CN100393748C - Human tumour necrosin antibody, its preparation and medicinal composition - Google Patents
Human tumour necrosin antibody, its preparation and medicinal composition Download PDFInfo
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- CN100393748C CN100393748C CNB2003101084400A CN200310108440A CN100393748C CN 100393748 C CN100393748 C CN 100393748C CN B2003101084400 A CNB2003101084400 A CN B2003101084400A CN 200310108440 A CN200310108440 A CN 200310108440A CN 100393748 C CN100393748 C CN 100393748C
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Abstract
The present invention relates to a full human anti-TNF-alpha monoclonal antibody. The full human anti-TNF-alpha monoclonal antibody has the characteristics of strong specificity and low immunogenicity. The present invention also provides a preparation method of the monoclonal antibody and a medical composition containing the monoclonal antibody.
Description
Technical field
The present invention relates to field of immunology; More specifically, the present invention relates to tnf antibody, its encoding sequence, preparation method and application thereof.
Background technology
Tumor necrosis factor alpha (Tumor Necrosis Factor α, TNF-α) be that the intravital a kind of multifunction immunity of machine is regulated molecule, it can play a role with the receptors bind on the cytolemma, often causes the death (its title promptly derives from this) of target cell or attracts immune effector cell in partial gathering.TNF-α is a kind of homotrimer subunit of solubility, and molecular weight is 17KD (Smith, et al., J.Biol.Chem.262:6951-6954,1987).Also find to stride membrane-bound precursor TNF-α, molecular weight is 26KD (Kriegler, et al., Cell53:45-53,1988).Mononuclear macrophage can TNF secretion-α and TNF-β after being subjected to intracellular toxin and some other stimulator and stimulating, and some other in addition cell also can TNF secretion-α.
TNF-α plays a key role in the pathology process of rheumatoid arthritis, bacterium or virus infection, chronic inflammatory diseases, autoimmune disease such as AIDS (AIDS), malignant tumour and/or nerve degenerative diseases etc.The TNF-alpha monoclonal antibodies can in and TNF-α, in vivo the activity of TNF-α has been played the effect of negative adjusting.And have in a large number and studies show that TNF-α still causes the main medium of septic shock syndromes.The TNF-alpha levels raises and is indicating that mortality ratio or disability rate raise among the septic shock syndromes patients serum.Use the TNF-Alpha antibodies clinically or its acceptor has certain curative effect to the septic shock syndromes.
In addition, TNF-α promotes HIV symptomless infection state to enter one of main medium of AIDS, and at the monoclonal antibody of TNF-G can in and the activity of TNF-α, in vivo the activity of TNF-α is born adjusting, can remove the patient from the inducement that the symptomless infection state enters AIDS, reach certain AIDS therapeutic purpose.With monoclonal antibody and the medication combined medication of other AIDS of TNF-α, antagonism will obviously improve drug effect by the too much caused side effect of TNF-α.
1987, people such as Cerami obtained the polyclone murine antibody (EPO patent publications on March 4th, 0212489,1987) of a kind of TNF-α, can be used for immunoassay of shock diagnostic and treatment that infectation of bacteria causes.Obtained the monoclonal antibody of TNF-α afterwards again in succession, as Rubin et al., people's such as EPO patent publication on April 22nd, 0218868,1987 and Yone EPO patent publications is described in 28,0288088,1988 on October.
The other investigator has described the specific monoclonal antibody to the reorganization humanTNF-, has among the TNF-α and active (Liang, et al., Biochem.Biophys.Res.Comm.137:847-854,1986 external; Meager, et al., Hybridoma 6:305-311,1987).
But because the mouse source that the mostly is antibody that obtains of above-mentioned research, all there is the low and/or stable problem such as not enough of in various degree immunogenicity height, specificity in these antibody, do not provide a kind of and can bring into play diagnosis or therapeutic action in vivo.
Therefore, this area presses for avidity and the specificity that foundation can either keep or improve antibody, can reduce again or the anti-TNF-Alpha antibodies of basically eliminate antibody mediated immunity originality, thereby can be used for clinical treatment, such as treatment rheumatoid arthritis and AIDS etc.
