CN105777905B - A kind of full source of people anti-tnf-alpha monoclonal antibody and its application - Google Patents

A kind of full source of people anti-tnf-alpha monoclonal antibody and its application Download PDF

Info

Publication number
CN105777905B
CN105777905B CN201610171500.0A CN201610171500A CN105777905B CN 105777905 B CN105777905 B CN 105777905B CN 201610171500 A CN201610171500 A CN 201610171500A CN 105777905 B CN105777905 B CN 105777905B
Authority
CN
China
Prior art keywords
tnf
seq
monoclonal antibody
cell
variable region
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CN201610171500.0A
Other languages
Chinese (zh)
Other versions
CN105777905A (en
Inventor
涂晶晶
李扬
杨彬
李文佳
孙文正
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guangdong HEC Pharmaceutical
Original Assignee
Guangdong HEC Pharmaceutical
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guangdong HEC Pharmaceutical filed Critical Guangdong HEC Pharmaceutical
Publication of CN105777905A publication Critical patent/CN105777905A/en
Application granted granted Critical
Publication of CN105777905B publication Critical patent/CN105777905B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • 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

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

The present invention relates to field of biotechnology, it is specifically related to a kind of full source of people anti-tnf-alpha monoclonal antibody, amino acid sequence of the heavy chain variable region with amino acid sequence shown in SEQ ID NO:1 or the homology for having at least 98% with it, amino acid sequence of the light chain variable region with amino acid sequence shown in SEQ IDNO:2 or the homology for having at least 98% with it.In addition invention also discloses a kind of DNA sequence dnas of above-mentioned novel full source of people anti-tnf-alpha monoclonal antibody, heavy chain variable region nucleotide coding is nucleotide sequence shown in SEQ ID NO:3, and light chain variable region nucleotide coding is nucleotide sequence shown in SEQ ID NO:4.The invention discloses application prospect of the source of people anti-tnf-alpha monoclonal antibody in preparation treatment autoimmune disease drug.

