CN1510052A - Tumour necrosis factor antibody preparing method and medicinal composition thereof - Google Patents
Tumour necrosis factor antibody preparing method and medicinal composition thereof Download PDFInfo
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Abstract
A monoclonal TNF antibody with high specificity and low immunogenicity for treating rheumatoid arthritis, its preparing process, and the medicinal composition containing it are disclosed.
Description
Technical field
The present invention relates to medical field.More specifically, the present invention relates to tnf antibody, its encoding sequence, preparation method and application thereof.
Background technology
Tumour necrosis factor (Tumor Necrosis Factor, TNF) 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.
TNF α is at rheumatoid arthritis (Rheumatoid Arthritis, RA) play a key role in the pathology process, TNF horizontal abnormality rising (Feldman M in patient or the animal model pathology joint cavity, Brennan FM, Maini RN.The role of cytokines in rheumatoid arthritis.AnnRev Immunol 1996; 14:397; Grom A, Murray KF, Luyrink L etc.).On the one hand, the receptors bind of it and synovium of joint cell causes this cell coup injury; On the other hand, it can convene immune effector cell to assemble so far, secretes more cytokine, produces stronger more persistent autoimmune response.At the RA patient part excessive TNF-alpha expression being arranged, is one of principal element that causes disease sites swelling and pain.And the TNF monoclonal antibody can in and TNF α, in vivo the activity of TNF has been played the effect of negative adjusting.And have in a large number and studies show that TNF 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.The medicable disease of TNF antibody also comprises bacterium or virus infection, chronic inflammatory diseases, autoimmune disease, malignant tumour and/or nerve degenerative diseases etc. in addition.
1987, people such as Cerami obtained the polyclone murine antibody (EPO patent publications on March 4th, 0212489,1987) of a kind of TNF, can be used for immunoassay of shock diagnostic and treatment that infectation of bacteria causes.Obtained the monoclonal antibody of TNF 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 recombinant human TNF, has among the TNF 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 antibodies of basically eliminate antibody mediated immunity originality, thereby can be used for clinical treatment.
Summary of the invention
Purpose of the present invention provides a specific specificity height, anti-TNF monoclonal antibody that immunogenicity is low.
Another object of the present invention provides described anti-TNF MONOCLONAL ANTIBODIES SPECIFIC FOR method.
Another object of the present invention provides a kind of pharmaceutical composition that contains described anti-TNF monoclonal antibody.
In a first aspect of the present invention, provide a kind of monoclonal antibody V
HChain, the aminoacid sequence with the group of being selected from down:
1-119 among the SEQ ID NO:10;
1-118 among the SEQ ID NO:12;
SEQ?ID?NO：14。
In a second aspect of the present invention, provide a kind of monoclonal antibody V
LChain has the aminoacid sequence that is selected from down the CDR that organizes:
135-241 among the SEQ ID NO:10;
134-244 among the SEQ ID NO:12;
SEQ?ID?NO：16。
In a third aspect of the present invention, provide a kind of monoclonal antibody, its V
HChain has the aminoacid sequence of the group of being selected from down:
1-119 among the SEQ ID NO:10;
1-118 among the SEQ ID NO:12;
SEQ?ID?NO：14；
And its V
LChain has the aminoacid sequence of the group of being selected from down:
135-241 among the SEQID NO:10;
134-244 among the SEQID NO:12;
SEQ?ID?NO：16。
In a preference, the Fc district of described monoclonal antibody is the constant region of human IgG.
In another preference, described antibody is single-chain antibody.
In another preference, described antibody has the aminoacid sequence shown in SEQ ID NO:10 or 12.
In a fourth aspect of the present invention, a kind of dna molecular is provided, its coding is selected from down the polypeptide of group: the monoclonal antibody V that the present invention is above-mentioned
HChain; The monoclonal antibody V that the present invention is above-mentioned
LChain; The monoclonal antibody that the present invention is above-mentioned.
Preferably, described dna molecular has the dna sequence dna of the group of being selected from down: SEQID NO:9,11,13 and 15.
In a fifth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains monoclonal antibody and pharmaceutically acceptable carrier, the V of wherein said antibody
HChain has the aminoacid sequence of the group of being selected from down:
1-119 among the SEQ ID NO:10;
1-118 among the SEQ ID NO:12;
SEQ?ID?NO：14；
And its V
LChain has the aminoacid sequence of the group of being selected from down:
135-241 among the SEQ ID NO:10;
134-244 among the SEQ ID NO:12;
SEQ?ID?NO：16。
In a sixth aspect of the present invention, the purposes of monoclonal antibody of the present invention is provided, it is used to prepare the medicine for the treatment of rheumatoid arthritis.
Description of drawings
Fig. 1 is the structure iron of plasmid pL101.What wherein, pA represented SV40 virus adds " poly A " tail signal; HCMV promotor: the promotor (containing enhancer sequence) of expression human cytomegalic inclusion disease virus; AmpR represents ampicillin resistance gene; IgG1 constant represents human IgG1's constant region.
Fig. 2 has shown that the monoclonal antibody of the present invention of various dose is to the treatment of rheumatoid arthritis effect.
Embodiment
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., total man of the present invention source and chimeric antibody preparation procedure advanced person are easy, cheap, 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 a kind of chimeric or anti-TNF Humanized monoclonal antibodies of recombinating, it comprises people source Heng Qu (as permanent district, people source IgG1-Fc), has unique prior art constructions that is different from through variable region of heavy chain after the artificial design and variable region of light chain.
The present invention also provides aminoacid sequence and its variable region chain thereof of inosculating antibody TNF monoclonal antibody, and other protein or fusion expressed product with these chains.Particularly, the present invention includes and have the hypervariable region of containing (complementary determining region, any protein of light chain CDR) and heavy chain or protein conjugate and fusion expressed product (being immune conjugate and fusion expressed product), as long as identical or at least 90% homology of hypervariable region of this hypervariable region and light chain of the present invention and heavy chain, preferably at least 95% homology.
The antigen binding characteristic of antibody can be described by 3 specific zones that are positioned at heavy chain and variable region of light chain, be called hypermutation zone (CDR), should intersegmentally be divided into 4 frame areas (FR), the aminoacid sequence of 4 FR is relatively conservative, does not participate in association reaction directly.These CDR form ring texturees, and the βZhe Die that the FR by therebetween forms is close mutually on space structure, and the CDR on CDR on the heavy chain and the corresponding light chain has constituted the antigen binding site of antibody.Can determine which amino acid has constituted FR or CDR zone by the aminoacid sequence of antibody more of the same type.
