Neutral antibodies

Abstract

The present invention relates to a neutral antibody. The neutral antibody comprises a CDR sequence selected from at least one of the following: light chain variable region CDR sequences: SEQ ID NOs: 1 to 3, SEQ ID NOs: 7 to 9, and SEQ ID NOs: 83 to 85, and heavy chain variable region CDR sequences: SEQ ID NOs: 4 to 6, SEQ ID NOs: 10 to 12, SEQ ID NOs: 86 to 88, and SEQ ID NOs: 89 to 91. The antibody of the present invention has low antigen binding activity and high expression level.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology and medicine, and in particular, to a neutral antibody, and more particularly, to a neutral antibody and its use, nucleic acid molecule, expression vector, recombinant cell and fusion protein. Background Art

[0002] The current fusion proteins are all made by connecting the ligand binding fragment and the Fc fragment, and the Fc fragment has no binding activity, so that the obtained fusion protein can specifically bind to the target ligand through the ligand binding fragment, and has specificity. However, the current full-length antibodies all have antigen binding activity, and it is not possible to fuse the full-length antibody with a specific ligand binding fragment to prepare a fusion protein. Therefore, the present invention provides a neutral antibody without antigen binding activity. This can be used as a skeleton to design an antibody fusion protein that binds to a specific ligand, and is used in pharmaceuticals, detection, adsorption, and other occasions. Summary of the invention

[0003] The present invention aims to solve one of the technical problems existing in the prior art to at least a certain extent. To this end, the present invention provides a neutral antibody, wherein the neutral antibody of the present invention has low antigen binding activity.

[0004] In the first aspect of the present invention, the present invention provides an antibody. According to an embodiment of the present invention, the antibody comprises: a light chain variable region CDR and a heavy chain variable region CDR; wherein the heavy chain variable region CDR1 sequence is shown as GX1X2X3X4X5X6X7X8X9, and the heavy chain variable region CDR2 sequence is shown as X 10 X 11 X 12 X 13 X 14 X 15 GX 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 The heavy chain variable region CDR3 sequence is shown in X 24 GGX 25 X 26 X 27 X 28 X 29 X 30 X 31 Y, X1 is G or F, X2 is G or N, X3 is G or I, X4 is G, K or S, X5 is G or D, X6 is G or T, X7 is G or Y, X8 is G or I, X9 is H or S, X 10 R or G, X 11 G or I, X12 G or Y, X 13 G or P, X 14 T or S, X 15 G or N, X 16 G or Y, X 17 G or T, X 18 G, S or R, X 19 G or Y, X 20 G or A, X 21 G or D, X 22 G or S, X 23 S or V, X 24 G or W, X 25 G or D, X 26 G or S, X 27 G or F, X 28 G or Y, X 29 G or A, X 30 G or M, X 31 is S, G or D; the condition is that X1 is F, X2 is N, X3 is I, X4 is K, X5 is D, X6 is T, X7 is Y, X8 is I, X9 is H, X 10 R, X 11 I, X 12 Y, X 13 P, X 14 For T, X 15 N, X 16 Y, X 17 For T, X 18 R, X 19 Y, X 20 A, X 21 D, X 22 For S, X 23 V, X 24 W, X 25 D, X 26 G, X 27 F, X 28 Y, X 29 A, X 30 M, X 31 D is not true at the same time; the light chain variable region CDR1 sequence is X 32 X 33 X 34 X 35 X 36 X 37 X 38 X 39 X 40 X 41 A, the light chain variable region CDR2 sequence is shown in X 42 X43 X 44 X 45 X 46 X 47 S, the light chain variable region CDR3 sequence is shown in X 48 X 49 X 50 X 51 X 52 X 53 X 54 X 55 X 56 As shown, X 32 G or R, X 33 G or A, X 34 G or S, X 35 G or Q, X 36 is G, D or S, X 37 G or V, X 38 G or N, X 39 G or T, X 40 G or A, X 41 S or V, X 42 G or S, X 43 G or A, X 44 G or S, X 45 G or F, X 46 For L or S, X 47 G or Y, X 48 G or Q, X 49 G or Q, X 50 G or H, X 51 G or Y, X 52 For S or T, X 53 G or T, X 54 G or P, X 55 G or P, X 56 G or T; the condition is X 32 R, X 33 A, X 34 For S, X 35 Q, X 36 D, X 37 V, X 38 N, X 39 For T, X 40 A, X 41 V, X 42 For S, X 43 A, X 44 For S, X 45 F, X 46 L, X 47 Y, X 48 Q, X 49 Q, X50 H, X 51 Y, X 52 For T, X 53 For T, X 54 P, X 55 P, X 56 The inventors have found through a large number of experiments that the above antibody has low antigen binding activity, and after forming an antibody fusion protein, it can effectively avoid non-specific binding of the antibody fusion protein. At the same time, more importantly, the expression level of the antibody is high, which is suitable for the development of antibody fusion protein drugs.

[0005] In the second aspect of the present invention, the present invention provides a first nucleic acid molecule. According to an embodiment of the present invention, the first nucleic acid molecule encodes the antibody described in the first aspect. The antibody encoded by the first nucleic acid molecule according to the embodiment of the present invention has low antigen binding activity, and after forming an antibody fusion protein, the non-specific binding of the antibody fusion protein can be effectively avoided. At the same time, the expression amount of the antibody is high, which is suitable for the development of antibody fusion protein drugs.

[0006] In the third aspect of the present invention, the present invention provides a first expression vector. According to an embodiment of the present invention, the first expression vector carries the first nucleic acid molecule described in the second aspect. After the first expression vector according to an embodiment of the present invention is introduced into a suitable recipient cell, the expression of the antibody with low antigen binding activity described in the first aspect can be effectively achieved under the mediation of the regulatory system, so as to obtain the antibody in large quantities.

[0007] In a fourth aspect of the present invention, the present invention provides a first recombinant cell. According to an embodiment of the present invention, the first recombinant cell comprises: 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 an 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.

[0008] In the fifth aspect of the present invention, the present invention proposes a use of the antibody described in the first aspect in the preparation of a drug. As mentioned above, the above-mentioned antibody of the present invention has no antigen binding activity, and it can be used as a neutral drug carrier to prepare a drug, and the drug can be used to treat or prevent a disease. For example, the above-mentioned antibody can be fused with an antigen epitope peptide to prepare a fusion protein. Since the above-mentioned antibody has no antigen binding activity, the prepared fusion protein specifically binds to the antibody corresponding to the antigen epitope peptide and does not bind to other antibodies, which can avoid the side effects of non-specific binding of the fusion protein, and can be used to treat or prevent related diseases caused by the antibody corresponding to the antigen epitope peptide.

[0009] According to an embodiment of the present invention, the drug includes a fusion protein or an ADC drug.

[0010] In the sixth aspect of the present invention, the present invention proposes a fusion protein. According to an embodiment of the present invention, the fusion protein includes: a neutral antibody, the neutral antibody includes the antibody described in the first aspect; one or more antigenic epitope peptides, the antigenic epitope peptides are connected to the neutral antibody. The inventors have found through experiments that by fusing the antigenic epitope peptide with the above-mentioned neutral antibody, the fusion protein obtained can competitively bind to the antibody corresponding to the antigenic epitope peptide. For example, the PLA2R antigenic epitope peptide, the antigenic epitope peptide in the fusion protein of the present invention binds to the antibody, the neutral antibody of the fusion protein contains an Fc fragment, the Fc fragment can bind to the FcγR receptor of the NK cell, thereby mediating the NK cell to directly kill the B cell expressing the membrane-type PLA2R antibody, the Fc fragment can also bind to the complement C1q and activate the complement system, and exert a lytic effect on the B cell expressing the membrane-type PLA2R antibody. Since the fusion protein of the present invention specifically binds to the PLA2R antibody and does not bind to other antibodies, it can specifically eliminate B cells and avoid the side effects of nonspecific immunity. In addition, the fusion protein of the present invention can also bind to and protect the liver FcRn receptor, thereby preventing the fusion protein from being degraded and having a very long plasma half-life. Therefore, as a drug, the fusion protein has the advantages of a 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.

[0011] In the sixth aspect of the present invention, the present invention proposes a use of the above-mentioned antibody as a drug carrier or a negative antibody. As mentioned above, the above-mentioned antibody of the present invention has no antigen binding activity, and it can be used as a neutral drug carrier to prepare drugs, or can be directly used as a negative antibody reference without antigen binding activity.

[0012] According to an embodiment of the present invention, the drug carrier or negative antibody has no antigen binding ability.

[0013] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0015] Figure 1 This is a schematic diagram of the structure of the expression framework in Example 1 of the present invention;

[0016] Figure 2 The electrophoresis result diagram of pcDNA3.4, trsH, trsL, RtxH and RtxL in Example 1 of the present invention;

[0017] Figure 3The electrophoresis result diagram of dTrs1H, dTrs2H and dTrs1L (dTrs2L) in Example 1 of the present invention;

[0018] Figure 4 The electrophoresis result diagram of dTrs3H and dTrs3L in Example 1 of the present invention;

[0019] Figure 5 The electrophoresis result diagram of dTrs4H and dTrs4L in Example 1 of the present invention;

[0020] Figure 6 The purification effect of Trs, dTrs1-4 and Rtx proteins detected by SDS-PAGE in Example 1 of the present invention;

[0021] Figure 7 The results of the detection of the binding of Trs, dTrs1-4 and Rtx proteins to Her2 in Example 1 of the present invention;

[0022] Figure 8 Schematic diagram of the structure of RC1-RC8 protein in Example 2 of the present invention;

[0023] Fig. 9 is a schematic diagram of the structure of PLA2R in Example 2 of the present invention;

[0024] Fig.10 The electrophoresis result of pcDNA3.4 in Example 2 of the present invention is shown;

[0025] Fig.11 The electrophoresis result diagram of pcDNA3.4, RC1H, RC2L, RC4H and RC5L in Example 2 of the present invention;

[0026] Fig.12 The electrophoresis result diagram of RC3 and RC6 in Example 2 of the present invention;

[0027] Fig.13 The purification effect of RC1-8 protein detected by SDS-PAGE in Example 2 of the present invention;

[0028] Fig.14 The neutralizing effect of RC1-RC8 in Example 2 of the present invention on PLA2R-Ab in the plasma of pMN-2 patients with membranous nephropathy positive;

[0029] Fig.15 The neutralizing effect of RC1-RC8 in Example 2 of the present invention on PLA2R-Ab in the plasma of pMN-4 patients with membranous nephropathy positive;

[0030] Fig.16 The neutralizing effect of RC1-RC8 in Example 2 of the present invention on PLA2R-Ab in the plasma of pMN-7, a patient with membranous nephropathy positive;

[0031] Fig.17 is the clearance rate of RC3 and RC6 in Example 2 of the present invention for the adsorption of PLA2R-Ab in the plasma samples of PMN-4 patients;

[0032] Fig.18 is the clearance rate of RC3 and RC6 in Example 2 of the present invention for the adsorption of PLA2R-Ab in the plasma samples of PMN-7 patients;

[0033] Fig.19 This is a weight trend chart of mice injected subcutaneously with RC1, RC2, RC4, RC5, RC7, and RC8 proteins in Example 2 of the present invention;

[0034] Fig. 20 This is a weight trend chart of mice injected subcutaneously with RC3 and RC6 proteins in Example 2 of the present invention;

[0035] Fig.21 The results of the detection of the binding of Trs, dTrs1-4 and Rtx proteins to IgA in Example 1 of the present invention;

[0036] Fig. 22 The results of the detection of the binding of Trs, dTrs1-4 and Rtx proteins to IgM in Example 1 of the present invention;

[0037] Fig.23 The results of the detection of the binding of Trs, dTrs1-4 and Rtx proteins to HSA in Example 1 of the present invention;

[0038] Fig.24 This is the electrophoresis result diagram of the stability study of RC1-RC8 in Example 2 of the present invention. DETAILED DESCRIPTION

[0039] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0040] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0041] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0042] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0043] In this article, the term "antibody" generally refers to an immunoglobulin molecule, which includes two light chains with a relatively low molecular weight and two heavy chains with a relatively high molecular weight. The heavy chain (H chain) and the light chain (L chain) are connected by disulfide bonds to form a tetrapeptide chain molecule. Among them, the amino acid sequence at the amino terminal (N-terminus) of the peptide chain varies greatly, which is called the variable region (V region), and the carboxyl terminal (C-terminus) is relatively stable and varies little, which is called the constant region (C region). The V regions of the L chain and H chain are called VL and VH, respectively. In the variable region, the amino acid composition and arrangement order of certain regions have a higher degree of variation, which is called the hypervariable region (HVR). The hypervariable region is the location where the antigen and antibody bind, so it is also called the complementarity-determining region (CDR). There are three CDRs on both the heavy chain variable region and the light chain variable region.

