Method for reducing level of immunoglobulin g in subject

Abstract

The present disclosure relates to a method for reducing the level of immunoglobulin G in a subject and particularly to a method for reducing the level of immunoglobulin G in a subject by means of continuous administration of an immunoglobulin G-degrading enzyme to the subject.

Description

Methods to reduce immunoglobulin G levels in subjects

[0001] Priority Statement

[0002] This disclosure claims priority to the following earlier applications:

[0003] An international application with application number PCT / CN2024 / 105695 and application date of July 16, 2024;

[0004] An international application with application number PCT / CN2024 / 119092 and application date of September 14, 2024;

[0005] Chinese invention patent application with application number CN2024112963954 and application date of September 14, 2024;

[0006] Chinese invention patent application with application number CN2024114750721 and application date of October 22, 2024;

[0007] Chinese invention patent application with application number CN2025101347718 and application date of February 6, 2025;

[0008] Chinese invention patent application with application number CN2025104891840 and application date of April 17, 2025;

[0009] Chinese invention patent application with application number CN2025106138634 and application date of May 12, 2025;

[0010] The full text of all the above applications is incorporated herein by reference. Technical Field

[0011] This disclosure relates to the field of biotechnology, and more specifically to a method for reducing immunoglobulin G levels in a subject using immunoglobulin-degrading enzymes. Background Technology

[0012] Immunoglobulin G (IgG) is an abundant protein in human serum, accounting for approximately 10%–20% of plasma proteins. IgG is crucial for normal immune function in the body. After infection or vaccination, the body produces IgG antibodies against viruses and bacteria. Cells in our own tissues also possess antigens. Normally, the immune system only reacts to foreign or dangerous substances, not to antigens in its own tissues. However, sometimes immune dysfunction occurs, causing the immune system to treat its own tissues as foreign, producing antibodies (called autoantibodies) or immune cells that attack its own cells or tissues. This reaction is called an autoimmune response, which leads to inflammation and tissue damage, and can also cause autoimmune diseases.

[0013] Anti-glomerular basement membrane (GBM) disease (anti-GBM disease) is an autoimmune disease characterized by the deposition of circulating anti-GBM antibodies in the glomerular and alveolar basement membranes, leading to rapidly progressive crescentic glomerulonephritis and pulmonary hemorrhage. Current treatment options include intensive plasma exchange combined with glucocorticoids and immunosuppressants (cyclophosphamide being the preferred choice). This treatment can improve patient and renal outcomes but often causes serious complications. On July 16, 2018, the U.S. Food and Drug Administration (FDA) approved Hansa Medical's IdeS (imlifidase) for clinical trials to treat anti-GBM disease.

[0014] IdeS is a cysteine ​​protease derived from Streptococcus pyogenes with the ability to specifically cleave immunoglobulin G (IgG). This enzyme breaks down IgG into F(ab')2 and Fc fragments, a property that suggests potential applications for IdeS in the treatment of autoimmune diseases and antibody-mediated transplant rejection. IdeS is a virulence factor of human pathogens, and due to early infection with Streptococcus pyogenes, a significant portion of the population has pre-existing IgG antibodies against IdeS (Anti-Drug Antibodies, ADAs). A clinical study of Imlifidase found that 10 out of 130 participants had IdeS-specific IgG levels below a cutoff (2.0 mg / L); the median anti-IdeS IgG level was 6.1 mg / L, and the 80th percentile was 15 mg / L. These anti-IdeS antibodies may increase the risk of hypersensitivity / infusion-like reactions to imlifidase (Winstedt L et al. (2015). PLoS ONE 10(7)), limiting the use of IdeS in patients with high-titer ADA. Furthermore, because anti-IdeS antibodies are widely present in patients, anti-drug antibodies are rapidly generated after the first administration of IdeS. For example, in another phase II clinical study of imlifidase (JAm Soc Nephrol. 2022 Apr; 33(4):829–838. doi:10.1681 / ASN.2021111460), subjects had varying ADA levels before administration, ranging from 3.5 to 16.7 mg / L. Despite the use of steroids and cyclophosphamide (CYC), ADA levels in samples on day 14 increased sharply. Therefore, although IdeS can be given a second time within 24 hours, the dosing interval should not exceed 24 hours. However, even continuous administration within 24 hours cannot effectively clear and prevent the formation of new immunoglobulins in the body, and thus cannot effectively deal with the repeated occurrence of pathogenic antibodies in the patient's body, or eliminate the negative effects caused by autoimmune reactions.

[0015] WO2024008943A1 relates to a pretreatment protocol for cell transplantation in sensitized subjects, comprising at least two administrations of an enzyme that inactivates serum IgG molecules in the subject. Given that many enzymes with this function are bacterial (often pathogenic) in origin, the second dose may present problems due to the patient's immune response to it. For example, IdeS is a bacterial enzyme derived from Streptococcus pyogenes, which most people have been exposed to and developed immune memory for. This can lead to rapid and sometimes very strong anti-drug antibody responses, making repeated dosing impractical. Therefore, alternative enzymes with lower immunogenicity in human subjects may be preferred.

[0016] On the other hand, during gene therapy (e.g., AAV gene therapy), AAV neutralizing antibodies (Nab) and total binding antibodies (Tab) significantly reduce the effectiveness of gene therapy. These antibodies primarily target the AAV capsid protein, neutralizing the virus and preventing it from effectively entering host cells, thus affecting the efficacy of gene therapy. The presence of these antibodies limits the applicable population for treatment. Studies have shown that approximately 50%–90% of the population (depending on serotype) already possesses anti-AAV antibodies, making this group unsuitable for gene therapy. Furthermore, the presence of these antibodies can lead to ineffective repeated treatments because the host has developed immune memory against AAV. Therefore, there is an urgent need to eliminate pre-existing AAV neutralizing and total binding antibodies in these populations to expand the pool of patients eligible for AAV gene therapy. WO2020102740A2 discloses that IdeS can reduce or eliminate neutralizing antibodies against the AAV capsid and can treat patients previously considered unsuitable for gene therapy or who have developed AAV antibodies after AAV gene therapy. However, this regimen does not involve repeated administration of IgG degrading enzymes and cannot reduce AAV neutralizing antibody levels in patients with pre-existing high titers to acceptable levels. Summary of the Invention

[0017] The inventors of this disclosure discovered through clinical studies that when subjects receive immunosuppressive therapy, the production of ADA by IgG degrading enzymes is effectively inhibited, reducing the risks associated with ADA. This makes repeated administration of IgG degrading enzymes possible, thereby further reducing the level of unwanted immunoglobulins in patients.

[0018] The first aspect of this disclosure provides a method for reducing immunoglobulin G levels in a subject, the method comprising: administering at least two doses of immunoglobulin G degrading enzyme to the subject; wherein the interval between the administration of at least one dose of immunoglobulin G degrading enzyme and the previous dose of immunoglobulin G degrading enzyme is greater than 24 hours; and the subject also receiving treatment with at least one immunosuppressant.

[0019] In an optional embodiment, the interval between the administration of the at least one dose of immunoglobulin G degrading enzyme and the previous dose of immunoglobulin G degrading enzyme shall not exceed 7 days to 12 months, including but not limited to 7 days, 10 days, 15 days, 1 month, 2 months, 3 months, 4 months, 6 months or 12 months.

[0020] In an optional embodiment, the method includes administering the subject at least 3 to 10 doses of immunoglobulin G degrading enzyme, optionally administering the subject at least 3, 4, 5, 6, 7, 8, 9, or 10 doses of immunoglobulin G degrading enzyme.

[0021] In an optional implementation, the pre-existing antibody against the immunoglobulin G degrading enzyme is absent or undetectable in the subject.

[0022] In an optional embodiment, the amount of the immunosuppressant is sufficient to ensure that the titer of anti-immunoglobulin G degrading enzyme antibody (ADA) in the subject during treatment is less than 1:100,000, 1:50,000, 1:15,000, 1:10,000, 1:4,000, 1:2,000, 1:1,000, 1:800, 1:600, 1:400, 1:200, 1:100, 1:50, 1:20, or 1:10. Optionally, the subject is negative for anti-immunoglobulin G degrading enzyme antibody (ADA) during treatment.

[0023] In an optional implementation, the subject is a human subject; optionally, the subject suffers from an autoantibody-mediated disease; optionally, the autoantibody-mediated disease is anti-GBM disease; optionally, the subject is a highly sensitized patient awaiting kidney transplantation.

[0024] In an optional implementation, the method is used to alleviate the immune rejection reaction of a subject or for desensitization treatment of highly sensitized patients awaiting kidney transplantation, wherein the immune rejection reaction is a transplant-induced rejection reaction.

[0025] In an optional embodiment, the method can reduce the immunoglobulin G level in the subject to about 50%, 40%, 30%, 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or about 0.5% below the level before administration 45 min to 6 h after administration; or, the method can reduce the immunoglobulin G level in the subject to about 5 g / L, about 4 g / L, about 3 g / L, about 2 g / L, about 1 g / L, or about 0.5 g / L below the level after administration 45 min to 6 h after administration.

[0026] In an optional embodiment, the immunosuppressant is selected from glucocorticoids, alkylating agents, nucleotide reductase or tyrosine kinase inhibitors, rapamycin target molecule inhibitors, herbal medicines, calcineurin inhibitors, antimetabolites, protein drugs, or monoclonal antibodies.

[0027] In an optional embodiment, the immunoglobulin G degrading enzyme is selected from IdeS, IdeE, IdeZ, IdeP, IdeSORK, or mutants thereof. The immunoglobulin G degrading enzyme is a low-immunogenic immunoglobulin G degrading enzyme; optionally, the immunogenic immunoglobulin G degrading enzyme has even lower immunogenicity than IdeS.

[0028] In an optional embodiment, the amino acid sequence of the immunoglobulin G degrading enzyme has at least 80%, at least 85%, optionally at least 90%, at least 95%, at least 98%, or at least 99% identity with the amino acid sequences shown in any one of SEQ ID NO:1 to 16. Optionally, the amino acid sequence of the immunoglobulin G degrading enzyme includes the amino acid sequence shown in any one of SEQ ID NO:1 to 17. Optionally, the amino acid sequence of the immunoglobulin G degrading enzyme includes the amino acid sequence shown in any one of SEQ ID NO:1 to 11, 14 to 17. Optionally, the amino acid sequence of the immunoglobulin G degrading enzyme is as shown in SEQ ID NO:2, SEQ ID NO:9, SEQ ID NO:13, or SEQ ID NO:17.

[0029] In optional embodiments, the immunoglobulin G degrading enzyme in each dose may be the same or different. Optionally, the dosage of the immunoglobulin G degrading enzyme in each dose is the same, ranging from about 0.01 mg / kg to about 1.0 mg / kg, for example 0.01 mg / kg, 0.04 mg / kg, 0.12 mg / kg, 0.15 mg / kg, 0.2 mg / kg, 0.25 mg / kg, 0.3 mg / kg, 0.35 mg / kg, 0.4 mg / kg, 0.45 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, and 1.0 mg / kg.

[0030] In an optional embodiment, the dosage of each dose of immunoglobulin G degrading enzyme may be the same or different; optionally, the dosage of at least one dose of immunoglobulin G degrading enzyme is about 100% to about 20% of the dosage of the previous dose of immunoglobulin G degrading enzyme, optionally about 80% to about 40%, optionally about 50% to about 70%, optionally about 60%.

[0031] In an optional embodiment, the dosage of the first dose of immunoglobulin G degrading enzyme is about 0.01 mg / kg to about 1.0 mg / kg; the dosage of subsequent doses of immunoglobulin G degrading enzyme is about 0.1 mg / kg to about 0.25 mg / kg; optionally, the dosage of the first dose is about 0.25 mg / kg, and the dosage of subsequent doses of immunoglobulin G degrading enzyme is about 0.15 mg / kg.

[0032] In an optional embodiment, the administration interval of the immunoglobulin G degrading enzyme is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 25 days, 28 days, 30 days, 35 days, 60 days, 90 days, or 180 days.

[0033] In an optional implementation, the method includes: a) administering a first dose of immunoglobulin G degrading enzyme to the subject on day 1; b) administering another dose of immunoglobulin G degrading enzyme to the subject during days 4 to 10; optionally, the method further includes administering additional doses of immunoglobulin G degrading enzyme to the subject every 3 to 9 days.

[0034] In optional embodiments, the method may or may not involve administering two doses of immunoglobulin G degrading enzyme consecutively within 24 hours. Optionally, the method includes administering another dose of immunoglobulin G degrading enzyme to the subject within 24 hours after administering the first dose. Optionally, the dosage of immunoglobulin G degrading enzyme in each dose may be the same or different; optionally, the dosage of the first dose of immunoglobulin G degrading enzyme is 0.25 mg / kg; the dosage of subsequent doses is 0.15 mg / kg. Optionally, the immunoglobulin G degrading enzyme is the immunoglobulin G degrading enzyme with the amino acid sequence SEQ ID NO:2, SEQ ID NO:9, SEQ ID NO:13, or SEQ ID NO:17; optionally, the immunoglobulin G degrading enzyme is the immunoglobulin G degrading enzyme with the amino acid sequence SEQ ID NO:2.

