Monoclonal antibody against NMP22 as well as preparation method and application of monoclonal antibody

Abstract

The invention belongs to the field of antibodies, and particularly relates to an anti-NMP22 monoclonal antibody as well as a preparation method and application thereof. The invention provides CDR sequences of a heavy chain variable region and a light chain variable region of an anti-NMP22 antibody, the antibody can specifically recognize nuclear matrix protein 22 (NMP22), has high affinity and high specificity to the protein, can be applied to diagnosis of diseases related to abnormal urine nuclear matrix protein 22 level, and especially can be applied to clinical diagnosis of bladder cancer.

Description

[0001] Cross-reference information

[0002] This application claims priority to a Chinese patent application with application number 202311871056X, filed on December 29, 2023, the entire content of which is incorporated herein by reference. Technical field

[0003] The present invention belongs to the field of antibodies, and particularly relates to monoclonal antibodies against NMP22, and methods for their preparation and use. Background art

[0004] Nuclear Matrix Protein 22 (NMP22) is an important member of the nuclear matrix protein family. It is a subunit of the nuclear mitotic apparatus protein (NuMA), which is related to the formation of the spindle during mitosis. Its main function is to coordinate the correct and uniform distribution of chromosomes into daughter cells during nuclear mitosis. NMP-22 is mostly distributed in tissues with relatively active mitosis, such as epithelial cells, especially urothelial cells. NMP22 plays an important role in maintaining nuclear function, DNA replication, RNA processing, and gene regulation (Miyake M, Goodison S, Giacoia EG, Rizwani W, Ross S, Rosser CJ. Influencing factors on the NMP-22 urine assay: an experimental model[J]. BMC Urol. 2012 Aug 28;12:23).

[0005] When bladder epithelial cells become cancerous, the distribution of nuclear genetic material is extremely abnormal during the telophase of cell division. When bladder cancer occurs, the synthesis of NMP22 in cancer cells surges and is released into the urine. The concentration of NMP22 in the urine of bladder cancer patients is 25 times that of normal people (Jamshidian H, Kor K, Djalali M. Urine concentration of nuclear matrix protein 22 for diagnosis of transitional cell carcinoma of bladder [J]. Urol J. 2008 Fall; 5(4): 243-7). Therefore, NMP22 can be used for the early diagnosis and monitoring of bladder cancer (Chen YT, CL Hayden, KJ Marchand and RW Makuch. 1997. Comparison of Urine Collection Methods for Evaluating Urinary Nuclear Matrix Protein, NMP22, as a Tumor Marker. J. Urol, 158: 1899).

[0006] In addition to bladder cancer, some literature reports have also shown that changes in the level of NMP22 are related to the occurrence of other urinary system diseases such as prostate cancer, urothelial intraepithelial neoplasia (such as urethral intraepithelial neoplasia or urothelial carcinoma in situ), and urinary system inflammation (such as cystitis, glomerulonephritis, and pyelonephritis). However, its current main clinical application is still related to the diagnosis and monitoring of bladder cancer.

[0007] Currently, the common methods for the clinical diagnosis of bladder cancer include cystoscopic tissue biopsy, urine exfoliated cell examination, imaging examination, etc. Among them, cystoscopic tissue biopsy is the gold standard for the diagnosis of bladder cancer, but its disadvantage is that it causes relatively large trauma. Urine exfoliated cytology is the most commonly used examination method in the clinic for bladder cancer, but its disadvantage is low sensitivity. Using NMP22 as a bladder tumor marker will facilitate the diagnosis of bladder cancer patients.

[0008] Currently, the main detection methods for NMP22 are ELISA and colloidal gold methods. The human NMP-22 immunoassay reagent of Abbott Laboratories in the United States occupies a large proportion in the market of major hospitals and clinical testing institutions. However, the market feedback shows that its sensitivity is low. Therefore, there is still an urgent clinical need for a human NMP-22 assay reagent with high specificity and sensitivity. Summary of the Invention

[0009] The first object of the present invention is to provide an antibody against NMP22 or its antigen-binding part, which comprises a heavy chain variable region and a light chain variable region; wherein,

[0010] i) A heavy chain variable region, which comprises VH CDR1, VH CDR2 and VH CDR3, wherein VH CDR1, VH CDR2 and VH CDR3 respectively comprise the amino acid sequences of TYTMH (SEQ ID NO:4), YINPNSGYTNYNQKFSD (SEQ ID NO:5), and DYTYDEFPY (SEQ ID NO:6), or amino acid sequences having 1-3 amino acid substitutions in each CDR as compared with the above amino acid sequences; and / or

[0011] ii) A light chain variable region, which comprises VL CDR1, VL CDR2 and VL CDR3, wherein VL CDR1, VL CDR2 and VL CDR3 respectively comprise the amino acid sequences of KSSQSLLNSRTRKNYLA (SEQ ID NO:9), WASTRES (SEQ ID NO:10) and KQSYNLYT (SEQ ID NO:11), or amino acid sequences having 1-3 amino acid substitutions in each CDR as compared with the above amino acid sequences.

[0012] As a preferred embodiment, the anti-NMP22 antibody or its antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 respectively comprise the amino acid sequences of TYTMH (SEQ ID NO:4), YINPNSGYTNYNQKFSD (SEQ ID NO:5), DYTYDEFPY (SEQ ID NO:6), KSSQSLLNSRTRKNYLA (SEQ ID NO:9), WASTRES (SEQ ID NO:10) and KQSYNLYT (SEQ ID NO:11).

[0013] As a preferred embodiment, the heavy chain variable region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:3.

[0014] As a preferred embodiment, the light chain variable region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:8.

[0015] As a preferred embodiment, the heavy chain variable region and the light chain variable region respectively comprise amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:3 and SEQ ID NO:8.

[0016] As a preferred embodiment, the antibody is a murine monoclonal antibody, a humanized monoclonal antibody, a chimeric monoclonal antibody or a fully human monoclonal antibody.

