An anti-Kim-1 antibody and its application

Abstract

The present invention discloses an anti-Kim-1 antibody and its application, belonging to the field of immunoglobulins. Specifically disclosed are anti-Kim-1 antibodies or antigen-binding fragments thereof comprising a heavy-chain variable region (positions 1-120 of SEQ ID NO: 1) and a light-chain variable region (positions 1-115 of SEQ ID NO: 2), including the murine monoclonal antibody CV18 and the humanized chimeric antibody hCV18 developed based on it. The antibodies CV18 and hCV18 of the present invention are characterized by high affinity and specificity, have good sensitivity and specificity for detecting the Kim-1 protein, and can be made into products for clinical Kim-1 detection, such as detection reagents or kits, etc. The present invention solves the problems of relatively short supply of antibody raw materials for the Kim-1 detection item in the current market, as well as poor specificity and low sensitivity, and can be used as an auxiliary diagnostic tool for kidney-related diseases (such as acute kidney injury, etc.).

Description

Technical Field

[0001] The present invention belongs to the field of immunoglobulins, and particularly relates to an anti-Kim-1 antibody and its application. Background Art

[0002] Kidney injury molecule 1 (Kim-1), also known as hepatitis A virus cellular receptor 1 (HAVCR1), is a transmembrane glycoprotein in renal proximal tubular epithelial cells and belongs to the immunoglobulin gene superfamily. Kim-1 is not expressed in healthy kidneys. When the kidney is damaged, the expression level of KIM-1 will increase significantly. This property makes Kim-1 a sensitive biomarker for diagnosing and monitoring kidney injury. Especially in the early stage of kidney injury, obvious changes in KIM-1 can be detected, which helps to timely detect and intervene in kidney diseases, reverse renal function injury, and avoid further deterioration of the condition. KIM-1 can rapidly, sensitively, and specifically reflect the injury and recovery processes of various kidney diseases. It is not only a reliable biomarker for detecting early kidney injury, but also can be used to evaluate the recovery of kidney diseases, providing a basis for doctors to formulate personalized treatment plans. By using specific antibodies against Kim-1 to monitor the change of KIM-1 level, early kidney injury can be detected, the progression and treatment effect of the disease can be evaluated, thus providing key information for timely treatment and a basis for doctors to adjust treatment plans. Although certain progress has been made in the current detection technology of Kim-1, there are still problems such as relatively short supply of antibody raw materials for Kim-1 detection items, as well as insufficient specificity and sensitivity.

[0003] In view of this, researching and developing anti-Kim-1 antibodies with high specificity and sensitivity, as well as their supporting immunological detection methods and immunoassay kits, helps to achieve early and accurate identification of kidney injury, improve the accuracy of clinical diagnosis, optimize patient management strategies, and at the same time provide strong support for the timely treatment and prognosis evaluation of kidney diseases. Summary of the Invention

[0004] One object of the present invention is to provide an anti-Kim-1 antibody and its application. The technical problems to be solved are not limited to the described technical topics, and those skilled in the art can clearly understand other technical topics not mentioned herein through the following description.

[0005] To achieve the above object, the present invention first provides an anti-Kim-1 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment comprises a heavy-chain variable region and a light-chain variable region. The heavy-chain variable region comprises CDR1 shown by the amino acid sequence at positions 31-35 of SEQ ID NO:1, CDR2 shown by the amino acid sequence at positions 50-66 of SEQ ID NO:1, and CDR3 shown by the amino acid sequence at positions 99-109 of SEQ ID NO:1. The light-chain variable region comprises CDR1 shown by the amino acid sequence at positions 24-39 of SEQ ID NO:2, CDR2 shown by the amino acid sequence at positions 55-61 of SEQ ID NO:2, and CDR3 shown by the amino acid sequence at positions 94-105 of SEQ ID NO:2.

[0006] Wherein the antigen-binding fragment includes Fab, Fab′, F(ab′) 2 , antibody variable region (Fv), disulfide-stabilized Fv (dsFv), single-chain antibody (ScFv), single-domain antibody (sdAb, i.e., nanobody), minibody, and minimal recognition unit (MRU), etc., but not limited thereto.

[0007] The sequences of the complementary determining regions (CDRs) described herein can be defined according to the Kabat numbering system.

[0008] Both the heavy-chain variable region and the light-chain variable region comprise framework regions (FR, the regions other than CDRs in the variable region). The framework region can be derived from human or non-human mammals (such as mice, rats, guinea pigs, rabbits, sheep, horses, donkeys, chickens, monkeys, camels, etc.).

[0009] Furthermore, the amino acid sequence of the heavy-chain variable region can be as shown at positions 1-120 of SEQ ID NO:1, and the amino acid sequence of the light-chain variable region can be as shown at positions 1-115 of SEQ ID NO:2.

[0010] Furthermore, the antibody or the antigen-binding fragment thereof may further include a heavy-chain constant region (CH) and a light-chain constant region (CL). The heavy-chain constant region can be selected from the heavy-chain constant regions of IgG, IgA, IgM, IgD, or IgE. The heavy-chain constant region can also be selected from the CH1, Fc, and CH3 domains. The light-chain constant region can be selected from the kappa (κ) or lambda (λ) type light-chain constant regions. The heavy-chain constant region and the light-chain constant region can be derived from human or non-human mammals (such as mice, rats, guinea pigs, rabbits, sheep, horses, donkeys, chickens, monkeys, camels, etc.).

[0011] Further, the heavy chain constant region may be selected from the heavy chain constant regions of human IgG subclasses such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant region may also be selected from the heavy chain constant regions of mouse IgG subclasses such as IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgG4, IgG5, and IgG6.

[0012] Further, the antibody or its antigen-binding fragment may further comprise a heavy chain constant region and a light chain constant region as shown in A1) or A2) below:

[0013] A1) The amino acid sequence of the heavy chain constant region is as shown in positions 121-444 of SEQ ID NO:1, and the amino acid sequence of the light chain constant region is as shown in positions 116-222 of SEQ ID NO:2;

[0014] A2) The amino acid sequence of the heavy chain constant region is as shown in positions 121-450 of SEQ ID NO:5, and the amino acid sequence of the light chain constant region is as shown in positions 116-222 of SEQ ID NO:6.

[0015] Further, the antibody or its antigen-binding fragment may comprise a heavy chain and a light chain as shown in B1) or B2) below:

[0016] B1) The amino acid sequence of the heavy chain is as shown in SEQ ID NO:1, and the amino acid sequence of the light chain is as shown in SEQ ID NO:2;

[0017] B2) The amino acid sequence of the heavy chain is as shown in SEQ ID NO:5, and the amino acid sequence of the light chain is as shown in SEQ ID NO:6.

[0018] The antibody containing the heavy chain and light chain described in B1) may be a murine monoclonal antibody, and its name may be CV18.

[0019] The antibody containing the heavy chain and light chain described in B2) may be a humanized chimeric antibody, and its name may be hCV18.

[0020] The present invention also provides a biological material, which may be any one of the following:

[0021] C1) A nucleic acid molecule encoding the heavy chain variable region and the light chain variable region of any antibody or its antigen-binding fragment described herein;

[0022] C2) A nucleic acid molecule encoding the heavy chain and the light chain of any antibody or its antigen-binding fragment described herein;

[0023] C3) An expression cassette containing the nucleic acid molecule described in C1) or C2);

[0024] C4) A recombinant vector containing the nucleic acid molecule described in C1) or C2);

[0025] C5) A recombinant microorganism containing the nucleic acid molecule described in C1) or C2);

[0026] C6) A recombinant host cell containing the nucleic acid molecule described in C1) or C2).

[0027] Furthermore, any of the above biological materials can express any of the antibodies or their antigen-binding fragments described herein.

[0028] Among the above biological materials, the recombinant vector can be a cloning vector or an expression vector.

[0029] Furthermore, the recombinant vector can be a recombinant expression vector obtained by cloning a nucleic acid molecule encoding any of the antibodies or their antigen-binding fragments described herein into an expression vector (such as a prokaryotic expression vector, a eukaryotic expression vector, and a viral expression vector).

[0030] The prokaryotic expression vector can be selected from Escherichia coli expression vectors (such as pET series vectors, etc.). The eukaryotic expression vector can be selected from yeast expression vectors (such as pYES2, pPICZaA, etc.), insect cell expression vectors (such as pFastBac1, pMT-Bip-V5-HisA, pAc5.1, etc.), and mammalian cell expression vectors (such as pCMV3, pcDNA3.1, pcDNA3.4, etc.). The viral expression vector can be selected from adeno-associated virus (AAV) vectors, adenovirus vectors, herpes simplex virus (HSV) vectors, lentivirus (LV) vectors, poxvirus vectors, retrovirus vectors, rhabdovirus (baculovirus) vectors, papillomavirus vectors, Sendai virus vectors, and Simian virus expression vectors.

[0031] Among the above biological materials, the nucleic acid molecule can be any of the following:

[0032] D1) A DNA molecule encoding a sequence shown as positions 1-360 of SEQ ID NO:3 and positions 1-345 of SEQ ID NO:4;

[0033] D2) A DNA molecule encoding a sequence shown as SEQ ID NO:3 and SEQ ID NO:4;

[0034] D3) A DNA molecule encoding a sequence shown as SEQ ID NO:7 and SEQ ID NO:8.

[0035] The DNA molecule shown at positions 1-360 of SEQ ID NO:3 may be the encoding gene for the heavy chain variable region of the murine monoclonal antibody CV18, and the amino acid sequence it encodes is the heavy chain variable region shown at positions 1-120 of SEQ ID NO:1.

