不同浓度的E97D_del18突变体和IdeZ切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图。
图10为E97D_del18突变体在小鼠血清和血浆中切割人IVIg产生的切割产物SDS-PAGE凝胶电泳图。
图11为E97D_del18突变体在小鼠和人血清中产生的切割产物SDS-PAGE凝胶电泳图。
图12A-12D为不同浓度的E97D_del18在比格犬、大鼠、小鼠、兔、猴和猪血清中切割IgG产生的切割产物SDS-PAGE凝胶电泳图。
图13为E97D_del18在小鼠体内不同时间内切割人IVIg产生的切割产物SDS-PAGE凝胶电泳图。
图14A和14B为具有不同突变组合的突变体和IdeE切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图(酶:底物＝1:2000)。
发明详述
I.具有免疫球蛋白降解酶活性的功能性多肽
本发明的第一方面，提供了一种功能性多肽，所述功能性多肽具有免疫球蛋白降解酶的活性并包含有基于SEQ ID NO:2所示的氨基酸序列的突变体，所述突变体选自：
(1)在SEQ ID NO:2的第8、10、24、59、97和280中的一位或者多位氨基酸进行替换后得到突变体；和/或，
(2)SEQ ID NO:2的N端的截短突变体，选自删除其N端的前1个、前2个、前3个、前4个、前5个、前6个、前7个、前8个、前9个、前10个、前11个、前12个、前13个、前14个、前15个、前16个、前17个、前18个或者前19个氨基酸序列；和/或，
(3)SEQ ID NO:2的C端的截短突变体，选自删除其C端的最后1个、最后2个、最后3个、最后4个、最后5个、最后6个、最后7个、最后8个、最后9个或者最后10个氨基酸序列。
本发明所述的突变体具有所述免疫球蛋白降解酶IdeE的功能，优选地还具有提高的IgG切割活性和热稳定性。
本发明中的术语“具有高于免疫球蛋白降解酶IdeE的活性”指的是所述突变体降解免疫球蛋白的能力优于野生型免疫球蛋白降解酶IdeE。
本发明中的术语“比IdeE具有更高的热稳定性“指的是所述突变体在某温度下维持一段时间后，降解免疫球蛋白的能力优于同等条件下的野生型免疫球蛋白降解酶IdeE。
本发明所述的突变体优选通过基因工程重组方式生产得到。
优选地，所述突变体与SEQ ID NO:2所示序列具有至少60％、至少65％、至少70％、至少75％、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％或至少99％的同一性。
更优选地，对所述位置8、10、24、59、97或者280的氨基酸进行替换，例如所得突变体的氨基酸序列如SEQ ID NO:3～17、SEQ ID NO:35中任一 所示；
或者删除所述免疫球蛋白降解酶IdeE N端的前15个、前16个、前17个、前18个或者前19个氨基酸，所得突变体的氨基酸序列如SEQ ID NO:18～22中任一所示；
或者删除所述免疫球蛋白降解酶IdeE C端的最后1个、最后5个、最后8个或者最后10个氨基酸，例如所得突变体的氨基酸序列如SEQ ID NO:23～24中任一所示；
或者对所述位置8、10、24、59、97或者280进行替换并同时删除所述免疫球蛋白降解酶IdeE N端的前15个、前16个、前17个、前18个或者前19个氨基酸，优选删除前18个氨基酸，例如所得突变体的氨基酸序列如SEQ ID NO:25～29中任一所示。
或者对所述位置8、10、24、59、97或者280进行替换并同时删除所述免疫球蛋白降解酶IdeE N端的前15个、前16个、前17个、前18个或者前19个氨基酸并同时删除所述免疫球蛋白降解酶IdeE C端的最后1个、最后5个、最后8个或者最后10个氨基酸，优选删除前18个氨基酸，优选删除后5个氨基酸，例如所得突变体的氨基酸序列如SEQ ID NO:30～34中任一所示。
在本发明一个优选的实施方案中，所述氨基酸的替换选自下组：
(1)SEQ ID NO:2的位置8处的苏氨酸被替换成半胱氨酸、苯丙氨酸、色氨酸、酪氨酸、天冬氨酸、谷氨酸、丙氨酸、甘氨酸、组氨酸、异亮氨酸、亮氨酸、蛋氨酸、天冬酰胺、脯氨酸、谷氨酰胺、丝氨酸、缬氨酸、精氨酸和赖氨酸中的任意一种；
(2)SEQ ID NO:2的位置10处的丙氨酸被替换成半胱氨酸、天冬氨酸、谷氨酸、苯丙氨酸、甘氨酸、组氨酸、异亮氨酸、赖氨酸、亮氨酸、蛋氨酸、天冬酰胺、脯氨酸、谷氨酰胺、精氨酸、丝氨酸、苏氨酸、缬氨酸、色氨酸和酪氨酸中的任意一种；
(3)SEQ ID NO:2的位置24处的苏氨酸被替换成丙氨酸、半胱氨酸、天冬氨酸、天冬酰胺、谷氨酸、苯丙氨酸、甘氨酸、组氨酸、异亮氨酸、赖氨酸、亮氨酸、蛋氨酸、脯氨酸、谷氨酰胺、精氨酸、丝氨酸、缬氨酸、色氨酸和酪氨酸中的任意一种；
(4)SEQ ID NO:2的位置59处的丙氨酸被替换成半胱氨酸、天冬氨酸、 谷氨酸、苯丙氨酸、甘氨酸、组氨酸、异亮氨酸、赖氨酸、亮氨酸、蛋氨酸、天冬酰胺、脯氨酸、谷氨酰胺、精氨酸、丝氨酸、苏氨酸、缬氨酸、色氨酸和酪氨酸中的任意一种；
(5)SEQ ID NO:2的位置97处的谷氨酸被替换成丙氨酸、半胱氨酸、天冬氨酸、苯丙氨酸、甘氨酸、组氨酸、异亮氨酸、赖氨酸、亮氨酸、蛋氨酸、天冬酰胺、脯氨酸、谷氨酰胺、精氨酸、丝氨酸、苏氨酸、缬氨酸、色氨酸和酪氨酸中的任意一种；
(6)SEQ ID NO:2的位置280处的精氨酸被替换成丙氨酸、天冬氨酸、谷氨酸、半胱氨酸、丝氨酸、苯丙氨酸、组氨酸、异亮氨酸、赖氨酸、亮氨酸、蛋氨酸、天冬酰胺、脯氨酸、谷氨酰胺、苏氨酸、缬氨酸、色氨酸和酪氨酸中的任意一种。
在本发明一个更优选的实施方案中，所述氨基酸的替换选自下组：
(1)SEQ ID NO:2的位置8处的苏氨酸被替换成天冬氨酸、谷氨酸、色氨酸或酪氨酸；
(2)SEQ ID NO:2的位置10处的丙氨酸被替换成赖氨酸或精氨酸；
(3)SEQ ID NO:2的位置24处的苏氨酸被替换成丙氨酸、甘氨酸或丝氨酸；
(4)SEQ ID NO:2的位置59处的丙氨酸被替换成异亮氨酸、亮氨酸或缬氨酸；
(5)SEQ ID NO:2的位置97处的谷氨酸被替换成天冬酰胺；和/或
(6)SEQ ID NO:2的位置280处的精氨酸被替换成组氨酸或赖氨酸。
在另一优选的实施方案中，基于SEQ ID NO:9、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:1、SEQ ID NO:5和SEQ ID NO:16这5个经氨基酸替换得到的序列进一步删除其N端的前18个氨基酸，所得突变体的氨基酸序列如序列表中SEQ ID NO:25～29所示。
在另一优选的实施方案中，基于SEQ ID NO:26～29这5个经氨基酸替换得到的序列进一步删除其C端的5个或10氨基酸，所得突变体的氨基酸序列如序列表中SEQ ID NO:30～34所示。
在另一优选的实施方案中，基于SEQ ID NO:14～16这3个突变体进一步进行组合突变，所得突变体的氨基酸序列如序列表中SEQ ID NO:35所示。在另一优选的实施方案中，基于SEQ ID NO:9、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:1、SEQ ID NO:5和SEQ ID NO:16这5个经氨基酸替换得到的 序列进一步删除其N端的前18个氨基酸，所得突变体的氨基酸序列如序列表中SEQ ID NO:25～29所示。
