CN105037548B - - 6 receptor β chain monoclonal antibody of antihuman interleukin, preparation method and use - Google Patents

Abstract

The present invention provides a monoclonal antibody against the free interleukin-6 receptor β chain on the surface of human cells or serum, the cDNA and amino acid sequence of the light chain variable region of the Fab fragment antigen-binding site of the antibody molecule such as sequence No. . 1 and 2 and the cDNA and amino acid sequences of the heavy chain variable region are shown in Sequence No. 3 and 4. The invention also provides the preparation method of the monoclonal antibody and its application in the medicine for detecting and intervening the diagnosis and treatment of rheumatoid arthritis. Experiments in vivo and in vitro prove that the monoclonal antibody of the present invention has a high affinity to interleukin-6 receptor β chain-positive cells and can regulate synovial fibroblast interleukin-6 receptor β in patients with rheumatoid arthritis by antagonism The molecular mechanism of the chain-RANKL-WNT5A signaling pathway achieves the results of alleviating the immune dysfunction in patients with rheumatoid arthritis and mediating the joint damage caused by the imbalance of bone metabolism.

Description

Anti-human interleukin-6 receptor β chain monoclonal antibody, its preparation method and use

technical field

The invention belongs to the field of biopharmaceuticals, and in particular relates to a monoclonal antibody, in particular to an anti-human interleukin-6 receptor beta chain monoclonal antibody and its preparation method and application.

Background technique

The molecular weight is 130kD, and its cell exterior is composed of 6 continuous β-sheet domains, which are immunoglobulin-like domain (functional domain 1), CHR molecule (functional domain 2, functional domain 3) and 3 fibronectin III-like domains (functional domains 4-6), different domains play an important role in ligand connection and signal transduction. Interleukin-6 receptor beta chain as interleukin-6 family cytokines - interleukin-6, cytostatic factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), interleukin-11, cardiotrophic Cardiotrophin-1 and interleukin-27 co-receptors are involved in various diseases by mediating the signal transduction of different inflammatory factors, such as rheumatoid arthritis (RA), giant lymphoproliferative disease, It plays an important role in the occurrence and development of diseases such as inflammatory bowel disease, multiple myeloma, psoriasis, diabetes, asthma, obesity and tumors. With the deepening of research, people gradually pay attention to the interleukin-6 receptor β chain-related signaling pathway may become a new target for clinical immune intervention and treatment. At the International Antibody Conference held in the United States in 2009, researchers have reported and proposed that diagnostic and therapeutic antibodies to the interleukin-6 receptor β chain have good application prospects. Therefore, whether it is from the perspective of immunopharmacology or drug development, targeting interleukin-6 The regulation and analysis of interleukin-6 receptor β chain, its corresponding ligand or signal transduction-related proteins in the receptor β chain system are currently research hotspots.

Interleukin-6 is one of the important ligands of interleukin-6 receptor β chain, which is closely related to the occurrence of chronic inflammatory diseases, autoimmune diseases and tumors. Interleukin-6 receptors include specific interleukin-6 receptor alpha chain (gp80) and interleukin-6 receptor beta chain (gp130). The expression of interleukin-6 receptor α chain is mainly limited to the surface of liver cells, leukocyte subsets, megakaryocytes and other cells, while the expression of interleukin-6 receptor β chain is on the surface of all cells. Interleukin-6 transmits signals to cells through two ways: the traditional pathway and the trans-signaling pathway. In the classical pathway, interleukin-6 first binds to the membrane-bound interleukin-6 receptor α chain, and then induces homodimerization of the interleukin-6 receptor β chain to form a high-affinity interleukin-6/interleukin-6 receptor α/interleukin- 6-receptor β-chain complex for signal transduction. In the trans pathway, the soluble form of free interleukin-6 receptor α chain binds to interleukin-6 and directly forms a complex with interleukin-6 receptor β chain, so cells that do not express membrane-bound interleukin-6 receptor α chain still remain Responds via interleukin-6. The researchers believe that the existence of free interleukin-6 receptor α chain and trans-signal makes interleukin-6 one of the most important cytokines involved in the inflammatory process, which is involved in the occurrence and development of various autoimmune diseases and tumors, especially interleukin-6 The mechanism of action of -6 in rheumatoid arthritis has always been a hot spot in clinical research.