Description

A kind of full source of people anti-tnf-alpha monoclonal antibody and its application
Technical field
The present invention relates to field of biotechnology, specifically disclose a kind of novel full human monoclonal antibody, including coding Sequence, expression vector, host cell and preparation method thereof and the anti-tnf-alpha human antibody are in preparation treatment autoimmune disease Application in medicine.
Background technique
Tumor necrosis factor α (Tumor necrosis factor α, TNF-α) is a kind of with extensive biological activity Cell factor, it is mainly immune by monocyte, macrophage, fat cell, B cell, T cell and natural kill (NK) cell etc. Cell generates.Internal TNF-α albumen has Transmembrane TNF α and Secretary TNF α.233 amino acid of cross-film TNF-α overall length, Middle 1-76 amino acids are signal peptide (including cross-film amino acid), and 77-233 amino acid is extracellular fragment.The 77-233 amino acid It is also the amino acid sequence of Secretary TNF α, size is 17KD (Smith, et al., J.Biol.Chem.262:6951- 6954,1987), with the combination of the TNF receptor (TNFR) of trimeric form and cell surface, various biological effect is mediated.TNF-α The receptor for acting on oncocyte surface enters lysosome by the identification to cell, combination, endocytosis, causes lysosome and protease Class advanced activation leads to cell death, it plays an important role in immune response, inflammation and injury response, and the main cell that influences increases The adjusting with Apoptosis is grown, not only has cytotoxicity to tumour cell, cell dissolution, induce cell apoptosis, Inhibit proliferaton etc. Effect, additionally it is possible to promote myeloid leukemia cell to macrophage differentiation, improve the phagocytic activity of neutrophil leucocyte.
There are many defects as therapeutic agent for source of mouse monoclonal antibody, since human body has strong immunogenicity, elimination in vivo Fastly, half-life short causes clinical efficacy limited, and side effect is big.It is anti-to mouse to overcome the problems, such as to generate in human body using mouse antibody Body carries out genetically engineered humanization, such as it is to utilize genetic engineering system that people mouse, which is fitted into anti-tnf-alpha monoclonal antibody (Infliximab), Standby acquisition, variable region is still derived from source of mouse TNF-α monoclonal antibody, remains and tumor necrosis factor soluble fragments and transmembrane region knot The specificity and affinity of conjunction, replaced constant region of the constant region by human IgG1, Half-life in vivo is greatly prolonged.However these are embedding It closes antibody and humanized antibody still may cause undesired immune response, especially when long term administration.In December, 2002 The Humira of Abbott company becomes first and is approved for mitigating in adult to severe Active Rheumatoid Arthritis (RA) Symptom and symptom and inhibit joint structure lesion progress human monoclonal antibodies.Due to being human antibody, avoiding can be produced The shortcomings that raw immune response.
Autoimmune disease is the third major class disease after cancer and cardiovascular disease.RA is the most common autoimmunity One of disease, China's rheumatoid arthritis illness rate is about 0.32-0.36%, 30% is up in 60 years old or more crowd's disease incidence~ 40%.In face of expensive import drug medical expense for each person every year, patient often can only long-term uncomplaining chemotherapy.Import Though antibody drug has entered Chinese market, its expensive price makes patient be difficult to receive, and therefore, there is an urgent need to domestic antibody medicines The exploitation of object lists.
To attempt to overcome relevant issues caused by using non-human source antibodies and humanized antibody, pass through the human antibody of building To reduce the immunity of organism originality of human anti-mouse antibody (HAMA) initiation, it has also become more effective therapeutic strategy.Under existing situation, At home, there is an urgent need in the art to establish to keep or improve antibody specificity, and it can reduce or eliminate antibody mediated immunity The anti-tnf-alpha monoclonal antibody of originality, so as to be used for clinical treatment RA and acquired immune deficiency syndrome (AIDS) etc..Therefore, The present invention provides a kind of safe and reliable, people's Half-life in vivo is longer, the significant human antibody of function.
Summary of the invention
The invention discloses a kind of full source of people anti-tnf-alpha monoclonal antibody, including heavy chain variable region and light chain variable region, Heavy chain variable region (VH) has SEQ ID NO:1 amino acid sequence, and light chain variable region (VL) has SEQ ID NO:2 amino acid Sequence.
The invention discloses a kind of full source of people anti-tnf-alpha monoclonal antibody, including heavy chain and light chain, heavy chain (H) is SEQ Amino acid sequence shown in ID NO:5, light chain (L) are amino acid sequence shown in SEQ ID NO:6.
The invention also discloses a kind of nucleotide sequences, encode above-mentioned full source of people anti-tnf-alpha monoclonal antibody.
