Anti-cyclophilin a monoclonal antibody and use thereof in treating viral pneumonia

Abstract

An anti-Cyclophilin A monoclonal antibody, and a use thereof in treating viral pneumonia. Provided is a hybridoma cell SQHC14 secreting an anti-CypA monoclonal antibody, having the preservation number CGMCC No. 45049; also provided is an anti-CypA monoclonal antibody SQHC14 secreted by the hybridoma cell SQHC14; the monoclonal antibody SQHC14 can specifically recognize CypA, and inhibit secretion of inflammatory factors and leukocyte migration induced by viral infection.

Description

Anti-Cyclophilin A monoclonal antibodies and their use in treating viral pneumonia

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application (application number 202311469423.3) filed on November 7, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention belongs to the field of biomedicine and relates to an anti-Cyclophilin A monoclonal antibody and an application thereof in treating viral pneumonia. Background Art

[0004] Cyclophilin A (CypA), a highly conserved protein with peptidyl-prolyl cis / trans isomerase activity, is ubiquitous in the biological world. CypA is closely associated with viral-induced inflammatory responses. Influenza A and B viruses, as well as the novel coronavirus, often trigger viral pneumonia and even cytokine storms, which can lead to acute respiratory distress syndrome. However, current antiviral therapies are insufficient to prevent or treat these complications. During viral infection, intracellular CypA enhances the stability of nuclear transcription factor-κB (NF-κB) p65 and promotes its phosphorylation and nuclear import, thereby upregulating the expression of proinflammatory cytokines such as IL-6 and IL-1β. Furthermore, CypA is secreted extracellularly in response to viral stimulation. Extracellular CypA (eCypA) plays a crucial regulatory role in the progression of inflammation. For example, after viral infection, the secretion of eCypA increases, and eCypA and CD147 may interact in the form of ligand-receptor, initiating downstream intracellular ERK and other related signaling pathways, thereby promoting the secretion of inflammatory factors and chemokines, and participating in regulating the host's inflammatory response.

[0005] Previously, a monoclonal antibody, QH01 (CN 113248617B), was obtained against Cyclophilin A and demonstrated some therapeutic efficacy against influenza A virus-induced pneumonia in mice. Given that different monoclonal antibodies bind to CypA at different sites, potentially leading to varying anti-inflammatory effects, there remains a need to screen for more effective CypA-targeting antibodies to promote their application in the treatment of viral pneumonia.

[0006] Therefore, finding new antibodies targeting CypA is promising for the treatment of viral pneumonia.

[0007] Invention Disclosure

[0008] The purpose of the present invention is to provide an anti-Cyclophilin A monoclonal antibody and an application thereof in treating viral pneumonia.

[0009] In a first aspect, the present invention provides a hybridoma cell line SQHC14 secreting an anti-CypA monoclonal antibody (abbreviated as CypA monoclonal antibody or anti-CypA monoclonal antibody), whose deposit number is CGMCC No.45049.

[0010] In a second aspect, the present invention provides an anti-CypA monoclonal antibody secreted by the hybridoma cell line SQHC14 described in the first aspect.

[0011] In a third aspect, the present invention provides a use of the anti-CypA monoclonal antibody according to the second aspect, wherein the use is selected from at least one of the following:

[0012] 1) For the preparation of products for the treatment of viral pneumonia;

[0013] 2) Preparation of products that inhibit inflammatory responses and / or leukocyte chemotaxis induced by viral infection;

[0014] The above inhibition of inflammatory response and / or leukocyte chemotaxis induced by viral infection is specifically manifested in reducing the content of interleukin 1β (IL-1β) and / or CC chemokine ligand 2 (CCL2) in cells.

[0015] 3) Preparation of products that inhibit inflammatory responses in the body caused by viral infection;

[0016] The above-mentioned inflammatory response of the body is further manifested as pneumonia, which is specifically manifested in a decrease in lung index or a decrease in the content of CCL2 and IL-1β in the body;

[0017] 4) preparing products for inhibiting leukocyte adhesion caused by inflammation;

[0018] 5) Preparation of products for inhibiting leukocyte migration caused by inflammation;

[0019] 6) Prepare a product for detecting cyclophilin A.

[0020] In the above-mentioned use, the virus is influenza virus or other pneumonia-inducing virus.

[0021] In a fourth aspect, the present invention provides a product for treating viral pneumonia, comprising the anti-CypA monoclonal antibody described in the second aspect.

[0022] In a fifth aspect, the present invention provides a product for inhibiting inflammatory response and / or monocyte chemotaxis induced by viral infection, which comprises the anti-CypA monoclonal antibody described in the second aspect.

[0023] In a sixth aspect, the present invention provides a product for inhibiting the body's inflammatory response caused by viral infection, which comprises the anti-CypA monoclonal antibody described in the second aspect.

[0024] In a seventh aspect, the present invention provides a product for inhibiting leukocyte adhesion caused by inflammation, comprising the anti-CypA monoclonal antibody described in the second aspect.

