A monoclonal antibody and its use in treating gout

Abstract

The present invention relates to a monoclonal antibody and its use in treating gout. The present invention prepares and obtains a recombinant IL-1β protein. Using this protein as an immunogen, mice are immunized and hybridoma technology is used to prepare and obtain an IL-1β monoclonal antibody. The antibody has good specificity and affinity, and gout model experiments have also confirmed that it can effectively inhibit the expression of IL-1β in cell models, providing an effective drug for treating gout.

Description

Technical Field

[0001] The present application relates to the biological field, and more specifically to a monoclonal antibody and its use in treating gout. Background Art

[0002] Gout is a common and complex type of arthritis that can affect people of all ages, with a higher incidence in men than in women. Gout patients often experience sudden joint pain at night, with an acute onset of pain, edema, redness, swelling, and inflammation in the joints. The pain gradually subsides until it disappears, lasting for several days or weeks. Gout attacks are related to the concentration of uric acid in the body. Gout can form urate deposits in the joint cavity and other places, leading to acute joint pain. Gout is a crystal-related arthritis caused by the deposition of monosodium urate. Gout mainly includes symptoms such as acute onset arthritis, tophi formation, tophi-induced chronic arthritis, urate nephropathy, uric acid urinary stones, joint disability, and renal failure. The main cause of gout is the consumption of foods rich in purine.

[0003] Gout is primarily treated with medication and dietary therapy. Early anti-inflammatory and analgesic treatment is recommended during an acute gout attack (generally within 24 hours). Nonsteroidal anti-inflammatory drugs (NSAIDs), colchicine, and glucocorticoids are effective in providing anti-inflammatory and analgesic treatment, improving patients' quality of life. Acid-lowering therapy is not recommended during an acute attack, but patients already taking acid-lowering medications should not discontinue them to avoid fluctuations in serum uric acid levels, which can prolong the attack or lead to a recurrence. NSAIDs are generally effective in relieving joint pain and swelling. Commonly used drugs include indomethacin, diclofenac, and etoricoxib. After pain and inflammation subside, NSAIDs should be continued to prevent recurrence of symptoms. Colchicine is a traditional treatment. During an acute gout attack, low-dose colchicine alone is recommended for patients with contraindications to NSAIDs. Low-dose colchicine (1.5-1.8 mg / day) is effective, has minimal adverse reactions, and is most effective when used within 48 hours. Glucocorticoids are mainly used for patients who are ineffective or contraindicated with nonsteroidal anti-inflammatory drugs and colchicine, and for patients with renal insufficiency. During the acute attack of gout, short-term use of glucocorticoids alone (30 mg / d, 3 days) has similar efficacy and safety to nonsteroidal anti-inflammatory drugs.

[0004] Studies have found high levels of IL-1β in the serum of patients with gouty arthritis and hyperuricemia. Studies have also shown elevated IL-1 mRNA expression in animal models of acute gouty arthritis, with significantly increased IL-1β activity compared to other stages of gout, suggesting a key role for IL-1β in the joint damage of gouty arthritis. Animal studies have also demonstrated that the NALP3 inflammasome and IL-1β play important roles in gout inflammation. In mouse models, urate crystals were injected into the peritoneal cavity. Results showed that neutrophils in the peritoneal cavity accumulated at sites of urate crystal release, rather than at sites lacking IL-1β receptors. Furthermore, IL-1 blockade prevented neutrophil accumulation in the peritoneal cavity. However, neutrophil accumulation was not affected in mice treated with a tumor necrosis factor α inhibitor. These findings suggest that IL-1β signaling is essential for sensing urate crystal-induced inflammation. Studies in animal models have also confirmed that IL-1 plays a crucial role in both acute and chronic gouty arthritis. Urate crystals stimulate monocytes and phagocytes in the blood and joint fluid, triggering the release of massive amounts of IL-1. Studies have shown that during acute gout attacks, IL-1β plays a key regulatory role in the production of numerous proinflammatory cytokines. As discussed in the previous section, mice lacking the IL-1β receptor or blocking key components of IL-1 signaling can avoid inflammatory attacks and the accumulation of neutrophils at sites where urate crystals are present.

