CN106397594B - A kind of agonist single-chain antibody of the anti-human death receptor 5 of full source of people and application - Google Patents

A kind of agonist single-chain antibody of the anti-human death receptor 5 of full source of people and application Download PDF

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CN106397594B
CN106397594B CN201610932535.1A CN201610932535A CN106397594B CN 106397594 B CN106397594 B CN 106397594B CN 201610932535 A CN201610932535 A CN 201610932535A CN 106397594 B CN106397594 B CN 106397594B
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chain antibody
death receptor
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acid sequence
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谭树华
雷高新
徐智盼
徐梦龙
陆陈晨
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China Pharmaceutical University
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Abstract

The single-chain antibody that human death receptor 5 (DR5) can be specifically bound and activated the present invention relates to one kind belongs to a kind of human antibody.The single-chain antibody is by being obtained through five wheel screenings from the full Large human naive scFv phage library of high capacity using human death receptor DR5 extracellular region protein as target antigen.It can purify to obtain by the inducing expression in Escherichia coli and through nickel ion affinity chromatograph and molecular exclusion chromatography.Pharmacodynamic experiment proves that the growth of the colon cancer cell COLO205 and breast cancer cell MDA-MB-231 of the DR5 positive can be effectively suppressed in the single-chain antibody.The single-chain antibody itself and other antibody formations, including overall length, bispecific or fusion protein etc. are transformed into, can be used as neoplasm targeted therapy of the drug for the death receptor DR5 positive.The single-chain antibody has many advantages, such as that low immunogenicity, high specificity, penetrability are strong, is a kind of ideal targeting therapy for tumor drug.

Description

A kind of agonist single-chain antibody of the anti-human death receptor 5 of full source of people and application
Technical field
The invention belongs to genetic engineering pharmaceutical field, be related to Human anti-human death receptor 5 (death receptor 5, DR5 single-chain antibody screening, identification, expression and purifying), and its application in neoplasm targeted therapy.
Background of invention
Tumor necrosin relative death inducing ligand TRAIL (TNF-related apoptosis-inducing Ligand, also referred to as APO2L) be tumor necrosis factor superfamily member, contain 281 amino acid, with other TNF superfamily members Equally, TRAIL is II type transmembrane protein, extracellular regions [the Nagane M., et of trans-membrane region and C-terminal comprising N-terminal Al.Neuro-oncology2010,12 (7): 687-700].TRAIL is in vivo only with the presence of film mating type, but extracellular regions can To be expressed by gene engineering method with soluble form and keep certain activity.TRAIL is currently known in vitro The apoptosis of induced various types of tumors cell, sphere of action are wide.Experiment in vitro show no matter film mating type TRAIL, or individually with can Extracellular region Truncated TRAIL existing for molten form can induce rapidly various kinds of cell surface to have the swollen of TRAIL specific receptor Apoptosis [Fan Q., et al.Sci Rep.2013,3:3565] occurs for oncocyte.Inside and outside experiment show TRAIL and it is dead by Body combines optionally induced various types of tumors Apoptosis, and to most of normal cells without apparent lethal effect [Subbiah V., et al.Mol Cancer Ther 2012,11 (11): 2541-2546], therefore receive significant attention.
TRAIL optionally induced various types of tumors Apoptosis, and most of normal cells are made without apparent killing Mechanism, Main Viewpoints are considered since TRAIL is there are four types of membrane-bound receptor at present, be respectively two kinds of death receptor DR4, DR5 is distributed in tumor cell surface mostly；Two kinds of Decoy receptors DcR1, DcR2, are distributed in normal cell surface mostly.It is dead The structure intracellular of receptor contains death domain, is capable of the apoptosis of mediated cell, and the structure intracellular of DcR1 is without dead knot The death domain in structure domain, DcR2 is truncated, thus two Decoy receptors be all unable to mediated cell apoptosis [SheridanJ., Et al.Science, 1997,277:818-821].But also exactly because it is with multiple bind receptors, so that it is easy to produce Drug resistance [Sheridan, J., et al.Science, 1997,277:818-821；Merino D., et al.Molecular , and its half-life short (about 30 minute) in vivo and cellular biology 2006,26 (19): 7046-7055] [Herbst RS., et al.Journal of clinical oncology:official journal of the American Society of Clinical Oncology 2010,28 (17): 2839-2846].The study found that TRAIL pairs In liver cell, there are certain toxicity, and the generation of this toxicity is mainly [Jo M., the et mediated due to death receptor DR4 al.Nat Med.2000 May；6 (5): 564-7；Secchiero P., et al.Blood.2004 Jan 15；103 (2): 517-22；Di PietroR., et al.Blood 2001,97 (9): 2596-2603].In order to overcome these disadvantages of TRAIL, The activity antibody for screening death receptor DR5 specificity is considered as a kind of effective ways [Holland PM.Cytokine& Growth factor reviews 2014,25 (2): 185-193].
