Antibodies against ck14 protein and uses thereof

Abstract

The application discloses an antibody against CK14 protein and application thereof, and relates to the technical field of antibodies. The antibody comprises a light chain complementarity determining region in a light chain variable region shown in SEQ ID NO: 15 and a heavy chain complementarity determining region in a heavy chain variable region shown in SEQ ID NO: 16. The antibody provided by the application can be used for diagnosing diseases taking CK14 as a marker, and the application provides more protein selection for detection of CK14 and diagnosis of diseases taking CK14 as a marker.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and more specifically, to an antibody against CK14 protein and its application. Background Technology

[0002] Currently, CK14 (cytoceratin 14) is a differentiation-specific protein belonging to the acidic cytokeratin family. CK14 expression is limited to basal cells of myoepithelial and keratinized stratified epithelium from various sources, and it is one of the proteins that make up the cytoskeleton. Studies have shown that CK14 is highly expressed in malignant tumors such as lung squamous cell carcinoma and invasive ductal carcinoma of the breast. Furthermore, research has indicated that high CK14 expression is associated with the process, development, and prognosis of esophageal squamous cell carcinoma and cervical cancer.

[0003] Immunohistochemistry (IHC) is a common clinical method for detecting protein expression in tumor cells. The accuracy and sensitivity of IHC testing depend heavily on the quality of the monoclonal antibody that specifically binds to the protein. Therefore, developing a highly specific monoclonal antibody against CK14 is crucial for detecting CK14 expression levels using IHC.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide an antibody against CK14 protein and its application to solve the above-mentioned technical problems.

[0006] This invention is implemented as follows:

[0007] In a first aspect, the present invention provides an antibody against CK14 protein or an antigen-binding fragment thereof, comprising a light chain complementarity-determining region in the light chain variable region as shown in SEQ ID NO: 15, and a heavy chain complementarity-determining region in the heavy chain variable region as shown in SEQ ID NO: 16.

[0008] Secondly, the present invention also provides the use of an antibody or its antigen-binding fragment in the preparation of any of the following products:

[0009] (1) Tumor diagnostic products;

[0010] (2) Products for evaluating the efficacy of tumor treatment;

[0011] (3) Tumor prognostic assessment products;

[0012] (4) Products for monitoring tumor recurrence and metastasis;

[0013] (5) CK14 protein detection products;

[0014] (6) CK14 protein isolation or enrichment products.

[0015] Thirdly, the present invention also provides a product for tumor diagnosis, efficacy evaluation, prognosis evaluation, or recurrence and metastasis monitoring, the product comprising: the above-mentioned antibody or its antigen-binding fragment.

[0016] Fourthly, the present invention also provides a CK14 protein detection product, a CK14 protein isolation product or an enrichment product, the product comprising: the above-mentioned antibody or its antigen-binding fragment.

[0017] Fifthly, the present invention also provides a cell, which is a non-plant cell, expressing the above-mentioned antibody or its antigen-binding fragment.

[0018] In a sixth aspect, the present invention also provides a method for preparing the above-mentioned antibody or antigen-binding fragment thereof, comprising: culturing the above-mentioned cells, and separating and purifying the antibody or antigen-binding fragment thereof from the culture product.

[0019] The present invention has the following beneficial effects:

[0020] This invention reveals that antibodies with specific six complementarity-determining regions exhibit good activity and can maintain high binding specificity and affinity to the CK14 antigen. This means that the binding protein can be used to detect CK14, helping to improve the sensitivity and specificity of detection. This antibody can be used to diagnose diseases using CK14 as a biomarker. This invention provides more protein options for the detection of CK14 and the diagnosis of diseases using CK14 as a biomarker.

[0021] The antibody that specifically binds to CK14 polypeptide provided by this invention can be used to prepare tumor diagnostic reagents, kits or chips. The tumor diagnostic marker is CK14 (cytoceratin). Based on the antibody's specific binding activity to CK14, the expression level of CK14 in relevant tumor tissues can be detected. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The image shows the results of immunohistochemical experiments on esophageal squamous cell carcinoma samples. (A) is CK14 monoclonal antibody 10E1, and (B) is CK14 antibody from Maixin Pharmaceutical Co., Ltd.

