CN104004080A - Humanized antibody targeted to RANKL (Receptor Activator Of Nuclear Factor Kappa B Ligand) and TNF-alpha (Tumor Necrosis Factor) and application of humanized antibody - Google Patents
Humanized antibody targeted to RANKL (Receptor Activator Of Nuclear Factor Kappa B Ligand) and TNF-alpha (Tumor Necrosis Factor) and application of humanized antibody Download PDFInfo
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Abstract
The invention relates to a humanized antibody targeted to RANKL (Receptor Activator Of Nuclear Factor Kappa B Ligand) and TNF-alpha (Tumor Necrosis Factor) and an application of the humanized antibody. The antibody provided by the invention is obtained by carrying out humanized modification on a region V of a murine monoclonal antibody aiming at RANKL-TNF sample region epitope through a CDR (Complementarity Determining Region) replacement technology. The invention further provides an expression vector for expressing the humanized antibody targeted to RANKL and TNF-alpha and a construction method of the expression vector. The humanized antibody provided by the invention can effectively neutralize TNF-alpha in vitro, inhibit apoptosis caused by TNF-alpha, neutralize RANKL and inhibit generation of osteoclast and meanwhile can antagonize inflammations caused by TNF-alpha and bone destructive effect caused by RANKL. The humanized antibody provides a base for research on novel biological preparations for treating rheumatoid arthritis.
Description
Technical field
The present invention relates to genetically engineered and field of immunology, particularly, relate to target in RANKL and TNF-α humanized antibody and application thereof.
Background technology
Rheumatoid arthritis (rheumatoid arthritis, RA) is a kind of common autoimmune disorder that chronic, carrying out property, aggressive sacroiliitis be main manifestations of take.Often can affect the many histoorgans of whole body, especially with joint, involve the most common.Persistence synovitis, synovial tissue's paraplasm that can show as the joint of getting involved, the damage of the formation of massive inflammatory cells infiltrated, pannus and bone, cartilage, ligament, finally causes joint deformity, dysfunction, even disables.
The pathology of RA is extremely complicated, does not also illustrate completely at present.But a large amount of research shows in the pathologic process of RA, the immune inflammation reaction of proinflammatory factor mediation is its important link.The biological action of TNF-α has all obtained research widely in laboratory animal and human body, and particularly it all shows obvious high expression level in the relevant many diseases of inflammation.TNF-α is a polyphenic cytokine, has mediated biological action widely, and in the whole pathologic process of RA, it also becomes an important target spot and be extensively concerned.In RA patient, TNF-α is except show as the same function showing in various diseases type, as mediated cell raise, breed and dead, and outside immunoregulatory effect, it also shows some special biological functions, as the generation of induction osteoclast.In the pathologic process of the autoimmune diseases such as RA, TNF-α plays an important role for the adjusting of pathologic inflammation waterfall stream, as it can induce rapidly inflammatory factor IL-1 β, IL-6, IL-17, and acute inflammation albumen---the generation of c reactive protein.TNF-α is bringing into play and is acting in inflammatory cell and the coefficient complex network of inflammatory factor at one, in this complex network, TNF-α is an early stage and important node, it triggers and has mediated the mechanism in downstream, and by various positive-negative feedback, is controlling generation and the development of inflammation pathology.In RA patient, the inflammation of synovium of joint has increased the supply of blood flow and the infiltration of inflammatory cell.Scavenger cell is the main source of TNF-α in synovial membrane inflammation, and its pathomechanism by contacting between cell and cell, cytokine, immunocomplex, complement, infected by microbes, endogenic ligand (Toll sample acceptor), anoxic affects the generation of TNF-α.In the process of whole disease, synovial tissue can continue to detect the expression of TNF-α.In the transgenic mice of high expression level TNF-α,, there is spontaneous arthritic performance in high expression level IL-1 β, IL-6 in its joint, has occurred being similar to clinical manifestation and the histo pathological change of RA.Cultivate in vitro in the process of RA patient synovial tissue, in and TNF-α can effectively suppress IL-1 and other cause the generation of the inflammatory factor of RA pathologic process.Also proof in the research of collagen-induced sacroiliitis mouse model, by and TNF-α or check its membrane receptor, for the improvement of disease, all there is obvious effect.Just be based in experimental model about the effect of TNF-α and checking the effect that it shows, make people start to be devoted to the monoclonal antibody research for TNF-α, being wherein applied to the earliest clinical is its a kind of human mouse chimeric antibody---Ying Fulixi (infliximab).Occurred based on this afterwards a large amount of correlation researchs, result all shows the application of anti-TNF-α antibody, can effectively alleviate the disease activity degree of RA, improves patient's symptom and sign.At present the TNF-alpha-2 antagonists one in the U.S. and European Union's listing has 5 kinds, is respectively Ying Fulixi (infliximab), etanercept (etanercept), adalimumab (adalimumab), matches appropriate Zhu's monoclonal antibody (certolizumab) and usury monoclonal antibody (golimumab).But in clinical study, find, although for the antagonism of TNF-α, can effectively alleviate synovial membrane inflammation and the arthroncus showing in RA process, its provide protection for the joint cartilage accompanying and osteoclasia corresponding a little less than.
Research now shows, osteoclast plays an important role in the bone of rheumatoid arthritis and the pathologic process of cartilage destruction, in RA clinical patients or experimental model, all can find the generation that the quantity increase of osteoclast and activation have directly caused bone loss pathologic reaction.And the acting in conjunction of the part (RANKL) of NF-kB activation factor acceptor, osteoprotegerin (OPG), NF-kB activation factor acceptor (RANK) is regulating osteoclast to raise and activate.RANKL is TNF superfamily member, and at the T cell surface expression of stroma cell, scleroblast or the activation of marrow, it can be combined with the RANK on scavenger cell or Meloid progenitor surface, and induction the latter is divided into ripe osteoclast.In the culture system in vitro of scavenger cell or Meloid progenitor, the exogenous restructuring RANKL that adds also can induce the generation of osteoclast.As knocked out RANKL or RANK gene in Mice Body, there will be osteoclast defect to cause the generation of osteopetrosis, also show that RANK and RANKL are the keys that osteoclast generates.OPG is the antagonist of RANKL, as it lacks in vivo, to the antagonistic action of RANKL, will weaken thereupon, and the quantity that the osteoclast of activation produces will increase thereupon, thereby occur osteoporosis clinically.If changing has appearred in RANK and OPG in visible body, the disorder of bone metabolism will be caused, simultaneously, the inflammatory molecule that the TNF-α of take is main representative respectively with its acceptor interaction after, destroyed the microenvironment of bone remodeling, and then making the RANKL up-regulated on scleroblast, the RANK on osteoclast is combined, and has activated the signal transduction pathway of osteoclast, make osteoclast abnormality proliferation, activation, further make inflammation increase the weight of to continue with osteoclasia.
