CN102250247B - Bispecific antibody to VEGF/ANG2 and application thereof - Google Patents
Bispecific antibody to VEGF/ANG2 and application thereof Download PDFInfo
- Publication number
- CN102250247B CN102250247B CN 201110160558 CN201110160558A CN102250247B CN 102250247 B CN102250247 B CN 102250247B CN 201110160558 CN201110160558 CN 201110160558 CN 201110160558 A CN201110160558 A CN 201110160558A CN 102250247 B CN102250247 B CN 102250247B
- Authority
- CN
- China
- Prior art keywords
- vegf
- ang2
- antibody
- ser
- tumor
- 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.)
- Active
Links
Images
Abstract
The invention relates to a medicine of a bispecific monoclonal antibody, and especially to a medicine of a bispecific monoclonal antibody to human vascular endothelial growth factor (VEGF/VEGF-A) and angiopoietin 2 (ANG2) for resistance to angiogenesis of tumors. The bispecific antibody to VEGF/ANG2 provided in the invention is characterized in that: a monoclonal antibody to VEGF is used as the base for the antibody and a single chain antibody to ANG2 is connected with the terminal of FC segment of the monoclonal antibody to VEGF to form the bispecific antibody to VEGF/ANG2. The bispecific antibody related to in the invention is obtained by employing technical means like gene engineering and constructing antibody segments which identify VEGF and ANG2 in a same antibody molecule that can be specifically bound with the two antibody segments; the effect of the bispecific antibody on inhibiting angiogenesis of tumor issue is obviously superior to that of a single antibody to VEGF; and the bispecific antibody has good activity in resisting tumors.
Description
Technical field
This patent relates to the bispecific monoclonal antibody medicine, particularly the bispecific monoclonal antibody medicine of antineoplastic vascular new life's human vessel endothelium growth factor resisting (VEGF/VEGF-A) and vectors containing human platelet-derived growth acceptor (PDGFR).
Background technology
Before century more than one, just there is bibliographical information to cross tumor growth and is accompanied by new vessel generation (Ferrara2002).But until nineteen thirty-nine, just proposed first by Ide and colleague thereof, the Angiogenesis related factor that may exist certain tumour source provides vascularity (Ide et al.1939) for the growth of tumour.After several years, can be prior to the Fast Growth of tumour owing to observing increasing of vessel density, the people such as Algire think " rapid diffusion of tumour depend on abundant blood vessel supply with " (Algire et al.1945).In the eighties of last century sixties, Greenblatt, Shubik(Greenblatt et al.1968) and Ehrmann, Knoth(Ehrmann et al.1968) experiment of two research groups in succession provides Prima Facie Evidence, but confirm that the vasculogenesis of tumour is some spreading factor mediation that is produced by tumour cell.
1971, American scientist Volkmann (Judah Folkman) proposed in " New England Journal of Medicine ", and angiogenesis inhibitor may be a kind of effective anticancer means (Folkman1971).From the seventies in early days, take this prospective hypothesis as the basis, Volkmann and research group thereof are devoted to separate certain ' tumor angiogenesis factor ' (Folkman et al.1971) from the tumour of human body and animal.1978, Gullino also proposed to suppress the viewpoint (Gullino1978) that vasculogenesis can be avoided cancer.Subsequently, the multiple blood vessel source factor (as, Urogastron EGF, transforming growth factor TGF-α, TGF-β, tumor growth factor TNF-α and angiogenine etc.) successively be found (Folkman et al.1987).
Vascular endothelial growth factor (VEGF)
Vascular endothelial growth factor (vascular endothelial growth factor, VEGF, also can write VEGF-A) be the crucial regulatory factor of a vasculogenesis, for VEGF gene family institute's role in vasculogenesis is regulated, people have furtherd investigate more than ten years (Ferrara2002).VEGF family mainly comprises prototype member (VEGF-A), placenta growth factor (placenta growth factor, PlGF) (Maglione et al.1991), VEGF-B(Olofsson et al.1996 at present), VEGF-C(Joukov et al.1996), VEGF-D(Orlandini et al.1996).Wherein VEGF-A is the angiogenesis factor that the induced tumor angiogenic action is the strongest, specificity is the highest.VEGF has the tyrosine kinase receptor (RTKs) of 3 high affinities, is respectively VEGFR-1(Flt-1) (Shibuya et al.1990; De Vries et al.1992), VEGFR-2(KDR, Flk-1) (Yoshiji et al.1996; Ellis et al.1998; Tomisawa et al.1999) and VEGFR3(Flt-4) (Joukov et al.1996).KDR is angiopoietic main regulatory molecule, has obvious chemical chemotactic and short splitting action, and is relevant with hematopoiesis with blood vessel island, vascularization.
The growth of tumour has two visibly different stages, and namely changing into from avascular slow growth phase has the fast breeding of the blood vessel stage, and vasculogenesis makes tumour can obtain enough nutritive substances, is the key link of facilitating above-mentioned transformation.If there is no vasculogenesis, the growth of primary tumo(u)r can not surpass 1～2mm
3It is the major cause of oncotherapy failure that tumor invasion shifts, and in the multi-step process of tumour generation Invasion and Metastasis, vasculogenesis all plays an important role.
In situ hybridization research has been found that VEGF mRNA has expression in multiple human tumor, comprises lung cancer (Volm et al.1997), mammary cancer (Yoshiji et al.1996), gastrointestinal cancer (Ellis et al.1998), kidney (Tomisawa et al.1999) and ovarian cancer (Sowter et al.1997).The Duo Jia laboratory uses the means of multiple anti-VEGF all to realize the inhibition of tumor growth, these methods comprise: for antibody, the soluble receptors of VEGF or its acceptor (VEGFRs), and the micromolecular inhibitor of VEGFRs Tyrosylprotein kinase and utilize the sudden change heterodimer of VEGF to seal its receptor binding site etc.
1993, Ferrara prepared the mouse antibody of VEGF, and in experiment in vitro, mouse antibody can significantly suppress several human cancer cell lines' growth.From then on, the clinical value of VEGF antibody begins to appear.In order to reduce the immunogenicity of mouse antibody, Ferrara changes with the skeleton of mouse source antibody the part of being human antibody IgG1, and Genentech company " cookle level " medicine--rhuMAb-VEGF (Avastin) thus has been born.It is the humanized antibody (IgG1) of a kind of anti-VEGF, people's source structure territory of 93% and 7% mouse source calmodulin binding domain CaM form, it is a monoclonal antibody drug that is approved for the inhibition angiogenic growth that beats the world, in February, 2004 U.S. food Drug Administration (Food and Drug Administration, FDA) ratifies this medicine and is used for treatment transitivity colorectal carcinoma (mCRC), minicell molecule lung cancer (GBM) and metastatic renal cell carcinoma.Arastin is different from existing cancer therapy drug, and take VEGF as drug target, the specific binding capacity of antibody, make its clinical efficacy remarkable in addition.In human trial, even for the cancer patients in late period, but also prolongs life several months of injection Arastin.The monoclonal antibody Cetuximab (Cetuximab) of ASCO meeting Souglakos report Avastin associating targeting EGFR in 2007 can treat the metastatic colorectal carcinoma in late period of chemotherapy failure safely and effectively.Genentech company carries out indication research with Avastin to surpassing 40 kinds of cancers, and hope can be produced more monoclonal antibody extension product.Simultaneously, they also after research is with whole antibody molecule I gG cutting, first with the albumen procaryotic cell expression, and then are connected to IgG with it by engineered means.