In addition, also find the dependency structure that is made of variable region of light chain recently, compare with corresponding variable region of heavy chain that its bonded kinetics is smaller, isolating weight chain variable zone self has antigen-binding activity.
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; Or chimeric antibody, as have the murine antibody binding specificity but keep antibody from people's antibody moiety.
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, these materials can be 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 change to some extent with being prepared 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 rheumatoid arthritis, and other need suppress the occasion of TNF.
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.
When making pharmaceutical composition, be that immune conjugate with safe and effective amount is applied to Mammals, wherein this safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
Outstanding advantage of the present invention is: the having very high avidity and be humanized antibody of monoclonal antibody of the present invention.The monoclonal antibody of this high-affinity has significant values clinically.
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:ColdSpring Harbor 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 ends (back), 3 ends (forward) primer.Wherein, in each primer sequence, introduced degeneracy site, many places (ambiguity site) in order further to strengthen the versatility of primer to people's antibody V district's gene amplification.Added suitable restriction site sequence according to used carrier pCANTAB5E (a kind of phasmid is Pharmacia company product).Primer sequence is as follows:
HVH back (containing the SfiI site)
5’-GTC?CTC?GCA?ACT?GCG?GCC?CAG?CCG?GCC?ATG?GCC?CAG?GTG?CAG?YTR?NDGSAG?TCD?GS-3’(SEQID?NO：1)
hVH?forward
5’-TGA?GGA?GAC?GGT?GAC?CRK?KGT?BCC-3’(SEQ?ID?NO：2)
hVL?back
5’-GAM?ATY?SWG?MTS?ACB?CAG?TCT?CC-3’(SEQ?ID?NO：3)
HVL forward (containing Not I site)
5’-GAG?TCA?TTC?TCG?ACT?TGC?GGC?CGC?ACG?TTT?GAT?YTC?CAS?YYK?KGT?CCC-3’(SEQID?NO：4)
Wherein, the ambiguity site code name in the primer sequence is to compile according to the standard scheme of NK of international biological chemistry association (IUB) (NC) announcement.Primer all entrusts Shanghai living worker company to synthesize.
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 4 U Taq enzymes (available from magnificent company) in 100 μ l total reaction volume, condition is: 94 ℃ of 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 ends, 5 end parts sequences Design and synthetic:
Joint template DNA sequence:
5’-GGT?GGA?GGC?GGT?TCA?GGC?GGA?GGT?GGC?TCT?GGC?GGT?GGC?GGA?TCG-3’(SEQID?NO：6)
Be used for the reverse VH sequence of ScFv joint
5’-GVA?CMM?YGG?TCA?CCG?TCT?CCT?CAG?GTGGAG?GCG?GTT?CAG?G-3’(SEQID?NO：7)
Be used for the reverse VL sequence of ScFv joint
5’-GGA?GAC?TGV?GTS?AKC?WSR?ATK?TCC?GAT?CCG?CCA?CCG?CCA?GAG-3’(SEQ?IDNO：8)
Wherein, the international standard that the affiliated NC of IUB announces carried out in ambiguity site code name in the primer sequence, all entrusts Shanghai to give birth to worker company and synthesize.
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 and obtain the joint sequence that length is about 100 bp.
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 centrifugal post (spun-column) chromatography purification, 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 l 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 TGl 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 10ml2 * 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 recombinant human TNF (rhTNF) 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, (Tween-20 0.5% for Tris 50mmol/L, NaCl150mmol/L for TBST, 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 TGl, 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 recombinant human TNF (rhTNF) that adds 50 μ l 200ng/ml, the BSA sealing, 37 ℃ of incubation 1h add (KCl2.7mmol/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 phage antibody is hatched 2 h for 37 ℃.Wash plate, add the goat-anti M13 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 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 joint sequence.The VH gene order is in the upstream of joint, and the VL gene order is in the downstream of joint.Result's following (wherein underscore partly is a joint sequence):
Clone 1:
GAGGTGAAGC?TGGAGGAGTC?CGGCGGCGGC?CTGGTGCAGC?CCGGCGGCTC?CATGAAGCTG??60
TCCTGCGTGG?CCACCGGCTT?CATCTTCTCC?AACCACTGGA?TGAACTGGGT?GCGCGAGACC??120
CCCGAGAAGG?GCCTGGAGTG?GGTGGCCGAG?ATCCGCTCCA?AGTCCATCAA?CTCCGTGACC??180
CACTACGCCG?AGTCCGTGAA?GGGCCGCTTC?CCCATCTCCC?GCGACGACTC?CAAGTCCGCC??240
GTGTACCTGC?AGCTGACCGA?CCTGCGCACC?GAGGACACCG?GCGCCTACTA?CTGCTCCCGC??300
AACTACTACG?GCTCCACCTA?CGACTACTGG?GGCCAGGGCA?CCACCCTGAC?CGTGTCCGGC??360
GGCGGCGGCT?CCGGCGGCGG?CGGCTCCGGC?GGCGGCGGCT?CCGACATCCT?GCTGACCCAG??420
TCCCCCGCCA?TCCTGTCCGT?GTCCCCCGGC?GAGCGCGTGT?CCCCCTCCTG?CCGCGCCTCC??480
CAGTTCGTGG?GCTCCACCAT?CCACTGGTAC?CAGCAGCGCA?CCAACGGCTC?CCCCCGCCTG??540
CTGATCAAGT?ACGTGTCCGA?GTCCATGTCC?GGCATCCCCT?CCCGCTTCAC?CGGCTCCGGC??600
TCCGGCACCG?ACTTCACCCT?GTCCATCAAC?ACCGCCGAGT?CCGAGGACGT?GGCCGACTAC??660
TACTGCCAGC?AGTCCCACTC?CTGGCCCTTC?ACCTTCGGCT?CCGGCACCAA?CCTGGAGGTG??720
AAG????????????????????????????????????????????????????????????????723
(723bp)(SEQ?ID?NO：9)
Wherein, variable region of heavy chain is 1-357 position among the SEQ ID NO:9, and variable region of light chain is the 403-723 position.
