Mutants of the immunoglobulin-degrading enzyme IdeE

Abstract

The present invention provides a mutant of the immunoglobulin-degrading enzyme IdeE, which comprises the amino acid sequence set forth in SEQ ID NO:2 in the Sequence Listing, and the mutant obtained by substituting at least one or more of positions 8, 10, 24, 59, 97, and 280 of the amino acid sequence, and the function of the mutant includes at least the function of the immunoglobulin-degrading enzyme IdeE. The activity and thermostability of the provided immunoglobulin-degrading enzyme mutant are higher than those of wild-type IdeE.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to mutants of immunoglobulin-degrading enzymes.

Background Art

[0002] Streptococcus pyogenes is a type of pathogen commonly found in humans and livestock, and widely exists in nature and in the oral pharynx, respiratory tract, and intestinal tract of humans or animals. Streptococcal infections cause related diseases. Examples of relatively mild diseases include, for example, pyogenic dermatitis and pharyngitis. Examples of relatively severe diseases include sepsis, necrotizing fasciitis, and toxic shock syndrome. The immunoglobulin G-degrading enzyme (IdeS) derived from Streptococcus pyogenes is a commonly seen cysteine protease of Group A Streptococcus pyogenes (GAS), and has endopeptidase activity for hydrolyzing IgG (Agniswamy J, Lei B, Musser J M et al., J Biol Chem, 2004, 279:52789-52796. Lei B, DeLeo F R, Reid S D et al., Infect Immun, 2002, 70:6880-6890. Von Pawel-Rammingen U, Johansson B P, Bjorck L.. EMBO J, 2002, 21:1607-1615.). As a virulence factor of the pathogen, it can specifically degrade IgG by identifying the lower hinge region CH1 and CH2 structural regions of the antibody, obtain the same F(ab)2 and Fc fragments, and by assisting GAS to avoid antibody-mediated phagocytosis and cytotoxicity, it can weaken the killing of GAS by the host immune system (Von Pawel-Rammingen U. J Innate Immunity, 2012, 4:132-140. Su, Y.-F. et al., Molecular Immunology, 2011, 49:134-142.).

[0003] Immunoglobulin G (IgG) is the main antibody component of serum, accounting for approximately 75% of serum immunoglobulins. It primarily plays a protective role in the body's immune system, effectively preventing infectious diseases. In addition to its protective function, IgG is also associated with disease. In some autoimmune diseases, IgG antibodies react with the body's own molecules, and in organ transplantation, IgG can cause acute transplant rejection. IdeS achieves immunosuppression by specifically degrading IgG, thereby rendering it unable to perform its intended functions.

[0004] Currently, IdeS used in clinical practice has problems such as poor activity and the large number of antibodies already present in the human body. IdeS is a toxic factor of human pathogens, and clinical studies have shown that in normal physiological conditions, the rate at which anti-IdeS antibodies are detected in most people is close to 100%. Therefore, the administration efficiency of IdeS is low, and there are safety concerns.

[0005] The IdeE protease, which has approximately 70% sequence homology to IdeS, originates from Streptococcus equi ssp. equi, a type of equine pathogen (Jonas Lannerg, Bengt Guss. FEMS Microbiol Lett, 2006, 262:230-235). Both IdeE and IdeS enzymes cleave IgG at the exact same site, exhibiting high reproducibility and specificity in their cleavage, and possessing very similar substrate ranges. Because IdeE originates from an equine pathogen, it is estimated that the pre-existing antibodies in the human body to IdeE may be far lower than those to IdeS, making it more suitable for the development of immunosuppressants for the treatment and prevention of IgG antibody-mediated diseases. However, wild-type IdeE, like IdeS, suffers from low activity. Therefore, by improving the activity of IdeE through molecular design and mutant selection, and reducing the clinically used dosage, the risks associated with high-dose bacterial-derived proteins can be reduced. This is the objective of the present invention. [Overview of the project]

[0006] A first aspect of the present invention is a mutant of the immunoglobulin-degrading enzyme IdeE, The immunoglobulin-degrading enzyme IdeE contains or is composed of the amino acid sequence indicated by SEQ ID NO:2 in the sequence listing. The aforementioned mutation is (1) Obtaining the mutant after substituting one or more of the amino acid sequences at positions 8, 10, 24, 59, 97, and 280, and / or (2) Shortening the immunoglobulin-degrading enzyme IdeE by deleting the first 1, first 2, first 3, first 4, first 5, first 6, first 7, first 8, first 9, first 10, first 11, first 12, first 13, first 14, first 15, first 16, first 17, first 18 or first 19 amino acid sequences of its N-terminus, and / or (3) Selected from the group consisting of shortening the immunoglobulin-degrading enzyme IdeE and deleting the last 1, last 2, last 3, last 4, last 5, last 6, last 7, last 8, last 9, or last 10 amino acids of its C-terminus, The present invention relates to a mutant of the immunoglobulin-degrading enzyme IdeE, characterized in that the mutant has higher activity and / or thermal stability than the immunoglobulin-degrading enzyme IdeE.

[0007] A second aspect of the present invention relates to a protein comprising a mutant of the present invention. The protein is characterized in that a secretory signal sequence and / or methionine is ligated to the N-terminus of the mutant, and / or a histidine tag is ligated to the C-terminus of the mutant.

[0008] Third to fifth aspects of the present invention relate to nucleotides encoding the mutant or protein of the present invention, expression vectors containing the nucleotides, and host cells containing the expression vector or expressing the mutant or protein of the present invention.

[0009] A sixth aspect of the present invention relates to a composition or kit comprising the mutant or protein of the present invention and any substance selected from the group consisting of a pharmaceutically acceptable vector or excipient, an antibody or Fc-containing protein, and a viral vector drug. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 shows the electrophoresis of SDS-PAGE gels (enzyme:substrate = 1:1000) of cleavage products produced by cleaving human IgG1 with seven single-site mutants and wild-type IdeE. [Figure 2] Figure 2 shows the electrophoresis of SDS-PAGE gels (enzyme:substrate = 1:2000) of cleavage products produced by cleaving human IgG1 with seven single-site mutants and wild-type IdeE. [Figure 3] Figure 3 shows the electrophoresis of SDS-PAGE gels (enzyme:substrate = 1:1000) of cleavage products produced by cleaving human IgG1 with five N-terminal truncated mutants. [Figure 4] Figure 4 shows the electrophoresis of SDS-PAGE gels (enzyme:substrate = 1:1000) of cleavage products produced by cleaving human IgG1 with five N-terminal truncated mutants and wild-type IdeE after incubation at 50°C for 1 hour. [Figure 5] Figure 5 shows the electrophoresis of the SDS-PAGE gel of the cleavage products produced by cleaving human IgG1 with two C-terminal truncated mutants (enzyme:substrate = 1:1000). [Figure 6] Figure 6 shows the electrophoresis of SDS-PAGE gels (enzyme:substrate = 1:2000) of cleavage products produced by cleaving human IgG1 with five combination mutants. [Figure 7] Figure 7 shows the electrophoresis of the SDS-PAGE gel of the cleavage products (enzyme:substrate = 1:2000) produced by cleaving human IgG1 with five combination mutants after incubation at 50°C for 1 hour. [Figure 8]Figure 8 shows the electrophoresis of SDS-PAGE gels of cleavage products produced by cleaving human IgG1 with different concentrations of the E97D_del18 mutant and IdeS. [Figure 9] Figure 9 shows the electrophoresis of SDS-PAGE gels of cleavage products produced by cleaving human IgG1 with different concentrations of the E97D_del18 mutant and IdeZ. [Figure 10] Figure 10 shows the electrophoresis of SDS-PAGE gels of cleavage products produced by cleaving human IVIg in mouse serum and plasma using the E97D_del18 mutant. [Figure 11] Figure 11 shows the electrophoresis of SDS-PAGE gels of cleavage products produced in mouse and human serum by the E97D_del18 mutant. [Figure 12A] Figures 12A to 12D are electrophoresis maps of SDS-PAGE gels of cleavage products produced by cleaving IgG in the serum of beagle dogs, rats, mice, rabbits, monkeys, and pigs with different concentrations of E97D_del18. [Figure 12B] Figures 12A to 12D are electrophoresis maps of SDS-PAGE gels of cleavage products produced by cleaving IgG in the serum of beagle dogs, rats, mice, rabbits, monkeys, and pigs with different concentrations of E97D_del18. [Figure 12C] Figures 12A to 12D are electrophoresis maps of SDS-PAGE gels of cleavage products produced by cleaving IgG in the serum of beagle dogs, rats, mice, rabbits, monkeys, and pigs with different concentrations of E97D_del18. [Figure 12D] Figures 12A to 12D are electrophoresis maps of SDS-PAGE gels of cleavage products produced by cleaving IgG in the serum of beagle dogs, rats, mice, rabbits, monkeys, and pigs with different concentrations of E97D_del18. [Figure 13] Figure 13 shows the electrophoresis of SDS-PAGE gels of cleavage products produced by cleaving human IVIg in mice at different time intervals using E97D_del18. [Figure 14A]Figures 14A and 14B are electrophoretograms of SDS-PAGE gels of cleavage products produced by cleaving human IgG1 with a mutant having different mutant combinations and IdeE (enzyme:substrate = 1:2000). [Figure 14B] Figures 14A and 14B are electrophoretograms of SDS-PAGE gels of cleavage products produced by cleaving human IgG1 with a mutant having different mutant combinations and IdeE (enzyme:substrate = 1:2000).

