An antibody against p53 protein, a tumor diagnostic product and application thereof

Abstract

The application discloses an antibody against P53 protein, a tumor diagnosis product and application thereof, and relates to the technical field of antibodies. The antibody comprises a heavy chain complementarity determining region in a heavy chain variable region shown in SEQ ID NO: 14 and a light chain complementarity determining region in a light chain variable region shown in SEQ ID NO: 15. The antibody can be used for diagnosing diseases with P53 as a marker. The application provides more protein options for the detection of P53 and the diagnosis of diseases with P53 as a marker, and enriches the types of monoclonal antibodies against P53 protein. In addition, the antibody provided by the application can be used for preparing a tumor efficacy evaluation product, a prognosis evaluation product or a recurrence and metastasis monitoring product.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and more specifically, to an antibody against P53 protein, a tumor diagnostic product, and its applications. Background Technology

[0002] P53 is a tumor suppressor gene that encodes a 393-amino acid nucleoprotein. Its protein band appears at 53 kDa, as indicated by the marker band, hence the name P53. The encoded protein exists in two subtypes: wild-type and mutant. The wild-type p53 induces apoptosis in cancer cells, thus preventing carcinogenesis, and also helps repair defects in cellular genes. The mutant p53 increases the risk of cancer. P53 is a very important anti-cancer gene.

[0003] The p53 protein is mainly distributed in the nucleus and cytoplasm, and can specifically bind to DNA. Its activity is regulated by post-translational modifications such as phosphorylation, acetylation, methylation, and ubiquitination. The normal biological function of p53 is to check for DNA damage sites during the G1 phase and monitor the integrity of the genome. If damage is found, the p53 protein inhibits DNA replication to provide sufficient time for repair. If repair fails, the p53 protein induces apoptosis. If the p53 gene mutates, its spatial conformation changes, and it loses its regulatory role in cell growth, apoptosis, and DNA repair. The p53 gene transforms from a tumor suppressor gene into an oncogene, leading to cell carcinogenesis.

[0004] The p53 gene is the gene most strongly associated with human tumors discovered to date. Studies have shown that more than half of solid tumor cells have gene mutations or loss of function in the p53 protein. If p53 gene mutations occur early in tumor development, it aids in early tumor diagnosis. Furthermore, individuals with p53 gene mutations often respond poorly to radiotherapy and chemotherapy and are more prone to metastasis, making it an indicator of treatment efficacy and prognosis. Therefore, immunohistochemical detection of mutant p53 plays a crucial role in the study of various malignant tumors.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide an antibody against P53 protein, a tumor diagnostic product, and its application to solve the above-mentioned technical problems.

[0007] This invention is implemented as follows:

[0008] In a first aspect, the present invention provides an antibody against P53 protein or an antigen-binding fragment thereof, comprising a heavy chain complementarity-determining region in the heavy chain variable region as shown in SEQ ID NO: 14, and a light chain complementarity-determining region in the light chain variable region as shown in SEQ ID NO: 15.

[0009] Secondly, the present invention also provides the use of antibodies or antigen-binding fragments thereof in the preparation of any of the following products:

[0010] (1) Tumor diagnostic products;

[0011] (2) Tumor auxiliary diagnostic products;

[0012] (3) Products for evaluating the efficacy of tumor treatment;

[0013] (4) Tumor prognostic assessment products;

[0014] (5) Products for monitoring tumor recurrence and metastasis;

[0015] (6) P53 protein detection products;

[0016] (7) P53 protein isolation or enrichment products.

[0017] Thirdly, the present invention also provides a product for tumor diagnosis, auxiliary diagnosis, efficacy evaluation, prognosis evaluation, or recurrence and metastasis monitoring, the product comprising: the above-mentioned antibody or its antigen-binding fragment.

[0018] Fourthly, the present invention also provides a P53 protein detection product, a P53 protein isolation product or an enrichment product, the product comprising: the above-mentioned antibody or its antigen-binding fragment.

[0019] Fifthly, the present invention also provides a cell, which is a non-plant cell, expressing the above-mentioned antibody or its antigen-binding fragment.

[0020] 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.

