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

Abstract

The present invention provides the monoclonal antibody of a kind of anti-human cell surface or the Interleukin-6 receptor β chains of free serum, the cDNA and amino acid sequence of the light chain variable region of the Fab section antigen-binding site of the antibody molecule are as shown in sequence No.1 and 2 and the cDNA of heavy chain variable region and amino acid sequence are as shown in sequence No.3 and 4.Purposes the present invention also provides the preparation method of the monoclonal antibody and its in the drug of detection and the diagnosing and treating for intervening rheumatoid arthritis.Inside and outside experiments have shown that, the monoclonal antibody of the present invention has the compatibility to interleukin 6 receptor β chain positive cell height and can regulate and control the molecular mechanism that patient with rheumatoid arthritis synovium ties up thin Interleukin-6 receptor β chains RANKL WNT5A signal paths by antagonism, reach rheumatoid arthritis patient immune function disorder and mediate the result of the unbalance caused patient articular's damage of bone metabolism.

Description

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

Technical field

The invention belongs to field of biological pharmacy more particularly to a kind of monoclonal antibody, a kind of specifically anti-human white Jie Plain -6 receptor β chain monoclonal antibodies and its preparation method and application.

Background technology

Molecular weight is 130kD, and outside is made of 6 continuous β lamellar structures domains, respectively immunoglobulin-like Structural domain (functional domain 1), CHR molecules (functional domain 2, functional domain 3) and 3 III spline structure domains (functional domain 4-6) of fibronectin, no Same structural domain plays a significant role in ligand connection and signal transduction.Interleukin-6 receptor β chain is as interleukin-6 man Race's cell factor --- interleukin-6, cytostatic factor (LIF), carcinophylin M (OSM), ciliary neurotrophic factor (CNTF), The co-receptor of interleukin-11, the myocardial nutrition factor 1 (cardiotrophin-1) and interleukin-27, by mediating different inflammation The signal transduction of the factor and participate in a variety of diseases, such as at rheumatoid arthritis (RA), huge lymphocytic hyperplasia disease, inflammation Property enteropathy, Huppert's disease, psoriasis, diabetes, asthma plays the part of important angle in the occurrence and development of diseases such as fat and tumour Color.With going deep into for research, people, which are gradually concerned about interleukin-6 receptor β chain associated signal paths, may become clinical immunization The novel targets intervened and treated.The international antibody meeting held in the U.S. in 2009 has researcher's report and proposes interleukin-6 The diagnosing and treating antibody of receptor β chain has a good application prospect, therefore either from ImmunopharmacologicaResearch or drug development Angle, for interleukin-6 receptor β chain in interleukin-6 receptor β catenary system, its respective ligand or signal transduction GAP-associated protein GAP Regulation and control analysis is all current research hotspot.

Interleukin-6 is one of the important ligand of interleukin-6 receptor β chain, with chronic inflammation disease, autoimmune disease The generation of sick and tumour etc. is closely related.Interleukin-6 receptor includes specific interleukin-6 receptor alpha chain (gp80) and interleukin- 6 receptor β chains (gp130).The expression of interleukin-6 receptor alpha chain is mainly limited to liver cell, and WBC sub-population, megacaryocyte etc. is carefully Cellular surface, and interleukin-6 receptor β chain is then expressed in all cell surfaces.Interleukin-6 passes through classical pathway and upside down signal way Diameter two ways is to intracellular transduction signal.Interleukin-6 is first combined with film connection interleukin-6 receptor alpha chain in classical pathway, it Induce interleukin-6 receptor β chain homodimerization afterwards, formed high-affinity interleukin-6/interleukin-6 receptor alpha/interleukin-6 by Body β chain complexs, to carry out signal transduction.The free interleukin-6 receptor alpha chain of soluble form is situated between with white in trans- approach Element -6 directly forms complex with interleukin-6 receptor β chain after combining, therefore connects interleukin-6 receptor alpha chain not expressing film Cell can still be reacted by interleukin-6.Researcher thinks that the presence of free interleukin-6 receptor alpha chain and upside down signal makes Interleukin-6, which becomes, participates in one of cell factor mostly important in inflammatory process, participates in various autoimmune disease and tumour Occurrence and development, especially mechanism of action of the interleukin-6 in rheumatoid arthritis is always the hot spot of clinical research.

