Application of combined umbilical cord mesenchymal stem cells and peripheral blood PRP in the treatment of knee osteoarthritis

Abstract

This invention provides the application of umbilical cord mesenchymal stem cells combined with peripheral blood PRP in the treatment of knee osteoarthritis. Specifically, it also provides a monoclonal antibody targeting IL-20, which can effectively inhibit the levels of inflammatory factors IL-6 and IL-20 and promote chondrocyte proliferation. Using the monoclonal antibody alone or in combination with umbilical cord mesenchymal stem cells and peripheral blood PRP can effectively reduce the OARSI score of knee cartilage in osteoarthritis rats, demonstrating good therapeutic effects and promising application prospects.

Description

Technical Field

[0001] This application relates to the field of biology, specifically to the application of umbilical cord mesenchymal stem cells combined with peripheral blood PRP in the treatment of knee osteoarthritis. Background Technology

[0002] Knee osteoarthritis (KOA) is a degenerative disease of the knee joint, characterized by synovitis, bone hyperplasia, osteophyte formation, and gradual loss of articular cartilage, leading to clinical symptoms such as joint pain, swelling, deformity, and limited mobility. If left untreated, KOA can cause loss of joint function, severely impacting patients' quality of life. Currently, commonly used clinical treatments include non-pharmacological therapies, pharmacological therapies, and surgical treatments. These treatments primarily aim to relieve pain, improve joint function, and slow the progression of KOA, but they cannot completely repair the damage to the articular cartilage, and patients may need to undergo multiple surgeries, undoubtedly increasing their financial burden. Mesenchymal stem cells, as a popular research area in regenerative medicine, have shown great potential in treating various diseases such as ischemic heart failure, skin trauma, transplant rejection, diabetes, liver disease, and nerve damage.

[0003] Umbilical cord mesenchymal stem cells (UC-MSCs) differentiate into chondrocytes, directly participating in cartilage synthesis. UC-MSCs can also enhance chondrocyte proliferation and differentiation by activating the WNt / β-catenin signaling pathway; secrete growth factors and cytokines, such as vascular endothelial growth factor (VEGF) and transforming growth factor β (TGF-β), promoting chondrocyte proliferation and differentiation, increasing cartilage matrix synthesis, and simultaneously increasing type II collagen while decreasing matrix metalloproteinase 13 and depolymerizing protein-like metalloproteinase-5 containing type I platelet-binding protein motifs, maintaining the homeostasis of the cartilage microenvironment to reduce cartilage damage; UC-MSCs can also upregulate the expression of cartilage formation-related genes, such as type X collagen, B-cell lymphoma-2 gene, and TSG-6 gene, promoting chondrocyte survival; and reduce chondrocyte apoptosis by inhibiting the mTOR pathway and activating autophagy. These mechanisms work synergistically to effectively increase cartilage thickness, promote cartilage matrix synthesis and deposition, and maintain the homeostasis of the cartilage microenvironment, fundamentally solving the knee-osteoarthritis (KOA) problem caused by cartilage loss. Regulating the inflammatory response is the basis for umbilical cord mesenchymal stem cells to delay the progression of KOA. The known specific mechanisms include: regulating inflammatory factors to exert anti-inflammatory effects: on the one hand, reducing the levels of pro-inflammatory factors such as nitric oxide, interleukin-6, IL-1β and tumor necrosis factor-α, and on the other hand, secreting anti-inflammatory factors such as IL-10 and IL-4, effectively reducing joint inflammation and cartilage damage; promoting macrophage polarization: by regulating the polarization state of macrophages, promoting the transformation of macrophages from a pro-inflammatory to an anti-inflammatory state, thereby inhibiting inflammation; and regulating the immune response.

