CN105175545A - VEGF-resistant and PD-1-resistant difunctional antibody and application thereof - Google Patents

Abstract

The invention discloses an anti-VEGF-anti-PD-1 bifunctional antibody and its application, belonging to the technical field of molecular immunology. The anti-VEGF-anti-PD-1 bifunctional antibody provided by the present invention comprises a light chain and a heavy chain, wherein the amino acid sequence of the light chain is as shown in SEQ? ID? As shown in NO.2, the amino acid sequence of the heavy chain is as SEQ? ID? NO.4 or SEQ? ID? Shown in NO.6. At the same time, the invention also provides the gene encoding the bifunctional antibody and the application of the bifunctional antibody. The bifunctional antibody provided by the present invention can bind to PD-1 and VEGF with high affinity, can effectively stimulate T cells to secrete IL2 and induce T cells to secrete IFN-γ, and can significantly inhibit the growth of tumors in mice. The application in the preparation of anti-tumor drugs has great potential.

Description

A kind of anti-VEGF-anti-PD-1 bifunctional antibody and its application

technical field

The invention relates to an anti-VEGF-anti-PD-1 bifunctional antibody and its application, belonging to the technical field of molecular immunology.

Background technique

According to the prediction of WHO experts, by 2020, the number of cancer cases in the global population will reach 20 million, and the death toll will reach 12 million. Therefore, tumor will become the biggest killer of human beings in this century and pose the most serious threat to human survival. . As China's population ages, more and more people will suffer from tumor diseases. Therefore, the development of anti-tumor drugs has become the focus of national health.

Vascular endothelial growth factor, also known as VEGF. VEGF protein was successfully purified and identified by scientists from two biotechnology companies in the United States in 1989, and its gene sequence was cloned and determined, proving that VPF and VEGF are the same protein encoded by the same gene. VEGF has six isoforms (isoforms): VEGF-A, -B, -C, -D, and -E; its molecular weight ranges from 35 to 44 kDa, and each isoform specifically interacts with three "vascular endothelial growth Factor receptors" (VEGFR-1, -2, and -3) in combination. VEGF is a highly conserved homodimeric glycoprotein. Two single chains with a molecular weight of 24 kDa each form a dimer with disulfide bonds. The monomer decomposed by VEGF is inactive, and the removal of N2 sugar group has no effect on biological effects, but it may play a role in cell secretion. Due to the different splicing methods of mRNA, at least five protein forms are produced, including VEGF121, VEGF145, VEGF165, VEGF185, and VEGF206. Among them, VEGF121, VEGF145, and VEGF165 are secreted soluble proteins that can directly act on vascular endothelial cells to promote vascular endothelial cells. Proliferation, increased vascular permeability. In 1990, Dr. Folkman of Harvard University proposed the famous Folkman theory, that is, the growth of tumor tissue must rely on angiogenesis to provide sufficient oxygen and nutrients for maintenance. It is considered to be the basis of clinical application of VEGF. Monoclonal antibody against VEGF and VEGFR can inhibit vascular endothelial growth factor and is used to treat various metastatic cancers.

Programmed death receptor 1 (programmed death1, PD-1) is a member of the CD28 superfamily. PD-1 is expressed in activated T cells, B cells and myeloid cells, and it has two ligands, namely programmed death ligand-1 (programmed death ligand 1, PD-L1) and PD-L2. PD-L1/L2 is expressed on antigen-presenting cells, and PD-L1 is also expressed in various tissues. The combination of PD-1 and PD-L1 mediates the co-inhibitory signal of T cell activation, regulates T cell activation and proliferation, and plays a negative regulatory role similar to CTLA-4. Chinese scientist Chen Lieping's laboratory first discovered that PD-L1 is highly expressed in tumor tissues and regulates the function of tumor-infiltrating CD8 T cells. Therefore, immune regulation targeting PD-1/PD-L1 is of great significance for anti-tumor. In recent years, clinical research on a variety of Anti-PD-1/PD-L1 antibodies in tumor immunotherapy has been carried out rapidly. Currently, Pembrolizumab and Nivolumab have been approved by the FDA for advanced melanoma, and recently Nivolumab has also been approved by the US FDA for the treatment of advanced squamous non-small cell lung cancer. In addition, MPDL3280A (anti-PD-L1 monoclonal antibody), Avelumab (anti-PD-L1 monoclonal antibody), etc. have also entered multiple late-stage clinical studies, covering non-small cell carcinoma, melanoma, bladder cancer and other tumor types . Due to the broad-spectrum anti-tumor prospects and amazing drug efficacy of PD-1 antibodies, it is generally believed in the industry that antibodies targeting the PD-1 pathway will bring breakthroughs in the treatment of various tumors: for the treatment of non-small cell lung cancer, Renal cell carcinoma, ovarian cancer, melanoma, leukemia and anemia, etc. After the clinical efficacy data of PD-1 antibody drugs announced at the American Cancer Society (AACR) annual meeting and the American Society of Clinical Oncology (ASCO) annual meeting in 2012 and 2013, PD-1 antibody has become the most popular drug in the global pharmaceutical industry. Hot research antibody drug.

