A monoclonal antibody, FHY-001, for the treatment of nasopharyngeal carcinoma and its applications.

Abstract

This invention belongs to the field of biotechnology, specifically relating to a monoclonal antibody FHY-001 for the treatment of nasopharyngeal carcinoma and its applications. The monoclonal antibody FHY-001 comprises heavy chain variable regions of VHCDR1, VHCDR2, and VHCDR3, and light chain variable regions comprising VLCDR1, VLCDR2, and VLCDR1. This invention focuses on the research and development of monoclonal antibodies targeting specific and effective therapeutic targets for nasopharyngeal carcinoma. Specifically, it utilizes abnormal proteins encoded by mutated genes in nasopharyngeal carcinoma tumor cells, which can be recognized by immune cells and activate the body's immune response, or neoantigens derived from non-human normal genomes, to prepare specific novel antibodies for the treatment of nasopharyngeal carcinoma. The monoclonal antibody of this invention exhibits high specificity, specifically binding to human nasopharyngeal carcinoma tumor cells, and significantly inhibits the growth of nasopharyngeal carcinoma cells in vitro. It also has a high target binding rate, can be mass-produced, and can serve as a potential drug for the immunotherapy of nasopharyngeal carcinoma.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a monoclonal antibody FHY-001 for the treatment of nasopharyngeal carcinoma and its applications. Background Technology

[0002] Immune checkpoint inhibitors have become a research focus in the field of tumor immunotherapy, with the most representative drugs being inhibitors of programmed death 1 (PD-1) and programmed death-ligand 1 (PD-L1). These two drugs can enhance T cell activity by blocking the PD-1 / PD-L1 pathway, exerting a broad-spectrum anti-cancer effect and significantly prolonging patient survival. PD-1 is mainly expressed in activated T / B lymphocytes, NK cells, monocytes, dendritic cells (DCs), and mesenchymal stem cells (BMSCs), and is an important immunosuppressive molecule for maintaining autoimmune tolerance. Under physiological conditions, PD-1 regulates immune function by modulating the differentiation direction of T cells in peripheral tissues, controlling the body's immune response to foreign or self-antigens. The ligands PD-L1 and PD-L2 are both highly expressed in placental tissue and lowly expressed in the spleen, lymph nodes, and thymus, which is significant for maintaining peripheral immune tolerance and reducing the occurrence of autoimmune diseases. PD-L1, as the main ligand of PD-1, is overexpressed on malignant tumor cells and also expressed in APCs, lymphocytes, hematopoietic cells, and epithelial cells. Blocking the PD-1 / PD-L1 signaling pathway can upregulate T cell activation, activate the endogenous anti-tumor immune response, and thus exert a therapeutic effect on tumors. Among PD-1 monoclonal antibodies, a total of 15 PD-1 / PD-L1 related drugs have been approved globally, of which 6 are domestically produced and marketed, and another 3 domestically produced PD-1 monoclonal antibodies are in the application stage.

[0003] As broad-spectrum immunosuppressants, PD-1 / PD-L1 monoclonal antibodies have achieved good clinical results in many tumors, but their overall immune response rate of around 20% is relatively low. Therefore, only a small percentage of patients can benefit from PD-1 monoclonal antibodies. Thus, identifying new immunotherapy targets and developing bispecific immunotherapies is a highly promising solution. On the other hand, with the increasing number of domestic PD-1 / L1 monoclonal antibody data, the worsening competitive landscape, coupled with repeated negotiations on expanded indications and medical insurance coverage, the price of domestically produced PD-1 / L1 monoclonal antibodies is expected to continue to decline, leading to a decrease in net profit margins and a gradual shift towards generic drugs.

