CN105777905A - Fully human anti-TNF (Tumor Necrosis Factor)-alpha monoclonal antibody and application thereof - Google Patents

Fully human anti-TNF (Tumor Necrosis Factor)-alpha monoclonal antibody and application thereof Download PDF

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CN105777905A
CN105777905A CN201610171500.0A CN201610171500A CN105777905A CN 105777905 A CN105777905 A CN 105777905A CN 201610171500 A CN201610171500 A CN 201610171500A CN 105777905 A CN105777905 A CN 105777905A
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tnf
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amino acid
monoclonal antibody
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CN105777905B (en
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涂晶晶
李扬
杨彬
李文佳
孙文正
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Guangdong HEC Pharmaceutical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

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Abstract

The invention relates to the field of biotechnology, in particular to a fully human anti-TNF (Tumor Necrosis Factor)-alpha monoclonal antibody. A heavy chain variable region is provided with an amino acid sequence as shown in an SEQ ID NO:1, or an amino acid sequence with at least 98 percent of homology; a light chain variable region is provided with an amino acid sequence as shown in an SEQ ID NO: 2, or an amino acid sequence with at least 98 percent of homology. In addition, the invention also discloses a DNA (Deoxyribonucleic Acid) sequence of the novel fully human anti-TNF-alpha monoclonal antibody. A heavy chain variable region nucleotide code is a nucleotide sequence as show in an SEQ ID NO: 3; a light chain variable region nucleotide code is a nucleotide sequence as show in an SEQ ID NO: 4. The invention discloses the application prospect of the fully human anti-TNF-alpha monoclonal antibody in preparing medicines for treating autoimmune diseases.

Description

A kind of full people source anti-tnf-alpha monoclonal antibody and application thereof
Technical field
The present invention relates to biological technical field, concrete discloses a kind of novel full human monoclonal antibody, including coded sequence, expression vector, place Chief cell and preparation method thereof and the application in preparation treatment autoimmune disease medicine of the described anti-tnf-alpha human antibody.
Background technology
Tumor necrosis factor α (Tumor necrosis factor α, TNF-α) is the cell factor that a class has extensive BA, mainly by list The immunocytes such as nucleus, macrophage, adipocyte, B cell, T cell and NKT (NK) cell produce.Internal TNF-α albumen There are Transmembrane TNF α and Secretary TNF α.233 amino acid of cross-film TNF-α total length, wherein 1-76 amino acids is that signal peptide (includes cross-film ammonia Base acid), 77-233 amino acid is extracellular fragment.The 77-233 amino acid is also the amino acid sequence of Secretary TNF α, its size be 17KD (Smith, Et al., J.Biol.Chem.262:6951-6954,1987), combine with the TNF acceptor (TNFR) of trimeric form with cell surface, mediation Various biological effect.TNF-α acts on the acceptor on oncocyte surface, by the identification of cell, combination, endocytosis are entered lysosome, causes lyase Body and protease advanced activation cause cell death, and it plays an important role in immune response, inflammation and injury response, mainly affect cell proliferation With apoptotic regulation, the dissolving of cytotoxicity, cell, inducing cell apoptosis, Inhibit proliferaton etc. is not only had to act on to tumour cell, additionally it is possible to promote Enter myeloid leukemia cell to macrophage differentiation, the phagocytic activity of raising neutrophil leucocyte.
There is a lot of defect as medicine in mouse source monoclonal antibody, owing to human body has strong immunogenicity, internal elimination is fast, and the half-life is short, causes Clinical efficacy is limited, and side effect is big.For overcoming the problem using mouse-anti body to produce at human body, mouse-anti body is carried out genetically engineered humanization, example If people mouse inosculating antibody TNF-α monoclonal antibody (Infliximab) is to utilize genetic engineering to prepare, mouse source TNF-α monoclonal antibody is still taken from its variable region, remains Specific and the affinity being combined with TNF soluble fragments and cross-film district, constant region is replaced by the constant region of human IgG1, internal partly declines Phase is greatly prolonged.But these chimeric antibodies and humanized antibody still may cause undesired immune response, particularly when long term administration.2002 Year December, the Humira of Abbott company became first symptom and disease being approved for alleviating in adult to severe Active Rheumatoid Arthritis (RA) Shape and the human monoclonal antibodies of suppression articulation structure lesion progress.Owing to being human antibody, it is to avoid immunoreactive shortcoming can be produced.
