CN102250249A

CN102250249A - Anti-VEGF/PDGFR (Vascular Endothelial Growth Factor/Platelet-Derived Growth Factor Receptor) beta double-specificity antibody and application thereof

Info

Publication number: CN102250249A
Application number: CN2011101648056A
Authority: CN
Inventors: 霍世元; 叶亚东; 滕凌; 朱文华; 潘鹂; 路易斯易格那罗
Original assignee: CHANGZHOU ADAM BIOTECH Inc
Current assignee: CHANGZHOU ADAM BIOTECH Inc
Priority date: 2011-06-18
Filing date: 2011-06-18
Publication date: 2011-11-23

Abstract

The invention relates to a double-specificity monoclonal antibody medicament, in particular to an anti-vascular endothelial growth factor (VEGF/VEGF-A) and anti-platelet-derived growth factor receptor (PDGFR) double-specificity monoclonal antibody medicament for resisting tumor angiogenesis; and the anti-VEGF/PDGFR beta double-specificity antibody is characterized by being constructed by connecting an anti-PDGFR beta single-chain antibody at the tail end of an FC segment based on an anti-VEGF monoclonal antibody.

Description

A kind of anti-VEGF/PDGFRbeta bi-specific antibody and application thereof

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 human blood platelets derived growth factor receptor (PDGFR);

Background technology

Before century more than one, just there is bibliographical information to cross tumor growth and is accompanied by new vessel generation (Ferrara 2002).But up to nineteen thirty-nine, just proposed first by Ide and colleague thereof, the angiogenic growth stimulating factor that may exist certain tumour source provides vascularity (Ide et al.1939) for growth of tumor.After several years, can be owing to observe increasing of vessel density prior to the quick growth of tumour, 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, the experiment of Greenblatt, Shubik (Greenblatt et al.1968) and two research groups of Ehrmann, Knoth (Ehrmann et al.1968) provides Prima Facie Evidence in succession, but confirms that the vasculogenesis of tumour is some the spreading factor mediation that is produced by tumour cell.

1971, U.S. scientist Volkmann (Judah Folkman) proposed in " New England Journal of Medicine ", and angiogenesis inhibitor may be a kind of effective anticancer means (Folkman 1971).From the seventies in early days, based on this prospective hypothesis, 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 (Gullino 1978) that vasculogenesis can be avoided cancer.Subsequently, the multiple blood vessel source factor (as, Urogastron EGF, transforming growth factor TGF-α, TGF-beta, 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 (Ferrara 2002).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), VEGF-C (Joukov et al.1996), VEGF-D (Orlandini et al.1996) at present.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 tangible chemical chemotactic and short splitting action, and is relevant with blood vessel island, vascularization and hematopoiesis.

Growth of tumor has two visibly different stages, and promptly 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 not 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 rapid process of multistep of tumour generation Invasion and Metastasis, vasculogenesis is all being brought into play important effect.

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).Duo Jia uses in the laboratory means of multiple anti-VEGF all to realize the inhibition of tumor growth, these methods comprise: at 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 endogenous antibody of VEGF, and in experiment in vitro, the mouse endogenous 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 endogenous antibody, Ferrara changes the part of being human antibody IgG1 with the skeleton of mouse source antibody, the Genentech company of being born thus " cookle level " medicine--rhuMAb-VEGF (Avastin).It is the humanized antibody (IgG1) of a kind of anti-VEGF, 93% people's source structure territory and 7% mouse source calmodulin binding domain CaM are formed, 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 to treat transitivity colorectal carcinoma (mCRC), minicell molecule lung cancer (GBM) and transitivity kidney.A Wasiting is different from existing cancer therapy drug, is drug target with VEGF, and the specific binding capacity of antibody makes 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 A Wasiting.The monoclonal antibody Cetuximab (Cetuximab) of ASCO meeting Souglakos report Avastin associating targeting EGFR in 2007 can treat the transitivity colorectal cancer 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 in research with after the whole antibody molecule I gG cutting, earlier with the albumen procaryotic cell expression, and then it is connected to IgG by engineered means.

Except cancer, VEGF also is that treatment comprises senile macular degeneration SMD (AMD), and the retinopathy that diabetes cause is in interior multiple ophthalmic diseases key.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 another medicine thus--the blue Buddhist nun's monoclonal antibody (Lucentis) of twin sister of Avastin.2006, blue Buddhist nun's monoclonal antibody is used for the treatment of age-related macular degeneration by the approval of U.S. Bureau of Drugs Supervision, and (age-related macular degeneration AMD), becomes the treatment senile macular degeneration SMD soon, the drug of first choice of the retinopathy that diabetes cause captures the share of North America market more than 80%.

Thr6 PDGF BB and acceptor thereof

Thr6 PDGF BB (Platelet-derived growth factor, PDGF) be ubiquitous a kind of somatomedin by sis proto-oncogene coding, from thrombocyte α, separate at first and obtain, mainly synthetic by megalokaryocyte, in the tissue injury reparation, play important effect (Heldin 1992) in vasculogenesis and the cell differentiation procedure.The PDGF molecule is a glycoprotein dimer, can be divided into-AA ,-BB and-three types of AB.PDGF and acceptor thereof (Platelet-derived growth factor receptor PDGFR) has in multiple human tumor than higher expression, as carcinoma of the pancreas, and small cell lung cancer, cancer of the stomach and mammary cancer etc.

