Mutant of immunoglobulin G degrading enzyme and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to a mutant of immunoglobulin G degrading enzyme and application of the mutant. The invention provides a mutant of immunoglobulin G degrading enzyme. The mutant has an amino acid sequence as shown in SEQ ID NO: 1-SEQ ID NO: 4. The invention discloses a mutant of immunoglobulin G degrading enzyme, which is used for treating diseases mediated by IgG antibodies, and the stability of the mutant is higher. In addition, when an anti-immune globulin G degrading enzyme antibody exists in the body in advance or drug resistance appears after the drug is used for a period of time, an immunoadsorption method is combined to remove the anti-immune globulin G degrading enzyme antibody so as to improve the curative effect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a mutant of immunoglobulin G degrading enzyme and application thereof. Background Art

[0002] Monoclonal antibody drugs have become an important part of biomedicine due to their strong specificity for therapeutic targets, high activity, controllable safety, and good efficacy. At present, monoclonal antibody drugs are one of the fastest growing segments in the global pharmaceutical field. With their high targeting, monoclonal antibody drugs can directly reach diseased cells, and have the unique advantages of reducing damage to normal cells and reducing side effects, and are widely used in clinical practice. They also have broad application prospects in medical treatment and have been successfully used to treat a variety of diseases such as tumors, autoimmune diseases, infectious diseases, and transplant rejection. However, long-term use of antibody drugs will produce drug resistance. Antibody drugs themselves are immunogenic. After infusion, anti-drug antibodies (ADA) may be produced. ADA may bind to the immune complex (IC) formed by the drug, thereby blocking the binding of the drug to the target, resulting in poor clinical effects, loss of drug effectiveness, changes in pharmacokinetics, and allergic reactions during infusion. After entering the body, monoclonal antibody drugs bind to antigens in the body to form immune complexes. With the widespread application of biotechnology drugs, the potential hazards of immune complex renal injury have received increasing attention. Immune complexes often exist in the form of circulation in the blood system and deposition in tissues. Incomplete clearance of immune complexes may also cause tissue damage at the deposition site, such as glomerulonephritis, vasculitis, and perivascular inflammation.

[0003] Immunoglubulin G-degrading enzyme of Streptococcus pyogenes (IdeS) is a typical cysteine ​​hydrolase that can perform enzymatic cleavage at specific sites in the hinge region of antibodies, hydrolyzing IgG into complete Fab2 fragments and Fc fragments. As a new method for eliminating pathogenic IgG, it has a rapid onset mode and can rapidly cleave IgG antibodies and inhibit their reactivity within hours after administration. Therefore, it can be used to treat diseases related to autoantibodies, such as a single intravenous infusion in highly sensitive renal transplant patients, and rapidly inactivate donor-specific antibodies (DSA). It can also be used to infuse and remove anti-drug antibodies in the body after antibody drugs develop resistance.

[0004] As research progresses, it is found that normal humans generally have pre-existing anti-IdeS antibodies, which reduces the efficacy of Ides drugs. For example, the disadvantages of the drug imlifidase include the rapid reappearance of donor-specific antibodies DSA, which brings the risk of antibody-mediated rejection reactions. The rapid development of anti-ides antibodies excludes the repeated use of imlifidase and its IgG degradation potential, limiting the use of therapeutic antibodies. Blood purification uses the principle of filtration or adsorption to specifically or highly selectively remove endogenous or exogenous toxins (pathogenic substances) in the body through blood purification materials, thereby achieving the purpose of curing diseases. It is an effective treatment for various difficult diseases (such as Figure 1 As shown). Protein A / G / L immunoadsorption belongs to immunoadsorption in blood purification. Protein A can directly and quickly remove IgG antibodies in the body by binding to the Fc and Fab fragments of IgG, antibodies and antigen-antibody complexes. Immunoadsorption belongs to physical adsorption, which has little interference with normal physiological components in the human body and is a safe and effective method for removing antibodies. Therefore, immunoadsorption can be used to remove anti-drug antibodies in the body, or immunoglobulin G degrading enzyme can be used for immunoadsorption before or after use to further enhance its efficacy. Summary of the invention

