Antibodies, fusion proteins and uses thereof

Abstract

The present invention provides an antibody, a fusion protein and use thereof. The antibody comprises at least one of the following CDR sequences: light chain variable region CDR sequences: SEQ ID NOs: 1-3, 7-9 and 83-85; heavy chain variable region CDR sequences: SEQ ID NOs: 4-6, 10-12, 86-88 and 89-91. The fusion protein comprises an antibody F C segment or a neutral antibody comprising the aforementioned antibody, and one or more PLA2R antigen epitope peptides linked to the neutral antibody. The provided antibody has low antigen binding activity and high expression level.

Description

[Technical field]

[0001] The present invention relates to the fields of molecular biology and medicine, specifically, the present invention relates to antibodies, fusion proteins and uses thereof, more specifically, the present invention relates to antibodies and their nucleic acid molecules, expression vectors, recombinant cells and fusion proteins and their nucleic acid molecules, expression vectors, recombinant cells, pharmaceutical compositions and uses. [Background technology]

[0002] Membranous nephropathy (MN), specifically, refers to a nephropathy syndrome caused by excessive deposition of immune complexes in glomerular capillaries. It is one of the most common pathological types in adult nephropathy syndromes, with a worldwide incidence of approximately 12 / million (Mc Grogan A, Franssen CF, de Vries CS. Nephrol Dial Transplant. 2011; 26(2): 414-430). In recent years, the incidence of MN has increased at a rate of 13% per year (Lwezaula BF, Ameh OI, Ekrikpo UE, et al. BMC Nephrol. 2021; 22(1): 15), becoming the second most common cause of chronic glomerulonephritis. Clinically, MN mainly presents with massive proteinuria, hypoproteinemia, edema and hyperlipidemia, and diagnosis depends on renal biopsy histopathological examination. Its pathological diagnostic features include thickening of the glomerular capillary walls, deposition of IgG and complement C3 along the capillary walls under immunofluorescence conditions, and formation of electron compacts beneath the glomerular epithelial cells under electron microscopy.

[0003] According to the etiology, MN can be divided into idiopathic membranous nephropathy (IMN) and secondary membranous nephropathy (SMN). IMN is the main type of MN, accounting for about 70% to 80% of cases, and its etiology is still unknown, so it is also called primary membranous nephropathy (PMN). Currently, the pathogenesis of PMN is not yet fully clear, but many scholars believe that PMN is an antibody-mediated autoimmune glomerular disease, in which target antigens in podocytes are recognized by autoantibodies, which bind to them to form immune complexes that are deposited under the basement membrane of podocytes, activating the complement system and causing podocyte damage and shedding, which increases the permeability of the basement membrane and results in the appearance of large amounts of proteinuria.

[0004] In 2009, Beck et al. reported for the first time that phospholipase A2 receptor (PLA2R) is one of the major antigens involved in PMN development, and anti-phospholipase A2 receptor antibody (PLA2R-Ab) could be detected in the blood and renal tissue eluates of 70% of PMN patients (Beck LH Jr, Bonegio RG, Lambeau G, et al. N Engl J Med. 2009; 361(1): 11-21), which attracted widespread attention to PLA2R in the medical community. PLA2R-Ab can specifically bind to the target antigen (i.e., PLA2R) on podocytes in kidney tissue and can be expressed in the sera of 70% to 80% of PMN patients (Bobart SA, De Vriese AS, Pawar AS, et al. Kidney Int. 2019; 95(2): 429-438). Meanwhile, the positive expression rate of PLA2R-Ab antibodies in the sera of healthy subjects and patients with SMN and other glomerular diseases is extremely low, so the specificity of PLA2R-Ab diagnosis of MN is high. According to the latest Meta analysis (Len Mingjian, Lu Rong, Zhang Jiaxin, et al. Journal of Clinical Laboratory Science, 2017, 35 (7):545-549), the sensitivity of PMN diagnosis by serum PLA2R-Ab detection was 69%, and the total specificity was 97%, explaining that serum PLA2R-Ab has good sensitivity and high specificity for PMN and can be used as a specific biomarker to diagnose PMN.

[0005] In addition, a large amount of research has shown that the serum PLA2R-Ab titer level of MN patients is negatively correlated with the patients' glomerular filtration rate, serum total protein and albumin and other indicators, and is also significantly negatively correlated with the spontaneous remission rate of PMN patients, indicating that mild patients with no PLA2R-Ab or low PLA2R-Ab levels can be naturally alleviated. During treatment with immunosuppressants, patients with serum PLA2R-Ab positive can respond to immunosuppressive treatment within a short time, but patients with serum PLA2R-Ab positive are less sensitive to immunosuppressants. After receiving immunosuppressive treatment, their serum PLA2R-Ab levels also decrease continuously, suggesting that PLA2R-Ab plays an important role in evaluating the therapeutic effect. After entering remission, failure to become negative for serum PLA2R-Ab or an increase in titer indicates that disease may relapse (Xu X, Nie S, Ding H, et al. Nat Rev Nephrol. 2018; 14(5): 313~324). For MN patients who underwent kidney transplantation, those who developed circulating PLA2R-Ab were more likely to experience early disease relapse (Passerini P, Malvica S, Tripodi F, et al. Front Immunol. 2019; 10: 1326), suggesting that postoperative relapse may also be related to PLA2R-Ab. In addition, the results of the study showed that the negativity rate of PLA2R-Ab showed a significant positive correlation with the total remission rate, and PLA2R-Ab predicted the sensitivity, specificity and accuracy rates of 12-month clinical remission events in MN patients were 69.51%, 76.35% and 80.26%, respectively (Zheng Na, Wang Meihua, An Zhi. Journal of Clinical Military Medicine 2021; 49(08): 942~944).

[0006] From the above, PLA2R-Ab has important reference value in the clinical diagnosis, disease condition judgment and prognosis evaluation of PMN patients. PLA2R-Ab is involved in the disease progression of PMN, and patients with low PLA2R-Ab titers have milder disease, more pronounced effects after immunosuppressant treatment, and may even undergo spontaneous remission. Therefore, it is suggested that monitoring PLA2R-Ab levels can help clinicians guide patients on the dosage and duration of immunosuppressants, avoid excessive immunosuppressive treatment, and at the same time, reduce the medical burden on patients. The higher the serum PLA2R-Ab level, the stronger the immune activity of the body, the more serious the disease, the worse the therapeutic effect of immunosuppression, and the worse the clinical prognosis. It shows that monitoring serum PLA2R-Ab is useful for guiding clinical treatment. Patients admitted with high levels of PLA2R-Ab should be treated with early and effective intervention.

[0007] At present, the treatment of MN in clinical practice mainly involves a low-fat, low-salt, low-protein diet, symptomatic supportive therapy to control proteinuria and hyperlipidemia, and supplemented with immunosuppressive therapy and some non-immunosuppressive therapy, but there is still no reliable treatment method. In addition, the treatment effect of patients with membranous nephropathy is poor, and only 40% of patients can achieve spontaneous remission, while 30% of patients have poor treatment effect and eventually progress to end-stage renal disease that can only be maintained by dialysis or transplantation, which affects the economic burden and quality of life of patients. For kidney transplant patients, about 40% of kidney transplant patients have poor treatment effect, and some of them lose the transplanted kidney. Based on the pathogenesis of PMN and the important value of serum PLA2R-Ab level in evaluating the treatment effect and clinical prognosis of PMN, drugs or technologies that can effectively treat PLA2R-Ab positive MN patients are urgently needed. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention solves at least to some extent one of the technical problems existing in the prior art. Therefore, the present invention provides an antibody and a fusion protein that binds to an autoimmune antigen of primary membranous nephropathy, the antibody of the present invention has low antigen binding activity, and the fusion protein of the present invention can specifically bind to a PLA2R antibody. [Means for solving the problem]

[0009] The present invention has been completed based on the following findings of the inventors.

[0010] One possible treatment method for PLA2R antibody-positive membranous nephropathy is to specifically neutralize PLA2R-Ab in serum.The inventors have surprisingly found that the fusion protein of PLA2R and antibody developed in this application (the fusion protein of antigen and antibody in this application is called antigenic antibody) can achieve the technical effect of specifically neutralizing PLA2R-Ab in serum.

[0011] PLA2R is a membrane protein that is highly expressed on the surface of glomerular podocytes and contains the structural domains shown in Figure 1. At the same time, there is a small amount of secreted PLA2R in the blood. According to a study by Liyo Kao et al. (Liyo Kao, Vinson Lam, et. al., J Am Soc Nephrol. 2015 Feb; 26(2):291-301), antibodies in the patient's plasma mainly recognize three structural domains in PLA2R: CysR, FNII, and CTLD1.

[0012] Based on this, the inventor has found that the fusion protein obtained by fusing the antigen region of PLA2R with an antibody can maintain the binding ability between PLA2R and PLA2R-Ab. In addition, in the course of research and development, the inventor has found that the CDR region of the mutated antibody makes the resulting antibody have extremely low antigen binding activity, avoiding side effects caused by subsequent non-specific binding, and has a higher expression level. Therefore, the fusion protein obtained by fusing the mutated antibody with the antigen region of PLA2R can be used to specifically neutralize PLA2R-Ab, and does not bind to other antibodies, avoiding side effects caused by non-specific immunity. In addition, the fusion protein has the advantage of a long half-life, which can reduce the frequency of administration.

[0013] In a first aspect of the present invention, the present invention provides an antibody. According to an embodiment of the present invention, the antibody comprises a light chain variable region CDR and a heavy chain variable region CDR, the heavy chain variable region CDR1 sequence is represented as GX1X2X3X4X5X6X7X8X9 and the heavy chain variable region CDR2 sequence is represented as X 10 X 11 X 12 X 13 X 14 X 15 GX 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 The heavy chain variable region CDR3 sequence is shown in 24 GGX 25 X 26 X 27 X 28 X 29 X 30 X 31 X1 is G or F, X2 is G or N, X3 is G or I, X4 is G, K or S, X5 is G or D, X6 is G or T, X7 is G or Y, X8 is G or I, X9 is H or S, X 10 is R or G, X 11 is G, I, X 12 is G, Y, X 13 is G, P, or X 14 is T, S, X 15is G, N, or X 16 is G, Y, X 17 is G, T, X 18 is G, S or R, X 19 is G, Y, X 20 is G, A, X 21 is G, D, X 22 is G, S, or X 23 is S, V, X 24 is G, W, or X 25 is G, D, X 26 is G, S, or X 27 is G, F, X 28 is G, Y, X 29 is G, A, X 30 is G, M, X 31 is S, G or D, and the conditions are: X1 is F, X2 is N, X3 is I, X4 is K, X5 is D, X6 is T, X7 is Y, X8 is I, X9 is H, X 10 is R, X 11 is I, X 12 is Y, X 13 P, X 14 is T, X 15 is N, X 16 is Y, X 17 is T, X 18 is R, X 19 is Y, X 20 A, X 21 D, X 22 is S, X 23 V, X 24 is W, X 25 D, X 26 G, X 27 F, X 28 is Y, X 29 A, X 30 is M, X 31 and D, and the light chain variable region CDR1 sequence is X 32 X 33 X 34 X 35 X 36 X 37 X 38 X 39 X 40 X 41 The light chain variable region CDR2 sequence is shown in A. 42 X 43 X44 X 45 X 46 X 47 The light chain variable region CDR3 sequence is shown in S. 48 X 49 X 50 X 51 X 52 X 53 X 54 X 55 X 56 As shown in X 32 is G, R, X 33 is G, A, X 34 is G, S, or X 35 is G, Q, or X 36 is G, D or S, X 37 is G, V, X 38 is G, N, or X 39 is G, T, X 40 is G, A, X 41 is S, V, X 42 is G, S, or X 43 is G, A, X 44 is G, S, or X 45 is G, F, X 46 is L, S, X 47 is G, Y, X 48 is G, Q, or X 49 is G, Q, or X 50 is G, H, X 51 is G, Y, X 52 is S, T, X 53 is G, T, X 54 is G, P, or X 55 is G, P, or X 56 is G or T, and the condition is X 32 is R, X 33 A, X 34 is S, X 35 Q, X 36 D, X 37 V, X 38 is N, X 39 is T, X 40 A, X 41 V, X 42 is S, X 43 A, X 44 is S, X 45 F, X 46 L, X47 is Y, X 48 Q, X 49 Q, X 50 H, X 51 is Y, X 52 is T, X 53 is T, X 54 P, X 55 P, X 56 However, it is not true that both are T. As a result of extensive experiments by the inventors, it is found that the above antibody has low antigen binding activity, and after forming an antigenic antibody, it can effectively avoid non-specific binding of the immune antibody, and more importantly, the expression amount of the antibody is high, which is suitable for the development of antigenic antibody drugs.

[0014] In a second aspect of the present invention, the present invention provides a first nucleic acid molecule. According to an embodiment of the present invention, the first nucleic acid molecule encodes an antibody according to the first aspect. The antibody encoded by the first nucleic acid molecule according to the embodiment of the present invention has low antigen-binding activity, and after forming an antigenic antibody, can effectively avoid non-specific binding of the immune antibody, and at the same time, the expression level of the antibody is high, which is suitable for the development of an antigenic antibody drug.

[0015] In a third aspect of the present invention, the present invention provides a first expression vector. According to an embodiment of the present invention, the first expression vector carries a first nucleic acid molecule according to the second aspect. After the first expression vector according to the embodiment of the present invention is introduced into a suitable recipient cell, the expression of the antibody with low antigen binding activity according to the first aspect can be effectively achieved through a control system, and the antibody can be obtained in large quantities.

[0016] In a fourth aspect of the present invention, the present invention provides a first recombinant cell. According to an embodiment of the present invention, the first recombinant cell carries a first nucleic acid molecule according to the second aspect or expresses an antibody according to the first aspect. The first recombinant cell according to an embodiment of the present invention can be used for in vitro expression and large-scale production of an antibody with low antigen-binding activity according to the first aspect.

[0017] In the fifth aspect of the present invention, the present invention provides the use of the antibody according to the first aspect in the preparation of a fusion protein drug.As can be seen from the above, the above antibody of the present invention has no antigen binding activity and is used as a neutral drug vector to prepare a drug, which is used to treat or prevent a disease.For example, the above antibody can be fused with an antigen epitope peptide to prepare a fusion protein, and since the above antibody has no antigen binding activity, the prepared fusion protein specifically binds to the antibody corresponding to the antigen epitope peptide and does not bind to other antibodies, thereby avoiding the side effects of non-specific binding of the fusion protein, and can be used to treat or prevent related diseases caused by the antibody corresponding to the antigen epitope peptide.

[0018] In the sixth aspect of the present invention, the present invention provides the use of the above-mentioned antibody as a drug vector or negative antibody.As can be seen from the above, the above-mentioned antibody of the present invention has no antigen binding activity, and can be used as a neutral drug vector to prepare drugs, or can be referred to as a negative antibody that has no direct antigen binding activity.

[0019] In a seventh aspect of the present invention, the present invention provides a fusion protein. According to an embodiment of the present invention, the fusion protein comprises a neutral antibody comprising an antibody FC segment or an antibody according to the first aspect, and one or more PLA2R antigen epitope peptides connected to the neutral antibody. The inventors have found through experiments that by fusing the PLA2R antigen epitope peptides with the neutral antibody, the resulting fusion protein can specifically bind to the PLA2R antibody and does not bind to other antibodies, avoiding the side effects of non-specific binding, and has the advantages of good expression and high production.

[0020] In an eighth aspect of the present invention, there is provided a second nucleic acid molecule, which according to an embodiment of the present invention encodes a fusion protein according to the seventh aspect, and the second nucleic acid molecule can be effectively used to express the fusion protein.

[0021] In a ninth aspect of the present invention, there is provided a second expression vector, which according to an embodiment of the present invention carries a second nucleic acid molecule according to the eighth aspect, which can be effectively used to express the fusion protein.

[0022] In a tenth aspect of the invention, the invention provides a second recombinant cell. According to an embodiment of the invention, the second recombinant cell carries a second nucleic acid molecule according to the eighth aspect or expresses a fusion protein according to the seventh aspect. The recombinant cell according to an embodiment of the invention may be used for the in vitro expression and large scale production of the fusion protein according to the fifth aspect.

[0023] In an eleventh aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises the fusion protein according to the seventh aspect. After extensive experiments, the inventors have found that the pharmaceutical composition can effectively prevent and treat PLA2R antibody-related diseases, such as PLA2R antibody-positive membranous nephropathy.

[0024] In a twelfth aspect of the present invention, there is provided an immunoadsorbent. According to an embodiment of the present invention, the immunoadsorbent comprises the fusion protein according to the seventh aspect. After extensive experiments, the inventors have found that the immunoadsorbent can adsorb PLA2R antibodies, and can effectively treat PLA2R antibody-related diseases, such as PLA2R antibody-positive membranous nephropathy, through immunoadsorption.

[0025] In the thirteenth aspect of the present invention, the present invention provides a reagent kit. According to an embodiment of the present invention, the reagent kit comprises the fusion protein according to the seventh aspect. After extensive experiments, the inventors have found that the reagent kit can effectively detect the content of PLA2R antibody, and can also be used to diagnose or treat PLA2R antibody-related diseases, such as PLA2R antibody-positive membranous nephropathy.