[0021] The present invention has the following beneficial effects:

[0022] The anti-P53 protein antibody provided by this invention exhibits high activity and a high affinity for the P53 protein. It can be used to develop corresponding detection products, such as reagents, kits, and chips, for detecting the expression level of P53 protein in relevant tumor tissues, thereby improving the sensitivity and specificity of detection. This antibody can be used to diagnose diseases that use P53 as a biomarker. This invention provides more protein options for the detection of P53 and the diagnosis of diseases using P53 as a biomarker, enriching the types of monoclonal antibodies against the P53 protein.

[0023] Furthermore, the antibody provided by this invention can be used to prepare tumor efficacy assessment products, prognostic assessment products, or recurrence and metastasis monitoring products. Based on the high specific binding affinity between the P53 protein and the antibody, and based on the level of the P53 protein in the subject's sample, tumor efficacy assessment, prognostic assessment, or recurrence and metastasis monitoring can be achieved. Attached Figure Description

[0024] 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.

[0025] Figure 1 This is the antibody affinity curve;

[0026] Figure 2 Image showing the results of immunohistochemical staining of breast and ovarian cancer sections;

[0027] Figure 3 The figure shows the experimental results of immunohistochemical staining of breast cancer sections with the antibody provided in this invention and the commercially available P53 antibody. Detailed Implementation

[0028] 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.

[0029] 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.

[0030] 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."

[0031] 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 p53 protein.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] LCDR1 is CDR-L1, LCDR2 is CDR-L2, and LCDR3 is CDR-L3.

[0037] In a first aspect, the present invention provides an antibody against P53 protein or an antigen-binding fragment thereof, comprising a heavy chain complementarity-determining region in the heavy chain variable region (VH) as shown in SEQ ID NO: 14, and a light chain complementarity-determining region in the light chain variable region (VL) as shown in SEQ ID NO: 15.

[0038] VH, SEQ ID NO:14:

[0039] VQLQESGGGLVQPGRSMKLSCVASGFTFGNYWMNWVRQSPEKGLEWL AQIRLKSDNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYFCTAL DY WGQGTTVTVSS.

[0040] VL, SEQ ID NO:15:

[0041] DIELTQSPSLASQVRASPVSISCKSSQSLLDSDGKTHFNWLLQRPGQSPKR LIYLVSKLDSGVPDRFTGSGSGTDFTLKISKVEAEDLGVYYCWQGTHFPGT FGGGTKLEIK.

[0042] Inputting the above SEQ ID NO:14 and SEQ ID NO:15 sequences into the CDR identification system will yield the corresponding CDR sequences.

[0043] 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 P53 protein. Based on the good binding activity and affinity of this antibody, it can be used to develop detection products for P53 protein, such as detection reagents and kits. Using the binding protein of this invention to detect P53 protein can improve the sensitivity and specificity of detection. Furthermore, this antibody can be used to diagnose diseases that use P53 protein as a biomarker, which is beneficial for early detection and early intervention. This invention provides more protein options for the detection of P53 protein and the diagnosis of diseases using P53 protein as a biomarker.

[0044] In a preferred embodiment of the present invention, the above-mentioned binding protein and P53 protein have a K ≤ 3.27 × 10⁻⁶ ratio. 9 Affinity binding at L / mol.

[0045] The affinity constant is calculated using the formula K = (N-1) / (N*AB'-AB). AB' and AB are the antibody concentrations (mol / L) that produce the half-maximum absorbance at the corresponding antigen concentration AG. N = AG / AG' (AG>AG').

[0046] 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 CDR-H1 and CDR-H2 are shown in SEQ ID NO: 1-2, and the amino acid sequence of CDR-H3 is LDY; 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: 3-5.

[0047] name sequence serial number CDR-H1 NYWMN SEQ ID NO:1 CDR-H2 QIRLKSDNYATHYAESVKG SEQ ID NO:2 CDR-H3 LDY CDR-L1 KSSQSLLDSDGKTHFN SEQ ID NO:3 CDR-L2 LVSKLDS SEQ ID NO:4 CDR-L3 WQGTHFPGT SEQ ID NO:5

[0048] 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:10-13; 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:10-13.

[0049] 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:6-9; 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:6-9.