Rheumatoid arthritis is an autoimmune disease characterized by synovial hyperplasia and progressive destruction of joints. The incidence rate in my country is about 0.4%-1.0%. Mainly all joints of the whole body are severely injured, and the disability rate is as high as 15%. Drugs such as traditional preparations methotrexate and leflunomide are not very specific, and the curative effect is often not satisfactory. In recent years, the synergy of new immunological intervention methods and clinical classic treatment methods has played a certain role in inhibiting the occurrence and development of rheumatoid arthritis. The immunosuppressants related to the treatment of rheumatoid arthritis that have entered preclinical or clinical research can be mainly divided into two categories: 1) Cytokine intervention agents: mainly tumor necrosis factor (TNF) blocking agents (adalimumab, etc.) ), interleukin-6 receptor α chain blocker (tacilizumab TCZ); 2) protein kinase inhibitors: such as JAK inhibitors, SYK inhibitors. However, due to the individual differences of patients with rheumatoid arthritis, they respond differently to the existing biological agents. Most patients still experience the development of osteoarthritis from osteoarthritis to rheumatoid arthritis, and finally suffer from severe joint damage and become disabled. . Therefore, it is urgent for us to continue in-depth research on the pathogenesis of rheumatoid arthritis and develop new drug targets.

A large number of studies have shown that there is a close relationship between the interleukin-6 signaling system and rheumatoid arthritis. The concentrations of interleukin-6 and free interleukin-6 receptor chain α in serum and synovial fluid of patients with rheumatoid arthritis increased, and the correlation between interleukin-6 level and disease activity factors (rheumatoid factor, erythrocyte sedimentation rate, C-reactive protein) The expression level is closely related to the clinical manifestations of patients with rheumatoid arthritis (such as X-ray changes, the number of affected joints, and the duration of morning stiffness). Alleviating joint bone destruction is the most critical issue in the treatment of patients with rheumatoid arthritis, and the increased proportion of osteoclasts is considered to be an important factor for bone and joint damage in patients with rheumatoid arthritis. In patients with rheumatoid arthritis, related inflammatory cytokines activate the proliferation and differentiation of osteoclast precursor cells, and the proportion of osteoclasts increases, resulting in the imbalance of bone metabolism in patients, and the interleukin-6 signaling system plays a role in the regulation of osteoclasts. important role.

Previous studies have proved that antagonists that inhibit the overexpression of interleukin-6 or the abnormality of the interleukin-6 signaling system can effectively regulate the immune disorders mediated by interleukin-6. Clinical studies have shown that antagonists targeting the interleukin-6 signaling system can alleviate the pathogenesis of rheumatoid arthritis patients/rheumatoid arthritis-like animal models to a certain extent. Antagonists targeting interleukin-6 (such as lokizumab) cannot be cleared from the systemic circulation due to the binding of anti-interleukin-6 antibodies to free interleukin-6, but side effects caused by accumulation of interleukin-6 (near milligram levels) occur , has been limiting and hindering its wide clinical application in China. Tocilizumab, a monoclonal antibody drug targeting interleukin-6 receptor chain α, is the only biological agent related to the interleukin-6 signaling pathway that has passed clinical phase III trials. In clinical trials, after treatment of rheumatoid arthritis patients with tocilizumab, it can be observed that the patient's disease activity index is significantly and continuously reduced, the local joint symptoms and signs are significantly improved, and the C-reactive protein quickly returns to normal levels, and it is effective for some tumors. Patients refractory to necrosis factor therapy are hyperresponsive and are therefore considered an effective treatment modality for patients with advanced rheumatoid arthritis. However, in-depth studies have found that tocilizumab acts on both classical and trans-signals, completely blocking the activity of interleukin-6, and thus brings a series of side effects, such as the occurrence of infection, elevated levels of alanine aminotransferase in the blood, Elevated levels of total blood cholesterol, high-density lipoprotein and low-density lipoprotein, and increased risk of cardiovascular disease have also hindered its widespread clinical application. Another monoclonal antibody preparation (sgp130) targeting free interleukin-6 receptor chain β is currently only in experimental research due to the formation of a large number of aggregates in vivo.