[0036] The DNA molecule shown at positions 1-345 of SEQ ID NO:4 may be the encoding gene for the light chain variable region of the murine monoclonal antibody CV18, and the amino acid sequence it encodes is the light chain variable region shown at positions 1-115 of SEQ ID NO:2.

[0037] The DNA molecules shown in SEQ ID NO:3 and SEQ ID NO:4 may be the encoding genes for the heavy and light chains of the antibody CV18.

[0038] The DNA molecules shown in SEQ ID NO:7 and SEQ ID NO:8 may be the encoding genes for the heavy and light chains of the humanized chimeric antibody hCV18.

[0039] The nucleic acid molecules described herein may also include nucleic acid molecules obtained by modifying the nucleotide sequences shown at positions 1-360 of SEQ ID NO:3, positions 1-345 of SEQ ID NO:4, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, and / or SEQ ID NO:8 based on codon preference. Considering the degeneracy of codons and the codon preferences of different species, those skilled in the art can use codons suitable for expression in a specific species as needed.

[0040] The present invention also provides an antibody conjugate, comprising an antibody portion and a conjugate portion, wherein the antibody portion comprises any of the antibodies or antigen-binding fragments thereof described herein.

[0041] Furthermore, the antibody portion and the conjugate portion may be directly linked or covalently linked through a linker (such as a hydrazone bond, a disulfide bond, a thioether bond, a peptide bond).

[0042] Furthermore, the conjugate portion may be a detectable label.

[0043] Furthermore, the detectable label includes enzymes (such as horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-galactosidase, etc.), chemiluminescent reagents (such as acridinium esters, acridinium sulfonamides, luminol and its derivatives, ruthenium derivatives such as tris(bipyridine)ruthenium, etc.), fluorescent dyes (such as AMCA, FITC, CFSE, GFP, DAPI, 7-AAD, Hoechst 33342, Pacific Blue, PE, PE-TR, PE-Cy7, PE-Cy5, PI, PerCP-Cy5.5, APC, APC-CY7, APC-H7, V500, Alexa 700, BV605, BV480, BV785, BV510, BV711, BV421, etc.), near-infrared dyes (such as cyanine dyes, BODIPY dyes, rhodamine dyes, squaric acid dyes, porphyrin dyes, etc.), radionuclides (such as 125I, 18F, 11C, 99mTc, 123I, etc.), biotin, nanoparticles for magnetic resonance imaging, quantum dots for magnetic resonance imaging, magnetic substances (such as magnetic beads, gadolinium complex-containing nanoparticles, superparamagnetic iron oxide nanoparticles), and colloidal gold, etc., but not limited thereto.

[0044] In one or more embodiments of the present invention, the coupling moiety is alkaline phosphatase (ALP).

[0045] The coupling moiety described herein can also be a tag protein.

[0046] In order to facilitate the separation, purification, detection, and / or localization of the antibody or its antigen-binding fragment described herein, a tag protein can be linked to the amino terminus or carboxyl terminus of the antibody or its antigen-binding fragment. The tag proteins include but are not limited to: GST (glutathione S-transferase) tag protein, Trx (thioredoxin) tag protein, nitrogen utilization substrate A (NusA) tag protein, His tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA (influenza hemagglutinin) tag protein, Myc tag protein, LacZ tag protein, CBD (cellulose-binding domain) tag protein, bacteriophage T7 protein kinase (T7PK) tag protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein), or AviTag tag protein. Those skilled in the art know how to select a suitable tag protein according to the desired purpose. The use of the tag does not change the function of the target protein, and its purpose is for separation, purification, detection, or tracing. Therefore, the tag proteins suitable for this application are not limited to specific types. The tag can be separated from the target protein by chemical cleavage methods or enzymatic methods known in the art (such as introducing a protease cleavage site and using TEV protease to cleave and remove the tag).

[0047] The present invention also provides the use of any of the antibodies or antigen-binding fragments thereof, or the biological material, or the antibody conjugate described herein in any of the following:

[0048] E1) Use in the preparation of a product for detecting Kim-1 protein;

[0049] E2) Use in the preparation of a product for diagnosing or screening diseases related to the Kim-1 target.

[0050] The diseases related to the Kim-1 target described herein may be kidney diseases or kidney injuries, including but not limited to acute kidney injury (AKI), diabetic nephropathy (DN), sepsis complicated with acute kidney injury, kidney fibrosis, and inflammation.

[0051] The products described herein may include reagents, kits, chips, test strips, test cards, and immunosensors (such as electrochemical immunosensors).

[0052] The detection of Kim-1 protein includes any in vivo or in vitro detection of Kim-1 protein based on the principle of specific antigen-antibody reaction. The detection of Kim-1 protein may be to detect whether the test sample contains Kim-1 protein and / or to detect the content of Kim-1 protein in the test sample.

[0053] The methods for detecting Kim-1 protein may be immunological detections, such as magnetic particle chemiluminescence detection methods, precipitation reactions, agglutination tests, immunoblotting (Western Blot), enzyme immunoassays (such as ELISA), sandwich ELISA methods, fluorescence-linked immunosorbent assays (FLISA), enzyme immunoassays (EIA), chemiluminescence immunoassays, fluorescence immunoassays, radioimmunoassays (RIA), colloidal gold immunoassays (GIC), colloidal gold immunochromatography assays (GICA), immunohistochemistry (IHC), complement fixation reactions, multiplex immunoassays, and fluorescence immunochromatography techniques, etc.

[0054] Furthermore, the method for detecting Kim-1 protein may be an ELISA method or a magnetic particle chemiluminescence detection method.

[0055] Furthermore, the method for detecting Kim-1 protein includes the step of coating a solid-phase carrier with any of the antibodies or antigen-binding fragments thereof or antibody conjugates described herein to obtain a solid-phase carrier coated with the antibody. The coating may refer to being bound to the solid-phase carrier through physical adsorption.

[0056] Further, the solid-phase carrier may be an enzyme-linked immunosorbent assay (ELISA) plate, a membrane carrier, microspheres, a biochip, magnetic beads, or the like, but is not limited thereto. The material of the solid-phase carrier may be polystyrene, cellulose, cross-linked dextran, polyacrylamide, polyethylene, polypropylene, polyvinyl chloride, cross-linked dextran, glass, silicone rubber, agarose gel, or the like, but is not limited thereto. The membrane carrier may be a nitrocellulose membrane, a glass cellulose membrane, a nylon membrane, or the like, but is not limited thereto.

[0057] The present invention also provides a reagent or a kit, which comprises any one of the antibodies or antigen-binding fragments thereof described herein, or the antibody conjugate.

[0058] In one or more embodiments of the present invention, the antibody or antigen-binding fragment thereof is labeled with alkaline phosphatase (ALP) and used as a component of a chemiluminescent immunoassay reagent for specifically recognizing Kim-1 on magnetic particles.

[0059] The reagent (detection reagent) or kit may have at least one of the following uses: (1) detecting Kim-1 protein; (2) diagnosing or screening diseases related to the Kim-1 target.

[0060] The test sample for the reagent or kit may be a blood sample (such as whole blood, plasma, serum), a urine sample, a tissue sample, a cell sample, or the like, but is not limited thereto.

[0061] The kit may be a chemiluminescent immunoassay kit (such as a chemiluminescent immunoassay kit on magnetic particles), an enzyme-linked immunosorbent assay (ELISA) kit, an immunoprecipitation assay kit, a Western blot assay kit, an immunochromatography assay kit, a flow cytometry assay kit, an immunohistochemistry assay kit, a colloidal gold immunoassay kit, a fluorescence immunoassay kit, or the like, but is not limited thereto.

[0062] Further, the kit may further include reagents required for immunoassay, such as labeled antibodies or antigens, magnetic particles, blocking solution, diluent, washing solution, chromogenic solution, termination solution, or the like, but is not limited thereto.

[0063] The various reagent components of the kit may be present in separate containers, or may be pre-combined in whole or in part into a reagent mixture.

[0064] The present invention also provides a method for preparing any one of the antibodies or antigen-binding fragments thereof described herein, the method comprising expressing any one of the antibodies or antigen-binding fragments thereof described herein in a host cell, and recovering or isolating the antibody or antigen-binding fragment thereof.

[0065] Further, the preparation method may include the following steps: cloning a nucleic acid molecule encoding any of the antibodies or antigen-binding fragments thereof described herein into an expression vector (such as a prokaryotic expression vector, a eukaryotic expression vector, and a viral expression vector) to obtain a recombinant expression vector; introducing the recombinant expression vector into a host cell to obtain a recombinant host cell expressing the antibody or antigen-binding fragment thereof; culturing the recombinant host cell, and recovering or isolating the antibody or antigen-binding fragment thereof from the culture of the recombinant host cell.

[0066] Further, the recovery or isolation may be carried out by precipitation methods (such as salting-out method, organic solvent precipitation method, octanoic acid-ammonium sulfate saturation precipitation method, isoelectric point precipitation method) or chromatography techniques (such as ion exchange chromatography, gel filtration chromatography, affinity chromatography) to separate from the culture (including all substances in the culture vessel).

