Human anti-human PLA2R antibody standard and its use

Abstract

The present invention provides a human anti-human PLA2R antibody or antigen-binding fragment thereof, which comprises a CDR sequence selected from at least one of a heavy chain variable region CDR sequence selected from SEQ ID NOs: 1 to 105 and a light chain variable region CDR sequence selected from SEQ ID NOs: 106 to 210, or an amino acid sequence having at least 80% identity thereto.

Description

[Technical Field]

[0001] The present invention relates to the field of bioengineering. Specifically, the present invention relates to a human anti-human PLA2R antibody standard and its use. More specifically, the present invention relates to an anti-human PLA2R antibody or its antigen-binding fragment, a nucleic acid molecule, an expression vector, a recombinant cell, a kit, and uses thereof. [Background technology]

[0002] Primary membranous nephropathy (PMN) is an important cause of renal failure and a major cause of nephrotic syndrome. While spontaneous remission may occur in less than 30% of cases, another 30% experience gradual progression of renal function over approximately 10 years, leading to renal failure and posing a serious threat to people's lives and health. Recent basic research has confirmed that PMN is an autoimmune disease, in which the body's autoimmune antigens activate the immune system under disease conditions, resulting in the production of autoimmune antibodies. Autoimmune antibodies bind to autoimmune antigens on glomerular podocytes, causing the immune system to attack the podocytes and surrounding tissues, resulting in inflammation and glomerular damage.

[0003] Current research has relatively clear evidence that primary membranous nephropathy autoimmune antigens are PLA2R and THSD7A. Approximately 70% to 80% of primary membranous nephropathy cases are PLA2R-type, while less than 10% are THSD7A-type.

[0004] PLA2R antibodies (PLA2R-Ab) play an important role in the development and progression of PLA2R membranous nephropathy. Serum PLA2R-Ab levels can be used as a predictive marker for PLA2R membranous nephropathy. High PLA2R-Ab titers indicate a lower rate of spontaneous remission. A decrease in PLA2R-Ab titers often accompanies spontaneous remission, reaching undetectable levels in patients with complete disease remission. Immunosuppressive treatment can reduce PLA2R-Ab titers.

[0005] PLA2R antibody concentrations can be qualitatively or quantitatively detected using diagnostic kits. PLA2R antibody diagnostic kits typically use immunological detection methods, such as ELISA, to detect antibody signals in samples and compare them with those of antibody standards to obtain relative concentrations. The standard used in current PLA2R antibody diagnostic kits is diluted human serum, but diluted human serum as a standard has several major drawbacks. First, human serum standards are obtained relative to the antibody concentration in standard human serum, meaning that absolute quantification of the antibody concentration in plasma is impossible; only relative units (RU) are obtained. This limits their value in clinical research and treatment, as well as in drug and therapeutic development. Second, while human serum standards have been used to detect several viruses, such as HIV and HCV, contamination by other viruses and pathogenic microorganisms cannot be ruled out, posing a risk of pathogen contamination. Finally, standards prepared from human serum are subject to limited sources, high production costs, and lot-to-lot variations due to variations in antibody production among different patients.

[0006] Therefore, there is an urgent need for a PLA2R antibody standard that is easy to prepare and less prone to lot-to-lot variations. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention aims to solve at least one of the technical problems present in the prior art to a certain extent, and therefore provides an anti-human PLA2R antibody or antigen-binding fragment thereof, which has high affinity for PLA2R protein, and has advantages such as easy preparation and little lot-to-lot variation, and can be used as an anti-human PLA2R antibody standard. [Means for solving the problem]

[0008] The present invention has been completed based on the following discoveries made by the inventors.

[0009] PLA2R is a membrane protein highly expressed on the surface of glomerular podocytes and has the structural domains shown in Figure 1. Antibodies in patient plasma primarily recognize three structural domains in PLA2R: CysR, FNII, and CTLD1. To this end, the inventors designed and recombinantly expressed these three structural domains, used them as antigens, and screened human antibodies targeting these three structural domains using a humanized recombinant phage display library. These antibodies were then used as anti-human PLA2R antibody standards, calibrated with commercially available diagnostic kits, and a correlation was established between the concentrations of the anti-human PLA2R antibody standards and those in human serum. Furthermore, the antibodies were obtained by recombinant expression in mammalian cells, enabling stable and mass production, thereby improving the production volume and quality of standard antibodies, reducing production costs, and eliminating the risk of pathogenic microbial contamination.

[0010] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof. According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises a CDR sequence selected from at least one of heavy chain variable region CDR sequences: SEQ ID NOs: 1-105 and light chain variable region CDR sequences: SEQ ID NOs: 106-210, or an amino acid sequence having at least 80% identity thereto. The antibody or antigen-binding fragment thereof has high affinity for PLA2R protein and can be used as an anti-human PLA2R antibody standard. The antibody or antigen-binding fragment thereof is obtained by recombinant expression in cells, which has advantages such as stability and mass production, and also eliminates the possibility of contamination with pathogenic microorganisms during preparation, thereby avoiding lot-to-lot variations and ensuring the purity and stability of the anti-human PLA2R antibody standard.

[0011] In another aspect, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the above-described antibody or antigen-binding fragment thereof. The nucleic acid molecule described in the present invention can effectively identify the above-described antibody or antigen-binding fragment thereof.

[0012] In a further aspect, the present invention provides an expression vector, which, according to an embodiment of the present invention, carries the nucleic acid molecule described above. The expression vector described in the present invention can effectively detect the antibody or antigen-binding fragment thereof described above.

[0013] In a further aspect, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell harbors the nucleic acid molecule described above or expresses the antibody or antigen-binding fragment thereof described above. The recombinant cell according to the embodiment of the present invention can be used for the in vitro expression and large-scale production of the antibody or antigen-binding fragment thereof described above.

[0014] In a further aspect of the present invention, the present invention provides the use of the aforementioned antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell as a PLA2R antibody standard. As can be seen from the above, the aforementioned antibody or antigen-binding fragment thereof has high affinity for PLA2R protein and is obtained by recombinant expression in mammalian cells, offering advantages such as stability and mass production, thereby eliminating the possibility of contamination with pathogenic microorganisms during preparation. This allows the aforementioned antibody or antigen-binding fragment thereof to be used as an anti-human PLA2R antibody standard, improving the accuracy of PLA2R antibody detection.

[0015] In a further aspect, the present invention provides a kit. According to an embodiment of the present invention, the kit includes the aforementioned antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell. As can be seen from the above, the aforementioned antibody or antigen-binding fragment thereof has high affinity for PLA2R protein, can be produced stably and in large quantities, can be free from contamination by pathogenic microorganisms, and can be used as an anti-human PLA2R antibody standard. Therefore, a kit including the aforementioned antibody or antigen-binding fragment thereof has advantages such as high accuracy in detecting PLA2R antibodies.

[0016] In a further aspect of the present invention, the present invention provides the use of the aforementioned antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell in the preparation of a kit for detecting PLA2R antibodies or diagnosing PLA2R antibody-positive membranous nephropathy. As can be seen from the above, the aforementioned antibody or antigen-binding fragment thereof has high affinity for PLA2R protein, can be produced stably and in large quantities, can be free from contamination by pathogenic microorganisms, and can be used as an anti-human PLA2R antibody standard. Therefore, a kit containing the aforementioned antibody or antigen-binding fragment thereof has advantages such as high accuracy in detecting PLA2R antibodies.

[0017] In a further aspect of the present invention, the present invention provides the use of the aforementioned antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell for detecting PLA2R antibodies or diagnosing PLA2R antibody-positive membranous nephropathy. As can be seen from the above, the aforementioned antibody or antigen-binding fragment thereof has high affinity for PLA2R protein, can be produced stably and in large quantities, can be free from contamination by pathogenic microorganisms, and can be used as an anti-human PLA2R antibody standard. Therefore, kits containing the aforementioned antibody or antigen-binding fragment thereof have advantages such as high accuracy in detecting PLA2R antibodies.

[0018] In a further aspect of the present invention, the present invention provides the aforementioned antibody or its antigen-binding fragment, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell for detecting PLA2R antibodies or diagnosing PLA2R antibody-positive membranous nephropathy. As can be seen from the above, the aforementioned antibody or its antigen-binding fragment has high affinity for PLA2R protein, can be produced stably and in large quantities, can be free from contamination by pathogenic microorganisms, and can be used as an anti-human PLA2R antibody standard. Therefore, a kit containing the aforementioned antibody or its antigen-binding fragment has advantages such as high accuracy in detecting PLA2R antibodies.

