A monoclonal antibody targeting keratin 5, its preparation method and application

Abstract

This invention provides a monoclonal antibody targeting keratin 5, its preparation method, and its applications. Specifically, this invention provides an antibody targeting keratin 5 (KRT5) or its antigen-binding fragment thereof, as well as a coding sequence encoding the antibody or its antigen-binding fragment, a corresponding expression vector, and a host cell capable of expressing the antibody or its antigen-binding fragment. The monoclonal antibody targeting keratin 5 of this invention can accurately recognize and specifically bind to the KRT5 protein in various applications (such as Western blotting, immunocytochemistry, flow cytometry, etc.), and can be used for the detection of KRT5.

Description

Technical Field

[0001] This invention relates to the field of biodiagnostic technology, specifically to a monoclonal antibody targeting keratin 5, its preparation method, and its application. Background Technology

[0002] Keratin 5 (KRT5) is a core member of the type II keratin family, primarily expressed in the basal cells of stratified squamous epithelium, and participates in maintaining epithelial cell structural stability, regulating stem cell stemness, and repairing damage. Pathologically, KRT5 gene mutations can lead to epidermolysis bullosa (EBS), and its abnormal expression is also closely related to the differential diagnosis of tumors such as basaloid subtypes of breast cancer and squamous cell carcinoma of the lung, making it a key biomarker in clinicopathological testing.

[0003] Currently, KRT5 detection relies heavily on immunological techniques such as immunohistochemistry, Western blotting, and immunofluorescence, with specific antibodies being the core tool for achieving these detections. For example, most existing antibodies are only effective on specific detection platforms, applicable only to Western blotting or paraffin-embedded tissue sections, lacking broad applicability validated across multiple technology platforms, thus limiting their consistent performance in research and clinical testing. Furthermore, current antibody preparation methods often use full-length proteins or single peptides as immunogens, making it difficult to simultaneously elicit immune responses against both linear and conformational epitopes. This results in relatively limited epitope types for the screened antibodies, affecting their adaptability in different application scenarios. Simultaneously, the antibody secretion capacity of some hybridoma cell lines decreases during long-term culture, and the batch-to-batch stability of purified products is insufficient, also affecting the reproducibility and reliability of the detection system.

[0004] In addition, existing methods for assessing the specificity of KRT5 rely on indirect methods such as ELISA or immunoblotting, which cannot effectively exclude the non-specific binding of antibodies to unknown intracellular proteins. This leads to prominent background signals and false positives in practical applications. Therefore, KRT5 is prone to false positives or localization bias in samples with low expression.

[0005] Therefore, the present invention urgently needs to develop an antibody that targets KRT5 with high specificity and high sensitivity. Summary of the Invention

[0006] The purpose of this invention is to provide an antibody that targets KRT5 with high specificity and high sensitivity.

[0007] In a first aspect of the invention, an antibody or antigen-binding fragment thereof targeting keratin 5 (KRT5) is provided, said antibody or antigen-binding fragment comprising: (1) The following three complementary determinant regions CDR (HCDR) for heavy chain variable regions and three complementary determinant regions CDR (LCDR) for light chain variable regions as defined by the IMGT rules: HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4 HCDR3 shown in SEQ ID NO: 5 The LCDR1 shown in SEQ ID NO: 6, The LCDR2 shown in SEQ ID NO: 7, LCDR3 as shown in SEQ ID NO: 8; or (2) The following three complementary determinant regions CDR (HCDR) for heavy chain variable regions and three complementary determinant regions CDR (LCDR) for light chain variable regions as defined by Kabat rules: HCDR1, as shown in SEQ ID NO:12, HCDR2 shown in SEQ ID NO: 13 HCDR3 shown in SEQ ID NO: 14 LCDR1 shown in SEQ ID NO: 15, LCDR2 shown in SEQ ID NO:16 LCDR3 shown in SEQ ID NO: 17.

[0008] In another preferred embodiment, the sequence further includes a sequence having keratin 5 binding affinity by adding, deleting, modifying and / or substituting at least one amino acid from any of the above-mentioned amino acid sequences.

[0009] In another preferred embodiment, the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

[0010] In another preferred embodiment, the antigen-binding fragment includes the Fab fragment, the F(ab')2 fragment, and the Fv fragment.

[0011] In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 1, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 2.

[0012] In another preferred embodiment, the amino acid sequence of the heavy chain variable region (VH) of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 1, or has an amino acid sequence having ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% homology with the sequence shown in SEQ ID NO: 1.

[0013] In another preferred embodiment, the amino acid sequence of the light chain variable region (VL) of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 2, or has an amino acid sequence having ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% homology with the sequence shown in SEQ ID NO: 2.

[0014] In another preferred embodiment, the complementarity-determining region (CDR) of the heavy chain variable region of the antibody is obtained according to the IMGT, Kabat, Chothia or Abm rules for the VH partition shown in SEQ ID NO: 1.

[0015] In another preferred embodiment, the complementarity-determining region (CDR) of the variable region of the light chain of the antibody is obtained according to the IMGT, Kabat, Chothia or Abm rules for the VL partition shown in SEQ ID NO: 2.

[0016] In another preferred embodiment, the heavy chain of the antibody or its antigen-binding fragment further includes a heavy chain constant region; and / or, the light chain of the antibody or its antigen-binding fragment further includes a light chain constant region.

[0017] In another preferred embodiment, the antibody is a single-chain antibody, a double-chain antibody, or an antigen-binding fragment.

[0018] In another preferred embodiment, the antibody is a humanized antibody, a murine antibody, or a chimeric antibody.

[0019] In another preferred embodiment, the antibody is a murine antibody, a murine-human chimeric antibody, or a humanized IgG1 antibody.

[0020] In another preferred embodiment, the heavy chain constant region is of human or mouse origin.

[0021] In another preferred embodiment, the light chain constant region is of human or mouse origin.

[0022] In another preferred embodiment, the antibody is a full-length antibody protein or an antigen-binding fragment.

[0023] In another preferred embodiment, the antibody is a monoclonal antibody.

[0024] In another preferred embodiment, the monoclonal antibody is a murine monoclonal antibody.

[0025] In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.

[0026] In another preferred embodiment, the antibody includes monospecific, bispecific, trispecific, or multispecific antibodies.

[0027] In another preferred embodiment, the antibody further comprises a linker peptide located between the heavy chain variable region and the light chain variable region.

[0028] In another preferred embodiment, the antibody targets and binds to the KRT5 protein within the intracellular or nuclear region of tumor cells.

[0029] In a second aspect of the invention, a fusion protein is provided, the fusion protein comprising: (i) an antibody or antigen-binding fragment thereof targeting keratin 5 as described in the first aspect of the invention; and (ii) A fusion portion fused with the antibody or its antigen-binding fragment.

[0030] In another preferred embodiment, the fusion portion is selected from the group consisting of: tag sequences, signal peptides, or membrane-penetrating elements, or combinations thereof.

[0031] In another preferred embodiment, the tag sequence includes a 6His tag and a FLAG tag.

[0032] In a third aspect of the invention, a polynucleotide is provided that encodes an antibody or antigen-binding fragment thereof targeting keratin 5 as described in the first aspect of the invention, or a fusion protein as described in the second aspect of the invention.

[0033] In another preferred embodiment, the polynucleotide includes DNA, RNA, or cDNA.

[0034] In a fourth aspect of the invention, an expression vector is provided, the expression vector containing the polynucleotide as described in the third aspect of the invention.

[0035] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof; preferably, the expression vector includes viral vectors such as lentiviruses, adenoviruses, AAV viruses, retroviruses, or combinations thereof.

[0036] In another preferred embodiment, the expression vector is selected from the group consisting of: pTomo lentiviral vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, or pLV, or combinations thereof.

[0037] In another preferred embodiment, the expression vector is selected from the group consisting of pcDNA3.1 vector, pMES4 vector, or pABG1 vector (including pABG1-Fc vector), or combinations thereof.

[0038] In another preferred embodiment, the expression vector further includes a selection from the group consisting of promoters, transcriptional enhancement elements (WPREs), long terminal repeat sequences (LTRs), or combinations thereof.

[0039] In a fifth aspect of the invention, a host cell is provided, the host cell containing an expression vector as described in the fourth aspect of the invention, or having a genome integrated with polynucleotides as described in the third aspect of the invention.

[0040] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0041] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or combinations thereof.

[0042] In another preferred embodiment, the prokaryotic cells are selected from the group consisting of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, or combinations thereof.

[0043] In another preferred embodiment, the eukaryotic cells are selected from the group consisting of mammalian cells, Pichia pastoris, Saccharomyces cerevisiae, Schizosoma, Trichoderma, or combinations thereof.

[0044] In another preferred embodiment, the host cell is a mammalian cell, such as a CHO cell or a 293 cell.

[0045] In a sixth aspect of the invention, an antibody conjugate is provided, the antibody conjugate comprising: (a) An antibody or antigen-binding fragment thereof targeting keratin 5 as described in the first aspect of the invention; and (b) Coupled part.

[0046] In another preferred embodiment, the coupling portion is a detectable marker.

[0047] In another preferred embodiment, the detectable marker is selected from the group consisting of fluorescent groups, chemiluminescent groups, radionuclides, or enzymes capable of generating detectable signals (such as horseradish peroxidase), or combinations thereof.

[0048] In another preferred embodiment, the (a) portion is coupled to the coupling portion by a chemical bond or a connector.

[0049] In another preferred embodiment, the radionuclide includes: (i) A diagnostic isotope selected from the group consisting of: Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or combinations thereof; and / or (ii) A therapeutic isotope selected from the group consisting of: Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or combinations thereof.

[0050] In another preferred embodiment, the coupling portion is selected from the group consisting of: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, or any form of nanoparticle.

[0051] In a seventh aspect of the invention, there is provided a use of an active ingredient selected from the group consisting of: antibodies targeting keratin 5 as described in the first aspect of the invention, or antigen-binding fragments thereof, or antibody conjugates as described in the sixth aspect of the invention, or combinations thereof, wherein the active ingredient is used to prepare reagents or kits for the in vitro or in vivo detection of keratin 5.

[0052] In another preferred embodiment, the reagent is a detection reagent.

[0053] In another preferred embodiment, the detection reagent is a contrast agent.

[0054] In another preferred embodiment, the kit is selected from the group consisting of test strips or test plates.

[0055] In another preferred embodiment, the reagent or kit is used for: (1) Detect the KRT5 protein or fragments thereof in the sample; and / or (2) Detect cells expressing KRT5 protein.

[0056] In another preferred embodiment, the KRT5 protein includes: human KRT5 protein, mouse KRT5 protein, or recombinant KRT5 protein.

[0057] In another preferred embodiment, the detection is used for the diagnosis or prognosis of KRT5-related diseases or conditions.

[0058] In another preferred embodiment, the KRT5-related disease or condition is a tumor.

[0059] In another preferred embodiment, the tumor disease is selected from the group consisting of: breast cancer (such as basal-like breast cancer), adenoid cystic carcinoma, pleomorphic adenoma, basal cell adenoma, lung cancer (such as squamous cell carcinoma of the lung, adenocarcinoma of the lung), adenomyoma, mesothelioma, basal cell carcinoma, transitional cell carcinoma, thymoma, papillary carcinoma, or combinations thereof.

[0060] In another preferred embodiment, the detection includes immunoblotting, immunohistochemistry, flow cytometry, immunocytochemistry, or ELISA.

[0061] In another preferred embodiment, the detection includes immunoblotting, flow cytometry, and immunocytochemistry.

[0062] In another preferred embodiment, the use is non-diagnostic and non-therapeutic; or therapeutic or diagnostic.

[0063] In an eighth aspect of the invention, a method for detecting keratin 5 in a sample in vitro without diagnostic or therapeutic purposes is provided, the method comprising the steps of: (1) Contact the sample with an antibody or antigen-binding fragment thereof that targets keratin 5 as described in the first aspect of the present invention, or an antibody conjugate or combination thereof as described in the sixth aspect of the present invention; (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of keratin 5 in the sample.

[0064] In a ninth aspect of the invention, an in vitro diagnostic reagent for detecting keratin 5 is provided, the diagnostic reagent comprising: (a) An antibody or antigen-binding fragment thereof targeting keratin 5 as described in the first aspect of the present invention, or an antibody conjugate as described in the sixth aspect of the present invention; (b) A carrier that is scientifically acceptable.

[0065] In another preferred embodiment, the method for detecting keratin 5 is selected from the group consisting of: Western blotting, immunofluorescence (IF), immunohistochemistry (IHC), flow cytometry, immunochromatography, chemiluminescence, or ELISA.

[0066] In another preferred embodiment, the detection-acceptable carrier is a non-toxic, inert aqueous carrier medium.

[0067] In another preferred embodiment, the detection reagent is one or more reagents selected from the group consisting of isotope tracers, contrast agents, flow cytometry reagents, cell immunofluorescence reagents, magnetic nanoparticles, and imaging agents.

[0068] In another preferred embodiment, the method is non-diagnostic and non-therapeutic; or therapeutic or diagnostic.

[0069] In another preferred embodiment, the test reagent is in liquid or powder form (e.g., aqueous solution, injection, lyophilized powder, tablet, aerosol).

[0070] In a tenth aspect of the present invention, a kit for in vitro detection of keratin 5 is provided, the kit comprising an antibody or antigen-binding fragment thereof targeting keratin 5 as described in the first aspect of the present invention, or an antibody-drug conjugate as described in the sixth aspect of the present invention, and an instruction manual.

[0071] In another preferred embodiment, the instruction manual states that the kit is used for non-invasive detection of keratin 5 expression levels in a test subject.

[0072] In an eleventh aspect of the present invention, a diagnostic method for KRT5-related diseases is provided, comprising the steps of: (i) Obtaining a sample from a diagnostic subject and contacting the sample with an antibody or antigen-binding fragment thereof targeting KRT5 as described in the first aspect of the invention, or an antibody-drug conjugate as described in the sixth aspect of the invention, or an in vitro diagnostic reagent as described in the ninth aspect of the invention, or a combination thereof; and (ii) Detect whether an antigen-antibody complex is formed, wherein the formation of a complex indicates that the subject is a confirmed patient with KRT5-related disease.

[0073] In another preferred embodiment, the KRT5-related disease is a disease with high KRT5 expression (such as breast cancer or lung cancer).

[0074] In another preferred embodiment, the sample is a blood sample or a throat swab sample, or a sample from other tissues or organs.

[0075] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0076] Figure 1The image shows SDS-PAGE electrophoresis of monoclonal antibodies targeting keratin 5 and a blank control (wherein the blank control was treated with PBS) produced by different hybridoma cell lines selected in one embodiment of the present invention, specifically recognizing the recombinant KRT5 protein (i.e., the KRT5 antigen, wherein the KRT5 antigen is truncated), showing protein purity and an expected molecular weight of approximately 38.6 kDa; wherein the hybridoma cell lines are, in order, 5-A1-F2, 9-D3-G10, 10-C2-G1, 18-F5-A4, 20-D1-B12, 21-E9-G3, 30-A12-D8, 33-G5-B6, 36-D12-H5, and 39-C2-A7.

[0077] Figure 2 This invention demonstrates a monoclonal antibody targeting keratin 5 (i.e., a monoclonal antibody secreted by the 5-A1-F2 hybridoma cell line). Figure 2 The kinetic fitting curves for the adsorption-elution-dissociation of antigen KRT5 (represented by "5-A1-F2") with a 1:1 ratio are shown in the figure. The colored curves refer to the raw, measured sensor data acquired in real time by the instrument at various concentrations (12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM). The black curves represent the kinetic fitting curves corresponding to the colored curves at various concentrations (12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM).

[0078] Figure 3 The diagram shows the results of the monoclonal antibody targeting keratin 5 of the present invention specifically recognizing the KRT5 protein at 53 kDa in cell lysates of HeLa cells (lane 1), HaCaT cells (lane 2), MCF7 cells (lane 3), DU145 cells (lane 4), H9c2 cells (lane 5), and C2C12 cells (lane 6) in one embodiment of the present invention.

[0079] Figure 4 The illustration shows, in one embodiment of the invention, the detection of KRT5 expression in wild-type cells and KRT5 knockout cells using the monoclonal antibody targeting keratin 5 of the invention.

[0080] Figure 5 The image shows flow cytometry (FCM) results of the isotype control group (green fluorescence) and the KRT5 staining experimental group (red fluorescence) in one embodiment of the present invention.

[0081] Figure 6 The image shows an immunocytochemical (ICC) fluorescence result of DAPI co-stained with the monoclonal antibody targeting keratin 5 of the present invention in one embodiment of the invention. Detailed Implementation

[0082] This invention, based on extensive and in-depth research, numerous experiments, and screening, has for the first time developed a monoclonal antibody targeting keratin 5 that specifically binds to the KRT5 protein (also known as the KRT5 antigen). It exhibits extremely high specificity for the KRT5 antigen in various applications of basic experiments (such as Western blotting, immunocytochemistry, and flow cytometry). This monoclonal antibody targeting keratin 5 accurately recognizes and specifically binds to the KRT5 protein, thus solving the long-standing technical problem of "false positives" in the antibody field. This technological breakthrough provides a reliable tool for the application of the KRT5 protein in cancer diagnosis and the development of related targeted drugs. This invention was completed based on this foundation.

[0083] the term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0084] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0085] KRT5 As used in this article, “keratin 5”, “keratin 5 antigen”, “KRT5 protein”, and “KRT5 antigen” can be used interchangeably.

[0086] As used herein, the terms "keratin 5" and "KRT5" are used interchangeably, referring to a representative component of the type II keratin family, encoded by the gene KRT5. Intracellularly, KRT5 assembles with type I keratin KRT14 to form a heterodimer, which further aggregates into intermediate filament structures. Its expression exhibits tissue selectivity, primarily localizing in the basal cells of stratified squamous epithelium, such as the basal layer of the epidermis, the outer root sheath of hair follicles, and the basal cells of mammary ducts. As a key structural component of basal cells, KRT5 not only participates in maintaining cellular mechanical stability but also plays a crucial role in the regulation of epithelial stem cell stemness, tissue damage repair, and related signal transduction. Under pathological conditions, abnormal KRT5 expression levels are closely related to the occurrence and development of various diseases. Genetic evidence indicates that KRT5 gene mutations can cause a series of hereditary skin diseases, including epidermolysis bullosa (EBS). In the field of tumor pathology, KRT5 is often used as an important marker molecule for squamous epithelial differentiation and basal-like phenotype. It is used to identify basal-like subtypes of breast cancer, differentiate between squamous cell carcinoma and adenocarcinoma of the lung, and mark basal cells in prostate tissue.

[0087] The antibody targeting KRT5 of this invention As used herein, the terms "anti-KRT5 antibody of the present invention", "anti-KRT5 monoclonal antibody of the present invention", "antibody of the present invention", "monoclonal antibody of the present invention", "antibody targeting KRT5 protein of the present invention", "antibody targeting keratin 5 of the present invention", "antibody targeting KRT5 of the present invention", etc., are used interchangeably and refer to the antibody described in the first aspect of the present invention.

[0088] As used herein, the term "antibody" or "immunoglobulin" is a heterotetramer composed of two light chains (L) and two heavy chains (H). The N-terminus of each heavy chain is a variable region (VH) connecting to the constant region of the heavy chain. The N-terminus of each light chain is a variable region (VL) connecting to the constant region of the light chain.

[0089] As used herein, the term "variable" refers to the fact that the variable regions of an antibody differ in a specific sequence, resulting in the affinity and specificity of a particular antibody for a specific antigen. Antibody variable regions include complementarity-determining regions (CDRs) or hypervariable regions, as well as more conserved framework regions (FRs). The primary sequence of the heavy and light chain variable regions consists of four FR sequences and three CDR sequences spaced apart (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The sequence and spatial conformation of the heavy and light chain variable regions determine the specific binding of the antibody to the antigenic epitope. Antibody constant regions do not directly participate in antibody-antigen binding, but they affect the performance of antibody capture and detection.

[0090] The "light chain" of vertebrate antibodies (immunoglobulins) can be classified according to the amino acid sequence of their constant region. and Immunoglobulins are a class of proteins. Based on the amino acid sequence of their heavy chain constant region, immunoglobulins can be divided into different types, mainly five classes: IgA, IgD, IgE, IgG, and IgM, as well as antibody subtypes (isotypes), such as mouse IgG, which includes IgG1, IgG2a, and IgG2b subtypes. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known to those skilled in the art.

[0091] As used herein, the term "monoclonal antibody (MABS)" refers to an antibody obtained from a largely homogeneous population, meaning that the individual antibodies contained in that population are identical. Monoclonal antibodies target a single antigenic determinant (epitope) with high specificity. The modifier "monoclonal" indicates the antibody's characteristic of being obtained from a largely homogeneous population of antibodies, which should not be interpreted as requiring any special methods to produce the antibody.

[0092] The present invention also includes monoclonal antibodies having the corresponding amino acid sequence of the anti-KRT5 monoclonal antibody, monoclonal antibodies having the variable region chain of the anti-KRT5 monoclonal antibody, and other proteins or protein conjugates and fusion expression products having these chains. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., antibody conjugate and fusion expression product) having a light chain and a heavy chain containing a variable region (complementarity-determining region, CDR), provided that the variable region is the same as or has at least 90% homology with the variable regions of the light chain and heavy chain of the present invention, preferably at least 95% homology.

[0093] As known to those skilled in the art, antibody conjugates and fusion expression products include conjugates formed by binding a detectable marker (e.g., a fluorescent or luminescent marker), a radiolabel, an enzyme capable of producing a detectable product, gold nanoparticles / nanoran and other detectable molecules to an antibody or antigen-binding fragment targeting the KRT5 protein.

[0094] The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that maintain its ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used for antigen-binding function. Examples of binding fragments included in the term "antigen-binding fragment of an antibody" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bonds on their chain regions; and (iii) scFv fragments consisting of the VH and VL domains of a single arm of the antibody.

[0095] This invention includes not only complete monoclonal antibodies, but also antibody fragments with binding activity, such as Fab or (Fab')2 fragments; antibody heavy chains; antibody light chains or scFv.

[0096] The term "epitope" or "antigenic determinant" refers to the site on an antigen where immunoglobulins or antibodies specifically bind. Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique spatial conformation.

[0097] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to an epitope on a pre-defined antigen.

[0098] As used herein, the term "antigen determinant" refers to a discontinuous three-dimensional spatial site on an antigen that is recognized by the antibody or antigen-binding fragment of the present invention.

[0099] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0100] In this invention, the antibody includes murine antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies can be prepared using DNA recombination techniques well known in the art.

[0101] In this invention, the antibody can be monospecific, bispecific, trispecific, or more multiple specific.

[0102] As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably. The terms "light chain variable region" and "VL" are used interchangeably.

[0103] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. One of the most commonly used definitions of these six CDRs is provided by Kabat EA et al., (1991) Sequences of proteins of immune interest. NIH Publication 91-3242, namely the Kabat numbering system. Alternatively, antibody CDRs can also be classified using other numbering systems in the field, such as the IMGT, Chothia, and Abm numbering systems.

[0104] In one aspect of the invention, an antibody targeting the KRT5 protein or an antigen-binding fragment thereof is provided. The function of the antibody of the present invention is determined by the variable region sequences of the antibody's light and heavy chains and its structural conformation, enabling it to specifically bind to the KRT5 protein. Using this antibody variable region gene or complementarity-determining region (CDR) gene, different forms of genetically engineered antibodies can be modified and produced in any expression system utilizing prokaryotic and eukaryotic cells.

[0105] In this invention, the terms "antibody of the present invention", "protein of the present invention" or "peptide of the present invention" are used interchangeably and all refer to antibodies that specifically bind to the KRT5 protein, such as proteins or peptides having the heavy chain variable region shown in SEQ ID NO: 1 and the light chain variable region shown in SEQ ID NO: 2.

[0106] The terms "fragment," "derivative," and "analyte" of an antibody refer to a polypeptide that substantially retains the same biological function or activity as the antibody of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by coupling a mature polypeptide with another compound (e.g., a compound used for chemiluminescence, such as acridinium ester); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence, secretion sequence, or tag protein sequence or other fusion protein sequence used to purify or detect this polypeptide). These fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0107] The antibody of this invention refers to a polypeptide having KRT5 protein-binding activity and including the aforementioned CDR region. This term also includes variants of the polypeptide containing the aforementioned CDR region that have the same function as the antibody of this invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) at the C-terminus and / or N-terminus. This term also includes active fragments and active derivatives of the antibody of this invention.

[0108] The variant forms of the polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, and polypeptides or proteins obtained using antiserum containing the antibody of this invention.

[0109] The present invention also includes fragments of the antibody of the present invention. Typically, the fragment has at least about 50 consecutive amino acids, preferably at least about 60 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids of the antibody of the present invention.

[0110] Polynucleotides, vectors and host cells This invention also provides polynucleotides encoding the aforementioned antibodies or fragments thereof or their fusion proteins. The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or synthetically produced DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be identical to the coding region sequence of the aforementioned anti-KRT5 antibody or a degenerate variant. As used herein, "degenerate variant" refers to a nucleic acid sequence encoding a sequence having the same amino acid sequence as the polypeptide of this invention, but with a different coding region sequence.

[0111] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0112] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include polynucleotides that include additional coding and / or non-coding sequences. The full-length nucleotide sequence or fragments of the antibody of the present invention can typically be obtained by PCR amplification, recombinant methods, or artificial synthesis. Furthermore, heavy or light chains can be fused with proteins or tag sequences (such as fluorescent proteins, flag tags) to form fusion proteins.

[0113] Once the antibody sequence is obtained, recombinant antibodies can be prepared using bioengineering methods. Typically, the gene encoding the antibody sequence is cloned into a vector, the expression vector is then transferred into cells for expression, the cells or expression supernatant are harvested, and the recombinant antibody is obtained through purification. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0114] Currently, the DNA sequence encoding the protein (or a fragment thereof, or a derivative thereof) of this invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing plasmids (or other expression vectors) known in the art. Furthermore, mutations can be introduced into the antibody sequence of this invention through chemical synthesis.

[0115] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0116] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0117] Transforming host cells with recombinant DNA to express antibodies is a routine technique well-known to those skilled in the art. The recombinantly expressed antibodies can be isolated and purified using routine techniques well-known to those skilled in the art, which will not be elaborated upon here.

[0118] Test reagents and kits The antibodies of this invention can be used in detection applications, such as for testing samples, to provide diagnostic information.

[0119] In this invention, the samples used include cells, tissue samples, and biopsy specimens. The term "biopsy" as used in this invention should include all types of biopsies known to those skilled in the art. Therefore, biopsies used in this invention can include tissue samples prepared, for example, by endoscopic methods or by puncture or needle biopsy of organs.

[0120] The samples used in this invention include fixed or preserved cell or tissue samples.

[0121] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be immobilized on a detection plate.

[0122] application As described above, the antibody of the present invention has broad biological and clinical application value, and its applications involve multiple fields such as the diagnosis and treatment of KRT5-related diseases, basic medical research, and biological research. A preferred application is for the clinical diagnosis, prevention, and targeted therapy of KRT5.

[0123] Experiments have demonstrated that the KRT5-targeting antibody of this invention can specifically bind to the KRT5 protein, exhibiting a certain binding affinity for KRT5 and thus higher sensitivity and specificity when used to detect KRT5 protein in samples. Based on the KRT5-targeting monoclonal antibody obtained through screening in this invention, detection reagents and kits for detecting KRT5 protein in samples can be prepared and applied to the monitoring of KRT5-related cancers and disease progression.

[0124] In this invention, gene knockout (KO) experiments are used as the "gold standard" for evaluating the specificity of monoclonal antibodies targeting KRT5. This involves specifically downregulating KRT5 protein expression at the cellular level and observing whether the antibody recognition signal disappears synchronously, allowing direct verification of the antibody-target protein correspondence against an intact cell background. This strategy efficiently eliminates cross-reactive clones, retains highly specific antibodies that truly recognize KRT5 protein, and significantly improves antibody reliability across various detection platforms.

[0125] The main advantages of this invention include: (a) The antibody targeting KRT5 of the present invention has extremely high specificity. Gene knockout (KO) verification is used as the core screening criterion for monoclonal antibodies to completely eliminate inferior clones that cross-react with homologous proteins or unknown antigens, thus overcoming the long-standing technical defect of "non-specific binding" in the field of antibodies.

[0126] (b) The KRT5-targeting antibody of the present invention has applicability to multiple scenarios, is suitable for validation of mainstream detection methods such as WB and ICC, has wide applicability, small batch-to-batch differences, and can be used as an important tool for basic research.

[0127] (c) The antibody sequence targeting KRT5 of the present invention is well-defined and stable, providing a definite material basis for subsequent recombinant expression, humanization and antibody-drug conjugate (ADC) development.

[0128] (d) In this invention, the recombinant expression technology of the eukaryotic expression system is used to produce the antibody targeting KRT5 of this invention, which is particularly suitable for large-scale industrial production and has great industrialization potential.

[0129] (e) Due to the high homology interference of the KRT5 target, no antibody with high specificity and high sensitivity targeting KRT5 has been developed in the prior art. However, the present invention is the first to develop an antibody targeting KRT5, which has extremely high KRT5 protein precise recognition and specific binding ability.

[0130] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0131] Example 1: Expression and purification of recombinant KRT5 protein 1) Experimental methods (1) Construction of eukaryotic plasmid expression vector The amino acid sequence of KRT5 (Uniport ID: P13647) from the N-terminus to the C-terminus, specifically threonine 165-leucine 473, was truncated for expression. A KOZAK expression enhancement sequence, a 6×His Tag, and a thrombin restriction site were added to the N-terminus of this sequence. After humanization codon optimization by Anhui General Company, the resulting compound was cloned into a eukaryotic expression vector (ThermoFisher, pcDNA3.1) for subsequent transfection and expression experiments (the amino acid sequence of the KRT5 recombinant protein expression vector is shown in SEQ ID NO:9).

[0132] Among them, SEQ ID NO.9: MGSSHHHHHHSSGLVPRGSHMTEEREQIKTLNNKFASFIDKVRFLEQQNKVLDTKWTLLQEQGTKTVRQNLEPLFEQYINNLRRQLDSIVGERGRLDSELRNMQDLVEDFKNKYEDEINKRTTAENEFVMLKKDVDAAYMNKVELEAKVDALMDEINFMKMFF DAELSQMQTHVSDTSVVLSMDNNRNLDLDSIIAEVKAQYEEIANRSRTEAESWYQTKYEELQQTAGRHGDDLRNTKHEISEMNRMIQRLRAEIDNVKKQCANLQNAIADAEQRGELALKDARNKLAELEEALQKAKQDMARLLREYQELMNTKLALDVEIATYRKLL; (2) Transfection of KRT5 plasmid After thawing HEK 293F cells from the liquid nitrogen tank, they were continuously passaged until the cell mass reached the specified density and the viability exceeded 90%. Then, the prepared HEK 293F cells were used for subsequent transfection experiments. The cells were transferred to a new 125mL shake flask, and 30mL of HEK 293F cells were added, resulting in a density of 0.5 × 10⁻⁶ cells / mL. 6 Cells / mL. Following the Lipo transfection reagent instructions, the eukaryotic expression vector was mixed with lipo2000 at a ratio of 1:3 and added to the prepared HEK293F cells, which were then cultured in a shaker at 37°C and 5% CO2. When the culture reached 10% of the feed medium (e.g., PolyTool™ 293 F), 0.5% enhancer was added, followed by 1% 293F cells. Cell viability was monitored during culture, and when viability approached 70%, cells were removed by centrifugation, and the supernatant was used for target protein purification.

[0133] (3) Purification of KRT5 recombinant protein The collected supernatant was filtered through a 0.45 μm filter membrane to remove residual cell debris. The pH was adjusted to 7.5-8.0, imidazole was added to a final concentration of 10 mM, and NaCl was adjusted to 150 mM.

[0134] Pour the nickel packing material into a gravity column, allow it to settle naturally, then wash with 5 column volumes of deionized water, followed by equilibration with 5 column volumes of binding buffer. Maintain a flow rate of 1-2 mL / min (natural flow rate for the gravity column is acceptable). Add the processed sample to the chromatography column at a flow rate of 1.0 mL / min and collect the flow-through. Wash with 10 column volumes of binding buffer to remove unbound proteins, then wash with 10 column volumes of wash buffer to remove non-specific bound proteins. Monitor the OD of the effluent during the washing process. 280 Continue eluting until the column reaches baseline. Elute in one step with 10 volumes of elution buffer (250-500 mM imidazole), collecting 1 mL of eluent per tube and labeling each tube. Wash away any remaining impurities in the nickel column with 5 volumes of deionized water, 5 volumes of 0.1 M EDTA, and 5 column volumes of deionized water. Regenerate the nickel column with 5 column volumes of 0.1 M NiSO4, 5 column volumes of deionized water, and 5 column volumes of binding buffer. Equilibrate the column with 20% ethanol and store at 4°C.

[0135] The purified protein was concentrated by ultrafiltration, then the N-segment tag was removed with thrombin, and the 6×His tag after enzyme digestion and any remaining protein with the tag removed were removed using a nickel column to obtain the recombinant antigen (sequence as shown in SEQ ID NO:18). The purified protein was concentrated by ultrafiltration, then filtered through a 0.45 μm filter membrane for sterilization, and the protein concentration was measured using nanodroplets before being stored at -20°C.

[0136] (4) KRT5 recombinant protein detection Take an appropriate amount of sample (2 μg) and mix it with 2× reducing loading buffer in the specified ratio. Heat at 100°C for 5 minutes, then briefly centrifuge before loading the sample. After electrophoresis, place the gel in Coomassie Brilliant Blue staining solution and stain on a shaker at room temperature for 2 hours. Then transfer it to destaining solution (40% methanol, 10% acetic acid) and destain overnight or change the destaining solution until the background is transparent. Take a picture in white light mode, save the image, and label the position of the molecular weight standard and sample information. Estimate the molecular weight of the target protein based on the molecular weight standard.

[0137] Example 2: Preparation and Identification of Mouse Hybridoma Antibodies (1) Immunity The KRT5 recombinant protein prepared in Example 1 was mixed with complete Freund's adjuvant (1:1) and emulsified. Three BALB / c mice were immunized subcutaneously. Two weeks later, the antigen containing the above recombinant protein (SEQ ID NO.18) was emulsified with incomplete Freund's adjuvant (1:1) for a second and third immunization. After four immunizations, blood was collected and serum titers were determined by serial dilution using ELISA.

[0138] The results are shown in Table 1. Further screening was conducted on mouse #3, which had the highest antibody titer against the SEQ ID NO.18 antigen, as a candidate mouse for the next step of cell fusion.

[0139] SEQ ID NO.18: GSHMTEEREQIKTLNNKFASFIDKVRFLEQQNKVLDTKWTLLQEQGTKTVRQNLEPLFEQYINNLRRQLDSIVGERGRLDSELRNMQDLVEDFKNKYEDEINKRTTAENEFVMLKKDVDAAYMNKVELEAKVDALMDEINFMKMFFDAELSQMQTHV SDTSVVLSMDNNRNLDLDSIIAEVKAQYEEIANRSRTEAESWYQTKYEELQQTAGRHGDDLRNTKHEISEMNRMIQRLRAEIDNVKKQCANLQNAIADAEQRGELALKDARNKLAELEEALQKAKQDMARLLREYQELMNTKLALDVEIATYRKLL; Table 1. ELISA titers of serum from quadruple-immunized mice at different dilutions. In the table, the blank control is mouse serum before immunization, and the negative control is PBS solution.

[0140] (2) Cell fusion Prepare sp2 / 0 myeloma cells from mouse #3 in advance, ensuring they are in logarithmic growth phase at the time of fusion. Take spleens from immunized mice and prepare a single-cell suspension of lymphocytes. Mix mouse spleen lymphocytes with the myeloma cells and perform electrofusion according to the program set on a BTX 2001 LITE cell fusion instrument. After fusion, allow the cells to stand for 4 minutes, then dilute them in a 50ml centrifuge tube into 40ml of RPMI 1640 medium containing HAT (100μmol / L hypoxanthine (H), 100nmol / L aminopterin (A), and 16μmol / L thymidine (T)). Incubate at 37℃ for 1 hour, then aliquot into 10 wells of 96-well plates and incubate at 37℃ in a 5% CO2 incubator. After 6-9 days of fusion, the fused cell status in the 96-well plate was observed. The medium was changed to RPMI 1640 medium containing HT (200 μmol / L hypoxanthine (H) and 16 μmol / L thymidine (T)) and the cells were cultured in a constant temperature incubator at 37°C and 5% CO2.

[0141] (3) Screening and cloning 1) Experimental methods Clonal cells were screened by ELISA using the above antigen (SEQ ID NO.18) 7-10 days after fusion.

[0142] Label the corresponding cell line numbers, and perform limiting dilutions on the positive wells until the entire 96-well plate is positive for ELISA. Select stable monoclonal lines with high positive values ​​to obtain 10 hybridoma cell lines that secrete specific monoclonal antibodies: 5-A1-F2, 9-D3-G10, 10-C2-G1, 18-F5-A4, 20-D1-B12, 21-E9-G3, 30-A12-D8, 33-G5-B6, 36-D12-H5, and 39-C2-A7.

[0143] The antibody ELISA titer and corresponding antibody subtype are shown in Table 2 below.

[0144] Table 2 ELISA values ​​and corresponding antibody subtypes for different monoclonal hybridoma cell lines In the table, the blank control is PBS, and the positive control is the serum of mice after immunization.

[0145] Western blot (WB) experiments were performed to verify the monoclonal antibodies secreted by the above-mentioned monoclonal hybridoma cell lines. ① To identify the specificity of the screening antibody, KRT5 recombinant protein (i.e., KRT5 recombinant antigen, SEQ ID NO:18) was specifically selected. The following steps were performed: gel preparation, sample loading, electrophoresis, and membrane transfer. Before transfer, the PVDF membrane was activated by activating it with methanol for 1 min, washing it twice with pure water, and then washing it three times with TBST. Blocking: The membrane was placed in blocking solution prepared with PBST containing 5% skim milk and shaken at room temperature for 1 h. ② Primary antibody incubation: Dilute the same concentration of anti-KRT5 monoclonal antibodies obtained from different hybridoma cell lines at a ratio of 1:5000 to 5 mL of antibody dilution buffer, place the blocked membrane into the corresponding diluted antibody, and incubate overnight at 4°C with shaking. ③ Remove the membrane and wash it in TBST solution 4 times (10 min × 4). ④ Secondary antibody incubation: Dilute HRP anti-mouse IgG (0.4 mg / mL, 1 μL) with antibody dilution buffer at a ratio of 1:5000, mix well, add to the membrane strip, and incubate at room temperature for 1 hour. ⑤ Remove the membrane strip and wash it in TBST solution 4 times (4×10min). ⑥ Development: Mix developer A and developer B in a 1:1 ratio and develop using a developing system.

[0146] 2) Experimental Results The results are shown in Table 2. Compared with the blank control, all 10 monoclonal hybridoma cell lines secreted monoclonal antibodies; in addition, the results are as follows: Figure 1 As shown, compared with the blank control, the monoclonal antibodies secreted by the above 10 monoclonal hybridoma cell lines all showed significant specificity; after further screening, the monoclonal antibody secreted by the high-performance hybridoma cell line 5-A1-F2 was selected as the verification antibody for the subsequent use of this invention.

[0147] (4) The monoclonal antibody secreted by the monoclonal hybridoma cell line 5-A1-F2 was sequenced: According to the TriZol reagent instructions, total RNA secreting the anti-KRT5 monoclonal antibody of this invention was extracted from hybridoma cells 5-A1-F2. According to the Vazyme first-strand cDNA synthesis kit instructions, the total RNA was reverse transcribed into cDNA. Using specific primers for the heavy chain variable region and the light chain variable region, the nucleotide sequences of the heavy chain variable region and the light chain variable region of the KRT5 monoclonal antibody were amplified. Then, the nucleotide sequences of the heavy chain variable region and the light chain variable region were cloned into the eukaryotic expression vector (Thermo Fisher, pcDNA3.1) in preparation for cell transfection.

[0148] (5) Cell transfection and screening Prepare the 293F cells to be transfected in advance. After centrifugation and replacement with fresh culture medium, transfer the cells into 6-well plates, 2 ml per well, at a density of 0.5 × 10⁻⁶ cells / well. 6 Cells / ml. The eukaryotic expression vector was mixed with lipo2000 at a ratio of 1:3 and added to the prepared 293F cells, which were then cultured in a shaker at 37°C and 5% CO2. After 7 days of culture, the transfected cell supernatant was subjected to ELISA to screen positive wells against the corresponding antigen. The cell supernatant from the positive wells was then subjected to immunocytochemistry. If the immunocytochemistry test was positive, the detected antibody sequence was confirmed to be correct.

[0149] (6) Preparation and purification of monoclonal antibodies on cells The confirmed positive expression vector was used to transfect a large number of cells. After culturing for 3-5 days, the cell suspension was collected, centrifuged, and the supernatant was purified using affinity chromatography. The purified monoclonal antibody concentration was determined, aliquoted, and stored at 4°C. Finally, the heavy chain variable region of the anti-KRT5 monoclonal antibody of the present invention is encoded by the DNA sequence shown in SEQ ID NO.10, and the light chain variable region of the anti-KRT5 monoclonal antibody of the present invention is encoded by the DNA sequence shown in SEQ ID NO.11.

[0150] SEQ ID NO.10: GAGGTCCAGTTGCAGCAGTCTGGACCTGAGCTAGTGAAGACTGGGGCTTCAGTGAAGATATCCTGTAAGGCTTCTGGTGACTCATTCATGGGTTACTACATACACTGGGTCAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGATATATTGGTTGTTACAATGGTGCTACTAGCTACAAC CAGAAGTTCAAGGGCAAGGCCACATTTACTGTAGACATATCCTCCAGCACAGCCTACATGCAGTTCAACAGCCTGACATCTGAAGACTCTGCGGTCTATTACTGTGCAGGAGAGAGGGAGATGATTACGACGAACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA; SEQ ID NO.11: GACATTTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAATCCAGTCAGGGTCTGCTCAACAGTAGAACCCGAAAGAACTACTTAGCTTGGTACCAACAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGG GCAGCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTTACTGCAAGGAATCTTCTAATCTGTACACGTTCGGAGGGGGGACCAAGCTGGAAAATAAAA.

[0151] The obtained base sequence was translated into amino acid sequence analysis, and the amino acid sequence of the heavy chain variable region of the anti-KRT5 monoclonal antibody of the present invention was obtained as shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region of the KRT5 monoclonal antibody of the present invention was shown in SEQ ID NO.2.

[0152] SEQ ID NO.1: EVQLQQSGPELVKTGASVKISCKASGDSFMGYYIHWVKQSHGKSLEWIGYIGCYNGATSYNQKFKGKATFTVDISSSTAYMQFNSLTSEDSAVYYCAGEREMITTNYAMDYWGQGTSVTVSS; SEQ ID NO.2: DIVMSQSPSSLAVSAGEKVTMSCKSSQGLLNSRTRKNYLAWYQQKPGQSPKLLIYWAATRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKESSNLYTFGGGTKLEIK.

[0153] Example 3: Affinity and kinetic tests of the anti-KRT5 monoclonal antibody of the present invention 1) Experimental methods (1) Preparation before the experiment Before the experiment, prepare 1×PBST buffer and ultrapure water. Place the buffer and deionized water in the right tray of the system and connect the corresponding inlet tubes. Replace the chip using the control software, pushing the Protein A chip into the slot in the direction of the arrow and securing it. The system will automatically enter standby mode. After replacing the solution, flush the flow path at a high flow rate for approximately 6 minutes.

[0154] (2) Sample testing process: Ligand (i.e. KRT5 recombinant antigen) concentration: Dilute the antibody to 0.5 μg / mL or 1 μg / mL with 1 × PBST buffer, 400 μl.

[0155] Analyte (i.e., the anti-KRT5 monoclonal antibody of the present invention) concentration: The antibody was diluted with 1 × PBST buffer to 0, 1.56, 3.125, 6.25, 12.5, 25, 50, and 100 nM.

[0156] (3) Parallel method for detecting affinity and kinetics The standard method was used for detection and analysis using Biacore 8K Control Software. The method is as follows: In the "Method Definition" interface, the sample chamber temperature was selected as 25℃, and the concentration unit was selected accordingly; other settings remained unchanged. In the "Analysis" window below, under the "Analyte" tab, the analysis temperature was set to the default 25℃, the incubation time to 120s, the dissociation time to 180s (for slow-dissociating samples, this can be set to 300s or longer), and the flow rate to 30 μl / min; under the "Capture" tab, the incubation time was set to 60s, and the flow rate to 10 μl / min. Eight cycles were set, running sequentially from low to high concentration, including zero concentration and repeat concentration.

[0157] (4) Data Analysis After data acquisition, analysis was performed using Biacore Insight Evaluation Software. The appropriate multi-cycle kinetic capture and evaluation method was selected, and the software automatically fitted the data. It was necessary to check if the binding signal was below 20% of the reference signal; if non-specific binding was present, the sample concentration could be reduced.

[0158] 2) Experimental Results Based on the above test analysis, the KD(1 / Ms) value of the KRT5-targeting monoclonal antibody of the present invention is 1.1 × 10⁻⁶. -7 Other test parameters are shown in Table 3, and the dynamic fitting curves are shown in Table 3. Figure 2 As shown.

[0159] Table 3. Affinity and kinetics results of the KRT5-targeting monoclonal antibody of the present invention. Example 4: Verification of the ability of the monoclonal antibody targeting KRT5 of the present invention to recognize the KRT5 protein using multiple cell lines. 1) Experimental methods In this embodiment, the anti-KRT5 monoclonal antibody of the present invention was used as the primary antibody. Western blotting was used to detect the KRT5 protein expression levels in six common cell lines to verify its ability to recognize the KRT5 protein. The method is as follows: (1) To identify the specificity of the screening antibodies, six cell types with different KRT5 expression levels, namely HeLa cells, HaCaT cells, MCF7 cells, DU145 cells, H9c2 cells, and C2C12 cells, were specifically selected for culture, protein was extracted, lysate was prepared, and gel preparation, spotting, electrophoresis, and membrane transfer were performed in sequence. Before membrane transfer, the PVDF membrane was activated with methanol for 1 min, washed twice with pure water, and then washed three times with TBST. Blocking: The membrane was placed in blocking solution prepared with PBST containing 5% skim milk and shaken at room temperature for 1 h. (2) Primary antibody incubation: The monoclonal antibody against KRT5 of the present invention (1 mg / mL, 1 μL) was diluted 1:5000 into 5 ml of antibody dilution solution. The blocked membrane was placed into the corresponding diluted antibody and incubated overnight at 4°C with shaking. (3) Take out the membrane and wash it in TBST solution 4 times (10min×4). (4) Secondary antibody incubation: HRP anti-mouse IgG (0.4 mg / mL, 1 μL) was diluted with antibody diluent at a ratio of 1:5000, mixed well and added to the membrane strip, and shaken at room temperature for 1 h; (5) Remove the membrane strip and wash it in TBST solution 4 times (4×10min). (6) Development: Mix color developer A and B in a 1:1 ratio and develop using a developing system.

[0160] 2) Experimental Results The results are as follows Figure 3 As shown, where, Lane 1: HeLa represents lysate from human cervical cancer cells; band size: approximately 53 kDa; Lane 2: HaCaT represents human immortalized keratinocyte lysate; band size: approximately 53 kDa; Lane 3: MCF7 represents lysate from human squamous cell carcinoma cells; band size: approximately 53 kDa; Lane 4: DU145 represents lysate from human prostate cancer cells, band size: approximately 53 kDa; Lane 5: H9c2 represents rat cardiomyocyte lysate, band size: approximately 53 kDa; Lane 6: C2C12 represents mouse myoblast lysate, band size: approximately 53 kDa; The above results indicate that the anti-KRT5 monoclonal antibody of the present invention can specifically recognize the endogenous KRT5 protein in the cell lysate, with a molecular weight of about 53 kDa. This shows that the anti-KRT5 monoclonal antibody of the present invention has good specificity in immunoblotting experiments, high specificity for binding to KRT5 protein, and no non-specific binding.

[0161] Example 5: Detection of the specificity of the anti-KRT5 monoclonal antibody of the present invention based on the KO assay. 1) Experimental methods In this embodiment, the anti-KRT5 monoclonal antibody of the present invention was used as the primary antibody. Western blotting was used to detect the expression level of KRT5 protein in wild-type C2C12 (mouse myoblast) cell lines or KRT5 gene knockout (KO stable transfection) C2C12 (mouse myoblast) cell lines, verifying its specific binding ability to endogenous KRT5 protein. The method is as follows: (1) Wild-type C2C12 (mouse myoblast cell line) cell line and KRT5 gene knockout C2C12 cell lysate (20 μg) were prepared and subjected to gel preparation, spotting, electrophoresis and membrane transfer in sequence. The PVDF membrane needs to be activated before the transfer. The membrane is activated with methanol for 1 min, washed twice with pure water and then washed three times with TBST. Blocking: The membrane is placed in the blocking solution prepared with PBST containing 5% skim milk and shaken at room temperature for 1 h. (2) Primary antibody incubation: Dilute the anti-KRT5 monoclonal antibody of the present invention at a ratio of 1:5000 to 5 ml of antibody dilution solution, place the blocked membrane into the corresponding diluted antibody, and incubate overnight at 4°C with shaking. (3) Take out the membrane and wash it in TBST solution 4 times (10min×4). (4) Secondary antibody incubation: HRP anti-mouse IgG (0.4 mg / mL, 1 μL) was diluted with antibody diluent at a ratio of 1:5000, mixed well and added to the membrane strip, and shaken at room temperature for 1 h; (5) Remove the membrane strip and wash it in TBST solution 4 times (4×10min). (6) Development: Mix color developer A and B in a 1:1 ratio and develop using a developing system.

[0162] Among them, the WT group represents the wild-type C2C12 (mouse myoblast cell line) cell line; the KO group represents the C2C12 (mouse myoblast cell line) cell line with KRT5 gene knockout.

[0163] 2) Experimental Results The results are as follows Figure 4 As shown, in lanes 1-2, the internal reference protein Hsp90α (molecular weight of approximately 84.7 kDa) was expressed in both wild-type C2C12 cells and the KRT5 gene knockout C2C12 (mouse myoblast cell line).

[0164] In lanes 3-4, the expression of KRT5 (molecular weight 53 kDa) in wild-type C2C12 cells and KRT5 knockout C2C12 (mouse myoblast cell line) was detected. The results showed that the anti-KRT5 monoclonal antibody of the present invention had a signal in lane 3 and no signal in lane 4. Since the KRT5 knockout cell line did not express KRT5, and no non-specific bands were generated after incubation with the anti-KRT5 monoclonal antibody of the present invention, the anti-KRT5 monoclonal antibody of the present invention could recognize and bind to the KRT5 protein with high specificity in the immunoblotting experiment.

[0165] Example 6: Flow cytometry verification of the specificity of the anti-KRT5 monoclonal antibody of the present invention 1) Experimental methods (1) Cell preparation Take HaCaT cells in the logarithmic growth phase, discard the old culture medium, and gently wash the cells twice with PBS. Add an appropriate amount of trypsin to digest the cells. After the cells have completely detached, add complete culture medium to stop the digestion, and transfer the cell suspension to a 15 mL centrifuge tube.

[0166] Centrifuge at 4℃ and 1000 rpm for 5 min, discard the supernatant, resuspend the cell pellet in pre-cooled PBS, and repeat the centrifugation and washing once. Resuspend the cells in flow cytometry staining buffer and adjust the cell concentration to 1×10⁻⁶. 6 100 μL of cells / 100 μL were dispensed into flow cytometry tubes, 100 μL per tube, and divided into experimental group and isotype control group, with 3 biological replicates in each group.

[0167] (2) Cell fixation and permeability (KRT5 is a nucleoprotein and requires permeability treatment) Add 1 mL of pre-chilled 4% paraformaldehyde to each tube of cells, vortex to mix, and fix at 4°C in the dark for 15 min. Centrifuge at 1000 rpm for 5 min at 4°C, discard the supernatant, add 1 mL of pre-chilled PBS to resuspend the cells, and wash once by centrifugation to remove residual fixative. Add 1 mL of pre-chilled 0.1% Triton X-100 permeabilization buffer to the cell pellet, vortex to mix, and permeabilize at 4°C in the dark for 10 min to allow the antibody to enter the cell nucleus and bind endogenous KRT5 protein. Centrifuge, discard the supernatant, and wash twice with pre-chilled PBS to completely remove the permeabilization buffer.

[0168] (3) Closed and primary antibody incubation Add 100 μL of 1% BSA-PBS blocking solution to each tube of cells, vortex to mix, and block at 4°C for 30 min to block non-specific binding sites. Centrifuge and discard the supernatant. ① Experimental group: Add 100 μL of the anti-KRT5 monoclonal antibody of the present invention diluted 1:2000 (diluted with 1% BSA-PBS), and vortex to mix; ② Isotype control group: Add 100 μL of homologous isotype control IgG diluted at the same concentration, and vortex to mix. Incubate at 4°C in the dark for 1 hour (or overnight at 4°C).

[0169] After each group has finished incubating, add 1 mL of pre-cooled flow cytometry staining buffer to each tube, centrifuge at 4°C and 1000 rpm for 5 min, discard the supernatant, and repeat the washing process 3 times to completely remove unbound primary antibody and reduce background fluorescence.

[0170] (4) Incubation with fluorescent secondary antibody Add 100 μL of Alexa Fluor® 647-labeled secondary antibody (diluted with 1% BSA-PBS) diluted according to the manufacturer's instructions to each cell pellet and vortex to mix. Incubate at 4°C in the dark for 30 min, strictly avoiding light throughout the process to prevent fluorescence quenching. After incubation, wash three times with pre-cooled flow cytometry staining buffer, centrifuging at 4°C and 1000 rpm for 5 min each time to completely remove unbound secondary antibody.

[0171] (5) On-machine testing and data analysis Add 300-500 μL of pre-chilled flow cytometry staining buffer to each tube of cells, resuspend the cells, vortex to mix, and analyze within 2 hours. Flow cytometer settings: Forward scatter (FSC) / Side scatter (SSC) gate, select intact viable cell populations, and exclude cell debris and clusters. Select the corresponding detection channel for the Alexa Fluor® 647 (usually the APC channel), adjust the voltage with an isotype control group, and set the negative gate. Collect ≥10,000 viable cells per sample and record fluorescence intensity data.

[0172] Data analysis: FlowJo software was used to analyze the data and plot fluorescence intensity histograms. The fluorescence signal shift between the KRT5-stained group (red) and the isotype control group (green) was calculated, along with the proportion of KRT5-positive cells and the mean fluorescence intensity (MFI). The green curve represents the isotype control, and the red curve represents the KRT5-stained group.

[0173] 2) Experimental Results The results are as follows Figure 5As shown, compared with the isotype control group (green), the fluorescence intensity of the KRT5 staining group (red) shifted significantly to the right, while the fluorescence signal of the isotype control group (green) was concentrated in the negative region, and the fluorescence peaks of the two groups did not overlap significantly. This indicates that the anti-KRT5 monoclonal antibody of the present invention can be applied to flow cytometry detection, has high specificity for KRT5 protein binding, and has almost no non-specific binding.

[0174] Example 7: Verification of the specificity of the anti-KRT5 monoclonal antibody of the present invention based on immunocytochemistry In this embodiment, the anti-KRT5 monoclonal antibody of the present invention (dilution ratio: 1:1000) was used to perform immunocytochemical staining on HaCaT cells.

[0175] 1) Experimental methods (1) Cell sample preparation Remove the HaCaT cell-inoculated spread / confocal culture dish from the CO2 incubator, disinfect the surface of the dish by wiping it with an alcohol swab, and place it in a laminar flow hood. Aspirate the old culture medium from the dish, and gently rinse the cell surface twice with PBS buffer. After each rinse, gently absorb excess liquid with absorbent paper (avoid vigorous rinsing to prevent cell detachment).

[0176] (2) Cell fixation Add an appropriate amount of 4% paraformaldehyde fixative to the culture dish, completely covering the cell surface, and incubate at room temperature for 15-20 minutes (do not fix for too long to avoid damaging the antigen epitopes). After fixation, aspirate the fixative, rinse the cells three times with PBS buffer for 5 minutes each time, and shake gently on a shaker to ensure complete removal of any residual fixative.

[0177] (3) Cell permeability Add an appropriate amount of 0.1% Triton X-100 permeabilization buffer to the culture dish, completely covering the cells, and incubate at room temperature for 10 minutes for permeabilization. After permeabilization, aspirate the permeabilization buffer, wash the cells three times with PBS buffer for 5 minutes each time, and shake gently on a shaker to remove any residual permeabilization buffer.

[0178] (4) Sealing treatment Discard the PBS buffer, add an appropriate amount of 5% BSA blocking solution to the culture dish, completely covering the cell surface, and incubate at room temperature for 30 minutes. After incubation, no rinsing is required; simply discard the blocking solution.

[0179] (5) Primary antibody incubation (specific binding of antigen) The experimental group and the blank control group were set up separately, and the groups were as follows: ① Experimental group: Slowly add 5 mL of the pre-diluted anti-KRT5 monoclonal antibody of this invention (dilution ratio: 1:1000) to the cell surface, ensuring complete coverage of the cells. Incubate overnight at 4°C in the dark (or at room temperature for 2 hours, or at 4°C overnight). During incubation, ensure uniform antibody coverage to avoid air bubbles or antibody loss. ② Blank control: Only blocking solution (5 mL) is added, without the above (5 mL) anti-KRT5 antibody (1:1000) of the present invention, to exclude non-specific binding of the secondary antibody.

[0180] After the two groups were incubated, the primary antibody was aspirated and the cells were washed three times with PBS buffer for 5 minutes each time, with the cells shaken at low speed to completely remove unbound primary antibody and avoid background fluorescence interference.

[0181] (6) Secondary antibody incubation (fluorescent labeling) Slowly add pre-diluted Alexa Fluor 647-labeled secondary antibody to the cell surface, completely covering the cells, and incubate at room temperature in the dark for 1 hour. During incubation, gently shake the cell on a shaker to ensure uniform binding of the secondary antibody; avoid excessively high concentrations of the secondary antibody, which can lead to nonspecific fluorescence enhancement. After incubation, discard the secondary antibody and wash the cells three times with PBS buffer for 5 minutes each time, shaking gently on a shaker to remove unbound secondary antibody and reduce background fluorescence.

[0182] (7) Nuclear staining (DAPI staining) Discard the PBS buffer, add an appropriate amount of diluted DAPI staining solution to the culture dish, completely covering the cells, and stain at room temperature in the dark for 5-10 minutes (the staining time should not be too long to avoid excessive nuclear fluorescence masking the target protein signal). After staining, discard the DAPI staining solution, rinse the cells twice with PBS buffer, 5 minutes each time, and shake gently on a shaker to remove residual DAPI staining solution and avoid fluorescence interference.

[0183] (8) Mounting and microscopic observation Gently blot away excess liquid from the surface of the slide with absorbent paper. Add 1-2 drops of anti-fluorescence quencher to the center of the slide, and gently cover the slide with the cell side down on the anti-fluorescence quencher, avoiding air bubbles. If using a confocal culture dish, the anti-fluorescence quencher can be added directly without mounting. Place the mounted slide / confocal culture dish in a light-proof box and immediately observe with a Leicastellaris 5 laser confocal microscope. Adjust the laser intensity and smart gain parameters to acquire fluorescence images.

[0184] Single-channel and merged images of DAPI (blue fluorescence, excitation wavelength 405nm) and Alexa Fluor 647 (magenta fluorescence, excitation wavelength 647nm) were acquired respectively.

[0185] 2) Experimental Results The results showed that the cell nucleus stained blue with DAPI ( Figure 6 (Left image); KRT5 stained with Alexa Fluor 647 turns magenta ( Figure 6 (The middle image); after co-staining with DAPI and Alexa Fluor 647, it exhibits a reddish-blue color. Figure 6 (See the right image). None of the three staining results showed nonspecific staining.

[0186] The above results demonstrate that the anti-KRT5 monoclonal antibodies of this invention exhibit high specificity in immunocytochemical experiments. Figure 6 ).

[0187] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0188] The sequence information of this invention is shown in Table A.

[0189] Table A

Claims

1. An antibody or its antigen-binding fragment targeting keratin 5, characterized in that, The antibody or its antigen-binding fragment includes: (1) The following three complementary determinant regions (CDRs) for the heavy chain variable region and the light chain variable region, as defined by the IMGT rule: HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4 HCDR3 shown in SEQ ID NO: 5 The LCDR1 shown in SEQ ID NO: 6, The LCDR2 shown in SEQ ID NO: 7, LCDR3 as shown in SEQ ID NO: 8; or (2) According to the Kabat rule, the following three complementary determinant regions (CDRs) for the variable region of the heavy chain and the three complementary determinant regions (CDRs) for the variable region of the light chain are defined as follows: HCDR1, as shown in SEQ ID NO:12, HCDR2 shown in SEQ ID NO: 13 HCDR3 shown in SEQ ID NO: 14 LCDR1 shown in SEQ ID NO: 15, LCDR2 shown in SEQ ID NO:16 LCDR3 shown in SEQ ID NO:

17.

2. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

2.

3. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The heavy chain of the antibody or its antigen-binding fragment further includes a heavy chain constant region; and the light chain of the antibody or its antigen-binding fragment further includes a light chain constant region.

4. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The antibody is a monoclonal antibody.

5. A polynucleotide, characterized in that, The polynucleotide encodes the antibody or antigen-binding fragment thereof that targets keratin 5 as described in claim 1.

6. An expression carrier, characterized in that, The expression vector contains the polynucleotide as described in claim 5.

7. A host cell, characterized in that, The host cell contains the expression vector as described in claim 6, or the genome is integrated with the polynucleotide as described in claim 5.

8. An antibody conjugate, characterized in that, The antibody conjugate comprises: (a) The antibody targeting keratin 5 as described in claim 1, or its antigen-binding fragment; and (b) The coupling portion, which is a detectable marker.

9. The use of an active ingredient, characterized in that, The active ingredient is selected from the group consisting of: the antibody targeting keratin 5 as described in claim 1 or its antigen-binding fragment, or the antibody conjugate as described in claim 8, or a combination thereof, wherein the active ingredient is used to prepare: a reagent or kit for in vitro detection of keratin 5.

10. A kit for in vitro detection of keratin 5, characterized in that, The kit contains an antibody targeting keratin 5 as described in claim 1 or an antigen-binding fragment thereof, or an antibody conjugate as described in claim 8.

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