Autoimmune disease is the third-largest class disease of cancer and angiocardiopathy of continuing.RA is one of modal autoimmunity disease, China's rheumatoid Arthritis illness rate is about 0.32-0.36%, and more than 60 years old, crowd's incidence of disease is up to 30%～40%.In the face of expensive import drug therapy for each person every year Expense, patient often can only long-term uncomplaining chemotherapy.Though import antibody drug has been enter into Chinese market, but the price of its costliness makes patient Being difficult to accept, therefore, the exploitation in the urgent need to domestic antibody drug lists.
For attempting to overcome the relevant issues using non-human source antibodies and humanized antibody to cause, reduce HAMA by the human antibody built (HAMA) the immunity of organism originality caused, it has also become more effective therapeutic strategy.Under existing situation, at home, this area is in the urgent need to setting up Antibody can either be kept or improve specific, the anti-tnf-alpha monoclonal antibody of antibody immunogenicity can be reduced or eliminate again, such that it is able to for clinical treatment RA and acquired immune deficiency syndrome (AIDS) etc..Therefore, the invention provides a kind of safe and reliable, longer at people's Half-life in vivo, The significant human antibody of function.
Summary of the invention
The invention discloses a kind of full people source anti-tnf-alpha monoclonal antibody, including variable region of heavy chain and variable region of light chain, its variable region of heavy chain (VH) Having SEQ ID NO:1 amino acid sequence, its variable region of light chain (VL) has SEQ ID NO:2 amino acid sequence.
The invention discloses a kind of full people source anti-tnf-alpha monoclonal antibody, including heavy chain and light chain, its heavy chain (H) is shown in SEQ ID NO:5 Amino acid sequence, its light chain (L) is the amino acid sequence shown in SEQ ID NO:6.
The invention also discloses a kind of nucleotide sequence, encode above-mentioned full people source anti-tnf-alpha monoclonal antibody.
The invention discloses above-mentioned nucleotide sequence, including variable region of heavy chain and variable region of light chain, wherein encode full people source anti-tnf-alpha monoclonal antibody Variable region of heavy chain for SEQ ID NO:3 nucleotide sequence, encode full people source anti-tnf-alpha monoclonal antibody variable region of light chain for SEQ ID NO: 4 nucleotide sequences.
The invention discloses above-mentioned nucleotide sequence, wherein encode full people source anti-TNF-α antibody heavy chain for the nucleotides sequence shown in SEQ ID NO:7 Row, encode full people source anti-TNF-α antibody light chain for the nucleotide sequence shown in SEQ ID NO:8；
The present invention not only includes complete monoclonal antibody, also includes immunocompetent antibody fragment, such as: Fab (antigen-binding fragment), Fragment；Heavy chain of antibody；Light chain of antibody；Genetically engineered ScFv (single chain Fv) molecule.
The invention discloses a kind of genetically engineered host cell, it is by many containing the described anti-human TNF-α monoclonal antibody heavy chain of coding and light chain The carrier of nucleotides is converted or is transduceed.Described host cell is eukaryotic, specifically may is that the gonad cell CHO of Chinese hamster, the kidney of monkey Dirty cell COS cell, the embryonic kidney cells HEK-293 of people, the cervical cancer cell HELA etc. of people.
The full people source anti-tnf-alpha monoclonal antibody that the invention discloses has the purposes in preparation treatment autoimmune disease medicine, autoimmunity Property disease is rheumatoid arthritis, ankylosing spondylitis, psoriasis.
Accompanying drawing explanation
Fig. 1 is a curve map, describes compared with adalimumab, full people source anti-TNF-α antibody HE03 (antibody prepared in the present invention) press down TNF-α processed and the Binding experiment of U-937 cell surface receptor.
Fig. 2 is a bar chart, describes compared with adalimumab, little with people anti-TNF-α antibody HE03 protection D-galactosamine sensitization by giving The lethal that mouse is induced from TNF-a.
