Antibodies against human keratin 8, antibody conjugates and uses thereof

Abstract

The application belongs to the technical field of antibody preparation, and particularly relates to an antibody against human keratin 8, an antibody conjugate and application thereof.The amino acid sequences of CDR1-3 on the light chain variable region of the antibody are respectively shown as SEQ ID NO.3-5, and the amino acid sequences of CDR1-3 on the heavy chain variable region are respectively shown as SEQ ID NO.8-10.The monoclonal antibody provided by the application has high specificity, good binding affinity and high recognition sensitivity to CK8 protein in the cytoplasm, and has no cross reaction and non-specific binding to non-target proteins in the cell, effectively ensuring that the detection signal comes from the target protein, being conducive to reducing background noise, improving the signal-to-noise ratio, improving the accuracy and reliability of the detection result, and having high consistency between the detection result and the actual situation, and being suitable for the construction of immunological biopsy technology of clinical pathological cells or tissues.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of antibody preparation, in particular to an antibody against human keratin 8, an antibody conjugate and application thereof. BACKGROUND

[0002] Keratin 8, also known as Keratin 8, KRT8, Cytokeratin 8, CK8, Type-II keratin, etc., is an intermediate filament protein belonging to the cytokeratin family. The cytokeratin family contains more than 20 members, which are divided into acidic proteins (class I, including CK9-CK20) and basic proteins (class II, including CK1-CK8) according to the isoelectric point. CK8 belongs to class II cytokeratin, with a molecular weight of about 40-67 kDa, which is composed of a central rod-shaped domain (Intermediate filament protein domain), an N-terminal head domain (Keratin type II head domain) and a C-terminal tail domain (HAUS2 domain). The central rod-shaped domain is highly conserved and is a characteristic shared by members of the intermediate filament protein family, which is responsible for forming heterodimers with other keratins (such as CK18), and the N-terminal and C-terminal domains are relatively variable, which are involved in keratin fiber assembly and interaction with other cellular structures. Under normal circumstances, CK8 forms a heterodimer with the corresponding acidic keratin at a ratio of 1:1 to construct an intermediate fiber network, maintain cell morphology and mechanical stability, and help cells resist external stress (such as stretching, compression and shear force). In addition, CK8 also acts on signal transduction and cell differentiation, interacts with other signal molecules in the cell, participates in intracellular signal transduction, and plays a regulatory role in cell growth, differentiation, apoptosis and other processes.

[0003] Studies have shown that CK8 is widely expressed in the human body, involving various tissues and organs, such as the glandular epithelium of the colon, prostate, and stomach, the renal tubules of the kidney tissue, and the cytoplasm of lung cells and hepatocytes in the lung tissue. These expression locations show the important role of CK8 in various epithelial cell types. The expression pattern of CK8 also makes it an important biomarker for studying various diseases, especially for distinguishing between epithelial-derived tumors and non-epithelial-derived tumors. In the study of lung adenocarcinoma, a group of alveolar intermediate cells (KACs) carrying KRAS oncogene mutations and expressing CK8 were found. These cells can eventually transform into lung adenocarcinoma cells. In the study of coronary heart disease, the gene mutation frequency of CK8 is significantly higher than that of healthy people, and the protein expression level is also relatively high. In eye diseases, CK8 is highly expressed in retinal ganglion cells (RGCs) and its expression increases after acute ocular hypertension (AOH) induction. The lack of CK8 exacerbates RGC damage under AOH, leading to loss of retinal and inner retinal thickness, reduction, apoptosis, and dysfunction of RGCs, and activation of glial cells. At the same time, CK8 is co-expressed with its complementary subunit CK18 in normal glandular epithelium, transitional cell epithelium, and hepatocytes, but not in squamous epithelium, making CK8 / 18 play a large role in tumor diagnosis, prognosis, and treatment. For example, in non-cancerous tissues, CK8 and CK18 are usually expressed in normal glandular epithelium, while in cancerous tissues such as human breast cancer or colorectal cancer, the expression of CK8 / 18 is reduced, which is associated with tumor progression and poor prognosis. On the contrary, in head and neck cancer, oral cancer, and urothelial cancer, the expression of CK8 / 18 is up-regulated and is associated with poor prognosis; in esophageal squamous cell carcinoma, CK8 is identified as an independent prognostic predictor of disease-free survival (DFS) and overall survival (OS) as a single marker, especially in pStage II / III tumor patients. If CK8 expression is combined with pathological TNM staging at this time, it can be used as a valuable guide for adjuvant therapy decision-making for OSCC patients. Postoperative chemotherapy may be beneficial for pStage II / III tumors expressing CK8 and pStage IV tumors regardless of CK8 expression, but not for pStage I tumors and CK8-negative pStage II / III tumors.

[0004] Due to the diagnostic and marker value of CK8, the demand for detecting CK8 protein is steadily increasing. Currently, the target protein in pathological cells / tissues is often detected by immunofluorescence, flow cytometry, etc. in clinical practice, and the core lies in the development of specific antibodies against the target protein. The performance of the antibody directly determines the accuracy and reliability of the detection results. However, there is no CK8 antibody with strong comprehensive performance such as specificity and sensitivity on the market. SUMMARY

[0005] In order to solve the problem of lack of anti-human CK8 antibody with excellent performance for cell / tissue immunological biopsy in the prior art, the application provides an antibody against human keratin 8 and an antibody conjugate thereof, and further provides application of the antibody and the antibody conjugate in preparation of a human keratin 8 immunological detection kit and a related immunological detection kit.

[0006] The application provides an antibody against human keratin 8, which comprises a light chain variable region and a heavy chain variable region, wherein the amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region are respectively shown as SEQ ID NO. 3-5, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are respectively shown as SEQ ID NO. 8-10.