Tumor immunotherapy is the application of immunological principles and methods to improve the immunogenicity of tumor cells and the sensitivity to effector cell killing, stimulate and enhance the body's anti-tumor immune response, and use immune cells and effector molecules to infuse the host into the body to cooperate with the body. The immune system kills tumors and inhibits tumor growth. Tumor immunotherapy has attracted much attention recently and is the focus in the field of tumor treatment. In recent years, the good news of tumor immunotherapy has continued. At present, it has demonstrated strong anti-tumor activity in the treatment of some tumor types such as melanoma and non-small cell lung cancer, and tumor immunotherapy drugs have been approved by the US FDA ( Food and Drug Administration, FDA) approved clinical application. Due to its excellent efficacy and innovation, tumor immunotherapy was named the most important scientific breakthrough of the year by Science magazine in 2013. Tumor immunotherapy is expected to become an innovation in the field of tumor treatment following surgery, chemotherapy, radiotherapy, and targeted therapy.

Bifunctional antibodies, also known as bispecific antibodies, are non-natural antibodies that can target two different targets or proteins at the same time. Bispecific antibodies, which can specifically bind two different antigens simultaneously, are increasingly important in tumor immunotherapy due to their specificity and bifunctionality. On December 3, 2014, the US FDA approved the marketing of the bispecific antibody Blincyto (Blinatumomab) developed by Amgen for the treatment of acute lymphoblastic leukemia. Blinatumomab is a CD19, CD3 bispecific antibody, and Blincyto (Blinatumomab) is the first bispecific antibody approved by the US FDA. At present, more than 40 forms of bifunctional antibodies have been developed, but due to problems such as low production efficiency and poor pharmacokinetic performance, the development of bispecific antibodies has always been difficult. At present, there is no anti-VEGF-anti-PD-1 bifunctional antibody at home and abroad.

Contents of the invention

In order to solve the above problems, the present invention provides an anti-VEGF-anti-PD-1 bifunctional antibody, and the adopted technical scheme is as follows:

The purpose of the present invention is to provide an anti-VEGF-anti-PD-1 bifunctional antibody. The bifunctional antibody comprises a light chain and a heavy chain, wherein the amino acid sequence of the light chain is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.4 or SEQ ID NO.6.

Preferably, the bifunctional antibody is anti-VEGF-anti-PD-1 bifunctional antibody BsAbB7, the amino acid sequence of the light chain is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.4.

Preferably, the bifunctional antibody is anti-VEGF-anti-PD-1 bifunctional antibody BsAbB8, the amino acid sequence of the light chain is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.6.

The present invention also provides a gene encoding the anti-VEGF-anti-PD-1 bifunctional antibody, the nucleotide sequence encoding the light chain of the gene is shown in SEQ ID NO.1, and the nucleotide sequence encoding the heavy chain is shown in SEQ ID NO. .3 or shown in SEQ ID NO.5.

Preferably, the nucleotide sequences SEQIDNO.1 and SEQIDNO.3 in the gene encode the light chain and heavy chain of the anti-VEGF-anti-PD-1 bifunctional antibody BsAbB7, respectively.

Preferably, the nucleotide sequences SEQIDNO.1 and SEQIDNO.5 in the gene encode the light chain and heavy chain of the anti-VEGF-anti-PD-1 bifunctional antibody BsAbB8, respectively.

The application of any antibody in the preparation of drugs for preventing, diagnosing, treating or adjuvantly treating tumors also falls within the protection scope of the present invention.