[0005] In recent years, the emergence of biologics has opened up new avenues for the treatment of rheumatic diseases and has significantly changed the prognosis of the disease. The new view is that gouty arthritis is not only a group of intra-articular diseases, but also a systemic autoinflammatory disease. A number of biologics have been used abroad to improve the joint symptoms of gout. Their target is IL-1, an important inflammatory mediator in the pathogenesis of gouty arthritis. Among IL-1 blockers, the most widely studied and researched drugs include anakinra, rilonacept, and canakinumab. Canakinumab is a fully humanized monoclonal antibody specific for IL-1β and was first used in mixed cryoglobulinemia. The effectiveness of canakinumab in treating acute gouty arthritis attacks was first reported in an 8-week Phase II clinical trial (multiple-dose controlled) in 2010. In the clinical trial to evaluate the effectiveness and safety of canakinumab in treating gout, 200 patients with refractory gouty arthritis were selected and randomly received subcutaneous injection of canakinumab (10, 25, 50, 90 or 150 mg, n=143) or intramuscular injection of triamcinolone acetonide (40 mg, n=57). After 72 hours of medication, the pain level of the subjects was evaluated with a 100mm visual analog scale (VAS score) and used as an efficacy indicator. The test results showed that the pain relief level of the subjects in the canakinumab dose group was higher than that in the intramuscular triamcinolone acetonide group, the gout recurrence rate was significantly reduced, and the quality of life was significantly improved compared with the triamcinolone acetonide group. During the entire trial, the overall incidence of adverse events in the two groups was similar, and the severity was mild to moderate, and the difference was not statistically significant.

[0006] However, at present, there is not enough research on IL-1β-specific monoclonal antibodies in China, and the types of optional alternative monoclonal antibodies available are not rich enough, which can easily lead to a bottleneck effect by foreign pharmaceutical companies. Therefore, developing a rich variety of domestic IL-1β-specific monoclonal antibodies is currently a top priority. Summary of the Invention

[0007] The present invention overcomes the defects of the prior art and provides a monoclonal antibody specific for IL-1β.

[0008] The monoclonal antibody, specifically 3A6, was prepared and obtained using hybridoma technology. This antibody, through specific identification, exhibited good specificity and affinity activity. Its variable region sequence was identified using a kit and sequenced, yielding the light chain variable region sequence shown in SEQ ID NO: 1 and the heavy chain variable region sequence shown in SEQ ID NO: 2.

[0009] Further, the monoclonal antibody of the present invention also includes functional variants." functional variant " refers to a polypeptide having substantially the same amino acid sequence as a naturally occurring sequence or being encoded by substantially the same nucleotide sequence and being able to have one or more activities of a naturally occurring sequence. In the context of the present application, the variant of any given sequence refers to a sequence in which the specific sequence of residues (whether amino acid or nucleotide residues) has been modified so that the polypeptide or polynucleotide substantially retains at least one endogenous function. Variant sequences can be obtained by adding, disappearing, replacing, modifying, substituting and / or varying at least one amino acid residue and / or nucleotide residue present in naturally occurring proteins and / or polynucleotides, as long as the original functional activity is maintained.

[0010] In one aspect, the present application provides a modified IL-1β specific monoclonal antibody that can be used to detect IL-1β with a K of 1E-08M or less in a Biacore assay. D Value (for example, the K D No more than about 1E-08 M, no more than about 9E-09 M, no more than about 8E-09 M, no more than about 7E-09 M, no more than about 6E-09 M, no more than about 5E-09 M, no more than about 4E-09 M, no more than about 3E-09 M, no more than about 2E-09 M, no more than about 1E-09 M or less) specifically binds to human IL-1β protein.

[0011] Furthermore, the present invention also provides a pharmaceutical composition containing IL-1β monoclonal antibody.

[0012] Furthermore, the pharmaceutical composition may comprise the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, the cell, and / or a pharmaceutically acceptable adjuvant and / or excipient as described herein. In the present application, the pharmaceutically acceptable adjuvant may include a buffer, an antioxidant, a preservative, a low molecular weight polypeptide, a protein, a hydrophilic polymer, an amino acid, a sugar, a chelating agent, a counterion, a metal complex, and / or a nonionic surfactant. Unless incompatible with the cells described herein, any conventional medium or reagent may be considered for use in the pharmaceutical composition of the present application. In the present application, the pharmaceutically acceptable excipient may include an additive other than the main drug in a pharmaceutical preparation, and may also be referred to as an excipient. For example, the excipient may include a binder, filler, disintegrant, or lubricant in a tablet. For example, the excipient may include wine, vinegar, or medicinal juice in a traditional Chinese medicine pill. For example, the excipient may include the matrix portion of a semisolid ointment or cream. For example, the excipients may include preservatives, antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, and colorants in liquid preparations.