[0067] The methods of introduction may include any of the following: (1) introducing the target gene or a recombinant vector containing the target gene into a host bacterium by chemical transformation methods (such as Ca ion-induced transformation method, polyethylene glycol-mediated transformation method, or metal cation-mediated transformation method, etc.) or physical transformation methods (such as electroporation transformation method). (2) Transducing the target gene into a host bacterium by phage transduction method. (3) Directly transferring the target gene into a plant receptor cell by physical or chemical methods, such as chemical stimulation method, electroshock method, liposome-mediated method, microinjection method, gene gun method, laser microbeam method, pollen tube pathway method, ultrasonic method, pneumatic gun method, and eddy current method, etc. (4) Transferring the target gene into a plant receptor cell using a vector as a medium, such as Agrobacterium Ti plasmid vector (including Ti plasmid-derived vectors such as co-integrated vector system and binary vector system) mediated method. (5) Introducing the target gene into ex vivo animal cells (transfection) by calcium phosphate co-precipitation method, cationic polymer method (such as DEAE-dextran transfection method), cationic liposome method, electroporation method (i.e., electroporation transfection method), microinjection, gene gun method, or virus-mediated method (such as adenovirus infection method, lentivirus infection method), etc.

[0068] The host cells include but are not limited to 293F cells, CHO cells, 293T cells. In one embodiment of the present invention, the host cell is ExpiCHO cell.

[0069] The present invention first screened and identified a murine monoclonal antibody CV18 with high affinity against Kim-1 through hybridoma technology. The titer of this antibody can reach 1:729000. After identification, the heavy chain of antibody CV18 is of IgG1 subtype and the light chain is of kappa subtype. When screening for antibodies, the present invention selected a variety of natural cell antigens to compare the specific detection of antibodies. After multiple rounds of screening, CV18 was obtained as an antibody with good specificity for Kim-1. During the animal immunization process, subcutaneous multiple-point injections were used, which improved the titer of the final animal serum and increased the positive clone rate. Delaying the addition of HAT screening reagent from the day of fusion to the second day significantly increased the cell positive rate. The present invention further performed humanization modification on the murine monoclonal antibody CV18 and designed and obtained a humanized chimeric antibody hCV18, avoiding the HAMA reaction in natural samples. The titer of the humanized chimeric antibody hCV18 can reach 1:7290000.

[0070] Experiments show that the antibodies CV18 and hCV18 of the present invention have the characteristics of high affinity and specificity, and have good sensitivity and specificity for detecting Kim-1 protein, and can be used in various immunological detection methods. The anti-Kim-1 antibodies (antibodies CV18 and hCV18) of the present invention can be used to detect Kim-1 on the surface of human peripheral blood lymphocytes, or substances containing Kim-1, as well as substances that can specifically bind to Kim-1 (such as anti-Kim-1 antibodies). The detection principle is mainly based on the specific recognition and binding of the Kim-1 antibody of the present invention to Kim-1, and chemical labeling techniques (such as labeled antibodies or labeled specific secondary antibodies) or physical detection techniques (such as light scattering techniques, surface plasmon resonance techniques, etc.) are used for detection. The present invention exemplarily provides the applications of antibodies CV18 and hCV18 in ELISA methods and magnetic particle chemiluminescence methods. In the indirect ELISA method, the lowest detection limit of CV18 antibody is 25 ng / mL; the hCV18 antibody is 12.5 ng / mL, both significantly higher than the control antibody (rabbit anti-Kim-1 antibody abcam, #228973). In addition, the antibodies CV18 and hCV18 of the present invention can also be used in magnetic particle chemiluminescence detection methods to detect Kim-1 protein in urine samples, and both show good specificity.

[0071] The present invention solves the problems of relatively short supply of antibody raw materials for Kim-1 detection items in the current market, as well as poor specificity and low sensitivity. The antibodies CV18 and hCV18 developed by the present invention can be expressed and produced in prokaryotic cells, eukaryotic cells and any recombinant system, and can be made into products clinically used for Kim-1 detection, such as detection reagents or kits, etc., and can be used as auxiliary diagnostic tools for kidney-related diseases, such as acute kidney injury (AKI), diabetic nephropathy (DN), sepsis complicated with acute kidney injury, kidney fibrosis and inflammation, and assessment of kidney treatment prognosis.

[0072] Term Definitions

[0073] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. At the same time, for a better understanding of the present invention, the definitions and explanations of related terms are provided below.

[0074] The term "antibody conjugate" in this article not only includes antibody-drug conjugates (ADCs) formed by conjugating an antibody with a drug (a substance for preventing, treating, or diagnosing diseases) through a linker, but also includes conjugates obtained by conjugating an antibody with a detectable label or tag.

[0075] The term "detectable label" generally refers to any chemical substance with a tracer effect. The conjugate obtained by conjugating an antibody with a detectable label can be called a labeled antibody.

[0076] The term "antigen-binding fragment" generally refers to the antigen-binding fragment of an antibody and antibody analogs, which usually include at least part of the antigen-binding region or variable region of the parental antibody (such as one or more CDRs). The antigen-binding fragment retains at least some binding specificities of the parental antibody. Generally, when expressed based on molarity, the antigen-binding fragment retains at least 10% of the parental binding activity. Specifically, the antigen-binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parental antibody to the target. As is well known to those skilled in the art, the antigen-binding fragment can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of a complete antibody.

[0077] The term "chimeric antibody" generally refers to an antibody in which the constant region (C region) in the antibody molecule is replaced with a human source, while retaining the murine variable regions (V regions) of the heavy and light chains. A chimeric antibody can also include an antibody in which the heavy-chain variable region and light-chain variable region of the antibody are from a first antibody, while the heavy-chain constant region and light-chain constant region of the antibody are from a second antibody.

[0078] The term "complementary determining region" or "CDR" generally refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. CDRs can be defined according to various numbering systems known in the art, such as those defined by the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system. For a given antibody, those skilled in the art can easily identify the CDRs defined by each numbering system. Moreover, the correspondence relationships between different numbering systems are well-known to those skilled in the art.

[0079] The term "comprising" is not intended to be restrictive, but rather is intended to be inclusive and means that there may be other elements in addition to the listed elements, and can be interpreted as "including but not limited to". The term "comprising" also encompasses the terms "consisting of" and "consisting essentially of". In this text, the terms "comprising" and "including" are used interchangeably. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 It is the construction map of the pCDNA3.1-Kim-1 plasmid.

[0081] Figure 2 It is for detecting the purity of the Kim-1 target protein through SDS-PAGE experiments.

[0082] Figure 3 It is the gel map for the subtype identification of the antibody expressed by the hybridoma cell line CV18 and the cloning of the variable region of the antibody.

[0083] Figure 4 It is the plasmid map of the humanized chimeric antibody expression plasmid pGmab-K-hCV18 constructed using the antibody CV18.

[0084] Figure 5 It is the plasmid map of the humanized chimeric antibody expression plasmid pGmab-H-hCV18 constructed using the antibody CV18.

[0085] Figure 6 It is the standard curve of Kim-1 protein concentration - luminescence value. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0086] The present invention will be further described in detail below in conjunction with the specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0087] The experimental methods in the following embodiments, unless otherwise specified, are all conventional methods, carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial sources.

[0088] Example 1: Preparation of Kim-1 Recombinant Protein

[0089] 1. Construction of Kim-1 Recombinant Protein Expression Plasmid

[0090] The nucleic acid sequence of Kim-1 is based on the sequence shown in NCBI Gene ID: NM_001173393.3 (Kim-1). When synthesizing the DNA sequence, the extracellular region sequence of Kim-1 (1Met-295Gly) was ligated with a protein purification tag sequence (6×His) at the 3´ end, and EcoRI and XbaI sites were designed at the upstream and downstream of the DNA sequence respectively. The designed DNA sequence was synthesized by a third-party gene synthesis company and constructed into the pUC57 plasmid. The constructed plasmid was named pUC57-Kim-1. Using the NEBCutsmart endonuclease system, 2 μg of pUC57-Kim-1 and pCDNA3.1 plasmids were respectively added with 2 U of EcoRI and 2 U of XbaI endonucleases, digested at 37°C for 2 hours, separated by 1% agarose gel, and the positive bands were recovered. The concentration of the recovered DNA fragment was detected by A260. 2 nmol of the double-digested fragments of pUC57-Kim-1 and pCDNA3.1 plasmids were respectively taken, added with 1 U of T4 ligase, made up to 20 μL with T4 ligase buffer and ultrapure water, ligated overnight at 16°C, transformed into DH5α competent cells, and spread on LB plates. Single colonies were picked and sequenced with FCMV and SV40 universal primers. The gene sequence was correct after sequencing. The constructed plasmid was named pCDNA3.1-Kim-1, and its structure is as Figure 1 shown.

[0091] 2. Expression and Identification of Kim-1 Recombinant Protein

[0092] First, the expression plasmid pCDNA3.1-Kim-1 was transfected into 293F cells. In the transfected suspension-cultured 293F cells, the transfection reagent used was Free Style MAX (Invitrogen, 16447100), and the transfection operation steps were carried out according to the manufacturer's product manual.

[0093] Subsequently, the transfected 293F cells were transferred to a 125 mL Erlenmeyer flask, in 30 mL of FreeStyle293 medium, and the cell density was 1×10 6 cells / mL. The cells were cultured in a CO 2 incubator (37°C, 5% CO 2(It was) cultured inside (at a) shaker speed of 130 rpm. The supernatant of the culture medium was collected 7 days after cell culture, and the concentration of the recombinant Kim-1 protein in the supernatant (expressed and secreted by 293F cells) was detected by the sandwich ELISA method.

