Antibodies or antigen-binding fragments thereof targeting nmp22 and uses thereof

Abstract

The application discloses an antibody or antigen binding fragment thereof targeting NMP22 and application thereof. The antibody or antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region, characterized in that the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are respectively shown as SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5; and the amino acid sequences of LCDR1, LCDR2 and LCDR of the light chain variable region are respectively shown as SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9. The antibody or antigen binding fragment thereof targeting NMP22 provided by the application can specifically recognize and combine NMP22 protein, has high specificity and high sensitivity, can be used for non-invasive, simple and rapid detection of bladder cancer, and has wide application prospects in detection of NMP22 protein and preparation of diagnosis, prevention and / or treatment of bladder cancer reagents and drugs.

Description

Technical Field

[0001] This invention belongs to the field of tumor immune detection, specifically relating to an antibody or its antigen-binding fragment targeting NMP22 and its applications. Background Technology

[0002] Bladder cancer is one of the most common malignant tumors of the urinary system. In 2018, there were 549,000 new cases of bladder cancer worldwide, with 200,000 deaths, ranking 11th in incidence among malignant tumors. Early diagnosis and treatment of bladder cancer are crucial for cancer patients. Cystoscopy with biopsy, urine cytology, and imaging examinations are currently the most commonly used diagnostic methods in clinical practice. Cystoscopy with biopsy is the gold standard for bladder cancer diagnosis, but it is relatively invasive. Urine cytology is currently the most commonly used examination method in clinical practice, but its sensitivity is low. Developing simple, rapid, and accurate methods for detecting bladder cancer has become a research hotspot in bladder cancer diagnosis.

[0003] Nuclear matrix protein 22 (NMP22) is a mitotic apparatus protein. It is primarily involved in the formation of the nuclear spindle. NMP22 participates in chromosome formation, playing a crucial role in the normal pairing and separation of chromosomes. Spindle abnormalities often lead to disordered chromosome pairing and separation, resulting in incorrect chromosome separation and ultimately cell death or malignant transformation. In the early stages of apoptosis, some NMP22 undergoes hydrolysis, causing DNA to detach from the nuclear matrix. During the malignant proliferation and division of bladder tumor cells, NMP22 is expressed in large quantities in the nuclear matrix. When cancer cells die, NMP22 is released into the urine, significantly increasing the concentration of NMP22 in the patient's urine. Therefore, NMP22 has potential application value in the diagnosis of bladder cancer, and there is an urgent need for a product and method that can rapidly, effectively, and with high sensitivity and specificity for its detection. Summary of the Invention

[0004] The technical problem this invention aims to solve is to overcome the shortcomings of existing technologies in lacking a highly sensitive, non-invasive, simple, and rapid method for detecting bladder cancer, and to provide an antibody targeting NMP22 or its antigen-binding fragment and its applications. The antibody targeting NMP22 or its antigen-binding fragment provided by this invention can specifically recognize and bind to the NMP22 protein, exhibiting high specificity and high sensitivity. It can be used for non-invasive, simple, and rapid detection of bladder cancer, and has broad application prospects in the detection of NMP22 protein and the preparation of reagents and drugs for the diagnosis, prevention, and / or treatment of bladder cancer.

[0005] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0006] The first aspect of the present invention provides an antibody or antigen-binding fragment thereof targeting NMP22, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are shown in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; and the amino acid sequences of LCDR1, LCDR2, and LCDR of the light chain variable region are shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively.

[0007] In some embodiments of the present invention, the amino acid sequences of HFR1, HFR2, HFR3 and HFR4 of the heavy chain variable region frame region are shown in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, respectively; and the amino acid sequences of LFR1, LFR2, LFR3 and LFR4 of the light chain variable region frame region are shown in SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17, respectively.

[0008] In some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:6.

[0009] In some embodiments of the present invention, the antibody or its antigen-binding fragment satisfies one or more of the following:

[0010] (1) The antibody is a full-length antibody and the antigen-binding fragment is Fab, Fab', F(ab')2 or scFv;

[0011] (2) The antibody or its antigen-binding fragment is a monospecific antibody, a bispecific antibody or a multispecific antibody;

[0012] (3) The antibody or its antigen-binding fragment is a monoclonal antibody or a polyclonal antibody;

[0013] (4) The antibody or its antigen-binding fragment is a mouse antibody or a humanized antibody;

[0014] In some preferred embodiments of the present invention, when the antibody is a full-length antibody, the full-length antibody includes a heavy chain constant region and a light chain constant region; the heavy chain constant region is selected from IgG, IgA, IgM, IgE and IgD, preferably IgG1, IgG2, IgG3 or IgG4; the light chain constant region is a κ or λ chain.

[0015] A second aspect of the present invention provides an isolated nucleic acid that encodes an antibody or an antigen-binding fragment thereof as described in the first aspect.

[0016] A third aspect of the present invention provides a recombinant expression vector comprising the isolated nucleic acid as described in the second aspect.

[0017] In some embodiments of the present invention, the recombinant expression vector is a plasmid, granule, bacteriophage, or viral vector.

[0018] A fourth aspect of the present invention provides a transformant comprising the nucleic acid as described in the second aspect or the recombinant expression vector as described in the third aspect;

[0019] In some embodiments of the present invention, the host cell of the transformant is a prokaryotic cell or a eukaryotic cell;

[0020] In some preferred embodiments of the present invention, the eukaryotic cells are yeast cells or mammalian cells; the mammalian cells are, for example, SP2 / 0 cells, 293T cells or CHO cells.

[0021] The fifth aspect of the present invention provides a method for preparing an antibody or antigen-binding fragment targeting NMP22, the method comprising the steps of: culturing a transformant as described in the fourth aspect, and obtaining the antibody or antigen-binding fragment targeting NMP22 from the culture.

[0022] A sixth aspect of the present invention provides a kit comprising an antibody or antigen-binding fragment thereof as described in the first aspect, a nucleic acid as described in the second aspect, a recombinant expression vector as described in the third aspect, and / or a transformant as described in the fourth aspect.

[0023] A seventh aspect of the present invention provides a method for detecting NMP22, the method comprising the following steps:

[0024] (1) Contact the sample with an antibody or antigen-binding fragment thereof as described in the first aspect or a kit as described in the sixth aspect;

[0025] (2) Quantitative or qualitative determination of NMP22 in the sample based on the binding of the antibody or its antigen-binding fragment to the sample.

[0026] The eighth aspect of the present invention provides the use of an antibody or antigen-binding fragment as described in the first aspect, a nucleic acid as described in the second aspect, a recombinant expression vector as described in the third aspect, or a transformant as described in the fourth aspect in the preparation of a reagent for detecting NMP22.

[0027] The ninth aspect of the present invention provides the use of an antibody or antigen-binding fragment thereof targeting NMP22 as described in the first aspect, a nucleic acid as described in the second aspect, a recombinant expression vector as described in the third aspect, and / or a transformant as described in the fourth aspect in the preparation of medicaments for the diagnosis, prevention and / or treatment of tumors.

[0028] In some embodiments of the present invention, the tumor is an NMP22 positive tumor.

[0029] In some preferred embodiments of the present invention, the tumor is bladder cancer.

[0030] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0031] The reagents and raw materials used in this invention are all commercially available.

[0032] The positive and progressive effects of this invention are as follows: the antibody or antigen-binding fragment targeting NMP22 provided by this invention can specifically recognize and bind to NMP22 protein (EC50 is 6.92 ng / mL), and has high specificity and high sensitivity. It has broad application prospects in the detection of NMP22 protein and the preparation of reagents and drugs for the diagnosis, prevention and / or treatment of bladder cancer. Attached Figure Description

[0033] Figure 1 The purity of NMP22 protein was determined by SDS-PAGE. The left band is the protein molecular weight marker, and the right band is the purified NMP22 protein.

[0034] Figure 2 To determine the serum titer of NMP22 protein in mice immunized by ELISA.

[0035] Figure 3 The purity of the purified mouse anti-NMP22 protein monoclonal antibody (1A5C10) was determined by SDS-PAGE. The left band is the protein molecular weight marker, and the right band is the mouse anti-NMP22 protein monoclonal antibody (1A5C10) under reducing conditions.

[0036] Figure 4 To identify the specific binding of mouse anti-NMP22 protein monoclonal antibody (1A5C10) to NMP22 protein using ELISA. Detailed Implementation

[0037] the term

[0038] Unless otherwise stated, the technical and scientific terms used in this invention have the meanings commonly understood by a person skilled in the art to which this invention pertains.

[0039] The term "antibody" as used in this article refers to immunoglobulin molecules, which are typically tetramers composed of two identical heavy chains and two identical light chains linked together by disulfide bonds. Based on differences in the conservation of their amino acid sequences, the heavy and light chains are divided into variable regions (V) located at the amino terminus and constant regions (C) located at the carboxyl terminus. Within the variable regions of both the heavy and light chains, three local regions exhibit a higher degree of variability in their amino acid composition and sequence, serving as key sites for antibody-antigen binding; these are also known as complementarity-determining regions (CDRs). In this article, the three heavy chain CDRs are designated HCDR1, HCDR2, and HCDR3, and the three light chain CDRs are designated LCDR1, LCDR2, and LCDR3. The interaction between the variable regions of one heavy chain and one light chain forms the antigen-binding site (Fv). Antibodies can be classified into different categories based on the amino acid sequence of their heavy chain constant regions. There are five main types of complete antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further subdivided into subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are known in the art. This invention is intended to include antibodies of any of the aforementioned classes or subclasses.

[0040] The term “antibody” as used herein is also intended to cover its digested fragments or functional variants, such as antibody fragments capable of binding to NMP22 or portions thereof, including but not limited to Fab (e.g., antibodies obtained by papain digestion), F(ab')2 (e.g., obtained by pepsin digestion), Fv, or scFv (e.g., obtained by molecular biology techniques).

[0041] As used herein, the term "monoclonal antibody" refers to a homogeneous antibody that targets only a specific antigenic epitope. In contrast to conventional polyclonal antibody formulations, which typically comprise different antibodies targeting different antigenic determinants (epitaxes), each monoclonal antibody targets a single antigenic determinant on the antigen. The modifier "monoclonal" indicates the homogeneity of the antibody and is not interpreted as requiring the antibody to be produced by any particular method. The monoclonal antibodies of the present invention are preferably produced by recombinant DNA methods or obtained by screening methods described elsewhere herein.

[0042] As used herein, the term "isolated polynucleotide" refers to polynucleotides not naturally occurring in nature, including polynucleotides isolated from nature (including organisms) through biological techniques, as well as artificially synthesized polynucleotides. Isolated polynucleotides can be genomic DNA, cDNA, mRNA, or other synthetic RNA, or combinations thereof. This document provides several nucleotide sequences for encoding the heavy chain variable region and light chain variable region of monoclonal antibodies against human NMP22 protein. It should be noted that those skilled in the art can design nucleotide sequences that are not entirely identical to the nucleotide sequences provided above, based on codon degeneracy, using the amino acid sequences of the heavy chain variable region and light chain variable region provided herein, but all encoding the same amino acid sequence. These modified nucleotide sequences are also included within the scope of this invention.

[0043] When referring to polynucleotides, the term "vector" as used herein refers to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer nucleotide-encoded information into a host cell. The terms "expression vector" or "expression cassette" refer to a vector suitable for expressing a target gene (the nucleotide sequence to be expressed) within a host cell, typically including the target gene, promoter, and terminator.

[0044] As used in this article, the term "host cell" refers to a cell that has been or can be transformed with a nucleic acid sequence to express the selected target gene. This term includes the offspring of the parent cell, regardless of whether the offspring are morphologically or genetically identical to the original parent cell, as long as the selected target gene is present in the offspring. Commonly used host cells include bacteria, yeast, and mammalian cells.

[0045] As used in this article, "transfection" refers to the uptake of foreign or exogenous DNA by cells. This technique can be used to introduce one or more portions of exogenous DNA into suitable host cells. Cells can be induced to a physiological state, i.e., "competent state," through physicochemical methods (e.g., treatment with calcium chloride).

[0046] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0047] Example 1: Preparation of Recombinant Human NMP22 Protein Fragment

[0048] The full-length gene information of human NMP-22 was obtained from GenBank (accession number BC043499.1), and the N-terminal fragment sequence of the NMP-22 protein was extracted. A His-tag was added to the C-terminus of the NMP-22 protein fragment for affinity purification and identification. The sequence of the recombinant human NMP22 protein fragment is shown in SEQ ID NO:1. The gene sequence of the recombinant human NMP22 protein fragment was optimized using GenScript codon optimization software, and the optimized gene sequence of the recombinant human NMP22 protein fragment was synthesized by chemical synthesis. The correctly sequenced human NMP22 protein fragment gene sequence was inserted into plasmid PET15b to construct the recombinant plasmid.

[0049] The plasmid PET15b-NMP22, containing the human NMP22 protein fragment gene, was transfected into E. coli BL21 competent cells. E. coli BL21 cells containing the PET15b-NMP22 plasmid were then cultured in a medium (1 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, and 100 mg / L ampicillin) at 37°C.

[0050] Rinse the constant flow pump thoroughly with distilled water, then rinse the glass chromatography column. Add approximately 200 mL of Ni-IDA (Genscript) to the column and allow the column material to settle naturally.

[0051] Equilibrate approximately 3 L of equilibration buffer (20 mM Tris, 300 mM NaCl) using a constant flow pump at a flow rate of 5 mL / min. Resuspend the E. coli containing recombinant human NMP22 protein in 200 mL of equilibration buffer (20 mM Tris, 300 mM NaCl), and then disrupt the cells using an ultrasonic disruptor (Ningbo Xinzhi Biotechnology Co., Ltd. JY98-IIIDH).

[0052] After centrifuging the lysate, load the supernatant onto the sample at a flow rate of 2 mL / min. After loading, wash with equilibration buffer until the absorbance remains constant at a flow rate of 5 mL / min.

[0053] After washing, elute with elution buffer (20 mM Tris, 300 mM NaCl, 250 mM Iminazole) at a flow rate of 5 mL / min and collect the eluent.

[0054] It was subjected to SDS-PAGE analysis at 4–20% gel concentration, and the results are as follows: Figure 1 As shown, the purified NMP22 recombinant protein has a purity of over 90%.

[0055] Example 2: Preparation of NMP22 Monoclonal Antibody

[0056] 1. Female Balb / c mice were subcutaneously immunized with a 1:1 emulsion containing 200 μL of Freund's complete adjuvant (Sigma-Aldrich) containing 50 μg of recombinant NMP22 protein.

[0057] 2. Mice were boosted with intraperitoneal / subcutaneous injections of a 1:1 emulsion containing 25 μg NMP22 Sigma-Aldrich every two weeks, up to three times.

[0058] 3. Four days before myeloma fusion, the highest antibody titer was observed (see...). Figure 2 Mice #1 (antibody titer determined by serum ELISA) were given an intraperitoneal booster immunization with 25 μg NMP22 (without adjuvant).

[0059] 4. Under aseptic conditions, spleen was extracted and ground to form a single-cell suspension. Simultaneously, a single-cell suspension of myeloma cells (SP2 / 0) was prepared. Electrofusion was used to ionize 8.1 × 10⁸ cells. 7 3.8 × 10⁸ spleen cells 7 Fusing SP2 / 0 mouse myeloma cells.

[0060] 5. Resuspend the fused cells in 100 mL of DMEM / 10% FBS selective medium containing thymidine pyrimidine, hypoxanthine and aminopterin. Transfer the cell suspension to 96-well plates using a pipette, 100 μL per well.

[0061] 6. Incubate at 37°C with 6% CO2 for 6 days. After 7 days of incubation, detect the presence of NMP22 antibody in each well using indirect ELISA.

[0062] 7. Subcloning was performed using limiting dilution. Cell counts were determined by serial dilutions of cells in DMEM / 10% FBS selective medium containing thymidine, hypoxanthine, and aminopterin until a cell density of 5–15 cells / mL was achieved.

[0063] 8. For each hybridoma, transfer 200 μL of cell solution to 96 wells using a pipette, at a density of 1–3 cells / well. Incubate at 37°C in 6% CO2 for 1 week, and then perform the above ELISA assay on the supernatant to assess the presence of antibodies against NMP22.

[0064] Example 3: Indirect ELISA screening of NMP22 positive clones

[0065] 1. Dilute recombinant NMP22 to 0.5 μg / mL with PBS buffer, coat ELISA plates (Nunc) with 100 μL / well, and incubate overnight at 4°C.

[0066] 2. Wash the wells once with PBS-T (0.05% Tween) and block them with 200 μL / well of PBS-T containing 1% BSA at 37°C for 0.5 hours.

[0067] 3. Then discard the blocking solution, add 100 μL of hybridoma cell culture supernatant to each well, and incubate at room temperature for 1 hour.

[0068] 4. Wash the plate three times with PBST and incubate it at 37°C for 0.5 hours with 100 μL / well of horseradish peroxidase-labeled goat anti-mouse IgG working solution (GenScript).

[0069] 5. Wash the plate five times with PBST, then add TMB chromogenic solution (GenScript) and incubate at room temperature in the dark for 15 minutes.

[0070] 6. Terminate the reaction by adding 50 μL of 1M HCl stop solution (Sigma). Read the plate at 450 nm using a microplate reader (TECAN).

[0071] Example 4: Sequencing of the variable region of NMP22 monoclonal antibody

[0072] 1. Antibody subtypes in hybridoma cell culture supernatant were identified using a rapid ELISA mouse antibody subtype identification kit (Clonotyping System-HRP SouthernBiotech).

[0073] 2. Using TRIzol (Ambion) from 3×10 6 ~5×10 6 Total RNA was extracted from hybridoma cells and analyzed using antibody subtype-specific primers and universal primers (PrimeScript). TM 1 st The Strand cDNA Synthesis Kit (Takara) reverse transcribes it into cDNA.

[0074] 3. Subsequently, the V-region fragments of mouse immunoglobulin heavy and light chains were amplified by RACE PCR (GenScript), and the resulting PCR fragments were subcloned into the pMD18-T vector system (Takara). The inserted fragments were then sequenced using vector-specific primers.

[0075] 4. Finally, the amino acid sequences of the heavy and light chain variable regions of the mouse anti-NMP22 monoclonal antibody (1A5C10) were obtained.

[0076] The CDR and frame region sequences of the mouse anti-NMP22 monoclonal antibody (1A5C10) are shown in Tables 1 and 2.

[0077] Table 1. CDR region sequence of antibody 1A5C10

[0078]

[0079] V H SEQ ID NO:2

[0080] V L Sequence (SEQ ID NO:6): DIVMTQSPSSLAMSVGQKVTMSCKSSQ SLLNSDNQKNYLAWYQQKPGQSPKLLVYFASTRESGVPDRFIGSGSGTDF TLTITTVQAEDLADYFCQQHYSTPWTFGGGTKLEIK

[0081] Table 2. Frame region sequence of antibody 1A5C10

[0082]

[0083] Example 5: Production of NMP22 Monoclonal Antibody

[0084] 1. After culturing the hybridoma cells in a shake flask at 37°C for 10 days, the supernatant was collected for antibody purification.

[0085] 2. Before purification, depyrogenize the tubing and Protein A column with 0.2M NaOH. Reequilibrate the column with a buffer containing 0.05M Tris and 1.5M NaCl (pH 8.0).

[0086] 3. The harvested cell culture supernatant was then diluted 1:1 with 2× the above buffer solution and filtered for sterilization.

[0087] 4. Incubate the filtered supernatant with the protein A column at room temperature for 2 hours. Wash the column with 1× the above buffer, then elute the IgG with sterile 0.1M sodium citrate (pH 3.5). Collect the eluent and neutralize it with one-ninth volume of sterile 1M Tris-HCl (pH 9).

[0088] 5. Replace the product buffer with PBS (pH 7.4) to remove any elution buffer and concentrate the sample. After concentration, measure the sample using an extinction coefficient Ec (0.1%) of 1.43 via OD. 280nm The antibodies were quantified.

[0089] 6. The purified antibody was analyzed by SDS-PAGE using a 10% precast gel (GenScript) via the BioRad electrophoresis system. The gel was stained with Estain 2.0 (GenScript), and antibody purity was calculated by comparing the staining bands with the protein marker (GenScript). The SDS-PAGE results are shown in the figure below. Figure 3 As shown.

[0090] Example 6: Binding of NMP22 monoclonal antibody / post-immunization antiserum to recombinant NMP22 protein

[0091] 1. Indirect ELISA was used to assess the binding ability of antiserum / purified antibody to NMP22. ELISA plates (Nunc) were coated overnight at 4°C with 0.5 μg / mL recombinant NMP22 in 100 μL / well of PBS.

[0092] 2. Wash the plate with PBS-T (0.05% Tween) and block it with 200 μL / well of PBST containing 1% BSA at 37°C for 2 hours.

[0093] 3. Discard the sealing solution and dry at 37°C for 2 hours.

[0094] 4. Then discard the blocking solution, add 100 μL of purified antibody (or antiserum diluted 1:1000) at 1 μg / mL to the first well, and then perform serial dilutions of 3-fold to obtain a total of 10 test concentration gradients.

[0095] 5. Then incubate at room temperature for 1 hour. Wash the plate four times with PBST and incubate at 37°C for 0.5 hours with 100 μL / well of horseradish peroxidase-conjugated goat anti-mouse IgG (GenScript).

[0096] 6. Wash the plate four times with PBST, then add TMB chromogenic solution (FcMACS) and incubate in the dark at room temperature for 15 minutes.

[0097] 7. Terminate the reaction by adding 50 μL of 1M HCl (Sigma) stop solution. Read the plate at 450 nm using a microplate reader.

[0098] The binding affinity of mouse antiserum to recombinant NMP22 protein after immunization is as follows: Figure 2 As shown in the figure. The X-axis represents the OD450 signal value read by the microplate reader. The Y-axis represents the dilution factor of the mouse antiserum after triple immunization, expressed as a logarithm with a base of 10. Figure 2 The results showed that after mice were immunized with recombinant NMP22 protein, specific NMP22 antibodies were produced in the mouse serum.

[0099] The binding affinity of mouse anti-NMP22 monoclonal antibody (1A5C10) to recombinant NMP22 protein is as follows: Figure 4 As shown in the figure. The X-axis represents the OD450 signal value of the microplate reader. The Y-axis represents the dilution factor of the purified antibody, expressed as the logarithm to the base 10. Figure 4 The results showed that the purified mouse anti-NMP22 monoclonal antibody (1A5C10) could specifically recognize the NMP22 recombinant protein, with an EC50 of 6.92 ng / mL, indicating that the NMP22 monoclonal antibody (1A5C10) had a high affinity for the NMP22 recombinant protein and could effectively bind to it.

Claims

1. An antibody or antigen-binding fragment thereof targeting NMP22, comprising a heavy chain variable region and a light chain variable region, characterized in that, The amino acid sequences of the heavy chain variable regions HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; the amino acid sequences of the light chain variable regions LCDR1, LCDR2, and LCDR are shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein, The amino acid sequences of HFR1, HFR2, HFR3, and HFR4 in the framework region of the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively; the amino acid sequences of LFR1, LFR2, LFR3, and LFR4 in the framework region of the light chain variable region are shown in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

6.

4. The antibody or antigen-binding fragment thereof as described in any one of claims 1-3, characterized in that, The antibody or its antigen-binding fragment satisfies one or more of the following: (1) The antibody is a full-length antibody, and the antigen-binding fragment is Fab, Fab', F(ab')2 or scFv; (2) The antibody or its antigen-binding fragment is a monospecific antibody; (3) The antibody or its antigen-binding fragment is a monoclonal antibody; (4) The antibody or its antigen-binding fragment is a mouse antibody or a humanized antibody.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein, When the antibody is a full-length antibody, the full-length antibody includes a heavy chain constant region and a light chain constant region; the heavy chain constant region is selected from IgG, IgA, IgM, IgE and IgD; the light chain constant region is a κ or λ chain.

6. The antibody or antigen-binding fragment thereof of claim 5, wherein, The heavy chain constant region is IgG1, IgG2, IgG3, or IgG4.

7. An isolated nucleic acid, comprising, The nucleic acid encodes the antibody or its antigen-binding fragment as described in any one of claims 1-6.

8. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the isolated nucleic acid as described in claim 7.

9. The recombinant expression vector of claim 8, wherein, The recombinant expression vector is a plasmid, granule, bacteriophage, or viral vector.

10. A transformant characterized in that, It comprises the nucleic acid as described in claim 7 or the recombinant expression vector as described in claim 8 or 9.

11. The transformant of claim 10, wherein The host cell of the transformant is a prokaryotic cell or a eukaryotic cell.

12. The transformant of claim 11, wherein, The eukaryotic cells are yeast cells or mammalian cells.

13. The transformant of claim 12, wherein, The mammalian cells are SP2 / 0 cells, 293T cells, or CHO cells.

14. A method of making an antibody or antigen-binding fragment thereof targeting NMP22, characterized in that, The method comprises the following steps: culturing the transformant as described in any one of claims 10-13, and obtaining the antibody or antigen-binding fragment targeting NMP22 from the culture.

15. A kit comprising, The kit comprises an antibody or antigen-binding fragment thereof as described in any one of claims 1-6, a nucleic acid as described in claim 7, a recombinant expression vector as described in claim 8 or 9, and / or a transformant as described in any one of claims 10-13.

16. A method for detecting NMP22, characterized in that, The method includes the following steps: (1) Contact the sample with the antibody or antigen-binding fragment thereof as described in any one of claims 1-6 or the kit as described in claim 15; (2) Quantitatively or qualitatively determine the NMP22 in the sample based on the binding of the antibody or its antigen-binding fragment to the sample; The method described is not for diagnostic purposes.

17. The use of an antibody or antigen-binding fragment as described in any one of claims 1-6, a nucleic acid as described in claim 7, a recombinant expression vector as described in claim 8 or 9, or a transformant as described in any one of claims 10-13 in the preparation of a reagent for detecting NMP22.

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