Rheumatoid arthritis is a kind of autoimmune disease characterized by synovial hyperplasia and joint progressive destroy, Incidence in China is about 0.4%-1.0%, and Severe Rheumatoid Arthritis patient finally suffers from based on extremities joint Each joint major injury of whole body, disability rate are up to 15%.The specific aim of the drugs such as conventional formulation methotrexate (MTX), leflunomide is not By force, curative effect is usually not fully up to expectations.The new intervention means of immunology and the classic and clinical therapy are cooperateed with for suppression in recent years Generation, the development of rheumatoid arthritis processed play certain effect.Enter preclinical or clinical research treatment class wind now Wet arthritis related immune inhibitor can be divided mainly into two major classes：1) cell factor intervenes preparation：Mainly tumor necrosis factor Sub (TNF) blocking agent (adalimumab etc.), interleukin-6 receptor alpha chain blocking agent (the anti-TCZ in tower Xidan)；2) protein kinase inhibits Agent：Such as JAK inhibitor, SYK inhibitor.However due to the individuation difference of patient with rheumatoid arthritis, to existing biology Reactions vary for preparation, and most of patient still experienced develops from osteoarthritic inflammation to rheumatoid arthritis, finally suffers from tight Weight joint injury and disable.Therefore it there is an urgent need for the pathogenesis that we continue deeper into research rheumatoid arthritis, develops new Drug target.

Numerous studies show：There are close associations with rheumatoid arthritis for interleukin-6 signal system.Rheumatoid The concentration of interleukin-6 and free interleukin-6 receptor chain α increase in arthritic's serum and synovia, and interleukin-6 With the expression of disease activity sex factor (rheumatoid factor, erythrocyte sedimentation rate, c reactive protein), patient with rheumatoid arthritis clinic Performance (such as x-ray changes, affected joints quantity, the duration of morning stiffness) is closely related.It is treatment class wind to alleviate joint osteoclasia Wet arthritis patient's most critical issue, and it is considered as patient with rheumatoid arthritis Bones and joints damage that osteoclast ratio, which increases, Harmful key factor.The proliferation of related inflammation cytokine activation osteoclast precursor cells point in patient with rheumatoid arthritis Change, osteoclast generates ratio and increases, and causes patient bone metabolic imbalance, and interleukin-6 signal system regulates and controls to osteoclast In play an important role.

Existing research work proves：Inhibit the antagonist of interleukin-6 overexpression or interleukin-6 signal system exception that can have The immunologic derangement disease that regulation and control interleukin-6 in effect ground mediates.Clinical studies show, for the antagonism of interleukin-6 signal system Agent, to a certain extent can rheumatoid arthritis patient/rheumatoid arthritis sample animal model Development process.With Interleukin-6 is the antagonist (such as lokizumab) of target spot, since anti-interleukin-6 antibody combines free interleukin-6 cannot It is eliminated from body circulation, however side effect caused by interleukin-6 accumulation (nearly milligram is horizontal) occurs, limited always and hinder Hindered its wide clinical application.Anti- as the monoclonal antibody medicine tower Xidan of target spot using interleukin-6 receptor chain α is uniquely by facing The interleukin-6 signal path associated biomolecule preparation that the bed III phases test.In clinical trial, patient with rheumatoid arthritis uses tower After the treatment-resistant of Xidan, the notable duration of patient disease activity index, which can be observed, to be reduced, and local joint symptoms and sign improvement are bright Aobvious, the fast quick-recovery of c reactive protein is to normal level, and the patient invalid to some Drug therapies has high reaction Property, therefore be considered as effective treatment means of advanced rheumatoid arthritis patient.However further investigation finds that tower Xidan is anti- Classical signals and upside down signal are acted on simultaneously, has blocked the activity of interleukin-6 comprehensively, thus is also brought a series of Side effect, such as generation of infection, gpt level increases in blood, blood total cholesterol, high density lipoprotein level bletilla low density lipoprotein Protein level increases, and angiocardiopathy occurrence risk increases etc., also therefore hinders its extensive use clinically.It is another Using free Interleukin-6 receptor chain β as the monoclonal antibody preparation (sgp130) of target spot, since a large amount of aggregations can be formed in vivo, at present Only rest on experimental study.