[0025] In an eighth aspect, the present invention provides a product for inhibiting leukocyte migration caused by inflammation, comprising the anti-CypA monoclonal antibody described in the second aspect.

[0026] In a ninth aspect, the present invention provides a product for detecting cyclophilin A, comprising the anti-CypA monoclonal antibody described in the second aspect.

[0027] In the above, the product is a kit, a medicine or a composition.

[0028] The products mentioned above are drugs, and also include any other substances that can serve as drug carriers.

[0029] In practical applications, the anti-CypA monoclonal antibody of the present invention can be directly administered to a patient as a drug, or mixed with a suitable carrier or excipient and administered to a patient to achieve the purpose of treating viral pneumonia. The carrier materials here include but are not limited to water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acids, etc.), insoluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Among them, water-soluble carrier materials are preferred. These materials can be used to make a variety of dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections, etc. It can be a conventional preparation, sustained-release preparation, controlled-release preparation and various particulate delivery systems. In order to make unit dosage forms into tablets, various carriers well known in the art can be widely used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants. , such as dried starch, alginate, agar powder, brown seaweed starch, sodium bicarbonate with citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; absorption enhancers, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further prepared as coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer tablets and multilayer tablets. To prepare the unit dosage form into a pill, a wide variety of carriers known in the art can be used. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinyl pyrrolidone, gelucine, kaolin, and talc; binders such as gum arabic, tragacanth gum, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dry starch, alginates, sodium lauryl sulfate, methylcellulose, and ethylcellulose. To prepare unit dosage forms as suppositories, a wide variety of carriers known in the art can be used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides.To prepare unit dosage forms into injectable preparations, such as solutions, emulsions, lyophilized powder injections, and suspensions, any diluent commonly used in the art can be used, including water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. Furthermore, to prepare isotonic injections, appropriate amounts of sodium chloride, glucose, or glycerol can be added to the injectable preparation. Conventional cosolvents, buffers, and pH adjusters can also be added. Furthermore, colorants, preservatives, fragrances, flavoring agents, sweeteners, or other materials may be added to the pharmaceutical preparations, if desired. These dosage forms can be administered by injection, including subcutaneous, intravenous, intramuscular, and intracavitary injections; cavitary administration, such as rectal and vaginal; respiratory administration, such as nasal; and mucosal administration. Injection is preferred among these routes of administration.

[0030] In a tenth aspect, the present invention provides a method for detecting cyclophilin A, comprising the following steps: using the anti-CypA monoclonal antibody described in the second aspect as an antibody to detect whether a sample to be tested contains cyclophilin A.

[0031] In an eleventh aspect, the present invention provides a method for treating viral pneumonia, comprising the steps of administering the anti-CypA monoclonal antibody or a pharmaceutically acceptable salt thereof according to the second aspect to a subject to treat and / or prevent viral pneumonia.

[0032] In a twelfth aspect, the present invention provides a method for inhibiting pneumonia induced by a virus-infected animal, comprising the following steps: administering the anti-CypA monoclonal antibody or a pharmaceutically acceptable salt thereof described in the second aspect to a subject to inhibit pneumonia induced by a virus-infected animal.

[0033] In the thirteenth aspect, the present invention provides a method for inhibiting the body's inflammatory response caused by viral infection, comprising the following steps: using the anti-CypA monoclonal antibody described in the second aspect to act on the body with an inflammatory response caused by viral infection to inhibit the body's inflammatory response caused by viral infection.

[0034] The viruses mentioned above are influenza viruses or other pneumonia-inducing viruses.

[0035] In the embodiment of the present invention, influenza A virus or influenza B virus is taken as an example.

[0036] As used herein, a "subject" is an animal, such as a mammal, including a primate (e.g., a human or non-human primate, such as a monkey or chimpanzee), a non-primate (e.g., a cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, horse, or whale), or a bird (e.g., a duck or goose). In one embodiment, the subject is a human.

[0037] Preservation Instructions

[0038] Strain name: SQHC14

[0039] Classification and nomenclature: Mouse hybridoma cells

[0040] Depository: General Microbiology Center of China Culture Collection Administration

[0041] Abbreviation of depository institution: CGMCC

[0042] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0043] Date of deposit: February 24, 2022

[0044] CGMCC registration number: CGMCC No.45049 BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 shows the titer of SQHC14 monoclonal antibody detected by ELISA.

[0046] Figure 2 shows the detection of CypA expression in cells and tissues by SQHC14.

[0047] FIG3 shows that SQHC14 inhibits THP1 cell adhesion and migration.

[0048] FIG4 shows that SQHC14 inhibits the expression of chemokines and cytokines in THP1 cells induced by IAV.

[0049] FIG5 shows the lung index of mice in the IAV-induced pneumonia model treated with SQHC14.