Target is in the application clinically of the biotechnological formulation of TNF-α and RANKL, changed RA patient's clinical treatment situation, nowadays the Di Nuosaimai of the adalimumab of TNF alpha antibody short of money, infliximab, etanercept and antagonism RANKL effect, successively put into clinical in and obtained good result for the treatment of.But the synovial membrane inflammation occurring for RA and the destruction of bone of following, a kind of biology preparation of single application all can not be solved completely.
In view of illustrating of RANKL-RANK and TNF-signal α Signal Transduction Pathways crosstalk mechanism, and the dependency of RANKL and TNF-α and the main pathological change of RA, the effect that the present invention's imagination suppresses RANKL and TNF-α simultaneously may more be conducive to control RA disease progression.Based on RANKL, be TNFSF member, core texture is 158 amino acid of the outer carboxyl terminal of born of the same parents, forms WeiTNF family homology structural domain by several β types are folding.The candidate epi-position of the high region of the amino acid sequence similarity of RANKL TNFYang district and TNF-α as antigen usingd in the present invention, select corresponding prokaryotic expression system, build expression of recombinant proteins carrier, and recombinant protein is expressed, by lot of experimental data, verified, this albumen stimulates the osteoclasia that the polyclonal antibody inflammation that TNF alpha antibody short of money causes simultaneously that body produces and RANKL cause and then reduced collagen-induced mouse arthritis sickness rate has alleviated disease degree.By monoclonal antibody triage techniques, obtained the specific mouse monoclonal antibody of a plant height, can be effectively the biological action of TNF alpha antibody short of money and RANKL simultaneously, alleviate the inflammatory reaction that occurs in lysis and the pathologic process of osteoclasia simultaneously.But due to the species specificity of mouse monoclonal antibody, can make it in the application process of mankind's rheumatoid arthritis disease clinical treatment, be restricted.Therefore need the antibody of a kind of humanized TNF alpha antibody short of money simultaneously of research and development and RANKL badly and it is reasonably applied in the treatment of mankind's inflammatory bone disease.
Summary of the invention
In order to solve the problems of the technologies described above, the object of the invention is to provide a kind of target in the Humanized monoclonal antibodies of RANKL and TNF-α.
Another object of the present invention is to provide expression vector of expressing said monoclonal antibody and preparation method thereof.
Another object of the present invention is to provide the application of said monoclonal antibody in preparation treatment inflammatory bone disease medicine.
For achieving the above object, the V district that the present invention is directed to the mouse resource monoclonal antibody (its preparation method is open in Chinese patent CN 103060274B) in contriver's early-stage Study achievement RANKL-TNF sample district is undertaken humanization modified by CDR replacement technology, retaining under the prerequisite of its specific function, realize the structure of recombinant humanized antibody, prepared target in the humanized antibody of TNF-α and RANKL, and its biological characteristics and function have been identified.
Target provided by the invention is in the humanized antibody of RANKL and TNF-α, and its light-chain amino acid sequence is as shown in SEQ ID NO.1, and its heavy chain amino acid sequence is as shown in SEQ ID NO.2.
Particularly, the present invention adopts CDR replacement technology to prepare target in the humanized antibody of TNF-α and RANKL, comprises the steps:
(1) clone of mouse resource monoclonal antibody variable region cDNA sequence and definite: extract the total RNA of hybridoma of the two target spot mouse of secretion source antibody, RT-PCR technology obtains the cDNA sequence of antibody variable region, mouse source;
(2) design of humanized antibody variable region and synthetic: choose respectively human antibody heavy chain that homology is the highest, light chain skeleton as an alternative, the skeleton district of antibody CDR district, mouse source and human antibody is spliced, complete the replacement of mouse source CDR;
(3) structure of recombinant humanized antibody expression vector: the humanized antibody heavy chain that above-mentioned structure completes, the sequence of light chain are spliced mutually with the expression vector that carries people's IgG antibody 1 and κ chain constant region respectively, has built the expression vector that carries complete humanized antibody weight, light chain gene;
(4) expression of recombinant humanized antibody and purification: by carrying out eukaryotic expression in above-mentioned carrier cotransfection cell, the secretory antibody in culture supernatant is purified by protein G affinity chromatography.The final target that obtains is in the humanized antibody of RANKL and TNF-α.
In aforesaid method, in the RT-PCR method of step (1), for increasing, heavy chain upstream primer is:
5'-CCGCTCGAGTCATGCTCTTCTTGGTAGCAACAGCT-3'
Downstream primer is: 5'-CTAGCTAGCTGCAGAGACAGTGAGAGTGG-3';
Upstream primer for the light chain that increases is:
5'-CATGCCATGGAAAATGGAGACAGACACACTCCTGC-3'
Downstream primer: 5'-CTACGTACGCCCGTTTGATTTCCAACTT-3'.
Wherein, the PCR condition of the heavy chain of step (1) is: 94 ℃, and 2min; 94 ℃, 30s, 58 ℃, 30s, 72 ℃, 1s, totally 35 circulations; 72 ℃ are extended 10min.
The PCR condition of light chain is: 94 ℃, and 2min; 94 ℃, 30 seconds, 55 ℃, 30s, 72 ℃, 1s, totally 35 circulations; 72 ℃ are extended 10min.
The described cell of step (4) is preferably Chinese hamster ovarian epithelial cell (Chinese hamster ovary, CHO).
The gene that the invention provides the above-mentioned humanized antibody of coding, its light chain nucleotide sequence is as shown in SEQ ID NO.3, and its heavy chain nucleotide sequence is as shown in SEQ ID NO.4.
The present invention also provides for expressing target in the restructuring heavy chain expression carrier hG1-hHv of the humanized antibody of RANKL and TNF-α and restructuring light chain expression vector hK-hLv.
