Uses of a mutant of the immunoglobulin-degrading enzyme IdeE

Abstract

A mutant of the immunoglobulin-degrading enzyme IdeE, a protein comprising said mutant, a composition, and a kit are provided for use in the preparation of a medicament for reducing IgG levels in subjects; wherein said mutant has an amino acid substitution, N-terminal truncation, and / or C-terminal truncation at one or more positions of amino acid positions 8, 10, 24, 59, 97, and 280 of the amino acid sequence shown in SEQ ID NO: 2, and said mutant possesses the function of the immunoglobulin-degrading enzyme IdeE and has higher activity and thermostability than wild-type IdeE.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a mutant of an immunoglobulin-degrading enzyme and a composition comprising the same for use in reducing IgG levels. Background Technology

[0002] Streptococcus pyogenes is one of the most common pathogens in humans and animals, widely distributed in nature and in the oropharynx, respiratory tract, and intestines of humans and animals. Streptococcal infections can cause a range of diseases, from milder conditions such as purulent dermatitis and pharyngitis to more serious conditions such as sepsis, necrotizing fasciitis, and toxic shock syndrome. Immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) is a common cysteine ​​protease from Group A Streptococcus pyogenes (GAS) and possesses intrapeptidase activity that hydrolyzes IgG (Agniswamy J, Lei B, Musser JM, et al., J Biol Chem, 2004, 279: 52789-52796. Lei B, DeLeo FR, Reid SD, et al., Infect Immun, 2002, 70: 6880-6890. Von Pawel-Rammingen U, Johansson BP, Bjorck L.. EMBO J, 2002, 21: 1607-1615.). As a virulence factor of pathogenic bacteria, it can recognize the CH1 and CH2 domains of the lower hinge region of antibodies and specifically degrade IgG to obtain homogeneous F(ab)2 and Fc fragments, helping GAS to evade antibody-mediated phagocytosis and cytotoxicity, thereby weakening the host immune system's killing of GAS (Von Pawel-Rammingen UJ 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 plays a primarily protective role in the body's immune system, effectively preventing infectious diseases. Besides its protective function, IgG is also associated with 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, thereby causing it to lose its proper function and achieving immunosuppression.

[0004] Currently, IdeS used clinically suffers from poor activity and high levels of pre-existing antibodies in the human body. IdeS is a virulence factor of human pathogens, and clinical studies have found that nearly 100% of healthy individuals under normal physiological conditions have detectable anti-IdeS antibodies, resulting in low drug delivery efficiency and safety concerns associated with its use.

[0005] Therefore, there is a need for an immunoglobulin-degrading enzyme that is safer while maintaining activity for the preparation of clinical drugs for related diseases. Summary of the Invention

[0006] A first aspect of the present invention relates to the use of a mutant of the immunoglobulin-degrading enzyme IdeE, preferably in the preparation of a medicament for treating an autoantibody-mediated condition in a subject, said immunoglobulin-degrading enzyme IdeE comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing; said mutation is selected from the group consisting of:

[0007] (1) The mutant is obtained by replacing one or more positions from positions 8, 10, 24, 59, 97, and 280 of the amino acid sequence; and / or,

[0008] (2) The immunoglobulin-degrading enzyme IdeE is truncated by deleting the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acid sequences at its N-terminus; and / or,

[0009] (3) The immunoglobulin degrading enzyme IdeE is truncated 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 at its C-terminus.

[0010] The mutants described herein have an activity and / or thermal stability greater than or equal to that of the immunoglobulin-degrading enzyme IdeE.

[0011] A first aspect of the invention also relates to the use of a protein comprising a mutant of the invention in the preparation of a medicament for treating an autoantibody-mediated condition in a subject. Preferably, the protein has a secretion signal sequence and / or a methionine residue linked to the N-terminus of the mutant; and / or the protein has a histidine tag linked to the C-terminus of the mutant. The first aspect of the invention also relates to the use of a composition comprising a mutant or protein of the invention, or a kit comprising a mutant or protein of the invention, in the preparation of a medicament for treating an autoantibody-mediated condition in a subject, preferably the composition further comprising an optional pharmaceutically acceptable carrier or excipient and / or an additional therapeutic agent selected from the group consisting of: (a) antibodies or Fc-containing proteins, preferably the antibody targeting a target selected from the group consisting of: cell surface proteins, cytokines, hormones, enzymes, intracellular messengers, intercellular messengers, and immune checkpoints; (b) viral vector medicaments, preferably the viral vector medicaments selected from the group consisting of: oncolytic viruses, gene therapy viruses, and viral vector vaccines; and (c) medicaments that reduce blood IgG levels, preferably the medicaments that reduce blood IgG levels selected from the group consisting of: FcRn antibodies, Fc fragment variants with high affinity for FcRn.

[0012] A second aspect of the invention relates to the use of the mutant of the invention, a protein comprising the mutant, or a composition or kit comprising the mutant or the protein in the preparation of a medicament for reducing IgG levels in a subject.

[0013] A third aspect of the invention relates to the use of the mutant of the invention, a protein comprising the mutant, or a composition or kit comprising the mutant or the protein in the preparation of a medicament for the prevention and / or treatment of autoantibody-mediated organ rejection in subjects following solid organ transplantation.

[0014] The fourth aspect of the invention relates to the use of the mutant of the invention, a protein comprising the mutant, or a composition or kit comprising the mutant or the protein in the preparation of a medicament for gene therapy, and in the preparation of a medicament for the preparation of a neutralizing antibody for the elimination of pre-existing antiviral vectors in vivo prior to viral vector-based gene therapy.

[0015] The fifth aspect of the invention relates to the use of the mutant of the invention, a protein comprising the mutant, or a composition or kit comprising the mutant or the protein in the preparation of a medicament for treating tumors in a subject.

[0016] The sixth aspect of the invention relates to the use of the mutant of the invention, a protein comprising the mutant, or a composition or kit comprising the mutant or the protein in the preparation of a medicament for clearing autoantibodies in a subject so that the Fc-containing agent can exert its therapeutic effect.

[0017] The seventh aspect of the invention relates to the use of the mutant of the invention, a protein comprising the mutant, or a composition or kit comprising the mutant or the protein in the preparation of a medicament for reducing serum levels of the Fc-containing agent in subjects who have been administered the Fc-containing agent. Attached Figure Description

[0018] Figure 1 SDS-PAGE gel electrophoresis images of the cleavage products of human IgG1 produced by cleavage of 7 single-point mutants and wild-type IdeE (enzyme:substrate = 1:1000).

[0019] Figure 2 SDS-PAGE gel electrophoresis images of the cleavage products of human IgG1 produced by cleavage of 7 single-point mutants and wild-type IdeE (enzyme:substrate = 1:2000).

[0020] Figure 3 SDS-PAGE gel electrophoresis images of the cleavage products of human IgG1 produced by cleavage of 5 N-terminal truncated mutants (enzyme:substrate = 1:1000).

[0021] Figure 4 SDS-PAGE gel electrophoresis images of the cleavage products of five N-terminal truncated mutants and wild-type IdeE cleavage of human IgG1 after incubation at 50℃ for 1 h (enzyme:substrate = 1:1000).

[0022] Figure 5 SDS-PAGE gel electrophoresis images of the cleavage products of human IgG1 produced by the cleavage of two C-truncation mutants (enzyme:substrate = 1:1000).

[0023] Figure 6 SDS-PAGE gel electrophoresis images of the cleavage products of human IgG1 produced by cleavage of 5 combined mutants (enzyme:substrate = 1:2000).

[0024] Figure 7 The image shows the SDS-PAGE gel electrophoresis results of the cleavage products of human IgG1 produced by the five combined mutants after incubation at 50℃ for 1 hour (enzyme:substrate = 1:2000).

[0025] Figure 8 SDS-PAGE gel electrophoresis images of the cleavage products of human IgG1 cleaved by different concentrations of E97D_del18 mutant and IdeS.

[0026] Figure 9 SDS-PAGE gel electrophoresis images of the cleavage products of human IgG1 cleaved by different concentrations of E97D_del18 mutant and IdeZ.

[0027] Figure 10 SDS-PAGE gel electrophoresis images of the cleavage products of human IVIg generated by the E97D_del18 mutant in mouse serum and plasma.

[0028] Figure 11 SDS-PAGE gel electrophoresis images of the cleavage products of the E97D_del18 mutant in mouse and human serum.

[0029] Figure 12A-12D SDS-PAGE gel electrophoresis images of cleavage products of IgG produced by different concentrations of E97D_del18 in the serum of beagle dogs, rats, mice, rabbits, monkeys and pigs.

[0030] Figure 13 SDS-PAGE gel electrophoresis images of the cleavage products of E97D_del18 cleaved from human IVIg at different time points in mice.

[0031] Figure 14A and 14B SDS-PAGE gel electrophoresis images of mutants with different mutation combinations and cleavage products of IdeE cleavage of human IgG1 (enzyme:substrate = 1:2000).

[0032] Figure 15A This is a spectrum for IDEEV2 activity detection. Lanes 1-5 represent undigested IgG1, IdeZ, IdeS, IdeE, and IDEEV2-digested IgG1 (non-reducing electrophoresis), respectively.

[0033] Figure 15B This is a spectrum of the thermostability and biological activity of the IDEEV2 mutant. Lanes 1-3 are IDEEV2, E97D_del18, and IgG1 after IdeE digestion (non-reducing electrophoresis), respectively.

[0034] Figure 16A SDS-PAGE gel electrophoresis images of the cleavage products of human IgG1–4 produced by IDEEV2 mutant and IdeS cleavage (enzyme:substrate = 1:200). Lanes 1–12 are, respectively, the non-enzyme digestion control of IgG1, IgG2, IgG3, and IgG4, and the IDEEV2:substrate digestion at a ratio of 1:200 (non-reduction electrophoresis).

[0035] Figure 16B SDS-PAGE gel electrophoresis images of cleavage products (enzyme:substrate = 1:200) of IDEEV2 mutant and IdeS cleavage of different human immunoglobulins. Lanes 1-12 are, respectively, the non-enzyme digestion control of IgG, IgM, IgA, IgE, and IgD, and the enzyme digestion (reduction electrophoresis) of IDEEV2:substrate at a ratio of 1:200.

[0036] Figure 17 This image shows the restriction enzyme digestion patterns of IDEEV2 mutant and purified IgG from different animal species. Lanes 1-12, respectively, show the non-restricted digestion patterns of rabbit IgG, canine IgG, rat IgG, mouse IgG, monkey IgG, human IgG, and human IgG1, as well as the restriction enzyme digestion patterns of IDEEV2. (IDEEV2:substrate ratio is 1:200).

[0037] Figure 18A New Zealand rabbits were given IDEEV2 via intravenous infusion once a week for two weeks. The changes in IgG levels in the animals at the 2 mg / kg dose group over time were analyzed (SDS-PAGE results).

[0038] Figure 18B New Zealand rabbits were given IDEEV2 via intravenous infusion once a week for two weeks. The changes in IgG levels in the animals at the 2 mg / kg dose group over time were recorded (ELISA results).

[0039] Figure 19A IDEEV2 was administered intravenously to Beagle dogs once a week for two weeks. The changes in IgG levels in animals over time in each dosage group were recorded (SDS-PAGE results): a. 0.2 mg / kg dosage group; b. 2 mg / kg dosage group; c. 20 mg / kg dosage group.

[0040] Figure 19B IDEEV2 was administered intravenously to Beagle dogs once a week for two weeks. The changes in IgG levels in animals of each dose group over time were recorded (ELISA results).

[0041] Figure 20A The platelet count of each group of animals was changed during the IDEEV2 model animal model by intraperitoneal injection of antiplatelet antibodies.

[0042] Figure 20B Animals were modeled by intraperitoneal injection of antiplatelet antibodies into IDEEV2, and the survival rates of each group were recorded.

[0043] Figure 21 Immunoglobulin-degrading enzyme mutants can eliminate the negative impact of immunoglobulins on the anti-tumor efficacy of monoclonal antibodies.

[0044] Figure 22A This image shows the systemic transduction effect of AAV9-Fluc infection in mice. From left to right: 1) Fluorescent control group (AAV9-Fluc); 2) Model group (IVIg+AAV9-Fluc); 3) Test sample group (IVIg+E97D_del18+AAV9-Fluc). Figure 22BThis demonstrates the systemic transduction effect of AAV9-Fluc infection in mice.

