CN103864935A - Targeted EGFR/KDR (Epidermal Growth Factor Receptor/Kinase Insert Domain Receptor) specific diabody - Google Patents
Targeted EGFR/KDR (Epidermal Growth Factor Receptor/Kinase Insert Domain Receptor) specific diabody Download PDFInfo
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Abstract
The invention belongs to the technical field of gene engineering antibodies, and in particular relates to a preparation method and an application of a bispecific diabody of an anti-tumor epidermal growth factor receptor (EGFR) and an anti-tumor vascular endothelial growth factor (VEGFR/KDR). The bispecific diabody formed by cross-linking and expressing heavy/light chain variable areas of an anti-EGFR high-affinity single-chain antibody E10 and an anti-KDR single-chain antibody AK404 by using genetic engineering technology can simultaneously act on EGFR with high expression on the surfaces of tumor cells and KDR with high expression on the surfaces of tumor angiogenesis vascular endothelial cells, and can achieve the purposes of simultaneously inhibiting or killing tumor cells or inhibiting or damaging tumor angiogenesis.
Description
Technical field
The invention belongs to bioengineering field, be specifically related to one can with the high-affinity total man source dual specific double-chain antibody of Human epidermal growth factor receptor (EGFR)/human vascular endothelial growth factor acceptor (KDR) specific combination, can suppress the activation of EGF-R ELISA (EGFR)/vascular endothelial growth factor (KDR) acceptor, can suppress the growth of high expression level EGF-R ELISA tumour cell A431 and the growth of high expression level vascular endothelial growth factor receptor human vascular endothelial HUVEC, it is a kind of specific double-strand genetic engineering antibody with targeting anti-tumor and angiogenic activity.
Background technology
Monoclonal antibody with its specificity, homogeneity, the advantage such as can produce in a large number, be widely used in diagnosis and the treatment of disease.Although antibody has powerful natural effector function, in oncotherapy, the clinical efficacy of two solely application antibody is mostly very limited.In order to strengthen the effector function of antibody, people attempt several different methods engineered antibody molecule.Bi-specific antibody (bispecific antibody, BsAb) is one of developing direction strengthening Antybody therapy effect, can block the signal transduction that two independent antigens trigger simultaneously, in addition, also increases the specificity of cancer target.Due to antibody molecule characteristic difference, the Quality and yield of recombinant antibodies is required to difference, can select different BsAb to build pattern.Small molecular antibody has the advantages such as the strong immunogenicity of tumor tissues penetration power is low compared with complete antibody, and the key of preparing small molecules BsAb is to make two bispecific antibody fragments be fused into the antibody molecule with dual specific.Double-chain antibody (Diabody, Db) is the length by shortening connection peptides between variable region, and the structural domain of two different polypeptide chains is matched mutually, forms the bsAb of heterodimer, and the antibody molecule of this structure can be in bacterium great expression.
In recent years, blocking-up EGF-R ELISA (Epidermal growth factor receptor, EGFR), vascular endothelial growth factor receptor (Vascular endothelial growth factor receptor, and platelet derived growth factor acceptor (Platelet derived growth factor receptor VEGFR), PDGFR) therapeutic strategy is subject to clinical attention increasingly, it can improve the susceptibility of tumour to radiotherapy effectively because of blocking-up EGFR and VEGFR, strengthens combination therapy curative effect.EGFR is a kind of transmembrane receptor with tyrosine activity, can comprise that Urogastron (EGF) and transforminggrowthfactor-α (TGF-α) activate by multiple aglucon, plays important effect in the formation of kinds of tumors with in sending out; Suppress the activity of VEGF, the growth to tumour, transfer, recurrence, prognosis all have obvious restraining effect simultaneously.In addition, VEGFR and two kinds of signal transduction pathway of EGFR can crosstalk, clinical these two kinds of conduction paths that simultaneously suppress can provide the foundation that suppresses tumor growth.
