A monoclonal antibody for treating gout and a corresponding composition thereof

Abstract

This invention relates to a monoclonal antibody for treating gout and a corresponding composition thereof. The invention prepares and obtains recombinant IL-1β protein, uses this protein as an immunogen to immunize mice, and prepares and obtains an IL-1β monoclonal antibody using hybridoma technology. The antibody exhibits good specificity and affinity, and its ability to effectively inhibit IL-1β expression in a gout model has been demonstrated in gout model experiments, providing an effective drug for treating gout.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biology, more particularly to a monoclonal antibody for treating gout and a corresponding composition thereof. BACKGROUND

[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 acute onset, joint pain, edema, redness and inflammation, and the pain gradually subsides until it disappears, lasting for several days or weeks. Gout is associated with the concentration of uric acid in the body, and gout can form urate deposits in the joint cavity and other places, thereby causing acute joint pain. Gout is a crystal-related arthropathy caused by monosodium urate deposition. Gout mainly includes acute gouty arthritis, gouty tophi formation, gouty tophi chronic arthritis, urate nephropathy, uric acid urinary calculi, joint disability, and renal failure. The main cause of gout is the consumption of foods rich in a large amount of purines.

[0003] There are two methods for treating gout, namely drug therapy and dietary therapy. It is recommended to perform anti-inflammatory analgesic treatment as soon as possible (usually within 24 hours) during the acute attack period of gout. Non-steroidal anti-inflammatory drugs (NSAIDs), colchicine and glucocorticoids can effectively relieve inflammation and pain and improve the quality of life of patients. No acid-lowering treatment is performed during the acute attack period, but those who have already taken acid-lowering drugs do not need to stop taking them to avoid causing fluctuations in blood uric acid, leading to prolonged or repeated attacks. Non-steroidal anti-inflammatory drugs are usually effective in relieving joint pain and swelling. Commonly used drugs include indomethacin, diclofenac, etoricoxib, etc. After the pain and inflammation are relieved, non-steroidal anti-inflammatory drugs should continue to be used to prevent the symptoms from reappearing. Colchicine is a traditional treatment drug. In the acute attack period of gout, low-dose colchicine is recommended for those who have contraindications to non-steroidal anti-inflammatory drugs. Low-dose colchicine (1.5 mg / d-1.8 mg / d) is effective and has fewer adverse reactions, and the effect is better within 48 hours. Glucocorticoids are mainly used for those who have contraindications to non-steroidal anti-inflammatory drugs and colchicine or have renal dysfunction. In the acute attack period of gout, short-term single use of glucocorticoids (30 mg / d for 3 days) has similar efficacy and safety to non-steroidal anti-inflammatory drugs.

[0004] It has been found that high levels of IL-1 β can be detected in the serum of patients with gouty arthritis and hyperuricemia. It has been found that high expression of IL-1 mRNA can be detected in animal experimental models of acute gouty arthritis, and that the activity of IL-1 β is significantly higher than that of other stages of gout. It can be seen that IL-1 β plays a key role in the process of joint damage in gouty arthritis. Animal experimental studies have also shown that NALP3 inflammasome and IL-1 β play an important role in the process of gout inflammation. In mouse model experiments, the peritoneal cavity of mice was injected with urate crystals, and the results showed that neutrophils in the peritoneal cavity gathered at the site where urate crystals were released, and did not gather at the site where the mice lacked IL-1 β receptors. Moreover, the application of IL-1 blockers can prevent the aggregation of neutrophils in the peritoneal cavity, but the aggregation of neutrophils in the peritoneal cavity of mice injected with tumor necrosis factor α inhibitors was not affected. The above shows that the IL-1 β signal is essential for sensing the inflammation caused by urate crystals. Animal model studies have also confirmed that IL-1 plays an important role in both acute and chronic gouty arthritis. Urate crystals stimulate monocytes and phagocytes in the blood and synovial fluid, causing the release of large amounts of IL-1. Studies have shown that IL-1 β plays a key regulatory role in the production of a large number of pro-inflammatory cytokines during acute gout attacks. As discussed in the previous section, mice lacking IL-1 β receptors or blocking key components of the IL-1 signal can avoid inflammatory attacks and the aggregation of neutrophils at the site of urate crystals.