[0005] In view of this, the present invention provides a mutant of immunoglobulin G degrading enzyme and its application. The present invention discloses a mutant of immunoglobulin G degrading enzyme for treating diseases mediated by IgG class antibodies, and the mutant has higher stability. In addition, when anti-immunoglobulin G degrading enzyme antibodies pre-exist in the body or drug resistance appears after a period of use of drugs, the anti-immunoglobulin G degrading enzyme antibodies can be removed by combined immunoadsorption method to improve the efficacy.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a mutant of an immunoglobulin G degrading enzyme having:

[0008] (1) the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 4; or

[0009] (2) An amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (1), and having the same or similar function as the amino acid sequence shown in (1); or

[0010] (3) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (1) or (2).

[0011] In some embodiments of the present invention, the mutants described above have:

[0012] (4) the amino acid sequence shown in SEQ ID NO: 1; or

[0013] (5) An amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (4), and having the same or similar function as the amino acid sequence shown in (4); or

[0014] (6) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (4) or (5).

[0015] In some embodiments of the present invention, the sequence of SEQ ID NO: 1 is: MDSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVATQGWYDITKTFNGKDDLLCGAATAGNMLHWWADQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVRPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADYDSNGNLKAIYVTDSDSNASIGMKKYFVGVQSAGKVAISAKEIKEDNIGAQVKGLFTLSTGQDSWNQTNHHHHHH.

[0016] In some embodiments of the present invention, the sequence of SEQ ID NO: 2 is: MDSFSAQQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDRTKTFNGKDDLLCGAATAGTMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFIRGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEAKALGLSHTYANVRIQHVINLWGADFDSNGNLKAKYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIAAQVLGLFTLSTGQDSWNQTNHHHHHH.

[0017] In some embodiments of the present invention, the sequence of SEQ ID NO: 3 is: MDSFSANQEIRYSEVAPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFREKNLKEISDLIKKELTEGKSLGLSHTYANVRINHVINLWGAEFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSTGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTNHHHHHH.

[0018] In some embodiments of the present invention, the sequence of SEQ ID NO:4 is: MDSFSANQEIRYSEVTPYHVNSVWTKGVTPPANFTQGEDVFDAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEKHPEKQKINFNGEQMFDVEEAVYTENHQLDSKLFEYFKEKAFPYLSTNHLGVFLDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDLSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDRNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTNHHHHHH.

[0019] The present invention also provides a nucleic acid molecule encoding the mutant.

[0020] In some embodiments of the present invention, the above-mentioned nucleic acid molecule has:

[0021] (7) the nucleotide sequence shown in SEQ ID NO: 5 to SEQ ID NO: 8; or

[0022] (8) A nucleotide sequence obtained by substituting, deleting or adding one or more bases of the nucleotide sequence shown in (7), and having the same or similar function as the nucleotide sequence shown in (7); or

[0023] (9) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (7) or (8).