The invention discloses above-mentioned nucleotide sequences, including heavy chain variable region and light chain variable region, wherein encoding full source of people Anti-tnf-alpha monoclonal antibody heavy variable region is SEQ ID NO:3 nucleotide sequence, encodes full source of people anti-tnf-alpha monoclonal Antibody's light chain variable region is SEQ ID NO:4 nucleotide sequence.
The invention discloses above-mentioned nucleotide sequences, wherein encoding full source of people anti-TNF-α antibody heavy chain is SEQ ID Nucleotide sequence shown in NO:7, encoding full source of people anti-TNF-α antibody light chain is nucleotides sequence shown in SEQ ID NO:8 Column；
The present invention not only includes complete monoclonal antibody, further includes immunocompetent antibody fragment, such as: Fab (antigen-binding fragment), segment；Heavy chain of antibody；Antibody light chain；Genetically engineered ScFv (single Chain Fv) molecule.
The invention discloses a kind of genetically engineered host cells, it is contained the coding anti-human TNF-α monoclonal The carrier of the polynucleotides of heavy chain of antibody and light chain converts or transduces.The host cell is eukaryocyte, specifically be may is that The gonad cell CHO of Chinese hamster, the kidney cell COS cell of monkey, the embryonic kidney cells HEK-293 of people, the cervix cancer of people are thin Born of the same parents HELA etc..
The invention discloses full source of people anti-tnf-alpha monoclonal antibody have preparation treat autoimmune disease drug In purposes, autoimmune disease be rheumatoid arthritis, ankylosing spondylitis, psoriasis.
Detailed description of the invention
Fig. 1 is a curve graph, describe compared with adalimumab, by full source of people anti-TNF-α antibody HE03 (present invention in institute The antibody of preparation) inhibit TNF-α and U-937 cell surface receptor Binding experiment.
Fig. 2 is a bar chart, is described compared with adalimumab, protects D- by giving people's anti-TNF-α antibody HE03 The lethal that galactosamine sensitized mice is induced from TNF-a.
Specific embodiment
Combined with specific embodiments below, this explanation is further described.Following embodiment, experimental example only carry out the present invention Further instruction should not be construed as limiting the invention.The test method of actual conditions is not specified in the following example, leads to Often according to normal conditions, such as Sambrook et al., molecular cloning: laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.
Used various common chemical reagent, are commercial product in example scheme.Used in embodiment A Damu antibody, that is, adalimumab monoclonal antibody is purchased from Abbott company；Actinomycin D is bought in the limited public affairs of magnificent bioengineering Department；CDNA reverse transcription reagent box is purchased from Dalian treasured bioengineering Co., Ltd；Trizol reagent is purchased from Invitrogen company；Greatly Enterobacteria 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；Recombinate humanTNF-α (rhTNF- α) antigen is purchased from Shenzhen Jing Mei company；Lymphocyte separation medium is the production of Tianjin Blood Research Institute.
One, the building of humanized's single-chain antibody library:
(1) preparation of cDNA
100 each 10ml of healthy human peripheral blood are collected, are mixed, separate mononuclearcell with lymphocyte separation medium.With Trizol reagent extracts the total serum IgE of cell from isolated human peripheral lymphocyte, with cDNA reverse transcription reagent box reverse transcription CDNA out.Above step is carried out according to the specification that producer provides.
(2) building of antibody library
Design of primers, it is anti-that bibliography (Immunotechnology, 1998,3:271-278) designs and synthesizes human cloning VHBack, VHFor, VLBack and the VLFor primer of body heavy chain variable region (VH) and light chain variable region (VL) gene.VHBack, The sequence of VHFor, VLBack and VLFor are shown in Immunotechnology, 1998,3:271-278.Wherein in VHBack primer 5 ' ends add the sequence atg gcc cag ccg gcc atg gcc containing Sfi I site, add at 5 ' ends of VHFor primer Sequence gcc aga acc acc gcc gcc gga gcc acc acc gcc adds sequence at 5 ' ends of VLBack primer Tccggc ggc ggt ggt tct ggc gga ggc gga tct is added at 5 ' ends of VLFor primer and is contained Not I site Sequence atg cgg ccg c (primer is synthesized by Invitrogen Corp.).
Referring to above-mentioned primer, heavy chain variable region and light-chain variable region gene are expanded from the first chain of cDNA；Preparation ScFv connects Head DNA；The building of ScFv gene library is carried out with ScFv connector PCR assembly VH and VL；Expand ScFv gene library again to add Restriction site is cloned；It is bis- that Sfi I and NOT I are carried out simultaneously to ScFv gene library and phagemid vector pCANTAB-5E Digestion；The connection of ScFv and carrier DNA；Attachment electrotransformation e. coli TG1.Then cell culture (is contained in SOB-AG The SOB culture medium of 100mg/l ampicillin and 2% glucose) on agar plate, 30 DEG C of cultivation temperature.Pass through 100 electricity repeatedly Conversion, makes the capacity of obtained antibody library reach 1x1010。
(3) phage antibody of the preparation for being selected on antigen