Except cancer, VEGF is also that treatment comprises senile macular degeneration SMD (AMD), and the retinopathy that diabetes cause is crucial in interior multiple ophthalmic diseases.In order to treat these diseases, on the basis of Avastin, Genentech company simplifies its whole antibody molecular structure again, reservation can neutralize VEGF antibody fragment, change route of administration into the vitreum direct injection by intravenous injection simultaneously, achieve thus another medicine--the twin sister ranibizumab (Lucentis) of Avastin.2006, ranibizumab is used for the treatment of age-related macular degeneration (age-related macular degeneration, AMD) by the approval of U.S. Bureau of Drugs Supervision, becomes soon the treatment senile macular degeneration SMD, the drug of first choice of the retinopathy that diabetes cause captures the share of North America market more than 80%.
ANG2 (Ang2)
American scientist Volkmann (J.Folkman) proposed in 1971, and the growth of tumour and transfer depend on the generation (Folkman1971) of blood vessel.This viewpoint was confirmed by many institutes in decades subsequently.Angiogenin (Angiopoietin, Ang) is that first people's tumor tissues that derives from that is determined has the cytokine that promotes angiogenic action.Ang is a kind of secretor type strand basic protein, is unique material with rnase (Rnase) activity in angiogenic factors.Known Ang comprises four hypotypes at present, Ang1, and Ang2, Ang3 and Ang4, wherein the generation relation of Ang1 and Ang2 and blood vessel is the closest, and to the research of Ang2 more (Lee state Hao et al.2004).
Mankind Ang2 gene is positioned at No. 8 the short arm of a chromosome districts (8p23.1), and the mRNA that transcribes generation is 1491bp, the albumen that 496 amino acid of encoding form, and molecular weight is 75kD.Ang2 can with Tie2(tyrosine kinase with immunoglobulin and epidermal growth factor homology domain2) combination, be its natural part.Ang2 is mainly synthetic by vascular endothelial cell, is combined with the Tie2 receptor-specific on self cytolemma by Autocrine, can be induced the unstable blood vessel that forms by Ang1 by competitive inhibition.Experiment showed, that the existence of VEGF can improve the expression of Ang2 (Gu et al.2006).
Ang2 participates in the vasculogenesis of kinds of tumors, and clinical stages and the Prognostic significance of the number that forms with tumor vessel, tumour are close.Use Ang2cDNA transfected with human colon cancer cell HT29 and set up the mouse-borne tumor model, the people such as Ahmad find that the gross tumor volume of Ang2 transfection obviously becomes large, the intratumoral vasculature increase in density, and the tumor cell proliferation index uprises (Ahmad et al.2001).Etoh etc. also find the research of cancer of the stomach case, in tissue the Ang2 high expression level simultaneously with vessel density increase, ripening degree descends, patient clinical TNM is evening by stages, survival rates low (Etoh et al.2001).Stomach cancer cell inoculation nude mice with the Ang2 transfection, found that the metastases rate increases, and the stomach cancer cell of transfection is under the condition that external VEGF exists, can promote more metal matrix proteolytic enzyme (the matrix metalloproteinases of production by human umbilical vessel endothelial cells, MMP) and the proteolytic ferment such as urokinase, this may promote tumor growth with Ang2, shift relevant.The people such as old yellow wren studies show that, Ang1 can suppress the generation of stomach organization blood vessel, and the function of Ang2 antagonism Ang1 has dual regulation to vasculogenesis, and are VEGF dependency (old yellow wren et al.2004).The people such as Zhang Zhenzhen think, the ratio of Ang2/Ang1 is the final factor that determines stomach organization vasculogenesis and tumor growth.As Ang2/Ang1〉1 the time, promote the stomach organization vasculogenesis, on the contrary suppress the generation of its blood vessel.Simultaneously they infer that angiogenin and acceptor thereof take (Zhang Zhenzhen et al.2006) in the conciliation effect that gastric cancer vascular generates as the leading factor with Ang2.Ang2 plays an important role in development in the generation of cancer of the stomach, can become a potential target of the anti-angiogenic treatment of cancer of the stomach.In addition, Ang2 is except the effect in the cancer of the stomach tumor-blood-vessel growth, and it is expressed also and glioma, colorectal carcinoma, the invasion and attack relevant with metastatic phenotype (Bach et al.2001, Imanishi et al.2001) of prostate cancer and mammary cancer etc.
Except above-mentioned cancer, Ang2 also has close relationship with angiogenesis and the growth of nonsmall-cell lung cancer (NSCLC) tumor tissues.Xing Lihua etc. and Takanami use the RT-PCR technology, have detected the expression level of ANG2 gene in the NSCLC tumour cell, and they find that the mRNA of ANG2 measures apparently higher than normal cell tissue (Xing Li magnificent et al.2003, Takanami2004) in contrast.In addition, the people such as the people such as Park and Xiao Shuhua detect 136 examples and 82 routine NSCLC patient bloods with elisa technique respectively, discovery is with respect to control group, and in the NSCLC patient blood, the concentration of ANG2 and VEGF is apparently higher than the former (Park et al.2007, desolate refined magnificent et al.2011).The people such as domestic Zhang Xuanbin have carried out the detection of immunohistochemical methods (ICH) to 37 routine patient's NSCLC tumor samples, they find ANG2 in patient's NSCLC tumor specimen, and VEGF and microvessel density (MVD) are all apparently higher than the healthy tissues that is used for contrast.Therefore they think Ang-2 and nonsmall-cell lung cancer infiltration, make progress closely related; It has promoter action to the nonsmall-cell lung cancer tumor-blood-vessel growth, and this effect has obvious VEGF dependency (Zhang Xuanbin et al.2000).In a research of delivering recently, the people such as Andersen have also proposed similar viewpoint.These researchists method of application chips (microarray) and immunohistochemical methods (ICH) have respectively carried out detection for ANG and VEGF-A to the 335 routine NSCLC patient tumors samples of randomly drawing, they find that the expression amount of ANG2 and VEGF-A has obvious growth in tumor tissues, and both expression amounts are closely related, have positively related relation (Andersen et al.2011).
Reference:
Ahmad A S,Lin W,Jung Y D,et al.The effects of angiopoietin1and2on tumor growth and angiogenesis in human colon cancer[J].Cancer Res,2001,61(4):12551259.
Algire,G.H.,Chalkley,H.W.,Legallais,F.Y.&Park,H.D.Vascular reactions of normal and malignant tissues in vivo.I.Vascular reactions of mice to wounds and to normal and neoplastic transplants.J.Natl Cancer Inst.1945,6,73–85.
Andersen S,Donnem T,Al-Shibli K,Al-Saad S,Stenvold H,Busund LT,Bremnes RM.Prognostic Impacts of Angiopoietins in NSCLC Tumor Cells and Stroma:VEGF-A Impact Is Strongly Associated with Ang-2.PLoS One.2011,6(5):e19773.
Bach F，Uddin FJ，Burke D．Angiopoietins in malignancy[J]．Ear J Surg Oncol，2007．33：7—15．
de Vries,C.et al.The fms-like tyrosine kinase,a receptor for vascular endothelial growth factor.Science.1992,255,989–991.
Ehrmann,R.L.&Knoth,M.Choriocarcinoma.Transfilter stimulation of vasoproliferation in the hamster cheek pouch.Studied by light and electron microscopy.J.Natl.Cancer Inst.1968,41,1329–1341.
Ellis,L.M.et al.Vessel counts and vascular endothelial growth factor expression in pancreatic adenocarcinoma.Eur.J.Cancer.1998,34,337–340.
Etoh T,Inoue H,Tanaka S,et al.Angiopoietin2is related to tumor angiogenesis in gastric carcinoma:possible in vivo regulation via induction of proteases[J].Cancer Res,2001,61(5):21452153.
Ferrara,N.VEGF and the quest for tumour angiogenesis factors.Nature Rev.Cancer.2002,2,795–803.