EVKLEESGGG?LVQPGGSMKL?SCVATGFIFS?NHMMNWVRET?PEKGLEWVAE?IRSKSINSVT??60
HYAESVKGRF?PISRDDSKSA?VYLQLTDLRT?EDTGAYYCSR?NYYGSTYDYW?GQGTTLTVSG??120
GGGSGGGGSG?GGGSDILLTQ?SPAILSVSPG?ERVSPSCRAS?QFVGSTIHWY?QQRTNGSPRL??180
LIKYVSESMS?GIPSRFTGSG?SGTDFTLSIN?TAESEDVADY?YCQQSHSWPF?TFGSGTNLEV??240
K??????????????????????????????????????????????????????????????????241
(SEQID?NO：10)
Wherein, the aminoacid sequence of variable region of heavy chain is SEQ ID NO:10 1-119 position, and variable region of light chain is the 135-241 position.
Clone 2:
ATGGCCAACG?TGCAGCTGGT?GCAGTCCGGC?GCCGAGGTGA?AGAAGCCCGG?CGAGTCCCTG??60
AAGATCTCCT?GCAAGGGCTC?CGGCTACTCC?TTCACCTCCT?ACTGGATCGG?CTGGGTGCGC??120
CAGATGCCCG?GCAAGGGCCT?GGAGTGGATG?GGCATCATCT?ACCCCGGCGA?CTCCGACACC??180
CGCTACTCCC?CCTCCTTCCA?GGGCCAGGTG?ACCATCTCCG?CCGACAAGTC?CATCTCCACC??240
GCCTACCTGC?AGTGGTCCTC?CCTGAAGGCC?TCCGACACCG?CCATGTACTA?CTGCGCCTCC??300
CACGGCTGGG?GCATGGACGT?GTGGGGCCAG?GGCACCCTGG?TGACCGTGTC?CTCCGGCGGC??360
GGCGGCTCCG?GCGGCGGCGG?CTCCGGCGGC?GGCGGCTCCG?CCGCCGTGCT?GACCCAGCCC??420
TCCTCCGTGT?CCGGCGCCCC?CGGCCAGCGC?GTGACCATCT?CCTGCACCGG?CTCCTCCTCC??480
AACATCGGCG?CCGGCTACGA?CGTGCACTGG?TACCAGCAGC?TGCCCGGCAC?CGCCCCCAAG??540
CTGCTGATCT?ACGGCAACTC?CAACCGCCCC?TCCGGCGTGC?CCGACCGCTT?CTCCGGCTCC??600
AAGTCCGGCA?CCTCCGCCTC?CCTGGCCATC?ACCGGCCTGG?CCGCCGAGGA?CGAGGCCGAC??660
TACTACTGCC?AGTCCTACGA?CTCCTCCCTG?TCCGGCTCCG?TGTTCGGCGG?CGGCACCAAG??720
CTGACCGTGC?TG??????????????????????????????????????????????????????732
(SEQ?ID?NO：11)
Wherein, variable region of heavy chain is 1-354 position among the SEQ ID NO:11, and variable region of light chain is the 400-732 position.
The aminoacid sequence of deriving from above-mentioned dna sequence dna is:
MANVQLVQSG?AEVKKPGESL?KISCKGSGYS?FTSYWIGWVR?QMPGKGLEWM?GIIYPGDSDT??60
RYSPSFQGQV?TISADKSIST?AYLQWSSLKA?SDTAMYYCAS?HGWGMDVWGQ?GTLVTVSSGG??120
GGSGGGGSGG?GGSAAVLTQP?SSVSGAPGQR?VTISCTGSSS?NIGAGYDVHW?YQQLPGTAPK??180
LLIYGNSNRP?SGVPDRFSGS?KSGTSASLAI?TGLAAEDEAD?YYCQSYDSSL?SGSVFGGGTK??240
LTVL???????????????????????????????????????????????????????????????244
(SEQ?ID?NO：12)
Wherein, the variable region of heavy chain corresponding amino acid sequence is SEQ ID NO:12 1-118 position, and variable region of light chain is the 134-244 position.
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/LTris-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 Wersten 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 respectively and the primer of synthetic heavy chain and light chain, add the XbaI/NheI restriction enzyme site at heavy chain primer front end; Add the HindIII/BsiWI restriction enzyme site at light chain primer front end.
Each design of primers is as follows:
Clone 1:
Primer A:5 '-CAGGCTAGCGAGGTGAAGCTGGAGGAGTCCG-3 ' (SEQ ID NO:17)
Primer a:5 '-CAG TCTAGAGGACACGGTCAGGGTGGTGCCCTG-3 ' (SEQ ID NO:18)
Primer B:5 '-CAGAAGCTTCCGACATCCTGCTGACCCAGTCC-3 ' (SEQID NO:19)
Primer b:5 '-CAGCGTACGCTTCACCTCCAGGTTGGTG-3 ' (SEQ ID NO:20)
Clone 2:
Primer C:5 '-CAGGCTAGCATGGCCAACGTGCAGCTGGTGCAG-3 ' (SEQID NO:21)
Primer ' c:5 '-CAGTCTAGAGCCGGAGGACACGGTCACCAGGG-3 ' (SEQ ID NO:22)
Primer D:5 '-CAGAAGCTTGCCGTGCTGACCCAGCCCTCC-3 ' (SEQ ID NO:23)
Primer d:5 '-CAGCGTACGCAGCACGGTCAGCTTGGTGC-3 ' (SEQ ID NO:24)
Use above-mentioned synthetic primer respectively, the ScFv fragment that obtains with embodiment 3 is a template, 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.
With above-mentioned variable region of heavy chain (1-357 position among the SEQ ID NO:9 (corresponding amino acid sequence is SEQ IDNO:10 1-119 position), or among the SEQ ID NO:11 1-354 position (corresponding amino acid sequence is SEQ IDNO:12 1-118 position)) be inserted in the XbaI/NheI site of expression vector pL101, use Hind III and Bsi WI with above-mentioned antibody chain variable region (403-723 position among the SEQID NO:9 (corresponding amino acid sequence is SEQID NO:10 135-241 position) again, or among the SEQ ID NO:11 400-732 position (corresponding amino acid sequence is SEQ ID NO:12 134-244 position)) be inserted in the HindIII/Bsi WI site of the pL101 that is inserted with the variable region of heavy chain encoding sequence, make up the expression vector of adult source TNF antibody.Expression vector pL101 is available from Invitrogen company, and its structure iron is seen Fig. 1.
