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, 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 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 Sfi I site)
5’-GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC CAG GTG CAG YTR NDGSAG TCD GS-3’(SEQ ID 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 4U Taq enzyme (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’(SEQ ID 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 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 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 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 M13KO7 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 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 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 2h 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 NHWMNWVRET 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
(SEQ ID 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 CGGCCGAGGA 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
(SEQID 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 '-CAGTCTAGAGGACACGGTCAGGGTGGTGCCCTG-3 ' (SEQ ID NO:18)
Primer B:5 '-CAGAAGCTTCCGACATCCTGCTGACCCAGTCC-3 ' (SEQ ID NO:19)
Primer b:5 '-CAGCGTACGCTTCACCTCCAGGTTGGTG-3 ' (SEQ ID NO:20)
Clone 2:
Primer C:5 '-CAGGCTAGCATGGCCAACGTGCAGCTGGTGCAG-3 ' (SEQ ID 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 SEQ ID NO:9 (corresponding amino acid sequence is SEQ ID 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 Sp2/0 (available from ATCC, preserving number CRL1581) were carried out PEG with 4: 1 to be merged.Hatched 6 hours at 37 ℃, change the 0.2ml fused cell over to 96 orifice plates, 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
(SEQ ID 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
(SEQ ID 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 DNAPurification 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.
<223〉the aminoacyl sequence of anti-TNF antibodies variable region of light chain