[0050] Figure 6 shows the expression of chemokines and cytokines in mice in the IAV-induced pneumonia model treated with SQHC14.

[0051] FIG7 shows that SQHC14 inhibits the expression of chemokines and cytokines induced by IBV in THP1 cells.

[0052] FIG8 shows the lung index of mice in the IBV-induced pneumonia model treated with SQHC14.

[0053] FIG9 shows the expression of chemokines and cytokines in mice treated with SQHC14 in an IBV-induced pneumonia model.

[0054] Best Mode for Carrying Out the Invention

[0055] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0056] Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent stores.

[0057] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention.

[0058] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0059] Female C57BL / 6 mice were purchased from Beijing Weitonglihua Laboratory Animal Co., Ltd.

[0060] The CypA monoclonal antibody QH01 in the embodiment is QH01 in CN 113248617 B (authorization announcement date 2021.10.01; application number 202110682376.5), and the preservation number of the hybridoma cell line secreting it is CGMCC No.21909.

[0061] The PBS (pH: 7.2-7.4) in the following examples was purchased from Beijing Solebow Technology Co., Ltd. with the product number P1020.

[0062] In the following examples, CypA protein was purchased from Suzhou Jinan Protein Technology Co., Ltd. with the product number CR15.

[0063] The reagents used in the ELISA method are as follows:

[0064] The coating solution (pH = 9.6) was purchased from Beijing Solebow Technology Co., Ltd. with the product number C1050.

[0065] The washing solution (pH: 7.2-7.4) was purchased from Beijing Solebow Technology Co., Ltd. with the product number P1031.

[0066] The blocking solution was a washing solution containing (mass volume percentage, g:ml) 2% skimmed milk powder;

[0067] The color developing solution is a mixture of 1% solution A and 10% solution B by volume, wherein solution A is 1% TMB in DMSO; solution B is citric acid buffer containing 0.1% CH4N2O·H2O2.

[0068] The stop solution was 0.5 M aqueous sulfuric acid solution.

[0069] Example 1. Preparation of anti-CypA monoclonal antibody

[0070] 1. Obtaining hybridoma cell lines secreting anti-CypA monoclonal antibodies

[0071] 1. Immunization of mice

[0072] Six SPF-grade BALB / c female mice were immunized with CypA protein by intramuscular injection for the first immunization, with an immunization dose of 20 μg protein per mouse; then six subsequent booster immunizations were performed in the same manner and dosage, with each immunization separated by one week; one week after the booster immunization, the mice were intraperitoneally injected with 50 μg CypA protein to complete the final immunization, and one week later, blood was collected from the mouse orbitals to determine the serum antibody titer.

[0073] 2. Screening SQHA4

[0074] ① Detect the serum titer of 6 immunized mice by ELISA, and select mice with higher titer for hybridoma fusion.

[0075] ②Cell fusion experiment: Mouse spleen cells were fused with SP2 / 0 cells using the PEG method. After fusion, the cells were screened and cultured in a semi-solid culture medium (containing HAT).

[0076] ③ Picking single clones: Pick 10 plates × 93 cell single clones and culture them in 96-well cell culture plates (previously plated with thymocytes, 100 μL / well).

[0077] ④ Primary screening of monoclonal cells: discard all the supernatants of monoclonal cells in the 96-well culture plate, add 200 μL / well of 20% newborn calf IMDM culture medium (containing HT) for the first screening.

[0078] ⑤ Secondary screening of monoclonal cells: Coat the plate with recombinant CypA protein, and perform a second screening on the selected clones using the ELISA method to obtain positive hybridoma cell lines.

[0079] ⑥ Three-stage screening of monoclonal cells: The positive cell lines obtained from the second screening were coated with recombinant CypA protein and "tag protein" respectively, and the ELISA method was used for the third screening, and finally the hybridoma cell line SQHC14 that secreted CypA monoclonal antibody (SQHC14) was obtained.

[0080] The hybridoma cell line secreting SQHC14 was deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101) on February 24, 2022, with the deposit number CGMCC No. 45049 and the classification name as mouse hybridoma cell.

[0081] 2. Preparation of anti-CypA monoclonal antibody from ascites

[0082] The hybridoma cell line SQHC14 secreting SQHC14 obtained above was injected into the peritoneal cavity of BALB / c mice at a cell concentration of 1.5×10 6 Ascites was collected after 10 to 14 days.

[0083] The antibodies in the ascites were purified using a Protein G-Sepharose 4B adsorption chromatography column to obtain the anti-CypA monoclonal antibody SQHC14. The antibodies were collected in 0.1 M glycine-hydrochloric acid buffer (pH = 2.7), and then replaced in PBS for storage and use in subsequent experiments.

[0084] The titer of anti-CypA monoclonal antibody SQHC14 was detected by ELISA and compared with that of CypA monoclonal antibody QH01.

[0085] 1. Dilute CypA protein with coating solution to a final concentration of 2 μg / ml, 100 μl / well, incubate at 4°C overnight, and then wash three times with washing solution.