[0004] With the application of PD1 / PDL1 monoclonal antibodies in tumor immunotherapy, an increasing number of tumor treatments have been approved for clinical use by my country's drug regulatory agencies and the FDA, such as malignant melanoma, lung cancer, head and neck cancer, bladder cancer, kidney cancer, and Hodgkin's lymphoma. However, immunotherapy for solid tumors still faces many unresolved challenges, such as the lack of effective means to identify individualized tumor-specific targets. This invention focuses on the research and development of monoclonal antibodies targeting specific and effective therapeutic targets for nasopharyngeal carcinoma. Specifically, it utilizes abnormal proteins encoded by mutated genes in nasopharyngeal carcinoma cells that can be recognized by immune cells and activate the body's immune response, or neoantigens derived from non-human normal genomes, to prepare specific new antibodies for the treatment of nasopharyngeal carcinoma. Summary of the Invention

[0005] The purpose of this invention is to provide a monoclonal antibody, FHY-001, for the treatment of nasopharyngeal carcinoma.

[0006] According to a specific embodiment of the present invention, the monoclonal antibody FHY-001 for treating nasopharyngeal carcinoma comprises heavy chain variable regions of VHCDR1, VHCDR2, and VHCDR3, and light chain variable regions comprising VLCDR1, VLCDR2, and VLCDR3.

[0007] The amino acid sequence of VHCDR1 is shown in SEQ ID NO.1: GYTFTT.

[0008] The amino acid sequence of VHCDR2 is shown in SEQ ID NO.2: NTYSGV.

[0009] The amino acid sequence of VHCDR3 is shown in SEQ ID NO.3: GNYYFDY.

[0010] The amino acid sequence of VLCDR1 is shown in SEQ ID NO.4: KSVSTSGYSY.

[0011] The amino acid sequence of VLCDR2 is shown in SEQ ID NO.5: LASSNLES.

[0012] The amino acid sequence of the VLCDR3 is shown in SEQ ID NO.6: QHSREL.

[0013] The monoclonal antibody of the present invention also includes functional variants of the antibody, all of which can specifically bind to the antigen.

[0014] Specifically, if the functional variant includes (but is not limited to) derivatives that are substantially similar in primary structural sequence but contain chemically and / or biochemically modified derivatives not present in the parental monoclonal antibody of the present invention, such as those modified in vitro or in vivo. These modifications include, for example, acetylation, acylation, covalent linking of nucleotides or nucleotide derivatives, covalent linking of lipids or lipid derivatives, crosslinking, disulfide bond formation, glycosylation, hydroxylation, methylation, oxidation, polyethylene glycolation, proteolytic treatment, phosphorylation, etc.

[0015] This invention also provides the encoding gene of the above-mentioned monoclonal antibody FHY-001 for treating nasopharyngeal carcinoma.

[0016] The heavy chain variable region encoding VHCDR1 has the nucleotide sequence shown in SEQ ID NO.7.

[0017] ggg tat acc ttc aca acc,

[0018] The heavy chain variable region encoding VHCDR2 has the nucleotide sequence shown in SEQ ID NO. 8.

[0019] aac acc tac tct gga gtg,

[0020] The heavy chain variable region encoding VHCDR3 has the nucleotide sequence shown in SEQ ID NO. 9.

[0021] ggt aac tac tac ttt gac tac,

[0022] The light chain variable region encoding VLCDR1 has the nucleotide sequence shown in SEQ ID NO.10.

[0023] aaa agt gtc agt aca tct ggc tat agt tat,

[0024] The light chain variable region encoding the VLCDR2 has the nucleotide sequence shown in SEQ ID NO.11.

[0025] ctt gca tcc aac cta gaa tct,

[0026] The light chain variable region encoding the VLCDR3 has the nucleotide sequence shown in SEQ ID NO.12.

[0027] cag cac agt agg gag ctt.

[0028] The present invention provides a vector containing the above-mentioned encoding gene.

[0029] The present invention also provides a host cell containing the above-described encoding gene or the above-described vector.

[0030] Preferably, the present invention provides the use of the above-mentioned monoclonal antibody FHY-001 in the preparation of drugs for treating nasopharyngeal carcinoma.

[0031] The present invention relates to a formulation for treating nasopharyngeal carcinoma, wherein the formulation uses the aforementioned monoclonal antibody FHY-001 as an active ingredient, and the formulation is a drug or a drug composition.