In view of the monoclonal of multiple action and interleukin-6 receptor chain α of the interleukin-6 signal in rheumatoid arthritis Antibody (tower Xidan is anti-) is to the improvement result of TNF antagonist inerts patient, advanced rheumatoid arthritis patient, anti-human white Jie The development of plain -6 receptor chain β functional domain monoclonal antibodies is likely to become treatment patient with rheumatoid arthritis new tool.With interleukin-6 The conjugation sites of receptor β chain and its ligand it is further clear, and confirm interleukin-6 family different cytokines need to by with After respective peculiar receptor connection structural domain different from interleukin-6 receptor chain β again in conjunction with and function, anti-interleukin-6 The monoclonal antibody in receptor β chain different function domain has different biological functions, also makes to prepare interleukin-6 receptor β chain single The antagonist of ligand signal is possibly realized, and is expected to provide powerful to treat the research and development of monoclonal antibody medicine.It is above-mentioned in order to verify Problem in science, we establish rheumatoid arthritis sample mouse model (collagen by anti-collagen II functional domain antibody inductions Antibody induce arthritis, CAIA), the results showed that -6 receptor β chain monoclonal antibody of antihuman interleukin can inhibit model mice Disease development, maintain mouse bone balance etc.；Using the monoclonal antibody of anti-human/anti-mouse of mouse, it is analyzed for anti-human white Potency, Competitive assays, affinity and the life for different Antigenic Targets in interleukin -6 receptor β chain monoclonal antibody different function domain and epitope Object characteristic；The monoclonal antibody completed for anti-human/anti-mouse of mouse carries out full genome sequencing；Tentatively disclose above-mentioned monoclonal antibody tune Control immunopathology mechanism rheumatoid arthritis original mold type mouse invasion and lapsed to；Established rheumatoid is applied simultaneously The cell-mediated osteoclast precursor cells of arthritic's lesion synovial membrane synovioblast are to osteoclast differentiation technique, just Step illustrates the regulation and control patient with rheumatoid arthritis immunologic function disorder of -6 receptor β chain monoclonal antibody of antihuman interleukin and bone metabolism loses The essence of weighing apparatus shows effective biological activity of -6 receptor β chain monoclonal antibody of antihuman interleukin of present patent application and with intervention class wind The potential potential applicability in clinical practice of wet arthritis morbidity.

Invention content

In order to solve the problems, such as above-mentioned Science and Technology, the present invention provides a kind of anti-human cell surface or the lists of free serum Clonal antibody, the cDNA and amino acid sequence of the light chain variable region of the antibody molecule Fab section antigen-binding site are as follows, The cDNA and amino acid sequence of heavy chain variable region are as follows.

The cDNA and amino acid sequence of -6 receptor β chain monoclonal antibody molecule of the antihuman interleukin are as follows and right respectively Answer the sequence 1,2,3 and 4 in sequence table：

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 said monoclonal antibodies in the diagnosing and treating for detecting and intervening rheumatoid arthritis Purposes in drug.The present invention also provides the preparation methods of above-mentioned monoclonal antibody.By with U266 cells and foundation Quasi-wind gateway sample mouse model and patient with rheumatoid arthritis inside and outside it is demonstrated experimentally that purifying and degerming after it is above-mentioned (10.51) monoclonal antibody has the compatibility to positive cell height and can regulate and control patient with rheumatoid arthritis by antagonism The molecular mechanism of synovioblast interleukin-6 receptor β chain-RANKL-WNT5A accesses reaches alleviation rheumatoid joint The result of scorching patient immune function's disorder and the unbalance caused patient articular's damage of bone metabolism mediated.

Further, above-mentioned monoclonal antibody (10.51) is a kind of immunoglobulin of mouse IgG hypotype.

The present invention also provides above-mentioned (10.51) monoclonal antibodies in diagnosis and Clinical intervention quasi-wind gateway drug Purposes.

Further, the rheumatoid arthritis includes but is not limited to different clinical early stages, mid-term or late period (including bone injury) patient with rheumatoid arthritis.

A kind of detection method is further provided, it is characterised in that：Anti-human cell surface or free expression interleukin-6 The marker that the monoclonal antibody of receptor β chain (10.51) combines, the marker are fluorescent marker or radioactivity mark Remember object or enzyme mark marker.

The monoclonal antibody of a kind of combination human cell surface or the free interleukin-6 receptor β chain expression positive can With in rheumatoid joint peripheral blood in patients, serum, synovial fluid combines in the tissue of synovial fluid cell and injured joint.

A kind of preparation side of the monoclonal antibody of the combination human cell surface or free interleukin-6 receptor β chain Method, it is characterised in that：Mouse is immunized using commercial standard human interleukin-6 receptor β chain multiple spot, normal immunological adds with primary three times Mouse spleen cells after being immunized by force are merged with murine myeloma cell SP2/0 by PEGylated, and energy can be secreted out of by filtering out Enough combine above-mentioned interleukin-6 receptor β chain antigen, the hybridoma cell strain (experiment numbers that the surface antigen of positive cell is combined 10.51), after this hybridoma cell strain is subcloned, to obtain cultivated hybridoma supernatant, through affinity purification to obtain the final product To the monoclonal antibody of the present invention.If by that can develop for rheumatoid after the monoclonal antibody human/mouse is chimerization or humanization The biological agent of property arthritic's diagnosing and treating.

Description of the drawings

Fig. 1 shows indirect enzyme-linked immunosorbent assay (ELISA) to the mice serum after interleukin-6 receptor β chain is immune And the monoclonal antibody of -6 receptor β chain of antihuman interleukin obtained after purification (is produced by the hybridoma cell strain of experiment numbers 10.51 Raw, the monoclonal antibody of the present invention being mentioned herein is herewith) it is reacted with antigen interleukin-6 receptor β chain titre.

Fig. 2 shows that flow cytometry (FCS) is anti-to (10.51) monoclonal of not -6 receptor β chain of homophyletic antihuman interleukin Body is reacted with U266 cell surface interleukin-6 receptor β chain combinations.

