CN117187190A - Anti-cyclophilin A monoclonal antibody and application thereof in treatment of rheumatoid arthritis - Google Patents

Abstract

The invention discloses an anti-cyclophilin A monoclonal antibody and application thereof in treatment of rheumatoid arthritis. The invention provides a hybridoma cell BQHA7 secreting anti-CypA monoclonal antibody, the preservation number of which is CGMCC No. 45101, and the anti-CypA monoclonal antibody secreted by the hybridoma cell BQHA7. The experiments of the invention prove that the invention provides a hybridoma cell strain capable of secreting anti-CypA monoclonal antibody, the hybridoma cell strain can secrete the monoclonal antibody BQHA7, the monoclonal antibody BQHA7 can specifically recognize CypA, and the hybridoma cell strain has good therapeutic effect on type II collagen-induced mouse rheumatoid arthritis (rheumatoid arthritis, RA).

Description

Anti-cyclophilin A monoclonal antibody and its application in the treatment of rheumatoid arthritis

Technical field

The invention belongs to the field of biomedicine and relates to anti-cyclophilin A monoclonal antibody and its application in the treatment of rheumatoid arthritis.

Background technique

Cyclophilin A (CypA, cyclophilin A) has peptidyl prolyl cis/trans isomerase activity and is a highly conserved protein that is widespread in the biological world. CypA is mainly distributed in the cytoplasm and is the main intracellular receptor for the immunosuppressive drug Cyclosporin A (CsA, cyclosporine A). CypA in cells is also involved in various biological processes such as apoptosis, immune regulation, bacterial infection, and viral replication. When stimulated by mechanical damage, reactive oxygen species, pathogenic microbial infection, etc., CypA can also be secreted out of the cell. Extracellular CypA (eCypA) plays an important role in the occurrence of various inflammatory diseases. For example, the connection between eCypA and inflammation can be observed in the occurrence of inflammation-related autoimmune diseases such as rheumatoid arthritis, psoriasis, and systemic lupus erythematosus.

Since anti-CypA monoclonal antibodies only act against eCypA outside cells, they will not affect the normal function of intracellular CypA. Therefore, the feasibility of anti-CypA monoclonal antibody as a drug to treat inflammatory diseases is much higher than that of other drugs, which will greatly enhance the specificity of the drug and reduce the occurrence of side effects. Chen Zhinan et al. obtained an anti-CypA camelid heavy chain VHH antibody (patent CN 102660551 B) and an anti-human cyclophilin protein A monoclonal antibody (CN 108893449 A); Liu Wenjun et al. obtained an anti-Cyclophilin A monoclonal antibody (CN 113248617 B) has a good therapeutic effect on virus-induced acute pneumonia. The above antibodies provide tools for the diagnosis and treatment of eCypA-related inflammation, but there are currently no reports on experimental data on the application of CypA antibodies in the treatment of autoimmune diseases such as rheumatoid arthritis.

Contents of the invention

The object of the present invention is to provide anti-cyclophilin A monoclonal antibodies and their application in the treatment of rheumatoid arthritis.

In a first aspect, the present invention provides a hybridoma cell line BQHA7 that secretes anti-CypA monoclonal antibody (referred to as CypA monoclonal antibody or anti-CypA monoclonal antibody), and its deposit number is CGMCC No. 45101.

In a second aspect, the present invention provides anti-CypA monoclonal antibodies secreted by the hybridoma cell line BQHA7 described in the first aspect.

In a third aspect, the present invention provides the use of the anti-CypA monoclonal antibody described in the second aspect in any of the following;

1) Preparation of products for the treatment of rheumatoid arthritis;

2) Prepare products that reduce the degree of damage or destruction of rheumatoid arthritis joints;

3) Preparing products that delay the disease process of rheumatoid arthritis;

4) Preparing products for alleviating the inflammatory response of rheumatoid arthritis;

5) Prepare products that improve joint swelling and/or bone erosion in rheumatoid arthritis;

6) Prepare products that reduce the number of inflammatory cell infiltrates;

7) Prepare products that reduce the expression of factors related to inflammatory response;

8) Prepare products for detecting cyclophilin A.