[0044] In this article, the term "framework region" or "framework region" is referred to as FR, which refers to the non-CDR part in the variable region of the heavy chain or light chain of the antibody. Among them, the near N-terminal of the H chain and L chain of the antibody has about 110 amino acid sequences that vary greatly, and the amino acid sequences of the other parts are relatively constant. Based on this, the light chain and heavy chain can be divided into a variable region (V region) and a constant region (C region). The variable region contains a hypervariable region HVR (hypervariable region) or a complementary determining region CDR (Complementarity-determining region) and a framework region, and the variability of FR is lower than that of CDR. There are four FR molecules, namely FR1, FR2, FR3 and FR4. When recognizing antibodies, the four FR molecules curl to make the CDR molecules close to each other.

[0045] Herein, the term "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.

[0046] As used herein, the term "diabody" refers to an antibody with a symmetrical structure, such as an antibody consisting of only two Fc fragments, or a complete antibody consisting of two heavy chains and two light chains, such as the antibody proposed in the first aspect of the present invention.

[0047] As used herein, the terms "identity", "homology" or "similarity" are used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see 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 There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment 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. 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. 85:24 ... .70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0048] As used herein, the term "nucleotide" generally refers to a ribonucleotide, a deoxynucleotide, or a modified form of either type of nucleotide, and combinations thereof.

[0049] As used herein, the term "at least 85% similarity" refers to at least 85%, and may be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% similarity to each reference sequence.

[0050] In this article, the term "expression vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and transfers the inserted nucleic acid molecule into and / or between host cells. The expression vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used for the expression of transcription and / or translation of DNA or RNA. The expression vector also includes vectors with multiple of the above functions. The expression vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.

[0051] In this article, the term "recombinant cell" generally refers to the use of genetic engineering technology or cell fusion technology to modify or reorganize the genetic material of the host cell to obtain a cell with a unique trait of stable inheritance. Among them, the term "host cell" refers to a prokaryotic cell or eukaryotic cell that can be introduced into a recombinant expression vector. The terms "transformed" or "transfected" used herein refer to the introduction of nucleic acids (such as vectors) into cells by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of the present invention, and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in the present invention include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), insect cells, PER.C6 cells and CoS cells.

[0052] In this article, the term "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, the expression product of two genes recombined by DNA recombination technology. In this article, it means that PLA2R and the neutral antibody of the double-chain structure are in a fusion state. It should be noted that PLA2R fusion protein does not mean that all parts of the protein are formed by fusion. For example, the two Fc segments in the double-chain structure can be connected together by chemical bonds such as disulfide bonds.

[0053] As used herein, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical field. All methods include the step of combining the active ingredient with a carrier that constitutes one or more accessory ingredients. Typically, the composition is prepared by uniformly and fully combining the active fusion protein with a liquid carrier, a finely divided solid carrier, or both.

[0054] In this article, the term "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 mammals treated therewith. Preferably, the "pharmaceutically acceptable" of the present invention refers to those approved by federal regulatory agencies or national governments or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeias for use in animals, particularly humans.

[0055] In this article, the term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. In addition to any conventional excipients incompatible with the fusion protein of the present invention, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of the present invention.

[0056] For other pharmaceutically acceptable excipients or other pharmaceutically acceptable carriers mentioned herein and their processes, reference can be made to a large number of literature on this topic, specifically see Handbook of Pharmaceutical Excipients, 3rd edition, edited by Arthur H. Kibbe, American Pharmaceutical Association, Washington, USA and Pharmaceutical Press, London; and Lexikon der Hilfsstoffe fürPharmazie, Kosmetik und angrenzende Gebiete, edited by HP Fiedler, 4th edition, edited by Cantor, Aulendorf and earlier versions.

[0057] In this article, the term "administration" refers to the introduction of a predetermined amount of material into a patient by a certain suitable mode. Fusion protein or pharmaceutical composition of the present invention can be administered by any common approach, as long as it can reach the expected tissue. The various modes of administration are expected to include peritoneal, intravenous, intramuscular, subcutaneous injections, etc., but the present invention is not limited to these exemplified modes of administration. Preferably, compositions of the present invention are administered by intravenous or subcutaneous injection.

[0058] In this article, the term "PLA2R antibody-induced 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. Among them, PLA2R is a membrane protein expressed in glomerular podocytes. Due to 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.

[0059] As used herein, the term "treatment" refers to the use of the term to refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or fusion protein to an individual to treat, cure, alleviate, improve, mitigate or inhibit an individual's disease, including but not limited to administering a drug containing the fusion protein described herein to an individual in need.

[0060] The present invention provides an antibody, a fusion protein and uses thereof, which will be described in detail below.

[0061] Antibody

[0062] The present invention mutates different sites of the CDR region of the Trastuzumab (Trs) antibody, and obtains the antibody of the present invention after a large number of experiments. The antibody is extremely weakly bound to its antigen Her2 and does not bind to any other antigens. The antigen binding activity of the antibody is extremely low, and the antibody can be used as a neutral antibody to fuse with a biologically active molecule to obtain a fusion protein. The fusion protein can bind to a specific binding substance of the biologically active molecule. For example, the fusion protein obtained by binding the neutral antibody to PLA2R can specifically bind to the PLA2R antibody and does not bind to other antibodies. When used as a drug, the side effects of non-specific binding are avoided. The fusion protein obtained by fusing the neutral antibody to the antigen is used as a drug, which can bind to the specific antibody of the antigen and does not bind to other antibodies, and can specifically eliminate the cells producing the specific antibody of the antigen.

[0063] Therefore, in the first aspect of the present invention, the present invention provides an antibody. According to an embodiment of the present invention, the antibody comprises: a light chain variable region CDR and a heavy chain variable region CDR; wherein the heavy chain variable region CDR1 sequence is shown as GX1X2X3X4X5X6X7X8X9, and the heavy chain variable region CDR2 sequence is shown as X 10 X 11 X 12 X 13 X 14 X 15 GX 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 The heavy chain variable region CDR3 sequence is shown in X 24 GGX 25 X 26 X 27 X 28 X 29 X 30 X 31 Y, X1 is G or F, X2 is G or N, X3 is G or I, X4 is G, K or S, X5 is G or D, X6 is G or T, X7 is G or Y, X8 is G or I, X9 is H or S, X 10 R or G, X 11 G or I, X 12 G or Y, X 13 G or P, X 14 T or S, X 15 G or N, X 16 G or Y, X 17 G or T, X 18 G, S or R, X 19 G or Y, X 20 G or A, X 21 G or D, X 22 G or S, X 23 S or V, X 24 G or W, X 25 G or D, X 26 G or S, X 27 G or F, X 28 G or Y, X 29 G or A, X 30 G or M, X 31 is S, G or D; the condition is that X1 is F, X2 is N, X3 is I, X4 is K, X5 is D, X6 is T, X7 is Y, X8 is I, X9 is H, X 10 R, X11 I, X 12 Y, X 13 P, X 14 For T, X 15 N, X 16 Y, X 17 For T, X 18 R, X 19 Y, X 20 A, X 21 D, X 22 For S, X 23 V, X 24 W, X 25 D, X 26 G, X 27 F, X 28 Y, X 29 A, X 30 M, X 31 D is not true at the same time; the light chain variable region CDR1 sequence is X 32 X 33 X 34 X 35 X 36 X 37 X 38 X 39 X 40 X 41 A, the light chain variable region CDR2 sequence is shown in X 42 X 43 X 44 X 45 X 46 X 47 S, the light chain variable region CDR3 sequence is shown in X 48 X 49 X 50 X 51 X 52 X 53 X 54 X 55 X 56 As shown, X 32 G or R, X 33 G or A, X 34 G or S, X 35 G or Q, X 36 is G, D or S, X 37 G or V, X 38 G or N, X 39 G or T, X 40 G or A, X 41 S or V, X 42 G or S, X 43 G or A, X 44G or S, X 45 G or F, X 46 For L or S, X 47 G or Y, X 48 G or Q, X 49 G or Q, X 50 G or H, X 51 G or Y, X 52 For S or T, X 53 G or T, X 54 G or P, X 55 G or P, X 56 G or T; the condition is X 32 R, X 33 A, X 34 For S, X 35 Q, X 36 D, X 37 V, X 38 N, X 39 For T, X 40 A, X 41 V, X 42 For S, X 43 A, X 44 For S, X 45 F, X 46 L, X 47 Y, X 48 Q, X 49 Q, X 50 H, X 51 Y, X 52 For T, X 53 For T, X 54 P, X 55 P, X 56 The inventors have found through a large number of experiments that the antigen binding activity of the above antibodies is extremely low and has the advantage of high expression level.

[0064] According to an embodiment of the present invention, the heavy chain variable region CDR and the light chain variable region CDR satisfy at least one of the following conditions: X4 is G or S, X5 is G, X 21 G, X 31 S or G, X 36 It is G or S.

[0065] According to an embodiment of the present invention, in the heavy chain variable region CDR, X1 is G or F, X2 is G or N, X3 is G or I, X4 is G or S, X5 is G, X6 is G or T, X7 is G or Y, X8 is G or I, X9 is H or S ...1 is G or F, X1 is G or F, X1 is G or F, X1 is G or F, X1 is G or F, X1 is G or F, X1 is G or F, X1 is G or F, X1 is G or F, X1 is G or F, X1 is G or F, X1 is G or F, X1 is G or F, X1 is G or F, X1 is G or F, X1 is 10 G, X 11 G or I, X 12 G or Y, X13 G or P, X 14 T or S, X 15 G or N, X 16 G, X 17 G or T, X 18 G or S, X 19 G or Y, X 20 G or A, X 21 G, X 22 G or S, X 23 S or V, X 24 G, X 25 G or D, X 26 G or S, X 27 G or F, X 28 G, X 29 G or A, X 30 G or M, X 31 is S or G; in the light chain variable region CDR, X 32 G or R, X 33 G or A, X 34 G or S, X 35 G or Q, X 36 G or S, X 37 G or V, X 38 G, X 39 G or T, X 40 G or A, X 41 S or V, X 42 G or S, X 43 G or A, X 44 G or S, X 45 G, X 46 For L or S, X 47 G or Y, X 48 G or Q, X 49 G or Q, X 50 G or H, X 51 G, X 52 For S or T, X 53 G or T, X 54 G or P, X 55 G or P, X 56 It is G or T.

[0066] According to an embodiment of the present invention, the antibody comprises a CDR sequence selected from at least one of the following: a 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; a 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.

[0067] RASQGVGTAVA (SEQ ID NO: 1).

[0068] SASGLYS (SEQ ID NO: 2).

[0069] QQHGTTPPT (SEQ ID NO: 3).

[0070] GFNIGGTGIH (SEQ ID NO: 4).

[0071] GIYPTNGGTGYAGSV (SEQ ID NO: 5).

[0072] GGGDGFGAMGY (SEQ ID NO: 6).

[0073] GGGGSGGGGSA (SEQ ID NO: 7).

[0074] GGGGSGS (SEQ ID NO: 8).

[0075] GGGGSGGGG (SEQ ID NO: 9).

[0076] GGGGSGGGGS (SEQ ID NO: 10).

[0077] GGGGSGGGGSGGGGS (SEQ ID NO: 11).

[0078] GGGGSGGGGSY (SEQ ID NO: 12).

[0079] RASQDVGTAVA (SEQ ID NO: 83).

[0080] SASGLYS (SEQ ID NO: 84).

[0081] QQHGTTPPT (SEQ ID NO: 85).

[0082] GFNIKDTYIH (SEQ ID NO: 86).

[0083] RIYPTNGGTRYADSV (SEQ ID NO:87).

[0084] WGGDGFGAMDY (SEQ ID NO: 88).

[0085] GFNIKDTGIH (SEQ ID NO: 89).

[0086] GIYPTNGGTGYADSV (SEQ ID NO:90).

[0087] GGGDGFGAMDY (SEQ ID NO:91).

[0088] In other embodiments, the antibodies provided by the present invention have conservative amino acid substitutions compared to the above-mentioned heavy and light chains. "Conservative amino acid substitution" refers to the replacement of an amino acid with another amino acid residue that is biologically, chemically or structurally similar. Biologically similar means that the replacement does not destroy the biological activity of the antibody's low antigen binding activity. Structurally similar means that the amino acids have side chains of similar length, such as alanine, glycine or serine, or have side chains of similar size. Chemical similarity means that the amino acids have the same charge or are hydrophilic or hydrophobic. For example, the hydrophobic residues isoleucine, valine, leucine or methionine replace each other. Or with polar amino acids, such as arginine replacing lysine, glutamic acid replacing aspartic acid, glutamine replacing asparagine, serine replacing threonine, etc.

[0089] According to an embodiment of the present invention, the light chain variable region CDR sequence includes at least one of the amino acid sequences having at least 85% similarity to SEQ ID NOs: 1 to 3, SEQ ID NOs: 7 to 9 and SEQ ID NOs: 83 to 85; the heavy chain variable region CDR sequence includes at least one of the amino acid sequences having at least 85% similarity to SEQ ID NOs: 4 to 6, SEQ ID NOs: 10 to 12, SEQ ID NOs: 86 to 88 and SEQ ID NOs: 89 to 91.