[0024] Figure 2 The images show the results of the second batch of immunohistochemical and neutralization experiments for esophageal squamous cell carcinoma samples. (A) is the CK14-10E1 antibody produced in this example, (B) is the CK14 antibody from Maixin Company, and (C) is the result of the neutralization experiment of the CK14-10E1 antibody. Detailed Implementation

[0025] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0026] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0028] The term "antigen-binding fragment" broadly refers to all proteins / protein fragments containing a CDR region, particularly antibodies or antibody functional fragments. "Antigen-binding fragment" includes antigen-binding fragments of the aforementioned antibodies, including Fab, F(ab')2, Fd, Fv, scFv, bispecific antibodies, multispecific antibodies, and the smallest antibody recognition unit, as well as single-chain derivatives of these antibodies and fragments. Antibody types can include IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD, etc. Furthermore, the term "antibody" includes both naturally occurring and non-naturally occurring antibodies, including, for example, chimeric, bifunctional, and humanized antibodies, as well as related synthetic isoforms. The term "antibody" is used interchangeably with "immunoglobulin."

[0029] The term “antibody” in this article is used in the broadest sense and can include full-length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments, as long as they exhibit the desired biological activity, such as specific binding to the CK14 protein.

[0030] In this invention, the terms "complementarity-determining region" or "CDR" refer to highly variable regions of the heavy and light chains of an immunoglobulin, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding affinity of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In specific embodiments of this invention, CDRs refer to highly variable regions of the heavy and light chains of the antibody.

[0031] In this invention, the heavy chain complementarity-determining region (CDR) is represented by HCDR, which includes HCDR1, HCDR2, and HCDR3; the light chain complementarity-determining region (LCDR) is represented by LCDR, which includes LCDR1, LCDR2, and LCDR3. Commonly used CDR labeling methods in the art include the Kabat numbering scheme, the IMGT numbering scheme, the Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of the immunoglobulin superfamily. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences led to the creation of the Kabat database, and the Kabat numbering scheme is generally considered the widely adopted standard for numbering antibody residues. This invention uses the Kabat annotation standard to label CDR regions, but CDR regions labeled by other methods are also within the scope of this invention.

[0032] Typically, the variable region (VH) of the antibody heavy chain is obtained by linking the following CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4. HCDR1 is synonymous with CDR-H1.

[0033] The variable region (VL) of the antibody light chain can be obtained by linking the following numbered CDRs with FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0034] LCDR1 is CDR-L1, LCDR2 is CDR-L2, and LCDR3 is CDR-L3.

[0035] In a first aspect, the present invention provides an antibody against CK14 protein or an antigen-binding fragment thereof, comprising a light chain complementarity-determining region in the light chain variable region as shown in SEQ ID NO: 15, and a heavy chain complementarity-determining region in the heavy chain variable region as shown in SEQ ID NO: 16.

[0036] Light chain variable region: SEQ ID NO:15:

[0037] DIVMSQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSN LESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWK.

[0038] Heavy chain variable region: SEQ ID NO:16:

[0039] EAQLQQSGAELVKPGASVKLSCRASGYIFRSYDIHWVRQRPEQGLEWIGWIFPGDGS TKYNEEFKGKATLTTDKSSSTAYMHLNRLTSEDSAVYFCARDGSRYIDYWGQGTTVTVSS.

[0040] Inputting the above SEQ ID NO:15 and SEQ ID NO:16 sequences into the CDR identification system will yield the corresponding CDR sequences.

[0041] The amino acid sequences of the heavy chain complementarity-determining regions and light chain complementarity-determining regions described above are newly discovered and disclosed in this invention, and can endow the binding protein with the ability to specifically bind to CK14 protein. Based on the good binding activity and affinity of this antibody, it can be used to develop detection products for CK14 protein, such as detection reagents and kits. Detection reagents include, but are not limited to, immunohistochemical staining (IHC) related reagents and reagents with detectable markers; kits include, but are not limited to, ELISA kits, Elisopt kits, microfluidic chip kits, etc. Using the binding protein of this invention to detect CK14 protein can improve the sensitivity and specificity of detection. Furthermore, this antibody can be used to diagnose diseases that use CK14 protein as a biomarker, which is beneficial for early detection and early intervention. This invention provides more protein options for the detection of CK14 protein and the diagnosis of diseases using CK14 protein as a biomarker.

[0042] In a preferred embodiment of the present invention, the binding protein and CK14 peptide are used to K D ≤3.2525×10 9 Affinity binding at L / mol.

[0043] For example, K D ≤8×10 10 L / mol affinity binding, such as K D ≤9×10 10 L / mol, K D ≤7×10 10 L / mol, K D ≤6×10 10 L / mol, K D ≤5×10 10 L / mol, K D ≤4×10 10 L / mol, K D ≤3×10 10 L / mol, K D ≤2×10 10 L / mol or K D ≤1×10 10 Affinity binding at L / mol.