In the above, factors related to inflammatory response are specifically IL-17A, TNF-α and/or IL-6 in serum.

In the above, detecting cyclophilin A means detecting whether the sample to be tested contains cyclophilin A; the sample to be tested is a cell or tissue.

In a fourth aspect, the present invention provides a product comprising the anti-CypA monoclonal antibody described in the second aspect.

The products mentioned above have at least one of the following functions:

1) Treat rheumatoid arthritis;

2) Reduce the degree of damage or destruction of rheumatoid arthritis joints;

3) Delay the disease process of rheumatoid arthritis;

4) Relieve the inflammatory response of rheumatoid arthritis;

5) Improve joint swelling and/or bone erosion in rheumatoid arthritis;

6) Reduce the number of inflammatory cell infiltrates;

7) Reduce the expression of factors related to inflammatory response;

8) Detect cyclophilin A.

The above example of rheumatoid arthritis is type II collagen-induced rheumatoid arthritis.

In the above, the product is a kit.

In a fifth aspect, the present invention provides a method for detecting cyclophilin A, which includes the following steps: using the anti-CypA monoclonal antibody described in the second aspect as an antibody, detecting whether the sample to be tested contains cyclophilin A.

Experiments of the present invention prove that the present invention provides a hybridoma cell line that can secrete anti-CypA monoclonal antibodies. This hybridoma cell line can secrete monoclonal antibody BQHA7. This monoclonal antibody BQHA7 can specifically recognize CypA and is effective against type II collagen. Induced rheumatoid arthritis (RA) in mice has good therapeutic effect.

Preservation instructions

Strain name: BQHA7

Classification and naming: mouse hybridoma cells

Collection institution: General Microbiology Center of China Committee for the Collection of Microbial Cultures

Abbreviation of depository institution: CGMCC

Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

Storage date: February 24, 2022

Collection center registration number: CGMCC No. 45101

Description of the drawings

Figure 1 shows the titer of BQHA7 monoclonal antibody detected by ELISA.

Figure 2 shows the detection of BQHA7’s specific recognition of CypA in cell lines and tissues.

Figure 3 shows the detection of the effect of BQHA7 on joint inflammation scores in mice.

Figure 4 shows the detection of the effect of BQHA7 on micro-CT imaging of mouse joint inflammation.

Figure 5 shows the detection of the therapeutic effect of BQHA7 on pathological injuries of mouse ankle joints.

Figure 6 shows the detection of the therapeutic effect of BQHA7 on ankle cartilage damage in mice.

Figure 7 shows the detection of the effect of BQHA7 on mouse serum cytokines.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The test materials used in the following examples were all purchased from conventional biochemical reagent stores unless otherwise specified.

The following examples facilitate a better understanding of the present invention, but do not limit the present invention.

The quantitative experiments in the following examples were repeated three times, and the results were averaged.

Male DBA/1 mice aged 7-8 weeks were purchased from Beijing Vitong Lever Experimental Animal Co., Ltd.

The CypA monoclonal antibody QH01 in the example is QH01 in CN 113248617 B (authorization announcement date 2021.10.01; application number 202110682376.5), and the deposit number of the hybridoma cell line that secretes it is CGMCC No. 21909.

The PBS (pH: 7.2-7.4) in the following examples was purchased from Beijing Solebao Technology Co., Ltd., and the product number is P1020.

In the following examples, the CypA protein was purchased from Suzhou Nearshore Protein Technology Co., Ltd., and the product number is CR15.

The reagents used in the ELISA method are as follows:

The coating solution (pH=9.6) was purchased from Beijing Solebao Technology Co., Ltd., the product number is C1050.

Washing liquid (pH: 7.2-7.4) was purchased from Beijing Solebao Technology Co., Ltd., the product number is P1031.

The blocking solution is a washing solution containing (mass volume percentage, g:ml) 2% skimmed milk powder;

The chromogenic solution is a mixture of 1% solution A and 10% solution B by volume, where solution A: 1% TMB in DMSO; solution B: citrate buffer containing 0.1% CH ₄ N ₂ O·H ₂ O ₂ .