Fig. 3 is that the monoclonal antibody of the present invention of various concentration is reacted with the interleukin-6 receptor β chain combination of U266 cell strains surface FACS is detected；Wherein, A is Isotype control；A concentration of 10ug/mL of monoclonal antibody of the present invention in B；A concentration of 5ug/ of monoclonal antibody of the present invention in C mL；A concentration of 2.5ug/mL of monoclonal antibody of the present invention in D；A concentration of 0.5ug/mL of monoclonal antibody of the present invention in E；F irrelevant antibodies.

Fig. 4 is that interaction of biomacromolecules analyzer (biacore X100) analysis 3-2.3 and 10.51 monoclonal antibodies are situated between with white The binding curve of plain -6 receptor β chains and affinity detection.

Fig. 5 is potency identification detection after the monoclonal antibodies such as elisa assay (10.51) label HRP.

Fig. 6 is that competition indirect enzyme-linked immunosorbent assay is analyzed, in the presence of the competition antibody of various concentration, horseradish peroxidase The binding ability of (10.51) monoclonal antibody and interleukin-6 receptor β chain antigen of enzyme (HRP) label.

Fig. 7 is the regulating and controlling effect of interleukin-6 various dose and induction time to U266 cell STAT3 phosphorylations.

Fig. 8 is Western of the immune serum to the U266 cell STAT3 phosphorylations of regulation and control interleukin-6 induction Blot is detected.

Fig. 9 is U266 cell of -6 receptor β chain (10.51) monoclonal antibody of antihuman interleukin to regulation and control interleukin-6 induction The Western Blot detections of STAT3 phosphorylations.

Figure 10 is immune serum and -6 receptor β chain (10.51) monoclonal antibody of antihuman interleukin to regulating and controlling interleukin - 6, the Western Blot detections of the U266 cell STAT3 phosphorylations of free interleukin-6 receptor alpha chain co-induction.

Figure 11 is indirect enzyme-linked immunosorbent assay to rheumatoid arthritis/osteoarthritis (OA) patient interleukin-6/white - 6 receptor alpha chains of interleukin/interleukin-6 receptor β chain expression and its ratio detection.

Figure 12 is that -6 receptor β chain (10.51) monoclonal antibody of antihuman interleukin is thin to the regulation and control single core of normal human peripheral blood Born of the same parents, peripheral blood mononuclear cells of rheumatoid arthritis patients, patient with rheumatoid arthritis synovial membrane mononuclearcell, rheumatoid Property arthritic's synovioblast cell interleukin-6 induction normal cell STAT3 phosphorylations Western Blot is detected.

Figure 13 is that the monoclonal antibody of -6 receptor β chain (10.51) of Flow cytometry antihuman interleukin is normal to regulating and controlling Human peripheral blood single nucleus cell, rheumatoid arthritis peripheral blood mononuclear cells, the single core of rheumatoid arthritis synovial are thin The effect of the normal cell STAT3 phosphorylations of the interleukin-6 induction of born of the same parents.

Following embodiment is only that the present invention is furture elucidated, has no the meaning for limiting the invention to the specific embodiment Figure.One skilled in the art would recognize that present invention encompasses in Claims scope all possible alternative, Improvement project and equivalent scheme.

Specific implementation mode

Following example use reagent and experimental apparatus be this field ordinary articles, can buy on the market or Person can be obtained by regular approach.

Embodiment 1：Antibody preparation：

2 male BALB/c mouses are chosen, interleukin-6 receptor β chain antigen is mixed with Freund's complete adjuvant, has been emulsified Full pneumoretroperitoneum injection, the immunizing dose of every mouse are 20ug, 2 weeks and one month after first immunisation, are repeated to be immunized twice, the After 3 times immune l weeks, mice serum, indirect enzyme-linked immunosorbent assay is taken to identify the combination of serum and interleukin-6 receptor β chain antigen Ability, normal BABL/c mice serums select the higher mouse of serum titer, carry out merging preceding booster immunization as negative control (being only injected intraperitoneally).Previous week is merged, with MD6 culture solution mass propgation myeloma cell SP2/0, makes cell growth state Well and it is in exponential phase, immune BALB/c mouse spleen is taken out in fusion same day sterile working, and grinding becomes slender Born of the same parents' suspension counts, spare.Later by splenocyte and SP2/0 according to 1:1 ratio mixing, addition polyethylene glycol (PEG), several points After clock be added MD6 terminate, centrifugation, with containing HAT (H-Hypoxanthine hypoxanthine, A-Aminopterin methopterins, T --- Thymidine thymidines) MD6 culture solutions be resuspended, be added in 96 orifice plates, per hole 200ul, cell number is about It is the every holes 2x104/, totally 50 blocks of plates, are cultivated 7-10 days in HAT selectivity culture solutions.Cell in 96 orifice plate of microscopically observation Growth conditions indirect ELISA is carried out using 96 boreliquid processing systems and Full-automatic plate-washing, liquid separation system if Colony forming High throughput intersects screening, selects the higher hole of positive value, and sub- to the positive hybridoma cell progress detected through limiting dilution Clone identification selects the high hole of positive value and expands culture and carry out cell cryopreservation.By the positive be worth higher hybridoma according to 6~10 × 106 cells are inoculated with per 150ml hybridizing tumour cell non-serum culture mediums, culture is enlarged 10~14 days, waits for 90% After above cell death, cell conditioned medium is collected, 3500 turns, 4 DEG C centrifuge 15 minutes, take supernatant, and Proclin 300 (1 is added： 2000) after mixing 4 DEG C save backup.

