WO2023185957A1 - 抗体、融合蛋白及其用途 - Google Patents

抗体、融合蛋白及其用途 Download PDF

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WO2023185957A1
WO2023185957A1 PCT/CN2023/084857 CN2023084857W WO2023185957A1 WO 2023185957 A1 WO2023185957 A1 WO 2023185957A1 CN 2023084857 W CN2023084857 W CN 2023084857W WO 2023185957 A1 WO2023185957 A1 WO 2023185957A1
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seq
antibody
pla2r
amino acid
acid sequence
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French (fr)
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赖兵
邱勇
张知明
龚石林
何玲
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成都地奥制药集团有限公司
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    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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Definitions

  • the present invention relates to the fields of molecular biology and medicine. Specifically, the present invention relates to an antibody, a fusion protein and its use. More specifically, the present invention relates to an antibody and its nucleic acid molecules, expression vectors, recombinant cells and fusion proteins. Its nucleic acid molecules, expression vectors, recombinant cells, pharmaceutical compositions and uses.
  • MN Membranous nephropathy
  • MN is membranous glomerulonephritis, which specifically refers to nephrotic syndrome caused by excessive deposition of immune complexes in glomerular capillaries. It is one of the most common pathological types of nephrotic syndrome in adults. 1.
  • the global incidence rate is approximately 12/1 million (Mc Grogan A, Franssen CF, de Vries CS. Nephrol Dial Transplant. 2011; 26(2): 414-430).
  • the incidence of MN has increased at an annual rate of 13% (Lwezaula BF, Ameh OI, Ekrikpo UE, et al. BMC Nephrol.
  • MN mainly manifests as massive proteinuria, hypoalbuminemia, edema and hyperlipidemia.
  • the diagnosis relies on histopathological examination of renal biopsy. Its pathological diagnostic features include glomerular capillary wall thickening, immunofluorescence conditions It can be seen that IgG and complement C3 are deposited along the capillary wall, and electron microscopy shows the formation of electron dense material under the glomerular epithelial cells.
  • MN can be divided into idiopathic membranous nephropathy (IMN) and secondary membranous nephropathy (SMN).
  • IFN idiopathic membranous nephropathy
  • SSN secondary membranous nephropathy
  • PMN primary membranous nephropathy
  • the target antigen located in podocytes is recognized by autoantibodies and combines with it to form an immune complex. The substance is deposited under the basement membrane podocytes, activating the complement system and causing podocyte damage and shedding, resulting in increased basement membrane permeability and subsequent massive proteinuria.
  • PHA2R phospholipase A2 receptor
  • PHA2R-Ab Phospholipase-A2 receptor antibody
  • PLA2R-Ab can specifically bind to the target antigen (ie, PLA2R) on podocytes in renal tissue and can be expressed in the serum of 70% to 80% of PMN patients (Bobart SA, De Vriese AS, Pawar AS, et al. Kidney Int.2019;95(2):429-438), and the positive expression rate of PLA2R-Ab antibody in the serum of healthy people, patients with SMN and other glomerular diseases is very low, so the specificity of PLA2R-Ab in diagnosing MN higher.
  • the latest Meta-analysis shows (Lian Mingjian, Lu Rong, Zhang Jiaqin, et al.
  • the sensitivity, specificity and accuracy of PLA2R-Ab in predicting 12-month clinical remission events in MN patients were 69.51% and 76.35, respectively. % and 80.26% (Zheng Na, Wang Meihua, An Zhi. Journal of Clinical Military Medicine. 2021;49(08):942-944).
  • PLA2R-Ab has important reference value for the clinical diagnosis, condition judgment and prognosis assessment of PMN patients.
  • PLA2R-Ab is involved in the disease progression of PMN. Patients with low titers of PLA2R-Ab have milder disease, more significant effects after immunosuppressive treatment, and may even experience spontaneous remission, suggesting that monitoring PLA2R-Ab levels can help guide clinicians.
  • the dosage and duration of immunosuppressive therapy for patients can avoid excessive immunosuppressive treatment and also help reduce the patient's medical burden.
  • the higher the serum PLA2R-Ab level the stronger the immune activity of the body, the more serious the condition, the worse the effect of immunosuppressive treatment, and the poorer clinical prognosis. It further shows that monitoring serum PLA2R-Ab can help guide clinical treatment. Early and effective intervention treatment should be carried out for patients with high PLA2R-Ab levels after admission.
  • the clinical treatment of MN is mainly based on a low-fat, low-salt, low-protein diet, symptomatic and supportive treatment to control proteinuria and hyperlipidemia, supplemented by immunosuppressive treatment and some non-immunosuppressive treatments, but there is no definite treatment. method.
  • the treatment effect of patients with membranous nephropathy is poor. Only 40% of patients can spontaneously resolve, and 30% of patients have poor curative effect and eventually progress to end-stage renal disease. They can only maintain their lives through dialysis or transplantation, which seriously increases the patient's financial situation. burden and impact on patients' quality of life. For kidney transplant patients, about 40% of kidney transplant patients have poor treatment results, and some of them will lose the transplanted kidney. Based on the pathogenesis of PMN and the important value of serum PLA2R-Ab levels in evaluating the therapeutic effect and clinical prognosis of PMN, an effective treatment for PLA2R-Ab positive patients is urgently needed. Drugs or technologies for MN patients.
  • the present invention aims to solve one of the technical problems existing in the prior art, at least to a certain extent.
  • the present invention provides an antibody and a fusion protein that combines the antibody with the 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 PLA2R. Antibody.
  • PLA2R antibody-positive membranous nephropathy One possible treatment for PLA2R antibody-positive membranous nephropathy is to specifically neutralize PLA2R-Ab in serum.
  • the inventors were surprised to find that the fusion protein of PLA2R and antibody developed in this application (in this application, the fusion protein of antigen and antibody is called antigenic antibody) can achieve the technology of specifically neutralizing PLA2R-Ab in serum. Effect.
  • PLA2R is a membrane protein highly expressed on the surface of glomerular podocytes and contains the structural domain shown in Figure 1; at the same time, there is a small amount of secreted PLA2R in the blood.
  • the antibodies in the patient's plasma mainly recognize CysR and FNII among PLA2R and three domains of CTLD1.
  • the inventors found that the fusion protein obtained by fusing the antigenic region of PLA2R with an antibody can maintain the binding ability of PLA2R and PLA2R-Ab. Moreover, during the research and development process, the inventor found that by mutating the CDR region of the original antibody, the antibody obtained had extremely low antigen-binding activity, which could avoid side effects caused by subsequent non-specific binding and have a higher expression level. Therefore, the fusion protein obtained by fusing the mutated antibody with the antigenic region of PLA2R specifically neutralizes PLA2R-Ab without binding to other antibodies, thus avoiding the side effects of non-specific immunity. In addition, the fusion protein also has the advantage of a long half-life, allowing for reduced dosing frequency.
  • the invention provides an antibody.
  • the antibody includes: light chain variable region CDR and heavy chain variable region CDR; wherein, the heavy chain variable region CDR1 sequence is such as GX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 is shown , and the heavy chain variable region CDR2 sequence is shown as X 10 X 11 X 12 X 13 X 14
  • the region CDR3 sequence is shown as X 24 GGX 25 X 26 X 27 X 28 X 29 X 30 X 31 Y,
  • X 1 is G or F
  • X 2 is G or N
  • X 3 is G or I
  • X 5 is G or D
  • X 6 is G or T
  • X 7 is G or Y
  • X 8 is G or I
  • X 9 is H or S
  • X 10 is R or G
  • X 11 is G Or I
  • X 12 is G or Y
  • X 13 is G or P
  • the inventor found through a large number of experiments that the above-mentioned antibody has low antigen-binding activity. After forming an antigenic antibody, it can effectively avoid non-specific binding of the antigenic antibody. At the same time, more importantly, the antibody has a high expression level and is suitable for antigenicity. Development of antibody drugs.
  • the invention provides a first nucleic acid molecule.
  • the first nucleic acid molecule encodes the antibody of 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. After forming an antigenic antibody, it can effectively avoid non-specific binding of the antigenic antibody. At the same time, the expression level of the antibody is high, which is suitable for antigenic antibodies. Drug development.
  • the invention provides a first expression vector.
  • the first expression vector carries the first nucleic acid molecule described in the second aspect.
  • the invention provides a first recombinant cell.
  • the first recombinant cell includes: carrying the first nucleic acid molecule described in the second aspect; or expressing the antibody described in the first aspect.
  • the first recombinant cell according to the embodiment of the present invention can be used for in vitro expression and large-scale acquisition of the antibody with low antigen-binding activity described in the first aspect.
  • the present invention provides the use of the antibody described in the first aspect in the preparation of fusion protein drugs.
  • the above-mentioned antibody has no antigen-binding activity and can be used as a neutral drug carrier to prepare drugs, which can be used to treat or prevent diseases.
  • the above-mentioned antibodies can be used to fuse the antigenic epitope peptides to prepare a fusion protein. Since the above-mentioned antibodies have no antigen-binding activity, the prepared fusion protein specifically binds to the antibody corresponding to the antigenic epitope peptide and does not bind to other antibodies. To avoid the side effects of non-specific binding of fusion proteins, it can be used to treat or prevent related diseases caused by antibodies corresponding to epitope peptides.
  • the present invention proposes the use of the aforementioned antibody as a pharmaceutical carrier or negative antibody. It can be known from the above that the above-mentioned antibody of the present invention has no antigen-binding activity, and can be used as a neutral drug carrier to prepare drugs, or can be directly used as a reference for negative antibodies without antigen-binding activity.
  • the present invention provides a fusion protein.
  • the fusion protein includes: a neutral antibody including an antibody FC segment or the antibody described in the first aspect; one or more PLA2R antigen epitope peptides, the PLA2R antigen The epitope peptide is linked to the neutral antibody.
  • the inventor found through experiments that by fusing the PLA2R epitope peptide with the above-mentioned 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 having the ability to express Good performance and high yield.
  • the invention provides a second nucleic acid molecule.
  • the nucleic acid molecule encodes the fusion protein described in the seventh aspect.
  • the second nucleic acid molecule can be effectively used to express the above-mentioned fusion protein.
  • the present invention provides a second expression vector.
  • the second expression vector carries the second nucleic acid molecule described in the eighth aspect. This second expression vector can be effectively used to express the above-mentioned fusion protein.
  • the present invention provides a second recombinant cell.
  • the second recombinant cell includes: carrying the second nucleic acid molecule described in the eighth aspect; or expressing the fusion protein described in the seventh aspect.
  • Recombinant cells according to embodiments of the present invention can be used for in vitro expression and large-scale acquisition of the fusion protein described in the fifth aspect.
  • the invention provides a pharmaceutical composition.
  • the pharmaceutical composition includes: the fusion protein described in the seventh aspect.
  • the inventor found through extensive experiments that the above pharmaceutical composition can effectively prevent and treat related diseases caused by PLA2R antibodies, such as PLA2R antibody-positive membranous nephropathy.
  • the present invention provides an immunoadsorbent.
  • the immunoadsorbent includes: the fusion protein described in the seventh aspect. The inventor found through extensive experiments that the above-mentioned immunoadsorbent can adsorb PLA2R antibodies, and it can effectively treat related diseases caused by PLA2R antibodies, such as PLA2R antibody-positive membranous nephropathy, through immunoadsorption.
  • the present invention provides a kit.
  • the kit includes: the fusion protein described in the seventh aspect.
  • the inventor found through extensive experiments that the above kit can effectively detect PLA2R antibody content, and can also be used to diagnose or treat related diseases caused by PLA2R antibodies, such as PLA2R antibody-positive membranous nephropathy.
  • the invention provides an adsorption product.
  • the adsorption product includes: the immunoadsorbent mentioned above; a solid phase carrier, and the immunoadsorbent is connected to the solid phase carrier.
  • the adsorption product of the present invention can be used to adsorb PLA2R antibodies, such as adsorbing PLA2R antibodies in blood or plasma. By adsorbing PLA2R antibodies in blood or plasma, it can be used to prevent or treat related diseases caused by PLA2R antibodies.
  • the present invention proposes the use of the immunoadsorbent described in the twelfth aspect in preparing an adsorption product, and the adsorption product is used to adsorb PLA2R antibodies.
  • the present invention provides a fusion protein described in the seventh aspect, a second nucleic acid molecule described in the eighth aspect, a second expression vector described in the ninth aspect, a fusion protein described in the tenth aspect,
  • the present invention provides a fusion protein described in the seventh aspect, a second nucleic acid molecule described in the eighth aspect, a second expression vector described in the ninth aspect, a fusion protein described in the tenth aspect, The use of the second recombinant cells or the pharmaceutical composition of the eleventh aspect in preparing an immunoadsorbent for adsorbing PLA2R antibodies.
  • the present invention provides 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, and a second expression vector according to the tenth aspect.
  • the present invention provides a method for preventing or treating related diseases caused by PLA2R antibodies.
  • the method includes: administering to the subject a pharmaceutically acceptable amount of the fusion protein according to the seventh aspect or the pharmaceutical composition according to the eleventh aspect.
  • related diseases caused by PLA2R antibodies can be effectively treated.
  • the present invention proposes an immunoadsorption treatment method for preventing or treating related diseases caused by PLA2R antibodies.
  • the method includes: treating the subject using the immunoadsorbent described in the twelfth aspect.
  • the present invention provides a method for diagnosing related diseases caused by PLA2R antibodies.
  • the The method includes: 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 eleventh aspect
  • the pharmaceutical composition described in the twelfth aspect, the immunosorbent described in the twelfth aspect, or the kit described in the thirteenth aspect is used to detect the PLA2R antibody in the sample to be tested; based on the detection results, determine the PLA2R antibody in the sample to be tested content.
  • the present invention proposes a method for assessing the staging of related diseases caused by PLA2R antibodies.
  • the method includes: 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 The second recombinant cell, the pharmaceutical composition described in the eleventh aspect, the immunoadsorbent described in the twelfth aspect or the kit described in the thirteenth aspect detects the PLA2R antibody in the test sample; based on the detection results, Determine the content of PLA2R antibodies in the sample to be tested.
  • the present invention proposes a prognostic method for evaluating related diseases caused by PLA2R antibodies.
  • the method includes: 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 The second recombinant cell, the pharmaceutical composition described in the eleventh aspect, the immunoadsorbent described in the twelfth aspect or the kit described in the thirteenth aspect detects the PLA2R antibody in the test sample; based on the detection results, Determine the content of PLA2R antibodies in the sample to be tested.
  • Figure 1 is a schematic structural diagram of the expression framework in Embodiment 1 of the present invention.
  • Figure 2 is an electrophoresis result diagram of pcDNA3.4, trsH, trsL, RtxH and RtxL in Example 1 of the present invention
  • Figure 3 is an electrophoresis result diagram of dTrs1H, dTrs2H and dTrs1L (dTrs2L) in Example 1 of the present invention
  • Figure 4 is an electrophoresis result diagram of dTrs3H and dTrs3L in Example 1 of the present invention
  • Figure 5 is an electrophoresis result diagram of dTrs4H and dTrs4L in Example 1 of the present invention
  • Figure 6 shows the purification effect of Trs, dTrs1-4 and Rtx proteins detected by SDS-PAGE in Example 1 of the present invention
  • Figure 7 is the detection results of the binding of Trs, dTrs1-4 and Rtx proteins to Her2 in Example 1 of the present invention.
  • Figure 8 is a schematic structural diagram of the RC1-RC8 protein in Example 2 of the present invention.
  • FIG. 9 is a schematic structural diagram of PLA2R in Embodiment 2 of the present invention.
  • Figure 10 is an electrophoresis result diagram of pcDNA3.4 in Example 2 of the present invention.
  • Figure 11 is an electrophoresis result diagram of pcDNA3.4, RC1H, RC2L, RC4H and RC5L in Example 2 of the present invention
  • Figure 12 is an electrophoresis result diagram of RC3 and RC6 in Example 2 of the present invention.
  • Figure 13 shows the purification effect of RC1-8 protein detected by SDS-PAGE in Example 2 of the present invention
  • Figure 14 shows the neutralizing effect of RC1-RC8 on PLA2R-Ab in the plasma of membranous nephropathy-positive patient pMN-2 in Example 2 of the present invention
  • Figure 15 shows the neutralizing effect of RC1-RC8 on PLA2R-Ab in the plasma of membranous nephropathy-positive patient pMN-4 in Example 2 of the present invention
  • Figure 16 shows the neutralizing effect of RC1-RC8 on PLA2R-Ab in the plasma of pMN-7, a positive patient with membranous nephropathy, in Example 2 of the present invention
  • Figure 17 shows the clearance rate of PLA2R-Ab adsorption in plasma samples of PMN-4 patients by RC3 and RC6 in Example 2 of the present invention
  • Figure 18 shows the clearance rate of PLA2R-Ab adsorption in PMN-7 patient plasma samples by RC3 and RC6 in Example 2 of the present invention
  • Figure 19 is a body weight trend chart of mice injected subcutaneously with RC1, RC2, RC4, RC5, RC7, and RC8 proteins in Example 2 of the present invention
  • Figure 20 is a body weight trend chart of mice injected subcutaneously with RC3 and RC6 proteins in Example 2 of the present invention.
  • Figure 21 is the detection result of Trs, dTrs1-4 and Rtx protein binding to IgA in Example 1 of the present invention.
  • Figure 22 is the detection result of Trs, dTrs1-4 and Rtx protein binding to IgM in Example 1 of the present invention
  • Figure 23 is the detection results of the binding of Trs, dTrs1-4 and Rtx proteins to HSA in Example 1 of the present invention.
  • Figure 24 is an electrophoresis result diagram of the stability investigation of RC1-RC8 in Example 2 of the present invention.
  • first and second are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Therefore, features defined as “first” and “second” may explicitly or implicitly include one or more of these features. Further, in the description of the present invention, unless otherwise stated, the meaning of "plurality” is two or more.
  • the terms “optionally,” “optionally,” or “optionally” generally mean that the subsequently described event or condition may but need not occur, and that the description includes instances in which the event or condition occurs, and A situation in which the event or condition did not occur.
  • the term "antibody” generally refers to an immunoglobulin molecule, including two light chains with a lighter molecular weight and two heavy chains with a heavier molecular weight.
  • the heavy chain (H chain) and the light chain (L chain) are composed of two Sulfur bonds connect to form a tetrapeptide chain molecule.
  • the amino-terminal (N-terminal) amino acid sequence of the peptide chain changes greatly and is called the variable region (V region).
  • the carboxyl-terminal (C-terminal) is relatively stable and changes very little, and is called the constant region (C region).
  • the V regions of L chain and H chain are called VL and VH respectively.
  • variable region certain regions have a higher degree of variation in amino acid composition and sequence, called the hypervariable region (HVR).
  • the hypervariable region is the location where antigen and antibody bind, so it is also called the complementarity determining region. (complementarity-determining region, CDR).
  • CDR complementarity-determining region
  • FR backbone region
  • HVR hypervariable region
  • CDR complementarity-determining region
  • neutral antibody refers to a protein structure with low antigen-binding activity, such as an Fc segment, or the antibody proposed in the first aspect of the present invention.
  • the term “diabody” refers to an antibody with a symmetrical structure, such as an antibody composed of only two Fc fragments, or a complete antibody composed of two heavy chains and two light chains, such as the antibody of the present invention. Antibodies presented in one aspect.
  • the terms “identity”, “homology” or “similarity” are used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, using conventional methods to determine two amino acid sequences or nucleic acid sequences.
  • the percentage of identical amino acids or nucleotides between each other see, for example, 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, D.C.).
  • ALIGN program Dayhoff ( 1978), Atlas of Protein Sequence and Structure 5: Suppl.3 (National Biomedical Research Foundation, Washington, D.C.).
  • There are many algorithms for comparing sequences and determining sequence identity including, Needleman et al.
  • 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, etc., above, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and from Intelligenetics, Mountain View, California CLUSTAL in the PC/Gene program.
  • GCG Genetics Computing Group
  • nucleotide generally refers to ribonucleotides, deoxynucleotides, or modified forms of either type of nucleotide, and combinations thereof.
  • the term "at least 85% similarity” means at least 85% similarity to each reference sequence, and may be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% similarity.
  • the term "expression vector” generally refers to a nucleic acid molecule capable of inserting into a suitable host to self-replicate, which transfers the inserted nucleic acid molecule into and/or between host cells.
  • the expression vectors may include vectors primarily used for insertion of DNA or RNA into cells, vectors primarily used for replication of DNA or RNA, and vectors primarily used for expression of transcription and/or translation of DNA or RNA.
  • the expression vectors also include vectors having a variety of the above-mentioned functions.
  • the expression vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell.
  • the expression vector can produce the desired expression product by culturing a suitable host cell containing the expression vector.
  • the term “recombinant cell” usually refers to the use of genetic engineering technology or cell fusion technology to modify or recombine the genetic material of the host cell to obtain cells with stable inherited unique traits.
  • the term "host cell” refers to a prokaryotic cell or eukaryotic cell into which a recombinant expression vector can be introduced.
  • the term “transformed” or “transfected” refers to the introduction of nucleic acid (eg, a vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of the invention and can be used for expression and/or secretion of the target protein.
  • suitable host cells include immortalized hybridoma cells, NS/0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (human amniotic fluid-derived cells), insect cells , PER.C6 cells and CoS cells.
  • fusion protein refers to a new type of protein formed by the fusion of at least two proteins or polypeptides.
