CN104231079A - Antibody targeting high affinity bFGF receptor binding site and application thereof - Google Patents

Abstract

The invention discloses a murine monoclonal antibody targeting human basic fibroblast growth factor (basic fibroblast growth factor, bFGF) high affinity receptor-fibroblast growth factor receptor (fibroblast growth factor receptor, FGFR) binding site and its application . The antibody variable region genes include heavy chain variable region genes and light chain variable region genes. The present invention successfully prepares a mouse-derived monoclonal antibody that can target the binding site of bFGF high-affinity receptor FGFR, can specifically bind to bFGF, hinder its binding to FGFR, and then block the transduction of bFGF/FGFR signal pathway, thereby Inhibit the proliferation of tumor cells, can inhibit tumor growth in vivo, and have high affinity and specificity, and can be used to develop antibody drugs for tumor diagnosis and treatment.

Description

Antibody targeting bFGF high-affinity receptor binding site and application thereof

technical field

The present invention belongs to the field of biotechnology. More specifically, it relates to an antibody targeting bFGF high-affinity receptor binding site and its application.

Background technique

Basic fibroblast growth factor (bFGF), also known as fibroblast growth factor-2 (fibroblast growth factor 2, FGF2), is one of the 21 structurally related fibroblast growth factor protein family members. The relative molecular weight of bFGF is 18KD, 22 KD, 22.5 KD, 24KD and 34KD. At present, the 18 KD subtype of bFGF has been studied more, which is also the main active form of bFGF. bFGF is one of the most important cytokines stimulating angiogenesis after vascular endothelial growth factor (VEGF), which is involved in a variety of tumors (such as glioma, rhabdomyoma, leukemia, kidney cancer, Pneumocytoma, melanoma, liver cancer, prostate cancer, colon cancer, etc.), a considerable part of the tumors have high expression of bFGF and its high-affinity receptor-fibroblast growth factor receptor (FGFR). Express. bFGF not only promotes local angiogenesis through paracrine and autocrine pathways, but also directly acts on tumor cells to promote their proliferation and accelerate tumor invasion and metastasis. In vivo and in vitro experiments have confirmed that bFGF can also promote the secretion of various growth factors, and has a synergistic effect with VEGF and platelet-derived growth factor (PDGF) to jointly promote tumorigenesis.

Members of the FGF family mainly transmit signals to cells by binding to their high-affinity receptor-fibroblast growth factor receptor (FGFR) to exert their biological activities. This receptor is also called receptor tyrosine kinase (Receptor tyrosine kinase, RTKs), and there are four proteins: FGFR-1, FGFR-2, FGFR-3, and FGFR-4. The complete FGFR molecule consists of an extracellular ligand-binding domain, a transmembrane domain, and an intracellular domain containing a catalytically active protein tyrosine center. The extracellular ligand-binding domain consists of three IgG-like domains, called D1, D2, and D3. FGF mainly binds to the D2 and D3 regions. In addition to not binding to FGFR-2b, bFGF can bind to several other FGFR subtypes, including FGFR-1b, FGFR-1c, FGFR-2c, FGFR-3b, FGFR-3c, and FGR-4. Different receptor subtypes play different roles in the process of tumorigenesis and development. Among them, FGFR-1 is highly expressed on the surface of vascular endothelial cells and various tumor cells, and is the main receptor of bFGF. The bFGF/FGFR signaling pathway plays an important role in the regulation of cell proliferation and growth. Studies have confirmed that a variety of tumors can promote tumor growth by altering the bFGF/FGFR signaling pathway through bFGF overexpression. The 407-433 amino acids of the juxtamembrane domain of FGFR-1 bind to the mooring proteins FRS2α and FRS2β of the FRS2 family when there is no bFGF stimulation. After being stimulated by bFGF, the two are phosphorylated to recruit the Grb2/Sos complex, thereby It leads to the activation of Ras/MAPK signaling pathway, and its downstream target proteins are some transcriptional proteins and anti-apoptotic proteins, which can regulate cell survival and proliferation. In addition, PI3K/Akt pathway and PKA pathway are also important signaling pathways involved in the function of bFGF, and multiple signaling pathways regulate each other and play a role together.

