CN100427504C - TNF alpha high-affinity chimeric antibody and use thereof - Google Patents

Abstract

The present invention relates to a new hihg-appetency chimeric antibody of human tumor necrosis factor (hTNF-alpha) and a usage thereof. The present invention has the technical scheme that a hihg-appetency chimeric antibody is obtained by sieving an antibody library and combining computer-aided analysis, and 141 to 146 positions of the hTNF are recognized; compared with the marketable antibody Remicade E, the hihg-appetency chimeric antibody has the advantages of high appetency, consistent neutralization activity and different recognition position. The hihg-appetency chimeric antibody can be used for curing diseases relevant to the TNF-alpha.

Description

TNF α high-affinity chimeric antibody and uses thereof

Technical field

The present invention relates to utilize phage antibody library technique to combine human tumor necrosis factor (hTNF-α) the high-affinity chimeric antibody (called after CH22 among the present invention) that screening obtains with area of computer aided appropriate design method.

Background technology

Huamn tumor necrosis factory alpha (hTNF α) be a kind of by monokaryon, hugely bite, cytokine that T lymph, neutral grain, hypertrophy or endotheliocyte secretion produce, can mediate the various biological activity.When hTNF-α a large amount of generations and release in vivo can cause the immunity of organism dysequilibrium, and can produce multiple pathology damage with other inflammatory factor, the disorderly syndromes multiple diseases such as (MDS) of the multiple organ dysfunction syndrome due to emaciation and the septic shock, rheumatoid arthritis (RA), ankylosing spondylitis (AS), multiple sclerosis (MS), inflammatory bowel (IBD), graft versus host disease (GVH disease) (GVHD) and marrow hemopoiesis for example.Therefore, capable of blocking by in using and the antibody of hTNF-alpha active because of the hTNF-alpha levels increases the body pathology damage that causes unusually, finally reach effective therapeutic purpose to disease.Anti-hTNF-α chimeric antibody (the trade(brand)name Remicade of U.S. Centocor company development and production

), U.S. FDA, European Union and Japanese approved in succession are used for the clinical treatment of RA, colon C rohn disease and AS.

This study group in last century the eighties prepared anti-hTNF α mouse resource monoclonal antibody (McAb) Z12 (Li Yan etc., the biotechnology journal, 1991,7 (2), 121-126), in having and hTNF α cytotoxicity function, its avidity is at K _a=8 * 10 ⁷L/mol.In its humanization process, the avidity of antibody has reduction significantly again.The present invention attempts replacing (Chain Shuffling) method by chain, with mouse source antibody Z12 is female parent, by the affine screening of phage antibody library technique, obtain the high-affinity chimeric antibody by the computer appropriate design, in the hope of reaching in keeping antibody and the active purpose that significantly improves affinity of antibody simultaneously.

Summary of the invention

The present invention is based on original maternal mouse source antibody Z12, adopt a kind of new strategy (being that phage antibody library chain replacement technology combines with the area of computer aided appropriate design) to obtain high-affinity antibody.

At first, on antibody Z12 basis, maternal mouse source, the phage antibody library that makes up is screened, obtain potential and have the active chimeric antibody of higher neutralization by chain replacement method.

Utilize homology mould construction method that the chimeric antibody variable region space conformation of screening is carried out reasonable analysis, under theory instructs, itself and maternal antibody Z12 space conformation are carried out the comparison of structure, apparent sex character; Make up antibody and the interactional mixture space conformation of hTNF α by the molecular docking method, to the stability of mixture, carry out theoretical analysis, determine to have the affine active chimeric antibody of height in conjunction with power and interaction zone by the correlation theory method.Obtain this antibody by biological test, its neutralization activity is verified by functional trial.

The present invention has obtained a plant height avidity anti-TNF alpha chimeric antibody by following steps.

1, utilize phage antibody library technique screening TNF α high-affinity chimeric antibody:

Make up for chain and replace the antibody library that (Chain Shuffling) uses, obtain the positive bacteriophage antibody cloning by affine enrichment screening, gene sequencing obtains the antibody variable gene sequence.

2, the anti-hTNF α of area of computer aided reasonable prediction high-affinity chimeric antibody:

Utilize the space conformation of the antibody variable region of homology mould construction method antagonist storehouse screening acquisition to simulate, under molecular mechanics and dynamic conditions, select CVFF, the Amber field of force that its preferred conformation is analyzed.Antibody variable region three-dimensional conformation and maternal antibody Z12 space conformation to screening on distance geometry, Fundamentals of Computer Graphics compare, the difference of initial analysis screening antibody variable region conformation and maternal antibody Z12 conformation.

Obtain antibody and the interactional mixture model of antigen hTNF α by the molecular docking method, forming theoretical antibody variable region and antigen TNF α binding mode to screening by computer graphics techniques, free energy of reaction theory, intermolecular hydrogen bonding compares, by maternal antibody Z12 and the interactional mathematical model of TNF α set up, determine to have the chimeric antibody of high-affinity simultaneously.

The present invention has obtained a strain by above-mentioned strategy and has had high-affinity (K _a=6 * 10 ¹⁰L/mol) anti-hTNF α chimeric antibody CH22.It is evident that the antigen-binding portion thereof of chimeric antibody of the present invention is also within protection scope of the present invention.

3, high-affinity chimeric antibody CH22 biological effect is estimated:

By the theoretical prediction result, by biological method to chimeric antibody CH22 biological function experiment Analysis: chimeric antibody CH22 can specificity in conjunction with hTNF α, the main epi-position hTNF α 141-146 epi-position of its antigen recognition, with respect to maternal antibody Z12, CH22 avidity improves 750 times, and the ability of the active TNF α that neutralizes improves 10 times; Compare Remicade

(Infliximab, the FDA approval is used for the chimeric antibody of clinical treatment rheumatoid arthritis) avidity is high 6 times.The functional epitope difference of their identification TNF α, but in ability with the TNF alpha active identical.Show that by function test antibody CH22 has the ability of identification reorganization simultaneously and natural hTNF α, has good potential applicability in clinical practice.

Description of drawings

Fig. 1 .3.1.1EILSA measures immune serum and tires.

The electrophoretogram of the total RNA of Fig. 1 .3.1.2 mouse spleen lymphocyte.

Fig. 1 .3.2.1RT-PCR amplification obtains the antibody gene library.

The segmental PCR product of 1:Fd; 2:DNA mark (DL2000); 3: light chain PCR product

Fig. 1 .3.3.1 phage antibody expression vector p3MH (synoptic diagram).

Lac: promotor;

OmpA and PelB: signal sequence;

9E10: the epi-position of coding McAb 9E10 identification;

His6: histidine tail

The evaluation of Fig. 1 .3.3.2 phage antibody expression vector p3MH restriction enzyme site.

1：p3MH； 2：p3MH/SacI；

3：p3MH/XbaI；4：p3MH/XhoI；

5：p3MH/SpeI

Fig. 1 .3.3.3p3MH/Z12L enzyme is cut the evaluation collection of illustrative plates.

1:DNA mark (DL2000);

2-4：p3MH/Z12L/SacI+XbaI；

5：p3MH/SacI+XbaI

Fig. 1 .3.3.4p3MH/Z12L sequencing result.