  • the above fusion operation can usually be achieved through genetic engineering and other technologies. For example, two genes obtained through DNA recombinant technology are recombined. expression product.
  • PLA2R is in a fusion state with the neutral antibody of the double-stranded structure.
  • PLA2R fusion protein does not mean that all parts of the protein are formed by fusion, for example, the double-stranded structure
  • the two Fc segments in the body can be connected together through chemical bonds such as disulfide bonds.
  • composition generally refers to unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. Generally, compositions are prepared by uniformly and thoroughly bringing into association the active fusion protein with a liquid carrier, a finely divided solid carrier, or both.
  • pharmaceutically acceptable means that the substance or composition must be chemically and/or toxicologically compatible with the other ingredients of the formulation and/or the mammal being treated therewith.
  • pharmaceutically acceptable in the present invention refers to those approved by federal regulatory agencies or national governments or listed in the United States Pharmacopeia or other generally recognized pharmacopeias for use in animals, especially humans.
  • the terms "pharmaceutically acceptable excipients” or “pharmaceutically acceptable carriers” may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the specific target dosage form. . Except to the extent that any conventional excipients are incompatible with the fusion proteins of the invention, such as resulting in any adverse biological effects or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, they The uses are also within the scope of consideration of the present invention.
  • the term "administration" refers to the introduction of a predetermined amount of a substance into a patient by some suitable means.
  • the fusion protein or pharmaceutical composition of the present invention can be administered by any common route as long as it can reach the intended tissue.
  • Various modes of administration are contemplated, including peritoneal, intravenous injection, intramuscular injection, subcutaneous injection, etc., but the present invention is not limited to these exemplified administration modes.
  • the compositions of the present invention are administered intravenously or subcutaneously.
  • PLA2R antibody-related diseases generally refers to diseases caused by abnormal PLA2R antibodies, such as related diseases caused by increased PLA2R antibodies, including but not limited to PLA2R antibody-positive membranous nephropathy.
  • PLA2R is a membrane protein expressed in glomerular podocytes. For unknown reasons, it becomes an antigen, causing the body to produce corresponding autoimmune antibodies (PLA2R antibodies).
  • PLA2R antibodies bind to PLA2R and deposit under the glomerular basement membrane, triggering downstream effects such as C3 deposition, leading to PLA2R antibody-positive membranous nephropathy.
  • treatment refers to treatment to obtain a desired pharmacological and/or physiological effect.
  • the effect may be prophylactic in terms of complete or partial prevention of the disease or its symptoms, and/or therapeutic in terms of partial or complete cure of the disease and/or adverse effects caused by the disease.
  • Treatment encompasses diseases of mammals, especially humans, including: (a) preventing the occurrence of a disease or disorder in individuals susceptible to the disease but who have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviate disease, such as reducing symptoms associated with a disease.
  • Treatment encompasses any administration of a fusion protein, pharmaceutical composition or drug to an individual to treat, cure, alleviate, ameliorate, alleviate or inhibit a disease in the individual, including but not limited to the administration of a drug containing the fusion protein described herein Give to individuals in need.
  • the present invention proposes an antibody, a fusion protein and their uses, which will be described in detail below.
  • Antibody first nucleic acid molecule, first expression vector, first recombinant cell and uses thereof
  • the present invention mutates different sites in the CDR region of the Trastuzumab (Trs) antibody, and obtains the antibody of the present invention through a large number of experiments.
  • the antibody has extremely weak binding to its antigen Her2 and does not bind to any other antigens.
  • the antibody has extremely high antigen-binding activity. Low, it can be used as a fusion protein by fusing a neutral antibody with a biologically active molecule.
  • the fusion protein can bind to the specific binder of the biologically active molecule.
  • the fusion protein obtained by combining the neutral antibody with PLA2R can specifically bind to the PLA2R antibody. , and does not bind to other antibodies.
  • the fusion protein obtained by fusing a neutral antibody with an antigen is used as a drug, which can bind to the specific antibody of the antigen without binding to other antibodies.
  • Specific antibody-producing cells that can specifically eliminate antigens.
  • the invention provides an antibody.
  • the antibody includes: light chain variable region CDR and heavy chain variable region CDR; wherein, the heavy chain variable region CDR1 sequence is such as GX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 is shown , and the heavy chain variable region CDR2 sequence is shown as X 10 X 11 X 12 X 13 X 14
  • the region CDR3 sequence is shown as X 24 GGX 25 X 26 X 27 X 28 X 29 X 30 X 31 Y,
  • X 1 is G or F
  • X 2 is G or N
  • X 3 is G or I
  • X 5 is G or D
  • X 6 is G or T
  • X 7 is G or Y
  • X 8 is G or I
  • X 9 is H or S
  • X 10 is R or G
  • X 11 is G Or I
  • X 12 is G or Y
  • X 13 is G or
  • the heavy chain variable region CDR and light chain variable region CDR satisfy at least one of the following conditions: X 4 is G or S, X 5 is G, X 21 is G, and X 31 is S or G, X 36 is G or S.
  • X 1 is G or F
  • X 2 is G or N
  • X 3 is G or I
  • X 4 is G or S
  • X 5 is G
  • X 6 is G or T
  • X 7 is G or Y
  • X 8 is G or I
  • X 9 is H or S
  • X 10 is G
  • X 11 is G or I
  • X 12 is G or Y
  • X 13 is G or P
  • X 14 is T or S
  • X 15 is G or N
  • X 16 is G
  • X 17 is G or T
  • X 18 is G or S
  • X 19 is G or Y
  • X 20 is G or A
  • X 21 is G
  • X 22 is G or S
  • X 23 is S or V
  • X 24 is G
  • X 25 is G or D
  • X 26 is G or S
  • X 27 is G or F
  • X 28 is G
  • X 29 is G or A
  • the antibody includes a CDR sequence selected from at least one of the following: light chain variable region CDR sequence: 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 sequence: 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.
  • the antibodies provided by the invention have conservative amino acid substitutions compared to the above-mentioned heavy chains and light chains.
  • Constant amino acid substitution refers to the substitution of an amino acid with a biologically, chemically or structurally similar residue of another amino acid. Biologically similar means that the substitution does not destroy the low biological activity of the antibody's antigen-binding activity.
  • Structurally similar refers to amino acids having side chains of similar length, such as alanine, glycine, or serine, or having side chains of similar size. Chemical similarity means that amino acids have the same charge or are both hydrophilic or hydrophobic. For example, the hydrophobic residues isoleucine, valine, leucine or methionine substitute for each other. Or use polar amino acids such as arginine instead of lysine, glutamic acid instead of aspartic acid, and glutamine instead of aspartate. Amides, serine instead of threonine, etc.
  • the light chain variable region CDR sequence includes an amino acid sequence having at least 85% similarity with SEQ ID NO: 1-3, SEQ ID NO: 7-9 and SEQ ID NO: 83-85 At least one of;
  • the heavy chain variable region CDR sequence includes SEQ ID NO:4 ⁇ 6, SEQ ID NO:10 ⁇ 12, SEQ ID NO:86 ⁇ 88 and SEQ ID NO:89 ⁇ 91 having at least the same At least one of the amino acid sequences has 85% similarity.
  • the antibody comprises: a light chain variable as shown in SEQ ID NO: 1, 2 and 3 respectively or an amino acid sequence having at least 85% similarity to SEQ ID NO: 1, 2 and 3 Region CDR1, CDR2, CDR3 sequence; alternatively, a light chain variable region CDR1 as set forth in SEQ ID NO:7, 8 and 9 respectively or an amino acid sequence having at least 85% similarity to SEQ ID NO:7, 8 and 9 , CDR2, CDR3 sequence; alternatively, a light chain variable region CDR1, CDR2 as set forth in SEQ ID NO:83, 84 and 85 respectively or an amino acid sequence having at least 85% similarity to SEQ ID NO:83, 84 and 85 , CDR3 sequence.
  • the antibody comprises: a heavy chain variable as shown in SEQ ID NO:4, 5 and 6 respectively or an amino acid sequence having at least 85% similarity to SEQ ID NO:4, 5 and 6 Region CDR1, CDR2, CDR3 sequence; alternatively, a heavy chain variable region CDR1 as set forth in SEQ ID NO: 10, 11 and 12 respectively or an amino acid sequence having at least 85% similarity to SEQ ID NO: 10, 11 and 12 , CDR2, CDR3 sequence; alternatively, a heavy chain variable region CDR1, CDR2 as set forth in SEQ ID NO:86, 87 and 88 respectively or an amino acid sequence having at least 85% similarity to SEQ ID NO:86, 87 and 88 , CDR3 sequence; alternatively, heavy chain variable regions CDR1, CDR2, CDR3 as set forth in SEQ ID NO:89, 90 and 91 respectively or an amino acid sequence having at least 85% similarity to SEQ ID NO:89, 90 and 91 sequence.
  • the antibody comprises: a light chain variable as shown in SEQ ID NO: 1, 2 and 3 respectively or an amino acid sequence having at least 85% similarity to SEQ ID NO: 1, 2 and 3 Region CDR1, CDR2, CDR3 sequences, and the heavy chain variable region CDR1, CDR1, CDR2, CDR3 sequence; or light chain variable region CDR1, CDR2, CDR3 as set forth in SEQ ID NO:7, 8 and 9 respectively or an amino acid sequence having at least 85% similarity to SEQ ID NO:7, 8 and 9 Sequences, and heavy chain variable region CDR1, CDR2, CDR3 sequences as set forth in SEQ ID NO: 10, 11, and 12, respectively, or an amino acid sequence that is at least 85% similar to SEQ ID NO: 10, 11, and 12; or Light chain variable region CDR1, CDR2, CDR3 sequences as set forth in SEQ ID NOs: 83, 84 and 85 respectively or an amino acid sequence having at least 85% similarity to SEQ ID NOs: 83, 84 and 85
  • the antibody contains: at least one of a heavy chain framework region sequence and a light chain framework region sequence; wherein at least one of the heavy chain framework region sequence and the light chain framework region sequence contains at least A part comes from at least one of murine antibodies, human antibodies, primate antibodies or mutants thereof, preferably human antibodies.
  • 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 has a light chain variable region such as SEQ ID NO: 14 or SEQ ID NO: 95;
  • the inventor can obtain the CDR region of the heavy chain variable region sequence of the above-mentioned antibody through antibody sequence comparison databases (NCBI, IMGT) or related software (as shown in SEQ ID NO: 4-6, SEQ ID NO: 10-12) and the CDR region of the light chain variable region sequence (as shown in SEQ ID NO: 1 to 3, SEQ ID NO: 7 to 9).
  • the light chain variable region sequence of the antibody has conservative amino acid substitutions compared with the amino acid sequence shown in SEQ ID NO: 13 or 14; or, the heavy chain variable region sequence of the antibody has a conservative amino acid substitution. Compared with the amino acid sequence shown in SEQ ID NO:15 or 16, it has conservative amino acid substitutions. Of course, these conservative amino acid substitutions will not change the biological function of the antibody. In some specific ways, these conservative amino acid substitutions may occur at amino acids in the heavy chain variable region and light chain variable region other than the CDR region.
  • the antibody has a light chain variable region with an amino acid sequence shown in SEQ ID NO: 13 and an amino acid sequence shown in SEQ ID NO: 15 Sequence of the heavy chain variable region.
  • the antibody has a light chain variable region with an amino acid sequence shown in SEQ ID NO: 14 and a heavy chain variable region with an amino acid sequence shown in SEQ ID NO: 16.
  • the antibody has a light chain variable region with an amino acid sequence shown in SEQ ID NO: 95 and a heavy chain variable region with an amino acid sequence shown in SEQ ID NO: 96.
  • the antibody has a light chain variable region with an amino acid sequence shown in SEQ ID NO: 95 and a heavy chain variable region with an amino acid sequence shown in SEQ ID NO: 97.
  • the antibody contains at least one of a heavy chain constant region and a light chain constant region, and at least a part of at least one of the heavy chain constant region and the light chain constant region is from a murine antibody, a human At least one of a human antibody, a primate antibody or a mutant thereof, preferably a human antibody.
  • the antibody has a heavy chain with an amino acid sequence as shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 33 or SEQ ID NO: 35 and has a heavy chain as shown in SEQ ID NO: 19 , the light chain of the amino acid sequence shown in SEQ ID NO:20 or SEQ ID NO:34.
  • the antibody has a heavy chain with an amino acid sequence shown in SEQ ID NO: 17 and a light chain with an amino acid sequence shown in SEQ ID NO: 19.
  • the antibody has a heavy chain with an amino acid sequence shown in SEQ ID NO: 18 and a light chain with an amino acid sequence shown in SEQ ID NO: 20.
  • the antibody has a heavy chain with an amino acid sequence shown in SEQ ID NO: 33 and a light chain with an amino acid sequence shown in SEQ ID NO: 34.
  • the antibody has a heavy chain with an amino acid sequence shown in SEQ ID NO: 35 and a light chain with an amino acid sequence shown in SEQ ID NO: 34.
  • the nucleic acid molecules expressing these antibodies can be connected to different vectors and then expressed in different cells to obtain the corresponding antibodies.
  • the invention provides a first nucleic acid molecule.
  • the first nucleic acid molecule encodes the antibody of the first aspect.
  • the antibody encoded by the first nucleic acid molecule according to the embodiment of the present invention has extremely low antigen-binding activity and has the advantage of high expression level.
  • the nucleic acid molecule is DNA.
  • 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.
  • 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.
  • 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.
  • 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.
  • the invention provides a first expression vector.
  • the first expression vector carries the first nucleic acid molecule described in the second aspect.
  • the polynucleotide can be directly or indirectly connected to the control elements on the first expression vector, as long as these control elements can control the translation and expression of the polynucleotide, etc. That’s it.
  • these control elements can come directly from the first expression vector itself, or they can be exogenous, that is, they do not come from the first expression vector itself.
  • the polynucleotide is operably linked to the control element.
  • operably connected refers to connecting an exogenous gene to a vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, can exert their intended effects on regulating the transcription and translation of the exogenous gene. function.
  • control elements in the vector such as transcription control sequences and translation control sequences
  • the polynucleotides used to encode the heavy chain and light chain of the antibody can be independently inserted into different vectors. Commonly, they are inserted into the same vector. Commonly used vectors can be, for example, plasmids, phages, etc.
  • the first expression vector is a eukaryotic expression vector.
  • the first expression vector is a lentiviral vector.
  • the invention provides a first recombinant cell.
  • the first recombinant cell includes: carrying the first nucleic acid molecule described in the second aspect; or expressing the antibody described in the first aspect.
  • the first expression vector can be introduced into mammalian cells, the first recombinant cells can be constructed, and then these first recombinant cells can be used to express the antibodies provided by the invention. By culturing the first recombinant cells, the corresponding antibody can be obtained.
  • These available mammalian cells can be, for example, CHO cells.
  • the recombinant cell is obtained by introducing a first expression vector into a host cell.
  • the recombinant cells are eukaryotic cells.
  • the recombinant cells are mammalian cells.
  • the present invention provides the use of the antibody described in the first aspect in the preparation of fusion protein drugs.
  • the above-mentioned antibody of the present invention has no antigen-binding activity, and can be used as a neutral pharmaceutical carrier to prepare drugs, and the drugs can be used to treat or prevent diseases.
  • the above-mentioned antibodies can be used to fuse the antigenic epitope peptides to prepare a fusion protein. Since the above-mentioned antibodies have no antigen-binding activity, the prepared fusion protein specifically binds to the antibody corresponding to the antigenic epitope peptide and does not bind to other antibodies. To avoid the side effects of non-specific binding of fusion proteins, it can be used to treat or prevent related diseases caused by antibodies corresponding to epitope peptides.
  • the drug includes a fusion protein or an ADC drug.
  • the present invention proposes the use of the aforementioned antibody as a pharmaceutical carrier or negative antibody. It can be known from the above that the above-mentioned antibody of the present invention has no antigen-binding activity, and can be used as a neutral drug carrier to prepare drugs, or can be directly used as a reference for negative antibodies without antigen-binding activity.
  • the drug carrier or negative antibody has no antigen-binding ability.
  • the present invention provides a fusion protein.
  • the fusion protein includes: a neutral antibody including an antibody FC segment or the antibody described in the first aspect; one or more PLA2R antigen epitope peptides, the PLA2R antigen The epitope peptide is linked to the neutral antibody.
  • the inventor found through experiments that by fusing the PLA2R epitope peptide with the above-mentioned neutral antibody, the resulting fusion protein can competitively bind to the PLA2R antibody, prevent the PLA2R antibody from binding to PLA2R in the body, and can effectively treat PLA2R antibody-induced diseases. related diseases.
  • the PLA2R epitope peptide in the fusion protein of the present invention combines with the PLA2R antibody.
  • the neutral antibody of the fusion protein contains an Fc fragment. The Fc fragment can bind to the Fc ⁇ R receptor of NK cells, thereby mediating NK cells to directly kill the expression membrane.
  • the Fc fragment of B cells expressing membrane-type PLA2R antibodies can also bind to complement C1q and activate the complement system, exerting a lytic effect on B cells expressing membrane-type PLA2R antibodies. Since the fusion protein of the present invention specifically binds to PLA2R antibodies and does not bind to Combined with other antibodies, it can specifically eliminate B cells and avoid the side effects of non-specific immunity. Moreover, the fusion protein of the present invention can also bind to liver FcRn receptors and be protected from degradation, and has a long plasma half-life. Therefore, as a drug, this fusion protein has the advantage of long half-life, can reduce the frequency of administration, and also has the advantages of good expression, high yield, high stability, high affinity, good solubility and good drugability.
  • amino acid fragments are usually divided into N-terminus and C-terminus.
  • the present invention does not strictly limit the connection method between the neutral antibody and the PLA2R epitope peptide, as long as the N-terminus of one fragment is connected to another fragment. Just connect to the C terminal.
  • the C-terminus of the PLA2R epitope peptide is connected to the N-terminus of the neutral antibody.
  • the PLA2R epitope peptide includes CysR, FnII, and PLA2R binding region 1 and/or PLA2R binding region 2.
  • the C-terminal of the CysR is connected to the N-terminal of the FnII, and the C-terminal of the FnII is connected to the N-terminal of the PLA2R binding region 1.
  • the C-terminal of the CysR is connected to the N-terminal of the FnII
  • the C-terminal of the FnII is connected to the N-terminal of the PLA2R binding region 1
  • the C-terminal of the PLA2R binding region 1 is connected to The N-terminus of PLA2R binding region 2 is connected.
  • the PLA2R epitope peptide has an amino acid sequence as shown in SEQ ID NO: 21 or 22 or an amino acid sequence that is at least 95% similar to SEQ ID NO: 21 or 22.
  • the neutral antibody is an antibody FC segment
  • the C-terminus of the PLA2R epitope peptide is connected to the N-terminus of at least one of the antibody FC segments.
  • the neutral antibody is the antibody described in the first aspect, and the C-terminus of the PLA2R epitope peptide is connected to the N-terminus of at least one light chain or heavy chain of the antibody.
  • the neutral antibody is a diabody, and the N-terminus of each chain of the antibody FC segment is respectively connected to the C-terminus of a PLA2R epitope peptide.
  • the fusion protein contains two PLA2R epitope peptides, which is a bivalent molecule. The inventor found through experiments that compared with monovalent natural PLA2R, the fusion protein of the present invention has a stronger binding ability to PLA2R antibodies, can effectively neutralize PLA2R antibodies, and protect the natural PLA2R on the surface of glomerular cells from being attacked by PLA2R antibodies. .
  • the N-terminus of each light chain of the antibody described in the first aspect is respectively connected to the C-terminus of a PLA2R epitope peptide.
  • the fusion protein contains two PLA2R epitope peptides, which is a bivalent molecule.
  • the inventor found through experiments that compared with monovalent natural PLA2R, the fusion protein of the present invention has a stronger binding ability to PLA2R antibodies, can effectively neutralize PLA2R antibodies, and protect the natural PLA2R on the surface of glomerular cells from being attacked by PLA2R antibodies. .
  • the N-terminus of each heavy chain of the antibody described in the first aspect is respectively connected to the C-terminus of a PLA2R epitope peptide.
  • the fusion protein contains two PLA2R epitope peptides, which is a bivalent molecule.
  • the inventor found through experiments that compared with monovalent natural PLA2R, the fusion protein of the present invention has a stronger binding ability to PLA2R antibodies, can effectively neutralize PLA2R antibodies, and protect the natural PLA2R on the surface of glomerular cells from being destroyed by PLA2R antibodies. attack.
  • the N-terminus of each heavy chain and each light chain of the antibody described in the first aspect is respectively connected to the C-terminus of a PLA2R epitope peptide.
  • the inventor found through experiments that the fusion protein contains four PLA2R epitope peptides. Compared with the monovalent natural PLA2R, the fusion protein of the present invention has a stronger binding ability to PLA2R antibodies and can effectively neutralize PLA2R antibodies and protect the kidneys. The native PLA2R on the cell surface of the pellet group was not attacked by PLA2R antibodies.
  • the fusion protein further includes a connecting peptide disposed between the PLA2R epitope peptide and the antibody.
  • the N-terminus of the connecting peptide is connected to the C-terminal of the PLA2R epitope peptide, and the C-terminal of the connecting peptide is connected to the N segment of the neutral antibody. Therefore, the PLA2R epitope peptide and the neutral antibody that can be fused with the above-mentioned linking peptide are used to improve the binding ability of the finally obtained fusion protein and the PLA2R antibody.
  • the amino acid sequence of the connecting peptide is (GGGGS) n , where n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the connecting peptide includes the amino acid sequence shown in SEQ ID NO: 23.
  • the antibody FC segment has an amino acid sequence such as SEQ ID NO:24 or at least 90% similar to SEQ ID NO:24.
  • the fusion protein has a first peptide segment with an amino acid sequence shown in SEQ ID NO: 57 and a second peptide segment with an amino acid sequence shown in SEQ ID NO: 19; or
  • the fusion protein has a first peptide segment with an amino acid sequence as shown in SEQ ID NO: 17 and a second peptide segment with an amino acid sequence as shown in SEQ ID NO: 59; or
  • amino acid sequence of the fusion protein is shown in SEQ ID NO: 61; or
  • the fusion protein has a first peptide segment with an amino acid sequence shown in SEQ ID NO: 63 and a second peptide segment with an amino acid sequence shown in SEQ ID NO: 19; or