[0007] Further, the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 7.

[0008] Further, the amino acid sequence of the antibody light chain is shown as SEQ ID NO. 1, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 6.

[0009] Further, the antibody is a full-length antibody or an antigen binding region thereof; and the antigen binding region is at least one selected from a Fab fragment, a F(ab)2 fragment, a Fv fragment, a (Fv)2 fragment, a scFv fragment and a sc(Fv)2 fragment.

[0010] The application provides an antibody conjugate, which comprises the antibody against human keratin 8 and a detection label connected to the antibody.

[0011] The application provides a nucleic acid molecule, a recombinant vector comprising the nucleic acid molecule or a host cell comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes the antibody against human keratin 8.

[0012] The application provides application of the antibody against human keratin 8 or the antibody conjugate in preparation of a human keratin 8 immunological detection kit.

[0013] Further, the kit is selected from an immunofluorescence kit or a flow cytometry kit.

[0014] The application provides a human keratin 8 immunological detection kit, which comprises the antibody against human keratin 8 or the antibody conjugate.

[0015] Further, the kit further comprises a fluorescent secondary antibody of the anti-human keratin 8 antibody, which is a fluorescein-coupled anti-rabbit IgG antibody.

[0016] The advantages and positive effects of the present application are:

[0017] The monoclonal antibody strain provided by the present application can specifically target human keratin 8 (CK8 or KRT8), has strong specificity, good binding affinity and high recognition sensitivity to CK8 protein in the cytoplasm, and has no cross reaction and non-specific binding to non-target proteins in the cell, effectively ensuring that the detection signal comes from the target protein, which is conducive to reducing background noise, improving signal-to-noise ratio, improving the accuracy and reliability of the detection result, and the detection result is highly consistent with the actual situation, and is suitable for the construction of immunological biopsy technology of clinical pathological cells or tissues, and has high practical value in the clinical diagnosis and scientific research detection of human CK8 protein expression level. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0019] Figure 1 The figure is the detection result of the immune serum titer of the Japanese big-eared white rabbit immunized by human keratin 8 in the present application embodiment 1;

[0020] Figure 2 The figure is the immunofluorescence imaging of keratin 8 in the positive cell sample detected by the immune serum of the Japanese big-eared white rabbit immunized by human keratin 8 in the present application embodiment 1;

[0021] Figure 3 The figure is the vector map of the expression vector pRB322 for constructing the anti-human keratin 8 antibody in the present application embodiment 1, from left to right, respectively, the vector pre-carries the antibody light chain constant region and the heavy chain constant region;

[0022] Figure 4 The figure is the immunofluorescence imaging of keratin 8 in the positive cell detected by the anti-human keratin 8 antibody in the present application embodiment 2;

[0023] Figure 5 The figure is the flow cytometry imaging of keratin 8 in the positive cell detected by the anti-human keratin 8 antibody in the present application embodiment 2. DETAILED DESCRIPTION

[0024] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the embodiments. The embodiments described herein are only used to explain the present application and should not be used to limit the present application.

[0025] In light of the information included herein, those skilled in the art will readily produce without undue experimentation a variety of modifications of the precise embodiment described herein, all of which are intended to be within the scope of the claims appended hereto. It will be understood that the scope of the present application is not limited to the processes, compositions or components specifically recited, as these are merely illustrative of specific aspects of the present application. Indeed, various modifications of the embodiments of the present application, all falling within the scope of the claims appended hereto, will readily occur to those skilled in the art or in related fields.

[0026] In order to better understand the present application without limiting the scope of the present application, all numbers expressing quantities of ingredients, percentages and other numerical values used in the specification and claims herein are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the present application. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0027] In addition, it should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, unless otherwise defined herein.

[0028] The terms "comprising", "containing", "including", "having" and the like are to be construed open-ended, i.e., to mean including, but not limited to.

[0029] The term "and / or" should be construed to mean either of the two specified features or components, or both, in either order.

[0030] The terms "rabbit monoclonal antibody", "monoclonal antibody", "rabbit-derived antibody" and "mAb" and the like have the same meaning and, unless otherwise specified, refer to an antibody that specifically binds to human (Human) Keratin 8. The terms "CK8", "KRT8", "Keratin 8", "Cytokeratin 8", "Keratin 8" and the like have the same meaning. The modifier "rabbit" indicates that the complementarity determining regions (CDRs) of the antibody are derived from rabbit-derived immunoglobulin sequences.

[0031] An antibody is a molecule of immunoglobulin that is capable of specific binding to a target antigen or epitope through at least one antigen recognition site located in a variable region of the immunoglobulin molecule. In the present application, the term "antibody" is to be interpreted in the broadest sense and includes different antibody structures, including but not limited to so-called full-length antibodies, antibody fragments, and their genetic or chemical modifications, as long as they exhibit the desired antigen binding activity. An antibody fragment can be one or more portions or fragments of a full-length antibody that retain the ability of the antibody to specifically bind to a target antigen.