Fig. 1 .3.3.5p3MH/Z12H enzyme is cut the evaluation collection of illustrative plates.

1:DNA mark (DL2000); 2:p3MH/XhoI+SpeI; 3:p3MH/Z12H/XhoI+SpeI

Fig. 1 .3.3.6p3MH/Z12H sequencing result.

Fig. 1 .3.5.1 phage antibody H22, Ke, Kg and Z12 are relatively to rh-TNF α relative affinity.

Fig. 2 .2.1.1pGEM-T Easy/ phage antibody clone enzyme is cut the evaluation collection of illustrative plates.

1:DNA mark (DL2000);

2-5:pGEM-T Easy/ phage antibody gene/EcoRI

Fig. 2 .2.1.2 antibody cloning H22 heavy chain Fd portion gene sequencer address.

Fig. 2 .2.1.3 antibody cloning Ke light chain gene sequencer address.

The translation of Fig. 2 .2.2.1 antibody variable gene.

Fig. 2 .2.2.2 antibody variable gene FR, CDR region class.

Fig. 2 .2.3.1 antibody A 2H variable region space conformation synoptic diagram.

Fig. 2 .2.3.2 antibody C2H variable region space conformation synoptic diagram.

Fig. 2 .2.3.3 antibody H22 variable region space conformation synoptic diagram.

The mixture space conformation of Fig. 2 .2.4.1 antibody A 2H variable region and TNF α effect.

The mixture space conformation of Fig. 2 .2.4.2 antibody A 4H variable region and TNF α effect.

The mixture space conformation of Fig. 2 .2.4.3 antibody H22 variable region and TNF α effect.

Fig. 3 .3.1.1 chimeric antibody expression vector PWS3 synoptic diagram.

Structure and the evaluation of Fig. 3 .3.1.2 recombinant expression plasmid PWS3H22.

(A): the enzyme of PWS3H22 is cut digestion

The PWS3H22 of 1:HindIII/XhoI digestion; The PWS3H22 of 2:BamHI/XbaI digestion; The 3:DL2000 mark

(B): PCR identifies PWS3H22 on 1% sepharose

1:H22 variable region of heavy chain product; 2:H22 variable region of light chain product; 3:DNA mark (DL2000)

Fig. 3 .3.2.1CH22 detects TNF-α binding specificity.

A:ELISA detects the specificity combination of CH22 to TNF-α, 1. positive control (Z12); 2.CH22; 3. negative control (human IgG);

B: western blotting detects the specificity combination of CH22 to TNF-α, 1.PBS; 2. negative control (human IgG); 3. positive control (Z12); 4.CH22

Fig. 3 .3.3.1 antibody purification molecule SDS-PAGE electrophorogram.

1.2.CH22; 3.Z12; 4.Remicade

5. human IgG

The anti-hTNF Alpha antibodies of several differences of Fig. 3 .3.4.1 antigen recognition site relatively.

Fig. 4 high-affinity chimeric antibody of the present invention makes up schema.

Embodiment

1, the structure of phage antibody library and affine screening

1.1 material

Bacterial strain and plasmid

The biomaterial that uses among the present invention comprises: e. coli jm109 bacterium, XLI-Blu bacterium (Invitrogen); Phage antibody library Fab segment table reach carrier p3MH (Barbas CF 3rd, KangAS, Lerner RA, Benkovic SJ.Proc Natl Acad Sci U S is Sep15 A.1991; 88 (18): 7978-82)

Toolenzyme, reagent

Restriction enzyme is available from New England Biolabs company;

T4 dna ligase, pGEM-T Easy support agent box, reversed transcriptive enzyme are available from Promega company;

Pyrobest archaeal dna polymerase, dna molecular amount standard DL2000 are available from TaKaRa company;

TRIzol Reagent is available from Invitrogen company;

Adenosine diphosphate (ADP) (ADP) is the Biopool product;

Dna fragmentation reclaims test kit available from OMEGABIO-TEK company;

It is Time Inc. available from the sky that plasmid reclaims test kit;

The anti-M13 antibody of horseradish peroxidase-labeled is available from amersham pharmacia company;

Other chemical reagent commonly used are import or homemade analytical pure;

1.2 method

1.2.1 mouse spleen separates the preparation lymphocyte

The eyeball of mouse dehematize, bubble is to 75% ethanol.Take out mouse and place on the flat board ventricumbent position.Pick up left side fur portion with tweezers, cut off skin, peritonaeum, separating spleen is placed in the plate.Softly cut off spleen both sides mesentery and surrounding tissue, in plate, add 1-2ml physiological saline, the washing of jog plate.Gently push the spleen coating with flat flat tooth tweezers, spleen cell is extruded onto in the plate, form cell suspension.In advance prior to adding 3ml mouse lymphocyte parting liquid in the test tube, add the 6ml cell suspension again and add and fashionablely slowly add along tube wall, form different liquid level layers.The centrifugal 2000rpm of room temperature 30 minutes gently around drawing the liquid level cell, adds the washing of physiological saline 6-8ml mixing.Centrifugal 1500rpm abandoned supernatant in 5 minutes, and the gained precipitation is lymphocyte.

1.2.2 the extraction of splenic lymphocyte cell total rna

Get lymphocyte suspension counting, by 1 * 10 ⁷The cell branch is packed in the EP pipe, and centrifugal 5 minutes of 1500rpm abandons physiological saline, stays cell precipitation.In every pipe, add 1ml TRIzol respectively, shake up to cell debris cleaved fully repeatedly with hand.After room temperature left standstill several minutes, every pipe added the 0.2ml chloroform, and vibration shakes up repeatedly in hand, and room temperature left standstill 10 minutes.The EP pipe is placed the high speed tabletop refrigerated centrifuge, 4 ℃, centrifugal 30 minutes of 12000rpm.The careful upper phase of drawing places another aseptic EP pipe, and Yu Guanzhong adds equal-volume Virahol, precipitation at room temperature 10 minutes.4 ℃, centrifugal 20 minutes of 12000rpm abandons supernatant, and precipitation is washed one time with 75% ethanol.Sedimentary RNA is sure not to drain at natural drying at room temperature.The resuspended dried RNA of the pure water that every effective 30ml does not have the RNA enzyme.It is synthetic or put-70 ℃ of prolonged preservation to place ice bath to be used for cDNA immediately.

1.2.3RNA denaturing formaldehyde electrophoresis

Running gel: 0.3g agarose, 21.6ml do not have the deionized water of RNA enzyme, after the microwave oven heating and melting, add 3ml 10 * mops again, 5.4ml formaldehyde.

Sample preparation: 5 μ l RNA samples, 5 μ l, 10 * mops, 25 μ l methane amides, behind the 9 μ l formaldehyde mixings, 56 ℃ of reactions added that the sample damping fluid carries out electrophoresis after 15 minutes.

Electrode buffer (10 *): 0.4M mops, 0.1M sodium-acetate, 0.01M EDTA.

After electrophoresis finishes, EB dyeing, ultraviolet visualization.

1.2.4PCR primer

Primer of the present invention is mainly used in the full-length gene of amplification IgG antibody heavy chain Fd part, light chain κ chain, and is specific as follows.