[0045] Figure 23 This demonstrates the effects of AAV9-Fluc infection on cardiac and hepatic transduction in mice. Invention Details

[0047] I. Mutants of the immunoglobulin-degrading enzyme IdeE

[0048] The IdeE protease, which shares approximately 70% sequence homology with IdeS, originates from *Streptococcus equi* ssp. equi, a pathogenic bacterium of horses (Jonas). Bengt Guss. FEMS Microbiol Lett., 2006, 262: 230-235. IdeE and IdeS cleave IgG at exactly the same location, exhibiting highly reproducible and specific cleavage, and have very similar substrate ranges. Since IdeE originates from equine pathogens, it is speculated that pre-existing antibodies in the human body may be far lower than those of IdeS, making it more suitable for developing immunosuppressants for the treatment and prevention of IgG antibody-mediated diseases. However, like IdeS, wild-type IdeE also suffers from low activity.

[0049] Therefore, the present invention relates to a mutant of the immunoglobulin-degrading enzyme IdeE, said mutant having immunoglobulin-degrading enzyme activity and selected from the group consisting of:

[0050] (1) A mutant is obtained by substituting one or more amino acids in positions 8, 10, 24, 59, 97, and 280 of SEQ ID NO: 2; and / or,

[0051] (2) A truncated mutant of the N-terminus of SEQ ID NO: 2, selected from the deletion of the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids from its N-terminus; and / or,

[0052] (3) A truncated mutant of the C-terminus of SEQ ID NO: 2, selected from 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 deleted from its C-terminus.

[0053] 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.

[0054] The term "having an activity greater than or equal to that of the immunoglobulin-degrading enzyme IdeE" in this invention means that the mutant's ability to degrade immunoglobulins is superior to or equal to that of the wild-type immunoglobulin-degrading enzyme IdeE.

[0055] The term "higher thermal stability than IdeE" in this invention refers to the mutant's ability to degrade immunoglobulins better than that of wild-type immunoglobulin-degrading enzyme IdeE under the same conditions after being maintained at a certain temperature for a period of time.

[0056] The mutant of the present invention is preferably produced by genetic engineering recombination.

[0057] 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 in SEQ ID NO: 2.

[0058] More preferably, the amino acids at positions 8, 10, 24, 59, 97 or 280 are replaced, for example, the amino acid sequence of the resulting mutant is shown in any one of SEQ ID NO: 3-17 or SEQ ID NO: 35;

[0059] Alternatively, the first 15, 16, 17, 18, or 19 amino acids of the N-terminus of the immunoglobulin degrading enzyme IdeE can be deleted, and the amino acid sequence of the resulting mutant is shown in any of SEQ ID NO: 18-22.

[0060] Alternatively, the last 1, last 5, last 8, or last 10 amino acids at the C-terminus of the immunoglobulin degrading enzyme IdeE may be deleted, for example, the amino acid sequence of the resulting mutant is shown in any of SEQ ID NO: 23-24;

[0061] Alternatively, positions 8, 10, 24, 59, 97, or 280 can be replaced, and the first 15, 16, 17, 18, or 19 amino acids at the N-terminus of the immunoglobulin degrading enzyme IdeE can be deleted, preferably the first 18 amino acids. For example, the amino acid sequence of the resulting mutant is shown in any of SEQ ID NO: 25 to 29.

[0062] Alternatively, positions 8, 10, 24, 59, 97, or 280 can be replaced, and the first 15, 16, 17, 18, or 19 amino acids at the N-terminus of the immunoglobulin degrading enzyme IdeE can be deleted, along with the last 1, 5, 8, or 10 amino acids at the C-terminus of the immunoglobulin degrading enzyme IdeE. Preferably, the first 18 amino acids can be deleted, and more preferably, the last 5 amino acids can be deleted. For example, the amino acid sequence of the resulting mutant is shown in any of SEQ ID NO: 30 to 34.

[0063] In a preferred embodiment of the present invention, the amino acid substitution is selected from the group consisting of:

[0064] (1) The threonine at position 8 of SEQ ID NO:2 is replaced with any one of cysteine, phenylalanine, tryptophan, tyrosine, aspartic acid, glutamic acid, alanine, glycine, histidine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, valine, arginine, and lysine.

[0065] (2) The alanine at position 10 of SEQ ID NO:2 is replaced with any one of cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan and tyrosine.

[0066] (3) The threonine at position 24 of SEQ ID NO: 2 is replaced with any one of alanine, cysteine, aspartic acid, asparagine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, proline, glutamine, arginine, serine, valine, tryptophan and tyrosine.

[0067] (4) The alanine at position 59 of SEQ ID NO:2 is replaced with any one of cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan and tyrosine.

[0068] (5) The glutamic acid at position 97 of SEQ ID NO:2 is replaced with any one of alanine, cysteine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan and tyrosine.

[0069] (6) The arginine at position 280 of SEQ ID NO: 2 is replaced with any one of alanine, aspartic acid, glutamic acid, cysteine, serine, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, threonine, valine, tryptophan and tyrosine.

[0070] In a more preferred embodiment of the invention, the amino acid substitution is selected from the group consisting of:

[0071] (1) Threonine at position 8 of SEQ ID NO: 2 is replaced with aspartic acid, glutamic acid, tryptophan or tyrosine;

[0072] (2) The alanine at position 10 of SEQ ID NO: 2 is replaced with lysine or arginine;

[0073] (,3) The threonine at position 24 of SEQ ID NO:2 is replaced with alanine, glycine or serine;

[0074] (4) The alanine at position 59 of SEQ ID NO: 2 is replaced with isoleucine, leucine or valine;

[0075] (5) The glutamic acid at position 97 of SEQ ID NO: 2 is replaced with asparagine; and / or

[0076] (6) The arginine at position 280 of SEQ ID NO: 2 is replaced with histidine or lysine.

[0077] In another preferred embodiment, the first 18 amino acids at the N-terminus of the 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 further deleted, and the amino acid sequence of the resulting mutant is shown in SEQ ID NO: 25 to 29 in the sequence listing.

[0078] In another preferred embodiment, based on the five amino acid substitution sequences obtained by SEQ ID NO: 26-29, five or ten amino acids at the C-terminus are further deleted, and the amino acid sequence of the resulting mutant is shown in SEQ ID NO: 30-34 in the sequence listing.

[0079] In another preferred embodiment, the three mutants SEQ ID NO: 14–16 are further subjected to combined mutations, and the amino acid sequence of the resulting mutant is shown in SEQ ID NO: 35 of the sequence listing. In another preferred embodiment, the first 18 amino acids at the N-terminus of the five sequences obtained by amino acid substitution in 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 further deleted, and the amino acid sequence of the resulting mutant is shown in SEQ ID NO: 25–29 of the sequence listing.

[0080] In another preferred embodiment, the amino acid sequence of the mutant is shown as SEQ ID NO: 36 in the sequence listing.

[0081] Preferably, the mutant described in this invention can be further mutated, and the sequence of the variant 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 at the same time has the function of immunoglobulin degrading enzyme IdeE.

[0082] The full-length IdeE sequence 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 residue, followed by a 33-amino acid secretion signal sequence, and then the IdeE coding sequence. The N-terminal methionine residue and the signal sequence are typically removed to form the mature IdeE protein, the sequence of which is provided herein as SEQ ID NO: 2. Unless otherwise stated, all references to the amino acid positions in the disclosed immunoglobulin-degrading enzyme sequences are based on the corresponding positions in SEQ ID NO: 2, starting from the N-terminus.

[0083] This invention also relates to proteins comprising mutants of this invention.

[0084] In a preferred embodiment, the protein contains a signal peptide at the N-terminus of the mutant; preferably, the protein has a secretion signal sequence attached to the N-terminus of the mutant and a methionine residue attached to the N-terminus of the secretion sequence and / or a histidine tag attached to the C-terminus of the mutant; more preferably, the protein comprises or consists of the following from the N-terminus to the C-terminus: methionine residue, secretion signal sequence, and the mutant.

[0085] II. Pharmaceutical Composition

[0086] This invention relates to a composition comprising the immunoglobulin-degrading enzyme mutant of the present invention or a protein containing said mutant, and optionally a pharmaceutically acceptable carrier or excipient.

[0087] In one specific embodiment, the composition of the present invention further comprises: an antibody, a small molecule targeted drug, and / or a protein containing Fc.

[0088] In one specific implementation, the antibody target is selected from the group consisting of: cell surface proteins, cytokines, hormones, enzymes, intracellular messengers, intercellular messengers, and immune checkpoints.

[0089] In one specific embodiment, the composition of the present invention further comprises: a viral vector drug or a gene therapy drug, preferably, the viral vector drug is selected from the group consisting of oncolytic viruses, gene therapy viruses and viral vector vaccines.

[0090] In one specific embodiment, the composition of the present invention further comprises: a drug capable of reducing blood IgG levels, preferably, said drug for reducing blood IgG levels is selected from the group consisting of: FcRn antibodies, Fc fragment variants with high affinity for FcRn.

[0091] 2.1 Antibodies

[0092] Preferably, in the composition described above, the target of the antibody may be a cell surface protein, including but not limited to: AFP, αv integrin, α4β7 integrin, BCMA, CD2, CD3, CD4, CD11a, CD19, CD20, CD22, CD25, CD30, CD32, CD33, CD36, CD38, CD40, CD46, CD47, CD52, CD56, CD64, and CD70. , CD74, CD79, CD80, CD86, CD105, CD121, CD123, CD133, CD138, CD174, CD205, CD227, CD326, CD340, CEA, c-Met, Cripto, CA1X, Claudin6, Claudin18.2, ED-B, EGFR, EpCAM, EphA2, EphB2, FAP, FOLR1, GD2, Globo H, GPC3, GPNMB, GPRC5D, HER-1, HER-2, HER-3, MAGE-A3, Mesothelin, MUC1, MUC4, MUC16, PSMA, TMEFF2, TAG-72, 5T4, ROR-1, Sca-1, SP, Trop-2, CD38, CGRP, IGF-1R, Nectin-4, P-Selectin, vWF, KLK, CCR4, SLAMF7, PCSK9, GD2, VEGFR2, BLyS, RANKL, α4β1 integrin, VEGFR, platelet glycoprotein Iib / IIIa, IFNAR1, TSLP, VEGF or WT1.

[0093] The target of the antibody may be one or more cytokines, including but not limited to: interleukins IL-1 to IL-13, IL-1β, IL-15, IL-17, IL-23p19, IL-2R, IL-5R, IL-6R, IL-17R, IL-2R, tumor necrosis factor α and β, interferon α, β and γ, VEGF, PDGF-α, FGF-23, sclerostin, 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 (edited by Aggrawal et al., Blackwell Scientific, Boston, MA 1991).

[0094] The target of the antibody may be a hormone, enzyme, intracellular and intercellular messenger, such as adenylate cyclase, guanylate cyclase or phospholipase C.

[0095] The target of the antibody may be one or more immune checkpoints, including but not limited to: CTLA-4, PD-1, PD-L1, TIM-3, LAG3, Siglec7, Siglec9, Siglec15, 4-1BB, GITR, OX40, CD40L, CD28, TIGIT, and VISTA.

[0096] The target of the antibody may be IgE, RSVF, SARS-CoV-2, Dabigatran, FIX / FX, Clostridium difficile toxin B, C2, C5, or PA of anthrax.

[0097] Satralizumab, Belantamab mafodotin, Tafasitamab, Inebilizumab, Sacituzumab goVitecan, Isatuximab, Eptinezumab, Teprotumumab, Trastuzumab, Trastucan, Extenfortumab Vedotin、Romosozumab、Crizanlizumab、Brolucizumab、Polatuzumab、Risankizumab、Caplacizumab、Emapalumab、Ravulizumab、Cemiplimab、galcanezumab、Fre manezumab、MoxetumomabPasudotox、Lanadelumab、Mogamulizumab、Erenumab、Burosumab、Tildrakizumab、Ibalizumab、Emicizumab、Benralizumab、inotuzumab ozogamicin、Sarilumab、Guselkumab、Durvalumab、Dupilumab、Ocrelizumab、Avelumab、Brodalumab、Daclizumab、Bezlotox umab、Olaratumab、Atezolizumab、Obiltoxaximab、Ixekizumab、Reslizumab、Idarucizumab、MepOlizumab、Elotuzumab、Neci tumamab、Evolocumab、Alirocumab、Daratumumab、Dinutuximab、Secukinumab、Blinatumomab、Pembrolizumab、Nivolumab、Ve dolizumab、Siltuximab、Ramucirumab、Obinutuzumab、Ado-Trastuzumabemtansine、Raxibacumab、Pertuzumab、Brentuximab vedotin、Belimumab、Ipilimumab、Tocilizumab、Denosumab、Denosumab、Ofatumumab、Canakinumab、Golimumab、Ustekinumab、Certolizumabpegol, Eculizumab, Panitumumab, Ranibizumab, Natalizumab, Bevacizumab, Cetuximab, Efalizumab, Tositumomab, Omalizumab, Adalimumab, Ibritumomab tiuxetan, Alemtuzumab, GemtuzumabOzogamicin, Trastuzumab, Infliximab, palivizumab, Basiliximab, Daclizumab, Rituximab, Abciximab, Catumaxomab, Muromomab.