[0017] As a preferred embodiment, the antigen-binding portion is Fab, Fab', F(ab')2, Fd, Fv, scFv, or SdAb.

[0018] As a preferred embodiment, the antibody further comprises a heavy chain constant region and a light chain constant region; the heavy chain constant region is any one of IgG type, IgA type, IgM type, IgE type, IgD type; the light chain constant region is a κ chain or a λ chain.

[0019] As a further preferred embodiment, the heavy chain constant region sequence is of IgG type and the light chain constant region is a κ chain.

[0020] As a preferred embodiment, the antibody or its antigen-binding portion comprises a heavy chain and a light chain; the heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:2;

[0021] the light chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:7.

[0022] The second object of the present invention is to provide a nucleic acid molecule encoding the anti-NMP22 antibody or its antigen-binding portion described above.

[0023] The third object of the present invention is to provide an expression vector comprising the nucleic acid molecule described in the present invention.

[0024] The fourth object of the present invention is to provide a host cell comprising the nucleic acid molecule described in the present invention or having the nucleic acid molecule integrated into its genome.

[0025] The fifth object of the present invention is to provide a complex comprising: i) the anti-NMP22 antibody or its antigen-binding portion described above; and ii) a detectable label.

[0026] The sixth object of the present invention is to provide a conjugate, which comprises: I) the anti-NMP22 antibody or its antigen-binding part, or the complex; and, II) a solid support.

[0027] The seventh object of the present invention is to provide the use of the anti-NMP22 antibody or its antigen-binding part in any of the following aspects: a1) detecting NMP22; a2) preparing a product for detecting NMP22.

[0028] The eighth object of the present invention is to provide the use of the anti-NMP22 antibody or its antigen-binding part in any of the following aspects: b1) diagnosing or assisting in diagnosing a disease associated with abnormal NMP22 levels; b2) preparing a product for diagnosing or assisting in diagnosing a disease associated with abnormal NMP22 levels.

[0029] As a preferred embodiment, the disease includes urinary system diseases associated with abnormal NMP22 levels.

[0030] As a more preferred embodiment, the disease includes bladder cancer.

[0031] The ninth object of the present invention is to provide a composition, which comprises the anti-NMP22 antibody or its antigen-binding part, or comprises the nucleic acid molecule, or comprises the expression vector, or comprises the host cell, or comprises the complex, or comprises the conjugate.

[0032] The tenth object of the present invention is to provide a method for preparing an anti-NMP22 antibody or its antigen-binding part, which comprises: expressing the nucleic acid molecule using a host cell, and then recovering the produced antibody or its antigen-binding part from the culture medium and / or the cultured host cell.

[0033] The antibody provided by the present invention can specifically recognize nuclear matrix protein 22 (NMP22), and has high affinity and high specificity for this protein, and can be applied to the diagnosis of diseases associated with abnormal urinary nuclear matrix protein 22 levels, especially can be applied to the clinical diagnosis of bladder cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0035] Figure 1 It is the SDS-PAGE identification diagram of the purity of recombinant human NMP22 protein in the embodiment of the present invention.

[0036] Figure 2 This is the detection result of the ELISA titer of the mouse antiserum after immunization with recombinant human NMP22 protein in the embodiment of the present invention.

[0037] Figure 3 This is the SDS-PAGE identification diagram of the purity of the anti-NMP22 protein monoclonal antibody (25B1C7) in the embodiment of the present invention. The left band is the protein molecular weight Marker, and the right band is the anti-NMP22 protein monoclonal antibody (25B1C7) under reducing conditions.

[0038] Figure 4 This is the curve graph of the specific binding of the anti-NMP22 monoclonal antibody (25B1C7) to the recombinant human NMP22 protein in the embodiment of the present invention.

[0039] Figure 5 This is the affinity determination of the anti-NMP22 monoclonal antibody (25B1C7) by Gator analysis in the embodiment of the present invention. Detailed implementation manners

[0040] The following details the specific implementation manners of the present invention. It should be understood that the specific implementation manners described herein are only for the purpose of illustrating and explaining the present invention, and are not used to limit the present invention. Those skilled in the art can make various modifications and changes to the present invention without departing from the scope or spirit of the present invention. For example, features described or illustrated as part of one implementation manner can be used in another implementation manner to produce a further implementation manner.

[0041] Unless otherwise specified, the meanings of all terms (including technical and scientific terms) used to disclose the present invention are the same as those commonly understood by those of ordinary skill in the art to which the present invention pertains. Through further guidance, the following definitions are used to better understand the teachings of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0042] The terms "and / or", "or / and", and "and / or" used in the present invention cover any one of two or more related listed items, as well as any and all combinations of the related listed items. The said any and all combinations include combinations of any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctive combinations selected from "and / or", "or / and", and "and / or", it should be understood that in this application, this technical solution undoubtedly includes the technical solution connected by "logical AND", and also undoubtedly includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another example, the technical solution of "A, B, C, and / or D" includes any one of A, B, C, and D (that is, the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, it includes combinations of any two or any three of A, B, C, and D, and also includes the combination of the four items A, B, C, and D (that is, the technical solution connected by "logical AND").

[0043] The terms "comprising", "containing", and "including" used in the present invention are synonyms, which are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0044] The numerical ranges expressed by endpoints in the present invention include all the numerical values and fractions included within the range, as well as the recited endpoints.

[0045] Regarding the concentration values involved in the present invention, their meanings include fluctuations within a certain range. For example, they can fluctuate within the corresponding precision range. For example, for 2%, fluctuations within the range of ±0.1% are allowed. For larger numerical values or those that do not require overly precise control, larger fluctuations are also allowed in their meanings. For example, for 100 mM, fluctuations within the ranges of ±1%, ±2%, ±5%, etc. are allowed. Regarding molecular weights, fluctuations within the range of ±10% are allowed in their meanings.

[0046] In the present invention, descriptions such as "a plurality of" and "a variety of", without special limitations, refer to a quantity greater than or equal to 2.

[0047] In the present invention, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the listed features, as well as an open technical solution containing the listed features.