Heavy chain of antibody Fd gene primer is used to increase:

A (justice) 5 '-CCAGTTCG GAGCTCGTTGTGACTCAGGAATCT-3 ' (sEQ ID NO:9)

B (justice) 5 '-CCAGTTCC GAGCTCGTGTTGACGCAGCCGCCC-3 ' (SEQ ID NO:10)

C (justice) 5 '-CCAGTTCC GAGCTCGTGCTCACCCAGTCTCCA-3 ' (SEQ ID NO:11)

D (justice) 5 '-CCAGTTCC GAGCTCCAGATGACCCAGTATCCA-3 ' (SEQ ID NO:12)

E (justice) 5 '-CCAGATGT GAGCTCGTGATGACCCAGACTCCA-3 ' (SEQ ID NO:13)

F (justice) 5 '-CCAGATGT GAGCTCGTCATGACCCAGTCTCCA-3 ' (SEQ ID NO:14)

G (justice) 5 '-CCAGTTCC GAGCTCGTGATGACACAGTCTCCA-3 ' (SEQ ID NO:15)

H (antisense) 5 '-CCG ACTAGTTCATTTCAGCTCCAGCCAGGTG-3 ' (SEQ ID NO:16)

I (antisense) 5 '-CTCGAC ACTAGTTTTGCGCTCAACTGTCTT-3 ' (SEQ IDNO:17)

J (antisense) 5 '-TGTGTG ACTAGTGTCACCAAGTGGGGTTTT-3 ' (SEQ ID NO:18)

K (antisense) 5 '-GCATGA ACTAGTTGGGGGACCATATTTGGA-3 ' (SEQ ID NO:19)

Light chain of antibody full-length gene primer is used to increase:

L (justice) 5 '-AGGT CTCGAGCTCGAGTCTGG-3 ' (SEQ ID NO:20)

M (justice) 5 '-AGGT CTCGAGCTCGAGTCAGG-3 ' (SEQ ID NO:21)

N (justice) 5 '-AGGT CTCGAGTCCGAGTCTGG-3 ' (SEQ ID NO:22)

O (justice) 5 '-AGGT CTCGAGTTCGAGTCAGG-3 ' (SEQ ID NO:23)

P (justice) 5 '-AGGT CTCGAGGCTGAGTCTGG-3 ' (SEQ ID NO:24)

Q (justice) 5 '-AGGT CTCGAGCGTAAGTCTGG-3 ' (SEQ ID NO:25)

R (justice) 5 '-AGGT CTCGAGGGAAAGTCAGG (SEQ ID NO:26)

S (antisense) 5 '-GACTTGG TCTAGACGAGACGGTGAC-3 ' (SEQ ID NO:27)

^*The line part is respectively the restriction enzyme site of XhoI, SpeI, SacI, XbaI.

1.2.5 antibody is light, the foundation (RT-PCR) in heavy chain gene library

Get the total RNA of 10 μ g, add AMV 5 * damping fluid 10 μ l successively, Oligo (dT) (500ng/ μ l) 2 μ l, 10mmol/L dNTPs4 μ l, Rnasin (50U/ μ l) 1 μ l, deionized water mend to 50 μ l, ThermoScript II 100U, and 42 ℃ were extended 1 hour.70 ℃ of deactivation reversed transcriptive enzymes 15 minutes are put in the ice bath, and product is cDNA first chain.In 100 μ l PCR reaction systems, add reverse transcription product 2 μ l respectively then, Pyrobest enzyme 10 * buffer 10 μ l, each 1 μ l of upstream and downstream primer, 10mmol/L dNTPs 1 μ l adds Pyrobest enzyme 2-5U, and deionized water is mended to 100 μ l.95 ℃ of sex change 5 minutes, loop parameter is: 94 ℃ 1 minute, 50 ℃ 1 minute, 72 ℃ 1 minute, totally 35 circulations were extended 10 minutes after 72 ℃.

1.2.6DNA segmental purifying

With the dna fragmentation purification kit of OMEGABIO-TEK company, purifying carries out to specifications.With the gel electrophoresis of 0.8% plain agar sugar, isolate the dna fragmentation that desire reclaims, under long wave ultraviolet light, downcut the blob of viscose that contains target DNA fragment, put into centrifuge tube, add the long-pending change glue of three times of colloids, blob of viscose is dissolved in 55 ℃ of water-baths fully.Add DNA then and reclaim post, room temperature was placed 2 minutes, and 10, centrifugal 30 seconds of 000rpm, lavation buffer solution 1ml washes twice to precipitation respectively, and the centrifugal waste liquid of abandoning is added in the post with 50 μ l deionized waters, room temperature was placed after 2 minutes, and centrifugal, the dna fragmentation of purifying is in the aqueous solution.

1.2.7 the structure of carrier P3MH/Z12L and P3MH/Z12H

From the carrier pGEM-Teasy/Z12L and pEM-Teasy/Z12H of clonal antibody Z12 light chain total length and heavy chain Fd section, the method by PCR regains antibody gene.After the PCR product of carrier P3MH and purifying cut digestion according to enzyme accordingly respectively, connect, transform XLI-Blu host bacterium, the evaluation after enzyme is cut and checked order successfully makes up P3MH/Z12L and P3MH/Z12H carrier.

1.2.8 the enzyme of carrier DNA and purified pcr product is cut

Double digestion in the antibody library gene clone

Endonuclease reaction carries out routinely, and generally 37 ℃ of water-baths 3-4 hour at least, but also digested overnight is the longest not above 20 hours.Carrier DNA consumption and PCR product amount ratio are 10: 1.Need carry out electrophoresis behind the endonuclease reaction with the required fragment of purifying, method is as described in the 1.2.6.

1.2.9 electricity changes the preparation of competence bacterium XLI-Blu

Select the single bacterium colony of an XLI-Blu in LB-tsiklomitsin plate, 2 * YT nutrient solution of 10ml tetracyclin resistance is gone in inoculation, and 37 ℃ of shaking culture are spent the night.Get above-mentioned bacterium liquid and do dilution in 1: 50,500ml2 * YT nutrient solution is gone in inoculation, wherein contains 10ml 20% glucose and 5ml1mol/L magnesium chloride, and 37 ℃ of shaking culture are to OD ₆₀₀=0.7-0.8.Above-mentioned microbial culture liquid was placed ice bath 15 minutes, and 4 ℃ of 4000rpm are centrifugal 20 minutes.Abandon supernatant, add 10% glycerine of 250ml precooling, 4 ℃ of 4000rpm are centrifugal 20 minutes.Abandon supernatant and repeat previous step.Abandoning supernatant, is 10% glycerine of 20ml precooling with total amount, the re-suspended cell precipitation, and ice bath left standstill 30 minutes, and 4 ℃ of 3500rpm are centrifugal 20 minutes.Abandon supernatant, add 1ml10% glycerine re-suspended cell, the packing tubule uses or-70 ℃ of prolonged preservation immediately.