In order to study the role of VEGF, PD-ECGF (platelet derived endothelial cell growth factor, platelet derived endothelial cell growth factor), bFGF and IL-8 in melanoma angiogenesis and metastasis, Erinar K et al. The antibody of the factor was studied, and it was found that the antibody could significantly inhibit the spontaneous metastasis of melanoma and the formation of lung clones. Studies by Loilome et al. have shown that bFGF monoclonal antibody can effectively inhibit the growth of glioma by blocking the FGF signaling pathway. Studies by Rofstad et al. have shown that bFGF antibody can inhibit the growth, angiogenesis and metastasis of transplanted melanoma in mice. Studies by Zeng Shibin and others have shown that bFGF antibodies can neutralize free bFGF and reduce the concentration of bFGF in tissues, and can also bind to bFGF/FGFR complexes that have bound to membrane receptors, thereby blocking the binding of bFGF to its receptors and subsequent Cell signal transduction, inhibit bFGF-induced melanoma cell proliferation and angiogenesis. Studies by Xie Qingxiang and others showed that bFGF monoclonal antibody inhibited the growth of bladder cancer in a concentration-dependent manner in nude mice. bFGF and FGFR-1 are highly expressed in a variety of tumor tissues including melanoma, lung cancer, and breast cancer, and are related to tumor metastasis, staging, and prognosis. Blocking the effect of bFGF has significant anti-tumor and anti-tumor angiogenesis As a result, the bFGF/FGFR system has become a new target for anti-tumor therapy.

Therefore, it is of great significance to explore the technology and method of blocking bFGF/FGFR signaling pathway for anti-tumor therapy.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method that can block the bFGF/FGFR signaling pathway, thereby inhibiting tumor cell proliferation and tumor growth in vivo.

The purpose of the present invention is to provide a mouse-derived monoclonal antibody targeting human basic fibroblast growth factor (basic fibroblast growth factor, bFGF) high-affinity receptor binding site, which can specifically bind fibroblasts Growth factor receptors compete for binding to human basic fibroblast growth factor (bFGF), thereby inhibiting tumor cell proliferation and tumor growth in vivo.

Another object of the present invention is to provide the application of the mouse-derived monoclonal antibody targeting bFGF high-affinity receptor binding site.

The above object of the present invention is achieved through the following technical solutions:

The invention discloses a mouse-derived monoclonal antibody targeting the high-affinity receptor binding site of human basic fibroblast growth factor (basic fibroblast growth factor, bFGF). The high-affinity receptor refers to fibroblast growth factor receptor (FGFR).

The variable region gene of the antibody comprises a heavy chain variable region gene and a light chain variable region gene; the gene sequence of the heavy chain variable region is shown in SEQ ID NO.1, and the light chain variable region gene sequence is shown in SEQ ID NO.1. The gene sequence is shown in SEQ ID NO.2.

The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4.

The heavy chain variable region consists of 366 bases, encoding 122 amino acids, and the variable region contains 3 CDR (complementary determinant) regions. CDR1 encodes 5 amino acids, CDR2 encodes 13 amino acids, and CDR3 encodes 14 amino acids. The framework region of the variable region has a homology of 91.9% with other murine antibodies, and the three CDR regions are specific sequences, which are different from the CDR regions of the heavy chain variable region of other murine antibodies.

The light chain variable region consists of 318 bases, encoding 106 amino acids, and the variable region contains 3 CDR (complementary determinant) regions. CDR1 encodes 11 amino acids, CDR2 encodes 11 amino acids, and CDR3 encodes 8 amino acids. The framework region of the variable region shares 92.6% homology with other murine antibodies, while the three CDR regions are specific sequences, which are different from the CDR regions of the light chain variable regions of other murine antibodies.

The function of the anti-human bFGF mouse-derived monoclonal antibody protein obtained in the present invention is composed of antibody light and heavy chain variable region complementarity determining regions (complementarity determining regions CDRs) CDR1, CDR2, CDR3 (the functional active region of the present invention) Determined by the specific nucleotide sequence, its corresponding amino acid sequence constitutes the region where the antibody specifically targets the binding site of the bFGF high-affinity receptor FGFR.