Considering the multiple roles of interleukin-6 signaling in rheumatoid arthritis and the improvement effect of monoclonal antibody to interleukin-6 receptor chain α (tacilizumab) on patients with TNF antagonist ineffectiveness and advanced rheumatoid arthritis, The development of anti-human interleukin-6 receptor chain β domain monoclonal antibody may become a new method for the treatment of patients with rheumatoid arthritis. As the coupling site of the interleukin-6 receptor β chain and its ligand is further clarified, it has been confirmed that different cytokines of the interleukin-6 family need to be connected with their own specific receptors to be different from the interleukin-6 receptor chain β Monoclonal antibodies against different functional domains of interleukin-6 receptor β chains have different biological functions, and it is also possible to prepare antagonists of single ligand signaling of interleukin-6 receptor β chains, And it is expected to provide a powerful tool for the development of therapeutic monoclonal antibody drugs. In order to verify the above scientific questions, we established a rheumatoid arthritis-like mouse model (collagenantibody induce arthritis, CAIA) by induction of anti-collagen II functional domain antibody, and the results showed that anti-human interleukin-6 receptor β chain monoclonal antibody can inhibit The occurrence and development of mouse diseases, maintenance of mouse bone balance, etc.; use mouse anti-human/anti-mouse monoclonal antibodies to analyze their potency and competition against different functional domains and epitopes of anti-human interleukin-6 receptor β chain monoclonal antibodies Inhibition, affinity, and biological characteristics of different antigen targets; completed the whole gene sequencing of mouse anti-human/anti-mouse monoclonal antibodies; preliminarily revealed that the above-mentioned monoclonal antibodies regulate the pathogenesis and The immunopathological mechanism of the outcome; at the same time, the application of the established synovial fibroblast cell-mediated differentiation technology of osteoclast precursor cells into osteoclasts in patients with rheumatoid arthritis preliminarily clarified the anti-human interleukin- The substance of the 6-receptor β-chain monoclonal antibody regulating the immunological dysfunction and bone metabolism imbalance in patients with rheumatoid arthritis shows that the anti-human interleukin-6 receptor β-chain monoclonal antibody of this patent application has effective biological activity and has the ability to intervene in rheumatoid arthritis Potential clinical application prospect of onset in patients with osteoarthritis.

Contents of the invention

In order to solve the above scientific and technical problems, the present invention provides a monoclonal antibody against human cell surface or free serum, the cDNA and amino acid sequence of the light chain variable region of the Fab fragment antigen binding site of the antibody molecule are as follows, The cDNA and amino acid sequences of the heavy chain variable region are shown below.

The cDNA and amino acid sequences of the anti-human interleukin-6 receptor beta chain monoclonal antibody molecule are as follows, and respectively Sequences 1, 2, 3 and 4 in the response list:

Light chain variable region ( cDNA sequence 1 and amino acid sequence 2 ):

Heavy chain variable region ( cDNA sequence 3 and amino acid sequence 4 ):

The present invention also provides the use of the above-mentioned monoclonal antibody in the detection and intervention of rheumatoid arthritis diagnosis and therapeutic drugs. The present invention also provides the preparation method of the above-mentioned monoclonal antibody. In vivo and in vitro experiments using U266 cells and established rheumatoid-like mouse models and patients with rheumatoid arthritis have proved that the above-mentioned (10.51) monoclonal antibody after purification and sterilization has a high affinity for positive cells and can Through antagonistic regulation of the molecular mechanism of interleukin-6 receptor β chain-RANKL-WNT5A pathway in synovial fibroblasts in patients with rheumatoid arthritis, the immune dysfunction and mediated bone metabolism imbalance in patients with rheumatoid arthritis can be alleviated result of joint damage in patients.

Further, the above-mentioned monoclonal antibody (10.51) is an immunoglobulin of murine IgG subtype.

The present invention also provides the use of the above-mentioned (10.51) monoclonal antibody in the diagnosis and interventional treatment of rheumatoid arthritis.

Further, the rheumatoid arthritis includes but is not limited to different clinical early, middle or late (including bone damage) patients with rheumatoid arthritis.

Further provide a detection method, characterized in that: anti-human cell surface or free expression of interleukin-6 receptor β chain (10.51) monoclonal antibody binding marker, the marker is a fluorescent marker, or Radioactive markers, or enzyme-labeled markers.

The monoclonal antibody that binds to the surface of human cells or free interleukin-6 receptor β-chain expresses positively can be used in peripheral blood, serum, synovial fluid, synovial fluid cells and damaged joints of patients with rheumatoid arthritis. combined in the organization.

The preparation method of a monoclonal antibody that binds to the surface of human cells or free interleukin-6 receptor β chain is characterized in that: multi-point immunization of mice with commercially available standard human interleukin-6 receptor β chain, three times After normal immunization and a booster immunization, mouse spleen cells and mouse myeloma cells SP2/0 are chemically fused with PEG, and the surface antigens that can secrete the above-mentioned interleukin-6 receptor β-chain antigens and positive cells are screened out. The hybridoma cell line (experiment number 10.51) was subcloned, and the cultured hybridoma cell supernatant was obtained, and the monoclonal antibody of the present invention was obtained through affinity purification. If the human/mouse chimerization or humanization of the monoclonal antibody can be used to develop biological agents for the diagnosis and treatment of patients with rheumatoid arthritis.

Description of drawings

Figure 1 shows the monoclonal antibody against human interleukin-6 receptor β chain obtained after indirect enzyme-linked immunosorbent assay (ELISA) on mouse serum immunized with interleukin-6 receptor β chain and after purification (numbered by experiment 10.51 produced by the hybridoma cell line, the monoclonal antibody of the present invention mentioned herein is the same) reacts with the antigen interleukin-6 receptor β chain titer.