  • the fusion protein has a first peptide segment with an amino acid sequence shown in SEQ ID NO: 17 and a second peptide segment with an amino acid sequence shown in SEQ ID NO: 65; or
  • amino acid sequence of the fusion protein is shown in SEQ ID NO: 67; or
  • the fusion protein has a first peptide segment with an amino acid sequence shown in SEQ ID NO: 57 and a second peptide segment with an amino acid sequence shown in SEQ ID NO: 59; or
  • the fusion protein has a first peptide segment with an amino acid sequence shown in SEQ ID NO: 63 and a second peptide segment with an amino acid sequence shown in SEQ ID NO: 65.
  • the first peptide segment of the fusion protein is a peptide segment including the heavy chain of the first antibody
  • the second peptide segment of the fusion protein is a peptide segment including the light chain of the first antibody.
  • the first peptide segment and the second peptide segment are connected through inter-chain disulfide bonds; when the neutral antibody is the above-mentioned antibody Fc fragment, the fusion protein is a peptide segment including the antibody Fc fragment.
  • the nucleic acid molecules expressing these fusion proteins can be connected to different vectors and then expressed in different cells to obtain the corresponding fusion proteins.
  • the invention provides a second nucleic acid molecule.
  • the nucleic acid molecule encodes the fusion protein described in the seventh aspect.
  • the second nucleic acid molecule can be effectively used to express the above-mentioned fusion protein, especially in prokaryotes or lower eukaryotes expression systems.
  • the second nucleic acid molecule here does not need to encode the entire sequence of the above-mentioned fusion protein.
  • it can include two types of nucleic acid molecules, encoding two monomers respectively, and further can be assembled in vitro to obtain a heterodimer, that is, Fusion proteins containing diabodies can be formed.
  • the second nucleic acid molecule is DNA.
  • the present invention provides a second expression vector.
  • the second expression vector carries the second nucleic acid molecule described in the eighth aspect.
  • This second expression vector can be effectively used to express the above fusion protein, especially in prokaryotes or lower eukaryotes.
  • the above fusion protein can be effectively expressed in the system.
  • the second expression vector is a eukaryotic expression vector.
  • the second expression vector is a lentiviral vector.
  • the present invention provides a second recombinant cell.
  • the second recombinant cell includes: carrying the second nucleic acid molecule described in the eighth aspect; or expressing the fusion protein described in the seventh aspect.
  • Recombinant cells according to embodiments of the present invention can be used for in vitro expression and large-scale acquisition of the fusion protein described in the seventh aspect.
  • the second recombinant cell is a eukaryotic cell.
  • the second recombinant cell is a mammalian cell.
  • the invention provides a pharmaceutical composition.
  • the pharmaceutical composition includes: the fusion protein described in the seventh aspect.
  • the inventor found through extensive experiments that the above pharmaceutical composition can effectively prevent and treat related diseases caused by PLA2R antibodies, such as PLA2R antibody-positive membranous nephropathy.
  • the pharmaceutical composition further includes pharmaceutically acceptable excipients.
  • the present invention provides an immunoadsorbent.
  • the immunoadsorbent includes: the fusion protein described in the seventh aspect. The inventor found through extensive experiments that the above-mentioned immunoadsorbent can adsorb PLA2R antibodies, and it can effectively treat related diseases caused by PLA2R antibodies, such as PLA2R antibody-positive membranous nephropathy, through immunoadsorption.
  • the present invention provides a kit.
  • the kit includes: the fusion protein described in the seventh aspect.
  • the inventor found through extensive experiments that the above kit can effectively detect PLA2R antibody content, and can also be used to diagnose or treat related diseases caused by PLA2R antibodies, such as PLA2R antibody-positive membranous nephropathy.
  • the invention provides an adsorption product.
  • the adsorption product includes: the immunoadsorbent described in the twelfth aspect; a solid phase carrier, and the immunoadsorbent is connected to the solid phase carrier.
  • the adsorption product of the present invention can be used to adsorb PLA2R antibodies, such as adsorbing PLA2R antibodies in blood or plasma. By adsorbing PLA2R antibodies in blood or plasma, it can be used to prevent or treat related diseases caused by PLA2R antibodies.
  • connection method between the immunosorbent and the solid phase carrier is not specifically limited, and they can be coupled or connected through chemical bonds.
  • the blood or plasma may be derived from any animal, such as mammals.
  • the blood or plasma is selected from human blood or plasma.
  • the adsorption product is selected from an immunoadsorption column or a blood purification agent.
  • the present invention proposes the use of the immunoadsorbent described in the twelfth aspect in preparing an adsorption product, and the adsorption product is used to adsorb PLA2R antibodies.
  • the adsorption product is selected from an immunoadsorption column or a blood purification agent.
  • the adsorption product is an immunoadsorption column, and the adsorption product is used to prevent or treat related diseases caused by PLA2R antibodies.
  • the above-mentioned immunoadsorbent can adsorb PLA2R antibodies. Therefore, the immunoadsorption column containing the above-mentioned immunoadsorbent can adsorb PLA2R antibodies through the immunoadsorbent. The immunoadsorption column can effectively treat related diseases caused by PLA2R antibodies.
  • the related disease caused by the PLA2R antibody is selected from PLA2R antibody-positive membranous nephropathy.
  • the adsorption product is a blood purification agent, and the adsorption product is used to adsorb PLA2R antibodies in blood or plasma.
  • the above-mentioned immunoadsorbent can adsorb PLA2R antibodies. Therefore, a blood purification agent containing the above-mentioned immunoadsorbent can adsorb PLA2R antibodies through the immunoadsorbent.
  • the blood purification agent can effectively adsorb PLA2R antibodies in blood or plasma.
  • the present invention provides a fusion protein described in the seventh aspect, a second nucleic acid molecule described in the eighth aspect, a second expression vector described in the ninth aspect, a fusion protein described in the tenth aspect, The use of the second recombinant cell or the pharmaceutical composition of the eleventh aspect in the preparation of medicines for preventing or treating related diseases caused by PLA2R antibodies.
  • the related disease caused by the PLA2R antibody is selected from PLA2R antibody-positive membranous nephropathy.
  • the dosage form of the traditional Chinese medicine composition includes injection.
  • the administration route of the traditional Chinese medicine composition includes subcutaneous injection or intravenous injection.
  • the present invention provides a fusion protein described in the seventh aspect, a second nucleic acid molecule described in the eighth aspect, a second expression vector described in the ninth aspect, a fusion protein described in the tenth aspect, The use of the second recombinant cells or the pharmaceutical composition of the eleventh aspect in preparing an immunoadsorbent for adsorbing PLA2R antibodies.
  • the present invention provides 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, and a second expression vector according to the tenth aspect.
  • the present invention provides a method for preventing or treating related diseases caused by PLA2R antibodies.
  • the method includes: administering to the subject a pharmaceutically acceptable amount of the fusion protein according to the seventh aspect or the pharmaceutical composition according to the eleventh aspect.
  • related diseases caused by PLA2R antibodies can be effectively treated.
  • the administration route of the method adopts subcutaneous injection or intravenous injection.
  • the related disease caused by the PLA2R antibody is selected from PLA2R antibody-positive membranous nephropathy.
  • the present invention proposes an immunoadsorption treatment method for preventing or treating related diseases caused by PLA2R antibodies.
  • the method includes: treating the subject using the immunoadsorbent described in the twelfth aspect.
  • the method uses extracorporeal blood purification to treat a subject.
  • the related disease caused by the PLA2R antibody is selected from PLA2R antibody-positive membranous nephropathy.
  • the present invention provides a method for diagnosing related diseases caused by PLA2R antibodies.
  • the method includes: 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 The second recombinant cell, the pharmaceutical composition described in the eleventh aspect, the immunoadsorbent described in the twelfth aspect or the kit described in the thirteenth aspect detects the PLA2R antibody in the test sample; based on the detection results, Determine the content of PLA2R antibodies in the sample to be tested.
  • the content of the PLA2R antibody in the sample to be tested is not less than 14 RU/ml, which is an indication that the sample to be tested is derived from patients suffering from related diseases caused by PLA2R antibodies.
  • the sample to be tested is The content of PLA2R antibodies in the sample is not less than 20RU/ml, which is an indication that the sample to be tested is derived from patients with related diseases caused by PLA2R antibodies.
  • the sample to be tested is blood.
  • the related disease caused by the PLA2R antibody is selected from PLA2R antibody-positive membranous nephropathy.
  • the present invention proposes a method for assessing the staging of related diseases caused by PLA2R antibodies.
  • the method includes: 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 The second recombinant cell, the pharmaceutical composition described in the eleventh aspect, the immunoadsorbent described in the twelfth aspect or the kit described in the thirteenth aspect detects the PLA2R antibody in the test sample; based on the detection results, Determine the content of PLA2R antibodies in the sample to be tested.
  • the stage of the patient to be tested suffering from related diseases caused by the PLA2R antibody is determined.
  • the related disease caused by the PLA2R antibody is selected from PLA2R antibody-positive membranous nephropathy.
  • PLA2R antibody-positive membranous nephropathy is divided into four stages.
  • the sample to be tested is blood.
  • the present invention proposes a prognostic method for evaluating related diseases caused by PLA2R antibodies.
  • the method includes: 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 The second recombinant cell, the pharmaceutical composition described in the eleventh aspect, the immunoadsorbent described in the twelfth aspect or the kit described in the thirteenth aspect detects the PLA2R antibody in the test sample; based on the detection results, Determine the content of PLA2R antibodies in the sample to be tested.
  • the sample to be tested is derived from patients suffering from related diseases caused by PLA2R antibodies before or after treatment.
  • the prognostic effect of related diseases caused by PLA2R antibodies is determined based on the content of PLA2R antibodies in test samples of patients suffering from related diseases caused by PLA2R antibodies before or after treatment.
  • a decrease in the content of PLA2R antibodies in the test samples of patients suffering from related diseases caused by PLA2R antibodies after treatment is an indication of a good prognosis of the patients.
  • the sample to be tested is blood.
  • the related disease caused by the PLA2R antibody is selected from PLA2R antibody-positive membranous nephropathy.
  • the plasmid vector uses the pcDNA3.4 transient expression vector, which contains the native full-length CMV promoter and the WPRE element downstream of the cloning site.
  • Vectors based on the CMV promoter usually have good expression for the CHO cell transient expression system.
  • the WPRE element is located downstream of the multiple cloning site, which can effectively improve the transcription and expression of genes.
  • E. coli it has an ampicillin resistance gene.
  • PCR technology is used to amplify the vector gene pcDNA3.4 and the target gene.
  • the PCR template is obtained through gene synthesis.
  • the vector fragment and the target gene fragment are connected through a homologous recombination kit.
  • a single clone is obtained by DH5 ⁇ competent transformation, and the single clone is amplified. After amplification, sequencing is performed. After confirmation by sequencing, the plasmid is extracted to obtain the recombinant expression plasmid of the target gene.
  • the DNA encoding the target sequence plus the DNA encoding the secretion expression signal peptide were genetically synthesized and cloned between the CMV promoter and WPRE genes of plasmid pcDNA3.4. See Figure 1 for details.
  • the name and sequence of the target gene are shown in Table 1.
  • the primers and templates of the pcDNA3.4 vector and the target gene (or target fragment) are shown in Table 2. Among them, RtxH and RtxL are used to synthesize the target gene.
  • Rituximab (Rtx for short).
  • dTrs1L and dTrs2L in this example are the same target genes.
  • the PCR reaction uses TAKARA’s KOD reaction system (Cat.KFX-101), reaction conditions, reagents and PCR target fragment information are shown in Tables 3 to 6.
  • the sequencing primer is CMV-F: CGCAAATGGGCGGTAGGCGTG (SEQ ID NO:79). After comparing the sequencing results, it was confirmed that all the test sequences were consistent with the original sequences. Add 100 ⁇ L of the monoclonal bacterial solution to 50 mL of LB liquid culture medium containing ampicillin (final concentration 50 ⁇ g/mL), and incubate at 37°C and 200 rpm. Cultivate overnight under the conditions, preserve the strain, extract the plasmid, and obtain the target gene expression plasmid.
  • Trs Trs, dTrs1-4 and Rtx proteins
  • FreeStyle TM Cells (cell density is 5-6 ⁇ 10 5 /mL, from FreeStyle TM MAX CHO expression system of American Gibco Company, product number: K900020), were cultured at 37°C, 5% CO 2 and 120-135 rpm/min.
  • the Trs, dTrs1-4 and Rtx culture supernatants were centrifuged at 4°C and 7000g for 15 minutes and then filtered and sterilized with a 0.22um syringe filter.
  • Elute with Buffer B1 0.1M Glycine-HCl, pH value 3.0
  • collect elution peak samples collect elution peak samples, and adjust the target protein eluate to neutrality with 1M Tris-HCl (pH value 9.0).
  • Ultrafilter into PBS buffer Ultrafilter into PBS buffer.
  • Identification of the target protein Select a 10-well SDS-PAGE precast gel and run the gel at 150V for 50 minutes. The sample was heated at 90°C for 5 min, and 10 ⁇ g was loaded into each well.
  • lane 4 is the elution and reduction of dTrs1
  • lane 5 is the elution and non-reduction of dTrs2
  • lane 6 is the elution and reduction of dTrs2
  • lane 7 is the elution and reduction of dTrs3
  • lane 8 is the elution and non-reduction of dTrs3
  • the lane 9 is the elution and non-reduction of dTrs4.
  • Lane 10 is dTrs4 elution and reduction
  • lane 11 is Rtx non-reduction
  • lane 12 is Rtx reduction.
  • Table 10 The expression levels of Trs, dTrs1-4 and Rtx proteins are shown in Table 10.
  • Trs, dTrs1-4 and Rtx proteins bind to Her2 ELISA
  • Trs (0.44mg/mL), dTrs1(0.38mg/mL), dTrs2(0.42mg/mL), dTrs3(0.115mg/mL), dTrs4(1.4mg/mL), Rtx(0.21mg/mL) , where Trs is the positive control and Rtx is the negative control.
  • HRP-conjugated goat anti-human IgG (Sangon Bioengineering (Shanghai) Co., Ltd., Cat. No. D110150).
  • Coating Dilute the culture supernatant with coating solution, add 0.1 ⁇ g/100 ⁇ L Her2 protein (34 ⁇ L mother solution + 8466 ⁇ L coating solution) to each well, seal the plate, and incubate at 37°C for 2 hours. Discard the liquid in the wells, wash the plate three times with 200 ⁇ L of washing solution each time, and let it stand for 2 minutes.
  • Blocking Add 100 ⁇ L of blocking solution to each well, keep overnight at 4°C, and discard the liquid in the wells.
  • Trs, dTrs1-4 or Rtx Primary antibody (Trs, dTrs1-4 or Rtx) incubation: dilute Trs, dTrs1-4 and Rtx with antibody diluent respectively, add 100 ⁇ L to each well, seal the plate, incubate at 37°C for 1 hour, discard the liquid in the well, and wash with Wash the plate three times with 200 ⁇ L each time and let it stand for 2 minutes.
  • Secondary antibody incubation Dilute the secondary antibody 1:2000 with antibody diluent, add 100 ⁇ L to each well, seal the plate, and incubate at 37°C for 1 hour. Discard the liquid in the well, wash the plate three times with washing solution, 200 ⁇ L each time, and let stand. Set for 2 minutes.
  • the preparation of lotion is as shown in the table below:
  • Trs, dTrs1-4 and Rtx proteins Binding of Trs, dTrs1-4 and Rtx proteins to IgA, IgM or HAS
  • IgA is derived from human serum (Sigma-Aldrich, I4036) with a concentration of 1 mg/mL
  • IgM is derived from humans. Serum (Sigma-Aldrich, I8260), concentration is 1 mg/mL
  • HSA Absin, abs47047388) concentration is 20 mg/mL;
  • step 2) Discard the solution from step 1), wash the plate with PBST (diluted Solebao 10*PBS 10 times with double distilled water (sterilized), +0.1% Tween), wash the plate 4 times with the plate washer, and turn the plate shaker on Spin dry for 1 minute at 600 ⁇ g and pat dry several times;
  • PBST diluted Solebao 10*PBS 10 times with double distilled water (sterilized), +0.1% Tween
  • the name, primers, sequence and composition of heavy chain, light chain and 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 primers and primers of the pcDNA3.4 vector and the target gene are as shown in Table 12.
  • the template is shown in Table 14, and the destination fragment information is shown in Table 15.
  • PLA2R signal peptide nucleotide sequence :
  • Trs heavy chain signal peptide nucleotide sequence Trs heavy chain signal peptide nucleotide sequence:
  • Trs light chain signal peptide nucleotide sequence
  • FIG. 10-12 The electrophoresis results of the purified PCR products are shown in Figure 10-12, in which lanes 1-5 from left to right in Figure 10 are DL5000, pcDNA3.4, pcDNA3.4, pcDNA3.4 and pcDNA3.4; Lanes 1-13 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; lanes 1-7 from left to right in Figure 12 are DL5000, unrelated, unrelated, RC3, RC3, RC6 and RC6.
  • the purified PCR bands were subjected to homologous recombination.
  • the system was consistent with Table 7.
  • the constructed plasmid table is shown in Table 16.
  • RC1H, RC2L, RC4H and RC5L are all spliced by two PCR fragments, that is, RC1H is spliced by RC1H-1 and RC1H-2, RC2L is spliced by RC2L-1 and RC2L-2, RC4H It is made of RC4H-1 and RC4H-2, and RC5L is made of RC5L-1 and RC5L-2.
  • FreeStyle TM Cells (cell density is 5-6 ⁇ 10 5 /mL, from FreeStyle TM MAX CHO expression system of American Gibco Company, product number: K900020), cultured at 37°C, 5% CO 2 , 120-135 rpm/min.
  • the RC1-RC8 culture supernatant was centrifuged at 4°C and 7000 g for 15 min, and then filtered and sterilized with a 0.22 ⁇ m syringe filter. Use 5 times the column volume of Buffer A1 to balance the Protein A chromatography medium (flow rate is 2mL/min), add the processed sample, rinse with Buffer A1 until the absorption value reaches the baseline, elute with Buffer B1, and collect the elution peak samples. The target protein eluate was adjusted to neutrality with 1M Tris-HCl (pH value 9.0). Super dex200 was filtered into PBS.
  • Identification of the target protein Select a 10-well SDS-PAGE precast gel and run the gel at 150V for 50 minutes. The sample was heated at 90°C for 5 min, and 3 ⁇ g was loaded into each well.
  • Lane M is a marker
  • lane 1 is RC1 elution and non-reduction
  • lane 2 is RC1 elution and reduction
  • lane 3 is RC2 elution and reduction.
  • 4 is RC2 elution and non-reduction
  • lane 5 is RC3 elution and non-reduction
  • lane 6 is RC3 elution and reduction
  • lane 7 is RC4 elution and reduction
  • lane 8 is RC4 elution and non-reduction
  • lane 9 is RC5 elution and non-reduction.
  • lane 10 is the elution and reduction of RC5
  • lane 11 is the elution and non-reduction of RC6
  • lane 12 is the elution and reduction of RC6
  • lane 13 is the elution and non-reduction of RC7
  • lane 14 is the elution and reduction of RC7
  • the lane 15 is the elution and non-reduction of RC8.
  • Reduction lane 16 is the elution and reduction of RC8.
  • the expression levels of RC1-RC8 proteins are shown in Table 18.
  • amino acid sequence and nucleotide sequence of CC1h are as follows:
  • the neutralization rate is calculated as follows:
  • the capture molecule in the diagnostic kit is PLA2R.
  • [PLA2R ⁇ Ab] 1 corresponds to the detection concentration of the antibody.
  • [PLA2R ⁇ Ab] 2 corresponds to the detection concentration of the antibody at this time.
  • PLA2R is greatly excessive as the capture antigen, so [PLA2R] 2 ⁇ [PLA2R] 1 , so That is, the proportion of PLA2R-Ab reduction in plasma caused by the addition of drug molecules. Can represent the neutralizing effect of drug molecules.
  • pMN-2 is a positive sample, and each group of drugs showed more than 95% neutralizing effect against autoimmune antibodies with a plasma concentration of around 44.79RU/mL.
  • pMN-4 is a positive sample, and each group of drugs showed more than 80% neutralizing effect against autoimmune antibodies with a plasma concentration of around 124.905RU/mL.
  • pMN-7 is a positive sample. Against autoimmune antibodies with a concentration in plasma of about 53.72RU/mL, except for RC5, which has a neutralization rate of 77.63%, all other groups of drugs show a neutralizing effect of more than 80%.
  • the ELISA method was used to detect the adsorption of PLA2R-Ab in plasma samples of PMN-4 and PMN-7 by RC3 and RC6 after immobilization, as shown in Part 4 of this example (neutralization of PLA2R-Ab in plasma samples by RC1-RC8).
  • the results are shown in Figures 17-18. The results show that after immobilization, the clearance rates of PLA2R-Ab in PMN-4 and PMN-7 plasma samples by RC3 and RC6 were both above 80%.
  • mice The purchased BALB/c female mice were adaptively raised for one week and randomly divided into 8 groups, divided into RC1 group, RC2 group, RC4 group, RC5 group, RC7 group, RC8 group, and placebo group (also known as "Buffer” group). ) and blank group (also known as "No treatment”), each group has 5 animals, weigh and mark. Administer at 10 times the dose of 40mg/person. The mice were weighed before administration, and then administered.
  • the RC1 group was subcutaneously injected with 0.1 mL of RC1 protein at a concentration of 1 mg/mL
  • the RC2 group was subcutaneously injected with 0.1 mL of RC2 protein at a concentration of 1 mg/mL
  • the RC4 group was subcutaneously injected.
  • the RC4 group was subcutaneously injected with 0.1mL of RC4 protein with a concentration of 1mg/mL
  • the RC5 group was subcutaneously injected with 0.1mL of RC5 protein with a concentration of 1mg/mL
  • the RC7 group was subcutaneously injected with 0.1mL of RC7 protein with a concentration of 1mg/mL
  • the RC8 group was subcutaneously injected with a concentration of 0.1mL.
  • the placebo group was injected with 0.1 mL PBS, and the blank group was not injected.
  • the day of administration was recorded as day 0. After the administration was completed, the mice were observed for 1 hour. No obvious adverse reactions such as dyspnea, reduced movement, and tremor were observed in the mice. The mice were weighed at 24h, 48h, and 72h, and then every 48h until the tenth day of administration.
  • the purchased BALB/c female mice were adaptively raised for one week and randomly divided into 4 groups, divided into RC3 group, RC6 group, placebo group (also known as "Buffer") and blank group (also known as "No treatment”) , 5 animals per group, weighed and marked. Administer at 10 times the dose of 40mg/person. Weigh the body weight of the mice before administration, and then administer the drug.
  • the RC3 group is injected subcutaneously with 0.1mL of RC3 protein with a concentration of 1mg/mL
  • the RC6 group is injected subcutaneously with 0.1mL of RC6 protein with a concentration of 1mg/mL
  • the placebo group is injected with 0.1 mLPBS and blank groups were not injected.
  • mice were observed for 1 hour. No obvious adverse reactions such as dyspnea, reduced movement, and tremor were observed in the mice. The mice were weighed at 24h, 48h, and 72h, and then every 48h until the 30th day of administration.
  • references to the terms “one embodiment,” “some embodiments,” “an example,” “specific examples,” or “some examples” or the like means that specific features are described in connection with the embodiment or example. , structures, materials or features are included in at least one embodiment or example of the invention. In this specification, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described in this specification unless they are inconsistent with each other.