[0050]

[0051]

[0052] 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.

[0053] 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.

[0054] In a preferred embodiment of the present invention, the species source of the constant region is mice.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] Secondly, the present invention also provides the use of antibodies or antigen-binding fragments thereof in the preparation of any of the following products:

[0059] (1) Tumor diagnostic products;

[0060] (2) Tumor auxiliary diagnostic products;

[0061] (3) Products for evaluating the efficacy of tumor treatment;

[0062] (4) Tumor prognostic assessment products;

[0063] (5) Products for monitoring tumor recurrence and metastasis;

[0064] (6) P53 protein detection products;

[0065] (7) P53 protein isolation or enrichment products.

[0066] In a preferred embodiment of the present invention, the tumor is a tumor with overexpression of p53 protein or p53 gene mutation.

[0067] In a preferred embodiment of the present invention, the tumor with p53 gene mutation is a tumor with p53 gene deletion or p53 missense mutation.

[0068] In a preferred embodiment of the present invention, the solid tumor is selected from epithelial tumors;

[0069] In a preferred embodiment of the present invention, the epithelial tumor is selected from gastrointestinal cancer, uterine cancer, ovarian cancer, cervical cancer, lung cancer, adenocarcinoma, breast cancer, adenoma, or squamous cell carcinoma.

[0070] In a preferred embodiment of the present invention, the gastrointestinal cancer is selected from esophageal cancer, gallbladder cancer, gastric cancer, liver cancer, pancreatic cancer, bile duct cancer, small bowel cancer, colon cancer, rectal cancer, colorectal cancer, and anal cancer; the squamous cell carcinoma is one or more of oral squamous cell carcinoma, pharyngeal squamous cell carcinoma, laryngeal cancer, esophageal squamous cell carcinoma, squamous cell carcinoma of the lip, squamous cell carcinoma of the uterus, squamous cell carcinoma of the vagina, and squamous cell carcinoma of the skin.

[0071] In one alternative implementation, adenocarcinoma includes, but is not limited to, lung adenocarcinoma, thyroid cancer, salivary gland cancer, or pancreatic cancer.

[0072] In one alternative implementation, the adenoma includes, but is not limited to, cystic adenoma, fibroadenoma, pleomorphic adenoma, or polypoid adenoma.

[0073] In one alternative implementation, breast cancer includes, but is not limited to, breast epithelial carcinoma;

[0074] Lung cancer, preferably including small cell lung cancer (preferably stage I to IIIb), non-small cell lung cancer (preferably poorly to moderately differentiated squamous and adenocarcinoma), and large cell lung cancer.

[0075] Squamous cell carcinoma includes one or more of the following: oral squamous cell carcinoma, pharyngeal squamous cell carcinoma, laryngeal squamous cell carcinoma, esophageal squamous cell carcinoma (e.g., esophageal squamous cell carcinoma), squamous cell carcinoma of the lip, uterine squamous cell carcinoma, vaginal squamous cell carcinoma, and skin squamous cell carcinoma.

[0076] Oral squamous cell carcinoma (OSCC) is also known as oral squamous cell carcinoma. Examples of oral squamous cell carcinoma include, but are not limited to, tongue squamous cell carcinoma.

[0077] In other embodiments, the aforementioned squamous cell carcinoma can also be squamous cell carcinoma formed by squamous metaplasia of the bronchus, bladder, renal pelvis, etc.

[0078] In a preferred embodiment of the present invention, the tumor is one or more of breast cancer, prostate cancer, liver cancer, ovarian cancer, colorectal cancer, stomach cancer, and colon cancer.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.).

[0083] 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.

[0084] 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、 105 Rh、 177 Lu、 172 Lu and 18 F.

[0085] 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.

[0086] 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.

[0087] In optional embodiments, the colloid includes, but is not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latexes.

[0088] In optional embodiments, colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0089] In a preferred embodiment of the present invention, in application (7), the P53 protein detection is performed by binding protein-labeled tissues and / or cells, and the labeled tissues and / or cells are then detected.

[0090] Antibody chips are chips formed by immobilizing the aforementioned antibodies or their antigen-binding fragments on a carrier.