Embodiment 2：The titration of -6 receptor β chain monoclonal antibody of antihuman interleukin

- 6 receptor β chain monoclonal antibody of antihuman interleukin is measured (by experiment numbers with indirect enzyme-linked immunosorbent assay 10.51 hybridoma cell strain generates) potency：With 96 orifice plate of interleukin-6 receptor β chain antigen coat of 0.1ug/ml, per hole 100ul is added, is placed in wet box and is coated with overnight, washed 2 times, be used in combination with the phosphate buffer (PBST) containing 0.05% polysorbas20 PBST room temperatures containing 1.5% bovine serum albumin(BSA) (BSA) are closed 1 hour, are diluted purified interleukin-6 receptor β chain antigen and are exempted from Mice serum after epidemic disease is added in plate, and 100ul is added per hole, is incubated at room temperature 1 hour, is washed 4 times with PBST.Horseradish mistake is added Sheep anti-mouse igg-Fc the secondary antibodies of oxide enzyme label, 100ul is added per hole, is incubated at room temperature 1 hour, and PBST is used in combination to wash 4 times.It 3,3', 5,5'- tetramethyl benzidine (TMB) Color Appearance System 150ul of substrate is added afterwards to develop the color 15-30 minutes, with sulfuric acid (H₂SO₄) It terminates, reads absorbance value in 30 minutes at 450nm.

The result shows that：No. 2 relatively high mice serums of immunoreactivity are reacted with the titre of interleukin-6 receptor β chain combination It is higher, the fusion experiment of the monoclonal antibody hybridoma generation for -6 receptor β chain of antihuman interleukin that this mouse boosting cell is used for.(such as Fig. 1 institutes Show).

Embodiment 3：The subgroup identification of monoclonal antibody

- 6 receptor β chain of antihuman interleukin is purified using the affinity column standard reagent box of Pall companies (U.S.) (10.51) monoclonal antibody, monoclonal antibody are concentrated through ultrafiltration membrane, MICON 50ml dialysis ultrafiltration apparatus and PBS dialysis rich in liquid, are finally used NANODROP 2000 measures the concentration of IgG containing monoclonal antibody solution after dialysis, is preserved after 0.22 μm of millipore filter filters. Monoclonal antibody after purification identifies antibody subtype using Roche companies mouse monoclonal antibody hypotype Rapid identification kit, identified Antihuman interleukin -6 receptor β chain (10.51) monoclonal antibody heavy chain belongs to IgG2b, and light chain is κ chains.

Embodiment 4：Sequencing

With the RNeasy kits of Qiagen (Valencia, California, USA) from (10.51) monoclonal antibody hybridoma cell strain Extracted total RNA, with the SuperScript III First-Strand of Invitrogen (Grand Island, New York, United States) Kit is by mRNA reverse transcriptions at the cDNA library of 10.51 monoclonal antibodies.Utilize the Progen Biotechnik companies of Germany " Mouse 23 primers of IgG Library Primer Set " (F2010) kit carry out 21 PCR reactions and (do not include the anti-of lambda light chain Answer), generated special light and heavy chain product carries out DNA sequencing, the translation of amino acid polypeptide sequence and CDRs (epiopes Region) and FW (backbone region) identification.

Sequence in the cDNA of the light chain variable region of antibody molecule Fab section antigen-binding site and amino acid sequence such as sequence table Shown in 1 and 2, in the cDNA and amino acid sequence such as sequence table of heavy chain variable region shown in sequence 3 and 4, wherein cDNA and amino acid Sequence is reciprocity.Underscore annotated sequence in amino acid sequence is the position for showing CDR region domain, is by CDR1, CDR2 It is ranked sequentially with CDR3 and regional sequence between them is skelemin sequence (FW).

Embodiment 5：Antibody is measured with antigen-binding affinity

- 6 receptor β chain (10.51) monoclonal antibody of antihuman interleukin after affinity purification illustrates described indirect enzyme-linked through embodiment 2 Immunoabsorption is tested, and is combined staining reaction to (10.51) hybridoma cell strain, and OD values are read in microplate reader.Its Its experimental procedure is same as Example 2.