[0032] The aforementioned formulations may include pharmaceutical excipients, diluents, or carriers, and may be used in any suitable manner for patients requiring treatment. Suitable methods include, but are not limited to, oral, rectal, nasal, topical (including oral and sublingual), subcutaneous, or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, and intrathecal) administration.

[0033] The present invention will be further described below: Unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. Additionally, to better understand the present invention, definitions and explanations of relevant terms are provided below.

[0034] The term "antibody" as used in this invention includes complete antibodies and any antigen-binding fragments (i.e., "antigen-binding portions") or single chains thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds, or its antigen-binding portion. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains CH1, CH2, and CH3. Each light chain consists of a light chain variable region and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are scattered in more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that can interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (C1q) of the classical complement system.

[0035] In this invention, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a formulation of an antibody molecule consisting of a single molecule. Monoclonal antibody compositions exhibit single-molecule binding specificity and affinity for a specific epitope.

[0036] The beneficial effects of this invention are:

[0037] The monoclonal antibody provided by this invention has good specificity, can specifically bind to some human nasopharyngeal carcinomas, has a high binding rate, and can be produced on a large scale, making it a potential drug for immunotherapy of solid tumors. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 Displays the CDC test results for HFY-001;

[0040] Figure 2 The results of the complement-induced kill rate test of HFY-001 are displayed;

[0041] Figure 3 The immunohistochemical results of HFY-001 are shown. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0043] The main reagents used in the experiment and their sources were as follows: RPMI 1640 culture medium (Hyclone), Australian fetal bovine serum (Gibco), HRP goat anti-mouse secondary antibody (Landu), TMB (Aladdin), trypsin (Solepro), additive factor (Biolong), PBS (Zhongke Maichen), lactate dehydrogenase cytotoxicity assay kit (Beyotime), HT (50X) (Sigma), HAT (Sigma), BSA (bovine serum albumin) (Solepro), immunohistochemistry kit (Proteintech), and routine chemical reagents were all purchased from Sinopharm.

[0044] Example 1: Preparation of Monoclonal Antibodies

[0045] 1. Preparation of antigens and immunization in mice

[0046] 1.1 Preparation of antigen: Tumor cells were incubated with pH 3.3 phosphate citrate buffer for 3 min to remove organic salts from the washing solution, and then separated by Sephadex G25 gel separation column and Centricon ultrafiltration equipment to obtain antigen.

[0047] 2. Immune Strategy

[0048] ① Three mice aged 6-8 weeks were selected from each group and subjected to live cell immunization and dead cell immunization, respectively;

[0049] ② Each group of mice was immunized once a week, for a total of three immunizations, injected into the thigh muscle and abdominal cavity, each dose was 50ug, the third dose was 100ug. One week after the last injection, the antiserum was tested. After passing the test, the cell fusion experiment was carried out.

[0050] 3. Preparation of hybridoma cells and screening of monoclonal antibodies

[0051] 3.1 Obtaining SP2 / 0 tumor cells (in vitro proliferation)

[0052] (1) The frozen tumor cells were thawed and cultured in a 37°C, 5% CO2 incubator;

[0053] (2) To be cultured to a suitable quantity (1×10) 6 - 5×10 6 Cells were collected using a cell scraper and centrifuged at 1100 rpm for 6 minutes.

[0054] (3) Discard the supernatant, add an appropriate amount (20 ml) of serum-free 1640 medium to resuspend the cells, count them with a counting chamber, and store them in a 4 ℃ refrigerator (or incubator) for later use.

[0055] 3.2 Obtaining spleen cells

[0056] (1) After the mice were euthanized by cervical dislocation, they were soaked in a beaker containing 75% alcohol for 5 minutes.

[0057] (2) Transfer the beaker to the clean bench, take out the mouse, place it on the foam board with its abdomen facing up, fix the limbs with a needle, and cut open the abdomen (first cut open the skin, then cut open the membrane with new scissors and tweezers).

[0058] (3) Replace the scissors and tweezers to remove the spleen (drip some alcohol on it before removing it).