[0019] In a further aspect, the present invention provides a method for detecting PLA2R antibodies. According to an embodiment of the present invention, the method includes determining the content of PLA2R antibodies in a sample to be measured based on the results of PLA2R antibody detection using the aforementioned antibody or its antigen-binding fragment as a PLA2R antibody standard. This improves the accuracy of PLA2R antibody detection. As can be seen from the above, the aforementioned antibody or its antigen-binding fragment has high affinity for PLA2R protein, can be produced stably and in large quantities, and can be used as an anti-human PLA2R antibody standard, eliminating contamination with pathogenic microorganisms. Therefore, the method of the present invention has advantages such as high accuracy in detecting PLA2R antibodies.

[0020] In a further aspect, the present invention provides a method for diagnosing PLA2R antibody-positive membranous nephropathy. According to an embodiment of the present invention, the method includes the steps of: determining the PLA2R antibody content in the test sample based on the PLA2R antibody detection result in the test sample using the aforementioned antibody or its antigen-binding fragment as a PLA2R antibody standard; and determining whether the patient corresponding to the test sample has PLA2R antibody-positive membranous nephropathy based on the PLA2R antibody content. This improves the accuracy of PLA2R antibody detection. As can be seen from the above, the aforementioned antibody or its antigen-binding fragment has high affinity for PLA2R protein, can be produced stably and in large quantities, and can be used as an anti-human PLA2R antibody standard to accurately detect PLA2R antibody content. Therefore, the method of the present invention has high accuracy in diagnosing PLA2R antibody-positive membranous nephropathy.

[0021] Additional aspects and advantages of the invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the invention.

[0022] The amino acid sequence listing of the present invention is shown below. [Table 0-01] [Table 0-02] [Table 0-03] [Table 0-04] [Table 0-05] [Table 0-06] [Table 0-07] [Table 0-08] [Table 0-09] [Table 0-10]

[0023] The nucleotide sequence listing of the present invention is shown below. [Table 0-11] [Table 0-12] [Table 0-13] [Table 0-14]

Table 0-15

Table 0-18

Table 0-19

Table 0-20

Table 0-23

Table 0-25

Table 0-27

[0024] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the following detailed description of the embodiments taken in conjunction with the drawings. [Figure 1] FIG. 1 is a schematic diagram of the structure of PLA2R in the present invention. [Figure 2] FIG. 1 is a schematic diagram showing the structure of an expression framework in Example 1 of the present invention. [Figure 3] 1 shows SDS-PAGE analysis of CC1H in Example 1 of the present invention, where lane 1 is a molecular weight reference, lane 2 is purified CC1H non-reduced, and lane 3 is purified CC1H reduced. [Figure 4A] 1 shows the results of the binding activity of each antibody to CC1h in Example 3 of the present invention. [Figure 4B] 1 shows the results of the binding activity of each antibody to CC1h in Example 3 of the present invention. [Figure 4C] 1 shows the results of the binding activity of each antibody to CC1h in Example 3 of the present invention. [Figure 5A] 1 shows the results of the binding activity of each antibody to CC1h in Example 3 of the present invention. [Figure 5B] 1 shows the results of the binding activity of each antibody to CC1h in Example 3 of the present invention. [Figure 6]1 is a protein standard curve (y=0.6849x+0.6325, R2=0.9925) measured by the Bradford method in Example 4 of the present invention. [Figure 7] FIG. 1 is a diagram showing the purification effect of CC1H-Biotin detected by SDS-PAGE in Example 4 of the present invention, where lane 1 is CC1H-Biotin in a DTT-containing reducing sample buffer, and lane 2 is CC1H-Biotin in a non-reducing sample buffer. [Figure 8] 1 shows standard curves prepared after diluting the A13 (P59368) and A13SP (P62297) antibodies in Example 5 of the present invention to a concentration equivalent to that of a standard at 1 RU / mL = 70 ng / mL. [Figure 9A] 1 shows standard curves prepared after diluting the nine antibodies in Example 5 of the present invention to a concentration equivalent to that of a standard at 1 RU / mL = 70 ng / mL. [Figure 9B] 1 shows a standard curve prepared after diluting the SP modified antibodies of the nine antibodies in Example 5 of the present invention to a concentration equivalent to that of the standard at 1 RU / mL = 70 ng / mL. DETAILED DESCRIPTION OF THE INVENTION

[0025] The following detailed description of the present invention will be given with reference to the examples. The examples described below are merely illustrative and are used to explain the present invention, but are not intended to limit the present invention.

[0026] It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be understood as expressing or implying relative importance or the number of technical features being presented. Thus, a feature qualified by "first" or "second" may explicitly or implicitly include one or more of the feature. Furthermore, in the description of the present invention, "plurality" means two or more than two, unless otherwise specified.

[0027] The endpoints of ranges and any value disclosed herein should be understood to be not limited to such exact ranges or values, but to include values ​​close to those ranges or values. In the case of ranges of numerical values, values ​​between the endpoints of each range, between each endpoint and any single point value, and between any single point value may be combined with each other to form one or more new numerical ranges, and these numerical ranges are deemed to be specifically disclosed in the specification.

[0028] To facilitate understanding of the present invention, certain technical and scientific terms are defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the meaning commonly understood by those of ordinary skill in the art. Abbreviations for amino acid residues refer to the standard three-letter and / or one-letter codes used in the art to denote one of the 20 commonly used L-amino acids.

[0029] In this specification, the terms "comprise" or "include" are non-limiting expressions, i.e., they include the contents shown in the present invention but do not exclude the contents of other embodiments.

[0030] As used herein, the terms "selectably," "selectable," "optionally," "any," or "optional" generally mean that a described event or circumstance may occur, but does not necessarily occur, and the description includes the occurrence of the event or circumstance and the absence of the event or circumstance.

[0031] As used herein, the terms "identity," "homology," or "similarity," when describing an amino acid sequence or a nucleic acid sequence relative to a reference sequence, refer to the conventional method of determining the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences, as described, for example, in 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 Institute)). Foundation, Washington, DC. Algorithms for comparing sequences and determining sequence identity include the homology comparison algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443, the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482, the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444, the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997)), and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410).Computer programs utilizing these algorithms are also available, and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)), or GAP, BESTFIT, BLAST Altschul, etc., FASTA, and TFASTA are available in the Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA, and in the CLUSTAL in PC / Gene program provided by Intelligenetics, Mountain View, California.

[0032] Those skilled in the art can obtain variants of the antibody or antigen-binding fragment sequences of the present invention by substituting, adding, and / or deleting one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the sequences of the present invention, provided that the antibody activity (retaining at least 90% activity) is not substantially affected. These are considered to be within the scope of protection of the present invention. For example, amino acids with similar properties may be substituted in the variable region. The variant sequences described in the present invention may have at least 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence. Sequence identity described in the present invention can be determined using sequence analysis software, such as the computer program BLAST, particularly BLASTP or TBLASTN, with default parameters. All amino acid sequences referred to in the present invention are presented from the N-terminus to the C-terminus.

[0033] As used herein, the term "at least 80% homology" refers to having at least 80% homology with the respective reference sequence, which may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% homology. The term "at least 90% homology" refers to having at least 90% homology with the respective reference sequence, which may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% homology.

[0034] As used herein, the term "mutant" or "variant" refers to any naturally occurring or artificially modified molecule that contains one or more nucleotide or amino acid mutations.

[0035] As used herein, the term "expression vector" generally refers to a nucleic acid molecule that can be inserted into an appropriate host and self-replicate, transferring the inserted nucleic acid molecule into and / or between host cells. Expression vectors include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcribing and / or translating DNA or RNA. Expression vectors further include vectors with various of the above functions. The expression vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into an appropriate host cell. Generally, the expression vector can produce a desired expression product by culturing an appropriate host cell containing the expression vector.

[0036] As used herein, the term "recombinant cell" refers to a cell that has a unique trait with stable inheritance, typically by modifying or recombining the genetic material of a host cell using genetic engineering or cell fusion techniques. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transformation" and "transfect" as used herein refer to the introduction of a nucleic acid (e.g., a vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of the present invention and used to express and / or secrete target proteins. Examples of suitable host cells that can be used in the present invention include permanent hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, CaP cells (human amniotic fluid-derived cells), and CoS cells.

[0037] The present invention provides an anti-human PLA2R antibody or its antigen-binding fragment, a nucleic acid molecule, an expression vector, a recombinant cell, a kit, and uses thereof, each of which is described in detail below.

[0038] Antibodies or antigen-binding fragments thereof

[0039] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof. According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises a CDR sequence selected from at least one of heavy chain variable region CDR sequences: SEQ ID NOs: 1-105 and light chain variable region CDR sequences: SEQ ID NOs: 106-210, or an amino acid sequence having at least 80% identity thereto. The antibody or antigen-binding fragment thereof has high affinity for PLA2R protein and can be used as an anti-human PLA2R antibody standard. The antibody or antigen-binding fragment thereof is obtained by recombinant expression in cells, which has advantages such as stability and mass production, and eliminates the possibility of contamination with pathogenic microorganisms during preparation, thereby ensuring the purity and stability of the anti-human PLA2R antibody standard.

[0040] As used herein, the term "antibody" is used in the broadest sense and can encompass full-length monoclonal antibodies, multispecific antibodies, and chimeric antibodies, without any specific structural limitations, as long as they exhibit the desired biological activity. Antibody molecules typically comprise a light chain with a low molecular weight and a heavy chain with a high molecular weight, with the heavy (H) and light (L) chains connected by disulfide bonds. The amino acid sequence at the amino terminus (N-terminus) of the peptide chain varies significantly and is called the variable region (V region), while the carboxyl terminus (C-terminus) is relatively stable and less variable and is called the constant region (C region). The V regions of the L chain and H chain are called VL and VH, respectively, and both VL and VH contain the regions of major amino acid residues responsible for binding affinity to a recognized antigen or epitope, i.e., CDRs.

[0041] As used herein, the term "complementarity determining region," "CDR," or "CDRs" refers to the highly variable regions of immunoglobulin heavy and light chains and refers to the region of major amino acid residues responsible for the binding affinity of one, more, or all antibodies or antigen-binding fragments thereof to their recognized antigen or epitope.

[0042] As used herein, heavy chain complementarity determining regions (heavy chain variable region CDRs) are referred to as "HCDRs" or "HCDRs" and include HCDR1 (also known as CDR-H1), HCDR2 (also known as CDR-H2), and HCDR3 (also known as CDR-H3); light chain complementarity determining regions (light chain variable region CDRs) are referred to as "LCDRs" or "LCDRs," and include LCDR1 (also known as CDR-L1), LCDR2 (also known as CDR-L2), and LCDR3 (also known as CDR-L3). Commonly used CDR definitions in the art include Kabat, Chothia, IMGT, Contact, and AbM. As used herein, "Kabat" refers to the definition system described in Kabat et al., U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). For the "Chothia definition," see Chothia et al., J Mol Biol 196:901-917 (1987). Exemplary defined CDRs are shown in Table A below. Definitions vary slightly depending on the literature. Given the amino acid sequence of an antibody variable region, one skilled in the art can routinely determine which residues comprise a particular CDR. The CDRs of the present invention include, but are not limited to, CDRs defined by other methods in Table A. CDRs determined using other rules disclosed in the art based on the heavy chain variable region and light chain variable region disclosed herein also fall within the scope of protection of the present disclosure.

[0043] [Table 0-A]

[0044] 1 The numbering of all CDR definitions in Table A follows the Kabat numbering system (see below), with amino acid numbers on the heavy chain designated as "H+number" and amino acid numbers on the light chain designated as "L+number."

[0045] 2"AbM" as used in Table A has a lower case "b" and refers to the CDRs as defined by Oxford Molecular's "AbM" antibody modeling software.

[0046] 3 When both H35A and H35B are absent, CDR-H1 ends at position 35; when only H35A is present, CDR-H1 ends at position 35A; when H35A and H35B are present together, CDR-H1 ends at position 35B.

[0047] 4 When both H35A and H35B are absent, CDR-H1 ends at position 32; when only H35A is present, CDR-H1 ends at position 33; and when both H35A and H35B are present, CDR-H1 ends at position 34.

[0048] Kabat et al. also define a numbering system that can be applied to any antibody variable region sequence. One skilled in the art can unambiguously apply the Kabat numbering system to any variable region sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to numbering based on the numbering system described in Kabat et al., U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). The HCDRs and LCDRs of the present antibody or antigen-binding fragment thereof are numbered using this numbering system, and specific numbering results are shown in Table A.

[0049] The CDRs of the present invention are labeled based on structural domain comparison (see, for example, Ehrenmann F, Kaas Q, Lefranc MP. IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF, and MhcSF. Nucleic Acids Res. 2010 Jan;38(Database issue):D301-7. PMID:19900967 and Ehrenmann F, Lefranc MP. IMGT / DomainGapAlign: IMGT standardized analysis of amino acid sequences of variable, constant, and groove domains (IG, TR, MH, IgSF, MhSF). Cold Spring Harb Protoc. 2011 Jun 1;2011(6):737-49. PMID:21632775). However, a person skilled in the art may completely convert and define the heavy and light chains in the sequence listing into other numbering systems (e.g., Kabat, Chothia, Contact, and AbM) based on the definition rules of Table A, and any CDRs obtained thereby are within the scope of the present invention.

[0050] As used herein, the terms "full length antibody," "full length monoclonal antibody," or "full length monoclonal antibody" all refer to an antibody that comprises at least two identical light chains and at least two identical heavy chains connected by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE).

[0051] As used herein, the terms "polyclonal antibody" and "multispecific antibody" are synonymous and refer to antibodies capable of recognizing multiple antigen epitopes, such as antibodies capable of recognizing two antigen epitopes (bispecific antibodies, abbreviated as "double antibodies"), antibodies capable of recognizing three antigen epitopes, or antibodies capable of recognizing four antigen epitopes. This is a broad interpretation, and there are no restrictions on the specific structure, as long as the antibody can recognize multiple antigen epitopes. In the present invention, at least one of the multiple antigen epitopes is derived from cTnI.

[0052] As used herein, the term "antigen-binding fragment" refers to a fragment containing part or all of an antibody, lacking at least some amino acids present in the full-length chain, but still retaining the ability to specifically bind to an antigen; for example, the fragment may contain part or all of the antibody CDRs. Such fragments retain biological activity, bind to antigen, and compete with other antigen-binding molecules (including complete antibodies) to bind to a predetermined epitope. Such fragments are selected from Fab, Fv, scFv, or single-domain antibodies. Such fragments can be produced by recombinant nucleic acid technology or by enzymatic or chemical degradation of antigen-binding molecules (including complete antibodies).

[0053] In some alternative embodiments of the present invention, the antibody or antigen-binding fragment thereof may further comprise at least one of the following additional technical features:

[0054] In some alternative embodiments of the present invention, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region CDR1 represented by an amino acid sequence selected from any one of SEQ ID NOs: 1 to 35 or an amino acid sequence having at least 80% identity thereto; a heavy chain variable region CDR2 represented by an amino acid sequence selected from any one of SEQ ID NOs: 36 to 70 or an amino acid sequence having at least 80% identity thereto; a heavy chain variable region CDR3 represented by an amino acid sequence selected from any one of SEQ ID NOs: 71 to 105 or an amino acid sequence having at least 80% identity thereto; a light chain variable region CDR1 represented by an amino acid sequence selected from any one of SEQ ID NOs: 106 to 140 or an amino acid sequence having at least 80% identity thereto; a light chain variable region CDR2 represented by an amino acid sequence selected from any one of SEQ ID NOs: 141 to 175 or an amino acid sequence having at least 80% identity thereto; It comprises a light chain variable region CDR3 represented by an amino acid sequence selected from any one of NOs: 176 to 210 or an amino acid sequence having at least 80% identity thereto.

[0055] In some alternative embodiments of the present invention, the antibody or antigen-binding fragment thereof comprises any one of the following heavy chain variable regions: [Table 0-33] and / or the antibody or antigen-binding fragment thereof comprises any one of the following light chain variable regions: [Table 0-34]

[0056] In some alternative embodiments of the invention, the antibody or antigen-binding fragment thereof comprises any one of the following combinations: [Table 0-35]

[0057] In some alternative embodiments of the invention, the antibody or antigen-binding fragment thereof specifically recognizes a PLA2R protein.

[0058] In some alternative embodiments of the present invention, the antibody or antigen-binding fragment thereof specifically recognizes a PLA2R antigen epitope peptide.

[0059] As used herein, the term "PLA2R antigen epitope peptide" refers to a polypeptide whose amino acid sequence is derived from PLA2R, and the "PLA2R antigen epitope peptide" comprises an antigenic epitope and can specifically bind to a PLA2R antibody; for example, the PLA2R antigen epitope peptide comprises CysR, FnII, and PLA2R binding region 1, the term "CysR" refers to the CysR structural domain in PLA2R, and the term "PLA2R binding region 1" refers to CTLD1 (or called CTLD structural domain 1) in PLA2R, the specific structure of which is shown in Figure 1.

[0060] In some alternative embodiments of the present invention, the antibody or antigen-binding fragment thereof specifically recognizes the CysR, FNII, and CTLD1 structural domains in the PLA2R protein.

[0061] In some alternative embodiments of the present invention, the antibody or antigen-binding fragment thereof further comprises at least one of a heavy chain framework region sequence and a light chain framework region sequence, wherein at least a portion of at least one of the heavy chain framework region sequence and the light chain framework region sequence is derived from at least one of a mouse-derived antibody, a human-derived antibody, a primate-derived antibody, or a variant thereof.

[0062] As used herein, the term "framework region" or "FR" region includes heavy chain framework regions and light chain framework regions and refers to the regions of an antibody heavy chain variable region (which can be denoted as VH) and light chain variable region (which can be denoted as VL) other than the CDRs; the heavy chain framework region is denoted as "HFR" and is further subdivided as adjacent regions separated by CDRs, including the framework regions HFR1, HFR2, HFR3, and HFR4; and the light chain framework region is denoted as "LFR" and is further subdivided as adjacent regions separated by CDRs, including the framework regions LFR1, LFR2, LFR3, and LFR4.

[0063] In some alternative embodiments of the present invention, at least a portion of the heavy chain framework region sequences and the light chain framework region sequences are derived from a human antibody or a variant thereof.

[0064] In some alternative embodiments of the present invention, the antibody or antigen-binding fragment thereof has a heavy chain variable region of the amino acid sequence set forth in any one of SEQ ID NOs: 211-245.

[0065] In some alternative embodiments of the present invention, the antibody or antigen-binding fragment thereof has a light chain variable region of the amino acid sequence set forth in any one of SEQ ID NOs: 246-280.

[0066] In some alternative embodiments of the invention, the antibody or antigen-binding fragment thereof comprises any one of the following combinations: [Table 0-36]

[0067] In some alternative embodiments of the present invention, the antibody comprises at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a mouse-derived antibody, a human-derived antibody, a primate-derived antibody, or a variant thereof.

[0068] In some alternative embodiments of the present invention, the heavy chain constant region comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD, or the light chain constant region comprises a light chain constant region selected from κ or λ.

[0069] In some alternative embodiments of the present invention, the light chain constant region and the heavy chain constant region are both derived from a human IgG4 antibody or a variant thereof.

[0070] In some alternative embodiments of the invention, the heavy chain constant region is wild-type human IgG4 or a human IgG4 variant.

[0071] In some alternative embodiments of the present invention, the human IgG4 variant (having the amino acid sequence shown in SEQ ID NO:282) has a S228P mutation compared to the heavy chain constant region of the wild-type human IgG4 antibody.

[0072] The numbering of the above-mentioned sites is based on the amino acid numbering of the wild-type human IgG4 Fc portion (having the amino acid sequence shown by SEQ ID NO:281) in accordance with the EU numbering system; for example, position 228 refers to position 228 as numbered according to the EU numbering system, and "S228P" indicates that the serine at position 228 as numbered according to the EU numbering system has been substituted with proline.

[0073] In some alternative embodiments of the invention, the heavy chain constant region has the amino acid sequence set forth in SEQ ID NO:281 or SEQ ID NO:282.

[0074] In some alternative embodiments of the invention, the light chain constant region has the amino acid sequence set forth in SEQ ID NO:357.

[0075] In some alternative embodiments of the present invention, the antibody or antigen-binding fragment thereof has a heavy chain having the amino acid sequence of any one of SEQ ID NOs: 283-317, SEQ ID NOs: 358-366, or the antibody or antigen-binding fragment thereof has a light chain having the amino acid sequence of any one of SEQ ID NOs: 318-352.

[0076] In some alternative embodiments of the invention, the antibody or antigen-binding fragment thereof comprises any one of the following combinations: [Table 0-37]

[0077] In some alternative embodiments of the present invention, the antibody comprises at least one selected from a monoclonal antibody, a polyclonal antibody, a multimeric antibody, and a CDR-grafted antibody.

[0078] In some alternative embodiments of the present invention, the antibody comprises at least one selected from a single chain antibody, a Fab antibody, a Fab' antibody, a F(ab')2 antibody, an Fv antibody, a single chain antibody, a single domain antibody, and a minimal recognition unit.

[0079] In some alternative embodiments of the invention, the antigen-binding fragment comprises at least one of a Fab fragment, a Fab' fragment, a F(ab)2 fragment, a F(ab')2 fragment, an Fv fragment, an scFv fragment, an scFv-Fc fusion protein, an scFv-Fv fusion protein, an Fv fragment, and a minimal recognition unit.

[0080] The antigen-binding fragment of the antibody generally has the same binding specificity as the antibody from which it was derived. As those skilled in the art can easily understand based on the contents of the present disclosure, the antigen-binding fragment of the antibody can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or cleavage of disulfide bonds by chemical reduction. Based on the structure of the complete antibody in the present disclosure, those skilled in the art can easily obtain the antigen-binding fragment.

[0081] Antigen-binding fragments of the above antibodies can be synthesized using recombinant genetic techniques known to those skilled in the art or automated peptide synthesizers such as those commercially available from Applied BioSystems.

[0082] As used herein, the terms "polyclonal antibody" and "multispecific antibody" are synonymous and refer to antibodies capable of recognizing multiple antigen epitopes, such as antibodies capable of recognizing two antigen epitopes (bispecific antibodies, abbreviated as double antibodies), antibodies capable of recognizing three antigen epitopes, or antibodies capable of recognizing four antigen epitopes. This is a broad interpretation, and there are no restrictions on the specific structure, as long as the antibody is capable of recognizing multiple antigen epitopes.

[0083] As used herein, the terms "CDR-grafted antibody" and "modified antibody" refer to the grafting of the CDRs of a monoclonal antibody onto the variable region of an antibody of another species. For example, by grafting the CDRs of a mouse-derived monoclonal antibody onto the variable region of a human-derived antibody, the CDRs of the human-derived antibody are replaced, thereby conferring the antigen-binding specificity of the mouse-derived monoclonal antibody to the human-derived antibody and reducing its heterogeneity. Note that both polyclonal and monoclonal antibodies in the present application may be CDR-grafted antibodies.

[0084] In this specification, the terms "single domain antibody", "nanobody" and "VHH antibody" can be used interchangeably, and antigen-binding immunoglobulin (variable) domains, originally described as "heavy chain antibodies" (i.e., "antibodies lacking light chains") (Hamers-Casterman C, Atharhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)), contain a heavy chain variable region (VH) and conventional CH2 and CH3 regions, and specifically bind to an antigen protein (e.g., cTnI) via the heavy chain variable region.

[0085] As used herein, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment containing only the Fab molecule, which consists of the VH and CH1 of a heavy chain and an intact light chain, with the light and heavy chains connected by a single disulfide bond.

[0086] As used herein, the term "F(ab')2 antibody" or "F(ab')2 fragment" has two antigen-binding F(ab') portions connected by disulfide bonds.

[0087] As used herein, the term "Fv antibody" or "Fv fragment" generally refers to an antibody or fragment in which only the light chain variable region (VL) and heavy chain variable region (VH) are non-covalently connected, and is the minimum functional fragment of an antibody molecule that retains an intact antigen-binding site.

[0088] As used herein, the terms "single-chain antibody" and "scFv fragment" refer to an antibody or fragment in which the antibody heavy chain variable region and light chain variable region are connected by a short peptide.

[0089] As used herein, the terms "minimal recognition unit" and "MRU" refer to an antibody or fragment consisting of only one CDR, which has a very small molecular weight, accounting for approximately 1% of the molecular weight of a complete antibody.

[0090] Nucleic acid molecules, expression vectors, recombinant cells and kits, and their uses

[0091] In the process of preparing or obtaining the aforementioned antibodies or antigen-binding fragments thereof, nucleic acid molecules expressing these antibodies or antigen-binding fragments thereof can be used to obtain the corresponding antibodies or antigen-binding fragments thereof by linking them to different carriers and then expressing them in different cells.

[0092] In another aspect, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the above-described antibody or antigen-binding fragment thereof. The nucleic acid molecule according to an embodiment of the present invention can be obtained by encoding the above-described antibody or antigen-binding fragment thereof.

[0093] In some alternative embodiments of the invention, the nucleic acid molecule is DNA.

[0094] It should be understood by those skilled in the art that the nucleic acid molecules referred to herein actually include either or both of the complementary strands. For convenience, in most cases, only one strand is provided herein, but the complementary strand is also disclosed. Furthermore, the molecular sequences of the present invention include DNA and RNA forms, and the disclosure of either one implies the disclosure of the other.

[0095] In a further aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the nucleic acid molecule described above is carried. When the nucleic acid molecule is attached to a carrier, the nucleic acid molecule can be directly or indirectly linked to regulatory elements on the expression vector, as long as these regulatory elements control the translation and expression of the nucleic acid molecule. Of course, these regulatory elements may be derived directly from the expression vector itself, or may be exogenous, i.e., not derived from the expression vector itself. Of course, the nucleic acid molecule may be operably linked to regulatory elements. As used herein, "operably linked" refers to the attachment of an exogenous gene to the carrier, allowing regulatory elements within the carrier, such as transcriptional regulatory sequences and translational regulatory sequences, to perform the function of regulating the transcription and translation of the exogenous gene in a predetermined manner. Commonly used carriers include, for example, plasmids and phages. After introducing the carrier according to some specific embodiments of the present invention into appropriate recipient cells, the expression of the antibody or its antigen-binding fragment can be effectively achieved through the mediation of a regulatory system, and the in vitro mass production of the antibody or its antigen-binding fragment can be achieved.

[0096] In some alternative embodiments of the present invention, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

[0097] In some alternative embodiments of the present invention, the expression vector is a plasmid expression vector.

[0098] In a further aspect, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell carries the above-described nucleic acid molecule or expresses the above-described antibody or antigen-binding fragment thereof. The cell can be used to efficiently express the above-described antibody or antigen-binding fragment thereof intracellularly under appropriate conditions.

[0099] In some alternative embodiments of the present invention, the recombinant cell is obtained by introducing the above-described expression vector into a host cell.

[0100] In some alternative embodiments of the present invention, the recombinant cell is a eukaryotic cell.

[0101] In some alternative embodiments of the present invention, the recombinant cell is a mammalian cell.

[0102] The cells of the present invention are not particularly limited and may be prokaryotic cells, eukaryotic cells, or phages. Examples of prokaryotic cells include Escherichia coli, Bacillus subtilis, Streptomyces, and Proteus mirabilis. Examples of eukaryotic cells include fungi such as Pasteurella yeast, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Trichoderma sp., insect cells such as those of the armyworm, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells.

[0103] In an alternative embodiment of the invention, the cell is a mammalian cell, including a BHK cell, a CHO cell, an NSO cell or a COS cell, and does not include an animal germ cell, a fertilized egg or an embryonic stem cell.

[0104] The term "appropriate conditions" used herein refers to conditions suitable for the expression of an antibody or antigen-binding fragment thereof. As those skilled in the art will readily understand, suitable conditions for the antibody or antigen-binding fragment thereof include, but are not limited to, a suitable transformation or transfection method, suitable transformation or transfection conditions, healthy cell conditions, suitable cell density, suitable cell culture environment, and suitable cell culture time. The "appropriate conditions" are not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the antibody or antigen-binding fragment thereof depending on the specific laboratory environment.

[0105] In a further aspect, the present invention provides the use of the aforementioned antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell as a PLA2R antibody standard. Methods according to some specific embodiments of the present invention can efficiently obtain the antibody or antigen-binding fragment thereof in large quantities.

[0106] Based on the amino acid sequence of an antibody or antigen-binding fragment thereof of the present disclosure, a person skilled in the art can easily conceive of obtaining the antibody or antigen-binding fragment thereof by preparing it using genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), for example, by separating and purifying the antibody or antigen-binding fragment thereof described in any one of the above from a culture product of a recombinant cell capable of recombinant expression, and obtaining the antibody or antigen-binding fragment thereof. This can be easily accomplished by a person skilled in the art, and any technique used to prepare the antibody or antigen-binding fragment thereof of the present disclosure based on this falls within the scope of protection of the present disclosure.

[0107] In a further aspect, the present invention provides a kit. According to an embodiment of the present invention, the kit includes the aforementioned antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell. As can be seen from the above, the aforementioned antibody or antigen-binding fragment thereof has high affinity for PLA2R protein, can be produced stably and in large quantities, and can be free from contamination by pathogenic microorganisms, making it suitable for use as an anti-human PLA2R antibody standard. Therefore, a kit including the aforementioned antibody or antigen-binding fragment thereof has advantages such as high accuracy in detecting PLA2R antibodies.

[0108] In an alternative embodiment of the invention, the kit may further comprise reagents for detecting PLA2R antibodies.

[0109] In a further aspect of the present invention, the present invention provides the use of the aforementioned antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell in the preparation of a kit for detecting PLA2R antibodies or diagnosing PLA2R antibody-positive membranous nephropathy. As can be seen from the above, the aforementioned antibody or antigen-binding fragment thereof has high affinity for PLA2R protein, can be produced stably and in large quantities, can be free from contamination by pathogenic microorganisms, and can be used as an anti-human PLA2R antibody standard. Therefore, a kit containing the aforementioned antibody or antigen-binding fragment thereof has advantages such as high accuracy in detecting PLA2R antibodies.

[0110] In a further aspect of the present invention, the present invention provides the use of the aforementioned antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell for detecting PLA2R antibodies or diagnosing PLA2R antibody-positive membranous nephropathy. As can be seen from the above, the aforementioned antibody or antigen-binding fragment thereof has high affinity for PLA2R protein, can be produced stably and in large quantities, can be free from contamination by pathogenic microorganisms, and can be used as an anti-human PLA2R antibody standard. Therefore, a kit containing the aforementioned antibody or antigen-binding fragment thereof has advantages such as high accuracy in detecting PLA2R antibodies.

[0111] In a further aspect of the present invention, the present invention provides the aforementioned antibody or its antigen-binding fragment, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell for detecting PLA2R antibodies or diagnosing PLA2R antibody-positive membranous nephropathy. As can be seen from the above, the aforementioned antibody or its antigen-binding fragment has high affinity for PLA2R protein, can be produced stably and in large quantities, and can be free from contamination by pathogenic microorganisms, making it suitable for use as an anti-human PLA2R antibody standard. Therefore, kits containing the aforementioned antibody or its antigen-binding fragment have advantages such as high accuracy in detecting PLA2R antibodies.

[0112] method

[0113] In a further aspect, the present invention provides a method for detecting PLA2R antibodies. According to an embodiment of the present invention, the method determines the content of PLA2R antibodies in a sample to be measured based on the results of PLA2R antibody detection, using the aforementioned antibody or its antigen-binding fragment as a PLA2R antibody standard. This improves the accuracy of PLA2R antibody detection. As can be seen from the above, the aforementioned antibody or its antigen-binding fragment has high affinity for PLA2R protein, can be produced stably and in large quantities, can be free from contamination by pathogenic microorganisms, and can be used as an anti-human PLA2R antibody standard. Therefore, the method of the present invention has advantages such as high accuracy in detecting PLA2R antibodies.

[0114] In a further aspect, the present invention provides a method for diagnosing PLA2R antibody-positive membranous nephropathy. According to an embodiment of the present invention, the method includes the steps of: determining the PLA2R antibody content in the test sample based on the PLA2R antibody detection result in the test sample using the aforementioned antibody or its antigen-binding fragment as a PLA2R antibody standard; and determining whether the patient corresponding to the test sample has PLA2R antibody-positive membranous nephropathy based on the PLA2R antibody content. This improves the accuracy of PLA2R antibody detection. As can be seen from the above, the aforementioned antibody or its antigen-binding fragment has high affinity for PLA2R protein, can be produced stably and in large quantities, and can be used as an anti-human PLA2R antibody standard, eliminating contamination with pathogenic microorganisms. The PLA2R antibody content can be accurately detected. Therefore, the method of the present invention has high diagnostic accuracy for PLA2R antibody-positive membranous nephropathy.

[0115] The following examples are used in combination to illustrate the present disclosure. Those skilled in the art can understand that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. If no specific techniques or conditions are shown in the examples, they should be carried out according to the techniques or conditions described in the literature of the field or according to the product specifications. If no manufacturer is shown for the reagents or equipment used, they are conventional products that can be purchased commercially.

[0116] Example 1: Preparation of PLA2R major antigen region CC1H

[0117] 1.1 Construction of PLA2R major antigen domain recombinant protein

[0118] The plasmid carrier was the pcDNA3.4 transient expression vector, which contained the native full-length CMV promoter and a WPRE element downstream of the cloning site. CMV promoter-based carriers typically exhibit good expression levels in CHO cell transient expression systems. The WPRE element, located downstream of the polyclonal site, can effectively improve gene transcription and expression. In E. coli, it also contains an ampicin resistance gene. Next, PCR was used to amplify and ligate the carrier gene pcDNA3.4 with a synthetic gene (the gene sequence linking the gene of interest and a signal peptide is referred to as the "synthetic gene"). The PCR template was obtained by gene synthesis, and the pcDNA3.4 carrier fragment and the synthetic gene fragment were ligated using a homologous recombination kit. DH5α competent cells were transformed to obtain monoclonal clones, which were amplified and sequenced. After confirmation by sequence analysis, the plasmid was extracted to obtain the synthetic recombinant expression plasmid. The synthetic gene was cloned between the CMV promoter and WPRE gene of the pcDNA3.4 plasmid, as shown in Figure 2.

[0119] The amino acid sequence of the signal peptide is MLLSPSLLLLLLLGAPRGCA (SEQ ID NO:353), The nucleotide sequence of the signal peptide is ATGCTGCTGTCGCCGTCGCTGCTGCTGCTGCTGCTGCTGGGGGCGCCGCGGGGCTGCGCC (SEQ ID NO:354).

[0120] The structural diagram of PLA2R is shown in Figure 1, and the target gene is the amino acid sequence 1 to 367 (Glu) of the PLA2R fragment. The constructed transient expression plasmid is named pcDNA3.4-CC1H.

[0121] The amino acid sequence of the synthetic gene (PLA2R major antigen region) is: MLLSPSLLLLLLLGAPRGCAEGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWH HECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVEGGHHHHHH(SEQ ID NO:355), The nucleotide sequence of the synthetic gene (PLA2R major antigen region) is:

[0122] 1.2 Temporal expression of CC1H Approximately 24 hours before transfection, TM CHO-S (R) Cells (cell density 5-6 × 10 5 / mL, and Gibco FreeStyle TM MAX CHO Expression System (catalog number: K900020) and cultured under conditions of 37°C, 5% CO2, and 120-135 rpm / min.

[0123] On the day of transfection, cells were cultured at a cell density of 1 × 10 6 10 mL of cells (cell viability ≥ 95%) were added to each shake flask.

[0124] FreeStyle TM Mix the MAX Reagent gently several times, being careful to avoid vortexing.

[0125] Take 12.5 μg of pcDNA3.4-CC1H and dilute it in OptiPRO until the total volume reaches 0.2 mL. TM Add SFM and mix gently.

[0126] 12.5 μL of FreeStyle TM Add MAX Reagent and add OptiPRO TM Add SFM and mix gently. After mixing evenly, add to the plasmid mixture, mix gently, and add 0.4 mL of DNA-FreeStyle TM The MAX mixture was taken and incubated at room temperature for 10 minutes to form the complex.

[0127] 0.4mL of DNA-FreeStyle TM The MAX complex was slowly added to the cells while gently rotating the shake flask.

[0128] The cells were cultured at 37°C, 5% CO2, and 120-135 rpm / min without medium replacement or replenishment, and the cell culture supernatant was collected after 7 days.

[0129] 1.3 Purification and identification of CC1H

[0130] Sample: 25 mL of the cell culture supernatant from step 1.2 was collected. It was centrifuged at 7000 rpm at 4°C for 10 minutes and passed through a 0.22 μm needle filter. It was loaded onto a Ni Sepharose 6FF column at 2 mL / min. It was equilibrated and washed with 20 mM phosphate buffer, 500 mM NaCl, and 20 mM imidazole, pH 6.8. It was eluted over 5 column volumes with 0-100% 20 mM phosphate buffer, 1 M NaCl, and 500 mM imidazole, pH 6.8. Aliquots were collected in 2 mL tubes.

[0131] The 30 kDa elution peak was ultrafiltered three times in an ultrafiltration centrifuge tube, and the solution was exchanged into 50 mM phosphate buffer and 150 mM NaCl, pH 7.4. Quantification was performed using the Bradford assay. The expression level of CC1H was measured to be 37 mg / L. CC1H was analyzed by SDS-PAGE, and the results are shown in Figure 3.

[0132] Example 2: CC1H biotin tagging and humanized phage display library screening

[0133] CC1H biotin-labeled and humanized phage display library screening was completed by Sanyu Biomedical (Shanghai) Co., Ltd. CC1H was first biotin-labeled, and ELISA confirmed that CC1H was biotin-labeled. Subsequently, the humanized recombinant antibody library constructed by Sanyu Biomedical (Shanghai) Co., Ltd. was screened using solid-phase and liquid-phase cross-screening with immunotubes and magnetic bead sorting, and specific Fab antibodies against CC1H were enriched using trypsin elution. The enrichment status of different output pools was detected by ELISA, and this screening yielded many output pools with good ELISA-level enrichment. After primary ELISA screening, a total of 1,748 clones were selected, and 834 positive clones were identified that specifically bound to CC1H. Of these, 98 clones were sequence-unique, and 34 molecules (see Table 1 for details) were selected for full-length construction. These 34 molecules belong to the IgG4 subtype.

[0134] Nine antibodies with good affinity and expression levels were selected at the phage level, and IgG4SP subtype antibodies (specifically, see P62295 to P62298 and P62300 to P62304 in Table 1, abbreviated as SP mutants) were constructed.

[0135] A total of 43 antibodies were selected for expression (see "List of Amino Acid Sequences of the Invention and List of Nucleotide Sequences of the Invention" in the Summary of the Invention for their amino acid and nucleotide sequences), and the corresponding heavy and light chains were cloned between the CMV promoter and WPRE gene of pcDNA3.4. CHO cells were transiently transfected and expression levels were detected (ExpiCHO Expression System Kit, ThermoFisher, Cat. No. A29133). Antibody numbers and expression levels are shown in Table 1.

[0136] [Table 1] Note: *IgG4SP is a point mutation of IgG4, with the S228P mutation site in its hinge region, which can improve antibody stability.

[0137] Example 3: Detection of human anti-human PLA2R antibodies ELISA

[0138] The human anti-human PLA2R antibody obtained through phage screening and expression in Example 2 was assayed for binding to CC1h by indirect ELISA.

[0139] 30 μL of CC1h (2 μg / mL, diluted in 1% PBS) was added to the reaction wells of an ELISA plate, sealed, and incubated at 4°C overnight. The plate was washed three times with PBST, discarded, and 5% PBSM was added to close the ELISA plate at room temperature for 2 h. The plate was washed three times with PBST. 30 μL of Abs (diluted in 1% PBS) was added to each well and incubated at room temperature for 60 min. The plate was washed three times with PBST, and 30 μL of anti-human IgG Fc HRP (diluted 1:8000 in 1% PBSM) was added to each well and incubated at room temperature for 60 min. The reaction was stopped with 2M TMB blocking solution and the OD was measured at 450 nm. Recombinant ipilimumab (Sanyu Biologics) was used as a negative control. The detection results are shown in Table 2, Figures 4A, 4B, 4C, and 5.

[0140] [Table 2]

[0141] As can be seen from the ELISA results, four antibodies, A22 (see Figure 4A), B44 (see Figure 4C), B88 (see Figure 4C), and B145 (see Figure 5A), did not clearly bind to CC1h, and the positive results obtained during the screening of the phage display library may be false positives. The other antibodies all showed different strengths of binding to CC1h, and the EC 50The concentrations ranged from 3.85 ng / mL (B29) to 27.6 ng / mL (A23). The results showed that most of the antibodies specifically bound to CC1h.

[0142] Example 4: Interaction kinetics and thermodynamic analysis of antibodies and CC1H

[0143] Sartorius Octet based on Bio-Layer Interferometry (BLI) (R) The N1 molecular interaction device was used to detect interactions between proteins and biological molecules, i.e., the binding constant (Ka), dissociation constant (Kd), and affinity constant (KD) of the interaction between the 43 PLA2R human antibodies in Example 2 and biotin-labeled CC1H were determined. The specific experimental steps are as follows:

[0144] 4.1 Biotin labeling of CC1H The CC1H protein purified in Example 1 was labeled with biotin using the EZ-Link Sulfo-NHS-LC-Biotin kit (Thermo Scientific, 21335), and the specific steps were as follows:

[0145] Allow the kit to equilibrate to room temperature. Prepare a 10 mM biotin reagent solution. Add 90 μL of sterile double-distilled water to 0.5 mg of biotin reagent and dissolve thoroughly. To the CC1H protein solution, add 20 molar equivalents of biotin reagent solution to the protein, The mixture is placed on ice and incubated for 2 hours, during which time it is inverted several times to ensure thorough and uniform mixing.

[0146] 4.2 Purification of CC1H-Biotin Biotin-labeled CC1H-Biotin was purified using a G-25 prepacked desalting column (Bogelong, EG001). A 20 mM PBS buffer solution at pH 7.2 was prepared according to Table 3.

[0147] [Table 3] Equilibration: Extract the PBS buffer with a 10 mL syringe, connecting the column and syringe drop-to-drop each time to prevent air bubbles. Cut off the outlet end, A total of 25 mL of PBS buffer was passed through the column at 5 mL / min (120 drops / min) to remove the ethanol in the column, and the column effluent was discarded. Sample Load: Load 1.5 mL of sample with a 3 mL syringe at 5 mL / min (if less than 1.5 mL, top up with PBS buffer), discard the column effluent, 3 mL of PBS buffer was injected followed by a total collection of approximately 2 mL. Column Cleaning and Storage: Pass 25 mL of PBS buffer through the column with a 10 mL syringe, then pass 25 mL of water through the column, and discard the column effluent. Pass 25 mL of 20% ethanol through a 10 mL syringe and store at 4 °C. Sample storage: The collected sample is sterilized by suction filtration through a 0.2 μm pore size filter, and then aliquoted and stored.

[0148] 4.3 Measurement of CC1H-Biotin protein concentration by Bradford method (Biyuntian, P0060) The protein standard (5 mg / ml BSA) was completely melted and mixed, then diluted with PBS buffer to prepare 0, 0.125, 0.25, 0.5, 0.75, 1, and 1.5 mg / ml protein standards, which were then thoroughly and uniformly mixed. 5 μL of different concentrations of protein standard was taken and added to the standard wells of a 96-well immunoplate (Thermo Scientific, 468667). Take 5 μL of sample and add it to the sample well of a 96-well immunoplate (if it is less than 5 μL, add PBS buffer to fill it up), Add 250 μL of G250 staining solution to each well. Measure A595 with an enzyme marker, The protein concentration in the samples was calculated from the standard curve, which is shown in Figure 6, where y = 0.6849x + 0.6325, R 2 =0.9925. The calculated CC1H-Biotin protein concentration was 2.36 mg / mL.

[0149] 4.4 Identification of sample purity and concentration by reducing / non-reducing SDS-PAGE The purity and concentration of the samples were determined by reducing / non-reducing SDS-PAGE. The purification effect of CC1H-Biotin is shown in Figure 7. The lanes from left to right are CC1H-Biotin in reducing sample buffer containing DTT and CC1H-Biotin in non-reducing sample buffer. The molecular weight of CC1H-Biotin is approximately 40 KDa and the purity is >98%.

[0150] 4.5 Pierce TM Biotin Quantitation Kit (HABA assay)(Thermo Scientific TM , 28005) Kit for detecting biotin contamination levels Allow the ABA / Avidin premix to equilibrate to room temperature. Add 100 μL of ultrapure water to the HABA / Avidin premix tube and blow evenly with a gun. Add 160 μL of PBS buffer into the wells of a 96-well immunoplate (Thermo Scientific, 468667). 20 μL of HABA / Avidin premixed solution was added to the well containing PBS buffer, and after shaking and mixing, A500 was measured. 20 μL of biotinylated sample was added to the wells containing HABA / Avidin, and the A500 was re-detected after shaking and mixing, and held constant for at least 15 seconds. The biotin contamination level was calculated based on Beer's law, and the results are shown in Table 4. The calculated biotin contamination level of CC1H-Biotin was 1.80 mol Biotin / mol protein.

[0151] [Table 4]

[0152] 4.6 Measurement of the interaction between CC1H-Biotin and PLA2R human antibodies screened by phage display library Sartorius Octet (R) The N1 molecular interaction device was used to detect SA sensors (Sartorius, Octet (R) Select Streptavidin (SA) Biosensor, 18-5019) The sensor was pre-wetted by immersing it in PBSTB buffer (PBS buffer plus 0.02% Tween-20 and 0.1% BSA) for 10 min or more, and then loaded onto the molecular interaction device. The sensor was immersed in different buffer solutions one after another and detected using a five-step method. Baseline 1 (300 μL PBSTB buffer, 1 min) → Curing (300 μL CC1H-biotin, 20 μg / mL, 2 min) → Baseline 2 (3 min PBSTB buffer) → Binding (76.6-8.5 nM, 300 μL PLA2R human anti-biotin prepared in Example 2 diluted in gradient with PBSTB buffer, 2 min) → Dissociation (300 μL PBSTB buffer, 5 min) (human anti-biotin sample was used for ExpiCHO TM Containing expression medium and dissociated with PBSTB buffer at a concentration corresponding to baseline 2, ExpiCHO TM Expression medium (Gibco, A2910001)), The association constant (Ka), dissociation constant (Kd) and affinity constant (KD) are calculated, specifically referring to Table 5.

[0153] [Table 5] As can be seen from the table above, all of the antibodies with detection molecular interactions other than P59390 (B88) bind well to CC1H and can be used as PLA2R antibody standards.

[0154] Example 5: Calibration of PLA2R antibody standards

[0155] 5.1 Metric Conversion The titers of A13 (P59368) and A13SP (P62297) prepared in Example 2 at different dilutions were detected using the Omun anti-PLA2R (IgG) kit (Euroimmun, EA 1254-9601 G (96)). The average values ​​are shown in Table 6 as a conversion relationship between RU and ng.

[0156] [Table 6] The metric conversion relationship obtained for A13 / A13SP is approximately 1RU = 70ng. Plot the A13 / A13SP standard curve at 1RU = 70ng and compare it with the Omn standard, see Figure 8.

[0157] As can be seen in Figure 8, the A13 / A13SP standard curve is slightly different from the Omun standard in the high concentration range (>750 RU / mL). This may be because the kit standard is a polyclonal antibody, a mixture of antibodies with significantly different affinities, and its ELISA curve may show a different expression from that of the screened monoclonal antibody.

[0158] 2. Standard curve comparison The nine antibodies prepared in Example 2 and their SP variants (see Figure 9 in particular) were detected and screened using the Omun ELISA kit, and standard curves were obtained and compared with the kit standard.

[0159] The experimental steps are as follows: Add 100 μL / well of Standards 1-5, human anti-samples, and negative controls, seal the plate, and incubate at room temperature (25°C) for 30 minutes. Discard the liquid in the wells and wash the plates with a plate washer (Bio-Rad, ImmunoWash TMThe plate was placed in a 1575 plate washer, washed four times with the plate washing solution, placed in a plate thawer (Hettich, Universal 16R), centrifuged at 600 g for 1 minute, and then tapped several times to dry. Add 100 μL of the matching enzyme conjugate to each well, seal the plate, and incubate at room temperature (25°C) for 30 min. Repeat the plate washing 100 μL of matching substrate color developing solution is added to each well, and after incubation at room temperature (25° C.) for 15 minutes in the dark, 100 μL of matching termination solution is added to each well and A450 is measured.

[0160] The detection results are shown in FIG. 9, and the screening antibody concentration units in FIG. 9 are converted to RU, with 1 RU=70 ng.

[0161] As can be seen in Figure 9A, P59390 (B88) could not be detected with the commercialized kit, which was consistent with the results of CC1h ELISA and BLI analysis. The other antibodies and their SP variants exhibited curves similar to the standard curves of the Omun ELISA kit. The curve shapes of the antibodies obtained through screening differed from those of the kit standard. This may be because the kit standard is a polyclonal antibody, a mixture of antibodies with significantly different affinities, and therefore its ELISA curves exhibited different profiles from those of the monoclonal antibodies screened.

[0162] In the description herein, a statement referring to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples" means that a particular feature, structure, material, or characteristic described with reference to that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, general references to the above terms do not necessarily refer to the same embodiment or example. In addition, a particular feature, structure, material, or characteristic described may be incorporated in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art can combine and combine the various embodiments or examples described herein and the features of the various embodiments or examples without mutual contradiction.

[0163] Although the embodiments of the present invention have been shown and described, the above embodiments are illustrative and should not be construed as limiting the present invention. Those skilled in the art will appreciate that various changes, modifications, substitutions and variations can be made to the above embodiments within the scope of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof, Heavy chain variable region CDR sequence: SEQ ID NO: 1-105, An antibody or antigen-binding fragment thereof, comprising a CDR sequence selected from at least one of light chain variable region CDR sequences: SEQ ID NO: 106 to 210, or an amino acid sequence having at least 80% identity thereto.

2. The antibody or antigen-binding fragment thereof A heavy chain variable region CDR1 represented by an amino acid sequence selected from any one of SEQ ID NOs: 1 to 35 or an amino acid sequence having at least 80% identity thereto; A heavy chain variable region CDR2 represented by an amino acid sequence selected from any one of SEQ ID NOs: 36 to 70 or an amino acid sequence having at least 80% identity thereto; A heavy chain variable region CDR3 represented by an amino acid sequence selected from any one of SEQ ID NOs: 71 to 105 or an amino acid sequence having at least 80% identity thereto; A light chain variable region CDR1 represented by an amino acid sequence selected from any one of SEQ ID NOs: 106 to 140 or an amino acid sequence having at least 80% identity thereto; a light chain variable region CDR2 having an amino acid sequence selected from any one of SEQ ID NOs: 141 to 175 or an amino acid sequence having at least 80% identity thereto; or The antibody or antigen-binding fragment thereof according to claim 1, characterized in that it comprises a light chain variable region CDR3 represented by an amino acid sequence selected from any one of SEQ ID NOs: 176 to 210 or an amino acid sequence having at least 80% identity thereto.

3. The antibody or antigen-binding fragment thereof comprises any one of the following sets of heavy chain variable regions: Table 1 Table 2 and / or the antibody or antigen-binding fragment thereof comprises any one of the following light chain variable regions: Table 3 The antibody or antigen-binding fragment thereof according to claim 1.

4. The antibody or antigen-binding fragment thereof comprises any one of the following combinations: Table 4 The antibody or antigen-binding fragment thereof according to claim 1.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof specifically recognizes a PLA2R protein.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof specifically recognizes the CysR, FNII and CTLD1 structural domains in the PLA2R protein.

7. The antibody or antigen-binding fragment thereof further comprises: at least one of a heavy chain framework region sequence and a light chain framework region sequence; The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein at least a portion of at least one of the heavy chain framework region sequence and the light chain framework region sequence is derived from at least one of a mouse-derived antibody, a human-derived antibody, a primate-derived antibody, or a mutant thereof.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein at least a portion of the heavy chain framework region sequence and the light chain framework region sequence are derived from a human antibody or a mutant thereof.

9. the antibody or antigen-binding fragment thereof has a heavy chain variable region of the amino acid sequence set forth in any one of SEQ ID NOs: 211 to 245; and / or The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof has a light chain variable region having an amino acid sequence set forth in any one of SEQ ID NOs: 246 to 280.

10. The antibody or antigen-binding fragment thereof comprises any one of the following combinations: Table 5 The antibody or antigen-binding fragment thereof according to claim 9.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody comprises at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a mouse-derived antibody, a human-derived antibody, a primate-derived antibody, or a mutant thereof.

12. The heavy chain constant region is a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD; or The antibody or antigen-binding fragment thereof according to claim 11, wherein the light chain constant region comprises a light chain constant region selected from a κ type or a λ type.

13. The antibody or antigen-binding fragment thereof according to claim 11, wherein the light chain constant region and the heavy chain constant region are both derived from a human IgG4 antibody or a variant thereof.

14. The antibody or antigen-binding fragment thereof according to claim 13, wherein the heavy chain constant region is wild-type human IgG4 or a human IgG4 variant.

15. The antibody or antigen-binding fragment thereof according to claim 14, characterized in that the human IgG4 variant has an S228P site mutation compared to the heavy chain constant region of the wild-type human wild-type IgG4 antibody.

16. the heavy chain constant region has the amino acid sequence set forth in SEQ ID NO: 281 or SEQ ID NO: 282; and / or The antibody or antigen-binding fragment thereof according to claim 11, characterized in that the light chain constant region has the amino acid sequence shown in SEQ ID NO:

357.

17. the antibody or antigen-binding fragment thereof has a heavy chain represented by the amino acid sequence of any one of SEQ ID NOs: 283-317, SEQ ID NOs: 358-366, or The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof has a light chain represented by the amino acid sequence of any one of SEQ ID NOs: 318 to 352.

18. The antibody or antigen-binding fragment thereof comprises any one of the following combinations: Table 6 18. The antibody or antigen-binding fragment thereof of claim 17.

19. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that the antibody is selected from at least one of a monoclonal antibody, a polyclonal antibody, a multimeric antibody, and a CDR-grafted antibody.

20. The antibody may be a single chain antibody, a Fab antibody, a Fab' antibody, or a F(ab') ２ and / or selected from at least one of an antibody, an Fv antibody, a single chain antibody, a single domain antibody, and a minimal recognition unit. The antigen-binding fragment of the antibody may be a Fab fragment, a Fab' fragment, or a F(ab) fragment. ２ Fragment, F(ab') ２ The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that it comprises at least one of an Fc fragment, an Fv fragment, an scFv fragment, an scFv-Fc fusion protein, an scFv-Fv fusion protein, an Fv fragment, and a minimum recognition unit.

21. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 20, Optionally, said nucleic acid molecule is DNA.

22. An expression vector carrying the nucleic acid molecule of claim 21, Optionally, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector; Preferably, the expression vector is a plasmid expression vector.

23. A recombinant cell, comprising: Carrying a nucleic acid molecule according to claim 21, or expressing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 20, Optionally, the recombinant cell is obtained by introducing the expression vector of claim 22 into a host cell, Optionally, the recombinant cell is a eukaryotic cell; Preferably, the recombinant cell is a mammalian cell.

24. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 20, the nucleic acid molecule according to claim 21, the expression vector according to claim 22, or the recombinant cell according to claim 13 as a PLA2R antibody standard.

25. A kit comprising: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 20.

22. The nucleic acid molecule of claim 21 .

23. The expression vector of claim 22, or A kit comprising the recombinant cell of claim 23.

26. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 20, the nucleic acid molecule according to claim 21, the expression vector according to claim 22, or the recombinant cell according to claim 23 in the preparation of a kit for detecting PLA2R antibodies or diagnosing PLA2R antibody-positive membranous nephropathy.

27. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 20, the nucleic acid molecule according to claim 21, the expression vector according to claim 22, or the recombinant cell according to claim 23 for detecting PLA2R antibodies or diagnosing PLA2R antibody-positive membranous nephropathy.

28. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 20, a nucleic acid molecule according to claim 21, an expression vector according to claim 22, or a recombinant cell according to claim 23, for detecting a PLA2R antibody or diagnosing PLA2R antibody-positive membranous nephropathy.

29. A method for detecting PLA2R antibodies or diagnosing PLA2R antibody-positive membranous nephropathy, comprising: Based on the detection result of the PLA2R antibody in the measurement sample, the antibody or antigen-binding fragment thereof according to any one of claims 1 to 20 is used as a PLA2R antibody standard, and the content of the PLA2R antibody in the measurement sample is obtained; and determining whether the patient corresponding to the sample to be measured has PLA2R antibody-positive membranous nephropathy based on the content of the PLA2R antibody.

30. 1. A method for detecting a PLA2R antibody, comprising: A method for detecting a PLA2R antibody, comprising the step of determining the content of the PLA2R antibody in a sample to be measured based on the detection result of the PLA2R antibody, using the antibody or antigen-binding fragment thereof described in any one of claims 1 to 20 as a PLA2R antibody standard.

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