[0035] In an optional implementation, the immunosuppressant may be administered before, after, or simultaneously with the administration of the immunoglobulin G degrading enzyme, and / or between any two doses of the immunoglobulin G degrading enzyme. For example, the immunosuppressant may be administered one week or three days before the administration of the immunoglobulin G degrading enzyme and may continue until the last dose of the immunoglobulin G degrading enzyme is administered.

[0036] In an optional embodiment, the subject has anti-GBM disease and is receiving treatment with cyclophosphamide and / or glucocorticoids; the method includes: a) a single intravenous infusion of 0.25 mg / kg of IdeE-2 on day 1; b) if the subject's anti-GBM antibody is higher than 20 RU / ml or positive during days 3 to 10, a single intravenous infusion of 0.15 mg / kg of IdeE-2 is administered, otherwise a single intravenous infusion of 0.15 mg / kg of IdeE-2 is administered on day 8; optionally, the glucocorticoid is prednisolone, administered at a dose of 0.25–7.5 mg / kg / day; optionally, the prednisolone dose is 1 mg / kg / day, reduced to 20 mg within 6 weeks, and gradually reduced to discontinue over 6–9 months. Optionally, methylprednisolone 7.5 mg / kg is administered intravenously three times over three consecutive days or every other day, followed by oral glucocorticoids, such as prednisone 40-80 mg, gradually tapering down until discontinuation. Optionally, the glucocorticoid treatment method includes first administering glucocorticoids intravenously, followed by oral glucocorticoid treatment. Optionally, the method of first administering glucocorticoids intravenously includes first administering 0.25-1.0 g of glucocorticoids intravenously. Optionally, the method of first administering glucocorticoids intravenously includes first administering 0.25-1.0 g of glucocorticoids intravenously daily or every other day. Optionally, the method of first administering glucocorticoids intravenously includes first administering 0.25-1.0 g of glucocorticoids intravenously daily or every other day for a total of three days. Optionally, the method of then administering oral glucocorticoids includes gradually reducing the oral dose for a duration of 6-9 months. Optionally, the method of re-administering oral glucocorticoid therapy includes administering 40-80 mg of glucocorticoid daily for the first week, and maintaining the dose at 5-10 mg in week 26. Optionally, the glucocorticoid used in the method of first administering intravenous glucocorticoid therapy followed by oral glucocorticoid therapy is independently selected from prednisolone, prednisone, or methylprednisolone, for example, first administering methylprednisolone intravenously, followed by oral prednisone. Optionally, the method of cyclophosphamide treatment includes oral administration of cyclophosphamide 1-5 mg / kg / day. Optionally, the method of cyclophosphamide treatment includes intravenous administration of 0.5-1.0 g / m². 2 / month; optionally, the treatment method of cyclophosphamide includes intravenous administration of 0.2g / day; optionally, the treatment method of cyclophosphamide includes intravenous administration of 0.2g / week; optionally, the treatment method of cyclophosphamide includes intravenous administration of 0.5g / two weeks; optionally, the treatment duration of cyclophosphamide is 3 months.

[0037] In an optional implementation, the subject has anti-GBM disease, and the method includes: a) a single intravenous infusion of 0.25 mg / kg of IdeE-2 on day 1; b) intravenous infusions of 0.15 mg / kg of IdeE-2 on days 4 and 7.

[0038] In an optional implementation, the subject has anti-GBM disease, and the method includes: a) a single intravenous infusion of 0.25 mg / kg of IdeE-2 on day 1; b) intravenous infusions of 0.15 mg / kg of IdeE-2 on days 4, 7, and 10.

[0039] In an optional embodiment, the subject suffers from anti-GBM disease and is treated with cyclophosphamide and / or glucocorticoids; the method includes: a) a single intravenous infusion of 0.25 mg / kg of IdeE-2 on day 1; b) intravenous infusions of 0.15 mg / kg of IdeE-2 on days 4 and 7; optionally, the glucocorticoid is prednisolone, administered at a dose of 0.5–2.0 mg / kg / day; optionally, the prednisolone dose is 1 mg / kg / day, reduced to 20 mg over 6 weeks, and gradually reduced to discontinue over 6–9 months; optionally, the glucocorticoid treatment method includes intravenous glucocorticoid treatment followed by oral glucocorticoid treatment; optionally, the method of intravenous glucocorticoid treatment includes intravenous glucocorticoid treatment of 0.25–1.0 g; optionally, the method of intravenous glucocorticoid treatment includes intravenous glucocorticoid treatment of 0.25–1.0 g first; Treatment with 25-1.0g; Optionally, the method of first administering intravenous glucocorticoids includes administering 0.25-1.0g of glucocorticoids intravenously daily or every other day for a total of three days; Optionally, the method of then administering oral glucocorticoids includes gradually reducing the oral dose for a duration of 6-9 months; Optionally, the method of then administering oral glucocorticoids includes administering 40-80mg of glucocorticoids daily for the first week and maintaining the dose at 5-10mg in the 26th week; Optionally, the glucocorticoids used in the method of first administering intravenous glucocorticoids and then administering oral glucocorticoids are independently selected from prednisolone, prednisone, or methylprednisolone, for example, administering methylprednisolone intravenously first and then administering prednisone orally; Optionally, the method of cyclophosphamide treatment includes oral administration of cyclophosphamide 1-5mg / kg / day; Optionally, the method of cyclophosphamide treatment includes intravenous administration of 0.5-1.0g / m 2 / month; optionally, the treatment method of cyclophosphamide includes intravenous administration of 0.2g / day; optionally, the treatment method of cyclophosphamide includes intravenous administration of 0.2g / week; optionally, the treatment method of cyclophosphamide includes intravenous administration of 0.5g / two weeks; optionally, the treatment duration of cyclophosphamide is 3 months.

[0040] In an optional embodiment, the subject has anti-GBM disease and is receiving treatment with cyclophosphamide and / or glucocorticoids; the method includes: a) a single intravenous infusion of 0.25 mg / kg of IdeE-2 on day 1; b) intravenous infusions of 0.15 mg / kg of IdeE-2 on days 4, 7, and 10; optionally, the glucocorticoid is prednisolone, administered at a dose of 0.5–2.0 mg / kg / day; optionally, the glucocorticoid treatment method includes first administering glucocorticoids intravenously, followed by oral glucocorticoid treatment; optionally, the method of first administering glucocorticoids intravenously includes first administering 0.25–1.0 g of glucocorticoids intravenously; optionally, the method of first administering glucocorticoids intravenously includes first administering 0.25–1.0 g of glucocorticoids intravenously daily or every other day; optionally, the method of first administering intravenous... The methods of glucocorticoid therapy include: first, administering 0.25–1.0 g of glucocorticoids intravenously daily or every other day for a total of three days; optionally, the method of subsequent oral glucocorticoid therapy includes gradually reducing the oral dose for a duration of 6–9 months; optionally, the method of subsequent oral glucocorticoid therapy includes administering 40–80 mg of glucocorticoids daily for the first week and maintaining the dose at 5–10 mg in the 26th week; optionally, the glucocorticoids used in the initial intravenous glucocorticoid therapy followed by oral glucocorticoid therapy are independently selected from prednisolone, prednisone, or methylprednisolone, for example, administering methylprednisolone intravenously first, followed by oral prednisone; optionally, the method of cyclophosphamide therapy includes oral administration of cyclophosphamide 1–5 mg / kg / day; optionally, the method of cyclophosphamide therapy includes intravenous administration of 0.5–1.0 g / m². 2 / month; optionally, the treatment method of cyclophosphamide includes intravenous administration of 0.2g / day; optionally, the treatment method of cyclophosphamide includes intravenous administration of 0.2g / week; optionally, the treatment method of cyclophosphamide includes intravenous administration of 0.5g / two weeks; optionally, the treatment duration of cyclophosphamide is 3 months.

[0041] In an optional embodiment, the subject is a highly sensitized kidney transplant patient who first receives anti-CD20 and / or anti-CD38 antibody treatment, followed by combination therapy with calcineurin inhibitors, antimetabolites, and glucocorticoids; optionally, the anti-CD20 antibody is rituximab, repertuzumab, or octotuzumab; and the anti-CD38 antibody is daratumumab or esartuximab.

[0042] In an optional embodiment, the subject is a patient intended to receive AAV gene therapy; optionally, the patient has pre-existing AAV neutralizing antibodies. Optionally, the serotype of the AAV gene therapy vector is selected from one or more of AAV1, AAV2, AAV3, AAV3B, AAV3S, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, and AAV-rh74; for example, the serotype of the AVV gene therapy vector is AAV2 or AAV8. Optionally, the AAV gene therapy may use engineered capsids, self-complementary, chimeric, or CRISPR-AAV type viral vectors. Optionally, the subject is given two doses of immunoglobulin G degrading enzyme; and the interval between the second dose of immunoglobulin G degrading enzyme and the previous dose is 1 to 10 days, for example, 2 to 7 days, or 2 to 4 days, or 3 days. Optionally, the method can reduce the AAV neutralizing antibody titer in the patient to below 50%, 40%, 30%, 20%, or 10%.

[0043] The second aspect of this disclosure provides an immunoglobulin G degrading enzyme for reducing immunoglobulin G levels in a subject, the method for reducing immunoglobulin G levels in a subject being as described in the first aspect of this disclosure.

[0044] The third aspect of this disclosure provides the use of an immunoglobulin G degrading enzyme in the preparation of a medicament for reducing immunoglobulin G levels in a subject, the method for reducing immunoglobulin G levels in a subject being as described in the first aspect of this disclosure.

[0045] This disclosure provides a fourth aspect of a pharmaceutical composition comprising an immunoglobulin G degrading enzyme sufficient to administer the method of reducing immunoglobulin G levels in a subject as described in the first aspect of this disclosure, and pharmaceutically acceptable excipients.

[0046] The fifth aspect of this disclosure provides a pharmaceutical combination comprising at least one immunoglobulin G degrading enzyme sufficient to administer the method for reducing immunoglobulin G levels in a subject as described in the first aspect of this disclosure, and at least one immunosuppressant.

[0047] In optional embodiments, the immunoglobulin G degrading enzyme described in any of the foregoing embodiments is selected from IdeS, IdeE, IdeZ, IdeP, IdeSORK, or mutants thereof; optionally, the immunoglobulin G degrading enzyme is selected from the immunoglobulin G degrading enzymes shown in any of the amino acid sequences SEQ ID NO:1 to 16; optionally, the immunoglobulin G degrading enzyme is selected from the immunoglobulin G degrading enzymes shown in the amino acid sequences SEQ ID NO:2, 9, or 13; optionally, the immunoglobulin G degrading enzyme has one or more unit doses; optionally, one unit dose of the immunoglobulin G degrading enzyme is about 8 mg to about 100 mg, optionally about 10 mg to about 80 mg, optionally about 8 mg to about 40 mg, optionally about 10 mg to about 25 mg, optionally about 10 mg to about 15 mg; optionally, another unit dose of the immunoglobulin G degrading enzyme is about 5 mg to about 20 mg, optionally about 20 mg.

[0048] This disclosure successfully reduced the levels of pathogenic IgG antibodies, neutralizing antibodies, or anti-drug antibodies in subjects by adjusting the administration method and / or selecting immunoglobulin G degrading enzymes with low immunogenicity, making repeated administration of IgG degrading enzymes possible. Attached Figure Description

[0049] Figure 1 shows the electrophoresis results of the expression products in the shake-flask fermentation supernatant of the IdeE-1 expression strain in Example 1;

[0050] Figure 2 shows the electrophoresis results of the expression product of the fermentation cell lysate of IdeE-2 expression in Example 2;

[0051] Figure 3a shows the OD values ​​of the IdeE-1 expression strain at different induction times during fermentation in a 5L fermenter. 600 Test results;

[0052] Figure 3b shows the OD values ​​of the IdeE-2 expression strain at different induction times during fermentation in a 5L fermenter. 600 Test results;

[0053] Figure 3c shows the OD values ​​of the IdeE-1 expression strain at different induction times during fermentation in a 5L fermenter. 600 Results of the detection of changes in turbidity in the fermentation supernatant;

[0054] Figure 4 shows the electrophoretic comparison results of the expression products of IdeE-1 and IdeE-2 strains after fermentation in a 5L fermenter at different induction times;

[0055] Figure 5 shows the enzymatic cleavage effect of IdeE-1 intravenous infusion on IgG in New Zealand rabbits in Example 4;

[0056] Figure 6 shows the mean blood concentration-time curves of IgG in different groups of subjects in Example 5; Figures 6A and 6B are the mean blood concentration-48 hours (h) curves of IgG in clinical subjects in China and New Zealand, respectively; Figures 6C and 6D are the mean blood concentration-63 days (D) curves of IgG in clinical subjects in China and New Zealand, respectively.

[0057] Figure 7 shows the ADA titers of subjects before IdeE-2 and IdeS administration in Example 5;

[0058] Figure 8a shows the trend of ADA changes in Chinese IdeE-2 subjects in Example 5;

[0059] Figure 8b shows the trend of ADA changes in New Zealand IdeE-2 subjects in Example 5;

[0060] Figure 9 shows the changes in neutralizing antibodies in different serotypes after administration to the subjects in Example 8;

[0061] Figure 10 shows the change in IdeE-2 antibody titer against AAV2 over time in the high-titer group (>1:1000) and medium-titer group (1:100 to 1:1000) in Example 8.