The monoclonal antibody has the characteristics of targeting the binding site of human bFGF high-affinity receptor FGFR, can compete with FGFR for binding bFGF, and can be clinically (1) used for tumor diagnosis; (2) inhibit tumor cell proliferation; (3) ) inhibit tumor growth and metastasis in vivo.

The application of the mouse-derived monoclonal antibody targeting the high-affinity receptor binding site of human basic fibroblast growth factor bFGF in the preparation of drugs for diagnosis and treatment of tumors is also within the protection scope of the present invention.

Preferably, the tumor is a tumor regulated or controlled by the bFGF/FGFR signaling pathway.

More preferably, the tumor is lung cancer, melanoma, liver cancer, head and neck tumor, prostate cancer, colon cancer, breast cancer, thyroid cancer, gastric cancer, human brain glioma, bladder cancer or meningioma.

The present invention has the following beneficial effects:

The invention discloses a murine monoclonal antibody targeting the binding site of human basic fibroblast growth factor bFGF high-affinity receptor, which can compete with fibroblast growth factor receptor for binding to human basic fibroblast Cell growth factor (bFGF) can block the combination of bFGF and FGFR expressed on the surface of tumor cells, and block the signal transduction of bFGF/FGFR pathway, thereby effectively inhibiting the proliferation of tumor cells.

The advantages of the development of monoclonal antibodies targeting the binding site of the bFGF high-affinity receptor FGFR are mainly reflected in: (1) specifically binding to the epitope of the binding site of the bFGF high-affinity receptor FGFR, which can block the bFGF/FGFR signaling pathway , to inhibit the proliferation of tumor cells and the growth and metastasis of tumors; (2) Since bFGF is a human protein, it cannot induce human immune response very well, and bFGF is closely related to tumors, so it cannot be directly used to develop vaccines; The application of the invented antibody well complements this deficiency.

Description of drawings

Figure 1 is the SDS-PAGE detection result of the purification of bFGF murine monoclonal antibody E12; Lane 1 is the Marker, and lanes 2 and 3 are the purified E12 monoclonal antibody.

Fig. 2 is a graph showing the titer detection ELISA results of bFGF mouse monoclonal antibody E12.

Figure 3 is a graph showing the results of competition ELISA between bFGF murine monoclonal antibody E12 and FGFR-1C.

Figure 4 shows the three CDR (complementarity determinant) regions of the antibody heavy chain variable region.

Figure 5 shows the three CDR (complementarity determinant) regions of the antibody light chain variable region.

Fig. 6 is a graph showing the results of bFGF murine monoclonal antibody E12 inhibiting tumor cell proliferation in vitro.

Fig. 7 is a diagram showing the inhibition results of bFGF murine monoclonal antibody E12 on LL/2 subcutaneous tumor in vivo.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the present invention are commercially available.

Example 1 Antigen Preparation

Recombinant human basic fibroblast growth factor (bFGF), purchased from PEPROTECH, Cat. No. #100-18B, expressed in Escherichia coli, has a molecular weight of 17.2kDa and consists of 154 amino acid residues. bFGF can promote cell proliferation by binding to FGFR 1b, 1c, 2c, 3c and 4 for signal transmission. Before use, dissolve powdered recombinant human basic fibroblast growth factor in 0.1M PBS to a concentration of 1.0mg/ml, and store at -20°C.

Example 2 Preparation of Monoclonal Antibody

The preparation method of anti-recombinant human basic fibroblast growth factor (bFGF) mouse-derived monoclonal antibody is as follows:

1. Animal immunity

(1) Initial immunization: subcutaneous injection of 60 μg antigen Freund's complete adjuvant in multiple points;

(2) Second immunization: 3 weeks later, the same amount of antigen as the first immunization plus Freund's incomplete adjuvant was injected subcutaneously at multiple points;

(3) Three times of immunization: 2 weeks later, the same amount of antigen as the first immunization plus Freund's incomplete adjuvant was injected subcutaneously at multiple points (blood was collected 10 days later to measure its potency);

(4) Booster immunization: 2 weeks later, the same amount of antigen as the initial immunization was injected intraperitoneally without adjuvant;

(5) After 3 days, the spleen was taken for fusion.