Summary of the invention
Technical problem to be solved by this invention is to provide a specific specificity height, the anti-TNF-alpha monoclonal antibodies in total man source that immunogenicity is low.The variable region of heavy chain of monoclonal antibody of the present invention has the described aminoacid sequence of SEQ ID NO:1, and variable region of light chain has the described aminoacid sequence of SEQ ID NO:2.
Technical problem to be solved by this invention also is to provide the nucleotide sequence of the anti-TNF-alpha monoclonal antibodies in described total man source.
Technical problem to be solved by this invention also is to provide a kind of pharmaceutical composition that contains the anti-TNF-alpha monoclonal antibodies in described total man source, and it contains monoclonal antibody of the present invention and pharmaceutically acceptable carrier.
Technical problem to be solved by this invention also is to provide the purposes of monoclonal antibody of the present invention, can be used for preparing the medicine for the treatment of AIDS, and can be used for preparing the medicine for the treatment of rheumatoid arthritis.
The present invention is based on phage antibody library technique clone and expressing tumor necrosin antibody, the antibody that obtains through screening is the total man source, has overcome the high shortcoming of mouse source antibody mediated immunity originality of present existence.That compares obtains antibody gene from hybridoma etc., Antibody Preparation program advanced person is easy, cheap in total man of the present invention source, the more important thing is this directly method of amplification antibody gene from human peripheral lymphocyte, is the fundamental way that solves insurmountable always humanized's antibody sources problem such as hybridoma technology.
The invention provides the anti-TNF-alpha monoclonal antibodies in a kind of total man source, its variable region of heavy chain and variable region of light chain have unique prior art constructions that is different from.
The present invention also provides the aminoacid sequence of the anti-TNF-alpha monoclonal antibodies in total man source, and other protein or fusion expressed product with these chains.
The present invention not only comprises complete monoclonal antibody, also comprises having immunocompetent antibody fragment, as Fab or (Fab ')
2Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule.
The present invention also provides coding said monoclonal antibody or its segmental dna molecular.The Nucleotide full length sequence of monoclonal antibody of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.A kind of feasible method is to synthesize relevant sequence, especially fragment length more in short-term with artificial synthetic method.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.In addition, also the encoding sequence of light chain and heavy chain can be merged, form single-chain antibody.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
The invention still further relates to the carrier that comprises above-mentioned suitable dna sequence dna and suitable promotor or control sequence.These carriers can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS7,293 cells etc.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
The present invention also provides a kind of pharmaceutical composition, and it contains above-mentioned monoclonal antibody or immune conjugate, and pharmaceutically acceptable carrier.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intravenously or topical.
Pharmaceutical composition of the present invention can be used for treating AIDS, rheumatoid arthritis, also can be used for the illness that other need suppress TNF-α.
Pharmaceutical composition of the present invention contains antibody of the present invention and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as injection, solution should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.
Outstanding advantage of the present invention is: the having very high avidity and be the antibody in total man source of monoclonal antibody of the present invention.The monoclonal antibody of this high-affinity has significant values clinically.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
The structure of humanized's single-chain antibody (scFv) gene pool
(1) preparation of .cDNA
Collect each 5ml of peripheral blood of 1000 people, mix, with lymphocyte separation medium (medical courses in general institute Tianjin blood grind produce) separation mononuclearcell.With the test kit of Invitrogen company, from isolating human peripheral lymphocyte, extract total mRNA of cell.With the mRNA purification kit purifying of GIBCO company, be template with the mRNA of above-mentioned acquisition, reverse transcription goes out cDNA first chain.Above step is carried out according to the specification sheets that producer provides.
(2) .PCR amplification
With reference to the conservative sector sequence of V district FR1 and FR4 in the various human antibody gene man family sequence of having delivered both at home and abroad, design and synthesize with servant's antibody VH gene (hVH), VL gene (hVL), 5 ' end (back), 3 ' end (forward) primer.Add suitable restriction site sequence (primer all entrusts Shanghai living worker company to synthesize) according to used carrier pCANTAB5E (a kind of phasmid is Pharmacia company product).