PDGFR is that (Receptor tyrosine kinase RTK), can be divided into two hypotypes to a kind of receptor tyrosine kinase that is positioned at cell surface: PDGFR α and PDGFRbeta (Matsui et al.1989).PDGFRbeta can be specific with PDGF-BB or-AB combines (Herdaran et al.1991) and is activated.After being activated, can form dimer between two PDGFR, and forward path to, to realize the function of its regulating cell differential growth by the signal in autophosphorylation activation downstream.

In recent years, at the target medicinal of VEGF treatment cancer and senile macular degeneration SMD (AMD) have been obtained the curative effect (Ferrara et al.2007) of highly significant.But still there are many deficiencies in this targeted therapy at VEGF, also has a lot of improved spaces.The patient who for example has has a kind of intrinsic resistance to this therapy, the other cancer patient is after treatment, obtained curative effect preferably in a short time, but this curative effect can only be kept the relatively shorter time, tumour (the Jain et al.2005 that grows again afterwards, Shojaei et al.2007, Gaur et al.2009).For this resistance to the VEGF targeted therapy, another viewpoint thinks that the secreted PDGFRbeta of new vessel pericyte (pericytes) has played important effect (Abramsson et al.2003 therein, Mancuso et al.2006, Bergers et al.2008).

Anti-VEGF/PDGFRbeta bi-specific antibody

The evidence that obtained of cancer cells model and the mouse model cultivated of chamber shows by experiment, the common function ratio that suppresses VEGF/VEGFR and PDGF/PDGFR separately at VEGF/VEGFR can more effective inhibition tumor tissues in growth (the Bergers et al.2003 of new vessel, Erber et al.2004, Pietras et al.2005, Jo et al.2006, Shen et al.2007).At present, some small molecules receptor tyrosine kinase inhibitors (tyrosine kinase inhibitors, TKIs) be proved can be used in the treatment cancer (Hiles et al.2008).These inhibitor can combine with kinases in a lot of cells that comprise VEGFR and PDGFR, suppress its function, thereby reach the purpose (Fabian et al.2005) of treatment cancer.But these micromolecular inhibitors have certain toxicity to human body, when particularly it combines with chemotherapy, and toxicity bigger (Sosman et al.2007, Roodhart et al.2008).Compare inhibitor therapy, use singlely, the humanized antibody with anti-VEGF and PDGF signal path dual specific has bigger advantage and brighter prospect as antitumor drug.The bi-specific antibody that this patent relates to is technique means such as utilization genetically engineered, and identification VEGF is structured in the same antibody molecule with the antibody fragment of identification PDGFRbeta, can specificly combine with the two.Its effect that suppresses the tumor tissues angiogenesis obviously is better than using separately VEGF antibody.

Reference:

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Summary of the invention

Goal of the invention

The invention provides anti-VEGF-PDGFR bi-specific antibody, technique means such as its utilization genetically engineered, the antibody fragment of identification VEGF and identification PDGFRbeta is structured in the same antibody molecule, can specificly combine with the two, its effect that suppresses the tumor tissues angiogenesis obviously is better than using separately VEGF antibody.

Technical scheme

Anti-VEGF/PDGFRbeta bi-specific antibody is characterized in that this antibody is connected with in turn by the N end of SEQ NO.5

SEQ NO.4, SEQ NO.3, SEQ NO.2 and SEQ NO.1, wherein the C in Fc district end is by (GGGGS) ₃Sealing.

The application of anti-VEGF/PDGFRbeta bi-specific antibody in preparation medicine for treating tumor thing.

Aminoacid sequence table SEQ NO.1 is anti-PDGFRbeta light chain Fab district (Anti-PDGFRbeta Fab VL-CL) specifically; Aminoacid sequence table SEQ NO.2 is that anti-PDGFRbeta heavy chain Fab district (Anti-PDGFRbeta Fab VH-CH) aminoacid sequence table SEQ NO.3 is anti-VEGF A heavy chain Fab district (Anti-VEGF A Fab VH-CH); Aminoacid sequence table SEQ NO.4 is anti-VEGF A light chain Fab district (Anti-VEGF A Fab VL-CL); Aminoacid sequence table SEQ NO.5 is Fc;

Beneficial effect

1, the bi-specific antibody that relates to of this patent is technique means such as utilization genetically engineered, the antibody fragment of identification VEGF and identification PDGFR β is structured in the same antibody molecule, can specificly combine with the two, its effect that suppresses the tumor tissues angiogenesis obviously is better than using VEGF antibody separately, and has the fine tumor promotion of seeing.

2, the bi-specific antibody that relates to of this patent can specificity in conjunction with VEGF-A and PDGFR beta molecule, suppress the biological function that it stimulates new vessel growth.Experiment shows, this bi-specific antibody is approximate in conjunction with the ability of VEGF-A or PDGFR β and anti-VEGF-A or anti-PDGFR β monoclonal antibody separately, and it has very strong the time and these two kinds of antigen bonded abilities.Experiment shows, suppress VEGF-A and PDGFR β simultaneously and can better suppress tumor vascular growth than suppressing separately a kind of among both, therefore use this bi-specific antibody can effectively suppress the growth of tumor tissues, the increase of dosage in the time of avoiding again using two kinds of monoclonal antibodies simultaneously, the unpredictable risk of bringing to the patient.