[0098] 2.2 Small molecule targeted drugs

[0099] Preferably, in the composition described above, the composition further comprises a targeted drug or a chemotherapeutic drug or an immune checkpoint inhibitor, wherein the targeted drug is selected from epigenetic drugs, inhibitors targeting the PI3K / Akt / mTOR signaling pathway, and tyrosine kinase inhibitors; the chemotherapeutic drug is selected from immunosuppressants, proteasome inhibitors, cytotoxic drugs, and cell cycle nonspecific drugs; 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 drug is, for example, gemcitabine or temozolomide; and the cell cycle nonspecific drug is, for example, mitoxantrone.

[0100] 2.3 Drugs that can lower blood IgG levels

[0101] Preferably, in the composition described above, the polypeptide drug that reduces blood IgG levels can block the binding of blood IgG and FcRn protein. Preferably, the polypeptide has a higher affinity for human FcRn protein than for blood IgG and human FcRn protein; the IgG is selected from IgG1, IgG2, IgG3, and IgG4. Preferably, the polypeptide comprises an antibody Fc fragment variant containing a mutation that increases the affinity for Fc and FcRn, preferably YTE, YTEKF, LS, or NHS, and the antibody Fc fragment is, for example, Efgartigimod. The variant can be a monomer, dimer, or multimer. The mutations YTE, YTEKF, LS, NHS, etc., used in this 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 mutation target is selected from human IgG, and the IgG is selected from IgG1, IgG2, IgG3, and IgG4.

[0102] Other Fc fragment variants that can be used in this invention include, but are not limited to, mutations described by Dall'Acqua et al. (WF, DA et al. (2002). Journal of Immunology (Baltimore, Md.: 1950) 169(9): 5171-5180.), mutations described by Shan et al. (Shan, L. et al. (2016). PLoS One 11(8): e0160345.), mutations described by Lee et al. (Lee, CH et al. (2019). Nat Commun 10(1): 5031.), mutations described by Mackness et al. (Mackness, BC et al. (2019). MAbs 11(7): 1276-1288.), and mutations described by Christophe et al. (Dumet Christophe, Pottier Jérémy, Gouilleux-Gruart Valérie et al., MAbs, 2019, 11: 1341-1350.).

[0103] Preferably, the polypeptide comprises an antibody Fc fragment variant containing mutations that enhance the affinity for Fc and FcγR. The variants are preferably S239D / I322E, S239D / I322E / A330L, K326W / E333S, or R214K mutations; the variants are preferably free of fucose modification. The variants can be monomers, dimers, or polymers. Other Fc fragment variants that can be used in this invention include, but are not limited to, the mutations 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).

[0104] Preferably, the variants that enhance the affinity for Fc and FcRn also include mutations that enhance the affinity for Fc and FcγR. The variants can be monomers, dimers, or polymers.

[0105] Preferably, in the drug combination described above, the polypeptide is selected from anti-FcRn antibodies, such as Nipocalimab, Rozanolixizumab, RVT-1401, HBM9161, ALXN1830, SYNT001, and Nirsevimab.

[0106] Preferably, in the drug combination as described above, the polypeptide is selected from small peptide fragments that can specifically bind to FcRn, and the small peptide fragments are 10-70 amino acids in length; the small peptide fragments are, for example, ABY-039.

[0107] Preferably, the polypeptide is selected from Fc polymers that can specifically bind to FcRn, such as GL-2045, M230, PRIM, and HexaGard. TM , CSL777, Hexavalent molecules by UCB.

[0108] Preferably, 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).

[0109] 2.4 Viral Vector Drugs

[0110] Preferably, in the composition described above, 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 selectively attenuated strains, gene-loaded virus strains, or gene transcription-targeted virus strains.

[0111] 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.

[0112] Preferably, the genetically engineered selectively attenuated strain achieves tumor selectivity for viral replication by artificially deleting key genes, such as a genetically modified human herpes simplex virus I (HSV-1) with thymidine kinase (TK) knockout. Examples of such genetically engineered selectively attenuated strains include ONYX-015 and G207. ONYX-015 has an 827bp deletion in the E1b region and a point mutation in the gene targeting the E1B55K protein, causing premature termination of its expression and preventing the expression of the E1B55K protein. G207 has the γ34.5 gene deleted, which is a determinant of the neurotoxicity of HSV-1.

[0113] Preferably, the gene-loaded viral strain is loaded with a foreign gene, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), and the gene-loaded viral strain is such as JX-594 or T-VEC.

[0114] Preferably, the gene transcription-targeting viral strain is a tissue- or tumor-specific promoter inserted before the essential viral gene to control the replication of oncolytic virus in tumor cells, and the gene transcription-targeting viral strain is, for example, G92A.

[0115] Preferably, in the drug combination described above, the ssDNA virus is selected from parvoviruses, and more preferably, the parvovirus is H-1PV virus.

[0116] Preferably, the dsDNA virus is selected from herpes simplex virus, adenovirus, or poxvirus; more preferably, the herpes simplex virus is preferably type I herpes simplex virus HSV-1, such as R3616, T-VEC, HF10, G207, NV1020, or OrienX010, and the poxvirus is selected from Pexa-Vec (vacciniaviruse), 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).

[0117] 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; the alphavirus is selected from M1, Sindbis AR339, and Semliki Forestvirus; 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.

[0118] Preferably, the dsRNA virus is selected from Reoviruses; 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.

[0119] Preferably, in the drug combination described above, the oncolytic virus expresses a foreign gene, which is preferably a bispecific T cell engager (BiTE), scFv fragment, cytokine, or chemokine. The BiTE can bind to molecules that activate T cells, such as CD3, and can also 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 include, for example, GM-CSF, interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interferon (IFN), tumor necrosis factor (TNF), soluble CD80, and CCL3.

[0120] In some implementations, the viral drug carrier is preferably an AAV virus, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV3B, AAV-2i8, Rh10, or Rh74.

[0121] In some implementation examples, the viral drug vector is preferably adenovirus, lentivirus, or retrovirus.

[0122] The viral vector drug and / or immunoglobulin degrading enzyme variants may be encapsulated or fused with liposomes, nanoparticles, lipid nanoparticles, polymers, microparticles, microcapsules, micelles or exosomes.

[0123] 2.5 Gene therapy drugs

[0124] Preferably, in the composition described above, the gene therapy virus expresses a foreign gene, the foreign gene encoding a protein required for gene defect diseases, the protein being selected from acid α-glucosidase, copper-transfer ATPase 2, α-galactosidase, arginine succinate synthase, β-glucocerebrosidase, β-hexosamine 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, and angiogenesis. Angiogenic inhibitor, granulocyte colony-stimulating factor, erythropoietin, connective tissue growth factor, basic fibroblast growth factor, acidic fibroblast growth factor, epidermal growth factor, transforming growth factor α, platelet-derived growth factor, insulin-like growth factor I and II, TGF, bone morphogenetic protein, nerve growth factor, brain-derived neurotrophic factor, neurotrophic proteins NT-3 and NT4 / 5, ciliary neurotrophic factor, glial cell line-derived neurotrophic factor, neurotrophins, lectins, netrin-1 and netrin-2, hepatocyte growth factor, Ephrins, tyrosine hydroxylase, thrombopoietin, interleukins (IL-1 to IL-36, etc.), monocyte chemoattractant proteins, leukemia inhibitory factor, granulocyte-macrophage protein 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 immunoglobulins, humanized antibodies, single-chain antibodies, T cell receptors, chimeric T cell receptors, single-chain T cell receptors, class I and II MHC molecules, cystic fibrosis transmembrane regulatory proteins, coagulation (coagulation) Factors (Factor XIII, Factor IX, Factor VIII, Factor X, Factor VII, Factor VIIa, Protein C, etc.), retinal pigment epithelium-specific 65kDa protein, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, β-globulin, α-globulin, spectroscopy, α-antitrypsin adenosine deaminase, metal transporter (ATP7A or ATP7), sulfonamide enzyme, enzyme involved in lysosomal storage diseases (ARSA), hypoxanthine-guanine phosphoribosyltransferase, β-25-glucocerebroside lipase, sphingomyelinase, lysosomal hexosaminease, branched-chain ketoacid dehydrogenase.

[0125] Preferably, in the composition described above, the gene therapy virus carries a foreign gene encoding a repressive nucleic acid selected from siRNA, antisense molecules, miRNA, RNAi, ribozymes, and shRNA. The repressive nucleic acid binds to a polynucleotide repeat disease-related gene, its transcript, or a polynucleotide repeat of the gene's transcript. The disease gene encodes a related protein selected from Huntington's protein (HTT), androgen receptor on the X chromosome of spinal bulbomuscular atrophy, human attaxin-1 / -2 / -3 / -7, Cav2.1P / Q voltage-dependent calcium channel (CACNA1A), TATA-binding protein, attaxin8 reverse strand (ATXN80S), serine / threonine protein phosphatase 2A 55kDa, subtypes B and β of spinocerebellar ataxia (types 1, 2, 3, 6, 7, 8, 12, 17), 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; troponin kinase (MT-PK) and Frataxin in myotonic dystrophy. The disease genes are selected from mutants of the superoxide dismutase 1 (SOD1) gene, 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 from influenza virus infection, severe acute respiratory syndrome (SARS) coronavirus genome / gene sequence from SARS infection, and respiratory syncytial virus genome / gene sequence from respiratory syncytial virus infection. Ebola virus genome / gene sequence in Ebola virus infection; genome / gene sequence of hepatitis B and C viruses in hepatitis B and C viruses; genome / gene sequence of herpes simplex virus (HSV) in HSV infection; genome / gene sequence of Coxsackievirus B3 in Coxsackievirus B3 infection; pathogenic alleles (allele-specific silencing) such as torsin A in silenced primary dystonia; specific pan-I and HLA alleles in transplantation; mutations and rhodopsin genes in autosomal dominant retinitis pigmentosa.

[0126] III. Products and Reagent Kits

[0127] The present invention also provides a product comprising the mutant, protein and / or composition of the present invention and additional therapeutic agents; said additional therapeutic agents are selected from viral vector drugs, antibodies, and polypeptide drugs that can reduce blood IgG levels.

[0128] The present invention also provides a kit or pharmaceutical kit comprising: 1) a therapeutically effective amount of a drug comprising the mutant, protein, and / or composition of the present invention; and 2) a therapeutically effective amount of an additional therapeutic agent; said therapeutic agent being selected from viral vector drugs, antibodies, and peptide drugs that can reduce blood IgG levels; said viral vector drug preferably being an oncolytic virus or a gene therapy virus. The kit may further include 3) a targeted drug or a chemotherapeutic drug or an immune checkpoint inhibitor. The targeted drug is selected from epigenetic drugs, inhibitors targeting the PI3K / Akt / mTOR signaling pathway, and tyrosine kinase inhibitors; the chemotherapeutic drug is selected from immunosuppressants, proteasome inhibitors, cytotoxic drugs, and cell cycle non-specific drugs; 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 drug is, for example, gemcitabine or temozolomide; and the cell cycle non-specific drug is, for example, mitoxantrone.

[0129] The kit or pharmacy kit includes pharmacy A and pharmacy B. Pharmacy A includes a therapeutically effective amount of the mutant, protein, and / or composition of the present invention, and pharmacy B includes a therapeutically effective amount of an additional therapeutic agent. The therapeutic agent is selected from viral vector drugs, antibodies, and peptide drugs that can reduce blood IgG levels. The viral vector drug is preferably an oncolytic virus or a gene therapy virus. The pharmacy kit may also include pharmacy C. Pharmacy C includes a targeted drug, a chemotherapy drug, or an immune checkpoint inhibitor. The targeted drug is selected from epigenetic drugs, inhibitors targeting the PI3K / Akt / mTOR signaling pathway, and tyrosine kinase inhibitors; the chemotherapeutic drug is selected from immunosuppressants, proteasome inhibitors, cytotoxic drugs, and cell cycle non-specific drugs; 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 drug is, for example, gemcitabine or temozolomide; and the cell cycle non-specific drug is, for example, mitoxantrone.

[0130] The kit may contain instructions for administering therapeutically effective amounts of the mutants, proteins, and / or compositions of the present invention, and additional therapeutically effective amounts of other therapeutic agents (e.g., dosage information, dosing interval information). The additional therapeutic agents are selected from viral vector drugs, antibodies, and peptide drugs that reduce blood IgG levels; the viral vector drugs are preferably oncolytic viruses or gene therapy viruses.

[0131] Mature and well-established expression systems can be used to manufacture viral vector drugs. Some examples of methods include using mammalian cell expression systems to produce viral particles, such as using HEK293 cells to produce adenovirus-like viral 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).

[0132] Drug carriers can be liquids, and drug compositions can be in solution form. 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, organic solvents, mixtures of both, or pharmaceutically acceptable oils or fats.