[0044] In a preferred embodiment of the present invention, the heavy chain complementarity-determining region includes CDR-H1, CDR-H2, and CDR-H3, the amino acid sequences of which are shown in SEQ ID NO: 9-11, respectively; the light chain complementarity-determining region includes CDR-L1, CDR-L2, and CDR-L3, the amino acid sequences of which are shown in SEQ ID NO: 12-14, respectively.

[0045] CDR-VH1: SYDIH; SEQ ID NO: 9;

[0046] CDR-VH2: WIFPGDGSTKYNEEFKG; SEQ ID NO: 10;

[0047] CDR-VH3: DGSRYIDY; SEQ ID NO: 11;

[0048] CDR-VL1:RASKSVSTSGYSYMH;SEQ ID NO:12;

[0049] CDR-VL2: LVSNLES; SEQ ID NO: 13;

[0050] CDR-VL3: QHIRELTRS; SEQ ID NO:14.

[0051] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment further includes a heavy chain framework region and / or a light chain framework region; the heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4 having at least 80% homology with the amino acid sequences shown in SEQ ID NO:5-8; for example, the heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology with the amino acid sequences shown in SEQ ID NO:5-8.

[0052] The light chain framework region includes LFR1, LFR2, LFR3 and LFR4, which have at least 80% homology with the amino acid sequences shown in SEQ ID NO:1-4; for example, the light chain framework region includes LFR1, LFR2, LFR3 and LFR4, which have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology with the amino acid sequences shown in SEQ ID NO:1-4.

[0053] The sequences of SEQ ID NO:1-8 are shown in the table below:

[0054]

[0055] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment further includes a constant region, which includes a heavy chain constant region and / or a light chain constant region. The heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0056] In a preferred embodiment of the present invention, the species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans;

[0057] In a preferred embodiment of the present invention, the species source of the constant region is mice;

[0058] In a preferred embodiment of the present invention, the antigen-binding fragment is selected from any one of the antibody F(ab')2, Fab', Fab, Fv, Fab'-SH and scFv.

[0059] The antigen-binding fragments of the aforementioned antibodies typically possess the same binding specificity as the antibodies from which they originate. Those skilled in the art will readily understand, based on the description herein, that the functional fragments of the aforementioned antibodies can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds.

[0060] The antigen-binding fragments of the aforementioned antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesizing, for example, automated peptide synthesizers sold by Applied BioSystems.

[0061] Secondly, the present invention also provides the use of an antibody or its antigen-binding fragment in the preparation of any of the following products:

[0062] (1) Tumor diagnostic products;

[0063] (2) Products for evaluating the efficacy of tumor treatment;

[0064] (3) Tumor prognostic assessment products;

[0065] (4) Products for monitoring tumor recurrence and metastasis;

[0066] (5) CK14 protein detection products;

[0067] (6) CK14 protein isolation or enrichment products;

[0068] In a preferred embodiment of the present invention, the tumor is a tumor that overexpresses the CK14 protein.

[0069] In a preferred embodiment of the present invention, the tumor is selected from squamous cell carcinoma of the lung, adenocarcinoma of the lung, breast cancer, or squamous cell carcinoma of the esophagus.

[0070] In a preferred embodiment of the present invention, the breast cancer is invasive ductal carcinoma of the breast.

[0071] In a preferred embodiment of the present invention, the products used in (1)-(5) are reagents, kits, test strips, antibody chips, antibody probes or detectors; the separation or enrichment products used in (6) are magnetic beads, kits or separation columns.

[0072] Antibody chips are chips formed by immobilizing the aforementioned antibodies or their antigen-binding fragments on a carrier.

[0073] In a preferred embodiment of the present invention, in applications (1)-(5), the antibody or its antigen-binding fragment is labeled with a detectable marker.

[0074] Detectable markers refer to substances that have properties that can be directly observed by the naked eye or detected or probing by instruments, such as luminescence, color development, radioactivity, etc. These properties can be used to qualitatively identify the corresponding target.

[0075] In optional embodiments, the detectable markers include, but are not limited to, fluorescent dyes, enzymes that catalyze substrate color development, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0076] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this invention.

[0077] In optional embodiments, fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy...). 5. Cy5.5, Cy3, etc. or similar), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or similar) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP), etc.).

[0078] In optional embodiments, the enzymes that catalyze substrate color development include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxygenase.