Take 2 × YT (containing the 100 μ g/mL ammonia benzyls and 2% glucose) dilution of part phage library bacterium solution, 37 DEG C of concussion trainings It supports 2 hours；It is small that infection 1 is carried out according to the ratio addition helper phage M13KO7 that the ratio between helper phage and bacterium are 10:1 When；Then with 3000r/ minutes centrifuged bacterial culture solutions, supernatant is abandoned.2 × YT of bacterial precipitation (is contained into 100 μ g/mL ammonia benzyls and 50 μ g/L kanamycins) culture medium be resuspended after, 37 DEG C concussion overnight；Supernatant is taken after overnight bacterium is centrifuged 30 minutes, 0.2 times of body is added PEG/NaCl (20%PEG 8000/2.5M NaCl) solution precipitating phage of product pre-cooling, ice bath 1 hour；Centrifugation 15 minutes, Supernatant is abandoned, precipitating is resuspended with sterile PBS, is added the PEG/NaCl solution precipitating phage of 0.2 volume again, ice bath 1 hour Afterwards；Centrifugation 15 minutes dissolves precipitating with the PBS of appropriate 1/10 sterile volume, and 4 DEG C store for future use.Two, the elutriation of antibody library and Positive clone identification:
The antibody in phage display library obtained for embodiment 1, selects high-affinity antibody by panning technique.
(1) enrichment isolation of phage antibody library
It is stayed overnight at 4 DEG C with rhTNF- α antigen coat culture plate；Coating plate is taken out, is washed 3 times with PBS, is taken off with containing 10% The PBS confining liquid room temperature of rouge milk powder is closed 2 hours；Liquid is outwelled, PBS is washed 3 times, adds phage antibody library, and 37 DEG C of cultures 2 are small When；It empties, is washed 5 times with PBS (containing 0.05%Tween 20), wash 5 (each washing of the 2nd wheel 10 times, the 3rd each washings of wheel with PBS 20 times, and wash time also increases)；It empties, is added 100mM triethylamine (pH=12), divides in room temperature elution phage particle 10 1M Tris (pH=7.4) is added after clock and mixes neutralization, is transferred in 15mL pipe；With the E.coli for being in logarithmic growth phase TG1(OD600=0.4) it is mixed with the bacteriophage of elution, stands 30 minutes in 37 DEG C of water-baths.200 μ L are taken to infect the thin of bacteriophage Bacterium carries out the measurement of the bacterial community total (cfu) in unit volume, remaining bacterium centrifugation (contains 100 μ g/ with 2 × YT ML ammonia benzyl and 2% glucose) it is resuspended, it is coated on plate, puts 37 DEG C of overnight incubations, preparation bacteriophage is sieved and washed for next round, 3 wheel screenings are carried out altogether.
(2) ELISA method identifies positive colony
Preferable colony inoculation, overnight incubation will be separated.Overnight with 4 DEG C of 96 hole elisa plate of rhTNF- α antigen coat；Next day The overnight culture of 20 μ l is taken to be seeded in 96 hole elisa plates, culture to OD600=0.5 or so, supernatant, each hole are abandoned in centrifugation Adding 200 2 × YT of μ L, (isopropylthiogalactoside of the 1mM containing final concentration, 37 DEG C, 250rpm is induced 3 hours；By induced product It is spare to collect supernatant for centrifugation 15 minutes；The elisa plate being coated with is washed three times with PBS, every hole adds 300 μ L confining liquids, room temperature Closing 2 hours；The elisa plate closed is washed three times with PBS, every hole adds 100 μ L Block buffers, then respectively adds 100 toward each hole μ L induces supernatant (ScFv containing secretion) to mix, and combines water-bath to combine 1 hour under 37 DEG C of water-baths；With Anti-E-Tag antibody (with the dilution proportion of 1:1000 in Block buffer), after washing three times with PBS, the Anti-E-Tag for adding 100 μ L to dilute is anti- Body combines 1 hour in 37 DEG C of water-baths, and PBS is washed three times, adds the antibody 100 μ L of the horseradish peroxidase-labeled diluted, and 37 DEG C Water-bath 1 hour；PBS is washed 3 times, adds (OPD) developing solution, and black out develops the color 30 minutes.2M H is added2SO4Reaction is terminated, microplate reader is surveyed Its OD490Value.
(3) the determined dna sequence analysis of positive colony
Select ELISA detection the higher independent cloning of positive colony ELISA value be sent to Shanghai give birth to work bioengineering it is limited Company carries out sequencing analysis.
One plant of anti-human TNF-α single-chain antibody ScFv is obtained after 3 elutriation antibody libraries, its gene sequence is obtained after sequencing Column.SEQ ID NO:3 and SEQ ID NO:1 respectively illustrates the nucleotide sequence and amino acid sequence of ScFv heavy chain variable region VH Column.SEQ ID NO:4 and SEQ ID NO:2 respectively illustrates the nucleotide sequence and amino acid sequence of ScFv light chain variable region VL Column.
Three, the building of anti-human TNF-α heavy chain carrier for expression of eukaryon and anti-human TNF-α light chain carrier for expression of eukaryon:
(1) full genome synthesizes