[0094] Specifically, in the sandwich ELISA, purified antibodies (abcam, # ab228973, # ab195534) were used as the coating antibody and the detection antibody in the sandwich ELISA method respectively. To determine the active concentration of Kim-1, Kim-1 control antigen (origene, # TP317289) with known concentration (detected by the A280 method) was used to make a concentration standard curve. Dilute the rabbit anti-Kim-1 antibody (abcam, # 228973) at 1 μg / mL with carbonate buffer solution at pH 9.5, coat a 96-well ELISA plate at 100 μL / well, and incubate overnight at 4°C; wash twice with PBST; add 200 µL of 1% BSA in PBS to each well, block at room temperature for 2 hours, and then pat dry on blotting paper; sample addition: Dilute the Kim-1 transfected supernatant and the control antigen with PTB (1% BSA added to 10 mM PBS buffer) at different ratios or different concentrations. Add 0.1 mL of the diluted sample to the reaction wells, incubate at 37°C for 1 hour, then wash, and at the same time make blank wells (without adding samples), negative control wells and positive control wells. Add 0.1 mL of the newly diluted antibody to each reaction well, incubate at 37°C for 1 hour, and then wash 3 times. Add enzyme-labeled secondary antibody: Add 0.1 mL of the newly diluted enzyme-labeled antibody to each reaction well. Incubate at 37°C for 1 hour and wash 3 times. Add substrate solution for color development: Add 0.1 mL of TMB substrate solution to each reaction well and let it stand at room temperature for 10 minutes. Add 1M H 2 SO 4 0.1 mL. Measure the OD value to judge the result. Use an enzyme-linked immunosorbent assay reader to measure the absorbance value (A450) at 450 nm. The results are shown in Table 1.

[0095] Finally, use ELISA CALC to calculate the 4-parameter logistic curve of the control protein as the standard curve, where R 2 = 0.990. The calculated expression concentration of the Kim-1 protein in the supernatant of the cells to be tested is about 20.8 ng / mL, and the results show that there is obvious expression of the target protein in the supernatant of 293F cells transfected with pCDNA3.1-Kim-1.

[0096]

[0097] 3. Screening and identification of the 293F stable cell line of Kim-1

[0098] To screen and obtain a cell line stably expressing Kim-1, ExpiCHO cells transfected with pCDNA3.1-Kim-1 were diluted to a density of 100 cells / well and added to the microwells of a 96-well plate, with 200 µL of CD-CHO medium / well. The medium contained 12.5 µM MSX reagent (glutamine synthetase GS inhibitor, used to screen for GS resistance gene). The microwell culture plate was cultured in a CO 2 incubator (37 °C, 5% CO 2 2) for about 20 days to allow the growth of resistant cells. The cells in the microwells with growing resistant cells were transferred to a 24-well cell culture plate for expansion culture (while quantitatively measuring the expression levels of Kim-1 protein in different cell lines). Approximately 1000 cells / well were transferred. After 7 days of culture, the cell supernatant in the plate was taken out for Kim-1 protein concentration detection.

[0099] The specific operation for Kim-1 protein concentration detection was as follows: Rabbit anti-Kim-1 antibody (abcam, #228973) diluted with carbonate buffer solution at pH 9.6 at 1 μg / mL, 100 μL / well was used to coat a 96-well ELISA plate and incubated overnight at 4 °C; washed 2 times with PBST; 200 µL of 1% BSA in PBS was added to each well, blocked at room temperature for 2 hours and then patted dry on three-fold filter paper; sample addition: The culture supernatants of different Kim-1 transfected ExpiCHO stable cell lines and control antigen (origene, #TP317289) were diluted at different ratios or different concentrations with PTB. 0.1 mL of the diluted sample was added to the reaction wells, incubated at 37 °C for 1 hour, then washed. At the same time, blank wells (without adding samples), negative control wells and positive control wells were set up. 0.1 mL of freshly diluted antibody was added to each reaction well, incubated at 37 °C for 1 hour, and then washed 3 times. Addition of enzyme-labeled secondary antibody: 0.1 mL of freshly diluted enzyme-labeled antibody was added to each reaction well. Incubated at 37 °C for 1 hour and washed 3 times. Addition of substrate solution for color development: 0.1 mL of TMB substrate solution was added to each reaction well and allowed to stand at room temperature for 10 minutes. 0.1 mL of 1 M H 2 SO 4 4 was added to each reaction well. The OD value was measured to judge the result. The absorbance value (A450) was measured at 450 nm using an enzyme-linked immunosorbent assay reader. The results are shown in Table 2.

[0100] Using ELISA CALC to calculate the 4-parameter logistic curve of the control protein as the standard curve, where R 2= 0.990. By calculating the expression concentration of Kim-1 protein in the supernatant of the cells to be tested, the results showed that there was obvious expression of the target protein, 228.1 ng / mL, in the supernatant of the 3F5 cell line of 293F transfected with pCDNA3.1-Kim-1. Combining the Kim-1 expression level and cell growth characteristics, the 3F5 cell line was finally selected for the subsequent production of Kim-1 protein.

[0101]

[0102] 4. Production and purification of recombinant Kim-1 protein

[0103] Cell culture stage: The stable cell line 3F5 of Kim-1 was cultured at a concentration of 2×10 5 cells / ml in a 1 L shake flask containing 400 mL of CD-CHO medium supplemented with 12.5 µM MSX reagent. The shake flask was placed in a CO 2 incubator (37 °C, 5% CO 2 ) on a shaker (130 rpm) for 10 days. The cell culture supernatant after 10 days of culture was taken out, centrifuged at 200 g for 5 minutes (to separate cells and coarse cell debris), and the supernatant was transferred to a new flask. Then it was centrifuged at 3000 g for 15 minutes (to separate fine cell debris). The separated supernatant was filtered through a 0.22 µM filter cup to remove bacteria and stored at 4 °C.

[0104] Protein purification stage: The cell supernatant after removing bacteria was equilibrated to room temperature and then purified using a Ni column. The Ni column packing (medium) was purchased from GE Healthcare, product number: #17-3712-02. A 30 mL chromatography column was packed, and the final volume of the packed medium was 10 mL. The sample loading volume of the cell supernatant was 400 mL, and the flow rate was 1.0 mL / min. Then it was eluted with elution buffers of different imidazole concentrations (20 mM Tris-HCl, pH 8.5, 50 - 1000 mM imidazole concentration gradient). The recombinant Kim-1 protein was eluted with the 100 mM imidazole elution buffer. Samples from each elution step of the purification experiment were taken and the purity of the target Kim-1 protein was detected by SDS-PAGE experiment. The results are shown in Figure 2 , where the samples loaded in lanes 1 - 9 were: supernatant, flow-through, wash, Marker, 10 mM imidazole elution buffer, 50 mM imidazole elution buffer, 100 mM imidazole elution buffer, 200 mM imidazole elution buffer, 500 mM imidazole elution buffer; the purified Kim-1 protein mainly existed in lane 7 (i.e., in the 100 mM imidazole elution buffer). As Figure 2The results showed that the purified recombinant Kim-1 protein was mainly present in Lane 7, with a protein molecular weight of 32.4 KDa, which was consistent with the theoretical value; the visual purity was 90%.

[0105] The eluate containing the recombinant Kim-1 protein (Lane 7) was concentrated using a 10 KDa ultrafiltration tube (Millipore, UFC900396) and dialyzed overnight using a 10 KDa dialysis bag. The dialysis buffer was 10 mM PBS buffer at pH 7.4. The protein concentration of the dialyzed recombinant protein was detected by the A280 method. The total protein concentration was 1.12 mg / mL. The purified recombinant Kim-1 protein was stored at 4°C.

[0106] Example 2: Preparation and Identification of Anti-Kim-1 Monoclonal Antibody

[0107] 1. Immunize mice with recombinant Kim-1 protein

[0108] BALB / c mice aged 6 - 8 weeks were selected for the following immunization protocol: At the primary immunization, 25 µg of the purified recombinant Kim-1 protein was mixed with an equal volume of Freund's complete adjuvant, emulsified, and injected subcutaneously at multiple points; 14 days after the first immunization, 12.5 µg of the purified recombinant Kim-1 protein was mixed with Freund's incomplete adjuvant, emulsified, and used for booster immunization. 14 days after the second immunization, 12.5 µg of the purified recombinant Kim-1 protein was mixed with Freund's incomplete adjuvant, emulsified, and used for booster immunization. 14 days after the third immunization, blood was collected and serum was separated. The ELISA plate was coated with the recombinant Kim-1 protein for indirect ELISA assay to determine the serum titer. The results showed that the titer of the prepared mouse antiserum was 1:72900. The detection results of the immunized mouse serum are shown in Table 3.

[0109]

[0110] 2. Cell fusion

[0111] After the above third immunization, the mice were given a booster immunization. The specific steps were as follows: 12.5 µg of the purified recombinant Kim-1 protein was mixed with PBS to a volume of 200 μL and injected intraperitoneally. Cell fusion was performed 3 days after the booster immunization of the mice. After collecting blood from the mouse eyeballs, the mouse was sacrificed by dislocation of the cervical vertebrae and placed in a 70% alcohol bottle for 2 minutes. Then, the mouse was fixed on a foam board in a biosafety cabinet. The abdominal skin was cut open to find the spleen, which was removed with forceps and gently ground in a 200-mesh stainless steel filter membrane. The cells were gently rinsed with DMEM medium (Thermo, 11965092), and then centrifuged at 200 g for 10 minutes in a centrifuge at room temperature. The supernatant was discarded, and the spleen cells were reserved for use.