[0090] According to an embodiment of the present invention, the antibody comprises: light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 1, 2 and 3, or amino acid sequences having at least 85% similarity to SEQ ID NO: 1, 2 and 3, respectively; or, light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 7, 8 and 9, or amino acid sequences having at least 85% similarity to SEQ ID NO: 7, 8 and 9, respectively; or, light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 83, 84 and 85, or amino acid sequences having at least 85% similarity to SEQ ID NO: 83, 84 and 85, respectively.

[0091] According to an embodiment of the present invention, the antibody comprises: heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO:4, 5 and 6, or amino acid sequences having at least 85% similarity to SEQ ID NO:4, 5 and 6, respectively; or, heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO:10, 11 and 12, or amino acid sequences having at least 85% similarity to SEQ ID NO:10, 11 and 12, respectively; or, heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO:86, 87 and 88, or amino acid sequences having at least 85% similarity to SEQ ID NO:86, 87 and 88, respectively; or, heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO:89, 90 and 91, or amino acid sequences having at least 85% similarity to SEQ ID NO:89, 90 and 91, respectively.

[0092] According to an embodiment of the present invention, the antibody comprises: light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 1, 2 and 3, or amino acid sequences having at least 85% similarity to SEQ ID NOs: 1, 2 and 3, respectively, and heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 4, 5 and 6, or amino acid sequences having at least 85% similarity to SEQ ID NOs: 4, 5 and 6, respectively; or light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 7, 8 and 9, or amino acid sequences having at least 85% similarity to SEQ ID NOs: 7, 8 and 9, respectively, and heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 10, 11 and 12, or amino acid sequences having at least 85% similarity to SEQ ID NOs: 10, 11 and 12, respectively; or SEQ ID NOs: 83, 84 and 85, or amino acid sequences having at least 85% similarity to SEQ ID NOs: 84 and 86, respectively. NO:83, 84 and 85, respectively, and the light chain variable region CDR1, CDR2, CDR3 sequences shown in SEQ ID NO:83, 84 and 85, respectively, and the heavy chain variable region CDR1, CDR2, CDR3 sequences shown in SEQ ID NO:86, 87 and 88, respectively, or the amino acid sequences with at least 85% similarity to SEQ ID NO:86, 87 and 88; or the light chain variable region CDR1, CDR2, CDR3 sequences shown in SEQ ID NO:83, 84 and 85, respectively, or the amino acid sequences with at least 85% similarity to SEQ ID NO:83, 84 and 85, respectively, and the heavy chain variable region CDR1, CDR2, CDR3 sequences shown in SEQ ID NO:89, 90 and 91, respectively, or the amino acid sequences with at least 85% similarity to SEQ ID NO:89, 90 and 91.

[0093] According to an embodiment of the present invention, the antibody contains: 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 from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof, preferably a human antibody.

[0094] According to an embodiment of the present invention, the antibody has a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 95; and / or, the antibody has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 96 or SEQ ID NO: 97. The inventors can obtain the CDR region of the heavy chain variable region sequence (as shown in SEQ ID NO: 4-6, SEQ ID NO: 10-12, SEQ ID NO: 86-88, SEQ ID NO: 89-91) and the CDR region of the light chain variable region sequence (as shown in SEQ ID NO: 1-3, SEQ ID NO: 7-9, SEQ ID NO: 83-85) of the above antibodies through the antibody sequence comparison database (NCBI, IMGT) or related software.

[0095] DIQMTQSPSSSLSASVGDRVTITCRASQGVGTAVAWYQQKPGKAPKLLIYSASGLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHGTTPPTFGQGTKVEIK (SEQ ID NO: 13).

[0096] DIQMTQSPSSSLSASVGDRVTITCGGGGSGGGGSAWYQQKPGKAPKLLIYGGGGSGSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCGGGGSGGGGFGQGTKVEIK (SEQ ID NO: 14).

[0097] EVQLVESGGGLVQPGGSLRLSCAASGFNIGGTGIHWVRQAPGKGLEWVAGIYPTNGGTGYAGSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRGGGDGFGAMGYWGQGTLVTV (SEQ ID NO: 15).

[0098] EVQLVESGGGLVQPGGSLRLSCAASGGGGSGGGGSWVRQAPGKGLEWVAGGGGSGGGGSGGGGSKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRGGGGSGGGGSYWGQGTLVTV (SEQ ID NO: 16).

[0099] DIQMTQSPSSSLSASVGDRVTITCRASQDVGTAVAWYQQKPGKAPKLLIYSASGLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHGTTPPTFGQGTKVEIK (SEQ ID NO: 95).

[0100] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGGTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFGAMDYWGQGTLVTV (SEQ ID NO: 96).

[0101] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTGIHWVRQAPGKGLEWVAGIYPTNGGTGYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRGGGDGFGAMDYWGQGTLVTV (SEQ ID NO: 97).

[0102] According to an embodiment of the present invention, the light chain variable region sequence of the antibody has a conservative amino acid substitution compared to 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 to the amino acid sequence shown in SEQ ID NO: 15 or 16. Of course, these conservative amino acid substitutions will not change the biological function of the antibody. In some specific embodiments, these conservative amino acid substitutions may occur in amino acids other than the CDR region in the heavy chain variable region and the light chain variable region.

[0103] According to an embodiment of the present invention, the antibody has a light chain variable region with an amino acid sequence as shown in SEQ ID NO:13 and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:15.

[0104] According to an embodiment of the present invention, the antibody has a light chain variable region with an amino acid sequence as shown in SEQ ID NO:14 and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:16.

[0105] According to an embodiment of the present invention, the antibody has a light chain variable region with an amino acid sequence as shown in SEQ ID NO:95 and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:96.

[0106] According to an embodiment of the present invention, the antibody has a light chain variable region with an amino acid sequence as shown in SEQ ID NO:95 and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:97.

[0107] According to an embodiment of the present invention, the antibody contains 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 murine antibody, a human antibody, a primate antibody or a mutant thereof, preferably a human antibody.

[0108] According to an embodiment of the present invention, the antibody has a heavy chain having 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 a light chain having an amino acid sequence as shown in SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:34.

[0109] EVQLVESGGGLVQPGGSLRLSCAASGFNIGGTGIHWVRQAPGKGLEWVAGIYPTNGGTGYAGSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRGGGDGFGAMGYWGQG TLVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:17).

[0110] EVQLVESGGGLVQPGGSLRLSCAASGGGGSGGGGSWVRQAPGKGLEWVAGGGGSGGGGSGGGGSKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRGGGGSGGGGSYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEKPKSCDKTH TCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK(SEQ ID NO:18).

[0111] DIQMTQSPSSLSASVGDRVTITCRASQGVGTAVAWYQQKPGKAPKLLIYSASGLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHGTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:19).

[0112] DIQMTQSPSSLSASVGDRVTITCGGGGSGGGGSAWYQQKPGKAPKLLIYGGGSGSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCGGGGSGGGGFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:20).

[0113] According to an embodiment of the present invention, the antibody has a heavy chain having an amino acid sequence as shown in SEQ ID NO:17 and a light chain having an amino acid sequence as shown in SEQ ID NO:19.

[0114] According to an embodiment of the present invention, the antibody has a heavy chain having an amino acid sequence as shown in SEQ ID NO: 18 and a light chain having an amino acid sequence as shown in SEQ ID NO: 20.

[0115] According to an embodiment of the present invention, the antibody has a heavy chain having an amino acid sequence as shown in SEQ ID NO:33 and a light chain having an amino acid sequence as shown in SEQ ID NO:34.

[0116] According to an embodiment of the present invention, the antibody has a heavy chain having an amino acid sequence as shown in SEQ ID NO:35 and a light chain having an amino acid sequence as shown in SEQ ID NO:34.

[0117] First nucleic acid molecule, first expression vector, first recombinant cell

[0118] In the process of preparing or obtaining these antibodies, nucleic acid molecules expressing these antibodies can be connected to different vectors and then expressed in different cells to obtain the corresponding antibodies.

[0119] To this end, in the second aspect of the present invention, the present invention provides a first nucleic acid molecule. According to an embodiment of the present invention, the first nucleic acid molecule encodes the antibody described in the first aspect. The antibody encoded by the first nucleic acid molecule according to an embodiment of the present invention has extremely low antigen binding activity and has the advantage of high expression level.

[0120] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0121] 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 an amino acid sequence shown in SEQ ID NO:17.

[0122]

[0123] 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 an amino acid sequence shown in SEQ ID NO: 19.

[0124] (SEQ ID NO:26).

[0125] 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 an amino acid sequence shown in SEQ ID NO:18.

[0126]

[0127] 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 an amino acid sequence shown in SEQ ID NO: 20.

[0128] (SEQ ID NO:28).

[0129] Those skilled in the art will appreciate that the features and advantages described above for antibodies are also applicable to the first nucleic acid molecule and will not be described in detail here.

[0130] In the third aspect of the present invention, the present invention proposes a first expression vector. According to an embodiment of the present invention, the first expression vector carries the first nucleic acid molecule described in the second aspect. When the above-mentioned isolated polynucleotide is connected to the first expression vector, 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. Of course, these control elements can come directly from the first expression vector itself, or they can be exogenous, that is, not from the first expression vector itself. Of course, the polynucleotide and the control element can be operably connected.

[0131] Herein, "operably linked" means connecting the foreign gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended functions of regulating the transcription and translation of the foreign gene. Of course, the polynucleotides encoding the heavy and light chains of the antibody can be inserted into different vectors independently, and are commonly inserted into the same vector. Commonly used vectors can be, for example, plasmids, bacteriophages, etc.

[0132] According to an embodiment of the present invention, the first expression vector is a eukaryotic expression vector, preferably, the first expression vector is a lentiviral vector.

[0133] Those skilled in the art will appreciate that the features and advantages described above for antibodies are also applicable to the first expression vector and will not be described in detail here.

[0134] In the fourth aspect of the present invention, the present invention provides a first recombinant cell. According to an embodiment of the present invention, the first recombinant cell comprises: 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 a mammalian cell to construct a first recombinant cell, and then the first recombinant cell is used to express the antibody provided by the present invention. By culturing the first recombinant cell, the corresponding antibody can be obtained. These available mammalian cells can be, for example, CHO cells, etc.

[0135] According to an embodiment of the present invention, the recombinant cell is obtained by introducing the first expression vector into the host cell.

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

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

[0138] Those skilled in the art will appreciate that the features and advantages described above for antibodies are also applicable to the first recombinant cell and will not be described in detail here.

[0139] Those skilled in the art will appreciate that the features and advantages described above for antibodies are also applicable to the first nucleic acid molecule, the first expression vector and the first recombinant cell, and will not be described in detail here.

[0140] Fusion Protein

[0141] The invention is based on the following discoveries by the inventors:

[0142] For PLA2R antibody-positive membranous nephropathy, a possible treatment method is to specifically neutralize PLA2R-Ab in serum. The inventors were pleasantly surprised to find that the fusion protein of PLA2R and antibody developed in the present application (in the present application, the fusion protein of antigen and antibody is referred to as antibody fusion protein) can achieve the technical effect of specifically neutralizing PLA2R-Ab in serum.

[0143] PLA2R is a membrane protein highly expressed on the surface of glomerular podocytes, including Figure 1 At the same time, there is a small amount of secretory PLA2R in the blood. According to the research of Liyo Kao et al. (Liyo Kao, Vinson Lam, et al., J Am Soc Nephrol. 2015 Feb; 26(2): 291-301), the antibodies in the patient's plasma mainly recognize the three domains of CysR, FNII and CTLD1 in PLA2R.

[0144] Based on this, the inventors found that the fusion protein obtained by fusing the antigen region of PLA2R with the antibody can maintain the binding ability of PLA2R to PLA2R-Ab. In addition, during the research and development process, the inventors found that by mutating the CDR region of the original antibody, the antigen binding activity of the antibody obtained was extremely low, which can avoid the side effects caused by subsequent non-specific binding and has a higher expression level. Therefore, the fusion protein obtained by fusing the mutated antibody with the antigen region of PLA2R specifically neutralizes PLA2R-Ab and does not bind to other antibodies, avoiding the side effects of non-specific immunity. In addition, the fusion protein also has the advantage of a long half-life, which can reduce the frequency of administration.

[0145] In the fifth aspect of the present invention, the present invention provides a fusion protein. According to an embodiment of the present invention, the fusion protein comprises: a neutral antibody, the neutral antibody comprises an antibody FC segment or the antibody described in the first aspect; and one or more PLA2R antigen epitope peptides, the PLA2R antigen epitope peptides being linked to the neutral antibody.