[0133] Pharmaceutical compositions intended for parenteral administration are sterile, substantially isotonic, pyrogen-free, and prepared according to GMP standards of the FDA or similar agencies. Viral vector drugs can be administered as injectable dosage forms of solutions or suspensions of the substance, wherein the substance is in physiologically acceptable diluents and drug carriers (which may be sterile liquids such as water, oil, saline, glycerol, or ethanol). Additionally, excipients may be present in the composition, such as wetting agents or emulsifiers, surfactants, and pH buffers. Other components of the pharmaceutical composition may be of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, and mineral oil. Typically, diols such as propylene glycol or polyethylene glycol are preferred liquid carriers, especially for injectable solutions. Viral vector drugs can be administered in the form of accumulated injectables or implantable formulations, which can be formulated to allow for sustained release of the active ingredient. Typically, compositions are prepared as injectables, i.e., liquid solutions or suspensions; they can also be prepared as solid forms suitable for dissolution or suspension in a liquid carrier prior to injection.

[0134] IV. Uses of mutants, proteins, pharmaceutical compositions, and kits

[0135] This invention also relates to the use of the mutants, proteins, compositions, and / or kits of the invention in the preparation of pharmaceuticals. In some embodiments, the pharmaceuticals are used to treat autoantibody-mediated conditions in subjects. In some embodiments, the pharmaceuticals are used to reduce IgG levels in subjects. In some embodiments, the pharmaceuticals are used to prevent and / or treat autoantibody-mediated organ rejection following solid organ transplantation in subjects. In some embodiments, the pharmaceuticals are used for gene therapy. In some embodiments, the pharmaceuticals are used to clear pre-existing neutralizing antibodies against antiviral vectors in vivo prior to viral vector-based gene therapy. In some embodiments, the pharmaceuticals are used to treat tumors in subjects. In some embodiments, the pharmaceuticals are used to clear autoantibodies in subjects to allow Fc-containing agents to exert better therapeutic effects. In some embodiments, the pharmaceuticals are used to reduce serum levels of Fc-containing agents in subjects who have been administered Fc-containing agents.

[0136] In a preferred embodiment, the mutants, proteins, compositions, and / or kits of the present invention can be used for the analysis of IgG. Specifically, the Fc and F(ab')2 fragments obtained by cleaving IgG using the mutants, proteins, compositions, and / or kits of the present invention can be used for, for example, mass spectrometry molecular weight analysis, glycoform modification analysis, ADC molecular analysis, etc.

[0137] In another preferred embodiment, the mutants, proteins, compositions, and / or kits of the present invention can also be used to prepare Fab antibody fragments. Fab fragments are prepared by reducing the F(ab')2 fragment generated after cleaving IgG using the mutants, proteins, compositions, and / or kits of the present invention.

[0138] 4.1 Autoantibody-mediated diseases

[0139] This invention relates to the use of the mutants, proteins, compositions, and / or kits of the present invention in the preparation of medicaments for treating autoantibody-mediated conditions in subjects. In some embodiments, the autoantibody-mediated condition is an autoimmune disease and / or a disease or condition mediated by a pathogenic antibody.

[0140] Preferably, the disease is a disease or condition mediated by pathogenic antibodies; including but not limited to autoimmune diseases or conditions mediated by pathogenic IgG, such as Addison's disease, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, aplastic anemia, anti-GBM glomerulonephritis, anti-NMDAR encephalitis, antiphospholipid syndrome, autoimmune gastritis, autoimmune deafness, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hypoparathyroidism, autoimmune hypophysitis, and autoimmune inner ear disease. Autoimmune lymphoproliferative syndrome, autoimmune myocarditis, autoimmune oophoritis, autoimmune bullous dermatitis, autoimmune orchitis, autoimmune multiple endocrine disorders, Bechtel's disease, bullous pemphigoid, cardiomyopathy, inflammatory demyelinating polyneuropathy, acute motor axononeuropathy, Tudor-Schönlein syndrome, abdominal diseases, autoimmune urticaria, Crohn's disease, CREST syndrome, celiac disease, Degos disease, antineutrophil cytoplasmic antibody-associated vasculitis, autoimmune neutropenia. Acquired epidermolysis bullosa, primary mixed cryoglobulin thrombosis, giant cell arteritis, glomerulonephritis, Goodpasser syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic thrombocytopenic purpura, inflammatory bowel disease, Kawasaki disease, Meniere's syndrome, mixed connective tissue disease, Molen's ulcer, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, stiff-person syndrome, complete congenital heart block, acquired epidermolysis bullosa, pemphigus foliaceus, pemphigus vulgaris, pernicious anemia, polydactyly nodular lesions Arteritis, types of polygonal autoimmune syndrome, primary biliary cirrhosis, types of polygonal autoimmune syndrome, polymyositis / dermatomyositis, psoriasis, psoriatic arthritis, Raynaud's syndrome, Leter syndrome, rheumatic heart disease, hemophilia-acquired FVIII deficiency, Lambert-Eaton myasthenia gravis, multiple myeloma, sarcoidosis, collagen stasis, Sjögren's syndrome, subacute thyroiditis, sympathetic ophthalmia, systemic lupus erythematosus, Goran's arteritis, Syndrome, type 1 diabetes, vitiligo, Vogt-Koyanagi-Harada syndrome or Wegener's granulomatosis, acute or chronic asthma, organ transplant rejection, primary progressive multiple sclerosis, systemic sclerosis, serum sickness, and immune complex hypersensitivity.

[0141] In organ transplantation, host IgG can cause acute transplant rejection. Transplant rejection is divided into two types: host-versus-graft reaction (HVGR) and graft-versus-host reaction (GVHR). In solid organ transplantation, HVGR is the primary type, while GVHR is rare. In bone marrow transplantation, GVHR is the most common type.

[0142] Graft-versus-host disease (GVHR) refers to the immune system's response to allogeneic tissue or organ transplantation. The transplanted tissue or organ is recognized as a foreign component and triggers an immune attack, destruction, and clearance of the graft. Patients highly sensitive to human lymphocyte antigen (HLA) are more prone to transplant rejection. The mechanisms of rejection primarily involve cellular and humoral immunity. Humoral immunity is the protective mechanism achieved through the production of IgG antibodies by effector B cells.

[0143] This invention relates to the use of the mutants, proteins, compositions, and / or kits of the present invention in the preparation of medicaments for the prevention and / or treatment of autoantibody-mediated organ rejection following solid organ transplantation in subjects. In some embodiments, the organ rejection includes, but is not limited to, allogeneic graft rejection in organ transplantation, such as: kidney transplant rejection, allogeneic islet transplant rejection, pancreas transplant rejection, heart transplant rejection, liver transplant rejection, lung transplant rejection, or small intestine transplant rejection.

[0144] Diseases or conditions mediated by pathogenic antibodies also include hyperglobulinemia. The hyperglobulin is produced by leukocytes, selected from B cells and abnormal B cells; the globulin includes gamma globulin; and the hyperglobulinemia includes primary monoclonal gammopathy, connective tissue diseases, liver diseases, infectious diseases, sarcoidosis, myasthenia gravis, Hodgkin's disease, Behcet's disease, nephritis, allergic purpura, immune (or spontaneous) thrombocytopenic purpura, and malignant monoclonal gammopathy (such as multiple myeloma, heavy chain disease, malignant lymphoma, chronic lymphocytic leukemia). This includes hematologic disorders, macroglobulinemia, secondary monoclonal gammopathy (such as non-lymphoreticular system tumors, monocytic leukemia, cryoglobulinemia, etc.), benign M-proteinemia, monoclonal gammopathy of undetermined significance (MGUS), Waldenström macroglobulinemia, AL amyloidosis, solitary plasmacytoma (bone or extraosseous), POMES syndrome, reactive plasmacytosis, osteolytic lesions of metastatic cancer, plasmablastic lymphoma, and monoclonal immunoglobulin-associated kidney damage (MGRS), etc. Multiple myeloma is the preferred disease among these.

[0145] This invention also relates to the use of the mutants, proteins, compositions, and / or kits of this invention in the preparation of medicaments for treating or preventing diseases. Preferably, the diseases include, but are not limited to, tumors, cancers, infectious diseases, genetic defects, and diseases or conditions mediated by pathogenic IgG antibodies. The infectious diseases include viral infections, bacterial infections, or fungal infections.

[0146] 4.2 Tumors or cancer

[0147] The tumors or cancers mentioned are selected from the group consisting of: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendiceal cancer, astrocytoma, cerebellar or cerebral cancer in children, basal cell carcinoma, extrahepatic bile duct cancer, bladder cancer, bone cancer, osteosarcoma / malignant fibrous histiocytoma, brainstem glioma, brain cancer, brain tumor - cerebellar astrocytoma, brain tumor - cerebral astrocytoma / malignant glioma, brain tumor - ependymoma, brain tumor - medulloblastoma, brain tumor - supratentorial primitive neuroectodermal tumor, brain tumor - visual pathway and hypothalamic glioma, breast cancer, bronchial adenoma / carcinoid, Burkitt lymphoma, carcinoid tumor, cancer of unknown primary origin, central nervous system lymphoma, cerebellar astrocytoma. Astrocytoma, cerebral astrocytoma / malignant glioma, cervical cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, connective tissue proliferative small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma in the Ewing tumor family, extracranial germ cell tumors, childhood extragonadal germ cell tumors, extrahepatic biliary tract cancer, ocular cancer - intraocular melanoma, ocular cancer - retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), extracranial, extragonadal, or ovarian germ cell tumors, gestational trophoblastoma, brainstem glioma, childhood cerebral astrocytoma / glioma, childhood visual pathway and hypothalamic glioma, gastric carcinoid, hair Leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, islet cell carcinoma (endocrine pancreas), Kaposi's sarcoma, renal cell carcinoma, laryngeal cancer, leukemia, acute lymphoblastic leukemia (also known as acute lymphoblastic leukemia), acute myeloid leukemia (also known as acute myeloid leukemia), chronic lymphocytic leukemia (also known as chronic lymphocytic leukemia), chronic myeloid leukemia (also known as chronic myeloid leukemia), hairy cell leukemia, lip and oral cancer, liposarcoma, primary liver cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, cutaneous T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (Old classification: all lymphomas except Hodgkin's lymphoma), primary central nervous system lymphoma, macroglobulinemia, malignant fibrous histiocytoma / osteosarcoma of bone, medulloblastoma, melanoma, intraocular (ocular) melanoma, Merkel cell carcinoma, mesothelioma, adult malignant mesothelioma, primary occult metastatic squamous neck carcinoma, oral cancer, multiple endocrine adenoma syndrome, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorders, chronic myeloid leukemia, adult acute myeloid leukemia, childhood acute myeloid leukemia, myeloproliferative disorders, nasal and paranasal sinus carcinoma, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone,Ovarian cancer, ovarian epithelial carcinoma (surface epithelial-stromal cell tumor), ovarian germ cell tumor, low-grade malignant potential ovarian tumor, pancreatic cancer, islet cell pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pheochromocytoma, pineal astrocytoma, pineal germ cell tumor, pineal cell carcinoma and supratentorial primitive neuroectodermal tumor, pituitary adenoma, plasmacytoma / multiple myeloma, pleural pulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma of the renal pelvis and ureter (renal cancer), transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Ewing family neoplastic sarcoma, Kaposi's sarcoma, soft tissue sarcoma, uterine sarcoma, Cezari Sézary syndrome, skin cancer (non-melanoma), skin cancer (melanoma), Merkel cell skin cancer, small cell lung cancer, small bowel cancer, soft tissue sarcoma, squamous cell carcinoma, primary occult squamous neck cancer, metastatic gastric cancer, supratentorial primitive neuroectodermal tumor, cutaneous T-cell lymphoma (see mycosis fungoides and Sézary syndrome), testicular cancer, pharyngeal cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor of the ureter and renal pelvis, transitional cell carcinoma of the urethra, endometrial uterine cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, macroglobulinemia and nephroblastoma (renal carcinoma).