Further, the invention provides the above-mentioned construction process that carries complete humanized antibody weight, light chain gene expression vector, comprise the following steps:
(1) build restructuring heavy chain expression carrier hG1-hHv;
(2) build restructuring light chain expression vector hK-hLv;
(3) above-mentioned two expression vectors are proceeded to respectively in competent cell, complete conversion.
Wherein, step (1) is that people source recombinant antibodies weight chain variabl area sequence and carrier pFUSEss-CHIg-hG1 are used respectively to XhoI and NheI double digestion, reclaims enzyme and cuts product, under the effect of T4 ligase enzyme, connects;
Wherein, step (2) is that people source recombinant antibodies light chain variable region sequence and carrier pFUSE2ss-CLIg-hk are used respectively to NcoI and BsiWI double digestion, reclaims enzyme and cuts product, under the effect of T4 ligase enzyme, connects.
Further, the enzyme blanking method of step (2) is first to add NcoI, fully mixes, the centrifugal several seconds, water-bath 1 hour, after having digested for the first time, in reaction system, add again 10 * Buffer3, BsiWI and deionized water fully to mix, centrifugal several seconds, water-bath 1 hour.Concrete enzyme blanking method is as follows:
(1) the endonuclease reaction system of heavy chain or carrier (20 μ l)
10 * Buffer4,2 μ l; People source recombinant antibodies weight chain variabl area sequence (or pFUSEss-CHIg-hG1), 16.8 μ l; 100 * BAS, 0.2 μ l; Each 0.5 μ l of XhoI and NheI, fully mixes centrifugal several seconds, water-bath 1 hour.
(2) the endonuclease reaction system of light chain or carrier:
10 * Buffer3,2 μ l; People source recombinant antibodies light chain variable region sequence (or pFUSE2ss-CLIg-hk), 17.5 μ l; NcoI 0.5 μ l, fully mixes, centrifugal several seconds, water-bath 1 hour.After having digested for the first time, in above-mentioned 20 μ l reaction systems, add: 10 * Buffer3,1 μ l; BsiWI, 0.75 μ l; Deionized water, 8.25 μ l, fully mix, centrifugal several seconds, water-bath 1 hour.
(3) above-mentioned enzyme is cut to correct fragment row T4 ligase enzyme and connected, build complete recombinant humanized antibody expression vector.(20 μ l) is as follows for linked system:
10 * T4 ligase enzyme reaction buffer, 2 μ l; T4 ligase enzyme, 1 μ l; Carrier segments 3 μ l; Variable region fragment 14 μ l, fully mix, and of short duration centrifugal, water-bath is spent the night.
(4) by above-mentioned expression vector hG1-hHv and the hK-hLv that is connected with respectively recombinant humanized antibody weight, light chain, proceed to respectively in the competent cell of DH5 α, by abovementioned steps, complete conversion.The competent cell coating Zeo that contains recombinant humanized heavy chain of antibody expression vector selects dull and stereotyped, and the competent cell that contains light chain chimeric vector is coated Bar and selected flat board, hatches incubated overnight.Choose the carrier that order-checking is correct.
The humanized antibody obtaining by the way is further carried out to biological assay, by goat anti-human igg (L+H) antibody test, proceed to the humanized antibody of pvdf membrane, its molecular weight is about respectively 23kDa and 50kDa, and molecular size range and theoretical antibody weight chain strand are similar.RT-PCR obtains and expresses the cDNA that has recombinant antibodies, with people's antibody upstream and downstream primer, carry out PCR, electrophoresis result shows, approximately about 700bp and 1400bp, there is positive band, match with theoretical length light chain 666bp, heavy chain 1356bp, further prove that the recombinant antibodies of this secretion is humanized antibody.
Result of indirect ELISA demonstration, this humanized antibody can be combined with RANKL and TNF-α respectively, although under equal conditions the ability of its combination will be weaker than corresponding commercially available antibody, the characteristic of its double combination is that current commercially available antibody is unexistent.Western blotting result also shows, this humanized antibody can with TNF-α and RANKL, special combination occur respectively.Cell toxicity test interpretation of result, TNF-α (0.25ng/ml) can induce L929 cell generation apoptosis, mean apoptotic rate is 48.65%, apply this humanized antibody through row neutralization test, when optimal inhibition result comes across 2 μ g/ml, now apoptosis rate is 22.01%, and osteoclast generates test and shows, humanized antibody can suppress the differentiation by the osteoclast of RANKL induction generation.
And then the present invention also provides the medicine that contains above-mentioned humanized antibody, described medicine also comprises pharmaceutically acceptable carrier and/or vehicle.This medicine can be used for alleviating inflammation and two pathological characters of osteoclasia of rheumatoid arthritis, and then is used for the treatment of the inflammatory bone diseases such as rheumatoid arthritis.
The present invention also provides the application of described humanized antibody in preparation treatment inflammatory bone disease medicine.Described inflammatory bone disease is followed osteoclasia.Described inflammatory bone disease is as rheumatoid arthritis.
The invention has the advantages that can be in vitro effectively in and humanTNF-α, its effect of antagonism, and also can in and people RANKL, effectively suppress differentiation, the maturation of osteoclast, the bone loss of releasing osteoporosis; Be a kind of can inflammation-inhibiting and two target spot humanized antibodies of osteoclasia, can become a kind of effectively for the unitary agent of two pathological characters of rheumatoid arthritis.
Accompanying drawing explanation
Fig. 1 is variable region, immunized mice source cDNA sequence PCR electrophorogram, swimming lane 1:Marker wherein, 2: comprise the mouse source weight chain variabl area sequence of leader sequence, 3: the mouse endogenous light chain variable region sequences that comprises leader sequence;
Fig. 2 is mouse source heavy chain signal peptide sequence prognostic chart, and SRVMLFLVATATGVHSQ is signal peptide sequence.
Fig. 3 is mouse endogenous light chain signal peptide sequence prognostic chart, and MEMETDTLLLWVLLLWVPGSTG is signal peptide sequence
Fig. 4 is mouse endogenous light chain CDR sequential analysis figure.
Fig. 5 is mouse source heavy chain CDR sequential analysis figure.