优选地，还可以对本发明中所述的突变体进行进一步的突变，进一步突变后所得的变体的序列与SEQ ID NO:2的序列具有至少60％、至少65％、至少70％、至少75％、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％或至少99％的同一性，同时具有免疫球蛋白降解酶IdeE的功能。
本发明使用的IdeE全序列作为GenBank登录号ABF57910.1是公众可获得的，其序列在本文中作为SEQ ID NO:1提供。该序列包括N末端甲硫氨酸，接着是33个氨基酸的分泌信号序列，接着是IdeE编码序列。N末端甲硫氨酸和信号序列通常被去除以形成成熟的IdeE蛋白，其序列在本文中作为SEQ ID NO:2提供。除非另有说明，对本文公开的免疫球蛋白降解酶序列中氨基酸位置的编号的所有提及均基于从N末端开始的SEQ ID NO:2中相应位置的编号。
本发明还提供一种包含如上所述的突变体的蛋白。
在一个优选的实施例中，所述蛋白为在上述突变体的N末端包含信号肽；优选地，所述蛋白在所述突变体的N末端连接有分泌信号序列并在所述分泌序列的N末端连接有甲硫氨酸和/或在所述突变体的C末端连接有组氨酸标签；更优选地，所述蛋白从N末端至C末端包含如下或由如下组成：甲硫氨酸、分泌信号序列和所述突变体。
本发明的第二方面，提供了一种编码如上所述的蛋白或者突变体的核苷酸。
本发明还提供一种包含所述核苷酸的表达载体。
本发明还提供一种包含如上所述的表达载体的宿主细胞，或者表达如上所述的蛋白或者突变体的宿主细胞。
所述的宿主细胞可为本领域常规的用于表达蛋白或者多肽的细胞，选自大肠杆菌细胞、酵母细胞等。
II.药物组合
本发明的第三方面，提供了一种组合物，其包括免疫球蛋白降解酶或其突变体或者包含免疫球蛋白降解酶或其突变体的蛋白，以及任选的药学上可接受的载体或赋形剂。在一个具体的实施方案中，所述免疫球蛋白降解酶选自IdeE、IdeS和IdeZ。在一个具体的实施方案中，所述免疫球蛋白降解酶的 突变体为如上所述的突变体，所述蛋白为包含如上所述的突变体的蛋白。在一个具体的实施方案中，本发明的组合物进一步包含：抗体或者含有Fc的蛋白。在一个具体的实施方案中，所述抗体的靶点选自下组：细胞表面蛋白、细胞因子、激素、酶、胞内信使、胞间信使和免疫检查点。在一个具体的实施方案中，本发明的组合物进一步包含：病毒载体药物，优选地，所述病毒载体药物选自下组：溶瘤病毒、基因治疗病毒和病毒载体疫苗。在一个具体的实施方案中，本发明的组合物进一步包含：能降低血液IgG水平的药物，优选的，所述降低血液IgG水平的药物选自下组：FcRn抗体、与FcRn高亲和力的Fc片段变体。
2.1抗体的靶点
优选地，如上所述的组合物，其中，所述抗体的靶点可为细胞表面蛋白，包括但不限于：AFP、αv整联蛋白(integrin)、α4β7整联蛋白、BCMA、CD2、CD3、CD19、CD20、CD22、CD25、CD30、CD32、CD33、CD36、CD40、CD46、CD52、CD56、CD64、CD70、CD74、CD79、CD80、CD86、CD105、CD121、CD123、CD133、CD138、CD174、CD205、CD227、CD326、CD340、CEA、c-Met、Cripto、CA1X、Claudin18.2、ED-B、EGFR、EpCAM、EphA2、EphB2、FAP、FOLR1、GD2、Globo H、GPC3、GPNMB、HER-1、HER-2、HER-3、MAGE-A3、Mesothelin、MUC16、GPNMB、PSMA、TMEFF2、TAG-72、5T4、ROR-1、Sca-1、SP、VEGF或WT1。
所述抗体的靶点可为细胞因子，包括但不限于：白介素IL-1至IL-13、肿瘤坏死因子α和β、干扰素α、β和γ、肿瘤生长因子β(TGF-β)、集落刺激因子(CSF)或粒细胞单核细胞集落刺激因子(GM-CSF)。见HumanCytokines:Handbook for Basic&Clinical Research(Aggrawal等编,Blackwell Scientific,Boston,MA 1991)。
所述抗体的靶点可为激素、酶、胞内和胞间信使，例如：腺苷环化酶、鸟苷环化酶或磷脂酶C。
所述抗体的靶点可为免疫检查点，所述的免疫检查点包括：CTLA-4、PD-1、PD-L1、TIM-3、LAG3、Siglec15、4-1BB、GITR、OX40、CD40L、CD28、TIGIT、VISTA。
2.2靶向药物
优选地，如上所述组合物，其中，所述组合物还包括靶向药物或化疗药 物或免疫检查点阻断剂，所述靶向药物选自表观遗传学药物、靶向PI3K/Akt/mTOR信号通路的抑制剂和酪氨酸激酶抑制剂，所述化疗药物选自免疫抑制剂、蛋白酶体抑制剂、细胞毒药物和细胞周期非特异性药物，所述免疫检查点阻断剂选自抗CTLA-4抗体、抗PD-1抗体、抗TIM-3抗体、抗LAG3抗体、抗Siglec15抗体、抗4-1BB抗体、抗GITR抗体、抗OX40抗体、抗CD40L抗体、抗CD28抗体、抗TIGIT抗体、抗VISTA抗体；所述表观遗传学药物例如组蛋白去乙酰化酶抑制剂，所述靶向PI3K/Akt/mTOR信号通路的抑制剂例如Tricibine，所述酪氨酸激酶抑制剂例如舒尼替尼，所述免疫抑制剂例如环磷酰胺，所述蛋白酶体抑制剂例如硼替佐米，所述免疫抑制剂例如沙利度胺、泊马度胺，所述细胞毒药物例如吉西他滨、替莫唑胺，所述细胞周期非特异性药物例如米托蒽醌。
2.3能降低血液IgG水平的药物
优选地，如上所述的组合物，其中，所述能降低血液IgG水平的多肽药物能够阻断血液IgG和FcRn蛋白的结合。优选地，所述多肽与人FcRn蛋白的亲和力高于血液IgG和人FcRn蛋白的亲和力；所述IgG选自IgG1、IgG2、IgG3、IgG4。优选地，所述多肽包含抗体Fc片段变体，所述变体包含能够提高Fc和FcRn亲和力的突变，所述突变优选为YTE、YTEKF、LS、NHS，所述抗体Fc片段例如Efgartigimod。所述变体可以为单体、二聚体、多聚体。可用于本发明的所述YTE、YTEKF、LS、NHS等突变，所述突变的位置分别如Dall'Acqua等所描述(WF,D.A.等(2002).Journal of immunology(Baltimore,Md.:1950)169(9):5171-5180.)、Lee等所描述(Lee,C.H.等(2019).Nat Commun 10(1):5031.)。所述突变对象选自人IgG，所述IgG选自IgG1、IgG2、IgG3、IgG4。
可用于本发明的其他Fc片段变体，所述变体包含包括但不限于Dall'Acqua等描述的突变(WF,D.A.等(2002).Journal of immunology(Baltimore,Md.:1950)169(9):5171-5180.)、Shan等描述的突变(Shan,L.等(2016).PLoS One 11(8):e0160345.)、Lee等描述的突变(Lee,C.H.等(2019).Nat Commun 10(1):5031.)、Mackness等描述的突变(Mackness,B.C.等(2019).MAbs 11(7):1276-1288.)、Christophe等描述的突变(Dumet Christophe,Pottier Jérémy,Gouilleux-Gruart Valérie等,MAbs,2019,11:1341-1350.)。