The stop solution is 0.5M sulfuric acid aqueous solution.

Example 1. Preparation of anti-CypA monoclonal antibody

1. Obtaining hybridoma cell lines secreting anti-CypA monoclonal antibodies

1. Immunized mice

Six SPF grade BALB/c female mice were immunized with CypA protein. The primary immunization was intramuscular injection with an immunization dose of 20 μg protein/mouse. Subsequently, 6 subsequent booster immunizations were carried out in the same way and dose, with a one-week interval between each immunization. ; One week after the booster immunization, the mice were intraperitoneally pulse-immunized with 50 μg of CypA protein to complete the final immunization. One week later, blood was taken from the mouse orbits to determine the serum antibody titer.

2. Screen BQHA7

① ELISA was used to detect the serum titers of the 6 immunized mice, and mice with higher titers were screened for hybridoma fusion.

② Cell fusion experiment: Take mouse spleen cells and SP2/0 cells and fuse them using the PEG method. After fusion, cells are screened and cultured in semi-solid medium (containing HAT).

③ Pick single clones: Pick 10 plates × 93 cell single clones and culture them in a 96-well cell culture plate (previously plated with thymocytes, 100 μL/well).

④Initial screening of monoclonal cells: Discard all monoclonal cell supernatants in the 96-well culture plate, add 200 μL/well, 20% newborn bovine IMDM medium (containing HT), and perform the first screening.

⑤ Secondary screening of monoclonal cells: Use recombinant CypA protein to coat the plate, use ELISA method for the selected clones, conduct a second screening, and obtain positive hybridoma cell lines.

⑥Third screening of monoclonal cells: The positive cell lines obtained from the secondary screening were plated with recombinant CypA protein and "tagged protein" respectively, and the third screening was performed using ELISA method, and finally the secreted CypA monoclonal antibody (BQHA7) was obtained The hybridoma cell line BQHA7.

The BQHA7-secreting hybridoma cell line BQHA7 has been deposited in the General Microbiology Center of the China Council for the Collection of Microbial Cultures (CGMCC, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, postal code 100101) , the deposit number is CGMCC No.45101, and the classification is named mouse hybridoma cells.

2. Preparation of anti-CypA monoclonal antibody from ascites

The BQHA7-secreting hybridoma cell line BQHA7 obtained above was injected into the abdominal cavity of BALB/c mice at a cell concentration of 1.5×10 ⁶ /mouse. Ascitic fluid was collected after 10 to 14 days.

The antibody in ascites was purified using a Protein G-Sepharose4B adsorption chromatography column to obtain anti-CypA monoclonal antibody BQHA7. The antibody was collected into 0.1 M glycine-hydrochloride buffer (pH=2.7), and then the antibody was replaced with PBS and stored for use. in subsequent experiments.

1. Dilute CypA protein with coating solution to a final concentration of 2ug/ml, 100ul/well, incubate at 4°C overnight, and then wash 3 times with washing solution;

2. Block with blocking solution, 200ul/well, incubate at 37°C for 2 hours, and then wash 3 times with washing solution;

3. Add primary antibody (anti-CypA monoclonal antibody BQHA7 or CypA monoclonal antibody QH01) or PBS (Blank), 10 μg/mL in the first well, and then perform 4-fold gradient dilution with PBS, 8 points, blank in the last well, 100 μL /well, incubate at 25°C for 1 hour, then wash 3 times with washing solution;

4. Add HRP-labeled goat anti-mouse IgG secondary antibody diluted 10,000 times in PBS (purchased from Aibotek Biotechnology Co., Ltd., catalog number: AS003), 100 μL/well, incubate at 25°C for 1 hour, remove and wash with washing solution for 3 Second-rate;

5. Add 100 μL/well of chromogenic solution, and the color development time is about 10 minutes;

6. Add 50 μL stop solution to each well to stop;

7. Measure the absorbance value at dual wavelengths (450nm, 630nm), record and save the data;

The analysis results are shown in Figure 1. The half effective concentration (EC50) of anti-CypA monoclonal antibody BQHA7 is 0.2629 μg/mL, and the EC50 of QH01 is 2.415 μg/mL.