2. Cell Fusion

(1) Take logarithmic growth myeloma cells SP2/0, use RPMI1640 basic medium (RPMI Medium 1640 basic, purchased from gibco, product number 8114056, add penicillin and streptomycin before use, add 1mL containing 100U penicillin per 100mL medium with streptomycin double antibody) to wash and count after blowing;

(2) Take the mouse spleen, wash and grind with RPMI1640 basal medium, prepare a single spleen cell suspension, and count;

(3) Mix myeloma cells and spleen cells at a ratio of 1:10, and centrifuge at 1000rpm for 7min;

(4) Discard the supernatant, suck up the remaining liquid with a dropper, add 1 mL of polyethylene glycol (PEG-2000) with a molecular weight of 2000 in a water bath at 37°C within 1 min, let it stand for 90 seconds, and add it within 2 to 4 min 15mL RPMI 1640 basal medium to terminate the reaction;

(5) Centrifuge at 1000rpm for 7min, discard the supernatant, and gently suspend with 100mL 10% fetal bovine serum RPMI 1640 (including HAT); add dropwise to a 96-well plate pre-coated with feeder cells, 100μL/well; 37°C, cultured in a 5% CO ₂ incubator.

3. Screening and cloning of fusion cells

(1) Take the cell culture supernatant on the sixth day after cell fusion, use the ELISA plate coated with 20ng/well bFGF for indirect ELISA detection, select the positive wells and use biotin-labeled FGFR1βⅢC as a competitor, and screen by competitive ELISA Positive wells with better competition effect;

(2) The hybridoma cells in the screened positive wells were cloned by the limiting dilution method, and a hybridoma cell line stably secreting bFGF monoclonal antibody was obtained after three times of subcloning screening.

4. Mass preparation of monoclonal antibodies

(1) 10-week-old female Balb/c mice were intraperitoneally injected with 0.5 mL of Freund's incomplete adjuvant;

(2) 1 week later, 4* ¹⁰⁵ hybridoma cells were inoculated into the peritoneal cavity of mice, and ascites could be induced 7 to 10 days after cell inoculation;

(3) Extract ascites when there is ascites as much as possible;

(4) After an interval of 1 to 2 days, after ascites regenerates and accumulates, pump again in the same way. After pumping, the ascites is centrifuged at 3,000 rpm for 10 minutes, and the supernatant is frozen and stored at -20°C.

5. Purification of monoclonal antibodies

(1) Centrifuge the ascites at 1200 rpm for 30 minutes at 4°C, and take the supernatant;

(2) Add an equal volume of saturated ammonium sulfate solution drop by drop with stirring, and let stand at 4°C for overnight precipitation;

(3) Centrifuge the pellet at 7500rpm, 4°C for 30min, discard the supernatant, and dissolve the pellet with 0.1M PBS;

(4) Put the ascites in a dialysis bag, dialyze in 0.1M PBS for 6 hours, and change the dialysate every 2 hours;

(5) Purify protein with Protein G affinity chromatography column.

The purification steps are as follows:

(1) Column packing

Equilibrate the column: Use 0.1M PBS (5-10 times the column volume) to equilibrate the column, wash out 20% ethanol in the column, and set the nucleic acid protein analyzer to zero;

(2) Sample loading: Before loading the sample, turn off the constant flow pump first, and then slowly load the sample. When the A value starts to rise, collect the liquid (miscellaneous protein) to prevent the protein from not binding to the column. Add it when the sample is about to be loaded 0.1M PBS dilution, so that the protein is almost completely loaded on the column;

(3) Elution: When the A value drops to "0", use the eluent to elute the target protein, and collect the liquid when the A value starts to rise (because the protein is negatively charged and weakly alkaline, add a certain Measure the neutralizing solution to keep the pH of the collected solution above 7.0);

(4) Equilibrate the column: when the A value is "0", use 0.1M PBS (more than 5 times the column volume) to pass through the column;

(5) Preservation: Pass through the column with 20% ethanol (5 times the column volume) and store the column.