[0086] 2. Block with blocking solution, 200ul / well, incubate at 37℃ for 2h, then wash 3 times with washing solution;

[0087] 3. Add primary antibody (anti-CypA monoclonal antibody SQHC14 or CypA monoclonal antibody QH01) or PBS (Blank) at 10 μg / mL in the first well, then perform a 4-fold serial dilution with PBS, with 8 points, and a blank in the last well, at 100 μL / well, incubate at 25°C for 1 hour, and then wash three times with washing solution;

[0088] 4. Add HRP-labeled goat anti-mouse IgG secondary antibody (purchased from Abotek Biotechnology Co., Ltd., catalog number: AS003) diluted 10,000-fold with PBS, 100 μL / well, incubate at 25°C for 1 hour, remove and wash three times with washing buffer;

[0089] 5. Add 100 μL / well of color development solution and the color development time is about 10 minutes;

[0090] 6. Add 50 μL stop solution to each well to stop the reaction;

[0091] 7. Measure absorbance at dual wavelengths (450nm, 630nm) and record and save data;

[0092] The analysis results are shown in FIG1 . The half effective concentration (EC50) of the anti-CypA monoclonal antibody SQHC14 is 0.02899 μg / mL, and the EC50 of QH01 is 2.415 μg / mL.

[0093] Example 2: Identification of subtypes of anti-CypA monoclonal antibody SQHC14

[0094] The subtype of anti-CypA monoclonal antibody SQHC14 was identified using a mouse monoclonal antibody subtype identification kit (purchased from Proteintech, catalog number PK20002). The steps were as described in the manufacturer's instructions.

[0095] The analysis results are shown in Table 1 . The heavy chain of the anti-CypA monoclonal antibody SQHC14 is IgG1, and the light chain is κ.

[0096] Table 1 shows the subtype identification of SQHC14

[0097] Example 3: Detection of the specific recognition of CypA by anti-CypA monoclonal antibody SQHC14 in cell lines and mouse tissues

[0098] Human monocytic leukemia cell line (THP1, purchased from ATCC, TIB-202) and lung, spleen and lymph node tissues of 6-8 week old female C57BL / 6 mice (purchased from Weitonglihua, hereinafter referred to as mice) were lysed using Lysis Buffer (150mM NaCl, 20mM HEPES, 1mM EDTA, 1% Triton100, 10% glycerol) containing Protease Inhibitor Cocktail (Roche, #5871, dosage refer to the instructions), and the lysates were collected.

[0099] An appropriate amount of 5× SDS-PAGE protein loading buffer was added to each lysate and heated at 98°C for 15 min. Conventional SDS-PAGE electrophoresis and Western blot analysis were performed using the anti-CypA monoclonal antibody SQHC14 and anti-GAPDH antibody (Santa Cruz, catalog number sc-1616-R) prepared from ascites fluid in Example 1 as primary antibodies and an HRP-labeled goat anti-mouse monoclonal antibody (Jackson, catalog number 115035003) as a secondary antibody.

[0100] The specific recognition results of anti-CypA monoclonal antibody SQHC14 for CypA in cells and mouse tissues are shown in Figure 2. 2A shows the specific recognition results of anti-CypA monoclonal antibody SQHC14 for CypA in cells, and 2B shows the specific recognition results of anti-CypA monoclonal antibody SQHC14 for CypA in mouse tissues. It can be seen that anti-CypA monoclonal antibody SQHC14 can specifically recognize CypA protein (molecular weight approximately 18 kDa) in cell lines and different mouse tissues.

[0101] Example 4: Detection of the effect of anti-CypA monoclonal antibody SQHC14 on THP1 cell adhesion and migration

[0102] Chemokine-induced excessive accumulation of immune cells in the lungs is a major factor in the development of viral pneumonia. Key steps in this accumulation process are leukocyte adhesion and migration. Using CCL2 to induce THP1 cell adhesion and migration, mimicking the immune cell accumulation process during pneumonia, we tested the effect of CypA antibodies.

[0103] In this experiment, CypA monoclonal antibody (QH01) was compared with the SQHC14 monoclonal antibody prepared in Example 1. Specifically, four groups were set up: blank group (PBS), chemokine group (CCL2), antibody group 1 (CCL2+SQHC14) and antibody group 2 (CCL2+QH01).

[0104] In the adhesion assay, THP1 cells were resuspended in 1640 medium (PBS group), 1640 medium containing 20 ng / mL CCL2 (purchased from MCE, catalog number HY-P700034AF) (chemokine group), 1640 medium containing 50 nM SQHC14 + 20 ng / mL CCL2 (antibody group 1), and 1640 medium containing 50 nM QH01 + 20 ng / mL CCL2 (antibody group 2), and added to a 96-well plate coated with 5 mg / mL ICAM1. After incubation in a cell culture incubator, non-adherent cells were collected, and the remaining adherent cells were collected with a cell scraper and counted. The relative adhesion of the cells was determined by the ratio of adherent cells to total cells (non-adherent cells + adherent cells).