[0026] In a fourteenth aspect of the present invention, the present invention provides an adsorption product. According to an embodiment of the present invention, the adsorption product includes the above-mentioned immunoadsorbent and a solid-phase vector connected to the immunoadsorbent. The adsorption product of the present invention can be used to adsorb PLA2R antibodies, for example, PLA2R antibodies in blood or plasma, and the adsorption of PLA2R antibodies in blood or plasma can be used to prevent or treat diseases associated with PLA2R antibodies.

[0027] In a fifteenth aspect of the invention, the invention provides the use of an immunoadsorbent according to the twelfth aspect in the preparation of an adsorption product for adsorbing PLA2R antibodies.

[0028] In a sixteenth aspect of the present invention, the present invention provides the use of a fusion protein according to the seventh aspect, a second nucleic acid molecule according to the eighth aspect, a second expression vector according to the ninth aspect, a second recombinant cell according to the tenth aspect, a pharmaceutical composition according to the eleventh aspect or an immunoadsorbent according to the twelfth aspect in the preparation of a medicament for preventing or treating a PLA2R antibody-related disease.

[0029] In a 17th aspect of the present invention, the present invention provides the use of a fusion protein according to the seventh aspect, a second nucleic acid molecule according to the eighth aspect, a second expression vector according to the ninth aspect, a second recombinant cell according to the tenth aspect or a pharmaceutical composition according to the eleventh aspect in the preparation of an immunoadsorbent for adsorbing PLA2R antibodies.

[0030] In an 18th aspect of the present invention, the present invention provides the use of a preparation of a fusion protein according to the seventh aspect, a second nucleic acid molecule according to the eighth aspect, a second expression vector according to the ninth aspect, a second recombinant cell according to the tenth aspect, a pharmaceutical composition according to the eleventh aspect or an immunoadsorbent according to the twelfth aspect in a reagent kit for detecting PLA2R antibodies.

[0031] In a nineteenth aspect of the present invention, the present invention provides a method for preventing or treating a PLA2R antibody-associated disease. According to an embodiment of the present invention, the method comprises administering to a subject a pharma- ceutically acceptable amount of the fusion protein according to the seventh aspect or the pharmaceutical composition according to the eleventh aspect. This allows for effective treatment of a PLA2R antibody-associated disease.

[0032] In a twentieth aspect of the present invention, there is provided an immunoadsorption therapeutic method for preventing or treating a PLA2R antibody-related disease, which method, according to an embodiment of the present invention, comprises treating a subject with an immunoadsorbent according to the twelfth aspect.

[0033] In the twenty-first aspect of the present invention, the present invention provides a method for diagnosing a disease associated with PLA2R antibody. According to an embodiment of the present invention, the method includes the steps of detecting PLA2R antibody in a sample to be measured using the fusion protein according to the seventh aspect, the second nucleic acid molecule according to the eighth aspect, the second expression vector according to the ninth aspect, the second recombinant cell according to the tenth aspect, the pharmaceutical composition according to the eleventh aspect, the immunoadsorbent according to the twelfth aspect, or the reagent kit according to the thirteenth aspect, and determining the content of PLA2R antibody in the sample to be measured based on the detection result.

[0034] In the twenty-second aspect of the present invention, the present invention provides a method for evaluating the stage of a disease associated with PLA2R antibody. According to an embodiment of the present invention, the method includes the steps of detecting PLA2R antibody in a sample to be measured using the fusion protein according to the seventh aspect, the second nucleic acid molecule according to the eighth aspect, the second expression vector according to the ninth aspect, the second recombinant cell according to the tenth aspect, the pharmaceutical composition according to the eleventh aspect, the immunoadsorbent according to the twelfth aspect, or the reagent kit according to the thirteenth aspect, and determining the content of PLA2R antibody in the sample to be measured based on the detection result.

[0035] In the twenty-third aspect of the present invention, the present invention provides a method for evaluating the prognosis of a related disease by PLA2R antibody. According to an embodiment of the present invention, the method includes the steps of detecting PLA2R antibody in a sample to be measured using the fusion protein described in the seventh aspect, the second nucleic acid molecule described in the eighth aspect, the second expression vector described in the ninth aspect, the second recombinant cell described in the tenth aspect, the pharmaceutical composition described in the eleventh aspect, the immunoadsorbent described in the twelfth aspect, or the reagent kit described in the thirteenth aspect, and determining the content of PLA2R antibody in the sample to be measured based on the detection result.

[0036] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present application. [Brief description of the drawings]

[0037] The above and / or additional aspects and advantages of the present invention will become apparent and will be readily understood from the following detailed description of the embodiments taken in conjunction with the drawings. [Figure 1] FIG. 1 is a schematic diagram showing the structure of an expression frame in Example 1 of the present invention. [Diagram 2] FIG. 2 shows the results of electrophoresis of pcDNA3.4, trsH, trsL, RtxH and RtxL in Example 1 of the present invention. [Diagram 3] FIG. 2 shows the results of electrophoresis of dTrs1H, dTrs2H, and dTrs1L (dTrs2L) in Example 1 of the present invention. [Figure 4] FIG. 2 shows the results of electrophoresis of dTrs3H and dTrs3L in Example 1 of the present invention. [Diagram 5] FIG. 2 shows the results of electrophoresis of dTrs4H and dTrs4L in Example 1 of the present invention. [Figure 6] 1 shows the purification effect of Trs, dTrs1 to 4, and Rtx proteins detected by SDS-PAGE in Example 1 of the present invention. [Figure 7] 1 shows the detection results of binding of Trs, dTrs1 to 4, and Rtx proteins to Her2 in Example 1 of the present invention. [Figure 8] FIG. 2 is a schematic diagram showing the structures of RC1 to RC8 proteins in Example 2 of the present invention. [Figure 9] FIG. 1 is a schematic diagram of the structure of PLA2R in Example 2 of the present invention. [Figure 10] FIG. 1 shows the results of electrophoresis of pcDNA3.4 in Example 2 of the present invention. [Figure 11] FIG. 1 shows the results of electrophoresis of pcDNA3.4, RC1H, RC2L, RC4H and RC5L in Example 2 of the present invention. [Figure 12] FIG. 1 shows the results of electrophoresis of RC3 and RC6 in Example 2 of the present invention. [Figure 13] 1 shows the purification effect of SDS-PAGE detection for RC1 to RC8 proteins in Example 2 of the present invention. [Figure 14] RC1 to RC8 in Example 2 of the present invention represent the neutralizing effect against PLA2R-Ab in pMN-2 plasma of a patient with positive membranous nephropathy. [Figure 15] RC1 to RC8 in Example 2 of the present invention represent the neutralizing effect against PLA2R-Ab in pMN-4 plasma of membranous nephropathy positive patients. [Figure 16] RC1 to RC8 in Example 2 of the present invention represent the neutralizing effect against PLA2R-Ab in the plasma of pMN-7 patients positive for membranous nephropathy. [Figure 17] RC3 and RC6 in Example 2 of the present invention are removal rates for PLA2R-Ab adsorption in plasma samples from PMN-4 patients. [Figure 18] RC3 and RC6 in Example 2 of the present invention are removal rates for PLA2R-Ab adsorption in plasma samples from PMN-7 patients. [Figure 19] This is a graph showing the weight trend of mice subcutaneously injected with RC1, RC2, RC4, RC5, RC7, and RC8 proteins in Example 2 of the present invention. [Figure 20] FIG. 13 is a graph showing the weight trend of mice subcutaneously injected with RC3 and RC6 proteins in Example 2 of the present invention. [Figure 21]1 shows the detection results of binding of Trs, dTrs1 to 4, and Rtx proteins to IgA in Example 1 of the present invention. [Figure 22] 1 shows the detection results of binding of Trs, dTrs1 to 4, and Rtx proteins to IgM in Example 1 of the present invention. [Figure 23] 1 shows the detection results of binding of Trs, dTrs1 to 4, and Rtx proteins to HSA in Example 1 of the present invention. [Figure 24] FIG. 1 shows the electrophoretic results of the stability investigation of RC1 to RC8 in Example 2 of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Hereinafter, the present invention will be described in detail with reference to the examples. The examples described below are illustrative and are intended only to explain the present invention, and should not be understood as limiting the present invention.

[0039] It should be noted that the terms "first" and "second" are used for descriptive purposes only and cannot be understood as expressing or implying a relative importance or as indicating the number of technical features shown. Thus, a feature defined by "first" or "second" can explicitly or implicitly include at least one of the feature. In the description of the present invention, "plurality" means two or more than two, unless otherwise specified.

[0040] In this specification, the terms "comprises" and "including" are non-limiting expressions, i.e., they include the subject matter set forth in the present invention but do not exclude the subject matter of other embodiments.

[0041] As used herein, the terms "optionally," "optional," or "arbitrary" generally mean that the event or condition described below may occur, but does not necessarily occur, and the description includes cases where the event or condition occurs and cases where the event or condition does not occur.

[0042] As used herein, the term "antibody" generally refers to an immunoglobulin protein molecule that includes two light chains and two heavy chains, with the heavy (H) and light (L) chains connected by disulfide bonds to form a tetrapeptide molecule. The amino acid sequence at the amino terminus (N-terminus) of the peptide chain is highly variable and is called the variable region (V-region), while the carboxyl terminus (C-terminus) is relatively stable and less variable and is called the constant region (C-region). The V-regions of the L and H chains are called VL and VH, respectively. Within the variable regions, there are regions with a higher degree of variation in amino acid composition and sequence order, called hypervariable regions (HVRs). The hypervariable regions are also called complementarity-determining regions (CDRs) because they are the sites where antigens bind to antibodies. Both the heavy and light chain variable regions have three CDRs.

[0043] In this specification, the term "framework region" or "framework region" is abbreviated as FR, and both refer to the portion of the variable region of the heavy or light chain of an antibody other than CDR. The amino acid sequences of about 110 amino acids near the N-terminus of the H and L chains of an antibody vary greatly, while the amino acid sequences of other portions are relatively constant, so that the light and heavy chains can be distinguished into a variable region (V region) and a constant region (C region). The variable region includes a hypervariable region HVR (hypervariable region) or a complementarity-determining region CDR (complementarity-determining region) and a framework region, and the variability of the FR must be lower than that of the CDR. There are four FR molecules in total, FR1, FR2, FR3, and FR4, respectively. When recognizing an antibody, the four FR molecules curl to bring the CDR molecules closer to each other.

[0044] As used herein, the term "neutral antibody" refers to a protein structure with low antigen-binding activity, such as an Fc segment, or an antibody as proposed in the first aspect of the invention.

[0045] As used herein, the term "double-chain antibody" refers to an antibody with a symmetric structure, an antibody consisting of only two Fc fragments, or a complete antibody consisting of two heavy chains and two light chains, such as the antibody proposed in the first aspect of the invention.

[0046] As used herein, the terms "identity," "homology," or "similarity," in describing an amino acid sequence or a nucleic acid sequence relative to a reference sequence, refer to the percentage of identical amino acids or nucleotides between two amino acid or nucleic acid sequences as determined by conventional methods, e.g., Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5:Suppl.3 (National Biomedical Research Foundation, Washington, DC. Methods for comparing sequences and determining sequence identity include the homology comparison algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410).Computer programs utilizing these algorithms are also available, and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)), or GAP, BESTFIT, BLAST Altschul et al., FASTA, and TFASTA are available in the Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA, and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0047] As used herein, the term "nucleotide" generally refers to ribonucleotides, deoxynucleotides, or modified forms of either type of nucleotide, and combinations thereof.

[0048] As used herein, the term "at least 85% similar" means having at least 85% similarity to the respective reference sequence, which may be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%.

[0049] As used herein, the term "expression vector" generally refers to a nucleic acid molecule capable of self-replicating when inserted into a suitable host, and transferring the inserted nucleic acid molecule into and / or between host cells. The expression vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcribing and / or translating DNA or RNA. The expression vector further includes vectors having a variety of the above functions. The expression vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.

[0050] As used herein, the term "recombinant cell" refers to the modification or recombination of the genetic material of a host cell, typically using genetic engineering or cell fusion techniques, to obtain a cell with a unique trait with stable inheritance. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transformation" or "transfect" as used herein refer to the introduction of a nucleic acid (e.g., a vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of the present invention and used to express and / or secrete a target protein. Examples of suitable host cells that can be used in the present invention include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), insect cells, PER.C6 cells, and CoS cells.

[0051] As used herein, the term "fusion protein" refers to a new protein in which at least two proteins or polypeptides are fused together, and the above fusion operation can usually be realized by techniques such as genetic engineering, for example, an expression product in which two genes obtained by DNA recombination techniques are recombined. As used herein, it refers to a state in which PLA2R is fused to a neutral antibody in a two-chain structure. Note that the PLA2R fusion protein does not mean that all parts of the protein are formed by fusion, and for example, the two Fc segment parts in the two-chain structure are connected by a chemical bond such as a disulfide bond.

[0052] As used herein, the term "pharmaceutical composition" generally refers to a unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with a vector that constitutes one or more accessory ingredients. Typically, the composition is prepared by uniformly and thoroughly combining the active fusion protein with a liquid vector, a finely divided solid vector, or both.

[0053] As used herein, the term "pharmaceutical acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other ingredients comprising the formulation and / or with the mammal being treated therewith. Preferably, "pharmaceutical acceptable" according to the present invention means for use in animals, particularly humans, as approved by a federal regulatory agency or national government or as listed in the United States Pharmacopeia or other generally recognized pharmacopoeias.

[0054] As used herein, the term "pharmaceutical acceptable excipient" or "pharmaceutical acceptable vector" can include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a particular target dosage form. To the extent that a conventional excipient is incompatible with the fusion protein of the present invention, e.g., causes any adverse biological effect or interacts in a deleterious manner with any other component of the pharmaceutical acceptable composition, it is also contemplated by the present invention.

[0055] For other pharma- ceutically acceptable auxiliaries or other pharma- ceutically acceptable vectors and processes mentioned in this text, reference can be made to the many publications on the subject, in particular to Handbook of Pharmaceutical Excipients, Version 3, edited by Arthur H. Kibbe, American Pharmaceutical Association, Washington, USA and Pharmaceutical Press, London, and Lexikon der Hilfsstoffe fur Pharmazie, Kosmetik und angrenzende Gebiete, edited by HP Fiedler, Version 4, edited by Cantor, Aulendorf and earlier versions.

[0056] As used herein, the term "administration" refers to the introduction of a given amount of a substance into a patient in a suitable manner. The recombinant cells or pharmaceutical compositions of the present invention can be administered in any common manner, provided that they can reach the desired tissue. Various modes of administration are possible, such as peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplary modes of administration. Preferably, the compositions of the present invention are administered by intravenous or subcutaneous injection.

[0057] As used herein, the term "PLA2R antibody-associated disease" generally refers to a disease caused by abnormality in PLA2R antibodies, such as an increase in PLA2R antibodies, including but not limited to PLA2R antibody-positive membranous nephropathy. PLA2R is a membrane protein expressed in glomerular podocytes, which becomes an antigen for unknown reasons, and the corresponding autoimmune antibody (PLA2R antibody) is produced in the body. PLA2R antibody binds to PLA2R and deposits under the glomerular basement membrane, causing downstream effects such as C3 deposition, resulting in PLA2R antibody-positive membranous nephropathy.

[0058] As used herein, the term "treatment" means used to obtain a desired pharmacological and / or physiological effect. The effect may be preventative, in that it completely or partially prevents a disease or its symptoms, and / or may be therapeutic, in that it partially or completely cures a disease and / or the adverse effects of a disease. As used herein, "treatment" covers mammalian, particularly human, diseases and includes (a) preventing the disease or onset of a disease in an individual who is susceptible to the disease but has not yet been diagnosed with the disease, (b) suppressing the disease, such as preventing the progression of the disease, or (c) alleviating the disease, e.g., reducing symptoms associated with the disease. As used herein, "treatment" includes any administration of a fusion protein, pharmaceutical composition, or drug to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a disease in an individual, including, but not limited to, administering a drug comprising a fusion protein as described herein to an individual in need thereof.

[0059] The present invention provides antibodies, fusion proteins and uses thereof, each of which is described in detail below.

[0060] [Antibody, First Nucleic Acid Molecule, First Expression Vector, First Recombinant Cell, and Use Thereof]

[0061] The present invention mutates different sites in the CDR region of the Trastuzμmab (Trs) antibody, and through a large amount of experiments, the antibody of the present invention is obtained. The antibody binds very weakly to its antigen, Her2, and does not bind to any other antigens. The antibody has very low antigen binding activity, and can be fused as a neutral antibody with a biologically active molecule to obtain a fusion protein. The fusion protein can bind to a specific binding substance of the biologically active molecule. For example, the fusion protein obtained by binding the neutral antibody to PLA2R can specifically bind to PLA2R antibody and does not bind to other antibodies. When used as a drug, the side effects of non-specific binding are avoided. When the fusion protein obtained by fusing the neutral antibody with an antigen is used as a drug, it can bind to the specific antibody of the antigen, not bind to other antibodies, and can exclusively eliminate the cells that produce the specific antibody of the antigen.