[0032] A typical antibody molecule (full-length antibody) is composed of two identical light (L) chains and two identical heavy (H) chains. Light chains can be classified into two types, kappa and lambda; heavy chains can be classified into five types, mu, delta, gamma, alpha, and epsilon, and define the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. The amino acid sequences of the heavy and light chains near the N-terminus vary greatly, while the amino acid sequences of the other parts are relatively constant. The region of the light and heavy chains near the N-terminal amino acid sequence that varies greatly is called the variable region (V), and the region near the C-terminal amino acid sequence that is relatively stable is called the constant region (C). The heavy chain variable region (VH) and the light chain variable region (VL) are usually the most variable parts of the antibody and contain the antigen recognition site. The VH and VL regions can be further subdivided into hypervariable regions (HVRs) and framework regions (FRs), which are also called complementarity determining regions (CDRs). The CDRs are looped structures, and the heavy chain CDRs and the light chain CDRs are closely held together and cooperate with each other through the FR regions to form a surface that is complementary to the three-dimensional structure of the target antigen or epitope, which determines the specificity of the antibody and is the site of recognition and binding of the antibody to the antigen. The FR regions are relatively conserved parts of the VH and VL, which are roughly in a beta-sheet conformation, connected by three CDRs forming connecting loops. Each VH and VL is usually composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0033] CDRs and FRs can be identified according to the Kabat definition, the Chothia definition, the accumulation of both the Kabat definition and the Chothia definition, the AbM definition, the contact definition, the IMGT unique numbering definition, and / or the conformational definition, or any CDR determination method well known in the art. As used in the present application, the numbering is defined by the Kabat numbering system.

[0034] The light chain constant region (CL) and the heavy chain constant region (CH) are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, for example, they are involved in antibody-dependent cellular cytotoxicity. The CL of different types of Ig (kappa or lambda) are essentially uniform in length, but the CH of different classes of Ig are different in length, such as IgG, IgA and IgD include CH1, CH2 and CH3, while IgM and IgE include CH1, CH2, CH3 and CH4. The amino acid sequences of the antibody heavy chain and light chain constant regions are well known in the art and can be obtained by querying the IMGT database.

[0035] The full-length antibody is the most complete antibody molecular structure, with a typical Y-shaped molecular structure, therefore, in the context of the present application, "full-length antibody", "complete antibody" and "Y-shaped antibody" have the same meaning and can be used interchangeably.

[0036] The antibody fragment is one or more parts or fragments of the full-length antibody, which essentially maintains the same biological function or activity as the full-length form, specifically, the antibody fragment at least includes the same CDR region as the full-length antibody, more preferably has the same variable region, thereby retaining the complete antigen recognition and binding site, which can bind to the same antigen as the full-length antibody, especially to the same epitope. In typical examples, the antibody fragment includes Fab, F(ab)2, Fab', F(ab')2, Fv, (Fv)2, scFv, sc(Fv)2, which can be obtained by conventional techniques in the art.

[0037] (i) Fab: Antigen-binding fragment (Antigen-binding fragment, Fab) is a monovalent fragment composed of a complete light chain (variable region and constant region) and a partial heavy chain (variable region and first constant region), which can be obtained by protease digestion of full-length antibodies. For example, under the action of papain, IgG can be degraded into two Fab fragments and one Fc fragment; under the action of pepsin, IgG can be degraded into one F(ab')2 fragment and one pFc' fragment. F(ab')2 fragment is further reduced to form two Fab' fragments. Since Fab has an antigen binding region and a partial constant region, it not only has the same antibody-antigen affinity as scFv, excellent tissue penetration, etc., but also has a more stable structure.

[0038] (ii) F(ab)2: A bivalent fragment composed of two Fabs connected by a hinge region disulfide bridge.

[0039] (iii) Fv: The variable fragment (Fv) is located at the N-terminus of the Fab fragment of the antibody, only contains the variable region, and is composed of the variable region of a light chain and a heavy chain, which is a dimer of a VH and a VL (VH-VL dimer) non-covalently bound, 3 CDRs of each variable region interact with each other to form an antigen binding site on the surface of the VH-VL dimer, which has the ability to recognize and bind to antigens, although the affinity is lower than that of the complete antibody.

[0040] (iv) (Fv)2: composed of two covalently linked Fv fragments.

[0041] (v) scFv: Single-chain variable fragment (scFv) is an Fv fragment composed of a single polypeptide chain, which is composed of a heavy chain variable region (VH) and a light chain variable region (VL) connected by a flexible linker (linker, generally composed of 10-25 amino acids), which retains the original antibody's binding specificity to antigens. The linker in the present application can be any linker that does not hinder the expression of the antibody variable regions connected at its two ends, and is not particularly limited. Compared with full-length antibodies, scFv has the characteristics of small molecular weight, so it has higher penetration and lower immune side reactions.

[0042] (vi) sc(Fv)2 fragment, which is composed of two heavy chain variable regions and two light chain variable regions connected by a linker.

[0043] In some embodiments, the full-length sequence of the antibody or antibody fragment of the present application can contain CDR regions and FR regions from rabbit-derived immunoglobulin sequences. In other embodiments, the antibody can contain amino acid residues encoded by non-rabbit-derived immunoglobulin sequences, such as humanized antibodies, chimeric antibodies, and the like, to reduce the body's rejection reaction while maintaining the desired specificity and affinity. The term "chimeric antibody" refers to an antibody whose part is derived from a specific source or species, while the rest is derived from a different source or species. The term "humanized antibody" refers to a chimeric antibody of the CDR region of a non-human antibody such as a rabbit-derived antibody and a FR region from a human, in some cases, the variable region of a non-human antibody is combined with the constant region of a human antibody, such as a human rabbit chimeric antibody; in other cases, the CDR region of a non-human antibody is grafted onto a human antibody framework (FR) sequence, which is derived from the FR sequence of a single or multiple other human antibody variable regions. In the present application, the CDR region in the chimeric antibody or humanized antibody is derived from the rabbit-derived CDR region.