Fig. 6 is restructuring humanized antibody and mouse From Template chain-ordering comparison diagram, wherein A figure is restructuring humanized antibody sequence of light chain and mouse From Template chain-ordering comparison diagram, have 18 site differences, be respectively 10 (Thr-Ser), 12 (Ser-Val), 15 (Pro-Leu), 23 (Cys-Tyr), 40 (Tyr-Asn), 49 (Lys-Arg), 78 (Thr-Asn), 80 (Ser-His), 81 (Ser-Pro), 84 (Pro-Glu), 87 (Phe-Ala), 105 (Gly-Pro), 106 (Thr-Ser), 107 (Lys-Trp), 108 (Leu-Lys), 109 (Glu-Ser), 110 (Ile-Asn), 111 (Lys-Gly).B figure is restructuring humanized antibody sequence of heavy chain and the comparison of mouse From Template, result shows to have 9 site differences, be respectively 1 (Glu-Gln), 3 (Lys-Gln), 9 (Pro-Ala), 40 (Lys-Thr), 62 (Glu-Gln), 72 (Ser-Ala), 82 (Glu-Gln), 83 (Leu-Val), 119 (Ser-Ala).
Fig. 7 is the expression vector collection of illustrative plates of the complete sequence of light chain of recombinant humanized antibody that contains human antibody κ constant region.
Fig. 8 is the expression vector collection of illustrative plates of the complete sequence of heavy chain of recombinant humanized antibody that contains human antibody γ 1 constant region.
Fig. 9 is the Antibody Results figure that sandwich ELISA is identified secreting, expressing in transfection expression support C HO cells and supernatant, when heavy chain light chain expression vector proceeds to simultaneously, just complete antibody expression can be detected.(***P＜0.001)
Figure 10 is that western blot analyzes that humanized antibody is light, heavy chain figure, is presented at 23kDa and there is positive band in 50kDa site.
Figure 11 is that RT-PCR obtains the electrophorogram of expressing the cDNA that has recombinant antibodies, the light chain product that wherein 1:PT-PCR obtains, about 700bp, the heavy chain product that 2:PT-PCR obtains, about 1400bp, 3:Marker.
Figure 12 is the binding ability figure of humanized antibody and TNF-α, passes through OD
450checking humanized antibody group is 1.376 ± 0.127, though effect is compared deficiency than commercially available restructuring TNF-Alpha antibodies 2.162 ± 0.084, is obviously better than negative control group 0.248 ± 0.030 (* * * P<0.001).
Figure 13 is the binding ability figure of humanized antibody and RANKL, passes through OD
450checking humanized antibody group is 1.725 ± 0.104, though effect is compared lower slightly (* * P<0.01) than commercially available restructuring RANKL antibody 2.470 ± 0.075, be obviously better than negative control group 0.231 ± 0.018 (* * * P<0.001).
Figure 14 is that western blot analysis humanized antibody is combined with TNF-α and RANKL simultaneously, A:TNF-α (17.5KD), B:RANKL (23KD).
Figure 15 is cell toxicity test interpretation of result figure, TNF-α (0.25ng/ml) induction 48.65%L929 cell generation apoptosis, and humanized antibody can drop to this apoptosis rate 22.01 (* P<0.05) when 2 μ g/ml.
Figure 16 is for restructuring humanized antibody is at osteoclast generation experiment effect figure, differentiation (the TRAP stained positive of the humanized antibody treatment group osteoclast that still visible RANKL induction produces when low dosage, ↑ mark site), when 1 μ g/ml and the processing of above dosage, can suppress the effect of RANKL completely.A: negative control (30ng/ml M-CSF); B: positive control (30ng/ml M-CSF+40ng/ml RANKL); C treats control group (30ng/ml M-CSF+40ng/ml RANKL+0.1 μ g/ml anti-RANKL); D: experimental group 1 (30ng/ml M-CSF+40ng/ml RANKL+0.1 μ g/ml humanized antibody); E: experimental group 2 (30ng/ml M-CSF+40ng/ml RANKL+1 μ g/ml humanized antibody); F: experimental group 3:(30ng/ml M-CSF+40ng/ml RANKL+10 μ g/ml humanized antibody)
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, the modification that the inventive method, step or condition are done or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art.Agents useful for same all can business be buied.The mouse resource monoclonal antibody in the RANKL-TNF sample district the present invention relates to (open in Chinese patent CN103060274B).
The clone of embodiment 1 variable region, animal immune mouse source cDNA sequence and definite
According to schedule of operation, extract total mRNA (SV Total RNA, Promega) of hybridoma (preserving number is CGMCC NO.6853); RT-PCR reverse transcription system (promega) obtains mouse source antibody variable region cDNA; According to follow procedure, carry out the antibody variable gene amplification of mouse source
1, primer (INVIVOGEN company is synthetic)
(1) heavy chain upstream primer:
5'-CCGCTCGAGTCATGCTCTTCTTGGTAGCAACAGCT-3'
(2) heavy chain downstream primer:
5'-CTAGCTAGCTGCAGAGACAGTGAGAGTGG-3'
(3) light chain upstream primer:
5'-CATGCCATGGAAAATGGAGACAGACACACTCCTGC-3'
(4) light chain downstream primer: 5'-CTACGTACGCCCGTTTGATTTCCAACTT-3'
2, PCR reaction cycle condition
The PCR condition of heavy chain is: 94 ℃, and 2min; 94 ℃, 30 seconds, 58 ℃, 30 seconds, 72 ℃, 1 second, totally 35 circulations; 72 ℃ are extended 10 minutes.The PCR condition of light chain is: 94 ℃, and 2min; 94 ℃, 30 seconds, 55 ℃, 30 seconds, 72 ℃, 1 second, totally 35 circulations; 72 ℃ are extended 10 minutes.
The results are shown in Figure 1 and be shown in about 400bp and have obvious positive band, (antibody variable region heavy chain and light chain size are generally between 350～450bp) conforms to theoretical value.
3,, to PCR positive products, cut after glue reclaims and check order.