[0005] In recent years, the emergence of biological agents for rheumatic disease treatment opened a new situation, greatly changed the outcome of the disease. New ideas that gouty arthritis is not only a group of intra-articular disease, but also a systemic inflammatory disease. Some biological agents have been used to improve the joint symptoms of gout abroad, and the target is IL-1, an important inflammatory mediator in the pathogenesis of gouty arthritis. Among the IL-1 blockers, the most widely concerned and studied drugs mainly include anakinra, rilonacept and canakinumab. Canakinumab is an IL-1β specific fully human monoclonal antibody, which is first applied to mixed cryoglobulinemia. The effectiveness of canakinumab in treating acute gouty arthritis was first reported in an 8-week phase II clinical trial (multiple dose control) in 2010. In the evaluation of 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) treatment. 72 hours after administration, the pain degree of the subjects was evaluated by 100mm visual analogue scale (VAS score) and used as the efficacy index. The results showed that the pain relief degree of the subjects in the canakinumab dose group was higher than that in the intramuscular injection of triamcinolone acetonide group, the recurrence rate of gout was significantly reduced, and the quality of life was significantly improved compared with the triamcinolone acetonide group. The overall incidence of adverse events in the two groups during the entire trial was similar, and the severity was mild to moderate, with no statistically significant difference.

[0006] However, at present, the research of IL-1β specific monoclonal antibody is not enough in China, and the types of alternative monoclonal antibodies are not enough, which is easy to form a neck effect by foreign pharmaceutical companies. Therefore, it is urgent to develop domestic IL-1β specific monoclonal antibodies with rich types. SUMMARY

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

[0008] The monoclonal antibody is prepared and obtained by hybridoma technology, and specifically relates to monoclonal antibody 4D14. The antibody has good specificity and affinity activity through specific identification. After the variable region sequence is identified and sequenced by the kit, the light chain variable region sequence is shown as SEQ ID NO: 1, and the heavy chain variable region sequence is shown as SEQ ID NO: 2.

[0009] Further, the monoclonal antibodies of the present application also include functional variants. "Functional variant" refers to a polypeptide having substantially identical amino acid sequence or encoded by substantially identical nucleotide sequence to a naturally occurring sequence and capable of having one or more activities of the naturally occurring sequence. In the context of the present application, a variant of any given sequence refers to a sequence in which the particular sequence of residues, whether amino acid or nucleotide residues, has been modified such that the polypeptide or polynucleotide substantially retains at least one endogenous function. Variant sequences can be obtained by addition, deletion, substitution, modification, replacement and / or mutation of at least one amino acid residue and / or nucleotide residue present in the naturally occurring protein and / or polynucleotide, as long as the original functional activity is maintained.

[0010] In one aspect, the present application provides a monoclonal antibody specific for IL-1β that binds specifically to human IL-1β protein with a K D value (e.g., the K D not higher than about 1E-08M, not higher than about 9E-09M, not higher than about 8E-09M, not higher than about 7E-09M, not higher than about 6E-09M, not higher than about 5E-09M, not higher than about 4E-09M, not higher than about 3E-09M, not higher than about 2E-09M, not higher than about 1E-09M or lower) in a Biacore assay.

[0011] Further, the present application also provides pharmaceutical compositions comprising the IL-1β monoclonal antibody.

[0012] Further, the pharmaceutical composition can 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 described herein. In the present application, the pharmaceutically acceptable adjuvant can include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes, and / or nonionic surfactants. Any conventional medium or reagent can be considered for use in the pharmaceutical composition of the present application, unless it is incompatible with the cell described herein. In the present application, the pharmaceutically acceptable excipient can include additional substances other than the main drug in the pharmaceutical preparation, which can also be referred to as auxiliary materials. For example, the excipient can include binders, fillers, disintegrants, lubricants in tablets. For example, the excipient can include wine, vinegar, medicinal juice, etc. in traditional Chinese medicine pills. For example, the excipient can include the base part in ointments, creams in semisolid preparations. For example, the excipient can include preservatives, antioxidants, flavorings, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants in liquid preparations.

[0013] Further, the present application also provides the use of the IL-1β monoclonal antibody in the preparation of a medicament for treating gout.

