CN101684156B

CN101684156B - Human TNF alpha monoclonal antibody, PEGylation nanoparticle and application thereof

Info

Publication number: CN101684156B
Application number: CN2008102006628A
Authority: CN
Inventors: 倪健
Original assignee: SUZHOU INDUSTRIAL PARK CHENJIAN ANTIBODY GROUP PHARMACEUTICAL DEVELOPMENT CO LTD
Current assignee: Youjian Biopharmaceutical Technology Shenzhen Co ltd
Priority date: 2008-09-27
Filing date: 2008-09-27
Publication date: 2011-12-28
Anticipated expiration: 2028-09-27
Also published as: CN101684156A

Abstract

The invention discloses an antihuman TNF alpha monoclonal antibody, where the light chain amino acid sequence is SEQ ID NO:3, heavy chain variable region amino acid sequence is SEQ ID NO:4, and heavy chain overall length sequence is coded by SEQ ID NO:6. The invention further discloses a Fab antibody of the antihuman TNF alpha monoclonal antibody; in addition, the invention adopts the PEGylation method, nanometre technology and the like for in vitro transformation and modification to obtain the PEGylation antibody and PEGylation antihuman TNF alpha monoclonal antibody nanoparticles; and the invention further discloses an application of the antibody or the antibody nanoparticles. The anti TNF monoclonal antibody provided by the invention is a total human antibody sequence and has low immunogenicity, small side effect and low production cost; the monoclonal antibody adopts the nanoparticle preparing technology, the surface modified technology, the micromolecule antibody bridged linkage technology and the like to prolong and realize the purpose of in vivo slow release, avoids the shortage of short half-life period of the micromolecule antibody and is more suitable for in vivo application under the condition of keeping anti TNF activity thereof.

Description

Human TNF alpha monoclonal antibody, its PEGization nano particle and application thereof

Technical field

The present invention relates to biotechnology and field of medicaments, be specifically related to anti-human TNF alpha monoclonal antibody and PEGization nano particle and application thereof.

Background technology

Autoimmune disorder is the third-largest class disease of cancer and cardiovascular disorder of continuing.Rheumatoid arthritis (RA) is one of modal autoimmune disease, and China's rheumatoid arthritis morbidity is about 0.32-0.36%, at crowd's sickness rate more than 60 years old up to 30%～40%.In the face of the import medicine medical expense of hundreds of thousands of unit for each person every year, the patient often can only long-term uncomplaining chemotherapy, but produces little effect.

The ill crowd of RA is many, and dosage is big, makes the market of its curative drug huge, and the world market reached 6,000,000,000 dollars in 2005.At present the scheme of clinical treatment RA is a lot, and is first-selected with heavy dose of NSAID (non-steroidal anti-inflammatory drug) may command symptom, and as invalid, then immunosuppressive drug such as clinical first-selected methotrexate is treated.About 1/4th patient falls ill first after treatment in time can no longer be recurred throughout one's life, and about 1/2nd patient symptom after treating is temporarily alleviated, but still can show effect repeatedly, and part finally develops into joint function disturbance; About 1/4th patient is all invalid at present all conventional treatmenies.

Tumor necrosis factor-alpha (TNF-α) is the cytokine that plays a major role in the rheumatoid arthritis change process, and experiment and clinical effectiveness confirm that all TNF is the suitable target spot of this disease of treatment.The pathogeny of RA has been carried out sufficient research abroad, find tumour necrosis factor (Tumor Necrosis Factor alpha, TNF-α) play a key role in the pathology process of this disease, the TNF horizontal abnormality in patient or the animal model pathology joint cavity raises.TNF α and TNF β are the class homology secretary proteins in the mammalian cell, and they have very high homology each other, and the two can cause many-sided influence to many cell types.These two kinds of albumen can by with cytolemma on receptor acting influence the biological function of cell.TNF (Tumor Necrosis Factor) alpha can cause the tumor tissues hemorrhagic necrosis and have many-sided function.Ripe molecule is made up of 157 amino acid, contains two disulfide linkage, does not have glycosylation site.Its precursor is the polypeptide that 233 amino acid are formed, and the polypeptide that 76 amino acid of N-end are formed is not a signal peptide, and it can be fixed on precursor on the cytolemma, and sophisticated secretor type is retained on the cytolemma.The ripe mRNA length of precursor is 1.7kb.Mainly produce in vivo by mononuclear macrophage.