DR5 belongs to I type transmembrane protein, the knot rich in cysteine as one and the death receptor of TRAIL specific binding Structure domain and death domain.Studies have shown that death receptor DR5 is expressed in many tumor tissues at a high level, such as colon cancer, mammary gland [Meng RD., the et al.Mol such as cancer, liver cancer, oophoroma, cervical carcinoma, lung cancer, cancer of pancreas, carcinoma of testis, prostate cancer Ther.2000 Feb；1 (2): 130-144；NavalJ., et al.Exp Cell Res.2007 Jul 1；313 (11): 2378-2388；El-Deiry WS., et al.Semin Cancer Biol.2003 Apr；13 (2): 135-147].Due to dead Receptor DR5 is died in cell distribution and the uniqueness of mechanism of action, becomes the promising target of anti-tumor drug exploitation.2001 Ichikawa prepares the special activated form monoclonal antibody TRA-8 of human death receptor DR5, this is specificity for people's cell film Surface death receptors DR5 and the monoclonal antibody synthesized, it can specifically be combined with death receptor DR5, play ligand Effect, to induce cell apoptosis, monoclonal antibody TRA-8 is also in clinical investigation phase as anti-tumor drug at present [Kendrick JE., et al.Gynecologic oncology 2008,108 (3): 591-597].
Summary of the invention
The object of the present invention is to provide Human anti-human death receptor DR5's a kind of new and with potential medical value Single-chain antibody.The present invention is by display technique of bacteriophage, with human death receptor DR5 extracellular region protein as target antigen from high capacity Full Large human naive scFv phage library in through five wheel screen, obtain feature be specific binding human death receptor DR5 extracellular region, The single-chain antibody TR2-3 of death receptor DR5 inducing apoptosis of tumour cell can be activated in vitro.
Specific technical solution of the present invention is as follows:
A kind of single-chain antibody of the anti-human death receptor 5 of full source of people, including heavy chain variable region and light chain variable region, heavy chain can Become area and connected with light chain variable region by flexible peptide, the heavy chain variable region includes the domain heavy chain CDR1, CDR2 and CDR3, described heavy The amino acid sequence in the domain chain CDR1 as shown in SEQ ID NO.3, the amino acid sequence of heavy chain CDR2 as shown in SEQ ID NO.4, The amino acid sequence of heavy chain CDR3 is as shown in SEQ ID NO.5；The light chain variable region includes light chain CDR1, CDR2 and CDR3 Domain, the amino acid sequence in the domain the light chain CDR1 is as shown in SEQ ID NO.6, the amino acid sequence in the domain light chain CDR2 such as SEQ ID Shown in NO.7, the amino acid sequence in the domain light chain CDR3 is as shown in SEQ ID NO.8；The flexibility peptide amino acid sequence such as SEQ ID Shown in NO.9.The domain described heavy chain CDR1, CDR2, CDR2 and/or the domain light chain CDR1, CDR2, CDR2 can be and set by amino acid The amino acid sequence for changing or being obtained after modifying.
Preferably, the amino acid sequence of the heavy chain variable region is SEQ ID NO:1, the amino acid sequence of light chain variable region One or more amino acid are substituted, are inserted into or lacked for SEQ ID NO:2 or the heavy chain variable region and light chain variable region Homologous sequence.
Anti- 735 nucleotide of death receptor DR5 single-chain antibody TR2-3 gene order overall length of full source of people of the present invention, in advance There are 245 amino acid in phase.Heavy chain variable region (SEQ ID NO:1, see Fig. 3) with 122 amino acid and 108 amino acid Light chain variable region (SEQ ID NO:2, see Fig. 4) passes through the flexible peptide SEQ ID NO.9 connection of 15 amino acid.