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Abstract

提供一种抗体、融合蛋白及其用途。所述抗体包括选自下列至少之一的CDR序列：轻链可变区CDR序列：SEQ ID NO:1～3、SEQ ID NO:7～9和SEQ ID NO:83～85，重链可变区CDR序列：SEQ ID NO:4～6、SEQ ID NO:10～12、SEQ ID NO:86～88和SEQ ID NO:89～91；所述融合蛋白包括：中性抗体，所述中性抗体包括抗体FC区段或前述的抗体，一个或多个PLA2R抗原表位肽，所述PLA2R抗原表位肽与所述中性抗体相连。提供的抗体的抗原结合活性低、表达量高。

Description

抗体、融合蛋白及其用途 技术领域
本发明涉及分子生物学及药物领域，具体地，本发明涉及一种抗体、融合蛋白及其用途，更具体地，本发明涉及一种抗体及其核酸分子、表达载体、重组细胞以及融合蛋白及其核酸分子、表达载体、重组细胞、药物组合物和用途。
背景技术
膜性肾病(membranous nephropathy,MN)即膜性肾小球肾炎，具体是指免疫复合物在肾小球毛细血管中过度沉积导致的肾病综合征，是成人肾病综合征中最常见的病理类型之一，全球发病率约为12/100万(Mc Grogan A,Franssen CF,de Vries CS.Nephrol Dial Transplant.2011；26(2):414-430)。近年来MN的发病率以每年13％的速度增长(Lwezaula BF,Ameh OI,Ekrikpo UE,et al.BMC Nephrol.2021；22(1):15)，已成为导致慢性肾小球肾炎的第二大原因。临床上MN主要表现为大量蛋白尿、低蛋白血症、水肿及高脂血症，诊断依赖于肾脏活检组织病理学检查，其病理诊断特征包括肾小球毛细血管壁增厚，免疫荧光条件下可见IgG、补体C3沿着毛细血管壁沉积，电镜下显示肾小球上皮细胞下电子致密物形成。
根据病因的不同，MN可分为特发性膜性肾病(idiopathic membranous nephropathy,IMN)与继发性膜性肾病(secondary membranous nephropathy,SMN)。其中，IMN是MN的主要类型，占比约70％～80％，其病因尚不明确，又名原发性膜性肾病(primary membranous nephropathy,PMN)。目前，PMN的发病机制尚不十分明确，但大多数学者认为PMN是一种抗体介导的自身免疫性肾小球疾病，位于足细胞的靶抗原被自身抗体识别，并与之结合形成免疫复合物沉积于基底膜足细胞下，激活补体系统引起足细胞损伤脱落，导致基底膜通透性增加，进而出现大量蛋白尿。
2009年Beck等首次报道磷脂酶A2受体(phospholipase A2 receptor,PLA2R)是导致PMN发生的主要抗原之一，在70％的PMN患者血液和肾组织洗脱液中可检测出抗磷脂酶A2受体抗体(phospholipase-A2 receptor antibody,PLA2R-Ab)(Beck LH Jr,Bonegio RG,Lambeau G,et al.N Engl J Med.2009；361(1):11-21)，这引起了医学界对PLA2R的广泛关注。PLA2R-Ab能够特异性地与肾脏组织中足细胞上的靶抗原(即PLA2R)结合，可在70％～80％的PMN患者血清中表达(Bobart SA,De Vriese AS,Pawar AS,et al.Kidney Int.2019；95(2):429-438)，而在健康人群、SMN及其他肾小球疾病患者的血清中PLA2R-Ab抗体阳性表达率极低，故PLA2R-Ab诊断MN的特异性较高。最新的一项Meta分析显示(练明建,卢榕,张加勤,等.临床检验杂志,2017,35(7)：545-549)，血清PLA2R-Ab检测对诊断PMN的总敏感度为69％，总特异度为97％，进一步说明血清PLA2R-Ab对PMN有较好的敏感性和很高的特异性，可以作为诊断PMN的特异性生物标志物。
此外，大量研究表明，MN患者血清PLA2R-Ab滴度水平与患者肾小球滤过率、血清总蛋白和白蛋白等指标均呈负相关，也与PMN患者的自发缓解率呈显著负相关，对于无PLA2R-Ab或PLA2R-Ab水平低的轻症患者可自然缓解。在使用免疫抑制剂治疗期间，血清PLA2R-Ab阴性者在较短的时间内即能够出现免疫抑制治疗反应，而血清PLA2R-Ab阳性者对于免疫抑制剂的灵敏度则较低。在接受免疫抑制治疗后，其血清PLA2R-Ab水平也在不断地降低，提示PLA2R-Ab对疗效评估具有重要作用。在进入缓解期后，若血清PLA2R-Ab未转阴或滴度增加，提示疾病有复发的可能(Xu X,Nie S,Ding H,et al.Nat Rev Nephrol.2018；14(5):313-324)。对于接受肾移植的MN患者，产生循环PLA2R-Ab的患者易较早出现疾病复发(Passerini P,Malvica S,Tripodi F,et al.Front Immunol.2019；10:1326)，提示术后复发可能也与PLA2R-Ab有关。另外，研究结果还显示，PLA2R-Ab转阴率与总缓解率呈显著正相关，PLA2R-Ab预测MN患者12个月的临床缓解事件的敏感度、特异度及准确率分别为69.51％、76.35％和80.26％(郑娜,汪梅花,安智.临床军医杂志.2021；49(08):942-944)。
综上所述，PLA2R-Ab对PMN患者的临床诊断和病情判断以及预后评估均具有重要的参考价值。PLA2R-Ab参与了PMN的疾病进展，PLA2R-Ab低滴度的患者病情较轻，免疫抑制剂治疗后效果更显著，甚至有可能自发缓解，提示通过监测PLA2R-Ab水平有助于指导临床医师对患者的免疫抑制剂的用量和使用时间，避免过度免疫抑制治疗，同时也有助于减轻患者的医疗负担。而血清PLA2R-Ab水平越高，反映机体免疫活动性越强，病情越严重，对免疫抑制治疗效果越差，临床预后也较差，也进一步说明监测血清PLA2R-Ab有助于指导临床治疗，对于入院PLA2R-Ab水平高的患者应早期进行有效的干预治疗。
目前，临床上MN的治疗方法主要以低脂低盐低蛋白饮食、控制蛋白尿和高脂血脂的对症支持治疗为主，辅以免疫抑制治疗和一些非免疫抑制治疗，但尚无确切的治疗方法。此外，膜性肾病患者的治疗效果较差，仅有40％的患者可自发缓解，30％的患者疗效不佳，最终进展为终末期肾病，只能通过透析或移植维持生命，严重增加患者经济负担和影响患者生活质量。对于肾移植的患者，约40％的肾移植患者治疗效果欠佳，其中一部分会失去移植肾。基于PMN的发病机制，以及血清PLA2R-Ab水平在评估PMN的治疗效果和临床预后中的重要价值，亟需一种有效治疗PLA2R-Ab阳性的 MN患者的药物或技术。
发明内容
本发明旨在至少在一定程度上解决现有技术中存在的技术问题之一。为此，本发明提供了一种抗体及此抗体与原发性膜性肾病的自身免疫抗原结合的融合蛋白，本发明的抗体的抗原结合活性低，本发明的融合蛋白可特异性地结合PLA2R抗体。
本发明是基于发明人的下列发现而完成的：
针对PLA2R抗体阳性膜性肾病，一种可能的治疗方法是特异性中和血清中的PLA2R-Ab。发明人惊喜地发现，本申请所开发的PLA2R和抗体的融合蛋白(在本申请中，抗原与抗体的融合蛋白，称为抗原性抗体)可实现特异性中和血清中的PLA2R-Ab的技术效果。
PLA2R是一种高表达于肾小球足细胞表面的膜蛋白，包含如图1所示的结构域；同时，血液中有少量分泌型的PLA2R。并且，根据Liyo Kao等人的研究(Liyo Kao,Vinson Lam,et.al.,J Am Soc Nephrol.2015Feb；26(2):291-301)，病人血浆中的抗体主要识别PLA2R当中CysR、FNII和CTLD1三个结构域。
基于此，发明人发现，采用PLA2R的抗原区与抗体融合得到的融合蛋白，可保持PLA2R与PLA2R-Ab的结合能力。并且，在研发过程中，发明人发现，通过突变原抗体的CDR区，得到的抗体的抗原结合活性极低，可避免后续非特异性结合造成副作用，且具有更高的表达量。因此，采用突变后的抗体与PLA2R的抗原区融合得到的融合蛋白，特异性地中和PLA2R-Ab，且不与其他抗体结合，避免了非特异性免疫的副作用。此外，该融合蛋白还具有长半衰期的优势，可降低给药频率。
在本发明的第一方面，本发明提出了一种抗体。根据本发明的实施例，所述抗体包括：轻链可变区CDR和重链可变区CDR；其中，重链可变区CDR1序列如GX1X2X3X4X5X6X7X8X9所示，重链可变区CDR2序列如X10X11X12X13X14X15GX16X17X18X19X20X21X22X23所示，重链可变区CDR3序列如X24GGX25X26X27X28X29X30X31Y所示，X1为G或F，X2为G或N，X3为G或I，X4为G、K或S，X5为G或D，X6为G或T，X7为G或Y，X8为G或I，X9为H或S，X10为R或G，X11为G或I，X12为G或Y，X13为G或P，X14为T或S，X15为G或N，X16为G或Y，X17为G或T，X18为G、S或R，X19为G或Y，X20为G或A，X21为G或D，X22为G或S，X23为S或V，X24为G或W，X25为G或D，X26为G或S，X27为G或F，X28为G或Y，X29为G或A，X30为G或M，X31为S、G或D；条件是X1为F、X2为N、X3为I、X4为K、X5为D、X6为T、X7为Y、X8为I、X9为H、X10为R、X11为I、X12为Y、X13为P、X14为T、X15为N、X16为Y、X17为T、X18为R、X19为Y、X20为A、X21为D、X22为S、X23为V、X24为W、X25为D、X26为G、X27为F、X28为Y、X29为A、X30为M、X31为D不同时成立；轻链可变区CDR1序列如X32X33X34X35X36X37X38X39X40X41A所示，轻链可变区CDR2序列如X42X43X44X45X46X47S所示，轻链可变区CDR3序列如X48X49X50X51X52X53X54X55X56所示，X32为G或R，X33为G或A，X34为G或S，X35为G或Q，X36为G、D或S，X37为G或V，X38为G或N，X39为G或T，X40为G或A，X41为S或V，X42为G或S，X43为G或A，X44为G或S，X45为G或F，X46为L或S，X47为G或Y，X48为G或Q，X49为G或Q，X50为G或H，X51为G或Y，X52为S或T，X53为G或T，X54为G或P，X55为G或P，X56为G或T；条件是X32为R、X33为A、X34为S、X35为Q、X36为D、X37为V、X38为N、X39为T、X40为A、X41为V、X42为S、X43为A、X44为S、X45为F、X46为L、X47为Y、X48为Q、X49为Q、X50为H、X51为Y、X52为T、X53为T、X54为P、X55为P、X56为T不同时成立。发明人经过大量实验发现，上述抗体抗原结合活性低，形成抗原性抗体后，可有效避免抗原性抗体的非特异性结合，同时，更为重要的是，该抗体的表达量高，适于抗原性抗体药物的开发。
在本发明的第二方面，本发明提出了一种第一核酸分子。根据本发明的实施例，所述第一核酸分子编码第一方面所述的抗体。根据本发明实施例的第一核酸分子所编码的抗体抗原结合活性低，形成抗原性抗体后，可有效避免抗原性抗体的非特异性结合，同时，该抗体的表达量高，适于抗原性抗体药物的开发。
在本发明的第三方面，本发明提出了一种第一表达载体。根据本发明的实施例，所述第一表达载体携带第二方面所述的第一核酸分子。根据本发明实施例的第一表达载体导入合适的受体细胞后，可在调控系统的介导下，有效实现第一方面所述的抗原结合活性低的抗体的表达，以便大量获得所述抗体。
在本发明的第四方面，本发明提出了一种第一重组细胞。根据本发明的实施例，所述第一重组细胞包括：携带第二方面所述的第一核酸分子；或，表达第一方面所述的抗体。根据本发明实施例的第一重组细胞可用于第一方面所述的抗原结合活性低的抗体的体外表达和大量获得。
在本发明的第五方面，本发明提出了一种第一方面所述的抗体在制备融合蛋白药物中的用途。由前可知，本发明的 上述抗体无抗原结合活性，其可作为中性的药物载体制备药物，该药物可用于治疗或预防疾病。例如，采用上述抗体可抗原表位肽融合制备成融合蛋白，由于上述抗体为无抗原结合活性，制备的融合蛋白特异性地与抗原表位肽相应的抗体结合，且不与其他抗体结合，可避免融合蛋白非特异性结合的副作用，可用于治疗或预防抗原表位肽相应的抗体引起的相关疾病。
在本发明的第六方面，本发明提出了一种前述的抗体在作为药物载体或阴性抗体中的用途。由前可知，本发明的上述抗体无抗原结合活性，其可作为中性的药物载体制备药物，或者可直接作为无抗原结合活性的阴性抗体参照。
在本发明的第七方面，本发明提出了一种融合蛋白。根据本发明的实施例，所述融合蛋白包括：中性抗体，所述中性抗体包括抗体FC区段或第一方面所述的抗体；一个或多个PLA2R抗原表位肽，所述PLA2R抗原表位肽与所述中性抗体相连。发明人经过实验发现，通过将PLA2R抗原表位肽与上述中性抗体融合，得到的融合蛋白可特异性地与PLA2R抗体结合，并且不与其他抗体结合，避免了非特异性结合的副作用，具有表达性好和产量高等优点。
在本发明的第八方面，本发明提出了一种第二核酸分子。根据本发明的实施例，所述核酸分子编码第七方面所述的融合蛋白。利用该第二核酸分子可以有效地用于表达上述融合蛋白。
在本发明的第九方面，本发明提出了一种第二表达载体。根据本发明的实施例，所述第二表达载体携带第八方面所述的第二核酸分子。利用该第二表达载体可以有效地用于表达上述融合蛋白。
在本发明的第十方面，本发明提出了一种第二重组细胞。根据本发明的实施例，所述第二重组细胞包括：携带第八方面所述的第二核酸分子；或，表达第七方面所述的融合蛋白。根据本发明实施例的重组细胞可用于第五方面所述的融合蛋白体外表达和大量获得。
在本发明的第十一方面，本发明提出了一种药物组合物。根据本发明的实施例，所述药物组合物包括：第七方面所述的融合蛋白。发明人经过大量实验发现，上述药物组合物可有效预防和治疗PLA2R抗体引起的相关疾病，例如PLA2R抗体阳性膜性肾病。
在本发明的第十二方面，本发明提出了一种免疫吸附剂。根据本发明的实施例，所述免疫吸附剂包括：第七方面所述的融合蛋白。发明人经过大量实验发现，上述免疫吸附剂可吸附PLA2R抗体，其可通过免疫吸附有效治疗PLA2R抗体引起的相关疾病，例如PLA2R抗体阳性膜性肾病。
在本发明的第十三方面，本发明提出了一种试剂盒。根据本发明的实施例，所述试剂盒包括：第七方面所述的融合蛋白。发明人经过大量实验发现，上述试剂盒可有效检测PLA2R抗体含量，并且还可以用于诊断或治疗PLA2R抗体引起的相关疾病，例如PLA2R抗体阳性膜性肾病。
在本发明的第十四方面，本发明提出了一种吸附产品。根据本发明的实施例，所述吸附产品包括：其述的免疫吸附剂；固相载体，所述免疫吸附剂与固相载体相连。本发明的吸附产品可用于吸附PLA2R抗体，例如吸附血液或血浆中的PLA2R抗体，通过吸附血液或血浆中的PLA2R抗体可用于预防或治疗PLA2R抗体引起的相关疾病。
在本发明的第十五方面，本发明提出了一种第十二方面所述的免疫吸附剂在制备吸附产品中的用途，所述吸附产品用于吸附PLA2R抗体。
在本发明的第十六方面，本发明提出了一种第七方面所述的融合蛋白、第八方面所述的第二核酸分子、第九方面所述的第二表达载体、第十方面所述的第二重组细胞、第十一方面所述的药物组合物或第十二方面所述的免疫吸附剂在制备药物中的用途，所述药物用于预防或治疗PLA2R抗体引起的相关疾病。
在本发明的第十七方面，本发明提出了一种第七方面所述的融合蛋白、第八方面所述的第二核酸分子、第九方面所述的第二表达载体、第十方面所述的第二重组细胞或第十一方面所述的药物组合物在制备免疫吸附剂中的用途，所述免疫吸附剂用于吸附PLA2R抗体。
在本发明的第十八方面，本发明提出了一种第七方面所述的融合蛋白、第八方面所述的第二核酸分子、第九方面所述的第二表达载体、第十方面所述的第二重组细胞、第十一方面所述的药物组合物或第十二方面所述的免疫吸附剂在制备试剂盒中的用途，所述试剂盒用于检测PLA2R抗体。
在本发明的第十九方面，本发明提出了一种预防或治疗PLA2R抗体引起的相关疾病的方法。根据本发明的实施例，所述方法包含：向受试者施用药学上可接受量的第七方面所述的融合蛋白或第十一方面所述的药物组合物。由此，可有效治疗PLA2R抗体引起的相关疾病。
在本发明的第二十方面，本发明提出了一种用于预防或治疗PLA2R抗体引起的相关疾病的免疫吸附治疗方法。根据本发明的实施例，所述方法包含：采用第十二方面所述的免疫吸附剂对受试者进行治疗。
在本发明的第二十一方面，本发明提出了一种诊断PLA2R抗体引起的相关疾病的方法。根据本发明的实施例，所 述方法包括：采用第七方面所述的融合蛋白、第八方面所述的第二核酸分子、第九方面所述的第二表达载体、第十方面所述的第二重组细胞、第十一方面所述的药物组合物、第十二方面所述的免疫吸附剂或第十三方面所述的试剂盒对待测样品中的PLA2R抗体进行检测；基于检测结果，确定待测样品中的PLA2R抗体的含量。
在本发明的第二十二方面，本发明提出了一种评估PLA2R抗体引起的相关疾病分期的方法。根据本发明的实施例，所述方法包括：采用第七方面所述的融合蛋白、第八方面所述的第二核酸分子、第九方面所述的第二表达载体、第十方面所述的第二重组细胞、第十一方面所述的药物组合物、第十二方面所述的免疫吸附剂或第十三方面所述的试剂盒对待测样品中的PLA2R抗体进行检测；基于检测结果，确定待测样品中的PLA2R抗体的含量。
在本发明的第二十三方面，本发明提出了一种评估PLA2R抗体引起的相关疾病的预后方法。根据本发明的实施例，所述方法包括：采用第七方面所述的融合蛋白、第八方面所述的第二核酸分子、第九方面所述的第二表达载体、第十方面所述的第二重组细胞、第十一方面所述的药物组合物、第十二方面所述的免疫吸附剂或第十三方面所述的试剂盒对待测样品中的PLA2R抗体进行检测；基于检测结果，确定待测样品中的PLA2R抗体的含量。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1为本发明实施例1中表达框架的结构示意图；
图2为本发明实施例1中pcDNA3.4、trsH、trsL、RtxH和RtxL的电泳结果图；
图3为本发明实施例1中dTrs1H、dTrs2H和dTrs1L(dTrs2L)的电泳结果图；
图4为本发明实施例1中dTrs3H和dTrs3L的电泳结果图；
图5为本发明实施例1中dTrs4H和dTrs4L的电泳结果图；
图6为本发明实施例1中SDS-PAGE检测Trs、dTrs1-4和Rtx蛋白的纯化效果；
图7为本发明实施例1中Trs、dTrs1-4和Rtx蛋白与Her2结合的检测结果；
图8为本发明实施例2中RC1-RC8蛋白的结构示意图；
图9为本发明实施例2中PLA2R的结构示意图；
图10为本发明实施例2中pcDNA3.4的电泳结果图；
图11为本发明实施例2中pcDNA3.4、RC1H、RC2L、RC4H和RC5L的电泳结果图；
图12为本发明实施例2中RC3和RC6的电泳结果图；
图13为本发明实施例2中SDS-PAGE检测RC1-8蛋白的纯化效果；
图14为本发明实施例2中RC1-RC8对膜性肾病阳性病人pMN-2血浆中PLA2R-Ab的中和效果；
图15为本发明实施例2中RC1-RC8对膜性肾病阳性病人pMN-4血浆中PLA2R-Ab的中和效果；
图16为本发明实施例2中RC1-RC8对膜性肾病阳性病人pMN-7血浆中PLA2R-Ab的中和效果；
图17为本发明实施例2中RC3和RC6对PMN-4病人血浆样品中PLA2R-Ab吸附的清除率；
图18为本发明实施例2中RC3和RC6对PMN-7病人血浆样品中PLA2R-Ab吸附的清除率；
图19为本发明实施例2中小鼠皮下注射RC1、RC2、RC4、RC5、RC7、RC8蛋白的体重趋势图；
图20为本发明实施例2中小鼠皮下注射RC3、RC6蛋白的体重趋势图；
图21为本发明实施例1中Trs、dTrs1-4和Rtx蛋白与IgA结合的检测结果；
图22为本发明实施例1中Trs、dTrs1-4和Rtx蛋白与IgM结合的检测结果；
图23为本发明实施例1中Trs、dTrs1-4和Rtx蛋白与HSA结合的检测结果；
图24为本发明实施例2中RC1-RC8的稳定性考察的电泳结果图。
具体实施方式
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
需要说明的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地，在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。
在本文中，术语“包含”或“包括”为开放式表达，即包括本发明所指明的内容，但并不排除其他方面的内容。
在本文中，术语“任选地”、“任选的”或“任选”通常是指随后所述的事件或状况可以但未必发生，并且该描述包括其中发生该事件或状况的情况，以及其中未发生该事件或状况的情况。
在本文中，术语“抗体”通常是指免疫球蛋白分子，包括两条分子量较轻的轻链和两条分子量较重的重链，重链(H链)和轻链(L链)由二硫键连接形成一个四肽链分子。其中，肽链的氨基端(N端)氨基酸序列变化很大，称为可变区(V区)，羧基端(C端)相对稳定，变化很小，称为恒定区(C区)。L链和H链的V区分别称为VL和VH。在可变区中某些区域氨基酸组成和排列顺序具有更高的变化程度，称为高变区(Hypervariable region,HVR)，高变区为抗原和抗体结合的位置，因此也称为互补决定区(complementarity-determining region,CDR)。重链可变区和轻链可变区上均有三个CDR。