[0091] The antibody provided by this invention can be used as a coating antibody or a detection antibody.

[0092] Thirdly, the present invention also provides a product for tumor diagnosis and auxiliary diagnosis, the product comprising: the above-mentioned antibody or its antigen-binding fragment.

[0093] In a preferred embodiment of the present invention, the product is a reagent or a reagent kit.

[0094] Fourthly, the present invention also provides a P53 protein detection product, a P53 protein isolation product or an enrichment product, the product comprising: the above-mentioned antibody or its antigen-binding fragment;

[0095] In a preferred embodiment of the present invention, the P53 protein detection product is a reagent or kit;

[0096] In a preferred embodiment of the present invention, the P53 protein isolation or enrichment product is a magnetic bead, a reagent kit, or a separation column.

[0097] For example, the antibodies described above can be coated onto magnetic beads for the separation and enrichment of P53 protein. In one embodiment, the antibodies are coated onto packing material and packed into a separation column for affinity separation and enrichment of P53 protein. Therefore, the antibodies described above or their antigen-binding fragments have promising applications in the preparation of P53 protein enrichment products.

[0098] Fifthly, the present invention also provides a cell, which is a non-plant cell, expressing the above-mentioned antibody or its antigen-binding fragment.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0103] Example 1

[0104] This embodiment describes the preparation of a monoclonal antibody specifically targeting the P53 protein.

[0105] 1. Preparation of antigen

[0106] First, primer sequences for the P53 protein were designed. Then, PCR was performed to obtain the target gene. The target gene was then embedded in a plasmid. The plasmid was then introduced into DH5α competent cells using electroporation. The competent cells were then inoculated onto antibiotic-containing plates and placed in a 37°C oven for incubation. This process allowed for the selection of E. coli that had been introduced with the target gene. The E. coli were then sonicated and purified by filtration using a His-tagged nickel column to obtain the P53 protein, which is the antigen.

[0107] 2. Immunize the mice.

[0108] Mice were immunized with an immunogen using a standard method. The immunogen was a His-tagged recombinant human P53 protein, 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 MRP1 antigen. An 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 solution was emulsified thoroughly on an emulsifier 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 immunized intraperitoneally for final immunization, followed by cell fusion.

[0109] 3. Perform hybridoma cell fusion and screening.

[0110] The following preparations need to be made before cell fusion:

[0111] (1) Culture mouse myeloma cells SP2 / 0 to the logarithmic growth phase;

[0112] (2) One negative mouse was sacrificed the day before fusion. The peritoneal trophoblast cells of the mouse were injected into the peritoneal cavity of the mouse in a sterile environment with HAT medium and plated on a 96-well plate at 100 μL per well. These cells have a promoting effect on the growth of hybridoma cells.

[0113] Immunized mice were sacrificed, and spleens were harvested under sterile conditions. Spleen B cells and SP2 / 0 myeloma cells were chemically fused using PEG. Appropriate HAT medium was added according to the number of cells to be plated. Finally, the fused cells were plated on feeder cell culture plates at 100 μL per well.

[0114] 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 the p53 protein antigen. The method is as follows:

[0115] Coat an ELISA plate with 100 μL of PBS solution containing 2 μg / ml P53 protein antigen and incubate at 37°C for two hours. Wash the plate three times with PBST, then 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 1:7000, incubate at 37°C for 45 minutes, wash three times, and blot dry. Add 100 μL / well of TMB chromogenic solution, incubate at room temperature for 5-10 minutes, stop the reaction with 2M sulfuric acid solution, and measure the absorbance at 450 nm for each well. Select positive hybridoma cells.

[0116] Multiple strains were selected from the ELISA-positive fusion wells and then subjected to immunohistochemical staining (IHC). The positive wells were then selected for subsequent experiments.

[0117] ELISA and IHC assays were used to select fusion cells that showed positive binding, and these cells were 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 and IHC to identify hybridomas that specifically recognize p53 protein and can block p53 binding. These hybridomas were then subcloned using the limiting dilution method to obtain a single-clone cell line, 4G5. 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 7-10 days, the mice produced ascites. The ascites was collected for antibody purification. After purification, a mouse monoclonal antibody specifically against P53 protein was obtained.