Folkman J.Tumor angiogenesis:therapeutic implication[J].N Engl J Med,1971,285(21):11821186.
Folkman,J.&Klagsbrun,M.Angiogenic factors.Science.1987,235,442–447.
Folkman,J.,Merler,E.,Abernathy,C.&Williams,G.Isolation of a tumor factor responsible for angiogenesis.J.Exp.Med.1971,133,275–288.
Greenblatt,M.&Shubick,P.Tumor angiogenesis:transfilter diffusion studies in the hamster by the transparent chamber technique.J.Natl Cancer Inst.1968,41,111–124.
Gu J,Yamamoto H,Ogawa M,et al.Hypoxiainduced upregulation of angipotietin2in colorectal cancer[J].Oncol Rep,2006,15(4):779783.
Gullino,P.M.Angiogenesis and oncogenesis.J.Natl Cancer Inst.1978,61,639–643.
Ide,A.G.,Baker,N.H.&Warren,S.L.Vascularization of theBrown Pearce rabbit epithelioma transplant as seen in thetransparent ear chamber.Am.J.Roentgenol.1939,42,891–899.
Imanishi Y, Ho B, Jmzynka MJ, et a1.Angiopoietin one 2stimulates breast cancer metastasis through the{alpha}5{beta}t integrin-mediated pathway[J] .Cancer Res, 2007.67:4254-4263.
Joukov,V.et al.A novel vascular endothelial growth factor,VEGF-C,is a ligand for the Flt4(VEGFR-3)and KDR(VEGFR–2)receptor tyrosine kinases.EMBO J.1996,15,1751.
Maglione,D.,Guerriero,V.,Viglietto,G.,Delli-Bovi,P.&Persico,M.G.Isolation of a human placenta cDNA coding for a protein related to the vascular permeability factor.Proc.Natl Acad.Sci.USA.1991,88,9267–9271.
Orlandini,M.,Marconcini,L.,Ferruzzi,R.&Oliviero,S.Identification of a c-fos-induced gene that is related to the platelet-derived growth factor/vascular endothelial growth factor family.Proc.Natl Acad.Sci.USA.1996,93,11675–11680.
Olofsson,B.et al.Vascular endothelial growth factor B,a novel growth factor for endothelial cells.Proc.Natl Acad.Sci.USA.1996,93,2576–2581.
Park JH,Park KJ,Kim YS,Sheen SS,Lee KS,Lee HN,Oh YJ,Hwang SC.Serum angiopoietin-2as a clinical marker for lung cancer.Chest.2007,132(1):200-6.
Shibuya,M.et al.Nucleotide sequence and expression of a novel human receptor-type tyrosine kinase(flt)closely related to the fms family.Oncogene.1990,8,519–527.
Sowter,H.M.et al.Expression and localization of the vascular endothelial growth factor family in ovarian epithelial tumors.Lab.Invest.1997,77,607–614.
Takanami I.Overexpression of Ang-2mRNA in non-small cell lung cancer:association with angiogenesis and poor prognosis.Oncol Rep.2004,12(4):849-53.
Tomisawa,M.et al.Expression pattern of vascular endothelial growth factor isoform is closely correlated with tumour stage and vascularisation in renal cell carcinoma.Eur.J.Cancer.1999,35,133–137.
Volm,M.,Koomagi,R.&Mattern,J.Prognostic value of vascular endothelial growth factor and its receptor Flt-1in squamous cell lung cancer.Int.J.Cancer.1997,74,64–68.
Yoshiji,H.,Gomez,D.E.,Shibuya,M.&Thorgeirsson,U.P.Expression of vascular endothelial growth factor,its receptor,and other angiogenic factors in human breast cancer.Cancer Res.1996,56,2013–2016.
Old yellow wren, Zhang Sheng, Lin Jianyin, Huang Peisheng.The two-way effect that ANP-2 generates gastric cancer vascular.Chinese Journal of Oncology, 2004; 26:161-164.
Li Guohao, Yang Weimin.The effect of angiogenin, express and with the relation [J] of tumor vascular growth.Clinical Surgery's magazine, 2004; 12(5): 311-3.
Xiao Shuhua, Rong Fu, Huang Jing, a day holder.The relation of NSCLC patients serum Ang2 and VEGF dynamic change.Treatment and prevention of tumour research, 1 phase in 2011.
Xing Lihua, Zhang Zhenxiang, Xu Yongjian.Expression of Angiopoietin-2Gene in Non-small Cell Lung Cancer. Central China University of Science and Technology journal: medical science English the German edition, the 4th phase in 2003.
Zhang Xuanbin, the summer prosperous Zheng.ANP-2 (Ang-2) in nonsmall-cell lung cancer (NSCLC) expression and the relation of tumor-blood-vessel growth.Shanghai Metallurgy Inst., Chinese Academy of Sciences; Materials Physics and Chemistry (specialty) 2000 years of Ph D dissertation.
Zhang Zhenzhen, Zhang Sheng, Lin Jianyin, Huang Peisheng, Chen Yupeng.The relation that the unconventionality expression of angiogenesis hormone and acceptor thereof and gastric cancer vascular generate.Chinese Journal of Oncology, 2006; 28:280-284.
Summary of the invention
Goal of the invention
The invention provides anti-VEGF-ANG2 bi-specific antibody, it uses the technique means such as genetically engineered, the antibody fragment of identification VEGF and identification ANG2 is structured in same antibody molecule, can be specific and the two combination, its successful that suppresses the tumor tissues angiogenesis is better than using separately VEGF antibody.
Technical scheme
Anti-VEGF/ANG2 bi-specific antibody is characterized in that this antibody is the basis by anti-VEGF monoclonal antibody, and passes through (GGGGS) at its FC section end
3Connecting anti-ANG2 single-chain antibody consists of.
Anti-VEGF monoclonal antibody is comprised of Anti-VEGF A Fab VH-CH, Anti-VEGF A Fab VL-CL and Fc; Wherein the aminoacid sequence table of Anti-VEGF A Fab VH-CH is that the aminoacid sequence table of SEQ NO.1, Anti-VEGF A Fab VL-CL is the aminoacid sequence table SEQ NO.3 of SEQ NO.2, Fc.
Described anti-ANG2 single-chain antibody passes through (GGGGS) by Anti-ANG2scFv VL and Anti-ANG2scFv VH
3Composition is formed by connecting; Wherein the aminoacid sequence table of Anti-ANG2scFv VL is that the aminoacid sequence table of SEQ NO.4, Anti-ANG2scFv VH is SEQ NO.5.
The application of anti-VEGF/ANG2 bi-specific antibody in preparation medicine for treating tumor thing.
Beneficial effect
1, the bi-specific antibody that relates to of this patent is to use the technique means such as genetically engineered, the antibody fragment of identification VEGF and identification ANG2 is structured in same antibody molecule, can be specific and the two combination, its successful that suppresses the tumor tissues angiogenesis is better than using separately VEGF antibody, and has the fine tumor promotion of seeing.
2, the bi-specific antibody that relates to of this patent can specific binding VEGF-A and ANG2 molecule, suppresses the biological function that it stimulates neovascularization growth.Experiment shows, this bi-specific antibody is approximate in conjunction with the ability of VEGF-A or ANG2 and anti-VEGF-A or anti-ANG2 monoclonal antibody separately, and it has very strong the time and these two kinds of abilities that antigen is combined.Experiment shows, suppress simultaneously VEGF-A and ANG2 and can better suppress tumor vascular growth than suppressing separately a kind of in both, therefore use this bi-specific antibody can effectively suppress the growth of tumor tissues, the increase of dosage in the time of avoiding using simultaneously two kinds of monoclonal antibodies again, the unpredictable risk of bringing to the patient.