[0048] In the present invention, "preferred", "better", "more preferred", and "preferably" are only used to describe the embodiments or examples with better effects, and it should be understood that they do not constitute a limitation on the protection scope of the present invention.

[0049] In the present invention, the terms "optionally", "optional", "option", "alternatively", "alternative", "alternate" mean that something is either present or absent, i.e., it refers to any one of two alternative scenarios of "present" or "absent". If the term "optionally" or "alternatively" appears multiple times in a technical solution, and there is no special indication, no contradiction or mutual restriction relationship, then each "optionally" or "alternatively" is independent of each other.

[0050] In the present invention, the term "antibody" refers to an immunoglobulin molecule, which is typically a tetramer composed of two identical heavy chains and two identical light chains interconnected by disulfide bonds. According to the differences in amino acid sequence conservation, 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 antibody binding to antigens, and are thus also called complementarity-determining regions (CDRs). In the present invention, the three heavy-chain complementarity-determining regions are respectively called VH CDR1, VH CDR2, and VH CDR3, and the three light-chain complementarity-determining regions are respectively called VL CDR1, VL CDR2, and VL CDR3. The variable regions of one heavy chain and one light chain interact to form an antigen-binding site (Fv). According to the amino acid sequence of their heavy-chain constant regions, antibodies can be classified 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.

[0051] In the present invention, the "antigen-binding portion" means an antigen-binding fragment of an antibody and an antibody analogue, covering digestion fragments or functional variants of the antibody, which generally include at least part of the antigen-binding region or variable region (such as one or more CDRs) of the parental antibody. For example, an antibody fragment capable of binding NMP22 or a part thereof, including but not limited to Fab, Fab', F(ab')2, Fd, Fv, scFv, or SdAb.

[0052] The term "monoclonal antibody" used in the present invention refers to an antibody that is homogeneous and specific for a particular antigen epitope. Compared with a typical polyclonal antibody preparation that typically includes different antibodies against different antigen determinants (epitopes), each monoclonal antibody targets a single antigen determinant on the antigen. The modifier "monoclonal" indicates the homogeneous nature of the antibody and is not construed to require an antibody produced by any specific method. The monoclonal antibodies of the present invention are preferably produced by recombinant DNA methods or obtained by screening methods described elsewhere in the present invention.

[0053] In the present invention, the "percentage (%) identity" of a peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a specific peptide or polypeptide sequence, after aligning the sequences being compared and introducing gaps if necessary to obtain the maximum percentage sequence identity, and without considering any conservative substitutions as part of the sequence identity. Sequence alignments can be performed in a variety of ways within the skill in the art to determine the percentage amino acid sequence identity, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for the alignment, including any algorithms required to obtain the maximum alignment over the full length of the sequences being compared.

[0054] antibody or its antigen-binding portion

[0055] The present invention relates to an antibody against NMP22 or an antigen-binding portion thereof, which comprises a heavy chain variable region and a light chain variable region; wherein,

[0056] The heavy chain variable region comprises VH CDR1, VH CDR2 and VH CDR3;

[0057] i) A heavy chain variable region, which comprises VH CDR1, VH CDR2 and VH CDR3, wherein VH CDR1, VH CDR2 and VH CDR3 comprise the amino acid sequences of TYTMH (SEQ ID NO:4), YINPNSGYTNYNQKFSD (SEQ ID NO:5), and DYTYDEFPY (SEQ ID NO:6), respectively, or comprise amino acid sequences having 1-3 amino acid substitutions in each CDR as compared with the above amino acid sequences; and / or

[0058] ii) A light chain variable region, which comprises VL CDR1, VL CDR2 and VL CDR3, wherein VL CDR1, VL CDR2 and VL CDR3 comprise the amino acid sequences of KSSQSLLNSRTRKNYLA (SEQ ID NO:9), WASTRES (SEQ ID NO:10) and KQSYNLYT (SEQ ID NO:11), respectively, or comprise amino acid sequences having 1-3 amino acid substitutions in each CDR as compared with the above amino acid sequences.

[0059] In the present invention, the substitutions include deletions, substitutions and / or insertions.

[0060] In some embodiments, the variant has one, two or three amino acid substitutions as compared with the parent.

[0061] In some embodiments, the variant has the same or similar performance as the parent in terms of specifically binding to a protein or polypeptide that binds NMP22, and the performance includes specificity and affinity.

[0062] In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO: 3;

[0063] the light chain variable region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO: 8.

[0064] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 3; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8.

[0065] In some embodiments, the heavy chain variable region comprises the amino acid sequence of a protein or polypeptide with the same function obtained by deleting, substituting and / or inserting one or more amino acids into the amino acid sequence shown in SEQ ID NO: 3; the light chain variable region comprises the amino acid sequence of a protein or polypeptide with the same function obtained by deleting, substituting and / or inserting one or more amino acids into the amino acid sequence shown in SEQ ID NO: 8.

[0066] In some embodiments, the antibody is a murine monoclonal antibody, a humanized monoclonal antibody, a chimeric monoclonal antibody or a fully human monoclonal antibody.

[0067] In some embodiments, the antigen-binding portion is Fab, Fab', F(ab')2, Fd, Fv, scFv or SdAb.

[0068] Among them, Fab (Fragment antigen binding) is usually obtained by enzymatically digesting a complete antibody. A common method is to use papain which can cleave the heavy chain of the antibody, generating two Fab fragments and one Fc fragment.

[0069] Fab'(Fragment antigen binding prime) is usually obtained by further enzymatic digestion of the Fab fragment. Pepsin can be used to cleave the hinge region of the antibody, generating an antibody fragment approximately the size of Fab.

[0070] F(ab')2(Fragment antigen binding 2) is usually obtained by using an enzymatic digestion method called Panning separation on the intact antibody. This method involves exposing the antibody to specific conditions that cause enzymatic digestion of the Fc fragment, resulting in two Fab fragments bound together to form the F(ab')2 fragment.