优选地，所述多肽包含抗体Fc片段变体，所述变体包含能够提高Fc和 FcγR亲和力的突变，所述变体优选为S239D/I322E、S239D/I322E/A330L、K326W/E333S、R214K突变；所述变体优选为无岩藻糖修饰。所述变体可以为单体、二聚体、多聚体。可用于本发明的其他Fc片段变体，所述变体包含包括但不限于Wang等描述的突变(Wang Xinhua.,Mathieu Mary.,Brezski Randall J.(2018).Protein Cell,9(1),63-73.doi:10.1007/s13238-017-0473-8)。
优选地，所述包含能够提高Fc和FcRn亲和力的变体同时包含能够提高Fc和FcγR亲和力的突变。所述变体可以为单体、二聚体、多聚体。
优选地，如上所述的药物组合，其中，所述多肽选自抗FcRn抗体，所述抗体例如Nipocalimab、Rozanolixizumab、RVT-1401、HBM9161、ALXN1830、SYNT001、Nirsevimab。
优选地，如上所述的药物组合，其中，所述多肽选自能特异性结合FcRn的小肽片段，所述小肽片段的长度为10-70个氨基酸；所述小肽片段例如ABY-039。
优选地，所述多肽选自能特异性结合FcRn的Fc多聚体，所述Fc多聚体例如GL-2045、M230、PRIM、HexaGard TM、CSL777、Hexavalent molecules by UCB。
优选地，所述多肽包括但不限于Sockolosky等描述的多肽片段(Sockolosky Jonathan T,Szoka Francis C.Adv.Drug Deliv.Rev.,2015,91:109-24)。
2.4病毒载体药物
优选地，如上所述的组合物，其中，所述病毒载体药物中，所述病毒载体药物所用的病毒选自ssDNA类病毒、dsDNA类病毒、ssRNA类病毒或dsRNA类病毒；和/或，所述病毒载体药物所用的病毒选自野生型病毒株或自然减毒株、基因工程选择性减毒株、基因加载型病毒株、基因转录靶向型病毒株。
优选地，所述野生型病毒株或自然减毒株选自新城疫病毒、呼肠孤病毒、流行性腮腺炎病毒、西尼罗河病毒、腺病毒、牛痘病毒等。
优选地，所述基因工程选择性减毒株通过人工方式删除关键基因而实现病毒复制的肿瘤选择性，例如胸苷激酶(Thymidinekinase,TK)敲除的基因改造人单纯疱疹病毒I(HSV-1)，所述基因工程选择性减毒株例如ONYX-015、G207。ONYX-015在E1b区域删除了827bp，并且在针对E1B55K蛋白的基因进行点突变，使其表达基因提前终止，无法表达E1B55K蛋白。G207删除了γ34.5基因，该基因为HSV-1的神经毒性决定因素。
优选地，所述基因加载型病毒株加载了外源基因，所述外源基因例如为 粒细胞巨噬细胞集落刺激因子(GM-CSF)，所述基因加载型病毒株例如为JX-594或T-VEC。
优选地，所述基因转录靶向型病毒株，即在病毒必需基因前插入组织或肿瘤特异性启动子来控制溶瘤病毒在肿瘤细胞内复制，所述基因转录靶向型病毒株例如为G92A。
优选地，如上所述的药物组合，其中，所述ssDNA类病毒选自细小病毒(parvovirus)，优选所述细小病毒为H-1PV病毒。
优选地，所述dsDNA类病毒选自单纯疱疹病毒(herpes simplex virus)、腺病毒(adeno virus)、poxvirus；更优选地，所述单纯疱疹病毒优选为I型单纯疱疹病毒HSV-1，例如为R3616、T-VEC、HF10、G207、NV1020、OrienX010，所述poxvirus选自Pexa-Vec(vaccinia viruse)、JX-594(vaccinia viruse)、GL-ONC1、Myxoma；所述腺病毒选自Enadenotucirev、DNX-2401、C-REV、NG-348、ProsAtak、CG0070、ADV-TK、EDS01、KH901、H101、H103、VCN-01、Telomelysin(OBP-301)。
优选地，所述ssRNA类病毒选自Picornavirus、alphavirus、Retroviruses、Paramyxoviruses、Rhabdoviruses；优选地，所述Picornavirus选自CAVATAK、PVS-RIPO、CVA21(enterovirus)、RIGVIR，所述alphavirus选自M1、Sindbis AR339、Semliki Forest virus，所述Retroviruses选自Toca511，所述Paramyxoviruses选自MV-NIS、PV701(Newcastle disease virus)，所述Rhabdoviruses选自VSV-IFNβ、MG1-MAGEA3、VSV-GP。
优选地，所述dsRNA类病毒选自Reoviruses；优选地，所述Reoviruses选自Pelareorep、呼肠孤病毒(Reolysin)、牛痘病毒(vaccinia virus)、腮腺炎病毒、人类免疫缺陷病毒(human immunodeficiency virus，HIV)；优选的，所述RNA类病毒选自呼肠孤病毒(reovirus)、柯萨奇病毒(coxsackievirus)、脊髓灰质炎病毒(polio virus)、猪塞内加谷病毒(seneca valley virus)、麻疹病毒(measles virus)、新城疫病毒(newcastle disease virus)、水泡性口炎病毒(vesicular stomatitis virus)、流感病毒。
优选地，如上所述的药物组合，其中，所述溶瘤病毒表达外源基因，所述外源基因优选双特异性T细胞结合子(Bispecific T cell engagers，BiTE)、scFv片段、细胞因子、趋化因子。所述BiTE能结合CD3等激活T细胞的分子，同时能结合癌细胞表面的抗原靶点；所述scFv靶向免疫检查点；所述免 疫检查点包括CTLA-4、PD-1、TIM-3、LAG3、Siglec15、4-1BB、GITR、OX40、CD40L、CD28、TIGIT、VISTA。所述细胞因子、趋化因子例如GM-CSF、白细胞介素-2(IL-2)、白细胞介素-12(IL-12)、干扰素(IFN)、肿瘤坏死因子(TNF)、可溶性CD80、CCL3。
2.5基因治疗药物
优选地，如上所述的组合物，其中，所述基因治疗病毒表达外源基因，所述外源基因编码基因缺陷疾病所需的蛋白，所述蛋白选自酸性α-葡糖苷酶、铜转运ATPase2、α半乳糖苷酶、精氨酸琥珀酸酯合酶、β-葡萄糖脑苷脂酶、β-己糖胺酶A、Cl蛋白酶抑制剂或Cl酯酶抑制剂、葡萄糖6磷酸酶、胰岛素、胰高血糖素、生长激素、甲状旁腺激素、生长激素释放因子、卵泡刺激素、黄体生成激素、人绒毛膜促性腺激素、血管内皮生长因子、血管生成素、血管生成抑制素、粒细胞集落刺激因子、促红细胞生成素、结缔组织生长因子、碱性成纤维细胞生长因子、酸性成纤维细胞生长因子、表皮生长因子、转化生长因子a、血小板衍生生长因子、胰岛素生长因子I和II、TGF、骨形态发生蛋白、神经生长因子、脑源性神经营养因子、神经营养蛋白NT-3和NT4/5、睫状神经营养因子、神经胶质细胞系衍生神经营养因子、神经营养素、凝集素、netrin-1和netrin-2、肝细胞生长因子、ephrins、酪氨酸羟化酶、血小板生成素、白介素(IL-1至IL-36等)、单核细胞趋化蛋白、白血病抑制因子、粒细胞巨噬细胞的蛋白质集落刺激因子、Fas配体、肿瘤坏死因子a和b、干扰素a/b/g、干细胞因子、flk-2/flt3配体、IgM，IgA，IgD和IgE、嵌合免疫球蛋白、人源化抗体、单个链抗体、T细胞受体、嵌合T细胞受体、单链T细胞受体、I类和II类MHC分子、囊性纤维化跨膜调节蛋白、凝血(凝血)因子(因子XIII，因子IX，因子VIII，因子X，因子VII，因子VIIa，蛋白C等)、视网膜色素上皮特异性65kDa蛋白、LDL受体、脂蛋白脂肪酶、鸟氨酸转氨甲酰酶、β-球蛋白、α-球蛋白、血影蛋白、α-抗胰蛋白酶腺苷脱氨酶、金属转运蛋白(ATP7A或ATP7)、磺酰胺酶、参与溶酶体贮积病的酶(ARSA)、次黄嘌呤鸟嘌呤磷酸核糖基转移酶、b-25葡糖脑苷脂酶、鞘磷脂酶、溶酶体己糖胺酶、支链酮酸脱氢酶。
