Novel anticancer application of monoclonal antibody FHY-001

Abstract

The invention provides novel anticancer application of a monoclonal antibody FHY-001, and belongs to the technical field of anticancer. The invention provides the application of the monoclonal antibody FHY-001 in treatment of oral cancer, esophageal cancer and lung squamous cell carcinoma, and solves the problem of lack of human malignant solid tumor immunotherapy methods in the prior art. The invention provides an effective way for treating more cancers, widens the treatment range of the monoclonal antibody FHY-001 in the solid tumor field, and provides a new way for tumor immunotherapy related to oral cancer, esophageal cancer and lung squamous cell carcinoma.

Description

Technical Field

[0001] The present invention relates to the field of anticancer technology, and in particular to a new anticancer application of monoclonal antibody FHY-001. Background Art

[0002] Cancer refers to a malignant tumor originating from epithelial tissue and is the most common type of malignant tumor. In contrast, malignant tumors originating from mesenchymal tissue are collectively referred to as sarcomas. A few malignant tumors are not named according to the above principles, such as Wilms tumor, malignant teratoma, etc. Generally speaking, "cancer" refers to all malignant tumors. Cancer has biological characteristics such as abnormal cell differentiation and proliferation, uncontrolled growth, invasiveness and metastasis. The occurrence of cancer is a complex process with multiple factors and steps, which is closely related to factors such as smoking, infection, occupational exposure, environmental pollution, unreasonable diet, and genetics.

[0003] At present, there are many drugs for treating cancer, among which doxorubicin, cyclophosphamide, 5-fluorouracil, methotrexate, mitomycin, cisplatin, vincristine, paclitaxel, docetaxel, etc. have better therapeutic effects. The effectiveness of these drugs against cancer is generally around 30%. There are only a few drugs that can achieve an effectiveness of 50% or more, and their toxicity is generally greater.

[0004] Chinese patent CN11584694A discloses a monoclonal antibody FHY-001 for treating nasopharyngeal carcinoma and its application, but the type of cancer it can treat is only nasopharyngeal carcinoma and the treatment range is not wide. Summary of the invention

[0005] The purpose of the present invention is to provide a new anti-cancer application of the monoclonal antibody FHY-001, which provides an effective way to treat more cancers.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides application of monoclonal antibody FHY-001 in preparing a drug for treating oral cancer.

[0008] The present invention also provides the use of the monoclonal antibody FHY-001 in preparing a drug for treating esophageal cancer.

[0009] The present invention also provides the use of the monoclonal antibody FHY-001 in preparing a drug for treating lung squamous cell carcinoma.

[0010] The present invention also provides the use of the monoclonal antibody FHY-001 in preparing an inhibitor of HB human oral cancer cell line.

[0011] The present invention also provides the use of the monoclonal antibody FHY-001 in preparing a CaES-17 human esophageal cancer cell line inhibitor.

[0012] The present invention also provides the use of the monoclonal antibody FHY-001 in preparing an inhibitor of A549 human lung squamous cell carcinoma cell line.

[0013] The present invention also provides a preparation for treating oral cancer, wherein the preparation uses the monoclonal antibody FHY-001 as an active ingredient, and the preparation is a medicine or a pharmaceutical composition.

[0014] The present invention also provides a preparation for treating esophageal cancer, wherein the preparation uses the monoclonal antibody FHY-001 as an active ingredient, and the preparation is a medicine or a pharmaceutical composition.

[0015] The present invention also provides a preparation for treating lung squamous cell carcinoma, wherein the preparation uses the monoclonal antibody FHY-001 as an active ingredient, and the preparation is a medicine or a pharmaceutical composition.

[0016] Preferably, the monoclonal antibody FHY-001 comprises a heavy chain variable region comprising VHCDR1, VHCDR2 and VHCDR3, and a light chain variable region comprising VLCDR1, VLCDR2 and VLCDR3.

[0017] The amino acid sequence of VHCDR1 is shown in SEQ ID NO.1: GYTFTT,

[0018] The amino acid sequence of VHCDR2 is shown in SEQ ID NO.2: NTYSGV,

[0019] The amino acid sequence of VHCDR3 is shown in SEQ ID NO.3: GNYYFDY,

[0020] The amino acid sequence of VLCDR1 is shown in SEQ ID NO.4: KSVSTSGYSY,

[0021] The amino acid sequence of the VLCDR2 is shown in SEQ ID NO.5: LASNLES,

[0022] The amino acid sequence of the VLCDR3 is shown in SEQ ID NO.6: QHSREL.