Description of drawings
Fig. 1. anti-VEGF-ANG2 bi-specific antibody structural representation.This antibody is the basis by anti-VEGF monoclonal antibody, and connects anti-ANG2 single-chain antibody (scFv) formation at its FC section end;
Fig. 2. anti-VEGF-ANG2 bi-specific antibody heavy chain expression carrier;
Fig. 3. anti-VEGF Fab light chain expression vector;
Fig. 4. anti-VEGF-ANG2 bi-specific antibody (bsAb), Anti-VEGF, ANG2 in conjunction with the VEGF antigen-antibody in conjunction with test result figure.
The anti-VEGF-ANG2 bi-specific antibody of Fig. 5 (bsAb), Anti-VEGF, Anti-ANG2 in conjunction with the ANG2 antigen-antibody in conjunction with test result figure;
Fig. 6 is anti-VEGF-ANG2 bi-specific antibody (bsAb), Anti-VEGF, Anti-ANG2 in conjunction with VEGF ANG2 antigen-antibody in conjunction with test result figure;
Fig. 7. the restructuring bispecific monoclonal antibody is expressed and production scheme in mammalian cell.After the expression vector that builds changes cell over to, cultivate the cell strain of screening stably express in 96 orifice plates.The cell strain that screening obtains is amplification culture step by step, carries out at last scale operation in bio-reactor.
Embodiment
1. anti-VEGF-ANG2 bi-specific antibody (bsAb) nucleotide sequence design and synthetic:
According to this bsAb heavy chain VH
VEGF-CH1
VEGF-CH2
VEGF-CH3
VEGF-(GGGGS)
3-VH
ANG2-(GGGGS)
3-VL
ANG2Aminoacid sequence and type of attachment design heavy chain nucleotide sequence, and add Sac I restriction enzyme site at this sequence 5 ' end, KOZAK sequence and leader sequence add Xho I restriction enzyme site at its 3 ' end.Design the Oligonucleolide primers that 33 ' ends contain EcoR I restriction enzyme site, respectively synthetic heavy chain VH
VEGF-CH1
VEGF, CH2
VEGF-CH3
VEGF-(GGGGS)
3And VH
ANG2-(GGGGS)
3-VL
ANG2Segment is used the bsAb heavy chain gene that PCR conjugation (SDL PCR) synthesizes total length 2118bp.
VEGF-A IgG1 light-chain amino acid sequence anti-according to the people, design 5 ' end contains Not I restriction enzyme site, KOZAK sequence and leader sequence, 3 ' end contains the PCR primer of Psi I restriction enzyme site, uses the anti-VEGF-A light chain gene of people of PCR method amplification total length 639bp.
2. the structure of anti-VEGF-ANG2bsAb expression vector:
This bsAb heavy chain gene with Sac I and Xho I double digestion, with light chain gene Not I, after Psi I double digestion, is connected to heavy chain gene in the carrier for expression of eukaryon of Sac I and Xho I processing with the T4 ligase enzyme; Light chain gene is connected in the carrier for expression of eukaryon of Not I and Psi I processing.
These two expression vectors (comprising the phoA promotor) are then changed over to BL21 competence coli strain.BL21 bacterial strain after conversion is chosen positive colony, in containing the LB substratum (2mL) of 50ug/mL in 37 ℃ of incubated overnight.Bacterial strain changes in the CRAP substratum that 2L contains 50ug/mL and cultivated 24 hours in 30 ℃ afterwards.
Bacterium liquid 3000 turns the centrifugal supernatant of abandoning of normal temperature, and the intestinal bacteria of gained are with the plasmid extraction kit cracking of Qiagen company and extract plasmid, obtain to contain the expression vector of heavy chain and light chain.
3. the expression of anti-VEGF-ANG2bsAb and separation and purification
The electroporation cotransfection Flp-In of two expression vectors after purifying
TMChinese hamster ovary celI (Invitrogen) is cultivated at the condition low suspension that 500ug/ml Liu Suanyan NEOMYCIN SULPHATE (neo) exists, the strain of screening positive cell.
Recombinant antibodies is the complete bi-specific antibody molecule of 200kDa left and right with affinity chromatography (Protein A) and sieve chromatography separating-purifying molecular weight.Each component after purification is identified by the SDS-PAGE method.
Concrete grammar:
1. loading is used Protein A-Sepharose CL-4B affinity chromatography monoclonal antibody purification, and A KTA explorer100 monitors.Collect the cell culture medium supernatant liquor 0.02M that the amplification culture screening obtains, the phosphate buffered saline buffer dilution of pH7.4 is with the flow velocity loading of 1ml/min.
2. wash-out carries out stream with sample-loading buffer and washes, and 10 times of column volumes, flow velocity are 1ml/min.Then use 0.02M, the citrate buffer solution wash-out antibody of pH4.0.Monitor with A KTA explorer100 simultaneously, when elution peak occurring, get clean centrifuge tube and collect.After every collection 3ml, use immediately 1M, the Tris-HCl damping fluid of pH9.0 is adjusted pH value to 7.0.
Antigen-antibody is in conjunction with testing (ELISA method):
1. coated: with the coated damping fluid of 0.05M pH9.0 You carbonate, antigen (hVEGF-A or ANG2) being diluted to protein content is 5 μ g/ml.Add the antigen after 0.1ml dilutes in the reacting hole of each polystyrene board, 4 ℃ are spent the night.Discard solution in the hole next day, washes 3 times each 3 minutes with lavation buffer solution.
2. application of sample: add antibody to be checked (anti-VEGF Fab, anti-ANG2 monoclonal antibody or the anti-VEGF-ANG2bsAb) 0.1ml of certain dilution in above-mentioned coated reacting hole, put 37 ℃ and hatched 1 hour.Then washing.(doing simultaneously blank well, negative control hole and positive control hole).
3. add enzyme labelled antibody (goat anti-human igg-gamma): in each reacting hole, add enzyme labelled antibody (extent of dilution after the titration) 0.1ml of fresh dilution.Hatched 0.5～1 hour washing for 37 ℃.
4. add substrate solution colour developing: add the tmb substrate solution 0.1ml of interim preparation in each reacting hole, 37 ℃ 10～30 minutes.
5. termination reaction: add 2M sulfuric acid 0.05ml in each reacting hole.
6. result is judged: can be on white background, and result directly detects by an unaided eye: in reacting hole, color is darker, and positive degree is stronger, and negative reaction is colourless or extremely shallow, according to the depth of be color, with "+", "-" number expression.Also can survey the OD value: on the ELISA detector, locate in 450nm (if with ABTS colour developing, 410nm), surveying each hole OD value after blank hole zeroing, if greater than 2.1 times of the negative control OD value of stipulating, namely positive.
Experimental result:
Separately when test bsAb and VEGF or ANG2 binding ability, the binding ability and the binding ability identical (Fig. 4, Fig. 5) of monospecific corresponding monoclonal antibody in antigen of this bi-specific antibody and antigen.Fig. 6 shows, this bi-specific antibody can the specific while in conjunction with VEGF and ANG2, and monospecific bevacizumab or anti-ANG2 monoclonal antibody do not have this ability.