EGF-R ELISA (EGFR)
Human epidermal growth factor receptor (epidermal growth factor receptor, also referred to as HER-1 or Erb-B1, be called " EGFR " herein) be the transmembrane glycoprotein of 170kDa, one of four members in Epidermal Growth Factor Receptor Family, it is encoded by c-erbB proto-oncogene, is made up of extracellular region, cross-film district and intracellular region 3 parts.Intracellular region shows intrinsic tyrosine kinase activity, and cross-film district is made up of a lipotropy polypeptide spiral; Extracellular region is divided into 4 subprovinces, is the position of EGFR and part (somatomedin etc.) combination.Under inactive state, after EGFR acceptor and its ligand binding, can form homology or heterodimer, acceptor intracellular region generation phosphorylation can start a series of intracellular signal cascades.EGFR combination comprises the part of the following: Urogastron (EGF), transforminggrowthfactor-α (TGF-α), amphiregulin, Heparin-binding EGF (hb-EGF), β tunicin and epiregulin.EGFR and part thereof are parts for cell signaling system, EGFR regulates many cell processes via tyrosine kinase mediated signal transduction pathway, includes but not limited to that active control cell proliferation, differentiation, cell survival, apoptosis, blood vessel occur, mitotic division occurs and the signal transduction pathway of transfer.Research shows, the increase of EGFR gene copy number or overexpression, and the equal transfer that can promote Normocellular conversion and malignant tumour, the signal conduction network of EGFR has accounted for consequence in the formation and development process of tumour.Have been found that its expression is at many human tumors, comprise in head and the cancer of neck, mammary gland, colon, prostate gland, lung and ovary and raising, the degree of overexpression is relevant to not good clinical prognosis.The tumor prognosis of EGFR high expression level is poor, easily shifts, and survival time is short.EGFR forms owing to relating to tumour, has become the specific target target of antineoplaston.
Targeting EGFR medicine mainly contains two classes: a class is to directly act on recipient cell inner section in case the synthetic tyrosine kinase inhibitor (TKI) of stop signal conduction mainly comprises Gefitinib, erlotinib etc.; Another kind of is the monoclonal antibody (MAb) that block ligand is attached to acceptor ectodomain, comprises Cetuximab (cetuximab) etc.
Vascular endothelial growth factor receptor (VEGFR/KDR)
Vascularization (angiogenesis) is a complicated process, be subject to the positive negative regulation of the multiple factor, its VEGF (vascular endothelial growth factor, VEGF) be to act on one of the strongest positivity regulatory factor, by with vascular endothelial growth factor receptor (vascular endothelial growth factor receptor, VEGFR) in conjunction with stimulating the formation of new vessel.The signal path of VEGFR mediation participates in migration, propagation, the existence of tumor neogenetic endotheliocyte, in tumour surrounding blood vessel Newborn Process, has important effect, has close relationship with the morbidity of the multiple kinds of tumor of body and the transfer of tumour.Therefore, VEGFR signal transduction pathway can be used as the desirable target spot of antineoplaston scheme.
There are three acceptor: VEGFR-1, VEGFR-2, VEGFR-3 in current known VEGF family, and respectively by fit-1, flk-1/KDR, fit-4 genes encoding, wherein VEGFR-2 is that KDR mainly participates in vascularization process.KDR belongs to Tyrosylprotein kinase receptor, is divided into extracellular region, cross-film district and film inner region three parts.3rd district in immunoglobulin-like district, 7 of extracellular regions (containing 97 amino acid) are the most outstanding to the combination contribution of VEGF.
Target VEGF signal path medicine mainly contains three classes: a class is to directly act on recipient cell inner section in case the synthetic tyrosine kinase inhibitor (TKI) of stop signal conduction, mainly comprise PTK787, Sutent, Sorafinib, Zactima and Recentin etc.; Another kind of is the monoclonal antibody (MAb) of anti-VEGF, block ligand is attached to acceptor ectodomain, comprise rhuMAb-VEGF (bevacizumab) and aflibercept etc., the 3rd class is the antibody that acts on VEGFR, as: Ramucirumab (IMC-1121B), CDP-791 is all the antibody that acts on VEGFR2.