PCR reaction: the cDNA product of transcribing that obtains with step (1) is a template, respectively with the VH primer to or the VL primer to carrying out pcr amplification.Add 4UTaq enzyme (available from magnificent company) in 100 μ l total reaction volume, condition is: 94C 60s, and 52 ℃ of 70s, 72 ℃ of 80s, totally 30 circulations, last extends 10min for 72 ℃.
(3) recovery of .PCR product and purifying
After carrying out PCR according to above-mentioned condition, product electrophoresis on 0.8% sepharose is identified, discovery has molecular weight respectively about 310bp and two bands about 300bp, reclaims test kit with the glue of Promega company and reclaims fragment, and operation is carried out according to the specification sheets of producer.
(4). the structure of humanized's single-chain antibody (scFv) gene
With (Gly
4Ser)
3The ScFv gene that connects VH and VL formation heavy chain-joint sequence-light chain structure for joint sequence (SEQ ID NO:5) respectively.The joint primer is according to joint sequence template and people's antibody V district gene order 3 ' end, 5 ' end parts sequences Design and synthetic.
Carry out pcr amplification and recovery, purifying (PCR condition and recovery, purification process are all the same) with above-mentioned joint template and primer, separate obtaining the joint sequence that length is about 100bp.
Embodiment 2
The ScFv fragment cloning enters the phagemid carrier
After carrying out PCR according to above-mentioned condition, obtain the ScFv fragment that two ends have SfiI and NotI restriction enzyme site, carry out the spin-column chromatography purifying, remove unnecessary joint primer and dNTP.The ScFv fragment that purifying finishes is carried out double digestion with SfiI and NotI (all available from Promega company), the sticky end that generation can be connected with carrier pCANTAB5E (Pharmacia company).Carry out the spin-column chromatography purifying again, removal may influence its little SfiI that is connected with carrier or NotI fragment.
Phagemid carrier pCANTAB5E (Pharmacia company) itself includes SfiI and the NotI enzyme is cut sticky end, can be connected with above-mentioned ScFv fragment.Adopt conventional dna ligase, the ScFv fragment cloning is entered corresponding site on the phagemid carrier.Its consecutive position is the g3p gene on the pCANTAB5E carrier, can express the ScFv-g3p fusion gene in the future.
1 liter of LB nutrient solution adds the TG1 cell (Pharmacia company) of 1ml incubated overnight, be cultured to OD value about 0.3～0.4, the 4000rpm centrifugal collecting cell, 10 times of volumes ice pure water washed twice, be resuspended in 1ml and contain 2% yeast powder, 1% peptone, 1mM MgCl
210mM Tris-HCl damping fluid (PH8.0) in.100 μ, 1 electric shock competent cell adds and connects the DNA sample 100ng that spends the night, and voltage is 10 kilovolts and shocks by electricity, and will comprise the segmental connection carrier transfection of ScFv and enter E.coli TG1 cell.Then with the E.coli cell cultures in SOBAG (the SOB substratum that contains 100mg/L penbritin and 2% glucose) agar plate, culture temperature is 30 ℃, wherein transforming has the Ecoli cell of pCANTAB5E carrier to survive.Carry out electricity repeatedly and transform, make the capacity of the antibody library that obtains reach 1 * 10
11
Dilute bacterium to OD600=0.2 with 2 * YTAG (2 * YT substratum that contains 100mg/L penbritin and 2% glucose); Be cultured to logarithmic phase with this suspension of 15ml again, add 10
11The M13K07 helper phage of pfu (available from Promega company), 1h is cultivated in 37 ℃ of joltings, be resuspended in 10ml 2 * YTAK (2 * YT substratum that contains 100mg/L penbritin and 70 μ g/ml kantlex) after centrifugal, the shaking table overnight incubation, in 4 ℃ with the centrifugal 15min of 1500 * g.Collect supernatant and be humanized ScFv phage display library.With after its packing in-20 ℃ of preservations, in order to next step with specific antigens to the library carry out " elutriation (panning) " screening.
Embodiment 3
The elutriation antibody library
Antibody in the phage display library that obtains for embodiment 2 is by the high antibody of panning technique selective affinity.