[0062] Thus, in a first aspect of the present invention, the present invention provides an antibody. According to an embodiment of the present invention, the antibody comprises a light chain variable region CDR and a heavy chain variable region CDR, wherein the heavy chain variable region CDR1 sequence is represented by GX1X2X3X4X5X6X7X8X9 and the heavy chain variable region CDR2 sequence is represented by X 10 X 11 X 12 X 13 X 14 X 15 GX 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 The heavy chain variable region CDR3 sequence is shown in 24 GGX 25 X 26 X 27 X 28 X 29 X 30 X 31 X1 is G or F, X2 is G or N, X3 is G or I, X4 is G, K or S, X5 is G or D, X6 is G or T, X7 is G or Y, X8 is G or I, X9 is H or S, X 10 is R or G, X 11 is G, I, X12 is G, Y, X 13 is G, P, or X 14 is T, S, X 15 is G, N, or X 16 is G, Y, X 17 is G, T, X 18 is G, S or R, X 19 is G, Y, X 20 is G, A, X 21 is G, D, X 22 is G, S, or X 23 is S, V, X 24 is G, W, or X 25 is G, D, X 26 is G, S, or X 27 is G, F, X 28 is G, Y, X 29 is G, A, X 30 is G, M, X 31 is S, G or D, and the conditions are: X1 is F, X2 is N, X3 is I, X4 is K, X5 is D, X6 is T, X7 is Y, X8 is I, X9 is H, X 10 is R, X 11 is I, X 12 is Y, X 13 P, X 14 is T, X 15 is N, X 16 is Y, X 17 is T, X 18 is R, X 19 is Y, X 20 A, X 21 D, X 22 is S, X 23 V, X 24 is W, X 25 D, X 26 G, X 27 F, X 28 is Y, X 29 A, X 30 is M, X 31 and D, and the light chain variable region CDR1 sequence is X 32 X 33 X 34 X 35 X 36 X 37 X 38 X 39 X40 X 41 The light chain variable region CDR2 sequence is shown in A. 42 X 43 X 44 X 45 X 46 X 47 The light chain variable region CDR3 sequence is shown in S and X 48 X 49 X 50 X 51 X 52 X 53 X 54 X 55 X 56 As shown in X 32 is G, R, X 33 is G, A, X 34 is G, S, or X 35 is G, Q, or X 36 is G, D or S, X 37 is G, V, X 38 is G, N, or X 39 is G, T, X 40 is G, A, X 41 is S, V, X 42 is G, S, or X 43 is G, A, X 44 is G, S, or X 45 is G, F, X 46 is L, S, X 47 is G, Y, X 48 is G, Q, or X 49 is G, Q, or X 50 is G, H, X 51 is G, Y, X 52 is S, T, X 53 is G, T, X 54 is G, P, or X 55 is G, P, or X 56 is G or T, and the condition is X 32 is R, X 33 A, X 34 is S, X 35 Q, X 36 D, X 37 V, X 38 is N, X 39 is T, X 40 A, X 41 V, X 42 is S, X43 A, X 44 is S, X 45 F, X 46 L, X 47 is Y, X 48 Q, X 49 Q, X 50 H, X 51 is Y, X 52 is T, X 53 is T, X 54 P, X 55 P, X 56 However, it is not possible that both are T. As a result of a large amount of experiments, the inventors have found that the above antibody has the advantages of extremely low antigen binding activity and high expression level.

[0063] According to an embodiment of the present invention, the heavy chain variable region CDR and the light chain variable region CDR are: X4 is G or S; X5 is G; 21 is G, X 31 is S or G, and X 36 satisfies at least one of the conditions that it is G or S.

[0064] According to an embodiment of the present invention, in the heavy chain variable region CDR, X1 is G or F, X2 is G or N, X3 is G or I, X4 is G or S, X5 is G, X6 is G or T, X7 is G or Y, X8 is G or I, X9 is H or S, and X 10 G, X 11 is G, I, X 12 is G, Y, X 13 is G, P, or X 14 is T, S, X 15 is G, N, or X 16 G, X 17 is G, T, X 18 is G, S, or X 19 is G, Y, X 20 is G, A, X 21 G, X 22 is G, S, or X 23 is S, V, X 24 G, X 25 is G, D, X 26 is G, S, or X 27 is G, F, X28 G, X 29 is G, A, X 30 is G, M, X 31 is S or G, and in the light chain variable region CDR, X 32 is G, R, X 33 is G, A, X 34 is G, S, or X 35 is G, Q, or X 36 is G, S, or X 37 is G, V, X 38 G, X 39 is G, T, X 40 is G, A, X 41 is S, V, X 42 is G, S, or X 43 is G, A, X 44 is G, S, or X 45 G, X 46 is L, S, X 47 is G, Y, X 48 is G, Q, or X 49 is G, Q, or X 50 is G, H, X 51 G, X 52 is S, T, X 53 is G, T, X 54 is G, P, or X 55 is G, P, or X 56 is G or T.

[0065] According to an embodiment of the present invention, the antibody comprises at least one of the following CDR sequences: light chain variable region CDR sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:83, SEQ ID NO:84 and SEQ ID NO:85; heavy chain variable region CDR sequences: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90 and SEQ ID NO:91. RASQGVGTAVA(SEQ ID NO:1)。 SASGLYS(SEQ ID NO:2)。 QQHGTTPPT(SEQ ID NO:3)。 GFNIGGTGIH(SEQ ID NO:4)。 GIYPTNGGTGYAGSV(SEQ ID NO:5)。 GGGDGFGAMGY(SEQ ID NO:6)。 GGGGSGGGGSA(SEQ ID NO:7)。 GGGGSGS(SEQ ID NO:8)。 GGGGSGGGG(SEQ ID NO:9)。 GGGGSGGGGS(SEQ ID NO:10)。 GGGGSGGGGSGGGGS(SEQ ID NO:11)。 GGGGSGGGGSY(SEQ ID NO:12)。 RASQDVGTAVA (SEQ ID NO:83)。 SASGLYS (SEQ ID NO:84)。 QQHGTTPPT (SEQ ID NO:85)。 GFNIKDTYIH (SEQ ID NO:86)。 RIYPTNGGTRYADSV (SEQ ID NO:87)。 WGGDGFGAMDY (SEQ ID NO:88)。 GFNIKDTGIH (SEQ ID NO:89)。 GIYPTNGGTGYADSV (SEQ ID NO:90)。 GGGDGFGAMDY (SEQ ID NO:91)。

[0066] In some other embodiments, the antibody provided by the present invention has conservative amino acid substitutions compared to the heavy and light chains. "Conservative amino acid substitution" refers to the substitution of an amino acid with a biologically, chemically or structurally similar residue to another amino acid. Biologically similar refers to the substitution not destroying the low antigen-binding biological activity of the antibody. Structurally similar refers to the amino acid having a side chain with a similar length, such as alanine, glycine or serine, or a side chain with a similar size. Chemical similarity refers to the amino acid having the same charge or being hydrophilic or hydrophobic. For example, hydrophobic residues isoleucine, valine, leucine or methionine are substituted for each other. Or polar amino acids such as arginine instead of lysine, glutamic acid instead of aspartic acid, asparagine instead of glutamine, and serine instead of threonine are used.

[0067] According to an embodiment of the present invention, the light chain variable region CDR sequence comprises at least one of amino acid sequences having at least 85% similarity to SEQ ID NOs:1-3, SEQ ID NOs:7-9 and SEQ ID NOs:83-85, and the heavy chain variable region CDR sequence comprises at least one of amino acid sequences having at least 85% similarity to SEQ ID NOs:4-6, SEQ ID NOs:10-12, SEQ ID NOs:86-88 and SEQ ID NOs:89-91.

[0068] According to an embodiment of the present invention, the antibody comprises light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs:1, 2 and 3, respectively, or amino acid sequences having at least 85% similarity to SEQ ID NOs:1, 2 and 3, respectively; or comprises light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs:7, 8 and 9, respectively, or amino acid sequences having at least 85% similarity to SEQ ID NOs:7, 8 and 9, respectively; or comprises light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs:83, 84 and 85, respectively, or amino acid sequences having at least 85% similarity to SEQ ID NOs:83, 84 and 85, respectively.

[0069] According to an embodiment of the present invention, the antibody comprises heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 4, 5 and 6, or amino acid sequences having at least 85% similarity to SEQ ID NOs: 4, 5 and 6, respectively; or comprises heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 10, 11 and 12, or amino acid sequences having at least 85% similarity to SEQ ID NOs: 10, 11 and 12, respectively; or comprises heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 86, 87 and 88, or amino acid sequences having at least 85% similarity to SEQ ID NOs: 86, 87 and 88, respectively; or comprises heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 89, 90 and 91, or SEQ ID NOs: 89, 90 and 91, and contains the heavy chain variable region CDR1, CDR2 and CDR3 sequences shown in the amino acid sequences having at least 85% similarity thereto.

[0070] According to an embodiment of the present invention, the antibody comprises light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 1, 2 and 3, respectively, or amino acid sequences having at least 85% similarity to SEQ ID NOs: 1, 2 and 3, and heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 4, 5 and 6, respectively, or amino acid sequences having at least 85% similarity to SEQ ID NOs: 4, 5 and 6, respectively; or light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 7, 8 and 9, respectively, or amino acid sequences having at least 85% similarity to SEQ ID NOs: 7, 8 and 9, respectively; and heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 10, 11 and 12, respectively, or amino acid sequences having at least 85% similarity to SEQ ID NOs: 10, 11 and 12, respectively; or comprising light chain variable region CDR1, CDR2, CDR3 sequences as set forth in SEQ ID NOs: 83, 84 and 85 or amino acid sequences having at least 85% similarity to SEQ ID NOs: 83, 84 and 85, respectively, and heavy chain variable region CDR1, CDR2, CDR3 sequences as set forth in SEQ ID NOs: 86, 87 and 88 or amino acid sequences having at least 85% similarity to SEQ ID NOs: 86, 87 and 88, respectively; or comprising light chain variable region CDR1, CDR2, CDR3 sequences as set forth in SEQ ID NOs: 83, 84 and 85 or amino acid sequences having at least 85% similarity to SEQ ID NOs: 83, 84 and 85, respectively, and heavy chain variable region CDR1, CDR2, CDR3 sequences as set forth in SEQ ID NOs: 89, 90 and 91 or amino acid sequences having at least 85% similarity to SEQ ID NOs: 89, 90 and 91, respectively.

[0071] According to an embodiment of the present invention, the antibody comprises at least one of a heavy chain skeletal region sequence and a light chain skeletal region sequence, and at least a portion of at least one of the heavy chain skeletal region sequence and the light chain skeletal region sequence is derived from at least one of a mouse-derived antibody, a human-derived antibody, a primate-derived antibody or a variant thereof, preferably a human-derived antibody.

[0072] According to an embodiment of the present invention, the antibody has a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:95, and / or the antibody has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:96 or SEQ ID NO:97. The inventors have found that the CDR regions of the heavy chain variable region sequences (e.g., as set forth in SEQ ID NOs:4-6, SEQ ID NOs:10-12) and the CDR regions of the light chain variable region sequences (e.g., as set forth in SEQ ID NOs:1-3, SEQ ID NOs:7-9) of the above antibodies can be obtained by using antibody sequence matching databases (NCBI, IMGT) or related software. DIQMTQSPSSLSASVGDRVTITCRASQGVGTAVAWYQQKPGKAPKLLIYSASGLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHGTTPPTFGQGTKVEIK(SEQ ID NO:13). DIQMTQSPSSLSASVGDRVTITCGGGGSGGGGSAWYQQKPGKAPKLLIYGGGGSGSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCGGGGSGGGGFGQGTKVEIK (SEQ ID NO:14). EVQLVESGGGLVQPGGSLRLSCAASGFNIGGTGIHWVRQAPGKGLEWVAGIYPTNGGTGYAGSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRGGGDGFGAMGYWGQGTLVTV(SEQ ID NO:15). EVQLVESGGGLVQPGGSLRLSCAASGGGGSGGGGSWVRQAPGKGLEWVAGGGGSGGGGSGGGGSKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRGGGGSGGGGSYWGQGTLVTV(SEQ ID NO:16). DIQMTQSPSSLSASVGDRVTITCRASQDVGTAVAWYQQKPGKAPKLLIYSASGLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHGTTPPTFGQGTKVEIK (SEQ ID NO:95). EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGGTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFGAMDYWGQGTLVTV (SEQ ID NO:96). EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTGIHWVRQAPGKGLEWVAGIYPTNGGTGYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRGGGDGFGAMDYWGQGTLVTV (SEQ ID NO:97).

[0073] According to an embodiment of the invention, the antibody light chain variable region sequence has conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 13 or 14, or the antibody heavy chain variable region sequence has conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 15 or 16. Of course, these conservative amino acid substitutions do not result in changes in the biological functions of the antibody. In some specific embodiments, these conservative amino acid substitutions can occur in amino acids other than the CDR regions in the heavy and light chain variable regions.

[0074] According to an embodiment of the invention, the antibody has a light chain variable region having the amino acid sequence shown in SEQ ID NO:13 and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:15.

[0075] According to an embodiment of the invention, the antibody has a light chain variable region having the amino acid sequence shown in SEQ ID NO:14 and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:16.

[0076] According to an embodiment of the invention, the antibody has a light chain variable region having the amino acid sequence shown in SEQ ID NO:95 and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:96.

[0077] According to an embodiment of the invention, the antibody has a light chain variable region having the amino acid sequence shown in SEQ ID NO:95 and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:97.

[0078] According to an embodiment of the present invention, the antibody comprises at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a mouse-derived antibody, a human-derived antibody, a primate-derived antibody or a variant thereof, preferably a human-derived antibody.

[0079] According to an embodiment of the present invention, the antibody has a heavy chain having an amino acid sequence as set forth in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:33 or SEQ ID NO:35 and a light chain having an amino acid sequence as set forth in SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:34. EVQLVESGGGLVQPGGSLRLSCAASGFNIGGTGIHWVRQAPGKGLEWVAGIYPTNGGTGYAGSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRGGGDGFGAMGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:17). EVQLVESGGGLVQPGGSLRLSCAASGGGGSGGGSWVRQAPGKGLEWVAGGGSGGGSGGGSGGGSKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRGGGGSGGGSGGGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:18). DIQMTQSPSSLSASVGDRVTITCRASQGVGTAVAWYQQKPGKAPKLLIYSASGLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHGTTPPTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:19). DIQMTQSPSSLSASVGDRVTITCGGGGSGGGGSAWYQQKPGKAPKLLIYGGGGSGSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCGGGGSGGGGFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:20).

[0080] According to an embodiment of the invention, the antibody has a heavy chain having the amino acid sequence shown in SEQ ID NO:17 and a light chain having the amino acid sequence shown in SEQ ID NO:19.

[0081] According to an embodiment of the invention, the antibody has a heavy chain having the amino acid sequence shown in SEQ ID NO:18 and a light chain having the amino acid sequence shown in SEQ ID NO:20.

[0082] According to an embodiment of the invention, the antibody has a heavy chain having the amino acid sequence shown in SEQ ID NO:33 and a light chain having the amino acid sequence shown in SEQ ID NO:34.

[0083] According to an embodiment of the invention, the antibody has a heavy chain having the amino acid sequence shown in SEQ ID NO:35 and a light chain having the amino acid sequence shown in SEQ ID NO:34.

[0084] In the process of preparing or obtaining these antibodies above, the nucleic acid molecules expressing these antibodies can be utilized to connect to different vectors and then expressed in different cells to obtain the corresponding antibodies.

[0085] Thus, in a second aspect of the present invention, the present invention provides a first nucleic acid molecule, which according to an embodiment of the present invention encodes an antibody according to the first aspect. The antibody encoded by the first nucleic acid molecule according to an embodiment of the present invention has the advantage of extremely low antigen binding activity and high expression level.

[0086] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0087] The nucleotide sequence comprises SEQ ID NO:25 or a nucleotide sequence having at least 80% similarity thereto. SEQ ID NO:25 is used to encode a heavy chain having the amino acid sequence shown in SEQ ID NO:17.

[0088] The nucleotide sequence comprises SEQ ID NO:26 or a nucleotide sequence having at least 80% similarity thereto. SEQ ID NO:26 is used to encode a light chain having the amino acid sequence shown in SEQ ID NO:19. (SEQ ID NO:26).

[0089] The nucleotide sequence comprises SEQ ID NO:27 or a nucleotide sequence having at least 80% similarity thereto. SEQ ID NO:27 is used to encode a heavy chain having the amino acid sequence shown in SEQ ID NO:18.

[0090] The nucleotide sequence comprises SEQ ID NO:28 or a nucleotide sequence having at least 80% similarity thereto. SEQ ID NO:28 is used to encode a light chain having the amino acid sequence shown in SEQ ID NO:20. (SEQ ID NO:28).

[0091] Those skilled in the art will appreciate that the features and advantages described above for antibodies are equally applicable to said first nucleic acid molecule and will not be described further.

[0092] In a third aspect of the present invention, the present invention provides a first expression vector. According to an embodiment of the present invention, the first expression vector carries a first nucleic acid molecule according to the second aspect. When the polynucleotide isolated above is connected to the first expression vector, the polynucleotide can be directly or indirectly connected to a control element on the first expression vector, and these control elements may control the translation, expression, etc. of the polynucleotide. Of course, these control elements may be directly derived from the first expression vector or may be exogenous, i.e., not derived from the first expression vector itself. Of course, the polynucleotide may be operably connected to the control element.