Anti-VEGF-ANG2 dual specific is anti-to people's lung cancer H460 nude mouse xenotransplantation tumor growth inhibition test
The tumor tissue of getting the growth animated period cuts into 1.5mm
3The left and right under aseptic condition, is inoculated in the nude mouse right side subcutaneous.Mice-transplanted tumor treats that with vernier caliper measurement transplanted tumor diameter tumor growth is to 100-200mm
3Rear animal random packet.Use the method for measuring the knurl footpath, dynamically observe the antineoplastic effect of tested animal.The measurement number of times of diameter of tumor is every 2 days 1 time, and each the measurement also needs the weighing mouse heavy simultaneously.The anti-VEGF-ANG2 dual specific of experimental group tail vein injection is anti-, and every day 1 time, negative group is given equivalent physiological saline simultaneously.The gross tumor volume calculation formula:
TV=0.52×a×b
2
Wherein a, b represent respectively length and width.Calculate relative tumour volume according to the result of measuring.The evaluation index of anti-tumor activity is relative tumor proliferation rate T/C (%), and calculation formula is as follows:
T/C(%)=T
RTV/C
RTV×100%
T
RTV: treatment group RTV; C
RTV: negative control group RTV
The anti-restraining effect to people's lung cancer H460 bare mouse different species Growth of Tumors Transplanted of the anti-VEGF-ANG2 dual specific of table 1.
Result: see Table 1, taxol group 10mg/kg is 70.05% to the tumour inhibiting rate of people's lung cancer H460 Nude Mice; RhEndostatin group 2.5mg/kg is 23.46% to the tumour inhibiting rate of people's lung cancer H460 Nude Mice; The inhibitory rate 75.88%, 60.64%, 34.32% of the anti-high, medium and low dosage group of anti-VEGF-ANG2 dual specific to people's lung cancer H460 Nude Mice.
Anti-VEGF-ANG2 dual specific is anti-to be shown people's lung cancer H460 Nude Mice growth inhibition test result, compare with negative control group, the anti-3mg/kg group of the anti-6mg/kg group of anti-VEGF-ANG2 dual specific and anti-VEGF-ANG2 dual specific has the restraining effect of significance to the growth of people's lung cancer H460 transplanted tumor.
Anti-VEGF-ANG2 dual specific is anti-to human esophagus cancer Ec109 nude mouse xenotransplantation tumor growth inhibition test
The tumor tissue of getting the growth animated period cuts into 1.5mm
3The left and right under aseptic condition, is inoculated in the nude mouse right side subcutaneous.Mice-transplanted tumor treats that with vernier caliper measurement transplanted tumor diameter tumor growth is to 100-200mm
3Rear animal random packet.Use the method for measuring the knurl footpath, dynamically observe the antineoplastic effect of tested animal.The measurement number of times of diameter of tumor is every 2 days 1 time, and each the measurement also needs the weighing mouse heavy simultaneously.The anti-VEGF-ANG2 dual specific of experimental group tail vein injection is anti-, and every day 1 time, negative group is given equivalent physiological saline simultaneously.The gross tumor volume calculation formula:
TV=0.52×a×b
2
Wherein a, b represent respectively length and width.Calculate relative tumour volume according to the result of measuring.The evaluation index of anti-tumor activity is relative tumor proliferation rate T/C (%), and calculation formula is as follows:
T/C(%)=T
RTV/C
RTV×100%
T
RTV: treatment group RTV; C
RTV: negative control group RTV
The anti-restraining effect to human esophagus cancer Ec109 bare mouse different species Growth of Tumors Transplanted of the anti-VEGF-ANG2 dual specific of table 2.
Result: see Table 2, taxol group 10mg/kg is 69.41% to the tumour inhibiting rate of human esophagus cancer Ec109 Nude Mice; RhEndostatin group 2.5mg/kg is 50.02% to the tumour inhibiting rate of human esophagus cancer Ec109 Nude Mice; The inhibitory rate 59.54%, 78.76%, 50.21%% of the anti-high, medium and low dosage group of anti-VEGF-ANG2 dual specific to human esophagus cancer Ec109 Nude Mice.
Anti-VEGF-ANG2 dual specific is anti-to be shown human esophagus cancer Ec109 Nude Mice growth inhibition test result, compare with negative control group, the anti-3mg/kg group of anti-VEGF-ANG2 dual specific has the restraining effect of significance to the growth of human esophagus cancer Ec109 transplanted tumor.
Anti-VEGF-ANG2 dual specific is anti-to human breast carcinoma MDA-MB-231 nude mouse xenotransplantation tumor growth inhibition test
The tumor tissue of getting the growth animated period cuts into 1.5mm
3The left and right under aseptic condition, is inoculated in the nude mouse right side subcutaneous.Mice-transplanted tumor treats that with vernier caliper measurement transplanted tumor diameter tumor growth is to 100-200mm
3Rear animal random packet.Use the method for measuring the knurl footpath, dynamically observe the antineoplastic effect of tested animal.The measurement number of times of diameter of tumor is every 2 days 1 time, and each the measurement also needs the weighing mouse heavy simultaneously.The anti-VEGF-ANG2 dual specific of experimental group tail vein injection is anti-, and every day 1 time, negative group is given equivalent physiological saline simultaneously.The gross tumor volume calculation formula:
TV=0.52×a×b
2
Wherein a, b represent respectively length and width.Calculate relative tumour volume according to the result of measuring.The evaluation index of anti-tumor activity is relative tumor proliferation rate T/C (%), and calculation formula is as follows:
T/C(%)=T
RTV/C
RTV×100%
T
RTV: treatment group RTV; C
RTV: negative control group RTV
The anti-restraining effect to human breast carcinoma MDA-MB-231 bare mouse different species Growth of Tumors Transplanted of the anti-VEGF-ANG2 dual specific of table 3.
Result: see Table 3, taxol group 10mg/kg is 75.29% to the tumour inhibiting rate of human breast carcinoma MDA-MB-231 Nude Mice; RhEndostatin group 2.5mg/kg is 65.45% to the tumour inhibiting rate of human breast carcinoma MDA-MB-231 Nude Mice; The inhibitory rate 70.71%, 81.57%, 45.57% of the anti-high, medium and low dosage group of anti-VEGF-ANG2 dual specific to human breast carcinoma MDA-MB-231 Nude Mice.
Anti-VEGF-ANG2 dual specific is anti-to be shown human breast carcinoma MDA-MB-231 Nude Mice growth inhibition test result, compare with negative control group, the anti-3mg/kg group of the anti-6mg/kg group of anti-VEGF-ANG2 dual specific and anti-VEGF-ANG2 dual specific all has the restraining effect of significance to the growth of human breast carcinoma MDA-MB-231 transplanted tumor.
Embodiment 6
Anti-VEGF-ANG2 dual specific is anti-to people's kidney A498 nude mouse xenotransplantation tumor growth inhibition test
People's kidney A498 cell strain of taking the logarithm vegetative period is prepared into 5 * 10 after under aseptic condition
7/ ml cell suspension is inoculated in nude mouse right side armpit with 0.1ml subcutaneous.Nude Mice treats that with vernier caliper measurement transplanted tumor diameter tumor growth is to 100-200mm
3After with the animal random packet.Use the method for measuring the knurl footpath, dynamically observe the antineoplastic effect of tested thing.The measurement number of times of diameter of tumor is survey in every 2 days 1 time.Administering mode all adopts tail vein injection.Negative control group injection equivalent physiological saline, every day 1 time; Taxol group 10mg/kg, administration is 1 time weekly; RhEndostatin group 2.5mg/kg, administration every day 1 time; Anti-VEGF-ANG2 dual specific resists high, normal, basic group respectively with 6,3,1.5mg/kg, administration every day 1 time.After administration finished, mouse was put to death, and operation strips the knurl piece and weighs.
The anti-restraining effect to people's kidney A498 bare mouse different species Growth of Tumors Transplanted of the anti-VEGF-ANG2 dual specific of table 4.
Result: see Table 4, taxol group 10mg/kg is 85.53% to the tumour inhibiting rate of people's kidney A498 Nude Mice; RhEndostatin group 2.5mg/kg is 32.57% to the tumour inhibiting rate of people's kidney A498 Nude Mice; The inhibitory rate 71.49%, 65.17%, 57.33% of the anti-high, medium and low dosage group of anti-VEGF-ANG2 dual specific to people's kidney A498 Nude Mice.