[0071] Fd(Fragment antigen binding) is usually obtained by enzymatic digestion or PCR amplification of the intact antibody. The enzymatic digestion method is similar to that of Fab preparation, except that it targets the heavy chain for enzymatic digestion.

[0072] Fv(Variable fragment) is usually obtained by synthesizing the variable regions of the two chains and joining them together, or by enzymatic digestion from existing antibodies.

[0073] scFv(Single-chain variable fragment) is usually obtained by linking the Fv of the heavy chain and the Fv of the light chain of the antibody to form a single-chain variable fragment.

[0074] SdAb(Single-domain antibody) can usually be obtained by immunizing animals or by screening in vitro display libraries.

[0075] In some embodiments, the antibody further comprises a heavy chain constant region and a light chain constant region; the heavy chain constant region is any one of IgG type, IgA type, IgM type, IgE type, IgD type; the light chain constant region is a κ chain or a λ chain.

[0076] In some preferred embodiments, the heavy chain constant region sequence is of IgG type and the light chain constant region is a κ chain.

[0077] In some embodiments, the antibody or its antigen-binding portion comprises a heavy chain and a light chain; the heavy chain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO:2; the light chain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO:7.

[0078] In some embodiments, the heavy chain comprises the amino acid sequence of a protein or polypeptide having the same function obtained by deleting, substituting and / or inserting one or more amino acids into the amino acid sequence shown in SEQ ID NO:2; the light chain comprises the amino acid sequence of a protein or polypeptide having the same function obtained by deleting, substituting and / or inserting one or more amino acids into the amino acid sequence shown in SEQ ID NO:7.

[0079] In some embodiments, the substitution of an amino acid is a "conservative substitution", also known as substitution by "homologous" amino acid residues, which refers to a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. For example, amino acids with basic side chains (such as lysine, arginine and histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), uncharged polar side chain amino acids (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chain amino acids (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chain amino acids (such as threonine, valine, isoleucine) and aromatic side chain amino acids (such as tyrosine, phenylalanine, tryptophan, histidine). Conservative amino acid substitutions generally have less effect on the activity of the resulting protein.

[0080] In a preferred embodiment, the "protein or polypeptide obtained by deleting, substituting and / or inserting one or more amino acids" still retains the activity of the antibody or its antigen-binding portion before being "deleted, substituted and / or inserted". For example, the high specificity and / or high affinity of the antibody or its antigen-binding portion for binding to NMP22.

[0081] nucleic acid molecule

[0082] The present invention also provides a nucleic acid molecule encoding the anti-NMP22 antibody or its antigen-binding portion.

[0083] In some embodiments, the isolated nucleic acid comprises a nucleotide sequence encoding the heavy chain variable region of the antibody or its antigen-binding portion, and a nucleotide sequence encoding the light chain variable region of the antibody or its antigen-binding portion.

[0084] As used herein, the term "nucleic acid molecule" refers to a polynucleotide that is not in a naturally occurring state in nature, including polynucleotides isolated from nature (including within organisms) by biological techniques, and also including synthetic polynucleotides.

[0085] In some embodiments, the isolated polynucleotide can be genomic DNA, cDNA, mRNA, or other synthetic RNA, or a combination thereof.

[0086] Those skilled in the art can design corresponding nucleotide sequences based on the amino acid sequences of the antibodies provided by the present invention, and prepare them in an isolated state by using standard genetic engineering methods, molecular biology methods, biochemical methods, etc. Based on codon degeneracy, those skilled in the art can design a variety of nucleotide sequences that are not exactly the same, but all encode the same amino acid sequence. These modified nucleotide sequences are also included within the scope of the present invention.

[0087] biological material

[0088] The present invention also provides a biological material comprising the isolated nucleic acid, and the biological material is selected from one of an expression cassette, a vector, a transposon, and a host cell.

[0089] As used herein, the term "vector" refers to any molecule (e.g., nucleic acid, plasmid, or virus, etc.) used to transfer nucleotide coding information into a host cell.

[0090] As used herein, the term "expression cassette" or "expression vector" refers to a vector suitable for expressing a target gene (nucleotide sequence to be expressed) in a host cell, and generally includes a target gene, a promoter, and a terminator.

[0091] As used herein, the term "transposon" is a DNA sequence capable of moving positions in a genome, and is also known as a jumping gene or transposon. They exist in a variety of organisms, including bacteria, plants, animals, and fungi. Transposons can move through positions in the genome, insert into new positions and cause changes in the genome structure. Using transposon systems, foreign genes can be introduced into specific sites in the host genome to achieve applications such as genome editing, gene therapy, and transgenic technology.

[0092] As used herein, the term "host cell" refers to a cell that has been or can be transformed with a nucleic acid sequence and thereby expresses a selected gene of interest. This term includes the progeny of the parental cell, whether or not the progeny are identical in morphology or genetic composition to the original parental cell, as long as the selected gene of interest is present in the progeny. Commonly used host cells include bacteria, yeast, mammalian cells, etc. A commonly used method for introducing an exogenous gene into a host cell is "transfection", which can cause foreign or exogenous DNA to be taken up by the host cell. The cell can be induced by physical and chemical methods (such as 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".

[0093] complex

[0094] The present invention also provides a complex, which comprises: i) the anti-NMP22 antibody or its antigen-binding portion described above; and ii) a detectable label.

[0095] In some embodiments, the detectable label can be selected from any one or more of chromophores, digoxigenin-labeled probes, electron-dense substances, colloidal gold, or enzymes. The following non-limiting examples list these labels:

[0096] i) Enzymes that produce a detectable signal, such as detected by colorimetry, fluorescence, and luminescence, such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, and glucose-6-phosphate dehydrogenase.

[0097] ii) Chromophores (fluorophores), such as fluorescence, quantum dots, fluorescent microspheres, luminescent compounds, and dyes, such as organic dyes like fluorescein, phenanthroline, rhodamine, or fluorescein dyes like FITC (fluorescein isothiocyanate), TRITC (tetramethylrhodamine isothiocyanate), Cy3 (cyanine dye 3), Cy5 (cyanine dye 5).