With reorganization humanTNF-(rhTNF-α) antigen (available from Shenzhen brilliant U.S. company) bag by elisa plate, the BSA sealing, incubation 2h adds 50 μ l phage antibody libraries (about 10 after washing plate
12CFU) incubation 2h, TBST (Tris 50mmol/L, NaCl 150mmol/L, Tween-200.5%, BSA 1%, pH7.5) liquid is washed (the 2nd takes turns and wash 5 times, and the 3rd washes each 5min 10 times after taking turns) 1 time, reclaim phage with 50 μ l elutriants, neutralization buffer is regulated pH to neutral, infects Ecoli TG1, carries out the next round screening.Carry out 3 altogether and take turns screening, remove the phagemid that does not have antigen binding capacity.
Carry out the sandwich ELISA experiment, measure the antigen-binding activity of antibody.The antigen coated elisa plate of reorganization humanTNF-(rhTNF-α) that adds 50 μ l 200ng/ml, the BSA sealing, 37 ℃ of incubation 1h add (KCl2.7mmol/L, Na with PBST
2HP0
410mmol/L, KH
2PO
41.8mol/L, NaCl 137mmol/L, Tween-20 0.5%, and pH7.4) two-fold dilution's phage antibody is hatched 2h for 37 ℃.Wash plate, add the goat-anti people monoclonal antibody (available from Pharmacia company) of HRP mark, 37 ℃ of 1h.PBS with 1%Tween-20 washes plate, adds the substrate solution colour developing, the photoabsorption of reading 595 nanometers on plate reading machine.According to calculating positive rate near 20%, determine the wherein the strongest clone of avidity, be used for next step research.
Pick out 2 clones that avidity is the strongest and infect E.coli HB2151 cell (Pharmacia company) above-mentioned, the dull and stereotyped cultivation, the single bacterium colony of picking on the reformer plate, in 30 ℃ of overnight incubation, in a small amount extract phagemid dna with alkaline lysis with 2 * YTAG (2 * YT nutrient solution that contains 100mg/L penbritin and 2% glucose), pcr amplification goes out the ScFv gene fragment, and be cloned into the pUC19 carrier, carry out sequencing, comprise the VH of expection, VL gene and linker sequence.The VH gene order is in the upstream of linker, and the VL gene order is in the downstream of linker.
The reorganization phagemid bacterial classification inoculation that contains the scFv gene that above-mentioned avidity is stronger is in 5ml LB substratum, incubated overnight.Add among the 50ml SBAG (the SB nutrient solution that contains 100mg/L penbritin and 2% glucose), 30 ℃ of shaking culture 1h, the centrifugal 15min of 5000rpm abandons supernatant.Precipitation is resuspended among the 50ml SBAI (the SB nutrient solution that contains 100mg/L penbritin and 1mmol/L IPTG), induces 3h for 37 ℃, centrifugal the same.Get the N,O-Diacetylmuramidase (1g/L) that precipitation is suspended from the new preparation of 5ml, 20% (w/v) sucrose, in 30mmol/L Tris-Cl (pH8.0) and 1mmol/L EDTA (pH8.0) solution, ice bath 10min, 4 ℃ with the centrifugal 5min of 12000rpm, and supernatant is outer pericentral siphon part.After its lyophilize, be stored in-20 ℃.Face with preceding and be dissolved to 500 μ l with 0.01mol/L PBS.
The single-chain antibody of above-mentioned acquisition carries out the test of Western trace according to ordinary method, and the result records it and has antigen-specific.
Embodiment 4
Again be cloned into the expression vector pL101 that contains people source Heng Qu, transform Chinese hamster ovary celI
Design the primer of heavy chain and light chain respectively, add the XbaI/NheI restriction enzyme site at heavy chain primer front end; Adding the HindIII/BsiWI restriction enzyme site at light chain primer front end, is template with the ScFv fragment then, carries out PCR according to a conventional method, obtains to have the variable region of heavy chain fragment and the variable region of light chain fragment of corresponding restriction enzyme site.
Above-mentioned variable region of heavy chain is inserted in the XbaI/NheI site of expression vector pL101, above-mentioned antibody chain variable region is inserted in the HindIII/Bsi WI site of the pL101 that is inserted with the variable region of heavy chain encoding sequence with Hind III and Bsi WI again, makes up the expression vector of adult source TNF-Alpha antibodies.Expression vector pL101 is available from Invitrogen company.