[0044] The terms "monoclonal antibody" or "monoclonal" (including "monoclonal" or "polyclonal antibody" or "polyclonal antibody" or "MAb" or "mAb"), and "specifically immunoreactive with," and like terms refer to a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translation modifications (e.g., isomerization, amidation) that can be present. "Monoclonal" antibodies are highly specific, being directed against a single antigenic site or epitope. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as a requirement for use of a particular methodology for their production. The antibodies can be made using a wide variety of methods known in the art, including but not limited to the hybridoma method, phage display methods, yeast display methods, recombinant DNA methods, single cell screening or single cell sequencing methods.

[0045] The term "specifically binds" is a term of art well known in the art, and a molecule exhibits "specific binding", "specifically binds" or is said to "preferentially bind" if it reacts, more frequently, more rapidly, with greater duration, and / or with greater affinity with a particular target antigen or epitope than with other target antigens or epitopes, and does not necessarily require (although can include) exclusive binding. In order to make the above-mentioned objects, features and advantages of the present application more obvious and comprehensible, specific embodiments of the present application are described in detail below with reference to the accompanying drawings.

[0046] The antibody against human keratin 8 provided by the embodiment of the present application comprises a light chain variable region and a heavy chain variable region, and the light chain variable region and the heavy chain variable region both comprise three complementarity determining regions (CDRs) named CDR1, CDR2 and CDR3 respectively, wherein the amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region are respectively shown as SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5; and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are respectively shown as SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10.

[0047] The monoclonal antibody strain provided by the present application can specifically target human keratin 8 (CK8 or KRT8), and is suitable for the construction of an immunological biopsy technology for clinical pathological cells or tissues, and especially meets the application of immunofluorescence, flow cytometry and the like. The antibody of the present application is used as a primary antibody to capture a target antigen, a fluorescein-labeled anti-rabbit IgG secondary antibody is used to develop an immunofluorescence and flow cytometry system, which shows that the antibody of the present application has strong specificity, good binding affinity and high recognition sensitivity for CK8 antigen in the cytoplasm, and has no cross reaction and non-specific binding to non-target proteins in the cell, effectively ensures that the detection signal comes from the target protein, is conducive to reducing background noise, improving the signal-to-noise ratio, improving the accuracy and reliability of the detection result, and has a high degree of consistency with the actual situation, and has high practical value in the clinical diagnosis and scientific research detection of the expression level of human CK8 protein.

[0048] Optionally, the light chain variable region and the heavy chain variable region each comprises four framework regions (FRs) interlaced with three CDRs in order, constituting the variable region. The amino acid sequence of the antibody light chain variable region (VL) of the present application is shown in SEQ ID NO. 2, and the amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO. 7.

[0049] Optionally, the antibody of the present application further comprises a light chain constant region (CL) and a heavy chain constant region (CH), the CL and VL constituting a complete light chain (FL), and the CH and VH constituting a complete heavy chain (FH). The constant region of the antibody can be obtained by public query, such as searching for CH by searching for rabbit IgG gamma C reign in the IMGT online database (www.imgt.org), and searching for CL by searching for rabbit IgG Kappa C reign.

[0050] Specifically, the amino acid sequence of the antibody light chain is shown in SEQ ID NO. 1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO. 6.

[0051] It should be noted that the antibody of the present application can be a full-length antibody (having a typical Y-shaped molecular structure) or an antigen binding region of the full-length antibody; the antigen binding region refers to a polypeptide substantially maintaining the same biological function or activity as the full-length form, specifically, the antigen binding region comprises the CDR region as described above, and more preferably has the variable region as described above, thereby retaining the complete antigen recognition and binding site, and being capable of binding to the same antigen as the full-length antibody, especially to the same epitope. Optionally, the antigen binding region is selected from at least one of Fab, F(ab)2, Fab', F(ab')2, Fv, (Fv)2, scFv and sc(Fv)2. These antigen binding regions can be obtained by conventional techniques in the art.

[0052] Another embodiment of the present application provides an antibody conjugate comprising the antibody against human keratin 8 as described above and a detection label connected to the antibody.

[0053] The detection label is used to produce a recognizable signal change, so as to recognize the antibody of the present application according to the signal change, and then recognize the expression of CK8 antigen in the sample to be detected through the specific reaction of antigen and antibody. The detection label includes but is not limited to biotin, fluorescent dye (such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride), fluorescent protein (such as isoallophycocyanin, phycoerythrin, PerCP and phycocyanin), enzyme (such as alkaline phosphatase, acid phosphatase, beta-galactosidase, glucose oxidase, horseradish peroxidase, acetylcholinesterase, avidin), colloidal gold, colored magnetic beads, latex particles, radionuclide, detection antibody or combination thereof.

[0054] It should be emphasized that the antibody of the present application can be used alone or connected (covalently or non-covalently) with a detection label to form an antibody conjugate. In some embodiments, the antibody of the present application is used as an antigen-binding (or capture) antibody which specifically recognizes and binds to CK8 in the sample to be detected, and then the CK8 protein is qualitatively or quantitatively detected by analyzing the signal of the detection label connected thereto; in other embodiments, the anti-CK8 antibody (as a primary antibody or a capture antibody) is not labeled, and a detection label is conjugated to a secondary antibody (as a detection antibody) or other molecules which can bind to the primary antibody, for example, if the anti-CK8 antibody is a rabbit-derived IgG antibody, the secondary antibody can be an anti-rabbit IgG antibody, so as to realize the qualitative or quantitative detection of CK8 by analyzing the signal change of the detection label generated after the specific binding of the secondary antibody to the antibody of the present application, for example, the detection systems established in Example 2 of the present application.

[0055] Another embodiment of the present application provides a nucleic acid molecule, a recombinant vector comprising the nucleic acid molecule or a host cell comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes the antibody against human keratin 8 as described above.