[0118] Example 2

[0119] The binding ability of purified antibody p53-4G5 to p53 protein was determined by immunohistochemistry (IHC).

[0120] Ovarian serous carcinoma or breast cancer slides were baked in a 60°C oven for 60 minutes. The slides were then soaked in xylene I for 15 minutes, followed by xylene II for another 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, and 75% ethanol for 5 minutes. Finally, they were soaked in ddH2O for 5 minutes and washed three 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 slides. The mixture was heated to boiling. The slides were placed on a heat-resistant slide rack and placed in the pressure cooker. The lid was closed, the pressure valve was engaged, and the mixture was heated. The pressure was maintained for 4 minutes. After the time was up, the vent valve was opened to release the pressure. Once the pressure was zero, the lid was opened, the inner pot was removed, and the mixture was allowed to cool to room temperature.

[0121] 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.

[0122] For comparison, Roche's p53 antibody was added to the processed tissue sections, while the remaining sections were added to purified P53-4G5 antibody. 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 Biotechnology secondary antibody (CAT#:) 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 placed in 1% hydrochloric acid-ethanol for decolorization for 2-3 seconds, quickly removed, and placed in distilled water to terminate the reaction, 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 and mount the slide, then cover with a coverslip; photograph under a microscope. Figure 2 , Figure 3 As shown.

[0123] Immunohistochemical neutralization assay: A certain amount of P53 antigen protein was mixed with purified antibody and neutralized at 37°C for 1 hour. Following the immunohistochemical assay procedure, the primary antibody was added to the lung adenocarcinoma section and incubated overnight at 4°C. Immunohistochemical staining and imaging were then performed using the same procedure.

[0124] Figure 2 , Figure 3 The immunohistochemical assay for P53 was performed on ovarian and breast cancer tissues from different patients. Figure 2 In the diagram, A represents ovarian cancer tissue, and B represents breast cancer tissue. The antibody used is the p53-4G5 antibody produced in this invention. Figure 3 The tissue in question is breast cancer tissue, and A represents the p53-4G5 antibody used in this invention. Figure 3 In the text, B represents a P53 antibody from a domestic biotechnology company. Figure 3 The staining results show that the antibody provided by this invention has a stronger staining effect compared to the antibody from a certain biotechnology company. It can be seen that the anti-P53 protein monoclonal antibody P53-4G5 provided in Example 1 of this invention can recognize and bind to the P53 protein in human cells. In subsequent IHC staining experiments, it caused the cell nuclei in ovarian cancer tissue to turn brown, confirming that the p53-4G5 antibody provided by this invention has the potential for application in preparing reagent kits.

[0125] Example 3

[0126] This embodiment tests the affinity and sensitivity of the monoclonal antibody prepared in Example 1 above.

[0127] 1. The recombinant human p53 protein from Example 1 was plated at concentrations of 3 mg / L, 1.5 mg / L, 0.75 mg / L, and 0.375 mg / L.

[0128] 2. Adjust the concentration of the monoclonal antibody obtained in Example 1 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.

[0129] 3. Add secondary antibody and develop TMB colorimetric assay. Measure the absorbance at 450 nm. The absorbance data are shown in Table 1.

[0130] 4. Based on the S-curve of antigen-antibody binding, determine the antibody concentration with the half-maximum absorbance at different antigen concentrations. This results in four antibody concentrations (mol / L). 9.924*10 -10 mol / L, 6.611*10 -10 mol / L, 4.32*10 -10 mol / L, 2.766*10-10 mol / L.

[0131] 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').

[0132] 6. When N=2, three K values ​​can be obtained (x10) 9 The kinematic coefficients (k / mol) are 3.032, 4.929, and 8.251. When N = 4, two kinematic values ​​are obtained: 4.078 and 6.737. When N = 8, a single kinematic value of 5.736 is obtained. The average of the six kinematic values ​​is 5.46 * 10^6. 9 .

[0133] The affinity curve for the P53 antibody is as follows: Figure 1 As shown.