[0112] When preparing feeder cells, the mice are sacrificed by cervical dislocation and placed in a 70% alcohol bottle for 2 minutes. Then, the mice are fixed on a foam board in a biosafety cabinet. The abdominal skin is cut open, and PBS is gently injected subcutaneously with a syringe. The liquid containing feeder cells is washed out from the other side. Then, it is centrifuged at 200 g for 10 minutes in a centrifuge at room temperature. After discarding the supernatant, the feeder cells are obtained and reserved for use. 2.0×10 7 FO myeloma cells (ATCC CRL-1646) are mixed with 2.0×10 8 spleen cells, centrifuged at 200 g for 10 minutes in a centrifuge, and the supernatant is discarded. The mixture is gently shaken and mixed evenly. In a 37°C water bath, 1 mL of an aqueous solution of PEG-1450 (Merk, P1458) with a volume concentration of 50% is added dropwise within 90 seconds, and then 20 mL of DMEM medium is added dropwise. It is centrifuged at 200 g for 10 minutes in a centrifuge, and the supernatant is discarded. The washing is repeated once, centrifuged at 200 g for 10 minutes in a centrifuge, and the supernatant is discarded to obtain hybridoma cells. The hybridoma cells are seeded into 10 96-well culture plates, 150 μL per well. 10,000 feeder cells per well are added to the above 10 96-well cell culture plates, 100 μL per well. After labeling the culture plates, they are placed in a cell culture incubator at 37°C containing 5% CO 2 . On the second day, HAT screening medium is added, and within 1 - 2 days of selection culture with HAT, a large number of tumor cells will die. After 3 - 4 days, the tumor cells disappear, and small colonies of hybrid cells are formed. After maintaining the HAT screening culture medium for 7 - 10 days, it is replaced with HT medium (Merk, H0137), and maintained for another 2 weeks. Then, it is changed to DMEM medium containing 20% FBS (ExCell, FSP500) and continued to be cultured. During the above selection culture period, when the hybridoma cells cover 1 / 10 of the bottom area of the well, the detection of specific antibodies can be started to screen out the required hybridoma cell line. During the selection culture period, half of the culture medium is changed every 2 - 3 days.

[0113] 3. Screening and subcloning culture of positive hybridoma cell lines

[0114] First, the optimal coating amount of Kim-1 recombinant protein (also simply referred to as Kim-1 protein in this article) as an antigen was determined by checkerboard titration. Kim-1 protein at 0.5, 1.0, 2.0, and 4.0 μg was coated on 96-well plates, with 6 wells for each concentration, and 3 of them were set as positive and 3 as negative. Square array titration was performed using mouse positive serum immunized with Kim-1 protein at different dilution multiples, and non-immunized mouse negative serum was used as a negative control. Coat a 96-well ELISA plate with 0.5 μg of purified Kim-1 protein per well and incubate overnight at 4°C; wash twice with PBST; add 200 μL of PBS containing 1% BSA to each well, block at room temperature for 2 hours, and then pat dry on filter paper; sample addition: add 0.1 mL of the sample to be tested to the reaction wells, incubate at 37°C for 1 hour, then wash, and at the same time, set up blank wells (without adding samples), negative control wells, and positive control wells. Add 0.1 mL of the newly diluted antibody to each reaction well, incubate at 37°C for 1 hour, and then wash 3 times. Add enzyme-labeled secondary antibody: Add 0.1 mL of the newly diluted enzyme-labeled antibody (Solarbio, #SE131) to each reaction well. Incubate at 37°C for 1 hour and wash 3 times. Add substrate solution for color development: Add 0.1 mL of TMB substrate solution to each reaction well and let it stand at room temperature for 10 minutes. Add 1 M H 2 SO 4 0.1 mL. Measure the OD value to judge the result, and use an enzyme-linked immunosorbent assay reader to measure the absorbance (A450) at 450 nm. A value more than 2.1 times the OD value of the negative control is considered positive (calculated after zeroing with the blank control well). Select monoclonal hybridoma cells against Kim-1.

[0115] According to the above method, the selected positive hybridoma cells were subcloned. The original wells were diluted with HAT selection medium by the limiting dilution method and then redistributed into 96-well culture plates, and then the cell morphology and quantity were observed. Adjust the cells to 3 - 10 cells / mL. Take the cell culture plate of feeder cells prepared the previous day, and add 100 μL of the diluted cells to each well. Incubate statically in an incubator at 37°C and 5% CO 2 . Change the medium on the 7th day, and then change the medium once every 2 - 3 days. Cell clones can be seen on the 8th - 9th day, and the antibody activity was detected in a timely manner. Transfer the cells in the positive wells to a 24-well plate for expansion culture. The binding activity of the antibody in the culture supernatant of different cell lines to Kim-1 protein was detected and compared by indirect ELISA, and the hybridoma cell line CV18 with the highest OD value (OD 450 = 1.856) was selected for later experiments. The cells should be cryopreserved as soon as possible, and finally, the hybridoma cell line with clone number CV18 was selected for antibody production. The monoclonal antibody secreted by the hybridoma cell line CV18 was named monoclonal antibody CV18 (also referred to as anti-Kim-1 antibody CV18 or antibody CV18 in this article).

[0116] 4. Large-scale Preparation of Monoclonal Antibodies and Determination of Antibody Titer

[0117] (1) Large-scale Preparation of Monoclonal Antibodies

[0118] Intraperitoneally inject 0.5 mL of Freund's incomplete adjuvant into 8-week-old BALB / C mice. Two weeks later, intraperitoneally inject 1×10 6 hybridoma cells CV18. Ascites can be produced 7 - 10 days after cell inoculation. Closely observe the health status of the animals and the signs of ascites. Before the mice are on the verge of death when the ascites is as much as possible, sacrifice the mice. Use a dropper to suck the ascites into a test tube. One mouse can yield 5 - 10 mL of ascites. Ascites can also be extracted with a syringe and collected repeatedly several times. Centrifuge the obtained ascites at 3000 g for 10 minutes, discard the upper grease and the bottom precipitate, and collect the supernatant. Aliquot the supernatant and store it at -20°C. After thawing and equilibrating the ascites supernatant to room temperature, add 1 / 10 volume of 1 M Tris–HCl pH 8.0 to adjust the sample pH to 8.0. Equilibrate a protein G affinity column with 20 column volumes of 100 mM pH 8.0 Tris–HCl. Load the ascites supernatant with adjusted pH to 8.0 onto the column, then wash it with 20 column volumes of 100 mM pH 8.0 Tris–HCl, and finally elute the antibody with 100 mM Glycine–HCL pH 2.5. Add the antibody eluate to a concentrator tube (Millipore, UFC801008, 10K) and centrifuge it at 3000×g for 20 minutes at room temperature using a centrifuge (Xiangyi, L550). Centrifuge in batches until the solution volume reaches 1 mL / concentrator tube (2 tubes). Add 4 mL of 10 mM PBS pH 7.4 buffer and continue to centrifuge at 3000×g for 20 minutes at room temperature. Repeat centrifugation 3 times to make the antibody buffer 10 mM PBS pH 7.4. Add 10 mM PBS pH 7.4 to a total volume of 10 mL. Finally, aliquot the concentrated antibody solution at 2 mL / tube into centrifuge tubes and store it at -80°C. Use a BCA kit (Solarbio, PC0020) to measure the antibody concentration. After measurement, the concentration of the purified monoclonal antibody CV18 is 0.89 mg / mL.

[0119] (2) Determination of Antibody Titer

[0120] The titer of anti-Kim-1 antibody CV18 was detected by the indirect ELISA method. The Kim-1 protein was diluted with PBS and coated on a 96-well ELISA plate at 0.2 µg / mL, 100 µL / well. After overnight incubation at 4°C, the ELISA plate was washed 2 times with PBST solution, 300 µL / well each time, and patted dry after washing. The ELISA plate was blocked with PBS solution containing 1% BSA, 200 µL / well, at room temperature for 2 hours, and patted dry after blocking; The anti-Kim-1 antibody CV18 diluted to 20 ng / mL with PTB was added and reacted in an incubator at 37°C for 1 hour. The ELISA plate was washed 2 times with PBST solution, 300 µL / well each time. After washing, it was patted dry. HRP-labeled goat anti-mouse antibody diluted 5000-fold with PTB was added and reacted in an incubator at 37°C for 1 hour. The ELISA plate was washed 2 times with PBST solution, 300 µL / well each time. After washing, it was patted dry. TMB substrate solution was added, 100 µL / well, and reacted at room temperature for 10 minutes. 100 µL / well of 1 M H 2 SO 4 The reaction was terminated. The absorbance (A450) was measured at 450 nm using an ELISA reader. The results are shown in Table 4. It can be seen that the titer of the purified anti-Kim-1 antibody is 1:729000.

[0121]

[0122] 5. Sequence and subtype identification of monoclonal antibodies

[0123] (1) Subtype identification of monoclonal antibodies

[0124] The hybridoma cell line CV18 was cultured in a 10 cm diameter cell culture dish with DMEM medium (GIBCO, #C11995500BT) supplemented with 10% serum (37°C, 5% CO 2 ). After 7 days of culture, the cells were transferred to a 15 mL centrifuge tube. After counting with a hemocytometer, 4×10 6 cells were taken out, centrifuged at 200 g for 5 minutes, the supernatant was discarded, and the centrifuge tube was inverted to drain the liquid in the tube. Total RNA was extracted from the cells in the tube and cDNA was synthesized using the reverse transcription kit (Qiagen, 74134) from QIAGEN. The antibody subtype was determined by PCR using subtype-specific primers. The synthesized cDNA above was used as the PCR reaction template. The primer sequence information for PCR is shown in Table 5.

[0125]

[0126] Note: In the table, S = C or G, M = A or C, R = A or G, W = A or T.