[0004] Platelet-Rich Plasma (PRP) is a concentrated platelet extract obtained from whole blood through centrifugation. It consists of platelets, plasma, and various bioactive substances in the plasma, including coagulation factors and growth factors. Once activated, PRP allows fibrinogen in the plasma to form a three-dimensional fibrin scaffold. Simultaneously, platelets release active substances, thereby promoting cell proliferation, differentiation, and migration, and promoting the regeneration of articular chondrocytes. The main mechanism by which PRP promotes bone and soft tissue repair lies in its high concentration of platelets. Upon activation, PRP releases various growth factors, such as platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), type I insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF). These growth factors are mainly found in the α-granules of platelets. When platelets are activated, the α-granules fuse with the cell membrane, resulting in degranulation. The released growth factors are converted into an active state and bind to the surface of target cell membranes through endocrine, autocrine, and paracrine pathways, inducing the activation of endogenous protein signals and secondary signal activation. This leads to the induction of intracellular gene sequence expression and mRNA transcription, the synthesis of various proteins required for tissue regeneration, and the guidance of cell proliferation, matrix synthesis, osteoid production, and collagen synthesis. This accelerates the differentiation of mesenchymal stem cells, promotes the proliferation of osteoblasts and fibroblasts, improves the inflammatory response at the disease site, promotes cell proliferation, differentiation, and tissue regeneration, and thus improves the patient's clinical symptoms or promotes damage repair.

[0005] IL-20 is a newly discovered member of the IL-10-related cytokine family. It is mainly produced by activated monocytes and shares 28% homology with IL-10. While it shares some amino acid sequence homology with other IL-10 family members such as IL-19, IL-22, and IL-24, their biological functions differ. Studies have found that IL-20 can promote the secretion and expression of pro-inflammatory factors such as TNF-A and monocyte chemoattractant protein (MCP), and promote the proliferation and differentiation of keratinocytes. Currently, IL-20 is found to be closely related to psoriasis. Recent studies have found that IL-20 levels in the synovial fluid of RA patients are significantly higher than those in patients with osteoarthritis and gout, but there was no difference in serum IL-20 levels among the three groups. Simultaneously, it was confirmed that IL-20R is expressed in the synovium of RA patients, and their synovial fibroblasts (RASFs) express the IL-20 receptor complex. IL-20 can activate ERK-1 / 2 (extracellular signal-regulated kinase) in RASFs. Studies have shown that the production of TNF-A in the synovium of rheumatoid arthritis (RA) patients is related to the activation of the ERK signaling pathway. In vitro experiments have confirmed that IL-20 can chemotactically attract neutrophils, promote the proliferation of synovial fibroblasts in rheumatoid arthritis patients, and secrete IL-6 and IL-8. Therefore, it is believed that IL-20 itself is a chemokine that can induce the production of other chemical factors, suggesting that IL-20 is related to the pathogenesis of RA.

[0006] However, there are not enough drugs available at present that can more effectively treat osteoarthritis, and further development and research are needed. Summary of the Invention

[0007] In one aspect, the present invention provides a monoclonal antibody that specifically binds to and inhibits the activity of IL-20.

[0008] The monoclonal antibody is a 2E15 monoclonal antibody, the sequence of which is shown in SEQ ID NO: 1 and the sequence of which is shown in SEQ ID NO: 2.

[0009] Specifically, the monoclonal antibody was able to significantly inhibit synovial inflammation and effectively improve joint function in a rat model.

[0010] In some embodiments, the light chain variable region of the present invention

[0011] i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 1; or

[0012] ii) Contains or consists of an amino acid sequence selected from or composed of said amino acid sequence; or

[0013] (ii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 1.

[0014] In some embodiments, the heavy chain variable region of the present invention

[0015] i) Contains or is composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 2; or

[0016] ii) Contains or consists of an amino acid sequence selected from or composed of said amino acid sequence; or

[0017] (ii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 2.

[0018] A "conservative amino acid substitution" is an amino acid substitution in which one amino acid residue is replaced by another amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, the substitution of tyrosine with phenylalanine is a conserved substitution. Methods for identifying conserved substitutions of nucleotides and amino acids that do not eliminate antigen binding are well known in the art.

[0019] Specifically, the light chain variable region SEQ ID NO: 1

[0020] DIQLTQSPAIMSASPGEKVTMTCSARMDQHNMYWYQQKAGSSPRLLIYNQWNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCYIVQVKLAMFGAGTKLEIK.

[0021] Heavy chain variable region SEQ ID NO:2

[0022] QVKLQESGTVLAGPGALVKMSCKASRAYKSCTRMHWIKQRPGQGLEWIGAFHVFCSYRSYNQKFKGKVKLTAVTSTSTAYMELSSLTNEDAAVYYCRGMLGKWGQGTTVTVSS.