[0056] The nucleic acid molecule can be in the form of DNA (such as cDNA or genomic DNA or synthetic DNA) or RNA (such as mRNA or synthetic RNA). The DNA can be single-stranded or double-stranded, and can be a coding strand or a non-coding strand.

[0057] The sequence of the nucleic acid molecule can be obtained by conventional means such as codon coding rules according to the AA sequence of the antibody. The full-length sequence of the nucleic acid molecule or a fragment thereof can be obtained by PCR amplification, recombination or artificial synthesis.

[0058] The original vector for constructing the recombinant vector is any of the various vectors conventionally used in the art, as long as it can accommodate the nucleic acid molecule. Typical vectors include plasmids (e.g., pBR322, pUC series, pET series, pGEX series), viral vectors, bacteriophages (e.g., λgt4 λB, λ-Charon, λΔz1 and M13), cosmids and minichromosomes. The vector can be a cloning vector (i.e., used to transfer the nucleic acid molecule into a host and propagate it in the host cell) or an expression vector (i.e., contains the necessary genetic elements to allow the nucleic acid molecule inserted into the vector to be expressed in a host cell). The nucleic acid molecule is inserted into a suitable vector to form a cloning vector or an expression vector carrying the nucleic acid molecule, which is then introduced into a host cell and cultured under specific conditions to express the antibody. This is a well-known technique in the art and will not be described in detail here.

[0059] The nucleic acid molecules encoding the antibodies FL and FH of the present application can be inserted into two vectors, respectively, which can be introduced into the same or different host cells. When the heavy chain and the light chain are expressed in different host cells, each chain can be isolated from the host cell expressing it and mixed together and incubated under suitable conditions to form the antibody. In other embodiments, the nucleic acid molecules encoding the antibodies FL and FH can also be cloned into one vector, with each nucleic acid sequence linked downstream of a suitable promoter; for example, each nucleic acid sequence encoding the heavy chain and the light chain can be operably linked to different promoters, or the nucleic acid sequences encoding the heavy chain and the light chain can be operably linked to a single promoter, such that both the heavy chain and the light chain can be expressed from the same promoter. The choice of expression vector / promoter depends on the type of host cell used to produce the antibody.

[0060] The transfection or transformation of the recombinant vector into the host cell is carried out using conventional techniques. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DNA are harvested after the exponential growth phase and treated with CaCl2or MgCl2; alternatively, microinjection, electroporation or liposome packaging can be used. When the host is a eukaryote, the following DNA transfection methods can be used to achieve gene introduction: calcium phosphate coprecipitation, microinjection, electroporation, liposome packaging or gene gun bombardment.

[0061] The host cell can be a prokaryotic or eukaryotic cell. Examples of prokaryotic host cells that can be used in the present application include, but are not limited to, Escherichia coli (e.g., DH5α, JM109, BL21, W3110), Bacillus sp. (e.g., Bacillus subtilis, Bacillus thuringiensis), and Enterobacteriaceae strains (e.g., Salmonella typhimurium, Serratia marcescens), and Pseudomonas sp. Examples of eukaryotic host cells that can be used for transformation include, but are not limited to, yeast, insect cells, and animal cells, such as Drosophila S2 or Sf9 cells, mammalian CHO, CHO DG44, CHO-S, COS-7, 293 series cells, HepG2, Huh7, 3T3, RIN, MDCK, and HEK293 cell lines. After obtaining the host cells transfected or transformed with the recombinant vector as described above, the antibody can be expressed under suitable conditions, and then isolated to obtain the purified antibody.

[0062] In a typical embodiment, the method for preparing the antibody comprises: connecting the heavy chain gene and the light chain gene of the antibody with a signal peptide in series, respectively loading the signal peptide on an expression vector pBR322, co-transfecting human kidney epithelial cells (293F), culturing the 293F cells, collecting the cell culture supernatant, and purifying the target antibody strain. The selection of the signal peptide is designed according to the host cell, and the present application does not have special limitations.

[0063] Another embodiment of the present application provides the use of the antibody or antibody conjugate against human keratin 8 as described above in the preparation of an immunodetection kit for human keratin 8.

[0064] The use of the antibody or antibody conjugate against human keratin 8 in the preparation of an immunodetection kit for human keratin 8 has the same advantages as the antibody against human keratin 8 described above relative to the prior art, and will not be repeated here.

[0065] Based on the same inventive concept, an embodiment of the present application also provides an immunodetection kit for human keratin 8, which comprises the antibody or antibody conjugate against human keratin 8 as described above.

[0066] The immunodetection methods mentioned above include, but are not limited to, enzyme linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISPOT), immunohistochemistry (IHC), immunofluorescence (IF), Western blot (WB), immunoprecipitation (IP) and flow cytometry (FC). The kit can be an enzyme-linked immunosorbent assay kit, an enzyme-linked immunospot kit, an immunohistochemistry kit, an immunofluorescence kit, a Western blot kit or a flow cytometry kit.

[0067] Preferably, the kit is a flow cytometry kit or an immunofluorescence kit.

[0068] Optionally, the kit further comprises a fluorescent secondary antibody of the anti-human keratin 8 antibody, which is a fluorescently conjugated anti-rabbit IgG antibody.

[0069] The application will be further described below in conjunction with specific examples. The experimental methods in the following examples, for which no specific conditions are indicated, are generally performed under conventional conditions, for example, the conditions described in Molecular Cloning: A Laboratory Manual (Fourth Edition) published by Cold Spring Harbor Laboratory, or generally under the conditions recommended by the manufacturer.