Figure 2 shows the binding reaction of different strains of anti-human interleukin-6 receptor β chain (10.51) monoclonal antibody to U266 cell surface interleukin-6 receptor β chain by flow cytometry (FCS).

Fig. 3 is the FACS detection of the binding reaction between different concentrations of the monoclonal antibody of the present invention and the interleukin-6 receptor β chain on the surface of U266 cell line; wherein, A is the isotype control; the concentration of the monoclonal antibody of the present invention in B is 10ug/mL; The concentration of the monoclonal antibody of the invention is 5ug/mL; the concentration of the monoclonal antibody of the invention in D is 2.5ug/mL; the concentration of the monoclonal antibody of the invention in E is 0.5ug/mL; F is an irrelevant antibody.

Fig. 4 is the binding curve and affinity detection of the 3-2.3 and 10.51 monoclonal antibodies and the interleukin-6 receptor β chain analyzed by the biomacromolecule interaction analyzer (biacore X100).

Figure 5 is the titer identification test after monoclonal antibody labeled HRP such as ELISA analysis (10.51).

Fig. 6 is the analysis of competitive indirect ELISA. When different concentrations of competing antibodies exist, the binding ability of horseradish peroxidase (HRP)-labeled (10.51) monoclonal antibody to interleukin-6 receptor β chain antigen.

Figure 7 shows the regulatory effect of different doses and induction times of interleukin-6 on the phosphorylation of STAT3 in U266 cells.

Figure 8 is the Western Blot detection of immunized mouse serum regulating interleukin-6-induced STAT3 phosphorylation in U266 cells.

Fig. 9 is a Western Blot detection of anti-human interleukin-6 receptor β chain (10.51) monoclonal antibody regulating interleukin-6-induced STAT3 phosphorylation in U266 cells.

Fig. 10 is a Western Blot detection of STAT3 phosphorylation in U266 cells induced by immunized mouse serum and anti-human interleukin-6 receptor β chain (10.51) monoclonal antibody in coordination with interleukin-6 and free interleukin-6 receptor α chain.

Figure 11 shows the detection of the expression level and ratio of interleukin-6/interleukin-6 receptor alpha chain/interleukin-6 receptor beta chain in patients with rheumatoid arthritis/osteoarthritis (OA) by indirect enzyme-linked immunosorbent assay.

Figure 12 shows the effect of anti-human interleukin-6 receptor β chain (10.51) monoclonal antibody on the regulation of normal human peripheral blood mononuclear cells, peripheral blood mononuclear cells of patients with rheumatoid arthritis, and synovial mononuclear cells of patients with rheumatoid arthritis. Western Blot detection of interleukin-6-induced phosphorylation of STAT3 in normal cells and synovial fibroblasts from patients with rheumatoid arthritis.

Figure 13 is flow cytometry detection anti-human interleukin-6 receptor beta chain (10.51) monoclonal antibody on the regulation of normal human peripheral blood mononuclear cells, rheumatoid arthritis peripheral blood mononuclear cells, rheumatoid arthritis Role of interleukin-6-induced phosphorylation of normal cellular STAT3 in synovial mononuclear cells.

The following examples are only to further illustrate the present invention, and are not intended to limit the present invention to the specific examples. Those skilled in the art will realize that the invention covers all possible alternatives, modifications and equivalents within the scope of the claims.

Detailed ways

The reagents and experimental equipment used in the following examples are common items in this field, which can be purchased in the market or obtained through regular channels.

Embodiment 1: Antibody preparation:

Select 2 male BALB/c mice, mix the interleukin-6 receptor β chain antigen with Freund's complete adjuvant, and inject it intraperitoneally after the emulsification is complete. The immunization dose for each mouse is 20ug. After 1 month, the immunization was repeated twice, and one week after the third immunization, the mouse serum was collected, and the binding ability of the serum to the interleukin-6 receptor β chain antigen was identified by indirect enzyme-linked immunosorbent assay, and the normal BABL/c mouse serum was used as As a negative control, mice with higher serum titers were selected for booster immunization before fusion (peritoneal injection only). One week before fusion, a large number of myeloma cells SP2/0 were cultured with MD6 medium, so that the cells grew well and were in the logarithmic growth phase. On the day of fusion, the spleens of immune BALB/c mice were aseptically removed and ground to make them single cells. Suspension, count, reserve. Afterwards, the splenocytes and SP2/0 were mixed according to the ratio of 1:1, polyethylene glycol (PEG) was added, and MD6 was added after a few minutes to stop, centrifuged, and the mixture containing HAT (H—H—Hypoxanthine, A—Aminopterin) Pterin, T——Thymidine) was resuspended in MD6 culture medium, added to 96-well plate, 200ul per well, the number of cells was about 2x104/well, a total of 50 plates, in HAT selective medium Cultivate for 7-10 days. Observe the growth state of the cells in the 96-well plate under a microscope. If colonies are formed, use a 96-well liquid handling system and a fully automatic plate washing and liquid separation system to perform indirect ELISA high-throughput cross-screening, select the wells with higher positive values, and The detected positive hybridoma cells were subcloned and identified by limiting dilution, and the wells with high positive values were selected for expansion and cultured and the cells were cryopreserved. The hybridoma cells with higher positive value were inoculated with 6 to 10×106 cells per 150ml hybridoma cell serum-free medium, and expanded for 10 to 14 days. After more than 90% of the cells died, the cell supernatant was collected. Centrifuge at 3500 rpm for 15 minutes at 4°C, take the supernatant, add Proclin 300 (1:2000) and mix well, then store at 4°C for later use.