[0146] The inventors have found through experiments that by fusing the PLA2R antigen epitope peptide with the above-mentioned neutral antibody, the obtained fusion protein can competitively bind to the PLA2R antibody, prevent the PLA2R antibody from binding to the PLA2R in the body, and effectively treat the related diseases caused by the PLA2R antibody. At the same time, the PLA2R antigen epitope peptide in the fusion protein of the present invention binds to the PLA2R antibody, and the neutral antibody of the fusion protein contains an Fc fragment, which can bind to the FcγR receptor of NK cells, thereby mediating the direct killing of B cells expressing membrane-type PLA2R antibodies by NK cells. The Fc fragment 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 the PLA2R antibody and does not bind to other antibodies, it can specifically eliminate B cells and avoid the side effects of nonspecific immunity. In addition, the fusion protein of the present invention can also bind to the liver FcRn receptor and be protected, avoiding the degradation of the fusion protein, and has a very long plasma half-life. Therefore, as a drug, the fusion protein has the advantages of a long half-life, which can reduce the frequency of administration. It also has the advantages of good expression, high yield, high stability, high affinity, good solubility and good drugability.

[0147] Those skilled in the art will appreciate that amino acid fragments are generally divided into an N-terminus and a C-terminus, and the present invention does not impose strict limitations on the connection method between the neutral antibody and the PLA2R antigen epitope peptide, as long as the N-terminus of one fragment is connected to the C-terminus of another fragment.

[0148] According to an embodiment of the present invention, the C-terminus of the PLA2R antigen epitope peptide is connected to the N-terminus of the neutral antibody.

[0149] According to an embodiment of the present invention, the PLA2R antigen epitope peptide includes CysR, FnII and PLA2R binding region 1 and / or PLA2R binding region 2.

[0150] It should be noted that the specific structures of CysR, FnII, PLA2R binding region 1 and PLA2R binding region 2 can be found in Fig. 9 .

[0151] According to an embodiment of the present invention, the C-terminus of the CysR is connected to the N-terminus of the FnII, and the C-terminus of the FnII is connected to the N-terminus of the PLA2R binding region 1.

[0152] According to an embodiment of the present invention, the C-terminus of the CysR is connected to the N-terminus of the FnII, the C-terminus of the FnII is connected to the N-terminus of the PLA2R binding region 1, and the C-terminus of the PLA2R binding region 1 is connected to the N-terminus of the PLA2R binding region 2.

[0153] According to an embodiment of the present invention, the PLA2R antigen epitope peptide has an amino acid sequence as shown in SEQ ID NO: 21 or 22 or an amino acid sequence having at least 95% similarity to SEQ ID NO: 21 or 22.

[0154] EGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTRE GREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVE(SEQ ID NO:21).

[0155] EGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRK YIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTL LSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVEKDAWKYYATHCEPGWNPYNRNCY KLQKEEKTWHEALRSCQADNSALIDITSLAEVEFLVTLLGDENASETWIGLSSNKIPVSFEWSNDSSVIFTNWHTLEPHIFPNRSQLCVSAEQSEGHWKVKNCEERLFYICKKAGHVLSD(SEQ ID NO:22).

[0156] According to an embodiment of the present invention, the neutral antibody is an antibody FC segment, and the C-terminus of the PLA2R antigen epitope peptide is connected to the N-terminus of at least one of the antibody FC segments.

[0157] According to an embodiment of the present invention, the neutral antibody is the antibody according to the first aspect, and the C-terminus of the PLA2R antigen epitope peptide is connected to the N-terminus of at least one light chain or heavy chain of the antibody.

[0158] According to an embodiment of the present invention, the neutral antibody is a double-chain antibody, and the N-terminus of each chain of the antibody FC segment is respectively connected to the C-terminus of a PLA2R antigen epitope peptide. The fusion protein contains two PLA2R antigen epitope peptides, that is, a bivalent molecule. The inventors have found through experiments that, compared with the monovalent natural PLA2R, the fusion protein of the present invention has a stronger binding ability with the PLA2R antibody, can effectively neutralize the PLA2R antibody, and protect the natural PLA2R on the surface of the glomerular group cells from being attacked by the PLA2R antibody.

[0159] According to an embodiment of the present invention, 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 antigen epitope peptide. The fusion protein contains two PLA2R antigen epitope peptides, that is, a bivalent molecule. The inventors have found through experiments that, compared with the monovalent natural PLA2R, the fusion protein of the present invention has a stronger binding ability with the PLA2R antibody, can effectively neutralize the PLA2R antibody, and protect the natural PLA2R on the surface of the glomerular group cells from being attacked by the PLA2R antibody.

[0160] According to an embodiment of the present invention, 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 antigen epitope peptide. The fusion protein contains two PLA2R antigen epitope peptides, that is, a bivalent molecule. The inventors have found through experiments that, compared with the monovalent natural PLA2R, the fusion protein of the present invention has a stronger binding ability with the PLA2R antibody, can effectively neutralize the PLA2R antibody, and protect the natural PLA2R on the surface of the glomerular group cells from being attacked by the PLA2R antibody.

[0161] According to an embodiment of the present invention, 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 antigen epitope peptide. The inventors have found through experiments that the fusion protein contains four PLA2R antigen epitope peptides. Compared with the monovalent natural PLA2R, the fusion protein of the present invention has a stronger binding ability with the PLA2R antibody, can effectively neutralize the PLA2R antibody, and protect the natural PLA2R on the surface of the glomerular group cells from being attacked by the PLA2R antibody.

[0162] According to an embodiment of the present invention, the fusion protein further comprises a connecting peptide, and the connecting peptide is disposed between the PLA2R antigen epitope peptide and the antibody.

[0163] According to an embodiment of the present invention, the N-terminus of the connecting peptide is connected to the C-terminus of the PLA2R antigen epitope peptide, and the C-terminus of the connecting peptide is connected to the N-segment of the neutral antibody. Thus, the PLA2R antigen epitope peptide and the neutral antibody that can be fused through the connecting peptide are used to improve the binding ability of the final fusion protein with the PLA2R antibody.

[0164] According to an embodiment of the present invention, the amino acid sequence of the connecting peptide is (GGGGS) n , wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0165] According to an embodiment of the present invention, the connecting peptide includes the amino acid sequence shown in SEQ ID NO:23.

[0166] GGGGSGGGGS (SEQ ID NO: 23).

[0167] According to an embodiment of the present invention, the antibody FC segment has an amino acid sequence as shown in SEQ ID NO: 24 or has at least 90% similarity to SEQ ID NO: 24.

[0168] THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:24).

[0169] According to an embodiment of the present invention, the fusion protein has a first peptide segment having an amino acid sequence as shown in SEQ ID NO: 57 and a second peptide segment having an amino acid sequence as shown in SEQ ID NO: 19; or

[0170] The fusion protein comprises a first peptide segment having an amino acid sequence as shown in SEQ ID NO: 17 and a second peptide segment having an amino acid sequence as shown in SEQ ID NO: 59; or

[0171] The amino acid sequence of the fusion protein is shown in SEQ ID NO:61; or

[0172] The fusion protein comprises a first peptide segment having an amino acid sequence as shown in SEQ ID NO: 63 and a second peptide segment having an amino acid sequence as shown in SEQ ID NO: 19; or

[0173] The fusion protein has a first peptide segment having an amino acid sequence as shown in SEQ ID NO: 17 and a second peptide segment having an amino acid sequence as shown in SEQ ID NO: 65; or

[0174] The amino acid sequence of the fusion protein is shown in SEQ ID NO: 67; or

[0175] The fusion protein has a first peptide segment having an amino acid sequence as shown in SEQ ID NO: 57 and a second peptide segment having an amino acid sequence as shown in SEQ ID NO: 59; or

[0176] The fusion protein has a first peptide segment having an amino acid sequence as shown in SEQ ID NO:63 and a second peptide segment having an amino acid sequence as shown in SEQ ID NO:65.

[0177] For example, reference Figure 8 When the neutral antibody is the antibody described above (or called the first antibody), the first peptide segment of the fusion protein is a peptide segment including the heavy chain of the first antibody, and the second peptide segment of the fusion protein is a peptide segment including the light chain of the first antibody, and the first peptide segment and the second peptide segment are connected by an interchain disulfide bond; when the neutral antibody is the antibody Fc fragment described above, the fusion protein is a peptide segment including the antibody Fc fragment.

[0178] Those skilled in the art will appreciate that the features and advantages described above for antibodies are also applicable to the fusion protein and will not be described in detail here.

[0179] Second nucleic acid molecule, second expression vector, second recombinant cell

[0180] In the process of preparing or obtaining these fusion proteins, 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.

[0181] To this end, in the sixth aspect of the present invention, the present invention proposes a second nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the fusion protein described in the fifth aspect. The second nucleic acid molecule can be effectively used to express the above-mentioned fusion protein, especially in a prokaryotic or lower eukaryotic expression system. It should be noted that the second nucleic acid molecule here does not need to encode the entire sequence of the above-mentioned fusion protein. For example, it can include two types of nucleic acid molecules, each encoding two monomers, which can be further assembled in vitro to obtain a heterodimer, thereby forming a fusion protein containing a double-chain antibody.

[0182] According to an embodiment of the present invention, the second nucleic acid molecule is DNA.

[0183] In the seventh aspect of the present invention, the present invention provides a second expression vector. According to an embodiment of the present invention, the second expression vector carries the second nucleic acid molecule described in the sixth aspect. The second expression vector can be effectively used to express the above-mentioned fusion protein, especially in a prokaryotic or lower eukaryotic expression system.

[0184] According to an embodiment of the present invention, the second expression vector is a eukaryotic expression vector, preferably, the second expression vector is a lentiviral vector.

[0185] In the eighth aspect of the present invention, the present invention provides a second recombinant cell. According to an embodiment of the present invention, the second recombinant cell comprises: carrying the second nucleic acid molecule described in the sixth aspect; or expressing the fusion protein described in the fifth aspect. The recombinant cell according to an embodiment of the present invention can be used for in vitro expression and mass production of the fusion protein described in the fifth aspect.

[0186] According to an embodiment of the present invention, the second recombinant cell is a eukaryotic cell.

[0187] According to an embodiment of the present invention, the second recombinant cell is a mammalian cell.

[0188] Those skilled in the art will appreciate that the features and advantages described above for the fusion protein are also applicable to the second nucleic acid molecule, the second expression vector and the second recombinant cell, and will not be described in detail here.

[0189] Pharmaceutical composition, immunoadsorbent, kit

[0190] In the ninth aspect of the present invention, the present invention proposes a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: the fusion protein described in the fifth aspect. The inventors have found through a large number of experiments that the pharmaceutical composition can effectively prevent and treat related diseases caused by PLA2R antibodies, such as PLA2R antibody-positive membranous nephropathy.

[0191] According to an embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0192] In the tenth aspect of the present invention, the present invention proposes an immunosorbent. According to an embodiment of the present invention, the immunosorbent includes: the fusion protein described in the fifth aspect. The inventors have found through a large number of experiments that the above-mentioned immunosorbent can adsorb PLA2R antibodies, which can effectively treat related diseases caused by PLA2R antibodies, such as PLA2R antibody-positive membranous nephropathy, through immunoabsorption.

[0193] In the eleventh aspect of the present invention, the present invention proposes a kit. According to an embodiment of the present invention, the kit comprises: the fusion protein described in the fifth aspect. The inventors have found through a large number of experiments that the above kit can effectively detect the content of PLA2R antibodies, and can also be used to diagnose or treat related diseases caused by PLA2R antibodies, such as PLA2R antibody-positive membranous nephropathy.

[0194] Those skilled in the art will appreciate that the features and advantages described above for the fusion protein, the second nucleic acid molecule, the second expression vector and the second recombinant cell are also applicable to the pharmaceutical composition, the immunosorbent and the kit, and will not be described in detail here.

[0195] Adsorption products and their uses

[0196] In another aspect of the present invention, the present invention provides an adsorption product. According to an embodiment of the present invention, the adsorption product comprises: the aforementioned immunosorbent; a solid phase carrier, wherein the immunosorbent 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, and can be used to prevent or treat diseases related to PLA2R antibodies by adsorbing PLA2R antibodies in blood or plasma.

[0197] It should be noted that the connection method between the immunoadsorbent and the solid phase carrier is not particularly limited, and they can be coupled or connected via chemical bonds.

[0198] Herein, the blood or plasma may be derived from any animal, such as a mammal.

[0199] Exemplarily, the blood or plasma is selected from human blood or plasma.

[0200] According to an embodiment of the present invention, the adsorption product is selected from an immunoadsorption column or a blood purifier.

[0201] In another aspect of the present invention, the present invention provides a use of the aforementioned immunosorbent in preparing an adsorption product, wherein the adsorption product is used for adsorbing PLA2R antibodies.

[0202] According to an embodiment of the present invention, the adsorption product is selected from an immunoadsorption column or a blood purifier.

[0203] According to an embodiment of the present invention, the adsorption product is an immunoadsorption column, and the adsorption product is used to prevent or treat diseases related to PLA2R antibodies. As mentioned above, the immunoadsorbent can adsorb PLA2R antibodies, and thus, the immunoadsorption column containing the immunoadsorbent can adsorb PLA2R antibodies through the immunoadsorbent, and the immunoadsorption column can effectively treat diseases related to PLA2R antibodies.