The application of any antibody in the preparation of anti-tumor drugs is also within the protection scope of the present invention.

Preferably, the application is specifically applied in drugs that bind to VEGF, block VEGF signaling pathways, and inhibit tumors.

Preferably, the application specifically includes drugs that bind to PD-1, drugs that block the combination of PD-1 and PDL-1, drugs that activate T lymphocytes, increase the expression of IL-2 and IFN-γ in T lymphocytes, Drugs used to suppress tumors.

Specifically, the tumor is selected from lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, kidney tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colorectal cancer, nasopharyngeal cancer, brain tumor, cervical cancer, cancer, blood cancer, bone cancer, lymphoma, pancreatic cancer, etc.

The beneficial effects that the present invention obtains are as follows:

The method used in the present invention is a new cutting-edge method for treating tumors at present, and tumor immunotherapy is expected to become an innovation in the field of tumor treatment after surgery, chemotherapy, radiotherapy and targeted therapy.

Both the newly discovered bifunctional antibodies BsAbB7 and BsAbB8 of the present invention can bind to PD-1, the affinity of bifunctional BsAbB7 is 0.24nM, and the affinity of bifunctional BsAbB8 is 0.24nM. At the same time, it can also be combined with VEGF, the affinity of the bifunctional antibody BsAbB7 is 0.15nM, and the affinity of the bifunctional antibody BsAbB8 is 0.12nM.

Description of drawings

Figure 1 is the SDS-PAGE electrophoresis results of PD-1 and VEGF antigens;

(Among them, A is VEGF antigen; B is PD-1 antigen).

Fig. 2 is the SDS-PAGE electrophoresis result figure of BsAb7 and BsAb8;

(Wherein, A is the SDS-PAGE electrophoresis result of BsAb7; B is the SDS-PAGE electrophoresis result of BsAb8).

Figure 3 is the results of the determination of the ability of bifunctional antibodies BsAb7 and BsAb8 to induce T lymphocytes to secrete IL2.

Fig. 4 is the results of determination of the ability of bifunctional antibodies BsAb7 and BsAb8 to induce T lymphocytes to secrete IFN-γ.

Detailed ways

The present invention will be further described below in conjunction with specific examples, but the present invention is not limited by the examples.

The materials, reagents, instruments and methods used in the following examples are conventional materials, reagents, instruments and methods in the art unless otherwise specified, and can be obtained through commercial channels.

Embodiment 1 Preparation of VEGF and PD1 protein

1. Construction of expression vector for PD-1 antigen

The cDNA of human PD-1 was synthesized from Nanjing GenScript Company, the GeneID is 5133, and the cDNAID is NM_005018.2. An Fc purification tag was added after the synthetic extracellular region PD-1 gene, and two restriction enzyme sites of XbaI and BamHI were introduced at both ends and connected to the pTT5 expression plasmid, which was verified to be correct by sequencing. The sequenced plasmid was transfected into Trans10 (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), and a single clone was picked and inoculated into 1 liter of LB liquid medium. When the OD ₆₀₀ was 1, the bacteria were collected by centrifugation and extracted with the plasmid. A kit (purchased from Qiagen) was used to extract plasmids.

2. Construction of expression vector for VEGF antigen

The amino acid corresponding to the gene VEGF (NCBIGeneID: 7422) was fused with the mouse IgG Fc protein fragment mFc (Iggamma-2AchainCregion) to obtain VEGF-mFc. In order to improve the expression efficiency of the target gene in the 293F cell expression system, the sequence was optimized, and two restriction enzyme sites of XbaI and BamHI were introduced at both ends to connect to the pTT5 expression plasmid, which was verified to be correct by sequencing. The sequenced plasmid was transfected into Trans10 (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), and a single clone was picked and inoculated into 1 liter of LB liquid medium. When the OD ₆₀₀ was 1, the bacteria were collected by centrifugation and extracted with the plasmid. A kit (purchased from Qiagen) was used to extract plasmids.