优选地，如上所述的组合物，其中，所述基因治疗病毒携带外源基因，所述外源基因编码选自siRNA，反义分子，miRNA，RNAi，核酶和shRNA的抑制性核酸。所述抑制性核酸结合至多核苷酸重复疾病相关的基因，该基 因的转录物或该基因的转录物的多核苷酸重复。所述疾病基因编码相关蛋白，所述蛋白选自选自亨廷顿蛋白(HTT)、脊髓球肌萎缩症X染色体上的雄激素受体、人Ataxin-1/-2/-3/-7、Cav2.1P/Q电压依赖性钙通道(CACNA1A)、TATA结合蛋白、Ataxin8反向链(ATXN80S)、丝氨酸/苏氨酸蛋白磷酸酶2A55kDa脊髓小脑性共济失调的亚型B亚型β亚型(1、2、3、6、7、8、1217型)、FMR1(脆性X综合征的脆弱性1)、脆性X相关性震颤/共济失调综合征的FMR1(脆性X智力障碍1)、脆性XE智力低下的FMR1(脆性X智力障碍2)或AF4/FMR2家庭成员2；肌强直性营养不良中的肌钙蛋白激酶(MT-PK)、Frataxin。所述疾病基因选自，超氧化物歧化酶1(SOD1)基因的突变体、与帕金森氏病和/或阿尔茨海默氏病发病机理有关的基因、载脂蛋白B(APOB)、PCSK9、HIV感染相关基因(HIVTat、TAR、HIVTAR、CCR5)、流感病毒感染中的甲型流感病毒基因组/基因序列、SARS感染中的严重急性呼吸综合征(SARS)冠状病毒基因组/基因序列、呼吸道合胞病毒感染中的呼吸道合胞病毒基因组/基因序列、埃博拉病毒感染中的埃博拉病毒基因组/基因序列、乙型和丙型肝炎病毒在乙型和丙型肝炎病毒中的基因组/基因序列、HSV感染的单纯疱疹病毒(HSV)基因组/基因序列、柯萨奇病毒B3感染的柯萨奇病毒B3基因组/基因序列、沉默原发性肌张力障碍中基因的致病性等位基因(等位基因特异性沉默)如torsinA、在移植中特异性泛I类和HLA等位基因、常染色体显性遗传性视网膜色素变性中的突变和视紫红质基因。
III.产品
本发明还提供了一种产品，所述产品含有如上所述的突变体或者蛋白和治疗剂；所述治疗剂选自病毒载体药物、抗体、能降低血液IgG水平的多肽药物。
本发明还提供了一种试剂盒或套装药盒，所述试剂盒包含：1)治疗有效量的包含如上所述的突变体的药物；和2)治疗有效量的治疗剂；所述治疗剂选自病毒载体药物、抗体、能降低血液IgG水平的多肽药物；所述病毒载体药物优选溶瘤病毒、基因治疗病毒。所述试剂盒还可以包括3)靶向药物或化疗药物或免疫检查点阻断剂。所述靶向药物选自表观遗传学药物、靶向PI3K/Akt/mTOR信号通路的抑制剂和酪氨酸激酶抑制剂，所述化疗药物选自免疫抑制剂、蛋白酶体抑制剂、细胞毒药物和细胞周期非特异性药物，所述免疫检查点阻断剂选自抗CTLA-4抗体、抗PD-1抗体、抗TIM-3抗体、抗 LAG3抗体、抗Siglec15抗体、抗4-1BB抗体、抗GITR抗体、抗OX40抗体、抗CD40L抗体、抗CD28抗体、抗TIGIT抗体、抗VISTA抗体；所述表观遗传学药物例如组蛋白去乙酰化酶抑制剂，所述靶向PI3K/Akt/mTOR信号通路的抑制剂例如Tricibine，所述酪氨酸激酶抑制剂例如舒尼替尼，所述免疫抑制剂例如环磷酰胺，所述蛋白酶体抑制剂例如硼替佐米，所述免疫抑制剂例如沙利度胺、泊马度胺，所述细胞毒药物例如吉西他滨、替莫唑胺，所述细胞周期非特异性药物例如米托蒽醌。
所述试剂盒或套装药盒包括药盒A和药盒B，所述药盒A包括治疗有效量的如上所述突变体或蛋白，所述药盒B包括治疗有效量的治疗剂；所述治疗剂选自病毒载体药物、抗体、能降低血液IgG水平的多肽药物；所述病毒载体药物优选溶瘤病毒、基因治疗病毒。所述套装药盒还可以包括药盒C。所述药盒C包括靶向药物或化疗药物或免疫检查点阻断剂。所述靶向药物选自表观遗传学药物、靶向PI3K/Akt/mTOR信号通路的抑制剂和酪氨酸激酶抑制剂，所述化疗药物选自免疫抑制剂、蛋白酶体抑制剂、细胞毒药物和细胞周期非特异性药物，所述免疫检查点阻断剂选自抗CTLA-4抗体、抗PD-1抗体、抗TIM-3抗体、抗LAG3抗体、抗Siglec15抗体、抗4-1BB抗体、抗GITR抗体、抗OX40抗体、抗CD40L抗体、抗CD28抗体、抗TIGIT抗体、抗VISTA抗体；所述表观遗传学药物例如组蛋白去乙酰化酶抑制剂，所述靶向PI3K/Akt/mTOR信号通路的抑制剂例如Tricibine，所述酪氨酸激酶抑制剂例如舒尼替尼，所述免疫抑制剂例如环磷酰胺，所述蛋白酶体抑制剂例如硼替佐米，所述免疫抑制剂例如沙利度胺、泊马度胺，所述细胞毒药物例如吉西他滨、替莫唑胺，所述细胞周期非特异性药物例如米托蒽醌。
该试剂盒可以包含涉及治疗有效量的包含如上所述的突变体或者蛋白和治疗有效量的治疗剂施用(例如，剂量信息、给药时间间隔信息)的说明书。所述治疗剂选自病毒载体药物、抗体、能降低血液IgG水平的多肽药物；所述病毒载体药物优选溶瘤病毒、基因治疗病毒。
可利用成熟完善的表达系统来制造病毒载体药物。一些方法示例包括利用哺乳动物细胞表达系统来产生病毒颗粒，例如使用HEK293细胞生产腺病毒类病毒载体药物(Freedman Joshua D,Duffy Margaret R,Lei-Rossmann Janet等,An Oncolytic Virus Expressing a T-cell Engager Simultaneously Targets Cancer and Immunosuppressive Stromal Cells.[J].Cancer Res.,2018,78:6852-6865)。
药物载剂可以是液体，并且药物组合物可以为溶液形式。液体载剂用于制备溶液、悬浮液、乳液、糖浆、酏剂和加压组合物。活性成分可以溶解或悬浮在药学上可接受的液体载剂中，例如水、有机溶剂、二者的混合物或药学上可接受的油或脂肪。
用于肠胃外施用的药物组合物是无菌的、基本上等渗的、无热原的，并根据FDA或类似机构的GMP来制备。病毒载体药物可以作为该物质的溶液或悬浮液的可注射剂型施用，其中，该物质处在生理上可接受的稀释剂和药物载剂(可以是无菌液体，例如水、油、盐水、甘油或乙醇)中。另外，组合物中可存在辅助物质，例如润湿剂或乳化剂、表面活性剂和pH缓冲物质等。药物组合物的其他组分有石油、动物、植物或合成来源的组分，例如花生油、大豆油和矿物油。通常，例如丙二醇或聚乙二醇等二醇是优选的液体载剂，对于可注射溶液尤其如此。病毒载体药物可以以积存注射剂或植入制剂的形式施用，这些形式能够被配制成允许活性成分持续释放。通常，将组合物制备成可注射物，即液体溶液或悬浮液；也可以制备成适合于在注射前溶解或悬浮在液体载质中的固体形式。
除非另外定义，否则本文所用的所有技术及科学术语都具有与本发明所属领域的普通技术人员通常所了解相同的含义。虽然在本发明的实践或测试中可使用与本文所述者类似或等效的任何方法、装置和材料，但现在描述优选方法、装置和材料。