[0013] Furthermore, the present invention also provides use of IL-1β monoclonal antibodies in the preparation of drugs for treating gout.

[0014] Further, the treatment means administering an internal or external therapeutic agent to a subject, such as an antibody or pharmaceutical composition thereof disclosed herein as a therapeutic agent, to a subject who has, is suspected of having, or is prone to having one or more proliferative diseases or symptoms thereof, and it is known that the therapeutic agent has a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered in an amount that effectively alleviates one or more symptoms of the disease in the treated subject or population, whether by inducing the regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent. The amount of the therapeutic agent that effectively alleviates any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to a variety of factors, such as the subject's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the disease symptoms have been alleviated can be evaluated by any clinical detection method commonly used by doctors or other professional health care personnel to evaluate the severity or progression of the symptoms. Although an embodiment of the present disclosure (e.g., a method of treatment or article of manufacture) may not be effective in alleviating the symptoms of the target disease in a certain subject, it should alleviate the symptoms of the target disease in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0015] Furthermore, an "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the method, route, and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects. The subject of the present disclosure may be an animal or a human subject.

[0016] Beneficial effects

[0017] The present invention prepares and obtains recombinant IL-1β protein, uses the protein as an immunogen, immunizes mice, and prepares and obtains IL-1β monoclonal antibodies through hybridoma technology. The antibody has good specificity and affinity, and gout model experiments have also confirmed that it can effectively inhibit the expression of IL-1β in cell models, providing an effective drug for treating gout. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1Results of SDS-PAGE method to verify the induced expression;

[0019] Figure 2 Monoclonal antibody specificity identification results diagram;

[0020] Figure 3 Figure 3. Effects of monoclonal antibodies on IL-1β levels. DETAILED DESCRIPTION

[0021] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive.

[0022] These and other aspects, features and advantages will be apparent to those of ordinary skill in the art upon reading the following detailed description and the appended claims. For the avoidance of doubt, any feature of one aspect of the present invention may be used in any other aspect of the present invention. The word "comprising" is intended to mean "including" but not necessarily "consisting of" or "composed of". In other words, the steps or options listed do not need to be exhaustive. It should be noted that the examples given in the following description are intended to illustrate the present invention and are not intended to limit the present invention to those examples themselves. Similarly, all percentages are weight / weight percentages unless otherwise indicated. Except in the operating and comparative examples, or where otherwise explicitly stated, all numbers in this description indicating the amount of material or reaction conditions, physical properties of materials and / or use should be understood to be modified by the word "about". The disclosure of the present invention as seen herein should be considered to cover all embodiments that can be seen in the claims that are multiple dependent on each other, regardless of the fact that it may be found that the claims do not have multiple dependencies or redundancy.

[0023] Example 1 Preparation of IL-1β recombinant protein

[0024] Molecular cloning, prokaryotic expression and purification of human IL-1β: Primers were designed based on the human IL-1β gene sequence using Primer5.0 primer design software. The upstream primer is: 5'-TCT GGATCC ATGGCAG AAGTACCTGAGC-3', downstream primer is 5'-ATCG CTCGAGGGAAGACACAAATTGC-3', underlined are the restriction sites of BamHI and XholI respectively. PCR reaction was carried out with human DNA as template. The PCR product was approximately 810 bp. After purification with a kit, the purified PCR product and the prokaryotic expression plasmid pET-32a(+) were simultaneously digested with BamHI and XholI. The PCR product and the prokaryotic expression plasmid pET-32a(+) after double digestion were ligated and transformed into competent bacteria DH5α. Positive clones were selected for sequencing. Plasmids were extracted from clones with correct sequencing and transformed into BL21(DE3) expression strain. IPTG was induced for expression. The results of induced expression were verified by SDS-PAGE. The results are shown in the figure. Figure 1 As shown, 0.5 mM IPTG was used to induce recombinant protein expression. The expression level of the recombinant protein was high at 12 hours of induction, as shown in the band of induction 1. The expression level of the recombinant protein was low at 4 hours of induction, as shown in the band of induction 2. The bacterial solution induced for 12 hours was used to purify the target protein and the protein concentration was adjusted to 2 mg / mL for later use.