3, anti-VEGF-PDGFR beta bi-specific antibody all produces effect to kinds of tumors, is the antitumor drug of wide spectrum.

Description of drawings

Fig. 1. anti-VEGF-PDGFR beta bi-specific antibody structural representation.；

Fig. 2 .Anti-VEGF-PDGFRbeta bi-specific antibody heavy chain expression carrier;

Fig. 3 .Anti-VEGF light chain of antibody expression vector;

Fig. 4, Anti-PDGFR beta light chain of antibody expression vector

Fig. 5 .ELISA measures antibody and antigen VEGF specific binding capacity.BsAb: anti-VEGF-PDGFR beta bi-specific antibody; The anti-VEGF eye is bright: anti-VEGF Fab segment; Anti-PDGFR beta: anti-PDGFR beta monoclonal antibody.

Fig. 6 .ELISA measures antibody and antigen PDGFR beta specific binding capacity.BsAb: anti-VEGF-PDGFR beta bi-specific antibody; The anti-VEGF eye is bright: anti-VEGF Fab segment; Anti-PDGFR beta: anti-PDGFR beta monoclonal antibody.

Fig. 7 .ELISA measures antibody while and antigen VEGF and PDGFR beta specific binding capacity.BsAb: anti-VEGF-PDGFR beta bi-specific antibody; The anti-VEGF eye is bright: anti-VEGF Fab segment; Anti-PDGFR beta: anti-PDGFR beta mono-clonal

Fig. 8 .HUVECs and HBVP cultivate altogether, test anti-VEGF-PDGFR beta bsAb to vascular endothelial cell growth, and endotheliocyte-pericyte bonded restraining effect: A.Bevacizumab, Bevacizumab+anti PDGFR beta and anti-VEGF-PDGFR beta bsAb (being all 25nM) are to the inhibition effect of new vessel growth; B. the Bevacizumab of different concns and anti-VEGF-PDGFR beta bsAb are to the inhibition effect (p＜0.01) of new vessel growth.

Fig. 9. the reorganization bispecific monoclonal antibody is expressed and production scheme in mammalian cell.After the expression vector that builds changes cell over to, in 96 orifice plates, cultivate the cell strain of screening stably express.The cell strain that screening obtains is amplification culture step by step, carries out scale operation at last in bio-reactor.

Embodiment

Embodiment 1

1. anti-VEGF-PDGFRbeta bi-specific antibody (bsAb) nucleotide sequence design and synthetic:

As shown in Figure 1, according to the heavy chain HC of this bsAb _VEGF-(GGGGS) ₃-HC _PDGFRbetaThe aminoacid sequence of (oppositely) and type of attachment design heavy chain nucleotide sequence, and at this sequence 5 ' end adding Sac I restriction enzyme site, 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, synthetic respectively heavy chain HC _VEGF-(GGGGS) ₃And HC _PDGFRbeta(oppositely) segment is used the bsAb heavy chain gene that the direct connection method of PCR (SDL PCR) is synthesized total length 2658bp.

According to people's anti-VEGF-A IgG1 light-chain amino acid sequence and the anti-PDGFRbeta IgG1 of people light-chain amino acid sequence, 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 and the anti-PDGFRbeta light chain gene of people of total length 657bp.

2. the structure of anti-VEGF-PDGFRbeta bsAb expression vector:

This bsAb heavy chain gene with Sac I and Xho I double digestion, with light chain gene Not I, behind the 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.

The expression vector (see figure 2) that contains heavy chain gene is then changed over to BL21 competence coli strain simultaneously with the expression vector (all comprising the phoA promotor) that contains light chain gene (anti-VEGF or anti-PDGFRbeta) (seeing Fig. 3,4).BL21 bacterial strain after the 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 changes 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, and acquisition contains the expression vector of heavy chain and light chain.

3. the expression of anti-VEGF-ANG2 bsAb and separation and purification

The electroporation cotransfection Flp-In of two expression vectors behind the purifying ^TMChinese hamster ovary celI (Invitrogen) is cultivated at the condition low suspension that 500ug/ml Xin Meisu (neo) exists, the strain of screening positive cell.

Recombinant antibodies separates the purifying molecules amount with affinity chromatography (Protein A) with sieve chromatography be complete bi-specific antibody molecule about 200kDa.Each component after the purification is identified by the SDS-PAGE method.

Concrete grammar:

1. go up sample and use Protein A-Sepharose CL-4B affinity chromatography monoclonal antibody purification, AKTA 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 sample on the flow velocity 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.Use 0.02M then, the citrate buffer solution wash-out antibody of pH4.0.Monitor with AKTA explorer100 simultaneously, when elution peak occurring, get clean centrifuge tube and collect.Behind every collection 3ml, use 1M immediately, the Tris-HCl damping fluid of pH9.0 is adjusted pH value to 7.0.