Example 2. Anti-CypA monoclonal antibody BQHA7 subtype identification

Use a mouse monoclonal antibody subtype identification kit (purchased from proteintech company, catalog number: PK20002) to identify the subtype of anti-CypA monoclonal antibody BQHA7. Refer to the instructions for steps.

The analysis results are shown in Table 1. The heavy chain of anti-CypA monoclonal antibody BQHA7 is IgG1 and the light chain is κ.

Example 3. Detection of specific recognition of CypA in cells and tissues by anti-CypA monoclonal antibody BQHA7

Lysis Buffer (150 mM NaCl, 20 mM HEPES, 1 mM EDTA, 1% Triton100, 10% glycerol) containing Protease Inhibitor Cocktail (Roche, #5871, refer to the instructions for dosage) was used to lyse the human monocytic leukemia cell line (THP1 , purchased from ATCC, TIB-202) and 7-8 week old male DBA/1 mice (purchased from Viton Lever) lung, spleen, lymph node and other tissues, and lysate products were collected.

Add an appropriate amount of 5×SDS-PAGE protein loading buffer to each lysate and heat at 98°C for 15 minutes. Anti-CypA monoclonal antibody BQHA7 and anti-GAPDH antibody (Santa Cruz Company, product catalog number: sc-47724) from ascites were prepared in Example 1 as the primary antibody, and HRP-labeled goat anti-mouse monoclonal antibody (Jackson, product Cat. No. 115035003) was used as a secondary antibody for routine SDS-PAGE electrophoresis and Western blot analysis.

The specific recognition results of anti-CypA monoclonal antibody BQHA7 on CypA in cells and various mouse tissues are shown in Figure 2. 2A is the specific recognition result of anti-CypA monoclonal antibody BQHA7 on CypA in cells. 2B is the specific recognition result of anti-CypA monoclonal antibody BQHA7 on CypA in cells. The results of the specific recognition of CypA in various mouse tissues by anti-BQHA7 show that the anti-CypA monoclonal antibody BQHA7 can specifically recognize the CypA protein (molecular weight approximately 18 kDa) in cell lines and different tissues of mice.

Example 4. Therapeutic effect of anti-CypA monoclonal antibody BQHA7 on type II collagen-induced rheumatoid arthritis in DBA/1 mice (hereinafter referred to as mice)

1. Grouping of mice and construction of mouse rheumatoid arthritis model

(1) Grouping of mice

Male mice aged 7-8 weeks were randomly divided into blank group (PBS, purchased from Dalian Meilun Biotech, MA0008), untreated group, BQHA7 treatment group, QH01 treatment group and methotrexate (MTX, USA Pfizer Pharmaceutical Company product) treatment group, 6 animals in each group.

(2) Constructing a mouse rheumatoid arthritis treatment model

First immunization: emulsify equal volumes of complete Freund's adjuvant (brand: chondrex, product number: 7008) and bovine type II collagen (brand: chondrex, product number: 20022) through a homogenizer for 2-3 minutes, so that the emulsion does not disperse in water The emulsification is considered successful. Use a 500 μL insulin syringe to subcutaneously inject 100 μL emulsion into the tail base of the mice in the untreated group, BQHA7 treatment group, QH01 treatment group and methotrexate treatment group, and the blank group was injected with 100 μL PBS.

Booster immunization: A booster immunization will be carried out 21 days after the first immunization. Equal volumes of incomplete Freund's adjuvant (brand: chondrex, product number: 7002) and bovine type II collagen are emulsified in an ice bath through a homogenizer for 2-3 minutes. . The mice in the untreated group, BQHA7 treatment group, QH01 treatment group and methotrexate treatment group were subcutaneously injected with 100 μL emulsion at the tail base, and the blank group was injected with 100 μL PBS. Obvious redness and swelling in the paws of DBA/1 mice within 1 week after the second immunization indicates that the model was successfully established.