Inhibitor at TNF-α becomes up to now the most successful RA medicine.Represent medicine such as the Enbrel of Wyeth/Amgen company (etanercept), Johnson ﹠amp; Johnson, the Schering Plough/Centocor Remicade of company (infliximab), the Humira (adalimumab) of Cambridge Antibody Technology/Abbott.Enbrel is the preparation of Amgen company exploitation, it is the fusion rotein of TNF-α acceptor and Fc, not only rheumatic arthritis there is very good curative effect, and psoriasis and psoriasis type sacroiliitis also there is the obvious treatment effect, in the research that promotes the spondylitis healing, Enbrel has also entered the second stage of clinical simultaneously.2004 annual sales amounts are 2,500,000,000 dollars, estimate can reach 3,400,000,000 dollars in 2008.Remicade is a kind of anti-TNF-alpha monoclonal antibodies of people mouse mosaic, but specificity in conjunction with solubility and film mating type TNF-α, its indication comprises Crohn ' disease, rheumatic arthritis (RA), severe ankylosing spondylitis (AS).At 2003 and global marketing volume in 2004 was respectively 22.7 hundred million and 28.9 hundred million dollars, was " cookle " of biomedicine field.The Humira of Abbott company became first and got permission to be used for to alleviate the adult to symptom and the symptom of severe active period RA and suppress total man's monoclonal antibody of articulation structure damage progress in December, 2002.Owing to be human antibody, avoided Remicade can produce immunoreactive shortcoming.Another advantage that Humira compares with Remicade and Enbrel is on the facility of medication.Humira only needed injection twice in every month, and Remicade and Enbrel must inject weekly twice.The Humira share of market improves just year by year, and the sales volume of 2003 and 2004 is respectively 2.8 hundred million and 8.5 hundred million dollars.

The curative effect of this Three Represents medicine is all more definite, and along with constantly the carrying out of extensive clinical trial, other autoimmune diseases such as the clone disease except that RA, ankylosing spondylitis, psoriatic also become new indication in succession, make its market huger.The specificity height of these biological products, target makes its side effect obviously reduce than other similar medicines well, little by little share a line medication as RA with methotrexate, and strengthen they at present to the infiltration in other autoimmune disease markets such as rheumatism of severe.The new drug that is in the exploitation stage in latter stage at present mainly contains the CDP-870 (PEGization humanization anti-TNF antibodies Fab fragment) and the CDP571 (the anti-TNF monoclonal antibody of humanization) of UCB/Celltech company, the CTLA4lg (Abatacept) of Bristol-Myers Squibb company, the Rituxan of Roche company and MRA etc.Wherein progress is CDP-870 the most rapidly, has entered the clinical III phase at present, estimates that CDP-870 will come into the market the end of the year 2006, and CDP-870 uses intestinal bacteria and produces, and makes its cost be reduced to 1/4th of other tnf inhibitor medicines.

Though it is all good than the effect of other any medicine that existing TNF antibody class medicine is considered to, also there is very big shortcoming in they.First, they are all from mammalian cell expression and purifying, because expression level is low and the limited demand that can not satisfy market far away of throughput, 500,000,000 U.S. dollars have been created 20 of global maximum even if Amgen furnishes funds for, still there be (according to U.S. biopharma report in 2004) in 000 liter of biological reactor for cell culture, this problem; Second, the sky high cost of these antibody makes greatly, and the patient is kept outside of the door, according to estimates, conventional application Enbrel, Remicade and the expense in 1 year of Humira treatment RA are respectively 11400,14200 and 16776 U.S. dollars, and need use repeatedly (according to U.S. biopharma report in 2004); The 3rd, the entrained Fc section of these antibody has conjugated complement and ADCC isoreactivity, and application can cause expressing the apoptosis of the cell of TNF in the body, can produce a series of side effects; The 4th, Remicade is a people mouse mosaic monoclonal antibody, contain 1/3 mouse source protein composition, injection can excite people's anti-mouse source protein antibody response or the anti-chimeric protein antibody response of people in the patient more than 10% continuously, influences result of treatment and may cause serious adverse effects.The CDP870 that is about to listing is a PEGization humanization anti-TNF antibodies Fab section [17], and its mouse source protein composition drops to 10%, but still immunogenicity is arranged.And the amino acid sequences of Humir and corresponding DNA sequence come from the human antibody library of CAT company exploitation, and the segmental coding region of its Fc is from people's IgG1.This shows that the variable region sequences of this product is not directly from the people, but from the antibody library that originates from human B cell.