[0014] Further, the treatment means administering to a subject who already has, is suspected of having, is predisposed to having, or is susceptible to one or more proliferative diseases or symptoms thereof, a therapeutic agent, e.g., a pharmaceutical composition comprising any of the antibodies of the present disclosure as a therapeutic agent, which is known to have a therapeutic effect on such symptoms. Generally, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated subject or population, whether by inducing regression of such symptoms or inhibiting progression of such symptoms to any clinically measurable extent. The amount of therapeutic agent effective to alleviate any particular symptom of the disease (also referred to as a "therapeutically effective amount") can vary according to factors such as the disease state, age, and weight of the subject, and the ability of the drug to elicit a desired effect in the subject. Whether a disease symptom has been alleviated can be assessed by any clinical detection method conventionally used by a physician or other health care professional to assess the severity or progression of the symptom. Although an embodiment of the present disclosure (e.g., a therapeutic method or article of manufacture) can not be effective in alleviating a target disease symptom in a particular subject, it should alleviate the target disease symptom in a statistically significant number of subjects as determined by any statistical test known in the art, such as the Student t-test, the chi-square test, the U-test according to Mann and Whitney, the H-test according to Kruskal-Wallis, the Jonckheere-Terpstra test, and the Wilcoxon test.

[0015] Further, an "effective amount" includes an amount which is enough to ameliorate or prevent the symptoms or conditions of a medical disorder. An effective amount also means an amount which is enough to allow or facilitate diagnosis. An effective amount for a subject can vary according to factors such as the condition to be treated, the overall health status of the subject, the method route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxic effects. A subject of the present disclosure can be an animal or a human subject.

[0016] Beneficial effects

[0017] The present application prepared and obtained a recombinant IL-1β protein, taking the protein as an immunogen, immunizing mice, and preparing and obtaining IL-1β monoclonal antibody through hybridoma technology, the antibody has good specificity and affinity, and the gout model experiment also proves that the expression of IL-1β in the cell model can be effectively inhibited, and an effective drug for treating gout is provided. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1SDS-PAGE method to verify the induction expression results chart;

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

[0020] Figure 3 Monoclonal antibody on IL-1β level of influence results chart. DETAILED DESCRIPTION

[0021] Other aspects and advantages of the present application will become apparent to those skilled in the art from the following detailed description in combination with the drawings. The detailed description is merely illustrative of the exemplary embodiments of the present application, and does not limit the scope thereof. As will be realized, the application is capable of modifications in various obvious aspects, all without departing from the spirit and scope of the inventive concepts presented herein.

[0022] These and other aspects, features, and advantages will become apparent to those of ordinary skill in the art from the detailed description and accompanying drawings. To the extent that certain details are set forth in the detailed description, it is understood that the disclosure is not limited to the particular embodiments set forth and is intended to cover any alternatives, modifications, and equivalents. The word "comprising" is used herein to mean "including", but not necessarily "consisting of" or "composed of". In other words, the listed steps or options need not be exhaustive. It is noted that the examples given in the description are intended to clarify the application and are not intended to limit the application to those examples per se. Similarly, all percentages are weight / weight percentages unless otherwise indicated. Except in the operating and comparative examples, or where otherwise explicitly indicated, all numerical quantities in this description are to be understood as being modified in all instances by the word "about". The disclosure of the application is not to be limited as set forth above but can be practiced with modification and alteration within the scope of the appended claims. Similarly, where the description indicates "examples," the disclosure is to be interpreted as an example of the described implementation and not as an enumeration of members of a collection of examples.

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

[0024] Molecular cloning, prokaryotic expression and purification of human IL-1β: According to the sequence of human IL-1β gene, the primer was designed by Primer 5.0 primer design software. The upstream primer is 5'-TCT GGATCC ATGGCAG AAGTACCTGAGC-3', the downstream primer is 5'-ATCG CTCGAGGGAAGACACAAATTGC-3', the underlined are the restriction sites of BamHI and XholI. The PCR reaction was performed with human DNA as template, and the PCR product was about 810 bp. The purified PCR product and the prokaryotic expression plasmid pET-32a(+) were digested with BamHI and XholI at the same time, and then the digested PCR product and the prokaryotic expression plasmid pET-32a(+) were ligated. The ligation product was transformed into the competent bacteria DH5a, and the positive clones were selected for sequencing. The plasmid of the clone with correct sequence was extracted and transformed into the BL21(DE3) expression strain. The expression was induced by IPTG, and the expression result was verified by SDS-PAGE method. The result is shown in Fig. 1. The expression of the recombinant protein was induced by 0.5 mM IPTG. The expression amount of the recombinant protein was higher at 12 h after induction, and the band is shown in Fig. 1 (induction 1). The expression amount of the recombinant protein was lower at 4 h after induction, and the band is shown in Fig. 1 (induction 2). The bacteria liquid at 12 h after induction was used for the purification of the target protein, and the protein concentration was adjusted to 2 mg / mL for standby. Figure 1