[0093] As used herein, "operably linked" refers to connecting a foreign gene to a vector so that control elements in the vector, such as transcriptional and translational control sequences, can function to regulate the transcription and translation of the desired foreign gene. Of course, polynucleotides encoding the heavy and light chains of an antibody can be inserted independently into different vectors, and are often inserted into the same vector. Commonly used vectors may be, for example, plasmids, bacteriophages, etc.

[0094] According to an embodiment of the present invention, the first expression vector is a eukaryotic expression vector, preferably, the first expression vector is a slow virus vector.

[0095] Those skilled in the art will understand that the features and advantages described above for the antibody are equally applicable to the first expression vector, and will not be described further.

[0096] In a fourth aspect of the present invention, the present invention provides a first recombinant cell. According to an embodiment of the present invention, the first recombinant cell carries a first nucleic acid molecule according to the second aspect or expresses an antibody according to the first aspect. The first expression vector is introduced into a mammalian cell to construct and obtain a first recombinant cell, and then these first recombinant cells are used to express the antibody provided by the present invention. The corresponding antibody can be obtained by culturing the first recombinant cell. These usable mammalian cells may be, for example, CHO cells.

[0097] According to an embodiment of the present invention, the recombinant cell is obtained by introducing a first expression vector into a host cell.

[0098] According to an embodiment of the present invention, the recombinant cell is a eukaryotic cell.

[0099] According to an embodiment of the present invention, the recombinant cell is a mammalian cell.

[0100] In the fifth aspect of the present invention, the present invention provides the use of the antibody according to the first aspect in the preparation of a fusion protein drug. As can be seen from the above, the above antibody of the present invention has no antigen binding activity and can be used as a neutral drug vector to prepare a drug, which can be used to treat or prevent a disease. For example, the above antibody is fused with an antigen epitope peptide to prepare a fusion protein, and the above antibody has no antigen binding activity, and the prepared fusion protein specifically binds to the antibody corresponding to the antigen epitope peptide and does not bind to other antibodies, so that the side effects of non-specific binding of the fusion protein can be avoided and the antibody corresponding to the antigen epitope peptide can be used to treat or prevent related diseases.

[0101] According to an embodiment of the present invention, the drug comprises a fusion protein or an ADC drug.

[0102] In the sixth aspect of the present invention, the present invention provides the use of the above-mentioned antibody as a drug vector or negative antibody.As can be seen from the above, the above-mentioned antibody of the present invention has no antigen binding activity, and can be used as a neutral drug vector to prepare drugs, or can be referred to as a negative antibody that has no direct antigen binding activity.

[0103] According to an embodiment of the present invention, the drug vector or negative antibody has no antigen binding ability.

[0104] Those skilled in the art will understand that the features and advantages described above for the antibody are equally applicable to the first nucleic acid molecule, the first expression vector, the first recombinant cell and the uses, and will not be described further.

[0105] [Fusion protein]

[0106] In a seventh aspect of the invention, the invention provides a fusion protein, according to an embodiment of the invention, comprising a neutral antibody comprising an antibody F C segment or an antibody according to the first aspect, and one or more PLA2R antigen epitope peptides linked to the neutral antibody.

[0107] The inventors have found through experiments that by fusing the PLA2R antigen epitope peptide with the neutral antibody, the resulting fusion protein can competitively bind to the PLA2R antibody, prevent the binding of the PLA2R antibody to PLA2R in vivo, and effectively treat the related diseases caused by the PLA2R antibody. At the same time, the PLA2R antigen epitope peptide in the fusion protein of the present invention binds to the PLA2R antibody. The neutral antibody of the fusion protein contains an Fc fragment. The Fc fragment can bind to the FcγR receptor of NK cells, and kill B cells expressing membrane-type PLA2R antibody through NK cells. The Fc fragment can bind to complement C1q to activate the complement system, and exert a lytic effect on B cells expressing membrane-type PLA2R antibody. The fusion protein of the present invention specifically binds to the PLA2R antibody and does not bind to other antibodies, so that it exclusively eliminates B cells and avoids the side effects of non-specific immunity. In addition, the fusion protein of the present invention can bind to and be protected by the hepatic FcRn receptor, preventing the fusion protein from being decomposed, and has a long plasma half-life, which means that the fusion protein has the advantage of a long half-life as a drug, reducing the frequency of administration, and also has the advantages of good expression, high production amount, high stability, high affinity, good solubility, and good drug development properties.

[0108] Those skilled in the art can understand that the amino acid fragment is usually divided into N-terminus and C-terminus. The present invention is not strictly limited to the manner of connecting the neutral antibody and the PLA2R antigen epitope peptide, as long as the N-terminus of one fragment is connected to the C-terminus of another fragment.

[0109] According to an embodiment of the present invention, the C-terminus of the PLA2R antigen epitope peptide is connected to the N-terminus of the neutral antibody.

[0110] According to an embodiment of the present invention, the PLA2R antigen epitope peptide comprises CysR, FnII and PLA2R binding domain 1 and / or PLA2R binding domain 2.

[0111] For specific structures of CysR, FnII, PLA2R binding region 1 and PLA2R binding region 2, see FIG.

[0112] According to an embodiment of the present invention, the C-terminus of the CysR is connected to the N-terminus of the FnII, and the C-terminus of the FnII is connected to the N-terminus of the PLA2R binding domain 1.

[0113] According to an embodiment of the present invention, the C-terminus of CysR is connected to the N-terminus of FnII, the C-terminus of FnII is connected to the N-terminus of PLA2R binding region 1, and the C-terminus of PLA2R binding region 1 is connected to the N-terminus of PLA2R binding region 2.

[0114] According to an embodiment of the present invention, the PLA2R antigen epitope peptide has an amino acid sequence shown in SEQ ID NO:21 or 22, or an amino acid sequence having at least 95% similarity to SEQ ID NO:21 or 22. EGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTRE GREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVE(SEQ ID NO:21). EGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRK YIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTL LSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVEKDAWKYYATHCEPGWNPYNRNCY KLQKEEKTWHEALRSCQADNSALIDITSLAEVEFLVTLLGDENASETWIGLSSNKIPVSFEWSNDSSVIFTNWHTLEPHIFPNRSQLCVSAEQSEGHWKVKNCEERLFYICKKAGHVLSD(SEQ ID NO:22).

[0115] According to an embodiment of the present invention, the neutral antibody is an antibody F C segment, and the C-terminus of the PLA2R antigen epitope peptide is connected to at least one N-terminus of the antibody F C segment.

[0116] According to an embodiment of the present invention, the neutral antibody is an antibody according to the first aspect, and the C-terminus of the PLA2R antigen epitope peptide is connected to the N-terminus of at least one light or heavy chain of the antibody.

[0117] According to an embodiment of the present invention, the neutral antibody is a double-chain antibody, and the N-terminus of each chain of the antibody FC segment is connected to the C-terminus of one PLA2R antigen epitope peptide. The fusion protein contains two PLA2R antigen epitope peptides, that is, it is a bivalent molecule. Through experiments, the inventors have found that the binding ability of the fusion protein of the present invention and PLA2R antibody is stronger than that of monovalent natural PLA2R, and can effectively neutralize PLA2R antibody and protect natural PLA2R on the surface of glomerular group cells from the attack of PLA2R antibody.

[0118] According to an embodiment of the present invention, the N-terminus of each light chain of the antibody according to the first aspect is connected to the C-terminus of one PLA2R antigen epitope peptide, respectively. The fusion protein contains two PLA2R antigen epitope peptides, that is, it is a bivalent molecule. Through experiments, the inventors have found that the binding ability of the fusion protein of the present invention with PLA2R antibody is stronger than that of monovalent native PLA2R, and can effectively neutralize PLA2R antibody and protect native PLA2R on the surface of glomerular group cells from the attack of PLA2R antibody.

[0119] According to an embodiment of the present invention, the N-terminus of each heavy chain of the antibody according to the first aspect is connected to the C-terminus of one PLA2R antigen epitope peptide, respectively. The fusion protein contains two PLA2R antigen epitope peptides, that is, it is a bivalent molecule. Through experiments, the inventors have found that the binding ability of the fusion protein of the present invention with PLA2R antibody is stronger than that of monovalent native PLA2R, and can effectively neutralize PLA2R antibody and protect native PLA2R on the surface of glomerular group cells from the attack of PLA2R antibody.

[0120] According to an embodiment of the present invention, the N-terminus of each heavy chain and each light chain of the antibody according to the first embodiment is connected to the C-terminus of one PLA2R antigen epitope peptide, respectively.The inventors have found through experiments that the fusion protein contains four PLA2R antigen epitope peptides, and compared with monovalent native PLA2R, the binding ability of the fusion protein of the present invention with PLA2R antibody is stronger, and the fusion protein can effectively neutralize PLA2R antibody and protect native PLA2R on the surface of glomerular group cells from the attack of PLA2R antibody.

[0121] According to an embodiment of the present invention, the fusion protein further comprises a connecting peptide, which is located between the PLA2R antigen epitope peptide and the antibody.

[0122] According to an embodiment of the present invention, the N-terminus of the connecting peptide is connected to the C-terminus of the PLA2R antigen epitope peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the neutral antibody, thereby improving the binding ability of the final fusion protein and the PLA2R antibody by adopting the PLA2R antigen epitope peptide and the neutral antibody that can be fused with the connecting peptide.

[0123] According to an embodiment of the present invention, the amino acid sequence of the connecting peptide is (GGGGS) n and n is an integer of 1 or more, and preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0124] According to an embodiment of the invention, the connecting peptide comprises the amino acid sequence shown in SEQ ID NO:23. GGGGSGGGGS (SEQ ID NO:23).

[0125] According to an embodiment of the invention, the antibody FC segment has an amino acid sequence of SEQ ID NO:24 or an amino acid sequence having at least 90% similarity to SEQ ID NO:24. THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:24).

[0126] According to an embodiment of the invention, the fusion protein comprises a first peptide segment having an amino acid sequence as set forth in SEQ ID NO:57 and a second peptide segment having an amino acid sequence as set forth in SEQ ID NO:19; or the fusion protein has a first peptide segment having an amino acid sequence as set forth in SEQ ID NO:17 and a second peptide segment having an amino acid sequence as set forth in SEQ ID NO:59; or The amino acid sequence of the fusion protein is shown in SEQ ID NO:61; or the fusion protein has a first peptide segment having an amino acid sequence as set forth in SEQ ID NO:63 and a second peptide segment having an amino acid sequence as set forth in SEQ ID NO:19; or the fusion protein has a first peptide segment having an amino acid sequence as set forth in SEQ ID NO:17 and a second peptide segment having an amino acid sequence as set forth in SEQ ID NO:65; or The amino acid sequence of the fusion protein is shown in SEQ ID NO:67; or the fusion protein has a first peptide segment of the amino acid sequence set forth in SEQ ID NO:57 and a second peptide segment of the amino acid sequence set forth in SEQ ID NO:59; or The fusion protein has a first peptide segment having an amino acid sequence set forth in SEQ ID NO:63 and a second peptide segment having an amino acid sequence set forth in SEQ ID NO:65.

[0127] Exemplarily, referring to FIG. 8, when the neutral antibody is the antibody described above, the first peptide segment of the fusion protein is a peptide segment comprising a first antibody heavy chain, the second peptide segment of the fusion protein is a peptide segment comprising a first antibody light chain, and the first peptide segment and the second peptide segment are connected by an interchain disulfide bond; when the neutral antibody is the antibody Fc fragment described above, the fusion protein is a peptide segment comprising an antibody Fc fragment.

[0128] Those skilled in the art will appreciate that the features and advantages described above for antibodies are equally applicable to the fusion proteins and will not be described further.

[0129] [Second nucleic acid molecule, second expression vector, second recombinant cell]

[0130] In the process of preparing or obtaining these fusion proteins, the nucleic acid molecules expressing these fusion proteins can be used to connect to different vectors and then expressed in different cells to obtain the corresponding fusion proteins.

[0131] Thus, in an eighth aspect of the present invention, the present invention provides a second nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes a fusion protein according to the seventh aspect. The second nucleic acid molecule can be used to effectively express the fusion protein, particularly in expression systems of prokaryotes and lower eukaryotes. Note that the second nucleic acid molecule does not need to encode the entire sequence of the fusion protein, and can, for example, comprise two types of nucleic acid molecules, each encoding two monomers, which can be further assembled in vitro to obtain an isodimer, forming a fusion protein comprising a double-chain antibody.

[0132] According to an embodiment of the present invention, the second nucleic acid molecule is DNA.

[0133] In a ninth aspect of the present invention, there is provided a second expression vector, which according to an embodiment of the present invention carries a second nucleic acid molecule according to the eighth aspect, which can be effectively used to express the fusion protein, particularly in prokaryotic and lower eukaryotic expression systems.

[0134] According to an embodiment of the present invention, the second expression vector is a eukaryotic expression vector, preferably, the second expression vector is a slow virus vector.

[0135] In a tenth aspect of the invention, the invention provides a second recombinant cell. According to an embodiment of the invention, the second recombinant cell carries a second nucleic acid molecule according to the eighth aspect or expresses a fusion protein according to the seventh aspect. The recombinant cell according to an embodiment of the invention may be used for the in vitro expression and large scale production of the fusion protein according to the seventh aspect.

[0136] According to an embodiment of the present invention, the second recombinant cell is a eukaryotic cell.

[0137] According to an embodiment of the present invention, the second recombinant cell is a mammalian cell.

[0138] Those skilled in the art will appreciate that the features and advantages described above for the fusion protein are equally applicable to the second nucleic acid molecule, the second expression vector and the second recombinant cell, and will not be described further.

[0139] [Pharmaceutical compositions, immunoadsorbents, reagent kits, adsorption products and their uses]

[0140] In an eleventh aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises the fusion protein according to the seventh aspect. After extensive experiments, the inventors have found that the pharmaceutical composition can effectively prevent and treat PLA2R antibody-related diseases, such as PLA2R antibody-positive membranous nephropathy.

[0141] According to an embodiment of the present invention, the pharmaceutical composition further comprises a pharma- ceutically acceptable adjuvant.

[0142] In a twelfth aspect of the present invention, there is provided an immunoadsorbent. According to an embodiment of the present invention, the immunoadsorbent comprises the fusion protein according to the seventh aspect. The inventors have found through extensive experiments that the immunoadsorbent can adsorb PLA2R antibodies, and can effectively treat PLA2R antibody-related diseases, such as PLA2R antibody-positive membranous nephropathy, through immunoadsorption.

[0143] In the thirteenth aspect of the present invention, a reagent kit is provided. According to an embodiment of the present invention, the reagent kit comprises the fusion protein according to the seventh aspect. After extensive experiments, the inventors have found that the reagent kit can effectively detect the content of PLA2R antibody, and can also be used to diagnose or treat PLA2R antibody-related diseases, such as PLA2R antibody-positive membranous nephropathy.

[0144] Those skilled in the art will understand that the features and advantages described for the fusion protein, the second nucleic acid molecule, the second expression vector and the second recombinant cell are equally applicable to the pharmaceutical composition, the immunoadsorbent and the reagent kit, and will not be described further.

[0145] In a fourteenth aspect of the present invention, the present invention proposes an adsorption product. According to an embodiment of the present invention, the adsorption product comprises an immunoadsorbent according to the twelfth aspect and a solid-phase vector connected to the immunoadsorbent. The adsorption product of the present invention can adsorb PLA2R antibodies, for example, PLA2R antibodies in blood or plasma, and can be used to prevent or treat diseases associated with PLA2R antibodies by adsorbing PLA2R antibodies in blood or plasma.

[0146] The method for connecting the immunoadsorbent and the solid-phase vector is not specifically limited, and they may be connected by coupling or chemical bonding.

[0147] As used herein, the blood or plasma may be from any animal, such as a mammal.

[0148] Exemplarily, said blood or plasma is selected from human blood or plasma.

[0149] According to an embodiment of the present invention, the adsorption product is selected from an immunoadsorption column or a blood purification agent.

[0150] Those skilled in the art will appreciate that the features and advantages described above for the immunosorbent are equally applicable to this use and will not be described further.

[0151] In a fifteenth aspect of the invention, the invention provides the use of an immunoadsorbent according to the twelfth aspect in the preparation of an adsorption product for adsorbing PLA2R antibodies.

[0152] According to an embodiment of the present invention, the adsorption product is selected from an immunoadsorption column or a blood purification agent.

[0153] According to an embodiment of the present invention, the adsorption product is an immunoadsorption column, and the adsorption product is used for preventing or treating diseases related to PLA2R antibodies. As can be seen from the above, the immunoadsorption agent can adsorb PLA2R antibodies. Thus, the immunoadsorption column containing the immunoadsorption agent can adsorb PLA2R antibodies by the immunoadsorption agent, and the immunoadsorption column can effectively treat diseases related to PLA2R antibodies.

[0154] According to an embodiment of the present invention, the PLA2R antibody-related disease is selected from PLA2R antibody-positive membranous nephropathy.

[0155] According to an embodiment of the present invention, the adsorption product is a blood purifier, and the adsorption product is used to adsorb PLA2R antibodies in blood or plasma. As can be seen from the above, the immunoadsorbent can adsorb PLA2R antibodies. Thus, the blood purifier containing the immunoadsorbent can adsorb PLA2R antibodies by the immunoadsorbent, and the blood purifier can effectively adsorb PLA2R antibodies in blood or plasma.

[0156] Those skilled in the art will appreciate that the features and advantages described above for the immunosorbent are equally applicable to this use and will not be described further.