3. Expression and purification of PD-1 and VEGF antigens

The correct expression vector identified by sequencing was transfected into 293F cells (purchased from Invitrogen), cultured at 37°C, 5% CO2, and 130 rpm/min for 7 days, and the supernatant was collected by centrifugation. The supernatant was centrifuged at 4000rpm for 10min, and then filtered with a 0.45μm filter membrane; 400mM NaCl was added to the filtrate; the pH was adjusted to 8.0. After the sample was filtered again through a 0.2μm filter membrane, the sample was loaded onto a 5ml HiTrapProteinA column equilibrated with PBS (137mMNaCl, 2.7mMKCl, 10mMNa ₂ HPO ₄ , 2mMKH ₂ PO ₄ , pH 7.4); rinse with PBS after the sample was loaded , flow rate 5ml/min, UV monitoring level. BufferB (1MGlycine, pH3.5) was eluted at a flow rate of 1ml/min. The collected effluent peaks were neutralized to pH7.5 with Tris and detected by SDS-PAGE. The results of SDS-PAGE electrophoresis are shown in Figure 1. Concentrate the eluted peak with an ultrafiltration concentrator tube and exchange the liquid into PBS, thereby obtaining the antigen.

Example 2 Preparation of bifunctional antibody BsAb7 and BsAb8 proteins

1. Construction of bifunctional antibody expression vector

The cDNA of the light chain of the artificially synthesized antibody BsAb7 (its sequence is shown in SEQ ID NO.1), the cDNA of the artificially synthesized antibody BsAb7 heavy chain (its sequence is shown in SEQ ID NO.3), the cDNA of the artificially synthesized antibody BsAb8 heavy chain (its sequence As shown in SEQ ID NO.5), the synthesized cDNAs were respectively cloned into pTT5 plasmids, and the correct construction of the plasmids was confirmed by sequencing. The sequenced plasmid was transfected into Trans10 (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), and a single clone was picked and inoculated into 1 liter of LB liquid medium. When the OD ₆₀₀ was 1, the bacteria were collected by centrifugation and extracted with the plasmid. A kit (purchased from Qiagen) was used to extract plasmids.

2. Protein expression and purification of bifunctional antibodies BsAb7 and BsAb8

The BsAb7 and BsAb8 heavy chain expression vectors and light chain expression vectors (1:1) identified correctly by sequencing were co-transfected into 293F cells, cultured at 37°C, 5% CO ₂ , 130rpm/min for 7 days, and the supernatant was collected by centrifugation. Centrifuge the supernatant at 4000rpm for 10min, and filter it with a 0.45μm filter membrane to collect the filtrate; add 400mM NaCl to the filtrate; adjust the pH to 8.0. After the sample was filtered again through a 0.2 μm filter membrane, the sample was loaded onto a 5ml HiTrapMabSelect column (purchased from GE) that had been equilibrated with PBS (137mMNaCl, 2.7mMKCl, 10mMNa ₂ HPO ₄ , 2mMKH ₂ PO ₄ , pH7.4); Rinse with PBS after application, the flow rate is 5ml/min, and the level is monitored by ultraviolet light. BufferB (1MGlycine, pH3.5) was eluted at a flow rate of 1ml/min. The collected effluent peaks were neutralized to pH7.5 with Tris and detected by SDS-PAGE. The results of SDS-PAGE non-reducing electrophoresis are shown in Figure 2. Concentrate the eluted peak with an ultrafiltration concentrator tube, and exchange the liquid into PBS with a desalting column, thereby obtaining antibody BsAb7 and BsAb8 proteins.

Example 3 Determination of the affinity of bifunctional antibodies BsAb7 and BsAb8 to PD-1 and VEGF

The characterization affinity and binding kinetics of anti-PD-1/VEGF antibodies BsAbB7 and BsAbB8 were analyzed by Biacore3000 instrument (purchased from GE). The recombinant fusion protein was covalently coupled to biotin using a biotin labeling kit (Pierce Company), and then flowed through an avidin-labeled SA chip (purchased from GE Company), so that the reaction value RU reached about 450. Binding was measured by flowing antibodies in PBS buffer at concentrations of 0.0133, 0.0266, 0.0532, 0.1064, 0.2128 μM and a flow rate of 50 μl/min. Antigen-antibody binding kinetics were followed for 3 minutes and dissociation kinetics were followed for 10 minutes. Association and dissociation curves were fitted to a 1:1 Langmuir binding model using BIAevaluation software. In order to minimize the role of avidity in estimating binding constants, only the initial data segments corresponding to the association and dissociation phases were used for fitting. The results of the determined Kd, Kon and Koff values are shown in Table 1.

Table 1 Determination results of bifunctional antibodies BsAb7 and BsAb8 for PD-1 and VEGF affinity constant (Kd)

Kn(10 ⁵ M ^-1 s ^-1 ) Koff(10 ^-5 s ^-1 ) Kd(nm) BsAb7-PD-1 1.02 2.42 0.24 BsAb8-PD-1 0.96 2.33 0.24 BsAb7-VEGF 1.44 2.10 0.15 BsAb8-VEGF 1.89 2.31 0.12

Example 4 Competition ELISA method to determine the competition of bifunctional antibodies BsAb7 and BsAb8 with PDL-1 for binding to antigen PD-1

Coat the ELISA plate with PD-1-mFc, block with 1% BSA, mix different concentrations of antibodies BsAb7 and BsAb8 with PDL-1-hFc, incubate at 37°C, add the enzyme-labeled secondary antibody and incubate at 37°C for 30 minutes. The absorbance value at 450 nm was detected on a microplate reader (see Table 2). The results of the binding of bifunctional antibodies BsAb7 and BsAb8 to the antigen PD-1 showed that both bifunctional antibodies BsAb7 and BsAb8 could effectively compete with PDL-1 for binding to PD-1 protein, and the binding efficiency was dose-dependent. Through the analysis of the competition ELISA results of the combined bifunctional antibodies BsAb7 and BsAb8, the binding efficiencies EC50 of the curve simulation bifunctional antibodies BsAb7 and BsAb8 were: 2.5nM and 2.9nM, respectively.

Table 2 The results of competition ELISA to determine the binding ability of bifunctional antibodies BsAb7 and BsAb8 to block PD-1/PDL-1

Antibody concentration _OD450 (BsAb7) _OD450 (BsAb8) (stock solution) 2μg/ml 0.096 0.107 1:3 0.101 0.110 1:9 0.323 0.323 1:27 0.733 0.802 1:81 0.852 0.831 1:243 0.857 0.854 1:729 0.898 0.866 control/0 0.901 0.899

Example 5 Bifunctional antibodies BsAb7 and BsAb8 induce T cells to secrete IL2 in vitro

Ficoll centrifugation (purchased from GE) and CD4+ T cell enrichment column (purchased from R&D Systems) were used to prepare fresh PBMCs and purify human T cells. The cells were plated into a 96-well flat-bottomed plate, and after culturing overnight, three different concentrations of bifunctional antibodies BsAb7 and BsAb8 (25nmol, 5nmol, and 1nmol) and 80ng/ml tetanus toxin (TT) were added, and three concentrations of 25nmol, 5nmol, and 1nmol were added. The concentration of the isotype control antibody was used as a negative control, and the supernatant was collected after 3 days of culture, and the secretion level of supernatant IL2 was detected with a Luminex instrument (purchased from LifeTechnology) and a cytokine IL2 detection kit (purchased from BD Biosciences). The results are shown in Figure 3. The results showed that both the bifunctional antibodies BsAb7 and BsAb8 could effectively stimulate the function of T cells and secrete IL2, which was related to the antibody concentration, while the isotype control antibody could not promote the proliferation of T cells and the secretion of IL2.

Example 6 Bifunctional antibodies BsAb7 and BsAb8 induce T cells to secrete IFN-γ in vitro

Fresh PBMCs were prepared by Ficoll centrifugal method (purchased from GE) and CD4+ T cell enrichment column (purchased from R&D Systems), and human T cells were purified. Monocytes were purified using the MiltenyiCD14 Monocyte Purification Kit, and DC cells were generated after the monocytes were cultured with GM-CSF and IL-4 (both purchased from PeproTech) for 7 days. Cells were plated into 96-well flat bottom plates and after overnight culture each culture contained 10e5 purified T cells and 10e4 dendritic cells in a total volume of 200 μl. Three different concentrations of bifunctional antibodies BsAb7 and BsAb8 were added in 25nmol, 5nmol and 1nmol, and isotype control antibodies in three concentrations of 25nmol, 5nmol and 1nmol were added as negative controls. Cells were cultured at 37°C for 5 days. After 5 days, 100 μl of medium was removed from each culture for cytokine IFN-γ measurement. The level of IFN-γ was determined by OptEIA ELISA kit (purchased from BD Biosciences). The results (as shown in Figure 4) show that the bifunctional antibodies BsAb7 and BsAb8 can effectively stimulate the function of T cells to secrete the cytokine IFN-γ, which is related to the concentration, while the isotype control antibody cannot promote the proliferation of T cells and the secretion of IFN-γ.

Example 7 Bifunctional antibodies BsAb7 and BsAb8 inhibit tumor growth in mice

Cultivate human Caki-1 cells, collect the cells in the logarithmic phase of the cells, make a concentration of (1.0×10 ⁷ )/ml cell suspension, take 6-8 weeks old nude mice, inject 1.0×10 ^Six (about 0.1ml) cell suspensions, the tumor grew to a diameter of about 5mm in about 10 days, and the tumor was successfully induced. They were randomly divided into 4 groups: negative control group (peritoneal injection of normal saline group), BsAb7 group (tail vein injection of 3mg /kg group), BsAb8 group (tail vein injection 3mg/kg), positive control group bevacizumab (tail vein injection 2mg/kg). Dosing once every 3 days for 21 consecutive days. After 21 days, the nude mice were killed and the tumor weight was weighed. The tumor inhibition rate=[1-average tumor weight of the experimental group (groups B, C, and D are the experimental group)/average tumor weight of the group A)]×100%. The experimental results are shown in Table 3, both BsAb7 and BsAb8 can inhibit tumor growth in mice.

Table 3 The results of bifunctional antibodies BsAb7 and BsAb8 inhibiting tumor growth in mice

group Average tumor weight (g) Average tumor inhibition rate (%) negative control 2.544±0.316 / BsAb7 0.932±0.224 63.4 BsAb8 0.867±0.255 65.9 Bevacizumab 0.912±0.213 64.2

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore Therefore, the protection scope of the present invention should be defined by the claims.

Claims (10)

1. anti-vegf-anti-PD-1 bifunctional antibody, comprises light chain and heavy chain, it is characterized in that, the aminoacid sequence of light chain is as shown in SEQIDNO.2, and the aminoacid sequence of heavy chain is as shown in SEQIDNO.4 or SEQIDNO.6.

2. a kind of anti-vegf-anti-PD-1 bifunctional antibody according to claim 1, it is characterized in that, be anti-vegf-anti-PD-1 bifunctional antibody BsAbB7, the aminoacid sequence of light chain is as shown in SEQIDNO.2, and the aminoacid sequence of heavy chain is as shown in SEQIDNO.4.

3. a kind of anti-vegf-anti-PD-1 bifunctional antibody according to claim 1, it is characterized in that, be anti-vegf-anti-PD-1 bifunctional antibody BsAbB8, the aminoacid sequence of light chain is as shown in SEQIDNO.2, and the aminoacid sequence of heavy chain is as shown in SEQIDNO.6.

4. encode the gene of a kind of anti-vegf described in claim 1-anti-PD-1 bifunctional antibody, it is characterized in that, the nucleotide sequence of coding light chain is as shown in SEQIDNO.1, and the nucleotide sequence of encoding heavy chain is as shown in SEQIDNO.3 or SEQIDNO.5.

5. gene according to claim 4, is characterized in that, described nucleotide sequence SEQIDNO.1 and SEQIDNO.3 encodes the light chain of anti-vegf-anti-PD-1 bifunctional antibody BsAbB7 and heavy chain respectively.

6. gene according to claim 4, is characterized in that, described nucleotide sequence SEQIDNO.1 and SEQIDNO.5 encodes the light chain of anti-vegf-anti-PD-1 bifunctional antibody BsAbB8 and heavy chain respectively.

7. arbitrary antibody described in claim 1-3 preparation prevention, diagnosis, treatment or adjuvant therapy of tumors medicine in application.

8. arbitrary antibody described in claim 1-3 is preparing the application in anti-tumor drug.

9. apply according to claim 8, it is characterized in that, apply in the medicine in conjunction with the medicine of VEGF, blocking VEGF signal path, Tumor suppression.

10. apply according to claim 8, it is characterized in that, in conjunction with PD-1 medicine, block medicine that PD-1 and PDL-1 combines, activated T lymphocytes medicine, improve IL-2, IFN-γ expression in T lymphocyte medicine in apply.