Detailed description of the invention
Below in conjunction with specific embodiment, this explanation is expanded on further.The present invention is only further detailed by following example, experimental example, no It is interpreted as limitation of the present invention.The test method of unreceipted actual conditions in the following example, generally according to normal condition, such as Sambrook Et al., molecular cloning: the condition described in laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989), or press According to the condition proposed by manufacturer.
Various conventional chemical reagent used in embodiment scheme, is commercially available prod.A Da wood antibody used in embodiment is i.e. Adalimumab monoclonal antibody, purchased from Abbott company；Actinomycin D is bought in magnificent bioengineering Co., Ltd；CDNA Reverse Transcription box is purchased from Dalian treasured bioengineering Co., Ltd；Trizol reagent is purchased from Invitrogen company；E. coli tg1 is purchased from Amersham company；Sfi I and NOT I double digestion is bought in Dalian treasured bioengineering Co., Ltd；ScFv gene library and phagemid vector pCANTAB-5E are purchased from Sweden Amersham Pharmacia company；Recombined human TNF-α (rhTNF-α) antigen is purchased from Shenzhen Jing Mei company；Lymphocyte separation medium is that Tianjin Blood Research Institute is raw Produce.
One, the structure of humanized's single-chain antibody library:
(1) preparation of cDNA
Collecting 100 each 10ml of healthy human peripheral blood, mixing, with lymphocyte separation medium separation mononuclearcell.With Trizol reagent from separation Human peripheral lymphocyte extracts the total serum IgE of cell, goes out cDNA with cDNA Reverse Transcription box reverse transcription.Above step provides according to producer Specification is carried out.
(2) structure of antibody library
Design of primers, bibliography (Immunotechnology, 1998,3:271-278) designs and synthesizes human cloning antibody heavy chain variable region (VH) VHBack, VHFor, VLBack and VLFor primer with variable region of light chain (VL) gene.VHBack, VHFor, VLBack and VLFor Sequence see Immunotechnology, 1998,3:271-278.Wherein 5 ' the ends at VHBack primer add the sequence containing Sfi I site Atg gcc cag ccg gcc atg gcc, the 5 ' ends at VHFor primer add sequence gcc aga acc acc gcc gcc gga gcc acc acc gcc, 5 ' ends of VLBack primer add containing Not plus sequence tccggc ggc ggt ggt tct ggc gga ggc gga tct, the 5 ' ends at VLFor primer Sequence atg cgg ccg c (primer is synthesized by Invitrogen Corp.) of I site.
With reference to above-mentioned primer, from cDNA the first chain, expand variable region of heavy chain and chain variable region gene；Preparation ScFv linker DNA；Use ScFv Joint PCR assembling VH and VL carries out the structure of ScFv gene library；Expand ScFv gene library again and clone to add restriction site； ScFv gene library and phagemid vector pCANTAB-5E are carried out Sfi I and NOT I double digestion simultaneously；ScFv and the connection of carrier DNA； Attachment electricity converts e. coli tg1.Then cell is incubated at SOB-AG (containing 100mg/l ampicillin and the SOB of 2% glucose Culture medium) on agar plate, cultivation temperature 30 DEG C.Converted by repeatedly 100 electricity, make the capacity of obtained antibody library reach 1x1010。
(3) phage antibody that preparation selects on antigen
Taking part phage library bacterium solution 2 × YT (containing 100 μ g/mL ammonia benzyl and 2% glucose) dilution, 37 DEG C of concussions are cultivated 2 hours；According to auxiliary Helper phage and the ratio that ratio is 10:1 of bacterium add helper phage M13KO7 carry out infecting 1 little time；Then centrifugal thin with 3000r/ minute Bacteria culture fluid, abandons supernatant.By bacterial precipitation with 2 × YT (containing 100 μ g/mL ammonia benzyls and 50 μ g/L kanamycins) after culture medium is resuspended, 37 DEG C of shakes Swing overnight；Overnight will take supernatant after being centrifuged 30 minutes by bacterium, add the PEG/NaCl (20%PEG 8000/2.5M NaCl) of 0.2 times of volume precooling Solution precipitating phage, ice bath 1 hour；Centrifugal 15 minutes, abandon supernatant, with the aseptic resuspended precipitation of PBS, again add the PEG/NaCl of 0.2 volume Solution precipitating phage, ice bath is after 1 hour；Centrifugal 15 minutes, with the PBS dissolution precipitation of 1/10 the most aseptic volume, 4 DEG C stored for future use. Two, the elutriation of antibody library and positive clone identification:
For the antibody in the phage display library that embodiment 1 obtains, select high-affinity antibody by panning technique.