[0148] 4.3 Cancer or infectious diseases

[0149] The cancer or infectious disease mentioned can be an animal disease or a human disease, for example as follows:

[0150]

[0151] 4.4 Diseases related to gene defects

[0152] The gene defect-related diseases include, but are not limited to, protein overexpression, protein loss, and heterologous protein expression caused by viral infection; preferably, the gene therapy drug is used to treat gene overexpression, gene low expression, gene defects, or infectious diseases; the diseases are selected from lung diseases (e.g., cystic fibrosis), hemorrhagic diseases (e.g., hemophilia A or hemophilia B with or without inhibitors), thalassemia, blood diseases (e.g., anemia), Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), epilepsy, lysosomal storage diseases (e.g., aspartate glucoseuria, Barten's disease, late-onset infantile neuronal lipofuscinosis type 2 (CLN2), cystinosis, Fabry disease, Gaucher's type I, II, and III, glycogen storage disease II (Pompe disease), type I GM2-gangliopathy (Tay Sachs disease), GM2-gangliopathy II (Sandhoff's disease), type I mucosal seborrheic disease (types I and II salivary intoxication, type II (I-cell disease), type III (pseudo-Hurler disease) and IV, mucopolysaccharidosis (Hurler disease and variants, Hunter, Sanfilippo A, B), C, D, Morquio Types A and B, Maroteaux-Lamy and Sly diseases, Niemann-Pick disease A / B, C1 and C2, and Schindler disease I and II, hereditary angioedema (HAE), copper or iron accumulation disorders (e.g., Wilson's disease or Menkes disease), lysosomal acid lipase deficiency, nervous system or neurodegenerative diseases, cancer, type 1 or type 2 diabetes, adenosine deaminase deficiency, metabolic defects (e.g., glycogen storage diseases), solid organ (e.g., brain, liver, kidney, heart) or infectious viruses (e.g., hepatitis B and C, HIV, etc.), bacterial or fungal diseases; coagulation disorders.

[0153] Preferably, the subject has hemophilia A, hemophilia A with inhibitory antibodies, hemophilia B, hemophilia B with inhibitory antibodies, any coagulation factor: VII, VIII, IX, X, XI, V, XII, II, von Willebrand factor or FV / FVIII combined deficiency, thalassemia, vitamin K cyclooxygenase Cl deficiency or γ-carboxylase deficiency.

[0154] Preferably, the disease caused by the genetic defect is anemia, bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC); or excessive anticoagulation associated with heparin, low molecular weight heparin, pentasaccharides, warfarin, small molecule antithrombotic agents (i.e., FXa inhibitors) or platelet disorders (such as Bernard Soulier syndrome, Glanzmann anemia, or depletion of platelets).

[0155] 4.5 Drug administration and treatment methods

[0156] This invention also relates to methods of applying the mutants, proteins, pharmaceutical compositions and / or kits of the invention, and methods of treating diseases or conditions by applying the mutants, proteins, pharmaceutical compositions and / or kits of the invention.

[0157] Preferably, the mutants, proteins, and / or compositions of the present invention can be administered before administration of other therapeutic agents that have already produced drug-resistant antibodies in the body or are prone to producing drug-resistant antibodies.

[0158] Preferably, the mutants, proteins, and / or compositions of the present invention can be administered before, simultaneously with, and / or after administration of other therapeutic agents that have already produced or are prone to producing drug-resistant antibodies in the body.

[0159] In the application of this invention, the mutants, proteins and / or compositions of this invention are present with additional therapeutic agents as combined formulations for simultaneous, separate or sequential use.

[0160] In some embodiments, the method includes the steps of: 1) administering the mutant, protein, and / or composition of the present invention to a subject; subsequently, 2) administering the additional therapeutic agent to the subject. Preferably, there is a time interval between the administration of the mutant, protein, and / or composition of the present invention and the additional therapeutic agent.

[0161] In some embodiments, the method includes the steps of: 1) administering the additional therapeutic agent to the subject; subsequently, 2) administering the mutant, protein, and / or composition of the present invention to the subject. Preferably, there is a time interval between the administration of the mutant, protein, and / or composition of the present invention and the additional therapeutic agent.

[0162] Preferably, the dosage and time interval of the mutants, proteins, and / or compositions of the present invention are sufficient to reduce the immunoglobulin levels in the subject to 60% of the initial level. More preferably, the dosage and time interval of the mutants, proteins, and / or compositions of the present invention are sufficient to reduce the immunoglobulin levels in the subject to 50%, 40%, 30%, 20%, or 10% below the initial level in the patient. The mutants, proteins, and / or compositions of the present invention may be administered at a single time point or over a set period of time.

[0163] Preferably, the mutants, proteins, and / or compositions of the present invention are administered via intravenous infusion, intraperitoneal injection, intramuscular injection, joint injection, intradermal injection, or subcutaneous injection, with intravenous infusion being the preferred method. Furthermore, / or the amount of the mutants, proteins, and / or compositions of the present invention administered is 0.01 mg / kg body weight to 2 mg / kg body weight, 0.04 mg / kg body weight to 2 mg / kg body weight, 0.12 mg / kg body weight to 2 mg / kg body weight, 0.24 mg / kg body weight to 2 mg / kg body weight, or 1 mg / kg body weight to 2 mg / kg body weight.

[0164] Preferably, the time interval between administration of the mutants, proteins and / or compositions of the present invention and the additional therapeutic agents is at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 4 hours, at least 5 hours, or at least 6 hours; and at most 35 days, at most 28 days, at most 21 days, at most 18 days, at most 14 days, at most 13 days, at most 12 days, at most 11 days, at most 10 days, at most 9 days, at most 8 days, at most 7 days, at most 6 days, at most 5 days, at most 4 days, at most 3 days, at most 2 days, at most 24 hours, at most 18 hours, at most 12 hours, at most 10 hours, at most 8 hours, at most 7 hours, or at most 6 hours.

[0165] Preferably, the time interval between the mutant, protein and / or composition of the present invention and the additional therapeutic agent is 30 minutes to 1 hour, 30 minutes to 2 hours, 30 minutes to 3 hours, 30 minutes to 4 hours, 30 minutes to 5 hours, 30 minutes to 6 hours, 1 to 2 hours, 1 to 3 hours, 1 to 4 hours, 1 to 5 hours, 1 to 6 hours, 2 to 3 hours, 2 to 4 hours, 2 to 5 hours, 2 to 6 hours, 3 to 4 hours, 3 to 5 hours, 3 to 6 hours, 4 to 5 hours, 4 to 6 hours, or 5 to 6 hours.

[0166] In another embodiment, the method includes the following steps: 1) ex vivo treatment of blood from the subject with the mutant, protein and / or composition of the present invention; 2) returning the blood to the subject; and 3) administering the additional therapeutic agent to the subject.

[0167] In another embodiment, the method includes the following steps: 1) administering the additional therapeutic agent to the subject; 2) ex vivo processing of blood from the subject with the mutant, protein and / or composition of the present invention; and 3) returning the blood to the subject.

[0168] In a preferred embodiment, the mutants, proteins, and / or compositions of the present invention are combined with additional therapeutic agents for the prevention and / or treatment of cancer.

[0169] In a preferred embodiment, the mutants, proteins, and / or compositions of the present invention are combined with additional therapeutic agents for the prevention and / or treatment of viral infections.

[0170] In a preferred embodiment, the mutants, proteins, and / or compositions of the present invention are combined with additional therapeutic agents for the prevention and / or treatment of bacterial infections.

[0171] In a preferred embodiment, the mutants, proteins, and / or compositions of the present invention are combined with additional therapeutic agents for the prevention and / or treatment of fungal infections.

[0172] In a preferred embodiment, the mutants, proteins, and / or compositions of the present invention are combined with additional therapeutic agents for the prevention and / or treatment of gene defect-related diseases.

[0173] In a preferred embodiment, the mutants, proteins, and / or compositions of the present invention are combined with additional therapeutic agents for the treatment of diseases or conditions mediated by pathogenic IgG antibodies.

[0174] The present invention also provides a method for administering the drug combination to a subject to treat or prevent gene defect-related diseases, cancer, infections, or diseases or conditions mediated by pathogenic IgG antibodies. The method results in a 20-50%, 50-75%, 75-90%, 90-95%, or 95% or more reduction in antibody binding to the viral vector; the method results in a 20-50%, 50-75%, 75-90%, 90-95%, or 95% or more reduction in pathogenic IgG antibodies. Preferably, the drug is used for a method of treating gene defect-related diseases. Preferably, the drug is used for a method of treating cancer, preventing cancer, or preventing infection. The infection is preferably a viral infection, bacterial infection, or fungal infection. Preferably, the drug is used for a treatment method for cancer. Preferably, the drug is used for a method of treating diseases or conditions mediated by pathogenic IgG antibodies.

[0175] Preferably, the additional therapeutic agent is a viral vector drug; preferably, the viral vector drug is an oncolytic virus, a viral vaccine, or a gene therapy virus.

[0176] Preferably, the additional therapeutic agent is an antibody.

[0177] Preferably, the additional therapeutic agent is a peptide drug that lowers blood IgG levels, such as Efgartigimod. More preferably, the peptide drug that lowers blood IgG levels is used for the treatment of the aforementioned disease mediated by pathogenic IgG antibodies.

[0178] Preferably, the components of the drug combination are administered individually or simultaneously.

[0179] These uses and methods described above can also be further combined with other therapeutic agents, such as anti-inflammatory agents.

[0180] In some embodiments, the additional therapeutic agent is a leukocyte-depleting agent.

[0181] In some embodiments, the additional therapeutic agent is a B-cell depleting agent.

[0182] In some embodiments, the B-cell depleting agent is an antibody, preferably an antibody for tumor treatment, and more preferably an antibody combined with CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD70, CD72, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD3, CD11a, CD14, CD25, CD28, CD30, C Specific binding to D33, CD36, CD38, CD40, CD44, CD52, CD55, CD59, CD56, CD103, CD134, CD137, CD138, CD152, CD3, IL-1β, IL-2R, IL-6, IL-6R, IL-12, IL-23, C5, BAFF, BLyS, BCMA, CXCR-4, ICAM-1, SLAMF7 / CS1, TNFα, IgE, CD85, or CD86.

[0183] In some embodiments, the additional therapeutic agents are rituximab, dacrolimus, balithimab, moromumab-CD3, infliximab, adalimumab, omalizumab, efalizumab, natezumab, tocilizumab, eculizumab, golimumab, canatumab, uterotumab, belimumab, daratumumab, isatuximab, or combinations thereof.

[0184] In some embodiments, the Fc-containing agent is a therapeutic agent or a diagnostic agent, and preferably the Fc-containing agent is an antibody or an Fc fusion protein antibody-drug conjugate.

[0185] V. Definitions

[0186] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods, apparatus, and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, preferred methods, apparatus, and materials are now described.

[0187] The terms "nucleotide" or "polynucleotide" refer to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and their polymers, either in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also refers to oligonucleotide analogs, including PNAs (peptide nucleic acids), DNA analogs (phosphate thioesters, phosphoramidites, etc.) used in antisense techniques. Unless otherwise specified, a specific nucleic acid sequence implicitly encompasses variants of its conserved modifications (including, but not limited to, degenerate codon substitutions) and complementary sequences, as well as explicitly specified sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more of the selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue (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)).

[0188] The terms “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. That is, the description of a peptide is equally applicable to the description of a peptide and the description of a protein, and vice versa. The terminology applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the terminology covers amino acid chains of any length, including full-length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds.

[0189] The term "host cell" refers to 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 may remain as a non-integrating vector, such as a plasmid, or may be integrated into the host genome. The host cell may be a prokaryotic or eukaryotic cell.

[0190] The term "transformation" refers to the method of introducing a heterologous DNA sequence into a host cell or organism. The term "expression" refers to the transcription and / or translation of an endogenous gene or transgene in a cell.

[0191] The positive and progressive effects of this invention are as follows: This invention provides an application of an immunoglobulin degrading enzyme mutant. When applied to the field described above, the mutant of this invention has the advantages of high activity, low pre-existing antibody levels, and high safety. Detailed Implementation

[0192] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0193] Example 1. Design and expression of mutant libraries

[0194] A mutant library of wild-type IdeE protein sequences was designed and constructed, and 40 mutant strains were obtained from it after screening.

[0195] After codon optimization, a polynucleotide sequence encoding the wild-type IdeE protein sequence (SEQ ID NO: 2) was synthesized, and an N-terminal signal peptide sequence and a C-terminal 6×histidine tag were added. The synthesized sequence was then inserted into the pET32a expression vector. After successful sequencing, a recombinant plasmid for expressing wild-type IdeE was obtained. Based on the wild-type IdeE expression plasmid, degenerate primers for the mutant library were designed to amplify the original wild-type sequence. The amplified sequence was then inserted into the vector to obtain the mutant library recombinant plasmid. The wild-type and mutant library recombinant plasmids were electroporated into *E. coli* BL21 Star(DE3) and inoculated onto LB agarose plates containing 100 μg / ml ampicillin. The plates were incubated overnight at 37°C until colonies appeared. Single colonies were picked and inoculated into 200 μL of LB medium containing 100 μg / ml ampicillin, and incubated overnight at 37°C and 250 rpm. The overnight culture was inoculated into 1 ml of LB medium containing 100 μg / ml ampicillin and incubated at 37°C for 4 h. Then, 0.1 mM IPTG was added, and the culture was incubated overnight at 30°C. The supernatant of the overnight culture was collected by centrifugation. SDS-PAGE was used to evaluate the concentration of mutant protein in the mutant expression supernatant.