Summary of the invention
Goal of the invention
The invention provides the specific double-strand antibody of a kind of total man source targeting EGFR/KDR with potential medical science and pharmacy value.The Bao Wai that is characterized as specific binding Human epidermal growth factor receptor extracellular region and KDR 3rd district of double-chain antibody of the present invention, can suppress in vitro the growth of epidermal carcinoma cell A431 and human vascular endothelial HUVEC, its effect that suppresses high expression level EGFR tumour is better than the single-chain antibody of the anti-EGFR of independent use.
Technical scheme
The double-chain antibody of the targeting EGFR/KDR in a kind of total man source, this antibody is take the single-chain antibody of anti-EGFR and anti-KDR as basis, gene recombination is built into the double-chain antibody of dual specific, and light (weight) chain variable domain of anti-EGFR is connected by flexible peptide with heavy (gently) territory, chain variable region of anti-KDR.
Wherein a chain is made up of the light chain of anti-EGFR single-chain antibody and the heavy chain of anti-KDR single-chain antibody, and its aminoacid sequence table is SEQNO.1; Another chain is made up of the light chain of anti-KDR single-chain antibody and the heavy chain of anti-EGFR single-chain antibody, and its aminoacid sequence table is SEQ NO.2.
A kind of osmotic shock method, it is for separating of the above-mentioned bi-specific antibody of purifying.
A nucleic acid for separation, is characterized in that the antibody that this nucleic acid encoding is above-mentioned.
A kind of expression vector, contains above-mentioned nucleic acid.
A kind of recombinant host cell, contains above-mentioned expression vector.
The antibody of above-mentioned any one or the application of antibody fragment, is characterized in that being optionally combined with EGFR/KDR or the transduction of its signal is blocked in the combination that suppresses EGFR/KDR and EGFR part/KDR part.
The antibody of above-mentioned any one or the conjugate of antibody fragment.
The application of anti-EGFR/KDR bi-specific antibody in medicine for treating tumor thing.
Invention further illustrates:
In the present invention, full human-derived anti-human EGFR/KDR bi-specific antibody is made up of two chains, and wherein light (weight) territory, chain variable region of anti-EGFR is connected by 5 amino acid whose flexible peptides (GGGGS) with heavy (gently) territory, chain variable region of anti-KDR.
The expression vector and the Host Strains that contain EGF-R ELISA/vascular endothelial growth factor receptor bi-specific antibody gene of the present invention all belong to protection scope of the present invention.
Another object of the present invention is to provide a kind of can expression and the method for the above-mentioned anti-epidermal growth factor receptor/vascular endothelial growth factor receptor of purifying double-chain antibody.
The present invention utilizes round pcr that the anti-KDR high-affinity single-chain antibody AK404 of the anti-EGFR high-affinity single-chain antibody E10 of screening acquisition and this laboratory screening acquisition is cloned to restructuring, build dual specific double-chain antibody (Db) recombinant vectors, after conversion intestinal bacteria, use 0.8mMIPTG abduction delivering; Utilize osmotic shock to extract bacterium periplasm protein, low-temperature centrifugation is removed cell debris, supernatant is crossed to nickel affinity chromatography post and carry out separation and purification; Western Blot identifies the antibody that separation and purification obtains; The affinity of SPR experimental analysis antibody and antigen; MTT detects the growth-inhibiting effect of double-chain antibody his-and-hers watches skin cancer cell A431 and human vascular endothelial HUVEC; Apoptosis experiment confirms that bi-specific antibody is to make its apoptosis by its physiological program to the inhibited proliferation of cell, but not cytotoxicity; Finally suppress to test by chicken embryonic implantation knurl and confirm that the tumor-inhibiting action of bi-specific antibody is better than the monoclonal antibody of the anti-EGFR of independent use.