According to document (Cell, 1980,22:197-207) and document (NucleicAcids Research, 1982,10: 4071-4079) sequence reported, SEQ ID NO:9 show the amino acid sequence of heavy chain constant region (CH).SEQ ID NO:10 Show the amino acid sequence of constant region of light chain (CL)；Heavy chain variable region (VH) amino for the human antibody that screening of phage antibody library obtains Acid sequence and light chain variable region (VL) amino acid sequence, are combined into complete heavy chain amino acid sequence (SEQ ID by VH+CH NO:5), VL+CL is combined into complete light-chain amino acid sequence (SEQ ID NO:6), human antibody entire heavy chain, complete light Chain gene contains 2 at 3 ' ends respectively in 5 ' end addition KOZAK sequence (GCCGCCACC), initiation codon, signal peptide gene sequence A translation termination codon.Signal peptide amino acid sequence is SEQ ID NO:11:MEFGLSWLFLVAILKGVQC (with reference to Ah reaching The wooden patent, WO_2007_014162_A2), with according to combinations of the above amino acid sequence, after carrying out base codon optimization, Quan Ji Because synthesizing above-mentioned two genetic fragments (by Shanghai, Invitrogen Corp. is synthesized).The nucleosides soda acid of heavy chain combinations segment after optimization Motif is classified as SEQ ID NO:7；The nucleotide base sequence of light chain combination segment after optimization is SEQ ID NO:8；
(2) building of expression vector
Above-mentioned two genetic fragments by specification step is passed through TA Cloning Kit (Invitrogen, Catalog no.K8300-01) andTA Vector Kit (Invitrogen, Catalog no.12744-017) TA clone is carried out respectively, heavy chain combinations segment TA is cloned into Vector, light chain combination segment TA are cloned intoVector successfully constructs 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 constructed is transferred to mammalian host cell line, it is anti-to express HTNF- α human antibody.In order to stablize high-caliber expression, preferred host cell line is dihyrofolate reductase (DHFR) Chinese hamster ovary (CHO) cell of deficiency (see, e.g. Chasin, the United States Patent (USP) of L. et al. 4818679).It is preferred that Transfection method be electroporation, other methods, including calcium phosphate co-sedimentation, fat transfection and Protoplast fusion etc. also can be used. In electroporation, with the electroporation apparatus Gene Pulser (Bio-Rad for being set as 300V electric field and 900 μ Fd capacitors Laboratories),
1.5 × 10 are added in cuvette7A cell is suspended in the PBS of 0.8mL, and (is purchased from containing 40 μ g with PvuI TakaRa) the expression vector Plasmid DNA linearized.After transfection 48 hours, Geneticin (G418) is carried out to phage library cell And methotrexate (methotrexate or MTX) pressurization screening, by take turns more various concentration MTX (100nm, 200nm, 500nm, 1000nm, 2000nm, 5000nm) pressurization screening after, with Method of Limited Dilution be subcloned transfectant, select high level expression people The cell strain of source antibody, overexpression cell line carry out further amplification fermented and cultured, are obtained by purifying a certain number of high Purity source of people anti-TNF-α antibody, is named as HE03, for subsequent antibody specificity and active assessment etc..
Five, the affinity of anti-human TNF-α monoclonal antibody and antigen binding:
It is measured using Biacore X100, Biacore X100 dynamics/affinity software analysis, using what is captured indirectly Method, by amine coupling kit (Amine Coupling Kit) goat anti-human igg Fc antibody coupling in CM5 chip surface As capture molecule, HE03 and control adalimumab are diluted to a certain concentration as ligand respectively by calculating, RhTNF- α is as analyte.Analyte dilutes 5 concentration, and each concentration is recycled as one, first uses HBS-EP (0.01M HEPES, pH7.4,0.15M NaCl, 3mM EDTA, 0.05% (v:v) surfactant P20) 3 circulations of buffer operation, if Counting an analyte concentration is that 0 concentration runs 2 circulations, finally designs a duplicate analyte concentration and runs 1 circulation.It is whole A process operation 11 circulations, each circulation can draw a curve, pass through Biacore X100 dynamics/affinity analysis Software is analyzed to obtain the dynamics/affinity data for surveying antibody HE03 and control group adalimumab of the present invention, and experiment repeats 3 It is secondary.
1 affinity experimental result of table
Note: Kd (M) is affinity constant；ka(M-1S-1) it is binding constant；Kd(S-1) it is dissociation constant.
The results are shown in Table 1, and it is anti-to show that the Kd value of human antibody HE03 obtained is substantially less than the full source of people of control group Body adalimumab illustrates that human antibody HE03 is higher than control group adalimumab to the affinity of TNF-α.
Six, the lethal effect of L929 cell is studied with TNF-α in antibody:
People recombinate TNF-α (rhTNF- α) cause the cytotoxicity to mouse L929 cell, such as it is following by total incubated antibodies with RhTNF- α evaluation antibody HE03 neutralization activity of the present invention in L929 detection.