术语“核苷酸”或者“多核苷酸”意指单股或双股形式的脱氧核糖核苷酸、脱氧核糖核苷、核糖核苷或核糖核苷酸及其聚合物。除非特定限制，否则所述术语涵盖含有天然核苷酸的已知类似物的核酸，所述类似物具有类似于参考核酸的结合特性并以类似于天然产生的核苷酸的方式进行代谢。除非另外特定限制，否则所述术语也意指寡核苷酸类似物，其包括PNA(肽核酸)、在反义技术中所用的DNA类似物(硫代磷酸酯、磷酰胺酸酯等等)。除非另外指定，否则特定核酸序列也隐含地涵盖其保守修饰的变异体(包括(但不限于)简并密码子取代)和互补序列以及明确指定的序列。特定而言，可通过产生其中一个或一个以上所选(或所有)密码子的第3位经混合碱基和/或脱氧肌苷残基取代的序列来实现简并密码子取代(Batzer等,Nucleic Acid Res.19:5081(1991)；Ohtsuka等,J.Biol.Chem.260:2605-2608(1985)；和Cassol等,(1992)；Rossolini等,Mol Cell.Probes8:91-98(1994))。
术语“多肽”和“蛋白”在本文中互换使用以意指氨基酸残基的聚合物。即，针对多肽的描述同样适用于描述肽和描述蛋白，且反之亦然。所述术语适用于天然产生氨基酸聚合物以及其中一个或一个以上氨基酸残基为非天然编码氨基酸的氨基酸聚合物。如本文中所使用，所述术语涵盖任何长度的氨基酸链，其包括全长蛋白(即抗原)，其中氨基酸残基经由共价肽键连接。
术语“宿主细胞”意指包含本发明核苷酸的细胞，而不管使用何种方法进行插入以产生重组宿主细胞，例如直接摄取、转导、配对或所属领域中已知的其它方法。外源性多核苷酸可保持为例如质粒的非整合载体或者可整合入宿主基因组中。宿主细胞可为原核细胞或真核细胞。
术语“转化”意指将异源性DNA序列引入到宿主细胞或有机体的方法。术语“表达”意指内源性基因或转基因在细胞中的转录和/或翻译。
本发明的积极进步效果在于：本发明提供一种免疫球蛋白降解酶突变体，其活性和/或热稳定性高于野生型IdeE，活性也高于IdeS和IdeZ(在切割人IgG上比IdeS和IdeZ更有效，活性接近IdeS的2倍，超过IdeZ的4倍)。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例1.突变体文库的设计和表达
设计构建野生型IdeE蛋白序列突变文库，经过筛选从中获得40个突变体菌株。
通过密码子优化后合成编码野生型IdeE蛋白序列(SEQ ID NO:2)的多核苷酸序列，并加入N端信号肽序列和C末端6×组氨酸标签，序列合成后插入到pET32a表达载体中，测序正确后获得用于表达野生型IdeE的重组质粒。基于野生型IdeE的表达质粒，设计突变文库所需兼并引物，对原始野生型序列进行扩增，扩增后序列插入到载体上获得突变文库重组质粒。将野生型和突变文库重组质粒电转化到大肠杆菌BL21 Star(DE3)中，并接种在含100ug/ml氨苄青霉素的LB琼脂糖平板上。37℃培养过夜至菌落长出。挑取单个菌落，接于200ul含100ug/ml氨苄青霉素的LB培养基中，37℃、250rpm培养过夜。过夜培养物接于1ml含100ug/ml氨苄青霉素的LB培养基中， 37℃培养4h后加入0.1mM IPTG，30℃继续培养过夜。过夜培养物通过离心收集上清。使用SDS-PAGE来评价突变体表达上清中突变体蛋白的浓度。
实施例2.突变体对人IgG1切割活性的评估
为了评价各突变体对人IgG1的切割活性，建立了基于ELSIA的活性测定方法。测定原理是用人IgG1特异性抗原包被酶标板，然后将含有相当浓度突变体蛋白的上清样品与人IgG1在孔中一起温育。使用针对抗体Fc部分具有特异性的人IgG1检测抗体来测量与孔结合的完整或未被完全切割的人IgG1的量。在孔中给定的上清中突变体蛋白浓度相同的情况下，突变体蛋白切割人IgG1活性越高，则越少的完整人IgG1抗体与孔结合，从而得到越低的信号。以不同浓度IgG1与对应检测信号之间的关系可制作IgG1标准曲线，根据标准曲线计算完整或未被完全切割的IgG1的量，进而计算被完全切割的IgG1的量。以被完全切割的IgG1占初始IgG1的比例来评价突变体活性的高低。
为了将实施例1中收获的上清中突变体蛋白浓度处于相当的水平，以相同的上样量进行SDS-PAGE检测，使用Quantity One对电泳图谱中目标蛋白条带的光密度值进行分析，在上样量相同的情况下，图谱中目标蛋白条带的光密度值越高，浓度越高。以IdeE上清作为对照，将其他突变体上清进行浓缩或稀释，使得突变体蛋白条带的光密度值都和IdeE对照的光密度值基本一致。
将上清中蛋白浓度调整至相当的水平后按照如下方法进行ELISA检测：将酶标板用2ug/ml人IgG1(曲妥珠单抗)特异性抗原(货号QRE-104，瑞安生物)在2～8℃包被过夜，然后用PBST(PBS+0.05％吐温20)洗涤，洗涤后的酶标板用2％BSA(PBS配制)在37℃封闭2h，封闭后PBST洗涤。
标准曲线制作：用200ng/ml曲妥珠单抗用反应缓冲液(10mM PB，10mM NaCl，pH6.5)以1:2比例进行梯度稀释直至3.125ng/ml，将100ul不同浓度的曲妥珠单抗加入酶标板的孔中，用于底物(曲妥珠单抗)标准曲线制作。
切割反应：将蛋白浓度调整后的上清用反应缓冲液(10mM PB，10mM NaCl，pH6.5)稀释5倍，将50ul 100ng/ml曲妥珠单抗和50ul稀释后的上清加入酶标板的孔中。
将酶标板放在37℃振荡温育1h，用PBST洗涤后再将40ng/ml Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody-HRP(货号31413，Thermo)加入孔板中，在37℃振荡温育1h，用PBST洗涤后加入TMB作为HRP的 显色底物温育15min，用2N H 2SO 4终止。用酶标仪检测在450nm处的吸光度。根据底物标准曲线计算不同测试孔中的完整或未被完全切割的曲妥珠单抗浓度，进而计算被完全切割的曲妥珠单抗占初始曲妥珠单抗的比例，以此来评价不同突变体的活性。
各突变体活性相对于野生型IdeE活性的倍数关系如表1所示。实施例1中筛选获得的40个突变体都具有高于野生型IdeE的活性，其中15个突变体的活性为野生型IdeE的2倍或2倍以上。
表1.通过ELISA测得的突变体相对于野生型IdeE活性的倍数关系
Figure PCTCN2021100844-appb-000002
实施例3.突变体热稳定性的评估
从表1中显示的15个活性是野生型IdeE的2倍或2倍以上的突变体中选取12个突变体来检测其热稳定性。检测方法活性检测方法如下：
将野生型或各突变体上清分两份，分别放置4℃和50℃条件下保温1h，保温后按照实施例2中的ELISA方法检测野生型或各突变体活性，用50℃保温的活性/4℃保温后的活性计算野生型或各突变体在50℃条件下保温1h的剩余活性百分比(％)，并以此来比较野生型和各突变体的热稳定性。
突变体活性相对于野生型IdeE的热稳定性的倍数关系如表2所示。表2显示12个突变体都具有高于野生型的热稳定性，其中有7个突变体热稳定性是野生型的3倍以上。
表2.突变体相对于野生型IdeE热稳定性的倍数关系
Figure PCTCN2021100844-appb-000003
Figure PCTCN2021100844-appb-000004
实施例4.单点突变体切割人IgG1活性的比较
检测如表1和表2中显示的T8D、T8W、T24A、A59L、A59V、E97D和R280H这7个“活性是野生型IdeE的2倍以上，热稳定性是野生型IdeE的3倍以上”的单点突变体切割人IgG1的活性。
1、突变体的表达和纯化
从实施例1中上述5个单点突变体的转化的平板上各挑1单菌落，接于3ml含100ug/ml氨苄青霉素的LB培养基中，37℃、250rpm培养过夜。过夜培养物接于50ml含100ug/ml氨苄青霉素的LB培养基中，37℃培养至OD600达到0.4～0.6，加入0.1mM IPTG，30℃继续培养过夜。过夜培养物通过离心收集上清。上清再用IDA-Ni琼脂糖磁珠纯化，纯化洗脱的蛋白用超滤离心管再换液至PBS缓冲体系中。使用SDS-PAGE来评价纯化后的突变体蛋白纯度。检测OD280，根据消光系数计算纯化后的突变体蛋白浓度。