[0070] Example 1 Preparation of rabbit-derived antibody against human keratin 8 (CK8, KRT8)

[0071] 1.1. Preparation of immunogen: The immunogen used is a 1-483 aa protein fragment of human recombinant CK8 protein expressed and purified from 293F cells. The amino acid sequence of the full-length CK8 is shown in Uniprot No. P05787 or NCBI Accession No. NP_002264.1, and the gene sequence is shown in NCBI Accession No. NM_002273.4. The gene sequence corresponding to CK8 1-483 aa was constructed into a pBR322 vector, and a recombinant protein with biological activity was expressed in 293F cells. The purity of the recombinant protein was greater than 90%.

[0072] 1.2, Animal immunization: Two Japanese white rabbits were immunized with 200 μg of human CK8 protein each. Before the first immunization, the antigen was mixed with an equal amount of complete Freund's adjuvant (purchased from Sigma Company) to prepare an emulsifier, which was injected subcutaneously at multiple points on the abdomen and back of the rabbit. After the first immunization, 100 μg of immunogen was mixed with an equal amount of incomplete Freund's adjuvant (purchased from Sigma Company) to prepare an emulsifier, which was injected subcutaneously at multiple points on the abdomen and back of the rabbit every 3 weeks for two times. After three immunizations, the serum was diluted 1:243000, and the titer of the serum against human CK8 was determined by enzyme-linked immunosorbent assay (ELISA). The rabbits with OD 450nm greater than 0.2 were immunized with 200 μg of immunogen for one time. Three to four days later, the spleen was taken out, and the final immunization serum was diluted and detected for the affinity and specific reaction of the serum with the target antigen in the cell sample to be tested by immunofluorescence (IF).

[0073] The determination of the titer of the immune serum by ELISA included the following steps: 1) coating: 25 μL / well of 1 μg / mL human CK8 protein was added to the enzyme-labeled plate, which was coated at 4°C overnight; 2) blocking: 75 μL / well of washing buffer (PBS containing 0.05% (v / v) Tween-20) was added for washing 5 times, and then 50 μL / well of blocking buffer (PBS containing 1% BSA, 0.5% gelatin and 5% sucrose) was added, which was incubated at room temperature for 1 h; 3) gradient dilution of the serum to be tested and sample addition: the hole plate was washed by repeating the plate washing process in 2), and then the serum to be tested was gradient diluted, which was diluted with dilution buffer (PBS containing 1% BSA) starting from 1:1000, and the dilution was three times, a total of 8 gradients; the serum diluent was added to the hole plate at 25 μL / well, which was incubated at room temperature for 1 h; 4) secondary antibody incubation: the hole plate was washed by repeating the plate washing process in 2), and then 25 μL / well of 1:5000 diluted horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (purchased from ABclonal Company, item number AS014) was added, which was incubated at room temperature for 1 h in the dark; 5) reaction termination and color development: the hole plate was washed by repeating the plate washing process in 2), and then 100 μL / well of TMB color developing agent was added, which was developed at 37°C in the dark for 10 min, and 100 μL / well of 0.5 M oxalic acid solution was added to terminate the reaction, and the optical absorption value at 450 nm was measured, the pre-immune rabbit serum was used as the negative control, the detection system without immune serum was used as the blank control (NC), and the ratio of the measured value to the control value ≥2.1 was considered as a positive immune serum.

[0074] The recognition specificity of the immune serum and the cell sample (human cervical cancer cells HeLa expressing CK8 protein) determined by the IF method includes the following steps: 1) cell treatment: prepare HeLa adherent cells, add 4% neutral formaldehyde fixing solution to fix at room temperature for 10-15 min, and after fixing, wash with PBS for 3 times, each time for 5 min; 2) cell blocking: add blocking buffer (1xPBS containing 5% BSA) to each well, and after blocking for 30-60 min, remove the blocking solution; 3) primary antibody incubation: add the immune serum (as the primary antibody) diluted with PBS to 100 μL / well, incubate at 2-8°C overnight; remove the primary antibody working solution after taking out and warming at room temperature for 15 min, then wash with 1xPBST for 5 min, and wash with 1xPBS for 2 times, each time for 5 min; 4) secondary antibody incubation: add the fluorescent secondary antibody (Cy3-conjugated Goat anti-Rabbit IgG (H+L) from ABclonal, product number AS007, the dilution ratio of the secondary antibody is 1:500) diluted with PBS to 100 μL / well, incubate at room temperature in the dark for 1 h; remove the secondary antibody working solution, then wash with 1xPBST for 5 min, and wash with 1xPBS for 2 times, each time for 5 min; 5) nuclear staining: add DAPI nuclear stain working solution to 100 μL / well, stain for 10-30 min, then wash with 1xPBST for 5 min, and wash with 1xPBS for 2 times, each time for 5 min; 6) immunofluorescence analysis: observe and take pictures for analysis by ABTK / G2023030034 wide-field fluorescence microscope.

[0075] The serum titer detection results are shown in Table 2. Figure 1 WA-63743D is the project number, and N16170 and N16171 are the rabbit numbers. The binding ability detection results of the immune serum and the CK8 protein expressed by the positive cell HeLa are shown in Table 3. Figure 2 The upper graph is the fluorescence imaging graph of the HeLa cell combined with the immune serum, and after the antibody in the serum binds with the CK8 protein, it is labeled as Cy3 red by the fluorescent secondary antibody, and the lower graph is the contrast graph of the red and blue two colors of the cell nucleus (blue) stained by DAPI, and the red and blue two colors are merged. From Figures 1-2 It can be seen that a strong immune response is generated in the rabbit body during the third immunization and the fourth booster immunization, and the antibody capable of specifically recognizing the CK8 protein is generated in the immune serum, which is manifested as obvious staining of the filamentous structure in the cytoplasm of the HeLa cell, and the antibody is located in the intermediate filament, which is consistent with the expectation. Therefore, the fourth immune serum can be used for subsequent isolation of the monoclonal antibody.