[0196] Example 2. Evaluation of the mutant's cleavage activity against human IgG1

[0197] To evaluate the cleavage activity of various mutants against human IgG1, an ELISA-based activity assay was established. The principle involves coating an ELISA plate with human IgG1-specific antigen, followed by incubation of a supernatant sample containing a suitable concentration of mutant protein and human IgG1 together in wells. Human IgG1 detection antibodies specific to the Fc region of the antibody are used to measure the amount of intact or incompletely cleaved human IgG1 bound to the wells. Given the same mutant protein concentration in the supernatant of each well, higher mutant protein cleavage activity against human IgG1 results in less intact human IgG1 antibody binding to the well, leading to a lower signal. An IgG1 standard curve can be constructed by analyzing the relationship between different concentrations of IgG1 and their corresponding detection signals. The amount of intact or incompletely cleaved IgG1 is calculated from the standard curve, and then the amount of completely cleaved IgG1 is calculated. The proportion of completely cleaved IgG1 to the initial IgG1 is used to evaluate the mutant activity.

[0198] To ensure a consistent concentration of mutant proteins in the supernatant harvested in Example 1, SDS-PAGE analysis was performed using the same loading volume. Quantity One was used to analyze the optical density values ​​of the target protein bands in the electrophoresis patterns. With the same loading volume, higher optical density values ​​of the target protein bands indicated higher concentrations. Using IdeE supernatant as a control, the supernatants of other mutants were concentrated or diluted to ensure that the optical density values ​​of the mutant protein bands were essentially consistent with those of the IdeE control.

[0199] After adjusting the protein concentration in the supernatant to a suitable level, perform ELISA detection as follows: Coat the ELISA plate with 2ug / ml human IgG1 (trastuzumab) specific antigen (catalog number QRE-104, Ruian Biotechnology) at 2-8℃ overnight, then wash with PBST (PBS + 0.05% Tween 20), and block the washed ELISA plate with 2% BSA (prepared in PBS) at 37℃ for 2h, and then wash with PBST.

[0200] Standard curve preparation: 200 ng / ml trastuzumab was serially diluted with reaction buffer (10 mM PB, 10 mM NaCl, pH 6.5) at a ratio of 1:2 until it reached 3.125 ng / ml. 100 μl of different concentrations of trastuzumab was added to the wells of the microplate for substrate (trastuzumab) standard curve preparation.

[0201] Cutting reaction: Dilute the supernatant after adjusting the protein concentration 5 times with reaction buffer (10mM PB, 10mM NaCl, pH 6.5), and add 50ul of 100ng / ml trastuzumab and 50ul of diluted supernatant to the wells of the microplate.

[0202] The microplate was incubated at 37°C with shaking for 1 hour. After washing with PBST, 40 ng / ml Goat anti-HumanIgG Fc Cross-Adsorbed Secondary Antibody-HRP (catalog number 31413, Thermo) was added to the wells, and the plate was incubated at 37°C with shaking for 1 hour. After washing with PBST, TMB was added as the chromogenic substrate for HRP, and the plate was incubated for 15 minutes. The incubation was terminated with 2N H2SO4. The absorbance at 450 nm was measured using a microplate reader. The concentrations of intact or incompletely cleaved trastuzumab in different test wells were calculated based on the substrate standard curve. The proportion of completely cleaved trastuzumab to the initial trastuzumab was then calculated to evaluate the activity of different mutants.

[0203] The fold relationship between the activity of each mutant and the activity of wild-type IdeE is shown in Table 1. All 40 mutants obtained in Example 1 had activities higher than or equal to wild-type IdeE, of which 15 mutants had activities that were 2 times or more than 2 times that of wild-type IdeE.

[0204] Table 1. Fold-ratio of mutant to wild-type IdeE activity as determined by ELISA

[0205]

[0206] Example 3. Evaluation of the thermal stability of the mutant

[0207] From the 15 mutants shown in Table 1 whose activity was 2 times or more that of wild-type IdeE, 12 mutants were selected to test their thermal stability. The activity detection method is as follows:

[0208] The supernatant of wild type or each mutant was divided into two portions and incubated at 4℃ and 50℃ for 1 hour respectively. After incubation, the activity of wild type or each mutant was detected by ELISA method in Example 2. The percentage of remaining activity of wild type or each mutant after incubation at 50℃ for 1 hour was calculated by dividing the activity after incubation at 50℃ by the activity after incubation at 4℃. This was used to compare the thermal stability of wild type and each mutant.

[0209] The fold relationship between mutant activity and the thermal stability of wild-type IdeE is shown in Table 2. Table 2 shows that all 12 mutants have higher thermal stability than the wild type, and 7 of them have thermal stability more than 3 times that of the wild type.

[0210] Table 2. Fold relationship of thermal stability of mutants relative to wild-type IdeE

[0211]

[0212] Example 4. Comparison of single-point mutant cleavage activity against human IgG1

[0213] The activity of seven single-point mutants, namely T8D, T8W, T24A, A59L, A59V, E97D, and R280H, which have "more than twice the activity of wild-type IdeE and more than three times the thermal stability of wild-type IdeE", as shown in Tables 1 and 2, in cleaving human IgG1 was tested.

[0214] 1. Expression and purification of mutants

[0215] One single colony was picked from each of the five single-point mutants transformed in Example 1 and inoculated into 3 ml of LB medium containing 100 μg / ml ampicillin. The culture was incubated overnight at 37°C and 250 rpm. The overnight culture was then 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 culture was incubated overnight at 30°C. The supernatant was collected by centrifugation. The supernatant was then purified using IDA-Ni agarose beads. The purified eluted protein was transferred to an ultrafiltration centrifuge tube and the medium was changed to a PBS buffer system. SDS-PAGE was used to evaluate the purity of the purified mutant protein. The OD280 was measured, and the concentration of the purified mutant protein was calculated based on the extinction coefficient.

[0216] 2. Comparison of mutant cleavage activity against human IgG1

[0217] The cleavage activity of different mutants relative to wild-type IdeE on human IgG1 was further evaluated by displaying the cleavage products of different concentrations of each mutant on SDS-PAGE. Purified mutant or wild-type IdeE was diluted to 0.002 mg / mL and 0.001 mg / mL, respectively. 50 μL of each concentration of mutant or wild-type IdeE was added to 50 μL of a reaction system containing 2 mg / mL trastuzumab to initiate the cleavage reaction. The reaction system was incubated at 37°C for 30 min. The sample was then mixed with an equal volume of 2×SDS loading buffer and incubated at 75°C for 5 min. The cleavage products were then detected by SDS-PAGE.

[0218] Figure 1 Electrophoretic images of the cleavage products of human IgG1 produced by cleavage of seven single-point mutants and wild-type IdeE are shown (enzyme:substrate = 1:1000). Figure 2 Electrophoretic images of the cleavage products of human IgG1 produced by seven single-point mutants and wild-type IdeE are shown (enzyme:substrate = 1:2000). The cleavage efficiency of the seven single-point mutants at a concentration of 0.001 mg / ml was no less than that of wild-type IdeE at 0.002 mg / ml, indicating that the cleavage activity of the seven single-point mutants in cleaving human IgG1 is at least twice that of wild-type IdeE.

[0219] Example 5. Comparison of N-terminal truncated mutant cleavage activity and thermostability of human IgG1

[0220] 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 from the wild-type IdeE, respectively (see Table 3).

[0221] Table 3. Design of truncated mutant sequences

[0222] mutant Modifications relative to wild-type or mutant sequences SEQ ID NO: WT_del15 Delete the first 15 amino acids of SEQ ID NO: 2 18 WT_del16 Delete the first 16 amino acids of SEQ ID NO: 2 19 WT_del17 Delete the first 17 amino acids of SEQ ID NO: 2 20 WT_del18 Delete the first 18 amino acids of SEQ ID NO: 2 21 WT_del19 Delete the first 19 amino acids of SEQ ID NO: 2 22

[0223] 1. Expression and purification of mutants

[0224] The mutant polynucleotide sequences in Table 3 were synthesized according to the method in Example 1, and the mutant expression recombinant plasmid was constructed and transformed into E. coli BL21 Star(DE3). The mutant purified protein was prepared according to the method in Example 4.

[0225] 2. Comparison of mutant cleavage activity against human IgG1

[0226] 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 mixture containing 2 mg / mL trastuzumab to initiate the cleavage reaction. The reaction mixture was incubated at 37°C for 30 min. The sample was then mixed with an equal volume of 2×SDS loading buffer and incubated at 75°C for 5 min. The cleavage products were then detected by SDS-PAGE.

[0227] Figure 3 Electrophoretic images of the cleavage products (enzyme:substrate = 1:1000) produced by five truncated mutants cleaving human IgG1 are shown. The cleavage activities of the five truncated mutants were not significantly different from those of wild-type IdeE.

[0228] 3. Comparison of the thermal stability of mutants

[0229] The purified mutant or wild-type IdeE was diluted to 0.1 mg / mL and incubated at 50°C for 1 h, then diluted again to 0.002 mg / mL. 50 μL of the diluted mutant or wild-type IdeE was added to 50 μL of a reaction mixture containing 2 mg / mL trastuzumab to initiate the cleavage reaction. The reaction mixture was incubated at 37°C for 30 min. The sample was then mixed with an equal volume of 2×SDS loading buffer and incubated at 75°C for 5 min. The cleavage products were then detected by SDS-PAGE.

[0230] Figure 4Electrophoretic images of the cleavage products of five truncated mutants and wild-type IdeE cleavage of human IgG1 after incubation at 50℃ for 1 h are shown (enzyme:substrate = 1:1000). The residual activity of the five truncated mutants after heat treatment at 50℃ was significantly higher than or equal to that of the wild type, indicating that the thermostability of the five truncated mutants was significantly improved compared with the wild type.

[0231] Example 6. Comparison of C-terminal truncated mutant activity against human IgG1

[0232] Two C-terminal truncated mutants were constructed by deleting the last 5 amino acids (W311-S315) and the last 10 amino acids (S306-S315) from the C-terminus of wild-type IdeE, respectively (see Table 4).

[0233] Table 4. Design of truncated mutant sequences

[0234] mutant Modifications relative to wild-type or mutant sequences SEQ ID NO: WT_delC5 Deleting the last 5 amino acids after SEQ ID NO: 2 23 WT_delC10 After deleting SEQ ID NO: 2, 10 amino acids were removed. 24

[0235] 1. Expression and purification of mutants

[0236] The mutant polynucleotide sequences in Table 4 were synthesized according to the method in Example 1, and the mutant expression recombinant plasmid was constructed and transformed into E. coli BL21 Star(DE3). The mutant purified protein was prepared according to the method in Example 4.

[0237] 2. Comparison of mutant cleavage activity against human IgG1

[0238] 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 mixture containing 2 mg / mL trastuzumab to initiate the cleavage reaction. The reaction mixture was incubated at 37°C for 30 min. The sample was then mixed with an equal volume of 2×SDS loading buffer and incubated at 75°C for 5 min. The cleavage products were then detected by SDS-PAGE.

[0239] Figure 5 Electrophoretic images of the cleavage products (enzyme:substrate = 1:1000) produced by two C-terminal truncated mutants cleaving human IgG1 are shown. The cleavage activities of both truncated mutants are more than 2 times higher than those of wild-type IdeE.

[0240] Example 7. Comparison of the cleavage activity and thermal stability of combined mutants of human IgG1

[0241] Five combined mutants were constructed by deleting the first 18 amino acids (D1-Q18) from five single-point mutants: T24A, A59L, A59V, E97D, and R280H (see Table 5).

[0242] Table 5. Combinatorial mutant sequence design

[0243] mutant Modifications relative to wild-type or mutant sequences SEQ ID NO: T24A_del18 Delete the first 18 amino acids of SEQ ID NO:9 25 A59L_del18 Delete the first 18 amino acids of SEQ ID NO: 13 26 A59V_del18 Delete the first 18 amino acids of SEQ ID NO: 14 27 E97D_del18 Delete the first 18 amino acids of SEQ ID NO: 15 28 R280H_del18 Delete the first 18 amino acids of SEQ ID NO: 16 29

[0244] 1. Expression and purification of mutants

[0245] The mutant polynucleotide sequences in Table 5 were synthesized according to the method in Example 1, and the mutant expression recombinant plasmid was constructed and transformed into E. coli BL21 Star(DE3). The mutant purified protein was prepared according to the method in Example 4.

[0246] 2. Comparison of mutant cleavage activity against human IgG1

[0247] The purified mutants were diluted to 0.001 mg / mL. 50 μL of the diluted mutant or wild-type IdeE was added to 50 μL of a reaction mixture containing 2 mg / mL trastuzumab to initiate the cleavage reaction. The reaction mixture was incubated at 37°C for 30 min. The sample was then mixed with an equal volume of 2×SDS loading buffer and incubated at 75°C for 5 min. The cleavage products were then detected by SDS-PAGE.