2、突变体切割人IgG1活性的比较
通过SDS-PAGE上显现有每种突变体的不同浓度对人IgG1所产生的切割产物来进一步评价不同突变体相对于野生型IdeE的切割人IgG1活性的高低。将纯化后的突变体或野生型IdeE分别稀释至0.002mg/mL和0.001mg/mL。分别取50ul不同浓度的突变体或野生型IdeE加入50ul含有2mg/ml曲妥珠单抗的反应体系中开始切割反应，反应体系置于37℃反应30min。将样品与同等体积的2×SDS上样缓冲液混合后75℃水浴5min，SDS-PAGE检测切割产物。
图1展示了7个单点突变体及野生型IdeE切割人IgG1产生的切割产物电泳图(酶:底物＝1:1000)。图2展示了7个单点突变体及野生型IdeE切割人IgG1产生的切割产物电泳图(酶:底物＝1:2000)。7个单点突变体在0.001mg/ml浓度下切割IgG1的效果都不差于0.002mg/ml野生型IdeE，说明7个单点突变体的切割人IgG1的活性不低于野生型IdeE的2倍。
实施例5.N端截短截短突变体切割人IgG1活性和热稳定性的比较
在野生型IdeE基础上分别删除N端前15个(D1-V15)、前16个(D1-P16)、前17个(D1-H17)、前18个(D1-Q18)和前19个氨基酸(D1-I19)， 构建5个N端截短突变体(见表3)。
表3.截短突变体序列设计
突变体 相对于野生型或突变体序列的修饰 SEQ ID NO:
WT_del15 删除SEQ ID NO:2前15个氨基酸 18
WT_del16 删除SEQ ID NO:2前16个氨基酸 19
WT_del17 删除SEQ ID NO:2前17个氨基酸 20
WT_del18 删除SEQ ID NO:2前18个氨基酸 21
WT_del19 删除SEQ ID NO:2前19个氨基酸 22
1、突变体的表达和纯化
按照实施例1的方法合成表3中的突变体多核苷酸序列，构建突变体表达重组质粒，转化大肠杆菌BL21 Star(DE3)。按照实施例4中的方法制备突变体纯化蛋白。
2、突变体切割人IgG1活性的比较
将纯化后的突变体或野生型IdeE分别稀释至0.002mg/mL。分别取50ul稀释后的突变体或野生型IdeE加入50ul含有2mg/ml曲妥珠单抗的反应体系中开始切割反应，反应体系置于37℃反应30min。将样品与同等体积的2×SDS上样缓冲液混合后75℃水浴5min，SDS-PAGE检测切割产物。
图3展示了5个截短突变体切割人IgG1产生的切割产物电泳图(酶:底物＝1:1000)。5个截短突变体切割活性都与野生型IdeE没有明显差别。
3、突变体热稳定性的比较
将纯化后的突变体或野生型IdeE分别稀释至0.1mg/ml，50℃条件下保温1h，保温后再稀释至0.002mg/mL。分别取50ul稀释后的突变体或野生型IdeE加入50ul含有2mg/ml曲妥珠单抗的反应体系中开始切割反应，反应体系置于37℃反应30min。将样品与同等体积的2×SDS上样缓冲液混合后75℃水浴5min，SDS-PAGE检测切割产物。
图4展示了50℃条件下保温1h后5个截短突变体及野生型IdeE切割人IgG1产生的切割产物电泳图(酶:底物＝1:1000)。50℃热处理后5个截短突变体的残余活性明显高于野生型，说明5个截短突变体热稳定性相对于野生型都有明显提高。
实施例6.C端截短截短突变体切割人IgG1活性的比较
在野生型IdeE基础上分别删除C端最后5个(W311-S315)和最后10 个氨基酸(S306-S315)，构建2个C端截短突变体(见表4)。
表4.截短突变体序列设计
突变体 相对于野生型或突变体序列的修饰 SEQ ID NO:
WT_delC5 删除SEQ ID NO:2后5个氨基酸 23
WT_delC10 删除SEQ ID NO:2后10个氨基酸 24
1、突变体的表达和纯化
按照实施例1的方法合成表4中的突变体多核苷酸序列，构建突变体表达重组质粒，转化大肠杆菌BL21 Star(DE3)。按照实施例4中的方法制备突变体纯化蛋白。
2、突变体切割人IgG1活性的比较
将纯化后的突变体或野生型IdeE分别稀释至0.002mg/mL。分别取50ul稀释后的突变体或野生型IdeE加入50ul含有2mg/ml曲妥珠单抗的反应体系中开始切割反应，反应体系置于37℃反应30min。将样品与同等体积的2×SDS上样缓冲液混合后75℃水浴5min，SDS-PAGE检测切割产物。
图5展示了2个C端截短突变体切割人IgG1产生的切割产物电泳图(酶:底物＝1:1000)。2个截短突变体切割活性都比野生型IdeE高出2倍以上。
实施例7.组合突变体切割人IgG1活性和热稳定性的比较
在T24A、A59L、A59V、E97D和R280H 5个单点突变体的基础上分别删除前18个(D1-Q18)氨基酸，构建5个组合突变体(见表5)。
表5.组合突变体序列设计
突变体 相对于野生型或突变体序列的修饰 SEQ ID NO:
T24A_del18 删除SEQ ID NO:9前18个氨基酸 25
A59L_del18 删除SEQ ID NO:13前18个氨基酸 26
A59V_del18 删除SEQ ID NO:14前18个氨基酸 27
E97D_del18 删除SEQ ID NO:15前18个氨基酸 28
R280H_del18 删除SEQ ID NO:16前18个氨基酸 29
1、突变体的表达和纯化
按照实施例1的方法合成表5中的突变体多核苷酸序列，构建突变体表达重组质粒，转化大肠杆菌BL21 Star(DE3)。按照实施例4中的方法制备突变体纯化蛋白。
2、突变体切割人IgG1活性的比较
将纯化后的突变体分别稀释至0.001mg/mL。分别取50ul稀释后的突变 体或野生型IdeE加入50ul含有2mg/ml曲妥珠单抗的反应体系中开始切割反应，反应体系置于37℃反应30min。将样品与同等体积的2×SDS上样缓冲液混合后75℃水浴5min，SDS-PAGE检测切割产物。
图6展示了5个组合突变体切割人IgG1产生的切割产物电泳图(酶:底物＝1:2000)。比较图6和图2的切割效果，5个截短突变体和单点组合突变体切割活性没有明显差别，说明组合突变体切割人IgG1的活性同样不低于野生型IdeE的2倍。
3、突变体热稳定性的比较
将纯化后的突变体分别稀释至0.1mg/ml，50℃条件下保温1h，保温后再稀释至0.001mg/mL。分别取50ul稀释后的突变体或野生型IdeE加入50ul含有2mg/ml曲妥珠单抗的反应体系中开始切割反应，反应体系置于37℃反应30min。将样品与同等体积的2×SDS上样缓冲液混合后75℃水浴5min，SDS-PAGE检测切割产物。
图7展示了50℃条件下保温1h后5个组合突变体切割人IgG1产生的切割产物电泳图(酶:底物＝1:2000)。比较图8和图7的切割效果，5个组合突变体50℃热处理后活性只是略有下降，说明5个组合突变体相对于野生型热稳定性也都有明显提高。
实施例8.E97D_del18突变体与IdeS和IdeZ活性比较
将实施例7中纯化的E97D_del18突变体依次稀释至20μg/mL，10μg/mL，5μg/mL，2.5μg/mL和1.25μg/ml。将IdeS(
Figure PCTCN2021100844-appb-000005