[0134] According to ELISA in OD 450 Based on the detection results, the half-absorbance values ​​and anti-N protein antibody concentrations at each antigen coating concentration were calculated, and the results are as follows:

[0135] Antigen coating concentration (AG) (ug / m³) <![CDATA[Half absorbance antibody concentration (AB) (10 -9 mol / L)]]> 3 0.9924 1.5 0.6611 0.75 0.432 0.3725 0.2766

[0136]

[0137] The affinity curve for the P53 antibody is as follows: Figure 1 As shown.

[0138] Table 1. Statistical table of absorbance values ​​at 450nm.

[0139]

[0140] Example 4

[0141] In this embodiment, the mouse monoclonal antibody screened in Example 1 above was cloned and sequenced to obtain the variable region gene sequence of the anti-human P53 monoclonal antibody.

[0142] 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 total RNA extraction reagent (Trizol) 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 and 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 obtained RNA solution was stored at -70°C or used for subsequent experiments.

[0143] 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.

[0144] 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 35 times):

[0145] Step 1 - Pre-denaturation: 94℃, 4 min;

[0146] Step 2 - Denaturation at 94°C for 30 seconds;

[0147] Step 3 - Annealing at 55°C for 45 seconds;

[0148] Step 4 - Extend at 72°C for 60 seconds;

[0149] Step 5: 72℃, 10 min;

[0150] Step 6 - Store at 4℃.

[0151] PCR products were analyzed by 1% agarose gel electrophoresis, and DNA bands of corresponding sizes were excised (approximately 355 bp for VH and approximately 335 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 dissolved in a 50°C water bath for 5-10 minutes; the dissolved gel 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.

[0152] The amino acid sequences of VH and VL of the mouse anti-human P53 protein monoclonal antibody secreted by the monoclonal cell line P53-4G5 are shown in the table below:

[0153]

[0154]

[0155] 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 P53 protein or an antigen-binding fragment thereof, 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. The amino acid sequences of CDR-H1 and CDR-H2 are shown in SEQ ID NO: 1-2, respectively, and the amino acid sequence of CDR-H3 is LDY. The light chain complementarity-determining region includes CDR-L1, CDR-L2, and CDR-L3. Their amino acid sequences are shown in SEQ ID NO: 3-5, respectively.

2. The antibody 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:10-13; 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:6-9.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO: 14, and the light chain variable region is shown in SEQ ID NO:

15.

4. The antibody or its antigen-binding fragment according to claim 1, 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 or its antigen-binding fragment according to claim 4, 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 or its antigen-binding fragment according to claim 4, characterized in that, The species source of the constant region is mice.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1-6, 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 auxiliary diagnostic product; the tumor is a tumor with p53 missense mutation; (2) Tumor efficacy assessment product; the tumor is a tumor with p53 missense mutation; (3) Tumor prognostic assessment product; the tumor is a tumor with a p53 missense mutation; (4) Products for monitoring tumor recurrence and metastasis; the tumor is a tumor with a p53 missense mutation; (5) P53 protein detection products; (6) P53 protein isolation or enrichment products; the products in the applications (1)-(5) are reagents, kits, test strips, antibody chips, antibody probes or detectors; the isolation or enrichment products in the application (6) are magnetic beads, kits or separation columns.

9. The application according to claim 8, characterized in that, In the applications (1)-(5), the antibody or its antigen-binding fragment is labeled with a detectable marker.

10. The application according to claim 8, characterized in that, In the application (6), the P53 protein detection is to label tissues and / or cells with the binding protein and to detect the labeled tissues and / or cells.

11. A product for tumor auxiliary diagnosis, efficacy evaluation, prognostic assessment, or recurrence and 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 tumor is a tumor with a p53 missense mutation; The products mentioned are reagents, kits, test strips, antibody chips, antibody probes, or detectors.

12. A P53 protein detection product, P53 protein isolation product, or enrichment product, characterized in that, The product includes: the antibody or its antigen-binding fragment as described in any one of claims 1-7; the P53 protein detection product is a reagent, kit, test strip, antibody chip, antibody probe or detector; The P53 protein isolation or enrichment product is a magnetic bead, a reagent kit, or a separation column.

13. 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.

14. 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 13 are cultured, and the antibody or its antigen-binding fragment is isolated and purified from the culture product.

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