[0098] iii) Groups having an electron density that can be detected by an electron microscope or by its electrical properties, such as conductivity, current analysis, voltage measurement, and resistance.

[0099] iv) Detectable groups, such that their molecular size is sufficient to induce a detectable modification in their physical and / or chemical properties; such detection can be achieved by optical methods (such as diffraction, surface plasmon resonance, surface variation, and contact variation angle) or physical methods (such as atomic force spectroscopy and tunneling).

[0100] v) Electron-dense substances, such as radioactive molecules (such as 32 P, 35 S or 125 I).

[0101] In some embodiments, the detectable label is selected from one or more of alkaline phosphatase, acridinium ester, horseradish peroxidase, ruthenium tris(bipyridine), isoluminol, or rare earth elements, preferably alkaline phosphatase, acridinium ester, or horseradish peroxidase.

[0102] In some embodiments, the detectable label is selected from fluorescein, phenanthroline, rhodamine, FITC (fluorescein isothiocyanate), TRITC (tetramethylrhodamine isothiocyanate), Cy3 (cyanine dye 3), or Cy5 (cyanine dye 5).

[0103] conjugate

[0104] The present invention also provides a conjugate comprising conjugated: Ⅰ) the anti-NMP22 antibody or antigen-binding portion thereof, or the complex; and, Ⅱ) a solid support.

[0105] As used herein, the term "solid support" refers to a medium to which an antibody is immobilized by physical or chemical means.

[0106] In some embodiments, the solid support may include polystyrene, polypropylene, polycarbonate, cycloolefin, glass, or quartz. In some embodiments, the solid support may be a microtiter (or "porous") plate or a microarray plate. In some embodiments, the solid support may be beads, such as magnetic beads.

[0107] application

[0108] The present invention also provides the use of the anti-NMP22 antibody or antigen-binding portion thereof in detecting NMP22.

[0109] The present invention also provides the use of the anti-NMP22 antibody or antigen-binding portion thereof in the preparation of a product for detecting NMP22.

[0110] The present invention also provides the anti-NMP22 antibody or antigen-binding portion thereof for diagnosing or assisting in the diagnosis of diseases associated with abnormal NMP22 levels.

[0111] The present invention also provides the use of the anti-NMP22 antibody or antigen-binding portion thereof in the preparation of a product for diagnosing or assisting in the diagnosis of diseases associated with abnormal NMP22 levels.

[0112] In some embodiments, the diseases include urinary system diseases associated with abnormal NMP22 levels. Such as: bladder cancer, prostate cancer, urothelial intraepithelial neoplasia (such as urethral intraepithelial neoplasia or urothelial carcinoma, etc.), urinary system inflammation (such as cystitis, glomerulonephritis, and pyelonephritis, etc.).

[0113] In some preferred embodiments, the disease includes bladder cancer.

[0114] In some embodiments, the detection sample in the application is a urine sample, bladder irrigation fluid, etc.

[0115] composition

[0116] The present invention also provides a composition, which comprises the anti-NMP22 antibody or its antigen-binding part, or comprises the nucleic acid molecule, or comprises the expression vector, or comprises the host cell, or comprises the complex, or comprises the conjugate.

[0117] In some embodiments, the composition further contains a buffer, which is used for diluting the antibody and the sample, adjusting the pH, etc. For example, Tris buffer, PBS buffer, etc.

[0118] In some embodiments, the composition further contains a preservative, which is used for protecting the antibody from degradation and prolonging the storage period of the antibody. For example, glycerol, gelatin, etc.

[0119] In some embodiments, the composition further contains a protective protein, which is used for preventing non-specific binding and reducing background interference. For example, bovine serum albumin (BSA), milk powder, etc.

[0120] In some embodiments, the composition further contains a blocker, which is used for blocking non-specific binding and reducing background interference. For example, skim milk powder, BSA, fish glue, etc.

[0121] In some embodiments, the composition further contains additives and adjuvants, which are used for adjusting the stability and performance of the antibody. For example, EDTA is used for chelating metal ions, and sodium benzoate is used for inhibiting microbial growth, etc.

[0122] In some embodiments, the composition further contains a catalytic reagent, which is used for enhancing the generation of signals. For example, a sensitizer is used in chemiluminescence experiments.

[0123] Those skilled in the art can set other components in the composition based on different purposes (including but not limited to protecting the antibody, enhancing the detection signal, reducing background interference, etc.) and common knowledge in the art.

[0124] detection product

[0125] The present invention also provides a detection product, which comprises the anti-NMP22 antibody or its antigen-binding part, or comprises the complex, or comprises the conjugate; the detection product includes reagents, test strips or kits.

[0126] In some specific embodiments, the detection products include, but are not limited to, ELISA (enzyme-linked immunosorbent assay) kits, immunohistochemical reagents, Western blot kits, immunoblotting kits, immunofluorescence staining kits, immunoprecipitation kits, immunochromatography reagents, immunohistochemical tissue staining kits, immunoprecipitation-mass spectrometry analysis kits, immunohistochemical flow cytometry analysis kits, magnetic bead immunoprecipitation kits, etc.

[0127] preparation method

[0128] The present invention also provides a method for preparing an antibody against NMP22 or an antigen-binding portion thereof, which comprises: expressing the nucleic acid molecule using a host cell, and then recovering the produced antibody or its antigen-binding portion from the culture medium and / or the cultured host cells.

[0129] In the present invention, the method for culturing host cells is generally a serum-free culture method, usually by suspending cells in serum-free medium. After culturing to produce the antibody of the present invention, it can be purified from the cell culture content according to standard procedures in the art, and the standard procedures include ammonium sulfate precipitation, affinity column, column chromatography, gel electrophoresis, etc. Such techniques are within the scope of those skilled in the art and are not limited to the several mentioned in the present invention. Another method for expressing antibodies can utilize expression in animals (especially transgenic animals or nude mice). This involves an expression system using an animal casein promoter, which, when transgenically incorporated into mammals, allows female animals to produce the desired recombinant protein in their milk. The culture medium secreting the antibody can be purified by conventional techniques. For example, it can be purified using an A or G Sepharose FF column containing adjusted buffer. Wash away the non-specifically bound components. Then elute the bound antibody using a pH gradient method, detect the antibody fragments by SDS-PAGE, and collect them. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The obtained product needs to be immediately frozen, such as at -70 °C, or lyophilized.