[0105] In the migration assay, THP1 cells were resuspended in 1640 medium (PBS group), 1640 medium containing 20 ng / mL CCL2 (purchased from MCE, catalog number HY-P700034AF) (chemokine group), 1640 medium containing 50 nM SQHC14 + 20 ng / mL CCL2 (antibody group 1), and 1640 medium containing 50 nM QH01 + 20 ng / mL CCL2 (antibody group 2), and added to 2×10 5 Human vascular endothelial cells (purchased from ATCC, CRL-1730) were cultured in the upper transwell chamber. After incubation in a cell culture incubator, the cells in the upper and lower transwell chambers were collected and counted. The relative migration of cells was determined by the ratio of the number of cells in the lower chamber to the total cells (upper chamber cells + lower chamber cells).

[0106] The experimental results are shown in Figure 3, with A showing the adhesion assay and B showing the migration assay. SQHC14 significantly inhibited CCL2-induced THP1 cell adhesion to ICAM1 and transendothelial migration (P < 0.01). Furthermore, QH01 had a weaker effect on CCL2-induced THP1 cell adhesion and transendothelial migration (P > 0.05). These results suggest that SQHC14 can inhibit leukocyte adhesion and / or migration during inflammation, thereby suppressing the inflammatory response.

[0107] Example 5: Detection of the effect of SQHC14 on chemokine and cytokine secretion after influenza A virus (IAV) infection of THP1 cells

[0108] CC chemokine ligand 2 (CCL2) and interleukin-1β (IL-1β) are crucial for the inflammatory response and leukocyte chemotaxis induced by viral infection, respectively.

[0109] In this experiment, CypA monoclonal antibody (QH01) was compared with SQHC14. The groups were divided into 4 groups: blank group (PBS), infection group (IAV), antibody group 1 (IAV + SQHC14), and antibody group 2 (IAV + QH01).

[0110] The details are as follows:

[0111] IAV (H1N1-WSN strain) was rescued and propagated by our laboratory. The strain is described in the following literature, Zheng et al., 2019, Cell Reports 27, 1875–1885, and is named Influenza virus A / WSN / 33 (H1N1) in the literature.

[0112] PBS group: PBS treatment for 2 h, and cells were suspended in 1640 medium and cultured for 12 h;

[0113] Infection group: IAV infected THP1 cells (infection dose 10 6 PFU, MOI value of 1.0) for 2 h, and the cells were suspended in 1640 medium and cultured for 12 h;

[0114] Antibody group 1 (IAV+SQHC14): IAV infected THP1 cells (infection dose 10 6 PFU, MOI value of 1.0) for 2 h, and then replaced with 1640 medium containing 50 nM SQHC14 and cultured for another 12 h;

[0115] Antibody group 2 (IAV+QH01): IAV infected THP1 cells (infection dose 10 6PFU, MOI value of 1.0) for 2 h, and then replaced with 1640 medium containing 50 nM QH01 and continued to culture for 12 h;

[0116] The cell supernatants of the above groups were collected for subsequent ELISA detection.

[0117] Human CCL2 (Hangzhou Lianke Biotechnology Co., Ltd., catalog number 70-EK187) and IL-1β (Hangzhou Lianke Biotechnology Co., Ltd., catalog number 70-EK101) ELISA kits were used to detect the effect of CypA monoclonal antibody on CCL2 and IL-1β secretion in the supernatant of IAV-infected THP1 cells. The steps were as described in the manufacturer's instructions.

[0118] The results are shown in Figure 4, where A is the CCL2 content detection result and B is the IL-1β content detection result. It can be seen that the CCL2 and IL-1β contents of antibody group 1 were significantly decreased compared with those of the infection group (P < 0.01). Although the CCL2 and IL-1β contents of antibody group 2 were also significantly decreased compared with those of the infection group and were statistically significant (P < 0.05), the downward trend of antibody group 2 was significantly smaller than that of antibody group 1, and there was a statistical difference between the two (P < 0.05).

[0119] Therefore, for IAV-infected THP1 cells, SQHC14 had a stronger inhibitory effect on the expression of CCL2 and IL-1β than QH01, indicating that it can inhibit the inflammatory response and leukocyte chemotaxis caused by viral infection.

[0120] Example 6: Therapeutic Effect of Anti-CypA Monoclonal Antibody SQHC14 on IAV-Induced Pneumonia in Mice

[0121] 1. Mouse grouping and establishment of IAV-induced mouse pneumonia model

[0122] (1) Grouping of mice

[0123] 7-8 week old C57BL / 6 mice were randomly divided into 4 groups: blank group (PBS, purchased from Dalian Meilun Biotechnology, MA0008), infection group (IAV), antibody group 1 (IAV+SQHC14) and antibody group 2 (IAV+QH01), with 6 mice in each group (weight 19±3 g).