[0248] Figure 6 Electrophoretic images of the cleavage products from human IgG1 produced by five combined mutants (enzyme:substrate = 1:2000) are shown. Comparison Figure 6 and Figure 2 The cleavage effect showed no significant difference between the 5 truncated mutants and the single-point combination mutant, indicating that the cleavage activity of the combination mutants in human IgG1 was no less than twice that of wild-type IdeE.

[0249] 3. Comparison of the thermal stability of mutants

[0250] The purified mutants were diluted to 0.1 mg / mL and incubated at 50°C for 1 h, then diluted again to 0.001 mg / mL. 50 μL of the diluted mutant or wild-type IdeE was added to 50 μL of a reaction mixture containing 2 mg / mL trastuzumab to initiate the cleavage reaction. The reaction mixture was incubated at 37°C for 30 min. The sample was then mixed with an equal volume of 2×SDS loading buffer and incubated at 75°C for 5 min. The cleavage products were then detected by SDS-PAGE.

[0251] Figure 7 Electrophoretic images of the cleavage products (enzyme:substrate = 1:2000) produced by five combined mutants cleaving human IgG1 after incubation at 50℃ for 1 hour are shown. Comparison Figure 6 and Figure 7 The cleavage effect was good; the activity of the five combined mutants decreased only slightly after heat treatment at 50℃, indicating that the thermal stability of the five combined mutants was significantly improved compared with that of the wild type.

[0252] Example 8. Comparison of E97D_del18 mutant activity with IdeS and IdeZ

[0253] The purified E97D_del18 mutant from Example 7 was successively diluted to 20 μg / mL, 10 μg / mL, 5 μg / mL, 2.5 μg / mL, and 1.25 μg / mL. IdeS( Catalog number: A0-FRI-020, Genovis) 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 markings. IdeZ ( 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 of each concentration of mutant, IdeS, or IdeZ was added to 50 μl of a reaction system containing 2 mg / ml trastuzumab to initiate the cleavage reaction. The reaction system was incubated at 37°C for 30 min. The sample was then mixed with an equal volume of 2×SDS loading buffer and incubated at 75°C for 5 min. The cleavage products were detected by SDS-PAGE.

[0254] Figure 8 Electrophoresis images of the cleavage products of human IgG1 produced by different concentrations of the E97D_del18 mutant and IdeS are shown. From the enzyme bands on the electrophoresis images, it can be determined that the IdeS enzyme concentration in lane 1 is between that in lanes 7 and 8 (E97D_del18 mutant enzyme concentration). Therefore, the IdeS enzyme concentration in lane 3 is between that in lanes 9 and 10 (E97D_del18 mutant enzyme concentration). The IgG1 cleavage efficiency in lane 3 is between that in lanes 10 and 11. Thus, it can be inferred that the E97D_del18 mutant has approximately twice the cleavage activity of IdeS in cleaving human IgG1.

[0255] Figure 9 Electrophoresis images of the cleavage products of human IgG1 produced by different concentrations of the E97D_del18 mutant and IdeZ are shown. From the enzyme-protein bands on the electrophoresis images, it can be determined that the IdeZ enzyme concentration in lane 1 is higher than the E97D_del18 mutant enzyme concentration in lane 7. Therefore, the IdeZ enzyme concentration in lane 3 is higher than the E97D_del18 mutant enzyme concentration in lane 9, which is four times higher than the E97D_del18 mutant enzyme concentration in lane 11. Since the IgG1 cleavage efficiency in lane 3 is similar to that in lane 11, it can be inferred that the E97D_del18 mutant has a cleavage activity of human IgG1 that is four times higher than that of IdeZ.

[0256] Example 9. In vitro detection of the cleavage activity of the E97D_del18 mutant against human IgG1

[0257] The in vitro cleavage activity of the E97D_del18 mutant against human IgG1 was evaluated by detecting the amount of intact or single-cleaved IVIg in the serum or plasma of mice treated with the E97D_del18 mutant and human IVIg. Enzyme digestion systems for different groups of mouse serum or plasma were prepared according to Table 6.

[0258] Table 6. Enzyme digestion systems of mouse serum or plasma

[0259]

[0260]

[0261] In the iodoacetic acid treatment group, the role of iodoacetic acid was to inhibit the activity of IgG degradation enzymes.

[0262] The system was reacted at 37℃ for 30 min. 20 μl of sample was mixed with an equal volume of 2×SDS non-reducing loading buffer and incubated in a water bath at 75℃ for 5 min. The cleavage products were then detected by SDS-PAGE.

[0263] Figure 10 Electrophoretic images of the cleavage products of human IVIg produced by the E97D_del18 mutant in mouse serum and plasma are shown. The results show that E97D_del18 can effectively cleave human IVIg in both mouse serum and plasma.

[0264] The in vitro cleavage activity of the E97D_del18 mutant against human IgG1 was evaluated by detecting the cleavage of mouse or human serum treated with the E97D_del18 mutant. Enzyme digestion systems for different groups of mouse or human serum were prepared according to Table 7.

[0265] Table 7. Enzyme digestion systems of mouse or human serum

[0266]

[0267] The system was reacted at 37℃ for 24 h. 20 μl of sample was mixed with an equal volume of 2×SDS reducing loading buffer, then diluted 20 times with 1×SDS reducing loading buffer, and incubated in a 75℃ water bath for 5 min. The cleavage products were then detected by SDS-PAGE.

[0268] Figure 11 Electrophoretic images of the cleavage products produced by the E97D_del18 mutant in mouse and human serum are shown. The results show that E97D_del18 produces a clearly visible 25kD Fc fragment in human serum, while this fragment is absent in mouse serum, indicating that E97D_del18 can effectively and specifically cleave IgG1 in human serum, while having very low or no cleavage activity against IgG1 in mouse serum.

[0269] Example 10. The E97D_del18 mutant cleaves immunoglobulins of the same species.

[0270] The in vitro cleavage activity of the E97D_del18 mutant on serum immunoglobulins of different animal species was evaluated by detecting the amount of intact or single-cut IgG in serum or plasma of different species after adding the E97D_del18 mutant. Serum or antibody enzymatic digestion systems for different species were prepared according to Tables 8 and 9.

[0271] Table 8. Enzymatic digestion systems of Beagle serum and antibodies from different species

[0272]

[0273] The system was placed at 37°C for 1 hour, and the enzyme digestion products were detected by SDS-PAGE.

[0274] Table 9. Serum enzyme digestion systems of different species

[0275]

[0276] Figure 12A-12D The effects of the E97D_del18 mutant on sera and antibodies from different species were demonstrated. Results showed that E97D_del18 effectively cleaved canine IgG, rabbit IgG, and mouse IgG2a, but not mouse IgG1. E97D_de118 effectively cleaved rabbit, canine, and monkey serum IgG, with the best cleavage effect on rabbit serum IgG, poorer cleavage on porcine serum IgG, and almost no cleavage on rat and mouse serum IgG.

[0277] Example 11. Low levels of pre-existing antibodies against the E97D_del18 mutant in the human body.

[0278] This assay is based on the competition between the E97D_del18 mutant and IdeS for binding to the anti-E97D_del18 / IdeS antibody. Pre-incubation of the test enzyme and human serum will allow both the anti-E97D_del18 / IdeS antibody and the E97D_del18 mutant to bind to IdeS.

[0279] The E97D_del18 mutant and IdeS were coated onto well plates overnight, washed with PBST, and blocked in 2% BSA blocking buffer for 1 hour. A mixed plate was prepared using stepwise dilutions of the test mutant, IdeS, and human serum. The mixed plate was incubated at room temperature with shaking for 1 hour. After washing with PBST, biotin-labeled E97D_del18 mutant and IdeS were added, followed by SA-HRP. The plate was then developed with TMB and the readings were taken. Parallel comparisons were performed to obtain the pre-existing antibodies against E97D_del18 and IdeS in approximately 80 human blood samples.

[0280] The results are shown in Table 10. The proportion of pre-existing antibodies in normal human serum for IdeS is as high as about 90%, while that for the E97D_del18 mutant is only about 20%. The pre-existing antibodies in vivo for the E97D_del18 mutant are significantly less than those for IdeS, proving that the E97D_del18 mutant has lower immunogenicity and is more conducive to in vivo drug administration.

[0281] Table 10. Comparison of pre-existing antibodies against E97D_del18 mutant and IdeS in human blood samples.

[0282] E97D_del18 mutant IdeS Total number of human blood samples (cases) 76 76 Percentage of pre-existing antibody-positive samples (%) 184 895

[0283] Example 12. In vivo detection of the cleavage activity of the E97D_del18 mutant against human IgG1

[0284] Two mice (mice 1 and 2) were intraperitoneally injected with human IVIg (intravenous immunoglobulin) under aseptic conditions at a dose of 1 g / kg. Twenty-four hours after the IVIg injection, the IgG degrading enzyme mutant (E97D_del18) was intravenously injected into the mice at a dose of 5 mg / kg. Blood samples were collected from both mice at 0 h, 15 min, 2 h, 6 h, and 24 h after E97D_del18 injection. 20 μl of serum sample was mixed with an equal volume of 2×SDS non-reducing loading buffer, then diluted 20-fold with 1×SDS non-reducing loading buffer, incubated at 75°C for 5 min, and analyzed by SDS-PAGE.

[0285] Figure 13 Electrophoretic images of the cleavage products of E97D_del18 at different time points in mice are shown. The results show that E97D_del18 has a significant effect on cleaving IVIg in mice, and almost complete cleavage is achieved within 15 minutes.

[0286] Example 13. Comparison of the cleavage activity of combined mutants against human IgG1

[0287] Based on the above mutants, six combined mutants were further constructed, and the sequences of the mutations are shown in Table 11.

[0288] Table 11. Combinatorial mutants

[0289]

[0290]

[0291] 1. Expression and purification of mutants

[0292] The mutant polynucleotide sequences in Table 11 were synthesized according to the method in Example 1, and the mutant expression recombinant plasmid was constructed and transformed into E. coli BL21 Star(DE3). The mutant purified protein was prepared according to the method in Example 4.

[0293] 2. Comparison of mutant cleavage activity against human IgG1

[0294] The purified mutants were diluted to 0.001 mg / mL. 50 μL of the diluted mutant or wild-type IdeE was added to 50 μL of a reaction mixture containing 2 mg / mL trastuzumab to initiate the cleavage reaction. The reaction mixture was incubated at 37°C for 30 min. The sample was then mixed with an equal volume of 2×SDS loading buffer and incubated at 75°C for 5 min. The cleavage products were then detected by SDS-PAGE.

[0295] Figure 14A and 14B Electrophoretic images of the cleavage products of human IgG1 produced by six combined mutants are shown (enzyme:substrate = 1:2000).

[0296] Example 14. Design and expression of novel mutants

[0297] Further analysis and screening of mutation data led to the selection of the IdeE mutant SEQ ID NO: 36 for evaluation, designated IdeEv2. After codon optimization, a polynucleotide sequence encoding this mutant was synthesized, and an N-terminal signal peptide sequence was added. The synthesized sequence was inserted into the pET32a expression vector, and after successful sequencing, a recombinant plasmid for expression was obtained. The mutant recombinant plasmid was electroporated into *E. coli* BL21 Star(DE3) and inoculated onto LB agarose plates containing 100 μg / ml ampicillin. Incubation was carried out overnight at 37°C until colonies appeared. Single colonies were picked and inoculated into 200 μL of LB medium containing 100 μg / ml ampicillin, and incubated overnight at 37°C and 250 rpm. The overnight culture was inoculated into 1 ml of LB medium containing 100 μg / ml ampicillin, incubated at 37°C for 4 h, and then 0.1 mM IPTG was added, followed by incubation at 30°C overnight. The supernatant from the overnight culture was collected by centrifugation. SDS-PAGE was used to evaluate the expression of mutant proteins in the mutant expression supernatant. The supernatant was purified sequentially by ion exchange chromatography and hydrophobic chromatography to obtain pure proteins. IdeS and IdeZ proteins were prepared using the same method. The purity of the obtained proteins was determined by SEC-HPLC, and both were found to be over 95%.

[0298] Example 15. Comparison of the cleavage activity and thermal stability of the IDEV2 mutant against human IgG1

[0299] 1. Comparison of mutant cleavage activity against human IgG1

[0300] The purified mutant and wild-type IdeE, IdeS, and IdeZ were diluted to 0.0025 mg / mL. 50 μL of each concentration of mutant, IdeE, IdeS, or IdeZ was added to 50 μL of a reaction mixture containing 2 mg / mL trastuzumab to initiate the cleavage reaction. The reaction mixture was incubated at 37°C for 30 min. The sample was then mixed with an equal volume of 2×SDS loading buffer and incubated at 75°C for 5 min. SDS-PAGE was used to detect the cleavage products. The results showed that the combined mutant exhibited similar cleavage activity against human IgG1 as wild-type IdeE. Figure 15A ).