The PCR positive products sequencing result of antibody variable region, mouse source heavy chain, light chain is carried out to sequence analysis (http://www.ncbi.nlm.nih.gov/) by blast program.Use signal peptide analysis software SignalP4.1 counterweight, variable region of light chain to analyze, signal peptide shearing site is predicted to (http://www.cbs.dtu.dk/services/SignalP/), SignalP4.1 interpretation of result, S value is positioned at baseline top, its corresponding amino acid sites is signal peptide composition, C value peak value place is the cleavage site of signal peptide and maturation protein, and Y value meaning is identical with C value, but more accurate.Mouse source heavy chain signal peptide sequence prediction as shown in Figure 2: SRVMLFLVATATGVHSQ is signal peptide sequence; Mouse endogenous light chain signal peptide sequence prediction as shown in Figure 3: MEMETDTLLLWVLLLWVPGSTG is signal peptide sequence.
VBASE2 (http://www.vbase2.org/) carries out domain analyses.As Fig. 4 mouse endogenous light chain CDR sequence is respectively CDR1:27 – 36, CDR2:52 – 56, CDR3:94 – 103 sites; The source of mouse shown in Fig. 5 heavy chain CDR sequence is respectively CDR1:26 – 33, CDR2:52 – 58CDR3:96 – 109 sites.
The design of embodiment 2 humanized antibody variable regions and synthetic
The design of recombinant humanized antibody: antibody variable region, the mouse source heavy chain respectively embodiment 1 being obtained is, the database information of human antibody heavy chain, light chain compare (http://www.ncbi.nlm.nih.gov/) in the sequence of light chain and blast program.Establish respectively and antibody variable region, mouse source heavy chain, the humanized antibody heavy chain (pdb:3DGG_B) that light chain homology is the highest, the sequence of light chain (pdb:1I9R_L).
To screen the humanized antibody heavy chain of gained, the sequence of light chain is carried out domain analyses by VBASE2 program (http://www.vbase2.org/) respectively, the CDR1 that establishes respectively QiCDR district and FR plot structure: 3DGG_B is GYTFTSYV, CDR2 is NPYNDDT, and CDR3 is CAREDYYGSRWGYW.The CDR1 of 1I9R_L is QRVSSSTYSY, and CDR2 is IKAS, and CDR3 is HSWEIPPTF.When fully guaranteeing humanized antibody FR plot structure integrity, the CDR structure of replacing antibody variable region, corresponding mouse source heavy chain, light chain, has built the humanized antibody (as shown in Figure 6) of restructuring.Humanized antibody heavy chain 5 ' upstream adds XhoI, and 3 ' downstream adds NheI restriction enzyme site; Humanized antibody light chain 5 ' upstream adds NcoI, and 3 ' downstream adds BsiWI restriction enzyme site.It is synthetic that INVITROGEN company carries out full gene.Obtain target of the present invention in the light chain nucleotide sequence of the recombinant humanized antibody of RANKL and TNF-α as shown in SEQ ID NO.3, its heavy chain nucleotide sequence is as shown in SEQ ID NO.4.
The construction and expression of embodiment 3 recombinant humanized antibody expression vectors
The structure of recombinant humanized antibody expression vector: recombinant antibodies weight chain variabl area sequence and pFUSEss-CHIg-hG1 (Catalog#pfusess-hchg1, purchased from American I nvivoGen company) application limitations restriction endonuclease XhoI, NheI37 ℃ water-bath are carried out to double digestion for 1 hour.Double digestion is carried out in recombinant antibodies light chain variable region sequence and pFUSE2ss-CLIg-hk (Catalog#pfuse2ss-hchlk, purchased from American I nvivoGen company) NcoI37 ℃ of water-bath of application limitations restriction endonuclease 1 hour, BsiWI55 ℃ water-bath for 1 hour.Above-mentioned enzyme is cut product row agarose gel electrophoresis (1.2% sepharose) respectively.Under ultraviolet lamp, cut positive band, press afterwards the capable gel of test kit read-me and reclaim (QIAquick Gel Extraction Kit, purchased from U.S. QIAGEN company).
Endonuclease bamhi T4 ligase enzyme is connected, build complete recombinant humanized antibody expression vector, carry the expression vector called after hG1-hHv (as shown in Figure 7) of recombinant humanized heavy chain of antibody, carry the expression vector called after hK-hLv (as shown in Figure 8) of recombinant humanized light chain of antibody.Respectively by above-mentioned hG1-hHv and hK-hLv carrier cotransfection Chinese hamster ovary celI, 48 hours post analysis transfection results.
Double antibodies sandwich ELISA method detects supernatant anti-body contg: by coated damping fluid 1:500 dilution for goat-anti people κ chain antibody, 100 μ l/ holes are added to (200ng/ hole) in 96 hole enzyme plates, and 4 ℃ are spent the night.PBST washing 3 times; Add 200 μ l/ hole confining liquids, 4 ℃ are spent the night.Discard confining liquid, add PBST washing 3 times.200 μ l/ holes add culture supernatant, and the multiple hole of 3 samples is set, and using normal untransfected Chinese hamster ovary celI supernatant as negative control, the positive contrast of human IgG standard substance (40ng/ml).37 ℃, 1 hour.PBST washing 3 times.The every hole 100 μ l of goat anti-human igg Fc section antibody (1:8,000) of HRP mark, 37 ℃, 1 hour.Add PBST washing 5 times.Developer 100 μ l colour developing 30 minutes, stops reagent 50 μ l and stops, and measures OD
450value.As shown in Figure 9, sandwich ELISA is identified the generation of the antibody of secreting, expressing in transfection expression support C HO cells and supernatant, while only having heavy chain light chain expression vector to proceed to, just complete antibody expression can be detected simultaneously.(* * * P < 0.001) target of the present invention is in the light-chain amino acid sequence of the recombinant humanized antibody of RANKL and TNF-α as shown in SEQ ID NO.1, and its heavy chain amino acid sequence is as shown in SEQ ID NO.2.
The stably express of embodiment 4 recombinant humanized antibody
The expression vector hG1-hHv of recombinant humanized heavy chain of antibody will be carried, the expression vector hK-hLv cotransfection of light chain enters in Chinese hamster ovary celI, DMEM substratum (10%FBS, 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates), 37 ℃, 5%CO
2incubator is hatched.Until transfection after 48 hours Chinese hamster ovary celI use BZ substratum (DMEM contains 10%PBS, 25 μ g/ml Zeo, 2.5 μ g/ml Bar) instead and cultivate, conventionally within 2～3 days, change liquid once, while changing liquid, PBS rinses, and removes dead cell.