The bi-specific antibody the present invention relates to can specific binding EGFR and KDR molecule, inhibition tumour and tumor neogenetic blood vessels growth.Experiment shows, this bi-specific antibody is approximate in conjunction with the ability of EGFR or KDR and anti-EGFR or anti-KDR single-chain antibody separately, and the propagation of tumour or vascular endothelial cell is suppressed to ability is similar to anti-EGFR or anti-KDR single-chain antibody.For explanation bi-specific antibody suppresses EGFR and the independent a kind of growth that can suppress better tumour suppressing in both of KDR ratio simultaneously, set up people's epidermal carcinoma cell A431 chick chorioallantoic membrane transplanted tumor model.
Accompanying drawing explanation
Fig. 1 is anti-EGFR/KDR double-chain antibody by single-chain antibody gene recombination analysis figure separately, and recombination size is about 1500bp.
Fig. 2 is the structural representation of anti-EGFR/KDR bi-specific antibody, this antibody is made up of two chains, wherein light (weight) territory, chain variable region of anti-EGFR is connected by flexible peptide (GGGGS) with heavy (gently) territory, chain variable region of anti-KDR, and heavy chain and the light chain of anti-EGFR (KDR) combine with non covalent bond in solution.
Fig. 3 is that SDS-PAGE protein electrophorese figure and Western Blot identify double-chain antibody figure.Fig. 3 A describes the bi-specific antibody Db of fermentation expression carries out separation and purification result by nickel post, and Fig. 3 B describes Western Blot and identifies the double-chain antibody result being separated to.The single-chain antibody AK404 that swimming lane 1 is anti-KDR, the single-chain antibody E10 that swimming lane 2 is anti-EGFR, swimming lane 3 is for separating the dual specific double-chain antibody Db obtaining.
Fig. 4 is antigen-antibody avidity composes curve figure, shows the combination test experiments (Biacore) of anti-EGFR/KDR bi-specific antibody and antigen KDR (Fig. 4 A) or antigen EGFR (Fig. 4 B).
Fig. 5 is graphic representation, total man source anti-EGFR/KDR dual specific double-chain antibody is described to HUVEC cell (Fig. 5 A, with the positive contrast of single-chain antibody AK404 of anti-KDR, using the single-chain antibody E10 of anti-EGFR as negative control) growth-inhibiting effect or growth-inhibiting effect (Fig. 5 B to A431 cell, using the single-chain antibody E10 of anti-EGFR as positive control, with the negative contrast of single-chain antibody AK404 of anti-KDR)
Fig. 6 is flow cytometer detection figure, describes total man source anti-EGFR/KDR dual specific double-chain antibody to HUVEC cell (using the single-chain antibody AK404 of anti-KDR as positive control) or the apoptotic effect to A431 cell (with the positive contrast of single-chain antibody E10 of anti-EGFR).
Fig. 7 is that chick chorioallantoic membrane A431 people epidermal carcinoma transplanted tumor suppresses experimental result column diagram, describes the growth-inhibiting effect of total man source anti-EGFR/KDR dual specific double-chain antibody to chick chorioallantoic membrane people epidermal carcinoma transplanted tumor.
Embodiment
The structure of embodiment 1 total man source anti-EGFG/KDR double-chain antibody Db
With scFv-E10, scFv-AK404R gene and pHEN2 plasmid are template respectively, design and synthesize primer and carry out pcr amplification.The structure of anti-EGFR/KDR double-chain antibody Db is take the variable region of first increasing independently as basis, then carries out overlapping PCR and extend amplification and obtain complete Db gene.PCR product detects with 1.0% agarose gel electrophoresis, and sepharose reclaims test kit and reclaims goal gene.Pcr amplification end product and plasmid pHEN2 carry out double digestion with restriction enzyme, after enzyme is cut product and cut glue and reclaim, with the connection of spending the night of 16 ℃ of T4 ligase enzymes.After connecting, transform intestinal bacteria HB2151 competence, spread plate, next day, picking mono-clonal double digestion and order-checking were identified.