The expression vector that has antibody gene of above-mentioned structure changes at e.colistraindh5, is inoculated in then in 100 milliliters of LB substratum to increase, with the ultrapure plasmid DNA purification kit extracting and purifying plasmid DNA of Qiagen company.The plasmid DNA of above-mentioned purifying is adopted the liposome method test kit transfection CHO cell of Invitrogen company, and operation is carried out with reference to the specification sheets of producer.
The Chinese hamster ovary celI that transforms carries out the extreme dilution at last and cultivates in the selection of selecting to carry out on the substratum continuous 9 weeks on 96 orifice plates, carry out continuously 3 times, carry out mono-clonalization.
The monoclonal cell of choosing ties up on RPMI 1641 substratum and cultivates, and supernatant is carried out the Western Blot experiment, judges expression intensity according to staining reaction, picks out the stronger clone of 8 expression as the candidate cell strain.
Embodiment 5
The high clone of expression intensity in the screening Chinese hamster ovary celI
The high expression level candidate clone that above-mentioned screening is obtained is incubated in the square cell bottle of 10ml, adopt the ELISA method to measure the expression amount of antibody: goat anti-human igg (Fc) bag quilt is in elisa plate, 4 ℃ are spent the night, in 37 ℃ of sealings 2 hours, add culture supernatant to be measured and standard substance (human IgG1) through 2%BSA, hatched 2 hours for 37 ℃, add HRP-goat anti-human igg (κ) and carry out association reaction, hatched 1 hour for 37 ℃, add TMB, use H at last in 37 ℃ of effects 10 minutes
2SO
4Termination reaction is surveyed A
450Value.The expression amount that records above-mentioned 8 candidate clones is as follows:
Table 1 antibody gene is expression intensity (mg/l) in Chinese hamster ovary celI
| Cell strain | 2A4 | 3H2 | 4A6 | 5D5 | 7B3 | 4H7 |
| Expression amount | 253.6 | 167.9 | 239.9 | 147.9 | 289.8 | 177.5 |
As can be seen from Table 1, the cell strain that is numbered 7B3 and 2A4 has very high expression level (250-290 mg/litre), and wherein the expression amount of 7B3 has reached 290.7 mg/litre.Select the high clone of antibody expression amount, can carry out mass cell and cultivate also purifying, thereby obtain the anti-TNF-Alpha antibodies in a large amount of total man sources.
Embodiment 6 total man source TNF-alpha monoclonal antibodies are for the external neutralizing effect of TNF-α
Carry out the ELISA experiment, it is active for the combination of TNF-α to measure total man source TNF-alpha monoclonal antibodies.Antigen coated elisa plate (the R﹠amp of reorganization humanTNF-(rhTNF-α) that adds 50 μ l 200ng/ml; D company product), the BSA sealing, 37 ℃ of incubation 1h add (KCl 2.7mmol/L, Na with PBST
2HPO
410mmol/L, KH
2PO
41.8mol/L, NaCl 137mmol/L, Tween-20 0.5%, and pH7.4) two-fold dilution's total man source TNF-alpha monoclonal antibodies is hatched 2h for 37 ℃.Wash plate, add the mouse-anti people monoclonal antibody (available from Pharmacia company) of HRP mark, 37 ℃ of 1h.PBS with 1%Tween-20 washes plate, adds the substrate solution colour developing, the photoabsorption of reading 595 nanometers on plate reading machine.According to calculating, positive rate illustrates that near 90% total man source TNF-alpha monoclonal antibodies has very high avidity for TNF-α.