1.2.10 phage antibody library (κ/Z12) and the structure of phage antibody library (H/Z12)

Get enzyme and cut the carrier DNA 2 μ g that digestion back purifying reclaims, pre-enzyme is cut light chain or each 500ng of heavy chain gene library, adds 2 μ l high density ligase enzymes, adds corresponding connection damping fluid, and 16 ℃ connect 12-16 hour.Getting the good electricity of prepared beforehand changes competence bacteria XLI-Blu 400 μ l and adds above-mentioned connection product and put in the ice bath.Other gets the space is one of 0.2cm electricity revolving cup, and above-mentioned every pipe mixture is added gently along tube wall, notes no bubble, puts in the sub-electroporation of electric revolving cup, and the voltage when the electricity commentaries on classics is set is 2.5KV, shocks by electricity 30 seconds-1 minute.Stand on after electricity changes and add 2ml SOC nutrient solution in each electric revolving cup, change over to immediately then in the bacteriological incubator, 37 ℃ of shaking culture 1 hour.Therefrom get 10 to 50 μ l nutrient solutions, be diluted to different concns after, be applied to respectively in the 2 * YT plate that contains Amp, to detect transformation efficiency and to calculate storage capacity, all the other bacterium liquid change a triangular flask over to, add the fresh 2 * YT nutrient solution that contains Amp of 10ml, 37 ℃ of shaking culture 1 hour.Add 100ml and contain Amp and the two anti-2 * YT nutrient solutions of tsiklomitsin, 37 ℃ of shaking culture 1 hour.Add 10 ¹²Pfu helper phage VSCM13,37 ℃ of shaking culture 2 hours.Add 50 μ g/ml kantlex, 37 ℃ of shaking culture are spent the night.4 ℃ of above-mentioned inoculum 4000rpm is centrifugal 20 minutes.Supernatant is put another aseptic triangular flask, after adding 4%PEG8000 and the 3%NaCl dissolving, be positioned in the ice bath, precipitate 30 minutes.4 ℃ of 9000rpm are centrifugal 20 minutes then.With the resuspended phage precipitation of the aseptic PBS of 2ml (pH=7.4), instantaneous centrifugal.Supernatant is phage antibody library, and this antibody library is distributed into tubule, and is stand-by or deposit-20 ℃.

1.2.11 the affine enrichment screening of phage antibody library

The antigen (rhTNF α) that will be used to screen specific antibody is made a certain amount of dilution back bag by 96 orifice plates with the 0.1mol/LpH9.6 soda-sodium bicarbonate solution, and 4 ℃ are spent the night.Adsorption antigen not in the next day flush away plate is with 3%BSA-PBS or with 37 ℃ of sealings of 10% calf serum 1 hour.Abandon confining liquid, in every hole, add 50-70 μ l phage antibody library, hatched 2 hours for 37 ℃.Abandon Kong Zhongwei in conjunction with phage, (0.5%Tween20) washing lotion is washed each hole for the 50mmol/Ltris-HCl of pH7.5,150mmol/L sodium-chlor, washes altogether 10～20 times, makes abundant flush away non-specific adsorption phage with TBS/Tween20.With distillation washing twice, the liquid in every hole is blotted only.Add 3～5 μ l 2mol/L tris alkali, the pH value that makes solution about 7, the phage solution that elutes with neutralization.The OD that adds the 2ml prepared fresh immediately ₆₀₀XLI-Blu bacterium about=1, incubated at room 15-20 minute.Change in the 200ml triangular flask, add 10ml and contain Amp and the two anti-2 * YT nutrient solutions of tsiklomitsin, get 10 μ l phage solutions immediately, be diluted to the phage that different concns elutes with titration, 37 ℃ of shaking culture of rest part 1 hour.Add 100ml and contain Amp and the two anti-2 * YT nutrient solutions of tsiklomitsin, 37 ℃ of shaking culture 1 hour.Add 10 ¹²Pfu helper phage VSCM13,37 ℃ of shaking culture 2 hours.Add 50 μ g/ml kantlex, 37 ℃ of shaking culture are spent the night.Phage after collect cultivating is as described in the 1.2.10.Repeat above enrichment screening step, no longer rise until input-output ratio, after many wheel enrichment screenings, with the XLI-Blu bacterium of the phage-infect prepared fresh that elutes, 37 ℃ of overnight incubation, preserving length has the plate of single bacterium colony stand-by in 4 ℃.

1.2.12 helper phage seed culture of viruses preparation

The single XLI-Blu colony inoculation of picking is gone into 10ml and is contained in 2 * YT nutrient solution of tsiklomitsin from plate.37 ℃ of shaking culture are spent the night.10ml is gone in above-mentioned incubated overnight bacterium 1: 500 dilution contain in 2 * YT nutrient solution of tsiklomitsin, 37 ℃ of shaking culture 1 hour.The single phage virus plaque of picking from the phage culture dish, inoculation are gone in the above-mentioned 10ml bacterium liquid, 37 ℃ of shaking culture 2 hours.Add the 2 * YT nutrient solution that contains 10 μ g/ml tsiklomitsins and 50 μ g/ml kantlex, total amount is to 500ml, and 37 ℃ of shaking culture are spent the night.4 ℃ of 3500-4000rpm are centrifugal 15 minutes.Get supernatant, 70 ℃ of cracking 20 minutes, recentrifuge.Get supernatant, in the aseptic subpackaged tubule, deposit 4 ℃ and can use half a year at least, need Using such method to prepare again after half a year.

1.2.13 the titration of phage seed culture of viruses

Prepare not contain any antibiotic LB culture dish 5-6.With common LB substratum system 0.7% agarose, be the top layer agarose, be chilled to 42 ℃ and 42 ℃ of preservations.Above-mentioned phage solution is done 10 ^-6, 10 ^-7, 10 ^-8, 10 ^-9, 10 ^-10Dilution.Get the XLI-Blu bacterium of dilution, OD ₆₀₀About=1, the packing small test tube, 100 μ l/ small test tubes indicate 10 ^-6To 10 ^-10Extent of dilution.Before adding in the step successively the phage of dilution in the dilution test tube of each corresponding standard, 37 ℃ of slow oscillatory reactions 20 minutes.Add top layer agarose 3ml to each test tube, change the previously prepd plate immediately over to and put 37 ℃ of overnight incubation.Calculate the plaque number in the plate, multiply each other, be tally-pfu (particle formingunit) of phage with extension rate.

1.2.14ELISA compare the antibody relative affinity

Rh-TNF α is dissolved in coating buffer (carbonate buffer solution pH 9.6,4 μ g/ml dilution), and wrap by affine 96 orifice plates of ELISA in 100 μ l/ holes, places after 8 hours 37 ℃ of sealings of 10% calf serum 1 hour for 4 ℃.PBST washes plate three times, the phage antibody that adds different concns by 50 μ l/ holes, room temperature 90 minutes, PBST washes plate three times, adds horseradish peroxidase labeling antibody (HRP-antiM13,50 μ l/ holes), after the room temperature 40 minutes, PBST washes plate three times, with the colour developing of OPD Color Appearance System, surveys the OD492nm absorbance.Obtaining combining activity with rh-TNF α according to the final screening of ELISA result clones greater than the phage antibody of Z12.