[0024] In some embodiments of the present invention, the sequence of SEQ ID NO:5 is as follows: CATATGGATAGCTTTAGCGCAAACCAGGAGATCCGCTATAGCGAGGTTACCCCGTATCACGTTACCAGCGTTTGGACCAAAGGCGTTACCCCGCCGGCCAACTTTACCCAGGGCGAAGACGTGTTTCATGCCCCGTATGTTGCCACTCAGGGCTGGTACGACATCACCAAAACCTTCAATGGCAAGGACGATCTGCTGTGCGGTGCCGCAACCGCCGGTAACATGCTGCACTGGTGGGCCGACCAGAATAAAGACCAGATCAAACGCTACCTGGAGGAACACCCGGAAAAACAGAAAATTAATTTCAACGGCGAACAGATGTTTGATGTGAAAGAAGCTATTGATACCAAGAACCACCAGCTGGACAGCAAGCTGTTCGAATATTTTAAGGAGAAAGCCTTCCCGTACCTGAGCACCAAACATCTGGGCGTGCGACCGGACCATGTGATCGACATGTTCATCAACGGCTATCGCCTGAGCCTGACCAATCATGGTCCGACCCCGGTGAAAGAAGGTAGCAAAGATCCGCGCGGTGGTATCTTCGATGCCGTGTTTACACGTGGCGATCAGAGTAAGCTGCTGACCAGCCGCCATGATTTTAAAGAGAAAAATCTGAAAGAAATCAGCGATCTGATTAAGAAGGAGCTGACCGAGGGCAAAGCCCTGGGCCTGAGCCACACCTACGCCAATGTGCGCATCAACCACGTGATCAACCTGTGGGGTGCCGACTATGATAGCAACGGCAACCTGAAGGCAATTTACGTGACCGACAGCGACAGCAATGCCAGTATTGGCATGAAAAAATACTTTGTTGGTGTGCAGAGCGCCGGCAAAGTGGCAATCAGTGCCAAGGAGATCAAAGAAGATAACATCGGCGCCCAGGTTAAAGGTCTGTTTACCCTGAGCACAGGTCAGGATAGCTGGAATCAGACCAATCATCATCACCACCATCACtaaCTCGAG。

[0025] In some embodiments of the present invention, the sequence of SEQ ID NO:6 is as follows: CATATGGATAGCTTTAGCGCACAGCAGGAGATCCGCTATAGCGAGGTTACCCCGTATCACGTTACCAGCGTTTGGACCAAAGGCGTTACCCCGCCGGCCAACTTTACCCAGGGCGAAGACGTGTTTCATGCCCCGTATGTTGCCAATCAGGGCTGGTACGACCGCACCAAAACCTTCAATGGCAAGGACGATCTGCTGTGCGGTGCCGCAACCGCCGGTACCATGCTGCACTGGTGGTTCGACCAGAATAAAGACCAGATCAAACGCTACCTGGAGGAACACCCGGAAAAACAGAAAATTAATTTCAACGGCGAACAGATGTTTGATGTGAAAGAAGCTATTGATACCAAGAACCACCAGCTGGACAGCAAGCTGTTCGAATATTTTAAGGAGAAAGCCTTCCCGTACCTGAGCACCAAACATCTGGGCGTGTTTCCGGACCATGTGATCGACATGTTCATCCGCGGCTATCGCCTGAGCCTGACCAATCATGGTCCGACCCCGGTGAAAGAAGGTAGCAAAGATCCGCGCGGTGGTATCTTCGATGCCGTGTTTACACGTGGCGATCAGAGTAAGCTGCTGACCAGCCGCCATGATTTTAAAGAGAAAAATCTGAAAGAAATCAGCGATCTGATTAAGAAGGAGCTGACCGAGGCCAAAGCCCTGGGCCTGAGCCACACCTACGCCAATGTGCGCATCCAACACGTGATCAACCTGTGGGGTGCCGACTTTGATAGCAACGGCAACCTGAAGGCAAAATACGTGACCGACAGCGACAGCAATGCCAGTATTGGCATGAAAAAATACTTTGTTGGTGTGAACAGCGCCGGCAAAGTGGCAATCAGTGCCAAGGAGATCAAAGAAGATAACATCGCCGCCCAGGTTCTGGGTCTGTTTACCCTGAGCACAGGTCAGGATAGCTGGAATCAGACCAATCATCATCACCACCATCACtaaCTCGAG。