[0079] In optional embodiments, radioactive isotopes include, but are not limited to, those mentioned above. 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188 Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105Rh、 177 Lu、 172 Lu and 18 F.

[0080] In optional embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0081] In optional embodiments, nanoparticle-based markers include, but are not limited to, nanoparticles and colloids. Nanoparticles include, but are not limited to, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0082] In optional embodiments, the colloid includes, but is not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latexes.

[0083] In optional embodiments, colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0084] In a preferred embodiment of the present invention, in steps (1)-(5), tissues and / or cells are labeled with antibodies or their antigen-binding fragments, and the labeled tissues and / or cells are detected.

[0085] In a preferred embodiment of the present invention, after the antibody or its antigen-binding fragment labels tissues and / or cells, the secondary antibody is incubated with the labeled tissues and / or cells, and the incubation product is detected.

[0086] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment is labeled with at least one of the following markers: fluorescent dye, enzyme that catalyzes substrate color development, radioactive isotope, chemiluminescent reagent, and nanoparticle marker.

[0087] Thirdly, the present invention also provides a product for tumor diagnosis, efficacy evaluation, prognosis evaluation, or recurrence and metastasis monitoring, the product comprising: the above-mentioned antibody or its antigen-binding fragment;

[0088] In a preferred embodiment of the present invention, the product is a reagent, reagent kit, test strip, antibody chip, antibody probe, or detector.

[0089] Reagents include, but are not limited to, immunohistochemical staining (IHC) related reagents and reagents with detectable markers; kits include, but are not limited to, ELISA kits, Elisopt kits, microfluidic chip kits, etc.

[0090] Fourthly, the present invention also provides a CK14 protein detection product, a CK14 protein isolation product or an enrichment product, the product comprising: the above-mentioned antibody or its antigen-binding fragment.

[0091] In one embodiment, the CK14 protein detection product, CK14 protein isolation product, or enrichment product includes a solid-phase support on which the aforementioned antibody or its antigen-binding fragment is coated. The solid-phase support includes, but is not limited to, glass, microplates, membranes, and substrates with activating groups.

[0092] In a preferred embodiment of the present invention, the CK14 protein detection product is a reagent, kit, test strip, antibody chip, antibody probe, or detector.

[0093] In a preferred embodiment of the present invention, the CK14 protein isolation or enrichment product is a magnetic bead, a reagent kit, or a separation column.

[0094] For example, the antibodies described above can be coated onto magnetic beads for the separation and enrichment of CK14 protein. In one embodiment, the antibodies are coated onto packing material and packed into a separation column for affinity separation and enrichment of CK14 protein. Therefore, the antibodies described above or their antigen-binding fragments have promising applications in the preparation of CK14 protein enrichment products.

[0095] Fifthly, the present invention also provides a cell, which is a non-plant cell, expressing the above-mentioned antibody or its antigen-binding fragment.

[0096] The host cells are selected from mammalian cells; the mammalian cells are selected from any one of 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, mouse L cells, LNCaP cells, 633 cells, Vero, BHK cells, CV1 cells, HeLa cells, MDCK cells, Hep-2 cells, and Per6 cells. Among them, the 293 series cells, Per6 cells, and CHO cells are commonly used mammalian cells for the production of antibodies or recombinant proteins and are well known to those skilled in the art.

[0097] In a sixth aspect, the present invention also provides a method for preparing the above-mentioned antibody or antigen-binding fragment thereof, comprising: culturing the above-mentioned cells, and separating and purifying the antibody or antigen-binding fragment thereof from the culture product.

[0098] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0099] Example 1

[0100] This embodiment describes the preparation of a monoclonal antibody specifically against CK14.

[0101] 1. Animal (mouse) immunization.

[0102] Mice were immunized with an immunogen using a standard method. The immunogen was a CK14 polypeptide synthesized by GIL Biotech, which also served as a detection antigen for serum titer and hybridoma screening. High-purity antigen increases the chance of obtaining the desired monoclonal antibody while reducing the screening workload. Five mice were immunized, each receiving 50 μg of CK14 antigen. The antigen protein solution was prepared using PBS. Appropriate amounts of antigen protein, PBS, and Freund's adjuvant were placed in a syringe, the syringe outlet was plugged, and the syringe was placed on an emulsifier and stirred thoroughly to form a stable water-in-oil solution. The first tail blood serum titer was measured 7-10 days after the primary and secondary immunizations. Good titers were obtained after 2-4 booster immunizations. Mice with high serum titers were selected for final intraperitoneal immunization followed by cell fusion.

[0103] 2. Hybridoma cell fusion and screening.

