CN115894694A - Monoclonal antibody FHY-001 for treating nasopharyngeal carcinoma and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to a monoclonal antibody FHY-001 for treating nasopharyngeal carcinoma and application thereof. The monoclonal antibody FHY-001 comprises a heavy chain variable region of VHCDR1, VHCDR2 and VHCDR3, and a light chain variable region comprising VLCDR1, VLCDR2 and VLCDR 1. The invention aims at researching and preparing the monoclonal antibody aiming at the specific and effective treatment target of the nasopharyngeal darcinoma, namely, the specific new antibody for treating the nasopharyngeal darcinoma is prepared by using the abnormal protein or the new antigen derived from the non-human normal genome which is generated by the coding of the tumor cell mutant gene of the nasopharyngeal darcinoma and can be identified by immune cells and activate the immune response of an organism. The monoclonal antibody of the invention has good specificity, can be specifically combined with human nasopharyngeal carcinoma tumor cells, can obviously inhibit the growth of in vitro nasopharyngeal carcinoma cells, has high binding rate with targets, can be produced in large scale, and can be used as a potential drug for nasopharyngeal carcinoma immunotherapy.

Description

Monoclonal antibody FHY-001 for treating nasopharyngeal carcinoma and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a monoclonal antibody FHY-001 for treating nasopharyngeal carcinoma and application thereof.

Background

Immune checkpoint inhibitors have been the focus of research in the field of tumor immunotherapy, with the most representative drugs being inhibitors of programmed death receptor 1 (PD-1) and programmed death ligand 1 (PD-L1). The two medicines can enhance the activity of T cells by blocking a PD-1/PD-L1 channel, play a broad-spectrum anticancer role and obviously prolong the life cycle of patients. PD ⁃ is expressed mainly 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 ⁃ regulates the immune function by regulating the differentiation direction of T cells in peripheral tissues, and regulates the immune response of organisms to foreign or self antigens, namely ligands PD ⁃ L1 and PD ⁃ L2, are highly expressed in the placenta tissues and are in a low expression state in spleen, lymph nodes and thymus, so that the method has important significance for maintaining peripheral immune tolerance and reducing the occurrence of diseases related to an autoimmune system. Among them, PD ⁃ L1 is overexpressed in malignant tumor cells and expressed in APCs, lymphocytes, hematopoietic cells and epithelial cells as a main ligand of PD ⁃ 1. Blocking the PD-1/PD-L1 signal channel can lead the activation of T cells to be up-regulated, and activate endogenous anti-tumor immune response, thereby playing the role of treating tumors. Among PD-1 monoclonal antibodies, 15 PD-1/PD-L1 related drugs are approved globally, 6 of them are sold in the market, and the other 3 are in the production stage.

As a broad-spectrum immunosuppressant, PD-1/PD-L1 monoclonal antibody has better clinical results in many tumors, but the overall immune response rate of about 20 percent is relatively low, so that a small number of patients who can benefit from the PD-1 monoclonal antibody are always present. Therefore, the search for new immunotherapeutic targets and the development of immune double antibodies are a potential solution. On the other hand, with the increasing of the data of the domestic PD-1/L1 monoclonal antibody, repeated negotiation of expanding medical insurance of the competitive format worsening superposition indication is carried out, the price of the domestic PD-1/L1 monoclonal antibody is expected to continuously decrease, and the net interest rate is reduced to gradually increase the dosage.

With the application of the PD1/PDL1 monoclonal antibody in tumor immunotherapy, more and more tumor therapies are approved by the drug administration and FDA of China for clinical treatment, such as: malignant melanoma, lung cancer, head and neck cancer, bladder cancer, kidney cancer, hodgkin lymphoma and the like, however, immunotherapy of solid tumors still has many unsolved problems, such as lack of effective means for searching for individualized tumor specific targets in immunotherapy and the like. The invention develops and prepares a monoclonal antibody aiming at a nasopharyngeal carcinoma specific and effective treatment target based on the basis, namely, a specific new antibody for treating the nasopharyngeal carcinoma is prepared by using an abnormal protein or a new antigen derived from a non-human normal genome, which is generated by coding a nasopharyngeal carcinoma cell mutant gene, can be identified by an immune cell and activates the immune response of an organism.