[0023] Beneficial effects of the present invention:

[0024] The present invention provides therapeutic applications of the monoclonal antibody FHY-001 in oral cancer, esophageal cancer and lung squamous cell carcinoma, broadens the therapeutic scope of FHY-001 in the field of solid tumors, and provides a new approach for immunotherapy of human malignant solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1This is the LDH test result of monoclonal antibody FHY-001 killing HB oral cancer cells;

[0026] Figure 2 This is the result of immunofluorescence detection of monoclonal antibody FHY-001 killing HB oral cancer cells;

[0027] Figure 3 This is the LDH detection result of monoclonal antibody FHY-001 killing CaES-17 esophageal cancer cells;

[0028] Figure 4 This is the result of immunofluorescence detection of monoclonal antibody FHY-001 killing CaES-17 esophageal cancer cells;

[0029] Figure 5 This is the LDH test result of monoclonal antibody FHY-001 killing A549 lung squamous cell carcinoma cells;

[0030] Figure 6 This is the immunofluorescence detection result of monoclonal antibody FHY-001 killing A549 lung squamous cell carcinoma cells. DETAILED DESCRIPTION

[0031] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0032] For the preparation method and related characterization of monoclonal antibody FHY-001, please refer to Chinese patent CN11584694A.

[0033] Example 1 HB human oral cancer cell anticancer experiment

[0034] Complement-dependent cytotoxicity (CDC): CDC refers to the cytotoxic effect of complement. Specific antibodies first bind to the corresponding antigens on the cell membrane surface. The Fc fragment of the antibody binds to C1q, activating various activation pathways of complement, forming C3 convertase and C5 convertase, and finally forming a membrane attack complex. The membrane attack complex inserted into the cell membrane forms a "leakage patch" by destroying the local phospholipid bilayer of the cell membrane, or forms a hydrophilic channel through the membrane, destroying the local phospholipid bilayer, and finally causing the target cell to disintegrate.

[0035] Lactate Dehydrogenase assay (LDH) activity analysis: Cell membrane structure destruction caused by cell apoptosis or necrosis will lead to the release of enzymes in the cytoplasm into the culture medium, including LDH with relatively stable enzyme activity. By detecting the activity of LDH released into the culture medium from cells with ruptured plasma membranes, quantitative analysis of cytotoxicity can be achieved. LDH release is regarded as an important indicator of cell membrane integrity and is widely used in cytotoxicity detection. LDH is an oxidoreductase. Under the action of LDH, NAD+ is reduced to form NADH. NADH and INT (2-p-iodophenl-3-nirophenyl tetrazolium chloride) are catalyzed by lipoamide dehydrogenase to generate NAD+ and formazan, a strong colorant, which produces an absorption peak at a wavelength of 490nm. Therefore, the activity of LDH can be quantified by colorimetry. The absorbance is linearly positively correlated with the activity of LDH.

[0036] The experimental steps for CDC and LDH detection are as follows:

[0037] 1. Plate the target cells in a 96-well plate. When the number of cells reaches 70-80% of the culture wells, remove the culture medium and add different concentrations of HFY-001 and complement diluted with serum-free culture medium. The complement is diluted 8 times of human serum stock solution, and 100 μL of antibody and complement are added to each well.

[0038] 2. Place the plate with added antibodies and complement in a 37°C, 5% CO2 incubator for overnight culture. 1 hour before the detection time point, add 20 μL of LDH release reagent to the LDH well and return it to continue culture for 1 hour.

[0039] 3. Pipette 120 μL of supernatant from each well into a new 96-well plate for testing.

[0040] 4. Dilute the INT solution (10×) in the kit to 1× with INT diluent. Take 100 μL of INT solution (10×), add 900 μL of INT diluent, and mix well to obtain INT solution (1×). INT solution (1×) should be prepared and used immediately.

[0041] 5. Prepare a certain volume of LDH detection working solution according to the ratio of lactic acid solution: INT solution (1×): enzyme solution = 1:1:1.

