Anti-CD70 monoclonal antibody and detection application thereof

Abstract

The invention belongs to the technical field of medicine, and relates to an anti-CD70 antibody or an antigen binding fragment thereof, which can specifically recognize and bind CD70 molecules. The CD70 antibody is obtained through a hybridoma technology and monoclonal screening, and can be used for identification, verification and the like of CD70 molecules.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to an antibody or its antigen-binding fragment, which specifically recognizes human cluster of differentiation 70 (CD70), can specifically recognize and bind to the CD70 antigen, so as to achieve the effect of detecting the expression of CD70. Technical Background

[0002] CD70, that is, cluster of differentiation 70, is a member of the tumor necrosis factor (TNF) superfamily, and is usually expressed in mature dendritic cells and a small number of activated B cells and T cells. In various types of tumors, such as certain lymphomas and solid tumors, the expression level of CD70 is abnormally increased, which is associated with poor prognosis of the disease. Therefore, CD70 is considered to be an important tumor marker, and the detection of its expression level has important value for the diagnosis, prognosis evaluation and treatment response monitoring of the disease.

[0003] CD70 participates in regulating the immune response and promoting the proliferation and survival of tumor cells through its interaction with its ligand CD27. In the tumor microenvironment, the expression of CD70 is related to the immune escape mechanism, and may promote the proliferation of malignant cells by activating the NF-κB and c-Jun kinase pathways. In addition, the expression of CD70 is also related to the activation of regulatory T cells (Tregs) and the inhibition of tumor immune surveillance.

[0004] Although the detection of CD70 is crucial for the management of various diseases, the current methods have some limitations. Traditional detection methods, such as immunohistochemistry (IHC) and flow cytometry, may have problems of insufficient sensitivity and specificity. In addition, these methods may require complex sample processing and professional operation skills, which limit their application in clinical practice. Therefore, developing new CD70 detection methods with high sensitivity and high specificity is of great significance for improving diagnostic accuracy and guiding clinical treatment.

[0005] In view of the key role of CD70 in tumor biology, developing an antibody or its antigen-binding fragment that specifically recognizes human CD70 has important clinical value for disease detection. Such a detection antibody can provide a rapid, accurate and cost-effective detection method, which is helpful for early diagnosis, disease monitoring and treatment effect evaluation. Summary of the Invention

[0006] One aspect of the present invention is to provide a new anti-CD70 monoclonal antibody and its applications. The anti-CD70 antibody or its antigen-binding fragment comprises heavy chain complementarity-determining regions HCDR1, HCDR2, HCDR3 and / or light chain complementarity-determining regions LCDR1, LCDR2, LCDR3; the anti-CD70 antibody or its antigen-binding fragment has the activity of binding to CD70 protein and can be used for ELISA detection of CD70 expression in the field of scientific research, etc.

[0007] Use

[0008] 1. One aspect of the present invention lies in using a primary antibody for ELISA to detect the expression of CD70 antigen.

[0009] 2. One aspect of the present invention lies in using it as a control to detect the binding ability to CD70 antigen. Brief Description of the Drawings

[0010] Figure 1 This is the result of using the anti-CD70 monoclonal antibody in the ELISA detection in the embodiments of the present invention. Detailed Description of the Invention

[0011] The present invention provides an anti-CD70 monoclonal antibody. The obtaining of this antibody includes animal immunization (using CD70 protein as an antigen), cell fusion, screening and cloning of positive hybridoma cells, and ELISA verification. The anti-CD70 monoclonal antibody provided by the present invention can effectively bind to CD70 protein, has stable performance, and can be used for ELISA detection of CD70 antigen.

[0012] The following examples are used to illustrate the present invention, but do not limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0013] Example 1 Obtaining of Anti-CD70 Monoclonal Antibody

[0014] The obtaining of the anti-CD70 monoclonal antibody provided by the present invention includes the following steps:

[0015] 1. Animal Immunization

[0016] Mix 100 μg of antigen protein with an equal volume of Freund's complete adjuvant, and after thoroughly emulsifying with a screw-capped syringe, inject it subcutaneously into each mouse at multiple points; 14 days later, mix 50 μg of antigen protein with an equal volume of Freund's complete adjuvant, and after thoroughly emulsifying with a screw-capped syringe, inject it subcutaneously into each mouse at multiple points; 21 days later, mix 50 μg of antigen protein with an equal volume of Freund's complete adjuvant, and after thoroughly emulsifying with a screw-capped syringe, inject it subcutaneously into each mouse at multiple points; select 1 mouse with the highest titer and inject 50 μg / mouse of antigen protein intraperitoneally. Take the spleen on the 4th day after immunization.

[0017] 2. Preparation of splenocytes

[0018] (1) Select 1 mouse with the highest titer, and after sacrificing, soak the mouse in 75% ethanol for 5 min for disinfection. Then, on the sterile operating table, fix the mouse and carefully remove the spleen.