[0104] Preparations are required before cell fusion: 1. Culture mouse myeloma cells SP2 / 0 to the logarithmic growth phase; 2. One negative mouse is sacrificed the day before fusion, and peritoneal trophoblast cells are extracted by injecting HAT medium into the peritoneal cavity under sterile conditions and seeded onto 96-well plates at 100 μL per well. These cells promote hybridoma cell growth. Immunized mice are sacrificed, and spleens are harvested under sterile conditions. Splenic B cells and SP2 / 0 myeloma cells are chemically fused using PEG. Appropriate amounts of HAT medium are added according to the number of cells to be seeded, and finally, the fused cells are seeded onto trophoblast cell culture plates at 100 μL per well.

[0105] After 7-10 days, the growth of surviving hybridoma cells can be observed under a microscope. Two weeks after plating, the supernatant from each well is collected, and hybridoma cells are screened using ELISA with CK14 peptide antigen. The method is as follows: Coat the ELISA plate with 100 μL of PBS solution containing 2 μg / ml CK14 peptide antigen and incubate at 37°C for two hours. After washing the plate three times with PBST, add 150 μL / well of PBS solution containing 3% skim milk powder and incubate overnight at 4°C. Wash the plate three more times, add 80 μL / well of hybridoma supernatant, incubate at 37°C for 1 hour, and then wash three more times. Add 100 μL / well of horseradish peroxidase-labeled goat anti-mouse secondary antibody diluted 1:8000, incubate at 37°C for 45 minutes, wash three times, and blot dry. Add 100 μL / well of TMB chromogenic solution, develop at room temperature for 5-10 minutes, stop with 2M sulfuric acid solution, and measure the absorbance at 450 nm for each well. Select positive hybridoma cells.

[0106] Select the ELISA-positive fusion wells and perform immunohistochemistry (IHC) to select the remaining positive wells for subsequent experiments. The experimental steps are as follows:

[0107] (1) Section processing: The esophageal squamous cell carcinoma sections were baked in a 60℃ constant temperature oven for 60 minutes. The sections were soaked in xylene I for 15 minutes, and then soaked in xylene II for 15 minutes. They were then soaked in anhydrous ethanol ① for 5 minutes, anhydrous ethanol ② for 5 minutes, 95% ethanol for 5 minutes, 85% ethanol for 5 minutes, 75% ethanol for 5 minutes, and ddH2O for 5 minutes. The sections were washed 3 times. Antigen retrieval was performed using a pressure cooker (boiling method). 10 mmol / L citrate buffer (pH 6.0) was added to the pressure cooker to submerge the sections. The pressure was heated to boiling. The sections were placed on a heat-resistant section rack and placed in the pot. The pot lid was closed and the pressure valve was closed. The heating was continued and the pressure was maintained for 4 minutes. After the time was up, the vent valve was opened to release the pressure. After the pressure returned to zero, the pot lid was opened and the inner pot was removed and placed to cool at room temperature. After the solution cools to room temperature, remove the sections (approximately 40 minutes); soak in ddH2O for 5 minutes, wash twice, soak in PBST for 5 minutes, wash twice; place the sections in 20 ml of 3% H2O2-methanol solution, protect from light, and treat at room temperature for 10 minutes; soak in PBST for 5 minutes, wash three times; add one drop (approximately 25 μl) of goat serum blocking solution to each tissue group, incubate in a humidified chamber at room temperature for 45 minutes; soak in PBST for 5 minutes, wash three times.

[0108] (2) Tissue sections are mixed with antibodies and incubated:

[0109] For comparison, the processed tissue sections were inoculated with CK14 antibody from Maixin Company, while the remaining sections were inoculated with antibodies secreted by the positive hybridoma cells. Incubation was performed overnight in a humidified chamber at 4°C; after removal from the 4°C freezer, incubation was carried out at room temperature for 60 minutes; after gentle rinsing with PBST, the sections were soaked for 5 minutes, and washed three times; 25 μL of HRP-labeled Long Island Company secondary antibody was added to each tissue group, and incubation was carried out at room temperature for 45 minutes; washing was performed; DAB chromogenic solution was prepared, and after reacting in the dark for 10-15 minutes, it was added to the sections, and chromogenic reaction was carried out for 1-5 minutes; the chromogenic reaction was terminated with distilled water; 50 μL of hematoxylin staining solution was added to each tissue group, and staining was carried out for 5-10 minutes, followed by rinsing with distilled water; the sections were then decolorized in 1% hydrochloric acid-ethanol for 2-3 seconds, quickly removed, and placed in distilled water to terminate the staining, followed by inversion in PBST (pH 8.0) for 5-10 minutes; and then soaked in 75% ethanol for 5 minutes, 85% ethanol for 5 minutes, 95% ethanol for 5 minutes, and anhydrous ethanol for 5 minutes respectively. Soak in xylene for 10 minutes, then replace with xylene and soak for another 10 minutes; add neutral resin to seal the slide, then cover with a coverslip; take a microscope image.