[0042] 6. Add the prepared LDH detection working solution to each well containing 120 μL of supernatant, 60 μL / well. Mix well, incubate at room temperature in the dark for 30 minutes, and wrap with aluminum foil and shake slowly on a shaker.

[0043] 7. Measure the absorbance at 490 nm in a microplate reader. Perform dual wavelength measurement using 600 nm as the reference wavelength.

[0044] 8. Calculation: Cell death rate (%) = (absorbance of experimental well - absorbance of target cell control well) / (absorbance of target cell maximum enzyme activity control well - absorbance of target cell control well) × 100.

[0045] LDH activity assay to test the killing effect of monoclonal antibody FHY-001 on HB oral cancer cell line Figure 1 As shown: The killing rate of FHY-001 on HB oral cancer cells is about 55%.

[0046] Immunofluorescence technology: Calcein-AM is a commonly used cell staining reagent for fluorescent labeling of living cells, emitting green fluorescence (Ex=490nm, Em=515nm). Because it introduces the acetyl methoxymethyl ester (AM) group on the basis of traditional Calcein (calcein), it increases the hydrophobicity and enables it to easily penetrate the membrane of living cells. Once entering the cell, Calcein-AM (which does not emit fluorescence itself) is cleaved by the esterase in the cell to form the membrane-impermeable polar molecule Calcein, which is retained in the cell and emits strong green fluorescence. Compared with other similar reagents (such as BCECF-AM and CFDA), Calcein-AM has extremely low cytotoxicity and is suitable for fluorescent probes for living cell staining. It also does not inhibit any cell functions, such as proliferation and chemotaxis of lymphocytes. Since dead cells lack esterase, Calcein-AM is only used for cell viability testing and short-term labeling of living cells. EthD-1 (Ethidium Homodimer-I) non-cell permeable viability indicator is a high-affinity nucleic acid stain that emits weak fluorescence before binding to DNA and red fluorescence after binding to DNA (maximum excitation / emission wavelengths are approximately 528 / 617nm, respectively). With a strong positive charge, the dye cannot pass through the cell membrane to stain live cells, so it is usually used to detect dead cells. EthD-1 is usually used in combination with Calcein AM to stain dead cells and live cells, respectively, to determine cell viability and toxicity. The detection experimental steps are as follows: Add complement and antibody reactions, remove the supernatant from the wells used for LDH detection, add dye diluted with PBS, dilute Calcein-AM 2000 times, dilute EthD 500 times, 50μL / well, stain for 20 minutes, and take pictures with a fluorescence microscope.

[0047] Immunofluorescence technology was used to test the killing effect of monoclonal antibody FHY-001 on HB oral cancer cell lines. The results are as follows Figure 2 As shown: the number of cell deaths changes gradually with the change of antibody concentration.

[0048] Example 2 Anticancer Experiment of CaES-17 Esophageal Cancer Cells

[0049] The killing effect of monoclonal antibody FHY-001 on CaES-17 esophageal cancer cell line was tested by LDH activity analysis. The experimental steps were referred to Example 1. The results are shown in FIG. Figure 3 As shown: The killing rate of FHY-001 on CaES-17 esophageal cancer cells is about 30%.

[0050] Immunofluorescence technology was used to test the killing effect of different concentrations of monoclonal antibody FHY-001 (concentrations: 11 μg / mL, 2.75 μg / mL, 0.69 μg / mL, 0.17 μg / mL, 0.04 μg / mL, 0.01 μg / mL, 0.0025 μg / mL) on CaES-17 esophageal cancer cell line. The experimental steps refer to Example 1. The results are as follows: Figure 4 As shown: the number of cell deaths changes gradually with the change of antibody concentration.

[0051] Example 3 Anticancer Experiment of A549 Lung Squamous Cell Carcinoma Cells

[0052] The killing effect of monoclonal antibody FHY-001 on A549 lung squamous cell carcinoma cell line was tested by LDH activity analysis. The experimental steps were referred to Example 1. The results are shown in FIG. Figure 5 As shown: The killing rate of FHY-001 on A549 lung squamous cell carcinoma cells is about 35%.