Expression, purifying, the evaluation of embodiment 2 total man source anti-EGFR/KDR double-chain antibody Db
By recombinant plasmid transformed, to intestinal bacteria HB2151 bacterial strain, picking transforms single bacterium colony, is inoculated in 2 × TY (100 μ g/ml Amp) liquid nutrient medium, 37 ℃ of activation of spending the night.Be connected to fresh 2 × TY substratum with 1% inoculum size next day, adds 0.4M sucrose in substratum, works as OD
600reach at 0.8 o'clock and add final concentration 0.8mM IPTG, in 25 ℃ of abduction delivering 18～24h.4 ℃, the centrifugal 15min of 8000rpm collects the fermented liquid thalline of abduction delivering, resuspended thalline in the hypertonic solution (50mM Tris-HCl, 20%sucrose, 1mM EDTA, pH8.0) of 5% ice-cold initial fermentation volume, mild stirring 10min.The centrifugal 30min of 12000rpm, collects supernatant, resuspended being deposited in isopyknic ice-cold 5mM MgSO4 simultaneously, mild stirring 15min on ice.Centrifugal supernatant is mixed with hypertonic solution supernatant, 0.22 μ m membrane filtration sample is purified with nickel affinity column chromatography, subsequently with the PBS buffer solution elution target protein containing different concns imidazoles.A.15%SDS-PAGE analyze and collect sample, select high purity protein and identify.B. high purity protein sample is carried out to Western Blot evaluation.4 ℃, 250mA constant current transfer printing 1.5h, is transferred to pvdf membrane (purchased from Millipore) by albumen; Transfer printing finishes, by film room temperature sealing 2h in 5% skimmed milk; PBS washes 3 times, add mouse-anti His antibody in the ratio of 1: 2000, hatch 1h for 37 ℃, with washing 3 times containing the PBS (TPBS) of 0.05% tween, add the sheep anti-mouse igg polyclonal antibody of HRP coupling in 1: 5000 ratio again, hatch 1h for 37 ℃, TBS washes 3 times, drip the luminous nitrite ion of ECL, the exposure of gel imaging instrument is taken pictures.Sample dialysis by final purifying and after identifying or ultrafiltration to PBS, liquid nitrogen flash freezer ,-70 ℃ of preservations.
The SPR experiment of embodiment 3 total man source anti-EGFR/KDR double-chain antibody Db
This experiment double center chain antibody Db and separately antigen are combined with biosensor that Biacore X100 relies on as SPR-and detect the interaction between Db and EGFR or KDR: A. and use and have the NTA chip on nitrilotriacetic acid surface, and connection is with the recombinant antibodies Db molecule of His-Tag mark.Respectively detectable level be 6.25,12.5,25,50,100, the KDR antigen of 200nmol/L, obtain in conjunction with and dissociation curve, with BIA assessment software analytical calculation acquisition equilibrium dissociation constant, Kd is about 34nmol/L.B. applying marking has the CM5 chip of anti-Fc antibody, connects the EGFR antigen molecule with Fc fragment.Detect respectively 1.56,3.125,6.25,12.5,25,50, the Db antibody of 100nmol/L, obtain in conjunction with and dissociation curve, with BIA assessment software analytical calculation acquisition equilibrium dissociation constant, Kd is about 11nmol/L.