(1) enrichment isolation of phage antibody library
With the antigen coated culture plate of rhTNF-α at 4 DEG C overnight；Taking-up is coated plate, washs 3 times with PBS, closes with the PBS containing 10% skimmed milk power Liquid chamber temperature is closed 2 hours；Outwelling liquid, PBS washs 3 times, adds phage antibody library, cultivates 2 hours for 37 DEG C；Turned letter, with PBS (containing 0.05%Tween 20) washing 5 times, wash 5 times (the 2nd takes turns each washing 10 times, and the 3rd takes turns each washing 20 times, and wash time also increases) with PBS；Turned letter, adds 100 MM triethylamine (pH=12), adds 1M Tris (pH=7.4) mixing after room temperature elution phage particle 10 minutes and neutralizes, transfer to 15mL Guan Zhong；With the E.coli TG1 (OD being in exponential phase600=0.4) and wash-out bacteriophage mixing, in 37 DEG C of water-baths stand 30 minutes.Take 200 μ L have infected the mensuration of bacterial community sum (cfu) that the bacterium of bacteriophage carries out in unit volume, remaining bacterium centrifugation, (contain with 2 × YT 100 μ g/mL ammonia benzyl and 2% glucose) resuspended, to coat on flat board, put 37 DEG C of overnight incubation, bacteriophage is sieved and washed for next round in preparation, carries out altogether 3 take turns screening.
(2) ELISA method identifies positive colony
Preferable colony inoculation, overnight incubation will be separated.With the antigen coated 96 hole elisa plate of rhTNF-α 4 DEG C overnight；Take 20 μ l next day overnight Culture is seeded in 96 hole elisa plates, cultivates to OD600About=0.5, centrifugation, abandon supernatant, each hole adds 200 μ L 2 × YT (containing the denseest The isopropylthiogalactoside of degree 1mM, 37 DEG C, 250rpm induces 3 hours；Induced product is centrifuged 15 minutes, collects supernatant standby； Being washed three times by the elisa plate PBS being coated, every hole adds 300 μ L confining liquids, and room temperature is closed 2 hours；The elisa plate PBS that will have closed Washing three times, every hole adds 100 μ L Block buffer, respectively adds 100 μ L induction supernatant (containing the ScFv of secretion) mixings, 37 DEG C of water toward each hole Combine water-bath under bath and combine 1 hour；Join Anti-E-Tag antibody (with the dilution proportion of 1:1000 in Block buffer), wash three times with PBS After, adding the Anti-E-Tag antibody that 100 μ L have diluted and combine 1 hour 37 DEG C of water-baths, PBS washes three times, adds the horseradish peroxidase diluted The antibody 100 μ L of enzyme labeling, 37 DEG C of water-baths 1 hour；PBS washes 3 times, adds (OPD) nitrite ion, and black out develops the color 30 minutes.Add 2M H2SO4 Terminating reaction, ELIASA surveys its OD490Value.
(3) the determined dna sequence analysis of positive colony
Select the higher independent cloning of positive colony ELISA value of ELISA detection to be sent to Shanghai Sheng Gong bioengineering Co., Ltd and carry out sequencing analysis.
After 3 elutriation antibody libraries, obtain a strain anti-human TNF-α single-chain antibody ScFv, after order-checking, obtain its gene order.SEQ ID NO:3 With nucleotide sequence and the amino acid sequence that SEQ ID NO:1 respectively illustrates ScFv variable region of heavy chain VH.SEQ ID NO:4 and SEQ ID NO:2 respectively illustrates nucleotide sequence and the amino acid sequence of ScFv variable region of light chain VL.