[0204] According to an embodiment of the present invention, the PLA2R antibody-induced related diseases are selected from PLA2R antibody-positive membranous nephropathy.

[0205] According to an embodiment of the present invention, the adsorption product is a blood purifier, and the adsorption product is used to adsorb PLA2R antibodies in blood or plasma. As can be seen from the above, the above-mentioned immunosorbent can adsorb PLA2R antibodies, and thus, the blood purifier containing the above-mentioned immunosorbent can adsorb PLA2R antibodies through the immunosorbent, and the blood purifier can effectively adsorb PLA2R antibodies in blood or plasma.

[0206] use

[0207] In the twelfth aspect of the present invention, the present invention proposes a use of the fusion protein described in the fifth aspect, the second nucleic acid molecule described in the sixth aspect, the second expression vector described in the seventh aspect, the second recombinant cell described in the eighth aspect or the pharmaceutical composition described in the ninth aspect in the preparation of a drug for preventing or treating related diseases caused by PLA2R antibodies.

[0208] According to an embodiment of the present invention, the PLA2R antibody-induced related diseases are selected from PLA2R antibody-positive membranous nephropathy.

[0209] According to an embodiment of the present invention, the dosage form of the Chinese medicine composition includes an injection.

[0210] According to an embodiment of the present invention, the administration route of the traditional Chinese medicine composition includes subcutaneous injection or intravenous injection.

[0211] Those skilled in the art will appreciate that the features and advantages described above for the pharmaceutical composition, immunosorbent and kit are also applicable to this use and will not be described in detail here.

[0212] In the thirteenth aspect of the present invention, the present invention proposes a use of the fusion protein described in the fifth aspect, the second nucleic acid molecule described in the sixth aspect, the second expression vector described in the seventh aspect, the second recombinant cell described in the eighth aspect or the pharmaceutical composition described in the ninth aspect in the preparation of an immunoadsorbent, wherein the immunoadsorbent is used to adsorb PLA2R antibodies.

[0213] Those skilled in the art will appreciate that the features and advantages described above for the pharmaceutical composition, immunosorbent and kit are also applicable to this use and will not be described in detail here.

[0214] In the fourteenth aspect of the present invention, the present invention proposes a use of the fusion protein described in the fifth aspect, the second nucleic acid molecule described in the sixth aspect, the second expression vector described in the seventh aspect, the second recombinant cell described in the eighth aspect, the pharmaceutical composition described in the ninth aspect or the immunoadsorbent described in the tenth aspect in the preparation of a kit for detecting PLA2R antibodies.

[0215] Those skilled in the art will appreciate that the features and advantages described above for the fusion protein, pharmaceutical composition, immunosorbent and kit are also applicable to the above-mentioned uses and will not be described in detail here.

[0216] method

[0217] In the fifteenth aspect of the present invention, the present invention provides a method for preventing or treating diseases related to PLA2R antibodies. According to an embodiment of the present invention, the method comprises: administering a pharmaceutically acceptable amount of the fusion protein described in the fifth aspect or the pharmaceutical composition described in the ninth aspect to a subject. According to an embodiment of the present invention, the method can effectively prevent or treat diseases related to PLA2R antibodies.

[0218] According to an embodiment of the present invention, the administration route of the method is subcutaneous injection or intravenous injection.

[0219] According to an embodiment of the present invention, the PLA2R antibody-induced related diseases are selected from PLA2R antibody-positive membranous nephropathy.

[0220] In the sixteenth aspect of the present invention, the present invention provides an immunoadsorption therapy method for preventing or treating diseases related to PLA2R antibodies. According to an embodiment of the present invention, the method comprises: treating a subject with the immunoadsorbent described in the tenth aspect.

[0221] According to an embodiment of the present invention, the method uses extracorporeal blood purification to treat the subject.

[0222] According to an embodiment of the present invention, the PLA2R antibody-induced related diseases are selected from PLA2R antibody-positive membranous nephropathy.

[0223] In the seventeenth aspect of the present invention, the present invention proposes a method for diagnosing a disease related to PLA2R antibodies. According to an embodiment of the present invention, the method comprises: using the fusion protein described in the fifth aspect, the second nucleic acid molecule described in the sixth aspect, the second expression vector described in the seventh aspect, the second recombinant cell described in the eighth aspect, the pharmaceutical composition described in the ninth aspect, the immunosorbent described in the tenth aspect, or the kit described in the eleventh aspect to detect the PLA2R antibodies in the sample to be tested; based on the detection result, determining the content of the PLA2R antibodies in the sample to be tested.

[0224] According to an embodiment of the present invention, the content of PLA2R antibodies in the test sample being not less than 14RU / ml is an indication that the test sample is derived from a patient suffering from a related disease caused by PLA2R antibodies. Preferably, the content of PLA2R antibodies in the test sample being not less than 20RU / ml is an indication that the test sample is derived from a patient suffering from a related disease caused by PLA2R antibodies.

[0225] According to an embodiment of the present invention, the sample to be tested is blood.

[0226] According to an embodiment of the present invention, the PLA2R antibody-induced related diseases are selected from PLA2R antibody-positive membranous nephropathy.

[0227] In the eighteenth aspect of the present invention, the present invention proposes a method for evaluating the staging of related diseases caused by PLA2R antibodies. According to an embodiment of the present invention, the method comprises: using the fusion protein described in the fifth aspect, the second nucleic acid molecule described in the sixth aspect, the second expression vector described in the seventh aspect, the second recombinant cell described in the eighth aspect, the pharmaceutical composition described in the ninth aspect, the immunosorbent described in the tenth aspect, or the kit described in the eleventh aspect to detect the PLA2R antibodies in the sample to be tested, wherein the sample to be tested is derived from the patient to be tested; based on the detection result, determining the content of the PLA2R antibodies in the sample to be tested.

[0228] According to an embodiment of the present invention, based on the content of PLA2R antibodies in the test sample, the stage of the disease caused by PLA2R antibodies in the test patient is determined.

[0229] According to an embodiment of the present invention, the PLA2R antibody-induced related diseases are selected from PLA2R antibody-positive membranous nephropathy.

[0230] According to an embodiment of the present invention, PLA2R antibody-positive membranous nephropathy is divided into four stages.

[0231] According to an embodiment of the present invention, the sample to be tested is blood.

[0232] In the nineteenth aspect of the present invention, the present invention proposes a method for evaluating the prognosis of a disease related to a PLA2R antibody. According to an embodiment of the present invention, the method comprises: using the fusion protein of the fifth aspect, the second nucleic acid molecule of the sixth aspect, the second expression vector of the seventh aspect, the second recombinant cell of the eighth aspect, the pharmaceutical composition of the ninth aspect, the immunosorbent of the tenth aspect, or the kit of the eleventh aspect to detect the PLA2R antibody in the sample to be tested; based on the detection result, determining the content of the PLA2R antibody in the sample to be tested.

[0233] According to an embodiment of the present invention, the sample to be tested is derived from a patient suffering from a disease related to PLA2R antibody before or after treatment.

[0234] According to an embodiment of the present invention, the prognostic effect of a disease related to PLA2R antibodies is determined based on the content of PLA2R antibodies in a test sample of a patient suffering from a disease related to PLA2R antibodies before or after treatment.

[0235] According to an embodiment of the present invention, a decrease in the content of PLA2R antibodies in a test sample of a patient suffering from a disease caused by PLA2R antibodies after treatment is an indication of a good prognosis for the patient.

[0236] According to an embodiment of the present invention, the sample to be tested is blood.

[0237] According to an embodiment of the present invention, the PLA2R antibody-induced related diseases are selected from PLA2R antibody-positive membranous nephropathy.

[0238] Those skilled in the art will appreciate that the features and advantages described above for the fusion protein, pharmaceutical composition, immunosorbent and kit are also applicable to the above-mentioned method and will not be described in detail here.

[0239] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0240] Example 1: Preparation and selection of neutral antibodies

[0241] 1. Expression Construction

[0242] First, the plasmid vector used is the pcDNA3.4 transient expression vector, which contains the native full-length CMV promoter and the WPRE element downstream of the cloning site. The vector based on the CMV promoter usually has a good expression level 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 the gene. In Escherichia coli, it has an ampicillin resistance gene. Then, the vector gene pcDNA3.4 and the target gene are amplified by PCR technology. The PCR template is obtained by gene synthesis. The vector fragment and the target gene fragment are connected by a homologous recombination kit. The DH5α competent cell is transformed to obtain a single clone. The single clone is amplified and sequenced. After sequencing confirmation, the plasmid is extracted to obtain the target gene recombinant expression plasmid. Among them, the target sequence encoding DNA plus the secretory expression signal peptide encoding DNA are gene synthesized and cloned between the CMV promoter and WPRE gene of the plasmid pcDNA3.4. For details, see Figure 1 .

[0243] 1.1 The name and sequence of the target gene are shown in Table 1, and the primers and templates of the pcDNA3.4 vector and the target gene (or target fragment) are shown in Table 2, where RtxH and RtxL are used to synthesize rituximab (Rtx for short).

[0244] Table 1: Names and sequences of target genes

[0245]

[0246] Note: dTrs1L and dTrs2L in this example are the same target gene.

[0247] Among them, the amino acid sequence of RtxH is:

[0248] QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:29).

[0249] Amino acid sequence of RtxL:

[0250] QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:30).

[0251] Amino acid sequence of TrsH:

[0252] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:31).

[0253] Amino acid sequence of TrsL:

[0254] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:32).

[0255] Amino acid sequence of dTrs1H:

[0256] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGGTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFGAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:33).

[0257] Amino acid sequence of dTrs1L:

[0258] DIQMTQSPSSLSASVGDRVTITCRASQDVGTAVAWYQQKPGKAPKLLIYSASGLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHGTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:34).

[0259] Amino acid sequence of dTrs2H:

[0260] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTGIHWVRQAPGKGLEWVAGIYPTNGGTGYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRGGGDGFGAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:35).

[0261] Nucleotide sequence of RtxH:

[0262]

[0263] Nucleotide sequence of RtxL:

[0264] CAGATCGTGTTGTCGCAATCCCCGGCCATCCTGTCCGCTTCGCCGGGAGAAAAGGTCACCATGACTTGTCGCGCCTCAAGCAGCGTGTCCTACATTCATTGGTTTCAGCAGAAGCCCGGGTCCAGCCCGAAGCCTTGGATCTACGCAACTAGCAACCTCGCCTCCGGCGTGCCAGTGCGCTTCTCCGGTTCCGGATCGGGAACGTCCTATAGCCTGACTATCTCGAGAGTGGAGGCCGAGGACGCTGCCACTTACTACTGCCAGCAGTGGACCTCGAATCCTCCAACTTTCGGTGGCGGCACCAAGCTGGAGATTAAGAGGACCGTCGCGGCCCCTAGCGTGTTCATTTTCCCCCCGTCCGACGAGCAGCTGAAGTCCGGGACAGCCTCAGTGGTCTGCCTGCTCAACAACTTCTACCCCCGGGAAGCCAAAGTGCAGTGGAAAGTGGATAACGCGCTTCAGAGCGGCAACTCACAAGAGTCCGTGACCGAACAGGACTCGAAGGACAGCACCTACAGCCTCTCCTCCACCCTGACCCTGTCAAAGGCGGATTACGAAAAGCACAAGGTCTACGCCTGCGAAGTGACCCACCAGGGGCTGTCCTCTCCCGTGACCAAGTCCTTCAACCGGGGAGAGTGC(SEQ ID NO:37).

[0265] Nucleotide sequence of TrsH:

[0266]

[0267] Nucleotide sequence of TrsL:

[0268] GATATACAGATGACGCAGAGCCCTAGCAGTCTAAGTGCAAGCGTTGGAGACCGTGTCACCATTACCTGCCGCGCTAGCCAGGATGTCAACACCGCAGTCGCCTGGTATCAGCAAAAACCGGGTAAGGCTCCAAAGCTGTTGATCTACAGTGCCAGCTTTCTGTACAGCGGTGTGCCAAGTAGGTTCAGCGGGAGCCGGAGTGGGACAGACTTTACACTCACTATTAGCAGTCTGCAGCCCGAGGACTTCGCCACCTACTACTGTCAGCAGCATTATACAACACCCCCAACATTCGGGCAGGGAACCAAAGTTGAGATAAAACGAACGGTTGCCGCGCCAAGCGTATTTATCTTTCCCCCCAGTGATGAGCAACTGAAGAGTGGTACAGCCAGCGTTGTCTGCCTTTTGAATAACTTCTATCCTCGGGAGGCAAAGGTCCAGTGGAAAGTGGATAATGCACTCCAAAGCGGTAACAGCCAGGAGAGTGTGACAGAACAGGACAGCAAAGATAGTACCTACAGCCTCAGTAGTACCCTCACACTGAGCAAAGCGGACTATGAAAAGCACAAGGTGTACGCTTGTGAAGTGACCCACCAGGGCCTAAGCAGCCCTGTTACTAAGAGCTTCAACAGAGGCGAATGT(SEQ ID NO:39).