[0110] Fusion cells showing positive binding were selected using ELISA and IHC assays and cloned using the limiting dilution method. Each positive cell line was seeded into 48 / 96-well plates and cultured further. A second round of screening was performed using ELISA to identify hybridomas that specifically recognize the CK14 peptide and block CK14 binding. These hybridomas were then subcloned using the limiting dilution method to obtain a single-clone cell line, 10E1. This single-clone cell line was then expanded, and approximately 1 × 10⁻⁶ cells were collected. 6 One cell was injected into selected mice (the mice needed to be injected with paraffin oil into their peritoneum one week in advance), and after a waiting period of 7-10 days, the mice produced ascites. The ascites was collected for antibody purification. After purification, a mouse monoclonal antibody specifically against CK14 peptide was obtained.

[0111] Figure 1 Immunohistochemical assay for CK14 monoclonal antibody 10E1 Figure 2 For the immunohistochemical neutralization assay of CK14, Figure 1 and Figure 2 All the specimens tested were esophageal squamous cell carcinoma tissues, the difference being that they came from different parts of the esophageal squamous cell carcinoma tissues. Figure 1 In this example, A is the CK14-10E1 antibody produced in this embodiment, and B is the CK14 antibody from Maixin Company. Figure 2 In this example, A is the CK14-10E1 antibody produced in this embodiment, and B is the CK14 antibody from Maixin Company.

[0112] Depend on Figure 1 and Figure 2 The staining results showed no significant difference in staining sites and intensity between CK14-10E1 antibody and Maixin antibody. This confirms that the CK14 provided by this invention has promising applications in preparing kits.

[0113] Example 2

[0114] DNA cloning and sequencing were performed, including sequencing of the variable region gene of the anti-CK14 monoclonal antibody.

[0115] Total RNA was extracted from mouse monoclonal cell lines using Trizol reagent. Cells cultured in 9cm dishes were transferred to 1.5ml centrifuge tubes, and the supernatant was aspirated. 1ml of Trizol reagent was added, and the cells were lysed by pipetting. The lysed sample or homogenate was incubated at room temperature for 5-10 minutes to allow complete separation of nucleoproteins and nucleic acids. 0.2ml of chloroform was added, and the mixture was vigorously vortexed for 15 seconds, then incubated at room temperature for 3 minutes. The mixture was centrifuged at 12000 rpm at 4°C for 10 minutes. The upper aqueous phase was transferred to a clean centrifuge tube, and an equal volume of isopropanol was added. The mixture was incubated at room temperature for 20 minutes. The mixture was centrifuged at 12000 rpm at 4°C for 10 minutes, and the supernatant was discarded. The precipitate was washed with 1ml of 75% ethanol. The mixture was centrifuged at 12000 rpm at 4°C for 3 minutes, and the supernatant was discarded. The mixture was dried at room temperature for 5-10 minutes. 30-50ul of RNase-free ddH2O was added. The resulting RNA solution was stored at -70°C or used for subsequent experiments.

[0116] Total RNA was reverse transcribed into cDNA using the AMV first-strand cDNA synthesis kit. The experimental configuration was as follows: 6 μL total RNA + 1 μL Oligo dT + 4 μL RNase-free water (total 11 μL). After gentle mixing, centrifuge for 3-5 seconds. The reaction mixture was pre-denatured at 65°C for 5 minutes, then incubated on ice for 30 seconds, centrifuged for 3-5 seconds, and then incubated on ice for 2 minutes. While in the ice bath, 4 μL of 5X buffer + 1 μL of dNTP mixture + 1 μL of RNase inhibitor + 1 μL of reverse transcriptase (total 20 μL) was added. After gentle mixing, centrifuge for 3-5 seconds, and then incubated on a PCR instrument at 42°C for 50 minutes and 85°C for 5 minutes to complete cDNA synthesis. Random primers are suitable for the synthesis of short-strand cDNAs under 500 bp. The transcribed RNA template does not require a poly(A) tail and can transcribe the 5' end region.