1.3 result

1.3.1rh-TNF the titration of alpha immunization mice serum, total RNA extract

Measuring the result (Fig. 1 .3.1.1) that immune serum tires from ELISA can see, the immunity of succeeding of five mouse after, the abdominal cavity multiple spot multiple injection subcutaneous through r-TNF α, and immune serum is tired and is on average reached about 1: 200000.

Extract cell total rna test handbook (Invitrogen company provides) according to mouse spleen lymphocyte separating experiment method and TRIzol Reagent, extract the total RNA of successful immune mouse splenic lymphocyte (Fig. 1 .3.1.2).

1.3.2 the acquisition of phage antibody gene library

From mouse spleen lymphocyte, extract total RNA, through reverse transcription, add special primer, with 35 circulations of high-fidelity DNA polymerase amplification, the clear band of about 660bp be can obtain, heavy chain of antibody Fd fragment gene library and light chain full-length gene library (Fig. 1 .3.2.1) are respectively.

1.3.3 the evaluation of phage antibody expression vector P3MH, P3MH/Z12L and P3MH/Z12H construction of recombinant plasmid

Structure synoptic diagram (Fig. 1 .3.3.1) by phage antibody expression vector P3MH, show by restriction enzyme digestion and electrophoresis result (Fig. 1 .3.3.2), carrier p3MH by SacI, XbaI, XhoI and four kinds of enzyme single endonuclease digestions of SpeI after, compare with empty carrier and all to be linearizing, show that SacI, XbaI, XhoI and four restriction enzyme sites of SpeI on the carrier p3MH are all excellent, can be used for Fd and light chain Cloning of Entire Gene.

With SacI, XbaI double digestion pGEM-T Easy/Z12 κ, obtain the light chain full-length gene of antibody.Its p3MH carrier with process SacI, XbaI double digestion is connected.Through transforming, choose the clone, the upgrading grain, carry out enzyme and cut evaluation (Fig. 1 .3.3.3), the result shows, behind the double digestion of the clone in the sample 2,3,4 (Fig. 1 .3.3.3) through SacI and XbaI, all obtains the light chain gene of 660bp, and do not obtain the fragment of 1.2Kb, illustrate that light chain gene has been cloned on the p3MH carrier; It is checked order (Fig. 1 .3.3.4), the result is correct, shows successfully to make up the P3MH/Z12L recombinant plasmid.

Adopt and use the same method, will cut the heavy chain of antibody Fd gene that obtains from pGEM-T Easy/Z12Fd through XhoI, SpeI enzyme and be connected with above-mentioned, make up prokaryotic expression recombinant plasmid P3MH/Z12H through the p3MH carrier of XhoI, SpeI double digestion.Through transforming, choose the clone, the upgrading grain is identified (Fig. 1 .3.3.5), and order-checking (Fig. 1 .3.3.6) success, shows successfully to make up the P3MH/Z12H recombinant plasmid.

Replace the method that (chain shuffling) principle is tested according to chain, will make up phage antibody library (H/Z12 κ) by heavy chain of antibody gene library and the recombinant plasmid p3MH/Z12L that the PT-PCR amplification obtains; Simultaneously, light chain of antibody gene library and recombinant plasmid p3MH/Z12H are made up phage antibody library (κ/Z12H).Determine the storage capacity of antibody library by the titration of phage seed culture of viruses, titration determines that (κ/Z12H) and (H/Z12 κ) have all reached 1 * 10 to phage antibody library according to the phage seed culture of viruses ⁷

1.3.4 phage antibody library (enrichment screening that κ/Z12H) and phage antibody library (H/Z12 κ) are affine

The affine enrichment screening of phage antibody library generally with input-output ratio (O/I ratio) as affine enrichment index.Good enrichment screening is taken turns after the screening through each, and input-output ratio can increase with exponential.When its input-output ratio no longer increases, perhaps even when negative growth occurring, affine enrichment screening process can stop, otherwise affine enrichment screening process can be carried out always, till above-mentioned situation occurring (table 1.3.4.1).

Enrichment screening that table 1.3.4.1 phage antibody library is affine

1.3.5 compare the relative affinity of phage selection antibody by ELISA

Single clone phage with obtaining after the affine enrichment screening obtains phage antibody by infecting the host bacterium, with phage antibody Z12 relatively to the bonding force of r-TNF α.The positive colony (the part positive findings is seen Fig. 1 .3.5.1) that adopts ELISA each phage antibody of comparison and r-TNF α relative affinity to obtain, screen 108 phage antibody clones by this method altogether, wherein obtained the phage antibody (seeing Table 1.3.5.1) that 18 antibody relative affinities are higher than Z12.

Table 1.3.5.1 phage antibody qualification result

2, the potential high-affinity chimeric antibody of computer-aided screening

2.1 method

2.1.1 the sequencing of phage antibody positive colony

P140Fab antibody gene pcr amplification product is cloned into respectively on the pGEM-T Easy carrier, through enzyme cut identify errorless after (Fig. 2 .2.1.1), give order-checking company to check order.

2.1.2 the simulation of chimeric antibody variable region three-dimensional conformation

(Protein Data Bank PDB), analyzes by sequence alignment (selecting Fasta3, Blastp program), Kabat, determines its maternal model to utilize the protein structure database.By homology mould (Homology) method of building the antibody variable region of measuring is carried out the simulation of space conformation.Under CVFF, the Amber field of force, select molecular mechanics, kinetics dynamic simulation that its conformation is carried out energy-optimisedly (passing through steepest descent (convergence criterion 0.05KJ/mol successively, convergence step 5000 step), conjugate gradient (convergence criterion 0.01KJ/mol, convergence step 10000 step)).

2.1.3 the simulation of chimeric antibody variable region and TNF α interaction mixture space conformation

By TNF alpha-crystal structural models (PDB number: 1tnf), utilize the molecular docking program that chimeric antibody and the interactional composite structure of TNF α are inquired into.Follow " complementary structure, static coupling " principle, under CVFF, the Amber field of force, the optimum matching degree of chimeric antibody and TNF α interaction pattern is carried out theoretical prediction through molecular mechanics, kinetics dynamic simulation.

2.1.4 determining of senior middle school and active chimeric antibody

Association reaction free energy theory, intermolecular hydrogen bonding form theory, by maternal antibody Z12 and the mutual recognition mode of TNF α, determine to have senior middle school and antibody active, high-affinity.

2.2 result

2.2.1 utilize clone, the order-checking of the positive antibody of phage antibody library screening

P140Fab antibody gene pcr amplification product is cloned into respectively on the pGEM-T Easy carrier, through enzyme cut identify errorless after (Fig. 2 .2.1.1), give order-checking company.Obtain 4 of brand-new light chain of antibody full-length gene order information altogether, 14 of heavy chain Fd fragment gene sequence informations.The part sequencing result is as follows: because the antibody gene sequence information is more, sequencing result is example (Fig. 2 .2.1.2 and 2.2.1.3) with antibody H22 heavy chain Fd fragment gene and antibody Ke light chain full-length gene order only.

2.2.2 antibody variable region sequential analysis

Utilize Www.expasy.chThe antibody variable region base sequence that order-checking obtains is translated, obtained its aminoacid sequence (Fig. 2 .2.2.1).Carry out FR, CDR classification analysis (Fig. 2 .2.2.2) by Kabat database antagonist variable region sequences.