6. After the protein was concentrated by ultrafiltration, take a small amount for SDS-PAGE gel electrophoresis to identify the purity (as shown in Figure 1), ELISA to detect the antibody titer (as shown in Figure 2), and compete with FGFR-1βⅢC by ELISA (as shown in Figure 1). As shown in Figure 3) other aliquots were stored at -20°C.

7. After research, the variable region gene of the obtained antibody includes a heavy chain variable region gene and a light chain variable region gene; the gene sequence of the heavy chain variable region is shown in SEQ ID NO.1, and the light chain variable region gene sequence is shown in SEQ ID NO.1. The gene sequence of the variable region is shown in SEQ ID NO.2.

The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4.

The heavy chain variable region consists of 366 bases, encoding 122 amino acids, and the variable region contains 3 CDR (complementary determinant) regions. CDR1 encodes 5 amino acids, CDR2 encodes 13 amino acids, and CDR3 encodes 14 amino acids (as shown in Figure 4). The framework region of the variable region has a homology of 91.9% with other murine antibodies, and the three CDR regions are specific sequences, which are different from the CDR regions of the heavy chain variable region of other murine antibodies.

The light chain variable region consists of 318 bases, encoding 106 amino acids, and the variable region contains 3 CDR (complementary determinant) regions. CDR1 encodes 11 amino acids, CDR2 encodes 11 amino acids, and CDR3 encodes 8 amino acids (as shown in Figure 5). The framework region of the variable region shares 92.6% homology with other murine antibodies, while the three CDR regions are specific sequences, which are different from the CDR regions of the light chain variable regions of other murine antibodies.

Example 3 The inhibitory effect of the monoclonal antibody of the present invention on tumor cells

1. Proliferation experiment of mouse lung cancer cell line LL/2 tumor cell

(1) Prepare cell suspension and count cells;

(2) Cells were seeded in 96-well plates at 2500 cells/well, and cultured in 10% DMEM medium for 24 h;

(3) Aspirate the medium, add E12 monoclonal antibody diluted with 1% FBS DMEM medium, non-immunized mouse IgG respectively, and 1% FBS DMEM medium as a control, 100 μL/well;

(4) Incubate at 37°C, 5% CO ₂ for 72 hours, add CCK-8 color developing solution (purchased from Nippon Dojin Chemical, Cat. No. C0038) to develop color for 1 hour;

(5) Detect the absorbance at 450nm.

The results are shown in Figure 6, the E12 monoclonal antibody of the present invention has obvious inhibitory effect on LL/2 tumor cells.

2. In vivo tumor inhibition experiment

(1) Ten 10-week-old BABL/c mice (purchased from the Animal Center of Southern Medical University) were randomly divided into control group and experimental group, with 5 mice in each group;

(2) Inoculate 5*10 ⁵ LL/2 cells subcutaneously on the shoulder of each mouse;

(3) After 6 days, the tumor began to form, and the body weight of each mouse was recorded, and the long and short diameters of the tumor were measured;

(4) The experimental group was injected with antibody (1mg/mouse) at 3 points around the tumor, and the control group was injected with an equal volume of 0.1M PBS at 3 points around the tumor;

(5) Antibody injections were performed every 3 days to measure body weight and tumor size, a total of 6 times;

(6) The mice were sacrificed 3 days after the sixth antibody injection, the tumor was removed, and the tumor growth rate inhibited by the antibody was calculated.

The tumor size measurement results are shown in Figure 7. Compared with the control group, the E12 monoclonal antibody of the present invention has a significant inhibitory effect on the growth of lung cancer tumors, and the inhibition rate on tumor growth is 41.93%.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

SEQUENCE LISTING

the

<110> Jinan University

the

<120> An antibody targeting bFGF high-affinity receptor binding site and its application

the

<130>

the

<160> 4

the

<170> PatentIn version 3.3

the

<210> 1

<211> 366

<212> DNA

<213> Gene sequence of antibody heavy chain variable region

the

<400> 1

gttcaactgc agcagtctgg agctgggctg gtgaaacccg ggacatcagt gaagctgtcc 60

the

tgcaaggctt ctggcttcac cttcactgag tatgttatac actgggtaaa acagaggtct 120

the

ggacagggtc ttgagtggat tgggtggttt taccctggaa gtggtactat aaagtacaat 180

the

gagaaattca aggacaaggc cagattgact gcggacaaat cctccagcac agtctatatg 240

the

gagcttaata gattgacatc tgaagactct ggggtttatt tctgtgcaag acacgaagac 300

the

gacgagtatg gtgacaccgg ctggtttggt tactggggcc aagggactct ggtcactgtc 360

the

tcctca 366

the

the

<210> 2

<211> 318

<212> DNA

<213> Gene sequence of antibody light chain variable region

the

<400> 2

gacatccaga tgacacagtc tccagcctcc ctatctgcat ctgtgggagc aactgtcacc 60

the

atcacatgtc gagcaagtga gaatattac agttattag catggtatca acagaaacag 120

the

ggaaaatctc ctcagctcct ggtctataat acaaaaatct taggagaagg tgtgccatca 180

the

aggttcagtg gcagtggatc aggcactcag ttttctctga agatcaacag cctgcagcct 240

the

gaagattttg ggacttatta ctgtcaacat cattttgata cttacacgtt cggagggggg 300

the

accaagctgg aaataaaa 318

the

the

<210> 3

<211> 122

<212> PRT

<213> Amino acid sequence of antibody heavy chain variable region

the

<400> 3

the

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

1 5 10 15

the

the

Val Lys Leu Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Glu Tyr Val

20 25 30

the

the

Ile His Trp Val Lys Gln Arg Ser Gly Gln Gly Leu Glu Trp Ile Gly

35 40 45 45

the

the

Trp Phe Tyr Pro Gly Ser Gly Thr Ile Lys Tyr Asn Glu Lys Phe Lys

50 55 60 60

the

the

Asp Lys Ala Arg Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Tyr Met

65 70 75 80

the

the

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

85 90 95

the

the

Arg His Glu Asp Asp Glu Tyr Gly Asp Thr Gly Trp Phe Gly Tyr Trp

100 105 110

the

the

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

the

the

<210> 4

<211> 106

<212> PRT

<213> Antibody light chain variable region amino acid sequence

the

<400> 4

the

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

1 5 10 15

the

the

Ala Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr

20 25 30

the

the

Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val

35 40 45 45

the

the

Tyr Asn Thr Lys Ile Leu Gly Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60 60

the

the

Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro

65 70 75 80

the

the

Glu Asp Phe Gly Thr Tyr Tyr Cys Gln His His Phe Asp Thr Tyr Thr

85 90 95

the

the

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

                                 

Claims (6)

1. a monoclonal antibody for targeted human Prostatropin high-affinity receptor binding site epi-position, is characterized in that, the variable region gene of described antibody comprises heavy chain variable region gene and chain variable region gene; The gene order of described variable region of heavy chain is as shown in SEQ ID NO.1, and the gene order of described variable region of light chain is as shown in SEQ ID NO.2.

2. monoclonal antibody according to claim 1, it is characterized in that, the aminoacid sequence of described variable region of heavy chain is as shown in SEQ ID NO.3, and described chain variable region amino acid sequence is as shown in SEQ ID NO.4.

3. monoclonal antibody described in claim 1 blocks the application in the medicine of bFGF/FGFR signal path in preparation.

4. monoclonal antibody described in claim 1 is preparing the application in tumor diagnosis and therapy medicine.

5. apply according to claim 4, it is characterized in that, described tumour is the tumour that bFGF/FGFR signal path regulates or controls.

6. apply according to claim 4, it is characterized in that, described tumour is lung cancer, melanoma, liver cancer, tumor of head and neck, prostate cancer, colorectal carcinoma, mammary cancer, thyroid carcinoma, cancer of the stomach, human glioma, bladder cancer or meningioma.