[0301] 2. Comparison of the thermal stability of mutants

[0302] The purified mutants were diluted to 0.1 mg / mL and incubated at 50 °C for 1 h, then diluted again to 0.0025 mg / mL. 50 μL of the diluted mutant or wild-type IdeE was added to 50 μL of a reaction mixture containing 2 mg / mL trastuzumab to initiate the cleavage reaction. The reaction mixture was incubated at 37 °C for 30 min. The sample was then mixed with an equal volume of 2×SDS loading buffer and incubated at 75 °C for 5 min. SDS-PAGE was used to detect the cleavage products. The results showed that the mutant IDEEV2 exhibited the least decrease in activity after heat treatment at 50 °C, indicating that the mutant IDEEV2 also showed significantly improved thermostability compared to the wild-type and other mutants.

[0303] Example 16. Evaluation of the cleavage specificity of the IDEEV2 mutant against different human immunoglobulins

[0304] The cleavage specificity of the IDEEV2 mutant for different substrates was further evaluated by displaying the cleavage products of the IDEEV2 mutant against different human IgGs on SDS-PAGE. The purified IDEEV2 mutant was diluted to 0.01 mg / mL. 50 μL of different concentrations of mutant or wild-type IdeE was added to 50 μL of a reaction system containing 2 mg / mL of different immunoglobulins (human IgG1–4, IgM, IgA, IgE, and IgD) to initiate the cleavage reaction. The reaction system was incubated at 37°C for 30 min. The sample was then mixed with an equal volume of 2×SDS loading buffer and incubated at 75°C for 5 min. The cleavage products were then detected by SDS-PAGE.

[0305] The results showed that IDEEV2 can effectively cleave human IgG1, IgG2, IgG3, and IgG4. Figure 16A It cannot cleave human immunoglobulins IgA, IgE, IgD, and IgM. Figure 16B This indicates that the variant has high substrate specificity.

[0306] Example 17. IDEEV2 mutant cleaves immunoglobulins from different species

[0307] The in vitro cleavage activity of the IDEEV2 mutant against immunoglobulins of different species was evaluated by detecting the amount of intact or single-cleaved IgG in the serum or plasma of different animal species after adding the IDEEV2 mutant. Rabbit, dog, rat, mouse, monkey, and human purified IgG were digested at a protein ratio of IDEEV2: different animal species or human purified IgG = 1:200, at 37°C for 1 h, and the digestion products were detected by non-reducing SDS-PAGE electrophoresis.

[0308] The results showed that IDEEV2 effectively cleaved IgG in rabbit, dog, monkey, and human serum with species specificity, but had no significant cleavage of IgG in rat and mouse serum. Figure 17 ).

[0309] Example 18. Experiment on the cleavage of IgG in New Zealand rabbits by the IDEEV2 mutant

[0310] The activity of IDEEV2 in cleaving IgG in New Zealand rabbits was studied. IDEEV2 was administered intravenously to New Zealand rabbits twice weekly at a dose of 2 mg / kg. Serum IgG levels were measured using ELISA or SDS-PAGE to evaluate the enzymatic effect of intravenous IDEEV2 on IgG cleavage in the animals.

[0311] The results showed that IDEEV2 could rapidly cleave IgG in rabbits: at the first blood sample collection point after the first and last administration (1 minute before the end of administration), the IgG content in rabbits at a dose of 2 mg / kg was generally below the detection limit. Approximately 1-2 days after the first and last administration, the IgG level in rabbits gradually recovered, and the content of the enzyme cleavage product gradually returned to the pre-administration level. After two administrations, the IgG content in rabbits returned to the normal range approximately 10 days later. Figure 18A )

[0312] IDEEV2 was administered intravenously to New Zealand rabbits once a week for four weeks. Blood samples from the 0.2, 2, and 20 mg / kg dose groups showed that IDEEV2 could rapidly cleave IgG in rabbits; the lowest dose (0.2 mg / kg) already exhibited complete pharmacological activity. Figure 18B )

[0313] The data above show that after intravenous infusion of IDEEV2 into New Zealand rabbits, IgG in rabbits was rapidly cleaved within a dose range of 0.2-20 mg / kg. Most of the IgG in the rabbits was cleaved within the first blood sample collection point after IDEEV2 administration (1 minute before the end of administration). Approximately 1-2 days after administration, the content of the cleaved products gradually returned to pre-administration levels. Approximately 10 days after administration, the IgG content in the rabbits returned to normal levels.

[0314] Example 19. Experiment on cleavage of IgG in beagle dogs by IDEEV2 mutant

[0315] IDEEV2 was administered intravenously to Beagle dogs once a week for two weeks. Blood samples from the 0.2, 2, and 20 mg / kg dose groups showed that IDEEV2 rapidly cleaved IgG in dogs. Approximately 3 days after the first and last administration, IgG levels in the dogs gradually recovered, and the content of the cleavage products returned to pre-administration levels. After two administrations, there were some individual differences in the recovery rate of IgG in the dogs (2 dogs / sex / group). At the 0.2 and 2 mg / kg doses, IgG levels returned to normal after approximately 10 days, while at the 20 mg / kg dose, levels generally returned to normal after 4 weeks. The rate of decrease and recovery of IgG levels generally showed a dose-response relationship with the administered dose.

[0316] As can be seen from the data above ( Figure 19A , 19B Following intravenous infusion of IDEEV2 into Beagle dogs, IgG in the dog's body is rapidly cleaved within a dose range of 0.2-20 mg / kg. Most of the IgG in the dog's body is cleaved within the first blood sample collection point after IDEEV2 administration (1 minute before the end of administration). Approximately 3 days after administration, the content of the cleaved products gradually returns to pre-administration levels. There is some individual variation in the recovery of IgG; the IgG content in the dog's body returns to normal levels approximately 10-28 days after administration.

[0317] Based on the data above, IDEEV2 can rapidly and effectively cleave IgG in both New Zealand rabbits and beagle dogs, demonstrating its advantages in antibody-mediated autoimmune diseases, especially acute, severe, and life-threatening antibody-mediated autoimmune diseases.

[0318] Example 20. IDEV2 can effectively reverse hypothrombocytopenia caused by antiplatelet antibodies.

[0319] A BALB / c mouse thrombocytopenia model was established by intraperitoneal injection of 10 mg / mouse modeling agent (rabbit IgG prepared by purification of rabbit antiserum against mouse platelets). The efficacy of intravenous injection of IDEEV2 on the thrombocytopenia model in BALB / c mice was evaluated.

[0320] Before modeling, platelet counts were similar in the model control group and the low- and high-dose IDEEV2 groups. 0.5 hours after modeling, platelet counts decreased significantly in all groups. Before IDEEV2 administration (approximately 2 hours after modeling), platelet counts in all groups continued to decrease. In the model control group, platelet counts remained consistently low after administration of the modeling agent. Figure 20A ), and all the animals in the group died within 3 days. Figure 20BIn both the low- and high-dose IDEEV2 groups, platelet counts began to recover after administration, reaching levels similar to pre-modeling levels at D20, indicating that IDEEV2 significantly improves thrombocytopenia in BALB / c mice. This further demonstrates that the variant of this invention exhibits good symptom-improving effects in autoimmune diseases caused by anti-autoantibodies.

[0321] Example 21. Immunoglobulin-degrading enzyme mutants effectively eliminate the effect of high immunoglobulin levels on the antitumor efficacy of monoclonal antibodies. Negative impact

[0322] The Daudi mouse model was used to evaluate the IDEEV2 anti-CD38 monoclonal antibody. The experiment was designed with four groups: 1) solvent control group; 2) anti-CD38 monoclonal antibody daratumumab; 3) anti-CD38 monoclonal antibody daratumumab + IVIg; 4) anti-CD38 monoclonal antibody daratumumab + IDEEV2 + IVIg. Specifically, logarithmic growth phase cells were harvested and subcutaneously inoculated into mice. Each CB-17SCID mouse received 5 × 10⁶ cells subcutaneously in the right axilla under sterile conditions. 6 Daudi cells were inoculated at a dose of 0.1 mL per cell. On the day of cell inoculation, a single intraperitoneal injection of 1 g / kg IVIg was administered. Twenty-four hours later, a single intravenous injection of 5 mg / kg of immunoglobulin-degrading enzyme E97D_del18 was administered. Ten days after IVIg administration, 2 mg / kg of anti-CD38 monoclonal antibody was administered intraperitoneally once a week for a total of four weeks. Tumor volume was measured twice a week after CD38 administration began for four weeks.

[0323] Experimental results show that ( Figure 21 The presence of IgG (IVIg) can inhibit the efficacy of anti-CD38 monoclonal antibody therapy for tumors. IDEEV2 can reduce the inhibitory effect of naturally occurring IgG on antibody efficacy and significantly enhance the therapeutic effect of therapeutic antibodies.

[0324] Example 22. Effects of IgG degrading enzyme mutants on AAV in vivo infection

[0325] C57BL / 6J mice received a single intraperitoneal administration of IVIg (1 g / kg), followed by a single intravenous administration of IDEEV2 (5 mg / kg) 30 min after IVIg administration. Twenty-four hours after IVIg administration, mice were given AAV9-Fluc (2 × 10⁻⁶ g / kg). 11 (vg / animal). Fluorescence imaging was performed at D8, D15, D22, D29, D36, and D43. Heart and liver tissue samples were collected on D44 to detect the copy number of the Fluc gene.

[0326] 1) Fluorescent control group (AAV9-Fluc);

[0327] 2) Model group (IVIg+AAV9-Fluc);

[0328] 3) Test sample group (IVIg+IDEEV2+AAV9-Fluc).

[0329] The results showed that AAV had no infectious effect on mice in the presence of IVIg, while E97D_del18 completely eliminated the influence of IVIg on AAV infection. Figure 22A , 22B The virus can be effectively transduced in both heart and liver tissues. Figure 23 The experimental results show that the mutant of the present invention can effectively degrade human immunoglobulin G in vivo and eliminate the interference of virus-neutralizing antibodies on drugs that use viruses as therapeutic carriers.

[0330] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

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Claims

1. A mutant of the immunoglobulin-degrading enzyme IdeE, wherein, The amino acid sequence of the mutant is shown in SEQ ID NO:

36.

2. A protein comprising the mutant of claim 1, wherein 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.

3. A protein as claimed in claim 2, wherein the protein has higher activity and / or thermal stability than the immunoglobulin-degrading enzyme IdeE with a sequence as shown in SEQ ID NO:

2.

4. A polynucleotide encoding a mutant as described in claim 1 or a protein as described in claim 2 or 3.

5. An expression vector comprising the polynucleotide as described in claim 4.

6. A host cell comprising the expression vector as described in claim 5, or expressing the mutant as described in claim 1 or the protein as described in claim 2 or 3; said host cell being an Escherichia coli cell or a yeast cell.

7. A composition comprising: a mutant as described in claim 1 or a protein as described in claim 2 or 3; and optionally a pharmaceutically acceptable carrier or excipient.

8. The composition of claim 7, further comprising: an antibody or a protein containing Fc.

9. The composition of claim 8, wherein the target of the antibody is selected from any one of the group consisting of cell surface proteins, cytokines, hormones, enzymes, intracellular messengers, intercellular messengers, and immune checkpoints.

10. The composition of any one of claims 7-9, further comprising: a viral vector drug, said viral vector drug being selected from any one of the group consisting of oncolytic viruses, gene therapy viruses, and viral vector vaccines.

11. The composition of any one of claims 7-10, further comprising: a drug capable of reducing blood IgG levels, said drug for reducing blood IgG levels being selected from FcRn antibodies or Fc fragment variants with high affinity for FcRn.

12. A kit comprising: (1) a mutant as claimed in claim 1 or a protein as claimed in claim 2 or 3; and (2) one or more selected from the group consisting of: (a) a pharmaceutically acceptable vector or excipient; (b) an antibody or a protein containing Fc; and / or (3) a viral vector drug selected from oncolytic viruses, gene therapy viruses, or viral vector vaccines; and / or (4) a drug that reduces blood IgG levels selected from FcRn antibodies or Fc fragment variants with high affinity for FcRn.

13. A reagent kit comprising: kit A and kit B, wherein, The kit A contains the mutant as described in claim 1 or the protein as described in claim 2 or 3, and the kit B contains one or more of the following: (1) a pharmaceutically acceptable carrier or excipient; (2) an antibody or a protein containing Fc; and / or (3) a viral vector drug; and / or (4) a drug that can reduce blood IgG levels; wherein the viral vector drug is selected from oncolytic viruses, gene therapy viruses or viral vector vaccines; and the drug that reduces blood IgG levels is selected from FcRn antibodies or Fc fragment variants with high affinity for FcRn.

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