2.2.3 the acquisition of antibody variable region space conformation

On graphics workstation Octane2 (R12000), utilize the InsightII2000 routine package to make up screening antibody variable region space conformation.Fig. 2 .2.3.1, Fig. 2 .2.3.2, Fig. 2 .2.3.3 have provided antibody variable region A2H, A4H, CH22 space conformation synoptic diagram respectively.

2.2.4 determining of the structure of Ag-Ab effect mixture, high-affinity antibody

Fig. 2 .2.4.1, Fig. 2 .2.4.2, Fig. 2 .2.4.3 have provided the interactional mixture model of antibody variable region A2H, A4H, H22 and TNF α respectively.Table 2.2.4.1 has listed antibody variable region and TNF α interaction pattern and identified region.Determine that by interactional free energy, intermolecular hydrogen bonding formation number and identified region CH22 has the desirable antibody that is higher than maternal antibody Z12 affinity.

Table 2.2.4.1 antibody-antigenic action pattern analysis

Interaction of hydrogen bond can (KJ) main identified region between antibody molecule

Z12 31 -413.88 135-150

H22 36 -425.37 136-149

A2H 19 -289.54 86-97

A4H 24 -345.21 137-145

Utilize the A Lunniusi formula to calculate, can tentatively draw the more maternal antibody Z12 of the affinity of CH22 antibody affinity height～120 times.

By computer-aided analysis, determine the hTNF-α functional epitope 141-146 position of chimeric antibody CH22 identification; Determine that simultaneously chimeric antibody CH22 keeps 56 important residue (antibody heavy chain variable region V of its biological effect _H: 26-31,48-52,90-95,66-68; Antibody chain variable region V _L: 26-33,52-65,72-76,98-106).

3, the biological function of high-affinity antibody CH22 is measured

3.1 material

Bacterial strain and plasmid

E. coli jm109 bacterium, XLI-Blu bacterium, BL21 bacterium (Invitrogen), single-chain antibody expression vector PET32-c (Novagen), Chinese hamster ovary celI (ATCC, the U.S.), PWS3 carrier (silver, Yu Lizhang, Lv Yingqian, Ai Junkui etc., Chinese Medical Journal, 2003,83 (4): 333-338)

Toolenzyme, reagent

Restriction enzyme: NcoI, BssHII, BstEII, XhoI, XbaI, BamHI, HindIII and EcoRV are available from New England Biolabs company; T4 dna ligase, WizardDNA purification kit, pGEM-T Easy support agent box are Promega company product; Dna fragmentation reclaims test kit, Taq archaeal dna polymerase, dna molecular amount standard DL2000 available from TaKaRa company; Coke diethyl phthalate (DEPC) is the Sigma product; HisTrap (nickel ion chelate column) is available from Novagen company; Lipfectamine2000 is a GIBCO company product; The goat anti-human igg Fc (U.S. Sigma) of DMEM substratum, foetal calf serum, sheep anti mouse Fab polyclonal antibody, horseradish peroxidase-labeled, other chemical reagent commonly used are import or homemade analytical pure.Restriction enzyme is available from New England Biolabs company; T4 dna ligase, pGEM-T Easy support agent box, reversed transcriptive enzyme are available from Promega company; PyrobestDNA polysaccharase, dna molecular amount standard DL2000 are available from TaKaRa company; TRIzolReagent is available from Invitrogen company; Adenosine diphosphate (ADP) (ADP) is the Biopool product; Dna fragmentation reclaims test kit available from OMEGABIO-TEK company; It is Time Inc. available from the sky that plasmid reclaims test kit; The anti-M13 antibody of horseradish peroxidase-labeled is available from amershampharmacia company; Other chemical reagent commonly used are import or homemade analytical pure.

3.2 method

3.2.1 the structure of carrier for expression of eukaryon PWS3H22 and expression

Because the antibody gene that obtains by antibody library does not have leader peptide sequences,, design optimized antibody leader peptide sequences so at first analyze according to BLAST.Behind the cis-acting elements of binding analysis expression vector PWS3, the redesign primer comes out by pcr amplification being cloned in pGEM-TEasy carrier H22 heavy chain and chain variable region gene respectively, is cloned in the PWS3 expression vector, make up the PWS3H22 carrier for expression of eukaryon, carry out the eucaryon transfection.The transfection basic parameter comprises: (1) Chinese hamster ovary celI density is 1 * 10 ⁵Individual/ml, be inoculated in the Tissue Culture Dish of diameter 100mm the day before yesterday in transfection, cell cultures is spent the night, and (2) reach 80% when adherent when cell, complete culture solution is removed, change serum free medium and hatched 2-8 hour, (3) will cut through enzyme and identify errorless expression vector 4 μ g, and 10 μ l liposomes are dissolved in respectively in the DMEM substratum of 250 μ l antibiotic-frees, serum-free, fully behind the mixing, room temperature was placed 5 minutes, and again with two kinds of abundant mixings of solution, room temperature was placed 30 minutes.Treat transfection culturing cell twice with antibiotic-free, serum-free DMEM washing simultaneously, in DNA-liposome mixture, add the DMEM substratum of 2.5ml antibiotic-free, serum-free, mix the back and add in the culturing cell.Culturing bottle is put into 37 ℃ of incubators hatch to inhale after 6 hours and remove nutrient solution, (4) discard transfection liquid, and (5) add the DMEM substratum that 5ml contains microbiotic and 10% calf serum, continue to cultivate about 24 hours.After treating that cell covers with, change the DMEM substratum of serum-free into.Received supernatant in (6) 60 hours, and changed cell over to stably express, the pressurization screening.

3.2.2 chimeric antibody CH22 purifying

Contain chimeric antibody CH22 nutrient solution with after the 0.1mol/LpH8.0PB dialysed overnight, be splined on Protein A Sephorase CL-4B chromatography column, behind same buffer wash-out foreign protein,, collect elution peak and concentrate with pH6.0 citrate buffer solution wash-out CH22.

3.2.3CH22 combine experiment with the TNF-alpha specific

Respectively hTNF-α is separated by affine 96 orifice plates of ELISA and SDS-PAGE with the finite concentration bag, ELISA and Western blot analysis of experiments identify that ScFv-H22 combines with the TNF-alpha specific.(" molecular cloning ") seen in ELISA, Western blot test elementary operation.

3.2.4CH22 the antigen recognition epi-position is determined experiment

The ultrasonic supernatant that will contain hTNF-α, hTNF-α D141-146 is diluted to 10 μ g/ml with coating buffer and wraps quilt.100 μ l/ holes bag is placed after 8 hours for 4 ℃ by affine 96 orifice plates of ELISA, and the 2%BSA sealing is spent the night.PBST washes plate three times, adds CH22 and carries out the ELISA detection by 100 μ l/ holes.Room temperature 90 minutes, PBST washes plate three times, adds horseradish peroxidase labeling antibody (HRP-GAH is available from PIERCE company) 50 μ l/ holes, and room temperature is after 40 minutes, and PBST washes plate three times, with the colour developing of OPD Color Appearance System, surveys A _492nmAbsorbance.