[0023] Furthermore, this invention provides the application of the 2E15 monoclonal antibody in the preparation of a medicament for treating knee osteoarthritis.

[0024] Furthermore, this invention provides the application of 2E15 monoclonal antibody combined with umbilical cord mesenchymal stem cells and PRP in the preparation of a medicament for treating knee osteoarthritis.

[0025] The term "joint" health refers to the health of one or more of the following joints: hand, elbow, wrist, axillary, sternoclavicular, vertebral, temporomandibular, sacroiliac, hip, knee, and foot.

[0026] Specifically, the drug or cassette may also contain a pharmaceutically acceptable carrier or excipient.

[0027] This disclosure includes, but is not limited to, improving joint movement and / or body function, maintaining joint health and mobility in old age, supporting, protecting or promoting joint comfort, reducing joint pain, reducing joint friction, reducing joint stiffness, improving joint range of motion and / or flexibility, promoting mobility, reducing inflammation, reducing oxidative stress, reducing and protecting joint wear and tear, managing and / or treating osteoarthritis and / or rheumatoid arthritis, preventing osteoarthritis and / or rheumatoid arthritis, or reversing the progression of osteoarthritis and / or rheumatoid arthritis; preventing and treating juvenile rheumatoid arthritis, Still's disease, psoriatic arthritis, reactive arthritis, septic arthritis, Reiter's syndrome, Behçet's syndrome or Felty's syndrome in mammals, etc.

[0028] Pharmaceutical compositions may also contain any pharmaceutically acceptable carrier. A “pharmaceutically acceptable carrier” is a pharmaceutically acceptable material, composition, or medium relating to the transport or delivery of a compound of interest from one tissue, organ, or body part to another. For example, a carrier can be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be “pharmaceutically acceptable” because it must be compatible with the other components of the formulation. It must also be suitable for contact with any tissue or organ it may come into contact with, meaning it must not carry the risk of toxicity, irritation, allergic reactions, immunogenicity, or any other complication that outweighs its therapeutic benefit.

[0029] In various embodiments, pharmaceutical compositions are provided comprising a pharmaceutically acceptable excipient and a therapeutically effective amount of anti-IL-20 antibody. "Pharmaceutically acceptable excipient" means an excipient used to prepare a generally safe, non-toxic, and desirable pharmaceutical composition, and includes excipients acceptable for both veterinary and human pharmaceutical use. The active ingredient may be mixed with an excipient that is pharmaceutically acceptable, compatible with the active ingredient, and in an amount suitable for the therapeutic methods described herein. Such excipients may be solid, liquid, semi-solid, or, in the case of an aerosol composition, gaseous. Suitable excipients are, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, water, saline, dextran, propylene glycol, glycerol, ethanol, mannitol, polysorbate, and combinations thereof. In addition, if desired, the composition may contain small amounts of excipients, such as wetting agents or emulsifiers, pH buffers, etc., to enhance or maintain the effectiveness of the active ingredient. Therapeutic compositions as described herein may contain pharmaceutically acceptable salts. Pharmaceutically acceptable salts include acid addition salts formed from inorganic acids such as hydrochloric acid or phosphoric acid, organic acids such as acetic acid, tartaric acid, or mandelic acid; salts formed from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide; and salts formed from organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc. Liquid compositions may also contain a liquid phase other than water and excluding water, such as glycerol, vegetable oils such as cottonseed oil, and water-oil emulsions. Physiologically tolerable carriers are well known in the art. The amount of active agent (i.e., antibody or fragment thereof) that will be effective in treating a particular condition or disease will depend on the nature of the condition or disease and can be determined by those skilled in the art using standard clinical techniques.

[0030] The pharmaceutical composition may also be encapsulated, tableted, or formulated as an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate its preparation. Liquid carriers include syrups, peanut oil, olive oil, glycerin, saline, alcohol, and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, kaolin, magnesium stearate or stearic acid, talc, pectin, gum arabic, agar, or gelatin. The carrier may also include a sustained-release substance, alone or with waxes, such as glyceryl monostearate or glyceryl distearate.