As a result indirect enzyme-linked immunosorbent assay confrontation human interleukin-6 receptor β chain (10.51) monoclonal antibody and interleukin-are shown The association reaction of 6 receptor β chain antigens, (10.51) reactivity highest (as shown in Figure 1).

Embodiment 6：Monoclonal antibody is detected with U266 cell strain binding affinities

The monoclonal antibody of -6 receptor β chain (10.51) of antihuman interleukin after affinity purification is through flow cytometer detection antibody and cell surface The binding ability of interleukin-6 receptor β chain.Every group is closed 30 points with 3x105 U266 cell, with 4 degree of the PBS containing 1.5%BSA - 6 receptor β chain monoclonal antibody of antihuman interleukin of various dose is added in Zhong Hou, and 4 degree are incubated 60 minutes.After being washed with PBS, add Enter the sheep anti mouse secondary antibody of FITC labels, 4 degree are protected from light incubation 30 minutes.1% paraformaldehyde 100ul is added after 2 washings, is resuspended thin Born of the same parents, through being detected with FACS Calibur instrument.

As a result streaming confrontation human interleukin-6 receptor β chain (10.51) monoclonal antibody and interleukin-6 receptor β chain antigen knot are shown Reaction is closed, (10.51) antibody and the binding ability of U266 cell surface interleukin-6 receptor β chains are consistent with 5 result of embodiment (such as Shown in Fig. 2).

Embodiment 7：(10.51) specificity and dose dependent of monoclonal antibody

(10.51) monoclonal antibody after affinity purification passes through the flow cytometer detection method illustrated with embodiment 7, to various concentration (10.51) staining reaction is combined to U266 cells, and reads average fluorescent strength (MFI) on FACS Calibur instrument Value.

FACS results show (10.51) monoclonal antibody can with U266 cell surface interleukin-6 receptor β chains specifically bind and Dose dependent is presented, wherein in Fig. 3, A is Isotype control；A concentration of 10ug/mL of monoclonal antibody of the present invention in B；The present invention is single in C Resist a concentration of 5ug/mL；A concentration of 2.5ug/mL of monoclonal antibody of the present invention in D；A concentration of 0.5ug/mL of monoclonal antibody of the present invention in E；F is unrelated Antibody (as shown in Figure 3).

Embodiment 8：Interaction of biomacromolecules analyzer analyzes affinity of antibody and binding kinetics

(10.51) anti-interleukin-6 is detected with GE companies interaction of biomacromolecules analyzer (biacore X100) The affinity and binding kinetics of receptor β chain monoclonal antibody.It regard sheep anti mouse Fc antibody (25 μ g/ml) as capture molecule first It is coupled on CM5 chip gold thin films surface, using -6 receptor beta chain antibody of (10.51) antihuman interleukin of preparation as ligand, later The concentration for going out required -6 receptor β chain monoclonal antibody of (10.51) antihuman interleukin according to formula to calculating carries out loading (19 μ g/ Ml contacts 180s), operation obtains the signal curve of monoclonal antibody and interleukin-6 receptor β chain antigen binding, with Langmuir models pair Curve is fitted, and is analyzed experimental result, is obtained the affinity constant of the monoclonal antibody.

The result shows that (10.51) monoclonal antibody and the affinity of antigen binding are 2.62E-10 (as shown in Figure 4).

Embodiment 9：The antigen binding site specificity of competitive indirect enzyme-linked immunosorbent assay detection antibody

It is that antibody is dissolved in carbonate buffer solution to carry out horseradish peroxidase-labeled to antibody, rear that peroxide containing horseradish is added The reaction solution of compound enzyme, and under the action of NaBH4, by horseradish peroxidase-labeled to antibody, finally utilize phosphate Buffer solution dialysis and concentrated antibody solution.

Enzyme linked immunosorbent assay detects between carrying out antibody：With 96 hole of interleukin-6 receptor β chain antigen coat of 0.1ug/ml Plate, coating overnight, are closed 1 hour after washing 2 times with the PBST room temperatures containing 1.5%BSA.It is added 1:3000 diluted label horseradishes The unlabelled monoclonal antibody that this liquid diluting is to be detected is used in combination in (10.51) monoclonal antibody of peroxidase, to be detected Antibody initial concentration is 100 μ g/ml, 1：3 gradient dilutions are added in ELISA Plate, per hole 100ul, using unlabelled antibody as Own control is incubated at room temperature 1 hour using dilution as unrelated control；PBST is washed 4 times；Tmb substrate Color Appearance System is added 150ul develops the color 15-30 minutes, is terminated with H2SO4；With in microplate reader at 450nm read absorbance value.