3.2.5L929 in the cell toxicant and the experiment

L929 is a l cell system, and it is extremely sensitive for TNF-α, exists the TNF-α of ng level promptly can cause the mass mortality of L929 cell in the growing environment.Eugonic L929 cell is at first got in this test, determines its activity unit (IU) according to TNF-α Determination of biological activity method, mix with the anti-TNF-Alpha antibodies of different concns with 10IUTNF-α then and with L929 cell (3 * 10 ⁴, the hole) together add 96 well culture plates, add dactinomycin (0.1 μ g/ hole) simultaneously, 37 ℃, 5%CO ₂Cultivated 20 hours under the condition, after violet staining, add 33% acetate, enzyme connection instrument is measured A automatically _570nm

3.2.6 chimeric antibody CH22 avidity is measured

Measure the method design experiment that affinity of antibody method-Scatchard analyzes according to classics,,, obtain antibody CH22 and hTNF-α bonded typical curve according to finite concentration dilution antibody (CH22) at first with antigen (hTNF-α) concentration fixed.On this basis, in antibody and antigen bonded saturation concentration scope, get the normal concentration point that some concentration are measured as affinity of antibody, measure binding antibody and reach the not concentration of binding antibody, according to formula B/F=Ka (S-B); B wherein: represent binding antibody concentration, F: represent free antibodies concentration, S: represent the antibody total concn

3.2.7 chimeric antibody CH22, Remicade

Recognition site, affinity, in and specific activity

In order further to determine the biological effect of chimeric antibody CH22, select the clinical treatment antibody Remicade that has ratified by FDA

Experimentize in contrast.Utilize hTNF deletion mutant (hTNF _23-89[expression disappearance 23-89 position residue], hTNF _53-92, hTNF _141-146) recognition site of the two is analyzed, utilize ELISA and cellulotoxic experiment that the two affinity, neutralization activity are compared.

3.3 result

3.3.1 the structure of recombinant expression plasmid PWS3H22 and evaluation

Get the correct clone of sequencing analysis, respectively with XbaI, BamHI digestion pGEM-TEasy/H22V κ S, with XhoI, HindIII digestion pGEM-T Easy/H22VHS, go into after glue reclaims purifying, H22V κ S and H22VHS fragment are cloned in respectively through enzyme cut on the PWS3 expression vector of (BamHI/XbaI, XhoI/HindIII) (Fig. 3 .3.1.1 and Fig. 3 .3.1.2).

3.3.2CH22 detect with hTNF-alpha specific bonded

Can combine with the hTNF-alpha specific by the CH22 that obtains behind ELISA and Western blot evidence process eukaryotic expression and the purifying respectively, prove that the antibody that obtains by above method screening has kept the identification specificity of maternal antibody Z12.(Fig. 3 .3.2.1)

3.3.3CH22 antibody molecule is identified

Contain chimeric antibody CH22 eukaryotic cell expression supernatant, behind Protein ASephorase CL-4B chromatography column purifying, the SDS-PAGE electrophoretic analysis, obtaining molecular weight is 155kDa protein band (Fig. 3 .3.3.1), with human IgG and Remicade

The chimeric antibody molecular weight is identical.

3.3.4CH22 antigen recognition epi-position confirmed test

The theoretical prediction result shows, the antigen recognition epi-position of antibody CH22 and maternal antigen Z12 are consistent, we carry out prokaryotic expression respectively with hTNF α D29-88 deletion mutant, hTNF α D53-91 deletion mutant, hTNF α D141-146 deletion mutant, hTNF, and will express supernatant and be coated in affine 96 orifice plates, detect several different anti-hTNF Alpha antibodies and its two Recognition Different by ELISA, thereby determine the antigen recognition site of antibody, and and Remicade

Antibody and Enbrel

Recognition site compares; The result shows that Z12 and CH22 antigen recognition site are consistent, and and Remicade

(through the anti-hTNF α chimeric antibody of FDA approval listing) and Enbrel

Different.(Fig. 3 .3.4.1)

3.3.5L929 in the cell toxicant and the experiment

Z12 have good in and the cytotoxic ability of hTNF-α, L929 cell toxicant neutralization test is the result show, (table 3.3.5.1) antibody CH22 kept Z12 in and function, and increase on this basis, point out it to have good potential applicability in clinical practice.

Compare with cell toxicant in the anti-TNF-Alpha antibodies of table 3.3.5.1

^*Titre is defined as the inverse of the highest diluted sample degree of the cellulotoxic effect that suppresses 10U/ml TNF-α.

3.3.6 chimeric antibody CH22 avidity is measured

According to the result that the classics mensuration affinity of antibody method that method-Scatchard analyzes is measured, the data fitting that records is in alignment, try to achieve this collinear negative slope and be antibody CH22 affinity costant (K _a) the results are shown in Figure 3.3.5.1.

Sequence table

＜110〉Beijing Tian-Guang Biotechnology Co., Ltd. Is

＜120〉TNF α high-affinity chimeric antibody and uses thereof

<130>1040003

<160>27

<170>PatentIn?version?3.2

<210>1

<211>223

＜212〉protein

＜213〉mouse

<400>1

Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Pro?Glu?Leu?Val?Lys?Pro?Gly?Ala

1 5 10 15

Ser?Val?Lys?Met?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Thr?Phe?Thr?Ser?Tyr

20 25 30

Val?Leu?His?Trp?Leu?Gln?Pro?Lys?Pro?Gly?Gln?Gly?Pro?Glu?Trp?Ile

35 40 45

Gly?Tyr?Val?Tyr?Pro?Tyr?Asn?Asp?Asp?Thr?Gln?Tyr?Asn?Glu?Arg?Phe

50 55 60

Arg?Gly?Lys?Ala?Thr?Leu?Thr?Ser?Asp?Lys?Ser?Ser?Thr?Thr?Ala?Tyr

65 70 75 80

Met?Asp?Leu?Ser?Gly?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Phe?Tyr?Cys

85 90 95

Ala?Arg?Gly?Trp?Leu?Gln?Arg?Ser?Tyr?Trp?Tyr?Phe?Asp?Val?Trp?Gly

100 105 110

Ala?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Ala?Lys?Thr?Thr?Ala?Pro?Ser

115 120 125

Val?Tyr?Pro?Leu?Ala?Pro?Val?Cys?Gly?Asp?Thr?Thr?Gly?Ser?Ser?Val

130 135 140

Thr?Leu?Gly?Cys?Leu?Val?Lys?Gly?Tyr?Phe?Pro?Glu?Pro?Val?Thr?Leu

145 150 155 160

Thr?Trp?Asn?Ser?Gly?Ser?Leu?Ser?Ser?Gly?Val?His?Thr?Phe?Pro?Ala

165 170 175

Val?Leu?Gln?Ser?Asp?Leu?Tyr?Thr?Leu?Ser?Ser?Ser?Val?Thr?Val?Thr

180 185 190

Ser?Ser?Thr?Trp?Pro?Ser?Gln?Ser?Ile?Thr?Cys?Asn?Val?Ala?His?Pro

195 200 205

Ala?Ser?Ser?Thr?Lys?Val?Asp?Lys?Lys?Ile?Glu?Pro?Ser?Gly?Pro

210 215 220

<210>2

<211>212

＜212〉protein

＜213〉mouse

<400>2

Glu?Leu?Val?Leu?Thr?Gln?Ser?Pro?Ala?Ile?Met?Ser?Val?Ser?Pro?Gly

1 5 10 15

Glu?Lys?Val?Thr?Met?Thr?Cys?Asn?Ala?Ser?Ser?Ser?Val?Ser?Tyr?Met

20 25 30

Tyr?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Ser?Ser?Pro?Arg?Leu?Leu?Ile?Tyr