[0124] (2) Establishment of a therapeutic model of viral pneumonia in mice

[0125] C57BL / 6 mice were anesthetized by intraperitoneal injection of avertin at a rate of 200 μL / 10 g body weight. After anesthesia, the following procedures were performed on each group of mice:

[0126] Blank group (PBS): Each mouse was injected with 50 μL PBS via nasal cavity; and then injected with 100 μL PBS at the same time as the corresponding antibody injection in the antibody group.

[0127] Infection group: Each mouse was intranasally instilled with 50 μL of PBS containing 2000 PFU of IAV-WSN. IAV-WSN was rescued and propagated in our laboratory. This strain is described in the following literature: Zheng et al., 2019, Cell Reports 27, 1875–1885, and is named Influenza virus A / WSN / 33 (H1N1) in the literature. At the same time as the antibody injection in the antibody group, each mouse was injected with 100 μL of PBS.

[0128] Antibody group 1: Each mouse was intranasally instilled with 50 μL of PBS containing 2000 PFU of IAV-WSN. Each mouse was intraperitoneally injected with the SQHC14 monoclonal antibody prepared in Example 1 (10 mg / kg body weight, dissolved in 100 μL PBS) 24 h after IAV infection.

[0129] Antibody group 2: Each mouse was intranasally instilled with 50 μL of PBS containing 2000 PFU of IAV-WSN, and each mouse was intraperitoneally injected with QH01 monoclonal antibody (10 mg / kg body weight, dissolved in 100 μL PBS) 24 h after IAV infection.

[0130] On the 7th day after treatment (the day of antibody injection was designated as day 0), the lung index and the expression levels of cytokines in lung tissue of the above groups were detected.

[0131] 2. Lung Index Test

[0132] Mice in each group were anesthetized with ether and weighed. Blood was collected from the mice, and the mice were placed in a supine position. The chest cavity was opened, the esophagus and heart were removed, and the lung tissue was isolated and weighed. The lungs were weighed to calculate the lung index (Lung Weight = Mouse Lung Weight / Mouse Body Weight). The Lung Index is a very important indicator of the severity of lung inflammation; inflammation can lead to an increase in the Lung Index.

[0133] The results are shown in Figure 5. The lung index of mice in antibody group 1 was significantly decreased compared to the infected group (P < 0.01). The lung index of mice in antibody group 2 also showed a downward trend compared to the infected group (P < 0.05), but the downward trend was not as significant as that in antibody group 1. The lung index of mice in antibody group 1 was significantly different from that in antibody group 2 (P < 0.05). Therefore, in the IAV-induced pneumonia model, SQHC14 demonstrated superior therapeutic efficacy compared to QH01.

[0134] Detection of chemokine and cytokine expression levels in lung tissue

[0135] (1) Obtain bronchoalveolar lavage fluid (BALF)

[0136] Mice in each group were killed by dislocating the neck; whole blood was collected by cardiac blood sampling, and the limbs of the mice were fixed with syringe heads; the chest cavity was opened, and a small incision was made perpendicularly at the thickest part of the trachea (near the Adam's apple) using ophthalmic scissors (curved), and then a T-shaped incision was made perpendicular to the incision using ophthalmic scissors (curved). The syringe head was removed from the cut syringe, and the syringe head was carefully inserted into the trachea and fixed with thread twice. After the syringe aspirated 1 mL of PBS, the syringe head slot was clamped with hemostats, the syringe was installed, and slowly pushed into the lungs. After 5-10 seconds, it was slowly aspirated (1 mL can recover about 0.8 mL), and the syringe head was clamped with hemostats, rotated and removed, and the alveolar lavage fluid was slowly and carefully injected into the EP tube; the lavage was repeated to obtain bronchoalveolar lavage fluid.

[0137] (2) Enzyme-linked immunosorbent assay (ELISA) detection of chemokines and cytokines

[0138] Mouse CCL2 and IL-1β are crucial for viral infection-induced inflammatory responses and monocyte tissue infiltration. Mouse BALF levels were measured using ELISA kits for mouse CCL2 (Hangzhou Lianke Biotechnology Co., Ltd., catalog number 70-EK287) and IL-1β (Hangzhou Lianke Biotechnology Co., Ltd., catalog number 70-EK201B). Follow the instructions in the manufacturer's manual.

[0139] The results are shown in Figure 6, where A shows the CCL2 content test results and B shows the IL-1β content test results. The CCL2 and IL-1β levels in the BALF of mice in antibody group 1 (referred to as treatment group 1 in the figure) were significantly decreased compared to those in the infection group (P < 0.01). Although the CCL2 and IL-1β levels in the BALF of mice in antibody group 2 (referred to as treatment group 2 in the figure) also showed a downward trend compared to the infection group (P < 0.05), the downward trend in antibody group 2 was significantly smaller than that in antibody group 1. Compared with antibody group 2, the CCL2 and IL-1β levels in antibody group 1 were significantly lower, with statistically significant differences (P < 0.05).