The growth-inhibiting effect of embodiment 4 total man source anti-EGFR/KDR double-chain antibody his-and-hers watches skin cancer cell A431/ vascular endothelial cell HUVEC
In this experiment, by the A431 cytosis of the HUVEC cell of bi-specific antibody Db and high expression level KDR antigen or high expression level EGFR antigen, and utilize SPSS software to analyze experimental data, to assess anti-new vessel and the anti-tumor activity of this albumen.A. by 3 × 10
3～5 × 10
3individual HUVEC cell is inoculated 96 porocyte culture plates, and 100 μ l/ holes, at 37 ℃ of 5%CO
2in incubator, cultivate.With containing the 1%ECM substratum of 20ng/ml VEGF, sample sets Db, positive control AK404, negative control E10 being diluted to respectively to 10 groups of different concns gradients (0,1,2.5,5,10,25,50,100,200,300nmol/L), after 24h, add the antibody of 100 μ l gradient dilutions, each concentration arranges three parallel holes, continues to cultivate 72h.B. by 1 × 10
4～2 × 10
4individual A431 cell is inoculated 96 porocyte culture plates, and 100 μ l/ holes, at 37 ℃ of 5%CO
2in incubator, cultivate.Sample sets Db, positive control E10, negative control AK404 are diluted to respectively to 10 groups of different concns gradients (0,1,2.5,5,10,25,50,100,200,500nmol/L) with 1% foetal calf serum substratum, after 24h, add the antibody of 100 μ l gradient dilutions, each concentration arranges three parallel holes, continues to cultivate 72h.
After observation of cell upgrowth situation, every hole adds the MTT of 11 μ L, and 37 ℃ are continued to cultivate 4h, the supernatant that carefully inclines, and every hole adds 150 μ LDMSO, measures absorbance value (OD value) at microplate reader 570nm/630nm wavelength place, calculates cell proliferation.The biological activity of Db is evaluated by the inhibiting rate of Growth of Cells, inhibiting rate=(1-experimental group OD value/control group OD value) × 100%.
The apoptosis experiment of the anti-EGFR/KDR double-chain antibody of embodiment 5 his-and-hers watches skin cancer cell A431/ vascular endothelial cell HUVEC
The anti-EGFR/KDR bi-specific antibody in total man source experimental results show that to HUVEC cell or to the apoptosis of A431 cell albumen is non-cytotoxicity death by programmed cell death to the inhibited proliferation of cell.A. the HUVEC cell 4 × 10 in vegetative period of taking the logarithm
5the individual 6 porocyte culture plates that are inoculated in, 1ml/ hole, separately supplements 1ml/ hole perfect medium, at 37 ℃ of 5%CO
2in incubator, cultivate.Sample sets Db, positive control AK404, negative control E10 are diluted to respectively to 6 groups of different concns gradients (0,2.5,5,10,20,50nmol/L) with 5%ECM substratum, after 48h, add the antibody of 1.5ml gradient dilution, continue to cultivate 36h.B. by A431 cell 2 × 10
5individual inoculation 96 porocyte culture plates, 1ml/ hole, separately supplements 1ml/ hole perfect medium, at 37 ℃ of 5%CO
2in incubator, cultivate.Sample sets Db, positive control E10, negative control AK404 are diluted to respectively to 6 groups of different concns gradients (0,2.5,5,10,20,50nmol/L) with 5% foetal calf serum substratum, after 48h, add the antibody of 1.5ml gradient dilution, continue to cultivate 36h.
After observation of cell upgrowth situation, inhale and abandon substratum, PBS washes 2 times, 0.25% trysinization orifice plate cell, twice of the gentle washed cell of cold PBS; With 1 × Binding Buffer re-suspended cell of 195 μ L, making cell density is 2～5 × 10
5individual/ml; The resuspended liquid of Annexin V-FITC to 195 μ L cell that adds 5 μ L, mixes rear lucifuge, incubated at room 10 minutes; 1 × Binding Buffer washed cell of 200 μ L once, is then resuspended in cell 1 × Binding Buffer of 190 μ L; Add 10 μ L Propidium Iodie, utilize flow cytometry analysis.Result showed cell apoptosis rate becomes positive correlation with antibody concentration, proves that antibody can induction of vascular endothelial cell and the apoptosis of epidermal carcinoma tumour cell.