It is a further object to provide the single-chain antibodies that above-mentioned anti-death receptor DR5 can be expressed and be purified to one kind Method.
Full human single chain variable fragments antibody TR2-3 gene is connected to expression vector PCANTAB 5E, is transferred to Bacillus coli expression host HB2151.Through 0.5mM IPTG, 30 DEG C of inducing expression 20h, using sonicated cells, low-temperature centrifugation removes cell fragment, will Supernatant nickel ion affinity chromatograph column and molecular exclusion chromatography column carry out isolating and purifying single-chain antibody TR2-3.Western Blot identifies the single-chain antibody TR2-3 isolated and purified.
It is a further object of the present invention to provide the single-chain antibodies for the anti-human death receptor 5 for expressing full source of people of the present invention Expression vector and/or host cell.
It is a further object of the present invention to provide the single-chain antibodies of the anti-human death receptor 5 of full source of people of the present invention to make The standby drug with antitumor action or the application in diagnostic reagent.The tumour is selected from colon cancer, breast cancer, liver cancer, ovary Cancer, cervical carcinoma, lung cancer, cancer of pancreas, carcinoma of testis, prostate cancer etc..
The invention has the advantages that
A kind of single-chain antibody of the anti-human death receptor 5 of full source of people provided by the invention shows the list by vitro test Chain antibody has apparent inhibiting effect to colon cancer cell COLO 205 and breast cancer cell MDA-MA-231, has obvious Anti-tumor activity.The single-chain antibody also achieves the solubility expression in Escherichia coli simultaneously, production simple with preparation process The advantages that product purity is high and easy expanding production.
Detailed description of the invention
The combination activity of Fig. 1: ELISA detection phage display single-chain antibody.NC, negative control；1-20, cloned sequence Number.OD450nmIt is positive colony that numerical value, which is greater than 3 times of negative control or more,.
Fig. 2: the single-chain antibody that mtt assay carries out is to colon cancer COLO205 cell activity qualification result figure.
The amino acid sequence of Fig. 3: TR2-3 heavy chain variable region.Under VH-CDR1, VH-CDR2 and VH-CDR3 sequence is used Underlining.
The amino acid sequence of Fig. 4: TR2-3 light chain variable region.Under VL-CDR1, VL-CDR2 and VL-CDR3 sequence is used Underlining.
Fig. 5: the expression vector schematic diagram of single-chain antibody TR2-3 gene.
Fig. 6: the SDS-PAGE electrophoresis that single-chain antibody TR2-3 is isolated and purified by nickel column.1, Escherichia coli are broken Supernatant；2, ni-sepharose purification penetrates liquid；3, the PBS buffer solution of the imidazoles containing 50mM elutes foreign protein；4, the PBS of the imidazoles containing 100mM is slow Fliud flushing elutes foreign protein；5, the PBS buffer solution of the imidazoles containing 500mM elutes destination protein TR2-3；M, Protein Marker.
Fig. 7: purifying gained TR2-3 albumen carries out purity analysis result figure using SEC-HPLC method.
The TR2-3 single-chain antibody that Fig. 8: Western Blot method purification Identification obtains.
Fig. 9: it is thin to colon cancer cell COLO205 and breast cancer cell MDA-MB-231 that mtt assay detects single-chain antibody TR2-3 Born of the same parents' increment inhibiting effect.
Figure 10: Western Blot method detects single-chain antibody TR2-3 to Caspase albumen in colon cancer cell COLO205 The effect of activation.
Specific embodiment
Illustrate specific steps of the invention by the following examples, but is not limited by the example.
Term as used in the present invention generally there are those of ordinary skill in the art usually to manage unless otherwise indicated The meaning of solution.
The present invention is described in further detail combined with specific embodiments below and referring to data.It should be understood that the embodiment is In order to demonstrate the invention, it rather than limits the scope of the invention in any way.
In the examples below, the various processes and method being not described in detail are conventional methods as known in the art.
The present invention is further described combined with specific embodiments below.
Material used in following examples, reagent, device, instrument, equipment etc. unless otherwise specified can be from business ways Diameter obtains.