[0031] Pharmaceutical compositions can be delivered in therapeutically effective amounts. The precise therapeutically effective amount is the quantity of the composition that will produce the most effective results in terms of therapeutic efficacy in a given subject. This amount will vary depending on a variety of factors, including, but not limited to, the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the subject's physiological condition (including age, sex, disease type and stage, general physical condition, responsiveness to a given dose, and type of drug), the nature of the pharmaceutically acceptable carrier in the formulation, and the route of administration. Those skilled in the art in the clinical and pharmacological fields will be able to determine the therapeutically effective amount through routine laboratory procedures.

[0032] For the treatment of a disease, the appropriate antibody dosage depends on the type of disease being treated, the severity and duration of the disease, the responsiveness of the disease, whether the antibody is administered for therapeutic or prophylactic purposes, prior treatment, and the patient's clinical history. The dosage may also be adjusted by an individual physician if any complications occur and at the discretion of the treating physician. The administering physician can determine the optimal dosage, method of administration, and repetition rate. TL1A antibodies can be administered once or as a series of treatments lasting from several days to several months, or until a cure or reduction in the disease state is achieved (e.g., treatment or improvement of IBD). The duration of treatment depends on the subject's clinical progress and response to treatment. In some embodiments, the dosage is from 0.01 μg to 100 mg per kilogram of body weight and can be administered once or more daily, weekly, monthly, or annually. For systemic administration, therapeutic doses may be administered to the subject, such as 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg or more.

[0033] The pharmaceutical compositions of this disclosure can be prepared by combining the compounds of this disclosure with suitable pharmaceutically or nutritionally acceptable carriers, diluents, or excipients, and can be formulated into solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalers, gels, creams, lotions, tinctures, capsules, ready-to-drink formulations, masks, microspheres, and aerosols. Typical routes of administration for such pharmaceutical compositions include oral, topical, transdermal, inhalation, parenteral, sublingual, oral, rectal, vaginal, or intranasal administration. The term parenteral, as used herein, includes subcutaneous injection, intravenous, intramuscular, intrasternal injection, or infusion techniques. The pharmaceutical compositions of this disclosure are formulated to allow the active ingredients contained therein to be bioavailable after administration to a patient. The compositions to be administered to a subject, patient, or mammal are in the form of one or more dose units, wherein, for example, tablets may be a single dose unit, and containers for compositions of the compounds or extracts of this disclosure or 2-3 plant extracts in aerosol form may contain multiple dose units. The actual methods for preparing such dosage forms are known to those skilled in the art.

[0034] Beneficial effects

[0035] This invention provides the application of umbilical cord mesenchymal stem cells combined with peripheral blood PRP in the treatment of knee osteoarthritis. Specifically, it also provides a monoclonal antibody targeting IL-20, which can effectively inhibit the levels of inflammatory factors IL-6 and IL-20 and promote chondrocyte proliferation. Using the monoclonal antibody alone or in combination with umbilical cord mesenchymal stem cells and peripheral blood PRP can effectively reduce the OARSI score of knee cartilage in osteoarthritis rats, demonstrating good therapeutic effects and broad application prospects. Attached Figure Description

[0036] Figure 1 Figure showing the results of specific identification of the 2E15 monoclonal antibody;

[0037] Figure 2 The results of the detection of inflammatory factors IL-6 and IL-20 in each group;

[0038] Figure 3 OARSI score results for knee cartilage in each group. Detailed Implementation

[0039] Specific embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While specific embodiments of the invention are shown in the drawings, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the invention and to fully convey the scope of the invention to those skilled in the art.