Anti-VEGF-ANG2 dual specific is anti-to be shown people's kidney A498 Nude Mice growth inhibition test result, compare with negative control group, the anti-3mg/kg group of the anti-6mg/kg group of anti-VEGF-ANG2 dual specific and anti-VEGF-ANG2 dual specific has the restraining effect of significance to the growth of people's kidney A498 transplanted tumor.
Embodiment 7
Anti-VEGF-ANG2 dual specific is anti-to Human gallbladder carcinoma GBC-SD nude mouse xenotransplantation tumor growth inhibition test
The Human gallbladder carcinoma GBC-SD cell strain of taking the logarithm vegetative period is prepared into 5 * 10 after under aseptic condition
7/ ml cell suspension is inoculated in nude mouse right side armpit with 0.1ml subcutaneous.Nude Mice treats that with vernier caliper measurement transplanted tumor diameter tumor growth is to 100-200mm
3After with the animal random packet.Use the method for measuring the knurl footpath, dynamically observe the antineoplastic effect of tested thing.The measurement number of times of diameter of tumor is survey in every 2 days 1 time.Administering mode all adopts tail vein injection.Negative control group injection equivalent physiological saline, every day 1 time; Taxol group 10mg/kg, administration is 1 time weekly; RhEndostatin group 2.5mg/kg, administration every day 1 time; Anti-VEGF-ANG2 dual specific resists high, normal, basic group respectively with 6,3,1.5mg/kg, administration every day 1 time.After administration finished, mouse was put to death, and operation strips the knurl piece and weighs.
The anti-restraining effect to Human gallbladder carcinoma GBC-SD bare mouse different species Growth of Tumors Transplanted of the anti-VEGF-ANG2 dual specific of table 5.
Result: see Table 5, taxol group 10mg/kg is 76.75% to the tumour inhibiting rate of Human gallbladder carcinoma GBC-SD Nude Mice; RhEndostatin group 2.5mg/kg, the tumour inhibiting rate that Human gallbladder carcinoma GBC-SD nude mouse is suppressed knurl is 28.53%; The anti-high, medium and low dosage group of anti-VEGF-ANG2 dual specific is 58.21%, 65.80%, 54.85% to the tumour inhibiting rate of Human gallbladder carcinoma GBC-SD Nude Mice.
Anti-VEGF-ANG2 dual specific resists the test-results to the tumour inhibiting rate of Human gallbladder carcinoma GBC-SD mice-transplanted tumor to show, compare with negative control group, the anti-3mg/kg group of anti-VEGF-ANG2 dual specific is the most obvious to the growth-inhibiting effect of Human gallbladder carcinoma GBC-SD transplanted tumor.
Embodiment 8
The anti-inhibition test to human colon carcinoma HT-29 nude mouse xenotransplantation tumor growth of anti-VEGF-ANG2 dual specific
The human colon carcinoma HT-29 cell strain of taking the logarithm vegetative period is prepared into 5 * 10 after under aseptic condition
7/ ml cell suspension is inoculated in nude mouse right side armpit with 0.1ml subcutaneous.Nude Mice treats that with vernier caliper measurement transplanted tumor diameter tumor growth is to 100-200mm
3After with the animal random packet.Use the method for measuring the knurl footpath, dynamically observe the antineoplastic effect of tested thing.The measurement number of times of diameter of tumor is survey in every 2 days 1 time.Administering mode all adopts tail vein injection.Negative control group injection equivalent physiological saline, every day 1 time; Taxol group 10mg/kg, administration is 1 time weekly; RhEndostatin group 2.5mg/kg, administration every day 1 time; Anti-VEGF-ANG2 dual specific resists high, normal, basic group respectively with 6,3,1.5mg/kg, administration every day 1 time.After administration finished, mouse was put to death, and operation strips the knurl piece and weighs.
The anti-restraining effect to human colon carcinoma HT-29 bare mouse different species Growth of Tumors Transplanted of the anti-VEGF-ANG2 dual specific of table 6
Result: see Table 6, taxol group 10mg/kg is 67.43% to the tumour inhibiting rate of human colon carcinoma HT-29 Nude Mice; RhEndostatin group 2.5mg/kg is 32.66% to the tumour inhibiting rate of human colon carcinoma HT-29 Nude Mice; The anti-high, medium and low dosage group of anti-VEGF-ANG2 dual specific is 40.74%, 62.04%, 51.33% to the tumour inhibiting rate of human colon carcinoma HT-29 Nude Mice
Therefore, anti-VEGF-ANG2 dual specific is anti-to be shown human colon carcinoma HT-29 Nude Mice growth inhibition test result, compare with negative control group, the anti-3mg/kg group of anti-VEGF-ANG2 dual specific has the restraining effect of significance to the growth of human colon carcinoma HT-29 transplanted tumor.
Embodiment 9
The anti-inhibition test to human ovarian cancer SK-OV-3 nude mouse xenotransplantation tumor growth of anti-VEGF-ANG2 dual specific
The human ovarian cancer SK-OV-3 cell strain of taking the logarithm vegetative period is prepared into 5 * 10 after under aseptic condition
7/ ml cell suspension is inoculated in nude mouse right side armpit with 0.1ml subcutaneous.Nude Mice treats that with vernier caliper measurement transplanted tumor diameter tumor growth is to 100-200mm
3After with the animal random packet.Use the method for measuring the knurl footpath, dynamically observe the antineoplastic effect of tested thing.The measurement number of times of diameter of tumor is survey in every 2 days 1 time.Administering mode all adopts tail vein injection.Negative control group injection equivalent physiological saline, every day 1 time; Cis-platinum group 10mg/kg, administration is 1 time weekly; RhEndostatin group 2.5mg/kg, administration every day 1 time; Anti-VEGF-ANG2 dual specific resists high, normal, basic group respectively with 6,3,1.5mg/kg, administration every day 1 time.After administration finished, mouse was put to death, and operation strips the knurl piece and weighs.
The anti-restraining effect to human ovarian cancer SK-OV-3 bare mouse different species Growth of Tumors Transplanted of the anti-VEGF-ANG2 dual specific of table 7.
Result: see Table 7, cis-platinum group 10mg/kg is 75.43% to the tumour inhibiting rate of human ovarian cancer SK-OV-3 Nude Mice; RhEndostatin group 2.5mg/kg is 22.62% to the tumour inhibiting rate of human ovarian cancer SK-OV-3 Nude Mice; The anti-high, medium and low dosage group of anti-VEGF-ANG2 dual specific is 59.59%, 70.12%, 50.08% to the tumour inhibiting rate of human ovarian cancer SK-OV-3 Nude Mice.
Therefore, anti-VEGF-ANG2 dual specific is anti-to be shown human ovarian cancer SK-OV-3 Nude Mice growth inhibition test result, compare with negative control group, the anti-3mg/kg group of anti-VEGF-ANG2 dual specific has the restraining effect of significance to the growth of human ovarian cancer SK-OV-3 transplanted tumor, the anti-6mg/kg group of anti-VEGF-ANG2 dual specific also has certain restraining effect to the growth of human ovarian cancer SK-OV-3 transplanted tumor.