The expression and purification of 1. human death receptor DR5 albumen of embodiment
With the cDNA (Divine Land Yi Qiao, Beijing Products) of human death receptor DR5 for template, PCR amplification death receptor DR5 The genetic fragment of extracellular region (1-130aa), is cloned on expression vector pET21b, is transferred in e. coli bl21 (DE3) and carries out Protein expression.Expression condition are as follows: seed liquor is inoculated in 2 × YT-AG culture medium 37 DEG C of shake cultures extremely by 2% inoculum concentration OD600nm=0.6, the IPTG of final concentration of 0.5mM, 20 DEG C of induction 20h is added.6000rpm, 4 DEG C of centrifugation 15min collect thallus； Thallus, ultrasonication (ultrasonic 3s, gap 3s, total 20min) is resuspended in PBS；12000rpm, 4 DEG C of centrifugation 30min collect supernatant；On Sorting does not pass through nickel ion affinity column (GE Products) and molecular exclusion chromatography column Superdex 75 (GE Products) is carried out It isolates and purifies, obtained death receptor DR5 extracellular region protein is screened as antigen for subsequent single-chain antibody.
The building in 2. bacteriophage human single chain variable fragments antibody library of embodiment
The building reference literature method in bacteriophage human single chain variable fragments antibody library carries out [Sblattero D., et al.Nat Biotechnol, 2000,18: 74-80；Sblattero D., et al.Immunotechnology, 1998,3:271-278]. Using 105 parts of healthy human peripheral bloods and 58 parts of tumour patient peripheral bloods as source, is separated and generated using the method for density gradient centrifugation The peripheral blood lymphocytes of antibody extracts total serum IgE, and reverse transcription synthesis cDNA (precious biotech firm's product), as template, choosing With under the heavy chain upstream primer of human-specific antibody variable region 6,4 heavy chain downstream primers, 7 λ chain upstream primers and 3 λ chains Swim primer carry out PCR amplification [Sblattero D., et al.Nat Biotechnol, 2000,18:74-80；Sblattero D., et al.Immunotechnology, 1998,3:271-278], Sfi I, two enzymes of Not I are added respectively then at its both ends Enzyme site sequence and Linker sequence obtain single-chain antibody scFv segment through SOE-PCR connection heavy chain and sequence of light chain.Routinely Single-chain antibody scFv segment is cloned into Sfi I, Not two enzymes of I of phagemid vector pCANTAB 5E by molecular cloning protocols Between enzyme site.The connection product of single-chain antibody scFv segment and phasmid pCANTAB 5E are transferred to greatly by the method for electrotransformation The super competent cell of enterobacteria TG1 (biorad Products) collects positive bacterium colony, obtains human single chain variable fragments antibody library.Pass through Titer determination shows that finally obtaining storage capacity is 1.1 × 108Bacteriophage human single chain variable fragments antibody library.
The screening of the anti-human death receptor DR5 single-chain antibody of the full source of people of embodiment 3.
To be coated with buffer (50mM NaHCO3, pH9.6) and death receptor DR5 extracellular region protein is diluted to 20ug/ml, it takes 2ml is added in immune pipe, and room temperature coating is overnight；Next day, supernatant is abandoned, PBS is washed rapidly immune pipe 3 times；2%MPBS (contains 2% The PBS of skim milk), 37 DEG C of closing 2h；Confining liquid is abandoned, is washed rapidly with PBS immune pipe 3 times；By phage antibody library (~ 1012Pfu it) is suspended in 4ml 2%MPBS and is added in immune pipe, be placed on rotary shaker and rotate 2h repeatedly, to contain 0.1% The PBS of Tween-20 is washed immune pipe 15 times, then is washed immune pipe 15 times with PBS；1ml 100mM triethylamine, room temperature rotation is added Turn to be incubated for 10min, carries out specific elution；0.5ml 1M Tris-HCl (pH7.4) buffer is eluted for neutralizing rapidly Bacteriophage；Bacteriophage after neutralization is used to infect the e. coli tg1 of logarithmic phase, is expanded and is prepared, and next round is used for Screening.
The antigen-binding activity of embodiment 4.ELISA identification single-chain antibody
To 96 hole Bacteria Culture plates, every hole is added 200ul and contains the bacteriophage monoclonal obtained after the wheel screening of picking the 5th 1092 × YT-AG culture medium of pfuM13KO7,37 DEG C of shake cultures 2h, 2000rpm are centrifuged 10min, abandon supernatant, every hole is resuspended In 200 μ 12 × YT-AK culture mediums, 30 DEG C of shaken cultivations are stayed overnight.2000rpm, 4 DEG C of centrifugation 10min, supernatant are saved in 4 DEG C, It is identified for antigen-binding activity ELISA.