Disclosure of Invention

The invention aims to provide a monoclonal antibody FHY-001 for treating nasopharyngeal carcinoma.

Monoclonal antibody FHY-001 for use in the treatment of nasopharyngeal carcinoma according to a specific embodiment of the present invention, monoclonal antibody FHY-001 comprising a heavy chain variable region of VHCDR1, VHCDR2 and VHCDR3 and a light chain variable region comprising VLCDR1, VLCDR2 and VLCDR3,

the amino acid sequence of the VHCDR1 is shown in SEQ ID NO. 1: the content of the GYTFTT is determined,

the amino acid sequence of the VHCDR2 is shown in SEQ ID NO. 2: the concentration of NTYSGV is determined by the concentration of NTYSGV,

the amino acid sequence of the VHCDR3 is shown in SEQ ID NO. 3: the GNYFDY is a natural substance,

the amino acid sequence of the VLCDR1 is shown in SEQ ID NO. 4: KSVSTSGYSY of the formula,

the amino acid sequence of the VLCDR2 is shown in SEQ ID NO. 5: the number of LASNLES cells is as follows,

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

The monoclonal antibodies of the invention also include functional variants of the antibodies, each of which specifically binds to an antigen.

Specifically, if the functional variants include (but are not limited to): substantially similar in primary structural sequence but comprising chemically and/or biochemically modified derivatives, e.g., in vitro or in vivo, not present in the parent monoclonal antibody of the invention. Such modifications include, for example, acetylation, acylation, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, cross-linking, formation of disulfide bonds, glycosylation, hydroxylation, methylation, oxidation, pegylation, proteolytic processing, phosphorylation, and the like.

The invention also provides the coding gene of the monoclonal antibody FHY-001 for treating nasopharyngeal carcinoma,

the nucleotide sequence of the heavy chain variable region of the coding VHCDR1 is shown in SEQ ID NO.7,

ggg tat acc ttc aca acc，

the nucleotide sequence of the heavy chain variable region for encoding the VHCDR2 is shown in SEQ ID NO.8,

aac acc tac tct gga gtg，

the nucleotide sequence of the heavy chain variable region for encoding the VHCDR3 is shown in SEQ ID NO.9,

ggt aac tac tac ttt gac tac，

the nucleotide sequence of the variable region of the light chain encoding the VLCDR1 is shown in SEQ ID NO.10,

aaa agt gtc agt aca tct ggc tat agt tat，

the variable region of the light chain encoding the VLCDR2 has the nucleotide sequence shown in SEQ ID NO.11,

ctt gca tcc aac cta gaa tct，

the variable region of the light chain encoding the VLCDR3 has a nucleotide sequence shown in SEQ ID NO.12,

cag cac agt agg gag ctt。

the invention provides a vector, which contains the coding gene.

The invention also provides a host cell containing the coding gene or the vector.

Preferably, the invention provides application of the monoclonal antibody FHY-001 in preparation of a medicine for treating nasopharyngeal carcinoma.

The preparation for treating nasopharyngeal carcinoma, which takes the monoclonal antibody FHY-001 as an active ingredient, is a medicament or a pharmaceutical composition.

The above formulations may include a pharmaceutical excipient, diluent or carrier and may be administered to a patient in need of treatment by any suitable means. Suitable means include, but are not limited to, oral, rectal, nasal, topical (including buccal and sublingual), subcutaneous or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal) administration, among others.

The invention is further described below: in the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms, and laboratory procedures used herein are all terms and conventional procedures used extensively in the relevant art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

The term "antibody" as referred to herein includes whole antibodies and any antigen binding fragment (i.e., "antigen binding portion") or single chain thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains that are linked to each other by disulfide bonds, or an antigen-binding portion thereof. 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 CH 3. Each light chain is composed 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 regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-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 the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q).

In the present invention, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

The invention has the beneficial effects that:

the monoclonal antibody provided by the invention has good specificity, can be specifically combined with part of nasopharyngeal carcinoma of human, has high combination rate, can be produced in a large scale, and can be used as a potential drug for immunotherapy of solid tumors.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 shows the CDC assay results for HFY-001;

FIG. 2 shows the results of the detection of the killing rate of HFY-001 induced complement;

FIG. 3 shows immunohistochemical results for HFY-001.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.

Main reagents and sources for experiments: RPMI1640 medium (Hyclone), australian fetal bovine serum (Gibco), HRP goat anti-mouse secondary antibody (Byedu), TMB (Aladdin), pancreatin (Solaibao), additive factor (Boolong), PBS (Zhongkemian), lactate dehydrogenase cytotoxicity detection kit (Biyuntian), HT (50X) (Sigma), HAT (Sigma), BSA (bovine serum albumin) (Solaibao), immunohistochemical kit (Proteintech) and conventional chemicals were purchased from Chinese medicines.

EXAMPLE 1 preparation of monoclonal antibodies

1. Preparation of antigen and immunization of mice

1.1 preparation of antigen: incubating with phosphate-citrate buffer solution (pH 3.3) and tumor cells for 3 min, removing organic salts in the acid washing solution, and separating with SephadexG25 gel column and Centricon ultrafilter to obtain antigen.

2. Immunization strategy

(1) 3 mice of 6-8 weeks old are selected in each group and are respectively subjected to live cell immunity and dead cell immunity;

(2) and immunizing each group of mice once every week and three times in total, injecting thigh muscle and abdominal cavity, wherein the dosage is 50ug each time, 100ug the third time, injecting for one week at the last time, detecting antiserum, and performing cell fusion experiment after the detection is qualified.

3. Preparation of hybridoma cells and screening of monoclonal antibodies

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

(1) Recovering frozen tumor cells with 5% CO at 37 deg.C ₂ Culturing in an incubator;

(2) Culturing to a proper amount (1 × 10) ⁶ - 5×10 ⁶ ) Collecting cells by a cell scraper, centrifuging at 1100 rpm for 6 min;

(3) Discarding the supernatant, adding an appropriate amount (20 ml) 1640 serum-free culture medium to resuspend the cells, counting the number by a counting plate, and keeping in a refrigerator (or an incubator) at 4 ℃ for later use;

3.2 Harvesting of splenocytes

(1) The mouse is placed into a beaker filled with 75% alcohol for soaking for 5min after the dislocation of the cervical vertebra.

(2) The beaker was transferred to a clean bench, the mouse was removed, the abdomen was placed on a foam plate with the abdomen facing up, the limbs were fixed with a needle, and the abdomen was dissected open (skin was first cut open, and the membrane was cut open with a new scissors forceps).

(3) The spleen was removed again by replacing the scissors with forceps (the alcohol was poured before removal).

(4) The spleen was placed in a dish containing 1640, pricked with a needle (to allow for cell spillage), and crushed using a plunger handle.

(5) Solid tumor cell fluid was pipetted through the cell sieve and filtered twice (plates were washed with 1640) with one pipette being changed for each filtration.

(6) The spleen cells dispersed 1640 culture solution was transferred to a centrifuge tube and supplemented with about 1640 to 35 mL. 1200 Centrifuging at rpm for 10min, removing supernatant, resuspending into 35 mL RPMI-1640 basic culture solution, centrifuging again, repeating for three times, and washing to remove erythrocyte and connective tissue.

3.3 Cell fusion

(1) The spleen cells were mixed with the tumor cells in proportion (5-10 times the spleen cells are tumor cells) and centrifuged at 1200rpm for 10min.

(2) The supernatant was discarded and resuspended in 1640, and then centrifuged at 1300 rpm for 10min.

(3) Pouring out the supernatant, controlling water by reversing, and sucking the residual culture medium on the tube wall by absorbent paper.

(4) The centrifuge tube was placed in a 37 ℃ water cup and incubated, PEG incubated at 37 ℃ was aspirated by a 1 mL gun and added to the mixed cells within 1min while shaking the centrifuge tube. 1 mL 1640 incubated at 37 ℃ was added over 1min. 3 mL 1640 incubated at 37 ℃ was added over 3 min. 30 mL of 1640 incubated at 37 ℃ was added slowly, and the mixture was placed in a 37 ℃ incubator and allowed to stand for 5min.

3.4 Indirect non-competitive ELISA assay

(1) Coating: spreading the prepared antigen in an ELISA plate, fixing 2-4 h by 2.5% glutaraldehyde at 37 ℃ in an environment of 4 ℃, and then washing 1 time by PBST buffer solution;

(2) and (3) sealing: adding L-tyrosine and BSA as blocking solution into micropores of an ELISA plate according to the amount of 100 mu L/hole, incubating at 37 ℃ for 1 h, washing the ELISA plate for 1 time by PBST, and drying;

(3) adding a primary antibody: after cell clones (or after subcloning) grow to a proper size after cell fusion, taking the supernatant and adding the supernatant into a corresponding ELISA plate, taking 100 mu L of the supernatant per hole, incubating for 1 h at 37 ℃, manually washing the ELISA plate for 2 times by PBST (softly, preventing fixed cells from floating away), and buckling to dry;

(4) adding a secondary antibody: adding goat anti-mouse enzyme-labeled secondary antibody diluted by PBST solution (1 mul goat anti-mouse enzyme-labeled secondary antibody is added into 10mL PBST), 100 mul/hole, incubating for 1 h at 37 ℃, washing the enzyme-labeled plate for 4 times by PBST, and drying;

(5) color development and termination: adding color development solution (citric acid buffer solution) for developing, using the color development solution in situ, 100 μ L/hole, keeping out of the sun, and incubating at 37 deg.C for 15 min; the color reaction was terminated by adding 2 mol/L H2SO4 in an amount of 50. Mu.L/well, and the absorbance, i.e., OD, of the reaction solution at 450 nm was measured immediately using a microplate reader. (if solution A and solution B are used, 50. Mu. L A solution and then 50. Mu. L B solution are added to each well).

(6) And (4) interpretation of results: and selecting 2-3 plates with the largest OD value as subclones for each plate until the results of the whole plate are consistent, selecting the best hole for single cell cloning, and determining the strain when the length of the hole is good and the detection data is qualified.

3.5 Preparation and purification of antibodies

And (4) beating the fixed strain cells into the body of the mouse, taking out the mouse after the mouse is full of ascites, and collecting and purifying the mouse for later use. Purification of antibody 5mL of HiTrap r-Protein A FF prepacked column was selected for antibody purification. Taking appropriate amount of ascites, centrifuging at 10,000r/min for 15min, taking supernatant, filtering with 0.22 μm microporous membrane, loading with 20mmol/L phosphate buffer solution, and eluting with 0.1mol/L glycine-hydrochloric acid buffer solution. And (4) inspecting and selecting the pH value and the ionic strength of the loading buffer solution and the pH value of the elution buffer solution, and controlling the flow rate of the sample.

The purity of the monoclonal antibody is identified by an SDS-PAGE method, the concentration of separation gel is 12 percent, the concentration of concentration gel is 4 percent, the sample is processed under the reduction state, the sample is dyed by Coomassie brilliant blue R250 after electrophoresis, ethanol acetic acid is used for decoloring, and then the purity of the monoclonal antibody is analyzed by scanning through a gel imaging system.

Cell culture for monoclonal antibody production: after monoclonal antibody cell cloning, the amount of fetal bovine serum added to the basal medium (RPMI 1640) was gradually decreased in a flask in the order of 20% → 15% → 10% → 7.5% → 5% → 2.5%, the cells were adapted to passage for 2 generations at each serum concentration, and serum-free medium (Hyber) was added from 2.5% ^TM -B100, shanghai Beizhi) are subjected to acclimation culture, and the addition ratio of a serum-free culture medium is as follows in sequence: 20% → 40% → 60% → 80% → 90% → 100%, and a serum-free medium addition ratioAt 80% the cells were transferred from the flasks to shake flasks and adapted to passage for 2 passages at each serum-free medium concentration, finally allowing the cells to grow in suspension in complete serum-free medium.

Culturing the suspension-grown cells in a serum-free culture medium by step-by-step amplification in a 3L shake flask for 1L, co-culturing in 5 bottles, stopping culturing when the cell viability is about 90%, and centrifugally collecting supernatant: 4.902kg, the supernatant was concentrated by ultrafiltration using a 30KD membrane pack to yield 373g of antibody solution with protein content: 6.38mg/ml, total protein amount: 2.38g, expression level: 0.47g/L.

4. Identification of monoclonal antibodies

Selecting hybridoma cells which grow well in a serum-free culture medium for sequencing, and showing through bioinformatics analysis that a nucleic acid sequence of the variable region of the encoding antibody heavy chain is shown as SEQ ID NO.13, and an amino acid sequence of the variable region of the encoding antibody heavy chain is shown as SEQ ID NO.14; the nucleotide sequence of the variable region of the encoding antibody light chain is shown as SEQ ID NO.15, and the amino acid sequence of the variable region of the encoding antibody light chain is shown as SEQ ID NO.16.

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

Cagatccagttggtacagtctggacctgagctgaagaagcctggagagacagtcaagatctcctgcaaggcttctgggtataccttcacaacctatggaatgagctgggtgaaacaggctccaggaaagggtttaaagtggatgggctggataaacacctactctggagtgccaacatatgctgatgacttcaagggacggtttgccttctctttggaaacctctgccagcactgcctatttgcagatcaacaacctcaaaaatgaggacacggctacatatttctgtgcaagaggtaactactactttgactactggggccaaggcaccactctcacagtctcctca

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

QIQLVQSGPELKKPGETVKISCKASGYTFTTYGMSWVKQAPGKGLKWMGWINTYSGVPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARGNYYFDYWGQGTTLTVSS

the nucleotide sequence SEQ ID NO.15 encoding the variable region of the antibody light chain is as follows:

Gacattgtgctgacacagtctcctgcttccttagctgtatctctggggcagagggccaccatctcatgcagggccagcaaaagtgtcagtacatctggctatagttatatgcactggtaccaacagaaaccaggacagccacccaaactcctcatctatcttgcatccaacctagaatctggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggatgctgcaacctattactgtcagcacagtagggagcttccgctcacgttcggtgctgggaccaagctggagctgaaa

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

DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLELK

EXAMPLE 2 monoclonal antibody FHY-001 functional assay

1. Detection of tumor cells in vitro

1.1 detection method: a quantitative assay method for determining cytotoxicity by detecting the activity of LDH released from cells with disrupted plasma membranes, wherein target cells carrying specific antigens are bound to antibodies and the target cell membranes are damaged by the participation of complement. The concrete steps are briefly described as follows:

1.1.1 Nasopharyngeal carcinoma cells CNE1, CNE2, 5-8F and 6-10B at 1.2X 10 ⁴ Laying 96-well plates in each hole, and incubating overnight in an incubator;

1.1.2 The medium was aspirated and washed 1 time with PBS. Adding antibodies (57.08 mug/mL, 28.54 mug/mL, 14.27 mug/mL, 7.135 mug/mL) with different concentrations and rabbit serum with different dilution times respectively, wherein the antibodies are diluted by serum-free culture medium, and the complement is diluted by PBS; adding 100ul of basal medium and 100ul of PBS into the control group and the maximum enzyme activity control group at the early stage, adding corresponding antibody and complement, incubating for 30 min in a 5% carbon dioxide incubator at 37 ℃, and then performing complement lysis experiment;

1.1.3 complement lysis experiment, adding 20ul LDH release reagent into the control group with maximum enzyme activity, repeatedly beating and uniformly mixing, and continuing incubation;

1.1.4, respectively taking 120ul of supernatant of each hole, adding the supernatant into a new 96-well plate, and then carrying out sample detection, wherein a background blank control hole needs to be added into the 96-well plate to be detected;

1.1.5 preparing LDH detection working solution according to product specifications, adding 60ul of working solution into each hole respectively, preparing the working solution as it is used, and keeping out of light during preparation and use; after mixing, the mixture was incubated at room temperature (about 25 ℃) in the dark for 30 min, and then the absorbance was measured at 490 nm, and a dual wavelength measurement was performed using either 600 nm or a wavelength greater than 600 nm as the reference wavelength.

1.1.6 calculation: cytotoxicity or mortality (%) = (treated sample absorbance-sample control well absorbance)/(absorbance for maximum enzyme activity of cells-sample control well absorbance) × 100.

1.2 The result of the detection

Antibodies can effectively induce complement dependent killing effects (CDC), causing strong killing of cells.

As shown in figure 1, after the tumor cells are treated by FHY-001 with different concentrations by adding fixed concentration of complement (rabbit serum), the killing rate of the nasopharyngeal carcinoma tumor cells is remarkably improved, which shows that FHY-001 can induce strong CDC effect, resulting in the death of the tumor cells.

Similarly, after the antibody treatment concentration was fixed, different dilutions of complement (rabbit serum) were added, and as shown in fig. 2, the tumor cell death rate decreased with the decrease in complement concentration.

EXAMPLE 3 monoclonal antibody FHY-001 immunohistochemical assay

Respectively arranging nasopharyngeal carcinoma cell C666-1 and cervical carcinoma cell Hela in an experimental group and a control group, wherein the experimental group is added with a primary antibody and a secondary antibody; control group did not add primary antibody.

1. Preparing a climbing sheet: soaking the glass slide in anhydrous alcohol for 6 hours, scrubbing the glass slide for 1 time by using 75% alcohol, drying the glass slide in a super clean bench, placing the glass slide in a 6-hole plate, treating the glass slide with 1X polylysine for 5 minutes, then sucking off the redundant polylysine, and drying the glass slide in the super clean bench (or drying the glass slide away from a remote point by using an alcohol lamp) for later use.

2. Preparing a cell slide:

2.1. cells were trypsinized, counted, and resuspended in complete medium.

2.2. And selecting proper cell density to be inoculated into the culture plate according to requirements. When adding cells, according to the size of the slide, firstly, dripping a small amount of culture medium at the position where the slide is to be placed in each hole, so that the slide and the culture dish are adhered together by the tension of the culture medium, then placing the slide, and preventing the slide from floating when adding cell suspension to cause double-layer cell patches. The whole process is carried out aseptically.

2.3. The next experiment was performed with the cells fully spread on a slide (24-48 h).

3. Fixing and immunohistochemical treatment of cell slide:

3.1. slides that have crawled cells are rinsed 3 times for 3 min each with PBS in the culture plate.

3.2. Fixing in 4% paraformaldehyde at room temperature for 20 min, washing with PBS for 3 times, each for 3 min.

3.3. And (3) sealing: the swatches were placed in a dry petri dish and 3% BSA-PBS was added dropwise to submerge the swatches completely in the wet box for 2h at room temperature (or overnight at 4 ℃).

3.4. Excess BSA-PBS solution was blotted off with absorbent paper, taking care not to dry the tablets.

3.5. The diluted primary antibody (diluted 400 times) was added dropwise to the sample, and the sample was completely immersed in the primary antibody (incubated at room temperature for 2 h) overnight at 4 ℃.

3.6. The swatches were washed 3 times 5min each time with PBS.

3.7. Spin-drying, wiping, dripping proper amount of secondary antibody, and incubating at room temperature for 1-2 h.

3.8 TBS washes 4-5 times, 30 s each time.

Spin-drying, wiping, dripping appropriate amount of 1X DAB solution, and rapidly washing with deionized water after 2-5 min. (DAB working solution for use in the present)

3.9. Dropping a drop of Mayer's hematoxylin (hematoxylin), counterstaining for 1.5 min-2 min, washing with TBS solution, and soaking in TBS solution for 5-10 min.

3.10. Washing with deionized water for 3 times, each for 1min.

3.11. Dewatering

a. Soaking in 60% ethanol for 5 min;

b. soaking in 80% ethanol for 5 min;

c. soaking in 95% ethanol for 5 min;

d. soaking in absolute ethanol I jar for 5 min;

e. soaking in absolute ethanol II jar for 5 min;

f. soaking in xylene I jar for 5 min;

g. soaking in xylene II jar for 5min.

3.12. Sealing: the slices were removed from the xylene IV jar, drained of xylene, and then blocked with neutral gum.

3.13. And (6) microscopic examination.

The immunohistochemical experiment result is shown in fig. 3, the nasopharyngeal carcinoma cell C666-1 control group has no positive signal around the cell, the cell around the experimental group has obvious positive signal, and the control group and the experimental group of the cervical carcinoma cell Hela have no positive signal, which indicates that FHY-001 and the target spot of the nasopharyngeal carcinoma cell have specific positive reaction.

The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims (7)

1. A monoclonal antibody FHY-001 for use in the treatment of nasopharyngeal carcinoma, wherein monoclonal antibody FHY-001 comprises a heavy chain variable region of VHCDR1, VHCDR2 and VHCDR3 and a light chain variable region comprising VLCDR1, VLCDR2 and VLCDR3,

the amino acid sequence of the VHCDR1 is shown in SEQ ID NO.1,

the amino acid sequence of the VHCDR2 is shown in SEQ ID NO.2,

the amino acid sequence of the VHCDR3 is shown in SEQ ID NO.3,

the amino acid sequence of the VLCDR1 is shown in SEQ ID NO.4,

the amino acid sequence of the VLCDR2 is shown in SEQ ID NO.5,

the amino acid sequence of the VLCDR3 is shown in SEQ ID NO. 6.

2. The gene encoding the monoclonal antibody FHY-001 of claim 1 for use in the treatment of nasopharyngeal carcinoma,

the heavy chain variable region of the encoding VHCDR1 has a nucleotide sequence shown in SEQ ID NO. 7;

the heavy chain variable region of the encoding VHCDR2 has a nucleotide sequence shown in SEQ ID NO. 8;

the heavy chain variable region of the coding VHCDR3 has a nucleotide sequence shown in SEQ ID NO. 9;

the variable region of the light chain of the VLCDR1 is coded, and the nucleotide sequence of the variable region is shown as SEQ ID NO. 10;

the variable region of the light chain encoding the VLCDR2 has a nucleotide sequence shown in SEQ ID NO. 11;

the variable region of the light chain encoding the VLCDR3 has the nucleotide sequence shown in SEQ ID NO. 12.

3. A vector comprising the coding gene of claim 2.

4. A host cell comprising the coding gene of claim 2.

5. A host cell comprising the vector of claim 3.

6. The use of monoclonal antibody FHY-001 of claim 1 in the preparation of a medicament for the treatment of nasopharyngeal carcinoma.

7. An agent for treating nasopharyngeal carcinoma, which comprises the monoclonal antibody FHY-001 of claim 1 as an active ingredient, and which is a pharmaceutical or pharmaceutical composition.

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