[0026] In some embodiments of the present invention, the sequence of SEQ ID NO:7 is as follows: CATATGGATAGCTTTAGCGCAAACCAGGAGATCCGCTATAGCGAGGTTgCCCCGTATCACGTTACCAGCGTTTGGACCAAAGGCGTTACCCCGCCGGCCAACTTTACCCAGGGCGAAGACGTGTTTCATGCCCCGTATGTTGCCAATCAGGGCTGGTACGACATCACCAAAACCTTCAATGGCAAGGACGATCTGCTGTGCGGTGCCGCAACCGCCGGTAACATGCTGCACTGGTGGTTCGACCAGAATAAAGACCAGATCAAACGCTACCTGGAGGAACACCCGGAAAAACAGAAAATTAATTTCAACGGCGAACAGATGTTTGATGTGAAAGAAGCTATTGATACCAAGAACCACCAGCTGGACAGCAAGCTGTTCGAATATTTTAAGGAGAAAGCCTTCCCGTACCTGAGCACCAAACATCTGGGCGTGTTTCCGGACCATGTGATCGACATGTTCATCAACGGCTATCGCCTGAGCCTGACCAATCATGGTCCGACCCCGGTGAAAGAAGGTAGCAAAGATCCGCGCGGTGGTATCTTCGATGCCGTGTTTACACGTGGCGATCAGAGTAAGCTGCTGACCAGCCGCCATGATTTTAgAGAGAAAAATCTaAAAGAAATCAGCGATCTGATTAAGAAGGAGCTGACCGAGGGCAAAtCCCTGGGCCTGAGCCACACCTACGCaAATGTGCgCATCAACCACGTGATCAACCTGTGGGGTGCCGAgTTTGATAGCAACGGCAACCTGAAGGCAATTTACGTGACCGACAGCGACAGCAATGCCAGTATTGGCATGAaAAAATACTTTGTTGGTGTGAACAGCaCCGGCAAAGTGGCAATCAGTGCCAAGGAGATCAAAGAAGATAACATCGGCGCCCAGGTTCTtGGTCTGTTTACCCTGAGCACAGGTCAGGATAGCTGGAATCAGACCAATCATCATCACCACCATCACtaaCTCGAG。

[0027] In some embodiments of the present invention, the sequence of SEQ ID NO:8 is as follows: CATATGGATAGCTTTAGCGCAAACCAGGAGATCCGCTATAGCGAGGTTACCCCGTATCACGTTAaCAGCGTTTGGACCAAAGGCGTTACCCCGCCGGCCAACTTTACCCAGGGCGAAGACGTGTTTgATGCCCCGTATGTTGCCAATCAGGGCTGGTACGACATCACCAAAACCTTCAATGGCAAGGACGATCTGCTGTGCGGTGCCGCAACCGCCGGTAACATGCTGCACTGGTGGTTCGACCAGAATAAAGACCAGATCAAACGCTACCTGGAGAAACACCCGGAAAAACAGAAAATTAATTTCAACGGCGAACAGATGTTTGATGTGGAAGAAGCTgTTtATACCgAGAACCACCAGCTGGACAGCAAGCTGTTCGAATATTTTAAGGAGAAAGCCTTCCCGTACCTGAGCACCAAcCATCTGGGCGTGTTTCtGGACCATGTGATaGACATGTTCATCAACGGCTATCGCCTGAGCCTGACCAATCATGGTCCGACCCCGGTGAAAGAAGGTAGCAAAGATCCGCGtGGTGGTATCTTCGATGCCGTGTTTACACGTGGCGATCtGAGTAAGCTGCTGACCAGCCGCCATGATTTTAAAGAGAAAAATCTGAAAGAAATCAGCGAcCTGATTAAGAAGGAGCTGACcGAGGGCAAAGCCCTGGGCCTGAGCCACACCTACGCCAATGTGCGCATCAACCACGTGATCAACCTGTGGGGTGCCGACTTTGATcGCAACGGCAACCTGAAGGCAATTTACGTGACCGAcAGCGACAGCAATGCCAGTATTGGCATGAAAAAATACTTTGTTGGTGTGAACAGCGCCGGCAAAGTGGCAATCAGTGCCAAGGAGATCAAAGAAGATAACATCGGCGCCCAGGTTCTGGGTCTGTTTACCCTGAGCACAGGTCAGGATAGCTGGAATCAGACCAATCATCATCACCACCATCACtaaCTCGAG。

[0028] In some embodiments of the present invention, the sequence of SEQ ID NO: 9 is: DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTNHHHHHHSSG. (A1)

[0029] The present invention also provides a recombinant plasmid, comprising: the above nucleic acid molecule.

[0030] The present invention also provides a host for transforming and / or transfecting the above recombinant plasmid.

[0031] The present invention also provides a method for preparing a mutant of immunoglobulin G degrading enzyme, and the mutant is obtained by inoculating, culturing, inducing, expressing and purifying the above host.

[0032] The present invention also provides the use of the mutant, the nucleic acid molecule, the recombinant plasmid, the host and / or the mutant obtained by the preparation method in any of the following items:

[0033] ( ), in the preparation of products for degradation and / or removal of therapeutic antibodies and their immune complexes in vivo; and / or

[0034] ( ), in the preparation of products for the prevention and / or treatment of autoantibody diseases; and / or

[0035] ( ), in the preparation of products that improve the efficacy of drugs.

[0036] In some embodiments of the present invention, in the above application, the degradation and / or removal comprises the following steps: administering an immunosorbent to a subject, removing the therapeutic antibody and its immune complex in the body, administering the mutant to the subject, and administering the immunosorbent again;

[0037] The immunosorbent is obtained by combining Staphylococcus aureus protein A with microspheres through covalent bonds.

[0038] The present invention also provides a product, including: the mutant, the nucleic acid molecule, the recombinant plasmid, the host and / or the mutant obtained by the preparation method.

[0039] The present invention discloses a mutant of immunoglobulin G degrading enzyme, which is used to treat diseases mediated by IgG antibodies, and the mutant has higher stability. In addition, when anti-immunoglobulin G degrading enzyme antibodies are pre-existing in the body or drug resistance occurs after a period of use of drugs, the anti-immunoglobulin G degrading enzyme antibodies can be removed by combined immunoadsorption to improve the efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0041] Figure 1 Schematic diagram of blood purification is shown;

[0042] Figure 2 The cytotoxicity test results of Example 5 are shown. DETAILED DESCRIPTION

[0043] The invention discloses a mutant of immunoglobulin G degrading enzyme and application thereof.

[0044] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0045] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0046] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0047] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0048] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0049] The first aspect of the present invention provides a mutant of immunoglobulin G degrading enzyme and its use. The sequence of the immunoglobulin G degrading enzyme is shown in SEQ ID NO: 1 to SEQ ID NO: 4. By mutating the amino acid sequence, its soluble expression amount and activity are enhanced.

[0050] The enzyme is used in preventing and / or treating autoantibody-related diseases by degrading autoantibodies.

[0051] The enzyme is used to improve the efficacy of drugs by degrading anti-drug autoantibodies and immune complexes.

[0052] On the other hand, a treatment model is provided for when anti-immunoglobulin G degrading enzyme antibodies pre-exist in the human body and affect the efficacy of drugs by combined immunoadsorption methods.

[0053] The protein A immunosorbent of the present invention was purchased from Kangbier (protein A immunosorbent column) produced by our company.

[0054] In Examples 1 to 4 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0055] The present invention will be further described below in conjunction with embodiments:

[0056] Example 1 Expression and purification of Ides endonuclease

[0057] The sequence of SEQ ID NO:9 was used as a comparative example, numbered A1, and the nucleic acid sequences of SEQ ID NO:5, 6, 7, and 8 were directly synthesized and named A2, A3, A4, and A5. The target gene sequence was connected to the plasmid PET30a, and then transferred into Escherichia coli BL21 (DE3) for culture. 50 mL of LB medium was placed in a 250 mL conical flask, the final concentration of kanamycin was 70 µg / mL, 30°C, 180 rpm, and after 4 hours of culture, IPTG with a final concentration of 1 mM was added to induce expression. After another 5 hours of culture, the bacteria were collected. The collected bacteria were suspended with PBS solution, and the bacterial suspension was ultrasonically crushed in an ultrasonic crusher in an ice bath, with the program of working for 1 s, stopping for 2 s, and a total of 5 minutes of crushing. The crushed liquid was centrifuged at 10,000 g / min for 15 minutes at 4°C, the supernatant was collected, and then purified with Ni ion filler, eluted with 500 mM imidazole, the elution peak was collected, and the molecular weight was determined by 12% separation gel electrophoresis. The molecular weight of the purified enzyme was about 35 KD. The protein concentration was determined using a BCA kit, and the results are shown in Table 1. The soluble expression level of strain A3 was higher.

[0058] Table 1 Protein expression levels of different strains

[0059]

[0060] Example 2 Experiment on the degradation of antibodies by Ides endonuclease

[0061] Take 0.5 mL of serum from a myasthenia gravis patient, take the purified Ides endonuclease expressed by the A3 strain and add it to the human serum at a ratio of 1:100 (g / g), react at 37°C for half an hour, and then use ELISA to detect the content of complete IgG. The antibody against human IgG Fc fragment was diluted to 5 μg / mL with 0.01M PBS, added at 100 μL / well, and incubated at 4°C overnight; the plate was washed with PBS containing 0.05% Tween20 (PBST), 300 μL / well, washed 5 times; blocked with 5% BSA, 200 μL / well, incubated at 37°C for 2 hours, and washed 5 times; the patient's serum before and after enzyme treatment was added at 100 μL / well, incubated at 37°C for 2 hours, and washed 5 times; the secondary antibody against human IgG Fab fragment (HRP) was diluted to the working concentration (1:5000) with 1% BSA-PBS, 100 μL / well, incubated at 37°C for 2 hours, and washed 5 times; the color developing solution was added at 100 μL / well, reacted at 37°C in the dark for 10 minutes, and then 1M H2SO4 was added at 100 μL / well to terminate the reaction, and the plate was read at 450nm with an enzyme reader. The content of residual IgG in serum after enzyme digestion was determined to be 10.02%.

[0062] Example 3 Clearance of anti-Ides antibodies by protein A immunosorbent

[0063] Take 0.1mL of filler from our company's Kangbier (protein A immunoadsorption column) into the chromatography column, add 0.5mL of anti-Ides antibody (about 0.54mg), shake the affinity chromatography column at 100rpm±10rpm at room temperature for 1h, then rinse with PBS buffer. The PBS buffer flows down naturally by gravity, and about 2mL of PBS buffer is rinsed and collected. The protein concentration in the solution before and after adsorption is detected with a micro BCA kit, and the clearance rate is calculated. The experiment is repeated twice. The clearance rates of the filler against anti-Ides antibodies are determined to be 95.02% and 94.62%, respectively.

[0064] Example 4 Combination of Protein A Filler and Degradation Enzyme

[0065] 0.5 mL of serum from a myasthenia gravis patient was mixed with 0.1 mL of protein A filler and placed on a decolorizing shaker for incubation for 30 minutes. The adsorbed serum was added with a degradation enzyme as in Example 2. Ides endonuclease was added to the serum at a ratio of 1:100 (g / g), 1:500 (g / g), and 1:1000 (g / g). After the reaction was completed, the remaining anti-IgG content in the serum after enzyme cleavage was determined to be 1.05%, 2.28%, and 2.34%, respectively. This indicates that the use of protein A to adsorb the pre-existing antibody IgG in the serum and then combined with the degradation enzyme can exert its effect at a lower drug concentration.

[0066] Example 5 Cytotoxicity Experiment

[0067] Preparation of test solution: Sample 1 - protein A immunosorbent extracted with MEM medium containing fetal bovine serum as the extract; Sample 2 - anti-Ides antibody solution with a concentration of 10 mg / mL; Sample 3 - a mixed solution of sample 1 and sample 2.

[0068] Mouse fibroblasts (L-929) were grown and digested with trypsin, and 100 μL of the cell suspension was inoculated into a 96-well plate (1×10 4cells / well), and the cells were incubated for 24 h (5% CO2, 37°C, >90% humidity) to form a semi-confluent monolayer. After 24 h of incubation, the culture medium was discarded, and physiological saline solution was added to the blank control group, and high-density polyethylene pellet extract was added to the negative control group. The positive control solution was 0.3% phenol solution. The test sample solution was added to the test group, 100 μL per well, and the CO2 incubator was placed for further 24 h. After the culture was completed, the cell culture medium in the plate was removed, and 50 μL of MTT solution with a mass concentration of 1 mg / mL was added to each well, and the culture was continued for 2 h. Then the MTT solution was discarded, 100 μL of isopropanol solution was added to each well, and the well was placed on a shaker for 10 min, and the absorbance was measured at a wavelength of 570 nm on a microplate reader. Cell viability = (100*OD570e) / OD570b

[0069] Where: OD570e--average optical density of 100% of the test sample;

[0070] OD570b--average blank optical density;

[0071] The cell survival rates of sample 1, sample 2 and sample 3 were determined to be 100.70%, 107.06% and 107.46%, respectively, all of which were greater than 70%, indicating that the adsorbent and antibody solution were non-cytotoxic whether used alone or in combination.

[0072] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A mutant of an immunoglobulin G degrading enzyme, characterized in that It has: (1) the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 4; or (2) An amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (1), and having the same or similar function as the amino acid sequence shown in (1); or (3) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (1) or (2).

2. The mutant according to claim 1, characterized in that It has: (4) the amino acid sequence shown in SEQ ID NO: 1; or (5) An amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (4), and having the same or similar function as the amino acid sequence shown in (4); or (6) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (4) or (5).

3. A nucleic acid molecule encoding the mutant according to claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that It has: (7) the nucleotide sequence shown in SEQ ID NO: 5 to SEQ ID NO: 8; or (8) A nucleotide sequence obtained by substituting, deleting or adding one or more bases of the nucleotide sequence shown in (7), and having the same or similar function as the nucleotide sequence shown in (7); or (9) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (7) or (8).

5. A recombinant plasmid, characterized in that: include: A nucleic acid molecule according to claim 3 or 4.

6. A host, characterized in that Transform and / or transfect the recombinant plasmid as claimed in claim 5.

7. A method for preparing a mutant of an immunoglobulin G degrading enzyme, characterized in that: The host according to claim 6 is inoculated, cultured, induced, expressed and purified to obtain the mutant.

8. Use of the mutant according to claim 1 or 2, the nucleic acid molecule according to claim 3 or 4, the recombinant plasmid according to claim 5, the host according to claim 6 and / or the mutant obtained by the preparation method according to claim 7 in any of the following items; ( ), in the preparation of products for degradation and / or removal of therapeutic antibodies and their immune complexes in vivo; and / or ( ), in the preparation of products for the prevention and / or treatment of autoantibody diseases; and / or ( ), in the preparation of products that improve the efficacy of drugs.

9. The use according to claim 8, characterized in that The degradation and / or clearance comprises the following steps: administering a protein A immunosorbent to a subject, clearing the in vivo therapeutic antibody and its immune complex, administering the mutant to the subject, and administering the protein A immunosorbent again.

10. A product, characterized in that include: The mutant according to claim 1 or 2, the nucleic acid molecule according to claim 3 or 4, the recombinant plasmid according to claim 5, the host according to claim 6 and / or the mutant obtained by the preparation method according to claim 7.

Images