[0025] Example 2 Preparation of IL-1β Monoclonal Antibody

[0026] The purified IL-1β recombinant protein of Example 1 was used as an immunogen. 60 μg of the protein was mixed with an equal volume of adjuvant (Freund's complete (first immunization) / incomplete (second and third immunization)) and then placed in a three-way valve glass emulsifier for emulsification until the oil droplets formed a mass and did not spread when dropped into water, thus completing the emulsification. The emulsified immunogen was immunized three times (day 1, day 14, day 28) and subcutaneously immunized in BALB / c mice of about 6 weeks of age. About seven days after the third immunization, tail blood was collected and the serum antibody titer was detected by indirect ELISA, and BALB / c mice No. 2 with high titer were selected for booster immunization. The amount of booster immunization antigen was 110 μg, and no adjuvant was added for subcutaneous immunization. Three days after immunization, the mice were killed by cervical dislocation after blood was collected from the eyeballs and immersed in 75% alcohol for 10 minutes for disinfection. The mice were fixed on a dissecting board, and the skin and peritoneum were cut open with sterile scissors to expose the spleen. Remove surrounding tissue from the spleen, rinse the removed spleen with serum-free 1640, and place it on a 200-mesh sterile copper mesh placed on a plate. Crush the spleen while rinsing the splenocytes with serum-free 1640 into the plate. Add the collected splenocyte suspension to a centrifuge tube and centrifuge at 1000 rpm for 10 minutes. Resuspend the pellet in serum-free 1640 and count the cells. Place the pellet into a 50 mL centrifuge tube at a ratio of 10:1 splenocytes to SP2 / 0 cells and centrifuge at 1000 rpm for 10 minutes. Discard the supernatant, gently flick the cell pellet at the bottom of the centrifuge tube, and place the tube in a 37°C water bath. Preheat the cell fusion agent and slowly add 1 mL of cell fusion agent at a rate of 1 mL / min. Gently shake the tube to ensure thorough contact and mixing between the cells and the agent. Incubate in a 37°C water bath for 90 seconds. Add 1 mL of serum-free 1640 dropwise at 1 mL / min while gently shaking the tube. Repeat this process once more. Then, add 1 mL of serum-free 1640 dropwise at 1 mL / 30 seconds. Finally, add 7 mL of serum-free 1640 over 2 minutes, gently shaking the tube. Centrifuge at 1000 rpm for 10 minutes and discard the supernatant. Resuspend the cells in prewarmed HAT medium and add 100 μL / well to a 96-well plate containing feeder cells. Incubate at 37°C in a 5% CO2 incubator. On day 7 post-fusion, perform a half-medium medium change with prewarmed HT medium. Observe the cells daily and mark the wells that have fused. When the hybridoma cells have grown to 1 / 3-1 / 2 of the bottom of the 96-well plate, assay the supernatant. Set up negative and positive control wells. Assay twice before subcloning, and subclone the wells containing hybridoma cells that test positive twice. After four rounds of subcloning screening, two hybridoma cell lines that could stably secrete IL-1β monoclonal antibodies were obtained and named 3A6 and 4D14 respectively.

[0027] Sterilized liquid paraffin was injected into the peritoneal cavity of 10-week-old BALB / c mice at a rate of 0.5 mL per mouse. After 10 days, the hybridoma cells were resuspended in serum-free 1640 and counted at a rate of 2 × 10 6 Hybridoma cells / mL were injected into the peritoneal cavity of mice at a volume of 0.5 mL / mouse. After inoculation, the mice were observed. Approximately one week later, the abdomen began to bulge. When the abdominal circumference of the mice expanded and their movements became sluggish, ascites was collected. The intraperitoneal fluid was aspirated with a syringe. Centrifuged at 2000 rpm for 10 minutes, the supernatant was aspirated, and fat and cells were removed. Purification was then performed using a column to obtain the purified two antibodies. Aliquots were stored at -20°C until further use.

[0028] Example 3 Specificity Identification of Monoclonal Antibodies 3A6 and 4D14

[0029] Western blot antigenicity identification was performed using the prepared purified monoclonal antibody. The Western blot method steps are as follows:

[0030] (1) Prepare SDS-PAGE, with a 15% separating gel at the bottom and a 5% stacking gel at the top;

[0031] (2) Processing protein samples: IL-1β recombinant protein sample or His-tagged protein: SDS: DTT = 5:4:1 was placed in a centrifuge tube and boiled in 100°C water for 10 min to denature the protein;

[0032] (3) Sample loading: Adjust the protein samples to the same concentration for loading;

[0033] (4) Electrophoresis: Run the stacking gel at 90V for about 30 minutes, and the separation gel at 120V until it reaches the bottom.