在本文中，术语“骨架区”或“框架区”简称FR，其均是指抗体的重链或轻链的可变区中非CDR部分。其中，抗体的H链和L链的近N端约有110个氨基酸序列的变化很大，其他部分的氨基酸序列相对恒定，据此可将轻链和重链区分为可变区(V区)和恒定区(C区)，可变区内包含超变区HVR(hypervariable region)或称互补决定区CDR(Complementarity-determining region)与骨架区，FR的可变性要低于CDR。FR分子共四个，分别为FRl、FR2、FR3和FR4。在识别抗体时，四个FR分子卷曲使CDR分子相互靠近。
在本文中，术语“中性抗体”是指抗原结合活性低的蛋白结构，如Fc区段，或者本发明第一方面所提出的抗体。
在本文中，术语“双链抗体”是指具有对称结构的抗体，如有且仅有两条Fc片段组成的抗体，或者两条重链和两条轻链组成的完整抗体，如本发明第一方面所提出的抗体。
在本文中，术语“同一性”、“同源性”或“相似性”均用于描述相对于参考序列的氨基酸序列或核酸序列时，采用通过常规的方法进行确定两个氨基酸序列或核酸序列之间的相同氨基酸或核苷酸的百分比，例如参见，Ausubel等，编著(1995)，Current Protocols in Molecular Biology，第19章(Greene Publishing and Wiley-Interscience，New York)；和ALIGN程序(Dayhoff(1978)，Atlas of Protein Sequence and Structure 5：Suppl.3(National Biomedical Research Foundation，Washington，D.C.)。关于比对序列和测定序列同一性有很多算法，包括，Needleman等(1970)J.Mol.Biol.48：443的同源性比对算法；Smith等(1981)Adv.Appl.Math.2：482的局部同源性算法；Pearson等(1988)Proc.Natl.Acad.Sci.85：2444的相似性搜索方法；Smith-Waterman算法(Meth.Mol.Biol.70：173-187(1997)；和BLASTP，BLASTN，和BLASTX算法(参见Altschul等(1990)J.Mol.Biol.215：403-410)。利用这些算法的计算机程序也是可获得的，并且包括但不限于：ALIGN或Megalign(DNASTAR)软件，或者WU-BLAST-2(Altschul等，Meth.Enzym.，266：460-480(1996))；或者GAP，BESTFIT，BLAST Altschul等，上文，FASTA，和TFASTA，在Genetics Computing Group(GCG)包，8版，Madison，Wisconsin，USA中可获得；和Intelligenetics，Mountain View，California提供的PC/Gene程序中的CLUSTAL。
在本文中，术语“核苷酸”通常是指可为核糖核苷酸、脱氧核苷酸或任一类型核苷酸的修饰形式，及其组合。
在本文中，术语“至少85％相似性”指与各参考序列至少为85％，可为85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％、99.9％的相似性。
在本文中，术语“表达载体”通常是指能够插入在合适的宿主中自我复制的核酸分子，其将插入的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述表达载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述表达载体还包括具有多种上述功能的载体。所述表达载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述表达载体的合适的宿主细胞，所述表达载体可以产生期望的表达产物。
在本文中，术语“重组细胞”通常是指采用基因工程技术或细胞融合技术对宿主细胞的遗传物质进行修饰改造或重组，获得具有稳定遗传的独特性状的细胞。其中，术语“宿主细胞”是指可导入重组表达载体的原核细胞或真核细胞。本文所用术语“转化的”或“转染的”是指通过本领域已知的各种技术将核酸(例如载体)引入细胞。合适的宿主细胞可以用本发明的DNA序列转化或转染，并且可以用于靶蛋白的表达和/或分泌。可用于本发明的合适宿主细胞的例子包括永生化杂交瘤细胞、NS/0骨髓瘤细胞、293细胞、中国仓鼠卵巢(CHO)细胞、HeLa细胞、Cap细胞(人羊水来源的细胞)、昆虫细胞、PER.C6细胞和CoS细胞。
在本文中，术语“融合蛋白”是指至少两个蛋白质或者多肽融合而成的新型蛋白，通常可以通过基因工程等技术实现上述融合操作，例如，通过DNA重组技术得到的两个基因重组后的表达产物。在本文中是指，PLA2R与双链结构体的中性抗体呈融合状态，需要说明的是，PLA2R融合蛋白并不意味着蛋白质的所有部分都是通过融合的方式形成的，例如，双链结构体中的两个Fc段部分是可以通过二硫键等化学键连接在一起的。
在本文中，术语“药物组合物”通常是指单位剂量形式，并且可以通过制药领域中熟知的方法的任何一种进行制备。 所有的方法包括使活性成分与构成一种或多种附属成分的载体相结合的步骤。通常，通过均匀并充分地使活性融合蛋白与液体载体、细碎固体载体或这两者相结合，制备组合物。
在本文中，术语“药学上可接受的”是指物质或组合物必须与包含制剂的其它成分和/或用其治疗的哺乳动物化学上和/或毒理学上相容。优选地，本发明所述的“药学上可接受的”是指联邦监管机构或国家政府批准的或美国药典或其他一般认可药典上列举的在动物中、特别是人体中使用的。
在本文中，术语“药学上可接受的辅料”或者“药学上可接受的载体”均可包括任何溶剂、固体赋形剂、稀释剂或其他液体赋形剂等等，适合于特有的目标剂型。除了任何常规的辅料与本发明的融合蛋白不相容的范围，例如所产生的任何不良的生物效应或与药学上可接受的组合物的任何其他组分以有害的方式产生的相互作用，它们的用途也是本发明所考虑的范围。
对本文所提及的其他药学上可接受的辅料或其他药学上可接受的载体以及其工艺可参考关于此主题的大量文献，具体而言参见Handbook of Pharmaceutical Excipients，第3版，Arthur H.Kibbe编辑，American Pharmaceutical Association,Washington,USA和Pharmaceutical Press,London；以及Lexikon der Hilfsstoffe für Pharmazie、Kosmetik和angrenzende Gebiete，H.P.Fiedler编辑，第4版，编辑Cantor、Aulendorf和早期版本。
在本文中，术语“给药”指将预定量的物质通过某种适合的方式引入病人。本发明的融合蛋白或药物组合物可以通过任何常见的途径被给药，只要它可以到达预期的组织。给药的各种方式是可以预期的，包括腹膜、静脉注射、肌肉注射、皮下注射等等，但是本发明不限于这些已举例的给药方式。优选地，本发明的组合物采用静脉注射或皮下注射方式被给药。
在本文中，术语“PLA2R抗体引起的相关疾病”通常是指由于PLA2R抗体异常引起的疾病，例如PLA2R抗体增多引起的相关疾病，包括但不限于PLA2R抗体阳性膜性肾病。其中，PLA2R是表达于肾小球足细胞的膜蛋白，由于未知的原因成为抗原，导致机体产生了相应的自身免疫抗体(PLA2R抗体)。PLA2R抗体与PLA2R结合并沉积到肾小球基底膜下，引发下游效应如C3沉积，导致PLA2R抗体阳性膜性肾病。
在本文中，术语“治疗”是指用于获得期望的药理学和/或生理学效果。所述效果就完全或部分预防疾病或其症状而言可以是预防性的，和/或就部分或完全治愈疾病和/或疾病导致的不良作用而言可以是治疗性的。本文使用的“治疗”涵盖哺乳动物、特别是人的疾病，包括：(a)在容易患病但是尚未确诊得病的个体中预防疾病或病症发生；(b)抑制疾病，例如阻滞疾病发展；或(c)缓解疾病，例如减轻与疾病相关的症状。本文使用的“治疗”涵盖将融合蛋白、药物组合物或药物给予个体以治疗、治愈、缓解、改善、减轻或抑制个体的疾病的任何用药，包括但不限于将含本文所述融合蛋白的药物给予有需要的个体。
本发明提出了一种抗体、融合蛋白及其用途，下面将分别对其进行详细描述。
抗体、第一核酸分子、第一表达载体、第一重组细胞及其用途
本发明对Trastuzumab(Trs)抗体的CDR区不同位点进行突变，经过大量实验得到本发明的抗体，该抗体与其抗原Her2的结合极弱，且不结合任何其它抗原，该抗体的抗原结合活性极低，可作为中性抗体与生物活性分子融合得到融合蛋白，该融合蛋白可结合该生物活性分子的特异性结合物，例如，该中性抗体与PLA2R结合得到的融合蛋白可特异性结合PLA2R抗体，且不与其他抗体结合，当作为药物时，避免了非特异性结合的副作用；将中性抗体与抗原融合得到的融合蛋白作为药物，其可结合该抗原的特异性抗体，不结合其他抗体，可专一性的清除抗原的特异性抗体的生成细胞。
因此，在本发明的第一方面，本发明提出了一种抗体。根据本发明的实施例，所述抗体包括：轻链可变区CDR和重链可变区CDR；其中，重链可变区CDR1序列如GX1X2X3X4X5X6X7X8X9所示，重链可变区CDR2序列如X10X11X12X13X14X15GX16X17X18X19X20X21X22X23所示，重链可变区CDR3序列如X24GGX25X26X27X28X29X30X31Y所示，X1为G或F，X2为G或N，X3为G或I，X4为G、K或S，X5为G或D，X6为G或T，X7为G或Y，X8为G或I，X9为H或S，X10为R或G，X11为G或I，X12为G或Y，X13为G或P，X14为T或S，X15为G或N，X16为G或Y，X17为G或T，X18为G、S或R，X19为G或Y，X20为G或A，X21为G或D，X22为G或S，X23为S或V，X24为G或W，X25为G或D，X26为G或S，X27为G或F，X28为G或Y，X29为G或A，X30为G或M，X31为S、G或D；条件是X1为F、X2为N、X3为I、X4为K、X5为D、X6为T、X7为Y、X8为I、X9为H、X10为R、X11为I、X12为Y、X13为P、X14为T、X15为N、X16为Y、X17为T、X18为R、X19为Y、X20为A、X21为D、X22为S、X23为V、X24为W、X25为D、X26为G、X27为F、X28为Y、X29为A、X30为M、X31为D不同时成立；轻链可变区CDR1序列如X32X33X34X35X36X37X38X39X40X41A所示，轻链可变区CDR2序列如X42X43X44X45X46X47S所示，轻链可变区CDR3序列如X48X49X50X51X52X53X54X55X56所示，X32为G或R，X33为G或A，X34为G或S，X35为G或Q，X36为G、D或S，X37 为G或V，X38为G或N，X39为G或T，X40为G或A，X41为S或V，X42为G或S，X43为G或A，X44为G或S，X45为G或F，X46为L或S，X47为G或Y，X48为G或Q，X49为G或Q，X50为G或H，X51为G或Y，X52为S或T，X53为G或T，X54为G或P，X55为G或P，X56为G或T；条件是X32为R、X33为A、X34为S、X35为Q、X36为D、X37为V、X38为N、X39为T、X40为A、X41为V、X42为S、X43为A、X44为S、X45为F、X46为L、X47为Y、X48为Q、X49为Q、X50为H、X51为Y、X52为T、X53为T、X54为P、X55为P、X56为T不同时成立。发明人经过大量实验发现，上述抗体的抗原结合活性极低，具有表达量高的优点。
根据本发明的实施例，所述重链可变区CDR和轻链可变区CDR满足如下至少之一的条件：X4为G或S，X5为G，X21为G，X31为S或G，X36为G或S。
根据本发明的实施例，所述重链可变区CDR中，X1为G或F，X2为G或N，X3为G或I，X4为G或S，X5为G，X6为G或T，X7为G或Y，X8为G或I，X9为H或S，X10为G，X11为G或I，X12为G或Y，X13为G或P，X14为T或S，X15为G或N，X16为G，X17为G或T，X18为G或S，X19为G或Y，X20为G或A，X21为G，X22为G或S，X23为S或V，X24为G，X25为G或D，X26为G或S，X27为G或F，X28为G，X29为G或A，X30为G或M，X31为S或G；所述轻链可变区CDR中，X32为G或R，X33为G或A，X34为G或S，X35为G或Q，X36为G或S，X37为G或V，X38为G，X39为G或T，X40为G或A，X41为S或V，X42为G或S，X43为G或A，X44为G或S，X45为G，X46为L或S，X47为G或Y，X48为G或Q，X49为G或Q，X50为G或H，X51为G，X52为S或T，X53为G或T，X54为G或P，X55为G或P，X56为G或T。
根据本发明的实施例，所述抗体包括选自下列至少之一的CDR序列：轻链可变区CDR序列：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和SEQ ID NO:85；重链可变区CDR序列：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和SEQ ID NO:91。
在另一些实施方案中，本发明所提供的抗体与上述重链和轻链相比，具有保守氨基酸取代。“保守氨基酸取代”指的是氨基酸被另一氨基酸发生生物学上、化学上或者结构上相似的残基所取代。生物学上相似的指的是该取代不破坏抗体的抗原结合活性低的生物学活性。结构上相似指的是氨基酸具有相似长度的侧链，如丙氨酸、甘氨酸或丝氨酸，或具有相似大小的侧链。化学相似性指的是氨基酸具有相同的荷电或者都是亲水或者疏水的。例如疏水残基异亮氨酸、缬氨酸、亮氨酸或者甲硫氨酸相互取代。或者用极性氨基酸例如用精氨酸取代赖氨酸、谷氨酸取代天冬氨酸、谷氨酰胺取代天冬 酰胺，丝氨酸取代苏氨酸等等。
根据本发明的实施例，所述轻链可变区CDR序列包括与SEQ ID NO:1～3、SEQ ID NO:7～9和SEQ ID NO:83～85具有至少85％相似性的氨基酸序列中的至少之一；所述重链可变区CDR序列包括与SEQ ID NO:4～6、SEQ ID NO:10～12、SEQ ID NO:86～88和SEQ ID NO:89～91具有至少85％相似性的氨基酸序列中的至少之一。
根据本发明的实施例，所述抗体包括：分别如SEQ ID NO:1、2和3或者与SEQ ID NO:1、2和3具有至少85％相似性的氨基酸序列所示的轻链可变区CDR1、CDR2、CDR3序列；或者，分别如SEQ ID NO:7、8和9或者与SEQ ID NO:7、8和9具有至少85％相似性的氨基酸序列所示的轻链可变区CDR1、CDR2、CDR3序列；或者，分别如SEQ ID NO:83、84和85或者与SEQ ID NO:83、84和85具有至少85％相似性的氨基酸序列所示的轻链可变区CDR1、CDR2、CDR3序列。
根据本发明的实施例，所述抗体包括：分别如SEQ ID NO:4、5和6或者与SEQ ID NO:4、5和6具有至少85％相似性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列；或者，分别如SEQ ID NO:10、11和12或者与SEQ ID NO:10、11和12具有至少85％相似性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列；或者，分别如SEQ ID NO:86、87和88或者与SEQ ID NO:86、87和88具有至少85％相似性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列；或者，分别如SEQ ID NO:89、90和91或者与SEQ ID NO:89、90和91具有至少85％相似性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列。
根据本发明的实施例，所述抗体包括：分别如SEQ ID NO:1、2和3或者与SEQ ID NO:1、2和3具有至少85％相似性的氨基酸序列所示的轻链可变区CDR1、CDR2、CDR3序列，以及分别如SEQ ID NO:4、5和6或者与SEQ ID NO:4、5和6具有至少85％相似性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列；或者分别如SEQ ID NO:7、8和9或者与SEQ ID NO:7、8和9具有至少85％相似性的氨基酸序列所示的轻链可变区CDR1、CDR2、CDR3序列，以及分别如SEQ ID NO:10、11和12或者与SEQ ID NO:10、11和12具有至少85％相似性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列；或者分别如SEQ ID NO:83、84和85或者与SEQ ID NO:83、84和85具有至少85％相似性的氨基酸序列所示的轻链可变区CDR1、CDR2、CDR3序列，以及分别如SEQ ID NO:86、87和88或者与SEQ ID NO:86、87和88具有至少85％相似性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列；或者分别如SEQ ID NO:83、84和85或者与SEQ ID NO:83、84和85具有至少85％相似性的氨基酸序列所示的轻链可变区CDR1、CDR2、CDR3序列，以及分别如SEQ ID NO:89、90和91或者与SEQ ID NO:89、90和91具有至少85％相似性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列。
根据本发明的实施例，所述抗体含有：重链骨架区序列和轻链骨架区序列中的至少之一；其中，所述重链骨架区序列和轻链骨架区序列的至少之一的至少一部分来自于鼠源抗体、人源抗体、灵长目源抗体或其突变体中的至少之一，优选为人源抗体。
根据本发明的实施例，所述抗体具有如SEQ ID NO:13、SEQ ID NO:14或SEQ ID NO:95所示氨基酸序列的轻链可变区；和/或者，所述抗体具有如SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:96或SEQ ID NO:97所示氨基酸序列的重链可变区。发明人通过抗体序列比对数据库(NCBI、IMGT)或者相关软件可得到上述抗体的重链可变区序列的CDR区(如SEQ ID NO:4～6、SEQ ID NO:10～12所示)和轻链可变区序列的CDR区(如SEQ ID NO:1～3、SEQ ID NO:7～9所示)。
根据本发明的实施例，所述抗体的轻链可变区序列与SEQ ID NO:13或14所示氨基酸序列相比，具有保守氨基酸取代；或者，所述抗体的重链可变区序列与SEQ ID NO:15或16所示氨基酸序列相比，具有保守氨基酸取代。当然，这些保守氨基酸取代不会对抗体的生物学功能带来改变。在一些具体方式中，这些保守氨基酸取代可以发生在重链可变区和轻链可变区中除了CDR区之外的氨基酸上。
根据本发明的实施例，所述抗体具有如SEQ ID NO:13所示氨基酸序列的轻链可变区和如SEQ ID NO:15所示氨基酸 序列的重链可变区。
根据本发明的实施例，所述抗体具有如SEQ ID NO:14所示氨基酸序列的轻链可变区和如SEQ ID NO:16所示氨基酸序列的重链可变区。
根据本发明的实施例，所述抗体具有如SEQ ID NO:95所示氨基酸序列的轻链可变区和如SEQ ID NO:96所示氨基酸序列的重链可变区。
根据本发明的实施例，所述抗体具有如SEQ ID NO:95所示氨基酸序列的轻链可变区和如SEQ ID NO:97所示氨基酸序列的重链可变区。
根据本发明的实施例，所述抗体含有重链恒定区和轻链恒定区的至少之一，所述重链恒定区和轻链恒定区的至少之一的至少一部分来自于鼠源抗体、人源抗体、灵长目源抗体或其突变体的至少之一，优选为人源抗体。
根据本发明的实施例，所述抗体具有如SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:33或SEQ ID NO:35所示氨基酸序列的重链和具有如SEQ ID NO:19、SEQ ID NO:20或SEQ ID NO:34所示氨基酸序列的轻链。
根据本发明的实施例，所述抗体具有如SEQ ID NO:17所示氨基酸序列的重链和具有如SEQ ID NO:19所示氨基酸序列的轻链。
根据本发明的实施例，所述抗体具有如SEQ ID NO:18所示氨基酸序列的重链和具有如SEQ ID NO:20所示氨基酸序列的轻链。
根据本发明的实施例，所述抗体具有如SEQ ID NO:33所示氨基酸序列的重链和具有如SEQ ID NO:34所示氨基酸序列的轻链。
根据本发明的实施例，所述抗体具有如SEQ ID NO:35所示氨基酸序列的重链和具有如SEQ ID NO:34所示氨基酸序列的轻链。
在制备或者获取上述这些抗体的过程中，可以利用表达这些抗体的核酸分子，与不同的载体连接，然后在不同细胞中表达，来获得相应抗体。
为此，在本发明的第二方面，本发明提出了一种第一核酸分子。根据本发明的实施例，所述第一核酸分子编码第一方面所述的抗体。根据本发明实施例的第一核酸分子所编码的抗体的抗原结合活性极低，具有表达量高的优点。
根据本发明的实施例，所述核酸分子为DNA。
所述核苷酸序列包含SEQ ID NO:25或与其具有至少80％相似性的核苷酸序列。SEQ ID NO:25用于编码具有SEQ ID NO:17所示氨基酸序列的重链。