In addition, though the human immunity system can produce the extremely huge antibody of kind, but people or human antibody technology have but run into bottleneck: the precursor cell abundance that produces specific antibody in the human body is very low, even in the individuality of antibody positive, the precursor cell that can produce specific antibody also can produce the specific antibody cell quantity well below people's hybridoma technology is needed.The B cell of therefore, separation, purifying and enrichment specific secretion antibody becomes one of gordian technique of setting up the human antibody platform.

Summary of the invention

The present invention has set up can be with the technology of purpose B cellular segregation, purifying, enrichment and propagation that can secretory antibody.The specificity of secreted antibody can be screened and identifies by means such as ELISPOT, ELISA behind the B cell subclone.From the strain of mono-clonal cultured cells, obtain the gene order of purpose antibody, in order to make up protokaryon or carrier for expression of eukaryon, the activity that can rebuild antibody after the expression.The present invention utilizes this platform to obtain anti-people TNFa monoclonal antibody from rheumatoid arthritis patient body's B cell, prokaryotic expression Fab and scFv small molecular antibody, behind fermentation, the purifying, adopt external transformation of method and modifications such as Pegylation (PEGization) and nanotechnology, keeping the active while of its anti-TNF, making it to be more suitable for to use in the body.

One aspect of the present invention discloses a kind of anti-people TNFa monoclonal antibody, and its light-chain amino acid sequence is SEQ ID NO:3, and the weight chain variable region amino acid sequence is SEQ ID NO:4, and the heavy chain full length sequence is coded by SEQ ID NO:6.Preferably, above-mentioned antibody is by PEGization.

Second aspect present invention discloses the Fab antibody of above-mentioned anti-people TNFa monoclonal antibody.Preferably, above-mentioned Fab antibody is by PEGization.

Third aspect present invention provides the anti-people TNFa of a kind of PEGization monoclonal antibody nano particle, is the nano particle of the above-mentioned PEGization of coupling anti-people TNFa monoclonal antibody or its Fab antibody.

Fourth aspect present invention discloses the preparation method of the anti-people TNFa of above-mentioned PEGization monoclonal antibody nano particle, comprises the following steps:

1) expression and the purifying of human TNF alpha monoclonal antibody or its Fab antibody;

2) PEGization of human TNF alpha monoclonal antibody or its Fab antibody;

3) coupling of the human TNF alpha monoclonal antibody of PEGization or its Fab antibody and nano particle.

Preferable, above-mentioned anti-human TNF alpha monoclonal antibody or its Fab antibody are through escherichia coli expression.

The present invention also further optimizes the top condition of the anti-human TNF alpha monoclonal antibody preparation of nanoparticles of PEGization, the mensuration that comprises particle diameter, encapsulation rate, Z-current potential etc., external activity, drug release feature investigation etc., and by active determination in vitro and animal experiment study this nano particle treatment validity in animal body.

Fifth aspect present invention, disclose the anti-human TNF alpha monoclonal antibody and the Fab antibody thereof of above-mentioned anti-human TNF alpha monoclonal antibody and Fab antibody thereof, PEGization, and the anti-human TNF alpha monoclonal antibody nano particle of PEGization is in the medicine of preparation treatment autoimmune disorder such as the application in the medicine for treating rheumatoid arthritis.