Mode for Carrying Out the Invention

[0011] I. Functional Polypeptide Having Immunoglobulin-Degrading Enzyme Activity The first aspect of the present invention provides a functional polypeptide, the functional polypeptide having the activity of an immunoglobulin-degrading enzyme and including a mutant based on the amino acid sequence represented by SEQ ID NO:2, the mutant being (1) a mutant obtained after substituting an amino acid at one or more positions among positions 8, 10, 24, 59, 97, and 280 of SEQ ID NO:2, and / or (2) an N-terminal truncated mutant of SEQ ID NO:2 obtained by deleting the first 1, first 2, first 3, first 4, first 5, first 6, first 7, first 8, first 9, first 10, first 11, first 12, first 13, first 14, first 15, first 16, first 17, first 18, or first 19 amino acid sequences of the N-terminus, and / or (3) a C-terminal truncated mutant of SEQ ID NO:2 obtained by deleting the last 1, last 2, last 3, last 4, last 5, last 6, last 7, last 8, last 9, or last 10 amino acid sequences of the C-terminus, and being selected from the group consisting of.

[0012] The mutant described in the present invention has the function of the immunoglobulin-degrading enzyme IdeE and preferably has improved IgG cleavage activity and thermal stability.

[0013] The term "having higher activity than immunoglobulin-degrading enzyme IdeE" in the present invention refers to that the immunoglobulin-degrading ability of the mutant is superior to that of wild-type immunoglobulin-degrading enzyme IdeE.

[0014] The term "having higher thermal stability than IdeE" in the present invention means that after the mutant is maintained at a certain temperature for a certain period of time, its immunoglobulin-degrading ability is superior to that of wild-type immunoglobulin-degrading enzyme IdeE under the same conditions.

[0015] The mutant described in the present invention is preferably obtained by a genetic engineering recombination method.

[0016] Preferably, 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 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the sequence shown by SEQ ID NO:2.

[0017] More preferably, the amino acids at positions 8, 10, 24, 59, 97 or 280 are substituted. For example, whether the amino acid sequence of the obtained mutant is as shown by any of SEQ ID NO:3 to 17, SEQ ID NO:35, or the first 15, first 16, first 17, first 18 or first 19 amino acids at the N-terminus of the immunoglobulin-degrading enzyme IdeE are deleted, and whether the amino acid sequence of the obtained mutant is as shown by any of SEQ ID NO:18 to 22, or the last 1, last 5, last 8 or last 10 amino acids at the C-terminus of the immunoglobulin-degrading enzyme IdeE are deleted. For example, whether the amino acid sequence of the obtained mutant is as shown by any of SEQ ID NO:23 to 24, Alternatively, substitutions may be made at positions 8, 10, 24, 59, 97, or 280, and the first 15, 16, 17, 18, or 19 amino acids of the N-terminus of the immunoglobulin-degrading enzyme IdeE may be deleted, preferably the first 18 amino acids, such that the amino acid sequence of the resulting mutant is as shown in any of SEQ ID NO: 25-29. Alternatively, substitutions may be made at positions 8, 10, 24, 59, 97, or 280, and the first 15, 16, 17, 18, or 19 amino acids of the N-terminus of the immunoglobulin-degrading enzyme IdeE may be deleted, and the last 1, 5, 8, or 10 amino acids of the C-terminus of the immunoglobulin-degrading enzyme IdeE may be deleted, preferably the first 18 amino acids, preferably the last 5 amino acids, for example, if the amino acid sequence of the resulting mutant is SEQ ID As indicated by one of the numbers 30-34.

[0018] In one preferred embodiment of the present invention, the amino acid substitution is (1) The threonine at position 8 of SEQ ID NO:2 is replaced by one of the following: cysteine, phenylalanine, tryptophan, tyrosine, aspartic acid, glutamic acid, alanine, glycine, histidine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, valine, arginine, and lysine. (2) The alanine at position 10 of SEQ ID NO:2 is replaced by one of the following: cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, and tyrosine. (3) The threonine at position 24 of SEQ ID NO:2 is replaced by one of the following: alanine, cysteine, aspartic acid, asparagine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, proline, glutamine, arginine, serine, valine, tryptophan, and tyrosine. (4) The alanine at position 59 of SEQ ID NO:2 is replaced by one of the following: cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, and tyrosine. (5) The glutamic acid at position 97 of SEQ ID NO:2 is replaced by one of the following: alanine, cysteine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, and tyrosine. (6) Arginine at position 280 of SEQ ID NO:2 is replaced with one of the following: alanine, aspartic acid, glutamic acid, cysteine, serine, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, threonine, valine, tryptophan, and tyrosine. Selected from the group consisting of [the specified group].

[0019] In one more preferred embodiment of the present invention, the amino acid substitution is: (1) The threonine at position 8 of SEQ ID NO:2 is substituted with aspartic acid, glutamic acid, tryptophan, or tyrosine, (2) Alanine at position 10 of SEQ ID NO:2 is replaced by lysine or arginine, (3) The threonine at position 24 of SEQ ID NO:2 is replaced by alanine, glycine or serine, (4) Alanine at position 59 of SEQ ID NO:2 is substituted with isoleucine, leucine, or valine, (5) The glutamic acid at position 97 of SEQ ID NO:2 is substituted with asparagine, and / or (6) Arginine at position 280 of SEQ ID NO:2 is replaced by histidine or lysine, Selected from the group consisting of [the specified group].

[0020] In another preferred embodiment, five sequences obtained by amino acid substitution, namely 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, are used as a base, and the first 18 amino acids of the N-terminus are further deleted to obtain the amino acid sequence of the mutant. This is as indicated by SEQ ID NO:25~29 in the sequence listing.

[0021] In another preferred embodiment, five or ten amino acids are deleted from the C-terminus of the five amino acid substitution sequences, SEQ ID NO: 26-29, and the resulting mutant amino acid sequences are as shown in SEQ ID NO: 30-34 in the sequence listing.

[0022] In another preferred embodiment, combination mutations are performed on three mutants with SEQ ID NO: 14-16, and the amino acid sequence of the resulting mutant is as shown in SEQ ID NO: 35 in the sequence listing. In another preferred embodiment, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID Based on the five sequences obtained by amino acid substitution (NO:14, SEQ ID NO:1, SEQ ID NO:5, and SEQ ID NO:16), the first 18 amino acids at the N-terminus were further deleted, and the resulting mutant amino acid sequences are shown as SEQ ID NO:25-29 in the sequence listing.

[0023] Preferably, further mutations can be performed on the mutant described in the present invention, and the sequence of the mutant obtained after further mutation 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 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO:2, and has the function of immunoglobulin-degrading enzyme IdeE.

[0024] The complete sequence of IdeE used in this invention is publicly available as GenBank accession number ABF57910.1, and is provided herein as SEQ ID NO:1. This sequence comprises an N-terminal methionine, a 33-amino acid secretory signal sequence, and an IdeE coding sequence in that order. The N-terminal methionine and signal sequence are typically removed to form a mature IdeE protein, and this sequence is provided herein as SEQ ID NO:2. Unless otherwise stated, all references to the numbering of amino acid positions in the immunoglobulin-degrading enzyme sequences described herein are based on the numbering of the corresponding positions in SEQ ID NO:2, starting from the N-terminus.

[0025] The present invention further provides proteins containing the above-described mutants.

[0026] In one preferred embodiment, the protein comprises a signal peptide at the N-terminus of the mutant, preferably the protein has a secretory signal sequence linked to the N-terminus of the mutant with methionine linked to the N-terminus of the secretory sequence, and / or a histidine tag linked to the C-terminus of the mutant, more preferably the protein comprises or consists of methionine, a secretory signal sequence, and the mutant from the N-terminus to the C-terminus.

[0027] A second aspect of the present invention provides a nucleotide encoding the above-mentioned protein or mutant.

[0028] The present invention further provides an expression vector comprising the aforementioned nucleotide.

[0029] The present invention further provides host cells containing the expression vector described above, or host cells expressing the protein or mutant described above.

[0030] The host cell may be any cell used to express a protein or polypeptide, which is common in this field, and the cell may be selected from E. coli cells, yeast cells, and the like.