[0059] (4) Place the spleen in a petri dish containing 1640, poke a hole with a needle (to allow the cells to overflow), and crush the spleen with the piston handle.

[0060] (5) Use a pipette to draw up solid tumor cell fluid and filter it through a cell sieve into another petri dish. Filter twice (wash the petri dish with 1640). Use a different pipette each time you filter.

[0061] (6) Transfer the 1640 culture medium containing spleen cells to a centrifuge tube and add 1640 to about 35 mL. Centrifuge at 1200 rpm for 10 min, discard the supernatant, resuspend in 35 mL of RPMI-1640 basal culture medium, centrifuge again, and repeat three times to wash away red blood cells and connective tissue from the cells.

[0062] 3.3 Cell Fusion

[0063] (1) Mix spleen cells and tumor cells in a certain ratio (5-10 times the number of spleen cells and tumor cells), and centrifuge at 1200 rpm for 10 min.

[0064] (2) Discard the supernatant, resuspend in 1640 rpm, and centrifuge at 1300 rpm for 10 min.

[0065] (3) Pour out the supernatant, invert the tube to drain the water, and use absorbent paper to remove the remaining culture medium from the tube wall.

[0066] (4) Place the centrifuge tube in a 37°C water glass for incubation. Use a 1 mL pipette to add PEG 1500 incubated at 37°C to the mixed cells within 1 min, while shaking the centrifuge tube. Add 1 mL of PEG 1500 incubated at 37°C within 1 min. Add 3 mL of PEG 1500 incubated at 37°C within 3 min. Slowly add 30 mL of PEG 1500 incubated at 37°C, place the tube in a 37°C incubator, and let it stand for 5 min.

[0067] 3.4 Indirect non-competitive ELISA detection

[0068] ① Coating: The prepared antigen is plated into an ELISA plate and fixed at 37°C with 2.5% glutaraldehyde at 4°C for 2-4 h, and then washed once with PBST buffer.

[0069] ② Blocking: Use L-tyrosine and BSA as blocking solution, add 100 μL / well to the microplate wells, incubate at 37℃ for 1 h, wash the plate once with PBST, and dry.

[0070] ③ Add primary antibody: After the cells are fused and grow into cell clones (or subclones) to a suitable size, take the supernatant and add it to the corresponding ELISA plate, 100 μL per well, incubate at 37°C for 1 h, manually wash the ELISA plate twice with PBST (gently to prevent the fixed cells from drifting away), and then tap dry.

[0071] ④ Add secondary antibody: Add goat anti-mouse enzyme-labeled secondary antibody diluted with PBST solution (1 μL of goat anti-mouse enzyme-labeled secondary antibody is added to 10 mL of PBST), 100 μL / well, incubate at 37℃ for 1 h, wash the enzyme-labeled plate 4 times with PBST, and then dry.

[0072] ⑤ Color development and termination: Add color development solution (citric acid buffer solution) to develop the color. The color development solution should be prepared fresh and used immediately, 100 μL / well, and incubate at 37°C for 15 min in the dark. Add 2 mol / L H2SO4 at a rate of 50 μL / well to terminate the color development reaction. Then immediately measure the absorbance (OD) value of the reaction solution at 450 nm using a microplate reader. (If using solutions A and B, add 50 μL of solution A first, followed by 50 μL of solution B to each well).

[0073] ⑥ Result interpretation: Select 2-3 wells with the highest OD values ​​from each plate for subcloning until the results of the entire plate are consistent. Select the best well for single-cell cloning. Once the cells have grown well and the test data are qualified, they can be used for identification.

[0074] 3.5 Preparation and purification of antibodies

[0075] Cell lines were injected into mice and allowed to accumulate ascites. The cells were then collected, purified, and used for later use. Antibody purification was performed using a 5 mL HiTrap r-Protein A FF pre-packed chromatography column. An appropriate amount of ascites fluid was collected, centrifuged at 10,000 rpm for 15 min, and the supernatant was collected. After filtration through a 0.22 μm microporous membrane, the supernatant was loaded with 20 mmol / L phosphate buffer and eluted with 0.1 mol / L glycine-hydrochloric acid buffer. The pH and ionic strength of the loading buffer and the pH of the elution buffer were carefully selected to control the sample flow rate.

[0076] The purity of monoclonal antibodies was determined by SDS-PAGE. The separating gel concentration was 12%, the stacking gel concentration was 4%, and the samples were treated in a reducing state. After electrophoresis, the samples were stained with Coomassie Brilliant Blue R250, destained with ethanol and acetic acid, and then the purity was analyzed by scanning with a gel imaging system.

[0077] Cell culture for monoclonal antibody production: After monoclonal antibody cell line establishment, the amount of fetal bovine serum (FBS) added to the basal culture medium (RPMI 1640) was gradually reduced in square flasks, in the following order: 20% → 15% → 10% → 7.5% → 5% → 2.5%. Cells were passaged twice at each serum concentration. Serum-free medium (Hyperbolic acid) was added starting at 2.5%. TM-B100 (Shanghai Bei'anji) was used for acclimatization culture. The proportion of serum-free medium added was as follows: 20%→40%→60%→80%→90%→100%. When the proportion of serum-free medium added was 80%, the cells were transferred from the square bottle to the shake flask for culture. The cells were adapted to two passages at each serum-free medium concentration, and finally the cells were able to grow in suspension in a completely serum-free medium.

[0078] Suspension-grown cells were cultured in serum-free medium through a stepwise scale-up process, with 1L of cultured in 3L shake flasks for a total of 5 flasks. Culture was stopped when the cell viability reached approximately 90%. The supernatant was collected by centrifugation, with a net weight of 4.902 kg. The supernatant was then concentrated by ultrafiltration through a 30KD membrane, yielding 373 g of antibody solution with a protein content of 6.38 mg / ml, a total protein content of 2.38 g, and an expression level of 0.47 g / L.

[0079] 4. Identification of monoclonal antibodies

[0080] Hybridoma cells that grew well in serum-free culture medium were selected for sequencing. Bioinformatics analysis showed that the nucleic acid sequence encoding the antibody heavy chain variable region is shown in SEQ ID NO.13, the amino acid sequence encoding the antibody heavy chain variable region is shown in SEQ ID NO.14, the nucleotide sequence encoding the antibody light chain variable region is shown in SEQ ID NO.15, and the amino acid sequence encoding the antibody light chain variable region is shown in SEQ ID NO.16.

[0081] The nucleotide sequence encoding the variable region of the antibody heavy chain, SEQ ID NO.13, is as follows:

[0082] Cagatccagttggtacagtctggacctgagctgaagaagcctggagagacagtcaagatctcctgcaaggcttctgggtataccttcacaacctatggaatgagctgggtgaaacaggctccaggaaagggtttaaagtggatgggctggataaacacctactctggagtgcca acatatgctgatgacttcaagggacggtttgccttctctttggaaacctctgccagcactgcctatttgcagatcaacaacctcaaaaatgaggacacggctacatatttctgtgcaagaggtaactactactttgactactggggccaaggcaccactctcacagtctcctca

[0083] The amino acid sequence encoding the variable region of the antibody heavy chain, SEQ ID NO.14, is as follows:

[0084] QIQLVQSGPELKKPGETVKISCKASGYTFTTYGMSWVKQAPGKGLKWMGWINTYSGVPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARGNYYFDYWGQGTTLTVSS

[0085] The nucleotide sequence encoding the variable region of the antibody light chain, SEQ ID NO.15, is as follows:

[0086] Gacattgtgctgacacagtctcctgcttccttagctgtatctctggggcagagggccaccatctcatgcagggccagcaaaagtgtcagtacatctggctatagttatatgcactggtaccaacagaaaccaggacagccacccaaactcctcatctatcttgcat ccaacctagaatctggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggatgctgcaacctattactgtcagcacagtagggagcttccgctcacgttcggtgctgggaccaagctggagctgaaa

[0087] The amino acid sequence encoding the variable region of the antibody light chain, SEQ ID NO.16, is as follows:

[0088] DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLELK

[0089] Example 2: Functional Detection of Monoclonal Antibody FHY-001

[0090] 1. Detection of tumor cells in vitro

[0091] 1.1 Detection Method: After target cells carrying specific antigens bind to antibodies, they induce cell membrane damage with the participation of complement. The activity of LDH released from the ruptured cell membrane is detected to determine the quantitative analysis method for cytotoxicity. The specific steps are briefly described below:

[0092] 1.1.1 Nasopharyngeal carcinoma cells CNE1, CNE2, 5-8F, and 6-10B were induced at a dose of 1.2 × 10⁻⁶. 4 / Place 96-well plates and incubate overnight in an incubator;

[0093] 1.1.2 Remove the culture medium and wash once with PBS. Add different concentrations of antibody (57.08 μg / mL, 28.54 μg / mL, 14.27 μg / mL, 7.135 μg / mL) and rabbit serum at different dilutions. The antibody was diluted with serum-free medium, and the complement was diluted with PBS. In the control group and the maximum enzyme activity control group, 100 μL of basal medium and 100 μL of PBS were added in the early stage. After adding the corresponding antibody and complement, the mixture was incubated at 37°C in a 5% CO2 incubator for 30 min, and then the complement dissolution experiment was performed.

[0094] 1.1.3 Complement dissolution experiment: Add 20 μL of LDH release reagent to the maximum enzyme activity control group, mix thoroughly by repeated pipetting, and continue incubation;

[0095] 1.1.4 Take 120 μL of the supernatant from each well and add it to a new 96-well plate. Then perform sample testing. At this time, a background blank control well needs to be added to the 96-well plate to be tested.

[0096] 1.1.5 Prepare the LDH detection working solution according to the product instructions. Add 60 μL of working solution to each well. The working solution should be prepared fresh for each use and protected from light during preparation and use. Mix well and incubate at room temperature (about 25°C) in the dark for 30 min. Then measure the absorbance at 490 nm. Use 600 nm or any wavelength greater than 600 nm as the reference wavelength for dual-wavelength measurement.

[0097] 1.1.6 Calculation: Cytotoxicity or mortality rate (%) = (Absorbance of treated sample - Absorbance of sample control well) / (Absorbance of maximum enzyme activity of cells - Absorbance of sample control well) × 100.

[0098] 1.2 Test Results

[0099] Antibodies can effectively induce complement-dependent cytotoxicity (CDC), causing strong cell damage.

[0100] like Figure 1 As shown, when a fixed concentration of complement (rabbit serum) was added, the killing rate of nasopharyngeal carcinoma tumor cells was significantly increased after treatment with different concentrations of FHY-001, indicating that FHY-001 can induce a strong CDC effect, leading to tumor cell death.

[0101] Similarly, after fixing the antibody treatment concentration, complement (rabbit serum) was added at different dilutions, and the results were as follows. Figure 2 As shown, the mortality rate of tumor cells decreases with decreasing complement concentration.

[0102] Example 3 Immunohistochemical Experiment of Monoclonal Antibody FHY-001

[0103] Nasopharyngeal carcinoma C666-1 and cervical carcinoma HeLa were divided into experimental and control groups, respectively. The experimental group received primary and secondary antibodies, while the control group did not receive primary antibodies.

[0104] 1. Preparation of slides: Soak the slides in anhydrous alcohol for 6 hours, then wipe them once with 75% alcohol, and air dry them in a clean bench. Place the slides in a 6-well plate, treat the slides with 1X polylysine for 5 minutes, then absorb off the excess polylysine, and air dry them in a clean bench (or dry them with an alcohol lamp at a distance) for later use.

[0105] 2. Preparation of cell slides:

[0106] 2.1. Digest cells with trypsin, count the cells, and resuspend the cells in complete culture medium.

[0107] 2.2. Select an appropriate cell density for inoculation into the culture plate as needed. When adding cells, depending on the size of the slide, first drop a small amount of culture medium into each well where you intend to place the slide. This is to allow the slide to adhere to the culture dish due to the surface tension of the culture medium. Then place the slide to prevent it from floating up when adding the cell suspension, thus avoiding a double-layer cell adhesion. Maintain aseptic technique throughout the entire process.