The present invention has used everybody hybridoma technology in conjunction with means such as phage display and computer assisted structural analysis designs, independent research anti-TNF human antibody sequence, it is minimum that immunogenicity is reduced to; Use escherichia expression system and express Fab and scFv small molecular antibody, production cost is reduced greatly, and the side effect that does not have effects such as complement combination and ADCC to cause; Adopt the nanoparticle technology of preparing, process for modifying surface and with technology such as small molecular antibody bridging, reach the interaction that alleviates nano drug-carrying particle and serum composition in vivo, reduce the probability that it is engulfed by endothelial system, the time of prolongation in blood circulation, the while is implemented in the purpose of sustained release profile in vivo test again, has avoided short shortcoming of small molecular antibody transformation period, keeping the active while of its anti-TNF, making it to be more suitable for to use in the body.

Embodiment

Below enumerate specific embodiment and further set forth the present invention, should be understood that embodiment is used to limit protection scope of the present invention.

The acquisition of the B cell of the anti-human TNF alpha antibody of embodiment 1 secretion

Get 5 milliliters of reactivity rheumatoid arthritis patient peripheral bloods, separate white corpuscle, cultivate, identify positive colony according to ELISA result with lymphocyte separation medium.

1. selection blood sample

In order to obtain to secrete the human B cell of anti-human TNF alpha, conventional bag is by 96 orifice plates at first to use human TNF alpha (available from Shenzhen brilliant U.S. company), is spent the night with this protein 25 0ng, bag in every hole.Then, with 5% skim-milk room temperature sealing 2 hours, milk powder was prepared with pH7.2 PBS.After the washing, add different patients serum's 100 microlitres, room temperature incubation 1 hour.The goat anti-human igg who adds peroxidase labelling then, room temperature was placed 1 hour.After washing at least 5 times, add and contain TMD or other developers, room temperature or 37 degree were handled 20 minutes.At last, add stop buffer.The superoxide chromogenic substrate that adds added after 10 minutes, and the sulfuric acid termination reaction with 50 μ l1N reads the 450nm optical density value.Choose the high serum of the positive and OD value as being selected blood sample.

2. the separation of people B-cell

(pH7.2,50mM) dilution are superimposed upon Ficoll and separate on the liquid layer (common every 50ml separator tube 20mlFicoll solution) with PBS will to be selected blood sample.4 ℃ of blood suspension, 3000rpm, centrifugal 30 minutes.Subsequently, collect the top layer solution that mainly comprises peripheral blood lymphocytes (PBMC, peripheral blood mononuclear cell), place new test tube.(thorough washing is three times at least for pH7.2,50mM) thorough washing with PBS with PBMC.Cell counting.

The enrichment of human B cell

In PBMC, a plurality of clones of human B cell are arranged, be exposed in the various immunogens before them.From the PBMC mixture that obtains, can filter out special B cell clone.Adopted bio panning method herein, this method is proved to be a kind of high-efficiency method, and per unit blood can obtain 4x10 ⁶Individual cell.

Bio pinning method: with every hole 2 μ g tumour necrosis factor bags by 24 orifice plates (GREINER, GERMANY), 4 ℃ spend the night or 37 ℃ hatched 2 hours.Remove the TNF supernatant, every hole adds the isolating PBMC of 2ml on 24 orifice plates of quilt wrapping,, hatched 2 hours for 37 ℃.The B cell of enrichment secretion anti-TNF antibodies, detailed step is as follows:

1) 500 μ l TRIS-HCL (pH9.5) solution, wherein rabbit anti-human igg 5 μ g/ml wrap by 24 orifice plates (NUNC), and 4 ℃ are spent the night.

2) second day, aforementioned PBMC is cultivated based on 37 ℃ of suspensions, CO with 10%FBS-RPMI-1640 ₂5%, in the culturing bottle of 75ml, cultivated 2 hours.

3) 24 orifice plates remove supernatant, and (pH7.2,50mM) washing once with the PBS that contains 5% foetal calf serum (FBS, fetal bovine serum).After removing monocyte, place aforementioned 24 orifice plates through the enrichment of cell of washing, 4 ℃ 1 hour, intermittently stir.

The cell microscopic inspection of adhering in the hole 4) subsequently.Remove suspension cell.

5) with behind the 5%FBS-RPMI-1640 substratum flush away suspension cell, attached cell is washed from the hole, collect.

6) microscopic counting and adjust cell count.