[0040] Example 1: Preparation of umbilical cord mesenchymal stem cells

[0041] Under aseptic conditions, the near-fetal umbilical cord segment was thoroughly washed with PBS containing 100 U / mL penicillin and 100 U / mL streptomycin to remove residual blood stains from the umbilical arteries and veins. The umbilical cord was divided into 0.5 cm segments, and the arteries and veins were carefully removed. The segments were cut into 1 mm × 1 mm × 1 mm pieces, with LG-DMEM culture medium added dropwise to keep the tissue moist during the cutting process. The tissue was washed twice with PBS containing 100 U / mL penicillin and 100 U / mL streptomycin. One segment per centrifuge tube was then placed in the centrifuge tube for later use. Twice the volume of enzyme solution (a mixture of 0.3% type II collagenase, 0.1% trypsin, 0.02% EDTA, 0.1% hyaluronidase, and 0.1% DNase) was added, and the umbilical cord tissue blocks were digested by shaking in a 37°C water bath for 2 hours. Digestion was terminated using LG-DMEM medium containing 10% FBS, 100 U / mL penicillin, and 0.1 g / L streptomycin. Cells were centrifuged at 1000 rpm for 8 min, the supernatant was discarded, and the cells were resuspended in LG-DMEM medium containing 10% FBS, 100 U / mL penicillin, and 0.1 g / L streptomycin. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 4 mL of LG-DMEM medium containing 10% FBS, 100 U / mL penicillin, and 100 U / mL streptomycin. The resulting primary cells were seeded into T-25 culture flasks and incubated at 37°C, 5% CO2, and 95% humidity. The medium was changed for the first time after 3 days to remove non-adherent cells, and thereafter every 48 hours. Once the primary cells have grown to approximately 80% confluence with the bottom of the culture flask, discard the culture medium. Wash once with PBS, then digest with 0.25% trypsin-EDTA at 37°C for about 2 minutes. Gently tap the flask wall to promote cell detachment from the bottom. Then, add LG-DMEM medium containing 10% FBS, 100 U / mL penicillin, and 100 U / mL streptomycin to terminate the digestion. Collect the cells obtained after pipetting, centrifuge at 600g for 5 minutes, discard the supernatant, and resuspend in culture medium at a 1:2 ratio for subculturing. Incubate at 37°C, 5% CO2, and 95% humidity, changing the medium every 2 days. When the cells have proliferated and covered approximately 80% of the bottom of the flask, digest and subculture using the same method.

[0042] Fourth-generation cells in the logarithmic growth phase were prepared into 1×10⁴ cells. 9L-1 suspension was incubated with 10 μL of mouse anti-human PE-labeled CD73 and isotype control at 4°C in the dark for 30 min, washed three times with PBS, and fixed with 10 g / L paraformaldehyde for flow cytometry analysis. The experiment was repeated three times. The results showed that the positive rate of human MSCs labeled antigen CD73 was (94.88±5.36)%, indicating that relatively pure umbilical cord mesenchymal stem cells were isolated.

[0043] Example 2: Preparation of anti-IL-20 monoclonal antibody

[0044] Five 8-week-old female BALC / c mice were intramuscularly injected with IL-20 (100 μg / mouse, catalog number LZ-D6953, Shanghai Lianzu Biotechnology Co., Ltd.). Immunization was repeated every two weeks, using the same method and dosage as the second immunization. One week after the fourth immunization, blood was collected from the orbital sinus of the mice to determine the titer. Mice with the highest titer (number 2) were selected and given a booster immunization one week later. Five days later, spleen cells from these mice were fused with SP2 / 0 cells using standard methods. The fused cells were screened using HAT (Hybridization Technology) and positive clones were selected using limiting dilution. After three rounds of screening, four good hybridoma cell lines were obtained. These four cell lines were further expanded and cultured. Hybridoma cell line 2E15 showed the strongest positive reaction and best stability and was used for cell line establishment and preservation.

[0045] Ascites was induced in vivo using conventional methods. Specifically, normal Balb / c mice were pretreated one week prior by intraperitoneal injection of 0.5 ml of liquid paraffin, followed by injection of prepared hybridoma cells into the pretreated mice at a concentration of 2 × 10⁻⁶ cells / mL. 6 / animal, ascites fluid was collected after 10 days of culture. The monoclonal antibody titer in the ascites fluid was determined by ELISA, with the highest antibody dilution (P / N ≥ 2.0) used as the final titer value. A titer of 8 × 10⁻⁶ was considered the highest value. -5 The monoclonal antibody was further purified using a Protein-G affinity chromatography column. After column purification, the concentration was adjusted to 5 mg / mL for later use.

[0046] The ELISA method was used to detect the 2E15 monoclonal antibody according to the instructions of the subclass reagent kit from Shanghai Youkewei Biotechnology Co., Ltd. The results showed that the 2E15 monoclonal antibody was of type IgG1 and the light chain was of type κ.

[0047] The 2E15 monoclonal antibody was identified by sequencing, and its light chain variable region sequence is shown in SEQ ID NO: 1, and its heavy chain variable region sequence is shown in SEQ ID NO: 2.