[0034] (5) Transfer: The order of stacking from top to bottom during transfer is: filter paper - protein gel - NC membrane - filter paper. Transfer conditions: 60 mA, about 350 min. After transfer, place the membrane in 20 mL of 5% skim milk blocking solution and block at 4°C overnight.

[0035] (6) Antibody incubation: Pour off the blocking solution, wash the membrane with PBST, wash 5 times, each time for 5 minutes, use the prepared monoclonal antibody as the primary antibody, dilute it with PBST at a ratio of 1:1000, and incubate at 4℃ for 8 hours; recover the primary antibody, wash it with PBST, wash it 5 times, each time for 5 minutes; add goat anti-mouse IgG-HRP secondary antibody, dilute it with PBST at a ratio of 1:5000, and incubate it on a shaker at 37℃ for 2 hours; recover the secondary antibody, wash it with PBST, wash it 5 times, each time for 5 minutes; store the prepared ECL exposure solution in the dark, place the membrane in the exposure box, and react with the exposure solution for 30 seconds for exposure imaging. The results are as follows Figure 2 As shown. Figure 2It can be seen that monoclonal antibodies 3A6 and 4D14 produce specific target bands in induction lanes 2 and 4, respectively. Monoclonal antibody 3A6 in lane 1 and monoclonal antibody 4D14 in lane 3 do not react with the control protein, and no bands are produced, indicating that the monoclonal antibodies of the present invention have good specificity.

[0036] Example 4 Affinity and subtype identification of monoclonal antibody 3A6

[0037] The binding affinity of mAb 3A6 to recombinant IL-1β was determined using Biacore. 100 mL of 10× HBS-EP+ buffer and 900 mL of Milli-Q water were mixed to obtain 1 L of 1× HBS-EP+ buffer. The surface of channels 1-3 of a CM5 chip was activated with a 1:1 mixture of 50 mM NHS and 200 mM EDC (NHS and EDC were obtained from the amino coupling kit) at a flow rate of 10 μL / min for 420 seconds. Anti-hFc or anti-mouse Fc antibodies (diluted in sodium acetate, pH 4.5, at a concentration of 20 μg / mL) were injected at a flow rate of 10 μL / min for 200 seconds. Finally, excess reactive carboxyl groups on the chip were blocked with 1 M ethanolamine hydrochloride (pH 8.5). The chip surface was rinsed with 1× HBS-EP+ at a flow rate of 10 μL / min for 2 hours to stabilize the baseline. The instrument temperature was set at 25°C. The initial cycle consisted of two steps, sampling and regeneration, and was repeated three times before measurement to stabilize the baseline. Sampling: Inject 1× HBS-EP+ buffer into channels 1-3 at a flow rate of 30 μL / min for 120 seconds, followed by a 60-second dissociation period. Regeneration: Inject 10 mM glycine, pH 1.5, into channels 1-3 at 30 μL / min for 30 seconds, followed by a 30-second stabilization period. Binding kinetic parameter determination experimental procedures: The running buffer for kinetic measurements was 1× HBS-EP+ (pH 7.4). Capture: Inject antibody into the test channels 1-3 of the Anti-hFc or Anti-mouse Fc chip at a flow rate of 10 μL / min for 60 seconds, followed by capture. Antigen IL-1β recombinant protein was diluted to 100 nM in 1× HBS-EP+ (pH 7.4). Sampling: Injection into channels 1-3 at a flow rate of 30 μL / min, with a 0-concentration sample used to remove background signals; the association and dissociation times of the antigen and antibody were 180 and 400 seconds, respectively. Regeneration: Injection of 10 mM glycine pH 1.5 into channels 1-3 at a flow rate of 30 μL / min for 30 seconds, followed by stabilization for 60 seconds. The equilibrium dissociation constant (K) of each antibody in this application was calculated using Biacore 8K analysis software. D The reference channel (FC1) was used for background subtraction. The results showed that the dissociation constant of monoclonal antibody 3A6 was (8.62±0.24) nM. The above experiments indicate that the monoclonal antibody of the present application has good binding affinity with the antigen.

[0038] The mouse monoclonal antibody subtype identification kit was used to identify the subtype of the 3A6 monoclonal antibody. The heavy chain of the prepared 3A6 monoclonal antibody was identified as IgG2b, and the light chain was identified as a kappa chain.