[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 immunizations)) and emulsified in a three-way valve glass emulsifier until the oil droplets formed a cluster that did not spread when dropped into water. The emulsified immunogen was used to immunize 6-week-old BALB / c mice subcutaneously three times (day 1, day 14, and day 28). Tail blood was collected about seven days after the third immunization, and the serum antibody titer was determined by indirect ELISA. A BALB / c mouse No. 2 with a high titer was selected for booster immunization. The booster immunization was performed subcutaneously with 110 μg of the antigen without adjuvant. Three days after the immunization, the mouse was sacrificed by eye bleeding and then immersed in 75% alcohol for 10 min for sterilization. The mouse was fixed on a dissection board, and the skin and peritoneum were cut with sterile scissors to expose the spleen. The spleen was isolated from the surrounding tissue, washed with serum-free 1640, and then placed in a 200-mesh sterilized copper mesh on a petri dish. The spleen was crushed, and the spleen cells were washed into the petri dish with serum-free 1640. The collected spleen cell suspension was transferred into a centrifuge tube and centrifuged at 1000 rpm for 10 min. The precipitate was resuspended in serum-free 1640 and counted. The cells were mixed with SP2 / 0 cells at a ratio of 10:1 and centrifuged at 1000 rpm for 10 min. The supernatant was discarded, and the cell precipitate at the bottom of the centrifuge tube was gently tapped. The centrifuge tube was placed in a 37°C water bath, and the cell fusion agent was preheated. One mL of the cell fusion agent was slowly added at a rate of 1 mL / min while gently shaking the centrifuge tube to ensure that the cells were in contact with the fusion agent. The mixture was placed in a 37°C water bath for 90 s. One mL of serum-free 1640 was added and slowly dropped at a rate of 1 mL / min while gently shaking the centrifuge tube. This process was repeated once. Then, 1 mL of serum-free 1640 was added and dropped at a rate of 1 mL / 30 s. Finally, 7 mL of serum-free 1640 was added within 2 min while gently shaking the centrifuge tube. The mixture was centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. The precipitate was resuspended in preheated HAT medium and transferred into a 96-well cell plate with a feeder layer at a volume of 100 μL / well. The plate was incubated in a 37°C 5% CO2 cell culture incubator. On the seventh day after the fusion, the medium was half-exchanged with preheated HT medium. The cell state was observed daily, and the fused cell wells were marked. When the hybridoma cells grew to 1 / 3-1 / 2 of the bottom of the 96-well cell plate, the cell supernatant was detected, and negative and positive control wells were set. The hybridoma cell wells that tested positive in two detections were subcloned. After four rounds of subcloning and screening, two hybridoma cell strains that could stably secrete IL-1β monoclonal antibodies were obtained and named 3A6 and 4D14, respectively.

[0027] BALB / c mice at the age of 10 weeks were injected with sterilized liquid paraffin into the abdominal cavity, 0.5 mL per mouse. After 10 days, the hybridoma cells were resuspended in serum-free 1640 and counted, and then injected into the abdominal cavity of the mice at the amount of 2 x 10 6 After inoculation, the state of the mice was observed, and about one week later, the mice began to have bulges on the abdomen. When the mice had swollen abdomen and moved slowly, the ascites was collected. The liquid in the abdominal cavity of the mice was extracted by a syringe. After centrifugation at 2000 rpm for 10 min, the supernatant was taken, and the oil and cells were removed. The purified two antibodies were obtained by column purification and stored at -20°C.

[0028] Example 3 Specificity identification of monoclonal antibodies 3A6 and 4D14

[0029] Western blot antigenicity identification was performed by using the prepared purified monoclonal antibodies, and the steps of the Western blot method were as follows:

[0030] (1) Preparation of SDS-PAGE, 15% separation gel in the lower layer and 5% concentration gel in the upper layer;

[0031] (2) Treatment of protein samples, IL-1β recombinant protein sample or His-tag protein: SDS: DTT = 5:4:1 in a centrifuge tube, denaturation of the protein by boiling in water at 100°C for 10 min;

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

[0033] (4) Electrophoresis: the concentration gel was first run at 90V for about 30 min, and the separation gel was run at 120V to the bottom end;