[0048] Example 3: Affinity Identification of 2E15 Monoclonal Antibody

[0049] The affinity of 2E15 monoclonal antibody for human IL-20 was detected using a Biacore T200 instrument, with all procedures performed at 25 °C. A commercial Protein A chip was used to immobilize an appropriate amount of IL-20 via a capture method at a flow rate of 10 μL / min. The antigen was serially diluted, and the instrument flow rate was switched to 30 μL / min. The antigen was flowed sequentially from low to high concentration (20 nmol / L, 1:2 serial dilution) through the reference channel and the channel containing the immobilized antibody. Flow through buffer served as a negative control. After each binding and dissociation cycle, the chip was regenerated using glycine at pH 1.5. The instrument's built-in analysis software was used to fit the 1:1 binding model in the Kinetics option, calculating the antibody binding rate constant ka, dissociation rate constant kd, and dissociation equilibrium constant KD (kd / ka). The results are shown in Table 1.

[0050] Table 1. Affinity identification results of 2E15 monoclonal antibody

[0051]

[0052] As can be seen from the results in Table 1, the affinity of 2E15 monoclonal antibody for human IL-20 was detected by BiacoreT200.

[0053] Example 4: Specificity Identification of 2E15 Monoclonal Antibody

[0054] Human recombinant IL-20, human recombinant IL-6, and BSA were subjected to SDS-PAGE according to the LAEMMLI method, with a separating gel concentration of 12% and a stacking gel concentration of 5%. The electrophoretic gels were stained with Coomassie Brilliant Blue and then destained until the protein bands were clear. Western blot analysis involved transferring the electrophoretic gel to a membrane at 200 mA for 20 min, followed by blocking in 5% skim milk for 1 h. After washing three times with PBST, 2E15 monoclonal antibody (20 ng / mL) was used as the primary antibody, and goat anti-mouse HRP-IgG antibody (1:2500) was used as the secondary antibody. After washing, ECL was used for color development. Results are as follows: Figure 1 As shown.

[0055] Depend on Figure 1 It is evident that the 2E15 monoclonal antibody can specifically bind to IL-20 with a distinct band, but does not bind to IL-6 or BSA, indicating that the 2E15 monoclonal antibody has a strong specific response.

[0056] Example 5: Effects of 2E15 monoclonal antibody on chondrocyte proliferation and inflammation.

[0057] HUM-CELL-0096 human chondrocytes (Wuhan Yuansheng Primary Biotechnology Co., Ltd.) were cultured in DMEM medium (10% fetal bovine serum) at 37℃, 5% CO2, and saturated humidity. Cells were passaged when they reached 80% confluence with the bottom of the flask. Chondrocytes in the logarithmic growth phase were treated with IL-1β (10 ng / mL) medium for 24 h to induce an inflammatory response and establish a chondrocyte inflammation model. The medium was then discarded, and DMEM containing different concentrations of 2E15 monoclonal antibody was added for further culture (the control group was cultured in complete medium) for 24 h. Experimental groupings: Groups AC consisted of antibody treatments at concentrations of 10 μg / ml, 50 μg / ml, and 100 μg / ml; the control group consisted of chondrocytes cultured in conventional complete medium; the model group consisted of chondrocytes cultured in medium containing IL-1β (10 ng / mL) without monoclonal antibody treatment.

[0058] After 24 h, cell viability was detected using CCK-8 reagent, and absorbance at 450 nm (A450nm) was measured using an ELISA reader. The results are shown in Table 2.

[0059] Table 2 Results of chondrocyte proliferation activity assay

[0060]

[0061] *Compared with the model group, P < 0.05.

[0062] As can be seen from the results in Table 2, the increase in antibody concentration has a greater impact on the activity of chondrocyte proliferation and promotes chondrocyte proliferation more effectively.

[0063] Cell culture supernatants from each group were collected, and the levels of inflammatory factors IL-6 and IL-20 were detected by ELISA. The experimental procedure was performed according to the kit instructions. The model group was considered as 1, and the relative content results of the other groups are shown below. Figure 2 As shown.