[0157] In a sixteenth aspect of the present invention, the present invention provides the use of a fusion protein according to the seventh aspect, a second nucleic acid molecule according to the eighth aspect, a second expression vector according to the ninth aspect, a second recombinant cell according to the tenth aspect or a pharmaceutical composition according to the eleventh aspect in the preparation of a medicament for preventing or treating a PLA2R antibody-related disease.

[0158] According to an embodiment of the present invention, the PLA2R antibody-related disease is selected from PLA2R antibody-positive membranous nephropathy.

[0159] According to an embodiment of the present invention, the dosage form of the herbal composition includes an injection.

[0160] According to an embodiment of the present invention, the administration route of the herbal composition includes subcutaneous injection or intravenous injection.

[0161] Those skilled in the art will understand that the features and advantages described above for the pharmaceutical composition, immunoadsorbent, and reagent kit are equally applicable to this use, and will not be described further.

[0162] In a 17th aspect of the present invention, the present invention provides the use of a fusion protein according to the seventh aspect, a second nucleic acid molecule according to the eighth aspect, a second expression vector according to the ninth aspect, a second recombinant cell according to the tenth aspect or a pharmaceutical composition according to the eleventh aspect in the preparation of an immunoadsorbent for adsorbing PLA2R antibodies.

[0163] Those skilled in the art will understand that the features and advantages described above for the pharmaceutical composition, immunoadsorbent, reagent kit, etc. are equally applicable to this use, and will not be described further.

[0164] In an 18th aspect of the present invention, the present invention provides the use of a fusion protein according to the seventh aspect, a second nucleic acid molecule according to the eighth aspect, a second expression vector according to the ninth aspect, a second recombinant cell according to the tenth aspect, a pharmaceutical composition according to the eleventh aspect or an immunoadsorbent according to the twelfth aspect in the preparation of a reagent kit for detecting PLA2R antibodies.

[0165] Those skilled in the art will understand that the features and advantages described above for the fusion protein, pharmaceutical composition, immunoadsorbent and reagent kit are equally applicable to the above uses, and will not be described further.

[0166] [method]

[0167] In a nineteenth aspect of the present invention, the present invention provides a method for preventing or treating a PLA2R antibody-associated disease. According to an embodiment of the present invention, the method comprises administering to a subject a pharma- ceutically acceptable amount of the fusion protein according to the seventh aspect or the pharmaceutical composition according to the eleventh aspect. This allows for effective treatment of a PLA2R antibody-associated disease.

[0168] According to an embodiment of the present invention, the administration route of the method is subcutaneous injection or intravenous injection.

[0169] According to an embodiment of the present invention, the PLA2R antibody-related disease is selected from PLA2R antibody-positive membranous nephropathy.

[0170] In a twentieth aspect of the present invention, there is provided an immunoadsorption therapeutic method for preventing or treating a PLA2R antibody-related disease, according to an embodiment of the present invention, said method comprising the step of treating a subject with an immunoadsorbent according to the twelfth aspect.

[0171] According to an embodiment of the invention, the method comprises treating a subject with extracorporeal blood purification.

[0172] According to an embodiment of the present invention, the PLA2R antibody-related disease is selected from PLA2R antibody-positive membranous nephropathy.

[0173] In a twenty-first aspect of the present invention, the present invention provides a method for diagnosing a disease associated with PLA2R antibodies. According to an embodiment of the present invention, the method includes the steps of detecting PLA2R antibodies in a sample to be measured using the fusion protein according to the seventh aspect, the second nucleic acid molecule according to the eighth aspect, the second expression vector according to the ninth aspect, the second recombinant cell according to the tenth aspect, the pharmaceutical composition according to the eleventh aspect, the immunoadsorbent according to the twelfth aspect, or the reagent kit according to the thirteenth aspect, and determining the content of PLA2R antibodies in the sample to be measured based on the detection result. According to an embodiment of the present invention, the content of PLA2R antibodies in the sample to be measured is 14 RU / ml or more, which is an indication that the sample to be measured is derived from a patient suffering from a disease associated with PLA2R antibodies, and preferably, the content of PLA2R antibodies in the sample to be measured is 20 RU / ml or more, which is an indication that the sample to be measured is derived from a patient suffering from a disease associated with PLA2R antibodies.

[0174] According to an embodiment of the present invention, the measurement sample is blood.

[0175] According to an embodiment of the present invention, the PLA2R antibody-related disease is selected from PLA2R antibody-positive membranous nephropathy.

[0176] In the twenty-second aspect of the present invention, the present invention provides a method for evaluating the stage of a disease associated with PLA2R antibody. According to an embodiment of the present invention, the method includes the steps of detecting PLA2R antibody in a sample to be measured using the fusion protein according to the seventh aspect, the second nucleic acid molecule according to the eighth aspect, the second expression vector according to the ninth aspect, the second recombinant cell according to the tenth aspect, the pharmaceutical composition according to the eleventh aspect, the immunoadsorbent according to the twelfth aspect, or the reagent kit according to the thirteenth aspect, and determining the content of PLA2R antibody in the sample to be measured based on the detection result.

[0177] According to an embodiment of the present invention, the stage of a PLA2R antibody-associated disease in a test patient is determined based on the content of PLA2R antibody in a test sample.

[0178] According to an embodiment of the present invention, the PLA2R antibody-related disease is selected from PLA2R antibody-positive membranous nephropathy.

[0179] According to an embodiment of the present invention, PLA2R antibody-positive membranous nephropathy is divided into four disease stages.

[0180] According to an embodiment of the present invention, the measurement sample is blood.

[0181] In the twenty-third aspect of the present invention, the present invention provides a method for evaluating the prognosis of a related disease by PLA2R antibody. According to an embodiment of the present invention, the method includes the steps of detecting PLA2R antibody in a sample to be measured using the fusion protein according to the seventh aspect, the second nucleic acid molecule according to the eighth aspect, the second expression vector according to the ninth aspect, the second recombinant cell according to the tenth aspect, the pharmaceutical composition according to the eleventh aspect, the immunoadsorbent according to the twelfth aspect, or the reagent kit according to the thirteenth aspect, and determining the content of PLA2R antibody in the sample to be measured based on the detection result.

[0182] According to an embodiment of the present invention, the measurement sample is derived from a patient suffering from a PLA2R antibody-related disease before or after treatment.

[0183] According to an embodiment of the present invention, the prognostic effect of a PLA2R antibody-associated disease is determined based on the content of PLA2R antibody in a measurement sample of a patient suffering from a PLA2R antibody-associated disease before or after treatment.

[0184] According to an embodiment of the present invention, a decrease in the amount of PLA2R antibodies in the measured sample of a patient suffering from a PLA2R antibody-related disease after treatment is indicative of a good prognosis for the patient.

[0185] According to an embodiment of the present invention, the measurement sample is blood.

[0186] According to an embodiment of the present invention, the PLA2R antibody-related disease is selected from PLA2R antibody-positive membranous nephropathy.

[0187] Those skilled in the art will appreciate that the features and advantages described above for the fusion protein, pharmaceutical composition, immunosorbent and reagent kit are equally applicable to the above method, and will not be further described.

[0188] The following examples are used to illustrate the present invention. Those skilled in the art can understand that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific techniques or conditions are shown in the examples, they should be carried out according to the techniques or conditions described in the literature of the field or according to the product specifications. If no manufacturer is shown for the reagents or equipment used, they are conventional products that can be purchased commercially.

[0189] Example 1: Preparation and selection of neutral antibodies

[0190] 1. Expression construct First, the plasmid vector adopts pcDNA3.4 instantaneous expression vector, which contains the native full-length CMV promoter and the WPRE element downstream of the cloning site, and the vector based on the CMV promoter usually has a good expression level for CHO cell instantaneous expression system, and the WPRE element is located downstream of the polycloning site, which can effectively affect gene transcription and expression, and in E. coli, there is an ampicin resistance gene. Then, the vector gene pcDNA3.4 and the target gene are amplified using PCR technology, the PCR template is obtained by gene synthesis, and the vector fragment and the target gene fragment are connected with the homologous recombination reagent kit, and the monoclonal is obtained by DH5α sensitive transformation, and the monoclonal is amplified and sequenced, and after sequencing and confirmation, the plasmid is extracted to obtain the target gene recombinant expression plasmid. The target sequence coding DNA and the coding DNA secreting the expression signal peptide are cloned between the CMV promoter and the WPRE gene of the plasmid pcDNA3.4 by gene synthesis, and the specific reference is made to FIG. 1.

[0191] 1.1 The name and sequence of the gene of interest are shown in Table 1. The pcDNA3.4 vector and the primers and templates for the gene of interest (or called the fragment of interest) are shown in Table 2. RtxH and RtxL are used to synthesize Rituximab (abbreviated as Rtx).

[0192] [Table 1] Note: dTrs1L and dTrs2L in this example are the same target gene. where the amino acid sequence of RtxH is: QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGA GTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:29). Amino acid sequence of RtxL: QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:30). The amino acid sequence of TrsH: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:31). The amino acid sequence of TrsL: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:32). Amino acid sequence of dTrs1H: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGGTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFGAMDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:33). Amino acid sequence of dTrs1L: DIQMTQSPSSLSASVGDRVTITCRASQDVGTAVAWYQQKPGKAPKLLIYSASGLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHGTTPPTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:34). Amino acid sequence of dTrs2H: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTGIHWVRQAPGKGLEWVAGIYPTNGGTGYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRGGGDGFGAMDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:35). The nucleotide sequence of RtxH: Nucleotide sequence of RtxL: CAGATCGTGTTGTCGCAATCCCCGGCCATCCTGTCCGCTTCGCCGGGAGAAAAGGTCACCATGACTTGTCGCGCCTCAAGCAGCGTGTCCTACATTCATTGGTTTCAGCAGAAGCCCGGGTCCAGCCCGAAGCCTTGGATCTACGCAACTAGCAACCTCGCCTCCGGCGTGCCAGTGCGCTTCTCCGGTTCCGGATCGGGAACGTCCTATAGCCTGACTATCTCGAGAGTGGAGGCCGAGGACGCTGCCACTTACTACTGCCAGCAGTGGACCTCGAATCCTCCAACTTTCGGTGGCGGCACCAAGCTGGAGATTAAGAGGACCGTCGCGGCCCCTAGCGTGTTCATTTTCCCCCCGTCCGACGAGCAGCTGAAGTCCGGGACAGCCTCAGTGGTCTGCCTGCTCAACAACTTCTACCCCCGGGAAGCCAAAGTGCAGTGGAAAGTGGATAACGCGCTTCAGAGCGGCAACTCACAAGAGTCCGTGACCGAACAGGACTCGAAGGACAGCACCTACAGCCTCTCCTCCACCCTGACCCTGTCAAAGGCGGATTACGAAAAGCACAAGGTCTACGCCTGCGAAGTGACCCACCAGGGGCTGTCCTCTCCCGTGACCAAGTCCTTCAACCGGGGAGAGTGC(SEQ ID NO:37). Nucleotide sequence of TrsH: Nucleotide sequence of TrsL: GATATACAGATGACGCAGAGCCCTAGCAGTCTAAGTGCAAGCGTTGGAGACCGTGTCACCATTACCTGCCGCGCTAGCCAGGATGTCAACACCGCAGTCGCCTGGTATCAGCAAAAACCGGGTAAGGCTCCAAAGCTGTTGATCTACAGTGCCAGCTTTCTGTACAGCGGTGTGCCAAGTAGGTTCAGCGGGAGCCGGAGTGGGACAGACTTTACACTCACTATTAGCAGTCTGCAGCCCGAGGACTTCGCCACCTACTACTGTCAGCAGCATTATACAACACCCCCAACATTCGGGCAGGGAACCAAAGTTGAGATAAAACGAACGGTTGCCGCGCCAAGCGTATTTATCTTTCCCCCCAGTGATGAGCAACTGAAGAGTGGTACAGCCAGCGTTGTCTGCCTTTTGAATAACTTCTATCCTCGGGAGGCAAAGGTCCAGTGGAAAGTGGATAATGCACTCCAAAGCGGTAACAGCCAGGAGAGTGTGACAGAACAGGACAGCAAAGATAGTACCTACAGCCTCAGTAGTACCCTCACACTGAGCAAAGCGGACTATGAAAAGCACAAGGTGTACGCTTGTGAAGTGACCCACCAGGGCCTAAGCAGCCCTGTTACTAAGAGCTTCAACAGAGGCGAATGT(SEQ ID NO:39). Nucleotide sequence of dTrs1H: Nucleotide sequence of dTrs1L (dTrs2L): GATATACAGATGACGCAGAGCCCTAGCAGTCTAAGTGCAAGCGTTGGAGACCGTGTCACCATTACCTGCCGCGCTAGCCAGGATGTCGGCACCGCAGTCGCCTGGTATCAGCAAAAACCGGGTAAGGCTCCAAAGCTGTTGATCTACAGTGCCAGCGGCCTGTACAGCGGTGTGCCAAGTAGGTTCAGCGGGAGCCGGAGTGGGACAGACTTTACACTCACTATTAGCAGTCTGCAGCCCGAGGACTTCGCCACCTACTACTGTCAGCAGCATGGCACAACACCCCCAACATTCGGGCAGGGAACCAAAGTTGAGATAAAACGAACGGTTGCCGCGCCAAGCGTATTTATCTTTCCCCCCAGTGATGAGCAACTGAAGAGTGGTACAGCCAGCGTTGTCTGCCTTTTGAATAACTTCTATCCTCGGGAGGCAAAGGTCCAGTGGAAAGTGGATAATGCACTCCAAAGCGGTAACAGCCAGGAGAGTGTGACAGAACAGGACAGCAAAGATAGTACCTACAGCCTCAGTAGTACCCTCACACTGAGCAAAGCGGACTATGAAAAGCACAAGGTGTACGCTTGTGAAGTGACCCACCAGGGCCTAAGCAGCCCTGTTACTAAGAGCTTCAACAGAGGCGAATGT(SEQ ID NO:41). Nucleotide sequence of dTrs2H:

[0193] [Table 2]

[0194] 1.2 For the PCR reaction, TAKARA's KOD reaction system (Cat. KFX-101) was used. The reaction conditions, reagents, and PCR target fragment information are shown in Tables 3 to 6.

[0195] [Table 3]

[0196] [Table 4]

[0197] [Table 5]

[0198] [Table 6]

[0199] 1.3 After PCR, the sample was subjected to 1.5% agarose gel electrophoresis, 300mL of 1*TAE was added to the electrophoresis tank, and electrophoresis was performed at 100V and 30min. From the results, the size of the agarose gel electrophoresis bands was consistent with the theoretical size. The electrophoresis results were recovered using Omega Gel Extraction Kit gel recovery reagent kit (Cat. D2500-01), and eluted with 20μL of elution solution to obtain the purified PCR product. The electrophoresis results are shown in Figures 2 to 5. Lanes 1 to 9 from left to right in Figure 2 are DL5000, pcDNA3.4, pcDNA3.4, pcDNA3.4, pcDNA3.4, trsH, trsL, RtxH, and RtxL, respectively. Lanes 1 to 7 from left to right in Fig. 3 are DL2000, dTrs1H, dTrs1H, dTrs2H, dTrs2H, dTrs1L, and dTrs1L, lanes 1 to 5 from left to right in Fig. 4 are DL2000, dTrs3H, dTrs3H, dTrs3L, and dTrs3L, lanes 1 to 5 from left to right in Fig. 5 are DL2000, dTrs4H, dTrs4H, dTrs4L, and dTrs4L, respectively.

[0200] 1.4 After cutting and purifying the PCR band obtained in 1.3, perform homologous recombination and use the NEB reagent kit (NEBuilder (R) Fragment assembly was performed using HiFi DNA Assembly Master Mix (Cat. E2621S), the homologous recombination reaction system was incubated at 50°C for 1 hour, and the resulting reaction product was purified using Omega Cycle Pure Kit (Cat. D6492-01) and eluted with 15μL of elution solution. The reaction system and plasmid table are shown in Tables 7 and 8.

[0201] [Table 7]

[0202] [Table 8]

[0203] 1.5 Transform into CaCl2 sensitive cells DH5a, add each plasmid sample obtained in 1.4 to the sensitive cells in a ratio of 1:10, place on ice for 10 min, then incubate in a metal bath at 42°C for 45s, then place on ice for 10 min, finally spread the obtained solution on LB solid medium containing ampicillin (Amp. final concentration 50μg / mL), culture overnight at 37°C, take it into 2ml of LB liquid medium (final concentration 50μg / mL Amp.) the next day, shake the bacteria at 37°C and 200 rpm, and perform Sanger sequencing of the obtained bacterial solution to obtain the pass-through sequence, and the sequencing primer is CMV-F: CGCAAATGGGCGGTAGGCGTG (SEQ ID NO:79). After comparing the sequencing results, all pass-through sequences were found to match the original sequences. 100 μL of the monoclonal bacteria solution was added to 50 mL of LB liquid medium containing ampicillin (final concentration 50 μg / mL), and cultured overnight at 37°C and 200 rpm. The bacteria were then preserved, and the plasmid was extracted to obtain the target gene expression plasmid.