[0076] 1.3, Isolation of B cells in the spleen and sorting of antigen-specific B cells: see the related methods in the published patent "Method for efficiently isolating single antigen-specific B lymphocytes from spleen cells (Publication No. CN110016462A, Publication Date: 2019-07-16)" and the patent "B lymphocyte in vitro culture system and application (Publication No. CN111518765A, Publication Date: 2020-08-11)".

[0077] 1.4, Cloning of rabbit monoclonal antibody genes: B lymphocytes capable of recognizing and binding human CK8 protein were detected by antigen-coated ELISA, and the aforementioned cells were collected, lysed, and then RNA was extracted using Quick-RNA TM Micro Prep kit (purchased from ZYMO company, item number R1100-250) and reverse transcribed into cDNA. With the aforementioned cDNA as a template, the naturally paired rabbit-derived antibody light chain variable region (VL) and heavy chain variable region (VH) were amplified and sequenced by PCR method. The PCR reaction system includes: 4 μL cDNA, 1 μL forward primer (10 mM), 1 μL reverse primer (10 mM), 12.5 μL 2x Gloria HiFi (from ABclonal, item number RK20717) and 6.5 μL H2O; the PCR amplification program includes: 98℃ for 30s, then 98℃ for 10s, 64℃ for 30s, 72℃ for 30s for 40 cycles, finally 72℃ for 5min, and the reaction solution is stored at 4℃. The primer sequences (5'-3') for amplifying VL and VH genes are as follows, F and R represent forward primer and reverse primer, respectively.

[0078] VL-F: tgaattcgagctcggtacccATGGACACGAGGGCCCCCAC (SEQ ID NO. 12);

[0079] VL-R: cacacacacgatggtgactgTTCCAGTTGCCACCTGATCAG (SEQ ID NO. 13);

[0080] VH-F: tgaattcgagctcggtacccATGGAGACTGGGCTGCGCTG (SEQ ID NO. 14);

[0081] VH-R: gtagcctttgaccaggcagcCCAGGGTCACCGTGGAGCTG (SEQ ID NO. 15).

[0082] The amplified DNA products were sequenced to obtain VL sequence as shown in SEQ ID NO. 2 and VH sequence as shown in SEQ ID NO. 7; then the IMGT online database (www.imgt.org) was queried to obtain the sequence of the constant region, obtaining the complete light chain (FL) of antibody 15H6 as shown in SEQ ID NO. 1 and the complete heavy chain (FH) as shown in SEQ ID NO. 6. The amino acid (aa) and nucleic acid (DNA) sequences of antibody 15H6 are shown in Table 1. For convenience of description, the light chain complementarity determining regions CDR1-3 are represented as LCDR1-3, and the heavy chain complementarity determining regions CDR1-3 are represented as HCDR1-3, and the CDRs are based on the Kabat numbering system.

[0083] Table 1 Amino acid sequences of rabbit-derived antibody 15H6 and immunogen of the present embodiment

[0084]

[0085]

[0086] 1.5, Expression and large-scale production of antibody 15H6: The obtained antibody heavy chain and light chain genes were respectively loaded into expression vectors. In the present embodiment, the light chain constant region (CL) and heavy chain constant region gene (CH) were inserted into the mammalian expression vector pBR322 in advance, and the expression map is shown in Figure 3 , in which pBR322 origin and f1 origin are replication promoters, Ampcillin is a resistance gene, CMV promoter is a transcription promoter, SV40 PA terminator is a tailing signal, light chain constant is the nucleic acid sequence of CL (left), and heavy chain constant is the nucleic acid sequence of CH (right). Then the amplified VL and VH genes were connected to the expression vector pBR322 carrying the CL and CH genes linearized by XbaI (955 bp) and NheI (949 bp) restriction endonucleases by homologous recombination, to obtain the complete light chain (FL) and heavy chain (FH) gene expression vectors, and the vector construction was verified by sequencing.

[0087] For more convenient purification of antibodies, the secretion expression of antibodies is realized by adding a signal peptide upstream of the VL and VH genes. The signal peptide can be used in the art, such as the signal peptide "MDTRAPTQLLGLLLLWLPGARC" upstream of the VL and the signal peptide "METGLRWLLLVAVLKGVQC" upstream of the VH in the patents "Rabbit monoclonal antibody against human interferon alpha 2 and its application (publication number: CN116063487A, publication date: 2023-05-05)" and "High affinity human IL-5 rabbit monoclonal antibody and its application (publication number: CN115819578A, publication date: 2023-03-21)". Of course, the skilled person in the art can also replace other signal peptides after obtaining the antibody sequence of the application to express the antibody. Therefore, the signal peptide sequence is not embodied in the antibody sequence of Table 1 in this embodiment.

[0088] The successfully constructed FL and FH expression vectors were co-transfected into 293F cells, and after transfection, the cells were cultured for 72-96h to obtain the antibody 15H6 recognizing human CK8 protein in the culture supernatant. The target antibody was purified from the culture supernatant using protein A affinity gel resin (purchased from Tiandiren, product number SA015100). The antibody purity was verified to be ≥95% using 12% polyacrylamide gel electrophoresis (SDS-PAGE), and the antibody concentration was 1.33mg / mL. The purified antibody was aliquoted and stored at -20℃ for standby.

[0089] Example 2: Establishment of immunodetection method of antibody 15H6 and evaluation of application effect

[0090] 1. Establishment of immunofluorescence (IF) detection method based on antibody 15H6

[0091] The IF detection method is the same as in Example 1, except that the primary antibody is antibody 15H6, and the working concentration of the primary antibody is 2.66μg / mL.

[0092] The IF detection results are shown in Figure 4 , wherein the upper image is a fluorescence imaging image of antibody 15H6 binding to CK8 protein in HeLa cells. After antibody 15H6 binds to CK8 protein, it is labeled with a fluorescent secondary antibody as red, and the lower image is an imaging image showing the red and blue colors Merge. As can be seen from the image, the intermediate filament fluorescence signal in the cytoplasm of HeLa cells is clear and bright, and the actual color development result is consistent with the theoretical positioning of the antibody, indicating that antibody 15H6 can specifically and sensitively bind to CK8 protein of intermediate filament.

[0093] 2. Establishment of flow cytometry (FC) detection method based on antibody 15H6

[0094] FC detection includes the following steps: 1) UV irradiation disinfection clean bench for 15-20 min, open the fan for 5 min, prepare for aseptic work; 2) collect and wash cells (HeLa cells with high expression of CK8 or human T lymphocyte leukemia cells (Jurkat) with low expression of CK8), determine the total number of cells, and check the cell viability at 90%-95%; 3) resuspend the cells with 1xPBS solution to about 3x10 6 -5x10 6 cells / mL, distribute the cells into a 96-well V-shaped plate at 100 μL / well, and wash once with 1xPBS; 4) use Live / Dead staining solution (Zombie NIR, L / D staining solution) of Biolegend's Zombie NIR Fixable Viability Kit kit (item number 423105) for staining, dilute the L / D staining solution with 1xPBS at a ratio of 1:1500, distribute the diluted L / D staining solution into the well plate at 100 μL / well, and resuspend the cells in the well; 5) cover with aluminum foil, gently mix on a microplate shaker for 15 min, then centrifuge at 400g for 5 min, discard the supernatant, and wash twice with 1xPBS; 6) distribute 1xIntracelluLar Fixation buffer into the 96-well plate at 100 μL / well, and resuspend the cells in each well; 7) repeat step 5), wash twice with 1xPermeablization buffer; 8) distribute 1xPermbuffer diluted primary antibody 15H6 (the final concentration of the primary antibody is 2 μg / mL) into the well plate at 100 μL / well, and resuspend the cells in each well; 9) distribute the fluorescence secondary antibody (Fluorescein (FITC) AffiniPure F(ab')2 Fragment Goat Anti-Rabbit IgG, purchased from jackson, item number 111-096-046) diluted with 1xPBS (dilution ratio 1:200) into the well plate at 100 μL / well, and resuspend the cells in the well; 10) repeat step 5), then resuspend the cells in each well with 200 μL 1xPBS, and store in the dark; 11) analyze according to the Beckman cytoflex flow cytometer usage and maintenance SOP-105-AND-CA-008 operation.

[0095] The FC detection results are shown in Table 1. Figure 5In which, from left to right are the detection results of Jurkat cells and HeLa cells, the horizontal coordinate of the figure represents the relative fluorescence intensity of the fluorescence signal / scattering light signal of the channel, and the vertical coordinate represents the corresponding cell number, the red curve is the blank control without adding any antibody, the blue curve is the isotype control (for eliminating the background staining due to the non-specific binding of the antibody to the cells), and the yellow curve is the antibody 15H6. The results show that the antibody 15H6 has specific binding in the cell samples with high and low expression of CK8, and the fluorescence signal transition difference is obvious, further proving that the antibody 15H6 can specifically target the CK8 protein expressed in the cells, has high binding sensitivity to the target antigen, no cross reaction with non-target antigens, and good anti-cell component interference ability, which is conducive to improving the accuracy and reliability of the tissue cell pathological biopsy of the CK8 protein.

[0096] The above merely describes preferred embodiments of the present application and is not intended to limit the present application, and any modifications, equivalent replacements and improvements made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An antibody against human keratin 8, characterized in that, It includes a light chain variable region and a heavy chain variable region. The amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region are shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively. The amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

2. The antibody against human keratin 8 according to claim 1, characterized in that, The amino acid sequence of the light chain variable region is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

7.

3. The antibody against human keratin 8 according to claim 2, characterized in that, The amino acid sequence of the antibody light chain is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.

6.

4. The antibody against human keratin 8 according to claim 1, characterized in that, The antibody is a full-length antibody or its antigen-binding region; the antigen-binding region is selected from at least one of the following fragments: Fab fragment, F(ab)2 fragment, Fv fragment, (Fv)2 fragment, scFv fragment, and sc(Fv)2 fragment.

5. An antibody conjugate, characterized in that, It comprises an antibody against human keratin 8 as described in any one of claims 1-4 and a detection marker linked to the antibody.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes an antibody against human keratin 8 as described in any one of claims 1-4.

7. The use of the antibody against human keratin 8 as described in any one of claims 1-4 or the antibody conjugate as described in claim 5 in the preparation of a human keratin 8 immunoassay kit.

8. The use of the antibody or antibody-conjugate against human keratin 8 according to claim 7 in the preparation of a human keratin 8 immunoassay kit, characterized in that, The kit is selected from immunofluorescence kits or flow cytometry kits.

9. A human keratin 8 immunoassay kit, characterized in that, Includes the antibody against human keratin 8 as described in any one of claims 1-4 or the antibody conjugate as described in claim 5.

10. The human keratin 8 immunoassay kit according to claim 9, characterized in that, It also includes a fluorescent secondary antibody against human keratin 8, wherein the fluorescent secondary antibody is a fluorescein-conjugated anti-rabbit IgG antibody.

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