The inhibition experiment of the anti-EGFR/KDR double-chain antibody of embodiment 6 to chick chorioallantoic membrane (CAM) A431 people epidermal carcinoma transplanted tumor model
Instar chicken embryo on the 8th, delimiting is the place of windowing apart from fetal head 0.5～1.0cm place, opens the window of 1cm × 1cm as center, punctures shell membrane also moistening by stroke-physiological saline solution with syringe, removes shell membrane exposure CAM and prepares false air chamber.Get the resuspended logarithmic phase A431 cell 8 × 10 of PBS
6individual/egg, 20 μ L are inoculated in territory, the relative avascular area of chorioallantoic membrane, aseptic scotch tape sealing, fixed temperature and humidity is cultivated.Observe chicken embryo every day and survive situation, hatch after 3 days the positive transplanted tumor of inoculating successfully (visual inspection has 2mm solid tumor) is carried out to drug intervention.Add and measure knurl body long (a), wide footpath (b) before medicine, by formula: volume V (mm
3)=a × b
2/ 2 calculate gross tumor volume V
0.Medicine Db, AK404, E10, AK404+E10 are diluted to 300nmol/L with PBS, drip and on filter paper carrier, be put in tumour shaping place, after 48h, remove carrier and observe knurl body and take pictures and calculate gross tumor volume V
1.Chicken embryonic implantation knurl inhibiting rate=(1-V
1/ V
0) × 100%.
Claims (9)
1. the dual specific double-chain antibody of a total man source targeting EGFR/KDR, it is characterized in that: this antibody is take the single-chain antibody of anti-EGFR and anti-KDR as basis, gene recombination is built into the double-chain antibody of dual specific, and light (weight) territory, chain variable region of anti-EGFR is connected by flexible peptide with heavy (gently) territory, chain variable region of anti-KDR.
2. the dual specific double-chain antibody of a kind of total man source targeting EGFR/KDR according to claim 1, is characterized in that: wherein a chain is made up of the light chain of anti-EGFR single-chain antibody and the heavy chain of anti-KDR single-chain antibody, and its aminoacid sequence is SEQ NO.1; Another chain is made up of the light chain of anti-KDR single-chain antibody and the heavy chain of anti-EGFR single-chain antibody, and its aminoacid sequence is SEQ NO.2.
3. an osmotic shock method, is characterized in that: for separating of the bi-specific antibody of purifying total man claimed in claim 1 source targeting EGFR/KDR.
4. a nucleic acid for separation, is characterized in that: the dual specific double-chain antibody of a kind of total man source targeting EGFR/KDR of this nucleic acid encoding claim 1.
5. an expression vector, contains nucleic acid claimed in claim 4.
6. a recombinant host cell, contains expression vector claimed in claim 5.
7. claim 1 or 2 antibody of any one or the application of antibody fragment, is characterized in that being optionally combined with EGFR/KDR or the transduction of its signal is blocked in the combination that suppresses EGFR/KDR and EGFR part/KDR part.
8. claim 1 or 2 antibody of any one or the conjugate of antibody fragment.
9. the application of targeting EGFR/KDR dual specific double-chain antibody claimed in claim 1 in medicine for treating tumor thing.
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| CN107987168A (en) * | 2017-12-26 | 2018-05-04 | 北京格根生物科技有限公司 | The antibody of the single chain bispeci-fic of a kind of anti-vegf and EGFR and its application |
| CN108059681A (en) * | 2017-12-26 | 2018-05-22 | 北京格根生物科技有限公司 | The bispecific antibody fusion protein and its application of a kind of anti-vegf and EGFR |
| CN107987168B (en) * | 2017-12-26 | 2020-07-28 | 北京格根生物科技有限公司 | Single-chain double-specific antibody for resisting VEGF and EGFR and application thereof |
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