The expression vector that has antibody gene of above-mentioned structure changes at e.colistraindh5, be inoculated in then in 100 milliliters of LB substratum and increase, with ultrapure plasmid DNA purification kit (Ultrapure Plasmid DNA Purification Kit) the 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 | 2B3 | ?3D9 | ?4C4 | ?5H3 | ?7A3 | ?4B5 | ?5A4 | ?6F5 |
| Expression amount | 255.7 | ?174.9 | ?242.8 | ?144.7 | ?262.3 | ?177.2 | ?253.4 | ?290.7 |
As can be seen from Table 1, the cell strain that is numbered 6F5,7A3 and 2B3 has very high expression level (140-290 mg/litre), and wherein the expression amount of 6F5 has reached 290.7 mg/litre.Select the high clone of expression amount and carry out mass cell cultivation and purifying, the preparation anti-TNF antibodies.
Embodiment 6
The chimeric TNF-alpha Study of Monoclonal Antibodies of people mouse process
The recombinant human TNF (rhTNF, the Invitrogen product is available from Shenzhen brilliant U.S. company) that adopts the 40mu.g purifying is as immunogen, add isopyknic complete Freund's adjuvant, fully be ground to conventional subcutaneous multi-point injection 10 age in days Balb/c mouse after the complete emulsification, injection 50 microlitres, totally 6 points at every.Then with 1 weekly interval totally 5 duplicate injections react with booster immunization, and the serum antibody level is monitored.Last intravenous injection 5 * 10
6New fresh cell (being the cell strain of TNF secretion antibody)/PBS mixed solution.
After 4 days, mouse is put to death, take out splenocyte, be prepared into suspension.Splenocyte and non-secretion hybridoma Sp2/O (available from ATCC, preserving number CRL1581) were carried out PEG with 4: 1 to be merged.Hatched 6 hours at 37 ℃, O.2ml fused cell changes 96 orifice plates over to, culturing cell.The nutrient solution that brings Selection In after 24 hours was changed fresh culture 1 time in per 3 days, to growing the clone.
Select mono-clonal to form the hole, took out supernatant liquor at the 14th day, carry out ripe bone-marrow-derived lymphocyte cell fluorescence dyeing (supernatant and bone-marrow-derived lymphocyte 37 degree incubations 30 minutes, 1XPBS washing 10 times, the rabbit anti-mouse antibody that adds the FITC mark is anti-as two, 1XPBS washing 10 times, and the 1XPBS that contains 1%Tween-20 washs 10 times, the 1XPBS that contains 0.5%Triton X-100 washs 10 times, reads the fluorescence intensity at 650nm place on microplate reader).Positive hole keeps, and selects the high expressing cell strain behind the subclone 3 times.
The method of employing 5 ' RACE is obtained the variable region gene of monoclonal antibody, be cloned in pGEM-T (Promega) carrier, at the dull and stereotyped enterprising row filter of IPTG/X-gal, picking white bacterial plaque is inoculated in the LB liquid nutrient medium that contains penbritin and increases behind the transformed into escherichia coli TG1 cell.Screening positive clone with qiagen plasmid extraction agent box extracting plasmid and check order, is determined heavy chain and light chain variable region sequence.
The dna sequence dna that the mouse source anti-people TNFa monoclonal antibody that obtains is measured is as follows:
Variable region of heavy chain
GAAGTGAAGC?TTGAGGAGTC?TGGAGGAGGC?TTGGTGCAAC?CTGGAGGATC?CATGAAACTC??60
TCCTGTGTTG?CCTCTGGATT?CATTTTCAGT?AACCACTGGA?TGAACTGGGT?CCGCCAGTCT??120
CCAGAGAAGG?GGCTTGAGTG?GGTTGCTGAA?ATTAGATCAA?AATCTATTAA?TTCTGCAACA??180
CATTATGCGG?AGTCTGTGAA?AGGGAGGTTC?ACCATCTCAA?GAGATGATTC?CAAAAGTGCT??240
GTCTACCTGC?AAATGACCGA?CTTAAGAACT?GAAGACACTG?GCGTTTATTA?CTGTTCCAGG??300
AATTACTACG?GTAGTACCTA?CGACTACTGG?GGCCAAGGCA?CCACTCTCAC?AGTCTCC?????357
(SEQ?ID?NO：13)
The aminoacid sequence of inferring is
EVKLEESGGG?LVQPGGSMKL?SCVASGFIFS?NHWMNWVRQS?PEKGLEWVAE?IRSKSINSAT?60
HYAESVKGRF?TISRDDSKSA?VYLQMTDLRT?EDTGVYYCSR?NYYGSTYDYW?GQGTTLTVS??119
(SEQID?NO：14)
Variable region of light chain
GACATCTTGC?TGACTCAGTC?TCCAGCCATC?CTGTCTGTGA?GTCCAGGAGA?AAGAGTCAGT??60
TTCTCCTGCA?GGGCCAGTCA?GTTCGTTGGC?TCAAGCATCC?ACTGGTATCA?GCAAAGAACA??120
AATGGTTCTC?CAAGGCTTCT?CATAAAGTAT?GCTTCTGAGT?CTATGTCTGG?GATCCCTTCC??180
AGGTTTAGTG?GCAGTGGATC?AGGGACAGAT?TTTACTCTTA?GCATCAACAC?TGTGGAGTCT??240
GAAGATATTG?CAGATTATTA?CTGTCAACAA?AGTCATAGCT?GGCCATTCAC?GTTCGGCTCG??300
GGGACAAATT?TGGAAGTAAA?A????????????????????????????????????????????321
(SEQID?NO：15)
The aminoacid sequence of inferring
DILLTQSPAI?LSVSPGERVS?FSCRASQFVG?SSIHWYQQRT?NGSPRLLIKY?ASESMSGIPS??60
RFSGSGSGTD?FTLSINTVES?EDIADYYCQQ?SHSWPFTFGS?GTNLEVK????????????107
(SEQ?ID?NO：16)
By being similar to program identical among the embodiment 4, the variable region of heavy chain of above-mentioned antibody is inserted in the XbaI/NheI site of expression vector pL101, again 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, constitutes the expression vector that contains chimeric antibody gene.This expression vector contains heavy chain and the variable region of light chain and the people Fc formation antibody gene in mouse source.
Change this expression vector over to e.colistraindh5, be inoculated in then in 100 milliliters of LB substratum and increase, with ultrapure plasmid DNA purification kit (Ultrapure Plasmid DNA Purification Kit) the 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.Chinese hamster ovary celI is carried out cultured continuously and mono-clonalization, the monoclonal cell of choosing ties up on the RPM1641 substratum and cultivates, supernatant is carried out Western Blotting experiment, judge expression intensity according to staining reaction, pick out and express strong clone, promptly obtain the higher candidate clone of expression intensity as the candidate cell strain.Filter out the high clone of expression intensity.
Culture supernatant is carried out centrifugal, remove cell and fragment, carry out the albumin A affinity chromatography, determine purity, determined to obtain purity greater than 95% monoclonal antibody with HPLC.
Embodiment 7
Total man source TNF monoclonal antibody is for the therapeutic action of collagen-induced rheumatoid arthritis B10.RIII mouse
100.mu.g pig II Collagen Type VI (CII) adds complete Freund's adjuvant, intradermal injection B10.RIII mouse, repeat once after 14 days again, at this moment occur collagen-induced rheumatoid arthritis (CIA) symptom in the mouse body, 92% mouse shows serious CIA symptom after 28 days.Mouse peritoneum is injected people of the present invention source TNF monoclonal antibody, observe its influence in the mouse body the symptom of CIA.Detect after 12 weeks.
Experimental mice 3 days weekly, per injection 64.mu.g (high dosage), the total man's resource monoclonal antibody that obtains among 16.mu.g (middle dosage) and 8.mu.g (low dosage) embodiment 5 was from the 1st day to 35 days; To control group mice injection PBS.Estimate the severity of rheumatoid arthritis according to a conventional method, the high more expression of score value is serious more.
The results are shown in Figure 2, this shows that TNF monoclonal antibody of the present invention compares with control group, all can significantly reduce rheumatoid severity.Therefore, monoclonal antibody of the present invention can be developed into the medicine into the treatment rheumatoid arthritis.
In addition, the people mouse chimeric mAb of embodiment 6 is also carried out identical animal model experiment, obtained similar result of treatment.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉horse, cyanines
＜120〉tnf antibody, its preparation method and pharmaceutical composition
<130>027424
<160>24
<170>PatentIn?version?3.1
<210>1
<211>56
<212>DNA
＜213〉artificial sequence
<220>
<221>misc?feature
<222>(46)..(46)
<223>n＝a，t，c，g
<220>
<221>misc?feature
<222>(1)..(56)
＜223〉primer
<400>1
gtcctcgcaa?ctgcggccca?gccggccatg?gcccaggtgc?agytrndgsa?gtcdgs????56
<210>2
<211>24
<212>DNA
＜213〉artificial sequence
<220>
<221>misc?feature
<222>(1)..(24)
＜223〉primer
<400>2
tgaggagacg?gtgaccrkkg?tbcc????????????????????????????????????????24
<210>3
<211>23
<212>DNA
＜213〉artificial sequence
<220>
<221>misc?feature
<222>(1)..(23)
＜223〉primer
<400>3
gamatyswgm?tsacbcagtc?tcc???????????????????????????????23
<210>4
<211>48
<212>DNA
＜213〉artificial sequence
<220>
<221>misc?feature
<222>(1)..(48)
＜223〉primer
<400>4
gagtcattct?cgacttgcgg?ccgcacgttt?gatytccasy?ykkgtccc????48
<210>5
<211>15
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC?FEATURE
<222>(1)..(15)
＜223〉joint
<400>5
Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser
1????????????????5???????????????????10?????????????????15
<210>6
<211>45
<212>DNA
＜213〉artificial sequence
<220>
<221>misc?feature
<222>(1)..(45)
＜223〉oligonucleotide
<400>6
ggtggaggcg?gttcaggcgg?aggtggctct?ggcggtggcg?gatcg????????45
<210>7
<211>40
<212>DNA
＜213〉artificial sequence
<220>
<221>misc?feature
<222>(1)..(40)
＜223〉primer
<400>7
gvacmmyggt?caccgtctcc?tcaggtggag?gcggttcagg?????40
<210>8
<211>42
<212>DNA
＜213〉artificial sequence
<220>
<221>misc?feature
<222>(1)..(42)
＜223〉primer
<400>8
ggagactgvg?tsakcwsrat?ktccgatccg?ccaccgccag?ag??42
<210>9
<211>723
<212>DNA
＜213〉artificial sequence
<220>
<221>misc?feature
<222>(1)..(723)
＜223〉encoding sequence of strand anti-TNF antibodies
<400>9
gaggtgaagc?tggaggagtc?cggcggcggc?ctggtgcagc?ccggcggctc?catgaagctg????60
tcctgcgtgg?ccaccggctt?catcttctcc?aaccactgga?tgaactgggt?gcgcgagacc????120
cccgagaagg?gcctggagtg?ggtggccgag?atccgctcca?agtccatcaa?ctccgtgacc????180
cactacgccg?agtccgtgaa?gggccgcttc?cccatctccc?gcgacgactc?caagtccgcc????240
gtgtacctgc?agctgaccga?cctgcgcacc?gaggacaccg?gcgcctacta?ctgctcccgc????300
aactactacg?gctccaccta?cgactactgg?ggccagggca?ccaccctgac?cgtgtccggc????360
ggcggcggct?ccggcggcgg?cggctccggc?ggcggcggct?ccgacatcct?gctgacccag????420
tcccccgcca?tcctgtccgt?gtcccccggc?gagcgcgtgt?ccccctcctg?ccgcgcctcc????480
cagttcgtgg?gctccaccat?ccactggtac?cagcagcgca?ccaacggctc?cccccgcctg??540
ctgatcaagt?acgtgtccga?gtccatgtcc?ggcatcccct?cccgcttcac?cggctccggc??600
tccggcaccg?acttcaccct?gtccatcaac?accgccgagt?ccgaggacgt?ggccgactac??660
tactgccagc?agtcccactc?ctggcccttc?accttcggct?ccggcaccaa?cctggaggtg??720
aag????????????????????????????????????????????????????????????????723
<210>10
<211>241
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
<222>(1)..(241)
＜223〉aminoacid sequence of strand anti-TNF antibodies
<400>10
Glu?Val?Lys?Leu?Glu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1????????????????5???????????????????10?????????????????15
Ser?Met?Lys?Leu?Ser?Cys?Val?Ala?Thr?Gly?Phe?Ile?Phe?Ser?Asn?His
20??????????????????25??????????????????30
Trp?Met?Asn?Trp?Val?Arg?Glu?Thr?Pro?Glu?Lys?Gly?Leu?Glu?Trp?Val
35??????????????????40???????????????????45
Ala?Glu?Ile?Arg?Ser?Lys?Ser?Ile?Asn?Ser?Val?Thr?His?Tyr?Ala?Glu
50??????????????????55???????????????????60
Ser?Val?Lys?Gly?Arg?Phe?Pro?Ile?Ser?Arg?Asp?Asp?Ser?Lys?Ser?Ala
65??????????????????70??????????????????75???????????????????80
Val?Tyr?Leu?Gln?Leu?Thr?Asp?Leu?Arg?Thr?Glu?Asp?Thr?Gly?Ala?Tyr
85?????????????????90??????????????????95
Tyr?Cys?Ser?Arg?Asn?Tyr?Tyr?Gly?Ser?Thr?Tyr?Asp?Tyr?Trp?Gly?Gln
100??????????????????105????????????????110
Gly?Thr?Thr?Leu?Thr?Val?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly
115?????????????????????120?????????????????125
Ser?Gly?Gly?Gly?Gly?Ser?Asp?Ile?Leu?Leu?Thr?Gln?Ser?Pro?Ala?Ile
130?????????????????135?????????????????140
Leu?Ser?Val?Ser?Pro?Gly?Glu?Arg?Val?Ser?Pro?Ser?Cys?Arg?Ala?Ser
145?????????????????150?????????????????155?????????????????160
Gln?Phe?Val?Gly?Ser?Thr?Ile?His?Trp?Tyr?Gln?Gln?Arg?Thr?Asn?Gly
165?????????????????170?????????????????175
Ser?Pro?Arg?Leu?Leu?Ile?Lys?Tyr?Val?Ser?Glu?Ser?Met?Ser?Gly?Ile
180?????????????????185?????????????????190
Pro?Ser?Arg?Phe?Thr?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Ser
195?????????????????200??????????????????205
Ile?Asn?Thr?Ala?Glu?Ser?Glu?Asp?Val?Ala?Asp?Tyr?Tyr?Cys?Gln?Gln
210?????????????????215?????????????????220
Ser?His?Ser?Trp?Pro?Phe?ThrPhe?Gly?Ser?Gly?Thr?Asn?Leu?Glu?Val
225?????????????????230????????????????235?????????????????240
Lys
<210>11
<211>732
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(732)
＜223〉encoding sequence of strand anti-TNF antibodies
<400>11
atggccaacg?tgcagctggt?gcagtccggc?gccgaggtga?agaagcccgg?cgagtccctg????60
aagatctcct?gcaagggctc?cggctactcc?ttcacctcct?actggatcgg?ctgggtgcgc????120
cagatgcccg?gcaagggcct?ggagtggatg?ggcatcatct?accccggcga?ctccgacacc????180
cgctactccc?cctccttcca?gggccaggtg?accatctccg?ccgacaagtc?catctccacc????240
gcctacctgc?agtggtcctc?cctgaaggcc?tccgacaccg?ccatgtacta?ctgcgcctcc????300
cacggctggg?gcatggacgt?gtggggccag?ggcaccctgg?tgaccgtgtc?ctccggcggc????360
ggcggctccg?gcggcggcgg?ctccggcggc?ggcggctccg?ccgccgtgct?gacccagccc??420
tcctccgtgt?ccggcgcccc?cggccagcgc?gtgaccatct?cctgcaccgg?ctcctcctcc??480
aacatcggcg?ccggctacga?cgtgcactgg?taccagcagc?tgcccggcac?cgcccccaag??540
ctgctgatct?acggcaactc?caaccgcccc?tccggcgtgc?ccgaccgctt?ctccggctcc??600
aagtccggca?cctccgcctc?cctggccatc?accggcctgg?ccgccgagga?cgaggccgac??660
tactactgcc?agtcctacga?ctcctccctg?tccggctccg?tgttcggcgg?cggcaccaag??720
ctgaccgtgc?tg??????????????????????????????????????????????????????732
<210>12
<211>244
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
<222>(1)..(244)
＜223〉aminoacid sequence of strand anti-TNF antibodies
<400>12
Met?Ala?Asn?Val?Gln?Leu?Val?Gln?Ser?Gly?Ala?Glu?Val?Lys?Lys?Pro
1???????????????5???????????????????10??????????????????15
Gly?Glu?Ser?Leu?Lys?Ile?Ser?Cys?Lys?Gly?Ser?Gly?Tyr?Ser?Phe?Thr
20???????????????????25??????????????????30
Ser?Tyr?Trp?Ile?Gly?Trp?Val?Arg?Gln?Met?Pro?Gly?Lys?Gly?Leu?Glu
35??????????????????40???????????????????45
Trp?Met?Gly?Ile?Ile?Tyr?Pro?Gly?Asp?Ser?Asp?Thr?Arg?Tyr?Ser?Pro
50?????????????????55??????????????????60
Ser?Phe?Gln?Gly?Gln?Val?Thr?Ile?Ser?Ala?Asp?Lys?Ser?Ile?Ser?Thr
65??????????????????70???????????????????75??????????????????80
Ala?Tyr?Leu?Gln?Trp?Ser?Ser?Leu?Lys?Ala?Ser?Asp?Thr?Ala?Met?Tyr
85??????????????????90???????????????????95
Tyr?Cys?Ala?Ser?His?Gly?Trp?Gly?Met?Asp?Val?Trp?Gly?Gln?Gly?Thr
100?????????????????105?????????????????110
Leu?Val?Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser
115?????????????????120?????????????????125
Gly?Gly?Gly?Gly?Ser?Ala?Ala?Val?Leu?Thr?Gln?Pro?Ser?Ser?Val?Ser
130????????????????135??????????????????140
Gly?Ala?Pro?Gly?Gln?Arg?Val?Thr?Ile?Ser?Cys?Thr?Gly?Ser?Ser?Ser
145????????????????150??????????????????155?????????????????160
Asn?Ile?Gly?Ala?Gly?Tyr?Asp?Val?His?Trp?Tyr?Gln?Gln?Leu?Pro?Gly
165?????????????????170?????????????????175
Thr?Ala?Pro?Lys?Leu?Leu?Ile?Tyr?Gly?Asn?Ser?Asn?Arg?Pro?Ser?Gly
180?????????????????185?????????????????190
Val?Pro?Asp?Arg?Phe?Ser?Gly?Ser?Lys?Ser?Gly?Thr?Ser?Ala?Ser?Leu
195??????????????????200?????????????????205
Ala?Ile?Thr?Gly?Leu?Ala?Ala?Glu?Asp?Glu?Ala?Asp?Tyr?Tyr?Cys?Gln
210?????????????????215?????????????????220
Ser?Tyr?Asp?Ser?Ser?Leu?Ser?Gly?Ser?Val?Phe?Gly?Gly?Gly?Thr?Lys
225?????????????????230?????????????????235?????????????????240
Leu?Thr?Val?Leu
<210>13
<211>357
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(357)
＜223〉the variable region of heavy chain encoding sequence of anti-TNF antibodies
<400>13
gaagtgaagc?ttgaggagtc?tggaggaggc?ttggtgcaac?ctggaggatc?catgaaactc????60
tcctgtgttg?cctctggatt?cattttcagt?aaccactgga?tgaactgggt?ccgccagtct????120
ccagagaagg?ggcttgagtg?ggttgctgaa?attagatcaa?aatctattaa?ttctgcaaca????180
cattatgcgg?agtctgtgaa?agggaggttc?accatctcaa?gagatgattc?caaaagtgct????240
gtctacctgc?aaatgaccga?cttaagaact?gaagacactg?gcgtttatta?ctgttccagg?????300
aattactacg?gtagtaccta?cgactactgg?ggccaaggca?ccactctcac?agtctcc????????357
<210>14
<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 antibodies
<400>14
Glu?Val?Lys?Leu?Glu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1???????????????5??????????????????10???????????????????15
Ser?Met?Lys?Leu?Ser?Cys?Val?Ala?Ser?Gly?Phe?Ile?Phe?Ser?Asn?His
20??????????????????25??????????????????30
Trp?Met?Asn?Trp?Val?Arg?Gln?Ser?Pro?Glu?Lys?Gly?Leu?Glu?Trp?Val
35??????????????????40?????????????????45
Ala?Glu?lle?Arg?Ser?Lys?Ser?lle?Asn?Ser?Ala?Thr?His?Tyr?Ala?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?Tyr?Leu?Gln?Met?Thr?Asp?Leu?Arg?Thr?Glu?Asp?Thr?Gly?Val?Tyr
85???????????????????90?????????????????95
Tyr?Cys?Ser?Arg?Asn?Tyr?Tyr?Gly?Ser?Thr?Tyr?Asp?Tyr?Trp?Gly?Gln
100?????????????????105?????????????????110
Gly?Thr?Thr?Leu?Thr?Val?Ser
115
<210>15
<211>321
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_teature
<222>(1)..(321)
＜223〉the variable region of light chain encoding sequence of anti-TNF antibodies
<400>15
gacatcttgc?tgactcagtc?tccagccatc?ctgtctgtga?gtccaggaga?aagagtcagt????60
ttctcctgca?gggccagtca?gttcgttggc?tcaagcatcc?actggtatca?gcaaagaaca????120
aatggttctc?caaggcttct?cataaagtat?gcttctgagt?ctatgtctgg?gatcccttcc????180
aggtttagtg?gcagtggatc?agggacagat?tttactctta?gcatcaacac?tgtggagtct????240
gaagatattg?cagattatta?ctgtcaacaa?agtcatagct?ggccattcac?gttcggctcg????300
gggacaaatt?tggaagtaaa?a??????????????????????????????????????????????321
<210>16
<211>107
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC-FEATURE
<222>(1)..(107)
＜223〉the aminoacyl sequence of anti-TNF antibodies variable region of light chain
<400>16
Asp?Ile?Leu?Leu?Thr?Gln?Ser?Pro?Ala?Ile?Leu?Ser?Val?Ser?Pro?Gly
1???????????????5???????????????????10??????????????????15
Glu?Arg?Val?Ser?Phe?Ser?Cys?Arg?Ala?Ser?Gln?Phe?Val?Gly?Ser?Ser
20??????????????????25??????????????????30
Ile?His?Trp?Tyr?Gln?Gln?Arg?Thr?Asn?Gly?Ser?Pro?Arg?Leu?Leu?Ile
35?????????????????40???????????????????45
Lys?Tyr?Ala?Ser?Glu?Ser?Met?Ser?Gly?Ile?Pro?Ser?Arg?Phe?Ser?Gly
50???????????????????55?????????????????60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Ser?Ile?Asn?Thr?Val?Glu?Ser
65??????????????????70??????????????????75??????????????????80
Glu?Asp?Ile?Ala?Asp?Tyr?Tyr?Cys?Gln?Gln?Ser?His?Ser?Trp?Pro?Phe
85??????????????????90???????????????????95
Thr?Phe?Gly?Ser?Gly?Thr?Asn?Leu?Glu?Val?Lys
100?????????????????105
<210>17
<211>31
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(31)
＜223〉primer
<400>17
caggctagcg?aggtgaagct?ggaggagtcc?g????31
<210>18
<211>33
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(33)
＜223〉primer
<400>18
cagtctagag?gacacggtca?gggtggtgcc?ctg??33
<210>19
<211>32
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(32)
＜223〉primer
<400>19
cagaagcttc?cgacatcctg?ctgacccagt?cc????32
<210>20
<211>28
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(28)
＜223〉primer
<400>20
cagcgtacgc?ttcacctcca?ggttggtg???????????28
<210>21
<211>33
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(33)
＜223〉primer
<400>21
caggctagca?tggccaacgt?gcagctggtg?cag????33
<210>22
<211>32
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(32)
＜223〉primer
<400>22
cagtctagag?ccggaggaca?cggtcaccag?gg??????32
<210>23
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(30)
＜223〉primer
<400>23
cagaagcttg?ccgtgctgac?ccagccctcc?????????30
<210>24
<211>29
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(29)
＜223〉primer
<400>24
cagcgtacgc?agcacggtca?gcttggtgc????29
Claims (10)
1. monoclonal antibody V
HChain is characterized in that, has the aminoacid sequence of the group of being selected from down:
1-119 among the SEQ ID NO:10;
1-118 among the SEQ ID NO:12;
SEQ?ID?NO：14。
2. monoclonal antibody V
LChain is characterized in that, has the aminoacid sequence of the CDR of the group of being selected from down:
135-241 among the SEQID NO:10;
134-244 among the SEQID NO:12;
SEQID?NO：16。
3. a monoclonal antibody is characterized in that, its V
HChain has the aminoacid sequence of the group of being selected from down:
1-119 among the SEQID NO:10;
1-118 among the SEQID NO:12;
SEQID?NO：14；
And its V
LChain has the aminoacid sequence of the group of being selected from down:
135-241 among the SEQID NO:10;
134-244 among the SEQID NO:12;
SEQID?NO：16。
4. monoclonal antibody as claimed in claim 3 is characterized in that, described antibody Fc district is the constant region of human IgG.