货号：A0-FRI-020，Genovis)按照其标识分别稀释至2U/μl，1U/μl，0.5U/μl，0.25U/μl和0.125U/μl。将IdeZ(
Figure PCTCN2021100844-appb-000006
货号：A0-FRZ-020，Genovis)分别稀释至0.4U/μl，0.2U/μl，0.1U/μl，0.05U/μl和0.025U/μl。分别取50μl不同浓度的突变体、IdeS或IdeZ加入50μl含有2mg/ml曲妥珠单抗的反应体系中开始切割反应，反应体系置于37℃反应30min。将样品与同等体积的2×SDS上样缓冲液混合后75℃水浴5min，SDS-PAGE检测切割产物。
图8展示了不同浓度E97D_del18突变体和IdeS切割人IgG1产生的切割产物电泳图。从电泳图上的酶蛋白条带可以判断1号泳道IdeS酶浓度介于7号和8号泳道E97D_del18突变体酶浓度之间，由此递推3号泳道IdeS酶浓度介于9号和10号泳道E97D_del18突变体酶浓度，而3号泳道的IgG1 的酶切效果介于10号和11号泳道，由此可以推断E97D_del18突变体切割人IgG1的活性接近IdeS的2倍。
图9展示了不同浓度E97D_del18突变体和IdeZ切割人IgG1产生的切割产物电泳图。从电泳图上的酶蛋白条带可以判断1号泳道IdeZ酶浓度高于7号泳道E97D_del18突变体酶浓度，由此递推3号泳道IdeZ酶浓度高于9号泳道E97D_del18突变体酶浓度，即高于11号泳道E97D_del18突变体酶浓度的4倍，而3号泳道的IgG1的酶切效果接近11号泳道，由此可以推断E97D_del18突变体切割人IgG1的活性高于IdeZ的4倍。
实施例9.E97D_del18突变体切割人IgG1活性的体外检测
通过检测加入E97D_del18突变体与人IVIg处理过的小鼠血清或血浆中完整或单切割IVIg的量来评价E97D_del18突变体对人IgG1的体外切割活性。按照表6配制不同组别的小鼠血清或血浆酶切体系。
表6.小鼠血清或血浆酶切体系
Figure PCTCN2021100844-appb-000007
碘乙酸处理组中碘乙酸的作用是抑制IgG降解酶的活性。
将体系置于37℃反应30min。取20μl样品与同等体积的2×SDS非还原上样缓冲液混合后75℃水浴5min，SDS-PAGE检测切割产物。
图10展示了E97D_del18突变体在小鼠血清和血浆中切割人IVIg产生的切割产物电泳图。结果显示E97D_del18在小鼠血清和血浆中都可以有效切割人IVIg。
通过检测加入E97D_del18突变体处理过的小鼠或人血清来评价E97D_del18突变体是否具有对人IgG1的体外切割活性。按照表7配制不同组别的小鼠或人血清酶切体系。
表7.小鼠或人血清酶切体系
Figure PCTCN2021100844-appb-000008
将体系置于37℃反应24h。取20μl样品与同等体积的2×SDS还原性上样缓冲液混合，再用1×SDS还原性上样缓冲液稀释20倍，75℃水浴5min，SDS-PAGE检测切割产物。
图11展示了E97D_del18突变体在小鼠和人血清中产生的切割产物电泳图。结果显示E97D_del18在人血清中切割产生明显可见的25kD的Fc片段，而在小鼠血清中则没有该片段，说明E97D_del18能有效特异性切割人血清中IgG1，而对小鼠血清中的IgG1切割活性很低或无切割活性。
实施例10.E97D_del18突变体切割不同种属的免疫球蛋白
通过检测加入E97D_del18突变体与不同种属动物血清或血浆中完整或单切割IgG的量来评价E97D_del18突变体对不同种属动物血清免疫球蛋白的体外切割活性。按照表8和表9配制不同种属的血清或抗体酶切体系。
表8 比格犬血清及不同物种抗体酶切体系
Figure PCTCN2021100844-appb-000009
将体系置于37℃反应1小时，酶切产物采用SDS-PAGE检测。
表9 不同物种血清酶切体系
Figure PCTCN2021100844-appb-000010
图12A-12D展示了E97D_del18突变体在不同物种血清和抗体的效果。结果显示E97D_del18可有效切割犬IgG、兔IgG以及小鼠IgG2a，无法切割小鼠IgG1；E97D_del18可有效切割兔、犬和猴血清IgG，其中兔血清IgG切割效果最好，猪血清IgG切割效果较差，大鼠和小鼠血清IgG则几乎未被切动。
实施例11.人体内针对E97D_del18突变体的预存抗体低
该测定是基于E97D_del18突变体与IdeS之间对于与抗E97D_del18/IdeS抗体结合的竞争。测试酶和人血清的预温育将使得抗E97D_del18/IdeS抗体与E97D_del18突变体与IdeS能够结合。
将E97D_del18突变体与IdeS在孔板上包被过夜，然后用PBST洗涤并在2％BSA封闭液中封闭1小时，用逐步稀释的待测突变体与IdeS和人血清制备混合板，将混合版在室温下振荡温育1小时，PBST洗涤之后加入生物素标记的E97D_del18突变体与IdeS，再加入SA-HRP，用TMB显色并读数。平行比对获得约80个人血样本中E97D_del18和IdeS预存抗体的情况。
结果如表10所示，IdeS在正常人血清中预存抗体的比例高达约90％，而E97D_del18突变体仅为约20％。E97D_del18突变体在体内的预存抗体明显少于IdeS，证明E97D_del18突变体的免疫原性更低，更有利于体内用药。
表10.E97D_del18突变体和IdeS在人血样本中预存抗体比对情况
  E97D_del18突变体 IdeS
人血样本总数(例) 76 76
预存抗体阳性样本比例(％) 18.4 89.5
实施例12.E97D_del18突变体切割人IgG1活性的体内检测
在无菌条件下用人IVIg(静注人免疫球蛋白)腹腔注射2只小鼠(两只小鼠为平行实验，小鼠编号1号和2号)，注射剂量为1g/kg。注射人IVIg 24h后，再将IgG降解酶(E97D_del18)以5mg/kg的剂量静脉注射小鼠，2只小鼠都分别在注射E97D_del18后0h、15min、2h、6h和24h采血并收集血清样品。取20μl血清样品与同等体积的2×SDS非还原上样缓冲液混合后，再用1×SDS非还原性上样缓冲液稀释20倍，75℃水浴5min，SDS-PAGE检测。
图13展示了E97D_del18在小鼠体内不同时间内切割人IVIg产生的切割产物电泳图。结果显示，E97D_del18在小鼠体内切割IVIg效果显著，15min基本已经达到完全酶切。
实施例13.组合突变体切割人IgG1活性的比较
在上述突变体的基础上，进一步构建了6种组合突变体，所述突变的序列如表11所示。
表11.组合突变体
突变体 相对于野生型或突变体序列的修饰 SEQ ID NO:
E97D_del18_delC5 删除SEQ ID NO:28后5个氨基酸 30
E97D_del18_delC10 删除SEQ ID NO:28后10个氨基酸 31
A59V_del18_delC5 删除SEQ ID NO:27后5个氨基酸 32
A59L_del18_delC5 删除SEQ ID NO:26后5个氨基酸 33
R280H_del18_delC5 删除SEQ ID NO:29后5个氨基酸 34
E97D_A59V_R280H E97D、A59V、R280H三突变组合 35
1、突变体的表达和纯化
按照实施例1的方法合成表11中的突变体多核苷酸序列，构建突变体表达重组质粒，转化大肠杆菌BL21 Star(DE3)。按照实施例4中的方法制备突变体纯化蛋白。
2、突变体切割人IgG1活性的比较