[0064] from Figure 2It can be seen that normally cultured HUM-CELL-0096 human chondrocytes basically do not secrete inflammatory cytokines IL-6 and IL-20. After adding IL-1β (10 ng / mL) for 24 h, HUM-CELL-0096 human chondrocytes secrete large amounts of IL-6 and IL-20. After treatment with monoclonal antibody, the secretion of IL-20 in HUM-CELL-0096 human chondrocytes was significantly inhibited, with a significant difference compared with the model group (P < 0.05). This indicates that the monoclonal antibody of the present invention can alleviate the inflammatory response of chondrocytes by inhibiting the expression of IL-20, and the expression of IL-6 is also downregulated to a certain extent. This demonstrates that the monoclonal antibody of the present invention has a good inhibitory effect on the inflammatory response of chondrocytes.

[0065] Example 6: Therapeutic effects of 2E15 monoclonal antibody and / or umbilical cord mesenchymal stem cells and PRP on osteoarthritis.

[0066] SD rats (purchased from the Department of Laboratory Animal Science, Peking University Health Science Center) were randomly divided into four groups after one week of acclimatization: a blank control group (n=10), a model group (n=10), a positive control group (n=10), a 2E15 monoclonal antibody treatment group (monoclonal antibody group, n=10), a PRP and umbilical cord mesenchymal stem cell group (dual group, n=10), and a PRP and umbilical cord mesenchymal stem cell combined with 2E15 monoclonal antibody group (triple group, n=10). The control group underwent sham surgery, with the joint cavity cut and then directly sutured. The model group and the drug group underwent anterior cruciate ligament transection surgery to establish an osteoarthritis model. Three weeks after modeling, the local joint response and joint deformity at the modeling site were assessed in each rat, following the relevant details in the Lequesne knee joint assessment method. Local joint response assessment: The modeled joint was squeezed with fingers, and the rats' responses were graded into four levels. Grade I: No obvious pain response; Grade II: Obvious contraction at the model site; Grade III: Contraction and local spasm in the patient, with mild systemic reaction; Grade IV: Severe contraction and trembling in the patient, with the experimental rats screaming and struggling violently. Model rats that met the Grade II-III criteria for local joint response in the Lequesne knee joint assessment method were selected for the experiment.

[0067] Specifically, the administration methods for each group are as follows:

[0068] The positive control group received celecoxib orally at a dose of 24 mg / kg, 200 μL, once daily for 4 weeks.

[0069] The 2E15 monoclonal antibody treatment group received intra-articular injections of 100 μL of 2E15 monoclonal antibody at a concentration of 5 mg / mL, once a week for 4 weeks.

[0070] The two-drug regimen was first administered via intra-articular injection of 100 μL of PRP solution (platelet-rich plasma (PRP) preparation solution, catalog number: M535-01, brand: Aipu Science), with a platelet count of 6.5 × 10⁻⁶. 9 / L), and after an interval of 4 hours, 100 μL (1*10) of umbilical cord mesenchymal stem cells prepared in Example 1 were injected again. 8 (number / mL). Once a week for 4 weeks;

[0071] The triple therapy group was first administered an intra-articular injection of 100 μL of PRP solution (platelet-rich plasma (PRP) preparation solution, catalog number: M535-01, brand: Aipu Science), with a platelet count of 6.5 × 10⁻⁶. 9 / L), and after an interval of 4 hours, inject 50 μL (1*10) of umbilical cord mesenchymal stem cells prepared in Example 1. 8 (Units / mL). Two hours later, administer 100 μL of 2E15 monoclonal antibody intra-articularly at a concentration of 2.5 mg / mL. Repeat once weekly for four weeks.

[0072] The blank control group and the model group were given 200 μL of physiological saline by gavage, with the same administration time and dosage as the experimental group.

[0073] Rats in each group were anesthetized intraperitoneally with 0.3% sodium pentobarbital (0.2 mL / 100 g). After anesthesia, blood was collected via the abdominal aorta, and serum was collected by centrifugation at 3000 r / min for 10 min. The levels of IL-1β and TNF-α in rat serum were measured using an ELISA kit. The results are shown in Table 3.