[0204] 2. Instantaneous expression of Trs, dTrs1-4 and Rtx proteins 2.1 Approximately 24 hours before transfection, TM CHO-S (R) Cells (cell density 5–6 × 10 5 / mL, Gibco FreeStyle TM MAX CHO expression system (product number: K900020) and culture under conditions of 37℃, 5% CO2 and 120-135 rpm / min. 2.2 On the day of transfection, cells were cultured at a cell density of 1 × 10 6 Dilute to 100 / mL and add 10 mL of cells (cell viability is above 95%) to each shake flask. 2.3 FreeStyle TM Mix the MAX Reagent gently several times, shaking without vortexing. 2.4 Take 12.5 μg of each plasmid corresponding to each protein in Table 9 and use it in OptiPRO TMAdd SFM to a total volume of 0.2 mL of each and mix gently. 2.5 25 μL FreeStyle TM Take MAX Reagent and Opti-Pro TM Add SFM to a total volume of 0.4 mL and mix gently (shake but do not vortex). After mixing evenly, add it evenly to the plasmid mixture and mix gently to separate two 0.4 mL DNA-FreeStyle TM The MAX mixture was taken and incubated at room temperature for 10 minutes to allow complex formation. 2.6 Add the two 0.4 mL tubes of DNA-FreeStyle TM The MAX complex is added slowly to the cells while gently swirling and shaking the flask. 2.7 Culture the cells at 37 °C, 5% CO2, 120-135 rpm / min, and collect the cell solution after 7 d after replacing or replenishing the medium.

[0205] [Table 9]

[0206] 3. Purification and identification of Trs, dTrs1-4 and Rtx proteins The Trs, dTrs1-4 and Rtx culture supernatants are centrifuged at 4 °C and 7000 g for 15 min, then filtered through a 0.22 μm syringe filter to sterilize. Equilibrate the Protein A chromatography medium (flow rate 2 mL / min) with 5 column volumes of Buffer A1 (20 mM phosphate buffer, pH value 7.4), wash the treated sample with Buffer A1 until the absorption value reaches the baseline, elute with Buffer B1 (0.1 M Glycine-HCl, pH value 3.0), collect the elution peak sample, and adjust the target protein eluate to neutral with 1 M Tris-HCl (pH value 9.0). Ultrafilter into PBS buffer.

[0207] Identification of target protein: Select a 10-well SDS-PAGE precast gel and run electrophoresis at 150 V for 50 min. Heat the sample at 90° C. for 5 min, and place 10 μg of sample in each well.

[0208] The purification effect of detecting Trs, dTrs1-4 and Rtx proteins by SDS-PAGE is shown in Figure 6, where lane 1 is Trs elution non-reduced, lane 2 is Trs elution reduced, lane 3 is DTrs1 elution non-reduced, lane 4 is DTrs1 elution reduced, lane 5 is DTrs2 elution non-reduced, lane 6 is DTrs2 elution reduced, lane 7 is DTrs3 elution reduced, lane 8 is DTrs3 elution non-reduced, lane 9 is DTrs4 elution non-reduced, lane 10 is DTrs4 elution reduced, lane 11 is Rtx non-reduced, lane 12 is Rtx reduced. The expression levels of Trs, dTrs1-4 and Rtx proteins are shown in Table 10.

[0209] [Table 10]

[0210] 4. ELISA of binding of Trs, dTrs1-4 and Rtx proteins to Her2 4.1 The purchased Her2 with binding activity (10004-HCCH-B, Yiqiao Shenzhou) was used as the substrate, Trs, dTrs1-4 or Rtx protein was used as the primary antibody, and HRP-coupled goat anti-human IgG was used as the secondary antibody to detect the binding status between Her2 protein and Trs, dTrs1-4 or Rtx protein by enzyme-linked immunosorbent method.

[0211] 20 μg of solid Her2 protein was dissolved in 80 μL of sterile water to prepare a 0.25 mg / mL mother solution, which was then dispensed into 0.5 mL EP tubes and stored at −80° C. at 10 μL / tube. Primary antibodies: Trs (0.44 mg / mL), dTrs1 (0.38 mg / mL), dTrs2 (0.42 mg / mL), dTrs3 (0.115 mg / mL), dTrs4 (1.4 mg / mL), Rtx (0.21 mg / mL), where Trs is a positive control and Rtx is a positive control. Secondary antibody: HRP-coupled goat anti-human IgG (Shanghai Seiko Biotechnology Co., Ltd., Cat. No. D110150). 4.2 Detection Methods: 1) Coating: Dilute the culture supernatant with the coating solution, add 0.1 μg / 100 μL of Her2 protein (34 μL mother solution + 8466 μL coating solution) to each well, seal the plate, and incubate for 2 h at 37 ° C. Discard the liquid in the wells, wash the plate three times with 200 μL of washing solution each time, and leave it for 2 min. 2) Blocking: Add 100 μL of blocking solution to each well, incubate at 4°C overnight, and discard the liquid in the wells. 3) Primary antibody (Trs, dTrs1-4 or Rtx) incubation: Dilute Trs, dTrs1-4 and Rtx with antibody dilution solution, add 100 μL to each well, close the plate, incubate at 37 °C for 1 h, seal the plate, wash the plate three times with washing solution, 200 μL each time, and leave it for 2 min. 4) Secondary antibody incubation: Dilute the secondary antibody 1:2000 with antibody diluent, add 100 μL to each well, close the plate, incubate at 37 °C for 1 h, discard the liquid in the wells, wash the plate three times with washing solution, 200 μL each time, and leave it for 2 min. 5) Detection: Add 100μL of TMB to each well, close the plate, and incubate at 37℃ in the dark for 15min, when the inside of the well turns blue, add 100μL of stop solution to each well, it turns yellow immediately, and detect the OD value at 450nm, the detection results are shown in Figure 7 and Table 11. As can be seen from the results, compared with Trs, the binding ability of dTrs1~dTrs4 to Her2 gradually decreases, and the binding ability of dTrs4 to Her2 is the lowest, close to the positive control Rtx, and the expression level of dTrs4 is high, so dTrs4 can be selected as a drug scaffold.

[0212] The preparation of the coating solution is shown in the table below. [Table 10A]

[0213] The preparation of the blocking solution is shown in the table below. [Table 10B]

[0214] The preparation of antibody dilutions is shown in the table below. [Table 10C] The preparation of the cleaning solution is shown in the table below. [Table 10D]

[0215] [Table 11] Note: Both values ​​below the lower limit and above the upper limit are outside the calculation range. If the value is below the lower limit, the binding ability is strong, and if the value is above the upper limit, the binding ability is weak.

[0216] 5. Binding of Trs, dTrs1-4 and Rtx proteins to IgA, IgM or HAS 5.1 The experimental steps are as follows: 1) Seal the plate with a coating solution containing 10 μg / mL IgA / IgM / HSA in 100 μL / hole and leave it at 4 °C overnight, IgA is derived from human serum (Sigma-Aldrich, I4036) and has a concentration of 1 mg / mL, IgM is derived from human serum (Sigma-Aldrich, I8260) and has a concentration of 1 mg / mL, and HSA (Absin, abs47047388) has a concentration of 20 mg / mL, 2) Discard the liquid from step 1) and wash the plate with PBST (dilute Solivao 10*PBS 10 times with double distilled water (sterile), + 0.1% Tween), wash 4 times with a plate washer, shake for 1 min on a plate shaker under 600 × g conditions, and dry by tapping several times. 3) 100 μL / well of blocking solution (0.5% Casein (pH 7.09, 1M NaOH >50 mg / mL prepared in small volume, PBST added, pH adjusted with HCl, and then the volume determined) was prepared. Incubate at 37°C for 60 min. Then, repeat the plate washing in step 2). 4) Add 100 μL / well of each set of samples (Trs, dTrs1-4 or Rtx) diluted 10-fold (10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001), close the plate, incubate at room temperature for 30 min, then repeat the plate washing in step 2). 5) Add 100μL of detection antibody (Goat Anti-Hμman IgG-Fc Secondary Antibody (HRPμg / mL), Sino Biological, SSA001, dilute with PBST 1:3K to a working concentration of 1.2μg / mL) to each well, close the plate, incubate at room temperature for 30min, then repeat the plate washing in step 2). 6) Add 100 μL of Soraya TMB substrate coloring solution to each well, incubate at 25°C in the dark for 6 minutes, then add 100 μL of Soraya stop solution to each well and measure the OD 450 Measure. 5.2 Results The results of ELISA detection using human IgA on the plate are shown in Figure 21, where ** indicates p<0.01 and * indicates p<0.05. The results show that the signal generated by dTrs4 as a detection molecule is very low, even lower than that of wild-type Trs and the unrelated antibody RTX, indicating that dTrs4 does not bind to human IgA.

[0217] The dissimilarity analysis of Figure 21 is shown in the table below. [Table 11A]

[0218] Human IgA was plated and p-values ​​are from t-tests between dTRS4 and other molecules at 10ug / mL. The results of ELISA detection using human IgM on the plate are shown in Figure 22, where ** indicates p<0.01 and * indicates p<0.05. The results show that the signal generated by dTrs4 as a detection molecule is very low, even lower than that of wild-type Trs and the unrelated antibody RTX, indicating that dTrs4 does not bind to human IgM.

[0219] The dissimilarity analysis of Figure 22 is shown in the table below. [Table 11B]

[0220] Human IgM was plated and p-values ​​are from t-tests between dTRS4 and other molecules at 10ug / mL.

[0221] The results of ELISA detection using human HSA on the plate are shown in Figure 23, where ** indicates p<0.01 and * indicates p<0.05. The results show that the signal generated by the irrelevant antibody RTX is slightly high, and the signals of other molecules are all low. The signal generated by dTrs4 as the detection molecule is the lowest, and is even lower than the wild-type Trs and the irrelevant antibody RTX. This indicates that dTrs4 does not bind to human HSA.

[0222] The dissimilarity analysis of Figure 23 is shown in the table below. [Table 11C]

[0223] The p-values ​​are from a t-test between dTRS4 and other molecules when plated with human HSA at 10ug / mL.

[0224] [Example 2: Preparation and selection of RC1 to RC8 proteins] 1. Expression construct: The steps of expression construct of RC1 to RC8 proteins are the same as those of expression construct in Example 1, with the difference being that the target genes are different. A schematic diagram of the structures of RC1 to RC8 proteins is shown in FIG. 8. A schematic diagram of the structure of PLA2R is shown in FIG. 9. The PLA2R antigen epitope peptide CFC1 (SEQ ID NO: 21) in the RC1 to RC8 proteins is the amino acid sequence of 1 to 367 (Glu) of the PLA2R fragment, and the PLA2R antigen epitope peptide CFC2 (SEQ ID NO: 22) is the amino acid sequence of 1 to 510 (Asp) of the PLA2R fragment.

[0225] The name, primer, sequence, and composition of the heavy chain, light chain, and its signal peptide of the target gene are shown in Table 12. The name and sequence of the target gene are shown in Table 13. The pcDNA3.4 vector and the primers and templates of the target gene are shown in Table 14. The target fragment information is shown in Table 15.

[0226] PLA2R signal peptide nucleotide sequence: ATGCTGCTGTCGCCGTCGCTGCTGCTGCTGCTGCTGCTGGGGGCGCCGCGGGGCTGCGCC (SEQ ID NO:80). Trs heavy chain signal peptide nucleotide sequence: ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGATGCCAGATGT(SEQ ID NO:81). Trs light chain signal peptide nucleotide sequence: ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACTACAGGTGTCCACTCC(SEQ ID NO:82).

[0227] The electrophoretic results of the purified PCR products are shown in Figures 10 to 12. Lanes 1 to 5 from left to right in Figure 10 are DL5000, pcDNA3.4, pcDNA3.4, pcDNA3.4, and pcDNA3.4, respectively. Lanes 1 to 13 from left to right in Figure 11 are DL5000, pcDNA3.4, pcDNA3.4, pcDNA3.4, pcDNA3.4, RC1H-1, RC1H-2, RC2L-1, RC2L-2, RC4H-1, RC4H-2, RC5L-1, and RC5L-2, respectively. Lanes 1 to 7 from left to right in Figure 12 are DL5000, irrelevant, irrelevant, RC3, RC3, RC6, and RC6, respectively.

[0228] The PCR bands after gel excision and purification were homologous recombination and the system was consistent with Table 7. The constructed plasmid table is shown in Table 16.

[0229] [Table 12]

[0230] [Table 13] Note: RC1H, RC2L, RC4H, and RC5L are each composed of two PCR fragments joined together: RC1H is composed of RC1H-1 and RC1H-2, RC2L is composed of RC2L-1 and RC2L-2, RC4H is composed of RC4H-1 and RC4H-2, and RC5L is composed of RC5L-1 and RC5L-2.

[0231] Amino acid sequence of RC1H: (SEQ ID NO:57). Nucleotide sequence of RC1H: Amino acid sequence of RC2L: (SEQ ID NO:59). Nucleotide sequence of RC2L: Amino acid sequence of RC3: (SEQ ID NO:61). Nucleotide sequence of RC3: The amino acid sequence of RC4H: (SEQ ID NO:63). Nucleotide sequence of RC4H: Amino acid sequence of RC5L: (SEQ ID NO:65). The nucleotide sequence of RC5L: Amino acid sequence of RC6: (SEQ ID NO:67). The nucleotide sequence of RC6:

[0232] [Table 14]

[0233] [Table 15]

[0234] [Table 16]

[0235] 2. Instantaneous expression of RC1-RC8 proteins 2.1 Approximately 24 hours before transfection, TM CHO-S (R) Cells (cell density 5–6 × 10 5 / mL, Gibco FreeStyle TM MAX CHO expression system (product number: K900020) and culture under conditions of 37℃, 5% CO2 and 120-135 rpm / min. 2.2 On the day of transfection, cells were cultured at a cell density of 1 × 10 6 Dilute to 100 / mL and add 10 mL of cells (cell viability is above 95%) to each shake flask. 2.3 FreeStyle TM Mix the MAX Reagent gently several times, shaking without vortexing. 2.4 Take 12.5 μg of each plasmid corresponding to each protein in Table 17 and use it in OptiPRO TM Add SFM to a total volume of 0.2 mL of each and mix gently. 2.5 25 μL FreeStyle TM Take MAX Reagent and Opti-Pro TMAdd SFM to a total volume of 0.4 mL and mix gently (shake but do not vortex). After mixing evenly, add it evenly to the plasmid mixture and mix gently to separate two 0.4 mL DNA-FreeStyle TM The MAX mixture was taken and incubated at room temperature for 10 minutes to allow complex formation. (RC3 and RC6 proteins were incubated in 12.5 μL of FreeStyle TM Take MAX Reagent and Opti-Pro TM Add SFM to a total volume of 0.2 mL and final volume is 0.4 mL of DNA-FreeStyle TM (Get MAX mixture) 2.6 Add the two 0.4 mL tubes of DNA-FreeStyle TM The MAX complex is slowly added to the cells while gently swirling and shaking the flask. 2.7 Culture the cells at 37 °C, 5% CO2, 120-135 rpm / min, and collect the cell solution after 7 d after replacing or replenishing the medium.

[0236] [Table 17]

[0237] 3. Purification and identification of RC1-RC8 proteins Centrifuge the RC1 to RC8 culture supernatants at 4 °C and 7000 g for 15 min, then filter them through a 0.22 μm syringe filter to sterilize them. Equilibrate Protein A chromatography medium (flow rate 2 mL / min) with 5 column volumes of Buffer A1, add the treated sample, wash the treated sample with Buffer A1 until the absorption value reaches the baseline, elute Buffer B1, collect the elution peak sample, and adjust the target protein eluate to neutral with 1 M Tris-HCl (pH value 9.0). Filter through Super dex200 into PBS.

[0238] Identification of target protein: Select a 10-well SDS-PAGE precast gel and run electrophoresis at 150 V for 50 min. Heat the sample at 90° C. for 5 min, and place 3 μg of sample in each well.

[0239] The purification effect of RC1-RC8 protein detection by SDS-PAGE is shown in Figure 13, lane M is marker, lane 1 is RC1 elution non-reduced, lane 2 is RC1 elution reduced, lane 3 is RC2 elution reduced, lane 4 is RC2 elution non-reduced, lane 5 is RC3 elution non-reduced, lane 6 is RC3 elution reduced, lane 7 is RC4 elution reduced, lane 8 is RC4 elution non-reduced, lane 9 is RC5 elution non-reduced, lane 10 is RC5 elution reduced, lane 11 is RC6 elution non-reduced, lane 12 is RC6 elution reduced, lane 13 is RC7 elution non-reduced, lane 14 is RC7 elution reduced, lane 15 is RC8 elution non-reduced, lane 16 is RC8 elution reduced. The expression amount of RC1-RC8 protein is shown in Table 18.

[0240] [Table 18]

[0241] 4. Stability investigation of RC1~RC8 Protein aggregation is a common phenomenon for many proteins, which affects the determination of mass standards and formulation components of protein drugs. As candidate drug molecules, there is a tendency to select molecules with low aggregation. For this reason, a stability investigation was carried out on RC1 to RC8 prepared in step 3 of this example, and the specific steps are as follows:

[0242] When RC2 was stored at -20°C, it was apparently decomposed and was not stable, so further experiments were not performed. RC1, RC3, RC4, RC5, RC6, RC7, RC8, and CC1h (the amino acid sequence is shown in SEQ ID NO:92, the nucleotide sequence is shown in SEQ ID NO:93, and prepared by a method conventional in the art) were taken and diluted to the same concentration with sample buffer (25 mM sodium phosphate + 75 mM NaCl + 40% glycerin (pH 7.0)), respectively, and then investigated at 37°C for 24 and 48 hours, and the results are shown in Figure 24.