所述核苷酸序列包含SEQ ID NO:26或与其具有至少80％相似性的核苷酸序列。SEQ ID NO:26用于编码具有SEQ ID NO:19所示氨基酸序列的轻链。
所述核苷酸序列包含SEQ ID NO:27或与其具有至少80％相似性的核苷酸序列。SEQ ID NO:27用于编码具有SEQ ID NO:18所示氨基酸序列的重链。

所述核苷酸序列包含SEQ ID NO:28或与其具有至少80％相似性的核苷酸序列。SEQ ID NO:28用于编码具有SEQ ID NO:20所示氨基酸序列的轻链。
本领域技术人员能够理解的是，前面针对抗体所描述的特征和优点，同样适用于该第一核酸分子，在此不再赘述。
在本发明的第三方面，本发明提出了一种第一表达载体。根据本发明的实施例，所述第一表达载体携带第二方面所述的第一核酸分子。在将上述分离的多核苷酸连接到第一表达载体上时，可以将多核苷酸与第一表达载体上的控制元件直接或者间接相连，只要这些控制元件能够控制多核苷酸的翻译和表达等即可。当然这些控制元件可以直接来自于第一表达载体本身，也可以是外源性的，即并非来自于第一表达载体本身。当然，多核苷酸与控制元件进行可操作地连接即可。
本文中“可操作地连接”是指将外源基因连接到载体上，使得载体内的控制元件，例如转录控制序列和翻译控制序列等等，能够发挥其预期的调节外源基因的转录和翻译的功能。当然用来编码抗体重链和轻链的多核苷酸，可以分别独立的插入到不同的载体上，常见的是插入到同一载体上。常用的载体例如可以为质粒、噬菌体等等。
根据本发明的实施例，所述第一表达载体为真核表达载体，优选地，所述第一表达载体为慢病毒载体。
本领域技术人员能够理解的是，前面针对抗体所描述的特征和优点，同样适用于该第一表达载体，在此不再赘述。
在本发明的第四方面，本发明提出了一种第一重组细胞。根据本发明的实施例，所述第一重组细胞包括：携带第二方面所述的第一核酸分子；或，表达第一方面所述的抗体。可以将第一表达载体导入到哺乳动物细胞中，构建获得第一重组细胞，然后利用这些第一重组细胞表达本发明提供的抗体。通过该第一重组细胞进行培养，即可以获得相应抗体。这些可用的哺乳动物细胞例如可以为CHO细胞等。
根据本发明的实施例，所述重组细胞是通过将第一表达载体引入至宿主细胞中而获得的。
根据本发明的实施例，所述重组细胞为真核细胞。
根据本发明的实施例，所述重组细胞为哺乳动物细胞。
在本发明的第五方面，本发明提出了一种第一方面所述的抗体在制备融合蛋白药物中的用途。由前可知，本发明的上述抗体无抗原结合活性，其可作为中性的药物载体制备药物，该药物可用于治疗或预防疾病。例如，采用上述抗体可抗原表位肽融合制备成融合蛋白，由于上述抗体为无抗原结合活性，制备的融合蛋白特异性地与抗原表位肽相应的抗体结合，且不与其他抗体结合，可避免融合蛋白非特异性结合的副作用，可用于治疗或预防抗原表位肽相应的抗体引起的相关疾病。
根据本发明的实施例，所述药物包括融合蛋白或ADC药物。
在本发明的第六方面，本发明提出了一种前述的抗体在作为药物载体或阴性抗体中的用途。由前可知，本发明的上述抗体无抗原结合活性，其可作为中性的药物载体制备药物，或者可直接作为无抗原结合活性的阴性抗体参照。
根据本发明的实施例，所述药物载体或阴性抗体无抗原结合能力。
本领域技术人员能够理解的是，前面针对抗体所描述的特征和优点，同样适用于该第一核酸分子、第一表达载体、第一重组细胞及用途，在此不再赘述。
融合蛋白
在本发明的第七方面，本发明提出了一种融合蛋白。根据本发明的实施例，所述融合蛋白包括：中性抗体，所述中性抗体包括抗体FC区段或第一方面所述的抗体；一个或多个PLA2R抗原表位肽，所述PLA2R抗原表位肽与所述中性抗体相连。
发明人经过实验发现，通过将PLA2R抗原表位肽与上述中性抗体融合，得到的融合蛋白可竞争性地与PLA2R抗体结合，防止PLA2R抗体与机体内的PLA2R结合，可有效治疗PLA2R抗体引起的相关疾病。同时，本发明的融合蛋白中的PLA2R抗原表位肽与PLA2R抗体结合，融合蛋白的中性抗体含有Fc片段，Fc片段可与NK细胞的FcγR受体结合，从而介导NK细胞直接杀伤表达膜型PLA2R抗体的B细胞，Fc片段还可以与补体C1q结合并激活补体系统，对表达膜型PLA2R抗体的B细胞发挥裂解效应，由于本发明的融合蛋白特异性地与PLA2R抗体结合，且不与其他抗体结合，其可专一性的清除B细胞，避免非特异性免疫的副作用。并且，本发明的融合蛋白还可以与肝脏FcRn受体结合并得到保护，避免融合蛋白被降解，具有很长的血浆半衰期。因此，该融合蛋白作为药物，具有长半衰期的优势，可降低给药频率，还具有表达性好、产量高、稳定性高、亲和性高、溶解性好和成药性好等优点。
本领域技术人员能够理解的是，氨基酸片段通常分为N端和C端，本发明对于中性抗体和PLA2R抗原表位肽的连接方式不作严格限定，只要是一个片段的N端与另一个片段的C端相连即可。
根据本发明的实施例，所述PLA2R抗原表位肽的C端与所述中性抗体的N端相连。
根据本发明的实施例，所述PLA2R抗原表位肽包括CysR、FnII以及PLA2R结合区1和/或PLA2R结合区2。
需要说明的是，CysR、FnII、PLA2R结合区1和PLA2R结合区2的具体结构参见图9。
根据本发明的实施例，所述CysR的C端与所述FnII的N端相连，所述FnII的C端与所述PLA2R结合区1的N端相连。
根据本发明的实施例，所述CysR的C端与所述FnII的N端相连，所述FnII的C端与所述PLA2R结合区1的N端相连，所述PLA2R结合区1的C端与PLA2R结合区2的N端相连。
根据本发明的实施例，所述PLA2R抗原表位肽具有如SEQ ID NO:21或22所示氨基酸序列或者与SEQ ID NO:21或22具有至少95％相似性的氨基酸序列。
根据本发明的实施例，所述中性抗体为抗体FC区段，所述PLA2R抗原表位肽的C端与所述抗体FC区段的至少一条的N端相连。
根据本发明的实施例，所述中性抗体为第一方面所述的抗体，所述PLA2R抗原表位肽的C端与所述抗体的至少一条轻链或重链的N端相连。
根据本发明的实施例，所述中性抗体为双链抗体，所述抗体FC区段的每条链的N端分别与一个PLA2R抗原表位肽的C端相连。该融合蛋白上含有两个PLA2R抗原表位肽，即为二价分子。发明人经过实验发现，与单价的天然PLA2R相比，本发明的融合蛋白与PLA2R抗体的结合能力较强，可有效中和PLA2R抗体，保护肾小球组细胞表面的天然PLA2R不被PLA2R抗体攻击。
根据本发明的实施例，第一方面所述的抗体的每条轻链的N端分别与一个PLA2R抗原表位肽的C端相连。该融合蛋白上含有两个PLA2R抗原表位肽，即为二价分子。发明人经过实验发现，与单价的天然PLA2R相比，本发明的融合蛋白与PLA2R抗体的结合能力较强，可有效中和PLA2R抗体，保护肾小球组细胞表面的天然PLA2R不被PLA2R抗体攻击。
根据本发明的实施例，第一方面所述的抗体的每条重链的N端分别与一个PLA2R抗原表位肽的C端相连。该融合蛋白上含有两个PLA2R抗原表位肽，即为二价分子。发明人经过实验发现，与单价的天然PLA2R相比，本发明的融合蛋白与PLA2R抗体的结合能力较强，可有效中和PLA2R抗体，保护肾小球组细胞表面的天然PLA2R不被PLA2R抗体 攻击。
根据本发明的实施例，第一方面所述的抗体的每条重链、每条轻链的N端分别与一个PLA2R抗原表位肽的C端相连。发明人经过实验发现，该融合蛋白上含有四个PLA2R抗原表位肽，与单价的天然PLA2R相比，本发明的融合蛋白与PLA2R抗体的结合能力较强，可有效中和PLA2R抗体，保护肾小球组细胞表面的天然PLA2R不被PLA2R抗体攻击。
根据本发明的实施例，所述融合蛋白进一步包括连接肽，所述连接肽设置于所述PLA2R抗原表位肽与所述抗体之间。
根据本发明的实施例，所述连接肽的N端与所述PLA2R抗原表位肽的C端相连，所述连接肽的C端与所述中性抗体的N段相连。由此，采用过上述连接肽可融合的PLA2R抗原表位肽和中性抗体，提高最终所得到的融合蛋白与PLA2R抗体的结合能力。
根据本发明的实施例，所述连接肽的氨基酸序列为(GGGGS)n，其中n为大于或等于1的整数，优选为1、2、3、4、5、6、7、8、9或10。
根据本发明的实施例，所述连接肽包括SEQ ID NO:23所示的氨基酸序列。
根据本发明的实施例，所述抗体FC区段具有如SEQ ID NO:24或者与SEQ ID NO:24具有至少90％相似性的氨基酸序列。
根据本发明的实施例，所述融合蛋白具有如SEQ ID NO:57所示氨基酸序列的第一肽段和具有如SEQ ID NO:19所示氨基酸序列的第二肽段；或者
所述融合蛋白具有如SEQ ID NO:17所示氨基酸序列的第一肽段和具有如SEQ ID NO:59所示氨基酸序列的第二肽段；或者
所述融合蛋白的氨基酸序列如SEQ ID NO:61所示；或者
所述融合蛋白具有如SEQ ID NO:63所示氨基酸序列的第一肽段和具有如SEQ ID NO:19所示氨基酸序列的第二肽段；或者
所述融合蛋白具有如SEQ ID NO:17所示氨基酸序列的第一肽段和具有如SEQ ID NO:65所示氨基酸序列的第二肽段；或者
所述融合蛋白的氨基酸序列如SEQ ID NO:67所示；或者
所述融合蛋白具有如SEQ ID NO:57所示氨基酸序列的第一肽段和具有如SEQ ID NO:59所示氨基酸序列的第二肽段；或者
所述融合蛋白具有如SEQ ID NO:63所示氨基酸序列的第一肽段和具有如SEQ ID NO:65所示氨基酸序列的第二肽段。
示例性地，参考图8，当中性抗体为上述所述的抗体时，融合蛋白的第一肽段为包括第一抗体重链的肽段，融合蛋白的第二肽段为包括第一抗体轻链的肽段，第一肽段和第二肽段通过链间二硫键相连；当中性抗体为上述所述的抗体Fc片段时，融合蛋白为包括抗体Fc片段的肽段。
本领域技术人员能够理解的是，前面针对抗体所描述的特征和优点，同样适用于该融合蛋白，在此不再赘述。
第二核酸分子、第二表达载体、第二重组细胞
在制备或者获取这些融合蛋白的过程中，可以利用表达这些融合蛋白的核酸分子，与不同的载体连接，然后在不同细胞中表达，来获得相应融合蛋白。
为此，在本发明的第八方面，本发明提出了一种第二核酸分子。根据本发明的实施例，所述核酸分子编码第七方面所述的融合蛋白。利用该第二核酸分子可以有效地用于表达上述融合蛋白，尤其是在原核生物或者低等真核生物表达体系中可以有效地表达上述融合蛋白。需要说明的是，这里的第二核酸分子不需要编码上述融合蛋白的全部序列，例如，可以包括两类核酸分子，分别编码两个单体，进一步可以在体外进行组装得到异二聚体，即可形成含有双链抗体的融合蛋白。
根据本发明的实施例，所述第二核酸分子为DNA。
在本发明的第九方面，本发明提出了一种第二表达载体。根据本发明的实施例，所述第二表达载体携带第八方面所述的第二核酸分子。利用该第二表达载体可以有效地用于表达上述融合蛋白，尤其是在原核生物或者低等真核生物表达 体系中可以有效地表达上述融合蛋白。
根据本发明的实施例，所述第二表达载体为真核表达载体，优选地，所述第二表达载体为慢病毒载体。
在本发明的第十方面，本发明提出了一种第二重组细胞。根据本发明的实施例，所述第二重组细胞包括：携带第八方面所述的第二核酸分子；或，表达第七方面所述的融合蛋白。根据本发明实施例的重组细胞可用于第七方面所述的融合蛋白体外表达和大量获得。
根据本发明的实施例，所述第二重组细胞为真核细胞。
根据本发明的实施例，所述第二重组细胞为哺乳动物细胞。
本领域技术人员能够理解的是，前面针对融合蛋白所描述的特征和优点，同样适用于该第二核酸分子、第二表达载体和第二重组细胞，在此不再赘述。
药物组合物、免疫吸附剂、试剂盒、吸附产品及其用途
在本发明的第十一方面，本发明提出了一种药物组合物。根据本发明的实施例，所述药物组合物包括：第七方面所述的融合蛋白。发明人经过大量实验发现，上述药物组合物可有效预防和治疗PLA2R抗体引起的相关疾病，例如PLA2R抗体阳性膜性肾病。
根据本发明的实施例，所述药物组合物进一步包括药学上可接受的辅料。
在本发明的第十二方面，本发明提出了一种免疫吸附剂。根据本发明的实施例，所述免疫吸附剂包括：第七方面所述的融合蛋白。发明人经过大量实验发现，上述免疫吸附剂可吸附PLA2R抗体，其可通过免疫吸附有效治疗PLA2R抗体引起的相关疾病，例如PLA2R抗体阳性膜性肾病。
在本发明的第十三方面，本发明提出了一种试剂盒。根据本发明的实施例，所述试剂盒包括：第七方面所述的融合蛋白。发明人经过大量实验发现，上述试剂盒可有效检测PLA2R抗体含量，并且还可以用于诊断或治疗PLA2R抗体引起的相关疾病，例如PLA2R抗体阳性膜性肾病。
本领域技术人员能够理解的是，前面针对融合蛋白、第二核酸分子、第二表达载体和第二重组细胞所描述的特征和优点，同样适用于该药物组合物、免疫吸附剂和试剂盒，在此不再赘述。
在本发明的十四方面，本发明提出了一种吸附产品。根据本发明的实施例，所述吸附产品包括：第十二方面所述的免疫吸附剂；固相载体，所述免疫吸附剂与固相载体相连。本发明的吸附产品可用于吸附PLA2R抗体，例如吸附血液或血浆中的PLA2R抗体，通过吸附血液或血浆中的PLA2R抗体可用于预防或治疗PLA2R抗体引起的相关疾病。
需要说明的是，免疫吸附剂和固相载体的连接方式不受具体限制，可偶联，或者可通过化学键相连。
在本文中，所述血液或血浆可来源于任何动物，例如哺乳动物。
示例性地，所述血液或血浆选自人血液或血浆。
根据本发明的实施例，所述吸附产品选自免疫吸附柱或血液净化剂。
本领域技术人员能够理解的是，前面针对免疫吸附剂所描述的特征和优点，同样适用于该用途，在此不再赘述。
在本发明的十五方面，本发明提出了一种第十二方面所述的免疫吸附剂在制备吸附产品中的用途，所述吸附产品用于吸附PLA2R抗体。
根据本发明的实施例，所述吸附产品选自免疫吸附柱或血液净化剂。
根据本发明的实施例，所述吸附产品为免疫吸附柱，所述吸附产品用于预防或治疗PLA2R抗体引起的相关疾病。由前可知，上述免疫吸附剂可吸附PLA2R抗体，由此，含有上述免疫吸附剂的免疫吸附柱，可通过免疫吸附剂吸附PLA2R抗体，该免疫吸附柱可有效治疗PLA2R抗体引起的相关疾病。
根据本发明的实施例，所述PLA2R抗体引起的相关疾病选自PLA2R抗体阳性膜性肾病。
根据本发明的实施例，所述吸附产品为血液净化剂，所述吸附产品用于吸附血液或血浆中的PLA2R抗体。由前可知，上述免疫吸附剂可吸附PLA2R抗体，由此，含有上述免疫吸附剂的血液净化剂，可通过免疫吸附剂吸附PLA2R抗体，该血液净化剂可有效吸附血液或血浆中的PLA2R抗体。
本领域技术人员能够理解的是，前面针对免疫吸附剂所描述的特征和优点，同样适用于该用途，在此不再赘述。
在本发明的第十六方面，本发明提出了一种第七方面所述的融合蛋白、第八方面所述的第二核酸分子、第九方面所述的第二表达载体、第十方面所述的第二重组细胞或第十一方面所述的药物组合物在制备药物中的用途，所述药物用于预防或治疗PLA2R抗体引起的相关疾病。
根据本发明的实施例，所述PLA2R抗体引起的相关疾病选自PLA2R抗体阳性膜性肾病。
根据本发明的实施例，所述中药组合物的剂型包括注射剂。
根据本发明的实施例，所述中药组合物的给药途径包括皮下注射或静脉注射。
本领域技术人员能够理解的是，前面针对药物组合物、免疫吸附剂和试剂盒等所描述的特征和优点，同样适用于该用途，在此不再赘述。
在本发明的第十七方面，本发明提出了一种第七方面所述的融合蛋白、第八方面所述的第二核酸分子、第九方面所述的第二表达载体、第十方面所述的第二重组细胞或第十一方面所述的药物组合物在制备免疫吸附剂中的用途，所述免疫吸附剂用于吸附PLA2R抗体。
本领域技术人员能够理解的是，前面针对药物组合物、免疫吸附剂和试剂盒等所描述的特征和优点，同样适用于该用途，在此不再赘述。
在本发明的第十八方面，本发明提出了一种第七方面所述的融合蛋白、第八方面所述的第二核酸分子、第九方面所述的第二表达载体、第十方面所述的第二重组细胞、第十一方面所述的药物组合物或第十二方面所述的免疫吸附剂在制备试剂盒中的用途，所述试剂盒用于检测PLA2R抗体。
本领域技术人员能够理解的是，前面针对融合蛋白、药物组合物、免疫吸附剂和试剂盒等所描述的特征和优点，同样适用于上述的用途，在此不再赘述。
方法
在本发明的第十九方面，本发明提出了一种预防或治疗PLA2R抗体引起的相关疾病的方法。根据本发明的实施例，所述方法包含：向受试者施用药学上可接受量的第七方面所述的融合蛋白或第十一方面所述的药物组合物。由此，可有效治疗PLA2R抗体引起的相关疾病。
根据本发明的实施例，所述方法的给药途径采用皮下注射或静脉注射。
根据本发明的实施例，所述PLA2R抗体引起的相关疾病选自PLA2R抗体阳性膜性肾病。
在本发明的第二十方面，本发明提出了一种用于预防或治疗PLA2R抗体引起的相关疾病的免疫吸附治疗方法。根据本发明的实施例，所述方法包含：采用第十二方面所述的免疫吸附剂对受试者进行治疗。
根据本发明的实施例，所述方法采用体外血液净化对受试者进行治疗。
根据本发明的实施例，所述PLA2R抗体引起的相关疾病选自PLA2R抗体阳性膜性肾病。
在本发明的第二十一方面，本发明提出了一种诊断PLA2R抗体引起的相关疾病的方法。根据本发明的实施例，所述方法包括：采用第七方面所述的融合蛋白、第八方面所述的第二核酸分子、第九方面所述的第二表达载体、第十方面所述的第二重组细胞、第十一方面所述的药物组合物、第十二方面所述的免疫吸附剂或第十三方面所述的试剂盒对待测样品中的PLA2R抗体进行检测；基于检测结果，确定待测样品中的PLA2R抗体的含量。根据本发明的实施例，所述待测样品中PLA2R抗体的含量不低于14RU/ml是待测样品来源于患有PLA2R抗体引起的相关疾病的患者的指示，优选为，所述待测样品中PLA2R抗体的含量不低于20RU/ml是待测样品来源于患有PLA2R抗体引起的相关疾病的患者的指示。
根据本发明的实施例，所述待测样品为血液。
根据本发明的实施例，所述PLA2R抗体引起的相关疾病选自PLA2R抗体阳性膜性肾病。
在本发明的第二十二方面，本发明提出了一种评估PLA2R抗体引起的相关疾病分期的方法。根据本发明的实施例，所述方法包括：采用第七方面所述的融合蛋白、第八方面所述的第二核酸分子、第九方面所述的第二表达载体、第十方面所述的第二重组细胞、第十一方面所述的药物组合物、第十二方面所述的免疫吸附剂或第十三方面所述的试剂盒对待测样品中的PLA2R抗体进行检测；基于检测结果，确定待测样品中的PLA2R抗体的含量。
根据本发明的实施例，基于待测样品中的PLA2R抗体的含量，确定待测患者患有PLA2R抗体引起的相关疾病的分期。
根据本发明的实施例，所述PLA2R抗体引起的相关疾病选自PLA2R抗体阳性膜性肾病。
根据本发明的实施例，PLA2R抗体阳性膜性肾病分为四期。
根据本发明的实施例，所述待测样品为血液。
在本发明的第二十三方面，本发明提出了一种评估PLA2R抗体引起的相关疾病的预后方法。根据本发明的实施例，所述方法包括：采用第七方面所述的融合蛋白、第八方面所述的第二核酸分子、第九方面所述的第二表达载体、第十方面所述的第二重组细胞、第十一方面所述的药物组合物、第十二方面所述的免疫吸附剂或第十三方面所述的试剂盒对待测样品中的PLA2R抗体进行检测；基于检测结果，确定待测样品中的PLA2R抗体的含量。
根据本发明的实施例，所述待测样品来源于治疗前或后的患有PLA2R抗体引起的相关疾病的患者。
根据本发明的实施例，基于治疗前或后的患有PLA2R抗体引起的相关疾病的患者的待测样品中的PLA2R抗体的含量，确定PLA2R抗体引起的相关疾病的预后效果。
根据本发明的实施例，治疗后的患有PLA2R抗体引起的相关疾病的患者的待测样品中的PLA2R抗体的含量下降是患者预后良好的指示。
根据本发明的实施例，所述待测样品为血液。
根据本发明的实施例，所述PLA2R抗体引起的相关疾病选自PLA2R抗体阳性膜性肾病。
本领域技术人员能够理解的是，前面针对融合蛋白、药物组合物、免疫吸附剂和试剂盒所描述的特征和优点，同样适用于上述的方法，在此不再赘述。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：中性抗体的制备和选择
1、表达构建
首先，质粒载体采用的是pcDNA3.4瞬时表达载体，该载体包含原生的全长CMV启动子和克隆位点下游的WPRE元件，基于CMV启动子的载体通常对CHO细胞瞬时表达系统具有良好的表达水平，WPRE元件位于多克隆位点下游，可以有效提高基因的转录和表达，在大肠杆菌中，具有氨苄抗性基因。然后，利用PCR技术扩增载体基因pcDNA3.4和目的基因，PCR模板经过基因合成得到，通过同源重组试剂盒将载体片段和目的基因片段连接，DH5α感受态转化得到单克隆，将单克隆扩增后进行测序，经测序确认后，提取质粒得到目的基因重组表达质粒。其中，目的序列编码DNA加上分泌表达信号肽的编码DNA经基因合成，克隆于质粒pcDNA3.4的CMV promoter和WPRE基因之间，具体参见图1。
1.1目的基因(gene of interest)的名称和序列如表1所示，pcDNA3.4载体和目的基因(或称目的片段)的引物与模板如表2所示，其中，RtxH和RtxL用于合成利妥昔(Rituximab，简称Rtx)。
表1：目的基因的名称和序列
注：本实施例中的dTrs1L和dTrs2L为相同的目的基因。
其中，其中，RtxH的氨基酸序列：
RtxL的氨基酸序列：
TrsH的氨基酸序列：
TrsL的氨基酸序列：
dTrs1H的氨基酸序列：
dTrs1L的氨基酸序列：
dTrs2H的氨基酸序列：
RtxH的核苷酸序列：

RtxL的核苷酸序列：
TrsH的核苷酸序列：
TrsL的核苷酸序列：
dTrs1H的核苷酸序列：

dTrs1L(dTrs2L)的核苷酸序列：
dTrs2H的核苷酸序列：
表2：pcDNA3.4载体和目的基因的引物与模板
1.2 PCR反应采用TAKARA的KOD反应体系(Cat.KFX-101)、反应条件、试剂和PCR目的片段信息如表3～6所示。
表3：目的基因或pcDNA3.4的KOD反应体系
表4：载体pcDNA3.4的PCR反应条件
表5：目的基因的PCR反应条件
表6：PCR目的片段信息