[0269] Nucleotide sequence of dTrs1H:

[0270]

[0271] Nucleotide sequence of dTrs1L (dTrs2L):

[0272] GATATACAGATGACGCAGAGCCCTAGCAGTCTAAGTGCAAGCGTTGGAGACCGTGTCACCATTACCTGCCGCGCTAGCCAGGATGTCGGCACCGCAGTCGCCTGGTATCAGCAAAAACCGGGTAAGGCTCCAAAGCTGTTGATCTACAGTGCCAGCGGCCTGTACAGCGGTGTGCCAAGTAGGTTCAGCGGGAGCCGGAGTGGGACAGACTTTACACTCACTATTAGCAGTCTGCAGCCCGAGGACTTCGCCACCTACTACTGTCAGCAGCATGGCACAACACCCCCAACATTCGGGCAGGGAACCAAAGTTGAGATAAAACGAACGGTTGCCGCGCCAAGCGTATTTATCTTTCCCCCCAGTGATGAGCAACTGAAGAGTGGTACAGCCAGCGTTGTCTGCCTTTTGAATAACTTCTATCCTCGGGAGGCAAAGGTCCAGTGGAAAGTGGATAATGCACTCCAAAGCGGTAACAGCCAGGAGAGTGTGACAGAACAGGACAGCAAAGATAGTACCTACAGCCTCAGTAGTACCCTCACACTGAGCAAAGCGGACTATGAAAAGCACAAGGTGTACGCTTGTGAAGTGACCCACCAGGGCCTAAGCAGCCCTGTTACTAAGAGCTTCAACAGAGGCGAATGT (SEQ ID NO:41).

[0273] Nucleotide sequence of dTrs2H:

[0274]

[0275] Table 2: Primers and templates for pcDNA3.4 vector and target gene

[0276]

[0277] 1.2 PCR reaction used TAKARA's KOD reaction system (Cat. KFX-101), reaction conditions, reagents and PCR target fragment information as shown in Tables 3 to 6.

[0278] Table 3: KOD reaction system of target gene or pcDNA3.4

[0279]

[0280]

[0281] Table 4: PCR reaction conditions of vector pcDNA3.4

[0282] program temperature time condition 1 94℃ 2min 2 98℃ 10s 3 60℃ 30s 4 68℃ 6min Goto Step 2: 30 cycles 5 10℃ permanent

[0283] Table 5: PCR reaction conditions for target genes

[0284] program temperature time condition 1 94℃ 2min 2 98℃ 10s 3 60℃ 30s 4 68℃ 2-6min Goto Step 2: 30 cycles 5 10℃ permanent

[0285] Table 6: PCR target fragment information

[0286] DNA Primers template Theoretical length pcDNA3.4 vector Vector-f,Vector-r Synthetic gene pcDNA3.4 vector 6000bp HkDJ RtxH-f,RtxH-r Synthetic gene RtxH 1500bp RxF RtxL-f,RtxL-r Synthetic gene RtxL 800bp TxD TrsH-f, TrsH-r Synthetic gene TrsH 1500bp TxD TrsL-f, TrsL-r Synthetic gene TrsL 800bp dT dTrsH-f,dTrsH-r Synthetic gene dTrs1H 1455bp dTrs1L(dTrs2L) dTrsL-f,dTrsL-r Synthetic gene dTrs1L (dTrs2L) 744bp dT dTrsH-f,dTrsH-r Synthetic gene dTrs2H 1455bp dT dTrsH-f,dTrsH-r Synthetic gene dTrs3H 1455bp dT dTrsL-f,dTrsL-r Synthetic gene dTrs3L 744bp dT dTrsH-f,dTrsH-r Synthetic gene dTrs4H 1455bp dT dTrsL-f,dTrsL-r Synthetic gene dTrs4L 744bp

[0287] 1.3 After PCR, the samples were subjected to 1.5% agarose gel electrophoresis, where 300 mL of 1*TAE was added to the electrophoresis tank and the gel was run at 100 V for 30 min. The results showed that the size of the agarose gel electrophoresis band was consistent with the theoretical size. The electrophoresis results were recovered using the Omega Gel Extraction Kit (Cat. D2500-01), and the purified PCR product was eluted with 20 μL of elution buffer. The electrophoresis results are shown in Figure 2-5 As shown, Figure 2 Lanes 1-9 from left to right are DL5000, pcDNA3.4, pcDNA3.4, pcDNA3.4, pcDNA3.4, trsH, trsL, RtxH, and RtxL; Figure 3 Lanes 1-7 from left to right are DL2000, dTrs1H, dTrs1H, dTrs2H, dTrs2H, dTrs1L, and dTrs1L; Figure 4 Lanes 1-5 from left to right are DL2000, dTrs3H, dTrs3H, dTrs3L, and dTrs3L; Figure 5Lanes 1-5 from left to right are DL2000, dTrs4H, dTrs4H, dTrs4L and dTrs4L.

[0288] 1.4 The PCR bands obtained in 1.3 after gel excision and purification were subjected to homologous recombination using the NEB kit ( HiFi DNA Assembly Master Mix, Cat. E2621S) was used for fragment assembly, the homologous recombination reaction system was incubated at 50°C for 1 hour, and the obtained reaction product was purified using Omega Cycle Pure Kit (Cat. D6492-01) and eluted with 15 μL of elution solution. The reaction system and plasmid table are shown in Tables 7 and 8.

[0289] Table 7: Reaction system for homologous recombination

[0290]

[0291]

[0292] Table 8: Plasmid table

[0293]

[0294] 1.5 Use CaCl2 competent cells DH5a for transformation, add each plasmid sample obtained in 1.4 to the competent cells at a ratio of 1:10, place on ice for 10 min, then incubate in a metal bath at 42°C for 45 s, and then place on ice for 10 min. Finally, apply the obtained solution to LB solid culture medium added with ampicillin (Amp. final concentration 50 μg / mL), and culture overnight at 37°C. On the next day, pick a single clone and place it in 2 ml LB liquid culture medium (final concentration 50 μg / mLAmp.), shake the bacteria at 37°C and 200 rpm, and perform Sanger sequencing on the obtained bacterial solution to obtain the sequencing sequence. The sequencing primer is CMV-F: CGCAAATGGGCGGTAGGCGTG (SEQ ID NO: 79). After comparison of the sequencing results, it was confirmed that all the tested sequences were consistent with the original sequences. 100 μL of the monoclonal bacterial solution was added to 50 mL of LB liquid culture medium with ampicillin (final concentration 50 μg / mL), and cultured overnight at 37°C and 200 rpm. The strain was preserved, the plasmid was extracted, and the target gene expression plasmid was obtained.

[0295] 2. Transient expression of Trs, dTrs1-4 and Rtx proteins

[0296] 2.1 About 24 hours before transfection, take FreeStyle TM Cells (cell density 5-6×10 5 / mL, from FreeStyle, Gibco, USA TM MAX CHO Expression System, Catalog No.: K900020), cultured at 37°C, 5% CO2 and 120-135 rpm / min.

[0297] 2.2 On the day of transfection, dilute the cells to a cell density of 1×10 6 / mL, add 10mL of cells to each shake flask (cell survival rate is above 95%).

[0298] 2.3 Gently Mix FreeStyle TM Add MAX Reagent several times, being careful not to vortex.

[0299] 2.4 Take 12.5 μg of each plasmid corresponding to each protein in Table 9 and add OptiPRO TM SFM to each total volume 0.2 mL and mix gently.

[0300] 2.5 Take 25 μL of FreeStyle TM MAX Reagent, added Opti-Pro TM SFM to a total volume of 0.4 mL, gently mix (note: do not vortex). After mixing evenly, add evenly to the plasmid mixture and gently mix to obtain 2 0.4 mL DNA-FreeStyle TM MAX mixture and incubate at room temperature for 10 min to allow complex formation.

[0301] 2.6 Slowly add the two 0.4 mL DNA-FreeStyle TM MAX complexes were added to the cells while slowly rotating the flask.

[0302] 2.7 Culture the cells at 37°C, 5% CO2, 120-135 rpm / min. There is no need to replace or supplement the culture medium. Collect the cell fluid after 7 days.

[0303] Table 9: Plasmids corresponding to different proteins

[0304]

[0305] 3. Purification and identification of Trs, dTrs1-4 and Rtx proteins

[0306] The culture supernatant of Trs, dTrs1-4 and Rtx was centrifuged at 4°C and 7000g for 15 min and then sterilized by filtration with a 0.22um syringe filter. The Protein A chromatography medium was balanced with 5 column volumes of Buffer A1 (20mM phosphate buffer, pH 7.4) (flow rate of 2mL / min), and the treated sample was added and rinsed with Buffer A1 until the absorbance reached the baseline. Buffer B1 (0.1MGlycine-HCl, pH 3.0) was used for elution, and the elution peak sample was collected. The target protein eluate was adjusted to neutral with 1M Tris-HCl (pH 9.0). Ultrafiltration was performed into PBS buffer.

[0307] Identification of target protein: Select a 10-well SDS-PAGE precast gel and run it at 150 V for 50 min. Heat the sample at 90°C for 5 min and load 10 μg per well.

[0308] The purification effects of Trs, dTrs1-4 and Rtx proteins were detected by SDS-PAGE. Figure 6 As shown, wherein lane 1 is Trs eluted non-reduced, lane 2 is Trs eluted reduced, lane 3 is dTrs1 eluted non-reduced, lane 4 is dTrs1 eluted reduced, lane 5 is dTrs2 eluted non-reduced, lane 6 is dTrs2 eluted reduced, lane 7 is dTrs3 eluted reduced, lane 8 is dTrs3 eluted non-reduced, lane 9 is dTrs4 eluted non-reduced, lane 10 is dTrs4 eluted reduced, lane 11 is Rtx non-reduced, and lane 12 is Rtx reduced. The expression levels of Trs, dTrs1-4 and Rtx proteins are shown in Table 10.

[0309] Table 10: Expression levels of Trs, dTrs1-4 and Rtx proteins

[0310] Protein samples Protein concentration in culture medium (mg / mL) Trs 0.0086 dTrs1 0.0317 dTrs2 0.0182 dTrs3 0.00017 dTrs4 0.172 Rx 0.025

[0311] 4. Trs, dTrs1-4 and Rtx proteins binding to Her2 ELISA

[0312] 4.1 The binding of Her2 protein to Trs, dTrs1-4 or Rtx protein was detected by enzyme-linked immunosorbent assay using purchased Her2 with binding activity (10004-HCCH-B, Sino Biological) as substrate, Trs, dTrs1-4 or Rtx protein as primary antibody, and HRP-conjugated goat anti-human IgG as secondary antibody.

[0313] 20 μg of Her2 protein solid was dissolved in 80 μL of sterile water to prepare a 0.25 mg / mL stock solution, which was dispensed into 0.5 mL EP tubes, 10 μL / tube, and stored at -80°C.

[0314] Primary antibody: Trs (0.44 mg / mL), dTrs1 (0.38 mg / mL), dTrs2 (0.42 mg / mL), dTrs3 (0.115 mg / mL), dTrs4 (1.4 mg / mL), Rtx (0.21 mg / mL), of which Trs was the positive control and Rtx was the negative control.

[0315] Secondary antibody: HRP-conjugated goat anti-human IgG (Shanghai Sangon Biotechnology Co., Ltd., Cat. No. D110150).

[0316] 4.2 Detection plan:

[0317] 1) Coating: Dilute the culture supernatant with coating solution, add 0.1μg / 100μL Her2 protein (34μL mother solution + 8466μL coating solution) to each well, seal the plate, and incubate at 37℃ for 2h. Discard the liquid in the well, wash the plate 3 times with 200μL of washing solution each time, and let it stand for 2min.

[0318] 2) Blocking: Add 100 μL of blocking solution to each well, incubate at 4°C overnight, and discard the liquid in the well.

[0319] 3) Incubation with primary antibody (Trs, dTrs1-4 or Rtx): Dilute Trs, dTrs1-4 and Rtx with antibody diluent, add 100 μL to each well, seal the plate, incubate at 37°C for 1 h, discard the liquid in the wells, wash the plate 3 times with washing solution, 200 μL each time, and let it stand for 2 min.

[0320] 4) Secondary antibody incubation: dilute the secondary antibody with antibody diluent at 1:2000, add 100 μL to each well, seal the plate, incubate at 37°C for 1 h, discard the liquid in the wells, wash the plate 3 times with washing solution, 200 μL each time, and let it stand for 2 min.

[0321] 5) Detection: Add 100 μL of TMB to each well, seal the plate, and incubate at 37°C in the dark for 15 min. At this time, the wells will turn blue. Add 100 μL of stop solution to each well, and the wells will turn yellow immediately. Detect the OD value at 450 nm. The detection results are as follows: Figure 7 As shown in Table 11. The results showed that compared with Trs, the binding ability of dTrs1-dTrs4 to Her2 gradually decreased, among which dTrs4 had the lowest binding ability to Her2, close to the negative control Rtx. In addition, it was found that the expression level of dTrs4 was high, so dTrs4 could be selected as the drug skeleton.