Administration and treatment: Start administration and treatment on the day of the second vaccination, as follows:

Blank group: no processing;

Untreated group: mice were injected intraperitoneally with 200 μL PBS;

BQHA7 treatment group: mice were injected intraperitoneally with 100 μg anti-CypA monoclonal antibody BQHA7 (5 mg antibody/kg body weight, dissolved in 200 μL PBS), administered once every 7 days;

QH01 treatment group: mice were injected intraperitoneally with 100 μg anti-CypA monoclonal antibody QH01 (5 mg antibody/kg body weight, dissolved in 200 μL PBS), administered once every 7 days;

Methotrexate (MTX) treatment group: mice were injected with 30 μg MTX (1.5 mg MTX/kg body weight, dissolved in 50 μL PBS) through the tail vein, once every 3 days.

The mice in each group above were sacrificed on the 42nd day after the first immunization, and samples were collected and tested.

2. Evaluation of phalanx and ankle osteoarthritis inflammation in mice

(1) Paw arthritis score of DBA/1 mice

Using electronic vernier calipers, the thickness of the same hind paws of the mice and the degree of redness and swelling were measured and scored every 3 days starting from the 24th day after the first immunization. The degree of swelling and clinical scoring of mouse paws are based on the standards of the European League Against Rheumatism and the American College of Rheumatology (EULAR/ACR). Each mouse paw is scored from 0 to 4, with 0 being no redness and swelling, and 1 being the ankle. Mild redness and swelling appear on the joints and fingertips, 2 means moderate redness and swelling on the ankle joint and fingertips, 3 means severe redness and swelling on the ankle joint and fingertips, and 4 means maximum redness and swelling on the limbs.

The scores of the hind paws of mice in different groups are shown in Figure 3. The scores of the thickness of the paws, redness and swelling of the mouse paws in the BQHA7 treatment group were significantly lower than those of the untreated group (P<0.01), which is equivalent to the therapeutic effect of MTX. And BQHA7 has better therapeutic effect than QH01 (P<0.05).

(2) Micro-CT imaging analysis of DBA/1 mouse paws

Micro-CT imaging analysis was performed as soon as possible after mice were sacrificed. The isolated mouse hind paws were stored in paraformaldehyde solution and then placed in a high-resolution micro-CT system for scanning as soon as possible. After scanning, a 3D slicer is used for 2D image analysis and 3D image reconstruction. The degree of joint damage caused by inflammation is analyzed based on micro-CT images. In micro-CT images, the bones of normal joint tissues are uniform and smooth, but the bone tissue near the joints of mice with rheumatoid arthritis is severely damaged, which is manifested as local deformation and swelling of the joints, partial bone erosion, and rough borders, etc. feature.

The micro-CT images of the hind paws of mice in different groups are shown in Figure 4. The degree of joint damage in the hind paws of mice in the BQHA7 treatment group was significantly reduced. Compared with the untreated group, the joint swelling and bone erosion of mice in the BQHA7-treated group were significantly improved, and the joint bone boundaries were also smoother. BQHA7 showed better therapeutic effect than MTX. QH01 did not show good therapeutic effect.

(3) H&E staining of ankle synovial tissue

The mice were sacrificed, and the hind limbs of the mice were fixed in 4% paraformaldehyde for 24 hours, and then decalcified with 10% EDTA for 21 days. After decalcification, routine paraffin embedding and sectioning were performed, and H&E staining was used to detect ankle joint inflammation. Indicators such as cell infiltration, synovial hyperplasia, and bone destruction are used to comprehensively analyze the inflammatory response and inflammatory damage of the ankle joint. Normal synovial tissue is smooth and uniform, with synovial cells generally in a single layer and neatly arranged, with only a small amount or no inflammatory cell infiltration, no vascular proliferation, and no papillary hyperplasia.

The H&E staining results of the ankle joints of mice in different groups are shown in Figure 5. Compared with the untreated group, the number of inflammatory cell infiltrates in the ankle joints of mice in the BQHA7-treated group was significantly reduced, with basically no synovial hyperplasia and papillary hyperplasia, and overall joint destruction. The degree was significantly lower than that of the untreated group, and even showed a better therapeutic effect than MTX. QH01 did not show good therapeutic effect.

(4) Safranin-fast green staining analysis of ankle joint

The mice were sacrificed, and the hind limbs of the mice were fixed in 4% paraformaldehyde for 24 hours, and then decalcified with 10% EDTA for 21 days. After decalcification, routine paraffin embedding and sectioning were performed, and the ankles were detected by safranin-fast green staining. The degree of cartilage damage in the joints, where cartilage tissue appears red after staining and bone tissue appears green. Normal cartilage tissue is relatively evenly distributed, with clear boundaries and no proliferation.

The results of safranin-fast green staining of mouse ankle joints in different groups are shown in Figure 6. The ankle joint cartilage edges of mice in the BQHA7 treatment group are neat and clear (red), with no obvious proliferation. The degree of damage is significantly lower than that of the untreated group, and the symptoms The therapeutic effect is better than that of MTX. Although QH01 showed a certain effect, the therapeutic effect was worse than BQHA7.

(5) Enzyme-linked immunosorbent assay (ELISA) to detect cytokines

After reaching the end point of the experiment, the mice were sacrificed, and their blood was collected and serum was prepared. The ELISA method was used to detect IL-17A (Hangzhou Lianke Biotechnology Co., Ltd., catalog number: EK217-01), TNF-α (Hangzhou Lianke Biotechnology Co., Ltd., catalog number: EK282-01) and IL- 6 (Hangzhou Lianke Biotechnology Co., Ltd., catalog number: EK206-01). The levels of IL-17A, TNF-α and IL-6 in serum are important indicators reflecting the disease process and systemic inflammatory response of rheumatoid arthritis. Please refer to the instructions for ELISA steps.

The levels of IL-17A, TNF-α and IL-6 in the serum of mice in different groups are shown in Figure 7. The levels of IL-17A, TNF-α and IL-6 in the serum of mice in the BQHA7 treatment group were significantly lower than those in the untreated group. treatment group, and showed a therapeutic effect comparable to MTX, indicating that BQHA7 has a good effect on alleviating the inflammatory response associated with rheumatoid arthritis. BQHA7 showed better therapeutic effect than QH01.

Claims (8)

1. Hybridoma cell strain BQHA7 secreting anti-CypA monoclonal antibody, and the preservation number is CGMCC No. 45101.

2. An anti-CypA monoclonal antibody secreted by the hybridoma cell line BQHA7 of claim 1.

3. Use of the anti-CypA monoclonal antibody of claim 2 in any one of the following;

1) Preparing a product for treating rheumatoid arthritis;

2) Preparing a product that reduces the extent of injury or destruction of rheumatoid arthritis joints;

3) Preparing a product for delaying the disease process of rheumatoid arthritis;

4) Preparing a product for alleviating inflammatory response of rheumatoid arthritis;

5) Preparing a product for improving the degree of arthrocele and/or bone erosion of rheumatoid arthritis;

6) Preparing a product for reducing the infiltration quantity of inflammatory cells;

7) Preparing a product for reducing the expression level of factors related to inflammatory reaction;

8) The product for detecting the cyclophilin A is prepared.

4. A use according to claim 3, characterized in that: the product is a kit.

5. A product comprising the anti-CypA monoclonal antibody of claim 2.

6. The product according to claim 5, wherein:

the product has at least one of the following functions:

1) Treating rheumatoid arthritis;

2) Reducing the degree of injury or destruction of rheumatoid arthritis joints;

3) Delay the disease course of rheumatoid arthritis;

4) Relieving inflammatory response of rheumatoid arthritis;

5) Improving the arthrocele and/or bone erosion of rheumatoid arthritis;

6) Reducing inflammatory cell infiltration quantity;

7) Reducing the expression level of factors associated with inflammatory response;

8) Detecting the cyclophilin A.

7. The product according to claim 5 or 6, characterized in that: the product is a kit.

8. A method of detecting cyclophilin a comprising the steps of: the anti-CypA monoclonal antibody according to claim 2 is used as an antibody to detect whether the sample to be detected contains cyclophilin a.