[0127] PCR reaction solution system: TAKARA Ex Taq (5 U / µL, TAKARA, RR001B), 0.25 µL; 10× ExTaq Buffer, 5 µL; dNTP mixture (each 2.5 mM), 4 µL; template cDNA, 1 µL; upstream primer (100 µM), 1 µL; downstream primer (100 µM), 1 µL; add double-distilled water to a total volume of 50 µL.

[0128] PCR reaction temperature program: pre-denaturation at 94°C for 5 minutes, 30 temperature cycles (94°C for 1 minute, 57°C for 1 minute, 72°C for 1 minute), extension at 72°C for 10 minutes.

[0129] After the reaction, 10 µL of each PCR product was loaded onto a 1% agarose gel for electrophoresis. The electrophoresis pattern is as Figure 3 shown. Through the analysis of the electrophoresis pattern, positive bands of 650 bp of IgG1 heavy chain and kappa light chain were amplified in Lane 2 and Lane 6 respectively. Thus, it was determined that the heavy chain of the monoclonal antibody CV18 obtained in the present invention is of IgG1 subtype and the light chain is of kappa subtype. According to the results of the PCR products, the subtype of the antibody can be inferred. The monoclonal antibody CV18 obtained in the present invention has a heavy chain of IgG1 and a light chain of kappa.

[0130] (2) Sequencing of the variable region (V region) of the antibody of hybridoma cell line CV18

[0131] The fragment obtained after PCR amplification of the V region of the antibody of cell line CV18 in the above step (1) was cut from the agarose gel and extracted using a DNA extraction kit (Qiagen, 74134). The extracted DNA fragment was ligated to the pEASY-T1 cloning vector and transformed into Trans1-T1 competent cells (Transgen, CT101-1). Transformed bacterial colonies were picked into LB medium and subjected to DNA sequencing after overnight culture. The sequencing results showed that the sequence of anti-Kim-1 antibody CV18 is as follows:

[0132] The amino acid sequence of the heavy chain of antibody CV18 is as shown in SEQ ID NO:1, and the nucleotide sequence of the heavy chain-encoding gene is as shown in SEQ ID NO:3;

[0133] The amino acid sequence of the light chain of antibody CV18 is as shown in SEQ ID NO:2, and the nucleotide sequence of the light chain-encoding gene is as shown in SEQ ID NO:4;

[0134] The amino acid sequence of the heavy chain variable region of antibody CV18 is shown as positions 1-120 of SEQ ID NO:1, and the nucleotide sequence of the gene encoding the heavy chain variable region is shown as positions 1-360 of SEQ ID NO:3;

[0135] The amino acid sequence of the light chain variable region of antibody CV18 is shown as positions 1-115 of SEQ ID NO:2, and the nucleotide sequence of the gene encoding the light chain variable region is shown as positions 1-345 of SEQ ID NO:4;

[0136] The amino acid sequence of CDR1 of the heavy chain variable region of antibody CV18 is shown as positions 31-35 of SEQ ID NO:1;

[0137] The amino acid sequence of CDR2 of the heavy chain variable region of antibody CV18 is shown as positions 50-66 of SEQ ID NO:1;

[0138] The amino acid sequence of CDR3 of the heavy chain variable region of antibody CV18 is shown as positions 99-109 of SEQ ID NO:1;

[0139] The amino acid sequence of CDR1 of the light chain variable region of antibody CV18 is shown as positions 24-39 of SEQ ID NO:2;

[0140] The amino acid sequence of CDR2 of the light chain variable region of antibody CV18 is shown as positions 55-61 of SEQ ID NO:2;

[0141] The amino acid sequence of CDR3 of the light chain variable region of antibody CV18 is shown as positions 94-105 of SEQ ID NO:2.

[0142] The above CDR sequences are obtained according to the IMGT database; the sequences of the CDRs are defined according to the Kabat numbering system.

[0143] Example 3, Humanization, Expression, and Purification of Antibody CV18

[0144] 1. Design and Sequence of Humanized Chimeric Antibody

[0145] For the humanization of murine monoclonal antibody CV18, the constant regions (C regions) in antibody CV18 were replaced with human ones, and the murine variable regions (V regions) of the heavy and light chains were retained. The designed humanized chimeric antibody was named hCV18, and its sequence is as follows:

[0146] The amino acid sequence of the heavy chain of antibody hCV18 is shown in SEQ ID NO:5, and the nucleotide sequence of the heavy chain-encoding gene is shown in SEQ ID NO:7; among them, positions 1-120 of SEQ ID NO:5 are the heavy chain variable region, and positions 121-450 of SEQ ID NO:5 are the heavy chain constant region;

[0147] The amino acid sequence of the light chain of antibody hCV18 is shown in SEQ ID NO:6, and the nucleotide sequence of the light chain-encoding gene is shown in SEQ ID NO:8; among them, positions 1-115 of SEQ ID NO:6 are the light chain variable region, and positions 116-222 of SEQ ID NO:6 are the light chain constant region.

[0148] 2. Construction of pGmab-K-hCV18 / pGmab-H-hCV18 plasmids

[0149] The genes encoding the variable region of the light chain (VL) of antibody CV18 (nucleotide sequence shown in positions 1-345 of SEQ ID NO:4) and the variable region of the heavy chain (VH) (nucleotide sequence shown in positions 1-360 of SEQ ID NO:3) were respectively constructed into the pUC57 plasmid. The constructed plasmids were named pUC57-CV18-VL and pUC57-CV18-VH. Using the NEB Cutsmart endonuclease system, 2 μg of pUC57-CV18-VL or pUC57-CV18-VH and pGmab-K (containing the constant region of the human antibody kappa chain) or pGmab-H (containing the constant region of the human antibody IgG1 chain) plasmids were respectively added with 2 U of XbaI and 2 U of BamHI endonucleases. After digestion at 37°C for 2 hours, the positive bands were separated by 1% agarose gel and recovered, and the concentration of the recovered DNA fragments was detected using A260. 2 nmol of each of the inserted fragments (CV18-VL or CV18-VH) and the double-digested fragments of the expression plasmid (pGmab-K or pGmab-H) were taken, added with 1 U of T4 ligase, and made up to 20 μL with T4 ligase buffer and ultrapure water. After overnight ligation at 16°C, they were transformed into DH5α competent cells and spread on LB plates. Single colonies were picked and the gene sequences were correctly sequenced using FCMV and SV40 universal primers. The constructed expression plasmids were named pGmab-K-hCV18 (the light chain expression plasmid of antibody hCV18) and pGmab-H-hCV18 (the heavy chain expression plasmid of antibody hCV18), and the plasmid maps are shown in Figure 4 and Figure 5 。

[0150] 3. Expression and purification of humanized chimeric antibody hCV18

[0151] 3-1. Expression and Titer Detection of Antibody hCV18: The two plasmids pGmab-K-hCV18 and pGmab-H-hCV18 constructed above were mixed at a ratio of 1:1 and transfected into ExpiCHO cells. For the transfected suspension-cultured ExpiCHO cells, the transfection reagent used was Free Style MAX (Invitrogen, 16447100), and the transfection operation steps were carried out according to the manufacturer's product manual. Subsequently, the transfected ExpiCHO cells were transferred to a 125 mL Erlenmeyer flask and cultured in 30 mL of FreeStyleCHO medium at a cell density of 1×10 6 cells / mL. The cells were cultured in a CO 2 incubator (37°C, 5% CO 2 2) with a shaker speed of 130 rpm. Finally, the culture supernatant was collected 7 days after cell culture, and the activity concentration of the humanized chimeric antibody hCV18 (expressed and secreted by ExpiCHO cells) in the supernatant was detected. The detection method was indirect ELISA, and the steps were as follows: The Kim-1 protein was diluted with PBS as an antigen and coated on a 96-well ELISA plate at a coating concentration of 1 µg / mL and a volume of 100 µL / well. After coating overnight at 4°C, the ELISA plate was washed 2 times with PBST solution, 300 µL / well each time. After washing and blotting dry, the ELISA plate was blocked with PBS solution containing 1% BSA, 200 µL / well, at room temperature for 2 hours. After blotting dry, the cell supernatant expressing hCV18 diluted with different ratios of PTB was added to the above-blocked ELISA plate, 100 µL / well, and reacted in a 37°C incubator for 1 hour. After blotting dry, the plate was washed 3 times with PBST, 300 µL / well each time. After blotting dry, HRP-labeled goat anti-human antibody diluted 5000 times with PTB was added to the ELISA plate and reacted in a 37°C incubator for 1 hour. The plate was washed 3 times with PBST, 300 µL / well. After blotting dry, TMB substrate solution was added to the ELISA plate, 100 µL / well, at room temperature for 10 minutes, and then 100 µL / well of 1 M H 2 2 4 SO4 was added to terminate the reaction. The absorbance value (A450) was measured at 450 nm using an ELISA reader. The results are shown in Table 6. The activity titer of the anti-Kim-1 humanized antibody hCV18 in the cell supernatant was detected, and the titer of the antibody hCV18 in the cell supernatant was found to be 1:72900.