Example 2: Titer determination of anti-human interleukin-6 receptor β chain monoclonal antibody

The titer of the anti-human interleukin-6 receptor β chain monoclonal antibody (produced by the hybridoma cell line of experiment number 10.51) was determined by indirect enzyme-linked immunosorbent assay: 0.1ug/ml interleukin-6 receptor β chain antigen Coat a 96-well plate, add 100ul per well, place in a wet box for overnight coating, wash twice with phosphate buffered saline (PBST) containing 0.05% Tween 20, and wash with 1.5% bovine serum albumin (BSA) Block with PBST at room temperature for 1 hour, dilute the purified mouse serum after immunization with interleukin-6 receptor β chain antigen, add to the plate, add 100ul to each well, incubate at room temperature for 1 hour, and wash 4 times with PBST. Add horseradish peroxidase-labeled goat anti-mouse IgG-Fc secondary antibody, add 100ul per well, incubate at room temperature for 1 hour, and wash 4 times with PBST. Then add the substrate 3,3',5,5'-tetramethylbenzidine (TMB) color development system 150ul for 15-30 minutes, stop with sulfuric acid (H ₂ SO ₄ ), read at 450nm within 30 minutes Absorbance values.

The results showed that the titer of the No. 2 mouse serum with higher immunoreactivity to the interleukin-6 receptor β chain was higher, and the monoclonal antibody against the human interleukin-6 receptor β chain used in the splenocytes of this mouse Fusion experiments for hybridoma generation. (As shown in Figure 1).

Example 3: Subclass Identification of Monoclonal Antibodies

Purify the anti-human interleukin-6 receptor β chain (10.51) monoclonal antibody using the affinity chromatography column standard kit from Pall (USA), and the monoclonal antibody-rich solution is passed through ultrafiltration membrane, MICON 50ml dialysis ultrafiltration device and PBS dialysis Concentrate, and finally use NANODROP 2000 to measure the IgG concentration of the solution containing the monoclonal antibody after dialysis, filter through a 0.22 μm microporous filter, and store it. The purified monoclonal antibody was identified by the mouse monoclonal antibody subtype rapid identification kit of Roche Company, and the anti-human interleukin-6 receptor β chain (10.51) monoclonal antibody heavy chain was identified as IgG2b, and the light chain was κ chain. .

Example 4: Sequencing

Total RNA was extracted from the (10.51) monoclonal antibody hybridoma cell line with the RNeasy kit of Qiagen (Valencia, California, USA), and the mRNA was reversed with the SuperScript III First-Strand kit of Invitrogen (Grand Island, New York, USA). A cDNA library of 10.51 mAb was recorded. Using the 23 primers of the "MouseIgG Library Primer Set" (F2010) kit from Progen Biotechnik in Germany, 21 PCR reactions (reactions that do not include the lambda light chain) were performed, and the specific light and heavy chain products generated were subjected to DNA sequencing, amino acid Translation of polypeptide sequences and identification of CDRs (epitope regions) and FW (framework regions).

The cDNA and amino acid sequences of the light chain variable region of the Fab fragment antigen binding site of the antibody molecule are shown in sequences 1 and 2 in the sequence listing, and the cDNA and amino acid sequences of the heavy chain variable region are shown in sequences 3 and 4 in the sequence listing, The cDNA and amino acid sequences are equivalent. The underlined sequence in the amino acid sequence indicates the position of the CDR region, which is arranged in the order of CDR1, CDR2 and CDR3 and the sequence of the region between them is the framework protein sequence (FW).

Example 5: Antibody and Antigen Binding Affinity Determination

The affinity-purified anti-human interleukin-6 receptor beta chain (10.51) monoclonal antibody was tested by the indirect enzyme-linked immunosorbent assay described in Example 2, and the (10.51) hybridoma cell line was subjected to binding staining reaction, and in Read the OD value on a microplate reader. Other experimental steps are the same as in Example 2.