[0019] (2) Transfer the spleen to a 70 μm cell strainer, and gently squeeze the splenocytes through the strainer into 1640 culture medium using the piston of a syringe. Centrifuge at 1000 rpm for 10 min, collect the precipitated cells, lyse the red blood cells with 7 mL of red blood cell lysate, resuspend in 40 mL of 1640 medium, centrifuge at 1500 rpm for 5 min, repeat the lysis of red blood cells once, and finally resuspend the cells in 1640 medium, mix well, and count.

[0020] 3. Preparation of sp2 / 0 myeloma cells

[0021] Take out the sp2 / 0-Ag14 cells from the liquid nitrogen tank, melt them in water at 37°C, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend with 1640 complete medium, transfer to a cell flask, and culture in an incubator at 37°C, 5% CO2, and saturated humidity. On the day of fusion, take sp2 / 0-Ag14 cells with uniform cell morphology, clear boundaries, and good growth state, discard the culture medium, wash twice with PBS, then resuspend the cells with 1 mL of 1640 medium, and then count and place at 4°C for standby.

[0022] 4. Cell fusion

[0023] (1) Mix the prepared sp2 / 0 myeloma cells and splenocytes at a ratio of 1:5 - 1:10, place them in a 50 mL centrifuge tube, wash three times with DMEM culture medium, centrifuge at 1000 rpm for 8 min, and after the last centrifugation, aspirate and dry the supernatant, and gently tap the bottom of the centrifuge tube with your finger to loosen the cells.

[0024] (2) Place the centrifuge tube in a 37°C water bath, aspirate 1 mL of PEG1450 preheated at 37°C and add it to the centrifuge tube at a uniform speed while gently stirring, finish adding within 1 min, and let it stand for 1 min.

[0025] (3) Slowly add 30 mL of DMEM culture medium. Add 1 mL in the 1st minute, 3 mL in the 2nd minute, 5 mL in the 3rd minute, 7 mL in the 4th minute, and add all of it in the 5th minute.

[0026] (4) Centrifuge at 1000 rpm for 8 minutes and discard the supernatant.

[0027] (5) Resuspend the fused cells gently with HAT culture medium to prevent the newly fused cells from being blown away. Add 60 - 100 mL of HAT culture medium and start plating on 96 - well cell culture plates, which can plate 6 - 10 plates.

[0028] (6) Place the cell culture plates in an incubator at 37 °C, 5% CO₂, and saturated humidity. After 3 days of fusion, replace half of the HAT culture medium, and replace it with HT culture medium 10 days after fusion.

[0029] 5. ELISA screening of hybridomas

[0030] (1) Coating antigen: Dilute the CD70 antigen with PBS to 1 μg / mL and add 50 μL to each well of the enzyme - labeled plate, then incubate overnight at 4 °C.

[0031] (2) Discard the antigen and wash the plate 3 times with PBST, 200 μL per well.

[0032] (3) Add 200 μL of blocking solution (blocking solution is 5% skim milk powder prepared with PBS) to each well and incubate at 37 °C for 1 h.

[0033] (4) Discard the blocking solution and wash the plate 3 times with PBST, 200 μL per well.

[0034] (5) Add the supernatant of hybridoma cells diluted with PBS, 100 μL per well, diluted 1:1000, and incubate at 37 °C for 1 h.

[0035] (6) Wash the plate 3 times with PBST, 200 μL per well.

[0036] (7) Add HRP - goat anti - mouse IgG secondary antibody solution (diluted 1:5000 with PBS), 100 μL per well, and incubate at 37 °C for 1 h.

[0037] (8) Wash the plate 3 times with PBST, 200 μL per well.

[0038] (9) Add 100 μL of TMB chromogenic solution to each well and wait at room temperature for 5 - 10 minutes, then add 100 μL of 1 M HCl to terminate in each well.

[0039] (10) Detect the OD450 value with an enzyme-linked immunosorbent assay (ELISA) reader, select the positive wells with a high OD450 ratio, and transfer all the cells in the wells to a 24-well cell culture plate. After observing that the cells in the 24-well plate have grown confluently, detect the binding ability of the supernatant of the selected positive wells to the CD70 protein by ELISA. Select 4 positive wells for monoclonalization and cryopreservation for seed storage.

[0040] Table 1: OD values (average) of the binding of monoclonal antibodies against CD70 to CD70 antigen detected by ELISA

[0041]

[0042] 6. Screening for monoclonal antibodies by limiting dilution method

[0043] (1) Add the selected positive clone hybridoma cells to a 96-well plate and dilute them 1:3 fold serially for 8 gradients. Incubate the cells in a 37 °C, 5% CO2 incubator overnight.

[0044] (2) Take 300 - 500 cells from the corresponding dilution and seed them into a 96-well plate.

[0045] (3) When the supernatant turns yellow or obvious clone clusters are seen, take the supernatant for antibody detection, and the detection method is as described in 2.2.3.

[0046] (4) Add the cells from the positive wells obtained by antibody detection to a 24-well plate for expansion culture; when the cells reach 80% confluence, cryopreserve the cells and simultaneously perform the next round of subcloning until the positive wells are single cell clusters visible to the naked eye.

[0047] 7. Sequencing of the variable regions of monoclonal antibodies

[0048] Centrifuge and collect the obtained monoclonal cell lines, wash them once with PBS, discard the supernatant; send the cell pellet to Nanjing Detai Biotechnology Co., Ltd. for sequencing to obtain the heavy chain and light chain sequences of the monoclonal antibodies.

[0049] 7. Detecting the binding activity of antibodies to proteins by ELISA

[0050] (1) Antigen coating: Dilute the protein to a concentration of 2 μg / mL with PBS (pH 7.4), and the coating volume is 50 μL / well. The coating condition is 4 °C for 12 h.

[0051] (2) Blocking: Wash the plate 3 times with PBST, 200 μL / well. The blocking solution is 5% skim milk powder prepared with PBS, and the blocking condition is to block at 37 °C for 2 h.

[0052] (3) Add the test sample: Wash the plate 3 times, add the antibody diluted with PBS, 100 μL per well. The concentration in the first well is 20 μg / mL, and then dilute it 1:3 serially for a total of 8 wells. Incubate at 37 °C for 2 h.

[0053] (4) Secondary antibody incubation: Use HRP-conjugated IgG secondary antibody, dilute the secondary antibody with PBS (1:5000), 100 μL per well, incubate at 37 °C for 1 h.

[0054] (5) Color development: Wash the plate 3 times, add 100 μL per well of TMB color development solution, react at 37 °C in the dark for 15 min.

[0055] (6) Termination: Add an equal volume (100 μL per well) of 1 M hydrochloric acid solution to terminate the reaction, and the reaction solution in the wells changes from blue to yellow.

[0056] (7) Read the results: Measure the OD450 value with an enzyme-linked immunosorbent assay (ELISA) reader within 15 min. Plot the binding curves of the OD values of antibodies at different concentrations, as shown in Figure 1 . Use Graphad Prism software for analysis, calculate the EC50 value, and the results are shown in Table 2.

[0057] Table 2: EC50 value of the binding of anti-CD70 monoclonal antibody to CD70 protein

[0058]

[0059] The ELISA results showed that the monoclonal antibody 2H1 targeting CD70 had the lowest EC50 value compared with the positive antibody or other clones of the same batch. It was the most sensitive anti-CD70 monoclonal antibody in the same batch and could be used as a standard antibody for ELISA detection to detect the binding activity of CD70 antigen protein.

[0060] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention. Antibody sequence (2H1)

[0061]

Claims

1. A CD70 monoclonal antibody or an antigen-binding fragment thereof, characterized in that: The amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 of the antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 1-3, respectively, and the amino acid sequences of the light chain complementary determining regions CDR1, CDR2 and CDR3 are shown in SEQ ID NOs: 4-6.

2. The antibody according to claim 1, characterized in that The heavy chain variable region of the antibody is: the amino acid sequence shown in SEQ ID NO:7; or a polypeptide with the same function obtained by substituting, deleting and / or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:7; preferably, a polypeptide with at least 80% similarity to the amino acid sequence shown in SEQ ID NO:7 and having the same function; the light chain variable region of the antibody is: the amino acid sequence shown in SEQ ID NO:8; or a polypeptide with the same function obtained by substituting, deleting and / or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:8; preferably, a polypeptide with at least 80% similarity to the amino acid sequence shown in SEQ ID NO:8 and having the same function.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody or antigen-binding fragment thereof is selected from any one of monoclonal antibodies, Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, and single-chain antibody.

4. A bispecific antibody or multispecific antibody comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. A nucleic acid molecule characterized in that The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

6. A biological material containing the nucleic acid molecule according to claim 5, characterized in that: The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector or engineered bacteria.

7. An antibody conjugate, characterized in that: It is obtained by coupling the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or the bispecific antibody or multispecific antibody according to claim 4 with a marker or protein; preferably, the marker is selected from one or more of chemiluminescent dye labeling, enzyme labeling, biotin labeling, fluorescent dye labeling, colloidal gold labeling, and radioactive labeling.

8. The method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: The method comprises: culturing a host cell capable of expressing the antibody or the antigen-binding fragment thereof, and obtaining the antibody or the antigen-binding fragment thereof by separation.

9. Any of the following uses of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the bispecific antibody or multispecific antibody according to claim 4, the nucleic acid molecule according to claim 5, the biomaterial according to claim 6, or the antibody conjugate according to claim 7: (1) Use in the preparation of a product for detecting the presence or level of CD70 protein or cells expressing CD70 protein in a sample; (2) Use in detecting the presence or level of CD70 protein in a sample; (3) Use in detecting the presence or level of cells expressing CD70 protein in a sample; the use is for non-disease diagnosis and treatment purposes.

10. A biological product, characterized in that: The biological product comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or comprises the bispecific antibody or multispecific antibody according to claim 4, or comprises the antibody conjugate according to claim 7; The product is a detection reagent or a pharmaceutical composition.

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