After indirect enzyme-linked immunosorbent assay result shows the monoclonal antibody label HRP of -6 receptor β chain of (10.51) antihuman interleukin Potency identification (as shown in Figure 5).Competitiveness enzyme-linked immunoabsorption between antibody the result shows that, the antigen site of multi-strain antibody is special Anisotropic difference (as shown in Figure 6).

Embodiment 10：Interleukin-6 stimulates U266 cell experiments

Carry out the U266 cell Stimulation Assays of 3 multiple holes of each condition, U266 Nature enemies 24 hours, interleukin-6 induction Group：It is separately added into the interleukin-6 of various concentration (0ng/ml, 1ng/ml, 2ng/ml, 5ng/ml, 10ng/ml, 20ng/ml), is pierced Swash different time (5min, 10min, 20min, 30min, 60min), collect cell, be added precooling containing protease inhibitors Supernatant is collected by centrifugation in lysate, ice bath 30min, after carrying out protein quantification to supernatant, carries out protein adhesive loading (per hole 20-50 μ G), the phosphorylation of the intracellular STAT3 of Western Blot experimental analyses U266 and interleukin-6 stimulation time and dosage are utilized Relationship.

Western Blot testing results show that the interleukin-6 of various concentration stimulates U266 cell 15min, all can induce The phosphorylation of the downstreams U266 STAT3, and phosphorylation degree and interleukin-6 concentration are in dose-dependence.It is white with 10ng/mL Interleukin -6 stimulates U266 cell different times, the STAT3 phosphorylations that experiment discloses interleukin-6 induction to have instantaneity, thorn respectively Sharp 20min has the maximum phosphorylation degree (as shown in Figure 7) of induction.

Embodiment 11：Western Blot detection immune serums and STAT3 phosphorylations

Such as the step of experimental example 10, in the U266 cells by Nature enemy, the immune mouse blood of various concentration is added (being control with Normal Mouse Serum) clearly, 37 DEG C are incubated 1 hour, and interleukin-6 (1ng/mL), which is then added, stimulates 15min, utilizes The variation of Western Blot experiment detection STAT3 phosphorylations, to which indirect analysis immune serum is to IL-6 downstream signals Regulating and controlling effect.

Western Blot testing results show immune serum 1:500 dilution can obviously inhibit U266 into the cell by The phosphorylation of the STAT3 of interleukin-6 induction, has hypersensitivity (as shown in Figure 8).

Embodiment 12：Regulation and control of the monoclonal antibody to interleukin-6 downstream STAT3 phosphorylations

- 6 receptor beta chain antibody of 50 μ g/mL (10.51) antihuman interleukin to the stimulation test such as embodiments 10 of U266 cells, Western Blot experiment detection STAT3 phosphorylation levels variations, to the monoclonal antibody of -6 receptor β chain of indirect proof antihuman interleukin To the regulating and controlling effect of interleukin-6 downstream signal.

As a result show that (10.51) have regulating and controlling effect, energy one to -6 downstream STAT3 phosphorylations of U266 interleukins Determine the STAT3 phosphorylations (as shown in Figure 9) that degree inhibits the U266 cells induced by interleukin-6.Such as free interleukin-6 receptor The phosphorylation of α chains, STAT3 slightly enhances, and -6 receptor beta chain antibody of (10.51) antihuman interleukin is added and also can inhibit interleukin-6, The phosphorylation of the U266 cells STAT3 of free interleukin-6 receptor chain α combined inductions discloses (10.51) monoclonal antibody for interleukin- The classical signals and upside down signal of 6 inductions have regulating and controlling effect as shown in Figure 10).

Embodiment 13：Patient with rheumatoid arthritis interleukin-6/free interleukin-6 receptor alpha chain/free interleukin-6 The detection of receptor β chain content

Cooperate with Guanghua Combined Traditional Chinese ＆. Western Medicine Hospital, Changning De, through Ethics Committee (ethics meeting file is shown in annex) batch It is accurate and " patient's informed consent form " is signed by patient or family members, select qualified rheumatoid arthritis to suffer from according to standard Person.Experimental group is other than meeting the basic standard of table 2-1, it is necessary to meet following condition：1. hemoglobin >=5.5mmol/L； 2. leucocyte >=3.5 × 109/L；3. serum creatinine≤150 μm ol/L；4. blood platelet >=100 × 109/L；5. serum bilirubin, Glutamic-pyruvic transaminase, alkaline phosphatase, glutamic-oxalacetic transaminease are at 1.5 times or less of Upper Limit of Normal Value.Collection patients blood plasma, synovial fluid, Synovial tissue.

According to R＆D companiesThe operating procedure of ELISA sandwich method detection kits, detection normal healthy controls, OA Interleukin-6, free interleukin-6 receptor alpha chain, free white Jie in the serum and synovia of control and patient with rheumatoid arthritis The level of plain -6 receptor β chains, microplate reader detect absorbance (450nm).