Sequence table
＜110〉Shanghai CP Guojian Pharmaceutical Co.,Ltd
＜120〉total man source tnf antibody, its preparation method and pharmaceutical composition
<160>2
<170>PatentIn version 3.1
<210>1
<211>119
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
<222>(1)..(119)
＜223〉the weight chain variable region amino acid sequence of anti-TNF-Alpha antibodies
<400>1
Gln Val Lys Ile Glu Glu Thr Thr Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Met Lys Ile Ser Cys Val Ala Ser Gly Phe Leu Phe Ser Asn His
20 25 30
Trp Met Asn Trp Val Arg Gln Ser Ile Glu Lys Gly Leu Glu Trp Ala
35 40 45
Ala Glu Leu Arg Ser Lys Thr Ile Asn Ser Ala Thr His Tyr Val Glu
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ala
65 70 75 80
Val Leu Leu Gln Pro Ser Asp Leu Arg Thr Glu Asp Ser Gly Val Tyr
85 90 95
Tyr Cys Ser Arg Ash Tyr Tyr Glu Thr Thr Tyr Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Leu Thr Val Ser
115
<210>2
<211>107
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
<222>(1)..(107)
＜223〉the aminoacyl sequence of anti-TNF-Alpha antibodies variable region of light chain
<400>2
Asn Ile Ile Leu Ser Gln Ser Pro Ala Leu Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Val Thr Phe Thr Cys Arg Ala Ser Pro Phe Val Gly Ser Ser
20 25 30
Ile His Trp Tyr Gln Pro Arg Thr Asn Gly Ser Pro Arg Ile Leu Ile
35 40 45
Lys Tyr Ala Ser Glu Thr Met Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Leu Phe Thr Leu Thr Phe Asn Thr Val Glu Thr
65 70 75 80
Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser His Ser Trp Leu Phe
85 90 95
Thr Phe Gly Ser Gly Ser Asn Leu Glu Val Lys
100 105
Claims (4)
1. an anti-TNF-alpha monoclonal antibodies is characterized in that, it is the total man source, and its variable region of heavy chain has the described aminoacid sequence of SEQ ID NO:1, and variable region of light chain has the described aminoacid sequence of SEQ ID NO:2.
2. the dna molecular of the described anti-TNF-alpha monoclonal antibodies of coding claim 1 is characterized in that the described sequence of its variable region of heavy chain nucleotide coding SEQ ID NO:1, the described nucleotide sequence of variable region of light chain nucleotide coding SEQ IDNO:2.
3. a pharmaceutical composition is characterized in that, it contains described monoclonal antibody of claim 1 and pharmaceutically acceptable carrier.
4. the purposes of the described monoclonal antibody of claim 1 is characterized in that, is used to prepare the medicine for the treatment of rheumatoid arthritis.
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| WO2008141511A1 (en) * | 2007-05-22 | 2008-11-27 | Human Antibodomics (Shanghai) Inc. | A HUMAN ANTI-TNFα MONOCLONAL ANTIBODY AND THE USE THEREOF |
| CN101684156B (en) * | 2008-09-27 | 2011-12-28 | 苏州工业园区晨健抗体组药物开发有限公司 | Human TNF alpha monoclonal antibody, PEGylation nanoparticle and application thereof |
| WO2010105446A1 (en) * | 2009-03-20 | 2010-09-23 | 上海宏源生物技术有限公司 | A human anti-tumor necrosis factor alpha monoclonal antibody and use thereof |
| CN101696243B (en) * | 2009-10-27 | 2012-05-16 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Human genetic engineering antibody TRD 109 as well as preparation method and application thereof |
| CN102127167B (en) * | 2010-01-15 | 2013-06-19 | 苏州工业园区晨健抗体组药物开发有限公司 | Whole human TNF alpha (tumor necrosis factor alpha) monoclonal antibody and preparation and application thereof |
| CN102167741B (en) * | 2010-02-25 | 2014-05-14 | 上海百迈博制药有限公司 | Fully human anti-TNF-alpha (Tumor Necrosis Factor-alpha) monoclonal antibody and preparation method as well as application thereof |
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| CN1383450A (en) * | 2000-06-06 | 2002-12-04 | 细胞技术研究及开发有限公司 | Antibody molecules having specificity for huamn tumor necrosis factory alpha, and use thereof |
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| A single dose, placebo controlled study of the fullyhumananti-tumor necrosis factor-alpha antibody adalimumab(D2E7)in patients with rheumatoid arthritis. DEN BROEDER A 等.J. Rheumatol,Vol.29 No.11. 2002 * |
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