The coating of ELISA Plate: death receptor DR5 extracellular region protein is diluted with coating buffer (50mM NaHCO3, pH9.6) To 5ug/ml, the every hole 100ul is added in 96 hole elisa Plates, and 4 DEG C of coatings are overnight.After taking-up, sealed with 37 DEG C of 2%MPBS confining liquid Close 2h, centrifugation removal confining liquid.
The monoclonal phage supernatant of above-mentioned acquisition is added in 96 hole elisa Plates for being coated with antigen, 37 DEG C of incubations 2h.TBST board-washing 10 times, the anti-M13 antibody (Divine Land Yi Qiao, Beijing product) of HRP label, 37 DEG C of incubation 1h are added in every hole. TBST board-washing 10 times, substrate TMB is added to develop the color, microplate reader measures OD450nmValue.Using the hole M13KO7 as negative control, to be higher than yin Property control value three times more than be used as positive colony (Fig. 1).Positive colony send gene sequencing company to carry out gene sequencing.
It the solubility expression of 5. anti-human death receptor DR5 single-chain antibody of embodiment and isolates and purifies
Routinely molecular cloning protocols mark 83 ' ends of single-chain antibody gene obtained in embodiment 4 plus 6 × His Subclone is transformed into E. coli expression strains HB2151 to expression vector PCANTAB 5E after signing sequence.Positive colony is selected, The IPTG of final concentration of 0.5mM, 30 DEG C of inducing expression 20h is added to OD600nm=0.6 in culture.6000rpm, 4 DEG C of centrifugations 15min collects thallus；Thallus, ultrasonication (ultrasonic 3s, gap 3s, total 30min) is resuspended in PBS；12000rpm, 4 DEG C of centrifugations 30min collects supernatant；Supernatant is isolated and purified through nickel ion affinity chromatograph column (GE Products), with containing different dense Spend the PBS buffer solution elution foreign protein and destination protein of imidazoles；The sample that 12%SDS-PAGE detection is collected, will contain has purpose egg The eluent of Bai Chengfen is concentrated by ultrafiltration, then is further purified by molecular exclusion chromatography method (Superdex75, GE Products) Single-chain antibody dispenses the protein sample of acquisition after 0.22um sterilised membrane filter filtration sterilization, and saves in -80 DEG C.As a result As shown in the table, 5 single chain antibody proteins realize solubility expression, remaining 3 single chain antibody protein is with inclusion bodies table It reaches.
Single-chain antibody title Expression-form
TR2-1 Solubility expression
TR2-2 Inclusion body
TR2-3 Solubility expression
TR2-4 Inclusion body
TR2-5 Solubility expression
TR2-6 Solubility expression
TR2-7 Solubility expression
TR2-8 Inclusion body
The activity identification of 6. anti-human death receptor DR5 single-chain antibody of embodiment
Select the highly expressed colon cancer COLO205 cell of cell surface death receptors DR5 to 5 single-chain antibody eggs of acquisition White carry out Activity determination.By COLO205 cell culture to logarithmic phase under conditions of 37 DEG C, 5%CO2, the digestion of 0.5% pancreatin is thin Cell is resuspended with cell culture fluid after born of the same parents, and with 5.0 × 104The density of cells/ml is inoculated in 96 porocyte culture plates, 200 μ Former culture medium is replaced with 2% blood serum medium after culture 20h in 37 DEG C, 5%CO2 incubator in the hole l/.Various concentration is added Single-chain antibody is incubated for 48h, and 5 multiple holes are arranged in each concentration, the cell of single-chain antibody incubation is not added as negative control；Often The MTT of 10 μ l is added in hole, and 37 DEG C are continued to cultivate 4h, abandon supernatant, and 150 μ 1DMSO are added in every hole, surveys at microplate reader 570nm wavelength Determine absorbance value (OD value).Cell proliferation inhibition rate calculation formula is as follows, cell inhibitory rate=(1- (ODsample- ODblank)/(ODcontrol-OD blank)) × 100%.