[0053] Immunofluorescence technology was used to test the killing effect of different concentrations of monoclonal antibody FHY-001 (concentrations: 11 μg / mL, 2.75 μg / mL, 0.69 μg / mL, 0.17 μg / mL, 0.04 μg / mL, 0.01 μg / mL, 0.0025 μg / mL) on A549 lung squamous cell carcinoma cell line. The experimental steps refer to Example 1. The results are as follows: Figure 6 As shown: the number of cell deaths changes gradually with the change of antibody concentration.

[0054] Example 4 Affinity detection of FHY-001 and tumor cells

[0055] Method: The antigen was coated and fixed in a 96-well plate, and different dilutions of monoclonal antibody FHY-001 and enzyme-labeled secondary antibody were added. After color development, the absorbance value of the reaction solution at 450nm, i.e., the OD value, was measured using an enzyme-labeled instrument. The average OD value of each dilution of the antibody was calculated, and then the antibody concentration was used as the horizontal axis, and the OD 450 The vertical axis was plotted using Origin software, and the EC value of the antibody was calculated simultaneously. 50 By comparing the EC values ​​of positive cells and negative cells 50Value, judge the affinity of the antibody, EC 50 The smaller the value, the higher the affinity.

[0056] The affinity test results are shown in Table 1:

[0057] Table 1 Results of affinity test between FHY-001 and tumor cells

[0058]

[0059] As shown in Table 1 , the monoclonal antibody FHY-001 has a high affinity for HB human oral cancer cells, CaES-17 esophageal cancer cells, and A549 lung squamous cell carcinoma cells;

[0060] From the description of the immunohistochemical experiment of monoclonal antibody FHY-001 in Example 3 of Chinese patent CN11584694A, it can be known that the experiment used nasopharyngeal carcinoma cell C666-1 and cervical cancer cell Hela to set up experimental and control groups respectively. The results showed that there was no positive signal in both the control group and the experimental group of cervical cancer cell Hela.

[0061] The experimental results prove that the monoclonal antibody FHY-001 of the present invention is not universally applicable in cancer cells, and has a high affinity with HB human oral cancer cells, CaES-17 esophageal cancer cells, and A549 lung squamous cell carcinoma cells, which is specific.

[0062] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

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Claims

1. Application of monoclonal antibody FHY-001 in the preparation of drugs for the treatment of oral cancer.

2. Application of monoclonal antibody FHY-001 in the preparation of drugs for the treatment of esophageal cancer.

3. Application of monoclonal antibody FHY-001 in the preparation of drugs for the treatment of squamous cell lung carcinoma.

4. Application of monoclonal antibody FHY-001 in the preparation of HB human oral cancer cell line inhibitors.

5. Application of monoclonal antibody FHY-001 in the preparation of CaES-17 human esophageal cancer cell line inhibitors.

6. Application of monoclonal antibody FHY-001 in the preparation of inhibitors of A549 human lung squamous cell carcinoma cell line.

7. A preparation for treating oral cancer, characterized in that: The preparation uses the monoclonal antibody FHY-001 as an active ingredient, and the preparation is a medicine or a pharmaceutical composition.

8. A preparation for treating esophageal cancer, characterized in that: The preparation uses the monoclonal antibody FHY-001 as an active ingredient, and the preparation is a medicine or a pharmaceutical composition.

9. A preparation for treating lung squamous cell carcinoma, characterized in that: The preparation uses the monoclonal antibody FHY-001 as an active ingredient, and the preparation is a medicine or a pharmaceutical composition.

10. The use according to any one of claims 1 to 6 or the preparation according to any one of claims 7 to 9, characterized in that: The monoclonal antibody FHY-001 comprises a heavy chain variable region comprising VHCDR1, VHCDR2 and VHCDR3, and a light chain variable region comprising VLCDR1, VLCDR2 and VLCDR3. The amino acid sequence of VHCDR1 is shown in SEQ ID NO.1, The amino acid sequence of VHCDR2 is shown in SEQ ID NO.2, The amino acid sequence of VHCDR3 is shown in SEQ ID NO.3, The amino acid sequence of VLCDR1 is shown in SEQ ID NO.4, The amino acid sequence of VLCDR2 is shown in SEQ ID NO.5, The amino acid sequence of the VLCDR3 is shown in SEQ ID NO.6.

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