35 40 45

Asp?Thr?Ser?Asn?Leu?Ala?Ser?Gly?Val?Pro?Val?Arg?Phe?Ser?Gly?Ser

50 55 60

Gly?Ser?Gly?Thr?Ser?Tyr?Ser?Leu?Thr?Ile?Ser?Arg?Met?Glu?Ala?Glu

65 70 75 80

Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Trp?Ser?Ser?Tyr?Ser?Pro?Thr

85 90 95

Phe?Gly?Ala?Gly?Thr?Lys?Leu?Glu?Lys?Arg?Ala?Asp?Ala?Ala?Pro?Thr

100 105 110

Val?Ser?Ile?Phe?Pro?Pro?Ser?Ser?Glu?Gln?Leu?Thr?Ser?Gly?Gly?Ala

115 120 125

Ser?Val?Val?Cys?Phe?Leu?Asn?Asn?Phe?Tyr?Pro?Lys?Asp?Ile?Asn?Val

130 135 140

Lys?Trp?Lys?Ile?Asp?Gly?Ser?Glu?Arg?Gln?Asn?Gly?Val?Leu?Asn?Ser

145 150 155 160

Trp?Thr?Asp?Gln?Asp?Ser?Lys?Asp?Ser?Thr?Tyr?Ser?Met?Ser?Ser?Thr

165 170 175

Leu?Thr?Leu?Thr?Lys?Asp?Glu?Tyr?Glu?Arg?His?Asn?Ser?Tyr?Thr?Cys

180 185 190

Glu?Ala?Thr?His?Lys?Thr?Ser?Thr?Ser?Pro?Ile?Val?Lys?Ser?Phe?Asn

195 200 205

Arg?Asn?Glu?Cys

210

<210>3

<211>10

＜212〉protein

＜213〉mouse

<400>3

Gly?Tyr?Thr?Phe?Thr?Ser?Tyr?Val?Leu?His

1 5 10

<210>4

<211>17

＜212〉protein

＜213〉mouse

<400>4

Tyr?Val?Tyr?Pro?Tyr?Asn?Asp?Asp?Thr?Gln?Tyr?Asn?Glu?Arg?Phe?Arg

1 5 10 15

Gly

<210>5

<211>12

＜212〉protein

＜213〉mouse

<400>5

Gly?Trp?Leu?Gln?Arg?Ser?Tyr?Trp?Tyr?Phe?Asp?Val

1 5 10

<210>6

<211>10

＜212〉protein

＜213〉mouse

<400>6

Asn?Ala?Ser?Ser?Ser?Val?Ser?Tyr?Met?Tyr

1 5 10

<210>7

<211>7

＜212〉protein

＜213〉mouse

<400>7

Asp?Thr?Ser?Asn?Leu?Ala?Ser

1 5

<210>8

<211>9

＜212〉protein

＜213〉mouse

<400>8

Gln?Gln?Trp?Ser?Ser?Tyr?Ser?Pro?Thr

1 5

<210>9

<211>32

<212>DNA

＜213〉synthetic

<400>9

ccagttcgga?gctcgttgtg?actcaggaat?ct 32

<210>10

<211>32

<212>DNA

＜213〉synthetic

<400>10

ccagttccga?gctcgtgttg?acgcagccgc?cc 32

<210>11

<211>32

<212>DNA

＜213〉synthetic

<400>11

ccagttccga?gctcgtgctc?acccagtctc?ca 32

<210>12

<211>32

<212>DNA

＜213〉synthetic

<400>12

ccagttccga?gctccagatg?acccagtatc?ca 32

<210>13

<211>32

<212>DNA

＜213〉synthetic

<400>13

ccagatgtga?gctcgtgatg?acccagactc?ca 32

<210>14

<211>32

<212>DNA

＜213〉synthetic

<400>14

ccagatgtga?gctcgtcatg?acccagtctc?ca 32

<210>15

<211>32

<212>DNA

＜213〉synthetic

<400>15

ccagttccga?gctcgtgatg?acacagtctc?ca 32

<210>16

<211>31

<212>DNA

＜213〉synthetic

<400>16

ccgactagtt?catttcagct?ccagccaggt?g 31

<210>17

<211>30

<212>DNA

＜213〉synthetic

<400>17

ctcgacacta?gttttgcgct?caactgtctt 30

<210>18

<211>30

<212>DNA

＜213〉synthetic

<400>18

tgtgtgacta?gtgtcaccaa?gtggggtttt 30

<210>19

<211>30

<212>DNA

＜213〉synthetic

<400>19

gcatgaacta?gttgggggac?catatttgga 30

<210>20

<211>21

<212>DNA

＜213〉synthetic

<400>20

aggtctcgag?ctcgagtctg?g 21

<210>21

<211>21

<212>DNA

＜213〉synthetic

<400>21

aggtctcgag?ctcgagtcag?g 21

<210>22

<211>21

<212>DNA

＜213〉synthetic

<400>22

aggtctcgag?tccgagtctg?g 21

<210>23

<211>21

<212>DNA

＜213〉synthetic

<400>23

aggtctcgag?ttcgagtcag?g 21

<210>24

<211>21

<212>DNA

＜213〉synthetic

<400>24

aggtctcgag?gctgagtctg?g 21

<210>25

<211>21

<212>DNA

＜213〉synthetic

<400>25

aggtctcgag?cgtaagtctg?g 21

<210>26

<211>21

<212>DNA

＜213〉synthetic

<400>26

aggtctcgag?ggaaagtcag?g 21

<210>27

<211>25

<212>DNA

＜213〉synthetic

<400>27

gacttggtct?agacgagacg?gtgac 25

Claims (4)

1. the antibody of anti-human tumor necrosis factor or its antigen-binding portion thereof, the 141-146 amino acids of its identification human tumor necrosis factor, standard body outside in the L929 detection to be higher than 1 * 10 ^-7Among the IC50 of M and people TNF cytotoxicity; Described antibody or its antigen-binding portion thereof comprise the aminoacid sequence of SEQ IDNO:1, and this sequence contains heavy chain CDR1, CDR3 territory; Described antibody or its antigen-binding portion thereof also comprise the aminoacid sequence of SEQ ID NO:2, and this sequence contains light chain CDR1, CDR3 territory.

2. the antibody of claim 1 or its antigen-binding portion thereof, it is chimeric antibody or its antigen-binding portion thereof.

3. claim 1 or 2 antibody or its antigen-binding portion thereof are used for and the purposes of human tumor necrosis factor external.

4. claim 1 or 2 antibody or its antigen-binding portion thereof purposes in the medicine of preparation treatment multiple organ dysfunction syndrome, rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, inflammatory bowel, graft versus host disease (GVH disease) and the disorderly syndromes of marrow hemopoiesis.

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