Embodiment 10
The anti-inhibition test to human cervical carcinoma HeLa nude mouse xenotransplantation tumor growth of anti-VEGF-ANG2 dual specific
The HeLa Cells strain of taking the logarithm vegetative period is prepared into 5 * 10 after under aseptic condition
7/ ml cell suspension is inoculated in nude mouse right side armpit with 0.1ml subcutaneous.Nude Mice treats that with vernier caliper measurement transplanted tumor diameter tumor growth is to 100-200mm
3After with the animal random packet.Use the method for measuring the knurl footpath, dynamically observe the antineoplastic effect of tested thing.The measurement number of times of diameter of tumor is survey in every 2 days 1 time.Administering mode all adopts tail vein injection.Negative control group injection equivalent physiological saline, every day 1 time; Taxol group 10mg/kg, administration is 1 time weekly; RhEndostatin group 2.5mg/kg, administration every day 1 time; Anti-VEGF-ANG2 dual specific resists high, normal, basic group respectively with 6,3,1.5mg/kg, administration every day 1 time.After administration finished, mouse was put to death, and operation strips the knurl piece and weighs.
The anti-restraining effect to human cervical carcinoma HeLa bare mouse different species Growth of Tumors Transplanted of the anti-VEGF-ANG2 dual specific of table 8.
Result: see Table 8, taxol group 10mg/kg is 65.34% to the tumour inhibiting rate of human cervical carcinoma HeLa Nude Mice; RhEndostatin group 2.5mg/kg is 52.42% to the tumour inhibiting rate of human cervical carcinoma HeLa Nude Mice; The anti-high, medium and low dosage group of anti-VEGF-ANG2 dual specific is 40.76%, 72.04%, 51.86% to the tumour inhibiting rate of human cervical carcinoma HeLa Nude Mice
Therefore, anti-VEGF-ANG2 dual specific is anti-to be shown human cervical carcinoma HeLa Nude Mice growth inhibition test result, compare with negative control group, the anti-3mg/kg group of anti-VEGF-ANG2 dual specific has the restraining effect of significance to the growth of human cervical carcinoma HeLa transplanted tumor.
Embodiment 11
The anti-inhibition test to human prostata cancer DU-145 nude mouse xenotransplantation tumor growth of anti-VEGF-ANG2 dual specific
The human prostata cancer DU-145 cell strain of taking the logarithm vegetative period is prepared into 5 * 107/ml cell suspension after under aseptic condition, is inoculated in nude mouse right side armpit with 0.1ml subcutaneous.Nude Mice is with vernier caliper measurement transplanted tumor diameter, until tumor growth to the 100-200mm3 with the animal random packet.Use the method for measuring the knurl footpath, dynamically observe the antineoplastic effect of tested thing.The measurement number of times of diameter of tumor is survey in every 2 days 1 time.Administering mode all adopts tail vein injection.Negative control group injection equivalent physiological saline, every day 1 time; Cis-platinum group 10mg/kg, administration is 1 time weekly; RhEndostatin group 2.5mg/kg, administration every day 1 time; Anti-VEGF-ANG2 dual specific resists high, normal, basic group respectively with 6,3,1.5mg/kg, administration every day 1 time.After administration finished, mouse was put to death, and operation strips the knurl piece and weighs.
The anti-restraining effect to human prostata cancer DU-145 bare mouse different species Growth of Tumors Transplanted of the anti-VEGF-ANG2 dual specific of table 9.
Result: see Table 9, cis-platinum group 10mg/kg is 71.38% to the tumour inhibiting rate of human prostata cancer DU-145 Nude Mice; RhEndostatin group 2.5mg/kg is 21.30% to the tumour inhibiting rate of human prostata cancer DU-145 Nude Mice; The anti-high, medium and low dosage group of anti-VEGF-ANG2 dual specific is 46.24%, 65.72%, 56.38% to the tumour inhibiting rate of human prostata cancer DU-145 Nude Mice.
Therefore, anti-VEGF-ANG2 dual specific is anti-to be shown human prostata cancer DU-145 Nude Mice growth inhibition test result, compare with negative control group, the anti-3mg/kg group of anti-VEGF-ANG2 dual specific has the restraining effect of significance to the growth of human prostata cancer DU-145 transplanted tumor.
Embodiment 12
The anti-inhibition test to human bladder cancer HT1376 nude mouse xenotransplantation tumor growth of anti-VEGF-ANG2 dual specific
The human bladder cancer HT1376 cell strain of taking the logarithm vegetative period is prepared into 5 * 10 after under aseptic condition
7/ ml cell suspension is inoculated in nude mouse right side armpit with 0.1ml subcutaneous.Nude Mice treats that with vernier caliper measurement transplanted tumor diameter tumor growth is to 100-200mm
3After with the animal random packet.Use the method for measuring the knurl footpath, dynamically observe the antineoplastic effect of tested thing.The measurement number of times of diameter of tumor is survey in every 2 days 1 time.Administering mode all adopts tail vein injection.Negative control group injection equivalent physiological saline, every day 1 time; Taxol group 10mg/kg, administration is 1 time weekly; RhEndostatin group 2.5mg/kg, administration every day 1 time; Anti-VEGF-ANG2 dual specific resists high, normal, basic group respectively with 6,3,1.5mg/kg, administration every day 1 time.After administration finished, mouse was put to death, and operation strips the knurl piece and weighs.
The anti-restraining effect to human bladder cancer HT1376 bare mouse different species Growth of Tumors Transplanted of the anti-VEGF-ANG2 dual specific of table 10.
Result: see Table 10, taxol group 10mg/kg is 67.58 to the tumour inhibiting rate of human bladder cancer HT1376 Nude Mice; RhEndostatin group 2.5mg/kg is 32.42% to the tumour inhibiting rate of human bladder cancer HT1376 Nude Mice; The anti-high, medium and low dosage group of anti-VEGF-ANG2 dual specific is 40.70%, 62.03%, 51.80% to the tumour inhibiting rate of human bladder cancer HT1376 Nude Mice.
Therefore, anti-VEGF-ANG2 dual specific is anti-to be shown human bladder cancer HT1376 Nude Mice growth inhibition test result, compare with negative control group, the anti-3mg/kg group of anti-VEGF-ANG2 dual specific has the restraining effect of significance to the growth of human bladder cancer HT1376 transplanted tumor.
SEQUENCE LISTING
＜110〉Changzhou Adam Bioisystech Co., Ltd
＜120〉a kind of anti-VEGF/ANG2 bi-specific antibody and application thereof
<130>
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 219
<212> PRT
＜213〉artificial sequence
<400> 1
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe
50 55 60
Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Pro
115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
210 215
<210> 2
<211> 213
<212> PRT
＜213〉artificial sequence
<400> 2
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile
35 40 45
Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Thr
115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
Asn Arg Gly Glu Cys
210
<210> 3
<211> 222
<212> PRT
＜213〉artificial sequence
<400> 3
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
1 5 10 15
Leu Phe Pro Pro Leu Pro Leu Asp Thr Leu Met Ile Ser Arg Thr Pro
20 25 30
Gly Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
35 40 45
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
50 55 60
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
65 70 75 80
Leu Thr Val Leu His Gln Asp Trp Leu Gln Gly Lys Glu Tyr Lys Cys
85 90 95
Lys Val Ser Gln Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
100 105 110
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
115 120 125
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
130 135 140
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
145 150 155 160
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
165 170 175
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
180 185 190
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
195 200 205
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
210 215 220
<210> 4
<211> 120
<212> PRT
＜213〉artificial sequence
<400> 4
Glu Val Glx Leu Val Glx Ser Gly Gly Gly Val Val Glx Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Gly Met His Trp Val Arg Glx Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Pro Thr Ile Ser Arg Asp Asx Ala Lys Asx Ser Leu Tyr
65 70 75 80
Leu Asx Met Asx Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Leu Leu Asp Tyr Asp Ile Leu Thr Gly Tyr Gly Tyr Trp
100 105 110
Gly Glx Gly Thr Leu Val Thr Val
115 120
<210> 5
<211> 109
<212> PRT
＜213〉artificial sequence
<400> 5
Asp Ile Val Met Thr Glx Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Glx Ser Leu Leu His Ser
20 25 30
Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Glx Lys Pro Gly Glx Ser
35 40 45
Pro Glx Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Pro Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Glx Gly
85 90 95
Thr His Trp Pro Pro Thr Phe Gly Glx Gly Thr Lys Leu
100 105
Claims (2)
1. an anti-VEGF/ANG2 bi-specific antibody, is characterized in that this antibody is the basis by anti-VEGF monoclonal antibody, and connect anti-ANG2 single-chain antibody formation at its FC section end by (GGGGS) 3; Described anti-VEGF monoclonal antibody is comprised of Anti-VEGF A Fab VH-CH, Anti-VEGF A Fab VL-CL and Fc; Wherein the aminoacid sequence table of Anti-VEGF A Fab VH-CH is that the aminoacid sequence table of SEQ NO.1, Anti-VEGF A Fab VL-CL is the aminoacid sequence table SEQ NO.3 of SEQ NO.2, Fc; Described anti-ANG2 single-chain antibody passes through (GGGGS) by Anti-ANG2 scFv VL and Anti-ANG2 scFv VH
3Composition is formed by connecting; Wherein the aminoacid sequence table of Anti-ANG2 scFv VL is that the aminoacid sequence table of SEQ NO.4, Anti-ANG2 scFv VH is SEQ NO.5.