[0034] (5) Membrane transfer: the transfer was performed by stacking from top to bottom as follows: filter paper-protein gel-NC membrane-filter paper, and the transfer conditions were as follows: 60 mA, about 350 min. After the transfer, the membrane was placed in 20 mL of 5% defatted milk blocking solution, and blocked at 4°C overnight;

[0035] (6) Antibody incubation: the blocking solution was discarded, the membrane was washed with PBST, and washed 5 times for 5 min each time. The prepared monoclonal antibody was used as the primary antibody, and diluted in PBST at a dilution ratio of 1:1000, and incubated at 4°C for 8 h. The primary antibody was recovered, and washed with PBST, and washed 5 times for 5 min each time. Goat anti-mouse IgG-HRP secondary antibody was added, and diluted in PBST at a dilution ratio of 1:5000, and incubated on a shaking table at 37°C for 2 h. The secondary antibody was recovered, and washed with PBST, and washed 5 times for 5 min each time. The prepared ECL exposure solution was used as needed, and the membrane was placed in an exposure box and exposed to the exposure solution for 30 s for exposure imaging. The results are shown in Figure 2 Figure 2 ​It can be seen that the monoclonal antibodies 3A6 and 4D14 respectively induce 2 and 4 specific bands, and the monoclonal antibody 3A6 in lane 1 and the monoclonal antibody 4D14 in lane 3 do not react with the control protein, no band is produced, which shows that the monoclonal antibodies of the application have good specificity.

[0036] Example 4 Affinity and subtype identification of monoclonal antibody 4D14

[0037] The binding affinity of the monoclonal antibody 4D14 to the antigen IL-1β recombinant protein was detected by Biacore. 100 mL of 10x HBS-EP+buffer and 900 mL of Milli-Q water were mixed to obtain 1 L of 1x HBS-EP+buffer. The surface of the CM5 chip channels 1-3 was activated with a 1:1 mixture of 50 mM NHS and 200 mM EDC (NHS and EDC from the amino coupling kit) at a flow rate of 10 μL / min for 420 seconds. Anti-hFc or anti-mouse Fc antibody (diluted in sodium acetate solution at pH 4.5, concentration 20 μg / mL) was injected at a flow rate of 10 μL / min for 200 seconds, and finally the chip was blocked with 1 M ethanolamine hydrochloride (pH 8.5) to block the excess active carboxyl groups on the chip. The chip surface was washed with 1x HBS-EP+at a flow rate of 10 μL / min for 2 hours to stabilize the baseline, and the instrument was set to a temperature of 25°C. The initial cycle consisted of two steps, measurement and regeneration, and was repeated 3 times to stabilize the baseline. Measurement: 1x HBS-EP+buffer was injected into channels 1-3 at a flow rate of 30 μL / min for 120 seconds, and dissociation was performed for 60 seconds. Regeneration: 10 mM glycine pH 1.5 was injected into channels 1-3 at a flow rate of 30 μL / min for 30 seconds, and stabilization was performed for 30 seconds. The experimental procedure for determining the binding kinetics parameters was as follows: The running buffer for kinetic determination was 1x HBS-EP+(pH 7.4) solution. Capture: The antibody was injected into the test channels of the Anti-hFc or anti-mouse Fc chip channels 1-3 at a flow rate of 10 μL / min for 60 seconds to perform capture. The antigen IL-1β recombinant protein was diluted to 100 nM with 1x HBS-EP+(pH 7.4). Measurement: The antigen was injected into channels 1-3 at a flow rate of 30 μL / min, 1 sample of 0 concentration was used to remove background signal; the binding and dissociation times of the antigen and antibody were 180 and 400 seconds, respectively. Regeneration: 10 mM glycine pH 1.5 was injected into channels 1-3 at a flow rate of 30 μL / min for 30 seconds, and then stabilized for 60 seconds. The Biacore 8K analysis software was used to calculate the equilibrium dissociation constant (K D value) of each antibody of the application. The reference channel (FC1) was used for background subtraction. The results show that the dissociation constant of the monoclonal antibody 4D14 is (4.25±0.17) nM, and the above experiment shows that the monoclonal antibody of the application has good binding affinity to the antigen.

[0038] Simultaneously, the isotype of the 4D14 monoclonal antibody was identified using a mouse monoclonal antibody isotype identification kit. The prepared 4D14 monoclonal antibody was identified as having an IgG2b heavy chain and a Kappa chain light chain.