3. the preparation of feeder cell

The microenvironment of human B cell in-vitro multiplication is essential.Human B cell propagation microenvironment in this foundation is made up of human bone marrow substrate cell layer and/or mouse peritoneal exudate cells at first, comprises scavenger cell and dendritic cell.The feeder cell in isolating people source or mouse source form unique monolayer cell, can produce the multiple somatomedin that comprises Hemopoietic factor, keep the propagation and the differentiation of human B cell.The feeder cell starting point concentration that is made of scavenger cell and the dendritic cell of people or mouse is 3x10 ⁵Individual/ml, 37 ℃ of cultivations.With RPMI-1640 (containing 10% foetal calf serum, every liter 100 unit penicillin and 100 μ g Streptomycin sulphates) substratum, CO ₂5%, culturing cell to cell compiles and reaches 80-90%.

4.EBV (chronic burkitt's lymphoma virus) transforms and long-term cultivation

With RPMI-1640 (containing 10%FBS, every liter 100 unit penicillin and 100 μ g Streptomycin sulphates) substratum, in 96 orifice plates, allow feeder cell cover with individual layer.In the hole of individual layer feeder cell of having grown, add the B cell.The amount that adds the B cell is about every hole 3-5 cell, every hole contains 200 μ l perfect mediums, 37 ℃ of co-cultivation simultaneously, contain 100 μ l in 24 microwell plates of individual layer feeder cell from the EBV adding of B958 cell (available from pathologic, physiologic teaching and research room of Medical University Of Chongqing) supernatant.Per 4 days, half supernatant in the hole is replaced to fresh perfect medium, to cultivate after 2 days, microscopically B cell colonial morphology is as seen.

5. antibody-reaction ELISA screening

Detect the secretion of anti-TNF alpha IgG with ELISA.Sample to the human B cell nutrient solution supernatant that transforms through EBV immortality method screens.Wherein, the positive colony that selection has best binding ability, and be IgG1.

The acquisition of embodiment 2 anti-human TNF alpha antibody genes

Get positive colony cell 5000-10000 that embodiment 1 obtains, separate total RNA with Trizol (GIBCO), with MMLV ThermoScript II (Promega company product) to obtain cDNA.Above-mentioned behaviour is all carrying out according to shop instruction.Carry out PCR with following primer and condition, the Pfu that used amplification enzyme is ClonTech is to guarantee reducing possible sudden change in the amplification procedure.

Light chain upstream primer: GACATCGAGCTGACCCAGTC (SEQ ID NO:7)

Light chain downstream primer: CTAACACTCTCCCCTGTTGAAGC (SEQ ID NO:8)

Heavy chain upstream primer: GAGGTGCAGCTGGTGGAGTC (SEQ ID NO:9)

Heavy chain downstream primer: CTAGCATGTGTGAGTTTTGTCACAAG (SEQ ID NO:10)

The PCR condition:

A) composition of reaction system:

B) reaction conditions:

Pre-sex change: 94 degree 2 minutes

Major cycle: 94 degree 1 minute, 55 degree 1 minute, 72 degree 3 minutes.

Cycle number: 20

Extend the back: 72 degree 5 minutes

The PCR process is carried out on PROGENE Genium thermal cycler.

C) electrophoresis is identified with glue and is reclaimed

Carry out PCR according to above-mentioned condition.After finishing product is identified on 1% agarose gel electrophoresis that the product of two PCR is respectively about 650bp and 670bp, conformed to theoretical size.Glue recovery test kit with Promega company reclaims this fragment, and operation is carried out according to the specification sheets of producer, each gets about 2 micrograms of DNA.

Clone and order-checking:

Above-mentioned product routine is cloned into pUC57, checks order after identifying correct clone.Cloning process adopts MBI company test kit, and operation is carried out to specifications.Positive colony is checked order, and the result shows that these two clones are respectively the heavy chain of human IgG1's antibody and the encoding gene of variable region of light chain.Sequencing result is as follows:

V2L

ATGGACATGCGGGTTCCAGCCCAGCTTCTCGGACTTCTGCTGTTGTGGCTGCGCGGAGCACGGTGCGAAATTGTGCTCACACAGTCACCAGACTTTCAGTCTGTCACCCCTAAGGAGAAAGTGACCATCACTTGCAGGGCCTCTCAGTTCGTCGGCTATAGTATCCACTGGTACCAGCAGAAACCCGATCAGTCCCCTAAACTGCTGATCAAGTACGCCTCTGAATCAAGGTCAGGTGTCCCCAGTCGATTTTCTGGATCAGGATCTGGTACCGACTTCACCCTCACCATCAATAGCTTGGAGGCCGAGGACGCTGCTACCTACTACTGCCAACAAAGCCACAGCTGGCACTTTACTTTTGGCCAGGGGACCAAGCTTGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGC

?	GAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGATTCAGATCCGTTAACGGTTACCAACTACCTAGACTGGATTCGTGACACATGCGGCCGTGATATCTACGT ATGATCAGCCTCGAGGGCCCACGCGTAA (SEQ ID NO：1)
		V2H	ATGGAGTTCGGACTCAGTTGGCTGTTCCTGGTGGCCATCCTGAAGGGTGTGCAGTGTGAAGTCCAGCTGGTCGAGAGCGGTGGCGGGCTGGTGCAACCCGGTGGATCACTGCGGCTCAGCTGCGCTGCTAGTGGCTTTCCCTTCTCTAACCACTGGATGAATTGGGTCCGGCAGGCTCCAGGAAAGGGTCTGGAGTGGGTGGGTGAGATCAGGAGTAAGTCTATGAACTCCGCCACACACTATGCTGAAAGCGTGAAAGGGCGCTTCACAATCTCTAGAGACGATTCAAAGAACTCTCTGTACCTGCAGATGAACAGTCTGAAAACAGAGGACACCGCTGTGTATTACTGTGCTCGGAACTACTACGGTTCAACTTACGACCACTGGGGCCAAGGTACACTGGTCACCGTCTCGAGTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT

?	CCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA(SEQ?ID?NO：2)

Its encoded polypeptides aminoacid sequence is respectively:

2L	MDMRVPAQLLGLLLLWLRGARCEIVLTQSPDFQSVTPKEKVTITCRASQFVGYSIHWYQQKPDQSPKLLIK YASESRSGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQSHSWHFTFGQGTKLEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO：3)
		2H	MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFPFSNHWMNWVRQAPGKGLEWVGEIR SKSMNSATHYAESVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARNYYGSTYDHWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO：4)

The preparation of embodiment 3 anti-human TNF alpha antibody

1. the acquisition of human IgG1's heavy chain Fc fragment coding sequence

In order to carry out the genetic expression of full length antibody, we have synthesized human IgG1's heavy chain Fc sequence, so that constitute complete anti-human TNF alpha antibody gene.According to document, the segmental dna encoding sequence of human IgG1's heavy chain Fc is as follows:

The segmental dna sequence dna of human IgG1 Fc (5 ' → 3 ') (SEQ ID NO:5)

ACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACC

CTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC

TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTG

GTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTC

CCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCC

CGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAG

TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCCCCTTCTTCCTC

TACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTG

CACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

Above-mentioned sequence is synthetic according to conventional method for synthesizing gene.

2. the acquisition of the anti-people TNF of total length monoclonal antibody gene and expression product

Above-mentioned Fc fragment and variable region of heavy chain coding region are spliced according to conventional Overlapping PCR method.Spliced heavy chain full length fragment length is: 1424bp.This fragment is the heavy chain coding region of total length.

SEQ?ID?NO：6

ATGGAGTTCGGACTCAGTTGGCTGTTCCTGGTGGCCATCCTGAAGGGTGTGCAGTGTGAAGTCCAGCTGGTCGAGAGC

GGTGGCGGGCTGGTGCAACCCGGTGGATCACTGCGGCTCAGCTGCGCTGCTAGTGGCTTTCCCTTCTCTAACCACTGG

ATGAATTGGGTCCGGCAGGCTCCAGGAAAGGGTCTGGAGTGGGTGGGTGAGATCAGGAGTAAGTCTATGAACTCCGCC

ACACACTATGCTGAAAGCGTGAAAGGGCGCTTCACAATCTCTAGAGACGATTCAAAGAACTCTCTGTACCTGCAGATG

AACAGTCTGAAAACAGAGGACACCGCTGTGTATTACTGTGCTCGGAACTACTACGGTTCAACTTACGACCACTGGGGC

CAAGGTACACTGGTCACCGTCTCGAGTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGC

ACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCA

GGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTG

ACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGAC

AAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCG

TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTG

GACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAG

CCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGC

AAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAG

CCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTG

GTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG

CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG

AACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT

AAAACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGAC

ACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTC

AACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGT

GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCC

CTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCA

TCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTG

GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCCCCTTCTTC

CTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT

CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAG

The full length coding region of above-mentioned heavy chain (SEQ ID NO:6) and the aforementioned coding region of light chain (SEQ ID NO:1) are cloned into respectively on p0ptiVec (the Invitrogen company product) carrier, according to ordinary method (reference: Yves Durocher, SylviePerret and Amine Kamen, 2002, High-level and high-throughput recombinant proteinproduction by transient transfection of suspension-growing human293-EBNA1cells, Nucleic Acid Research, 2002,30 (2): E9) transfection CHO cell.

3.CHO the transfection of cell and screening

Liposome method is adopted in this research, and (LipoFemine 2000, Invitrogen) Chinese hamster ovary celI is carried out transfection, the transfection reagent box is available from Invitrogene company, get the plasmid that has heavy chain and light chain respectively 100 micrograms of above-mentioned purifying during transfection respectively and as the DNA sample Chinese hamster ovary celI is carried out transfection, the transfection schedule of operation is carried out according to the specification sheets of producer.

Chinese hamster ovary celI after the transfection is through continuous 3 months methotrexate (MTX) screening, and to 10 μ M, per two weeks increase a concentration to its concentration from 0.05 μ M, and the consumption of each MTX is about previous 2 times, concrete visual cell's growing state and deciding.Cell cultures is carried out according to routine, and substratum is: 15% foetal calf serum (Gibco)+RPM1640/DEME.In 37 degree 5%CO ₂Cultivate in the incubator.Carry out mono-clonalization according to routine extreme dilution method then.Use the expression amount that the ELISA method detects its antibody respectively, obtain 87 clones altogether.

4. the research of gene expression dose

Expression intensity through the above-mentioned part clone of ELISA is studied, and data presentation by the MTX screening, has obtained the antibody expression amount of obvious raising.

According to expression amount, the clone who determines to have growth characteristics and expression amount preferably is as the object that continues research.

Above-mentioned expression cell line is cultivated the back carry out affinity chromatography with Protein A-Sepharose, acquisition purity reaches the antibody protein more than 90%.

The cloning and expression of embodiment 4 anti-human TNF alpha-Fab

10 μ g above-mentioned dna fragmentation SEQID NO:1 and SEQ ID NO:2 are cloned into pCOM3H carrier (WuSC according to ordinary method, Lin YJ, Chou JW, Lin CW.2004, Construction and characterization of a Fabrecombinant protein for Japanese encephalitis virus neutralization.Vaccine.25; 23 (2): 163-71), after electricity transforms 5ml escherichia coli DH5a (from New England Biolabs or Takara) cell, product carries out preliminary screening on the IPTG/X-gal flat board identifies, getting 20 bacterial plaques is inoculated in the liquid LB substratum that contains penbritin and increases, after carrying out the ELISA evaluation after IPTG induces, carry out a small amount of Fab Antibody Preparation, b.The Fab antibody of preparation carries out the protectiveness test with L929 cell (ECACC 85011425), finds that Fab antibody has good provide protection.

The PEGization and the nanometer of embodiment 5 anti-human TNF alpha monoclonal antibodies and Fab antibody thereof

With embodiment 3 and 4 anti-human TNF alpha monoclonal antibody and the Fab antibody PEGization thereof that obtain, the PEGization of Fab and purification process are with reference to [reference: Therapeutic antibody fragments with prolongedin vivo half life.Nature Biotech.V17 1999 August such as AP Champman, Page 780-783] method carry out, obtain the anti-human TNF alpha monoclonal antibody and the Fab antibody thereof of PEGization.Further again with the antibody coupling of above-mentioned PEGization to the nano-carrier surface, obtain the nano particle of anti-human TNF alpha monoclonal antibody of PEGization and Fab antibody thereof.