[0243] The molecular weights of RC1, RC3, RC4, RC5, RC6, RC7, RC8 and CC1h are shown in the table below. [Table 18A]

[0244] The results showed that CC1h aggregated (about 82KD) after 24h at 37℃, and the aggregation increased after 48h. RC3 and RC6 had almost no aggregation, while RC1 and RC4 showed a small amount of double molecular weight band, and the aggregation was stronger after 48h than after 24h, but less than CC1h, RC1, and RC4. Therefore, RC3, RC6, RC1, and RC4 have a tendency to aggregate more than CC1h, and may have good drug discovery properties.

[0245] The amino acid and nucleotide sequences of CC1h are as follows: EGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVEGGHHHHHH(SEQ ID NO:92);

[0246] 5. Neutralization of PLA2R-Ab in plasma samples from RC1 to RC8 Currently, the only reagent kit approved by the US FDA for use in the diagnosis of PLA2R antibody-positive membranous nephropathy is used (Ommon, Germany, quantitatively detects PLA2R-Ab concentration in blood samples by ELISA method), and all steps are performed according to the specifications. The plasma samples of the three PLA2R antibody-positive membranous nephropathy-positive patients are numbered pMN-2, pMN-4, and pMN-7, respectively.

[0247] RC1~RC8 detect the neutralizing effect on PLA2R-Ab in the plasma of these three membranous nephropathy positive patients, and obtain the neutralization ratios in Figures 14~16 and Table 19. The neutralization ratio in Figures 14~16 is (1-(PLA2R-Ab detection value after adding inhibitor / PLA2R-Ab detection value without adding inhibitor))*100%, and the larger the value, the fewer the detectable autoimmune antibodies, and the better the corresponding inhibitory effect. (Assuming intravenous administration, 100% bioavailability, and 8L distribution volume of this inhibitor in humans, the concentration of the inhibitor in this study corresponds to 5μg / mL*8L=40mg / person dose.)

[0248] The calculation of the neutralization rate is as follows: The capture molecule in the reagent kit is PLA2R. When no drug molecules are added,

number

number

number

number

number

[0249] Neutralization ratio (NR):

number

[0250] In ELISA detection, PLA2R as a capture antigen was in large excess,

number

number

[0251] [Table 19]

[0252] The dose of RC1 to RC8 proteins in the three sets of experiments was 5 μg / mL. pMN-2 was a positive sample, and the drugs in each set showed a neutralizing effect of 95% or more against autoimmune antibodies with a plasma concentration of about 44.79 RU / mL. pMN-4 was a positive sample, and the drugs in each set showed a neutralizing effect of 80% or more against autoimmune antibodies with a plasma concentration of about 124.905 RU / mL. pMN-7 was a positive sample, and the drugs in each set showed a neutralizing effect of 80% or more against autoimmune antibodies with a plasma concentration of about 53.72 RU / mL, except for RC5, whose neutralizing rate was 77.63%.

[0253] 6. Fixation of RC3 and RC6 Take a certain amount of NHS-Bestarose (AA0031, Bogorong (Shanghai) Biotechnology Co., Ltd.) media stored in isopropanol into a gravity column and wash it with pre-cooled coupling solution A3 (1 mM hydrochloric acid) (0-4 °C) for at least 30 min. Wash 1 mL of media with 20 mL of coupling solution A3 solution, RC3, RC6, and replace its buffer environment with coupling solution B3 (0.1 M sodium bicarbonate, 0.5 M sodium chloride, pH 8.3) by ultrafiltration (ligand coupling concentration 10 mg / mL filler). Dilute the selected media with coupling solution A3 (1 mL media approximately 0.5 mL coupling solution A3), mix with the ligand solution to an equal volume, and shake at 4 °C overnight. The coupling supernatant was removed, blocking solution (0.1M Tris-HCl, pH 8.5) was added, blocking treatment was performed for 4 hours at room temperature, and the blocking solution and 0.2M acetic acid were alternately washed five times under the condition of pH 3.5, with 3 times the filler volume each time. After washing with PBS, the beads were used or stored in 0.1M phosphate buffer to obtain RC3-NHS Bestarose and RC6-NHS Bestarose.

[0254] 7. Adsorption of PLA2R-Ab in plasma samples by immobilized RC3 and RC6 RC3-NHS Bestarose and RC6-NHS Bestarose are thoroughly resuspended and 100 μL are added to the corresponding EP tubes, centrifuged at 1000 g for 5 min, and the supernatant is carefully aspirated. 500 μL of PBS is added and the supernatant is washed by resuspending-centrifuging-aspirating twice. 100 μL of the patient's plasma is added to the EP tube and resuspended, gently shaken for 30 min, and then centrifuged at 1000 g for 5 min. The supernatant is carefully aspirated and collected in three EP tubes, which are designated as RC3-pMN and RC6-pMN, respectively. The ELISA method detects the adsorption of PLA2R-Ab in PMN-4 and PMN-7 plasma samples by RC3 and RC6 after immobilization, and the method refers to the fourth part of this example (neutralization of PLA2R-Ab in plasma samples by RC1-RC8). The results refer to Figures 17-18. The results show that the removal rates of PLA2R-Ab in PMN-4 and PMN-7 plasma samples by RC3 and RC6 after fixation were both greater than 80%.

[0255] Removal rate of PLA2R-Ab in PMN-4 plasma samples by RC3 and RC6 after immobilization in Figure 17: RC3-NHS Bestarose=(225.55-43.16) / 225.55*100%=80.86%, RC6-NHS Bestarose=(225.55-13.82) / 225.55*100%=93.87%. Removal rate of PLA2R-Ab in PMN-7 plasma samples by RC3 and RC6 after immobilization in Figure 18: RC3-NHS Bestarose=(52.03-6.26) / 52.03*100%=87.96%, RC6-NHS Bestarose=(52.03-4.67) / 52.03*100%=91.02%.

[0256] 8. Acute toxicity test in RC1-RC8 mice Purchased BALB / c female mice were adapted and reared for one week, then randomly divided into eight groups: RC1 group, RC2 group, RC4 group, RC5 group, RC7 group, RC8 group, comfort group (also called "Buffer") and blank group (also called "No treatment"), with five mice in each group, weighed and marked, and administered at a dose of 10 times 40 mg / person. Before administration, the mice were weighed and then administered. Specifically, RC1 group was subcutaneously injected with 0.1mL of RC1 protein at a concentration of 1mg / mL, RC2 group was subcutaneously injected with 0.1mL of RC2 protein at a concentration of 1mg / mL, RC4 group was subcutaneously injected with 0.1mL of RC4 protein at a concentration of 1mg / mL, RC5 group was subcutaneously injected with 0.1mL of RC5 protein at a concentration of 1mg / mL, RC7 group was subcutaneously injected with 0.1mL of RC7 protein at a concentration of 1mg / mL, RC8 group was subcutaneously injected with 0.1mL of RC8 protein at a concentration of 1mg / mL, comfort group was injected with 0.1mL of PBS, and blank group was not injected. The day of administration was set as day 0, and after the end of administration, the mice were observed for 1 hour, and no obvious side effects such as difficulty in breathing, decreased movement, or tremors were observed. Mice are weighed at 24 h, 48 h, 72 h, and then every 48 h thereafter until day 10 after dosing.

[0257] The trend of body weight of mice subcutaneously injected with RC1, RC2, RC4, RC5, RC7, and RC8 proteins is shown in Figure 19. The results showed that there was no obvious difference in body weight between the experimental and control groups.

[0258] The purchased BALB / c female mice were adapted and reared for one week, then randomly divided into four groups: RC3 group, RC6 group, comfort group (also called "Buffer") and blank group (also called "No treatment"); each group had five mice, which were weighed and marked. The mice were administered at a dose of 10 times 40 mg / person. The mice were weighed before administration, and then administered. Specifically, the RC3 group was subcutaneously injected with 0.1 mL of RC3 protein at a concentration of 1 mg / mL, the RC6 group was subcutaneously injected with 0.1 mL of RC6 protein at a concentration of 1 mg / mL, the comfort group was injected with 0.1 mL of PBS, and the blank group was not injected. The day of administration was set as day 0, and the mice were observed for 1 hour after the end of administration, and no obvious side effects such as difficulty in breathing, decreased movement, or tremors were observed. The mice were weighed at 24 h, 48 h, and 72 h, and then weighed once every 48 h until the 30th day of administration.

[0259] The trend of body weight of mice subcutaneously injected with RC3 and RC6 proteins is shown in Figure 20. The results showed that there was no obvious difference in body weight between the experimental and control groups.

[0260] In the present description, a statement referring to terms such as "one embodiment," "some embodiments," "examples," "specific examples," or "some examples" means that a particular feature, structure, material, or characteristic described with reference to the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, general expressions of the above terms do not necessarily refer to the same embodiment or example. In addition, a particular feature, structure, material, or characteristic described may be incorporated in any one or more embodiments or examples in an appropriate manner. Furthermore, a person skilled in the art may combine and combine the various embodiments or examples described herein and the features of the various embodiments or examples without mutual inconsistency.

[0261] Although embodiments of the present invention have been presented and described, the above embodiments are illustrative and should not be construed as limiting the present invention. Those skilled in the art will appreciate that various changes, modifications, substitutions and variations are possible in the above embodiments within the scope of the present invention.

Claims

1. An antibody comprising a light chain variable region CDR and a heavy chain variable region CDR, The heavy chain variable region CDR1 sequence is GX １ X ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ As shown in The heavy chain variable region CDR2 sequence is X １０ X １１ X １２ X １３ X １４ X １５ GX １６ X １７ X １８ X １９ X ２０ X ２１ X ２２ X ２３ As shown in The heavy chain variable region CDR3 sequence is X ２４ G.G.X. ２５ X ２６ X ２７ X ２８ X ２９ X ３０ X ３１ As shown in Y, X １ is G or F, X ２ is G, N, or X ３ is G, I, X ４ is G, K or S, X ５ is G, D, or X ６ is G, T, or X ７ is G or Y, X ８ is G, I, X ９ is H, S, or X １０ is R or G, X １１ is G, I, X １２ is G or Y, X １３ is G, P, or X １４ is T, S, or X １５ is G, N, or X １６ is G or Y, X １７ is G, T, or X １８ is G, S or R, X １９ is G or Y, X ２０ is G, A, X ２１ is G, D, or X ２２ is G, S, or X ２３ is S, V, X ２４ is G, W, or X ２５ is G, D, or X ２６ is G, S, or X ２７ is G or F, X ２８ is G or Y, X ２９ is G, A, X ３０ is G, M, or X ３１ is S, G or D, The condition is X １ is F, X ２ is N,X ３ is I, X ４ is K, X ５ D, X ６ is T, X ７ is Y, X ８ is I, X ９ is H, X １０ is R, X １１ is I, X １２ is Y, X １３ is P, X １４ is T, X １５ is N,X １６ is Y, X １７ is T, X １８ is R, X １９ is Y, X ２０ A, X ２１ D, X ２２ is S, X ２３ V, X ２４ is W, X ２５ D, X ２６ is G, X ２７ is F, X ２８ is Y, X ２９ A, X ３０ is M,X ３１ is not true that D is true at the same time, The light chain variable region CDR1 sequence is X ３２ X ３３ X ３４ X ３５ X ３６ X ３７ X ３８ X ３９ X ４０ X ４１ As shown in A, The light chain variable region CDR2 sequence is X ４２ X ４３ X ４４ X ４５ X ４６ X ４７ As shown in S. The light chain variable region CDR3 sequence is X ４８ X ４９ X ５０ X ５１ X ５２ X ５３ X ５４ X ５５ X ５６ As shown in X ３２ is G, R, X ３３ is G, A, X ３４ is G, S, or X ３５ is G, Q, or X ３６ is G, D or S, X ３７ is G, V, X ３８ is G, N, or X ３９ is G, T, or X ４０ is G, A, X ４１ is S, V, X ４２ is G, S, or X ４３ is G, A, X ４４ is G, S, or X ４５ is G or F, X ４６ is L, S, X ４７ is G or Y, X ４８ is G, Q, or X ４９ is G, Q, or X ５０ is G, H, X ５１ is G or Y, X ５２ is S, T, X ５３ is G, T, or X ５４ is G, P, or X ５５ is G, P, or X ５６ is G or T, The condition is X ３２ is R, X ３３ A, X ３４ is S, X ３５ Q, X ３６ D, X ３７ V, X ３８ is N, X ３９ is T, X ４０ A, X ４１ V, X ４２ is S, X ４３ A, X ４４ is S, X ４５ is F, X ４６ L, X ４７ is Y, X ４８ Q, X ４９ Q, X ５０ is H, X ５１ is Y, X ５２ is T, X ５３ is T, X ５４ is P, X ５５ is P, X ５６ and T are not simultaneously satisfied.

2. The heavy chain variable region CDR and the light chain variable region CDR satisfy at least one of the following conditions: X ４ is G, S, or X ５ is G, X ２１ is G, X ３１ is S or G, and X ３６ The antibody of claim 1, wherein is G or S.

3. In the heavy chain variable region CDR, X １ is G or F, X ２ is G, N, or X ３ is G, I, X ４ is G, S, or X ５ is G, X ６ is G, T, or X ７ is G or Y, X ８ is G, I, X ９ is H, S, or X １０ is G, X １１ is G, I, X １２ is G or Y, X １３ is G, P, or X １４ is T, S, or X １５ is G, N, or X １６ is G, X １７ is G, T, or X １８ is G, S, or X １９ is G or Y, X ２０ is G, A, X ２１ is G, X ２２ is G, S, or X ２３ is S, V, X ２４ is G, X ２５ is G, D, or X ２６ is G, S, or X ２７ is G or F, X ２８ is G, X ２９ is G, A, X ３０ is G, M, or X ３１ is S or G, In the light chain variable region CDR, X ３２ is G, R, X ３３ is G, A, X ３４ is G, S, or X ３５ is G, Q, or X ３６ is G, S, or X ３７ is G, V, X ３８ is G, X ３９ is G, T, or X ４０ is G, A, X ４１ is S, V, X ４２ is G, S, or X ４３ is G, A, X ４４ is G, S, or X ４５ is G, X ４６ is L, S, X ４７ is G or Y, X ４８ is G, Q, or X ４９ is G, Q, or X ５０ is G, H, X ５１ is G, X ５２ is S, T, X ５３ is G, T, or X ５４ is G, P, or X ５５ is G, P, or X ５６ The antibody of claim 2, wherein is G or T.

4. The antibody comprises at least one of the following CDR sequences: Light chain variable region CDR sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:83, SEQ ID NO:84 and SEQ ID NO:85; The antibody of claim 1, characterized in that the heavy chain variable region CDR sequences are: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90 and SEQ ID NO:

91.

5. the light chain variable region CDR sequence comprises at least one of the amino acid sequences having at least 85% similarity to SEQ ID NOs: 1-3, 7-9, and 83-85; The antibody of claim 4, wherein the heavy chain variable region CDR sequence comprises at least one of the amino acid sequences having at least 85% similarity to SEQ ID NOs: 4-6, 10-12, 86-88, and 89-91.

6. The antibody is comprising the light chain variable region CDR1, CDR2, CDR3 sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, or an amino acid sequence having at least 85% similarity to SEQ ID NOs: 1, 2, and 3; or comprising the light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, or an amino acid sequence having at least 85% similarity to SEQ ID NOs: 7, 8, and 9, respectively; or The antibody of claim 4, comprising light chain variable region CDR1, CDR2, CDR3 sequences shown in SEQ ID NOs: 83, 84 and 85, respectively, or amino acid sequences having at least 85% similarity to SEQ ID NOs: 83, 84 and 85, respectively.

7. The antibody is or comprising the heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively, or an amino acid sequence having at least 85% similarity to SEQ ID NOs: 4, 5, and 6; or or comprising the heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively, or an amino acid sequence having at least 85% similarity to SEQ ID NOs: 10, 11, and 12; or comprising the heavy chain variable region CDR1, CDR2, CDR3 sequences shown in SEQ ID NOs: 86, 87, and 88, or an amino acid sequence having at least 85% similarity to SEQ ID NOs: 86, 87, and 88, respectively; The antibody of claim 4, comprising heavy chain variable region CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 89, 90, and 91, respectively, or amino acid sequences having at least 85% similarity to SEQ ID NOs: 89, 90, and 91.