[0117] PCR amplification of the light and heavy chains. For amplifying the variable region sequence of the antibody light chain, the PCR reaction system was prepared as follows: 25 μL 2x Taq enzyme buffer + 1 μL FP-VL + 1 μL RP-VL + 2 μL cDNA + 21 μL ddH2O. For amplifying the variable region sequence of the antibody heavy chain, the PCR reaction system was prepared as follows: 25 μL 2x Taq enzyme buffer + 1 μL FP-VH + 1 μL RP-VH + 2 μL cDNA + 21 μL ddH2O. The temperature cycling for PCR amplification of the variable regions of the heavy and light chains was as follows (steps 2 to 4 were repeated for 35 cycles):

[0118] Step 1 - Pre-denaturation: 94℃, 4 min;

[0119] Step 2 - Denaturation at 94°C for 30 seconds;

[0120] Step 3 - Annealing at 55°C for 45 seconds;

[0121] Step 4 - Extend at 72°C for 60 seconds;

[0122] Step 5: 72℃, 10 min;

[0123] Step 6 - Store at 4℃.

[0124] PCR products were analyzed by 1% agarose gel electrophoresis, and DNA bands of corresponding sizes were excised (approximately 375 bp for VH and approximately 325 bp for VL). DNA extraction was performed using the SanPrep DNA Gel Extraction Kit. The procedure is briefly described as follows: A gel block containing the target fragment was excised from the agarose gel and weighed; 3-6 times the weight of the gel block was added to buffer B2, and the gel was incubated at 50°C for 5-10 minutes to dissolve; the solution was transferred to an adsorption column and centrifuged at 8000g for 30 seconds; the liquid in the collection tube was discarded; 500 μL of wash solution was added to the column, and the column was centrifuged at 9000g for 30 seconds, and the liquid in the collection tube was discarded; the wash solution was added again, and the liquid was discarded; the adsorption column was centrifuged at 9000g for 1 minute; the adsorption column was placed in a clean 1.5 ml centrifuge tube, and 15-40 μL of Elution Buffer was added to the center of the adsorption membrane. After standing at room temperature for 1 minute, the column was centrifuged for 1 minute. The prepared DNA solution was obtained, and the PCR product was purified and sequenced to obtain the variable region sequence of the antibody.

[0125] The variable region of the light chain is shown in SEQ ID NO:15, and the variable region of the heavy chain is shown in SEQ ID NO:16.

[0126] Experimental Example 1

[0127] This experimental example tests the affinity and sensitivity of the antibody prepared in Example 1 above.

[0128] 1. The antigens in Example 1 were packaged into plates at concentrations of 3 mg / L, 1.5 mg / L, 0.75 mg / L, and 0.375 mg / L, respectively.

[0129] 2. Adjust the antibody concentration to 10. -7 mol / L level (1*10) -7 Up to 5*10 -7 (Mol / L is acceptable). Then serially dilute 1:2 to 1:256 and add to wells with different antigen coating amounts.

[0130] 3. Add secondary antibody and develop TMB colorimetric assay. Measure the absorbance at 450 nm; the data are shown in Table 1.

[0131] 4. Based on the antigen-antibody binding S-curve, determine the antibody concentration with the half-maximum absorbance at different antigen concentrations. This will result in four antibody concentrations (mol / L).

[0132] 5. Substitute the values ​​into the formula K = (N-1) / (N*AB'-AB) to calculate the affinity constant. AB' and AB are the antibody concentrations that produce the half-maximum absorbance at the corresponding antigen concentrations AG (3 mg / L, 1.5 ml / L, 0.75 mg / L, 0.375 mg / L). N = AG / AG' (AG > AG').

[0133] 6. When N=2, we get three K values: 1.385, 2.702, and 5.882. When N=4, we get two K values: 2.051 and 4.225. When N=8, we get one K value: 3.267. The average of the six K values ​​is 3.2525 × 10⁻⁶. 9 L / mol.

[0134] Table 1. Statistical table of absorbance values ​​under different treatments.

[0135]

[0136] Experiment Example 2

[0137] In this experimental example, an immunohistochemical neutralization experiment was performed: a certain amount of CK14 antigen was mixed with the purified CK14-10E1 antibody obtained from screening in Example 1, and the mixture was neutralized at 37°C for 1 hour. Following the immunohistochemical experimental procedure, the antibody was added as the primary antibody to the esophageal squamous cell carcinoma section and incubated overnight at 4°C. Afterward, immunohistochemical staining was performed according to the same procedure as in Example 1, and the slides were photographed.