The anti-human TNF alpha monoclonal antibody nano particle protection of embodiment 6 PEGization human TNF alpha is attacked mouse test

1. tentatively grope TNF α lethal dose:

Reagent, material: human TNF alpha, 8 of Balb/c mouse

Test method: 8 of alb/c mouse are divided into four groups (2/group) at random, give human TNF alpha 100ug/ mouse, 30ug/ mouse, 10ug/ mouse, 0ug/ mouse through tail vein/abdominal cavity, observe the mouse situation in the week: death, hair luster, urinate and defecate etc., and the record death time.

2. the lethal dose human TNF alpha is groped in refinement:

Test method: 15 of Balb/c mouse are divided into 3 groups at random, according to all dead dosage of experiment mice last time, design 2 dosage groups 100/50,30/15,10/5ug/ mouse, give the human TNF alpha of given dose through vein/abdominal cavity, observe the mouse situation in the week: death, hair luster, the record death time such as urinate and defecate.

3.PEG changing anti-human TNF alpha monoclonal antibody nano particle protection mouse lethal dose human TNF alpha attacks

Test method: 100 of Balb/c mouse are divided into 9 groups (10/group) at random; Give HAI278B20mg/kg, 10mg/kg, 5mg/kg, 1mg/kg respectively through tail vein/abdominal cavity; Give Humira20mg/kg, 10mg/kg, 5mg/kg, 1mg/kg, 0mg/kg respectively through tail vein/abdominal cavity; Behind the 2hr, give lethal dose human TNF alpha (preliminary experiment is determined) through tail vein/abdominal cavity; Observe the mouse situation in one week: death, hair luster is urinated and defecated etc.; The record death time.

The result shows: the anti-human TNF alpha monoclonal antibody nano particle of PEGization can effectively be protected the attack of human TNF alpha, and mouse death rate is obviously descended.

Sequence table

＜110〉Suzhou Industrial Park Chenjian Antibody Drug Development Co., Ltd.

＜120〉human TNF alpha monoclonal antibody, its PEGization nano particle and application thereof

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Claims

1. anti-people TNFa monoclonal antibody, its light-chain amino acid sequence is SEQ ID NO:3, and the aminoacid sequence of variable region of heavy chain is SEQ ID NO:4, and the full length sequence of heavy chain is coded by SEQ ID NO:6.

2. anti-according to claim 1 people TNFa monoclonal antibody is characterized in that, described anti-people TNFa monoclonal antibody is by PEGization.

3. the application of anti-as claimed in claim 1 or 2 people TNFa monoclonal antibody in preparation treatment autoimmune disorder medicine.

4. the Fab antibody of anti-according to claim 1 people TNFa monoclonal antibody.

5. as the Fab antibody of anti-people TNFa monoclonal antibody as described in the claim 4, it is characterized in that described Fab antibody is by PEGization.

6. as the application of Fab antibody in preparation treatment autoimmune disorder medicine of anti-people TNFa monoclonal antibody as described in claim 4 or 5.

7. the anti-people TNFa of PEGization monoclonal antibody nano particle, nano particle for surperficial coupling PEGization anti-people TNFa monoclonal antibody or its Fab antibody, wherein, the light-chain amino acid sequence of described anti-people TNFa monoclonal antibody is SEQ IDNO:3, the aminoacid sequence of variable region of heavy chain is SEQ ID NO:4, and the full length sequence of heavy chain is coded by SEQ ID NO:6.

8. as the preparation method of the anti-people TNFa of PEGization monoclonal antibody nano particle as described in the claim 7, comprise the following steps:

1) expression and the purifying of people TNFa monoclonal antibody or its Fab antibody;

2) PEGization of people TNFa monoclonal antibody or its Fab antibody;

3) coupling of the people TNFa monoclonal antibody of PEGization or its Fab antibody and nano particle.

9. as the preparation method of the anti-people TNFa of PEGization monoclonal antibody nano particle as described in the claim 8, it is characterized in that described anti-people TNFa monoclonal antibody or its Fab antibody are through escherichia coli expression.

10. as the application of the anti-people TNFa of PEGization monoclonal antibody nano particle as described in the claim 7 in preparation treatment autoimmune disorder medicine.