[0108] 2.3. Once the cells have spread evenly on the slide (24-48 h), the next experiment can be carried out.

[0109] 3. Fixation and immunohistochemical treatment of cell smears:

[0110] 3.1. Wash the glass slides in the culture plate with PBS three times for 3 minutes each time.

[0111] 3.2. Fix in 4% paraformaldehyde at room temperature for 20 min, wash 3 times with PBS for 3 min each time.

[0112] 3.3. Sealing: Place the sample in a dry petri dish, add 3% BSA-PBS to completely immerse the sample in the humidified chamber, and incubate at room temperature for 2 hours (or overnight at 4°C).

[0113] 3.4. Use absorbent paper to remove excess BSA-PBS solution, being careful not to let the slide dry.

[0114] 3.5. Add diluted primary antibody (400-fold dilution) to the sample, and completely immerse the sample in the primary antibody (incubate at room temperature for 2 hours) and then incubate overnight at 4°C.

[0115] 3.6. Wash the sample slides with PBS, three times, five minutes each time.

[0116] 3.7. Shake dry, wipe clean, add appropriate amount of secondary antibody, and incubate at room temperature for 1-2 hours.

[0117] 3.8. Rinse with TBS 4-5 times, 30 seconds each time.

[0118] Shake dry and wipe clean. Add an appropriate amount of 1X DAB solution and rinse thoroughly with deionized water after 2-5 minutes. (Prepare DAB working solution immediately before use.)

[0119] 3.9. Add one drop of Mayer's hematoxylin, counterstain for 1.5 min-2 min, rinse thoroughly with TBS solution, and then soak in TBS solution for 5-10 min.

[0120] 3.10. Rinse with deionized water 3 times, 1 minute each time.

[0121] 3.11. Dehydration

[0122] a. Soak in 60% ethanol for 5 minutes;

[0123] b. Soak in 80% ethanol for 5 minutes;

[0124] c. Soak in 95% ethanol for 5 minutes;

[0125] d. Immerse in anhydrous ethanol in tank #1 for 5 min;

[0126] e. Immerse in anhydrous ethanol (Vat II) for 5 minutes;

[0127] f. Soak in xylene for 5 minutes;

[0128] g. Soak in xylene II solution for 5 minutes.

[0129] 3.12. Mounting: Remove the slide from the xylene IV container, drain the xylene, and then mount the slide with neutral resin.

[0130] 3.13. Microscopic examination.

[0131] Immunohistochemical results as follows Figure 3As shown, the nasopharyngeal carcinoma C666-1 control group showed no positive signal around the cells, while the experimental group showed a significant positive signal around the cells. In contrast, neither the control nor the experimental group of cervical carcinoma HeLa showed any positive signal, indicating that FHY-001 specifically reacts positively with the target site of nasopharyngeal carcinoma cells.

[0132] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A monoclonal antibody, FHY-001, for the treatment of nasopharyngeal carcinoma, characterized in that, The monoclonal antibody FHY-001, which targets nasopharyngeal carcinoma cells, contains heavy chain variable regions and light chain variable regions. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

14. The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.

16.

2. The encoding gene of the monoclonal antibody FHY-001 for treating nasopharyngeal carcinoma as described in claim 1, characterized in that, The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.13; The nucleotide sequence of the variable region of the light chain is shown in SEQ ID NO.

15.

3. A carrier, characterized in that, The vector contains the encoding gene as described in claim 2.

4. A host cell, characterized in that, The host cell contains the encoding gene as described in claim 2.

5. A host cell, characterized in that, The host cell contains the vector as described in claim 3.

6. The use of the monoclonal antibody FHY-001 according to claim 1 in the preparation of drugs for treating nasopharyngeal carcinoma.

7. A preparation for treating nasopharyngeal carcinoma, characterized in that, The formulation uses the monoclonal antibody FHY-001 as the active ingredient according to claim 1, and the formulation is a drug or a drug composition.

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