MTT is analyzed the results show that single-chain antibody TR2-3 has apparent inhibiting effect simultaneously to colon cancer cell COLO 205 There are dose-dependences, remaining 4 single-chain antibody activity is weaker (Fig. 2), therefore preferably obtain human death receptor DR5 excitement Agent single-chain antibody TR2-3.
The purity analysis and Western Blot of 7. anti-human death receptor DR5 single-chain antibody TR2-3 of embodiment is identified
To the TR2-3 albumen purified in embodiment 5 using molecular exclusion method HPLC system (LC-2010A HT, SHIMADZU Corp., Japan) on carry out purity analysis, chromatographic column used is Shodex PROTEIN KW-802.5 (SHOWA DENKO K.K., Japan), mobile phase is 0.2M phosphate buffer (pH 7.6), Na2SO4 containing 0.1M, flow velocity 0.7ml/ min.The results show that TR2-3 purity of protein after purification is > 96% (Fig. 7).Determining the protein quantity is carried out with BCA method (biouniquer Products) realize single-chain antibody TR2-3 in Escherichia coli the results show that TR2-3 yield reaches 12mg/L Solution expression with high efficiency with isolate and purify.
Peak # Retention time Area Highly Area % Height %
1 0.192 1346 132 0.098 0.257
2 9.290 2322 125 0.169 0.244
3 10.897 43962 1366 3.194 2.661
4 11.868 1322561 49470 96.099 96.345
5 15.074 6061 254 0.440 0.494
It amounts to 1376252 51347 100.000 100.000
The single-chain antibody that purifying is obtained carries out SDS-PAGE electrophoresis, and 4 DEG C, 100mA constant current transfers 100min, and albumen is turned Print to pvdf membrane (Millipore Products)；Transfer terminates, by film be placed in 5%Milk TBST (containing 5% skim milk and The TBS of 0.1% polysorbas20) in room temperature close 1h；With 5%Milk TBST by 1: 5000 dilution Anti-6 × His rabbit Polyclonal antibody antibody (the raw chemical product in Shanghai), is incubated at room temperature 2h, TBST is washed 3 times, each 10min；With 5% Milk TBST is by 1: 2500 dilution HRP-conjugated Goat Anti-Rabbit IgG secondary antibody (the raw chemical product in Shanghai), room Temperature is incubated for 1h, and TBST is washed 3 times, each 10min；It is developed the color with ECL.Western Blot analysis the results show that 25KD with There is a specific band (Fig. 8) between 35KD, it is in the same size with single-chain antibody TR2-3 theoretical molecular weight 27.5KD, it was demonstrated that pure Changing obtained albumen is single-chain antibody TR2-3.
Inhibition of the 8. anti-human death receptor DR5 single-chain antibody of embodiment to colon cancer, Cells Proliferation of Human Breast Cancer
Colon cancer cell COL0205 and breast cancer cell MDA-MA-231 is selected, is cultivated under conditions of 37 DEG C, 5%CO2 To logarithmic phase, cell is resuspended with cell culture fluid after 0.5% trypsin digestion cell, and with 5.0 × 104The density of cells/ml connects Kind in 96 porocyte culture plates, 200 holes μ l/, at 37 DEG C, 5%CO2It is replaced after cultivating 20h in incubator with 2% blood serum medium Former culture medium.The single-chain antibody of various concentration is added, is incubated for 48h, each concentration is arranged 5 multiple holes, is incubated so that single-chain antibody is not added The cell educated is as negative control；The MTT of 10 μ l is added in every hole, and 37 DEG C are continued to cultivate 4h, abandon supernatant, and 150 μ l are added in every hole DMSO measures absorbance value (OD value) at microplate reader 570nm wavelength.Cell proliferation inhibition rate calculation formula is as follows, cell suppression Rate processed=(1- (ODsample-ODblank)/(ODcontrol-OD blank)) × 100%.
MTT is analyzed the results show that single-chain antibody TR2-3 is to colon cancer cell COLO 205 and breast cancer cell MDA-MA- 231 have apparent inhibiting effect and there are dose-dependence (Fig. 9), the results showed that, single-chain antibody TR2-3 has obvious Anti-tumor activity.