[0031] II. Drug Compositions A third aspect of the present invention provides a composition comprising an immunoglobulin-degrading enzyme or a mutant thereof, or a protein containing an immunoglobulin-degrading enzyme or a mutant thereof, and an optional pharmaceutically acceptable vector or excipient. In one specific embodiment, the immunoglobulin-degrading enzyme is selected from IdeE, IdeS, and IdeZ. In one specific embodiment, the mutant of the immunoglobulin-degrading enzyme is the mutant described above, and the protein is a protein containing the mutant described above. In one specific embodiment, the composition of the present invention further comprises an antibody or an Fc-containing protein. In one specific embodiment, 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. In one specific embodiment, 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. In one specific embodiment, the composition of the present invention further comprises a drug capable of lowering blood IgG levels, preferably the drug capable of lowering blood IgG levels is selected from the group consisting of FcRn antibodies and Fc fragment variants with high affinity for FcRn.

[0032] 2.1 Antibody Targets Preferably, in the above-described composition, the target of the antibody may be a cell surface protein, such as AFP, αv integrin, α4β7 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 Examples include, but are not limited to, 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, or WT1.

[0033] The targets of the aforementioned antibodies may be cytokines, including but not limited to interleukins IL-1 to IL-13, tumor necrosis factor α and β, interferon α, β and γ, tumor growth factor β (TGF-β), colony-stimulating factor (CSF), or granulocyte-monocyte colony-stimulating factor (GM-CSF) (see Human Cytokines: Handbook for Basic & Clinical Research (Aggrawal et al., Blackwell Scientific, Boston, MA 1991)).

[0034] The target of the antibody may be a hormone, enzyme, intracellular messenger, or intercellular messenger, for example, adenylyl cyclase, guanylate cyclase, or phospholipase C.

[0035] The target of the antibody may be an immune checkpoint, and examples of such immune checkpoints include CTLA-4, PD-1, PD-L1, TIM-3, LAG3, Siglec15, 4-1BB, GITR, OX40, CD40L, CD28, TIGIT, and VIST. A is one example.

[0036] 2.2 Targeted Drugs Preferably, the composition described above further comprises a target drug, a chemotherapeutic agent, or an immune checkpoint blocker, wherein the target drug is selected from epigenetic drugs, inhibitors targeting the PI3K / Akt / mTOR signaling pathway, and tyrosine kinase inhibitors; the chemotherapeutic agent is selected from immunosuppressants, proteasome inhibitors, cytotoxic agents, and cell cycle nonspecific drugs; and the immune checkpoint blocker is an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-TIM-3 antibody, an anti-LAG3 antibody, an anti-Siglec15 antibody, an anti-4-1BB antibody, an anti-GITR antibody, an anti-OX40 antibody, an anti-CD40L antibody, an anti-CD28 antibody, or an anti- Selected from TIGIT antibodies and anti-VISTA antibodies, the epigenetic drug is, for example, a histone deacetylase inhibitor, the inhibitor targeting the PI3K / Akt / mTOR signaling pathway is, for example, Tricibine, the tyrosine kinase inhibitor is, for example, sunitinib, the immunosuppressant is, for example, cyclophosphamide, the proteasome inhibitor is, for example, bortezomib, the immunosuppressant is, for example, thalidomide or pomalidomide, the cytotoxic agent is, for example, gemcitabine or temozolomide, and the cell cycle nonspecific drug is, for example, mitoxantrone.

[0037] 2.3 Drugs that can lower blood IgG levels Preferably, in the above-described composition, the polypeptide drug capable of lowering the blood IgG level can block the binding of blood IgG to the FcRn protein. Preferably, the affinity of the polypeptide to the human FcRn protein is higher than the affinity of blood IgG to the human FcRn protein. The IgG is selected from IgG1, IgG2, IgG3, and IgG4. Preferably, the polypeptide comprises an antibody Fc fragment variant, the variant comprising a mutation capable of improving the affinity between Fc and FcRn, the mutation being preferably YTE, YTEKF, LS, or NHS, and the antibody Fc fragment being, for example, Efgartigimod. The variant may be a monomer, a dimer, or a polymer. The YTE, YTEKF, LS, NHS, and other mutations available for use in the present invention are located as described by Dall'Acqua et al. (WF, DA et al. (2002). Journal of immunology (Baltimore, Md.: 1950) 169(9): 5171-5180.) and Lee et al. (Lee, CH et al. (2019). Nat Commun 10(1): 5031.). The target of the mutation is selected from human IgG, and the IgG is selected from IgG1, IgG2, IgG3, and IgG4.

[0038] Other Fc fragment mutants available for use in the present invention include the mutations described by Dall'Acqua et al. (WF, DA et al. (2002). Journal of immunology (Baltimore, Md.: 1950) 169(9): 5171-5180.), the mutations described by Shan et al. (Shan, L. et al. (2016). PLoS One 11(8): e0160345.), the mutations described by Lee et al. (Lee, CH et al. (2019). Nat Commun 10(1): 5031.), the mutations described by Mackness et al. (Mackness, BC et al. (2019). MAbs 11(7): 1276-1288.), and the mutations described by Christophe et al. (Dumet Christophe, Pottier Jeremy, Gouilleux-Gruart This includes, but is not limited to, Valerie et al. (MAbs, 2019, 11:1341-1350.).

[0039] Preferably, the polypeptide comprises an antibody Fc fragment variant, the variant comprises a mutation that can improve the affinity between Fc and FcγR, and the variant is preferably S239D / I322E, S239D / I322E / A330L, K326W / E333S, R21 The mutant is a 4K mutation, and the mutant is preferably not modified with fucose. The mutant may be a monomer, dimer, or polymer. Other Fc fragment mutants available for use in the present invention include, but are not limited to, the mutant described by Wang et al. (Wang Xinhua, Mathieu Mary, Brezski Randall J. (2018). Protein Cell, 9(1), 63-73. doi:10.1007 / s13238-017-0473-8).

[0040] Preferably, the mutant capable of improving the affinity between Fc and FcRn, as described above, simultaneously contains a mutation that improves the affinity between Fc and FcγR. The mutant may be a monomer, a dimer, or a polymer.

[0041] Preferably, in the drug composition, the polypeptide is selected from anti-FcRn antibodies, such as Nipocalimab, Rozanolixizumab, RVT-1401, HBM9161, ALXN1830, SYNT001, and Nirsevimab.

[0042] Preferably, in the drug composition, the polypeptide is selected from small peptide fragments that can specifically bind to FcRn, the length of the small peptide fragment is 10 to 70 amino acids, and the small peptide fragment is, for example, ABY-039.

[0043] Preferably, the polypeptide is selected from Fc polymers that can specifically bind to FcRn, and the Fc polymers are, for example, GL-2045, M230, PRIM, HexaGard®, CSL777, and Hexavalent molecules by UCB.

[0044] Preferably, the polypeptide includes, but is not limited to, the polypeptide fragment described by Sockolosky et al. (Sockolosky Jonathan T, Szoka Francis C. Adv. Drug Deliv. Rev., 2015, 91:109-24).

[0045] 2.4 Viral vector drugs Preferably, in the above-described composition, the virus used in the viral vector drug is selected from ssDNA viruses, dsDNA viruses, ssRNA viruses or dsRNA viruses, and / or the virus used in the viral vector drug is selected from wild-type virus strains or naturally attenuated strains, genetically engineered selective attenuated strains, gene-loaded virus strains or gene transcription-targeted virus strains.

[0046] Preferably, the wild-type virus strain or naturally attenuated strain is selected from Newcastle disease virus, reovirus, mumps virus, West Nile virus, adenovirus, vaccinia virus, etc.

[0047] Preferably, the genetically engineered selective attenuated strain achieves tumor selectivity in viral replication by artificially deleting key genes, and is, for example, herpes simplex virus I (HSV-1) manipulated with a gene knocked out by thymidine kinase (TK). Examples of such genetically engineered selective attenuated strains are ONYX-015 and G207. In ONYX-015, 827 bp is deleted in the E1b region, and a point mutation is made in the gene for the E1B55K protein, which terminates the expression of that gene prematurely and prevents the expression of the E1B55K protein. In G207, the γ34.5 gene is deleted, and this gene is a determinant of the neurotoxicity of HSV-1.

[0048] Preferably, the gene-loaded virus strain is loaded with an exogenous gene, the exogenous gene being, for example, granulocyte-macrophage colony-stimulating factor (GM-CSF), and the gene-loaded virus strain being, for example, JX-594 or T-VEC.

[0049] Preferably, the gene transcription-targeted virus strain controls the replication of the oncolytic virus within tumor cells by inserting a tissue or tumor-specific promoter before the essential viral gene, and the gene transcription-targeted virus strain is, for example, G92A.

[0050] Preferably, in the drug composition described above, the ssDNA virus is selected from parvoviruses, and preferably, the parvovirus is an H-1PV virus.

[0051] Preferably, the dsDNA virus is selected from herpes simplex virus, adenovirus, and poxvirus. More preferably, the herpes simplex virus is type I herpes simplex virus HSV-1, such as R3616, T-VEC, HF10, G207, NV1020, or OrienX010. The poxvirus is selected from Pexa-Vec (vaccinia viruse), JX-594 (vaccinia viruse), GL-ONC1, or Myxoma. The adenovirus is selected from Enadenotucirev, DNX-2401, C-REV, NG-348, ProsAtak, CG0070, ADV-TK, EDS01, KH901, H101, H103, VCN-01, or Telomelysin (OBP-301).

[0052] Preferably, the ssRNA virus is selected from Picornavirus, alphavirus, Retroviruses, Paramyxoviruses, and Rhabdoviruses. Preferably, the Picornavirus is selected from CAVATAK, PVS-RIPO, CVA21 (enterovirus), and RIGVIR, and the alphavirus is M1, Sindbis AR339, and Semliki. The Forest virus is selected, the Retroviruses are selected from Toca511, the Paramyxoviruses are selected from MV-NIS and PV701 (Newcastle disease virus), and the Rhabdoviruses are selected from VSV-IFNβ, MG1-MAGEA3, and VSV-GP.

[0053] Preferably, the dsRNA virus is selected from Reoviruses, and preferably, the Reoviruses are selected from Pelareorep, Reolysin, Vaccinia virus, Mumps virus, and Human Immunodeficiency Virus (HIV). Preferably, the RNA virus is selected from Reovirus, Coxsackievirus, Poliovirus, Seneca Valley virus, Measles virus, Newcastle disease virus, Vesicular stomatitis virus, and Influenza virus.

[0054] Preferably, in the drug composition described above, the oncolytic virus expresses an exogenous gene, which is preferably a bispecific T cell engager (BiTE), an scFv fragment, a cytokine, or a chemokine. The BiTE binds to molecules that activate T cells, such as CD3. It can do so and simultaneously bind to antigen targets on the surface of cancer cells. The scFv targets immune checkpoints. The immune checkpoints include CTLA-4, PD-1, TIM-3, LAG3, Siglec15, 4-1BB, GITR, OX40, CD40L, CD28, TIGIT, and VISTA. The cytokines and chemokines are, for example, GM-CSF, interleukin-2 (IL-2), interleukin-12 (IL-12), interferon (IFN), tumor necrosis factor (TNF), soluble CD80, and CCL3.

[0055] 2.5 Gene therapy drugs Preferably, in the above-described composition, the gene therapy virus expresses an exogenous gene, the exogenous gene codes for a protein necessary for a genetic defect disease, and the protein is acid α-glucosidase, copper transport ATPase 2, α-galactosidase, arginosuccinate synthase, β-glucocerebrosidase, β-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, acid fibroblast growth factor, epidermal growth factor, transforming growth factor Child a, platelet-derived growth factor, insulin growth factor I and II, TGF, bone morphogenesis protein, nerve growth factor, brain-derived neurotrophic factor, neurotrophin NT-3 and NT4 / 5, ciliary neurotrophic factor, glial cell-derived neurotrophic factor, neurotrophin, lectin, netrin-1 and netrin-2, hepatocyte growth factor, ephrins, tyrosine hydroxylase, thrombopoietin, interleukins (IL-1 to IL-36, etc.), monocyte chemotactic protein, leukemia inhibitor, granulocyte-macrophage colony-stimulating factor, Fas ligand, tumor necrosis factor a and b, interferon a / b / g, stem cell factor, flk-2 / flt3 ligand, IgM, IgA, IgD and IgE, chimeric immunoglobulin, humanized antibody, single-chain antibody, T cell receptor, chimeric T cell receptor, single-chain T cell receptor, class I and class IIThe enzymes selected are MHC molecules, cystic fibrosis transmembrane regulatory proteins, coagulation factors (factors XIII, IX, VIII, X, VII, VIIa, protein C, etc.), retinal pigment epithelium-specific 65kDa proteins, LDL receptors, lipoprotein lipases, ornithine transcarbamylase, β-globulin, α-globulin, spectrin, α-antitrypsin, adenosine deaminase (ADA), metal transporters (ATP7A or ATP7), sulfamidases, enzymes involved in lysosomal storage disease (ARSA), hypoxanthine guanine phosphoribosyltransferase, β-25 glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, and branched-chain keto hormones.

[0056] Preferably, in the composition described above, the gene therapy virus has an exogenous gene, and the exogenous gene code is selected from inhibitory nucleic acids such as siRNA, antisense molecules, miRNA, RNAi, ribozymes, and shRNA. The inhibitory nucleic acid binds to a gene associated with polynucleotide repeat disease, and the transcript of the gene or the polynucleotides of the transcript of the gene repeat. The aforementioned disease gene coding-related proteins include Huntington's protein (HTT), the androgen receptor on the X chromosome in spinal and bulbar muscular atrophy, human ataxin-1 / -2 / -3 / -7, Cav2.1P / Q voltage-gated calcium channel (CACNA1A), TATA-binding protein, ataxin 8 reverse chain (ATXN80S), serine / threonine protein phosphatase 2A 55kDa regulatory subunit B beta isoforms (types 1, 2, 3, 6, 7, 8, 1217) in spinocerebellar ataxia, FMR1 (fragility 1 in fragile X syndrome), FMR1 (fragile X intellectual disability 1) in fragile X-related tremor / ataxia syndrome, FMR1 (fragile X intellectual disability 2) in fragile XE intellectual disability, or AF4 / FMR2 family member 2, myotonia The disease genes are selected from myotonin-protein kinase (MT-PK) and Frataxin in sexual dystrophy. The disease genes are mutants of the superoxide dismutase 1 (SOD1) gene, genes involved in 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 syncytial virus genome / gene sequence in respiratory syncytial virus infection, and Ebola infection. The genomes / gene sequences are selected from the following: Ebolafirovirus genome / gene sequences in hepatitis B and C infections, herpes simplex virus (HSV) genome / gene sequences in HSV infections, coxsackievirus B3 genome / gene sequences in coxsackievirus B3 infections, allele-specific silencing of pathogenic alleles such as torsinA (TOR1A) in primary dystonia, pan-class I and HLA alleles in transplantation, mutants in autosomal dominant retinitis pigmentosa, and rhodopsin genes.

[0057] III.Product The present invention further provides a product comprising the above-mentioned mutant or protein and a therapeutic agent, wherein the therapeutic agent is selected from viral vector drugs, antibodies, and polypeptide drugs capable of reducing blood IgG levels.

[0058] The present invention further provides a kit or pharmaceutical pack, the kit comprising: 1) a therapeutically effective amount of a drug containing the mutant; and 2) a therapeutically effective amount of a therapeutic agent selected from a viral vector drug, an antibody, or a polypeptide drug capable of reducing blood IgG levels, wherein the viral vector drug is preferably an oncolytic virus or a gene therapy virus. The kit may further comprise 3) a targeted drug or a chemotherapeutic agent or an immune checkpoint blocker. The target drug is selected from epigenetic drugs, inhibitors targeting the PI3K / Akt / mTOR signaling pathway, and tyrosine kinase inhibitors; the chemotherapeutic agent is selected from immunosuppressants, proteasome inhibitors, cytotoxic agents, and cell cycle nonspecific drugs; the immune checkpoint blocker is 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, and anti-VISTA antibody; Epigenetic drugs include, for example, histone deacetylase inhibitors; inhibitors targeting the PI3K / Akt / mTOR signaling pathway include, for example, tricibine; tyrosine kinase inhibitors include, for example, sunitinib; immunosuppressants include, for example, cyclophosphamide; proteasome inhibitors include, for example, bortezomib; immunosuppressants include, for example, thalidomide and pomalidomide; cytotoxic drugs include, for example, gemcitabine and temozolomide; and cell cycle-nonspecific drugs include, for example, mitoxantrone.

[0059] The kit or pharmaceutical pack comprises pack A and pack B, wherein pack A comprises a therapeutically effective amount of the above-mentioned mutant or protein, and pack B comprises a therapeutically effective amount of a therapeutic agent, the therapeutic agent being selected from viral vector drugs, antibodies, and polypeptide drugs capable of reducing blood IgG levels, the viral vector drug being preferably an oncolytic virus or a gene therapy virus. The pharmaceutical pack may further comprise pack C, which comprises a targeted drug or chemotherapeutic agent or immune checkpoint blocker. The targeted drug is selected from epigenetic drugs, inhibitors targeting the PI3K / Akt / mTOR signaling pathway, and tyrosine kinase inhibitors, the chemotherapeutic agent is selected from immunosuppressants, proteasome inhibitors, cytotoxic agents, and cell cycle nonspecific drugs, and the immune checkpoint blocker is an anti-CTLA-4 antibody, an anti-PD-1 antibody, or an anti-TIM-3 antibody. The following antibodies are selected: 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, and anti-VISTA antibody. The epigenetic drug is, for example, a histone deacetylase inhibitor; the inhibitor targeting the PI3K / Akt / mTOR signaling pathway is, for example, Tricibine; the tyrosine kinase inhibitor is, for example, sunitinib; the immunosuppressant is, for example, cyclophosphamide; the proteasome inhibitor is, for example, bortezomib; the immunosuppressant is, for example, thalidomide or pomalidomide; the cytotoxic agent is, for example, gemcitabine or temozolomide; and the cell cycle nonspecific drug is, for example, mitoxantrone.

[0060] The kit may include instructions for administering a therapeutically effective amount of the aforementioned mutant or protein and a therapeutically effective amount of the therapeutic agent (e.g., dosage information, administration time interval information). The therapeutic agent is selected from viral vector drugs, antibodies, and polypeptide drugs that can reduce blood IgG levels, and the viral vector drug is preferably an oncolytic virus.

[0061] Viral vector drugs can be manufactured using well-established expression systems. Examples of such methods include utilizing mammalian cell expression systems to generate viral particles, for example, using HEK293 cells to produce adenovirus vector drugs (Freedman Joshua D, Duffy Margaret R, Lei-Rossmann Janet et al., An Oncolytic Virus Expressing a T-cell Engager Simultaneously Targets Cancer and Immunosuppressive Stromal Cells.[J] .Cancer Res., 2018, 78:6852-6865.).

[0062] The drug vector may be a liquid, and the drug composition may be in the form of a solution. Liquid vectors are used in the preparation of solutions, suspensions, emulsions, syrups, elixirs, and compressed compositions. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid vector, such as water, an organic solvent, a mixture of both, or a pharmaceutically acceptable oil or fat.

[0063] Drug compositions for parenteral administration are sterile, substantially isotonic, pyrogenic-free, and prepared in accordance with FDA or similar organization GMP. Viral vector drugs can be administered as injectable doses of solution or suspension in a physiologically acceptable diluent containing the drug vector, which may be a sterile solution such as water, oil, saline, glycerol, or ethanol. Furthermore, auxiliary substances such as wetting agents or emulsifiers, surfactants, and pH buffers may be present in the composition. Other components of the drug composition are petroleum, animal, plant, or synthetic components, such as peanut oil, soybean oil, and mineral oil. Generally, glycols such as propylene glycol or polyethylene glycol are preferred liquid vectors, especially for injectable solutions. Viral vector drugs can be administered in the form of depot injections or implant preparations, which can be formulated in a way that allows for sustained release of the active ingredient. Typically, compositions are prepared for injection as either a liquid solution or a suspension, and may also be prepared into a solid form suitable for use in solution, suspension, or liquid excipients before injection.

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by an ordinary person skilled in the art to which this invention pertains. Any methods, apparatus and materials similar to or equivalent to those described herein may be used in carrying out or measuring the present invention, but preferred methods, apparatus and materials are described below.

[0065] The term "nucleotide" or "polynucleotide" refers to a single-stranded or double-stranded form of deoxygenated molecules. This term refers to siribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and their polymers. Unless otherwise specified, this term encompasses nucleic acids containing known analogues of native nucleotides that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to native nucleotides. Unless otherwise specified, this term also refers to oligonucleotide analogues, including PNAs (peptide nucleic acids) and DNA analogues used in antisense technology (such as phosphorothioates and phosphoramidates). Unless otherwise specified, a nucleic acid sequence implicitly includes its conservatively modified variants (including, but not limited to, degenerate codon substitutions) and complementary sequences, as well as explicitly stated sequences. Specifically, degenerate codon substitution can be achieved by generating sequences in which the third position of one or more (or all) selected codons is replaced with a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0066] The terms “polypeptide” and “protein” are used herein to mean polymers of amino acid residues. That is, descriptions relating to polypeptides also apply to descriptions relating to peptides and proteins, and vice versa. The terms apply to spontaneously occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are amino acids not naturally encoded. As used herein, the terms encompass amino acid chains of any length, including full-length proteins (i.e., antigens) in which amino acid residues are linked by peptide covalent bonds.

[0067] The term "host cell" refers to a cell containing the nucleotides of the present invention, regardless of the method used for insertion, e.g., direct incorporation, transduction, f-mating, or any other method known in the art for producing recombinant host cells. The exogenous polynucleotides may be maintained as an unintegrated vector, e.g., a plasmid, or they may be integrated into the host genome.

[0068] The term "transformation" refers to a method of introducing a different DNA sequence into a host cell or living organism.

[0069] The term "expression" refers to the transcription and / or translation of an endogenous gene or a transgenic gene in a cell.

[0070] The positive effects of the present invention are as follows: The present invention provides an immunoglobulin-degrading enzyme mutant that has higher activity and / or thermal stability than wild-type IdeE, and higher activity than IdeS and IdeZ (more effective than IdeS and IdeZ when cleaving human IgG, with activity nearly twice that of IdeS and more than four times that of IdeZ). [Examples]

[0071] The present invention will be further described below with reference to examples, but the present invention is not limited to the scope of the examples described above. In the following examples, test methods for which specific conditions are not specified will be selected according to conventional methods and conditions or according to the product description.

[0072] Example 1. Design and expression of a mutant library We designed and constructed a mutant library of wild-type IdeE protein sequences, and selected 40 mutant strains from it.

[0073] After codon optimization, the wild-type IdeE protein sequence (SEQ ID NO:2) is used. A polynucleotide sequence was synthesized, an N-terminal signal peptide sequence and a C-terminal 6× histidine tag were added, and the sequence was synthesized. This sequence was then inserted into a pET32a expression vector, and after correct sequencing, a recombinant plasmid for wild-type IdeE expression was obtained. Based on the plasmid for wild-type IdeE expression, degenerate primers necessary for the mutant library were designed, the original wild-type sequence was amplified, and after amplification, the sequence was inserted into a vector to obtain a recombinant plasmid for the mutant library. The recombinant plasmids for the wild-type and mutant libraries were electrically transformed into E. coli BL21 Star (DE3) and inoculated into LB agarose plates containing 100 μg / ml ampicillin. The cells were cultured overnight at 37°C until colonies grew. Single colonies were picked and inoculated into 200 μl of LB medium containing 100 μg / ml ampicillin, and cultured overnight at 37°C and 250 rpm. After overnight culture, the cells were cultured in LB medium containing 100 μg / ml ampicillin. The sample was inoculated into 1 ml of culture solution and incubated at 37°C for 4 hours. Then, 0.1 mM IPTG was added, and the culture was continued overnight at 30°C. After overnight incubation, the supernatant was collected by centrifugation. The concentration of mutant protein in the mutant-expressing supernatant was evaluated using SDS-PAGE.

[0074] Example 2. Evaluation of the human IgG1 cleavage activity of mutants. To evaluate the human IgG1 cleavage activity of each mutant, an ELSIA-based activity measurement method was developed. The measurement principle involved coating an ELISA plate with a human IgG1-specific antigen, and then incubating a supernatant sample containing a corresponding concentration of mutant protein in the wells with human IgG1. Using a human IgG1 detection antibody specific to the antibody Fc portion, the amount of complete or incompletely cleaved human IgG1 bound to the wells was measured. When the concentration of mutant protein in the supernatant applied to the wells was the same, the higher the human IgG1 cleavage activity of the mutant protein, the less complete human IgG1 antibody bound to the wells, resulting in a weaker signal. An IgG1 calibration curve could be constructed based on the relationship between different concentrations of IgG1 and the corresponding detection signals. Based on the calibration curve, the amount of complete or incompletely cleaved IgG1 was calculated, and further, the amount of completely cleaved IgG1 was calculated. The level of mutant activity was evaluated by the ratio of completely cleaved IgG1 to the original IgG1.

[0075] In order to achieve a substantial concentration of mutant protein in the supernatant obtained in Example 1, SDS-PAGE detection was performed with the same loading volume, and the optical density values ​​of the target protein band in the electrophoresis profile were analyzed using Quantity One. When the loading volume is the same, a higher optical density value of the target protein band in the profile indicates a higher concentration. Using the IdeE supernatant as a control, the supernatants of the other mutants were concentrated or diluted until the optical density values ​​of the mutant protein bands were nearly identical to those of the IdeE control.

[0076] After adjusting the protein concentration in the supernatant to a suitable level, ELISA detection was performed according to the following method: ELISA plates were coated with 2 μg / ml human IgG1 (trastuzumab) specific antigen (Catalog No.: QRE-104, Suan Bio) at 2-8°C and left overnight. Subsequently, they were washed with PBST (PBS + 0.05% Tween 20), and the washed ELISA plates were sealed and incubated with 2% BSA (prepared in PBS) at 37°C for 2 hours. After sealing, they were washed with PBST again.

[0077] Calibration curve preparation: 200 ng / ml trastuzumab was gradient diluted in a 1:2 ratio with reaction buffer (10 mM PB, 10 mM NaCl, pH 6.5) until it reached approximately 3.125 ng / ml. 100 μl of trastuzumab at different concentrations was added to the wells of an ELISA plate, and a substrate (trastuzumab) calibration curve was prepared.

[0078] Cutting reaction: After adjusting the protein concentration, the supernatant was diluted fivefold with reaction buffer (10 mM PB, 10 mM NaCl, pH 6.5), and 50 μl of 100 ng / ml trastuzumab and 50 μl of the diluted supernatant were added to the wells of an ELISA plate.

[0079] ELISA plates were incubated at 37°C for 1 hour with shaking, washed with PBST, and then 40 ng / ml of Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody-HRP (Catalog No.: 31413, Thermo) was added to the well plates. The plates were incubated at 37°C for 1 hour with shaking, washed with PBST, and then TMB was added as a chromogenic substrate for HRP. The plates were incubated for 15 minutes and terminated in 2N H2SO4. Absorbance at 450 nm was detected using an ELISA. Based on the substrate calibration curve, the concentrations of complete or incompletely cleaved trastuzumab in different measurement wells were calculated, and the proportion of completely cleaved trastuzumab to the original trastuzumab was also calculated to evaluate the activity of different mutants.

[0080] Table 1 shows the plicative relationship of each mutant's activity to wild-type IdeE activity. In Example 1, all 40 mutants obtained by selection had higher activity than wild-type IdeE, and of these, 15 mutants had twice or more the activity of wild-type IdeE.

[0081] [Table 1]

[0082] Example 3 Evaluation of the thermal stability of mutants From the 15 mutants shown in Table 1, whose activity was twice or more than twice that of wild-type IdeE, 12 mutants were selected and their thermal stability was detected. The method for detecting activity is described below.

[0083] The supernatant of the wild-type or each mutant was divided into two parts and incubated for 1 hour under conditions of 4°C and 50°C, respectively. After incubation, the activity of the wild-type or each mutant was detected according to the ELISA method in Example 2. The percentage of residual activity (%) after incubation at 50°C or each mutant after incubation at 50°C was calculated using the ratio of the activity after incubation at 4°C. The thermal stability of the wild type and each mutant was calculated and used to compare them.

[0084] Table 2 shows the relationship between the thermal stability of mutant activity and wild-type IdeE. As shown in Table 2, all 12 mutants had higher thermal stability than the wild type, and of these, seven mutants had more than three times the thermal stability of the wild type.

[0085] [Table 2]

[0086] Example 4: Comparison of human IgG1 cleavage activity of single-site mutants We detected the human IgG1 cleavage activity of seven single-site mutants—T8D, T8W, T24A, A59L, A59V, E97D, and R280H—shown in Tables 1 and 2, that exhibited "activity more than twice that of wild-type IdeE and thermal stability more than three times that of wild-type IdeE."

[0087] 1. Expression and purification of mutants One single colony was selected from each of the five single-site mutant transformation plates described above in Example 1, inoculated into 3 ml of LB medium containing 100 μg / ml ampicillin, and incubated overnight at 37°C and 250 rpm. After overnight incubation, the cells were inoculated into 50 ml of LB medium containing 100 μg / ml ampicillin and incubated at 37°C until the OD600 reached 0.4-0.6. 0.1 mM IPTG was added, and the cells were incubated overnight at 30°C. After overnight incubation, the supernatant was collected by centrifugation. The supernatant was further purified using IDA-Ni magnetic agarose beads, and the purified and eluted proteins were transferred to a PBS buffer system using an ultrafiltration centrifuge tube. The purity of the purified mutant proteins was evaluated by SDS-PAGE. OD280 was detected, and the concentration of the purified mutant proteins was calculated based on the extinction coefficient.

[0088] 2. Comparison of human IgG1 cleavage activity of mutants The level of human IgG1 cleavage activity of different mutants against wild-type IdeE was further evaluated by examining the cleavage products produced against human IgG1 at different concentrations of various mutants as displayed by SDS-PAGE. Purified mutants or wild-type IdeE were diluted to 0.002 mg / mL and 0.001 mg / mL, respectively. 50 μl of each mutant or wild-type IdeE at different concentrations was added to 50 μl of a reaction system containing 2 mg / mL of trastuzumab to initiate the cleavage reaction, and the reaction system was incubated at 37°C for 30 min. After mixing the samples with the same volume of 2×SDS loading buffer, the samples were bathed in water at 75°C for 5 min, and the cleavage products were detected by SDS-PAGE.

[0089] Figure 1 shows the electrophoresis (enzyme:substrate = 1:1000) of the cleavage products produced by cleaving human IgG1 with seven single-site mutants and wild-type IdeE. Figure 2 shows the electrophoresis (enzyme:substrate = 1:2000) of the cleavage products produced by cleaving human IgG1 with seven single-site mutants and wild-type IdeE. The body found that the ability to cleave IgG1 at a concentration of 0.001 mg / ml was no less effective than that of wild-type IdeE at 0.002 mg / ml, and that the human IgG1 cleavage activity of the seven single-site mutants was more than twice that of wild-type IdeE.

[0090] Example 5: Comparison of human IgG1 cleavage activity and thermal stability of N-terminal truncated mutants Based on wild-type IdeE, five N-terminal truncated mutants were constructed by deleting the first 15 (D1-V15), first 16 (D1-P16), first 17 (D1-H17), first 18 (D1-Q18), and first 19 (D1-I19) amino acids of the N-terminus, respectively (see Table 3).

[0091] [Table 3]

[0092] 1. Expression and purification of mutants The mutant polynucleotide sequences in Table 3 were synthesized according to the method of Example 1, a recombinant plasmid for mutant expression was constructed, and E. coli BL21 Star(DE3) was transformed. Purified mutant proteins were produced according to the method of Example 4.

[0093] 2. Comparison of human IgG1 cleavage activity of mutants The purified mutant or wild-type IdeE was diluted to 0.002 mg / mL. 50 μl of the diluted mutant or wild-type IdeE was added to 50 μl of a reaction system containing 2 mg / mL of trastuzumab to initiate the cleavage reaction, and the reaction system was incubated at 37°C for 30 minutes. The samples were mixed with the same volume of 2×SDS loading buffer and then incubated in a water bath at 75°C for 5 minutes. The cleavage products were detected by SDS-PAGE.

[0094] Figure 3 shows the electrophoretic graph (enzyme:substrate = 1:1000) of the cleavage products produced by cleaving human IgG1 with five truncated mutants. The cleavage activity of all five truncated mutants was not significantly different from that of wild-type IdeE.

[0095] 3. Comparison of the thermal stability of mutants The purified mutant or wild-type IdeE was diluted to 0.1 mg / ml, incubated at 50°C for 1 hour, and then further diluted to 0.002 mg / mL. 50 μl of the diluted mutant or wild-type IdeE was selected and added to 50 μl of a reaction system containing 2 mg / ml of trastuzumab to initiate the cleavage reaction. The reaction system was incubated at 37°C for 30 minutes. The samples were mixed with the same volume of 2×SDS loading buffer and then incubated in a water bath at 75°C for 5 minutes. The cleavage products were detected by SDS-PAGE.

[0096] Figure 4 shows the electrophoresis (enzyme:substrate = 1:1000) of the cleavage products produced by cleaving human IgG1 with five shortened mutants and wild-type IdeE after incubation at 50°C for 1 hour. After heat treatment at 50°C, the residual activity of the five shortened mutants was clearly higher than that of the wild-type, and it was found that the thermal stability of all five shortened mutants was significantly improved compared to the wild-type.

[0097] Example 6: Comparison of human IgG1 cleavage activity of C-terminal truncated mutants Based on wild-type IdeE, two C-terminal truncated mutants were constructed by deleting the last five amino acids (W311-S315) and the last ten amino acids (S306-S315), respectively (see Table 4).

[0098] [Table 4]

[0099] 1. Expression and purification of mutants The mutant polynucleotide sequences in Table 4 were synthesized according to the method of Example 1, a recombinant plasmid for mutant expression was constructed, and E. coli BL21 Star(DE3) was transformed. Purified mutant proteins were produced according to the method of Example 4.

[0100] 2. Comparison of human IgG1 cleavage activity of mutants The purified mutant or wild-type IdeE was diluted to 0.002 mg / mL. 50 μl of the diluted mutant or wild-type IdeE was added to 50 μl of a reaction system containing 2 mg / mL of trastuzumab to initiate the cleavage reaction, and the reaction system was incubated at 37°C for 30 minutes. The sample was mixed with the same volume of 2×SDS loading buffer and then incubated in a water bath at 75°C for 5 minutes. The cleavage product was detected by SDS-PAGE.

[0101] Figure 5 shows the electrophoretic graph (enzyme:substrate = 1:1000) of the cleavage products produced by cleaving human IgG1 with two C-terminal truncated mutants. The cleavage activity of both truncated mutants was more than twice as high as that of wild-type IdeE.

[0102] Example 7: Comparison of human IgG1 cleavage activity and thermal stability of combined mutants Based on five single-site mutants, T24A, A59L, A59V, E97D, and R280H, five combination mutants were constructed by deleting the first 18 amino acids (D1-Q18) from each mutant (see Table 5).

[0103] [Table 5]

[0104] 1. Expression and purification of mutants The mutant polynucleotide sequences in Table 5 were synthesized according to the method of Example 1, a recombinant plasmid for mutant expression was constructed, and E. coli BL21 Star(DE3) was transformed. Purified mutant proteins were produced according to the method of Example 4.

[0105] 2. Comparison of human IgG1 cleavage activity of mutants The purified mutants were each diluted to 0.001 mg / mL. 50 μl of either the diluted mutant or wild-type IdeE was selected and added to 50 μl of a reaction system containing 2 mg / mL of trastuzumab to initiate the cleavage reaction. The reaction system was incubated at 37°C for 30 minutes. The samples were mixed with the same volume of 2×SDS loading buffer and then incubated in a water bath at 75°C for 5 minutes. The cleavage products were detected by SDS-PAGE.

[0106] Figure 6 shows the electrophoretic graph (enzyme:substrate = 1:2000) of the cleavage products produced by cleaving human IgG1 with five combination mutants. A comparison of the cleavage effects in Figure 6 and Figure 2 revealed no significant difference in the cleavage activity of the five truncated mutants and the single-site combination mutants, and the human IgG1 cleavage activity of the combination mutants was similarly more than twice that of wild-type IdeE.

[0107] 3. Comparison of the thermal stability of mutants Each purified mutant was diluted to 0.1 mg / mL, incubated at 50°C for 1 hour, and then further diluted to 0.001 mg / mL. 50 μl of either the diluted mutant or wild-type IdeE was selected and added to reaction system 5 containing 2 mg / mL of trastuzumab. The cleavage reaction was initiated by adding the solution to 0 μl of water, and the reaction system was incubated at 37°C for 30 minutes. The sample was then mixed with the same volume of 2×SDS loading buffer and incubated in a water bath at 75°C for 5 minutes. The cleavage products were detected by SDS-PAGE.

[0108] Figure 7 shows the electrophoretic graph (enzyme:substrate = 1:2000) of the cleavage products produced by cleaving human IgG1 with five combination mutants after incubation at 50°C for 1 hour. A comparison of the cleavage effects in Figure 8 and Figure 7 revealed that the activity of the five combination mutants decreased only slightly after heat treatment at 50°C, and that the thermal stability of the five combination mutants was clearly improved compared to the wild type.

[0109] Example 8: Comparison of the activity of the E97D_del18 mutant with that of IdeS and IdeZ. The E97D_del18 mutant purified in Example 7 was sequentially diluted to ~20 μg / mL, 10 μg / mL, 5 μg / mL, 2.5 μg / mL, and 1.25 μg / mL. IdeS (FabRICATOR®, catalog number: A0-FRI-020, Genovis) was diluted to ~2 U / μl, 1 U / μl, 0.5 U / μl, 0.25 U / μl, and 0.125 U / μl, respectively, according to its labeling. IdeZ (FabRICATOR-Z®, catalog number: A0-FRZ-020, Genovis) was diluted to 0.4 U / μl, 0.2 U / μl, 0.1 U / μl, 0.05 U / μl, and 0.025 U / μl, respectively. 50 μl each of mutants at different concentrations, IdeS or IdeZ, were selected and added to 50 μl of a reaction system containing 2 mg / ml trastuzumab to initiate the cleavage reaction. The reaction system was then incubated at 37°C for 30 minutes. The samples were mixed with the same volume of 2×SDS loading buffer and incubated in a water bath at 75°C for 5 minutes. The cleavage products were detected by SDS-PAGE.

[0110] Figure 8 shows the electrophoresis of cleavage products produced by cleaving human IgG1 with different concentrations of the E97D_del18 mutant and IdeS. From the enzyme protein bands in the electrophoresis, the concentration of the IdeS enzyme in lane 1 is between the concentrations of the E97D_del18 mutant enzyme in lanes 7 and 8. This leads to the conclusion that the concentration of the IdeS enzyme in lane 3 is between the concentrations of the E97D_del18 mutant enzyme in lanes 9 and 10, and the enzyme cleavage effect of IgG1 in lane 3 is between lanes 10 and 11. Thus, it can be estimated that the human IgG1 cleavage activity of the E97D_del18 mutant is nearly twice that of IdeS.

[0111] Figure 9 shows the electrophoresis of cleavage products produced by cleaving human IgG1 with E97D_del18 mutants and IdeZ at different concentrations. From the enzyme protein bands in the electrophoresis, it can be determined that the concentration of IdeZ enzyme in lane 1 is higher than the concentration of E97D_del18 mutant enzyme in lane 7. This leads to the conclusion that the concentration of IdeZ enzyme in lane 3 is higher than the concentration of E97D_del18 mutant enzyme in lane 9, i.e., more than four times the concentration of E97D_del18 mutant enzyme in lane 11. Furthermore, it can be inducted that the enzyme cleavage effect of IgG1 in lane 3 is similar to that of lane 11. Thus, it was found that the human IgG1 cleavage activity of the E97D_del18 mutant is more than four times that of IdeZ.

[0112] Example 9: In vitro detection of human IgG1 cleavage activity of the E97D_del18 mutant. The in vitro cleavage activity of the E97D_del18 mutant against human IgG1 was evaluated by detecting the amount of complete or single-cleaved IVIg in mouse serum or plasma treated with the E97D_del18 mutant and human IVIg. Different groups of mouse serum or plasma enzyme cleavage systems were prepared according to Table 6.

[0113] [Table 6]

[0114] In the group treated with iodoacetic acid, the effect of iodoacetic acid is to suppress the activity of IgG-degrading enzymes.

[0115] The system was reacted at 37°C for 30 minutes. A 20 μl sample was taken and mixed with the same volume of 2 × SDS non-reducing loading buffer, then incubated in a water bath at 75°C for 5 minutes, and the cleavage products were detected by SDS-PAGE.

[0116] Figure 10 shows the electrophoretic maps of the cleavage products 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 both mouse serum and plasma.

[0117] The E97D_del18 mutant was used to evaluate whether it possessed in vitro cleavage activity against human IgG1 by detecting mouse or human serum treated with the E97D_del18 mutant. Different groups of mouse or human serum enzyme cleavage systems were constructed according to Table 7.

[0118] [Table 7]

[0119] The system was reacted at 37°C for 24 hours. A 20 μl sample was taken and mixed with the same volume of 2×SDS reducing loading buffer, then diluted 20-fold with 1×SDS reducing loading buffer, and incubated in a water bath at 75°C for 5 minutes. The cleavage products were detected by SDS-PAGE.

[0120] Figure 11 shows the electrophoretic maps of the cleavage products produced by the E97D_del18 mutant in mouse and human serum. The results show that E97D_del18 significantly produced a 25kD Fc fragment when cleaved in human serum, whereas this fragment was not observed in mouse serum. This indicates that E97D_del18 can effectively and specifically cleave IgG1 in human serum, but its cleavage activity against IgG1 in mouse serum is very low or nonexistent.

[0121] Example 10: E97D_del18 mutant cleaves immunoglobulins of different species. The in vitro cleavage activity of the E97D_del18 mutant against immunoglobulins in the serum or plasma of different animal species was evaluated by detecting the amount of IgG that was completely or once cleaved by adding the E97D_del18 mutant to the serum or plasma of different animal species. Different serum or antibody enzyme cleavage systems were constructed according to Tables 8 and 9.

[0122] [Table 8]

[0123] The system was reacted at 37°C for 1 hour, and the enzyme cleavage products were detected by SDS-PAGE.

[0124] [Table 9]

[0125] Figures 12A-12D show the effects of the E97D_del18 mutant on serum and antibodies from different species. The results showed that E97D_del18 could effectively cleave canine IgG, rabbit IgG, and mouse IgG2a, but could not cleave mouse IgG1. E97D_del18 could effectively cleave rabbit, canine, and monkey serum IgG, with the best cleavage effect on rabbit serum IgG, poor cleavage effect on pig serum IgG, and almost no cleavage of rat and mouse serum IgG.

[0126] Example 11: There are few existing antibodies against the E97D_del18 mutant in the human body. This measurement is based on the competition for binding of anti-E97D_del18 / IdeS antibodies between the E97D_del18 mutant and IdeS. Pre-incubation of the measurement enzyme and human serum enabled the anti-E97D_del18 / IdeS antibody and the E97D_del18 mutant to bind to IdeS.

[0127] The E97D_del18 mutant and IdeS were coated in well plates and left overnight. Then, they were washed with PBST and sealed in 2% BSA sealed solution for 1 hour. Gradually diluted mutants to be measured were mixed with IdeS and human serum to prepare mixed plates. The reagents were incubated at room temperature for 1 hour with shaking, washed with PBST, and then biotin-marked E97D_del18 mutants and IdeS were added. SA-HRP was then added, and the reagents were colorimetrically analyzed using TMB and read. The status of existing E97D_del18 and IdeS antibodies in the blood samples obtained from approximately 80 individuals was compared in parallel.

[0128] As a result, as shown in Table 10, IdeS had a high proportion of pre-existing antibodies in normal human serum, approximately 90%, while the E97D_del18 mutant had only about 20%. The pre-existing antibodies of the E97D_del18 mutant were significantly lower in the body than those of IdeS, demonstrating that the E97D_del18 mutant has lower immunogenicity and is more advantageous for administration in the body.

[0129] [Table 10]

[0130] Example 12: In vivo detection of human IgG1 cleavage activity of the E97D_del18 mutant. Human IVIg (human immunoglobulin) was intraperitoneally injected into two mice (the two mice were in a parallel study, numbered 1 and 2) under sterile conditions, with an injection dose of 1 g / kg. Twenty-four hours after the injection of human IVIg, the mice were further intravenously injected with IgG-degrading enzyme (E97D_del18) at a dose of 5 mg / kg. Serum samples were collected from the two mice at 0h, 15 min, 2h, 6h, and 24h after the injection of E97D_del18. 20 μl of serum sample was mixed with the same volume of 2×SDS non-reducing loading buffer, then further diluted 20-fold with 1×SDS non-reducing loading buffer, incubated in a water bath at 75°C for 5 min, and detected by SDS-PAGE.

[0131] Figure 13 shows the electrophoretic maps of the cleavage products produced by E97D_del18 cleaving human IVIg within different timeframes in the mouse body. The results show that E97D_del18 exhibits a significant IVIg cleavage effect in the mouse body, completely cleaving the enzyme in 15 minutes.

[0132] Example 13: Comparison of human IgG1 cleavage activity of combination mutants Based on the mutants described above, six more combination mutants were constructed, and the sequences of these mutants are shown in Table 11.

[0133] [Table 11]

[0134] 1. Expression and purification of mutants The mutant polynucleotide sequences in Table 11 were synthesized according to the method of Example 1, a recombinant plasmid for mutant expression was constructed, and E. coli BL21 Star(DE3) was transformed. Purified mutant proteins were produced according to the method of Example 4.

[0135] 2. Comparison of human IgG1 cleavage activity of mutants The purified mutants were each diluted to 0.001 mg / mL. 50 μl of either the diluted mutant or wild-type IdeE was selected and added to 50 μl of a reaction system containing 2 mg / mL of trastuzumab to initiate the cleavage reaction. The reaction system was incubated at 37°C for 30 minutes. The samples were mixed with the same volume of 2×SDS loading buffer and then incubated in a water bath at 75°C for 5 minutes. The cleavage products were detected by SDS-PAGE.

[0136] Figures 14A and 14B show the electrophoretic maps (enzyme:substrate = 1:2000) of the cleavage products produced by cleaving human IgG1 with six combination mutants.

[0137] Although the present invention has been described in detail by the above embodiments, the applicant declares that the present invention is not limited to the above detailed methods, that is, it does not mean that it must be carried out only by the above detailed methods. Those skilled in the art will understand that any improvements to the present invention, such as the equivalence substitution of each raw material in the product according to the present invention, the addition of auxiliary components, and the selection of specific forms, all fall within the scope of protection and disclosure of the present invention.

Claims

1. A mutant of the immunoglobulin-degrading enzyme IdeE, The immunoglobulin-degrading enzyme IdeE consists of the amino acid sequence shown in Sequence ID No. 2 in the sequence listing, The aforementioned mutant is Deletion of the first 15, first 16, first 17, first 18, or first 19 amino acids at the N-terminus of the aforementioned amino acid sequence, and, (1) Substitution of one or more positions among positions 24, 59, 97 and 280 of the amino acid sequence, (2) Deletion of the last five or last ten amino acids at the C-terminus of the aforementioned amino acid sequence, A mutant of Sequence ID No. 2, modified by one or both of the following: The mutant is a mutant of immunoglobulinase ideE having higher activity than immunoglobulinase ideE and / or higher thermal stability than immunoglobulinase ideE.

2. The aforementioned mutant, Deletion of the first 18 amino acids at the N-terminus of the aforementioned amino acid sequence, and, (1) Substitution of one or more positions among positions 24, 59, 97 and 280 of the amino acid sequence, (2) Deletion of the last five or last ten amino acids at the C-terminus of the aforementioned amino acid sequence, The mutant according to claim 1, which is a mutant of Sequence ID No. 2, modified by one or both of the above.

3. The substitution described above is (1) The threonine at position 24 is replaced by one of the following: alanine, cysteine, aspartic acid, asparagine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, proline, glutamine, arginine, serine, valine, tryptophan, and tyrosine. (2) The alanine at position 59 is cysteine, aspartic acid, glutamic acid, fe It is substituted with one of the following: nyalanin, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, and tyrosine. (3) The glutamic acid at position 97 is substituted with one of the following: alanine, cysteine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, and tyrosine, and (4) The arginine at position 280 is substituted with one of the following: alanine, aspartic acid, glutamic acid, cysteine, serine, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, tryptophan, threonine, valine, and tyrosine. A mutant according to claim 1 or 2, selected from the group consisting of the following.

4. The aforementioned mutant, Deletion of the first 18 amino acids at the N-terminus of the aforementioned amino acid sequence, and, Substitution of positions 97 and / or 280 in the aforementioned amino acid sequence The mutant according to claim 2, which is a mutant of sequence number 2 modified by [the specified method].

5. The aforementioned mutant, Deletion of the first 18 amino acids at the N-terminus of the aforementioned amino acid sequence, and The substitution at position 97 of the amino acid sequence, wherein the glutamic acid at position 97 is replaced by aspartic acid. The mutant according to claim 4, which is a mutant of sequence number 2 modified by [the specified method].

6. The mutant according to claim 3, wherein the mutant is represented by any of sequence numbers 25 to 34 in the sequence listing.

7. A protein comprising a mutant according to any one of claims 1 to 6, wherein the protein comprises the mutant and the N-terminal signal peptide of the mutant.

8. A protein comprising a mutant according to any one of claims 1 to 6, The aforementioned mutant, and, (1) The secretory signal sequence and / or methionine at the N-terminus of the mutant, and (2) The histidine tag at the C-terminus of the mutant, The protein comprising one or both of the following.

9. The protein according to claim 8, wherein the protein comprises the mutant, the N-terminal secretory signal sequence of the mutant, the N-terminal methionine of the secretory signal sequence, and / or the C-terminal histidine tag of the mutant.

10. The protein according to claim 9, wherein the protein comprises, in order from the N-terminus to the C-terminus, methionine, a secretory signal sequence, and the mutant.

11. A mutant according to any one of claims 1 to 6, or a nucleotide encoding a protein according to any one of claims 7 to 10.

12. An expression vector comprising the nucleotide described in claim 11.

13. A host that expresses the expression vector described in claim 12, or the mutant described in any one of claims 1 to 6, or the protein described in any one of claims 7 to 10. cell.

14. A mutant of an immunoglobulin-degrading enzyme, or a protein containing the mutant of the immunoglobulin-degrading enzyme, With any pharmaceutically acceptable vector or excipient, A composition containing, The composition wherein the mutant of the immunoglobulin-degrading enzyme is the mutant according to any one of claims 1 to 6, or the protein containing the mutant of the immunoglobulin-degrading enzyme is the protein according to any one of claims 7 to 10.

15. The composition according to claim 14, further comprising an antibody or an Fc-containing protein.

16. The composition according to claim 15, wherein 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.

17. The composition according to any one of claims 14 to 16, further comprising a viral vector drug.

18. The composition according to any one of claims 14 to 16, further comprising a drug capable of lowering blood IgG levels.

19. (1) A mutant according to any one of claims 1 to 6, or a protein according to any one of claims 7 to 10, (2) One or more selected from the group consisting of (a) a pharmaceutically acceptable vector or excipient and (b) an antibody or Fc-containing protein, and / or (3) Viral vector drugs selected from oncolytic viruses, gene therapy viruses and viral vector vaccines, and / or (4) A drug that can lower blood IgG levels, selected from FcRn antibodies and Fc fragment variants with high affinity for FcRn, A kit that includes this.

20. A kit that includes pack A and pack B, The aforementioned pack A contains the mutant according to any one of claims 1 to 6, or the protein according to any one of claims 7 to 10. The aforementioned pack B is, It contains one or more selected from the group consisting of (1) a pharmaceutically acceptable vector or excipient, (2) an antibody or Fc-containing protein, and / or (3) a viral vector drug, and / or (4) a drug capable of lowering blood IgG levels. The kit wherein the viral vector drug is selected from oncolytic viruses, gene therapy viruses, and viral vector vaccines, and the drug capable of lowering blood IgG levels is selected from FcRn antibodies and Fc fragment variants with high affinity for FcRn.

Images