2. the application of anti-VEGF/ANG2 bi-specific antibody according to claim 1 in preparation medicine for treating tumor thing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110160558 CN102250247B (en) | 2011-06-15 | 2011-06-15 | Bispecific antibody to VEGF/ANG2 and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110160558 CN102250247B (en) | 2011-06-15 | 2011-06-15 | Bispecific antibody to VEGF/ANG2 and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102250247A CN102250247A (en) | 2011-11-23 |
| CN102250247B true CN102250247B (en) | 2013-06-19 |
Family
ID=44977845
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110160558 Active CN102250247B (en) | 2011-06-15 | 2011-06-15 | Bispecific antibody to VEGF/ANG2 and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102250247B (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250248A (en) * | 2011-06-15 | 2011-11-23 | 常州亚当生物技术有限公司 | Anti-VEGF/ANG2 bispecific antibody, and application thereof |
| HRP20211641T1 (en) | 2012-07-13 | 2022-02-04 | Roche Glycart Ag | Bispecific anti-vegf/anti-ang-2 antibodies and their use in the treatment of ocular vascular diseases |
| ES2649991T3 (en) * | 2012-11-19 | 2018-01-16 | Baliopharm Ag | Recombinant bispecific antibody that binds to CD20 and CD95 |
| SG11201609168PA (en) * | 2014-05-07 | 2016-12-29 | Medimmune Llc | Methods of using anti-ang2 antibodies |
| SG11201704741PA (en) * | 2014-12-22 | 2017-07-28 | Systimmune Inc | Bispecific tetravalent antibodies and methods of makiing and using thereof |
| US20220185875A1 (en) | 2019-03-18 | 2022-06-16 | Jiangsu Hengrui Medicine Co., Ltd. | Bispecific antibody specifically bound to vegf and ang2 |
| CN112168960A (en) * | 2019-07-03 | 2021-01-05 | 义慧科技(深圳)有限公司 | Use of anti-VEGF antibody and anti-ANG2 antibody for the preparation of a medicament for the treatment of tumors with Ras mutations |
| US20230312698A1 (en) * | 2020-08-07 | 2023-10-05 | Nanjing GenScript Biotech Co., Ltd. | Antibodies against human angiopoietin-2 and uses thereof |
| WO2022057888A1 (en) * | 2020-09-17 | 2022-03-24 | 江苏恒瑞医药股份有限公司 | Bispecific antigen binding molecule specifically binding to vegf and ang-2 |
-
2011
- 2011-06-15 CN CN 201110160558 patent/CN102250247B/en active Active
Non-Patent Citations (6)
| Title |
|---|
| 双特异性抗体及其在肿瘤临床上的应用;许元富等;《科学通报》;20001231;第45卷(第17期);1808-1815 * |
| 双特异性抗体的制备和应用;徐晨;《国外医学免疫学分册》;19991231;第22卷(第4期);184-188 * |
| 徐晨.双特异性抗体的制备和应用.《国外医学免疫学分册》.1999,第22卷(第4期),184-188. |
| 抗人喉癌/抗VEGF双特异性单克隆抗体的制备及初步鉴定;盛力;《中国优秀硕士学位论文全文数据库医药卫生科技辑》;20041231;E072-798 * |
| 盛力.抗人喉癌/抗VEGF双特异性单克隆抗体的制备及初步鉴定.《中国优秀硕士学位论文全文数据库医药卫生科技辑》.2004,E072-798. |
| 许元富等.双特异性抗体及其在肿瘤临床上的应用.《科学通报》.2000,第45卷(第17期),1808-1815. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102250247A (en) | 2011-11-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102250247B (en) | Bispecific antibody to VEGF/ANG2 and application thereof | |
| CN104870476B (en) | Target double targeting antibodies of VEGFR 2 and DLL4 and include its pharmaceutical composition | |
| CN102250246A (en) | Bispecific antibody to VEGF/PDGFR beta and application thereof | |
| ES2523457T3 (en) | Antibodies against vascular endothelial growth factor receptor 1 | |
| EA025148B1 (en) | BISPECIFIC BINDING MOLECULES BINDING TO VEGF AND Ang2 | |
| ES2718399T3 (en) | Molecules that attach to human bispecific EGFRVIII antibodies | |
| CN105263961B (en) | For preventing, diagnosing or the anti-VEGF antibody for the treatment of cancer or angiogenesis related disease and pharmaceutical composition containing the antibody | |
| AU2013271428B2 (en) | Human bispecific EGFRvIII antibody engaging molecules | |
| CN104225594A (en) | HER2-resistant humanized antibody and relevant tumor-resistant composition thereof | |
| US10280218B2 (en) | Antibodies binding to BMP4, and uses thereof | |
| Zhang et al. | Suppression of angiogenesis and tumor growth in vitro and in vivo using an anti‑angiopoietin‑2 single‑chain antibody | |
| CN112961250B (en) | Antibody fusion proteins and uses thereof | |
| CN102250249A (en) | Anti-VEGF/PDGFR (Vascular Endothelial Growth Factor/Platelet-Derived Growth Factor Receptor) beta double-specificity antibody and application thereof | |
| Li et al. | A novel multifunctional anti-PD-L1-CD16a-IL15 induces potent cancer cell killing in PD-L1-positive tumour cells | |
| CN102250248A (en) | Anti-VEGF/ANG2 bispecific antibody, and application thereof | |
| WO2023125842A1 (en) | Development of novel upar single-domain antibody | |
| WO2013007053A1 (en) | Fully human antibodies against human vegf | |
| WO2021083248A1 (en) | Anti-tspan8 monoclonal antibody and use thereof | |
| CN110627905B (en) | VEGF (vascular endothelial growth factor) -EGFR (epidermal growth factor receptor) -targeted bifunctional fusion protein and application thereof | |
| WO2016059057A1 (en) | Vegfr-2 binding polypeptides | |
| CN112830968B (en) | anti-VEGF single domain antibody, humanization thereof, fusion protein constructed by single domain antibody and IgG1-Fc and application | |
| EP3082858A1 (en) | Adrenomedullin binder for use in therapy of cancer | |
| CN110004167A (en) | Target VEGFR-2 and/or VEGFR-3 Chimeric antigen receptor and its application | |
| CN102276723A (en) | Anti-vascular endothelial growth factor (VEGF) monoclonal antibody Vancolizumab and application thereof | |
| CN109529040A (en) | LGR4 and R-spondin binding inhibitors and its purposes in oncotherapy |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |