Antibody targeting NMP22 or antigen binding fragment thereof and application thereof

Abstract

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

Description

Technical Field

[0001] The present invention belongs to the field of tumor immune detection, and particularly relates to an antibody targeting NMP22 or an antigen-binding fragment thereof and its application. Background Art

[0002] Bladder cancer is one of the most common malignant tumors in the urinary system. In 2018, the number of newly diagnosed bladder cancer patients globally was 549,000, and the number of deaths reached as high as 200,000, ranking 11th among malignant tumors in terms of incidence. Early diagnosis and treatment of bladder cancer are crucial for cancer patients. Cystoscopic tissue biopsy, urine exfoliated cell examination, and imaging examination are currently commonly used clinical diagnostic methods. Cystoscopic tissue biopsy is the gold standard for the diagnosis of bladder cancer, but it is highly invasive. Urine exfoliated cytology is the most commonly used clinical examination method at present, but its sensitivity is low. Simple, rapid, and accurate detection methods for bladder cancer have become a research hotspot in bladder cancer diagnosis.

[0003] Urine nuclear matrix protein 22 (NMP22) is a mitotic apparatus protein. It is mainly involved in the formation of the nuclear spindle of cells. NMP22 participates in chromosome formation and plays a key role in the normal pairing and separation of chromosomes. Abnormal spindles often cause disorders in chromosome pairing and separation, and chromosomes cannot be correctly separated, leading to cell death or malignancy. In the early stage of apoptosis, some NMP22 will be hydrolyzed, resulting in the detachment of DNA from the nuclear matrix. When bladder tumor cells proliferate and divide malignantly, NMP22 in the nuclear matrix is highly expressed. When cancer cells die, NMP22 is released into the urine, causing a significant increase in the concentration of NMP22 in the urine of patients. 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 and effectively detect NMP22 with high sensitivity and high specificity. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defect in the prior art of lacking a method for highly sensitive, non-invasive, simple, and rapid detection of bladder cancer, and to provide an antibody targeting NMP22 or an antigen-binding fragment thereof and its application. The antibody targeting NMP22 or the antigen-binding fragment thereof provided by the present invention can specifically recognize and bind to the NMP22 protein, has high specificity and high sensitivity, 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 technical problems through the following technical solutions.

[0006] In the first aspect of the present invention, there is provided an antibody or an antigen-binding fragment thereof targeting NMP22, which comprises a heavy chain variable region and a light chain variable region. The amino acid sequences of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are shown as SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5 respectively; the amino acid sequences of LCDR1, LCDR2, and LCDR of the light chain variable region are shown as 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 framework region of the heavy chain variable region are shown as 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 of the framework region of the light chain variable region are shown as 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 as SEQ ID NO:2, and the amino acid sequence of the light chain variable region is shown as 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, bispecific antibody or multispecific antibody;

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

[0013] (4) The antibody or its antigen-binding fragment is a murine 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 comprises 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] In a second aspect of the present invention, there is provided an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof as described in the first aspect.

[0016] In a third aspect of the present invention, there is provided 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, cosmid, phage or viral vector.

[0018] In a fourth aspect of the present invention, there is provided 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 cell is a yeast cell or a mammalian cell; the mammalian cell is, for example, an SP2 / 0 cell, a 293T cell or a CHO cell.

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

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

[0023] In a seventh aspect of the present invention, there is provided a method for detecting NMP22, the method comprising the following steps:

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

[0025] (2) making a quantitative or qualitative determination of NMP22 in the sample according to the binding of the antibody or antigen-binding fragment thereof to the sample.

[0026] In an eighth aspect of the present invention, there is provided the use of the antibody or antigen-binding fragment as described in the first aspect, the nucleic acid as described in the second aspect, the recombinant expression vector as described in the third aspect or the 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 an application of an antibody targeting NMP22 or an 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 in the preparation of a drug for diagnosing, preventing, and / or treating 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 general knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

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

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

[0033] Figure 1 For SDS-PAGE identification of the purity of NMP22 protein, the left band is the protein molecular weight Marker, and the right band is the purified NMP22 protein.

[0034] Figure 2 For ELISA identification of the serum titer of NMP22 protein-immunized mice.

[0035] Figure 3 For SDS-PAGE identification of the purity of the mouse anti-NMP22 protein monoclonal antibody (1A5C10) after purification. 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 For ELISA identification of the specific binding of the mouse anti-NMP22 protein monoclonal antibody (1A5C10) to the NMP22 protein. Detailed Embodiments

[0037] Term

[0038] Unless otherwise specified, the technical and scientific terms used in the present invention have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0039] As used herein, the term "antibody" refers to an immunoglobulin molecule, which is typically a tetramer composed of two identical heavy chains and two identical light chains linked to each other by disulfide bonds. According to the differences in the conservation of amino acid sequences, the heavy and light chains are divided into a variable region (V) at the amino terminus and a constant region (C) at the carboxyl terminus. Within the variable regions of the heavy and light chains, there are three local regions with a higher degree of variation in amino acid composition and arrangement order, which are the key positions for the antibody to bind to the antigen, and thus are also called complementarity-determining regions (CDRs). In this article, the three heavy-chain complementarity-determining regions are respectively called HCDR1, HCDR2, and HCDR3, and the three light-chain complementarity-determining regions are respectively called LCDR1, LCDR2, and LCDR3. The variable regions of one heavy chain and one light chain interact to form the antigen-binding site (Fv). According to the amino acid sequences of their heavy-chain constant regions, antibodies can be divided into different classes. There are five main types of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses, for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are known in the art. The present invention is intended to include antibodies of any of the foregoing classes or subclasses.

[0040] The term "antibody" as used herein is also intended to cover its digestive fragments or functional variants, for example, antibody fragments capable of binding to NMP22 or a part thereof, including but not limited to Fab (e.g., obtained by papain digestion of an antibody), F(ab’)2 (e.g., obtained by pepsin digestion), Fv, or scFv (e.g., obtained by molecular biology techniques).

[0041] The term "monoclonal antibody" as used herein refers to an antibody that is homogeneous and specific for a particular antigenic epitope. Compared with a typical polyclonal antibody preparation that typically includes different antibodies against different antigenic determinants (epitopes), each monoclonal antibody is directed against a single antigenic determinant on the antigen. The modifier "monoclonal" indicates the homogeneous nature of the antibody and is not to be construed as requiring an antibody 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 a polynucleotide that does not exist in its natural state in nature, including polynucleotides isolated from nature (including within organisms) by biological techniques and also including synthetic polynucleotides. The isolated polynucleotide can be genomic DNA, cDNA, mRNA, or other synthetic RNA, or a combination thereof. Multiple nucleotide sequences are provided herein for encoding the heavy-chain variable region and the light-chain variable region of a monoclonal antibody against human NMP22 protein. It should be noted that those skilled in the art can, based on codon degeneracy, design nucleotide sequences that are not exactly the same as the nucleotide sequences provided above according to the amino acid sequences of the heavy-chain variable region and the light-chain variable region provided herein, but all encode the same amino acid sequence. These modified nucleotide sequences are also included within the scope of the present invention.

[0043] When referring to polynucleotides, the term "vector" as used herein refers to any molecule (e.g., nucleic acid, plasmid, or virus, etc.) used to transfer nucleotide coding information into a host cell. The term "expression vector" or "expression cassette" refers to a vector suitable for expressing a gene of interest (nucleotide sequence to be expressed) in a host cell and generally includes the gene of interest, a promoter, and a terminator.

[0044] The term "host cell" as used herein refers to a cell that has been or can be transformed with a nucleic acid sequence and thus expresses the selected gene of interest. This term includes the progeny of the parental cell, regardless of whether the progeny is identical to the original parental cell in terms of morphology or genetic composition, as long as the selected gene of interest is present in the progeny. Commonly used host cells include bacteria, yeast, and mammalian cells, etc.

[0045] The term "transfection" as used herein refers to the uptake of foreign or exogenous DNA by a cell, and this technique can be used to introduce one or more exogenous DNA moieties into a suitable host cell. Cells can be induced by physical and chemical methods (e.g., by treatment with calcium chloride) to be in a physiological state that is optimal for the uptake and accommodation of foreign DNA, i.e., "competent state".

[0046] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions noted in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product instructions.

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

[0048] Retrieve the full-length gene information of human NMP-22 from GenBank (accession number BC043499.1), and intercept the N-terminal fragment sequence of the NMP-22 protein. Add a His-tag at 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. Use GenScript codon optimization software to optimize the gene sequence of the recombinant human NMP22 protein fragment, and synthesize the optimized gene sequence of the recombinant human NMP22 protein fragment by chemical synthesis. Insert the correctly sequenced gene sequence of the human NMP22 protein fragment into plasmid PET15b to construct a recombinant plasmid.

[0049] Transfect competent Escherichia coli BL21 cells with plasmid PET15b-NMP22 containing the gene of the human NMP22 protein fragment. Put Escherichia coli BL21 containing plasmid PET15b-NMP22 into a medium (1 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, and 100 mg / L Ampicillin) and culture at 37°C.

[0050] Rinse the constant flow pump with distilled water, and then rinse the glass chromatography column. Add about 200 mL of Ni-IDA (Genscript) to the column to pack the column, and wait for all the column materials to naturally precipitate.

[0051] Equilibrate with about 3 L of equilibration buffer (20 mM Tris, 300 mM NaCl) through a constant flow pump at a flow rate of 5 mL / min; resuspend Escherichia coli containing recombinant human NMP22 protein with 200 mL of equilibration buffer (20 mM Tris, 300 mM NaCl), and break the cells with an ultrasonic cell disruptor (JY98-IIIDH, Ningbo Xinzhi Biotechnology Co., Ltd.).

[0052] Load the supernatant after centrifugation of the disrupted solution at a flow rate of 2 mL / min; after loading, wash away impurities with the equilibration buffer until the absorbance remains unchanged, at a flow rate of 5 mL / min.

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

[0054] Perform SDS-PAGE detection with a gel concentration of 4 - 20%, and the results are as Figure 1 shown. The purity of the purified NMP22 recombinant protein reaches over 90%.

[0055] Example 2 Preparation of NMP22 Monoclonal Antibody

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

[0057] 2. The mice were boosted by injecting intraperitoneally / subcutaneously every two weeks with a 1:1 emulsion of Freund's incomplete adjuvant (Sigma-Aldrich) containing 25 μg of NMP22 up to 3 times.

[0058] 3. Four days before myeloma fusion, Mouse #1 showing the highest antibody titer (see Figure 2 Antibody titer was determined by serum ELISA) was boosted intraperitoneally with 25 μg of NMP22 (without adjuvant).

[0059] 4. The spleen was extracted and ground under sterile conditions to form a single-cell suspension, and at the same time, a single-cell suspension of myeloma cells (SP2 / 0) was prepared. 8.1×10 7 spleen cells were fused with 3.8×10 7 SP2 / 0 mouse myeloma cells using electrofusion.

[0060] 5. The fused cells were resuspended in 100 mL of DMEM / 10% FBS selection medium containing thymidine, hypoxanthine, and aminopterin, and the cell suspension was pipetted into a 96-well plate at a volume of 100 μL per well.

[0061] 6. Cultured for 6 days at 6% CO2 and 37°C. After 7 days of incubation, the presence of NMP22 antibody in each well was detected by indirect ELISA.

[0062] 7. Subcloning was performed using the limiting dilution method. A hemocytometer was used and the cells were serially diluted in DMEM / 10% FBS selection medium containing thymidine, hypoxanthine, and aminopterin to determine the cell number until the cell density reached 5 - 15 cells / mL.

[0063] 8. For each hybridoma, 200 μL of the cell solution was pipetted into 96 wells at a density of 1 - 3 cells / well. Cultured at 37°C in 6% CO2 for 1 week, and the presence of antibody against NMP22 in the supernatant was evaluated by the above ELISA.

[0064] Example 3 Indirect ELISA Screening for NMP22-Positive Clones

[0065] 1. Recombinant NMP22 was diluted to 0.5 μg / mL with PBS buffer and used to coat an ELISA plate (Nunc) at 100 μL / well overnight at 4°C.

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

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

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

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

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

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

[0072] 1. Use a Quick ELISA Mouse Antibody Subtype Identification Kit (Clonotyping System-HRP SouthernBiotech) to identify the subtype of the antibody in the hybridoma cell culture supernatant.

[0073] 2. Use TRIzol (Ambion) to extract total RNA from 3×10 6 ~5×10 6 hybridoma cells, and reverse transcribe it into cDNA using antibody subtype-specific primers and universal primers (PrimeScript TM 1 st Strand cDNA Synthesis Kit, Takara).

[0074] 3. Subsequently, amplify the murine immunoglobulin heavy and light chain V-region fragments by RACE PCR (GenScript), subclone the obtained PCR fragments into the pMD18-T vector system (Takara), and sequence the inserted fragments 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 region and framework region sequences of the mouse anti-NMP22 monoclonal antibody (1A5C10) are shown in Table 1 and Table 2.

[0077] Table 1 CDR Region Sequences of Antibody 1A5C10

[0078]

[0079] V H Sequence (SEQ ID NO:2): EVKLVESGGGLVQPGDSLRLSCATSGFTFTDYYLSWVRQPPGKALEWLGFIRNKANSYTTEYSSSVRGRFTISRDNSQSILYLQMNTLRAEDSATYYCARDALDYWGQGTSVTVSS

[0080] V L Sequence (SEQ ID NO:6): DIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSDNQKNYLAWYQQKPGQSPKLLVYFASTRESGVPDRFIGSGSGTDFTLTITTVQAEDLADYFCQQHYSTPWTFGGGTKLEIK

[0081] Table 2 Framework Region Sequences of Antibody 1A5C10

[0082]

[0083] Example 5 Production of NMP22 Monoclonal Antibody

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

[0085] 2. Before purification, the pipelines and the Protein A column were depyrogenated with 0.2 M NaOH. The column was re-equilibrated with a buffer containing 0.05 M Tris and 1.5 M NaCl (pH 8.0).

[0086] 3. Subsequently, the harvested cell culture supernatant was diluted 1:1 with 2× the above buffer and filtered through a sterile filter.

[0087] 4. The filtered supernatant was incubated with the Protein A column at room temperature for 2 hours. After washing the column with 1× the above buffer, IgG was eluted using sterile 0.1 M sodium citrate (pH 3.5). The eluate was collected and neutralized with one-ninth volume of sterile 1 M Tris-HCl (pH 9).

[0088] 5. The product buffer was exchanged to PBS (pH 7.4) to remove any elution buffer and concentrate the sample. After concentration, the antibody was quantified by OD using an extinction coefficient Ec (0.1%) of 1.43 280nm for the antibody.

[0089] 6. The purified antibody was analyzed by SDS-PAGE using a 10% precast gel (GenScript) on a BioRad electrophoresis system. The gel was stained with Estain 2.0 (GenScript) and the antibody purity was calculated by comparing the stained bands with a protein Marker (GenScript). The SDS-PAGE result diagram is as shown in Figure 3 shown.

[0090] Example 6 Binding of NMP22 Monoclonal Antibody / Immune Serum to NMP22 Recombinant Protein

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

[0092] 2. The plate was washed with PBS-T (0.05% Tween) and blocked with PBST containing 1% BSA at 200 μL / well for 2 hours at 37°C.

[0093] 3. The blocking solution was discarded and the plate was dried at 37°C for 2 hours.

[0094] 4. Subsequently, the blocking solution was discarded. 100 μL of 1 μg / mL purified antibody (or immune serum diluted 1:1000) was added to the first well and serially diluted 3-fold, for a total of 10 test concentration gradients.

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

[0096] 6. The plate was washed four times with PBST, then TMB chromogenic solution (FcMACS) was added and incubated at room temperature in the dark for 15 minutes.

[0097] 7. The reaction was terminated by adding 50 μL of 1 M HCl termination solution (Sigma). The plate was read at 450 nm using a microplate reader.

[0098] The binding ability of the immune serum of immunized mice to NMP22 recombinant protein is as shown in Figure 2 shown. Among them, the X-axis represents the OD450 signal value read by the microplate reader. The Y-axis represents the dilution factor of the immune serum of mice after the third immunization, which is expressed as the logarithm to the base 10. Figure 2 The results showed that specific NMP22 antibodies had been produced in the sera of mice after immunization with NMP22 recombinant protein.

[0099] The binding ability of mouse anti-NMP22 monoclonal antibody (1A5C10) to NMP22 recombinant protein is as Figure 4 shown. Among them, the X-axis represents the OD450 signal value read by the microplate reader. The Y-axis represents the dilution factor of the purified antibody, which is expressed as the logarithm to the base 10. Figure 4 The results show that the purified mouse anti-NMP22 monoclonal antibody (1A5C10) can specifically recognize NMP22 recombinant protein, and the EC50 of the binding curve is 6.92 ng / mL, indicating that the NMP22 monoclonal antibody (1A5C10) has a high affinity for NMP22 recombinant protein and can effectively bind to NMP22 recombinant protein.

Claims

1. An antibody or antigen-binding fragment thereof targeting NMP22, which 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 shown as SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5 respectively; the amino acid sequences of LCDR1, LCDR2, and LCDR of the light chain variable region are shown as SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9 respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequences of HFR1, HFR2, HFR3, and HFR4 of the framework region of the heavy chain variable region are shown as 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 of the framework region of the light chain variable region are shown as 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 according to claim 1 or 2, wherein The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:2, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO:

6.

4. The antibody or antigen-binding fragment thereof according to 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, bispecific antibody or multispecific antibody; (3) The antibody or its antigen-binding fragment is a monoclonal antibody or polyclonal antibody; (4) The antibody or its antigen-binding fragment is a murine antibody or a humanized antibody; Preferably, when the antibody is a full-length antibody, the full-length antibody comprises 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 κ or λ chain.

5. An isolated nucleic acid, characterized in that, The nucleic acid encodes the antibody or its antigen-binding fragment according to any one of claims 1-4.

6. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the isolated nucleic acid according to claim 5; Preferably, the recombinant expression vector is a plasmid, cosmid, phage or viral vector.

7. A transformant, characterized in that, It comprises the nucleic acid according to claim 5 or the recombinant expression vector according to claim 6; Preferably, the host cell of the transformant is a prokaryotic cell or a eukaryotic cell; More preferably, the eukaryotic cell is a yeast cell or a mammalian cell; the mammalian cell is, for example, SP2 / 0 cell, 293T cell or CHO cell.

8. A method for preparing an antibody or an antigen-binding fragment thereof targeting NMP22, characterized in that, The method comprises the following steps: culturing the transformant according to claim 7, and obtaining the antibody or its antigen-binding fragment targeting NMP22 from the culture.

9. A kit, characterized in that, The kit comprises the antibody or its antigen-binding fragment according to any one of claims 1-4, the nucleic acid according to claim 5, the recombinant expression vector according to claim 6 and / or the transformant according to claim 7.

10. A method for detecting NMP22, characterized in that, The method includes the following steps: (1) Contact a sample with the antibody or its antigen-binding fragment as described in any one of claims 1-4 or the kit as described in claim 9; (2) Judge quantitatively or qualitatively the NMP22 in the sample according to the binding situation between the antibody or its antigen-binding fragment and the sample.

11. Use of the antibody or antigen-binding fragment as described in any one of claims 1-4, the nucleic acid as described in claim 5, the recombinant expression vector as described in claim 6, or the transformant as described in claim 7 in the preparation of a reagent for detecting NMP22.

12. Use of the antibody or its antigen-binding fragment targeting NMP22 as described in any one of claims 1-4, the nucleic acid as described in claim 5, the recombinant expression vector as described in claim 6, and / or the transformant as described in claim 7 in the preparation of a drug for diagnosing, preventing, and / or treating tumors; preferably, the tumor is an NMP22-positive tumor; more preferably, the tumor is bladder cancer.

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