[0039] Example 5: Application of 4D14 monoclonal antibody in a gout model

[0040] Weigh 40 mg of MSU white powder, add 1 mL of Tween 80, and bring the volume to 100 mL with RMPI-1640 medium. Heat and stir with a magnetic stirrer until the crystals are completely dissolved to prepare a suspension with a concentration of 400 μg·mL⁻¹ MSU. Autoclave, seal, and dispense into containers, then store at 4 °C for later use.

[0041] THP-1 cells were quickly removed from the liquid nitrogen tank and placed in a preheated 37°C water bath for rapid thawing. The cell suspension was transferred to a 15mL sterile centrifuge tube; RPMI 1640 medium containing 1% penicillin + streptomycin and 10% FBS was added for a 10-fold dilution. The cell suspension was centrifuged at 1000rpm for 5min, the supernatant was discarded, and 1mL of fresh medium was added to the cell pellet. The cells were gently resuspended by pipetting and transferred to a culture flask, and another 5mL of fresh medium was added. The cells were cultured in a 37°C, 5% CO2 cell culture incubator. Cell growth was observed using an inverted microscope; the cells appeared bright, round, and suspended in the medium. The medium was changed every 2-3 days. When the cells reached 80-90% confluence, the cell suspension was transferred to a 15mL centrifuge tube and centrifuged at 1000rpm for 5min. The supernatant was discarded, and an appropriate amount of medium was added. The cells were gently resuspended by pipetting and transferred to a cell concentration of 2.0×10⁶ cells / mL. 5 The cells were passaged at 1.0 × 10⁹ / mL, and then cultured in a cell culture incubator at 37°C and 5% CO₂ saturated humidity. 6 THP-1 cells were seeded in 6-well plates and stimulated with 100 ng / mL PMA for 24 h to induce their differentiation 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, suggesting that 100 ng·mL-1 PMA can successfully induce THP-1 monocytes to differentiate into macrophages.

[0042] Monoclonal antibody activity validation: Macrophages were used as target cells. No monoclonal antibody was added to the normal and model groups, while different concentrations (1, 10, 50, 100 μg / mL) of 4D14 monoclonal antibody were added to the other groups. The positive control was 50 μg / mL of cannabinoids. After 6 hours, MSU was added to a final concentration of 400 μg / mL. Each concentration gradient was used in 5 replicates, and the cells were cultured for another 24 hours. The cell supernatant was collected, and the level of the inflammatory cytokine IL-1β in the cell culture supernatant was measured according to the ELISA kit instructions.

[0043] Results as shown in Figure 3 The results showed that compared with the normal group, the secretion level of inflammatory factor IL-1β in the cell supernatant was significantly increased after the THP-1 derived macrophages were stimulated by MSU in the model group (P<0.01). Compared with the model group, the content of IL-1β in the range of 1-100 μg / mL was significantly reduced after treatment with 4D14 monoclonal antibody, and the secretion level of IL-1β was most obvious in the treatment of 4D14 monoclonal antibody at a concentration of 50 μg / mL compared with the model group (P<0.01). Compared with the positive control group, the effect was similar, and the expression of IL-1β was only (25.7±4.3) pg / L, which was also significantly different compared with the normal group, and the treatment effect was better. Through this experiment, it was proved that the IL-1β monoclonal antibody 4D14 could effectively inhibit the expression of IL-1β in the gout model cells, and then realize the treatment of gout.

[0044] The foregoing detailed description has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the claims to the precise form disclosed. Many modifications and variations are possible in light of the above teaching. It is intended that the scope of claims and the patent be limited only by the appended claims and equivalents thereof.

Claims

1. A monoclonal antibody 4D14 specific for IL-1 β, characterized in that The sequence of the light chain variable region of the antibody is shown as SEQ ID NO: 1, and the sequence of the heavy chain variable region is shown as SEQ ID NO:

2.

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

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

4. The pharmaceutical composition of claim 3, wherein The pharmaceutically acceptable carrier includes a buffer, an antioxidant, a preservative or a non-ionic surfactant.

5. The use of the monoclonal antibody of IL-1β according to claim 1 in the preparation of a medicament for treating gout by inhibiting the expression of IL-1β.

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

7. Use according to claim 6, wherein The pharmaceutically acceptable carrier includes a buffer, an antioxidant, a preservative or a non-ionic surfactant.

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