L929 cell culture is in the RPMI-1640 culture solution (GIBCO) containing 10% fetal calf serum.Logarithmic growth phase cell After digestion counts, 200g being taken to be centrifuged 5 minutes, abandons supernatant, cell precipitation is resuspended with aforementioned culture solution, adjustment cell density to 2 × 105/ ml adds to cell in 96 well culture plates, and 100 holes μ l/ set 37 DEG C, 5%CO2Incubator overnight incubation.100 μ l will be contained Antibody HE03, positive control A Damu antibody 96 orifice plates carry out 2 times with the RPMI1640 culture medium containing 10% fetal calf serum Gradient serial dilution.50 μ l rhTNF- α, which are added, makes the ultimate density 500pg/ml of each sample well.Then by above-mentioned plate In incubation at room temperature 30 minutes, the L929 cell of the 50 μ l overnight incubations containing 1.0 μ g/ml actinomycin Ds is added, adjustment cell is whole Concentration is every hole 5x104It is a, multiple holes are set.In CO2Overnight incubation in the incubator that concentration is 5%, temperature is 37 DEG C.It is fresh to match The heterotope cell Proliferation detection examination (Promega) of system, is added in 96 well culture plates, and be placed in incubator by 20 holes μ l/ Inside continue culture 3 hours, microplate reader detects the light absorption at 490nm, reference wavelength 630nm.Using sample concentration as abscissa, Absorbance value is ordinate, measures IC50 value, the results are shown in Table 2.
Table 2: with TNF-α to the lethal effect of L929 cell in antibody
Experimental result shows, the L929 cell that the full source of people anti-tnf-alpha monoclonal antibody HE03 antagonism TNF-α of the present invention mediates The IC50 of lethal effect is slightly lower compared to the IC50 of control group adalimumab, illustrates in HE03 and the ability of TNF-α is even more than Control group adalimumab.
Seven, anti-TNF-α antibody HE03 inhibits the Binding experiment of TNF-α and U-937 cell surface receptor:
It is anti-with system, human tissue cell U-937 cell line (ATCC NO.CRL1593) the detection people of expression hTNF- α receptor TNF-α antibody inhibits ability of the hTNF- α in conjunction with cell surface hTNF- α receptor.U937 cell in good condition is taken, cell is living Cell concentration is adjusted to 5 × 10 with the RPMI1640 culture medium of 10% fetal calf serum after the counting of power calculating instrument5Unit is write, it will be thin Born of the same parents are by 100 μ l/ pipe point into Flow cytometry pipe.Fluorescein isothiocynate (FITC, Amresco) is marked with PBS RhTNF- α (for R&D Products 210-TA-050, marked and obtained using dialysis) is diluted to 100ng/ml, with the dilution Full source of people anti-tnf-alpha monoclonal antibody HE03, adalimumab are diluted to 100 μ g/ml, and continuous 2 times of gradient dilutions respectively, The sample diluted and reference substance are added in streaming pipe by 100 μ l/ pipes, are protected from light at 4 DEG C 1 hour；Again with containing 1% tire ox The PBS of serum is washed cell 2 times, and each 200g is centrifuged 5 minutes, abandons supernatant, and cell precipitation is resuspended in 300 μ l containing 1% tire ox blood In clear PBS, the fluorescence intensity of the every pipe of flow cytomery.Using sample concentration as abscissa, absorbance value is ordinate, is surveyed IC50 value is obtained, the results are shown in Table 3 and Fig. 1.
Table 3:HE03 inhibits TNF-α receptor on U-937 cell to combine
Experimental result shows that the full source of people anti-tnf-alpha monoclonal antibody HE03 of the present invention blocks TNF-α and U937 cell surface The IC50 of the combination of receptor blocks the IC50 of the combination of TNF-α and U937 cell surface receptor compared to control group adalimumab It is worth slightly lower, therefore illustrates in monoclonal antibody HE03 of the present invention and the ability of TNF-α is better than control group adalimumab.
Eight, the death assay of anti-TNF-α antibody HE03 antagonism TNF-α inducing mouse:
Lead to the lethal in 24 hours to the mouse injection rhTNF- α of D-galactosamine sensitization, to show in TNF-α Lethal can be prevented in this mode with agent.The intraperitoneal injection of 1 μ g rhTNF- α and 20mg D-galactosamine (Amresco), can Induce 80-90%C57BL/6 dead mouse.For detection people's anti-TNF-α antibody HE03 in this model in vivo in and TNF-α energy Power, in this experimental example, control group equally select adalimumab, first give a certain amount of various antibody and are injected intraperitoneally, It after 30 minutes, then gives 1 μ g rhTNF- α and 20mg D-galactosamine and is injected intraperitoneally, various antibody are observed after 24 hours Protecting effect.Each group injection dosage and result see the table below 4 and Fig. 2.
Table 4: each group mouse survival rate
The above experimental result shows that the full source of people anti-tnf-alpha monoclonal antibody HE03 antagonism TNF-α induction in the present invention is small The effect of mouse death is better than control group adalimumab, thus the antibody in Mice Body in and TNF-α ability ratio A Damu Monoclonal antibody is strong.
The above description of the embodiment is only used to help understand the method for the present invention and its core ideas.It should be pointed out that pair For those skilled in the art, without departing from the principle of the present invention, the present invention can also be carried out Some improvements and modifications, these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims (8)

1. a kind of full source of people anti-tnf-alpha monoclonal antibody, which is characterized in that heavy chain variable region amino as shown in SEQ ID NO:1 Acid sequence, light chain variable region amino acid sequence as shown in SEQ IDNO:2.
2. full source of people anti-tnf-alpha monoclonal antibody described in claim 1, which is characterized in that heavy chain is shown in SEQ ID NO:5 Amino acid sequence, light chain are amino acid sequence shown in SEQ ID NO:6.
3. encoding the DNA sequence dna of full source of people anti-tnf-alpha monoclonal antibody described in claim 1, which is characterized in that encoding heavy chain Variable region nucleotide sequence is as shown in SEQ ID NO:3, and coding light chain variable region nucleotide sequence is as shown in SEQ ID NO:4.
4. encoding the DNA sequence dna of full source of people anti-tnf-alpha monoclonal antibody as claimed in claim 2, which is characterized in that encoding heavy chain Nucleotide sequence encodes light chain nucleotide sequence as shown in SEQ IDNO:8 as shown in SEQ ID NO:7.
5. the expression vector containing DNA sequence dna described in claim 3 or 4 is protokaryon or carrier for expression of eukaryon.
6. the host cell containing the expression vector described in claim 5 is eukaryocyte.
7. host cell according to claim 6, the host cell is the gonad cell CHO of Chinese hamster, young storehouse The kidney cell BHK of mouse, the kidney cell COS cell of monkey, the embryonic kidney cells HEK-293 of people, people cervical cancer cell HELA.
8. purposes of the source of people anti-tnf-alpha monoclonal antibody described in claim 1 in preparation treatment autoimmune disease drug, The autoimmune 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)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201510131817 2015-03-24
CN2015101318177 2015-03-24