[0140] Therefore, SQHC14 exhibited superior therapeutic efficacy compared to QH01 in the IAV-induced pneumonia model.

[0141] Example 7: Detection of the effect of SQHC14 on chemokine and cytokine secretion in THP1 cells infected with influenza B virus (IBV)

[0142] In this experiment, CypA monoclonal antibody (QH01) was compared with SQHC14 monoclonal antibody obtained in Example 1. The groups were divided into 4 groups: blank group (PBS), infection group (IBV), antibody group 1 (IBV+SQHC14) and antibody group 2 (IBV+QH01).

[0143] The details are as follows:

[0144] IBV, this strain is recorded in the following literature, Zheng et al., 2019, Cell Reports 27, 1875–1885, and is named Influenza virus B / Guangxi / JZ1352 / 2018 in the literature.

[0145] PBS group: PBS treatment for 2 h, and cells were suspended in 1640 medium and cultured for 12 h;

[0146] Infection group: IBV infected THP1 cells (infection dose 10 6 PFU, MOI value of 1.0) for 2 h, and the cells were suspended in 1640 medium and cultured for 12 h;

[0147] Antibody group 1 (IBV+SQHC14): IBV infected THP1 cells (infection dose 10 6 PFU, MOI value of 1.0) for 2 h, and then replaced with 1640 medium containing 50 nM SQHC14 and cultured for another 12 h;

[0148] Antibody group 2 (IBV+QH01): IBV infected THP1 cells (infection dose 10 6 PFU, MOI value of 1.0) for 2 h, and then replaced with 1640 medium containing 50 nM QH01 and cultured for another 12 h;

[0149] The cell supernatants of the above groups were collected for subsequent ELISA detection.

[0150] Human CCL2 (Hangzhou Lianke Biotechnology Co., Ltd., catalog number 70-EK187) and IL-1β (Hangzhou Lianke Biotechnology Co., Ltd., catalog number 70-EK101) ELISA kits were used to detect the effect of CypA monoclonal antibody on CCL2 and IL-1β secretion in the supernatant of IBV-infected THP1 cells. The steps were as described in the manufacturer's instructions.

[0151] The results are shown in Figure 7, where A is the CCL2 content detection result, and B is the IL-1β content detection result. The CCL2 and IL-1β contents of antibody group 1 were significantly decreased compared with those of the infection group (P < 0.01), and the CCL2 and IL-1β contents of antibody group 2 were also significantly decreased compared with those of the infection group and were statistically significant (P < 0.05). However, the downward trend of antibody group 2 was significantly smaller than that of antibody group 1, and there was a statistically significant difference between the two (P < 0.05).

[0152] Therefore, SQHC14 has a stronger inhibitory effect on CCL2 and IL-1β than QH01 in IBV-infected THP1 cells.

[0153] Example 8: Therapeutic Effect of SQHC14 on IBV-Induced Pneumonia in Mice

[0154] 1. Mouse grouping and establishment of IBV-induced mouse pneumonia model

[0155] (1) Grouping of mice

[0156] Female C57BL / 6 mice aged 6-8 weeks were randomly divided into four groups: blank group (PBS), infection group (IAV), antibody group 1 (IAV + SQHC14), and antibody group 2 (IAV + QH01), with 6 mice in each group (weight 19 ± 3 g).

[0157] (2) Establishment of a therapeutic model of viral pneumonia in mice

[0158] C57BL / 6 mice were anesthetized by intraperitoneal injection of avertin at a rate of 200 μL / 10 g body weight. After anesthesia, the following procedures were performed on each group of mice:

[0159] Blank group (PBS): Each mouse was injected with 50 μL PBS via nasal cavity; and then injected with 100 μL PBS at the same time as the corresponding antibody injection in the antibody group.

[0160] Infection group: Each mouse was intranasally instilled with 50 μL of PBS containing 2000 PFU of IBV (described in the literature (Zheng et al., 2019, Cell Reports 27, 1875–1885, referred to as Influenza virus B / Guangxi / JZ1352 / 2018) and provided by Professor Wang Dayan of the Chinese Center for Disease Control and Prevention); the antibody group was then injected with 100 μL of PBS at the same time as the antibody injection.

[0161] Antibody group 1: Each mouse was intranasally instilled with 50 μL of PBS containing 2000 PFU of IBV. Each mouse was intraperitoneally injected with the SQHC14 monoclonal antibody prepared in Example 1 (10 mg / kg body weight, dissolved in 100 μL of PBS) 24 h after IBV infection.

[0162] Antibody group 2: Each mouse was intranasally instilled with 50 μL of PBS containing 2000 PFU of IBV, and each mouse was intraperitoneally injected with QH01 monoclonal antibody (10 mg / kg body weight, dissolved in 100 μL PBS) 24 h after IBV infection.

[0163] On the 7th day after treatment (the day of antibody injection was designated as day 0), the lung index and the expression levels of cytokines in lung tissue of the above groups were detected.

[0164] 2. Lung Index Test

[0165] The method is the same as Example 6.

[0166] The results are shown in Figure 8. The lung index of mice in antibody group 1 was significantly decreased compared to the infected group (P < 0.01). The lung index of mice in antibody group 2 also decreased compared to the infected group (P < 0.05), but the downward trend was not as obvious as that in antibody group 1. The lung index of mice in antibody group 1 was significantly different from that in antibody group 2 (P < 0.05).

[0167] Therefore, SQHC14 exhibited superior therapeutic efficacy to QH01 in the IBV-induced pneumonia model.

[0168] Detection of chemokine and cytokine expression levels in lung tissue

[0169] The method is the same as Example 6.

[0170] The results are shown in Figure 9, where A shows the CCL2 content test results and B shows the IL-1β content test results. The CCL2 and IL-1β levels in the BALF of mice in antibody group 1 (referred to as treatment group 1 in the figure) were significantly decreased compared to those in the infection group (P < 0.01). Although the CCL2 and IL-1β levels in the BALF of antibody group 2 also showed a downward trend compared to the infection group (P < 0.05), the downward trend in antibody group 2 (referred to as treatment group 2 in the figure) was significantly less than that in antibody group 1. Compared with antibody group 2, the CCL2 and IL-1β levels in antibody group 1 were statistically lower (P < 0.05). Therefore, in the IBV-induced pneumonia model, SQHC14 exhibited superior therapeutic effects to QH01.

[0171] Industrial Applications

[0172] Experiments of the present invention have shown that the present invention provides a hybridoma cell line capable of secreting anti-CypA monoclonal antibody. The hybridoma cell line can secrete a monoclonal antibody (SQHC14), which can specifically recognize CypA and inhibit the secretion of inflammatory factors and leukocyte migration induced by viral infection.

Claims

1. The hybridoma cell line SQHC14 secreting anti-CypA monoclonal antibody has a deposit number of CGMCC No.45049.

2. The anti-CypA monoclonal antibody secreted by the hybridoma cell line SQHC14 according to claim 1.

3. The use of the anti-CypA monoclonal antibody according to claim 2, wherein the use is selected from at least one of the following; 1) For the preparation of products for the treatment of viral pneumonia; 2) Preparation of products that inhibit inflammatory responses and / or leukocyte chemotaxis induced by viral infection; 3) Preparation of products that inhibit inflammatory responses in the body caused by viral infection; 4) preparing products for inhibiting leukocyte adhesion caused by inflammation; 5) Preparation of products for inhibiting leukocyte migration caused by inflammation; 6) Prepare a product for detecting cyclophilin A.

4. The use according to claim 3, characterized in that: The virus is influenza virus or other pneumonia-inducing virus.

5. The use according to claim 3 or 4, characterized in that: The product is a kit, a medicine or a composition.

6. A product for treating viral pneumonia, comprising the anti-CypA monoclonal antibody according to claim 2.

7. A product for inhibiting inflammatory response and / or monocyte chemotaxis induced by viral infection, comprising the anti-CypA monoclonal antibody according to claim 2.

8. A product for inhibiting the inflammatory response of an organism caused by viral infection, comprising the anti-CypA monoclonal antibody according to claim 2.

9. A product for inhibiting leukocyte adhesion caused by inflammation, comprising the anti-CypA monoclonal antibody according to claim 2.

10. A product for inhibiting leukocyte migration caused by inflammation, comprising the anti-CypA monoclonal antibody according to claim 2.

11. A product for detecting cyclophilin A, comprising the anti-CypA monoclonal antibody according to claim 2.

12. The product according to any one of claims 6 to 11, characterized in that: The product is a kit, a medicine or a composition.

13. A method for detecting cyclophilin A, comprising the following steps: using the anti-CypA monoclonal antibody according to claim 2 as an antibody to detect whether a sample to be tested contains cyclophilin A.

14. A method for treating and / or preventing viral pneumonia, comprising the following steps: administering the anti-CypA monoclonal antibody or a pharmaceutically acceptable salt thereof according to claim 2 to a subject to treat and / or prevent viral pneumonia.

15. A method for inhibiting pneumonia induced by virus infection in animals, comprising the following steps: administering the anti-CypA monoclonal antibody or a pharmaceutically acceptable salt thereof according to claim 2 to a subject to inhibit pneumonia induced by virus infection in animals.

16. The method according to claim 14 or 15, characterized in that: The virus is influenza virus or other pneumonia-inducing virus.

17. A method for inhibiting an inflammatory response in an organism caused by a viral infection, comprising the following steps: using the anti-CypA monoclonal antibody of claim 2 to act on an organism caused by an inflammatory response in an organism caused by a viral infection, so as to inhibit the inflammatory response in an organism caused by the viral infection.

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