Each porocyte of 15 days of above-mentioned screening and culturing, the capable double antibodies sandwich ELISA of its supernatant method detects, and selects 3 hole row limiting dilution assay subclones that expression amount is the highest and cultivate.Use BZ substratum that cell is resuspended and be progressively diluted to 5/ml.In 96 orifice plates, every hole adds 200 μ l cell suspensions, and now 1 cell is only contained in every hole in theory, 5, bed board, 37 ℃, 5%CO
2cultivate.Within 2～3 days, routine is changed liquid, chooses mono-clonal hole and carries out follow-up cultivation.At the bottom of monoclonal cell is paved with plate, 60% when above, gets culture supernatant row double antibodies sandwich ELISA method and detect, and sustainable secreting, expressing humanized antibody and continuous 2 higher clones of expression amount are proceeded to amplification cultivation in 24 orifice plates, build be, frozen.
The purifying of embodiment 5 recombinant humanized antibody
Application affinity chromatography carries out purifying to humanized antibody: at room temperature, with the initial damping fluid stream of 5～10 times of column volumes, wash pillar, stream scooter 1ml/cm
2.min.With the 0.1mol/L glycine-hydrochloride buffer of 3～5 times of volume column volumes, wash away the pollutent in post.Initial damping fluid rebalancing pillar with 5～10 times of column volumes.12,000rpm after the culture supernatant that contains recombinant humanized antibody is collected, 4 ℃, 30min, removes cell debris.0.45 μ m membrane filtration, the Hollow Fiber Ultrafiltration post that to remove polymkeric substance be 100kD with molecular weight cut-off is concentrated into 1/10 of original volume.With the initial damping fluid dilute sample of equal-volume.Sample is added to chromatographic column, until sample, flow through completely after chromatographic column, with 5～10 times of initial damping fluid Elution chromatography of column volume posts.
0.1mol/L glycine-hydrochloride buffer (pH2.5～3.0) wash-out with 5～10 times of column volumes.The container of accepting flowing lotion injects the 1mol/L Tris-HCl (pH8.0) that is about flowing lotion 1/10 volume in advance.Each connects sample pipe and meets flowing lotion 1ml, and pipe planted agent places 0.1ml1mol/L Tris-HCl (pH8.0) in advance.Merge with G280 and be reacted into each positive collection tube.The clean chromatographic column of 0.1mol/L glycine-hydrochloride buffer (pH2.5～3.0) with 5 times of column volumes.Initial damping fluid rebalancing pillar with 10 times of column volumes.While depositing chromatographic column, in the initial damping fluid of balance pillar, should add 0.02% sodium azide or 20% ethanol.
The dialysis of protein sample: dialysis tubing is cut into 15cm length segment; In dialysis tubing treatment solution, dialysis tubing is boiled to 10min; With distilled water, thoroughly clean dialysis tubing; Put into 1mmol/L EDTA (pH8.0) and boil 10min; With distilled water, thoroughly clean up; After cooling, be soaked in PBS, be placed on 4 ℃, standby; Pretreated protein sample is loaded in dialysis tubing, and two ends, with after clamp, are placed in dialysis tubing in the beaker of PBS solution.PBS liquor capacity is greater than 50 times of protein sample volumes.Beaker is placed in to 4 ℃ of refrigerators, continues magnetic agitation.Within every 4 hours, change liquid once, continue dialysis 24～48 hours.
The physical and chemical property determining of embodiment 6 recombinant humanized antibody
1, antibodies specific is measured:
Respectively TNF-α, RANKL, DKK1 and IL-17 recombinant cytokine being coated in to 4 ℃ of enzyme plates (100ng/ hole) spends the night.PBST washing 3 times.5% skimmed milk sealing, room temperature shaking table is hatched 1h or 4 ℃ and is spent the night, PBST washing 3 times, 100 μ l/ holes add antibody purification (100 μ g/ml), and the multiple hole of 3 samples is set, and hatch 2h for 37 ℃.PBST washing 3 times.The every hole of the goat anti-human igg of HRP mark (H+L) antibody (1:8,000) adds 100 μ l, hatches 1h for 37 ℃.PBST washing 3 times.Add developer (A, B liquid equal-volume mix) 100 μ l, lucifuge colour developing 30min, stops reagent 50 μ l and stops, and measures OD
450value.
2, BCA method is measured antibody concentration
(1) preparation of BSA concentration gradient reference liquid, in Table 1.
Table 1
(2) each sample of the preparation of BCA working fluid: BCA solution 200 μ l+4% copper sulfate 4 μ l
(3) operating process
1. the BSA concentration gradient reference liquid of configuration and testing protein sample 25 μ l/ holes are placed in to 96 orifice plates, 3 multiple holes are set respectively.
2. every hole adds 200 μ l BCA working fluids, hatches 30 minutes for 37 ℃.
3. in 10 minutes, measure and record responded light absorption value A
562.
4. according to the result drawing standard curve of BSA concentration gradient reference liquid.
5. referring to typical curve, according to protein sample light absorption value, in typical curve linearity range, read the protein concentration of sample.
6. the antibody of purifying is adjusted to concentration to 1.0mg/ml by ultrafiltration or dilution, packing leave in-20 ℃ standby.
The humanization of embodiment 7 recombinant humanized antibody is identified
1, sandwich ELISA identifier source engineered antibody: as previously shown: goat-anti people κ chain antibody dilution is coated in to enzyme plate (200ng/ hole), the sealing of spending the night of 5% skim-milk, PBST washs, and adds testing sample, adds HRP-goat anti-human igg Fc after washing again.DAB colour developing, measures OD
450value.As shown in Figure 9, the antibody of secreting, expressing can be combined with goat-anti people κ chain antibody and goat anti-human igg Fc simultaneously, and it is really humanized antibody.
2, RT-PCR detects the mRNA of people source engineered antibody:
(1) collect the hybridoma of secretion humanized antibody, shown in front, according to test kit explanation, extract total RNA.
(2) as previously shown, according to test kit explanation, utilize RT-PCR technology to obtain cDNA.