Embodiment 2

Antigen-antibody is in conjunction with testing (ELISA method):

1. bag quilt: being cushioned liquid with 0.05M PH9. You carbonate bag, antigen (hVEGF-A or PDGFRbeta) is diluted to protein content is 5 μ g/ml.Add 0.1ml 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: antibody to be checked (anti-VEGF Fab, anti-PDGFR beta monoclonal antibody or the anti-VEGF-PDGFR beta bsAb) 0.1ml that adds certain dilution in above-mentioned wrapped by reacting hole in, put 37 ℃ and hatched 1 hour.Washing then.(doing blank well simultaneously, 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: in each reacting hole, add the tmb substrate solution 0.1ml of interim preparation, 37 ℃ 10～30 minutes.

5. termination reaction: in each reacting hole, add 2M sulfuric acid 0.05ml.

6. the result judges: can be on white background, and the result directly detects by an unaided eye: color is dark more in the reacting hole, and positive degree is strong more, 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, survey each hole OD value with zeroing back, blank hole in 450nm (if with ABTS colour developing, then 410nm), if it is greater than 2.1 times of the negative control OD value of stipulating, promptly positive.

Experimental result:

Separately when test bsAb and VEGF or PDGFR beta binding ability, this bi-specific antibody and antigenic binding ability and monospecific corresponding monoclonal antibody in antigenic binding ability identical (Fig. 6, Fig. 7).Fig. 8 shows, this bi-specific antibody can the specific while in conjunction with VEGF and PDGFR beta, and monospecific bevacizumab or anti-PDGFR beta monoclonal antibody do not have this ability.

The HUVECs cell attachment is incubated overnight on Cytodex 3 microcarriers, inoculates human world matter stem cell afterwards, and microcarrier is placed 12 orifice plates (200 microcarrier/holes) of fibre-bearing albumin glue (fibrin gel).Add the EGM-2 substratum that contains 2ng/mL people HGF, per two days replacing fresh cultures.Since the 8th day, add the bevacizumab of different concns, anti-PDGFR beta monoclonal antibody or anti-VEGF-PDGFR beta bsAb.Cultivate after 7 days, cell fixedly spends the night in 4 ℃ with 4% PFA.The HUVECs cell has two anti-dyeing of fluorescence group with anti-PECAM antibody and corresponding coupling; Week cell plastid anti-α SMA-Cy3 dyeing.Fig. 9 A shows that (control) compares with negative control group, bevacizumab, bev+anti-PDGFR beta or anti-VEGF-PDGFR beta bsAb can both significantly suppress endotheliocyte sprout growth and with being connected of pericyte.Simultaneously, two kinds of monoclonal antibodies mix use or use bi-specific antibody, use the bevacizumab effect better obviously separately to the rejection ratio that sprouting of endotheliocyte grown.Fig. 9 B explanation, bevacizumab is relevant with concentration with the inhibition of anti-VEGF-PDGFR beta bsAb cell growth, and under lower concentration (1nM), bsAb still is better than bevacizumab to the restraining effect of endothelial cell growth.

Embodiment 3

Anti-VEGF/PDGFRbeta dual specific is anti-to human nasopharyngeal carcinoma CNE nude mouse xenotransplantation tumor growth inhibition test

The tumor tissue of getting the growth animated period cuts into 1.5mm ³About, under aseptic condition, it is subcutaneous to be inoculated in the nude mouse right side.Mice-transplanted tumor treats that with vernier caliper measurement transplanted tumor diameter tumor growth is to 100-200mm ³Back animal random packet.Use the method for measuring the knurl footpath, dynamic observe the antineoplastic effect of tested animal.The measurement number of times of diameter of tumor is per 2 days 1 time, and each the measurement also needs the weighing mouse heavy simultaneously.The anti-VEGF/PDGFRbeta 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 length and width respectively.Calculate relative tumour volume according to the result who measures.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 of the anti-VEGF/PDGFRbeta dual specific of table 3. to the growth of human nasopharyngeal carcinoma CNE bare mouse different species transplantation tumor

The result: seeing Table 3 cis-platinum group 10mg/kg, is 73.68% to the tumour inhibiting rate of human nasopharyngeal carcinoma CNE Nude Mice, but the influence that the body weight of laboratory animal is had significance; Grace degree group 2.5mg/kg is 45.10% to the tumour inhibiting rate of human nasopharyngeal carcinoma CNE Nude Mice; The anti-high, medium and low dosage group of anti-VEGF/PDGFRbeta dual specific is 55.17%, 61.25%, 50.45% to the tumour inhibiting rate of human nasopharyngeal carcinoma CNE Nude Mice, and the laboratory animal body weight is not had the significance influence.

Anti-VEGF/PDGFRbeta dual specific is anti-to be shown human nasopharyngeal carcinoma CNE Nude Mice growth inhibition test result, compare with negative control group, the anti-3mg/kg group of anti-VEGF/PDGFRbeta dual specific has best restraining effect to the growth of human nasopharyngeal carcinoma CNE transplanted tumor; Compare with positive control cis-platinum group,, do not see tangible toxic side effects the not influence of body weight of laboratory animal.

Embodiment 4

Anti-VEGF/PDGFRbeta dual specific is anti-to human thyroid cancer SW-579 nude mouse xenotransplantation tumor growth inhibition test

TV＝0.52×a×b ²

T/C(％)＝T _RTV/C _RTV×100％

T _RTV: treatment group RTV; C _RTV: negative control group RTV

The anti-restraining effect of the anti-VEGF/PDGFRbeta dual specific of table 4. to the growth of human thyroid cancer SW-579 bare mouse different species transplantation tumor

The result: see Table 4,5-Fu (5 FU 5 fluorouracil) organizes 10mg/kg, be 80.95% to the tumour inhibiting rate of human thyroid cancer SW-579 Nude Mice, but 5-Fu toxicity is bigger, and the weight of animals descends, and animal has death in the experimentation; Grace degree group 2.5mg/kg is 19.84% to the tumour inhibiting rate of human thyroid cancer SW-579 Nude Mice; The anti-high, medium and low dosage group of anti-VEGF/PDGFRbeta dual specific does not have the significance influence to the inhibitory rate 40.48%, 69.84%, 55.56% of human thyroid cancer SW-579 Nude Mice to the nude mice body weight.

Anti-VEGF/PDGFRbeta dual specific is anti-to be shown human thyroid cancer SW-579 Nude Mice growth inhibition test result, compare with negative control group, the anti-3mg/kg group of anti-VEGF/PDGFRbeta dual specific has the restraining effect of significance to the growth of human thyroid cancer SW-579 transplanted tumor; Compare with positive control cis-platinum group,, do not see tangible toxic side effects the not influence of body weight of laboratory animal.

Embodiment 5

Anti-VEGF/PDGFRbeta dual specific is anti-to human pancreas cancer SW-1990 nude mouse xenotransplantation tumor growth inhibition test

TV＝0.52×a×b ²

T/C(％)＝T _RTV/C _RTV×100％

T _RTV: treatment group RTV; C _RTV: negative control group RTV

The anti-restraining effect of the anti-VEGF/PDGFRbeta dual specific of table 5 to the growth of human pancreas cancer SW-1990 bare mouse different species transplantation tumor

The result: see Table 5,5-Fu organizes 10mg/kg, is 78.52% to the tumour inhibiting rate of human pancreas cancer SW-1990 Nude Mice; Grace degree group 2.5mg/kg is 35.16% to the tumour inhibiting rate of human pancreas cancer SW-1990 Nude Mice; The anti-high, medium and low dosage group of anti-VEGF/PDGFRbeta dual specific is to the inhibitory rate 77.09%, 64.30%, 48.77% of human pancreas cancer SW-1990 Nude Mice.

Anti-VEGF/PDGFRbeta dual specific is anti-to be shown human pancreas cancer SW-1990 Nude Mice growth inhibition test result, compare with negative control group, the anti-3mg/kg group of anti-6mg/kg group of anti-VEGF/PDGFRbeta dual specific and anti-VEGF/PDGFRbeta dual specific all has the restraining effect of significance to the growth of human pancreas cancer SW-1990 transplanted tumor.

Embodiment 6

Anti-VEGF/PDGFRbeta dual specific is anti-to people's lung cancer H460 nude mouse xenotransplantation tumor growth inhibition test

TV＝0.52×a×b ²

T/C(％)＝T _RTV/C _RTV×100％

T _RTV: treatment group RTV; C _RTV: negative control group RTV

The anti-restraining effect of the anti-VEGF/PDGFRbeta dual specific of table 6. to the growth of people's lung cancer H460 bare mouse different species transplantation tumor

The result: see Table 6, taxol group 10mg/kg is 70.05% to the tumour inhibiting rate of people's lung cancer H460 Nude Mice; Grace degree group 2.5mg/kg is 23.46% to the tumour inhibiting rate of people's lung cancer H460 Nude Mice; The anti-high, medium and low dosage group of anti-VEGF/PDGFRbeta dual specific is to the inhibitory rate 75.88%, 60.64%, 34.32% of people's lung cancer H460 Nude Mice.

Anti-VEGF/PDGFRbeta 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 anti-6mg/kg group of anti-VEGF/PDGFRbeta dual specific and anti-VEGF/PDGFRbeta dual specific has the restraining effect of significance to the growth of people's lung cancer H460 transplanted tumor.

Embodiment 7

Anti-VEGF/PDGFRbeta dual specific is anti-to human esophagus cancer Ec109 nude mouse xenotransplantation tumor growth inhibition test

TV＝0.52×a×b ²

T/C(％)＝T _RTV/C _RTV×100％

T _RTV: treatment group RTV; C _RTV: negative control group RTV

The anti-restraining effect of the anti-VEGF/PDGFRbeta dual specific of table 7. to the growth of human esophagus cancer Ec109 bare mouse different species transplantation tumor

The result: see Table 7, taxol group 10mg/kg is 69.41% to the tumour inhibiting rate of human esophagus cancer Ec109 Nude Mice; Grace degree group 2.5mg/kg is 50.02% to the tumour inhibiting rate of human esophagus cancer Ec109 Nude Mice; The anti-high, medium and low dosage group of anti-VEGF/PDGFRbeta dual specific is to the inhibitory rate 59.54%, 78.76%, 50.21%% of human esophagus cancer Ec109 Nude Mice.

Anti-VEGF/PDGFRbeta dual specific is anti-to be shown human esophagus cancer Ec 109 Nude Mice growth inhibition test results, compare with negative control group, the anti-3mg/kg group of anti-VEGF/PDGFRbeta dual specific has the restraining effect of significance to the growth of human esophagus cancer Ec109 transplanted tumor.

Embodiment 8

Anti-VEGF/PDGFRbeta dual specific is anti-to human breast carcinoma MDA-MB-231 nude mouse xenotransplantation tumor growth inhibition test

TV＝0.52×a×b ²

T/C(％)＝T _RTV/C _RTV×100％

T _RTV: treatment group RTV; C _RTV: negative control group RTV

The anti-restraining effect of the anti-VEGF/PDGFRbeta dual specific of table 8. to the growth of human breast carcinoma MDA-MB-231 bare mouse different species transplantation tumor

The result: see Table 8, taxol group 10mg/kg is 75.29% to the tumour inhibiting rate of human breast carcinoma MDA-MB-231 Nude Mice; Grace degree group 2.5mg/kg is 65.45% to the tumour inhibiting rate of human breast carcinoma MDA-MB-231 Nude Mice; The anti-high, medium and low dosage group of anti-VEGF/PDGFRbeta dual specific is to the inhibitory rate 70.71%, 81.57%, 45.57% of human breast carcinoma MDA-MB-231 Nude Mice.

Anti-VEGF/PDGFRbeta 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 anti-6mg/kg group of anti-VEGF/PDGFRbeta dual specific and anti-VEGF/PDGFRbeta dual specific all has the restraining effect of significance to the growth of human breast carcinoma MDA-MB-231 transplanted tumor.

Embodiment 9

Anti-VEGF/PDGFRbeta 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, it is subcutaneous to be inoculated in nude mouse right side armpit with 0.1ml.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, dynamic observe the antineoplastic effect of tested thing.The measurement number of times of diameter of tumor is survey in per 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; Grace degree group 2.5mg/kg, administration every day 1 time; Anti-VEGF/PDGFRbeta 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 of the anti-VEGF/PDGFRbeta dual specific of table 9. to the growth of people's kidney A498 bare mouse different species transplantation tumor

The result: see Table 9, taxol group 10mg/kg is 85.53% to the tumour inhibiting rate of people's kidney A498 Nude Mice; Grace degree group 2.5mg/kg is 32.57% to the tumour inhibiting rate of people's kidney A498 Nude Mice; The anti-high, medium and low dosage group of anti-VEGF/PDGFRbeta dual specific is to the inhibitory rate 71.49%, 65.17%, 57.33% of people's kidney A498 Nude Mice.

Anti-VEGF/PDGFRbeta 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 anti-6mg/kg group of anti-VEGF/PDGFRbeta dual specific and anti-VEGF/PDGFRbeta dual specific has the restraining effect of significance to the growth of people's kidney A498 transplanted tumor.

Embodiment 10

Anti-VEGF/PDGFRbeta dual specific is anti-to people's carcinoma of gallbladder GBC-SD nude mouse xenotransplantation tumor growth inhibition test

People's carcinoma of gallbladder GBC-SD cell strain of taking the logarithm vegetative period is prepared into 5 * 10 after under aseptic condition ⁷/ ml cell suspension, it is subcutaneous to be inoculated in nude mouse right side armpit with 0.1ml.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, dynamic observe the antineoplastic effect of tested thing.The measurement number of times of diameter of tumor is survey in per 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; Grace degree group 2.5mg/kg, administration every day 1 time; Anti-VEGF/PDGFRbeta 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 of the anti-VEGF/PDGFRbeta dual specific of table 10. to the growth of people's carcinoma of gallbladder GBC-SD bare mouse different species transplantation tumor

The result: see Table 10, taxol group 10mg/kg is 76.75% to the tumour inhibiting rate of people's carcinoma of gallbladder GBC-SD Nude Mice; Grace degree group 2.5mg/kg, the tumour inhibiting rate that people's carcinoma of gallbladder GBC-SD nude mouse is suppressed knurl is 28.53%; The anti-high, medium and low dosage group of anti-VEGF/PDGFRbeta dual specific is 58.21%, 65.80%, 54.85% to the tumour inhibiting rate of people's carcinoma of gallbladder GBC-SD Nude Mice.

Anti-VEGF/PDGFRbeta dual specific resists the test-results to the tumour inhibiting rate of people's carcinoma of gallbladder GBC-SD mice-transplanted tumor to show, compare with negative control group, the anti-3mg/kg group of anti-VEGF/PDGFRbeta dual specific is the most obvious to the growth-inhibiting effect of people's carcinoma of gallbladder GBC-SD transplanted tumor.

Embodiment 11

The anti-inhibition test of anti-VEGF/PDGFRbeta dual specific to human colon carcinoma HT-29 nude mouse xenotransplantation tumor growth

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, it is subcutaneous to be inoculated in nude mouse right side armpit with 0.1ml.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, dynamic observe the antineoplastic effect of tested thing.The measurement number of times of diameter of tumor is survey in per 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; Grace degree group 2.5mg/kg, administration every day 1 time; Anti-VEGF/PDGFRbeta 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 of the anti-VEGF/PDGFRbeta dual specific of table 11 to the growth of human colon carcinoma HT-29 bare mouse different species transplantation tumor

The result: see Table 11, taxol group 10mg/kg is 67.43% to the tumour inhibiting rate of human colon carcinoma HT-29 Nude Mice; Grace degree 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/PDGFRbeta 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/PDGFRbeta 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/PDGFRbeta dual specific has the restraining effect of significance to the growth of human colon carcinoma HT-29 transplanted tumor.

Embodiment 12

The anti-inhibition test of anti-VEGF/PDGFRbeta dual specific to human ovarian cancer SK-OV-3 nude mouse xenotransplantation tumor growth

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, it is subcutaneous to be inoculated in nude mouse right side armpit with 0.1ml.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, dynamic observe the antineoplastic effect of tested thing.The measurement number of times of diameter of tumor is survey in per 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; Grace degree group 2.5mg/kg, administration every day 1 time; Anti-VEGF/PDGFRbeta 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 of the anti-VEGF/PDGFRbeta dual specific of table 12. to the growth of human ovarian cancer SK-OV-3 bare mouse different species transplantation tumor

The result: see Table 12, cis-platinum group 10mg/kg is 75.43% to the tumour inhibiting rate of human ovarian cancer SK-OV-3 Nude Mice; Grace degree 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/PDGFRbeta 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/PDGFRbeta 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 of anti-VEGF/PDGFRbeta dual specific organizes the restraining effect that the growth of human ovarian cancer SK-OV-3 transplanted tumor is had significance, and the anti-6mg/kg group of anti-VEGF/PDGFRbeta dual specific also has certain restraining effect to the growth of human ovarian cancer SK-OV-3 transplanted tumor.

Embodiment 13

The anti-inhibition test of anti-VEGF/PDGFRbeta dual specific to human cervical carcinoma HeLa nude mouse xenotransplantation tumor growth

The HeLa Cells strain of taking the logarithm vegetative period is prepared into 5 * 10 after under aseptic condition ⁷/ ml cell suspension, it is subcutaneous to be inoculated in nude mouse right side armpit with 0.1ml.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, dynamic observe the antineoplastic effect of tested thing.The measurement number of times of diameter of tumor is survey in per 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; Grace degree group 2.5mg/kg, administration every day 1 time; Anti-VEGF/PDGFRbeta 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 of the anti-VEGF/PDGFRbeta dual specific of table 13. to the growth of human cervical carcinoma HeLa bare mouse different species transplantation tumor

The result: see Table 13, taxol group 10mg/kg is 65.34% to the tumour inhibiting rate of human cervical carcinoma HeLa Nude Mice; Grace degree 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/PDGFRbeta dual specific is 40.76%, 72.04%, 51.86% to the tumour inhibiting rate of human cervical carcinoma HeLa Nude Mice

Therefore, anti-VEGF/PDGFRbeta 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/PDGFRbeta dual specific has the restraining effect of significance to the growth of human cervical carcinoma HeLa transplanted tumor.

Embodiment 14

The anti-inhibition test of anti-VEGF/PDGFRbeta dual specific to human prostata cancer DU-145 nude mouse xenotransplantation tumor growth

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, and it is subcutaneous to be inoculated in nude mouse right side armpit with 0.1ml.Nude Mice is with vernier caliper measurement transplanted tumor diameter, treat tumor growth to the 100-200mm3 with the animal random packet.Use the method for measuring the knurl footpath, dynamic observe the antineoplastic effect of tested thing.The measurement number of times of diameter of tumor is survey in per 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; Grace degree group 2.5mg/kg, administration every day 1 time; Anti-VEGF/PDGFRbeta 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 of the anti-VEGF/PDGFRbeta dual specific of table 14. to the growth of human prostata cancer DU-145 bare mouse different species transplantation tumor

The result: see Table 14, cis-platinum group 10mg/kg is 71.38% to the tumour inhibiting rate of human prostata cancer DU-145 Nude Mice; Grace degree 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/PDGFRbeta 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/PDGFRbeta 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/PDGFRbeta dual specific has the restraining effect of significance to the growth of human prostata cancer DU-145 transplanted tumor.

Embodiment 15

The anti-inhibition test of anti-VEGF/PDGFRbeta dual specific to human bladder cancer HT1376 nude mouse xenotransplantation tumor growth

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, it is subcutaneous to be inoculated in nude mouse right side armpit with 0.1ml.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, dynamic observe the antineoplastic effect of tested thing.The measurement number of times of diameter of tumor is survey in per 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; Grace degree group 2.5mg/kg, administration every day 1 time; Anti-VEGF/PDGFRbeta 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 of the anti-VEGF/PDGFRbeta dual specific of table 15. to the growth of human bladder cancer HT1376 bare mouse different species transplantation tumor

The result: see Table 15, taxol group 10mg/kg is 67.58 to the tumour inhibiting rate of human bladder cancer HT1376 Nude Mice; Grace degree 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/PDGFRbeta dual specific is 40.70%, 62.03%, 51.80% to the tumour inhibiting rate of human bladder cancer HT1376 Nude Mice.

Therefore, anti-VEGF/PDGFRbeta 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/PDGFRbeta dual specific has the restraining effect of significance to the growth of human bladder cancer HT1376 transplanted tumor.