[0062] Figure 11 shows the changes in AAV neutralizing antibody, IgG and F(ab')2 in subjects after administration of IdeE-2 in Example 8;

[0063] Figure 12 shows the antibody titer ratio of AAV8 neutralizing antibody in the serum of subjects in Example 8 after administration of 0.4 mg / kg IdeE-2;

[0064] Figure 13 shows the changes in neutralizing antibodies against AVV2 and AAV8 in subjects after administration of IdE-2 in Example 8;

[0065] Figure 14 shows the ADA titer detection results of the subjects in Example 6 after administration of IdeE-2;

[0066] Figure 15 shows the ADA titer test results after Imlifidase administration, with each line representing one patient;

[0067] Figure 16 shows the detection results of IgG concentration in the subjects during the two-dose regimen in Example 7;

[0068] Figure 17 shows the results of detecting the average IgG concentration in the subjects during the two-dose regimen in Example 7;

[0069] Figure 18 shows the detection results of ADA titer in the subjects during the two-dose regimen in Example 7;

[0070] Figure 19 shows the detection results of anti-GBM antibody concentration in the subjects during the two-dose regimen in Example 7;

[0071] Figure 20 shows the detection results of IgG concentration in the subjects during the three-dose regimen in Example 9;

[0072] Figure 21 shows the results of detecting the average IgG concentration in the subjects during the three-dose regimen in Example 9;

[0073] Figure 22 shows the detection results of ADA titer in subjects in the three-dose regimen of Example 9;

[0074] Figure 23 shows the detection results of anti-GBM antibody concentration in the subjects during the three-dose regimen in Example 9;

[0075] Figure 24 shows the changes in ADA (indicated by SNR values) in animals under the regimens of IdeE-2 and HIGES2 alone and in combination with immunosuppressive drugs in Example 13. Detailed Implementation

[0076] Based on the above content of this disclosure, and in accordance with common technical knowledge and practices in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical ideas of this disclosure.

[0077] I. Definition

[0078] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0079] The terms “subject” and “patient” are used interchangeably and generally refer to humans.

[0080] The term "immunoglobulin G degrading enzyme" or "IgG degrading enzyme" refers to a functional polypeptide that possesses immunoglobulin G degrading enzyme activity. Common immunoglobulin G degrading enzymes are cysteine ​​proteases derived from Streptococcus, including but not limited to IdeE (Streptococcus Equi), IdeZ (Streptococcus zooepidemicus), IdeP, and IdeS (Streptococcus pyogenes). Without limitation, the immunoglobulin G degrading enzymes disclosed herein also include engineered IdeE, IdeZ, IdeP, IdeS, or mutants thereof. Known IgG degrading enzymes include those from the literature (Lannergard, J. and B. Guss. 2006. FEMS Microbiol Lett 262(2):230-235; Hulting, G. et al., (2009). FEMS Microbiol Lett 298(1):44-50), as well as immunoglobulin degrading enzymes disclosed in WO2016128559A1, WO2016128558A1, WO2022223818A1, WO2021254479A1, WO2023116817A1, and all enzymes and their variants that have the aforementioned immunoglobulin-cleaving activities. Variants of these known enzymes can also perform the same function. Optionally, immunoglobulin G degrading enzymes may have low immunogenicity, for example, in the absence or low presence of pre-existing antibodies in the subject. Optionally, low-immunogenic immunoglobulin G degrading enzymes include IdeE, IdeZ, IdeP, and their mutants, such as the immunoglobulin G degrading enzymes shown in sequences 1–17. Optionally, low-immunogenic immunoglobulin G degrading enzymes may be derived from patents WO2024036324A1, CN115698279A, CN115698279A, WO2025030053A1, US20250027068A1, or WO2024206953A2. The term "IdeS" or "IDES" refers to a cysteine ​​protease derived from Streptococcus pyogenes that has the ability to specifically cleave immunoglobulin G (IgG). Without limitation, the IdeS described in this disclosure refers to wild-type IdeS or its mutants. An exemplary IdeS is imlifidase (Idefirix), whose amino acid sequence is shown in SEQ ID NO:13.

[0081] The term "IdeE" or "IdeS" comes from *Streptococcus equi* ssp. equi, a pathogenic bacterium in horses, and shares approximately 70% sequence homology with IdeS. Both IdeE and IdeS enzymes cleave IgG at exactly the same location, exhibiting highly reproducible and specific cleavage, and have very similar substrate ranges.

[0082] The term "mutant" includes amino acid insertion mutants, amino acid addition mutants, amino acid deletion mutants, and / or amino acid substitution mutants. The variant sequence may have at least 80%, at least 85%, preferably at least 90%, at least 95%, at least 98%, or at least 99% identity with the template sequence.

[0083] Amino acid identity can be calculated using any suitable algorithm. For example, the PILEUP and BLAST algorithms can be used to calculate identity or alignment sequences (such as identifying equivalent or corresponding sequences, typically at their default settings). Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (http: / / www.ncbi.nim.nih.gov / ). The algorithm involves first identifying high-scoring sequence pairs (HSPs) by recognizing short characters of length W in the query sequence that match or satisfy some positive threshold score T when aligned with characters of the same length in the database sequence. T is called the neighborhood word score threshold. These initial neighboring words serve as seeds to initiate a search to find HSPs containing them. As long as the cumulative alignment score can be increased, the number of hit characters (words) will increase. The hit character expands in both directions along each sequence. Expansion in each direction stops when: the cumulative alignment score decreases by an amount X from its maximum realized value; the cumulative score reaches zero or below due to the accumulation of one or more negative score residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses a word length (W) of 11, a BLOSUM62 score matrix alignment (B) of 50, an expected value (E) of 10, M=5, N=4, and two-strand comparisons as default values.

[0084] The BLAST algorithm performs a statistical analysis of the similarity between two sequences. One similarity metric provided by the BLAST algorithm is the minimum sum probability (P(N)), which indicates the probability that a match will occur accidentally between two polynucleotide or amino acid sequences. For example, a sequence is considered similar to another sequence if the minimum sum probability of comparing the first sequence with the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. Alternatively, the UWGCG software package provides the BESTFIT procedure (e.g., on its default settings) for calculating identity.

[0085] Optionally, "identity" can mean that two peptide sequences share at least 70%, 75%, or 80% sequence identity when optimally aligned using, for example, a GAP or BESTFIT procedure, preferably at least 90% or 95% sequence identity, more preferably at least 97%, 98%, or 99% sequence identity, using the default gap weight parameters provided by the procedure. In some embodiments, the positional differences of different residues are due to conserved amino acid substitutions. A "conserved amino acid substitution" refers to replacing or substituting one amino acid residue with an amino acid residue having similar chemical properties (e.g., electrochemical or hydrophobic properties). Typically, conserved amino acid substitutions do not significantly alter the functional properties of a protein. In cases where two or more amino acid sequences differ from each other due to conserved substitutions, the percentage of sequence identity can be adjusted upwards to correct for the conservatism of the substitution. Such adjustments are well known to those skilled in the art. See, for example, Pearson, Methods Mol. Biol. 243:307-31 (1994). Examples of amino acid groups with chemically similar side chains include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aryl side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Conserved amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0086] The term "treatment" includes the suppression, relief, prevention, or elimination of one or more symptoms or side effects associated with the disease, condition, or disorder being treated. The term "effective dose" or "therapeutic effective dose" refers to a dose sufficient to treat, suppress, or reduce one or more symptoms of the treated disease state or otherwise provide the desired pharmacological and / or physiological effect. Precise dosages will vary depending on a variety of factors, such as subject-dependent variables (e.g., age, immune system health, etc.), the disease or disorder, and the treatment administered. The effect of an effective dose can be relative to a control. These controls are known in the art and discussed herein, and can be, for example, the condition of the subject before or without administration of the drug or combination of drugs, or, in the case of a combination of drugs, the combined effect can be compared to the effect of administration of only one drug.

[0087] The term "pharmaceutical composition" means a composition comprising the therapeutic agents described in this disclosure, such as immunoglobulin G degrading enzymes, and at least one pharmaceutically acceptable ingredient selected from the following, depending on the manner of administration and the nature of the dosage form: including, but not limited to, carriers, diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, thermosensitive materials, temperature regulators, adhesives, stabilizers, suspending agents, etc. Without limitation, the pharmaceutical composition may also comprise a second therapeutic agent.

[0088] The term "immunogenicity" refers to the property of an antigen to elicit an immune response; that is, the ability of an antigen to stimulate specific immune cells, causing them to activate, proliferate, and differentiate, ultimately producing immune effector substances such as antibodies and sensitized lymphocytes. Low immunogenicity refers to an antigen eliciting a weak immune response in the immune system, making it difficult to trigger an effective immune reaction, or the antigen having a low pre-existing antibody level.

[0089] The term "autoantibody-mediated disease" refers to a group of illnesses caused by the immune system mistakenly attacking the body's own tissues; these diseases are collectively known as autoimmune diseases (AD). Autoimmune diseases are characterized by the presence of antibodies against the body's own components. These antibodies can cause damage or dysfunction of organs and tissues; these antibodies are also called "pathogenic IgG antibodies." The level of pathogenic IgG antibodies can usually be detected by methods such as ELISA or SDS-PAGE. (Kühnl, Alexander et al. "Serodiagnosis of Anti-glomerular Basement Membrane Disease Using a Newly Developed Chemiluminescence Immunoassay." Frontiers in Medicine vol.9 915754.4 Jul.2022).

[0090] The term "immune rejection" refers to the body's immune defense response to foreign objects or abnormal cells, primarily carried out by the immune system. In this process, the immune system recognizes and attacks tissues perceived as foreign objects or abnormal cells to maintain a stable physiological state within the body. Immune rejection is a common problem in organ, tissue, and cell transplantation, involving immune incompatibility between the donor and recipient. This reaction can lead to transplant failure.

[0091] The term "hypersensitization" refers to the presence of high levels of anti-human leukocyte antigen (HLA) antibodies in a patient's body, typically manifested as a calculated population reactive antibody (cPRA) level ≥80% or a PRA ≥80%. Hypersensitization is often caused by sensitization events such as multiple blood transfusions, pregnancy, or previous transplantation, significantly increasing the risk of antibody-mediated rejection (AMR) after kidney transplantation and reducing transplant success rates.

[0092] The term "desensitization therapy" refers to a treatment strategy for highly sensitized patients. It aims to reduce the risk of antibody-mediated rejection (AMR) after transplantation by lowering the level of anti-HLA antibodies in the body, thereby improving the success rate of kidney transplantation. Desensitization therapy typically includes methods such as plasma exchange, immunoadsorption, intravenous immunoglobulin (IVIG), and rituximab, which can be used alone or in combination to clear circulating antibodies, inhibit antibody production, and improve donor-recipient matching.

[0093] The term "approximately" refers to a given number within a range of plus or minus 1% to 10%.

[0094] The term "dosing interval" refers to the time interval between two administrations. Dosing intervals can be measured in days or hours. Without limitation, when the dosing interval is measured in days, only the date difference between administrations is calculated, not the specific time difference. For example, a 1-day interval means administration on D1 and D3 respectively. In this disclosure, the dosing interval can be determined based on the level of pathogenic IgG antibodies in the patient's body. For example, if the level of pathogenic IgG antibodies in the patient's body is greater than a certain risk value, the next dose of IgG degrading enzyme can be administered. For example, for a patient clinically diagnosed with anti-GBM disease, the next dose of IgG degrading enzyme can be administered after a positive anti-GBM antibody test or a level higher than approximately 15 RU / ml, approximately 20 RU / ml, or approximately 30 RU / ml.

[0095] The term "drug combination" or "drug composition" includes drug packets, drug boxes, or combination drugs. Taking a two-component drug as an example, components A and B can be mixed together to form a single dosing unit, or they can be separate dosing units. A and B can be used together as a single drug, for example, having the same marketing authorization, or they can be used separately as independent drugs, for example, having different marketing authorizations. The two components can be used together, simultaneously, continuously, or separately. Unrestricted, the drug combinations disclosed herein comprise two identical or different IgG degrading enzymes, for example, the unit dose of the first dose of IgG degrading enzyme is about 8 mg to about 40 mg, optionally about 8 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 18 mg, 20 mg, 22 mg, 24 mg, 26 mg, 28 mg, 30 mg, 32 mg, 34 mg, 36 mg, 38 mg or 40 mg; for example, the unit dose of the second dose of IgG degrading enzyme is about 5 mg to about 20 mg, optionally about 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg or 20 mg.

[0096] The pharmaceutical compositions disclosed herein comprise IgG degrading enzymes and one or more other therapeutic immunosuppressant drugs.

[0097] Immunosuppressants are drugs that inhibit the body's immune response. They suppress the proliferation and function of cells involved in the immune response (such as T cells, B cells, and macrophages), thereby reducing antibody-mediated immune responses and inducing immune tolerance. These include glucocorticoids, alkylating agents, antimetabolites, nucleotide reductase or tyrosine kinase inhibitors, herbal medicines, calcineurin inhibitors, rapamycin target molecule inhibitors, protein drugs, and monoclonal antibodies.

[0098] The glucocorticoids include prednisone, prednisolone, methylprednisolone, methylprednisolone, or dexamethasone; the alkylating agents include cyclophosphamide, busulfan, thiotepa, or chlorambucil; the antimetabolites include mycophenolate mofetil, methotrexate, mycophenolate mofetil, azathioprine, mercaptopurine, or imidazolidin; the nucleotide reductase or tyrosine kinase inhibitors include hydroxyurea, penicillamine, or leflunomide; the herbal medicines mainly include Tripterygium wilfordii glycosides or total glucosides of paeony; calcineurin inhibitors such as cyclosporine, tacrolimus, or mycophenolate mofetil; rapamycin target molecule inhibitors such as rapamycin, sirolimus, or guanitolimus; and the protein drugs include complement inhibitors, cytokine inhibitors, innate immunosuppressants, immune cell and plasma cell inhibitors, or tolerance inducers. Further protein-based drugs include antibodies against CD28, CD40, CD154, CD20, CD38, CD19, CD27, TNF-α, and IL-6. More specifically, the antibodies are rituximab, ofatumumab, ocrelizumab, obinutuzumab, ublituximab, zuberitamab, ribertamab, iributumomab tiuxetan, MIL62, repertoire, daratumumab, isatuximab, felzartamab, mezagitamab, basiliximab, TAK-079, CM313, SC301SC, infliximab, adalimumab, cetozumab, golimumab, tocilizumab, or clazakizumab.

[0099] In some aspects of this disclosure, the immunosuppressants also include fingolimod, thalidomide, pomalidomide, mycophenolic acid analogues, etanercept, recombinant human type II tumor necrosis factor receptor-antibody fusion protein, anti-human thymocyte immunoglobulin, anti-lymphocyte globulin, i.e., anti-lymphocyte immunoglobulin ALG, tofacitinib, beracide, baricitinib, ruxolitinib, or everolimus.

[0100] The immunosuppressants applicable to this disclosure can be determined by a physician based on the patient's clinical symptoms. Unrestricted use of glucocorticoids and cyclophosphamide is common in anti-GBM treatment. The recommended dose of cyclophosphamide is 2–3 mg / kg / day orally for 2–3 months, with the dose reduced to 2 mg / kg for patients >55 years of age. The recommended glucocorticoid is prednisolone 1 mg / kg / day orally (maximum dose 60 mg), reduced to 20 mg within 6 weeks, and gradually tapered off over 6–9 months until discontinuation. Kidney transplantation typically includes at least immune induction therapy and immune maintenance therapy. Commonly used immune inducing agents include IL-2RA and lymphocyte-clearing antibodies. Lymphocyte-clearing antibodies include ATG and anti-human T lymphocyte immunoglobulin (ALG). The former includes rabbit anti-human thymocyte immunoglobulin (rATG), and the latter includes rabbit anti-human T cell immunoglobulin (ATG-F). Commonly used immunosuppressive regimens generally employ a triple immunosuppressive regimen consisting of CNI (combined immunosorbent inhibitors) + antiproliferative drugs + hormones. CNIs primarily include tacrolimus and cyclosporine; commonly used MPA (myocophenolate mofetil, MMF) and mycophenolate sodium (MPS) in China are also commonly used. MPS is available in enteric-coated mycophenolate sodium (EC-MPS). Other immunosuppressive therapy regimens include CNI + mizoribine + glucocorticoids, and CNI + mTORi + glucocorticoids. In cases of high risk of acute transplant renal failure, adjuvant therapy may be added, including: anti-CD20 monoclonal antibodies (such as rituximab), anti-plasma cell activity agents (proteasome inhibitors: bortezomib; anti-CD38 monoclonal antibodies: datumumab), terminal complement inhibitors (anti-C5 monoclonal antibodies: eculizumab), and anti-IL-6 / anti-IL-6R monoclonal antibodies (such as tocilizumab).

[0101] The term "anti-drug antibodies (ADAs)" refers to antibodies produced by the human immune system in response to exogenous drugs (such as biologics). These antibodies may affect the efficacy and safety of the drug.

[0102] The term "pre-existing antibody" refers to a component of the host that has been previously exposed to a foreign substance (such as a biological drug) and the long-lasting memory immune response that may result from the initial exposure.

[0103] The term "total binding antibody" (Tab) refers to antibodies that can bind to pathogens, including neutralizing antibodies (NAb) and non-neutralizing antibodies (Non-NAb).

[0104] The term "neutralizing antibody" refers to an antibody with antiviral activity that recognizes viral surface proteins and blocks the binding of the virus to specific receptors on the cell surface. It binds to its target and counteracts downstream cellular effects, such as cell proliferation or chemotaxis.

[0105] There are many methods for detecting ADA. These methods include, but are not limited to: a) immunoassays based on bridging ELISA; b) highly sensitive binding assays based on electrochemiluminescence (ECL) or surface plasmon resonance (SPR); c) neutralizing antibody function assays evaluated using reporter gene assays (RGA) or competitive ELISA; d) and supplementary validation methods such as liquid chromatography-mass spectrometry (LC-MS) or radioimmunoassay (RIA); e) other novel detection methods, such as ImmunoCAP, Winstedt, Lena et al. “Complete Removal of Extracellular IgG Antibodies in a Randomized Dose-Escalation Phase I Study with the Bacterial Enzyme IdeS--A Novel Therapeutic Opportunity.” PLOS 1 vol.10, 7 e013 2011.15 Jul.2015.

[0106] Autoimmune diseases are disease states caused by an immune response of the body's own immune system against its own components. While an immune response to foreign antigens usually results in the clearance of those antigens, an immune response to antigens affecting one's own cells or tissues is not easily and completely cleared by the immune system's effector cells; instead, these cells or tissues are continuously attacked, leading to a disease state. Autoimmune diseases are classified into organ-specific autoimmune diseases and systemic autoimmune diseases.

[0107] Organ-specific autoimmune diseases refer to diseases in which the lesions are generally limited to a specific organ and are caused by an autoimmune reaction against target antigens of that specific organ. In addition, certain autoantibodies can cause organ-specific functional disorders by overstimulating or inhibiting the normal function of the target organ. These include chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, chronic ulcerative colitis, pernicious anemia with chronic atrophic gastritis, goodpasture syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple cerebral sclerosis, and acute idiopathic polyneuritis, among others. Common examples will be described in the sections on diseases of each system.

[0108] Systemic autoimmune diseases, also known as generalized autoimmune disorders, are caused by autoimmune reactions against target antigens in multiple organs and tissues. The lesions can occur in various organs and tissues, with a wide distribution, including the skin, kidneys, and joints, manifesting as a variety of related clinical signs and symptoms. Common systemic autoimmune diseases include ankylosing spondylitis, cryoglobulinemia, rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis.

[0109] The autoantibody-mediated conditions are autoimmune diseases and / or diseases or conditions mediated by pathogenic antibodies.

[0110] 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 antibody-mediated rejection (AMR), Addison's disease, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, aplastic anemia, anti-GBM disease, anti-NMDAR encephalitis, antiphospholipid syndrome, autoimmune gastritis, autoimmune deafness, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hypoparathyroidism, autoimmune hypophysitis, and autoimmune diseases. Immune inner ear disorders, 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 neutrophil leukemia Cytopenia, Acquired bullous epidermolysis, 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 bullous epidermolysis, Pemphigus foliaceus, Pemphigus vulgaris, Pernicious anemia, and other related conditions. Jointed polyarteritis, 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.

[0111] 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, host versus graft reaction is the primary reaction, while graft versus host reaction is rare. In bone marrow transplantation, graft versus host reaction is the most common.

[0112] 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 involves activated B cells differentiating into plasma cells (terminally differentiated antibody-secreting cells), which produce large amounts of specific antibodies (such as IgG) to neutralize pathogens or promote their clearance.

[0113] Diseases or conditions mediated by pathogenic antibodies also include hyperglobulinemia. The hyperglobulins are produced by leukocytes, selected from B cells and abnormal B cells; the globulins include gamma globulins; and the hyperglobulinemia includes primary monoclonal gammopathy, connective tissue diseases, liver diseases, infectious diseases, sarcoidosis, myasthenia gravis (MG), 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 conditions such as leukemia, macroglobulinemia, secondary monoclonal gammopathy (e.g., non-lymphoreticular system tumors, monocytic leukemia, cryoglobulinemia), 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).

[0114] For these indications, the appropriate dosage will, of course, vary depending on, for example, the specific IdeE-2 of this disclosure to be used, the host, the route of administration, and the nature and severity of the condition being treated. Typically, dosages range from 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. The IdeE-2 of this disclosure can be administered via intravenous infusion, intraperitoneal injection, intramuscular injection, joint injection, intradermal injection, or subcutaneous injection, optionally via intravenous infusion.

[0115] One aspect of this disclosure is a method for reducing levels of autoantibodies, pre-existing antibodies, donor-specific antibodies (DSA), and drug-resistant antibodies (ADA), the method comprising administering multiple doses of an effective amount of IdeE-2 to a patient in need. Optionally, multiple administrations of IdeE-2 can be made when drug-resistant antibodies are negative or below a certain limit. Dosing intervals can be 2 times / day, 1 time / day, 1 time / 2 days, 1 time / 3 days, 1 time / 4 days, 1 time / 5 days, 1 time / 6 days, 1 time / 7 days, 1 time / 2 weeks, 1 time / 2 weeks, 1 time / 3 weeks, or 1 time / 4 weeks.

[0116] The IdeE-2 disclosed herein can be repeatedly administered at intervals of at least 30 minutes, or at least 1 hour, or at least 2 hours, or at least 3 hours, or at least 4 hours, or at least 5 hours, or at least 6 hours, or at least 12 hours, or at least 24 hours, or at least 2 days, or at least 3 days, or at least 4 days, or at least 5 days, or at least 6 days, or at least 7 days, or at least 10 days, or at least 15 days, or at least 20 days, or at least 30 days; and for a maximum of 12 months, or at most 10 months, or at most 8 months, or at most 6 months, or at most 4 months, or at most 2 months, or at most 35 days, or at most 28 days, or at most 21 days, or at most 18 days, or at most 14 days, or at most 13 days, or at most 12 days, or at most 11 days, or at most 10 days, or at most 9 days, or at most 8 days, or at most 7 days, or at most 6 days, or at most 5 days, or at most 4 days, or at most 3 days, or at most 2 days.

[0117] Another aspect of this disclosure is that IdeE-2 can be combined with various treatment methods such as plasma exchange, hemodialysis, immunosuppressant therapy, and AAV gene therapy for treatment and administration.

[0118] Furthermore, the immunosuppressant or AAV gene therapy drug can be administered before, during, or after IdeE-2 administration, with the specific timing of administration determined by an experienced physician.

[0119] Repeated administration of IdeE-2 and the dosage depend on the treatment regimen of the patient to be treated (age, weight, treatment history, etc.), which can be determined by a skilled physician. In one aspect of this disclosure, IdeE-2 is administered to the patient via an intermittent schedule, such as intravenous infusion, intraperitoneal injection, intramuscular injection, joint injection, intradermal injection, or subcutaneous injection, with intervals between administrations ranging from 0 to 14 days, which can be extended to 5 weeks depending on tolerance to previous administrations. Like cyclophosphamide and glucocorticoids, it can be administered daily throughout the treatment period, for example, in a once-daily dose. For example, for patients who are to receive or have already received AAV gene therapy, and who have pre-existing AAV neutralizing antibodies in their bodies, IdeE-2 can be administered twice, with an interval of 2 to 3 days between the two administrations. The effective dose for the two administrations is 0.01 mg / kg to 5 mg / kg, including but not limited to 0.01 mg / kg, 0.04 mg / kg, 0.12 mg / kg, 0.25 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, and 5 mg / kg. The serotype of the AAV gene therapy vector can be one or more of AAV1, AAV2, AAV3, AAV3B, AAV3S, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, and AAV-rh74; for example, the serotype of the AAV gene therapy vector is AAV2 or AAV8; optionally, the AAV gene therapy can use engineered capsids, self-complementary, chimeric, or CRISPR-AAV type viral vectors. Optionally, the subject is given two doses of immunoglobulin G degrading enzyme; and the interval between the second dose of immunoglobulin G degrading enzyme and the previous dose is 1–10 days, for example, 2–7 days, 2–4 days, or 3 days; optionally, the method can reduce the AAV neutralizing antibody titer in the patient to below 50%, 40%, 30%, 20%, or 10%.

[0120] On the other hand, plasma exchange therapy can be performed during the treatment process, with the timing and frequency determined by a skilled physician.

[0121] Goodpasture disease (GBM) is a rare autoimmune disease that affects glomerular capillaries (leading to glomerular necrosis and crescent formation) and / or pulmonary capillaries (causing alveolar hemorrhage). Most patients present with rapidly progressive (crescentic) glomerulonephritis, but some may experience relatively mild kidney damage. This disease often causes severe kidney injury and, if left untreated, rapidly progresses to end-stage renal disease (ESKD).

[0122] In some aspects of this disclosure, the diagnostic methods for the patients to be treated are those selected by the local medical institution, including but not limited to kidney biopsy and serological testing, and may further include indirect immunofluorescence assay, ELISA, chemiluminescence, radioimmunoassay, multiplex microbead assay, Western blotting, and biosensor systems.

[0123] The method for detecting anti-GBM antibodies can be determined by the local medical institution. The ELISA method can be used to detect serum anti-GBM antibodies at levels of 5RU / ml, 10RU / ml, 20RU / ml, 30RU / ml, 40RU / ml, or 50RU / ml. The detection limit can be set to above 20RU / ml.

[0124] II. Examples

[0125] The present disclosure is further illustrated below with reference to embodiments. The description of specific exemplary embodiments of the present disclosure is for illustrative and explanatory purposes. These descriptions are not intended to limit the present disclosure to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the teachings of this specification. The exemplary embodiments were chosen and described in order to explain the specific principles of the present disclosure and their practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the present disclosure, as well as various different choices and variations.

[0126] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0127] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0128] Example 1. Construction of IdeE-1 expression strain

[0129] The polynucleotide sequence of the IdeE-1 protein (SEQ ID NO:1) was synthesized after codon optimization, and an N-terminal signal peptide sequence and a C-terminal 6×histidine tag were added. The sequence was then inserted into the pET32a expression vector. After successful sequencing, a recombinant plasmid for expressing the mutant IdeE 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. The plates were incubated overnight at 37°C until colonies appeared. A single colony was picked and inoculated into 3 ml of LB medium containing 100 μg / ml ampicillin, and incubated overnight at 37°C and 250 rpm. 500 μl of the overnight culture was inoculated into 50 ml of LB medium containing 100 μg / ml ampicillin, and incubated at 37°C for 4 hours. 0.1 mM IPTG was then added for induction, and the culture was continued overnight. The culture supernatant and bacterial cells were collected by centrifugation after overnight induction. The content of mutant protein IdeE-1 in culture supernatant and bacterial cells was detected by SDS-PAGE, as shown in Figure 1. Lane 1 corresponds to bacterial cells after induction culture, and lane 2 corresponds to supernatant after induction culture. It can be seen that IdeE-1 is expressed in the supernatant.

[0130] Example 2. Construction of IdeE-2 expression strain

[0131] The polynucleotide sequence of the IdeE-2 protein (SEQ ID NO:2) was synthesized after codon optimization and inserted into the pET32a expression vector to obtain a recombinant plasmid for expressing the mutant IdeE. The mutant recombinant plasmid was 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. A single colony was picked and inoculated into 3 ml of LB medium containing 100 μg / ml ampicillin, and incubated overnight at 37°C and 250 rpm. 500 μl of the overnight culture was inoculated into 50 ml of LB medium containing 100 μg / ml ampicillin, incubated at 37°C for 4 hours, and then induced overnight with 0.1 mM IPTG. The bacterial culture that had been induced overnight was centrifuged to collect the cells, which were then disrupted by an ultrasonic cell disruptor. The content of the protein IdeE in the pre-disruption cells, the precipitate after cell disruption, and the supernatant after cell disruption was detected by SDS-PAGE, as shown in Figure 2. In the figure, lane 1 represents the cells before cell disruption after induction culture, lane 2 represents the precipitate after cell disruption after induction culture, and lane 3 represents the supernatant after cell disruption after induction culture. It can be seen that IdeE-2 is expressed intracellularly.

[0132] Example 3. Fermentation culture of IdeE-1 and IdeE-2 expression strains

[0133] The secretory and intracellular expression strains constructed in Examples 1 and 2 were gradually scaled up to a 5L fermentation tank after shake-flask culture. Fermentation was conducted using a fed-batch and submerged aeration method. Glycerol or glucose was selected as the pre-induction limiting carbon source for controlled feeding, while IPTG / lactose was selected as the inducer and post-induction limiting carbon source for controlled feeding. The fermentation temperature was 37℃, and the pH was controlled to 6.0–7.2 by adding 25% ammonia. The fermentation tank aeration rate was selected to be 0.5–2.0 vvm, and the dissolved oxygen level was maintained at approximately 30% by controlling the feeding rate, stirring rate, and aeration rate. During the fermentation process, samples were taken periodically to measure the OD (oxidative stress). 600 When OD 600 Once a certain value is reached, IPTG / lactose is added for induction, and fermentation is terminated after 8–16 hours of induction culture. The secretory expression strain shows an OD value in the later stages of induction. 600 The turbidity of the bacterial solution gradually decreased (Figure 3c), becoming viscous and increasing significantly. The final fermentation solution was so viscous that effective solid-liquid separation by centrifugation was impossible. This phenomenon is typical of E. coli autolysis, and multiple experiments confirmed that this late-stage autolysis induced by the secretory expression strain was not accidental (Figure 3a). The OD during fermentation of the IdeE-2 expression strain... 600 The protein expression level in the cells continued to rise (Figure 3b), and the level of protein expression in the cells also increased continuously with the induction time. After being removed from the fermentation tank, the bacterial solution was not sticky, and the cells could be harvested by centrifugation. Therefore, it was determined that the secretory expression of the IdeE-1 mutant protein in E. coli would cause cell autolysis, making it impossible to purify from the fermentation supernatant and thus preventing large-scale production.

[0134] Figure 4 shows the protein expression results of the IdeE-1 expression strain in the fermentation supernatant obtained in a 5L tank. M is the marker, lane 1 corresponds to the sample induced for 0 h, lane 2 for 2 h, lane 3 for 4 h, lane 4 for 6 h, and lane 5 for 8 h. Figure 4 shows the protein expression results of the IdeE-2 expression strain after cell disruption in a 5L tank. M is the marker, lane 1 corresponds to the sample induced for 0 h, lane 2 for 2 h, lane 3 for 4 h, lane 4 for 6 h, and lane 5 for 8 h. It can be seen that the expression level of IdeE-2 (amino acid sequence SEQ ID NO:2) is significantly higher than that of IdeE-1 (amino acid sequence SEQ ID NO:1). Furthermore, the strain encoding the protein IdeE-1 by SEQ ID NO:1 is less stable during the production process, and the cells exhibit autolysis (Figure 3a). In addition, the viscosity of the fermentation broth expressing IdeE-1 increases, making it impossible to effectively separate solids and liquids. Ultrafiltration requires a larger volume of liquid exchange and is more difficult, thus making purification more challenging.

[0135] Table 1 Comparison of IdeE-1 and IdeE-2 induced expression results

[0136] Example 4. IdeE-1 cleavage experiment of IgG in New Zealand rabbits

[0137] The activity of IdeE-1 in cleaving IgG in New Zealand rabbits was studied. IdeE-1 was administered intravenously to New Zealand rabbits twice a week at a dose of 2 mg / kg. Serum IgG levels were measured using ELISA or SDS-PAGE to evaluate the enzymatic effect of intravenous IdeE-1 on IgG cleavage in animals.

[0138] The results showed that IdeE-1 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 after approximately 10 days (as shown in Figure 5).

[0139] The data above show that after intravenous infusion of IdeE-1 into New Zealand rabbits, most of the IgG in the rabbits was cleaved by the enzyme at the first blood sampling point after IdeE-1 administration (1 minute before the end of administration). The levels remained low for approximately 1–2 days post-administration, then the content of the enzyme cleavage products gradually returned to pre-administration levels. Approximately 10 days post-administration, the IgG levels in the rabbits returned to normal range.

[0140] Example 5

[0141] The safety, tolerability, pharmacokinetic characteristics, pharmacodynamic characteristics, and immunogenicity of IdeE-2 were evaluated in healthy subjects in China and New Zealand. A total of 68 healthy subjects were included in the study, and their specific groupings are shown in Table 2. All subjects completed the study. The results of the Phase I clinical trials conducted in China and New Zealand were consistent, demonstrating that IdeE-2 has good safety and tolerability in healthy subjects. The pharmacokinetic characteristics of IdeE-2 were significant in each dose group, conforming to a two-compartment model, with an optimal dose-response ratio of 0.25 mg / kg. IdeE-2 can efficiently, rapidly, and specifically cleave human IgG. At a dose of 0.25 mg / kg, IdeE-2 cleaves over 90% of IgG within 45 minutes after administration and maintains a low IgG level (average reduction of over 70%) for one week. The proportion and titer of pre-existing anti-IdeE-2 antibodies were both low, showing a significant clinical advantage compared to similar foreign products like Imlifidase, as shown in Figure 6. This demonstrates that using IdeE-2 can improve clinical safety and efficacy.

[0142] Furthermore, as shown in Figure 7, prior to administration, the number of volunteers in both countries with pre-existing antibodies against IdeE-2 (Figure 7, left panel, sample population used in this clinical trial) was significantly less than the number with pre-existing antibodies against IdeS (Anti-IdeS) (Figure 7, right panel, Phase I clinical trial sample population of Imlifidase). Two weeks and two months after IdeE-2 administration, Chinese volunteers showed a wider range of ADA titer changes than New Zealand volunteers in the 0.25 mg / kg and 0.40 mg / kg dose groups. There were no significant differences in the median and range of ADA titers between the two countries before IdeE-2 administration and at 1 week and 6 months post-administration. Most volunteers in both studies began experiencing ADA changes on day 14 after IdeE-2 administration, peaking at approximately two weeks and then gradually declining. Six months after IdeE-2 administration, 56.86% (29 / 51) of the volunteers' ADA levels returned to baseline (Figures 8a and 8b).

[0143] Table 2 Clinical Trial Design Table

[0144] Example 6

[0145] This study evaluated the efficacy, safety, pharmacokinetic characteristics, and immunogenicity of IdeE-2 for pre-transplant immunosuppression in highly sensitized kidney transplant patients. The primary objective was to assess the preliminary efficacy of IdeE-2 in desensitization therapy for highly sensitized kidney transplant patients awaiting kidney transplantation.

[0146] Treatment regimen: On day 1, patients receive a single intravenous infusion of 0.25 mg / kg IdeE-2, with a body weight dose of 0.25 mg / kg on day 1. This dose can be supplemented with 0.15 mg / kg to bring the total dose to 0.4 mg / kg within 24 hours of the initial administration. Immunosuppressant therapy will be administered according to the clinical practice of each research center. In addition to IdeE-2 desensitization therapy, patients may receive anti-CD20 monoclonal antibody therapy such as rituximab and anti-CD38 monoclonal antibody therapy such as daratumumab. Once HLA levels decrease to transplantable levels after administration, kidney transplantation can be performed immediately. Post-transplantation, oral immunosuppressants are typically administered, generally with the first choice being a triple regimen of calcineurin inhibitor + antimetabolite + glucocorticoid, such as tacrolimus + mycophenolate mofetil + prednisolone acetate tablets. Cyclosporine capsules may be used for some patients with poor glycemic control or according to their preference. Anti-CD38 monoclonal antibodies may be administered on day 5 after IdeE-2 administration.

[0147] Methods for detecting anti-drug antibodies. This experiment uses an ELISA bridging method to determine anti-IdeE-2 antibodies in human serum. In the screening experiment, a certain amount of IdeE-2 antigen is coated on the ELISA plate to capture anti-IdeE-2 antibodies in the serum. The captured antibodies then bind to biotinylated IdeE-2, and subsequently react with Streptavidin-Peroxidase from Streptomyces avidinii. The signal value is positively correlated with the antibody concentration in the sample. Samples with high signal values ​​are considered to have potential anti-IdeE-2 antibodies in their serum. In the confirmatory experiment, IdeE-2 is added externally to inhibit the anti-drug antibodies in the serum. This confirmatory test is performed on samples that are potentially antibody-positive. The higher the signal inhibition rate, the higher the titer of anti-IdeE-2 antibodies in human serum.

[0148] Nine highly sensitized subjects were included, numbered S01–S09. Eight subjects (S02–S09) received 0.25 mg / kg, while one subject (S01) received 0.25 mg / kg followed by a supplemental 0.15 mg / kg within 24 hours. IdeE-2 rapidly and efficiently reduced or eliminated pre-existing HLA-I and HLA-II antibodies after administration, achieving a 100% success rate in desensitization within 24 hours. Three patients who successfully underwent desensitization did not receive a kidney transplant due to the lack of donor kidneys, while six patients who successfully underwent desensitization underwent kidney transplantation (without hyperacute rejection). The survival rate of the six transplant recipients was 100% during the trial, with 100% graft survival at both 2 and 6 months. Serum IgG levels began to recover approximately 3–7 days post-transplant. The proportion and titer of pre-existing anti-IdeE-2 antibodies were low, and no infusion reactions or hypersensitivity reactions occurred among the subjects during the study. Compared with similar foreign drugs like Imlifidase, IdeE-2 has significant clinical advantages, improving clinical safety and efficacy. Safety results showed a good safety profile, with no adverse events related to IdeE-2, no hyperacute rejection in kidney transplant patients, and no antibody-mediated rejection within 6 months.

[0149] In desensitization therapy of highly sensitized kidney transplant patients, the dosage of similar foreign product Imlifidase is 0.25 mg / kg. If desensitization is not successful within 24 hours, a second dose of 0.25 mg / kg can be administered, as shown in Figure 15. Compared with similar foreign products Imlifidase, this product has a lower proportion and titer of pre-existing anti-IdeE-2 antibodies (Figure 14 Uhlin, Fredrik et al. “Endopeptidase Cleavage of Anti-Glomerular Basement Membrane Antibodies in vivo in Severe Kidney Disease: An Open-Label Phase 2a Study.” Journal of the American Society of Nephrology: JASN vol.33,4(2022):829-838.), which has significant clinical advantages and can improve clinical safety and efficacy. At the same dose, IdeE-2 enzyme digestion of IgG has a faster onset of action and digestion rate than Imlifidase, which can create an earlier transplantable window in highly sensitized organ transplant patients, avoiding the situation where organs "wait for others" (Note: organ transplantation can only be performed after successful desensitization treatment).

[0150] Further evaluation of the efficacy of IdeE-2 in treating highly sensitized kidney transplant patients. Dosage regimen: Patients received IdeE-2 at a dose of 0.25 mg / kg. If necessary, a second dose of 0.15 mg / kg IdeE-2 could be administered within 24 hours to bring the total dose to 0.4 mg / kg. Kidney transplantation could proceed if HLA antibody levels decreased within 24 hours of the first IdeE-2 dose. Optionally, tacrolimus plus methylprednisolone could be started during the screening period (around D-35) before the first IdeE-2 dose. Anti-CD20 (<80 kg 200 mg; ≥80 kg 300 mg) or anti-CD38 antibody (<60 kg 400 mg; ≥60 kg 800 mg) could be administered from D-7 to D-3. Rabbit ATG (300 mg) could be administered 24 hours after anti-CD20 / CD38 antibody administration or on D-2. During and after surgery, a triple regimen of tacrolimus + methylprednisolone + prednisolone acetate tablets can be used. Anti-CD38 antibody treatment (400 mg for <60 kg; 800 mg for ≥60 kg) can be administered on day 5 after the first IdeE-2 dose.

[0151] Inclusion criteria: Patients must meet all of the following inclusion criteria to be eligible for enrollment:

[0152] (1) Males or females aged 18-65 (including the cutoff value) at the time of screening;

[0153] (2) During screening, the HLA antibody MFI ≥ 8000 and the calculated population response antibody (cPRA) ≥ 80%;

[0154] (3) ABO-compatible kidney transplant recipients;

[0155] (4) The patient is able to understand and sign the informed consent form.

[0156] Exclusion criteria: Patients meeting any of the following criteria are not eligible for enrollment in this study:

[0157] (1) Has received IdeE-2 or similar product Imlifidase treatment within the past year;

[0158] (2) Had received high-dose IVIG treatment (2g / kg) within 28 days prior to administration;

[0159] (3) Breastfeeding women, pregnant women, patients or their partners who plan to have children or do not wish to take effective contraceptive measures during or within 6 months after the trial;

[0160] (4) HIV-positive patients;

[0161] (5) Patients with active HBV or HCV;

[0162] (6) Patients with CMV or EBV infection (except those whose participation in the trial is deemed not to be affected by the investigator's assessment);

[0163] (7) Patients with active tuberculosis;

[0164] (8) Patients with abnormal laboratory test results that are clinically significant (except those whose participation in the trial is deemed not to be affected by the investigator's assessment);

[0165] (9) Hemoglobin (Hb) < 6.0 g / dL;

[0166] (10) Other serious illnesses requiring treatment and close monitoring, such as heart failure (>NYHA class 3), unstable coronary artery disease, or chronic obstructive pulmonary disease (COPD);

[0167] (11) Patients who are unable to comply with the trial protocol;

[0168] (12) Patients with a history of major thrombotic diseases, active peripheral vascular disease, or confirmed hypercoagulable state;

[0169] (13) Patients currently suffering from malignant tumors or with a history of malignant tumors within the past 5 years or with a history of lymphoma at any time. Patients with a history of squamous or basal cell carcinoma that has been surgically removed or excised by curettage and electrocautery are eligible for this trial;

[0170] (14) If the patient received other investigational drugs (or investigational devices) within 28 days or 5 half-lives prior to screening (if known), whichever is longer;

[0171] (15) History of obvious allergy to IdeE-2 ingredients or any related products (including excipients in the formulation), or severe allergic reaction to any drug (such as angioedema);

[0172] (16) Karnofsky (KPS) functional status score < 70;

[0173] (17) The researchers believe there are other circumstances that make it unsuitable for participation in this clinical study.

[0174] Example 7

[0175] Clinical studies evaluating IdeE-2 injection for the treatment of acute severe autoimmune diseases mediated by pathogenic immunoglobulin G (IgG) autoantibodies (e.g., anti-glomerular basement membrane antibody-associated diseases, anti-GBM disease) were conducted, with acute remission in each study cohort as the primary efficacy endpoint.

[0176] Treatment regimen: On day 1, administer 0.25 mg / kg of IdeE-2 intravenously. To prevent rebound of anti-GBM antibody levels, administer an additional 0.15 mg / kg of IdeE-2 on day 8 (the investigator may decide to advance or postpone the administration of 0.15 mg / kg of IdeE-2 between days 3 and 10, depending on the level of anti-GBM antibody). Patients will receive immunosuppressants (such as glucocorticoids, cyclophosphamide, etc.) or plasma exchange / immunoadsorption as prescribed according to the clinical practice of each research center.

[0177] Inclusion criteria:

[0178] 1. Age ≥ 18 years old, both male and female are acceptable;

[0179] 2. Clinically diagnosed with anti-GBM disease. Screening showed positive anti-GBM antibodies, with / without concurrent ANCA antibody positivity.

[0180] The detection method for ADA is as follows: This experiment uses an ELISA bridging method to determine anti-IdeE-2 antibodies in human serum. In the screening experiment, a certain amount of IdeE-2 antigen is coated on the ELISA plate to capture anti-IdeE-2 antibodies in the serum. The captured antibodies then bind to biotinylated IdeE-2, and subsequently react with Streptavidin-Peroxidase from Streptomyces avidinii. The signal value is positively correlated with the antibody concentration in the sample. Samples with high signal values ​​are considered to have potential anti-IdeE-2 antibodies in their serum. In the confirmatory experiment, IdeE-2 is added externally to inhibit the anti-drug antibodies in the serum. This confirmatory test is performed on samples that are potentially antibody-positive. The higher the signal inhibition rate, the higher the titer of anti-IdeE-2 antibodies in human serum.

[0181] Six subjects who met the inclusion criteria were numbered S01 to S06 and received two-dose administration on days 1 and 8. No infection or other infusion-related reactions were reported by any of the subjects, indicating that IdeE-2 has good tolerability and safety.

[0182] Figures 16-19 show that after two repeated doses of IdeE-2, serum IgG levels significantly decreased to near zero 6 hours after the first dose, remained at a low level for the following 7 to 10 days, and then gradually recovered slowly. Serum anti-GBM antibodies rapidly decreased to near zero 6 hours after the first dose. In some patients, anti-GBM antibody levels remained at zero for 14 days of follow-up, indicating a relatively ideal treatment effect; only one case showed a significant rebound in anti-GBM antibodies. Furthermore, during the follow-up period, anti-drug antibodies (ADA) remained negative in 3 patients, while ADA production was detected in the other 3 patients on days 8 or 10, respectively.

[0183] This demonstrates that repeated dosing of IdeE-2 plays a crucial role in diseases requiring a prolonged therapeutic window. In the treatment of autoantibody-mediated conditions, repeated dosing of IdeE-2 can reduce pre-existing IgG levels to an acceptable range. Furthermore, repeated dosing of IdeE-2 can provide a longer low IgG window, allowing for more thorough pretreatment assessment before administration of autoimmune therapies. Moreover, this approach could offer a potential solution for overcoming the neutralizing antibody barrier, enabling multi-round autoimmune therapy.

[0184] Example 8

[0185] In this embodiment, 32 healthy subjects from China were selected from the subjects provided in Example 5. The pre-existing titer of AAV neutralizing antibody (Nab) in the subjects' serum was measured and defined as 100%. Subsequently, the AAV neutralizing antibody titer, total IgG, and the titer of F(ab')2 derived from IgG were measured at 6 hours, 2 days, 3 days, 4 days, 7 days, 14 days, and 21 days after IdeE-2 administration to explore the optimal time window for AAV gene therapy after IdeE-2 administration. This study selected AAV2 and AAV8 serotypes as indicators for detecting neutralizing antibody titers. These two serotypes are widely used in gene therapy and are known to have relatively high pre-existing neutralizing antibody levels in more than 50% of the population.

[0186] IdeE-2 reduces serum total IgG, AAV2, and AAV8 neutralizing antibodies (Nab) by more than 90% by cleaving IgG into F(ab')2 and Fc fragments (Figure 9). Initial pre-stored AAV2 neutralizing antibody titers in all subjects in the 0.25 mg / kg and 0.40 mg / kg serum AAV2 groups were further analyzed by dividing them into two groups (>1:1000, 5 subjects; 1:100–1:1000, 4 subjects). The results are shown in Figure 10.

[0187] To assess how IdeE-2 might alter AAV2 antibody titers and the likelihood of a second dose, this study subsequently tested changes in F(ab')2 in these nine volunteers (Figure 11). IdeE-2 degradation of IgG led to the rapid generation of F(ab')2. F(ab')2 was gradually metabolized and almost completely eliminated by day 7. The trend in AAV8 antibody changes was similar to that of AAV2 antibody, as shown in Figure 12.

[0188] Figure 13 shows the changes in serum AAV neutralizing antibody (Nab) levels after IdeE-2 administration in AAV2 and AAV8 serotypes with different neutralizing antibody titers. A single dose of IdeE-2 effectively and significantly reduced neutralizing antibodies in both AAV2 and AAV8. With a Nab cutoff value of 1:100 for AAV2, a single dose of IdeE-2 reduced neutralizing antibody titers of 1:1000 and below to below the cutoff value. With a Nab cutoff value of 1:5 for AAV8, a single dose of IdeE-2 reduced neutralizing antibody titers of 1:50 and below to below the cutoff value.

[0189] Comparing Figures 9-13, we can see that:

[0190] 1. IdeE-2 at doses of 0.25 mg / kg and 0.40 mg / kg can reduce AAV2 and AAV8 Nab titers to approximately 10% of pre-dose levels.

[0191] 2. The rate of decrease in Nab titer was independent of the initial Nab titer of AAV. The titer decrease rate after administration was 90% in all cases, as shown in Figures 9-13.

[0192] 3. A single dose of IdeE-2 at 0.25 mg / kg or 0.40 mg / kg has the potential to expand the patient population eligible for gene therapy. It could potentially increase the Nab titer inclusion threshold for AAV-based gene therapy by at least one order of magnitude.

[0193] 4. For patients with high initial Nab levels, a single dose may not be sufficient to reduce Nab below the AAV treatment threshold, thus requiring additional doses or multiple administrations.

[0194] 5. F(ab')2 has neutralizing activity, but it is not as effective as IgG, which is similar to data published elsewhere.

[0195] 6. IgG levels reach their lowest point one day after administration and begin to rise on day 2. Nab levels reach their lowest point three days after administration and rise rapidly after day 7. Therefore, if further reduction of Nab titers is required, repeat administration of IdeE-2 should be feasible. The appropriate window for a second dose is 2 to 7 days.

[0196] Example 9

[0197] Clinical studies evaluating IdeE-2 injection for the treatment of acute severe autoimmune diseases mediated by pathogenic immunoglobulin G (IgG) autoantibodies (e.g., anti-glomerular basement membrane antibody-associated diseases, anti-GBM disease) were conducted, with acute remission in each study cohort as the primary efficacy endpoint.

[0198] Inclusion criteria:

[0199] 1. Age ≥ 18 years old, both male and female are acceptable;

[0200] 2. Clinically diagnosed with anti-GBM disease. Screening showed positive anti-GBM antibodies, with / without concurrent ANCA antibody positivity.

[0201] Four subjects met the inclusion criteria and were numbered S07 to S10.

[0202] Treatment regimen: IdeE-2 was administered intravenously at a dose of 0.25 mg / kg on day 1, and intravenously at a dose of 0.15 mg / kg on days 4 and 7. Patients will receive immunosuppressants (such as glucocorticoids, cyclophosphamide, etc.) as prescribed according to the clinical practice of each research center. No infections or other infusion-related reactions were reported in any of the subjects, indicating that IdeE-2 is well-tolerated and has a good safety profile.

[0203] The detection method for ADA is as follows: This experiment uses an ELISA bridging method to determine anti-IdeE-2 antibodies in human serum. In the screening experiment, a certain amount of IdeE-2 antigen is coated on the ELISA plate to capture anti-IdeE-2 antibodies in the serum. The captured antibodies then bind to biotinylated IdeE-2, and subsequently react with Streptavidin-Peroxidase from Streptomyces avidinii. The signal value is positively correlated with the antibody concentration in the sample. Samples with high signal values ​​are considered to have potential anti-IdeE-2 antibodies in their serum. In the confirmatory experiment, IdeE-2 is added externally to inhibit the anti-drug antibodies in the serum. This confirmatory test is performed on samples that are potentially antibody-positive. The higher the signal inhibition rate, the higher the titer of anti-IdeE-2 antibodies in human serum.

[0204] Figures 20-23 show that after three repeated doses of IdeE-2, serum IgG levels significantly decreased to near zero 6 hours after the first dose and remained at very low levels for the following 10 days, before gradually and slowly recovering. Serum anti-GBM antibodies in all patients also rapidly decreased to near zero 6 hours after the first dose and remained at this level throughout the follow-up period, demonstrating significant efficacy. Furthermore, some patients began to develop ADA on day 10 or day 14.

[0205] The above results indicate that IdeE-2 has a rapid onset of action and can effectively clear pathogenic IgG antibodies in a short period of time, thereby significantly reducing further damage to organs and tissues caused by autoimmune responses. Furthermore, the clearance effect of pathogenic antibodies was continuously observed in the three-dose regimen, which can play a crucial role in diseases requiring a prolonged treatment window, providing a more stable and reliable therapeutic window.

[0206] Similarly, in the context of AAV gene therapy, repeated doses of IdeE-2 can reduce pre-existing high anti-AAV IgG levels to within an acceptable range for AAV, and can also provide a longer low IgG window, allowing for more thorough pretreatment assessment before AAV administration. Furthermore, this approach could offer a potential solution for overcoming the neutralizing antibody barrier, enabling multi-round AAV gene therapy. Multiple administrations (e.g., two or three times) can further reduce anti-AAV IgG levels during AAV gene therapy, making more patients suitable for AAV treatment.

[0207] The results of the above embodiments demonstrate that multiple dosing (e.g., two or three times) provides clinical evidence for using multiple doses of IdeE-2 to reduce IgG levels, overcoming the antibody-mediated challenges of multiple dosing in the treatment of autoantibody-mediated diseases and AAV gene therapy. This provides a potential strategy for ultimately improving the treatment of autoantibody-mediated diseases and AAV gene therapy. Furthermore, this method can provide a potential solution for overcoming the neutralizing antibody barrier, enabling multi-round dosing therapy and multi-round AAV gene therapy for autoantibody-mediated diseases, thereby improving overall treatment efficacy.

[0208] IdeE-2 has a rapid onset of action and can effectively clear pathogenic IgG antibodies in a short period of time, thereby significantly reducing further damage to organs and tissues caused by autoimmune responses. Furthermore, the clearance of pathogenic antibodies was consistently observed in a three-dose regimen, indicating that IdeE-2 has good clinical benefits.

[0209] Example 10

[0210] To evaluate the clinical trials of injectable IdeE-2 for the treatment of acute severe autoimmune diseases mediated by pathogenic immunoglobulin G (IgG) autoantibodies, with acute remission in each study cohort as the primary efficacy endpoint.

[0211] Treatment regimen: IdeE-2 administered intravenously at a dose of 0.25 mg / kg on day 1, and intravenously at a dose of 0.15 mg / kg on days 4, 7, and 10. Patients will receive immunosuppressants (such as glucocorticoids, cyclophosphamide, etc.) as prescribed by the physician according to the clinical practice of each research center.

[0212] Inclusion criteria:

[0213] 1. Age ≥ 18 years old, both male and female are acceptable;

[0214] 2. Clinically diagnosed with anti-GBM disease. Screening showed positive anti-GBM antibodies, with / without concurrent ANCA antibody positivity.

[0215] Example 11

[0216] To further evaluate the clinical trials of IdeE-2 injection for the treatment of acute severe autoimmune diseases mediated by pathogenic immunoglobulin G (IgG) autoantibodies (e.g., anti-glomerular basement membrane antibody-positive diseases, anti-GBM disease), the acute phase remission of each study cohort was used as the primary efficacy endpoint.

[0217] Subsequent treatment options include:

[0218] (1) Dosing regimen 1: 0.25 mg / kg of IdeE-2 was administered intravenously once on day 1, and 0.15 mg / kg of IdeE-2 was administered again on day 4 and day 7;

[0219] (2) Dosing regimen 2: 0.25 mg / kg of IdeE-2 was administered intravenously once on day 1, followed by a supplemental dose of 0.25 mg / kg of IdeE-2 on day 4, and a supplemental dose of 0.15 mg / kg of IdeE-2 on day 7;

[0220] (3) Dosage regimen 3: 0.25 mg / kg of IdeE-2 was administered intravenously once on day 1, and 0.15 mg / kg of IdeE-2 was administered again on day 4, day 7 and day 10.

[0221] (4) Based on the obtained clinical data, the researchers and the sponsor will discuss and decide whether to explore other dosing regimens.

[0222] Patients will receive immunosuppressant treatment (such as glucocorticoids, cyclophosphamide, etc.) as prescribed by their doctor, based on the clinical practice of each research center.

[0223] Inclusion criteria:

[0224] (1) Age ≥ 18 years old, both male and female are acceptable;

[0225] (2) Clinically diagnosed with anti-GBM disease. The candidate is positive for anti-GBM antibodies at screening, and may or may not be positive for ANCA antibodies.

[0226] Example 12

[0227] IgG degrading enzyme activity was detected by enzymatic digestion of purified protein followed by capillary electrophoresis. Trastuzumab substrate was diluted to 10 mg / ml with water for injection, and the enzyme sample was diluted to 0.01 mg / ml. The pH was adjusted to 7.0 ± 0.1 using 5× phosphate buffer. A 200 μl reaction mixture of enzyme and substrate was prepared at a 1:5000 mass ratio using 5× phosphate buffer and incubated at 37 ± 1 °C for 60 ± 10 min. The digestion reaction was terminated by water bath at 70 °C for 10 min to obtain the pre-treated sample.

[0228] Take 20 μl of the pre-treated sample, add 75 μl of SDS-MW Sample Buffer and 5 μl of 0.25 mol / L iodoacetamide aqueous solution, and vortex to mix. Incubate at 65 °C for 4 minutes, cool to room temperature, vortex to mix again, and centrifuge at 4500 g for 1 minute. Take 90 μl of the sample to be tested for non-reducing CE-SDS separation, record the chromatogram at 220 nm wavelength, and record data for 40 minutes. Calculate the proportions of Fc, F(ab')2, scIgG, and IgG within the integration window using the area normalization method.

[0229] The test results showed that the activities of IdeE-2, Ides, HIGES2, and IM3 were comparable, the remaining amount of scIgG was not significantly different, IM1 and IM2 could completely cleave the substrate, and ISK enzyme activity was the lowest.

[0230] Table 3 Results of IgG Degrading Enzyme Activity Detection

[0231] Example 13

[0232] To evaluate the effect of IgG degrading enzymes on ADA in the presence of immunosuppressants. Male New Zealand rabbits were selected and administered the enzyme test product intravenously, while the immunosuppressant, cyclophosphamide, a commonly used clinical drug, was administered via gavage. Groups 1-4 received the enzyme test product once on days 1 and 8, while groups 3-4 received cyclophosphamide once daily from day 3 to day 15. Blood samples were collected before administration on day 1, 1 hour after administration on day 1, before administration on day 8, 1 hour after administration on day 8, and on day 15. The levels of ADA corresponding to IdeE-2 and HIGES2 in the blood were measured using ELISA.

[0233] Anti-drug antibodies in serum were measured using an ELISA bridging method. A certain amount of drug was coated on an enzyme-labeled plate to capture anti-drug antibodies in serum. The captured antibodies then combined with biotinylated drug, and subsequently reacted with Streptavidin-Peroxidase from Streptomyces avidinii. The SNR value was positively correlated with the antibody concentration in the sample.

[0234] The results showed that both groups 1 and 2, which did not receive immunosuppressants, produced relatively higher levels of ADA by day 15 compared to pre-administration levels, while the immunosuppressant groups showed suppressed ADA production. Even the more immunogenic enzyme HIGES2 could suppress high ADA production under the action of immunosuppressants.

[0235] Table 4. Experimental groups showing the effect of IgG degrading enzymes on ADA in the presence of immunosuppressants.

[0236] sequence list

Claims

1. A method of reducing immunoglobulin G levels in a subject, wherein, The method comprises: administering to the subject at least two doses of an immunoglobulin G-degrading enzyme; and the interval between the at least one dose of the immunoglobulin G-degrading enzyme and the previous dose of the immunoglobulin G-degrading enzyme is greater than 24 hours; the subject also receives treatment with at least one immunosuppressive agent.

2. The method of claim 1, wherein, The interval between the at least one dose of the immunoglobulin G-degrading enzyme and the previous dose of the immunoglobulin G-degrading enzyme is no more than 7 days to 12 months.

3. The method of claim 2, wherein, The interval between the at least one dose of the immunoglobulin G-degrading enzyme and the previous dose of the immunoglobulin G-degrading enzyme is no more than 7 days, 10 days, 15 days, 1 month, 2 months, 3 months, 4 months, 6 months or 12 months.

4. The method of any one of claims 1 to 3, wherein, The method comprises administering to the subject at least 2-10 doses of an immunoglobulin G-degrading enzyme, optionally, at least 2 doses, 3 doses, 4 doses, 5 doses, 6 doses, 7 doses, 8 doses, 9 doses or 10 doses of an immunoglobulin G-degrading enzyme.

5. The method of any one of claims 1 to 4, wherein, Pre-existing antibodies against the immunoglobulin G-degrading enzyme are not present or are not detectable in the subject.

6. The method of any one of claims 1 to 5, wherein, The amount of the immunosuppressive agent is sufficient to cause the subject to have a titre of anti-immunoglobulin G-degrading enzyme antibodies (ADA) of less than 1:100000, 1:50000, 1:15000, 1:10000, 1:4000, 1:2000, 1:1000, 1:800, 1:600, 1:400, 1:200, 1:100, 1:50, 1:20 or 1:10 during the course of treatment; optionally, the subject is negative for anti-immunoglobulin G-degrading enzyme antibodies (ADA) during the course of treatment.

7. The method of any one of claims 1 to 6, wherein, The subject is a human subject; Optionally, the subject has an autoantibody-mediated disorder, optionally, the autoantibody-mediated disorder is anti-GBM disease; Optionally, the subject is a highly sensitized patient awaiting a kidney transplant.

8. The method of any one of claims 1-7, wherein, The method is for alleviating an immune rejection response in the subject or for desensitization treatment of a highly sensitized patient awaiting a kidney transplant, optionally, the immune rejection response is a transplant-induced rejection response.

9. The method of any one of claims 1-8, wherein, The first dose of the immunoglobulin G-degrading enzyme of the method is capable of reducing the level of immunoglobulin G in the subject to less than about 50%, 40%, 30%, 20%, 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1% or about 0.5% of the level before administration within 45 minutes to 6 hours after administration of the first dose.

10. The method of any one of claims 1 to 9, wherein, The first dose of the immunoglobulin G-degrading enzyme of the method is capable of reducing the level of immunoglobulin G in the subject to less than about 5 g / L, about 4 g / L, about 3 g / L, about 2 g / L, about 1 g / L or about 0.5 g / L within 45 minutes to 6 hours after administration of the first dose.

11. The method of any one of claims 1-10, wherein, The immunosuppressive agent is selected from a glucocorticoid, an alkylating agent, a nucleotide reductase or tyrosine kinase inhibitor, a rapamycin target molecule inhibitor, a botanical drug, a calcineurin inhibitor, an antimetabolite, a protein drug or a monoclonal antibody.

12. The method of any one of claims 1-11, wherein, The immunoglobulin G-degrading enzyme is selected from IdeS, IdeE, IdeZ, IdeP, IdeSORK or a mutant thereof.

13. The method of any one of claims 1-12, wherein, The immunoglobulin G-degrading enzyme is a low immunogenic immunoglobulin G-degrading enzyme, optionally, the immunogenic immunoglobulin G-degrading enzyme has lower immunogenicity than IdeS.

14. The method of any one of claims 1-13, wherein, The amino acid sequence of the immunoglobulin G-degrading enzyme has at least 80%, at least 85%, optionally at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence of any one of SEQ ID NOs: 1-17.

15. The method of any one of claims 1-14, wherein, The amino acid sequence of the immunoglobulin G-degrading enzyme comprises the amino acid sequence of any one of SEQ ID NOs: 1-17.

16. The method of any one of claims 1-15, wherein, The amino acid sequence of the immunoglobulin G-degrading enzyme comprises the amino acid sequence of any one of SEQ ID NOs: 1-11, 14-17.

17. The method of any one of claims 1-16, wherein, The amino acid sequence of the immunoglobulin G-degrading enzyme is as set forth in SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 13, or SEQ ID NO:

17.

18. The method of any one of claims 1-17, wherein, The immunoglobulin G-degrading enzyme of each dose is the same or different.

19. The method of any one of claims 1-18, wherein, The administration dose of the immunoglobulin G-degrading enzyme of each dose is the same or different. Optionally, the administration dose of the immunoglobulin G-degrading enzyme of each dose is the same, and is about 0.01 mg / kg to about 1.0 mg / kg, for example, 0.01 mg / kg, 0.04 mg / kg, 0.12 mg / kg, 0.15 mg / kg, 0.2 mg / kg, 0.25 mg / kg, 0.3 mg / kg, 0.35 mg / kg, 0.4 mg / kg, 0.45 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9, 1.0 mg / kg. Optionally, the administration dose of at least one dose of the immunoglobulin G-degrading enzyme is about 100% to about 20% of the administration dose of the previous dose of the immunoglobulin G-degrading enzyme, optionally about 80% to about 40%, optionally about 50% to about 70%, optionally about 60%.