The results showed that the indirect enzyme-linked immunosorbent assay (ELISA) had the highest reactivity of the anti-human interleukin-6 receptor β chain (10.51) monoclonal antibody to the interleukin-6 receptor β chain antigen (10.51) (as shown in Figure 1).

Example 6: Detection of binding affinity between monoclonal antibody and U266 cell line

The affinity-purified monoclonal antibody against human interleukin-6 receptor β chain (10.51) was tested by flow cytometry for the binding ability of the antibody to the cell surface interleukin-6 receptor β chain. Each group used 3x105 U266 cells, blocked with PBS containing 1.5% BSA at 4 degrees for 30 minutes, added different doses of anti-human interleukin-6 receptor β chain monoclonal antibodies, and incubated at 4 degrees for 60 minutes. After washing with PBS, add FITC-labeled goat anti-mouse secondary antibody and incubate at 4 degrees for 30 minutes in the dark. After washing twice, 100ul of 1% paraformaldehyde was added, the cells were resuspended, and the cells were detected by a FACS Calibur instrument.

The results showed that the flow cytometric anti-human interleukin-6 receptor β chain (10.51) monoclonal antibody combined with the interleukin-6 receptor β chain antigen, the binding ability of the (10.51) antibody to the U266 cell surface interleukin-6 receptor β chain and The result of embodiment 5 is consistent (as shown in Figure 2).

Example 7: Specificity and dose dependence of (10.51) monoclonal antibody

The (10.51) monoclonal antibody after affinity purification was subjected to the flow detection method described in Example 7, and different concentrations of (10.51) were used to bind and stain U266 cells, and the mean fluorescence intensity (MFI) was read on the FACS Calibur instrument. )value.

FACS results showed that the (10.51) monoclonal antibody can specifically bind to the U266 cell surface interleukin-6 receptor β chain in a dose-dependent manner, wherein, in Figure 3, A is the isotype control; the concentration of the monoclonal antibody of the present invention in B is 10ug In C, the monoclonal antibody concentration of the present invention is 5ug/mL; in D, the monoclonal antibody concentration of the present invention is 2.5ug/mL; in E, the monoclonal antibody concentration of the present invention is 0.5ug/mL; F irrelevant antibody (as shown in Figure 3 ).

Example 8: Biomacromolecule Interaction Analyzer Analysis of Antibody Affinity and Binding Kinetics

The affinity and binding kinetics of (10.51) anti-interleukin-6 receptor β-chain monoclonal antibody were detected by GE Biomacromolecule Interaction Analyzer (biacore X100). First, the goat anti-mouse Fc antibody (25 μg/ml) was coupled to the gold film surface of the CM5 chip as a capture molecule, and the prepared (10.51) anti-human interleukin-6 receptor β chain antibody was used as a ligand, and then calculated according to the formula Load the desired (10.51) concentration of anti-human interleukin-6 receptor β chain monoclonal antibody (19 μg/ml, contact for 180 s), run to obtain the signal curve of the binding of monoclonal antibody to interleukin-6 receptor β chain antigen, The curve was fitted with the Langmuir model, and the experimental results were analyzed to obtain the affinity constant of the monoclonal antibody.

The results showed that the binding affinity of the (10.51) monoclonal antibody to the antigen was 2.62E-10 (as shown in FIG. 4 ).

Example 9: Antigen-binding site specificity of antibodies detected by competitive indirect enzyme-linked immunosorbent assay

The antibody is labeled with horseradish peroxidase by dissolving the antibody in carbonate buffer, then adding the reaction solution containing horseradish peroxidase, and under the action of NaBH4, the horseradish peroxidase is labeled to Finally, the antibody solution was dialyzed against phosphate buffered saline and concentrated.

Antibody enzyme-linked immunosorbent assay detection: 96-well plate was coated with 0.1ug/ml interleukin-6 receptor β chain antigen, coated overnight, washed twice and blocked with PBST containing 1.5% BSA for 1 hour at room temperature. Add the (10.51) monoclonal antibody labeled horseradish peroxidase diluted 1:3000, and use this solution to dilute the unlabeled monoclonal antibody to be detected. The initial concentration of the antibody to be detected is 100 μg/ml, 1:3 gradient Dilute, add to microplate, 100ul per well, use unlabeled antibody as self-control, diluent as irrelevant control, incubate at room temperature for 1 hour; wash 4 times with PBST; add 150ul of TMB substrate color development system to develop color 15- After 30 minutes, stop with H2SO4; read the absorbance value at 450nm on a microplate reader.