[0152]

[0153] 3-2. Purification of antibody hCV18 and detection of the titer of the purified antibody: The cell supernatant was centrifuged at 3000×g for 10 minutes, and 1 / 10 volume of 1 M Tris–HCl pH8.0 was added to adjust the pH of the sample to 8.0. The protein G affinity column was equilibrated with 20 column volumes of 100 mM Tris–HCl pH8.0. The cell supernatant with adjusted pH to 8.0 was loaded onto the column, then washed with 20 column volumes of 100 mM Tris–HCl pH 8.0, and finally the antibody was eluted with 100 mM Glycine–HCL pH2.5. The antibody eluate was added to a concentrator tube (Millipore, UFC801008, 10K), and centrifuged at 3000×g for 20 minutes at room temperature using a centrifuge (Xiangyi, L550). Centrifugation was carried out in batches until the solution volume reached 1 mL / concentrator tube (2 tubes). 4 mL of 10 mM PBS pH 7.4 buffer was added, and centrifugation was continued at 3000×g for 20 minutes at room temperature. Centrifugation was repeated 3 times to make the antibody buffer 10 mM PBS pH7.4, and 10 mM PBS pH 7.4 was added to a total volume of 10 mL. Finally, the concentrated antibody solution was aliquoted at 2 mL / tube into centrifuge tubes and stored at -80℃. The antibody concentration was determined using a BCA kit (Solarbio, PC0020). The concentration of the purified human chimeric antibody hCV18 was determined to be 4.1 mg / mL. The activity titer of the purified anti-Kim-1 humanized antibody hCV18 was detected by indirect ELISA. The steps were as follows: Kim-1 protein was diluted with PBS as an antigen and coated on a 96-well ELISA plate at a coating concentration of 1 µg / mL and a volume of 100µL / well. After coating overnight at 4℃, the ELISA plate was washed 2 times with PBST solution, 300 µL / well each time. After washing, it was patted dry. The ELISA plate was blocked with PBS solution containing 1% BSA, 200 µL / well, for 2 hours at room temperature. After patting dry, different concentrations of antibody hCV18 diluted with PTB were added to the above-blocked ELISA plate, 100 µL / well, and reacted in a 37℃ incubator for 1 hour. After patting dry, the plate was washed 3 times with PBST, 300 µL / well each time. After patting dry, HRP-labeled goat anti-human antibody diluted 5000 times with PTB was added to the ELISA plate and reacted in a 37℃ incubator for 1 hour. The plate was washed 3 times with PBST, 300 µL / well. After patting dry, TMB substrate solution was added to the ELISA plate, 100 µL / well, for 10 minutes at room temperature, and then 100 µL / well of 1 M H 2 SO 4 was added to terminate the reaction. The absorbance value (A450) was measured at 450 nm using an ELISA reader. The results are shown in Table 7. The titer of hCV18 in the cell supernatant was detected to be 1:7290000.

[0154]

[0155] Example 4. Identification of the Sensitivity and Specificity of Antibody hCV18 and CV18 for Detecting Kim-1

[0156] (1)Identification of the Sensitivity of Antibody hCV18 and CV18 for Detecting Kim-1

[0157] The sensitivity of antibody hCV18 and antibody CV18 for detecting Kim-1 was evaluated by indirect ELISA. And the difference in sensitivity with the control antibody (rabbit anti-Kim-1 antibody abcam, #228973) was compared. The specific steps are as follows: The Kim-1 protein was diluted with PBS as an antigen and coated on a 96-well ELISA plate at a coating concentration of 1 μg / mL and a volume of 100 μL / well. After overnight coating at 4°C, the ELISA plate was washed 2 times with PBST solution, 300 μL / well each time. After washing and blotting dry, the ELISA plate was blocked with PBS solution containing 1% BSA, 200 μL / well, for 2 hours at room temperature. After blotting dry, different concentrations of antibody CV18, hCV18, and the control antibody diluted with PTB were added to the above-blocked ELISA plate, 100 μL / well, and reacted in an incubator at 37°C for 1 hour. After blotting dry, the plate was washed 3 times with PBST, 300 μL / well each time. After blotting dry, HRP-labeled goat anti-human antibody diluted 5000 times with PTB was added to the ELISA plate and reacted in an incubator at 37°C for 1 hour. The plate was washed 3 times with PBST, 300 μL / well. After blotting dry, TMB substrate solution was added to the ELISA plate, 100 μL / well, for 10 minutes at room temperature, and then 100 μL / well of 1 M H 2 SO 4 4 was added to terminate the reaction. The absorbance value (A450) was measured at 450 nm using an ELISA reader.

[0158] The experimental results are shown in Table 8. The sensitivity of antibody CV18 and antibody hCV18 for detecting Kim-1 protein is better than that of the control antibody, and the background value of hCV18 is lower (0.052) compared with the Kim-1 protein. Through result analysis, the standard curve R 2 2 of the Kim-1 protein = 0.981, and the IC50 = 25 ng / mL. The range of the standard curve of this method is 0 - 1000 ng / mL. The lowest detection limit of the control antibody is 100 ng / mL; the CV18 antibody is 25 ng / mL; the hCV18 antibody is 12.5 ng / mL. The results are shown in Table 8.

[0159]

[0160] (2)Identification of the Specificity of Antibody hCV18 and CV18 for Detecting Kim-1

[0161] Preparation of Kim-1 overexpressing 293T cells: The coding gene of Kim-1 protein (nucleotide sequence as shown in GenBank accession number: AF043724.1) was cloned into the vector pCDNA3.1 (purchased from Thermo, catalog number V79520) to obtain a recombinant vector. The recombinant vector was transfected into 293T cells (ATCC CRL-11268) to obtain Kim-1 overexpressing 293T cells.

[0162] The ability of antibodies hCV18 and CV18 to detect the specificity of Kim-1 was evaluated by indirect ELISA. The specific steps were as follows: Cells (293T, used as a negative control in this experiment) were cultured. After culturing for 3 generations in DMEM medium containing 10% serum, the cells were lysed using a cell lysate (Beyotime, #P0013); Kim-1 overexpressing 293T cells (used as positive cells in this experiment) were lysed using a lysate (abcam, #ab94224). The lysates obtained by lysing the above two kinds of cell lysates were diluted to 1 μg / mL with a carbonate buffer solution at pH 9.5 and transferred to a 96-well cell culture plate, with a volume of 100 μL / well, and coated overnight. The plate was washed 2 times with PBST solution, 300 μL / well each time. After washing, it was patted dry. The 96-well plate was blocked with PBS solution containing 1% BSA, 200 μL / well, and patted dry after 2 hours at room temperature. Different concentrations of antibodies hCV18 and CV18 diluted with PTB, and a control antibody (rabbit anti-Kim-1 antibody abcam, #228973) were added at 100 μL / well, and reacted in an incubator at 37 °C for 1 hour. Patted dry, the plate was washed 3 times with PBST, 300 μL / well each time. Patted dry, HRP-labeled secondary antibody diluted 5000 times with PTB was added to the enzyme-linked immunosorbent assay (ELISA) plate and reacted in an incubator at 37 °C for 1 hour. The plate was washed 3 times with PBST, 300 μL / well. Patted dry, TMB substrate solution was added to the ELISA plate, 100 μL / well, for 10 minutes at room temperature, and then 100 μL / well of 1M H 2 SO 4 4 was added to terminate the reaction. The absorbance value (A450) was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The experimental results are shown in Table 9. Compared with the control antibody, both antibody CV18 and antibody hCV18 have good specificity, and antibody hCV18 has better specificity than antibody CV18 in detecting Kim-1 protein.

[0163]

[0164] Example 5. Application of antibodies hCV18 and CV18 in detecting Kim-1 in human urine by magnetic particle chemiluminescence detection method

[0165] (1) Alkaline Phosphatase (ALP) Labeling of Antibodies hCV18 and CV18

[0166] Respectively take antibodies hCV18 and CV18 (20 nmol, 3 mg) and make up the volume to 0.5 mL, then add them into a dialysis bag (10 KDa, width 1 cm), and dialyze overnight at 4°C in 2 L of 10 mM PBS solution (pH 7.4). The next day, put the dialysis bag containing the antibody solution into 1 L of 10 mM carbonate buffer (pH 9.5) and stir and dialyze at room temperature for 2 hours to prepare for coupling with the activated ALP.

[0167] Meanwhile, accurately weigh 0.1 mg of ALP using an analytical balance, add it to the above-mentioned dialyzed antibody solution, add ALP while stirring to avoid powder adhering to the wall, and place it in a 4°C refrigerator or ice storage and continue to stir for 12 - 18 hours. After the binding is completed, first centrifuge the conjugate at 3000×g for 20 minutes, remove a small amount of precipitate, then load it into a dialysis bag and place it in a beaker of 10 mM PBS solution (pH 7.4) and dialyze overnight. Take the dialyzed label, pass it through a Sephadex G-25 column to separate the free ALP, and collect the labeled antibody and store it at 4°C.

[0168] (2) Composition of the Magnetic Particle Chemiluminescence Detection Reagent Constructed by Using ALP-Labeled Antibodies hCV18 and CV18

[0169] In this experiment, the magnetic particle chemiluminescence detection reagent includes R1 reagent, magnetic separation reagent, calibration solution series, and chemiluminescence substrate solution.

[0170] The composition of the magnetic particle chemiluminescence detection reagent constructed by using ALP-labeled antibody CV18 is as follows:

[0171] Among them, the R1 reagent includes:

[0172] 1) R1 antibody: Monoclonal antibody CV18 against Kim-1 labeled with alkaline phosphatase (ALP), with a concentration of 0.6 μg / ml;

[0173] 2) Buffer: It includes Tris with a concentration of 12.0 g / L; sodium azide with a concentration of 1.98 g / L; sodium chloride with a concentration of 5.9 g / L; magnesium chloride solution with a molar concentration of 1 M, 1.0 mL / L; zinc chloride solution with a molar concentration of 0.1 M, 1.0 mL; fish skin gelatin with a concentration of 10 g / L; bovine serum albumin, 5 g / L; newborn bovine serum, 30 g / L; the rest is deionized water. The pH value of the buffer of the R1 reagent is 8.0.