20. The method of any one of claims 1-19, wherein, The administration amount of the first dose of the immunoglobulin G-degrading enzyme is about 0.01 mg / kg to about 1.0 mg / kg; and the administration amount of the subsequent dose of the immunoglobulin G-degrading enzyme is about 0.1 mg / kg to about 0.25 mg / kg. Optionally, the administration amount of the first dose is about 0.25 mg / kg, and the administration amount of the subsequent dose of the immunoglobulin G-degrading enzyme is about 0.15 mg / kg.

21. The method of any one of claims 1-20, wherein, The administration interval of the immunoglobulin G-degrading enzyme is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 25 days, 28 days, 30 days, 35 days, 60 days, 90 days, or 180 days.

22. The method of any one of claims 1-21, wherein, The method comprises a) administering to the subject a first dose of the immunoglobulin G-degrading enzyme on the first day; and b) administering to the subject another dose of the immunoglobulin G-degrading enzyme during the period of the 4th to 10th day; optionally, the method further comprises administering to the subject an additional dose of the immunoglobulin G-degrading enzyme every 3-9 days.

23. The method of any one of claims 1-22, wherein, The method is administered with or without 24 hours of continuous administration of two doses of immunoglobulin G-degrading enzyme, optionally, the method comprises administering to the subject another dose of immunoglobulin G-degrading enzyme within 24 hours after administering to the subject the first dose of immunoglobulin G-degrading enzyme.

24. The method of claim 23, wherein, The method comprises administering the same or different dose of immunoglobulin G-degrading enzyme in each dose, optionally, the first dose of immunoglobulin G-degrading enzyme is administered at a dose of 0.25 mg / kg, and the subsequent dose of immunoglobulin G-degrading enzyme is administered at a dose of 0.15 mg / kg.

25. The method of claim 23 or 24, wherein, The immunoglobulin G-degrading enzyme is an enzyme having an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 13, or SEQ ID NO: 17, optionally, the immunoglobulin G-degrading enzyme is an enzyme having an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:

17.

26. The method of any one of claims 1-22, wherein, The immunosuppressive agent is administered before, after or simultaneously with the administration of the immunoglobulin G-degrading enzyme, and / or between any two doses of the immunoglobulin G-degrading enzyme.

27. The method of any one of claims 1-26, wherein, The subject has anti-GBM disease and is receiving cyclophosphamide and / or glucocorticoid treatment, and the method comprises: a) administering a single intravenous infusion of 0.25 mg / kg of IdeE-2 on day 1; b) if the subject has anti-GBM antibody higher than 20 RU / ml or positive during day 3-10, administering a single intravenous infusion of 0.15 mg / kg of IdeE-2, otherwise administering a single intravenous infusion of 0.15 mg / kg of IdeE-2 on day 8; Optionally, the glucocorticoid is prednisolone, and the dose is 0.5-2.0 mg / kg / d; Optionally, the dose of prednisolone is 1 mg / kg / d, which is reduced to 20 mg within 6 weeks, and gradually reduced to stop within 6-9 months; Optionally, the glucocorticoid treatment method comprises intravenous administration of glucocorticoid treatment first, and then oral administration of glucocorticoid treatment; Optionally, the method of intravenous administration of glucocorticoid treatment first comprises intravenous administration of 0.25-1.0 g of glucocorticoid treatment first; Optionally, the method of intravenous administration of glucocorticoid treatment first comprises intravenous administration of 0.25-1.0 g of glucocorticoid treatment every day or every other day continuously; Optionally, the method of intravenous administration of glucocorticoid treatment first comprises intravenous administration of 0.25-1.0 g of glucocorticoid treatment every day or every other day continuously for a total of three days; Optionally, the method of oral administration of glucocorticoid treatment comprises gradually reducing the oral administration dose, and the oral administration lasts for 6-9 months; Optionally, the method of oral administration of glucocorticoid treatment comprises administering 40-80 mg of glucocorticoid per day in the first week, and maintaining the dose to 5-10 mg at week 26. Optionally, the glucocorticoid used in the intravenous administration of glucocorticoid therapy followed by oral administration of glucocorticoid therapy is independently selected from prednisolone, prednisone or methylprednisolone, for example, intravenous administration of methylprednisolone followed by oral administration of prednisone; Optionally, the treatment method of cyclophosphamide comprises oral administration of cyclophosphamide at 1-5 mg / kg / d; Optionally, the method of treatment with cyclophosphamide comprises intravenous administration of 0.5 to 1.0 g / m 2 / month. Optionally, the treatment method of cyclophosphamide comprises intravenous administration of 0.2 g / day; Optionally, the treatment method of cyclophosphamide comprises intravenous administration of 0.2 g / week; Optionally, the treatment method of cyclophosphamide comprises intravenous administration of 0.5 g / two weeks; Optionally, the treatment duration of cyclophosphamide is 3 months.

28. The method of any one of claims 1-27, wherein, The subject has anti-GBM disease, and the method comprises a) single intravenous infusion of 0.25 mg / kg of IdeE-2 on day 1; b) intravenous infusion of 0.15 mg / kg of IdeE-2 on day 4, day 7.

29. The method of any one of claims 1-27, wherein, The subject has anti-GBM disease, and the method comprises a) single intravenous infusion of 0.25 mg / kg of IdeE-2 on day 1; b) intravenous infusion of 0.15 mg / kg of IdeE-2 on day 4, day 7, day 10.

30. The method of any one of claims 1-27, wherein, The subject has anti-GBM disease, and the method comprises a) single intravenous infusion of 0.25 mg / kg of IdeE-2 on day 1; b) intravenous infusion of 0.15 mg / kg of IdeE-2 on day 4, day 7, day 10. Optionally, the glucocorticoid is prednisolone, and the administration dose is 0.5-2.0 mg / kg / d; Optionally, the administration dose of prednisolone is 1 mg / kg / d, which is reduced to 20 mg within 6 weeks, and gradually reduced to stop within 6-9 months; Optionally, the treatment method of glucocorticoid comprises intravenous administration of glucocorticoid therapy followed by oral administration of glucocorticoid therapy; Optionally, the method of intravenous administration of glucocorticoid therapy comprises intravenous administration of glucocorticoid at 0.25-1.0 g; Optionally, the method of intravenous administration of glucocorticoid therapy comprises continuous intravenous administration of glucocorticoid at 0.25-1.0 g every day or every other day; Optionally, the method of intravenous administration of glucocorticoid therapy comprises continuous intravenous administration of glucocorticoid at 0.25-1.0 g every day or every other day for a total of three days; Optionally, the method of oral administration of glucocorticoid therapy comprises gradually reducing the oral administration dose, and the oral administration duration is 6-9 months; Optionally, the method of oral administration of glucocorticoid therapy comprises daily administration of 40-80 mg of glucocorticoid in the first week, and maintaining the dose to 5-10 mg in the 26th week; Optionally, the glucocorticoid used in the intravenous administration of glucocorticoid therapy followed by oral administration of glucocorticoid therapy is independently selected from prednisolone, prednisone or methylprednisolone, for example, intravenous administration of methylprednisolone followed by oral administration of prednisone; Optionally, the method of treating the cyclophosphamide comprises administering the cyclophosphamide orally at 1-5 mg / kg / d; Optionally, the method of treatment with cyclophosphamide comprises intravenous administration of 0.5 to 1.0 g / m 2 / month. Optionally, the method of treating the cyclophosphamide comprises administering the cyclophosphamide intravenously at 0.2 g / day; Optionally, the method of treating the cyclophosphamide comprises administering the cyclophosphamide intravenously at 0.2 g / week; Optionally, the method of treating the cyclophosphamide comprises administering the cyclophosphamide intravenously at 0.5 g / two weeks; Optionally, the method of treating the cyclophosphamide lasts for 3 months.

31. The method of any one of claims 1-27, wherein, The subject has anti-GBM disease and receives treatment of cyclophosphamide and / or glucocorticoid; the method comprises, a) a single intravenous infusion of 0.25 mg / kg IdeE-2 on day 1; b) intravenous infusion of 0.15 mg / kg IdeE-2 on day 4, day 7, and day 10; Optionally, the glucocorticoid is prednisolone, and the administration dose is 0.5-2.0 mg / kg / d; Optionally, the method of treating the glucocorticoid comprises intravenous administration of glucocorticoid treatment first, and then oral administration of glucocorticoid treatment; Optionally, the method of intravenous administration of glucocorticoid treatment first comprises intravenous administration of glucocorticoid 0.25-1.0 g treatment first; Optionally, the method of intravenous administration of glucocorticoid treatment first comprises continuous intravenous administration of glucocorticoid 0.25-1.0 g treatment every day or every other day first; Optionally, the method of intravenous administration of glucocorticoid treatment first comprises continuous intravenous administration of glucocorticoid 0.25-1.0 g treatment every day or every other day first, for a total of three days; Optionally, the method of oral administration of glucocorticoid treatment again comprises gradually reducing the oral administration dose, and the oral administration lasts for 6-9 months; Optionally, the method of oral administration of glucocorticoid treatment again comprises daily administration of 40-80 mg of glucocorticoid in the first week, and maintaining the dose to 5-10 mg in the 26th week; Optionally, the glucocorticoid used in the method of intravenous administration of glucocorticoid treatment first, and then oral administration of glucocorticoid treatment is independently selected from prednisolone, prednisone, or methylprednisolone, for example, intravenous administration of methylprednisolone first, and oral administration of prednisone then; Optionally, the method of treating the cyclophosphamide comprises administering the cyclophosphamide orally at 1-5 mg / kg / d; Optionally, the method of treatment with cyclophosphamide comprises intravenous administration of 0.5 to 1.0 g / m 2 / month. Optionally, the method of treating the cyclophosphamide comprises administering the cyclophosphamide intravenously at 0.2 g / day; Optionally, the method of treating the cyclophosphamide comprises administering the cyclophosphamide intravenously at 0.2 g / week; Optionally, the method of treating the cyclophosphamide comprises administering the cyclophosphamide intravenously at 0.5 g / two weeks; Optionally, the method of treating the cyclophosphamide lasts for 3 months.

32. The method of any one of claims 1-26, wherein, The subject is a highly sensitized kidney transplant patient, and the subject first receives anti-CD20 and / or anti-CD38 antibody treatment, and then receives combined treatment of calcineurin inhibitor, anti-metabolite drug, and glucocorticoid; Optionally, the anti-CD20 antibody is rituximab, veltuzumab, or obinutuzumab; the anti-CD38 antibody is daratumumab or isatuximab.

33. The method of any one of claims 1-26, wherein, The subject is a patient who is to receive AAV gene therapy; Optionally, the patient has pre-existing AAV neutralizing antibodies and total binding antibodies in the body; Optionally, the AAV gene therapy vector is of a serotype selected from one or more of AAV1, AAV2, AAV3, AAV3B, AAV3S, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, and AAV-rh74; for example, the AAV gene therapy vector is of serotype AAV2 or AAV8. Optionally, the subject is administered two doses of the immunoglobulin G-degrading enzyme; and the second dose of the immunoglobulin G-degrading enzyme is administered 1-10 days, for example 2-7 days, or 2-4 days, or 3 days, after the previous dose of the immunoglobulin G-degrading enzyme. Optionally, the method is capable of reducing the AAV neutralizing antibody or total binding antibody level in the patient to 50%, 40%, 30%, 20%, 10% or less.

34. An immunoglobulin G-degrading enzyme for use in reducing the level of immunoglobulin G in a subject, the method of reducing the level of immunoglobulin G in a subject being as claimed in any one of claims 1-33.

35. Use of an immunoglobulin G-degrading enzyme in the manufacture of a medicament for reducing the level of immunoglobulin G in a subject, the method of reducing the level of immunoglobulin G in a subject being as claimed in any one of claims 1-33.

36. A pharmaceutical composition, wherein, The pharmaceutical composition comprises an immunoglobulin G-degrading enzyme and a pharmaceutically acceptable excipient in an amount sufficient to administer the method of reducing the level of immunoglobulin G in a subject of any one of claims 1-33.

37. A pharmaceutical combination wherein, The pharmaceutical composition comprises an immunoglobulin G-degrading enzyme and a pharmaceutically acceptable excipient in an amount sufficient to administer the method of reducing the level of immunoglobulin G in a subject of any one of claims 1-33.

38. The immunoglobulin G-degrading enzyme of claim 34, the use of claim 35, the pharmaceutical composition of claim 36, or the medicament of claim 37, wherein, The immunoglobulin G-degrading enzyme is selected from IdeS, IdeE, IdeZ, IdeP, IdeSORK, or a mutant thereof; Optionally, the immunoglobulin G-degrading enzyme is selected from an immunoglobulin G-degrading enzyme having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-17; Optionally, the immunoglobulin G-degrading enzyme is selected from an immunoglobulin G-degrading enzyme having an amino acid sequence as set forth in any one of SEQ ID NOs: 2, 9, 13, or 17; Optionally, the immunoglobulin G-degrading enzyme has one or more unit doses; Optionally, the unit dose of the immunoglobulin G-degrading enzyme is about 8 mg to about 100 mg, optionally about 10 mg to about 80 mg, optionally about 8 mg to about 40 mg, optionally about 10 mg to about 25 mg, optionally about 10 mg to about 15 mg; Optionally, the unit dose of the immunoglobulin G-degrading enzyme is about 5 mg to about 20 mg, optionally about 20 mg.

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