The results of indirect enzyme-linked immunosorbent assay showed (10.51) titer identification after HRP was labeled with monoclonal antibody against human interleukin-6 receptor β chain (as shown in Figure 5). The results of the competitive ELISA among antibodies showed that the antigenic site specificity of multiple strains of antibodies was different (as shown in FIG. 6 ).

Example 10: Interleukin-6 stimulates U266 cell experiment

The U266 cell stimulation experiment of 3 duplicate wells for each condition was carried out, U266 was starved for 24 hours, and the interleukin-6 induction group: different concentrations (0ng/ml, 1ng/ml, 2ng/ml, 5ng/ml, 10ng/ml, 20ng/ml) of interleukin-6, stimulated for different times (5min, 10min, 20min, 30min, 60min), collected the cells, added pre-cooled lysate containing protease inhibitors, centrifuged in ice bath for 30min to collect the supernatant, and the supernatant After protein quantification, the protein gel was loaded (20-50 μg per well), and the relationship between the phosphorylation of STAT3 in U266 cells and the stimulation time and dose of interleukin-6 was analyzed by Western Blot.

The results of Western Blot showed that stimulating U266 cells with different concentrations of interleukin-6 for 15 minutes could induce the phosphorylation of STAT3 downstream of U266, and the degree of phosphorylation was dose-dependent with the concentration of interleukin-6. U266 cells were stimulated with 10 ng/mL interleukin-6 for different periods of time. The experiment revealed that the phosphorylation of STAT3 induced by interleukin-6 was transient, and the maximum degree of phosphorylation was induced after 20 min of stimulation (as shown in Figure 7).

Example 11: Detection of immune mouse serum and STAT3 phosphorylation by Western Blot

As in Experimental Example 10, add different concentrations of immunized mouse serum (with normal mouse serum as the control) to U266 cells that have been starved, incubate at 37°C for 1 hour, and then add interleukin-6 (1ng/mL) After stimulation for 15 minutes, Western Blot was used to detect the phosphorylation changes of STAT3, so as to indirectly analyze the regulatory effect of immunized mouse serum on IL-6 downstream signaling.

The results of Western Blot detection showed that the 1:500 dilution of immunized mouse serum could significantly inhibit the phosphorylation of STAT3 induced by interleukin-6 in U266 cells, with high sensitivity (as shown in Figure 8).

Example 12: Regulation of Monoclonal Antibody on Phosphorylation of STAT3 Downstream of Interleukin-6

50 μg/mL (10.51) anti-human interleukin-6 receptor beta chain antibody stimulates U266 cells as in Example 10, Western Blot assay detects changes in STAT3 phosphorylation levels, thereby indirectly proving the effect of anti-human interleukin-6 receptor beta chain Regulation of interleukin-6 downstream signaling by monoclonal antibodies.

The results show that (10.51) has a regulatory effect on the phosphorylation of STAT3 downstream of interleukin-6 in U266 cells, and can inhibit the phosphorylation of STAT3 in U266 cells induced by interleukin-6 to a certain extent (as shown in Figure 9). For example, the phosphorylation of free interleukin-6 receptor α chain and STAT3 is slightly enhanced, adding (10.51) anti-human interleukin-6 receptor β chain antibody can also inhibit the joint induction of interleukin-6 and free interleukin-6 receptor chain α Phosphorylation of STAT3 in U266 cells reveals that (10.51) monoclonal antibody has a regulatory effect on both canonical and trans-signals induced by interleukin-6 (as shown in Figure 10).

Example 13: Detection of content of interleukin-6/free interleukin-6 receptor α chain/free interleukin-6 receptor β chain in patients with rheumatoid arthritis

In cooperation with Guanghua Hospital of Integrated Traditional Chinese and Western Medicine in Changning District, Shanghai, approved by the Ethics Committee (see the appendix for the documents of the Ethics Committee) and signed by the patients or their family members on the "Patient Informed Consent Form", eligible patients with rheumatoid arthritis were selected according to the criteria. In addition to meeting the basic criteria in Table 2-1, the experimental group must also meet the following conditions: ① Hemoglobin ≥ 5.5mmol/L; ② White blood cells ≥ 3.5×109/L; ③ Serum creatinine ≤ 150 μmol/L; ④ Platelets ≥ 100×109 /L; ⑤ Serum bilirubin, alanine aminotransferase, alkaline phosphatase, and aspartate aminotransferase are below 1.5 times the upper limit of normal value. Collect patient plasma, synovial fluid, and synovial tissue.

According to R&D company The operation steps of the ELISA sandwich method detection kit to detect interleukin-6, free interleukin-6 receptor α chain, and free interleukin-6 receptor β chain in serum and synovial fluid of healthy controls, OA controls and patients with rheumatoid arthritis Level, the microplate reader detects the absorbance (450nm).