将纯化后的突变体分别稀释至0.001mg/mL。分别取50ul稀释后的突变体或野生型IdeE加入50ul含有2mg/ml曲妥珠单抗的反应体系中开始切割反应，反应体系置于37℃反应30min。将样品与同等体积的2×SDS上样缓冲液混合后75℃水浴5min，SDS-PAGE检测切割产物。
图14A和14B展示了6个组合突变体切割人IgG1产生的切割产物电泳图(酶:底物＝1:2000)。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (18)

  1. 免疫球蛋白降解酶IdeE的突变体，其特征在于，所述的免疫球蛋白降解酶IdeE包含如序列表中SEQ ID NO:2所示的氨基酸序列或由所述氨基酸序列组成；所述突变选自下组：
    (1)对所述氨基酸序列的位置8、10、24、59、97和280中的一位或者多位进行替换后得到所述突变体；和/或，
    (2)对所述免疫球蛋白降解酶IdeE进行截短，删除其N端的前1个、前2个、前3个、前4个、前5个、前6个、前7个、前8个、前9个，前10个、前11个、前12个、前13个、前14个、前15个、前16个、前17个、前18个或者前19个氨基酸序列；和/或，
    (3)对所述免疫球蛋白降解酶IdeE进行截短，删除其C端的最后1个、最后2个、最后3个、最后4个、最后5个、最后6个、最后7个、最后8个、最后9个或者最后10个氨基酸序列；
    其中所述的突变体具有高于所述免疫球蛋白降解酶IdeE的活性，和/或具有高于所述免疫球蛋白降解酶IdeE的热稳定性。
  2. 如权利要求1所述的突变体，其特征在于所述突变选自下组：
    (1)对SEQ ID NO:2所示的氨基酸序列的位置8、10、24、59、97或者280进行替换；和/或
    (2)删除所述免疫球蛋白降解酶IdeE N端的前15个、前16个、前17个、前18个或者前19个氨基酸；优选删除前18个氨基酸；和/或
    (3)删除所述免疫球蛋白降解酶IdeE C端的最后5个或者最后10个氨基酸；优选删除最后5个氨基酸。
  3. 如权利要求1或2所述的突变体，其特征在于所述替换选自下组：
    (1)所述位置8的苏氨酸被替换成半胱氨酸、苯丙氨酸、色氨酸、酪氨酸、天冬氨酸、谷氨酸、丙氨酸、甘氨酸、组氨酸、异亮氨酸、亮氨酸、蛋氨酸、天冬酰胺、脯氨酸、谷氨酰胺、丝氨酸、缬氨酸、精氨酸和赖氨酸中的任意一种；
    (2)所述位置10的丙氨酸被替换成半胱氨酸、天冬氨酸、谷氨酸、苯丙氨酸、甘氨酸、组氨酸、异亮氨酸、赖氨酸、亮氨酸、蛋氨酸、天冬酰胺、脯氨酸、谷氨酰胺、精氨酸、丝氨酸、苏氨酸、缬氨酸、色氨酸和酪氨酸中 的任意一种；
    (3)所述位置24的苏氨酸被替换成丙氨酸、半胱氨酸、天冬氨酸、天冬酰胺、谷氨酸、苯丙氨酸、甘氨酸、组氨酸、异亮氨酸、赖氨酸、亮氨酸、蛋氨酸、脯氨酸、谷氨酰胺、精氨酸、丝氨酸、缬氨酸、色氨酸和酪氨酸中的任意一种；
    (4)所述位置59的丙氨酸被替换成半胱氨酸、天冬氨酸、谷氨酸、苯丙氨酸、甘氨酸、组氨酸、异亮氨酸、赖氨酸、亮氨酸、蛋氨酸、天冬酰胺、脯氨酸、谷氨酰胺、精氨酸、丝氨酸、苏氨酸、缬氨酸、色氨酸和酪氨酸中的任意一种；
    (5)所述位置97的谷氨酸被替换成丙氨酸、半胱氨酸、天冬氨酸、苯丙氨酸、甘氨酸、组氨酸、异亮氨酸、赖氨酸、亮氨酸、蛋氨酸、天冬酰胺、脯氨酸、谷氨酰胺、精氨酸、丝氨酸、苏氨酸、缬氨酸、色氨酸和酪氨酸中的任意一种；和
    (6)所述位置280的精氨酸被替换成丙氨酸、天冬氨酸、谷氨酸、半胱氨酸、丝氨酸、苯丙氨酸、组氨酸、异亮氨酸、赖氨酸、亮氨酸、蛋氨酸、天冬酰胺、脯氨酸、谷氨酰胺、色氨酸、苏氨酸、缬氨酸和酪氨酸中的任意一种。
  4. 如权利要求3所述的突变体，其特征在于，所述的突变体如序列表中SEQ ID NO:3～35任一个所示。
  5. 一种包含如权利要求1～4任一项所述的突变体的蛋白。
  6. 如权利要求5所述的蛋白，其特征在于，所述蛋白在所述突变体的N末端包含信号肽；优选地，所述蛋白在所述突变体的N末端连接有分泌信号序列并在所述分泌序列的N末端连接有甲硫氨酸和/或在在所述突变体C末端连接有组氨酸标签；更优选地，所述蛋白从N末端至C末端由如下组成：甲硫氨酸、分泌信号序列和所述突变体。
  7. 一种编码如权利要求1～4任一项所述的突变体或者如权利要求5或6所述的蛋白的核苷酸。
  8. 一种包含如权利要求7所述的核苷酸的表达载体。
  9. 一种包含如权利要求8所述的表达载体或者表达如权利要求1～4任一项所述的突变体或者如权利要求5或6所述的蛋白的宿主细胞；所述宿主细胞优选为大肠杆菌细胞或者酵母细胞。
  10. 一种组合物，其包含：
    免疫球蛋白降解酶或其突变体或者包含所述免疫球蛋白降解酶或其突变体的蛋白；和
    任选的药学上可接受的载体或赋形剂。
  11. 如权利要求10所述的组合物，其中所述免疫球蛋白降解酶选自IdeE、IdeS和IdeZ。
  12. 如权利要求10或11所述的组合物，其中所述免疫球蛋白降解酶的突变体为如权利要求1～4任一项所述的突变体，或者所述包含所述免疫球蛋白降解酶或其突变体的蛋白为如权利要求5或6所述的蛋白。
  13. 如权利要求10-12中任一项所述的组合物，其进一步包含：
    抗体或者含有Fc的蛋白。
  14. 如权利要求13所述的组合物，其中所述抗体的靶点选自下组：细胞表面蛋白、细胞因子、激素、酶、胞内信使、胞间信使和免疫检查点。
  15. 如权利要求10-14中任一项所述的组合物，其进一步包含：
    病毒载体药物，优选地，所述病毒载体药物选自下组：溶瘤病毒、基因治疗病毒和病毒载体疫苗。
  16. 如权利要求10-15中任一项所述的组合物，其进一步包含：
    能降低血液IgG水平的药物，优选的，所述降低血液IgG水平的药物选自下组：FcRn抗体、与FcRn高亲和力的Fc片段变体。
  17. 一种试剂盒，其包含：
    (1)如权利要求1～4任一项所述的突变体或者如权利要求5或6所述的蛋白；和
    (2)选自下组的一种或多种：(a)药学上可接受的载体或赋形剂；(b)抗体或者包含Fc的蛋白；和/或
    (3)病毒载体药物，所述病毒载体药物选自溶瘤病毒、基因治疗病毒和病毒载体疫苗；和/或
    (4)能降低血液IgG水平的药物，所述降低血液IgG水平的药物选自FcRn抗体、与FcRn高亲和力的Fc片段变体。
  18. 一种试剂盒，其包含：药盒A和药盒B，其特征在于，
    所述的药盒A含有如权利要求1～4任一项所述的突变体或者如权利要求5或6所述的蛋白，
    所述的药盒B含有选自下组的一种或多种：
    (1)药学上可接受的载体或赋形剂；(2)抗体或者包含Fc的蛋白；和/或(3)病毒载体药物；和/或(4)能降低血液IgG水平的药物；
    其中，所述病毒载体药物选自溶瘤病毒、基因治疗病毒和病毒载体疫苗；所述降低血液IgG水平的药物选自FcRn抗体、与FcRn高亲和力的Fc片段变体。
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CN114875003A (zh) * 2022-04-06 2022-08-09 浙江大学 一种短链脱氢酶的突变体、编码基因及编码基因获得方法、突变体的应用
CN114875003B (zh) * 2022-04-06 2024-05-24 浙江大学 一种短链脱氢酶的突变体、编码基因及编码基因获得方法、突变体的应用
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