1.3 PCR结束后将样品进行1.5％琼脂糖凝胶电泳，其中，电泳槽加1*TAE 300mL，于100V、30min跑胶。结果显示，琼脂糖凝胶电泳条带大小与理论大小一致。将电泳结果利用Omega Gel Extraction Kit胶回收试剂盒(Cat.D2500-01)进行回收，20μL洗脱液洗脱得到纯化后的PCR产物。电泳结果如图2-5所示，其中，图2的从左到右的泳道1-9依次为DL5000、pcDNA3.4、pcDNA3.4、pcDNA3.4、pcDNA3.4、trsH、trsL、RtxH和RtxL；图3的从左到右的泳道1-7依次为DL2000、dTrs1H、dTrs1H、dTrs2H、dTrs2H、dTrs1L和dTrs1L；图4的从左到右的泳道1-5依次为DL2000、dTrs3H、dTrs3H、dTrs3L和dTrs3L；图5的从左到右的泳道1-5依次为DL2000、dTrs4H、dTrs4H、dTrs4L和dTrs4L。
1.4将1.3中得到的切胶纯化后的PCR条带进行同源重组，利用NEB的试剂盒(HiFi DNA Assembly Master Mix，Cat.E2621S)进行片段组装，将同源重组的反应体系在50℃条件下孵育1小时，将得到的反应产物利用Omega Cycle Pure Kit(Cat.D6492-01)进行纯化，用15μL洗脱液进行洗脱。反应体系和质粒表如表7和表8所示。
表7：同源重组的反应体系
表8：质粒表
1.5利用CaCl2感受态细胞DH5a转化，将1.4得到的各个质粒样品分别以1:10的比例加入到感受态细胞中，置于冰上10min，然后金属浴42℃条件下孵育45s，再置于冰上10min，最后将得到的溶液涂布于加入了氨苄青霉素(Amp.终浓度50μg/mL)的LB固体培养基中，37℃条件下过夜培养，第二天挑取单克隆于2ml LB液体培养基中(终浓度50μg/mL Amp.)，在37℃、200rpm条件下摇菌，将得到的菌液进行Sanger测序，得到测通序列，测序引物为CMV-F: CGCAAATGGGCGGTAGGCGTG(SEQ ID NO:79)。经测序结果比对后，确认所有测通序列与原序列一致，将单克隆菌液取100μL加入到50mL加入了氨苄青霉素(终浓度50μg/mL)的LB液体培养基中，在37℃、200rpm条件下过夜培养，保存菌种，提取质粒，得到目的基因表达质粒。
2、Trs、dTrs1-4和Rtx蛋白的瞬时表达
2.1转染前约24小时，取FreeStyleTM 细胞(细胞密度为5-6×105/mL，来自于美国Gibco公司FreeStyleTMMAX CHO表达系统，货号：K900020)，在37℃、5％CO2和120-135rpm/min条件下培养。
2.2转染当日，稀释细胞至细胞密度为1×106/mL，每个摇瓶中加入10mL细胞(细胞的存活率在95％以上)。
2.3轻轻混合FreeStyleTMMAX Reagent几次，注意不能漩涡震荡。
2.4分别取表9中各蛋白对应质粒各12.5μg，加入OptiPROTMSFM至各自总体积0.2mL并轻轻混合。
2.5取25μL的FreeStyleTMMAX Reagent，加入Opti-ProTMSFM至总体积0.4mL，轻轻混合(注意不能漩涡震荡)。混合均匀后平均加入质粒混合液中，轻轻混合以获得2支0.4mL DNA-FreeStyleTMMAX混合物，并在室温下孵育10分钟，使复合物形成。
2.6慢慢将上述2支0.4mL DNA-FreeStyleTMMAX复合物加入到细胞中，同时缓慢旋转摇瓶。
2.7在37℃、5％CO2，120-135rpm/min培养细胞，不需要更换或补充培养基，7d后收取细胞液。
表9：不同蛋白所对应质粒
3、Trs、dTrs1-4和Rtx蛋白的纯化与鉴定
Trs、dTrs1-4和Rtx培养上清经4℃、7000g离心15min后用0.22um针头滤器过滤除菌。以5倍柱体积的Buffer A1(20mM磷酸缓冲液，pH值为7.4)以平衡Protein A层析介质(流速为2mL/min)，加入处理好的样品，Buffer A1冲洗，直到吸收值达到基线，Buffer B1(0.1M Glycine-HCl，pH值为3.0)洗脱，收集洗脱峰样品，目的蛋白洗脱液用1M Tris-HCl(pH值为9.0)调至中性。超滤至PBS缓冲液中。
目的蛋白的鉴定：选择10孔的SDS-PAGE预制胶，于150V条件下跑胶50min。样品90℃加热5min，每孔上样10μg。
SDS-PAGE检测Trs、dTrs1-4和Rtx蛋白的纯化效果如图6所示，其中，泳道1为Trs洗脱非还原，泳道2为Trs洗脱还原，泳道3为dTrs1洗脱非还原，泳道4为dTrs1洗脱还原，泳道5为dTrs2洗脱非还原，泳道6为dTrs2洗脱还原，泳道7为dTrs3洗脱还原，泳道8为dTrs3洗脱非还原，泳道9为dTrs4洗脱非还原，泳道10为dTrs4洗脱还原，泳道11为Rtx非还原，泳道12为Rtx还原。Trs、dTrs1-4和Rtx蛋白的表达量如表10所示。
表10：Trs、dTrs1-4和Rtx蛋白的表达量
4、Trs、dTrs1-4和Rtx蛋白与Her2结合ELISA
4.1以购买的具有结合活性的Her2(10004-HCCH-B，义翘神州)作为底物，Trs、dTrs1-4或Rtx蛋白作为一抗，HRP偶联的山羊抗人IgG为二抗，用酶联免疫吸附方法检测Her2蛋白与Trs、dTrs1-4或Rtx蛋白的结合情况。
Her2蛋白固体20μg溶于80μL无菌水中，配成0.25mg/mL母液，分装于0.5mL EP管，10μL/管，存放于-80℃。
一抗：Trs(0.44mg/mL)，dTrs1(0.38mg/mL)，dTrs2(0.42mg/mL)，dTrs3(0.115mg/mL)，dTrs4(1.4mg/mL)，Rtx(0.21mg/mL)，其中Trs为阳性对照，Rtx为阴性对照。
二抗：HRP偶联的山羊抗人IgG(生工生物工程(上海)股份有限公司，Cat.No.D110150)。
4.2检测方案：
1)包被：用包被液稀释培养上清，每孔加入0.1μg/100μL Her2蛋白(34μL母液+8466μL包被液)，封板，37℃孵育2h。弃去孔内液体，洗液洗板3次，每次200μL，静置2min。
2)封闭：每孔加入封闭液100μL，4℃过夜，弃去孔内液体。
3)一抗(Trs、dTrs1-4或Rtx)孵育：用抗体稀释液分别稀释Trs、dTrs1-4和Rtx，每孔加入100μL，封板，37℃孵育1h，弃去孔内液体，用洗液洗板3次，每次200μL，静置2min。
4)二抗孵育：用抗体稀释液按照1:2000稀释二抗，每孔加入100μL，封板，37℃孵育1h，弃去孔内液体，用洗液洗板3次，每次200μL，静置2min。
5)检测：每孔加入TMB 100μL，封板，37℃避光孵育15min，此时孔内呈蓝色，每孔加入终止液100μL，立即变黄，于450nm下检测OD值，检测结果如图7和表11所示。结果发现，和Trs相比，dTrs1-dTrs4与Her2的结合能力逐渐降低，其中dTrs4与Her2结合能力最低，接近于阴性对照Rtx，此外，还发现dTrs4的表达量高，因此可选择dTrs4作为药物骨架。
其中，包被液的配制如下表所示：
封闭液的配制如下表所示：
抗体稀释液的配制如下表所示：
洗液的配制如下表所示：

表11：Trs、dTrs1-4和Rtx蛋白与Her2结合结果
注：低于下限和高于上限均超出计算范围，其中，低于下限表示结合能力很强，高于上限表示结合能力很弱。
5、Trs、dTrs1-4和Rtx蛋白与IgA、IgM或HAS的结合
5.1实验步骤如下：
1)采用100μL/孔含10μg/mL IgA/IgM/HSA包被液封板，4℃过夜；其中，IgA来源于人类血清(Sigma-Aldrich，I4036)、浓度为1mg/mL，IgM来源于人类血清(Sigma-Aldrich，I8260)、浓度为1mg/mL，HSA(Absin，abs47047388)浓度为20mg/mL；
2)将步骤1)弃液，用PBST(以双蒸水(灭菌)稀释索莱宝10*PBS 10倍，+0.1％吐温)洗板，洗板机洗4次，甩板机在600×g条件下甩干1min，并拍干若干次；
3)加入100μL/孔封闭液(0.5％Casein(pH 7.09，1M NaOH>50mg/mL配制小份，加PBST，HCl调pH后定容配制)，37℃温育60min；然后重复步骤2)洗板；
4)加入100μL/孔10倍梯度稀释(10、1、0.1、0.01、0.001、0.0001、0.00001)的各组样品(Trs、dTrs1-4或Rtx)，封板，室温温育30min；然后重复步骤2)洗板；
5)每孔加入100μL检测抗体(Goat Anti-Human IgG-Fc Secondary Antibody(HRPμg/mL)，Sino Biological、SSA001，用PBST进行1:3K稀释至使用浓度为1.2μg/mL)，封板，室温温育30min；然后重复步骤2)洗板；
6)每孔加入100μL索莱宝TMB底物显色液，25℃避光温育6min后，每孔加入100μL索莱宝终止液，测OD450。
5.2结果
以人IgA铺板进行ELISA检测的结果如图21所示，其中p<0.01**、p<0.05*。结果表明，dTrs4作为检测分子产生的信号非常低，甚至低于野生型的Trs，以及无关抗体RTX。因此，可表明dTrs4不与人IgA结合。
其中，图21的差异性分析如下表所示：
以人IgA铺板，10ug/mL时dTRS4与其它分子进行t检验的p值。
以人IgM铺板进行ELISA检测的结果如图22所示，其中p<0.01**、p<0.05*。结果表明，dTrs4作为检测分子产生的信号非常低，甚至低于野生型的Trs，以及无关抗体RTX。因此，可表明dTrs4不与人IgM结合。
其中，图22的差异性分析如下表所示：
以人IgM铺板，10ug/mL时dTRS4与其它分子进行t检验的p值。
以人HSA铺板进行ELISA检测的结果如图23所示，其中p<0.01**、p<0.05*。结果表明，除无关抗体RTX产生的 信号略高，其它分子信号均很低。其中，dTrs4作为检测分子产生的信号最低，甚至低于野生型的Trs，以及无关抗体RTX。因此，可表明dTrs4不与人HSA结合。
其中，图23的差异性分析如下表所示：
以人HSA铺板，10ug/mL时dTRS4与其它分子进行t检验的p值。
实施例2：RC1-RC8蛋白的制备和选择
1、表达构建：RC1-RC8蛋白的表达构建的步骤与实施例1中表达构建相同，区别仅在于目的基因的不同。RC1-RC8蛋白的结构示意图如图8所示，PLA2R的结构示意图如图9所示，其中，RC1-RC8蛋白中的PLA2R抗原表位肽CFC1(SEQ ID NO:21)为PLA2R片段的1至367(Glu)氨基酸序列，PLA2R抗原表位肽CFC2(SEQ ID NO:22)为PLA2R片段的1至510(Asp)氨基酸序列。
其中，目的基因的名称、引物、序列和重链、轻链及其信号肽的组成如表12所示，目的基因的名称和序列如表13所示，pcDNA3.4载体和目的基因的引物与模板如表14所示，目的片段信息如表15所示。
其中，
PLA2R信号肽核苷酸序列：
Trs重链信号肽核苷酸序列：
Trs轻链信号肽核苷酸序列：
纯化后的PCR产物的电泳结果如图10-12所示，其中，图10的从左到右的泳道1-5依次为DL5000、pcDNA3.4、pcDNA3.4、pcDNA3.4和pcDNA3.4；图11的从左到右的泳道1-13依次为DL5000、pcDNA3.4、pcDNA3.4、pcDNA3.4、pcDNA3.4、RC1H-1、RC1H-2、RC2L-1、RC2L-2、RC4H-1、RC4H-2、RC5L-1和RC5L-2；图12的从左到右的泳道1-7依次为DL5000、无关、无关、RC3、RC3、RC6和RC6。
将切胶纯化后的PCR条带进行同源重组，体系与表7一致，构建的质粒表如表16所示。
表12：RC1-RC8蛋白的重链和轻链组成
表13：目的基因的名称和序列

注：RC1H、RC2L、RC4H和RC5L都是通过两段PCR片段拼接而成，即为，RC1H由RC1H-1和RC1H-2拼接而成，RC2L由RC2L-1和RC2L-2拼接而成，RC4H由RC4H-1和RC4H-2拼接而成，RC5L由RC5L-1和RC5L-2拼接而成。
其中，RC1H的氨基酸序列：
RC1H的核苷酸序列：

RC2L的氨基酸序列：
RC2L的核苷酸序列：
RC3的氨基酸序列：

RC3的核苷酸序列：
RC4H的氨基酸序列：
RC4H的核苷酸序列：

RC5L的氨基酸序列：

RC5L的核苷酸序列：
RC6的氨基酸序列：
RC6的核苷酸序列：

表14：pcDNA3.4载体和目的基因的引物与模板
表15：PCR目的片段信息

表16：质粒表
2、RC1-RC8蛋白的瞬时表达
2.1转染前约24小时，取FreeStyleTM 细胞(细胞密度为5-6×105/mL，来自于美国Gibco公司FreeStyleTMMAX CHO表达系统，货号：K900020)，37℃、5％CO2，120-135rpm/min培养。
2.2转染当日，稀释细胞至细胞密度为1×106/mL，每个摇瓶中加入10mL细胞(细胞的存活率在95％以上)。
2.3轻轻混合FreeStyleTMMAX Reagent几次，注意不能漩涡震荡。
2.4分别取表17中各蛋白对应质粒各12.5μg，加入OptiPROTMSFM至各自总体积0.2mL并轻轻混合。
2.5取25μL的FreeStyleTMMAX Reagent，加入Opti-ProTMSFM至总体积0.4mL，轻轻混合(注意不能漩涡震荡)。混合均匀后平均加入质粒混合液中，轻轻混合以获得2支0.4mL DNA-FreeStyleTMMAX混合物，并在室温下孵育10分钟，使复合物形成(RC3、RC6蛋白取12.5μL FreeStyleTMMAX Reagent，加入Opti-ProTMSFM至总体积0.2mL，最终获得1支0.4mL DNA-FreeStyleTMMAX混合物)。
2.6慢慢将上述2支0.4mL DNA-FreeStyleTMMAX复合物加入到细胞中(RC3、RC6蛋白只有1支DNA-FreeStyleTM MAX复合物)，同时缓慢旋转摇瓶。
2.7在37℃、5％CO2和120-135rpm/min条件下培养细胞，不需要更换或补充培养基，7d后收取细胞液。
表17：RC1-RC8蛋白所对应的质粒
3、RC1-RC8蛋白的纯化与鉴定
RC1-RC8培养上清经4℃、7000g条件下离心15min后，用0.22μm针头滤器过滤除菌。以5倍柱体积的Buffer A1以平衡Protein A层析介质(流速为2mL/min)，加入处理好的样品，Buffer A1冲洗，直到吸收值达到基线，Buffer B1洗脱，收集洗脱峰样品，目的蛋白洗脱液用1M Tris-HCl(pH值为9.0)调至中性。Super dex200过滤至PBS中。
目的蛋白的鉴定：选择10孔的SDS-PAGE预制胶，于150V条件下跑胶50min。样品90℃加热5min，每孔上样3μg。
SDS-PAGE检测RC1-RC8蛋白的纯化效果如图13所示，其中，泳道M为marker，泳道1为RC1洗脱非还原，泳道2为RC1洗脱还原，泳道3为RC2洗脱还原，泳道4为RC2洗脱非还原，泳道5为RC3洗脱非还原，泳道6为RC3洗脱还原，泳道7为RC4洗脱还原，泳道8为RC4洗脱非还原，泳道9为RC5洗脱非还原，泳道10为RC5洗脱还原，泳道11为RC6洗脱非还原，泳道12为RC6洗脱还原，泳道13为RC7洗脱非还原，泳道14为RC7洗脱还原，泳道15为RC8洗脱非还原，泳道16为RC8洗脱还原。RC1-RC8蛋白的表达量如表18所示。
表18：RC1-RC8蛋白的表达量
4、RC1-RC8的稳定性考察
蛋白质聚集是许多球蛋白的常见现象，会影响蛋白质药物质量标准的确定以及制剂成分。作为候选药物分子，通常倾向于选择聚集程度较低的分子。因此，对本实施例步骤3制备得到的RC1-RC8进行稳定性考察，具体步骤如下：
RC2在-20℃储存时有明显降解，稳定性较差，未进行后续实验。
取RC1、RC3、RC4、RC5、RC6、RC7和RC8、以及CC1h(氨基酸序列如SEQ ID NO:92所示、核苷酸序列如SEQ ID NO:93所示，采用本领域常规方法制备获得)分别采用样品缓冲液(25mM磷酸钠+75mM NaCl+40％甘油(pH 7.0))稀释至相同的浓度，然后在37℃下考察24小时和48小时，结果参见图24。
其中，RC1、RC3、RC4、RC5、RC6、RC7、RC8和CC1h的分子量如下表所示：
结果发现，CC1h在37℃放置24h后出现聚集(约82KD)，且48h后聚集增加。RC3,RC6几乎无聚集；RC1,RC4出现少量的二倍分子量条带，且48h强于24h，但相较于CC1h，RC1,RC4聚集程度较轻。因此，进一步说明RC3、RC6、RC1和RC4相对CC1h具有较轻的聚集倾向，可能具有较好的成药性。
其中，CC1h的氨基酸序列和核苷酸序列如下所示：
5、RC1-RC8对血浆样品中PLA2R-Ab的中和
采用当前唯一获美国FDA批准用于PLA2R抗体阳性膜性肾病诊断的试剂盒(德国欧蒙，ELISA法定量检测血液样品中PLA2R-Ab浓度)进行检测，所有步骤按照说明书进行。对3例PLA2R抗体阳性膜性肾病阳性患者的血浆分别编号为pMN-2、pMN-4及pMN-7。
检测了RC1-RC8对这3例膜性肾病阳性病人血浆中PLA2R-Ab的中和效果，得到了如图14-16和表19的中和率。其中，图14-16中的中和率neutralization ratio＝(1-(加入抑制剂后的PLA2R-Ab检测值/不加抑制剂的PLA2R-Ab检测值))*100％，该值越大，表明能够检测到的自身免疫抗体越少，相应的抑制效果越好。(假定静脉给药，生物利用度100％，本抑制剂在人身上的分布容积为8L，则本研究中抑制剂浓度相当于5μg/mL*8L＝40mg/人的剂量)。
中和率的计算如下所示：
诊断试剂盒中的捕获分子为PLA2R。
未加药物分子时：

其中，[PLA2R·Ab]1对应了抗体的检测浓度。
加入药物分子后：
其中，[PLA2R·Ab]2对应了此时抗体的检测浓度。
中和率neutralization ratio(NR)：
在ELISA检测中，PLA2R作为捕获抗原是大大过量的，因此[PLA2R]2≈[PLA2R]1，故即加入药物分子导致PLA2R-Ab在血浆中减少的比例。可代表药物分子的中和效果。
表19：RC1-RC8对这3例膜性肾病阳性病人血浆中PLA2R-Ab的中和率
3组实验中RC1-RC8蛋白的给药量均为5μg/mL。pMN-2为阳性样品，针对血浆中浓度在44.79RU/mL左右的自身免疫抗体，各组药物都表现出超过95％的中和效果。pMN-4为阳性样品，针对血浆中浓度在124.905RU/mL左右的自身免疫抗体，各组药物都表现出超过80％的中和效果。pMN-7为阳性样品，针对血浆中浓度在53.72RU/mL左右的自身免疫抗体，除了RC5中和率为77.63％外，其余各组药物都表现出超过80％的中和效果。
6、RC3和RC6的固定化
取一定体积保存于异丙醇中的NHS-Bestarose(AA0031，博格隆(上海)生物技术有限公司)介质于重力柱中，用预冷的偶联溶液A3(1mM盐酸)(0～4℃)至少洗涤30min，1mL介质用20mL的偶联溶液A3溶液洗涤；RC3、RC6经 超滤将其缓冲环境置换为偶联溶液B3(0.1M碳酸氢钠，0.5M氯化钠，pH 8.3)中(配基偶联浓度10mg/mL填料)。将洗好的介质用偶联溶液A3(1mL介质约0.5mL偶联溶液A3)稀释，并与配基溶液等体积混合，4℃震荡过夜。抽去偶联上清并加入封闭液(0.1M Tris-HCl，pH8.5)，室温封闭处理4h；采用封闭液与0.2M醋酸，在pH3.5的条件下交替洗涤5次，每次用3倍填料体积洗涤。用PBS清洗后使用或保存于0.1M磷酸缓冲液中，得到RC3-NHS Bestarose和RC6-NHS Bestarose。
7、固定化RC3和RC6对血浆样品中PLA2R-Ab的吸附
将RC3-NHS Bestarose、RC6-NHS Bestarose充分重悬取100μL分别加入对应EP管，1000g离心5min后小心吸去上清。加入500μL PBS重悬-离心-吸去上清2次以清洗填料。EP管中加入100μL患者血浆重悬，轻微震荡30min后1000g离心5min。小心吸取上清收集到3只EP管中，分别标记为RC3-pMN和RC6-pMN。ELISA方法检测固定化后RC3和RC6对PMN-4和PMN-7血浆样品中PLA2R-Ab的吸附，方法如本实例的第4部分(RC1-RC8对血浆样品中PLA2R-Ab的中和)。结果参见图17-18，结果表明，固定化后RC3和RC6对PMN-4和PMN-7血浆样品中PLA2R-Ab的清除率均在80％以上。
其中，图17中固定化后RC3和RC6对PMN-4血浆样品中PLA2R-Ab的清除率：
RC3-NHS Bestarose＝(225.55-43.16)/225.55*100％＝80.86％，
RC6-NHS Bestarose＝(225.55-13.82)/225.55*100％＝93.87％。
图18中固定化后RC3和RC6对PMN-7血浆样品中PLA2R-Ab的清除率：
RC3-NHS Bestarose＝(52.03-6.26)/52.03*100％＝87.96％，
RC6-NHS Bestarose＝(52.03-4.67)/52.03*100％＝91.02％。
8、RC1-RC8小鼠急性毒性试验
将购买回来的BALB/c雌性小鼠进行适应性饲养一周，随机分为8组，分为RC1组、RC2组、RC4组、RC5组、RC7组、RC8组、安慰组(又称“Buffer”)和空白组(又称“No treatment”)，每组5只，称重并做标记。按10倍40mg/人剂量进行给药。在给药前称量小鼠体重，然后进行给药，具体为，RC1组皮下注射0.1mL浓度为1mg/mL的RC1蛋白、RC2组皮下注射0.1mL浓度为1mg/mL的RC2蛋白、RC4组皮下注射0.1mL浓度为1mg/mL的RC4蛋白、RC5组皮下注射0.1mL浓度为1mg/mL的RC5蛋白、RC7组皮下注射0.1mL浓度为1mg/mL的RC7蛋白、RC8组皮下注射0.1mL浓度为1mg/mL的RC8蛋白、安慰组注射0.1mL PBS、空白组不注射。给药当天记为第0天，给药完成后，留观1h，未观察到小鼠存在有呼吸困难、运动减少、震颤等明显不良反应。并在24h、48h、72h对小鼠进行称重，此后每48h对小鼠称重一次至给药第十天。
小鼠皮下注射RC1、RC2、RC4、RC5、RC7、RC8蛋白体重趋势图，由图19所示，结果表明，实验组与对照组小鼠体重无明显差异。
将购买回来的BALB/c雌性小鼠进行适应性饲养一周，随机分为4组，分为RC3组、RC6组、安慰组(又称“Buffer”)和空白组(又称“No treatment”)，每组5只，称重并做标记。按10倍40mg/人剂量进行给药。在给药前称量小鼠体重，然后进行给药，具体为，RC3组皮下注射0.1mL浓度为1mg/mL RC3蛋白、RC6组皮下注射0.1mL浓度为1mg/mL RC6蛋白、安慰组注射0.1mLPBS、空白组不注射。给药当天记为第0天，给药完成后，留观1h，未观察到小鼠存在有呼吸困难、运动减少、震颤等明显不良反应。并在24h、48h、72h对小鼠进行称重，此后每48h对小鼠称重一次至给药第三十天。
小鼠皮下注射RC3、RC6蛋白体重趋势图，由图20所示，结果表明，实验组与对照组小鼠体重无明显差异。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (92)

  1. 一种抗体，其特征在于，包括：轻链可变区CDR和重链可变区CDR；
    其中，重链可变区CDR1序列如GX1X2X3X4X5X6X7X8X9所示，
    重链可变区CDR2序列如X10X11X12X13X14X15GX16X17X18X19X20X21X22X23所示，
    重链可变区CDR3序列如X24GGX25X26X27X28X29X30X31Y所示，
    X1为G或F，X2为G或N，X3为G或I，X4为G、K或S，X5为G或D，X6为G或T，X7为G或Y，X8为G或I，X9为H或S，X10为R或G，X11为G或I，X12为G或Y，X13为G或P，X14为T或S，X15为G或N，X16为G或Y，X17为G或T，X18为G、S或R，X19为G或Y，X20为G或A，X21为G或D，X22为G或S，X23为S或V，X24为G或W，X25为G或D，X26为G或S，X27为G或F，X28为G或Y，X29为G或A，X30为G或M，X31为S、G或D；
    条件是X1为F、X2为N、X3为I、X4为K、X5为D、X6为T、X7为Y、X8为I、X9为H、X10为R、X11为I、X12为Y、X13为P、X14为T、X15为N、X16为Y、X17为T、X18为R、X19为Y、X20为A、X21为D、X22为S、X23为V、X24为W、X25为D、X26为G、X27为F、X28为Y、X29为A、X30为M、X31为D不同时成立；
    轻链可变区CDR1序列如X32X33X34X35X36X37X38X39X40X41A所示，
    轻链可变区CDR2序列如X42X43X44X45X46X47S所示，
    轻链可变区CDR3序列如X48X49X50X51X52X53X54X55X56所示，
    X32为G或R，X33为G或A，X34为G或S，X35为G或Q，X36为G、D或S，X37为G或V，X38为G或N，X39为G或T，X40为G或A，X41为S或V，X42为G或S，X43为G或A，X44为G或S，X45为G或F，X46为L或S，X47为G或Y，X48为G或Q，X49为G或Q，X50为G或H，X51为G或Y，X52为S或T，X53为G或T，X54为G或P，X55为G或P，X56为G或T；
    条件是X32为R、X33为A、X34为S、X35为Q、X36为D、X37为V、X38为N、X39为T、X40为A、X41为V、X42为S、X43为A、X44为S、X45为F、X46为L、X47为Y、X48为Q、X49为Q、X50为H、X51为Y、X52为T、X53为T、X54为P、X55为P、X56为T不同时成立。
  2. 根据权利要求1所述的抗体，其特征在于，所述重链可变区CDR和轻链可变区CDR满足如下至少之一的条件：
    X4为G或S，X5为G，X21为G，X31为S或G，X36为G或S。
  3. 根据权利要求2所述的抗体，其特征在于，所述重链可变区CDR中，X1为G或F，X2为G或N，X3为G或I，X4为G或S，X5为G，X6为G或T，X7为G或Y，X8为G或I，X9为H或S，X10为G，X11为G或I，X12为G或Y，X13为G或P，X14为T或S，X15为G或N，X16为G，X17为G或T，X18为G或S，X19为G或Y，X20为G或A，X21为G，X22为G或S，X23为S或V，X24为G，X25为G或D，X26为G或S，X27为G或F，X28为G，X29为G或A，X30为G或M，X31为S或G；
    所述轻链可变区CDR中，X32为G或R，X33为G或A，X34为G或S，X35为G或Q，X36为G或S，X37为G或V，X38为G，X39为G或T，X40为G或A，X41为S或V，X42为G或S，X43为G或A，X44为G或S，X45为G，X46为L或S，X47为G或Y，X48为G或Q，X49为G或Q，X50为G或H，X51为G，X52为S或T，X53为G或T，X54为G或P，X55为G或P，X56为G或T。
  4. 根据权利要求1所述的抗体，其特征在于，所述抗体包括选自下列至少之一的CDR序列：
    轻链可变区CDR序列：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和SEQ ID NO:85；
    重链可变区CDR序列：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和SEQ ID NO:91。
  5. 根据权利要求4所述的抗体，其特征在于，所述轻链可变区CDR序列包括与SEQ ID NO:1～3、SEQ ID NO:7～9和SEQ ID NO:83～85具有至少85％相似性的氨基酸序列中的至少之一；
    所述重链可变区CDR序列包括与SEQ ID NO:4～6、SEQ ID NO:10～12、SEQ ID NO:86～88和SEQ ID NO:89～91具有至少85％相似性的氨基酸序列中的至少之一。
  6. 根据权利要求4所述的抗体，其特征在于，所述抗体包括：
    分别如SEQ ID NO:1、2和3或者与SEQ ID NO:1、2和3具有至少85％相似性的氨基酸序列所示的轻链可变区CDR1、 CDR2、CDR3序列；或者
    分别如SEQ ID NO:7、8和9或者与SEQ ID NO:7、8和9具有至少85％相似性的氨基酸序列所示的轻链可变区CDR1、CDR2、CDR3序列；或者
    分别如SEQ ID NO:83、84和85或者与SEQ ID NO:83、84和85具有至少85％相似性的氨基酸序列所示的轻链可变区CDR1、CDR2、CDR3序列。
  7. 根据权利要求4所述的抗体，其特征在于，所述抗体包括：
    分别如SEQ ID NO:4、5和6或者与SEQ ID NO:4、5和6具有至少85％相似性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列；或者
    分别如SEQ ID NO:10、11和12或者与SEQ ID NO:10、11和12具有至少85％相似性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列；或者
    分别如SEQ ID NO:86、87和88或者与SEQ ID NO:86、87和88具有至少85％相似性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列；或者
    分别如SEQ ID NO:89、90和91或者与SEQ ID NO:89、90和91具有至少85％相似性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列。
  8. 根据权利要求4所述的抗体，其特征在于，所述抗体包括：
    分别如SEQ ID NO:1、2和3或者与SEQ ID NO:1、2和3具有至少85％相似性的氨基酸序列所示的轻链可变区CDR1、CDR2、CDR3序列，以及分别如SEQ ID NO:4、5和6或者与SEQ ID NO:4、5和6具有至少85％相似性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列；或者
    分别如SEQ ID NO:7、8和9或者与SEQ ID NO:7、8和9具有至少85％相似性的氨基酸序列所示的轻链可变区CDR1、CDR2、CDR3序列，以及分别如SEQ ID NO:10、11和12或者与SEQ ID NO:10、11和12具有至少85％相似性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列；或者
    分别如SEQ ID NO:83、84和85或者与SEQ ID NO:83、84和85具有至少85％相似性的氨基酸序列所示的轻链可变区CDR1、CDR2、CDR3序列，以及分别如SEQ ID NO:86、87和88或者与SEQ ID NO:86、87和88具有至少85％相似性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列；或者
    分别如SEQ ID NO:83、84和85或者与SEQ ID NO:83、84和85具有至少85％相似性的氨基酸序列所示的轻链可变区CDR1、CDR2、CDR3序列，以及分别如SEQ ID NO:89、90和91或者与SEQ ID NO:89、90和91具有至少85％相似性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列。
  9. 根据权利要求1～8任一项所述的抗体，其特征在于，含有：
    重链骨架区序列和轻链骨架区序列中的至少之一；
    其中，所述重链骨架区序列和轻链骨架区序列的至少之一的至少一部分来自于鼠源抗体、人源抗体、灵长目源抗体或其突变体中的至少之一，优选为人源抗体。
  10. 根据权利要求1～8任一项所述的抗体，其特征在于，所述抗体具有如SEQ ID NO:13、SEQ ID NO:14或SEQ ID NO:95所示氨基酸序列的轻链可变区；和/或者，
    所述抗体具有如SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:96或SEQ ID NO:97所示氨基酸序列的重链可变区。
  11. 根据权利要求10所述的抗体，其特征在于，所述抗体具有如SEQ ID NO:13所示氨基酸序列的轻链可变区和如SEQ ID NO:15所示氨基酸序列的重链可变区；或者
    所述抗体具有如SEQ ID NO:14所示氨基酸序列的轻链可变区和如SEQ ID NO:16所示氨基酸序列的重链可变区；或者
    所述抗体具有如SEQ ID NO:95所示氨基酸序列的轻链可变区和如SEQ ID NO:96所示氨基酸序列的重链可变区；或者
    所述抗体具有如SEQ ID NO:95所示氨基酸序列的轻链可变区和如SEQ ID NO:97所示氨基酸序列的重链可变区。
  12. 根据权利要求1～8任一项所述的抗体，其特征在于，所述抗体含有重链恒定区和轻链恒定区的至少之一，所述重链恒定区和轻链恒定区的至少之一的至少一部分来自于鼠源抗体、人源抗体、灵长目源抗体或其突变体的至少之一。
  13. 根据权利要求12所述的抗体，其特征在于，所述重链恒定区和轻链恒定区的至少之一的至少一部分来自于人源抗体。
  14. 根据权利要求1～8任一项所述的抗体，其特征在于，所述抗体具有如SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:33或SEQ ID NO:35所示氨基酸序列的重链和具有如SEQ ID NO:19、SEQ ID NO:20或SEQ ID NO:34所示氨基酸序列的轻 链。
  15. 根据权利要求14所述的抗体，其特征在于，所述抗体具有如SEQ ID NO:17所示氨基酸序列的重链和具有如SEQ ID NO:19所示氨基酸序列的轻链；或者
    所述抗体具有如SEQ ID NO:18所示氨基酸序列的重链和具有如SEQ ID NO:20所示氨基酸序列的轻链；或者
    所述抗体具有如SEQ ID NO:33所示氨基酸序列的重链和具有如SEQ ID NO:34所示氨基酸序列的轻链；或者
    所述抗体具有如SEQ ID NO:35所示氨基酸序列的重链和具有如SEQ ID NO:34所示氨基酸序列的轻链。
  16. 一种核酸分子，其特征在于，所述核酸分子编码权利要求1～15任一项所述的抗体。
  17. 根据权利要求16所述的核酸分子，其特征在于，所述核酸分子为DNA。
  18. 一种表达载体，其特征在于，携带权利要求16或17所述的核酸分子。
  19. 根据权利要求18所述的表达载体，其特征在于，所述表达载体为真核表达载体或原核表达载体。
  20. 根据权利要求19所述的表达载体，其特征在于，所述表达载体为慢病毒载体或质粒表达载体。
  21. 一种重组细胞，其特征在于，所述重组细胞包括：
    携带权利要求16或17所述的核酸分子；或，
    表达权利要求1～15任一项所述的抗体。
  22. 根据权利要求21所述的重组细胞，其特征在于，所述重组细胞是通过将权利要求18～20任一项所述的表达载体引入至宿主细胞中而获得的。
  23. 根据权利要求21或22所述的重组细胞，其特征在于，所述重组细胞为真核细胞。
  24. 根据权利要求23所述的重组细胞，其特征在于，所述重组细胞为哺乳动物细胞。
  25. 权利要求1～15任一项所述的抗体在制备药物中的用途。
  26. 根据权利要求25所述的用途，其特征在于，所述药物包括融合蛋白或ADC药物。
  27. 权利要求1～15任一项所述的抗体在作为药物载体或阴性抗体中的用途。
  28. 根据权利要求27所述的用途，其特征在于，所述药物载体或阴性抗体无抗原结合能力。
  29. 一种融合蛋白，其特征在于，包括：
    中性抗体，所述中性抗体包括抗体FC区段或权利要求1～15任一项所述的抗体；
    一个或多个PLA2R抗原表位肽，所述PLA2R抗原表位肽与所述中性抗体相连。
  30. 根据权利要求29所述的融合蛋白，其特征在于，所述PLA2R抗原表位肽的C端与所述中性抗体的N端相连。
  31. 根据权利要求30所述的融合蛋白，其特征在于，所述PLA2R抗原表位肽包括CysR、FnII、以及PLA2R结合区1和/或PLA2R结合区2。
  32. 根据权利要求31所述的融合蛋白，其特征在于，所述CysR的C端与所述FnII的N端相连，所述FnII的C端与所述PLA2R结合区1的N端相连。
  33. 根据权利要求32所述的融合蛋白，其特征在于，所述CysR的C端与所述FnII的N端相连，所述FnII的C端与所述PLA2R结合区1的N端相连，所述PLA2R结合区1的C端与PLA2R结合区2的N端相连。
  34. 根据权利要求30所述的融合蛋白，其特征在于，所述PLA2R抗原表位肽具有如SEQ ID NO:21或22所示氨基酸序列或者与SEQ ID NO:21或22具有至少95％相似性的氨基酸序列。
  35. 根据权利要求29～34任一项所述的融合蛋白，其特征在于，所述中性抗体为抗体FC区段，所述PLA2R抗原表位肽的C端与所述抗体FC区段的至少一条的N端相连。
  36. 根据权利要求35所述的融合蛋白，其特征在于，所述中性抗体为权利要求1～15任一项所述的抗体，所述PLA2R抗原表位肽的C端与所述抗体的至少一条轻链或重链的N端相连。
  37. 根据权利要求29～34任一项所述的融合蛋白，其特征在于，所述中性抗体为双链抗体，所述抗体FC区段的每条链的N端分别与一个PLA2R抗原表位肽的C端相连。
  38. 根据权利要求37所述的融合蛋白，其特征在于，权利要求1～15任一项所述的抗体的每条轻链的N端分别与一个PLA2R抗原表位肽的C端相连。
  39. 根据权利要求38所述的融合蛋白，其特征在于，权利要求1～15任一项所述的抗体的每条重链的N端分别与一个PLA2R抗原表位肽的C端相连。
  40. 根据权利要求39所述的融合蛋白，其特征在于，权利要求1～15任一项所述的抗体的每条重链、每条轻链的N端分别与一个PLA2R抗原表位肽的C端相连。
  41. 根据权利要求29～34任一项所述的融合蛋白，其特征在于，进一步包括连接肽，所述连接肽设置于所述PLA2R抗原表位肽与所述抗体之间。
  42. 根据权利要求41所述的融合蛋白，其特征在于，所述连接肽的N端与所述PLA2R抗原表位肽的C端相连，所述连接肽的C端与所述中性抗体的N段相连。
  43. 根据权利要求42所述的融合蛋白，其特征在于，所述连接肽的氨基酸序列为(GGGGS)n，其中n为大于或等于1的整数。
  44. 根据权利要求43所述的融合蛋白，其特征在于，n为1、2、3、4、5、6、7、8、9或10。
  45. 根据权利要求41所述的融合蛋白，其特征在于，所述连接肽包括SEQ ID NO:23所示的氨基酸序列。
  46. 根据权利要求29～34任一项所述的融合蛋白，其特征在于，所述抗体FC区段具有如SEQ ID NO:24或者与SEQ ID NO:24具有至少90％相似性的氨基酸序列。
  47. 根据权利要求29～34任一项所述的融合蛋白，其特征在于，所述融合蛋白具有如SEQ ID NO:57所示氨基酸序列的第一肽段和具有如SEQ ID NO:19所示氨基酸序列的第二肽段；或者
    所述融合蛋白具有如SEQ ID NO:17所示氨基酸序列的第一肽段和具有如SEQ ID NO:59所示氨基酸序列的第二肽段；或者
    所述融合蛋白的氨基酸序列如SEQ ID NO:61所示；或者
    所述融合蛋白具有如SEQ ID NO:63所示氨基酸序列的第一肽段和具有如SEQ ID NO:19所示氨基酸序列的第二肽段；或者
    所述融合蛋白具有如SEQ ID NO:17所示氨基酸序列的第一肽段和具有如SEQ ID NO:65所示氨基酸序列的第二肽段；或者
    所述融合蛋白的氨基酸序列如SEQ ID NO:67所示；或者
    所述融合蛋白具有如SEQ ID NO:57所示氨基酸序列的第一肽段和具有如SEQ ID NO:59所示氨基酸序列的第二肽段；或者
    所述融合蛋白具有如SEQ ID NO:63所示氨基酸序列的第一肽段和具有如SEQ ID NO:65所示氨基酸序列的第二肽段。
  48. 一种核酸分子，其特征在于，所述核酸分子编码权利要求29～47任一项所述的融合蛋白。
  49. 根据权利要求48所述的核酸分子，其特征在于，所述核酸分子为DNA。
  50. 一种表达载体，其特征在于，携带权利要求48或49所述的核酸分子。
  51. 根据权利要求50所述的表达载体，其特征在于，所述表达载体为真核表达载体或原核表达载体。
  52. 根据权利要求51所述的表达载体，其特征在于，所述表达载体为慢病毒载体或质粒表达载体。
  53. 一种重组细胞，其特征在于，所述重组细胞包括：
    携带权利要求48或49所述的核酸分子；或，
    表达权利要求29～47任一项所述的融合蛋白。
  54. 根据权利要求53所述的重组细胞，其特征在于，所述重组细胞是通过将权利要求50～52任一项所述的表达载体引入至宿主细胞中而获得的。
  55. 根据权利要求53或54所述的重组细胞，其特征在于，所述重组细胞为真核细胞。
  56. 根据权利要求55所述的重组细胞，其特征在于，所述重组细胞为哺乳动物细胞。
  57. 一种药物组合物，其特征在于，包括：
    权利要求29～47任一项所述的融合蛋白。
  58. 根据权利要求57所述的药物组合物，其特征在于，进一步包括药学上可接受的辅料。
  59. 一种免疫吸附剂，其特征在于，包括：
    权利要求29～47任一项所述的融合蛋白。
  60. 一种试剂盒，其特征在于，包括：
    权利要求29～47任一项所述的融合蛋白或权利要求59所述的免疫吸附剂。
  61. 一种吸附产品，其特征在于，包括：
    权利要求59所述的免疫吸附剂；
    固相载体，所述免疫吸附剂与固相载体相连。
  62. 根据权利要求61所述的吸附产品，其特征在于，所述吸附产品选自免疫吸附柱或血液净化剂。
  63. 权利要求59所述的免疫吸附剂在制备吸附产品中的用途，所述吸附产品用于吸附PLA2R抗体。
  64. 根据权利要求63所述的用途，其特征在于，所述吸附产品选自免疫吸附柱或血液净化剂。
  65. 根据权利要求64所述的用途，其特征在于，所述吸附产品为免疫吸附柱，所述吸附产品用于预防或治疗PLA2R抗体引起的相关疾病。
  66. 根据权利要求65所述的用途，其特征在于，所述PLA2R抗体引起的相关疾病选自PLA2R抗体阳性膜性肾病。
  67. 根据权利要求64所述的用途，其特征在于，所述吸附产品为血液净化剂，所述吸附产品用于吸附血液或血浆中的PLA2R抗体。
  68. 权利要求29～47任一项所述的融合蛋白、权利要求48～49任一项所述的核酸分子、权利要求50～52任一项所述的表达载体、权利要求53～56任一项所述的重组细胞、权利要求57～58任一项所述的药物组合物或权利要求59所述的免疫吸附剂在制备药物中的用途，所述药物用于预防或治疗PLA2R抗体引起的相关疾病。
  69. 根据权利要求68所述的用途，其特征在于，所述PLA2R抗体引起的相关疾病选自PLA2R抗体阳性膜性肾病。
  70. 权利要求29～47任一项所述的融合蛋白、权利要求48～49任一项所述的核酸分子、权利要求50～52任一项所述的表达载体、权利要求53～56任一项所述的重组细胞或权利要求57～58任一项所述的药物组合物在制备免疫吸附剂中的用途，所述免疫吸附剂用于吸附PLA2R抗体。
  71. 权利要求29～47任一项所述的融合蛋白、权利要求48～49任一项所述的核酸分子、权利要求50～52任一项所述的表达载体、权利要求53～56任一项所述的重组细胞、权利要求57～58任一项所述的药物组合物或权利要求59所述的免疫吸附剂在制备试剂盒中的用途，所述试剂盒用于检测PLA2R抗体。
  72. 一种预防或治疗PLA2R抗体引起的相关疾病的方法，其特征在于，包含：
    向受试者施用药学上可接受量的权利要求29～47任一项所述的融合蛋白或权利要求57～58任一项所述的药物组合物。
  73. 根据权利要求72所述的方法，其特征在于，所述方法的给药途径采用皮下注射或静脉注射。
  74. 根据权利要求73所述的方法，其特征在于，所述PLA2R抗体引起的相关疾病选自PLA2R抗体阳性膜性肾病。
  75. 一种用于预防或治疗PLA2R抗体引起的相关疾病的免疫吸附治疗方法，其特征在于，包含：
    采用权利要求59所述的免疫吸附剂对受试者进行治疗。
  76. 根据权利要求75所述的方法，其特征在于，采用体外血液净化对受试者进行治疗。
  77. 根据权利要求75所述的方法，其特征在于，所述PLA2R抗体引起的相关疾病选自PLA2R抗体阳性膜性肾病。
  78. 一种诊断PLA2R抗体引起的相关疾病的方法，其特征在于，包括：
    采用权利要求29～47任一项所述的融合蛋白、权利要求48～49任一项所述的核酸分子、权利要求50～52任一项所述的表达载体、权利要求53～56任一项所述的重组细胞、权利要求57～58任一项所述的药物组合物、权利要求59所述的免疫吸附剂或权利要求60所述的试剂盒对待测样品中的PLA2R抗体进行检测；
    基于检测结果，确定待测样品中的PLA2R抗体的含量。
  79. 根据权利要求78所述的方法，其特征在于，所述待测样品中PLA2R抗体的含量不低于14RU/ml是待测样品来源于患有PLA2R抗体引起的相关疾病的患者的指示。
  80. 根据权利要求79所述的方法，其特征在于，所述待测样品中PLA2R抗体的含量不低于20RU/ml是待测样品来源于患有PLA2R抗体引起的相关疾病的患者的指示。
  81. 根据权利要求78～80任一项所述的方法，其特征在于，所述待测样品为血液。
  82. 根据权利要求78～80任一项所述的方法，其特征在于，所述PLA2R抗体引起的相关疾病选自PLA2R抗体阳性膜性肾病。
  83. 一种评估PLA2R抗体引起的相关疾病分期的方法，其特征在于，包括：
    采用权利要求29～47任一项所述的融合蛋白、权利要求48～49任一项所述的核酸分子、权利要求50～52任一项所述的表达载体、权利要求53～56任一项所述的重组细胞、权利要求57～58任一项所述的药物组合物、权利要求59所述的免疫吸附剂或权利要求60所述的试剂盒对待测样品中的PLA2R抗体进行检测，所述待测样品来源于待测患者；
    基于检测结果，确定待测样品中的PLA2R抗体的含量。
  84. 根据权利要求83所述的方法，其特征在于，基于待测样品中的PLA2R抗体的含量，确定待测患者患有PLA2R抗体引起的相关疾病的分期。
  85. 根据权利要求83或84所述的方法，其特征在于，所述PLA2R抗体引起的相关疾病选自PLA2R抗体阳性膜性肾病。
  86. 根据权利要求83或84所述的方法，其特征在于，所述待测样品为血液。
  87. 一种评估PLA2R抗体引起的相关疾病的预后方法，其特征在于，包括：
    采用权利要求29～47任一项所述的融合蛋白、权利要求48～49任一项所述的核酸分子、权利要求50～52任一项所述的表达载体、权利要求53～56任一项所述的重组细胞、权利要求57～58任一项所述的药物组合物、权利要求59所述的免疫吸附剂或权利要求60所述的试剂盒对待测样品中的PLA2R抗体进行检测；
    基于检测结果，确定待测样品中的PLA2R抗体的含量。
  88. 根据权利要求87所述的方法，其特征在于，所述待测样品来源于治疗前或后的患有PLA2R抗体引起的相关疾病的患者。
  89. 根据权利要求87所述的方法，其特征在于，基于治疗前或后的患有PLA2R抗体引起的相关疾病的患者的待测样品中的PLA2R抗体的含量，确定PLA2R抗体引起的相关疾病的预后效果。
  90. 根据权利要求87所述的方法，其特征在于，治疗后的患有PLA2R抗体引起的相关疾病的患者的待测样品中的PLA2R抗体的含量下降是患者预后良好的指示。
  91. 根据权利要求87～90任一项所述的方法，其特征在于，所述待测样品为血液。
  92. 根据权利要求87～90任一项所述的方法，其特征在于，所述PLA2R抗体引起的相关疾病选自PLA2R抗体阳性膜性肾病。
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