Publications (2)

Publication Number Publication Date
CN105777905A CN105777905A (en) 2016-07-20
CN105777905B true CN105777905B (en) 2019-06-25

Family

ID=56390779

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610171500.0A Expired - Fee Related CN105777905B (en) 2015-03-24 2016-03-23 A kind of full source of people anti-tnf-alpha monoclonal antibody and its application

Country Status (1)

Country Link
CN (1) CN105777905B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107903323B (en) * 2017-11-15 2020-04-17 中国药科大学 anti-hTNF- α fully human antibody and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1215407A (en) * 1996-02-09 1999-04-28 Basf公司 Human antibodies that bind huma TNF 'alpha'
CN102167741A (en) * 2010-02-25 2011-08-31 百迈博药业有限公司 Fully human anti-TNF-alpha (Tumor Necrosis Factor-alpha) monoclonal antibody and preparation method as well as application thereof
CN104341502A (en) * 2013-08-09 2015-02-11 北京天成新脉生物技术有限公司 Anti-TNF-alpha fully human monoclonal antibodies with low immunogenicity and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1215407A (en) * 1996-02-09 1999-04-28 Basf公司 Human antibodies that bind huma TNF 'alpha'
CN102167741A (en) * 2010-02-25 2011-08-31 百迈博药业有限公司 Fully human anti-TNF-alpha (Tumor Necrosis Factor-alpha) monoclonal antibody and preparation method as well as application thereof
CN104341502A (en) * 2013-08-09 2015-02-11 北京天成新脉生物技术有限公司 Anti-TNF-alpha fully human monoclonal antibodies with low immunogenicity and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
抗hTNF-a单克隆抗体的制备及其抗体基因克隆研究;黄蕊;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20101015;E059-111

Also Published As

Publication number Publication date
CN105777905A (en) 2016-07-20

Similar Documents

Publication Publication Date Title
US11365255B2 (en) PD-1 antibody, antigen-binding fragment thereof, and medical application thereof
KR102037016B1 (en) Anti PD-L1 Nano Antibody and Its Application
TWI823895B (en) Anti-b7-h4 antibody, antigen binding fragment thereof and medical use thereof
CN106939050B (en) anti-PD 1 and CD19 bispecific antibodies and uses thereof
US8597648B2 (en) Fully human anti-TNF-alpha monoclonal antibody, preparation method and use thereof
CN107405397A (en) The anti-antibody of TIM 3
AU2001283903B2 (en) Antibodies to human MCP-1
TW201819413A (en) CD47 antibody, antigen-binding fragments and pharmaceutical use thereof
KR20200012920A (en) Anti-CD40 Antibody, Antigen Binding Fragment thereof and Medical Use thereof
CN109776678A (en) A kind of humanization PD-L1 monoclonal antibody, preparation method and application
TW201932491A (en) Anti 4-1BB antibody, antigen binding fragment and pharmaceutical use thereof
US20220213195A1 (en) BIFUNCTIONAL FUSION PROTEIN AGAINST PDL1 AND TGFß AND USE THEREOF
US20220119540A1 (en) Humanized anti-human ox40 monoclonal antibody, preparation method therefor and use thereof
TWI685504B (en) Anti-gitr antibody, antigen-binding fragments and pharmaceutical use thereof
CN103649326A (en) Antibody-like proteins for therapeutic and diagnostic use
CN105777905B (en) A kind of full source of people anti-tnf-alpha monoclonal antibody and its application
Tong et al. A novel human anti-BAFF neutralizing monoclonal antibody derived from in vitro immunization
CN105061596B (en) The monoclonal antibody and its application of human B lymphocyte stimulating factor
TW202227504A (en) Anti-erbb3 antibody, antigen binding fragment thereof and medical use thereof
CN107236041B (en) I L-17 antibody
US20220411523A1 (en) Molecule capable of binding to human 4-1bb and its application thereof
CN114456267B (en) anti-CD 73 humanized monoclonal antibody and application thereof
CN109311988A (en) A kind of BlyS antibody and its preparation method and application
WO2023030311A1 (en) Antigen-binding protein targeting siglec15 and use thereof
CN116554324A (en) Antibody capable of specifically recognizing 4-1BB, preparation method and application thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20190625

Termination date: 20210323