[0074] Table 3. Results of serum IL-1β and TNF-α levels in rats of each group

[0075]

[0076] The ELISA results in Table 3 showed that the levels of IL-1β and TNF-α inflammatory factors in the serum of rats in the model group were significantly higher than those in the blank group (P<0.01). However, after treatment, the levels of IL-1β and TNF-α inflammatory factors in the serum of rats in the positive control group, the 2E15 monoclonal antibody treatment group (monoclonal antibody group), the PRP and umbilical cord mesenchymal stem cell group (dual group), and the PRP and umbilical cord mesenchymal stem cell group combined with 2E15 monoclonal antibody group (triple group) were lower than those in the model group (#, P<0.05), with the triple treatment showing the best effect.

[0077] Further, tissue samples were harvested from the knee joints of rats in each group, 2 cm above and below the knee joint. Skin was removed, and the samples were fixed in 4% paraformaldehyde solution at 4°C for 24 h. Surrounding soft tissue and ligaments were then removed. The samples were washed with PBS, rinsed with running water for 2 h, and then decalcified with 10% EDTA solution using microwave for 2 weeks. After rinsing with running water for 24 h, the tibia was harvested, the tissue blocks were trimmed, and the sections were embedded in paraffin. Sagittal discontinuous sections with a thickness of 5 μm were prepared and stained with azin blue / hematoxylin-acid orange G. The specific steps were as follows: sections were sequentially immersed in xylene and graded ethanol to dewax to water; rinsed with tap water; placed in 1% hydrochloric acid ethanol for 30 s, drained; placed in azin blue / hematoxylin neutralization for 30 min; thoroughly rinsed with deionized water; placed in 0.5% ammonia solution for 15 s; thoroughly rinsed with deionized water; placed in 95% ethanol for 1 min, drained; placed in Acid Orange G for 1.5 min; washed three times with 95% ethanol; dehydrated; cleared with xylene; and mounted with neutral resin. The sections were observed under a microscope. A double-blind method was used, referencing the Osteoarthritis International Research Society (OARSI) scoring criteria, to assess the degree of cartilage degeneration based on four aspects: matrix staining, cartilage tissue structure, chondrocyte clusters, and tidal line integrity. Results are as follows: Figure 3 As shown.

[0078] from Figure 3 As can be seen, the OARSI score of the model group was significantly higher than that of the blank control group, with a statistically significant difference (P<0.01). After treatment, the OARSI scores of the positive control group, the 2E15 monoclonal antibody treatment group (monoclonal antibody group), the PRP and umbilical cord mesenchymal stem cell group (dual therapy group), and the PRP and umbilical cord mesenchymal stem cell group combined with 2E15 monoclonal antibody group (triple therapy group) were all significantly lower than those of the model group, with statistically significant differences (P<0.01). In particular, the effect of 2E15 monoclonal antibody in reducing OARSI scores was even better than that of the positive control group. After the combined use of 2E15 monoclonal antibody, PRP, and umbilical cord mesenchymal stem cells, the OARSI score of the knee cartilage in osteoarthritis rats was basically similar to that of the blank control group, indicating that the combination of the three treatments has a significant synergistic therapeutic effect. Figure 3 ).

[0079] Although embodiments of the present invention have been described above in conjunction with the accompanying drawings, the present invention is not limited to the specific embodiments and application fields described above. The specific embodiments described above are merely illustrative and instructive, and not restrictive. Those skilled in the art can make many other forms based on the guidance of this specification and without departing from the scope of protection of the claims of the present invention, and all of these are within the scope of protection of the present invention.

Claims

1. A monoclonal antibody that specifically binds to and inhibits the activity of IL-20, characterized in that The monoclonal antibody is 2E15, the sequence of its light chain variable region is shown in SEQ ID NO: 1, and the sequence of its heavy chain variable region is shown in SEQ ID NO:

2.

2. Use of the monoclonal antibody as described in claim 1 in the preparation of a medicament for treating knee osteoarthritis.

3. Use according to claim 2, characterized in that The drug in question is an injectable dosage form.

4. The use of the monoclonal antibody as described in claim 1 in combination with umbilical cord mesenchymal stem cells and PRP (platelet-rich plasma) in the preparation of a medicament for treating knee osteoarthritis.

5. The use according to claim 4, wherein The umbilical cord mesenchymal stem cells and PRP (platelet-rich plasma) mentioned above are either artificially isolated or commercially purchased.

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