[0130] example

[0131] The embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, priority should be given to the guidance given in the present invention, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or refer to other known experimental methods in the art, or according to the conditions recommended by the manufacturer.

[0132] In the following specific embodiments, regarding the measurement parameters of raw material components, without special instructions, there may be slight deviations within the weighing accuracy range. Regarding temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operation accuracy are allowed.

[0133] Example 1: Preparation of recombinant human NMP22 protein fragment

[0134] Obtain 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 as follows:

[0135] NMP22-His protein fragment (SEQ ID NO:1):

[0136] MTLHATRGAALLSWVNSLHVADPVEAVLQLQDCSIFIKIIDRIHGTEEGQQILKQPVSERLDFVCSFLQKNRKHPSSPECLVSAQKVLEGSELELAKMTMLLLYHSTMSSKSPRDWEQFEYKIQAELAVILKFVLDHEDGLNLNEDLENFLQKAPVPSTCSSTFPEELSPPSHQAKREIRFLELQKVASSSSGNNFLSGSPASPMGDILQTPQFQMRRLKKQLADERSNRDELELELAENRKLLTEKDAQIAMMQQRIDRLALLNEKQAASPLEPKELEELRDKNESLTMRLHETLKQCQDLKTEKHHHHHH

[0137] Use the 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 method. Insert the gene sequence of the correctly sequenced human NMP22 protein fragment into the plasmid PET15b to construct a recombinant plasmid.

[0138] Transfect the plasmid PET15b-NMP22 containing the gene of the human NMP22 protein fragment into Escherichia coli BL21 competent cells. Put the Escherichia coli BL21 containing the PET15b-NMP22 plasmid into the culture medium (1 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl and 100 mg / L Ampicillin) and culture it at 37 °C.

[0139] Rinse the peristaltic pump thoroughly with distilled water, and then rinse the glass chromatography column thoroughly. Add approximately 200 ml of Ni-IDA (Genscript) to the column to pack the column, and wait for all the column material to settle naturally. Equilibrate with about 3 L of equilibration buffer (20 mM Tris, 300 mM NaCl) using a peristaltic pump at a flow rate of 5 ml / min; resuspend Escherichia coli containing recombinant human NMP22 protein in 200 ml of equilibration buffer (20 mM Tris, 300 mM NaCl), break the cells using an ultrasonic cell disruptor (JY98-IIIDH, Ningbo Xinzhi Biotechnology Co., Ltd.), load the supernatant after centrifugation of the disrupted solution at a flow rate of 2 ml / min; after loading, wash away impurities with equilibration buffer until the absorbance remains unchanged at a flow rate of 5 ml / min; after washing away impurities, start elution with Elution buffer (20 mM Tris, 300 mM NaCl, 250 mM imidazole) at a flow rate of 5 ml / min, and collect the eluate.

[0140] Perform SDS-PAGE detection with a gel concentration of 4-20%. As Figure 1 shown, the purity of the recombinant human NMP22 protein reaches over 90% after purification.

[0141] Example 2: Obtaining of NMP22 hybridoma cell line

[0142] 1) Animal immunization

[0143] The antigen is recombinant human NMP22 protein. Subcutaneously immunize female Balb / c mice with a 1:1 emulsion of 200 μl of Freund's complete adjuvant (Sigma-Aldrich) containing 50 μg of recombinant human NMP22 protein. Subsequently, alternately inject intraperitoneally / subcutaneously with a 1:1 emulsion of Freund's incomplete adjuvant (Sigma-Aldrich) containing 25 μg of recombinant human NMP22 protein every two weeks for up to 3 times to boost the immunity of the mice. Four days before myeloma fusion, mouse #1 showing the highest antibody titer (see Figure 2 the detection result of antibody titer determination by serum ELISA method) was intraperitoneally boosted with 25 μg of recombinant human NMP22 protein (without adjuvant). Figure 2 In the figure, the X-axis represents the OD450 signal value of the microplate reader reading the plate. The Y-axis represents the dilution factor of the mouse antiserum after the third immunization, which is expressed as the logarithm with base 10. Figure 2 The results show that after immunizing mice with NMP22 recombinant protein, specific NMP22 antibodies have been produced in the mouse serum.

[0144] 2) Hybridoma fusion and screening

[0145] The spleen was extracted under aseptic conditions and ground to form a single-cell suspension. Meanwhile, a single-cell suspension of myeloma cells (SP2 / 0) was prepared. 8.1×10⁷ spleen cells were fused with 3.8×10⁷ SP2 / 0 mouse myeloma cells using electrofusion. 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 with a volume of 100 μl per well. The cells were 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.

[0146] The 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. The plate wells were washed once with PBS-T (0.05% Tween), and blocked with 200 μl / well of PBST containing 1% BSA at 37 °C for 0.5 h. Subsequently, the blocking solution was discarded, 100 μl of hybridoma cell culture supernatant was added to each plate well, and then incubated at room temperature for 1 h. The plate was washed three times with PBST, and incubated with 100 μl / well of horseradish peroxidase-labeled goat anti-mouse IgG (Fab-specific) working solution (GenScript) at 37 °C for 0.5 h. The plate was washed five times with PBST, then TMB chromogenic solution (GenScript) was added and incubated in the dark at room temperature for 15 min. 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 (TECAN).

[0147] 3) Hybridoma subcloning

[0148] Subcloning was performed using the limiting dilution method. A hemocytometer was used to serially dilute the cells 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. For each hybridoma, 200 μl of the cell solution was pipetted into 96 wells at a density of 1 - 3 cells / well. The cells were cultured at 37 °C in 6% CO₂ for 1 week, and the supernatant was evaluated for the presence of antibodies against NMP22 by the above ELISA assay.