Indirect enzyme-linked immunosorbent assay is the result shows that interleukin-6 expression quantity is notable in patient with rheumatoid arthritis synovia Interleukin-6 receptor alpha chain is significantly higher than OA groups in expression quantity and rheumatoid arthritis synovial liquid in higher than same group serum, and class The free interleukin-6 receptor β chain expression of the natural agonist of interleukin-6 signal system is insufficient in rheumatic arthritis serum, bright It is aobvious to be less than OA control groups, Normal group.The further free interleukin-6 receptor alpha chain of more pairs of sample/free interleukin- 6 receptor β chain ratios find compared with OA groups and Normal group there is trip in rheumatoid arthritis synovial liquid and serum It is increased extremely from interleukin-6 receptor alpha chain/free interleukin-6 receptor β chain ratio, prompts patient with rheumatoid arthritis free Interleukin-6 receptor alpha chain/free interleukin-6 receptor β chain out-of-balance conditions, and synovial fluid is serious (refering to fig. 1 1) compared with serum.

Embodiment 14：The effect of the phosphorylation of Western Blot detection antibody on cell STAT3

Method such as embodiment 11, testing result show that -6 receptor beta chain antibody of 50 μ g/mL (10.51) antihuman interleukin can press down Make PBMC of healthy people, rheumatoid arthritis peripheral blood mononuclear cells, the class wind induced by interleukin-6 Wet arthritis synovial membrane mononuclearcell, rheumatoid arthritis synovial fibroblast cell STAT3 phosphorylation (refering to Figure 12).

Embodiment 15：The effect of the phosphorylation of flow cytometer detection antibody on cell STAT3

Flow cytometer detection detects intracellular STAT3 signals, in PBMC of healthy people, patient with rheumatoid arthritis 50ug/mL antibody is added in peripheral blood mononuclear cells and synovial membrane mononuclearcell, 37 DEG C are incubated 1 hour, and white be situated between then is added - 6 (10ng/mL) of element are incubated 20 minutes, collect cell, after washing twice, cell is collected by centrifugation, and are dyed through rupture of membranes and intracellular, are used FACS Calibur instrument is detected.

Streaming is the result shows that -6 receptor beta chain antibody of 50 μ g/mL (10.51) antihuman interleukin can inhibit and be induced by interleukin-6 PBMC of healthy people, rheumatoid arthritis peripheral blood mononuclear cells, rheumatoid arthritis synovial list A nucleus, rheumatoid arthritis synovial fibroblast cell STAT3 phosphorylation (refering to fig. 1 3).

Claims (10)

1. a kind of monoclonal antibody of the interleukin-6 receptor β chain of anti-human cell surface or free serum, the antibody molecule The cDNA sequence of the light chain variable region of Fab section antigen-binding site is as shown in cDNA sequence No.1 and the cDNA sequences of heavy chain variable region Row are as shown in cDNA sequence No.3.

2. monoclonal antibody as described in claim 1, the light chain variable region of the Fab section antigen-binding site of the antibody molecule Amino acid sequence is as shown in amino acid sequence No.2 and the amino acid sequence of heavy chain variable region is as shown in amino acid sequence No.4.

3. monoclonal antibody as described in claim 1, it is characterised in that：It is a kind of immunoglobulin of mouse IgG hypotype.

4. use of the monoclonal antibody described in claim 1 in preparing diagnosis and Clinical intervention medicine for treating rheumatoid arthritis On the way.

5. purposes as claimed in claim 4, the rheumatoid arthritis include but is not limited to different clinical early stages, Mid-term or late period include the rheumatoid arthritis of bone injury.

6. a kind of detection kit for detecting the cell and serum of the interleukin-6 receptor β chain antigen presentation positive, feature It is：The monoclonal antibody of interleukin-6 receptor β chain containing combination cell surface described in claim 1 or free serum.

7. detection kit as claimed in claim 6, it is characterised in that：Also contain and the anti-human cell surface or serum The marker that the monoclonal antibody of free interleukin-6 receptor β chain combines, the marker are fluorescent marker, radioactivity Any one in marker and enzyme mark marker.

8. a kind of pharmaceutical composition, which is characterized in that white containing anti-human cell surface described in claim 1 or free serum The monoclonal antibody of -6 receptor β chain of interleukin.

9. pharmaceutical composition as claimed in claim 8, which is characterized in that described pharmaceutical composition also contains pharmaceutically acceptable Carrier, excipient or intermixture.

10. a kind of for detecting the peripheral blood in rheumatoid joint patient, serum, synovial fluid, synovial fluid cell and damage The detection kit of interleukin-6 receptor β chain antigen in the tissue in joint, which is characterized in that containing described in claim 1 The monoclonal antibody of the interleukin-6 receptor β chain of combination cell surface or free serum.