Inspection of the 9. anti-human death receptor DR5 single-chain antibody of embodiment to colon cancer cell COLO205caspase signal pathway activated It surveys
Colon cancer cell COLO205 is cultivated under conditions of 37 DEG C and 5%CO2 to logarithmic phase, 0.5% trypsin digestion cell Cell is resuspended with cell culture fluid afterwards, and with 5.0 × 105The density in the hole cells/ is inoculated in 6 porocyte culture plates, 500ul/ Hole, in 37 DEG C, 5%CO2Former culture medium is replaced with serum free medium after culture 20h in incubator.The single-stranded of 1uM concentration is added Antibody is incubated for 1,2,4h respectively, not add the cell of drug-treated as negative control, is labeled as 0h；It will be thin after drug-treated Born of the same parents digest from 6 orifice plates, and suitable RIPA lysis buffer is added and cracks 30min on ice, 12000rpm is centrifuged 10min, obtains The supernatant arrived is used for SDS-PAGE electrophoresis, and 4 DEG C, 100mA constant current transfers 2h, and albumen is transferred to pvdf membrane, and (Millipore is public Take charge of product)；Transfer terminates, and film is placed in room temperature in 5%Milk TBST (TBS containing 5% skim milk) and closes 1h；With 5% Milk TBST is by the anti-capase 3 of 1: 500 dilution, caspase 8 and β-actin antibody (the raw chemical product in Shanghai), incubation at room temperature 2h, TBS are washed 3 times, each 10min；With 5%Milk TBST by 1: 2500 dilution HRP-conjugated Goat Anti- Rabbit IgG secondary antibody (the raw chemical product in Shanghai), is incubated at room temperature 1h, TBS is washed 3 times, each 10min；ECL colour developing.
Western Blot analysis the results show that caspase 3 and caspase 8 detect apparent cutting rod band, And with the extension of single-chain antibody TR2-3 action time, cutting rod band is more obvious.The result shows that single-chain antibody TR2-3 can live Change key protein caspase 3 and caspase 8 (Figure 10) in caspase access, to trigger Apoptosis.
SEQUENCE LISTING
<110>China Medicine University
<120>a kind of agonist single-chain antibody of anti-human death receptor 5 of full source of people and application
<130> 1
<160> 8
<170> PatentIn version 3.5
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Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly Ser Ile Ser Ser Ser
Asn Trp Trp Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
Ile Gly Glu Ile Tyr His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu
Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
Ala Arg Gly Ala Ala Ala Gly Thr Ala Asn Asp Ala Phe Asp Ile Trp
Gly Gln Gly Thr Met Val Thr Val Ser Ser
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<211> 108
<212> PRT
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Glu Thr Thr Leu Thr Gln Ser Pro Gly Ile Leu Ser Leu Ser Pro Gly
Glu Arg Ala Ser Leu Ser Cys Arg Ala Ser Gln Ser Val Pro His Asn
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
Ile Tyr Gly Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
Gly Ser Gly Ser Glu Thr Asp Phe Thr Leu Thr Val Thr Arg Leu Ala
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Arg Ser Leu
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg
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Gly Gly Ser Ile Ser Ser Ser Asn Trp Trp Ser
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Glu Ile Tyr His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser
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Ala Arg Gly Ala Ala Ala Gly Thr Ala Asn Asp Ala Phe Asp Ile
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Gln Ser Val Pro His Asn Tyr
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Gly Ala Ser Asn Arg Ala Thr
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Gln Gln Tyr Gly Arg Ser Leu Thr
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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

Claims (5)

1. a kind of single-chain antibody of the anti-human death receptor 5 of full source of people, it is characterised in that including heavy chain variable region and light chain variable Area, heavy chain variable region and light chain variable region are connected by flexible peptide, and the heavy chain variable region includes heavy chain CDR1, CDR2 and CDR3 Domain, the amino acid sequence in the domain the heavy chain CDR1 is as shown in SEQ ID NO.3, the amino acid sequence of heavy chain CDR2 such as SEQ ID Shown in NO.4, the amino acid sequence of heavy chain CDR3 is as shown in SEQ ID NO.5；The light chain variable region include light chain CDR1, The domain CDR2 and CDR3, the amino acid sequence in the domain the light chain CDR1 is as shown in SEQ ID NO.6, the amino acid sequence in the domain light chain CDR2 Column are as shown in SEQ ID NO.7, and the amino acid sequence in the domain light chain CDR3 is as shown in SEQ ID NO.8；The flexibility peptide ammino acid Sequence is as shown in SEQ ID NO.9.