Embodiment 16

The anti-inhibition test of anti-VEGF/PDGFRbeta dual specific to people's carcinoma of testis 5637 nude mouse xenotransplantation tumor growths

People's carcinoma of testis 5637 cell strains of taking the logarithm vegetative period are prepared into 5 * 10 after under aseptic condition ⁷/ ml cell suspension, it is subcutaneous to be inoculated in nude mouse right side armpit with 0.1ml.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, dynamic observe the antineoplastic effect of tested thing.The measurement number of times of diameter of tumor is survey in per 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; Grace degree group 2.5mg/kg, administration every day 1 time; Anti-VEGF/PDGFRbeta 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 of the anti-VEGF/PDGFRbeta dual specific of table 16. to the growth of people's carcinoma of testis 5637 bare mouse different species transplantation tumors

The result: see Table 16, cis-platinum group 10mg/kg is 80.54% to the tumour inhibiting rate of people's carcinoma of testis 5637 Nude Mice; Grace degree group 2.5mg/kg is 30.62% to the tumour inhibiting rate of people's carcinoma of testis 5637 Nude Mice; The anti-high, medium and low dosage group of anti-VEGF/PDGFRbeta dual specific is 58.22%, 68.94%, 39.56% to the tumour inhibiting rate of people's carcinoma of testis 5637 Nude Mice.

Therefore, anti-VEGF/PDGFRbeta dual specific is anti-to be shown people's carcinoma of testis 5637 Nude Mice growth inhibition test results, compare with negative control group, the anti-3mg/kg group of anti-VEGF/PDGFRbeta dual specific has the restraining effect of significance to the growth of people's carcinoma of testis 5637 transplanted tumors.

Embodiment 17

The anti-inhibition test of anti-VEGF/PDGFRbeta dual specific to sarcoma HT-1080 nude mouse xenotransplantation tumor growth

The sarcoma HT-1080 cell strain of taking the logarithm vegetative period is prepared into 5 * 10 after under aseptic condition ⁷/ ml cell suspension, it is subcutaneous to be inoculated in nude mouse right side armpit with 0.1ml.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, dynamic observe the antineoplastic effect of tested thing.The measurement number of times of diameter of tumor is survey in per 2 days 1 time.Administering mode all adopts tail vein injection.Negative control group injection equivalent physiological saline, every day 1 time; Endoxan group 15mg/kg, administration is 1 time weekly; Anti-VEGF/PDGFRbeta dual specific is anti-with 3mg/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 of the anti-VEGF/PDGFRbeta dual specific of table 17. to the growth of sarcoma HT-1080 bare mouse different species transplantation tumor

The result: see Table 1, endoxan group 10mg/kg is 72.65% to the tumour inhibiting rate of sarcoma HT-1080 Nude Mice; The anti-group of anti-VEGF/PDGFRbeta dual specific is 60.32% to the tumour inhibiting rate of sarcoma HT-1080 Nude Mice.

SEQUENCE?LISTING

＜110〉Changzhou Adam Bioisystech Co., Ltd

＜120〉a kind of anti-VEGF/PDGFRbeta bi-specific antibody and application thereof

<130>

<160> 5

<170> PatentIn?version?3.3

<210> 1

<211> 113

<212> PRT

＜213〉artificial sequence

<400> 1

Ala?Ile?Gln?Met?Thr?Gln?Ser?Pro?Asp?Ser?Leu?Ala?Val?Ser?Leu?Gly

1 5 10 15

Glu?Arg?Ala?Thr?Ile?Asn?Cys?Lys?Ser?Ser?Gln?Ser?Val?Leu?Tyr?Ser

20 25 30

Ser?Asn?Asn?Lys?Asn?Tyr?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln

35 40 45

Pro?Pro?Lys?Leu?Leu?Ile?Tyr?Trp?Ala?Ser?Thr?Arg?Glu?Ser?Gly?Val

50 55 60

Pro?Asp?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr

65 70 75 80

Ile?Ser?Ser?Leu?Gln?Ala?Glu?Asp?Val?Ala?Val?Tyr?Tyr?Cys?Gln?Gln

85 90 95

Tyr?Tyr?Ser?Thr?Pro?Ile?Thr?Phe?Gly?Gln?Gly?Thr?Arg?Leu?Glu?Ile

100 105 110

Lys

<210> 2

<211> 122

<212> PRT

＜213〉artificial sequence

<400> 2

Glu?Val?Gln?Leu?Leu?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?Phe?Thr?Phe?Ser?Met?Tyr

20 25 30

Phe?Met?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val

35 40 45

Ser?Gly?Ile?Ser?Pro?Ser?Gly?Gly?Met?Thr?Phe?Tyr?Ala?Asp?Ser?Val

50 55 60

Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?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?Asp?Gly?Ile?Pro?Leu?Ser?Ile?Ala?Ala?Pro?Ile?Asp?Tyr?Trp

100 105 110

Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser

115 120

<210> 3

<211> 219

<212> PRT

＜213〉artificial sequence

<400> 3

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> 4

<211> 213

<212> PRT

＜213〉artificial sequence

<400> 4

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> 5

<211> 222

<212> PRT

＜213〉artificial sequence

<400> 5

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

Claims

1. anti-VEGF/PDGFRbeta bi-specific antibody is characterized in that this antibody is connected with in turn by the N end of SEQ NO.5

SEQ NO.4, SEQ NO.3, SEQ NO.2 and SEQ NO.1, wherein the C of SEQ NO.5 end is by (GGGGS) ₃Sealing.

2. the application of anti-VEGF/PDGFRbeta bi-specific antibody in preparation medicine for treating tumor thing.