(3) design primer, respectively at human antibody heavy and light chain variable region FR1 end design 5' primer, in constant region C end design 3' primer, specific as follows:
People γ chain 5' end primer 5'-GTCTGGGCCTGAGCTGGTGA-3'
People γ chain 3' end primer 5'-TCTTCTGCGTGTAGTGGTTG-3'
People κ chain 5' end primer 5'-AGTCTCCTGCTACCTTATCT-3'
People κ chain 3' end primer 5'-TGAAGCTCTTTGTGACGG-3'
(4) by mix requirement, add reaction system, fully mix, be placed in PCR instrument and react.
The PCR condition of heavy chain is: 94 ℃, and 2min; 94 ℃, 30s, 58 ℃, 30s, 72 ℃, 1s, totally 35 circulations; 72 ℃ are extended 10min.
The PCR condition of light chain is: 94 ℃, and 2min; 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 1s, totally 35 circulations; 72 ℃ are extended 10min.
(5) by 1% agarose electrophoresis, detect corresponding amplified band.
Result is as shown in figure 11: RT-PCR obtains and expresses the cDNA that has recombinant antibodies, with people's antibody upstream and downstream primer, carry out PCR, electrophoresis result shows, approximately about 700bp and 1400bp, there is positive band, match with theoretical length light chain 666bp, heavy chain 1356bp, further prove that the recombinant antibodies of this secretion is humanized antibody.
3, Western blot analyst source chemical industry engineered antibody:
Humanized antibody (500ng/ hole) is separated by 10%SDS-PAGE; Semidrying proceeds on pvdf membrane, 5% skim-milk sealing, and TBST cleans three times, HRP-goat anti-human igg (H+L) (1:2,000) antibody incubated at room 1h, TBST cleans three times, chemical luminous substrate mulch film colour developing 1 minute.Exposure in darkroom, development, photographic fixing.Result is as shown in figure 10: by goat anti-human igg (L+H) antibody test, proceed to the humanized antibody of pvdf membrane, its molecular weight is about respectively 23kDa and 50kDa, and molecular size range and theoretical antibody are light, heavy chain strand is similar.
The evaluation of the binding specificity of embodiment 8 recombinant humanized antibody and TNF-α and RANKL
1, indirect ELISA is measured the binding ability of recombinant humanized antibody and TNF-α and RANKL
Be coated with respectively TNF-α and RANKL, 5% skimmed milk sealing, PBST cleans three times, and purifying humanized antibody is primary antibodie, 4 ℃ of night incubation, PBST cleans three times, HRP-goat anti-human igg (H+L) (1:8,000) antibody is two anti-, incubated at room 1h, and PBST cleans three times; Anti-goat-anti humanTNF-α and the positive contrast of anti-RANKL antibody (1:2,000) are set simultaneously, and PBST cleans three times, and the anti-sheep IgG of HRP-donkey (H+L) (1:2,000) antibody is two anti-; Nitrite ion lucifuge colour developing 30min, stop buffer termination reaction, measures A
450.Result of indirect ELISA is as Figure 12, Figure 13 demonstration, this humanized antibody can be combined with RANKL and TNF-α respectively, although under equal conditions the ability of its combination will be weaker than corresponding commercially available antibody, the characteristic of its double combination is that current commercially available antibody is unexistent.
2, Western blot analyzes the binding ability of recombinant humanized antibody and TNF-α and RANKL
TNF-α is separated by 10%SDS-PAGE with RANKL (500ng/ hole); Semidrying proceeds on pvdf membrane, 5% skim-milk sealing, TBST cleans three times, and purifying humanized antibody is primary antibodie, 4 ℃ of night incubation, TBST cleans three times, HRP-goat anti-human igg (H+L) (1:2,000) antibody is two anti-, incubated at room 1h, TBST cleans three times, chemical luminous substrate mulch film colour developing 1 minute.Exposure in darkroom, development, photographic fixing.Result is as shown in figure 14: humanized antibody can combine with TNF-α (17.5KD) and RANKL (23KD) simultaneously.
Embodiment 9 humanized antibodies are for the antagonistic action of TNF-α
L929 apoptosis experimental verification recombinant humanized antibody is for the antagonistic action of TNF-α: L929 cell routine is cultivated, 0.25% pancreatin (containing 0.02%EDTA) digestion, 1,000rpm is centrifugal, α-MEM (10%FBS, 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates) is resuspended, cell counting, adjusting cell concn is 5 * 10
5individual/ml, is inoculated in 96 porocyte culture plates, and 100 μ l cell suspensions are inoculated in every hole.The recombinant humanized of preparing gained α-MEM (10%FBS for antibody, 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates) dilute, final concentration is respectively 2 μ g/ml, 1 μ g/ml, 0.5 μ g/ml, 0.1 μ g/ml, 0.01 μ g/ml, fully mix with TNF-α (0.25ng/ml) and dactinomycin (1 μ g/ml) respectively, final volume is 1ml, jointly hatches 1h for 37 ℃.And negative control is set.After L929 cell is completely adherent, add above-mentioned mix reagent, 5%CO
2, hatch 12h for 37 ℃.
Row violet staining afterwards: discard cells and supernatant, PBS rinses cell 1 time.10% methyl alcohol room temperature fixed cell 30min, abandons supernatant.0.25% Viola crystallina dye liquor (20% methyl alcohol dilution is dissolved), every hole adds 100 μ l, hatches 20min for 37 ℃.Discard dye liquor, deionized water rinsing cell 2 times.After to be dried, 33% formic acid solution dissolves, and microplate reader reads A
560light absorption value.Apoptosis rate calculates, apoptosis rate=(negative hole OD-experimental port OD)/negative hole OD * 100%.Cell toxicity test interpretation of result is as shown in figure 15: TNF-α (0.25ng/ml) can induce L929 cell generation apoptosis, mean apoptotic rate is 48.65%, apply this humanized antibody and carry out neutralization test, when optimal inhibition result comes across 2 μ g/ml, now apoptosis rate is 22.01%.Presentation of results, the target that the present invention obtains has antagonistic action in the recombinant humanized antibody of RANKL and TNF-α for TNF-α, can suppress the apoptosis due to TNF-α, the inflammation that TNF alpha antibody short of money causes.