Three, anti-human TNF-α heavy chain carrier for expression of eukaryon and the structure of anti-human TNF-α light chain carrier for expression of eukaryon:
(1) full genome synthesis
Report according to document (Cell, 1980,22:197-207) and document (NucleicAcids Research, 1982,10:4071-4079) Sequence, SEQ ID NO:9 shows the amino acid sequence of CH (CH).SEQ ID NO:10 shows constant region of light chain (CL) Amino acid sequence；Variable region of heavy chain (VH) amino acid sequence of the human antibody that screening of phage antibody library obtains and variable region of light chain (VL) amino acid Sequence, is combined into complete heavy chain amino acid sequence (SEQ ID NO:5) by VH+CH, and VL+CL is combined into complete light chain amino acid Sequence (SEQ ID NO:6), human antibody entire heavy chain, Whole light chains gene, respectively 5 ' end add KOZAK sequence (GCCGCCACC), Initiation codon, signal peptide gene sequence, at 3 ' ends containing 2 translation termination codons.Signal peptide amino acid sequence is SEQ ID NO:11: MEFGLSWLFLVAILKGVQC (with reference to A Da wood patent, WO_2007_014162_A2), with according to combinations of the above amino acid sequence, After carrying out base codon optimization, full genome synthesizes above-mentioned two genetic fragments (Invitrogen Corp. synthesizes by Shanghai).Heavy chain combinations after optimization The nucleotide base sequence of fragment is SEQ ID NO:7；The nucleotide base sequence of the light chain combination fragment after optimization is SEQ ID NO:8；
(2) structure of expression vector
Above-mentioned two genetic fragment by specification steps are passed throughTA Cloning Kit (Invitrogen, Catalog no. K8300-01) andTA Vector Kit (Invitrogen, Catalog no.12744-017) carries out TA clone respectively, Heavy chain combinations fragment TA is cloned intoVector, light chain combination fragment TA is cloned intoVector, Success builds heavy chain expression vector and light chain expression vector.
Four, the expression of anti-human TNF-α complete antibody:
The above-mentioned recombinant expression carrier plasmid co-transfection built is proceeded to mammalian host cell line, to express anti-hTNF-α human antibody. In order to stablize high-caliber expression, preferred host cell system is Chinese hamster ovary (CHO) cell of dihyrofolate reductase (DHFR) deficiency (see, e.g. the United States Patent (USP) 4818679 of Chasin, L. et al.).Preferably transfection method is electroporation, it is possible to use additive method, Including calcium phosphate cosedimentation, fat transfection and Protoplast fusion etc..In electroporation, with being set to 300V electric field and the electroporation apparatus of 900 μ Fd electric capacity Gene Pulser (Bio-Rad Laboratories),
1.5 × 10 are added in cuvette7Individual cell is suspended in the PBS of 0.8mL, and containing 40 μ g PvuI (being purchased from TakaRa) linearisations Expression vector DNA.After transfecting 48 hours, phage library cell is carried out Geneticin (G418) and methotrexate (methotrexate Or MTX) pressurization screening, added by many wheels variable concentrations MTX (100nm, 200nm, 500nm, 1000nm, 2000nm, 5000nm) After pressure screening, with Method of Limited Dilution subclone transfectant, selecting the cell line of high level expression human antibody, overexpression cell line is further put Big fermented and cultured, obtains a number of high-purity people source anti-TNF-α antibody by purifying, and named HE03 is specific for follow-up antibody And the assessment etc. of activity.
Five, the affinity that anti-human TNF-α monoclonal antibody is combined with antigen:
Application Biacore X100 measures, Biacore X100 dynamics/affinity software analysis, and the method using capture indirectly is tried by amine coupling Agent box (Amine Coupling Kit) goat anti-human igg's Fc antibody coupling at CM5 chip surface as capture molecule, by calculate respectively HE03 and comparison adalimumab are diluted to finite concentration as part, using rhTNF-α as analyte.Analyte 5 concentration of dilution, each Concentration is as a circulation, first with HBS-EP (0.01M HEPES, pH7.4,0.15M NaCl, 3mM EDTA, 0.05% (v:v) surfactant P20) buffer solution runs 3 circulations, and designing an analyte concentration is that 0 concentration runs 2 circulations, and the analyte that finally design one repeats is dense Degree runs 1 circulation.Whole process operation 11 circulation, each circulation can draw a curve, by Biacore X100 dynamics/affine Power analysis software obtains surveyed antibody HE03 of the present invention and the dynamics/affinity data of control group adalimumab, and experiment is repeated 3 times.
Table 1 affinity experimental result
Note: Kd (M) is affinity constant；ka(M-1S-1) it is binding constant；Kd(S-1) it is dissociation constant.
Result is as shown in table 1, and the Kd value of the human antibody HE03 that display is obtained is substantially less than control group human antibody adalimumab, explanation Human antibody HE03 is higher than control group adalimumab to the affinity of TNF-α.
Six, the lethal effect of L929 cell is studied by antibody with TNF-α:
People's TNF-α (rhTNF-α) of recombinating causes the cytotoxicity to mouse L929 cell, is examined at L929 with rhTNF-α by common incubated antibodies as following Survey is evaluated in antibody HE03 of the present invention and activity.
L929 cell is incubated in the RPMI-1640 nutrient solution (GIBCO) containing 10% hyclone.After exponential phase cell dissociation counting, take 200g is centrifuged 5 minutes, abandons supernatant, and cell precipitation is resuspended with aforementioned nutrient solution, adjusts cell density to 2 × 105/ ml, adds to 96 well culture plates by cell In, 100 μ l/ holes, put 37 DEG C, 5%CO2Incubator overnight incubation.By containing 100 μ l antibody HE03,96 orifice plates of positive control A Da wood antibody 2 times of gradient serial dilutions are carried out with the RPMI1640 culture medium containing 10% hyclone.Add 50 μ l rhTNF-α and make the final of each sample well Concentration is 500pg/ml.Then by above-mentioned plate in incubation at room temperature 30 minutes, the L929 of the 50 μ l overnight incubation containing 1.0 μ g/ml actinomycin Ds is added Cell, adjusting final concentration of cells is every hole 5x104Individual, multiple hole is set.At CO2Concentration is 5%, temperature is overnight incubation in the incubator of 37 DEG C. Freshly prepared heterotope cell proliferation detection examination (Promega), is added in 96 well culture plates by 20 μ l/ holes, and is positioned over continuation cultivation in incubator 3 hours, the light at ELIASA detection 490nm absorbed, and reference wavelength is 630nm.With sample concentration as abscissa, absorbance value is ordinate, surveys Obtain IC50 value, the results are shown in Table 2.
Table 2: with the TNF-α lethal effect to L929 cell in antibody
Experimental result shows, the IC50 phase of the L929 killing functions of immunocytes of the present invention full people source anti-tnf-alpha monoclonal antibody HE03 antagonism TNF-α mediation More lower slightly than the IC50 in control group adalimumab, illustrate in HE03 and the ability of TNF-α is even more than control group adalimumab.
Seven, anti-TNF-α antibody HE03 suppression TNF-α and the Binding experiment of U-937 cell surface receptor:
With system of human tissue cell U-937 clone (ATCC NO.CRL1593) the detection people's anti-TNF-α antibody suppression expressing hTNF-α acceptor The ability that hTNF-α is combined with cell surface hTNF-α acceptor.Take U937 cell in good condition, with 10% tire ox after cell viability calculating instrument counting The RPMI1640 culture medium of serum adjusts cell concentration to 5 × 105Write unit, by cell by 100 μ l/ pipes point to Flow cytometry pipe.With The rhTNF-α that fluorescein isothiocynate (FITC, Amresco) is marked by PBS (for R&D Products 210-TA-050, uses dialysis mark Note obtains) it is diluted to 100ng/ml, respectively full people source anti-tnf-alpha monoclonal antibody HE03, adalimumab are diluted to 100 μ g/ml with this dilution, And continuous 2 times of gradient dilutions, the sample diluted and reference substance are added in streaming pipes by 100 μ l/ pipes, reacts 1 hour 4 DEG C of lucifuges；Again with containing 1% The PBS washed cell of hyclone 2 times, each 200g is centrifuged 5 minutes, abandons supernatant, and cell precipitation is resuspended in the 300 μ l PBS containing 1% hyclone In, the fluorescence intensity of flow cytomery often pipe.With sample concentration as abscissa, absorbance value is ordinate, records IC50 value, the results are shown in Table 3 and Fig. 1.
On table 3:HE03 suppression U-937 cell, TNF-α acceptor combines
Experimental result shows, the present invention full people source anti-tnf-alpha monoclonal antibody HE03 blocks the IC50 of TNF-α and the combination of U937 cell surface receptor Block TNF-α compared to control group adalimumab lower slightly with the IC50 value of the combination of U937 cell surface receptor, therefore monoclonal antibody HE03 of the present invention is described The ability neutralizing TNF-α is better than control group adalimumab.
Eight, the death assay of anti-TNF-α antibody HE03 antagonism TNF-α inducing mouse:
Cause 24 hours interior lethal to the injected in mice rhTNF-α of D-galactosamine sensitization, to show that TNF-α nertralizer can be in this mode Prevent lethal.1 μ g rhTNF-α and 20mg D-galactosamine (Amresco) lumbar injection, can induce 80-90%C57BL/6 dead mouse.For inspection Surveying people anti-TNF-α antibody HE03 ability of internal neutralization TNF-α in this model, in this experimental example, control group selects adalimumab equally, First give a certain amount of various antibody and carry out lumbar injection, after 30 minutes, then give 1 μ g rhTNF-α and 20mg D-galactosamine carries out lumbar injection, The protected effect of various antibody is observed after 24 hours.Each group injection dosage and result see table 4 and Fig. 2.
Table 4: each group mouse survival rate
Above experimental result shows, the effect of the full people source anti-tnf-alpha monoclonal antibody HE03 antagonism TNF-α inducing mouse death in the present invention is better than Control group adalimumab, therefore to neutralize the energy force rate adalimumab of TNF-α in Mice Body strong for this antibody.
The explanation of above example is only intended to help to understand method and the core concept thereof of the present invention.It should be pointed out that, for the art is common For technical staff, under the premise without departing from the principles of the invention, it is also possible to the present invention is carried out some improvement and modification, these improve and modify also Fall in the protection domain of the claims in the present invention.

Claims (8)

1. a full people source anti-tnf-alpha monoclonal antibody, it is characterised in that variable region of heavy chain have amino acid sequence shown in SEQ ID NO:1 or with It has the amino acid sequence of homology of at least 98%, and variable region of light chain has amino acid sequence shown in SEQ IDNO:2 or has at least 98% with it The amino acid sequence of homology.
2. the novel full people source anti-tnf-alpha monoclonal antibody described in claim 1, it is characterised in that heavy chain is amino acid shown in SEQ ID NO:5 Sequence, light chain is amino acid sequence shown in SEQ ID NO:6.
3. the DNA sequence dna of the coding novel full people source anti-tnf-alpha monoclonal antibody described in claim 1, it is characterised in that variable region of heavy chain nucleosides Acid encoding is the nucleotide sequence shown in SEQ ID NO:3, and variable region of light chain nucleotide coding is the nucleotide sequence shown in SEQ ID NO:4.
4. the DNA sequence dna of the coding novel full people source anti-tnf-alpha monoclonal antibody described in claim 2, it is characterised in that heavy chain nucleotide acid encoding For the nucleotide sequence shown in SEQ ID NO:7, light chain nucleotide is encoded to the nucleotide sequence shown in SEQ IDNO:8.
5. containing the expression vector of DNA sequence dna described in claim 3 or 4, it is protokaryon or carrier for expression of eukaryon.
6. containing the host cell of expression vector described in claim 5, it is eukaryotic.
Host cell the most according to claim 6, described host cell is the gonad cell CHO of Chinese hamster, the kidney cell of young hamster BHK, the kidney cell COS cell of monkey, the embryonic kidney cells HEK-293 of people, the cervical cancer cell HELA of people.
8. the purposes in preparation treatment autoimmune disease medicine of the people source anti-tnf-alpha monoclonal antibody described in claim 1, described autoimmunity Property disease is rheumatoid arthritis, ankylosing spondylitis, psoriasis.
CN201610171500.0A 2015-03-24 2016-03-23 A kind of full source of people anti-tnf-alpha monoclonal antibody and its application Expired - Fee Related CN105777905B (en)

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