[0174] The R1 reagent is prepared with the buffer (pH value 8.0) described in 2) above as the solvent and the R1 antibody as the solute.

[0175] Among them, the magnetic separation reagent includes:

[0176] 1) Magnetic particles: Magnetic particles coated with anti-Kim-1 monoclonal antibody (abcam, #228973), with a concentration of 1 mg / ml;

[0177] 2) Buffer: including Tris with a concentration of 11.08 g / L; sodium azide with a concentration of 1.917 g / L; sodium chloride with a concentration of 5.56 g / L; magnesium chloride solution with a molar concentration of 1 M, 1.0 mL / L; zinc chloride solution with a molar concentration of 0.1 M, 1.0 mL / L; bovine serum albumin with a concentration of 4.81 g / L; special-grade horse serum with a concentration of 4.91 g / L, and the remaining components are deionized water. The pH value of the buffer of the magnetic separation reagent is 8.0.

[0178] The magnetic separation reagent is prepared with the buffer (pH value 8.0) described in 2) above as the solvent and magnetic particles as the solute.

[0179] Among them, the calibration solution series includes:

[0180] 1) Kim-1 recombinant protein antigen;

[0181] 2) Buffer: including Tris with a concentration of 9.1 g / L; sodium chloride with a concentration of 12.9 g / L; tetracycline hydrochloride with a concentration of 0.01 g / L; neomycin sulfate with a concentration of 0.01 g / L; sodium azide with a concentration of 2.0 g / L; casein 10.0 g / L; Tween 20 with a concentration of 3.1 g / L. The pH value of the buffer of the calibration solution series is 7.6. The calibration solution series includes calibration products containing Kim-1 recombinant protein antigen at different concentrations (0 pg / mL, 10 pg / mL, 50 pg / mL, 100 pg / mL, 500 pg / mL, 1000 pg / mL).

[0182] Among them, the chemiluminescent substrate solution is a chemiluminescent substrate solution containing 0.3 mg / mL of dioxane compound (APCL) diluted with a Tris-HCl buffer with a pH value of 9.3 and a molar concentration of 0.2 M.

[0183] The composition of the magnetic particle chemiluminescent detection reagent constructed by using ALP-labeled antibody hCV18 is as follows: Except that the antibody CV18 in the R1 reagent is replaced with antibody hCV18, other components and preparation methods remain unchanged.

[0184] (3) Application of the magnetic particle chemiluminescent detection reagent constructed by antibodies hCV18 and CV18 in detecting Kim-1 in human urine

[0185] The content of Kim-1 protein in human urine was detected by a chemiluminescence immunoassay kit based on magnetic microparticles conjugated with antibody CV18. The steps are as follows (where the R1 reagent is based on antibody CV18):

[0186] 1) 50 μL of a series of calibration solutions (with Kim-1 recombinant protein antigen concentrations of 0 pg / mL, 10 pg / mL, 50 pg / mL, 100 pg / mL, 500 pg / mL, and 1000 pg / mL respectively) were added to reaction tubes, followed by 50 μL of R1 reagent and 50 μL of magnetic separation reagent. The mixture was incubated at 37°C for 15 minutes.

[0187] 2) The reagents obtained in step 1) were each combined with 25 μL of magnetic separation reagent and incubated at 37°C for an additional 5 minutes.

[0188] 3) The mixture was washed three times with a washing solution (0.1 M TrisHCl buffer with 0.02% Tween 20 and 15 w / w% sodium chloride, pH 8) to remove unbound antibodies and impurities.

[0189] 4) 150 μL of chemiluminescent substrate solution was added. After ALP catalyzed the substrate to emit light, the relative luminescence intensity (RLU) was measured using a chemiluminescence detector. The results are shown in Table 10 below:

[0190]

[0191] 5) Curve fitting was performed based on the values in Table 10 to obtain Figure 6 the standard curve of Kim-1 protein concentration vs. luminescence value as shown

[0192] 6) 50 μL of the test sample (urine samples of different disease types, with negative samples selected from physical examination samples and positive samples being acute kidney injury and diabetic nephropathy samples) and 50 μL of R1 reagent were added to reaction tubes in sequence. The mixture was incubated at 37°C for 15 minutes.

[0193] 7) The reagent obtained in step 6) was combined with 25 μL of magnetic separation reagent and incubated at 37°C for an additional 5 minutes.

[0194] 8) The mixture was washed three times with a washing solution (0.1 M TrisHCl buffer with 0.02% Tween 20 and 15 w / w% sodium chloride, pH 8) to remove unbound antibodies and impurities.

[0195] 9) 150 μL of chemiluminescent substrate solution was added. After ALP catalyzed the substrate to emit light, the relative luminescence intensity RLU was measured using a chemiluminescence detector.

[0196] 10) Calculate the Kim-1 protein concentration value corresponding to the sample RLU according to the Kim-1 recombinant protein concentration-luminescence value standard curve obtained in step 5).

[0197] Steps for detecting the content of Kim-1 protein in human urine using a chemiluminescence immunoassay reagent based on magnetic microparticles labeled with ALP-conjugated antibody hCV18: Except for using the R1 reagent based on antibody hCV18, the steps are the same as the above steps 1)-10).

[0198] Construct chemiluminescence immunoassay reagents based on antibodies hCV18 and CV18 respectively, and use them to detect the content of Kim-1 protein in human urine, and compare the differences between the two. The results are shown in Table 11 (the data in the table are the content of Kim-1 protein, unit: pg / ml).

[0199]

[0200] As shown in Table 11, both antibodies hCV18 and CV18 can significantly detect Kim-1 protein in human urine by chemiluminescence immunoassay, and the specificity of hCV18 antibody is significantly higher than that of CV18 antibody. It is thus judged that in the chemiluminescence immunoassay using hCV18 antibody magnetic microparticles, there are good advantages in specifically identifying Kim-1 protein in human urine.

[0201] The present invention provides monoclonal antibodies CV18 and / or hCV18 against Kim-1, which have the characteristics of high affinity and specificity. At the same time, a method for detecting Kim-1 in human urine by chemiluminescence immunoassay based on the above hCV18 antibody is also provided. The present invention solves the problem of relatively short supply of antibody raw materials for the Kim-1 detection item in the current market.

[0202] The above has detailed the present invention. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. An anti-Kim-1 antibody or an antigen-binding fragment thereof, characterized in that The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a CDR1 as shown in positions 31-35 of SEQ ID NO:1, a CDR2 as shown in positions 50-66 of SEQ ID NO:1, and a CDR3 as shown in positions 99-109 of SEQ ID NO:1; the light chain variable region comprises a CDR1 as shown in positions 24-39 of SEQ ID NO:2, a CDR2 as shown in positions 55-61 of SEQ ID NO:2, and a CDR3 as shown in positions 94-105 of SEQ ID NO:

2.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown at positions 1-120 of SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown at positions 1-115 of SEQ ID NO:

2.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region as shown in A1) or A2) below: A1) the amino acid sequence of the heavy chain constant region is as shown in positions 121-444 of SEQ ID NO: 1, and the amino acid sequence of the light chain constant region is as shown in positions 116-222 of SEQ ID NO: 2; A2) The amino acid sequence of the heavy chain constant region is shown at positions 121-450 of SEQ ID NO:5, and the amino acid sequence of the light chain constant region is shown at positions 116-222 of SEQ ID NO:

6.

4. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody or antigen-binding fragment thereof comprises the heavy chain and light chain shown in B1) or B2) below: B1) the amino acid sequence of the heavy chain is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain is shown in SEQ ID NO: 2; B2) The amino acid sequence of the heavy chain is shown in SEQ ID NO:5, and the amino acid sequence of the light chain is shown in SEQ ID NO:

6.

5. Biomaterial, characterized in that The biological material is any of the following: C1) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4; C2) an expression cassette containing the nucleic acid molecule described in C1); C3) a recombinant vector containing the nucleic acid molecule described in C1); C4) A recombinant host cell containing the nucleic acid molecule described in C1).

6. The biomaterial according to claim 5, characterized in that The nucleic acid molecule is any of the following: D1) comprising a DNA molecule encoding a heavy chain variable region and a DNA molecule encoding a light chain variable region, wherein the DNA molecule encoding the heavy chain variable region is as shown in positions 1-360 of SEQ ID NO:3, and the DNA molecule encoding the light chain variable region is as shown in positions 1-345 of SEQ ID NO:4; D2) comprising a DNA molecule encoding a heavy chain and a DNA molecule encoding a light chain, wherein the DNA molecule encoding the heavy chain is as shown in SEQ ID NO:3, and the DNA molecule encoding the light chain is as shown in SEQ ID NO:4; D3) comprises a DNA molecule encoding a heavy chain and a DNA molecule encoding a light chain, wherein the DNA molecule encoding the heavy chain is as shown in SEQ ID NO:7, and the DNA molecule encoding the light chain is as shown in SEQ ID NO:

8.

7. An antibody conjugate comprising an antibody portion and a conjugate portion, characterized in that: The antibody portion comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, and the coupling portion is alkaline phosphatase.

8. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the biomaterial according to claim 5 or 6, or the antibody conjugate according to claim 7 in any of the following: E1) Use in preparing a reagent or a kit for detecting Kim-1 protein; E2) Use in the preparation of reagents or kits for diagnosing or screening Kim-1 target related diseases.

9. A reagent or a kit, characterized in that The reagent or kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the antibody conjugate according to claim 7.

10. The method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that: The preparation method comprises expressing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 in a host cell, and recovering or isolating the antibody or antigen-binding fragment thereof.

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