CN106892980B

CN106892980B - anti-VEGFR 2 monoclonal antibody and application thereof

Info

Publication number: CN106892980B
Application number: CN201710243886.6A
Authority: CN
Inventors: 冯晓; 金磊; 王涛
Original assignee: GeneScience Pharmaceuticals Co Ltd
Current assignee: GeneScience Pharmaceuticals Co Ltd
Priority date: 2017-01-25
Filing date: 2017-04-14
Publication date: 2020-08-04
Anticipated expiration: 2037-04-14
Also published as: CN106892980A

Abstract

The invention belongs to the field of antibody drugs, and discloses a fully human anti-human VEGFR2 monoclonal antibody prepared by screening by using a phage antibody library technology and using a genetic engineering method, and a vector and a host cell containing polynucleotide for encoding the monoclonal antibody and application thereof. The monoclonal antibody of the invention blocks the binding of VEGF and VEGFR2 by binding VEGFR2, does not induce receptor dimerization any more, and then phosphorylates tyrosine residues of intracellular tyrosine kinase domains and activates downstream signal paths, including activation of phospholipase C, increase of intracellular calcium ion concentration and the like, so as to trigger the proliferation, survival, cytoskeletal rearrangement, cell migration, gene expression and the like of vascular endothelial cells, and finally blocks the vascular proliferation caused by the binding of VEGF to VEGFR2 and the induction of dimerization thereof. The monoclonal antibody can be used for treating diseases caused by tumor neovascularization.

Description

anti-VEGFR 2 monoclonal antibody and application thereof

The present application claims priority from chinese patent application having application number 201710060867.X entitled "anti-VEGFR 2 monoclonal antibody and its use" filed by chinese patent office in 2017, month 1 and 25, the entire contents of which are incorporated herein by reference.

Technical Field

The invention belongs to the field of antibody medicines, and particularly relates to a fully human anti-VEGFR 2 monoclonal antibody and application thereof.

Background

Angiogenesis refers to the generation of new blood vessels by budding or dividing existing blood vessels (capillaries and venules), which play an important role in many physiological processes, such as embryogenesis, wound healing, inflammatory response, and menstruation. At the same time, angiogenesis also plays an important role in several pathological processes, such as uncontrolled angiogenesis being the main cause of tumor proliferation and metastasis. Angiogenesis is also closely related to other diseases such as diabetic retinopathy, rheumatoid arthritis, psoriasis and the like. Inhibition of angiogenesis is therefore a viable approach to the treatment of these diseases.

Vascular endothelial cell growth factor (VEGF) plays an important role in the angiogenesis process, strongly stimulating vascular endothelial cell proliferation. Receptors to which VEGF binds include VEGFR1 (also known as Flt-1) and VEGFR2 (also known as Flk-1). VEGFR1 and VEGFR2 belong to the class III receptor tyrosine kinase family, and the extracellular domain consists of 7 immunoglobulin-like domains, including a ligand binding domain and a receptor dimerization domain, and includes a cellular transmembrane region and an intracellular tyrosine kinase domain. VEGFR2 mediates many effects of VEGF including endothelial cell proliferation, vascular proliferation and infiltration, while VEGFR1 does not appear to be directly involved in endothelial cell proliferation and vascular proliferation. Binding of VEGF dimer to VEGFR2 induces receptor dimerization, phosphorylation of tyrosine residues of intracellular tyrosine kinase domains, thereby activating downstream signaling pathways, including activation of phospholipase C, increase of intracellular calcium ion concentration, etc., triggering of vascular endothelial cell proliferation, survival, cytoskeletal rearrangement, cell migration, gene expression, etc., ultimately leading to vascular proliferation.

Therefore, VEGF and VEGFR2 are good therapeutic targets, and inhibition of this signaling pathway theoretically inhibits angiogenesis, thereby treating angiogenesis-related diseases. The US FDA approved bevacizumab (Avastin) targeting VEGF in 2004, which can obviously prolong the life of patients when clinically combined with chemotherapeutic drugs to treat colon cancer, but simultaneously inhibits the normal physiological function of VEGF, so that the patients have side effects of hypertension, hemorrhage, thrombosis and the like after using.

Given the important role of VEGFR2 in the VEGF signaling pathway, inhibition of VEGFR2 is also a strategy to inhibit angiogenesis. Compared with VEGF, the VEGFR2 targeting drug is more targeted and safer. Ramucirumab (IMC-1121B), approved by the FDA on the market in 2014, is an antibody drug targeting VEGFR 2.

Antibodies are immunoglobulins (igs) that specifically bind to an antigen when the antigen stimulates the body. Immunoglobulin class 5 such as IgG, IgA, IgM, IgD, and IgE are known and are commonly present in blood, body fluid, and external secretion in living bodies and on cell membranes of some cells (e.g., lymphocytes). As an important component of biopharmaceuticals, antibodies have become the largest and fastest growing class of biopharmaceuticals. The monoclonal antibody is more successfully used for treating various diseases such as tumor, autoimmune disease, infectious disease, transplant rejection and the like in clinic due to the high specificity of the monoclonal antibody aiming at the target.

At the early stage of antibody drug development, the mouse-derived monoclonal antibody plays a great role in promoting the research of human diseases, but because the mouse-derived monoclonal antibody is of mouse origin, severe immune response is caused in patients, and the development of antibody drugs is seriously hindered. With the development of antibody humanization technology, the bottleneck of antibody drug industrialization is broken. Therapeutic monoclonal antibodies currently used clinically include human-murine chimeric antibodies, humanized antibodies and fully human antibodies, of which fully human monoclonal antibodies are favored for their high affinity and low immunogenicity.

The fully human monoclonal antibody can be obtained by phage display technology, ribosome display technology, antibody humanization mouse technology and the like. The Phage Display Technology (PDT) of the antibody is to display the antibody V region fragment fused with phage surface protein (g3) on the phage surface, and then to screen the phage library for different antigens, finally to obtain the specific antibody. The technology simulates immune screening of organisms and obtains antibodies by screening from artificially established phage libraries. The antibody phage display technology can screen specific antibodies aiming at different antigens and can further mutate hypervariable regions of the antibodies so as to improve the antigen affinity of the antibodies.

Although some humanized monoclonal antibodies having great therapeutic advantages have been obtained by the above-described techniques, it is at no easy task to screen humanized monoclonal antibodies having desired properties and functions, and there is still a pressing need for such humanized monoclonal antibodies in reality.

Disclosure of Invention

In view of the above, the present invention aims to provide a fully human anti-human VEGFR2 monoclonal antibody, a vector of a polynucleotide encoding the monoclonal antibody, a host cell and uses thereof. The sequence of the antibody gene variable region related by the invention can construct a full-length antibody molecule as a medicine for clinically using the antibody molecule for indications caused by angiogenesis.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention screens the humanized antibody aiming at VEGFR2 by a phage display technology, and obtains a complete antibody by a genetic engineering method, thereby providing a medicament for treating angiogenesis-related diseases. The specific antibody preparation and related detection methods are as follows: 10 is constructed by extracting peripheral blood lymphocytes of different people, extracting total RNA and performing reverse transcription to form cDNA, then performing amplification of antibody variable region genes by using degenerate primers, performing enzyme digestion and connection to a phage display vector pCANTAB5E, cloning and transforming to TG1 cells for phage display⁸The single-chain antibody library combined with the human heavy chain variable region and the light chain variable region is characterized in that a single-chain antibody is displayed on the surface of a phage by a phage display technology, then VEGFR2 antigen is used for panning and enrichment, a large number of single-chain antibody genes combined with VEGFR2 antigen are screened, then the single-chain antibody is induced and expressed, VEGFR2 is used for E L ISA screening, a brand-new human antibody heavy chain variable region combined with VEGFR2 is screened out, then a full-length IgG antibody is constructed and converted, a small amount of purified antibody is prepared by using transient transfection, culture and small amount of affinity purification (Protein A), and a cytotoxic function test (ADCC, CDC and apoptosis test) is used for screening and identifying, a group of antibody molecules with good cytotoxic efficacy screened out by the phage display technology, and the sequence of the antibody variable regions, particularly CDRs region, is searched, and the group of antibody with the cytotoxic efficacy of VEGFR2 specificity is determined as a new.

The method constructs a high-capacity high-diversity antibody library, and selects an antibody which is combined with VEGFR2 and can block the combination of the antibody and ligand VEGF from the high-capacity antibody library, wherein the selected antibody has cell activity.

The anti-human VEGFR2 monoclonal antibody provided by the invention has a heavy chain variable region and a light chain variable region:

the amino acid sequence of the heavy chain variable region is shown as any one of SEQ ID No. 1-6; or an amino acid sequence which is at least 80 percent homologous with the amino acid sequence shown in the (I) and has the same or similar functions by substituting, deleting or adding one or more amino acids;

and/or

(II) the variable region of the light chain has an amino acid sequence as shown in any one of SEQ ID Nos. 7-12; or (b) one or more amino acid sequence(s) which has at least 80% homology with the amino acid sequence shown in the (II) and has the same or similar functions by substituting, deleting or adding one or more amino acids.

In some embodiments of the invention, the plurality is 2,3,4,5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32.

In some embodiments of the invention, the anti-VEGFR 2 monoclonal antibody has:

a heavy chain variable region having an amino acid sequence of

SEQ ID NO

1,2,3,4,5,6 and a light chain variable region having an amino acid sequence of SEQ ID NO 7; or

(ii) A heavy chain variable region having an amino acid sequence of SEQ ID NO. 1,2,3,4,5,6 and a light chain variable region having an amino acid sequence of SEQ ID NO. 8; or

(iii) the heavy chain variable region having the amino acid sequence of SEQ ID NO:1,2,3,4,5,6 and the light chain variable region having the amino acid sequence of SEQ ID NO: 9; or

(iv) the heavy chain variable region having an amino acid sequence of SEQ ID NO:1,2,3,4,5,6 and the light chain variable region having an amino acid sequence of SEQ ID NO: 10; or

(V) heavy chain variable region having amino acid sequence of SEQ ID NO:1,2,3,4,5,6 and light chain variable region having amino acid sequence of SEQ ID NO: 11; or

(VI) a heavy chain variable region having the amino acid sequence of SEQ ID NO:1,2,3,4,5,6 and a light chain variable region having the amino acid sequence of SEQ ID NO: 12.

Further, preferably, the anti-VEGFR 2 monoclonal antibody has any one of the following groups of heavy chain variable regions and light chain variable regions:

1) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQID NO. 9;

2) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2, and the amino acid sequence of the light chain variable region is shown as SEQID NO. 9;

3) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 12;

4) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 5, and the amino acid sequence of the light chain variable region is shown as SEQID NO. 9;

5) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 5, and the amino acid sequence of the light chain variable region is shown as SEQID NO. 11;

6) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 6, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 12.

The monoclonal antibody of the present invention includes not only the variable region but also the constant region. Preferably, the constant region is any one of human IgG1, IgG2, IgG3 or IgG 4.

More preferably, the constant region is human IgG 1.

The monoclonal antibodies of the invention are fully human.

The invention also provides a nucleotide for encoding the anti-CD 47 monoclonal antibody.

The invention provides an expression vector, which comprises nucleotide for coding the monoclonal antibody.

The invention provides a host cell which is transformed or transfected by the expression vector and is a prokaryotic cell or a eukaryotic cell.

The invention also provides a conjugate comprising the anti-human VEGFR2 monoclonal antibody covalently linked to a chemical or biological marker.

Wherein, the chemical label includes but is not limited to isotope, immunotoxin, chemical drug.

Preferably, the immunotoxin is aflatoxin, diphtheria toxin, pseudomonas aeruginosa exotoxin, ricin, abrin, mistletoe agglutinin, calycosin, PAP, nystatin, gelonin or luffa toxin.

The biomarkers include, but are not limited to, biotin, avidin, or enzyme markers.

Preferably, the enzyme is labeled horseradish peroxidase or alkaline phosphatase.

The invention also provides a conjugate formed by coupling the anti-human VEGFR2 monoclonal antibody and/or the conjugate with a solid medium or a semi-solid medium.

Preferably, the solid medium or non-solid medium is selected from colloidal gold, polystyrene plates or beads.

The invention also provides a pharmaceutical composition comprising the anti-human VEGFR2 monoclonal antibody and/or the conjugate.

The invention also provides a kit comprising the anti-human VEGFR2 monoclonal antibody and/or the conjugate.

The invention also provides application of the anti-human VEGFR2 monoclonal antibody and/or the conjugate and/or the pharmaceutical composition in preparation of drugs for treating diseases.

Wherein the disease is angiogenesis-related disease, including but not limited to non-small cell lung cancer, metastatic non-small cell lung cancer, glioma, colorectal cancer, hepatocellular carcinoma, metastatic hepatocellular carcinoma, HER2 negative metastatic breast cancer, metastatic gastric adenocarcinoma, metastatic colorectal cancer, metastatic melanoma, metastatic renal cell carcinoma.

The invention discloses a fully human anti-human VEGFR2 monoclonal antibody prepared by screening by using a phage antibody library technology and using a genetic engineering method, and discloses a vector containing a polynucleotide for coding the monoclonal antibody, a host cell and application. The monoclonal antibody of the invention blocks the binding of VEGF and VEGFR2 by binding VEGFR2, does not induce receptor dimerization any more, and then phosphorylates tyrosine residues of intracellular tyrosine kinase domains and activates downstream signal paths, including activation of phospholipase C, increase of intracellular calcium ion concentration and the like, so as to trigger the proliferation, survival, cytoskeletal rearrangement, cell migration, gene expression and the like of vascular endothelial cells, and finally blocks the vascular proliferation caused by the binding of VEGF to VEGFR2 and the induction of dimerization thereof. The monoclonal antibodies of the invention are useful for treating diseases caused by tumor neovascularization, including, but not limited to, the following tumors: non-small cell lung cancer, metastatic non-small cell lung cancer, hepatocellular carcinoma, metastatic hepatocellular carcinoma, HER 2-negative metastatic breast cancer, metastatic gastric adenocarcinoma, metastatic colorectal cancer, metastatic melanoma, metastatic renal cell carcinoma.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

Figure 1 is a SDS-PAGE electrophoretic image of the PCAb antibody, wherein lane M: protein tag PageRulerPrestained Protein ladder (manufacturer: Thermo, cat # 26616); lane 1 is reduced PCAb; lane 2 is non-reducing PCAb;

FIG. 2 is a map of a phagemid vector pCANTAB5E-SF vector;

FIG. 3 is a photograph of human PBMC for total RNA electrophoretic detection, in lane M DNA marker D L2000, lane 1 total RNA;

FIG. 4 is an electrophoresis test chart of PCR amplification products of human VH and V L, wherein, Lane M is DNA marker D L2000, Lanes 1-4 are PCR amplification products of human VH, Lane 5-8 are PCR amplification products of human V L;

FIG. 5 is a SDS-PAGE detection of purified products of the transient transfection expression of 300ml of 6 preferred antibodies, wherein lane M: protein tag PageRuler Prestained Protein ladder (manufacturer: Thermo, cat # 26616); lanes 1-6 are sequentially reduced 043 HAb-1-043 HAb-6; lanes 7-12 are sequentially non-reduced 043 HAb-1-043 HAb-6;

FIG. 6 shows the results of the preferred antibody epitope competition E L ISA;

FIG. 7 is a test result of the cell activity of a preferred antibody, wherein 7-A is a test result of the cell activity of a preferred antibody 043 HAb-1; 7-B is the detection result of the activity of the preferred antibody 043HAb-2 cells; 7-C is the cell activity detection result of a preferred antibody 043 HAb-3; 7-D is the detection result of the activity of the preferred antibody 043HAb-4 cells; 7-E is the detection result of the activity of the preferred antibody 043HAb-5 cells; 7-F is the result of the detection of the activity of the preferred antibody 043HAb-6 cell.

Detailed Description

The invention discloses a fully human anti-VEGFR 2 monoclonal antibody and application thereof, and a person skilled in the art can realize the antibody by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The invention will be further illustrated with reference to the following examples. The following experimental methods are conventional methods unless otherwise specified, and the raw materials and reagents used therefor are readily available from the market unless otherwise specified.

Example 1: positive antibody (PCAb) gene synthesis, expression vector construction and antibody preparation

1. Referring to patent CN 1345334a, an antibody with good biological activity is selected as a positive antibody (PCAb), and the amino acid sequence of the PCAb is as follows:

PCAbH, shown as SEQ ID NO: 13:

QVKLQQSGAELVGSGASVKLSCTTSGFNIKDFYMHWVKQRPEQGLEWIGWIDPENGDSDYAPKFQGKATMTADSSSNTAYLQLSSLTSEDTAVYYCNAYYGDYEGYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

PCAb L, as shown in SEQ ID NO: 14:

DIELTQSPAIMSASPGEKVTITCSASSSVSYMHWFQQKPGTSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

2. the amino acid sequence corresponding to the above antibody sequence was artificially optimized by codon, and then synthesized and optimized DNA was cloned into pUC57-Simple (available from Kinsley Biotechnology Co., Ltd.) vector to obtain pUC57-Simple-PCAbH and pUC57-Simple-PCAb L plasmids.

3. Plasmid pUC57 single-PCAbH and pUC57 single-PCAb L are digested by Xba I and BamH I, and then gene fragments PCAbH and PCAb L are recovered by agarose gel electrophoresis and are respectively connected with pGS003 vectors to be recombined and constructed to obtain pGS003-PCAbH and pGS003-PCAb L expression vectors.

4. After transfecting the recombinant plasmids pGS003-PCAbH and pGS003-PCAb L to FreeStyleTM293-F cells for 7 days, the culture solution was centrifuged at high speed, vacuum filtered through a microporous membrane, and purified using a Protein A column (Protein purification liquid chromatography system/AKTA Purifier 10, GE) according to the manufacturer's protocol to obtain the purified antibody PCAb, as shown in lane 2 of FIG. 1. the purified antibody was taken and the purified product was analyzed by SDS-PAGE under reducing conditions to identify the nature and purity of the antibody, the heavy chain under reducing conditions was 50KD and the light chain was 25KD, as shown in lane 1 of FIG. 1.

Example 2: construction of natural humanized single-chain antibody phage display library

1. Construction of phagemid vectors

Selecting pCANTAB5E as a phage display vector, carrying out vector modification according to cloning and phage display requirements, wherein the modification result is shown in figure 2. SfiI-NcoI-XhoI + L inker + NheI-NotI sequence (SED ID NO:15) is subjected to gene synthesis, then enzyme digestion is carried out by SfiI and NotI, and the modified vector pCANTAB5E-SF is constructed by carrying out ligation reaction recombination with pCANTAB5E vector.

2. PBMC isolation and mRNA extraction

Fresh peripheral blood was aseptically collected from healthy volunteers, and lymphocytes were separated from the blood using a lymphocyte separation medium (GE) and used by Invitrogen corporation

Extraction of 100 × 10 from reagent (15596-⁶Total RNA of individual cells, the results are shown in FIG. 3.

3. Antibody library primer design, synthesis and RT-PCR

Designing primers for amplifying heavy chains and light chains of the human antibodies and correct enzyme cutting sites according to antibody gene sequence information published by Kabat, V-base2 and IMGT websites, synthesizing primers by Kinsley Biotechnology Ltd, and performing PAGE purification, wherein the primer sequences are shown in tables 1 and 2:

TABLE 1 primers for amplifying human heavy chain variable region

Note: r ═ a/G, Y ═ C/T, M ═ a/C, K ═ G/T, S ═ C/G, W ═ a/T, H ═ a/C/T, B ═ C/G/T, V ═ a/C/G, D ═ a/G/T

TABLE 2 primers for amplifying the variable region of human light chain

Primer name	Sequence (5 '→ 3')
		h-k_F1	ggtggcggtggtagtgctagcGAWAYWGTGATGACMCAGYMTC
h-k_F2	ggtggcggtggtagtgctagcGAAGTTATGTTGATGCAGTCTCT
		h-k_F3	ggtggcggtggtagtgctagcGAWGTTGTGMTGACAYRGTC
h-k_F4	ggtggcggtggtagtgctagcRHCATCYRGATGACCCAGTCTSCA
		h-k_F5	ggtggcggtggtagtgctagcRACATCCAGATGAATTCAGTCTCC
h-k_F6	ggtggcggtggtagtgctagcGAMATTSTGYTGACHCAGWCTCCA
		h-k_F7	ggtggcggtggtagtgctagcGABAYTGTGATSRCCCAGACTCCA
h-k_F8	ggtggcggtggtagtgctagcRMCATCCAGDTGAYBCAGYCTCC
		h-k_F9	ggtggcggtggtagtgctagcGATGCTGYGAWGACCCAACCTCCA
h-k_R1	ATGAGTTTTTGTTCTGCGGCCGCTTTGATHTCCASYTTGGTCCCH
		h-k_R2	ATGAGTTTTTGTTCTGCGGCCGCTTTAATCTCCAGTCGTGTCCCT
h-λ_F1	ggtggcggtggtagtgctagcCAGYCTGYKCTGACYCAGV
		h-λ_F2	ggtggcggtggtagtgctagcCAGGCAGGGCWGACTCAGCMMC
h-λ_F3	ggtggcggtggtagtgctagcCAGCYTGTGCTGACTCARTCRYCC
		h-λ_F4	ggtggcggtggtagtgctagcCACGTTATACTGACTCAACCGCCC
h-λ_F5	ggtggcggtggtagtgctagcCRGCCYGTGCTGACTCARCYGCC
		h-λ_F6	ggtggcggtggtagtgctagcCTGSCTGTGCTRACTCAGGCCCC
h-λ_F7	ggtggcggtggtagtgctagcCAGBCTGTGCTGACTCAGCCR
		h-λ_F8	ggtggcggtggtagtgctagcCAGTCTGTSBTGACGCAGCCGCC
h-λ_R1	ATGAGTTTTTGTTCTGCGGCCGCKAGGACGGTSACCTTGGTSCCAST
		h-λ_R2	ATGAGTTTTTGTTCTGCGGCCGCTAGGACGGTCAGCTCSGTCCCCTC
h-λ_R3	ATGAGTTTTTGTTCTGCGGCCGCKAGGRCGGTCAGCTKGGTSCCTCC
		h-λ_R4	ATGAGTTTTTGTTCTGCGGCCGCTAAAATGATCAGCTGGGTTCCTCC

Using OligodT primer in PrimeScriptTMII first strand cDNA Synthesis kit (6210A) from Takara as template, first strand cDNA was synthesized by reverse transcription, and the heavy chain variable region VH and light chain variable region V L of antibody gene were amplified by PCR technique using the primers in the above Table 1 and Table 2 under conditions of 95 ℃ for 5min, and 25 cycles of denaturation at 95 ℃ for 30S, annealing at 56 ℃ for 30S, extension at 72 ℃ for 1min, and extension at 72 ℃ for 3min in the last cycle, and the detection by electrophoresis was carried out after completion of amplification, as shown in FIG. 4.

4. Construction of human VH and V L Single chain antibody libraries

V L was first cloned into pCANTAB5E-SF vector by NheI and NotI, and then VH was cloned into the vector of the previous step by NcoI and XhoI.

Cutting the pCANTAB5E-SF carrier and the amplified and purified PCR product of the human light chain variable region by NheI and NotI, connecting the cut 4.4 mu g carrier with 1 mu g fragment, purifying the connection product, transferring the connection product to 10 200 mu l TG1 electric transfer competent cells (2.5kV, 2cm specification electric shock cup), adding SOC, culturing at 37 ℃ for 1h, taking a small amount of resuscitating bacteria to calculate the storage capacity, coating the rest bacteria liquid on 10 phi 15cm plates, culturing overnight, calculating the storage capacity at the next day to be 2.2 × 10⁸And (3) carrying out single cloning, scraping and washing a phi 15cm flat plate by using 50ml of 2YT to obtain a light chain antibody library, taking part of bacterial liquid to prepare small quality-improved particles, adding glycerol with the final concentration of 30% (V/V) into the residual library bacterial liquid, and storing at-80 ℃.

The light chain antibody library plasmid and the amplified and purified human heavy chain variable region PCR product were digested with NcoI and XhoI. Carrying out enzyme digestion on 5.8 mu g of vector and 1.2 mu g of fragmentConnecting, electrically transferring the purified connecting product to 20 200 μ lTG1 cells (2.5kV, 2cm electric shock cup), adding SOC, culturing at 37 deg.C for 1h, collecting part of resuscitating bacteria, diluting, applying to 20 plates (phi 15 cm), culturing overnight, and calculating the storage capacity of 5.6 × 10⁸And (3) carrying out single cloning, scraping and washing a phi 15cm flat plate by using 50ml of 2YT to obtain humanized VH and V L single-chain antibody libraries, preparing small quality-improved particles by taking part of bacterial liquid, adding glycerol with the final concentration of 30% (V/V) into the residual library bacterial liquid, and storing at-80 ℃.

Example 3: phage display and screening of humanized antibody libraries

1. Phage display and panning of antibody libraries

The human VH and V L single-chain antibody library bacterial liquid with 100 times of library capacity is taken to inoculate 880ml of 2YT-AG culture medium (containing 100 mu g/ml ampicillin and 2% glucose), cultured at 37 ℃ and 200rpm until OD600 is 0.5-0.6, added with helper phage with 100 times of cell density, infected for 1.5h, centrifuged to collect thalli, resuspended in 400ml of 2YT-AK culture medium (containing 100 mu g/ml ampicillin and 75 mu g/ml kanamycin), and cultured at 30 ℃ and 200rpm overnight.

Centrifuging the culture of the previous step at 10000g and 4 ℃ for 20min, collecting supernatant, adding 1/4 volumes of PEG/NaCl, mixing, standing on ice for 1h, centrifuging at 12000g and 4 ℃ for 25min, discarding supernatant, reversely covering a centrifuge tube on flat paper to remove liquid, re-suspending phage precipitate with 2ml of precooled 1 × PBS, centrifuging at 12000g and 4 ℃ for 10min, transferring supernatant to a new 15ml centrifuge tube, adding 3% BSA (bovine serum albumin) to obtain a first round of initial phage, coating an immune tube with VEGFR2-His (Sino biological) as antigen, sealing with 2% M-PBS, and adding 10% BSA¹³The first round of initial phage was antibody antigen binding, PBST was used to wash away unbound phage, 1ml of Glycine-HCl (pH2.2) was used to elute phage, the eluted phage was re-infected with TG1, amplification of the eluted product was performed, and PEG/NaCl precipitation was used to purify phage for the next round of screening. And 4 rounds of enrichment screening of the phage library are carried out, the antigen amount is sequentially reduced, the washing strength is sequentially enhanced, and the titer of each round of elution products is determined.

2. Induced expression of monoclonal and E L ISA screening

Performing limited dilution and plate coating on the bacterial liquid subjected to the 1 st to 4 th rounds of elutriation, and culturing overnight; selecting the monoclonal to be cultured in a 96-hole deep-hole plate which is subpackaged with 0.5 ml/hole of 2YT-AG culture medium for overnight; the overnight culture was then transferred to a 96-well deep-well plate containing 0.5 ml/well of 2YT-AG medium at a ratio of 1:10, cultured until OD600 became 0.5-0.6, and the cells were collected by centrifugation at 3000g, resuspended in 2YT-AI medium (containing 100. mu.g/ml ampicillin and 1mM IPTG), induced overnight at 30 ℃, and the supernatant was transferred by centrifugation the next day to a clean 96-well deep-well plate and added with 3% BSA at final concentration to obtain a monoclonal phage sample.

VEGFR2-His is used as an antigen to coat a 96-hole enzyme label plate, after being sealed, 50 mu l of monoclonal phage sample is added into each hole, and the mixture is incubated for 1h at 25 ℃; then adding 200 mul PBST into each hole, oscillating for 5-10S, discarding the solution, and repeating for 3-5 times; adding 50 mu l of PBS (PBS) diluent of the anti-M13-HRP antibody into each well, and incubating for 1h at 25 ℃; then adding 200 mul PBST into each hole, oscillating for 5-10S, discarding the solution, and repeating for 5 times; adding 50 μ l of TMB color developing solution into each well, developing for 3-10 min (the specific color developing time depends on the color developing speed), and adding 50 μ l of 1M H into each well₂SO₄The method comprises the steps of terminating color development, measuring an OD450 value by using an enzyme-labeling instrument, selecting 360E L ISA test positive samples according to the data of monoclonal phage E L ISA, carrying out competitive E L ISA screening, diluting a positive control antibody to 200 mu g/ml, mixing 50 mu l of diluent with 50 mu l of monoclonal phage samples, carrying out E L ISA of the above steps, displaying that 30 monoclonal antibodies compete with the positive control antibody in epitope, selecting the clones for sequencing, taking overnight culture bacterial liquid in a 96-well plate deep-well plate 2YT-AG culture medium, carrying out sequencing analysis, and finally obtaining a unique variable region sequence of the monoclonal antibody as shown in SEQ ID NO: 1-12.

Example 4: full Length antibody preparation

1. Construction of full-Length antibody transient transfection expression vector

pGS003-hIgG1CH and pGS003-hIgKC L were selected as expression vectors for constructing heavy and light chains of the full-length anti-human VEGFR2 antibody, respectively, primers were designed based on the gene sequences of VH and V L and the multiple cloning sites in the vectors, after PCR amplification, 6 VH and 6V L antibody genes were cloned into pGS003-hIgG1CH and pGS003-hIgKC L, respectively, using an in vitro recombination method (iMulli multi-fragment recombinant cloning kit), as shown in Table 3, after identifying the correct insertion of the antibody genes by sequencing, the recombinant expression vector was transformed into E.coli TOP10F ', a single colony was selected and inoculated into L B medium containing 100. mu.g/ml ampicillin, shaking cultured for 16 hours at 37 ℃, plasmid was extracted using Zymo Research's endotoxin removal large-extraction kit, finally the plasmid was dissolved in 1ml of ultrapure water, and the plasmid concentration was determined by spectrophotometry and OD 260/280. OD 260.260/OD 9.8. OD 9. to OD 8. the plasmid.

TABLE 3 list of heavy and light chain transient transfection expression vectors

Heavy chain expression vector name	Heavy chain variable region sequence	Light chain expression vector name	Light chain variable region sequence
				H1	SEQ ID NO:1	L1	SEQ ID NO:7
H2	SEQ ID NO:2	L2	SEQ ID NO:8
				H3	SEQ ID NO:3	L3	SEQ ID NO:9
H4	SEQ ID NO:4	L4	SEQ ID NO:10
				H5	SEQ ID NO:5	L5	SEQ ID NO:11
H6	SEQ ID NO:6	L6	SEQ ID NO:12

2. Transfection, expression and detection in mammalian cells 293E

After the 6 heavy chain expression vectors and the 6 light chain expression vectors are combined in pairs (36 combinations in total), transient transfection expression evaluation of a 2ml293E system is carried out, expression quantity and detection value of E L ISA of the antibody combined with VEGFR2 are detected, and the results are shown in Table 4. according to the expression quantity and the detection value of E L ISA of the antibody combined with VEGFR2, 6 full-length antibodies of relatively low EC50 value and relatively high E L ISARmax value, preferably H1L 3, H2L 3, H3L 6, H5L 3, H5L 5 and H6L 6 are selected and named as 043HAb-1, 043HAb-2, 043HAb-3, 043HAb-4, 043HAb-5 and 043 HAb-6.

Tables 4 and 6 × 6 combine expression levels of transient transfection expression of full-length antibody minibody lines, EC50 values and E L ISA Rmax values

Transient transfection expression of 6 preferred antibodies using 293E in Freestyle Medium 24 hours prior to transfection, 0.5 × 10 was seeded in 1L cell culture flasks⁶Cell/ml 293E cells 300ml, 5% CO at 37 ℃₂Shaking and culturing at 120rpm in an incubator. When in transfection, 300 mu l of 293fectin is firstly added into 5.7ml of OPTIMEM, and after fully and uniformly mixing, the mixture is incubated for 2 minutes at room temperature; at the same time, 300. mu.g each of the heavy and light chain expression plasmids was diluted to 6ml using OptiMEM. Mixing the diluted transfection reagent and plasmid, incubating at room temperature for 15 min, adding the mixture into cells, mixing, and adding 5% CO at 37 deg.C₂Shaking at 120rpm in incubator for 7 days.

3. Purification and detection of antibodies

The cell culture broth was centrifuged at 2000g for 20min, the supernatant was collected, and the amount of antibody expression in the supernatant was determined by Octet, see Table 5.

TABLE 5,6 detection of expression level of 300ml of preferred antibody transient transfection expression

The supernatant was filtered through a 0.22 micron filter, then passed through a MabSelect Sure affinity chromatography column (GE), 20mM citric acid-sodium citrate, pH3.0, eluted, pH adjusted to neutral with 1M Tris base, and adjusted to an isotonic solution by addition of 10 xPBS. The protein was purified by SDS-PAGE using 4-20% gradient gel (Kinsley Biotechnology Ltd.) as shown in lanes 7-12 of FIG. 5. And (3) taking the purified antibody, analyzing the purified product by SDS-PAGE under a reducing condition, and identifying the property and purity of the antibody, wherein the heavy chain is 50KD and the light chain is 25KD under the reducing condition, and the result is shown in lanes 1-6 of figure 1.

Example 5 preferred antibody epitope Competition E L ISA

1. Coating: human VEGFR2-His was diluted to 1. mu.g/ml with PBS and added to a 96-well microplate in an amount of 50. mu.l per well and incubated overnight at 4 ℃.

2. And (3) sealing: after washing the plate three times, it was blocked with 3% BSA, 250. mu.l per well and incubated at 37 ℃ for 2 hours.

3. Adding a candidate antibody: before washing the plate, a positive antibody marked by 0.5 mu g/ml Biotin (Biotin) is mixed with 12 gradient samples diluted 2 times with the initial concentration of 0.15mg/ml (the initial concentration of the positive antibody is 0.3mg/ml), and after washing the plate 3 times, a candidate antibody sample or a positive control or a negative control is added. 50 μ l per well at 25 ℃ for 1 hour.

4. Adding a secondary antibody: after washing the plate 3 times, HRP-labeled streptavidin (1:10000) was added thereto in an amount of 50. mu.l per well, and the reaction was carried out at 25 ℃ for 1 hour.

5. Color development: after washing the plate 4 times, adding TMB developing solution, 50 μ l per well, and developing for 10 minutes in the dark at room temperature.

6. And (4) terminating: the reaction was stopped by directly adding 50. mu.l of stop solution per well.

7. And (3) detection: immediately after the reaction is terminated, the microplate is placed into a microplate reader, the OD value is measured at 450nm, and the original data is stored and collated as shown in FIG. 6. 043HAb-1 ~ 43HAb-6 preferred antibody and positive antibody have antigen epitope competition phenomenon.

Example 6: preferred antibody affinity assays

The affinity of the full length anti-human VEGFR2 antibody was measured using a Biacore T200 instrument. The specific method comprises the following steps: human antibodies were coupled to a CM5 biosensing chip (GE) using the human antibody capture kit from GE Healthcare, on which anti-human Fc antibodies capture either the preferred or positive antibodies, human VEGFR2 at different concentrations flowed through the candidate antibody on the chip at a flow rate of 30. mu.l/min, human VEGFR2 bound to the candidate antibody for a binding time of 120s and a dissociation time of 300 s. The results of the affinity constants obtained using biaevaluation software (GE) for the kinetic fit are shown in table 6 below, which shows that all 6 preferred antibodies have good binding activity to human VEGFR 2.

TABLE 6 results of affinity determination of candidate antibodies with human VEGFR2

Name of antibody	Ka(1/Ms)	Kd(1/s)	KD(M)
				043HAb-1	1.19E+05	6.72E-05	5.67E-10
043HAb-2	2.33E+05	4.92E-05	2.11E-10
				043HAb-3	1.75E+05	8.71E-05	4.98E-10
043HAb-4	1.53E+05	6.48E-05	4.25E-10
				043HAb-5	1.75E+05	8.69E-05	4.97E-10
043HAb-6	2.71E+05	7.51E-05	2.77E-10
				Positive antibody	1.03E+05	4.68E-05	4.57E-10

Note E + 05: × 10⁵；E-05：×10^-5；E-10：×10^-10

Example 7: preferred antibody cell Activity assays

Human umbilical vein vascular endothelial cells (HUVECs) express VEGFR2, and preferably the antibody or positive antibody blocks the interaction of VEGFR2 with VEGF by specifically binding to VEGFR2, thereby inhibiting the proliferation of HUVECs. Therefore, the cell activity can be detected by observing the proliferation of the HUVEC cells inhibited by the drug, and the specific steps are as follows.

1. Preparing a reference substance solution and a test solution: taking a reference substance (positive antibody) and a test substance (preferably antibody), and pre-diluting the reference substance and the test substance to 800 mu g/ml by using an EBMTM-2 basic culture medium according to the protein labeling amount, wherein the maximum dilution multiple does not exceed 25 times each time. The control and test samples were diluted in 96-well cell culture plates with a gradient of 800 μ g/ml as the maximum concentration: the first 8 concentrations were diluted by 4-fold gradient and the second 2 concentrations were diluted by 10-fold gradient, each concentration was at least 2 replicates, and the final concentrations were 200000, 50000, 12500, 3125, 781.25, 195.31, 48.83, 12.20, 1.22, 0.12ng/ml respectively.

2. Cell plating, taking HUVEC cells with cell viability rate greater than or equal to 90% within 5 generations (including 5 generations), and preparing into cell plating solution containing 2.5 × 10 per 1ml by EBM analysis culture medium⁴～3.5×10⁴The cell suspension of each cell was added to a 96-well cell culture plate-1 at 100. mu.l/well, and the mixture was incubated at 37 ℃ with 5% CO₂The culture was carried out in an incubator for about 3.5 h.

3. Reference solution and test solutionLoading sample: and taking the 96-well cell culture plate-1 after the cells are attached to the wall, sequentially adding the diluent in the 96-well cell culture plate in the step of preparing the reference substance solution and the test substance solution into the 96-well cell culture plate-1 by 50 mu l/well, and adding the EBMTM-2 basal medium into the rest wells by 50 mu l/well. After the sample addition operation was completed, the cell culture plate-1 was placed at 37 ℃ in 5% CO₂The culture was carried out in an incubator for about 1.5 h.

4. Loading of VEGF: VEGF working solution is added into the corresponding holes of the B th to G th rows of the 96-hole cell culture plate-1 by 50 mul/hole, and EBMTM-2 basal medium is added into the rest holes by 50 mul/hole. After the sample loading operation was completed, the 96-well cell culture plate-1 was placed at 37 ℃ and 5% CO₂Culturing for 92-94 h in an incubator.

5. Color development and plate reading operation: adding 20 μ l CCK-8 color developing solution into each well, placing 96-well cell culture plate-1 at 37 deg.C and 5% CO₂The incubator continues to incubate for about 3.5 h. And taking out the 96-well cell culture plate-1, placing the plate in an enzyme labeling instrument, setting vibration for 30S before reading the plate, and measuring absorbance by taking 450nm as a detection wavelength and 620nm as a reference wavelength.

Taking the concentration of the reference substance or the test substance as an abscissa and the average absorbance as an ordinate, and adopting a computer program or a four-parameter regression calculation method to process, calculating the semi-effective inhibition concentration (IC50) of the reference substance and the test substance, wherein the specific calculation formula is as follows:

specific assay results are shown in Table 7 and FIG. 7, and under the present experimental conditions, 50% inhibitory concentration of 043HAb-1, 043HAb-2, 043HAb-3, 043HAb-4, 043HAb-5, 043HAb-6, i.e., IC50 values were 5.57, 5.42, 4.86, 4.13, 2.72, 0.965. mu.g/ml, respectively, for VEGF-induced HUVEC cell proliferation, whereas under the same experimental conditions, IC50 values of positive antibody were 2.92, 2.75, 2.47, 2.06, 3.07, 2.76. mu.g/ml, respectively, showing that 043HAb-6 has higher in vitro anti-vascular endothelial cell proliferation activity than positive antibody.

TABLE 7 results of detection of cell Activity of preferred antibodies

Reference substance	IC50(nM？)	Sample (I)	IC50	Activity of
					Positive antibody	2.92	043HAb-1	5.57	52.4％
Positive antibody	2.75	043HAb-2	5.42	50.7％
					Positive antibody	2.47	043HAb-3	4.86	50.8％
Positive antibody	2.06	043HAb-4	4.13	49.9％
					Positive antibody	3.07	043HAb-5	2.72	112.9％
Positive antibody	2.76	043HAb-6	0.965	286.0％

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

SEQUENCE LISTING

<110> Changchun golden race pharmaceutical Limited liability company

<120> anti-VEGFR 2 monoclonal antibody and application thereof

<130>MP1706486

<160>15

<170>PatentIn version 3.3

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Gln Val Lys Leu Leu Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Thr Ser Gly Ser Asn Ile Glu Asp Phe

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asp Pro Glu Asp Gly Asp Ser Asp Tyr Ala Pro MetPhe

50 55 60

Gln Gly Arg Val Thr Met Thr Ala Asp Thr Ser Ile Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Gly Leu Arg Phe Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Tyr Tyr Gly Glu Tyr Glu Gly Tyr Trp Gly Gln Gly Thr Ala

100 105 110

Val Thr Val Ser Ser

115

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Gln Val Lys Leu Leu Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

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Ser Val Lys Val Ser Cys Lys Thr Ser Gly Ser Asn Ile Glu Asp Phe

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Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asp Pro Glu Asp Gly Asp Ser Asp Tyr Ala Pro Met Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Ala Asp Thr Ser Ile Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Gly Leu Arg Phe Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Tyr Tyr Gly Glu Tyr Glu Gly Tyr Trp Gly Gln Gly Thr Ala

100 105 110

Val Thr Val Ser Ser

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Gln Val Lys Leu Leu Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

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Ser Val Lys Val Ser Cys Lys Thr Ser Gly Ser Asn Ile Lys Asp Ile

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Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asp Pro Gly Asp Gly Asp Ser Asp Tyr Ala Pro Met Phe

50 55 60

Gln Asp Arg Val Thr Met Thr Ala Asp Ala Ser Ile Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Gly Leu Arg Phe Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ile Ala Tyr Tyr Gly Glu Tyr Glu Gly Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

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Gln Val Lys Leu Leu Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

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Ser Val Lys Val Ser Cys Lys Thr Ser Gly Phe Asn Ile Glu Asp Ile

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Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asp Pro Glu Asp Gly Asp Ser Asp Tyr Ala Pro Lys Phe

50 55 60

Gln Asp Arg Val Thr Met Thr Ala Asp Thr Ser Ile Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Gly Leu Arg Phe Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Tyr Tyr Gly Glu Tyr Glu Gly Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

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Gln Val Lys Leu Leu Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

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Ser Val Lys Val Ser Cys Lys Thr Ser Gly Ser Asn Ile Glu Asp Phe

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Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asp Pro Gly Asp Gly Asp Ser Asp Tyr Ala Pro Met Phe

50 55 60

Gln Asp Arg Val Thr Met Thr Ala Asp Thr Ser Ile Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Gly Leu Arg Phe Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ile Ala Tyr Tyr Gly Glu Tyr Glu Gly Tyr Trp Gly Gln Gly Thr Thr

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Val Thr Val Ser Ser

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Gln Val Lys Leu Leu Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

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Ser Val Lys Val Ser Cys Lys Thr Ser Gly Pro Asn Ile Glu Asp Ile

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Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

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Gly Trp Ile Asp Pro Gly Tyr Gly Asp Ser Asp Tyr Ala Pro Lys Phe

50 55 60

Gln Asp Arg Val Thr Met Thr Ala Asp Thr Ser Ile Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Gly Leu Arg Phe Asp Asp Thr Ala Val Tyr Tyr Cys

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Ile Ala Tyr Tyr Gly Glu Tyr Asp Gly Tyr Trp Gly Gln Gly Thr Thr

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Val Thr Val Ser Ser

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Asp Ile Glu Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

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Asp His Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Asn Val

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His Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Phe

35 40 45

Ser Thr Arg Asn Leu Val Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Ser Leu Gln Ser Glu

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Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ile His Pro Tyr Thr

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Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

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Asp Ile Glu Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

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Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Gly Val Ser Asn Thr

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His Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Ser Thr Arg Asn Leu Ala Asn Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ile Tyr Pro Tyr Thr

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Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

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Asp Ile Glu Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

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Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Gly Val Ser Asn Thr

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His Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

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Ser Thr Arg Asn Leu Val Asn Gly Val Pro Ser Arg Phe Ser Gly Ser

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Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Ser Leu Gln Pro Glu

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Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser His Pro Tyr Thr

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Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

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Asp Ile Glu Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

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Asp Arg Val Thr Ile Thr Cys Ile Ala Ser Ser Gly Val Ser Asn Thr

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His Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Ser Thr Arg Asn Leu Ala Asn Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Pro Glu

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Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Tyr Thr

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Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

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Asp Ile Glu Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

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Asp Arg Val Thr Ile Thr Cys Ile Ala Ser Ser Gly Val Ser Asn Thr

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His Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Ser Thr Ser Ser Leu Ala Asn Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

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Asp Ile Glu Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

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Val Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Gly Val Ser Tyr Thr

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His Arg Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Ser Thr Arg Asn Leu Val Asn Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp His Ser Leu Thr Ile Ser Ser Leu Gln Pro Asp

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Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ile His Pro Tyr Thr

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Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

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Gln Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Gly Ser Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Thr Ser Gly Phe Asn Ile Lys Asp Phe

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Ser Asp Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Lys Ala Thr Met Thr Ala Asp Ser Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Tyr Tyr Gly Asp Tyr Glu Gly Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

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Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Phe Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 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>15

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<212>DNA

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ggcccagccg gccatggcct aaggatccta aaccgtctcg agcggtggtg gcggtagtgg 60

cggtggtggt agcggtggcg gtggtagtgc tagcgacatc ctgcagtgaa aggcggccgc 120

Claims

1. An anti-VEGFR 2 monoclonal antibody having the following heavy chain variable region and light chain variable region:

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 6, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 12.

2. The anti-VEGFR 2 monoclonal antibody of claim 1, further comprising a constant region that is any one of human IgG1, IgG2, IgG3, or IgG 4.

3. An expression vector comprising nucleotides encoding the monoclonal antibody of claim 1 or 2.

4. A host cell transformed or transfected with the expression vector of claim 3, which is a prokaryotic cell or a eukaryotic cell.

5. A conjugate comprising the anti-VEGFR 2 monoclonal antibody of claim 1 or 2 covalently linked to a chemical or biological marker.

6. A conjugate formed by coupling the anti-VEGFR 2 monoclonal antibody of claim 1 or 2 and/or the conjugate of claim 5 to a solid or semi-solid medium.

7. A pharmaceutical composition comprising the anti-VEGFR 2 monoclonal antibody of claim 1 or 2 and/or the conjugate of claim 5 and/or the conjugate of claim 6.

8. Use of the anti-VEGFR 2 monoclonal antibody of claim 1 or 2 and/or the conjugate of claim 5 and/or the conjugate of claim 6 and/or the pharmaceutical composition of claim 7 for the manufacture of a medicament for the treatment of a disease associated with angiogenesis, said disease being non-small cell lung cancer, metastatic non-small cell lung cancer, hepatocellular carcinoma, metastatic hepatocellular carcinoma, HER 2-negative metastatic breast cancer, metastatic gastric adenocarcinoma, metastatic colorectal cancer, metastatic melanoma and/or metastatic renal cell carcinoma.