[0039] Example 5 Application of 3A6 mAb in Gout Model

[0040] Weigh 40 mg of white MSU powder, add 1 mL of Tween 80, and dilute to 100 mL with RMPI-1640 medium. Heat and stir with a magnetic stirrer until the crystals are completely dissolved to create a 400 μg mL MSU suspension. Autoclave, seal, and store at 4°C until needed.

[0041] The THP-1 cells were quickly taken out of the liquid nitrogen tank and placed in a constant temperature water bath preheated to 37°C and shaken quickly to thaw. The cell suspension was transferred to a 15mL sterile centrifuge tube; RPMI1640 medium containing 1% penicillin + streptomycin double antibody and 10% FBS was added to dilute 10 times, the cell suspension was centrifuged at 1000rpm for 5min, the supernatant was discarded, 1mL of fresh culture medium was added to the cell pellet, and the cells were gently blown to resuspend and then transferred to a culture flask. 5mL of fresh culture medium was added and cultured in a 37°C, 5% CO2 cell culture incubator. The cell growth status was observed under an inverted microscope, and the cells were bright round and suspended in the culture medium. The medium was changed every 2-3 days. When the cell growth fusion reached 80-90%, the cell suspension was transferred to a 15mL centrifuge tube, centrifuged at 1000r / min for 5min, the supernatant was discarded, and an appropriate amount of culture medium was added. The cells were gently blown to resuspend them, and the cell concentration was 2.0×10 5 / mL for subculture, add appropriate amount of culture medium and continue to culture in a cell culture incubator at 37℃ and 5% CO2 saturated humidity. THP-1 cells were cultured at 1.0×10 6 / well were seeded in 6-well plates and stimulated with 100 ng / mL PMA for 24 h to induce THP-1 cells to differentiate into macrophages. Compared with the blank control group, the expression of macrophage-specific protein CD11b was significantly increased after stimulation with 100 ng / mL PMA, indicating that 100 ng·mL-1 PMA can successfully induce THP-1 monocytes to differentiate into macrophages.

[0042] Verification of monoclonal antibody activity: Macrophages were used as target cells. No monoclonal antibody was added to the normal and model groups. The remaining groups were treated with different concentrations of monoclonal antibody (1, 10, 50, and 100 μg / mL). The positive control was 50 μg / mL of connexin. After 6 hours, MSU was added to a final concentration of 400 μg / mL. Five replicates were set for each concentration gradient, and the cells were cultured for another 24 hours. The cell supernatant was collected and the level of the inflammatory factor IL-1β in the cell culture supernatant was determined according to the ELISA kit instructions.

[0043] The results are as follows Figure 3 As shown in the results, compared with the normal group, the secretion level of the inflammatory factor IL-1β in the cell supernatant of THP-1-derived macrophages in the model group after MSU stimulation was significantly increased (P<0.01). Compared with the model group, after monoclonal antibody treatment, the content of IL-1β was significantly reduced in the range of 1-100μg / mL. Among them, when the monoclonal antibody was treated with a concentration of 50μg / mL, the IL-1β secretion level was the most cost-effective compared with the model group (P<0.01). Compared with the positive control group, the effect was similar, and the IL-1β expression level was only (32.3±3.1)pg / L, which was also significantly different from the normal group, indicating a good therapeutic effect. This experiment proved that the IL-1β monoclonal antibody can effectively inhibit the expression of IL-1β in gout model cells, thereby achieving the treatment of gout.

[0044] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various changes to the embodiments listed in the present application are obvious to those skilled in the art and are intended to fall within the scope of the appended claims and their equivalents.

Claims

1. A monoclonal antibody 3A6 specific for IL-1β, characterized in that The light chain variable region sequence of the antibody is shown in SEQ ID NO: 1, and the heavy chain variable region sequence is shown in SEQ ID NO:

2.

2. A pharmaceutical composition for inhibiting IL-1β expression, characterized in that The pharmaceutical composition contains the monoclonal antibody against IL-1β according to claim 1.

3. The pharmaceutical composition according to claim 2, wherein The pharmaceutical composition contains a pharmaceutically acceptable carrier.

4. The pharmaceutical composition according to claim 3, wherein Pharmaceutically acceptable carriers include buffers, antioxidants, preservatives or nonionic surfactants.

5. Use of the monoclonal antibody against IL-1β according to claim 1 in the preparation of a drug for treating gout by inhibiting the expression of IL-1β.

6. The use according to claim 5, characterized in that The drug contains a pharmaceutically acceptable carrier.

7. The use according to claim 6, characterized in that Pharmaceutically acceptable carriers include buffers, antioxidants, preservatives or nonionic surfactants.

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