[0322] The coating solution is prepared as shown in the following table:

[0323]

[0324]

[0325] The preparation of blocking solution is shown in the following table:

[0326]

[0327] The preparation of antibody diluent is shown in the following table:

[0328]

[0329] The preparation of the washing solution is shown in the following table:

[0330]

[0331] Table 11: Binding results of Trs, dTrs1-4 and Rtx proteins to Her2

[0332] Trs dTrs1 dTrs2 dTrs3 dTrs4 RTX <![CDATA[EC 50 ]]> Below the lower limit 0.4655 to 0.6385 1.997 to 2.747 Above the limit Above the limit Above the limit

[0333] Note: Values ​​below the lower limit and above the upper limit are beyond the calculation range. Values ​​below the lower limit indicate strong binding ability, while values ​​above the upper limit indicate weak binding ability.

[0334] 5. Binding of Trs, dTrs1-4 and Rtx proteins to IgA, IgM or HSA

[0335] 5.1 The experimental steps are as follows:

[0336] 1) Seal the plate with 100 μL / well of 10 μg / mL IgA / IgM / HSA coating solution at 4°C overnight; IgA is derived from human serum (Sigma-Aldrich, I4036) at a concentration of 1 mg / mL, IgM is derived from human serum (Sigma-Aldrich, I8260) at a concentration of 1 mg / mL, and HSA (Absin, abs47047388) at a concentration of 20 mg / mL;

[0337] 2) Discard the solution from step 1) and wash the plate with PBST (10 times dilution of Solebold 10*PBS with double distilled water (sterile), +0.1% Tween), wash 4 times with a plate washer, spin dry with a plate spinner at 600×g for 1 min, and pat dry several times;

[0338] 3) Add 100 μL / well blocking solution (0.5% Casein (pH 7.09, 1M NaOH>50 mg / mL, prepare in small aliquots, add PBST, adjust pH with HCl and make up to volume), incubate at 37°C for 60 min; then repeat step 2) to wash the plate;

[0339] 4) Add 100 μL / well of 10-fold gradient dilution (10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001) of each group of samples (Trs, dTrs1-4 or Rtx), seal the plate, and incubate at room temperature for 30 min; then repeat step 2) to wash the plate;

[0340] 5) Add 100 μL of detection antibody (Goat Anti-Human IgG-Fc Secondary Antibody (HRP μg / mL), Sino Biological, SSA001, diluted 1:3K with PBST to a working concentration of 1.2 μg / mL) to each well, seal the plate, and incubate at room temperature for 30 min; then repeat step 2) to wash the plate;

[0341] 6) Add 100 μL of Solebol TMB substrate colorimetric solution to each well, incubate at 25°C in the dark for 6 min, then add 100 μL of Solebol stop solution to each well and measure OD 450 .

[0342] 5.2 Results

[0343] The results of ELISA test using human IgA plate are as follows Fig.21 As shown, p<0.01**, p<0.05*. The results show that the signal generated by dTrs4 as a detection molecule is very low, even lower than that of wild-type Trs and irrelevant antibody RTX. Therefore, it can be shown that dTrs4 does not bind to human IgA.

[0344] in, Fig.21 The difference analysis is shown in the following table:

[0345]

[0346] The p value of t-test between dTRS4 and other molecules at 10ug / mL was obtained by plating human IgA.

[0347] The results of ELISA test using human IgM plate are as follows Fig. 22 As shown, p<0.01**, p<0.05*. The results show that the signal generated by dTrs4 as a detection molecule is very low, even lower than that of wild-type Trs and irrelevant antibody RTX. Therefore, it can be shown that dTrs4 does not bind to human IgM.

[0348] in, Fig. 22 The difference analysis is shown in the following table:

[0349]

[0350] The p value of t-test between dTRS4 and other molecules at 10ug / mL was obtained by plating with human IgM.

[0351] The results of ELISA test using human HSA as a plate are as follows Fig.23 As shown, p<0.01**, p<0.05*. The results show that except for the slightly higher signal generated by the irrelevant antibody RTX, the signals of other molecules are very low. Among them, dTrs4 as a detection molecule generates the lowest signal, even lower than the wild-type Trs and the irrelevant antibody RTX. Therefore, it can be shown that dTrs4 does not bind to human HSA.

[0352] in, Fig.23 The difference analysis is shown in the following table:

[0353]

[0354] The p value of t-test between dTRS4 and other molecules at 10ug / mL was obtained by plating human HSA.

[0355] Example 2: Preparation and selection of RC1-RC8 proteins

[0356] 1. Expression construction: The steps of expression construction of RC1-RC8 proteins are the same as those of expression construction in Example 1, except that the target gene is different. The structural diagram of RC1-RC8 proteins is shown in Figure 8 The structural diagram of PLA2R is shown in Fig. 9 As shown, the PLA2R antigen epitope peptide CFC1 (SEQ ID NO: 21) in the RC1-RC8 protein is the amino acid sequence from 21 to 367 (Glu) of the PLA2R fragment, and the PLA2R antigen epitope peptide CFC2 (SEQ ID NO: 22) is the amino acid sequence from 21 to 510 (Asp) of the PLA2R fragment.

[0357] Among them, the name, primer, sequence and composition of the heavy chain, light chain and its signal peptide of the target gene are shown in Table 12, the name and sequence of the target gene are shown in Table 13, the primers and templates of the pcDNA3.4 vector and the target gene are shown in Table 14, and the target fragment information is shown in Table 15.

[0358] in,

[0359] PLA2R signal peptide nucleotide sequence:

[0360] ATGCTGCTGTCGCCGTCGCTGCTGCTGCTGCTGCTGCTGGGGGCGCCGGGGCTGCGCC (SEQ ID NO: 80).

[0361] Trs heavy chain signal peptide nucleotide sequence:

[0362] ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGATGCCAGATGT (SEQ ID NO: 81).

[0363] Trs light chain signal peptide nucleotide sequence:

[0364] ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACTACAGGTGTCCACTCC (SEQ ID NO: 82).

[0365] The electrophoresis results of the purified PCR products are shown in Figure 10-12 As shown, Fig.10 Lanes 1-5 from left to right are DL5000, pcDNA3.4, pcDNA3.4, pcDNA3.4, and pcDNA3.4; Fig.11 Lanes 1-13 from left to right 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; Fig.12 Lanes 1-7 from left to right are DL5000, irrelevant, irrelevant, RC3, RC3, RC6, and RC6.

[0366] The PCR bands after gel excision and purification were subjected to homologous recombination, and the system was consistent with Table 7. The constructed plasmid table is shown in Table 16.

[0367] Table 12: Heavy and light chain composition of RC1-RC8 proteins

[0368]

[0369] Table 13: Name and sequence of target gene

[0370]

[0371]

[0372] Note: RC1H, RC2L, RC4H and RC5L are all formed by splicing two PCR fragments, that is, RC1H is formed by splicing RC1H-1 and RC1H-2, RC2L is formed by splicing RC2L-1 and RC2L-2, RC4H is formed by splicing RC4H-1 and RC4H-2, and RC5L is formed by splicing RC5L-1 and RC5L-2.

[0373] Among them, the amino acid sequence of RC1H is:

[0374] EGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVEGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIGGTGIHWVRQAPGKGLEWVAGIYPTNGGTGYAGSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRGGGDGFGAMGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:57).

[0375] Nucleotide sequence of RC1H:

[0376]

[0377] Amino acid sequence of RC2L:

[0378] EGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVEGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGVGTAVAWYQQKPGKAPKLLIYSASGLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHGTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:59).

[0379] Nucleotide sequence of RC2L:

[0380]

[0381] Amino acid sequence of RC3:

[0382] EGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVEGGGGSGGGGSTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:61).

[0383] Nucleotide sequence of RC3:

[0384]

[0385] Amino acid sequence of RC4H:

[0386] EGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVEKDAWKYYATHCEPGWNPYNRNCYKLQKEEKTWHEALRSCQADNSALIDITSLAEVEFLVTLLGDENASETWIGLSSNKIPVSFEWSNDSSVIFTNWHTLEPHIFPNRSQLCVSAEQSEGHWKVKNCEERLFYICKKAGHVLSDGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIGGTGIHWVRQAPGKGLEWVAGIYPTNGGTGYAGSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRGGGDGFGAMGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:63)。

[0387] Nucleotide sequence of RC4H:

[0388]

[0389] Amino acid sequence of RC5L:

[0390] EGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVEKDAWKYYATHCEPGWNPYNRNCYKLQKEEKTWHEALRSCQADNSALIDITSLAEVEFLVTLLGDENASETWIGLSSNKIPVSFEWSNDSSVIFTNWHTLEPHIFPNRSQLCVSAEQSEGHWKVKNCEERLFYICKKAGHVLSDGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGVGTAVAWYQQKPGKAPKLLIYSASGLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHGTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:65).

[0391] Nucleotide sequence of RC5L:

[0392]

[0393] Amino acid sequence of RC6:

[0394] EGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVEKDAWKYYATHCEPGWNPYNRNCYKLQKEEKTWHEALRSCQADNSALIDITSLAEVEFLVTLLGDENASETWIGLSSNKIPVSFEWSNDSSVIFTNWHTLEPHIFPNRSQLCVSAEQSEGHWKVKNCEERLFYICKKAGHVLSDGGGGSGGGGSTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:67).

[0395] Nucleotide sequence of RC6:

[0396]

[0397] Table 14: Primers and templates for pcDNA3.4 vector and target gene

[0398]

[0399] Table 15: PCR target fragment information

[0400]

[0401]

[0402] Table 16: Plasmid table

[0403] Plasmid name Purpose fragment Target fragment volume Deionized water volume pcDNA3.4-RC1H RC1H-1,RC1H-2 0.5 μL each 6μL pcDNA3.4-RC2L RC2L-1,RC2L-2 0.5 μL each 6μL pcDNA3.4-RC3 RC3 0.5μL 6.5μL pcDNA3.4-RC4H RC4H-1,RC4H-2 0.5 μL each 6μL pcDNA3.4-RC5L RC5L-1,RC5L-2 0.5 μL each 6μL pcDNA3.4-RC6 RC6 0.5μL 6.5μL

[0404] 2. Transient expression of RC1-RC8 proteins

[0405] 2.1 About 24 hours before transfection, take FreeStyle TM Cells (cell density 5-6×10 5 / mL, from FreeStyle, Gibco, USA TM MAX CHO Expression System, Catalog No.: K900020), cultured at 37°C, 5% CO2, 120-135 rpm / min.

[0406] 2.2 On the day of transfection, dilute the cells to a cell density of 1×10 6 / mL, add 10mL of cells to each shake flask (cell survival rate is above 95%).

[0407] 2.3 Gently Mix FreeStyle TM Add MAX Reagent several times, being careful not to vortex.

[0408] 2.4 Take 12.5 μg of each plasmid corresponding to each protein in Table 17 and add OptiPRO TM SFM to each total volume 0.2 mL and mix gently.

[0409] 2.5 Take 25 μL of FreeStyle TM MAX Reagent, added Opti-Pro TM SFM to a total volume of 0.4 mL, gently mix (note that you cannot vortex). After mixing evenly, add evenly to the plasmid mixture and gently mix to obtain 2 0.4 mL DNA-FreeStyle TMMAX mixture and incubate at room temperature for 10 minutes to allow complex formation (12.5 μL FreeStyle TM MAX Reagent, added Opti-Pro TM SFM to a total volume of 0.2 mL, and finally obtain a 0.4 mL DNA-FreeStyle TM MAX mixture).

[0410] 2.6 Slowly add the two 0.4 mL DNA-FreeStyle TM MAX complex added to cells (RC3 and RC6 proteins only have 1 DNA-FreeStyle TM MAX complex) while slowly rotating the shake bottle.

[0411] 2.7 The cells were cultured at 37°C, 5% CO2 and 120-135 rpm / min. There was no need to replace or supplement the culture medium. The cell fluid was collected after 7 days.

[0412] Table 17: Plasmids corresponding to RC1-RC8 proteins

[0413]

[0414] 3. Purification and identification of RC1-RC8 proteins

[0415] The supernatant of RC1-RC8 culture was centrifuged at 4°C and 7000g for 15 min, and then sterilized by filtration with a 0.22μm syringe filter. The Protein A chromatography medium was balanced with 5 column volumes of Buffer A1 (flow rate of 2mL / min), and the treated sample was added. Buffer A1 was used for washing until the absorbance reached the baseline, and Buffer B1 was used for elution. The elution peak sample was collected, and the target protein eluate was adjusted to neutral with 1M Tris-HCl (pH 9.0). Super dex200 was filtered into PBS.

[0416] Identification of target protein: Select a 10-well SDS-PAGE precast gel and run it at 150 V for 50 min. Heat the sample at 90°C for 5 min and load 3 μg per well.