[0149] Example 3: Variable region sequencing of monoclonal antibodies and antibody production

[0150] 1) The subtype of the antibody in the hybridoma cell culture supernatant was identified using a Quick ELISA Mouse Antibody Subtype Identification Kit (Clonotyping System-HRP SouthernBiotech). TRIzol (Ambion) was used to extract from 3×106 ~5×10 6 Total RNA was extracted from hybridoma cells and reverse-transcribed into cDNA using antibody subtype-specific primers and universal primers (PrimeScriptTM 1st Strand cDNA Synthesis Kit, Takara).

[0151] 2) Subsequently, the V-region fragments of murine immunoglobulin heavy and light chains were amplified by RACE PCR (GenScript), the resulting PCR fragments were subcloned into the pMD18-T vector system (Takara), and the inserted fragments were sequenced using vector-specific primers.

[0152] 3) Finally, the unique V-region amino acid sequence of clone 25B1C7 was obtained. As follows:

[0153] Full-length amino acid sequence of the 25B1C7 heavy chain (SEQ ID NO: 2):

[0154] QVQLQQSGAELARPGASVKMSCKASGYTFTTYTMHWIKQRPGQGLEWIGYINPNSGYTNYNQKFSDKATLTADKSSSTAYIQLSSLTSEDSAVYYCTRDYTYDEFPYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0155] Amino acid sequence of the variable region of the 25B1C7 heavy chain (SEQ ID NO: 3):

[0156] QVQLQQSGAELARPGASVKMSCKASGYTFTTYTMHWIKQRPGQGLEWIGYINPNSGYTNYNQKFSDKATLTADKSSSTAYIQLSSLTSEDSAVYYCTRDYTYDEFPYWGQGTLVTVSA

[0157] 25B1C7 VH CDR1 (SEQ ID NO:4):

[0158] TYTMH

[0159] 25B1C7 VH CDR2 (SEQ ID NO:5):

[0160] YINPNSGYTNYNQKFSD

[0161] 25B1C7 VH CDR3 (SEQ ID NO:6):

[0162] DYTYDEFPY

[0163] 25B1C7 full-length light chain amino acid sequence (SEQ ID NO:7):

[0164] DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0165] 25B1C7 light chain variable region amino acid sequence (SEQ ID NO:8):

[0166] DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLYTFGGGTKLEIK

[0167] 25B1C7 VL CDR1 (SEQ ID NO:9):

[0168] KSSQSLLNSRTRKNYLA

[0169] 25B1C7 VL CDR1(SEQ ID NO:10):

[0170] WASTRES

[0171] 25B1C7 VL CDR1(SEQ ID NO:11):

[0172] KQSYNLYT

[0173] 4) Synthesize DNA fragments corresponding to the full-length amino acid sequences of the heavy chain and the light chain respectively, and insert them into the pTT5 expression vector ((NRC Biotechnology Research Institute, National Research Council of Canada)) respectively to form expression plasmids.

[0174] 5) After culturing the above-mentioned hybridoma cells in a shaking flask at 37 °C for 10 days, collect the supernatant for antibody purification.

[0175] 6) Depyrogenate the pipelines and the Protein A column with 0.2 M NaOH before purification. Re-equilibrate the column with a buffer containing 0.05 M Tris and 1.5 M NaCl (pH 8.0). Subsequently, dilute the harvested cell culture supernatant 1:1 with 2× the above buffer and filter sterilize it.

[0176] 7) Incubate the filtered supernatant with the Protein A column at room temperature for 2 hours. After washing the column with 1× the above buffer, elute IgG with sterile 0.1 M sodium citrate (pH 3.5), collect the eluate and neutralize it with one-ninth volume of sterile 1 M Tris-HCl (pH 9).

[0177] 8) Under sterile conditions, exchange the buffer of the product to PBS (pH 7.4) to remove any elution buffer and concentrate the sample. After concentration, quantify the antibody by OD280nm using an extinction coefficient Ec (0.1%) of 1.43.

[0178] 9) Analyze the purified antibody by SDS-PAGE using a 10% precast gel (GenScript) on a BioRad electrophoresis system. Stain the gel with Estain2.0 (GenScript) and estimate the molecular size and purity by comparing the stained bands with Protein Ladder (GenScript). The SDS-PAGE identification results are as Figure 3 shown.

[0179] Example 4: Binding of Monoclonal Antibody to Recombinant Human NMP22 Protein

[0180] 1) Indirect ELISA was used to evaluate the binding ability of the purified antibody to NMP22. The ELISA plates (Nunc) were coated with 0.5 μg / ml of recombinant NMP22 in PBS at 100 μl / well overnight at 4 °C.

[0181] 2) The plates were washed with PBS-T (0.05% Tween) and blocked with PBST containing 1% BSA at 200 μl / well for 2 hours at 37 °C.

[0182] 3) The blocking solution was discarded and the plates were dried at 37 °C for 2 hours.

[0183] 4) Subsequently, the blocking solution was discarded, 100 μl of the purified antibody at 10 μg / ml was added to the first well, and it was diluted in a 3-fold gradient, with a total of 11 test concentration gradients.

[0184] 5) Then it was incubated at room temperature for 1 hour. The plates were washed four times with PBST and incubated with 100 μl / well of goat anti-mouse IgG (Fab-specific) conjugated with horseradish peroxidase (GenScript) at 37 °C for 0.5 hour. The plates were washed four times with PBST, then TMB chromogenic solution (FcMACS) was added and incubated at room temperature in the dark for 15 minutes.

[0185] 6) The reaction was terminated by adding 50 μl of 1 M HCl termination solution (Sigma). The plates were read using a microplate reader at 450 nm. The binding ability of clone 25B1C7 to the recombinant human NMP22 protein was as Figure 4 , Figure 4 shown, indicating that clone 25B1C7 can specifically bind to the recombinant human NMP22 protein. Figure 4 In, 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 showed that the purified mouse anti-NMP22 monoclonal antibody (25B1C7) could specifically recognize the NMP22 recombinant protein.