5. monoclonal antibody as claimed in claim 3 is characterized in that described antibody is single-chain antibody.
6. monoclonal antibody as claimed in claim 5 is characterized in that described antibody has the aminoacid sequence shown in SEQID NO:10 or 12.
7. a dna molecular is characterized in that, its coding is selected from down the polypeptide of group:
The described monoclonal antibody V of claim 1
HChain;
The described monoclonal antibody V of claim 2
LChain;
The described monoclonal antibody of claim 3.
8. dna molecular as claimed in claim 7 is characterized in that, it has the dna sequence dna of the group of being selected from down: SEQ ID NO:9,11,13 and 15.
9. a pharmaceutical composition is characterized in that, it contains monoclonal antibody and pharmaceutically acceptable carrier, the V of wherein said antibody
HChain has the aminoacid sequence of the group of being selected from down:
1-119 among the SEQ ID NO:10;
1-118 among the SEQ ID NO:12;
SEQ?ID?NO：14；
And its V
LChain has the aminoacid sequence of the group of being selected from down:
135-241 among the SEQ ID NO:10;
134-244 among the SEQ ID NO:12;
SEQ?ID?NO：16。
10. the purposes of the described monoclonal antibody of claim 3 is characterized in that, is used to prepare the medicine for the treatment of rheumatoid arthritis.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02157659 CN1225479C (en) | 2002-12-23 | 2002-12-23 | Tumour necrosis factor antibody preparing method and medicinal composition thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02157659 CN1225479C (en) | 2002-12-23 | 2002-12-23 | Tumour necrosis factor antibody preparing method and medicinal composition thereof |
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| Publication Number | Publication Date |
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| CN1510052A true CN1510052A (en) | 2004-07-07 |
| CN1225479C CN1225479C (en) | 2005-11-02 |
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| CN 02157659 Expired - Lifetime CN1225479C (en) | 2002-12-23 | 2002-12-23 | Tumour necrosis factor antibody preparing method and medicinal composition thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101111521B (en) * | 2004-12-29 | 2011-08-24 | 株式会社柳韩洋行 | Humanized antibody specific for tumor necrosis factor-alpha |
| CN102229653B (en) * | 2009-10-23 | 2012-05-23 | 山东省医药生物技术研究中心 | Polypeptide resistant to rheumatoid arthritis and application of polypeptide in pharmacy |
| CN102229658B (en) * | 2009-10-23 | 2012-05-23 | 山东省医药生物技术研究中心 | Anti-rheumatoid arthritis polypeptide and its application in pharmacy |
| CN102229654B (en) * | 2009-10-23 | 2012-05-23 | 山东省医药生物技术研究中心 | Polypeptide resistant to rheumatoid arthritis and application of polypeptide in pharmacy |
| CN102229656B (en) * | 2009-10-23 | 2012-05-23 | 山东省医药生物技术研究中心 | Polypeptide against rheumatoid arthritis and application thereof in pharmacy |
| CN102229655B (en) * | 2009-10-23 | 2012-05-23 | 山东省医药生物技术研究中心 | Polypeptide resistant to rheumatoid arthritis and application of polypeptide in pharmacy |
| CN102229657B (en) * | 2009-10-23 | 2012-05-23 | 山东省医药生物技术研究中心 | Polypeptide against rheumatoid arthritis and application thereof in pharmacy |
| WO2012116595A1 (en) * | 2011-02-28 | 2012-09-07 | 珠海市丽珠单抗生物技术有限公司 | Tumor necrosis factor-α humanized antibody |
| CN103336129A (en) * | 2005-11-01 | 2013-10-02 | 阿布维生物技术有限公司 | Methods and compositions for diagnosing ankylosing spondylitis using biomarkers |
| US8658171B2 (en) | 2011-02-28 | 2014-02-25 | Livzon Mabpharm Inc. | Humanized anti-TNFα antibodies |
| CN111153994A (en) * | 2019-12-31 | 2020-05-15 | 武汉班科生物技术股份有限公司 | Human monoclonal antibodies to human tumor necrosis factor |
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2002
- 2002-12-23 CN CN 02157659 patent/CN1225479C/en not_active Expired - Lifetime
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101111521B (en) * | 2004-12-29 | 2011-08-24 | 株式会社柳韩洋行 | Humanized antibody specific for tumor necrosis factor-alpha |
| CN103336129A (en) * | 2005-11-01 | 2013-10-02 | 阿布维生物技术有限公司 | Methods and compositions for diagnosing ankylosing spondylitis using biomarkers |
| CN102229653B (en) * | 2009-10-23 | 2012-05-23 | 山东省医药生物技术研究中心 | Polypeptide resistant to rheumatoid arthritis and application of polypeptide in pharmacy |
| CN102229658B (en) * | 2009-10-23 | 2012-05-23 | 山东省医药生物技术研究中心 | Anti-rheumatoid arthritis polypeptide and its application in pharmacy |
| CN102229654B (en) * | 2009-10-23 | 2012-05-23 | 山东省医药生物技术研究中心 | Polypeptide resistant to rheumatoid arthritis and application of polypeptide in pharmacy |
| CN102229656B (en) * | 2009-10-23 | 2012-05-23 | 山东省医药生物技术研究中心 | Polypeptide against rheumatoid arthritis and application thereof in pharmacy |
| CN102229655B (en) * | 2009-10-23 | 2012-05-23 | 山东省医药生物技术研究中心 | Polypeptide resistant to rheumatoid arthritis and application of polypeptide in pharmacy |
| CN102229657B (en) * | 2009-10-23 | 2012-05-23 | 山东省医药生物技术研究中心 | Polypeptide against rheumatoid arthritis and application thereof in pharmacy |
| WO2012116595A1 (en) * | 2011-02-28 | 2012-09-07 | 珠海市丽珠单抗生物技术有限公司 | Tumor necrosis factor-α humanized antibody |
| US8658171B2 (en) | 2011-02-28 | 2014-02-25 | Livzon Mabpharm Inc. | Humanized anti-TNFα antibodies |
| CN111153994A (en) * | 2019-12-31 | 2020-05-15 | 武汉班科生物技术股份有限公司 | Human monoclonal antibodies to human tumor necrosis factor |
| CN111153994B (en) * | 2019-12-31 | 2021-10-15 | 武汉班科生物技术股份有限公司 | Human monoclonal antibodies to human tumor necrosis factor |
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|---|---|
| CN1225479C (en) | 2005-11-02 |
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