[0417] The purification effect of RC1-RC8 protein was detected by SDS-PAGE. Fig.13As shown, wherein lane M is a marker, lane 1 is RC1 eluted non-reduced, lane 2 is RC1 eluted reduced, lane 3 is RC2 eluted reduced, lane 4 is RC2 eluted non-reduced, lane 5 is RC3 eluted non-reduced, lane 6 is RC3 eluted reduced, lane 7 is RC4 eluted reduced, lane 8 is RC4 eluted non-reduced, lane 9 is RC5 eluted non-reduced, lane 10 is RC5 eluted reduced, lane 11 is RC6 eluted non-reduced, lane 12 is RC6 eluted reduced, lane 13 is RC7 eluted non-reduced, lane 14 is RC7 eluted reduced, lane 15 is RC8 eluted non-reduced, and lane 16 is RC8 eluted reduced. The expression levels of RC1-RC8 proteins are shown in Table 18.

[0418] Table 18: Expression levels of RC1-RC8 proteins

[0419] sample Protein concentration in culture medium (mg / mL) RC1 0.363 RC2 0.333 RC3 0.0234 RC4 0.19 RC5 0.243 RC6 0.087 RC7 0.123 RC8 0.103

[0420] 4. Stability investigation of RC1-RC8

[0421] When a globulin molecule exists alone in an aqueous solution, it is in a low-energy state, forming a folded state in which the hydrophobic amino acids are wrapped inside and the hydrophilic amino acids are exposed outside. However, when multiple or a large number of molecules exist, multiple molecules interact with each other, changing the folded state to form macromolecular aggregates with lower energy. Increased temperature promotes protein aggregation. Protein aggregation is a common phenomenon of many globulins. Protein aggregation can cause proteins to lose their original higher-order structures and functions, which will affect the determination of protein drug quality standards and formulation components, and is usually avoided in protein drugs or various application scenarios. As candidate drug molecules, it is generally preferred to select molecules with a lower degree of aggregation. Therefore, the stability of RC1-RC8 prepared in step 3 of this embodiment was investigated, and the specific steps are as follows:

[0422] RC2 showed obvious degradation when stored at -20°C and had poor stability, so no subsequent experiments were performed.

[0423] RC1, RC3, RC4, RC5, RC6, RC7 and RC8, and CC1h (amino acid sequence as shown in SEQ ID NO: 92, nucleotide sequence as shown in SEQ ID NO: 93, prepared by conventional methods in the art) were diluted to the same concentration using sample buffer (25 mM sodium phosphate + 75 mM NaCl + 40% glycerol (pH 7.0)), and then observed at 37°C for 24 hours and 48 hours. The results are shown in Fig.24 .

[0424] Among them, the molecular weights of RC1, RC3, RC4, RC5, RC6, RC7, RC8 and CC1h are shown in the following table:

[0425] protein C1h RC1 RC2 RC3 RC4 RC5 RC6 RC7 RC8 Molecular weight (KD) 41 223 223 130 257 257 164 304 371

[0426] The results showed that CC1h aggregated (about 82KD) after being placed at 37°C for 24 hours, and the aggregation increased after 48 hours. RC3 and RC6 had almost no aggregation; RC1 and RC4 had a small amount of double molecular weight bands, and the 48h bands were stronger than the 24h bands, but compared with CC1h, the aggregation of RC1 and RC4 was lighter. Therefore, it further shows that RC3, RC6, RC1 and RC4 have a lighter aggregation tendency than CC1h, and may have better drugability.

[0427] Among them, the amino acid sequence and nucleotide sequence of CC1h are as follows:

[0428] EGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGRED DLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVEGGHHHHHH(SEQ ID NO:92);

[0429]

[0430] 5. Neutralization of PLA2R-Ab in plasma samples by RC1-RC8

[0431] The only FDA-approved kit for the diagnosis of PLA2R antibody-positive membranous nephropathy (Euromon, Germany, ELISA method for quantitative detection of PLA2R-Ab concentration in blood samples) was used for detection, and all steps were carried out according to the instructions. The plasma of the three patients with PLA2R antibody-positive membranous nephropathy was numbered pMN-2, pMN-4, and pMN-7.

[0432] The neutralization effects of RC1-RC8 on the PLA2R-Ab in the plasma of the three membranous nephropathy-positive patients were tested, and the following results were obtained: Figure 14-16 And the neutralization rate of Table 19. Among them, Figure 14-16 Neutralization ratio = (1-(PLA2R-Ab detection value after adding inhibitor / PLA2R-Ab detection value without inhibitor))*100%. The larger the value, the less autoimmune antibodies can be detected, and the better the corresponding inhibitory effect. (Assuming intravenous administration, bioavailability 100%, and the distribution volume of this inhibitor in humans is 8L, the inhibitor concentration in this study is equivalent to a dose of 5μg / mL*8L=40mg / person).

[0433] The calculation of the neutralization rate is as follows:

[0434] The capture molecule in the diagnostic kit is PLA2R.

[0435] When no drug molecules are added:

[0436]

[0437]

[0438] Among them, [PLS2R·Ab]1 corresponds to the detection concentration of the antibody.

[0439] After adding drug molecules:

[0440]

[0441] Among them, [PLS2R·Ab]2 corresponds to the detection concentration of the antibody at this time.

[0442] Neutralization ratio(NR):

[0443]

[0444] In the ELISA test, PLA2R is in great excess as a capture antigen, so [PLA2R]2≈[PLA2R]1, That is, the ratio of PLA2R-Ab in plasma reduced by the addition of drug molecules, which can represent the neutralizing effect of drug molecules.

[0445] Table 19: Neutralization rate of RC1-RC8 on the plasma PLA2R-Ab of the three membranous nephropathy-positive patients

[0446]

[0447]

[0448] In the three groups of experiments, the dosage of RC1-RC8 protein was 5μg / mL. pMN-2 was a positive sample, and all groups of drugs showed a neutralization effect of more than 95% against autoimmune antibodies with a plasma concentration of about 44.79RU / mL. pMN-4 was a positive sample, and all groups of drugs showed a neutralization effect of more than 80% against autoimmune antibodies with a plasma concentration of about 124.905RU / mL. pMN-7 was a positive sample, and all groups of drugs showed a neutralization effect of more than 80% against autoimmune antibodies with a plasma concentration of about 53.72RU / mL, except for RC5 with a neutralization rate of 77.63%.

[0449] 6. Immobilization of RC3 and RC6

[0450] A certain volume of NHS-Bestarose (AA0031, Boglon (Shanghai) Biotechnology Co., Ltd.) medium stored in isopropanol was placed in a gravity column and washed with precooled coupling solution A3 (1mM hydrochloric acid) (0-4°C) for at least 30 minutes. 1mL of medium was washed with 20mL of coupling solution A3 solution; RC3 and RC6 were replaced by ultrafiltration with coupling solution B3 (0.1M sodium bicarbonate, 0.5M sodium chloride, pH 8.3) (ligand coupling concentration 10mg / mL filler). The washed medium was diluted with coupling solution A3 (about 0.5mL coupling solution A3 for 1mL medium), mixed with an equal volume of ligand solution, and shaken at 4°C overnight. The coupling supernatant was removed and blocking solution (0.1M Tris-HCl, pH8.5) was added, and the column was blocked at room temperature for 4 hours; the blocking solution and 0.2M acetic acid were used to wash alternately 5 times at pH3.5, each time with 3 times the volume of filler. After washing with PBS, the mixture was used or stored in 0.1 M phosphate buffer to obtain RC3-NHS Bestarose and RC6-NHS Bestarose.

[0451] 7. Adsorption of PLA2R-Ab in plasma samples by immobilized RC3 and RC6

[0452] Resuspend RC3-NHS Bestarose and RC6-NHS Bestarose thoroughly, take 100 μL and add them to the corresponding EP tubes respectively. Centrifuge at 1000g for 5 minutes and carefully remove the supernatant. Add 500 μL PBS, resuspend - centrifuge - remove the supernatant twice to wash the filler. Add 100 μL of patient plasma to the EP tube and resuspend. After slight shaking for 30 minutes, centrifuge at 1000g for 5 minutes. Carefully aspirate the supernatant and collect it into 3 EP tubes, labeled as RC3-pMN and RC6-pMN respectively. The ELISA method was used to detect the adsorption of PLA2R-Ab in PMN-4 and PMN-7 plasma samples by RC3 and RC6 after immobilization, as described in Part 4 of this example (neutralization of PLA2R-Ab in plasma samples by RC1-RC8). For results, see Figure 17-18 ,The results showed that the clearance rates of PLA2R-Ab in PMN-4 and PMN-7 plasma samples by RC3 and RC6 after immobilization were both above 80%.

[0453] in, Fig.17 Clearance of PLA2R-Ab in PMN-4 plasma samples by RC3 and RC6 after immobilization:

[0454] RC3-NHS Bestarose=(225.55-43.16) / 225.55*100%=80.86%,

[0455] RC6-NHS Bestarose=(225.55-13.82) / 225.55*100%=93.87%.

[0456] Fig.18 Clearance of PLA2R-Ab in PMN-7 plasma samples by RC3 and RC6 after immobilization:

[0457] RC3-NHS Bestarose=(52.03-6.26) / 52.03*100%=87.96%,

[0458] RC6-NHS Bestarose=(52.03-4.67) / 52.03*100%=91.02%.

[0459] 8. RC1-RC8 mouse acute toxicity test

[0460] The purchased BALB / c female mice were adaptively raised for one week and randomly divided into 8 groups, namely RC1 group, RC2 group, RC4 group, RC5 group, RC7 group, RC8 group, placebo group (also known as "Buffer") and blank group (also known as "Notreatment"), with 5 mice in each group, weighed and marked. The drug was administered at 10 times the 40 mg / person dose. The mice were weighed before administration, and then the drugs were administered. Specifically, the RC1 group was subcutaneously injected with 0.1 mL of 1 mg / mL RC1 protein, the RC2 group was subcutaneously injected with 0.1 mL of 1 mg / mL RC2 protein, the RC4 group was subcutaneously injected with 0.1 mL of 1 mg / mL RC4 protein, the RC5 group was subcutaneously injected with 0.1 mL of 1 mg / mL RC5 protein, the RC7 group was subcutaneously injected with 0.1 mL of 1 mg / mL RC7 protein, the RC8 group was subcutaneously injected with 0.1 mL of 1 mg / mL RC8 protein, 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, the mice were observed for 1 hour, and no obvious adverse reactions such as dyspnea, decreased movement, and tremor were observed in the mice. The mice were weighed at 24h, 48h, and 72h, and then weighed every 48h until the tenth day of administration.

[0461] Body weight trend chart of mice injected subcutaneously with RC1, RC2, RC4, RC5, RC7, and RC8 proteins, Fig.19 As shown, the results showed that there was no significant difference in body weight between the experimental group and the control group.

[0462] The purchased BALB / c female mice were adaptively raised for one week and randomly divided into 4 groups, namely RC3 group, RC6 group, placebo group (also known as "Buffer") and blank group (also known as "No treatment"), with 5 mice in each group, weighed and marked. The mice were administered at 10 times the 40 mg / person dose. The mice were weighed before administration and then administered. Specifically, the RC3 group was subcutaneously injected with 0.1 mL of 1 mg / mL RC3 protein, the RC6 group was subcutaneously injected with 0.1 mL of 1 mg / mL RC6 protein, 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, and no obvious adverse reactions such as dyspnea, decreased movement, and tremor were observed in the mice. The mice were weighed at 24h, 48h, and 72h, and then weighed every 48h until the thirtieth day of administration.

[0463] Body weight trend of mice injected subcutaneously with RC3 and RC6 proteins, Fig. 20 As shown, the results showed that there was no significant difference in body weight between the experimental group and the control group.

[0464] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0465] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

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

34.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleic acid molecule is DNA.

4. An expression vector, characterized in that: Carrying the nucleic acid molecule according to claim 2 or 3.

5. The expression vector according to claim 4, characterized in that The expression vector is a eukaryotic expression vector.

6. The expression vector according to claim 4, characterized in that The expression vector is a lentiviral vector.

7. A recombinant cell, characterized in that The recombinant cell comprises: Carrying the nucleic acid molecule of claim 2 or 3; or, Express the antibody of claim 1.

8. The recombinant cell according to claim 7, characterized in that The recombinant cell is obtained by introducing the expression vector according to any one of claims 4 to 6 into a host cell.

9. The recombinant cell according to claim 7, characterized in that The recombinant cell is a eukaryotic cell.

10. The recombinant cell according to claim 7, characterized in that The recombinant cell is a mammalian cell.

11. A drug carrier, characterized in that Comprising the antibody of claim 1.

12. A fusion protein, characterized in that: include: The antibody according to claim 1; One or more antigenic epitope peptides, wherein the antigenic epitope peptides are linked to the antibody.

13. Use of the antibody according to claim 1 in preparing a fusion protein or an ADC drug.

14. Use of the antibody according to claim 1 in preparing a drug carrier or a negative antibody.

15. The use according to claim 14, characterized in that The drug carrier or negative antibody has no antigen binding ability.

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