[0186] Example 5: Determination of Antibody Affinity

[0187] 1) The probe with surface-coupled His protein was soaked in 250 μL of buffer K (PBS + 0.002% Tween 20 + 0.02% BSA) for 10 minutes;

[0188] 2) The recombinant human NMP22 protein was prepared into a working solution of 5 μg / mL with buffer K;

[0189] 3) Prepare the 25B1C7 clone in buffer K at four working concentrations: 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, and 0 μg / mL;

[0190] 4) Add reagents according to the instructions of the Gator label-free analyzer (Gator Bio, CAT#: Gator Prime). The affinity data is shown in Table 1 below, and some binding curves are as Figure 5 shown. The results show that the antibody of the 25B1C7 clone has a high affinity level for recombinant human NMP22 protein.

[0191] Table 1

[0192] clone number koff (1 / s) kon (1 / Ms) KD (M) 25B1C7 7.94E-06 5.83E+05 1.36E-11

Claims

1. An anti-NMP22 antibody or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region; wherein: i) a heavy chain variable region comprising VH CDR1, VH CDR2 and VH CDR3, wherein VH CDR1, VH CDR2 and VH CDR3 respectively comprise the amino acid sequences of TYTMH (SEQ ID NO: 4), YINPNSGYTNYNQKFSD (SEQ ID NO: 5), and DYTYDEFPY (SEQ ID NO: 6), or comprise an amino acid sequence having 1-3 amino acid substitutions in each CDR compared to the above amino acid sequences; and / or ii) a light chain variable region comprising VL CDR1, VL CDR2 and VL CDR3, wherein VL CDR1, VL CDR2 and VL CDR3 respectively comprise the amino acid sequences of KSSQSLLNSRTRKNYLA (SEQ ID NO: 9), WASTRES (SEQ ID NO: 10) and KQSYNLYT (SEQ ID NO: 11), or comprise an amino acid sequence having 1-3 amino acid substitutions in each CDR compared to the above amino acid sequences.

2. The anti-NMP22 antibody or antigen-binding portion thereof according to claim 1, comprising a heavy chain variable region and a light chain variable region, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 respectively comprise the amino acid sequences of TYTMH (SEQ ID NO: 4), YINPNSGYTNYNQKFSD (SEQ ID NO: 5), DYTYDEFPY (SEQ ID NO: 6), KSSQSLLNSRTRKNYLA (SEQ ID NO: 9), WASTRES (SEQ ID NO: 10) and KQSYNLYT (SEQ ID NO: 11).

3. The anti-NMP22 antibody or antigen-binding portion thereof according to claim 1 or 2, wherein The heavy chain variable region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:

3.

4. The anti-NMP22 antibody or antigen-binding portion thereof according to any one of claims 1 to 3, wherein The light chain variable region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:

8.

5. The anti-NMP22 antibody or antigen-binding portion thereof according to any one of claims 1 to 4, wherein the heavy chain variable region and the light chain variable region comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 3 and SEQ ID NO: 8, respectively.

6. The anti-NMP22 antibody or antigen-binding portion thereof according to claim 1, wherein The antibody is a mouse monoclonal antibody, a humanized monoclonal antibody, a chimeric monoclonal antibody or a fully human monoclonal antibody.

7. The anti-NMP22 antibody or antigen-binding portion thereof according to claim 1, wherein The antigen binding portion is Fab, Fab', F(ab')2, Fd, Fv, scFv or SdAb.

8. The anti-NMP22 antibody or antigen-binding portion thereof according to claim 1, wherein The antibody or antigen-binding portion thereof further comprises a heavy chain constant region and a light chain constant region; The heavy chain constant region is any one of IgG type, IgA type, IgM type, IgE type, and IgD type; The light chain constant region is a κ chain or a λ chain; Preferably, the heavy chain constant region sequence is of IgG type, and the light chain constant region is a kappa chain.

9. The anti-NMP22 antibody or antigen-binding portion thereof according to claim 1, wherein The antibody or antigen-binding portion thereof comprises a heavy chain and a light chain; The heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:2; The light chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:

7. 10 . A nucleic acid molecule encoding the anti-NMP22 antibody or antigen-binding portion thereof according to claim 1 . An expression vector comprising the nucleic acid molecule according to claim 10 .

12. A host cell comprising the expression vector according to claim 11, or having the nucleic acid molecule according to claim 10 integrated into its genome.

13. A composite comprising: i) the anti-NMP22 antibody or antigen-binding portion thereof according to any one of claims 1 to 9; and, ii) a detectable label.

14. A conjugate comprising: Ⅰ) the anti-NMP22 antibody or a functional fragment thereof according to any one of claims 1 to 9, or the complex according to claim 13; and, II) a solid support.

15. Use of the anti-NMP22 antibody or antigen-binding portion thereof according to any one of claims 1 to 9 in any of the following aspects: a1) Detection of NMP22; a2) Preparation of products for detecting NMP22.

16. Use of the anti-NMP22 antibody or antigen-binding portion thereof according to any one of claims 1 to 9 in any of the following aspects: b1) Diagnosis or auxiliary diagnosis of diseases related to abnormal NMP22 levels; b2) preparing products for diagnosis or auxiliary diagnosis of diseases associated with abnormal NMP22 levels; Preferably, the disease includes a urinary system disease associated with abnormal NMP22 levels, and more preferably, the disease includes bladder cancer.

17. A composition comprising the anti-NMP22 antibody or antigen-binding portion thereof according to any one of claims 1 to 9, the nucleic acid molecule according to claim 10, the expression vector according to claim 11, the host cell according to claim 12, the complex according to claim 13, or the conjugate according to claim 14.

18. A method for preparing an anti-NMP22 antibody or an antigen-binding portion thereof, comprising: The nucleic acid molecule of claim 10 is expressed in a host cell, and the produced antibody or antigen-binding portion thereof is then recovered from the culture medium and / or the cultured host cell.

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