Embodiment 10 humanized antibodies are for the antagonistic action of RANKL
Osteoclast induction experimental verification recombinant humanized antibody of the present invention is for the antagonistic action of RANKL: the scavenger cell (BMM of derived from bone marrow, bone marrow macrophage) acquisition: 4～6 week age BALB/c mouse, de-neck is put to death, 75% alcohol-pickled 5min, in Bechtop, separated femur under aseptic condition, expose medullary space, α-MEM (α-minimal essential medium contains 100U/ml penicillin and 100 μ g/ml Streptomycin sulphates) goes out marrow, be inoculated in 100mm Tissue Culture Dish, contain 10% foetal calf serum (FBS, fetal bovine serum) α-MEM, add 5ng/ml M-CSF, 5%CO
2, cultivate 16～24h for 37 ℃, remove not attached cell completely, attached cell is with containing 5ng/ml M-CSF, and the α of 10%FBS-MEM continues to cultivate 3d.
BMM0.25% trysinization, cell counting is also adjusted concentration, according to 5 * 10
5/ hole is inoculated in 24 orifice plates of putting in advance cell climbing sheet, and according to corresponding substratum, the 5%CO of adding as follows
2, cultivate 6d for 37 ℃.
Table 2
TRAP dyeing
1. remove cells and supernatant, TRAP dyeing stationary liquid 300 μ l are 30s fixedly, and rinsed with deionized water once.
2. prepare diazotized GBC solution (10T), slightly mix 30 seconds, the standing 2min of room temperature, standby.
Table 3
3. prepare TRAP staining fluid (10T), add in the following order, slightly mix simultaneously.
Table 4
4. every hole drips TRAP staining fluid 600 μ l, hatches 1h for 37 ℃.
5. remove dye liquor, rinsed with deionized water once.
6. 10s is redyed in Hematorylin 200 μ l/ holes, and tap water returns blue 5min.
7. carefully cell climbing sheet is taken out from 24 orifice plates, with neutral gum, be fixed on slide glass, micro-Microscopic observation is also taken pictures.
Osteoclast generates tests the demonstration as Figure 16: differentiation (the TRAP stained positive of the humanized antibody treatment group osteoclast that still visible RANKL induction produces when low dosage, ↑ mark site), when 1 μ g/ml and the processing of above dosage, can suppress the effect of RANKL completely.Illustrate that the target that the present invention obtains can suppress the differentiation by the osteoclast of RANKL induction generation, the osteoclasia effect that antagonism RANKL causes in the recombinant humanized antibody of RANKL and TNF-α.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. target, in the humanized antibody of RANKL and TNF-α, is characterized in that, the aminoacid sequence of its light chain is as shown in SEQ ID NO.1, and the aminoacid sequence of its heavy chain is as shown in SEQ ID NO.2.
2. the gene of humanized antibody described in coding claim 1, is characterized in that, its light chain nucleotide sequence is as shown in SEQ ID NO.3, and its heavy chain nucleotide sequence is as shown in SEQ ID NO.4.
3. the preparation method of humanized antibody described in claim 1, comprises the steps:
(1) clone of mouse resource monoclonal antibody variable region cDNA sequence and definite: extract the total RNA of hybridoma of the two target spot mouse of secretion source antibody, RT-PCR method obtains the cDNA sequence of antibody variable region, mouse source;
(2) design of humanized antibody variable region and synthetic: choose respectively human antibody heavy chain that homology is the highest, light chain skeleton as an alternative, the skeleton district of antibody CDR district, mouse source and human antibody is spliced, complete the replacement of mouse source CDR;
(3) structure of recombinant humanized antibody expression vector: the humanized antibody heavy chain that above-mentioned structure completes, the sequence of light chain are spliced mutually with the expression vector that carries people's IgG antibody 1 and κ chain constant region respectively, has built the expression vector that carries complete humanized antibody weight, light chain gene;
(4) expression of recombinant humanized antibody and purification: will in above-mentioned carrier cotransfection cell, carry out eukaryotic expression, secretory antibody in culture supernatant is purified by protein G affinity chromatography, finally obtain target in the humanized antibody of RANKL and TNF-α.
4. preparation method as claimed in claim 3, is characterized in that, in the RT-PCR method of step (1), for the upstream primer of the mouse resource monoclonal antibody heavy chain that increases, is:
5'-CCGCTCGAGTCATGCTCTTCTTGGTAGCAACAGCT-3'；
Downstream primer is:
5'-CTAGCTAGCTGCAGAGACAGTGAGAGTGG-3'；
Upstream primer for the mouse resource monoclonal antibody light chain that increases is:
5'-CATGCCATGGAAAATGGAGACAGACACACTCCTGC-3'；
Downstream primer: 5'-CTACGTACGCCCGTTTGATTTCCAACTT-3'.
5. for expressing described in claim 1 target in the recombinant expression vector of the humanized antibody of RANKL and TNF-α.
6. the method for preparing recombinant expression vector described in claim 5, is characterized in that, comprises the following steps:
(1) build restructuring heavy chain expression carrier hG1-hHv;
(2) build restructuring light chain expression vector hK-hLv;
(3) above-mentioned two expression vectors are proceeded to respectively in competent cell, complete conversion.
7. method as claimed in claim 6, is characterized in that, step (1) is that people source recombinant antibodies weight chain variabl area sequence and carrier pFUSEss-CHIg-hG1 are used respectively to XhoI and NheI double digestion, reclaims enzyme and cuts product, under the effect of T4 ligase enzyme, connects;
Step (2) is that people source recombinant antibodies light chain variable region sequence and carrier pFUSE2ss-CLIg-hk are used respectively to NcoI and BsiWI double digestion, reclaims enzyme and cuts product, under the effect of T4 ligase enzyme, connects.
8. contain target claimed in claim 1 in the medicine of the humanized antibody of RANKL and TNF-α.
9. described in claim 1, target is treated the application in inflammatory bone disease medicine in the humanized antibody of RANKL and TNF-α in preparation.
10. application according to claim 9, is characterized in that, described inflammatory bone disease is rheumatoid arthritis.
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| CN104497141A (en) * | 2014-12-31 | 2015-04-08 | 百泰生物药业有限公司 | Therapeutic antibody of anti-human CD6 molecule |
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| WO2019217450A1 (en) * | 2018-05-08 | 2019-11-14 | Rhode Island Hospital | Anti-chi3l1 antibodies for the detection and/or treatment of nonalcoholic fattly liver disease/nonalcoholic steatohepatitis and subsequent complications |
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