[0138] Figure 2 C represents the results of the neutralization experiment. In the neutralization experiment, the antigen reaction bound the CK14-10E1 antibody, which could not bind to the antigen in the cancer tissue, resulting in almost no staining. It can be seen that the anti-CK14 polypeptide monoclonal antibody CK14-10E1 provided in this embodiment can recognize and bind to the CK14 polypeptide in human cells, causing the cytoplasm of lung adenocarcinoma tissue to turn brown in subsequent IHC staining experiments, and showing no significant difference from the CK14 antibody from Maixin Company.

[0139] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antibody against CK14 protein or its antigen-binding fragment, characterized in that, It includes a heavy chain complementarity-determining region (CDR-H1) and a light chain complementarity-determining region (CDR-H2). The heavy chain complementarity-determining region includes CDR-H1, CDR-H2, and CDR-H3, and the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are shown in SEQ ID NO: 9-11, respectively. The light chain complementarity-determining region includes CDR-L1, CDR-L2, and CDR-L3, and their amino acid sequences are shown in SEQ ID NO: 12-14, respectively.

2. The antibody against CK14 protein or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment further includes a heavy chain framework region and a light chain framework region; the heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4, which have at least 80% homology with the amino acid sequences shown in SEQ ID NO:5-8; the light chain framework region includes LFR1, LFR2, LFR3 and LFR4, which have at least 80% homology with the amino acid sequences shown in SEQ ID NO:1-4.

3. The antibody against CK14 protein or its antigen-binding fragment according to claim 1, characterized in that, The light chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO: 15, and the heavy chain variable region is shown in SEQ ID NO:

16.

4. The antibody against CK14 protein according to claim 1, or its antigen-binding fragment, is characterized in that, The antibody or its antigen-binding fragment further includes a constant region, which includes a heavy chain constant region and / or a light chain constant region. The heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ-type or λ-type light chain constant region.

5. The antibody against CK14 protein according to claim 4, or its antigen-binding fragment, is characterized in that, The species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

6. The antibody against CK14 protein according to claim 4, or its antigen-binding fragment, is characterized in that, The species source of the constant region is mice.

7. The antibody against CK14 protein according to any one of claims 1-6, or its antigen-binding fragment, is characterized in that, The antigen-binding fragment is selected from any one of the antibody's F(ab')2, Fab', Fab, Fv, Fab'-SH, and scFv.

8. The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-7 in the preparation of any one of the following products: (1) Tumor prognostic assessment product; the tumor is esophageal squamous cell carcinoma; (2) CK14 protein detection products; (3) CK14 protein isolate or enrichment products; The products in applications (1)-(2) are reagents, kits, test strips, antibody chips, antibody probes or detectors; the separation or enrichment products in application (3) are magnetic beads, kits or separation columns.

9. The application according to claim 8, characterized in that, In the applications (1)-(2), the antibody or its antigen-binding fragment is labeled with a detectable marker.

10. The application according to claim 9, characterized in that, In the applications (1)-(2), tissues and / or cells are labeled with the antibody or its antigen-binding fragment, and the labeled tissues and / or cells are detected.

11. The application according to claim 10, characterized in that, After the antibody or its antigen-binding fragment labels tissues and / or cells, the secondary antibody is incubated with the labeled tissues and / or cells, and the incubation product is detected.

12. The application according to claim 11, characterized in that, The antibody or its antigen-binding fragment is labeled with at least one of the following markers: fluorescent dyes, enzymes that catalyze substrate color development, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

13. A product for tumor diagnosis, efficacy evaluation, prognostic assessment, or recurrence / metastasis monitoring, characterized in that, The product comprises: the antibody or antigen-binding fragment thereof as described in any one of claims 1-7; The products mentioned are reagents, kits, test strips, antibody chips, antibody probes, or detectors.

14. A CK14 protein detection product, CK14 protein isolation product, or enrichment product, characterized in that, The product comprises: the antibody or antigen-binding fragment thereof as described in any one of claims 1-7; The CK14 protein detection products include reagents, kits, test strips, antibody chips, antibody probes, or detectors. The CK14 protein isolation or enrichment product is a magnetic bead, a kit, or a separation column.

15. A cell characterized in that, The cells are non-plant cells that express the antibody or antigen-binding fragment of any one of claims 1-7.

16. A method for preparing an antibody or antigen-binding fragment thereof as described in any one of claims 1-7, characterized in that, It includes: The cells of claim 15 are cultured, and the antibody or its antigen-binding fragment is isolated and purified from the culture product.

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