2. the single-chain antibody of the anti-human death receptor 5 of full source of people as described in claim 1, it is characterised in that the heavy chain can Become the amino acid sequence in area as SEQ ID NO:1, the amino acid sequence of light chain variable region is SEQ ID NO:2.
3. a kind of expression vector and/or host cell of the single-chain antibody for the anti-human death receptor 5 for expressing full source of people, feature It is to express the single-chain antibody of the anti-human death receptor 5 of full source of people as claimed in claim 1 or 2.
4. the single-chain antibody of the anti-human death receptor 5 of full source of people as claimed in claim 1 or 2 has antitumor work in preparation Application in drug or diagnostic reagent.
5. application as claimed in claim 4, it is characterised in that tumour is selected from colon cancer, breast cancer, liver cancer, oophoroma, uterine neck Cancer, lung cancer, cancer of pancreas, carcinoma of testis or prostate cancer.
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WO2019100194A1 (en) * 2017-11-21 2019-05-31 深圳先进技术研究院 Anti-dr5 antibody and preparation method therefor and use thereof
WO2019100193A1 (en) * 2017-11-21 2019-05-31 深圳先进技术研究院 Anti-dr5 antibody and preparation method therefor and use thereof
CN109836500A (en) * 2017-11-25 2019-06-04 深圳宾德生物技术有限公司 It is a kind of to target the single-chain antibody of DR5, Chimeric antigen receptor T cell and its preparation method and application
CN109957025A (en) * 2017-12-25 2019-07-02 深圳宾德生物技术有限公司 It is a kind of to target the single-chain antibody of DR5, Chimeric antigen receptor T cell and its preparation method and application
CN109957020A (en) * 2017-12-25 2019-07-02 深圳宾德生物技术有限公司 It is a kind of to target the single-chain antibody of DR5, Chimeric antigen receptor T cell and its preparation method and application
CN109957019A (en) * 2017-12-25 2019-07-02 深圳宾德生物技术有限公司 It is a kind of to target the single-chain antibody of DR5, Chimeric antigen receptor T cell and its preparation method and application
CN109957024A (en) * 2017-12-25 2019-07-02 深圳宾德生物技术有限公司 It is a kind of to target the single-chain antibody of DR5, Chimeric antigen receptor T cell and its preparation method and application
CN118001381A (en) * 2024-01-24 2024-05-10 首都医科大学附属北京地坛医院 Pharmaceutical composition and application thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008154439A1 (en) * 2007-06-08 2008-12-18 Irm Llc Methods and compositions for inducing apoptosis in cancer cells
CN101717775A (en) * 2009-11-13 2010-06-02 厦门大学 Single-chain antibody gene of anti-human death receptor 5
CN102504026A (en) * 2011-11-21 2012-06-20 中国药科大学 Full human single chain antibody capable of resisting epidermal growth-factor receptor and application thereof
CN102924600A (en) * 2012-11-14 2013-02-13 河南大学 Death receptor-5 agitated polyvalent antibody and application thereof in preparation of anti-tumor medicines
CN104619725A (en) * 2012-07-09 2015-05-13 国际生物技术药物开发公司 Anti-DR5 family antibodies, bispecific or multivalent ANTI-DR5 family antibodies and methods of use thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008154439A1 (en) * 2007-06-08 2008-12-18 Irm Llc Methods and compositions for inducing apoptosis in cancer cells
CN101717775A (en) * 2009-11-13 2010-06-02 厦门大学 Single-chain antibody gene of anti-human death receptor 5
CN102504026A (en) * 2011-11-21 2012-06-20 中国药科大学 Full human single chain antibody capable of resisting epidermal growth-factor receptor and application thereof
CN104619725A (en) * 2012-07-09 2015-05-13 国际生物技术药物开发公司 Anti-DR5 family antibodies, bispecific or multivalent ANTI-DR5 family antibodies and methods of use thereof
CN102924600A (en) * 2012-11-14 2013-02-13 河南大学 Death receptor-5 agitated polyvalent antibody and application thereof in preparation of anti-tumor medicines

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
抗人DR5单链抗体噬菌体展示文库的建立和筛选;刘瑞敏等;《免疫学杂志》;20111130(第11期);第945-948页
抗人死亡受体5单链抗体特性分析;程小峰等;《免疫学杂志》;20110131(第1期);第70-72页

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