The results of indirect enzyme-linked immunosorbent assay showed that the expression of interleukin-6 in the synovial fluid of patients with rheumatoid arthritis was significantly higher than that in the serum of the same group, and the expression of interleukin-6 receptor α chain in the synovial fluid of patients with rheumatoid arthritis was significantly higher In the OA group, the expression of free interleukin-6 receptor β chain, a natural antagonist of the interleukin-6 signaling system in the serum of rheumatoid arthritis, was insufficient, which was significantly lower than that of the OA control group and the normal control group. Further comparing the ratio of free interleukin-6 receptor α chain/free interleukin-6 receptor β chain in paired specimens, it was found that compared with OA group and normal control group, there were free The ratio of interleukin-6 receptor α chain/free interleukin-6 receptor β chain was abnormally increased, indicating the imbalance of free interleukin-6 receptor α chain/free interleukin-6 receptor β chain in patients with rheumatoid arthritis, and slippery Membrane fluid is more severe than serum (see Figure 11).

Example 14: Western Blot detection of the effect of antibodies on the phosphorylation of cellular STAT3

The method is as in Example 11, and the detection results show that 50 μg/mL (10.51) anti-human interleukin-6 receptor beta chain antibody can inhibit normal human peripheral blood mononuclear cells and rheumatoid arthritis peripheral blood mononuclear cells induced by interleukin-6. Phosphorylation of STAT3 in cells, rheumatoid arthritis synovial mononuclear cells, rheumatoid arthritis synovial fibroblast cells (see Figure 12).

Example 15: Flow cytometric detection of the effect of antibodies on the phosphorylation of cellular STAT3

To detect intracellular STAT3 signal by flow cytometry, add 50ug/mL antibody to peripheral blood mononuclear cells of normal people, peripheral blood mononuclear cells of patients with rheumatoid arthritis and synovial mononuclear cells, incubate at 37°C for 1 hour, and then add Interleukin-6 (10ng/mL), incubated for 20 minutes, collected cells, washed twice, centrifuged to collect cells, membrane rupture and intracellular staining, detected by FACS Calibur instrument.

The results of flow cytometry showed that 50 μg/mL (10.51) anti-human interleukin-6 receptor β-chain antibody could inhibit normal human peripheral blood mononuclear cells, rheumatoid arthritis peripheral blood mononuclear cells, and rheumatoid arthritis induced by interleukin-6. Phosphorylation of STAT3 in arthritic synovial mononuclear cells, rheumatoid arthritis synovial fibroblast cells (see FIG. 13 ).

Claims (10)

1. A monoclonal antibody against the free interleukin-6 receptor β chain on the surface of human cells or serum, the cDNA sequence of the light chain variable region of the Fab fragment antigen-binding site of the antibody molecule is as shown in cDNA sequence No.1 The cDNA sequence of the heavy chain variable region is shown in cDNA sequence No.3. 2. The monoclonal antibody according to claim 1, the amino acid sequence of the light chain variable region of the Fab fragment antigen binding site of the antibody molecule is as shown in amino acid sequence No. 2 and the amino acid sequence of the heavy chain variable region is as shown in The amino acid sequence No.4 is shown. 3. The monoclonal antibody according to claim 1, characterized in that it is an immunoglobulin of the murine IgG subtype. 4. The use of the monoclonal antibody according to claim 1 in the preparation of drugs for diagnosis and interventional treatment of rheumatoid arthritis. 5. The use according to claim 4, said rheumatoid arthritis includes but is not limited to rheumatoid arthritis in different clinical early, middle or late stages including bone damage. 6. A detection kit for detecting cells and serum positive for interleukin-6 receptor beta chain antigen expression, characterized in that: it contains the interleukin-6 receptor beta bound to the cell surface or free in serum according to claim 1 chain monoclonal antibodies. 7. detection kit as claimed in claim 6, is characterized in that: also contain the mark that is combined with the monoclonal antibody of the interleukin-6 receptor beta chain of described anti-human cell surface or serum free, described The marker is any one of fluorescent markers, radioactive markers and enzyme-labeled markers. 8. A pharmaceutical composition, characterized in that it contains the monoclonal antibody against human cell surface or serum free interleukin-6 receptor beta chain according to claim 1. 9. The pharmaceutical composition according to claim 8, characterized in that, the pharmaceutical composition also contains a pharmaceutically acceptable carrier, excipient or mixture. 10. A detection kit for detecting interleukin-6 receptor beta chain antigen in peripheral blood, serum, synovial fluid, synovial fluid cells and tissue of damaged joints of patients with rheumatoid arthritis, characterized in that , comprising the monoclonal antibody of claim 1 binding to the interleukin-6 receptor beta chain free on the cell surface or serum.