8. The antibody is light chain variable region CDR1, CDR2, CDR3 sequences as set forth in SEQ ID NOs: 1, 2 and 3, respectively, or an amino acid sequence having at least 85% similarity to SEQ ID NOs: 1, 2 and 3, respectively, and heavy chain variable region CDR1, CDR2, CDR3 sequences as set forth in SEQ ID NOs: 4, 5 and 6, respectively, or an amino acid sequence having at least 85% similarity to SEQ ID NOs: 4, 5 and 6, respectively; or light chain variable region CDR1, CDR2, CDR3 sequences as set forth in SEQ ID NOs: 7, 8 and 9, or amino acid sequences having at least 85% similarity to SEQ ID NOs: 7, 8 and 9, respectively, and heavy chain variable region CDR1, CDR2, CDR3 sequences as set forth in SEQ ID NOs: 10, 11 and 12, or amino acid sequences having at least 85% similarity to SEQ ID NOs: 10, 11 and 12, respectively; or light chain variable region CDR1, CDR2, CDR3 sequences as set forth in SEQ ID NOs: 83, 84 and 85, or amino acid sequences having at least 85% similarity to SEQ ID NOs: 83, 84 and 85, respectively, and heavy chain variable region CDR1, CDR2, CDR3 sequences as set forth in SEQ ID NOs: 86, 87 and 88, or amino acid sequences having at least 85% similarity to SEQ ID NOs: 86, 87 and 88, respectively; The antibody described in claim 4, characterized in that it comprises light chain variable region CDR1, CDR2, CDR3 sequences shown in SEQ ID NOs: 83, 84 and 85 or amino acid sequences having at least 85% similarity to SEQ ID NOs: 83, 84 and 85, respectively, and heavy chain variable region CDR1, CDR2, CDR3 sequences shown in SEQ ID NOs: 89, 90 and 91 or amino acid sequences having at least 85% similarity to SEQ ID NOs: 89, 90 and 91, respectively.

9. at least one of a heavy chain framework region sequence and a light chain framework region sequence; The antibody according to any one of claims 1 to 8, characterized in that at least a portion of at least one of the heavy chain framework region sequence and the light chain framework region sequence is derived from at least one of a mouse-derived antibody, a human-derived antibody, a primate-derived antibody or a mutant thereof, preferably a human-derived antibody.

10. The antibody has a light chain variable region of the amino acid sequence shown in SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:95, and / or The antibody according to any one of claims 1 to 8, characterized in that the antibody has a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 96 or SEQ ID NO:

97.

11. the antibody has a light chain variable region having the amino acid sequence shown in SEQ ID NO:13 and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:15; or the antibody has a light chain variable region having the amino acid sequence shown in SEQ ID NO:14 and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:16; or the antibody has a light chain variable region having the amino acid sequence shown in SEQ ID NO:95 and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:96; or The antibody of claim 10, wherein the antibody has a light chain variable region having the amino acid sequence shown in SEQ ID NO:95 and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:

97.

12. The antibody according to any one of claims 1 to 8, characterized in that the antibody comprises at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a mouse-derived antibody, a human-derived antibody, a primate-derived antibody, or a mutant thereof.

13. The antibody of claim 12, wherein at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from a human antibody.

14. The antibody according to any one of claims 1 to 8, characterized in that the antibody has a heavy chain having the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 33 or SEQ ID NO: 35 and a light chain having the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO:

34.

15. the antibody has a heavy chain having the amino acid sequence shown in SEQ ID NO:17 and a light chain having the amino acid sequence shown in SEQ ID NO:19; or the antibody has a heavy chain having the amino acid sequence shown in SEQ ID NO:18 and a light chain having the amino acid sequence shown in SEQ ID NO:20; or the antibody has a heavy chain having the amino acid sequence shown in SEQ ID NO:33 and a light chain having the amino acid sequence shown in SEQ ID NO:34; or The antibody of claim 14, characterized in that the antibody has a heavy chain having the amino acid sequence shown in SEQ ID NO: 35 and a light chain having the amino acid sequence shown in SEQ ID NO:

34.

16. A nucleic acid molecule encoding the antibody according to any one of claims 1 to 15.

17. The nucleic acid molecule of claim 16, wherein the nucleic acid molecule is DNA.

18. An expression vector, characterized in that it carries a nucleic acid molecule according to claim 16 or 17.

19. The expression vector of claim 18, wherein the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

20. The expression vector of claim 19, wherein the expression vector is a slow virus vector or a plasmid expression vector.

21. 1. A recombinant cell comprising: Carrying a nucleic acid molecule according to claim 16 or 17, or A recombinant cell expressing the antibody according to any one of claims 1 to 15.

22. The recombinant cell according to claim 21, characterized in that the recombinant cell is obtained by introducing an expression vector according to any one of claims 18 to 20 into a host cell.

23. 23. The recombinant cell of claim 21 or 22, wherein the recombinant cell is a eukaryotic cell.

24. 24. The recombinant cell of claim 23, wherein the recombinant cell is a mammalian cell.

25. Use of an antibody according to any one of claims 1 to 15 in the preparation of a medicament.

26. 26. The use of claim 25, wherein the drug comprises a fusion protein or an ADC drug.

27. Use of an antibody according to any one of claims 1 to 15 as a drug vector or negative antibody.

28. The use according to claim 27, characterized in that the drug vector or negative antibody has no antigen binding ability.

29. A fusion protein comprising: A neutral antibody comprising an antibody F C segment or an antibody according to any one of claims 1 to 15; and one or more PLA2R antigen epitope peptides connected to the neutral antibody.

30. The fusion protein of claim 29, wherein the C-terminus of the PLA2R antigen epitope peptide is connected to the N-terminus of the neutral antibody.

31. The fusion protein of claim 30, wherein the PLA2R antigen epitope peptide comprises CysR, FnII, and PLA2R binding region 1 and / or PLA2R binding region 2.

32. The fusion protein of claim 31, wherein the C-terminus of CysR is connected to the N-terminus of FnII and the C-terminus of FnII is connected to the N-terminus of the PLA2R binding domain 1.

33. The fusion protein described in claim 32, characterized in that the C-terminus of CysR is connected to the N-terminus of FnII, the C-terminus of FnII is connected to the N-terminus of PLA2R binding region 1, and the C-terminus of PLA2R binding region 1 is connected to the N-terminus of PLA2R binding region 2.

34. The fusion protein of claim 30, wherein the PLA2R antigen epitope peptide has an amino acid sequence shown in SEQ ID NO: 21 or 22 or an amino acid sequence having at least 95% similarity to SEQ ID NO: 21 or 22.

35. A fusion protein according to any one of claims 29 to 34, characterized in that the neutral antibody is an antibody F C segment, and the C-terminus of the PLA2R antigen epitope peptide is connected to the N-terminus of at least one of the antibody F C segments.

36. The fusion protein of claim 35, wherein the neutral antibody is an antibody described in any one of claims 1 to 15, and the C-terminus of the PLA2R antigen epitope peptide is connected to the N-terminus of at least one light chain or heavy chain of the antibody.

37. A fusion protein described in any one of claims 29 to 34, characterized in that the neutral antibody is a double-chain antibody, and the N-terminus of each chain of the antibody FC segment is connected to the C-terminus of one PLA2R antigen epitope peptide.

38. The fusion protein described in claim 37, characterized in that the N-terminus of each light chain of an antibody described in any one of claims 1 to 15 is connected to the C-terminus of one PLA2R antigen epitope peptide.

39. The fusion protein described in claim 38, characterized in that the N-terminus of each heavy chain of an antibody described in any one of claims 1 to 15 is connected to the C-terminus of one PLA2R antigen epitope peptide.

40. The fusion protein described in claim 39, characterized in that the N-terminus of each heavy chain and each light chain of an antibody described in any one of claims 1 to 15 is connected to the C-terminus of one PLA2R antigen epitope peptide.

41. The fusion protein according to any one of claims 29 to 34, further comprising a connecting peptide between the PLA2R antigen epitope peptide and the antibody.

42. The fusion protein of claim 41, wherein the N-terminus of the connecting peptide is connected to the C-terminus of the PLA2R antigen epitope peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the neutral antibody.

43. The amino acid sequence of the connecting peptide is (GGGGS). ｎ 43. The fusion protein of claim 42, wherein n is an integer of 1 or greater.

44. 44. The fusion protein of claim 43, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

45. The fusion protein of claim 41, wherein the connecting peptide comprises the amino acid sequence shown in SEQ ID NO:

23.

46. The fusion protein of any one of claims 29 to 34, wherein the antibody F C segment has an amino acid sequence of SEQ ID NO:24 or an amino acid sequence having at least 90% similarity to SEQ ID NO:

24.

47. the fusion protein has a first peptide segment having an amino acid sequence as set forth in SEQ ID NO:57 and a second peptide segment having an amino acid sequence as set forth in SEQ ID NO:19; or the fusion protein has a first peptide segment having an amino acid sequence as set forth in SEQ ID NO:17 and a second peptide segment having an amino acid sequence as set forth in SEQ ID NO:59; or The amino acid sequence of the fusion protein is shown in SEQ ID NO: 61; or the fusion protein has a first peptide segment having an amino acid sequence as set forth in SEQ ID NO:63 and a second peptide segment having an amino acid sequence as set forth in SEQ ID NO:19; or the fusion protein has a first peptide segment having an amino acid sequence as set forth in SEQ ID NO:17 and a second peptide segment having an amino acid sequence as set forth in SEQ ID NO:65; or The amino acid sequence of the fusion protein is shown in SEQ ID NO: 67; or the fusion protein has a first peptide segment having an amino acid sequence as set forth in SEQ ID NO:57 and a second peptide segment having an amino acid sequence as set forth in SEQ ID NO:59; or The fusion protein according to any one of claims 29 to 34, characterized in that the fusion protein has a first peptide segment having an amino acid sequence as shown in SEQ ID NO: 63 and a second peptide segment having an amino acid sequence as shown in SEQ ID NO:

65.

48. A nucleic acid molecule encoding the fusion protein according to any one of claims 29 to 47.

49. 49. The nucleic acid molecule of claim 48, wherein the nucleic acid molecule is DNA.

50. 50. An expression vector, characterized in that it carries a nucleic acid molecule according to claim 48 or 49.

51. 51. The expression vector of claim 50, wherein the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

52. 52. The expression vector of claim 51, wherein the expression vector is a slow viral vector or a plasmid expression vector.

53. A recombinant cell, comprising: Carrying a nucleic acid molecule according to claim 48 or 49, or A recombinant cell expressing the fusion protein according to any one of claims 29 to 47.

54. The recombinant cell according to claim 53, characterized in that the recombinant cell is obtained by introducing an expression vector according to any one of claims 50 to 52 into a host cell.

55. 55. The recombinant cell of claim 53 or 54, wherein the recombinant cell is a eukaryotic cell.

56. 56. The recombinant cell of claim 55, wherein the recombinant cell is a mammalian cell.

57. A pharmaceutical composition comprising the fusion protein according to any one of claims 29 to 47.

58. 58. The pharmaceutical composition of claim 57, further comprising a pharma- ceutically acceptable adjuvant.

59. An immunoadsorbent comprising the fusion protein according to any one of claims 29 to 47.

60. A reagent kit comprising the fusion protein according to any one of claims 29 to 47 or the immunoadsorbent according to claim 59.

61. 60. The immunosorbent of claim 59 ; and a solid phase vector connected to said immunosorbent.

62. 62. The adsorption product of claim 61, wherein the adsorption product is selected from an immunoadsorption column or a blood purification agent.

63. 60. Use of the immunoadsorbent of claim 59 in the preparation of an adsorption product for adsorbing PLA2R antibodies.

64. 64. The use according to claim 63, characterized in that the adsorption product is selected from an immunoadsorption column or a blood purification agent.

65. The use according to claim 64, characterized in that the adsorption product is an immunoadsorption column and is used to prevent or treat diseases associated with PLA2R antibodies.

66. The use according to claim 65, characterized in that the PLA2R antibody-related disease is selected from PLA2R antibody-positive membranous nephropathy.

67. 65. The use according to claim 64, wherein the adsorption product is a blood purifying agent and the adsorption product is used to adsorb PLA2R antibodies in blood or plasma.

68. Use of a fusion protein according to any one of claims 29 to 47, a nucleic acid molecule according to any one of claims 48 to 49, an expression vector according to any one of claims 50 to 52, a recombinant cell according to any one of claims 53 to 56, a pharmaceutical composition according to any one of claims 57 to 58 or an immunoadsorbent according to claim 59 in the preparation of a medicament for preventing or treating a disease associated with PLA2R antibodies.

69. The use according to claim 68, characterized in that the PLA2R antibody-related disease is selected from PLA2R antibody-positive membranous nephropathy.

70. Use of a fusion protein according to any one of claims 29 to 47, a nucleic acid molecule according to any one of claims 48 to 49, an expression vector according to any one of claims 50 to 52, a recombinant cell according to any one of claims 53 to 56 or a pharmaceutical composition according to any one of claims 57 to 58 in the preparation of an immunoadsorbent for adsorbing PLA2R antibodies.

71. Use of a fusion protein according to any one of claims 29 to 47, a nucleic acid molecule according to any one of claims 48 to 49, an expression vector according to any one of claims 50 to 52, a recombinant cell according to any one of claims 53 to 56, a pharmaceutical composition according to any one of claims 57 to 58 or an immunoadsorbent according to claim 59 in the preparation of a reagent kit for detecting PLA2R antibodies.

72. A method for preventing or treating a PLA2R-related disease by an antibody, comprising: A method for preventing or treating a PLA2R antibody-related disease, comprising administering to a subject a pharma- ceutical acceptable amount of a fusion protein according to any one of claims 29 to 47 or a pharmaceutical composition according to any one of claims 57 to 58.

73. 73. The method of claim 72, wherein the route of administration of said method employs subcutaneous injection or intravenous injection.

74. The method of claim 73, wherein the PLA2R antibody-related disease is selected from PLA2R antibody-positive membranous nephropathy.

75. 1. An immunoadsorption therapeutic method for preventing or treating a PLA2R antibody-related disease, comprising:

60. A method of immunoadsorption therapy comprising the step of treating a subject with the immunoadsorbent of claim 59.

76. 76. The method of claim 75, wherein the subject is treated using extracorporeal blood purification.

77. The method of claim 75, wherein the PLA2R antibody-related disease is selected from PLA2R antibody-positive membranous nephropathy.

78. A method for diagnosing a PLA2R-related disease using an antibody, comprising: Detecting a PLA2R antibody in a sample to be measured using a fusion protein according to any one of claims 29 to 47, a nucleic acid molecule according to any one of claims 48 to 49, an expression vector according to any one of claims 50 to 52, a recombinant cell according to any one of claims 53 to 56, a pharmaceutical composition according to any one of claims 57 to 58, an immunoadsorbent according to claim 59, or a reagent kit according to claim 60; A method for diagnosing a disease associated with PLA2R antibodies, comprising the step of determining the amount of PLA2R antibodies in the sample to be measured based on the detection results.

79. The method of claim 78, characterized in that the content of PLA2R antibodies in the sample to be measured is 14 RU / ml or less, which is an indication that the sample to be measured is derived from a patient suffering from a disease associated with PLA2R antibodies.

80. The method of claim 79, characterized in that the content of PLA2R antibodies in the sample to be measured is 20 RU / ml or less, which is an indication that the sample to be measured is derived from a patient suffering from a disease associated with PLA2R antibodies.

81. The method according to any one of claims 78 to 80, wherein the sample to be measured is blood.

82. The method according to any one of claims 78 to 80, wherein the PLA2R antibody-related disease is selected from PLA2R antibody-positive membranous nephropathy.

83. 1. A method for assessing the stage of a PLA2R-related disease using an antibody, comprising: Detecting a PLA2R antibody in a measurement sample derived from a subject patient using the fusion protein according to any one of claims 29 to 47, the nucleic acid molecule according to any one of claims 48 to 49, the expression vector according to any one of claims 50 to 52, the recombinant cell according to any one of claims 53 to 56, the pharmaceutical composition according to any one of claims 57 to 58, the immunoadsorbent according to claim 59, or the reagent kit according to claim 60; A method for evaluating the stage of a disease associated with PLA2R antibodies, comprising the step of determining the amount of PLA2R antibodies in the sample to be measured based on the detection results.

84. The method according to claim 83, characterized in that the stage of a disease in which a test patient is suffering from a PLA2R antibody-related disease is determined based on the content of PLA2R antibody in the measured sample.

85. The method of claim 83 or 84, wherein the PLA2R antibody-related disease is selected from PLA2R antibody-positive membranous nephropathy.

86. The method according to claim 83 or 84, wherein the sample to be measured is blood.

87. A method for evaluating the prognosis of a PLA2R-related disease using an antibody, comprising: Detecting a PLA2R antibody in a sample to be measured using a fusion protein according to any one of claims 29 to 47, a nucleic acid molecule according to any one of claims 48 to 49, an expression vector according to any one of claims 50 to 52, a recombinant cell according to any one of claims 53 to 56, a pharmaceutical composition according to any one of claims 57 to 58, an immunoadsorbent according to claim 59, or a reagent kit according to claim 60; A method for evaluating the prognosis of a disease associated with PLA2R antibodies, comprising the step of determining the content of PLA2R antibodies in the sample to be measured based on the detection results.

88. The method of claim 87, wherein the sample to be measured is derived from a patient suffering from a disease associated with PLA2R antibodies before or after treatment.

89. The method described in claim 87, characterized in that the prognostic effect of a disease associated with PLA2R antibodies is determined based on the content of PLA2R antibodies in a measured sample of a patient suffering from a disease associated with PLA2R antibodies before or after treatment.

90. The method of claim 87, wherein a decrease in the content of PLA2R antibodies in the measured sample of a patient suffering from a disease associated with PLA2R antibodies after treatment is indicative of a good prognosis for the patient.

91. The method according to any one of claims 87 to 90, wherein the sample to be measured is blood.

92. The method according to any one of claims 87 to 90, wherein the PLA2R antibody-related disease is selected from PLA2R antibody-positive membranous nephropathy.

Images